U.S. patent application number 11/691350 was filed with the patent office on 2008-01-31 for humanized antibodies against cd3.
This patent application is currently assigned to Protein Design Labs, Inc.. Invention is credited to Roger Gingrich, Brian K. Link, J. Yun Tso, George Weiner.
Application Number | 20080025975 11/691350 |
Document ID | / |
Family ID | 31999180 |
Filed Date | 2008-01-31 |
United States Patent
Application |
20080025975 |
Kind Code |
A1 |
Weiner; George ; et
al. |
January 31, 2008 |
HUMANIZED ANTIBODIES AGAINST CD3
Abstract
The invention provides bispecific antibodies with selective
cytotoxicity against malignant B-cells. The bispecific antibodies
bind to an effector cell antigen and to a 28/32 kDa heterodimeric
protein on the surface of malignant B-cells. The invention also
includes the monospecific components of the bispecific antibodies,
humanized versions thereof, and humanized bispecific antibodies.
The invention further provides therapeutic and diagnostic methods
employing these antibodies.
Inventors: |
Weiner; George; (Iowa City,
IA) ; Gingrich; Roger; (Iowa City, IA) ; Link;
Brian K.; (Coralville, IA) ; Tso; J. Yun;
(Menlo Park, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Protein Design Labs, Inc.
Fremont
CA
Iowa Immunotherapy Investigators
Iowa City
IA
|
Family ID: |
31999180 |
Appl. No.: |
11/691350 |
Filed: |
March 26, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10435299 |
May 9, 2003 |
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11691350 |
Mar 26, 2007 |
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09618380 |
Jul 18, 2000 |
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10435299 |
May 9, 2003 |
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08397411 |
Mar 1, 1995 |
6129914 |
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09618380 |
Jul 18, 2000 |
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07859583 |
Mar 27, 1992 |
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08397411 |
Mar 1, 1995 |
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Current U.S.
Class: |
424/133.1 ;
530/387.3 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 2319/00 20130101; A61K 38/00 20130101; A61P 31/00 20180101;
C07K 2317/54 20130101; C07K 2317/24 20130101; C07K 16/2809
20130101; C07K 2317/31 20130101; C07K 16/3061 20130101; C07K
2317/74 20130101 |
Class at
Publication: |
424/133.1 ;
530/387.3 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 31/00 20060101 A61P031/00; C07K 16/00 20060101
C07K016/00 |
Claims
1. A bispecific antibody that binds to: (a) a first antigen on the
surface of effector cells selected from the group consisting of
T-cells and natural killer cells, and (b) a second antigen on a
28/32 kDa heterodimeric protein on the surface of malignant B
cells, which second antigen specifically binds to an antibody
designated 1D10, wherein the binding of the bispecific antibody to
the first and second antigens results in killing of the malignant B
cells.
2-15. (canceled)
16. A humanized antibody, the antibody comprising a humanized heavy
chain and a humanized light chain: (1) the humanized light chain
comprising three complementarity determining regions (CDR1, CDR2
and CDR3) having amino acid sequences from the corresponding
complementarity determining regions of the mouse M291
immunoglobulin light chain, and a variable region framework from a
human kappa light chain variable region framework sequence, and (2)
the humanized heavy chain comprising three complementarity
determining regions (CDR1, CDR2 and CDR3) having amino acid
sequences from the corresponding complementarity determining
regions of the mouse M291 immunoglobulin heavy chain, and a
variable region framework from a human heavy chain variable region
framework sequence except in at least one position selected from a
second group consisting of H30, H67, H68, H70, H72 and H74 wherein
the amino acid position is occupied by the same amino acid present
in the equivalent position of the mouse M291 immunoglobulin heavy
chain variable region framework; wherein the immunoglobulin
specifically binds to a CD3 antigen on the surface of T cells with
a binding affinity having a lower limit of about 10.sup.7 M.sup.-1
and an upper limit of about five-times the binding affinity of the
M291 immunoglobulin.
17. The humanized antibody of claim 16, wherein the humanized light
chain variable region framework is from the light chain variable
region framework of the HF2-1/17 antibody in subgroup I; the
humanized heavy chain region framework is from the heavy chain
region variable framework of the 21/28 antibody except in at least
one position selected from the second group, and except at position
44, wherein the amino acid position is occupied by the same amino
acid present in the equivalent position of a human immunoglobulin
subgroup I consensus sequence.
18. The humanized antibody of claim 17, wherein the humanized light
chain comprises the amino acid sequence of FIG. 5A (upper) and the
humanized heavy chain comprises the amino acid sequence of FIG. 5B
(upper).
19-33. (canceled)
34. A method of treating or preventing a disorder of the immune
system, comprising administering a therapeutically or
prophylactically effective dose of a humanized anti-CD3 antibody to
a patient affected by or at risk of the disorder.
35. The method according to claim 34, wherein the disorder of the
immune system is an autoimmune disease, inflammation, graft vs.
host disease, or host vs. graft disease.
36. The method of claim 35, wherein the disorder of the immune
system is graft vs. host disease.
37. The method according to claim 34, wherein the humanized
antibody comprises a pair of humanized heavy chains and humanized
light chains, wherein the humanized light chain variable region
comprises the amino acid sequence of FIG. 5A (upper lines) (SEQ.
ID. No. 8) and the humanized heavy chain variable region comprises
the amino acid sequence of FIG. 5B (upper lines) (SEQ. ID. No.
10)
38. The method according to claim 34, wherein the antibody
comprises a constant domain of the IgG2 isotype.
39. The method according to claim 34, wherein the antibody has an
affinity for the CD3 antigen having a lower limit of about 10.sup.7
M.sup.-1 and an upper limit of five-times the binding affinity of
the M291 immunoglobulin, wherein the mouse M291 antibody has a
heavy chain with a variable region of sequence SEQ. ID. No. 11 and
a light chain with a variable region of sequence SEQ. ID. No.
9.
40. The method according to claim 34, wherein the antibody
comprises a humanized heavy chain and a humanized light chain: (1)
the humanized light chain comprising three complementarity
determining regions, CDR1, CDR2 and CDR3, of the mouse M291
immunoglobulin light chain, and a variable region framework from a
human kappa light chain variable region framework sequence, and (2)
the humanized heavy chain comprising three complementarity
determining regions, CDR1, CDR2 and CDR3, of the mouse M291
immunoglobulin heavy chain, and a variable region framework from a
human heavy chain variable region framework sequence provided that
at least one position selected from a group consisting of H30, H67,
H68, H70, H72 and H74 is occupied by the same amino acid present in
the equivalent position of the mouse M291 immunoglobulin heavy
chain variable region framework; wherein the immunoglobulin
specifically binds to a CD3 antigen on the surface of T cells with
a binding affinity having a lower limit of about 10.sup.7M.sup.-1
and an upper limit of about five-times the binding affinity of the
M291 immunoglobulin wherein the mouse antibody has an IgG1 heavy
chain with a variable domain designated SEQ. ID. No. 11 and a kappa
light chain with a variable domain designated SEQ. ID. No. 9.
41. The method according to claim 34, wherein the antibody is
administered subcutaneously, intramuscularly or intravenously.
42. The method according to claim 34, wherein the patient is
affected by the disorder and the dose is 0.01 to 100 mg
43. The method according to claim 34, wherein the patient is
affected by the disorder and the dose is 1-10 mg.
44. The method according to claim 34, wherein the patient is at
risk of the disorder and the dose is 0.1 to 100 mg.
45. The method according to claim 34, wherein the patient is at
risk of the disorder and the dose is 1 to 10 mg.
46. The method of claim 34, wherein the humanized antibody is a
bispecific antibody comprising: a first Fab'fragment comprising the
humanized heavy chain variable region comprises the amino acid
sequence of FIG. 5B (upper lines) (SEQ. ID. No. 10) and the
humanized light chain variable region comprises the amino acid
sequence of FIG. 5A (upper lines) (SEQ. ID. No. 8); a second Fab'
fragment comprising the heavy chain variable region shown in FIG.
4B (upper) (SEQ ID NO. 3) and the light chain variable region shown
in FIG. 4A (upper) (SEQ ID NO. 1); wherein the first Fab'fragment
specifically binds to the CD3 antigen and the second Fab' fragment
specifically binds to the 28/32 kDa heterodimeric antigen on the
surface of the malignant B cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
07/859,583 filed Mar. 27, 1992, which is incorporated by reference
in its entirety for all purposes.
BACKGROUND OF THE INVENTION
[0002] Administration of monoclonal antibodies (MoAb) has shown
promise as a new treatment modality for human malignancy. However,
destruction of malignant cells by MoAb does not always occur, even
after successful binding of the antibody to the target cell. A
second approach to immunotherapy of malignancy involves the
manipulation of the cellular immune system. Lymphokines, such as
IL-2, can be used to activate both NK cells and T cells isolated
from the blood, spleen, or malignant tumors themselves. The
anti-tumor effects of such cells have been well documented both in
vitro and in vivo. Toxicity of therapy based on IL-2 alone can be
severe and may well limit the clinical utility of this therapy.
[0003] Immunotherapy of malignancy that attempts to combine the
specificity of antibodies with the power of activated lymphocytes
might be more effective and less toxic. One such approach is the
use of bispecific antibodies to redirect activated T cell toxicity
toward tumor cells expressing the target antigen (Ag.)
[0004] Various forms of bispecific antibodies have been produced.
These include BSIgG, which are IgG molecules comprising two
distinct heavy chains and two distinct light chains that hare
secreted by so-called "hybrid hybridomas", and heteroantibody
conjugates produced by the chemical conjugation of antibodies or
antibody fragments of different specificities.
[0005] Several investigators have evaluated anti-CD3/anti-tumor
bispecific antibody structures as immunotherapeutic agents. Such
studies have reported in vitro cytolysis of renal cell carcinoma,
melanoma, glioma, lymphoma, leukemia and cells expressing the
multidrug-resistance-related glycoprotein. IL-2-activated human
peripheral lymphocytes directed by certain
anti-CD3/anti-tumor-specific heteroantibody conjugates have also
been reported to prevent the growth of human cancer xenografts in
nude mice. Studies in vitro, and in vivo in immunodeficient mice
bearing human xenografts have reported that certain bispecific
antibodies are capable of blocking the growth of both tumor cells
bearing certain target antigens and, to some extend, bystander
tumor cells that are not recognized by the therapeutic
antibody.
[0006] The cell membranes of lymphocytes are uniquely constructed
and determine such diverse cellular phenotypic characteristics as
the suppressor, inducer, or cytolytic function of the cell, the
state of activation or stage of differentiation of the cell, and
whether the cell belongs to a population that is monoclonal or
polyclonal. The vast majority of cellular membrane antigens thus
far described on malignant lymphocytes are represented on
nonmalignant lymphocytes at some stage of differentiation or
activation.
[0007] From the foregoing, it is apparent that a need exists for
therapeutic agents that are targeted to an antigen found
predominantly or exclusively on malignant cells, and which are
capable of inducing strong cytolytic activity against such cells.
The present invention fulfills this and other needs.
SUMMARY OF THE INVENTION
[0008] The present invention is premised on the realization that a
bispecific monoclonal antibody which binds to malignant B-cell
lymphomas and to T cells can be formed which effectively binds only
to malignant B-cells and does not bind to normal B-cells.
[0009] Further, the present invention is premised on the
realization that a bispecific antibody can be formed from a cell
line obtained from peripherally diffuse large cell lymphoma to
produce a monoclonal antibody that is specific only to malignant
B-cells and that this monoclonal antibody can be modified to form a
bispecific antibody which also binds to killer T cells or NK
cells.
[0010] The present invention is further premised on the realization
that a cell line formed from a fusion of cell lines which produces
an IgG antibody specific to the T cells or NK cells and a cell line
which produces the IgG antibody specific to B-cell malignancies in
turn produce a unique bispecific antibody that effectively binds to
both malignant B-cells and T cells or NK cells thereby effectuating
the lysis or destruction of the malignant B-cells.
[0011] In the preferred embodiment the cell line is derived from
the fusion of a cell line producing an antibody specific to the CD3
antigen of the T cell in combination with a cell line specific to a
heterodimer on the cell membrane of the malignant B-cells as
explained further below.
[0012] In a further aspect, the invention provides the 1D10
antibody, which is specific for the 28/32 kDa heterodimeric protein
on the surface of malignant B-cells.
[0013] The invention further provides a humanized version of the
1D10 antibody. The humanized antibody comprises a humanized heavy
chain and a humanized light chain. The humanized light chain
comprises three complementarity determining regions (CDR1, CDR2 and
CDR3) having amino acid sequences from the corresponding
complementarity determining regions of the 1D10 immunoglobulin
light chain, and a variable region framework from a human kappa
light chain variable region framework sequence except in at least
one position selected from a first group consisting of L48, L49,
L69, and L70 wherein the amino acid position is occupied by the
same amino acid present in the equivalent position of the 1D10
immunoglobulin light chain variable region framework. The humanized
heavy chain comprising three complementarity determining regions
(CDR1, CDR2 and CDR3) having amino acid sequences from the
corresponding complementarity determining regions of 1D10
immunoglobulin heavy chain, and a variable region framework from a
human heavy chain variable region framework sequence except in at
least one position selected from a second group consisting of H27,
H29, H30, H37, H67, H71, H78 and H83, wherein the amino acid
position is occupied by the same amino acid present in the
equivalent position of the mouse 1D10 immunoglobulin heavy chain
variable region framework. The humanized antibody specifically
binds the 28/32 kDa heterodimeric protein cells with a binding
affinity having a lower limit of about 10.sup.7 M.sup.-1 and an
upper limit of about five-times the binding affinity of the 1D10
immunoglobulin. Preferably, the humanized light chain variable
region framework is from the R3.5H5G antibody. In this case,
position L43 can be substituted with the amino acid present in the
equivalent position of a human kappa subgroup I consensus sequence.
Preferably, the humanized heavy chain is from the heavy chain
region variable framework of the IC4 antibody. In this case,
position H73 can be substituted by the same amino acid present in
the equivalent position of a human immunoglobulin subgroup II or IV
consensus sequence.
[0014] In a further aspect, the invention provides humanized
antibodies specific for the CD3 antigen. The antibodies comprise
humanized heavy and light chains. The humanized light chain
comprises three complementarity determining regions (CDR1, CDR2 and
CDR3) having amino acid sequences from the corresponding
complementarity determining regions of the M291 immunoglobulin
light chain, and a variable region framework from a human kappa
light chain variable region framework sequence. The humanized heavy
chain comprises three complementarity determining regions (CDR1,
CDR2 and CDR3) having amino acid sequences from the corresponding
complementarity determining regions of M291 immunoglobulin heavy
chain, and a variable region framework from a human heavy chain
variable region framework sequence except in at least one position
selected from a second group consisting of H30, H67, H68, H70, H72
and H74 wherein the amino acid position is occupied by the same
amino acid present in the equivalent position of the mouse M291
immunoglobulin heavy chain variable region framework. The
immunoglobulin specifically binds to a CD3 antigen on the surface
of T cells with a binding affinity having a lower limit of about
10.sup.7 M.sup.-1 and an upper limit of about five-times the
binding affinity of the M291 immunoglobulin. Preferably, the
humanized light chain variable region framework is from the light
chain variable region framework of the HF2-1/17 antibody in
subgroup I. Preferably, the humanized heavy chain region framework
is from the heavy chain region variable framework of the 21/28
antibody. In this case, position H44 can be substituted with the
same amino acid present in the equivalent position of a human
immunoglobulin subgroup I consensus sequence.
[0015] In a further aspect, the invention provides humanized
bispecific antibodies comprising a first binding fragment that
specifically binds to the CD3 antigen and a second binding fragment
that specifically binds to the 28/32 kDa heterodimeric antigen on
the surface of the malignant B cells. The first binding fragment
comprises a humanized form of the heavy chain variable region of
the M291 antibody and a humanized form of the light chain variable
region of the M291 antibody. The second binding fragment, which is
linked to the first binding fragment, comprising: a humanized form
of the heavy chain variable region from the 1D10 antibody and a
humanized form of the light chain variable region from the 1D10
antibody.
[0016] Preferably, the first and second binding fragments each
further comprises a segment of a constant region fused to the
respective heavy chain variable regions, and the binding fragments
are linked by association of the constant regions. For, example,
the binding fragments can be Fab or Fab'. When both binding
fragments are Fab', the bispecific antibody is a F(ab').sub.2.
Optionally, the first and second binding fragments further comprise
first and second leucine zippers fused to the respective constant
regions.
[0017] The invention further provides pharmaceutical compositions
comprising the antibodies described above. Also provided are
methods of treating patients suffering from malignant B-cells
employing a therapeutically effective amount of bispecific antibody
as described above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a graph depicting lysis of malignant B-cells by
the bispecific antibody of the present invention;
[0019] FIG. 2 is a graph depicting lysis of Raji cells caused by
different concentrations of the antibody of the present
invention;
[0020] FIG. 3 is a graph depicting lysis of KH cells over a period
of time by the bispecific antibody of the present invention also
depicting a comparative study.
[0021] FIG. 4. Amino acid sequences of the light chain (A) (SEQ ID
NOS. 1 and 2) and the heavy chain (B) (SEQ ID NOS. 3 and 4)
variable regions of the humanized 1D10 antibody (upper lines) and
mouse 1D10 antibody (lower lines), not including the signal
sequences. The three CDRs in each chain are underlined Residues in
the human framework that have been replaced with mouse amino acids
or consensus human amino acids are doubly underlined. Amino acid
sequences of the complete light chain and the heavy chain of the
humanized 1D10 are showed in (C) (SEQ ID NO. 5) and (E) (SEQ ID NO.
7), respectively. The V.sub.L domain consists of residues 1-107,
and the C.sub.K 108-214. The V.sub.H domain consists of residues
1-116, the C.sub.H 1 117-214, the hinge 215-229, the C.sub.H 2
230-339, and the C.sub.H 3 domain 340-446. Amino acid sequence of
the Fd-Jun in the humanized F(ab'-zipper).sub.2 of 1D10 is shown in
(D) (SEQ ID NO. 6). The V.sub.H domain consists of residues 1-116,
the C.sub.H 1 domain 117-214, the modified hinge 215-234, and the
Fos leucine zipper 235-273.
[0022] FIG. 5. Amino acid sequences of the light chain (A) (SEQ ID
NOS. 8 and 9) and the heavy chain (B) (SEQ ID NOS. 10 and 11)
variable regions of the humanized M291 antibody (upper lines) and
mouse M291 antibody (lower lines), not including the signal
sequences. The three CDRs in each chain are underlined. Residues in
the human framework that have been replaced with mouse amino acids
or consensus human amino acids are doubly underlined. Amino acid
sequences of the complete light chain of the humanized M291 are
showed in (C) (SEQ ID NO. 12). The V.sub.L domain consists of
residues 1-106, and the human C.sub.K domain 107-213. Amino acid
sequence of the Fd-Fos in the humanized F(ab'-zipper)2 of M291 is
shown in (D) (SEQ ID NO. 13). The V.sub.H domain consists of
residues 1-120, the C.sub.H 1 domain 121-218, the modified hinge
219-238, and the Fos leucine zipper 239-279.
[0023] FIG. 6. Construction of the plasmid pHu1D10.IgG1.rG.dE used
for the expression of the humanized 1D10 IgG1.
[0024] FIG. 7. (A). Displacement assay to compare the relative
affinity of humanized 1D10 and murine 1D10 for the antigen.
Subsaturation amounts of murine 1D10-IgG2a-FITC on Raji cells were
displaced by increasing amounts of murine 1D10-IgG2a or humanized
1D10-IgG1. Raji cells were resuspended in complete media at
2.5.times.10.sup.6/ml. Dilutions of the test (humanized 1D10-IgG1)
or control (murine 1D10-IgG2a) antibody were added and incubated at
4.degree. C. for 1 hour. A fixed, subsaturation amount of murine
1D10-IgG2a-FITC was added, and the cells were incubated at
4.degree. C. for 1 hour, washed, and resuspended in 1%
paraformaldehyde. The cells were then analyzed using flow
cytometry. Values expressed in % inhibition of fluorescence
intensity compared to no competitive antibody control. (B).
Scatchard plot analysis of the binding of .sup.125I-labeled
humanized 1D10-IgG1 to Raji cells. Scatchard analysis was made by
binding dilutions of labeled antibody to 4.times.10.sup.5 Raji
cells in 0.2 ml for 90 min at 0.degree. C. The cells were washed in
binding buffer (2% horse serum in PBS containing 0.1% sodium azide)
and counted. Nonspecific binding was determined by inhibiting the
specific binding with an excess of nonlabeled humanized 1D10-IgG1.
The apparent Ka and the number of binding sites were calculated
from the slope and the X axis intercept, respectively, of the
Scatchard plot.
[0025] FIG. 8. (A). Antibody-dependent cell-mediated cytotoxicity
(ADCC) capability by various 1D10 isotypes. .sup.51Cr-labeled Raji
human lymphoma cells were used as targets for (.tangle-solidup.)
murine 1D10-IgG1, (.circle-solid.) murine 1D10-IgG2a, or
(.box-solid.) humanized 1D10-IgG1 and human peripheral mononuclear
as effector cells. The effector:target ratio was 40:1. Spontaneous
release was less than 20% of total release. Bars represent SEM.
(B). Complement-mediated cytotoxicity by various 1D10 isotypes.
.sup.51Cr-labeled Raji human lymphoma cells were used as targets
for (.tangle-solidup.) murine 1D10-IgG1, (.circle-solid.) murine
1D10-IgG2a, or (.box-solid.) humanized 1D10-IgG1 and human sera
from a normal subject as complement. Spontaneous release was less
than 20% of total release. Bars represent SEM.
[0026] FIG. 9. Schematic diagrams of the plasmids pHu1D10-Jun.rG.dE
and pHuM291-Fos.rG.dE for the expression of Hu1D10-Jun and
HuM291-Fos F(ab'-zipper).sub.2. The constructions of these two
plasmids were similar to that of pHu1D10.IgG1 in FIG. 6 except for
the replacement of the C.sub.H2 and C.sub.H3 exons by the leucine
zipper sequences Jun and Fos. The polyadenylation signal for the
Fd-zipper transcript is from the 3' noncoding sequence of mouse
IgG2a gene (see Kostelny et al., J. Immunol. 148, 1547 (1992)).
[0027] FIG. 10. (A). The sequence of the modified human IgG1 hinge
used in the hinge-zipper fusion. Two residues Lys-Cys (underlined)
were inserted in the modified hinge. The fist Cys in this modified
hinge forms disulfide bond with the light chain, and the last three
Cys residues form inter-heavy chain disulfides. For comparison,
hinge sequences of the human IgG1 (B) and the mouse IgG2a (C) are
also shown. All three Cys residues in the mouse IgG2a hinge are
used for inter-heavy chain disulfides. After the insertion of
Lys-Cys, the modified hinge and the mouse IgG2a hinge have
extensive sequence homology near the COOH-terminus.
[0028] FIG. 11. (A). Displacement assay to compare the relative
affinity of HuM291-Fos and M291 for their antigen. Subsaturation
amounts of murine M291-FITC on human T cells were displaced by
increasing amounts of murine M291 or HuM291-Fos. T cells were
resuspended in complete media at 2.5.times.10.sup.6/ml. Dilutions
of the test (HuM291-Fos) or control (murine M291) antibody were
added and incubated at 4.degree. C. for 1 hour. A fixed,
subsaturation amount of murine M291-FITC was added, and the cells
were incubated at 4.degree. C. for 1 hour, washed, and resuspended
in 1% paraformaldehyde. The cells were then analyzed using flow
cytometry. Values expressed in % inhibition of fluorescence
intensity compared to no competitive antibody control. (B).
Scatchard plot analysis of the binding of .sup.125I-labeled
HuM291-Fos to activated human T cells. Scatchard analysis was made
by binding dilutions of labeled antibody to 4.times.10.sup.5 T
cells in 0.2 ml for 90 min at 0.degree. C. The cells were washed in
binding buffer (2% horse serum in PBS containing 0.1% sodium azide)
and counted. Nonspecific binding was determined by inhibiting the
specific binding with an excess of nonlabeled HuM291-Fos. The
apparent K.sub.a and the number of binding sites were calculated
from the slope and the X axis intercept, respectively, of the
Scatchard plot.
[0029] FIG. 12. Bispecific antibody induced T cell mediated lysis
of 1D10 positive cells. T cells in human PBL were activated by
anti-CD3 antibody OKT3 and expanded by culturing them in IL-2.
Target cells were labeled with .sup.51Cr and washed. T cells and
labeled target cells at effector: target ratio of 25:1 were plated
in V bottom microtiter plates. Antibodies at desired concentration
were added. Antibodies used were: Hu1D10-Jun, HuM291-Fos, the mouse
bispecific IgG 1DT3-D, and the humanized bispecific
F(ab'-zipper).sub.2 Hu1D10-Jun.times.HuM291-Fos. Plates were
incubated at 37.degree. C. for 4 hours, centrifuged, and target
cell lysis was measured by determining the amount of .sup.51Cr
released. Percentages of specific release in this cytotoxicity
assay were calculated as: {Counts released by antibody minus counts
released without added antibody}/{Counts released by 0.1% SDS minus
counts released without added antibody}.times.100.
DEFINITIONS
[0030] The term "substantial identity" or "substantial homology"
means that two peptide sequences, when optimally aligned, such as
by the programs GAP or BESTFIT using default gap weights, share at
least 65 percent sequence identity, preferably at least 80 or 90
percent sequence identity, more preferably at least 95 percent
sequence identity or more (e.g., 99 percent sequence identity).
Preferably, residue positions which are not identical differ by
conservative amino acid substitutions.
[0031] For purposes of classifying amino acids substitutions as
conservative or nonconservative, amino acids are grouped as
follows: Group I (hydrophobic sidechains): norleucine, met, ala,
val, leu, ile; Group II (neutral hydrophilic side chains): cys,
ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic
side chains): asn, gln, his, lys, arg; Group V (residues
influencing chain orientation): gly, pro; and Group VI (aromatic
side chains): trp, tyr, phe. Conservative substitutions involve
substitutions between amino acids in the same class.
Non-conservative substitutions constitute exchanging a member of
one of these classes for a member of another.
[0032] Amino acids from the variable regions of the mature heavy
and light chains of immunoglobulins are designated Hx and Lx
respectively, where x is a number designating the position of an
amino acids according to the scheme of Kabat, Sequences of Proteins
of Immunological Interest (National Institutes of Health, Bethesda,
Md., 1987 and 1991). Kabat lists many amino acid sequences for
antibodies for each subclass, and lists the most commonly occurring
amino acid for each residue position in that subclass. Kabat uses a
method for assigning a residue number to each amino acid in a
listed sequence, and this method for assigning residue numbers has
become standard in the field. Kabat's scheme is extendible to other
antibodies not included in his compendium by aligning the antibody
in question with one of the consensus sequences in Kabat. The use
of the Kabat numbering system readily identifies amino acids at
equivalent positions in different antibodies. For example, an amino
acid at the L50 position of a human antibody occupies the
equivalent position to an amino acid position L50 of a mouse
antibody.
[0033] From N-terminal to C-terminal, both light and heavy chains
comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The
assignment of amino acids to each domain is in accordance with the
definitions of Kabat (1987) and (1991), supra, or Chothia &
Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature
342:878-883 (1989).
[0034] The basic antibody structural unit is known to comprise a
tetramer. Each tetramer is composed of two identical pairs of
polypeptide chains, each pair having one "light" (about 25 kDa) and
one "heavy" chain (about 50-70 kDa). The amino-terminal portion of
each chain includes a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
carboxy-terminal portion of each chain defines a constant region
primarily responsible for effector function. The variable regions
of each light/heavy chain pair form the antibody binding site.
Thus, an intact antibody has two binding sites.
[0035] Light chains are classified as either kappa or lambda. Heavy
chains are classified as gamma, mu, alpha, delta, or epsilon, and
define the antibody's isotype as IgG, IgM, IgA, IgD and IgE,
respectively. Within light and heavy chains, the variable and
constant regions are joined by a "J" region of about 12 or more
amino acids, with the heavy chain also including a "D" region of
about 10 more amino acids. (See generally, Fundamental Immunology
(Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989), Ch. 7
(incorporated by reference in its entirety for all purposes).
[0036] The term epitope includes any protein determinant capable of
specific binding to an immunoglobulin or T-cell receptor. Epitopic
determinants usually consist of chemically active surface groupings
of molecules such as amino acids or sugar side chains and usually
have specific three dimensional structural characteristics, as well
as specific charge characteristics.
[0037] The term patient includes human and veterinary subjects.
DETAILED DESCRIPTION
[0038] The present invention provides bispecific antibodies, which
are specific to both effector cells (T cells or natural killer
cells) and to a 28/32 kDa heterodimeric antigen present on the
surface of malignant B-cells. The present invention further
provides hybridomas and other cells lines producing the claimed
antibodies.
[0039] The 28/32 kDa antigen is found predominantly on the surface
of malignant B lymphocytes and is not expressed on resting
lymphocytes or B and T cells activated in vitro by a variety of
inductive stimuli. See Gingrich et al., Blood 75, 2375-2387 (1990).
The antigen can be expressed when lymphocytes undergo malignant
transformation or, in some cases, when they are perturbed by the
Epstein-Barr virus (EBV). Normal resting and stimulated lymphocytes
do not express the antigen. The antigen is also absent on
hemopoietic stem cells. Although the scientific basis for the 28/32
kDa antigen being expressed predominantly or exclusively on
malignant B-cells is not critical to the practice of the invention,
it is believed that the antigen may represent an aberrant
post-translational processing variant of the HLA-Dr antigen.
[0040] To produce the antibodies specific to malignant B-cells, a
lymphoma cell line derived from a patient with peripheralizing
diffuse large cell lymphoma labeled HO-85 was grown in suspension
culture RPMI 1640 with 10% fetal calf serum with a doubling time of
approximately 24 hours. The cell line is CD20, mu, delta (weakly),
kappa, HLA Class I and II antigen positive. It does not react with
monoclonal antibodies detecting CALLA, T cell, myeloid or monocytic
cell antigens. The cells react with the SFR7, DR7 and B7/21
monoclonal antibodies indicating that they express DR7 and DP
antigens respectively.
[0041] Female BALB/c mice, age 6-10 weeks, were given 4 to 6
intraperitoneal inoculations at two week intervals with
5.times.10.sup.6 cells from the human large cell lymphoma line as
described above. The animals were killed five days after last
inoculation and the spleen cells were fused with the nonsecretory
murine myeloma cell line N-1. Hybridomas were selected in
hypoxanthine-aminopterin-thymidine (HAT) medium after being plated
in 96 well cell culture trays. After 10 days, 25 microliter
aliquots were taken from each well for determination of malignant
B-cell (anti-HO-85) antibody binding activity.
[0042] Malignant B-cell (anti-HO-85) antibody binding activity was
determined by a whole cell, indirect radio immune assay using fresh
HO-85 cells as targets. The identical assay was done using as
targets RAJI (ATCC CCL86), MOLT-3 (ATCC CRL1582), HL-60 (ATCC
CCL240) and fresh peripheral blood mononuclear cells. Wells that
showed binding activity greater than 5 times that of tissue culture
medium alone to HO-85 and Raji but were not reactive with MOLT-3,
HL-60 and peripheral blood mononuclear cells were harvested.
[0043] Cells meeting the above criteria were found to produce an
antibody referred to as 1D10 and were subsequently cloned by
limiting dilution. The hybridoma grows well in vitro and ascites of
pristine-primed BALB/c mice.
[0044] The portion of the malignant B-cells to which 1D10 binds is
a heterodimeric polypeptide which contains two proteins with a
molecular weight of the alpha and beta chains being 32 kDa and 28
kDa respectively. The proteins can be obtained by solubilizing
malignant B-cells such as Raji cells with detergent. Molecular
weight determination is made by using iodinated cells and single
dimension SDS-PAGE analysis of the MoAb6-antigen precipitate. The
formation of the 1D10 antibody is discussed by Gingrich et al.,
Blood 75, 2375-2387 (1990). Other antibodies having the same or
similar binding specificity to 1D10 are screened by competition
binding with 1D10 to the 28/32 kDa heterodimeric antigen. Numerous
types of competitive binding assays are known, for example: solid
phase direct or indirect radioimmunoassay (RIA), solid phase direct
or indirect enzyme immunoassay (EIA), sandwich competition assay
(see Stahli et al., Methods in Enzymology 9, 242-253 (1983)); solid
phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol.
137, 3614-3619 (1986)); solid phase direct labeled assay, solid
phase direct labeled sandwich assay (see Harlow & Lane,
"Antibodies, A Laboratory Manual," Cold Spring Harbor Press
(1988)); solid phase direct label RIA using I-125 label (see Morel
et al., Molec. Immunol. 25, 7-15 (1988)); solid phase direct
biotin-avidin EIA (Cheung et al., Virology 176, 546-552 (1990));
and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol. 32,
77-82 (1990)). Typically, such an assay involves the use of cells
bearing the 28/32 kDa antigen, an unlabelled test immunoglobulin
and a labelled reference immunoglobulin (1D10). Competitive
inhibition is measured by determining the amount of label bound to
the cells in the presence of the test immunoglobulin. Usually the
test immunoglobulin is present in excess. Antibodies identified by
competition assay (competing antibodies) include antibodies binding
to the same epitope as the reference antibody and antibodies
binding to an adjacent epitope sufficiently proximal to the epitope
bound by the reference antibody for steric hindrance to occur.
[0045] The second component for the bispecific antibodies of the
invention is an antibody having specificity for an antigen on the
surface of T-cells or NK cells. Human T-cell antigens likely to be
suitable include CD3, CD2, CD28, CD44, C69, A13 and G1. Suitable
antigens on natural killer cells include FC Gamma receptors (3G8,
B73.1, LEUL1, VEP13, and AT10). Human T-cell antigens that are
probably unsuitable include MHC Class 1, CD4, CD8, CD18 and
CD71.
[0046] Cell lines producing IgG specific to the effector cell
antigens described above are commercially available or can be
produced de novo (see Example 3). The OKT3 cell (ATCC CRL 8001) is
a suitable source of antibodies for the CD3 antigen. Other
antibodies to the CD3 antigen include WT31, WT32, anti-leu-4,
UCHT-1, SPV-3TA and SPV-T3B. The CD3 site is preferred because of
its presence in all T cells.
[0047] The antibodies of the present invention can be produced by a
cell line formed by the fusion of a first component cell line
producing antibodies specific for the 28/32 kDa heterodimeric
antigen with a second cell line which produces an antibody specific
for either T cells or natural killer cells. For example, the
hybridoma producing 1D10 was fused with OKT3 as follows.
[0048] The OKT3 hybridoma cell line was selected by growing OKT3
cells sequentially in media containing 0.13 mM 8-azaguanine, then
1.0 mM ouabain. Hybrid-hybridomas were produced by fusion (using
38% polyethylene glycol) of 10.sup.6 HAT resistant, ouabain
sensitive, 1D10-secreting hybridomas with 106 HAT sensitive,
ouabain resistant OKT3-secreting hybridomas.
[0049] Fused cells were plated in HAT-ouabain media to select for
hybrid-hybridomas. The HAT in this media prevented the growth of
unfused OKT3 cells and the ouabain prevented the growth of unfused
1D10 cells. Thus, only hybrid-hybridomas containing genetic
material from both parental hybridomas survived. Twelve
hybrid-hybridomas were isolated using this technique.
[0050] Cell lines secreting bispecific antibodies can be identified
by a three-step screening procedure. For example, in analysis of
hybridomas formed from fusion of 1D10 and OKT3, an initial screen
was performed in which hybrid-hybridoma supernatant was added to
ELISA plates coated with goat anti-mouse IgG1 antibody. After
washing, alkaline phosphatase labeled goat anti-mouse IgG2a was
added. Reactivity indicated the hybrid-hybridoma supernatant
contained single antibody molecules with both IgG1 and IgG2a heavy
chains.
[0051] An indirect immunofluorescent assay was used as a second
screen for all samples that were positive on ELISA. In this second
screen, hybrid-hybridoma supernatant was added separately to HO-85
(1D10 reactive) and Jurkat (OKT3 reactive) cells. Goat anti-mouse
IgG-FITC was added after washing to detect the presence of bound
antibody. All twelve hybrid-hybridomas secreted antibody which was
capable of binding to both HO-85 and Jurkat cells. One of these
hybrid-hybridomas was selected for further study. It was subcloned
by limiting dilution .times.2, and designated 1DT3-D. This cell
line was deposited on Mar. 24, 1992 under the Budapest Treat at the
American Type Culture collection, 12301 Parklawn Drive, Rockville,
Md. 20852 and assigned the number ATCC HB 10993.
[0052] IDT3-D was cultured in vitro in HB 101 media supplemented
with 100 .mu.g L-glutamine and 100 U/ml penicillin-streptomycin.
These cells were transferred to a Mini Flo-path Bioreactor hollow
fiber apparatus. Antibody obtained from spent media was
fractionated by HPLC cation exchange using a gradient of 0.18 to
0.5M NaCl. The peak containing bispecific reactivity, as
demonstrated by the above assays, was isolated, dialyzed against
phosphate buffered saline, concentrated and used in further
studies.
[0053] The bispecific antibody formed by fusion of 1D10 and OKT3 is
a mouse derived monoclonal. Humanized versions of this antibody and
other bispecific antibodies of the invention can also be employed
as discussed in more detail below.
Humanized Antibodies
[0054] The invention further provides humanized immunoglobulins (or
antibodies). Some humanized antibodies are specific for the T-cell
antigen CD3. Other humanized antibodies are specific for the 28/32
kDa heterodimer on malignant B-cells. These humanized antibodies
are useful as therapeutic and diagnostic reagents in their own
right or can be combined to form a humanized bispecific antibody
possessing both of the binding specificities of its components. The
humanized forms of immunoglobulins have variable framework
region(s) substantially from a human immunoglobulin (termed an
acceptor immunoglobulin) and complementarity determining regions
substantially from a mouse immunoglobulin (referred to as the donor
immunoglobulin). The constant region(s), if present, are also
substantially from a human immunoglobulin. The humanized antibodies
exhibit a specific binding affinity for their respective antigens
of at least 10.sup.7, 10.sup.8, 10.sup.9, or 10.sup.10 M.sup.-1.
Often the upper and lower limits of binding affinity of the
humanized antibodies are within a factor of three or five or ten of
that of the mouse antibody from which they were derived.
[0055] (1) Mouse Antibodies for Humanization
[0056] The starting material for production of humanized antibody
specific for the 28/32 kDa heterodimer is preferably the 1D10 mouse
antibody, although other mouse antibodies, which compete with 1D10
for binding to the 28/32 kDa heterodimer can also be used. A
suitable starting material for production of humanized antibody
specific for CD3 is the M291 antibody whose isolation is described
in Example 3.
[0057] (2) Selection of Human Antibodies to Supply Framework
Residues
[0058] The substitution of mouse CDRs into a human variable domain
framework is most likely to result in retention of their correct
spatial orientation if the human variable domain framework adopts
the same or similar conformation to the mouse variable framework
from which the CDRs originated. This is achieved by obtaining the
human variable domains from human antibodies whose framework
sequences exhibit a high degree of sequence identity with the
murine variable framework domains from which the CDRs were derived.
The heavy and light chain variable framework regions can be derived
from the same or different human antibody sequences. The human
antibody sequences can be the sequences of naturally occurring
human antibodies or can be consensus sequences of several human
antibodies.
[0059] Suitable human antibody sequences are identified by computer
comparisons of the amino acid sequences of the mouse variable
regions with the sequences of known human antibodies. The
comparison is performed separately for heavy and light chains but
the principles are similar for each.
[0060] (3) Computer Modelling
[0061] The unnatural juxtaposition of murine CDR regions with human
variable framework region can result in unnatural conformational
restraints, which, unless corrected by substitution of certain
amino acid residues, lead to loss of binding affinity. The
selection of amino acid residues for substitution is determined, in
part, by computer modelling. Computer hardware and software for
producing three-dimensional images of immunoglobulin molecules are
widely available. In general, molecular models are produced
starting from solved structures for immunoglobulin chains or
domains thereof. The chains to be modelled are compared for amino
acid sequence similarity with chains or domains of solved three
dimensional structures, and the chains or domains showing the
greatest sequence similarity is/are selected as starting points for
construction of the molecular model. The solved starting structures
are modified to allow for differences between the actual amino
acids in the immunoglobulin chains or domains being modelled, and
those in the starting structure. The modified structures are then
assembled into a composite immunoglobulin. Finally, the model is
refined by energy minimization and by verifying that all atoms are
within appropriate distances from one another and that bond lengths
and angles are within chemically acceptable limits.
[0062] (4) Substitution of Amino Acid Residues
[0063] As noted supra, the humanized antibodies of the invention
comprise variable framework region(s) substantially from a human
immunoglobulin and complementarity determining regions
substantially from a mouse immunoglobulin (e.g., 1D10 or M291).
Having identified the complementarity determining regions of mouse
antibodies and appropriate human acceptor immunoglobulins, the next
step is to determine which, if any, residues from these components
should be substituted to optimize the properties of the resulting
humanized antibody. In general, substitution of human amino acid
residues with murine should be minimized, because introduction of
murine residues increases the risk of the antibody eliciting a HAMA
response in humans. Amino acids are selected for substitution based
on their possible influence on CDR conformation and/or binding to
antigen. Investigation of such possible influences is by modelling,
examination of the characteristics of the amino acids at particular
locations, or empirical observation of the effects of substitution
or mutagenesis of particular amino acids.
[0064] When an amino acid differs between a mouse variable
framework region and an equivalent human variable framework region,
the human framework amino acid should usually be substituted by the
equivalent mouse amino acid if it is reasonably expected that the
amino acid:
[0065] (1) noncovalently contacts antigen directly, or
[0066] (2) is adjacent to a CDR region or otherwise interacts with
a CDR region (e.g., is within about 4-6 .ANG. of a CDR region).
[0067] Other candidates for substitution are acceptor human
framework amino acids that are unusual for a human immunoglobulin
at that position. These amino acids can be substituted with amino
acids from the equivalent position of more typical human
immunoglobulins. Alternatively, amino acids from equivalent
positions in the mouse antibody can be introduced into the human
framework regions when such amino acids are typical of human
immunoglobulin at the equivalent positions.
[0068] In general, substitution of all or most of the amino acids
fulfilling the above criteria is desirable. Occasionally, however,
there is some ambiguity about whether a particular amino acid meets
the above criteria, and alternative variant immunoglobulins are
produced, one of which has that particular substitution, the other
of which does not.
[0069] The humanized antibodies of the invention that are derived
from the mouse 1D10 antibody usually contain a substitution of a
human kappa light chain framework residue with a corresponding mu
MAb 1D10 residue in at least 1, 2, 3 or 4 of the following
positions: L48, L49, L69 and L70. The humanized antibodies also
usually contain a substitution of a human heavy chain framework
residue in at least 1, 2, 3, 4, 5, 6, 7, or 8 of the following
positions H27, H29, H30, H37, H67, H71, H78 and H83. In preferred
embodiments when the human light chain acceptor immunoglobulin is
R3.5HG, the light chain also contains a substitution at position
43. This position is substituted with the amino acid from the
equivalent position of a human immunoglobulin having a more typical
amino acid residues or from a consensus sequence of such human
immunoglobulins. Similarly, when the human heavy chain acceptor
immunoglobulin is IC4, the heavy chain also contains a substitution
at position 73.
[0070] The humanized antibodies of the invention that are derived
from mouse M291 antibody contain no substitution of a human kappa
light chain framework residue if the light chain acceptor is
HF2-1/17. The humanized antibodies also usually contain a
substitution of a human heavy chain framework in at least 1, 2, 3,
4, 5 and 6 of the following positions: H30, H67, H68, H70, H72 and
H74. In preferred embodiments, when the heavy chain acceptor
immunoglobulin is 21/28, the light chain also contains a
substitution at position 44. This position is substituted with the
amino acid from the equivalent position of a human immunoglobulin
having a more typical amino acid residue or from a consensus
sequence of such human immunoglobulin.
[0071] Usually the CDR regions in humanized antibodies are
substantially identical, and more usually, identical to the
corresponding CDR regions in the mouse antibody from which they
were derived. Although not usually desirable, it is sometimes
possible to make one or more conservative amino acid substitutions
of CDR residues without appreciably affecting the binding affinity
of the resulting humanized immunoglobulin. Occasionally,
substitutions of CDR regions can enhance binding affinity.
[0072] Other than for the specific amino acid substitutions
discussed above, the framework regions of humanized immunoglobulins
are usually substantially identical, and more usually, identical to
the framework regions of the human antibodies from which they were
derived. Of course, many of the amino acids in the framework region
make little or no direct contribution to the specificity or
affinity of an antibody. Thus, many individual conservative
substitutions of framework residues can be tolerated without
appreciable change of the specificity or affinity of the resulting
humanized immunoglobulin. However, in general, such substitutions
are undesirable.
[0073] (5) Production of Variable Regions
[0074] Having conceptually selected the CDR and framework
components of humanized immunoglobulins, a variety of methods are
available for producing such immunoglobulins. Because of the
degeneracy of the code, a variety of nucleic acid sequences will
encode each immunoglobulin amino acid sequence. The desired nucleic
acid sequences can be produced by de novo solid-phase DNA synthesis
or by PCR mutagenesis of an earlier prepared variant of the desired
polynucleotide. All nucleic acids encoding the antibodies described
in this application are expressly included in the invention.
[0075] (6) Selection of Constant Region
[0076] The variable segments of humanized antibodies produced as
described supra are typically linked to at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. Human constant region DNA sequences can be isolated
in accordance with well-known procedures from a variety of human
cells, but preferably immortalized B-cells (see Kabat et al.,
supra, and WO87/02671). Ordinarily, the antibody will contain both
light chain and heavy chain constant regions. The heavy chain
constant region usually includes CH1, hinge, CH2, CH3, and,
sometimes, CH4 regions.
[0077] The humanized antibodies include antibodies having all types
of constant regions, including IgM, IgG, IgD, IgA and IgE, and any
isotype, including IgG1, IgG2, IgG3 and IgG4. When it is desired
that the humanized antibody exhibit cytotoxic activity, the
constant domain is usually a complement-fixing constant domain and
the class is typically IgG.sub.1. When such cytotoxic activity is
not desirable, the constant domain may be of the IgG.sub.2 class.
The humanized antibody may comprise sequences from more than one
class or isotype.
[0078] (7) Expression Systems
[0079] Nucleic acids encoding humanized light and heavy chain
variable regions, optionally linked to constant regions, are
inserted into expression vectors. The light and heavy chains can be
cloned in the same or different expression vectors. The DNA
segments encoding immunoglobulin chains are operably linked to
control sequences in the expression vector(s) that ensure the
expression of immunoglobulin polypeptides. Such control sequences
include a signal sequence, a promoter, an enhancer, and a
transcription termination sequence (see Queen et al., Proc. Natl.
Acad. Sci. USA 86, 10029 (1989); WO 90/07861; Co et al., J.
Immunol. 148, 1149 (1992), which are incorporated herein by
reference in their entirety for all purposes).
[0080] C. Fragments of Humanized Antibodies
[0081] The humanized antibodies of the invention include fragments
as well as intact antibodies. Typically, these fragments compete
with the intact antibody from which they were derived for antigen
binding. The fragments typically bind with an affinity of at least
10.sup.7 M.sup.-1, and more typically 10.sup.8 or 10.sup.9 M.sup.-1
(i.e., within the same ranges as the intact antibody). Humanized
antibody fragments include separate heavy chains, light chains Fab,
Fab.degree. F(ab').sub.2, and Fv. Fragments are produced by
recombinant DNA techniques, or by enzymic or chemical separation of
intact immunoglobulins.
Recombinant Bispecific Antibodies
[0082] The methods discussed above for forming bispecific
antibodies from antibodies produced by hybridoma cells can also be
applied or adapted to production of bispecific antibodies from
recombinantly expressed antibodies such as the humanized versions
of 1D10 and M291. For example, bispecific antibodies can be
produced by fusion of two cell lines respectively expressing the
component antibodies. Alternatively, the component antibodies can
be co-expressed in the same cell line. Bispecific antibodies can
also be formed by chemical crosslinking of component recombinant
antibodies.
[0083] Component recombinant antibodies can also be linked
genetically. In one approach, a bispecific antibody is expressed as
a single fusion protein comprising the four different variable
domains from the two component antibodies separated by spacers. For
example, such a protein might comprise from one terminus to the
other, the VL region of the first component antibody, a spacer, the
VH domain of the first component antibody, a second spacer, the VH
domain of the second component antibody, a third spacer, and the VL
domain of the second component antibody. See, e.g., Segal et al.,
Biologic Therapy of Cancer Updates 2, 1-12 (1992).
[0084] In a further approach, bispecific antibodies are formed by
linking component antibodies to leucine zipper peptides. See
generally copending application 11823-003200 (Ser. No. 07/801,798,
filed Nov. 29, 1991; Kostelny et al., J. Immunol. 148, 1547-1553
(1992) (incorporated by reference in their entirety for all
purposes). Leucine zippers have the general structural formula
(Leucine-X.sub.1--X.sub.2--X.sub.3--X.sub.4--X.sub.5--X.sub.6).sub.n
(SEQ ID NO. 14), where X may be any of the conventional 20 amino
acids (Proteins, Structures and Molecular Principles, (1984)
Creighton (ed.), W. H. Freeman and Company, New York), but are most
likely to be amino acids with high .alpha.-helix forming potential,
for example, alanine, valine, aspartic acid, glutamic acid, and
lysine (Richardson and Richardson, Science 240, 1648 (1988)), and n
may be 3 or greater, although typically n is 4 or 5. The leucine
zipper occurs in a variety of eukaryotic DNA-binding proteins, such
as GCN4, C/EBP, c-fos gene product (Fos), c-jun gene product (Jun),
and c-myc gene product. In these proteins, the leucine zipper
creates a dimerization interface wherein proteins containing
leucine zippers may form stable homodimers and/or heterodimers.
[0085] The leucine zippers for use in the present invention
preferably have pairwise affinity. Pairwise affinity is defined as
the capacity for one species of leucine zipper, for example, the
Fos leucine zipper, to predominantly form heterodimers with another
species of leucine zipper, for example, the Jun leucine zipper,
such that heterodimer formation is preferred over homodimer
formation when two species of leucine zipper are present in
sufficient concentrations. See Schuemann et al., Nucleic Acids Res.
19, 739 (1991). Thus, predominant formation of heterodimers leads
to a dimer population that is typically 50 to 75 percent,
preferentially 75 to 85 percent, and most preferably more than 85
percent heterodimers. When amino-termini of the synthetic peptides
each include a cysteine residue to permit intermolecular disulfide
bonding, heterodimer formation occurs to the substantial exclusion
of homodimerization.
[0086] In the formation of bispecific antibodies, binding fragments
of the component antibodies are fused in-frame to first and second
leucine zippers. Suitable binding fragments including Fv, Fab,
Fab', or the heavy chain. The zippers can be linked to the heavy or
light chain of the antibody binding fragment and are usually linked
to the C-terminal end. If a constant region or a portion of a
constant region is present, the leucine zipper is preferably linked
to the constant region or portion thereof. For example, in a
Fab'-leucine zipper fusion, the zipper is usually fused to the
C-terminal end of the hinge. The inclusion of leucine zippers fused
to the respective component antibody fragments promotes formation
of heterodimeric fragments by annealing of the zippers. When the
component antibodies include portions of constant regions (e.g.,
Fab' fragments), the annealing of zippers also serves to bring the
constant regions into proximity, thereby promoting bonding of
constant regions (e.g., in a F(ab')2 fragment). Typical human
constant regions bond by the formation of two disulfide bonds
between hinge regions of the respective chains. This bonding can be
strengthened by engineering additional cysteine residue(s) into the
respective hinge regions allowing formation of additional disulfide
bonds.
[0087] Leucine zippers linked to antibody binding fragments can be
produced in various ways. For example, polynucleotide sequences
encoding a fusion protein comprising a leucine zipper can be
expressed by a cellular host or in vitro translation system.
Alternatively, leucine zippers and/or antibody binding fragments
can be produced separately, either by chemical peptide synthesis,
by expression of polynucleotide sequences encoding the desired
polypeptides, or by cleavage from other proteins containing leucine
zippers, antibodies, or macromolecular species, and subsequent
purification. Such purified polypeptides can be linked by peptide
bonds, with or without intervening spacer amino acid sequences, or
by non-peptide covalent bonds, with or without intervening spacer
molecules, the spacer molecules being either amino acids or other
non-amino acid chemical structures. Regardless of the method or
type of linkage, such linkage can be reversible. For example, a
chemically labile bond, either peptidyl or otherwise, can be
cleaved spontaneously or upon treatment with heat, electromagnetic
radiation, proteases, or chemical agents. Two examples of such
reversible linkage are: (1) a linkage comprising a Asn-Gly peptide
bond which can be cleaved by hydroxylamine, and (2) a disulfide
bond linkage which can be cleaved by reducing agents.
[0088] Component antibody fragment-leucine zippers fusion proteins
can be annealed by co-expressing both fusion proteins in the same
cell line. Alternatively, the fusion proteins can be expressed in
separate cell lines and mixed in vitro. If the component antibody
fragments include portions of a constant region (e.g., Fab'
fragments), the leucine zippers can be cleaved after annealing has
occurred. The component antibodies remain linked in the bispecific
antibody via the constant regions.
Therapeutic Methods
[0089] Pharmaceutical compositions comprising bispecific antibodies
of the present invention are useful for parenteral administration,
i.e., subcutaneously, intramuscularly and particularly,
intravenously. The compositions for parenteral administration
commonly comprise a solution of the antibody or a cocktail thereof
dissolved in an acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers can be used, e.g., water, buffered
water, 0.4% saline, 0.3% glycine and the like. These solutions are
sterile and generally free of particulate matter. The compositions
may contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions such as pH
adjusting and buffering agents, toxicity adjusting agents and the
like, for example sodium acetate, sodium chloride, potassium
chloride, calcium chloride, sodium lactate. The concentration of
the bispecific antibodies in these formulations can vary widely,
i.e., from less than about 0.01%, usually at least about 0.1% to as
much as 5% by weight and will be selected primarily based on fluid
volumes, and viscosities in accordance with the particular mode of
administration selected.
[0090] A typical composition for intravenous infusion can be made
up to contain 250 ml of sterile Ringer's solution, and 10 mg of
bispecific antibody. See Remington's Pharmaceutical Science (15th
Ed., Mack Publishing Company, Easton, Pa., 1980).
[0091] The compositions containing the present bispecific
antibodies or a cocktail thereof can be administered for
prophylactic and/or therapeutic treatments. In therapeutic
application, compositions are administered to a patient already
affected by malignant B-cells (e.g., acute lymphoblastic leukemia,
B-cell lymphoma, chronic lymphocytic leukemia and multiple myeloma)
in an amount sufficient to cure or at least partially arrest the
condition and its complications. An amount adequate to accomplish
this is defined as a "therapeutically effective dose." Amounts
effective for this use will depend upon the severity of the
condition and the general state of the patient's own immune system,
but generally range from about 0.01 to about 100 mg of bispecific
antibody per dose, with dosages of from 0.1 to 50 mg and 1 to 10 mg
per patient being more commonly used. Single or multiple
administrations on a daily, weekly or monthly schedule can be
carried out with dose levels and pattern being selected by the
treating physician.
[0092] In prophylactic applications, compositions containing the
bispecific antibodies or a cocktail thereof are administered to a
patient who is at risk of developing the disease state to enhance
the patient's resistance. Such an amount is defined to be a
"prophylactically effective dose." In this use, the precise amounts
again depend upon the patient's state of health and general level
of immunity, but generally range from 0.1 to 100 mg per dose,
especially 1 to 10 mg per patient.
[0093] In some methods of treatment, bispecific antibodies are
administered with a second agent (e.g., an interleukin) in an
amount sufficient to active effector cells thereby augmenting their
cytotoxicity to malignant B-cells compared with the administration
of bispecific antibody alone. Interleukin-2 at a dosage of about
500,000 U/kg is suitable. Combination therapy is particularly
appropriate when the bispecific antibody being administered is a
F(ab')2 fragment.
[0094] The monospecific 1D10 antibody (particularly the humanized
form) is also suitable for therapeutic administration to patients
suffering from, or at risk of, B-cell malignancies. Optionally, the
antibody is conjugated to a radiolabel or toxin. The monospecific
M291 antibody (particularly the humanized form) can be used as an
immunosuppressant in treatment of diseases and disorders of the
immune system such as host vs. graft disease, graft vs. host
disease, autoimmune diseases, and inflammation. See, e.g., Cosimi
et al., N. Engl. J. Med. 305, 308 (1981); Russel et al., Annu. Rev.
Med. 35, 63 (1984). The dosages and pharmaceutical excipients for
administration of monospecific antibodies are similar to those for
the bispecific antibodies.
Diagnostic Methods
[0095] The M291 and 1D10 antibodies (both mouse and humanized
forms) are also useful in diagnostic methods. The 1D10 antibody
(and other antibodies binding to the same or similar epitope) is
useful for diagnosing the presence of malignant B cells and
monitoring the efficacy of treatments thereto. The antibody is also
useful for research purposes to identify and type cells of certain
lineages and developmental origins. The M291 antibody is useful for
diagnostic purposes in immunologically monitoring of patients (see,
e.g., Cosimi et al., supra) and for research purposes in
classifying leukocyte subtypes, e.g., as part of an antibody panel.
Methods of diagnosis can be performed in vitro using a cellular
sample (e.g., blood sample, lymph node biopsy or tissue) from a
patient or can be performed by in vivo imaging.
EXAMPLE 1
[0096] The ability of IDT3-D to induce the elimination of malignant
B cells by T cells was evaluated in vitro. The assay used was a
.sup.51 chromium-release cytotoxicity assay. Target malignant B
cells (10.sup.7 cells in 1 ml) were labeled during a 1 hour
incubation with 100 .mu.Ci .sup.51Cr. T cells from normal donors
were incubated in vitro with IL-2 or IL-2 and anti CD3 antibody for
3-7 days before use as effector cells. T cells were added to
.sup.51Cr-labeled malignant B cells along with antibody. This
mixture was incubated for 4 hours, and cell free supernatant was
removed and evaluated for the presence of released .sup.51Cr Cr by
gamma counting. Maximum release was determined by evaluating
supernatant obtained from wells that had been treated with
detergent (NP-40) that induces the lysis of all cells. Background
release was determined by evaluating .sup.51Cr levels from samples
that had target malignant B cells and T cells but no antibody.
Specific release of .sup.51Cr indicated lysis of the
.sup.51Cr-containing target cells, and was calculated using the
following formula. Sample .times. .times. Release - Background
.times. .times. Release Maximum .times. .times. Release -
Background .times. .times. Release .times. 100 ##EQU1##
[0097] FIG. 1 shows 1DT3-D induced the lysis of a large number of
different malignant B cells including Raji (a cell line established
from a patient with Burkitt's lymphoma), HO-85 (a large cell
lymphoma cell line), 697 (a pre-B acute lymphoblastic leukemia cell
line) and KH (fresh lymphocytes obtained from a patient with
chronic lymphocytic leukemia). The T-cell target cell ratio was
10:1 and the antibody concentration was 5 .mu.g/ml. Target lysis
was not seen when monospecific antibody was used.
[0098] FIG. 2 shows 1DT3-D can induce significant lysis of raji
cells at low T cell: Raji cell ratios (less than 1:1) and at low
antibody concentrations (less than 0.1 ug/ml. Similar results were
seen with other target cell lines.
[0099] FIG. 3 shows 1DT3-D-induced T-cell-mediated lysis of fresh
KH cells was noted after long incubation times.
[0100] The bispecific antibody of the present invention can also be
produced simply by taking the Fab or F(ab').sub.2 fragments of the
1D10 antibody fusing these with portions of the OKT3 antibody to
form a bispecific antibody of the present invention. Alternatively,
bispecific antibodies, recognizing 1D10 and an antigen on natural
killer cells or T cells, can be produced, by synthetic or genetic
engineering techniques.
[0101] A benefit of the claimed bispecific antibodies is their
ability to recognize malignant B-cells and distinguish these from
non-malignant B-cells. Thus, therapy using the bispecific
antibodies of the present invention is significantly less damaging
than therapy using, for example, a non-specific antibody such as
anti-CD 19 antibody B4.
[0102] Further, as shown by the data described in the example, the
antibody of the present invention induces significant lysis of
malignant B-cells at relatively low T cell ratios. FIG. 2 shows
that malignant to T cell ratios of less than 1:1 with relatively
low antibody concentrations of less than 0.1 micrograms per ml
provides significant destruction of the malignant cells. This is
particularly important since it reduces the dependency on the
concentration of T cells available in the patient. Further, it also
reduces the amount of antibody required, thereby limiting any
potential side effects.
EXAMPLE 2
In Vivo Efficacy of 1DT3-D Bispecific Antibody
[0103] This example describes an in vivo trial of the bispecific
antibody 1DT3-D. Normal donor human peripheral blood lymphocytes
were activated in vitro in the presence of OKT3 (2 .mu.g/ml), and
recombinant IL-2 (300 .mu.g/ml). CB-17 scid/scid mice (Itoh et al.,
Cancer 72, 2686-2694 (1993)) were injected subcutaneously with
5.times.10.sup.6 Raji cells mixed with 5.times.10.sup.6 activated
lymphocytes. 24-hr later, mice were injected with bispecific
antibody, a monospecific antibody component of the bispecific
antibody or no antibody. Mice were examined daily for the
development of tumors of at least 0.5 cm at the site of tumor
injection. Mice remaining tumor-free after 60 days were scored as
negative and mice developing tumors within 60 days as positive.
Control untreated mice always developed tumors within 21-28
days.
[0104] In a first experiment, 5 mice were treated with 10
.mu.g/mouse of bispecific antibody 24 hours after inoculation with
the mixture of malignant cells and activated human T-cells. A
control group of 5 mice was inoculated with vehicle only. The tumor
occurrence (i.e., development of a tumor of at least 0.5 cm within
at least eight weeks) in the treated and control groups was as
follows: TABLE-US-00001 Group No tumors Tumors Total Treatment 4 1
5 Control 0 5 5
[0105] Using the Fischer's one-sided exact test, bispecific
antibody treatment prolonged disease-free survival with a p value
of 0.024.
[0106] A second experiment was designed to compare the anti-tumor
effects of bispecific antibody with monospecific anti-CD3 and
monospecific 1D10 at a dose of 10 .mu.g/mouse in mice inoculated
with tumor and T-cells as outlined above. Group 2 mice received
monospecific 1D10 and monospecific OKT3, Group 3 received
bispecific antibody and the control group received vehicle only.
Group 4 mice also received bispecific antibody at a concentration
of 10 .mu.g/mouse, but these mice had previously been inoculated
with unactivated T-cells as distinct from all other groups which
received activated T-cells. TABLE-US-00002 Group No tumors Tumors
Total Control 0 5 5 2 5 0 5 3 5 0 5 4 2 3 5
[0107] Fisher's exact test for general two-way tables (Agresti,
Categorical Data Analysis (Wiley, N.Y., 1990), pp. 64-65) was used
to test the null hypothesis that the occurrence rates in the four
groups are equal. There is a highly significant difference among
the groups (p=0.001). Pairwise exact tests comparing the control
group to each of groups 2, 3, and 4 were also carried out. The
corresponding one-sides p-values are 0.004, 0.004, and 0.222. Thus,
groups 2 and 3 are both significantly different from the control
group. It was concluded that at a dose of 10 .mu.g/mouse treatment
with bispecific antibody or a combination of both component
monospecific antibodies prolonged tumor-free survival.
[0108] In a third experiment, a dose-response study was performed
to test the anti-tumor effects of varying dosages of bispecific
antibody. Separate groups of mice were respectively treated with
dosages of 0.4, 2 or 10 .mu.g/mouse bispecific antibody or vehicle.
TABLE-US-00003 Group No tumors Tumors Total Control 0 5 5 0.4 1 4 5
2 5 0 5 10 4 1 5
[0109] The Cochran-Armitage trend test, (Agresti, Categorical Data
Analysis (Wiley, N.Y., 1990), pp. 100-102, 118-119) was used to
test the null hypothesis that the occurrence rates in the four
groups are equal, versus the alternative hypotheses of a linear
trend. Using equally-spaced scores, the p-value is 0.001; using the
scores 0, 0.4, 2, and 10, the p-value is 0.0164. Both sets of
scores indicate a significant trend in the proportions. These
results show that the bispecific antibody is effective to prolong
survival time and that mice receiving larger doses (10 .mu.g and 2
.mu.g) have improved tumor-free survival.
[0110] A fourth experiment was designed to compare monospecific
OKT3 and 1D10 to bispecific antibody at a dose of 2 .mu.g
antibody/mouse. TABLE-US-00004 Group No tumors Tumors Total Control
0 5 5 2 0 5 5 3 1 4 5 4 4 1 5
[0111] Tumor-free survival of mice treated with monospecific OKT3
(Group 2) and monospecific 1D10 (Group 3) was not significantly
different from control, whereas mice treated with bispecific
antibody (Group 4) had prolonged survival using the Fisher's exact
test for general two-way tables.
[0112] These data indicate that systemic administration of 1DT3-D
kills and/or prevents the development of malignant B-cells in vivo
and that a dose of 2 .mu.g/animal, bispecific antibody therapy is
more effective than monospecific antibody therapy.
EXAMPLE 3
Generation of a Monoclonal Antibody Against the Human CD3
Antigen
[0113] The 1DT3-D antibody described in Example 1 incorporated OKT3
as the binding moiety having an affinity for effector cells. The
present example describes the isolation of an alternative antibody,
M291, for use as the effector-cell binding component in a
bispecific antibody.
[0114] Human peripheral blood mononuclear cells (PBMC) were
activated with PHA and IL-2 to expand T cells. Activated T cells
were used as immunogens in Balb/C mice. Hybridomas were generated
from the spleens of these mice by standard methods. These
hybridomas were screened for antibodies that could stimulate PBMC
to proliferate in vitro. Anti-CD3 antibodies with the appropriate
Fc cause T cells in PBMC to proliferate. One of these hybridomas,
M291, was isolated and found to secrete an antibody of the isotype
IgG2a/kappa that could activate T cells to proliferate. The
purified antibody M291 competes with another anti-human CD3
antibody, OKT3, (IgG2a/kappa) for binding to human T cells, showing
that the epitopes recognized by the respective antibodies are
closely spaced. M291 is thus an antibody having the specificity
against the human CD3 complex.
EXAMPLE 4
Humanization of 1D10 and M291 Antibodies
[0115] This example describes the separate humanization procedures
for the 1D10 and M291 antibodies.
[0116] (1) Cloning of 1D10 and M291 V Region cDNAs
[0117] Heavy and light V domain cDNAs for 1D10 and M291 were cloned
using an anchored PCR method (see Loh et al., Science 243, 217
(1989)). cDNAs were first synthesized by reverse transcriptase
after priming polyA+ RNAs from the hybridoma cells with oligo dT. A
tail of dGs was added to the 3' terminus of the cDNA by terminal
deoxynucleotidyl transferase. The V domains were then amplified by
PCR with 3' primers that hybridized to the C regions and 5' primers
that hybridized to the G-tails. Several independent H and L chain
clones were sequenced to ensure no sequence mistakes were
introduced by PCR. For 1D10, the V domains were expressed as an
antibody of the mouse isotype IgG2a/kappa by transfecting the genes
in suitable vectors into the myeloma cell line SP2/0 to confirm
they coded for the binding site of 1D10. The expression vectors
used in the transfection are similar to the plasmids pVk.G and
pVg.D described by Co et al. (see Co et al., J. Immunol. 148, 1149
(1992)), except that genes for the constant regions were derived
from mouse sequences. Antibody isolated from the transfected cells
was found by flow cytometry to bind to Raji cells in a pattern
indistinguishable from that of the parent mouse IgG1/kappa 1D10
antibody. The V domains of M291 were cloned similarly and they were
expressed as mouse F(ab'-zipper).sub.2 (see Kostelny et al., J.
Immunol. 148, 1547 (1992)). Flow cytometry assay indicated M291-Fos
F(ab'zipper).sub.2 binds to human T cells with similar or identical
affinity as the parent antibody. This observation confirmed that
the correct V domains of M291 were cloned.
[0118] (2) Modelling and Design of Humanized Sequences
[0119] The sequences of human V domains most similar to murine 1D10
and M291 were selected to serve as the framework of the humanized
antibody. For 1D10, the best human V.sub.k sequence was R3.5H5G of
human subgroup I with only sixteen differences from 1D10 in
framework regions. Manheimer-Lory et al., J. Exp. Med. 174,
1639-1652 (1991). The best V.sub.H sequence was IC4 of Kabat's
subgroup II or subgroup IV (see Id.), with twenty-six differences.
For M291, the best human V.sub.k sequence is HF2-1/17 of human
subgroup I with twenty-six amino acid differences from M291 in
framework regions (Athison et al., J. Clin. Invest. 75, 1138
(1985); Lampman Blood 74, 262 (1989)); the best human V.sub.H
sequence is 21/28 of human subgroup I with twenty amino acid
differences. Dersimonian et al., J. Immunol. 139, 2496-2501 (1987).
With the help of the 3-dimensional model, an additional number of
framework positions that differed between the murine antibodies and
the chosen human sequences were identified. The location of those
amino acid residues in 3-dimensional space relative to the
hypervariable regions, or CDRs, indicated they were likely to
influence CDR conformation, and thus binding affinity. Murine
sequences were used in these positions. A number of positions were
identified in the human sequences that differed from the consensus
of their respective subgroups. These amino acids were changed to
correspond to consensus sequences. V.sub.H and V.sub.L sequence
comparisons between the murine and humanized 1D10, and between the
murine and humanized M291, are shown in FIG. 4 and FIG. 5,
respectively.
[0120] (3) Synthesis and Expression of Humanized 1D10 Antibody
[0121] DNA segments encoding the humanized 1D10 L and H chain V
regions were constructed by total gene synthesis from overlapping
oligonucleotides. These mini exons included signal sequences, J
segments and splice donor sequences and were surrounded by XbaI
sites. The DNA segments were incorporated in an expression vector
using the scheme outlined in FIG. 6.
[0122] The humanized V domains were cloned into the XbaI sites of
the corresponding heavy and light chain expression plasmids
pV.sub.g 1.D.Tt and pVk.rG.dE. The resulting plasmids are called
pHu1D10.Vg1.D.Tt and pHu1D10.V.sub.k.rG.dE. The heavy chain
expression vector, pVg1.D.Tt, which contains the mutant
dihydrofolate reductase gene (mdhfr) as the selectable marker (see
Simonsen & Levinson, Proc. Natl. Acad. Sci. USA, 80, 2495,
(1983)), the human cytomegalovirus (hCMV) major immediate early
promoter and enhancer for transcription initiation (see Boshart et
al., Cell 41, 521 (1985)), and the human IgG1 constant regions was
constructed from the respective fragments by standard methods. It
differs from the vector pV.sub.g1.D described by Co et al, J.
Immunol. 148, 1149 (1992) by having a transcription termination
site 3' to the gamma. 1 gene poly(A) site. The transcription
termination site (Tt) was derived from the sequence located
downstream from the human complement gene C2 (+37 to +162 bp from
the C2 poly(A) site) (see Ashfield et al., EMBO J. 10, 4197 (1991))
and was synthesized entirely by using overlapping
oligonucleotides.
[0123] For light chain expression, a vector was constructed from
the hCMV promoter and enhancer, the human C.sub.K gene including
part of the preceding intron, and the xanthine-guanine
phosphoribosyltransferase (gpt) gene (see Mulligan & Berg,
Proc. Natl. Acad. Sci. USA, 78, 2072 (1981)) for selection. The
vector, pVk.rG.dE, is similar to pV.sub.k described by Co et al.
(see Co et al., J. Immunol. 148, 1149 (1992)) except for the
orientation of the gpt gene. In addition, one of the two repeated
sequences in the enhancer region of the SV40 promoter used to
transcribe the gpt gene was deleted by SphI digestion.
[0124] For coexpression of heavy and light chains in one plasmid,
an EcoRI fragment containing the hCMV promoter, the V.sub.H exon,
the C.sub.H 1, C.sub.H 2 and C.sub.H 3 exons, the polyA signal, and
the transcription termination signal was taken from the heavy chain
expression vector and cloned into the unique EcoRI site of the
corresponding light chain expression plasmid. Due to the presence
of the transcription termination signal situated between them, the
two genes are transcribed independently by the hCMV promoter. After
transcription the humanized V.sub.H exon is spliced to the human
.gamma.1 C.sub.H 1, hinge, C.sub.H 2 and C.sub.H 3 exons, and then
polyadenylated. Similarly the V.sub.L exon is spliced to the human
C.sub.K exon. The predicted amino acid sequences of the mature
light and heavy chains of humanized 1D10 are shown in FIGS. 4C and
4E, respectively.
[0125] Plasmid pHu1D10.IgG1.rG.dE, was used for transfection into
mouse myeloma cell line TSO by electroporation. TSO cells are
derivative of mouse myeloma NSO cells (ECACC 85110503) selected for
their ability to grow in serum-free media according to the
procedure of Sato et al., J. Exp. Med. 165, 1761 (1987). The cells
from each transfection were selected for gpt expression. Because
the SV40 promoter/enhancer for the gpt gene has been crippled, only
few transfectants can express gpt high enough to survive the
selection (see Jasin & Berg, Genes Dev. 2, 1353 (1988)).
Transfection efficiency is about 0.5-1.0.times.10.sup.-6; compared
to the efficiency of 10-50.times.10.sup.-6 from transfection using
near identical plasmid containing the wild type SV40 promoter for
gpt. When screened for production of humanized antibodies by
standard ELISA, the average surviving cells also gave higher levels
of antibody compared to those transfected with plasmid containing
the wild type SV40 promoter. The best antibody producer was then
subcloned for the production of the humanized 1D10. The antibody,
Hu1D10, was purified from the serum-free spent medium by Protein A
affinity chromatography.
[0126] (4) Properties of Hu1D10
[0127] Murine 1D10-IgG2a and humanized 1D10 had identical spectrums
of reactivity with 1D10 positive and 1D10 negative cell lines. The
affinity of murine 1D10-IgGa and humanized 1D10 for cells bearing
the target antigen was evaluated using a displacement assay (see
Woodle et al., J. Immunol. 148 2756 (1992)). In this assay, the
ability of prebound humanized 1D10 or murine 1D10-IgG2a to inhibit
the binding of FITC-labeled murine 1D10-IgG2a was quantitated by
FACS analysis. Humanized 1D10 competitively inhibited the binding
of murine 1D10-IgG2a to a degree similar to that seen with the
parent antibody (FIG. 7A). These data indicated that the humanized
antibody binds with similar affinity as the murine antibody.
Scatchard analysis was used to better estimate the apparent
affinity of humanized 1D10. Humanized 1D10-IgG1 was found to have
an apparent K.sub.a of 2.3.times.10.sup.8 M.sup.-1, and there are
about 5.times.10.sup.5 sites per cell in the Raji cell line (FIG.
7B). In addition, humanized 1D10 has the ability to direct ADCC and
complement mediated lysis, two effector functions that are not
present in the original murine 1D10 (FIGS. 8A and 8B).
[0128] (5) Synthesis and Expression of Humanized M291 and 1D10
(ab'-Zipper).sub.2
[0129] Leucine zipper genes, Jun and Fos, were synthesized as
described by Kostelny et al., J. Immunol. 148, 1547 (1992). The
resulting PCR products were 179 bp PstI-SalI fragments,
encompassing the entire hinge zipper gene fusion. The PstI site is
the natural restriction site located at the beginning of the hinge
exon, but the SalI site was added to the end of the zipper
sequences during PCR. The hinge/zipper exon was inserted with a 162
bp SalI-BamHI fragment containing the 3' noncoding sequence of the
mouse IgG2a gene into the heavy chain expression vector pVg1.D.Tt,
replacing the hinge, C.sub.H 2 and C.sub.H 3 exons in the plasmid.
Coexpression of the truncated heavy chain (Fd) gene with light
chain gene in one plasmid is essentially the same as described
above for pHu1D10.IgG1.rG.dE (FIG. 6). The expression plasmids are
called pHu1D10-Jun.rG.dE and pHuM291-Fos.rG.dE (FIG. 9). The
differences between these plasmids and those used to express the
whole antibody are: (1) the human .gamma.1 C.sub.H 1 exon is now
spliced to the hinge/zipper fusion exon instead of the hinge,
C.sub.H 2 and C.sub.H 3 exons, and (2) the transcript is
polyadenylated by a heterologous signal. The leucine zipper Jun is
used for the Fd of Hu1D10, and Fos for Fd of HuM291. When combined
with the corresponding light chain, the Fd-zipper would form
F(ab'-zipper).sub.2. The humanized F(ab'-zipper).sub.2 fragments
for 1D10 and M291 are called Hu1D10-Jun and HuM291-Fos,
respectively. The predicted amino acid sequences of the heavy chain
Fd-zipper in Hu1D10-Jun and HuM291-Fos are shown in FIGS. 4D and
5D, respectively. In both cases there were modifications of the
human IgG1 hinge at the region of hinge/zipper fusion (FIG. 10). An
insertion of two amino acid residues Lys-Cys derived from the mouse
IgG2a hinge was introduced to the hinge exon to provide an
additional inter-heavy chain disulfide bond. The insertion of these
two residues in the human IgG1 hinge renders its COOH-terminal half
homologous to that of the mouse IgG2a hinge. The modified hinge
would have three inter-heavy chain disulfide bonds compared to two
in the wild type human IgG1. In addition an Ala residue (first
residue of the CH 2 domain) and two Gly residues were introduced at
the fusion junction to make the joints more flexible. The
expression plasmids, pHu1D10-Jun.rG.dE and pHuM291-Fos.rG.dE, were
separately transfected into mouse myeloma cell line TSO by
electroporation. Transfectants were screened for the presence and
the quantity of secreted F(ab'-zipper).sub.2 fragments by ELISA.
F(ab'-zipper).sub.2 fragments were purified using Protein G
affinity chromatography.
[0130] (5) Properties of HuM291-Fos
[0131] The relative affinity of murine M291 and HuM291-Fos
F(ab'-zipper).sub.2 for T cells was evaluated using the
displacement assay described above. HuM291-Fos blocks the binding
of FITC-labeled murine M291 IgG2a as well as the unlabeled M291
(FIG. 11A). The affinity of HuM291 for CD3 is estimated to be
within 2-3 fold of M291's. Scatchard analysis indicated the
apparent affinity of HuM291-Fos was K.sub.a
.sup..about.1.1.times.10.sup.9 M.sup.-1, and there are about
6.6.times.10.sup.4 sites per cell in activated human T cells (FIG.
11B).
[0132] (6) Formation of the Bispecific Hu1D10-Jun.times.HuM291-Fos
F(ab'-zipper).sub.2 In Vitro
[0133] Hu1D1-Jun and HuM291-Fos were mixed in equal molar at
concentrations between 0.5 to 3.0 mg/ml and reduced with 10 mM DTT
in PBS at 37.degree. C. for 1 hour to form Fab'-zippers. They were
passed through Sepharose G-50 column in PBS to remove DTT. The
desalted protein was incubated at 4.degree. C. for 48 hours to
allow formation of heterodimeric bispecific
Hu1D10-Jun.times.HuM291-Fos. The bispecific molecules were further
purified by hydrophobic interaction chromatography (HIC) on a
Phenyl Sepharose column.
EXAMPLE 5
T Cell-Mediated Cytotoxicity by Humanized Bispecific Antibodies
[0134] The ability of Hu1D10-Jun.times.HuM291-Fos to direct T
cell-mediated lysis was tested in a chromium-release assay. Human T
cells derived from PBMC after OKT3 and IL-2 treatment were used as
effector cells. Dawo, which is a cell line developed from a patient
with large B cell lymphoma, was used as target cells. FIG. 12 shows
that the bispecific Hu1D10-Jun.times.HuM291-Fos, as well as the
mouse bispecific IgG 1DT3-D directed T cells to lyse target cells.
The two bispecific molecules seemed to have similar activities at
low antibody concentrations. The two parent antibodies, HuM291-Fos
and Hu1D10-Jun, were not effective in this assay, either singly or
in combination.
[0135] At high concentrations (10 .mu.g/ml, 1DT3-D had higher
activity than Hu1D10-Jun.times.HuM291-Fos in mediating target cell
lysis. This was because of low affinity Fc receptors on the surface
of the target cells. At high antibody concentration, bispecific
IgG's Fc could bind to these receptors and direct T cell to lyse
target cells independent of the target antigen, a mechanism known
as reverse lysis (see Weiner et al., J. Immunol. 152, 2385 (1994)).
Because Hu1D10-Jun.times.HuM291-Fos is an F(ab').sub.2-like
molecule without an Fc, it cannot initiate lysis by binding to an
Fc receptor. In some therapeutic application, the property of the
humanized antibody is advantageous in increasing selective toxicity
of the antibody.
[0136] All publications and patent applications cited above are
herein incorporated by reference in their entirety for all purposes
to the same extent as if each individual publication or patent
application were specifically and individually indicated to be so
incorporated by reference. Although the present invention has been
described in some detail by way of illustration and example for
purposes of clarity and understanding, it will be apparent that
certain changes and modifications may be practiced within the scope
of the appended claims.
Sequence CWU 1
1
14 1 107 PRT Artificial Sequence Light chain of Humanized 1D10 Ab
minus signal sequence 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Val 35 40 45 Ser Asn Ala Lys Thr
Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Lys Gln Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Asn Ser Tyr 85 90
95 Pro Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 2 107 PRT
Mus sp. 2 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn
Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys
Ser Pro Gln Leu Leu Val 35 40 45 Ser Asn Ala Lys Thr Leu Ala Glu
Gly Val Thr Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Lys Gln
Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly
Asn Tyr Tyr Cys Gln His His Tyr Gly Asn Ser Tyr 85 90 95 Pro Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 3 116 PRT Artificial
Sequence Heavy chain of Humanized 1D10 Ab minus signal sequence 3
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5
10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn
Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu
Glu Trp Ile 35 40 45 Gly Val Lys Trp Ser Gly Gly Ser Thr Glu Tyr
Asn Ala Ala Phe Ile 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Thr
Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Asn Ser Leu Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asn Asp Arg Tyr
Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser
Ser 115 4 116 PRT Mus sp. 4 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly
Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Gly
Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg
Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Lys Trp
Ser Gly Gly Ser Thr Glu Tyr Asn Ala Ala Phe Ile 50 55 60 Ser Arg
Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Phe 65 70 75 80
Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Met Tyr Tyr Cys Ala 85
90 95 Arg Asn Asp Arg Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
Val 100 105 110 Thr Val Ser Ser 115 5 214 PRT Artificial Sequence
Complete light chain of Humanized 1D10 Ab 5 Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val 35 40 45
Ser Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Lys Gln Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly
Asn Ser Tyr 85 90 95 Pro Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180
185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 6 273 PRT Artificial
Sequence Fd-jun in F(ab'-zipper)2 of humanized 1D10 antibody 6 Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10
15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr
20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu
Trp Ile 35 40 45 Gly Val Lys Trp Ser Gly Gly Ser Thr Glu Tyr Asn
Ala Ala Phe Ile 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser
Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Asn Ser Leu Thr Ala Ala
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asn Asp Arg Tyr Ala
Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145
150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Lys Cys
Pro Ala Gly Gly Arg Ile Ala Arg Leu Glu 225 230 235 240 Glu Lys Val
Lys Thr Leu Lys Ala Gln Asn Ser Glu Leu Ala Ser Thr 245 250 255 Ala
Asn Met Leu Arg Glu Gln Val Ala Gln Leu Lys Gln Lys Val Met 260 265
270 Asn 7 446 PRT Artificial Sequence Complete heavy chain of
Humanized 1D10 Ab 7 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ser
Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Lys Trp Ser Gly
Gly Ser Thr Glu Tyr Asn Ala Ala Phe Ile 50 55 60 Ser Arg Leu Thr
Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu
Asn Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95
Arg Asn Asp Arg Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100
105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225
230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345
350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 8 106 PRT
Artificial Sequence Light chain of Humanized M291 Ab minus signal
sequence 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser
Val Ser Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Arg Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly
Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr 85 90 95 Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys 100 105 9 106 PRT Mus sp. 9 Gln Ile
Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20
25 30 Asn Trp Tyr Lys Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Thr
Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe
Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser
Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln
Trp Ser Ser Asn Pro Pro Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu
Glu Ile Lys 100 105 10 120 PRT Artificial Sequence Heavy chain of
Humanized M291 Ab minus signal sequence 10 Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr 20 25 30 Thr Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Thr His Tyr Asn Gln Lys Leu 50
55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ala Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Ser Ala Tyr Tyr Asp Tyr Asp Gly Phe
Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115
120 11 120 PRT Mus sp. 11 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu
Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Ile Ser Tyr 20 25 30 Thr Met His Trp Val Lys
Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn
Pro Arg Ser Gly Tyr Thr His Tyr Asn Gln Lys Leu 50 55 60 Lys Asp
Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Ser Ala Tyr 65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Ser Ala Tyr Tyr Asp Tyr Asp Gly Phe Ala Tyr Trp Gly
Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ala 115 120 12 213 PRT
Artificial Sequence Complete light chain of Humanized M291 Ab 12
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr
Met 20 25 30 Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg
Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Trp Ser Ser Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135
140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 13
279 PRT Artificial Sequence Complete heavy chain of Humanized M291
Ab 13 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Ile Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Arg Ser Gly Tyr
Thr His Tyr Asn Gln Lys Leu 50 55 60 Lys Asp Lys Ala Thr Leu Thr
Ala Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser
Ala Tyr Tyr Asp Tyr Asp Gly Phe Ala Tyr Trp Gly Gln 100 105 110 Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120
125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His
Thr Cys Pro Pro Cys Lys Cys Pro Ala Gly Gly Leu Thr 225 230 235 240
Asp Thr Leu Gln Ala Glu Thr Asp Gln Leu Glu Asp Lys Lys Ser Ala 245
250 255 Leu Gln Thr Glu Ile Ala Asn Leu Leu Lys Glu Lys Glu Lys Leu
Glu 260 265 270 Phe Ile Leu Ala Ala Thr Ser 275 14 7 PRT Artificial
Sequence Leucine zipper motif MISC_FEATURE (2)..(7) Xaa is any
amino acid 14 Leu Xaa Xaa Xaa Xaa
Xaa Xaa 1 5
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