U.S. patent application number 12/214304 was filed with the patent office on 2009-01-08 for humanized immunoglubulin reactive with alph4beta7 integrin.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Mary M. Bendig, S. Tarran Jones, Walter Newman, Paul D. Ponath, Douglas J. Ringler, Jose Saldanha.
Application Number | 20090011464 12/214304 |
Document ID | / |
Family ID | 24814673 |
Filed Date | 2009-01-08 |
United States Patent
Application |
20090011464 |
Kind Code |
A1 |
Ponath; Paul D. ; et
al. |
January 8, 2009 |
Humanized immunoglubulin reactive with alph4beta7 integrin
Abstract
The present invention relates to isolated nucleic acids encoding
humanized immunoglobulins having binding specificity for
.alpha.4.beta.7 integrin, isolated nucleic acids encoding a
humanized immunoglobulin heavy chain and isolated nucleic acids
encoding a humanized light chain having binding specificity for
.alpha.4.beta.7 integrin. The invention also relates to expression
vectors and host cells comprising a nucleotide sequence which
encodes a humanized immunoglobulin or antigen-binding fragment
thereof having binding specificity for .alpha.4.beta.7. The
invention further relates to methods of preparing a humanized
immunoglobulin, humanized immunoglobulin heavy chain and humanized
immunoglobulin light chain that has binding specificity for
.alpha.4.beta.7 integrin.
Inventors: |
Ponath; Paul D.; (Boston,
MA) ; Ringler; Douglas J.; (Revere, MA) ;
Jones; S. Tarran; (Radlett, GB) ; Newman; Walter;
(Boston, MA) ; Saldanha; Jose; (Enfield, GB)
; Bendig; Mary M.; (London, GB) |
Correspondence
Address: |
MCDERMOTT WILL & EMERY LLP
28 STATE STREET
BOSTON
MA
02109-1775
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
Cambridge
MA
|
Family ID: |
24814673 |
Appl. No.: |
12/214304 |
Filed: |
June 18, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11511164 |
Aug 28, 2006 |
7402410 |
|
|
12214304 |
|
|
|
|
08700737 |
Aug 15, 1996 |
7147851 |
|
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11511164 |
|
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|
Current U.S.
Class: |
435/69.6 ;
435/320.1; 435/328; 536/23.53 |
Current CPC
Class: |
A61P 37/00 20180101;
C07K 16/2839 20130101; A61K 38/00 20130101; A61P 1/04 20180101;
A61P 29/00 20180101; C07K 2317/24 20130101; C07K 2319/00 20130101;
C07K 14/70503 20130101; A61P 1/00 20180101 |
Class at
Publication: |
435/69.6 ;
536/23.53; 435/320.1; 435/328 |
International
Class: |
C12P 21/00 20060101
C12P021/00; C07H 21/00 20060101 C07H021/00; C12N 15/63 20060101
C12N015/63; C12N 5/00 20060101 C12N005/00 |
Claims
1. An isolated nucleic acid molecule encoding a humanized
immunoglobulin or antigen-binding fragment thereof having binding
specificity for .alpha.4.beta.7 integrin, wherein said humanized
immunoglobulin or antigen-binding fragment thereof comprises a
heavy chain and a light chain, said heavy chain comprising the
variable region of SEQ ID NO:19, and said light chain comprising
the variable region of SEQ ID NO:21.
2. The isolated nucleic acid molecule of claim 1, wherein said
isolated nucleic acid molecule comprises a first nucleotide
sequence encoding the variable region of SEQ ID NO:19, and a second
nucleotide sequence encoding the variable region of SEQ ID
NO:21.
3. The isolated nucleic acid molecule of claim 2, wherein said
first nucleotide sequence further encodes an IgG constant region or
a modified IgG constant region with reduced Fc receptor binding
activity, reduced complement fixing activity, or reduced Fc
receptor binding activity and reduced complement fixing
activity.
4. An expression vector comprising a nucleotide sequence which
encodes a humanized immunoglobulin or antigen-binding fragment
thereof having binding specificity for .alpha.4.beta.7 integrin,
wherein said humanized immunoglobulin or antigen-binding fragment
thereof comprises a heavy chain and a light chain, said heavy chain
comprising the variable region of SEQ ID NO:19, and said light
chain comprising the variable region of SEQ ID NO:21.
5. The expression vector of claim 4, wherein said expression vector
comprises a first nucleotide sequence that encodes the variable
region of SEQ ID NO:19, and a second nucleotide sequence that
encodes the variable region of SEQ ID NO:21.
6. The expression vector of claim 5, wherein said first nucleotide
sequence further encodes an IgG constant region or a modified IgG
constant region with reduced Fc receptor binding activity, reduced
complement fixing activity, or reduced Fc receptor binding activity
and reduced complement fixing activity.
7. A host cell comprising the expression vector of claim 4.
8. A method of preparing a humanized immunoglobulin or
antigen-binding fragment thereof, comprising maintaining a host
cell of claim 7 under conditions suitable for expression of a
humanized immunoglobulin or antigen-binding fragment thereof,
whereby a humanized immunoglobulin or antigen-binding fragment
thereof is produced.
9. The method of claim 8, further comprising isolating the
humanized immunoglobulin or antigen-binding fragment.
10. An isolated nucleic acid molecule encoding a humanized
immunoglobulin heavy chain or antigen-binding portion thereof,
wherein said heavy chain or antigen-binding portion thereof
comprises the variable region of SEQ ID NO:19.
11. The isolated nucleic acid molecule of claim 10, wherein said
heavy chain comprises an IgG constant region or a modified IgG
constant region with reduced Fc receptor binding activity, reduced
complement fixing activity, or reduced Fc receptor binding activity
and reduced complement fixing activity.
12. An expression vector comprising a nucleotide sequence that
encodes a humanized immunoglobulin heavy chain or antigen-binding
portion thereof, wherein said heavy chain or antigen-binding
portion thereof comprises the variable region of SEQ ID NO:19.
13. The expression vector of claim 12, wherein said heavy chain
comprises an IgG constant region or a modified IgG constant region
with reduced Fc receptor binding activity, reduced complement
fixing activity, or reduced Fc receptor binding activity and
reduced complement fixing activity.
14. A host cell comprising the expression vector of claim 12.
15. A method of preparing a humanized immunoglobulin heavy chain or
antigen-binding portion thereof, comprising maintaining a host cell
of claim 14 under conditions suitable for expression of a humanized
immunoglobulin heavy chain or antigen-binding portion thereof,
whereby a humanized immunoglobulin heavy chain or antigen-binding
portion thereof is produced.
16. The method of claim 15, further comprising isolating the
humanized immunoglobulin heavy chain or antigen-binding portion
thereof.
17. An isolated nucleic acid molecule encoding a humanized
immunoglobulin light chain or antigen-binding portion thereof,
wherein said light chain or antigen-binding portion thereof
comprises the variable region of SEQ ID NO:21.
18. The isolated nucleic acid molecule of claim 17, wherein said
humanized immunoglobulin light chain further comprises a constant
region.
19. An expression vector encoding a humanized immunoglobulin light
chain or antigen-binding portion thereof, wherein said light chain
or antigen-binding portion thereof comprises the variable region of
SEQ ID NO:21.
20. The expression vector of claim 19, wherein said humanized
immunoglobulin light chain further comprises a constant region.
21. A host cell comprising the expression vector of claim 19.
22. A method of preparing a humanized immunoglobulin light chain or
antigen-binding portion thereof, comprising maintaining a host cell
of claim 21 under conditions suitable for expression of a humanized
immunoglobulin light chain or antigen-binding portion thereof,
whereby a humanized immunoglobulin light chain or antigen-binding
portion thereof is produced.
23. The method of claim 22, further comprising isolating the
humanized immunoglobulin light chain or antigen-binding portion
thereof.
Description
RELATED APPLICATION(S)
[0001] This application is a continuation of U.S. application Ser.
No. 11/511,164, filed Aug. 28, 2006, which is a divisional of U.S.
application Ser. No. 08/700,737, filed Aug. 15, 1996. The entire
teachings of the above applications are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] Integrin receptors are important for regulating both
lymphocyte recirculation and recruitment to sites of inflammation
(Carlos, T. M. and Harlan, J. M., Blood, 84:2068-2101 (1994)). The
human .alpha.4.beta.7 integrin has several ligands, one of which is
the mucosal vascular addressin MAdCAM-1 (Berlin, C., et al., Cell
74:185-195 (1993); Erle, D. J., et al., J. Immunol. 153:517-528
(1994)) expressed on high endothelial venules in mesenteric lymph
nodes and Peyer's patches (Streeter, P. R., et al., Nature
331:41-46 (1988)). As such, the .alpha.4.beta.7 integrin acts as a
homing receptor that mediates lymphocyte migration to intestinal
mucosal lymphoid tissue (Schweighoffer, T., et al., J. Immunol.
151:717-729 (1993)). In addition, the .alpha.4.beta.7 integrin
interacts with fibronectin and vascular cell adhesion molecule-1
(VCAM-1).
[0003] Inflammatory bowel disease (IBD), such as ulcerative colitis
and Crohn's disease, for example, can be a debilitating and
progressive disease involving inflammation of the gastrointestinal
tract. Affecting an estimated two million people in the United
States alone, symptoms include abdominal pain, cramping, diarrhea
and rectal bleeding. IBD treatments have included anti-inflammatory
drugs (such as, corticosteroids and sulfasalazine),
immunosuppressive drugs (such as, 6-mercaptopurine, cyclosporine
and azathioprine) and surgery (such as, colectomy). Podolsky, New
Engl. J. Med., 325:928-937 (1991) and Podolsky, New Engl. J. Med.,
325:1008-1016 (1991).
[0004] Antibodies against human .alpha.4.beta.7 integrin, such as
murine monoclonal antibody (mAb Act-1), interfere with
.alpha.4.beta.7 integrin binding to mucosal addressin cell adhesion
molecule-1 (MAdCAM-1) present on high endothelial venules in
mucosal lymph nodes. Act-1 was originally isolated by Lazarovits,
A. I., et al., J. Immunol. 133:1857-1862 (1984), from mice
immunized with human tetanus toxoid-specific T lymphocytes and was
reported to be a mouse IgG1/K antibody. More recent analysis of the
antibody by Schweighoffer, T., et al., J. Immunol. 151:717-729
(1993) demonstrated that it can bind to a subset of human memory
CD4+ T lymphocytes which selectively express the .alpha.4.beta.7
integrin. However, a serious problem with using murine antibodies
for therapeutic applications in humans is that they are highly
immunogenic in humans and quickly induce a human anti-murine
antibody response (HAMA), which reduces the efficacy of the mouse
antibody in patients and can prevent continued administration. The
HAMA response results in rapid clearance of the mouse antibody,
severely limiting any therapeutic benefit.
[0005] Thus, a need exists for improved therapeutic approaches to
inflammatory bowel diseases.
SUMMARY OF THE INVENTION
[0006] The present invention relates to a humanized immunoglobulin
having binding specificity for .alpha.4.beta.7 integrin, said
immunoglobulin comprising an antigen binding region of nonhuman
origin (e.g., rodent) and at least a portion of an immunoglobulin
of human origin (e.g., a human framework region, a human constant
region of the gamma type). In one embodiment, the humanized
immunoglobulin described herein can compete with murine Act-1 or
LDP-02 (see, e.g., Example 4) for binding to .alpha.4.beta.7
integrin. In a preferred embodiment, the antigen binding region of
the humanized immunoglobulin is derived from Act-1 monoclonal
antibody (e.g., LDP-02, an immunoglobulin comprising the variable
regions of the light and heavy chains shown in FIG. 11 (SEQ ID
NO:19) and FIG. 12 (SEQ ID NO:21), respectively).
[0007] For example, the humanized immunoglobulin can comprise an
antigen binding region comprising a complementarity determining
region (CDR) of nonhuman origin, and a framework region (FR)
derived from a human framework region. In one aspect, the humanized
immunoglobulin having binding specificity for .alpha.4.beta.7
integrin, comprises a light chain comprising a CDR derived from an
antibody of nonhuman origin which binds .alpha.4.beta.7 and a FR
derived from a light chain of human origin (e.g., GM607'CL), and a
heavy chain comprising a CDR derived from an antibody of nonhuman
origin which binds .alpha.4.beta.7 and a FR derived from a heavy
chain of human origin (e.g., 21/28'CL). In another aspect, the
light chain comprises three CDRs derived from the light chain of
the Act-1 antibody, and the heavy chain comprises three CDRs
derived from the heavy chain of the Act-1 antibody.
[0008] The present invention also relates to humanized
immunoglobulin light chains (e.g., comprising CDR1, CDR2 and CDR3
of the light chain of the Act-1 antibody, and a human light chain
FR), and to humanized immunoglobulin heavy chains (e.g., comprising
CDR1, CDR2 and CDR3 of the heavy chain of the Act-1 antibody, and a
human heavy chain FR). In a preferred embodiment, the invention
relates to humanized heavy and light chains described herein (e.g.,
a humanized light chain comprising the variable region of the light
chain shown in FIG. 7 (SEQ ID NO:12), a humanized heavy chain
comprising the variable region of the heavy chain shown in FIG. 9
(SEQ ID NO:15), a humanized light chain comprising the variable
region of the light chain shown in FIG. 12 (SEQ ID NO:21), a
humanized heavy chain comprising the variable region of the heavy
chain shown in FIG. 11 (SEQ ID NO:19)). Also encompassed are
humanized immunoglobulins comprising one or more humanized light
and/or heavy chains.
[0009] The invention further relates to isolated nucleic acids
comprising a sequence which encodes a humanized immunoglobulin of
the present invention (e.g., a single chain antibody), as well as
to isolated nucleic acids comprising a sequence which encodes a
humanized immunoglobulin light chain (e.g., SEQ ID NO:20) or heavy
chain (e.g., SEQ ID NO:18) of the present invention. For example,
the present invention provides a fused gene encoding a humanized
immunoglobulin light or heavy chain comprising a first nucleic acid
sequence encoding an antigen binding region derived from murine
Act-1 monoclonal antibody; and a second nucleic acid sequence
encoding at least a portion of a constant region of an
immunoglobulin of human origin.
[0010] The present invention further relates to a construct
comprising a nucleic acid encoding a humanized immunoglobulin
having binding specificity for .alpha.4.beta.7 integrin or a chain
of such an immunoglobulin. For example, an expression vector
comprising a fused gene encoding a humanized immunoglobulin light
chain, comprising a nucleotide sequence encoding a CDR derived from
a light chain of a nonhuman antibody having binding specificity for
.alpha.4.beta.7 integrin, and a framework region derived from a
light chain of human origin, is provided. An expression vector
comprising a fused gene encoding a humanized immunoglobulin heavy
chain, comprising a nucleotide sequence encoding a CDR derived from
a heavy chain of a nonhuman antibody having binding specificity for
.alpha.4.beta.7 integrin, and a framework region derived from a
heavy chain of human origin is another example of such a
construct.
[0011] The present invention also relates to a host cell comprising
a nucleic acid of the present invention, including one or more
constructs comprising a nucleic acid of the present invention. In
one embodiment, the invention relates to a host cell comprising a
first recombinant nucleic acid encoding a humanized immunoglobulin
light chain, and a second recombinant nucleic acid encoding a
humanized immunoglobulin heavy chain, said first nucleic acid
comprising a nucleotide sequence encoding a CDR derived from the
light chain of murine Act-1 antibody and a framework region derived
from a light chain of human origin; and said second nucleic acid
comprising a nucleotide sequence encoding a CDR derived from the
heavy chain of murine Act-1 antibody and a framework region derived
from a heavy chain of human origin.
[0012] The present invention also provides a method of preparing a
humanized immunoglobulin comprising maintaining a host cell of the
present invention under conditions appropriate for expression of a
humanized immunoglobulin, whereby a humanized immunoglobulin
chain(s) is expressed and a humanized immunoglobulin is produced.
The method can further comprise the step of isolating the humanized
immunoglobulin.
[0013] The humanized immunoglobulins of the present invention can
be less immunogenic than their murine or other nonhuman
counterparts. Thus, the humanized immunoglobulins described herein
can be used as therapeutic agents in humans, for example to control
lymphocyte homing to mucosal lymphoid tissue, thereby, reducing
inflammatory responses in the gut.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is an illustration of a consensus DNA sequence (SEQ
ID NO:1) and deduced amino acid sequence (SEQ ID NO:2) comprising
the variable region determined from several independent mouse heavy
chain variable region clones.
[0015] FIG. 2 is an illustration of a nucleotide sequence (SEQ ID
NO:3) and deduced amino acid sequence (SEQ ID NO:4) comprising a
portion of the variable region sequence determined from an
independent mouse heavy chain variable region clone designated
H2B#34.
[0016] FIG. 3 is an illustration of a nucleotide sequence (SEQ ID
NO: 5) and deduced amino acid sequence (SEQ ID NO:6) comprising the
variable region of several independent mouse light chain variable
region clones. The position of two mutations made to introduce a
KasI site for cloning are indicated.
[0017] FIG. 4A is a fluorescence plot illustrating the ability of
the murine Act-1 mAb and a mouse isotype-matched irrelevant control
antibody (MOPC 21; IgG1, kappa) to stain HuT 78 cells which express
.alpha.4.beta.7 integrin.
[0018] FIG. 4B is a fluorescence plot illustrating the ability of
(i) chimeric Act-1 antibody, (ii) a human isotype-matched
irrelevant control antibody (IgG1, kappa), and (iii) a COS-7 cell
supernatant, to stain HuT 78 cells which express .alpha.4.beta.7
integrin.
[0019] FIG. 5 is an alignment of the amino acid sequences of the
mouse Act-1 light chain variable region ("Act-1.v1") (SEQ ID NO:7)
and of the human GM 607'CL light chain variable region (SEQ ID
NO:8). Identical amino acids are indicated by a vertical line and
similar amino acids are indicated by four or two dots, depending on
the degree of similarity. CDRs are bracketed and labelled, and
residues are numbered sequentially.
[0020] FIG. 6 is an alignment of the amino acid sequences of the
mouse Act-1 heavy chain variable region ("Act-1.vh") (SEQ ID NO:9)
and of the human 21/28'CL heavy chain variable region (SEQ ID
NO:10). Identical amino acids are indicated by a vertical line and
similar amino acids are indicated by four or two dots, depending on
the degree of similarity. CDRs are bracketed and labelled, and
residues are numbered sequentially.
[0021] FIG. 7 is an illustration of the nucleotide sequence of a
double stranded nucleic acid (coding strand, SEQ ID NO:11;
non-coding strand, SEQ ID NO:64) encoding the mouse Act-1 light
chain variable region joined to the mouse Act-1 light chain signal
peptide sequence, and the deduced amino acid sequence of the Act-1
light chain variable region joined to the mouse Act-1 light chain
signal peptide sequence (SEQ ID NO:12).
[0022] FIG. 8 is an illustration of the nucleotide sequence of a
double stranded nucleic acid (coding strand, SEQ ID NO:13;
non-coding strand, SEQ ID NO:65) encoding the mature human GM607'CL
antibody kappa light chain variable region, and the deduced amino
acid sequence of the mature human GM607'CL antibody kappa light
chain variable region (SEQ ID NO:8).
[0023] FIG. 9 is an illustration of the nucleotide sequence of a
double stranded nucleic acid (coding strand SEQ ID NO:14;
non-coding strand, SEQ ID NO: 66) encoding the mouse Act-1 antibody
heavy chain and signal peptide, and the deduced amino acid sequence
of the mouse Act-1 antibody heavy chain variable region and heavy
chain signal peptide (SEQ ID NO:15). The nucleotide sequence of the
variable region is joined to a nucleotide sequence which encodes a
deduced mouse Act-1 heavy chain signal peptide sequence, to yield a
composite sequence. (The identity of the primer which amplified the
heavy chain region was deduced from the degenerate sequence, and an
amino acid sequence for the signal peptide was derived from the
primer, downsteam sequence and sequences of other signal peptides.
The signal peptide shown may not be identical to that of the Act-1
hybridoma.)
[0024] FIG. 10 is an illustration of the nucleotide sequence of a
double stranded nucleic acid (coding strand SEQ ID NO:16;
non-coding strand, SEQ ID NO: 67) encoding the human 21/28'CL
antibody heavy chain and signal peptide, and the deduced amino acid
sequence of the human 21/28'CL antibody heavy chain variable region
and heavy chain signal peptide (SEQ ID NO:17). The nucleotide
sequence encoding the variable region is joined to a nucleotide
sequence which encodes a signal peptide sequence derived from the
V.sub.H of human antibody HG3'CL (Rechavi, G., et al., Proc. Natl.
Acad. Sci., USA 80:855-859 (1983)), to yield a composite
sequence.
[0025] FIG. 11 is an illustration of the nucleotide sequence (SEQ
ID NO:18) and amino acid sequence (SEQ ID NO:19) of a portion of
the heavy chain of a humanized Act-1 antibody (LDP-02) with a heavy
chain signal peptide.
[0026] FIG. 12 is an illustration of the nucleotide sequence (SEQ
ID NO:20) and amino acid sequence (SEQ ID NO:21) of a portion of
the light chain of a humanized Act-1 antibody (LDP-02) with a light
chain signal peptide.
[0027] FIG. 13 is an illustration of the nucleotide sequences of
overlapping, complementary oligonucleotides designated L1-L6 (SEQ
ID NOS:22-27), which were used to make the light chain of a
humanized Act-1 immunoglobulin (LDP-02), and the nucleotide
sequences of overlapping, complementary oligonucleotides designated
H1-H10 (SEQ ID NOS:28-37), which were used to make the heavy chain
of the humanized Act-1 immunoglobulin.
[0028] FIG. 14 is a fluorescence plot illustrating the staining of
HuT 78 cells using a mouse-human Act-1 chimeric immunoglobulin, a
humanized Act-1 immunoglobulin or an irrelevant, human
isotype-matched control antibody (IgG1, kappa).
[0029] FIG. 15 is a graph illustrating the results of a titration
of biotinylated murine Act-1 and humanized Act-1 (LDP-02/3A9/LOT#1,
Example 4) performed by flow cytometry on Hut-78 cells.
[0030] FIG. 16 is a graph illustrating the competitive inhibition
of binding of biotinylated murine Act-1 by murine Act-1 or a
humanized Act-1 immunoglobulin (LDP-02/3A9/LOT#1, Example 4),
compared with control murine IgG1 or human IgG1.
[0031] FIG. 17 is a graph illustrating the results of a
.sup.51chromium release assay for complement mediated cell lysis of
human peripheral blood mononuclear cells in the presence of (a)
CAMPATH-1H, (b) CAMPATH-1G, (c) human IgG1, (d) LDP-02/3A9/Lot#1
(Example 4), or (e) LDP-01 (humanized anti-CD18, Fc-mutated) at
concentrations of 50, 25, 5, 2.5, and 0.5 .mu.g/ml.
[0032] FIGS. 18A-18B are graphs illustrating the results of an
adhesion assay monitoring the inhibition of adhesion by murine
Act-1 (FIG. 18A), murine IgG1 (FIG. 18A), LDP-02/3A9/Lot#1 (FIG.
18B) or human IgG1 (FIG. 18B) of .alpha.4.beta.7-bearing cells
(RPMI 8866) and a human MAdCAM-1-Ig chimera (immunoadhesin).
[0033] FIG. 19 is a graph comparing the staining of HuT 78 cells
using (a) LDP-02 (Fc-mutated), (b) a derivative of LDP-02
(Fc-mutated) having a mutation in the light chain (MV4) plus a
double mutation in the heavy chain (R38K, A40R), or (c) an
irrelevant, human isotype matched control antibody (IgG1,
kappa).
DETAILED DESCRIPTION OF THE INVENTION
[0034] The present invention relates to a humanized immunoglobulin
having binding specificity for .alpha.4.beta.7 integrin, comprising
an antigen binding region of nonhuman origin and at least a portion
of an immunoglobulin of human origin. Preferably, the humanized
immunoglobulins can bind .alpha.4.beta.7 integrin with an affinity
of at least about 10.sup.7 M.sup.-1, preferably at least about
10.sup.8 M.sup.-1, and more preferably at least about 10.sup.9
M.sup.-1. In one embodiment, the humanized immunoglobulin includes
an antigen binding region of nonhuman origin which binds
.alpha.4.beta.7 integrin and a constant region derived from a human
constant region. In another embodiment, the humanized
immunoglobulin which binds .alpha.4.beta.7 integrin comprises a
complementarity determining region of nonhuman origin and a
variable framework region of human origin, and optionally, a
constant region of human origin. For example, the humanized
immunoglobulin can comprise a heavy chain and a light chain,
wherein the light chain comprises a complementarity determining
region derived from an antibody of nonhuman origin which binds
.alpha.4.beta.7 integrin and a framework region derived from a
light chain of human origin, and the heavy chain comprises a
complementarity determining region derived from an antibody of
nonhuman origin which binds .alpha.4.beta.7 integrin and a
framework region derived from a heavy chain of human origin.
[0035] The present invention also relates to a humanized
immunoglobulin light chain or a humanized immunoglobulin heavy
chain. In one embodiment, the invention relates to a humanized
light chain comprising a light chain CDR (i.e., one or more CDRs)
of nonhuman origin and a human light chain framework region. In
another embodiment, the present invention relates to a humanized
immunoglobulin heavy chain comprising a heavy chain CDR (i.e., one
or more CDRs) of nonhuman origin and a human heavy chain framework
region. The CDRs can be derived from a nonhuman immunoglobulin.
[0036] Naturally occurring immunoglobulins have a common core
structure in which two identical light chains (about 24 kD) and two
identical heavy chains (about 55 or 70 kD) form a tetramer. The
amino-terminal portion of each chain is known as the variable (V)
region and can be distinguished from the more conserved constant
(C) regions of the remainder of each chain. Within the variable
region of the light chain is a C-terminal portion known as the J
region. Within the variable region of the heavy chain, there is a D
region in addition to the J region. Most of the amino acid sequence
variation in immunoglobulins is confined to three separate
locations in the V regions known as hypervariable regions or
complementarity determining regions (CDRs) which are directly
involved in antigen binding. Proceeding from the amino-terminus,
these regions are designated CDR1, CDR2 and CDR3, respectively. The
CDRs are held in place by more conserved framework regions (FRs).
Proceeding from the amino-terminus, these regions are designated
FR1, FR2, FR3, and FR4, respectively. The locations of CDR and FR
regions and a numbering system have been defined by Kabat et al.
(Kabat, E. A. et al., Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, U.S. Government Printing Office (1991); see also Tables 3
and 4).
[0037] Human immunoglobulins can be divided into classes and
subclasses, depending on the isotype of the heavy chain. The
classes include IgG, IgM, IgA, IgD and IgE, in which the heavy
chains are of the gamma (.gamma.), mu (.mu.), alpha (.alpha.),
delta (.delta.) or epsilon (.epsilon.) type, respectively.
Subclasses include IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, in which
the heavy chains are of the .gamma.1, .gamma.2, .gamma.3, .gamma.4,
.alpha.1 and .alpha.2 type, respectively. Human immunoglobulin
molecules of a selected class or subclass may contain either a
kappa (.kappa.) or lambda (.lamda.) light chain. See e.g., Cellular
and Molecular Immunology, Wonsiewicz, M. J., Ed., Chapter 45, pp.
41-50, W. B. Saunders Co, Philadelphia, Pa. (1991); Nisonoff, A.,
Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp.
45-65, Sinauer Associates, Inc., Sunderland, Mass. (1984).
[0038] The term "immunoglobulin" as used herein includes whole
antibodies and biologically functional fragments thereof. Such
biologically functional fragments retain at least one antigen
binding function of a corresponding full-length antibody (e.g.,
specificity for .alpha.4.beta.7 of Act-1 antibody), and preferably,
retain the ability to inhibit the interaction of .alpha.4.beta.7
with one or more of its ligands (e.g., MAdCAM-1, fibronectin). In a
particularly preferred embodiment, biologically functional
fragments can inhibit binding of .alpha.4.beta.7 to the mucosal
addressin (MAdCAM-1). Examples of biologically functional antibody
fragments which can be used include fragments capable of binding to
an .alpha.4.beta.7 integrin, such as single chain antibodies, Fv,
Fab, Fab' and F(ab').sub.2 fragments. Such fragments can be
produced by enzymatic cleavage or by recombinant techniques. For
instance, papain or pepsin cleavage can be used to generate Fab or
F(ab').sub.2 fragments, respectively. Antibodies can also be
produced in a variety of truncated forms using antibody genes in
which one or more stop codons have been introduced upstream of the
natural stop site. For example, a chimeric gene encoding the heavy
chain of an F(ab').sub.2 fragment can be designed to include DNA
sequences encoding the CH.sub.1 domain and hinge region of the
heavy chain.
[0039] The term "humanized immunoglobulin" as used herein refers to
an immunoglobulin comprising portions of immunoglobulins of
different origin, wherein at least one portion is of human origin.
For example, the humanized antibody can comprise portions derived
from an immunoglobulin of nonhuman origin with the requisite
specificity, such as a mouse, and from immunoglobulin sequences of
human origin (e.g., chimeric immunoglobulin), joined together
chemically by conventional techniques (e.g., synthetic) or prepared
as a contiguous polypeptide using genetic engineering techniques
(e.g., DNA encoding the protein portions of the chimeric antibody
can be expressed to produce a contiguous polypeptide chain).
Another example of a humanized immunoglobulin of the present
invention is an immunoglobulin containing one or more
immunoglobulin chains comprising a CDR derived from an antibody of
nonhuman origin and a framework region derived from a light and/or
heavy chain of human origin (e.g., CDR-grafted antibodies with or
without framework changes). Chimeric or CDR-grafted single chain
antibodies are also encompassed by the term humanized
immunoglobulin. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567;
Cabilly et al., European Patent No. 0,125,023 B1; Boss et al., U.S.
Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1;
Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al.,
European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539;
Winter, European Patent No. 0,239,400 B1; Padlan, E. A. et al.,
European Patent Application No. 0,519,596 A1. See also, Ladner et
al., U.S. Pat. No. 4,946,778; Huston, U.S. Pat. No. 5,476,786; and
Bird, R. E. et al., Science, 242: 423-426 (1988)), regarding single
chain antibodies.
[0040] The antigen binding region of the humanized immunoglobulin
(the nonhuman portion) can be derived from an immunoglobulin of
nonhuman origin (referred to as a donor immunoglobulin) having
binding specificity for .alpha.4.beta.7 integrin. For example, a
suitable antigen binding region can be derived from the murine
Act-1 monoclonal antibody (Lazarovits, A. I. et al., J. Immunol.,
133(4): 1857-1862 (1984)); see e.g., Examples 1-3). Other sources
include .alpha.4.beta.7 integrin-specific antibodies obtained from
nonhuman sources, such as rodent (e.g., mouse, rat), rabbit, pig
goat or non-human primate (e.g., monkey). Other polyclonal or
monoclonal antibodies, such as antibodies which bind to the same or
similar epitope as the Act-1 antibody, can be made (e.g., Kohler et
al., Nature, 256:495-497 (1975); Harlow et al., 1988, Antibodies: A
Laboratory Manual, (Cold Spring Harbor, N.Y.); and Current
Protocols in Molecular Biology, Vol. 2 (Supplement 27, Summer '94),
Ausubel et al., Eds. (John Wiley & Sons: New York, N.Y.),
Chapter 11 (1991)).
[0041] For example, antibodies can be raised against an appropriate
immunogen in a suitable mammal (e.g., a mouse, rat, rabbit or
sheep). Cells bearing .alpha.4.beta.7, membrane fractions
containing .alpha.4.beta.7, immunogenic fragments .alpha.4.beta.7,
a .beta.7 peptide conjugated to a suitable carrier are examples of
suitable immunogens. Antibody-producing cells (e.g., a lymphocyte)
can be isolated from, for example, the lymph nodes or spleen of an
immunized animal. The cells can then be fused to a suitable
immortalized cell (e.g., a myeloma cell line), thereby forming a
hybridoma. Fused cells can be isolated employing selective
culturing techniques. Cells which produce antibodies with the
desired specificity can be selected by a suitable assay (e.g.,
ELISA). Immunoglobulins of nonhuman origin having binding
specificity for .alpha.4.beta.7 integrin can also be obtained from
antibody libraries (e.g., a phage library comprising nonhuman Fab
molecules).
[0042] In one embodiment, the antigen binding region of the
humanized immunoglobulin comprises a CDR of nonhuman origin. In
this embodiment, the humanized immunoglobulin having binding
specificity for .alpha.4.beta.7 integrin comprises at least one CDR
of nonhuman origin. For example, CDRs can be derived from the light
and heavy chain variable regions of immunoglobulins of nonhuman
origin, such that humanized immunoglobulin includes substantially
heavy chain CDR1, CDR2 and/or CDR3, and/or light chain CDR1, CDR2
and/or CDR3, from one or more immunoglobulins of nonhuman origin,
and the resulting humanized immunoglobulin has binding specificity
for .alpha.4.beta.7 integrin. Preferably, all three CDRs of a
selected chain are substantially the same as the CDRs of the
corresponding chain of a donor, and more preferably, all three CDRs
of the light and heavy chains are substantially the same as the
CDRs of the corresponding donor chain.
[0043] The portion of the humanized immunoglobulin or
immunoglobulin chain which is of human origin (the human portion)
can be derived from any suitable human immunoglobulin or
immunoglobulin chain. For example, a human constant region or
portion thereof, if present, can be derived from the .kappa. or
.lamda. light chains, and/or the g (e.g., .gamma.1, .gamma.2,
.gamma.3, .gamma.4), .mu., .pi. (e.g., .alpha.1, .alpha.2), .delta.
or .epsilon. heavy chains of human antibodies, including allelic
variants. A particular constant region (e.g., IgG1), variant or
portions thereof can be selected in order to tailor effector
function. For example, an mutated constant region (variant) can be
incorporated into a fusion protein to minimize binding to Fc
receptors and/or ability to fix complement (see e.g., Example 3;
see also, Winter et al., GB 2,209,757 B; Morrison et al., WO
89/07142; Morgan et al., WO 94/29351, Dec. 22, 1994).
[0044] If present, human framework regions (e.g., of the light
chain variable region) are preferably derived from a human antibody
variable region having sequence similarity to the analogous or
equivalent region (e.g., light chain variable region) of the
antigen binding region donor. Other sources of framework regions
for portions of human origin of a humanized immunoglobulin include
human variable consensus sequences (see e.g., Example 2; see also,
Kettleborough, C. A. et al., Protein Engineering 4:773-783 (1991);
Carter et al., WO 94/04679, published Mar. 3, 1994)). For example,
the sequence of the antibody or variable region used to obtain the
nonhuman portion can be compared to human sequences as described in
Kabat, E. A., et al., Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, U.S. Government Printing Office (1991). In a particularly
preferred embodiment, the framework regions of a humanized
immunoglobulin chain are derived from a human variable region
having at least about 65% overall sequence identity, and preferably
at least about 70% overall sequence identity, with the variable
region of the nonhuman donor (e.g., mouse Act-1 antibody). A human
portion can also be derived from a human antibody having at least
about 65% sequence identity, and preferably at least about 70%
sequence identity, within the particular portion (e.g., FR) being
used, when compared to the equivalent portion (e.g., FR) of the
nonhuman donor. For example, as described in Example 2, the overall
sequence identity between the mouse Act-1 and human GM607'CL light
chain variable regions was 71.4%, and the overall sequence identity
between the mouse Act-1 and human 21/28'CL heavy chain variable
regions was 68.1%.
[0045] In one embodiment, the humanized immunoglobulin comprises at
least one of the framework regions (FR) derived from one or more
chains of an antibody of human origin. Thus, the FR can include a
FR1 and/or FR2 and/or FR3 and/or FR4 derived from one or more
antibodies of human origin. Preferably, the human portion of a
selected humanized chain includes FR1, FR2, FR3 and FR4 derived
from a variable region of human origin (e.g., from a human
immunoglobulin chain, from a human consensus sequence).
[0046] The immunoglobulin portions of nonhuman and human origin for
use in the present invention have sequences identical to
immunoglobulins or immunoglobulin portions from which they are
derived or to variants thereof. Such variants include mutants
differing by the addition, deletion, or substitution of one or more
residues. As indicated above, the CDRs which are of nonhuman origin
are substantially the same as in the nonhuman donor, and preferably
are identical to the CDRs of the nonhuman donor. As described in
Example 2, changes in the framework region, such as those which
substitute a residue of the framework region of human origin with a
residue from the corresponding position of the donor, can be made.
One or more mutations in the framework region can be made,
including deletions, insertions and substitutions of one or more
amino acids. Several such substitutions are described in the design
of a humanized Act-1 antibody in Example 2. For a selected
humanized antibody or chain, framework mutations can be designed as
described herein. Preferably, the humanized immunoglobulins can
bind .alpha.4.beta.7 integrin with an affinity similar to or better
than that of the nonhuman donor. Variants can be produced by a
variety of suitable methods, including mutagenesis of nonhuman
donor or acceptor human chains.
[0047] The humanized immunoglobulins of the present invention have
binding specificity for human .alpha.4.beta.7 integrin, and include
humanized immunoglobulins (including fragments) which can bind
determinants of the .alpha.4 and/or .beta.7 chains of the
heterodimer. In a preferred embodiment, the humanized
immunoglobulin of the present invention has at least one function
characteristic of murine Act-1 antibody, such as binding function
(e.g., having specificity for .alpha.4.beta.7 integrin, having the
same or similar epitopic specificity), and/or inhibitory function
(e.g., the ability to inhibit .alpha.4.beta.7-dependent adhesion in
vitro and/or in vivo, such as the ability to inhibit
.alpha.4.beta.7 integrin binding to MAdCAM-1 in vitro and/or in
vivo, or the ability to inhibit the binding of a cell bearing
.alpha.4.beta.7 integrin to a ligand thereof (e.g., a cell bearing
MAdCAM-1)). Thus, preferred humanized immunoglobulins can have the
binding specificity of the murine Act-1 antibody, the epitopic
specificity murine Act-1 antibody (e.g., can compete with murine
Act-1, a chimeric Act-1 antibody (see e.g., Example 1), or
humanized Act-1 (e.g., LDP-02) for binding to .alpha.4.beta.7
(e.g., on a cell bearing .alpha.4.beta.7 integrin)), and/or
inhibitory function.
[0048] The binding function of a humanized immunoglobulin having
binding specificity for .alpha.4.beta.7 integrin can be detected by
standard immunological methods, for example using assays which
monitor formation of a complex between humanized immunoglobulin and
.alpha.4.beta.7 integrin (e.g., a membrane fraction comprising
.alpha.4.beta.7 integrin, on a cell bearing .alpha.4.beta.7
integrin, such as a human lymphocyte (e.g., a lymphocyte of the
CD4+.alpha.4.sup.hi,.beta.1.sup.lo subset), human lymphocyte cell
line or recombinant host cell comprising nucleic acid encoding
.alpha.4 and/or .beta.7 which expresses .alpha.4.beta.7
integrin).
[0049] Binding and/or adhesion assays or other suitable methods can
also be used in procedures for the identification and/or isolation
of humanized immunoglobulins (e.g., from a library) with the
requisite specificity (e.g., an assay which monitors adhesion
between a cell bearing an .alpha.4.beta.7 integrin and a ligand
thereof (e.g., a second cell expressing MAdCAM, a MAdCAM-Ig chimera
(see e.g., Example 4), or other suitable methods.
[0050] The immunoglobulin portions of nonhuman and human origin for
use in the present invention include light chains, heavy chains and
portions of light and heavy chains. These immunoglobulin portions
can be obtained or derived from immunoglobulins (e.g., by de novo
synthesis of a portion), or nucleic acids encoding an
immunoglobulin or chain thereof having the desired property (e.g.,
binds .alpha.4.beta.7 integrin, sequence similarity) can be
produced and expressed. Humanized immunoglobulins comprising the
desired portions (e.g., antigen binding region, CDR, FR, C region)
of human and nonhuman origin can be produced using synthetic and/or
recombinant nucleic acids to prepare genes (e.g., cDNA) encoding
the desired humanized chain. To prepare a portion of a chain, one
or more stop codons can be introduced at the desired position. For
example, nucleic acid (e.g., DNA) sequences coding for newly
designed humanized variable regions can be constructed using PCR
mutagenesis methods to alter existing DNA sequences (see e.g.,
Kamman, M., et al., Nucl. Acids Res. 17:5404 (1989)). PCR primers
coding for the new CDRs can be hybridized to a DNA template of a
previously humanized variable region which is based on the same, or
a very similar, human variable region (Sato, K., et al., Cancer
Research 53:851-856 (1993)). If a similar DNA sequence is not
available for use as a template, a nucleic acid comprising a
sequence encoding a variable region sequence can be constructed
from synthetic oligonucleotides (see e.g., Kolbinger, F., Protein
Engineering 8:971-980 (1993)). A sequence encoding a signal peptide
can also be incorporated into the nucleic acid (e.g., on synthesis,
upon insertion into a vector). If the natural signal peptide
sequence is unavailable, a signal peptide sequence from another
antibody can be used (see, e.g., Kettleborough, C. A., Protein
Engineering 4:773-783 (1991)). Using these methods, methods
described herein or other suitable methods, variants can be readily
produced (see e.g., Example 5). In one embodiment, cloned variable
regions (e.g., of LDP-02) can be mutagenized, and sequences
encoding variants with the desired specificity can be selected
(e.g., from a phage library; see e.g., Krebber et al., U.S. Pat.
No. 5,514,548; Hoogengoom et al., WO 93/06213, published Apr. 1,
1993)).
Nucleic Acids and Constructs Comprising Same
[0051] The present invention also relates to isolated and/or
recombinant (including, e.g., essentially pure) nucleic acids
comprising sequences which encode a humanized immunoglobulin or
humanized immunoglobulin light or heavy chain of the present
invention.
[0052] Nucleic acids referred to herein as "isolated" are nucleic
acids which have been separated away from the nucleic acids of the
genomic DNA or cellular RNA of their source of origin (e.g., as it
exists in cells or in a mixture of nucleic acids such as a
library), and include nucleic acids obtained by methods described
herein or other suitable methods, including essentially pure
nucleic acids, nucleic acids produced by chemical synthesis, by
combinations of biological and chemical methods, and recombinant
nucleic acids which are isolated (see e.g., Daugherty, B. L. et
al., Nucleic Acids Res., 19(9): 2471-2476 (1991); Lewis, A. P. and
J. S. Crowe, Gene, 101: 297-302 (1991)).
[0053] Nucleic acids referred to herein as "recombinant" are
nucleic acids which have been produced by recombinant DNA
methodology, including those nucleic acids that are generated by
procedures which rely upon a method of artificial recombination,
such as the polymerase chain reaction (PCR) and/or cloning into a
vector using restriction enzymes. "Recombinant" nucleic acids are
also those that result from recombination events that occur through
the natural mechanisms of cells, but are selected for after the
introduction to the cells of nucleic acids designed to allow and
make probable a desired recombination event.
[0054] The present invention also relates more specifically to
isolated and/or recombinant nucleic acids comprising a nucleotide
sequence which encodes a humanized Act-1 immunoglobulin (i.e., a
humanized immunoglobulin of the present invention in which the
nonhuman portion is derived from the murine Act-1 monoclonal
antibody) or chain thereof. In one embodiment, the light chain
comprises three complementarity determining regions derived from
the light chain of the Act-1 antibody, and the heavy chain
comprises three complementarity determining regions derived from
the heavy chain of the Act-1 antibody. Such nucleic acids include,
for example, (a) a nucleic acid comprising a sequence which encodes
a polypeptide comprising the amino acid sequence of the heavy chain
variable region of a humanized Act-1 immunoglobulin (e.g., heavy
chain variable region of FIG. 11 (SEQ ID NO:19), heavy chain
variable region of FIG. 9 (SEQ ID NO:15)), (b) a nucleic acid
comprising a sequence which encodes a polypeptide comprising the
amino acid sequence of the light chain variable region of a
humanized Act-1 immunoglobulin (e.g., light chain variable region
of FIG. 12 (SEQ ID NO:21), light chain variable region of FIG. 7
(SEQ ID NO:12)), (c) a nucleic acid comprising a sequence which
encodes at least a functional portion of the light or heavy chain
variable region of a humanized Act-1 immunoglobulin (e.g., a
portion sufficient for antigen binding of a humanized
immunoglobulin which comprises said chain). Due to the degeneracy
of the genetic code, a variety of nucleic acids can be made which
encode a selected polypeptide. In one embodiment, the nucleic acid
comprises the nucleotide sequence of the variable region as set
forth or substantially as set forth in FIG. 11 (SEQ ID NO:18), or
as set forth or substantially as set forth in FIG. 12 (SEQ ID
NO:20), including double or single-stranded polynucleotides.
(Although various figures may illustrate polypeptides which are
larger than the variable region (i.e., include a signal peptide
coding sequence or a portion of a constant region coding sequence),
reference to the variable region of a particular figure is meant to
include the variable region portion of the sequence shown.)
Isolated and/or recombinant nucleic acids meeting these criteria
can comprise nucleic acids encoding sequences identical to
sequences of humanized Act-1 antibody or variants thereof as
discussed above.
[0055] Nucleic acids of the present invention can be used in the
production of humanized immunoglobulins having binding specificity
for .alpha.4.beta.7 integrin. For example, a nucleic acid (e.g.,
DNA) encoding a humanized immunoglobulin of the present invention
can be incorporated into a suitable construct (e.g., a vector) for
further manipulation of sequences or for production of the encoded
polypeptide in suitable host cells.
Method of Producing Humanized Immunoglobulins Having Specificity
for .alpha.4.beta.7 Integrin
[0056] Another aspect of the invention relates to a method of
preparing a humanized immunoglobulin which has binding specificity
for .alpha.4.beta.7 integrin. The humanized immunoglobulin can be
obtained, for example, by the expression of one or more recombinant
nucleic acids encoding a humanized immunoglobulin having binding
specificity for .alpha.4.beta.7 integrin in a suitable host cell,
for example.
[0057] Constructs or expression vectors suitable for the expression
of a humanized immunoglobulin having binding specificity for
.alpha.4.beta.7 integrin are also provided. The constructs can be
introduced into a suitable host cell, and cells which express a
humanized immunoglobulin of the present invention, can be produced
and maintained in culture. Suitable host cells can be procaryotic,
including bacterial cells such as E. coli, B. subtilis and or other
suitable bacteria, or eucaryotic, such as fungal or yeast cells
(e.g., Pichia pastoris, Aspergillus species, Saccharomyces
cerevisiae, Schizosaccharomyces pombe, Neurospora crassa), or other
lower eucaryotic cells, and cells of higher eucaryotes such as
those from insects (e.g., Sf9 insect cells (WO 94/26087, O'Connor,
published Nov. 24, 1994)) or mammals (e.g., COS cells, NSO cells,
SP2/0, Chinese hamster ovary cells (CHO), HuT 78 cells, 293 cells).
(See, e.g., Ausubel, F. M. et al., eds. Current Protocols in
Molecular Biology, Greene Publishing Associates and John Wiley
& Sons Inc., (1993)).
[0058] Host cells which produce a humanized immunoglobulin having
binding specificity for .alpha.4.beta.7 integrin can be produced as
follows. For example, a nucleic acid encoding all or part of the
coding sequence for the desired humanized immunoglobulin can be
inserted into a nucleic acid vector, e.g., a DNA vector, such as a
plasmid, virus or other suitable replicon for expression. A variety
of vectors are available, including vectors which are maintained in
single copy or multiple copy, or which become integrated into the
host cell chromosome.
[0059] Suitable expression vectors can contain a number of
components, including, but not limited to one or more of the
following: an origin of replication; a selectable marker gene; one
or more expression control elements, such as a transcriptional
control element (e.g., a promoter, an enhancer, terminator), and/or
one or more translation signals; a signal sequence or leader
sequence for membrane targeting or secretion. In a construct, a
signal sequence can be provided by the vector or other source. For
example, the transcriptional and/or translational signals of an
immunoglobulin can be used to direct expression.
[0060] A promoter can be provided for expression in a suitable host
cell. Promoters can be constitutive or inducible. For example, a
promoter can be operably linked to a nucleic acid encoding a
humanized immunoglobulin or immunoglobulin chain, such that it
directs expression of the encoded polypeptide. A variety of
suitable promoters for procaryotic (e.g., lac, tac, T3, T7
promoters for E. coli) and eucaryotic (e.g., yeast alcohol
dehydrogenase (ADH1), SV40, CMV) hosts are available.
[0061] In addition, the expression vectors typically comprise a
selectable marker for selection of host cells carrying the vector,
and, in the case of replicable expression vector, an origin or
replication. Genes encoding products which confer antibiotic or
drug resistance are common selectable markers and may be used in
procaryotic (e.g., .beta.-lactamase gene (ampicillin resistance),
Tet gene for tetracycline resistance) and eucaryotic cells (e.g.,
neomycin (G418 or geneticin), gpt (mycophenolic acid), ampicillin,
or hygromycin resistance genes). Dihydrofolate reductase marker
genes permit selection with methotrexate in a variety of hosts.
Genes encoding the gene product of auxotrophic markers of the host
(e.g., LEU2, URA3, HIS3) are often used as selectable markers in
yeast. Use of viral (e.g., baculovirus) or phage vectors, and
vectors which are capable of integrating into the genome of the
host cell, such as retroviral vectors, are also contemplated. The
present invention also relates to cells carrying these expression
vectors.
[0062] For example, a nucleic acid (i.e., one or more nucleic
acids) encoding the heavy and light chains of a humanized
immunoglobulin having binding specificity for .alpha.4.beta.7
integrin, or a construct (i.e., one or more constructs) comprising
such nucleic acid(s), can be introduced into a suitable host cell
by a method appropriate to the host cell selected (e.g.,
transformation, transfection, electroporation, infection), such
that the nucleic acid(s) are operably linked to one or more
expression control elements (e.g., in a vector, in a construct
created by processes in the cell, integrated into the host cell
genome). Host cells can be maintained under conditions suitable for
expression (e.g., in the presence of inducer, suitable media
supplemented with appropriate salts, growth factors, antibiotic,
nutritional supplements, etc.), whereby the encoded polypeptide(s)
are produced. If desired, the encoded protein (e.g., humanized
Act-1 antibody) can be isolated from (e.g., the host cells, medium,
milk). This process encompasses expression in a host cell of a
transgenic animal (see e.g., WO 92/03918, GenPharm International,
published Mar. 19, 1992).
[0063] Fusion proteins can be produced in which a humanized
immunoglobulin or immunoglobulin chain is linked to a
non-immunoglobulin moiety (i.e., a moiety which does not occur in
immunoglobulins as found in nature) in an N-terminal location,
C-terminal location or internal to the fusion protein. For example,
some embodiments can be produced by the insertion of a nucleic acid
encoding immunoglobulin sequences into a suitable expression
vector, such as a pET vector (e.g., pET-15 b, Novagen), a phage
vector (e.g., pCANTAB 5 E, Pharmacia), or other vector (e.g.,
pRIT2T Protein A fusion vector, Pharmacia). The resulting construct
can be introduced into a suitable host cell for expression. Upon
expression, some fusion proteins can be isolated or purified from a
cell lysate by means of a suitable affinity matrix (see e.g.,
Current Protocols in Molecular Biology (Ausubel, F. M. et al.,
eds., Vol. 2, Suppl. 26, pp. 16.4.1-16.7.8 (1991)).
Therapeutic Methods and Compositions
[0064] The present invention provides humanized immunoglobulins
which (1) can bind .alpha.4.beta.7 integrin in vitro and/or in
vivo; and/or (2) can modulate an activity or function of an
.alpha.4.beta.7 integrin, such as (a) binding function (e.g., the
ability of .alpha.4.beta.7 integrin to bind to MAdCAM-1,
fibronectin and/or VCAM-1) and/or (b) leukocyte infiltration
function, including recruitment and/or accumulation of leukocytes
in tissues (e.g., the ability to inhibit lymphocyte migration to
intestinal mucosal tissue). Preferably the humanized
immunoglobulins are capable of selectively binding .alpha.4.beta.7
in vitro and/or in vivo, and inhibiting .alpha.4.beta.7-mediated
interactions. In one embodiment, a humanized immunoglobulin can
bind an .alpha.4.beta.7 integrin, and can inhibit binding of the
.alpha.4.beta.7 integrin to one or more of its ligands (e.g.,
MAdCAM-1, VCAM-1, fibronectin), thereby inhibiting leukocyte
infiltration of tissues (including recruitment and/or accumulation
of leukocytes in tissues), preferably selectively. Such humanized
immunoglobulins can inhibit cellular adhesion of cells bearing an
.alpha.4.beta.7 integrin to vascular endothelial cells in mucosal
tissues, including gut-associated tissues, lymphoid organs or
leukocytes (especially lymphocytes such as T or B cells) in vitro
and/or in vivo. In a particularly preferred embodiment, a humanized
immunoglobulin (e.g., Act-1) can inhibit the interaction of
.alpha.4.beta.7 with MAdCAM-1 and/or fibronectin.
[0065] The humanized immunoglobulins of the present invention are
useful in a variety of processes with applications in research,
diagnosis and therapy. For instance, they can be used to detect,
isolate, and/or purify .alpha.4.beta.7 integrin or variants thereof
(e.g., by affinity purification or other suitable methods), and to
study .alpha.4.beta.7 integrin structure (e.g., conformation) and
function.
[0066] The humanized immunoglobulins of the present invention can
also be used in diagnostic applications (e.g., in vitro, ex vivo)
or to modulate .alpha.4.beta.7 integrin function in therapeutic
(including prophylactic) applications.
[0067] For example, the humanized immunoglobulins of the present
invention can be used to detect and/or measure the level of an
.alpha.4.beta.7 integrin in a sample (e.g., tissues or body fluids,
such as an inflammatory exudate, blood, serum, bowel fluid, on
cells bearing an .alpha.4.beta.7 integrin). For example, a sample
(e.g., tissue and/or body fluid) can be obtained from an individual
and a suitable immunological method can be used to detect and/or
measure .alpha.4.beta.7 integrin expression, including methods such
as enzyme-linked immunosorbent assays (ELISA), including
chemiluminescence assays, radioimmunoassay, and immunohistology. In
one embodiment, a method of detecting a selected .alpha.4.beta.7
integrin in a sample is provided, comprising contacting a sample
with a humanized immunoglobulin of the present invention under
conditions suitable for specific binding of the humanized
immunoglobulin to the .alpha.4.beta.7 integrin and detecting
antibody-.alpha.4.beta.7 integrin complexes which are formed. In an
application of the method, humanized immunoglobulins can be used to
analyze normal versus inflamed tissues (e.g., from a human) for
.alpha.4.beta.7 integrin reactivity and/or expression (e.g.,
immunohistologically)) to detect associations between IBD or other
conditions and increased expression of .alpha.4.beta.7 (e.g., in
affected tissues). The humanized immunoglobulins of the present
invention permit immunological methods of assessment of the
presence of .alpha.4.beta.7 integrin in normal versus inflamed
tissues, through which the presence of disease, disease progress
and/or the efficacy of anti-.alpha.4.beta.7 integrin therapy in
inflammatory disease can be assessed.
[0068] The humanized immunoglobulins of the present invention can
also be used to modulate (e.g., inhibit (reduce or prevent))
binding function and/or leukocyte (e.g., lymphocyte, monocyte)
infiltration function of .alpha.4.beta.7 integrin. For example,
humanized immunoglobulins which inhibit the binding of
.alpha.4.beta.7 integrin to a ligand (i.e., one or more ligands)
can be administered according to the method in the treatment of
diseases associated with leukocyte (e.g., lymphocyte, monocyte)
infiltration of tissues (including recruitment and/or accumulation
of leukocytes in tissues), particularly of tissues which express
the molecule MAdCAM. An effective amount of a humanized
immunoglobulin of the present invention (i.e., one or more) is
administered to an individual (e.g., a mammal, such as a human or
other primate) in order to treat such a disease. For example,
inflammatory diseases, including diseases which are associated with
leukocyte infiltration of the gastrointestinal tract (including
gut-associated endothelium), other mucosal tissues, or tissues
expressing the molecule MAdCAM-1 (e.g., gut-associated tissues,
such as venules of the lamina propria of the small and large
intestine; and mammary gland (e.g., lactating mammary gland)), can
be treated according to the present method. Similarly, an
individual having a disease associated with leukocyte infiltration
of tissues as a result of binding of leukocytes to cells (e.g.,
endothelial cells) expressing MAdCAM-1 can be treated according to
the present invention.
[0069] In a particularly preferred embodiment, diseases which can
be treated accordingly include inflammatory bowel disease (IBD),
such as ulcerative colitis, Crohn's disease, ileitis, Celiac
disease, nontropical Sprue, enteropathy associated with
seronegative arthropathies, microscopic or collagenous colitis,
eosinophilic gastroenteritis, or pouchitis resulting after
proctocolectomy, and ileoanal anastomosis.
[0070] Pancreatitis and insulin-dependent diabetes mellitus are
other diseases which can be treated using the present method. It
has been reported that MAdCAM-1 is expressed by some vessels in the
exocrine pancreas from NOD (nonobese diabetic) mice, as well as
from BALB/c and SJL mice. Expression of MAdCAM-1 was reportedly
induced on endothelium in inflamed islets of the pancreas of the
NOD mouse, and MAdCAM-1 was the predominant addressin expressed by
NOD islet endothelium at early stages of insulitis (Hanninen, A.,
et al., J. Clin. Invest., 92: 2509-2515 (1993)). Further,
accumulation of lymphocytes expressing .alpha.4.beta.7 within
islets was observed, and MAdCAM-1 was implicated in the binding of
lymphoma cells via .alpha.4.beta.7 to vessels from inflamed islets
(Hanninen, A., et al., J. Clin. Invest., 92: 2509-2515 (1993)).
[0071] Examples of inflammatory diseases associated with mucosal
tissues which can be treated according to the present method
include mastitis (mammary gland), cholecystitis, cholangitis or
pericholangitis (bile duct and surrounding tissue of the liver),
chronic bronchitis, chronic sinusitis, asthma, and graft versus
host disease (e.g., in the gastrointestinal tract). As seen in
Crohn's disease, inflammation often extends beyond the mucosal
surface, accordingly chronic inflammatory diseases of the lung
which result in interstitial fibrosis, such as hypersensitivity
pneumonitis, collagen diseases, sarcoidosis, and other idiopathic
conditions can be amenable to treatment.
[0072] The humanized immunoglobulin is administered in an effective
amount which inhibits binding .alpha.4.beta.7 integrin to a ligand
thereof. For therapy, an effective amount will be sufficient to
achieve the desired therapeutic (including prophylactic) effect
(such as an amount sufficient to reduce or prevent .alpha.4.beta.7
integrin-mediated binding and/or signalling, thereby inhibiting
leukocyte adhesion and infiltration and/or associated cellular
responses). The humanized immunoglobulin can be administered in a
single dose or multiple doses. The dosage can be determined by
methods known in the art and can be dependent, for example, upon
the individual's age, sensitivity, tolerance and overall
well-being. Suitable dosages for antibodies can be from about 0.1
mg/kg body weight to about 10.0 mg/kg body weight per
treatment.
[0073] According to the method, the humanized immunoglobulin can be
administered to an individual (e.g., a human) alone or in
conjunction with another agent. A humanized immunoglobulin can be
administered before, along with or subsequent to administration of
the additional agent. In one embodiment, more than one humanized
immunoglobulin which inhibits the binding of .alpha.4.beta.7
integrin to its ligands is administered. In another embodiment, a
monoclonal antibody, such as an anti-MAdCAM-1, anti-VCAM-1, or
anti-ICAM-1 antibody, which inhibits the binding of leukocytes to
an endothelial ligand is administered in addition to a humanized
immunoglobulin of the present invention. In yet another embodiment,
an additional pharmacologically active ingredient (e.g., an
antiinflammatory compound, such as sulfasalazine, another
non-steroidal antiinflammatory compound, or a steroidal
antiinflammatory compound) can be administered in conjunction with
a humanized immunoglobulin of the present invention.
[0074] A variety of routes of administration are possible,
including, but not necessarily limited to, parenteral (e.g.,
intravenous, intraarterial, intramuscular, subcutaneous injection),
oral (e.g., dietary), topical, inhalation (e.g., intrabronchial,
intranasal or oral inhalation, intranasal drops), or rectal,
depending on the disease or condition to be treated. Parenteral
administration is a preferred mode of administration.
[0075] Formulation will vary according to the route of
administration selected (e.g., solution, emulsion). An appropriate
composition comprising the humanized antibody to be administered
can be prepared in a physiologically acceptable vehicle or carrier.
For solutions or emulsions, suitable carriers include, for example,
aqueous or alcoholic/aqueous solutions, emulsions or suspensions,
including saline and buffered media. Parenteral vehicles can
include sodium chloride solution, Ringer's dextrose, dextrose and
sodium chloride, lactated Ringer's or fixed oils. Intravenous
vehicles can include various additives, preservatives, or fluid,
nutrient or electrolyte replenishers (See, generally, Remington's
Pharmaceutical Sciences, 17th Edition, Mack Publishing Co., PA,
1985). For inhalation, the compound can be solubilized and loaded
into a suitable dispenser for administration (e.g., an atomizer,
nebulizer or pressurized aerosol dispenser).
EXEMPLIFICATION
[0076] The present invention will now be illustrated by the
following Examples, which are not intended to be limiting in any
way.
[0077] As described in Example 1, murine Act-1 antibody was
purified and sequence analysis of the antibody was performed. cDNAs
encoding the light and heavy chain variable regions of mouse Act-1
antibody were PCR-cloned and sequenced. The amino acid sequence of
the kappa light chain variable region (V.sub.L) of Act-1 was also
determined by protein sequencing and found to match exactly the
amino acid sequence derived from the DNA sequence of the V.sub.L
gene. Most of the amino acid sequence of the heavy chain variable
region (V.sub.H) has been determined by protein sequence, and this
sequence also matches the amino acid sequence deduced from the DNA
sequence of the V.sub.H gene. These results indicate that the
correct mouse Act-1 variable regions were cloned from the hybridoma
cell line. Functional chimeric Act-1 antibodies were produced which
confirmed that the correct sequences have been cloned. In
particular, the DNAs encoding mouse Act-1 light and heavy chain
variable regions were joined to DNAs encoding human kappa light
chain and human gamma-1 or gamma-4 heavy chain constant regions,
respectively. The chimeric antibody was also used in a comparative
analysis with a humanized Act-1 mAb (reshaped Act-1 mAb
LDP-02).
[0078] To create a humanized Act-1 antibody that binds well to
.alpha.4.beta.7 integrin, reshaped human variable regions were
designed (Example 2). In order to assist in the design process, a
molecular model of the mouse Act-1 variable regions was built. The
regions of the murine Act-1 antibody directly involved in binding
to antigen, the complementarity determining region or CDRs, were
grafted into selected human variable regions. A few amino acid
changes at positions within the framework regions (FRs) of the
human variable regions were made. The reshaped human Act-1 variable
regions, included a single amino acid change in the FRs of the
selected human light chain variable region and five amino acid
changes in the FRs of the selected human heavy chain variable
region, each changing the original human residue to the
corresponding murine residue.
[0079] As described in Example 3, DNA sequences encoding these
reshaped human Act-1 variable regions were constructed and joined
to DNA sequences encoding human constant regions, and the resulting
nucleic acids were used to produce humanized Act-1 immunoglobulin.
Humanized Act-1 antibody was expressed in mammalian cells (Example
3), and was tested for binding to human .alpha.4.beta.7 integrin in
comparison with mouse Act-1 antibody (Example 4). As shown in Table
5, the humanized Act-1 antibody retained specificity for the
epitope recognized by murine Act-1, and displayed unexpectedly
improved binding affinity as compared with the native murine
antibody.
[0080] Several variants of the humanized Act-1 antibody were
identified in the design process (Examples 2 and 5). For example,
additional changes at one or more of the following positions can be
made: light chain mutant M4V (Met.fwdarw.Val mutation at position
4), heavy chain mutant R38K (Arg.fwdarw.Lys mutation at position
38), heavy chain mutant A40R (Ala.fwdarw.Arg mutation at position
40). In addition, a heavy chain mutant 173T (Ile.fwdarw.Thr
back-mutation at position 73), restoring position 73 to the human
threonine residue found at this position in the human framework
region. Introduction of one or more of these changes in a single
chain or various combinations of these changes in more than one
chain can be made.
[0081] Murine Act-1 Hybridoma cell line, which produces the murine
Act-1 monoclonal antibody, was deposited under the provisions of
the Budapest Treaty on Aug. 22, 2001, on behalf Millennium
Pharmaceuticals, Inc., 75 Sidney Street, Cambridge, Mass. 02139,
U.S.A., at the American Type Culture Collection, 10801 University
Boulevard, Manassas, Va. 20110, U.S.A., under Accession No.
PTA-3663.
Example 1
Cloning of Act-1 VH and VL Regions and Construction and Expression
of a Murine-Human Act-1 Chimeric Immunoglobulin
Cloning of Act-1 VH and VL Regions
[0082] RNA was obtained from hybridoma cells which produce Act-1
monoclonal antibody (Lazarovits, A. I. et al., J. Immunol., 133(4):
1857-1862 (1984); provided by A. I. Lazarovits and R. B. Colvin))
using TRIzol Reagent (Gibco/BRL) following the manufacturer's
suggested protocol.
[0083] Transcribed heavy and light chain variable regions were
amplified by polymerase chain reaction (PCR) using an Ig-Prime kit
(Novagen) according to the maufacturer's suggested protocol.
Briefly, 1.5 .mu.g of total RNA was reverse transcribed to cDNA in
a reaction containing 2.0 .mu.l 5.times.MMLV Buffer (5.times.=250
mM Tris-HCl, pH 8.3 at 25.degree. C., 375 mM KCl, 15 mM MgCl2), 1.0
.mu.l 100 mM DTT (dithiothreitol), 0.5 .mu.l 10 .mu.M dNTP mix (10
mM each dATP, dCTP, dTTP, dGTP), 0.5 .mu.l oligo dT (1 mg/ml), 0.25
.mu.l acetylated BSA (4 mg/ml), 1.0 .mu.l of appropriate Ig-3'
primer (10 pmol/.mu.l), 0.5 .mu.l MMLV Reverse Transcriptase (200
units/.mu.l) and RNase-free water added to a total volume of 10
.mu.l. The mixture was incubated for 5 minutes at 37.degree. C., 30
minutes at 42.degree. C., and 5 minutes at 99.degree. C. Each Ig-3'
primer was used in a separate reaction.
[0084] Variable regions were amplified from the reverse transcribed
material according to the manufacturer's protocol. Briefly, 8 .mu.l
of the reverse transcribed material was mixed with 4 .mu.l of 2.5
mM dNTPs, 5 .mu.l 10.times. reaction buffer (10.times.=100 mM
Tris-HCl, pH 8.8 at 25.degree. C., 500 mM KCl, 15 mM MgCl.sub.2, 1%
Triton X-100), 2.5 .mu.l Ig-5' leader primer (10 pmol/.mu.l) (each
Ig-5' leader primer was used in a separate PCR reaction), 0.25
.mu.l (1.25 units) AmpliTaq.RTM. DNA polymerase (Perkin-Elmer), and
water to a total volume of 50 .mu.l.
[0085] For amplifications with 5' primers MuIg V.sub.H5'-A,
MuIgV.sub.H5'-B, MuIgkV.sub.L5'-A, and MugIg.kappa.V.sub.L5'-B, the
cycle parameters were 35 cycles of 1 minute, 94.degree. C.; 1
minute, 50.degree. C.; 2 minutes, 72.degree. C.; followed by a
final 6 minute extension at 72.degree. C. The same reaction
conditions were used for all other 5' primers, except that the
annealing temperature was raised to 60.degree. C.
[0086] The heavy chain variable region was successfully amplified
using either MuIgGV.sub.H3'-2 or MuIgMV.sub.H3'-1 as the 3' primer,
and either MuIgV.sub.H5'-B or MuIgV.sub.H5'-E as the 5' primers.
The light chain variable region was successfully amplifed using
MuIgkV.sub.L3'-1 as the 3' primer and MuIgkV.sub.L5'-G as the 5'
primer.
[0087] The sequences of these primers were as follows:
TABLE-US-00001 MuIgGV.sub.H3'-2 (SEQ ID NO: 56): 5'-CCC AAG CTT CCA
GGG RCC ARK GGA TAR ACI GRT GG MuIgMV.sub.H3'-1 (SEQ ID NO: 57):
5'-CCC AAG CTT ACG AGG GGG AAG ACA TTT GGG AA MuIgV.sub.H5'-B (SEQ
ID NO: 58): 5'-GGG AAT TCA TGR AAT GSA SCT GGG TYW TYC TCT T
MuIgV.sub.H5'-E (SEQ ID NO: 59): 5'-ACT AGT CGA CAT GAA GWT GTG GBT
RAA CTG GRT MuIgkV.sub.L3'-1 (SEQ ID NO: 60): 5'-CCC AAG CTT ACT
GGA TGG TGG GAA GAT GGA MuIgkV.sub.L5'-G (SEQ ID NO: 61): 5'-ACT
AGT CGA CAT GGA TTT WCA RGT GCA GAT TWT CAG CTT
[0088] Amplified fragments were agarose gel purified and ligated
into the pT7Blue T vector (Novagen) supplied with the Ig-Prime kit,
and the ligation mixture was used to transform NovaBlue competent
cells provided with the kit, according to the manufacturer's
protocol.
[0089] White colonies containing inserts of the appropriate size
were sequenced using T7 promoter primer and U-19mer primer which
anneal on opposite sides of the insert just outside of the
polycloning site of pT7Blue vector. Sequencing was performed on
miniprep DNA using a Sequenase T7 DNA polymerase kit (USB/Amersham
Life Science) according to manufacturer's recommended protocol.
[0090] The consensus DNA sequence (SEQ ID NO:1) from several
independent heavy chain variable region clones and deduced amino
acid sequence (SEQ ID NO:2) is shown in FIG. 1. Degenerate primers
led to some degeneracy in sequence. The initiation codon is the Met
encoded by nucleotides 13-15, the predicted leader peptidase
cleavage site is between the Ser encoded by nucleotides 67-69 and
the Gln encoded by nucleotides 70-72 (nucleotides 13-69 encoding
the leader peptide). A portion of the murine constant region,
beginning with the alanine encoded by residues 433-435, is
shown.
[0091] The DNA sequence (SEQ ID NO:5) and amino acid sequence (SEQ
ID NO:6) of several independent light chain variable region clones
is shown in FIG. 3. Unlike the heavy chain variable region, the
amplified sequences were not degenerate, probably because the
primers used were not very degenerate and the variable region was
amplified from only a single primer pair.
Construction of a Chimeric Heavy Chain Gene
[0092] A gene encoding a chimeric mouse-human heavy chain gene was
produced. The source of the human heavy chain constant region was a
clone containing a wild type human gamma one (.gamma.1) constant
region (obtained from Dr. Herman Waldmann (University of Oxford); a
construct designated 3818 comprising a humanized anti-CD18 heavy
chain gene in a pEE6 expression vector (Celltech). The constant
region corresponds to that of the humanized CD 18 heavy chain gene
cloned into pEE6.hCMV as described in Sims, M. J. et al., J.
Immunol., 151 (4): 2296-2308 (1993) and WO 93/02191, published Feb.
4, 1993, the teachings of which are each incorporated herein by
reference in their entirety. The sequences encoding the heavy chain
variable and constant region (wild-type gamma one) of the humanized
anti-CD18 antibody were released from the expression vector by
digestion with HindIII and EcoRI. The 1.421 bp fragment containing
the heavy chain gene was recovered and subcloned into the HindIII
and EcoRI sites of pCR-Script.TM. (Stratagene) to yield a plasmid
designated pCR-CD18H. An Spe I restriction site is located at the
junction between the variable region and constant region in the
anti-CD18 heavy chain gene. pCR-CD18H was restriction digested with
HindIII and Spe I to release the heavy chain variable region. This
variable region was replaced with the mouse Act-1 variable region
generated as follows.
[0093] Two primers were synthesized to incorporate new restriction
sites. These primers were:
TABLE-US-00002 5'-primer (SEQ ID NO: 41): Hind III 5'-T[AA GCT T]CC
GCC ATG GGA TGG AGC 3'-primer (SEQ ID NO: 42): Spe I 5'- GGT GAC
[ACT AGT] GCC TTG ACC CCA G
Boldface type indicates a nucleotides in the primers which differ
from the template sequence. An independent mouse Act-1 heavy chain
clone designated H2B#34, with the nucleotide sequence (SEQ ID NO:3)
and amino acid sequence (SEQ ID NO:4) presented in FIG. 2, was used
as a template with the 5' and 3' primers above to amplify a mouse
variable region concomitantly introducing a HindIII site 5' of the
initiation codon and a Spe I site just 3' of the J region. The PCR
fragment was directly subcloned into pCR-ScriptT giving rise to
plasmid pCR-mACT1HV, and the correct sequence was confirmed. The
fragment was then released from pCR-mACT1HV by digestion with
HindIII and Spe I, and inserted into the HindIII and Spe I sites of
pCR-CD 18H in place of the anti-CD18 variable region to yield
pCR-mhACT1Hchi. The chimeric heavy chain (mouse Act-1 variable plus
human gamma one constant) gene was then released from
pCR-mhACT1Hchi with HindIII and EcoRI and cloned back into the
pEE6hCMV-B vector, containing the hCMV promoter, to yield a
construct designated pEE6 mhACT1Hchi.
Construction of a Chimeric Light Chain Gene
[0094] A chimeric mouse-human light chain gene was constructed in a
similar fashion as for the heavy chain. However, in the case of the
chimeric light chain, a new restriction site, Kas I, was engineered
into the construct by PCR amplification of a variable region
fragment using one of the mouse Act-1 light chain variable region
clones designated KG#87 as a template, and by PCR amplification of
a kappa light chain constant region using a construct containing a
humanized anti-CD18 kappa light chain gene as template (obtained
from Dr. Herman Waldmann (University of Oxford); construct
designated 3819 containing a humanized anti-CD18 light chain in the
pEE12 expression vector). The constant region corresponds to that
of the humanized CD18 light chain gene cloned into pEE12 as
described in Sims, M. J. et al., J. Immunol., 151 (4): 2296-2308
(1993) and WO 93/02191, published Feb. 4, 1993.
[0095] The primers for the variable region were:
TABLE-US-00003 5'-primer (SEQ ID NO: 43): HindIII 5'-T[AA GCT T]CC
GCC ATG AAG TTG CCT 3'-primer (SEQ ID NO: 44): Kas I 5'-[GGC GCC]
GCA TCA GCC CGT TTT
Boldface type indicates nucleotides in the primer which differ from
those in the template. The two nucleotide changes within the coding
region, T.fwdarw.G at position 423 and A.fwdarw.G at position 426
in FIG. 3 to create the Kas I site are silent, and do not change
the amino acid sequence.
[0096] The primers for the kappa constant region were:
TABLE-US-00004 5'- primer (SEQ ID NO: 45): Kas I 5'-C[GG CGC C]AT
CTG TCT TCA TC 3'-primer (SEQ ID NO: 46): HindIII 5'-[AAG CTT] CTA
ACA CTC TCC
[0097] The light chain variable and constant regions were amplified
separately with respective templates and primers, and the PCR
products were individually subcloned into pCR-Script.TM. to confirm
the sequence. Each fragment was then released from the vector by
digestion with HindIII and KasI, gel purified and triple ligated
into the HindIII site of the 3819 pEE12 expression vector from
which the humanized anti-CD18 light chain gene had been removed by
HindIII digestion. The resulting construct is designated pEE12
mhACT1Lchi.
Expression of a Chimeric Immunoglobulin
[0098] For construction of an expression vector containing both
chimeric heavy and light chain genes, the entire heavy chain gene
plus CMV promoter was released from the pEE6 expression vector
(pEE6 mhACT1Hchi) by digestion with BglII and BamHI. This fragment
was then ligated into the BamHI site of the pEE12 light chain gene
expression vector (pEE12 mhACT1Lchi) giving rise to a single
plasmid designated pEE12 mhLHchi, which contains both the chimeric
light chain gene and chimeric heavy chain gene each under the
transcription control of a separate CMV promoter.
[0099] The pEE6hCMV-B and pEE12 expression vectors and the Celltech
glutamine synthetase gene amplification system have been described
previously (see e.g., WO 86/05807 (Celltech), WO 87/04462
(Celltech), WO 89/01036 (Celltech), EP 0 323 997 B1 (Celltech), and
WO 89/10404 (Celltech), the teachings of which are each
incorporated herein by reference in their entirety).
[0100] For transient expression of the chimeric antibody, 20 .mu.g
of pEE12 mhLHchi was transfected into COS-7 cells (American Type
Culture Collection, 10801 University Boulevard, Manassas, Va.
20110-2209) by electroporation as follows. COS-7 cells growing in
log phase were harvested from tissue culture flasks by treatment
with trypsin-EDTA. The cells were washed once in Phosphate Buffered
Saline (PBS), once with Hank's Balanced Salts Solution (HBSS), and
resusended at a concentration of 1.5.times.10.sup.7 cells per ml of
HBSS. 1.2.times.10.sup.7 cells in 0.8 ml HBSS was mixed with 20
.mu.g of the plasmid DNA and incubated for 10 minutes at room
temperature. The DNA/cell mixture was then transferred to a 0.4 cm
electroporation cuvette and current applied at 250 V, 960 .mu.F
with a Bio-Rad GenePulser. After a 10 minute post-electroporation
incubation at room temperature, the cells were transferred to 20
mls of culture medium (Dulbecco's Modified Eagle's Medium (DMEM)
plus 10% FCS) and cultured in a 162 cm.sup.2 tissue culture flask
(Costar). After 5 days, the cell culture supernatant was harvested
and tested for the ability to stain HuT 78 cells which express the
.alpha.4.beta.7 integrin. HuT 78 cells (a human T cell lymphoma
line) are available from the American Type Culture Collection,
10801 University Boulevard, Manassas, Va. 20110-2209, Accession No.
ATCC TIB 161.
[0101] 100 .mu.l of transiently transfected COS-7 cell culture
supernatant, mock transfected COS-7 cell supernatant, purified
murine Act-1 antibody (10 .mu.g/ml), or the respective purified
irrelevant isotype-matched control antibodies for mouse (mouse
IgG1, Kappa (MOPC21), 10 .mu.g/ml from Sigma) and for human (human
IgG1, Kappa, 10 .mu.g/ml from Sigma) were incubated with
1.times.10.sup.5HuT 78 cells on ice for 30 minutes. The cells were
washed twice with ice cold buffer consisting of PBS containing 2%
fetal calf serum (FCS) and 0.01% sodium azide (FACS buffer). The
cells were then incubated for 30 minutes on ice with the
appropriate fluorescent secondary antibody (either fluorescein
(FITC)-conjugated AffiniPure F(ab').sub.2 fragment goat anti-mouse
IgG(H+ L) (Jackson ImmunoResearch) or fluorescein (FITC)-conjugated
AffiniPure F(ab').sub.2 fragment goat anti-human IgG(H+ L) (Jackson
ImmunoResearch)). After 30 minutes on ice, the cells were washed
twice with FACS buffer, resuspended in 300 ml of the same buffer,
and analyzed by flow cytometry on a Becton Dickinson FACscan. FIG.
4A shows staining of the murine Act-1 mAb compared to a mouse
isotype matched irrelevant control antibody, MOPC 21 (IgG1, kappa).
FIG. 4B shows chimeric Act-1 antibody staining of HuT 78 cells
compared to a human isotype matched irrelevant control antibody
(IgG1, kappa), and mock transfected COS-7 cell supernatant. Thus,
compared to the stain produced by the murine Act-1 antibody, the
chimeric antibody stained HuT 78 cells similarly. Collectively,
these date demonstrate that the appropriate sequences for mouse
Act-1 variable regions were successfully cloned and expressed.
Amino Acid Sequence Analysis
[0102] Amino acid sequence analysis was performed on purified
murine Act-1 heavy and light chains to confirm the identities of
the cDNAs for the light and heavy chain variable regions isolated
from the hybridoma. This was accomplished for the light chain as
follows:
[0103] Murine Act-1 (5 mg/ml) was reduced with 2 mM DTT for 2 hours
at 37.degree. C. in 0.3 M sodium borate, 0.15 M sodium chloride
under nitrogen. The solution was then made 10 mM in iodoacetamide
and incubated for 4 hr at room temperature. SDS-PAGE analysis under
non-denaturing conditions confirmed that the proteins were reduced
quantitatively. The protein solution was then extensively dialyzed
in PBS and an aliquot applied to a Superdex 75 column (16/60,
Pharmacia) (run 1). Heavy and light chain coeluted from this column
with an elution volume corresponding to that of the exclusion
volume indicating that the two chains were still held together.
Another aliquot was then made 8M urea and ran on a superdex 75
column under denaturing conditions (6M urea) (run 2). Both chains
again coeluted in the void volume probably due to unfolding.
SDS-PAGE analysis confirmed the presence of both chains in the two
samples eluted from the 2 gel filtration runs. These samples were
subjected to N-terminal sequence analysis (Commonwealth
Biotechnologies, Inc.) with the following result:
TABLE-US-00005 Sample 2: DVVVTQTPLSLPVSFDGQV (SEQ ID NO: 47) Sample
1: DVVVTQTPLSL (SEQ ID NO: 48)
[0104] The sequence that was obtained corresponds to the N-terminus
of the mature light chain as deduced from the DNA sequence. This
and other attempts to obtain sequence of the heavy chain indicated
that its N-terminus was likely blocked. Therefore, amino acid
sequence analysis of internal peptide fragments was performed on
the heavy chain.
[0105] To simplify internal amino acid sequencing F(ab)'2 fragments
from the antibody were produced by cleaving with pepsin. Murine
Act-1 was cleaved with pepsin at a ratio of antibody:pepsin of
1:200 for 2 hr at 37.degree. C. in 0.1 M sodium citrate, pH 3.0.
The reaction was complete as assessed by SDS-PAGE analysis. The
protein was then purified through protein G and protein A columns.
The sample was then reduced and alkylated as described above, and
the heavy chain fragment was separated from the light chain by
preparative SDS-PAGE (15%). The heavy chain fragment was excised,
and electroleuted in 1 ml of 0.1% SDS with running buffer for 2
hours. This sample was cleaved with 2 ng of Asp-N endoproteinase
for 30 minutes and the fragments were separated by SDS-PAGE
(17.5%). The digestion products were passively eluted in 0.1 M
Hepes pH 8.0, 0.1% SDS overnight and subjected to N-terminal
sequence analysis (Commonwealth Biotechnologies, Inc.).
[0106] The sequence obtained from a 17 Kda fragment was DYAIDYWG
(SEQ ID NO:49), which was present in the clone for the heavy chain
(FIG. 1; the sequence AIDY corresponds to the beginning of the JH4
region).
Example 2
Molecular Modelling of the Mouse Act-1 Variable Regions
[0107] In order to assist in the design of the CDR-grafted variable
regions, a molecular model of the mouse Act-1 variable regions was
produced. Modeling the structures of well-characterized protein
families with immunoglobulins was done using the established
methods for modeling by homology. Molecular modeling was carried
out using a Silicon Graphics IRIS 4D workstation running under the
UNIX operating system, the molecular modelling package QUANTA
(Polygen Corp., Waltham, Mass.), and the Brookhaven
crystallographic database of solved protein structures. As a first
step, the framework regions (FRs) of the new variable regions were
modeled on FRs from similar, structurally-solved immunoglobulin
variable regions. While identical amino acid side chains were kept
in their original orientation, mutated side chains were substituted
using the maximum overlap procedure to maintain chi angles as in
the original mouse Act-1 antibody. Most of the CDRs of the new
variable regions were modeled based on the canonical structures for
CDRs (Chothia, C., and A. M. Lesk, J. Mol. Biol. 196:901-917
(1987); Chothia, C., et al., Nature 342:877-883 (1989); Tramontano,
A., et al., J. Mol. Biol. 215:175-182 (1990); Chothia, C., et al.,
J. Mol. Biol. 227:799-817 (1992)). In cases such as CDR3 of the
heavy chain variable region, where there are no known canonical
structures, the CDR loop was modelled based on a similar loop
structure present in any structurally-solved protein. Finally, in
order to relieve unfavourable atomic contacts and to optimize Van
der Waals and electrostatic interactions, the model was subjected
to energy minimization using the CHARMm potential (Brooks, B. R.,
J. Comp. Chem. 4:187-217 (1983)) as implemented in QUANTA.
[0108] For the mouse Act-1 variable regions, the FRs from the light
chain variable region were modeled on the FRs from the Fab fragment
of mouse monoclonal antibody 4-4-20 (Herron, J. N., et al.,
Proteins. Structure, Function and Genetics 5:271-280 (1989)). The
FRs from the heavy chain variable region were modeled on the FRs
from the Fab fragment of mouse monoclonal antibody D11.5 (Chitarra,
V., et al., Proc. Natl. Acad. Sci., USA 90:7711-7715 (1993)). Those
amino acid side chains which differed between the mouse Act-1
antibody and the variable regions upon which the model was based
were substituted. The light chain of Fab 4-4-20 antibody was then
superimposed onto the light chain of D11.15 by aligning in space
residues 35-39, 43-47, 84-88 and 98-102 (as defined by Kabat, E.
A., et al., Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, U.S.
Government Printing Office (1991)), in order to place the two
heterologous variable regions (i.e. the 4-4-20-based kappa light
chain variable region and the D11.15-based heavy variable region)
into the correct orientation with respect to each other.
[0109] CDR1 (L1) of the light chain variable region of mAb Act-1
fitted into the L1 canonical subgroup 4, as proposed by Chothia,
C., et al., Nature 342:877-883 (1989). The L1 loop of mouse Fab
4-4-20 (see above) was identical in amino acid length, similar in
amino acid sequence, and also matched canonical subgroup 4.
Consequently the L1 loop was modeled on the L1 loop of Fab 4-4-20.
Similarly, CDR2 (L2) and CDR3 (L3) of the light chain variable
region of mAb Act-1 matched both their respective canonical
subgroup 1 loop structures and the corresponding CDRs of Fab
4-4-20. Accordingly, the L2 and L3 loops of the Act-1 kappa light
chain variable region were modeled on CDRs L2 and L3 of Fab
4-4-20.
[0110] CDR1 (H1) of the heavy chain variable region of mAb Act-1
fitted the H1 canonical subgroup 1, defined by Chothia, C., et al.,
Nature 342:877-883 (1989), as did the corresponding H1 loop of
mouse mAb D11.15 (see above). Moreover, mAb D11.15 CDR1 loop was
identical in length and very similar in amino acid sequence to H1
of mAb Act-1. Consequently, as with the light chain, this loop was
modeled on the CDR1 loop of the heavy variable region upon which
the model was based. CDR2 of the heavy chain variable region (H2)
was more difficult to define, but appeared to correspond to H2
canonical subgroup 2. Again, the H2 loop of the D11.15 antibody
also matched the same canonical subgroup and was very similar in
amino acid sequence, and so the H2 loop of mAb Act-1 was modeled on
the H2 loop of D11.15.
[0111] As discussed above, CDR3s of heavy chain variable regions
are highly variable and cannot be divided into identifiable
structural groups. For modelling H3 loops, loops of identical
length and similar amino acid sequence--preferably from another
antibody--are identified and used as a basis for the modeled loop.
There were three loops, all H3 loops from three antibodies, which
matched the Act-1 CDR3 for loop size. After testing all three loop
structures for steric clashes on the model, the H3 loop from the
human antibody Pot (Fan, Z. C., et al., J. Mol. Biol. 228:188-207
(1992)) was chosen to model the H3 loop of mAb Act-1. After
adjusting the whole of the model for obvious steric clashes it was
subjected to energy minimization as implemented in QUANTA.
Designing the CDR-Grafted Variable Regions
[0112] The first step in designing CDR-grafted variable regions is
the selection of the human light and heavy chain variable regions
that will serve as the basis of the humanized variable regions. Two
approaches for selecting the human variable regions were tested and
compared. In one approach, the human variable regions were selected
from the consensus sequences for the different subgroups of human
variable regions (Kabat, E. A., et al., Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, U.S. Government Printing Office (1991)). The
rodent light and heavy chain variable regions were compared to the
human consensus sequences and the most similar human light and
heavy chain consensus sequences were selected from among the six
subgroups of human lambda light chain variable regions, the four
subgroups of human kappa light chain variable regions, and the
three subgroups of human heavy chain variable regions (see
Kettleborough, C. A., Protein Engineering 4:773-783 (1991)). In
another approach, the human variable regions were selected from all
published sequences for human variable regions (Kabat, E. A., et
al., Sequences of proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, U.S.
Government Printing Office (1991)). The amino acid sequences of
rodent light and heavy chain variable regions were compared to
human sequences, and human variable regions with a high degree of
similarity to the rodent variable regions were selected. Human
light and heavy chain variable regions from the same human antibody
can be used in order to ensure that the two variable regions will
assemble properly (Queen, C., et al., Proc. Natl. Acad. Sci., USA
86:10029-10033 (1989)). However, as described herein, the human
light and heavy chain variable regions selected as the templates
were derived from two different human antibodies. In this way, it
was possible to select for human variable regions with a higher
degree of similarity to the rodent variable regions. There are many
successful examples of CDR-grafted antibodies based on variable
regions derived from two different human antibodies. One of the
best studied examples is reshaped human CAMPATH-1 antibody
(Riechmann, L., et al., Nature 332:323-327 (1988)).
[0113] To design reshaped human ACT-1 variable regions, the mouse
ACT-1 variable regions were compared to the consensus sequences for
all subgroups of mouse and human variable regions (Kabat, E. A., et
al., Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, U.S.
Government Printing Office (1991)). The results are summarized in
Tables 1 and 2.
[0114] The mouse Act-1 light chain variable region was most similar
to the consensus sequence for mouse kappa light chain subgroup II
with a 83.9% identity overall and a 87.5% identity within the FRs
only (Table 1). With respect to human antibody sequences, the mouse
Act-1 light chain variable region was most similar to the consensus
sequence for human kappa light chain subgroup II with a 72.3%
identity overall and a 78.8% identity within the FRs only (Table
1).
TABLE-US-00006 TABLE 1 Comparison of mouse Act-1 kappa light chain
variable region to the consensus sequences for the subgroups of
mouse and human kappa light chain variable regions. The amino acid
sequence of the mouse Act-1 kappa light chain variable region was
compared, with and without the sequences of the CDRs, to the
consensus sequences of the different subgroups of mouse and human
kappa light chain variable regions, with and without the sequences
of the CDRs. The percents similarity and identity to the most
similar mouse and human subgroups are listed. Mouse or Human
Complete Variable Variable Kabat Region or FRs Percent Percent
Region Subgroup only Similarity Identity Mouse II Complete 91.07
83.93 FRs only 95.00 87.50 Human II Complete 83.93 72.32 FRs only
90.00 78.75
[0115] The mouse Act-1 heavy chain variable region was most similar
to the consensus sequence for mouse heavy chain subgroup IIB with a
83.5% identity overall and a 94.3% identity within the FRs only
(Table 2). With respect to human antibody sequences, the mouse
Act-1 heavy chain variable region was most similar to the consensus
sequence for human heavy chain subgroup I with a 68.6% identity
overall, and a 75.9% identity within the FRs only (Table 2). These
results confirm that the mouse Act-1 variable regions appear to be
typical of mouse variable regions. The results also indicate
subgroups of human variable regions which can serve as good sources
for human variable region templates or acceptors for
CDR-grafting.
TABLE-US-00007 TABLE 2 Comparison of mouse Act-1 heavy chain
variable region to the consensus sequences for the subgroups of
mouse and human heavy chain variable regions. The amino acid
sequence of the mouse Act-1 heavy chain variable region was
compared, with and without the sequences of the CDRs, to the
consensus sequences of the different subgroups of mouse and human
heavy chain variable regions, with and without the sequences of the
CDRs. The percents similarity and identity to the most similar
mouse and human subgroups are listed. Complete Mouse or Human Kabat
Variable Region Percent Percent Variable Region Subgroup or FRS
only Similarity Identity Mouse IIB Complete 89.26 83.47 FRs only
95.40 94.25 Human I Complete 81.82 68.60 FRs only 85.06 75.86
[0116] The mouse Act-1 variable regions were also compared to the
individual sequences of all recorded examples of mouse and human
variable regions (Kabat, E. A., et al., Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, U.S. Government Printing Office (1991); UW GCG
package (University of Wisconsin)). With respect to human antibody
sequences, the mouse Act-1 light chain variable region was very
similar to the sequence for the human kappa light chain variable
region from human antibody GM607'CL (Klobeck, H.-G., et al., Nature
309:73-76 (1984)). FIG. 5 shows an alignment of the amino acid
sequences of the mouse Act-1 light chain variable region (SEQ ID
NO:7) and of the human GM607'CL light chain variable region (SEQ ID
NO:8). As expected, the light chain variable region of human
GM607'CL is a member of subgroup II of human kappa light chain
variable regions. The overall sequence identity between the mouse
Act-1 and human GM607'CL light chain variable regions was
calculated to be 71.4%. The mouse Act-1 heavy chain variable region
was very similar to the sequence for the human heavy chain variable
region from human antibody 21/28'CL (Dersimonian, H., et al., J.
Immunol. 139:2496-2501 (1987)). FIG. 6 shows an alignment of the
amino acid sequences of the mouse Act-1 heavy chain variable region
(SEQ ID NO:9) and of the human 21/28'CL heavy chain variable region
(SEQ ID NO:10). As expected, the heavy chain variable region of
human 21/28'CL is a member of subgroup I of human heavy chain
variable regions. The overall sequence identity between the mouse
Act-1 and human 21/28'CL heavy chain variable regions was
calculated to be 68.1%. Based on these comparisons, human GM607'CL
light chain variable region was selected as the human template for
the design of reshaped human Act-1 light chain variable region, and
human 21/28'CL heavy chain variable region was selected as the
human template for the design of reshaped human Act-1 heavy chain
variable region.
[0117] The second step in the design process was to insert the
rodent CDRs into the selected human light and heavy chain variable
regions. The entire rodent CDRs, as defined by Kabat, E. A., et
al., Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, U.S.
Government Printing Office (1991)), were joined to the human FRs to
create a simple CDR-graft. In some cases, a rodent antibody that is
humanized in a simple CDR graft will show little or no binding to
antigen. It is important to study the amino acid sequences of the
human FRs to determine if any of these amino acid residues are
likely to adversely influence binding to antigen, either directly
through interactions with antigen, or indirectly by altering the
positioning of the CDR loops.
[0118] In the third step, decisions were made as to which amino
acid residues in the human FRs should be altered in order to
achieve good binding to antigen. At this stage, the model of the
rodent variable regions becomes most useful in the design process.
Also useful are the canonical structures for the CDRs as defined by
Chothia, C., et al., Nature 342:877-883 (1989). It is important to
conserve in the humanized variable regions any of the rodent amino
acid residues that are part of the canonical structures. It is
helpful to compare the sequence of the rodent antibody to be
humanized to similar sequences from other rodent antibodies to
determine if the amino acids at certain positions are unusual or
rare. This might indicate that the rodent amino acid at that
position has an important role in antigen binding. By studying the
model of the rodent variable regions, it is possible to predict
whether amino acids at particular positions could or could not
influence antigen binding. When human variable regions from
individual human antibodies are being used as the basis of the
design, then it is advisable to compare the individual human
sequence to the consensus sequence for that subgroup of human
variable regions. Any amino acids that are particularly unusual
should be noted. In most cases, a few amino acids in the human FRs
are identified that should be changed from the amino acid present
at that position in the human variable region to the amino acid
present at that position in the rodent variable region.
[0119] Tables 3 and 4 summarize how the reshaped human Act-1
variable regions were designed. Table 3 is an alignment of amino
acid sequences used in the design of reshaped human mAb Act-1
V.sub.L regions, and lists the amino acid sequence of the mouse
Act-1 light chain variable region to be humanized (SEQ ID NO:7) in
column 4, the consensus sequence for the subgroup of mouse variable
regions to which the mouse Act-1 variable region belongs (SEQ ID
NO:50) in column 5 (Mouse .kappa.-II), the consensus sequence for
the subgroup of human variable regions to which the mouse Act-1
variable is most similar (SEQ ID NO:51) in column 6 (Human
.kappa.-II), the amino acid sequence of the human variable region
that is serving as a template (i.e., GM607'CL) (SEQ ID NO:8) in
column 7, and the amino acid sequence of the reshaped human Act-1
variable region (SEQ ID NO:52) as designed in column 8 (Act-1
RHV.sub.H). Table 4 an the alignment of amino acid sequences used
in the design of reshaped human mAb Act-1 V.sub.H regions and lists
the amino acid sequence of the mouse Act-1 heavy chain variable
region to be humanized (SEQ ID NO:9) in column 4, the consensus
sequence for the subgroup of mouse variable regions to which the
mouse Act-1 variable region belongs (SEQ ID NO:53) in column 5
(Mouse IIB), the consensus sequence for the subgroup of human
variable regions to which the mouse Act-1 is most similar (SEQ ID
NO:54) in column 6 (Human I), the amino acid sequence of the human
variable region that is serving as a template (i.e., 21/28'CL) (SEQ
ID NO:10) in column 7, and the amino acid sequence of the reshaped
Act-1 variable region (SEQ ID NO:55) as designed in column 8 (Act-1
RHV.sub.H). The penultimate column in Tables 3 and 4 indicates the
position (surface or buried) of residues in the FRs that differ
between the mouse Act-1 and the selected human FRs. The final
column in Tables 3 and 4 lists comments relevant to that position
in the variable region.
[0120] In Table 3, the following symbols are used: (*) invariant
residues as defined either by the Kabat consensus sequences i.e.
95% or greater occurrence within Kabat subgroup (Kabat, E. A., et
al., Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, U.S.
Government Printing Office (1991)) (in the case of columns 5 and 6)
or as part of the canonical structure for the CDR loops (in the
case of columns 5 and 6) or as part of the canonical structure for
the CDR loops (in the case of column 8) as defined by Chothia, C.,
et al., Nature 342:877-883 (1989); (BOLD) positions in FRs and CDRs
where the human amino acid residue was replaced by the
corresponding mouse residue; (UNDERLINE) positions in FRs where the
human residue differs from the analogous mouse residue number;
(.DELTA.) numbering of changes in the human FRs; (mouse Ab Act-1)
amino acid sequence of the V.sub.L region from mouse Act-1
antibody; (mouse .kappa.-II) consensus sequence of mouse kappa
V.sub.L regions from subgroup II (Kabat, E. A., et al., supra);
(human .kappa.-II) consensus sequence of human V.sub.L regions from
subgroup II (Kabat, E. A., et al., supra); (GM607'CL) amino acid
sequence from human GM607'CL antibody (Klobeck, H.-G., et al.,
Nature 309:73-76 (1984)); (Surface or Buried) position of amino
acid in relation to the rest of the residues in both chains of the
antibody variable regions; (Act-1 RH V.sub.K) amino acid sequence
of the reshaped human mAb Act-1 V.sub.L region.
TABLE-US-00008 TABLE 3 Alignment of amino acid sequences used in
the design of reshaped human mAb Act-1 VL regions. Mouse Mouse
Human GM 607 Act-1 k-II k-II CL FR or (SEQ ID (SEQ ID (SEQ ID (SEQ
ID Act-1 or RH Vk Surface Kabat # CDR NO: 7) NO: 50) NO: 51) NO: 8)
(SEQ ID NO: 52) or Buried Comment 1 1 FR1 D D* D* D D 2 2 | V V I*
I V buried Canonical AA for L1 loop (D1). 3 3 | V V V* V V 4 4 | V
M M M M buried Buried between L1 and L2. V = 9/245, M = 202/245 in
mouse k-II. M = 42/45, V not seen in human k-II. If binding is
poor, consider changing this to Val in second version. 5 5 | T T*
T* T T 6 6 | Q Q* Q* Q Q 7 7 | T T S S S surface Distal to binding
site (BS). T = 164/245 in mouse k-II. T = 10/37, S = 27/37 in human
k-II. 8 8 | P P P* P P 9 9 | L L L* L L 10 10 | S S S* S S 11 11 |
L L L* L L 12 12 | P P P P P 13 13 | V V* V* V V 14 14 | S S T* T T
surface Distal to BS. S = 151/248 in mouse .kappa.-II. T alone
(30/30) seen in human .kappa.-II. 15 15 | F L P P P surface Distal
to BS. F = 9/253 in mouse .kappa.-II, F not seen in human
.kappa.-II. P = 29/31 in human .kappa.-II. 16 16 | G G* G* G G 17
17 | D D E E E surface Distal to BS. E = 18/30, D not seen in human
.kappa.-II. 18 18 | Q Q P* P P surface Distal to BS and on a turn.
P alone (31/31) seen in human .kappa.-II. 19 19 | V A A* A A buried
Pointing into core, but standard mouse to human change. V = 66/253,
A = 187/253 in mouse k-II. A alone (30/30) seen in human
.kappa.-II. 20 20 | S S* S* S S 21 21 | I I* I* I I 22 22 | S S* S*
S S 23 23 FR1 C C* C* C C 24 24 CDR1 R R R R R 25 25 | S S* S* S S
Canonical AA for L1 loop. 26 26 | S S* S* S S Canonical AA for L1
loop. 27 27 | Q Q Q Q Q Canonical AA for L1 loop. 27A 28 | S S S S
S Canonical AA for L1 loop. 27B 29 | L L L* L L Canonical AA for L1
loop. 27C 30 | A V L L A Canonical AA for L1 loop. 27D 31 | K H H H
K Canonical AA for L1 loop. 27E 32 | S S S S S Canonical AA for L1
loop. 27F | -- -- X -- -- 28 33 | Y N D N Y Canonical AA for L1
loop. 29 34 | G G* G G G Canonical AA for L1 loop. 30 35 | N N N Y
N Canonical AA for L1 loop. 31 36 | T T* N N T Canonical AA for L1
loop. 32 37 | Y Y* Y* Y Y Canonical AA for L1 loop. 33 38 | L L* L*
L L Canonical SS for L1 loop. 34 39 CDR1 S E N D S Packing AA.
Unusual (117/1365). A, H and N most commonly seen here. 35 40 FR2 W
W* W* W W 36 41 | Y Y Y Y Y Packing AA. Most common AA. 37 42 | L
L* L L L 38 3 | H Q* Q Q Q buried Packing AA. H is unusual
(31/1312). Q is most common AA (1158/1312). H = 6/225, Q = 219/225
in mouse k-II. Q = 15/17, H not seen in human .kappa.-II. 39 44 | K
K K K K 40 45 | P P* P P P 41 46 | G G* G* G G 42 47 | Q Q* Q Q Q
43 48 | S S* S S S 44 49 | P P* P* P P Packing AA. Most common AA.
45 50 | Q K Q Q Q 46 51 | L L L L L Packing AA. Most common AA. 47
52 | L L* L L L 48 53 | I I* I* I I Canonical AA for L2 loop. 49 54
FR2 Y Y Y* Y Y 50 55 CDR2 G K L L G Canonical AA for L2 loop. 51 56
| I V V G I Canonical AA for L2 loop. 52 57 | S S* S* S S Canonical
AA for L2 loop. 53 58 | N N N N N 54 59 | R R R* R R 55 60 | F F A
A F 56 61 CDR2 S S* S* S S 57 62 FR3 G G* G* G G 58 63 | V V* V* V
V 59 64 | P P P* P P 60 65 | D D* D D D 61 66 | R R* R R R 62 67 |
F F* F* F F 63 68 | S S S* S S 64 69 | G G* G G G Canonical AA for
L2 loop. 65 70 | S S* S* S S 66 71 | G G* G* G G 67 72 | S S* S S S
68 73 | G G* G G G 69 74 | T T* T* T T 70 75 | D D D D D 71 76 | F
F* F* F F Canonical AA for L1 loop. 72 77 | T T* T* T T 73 78 | L
L* L* L L 74 79 | K K K K K 75 80 | I I* I* I I 76 81 | S S S S S
77 82 | T R* R R R surface Distal to BS. T = 6/221, R = 211/221 in
mouse .kappa.-II. R = 11/12, T not seen in human .kappa.-II. 78 83
| I V V* V V buried Pointing into core, but standard mouse to human
change. I = 6/213, V = 195/213 in mouse .kappa.-II. V alone (12/12)
seen in human .kappa.-II. 79 84 | K E E E E surface Distal to BS. K
= 20/215, E = 191/215 in mouse .kappa.-II. E = 9/12, K not seen in
human .kappa.-II. 80 85 | P A* A A A surface Distal to BS. P =
6/183, A = 175/183 in mouse .kappa.-II. P = 1/12, A = 11/12 in
human .kappa.-II. 81 86 | E E* E E E 82 87 | D D* D D D 83 88 | L L
V* V V half Distal to BS. V alone buried (12/12) seen in human
k-II. 84 89 | G G* G* G G 85 90 | M V V* V V half Distal to BS. M =
6/212, buried V = 196/212 in mouse k-II. V alone (12/12) seen in
human k-II. 86 91 | Y Y* Y* Y Y 87 92 | Y Y Y* Y Y Packing AA. Most
common AA. 88 93 FR3 C C* C* C C 89 94 CDR3 L F M* M L Packing AA.
L is unusual (93/1238). Q is most common AA (654/1238). 90 95 | Q
Q* Q Q Q Canonical AA for L3 loop. 91 96 | G G A A G Canonical for
L3/Packing AA. 3rd most common AA. 92 97 | T T L L T Canonical AA
for L3 loop. 93 98 | H H Q Q H Canonical AA for L3 loop. 94 99 | Q
V X T Q Canonical AA for L3 loop. 95 100 | P P* P P P Canonical AA
for L3 loop. 95A | -- P R* -- 95B | -- -- -- -- 95C | -- -- -- --
95D | -- -- -- -- 95E | -- -- -- -- 95F | -- -- -- -- 96 101 | Y Y
X Q Y Packing AA. 2nd most common AA. 97 102 CDR3 T T* T* T T
Canonical for L3. 98 103 FR4 F F* F* F F Packing AA. Most common
AA. 99 104 | G G* G* G G 100 105 | G G Q Q Q half Distal to BS. Q =
12/13, buried G = 1/12 in human .kappa.-II. 101 106 | G G* G* G G
102 107 | T T* T* T T 103 108 | K K* K K K 104 109 | L L* V V V
half Distal to BS. L = 5/14, buried V = 9/14 in human .kappa.-II.
105 110 | E E* E E E 106 111 | I I I* I I 106A | -- -- -- -- 107
112 FR4 K K* K K K
[0121] In Table 4, the following symbols are used: (*) invariant
residues as defined either by the Kabat consensus sequences i.e.
95% or greater occurrence within Kabat subgroup (Kabat, E. A., et
al., Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, U.S.
Government Printing Office (1991)) (in the case of columns 5 and 6)
or as part of the canonical structure for the CDR loops (in the
case of column 8) as defined by Chothia, C., et al., Nature
342:877-883 (1989); (BOLD) positions in FRs and CDRs where the
human amino acid residue was replaced by the corresponding mouse
residue; (UNDERLINE) positions in FRs where the human residue
differs from the analogous mouse residue number; (D) numbering of
changes in the human FRs; (mouse Ab Act-1) amino acid sequence of
the V.sub.H region from mouse Act-1 antibody; (mouse IIB) consensus
sequence of mouse V.sub.H regions from subgroup IIB (Kabat, E. A.,
et al., supra); (human I) consensus sequence of human V.sub.H
regions from subgroup I (Kabat, E. A., et al., supra); (human
21/28'CL amino acid sequence from human antibody 21/28'CL
(Dersimonian, H., et al., J. Immunol. 139:2496-2501 (1987));
(Surface or Buried) position of amino acid in relation to the rest
of the residues in both chains of the antibody variable regions;
(Act-1 RH V.sub.H) amino acid sequence of the reshaped human mAb
Act-1 V.sub.H region.
TABLE-US-00009 TABLE 4 Alignment of amino acid sequences used in
the design of reshaped human mAb Act-1 VH regions. Mouse Act-1
Mouse IIB Human I human donor FR or (SEQ ID (SEQ ID (SEQ ID 21/28CL
Act-1 RH VH Surface Kabat # CDR NO: 9) NO: 53) NO: 54) (SEQ ID NO:
10) (SEQ ID NO: 55) or Buried Comment 1 1 FR1 Q Q Q Q Q 2 2 | V V*
V V V 3 3 | Q Q* Q* Q Q 4 4 | L L* L* L L 5 5 | Q Q V V V surface
Distal to binding site (BS). Q = 135/143 in mouse IIB. V = 49/53, Q
= 1/53 in human I. 6 6 | Q Q Q Q Q 7 7 | P P S* S S half buried
Distal to BS. P = 102/150 in mouse IIB. P not seen in human I. 8 8
| G G* G* G G 9 9 | A A A A A 10 10 | E E E E E 11 11 | L L* V V V
surface Distal to BS. V = 50/54, L = 4/54 in human I. 12 12 | V V*
K K K buried Pointing into core, but standrd mouse to human change.
K = 41/55, V = 3/55 in human I. 13 13 | K K K* K K 14 14 | P P* P*
P P 15 15 | G G* G* G G 16 16 | T A A A A surface Distal to BS. T =
12/139, A = 117/139 in mouse IIB. T = 1/52, A = 23/52 in human I.
17 17 | S S* S* S S 18 18 | V V* V V V 19 19 | K K K K K 20 20 | L
L V V V buried Pointing into core, but standrd mouse to human
change. L = 138/179 in mouse IIB. V = 36/52, L = 1/52 in human I.
21 21 | S S* S S S 22 22 | C C* C* C C 23 23 | K K* K K K 24 24 | G
A* A A G buried Canonical AA for H1 loop (.DELTA.1). G not seen in
mouse IIB. G = 12/51, A = 34/51 in human I. 25 25 | Y S* S* S S
surface Pointing away from BS and so does not appear to bind
antigen (Ag). Y = 1/185 in mouse IIB. S = 48/50, Y not seen in
human I. 26 26 | G G* G* G G Canonical AA for H1 loop. 27 27 | Y Y
Y Y Y Canonical AA for H1 loop. 28 28 | T T T T T Canonical AA for
H1 loop. 29 29 | F F* F* F F Canonical AA for H1 loop. 30 30 FR1 T
T T T T Canonical AA for H1 loop. 31 31 CDR1 S S S S S Canonical AA
for H1 loop. 32 32 | Y Y Y Y Y Canonical AA for H1 loop. 33 33 | W
W A A W 34 34 | M M I M M Canonical AA for H1 loop. 35 35 | H H S H
H Packing AA. Most common AA. 35A | -- -- -- -- 35B CDR1 -- -- --
-- 36 36 FR2 W W* W* W W 37 37 | V V V V V Packing AA. Most common
AA. 38 38 | K K R* R R buried Pointing into core, but standard
mouse to human change. K = 177/188 in mouse IIB. R = 48/49, K not
seen in human I. However, Lys maybe packing H2 loop, therefore
consider changing in second version, in conjunction with A40R, if
binding poor. 39 39 | Q Q Q* Q Q Packing AA. Most common AA. 40 40
| R R A A A buried Pointing into core, but standard mouse to human
change. R = 160/177 in mouse IIB. A = 37/49, R = 0/49 in human I.
However, Arg maybe packing H2 loop, therefore consider changing in
second version, in conjunction with A38K, if binding poor. 41 41 |
P P P P P 42 42 | G G G G G 43 43 | Q Q Q Q Q 44 44 | G G G R R
buried Pointing into core, but standard mouse to human change. G =
43/48, R = 5/48 in human I. 45 45 | L L* L* L L Packing AA. Most
common AA. 46 46 | E E* E* E E 47 47 | W W* W* W W Packing AA. Most
common AA. 48 48 | I I* M M I buried Ile Underneath and supporting
H2 loop (.DELTA.2). Met = 41/48, Ile = 1/48 in human I. 49 49 FR2 G
G* G G G 50 50 CDR2 E R W W E 51 51 | I I I I I 52 52 | D D N N D
52A 53 | P P* P A P Canonical AA for H2 loop. 52B | -- -- Y -- 52C
| -- -- -- -- 53 54 | S N G G S Canonical AA for H2 loop. 54 55 | E
S N N E Canonical AA for H2 loop. 55 56 | S G G G S Canonical AA
for H2 loop. 56 57 | N G D N N 57 58 | T T T T T 58 59 | N N N K N
59 60 | Y Y Y Y Y 60 61 | N N A S N 61 62 | Q E Q Q Q 62 63 | K K*
K K K 63 64 | F F* F F F 64 65 | K K Q Q K 65 66 CDR2 G S G G G 66
67 FR3 K K* R R R surface Distal to BS. R = 39/49, K not seen in
human I. 67 68 | A A* V V V half buried Distal to BS. V = 45/48, A
not seen in human I. 68 69 | T T* T T T 69 70 | L L* I I L buried
Leu Underneath and supporting H2 loop (.DELTA.3). Ile = 26/49, Leu
= 1/49 in human I. 70 71 | T T* T T T 71 72 | V V A R V buried
Canonical AA for H2 loop (.DELTA.4). 72 73 | D D* D D D 73 74 | I K
T T I surface Behind H2 loop and may play a direct part in Ag
binding (.DELTA.5). Ile not seen in mouse IIB or human I. T = 21/49
in human I. 74 75 | S S S* S S 75 76 | S S* T A A surface Distal to
BS. T = 26/50, A = 4/50, S not seen in human I. 76 77 | S S S S S
77 78 | T T* T T T 78 79 | A A A A A 79 80 | Y Y* Y Y Y 80 81 | M M
M M M 81 82 | Q Q E E E half buried Distal to BS. Q = 163/194 in
mouse IIB. E = 35/50, Q = 11/50 in human I. 82 83 | L L* L L L 82A
84 | S S S S S 82B 85 | S S S S S 82C 86 | L L* L* L L 83 87 | T T*
R R R surface Distal to BS. R = 33/51, T = 4/51 in human I. 84 88 |
S S* S S S 85 89 | E E E E E 86 90 | D D* D* D D 87 91 | S S* T T T
surface Distal to BS. T = 48/51, S = 2/51 in human I. 88 92 | A A*
A A A 89 93 | V V* V V V 90 94 | Y Y* Y* Y Y 91 95 | Y Y Y Y Y
Packing AA. Most common AA. 92 96 | C C* C* C C 93 97 | A A A* A A
Packing AA. Most common AA. 94 98 FR3 R R R R R Canonical AA for H1
loop. 95 99 CDR3 G Y A G G Packing AA. 2nd most common residue seen
at this point --OK. 96 100 | G Y P -- G 97 101 | Y Y G -- Y 98 102
| D G Y -- D 99 103 | G G G G G 100 104 | W S S -- W 100A 105 | D S
G D 100B 106 | Y X G -- Y 100C 107 | A X G -- A 100D 108 | I V C --
I Packing AA. I = 26/1211. F and M are most commonly seen. 100E |
-- Y Y -- 100F | -- X R Y 100G | -- -- G Y 100H | -- Y* D G 100I |
-- W Y S 100J | -- Y X G 100K | -- F F S 101 109 | D D D N D 102
110 CDR3 Y Y Y Y Y 103 111 FR4 W W* W* W W Packing AA. Most common
AA. 104 112 | G G* G G G 105 113 | Q Q Q Q Q 106 114 | G G* G* G G
107 115 | T T* T T T 108 116 | S T L L L surface Distal to BS. T =
88/149 in mouse IIB. L = 25/39, T = 7/39 in human I. 109 117 | V V
V* V V 110 118 | T T* T T T 111 119 | V V* V V V 112 120 | S S* S*
S S 113 121 FR4 S S S* S S
[0122] With respect to the design of reshaped human Act-1 light
chain variable region (Table 3), one residue in the human FRs was
changed from the amino acid present in the human FRs to the amino
acid present in the original mouse FRs. This change was at position
2 in FR1 (as defined by Kabat, E. A., et al., Sequences of Proteins
of Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, U.S. Government Printing Office (1991)). In
particular, the isoleucine found in human GM607'CL light chain
variable region was changed to valine as found in mouse Act-1 light
chain variable region. This position in the kappa light chain
variable region has been identified by Chothia, C., et al., Nature
342:877-883 (1989) as one of the locations that is critical for the
correct orientation and structure of the L1 loop and, as such, is
known as one of the "canonical amino acids". Due to their important
role in loop conformation, such mouse framework residues are
generally always conserved in the reshaped variable region.
[0123] At position 4 in FR1, there is a valine in the mouse
sequence and a methionine in the human sequence. A change from a
valine to a methionine is not a drastic change in itself as both
amino acids are non-polar, hydrophobic residues, so the methionine
present in the human sequence was used in the reshaped human Act-1
variable region. However, the model indicates that the valine is
buried between the L1 and L2 loops and the mean volume of valine
when buried in proteins is 142 .ANG..sup.3, whereas methionine
occupies approximately 171 .ANG..sup.3 of space. The larger
methionine residue could cause a change in the conformation of
either, or both, of the L1 and L2 loops. Antigen binding of the
reshaped human Act-1 may be improved by an additional change at
position 4 from methionine to a valine in the reshaped human Act-1
light chain variable region.
[0124] With respect to the design of reshaped human Act-1 heavy
chain variable region (Table 4), there were five residues in the
human FRs which were changed from the amino acids present in the
human FRs to the amino acids present in the original mouse FRs. At
positions 24 in FR1 and 71 in FR3 (as defined by Kabat, E. A., et
al., Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, U.S.
Government Printing Office (1991)), the amino acid residues as
present in the mouse sequence were retained in the reshaped human
Act-1 heavy chain variable region because these positions are part
of the canonical structures for the H1 and H2 loops, respectively
(Chothia, C., et al., Nature 342:877-883 (1989)). Since any amino
acid changes at these positions could disrupt the packing and the
final structures of the H1 and H2 loops, mouse residues at these
critical locations are routinely conserved in the humanized heavy
chain variable region.
[0125] At position 48 in FR2, the methionine in the human sequence
was changed to an isoleucine as present in the mouse Act-1
sequence. The substitution of a methionine for an isoleucine is
unusual. More importantly, the model shows that the isoleucine
residue is buried underneath the H2 loop. As a result, changes at
this buried position may have influenced the structure of the H2
loop and hence interfered with antigen binding.
[0126] At position 69 in FR3, the isoleucine in the human sequence
was changed to a leucine as present in the mouse Act-1 sequence.
Although the substitution of a leucine for an isoleucine is not
unusual, the model shows that the leucine is buried under the H2
loop. Consequently, like the residue at position 48, changes at
this location could influence the conformation of the H2 loop and
thereby disrupt antigen binding.
[0127] Finally, at position 73 in FR3, the threonine in the human
sequence was changed to an isoleucine as present in the mouse
sequence. An isoleucine at this position in FR3 has never been seen
previously in mouse subgroup IIB, or human subgroup I (as defined
by Kabat, E. A., et al., Fifth Edition, U.S. Department of Health
and Human Services, U.S. Government Printing Office (1991)), which
suggests that the isoleucine at this location may have an important
role in antigen binding.
[0128] In the model, the leucine at position 73 appears to be on
the surface near the edge of the binding site and, depending on the
size and orientation of the epitope on the .alpha.4.beta.7
integrin, may possibly play a direct part in antigen binding.
However, as a surface residue position, the antibody as a whole
would have less immunogenic potential if the mouse amino acid was
not present in the reshaped human antibody. The isoleucine could be
replaced with the human threonine residue in derivatives of the
reshaped antibody, and the new construct re-tested to determine
whether the second version maintains a similar level of antigen
binding.
[0129] In addition to the five changes in the human FRs made in the
original design of the reshaped human Act-1 heavy chain variable
region, there were two other changes that could be made which may
improve antigen binding. The model suggests that residues 38Lys and
40Arg in the heavy variable region of mouse mAb Act-1 are
positioned underneath the H2 loop and pack close to 63Phe in CDR2
(numbering as in Table 4). However, these residues are also located
in the core of the heavy chain variable region and may have other,
possibly detrimental, effects if they were used to replace their
corresponding human amino acids (38 Arg and 40 Ala, respectively).
Therefore, the changes to positions 38 and 40 in FR2 were not
incorporated into the reshaped human heavy variable region of mAb
Act-1. However, either or both modifications of the reshaped heavy
chain may be used in derivatives to improve antigen binding.
Conclusions
[0130] A model of the mouse Act-1 variable regions was built based
mainly on the solved structures of other antibody variable regions.
The model was used in the design of humanized Act-1 variable
regions. Particular emphasis was put on retaining the structure of
the antigen-binding site in the reshaped human variable
regions.
[0131] A reshaped human Act-1 light chain variable region and a
reshaped human Act-1 heavy chain variable regions were designed
(Tables 3 and 4). The reshaped human Act-1 light chain variable
region was based on the CDRs of mouse Act-1 light chain variable
region and on the FRs from the light chain variable region of human
GM607'CL antibody. One amino acid change was made in the human FRs
at position 2. The reshaped human Act-1 heavy chain variable region
was based on the CDRs of mouse Act-1 heavy chain variable region
and on the FRs from the heavy chain variable region of human
21/28'CL antibody. Five amino acid changes were made in the human
FRs at positions 24, 48, 69, 71 and 73.
[0132] In addition, a single site at position 4 in FR1 of the kappa
light chain and two sites at positions 38 and 40 in FR2 of the
heavy chain were noted that might be considered in the design of
additional versions of reshaped human Act-1 variable regions. Also,
a single residue at position 73 in FR3 of the heavy chain was also
identified as a candidate for back-mutation from the mouse to the
human amino acid, in view of its location on the surface of the
antibody.
Example 3
Construction of Nucleic Acids Encoding Reshaped Variable
Regions
[0133] After confirming that the correct heavy chain and light
chain variable regions had been cloned biochemically (partial amino
acid sequence) and functionally (chimeric antibody staining of HuT
78 cells), a reshaped amino acid sequence was designed as described
above. Next, genes encoding the reshaped antibody chains were
designed and prepared.
Design, Construction, and Expression of Humanized ACT-1
[0134] After determining the primary amino acid sequence of the
humanized antibody as described in Example 2, the sequence was
reverse-translated into a degenerate nucleic acid sequence and
analyzed for potential restriction enzyme sites using MacVector
(Kodak, Scientific Imaging Systems) version 4.5.3. A nucleic acid
sequence was then selected which incorporated restriction enzyme
cleavage sites but conserved the primary amino acid sequence. The
heavy chain nucleic acid sequence (SEQ ID NO:18) and amino acid
sequence (SEQ ID NO:19) are shown in FIG. 11, and the light chain
nucleic sequence (SEQ ID NO:20) and amino acid sequence (SEQ ID
NO:21) are shown in FIG. 12 with restriction enzyme sites noted
which were used in subcloning.
[0135] The humanized Act-1 heavy and light chain variable region
genes were constructed as follows. Overlapping, complementary
oligonucleotides, designated L1-L6 (SEQ ID NOS:22-27, respectively)
for the light chain, and H1-H10 (SEQ ID NOS:28-37, respectively)
for the heavy chain were synthesized using an Applied Biosystems
DNA Synthesizer Model 392 (FIG. 13). After deprotection overnight
at 55.degree. C., oligos were dried in a Speed-Vac, resuspended in
100 ml of water and desalted over Bio-Spin 6 columns (Bio-Rad). The
oligo concentration was determined by measuring absorbance at 260
nm, and the oligos were purified by denaturing polyacrylamide gel
electrophoresis.
[0136] 100 .mu.g of each oligo was mixed with 2 volumes of loading
dye (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05%
xylene cyanol FF), heated for 2 minutes at 65.degree. C., and run
in 1.times.TBE for approximately 3 hours at 250 V. The gel was
stained with ethidium bromide and observed under ultraviolet light.
Oligos of correct length were then cut out of the gel, placed in
dialysis tubing with water and electroeluted. The oligos were twice
extracted with equal volumes of phenol/chloroform/isoamylalcohol
(25:24:1 v/v) (Gibco/BRL) and precipitated by adding 0.1 volumes of
3.0 M potassium acetate (pH 6) and 2 volumes of cold ethanol. After
centrifugation, the pellets were washed once with 70% ethanol,
vacuum dried, and resuspended in 50 .mu.l water.
[0137] Complementary oligos were annealed by mixing equal molar
quantities (approximately 100 .mu.g in 50 .mu.l water) of the
purified oligo with an equal volume (100 .mu.l) of 2.times.
annealing buffer (2.times.=1M NaCl, 40 mM Tris-HCl at pH 7.5, 2 mM
EDTA). Oligos were denatured by heating to 95.degree. C. for 10
minutes followed by an 8 hour incubation at 65.degree. C. Annealed
oligos were then ethanol precipitated as described previously and
resuspended in 40 .mu.l water.
[0138] Extension of the annealed oligos was accomplished by adding
2 .mu.l Large Fragment DNA Polymerase I (Klenow), 5 .mu.l 2.5 mM
dNTPs and 5 .mu.l 10.times. Buffer (10.times.=10 mM Tris-HC1, 10 mM
MgCl.sub.2, 1 mM DTT, pH 7.9 at 25.degree. C.) bringing the final
volume up to 52 .mu.l. The mixture was incubated for one hour at
room temperature. An additional 1 .mu.l of dNTPs and 1 .mu.l of
Klenow were added with a half hour incubation at 37.degree. C. Note
that heavy chain fragment A did not have to be extended.
[0139] Annealed and extended fragments were purified from single
stranded, unannealed material by electrophoresis through a 12%
native polyacrylamide gel. The gel was stained with ethidium
bromide and observed under ultraviolet light. The correct length
fragments were cut out and recovered by electroelution in dialysis
tubing as described above. The fragments were washed twice with
equal volumes of phenol/chloroform/isoamyl alcohol, ethanol
precipitated and resuspended in 10 .mu.l water.
[0140] The three light chain fragments (LA, LB & LC) and five
heavy chain fragments (designated HA-HE) were independently ligated
into pCR-Script.TM. and transformed, except as described below,
into XL-1 Blue Supercompetent Cells using a pCR-Script kit
(Stratagene) according to the manufacturer's recommended protocol.
Fragments pCR-LA and pCR-LB were transformed into DM1 (Gibco/BRL)
competent cells to avoid the Dcm methylase which would block
digestion with restriction enzyme Msc I. White colonies were
picked, and miniprep DNA was sequenced using Sequenase T7 DNA
polyemerase kit according to the manufacturer's recommended
protocol. T3 and T7 primers, which anneal on opposite sides of the
insert, were used for sequencing.
[0141] Compilation subcloning of the humanized heavy chain variable
region and light chain variable region fragments was accomplished
using specific restriction sites incorporated into the sequence
during synthesis. Heavy chain fragments HA-HD include an additional
Age I restriction site at the end of each sequence allowing for
sequential subcloning of the fragments as described below.
[0142] Miniprep DNA from pCR-HA and pCR-HB were digested with
restriction enzymes Spe I and Age I. DNA was electrophoresed on a
1% agarose gel. The 141 bp fragment HB was recovered from the gel
and ligated into pCR-HA at the Spe I and Age I sites giving rise to
pCR-HAB. Next, 112 bp fragment HC was released from pCR-HC using
Xba I and Age I and ligated into the Xba I and Age I sites in
pCR-HAB resulting in the plasmid pCR-AC. Fragments HD (141 bp) and
HC (130 bp) were ligated in the same sequential fashion using
restriction sites Nhe I and Age I for Fragment HD, and BstE II and
Age I for fragment E. The final plasmid containing all five heavy
chain variable region fragments in pCR-script was designated
pCR-HAE. All digests were performed using miniprep DNA with
incubations at 37.degree. C. for at least two hours except for
those using BstE II, which has an optimal incubation temperature of
65.degree. C. Ligations were done overnight at 16.degree. C. using
T4 DNA ligase with a 1:10 vector to insert ratio and transformed
the following day into DH5a subcloning efficiency competent cells
(Gibco/BRL) following the manufacturer's recommended protocol.
[0143] The Act-1 humanized heavy chain variable region in
pCR-Script was released by digestion of pCR-HAE with HindIII and
Age I. This 411 bp fragment was used to replace the mouse variable
region sequences of pEE6 mhACT1Hchi (Example 1) which had been
digested with HindIII and Age I generating the humanized ACT-1
heavy chain gene in pEE6hCMV-B. The resulting plasmid is designated
pEE6hACT1H. Correct DNA sequence was determined by sequencing.
[0144] Light chain fragment A in pCR-Script.TM. was digested with
BspE I and MscI. This 153 bp fragment was then used to replace the
mouse portion from BspE I to MscI of the mouse variable light chain
in pCR-script.TM.. This plasmid is designated pCR-LhAmBC. Light
chain fragment B, digested with Msc I and Nru I, and light chain
fragment C, digested with Nru I and Kas I, were triple ligated into
the MscI and Kas I sites of pCR-LhAmBC replacing the remaining
mouse sequence. Digestions, ligations and transformations used the
same procedures as previously stated except DM1 competent cells
were used in all except the final transformation.
[0145] The humanized light chain variable region in pCR-Script.TM.
and the plasmid pEE12 mhACT1Lchi (Example 1) were digested with
Hind III and Kas I. The 360 bp light chain variable region fragment
and the 315 bp light chain constant region were gel purified and
triple ligated into the Hind III restriction site of pEE12 to yield
pEE12hACT1L. Sequencing was performed to confirm correct
orientation and nucleic acid sequence.
[0146] An expression vector containing both the humanized heavy and
light chain genes was constructed using the same method as
described for the chimeric antibody (see Example 1, Expression of a
Chimeric Immunoglobulin) with the following exception. Due to an
additional Bgl II restriction site in the humanized variable heavy
chain region, a partial digest was used when cutting with Bgl II to
obtain the correct fragment. The vector containing both humanized
heavy and light chain genes is designated pEE12hACT1LH.
[0147] Transient expression of all humanized antibody constructs
and cell staining was performed using the same protocols as those
used for the chimeric antibody (see Example 1, Expression of a
Chimeric Immunoglobulin). FIG. 14 shows the results of HuT 78
staining using the mouse-human chimeric Act-1 antibody or humanized
Act-1 antibody compared to an irrelevant isotype-matched control
antibody (IgG1, kappa).
[0148] Stable transfectants of NSO cells, a myeloma cell line
(Methods in Enzymol. 73 (B):3-46 (1981); European Collection of
Animal Cell Cultures, PHLS CAMR, Porton Down, Salisbury, Wiltshire
SP4 OJG, U.K., ECACC No. 85110503) were obtained by electroporation
of NSO cells with pEE12hACT1LH.
Stable Expression in NSO Cells
[0149] 40 .mu.g of pEE 12hACT1LH for stable transfection was
linearized by digestion with SalI restriction enzyme, which cuts
within the bacterial plasmid portion of the construct. The
linearized DNA was precipitated from solution using two volumes
ethanol plus 1/10 volume sodium acetate, washed in 70% ethanol,
dried and resuspended in sterile water.
[0150] Exponentially growing NSO cells were maintained in
Non-Selective Medium (Dulbecco's Modified Eagles' Medium (high
glucose), with 2 mM L-glutamine, without sodium pyruvate, with 4500
mg/L glucose, and with 25 mM HEPES buffer (GIBCO/BRL, Catalog No.
12430-021), plus 10% Fetal Bovine Serum (Gibco/BRL, Catalog No.
16000-044)). NSO cells were centrifuged, washed and resuspended in
cold PBS, such that after the addition of the DNA the cells would
be at a concentration of 10.sup.7 cells/ml. The linearized plasmid
DNA (40 .mu.g) was added to 10.sup.7 cells in an electroporation
cuvette on ice. The cells and DNA were mixed gently so as to avoid
generating bubbles and the mixture was left on ice for 10 minutes.
The outside of the cuvette was wiped dry and two consecutive pulses
at 1500V, 3 .mu.F were delivered using a Gene Pulser (Bio-Rad). The
cuvette was returned to ice for 10 minutes.
[0151] Transfected cells were transferred to 96 well plates at
densities of 3.times.10.sup.5, 7.5.times.10.sup.4 and
1.5.times.10.sup.4 cells/ml in 50 .mu.l of non-selective medium and
incubated at 37.degree. C. for 24 hours. Subsequently 150 .mu.l of
Selective Medium (Glutamine Free Dulbecco's Modified Eagle's
Medium, with 4500 mg/L glucose, with 4 mg/L pyridoxine HCl, with
110 mg/L sodium pyruvate, without ferric nitrate, without
L-glutamine (JRH BioSciences, Catalog No. 51435-78P), plus
1.times.GS Supplement (50.times.GS Supplement obtained from JRH
Bioscience, Catalog No. 58672-77P), plus 10% Dialyzed Fetal Bovine
Serum (Gibco/BRL, Catalog No. 26300-061)) was added to all wells.
The plates were returned to the incubator until substantial cell
death had occurred and discrete surviving colonies had appeared.
Once colonies of glutamine-independent transfectants could be seen,
wells with single colonies were selected and spent tissue culture
supernatants were collected and assayed for human IgG secretion by
ELISA as described below. An antibody-producing clone designated
3A9, which was used in subsequent studies, was obtained in this
manner. A second transfection was performed as described above,
except that selection was conducted in the presence of L-methionine
sulphoximine (MSX, a glutamine synthetase inhibitor).
[0152] Positive colonies were screened by ELISA for human IgG
secretion as follows. ELISA plates (NUNC Maxisorp) were coated
overnight at 4.degree. C. with 100 .mu.l of AffiniPure F(ab').sub.2
fragment donkey anti-human IgG (H+ L) (Jackson ImmunoResearch
Laboratories) at 2.5 .mu.g/ml in carbonate buffer pH 9.5. Plates
were washed four times with PBS Tween 20 and blocked for 2 hrs at
37.degree. C. with 200 .mu.l PBS, 1% BSA. Plates were washed and
incubated 15 min at 37.degree. C. with 100 .mu.l stable transfected
NSO supernatant. Human IgG1 kappa at 1 mg/ml in PBS 1% BSA was used
as a standard. Fresh NSO media (DME+GS supplement) was used as a
negative control. Plates were washed and incubated 15 min at
37.degree. C. with 100 .mu.l peroxidase-conjugated AffiniPure
F(ab').sub.2 fragment donkey anti-human IgG (H+ L) (Jackson
ImmunoResearch Laboratories) at 0.05 .mu.g/ml in PBS (no
Ca.sup.2+/Mg.sup.2+). One 5 mg O-phenylenediamine dihydrochloride
(OPD) tablet (Sigma) was dissolved in 12 ml citrate buffer (0.1 M,
pH 5.0), and 12 .mu.l 30% hydrogen peroxide was added after the
tablet was dissolved. After washing to remove the secondary
antibody, 100 .mu.l of dissolved OPD substrate was added. The
reaction was stopped with 12.5% sulfuric acid and plates were read
on a Dynatech Plate Reader at 490 nm. Positive wells were cloned by
limiting dilution at 2, 1, and 0.5 cells per well. When all wells
from a single cloning tested positive for antibody production by
ELISA, the line was considered cloned.
[0153] Purification of humanized ACT-1 antibody from cell culture
supernatants of transient or stable cell transfectant cultures were
carried out by Protein A affinity chromatography (Poros A/M 4.6/100
mm, 5 mL/min using a Bio-Cad workstation (Perseptive Biosystems,
Inc.). The column was equilibrated with PBS followed by the
application of the cell culture supernatant which had previously
been filtered through 0.2 micron filters. The volume of cell
culture supernatant applied per run varied according to the
concentration of antibody. Normally no more than 15 mg of antibody
were applied to the column in one given run. Flow rate was 5 ml/min
throughout the purification procedure. After binding, the column
was washed first extensively with PBS until OD.sub.280 nm=0. The
column was then further washed with a minimum of 50 column volumes.
The column was then subsequently washed with 0.1 M sodium acetate,
pH 5.0. Elution was accomplished by washing with 0.1 M NaCltrate,
pH 3.5. The eluate was collected in 5 ml fractions and the pH
neutralized by addition of 200 .mu.ls of 1.5 M Na.sub.2CO.sub.3 pH
12. Antibody containing fractions were then pooled and concentrated
to the desired concentration by ultrafiltration (centricon, 30,000
KDa cut off, Amicon).
Construction of an Fc-Mutated Variant
[0154] A non-Fc binding (Fc-mutated) version of the humanized Act-1
antibody was also constructed. This antibody has the same variable
regions as the humanized Act-1 antibody (FIG. 11 and FIG. 12), and
an identical human IgG1 constant region, with the exception of two
amino acid substitutions in the IgG1 heavy chain constant region
designed to abrogate FcR recognition and eliminate Fc binding
(i.e., a Leu.sup.234.fwdarw.Ala.sup.234 substitution and a
Gly.sup.237.fwdarw.Ala.sup.237 substitution). The nucleic acid
encoding the heavy chain of the Fc-mutated derivative was
constructed as follows. A construct designated 3678 (obtained from
Dr. Herman Waldmann, University of Oxford), which encodes the light
chain and heavy chain of a humanized anti-CD18 antibody (WO
93/02191 (published Feb. 4, 1993); Sims, M. J., et al., J. Immunol.
151(4): 2296-2308 (1993)) in a pEE12 expression vector, but in
which two amino acid substitutions were introduced into the IgG1
heavy chain constant region by site-directed mutagenesis
(Leu.sup.234.fwdarw.Ala.sup.234 and
Gly.sup.237.fwdarw.Ala.sup.237), was digested with Age I and EcoRI
to release a 900 bp fragment containing the gamma constant region
mutant. This fragment was then used to replace the heavy chain wild
type gamma one constant region at the Agel/EcoRI sites in
pEE6hACT1H giving rise to pEE6hACT1H/FCmut. In a manner analagous
to that described above for other constructs comprising both
chains, a single construct (pEE12hACT1LH/FCmut) which contains the
reshaped light chain gene and the Fc-mutated reshaped heavy chain
gene was prepared.
Example 4
Characterization of LDP-02, a Humanized ACT-1 Antibody
[0155] Initial characterization studies were performed using
antibody produced from COS-7 cells transiently transfected with
pEE12hACT1LH/FCmut. This antibody preparation was produced and
purified as described above, and is referred to below as "1.degree.
HUM ACT-1" followed by the appropriate lot number.
[0156] Additional assays were performed using antibody produced
from a stable transfectant of murine cell line NSO as described
above (transfected with linearized pEE12hACT1LH/FCmut). This
antibody preparation is referred to below as "LDP-02/3A9/Lot
1".
[0157] "LDP-02/3A9/Lot #1" antibody was used in the following
studies described below: SDS-PAGE, Western Blot Analysis,
Isoelectric Focusing, Amino Acid Composition Analysis, Species
Cross-reactivity, Titration, Complement Mediated Lysis Assays, ADCC
Assays, and Binding Inhibition Assays. "1.degree. HUM ACT-1 Lot #7
was used in Affinity Assays #1-2, 1.degree. HUM ACT-1 Lot #8/9 was
used in Affinity Assays #3-5, and 1.degree. HUM ACT-1 Lot #8/9 was
used in C1q Binding Assays.
A. Physico-Chemical Properties
[0158] 1. SDS-PAGE
[0159] In order to assist in establishing identity, characterize
the first preparation, and assess purity LDP-02/3A9/Lot#1 was
subjected to sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) under non-reducing and reducing
conditions and stained with Colloidal Coomassie Blue.
[0160] 80 .mu.l of LDP-02/3A9/Lot#1 at a nominal concentration of
0.82 mg/ml was added to a microconcentrator. The citrate buffer, in
which the antibody was dissolved, was exchanged three times with
160 .mu.l of Tris buffer (0.5 mM, pH 8.8). The final volume of the
sample after buffer exchange was 135 .mu.l, yielding a protein
concentration of 0.486 mg/ml. This solution was diluted two-fold
with both non-reducing and reducing buffers to obtain a
concentration of 0.243 mg/ml. A 13 .mu.l aliquot of the 0.243 mg/ml
solution, containing 3.16 .mu.g of protein, was loaded onto the
designated sample lanes of the SDS gel. SDS-PAGE was performed, and
control articles included Mark 12 Molecular Weight Standards
(Novex, #LC5677).
[0161] Under non-reducing conditions, a major band with an apparent
molecular weight of slightly lower than 200,000 Daltons was present
in LDP-02/3A9/Lot#1. Several minor components were observed between
116,300 and 200,000 Daltons. Three additional minor components with
approximate molecular weights of 97,400 Daltons, slightly greater
than 55,400 Daltons, and less than 31,000 Daltons were also
observed. Scanning the gel using a laser densitometry allowed for
the quantitative analysis of the stained polypeptide bands and then
calculation of percent area associated with each visible band
(Table 5). The obtained data from the quantitative analysis
indicates that the major component observed at approximately
200,000 Daltons represented 84.4% of the total stained bands in the
test sample lane. This major band represented the intact antibody,
while the other bands at 55,000 and 31,000 Daltons represented
single heavy and light chains respectively.
[0162] Under reducing conditions, two major components were
observed on the electrophoresis gel. The molecular weight of one of
the components was approximately 55,400 Daltons and represented
68.6% of the total stained bands visualized in the gel lane, while
the second component corresponding to slightly less than 31,000
Daltons, represented 30.5% of the total stained bands (Table 5).
The molecular weights of these two components agree well with the
expected molecular weights of the heavy and light chains of an
immunoglobulin G. These data indicate that approximately 99% of the
preparation consisted of either intact antibody or single heavy or
light chain immunoglobulin chains. Besides the two major
components, one minor component at slightly less than 66,300
Daltons was also observed.
[0163] From this analyses, a high molecular weight species
consistent with that for intact immunoglobulin G is present as the
major band in LDP-02/3A9/Lot#1. Several minor bands are also
present in LDP-02/3A9/Lot# 1. Following reduction, two major bands
were observed which show electrophoretic migrations consistent with
those for the heavy and light chains of an immunoglobulin G
molecule.
TABLE-US-00010 TABLE 5 PURITY DATA SUMMARY COLLOIDAL COOMASSIE
BLUE, NON-REDUCING CONDITIONS Sample Lane Area Percent (Main
Component) LDP-02/3A9/Lot #1 5 84.4% PURITY DATA SUMMARY COLLOIDAL
COOMASSIE BLUE, REDUCING CONDITIONS Low M.W. Area Low M.W. Sample
Lane Percent Area Percent LDP-02/3A9/Lot #1 9 68.6% 30.5%
[0164] 2. Western Blot Analysis
[0165] Samples and standards were analyzed by SDS-PAGE as described
above. Briefly, nonreduced and reduced samples were analyzed on a
4-20% Tris-Glycine gel. Novex Mark 12 Molecular Weight Standards
were also run on the gel. Volumes of 2.1 ml and 4.5 ml aliquots of
the 0.2143 mg/ml solution, yielding 0.51 and 1.09 .mu.g of protein,
respectively, were loaded onto the designated sample lanes of the
SDS gel.
[0166] Following SDS-PAGE, sample protein were transferred from the
gel to nitrocellulose as per Novex Western Transfer Apparatus
instructions. The transfer buffer used was 1.times.Tris-Glycine
buffer in 20% Methanol. After approximately 2 hours, the
nitrocellulose blot was removed from the transfer apparatus and
rinsed with DDI water. The nitrocellulose blot was then blocked at
37.degree. C. for 35 minutes in Tris buffer (20 mM), containing 3%
gelatin and 0.1% Tween 20. The blot was removed from the blocking
solution and washed twice with Tris buffer. Goat anti-mouse IgG
solution, which was prepared by diluting anti-mouse IgG antibody
stock solution by 1000-fold with 20 mM Tris-3% BSA solution, was
added to the blot and incubated at 2-8.degree. C. overnight.
Following incubation, the blot was washed with four changes of Tris
buffer for 5 minutes each. Anti-goat IgG alkaline-phosphatase
conjugate solution, prepared by diluting anti-goat IgG
alkaline-phosphatase conjugate 5000-fold with 20 mM Tris-3% BSA
solution, was added to the blot and incubated at room temperature
for 2 hours. Following incubation, the blot was washed with four
changes of Tris buffer for 5 minutes each. BCIP/NBT
(5-Bromo-4-Chloro-3'-Indolyl Phosphatep-Toluidine salt/Nitro-Blue
Tetrazolium Chloride) substrate was added 10 ml at a time to the
blot. Blot was developed at room temperature with agitation.
Reaction was stopped by rinsing blot with Tris buffer. The above
procedure was then repeated using goat anti-human IgG instead of
goat anti-mouse IgG.
[0167] Under both non-reducing and reducing conditions using the
anti-mouse IgG reagent, the 0.51 .mu.g and the 1.09 .mu.g IgG
samples were clearly detected on the nitrocellulose blot. The
intensity of the bands increased with increasing concentration.
Under non-reducing conditions a major band, migrating slightly
faster than 200,000 Daltons marker, was detected. Several fainter
bands were also detected. Two of these bands migrated slower than
the major band and approximately three other bands migrated faster.
Under reducing conditions, two bands, characteristic of the heavy
and light chains of immunoglobulin G, were detected.
[0168] Using the anti-human IgG reagent under both non-reducing and
reducing conditions, the 0.51 .mu.g and the 1.09 .mu.g IgG samples
were clearly detected on the nitrocellulose blot. The intensity of
the bands increased with increasing concentration. Under
non-reducing conditions a major band, corresponding to a species
with an apparent molecular weight marker slightly lower than
200,000 Daltons, was detected. The fainter bands observed in the
blot, detected with antimouse IgG, were also detected. The
intensity of the immunostaining was greater for all bands when
detected with anti-human IgG. Several additional bands, not
observed in the other blot, were detected. It is likely that these
bands correspond to IgG fragments lacking epitopes which are
recognized by the anti-mouse IgG. Under reducing conditions a band
characteristic of the heavy chain of an immunoglobulin G was
detected. Because the antibody was specific for the Fc portion of
human IgG, the light chain was not detected. Several minor bands,
not seen in the blot developed with anti-mouse IgG, were observed
when detection was performed with the anti-human IgG. This
difference between the two blots may be the result of the presence
of IgG fragments which lack epitopes for anti-mouse IgG
binding.
[0169] 3. Isoelectric Focusing
[0170] LDP-02/3A9/Lot#1 was subjected to Isoelectric Focusing (IEF)
and stained with Colloidal Coomassie blue. The results obtained for
LDP-02/3A9/Lot#1 were compared to IEF standards which were focused
on the same gel.
[0171] 80 .mu.l of LDP-02/3A9/Lot#1 at a nominal concentration of
0.82 mg/ml was added to a microconcentrator. The citrate buffer
that the antibody was in, was exchanged three times with 160 .mu.l
of Tris buffer (0.5 mM, pH 8.8). The final volume of the sample was
135 .mu.l. The final concentration was calculated to be 0.486
mg/ml. This solution was diluted two-fold with 2.times.IEF sample
buffer to obtain a concentration of 0.243 mg/ml. A 13 .mu.l aliquot
to the 0.243 mg/ml solution, yielding 3.16 .mu.g of protein, was
loaded onto the designated sample of the IEF gel. Control articles
included IEF Standards pI 3.6-9.3 (Sigma, Cat #I-3018).
[0172] A standard plot was generated by graphing the average of
relative distance migration of eight IEF Standards versus the known
pI for each of these standard proteins. The linear regression fit
of these data yielded a negative slope of 0.03459 and an intercept
of 8.91857. The R.sup.2 of the fit equaled 0.99206.
[0173] Table 6 contains the average distances migrated by the six
IEF standards and by LDP-02/3A9/Lot#1. The calculated pIs for
LDP-02/3A9/Lot#1 are also shown in this table.
[0174] Using the linear regression parameters from the standard
plot, the approximate pIs of the five bands for LDP-02/3A9/Lot#1
were calculated to be 7.88, 7.95, 8.09, 8.26, and 8.43, with the
predominant peak represented by a pI of 8.09 (Table 6). The pI of
this major peak compares favorably with a predicted pI of 7.91
based upon the primary amino acid sequence.
TABLE-US-00011 TABLE 6 Standard Distance Migrated* pI.sup.1 Lectin
3.3 mm 8.8 Lectin 9.5 mm 8.6 Lectin 17.88 mm 8.2 Myoglobin 59.3 mm
6.8 Carbonic Anhydrase I 74.0 mm 6.6 Carbonic Anhydrase II 92.5 mm
5.9 b-Lactoglobulin A 105.8 mm 5.1 Trypsin Inhibitor 122.0 mm 4.6
Sample Distance Migrated pI.sup.1 LDP-02/3A9/Lot #1 (Band 1) 14.0
mm 8.43 LDP-02/3A9/Lot #1 (Band 2) 19.0 mm 8.26 LDP-02/3A9/Lot #1
(Band 3) 24.0 mm 8.09 LDP-02/3A9/Lot #1 (Band 4) 28.0 mm 7.95
LDP-02/3A9/Lot #1 (Band 5) 30.0 mm 7.88 *Average .sup.1Based on
standard curve (pI vs. Migration distance) where: Sample pI =
Intercept - Slope (Sample migration distance).
[0175] 4. Amino Acid Composition Analysis
[0176] Amino acid composition analysis was performed to determine
the protein content and amino acid composition of LDP-02/3A9/Lot#1
and confirm identity.
[0177] Triplicate 45 .mu.l aliquots were first removed for
hydrolysis. Hydrolysis was performed at 165.degree. C. for 60
minutes using 6N HCl vapors. As a control, the hydrolysis vessel
contained a standard protein which was hydrolyzed simultaneously
with the LDP-02/3A9/Lot#1. Amino acid standards were also
chromatographed before and after LDP-02/3A9/Lot#1 analysis. Control
articles included Bovine Serum Albumin (Tektagen Solution
Control:310:197 A) as the standard protein and Amino Acid
Hydrolysate Mixture (Tektagen Solution Control:310:199 A) as the
amino acid standard.
[0178] The test method employed analysis of resuspended protein
hydrolysate or free amino acid solution by ion exchange HPLC with
post-column ninhydrin reaction and absorbance monitoring at two
wavelengths. Absorbance at both wavelengths was quantified by
comparison to a calibration table obtained by analyzing amino acid
standards in triplicate.
[0179] Amino acid composition is presented in Table 7. The protein
concentration of LDP-02/3A9/Lot#1 was determined to be 0.709 mg/mL.
Upon correction for lack of quantitation of W and C, the protein
concentration was revised to 0.740 mg/mL. The data and pertinent
calculations are summarized in Table 8.
[0180] For LDP-02/3A9/Lot#1, a single hydrolysis time point (60
min) was performed at 165.degree. C. using 6N HCl vapors.
Correction factors, which have been derived from the standard
protein (BSA), were applied to the determinations of protein
content (Table 8).
[0181] Under conditions of this method, the mole percent values
obtained for proline (Table 7) may be slightly elevated, due to the
presence of a coeluting cysteine peak. Consequently, the accuracy
of proline quantitation is sample dependent, based upon the amount
of cysteine present in the sample hydrolysates. For this analysis,
the proline content has been corrected using a BSA derived
correction factor (Table 8). The accuracy of this correction is
sample dependent, based on the relative amounts of cysteine in the
BSA (6.0%) and in the sample.
[0182] The predicted amino acid composition of LDP-02 as relative
percent (frequency or mole percent) based upon the nucleotide
sequence of the heavy and light chains (Predicted %), and the
actual results of the amino acid analysis (Actual %) are presented
in Table 9. Comparison of predicted versus actual values shows good
correlation except for proline, which as previously described, is
likely artifactually high due to a coeluting cysteine peak.
TABLE-US-00012 TABLE 7 Sample: LDP-02/ LDP- LDP-02/3A9/LOT #1
3A9/LOT #1 02/3A9/LOT #1 With correction With correction for
Without factors derived from W/C.sup.1 and BSA correction BSA
derived factors AA % mole % mole % mole N/D 9.1 9.0 8.6 T 6.5 7.5
7.2 S 9.2 13.3 12.7 Q/E 11.4 11.3 10.8 P 8.2 9.8 9.4 G 7.8 7.4 7.1
A 5.9 5.8 5.6 V 10.2 9.5 9.1 M 0.3 0.7 0.7 I 2.5 2.6 2.5 L 8.2 7.9
7.6 Y 5.2 5.0 4.8 F 3.4 3.4 3.3 H 2.2 2.2 2.1 K 7.0 6.9 6.6 R 2.9
2.9 2.8 TOTAL 100 .sup.1Correlation factor is 0.958, which is based
on the W and C content of 1.8% and 2.4%, respectively.
TABLE-US-00013 TABLE 8 Protein Content Determination Mean
.sup.2Correction Corrected Quantity AA nmols Factor nmoles Residue
MW found (ng) N/D 5.954 0.991 5.900 115.1 679 T 4.243 1.156 4.905
101.1 496 S 6.054 1.448 8.766 87.1 764 Q/E 7.436 0.991 7.369 128.1
944 P 5.365 0.830 4.453 97.1 432 G 5.080 0.951 4.831 57.1 276 A
3.884 0.983 3.818 71.1 271 V 6.681 0.930 6.213 99.1 616 M 0.221
2.433 0.538 131.2 71 I 1.606 1.036 1.664 113.2 188 L 5.379 0.961
5.169 113.2 585 Y 3.374 0.954 3.219 163.2 525 F 2.229 0.992 2.211
147.2 325 H 1.442 0.981 1.415 137.2 194 K 4.616 0.984 5.542 125.2
582 R 1.922 1.005 1.392 156.2 302 Total Quantity Injected on column
(ng): 7250 Reconstitution Volume (.mu.l): 220 .sup.3Total Quantity
Hydrolyzed (ng): 31900 Total Quantity Hydrolyzed (.mu.g): 31.9
Original Sample Volume (.mu.l): 45 Diluted Sample Volume (.mu.l):
45 Aliquot Value for Hydrolysis (.mu.l): 45 Protein concentration
(mg/ml): 0.709 Protein Concentration (mg/ml) after correction for
W/C: 0.740 .sup.1Protein content is not corrected for cysteine and
tryptophan. .sup.2A BSA derived correction factor has been applied
to each amino acid detected. .sup.3Total ng hydrolyzed = (Total ng
injected .times. reconstitution volume)/Injection volume (50
.mu.l).
TABLE-US-00014 TABLE 9 Amino Acid Composition Amino Acid Amino
Predicted Actual Symbol Acid Number % % A Ala 68 5.06 5.6 C Cys 32
2.38 -- D Asp 56 4.17 -- E Glu 68 5.06 -- F Phe 40 2.98 3.3 G Gly
90 6.70 7.1 H His 28 2.08 2.1 I Ile 30 2.23 2.5 K Lys 96 7.14 6.6 L
Leu 98 7.29 7.6 M Met 10 0.74 0.7 N Asn 50 3.72 -- P Pro 94 6.99
9.4 Q Gln 64 4.76 -- R Arg 36 2.68 2.8 S Ser 170 12.65 12.7 T Thr
100 7.44 7.2 V Val 126 9.38 9.1 W Trp 24 6.98 -- Y Tyr 64 4.76 4.8
N/D Asn/Asp 106 7.89 8.6 Q/E Gln/Glu 132 9.82 10.8
[0183] 5. MALDI-TOF MS Analysis
[0184] LDP-02/3A9/Lot#1 was analyzed by MALDI-TOF MS to determine
the molecular weight. A main peak with a mass centered at 149,808
Da was detected. The peak centered at 74,927 Da represented the +2
ion of the species found in the main peak. It should be noted that
the mass of +2 ion is not exactly half of the M+H ion; this slight
disparity is likely caused by experimental inaccuracy, which is
within +/-0.2% of the measured value.
[0185] Based on the primary predicted sequence of the antibody, the
expected molecular mass should be 147,154 Da. The mass difference
of 2,654 Da between the observed and the predicted IgG molecular
masses, most probably, can be attributed to glycosylation of the
molecule. This observed difference would represent a glycosylation
level of approximately 1.8%.
B. Affinity
[0186] First, titration of LDP-02/3A9/Lot#1 and murine ACT-1
(Lot#2) was performed using flow cytometry on human derived HUT-78
cells. Briefly, 1.0.times.10.sup.6 HUT-78 cells were suspended in a
volume of 100 .mu.of either biotinylated murine ACT-1 (Lot#2),
biotinylated murine IgG1 (Lot#1 made at LeukoSite, Inc.),
biotinylated LDP-02/3A9/Lot#1, or biotinylated human IgG (Jackson
ImmunoResearch, Avondale, Pa.; Lot 25794) for 20 minutes at
4.degree. C., after which the antibodies were removed. Unless
otherwise indicated, all reagents were diluted in 0.15 M PBS/1.0%
FCS/0.1% sodium azide. The varying concentrations for both
antibodies included 30 .mu.g/ml (murine ACT-1 only), 15 .mu.g/ml,
7.5 .mu.g/ml, and subsequent 1:10 dilutions of each. After removal
of the primary antibodies, the cells were then suspended in 100
.mu.l streptavidin phycoerthrin (Dako Corp., Carpinteria, Calif.)
diluted 1:200. After washing in 200 .mu.l PBS, cells were
resuspended in 0.5 ml of PBS/1% formalin and refrigerated until
analyzed. Samples were analyzed on a FACScan (Becton Dickinson
Corp., San Jose, Calif.) using a 488 nm laser to excite
phycoerythrin. For each sample, a minimum of 10,000 cells was
analyzed and half-maximal mean channel fluorescence (MCF) was
calculated. All samples were performed in duplicate.
[0187] These titration studies indicated that at concentrations of
approximately 1.0 mg/ml, maximal fluorescence was approached using
both murine ACT-1 and LDP-02/3A9/Lot#1 (FIG. 15). Half-maximal mean
channel fluorescence was achieved at lower concentrations of LDP-02
than for murine ACT-1 (0.1 .mu.g/ml for biotinylated murine ACT-1
Lot#2, and 0.02 .mu.g/ml for LDP-02/3A9/Lot#, respectively).
[0188] Relative assessments of affinity (and specificity) were
performed using flow cytometry and cross-competitive binding of
LDP-02 and the murine Act-1 antibody, and vice versa on
human-derived HuT-78 cells. Briefly, 1.0.times.10.sup.6 HuT-78
cells were suspended in either 100 .mu.l of biotinylated murine
Act-1 (Lot#2) at 0.1 .mu.g/ml with varying concentrations of
unconjugated 1.degree. HUM ACT-1 or unconjugated murine Act-1 for
20 minutes at 4.degree. C., after which the antibodies were
removed. In a separate experiment, 100 .mu.l of biotinylated
LDP-02/3A9/Lot#1 at 0.02 .mu.g/ml was used with varying
concentrations of unconjugated murine ACT-1 (Lot#2) and
unconjugated LDP-02/3A9/Lot# 1. The concentration of biotinylated
antibodies held constant were the concentrations resulting in
half-maximal mean channel fluorescence (MCF) on HUT-78 cells
stained under identical conditions, as demonstrated above. Unless
otherwise indicated, all reagents were diluted in 0.15 M PBS/1.0%
FCS/0.1% sodium azide. The varying concentrations for both
antibodies ranged in half-log increments from 2.0.times.10.sup.-6M
to 5.0.times.10.sup.-11M. After removal of the primary antibodies,
the cells were then suspended in 100 .mu.l streptavidin
phycoerythrin (Dako Corp., Carpinteria, Calif.) diluted 1:200.
After washing in 200 .mu.l PBS, cells were resuspended in 0.5 ml of
PBS/1% formalin and refrigerated until analyzed. Samples were
analyzed on a FACScan (Becton Dickinson Corp., San Jose, Calif.)
using a 488 nm laser to excite phycoerythrin. For each sample, a
minimum of 10,000 cells was analyzed and MCF calculated. All
samples were performed in duplicate. The IC.sub.50 was determined
as the concentration of unconjugated antibody producing a 50%
reduction in the MCF from the biotinylated homologue antibody.
[0189] Estimates of affinity were performed in five independent
cross-competitive experiments between LDP-02 (1.degree. HUM ACT-1)
and murine ACT-1. When biotinylated murine Act-1 was used as the
antibody held constant in the assay, mean IC.sub.50 values (.+-.1
SEM) for LDP-02 (5.43.+-.0.86 nM) were statistically lower than
that for murine ACT-1 (7.94.+-.1.17 nM; p=0.02, two-tail t-test:
paired two sample for means), while irrelevant human IgG1 or murine
IgG1 had no competitive effect (all experiments summarized in Table
10; one experiment shown in FIG. 16). Similarly, when biotinylated
LDP-02/3A9/Lot#1 was the antibody held constant in the assay, a
greater concentration of unconjugated murine Act-1 than of
LDP-02/3A9/Lot#1 was required to compete LDP-02 off HuT-78 cell
membranes (IC.sub.50=6.3 nM vs. 4.3 nM, respectively). In each
experiment, LDP-02 had a lower IC.sub.50 than did murine Act-1.
These results demonstrate that LDP-02 was specific for the epitope
recognized by murine Act-1, and that its binding affinity was
better than that of the murine antibody.
TABLE-US-00015 TABLE 10 Murine ACT-1 and Humanized ACT-1 (LDP-02)
Affinity Assessment Experiment # Antibody Lot # IC.sub.50 (nM) 1
ACT-1 (murine) 2 7.57 2 2 10.95 3 2 6.02 4 2 4.91 5 2 10.24 MEAN
.+-. SEM 7.94 .+-. 1.17 1 LDP-02 (humanized) 7 4.34 2 7 6.13 3 8/9
4.71 4 8/9 3.53 5 8/9 8.44 MEAN .+-. SEM 5.43 .+-. 0.86 p = 0.02
Two-tail t-Test: Paired Two Sample for Means
C. Species Cross-Reactivity
[0190] Flow cytometry was used to evaluate species
cross-reactivity. 100 .mu.l of EDTA-anticoagulated blood drawn from
either a human, dog, cat, guinea pig, or rat was added to FACS
tubes. Plasma was removed and blood pellets were then resuspended
in 100 .mu.l of either biotinylated LDP-02/3A9/LOT#1, irrelevant
biotinylated human IgG (Jackson ImmunoResearch, Avondale, Pa.),
biotinylated murine Act-1 Lot#2, or irrelevant biotinylated Murine
IgG1 (Dako Corp., Carpinteria, Calif.) at a concentration of 15
.mu.g/ml. Unless otherwise indicated, all reagents were diluted in
0.15 M PBS/1.0% FCS/0.1% sodium azide. Samples were incubated with
antibodies for 20 minutes at 4.degree. C. after which the
antibodies were removed by washing. Cells were then incubated with
100 .mu.l of strepavidin phycoerythrin diluted 1:200 (Southern
Biotechnology Associates, Inc., Birmingham, Ala.) for 20 minutes at
4.degree. C. Red blood cells were then lysed using a commercial
lysing reagent (FACS Lysing Solution, Becton Dickinson, San Jose,
Calif.) according to manufacturer's protocol. After washing in PBS,
cells were resuspended in 0.5 ml of PBS/1% formalin and
refrigerated until analyzed. Samples were analyzed on a FACScan
(Becton Dickinson Corp., San Jose, Calif.) using a 488 nm laser to
excite phycoerythrin. Lymphocyte acquisition gate was set on
forward and 90 degree light scatter parameters. For each sample,
10,000 cells were analyzed.
[0191] Biotinylated LDP-02/3A9/Lot#1 recognized a subpopulation of
human lymphocytes with a heterogenous staining pattern, similar to
that produced with murine Act-1, and distinct from the pattern
produced by staining with human or murine isotype-matched controls.
In addition, when examined on lymphocytes from dog or cat, both
LDP-02/3A9/Lot#1 and murine Act-1 produced a similar heterogenous
staining pattern as that derived using human lymphocytes.
LDP-02/3A9/Lot#1 or murine ACT-1 did not recognize lymphocytes from
rat or guinea pig under these conditions.
D. C1q Binding
[0192] Flow cytometry was used to assess the potential of LPD-02 to
bind human complement component C1q, using a technique previously
described (Sims, M. J. et al., J. Immunol. 151: 2296-2308 (1993)).
Human peripheral blood mononuclear cells (PBMCs) were isolated by
standard Ficoll density separation. 375,000 cells were first
blocked with 10% normal rabbit serum/PBS for 10 minutes at
4.degree. C. After removal by washing, cells were incubated with
100 .mu.l of either (a) CAMPATH-1H (Therapeutic Antibody Center,
Cambridge, U.K.), (b) human IgG1 (Sigma Chemical Co., St. Louis,
Mo.), (c) LDP-01 (a derivative of the anti-CD18 antibody described
in WO 93/02191 (published Feb. 4, 1993) and Sims, M. J., et al., J.
Immunol. 151(4): 2296-2308 (1993), which contains two amino acid
substitutions in the IgG1 heavy chain constant region
(Leu.sup.234.fwdarw.Ala.sup.234 and
Gly.sup.237.fwdarw.Ala.sup.237), also referred to as "FcRmut CD18",
Therapeutic Antibody Center, Cambridge, U.K.), or (d) LDP-02
(1.degree. C. hum ACT-1 Lot#8/9) at 10 .mu.g/ml for 20 minutes at
4.degree. C. CAMPATH-1H served as a positive control antibody,
while LDP-01 and human IgG1 were used as negative control
antibodies. All reagents were diluted in 2% BSA/PBS. As an
additional negative control, 2% BSA/PBS was also added alone.
Antibody was then removed by washing, and cells were resuspended in
50 .mu.l human complement component C1q (Sigma Chemical Co., St.
Louis, Mo.) at 10 .mu.g/ml for 30 minutes at 4.degree. C. Cells
were then washed and resuspended in 100 .mu.l FITC-conjugated
rabbit anti-human C1q (Dako Corp., Carpinteria, Calif.) antibody at
20 .mu.g/ml for 20 minutes at 4.degree. C. After washing in 200
.mu.l PBS, cells were resuspended in 0.5 ml of PBS/1% formalin and
refrigerated until analyzed. Samples were analyzed on a FACScan
(Becton Dickinson Corp., San Jose, Calif.) using a 488 nm laser to
excite FITC. For each sample, a minimum of 10,000 cells were
analyzed and mean channel fluorescence (MCF) calculated.
[0193] Human PBMCs incubated with CAMPATH-1H bound human C1q,
resulting in a significant shift in MCF, while the staining
patterns elicited by incubation of PBMCs with LDP-01, BSA, or human
IgG1 were all similar and characterized by relatively low
background staining. The pattern of staining produced by PMBC
preincubation with LDP-02 was identical to that produced in these
negative control samples, demonstrating that LDP-02 does not bind
C1q under these conditions.
E. Complement-Mediated Lysis
[0194] The ability of LDP-02/3A9/Lot#1 to participate in complement
mediated cell lysis was examined using a protocol previously
described in Bindon, C. I., et al. (Transplantation, 40: 538-544
(1985)). Heparinized human blood was drawn aseptically, and plasma
was collected and immediately placed on ice. Peripheral blood
mononuclear cells (PBMCs) were isolated by centrifugation for 15
minutes over a layer of Ficoll-Hypaque, density 1.077 g/ml, and
were washed twice in complete medium consisting of RPMI 1640/10%
FCS/100 U/ml penicillin/100 .mu.g/ml streptomycin/2.0 mM
L-glutamine. 25 million cells were then incubated at 37.degree. C.
for 1 hr in 150 .mu.Ci sodium .sup.51chromate in sterile saline
(E.I. du Pont de Nemours & Co. Inc., Wilmington, Del.). Cells
were washed twice in medium and resuspended at 10.sup.6/ml. 50
.mu.l of the suspension (5.0.times.10.sup.4 cells) were then added
to wells of a U-bottom microtiter plate containing 100 .mu.l of
either (a) CAMPATH-1H (Therapeutic Center, Cambridge, U.K.), (b)
CAMPATH-1G (Therapeutic Center, Cambridge, U.K.), (c) human IgG1
(Sigma Chemical Co., St. Louis, Mo.), (d) LDP-02/3A9/Lot#1, or (e)
LDP-01 (FcRmut CD18, Therapeutic Antibody Center, Cambridge, U.K.
(see above)) at concentrations of 50, 25, 5, 2.5, and 0.5 .mu.g/ml
in medium. CAMPATH-1 antibodies were used as positive control
antibodies in the assay, while human IgG1 and LDP-01 were used as
negative controls. Additional wells contained cells suspended in
100 .mu.l of 0.1% Triton-X-100 (Fisher Scientific, Fair Lawn, N.J.)
in complete medium. Cells incubated with Triton-X-100 were used to
measure total release, while control wells with no antibody were
used to measure spontaneous release. After incubation for 15
minutes at room temperature, 50 .mu.l of autologous plasma as a
complement source was added to each well to a final concentration
of 20%. The cells were incubated for 45 minutes at 37.degree. C.,
then centrifuged at 100 g for 2 minutes, and 100 ul of the
supernatants were collected. Released .sup.51Cr was measured on a
Cobra II gamma counter (Packard Instruments, Downers Grove, Ill.).
All samples were performed in duplicate. The percentage of specific
.sup.51Cr release was calculated using the formula:
specific release = ( test - spontaneous ) .times. 100 % total -
spontaneous ##EQU00001##
[0195] As previously reported by Bindon et al. (Transplantation,
40: 538-544 (1985)), both CAMPATH-1H and CAMPATH-1G induced up to
35% complement-mediated lysis of human PBMCs in a dose-dependent
manner. In addition, as expected, human IgG1 and LDP-01 (Fc-mut CD
18) controls did not induce any detectable cell lysis. LDP-02 did
not mediate cell lysis at any of the concentrations examined, up to
and including 25 .mu.g/ml (FIG. 17).
F. Antibody Dependent Cell-mediated Cytotoxicity (ADCC)
[0196] Human CD3+ blasts were used as target cells to assess the
ability of LDP-02 to participate in antibody dependent
cell-mediated cytotoxicity (ADCC). CD3+ blasts were generated in
24-well plates coated with the anti-CD3 antibody RT66 at a
concentration of 5 .mu.g/ml diluted in PBS. Human peripheral blood
mononuclear cells (PBMCS) were isolated by centrifugation for 15
minutes over a layer of Ficoll-Hypaque, density 1.077 g/ml, washed
and resuspended in complete medium, as described in the previous
section. 2 million cells were then added to each well of the
24-well plate and incubated at 37.degree. C., 5% CO.sub.2 for 4
days. Cells were then transferred to a culture flask and incubated
at 37.degree. C., 5% CO.sub.2 in medium with human recombinant IL-2
(Genzyme Corp., Cambridge, Mass.) at a concentration of 10
units/ml. After three days in culture, 10.0.times.10.sup.6 CD3
blasts were then incubated at 37.degree. C. for 45 minutes in 150
.mu.Ci sodium .sup.51chromate in sterile saline (E.I. du Pont de
Nemours & Co. Inc., Wilmington, Del.; Lot#95M682). After two
washes in complete medium, cells were resuspended to
2.times.10.sup.5 cells/ml, and 50 .mu.l (10,000 cells) of the
suspension was added to wells of a U-bottom 96 well microtiter
plate. The wells contained 50 .mu.l of either CAMPATH-1H
(Therapeutic Antibody Center, Cambridge, U.K.) or LDP-02/3A9/Lot#1
at final concentrations of 50, 5, 2.5, 0.5, 0.25, or 0.05 .mu.g/ml
in medium. Cells were incubated with antibodies for 30 minutes at
room temperature after which 0.5.times.10.sup.6 freshly isolated
PBMC's (ficoll-hypaque gradient, 2 washes in complete medium at
37.degree. C.) from a different donor were added to each well as
effector cells (effector:target ratio of 50:1). To additional
wells, 100 .mu.l of 5% Triton-X-100 in medium (Fisher Scientific,
Fair Lawn, N.J.) was added. Cells incubated with Triton-X-100 were
used to measure total release, while controls with no antibody and
effector cells were included to measure spontaneous radioactivity
release. Cells were centrifuged at 100 g for 2 minutes at room
temperature and incubated for 20 hours at 37.degree. C., 5%
CO.sub.2 after which cells were transferred to a V-bottom 96-well
plate and pelleted at room temperature. 100 .mu.l of supernatants
were collected, and released radioactivity was measured on a Cobra
II gamma counter (Packard Instruments, Downers Grove, Ill.). All
samples were performed in duplicate. The percentage of specific
.sup.51Cr release was calculated using the formula:
specific release = ( test - spontaneous ) .times. 100 % total -
spontaneous ##EQU00002##
[0197] As previously demonstrated by Sims, M. J. et al., J.
Immunol., 151(4): 2296-2308 (1993), CAMPATH-1H participated in ADCC
in a dose-dependent manner, eliciting up to approximately 30%
specific .sup.51Cr release at concentrations .gtoreq.5.0 .mu.g/ml.
No specific release was detected in wells containing LDP-02 at any
of the concentrations examined.
G. Inhibition of Adhesion to MAdCAM-1
[0198] The ability of LDP-02 to inhibit binding of .alpha.4.beta.7
to MAdCAM-1 was assessed using fluorescently labeled
.alpha.4.beta.7+RPMI 8866 cells (a human B cell lymphoma) and a
MAdCAM-1 chimera comprising the entire extracellular domain of
human MAdCAM-1 fused to the Fc region of a human IgG1 (a constant
region derived from the same construct used to make the constant
region of Fc-mutated LDP-02).
[0199] 1. Construction of MAdCAM-IgG Chimera
[0200] A Human MAdCAM-1 clone designated pcDhuMAd4 (clone 4 cDNA in
pcDNA3; Shyjan, A. M. et al., J. Immunol., 156: 2851-2857 (1996);
the teachings of which are incorporated herein by reference in
their entirety) was used as a template for PCR amplification of
extracellular regions of human MAdCAM-1 to be fused with the
constant region of human IgG1, as described in International
Appplication No. PCT/US96/02153 (designating the U.S.), filed Feb.
12, 1996, which is a continuation-in-part of U.S. Ser. No.
08/523,004, filed Sep. 1, 1995, which is a continuation-in-part of
U.S. Ser. No. 08/386,857, filed Feb. 10, 1995, the teachings of
which are each incorporated herein by reference in their entirety.
To construct the MAdCAM-IgG chimera, primer HUMADIG4/2 (SEQ ID
NO:62), which contains the 5' end of human MAdCAM-1 coding sequence
(ATG codon, bold), was synthesized:
TABLE-US-00016 HindIII 5'-GGAAGCTTCCACCATGGATTTCGGACTGGCCC-3'
This 5' primer was used in conjunction with a 3' primer designated
HUMADIG3 to amplify a region encoding the entire extracellular
domain of human MAdCAM-1. The 3' primer HUMADIG3 (SEQ ID NO:63) has
the following sequence:
TABLE-US-00017 SpeI 5'-GGACTAGTGGTTTGGACGAGCCTGTTG-3'
[0201] The primers were designed with a 5' HindIII site or 3' SpeI
sites as indicated. These primers were used to PCR amplify a MAdCAM
fragment using a PCR optimizer kit from Invitrogen (San Diego,
Calif.). The PCR products were digested with the enzymes HindIII
and SpeI to generate ends for cloning, and were purified by gel
electrophoresis using the Glassmax DNA isolation system (Gibco,
Bethesda, Md.).
[0202] A 1 kb fragment encompassing the CH1, H (hinge), CH2 and CH3
regions was excised by digestion with SpeI and EcoRI from a
construct encoding a human immunoglobulin .gamma.1 heavy chain
having an Fc-mutated human constant region. The human constant
region in this construct was originally obtained by PCR
amplification of the CAMPATH-1H heavy chain (Reichmann, L. et al.,
Nature, 322: 323-327 (1988)) as described by Sims, M. J. et al. (J.
Immunol., 151: 2296-2308 (1993)) and Waldmann et al. (WO 93/02191,
Feb. 4, 1993 (page 23)), the teachings of which are each
incorporated herein by reference in their entirety. The mutations
in the constant region of this construct
(Leu.sup.234.fwdarw.Ala.sup.234 and Gly.sup.237.fwdarw.Ala.sup.237)
were designed to reduce binding to human Fc.gamma. receptors, and
were produced by oligonucleotide-directed mutagenesis. Thus, the
MAdCAM-Ig fusion produced contains the SpeI-EcoRI constant region
fragment described by Sims et al. (J. Immunol., 151: 2296-2308
(1993)) and Waldmann et al. (WO 93/02191), except for the
introduction of Leu.sup.234.fwdarw.Ala.sup.234 and
Gly.sup.237.fwdarw.Ala.sup.237 mutations.
[0203] The 1 kb SpeI-EcoRI fragment encoding the Fc-mutated IgG1
constant region was isolated by gel electrophoresis using the
Glassmax DNA isolation system (Gibco, Bethesda Md.). This constant
region fragment and the HindIII-SpeI fragment containing the entire
extracellular domain of MAdCAM were ligated in a three-way ligation
to vector pEE12 (Stephens, P. L. and M. L. Cockett, Nucl. Acids
Res., 17: 7110 (1989) and Bebbington, C. R. and C. C. G. Hentschel,
1987, The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells, (Academic Press,
N.Y.), which had been digested with HindIII and EcoRI.
Transformants of the bacterial strain DH10B were obtained. Colonies
were grown and mini-plasmid preps were analyzed by restriction
mapping. A construct which encodes a fusion protein comprising the
entire extracellular domain of MAdCAM-1 (construct HuMAdIg21) fused
to the Fc-mutated IgG1 constant region, was sequenced across the
entire MAdCAM-1 portion, confirming proper fusion of segments and
the absence of PCR induced mutations. The chimera was produced in
NSO cells and purified by standard protein A affinity
chromatography.
[0204] 2. Adhesion Assay
[0205] A high binding flat-bottom 96-well plate (Costar) was coated
for 1 hr at 37.degree. C. with 50 .mu.l of MAdCAM-1 chimera diluted
to 2.5 .mu.g/ml in carbonate buffer, pH 9.5. Wells were then washed
once with wash buffer (50 mM Tris HCl, 0.14 M NaCl, 1 mM MnC12, pH
7.2) using a microplate autowasher (Bio-Tek Instruments, Winooski,
Vt.) and blocked for 1.5 hrs at 37.degree. C. with 100 .mu.L of 10%
FBS diluted in PBS.
[0206] RPMI 8866 cells (a human B cell lymphoma line which
expresses .alpha.4.beta.7 (and not .alpha.4.beta.7) (Erle, D. J.,
et al., J. Immunol., 153:517 (1994); a gift from D. Erle)) were
first washed in 20 ml PBS (4.degree. C.) and resuspended to
4.0.times.10.sup.6 cells/ml in PBS. BCECF
(2',7'-bis-(2-carboxyethyl)-5-(and 6)-carboxy fluorescein,
acetoxymethyl ester; Molecular Probes, Inc., Eugene, Oreg.) was
reconstituted to 50 .mu.g/ml in DMSO and added to the cell
suspension to a final dilution of 1:500. After incubating for 30
minutes at 37.degree. C., cells were then washed in assay buffer
(HBSS with 2% Fetal Bovine Serum, 25 mM HEPES,
penicillin/streptomycin, pH 7.2), and 50,000 cells were added to
each well of a V-bottom 96-well plate. Cells were then resuspended
in 100 .mu.l of either (a) murine Act-1, (b) murine IgG1 (Sigma
Chemical Co., St. Louis, Mo.), (c) LDP-02/3A9/Lot#1, or (d) human
IgG1 (Sigma Chemical Co., St. Louis, Mo.) at concentrations from
15.0 to 0.00075 .mu.g/ml in assay buffer for 10 minutes at room
temperature. The plate coated with MAdCAM-1 chimera was washed to
remove blocking buffer, and these fluorescently labeled RPMI 8866
cells were then transferred to each well. The plate was placed on a
platform shaker (New Brunswick Scientific Co., Inc., Edison, N.J.)
at 40 RPM for 30 minutes at room temperature wrapped in aluminum
foil. Unbound cells were removed by a single wash step and
fluorescence subsequently measured (excite at 485 nm, read at 535
nm) with a Fluorescence Concentrator Analyzer (IDEXX Laboratories,
Inc., Westbrook, Me.) before and after washing. The percent of
bound cells for each well was calculated from Relative fluorescent
units (RFU) using the formula:
% bound cells = R F U before wash R F U after wash .times. 100
##EQU00003##
[0207] Both LDP-02 and murine Act-1 inhibited adhesion of RPMI 8866
cells to human MAdCAM in a dose dependent manner (FIGS. 18A-18B).
The concentrations which inhibited adhesion by 50% (IC.sub.50) were
relatively similar for murine Act-1 (0.0018 .mu.g/ml) and LDP-02
(0.0014 .mu.g/ml). Therefore, LDP-02 functionally inhibited
.alpha.4.beta.7-mediated adhesion to MAdCAM-1 at least as
effectively as murine Act-1.
Example 5
Additional Humanized Antibodies
[0208] As described above, several variations of the reshaped
antibody designed in Example 2 can be made to improve affinity
and/or to decrease the antigenicity of the reshaped antibody. Such
constructs include, but are not limited to, those having one or
more of the following mutations: M4V mutation in the light chain,
R38K mutation in the heavy chain, A40R mutation in the heavy chain,
and 173T back-mutation in the heavy chain. Mutants can be produced
individually (e.g., one mutation in one chain), or in various
combinations.
[0209] For example, FIG. 19 shows the results of HuT 78 staining
using the reshaped antibody (designed in Example 2) or a derivative
having an additional mutation in the light chain (MV4) and two
additional mutations in the heavy chain (R38K, A40R). These two
antibodies show similar staining patterns on HuT 78 cells (FIG.
19). The mutations were made by changing the nucleic acid sequence
using a Transformer Site-Directed Mutagenesis Kit (Clontech)
according to manufacturer's suggested protocol. Mutations of both
heavy chain and light chain variable regions were made with
variable fragments cloned into pCR-Script.TM.. The trans oligo Sca
I/Stu I (Clontech) was used for the trans oligo. The sequence of
the mutagenic oligos (SEQ ID NOS:38-40) were as follows:
TABLE-US-00018 H/R38K (SEQ ID NO: 38): 5'-C TGG CCA ACG H/173T (SEQ
ID NO: 39): 5'-CAC ATT GAC TGT AGA CAC TTC CGC TAG CAC AGC C L/M4V
(SEQ ID NO: 40): 5'-CCG GAG GTG ATG TTG TGG TGA CTC
All other manipulations, including subcloning into expression
vectors pEE6hCMV-B and pEE12, and construction of expression
plasmids containing both heavy and light chain genes, were as
described for the primary reshaped antibody. Transient
transfections and cell staining were also done as described for the
primary reshaped antibody.
[0210] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
specifically herein. Such equivalents are intended to be
encompassed in the scope of the following claims.
Sequence CWU 1
1
671494DNAArtificial SequenceMouse consensus sequence 1ttackrgwmk
wcatgrratg sasctrkrtc atyytcttct tggtatcaac agctacaagt 60gtccactccc
aggtccaact gcagcagcct ggggctgagc ttgtgaagcc tgggacttca
120gtgaagctgt cctgcaaggg ttatggctac accttcacca gctactggat
gcactgggtg 180aagcagaggc ctggacaagg ccttgagtgg atcggagaga
ttgatccttc tgagagtaat 240actaactaca atcaaaaatt caagggcaag
gccacattga ctgtagacat ttcctccagc 300acagcctaca tgcagctcag
cagcctgaca tctgaggact ctgcggtcta ctattgtgca 360agagggggtt
acgacggatg ggactatgct attgactact ggggtcaagg cacctcagtc
420accgtctcct cagccaaaac gacaccrycn csyktmtmyc yysbdnnccc
ykgrwscytg 480gnngaagctt ggga 4942144PRTArtificial SequenceMouse
consensus sequence 2Met Xaa Xaa Xaa Xaa Xaa Ile Xaa Phe Leu Val Ser
Thr Ala Thr Ser 1 5 10 15Val His Ser Gln Val Gln Leu Gln Gln Pro
Gly Ala Glu Leu Val Lys 20 25 30Pro Gly Thr Ser Val Lys Leu Ser Cys
Lys Gly Tyr Gly Tyr Thr Phe 35 40 45Thr Ser Tyr Trp Met His Trp Val
Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly Glu Ile Asp
Pro Ser Glu Ser Asn Thr Asn Tyr Asn65 70 75 80Gln Lys Phe Lys Gly
Lys Ala Thr Leu Thr Val Asp Ile Ser Ser Ser 85 90 95Thr Ala Tyr Met
Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110Tyr Tyr
Cys Ala Arg Gly Gly Tyr Asp Gly Trp Asp Tyr Ala Ile Asp 115 120
125Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr
130 135 1403428DNAUnknownMouse 3ttacttgacg actcggg atg gga tgg agc
tat atc atc ttc ttc ttg gta 50 Met Gly Trp Ser Tyr Ile Ile Phe Phe
Leu Val 1 5 10tca aca gct aca agt gtc cac tcc cag gtc caa ctg cag
cag cct ggg 98Ser Thr Ala Thr Ser Val His Ser Gln Val Gln Leu Gln
Gln Pro Gly 15 20 25gct gag ctt gtg aag cct ggg act tca gtg aag ctg
tcc tgc aag ggt 146Ala Glu Leu Val Lys Pro Gly Thr Ser Val Lys Leu
Ser Cys Lys Gly 30 35 40tat ggc tac acc ttc acc agc tac tgg atg cac
tgg gtg aag cag agg 194Tyr Gly Tyr Thr Phe Thr Ser Tyr Trp Met His
Trp Val Lys Gln Arg 45 50 55cct gga caa ggc ctt gag tgg atc gga gag
att gat cct tct gag agt 242Pro Gly Gln Gly Leu Glu Trp Ile Gly Glu
Ile Asp Pro Ser Glu Ser 60 65 70 75aat act aac tac aat caa aaa ttc
aag ggc aag gcc aca ttg act gta 290Asn Thr Asn Tyr Asn Gln Lys Phe
Lys Gly Lys Ala Thr Leu Thr Val 80 85 90gac att tcc tcc agc aca gcc
tac atg cag ctc agc agc ctg aca tct 338Asp Ile Ser Ser Ser Thr Ala
Tyr Met Gln Leu Ser Ser Leu Thr Ser 95 100 105gag gac tct gcg gtc
tac tat tgt gca aga ggg ggt tac gac gga tgg 386Glu Asp Ser Ala Val
Tyr Tyr Cys Ala Arg Gly Gly Tyr Asp Gly Trp 110 115 120gac tat gct
att gac tac tgg ggt caa ggc aca tca gtc acc 428Asp Tyr Ala Ile Asp
Tyr Trp Gly Gln Gly Thr Ser Val Thr 125 130 1354137PRTUnknownMouse
4Met Gly Trp Ser Tyr Ile Ile Phe Phe Leu Val Ser Thr Ala Thr Ser 1
5 10 15Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val
Lys 20 25 30Pro Gly Thr Ser Val Lys Leu Ser Cys Lys Gly Tyr Gly Tyr
Thr Phe 35 40 45Thr Ser Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly
Gln Gly Leu 50 55 60Glu Trp Ile Gly Glu Ile Asp Pro Ser Glu Ser Asn
Thr Asn Tyr Asn65 70 75 80Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr
Val Asp Ile Ser Ser Ser 85 90 95Thr Ala Tyr Met Gln Leu Ser Ser Leu
Thr Ser Glu Asp Ser Ala Val 100 105 110Tyr Tyr Cys Ala Arg Gly Gly
Tyr Asp Gly Trp Asp Tyr Ala Ile Asp 115 120 125Tyr Trp Gly Gln Gly
Thr Ser Val Thr 130 1355535DNAArtificial SequenceMouse consensus
sequence 5cgattactag tcgac atg aag ttg cct gtt agg ctg ttg gtg ctt
ctg ttg 51 Met Lys Leu Pro Val Arg Leu Leu Val Leu Leu Leu 1 5
10ttc tgg att cct gtt tcc gga ggt gat gtt gtg gtg act caa act cca
99Phe Trp Ile Pro Val Ser Gly Gly Asp Val Val Val Thr Gln Thr Pro
15 20 25ctc tcc ctg cct gtc agc ttt gga gat caa gtt tct atc tct tgc
agg 147Leu Ser Leu Pro Val Ser Phe Gly Asp Gln Val Ser Ile Ser Cys
Arg 30 35 40tct agt cag agt ctt gca aag agt tat ggg aac acc tat ttg
tct tgg 195Ser Ser Gln Ser Leu Ala Lys Ser Tyr Gly Asn Thr Tyr Leu
Ser Trp 45 50 55 60tac ctg cac aag cct ggc cag tct cca cag ctc ctc
atc tat ggg att 243Tyr Leu His Lys Pro Gly Gln Ser Pro Gln Leu Leu
Ile Tyr Gly Ile 65 70 75tcc aac aga ttt tct ggg gtg cca gac agg ttc
agt ggc agt ggt tca 291Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe
Ser Gly Ser Gly Ser 80 85 90ggg aca gat ttc aca ctc aag atc agc aca
ata aag cct gag gac ttg 339Gly Thr Asp Phe Thr Leu Lys Ile Ser Thr
Ile Lys Pro Glu Asp Leu 95 100 105gga atg tat tac tgc tta caa ggt
aca cat cag ccg tac acg ttc gga 387Gly Met Tyr Tyr Cys Leu Gln Gly
Thr His Gln Pro Tyr Thr Phe Gly 110 115 120ggg ggg acc aag ctg gaa
ata aaa cgg gct gat gct gca cca act gta 435Gly Gly Thr Lys Leu Glu
Ile Lys Arg Ala Asp Ala Ala Pro Thr Val125 130 135 140tccatcttcc
caccatccag taagcttggg aatccatatg actagtagat cctctagagt
495cgacctgcag gcatgcaagc ttccctatag tgagtcgtat 5356140PRTArtificial
SequenceMouse consensus sequence 6Met Lys Leu Pro Val Arg Leu Leu
Val Leu Leu Leu Phe Trp Ile Pro 1 5 10 15Val Ser Gly Gly Asp Val
Val Val Thr Gln Thr Pro Leu Ser Leu Pro 20 25 30Val Ser Phe Gly Asp
Gln Val Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45Leu Ala Lys Ser
Tyr Gly Asn Thr Tyr Leu Ser Trp Tyr Leu His Lys 50 55 60Pro Gly Gln
Ser Pro Gln Leu Leu Ile Tyr Gly Ile Ser Asn Arg Phe65 70 75 80Ser
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90
95Thr Leu Lys Ile Ser Thr Ile Lys Pro Glu Asp Leu Gly Met Tyr Tyr
100 105 110Cys Leu Gln Gly Thr His Gln Pro Tyr Thr Phe Gly Gly Gly
Thr Lys 115 120 125Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val
130 135 1407112PRTUnknownMouse 7Asp Val Val Val Thr Gln Thr Pro Leu
Ser Leu Pro Val Ser Phe Gly 1 5 10 15Asp Gln Val Ser Ile Ser Cys
Arg Ser Ser Gln Ser Leu Ala Lys Ser 20 25 30Tyr Gly Asn Thr Tyr Leu
Ser Trp Tyr Leu His Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile
Tyr Gly Ile Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Thr
Ile Lys Pro Glu Asp Leu Gly Met Tyr Tyr Cys Leu Gln Gly 85 90 95Thr
His Gln Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
1108112PRTHomo sapiens 8Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu
Pro Val Thr Pro Gly 1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Leu His Ser 20 25 30Asn Gly Tyr Asn Tyr Leu Asp Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu
Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95Leu Gln Thr
Pro Gln Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
1109121PRTUnknownMouse 9Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu
Val Lys Pro Gly Thr 1 5 10 15Ser Val Lys Leu Ser Cys Lys Gly Tyr
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Lys Gln Arg
Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asp Pro Ser Glu
Ser Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu
Thr Val Asp Ile Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Gly Tyr Asp Gly Trp Asp Tyr Ala Ile Asp Tyr Trp Gly 100 105 110Gln
Gly Thr Ser Val Thr Val Ser Ser 115 12010119PRTHomo sapiens 10Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp
Met 35 40 45Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln
Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Tyr Tyr Gly Ser Gly Ser
Asn Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11511396DNAUnknownMouse 11atg aag ttg cct gtt agg ctg ttg gtg ctt
ctg ttg ttc tgg att cct 48Met Lys Leu Pro Val Arg Leu Leu Val Leu
Leu Leu Phe Trp Ile Pro 1 5 10 15gtt tcc gga ggt gat gtt gtg gtg
act caa act cca ctc tcc ctg cct 96Val Ser Gly Gly Asp Val Val Val
Thr Gln Thr Pro Leu Ser Leu Pro 20 25 30gtc agc ttt gga gat caa gtt
tct atc tct tgc agg tct agt cag agt 144Val Ser Phe Gly Asp Gln Val
Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45ctt gca aag agt tat ggg
aac acc tat ttg tct tgg tac ctg cac aag 192Leu Ala Lys Ser Tyr Gly
Asn Thr Tyr Leu Ser Trp Tyr Leu His Lys 50 55 60cct ggc cag tct cca
cag ctc ctc atc tat ggg att tcc aac aga ttt 240Pro Gly Gln Ser Pro
Gln Leu Leu Ile Tyr Gly Ile Ser Asn Arg Phe 65 70 75 80tct ggg gtg
cca gac agg ttc agt ggc agt ggt tca ggg aca gat ttc 288Ser Gly Val
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95aca ctc
aag atc agc aca ata aag cct gag gac ttg gga atg tat tac 336Thr Leu
Lys Ile Ser Thr Ile Lys Pro Glu Asp Leu Gly Met Tyr Tyr 100 105
110tgc tta caa ggt aca cat cag ccg tac acg ttc gga ggg ggg acc aag
384Cys Leu Gln Gly Thr His Gln Pro Tyr Thr Phe Gly Gly Gly Thr Lys
115 120 125ctg gaa ata aaa 396Leu Glu Ile Lys
13012132PRTUnknownMouse 12Met Lys Leu Pro Val Arg Leu Leu Val Leu
Leu Leu Phe Trp Ile Pro 1 5 10 15Val Ser Gly Gly Asp Val Val Val
Thr Gln Thr Pro Leu Ser Leu Pro 20 25 30Val Ser Phe Gly Asp Gln Val
Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45Leu Ala Lys Ser Tyr Gly
Asn Thr Tyr Leu Ser Trp Tyr Leu His Lys 50 55 60Pro Gly Gln Ser Pro
Gln Leu Leu Ile Tyr Gly Ile Ser Asn Arg Phe65 70 75 80Ser Gly Val
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95Thr Leu
Lys Ile Ser Thr Ile Lys Pro Glu Asp Leu Gly Met Tyr Tyr 100 105
110Cys Leu Gln Gly Thr His Gln Pro Tyr Thr Phe Gly Gly Gly Thr Lys
115 120 125Leu Glu Ile Lys 13013336DNAHomo sapiens 13gatattgtga
tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60atctcctgca
ggtctagtca gagcctcctc catagtaatg gatcaaacta tttggattgg
120tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc
taatcgggcc 180tccggggtcc ctgacaggtt cagtggcagt ggatcaggca
cagattttac actgaaaatc 240agcagagtgg aggctgagga tgttggggtt
tattactgca tgcaagctct accaactcct 300cagacgttcg gccaagggac
caaggtggaa atcaaa 33614420DNAArtificial SequenceMouse Act-1
antibody heavy chain variable region with a signal peptide sequence
14atg gga tgg agc tgt atc atc ctc ttc ttg gta tca aca gct aca agt
48Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ser Thr Ala Thr Ser 1
5 10 15gtc cac tcc cag gtc caa ctg cag cag cct ggg gct gag ctt gtg
aag 96Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val
Lys 20 25 30cct ggg act tca gtg aag ctg tcc tgc aag ggt tat ggc tac
acc ttc 144Pro Gly Thr Ser Val Lys Leu Ser Cys Lys Gly Tyr Gly Tyr
Thr Phe 35 40 45acc agc tac tgg atg cac tgg gtg aag cag agg cct gga
caa ggc ctt 192Thr Ser Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly
Gln Gly Leu 50 55 60gag tgg atc gga gag att gat cct tct gag agt aat
act aac tac aat 240Glu Trp Ile Gly Glu Ile Asp Pro Ser Glu Ser Asn
Thr Asn Tyr Asn 65 70 75 80caa aaa ttc aag ggc aag gcc aca ttg act
gta gac att tcc tcc agc 288Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr
Val Asp Ile Ser Ser Ser 85 90 95aca gcc tac atg cag ctc agc agc ctg
aca tct gag gac tct gcg gtc 336Thr Ala Tyr Met Gln Leu Ser Ser Leu
Thr Ser Glu Asp Ser Ala Val 100 105 110tac tat tgt gca aga ggg ggt
tac gac gga tgg gac tat gct att gac 384Tyr Tyr Cys Ala Arg Gly Gly
Tyr Asp Gly Trp Asp Tyr Ala Ile Asp 115 120 125tac tgg ggt caa ggc
acc tca gtc acc gtc tcc tca 420Tyr Trp Gly Gln Gly Thr Ser Val Thr
Val Ser Ser 130 135 14015140PRTArtificial SequenceMouse Act-1
antibody heavy chain variable region with a signal peptide sequence
15Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ser Thr Ala Thr Ser 1
5 10 15Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val
Lys 20 25 30Pro Gly Thr Ser Val Lys Leu Ser Cys Lys Gly Tyr Gly Tyr
Thr Phe 35 40 45Thr Ser Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly
Gln Gly Leu 50 55 60Glu Trp Ile Gly Glu Ile Asp Pro Ser Glu Ser Asn
Thr Asn Tyr Asn65 70 75 80Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr
Val Asp Ile Ser Ser Ser 85 90 95Thr Ala Tyr Met Gln Leu Ser Ser Leu
Thr Ser Glu Asp Ser Ala Val 100 105 110Tyr Tyr Cys Ala Arg Gly Gly
Tyr Asp Gly Trp Asp Tyr Ala Ile Asp 115 120 125Tyr Trp Gly Gln Gly
Thr Ser Val Thr Val Ser Ser 130 135 14016414DNAArtificial
SequenceHuman 21/28'CL antibody heavy chain variable region with a
signal peptide sequence 16atg gag ttt ggg ctg agc tgg ctt ttt ctt
gtg gct att tta aaa ggt 48Met Glu Phe Gly Leu Ser Trp Leu Phe Leu
Val Ala Ile Leu Lys Gly 1 5 10 15gtc cag tgt cag gtg cag ctt gtg
cag tct ggg gct gag gtg aag aag 96Val Gln Cys Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys 20 25 30cct ggg gcc tca gtg aag gtt
tcc tgc aag gct tct gga tac acc ttc 144Pro Gly Ala Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45act agc tat gct atg cat
tgg gtg cgc cag gcc ccc gga caa agg ctt 192Thr Ser Tyr Ala Met His
Trp Val Arg Gln Ala Pro Gly Gln Arg Leu 50 55 60gag tgg atg gga tgg
atc aac gct ggc aat ggt aac aca aaa tat tca 240Glu Trp Met Gly Trp
Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser 65 70 75 80cag aag ttc
cag ggc aga gtc acc att acc agg gac aca tcc gcg agc 288Gln Lys Phe
Gln
Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser 85 90 95aca gcc tac
atg gag ctg agc agc ctg aga tct gaa gac acg gct gtg 336Thr Ala Tyr
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110tat
tac tgt gcg aga gga ggt tac tat ggt tcg ggg agc aac tac tgg 384Tyr
Tyr Cys Ala Arg Gly Gly Tyr Tyr Gly Ser Gly Ser Asn Tyr Trp 115 120
125ggc cag gga acc ctg gtc acc gtc tcc tca 414Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 130 13517138PRTArtificial SequenceHuman
21/28'CL antibody heavy chain variable region with a signal peptide
sequence 17Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu
Lys Gly 1 5 10 15Val Gln Cys Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys 20 25 30Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe 35 40 45Thr Ser Tyr Ala Met His Trp Val Arg Gln
Ala Pro Gly Gln Arg Leu 50 55 60Glu Trp Met Gly Trp Ile Asn Ala Gly
Asn Gly Asn Thr Lys Tyr Ser65 70 75 80Gln Lys Phe Gln Gly Arg Val
Thr Ile Thr Arg Asp Thr Ser Ala Ser 85 90 95Thr Ala Tyr Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ala
Arg Gly Gly Tyr Tyr Gly Ser Gly Ser Asn Tyr Trp 115 120 125Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 130 13518540DNAArtificial
SequencePortion of humanized Act-1 antibody heavy chain with a
heavy chain signal peptide sequence 18atg aaa tgc acc tgg gtc att
ctc ttc ttg gta tca aca gct aca agt 48Met Lys Cys Thr Trp Val Ile
Leu Phe Leu Val Ser Thr Ala Thr Ser 1 5 10 15gtc cac tcc cag gtc
caa cta gtg cag tct ggg gct gag gtt aag aag 96Val His Ser Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30cct ggg gct tca
gtg aag gtg tcc tgc aag ggt tct ggc tac acc ttc 144Pro Gly Ala Ser
Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe 35 40 45acc agc tac
tgg atg cat tgg gtg agg cag gcg cct ggc caa cgt cta 192Thr Ser Tyr
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu 50 55 60gag tgg
atc gga gag att gat cct tct gag agt aat act aac tac aat 240Glu Trp
Ile Gly Glu Ile Asp Pro Ser Glu Ser Asn Thr Asn Tyr Asn 65 70 75
80caa aaa ttc aag gga cgc gtc aca ttg act gta gac att tcc gct agc
288Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Val Asp Ile Ser Ala Ser
85 90 95aca gcc tac atg gag ctc agc agc ctg aga tct gag gac act gcg
gtc 336Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val 100 105 110tac tat tgt gca aga ggg ggt tac gac gga tgg gac tat
gct att gac 384Tyr Tyr Cys Ala Arg Gly Gly Tyr Asp Gly Trp Asp Tyr
Ala Ile Asp 115 120 125tac tgg ggt caa ggc acc ctg gtc acc gtc tcc
tca gcc tcc acc aag 432Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys 130 135 140ggc cca tcg gtc ttc ccc ctg gca ccc
tcc tcc aag agc acc tct ggg 480Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly145 150 155 160ggc aca gcg gcc ctg ggc
tgc ctg gtc aag gac tac ttc ccc gaa ccg 528Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175gtg acg gtg tcg
540Val Thr Val Ser 18019180PRTArtificial SequencePortion of
humanized Act-1 antibody heavy chain with a heavy chain signal
peptide sequence 19Met Lys Cys Thr Trp Val Ile Leu Phe Leu Val Ser
Thr Ala Thr Ser 1 5 10 15Val His Ser Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Ala Ser Val Lys Val Ser Cys
Lys Gly Ser Gly Tyr Thr Phe 35 40 45Thr Ser Tyr Trp Met His Trp Val
Arg Gln Ala Pro Gly Gln Arg Leu 50 55 60Glu Trp Ile Gly Glu Ile Asp
Pro Ser Glu Ser Asn Thr Asn Tyr Asn65 70 75 80Gln Lys Phe Lys Gly
Arg Val Thr Leu Thr Val Asp Ile Ser Ala Ser 85 90 95Thr Ala Tyr Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110Tyr Tyr
Cys Ala Arg Gly Gly Tyr Asp Gly Trp Asp Tyr Ala Ile Asp 115 120
125Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
130 135 140Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly145 150 155 160Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro 165 170 175Val Thr Val Ser
18020413DNAArtificial SequencePortion of humanized Act-1 antibody
light chain with a light chain signal peptide sequence 20atgaagttgc
ctgttaggct gttggtgctt ctgttgttct ggattcctgt ttccggaggt 60gatgttgtga
tgactcaaag tccactctcc ctgcctgtca cccctggaga accagcttct
120atctcttgca ggtctagtca gagtcttgca aagagttatg ggaacaccta
tttgtcttgg 180tacctgcaga agcctggcca gtctccacag ctcctcatct
atgggatttc caacagattt 240tctggggtgc cagacaggtt cagtggcagt
ggttcaggga cagatttcac actcaagatc 300tcgcgagtag aggctgagga
cgtgggagtg tattactgct tacaaggtac acatcagccg 360tacacgttcg
gacaggggac caaggtggaa ataaaacggg ctgatgcggc gcc
41321138PRTArtificial SequencePortion of humanized Act-1 antibody
light chain with a light chain signal peptide sequence 21Met Lys
Leu Pro Val Arg Leu Leu Val Leu Leu Leu Phe Trp Ile Pro 1 5 10
15Val Ser Gly Gly Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro
20 25 30Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser 35 40 45Leu Ala Lys Ser Tyr Gly Asn Thr Tyr Leu Ser Trp Tyr Leu
Gln Lys 50 55 60Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gly Ile Ser
Asn Arg Phe65 70 75 80Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe 85 90 95Thr Leu Lys Ile Ser Arg Val Glu Ala Glu
Asp Val Gly Val Tyr Tyr 100 105 110Cys Leu Gln Gly Thr His Gln Pro
Tyr Thr Phe Gly Gln Gly Thr Lys 115 120 125Val Glu Ile Lys Arg Ala
Asp Ala Ala Pro 130 1352294DNAArtificial SequenceSynthetic
oligonucleotide 22tttccggagg tgatgttgtg atgactcaaa gtccactctc
cctgcctgtc acccctggag 60aaccagcttc tatctcttgc aggtctagtc agag
942394DNAArtificial SequenceSynthetic oligonucleotide 23actggccagg
cttctgcagg taccaagaca aataggtgtt cccataactc tttgcaagac 60tctgactaga
cctgcaagag atagaagctg gttc 942483DNAArtificial SequenceSynthetic
oligonucleotide 24cctggccagt ctccacagct cctcatctat gggatttcca
acagattttc tggggtgcca 60gacaggttca gtggcagtgg ttc
832584DNAArtificial SequenceSynthetic oligonucleotide 25actcgcgaga
tcttgagtgt gaaatctgtc cctgaaccac tgccactgaa cctgtctggc 60accccagaaa
atctgttgga aatc 842667DNAArtificial SequenceSynthetic
oligonucleotide 26tctcgcgagt agaggctgag gacgtgggag tgtattactg
cttacaaggt acacatcagc 60cgtacac 672786DNAArtificial
SequenceSynthetic oligonucleotide 27atggcgccgc atcagcccgt
tttatttcca ccttggtccc ctgtccgaac gtgtacggct 60gatgtgtacc ttgtaagcag
taatac 862893DNAArtificial SequenceSynthetic oligonucleotide
28ataagcttcg ccatgaaatg cacctgggtc attctcttct tggtatcaac agctacaagt
60gtccactccc aggtccaact agtgcaccgg tta 932993DNAArtificial
SequenceSynthetic oligonucleotide 29taaccggtgc actagttgga
cctgggagtg gacacttgta gctgttgata ccaagaagag 60aatgacccag gtgcatttca
tggcgaagct tat 933087DNAArtificial SequenceSynthetic
oligonucleotide 30caactagtgc agtctggggc tgaggttaag aagcctgggg
cttcagtgaa ggtgtcctgc 60aagggttctg gctacacctt caccagc
873188DNAArtificial SequenceSynthetic oligonucleotide 31taaccggtac
tctagacgtt ggccaggcgc ctgcctcacc caatgcatcc agtagctggt 60gaaggtgtag
ccagaaccct tgcaggac 883276DNAArtificial SequenceSynthetic
oligonucleotide 32cgtctagagt ggatcggaga gattgatcct tctgagagta
atactaacta caatcaaaaa 60ttcaagggac gcgtca 763376DNAArtificial
SequenceSynthetic oligonucleotide 33taaccggtgt gctagcggaa
atgtctacag tcaatgtgac gcgtcccttg aatttttgat 60tgtagttagt attact
763488DNAArtificial SequenceSynthetic oligonucleotide 34ccgctagcac
agcctacatg gagctcagca gcctgagatc tgaggacact gcggtctact 60attgtgcaag
agggggttac gacggatg 883588DNAArtificial SequenceSynthetic
oligonucleotide 35tcaccggtgc ggtgaccagg gtgccttgac cccagtagtc
aatagcatag tcccatccgt 60cgtaaccccc tcttgcacaa tagtagac
883685DNAArtificial SequenceSynthetic oligonucleotide 36ctggtcaccg
tctcctcagc ctccaccaag ggcccatcgg tcttccccct ggcaccctcc 60tccaagagca
cctctggggg cacag 853785DNAArtificial SequenceSynthetic
oligonucleotide 37tcaccggttc ggggaagtag tccttgacca ggcagcccag
ggccgctgtg cccccagagg 60tgctcttgga ggagggtgcc agggg
853810DNAArtificial SequenceSynthetic oligonucleotide 38ctggccaacg
103934DNAArtificial SequenceSynthetic oligonucleotide 39cacattgact
gtagacactt ccgctagcac agcc 344024DNAArtificial SequenceSynthetic
oligonucleotide 40ccggaggtga tgttgtggtg actc 244124DNAArtificial
SequenceSynthetic oligonucleotide 41taagcttccg ccatgggatg gagc
244225DNAArtificial SequenceSynthetic oligonucleotide 42ggtgacacta
gtgccttgac cccag 254324DNAArtificial SequenceSynthetic
oligonucleotide 43taagcttccg ccatgaagtt gcct 244421DNAArtificial
SequenceSynthetic oligonucleotide 44ggcgccgcat cagcccgttt t
214520DNAArtificial SequenceSynthetic oligonucleotide 45cggcgccatc
tgtcttcatc 204618DNAArtificial SequenceSynthetic oligonucleotide
46aagcttctaa cactctcc 184719PRTUnknownMouse 47Asp Val Val Val Thr
Gln Thr Pro Leu Ser Leu Pro Val Ser Phe Asp 1 5 10 15Gly Gln
Val4811PRTUnknownMouse 48Asp Val Val Val Thr Gln Thr Pro Leu Ser
Leu 1 5 10498PRTUnknownMouse 49Asp Tyr Ala Ile Asp Tyr Trp Gly 1
550113PRTArtificial SequenceMouse consensus sequence 50Asp Val Val
Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15Asp
Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25
30Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val
Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr
Cys Phe Gln Gly 85 90 95Thr His Val Pro Pro Tyr Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile 100 105 110Lys51114PRTArtificial SequenceHuman
consensus sequence 51Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu
Pro Val Thr Pro Gly 1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Leu His Ser 20 25 30Xaa Asp Gly Asn Asn Tyr Leu Asn
Trp Tyr Leu Gln Lys Pro Gly Gln 35 40 45Ser Pro Gln Leu Leu Ile Tyr
Leu Val Ser Asn Arg Ala Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys65 70 75 80Ile Ser Arg Val
Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln 85 90 95Ala Leu Gln
Xaa Pro Arg Xaa Thr Phe Gly Gln Gly Thr Lys Val Glu 100 105 110Ile
Lys52112PRTArtificial SequenceReshaped humanized sequence 52Asp Val
Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10
15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Ala Lys Ser
20 25 30Tyr Gly Asn Thr Tyr Leu Ser Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45Pro Gln Leu Leu Ile Tyr Gly Ile Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Leu Gln Gly 85 90 95Thr His Gln Pro Tyr Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 11053127PRTArtificial SequenceMouse
consensus sequence 53Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu
Val Lys Pro Gly Ala 1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Lys Gln Arg
Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Arg Ile Asp Pro Asn Ser
Gly Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60Lys Ser Lys Ala Thr Leu
Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Tyr
Tyr Tyr Gly Gly Ser Ser Xaa Xaa Val Tyr Xaa Tyr Trp 100 105 110Tyr
Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
12554129PRTArtificial SequenceHuman consensus sequence 54Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45Gly Trp Ile Asn Pro Tyr Gly Asn Gly Asp Thr Asn Tyr Ala
Gln Lys 50 55 60Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr
Ser Thr Ala65 70 75 80Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr 85 90 95Cys Ala Arg Ala Pro Gly Tyr Gly Ser Gly
Gly Gly Cys Tyr Arg Gly 100 105 110Asp Tyr Xaa Phe Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser 115 120 125Ser55121PRTArtificial
SequenceReshaped humanized sequence 55Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15Ser Val Lys Val Ser
Cys Lys Gly Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His Trp
Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45Gly Glu Ile
Asp Pro Ser Glu Ser Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Gly
Arg Val Thr Leu Thr Val Asp Ile Ser Ala Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Gly Tyr Asp Gly Trp Asp Tyr Ala Ile Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
1205635DNAArtificial SequenceSynthetic oligonucleotide 56cccaagcttc
cagggrccar kggataracn grtgg 355732DNAArtificial SequenceSynthetic
oligonucleotide 57cccaagctta cgagggggaa gacatttggg aa
325834DNAArtificial SequenceSynthetic oligonucleotide
58gggaattcat
graatgsasc tgggtywtyc tctt 345933DNAArtificial SequenceSynthetic
oligonucleotide 59actagtcgac atgaagwtgt ggbtraactg grt
336030DNAArtificial SequenceSynthetic oligonucleotide 60cccaagctta
ctggatggtg ggaagatgga 306139DNAArtificial SequenceSynthetic
oligonucleotide 61actagtcgac atggatttwc argtgcagat twtcagctt
396232DNAArtificial SequenceSynthetic oligonucleotide 62ggaagcttcc
accatggatt tcggactggc cc 326327DNAArtificial SequenceSynthetic
oligonucleotide 63ggactagtgg tttggacgag cctgttg
2764396DNAUnknownMouse 64ttttatttcc agcttggtcc cccctccgaa
cgtgtacggc tgatgtgtac cttgtaagca 60gtaatacatt cccaagtcct caggctttat
tgtgctgatc ttgagtgtga aatctgtccc 120tgaaccactg ccactgaacc
tgtctggcac cccagaaaat ctgttggaaa tcccatagat 180gaggagctgt
ggagactggc caggcttgtg caggtaccaa gacaaatagg tgttcccata
240actctttgca agactctgac tagacctgca agagatagaa acttgatctc
caaagctgac 300aggcagggag agtggagttt gagtcaccac aacatcacct
ccggaaacag gaatccagaa 360caacagaagc accaacagcc taacaggcaa cttcat
39665336DNAHomo sapiens 65tttgatttcc accttggtcc cttggccgaa
cgtctgagga gttggtagag cttgcatgca 60gtaataaacc ccaacatcct cagcctccac
tctgctgatt ttctgtgtaa aatctgtgcc 120tgatccactg ccactgaacc
tgtcagggac cccggaggcc cgattagaac ccaaatagat 180caggagctgt
ggagactgcc ctggcttctg caggtaccaa tccaaatagt ttgatccatt
240actatggagg aggctctgac tagacctgca ggagatggag gccggctctc
caggggtgac 300gggcagggag agtggagact gagtcatcac aatatc
33666420DNAArtificial SequenceMouse Act-1 antibody heavy chain
variable region with a signal peptide sequence-antisense
66tgaggagacg gtgactgagg tgccttgacc ccagtagtca atagcatagt cccatccgtc
60gtaaccccct cttgcacaat agtagaccgc agagtcctca gatgtcaggc tgctgagctg
120catgtaggct gtgctggagg aaatgtctac agtcaatgtg gccttgccct
tgaatttttg 180attgtagtta gtattactct cagaaggatc aatctctccg
atccactcaa ggccttgtcc 240aggcctctgc ttcacccagt gcatccagta
gctggtgaag gtgtagccat aacccttgca 300ggacagcttc actgaagtcc
caggcttcac aagctcagcc ccaggctgct gcagttggac 360ctgggagtgg
acacttgtag ctgttgatac caagaagagg atgatacagc tccatcccat
42067414DNAArtificial SequenceHuman 21/28'CL antibody heavy chain
variable region with a signal peptide sequence-antisense
67tgaggagacg gtgaccaggg ttccctggcc ccagtagttg ctccccgaac catagtaacc
60tcctctcgca cagtaataca cagccgtgtc ttcagatctc aggctgctca gctccatgta
120ggctgtgctc gcggatgtgt ccctggtaat ggtgactctg ccctggaact
tctgtgaata 180ttttgtgtta ccattgccag cgttgatcca tcccatccac
tcaagccttt gtccgggggc 240ctggcgcacc caatgcatag catagctagt
gaaggtgtat ccagaagcct tgcaggaaac 300cttcactgag gccccaggct
tcttcacctc agccccagac tgcacaagct gcacctgaca 360ctggacacct
tttaaaatag ccacaagaaa aagccagctc agcccaaact ccat 414
* * * * *