U.S. patent application number 13/591024 was filed with the patent office on 2013-03-07 for bispecific antibodies specific for t-cell activating antigens and a tumor antigen and methods of use.
The applicant listed for this patent is Peter Bruenker, Tanja Fauti, Christiane Jaeger, Christian Klein, Pablo Umana. Invention is credited to Peter Bruenker, Tanja Fauti, Christiane Jaeger, Christian Klein, Pablo Umana.
Application Number | 20130058936 13/591024 |
Document ID | / |
Family ID | 46704675 |
Filed Date | 2013-03-07 |
United States Patent
Application |
20130058936 |
Kind Code |
A1 |
Bruenker; Peter ; et
al. |
March 7, 2013 |
BISPECIFIC ANTIBODIES SPECIFIC FOR T-CELL ACTIVATING ANTIGENS AND A
TUMOR ANTIGEN AND METHODS OF USE
Abstract
The present invention relates to bispecific antibodies that
specifically bind a T-cell activating antigen and a Tumor Antigen
(TA), comprising a first Fab fragment and a second Fab fragment,
wherein either the variable regions or the constant regions of the
second Fab heavy and light chain are exchanged; and wherein the
bispecific antibody does not comprise a Fc domain; methods for
their production, pharmaceutical compositions containing said
antibodies, and uses thereof.
Inventors: |
Bruenker; Peter; (Hittnau,
CH) ; Fauti; Tanja; (Zuerich, CH) ; Jaeger;
Christiane; (Wallisellen, CH) ; Klein; Christian;
(Bonstetten, CH) ; Umana; Pablo; (Wollerau,
CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bruenker; Peter
Fauti; Tanja
Jaeger; Christiane
Klein; Christian
Umana; Pablo |
Hittnau
Zuerich
Wallisellen
Bonstetten
Wollerau |
|
CH
CH
CH
CH
CH |
|
|
Family ID: |
46704675 |
Appl. No.: |
13/591024 |
Filed: |
August 21, 2012 |
Current U.S.
Class: |
424/136.1 ;
435/252.33; 435/254.11; 435/254.2; 435/328; 435/69.6;
530/387.3 |
Current CPC
Class: |
C07K 16/468 20130101;
A61P 35/00 20180101; C07K 2317/55 20130101; C07K 16/3053 20130101;
C07K 16/2809 20130101; C07K 2317/52 20130101; C07K 2317/66
20130101; C07K 2317/31 20130101; C07K 16/30 20130101 |
Class at
Publication: |
424/136.1 ;
530/387.3; 435/69.6; 435/252.33; 435/328; 435/254.11;
435/254.2 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00; C12N 1/19 20060101
C12N001/19; C12N 1/21 20060101 C12N001/21; C12N 5/10 20060101
C12N005/10; C12N 1/15 20060101 C12N001/15; C07K 16/46 20060101
C07K016/46; C12P 21/00 20060101 C12P021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 23, 2011 |
EP |
11178410.4 |
Claims
1. A bispecific antibody that specifically binds a T-cell
activating antigen and a Tumor Antigen (TA), comprising a first Fab
fragment and a second Fab fragment, wherein either the variable
regions or the constant regions of the second Fab heavy and light
chain are exchanged; and wherein the bispecific antibody does not
comprise a Fc domain.
2. The bispecific antibody of claim 1, wherein the first fragment
comprises at least one antigen binding site specific for a Tumor
Antigen; and the second Fab fragment comprises at least one antigen
binding site specific for a T-cell activating antigen.
3. The bispecific antibody of claim 1, wherein the T-cell
activating antigen is a CD3 T-Cell Co-Receptor (CD3) antigen.
4. The bispecific antibody of claim 1, wherein the N-terminus of
the second Fab fragment is connected to the C-terminus of the first
Fab fragment.
5. The bispecific antibody of claim 1, additionally comprising a
third Fab fragment.
6. The bispecific antibody of claim 5, wherein the third Fab
fragment comprises at least one antigen binding site specific for a
Tumor Antigen.
7. The bispecific antibody of claim 5, wherein the third Fab
fragment is connected to the first Fab fragment.
8. The bispecific antibody of claim 7, wherein the C-terminus of
the third Fab fragment is connected to the N-terminus of the first
Fab fragment.
9. The bispecific antibody of claim 5, wherein the third Fab
fragment is connected to the second Fab fragment.
10. The bispecific antibody of claim 9, wherein the N-terminus of
the third Fab fragment is connected to the C-terminus of the second
Fab fragment.
11. The bispecific antibody of claim 1 or claim 5, wherein the Fab
fragments are connected via a peptide linker.
12. The bispecific antibody of claim 11, wherein the peptide linker
is a (G4S)2 linker.
13. The bispecific antibody of claim 1 or claim 5, wherein the
Tumor Antigen is selected from the group consisting of
Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP),
Epidermal Growth Factor Receptor (EGFR), Carcinoembryonic Antigen
(CEA), Fibroblast Activation Protein (FAP) and CD33.
14. The bispecific antibody of claim 13, wherein the Tumor Antigen
is MCSP.
15. A pharmaceutical composition comprising the bispecific antibody
of claim 1 or claim 5.
16. (canceled)
17. (canceled)
18. A method of treating cancer comprising administering to a
patient in need thereof an effective amount of the bispecific
antibody of claim 1 or claim 5.
19. (canceled)
20. A prokaryotic or eukaryotic host cell comprising vectors
comprising nucleic acid molecules encoding the light chains and
heavy chains of the bispecific antibody of claim 1 or claim 5.
21. A method of producing an antibody comprising culturing the host
cell of claim 20 so that the antibody is produced.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of European Patent
Application No. 11178410.4, filed Aug. 23, 2011 which is
incorporated herein by reference in its entirety.
SEQUENCE LISTING
[0002] The present invention contains a Sequence Listing which has
been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Aug. 14, 2012, is named P4743_SequenceListing.txt and is 149,782
bytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates to bispecific antibodies that
specifically bind a T-cell activating antigen and a Tumor Antigen
(TA), comprising a first Fab fragment and a second Fab fragment,
wherein either the variable regions or the constant regions of the
second Fab heavy and light chain are exchanged; and wherein the
bispecific antibody does not comprise a Fc domain; methods for
their production, pharmaceutical compositions containing said
antibodies, and uses thereof.
BACKGROUND
[0004] The selective destruction of an individual cell or a
specific cell type is often desirable in a variety of clinical
settings. For example, it is a primary goal of cancer therapy to
specifically destroy tumor cells, while leaving healthy cells and
tissues undamaged. One approach is to selectively induce an immune
response against the tumor, which triggers the attack and
subsequent destruction of tumor cells by immune effector cells such
as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs).
CTLs constitute the most potent effector cells of the immune
system, however they cannot be activated by the effector mechanism
mediated by the Fc domain of conventional therapeutic antibodies.
In this regard, bispecific antibodies which are able to bind to a
surface antigen on cancer cells and to an activating invariant
component of the T cell receptor (TCR) complex have become of
interest in recent years. The simultaneous binding of the
bispecific antibody to both of its targets forces a temporary
interaction between cancer cell and T cell, causing activation of
cytotoxic T cells and subsequent lysis of the tumor cell.
[0005] Several bispecific antibody formats have been developed and
their suitability for T cell mediated cancer immunotherapy
investigated. Out of these, the so-called BiTE (bispecific T cell
engager) molecules have been very well characterized and already
shown some promising results in the clinic (reviewed in Nagorsen
and Bauerle, Exp Cell Res 317, 1255-1260 (2011)). BiTEs are tandem
scFv molecules wherein two scFv molecules are fused by a flexible
linker. Further bispecific formats being evaluated for T cell
engagement include diabodies (Holliger et al., Prot Eng 9, 299-305
(1996)) and derivatives thereof, such as tandem diabodies
(Kipriyanov et al., J Mol Biol 293, 41-66 (1999)). A more recent
development are the so-called DART (dual affinity retargeting)
molecules, which are based on the diabody format but feature a
C-terminal disulfide bridge for additional stabilization (Moore et
al., Blood 117, 4542-51 (2011)). The so-called triomabs, which are
whole hybrid mouse/rat IgG molecules and also currently being
evaluated in clinical trials, represent a larger sized format
(reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467
(2010)).
[0006] However, the bispecific antibodies developed for T cell
mediated cancer immunotherapy known so far have major drawbacks
relating to their efficacy, toxicity and applicability. Small
constructs such as, for example, BiTE molecules--while being able
to efficiently crosslink effector and target cells--have a very
short serum half life requiring them to be administered to patients
by continuous infusion. IgG-like formats on the other hand--while
having the great benefit of a long half life--suffer from toxicity
associated with the native effector functions inherent to IgG
molecules. This immunogenic potential constitutes another
unfavorable feature of IgG-like bispecific antibodies, for
successful therapeutic development. Finally, a major challenge in
the general development of bispecific antibodies remains the
production of bispecific antibody constructs at a clinically
sufficient quantity and purity. The mispairing of antibody heavy
and light chains of different specificities upon co-expression,
decreases the yield of the correctly assembled construct and
results in a number of non-functional side products.
[0007] Given the difficulties and disadvantages associated with
currently available bispecific antibodies for T cell mediated
cancer immunotherapy, there remains a need for novel, improved
formats of such molecules. These drawbacks have now been overcome
with the new bispecific antibodies of the invention. The new
bispecific antibodies can be easily produced with an increased
yield due to a decreased amount of mispaired side-products, which
show less aggregation than bispecific antibody fragments known in
the art. Using the crossover approach correct LC association can be
enforced without the need for the generation of a common light
chain. In addition, the new the new bispecific antibodies has a
higher molecular weight compared to many conventional bispecific
antibody fragments, thus preventing excessive kidney clearance and
leading to an improved half-life in vivo. The new bispecific
antibodies are fully functional and have comparable or improved
binding and activity as corresponding conventional bispecific
antibodies.
[0008] The present invention provides bispecific antigen binding
molecules designed for T cell activation and re-direction that
combine good efficacy and produceability with low toxicity and
favorable pharmacokinetic properties.
SUMMARY
[0009] The present invention relates to bispecific antibodies that
specifically bind a T-cell activating antigen and a Tumor Antigen
(TA), comprising a first Fab fragment and a second Fab fragment,
wherein either the variable regions or the constant regions of the
second Fab heavy and light chain are exchanged; and wherein the
bispecific antibody does not comprise a Fc domain.
[0010] The antibodies of the invention specifically bind to a Tumor
Antigen on the surface of a tumor cell and at the same time bind to
T-cell activating antigen. By that the bispecific antibody is
capable to elecit an immune response specifically at the site of
the tumor, subsequently resulting in apoptosis of the target
cell.
[0011] In one aspect, a bispecific antibody that specifically binds
a T-cell activating antigen and a Tumor Antigen (TA) is provided,
comprising at least two fab fragments, wherein the first Fab
fragment comprises at least one antigen binding site specific for a
Tumor Antigen (TA); and the second Fab fragment comprises at least
one antigen binding site specific for a T-cell activating antigen,
wherein either the variable regions or the constant regions of the
second Fab heavy and light chain are exchanged; and wherein the
bispecific antibody is devoid of a Fc domain.
[0012] In particular, the present invention relates to bispecific
antibodies wherein the T-cell activating antigen is a CD3 T-Cell
Co-Receptor (CD3) targeting antigen.
[0013] In one aspect, a bispecific antibody that specifically binds
CD3 T-Cell Co-Receptor (CD3) antigen and a Tumor Antigen (TA) is
provided, comprising at least two fab fragments, wherein the first
Fab fragment comprises at least one antigen binding site specific
for a Tumor Antigen (TA); and the second Fab fragment comprises at
least one antigen binding site specific for a CD3 T-Cell
Co-Receptor (CD3) wherein either the variable regions or the
constant regions of the second Fab heavy and light chain are
exchanged; and wherein the bispecific antibody is devoid of a Fc
domain. In one embodiment the first and second Fab fragments are
connected via a peptide linker. Preferably said peptide linker is a
(G4S)2 linker.
[0014] In one embodiment said antibody additionally comprises a
third Fab fragment. In another embodiment said third Fab fragment
comprises at least one antigen binding site specific for a Tumor
Antigen. In one embodiment the third Fab fragment is connected to
the N or C-terminus of the light chain or the heavy chain of the
first Fab fragment. In another embodiment the third Fab fragment is
connected to the N or C-terminus of the light chain or the heavy
chain of the second Fab fragment. In one embodiment the third Fab
fragment is connected to the first or second Fab fragment via a
peptide linker. Preferably said peptide linker is a (G4S)2
linker.
[0015] The bispecific antibodies according to the invention are at
least bivalent and can be trivalent or multivalent e.g. tetravalent
or hexavalent. In one embodiment said bispecific antibodies are
bivalent (1+1 format) with one binding site each targeting a Tumor
Antigen (TA) and a T-cell activating antigen, respectively. In
another embodiment said bispecific antibodies are trivalent (2+1
format) with two binding sites each targeting a Tumor Antigen (TA)
and one binding site targeting a T-cell activating antigen, as
detailed in the following section. In a preferred embodiment said a
T-cell activating antigen is CD3.
[0016] In a second object the present invention relates to a
pharmaceutical composition comprising a bispecific antibody of the
present invention.
[0017] In a third object the present invention relates to a
bispecific antibody of the present invention for the treatment of
cancer. In another embodiment, use of the bispecific antibody as a
medicament is provided. Preferably said use is for the treatment of
cancer.
[0018] In further objects the present invention relates to a
nucleic acid sequence comprising a sequence encoding a heavy chain
of a bispecific antibody of the present invention, a nucleic acid
sequence comprising a sequence encoding a light chain of a
bispecific antibody of the present invention, an expression vector
comprising a nucleic acid sequence of the present invention and to
a prokaryotic or eukaryotic host cell comprising a vector of the
present invention. In addition a method of producing an antibody
comprising culturing the host cell so that the antibody is produced
is provided.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1: Schematic illustration of exemplary bispecific
antibody formats of the invention. a) Fab-Crossfab molecule
C-terminal, b) Fab-Crossfab molecule N-terminal c) (Fab)2-Crossfab
molecule C-terminal d) (Fab)2-Crossfab molecule N-terminal e)
Fab-Crossfab-Fab molecule.
[0020] FIG. 2: Analysis of hu Fab(MCSP)-Crossfab(CD3) production
and purification: SDS-Page: 4-12% Bis/Tris (NuPage [invitrogen];
coomassie stained): a) 1--Mark 12 (invitrogen), 2--hu
Fab(MCSP)-Crossfab(CD3) non reduced; b) 1--Mark 12 (invitrogen),
2--hu Fab(MCSP)-Crossfab(CD3) reduced.
[0021] FIG. 3: Analysis Fab(MCSP)-Crossfab(CD3) production and
purification. Analytical size exclusion chromatography,
Chromatogram A280 (Superdex 200 10/300 GL [GE Healthcare]; 2 mM
MOPS pH 7.3, 150 mM NaCl, 0.02% (w/v) NaCl; 50 .mu.g sample were
injected).
[0022] FIG. 4: Analysis of hu Fab(MCSP)-Fab(MCSP)-Crossfab(CD3)
production and purification: SDS-Page: 4-12% Bis/Tris (NuPage
[invitrogen]; coomassie stained): a) 1--Mark 12 (invitrogen), 2--hu
Fab(MCSP)-Fab(MCSP)-Crossfab(CD3) non reduced; b) 1--Mark 12
(invitrogen), 2--hu Fab(MCSP)-Fab(MCSP)-Crossfab(CD3) reduced.
[0023] FIG. 5: Analysis of hu Fab(MCSP)-Fab(MCSP)-Crossfab(CD3)
production and purification. Analytical size exclusion
chromatography, Chromatogram A280 (Superdex 200 10/300 GL [GE
Healthcare]; 2 mM MOPS pH 7.3, 150 mM NaCl, 0.02% (w/v) NaCl; 50
.mu.g sample were injected).
[0024] FIG. 6: Analysis of hu Fab(MCSP)--Crossfab(CD3)-Fab(MCSP)
production and purification. SDS-Page: 4-12% Bis/Tris (NuPage
[invitrogen]; coomassie stained): a) 1--Mark 12 (invitrogen), 2--hu
Fab(MCSP)--Crossfab(CD3)-Fab(MCSP) non reduced; b) 1--Mark 12
(invitrogen), 2--hu Fab(MCSP)-Crossfab(CD3)-Fab(MCSP) reduced.
[0025] FIG. 7: Analysis of hu Fab(MCSP)-Crossfab(CD3)-Fab(MCSP)
production and purification. Analytical size exclusion
chromatography, Chromatogram A280 (Superdex 200 10/300 GL [GE
Healthcare]; 2 mM MOPS pH 7.3, 150 mM NaCl, 0.02% (w/v) NaCl; 50
.mu.g sample were injected).
[0026] FIG. 8: Analysis of murine Crossfab(CD3)-Fab(MCSP)-Fab(MCSP)
production and purification. SDS-Page: 4-12% Bis/Tris (NuPage
[invitrogen]; coomassie stained): a) 1--Mark 12 (invitrogen),
2--murine Crossfab(CD3)-Fab(MCSP)-Fab(MCSP) non reduced; b) 1--Mark
12 (invitrogen), 2--murine Crossfab(CD3)-Fab(MCSP)-Fab(MCSP)
reduced.
[0027] FIG. 9: Analysis of murine Crossfab(CD3)-Fab(MCSP)-Fab(MCSP)
production and purification. Analytical size exclusion
chromatography, Chromatogram A280 (Superdex 200 10/300 GL [GE
Healthcare]; 2 mM MOPS pH 7.3, 150 mM NaCl, 0.02% (w/v) NaCl; 50
.mu.g sample were injected).
[0028] FIG. 10: Killing (as measured by LDH release) of MDA-MB-435
tumor cells upon co-culture with human pan T cells (E:T ratio=5:1)
and activation for 20 hours by different concentrations of the hu
Fab(MCSP)-Crossfab(CD3) (="Fab-Crossfab"), hu
Fab(MCSP)-Crossfab(CD3)-Fab(MCSP) (="Fab-Crossfab-Fab"), hu
Fab(MCSP)-Fab(MCSP)-Crossfab(CD3) (="(Fab)2-Crossfab"), as well as
the (scFv)2 (antiMCSP/anti huCD3e) (="(scFv)2") bispecific
molecules. The constructs with bivalent MCSP-targeting show
comparable cytotoxic activity compared to the "(scFv)2" construct,
whereas the "Fab-Crossfab" construct with monovalent MCSP binding
is clearly less potent.
[0029] FIG. 11: Comparison of the hu
Fab(MCSP)-Fab(MCSP)-Crossfab(CD3) (="(Fab)2-Crossfab") and the
(scFv)2 (antiMCSP/anti huCD3e) (="(scFv)2") construct, Depicted is
the LDH release from MDA-MB-435 tumor cells upon co-culture with
human pan T cells (E/T ratio=5:1), and activation for 21 hours by
different concentrations of the bispecific constructs and
corresponding IgGs. The "(Fab)2-Crossfab" induces apoptosis in
target cells at least comparably good as the (scFv)2 molecule.
[0030] FIG. 12: Comparison of the hu
Fab(MCSP)-Fab(MCSP)-Crossfab(CD3) (="(Fab)2-Crossfab") and the
(scFv)2 (antiMCSP/anti huCD3e) (="(scFv)2") construct. Depicted is
the LDH release from MV-3 human melanoma tumor cells upon
co-culture with human PBMCs (E/T ratio=10:1), and activation for 26
hours by different concentrations of the bispecific constructs and
corresponding IgGs. The "(Fab)2-Crossfab" induces apoptosis in
target cells at least comparably good as the (scFv)2 molecule.
[0031] FIG. 13: LDH release from B16/F10-huMCSP Fluc2, clone 48
tumor cells, induced by primary murine T cell activation with the
murine Crossfab(CD3)-Fab(MCSP)-Fab(MCSP) construct
(=(Fab)2-CrossFab), targeting human MCSP, as well as the murine
CD3. The effector to target cell ratio was 5:1. The assay was
analyzed after incubation for 23.5 hours at 37.degree. C., 5% CO2.
The construct induces concentration-dependent, T cell-mediated
apoptosis of human MCSP-expressing target cells.
[0032] FIG. 14: LDH release from B16/F10-huMCSP Fluc2, clone 48
tumor cells, induced by primary murine T cell activation with 50 nM
of the murine Crossfab(CD3)-Fab(MCSP)-Fab(MCSP) construct
(=(Fab)2-CrossFab), targeting human MCSP, as well as the murine
CD3. The effector to target cell ratio was 5:1. The assay was
analyzed after incubation for 23.5 hours at 37.degree. C., 5% CO2.
The construct induces T cell-mediated apoptosis of human
MCSP-expressing target cells. There is only weak hyperactivation of
T cells at this concentration of the construct.
[0033] FIG. 15: Different cytokine levels measured in the
supernatant of whole blood after treatment with 1 nM of different
CD3-MCSP bispecific constructs (hu
Fab(MCSP)-Fab(MCSP)-Crossfab(CD3) (="(Fab)2-Crossfab") and
(scFv).sub.2 (antiMCSP/anti huCD3e) (="(scFv)2")) in the presence
(A, B) or absence (C,D) of Colo-38 tumor cells for 24 hours. 280
.mu.l whole blood were plated per well of a 96-well plate and 30
000 Colo-38 cells added, as indicated. The main cytokine that was
secreted upon activation of T cells in the presence of Colo-38
tumor cells, is IL-6, followed by IFNgamma. In addition, also the
levels of granzyme B increased enormously upon activation of T
cells in the presence of target cells. In general, the "(scFv)2"
construct elevated the levels of TNF and IFNgamma, as well as
granzyme B in the presence of target cells (A and B) a bit more
compared to the other bispecific construct.
[0034] There was no significant secretion of Th2 cytokines (IL-10
and IL-4) upon activation of T cells by the bispecific constructs
in the presence (or absence) of target cells. In this assay there
was also a weak secretion of IFNgamma, induced by the
"(Fab)2-Crossfab" construct in the absence of target cells.
[0035] FIG. 16: Surface expression level of the late activation
marker CD25 on murine pan T cells, isolated from splenocytes.
Murine pan T cells were incubated with 50 nM of the murine
Crossfab(CD3)-Fab(MCSP)-Fab(MCSP) construct (=(Fab)2-CrossFab)
bispecific construct (targeting murine CD3, as well as human MCSP),
in the presence or absence of B16/F10-huMCSP Fluc2 clone 48 tumor
target cells, as indicated (E:T ratio is 10:1). Depicted is the
expression level of the late activation marker CD25 on CD8+ T cells
after 70 hours. Up-regulation of CD25 on CD8+ T cells with the
(Fab)2-CrossFab construct occurs only in the presence of target
cells. The reference IgGs, used adjusted to the same molarity, were
not able to up-regulate CD25.
[0036] FIG. 17: Analysis of Fab(CD33)-CrossFab (CD3) production and
purification. SDS-Page: a) 3-8% Tris/Acetate (NuPage [invitrogen];
coomassie stained): a) 1--HiMark (invitrogen),
2--Fab(CD33)-CrossFab (CD3).non reduced; b) 4-12% Bis/Tris (NuPage
[invitrogen]: 1--Mark 12 (invitrogen), 2--Fab(CD33)-CrossFab
(CD3).reduced.
[0037] FIG. 18: Analysis of Fab(CD33)-CrossFab (CD3) production and
purification. Analytical size exclusion chromatography,
Chromatogram A280 (Superdex 200 10/300 GL [GE Healthcare]; 2 mM
MOPS pH 7.3, 150 mM NaCl, 0.02% (w/v) NaCl; 50 .mu.g sample were
injected).
[0038] FIG. 19: Killing (as measured by LDH release) of MV-3 tumor
cells upon co-culture with human PBMCs (E:T ratio=10:1) and
activation for 24 hours by different concentrations of CD3-MCSP
bispecific constructs (hu Fab(MCSP)-Crossfab(CD3); designated as
"1+1 non-Fc", and the (scFv)2 (antiMCSP/anti huCD3e) (="(scFv)2")
reference molecule). The "1+1 non-Fc" construct induces apoptosis
in MV-3 target cells with a calculated EC50 of 25.4 pM, whereas the
calculated EC50 for the "(scFv)2" reference molecule is 57 pM,
showing a slight better potency of the "1+1 non-Fc" molecule in
terms of EC50.
[0039] FIG. 20: Activation of CD4+ or CD8+ T cells, as measured by
up-regulation of CD69 (A), respective increase of CD69-positive
cells (B) in the presence of huMCSP-positive MV-3 tumor cells upon
co-culture with human PBMCs (E:T ratio=10:1), treated with the
CD3-MCSP bispecific constructs (hu Fab(MCSP)-Crossfab(CD3);
designated as "1+1 non-Fc", and the (scFv)2 (antiMCSP/anti huCD3e)
(="(scFv)2") reference molecule, respectively) for .about.24 hours.
In general, the CD69 median values are higher on CD8+ T cells
compared to CD4+ T cells. There is a clear concentration-dependent
increase in both, CD69 median values, as well percentage of CD69
positive cells for both constructs.
[0040] FIG. 21: Illustration of (scFv)2 reference molecule.
[0041] FIG. 22: Analysis of (scFv)2 (antiMCSP/anti huCD3e)
production and purification. SDS-Page: 4-12% Bis/Tris (NuPage
[invitrogen]; coomassie stained): 1--Mark 12 (invitrogen),
2--(scFv)2 (antiMCSP/anti huCD3e) reduced; 3-(scFv)2 (antiMCSP/anti
huCD3e), non reduced.
[0042] FIG. 23: Analysis of (scFv)2 (antiMCSP/anti huCD3e)
production and purification Analytical size exclusion
chromatography, Chromatogram A280 (Superdex 75 10/300 GL [GE
Healthcare]; 2 mM MOPS pH 7.3, 150 mM NaCl, 0.02% (w/v) NaCl; 50
.mu.g sample ((scFv)2 (antiMCSP/anti huCD3e)) were injected).
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
I. Definitions
[0043] "Framework" or "FR" refers to variable domain residues other
than hypervariable region (HVR) residues. The FR of a variable
domain generally consists of four FR domains: FR1, FR2, FR3, and
FR4. Accordingly, the HVR and FR sequences generally appear in the
following sequence in VH (or VL):
FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
[0044] An "acceptor human framework" for the purposes herein is a
framework comprising the amino acid sequence of a light chain
variable domain (VL) framework or a heavy chain variable domain
(VH) framework derived from a human immunoglobulin framework or a
human consensus framework, as defined below. An acceptor human
framework "derived from" a human immunoglobulin framework or a
human consensus framework may comprise the same amino acid sequence
thereof, or it may contain amino acid sequence changes. In some
embodiments, the number of amino acid changes are 10 or less, 9 or
less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or
less, or 2 or less. In some embodiments, the VL acceptor human
framework is identical in sequence to the VL human immunoglobulin
framework sequence or human consensus framework sequence.
[0045] A "human consensus framework" is a framework which
represents the most commonly occurring amino acid residues in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, NIH
Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one
embodiment, for the VL, the subgroup is subgroup kappa I as in
Kabat et al., supra. In one embodiment, for the VH, the subgroup is
subgroup III as in Kabat et al., supra.
[0046] The term "hypervariable region" or "HVR," as used herein,
refers to each of the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops ("hypervariable loops"). Generally, native four-chain
antibodies comprise six HVRs; three in the VH(H1, H2, H3), and
three in the VL (L1, L2, L3). HVRs generally comprise amino acid
residues from the hypervariable loops and/or from the
"complementarity determining regions" (CDRs), the latter being of
highest sequence variability and/or involved in antigen
recognition. Exemplary hypervariable loops occur at amino acid
residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55
(H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917
(1987).) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2,
and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2,
89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991).) The terms hypervariable regions (HVRs) and complementarity
determining regions (CDRs), are used herein interchangeably in
reference to portions of the variable region that form the antigen
binding regions. This particular region has been described by Kabat
et al., U.S. Dept. of Health and Human Services, "Sequences of
Proteins of Immunological Interest" (1983) and by Chothia et al.,
J. Mol. Biol. 196:901-917 (1987), where the definitions include
overlapping or subsets of amino acid residues when compared against
each other. Nevertheless, application of either definition to refer
to a CDR of an antibody or variants thereof is intended to be
within the scope of the term as defined and used herein. The
appropriate amino acid residues which encompass the CDRs as defined
by each of the above cited references are set forth below in Table
1 as a comparison. The exact residue numbers which encompass a
particular CDR will vary depending on the sequence and size of the
CDR. Those skilled in the art can routinely determine which
residues comprise a particular CDR given the variable region amino
acid sequence of the antibody.
TABLE-US-00001 TABLE 1 CDR Definitions.sup.1 CDR Kabat Chothia
AbM.sup.2 V.sub.H CDR1 31-35 26-32 26-35 V.sub.H CDR2 50-65 52-58
50-58 V.sub.H CDR3 95-102 95-102 95-102 V.sub.L CDR1 24-34 26-32
24-34 V.sub.L CDR2 50-56 50-52 50-56 V.sub.L CDR3 89-97 91-96 89-97
.sup.1Numbering of all CDR definitions in Table 1 is according to
the numbering conventions set forth by Kabat et al. (see below).
.sup.2"AbM" with a lowercase "b" as used in Table 1 refers to the
CDRs as defined by Oxford Molecular's "AbM" antibody modeling
software.
[0047] Kabat et al. also defined a numbering system for variable
region sequences that is applicable to any antibody. One of
ordinary skill in the art can unambiguously assign this system of
"Kabat numbering" to any variable region sequence, without reliance
on any experimental data beyond the sequence itself. As used
herein, "Kabat numbering" refers to the numbering system set forth
by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence
of Proteins of Immunological Interest" (1983). Unless otherwise
specified, references to the numbering of specific amino acid
residue positions in an antibody variable region are according to
the Kabat numbering system.
[0048] With the exception of CDR1 in VH, CDRs generally comprise
the amino acid residues that form the hypervariable loops. CDRs
also comprise "specificity determining residues," or "SDRs," which
are residues that contact antigen. SDRs are contained within
regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary
a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and
a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2,
89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See
Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008).) Unless
otherwise indicated, HVR residues and other residues in the
variable domain (e.g., FR residues) are numbered herein according
to Kabat et al., supra.
[0049] The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired antigen-binding activity. In
particular the term "antibody" includes the bispecific antibodies
of the invention comprising at least two fab fragments but no Fc
domain.
[0050] The term "bispecific" means that the antigen binding
molecule is able to specifically bind to at least two distinct
antigenic determinants. In certain embodiments the bispecific
antigen binding molecule is capable of simultaneously binding two
antigenic determinants, particularly two antigenic determinants
expressed on two distinct cells.
[0051] The term "monovalent binding to an antigen" means that not
more than one antigen comprised in the antibody specifically binds
to that antigen.
[0052] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human or a human cell or derived from a non-human source that
utilizes human antibody repertoires or other human
antibody-encoding sequences. This definition of a human antibody
specifically excludes a humanized antibody comprising non-human
antigen-binding residues.
[0053] The term "recombinant human antibody", as used herein, is
intended to include all human antibodies that are prepared,
expressed, created or isolated by recombinant means, such as
antibodies isolated from a host cell such as a NS0 or CHO cell or
from an animal (e.g. a mouse) that is transgenic for human
immunoglobulin genes or antibodies expressed using a recombinant
expression vector transfected into a host cell. Such recombinant
human antibodies have variable and constant regions in a rearranged
form. The recombinant human antibodies according to the invention
have been subjected to in vivo somatic hypermutation. Thus, the
amino acid sequences of the VH and VL regions of the recombinant
antibodies are sequences that, while derived from and related to
human germ line VH and VL sequences, may not naturally exist within
the human antibody germ line repertoire in vivo.
[0054] A "humanized" antibody refers to a chimeric antibody
comprising amino acid residues from non-human HVRs and amino acid
residues from human FRs. In certain embodiments, a humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the HVRs (e.g., CDRs) correspond to those of a non-human
antibody, and all or substantially all of the FRs correspond to
those of a human antibody. A humanized antibody optionally may
comprise at least a portion of an antibody constant region derived
from a human antibody. A "humanized form" of an antibody, e.g., a
non-human antibody, refers to an antibody that has undergone
humanization. Other forms of "humanized antibodies" encompassed by
the present invention are those in which the constant region has
been additionally modified or changed from that of the original
antibody to generate the properties according to the invention,
especially in regard to C1q binding and/or Fc receptor (FcR)
binding.
[0055] The term "chimeric" antibody refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or species,
usually prepared by recombinant DNA techniques. Chimeric antibodies
comprising a murine variable region and a human constant region are
preferred. Other preferred forms of "chimeric antibodies"
encompassed by the present invention are those in which the
constant region has been modified or changed from that of the
original antibody to generate the properties according to the
invention, especially in regard to C1q binding and/or Fc receptor
(FcR) binding. Such chimeric antibodies are also referred to as
"class-switched antibodies". Chimeric antibodies are the product of
expressed immunoglobulin genes comprising DNA segments encoding
immunoglobulin variable regions and DNA segments encoding
immunoglobulin constant regions. Methods for producing chimeric
antibodies involve conventional recombinant DNA and gene
transfection techniques are well known in the art. See e.g.
Morrison, S. L., et al., Proc. Natl. Acad. Sci. USA 81 (1984)
6851-6855; U.S. Pat. Nos. 5,202,238 and 5,204,244.
[0056] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variant antibodies, e.g., containing naturally occurring
mutations or arising during production of a monoclonal antibody
preparation, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody of a monoclonal
antibody preparation is directed against a single determinant on an
antigen. Thus, the modifier "monoclonal" indicates the character of
the antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by a variety of techniques, including but not
limited to the hybridoma method, recombinant DNA methods,
phage-display methods, and methods utilizing transgenic animals
containing all or part of the human immunoglobulin loci, such
methods and other exemplary methods for making monoclonal
antibodies being described herein.
[0057] An "antibody fragment" refers to a molecule other than an
intact antibody that comprises a portion of an intact antibody that
binds the antigen to which the intact antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab',
Fab'-SH, F(ab').sub.2; diabodies; linear antibodies; single-chain
antibody molecules (e.g. scFv); and multispecific antibodies formed
from antibody fragments. scFv antibodies are, e.g. described in
Houston, J. S., Methods in Enzymol. 203 (1991) 46-96). In addition,
antibody fragments comprise single chain polypeptides having the
characteristics of a VH domain, namely being able to assemble
together with a VL domain, or of a VL domain, namely being able to
assemble together with a VH domain to a functional antigen binding
site and thereby providing the antigen binding property of full
length antibodies.
[0058] As used herein, "Fab fragment" refers to an antibody
fragment comprising a light chain fragment comprising a VL domain
and a constant domain of a light chain (CL), and a VH domain and a
first constant domain (CH1) of a heavy chain. The bispecific
antibodies of the invention comprise at least two Fab fragments,
wherein either the variable regions or the constant regions of the
heavy and light chain of the second Fab fragment are exchanged. Due
to the exchange of either the variable regions or the constant
regions, said second Fab fragment is also referred to as
"cross-Fab" fragment or "xFab" fragment or "crossover Fab"
fragment. Two different chain compositions of a crossover Fab
molecule are possible and comprised in the bispecific antibodies of
the invention: On the one hand, the variable regions of the Fab
heavy and light chain are exchanged, i.e. the crossover Fab
molecule comprises a peptide chain composed of the light chain
variable region (VL) and the heavy chain constant region (CH1), and
a peptide chain composed of the heavy chain variable region (VH)
and the light chain constant region (CL). This crossover Fab
molecule is also referred to as CrossFab.sub.(VLVH). On the other
hand, when the constant regions of the Fab heavy and light chain
are exchanged, the crossover Fab molecule comprises a peptide chain
composed of the heavy chain variable region (VH) and the light
chain constant region (CL), and a peptide chain composed of the
light chain variable region (VL) and the heavy chain constant
region (CH1). This crossover Fab molecule is also referred to as
CrossFab.sub.(CLCH1).
[0059] In one embodiment said Fab fragments are connected via a
peptide linker. By "connected" is meant that the Fab fragments are
linked by peptide bonds, either directly or via one or more peptide
linker.
The term "peptide linker" as used within the invention denotes a
peptide with amino acid sequences, which is preferably of synthetic
origin. These peptide linkers according to invention are used to
connect one of the Fab fragments to the C- or N-terminus of the
other Fab fragment to form a multispecific antibody according to
the invention. Preferably said peptide linkers are peptides with an
amino acid sequence with a length of at least 5 amino acids,
preferably with a length of 5 to 100, more preferably of 10 to 50
amino acids. In one embodiment said peptide linker is (G.times.S)n
or (G.times.S)nGm with G=glycine, S=serine, and (x=3, n=3, 4, 5 or
6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5 and m=0, 1, 2 or 3),
preferably x=4 and n=2 or 3, more preferably with x=4, n=2.
Additionally, linkers may comprise (a portion of) an immunoglobulin
hinge region. In one embodiment said peptide linker is
(G.sub.4S).sub.2 (SEQ ID: NO 28). Other peptide linkers suitable
for connecting the Fab fragments, for example, (G.sub.4S).sub.6-GG
(SEQ ID NO: 147) or
(SG.sub.3).sub.2-(SEG.sub.3).sub.4-(SG.sub.3)-SG (SEQ ID NO: 148),
or EPKSC(D)-(G.sub.4S).sub.2 (SEQ ID NOs 145 and 146).
[0060] The terms "antigen binding domain" refers to the part of an
antigen binding molecule that comprises the area which specifically
binds to and is complementary to part or all of an antigen. Where
an antigen is large, an antigen binding molecule may only bind to a
particular part of the antigen, which part is termed an epitope. An
antigen binding domain may be provided by, for example, one or more
antibody variable domains (also called antibody variable regions).
Preferably, an antigen binding domain comprises an antibody light
chain variable region (VL) and an antibody heavy chain variable
region (VH).
[0061] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable domains of the heavy
chain and light chain (VH and VL, respectively) of a native
antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three
hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby
Immunology, 6.sup.th ed., W.H. Freeman and Co., page 91 (2007).) A
single VH or VL domain may be sufficient to confer antigen-binding
specificity. Furthermore, antibodies that bind a particular antigen
may be isolated using a VH or VL domain from an antibody that binds
the antigen to screen a library of complementary VL or VH domains,
respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887
(1993); Clarkson et al., Nature 352:624-628 (1991).
[0062] The term "antigen-binding site of an antibody" when used
herein refers to the amino acid residues of an antibody which are
responsible for antigen-binding. The antigen-binding portion of an
antibody comprises amino acid residues from the "complementary
determining regions" or "CDRs". "Framework" or "FR" regions are
those variable domain regions other than the hypervariable region
residues as herein defined. Therefore, the light and heavy chain
variable domains of an antibody comprise from N- to C-terminus the
domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Especially, CDR3
of the heavy chain is the region which contributes most to antigen
binding and defines the antibody's properties. CDR and FR regions
are determined according to the standard definition of Kabat et
al., Sequences of Proteins of Immunological Interest, 5th ed.,
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991) and/or those residues from a "hypervariable loop".
[0063] The term "epitope" includes any polypeptide determinant
capable of specific binding to an antibody. In certain embodiments,
epitope determinant include chemically active surface groupings of
molecules such as amino acids, sugar side chains, phosphoryl, or
sulfonyl, and, in certain embodiments, may have specific three
dimensional structural characteristics, and or specific charge
characteristics. An epitope is a region of an antigen that is bound
by an antibody.
[0064] The term "Fc domain" herein is used to define a C-terminal
region of an immunoglobulin heavy chain that contains at least a
portion of the constant region. For example in natural antibodies,
the Fc domain is composed of two identical protein fragments,
derived from the second and third constant domains of the
antibody's two heavy chains in IgG, IgA and IgD isotypes; IgM and
IgE Fc domains contain three heavy chain constant domains (C.sub.H
domains 2-4) in each polypeptide chain. The bispecific antibodies
of the invention are devoid of the Fc domain. "Devoid of the Fc
domain" as used herein means that the bispecific antibodies of the
invention do not comprise a CH2, CH3 or CH4 domain; i.e. the
constant heavy chain consists solely of one or more CH1
domains.
[0065] "Affinity" refers to the strength of the sum total of
noncovalent interactions between a single binding site of a
molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (KD).
Affinity can be measured by common methods known in the art,
including those described herein. Specific illustrative and
exemplary embodiments for measuring binding affinity are described
in the following.
[0066] As used herein, the term "binding" or "specifically binding"
means that the binding is selective for the antigen and can be
discriminated from unwanted or non-specific interactions. The
ability of an antigen binding moiety to bind to a specific
antigenic determinant can be measured either through an
enzyme-linked immunosorbent assay (ELISA) or other techniques
familiar to one of skill in the art, e.g. surface plasmon resonance
(SPR) technique (analyzed on a BIAcore instrument) (Liljeblad et
al., Glyco J 17, 323-329 (2000)), and traditional binding assays
(Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the
extent of binding of an antibody to an unrelated protein is less
than about 10% of the binding of the antibody to the antigen as
measured, e.g., by SPR. In certain embodiments, an antigen binding
moiety that binds to the antigen, or an antigen binding molecule
comprising that antigen binding moiety, has a dissociation constant
(K.sub.D) of .ltoreq.1 .mu.M, .ltoreq.100 nM, .ltoreq.10 nM,
.ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or .ltoreq.0.001 nM
(e.g. 10.sup.-8 M or less, e.g. from 10.sup.-8 M to 10.sup.-13 M,
e.g., from 10.sup.-9M to 10.sup.-13 M).
[0067] In one embodiment, the extent of binding of a bispecific
antibody that specifically binds to a T-cell activating antigen and
a Tumor Antigen (TA) to an unrelated protein is less than about 10%
of the binding of the antibody to a T-cell activating antigen or a
Tumor Antigen (TA) as measured, e.g., by a radioimmunoassay (RIA)
or flow cytometry (FACS). In certain embodiments, a bispecific
antibody that specifically binds T-cell activating antigen and a
Tumor Antigen (TA) has a dissociation constant (KD) of .ltoreq.1
.mu.M, .ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM,
.ltoreq.0.01 nM, or .ltoreq.0.001 nM (e.g. 10.sup.-8M or less, e.g.
from 10.sup.-8M to 10.sup.-13M, e.g., from 10.sup.-9M to 10.sup.-13
M). In certain embodiments, a bispecific antibody that specifically
binds a T-cell activating antigen and a Tumor Antigen (TA) binds to
an epitope of a T-cell activating antigen or a Tumor Antigen (TA)
that is conserved among a T-cell activating antigen or a Tumor
Antigen (TA) from different species.
[0068] An "affinity matured" antibody refers to an antibody with
one or more alterations in one or more hypervariable regions
(HVRs), compared to a parent antibody which does not possess such
alterations, such alterations resulting in an improvement in the
affinity of the antibody for antigen.
[0069] The terms "A bispecific antibody that specifically binds a T
cell activating antigen and a Tumor Antigen (TA)" refers to a
bispecific antibody that is capable of binding a T cell activating
antigen and a Tumor Antigen with sufficient affinity such that the
antibody is useful in mediating a T-cell mediated immune response
in or near cells expressing a Tumor Antigen. In a particular
embodiment the T cell activating antigen is the CD3 T-Cell
Co-Receptor (CD3) antigen, particularly human or cynomolgus CD3,
most particularly human CD3. In some embodiments, the T cell
activating antigen is the epsilon subunit of CD3. In other
embodiments, the T cell activating antigen is the alpha or beta
subunit of CD3.
[0070] In one embodiment, the bispecific antibody that specifically
binds a T cell activating antigen and a Tumor Antigen (TA) can
compete with monoclonal antibody H2C (described in PCT publication
no. WO2008/119567) for binding an epitope of CD3. In another
embodiment, the bispecific antibody that specifically binds a T
cell activating antigen and a Tumor Antigen (TA) can compete with
monoclonal antibody V9 (described in Rodrigues et al., Int J Cancer
Suppl 7, 45-50 (1992) and U.S. Pat. No. 6,054,297) for binding an
epitope of CD3. In yet another embodiment, the bispecific antibody
that specifically binds a T cell activating antigen and a Tumor
Antigen (TA) can compete with monoclonal antibody FN18 (described
in Nooij et al., Eur J Immunol 19, 981-984 (1986)) for binding an
epitope of CD3.
[0071] An "activating T cell antigen" as used herein refers to an
antigenic determinant expressed on the surface of a T lymphocyte,
particularly a cytotoxic T lymphocyte, which is capable of inducing
T cell activation upon interaction with an antigen binding
molecule. Specifically, interaction of an antigen binding molecule
with an activating T cell antigen may induce T cell activation by
triggering the signaling cascade of the T cell receptor complex. In
a particular embodiment the activating T cell antigen is CD3.
[0072] "T cell activation" as used herein refers to one or more
cellular response of a T lymphocyte, particularly a cytotoxic T
lymphocyte, selected from: proliferation, differentiation, cytokine
secretion, cytotoxic effector molecule release, cytotoxic activity,
and expression of activation markers. The T cell activating
bispecific antigen binding molecules of the invention are capable
of inducing T cell activation. Suitable assays to measure T cell
activation are known in the art described herein.
[0073] The term "CD3 T-Cell Co-Receptor (CD3)", as used herein,
refers to a protein complex and is composed of four distinct
chains. In mammals, the complex contains a CD3.gamma. chain, a
CD3.delta. chain, and two CD3.epsilon. chains. These chains
associate with a molecule known as the T cell receptor (TCR) and
the .zeta.-chain to generate an activation signal in T lymphocytes.
The term "CD3 T-Cell Co-Receptor (CD3)" includes any native CD3
from any vertebrate source, including mammals such as primates
(e.g. humans) and rodents (e.g., mice and rats), unless otherwise
indicated, preferably from a human source. The term encompasses
"full-length," unprocessed CD3 as well as any form of CD3 that
results from processing in the cell. The term also encompasses
naturally occurring variants of CD3, e.g., splice variants or
allelic variants. In a preferred embodiment, the term CD3 T-Cell
Co-Receptor refers to human or cynomolgus CD3, particularly human
CD3. In some embodiments, the T cell activating antigen is the
epsilon subunit of CD3. In other embodiments, the T cell activating
antigen is the alpha or beta subunit of CD3. An exemplary sequence
of human CD3 is given in SEQ ID NO.: 103.
[0074] The term "Tumor Antigen (TA)", as used herein, refers to
tumor-associated antigens as well as tumor-specific antigens, i.e.
any immunogenic epitope (e.g., protein) expressed by a tumor cell.
The protein may be expressed by non tumor cells but be immunogenic
only when expressed by a tumor cell. Alternatively, the protein may
be expressed by tumor cells, but not normal cells. Preferably, an
anti-TA antibody of the invention binds to the extracellular domain
of TA. In one preferred embodiment said Tumor Antigen is a human
Tumor Antigen. Exemplary Tumor Antigens include but are not limited
to Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP,
UniProt Q6UVK1, NCBI Accession NP.sub.--001888), Fibroblast
Activation Protein (FAP, Uni Prot Q12884, Q86Z29, Q99998; NCBI
Accession NP.sub.--004451), Epidermal Growth Factor Receptor (EGFR,
also known as ErbB1 and Her1, UniProt P00533; NCBI Accession
NP.sub.--958439, NP.sub.--958440), Carcinoembryonic Antigen (CEA,
also known as Carcinoembryonic antigen-related cell adhesion
molecule 5 or CD66e; UniProt P06731, NCBI Accession
NP.sub.--004354) and CD33 (also known as gp76 or Sialic
acid-binding Ig-like lectin 3 (Siglec-3), UniProt P20138, NCBI
Accession NP.sub.--001076087, NP.sub.--001171079).
[0075] In one embodiment the bispecific antibody of the invention
comprises at least one antigen binding site that is specific for
Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP).
[0076] In one embodiment the bispecific antibody of the invention
comprises at least one antigen binding site that is specific for
CD33.
[0077] Antibody specificity refers to selective recognition of the
antibody for a particular epitope of an antigen. Natural
antibodies, for example, are monospecific. "Bispecific antibodies"
according to the invention are antibodies which have two different
antigen-binding specificities. Antibodies of the present invention
are specific for two different antigens, i.e. a T-Cell activating
antigen as first antigen and a Tumor Antigen as second antigen.
[0078] The term "monospecific" antibody as used herein denotes an
antibody that has one or more binding sites each of which bind to
the same epitope of the same antigen.
[0079] The term "bispecific" antibody as used herein denotes an
antibody that has at least two binding sites each of which bind to
different epitopes of the same antigen or a different antigen.
[0080] The antibody provided herein is a multispecific antibody,
e.g. a bispecific antibody. Multispecific antibodies are monoclonal
antibodies that have binding specificities for at least two
different sites. Provided herein is a bispecific antibody, with
binding specificities for a Tumor Antigen (TA) and a T-cell
activating antigen. In certain embodiments, bispecific antibodies
may bind to two different epitopes of TA. Bispecific antibodies may
also be used to localize cytotoxic agents to cells which express
TA.
[0081] The term "valent" as used within the current application
denotes the presence of a specified number of binding sites in an
antibody molecule. As such, the terms "bivalent", "tetravalent",
and "hexavalent" denote the presence of two binding sites, four
binding sites, and six binding sites, respectively, in an antibody
molecule. The bispecific antibodies according to the invention are
at least "bivalent" and may be "trivalent" or "multivalent"
(e.g."tetravalent" or "hexavalent").
[0082] Antibodies of the present invention have two or more binding
sites and are bispecific. That is, the antibodies may be bispecific
even in cases where there are more than two binding sites (i.e.
that the antibody is trivalent or multivalent).
[0083] An "antibody that binds to the same epitope" as a reference
antibody refers to an antibody that blocks binding of the reference
antibody to its antigen in a competition assay by 50% or more, and
conversely, the reference antibody blocks binding of the antibody
to its antigen in a competition assay by 50% or more. An exemplary
competition assay is provided herein.
[0084] "No substantial cross-reactivity" means that a molecule
(e.g., an antibody) does not recognize or specifically bind an
antigen different from the actual target antigen of the molecule
(e.g. an antigen closely related to the target antigen),
particularly when compared to that target antigen. For example, an
antibody may bind less than about 10% to less than about 5% to an
antigen different from the actual target antigen, or may bind said
antigen different from the actual target antigen at an amount
selected from the group consisting of less than about 10%, 9%, 8%
7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%, preferably less
than about 2%, 1%, or 0.5%, and most preferably less than about
0.2% or 0.1% antigen different from the actual target antigen.
[0085] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the reference polypeptide sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. For purposes herein, however, % amino acid sequence
identity values are generated using the sequence comparison
computer program ALIGN-2. The ALIGN-2 sequence comparison computer
program was authored by Genentech, Inc., and the source code has
been filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available from Genentech, Inc., South San Francisco, Calif., or may
be compiled from the source code. The ALIGN-2 program should be
compiled for use on a UNIX operating system, including digital UNIX
V4.0D. All sequence comparison parameters are set by the ALIGN-2
program and do not vary.
[0086] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical
matches by the sequence alignment program ALIGN-2 in that program's
alignment of A and B, and where Y is the total number of amino acid
residues in B. It will be appreciated that where the length of
amino acid sequence A is not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not
equal the % amino acid sequence identity of B to A. Unless
specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained as described in the immediately
preceding paragraph using the ALIGN-2 computer program.
[0087] An "isolated" antibody is one which has been separated from
a component of its natural environment. In some embodiments, an
antibody is purified to greater than 95% or 99% purity as
determined by, for example, electrophoretic (e.g., SDS-PAGE,
isoelectric focusing (IEF), capillary electrophoresis) or
chromatographic (e.g., ion exchange or reverse phase HPLC). For
review of methods for assessment of antibody purity, see, e.g.,
Flatman et al., J. Chromatogr. B 848:79-87 (2007).
[0088] An "isolated" nucleic acid refers to a nucleic acid molecule
that has been separated from a component of its natural
environment. An isolated nucleic acid includes a nucleic acid
molecule contained in cells that ordinarily contain the nucleic
acid molecule, but the nucleic acid molecule is present
extrachromosomally or at a chromosomal location that is different
from its natural chromosomal location.
[0089] "Isolated nucleic acid encoding a bispecific antibody that
specifically binds a T-Cell activating antigen and a Tumor Antigen
(TA)" refers to one or more nucleic acid molecules encoding
antibody heavy and light chains (or fragments thereof), including
such nucleic acid molecule(s) in a single vector or separate
vectors, and such nucleic acid molecule(s) present at one or more
locations in a host cell.
[0090] The term "amino acid" as used within this application
denotes the group of naturally occurring carboxy .alpha.-amino
acids comprising alanine (three letter code: ala, one letter code:
A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D),
cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E),
glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine
(leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe,
F), proline (pro, P), serine (ser, S), threonine (thr, T),
tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).
[0091] The term "vector," as used herein, refers to a nucleic acid
molecule capable of propagating another nucleic acid to which it is
linked. The term includes the vector as a self-replicating nucleic
acid structure as well as the vector incorporated into the genome
of a host cell into which it has been introduced. Certain vectors
are capable of directing the expression of nucleic acids to which
they are operatively linked. Such vectors are referred to herein as
"expression vectors."
[0092] As used herein, the expressions "cell", "cell line", and
"cell culture" are used interchangeably and all such designations
include progeny. Thus, the words "transfectants" and "transfected
cells" include the primary subject cell and cultures derived there
from without regard for the number of transfers. It is also
understood that all progeny may not be precisely identical in DNA
content, due to deliberate or inadvertent mutations. Variant
progeny that have the same function or biological activity as
screened for in the originally transformed cell are included.
[0093] The terms "host cell," "host cell line," and "host cell
culture" are used interchangeably and refer to cells into which
exogenous nucleic acid has been introduced, including the progeny
of such cells. Host cells include "transformants" and "transformed
cells," which include the primary transformed cell and progeny
derived therefrom without regard to the number of passages. Progeny
may not be completely identical in nucleic acid content to a parent
cell, but may contain mutations. Mutant progeny that have the same
function or biological activity as screened or selected for in the
originally transformed cell are included herein.
[0094] A "naked antibody" refers to an antibody that is not
conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or
radiolabel. The naked antibody may be present in a pharmaceutical
formulation.
[0095] An "immunoconjugate" is an antibody conjugated to one or
more heterologous molecule(s), including but not limited to a
cytotoxic agent.
[0096] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents a cellular function and/or
causes cell death or destruction. Cytotoxic agents include, but are
not limited to, radioactive isotopes (e.g., At.sup.211, I.sup.131,
I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153,
Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive isotopes of Lu);
chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin,
vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,
melphalan, mitomycin C, chlorambucil, daunorubicin or other
intercalating agents); growth inhibitory agents; enzymes and
fragments thereof such as nucleolytic enzymes; antibiotics; toxins
such as small molecule toxins or enzymatically active toxins of
bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof; and the various antitumor or anticancer
agents disclosed below.
[0097] The term "N-terminus" denotes the last amino acid of the
N-terminus, the term "C-terminus" denotes the last amino acid of
the C-terminus.
[0098] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the formulation would be
administered.
[0099] A "pharmaceutically acceptable carrier" refers to an
ingredient in a pharmaceutical formulation, other than an active
ingredient, which is nontoxic to a subject. A pharmaceutically
acceptable carrier includes, but is not limited to, a buffer,
excipient, stabilizer, or preservative.
[0100] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to clinical
intervention in an attempt to alter the natural course of the
individual being treated, and can be performed either for
prophylaxis or during the course of clinical pathology. Desirable
effects of treatment include, but are not limited to, preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some embodiments, antibodies of
the invention are used to delay development of a disease or to slow
the progression of a disease.
[0101] An "individual" or "subject" is a mammal. Mammals include,
but are not limited to, domesticated animals (e.g., cows, sheep,
cats, dogs, and horses), primates (e.g., humans and non-human
primates such as monkeys), rabbits, and rodents (e.g., mice and
rats). In certain embodiments, the individual or subject is a
human.
[0102] An "effective amount" of an agent, e.g., a pharmaceutical
formulation, refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired therapeutic or
prophylactic result.
[0103] The term "cancer" as used herein refers to proliferative
diseases, such as lymphomas, lymphocytic leukemias, lung cancer,
non small cell lung (NSCL) cancer, bronchioloalviolar cell lung
cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the
head or neck, cutaneous or intraocular melanoma, uterine cancer,
ovarian cancer, rectal cancer, cancer of the anal region, stomach
cancer, gastric cancer, colon cancer, breast cancer, uterine
cancer, carcinoma of the fallopian tubes, carcinoma of the
endometrium, carcinoma of the cervix, carcinoma of the vagina,
carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus,
cancer of the small intestine, cancer of the endocrine system,
cancer of the thyroid gland, cancer of the parathyroid gland,
cancer of the adrenal gland, sarcoma of soft tissue, cancer of the
urethra, cancer of the penis, prostate cancer, cancer of the
bladder, cancer of the kidney or ureter, renal cell carcinoma,
carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer,
biliary cancer, neoplasms of the central nervous system (CNS),
spinal axis tumors, brain stem glioma, glioblastoma multiforme,
astrocytomas, schwanomas, ependymonas, medulloblastomas,
meningiomas, squamous cell carcinomas, pituitary adenoma and Ewings
sarcoma, including refractory versions of any of the above cancers,
or a combination of one or more of the above cancers.
[0104] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
II. Compositions and Methods
[0105] In one aspect, the invention is based, in part, on
bispecific antibodies comprising a first antigen binding site
specific for a T-cell activating antigen and a second antigen
binding site specific for a Tumor Antigen (TA). Antibodies of the
invention are useful, e.g., for the treatment of cancer.
[0106] A. Exemplary Bispecific Antibodies that Bind to a T-Cell
Activating Antigen and a Tumor Antigen (TA)
[0107] The present invention relates to bispecific antibodies
combining a T-cell activating antigen binding site with a second
antigen binding site that targets a Tumor Antigen (TA). The
antibodies of the invention specifically bind to a Tumor Antigen on
the surface of a tumor cell and at the same time bind to an antigen
on the surface of cytotoxic T lymphocytes. Preferably said antigen
is a CD3 T-Cell Co-Receptor (CD3) antigen. The bispecific antibody
is capable to elicit an immune response specifically at the site of
the tumor, subsequently resulting in apoptosis of the target
cell.
[0108] In a particular embodiment according to the invention, the T
cell activating bispecific antibody is capable of simultaneous
binding to a tumor cell antigen, and an activating T cell antigen.
In one embodiment, the T cell activating bispecific antibody is
capable of crosslinking a T cell and a tumor cell by simultaneous
binding to a tumor cell antigen and an activating T cell antigen.
In an even more particular embodiment, such simultaneous binding
results in lysis of the tumor cell. In one embodiment, such
simultaneous binding results in activation of the T cell. In other
embodiments, such simultaneous binding results in a cellular
response of a T lymphocyte, particularly a cytotoxic T lymphocyte,
selected from the group of: proliferation, differentiation,
cytokine secretion, cytotoxic effector molecule release, cytotoxic
activity, and expression of activation markers. In one embodiment,
binding of the T cell activating bispecific antibody to the
activating T cell antigen without simultaneous binding to the
target cell antigen does not result in T cell activation.
In one embodiment, the T cell activating bispecific antibody is
capable of re-directing cytotoxic activity of a T cell to a target
cell. In a particular embodiment, said re-direction is independent
of MHC-mediated peptide antigen presentation by the target cell
and/or specificity of the T cell. Particularly, a T cell according
to any of the embodiments of the invention is a cytotoxic T cell.
In some embodiments the T cell is a CD4.sup.+ or a CD8.sup.+ T
cell, particularly a CD8.sup.+ T cell.
[0109] In one embodiment bispecific antibodies are provided that
specifically bind a T-cell activating antigen and a Tumor Antigen
(TA), comprising a first Fab fragment and a second Fab fragment,
wherein either the variable regions or the constant regions of the
second Fab heavy and light chain are exchanged; and wherein the
bispecific antibody does not comprise a Fc domain.
[0110] In one aspect, a bispecific antibody that specifically binds
a T-cell activating antigen and a Tumor Antigen (TA) is provided,
comprising at least two fab fragments, wherein the first Fab
fragment comprises at least one antigen binding site specific for a
Tumor Antigen (TA); and the second Fab fragment comprises at least
one antigen binding site specific for a T-cell activating antigen,
wherein either the variable regions or the constant regions of the
second Fab heavy and light chain are exchanged; and wherein the
bispecific antibody is devoid of a Fc domain.
[0111] In a particular embodiment the T cell activating antigen is
the CD3 T-Cell Co-Receptor (CD3) antigen, particularly human or
cynomolgus CD3, most particularly human CD3. In some embodiments,
the T cell activating antigen is the epsilon subunit of CD3. In
other embodiments, the T cell activating antigen is the alpha or
beta subunit of CD3.
[0112] In one aspect, a bispecific antibody that specifically binds
CD3 T-Cell Co-Receptor (CD3) antigen and a Tumor Antigen (TA) is
provided, comprising at least two fab fragments, wherein the first
Fab fragment comprises at least one antigen binding site specific
for a Tumor Antigen (TA); and the second Fab fragment comprises at
least one antigen binding site specific for a CD3 T-Cell
Co-Receptor (CD3) wherein either the variable regions or the
constant regions of the second Fab heavy and light chain are
exchanged; and wherein the bispecific antibody is devoid of a Fc
domain.
[0113] In one embodiment the first and second Fab fragments are
connected via a peptide linker. Preferably said peptide linker is a
peptide with an amino acid sequence with a length of at least 5
amino acids, preferably with a length of 5 to 100, more preferably
of 10 to 50 amino acids. In one embodiment said peptide linker is
(G.times.S)n or (G.times.S)nGm with G=glycine, S=serine, and (x=3,
n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5 and
m=0, 1, 2 or 3), preferably x=4 and n=2 or 3, more preferably with
x=4, n=2. In one embodiment said peptide linker is
(G.sub.4S).sub.2. The peptide linker is used to connect the first
and the second Fab fragment.
[0114] In one embodiment the first Fab fragment is connected to the
C- or N-terminus of the second Fab fragment.
[0115] In one embodiment the first Fab fragment is connected to the
N-terminus of the second Fab fragment. Depending on whether the
variable or the constant domains of the heavy and the light chains
of the second Fab fragment are exchanged, different bispecific
antibody molecules are possible when the first Fab fragment is
connected to the N-terminus of the second Fab fragment.
[0116] In one embodiment the variable domains of the second Fab
fragment are exchanged (i.e. the second Fab fragment is a
CrossFab.sub.(VHVL)), and the C-terminus of the heavy or light
chain of the first Fab fragment is connected to the N-terminus of
the VLCH1 chain of the second Fab fragment. Preferably, the
C-terminus heavy chain of the first Fab fragment is connected to
the N-terminus of the VLCH1 chain of the second Fab fragment. Thus,
in one embodiment the bispecific antibody comprises three chains: a
light chain (VLCL) of the first Fab fragment, the heavy chain of
the first Fab fragment connected to the VLCH1 chain of the second
Fab fragment via a peptide linker (VHCH1-linker-VLCH1) and a VHCL
chain of the second Fab fragment.
[0117] In another embodiment the constant domains of the second Fab
fragment are exchanged (i.e the second Fab fragment is a
CrossFab.sub.(CLCH1)) and the C-terminus of the heavy or light
chain of the first Fab fragment is connected to the N-terminus of
the VHCL chain of the second Fab fragment. Preferably, the
C-terminus of the heavy chain of the first Fab fragment is
connected to the N-terminus of the VHCL chain of the second Fab
fragment. Thus, in one embodiment the bispecific antibody comprises
three chains: a light chain (VLCL) of the first Fab fragment, the
heavy chain of the first Fab fragment connected to the VHCL chain
of the second Fab fragment via a peptide linker (VHCH1-linker-VHCL)
and a VLCH1 chain of the second Fab fragment.
[0118] In one embodiment the first Fab fragment is connected to the
C-terminus of the second Fab fragment. Depending on whether the
variable or the constant domains of the heavy and the light chains
of the second Fab fragment are exchanged different bispecific
antibody molecules are possible when the first Fab fragment is
connected to the C-terminus of the second Fab fragment.
[0119] In one embodiment the variable domains of the second Fab
fragment are exchanged (i.e. the second Fab fragment is a
CrossFab.sub.(VHVL)), and the CH1 domain of the second Fab fragment
is connected to the N-terminus of the heavy or light chain of the
first Fab fragment. Preferably, the CH1 domain of the second Fab
fragment is connected to the N-terminus of the heavy chain of the
first Fab fragment. Thus, in one embodiment the bispecific antibody
comprises three chains: a light chain (VLCL) of the first Fab
fragment, the VLCH1 chain of the second Fab fragment connected to
the heavy chain of the first Fab fragment via a peptide linker
(VLCH1-linker-VHCH1) and a VHCL chain of the second Fab
fragment.
[0120] In another embodiment the constant domains of the second Fab
fragment are exchanged (i.e. the second Fab fragment is a
CrossFab.sub.(CLCH1)), and the CL domain of the second Fab fragment
is connected to the N-terminus of the heavy of light chain of the
first Fab fragment. Preferably, the CL domain of the second Fab
fragment is connected to the N-terminus of the heavy chain of the
first Fab fragment. Thus, in one embodiment the bispecific antibody
comprises three chains: a light chain (VLCL) of the first Fab
fragment, the VHCL chain of the second Fab fragment connected to
the heavy chain of the first Fab fragment via a peptide linker
(VLCH1-linker-VHCH1) and a VLCH1 chain of the second Fab
fragment.
[0121] The bispecific antibodies according to the invention are at
least bivalent and can be trivalent or multivalent e.g. tetravalent
or hexavalent. In one embodiment said bispecific antibodies are
bivalent (1+1 format) with one binding site each targeting a Tumor
Antigen (TA) and a T-cell activating antigen, respectively. In
another embodiment said bispecific antibodies are trivalent (2+1
format) with two binding sites each targeting a Tumor Antigen (TA)
and one binding site targeting a T-cell activating antigen, as
detailed in the following section.
[0122] In one embodiment said antibody additionally comprises a
third Fab fragment. In one embodiment said third Fab fragment
comprises at least one antigen binding site specific for a Tumor
Antigen. In one embodiment the antigen binding site of said third
Fab fragment is specific for the same Tumor Antigen as the antigen
binding site of the first Fab fragment.
[0123] In one embodiment the third Fab fragment is connected to the
N or C-terminus of the first Fab fragment. In one embodiment the
third Fab fragment is connected to the first Fab fragment via a
peptide linker. Preferably said peptide linker is a (G4S)2
linker.
[0124] In one embodiment the third Fab fragment is connected to the
N or C-terminus of the light chain or the heavy chain of the first
Fab fragment. Depending on which terminus of the first Fab fragment
is connected to the second Fab fragment (as detailed above), the
third Fab fragment is connected on the opposite (free) terminus of
the first fragment.
[0125] In one embodiment, the bispecific antibody of the invention
comprises three Fab fragments wherein said Fab fragments and said
linker are connected in the following order from N-terminal to
C-terminal direction: Fab fragment 3-linker-Fab fragment
1-linker-Fab fragment 2, wherein either the variable regions or the
constant regions of the heavy and light chain of the second Fab
fragment are exchanged. In this embodiment the C-terminus of the
third Fab fragment is connected to the N-terminus of the first Fab
fragment. As detailed above, the Fab fragments can be connected to
each other via the heavy or the light chains. In one embodiment the
C-terminus of the heavy chain of the third Fab fragment is
connected to the N-terminus of the heavy chain of the first Fab
fragment via a peptide linker; and the C-terminus of the first Fab
fragment is connected to the N-terminus of the second Fab fragment,
wherein either the variable regions or the constant regions of the
heavy and light chain of the second Fab fragment are exchanged.
Depending on whether the variable or the constant domains of the
heavy and the light chains of the second Fab fragment are exchanged
different bispecific antibody molecules are possible.
[0126] In one embodiment the variable domains of the second Fab
fragment are exchanged (i.e. the second Fab fragment is a
CrossFab.sub.(VHVL)), and the chains of the three Fab fragments are
connected in the following order from N-terminal to C-terminal
direction: VHCH1-linker-VHCH1-linker-VLCH1. In one embodiment the
bispecific antibody comprises four chains: a light chain (VLCL) of
the third Fab fragment, a light chain (VLCL) of the first Fab
fragment, the heavy chain of the third fragment connected to the
heavy chain of the first Fab fragment which itself is connected to
the VLCH1 chain of the second Fab fragment via a peptide linker
(VHCH1-linker-VHCH1-linker-VLCH1) and a VHCL chain of the second
Fab fragment.
[0127] In one embodiment the constant domains of the second Fab
fragment are exchanged (i.e. the second Fab fragment is a
CrossFab.sub.(CLCH1)), and the chains of the three Fab fragments
are connected in the following order from N-terminal to C-terminal
direction: VHCH1-linker-VHCH1-linker-VHCL. In one embodiment the
bispecific antibody comprises four chains: a light chain (VLCL) of
the third Fab fragment, a light chain (VLCL) of the first Fab
fragment, the heavy chain of the third fragment connected to the
heavy chain of the first Fab fragment which itself is connected to
the VHCL chain of the second Fab fragment via a peptide linker
(VHCH1-linker-VHCH1-linker-VHCL) and a VLCH1 chain of the second
Fab fragment.
[0128] In one embodiment the bispecific antibody of the invention
comprises three Fab fragments wherein said Fab fragments and said
linker are connected in the following order from N-terminal to
C-terminal direction: Fab fragment 2-linker-Fab fragment
1-linker-Fab fragment 3, wherein either the variable regions or the
constant regions of the heavy and light chain of the second Fab
fragment are exchanged. In this embodiment the N-terminus of the
third Fab fragment is connected to the C-terminus of the first Fab
fragment. As detailed above, the Fab fragments can be connected to
each other via the heavy or the light chains. In one embodiment the
N-terminus of the heavy chain of the third Fab fragment is
connected to the C-terminus of the heavy chain of the first Fab
fragment via a peptide linker; and the N-terminus of the first Fab
fragment is connected to the C-terminus of the second Fab fragment,
wherein either the variable regions or the constant regions of the
heavy and light chain of the second Fab fragment are exchanged.
Depending on whether the variable or the constant domains of the
heavy and the light chains of the second Fab fragment are exchanged
different bispecific antibody molecules are possible.
[0129] In one embodiment the variable domains of the second Fab
fragment are exchanged (i.e. the second Fab fragment is a
CrossFab.sub.(VHVL)), and the chains of the three Fab fragments are
connected in the following order from N-terminal to C-terminal
direction: VLCH1-linker-VHCH1-linker-VHCH1. In one embodiment the
bispecific antibody comprises four chains: a light chain (VLCL) of
the third Fab fragment, a light chain (VLCL) of the first Fab
fragment, the VLCH1 chain of the second Fab fragment connected to
the heavy chain of the first fragment which itself is connected to
the heavy chain of the first Fab fragment via a peptide linker
(VLCH1-linker-VHCH1-linker-VHCH1) and a VHCL chain of the second
Fab fragment.
[0130] In one embodiment the constant domains of the second Fab
fragment are exchanged (i.e. the second Fab fragment is a
CrossFab.sub.(CLCH1)), and the chains of the three Fab fragments
are connected in the following order from N-terminal to C-terminal
direction: VHCL-linker-VHCH1-linker-VHCH1. In one embodiment the
bispecific antibody comprises four chains: a light chain (VLCL) of
the third Fab fragment, a light chain (VLCL) of the first Fab
fragment, the VHCL chain of the second Fab fragment connected to
the heavy chain of the first fragment which itself is connected to
the heavy chain of the first Fab fragment via a peptide linker
(VHCL-linker-VHCH1-linker-VHCH1) and a VLCH1 chain of the second
Fab fragment.
[0131] In another embodiment the third Fab fragment is connected to
N or C-terminus of the light chain or the heavy chain of the second
Fab fragment. In one embodiment the third Fab fragment is connected
to the second Fab fragment via a peptide linker. Preferably said
peptide linker is a (G4S)2 linker. As detailed above, the Fab
fragments can be connected to each other via the heavy or the light
chains.
[0132] In one embodiment the bispecific antibody of the invention
comprises three Fab fragments wherein said Fab fragments and said
linker are connected in the following order from N-terminal to
C-terminal direction: Fab fragment 1-linker-Fab fragment
2-linker-Fab fragment 3, wherein either the variable regions or the
constant regions of the heavy and light chain of the second Fab
fragment are exchanged. In one embodiment the N-terminus of the
third Fab fragment is connected to the C-terminus of the second Fab
fragment.
[0133] In another embodiment the C-terminus of the heavy chain of
the third Fab fragment is connected to the N-terminus of the second
Fab fragment via a peptide linker; and the N-terminus of the first
Fab fragment is connected to the C-terminus of the second Fab
fragment, wherein either the variable regions or the constant
regions of the heavy and light chain of the second Fab fragment are
exchanged.
[0134] Depending on whether the variable or the constant domains of
the heavy and the light chains of the second Fab fragment are
exchanged different bispecific antibody molecules are possible.
[0135] In one embodiment the variable domains of the second Fab
fragment are exchanged (i.e. the second Fab fragment is a
CrossFab.sub.(VHVL)), and the chains of the three Fab fragments are
connected in the following order from N-terminal to C-terminal
direction: VHCH1-linker-VLCH1-linker-VHCH1. In one embodiment the
bispecific antibody comprises four chains: a light chain (VLCL) of
the third Fab fragment, a light chain (VLCL) of the first Fab
fragment, the heavy chain of the third fragment connected to to the
N-terminus of the VLCH1 chain of the second Fab fragment, and the
C-terminus of said VLCH1 chain connected to the N-terminus of the
heavy chain of the first Fab fragment via a peptide linker
(VHCH1-linker-VLCH1-linker-VHCH1) and a VHCL chain of the second
Fab fragment.
[0136] In one embodiment the constant domains of the second Fab
fragment are exchanged (i.e. the second Fab fragment is a
CrossFab.sub.(CLCH1)), and the chains of the three Fab fragments
are connected in the following order from N-terminal to C-terminal
direction: VHCH1-linker-VHCL-linker-VHCH1. In one embodiment the
bispecific antibody comprises four chains: a light chain (VLCL) of
the third Fab fragment, a light chain (VLCL) of the first Fab
fragment, the heavy chain of the third fragment connected to to the
N-terminus of the VHCL chain of the second Fab fragment, and the
C-terminus of said VHCL chain connected to the N-terminus of the
heavy chain of the first Fab fragment via a peptide linker
(VHCH1-linker-VHCL-linker-VHCH1) and a VLCH1 chain of the second
Fab fragment.
[0137] In one embodiment, the antigen binding site of said third
Fab fragment is specific for the same Tumor Antigen as the antigen
binding site of the first Fab fragment, and the bispecific antibody
of the invention comprises three Fab fragments connected via a
peptide linker in the following order (either from N-terminal to
C-terminal direction or from C-terminal to N-terminal direction):
Fab.sub.(TA)-linker-Fab.sub.(TA)-linker-xFab.sub.(T-cell activating
antigen), wherein Fab.sub.(TA) denotes a Fab fragment with antigen
binding site specific for a Tumor Antigen and xFab.sub.(T-cell
activating antigen) denotes a Fab fragment with antigen binding
site specific for a T-cell activating antigen, wherein either the
variable regions or the constant regions of the heavy and light
chain are exchanged.
[0138] In one embodiment, the antigen binding site of said third
Fab fragment is specific for the same Tumor Antigen as the antigen
binding site of the first Fab fragment, and the bispecific antibody
of the invention comprises three Fab fragments connected via a
peptide linker in the following order (either from N-terminal to
C-terminal direction or from C-terminal to N-terminal direction):
Fab.sub.(TA)-linker-xFab.sub.(T-cell activating
antigen)-linker-Fab.sub.(TA), wherein Fab.sub.(TA) denotes a Fab
fragment with antigen binding site specific for a Tumor Antigen and
xFab.sub.(T-cell activating antigen) denotes a Fab fragment with
antigen binding site specific for a T-cell activating antigen,
wherein either the variable regions or the constant regions of the
heavy and light chain are exchanged.
[0139] In one embodiment the bispecific antibody comprises an
antigen binding moiety that can compete with monoclonal antibody V9
for binding to an epitope of CD3. See for example Rodigues et al.,
Int J Cancer Suppl 7 (1992), 45-50; U.S. Pat. No. 6,054,297,
incorporated herein by reference in its entirety.
[0140] In one embodiment the bispecific antibody comprises an
antigen binding moiety that can compete with monoclonal antibody
FN18 for binding to an epitope of CD3. See Nooij et al., Eur J
Immunol 19 (1986), 981-984, incorporated herein by reference in its
entirety.
[0141] In one embodiment the bispecific antibody comprises an
antigen binding moiety that can compete with monoclonal antibody
CH2527 (Sequence ID 157 and 158) or an affinity matured variant
thereof for binding to an epitope of CD3.
[0142] In one embodiment the bispecific antibody comprises a second
Fab fragment specifically binding to CD3, wherein the heavy chain
variable region comprises a CDR1 of SEQ ID. NO. 10 or SEQ ID. NO.
32, a CDR2 of SEQ ID. NO. 11 or SEQ ID. NO. 33, and a CDR3 of SEQ
ID. NO. 12 or SEQ ID. NO. 34; and wherein the light chain variable
region comprises a CDR1 of SEQ ID. NO. 7 or SEQ ID. NO. 29, a CDR2
of SEQ ID. NO. 8 or SEQ ID. NO. 30, and a CDR3 of SEQ ID. NO. 9 or
SEQ ID. NO. 31.
[0143] In one embodiment the bispecific antibody comprises a second
Fab fragment specifically binding to CD3, wherein the heavy chain
variable region comprises a CDR1 of SEQ ID. NO. 10, a CDR2 of SEQ
ID. NO. 11, and a CDR3 of SEQ ID. NO. 12; and wherein the light
chain variable region comprises a CDR1 of SEQ ID. NO. 7, a CDR2 of
SEQ ID. NO. 8 and a CDR3 of SEQ ID. NO. 9.
[0144] In one embodiment the bispecific antibody comprises a second
Fab fragment specifically binding to CD3, wherein the heavy chain
variable region comprises a CDR1 of SEQ ID. NO. 32, a CDR2 SEQ ID.
NO. 33, and a CDR3 of SEQ ID. NO. 34; and wherein the light chain
variable region comprises a CDR1 of SEQ ID. NO. 29, a CDR2 of SEQ
ID. NO. 30, and a CDR3 of SEQ ID. NO. 31.
[0145] In one embodiment the bispecific antibody comprises a light
chain and a heavy chain of a second Fab fragment specifically
binding to CD3, wherein the heavy chain variable region sequence is
at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID. NO. 20 or SEQ ID. NO. 36; wherein the light
chain variable region sequence is at least about 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID. NO. 19. or SEQ
ID. NO 35, or variants thereof that retain functionality. In one
embodiment the bispecific antibody comprises a light chain and a
heavy chain of a second Fab fragment specifically binding to CD3,
wherein the heavy chain variable region comprises an amino acid
sequence of SEQ ID. NO. 20; and a light chain variable region
comprising an amino acid sequence of SEQ ID. NO. 19 or variants
thereof that retain functionality.
[0146] In one embodiment the bispecific antibody comprises a light
chain and a heavy chain of a second Fab fragment specifically
binding to CD3, wherein the heavy chain variable region comprises
an amino acid sequence of SEQ ID. NO. 36; and a light chain
variable region comprising an amino acid sequence of SEQ ID. NO. 35
or variants thereof that retain functionality.
[0147] In one embodiment the bispecific antibody comprises a light
chain and a heavy chain of a second Fab fragment specifically
binding to CD3, wherein the heavy chain variable region comprises
an amino acid sequence of SEQ ID. NO. 158; and a light chain
variable region comprising an amino acid sequence of SEQ ID. NO.
157 or variants thereof that retain functionality. In one
embodiment the bispecific antibody comprises a light chain and a
heavy chain of a second Fab fragment specifically binding to CD3,
wherein the heavy chain variable region sequence is at least about
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID.
NO. 158; wherein the light chain variable region sequence is at
least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID. NO. 157, or variants thereof that retain
functionality. In one embodiment the bispecific antibody comprises
a light chain and a heavy chain of a second Fab fragment
specifically binding to CD3, wherein the heavy chain variable
region sequence is an affinity matured variant of SEQ ID. NO. 158
and wherein the light chain variable region sequence is an affinity
matured variant of SEQ ID. NO. 157. Affinity matured variants in
this embodiment means that independently 1, 2, 3 or 4 amino acids
of SEQ ID. NO. 158 and/or SEQ ID. NO. 157 are exchanged.
[0148] In one embodiment, the bispecific antibody comprises a light
chain and a heavy chain of second Fab fragment specifically binding
to CD3, wherein said heavy chain comprises a constant region
comprising the amino acid sequence of SEQ ID NO: 22 or SEQ ID. NO.
38 or variants thereof that retain functionality. In one
embodiment, the bispecific antibody comprises a light chain and a
heavy chain of a second Fab fragment specifically binding to CD3,
wherein said heavy chain comprises a constant region comprising the
amino acid sequence of SEQ ID NO: 22 or SEQ ID. NO 38, and a light
chain and a heavy chain of first Fab fragment specific for a Tumor
Antigen (TA) comprising one or more amino acid sequences as defined
in any of the embodiments described herein.
[0149] In one embodiment, the bispecific antibody comprises a light
chain and a heavy chain of second Fab fragment specifically binding
to CD3, wherein said heavy chain comprises a constant region
comprising the amino acid sequence of SEQ ID NO: 22. In one
embodiment, the bispecific antibody comprises a light chain and a
heavy chain of a second Fab fragment specifically binding to CD3,
wherein said heavy chain comprises a constant region comprising the
amino acid sequence of SEQ ID NO: 22, and a light chain and a heavy
chain of first Fab fragment specific for a Tumor Antigen (TA)
comprising one or more amino acid sequences as defined in any of
the embodiments described herein.
[0150] In one embodiment, the bispecific antibody comprises a light
chain and a heavy chain of a second Fab fragment specifically
binding to CD3, wherein said light chain comprises a constant
region comprising the amino acid sequence of SEQ ID NO: 21 or SEQ
ID. NO. 37. In one embodiment, the bispecific antibody comprises a
light chain and a heavy chain of a second Fab fragment specifically
binding to CD3, wherein said light chain comprises a constant
region comprising the amino acid sequence of SEQ ID NO: 21 or SEQ
ID. NO. 37, and a light chain and a heavy chain of a first Fab
fragment specific for a Tumor Antigen (TA) comprising one or more
amino acid sequences as defined in any of the embodiments described
herein.
[0151] In one embodiment, the bispecific antibody comprises a light
chain and a heavy chain of a second Fab fragment specifically
binding to CD3, wherein said light chain comprises a constant
region comprising the amino acid sequence of SEQ ID NO: 21. In one
embodiment, the bispecific antibody comprises a light chain and a
heavy chain of a second Fab fragment specifically binding to CD3,
wherein said light chain comprises a constant region comprising the
amino acid sequence of SEQ ID NO: 21, and a light chain and a heavy
chain of a first Fab fragment specific for a Tumor Antigen (TA)
comprising one or more amino acid sequences as defined in any of
the embodiments described herein.
[0152] In yet another specific embodiment, a bispecific antibody of
the invention comprises a light chain and a heavy chain of a second
Fab fragment specifically binding to CD3, said heavy chain
comprising a heavy chain constant region comprising the amino acid
sequence of SEQ ID NO: 22 or SEQ ID. NO. 38; and said light chain
comprising a light chain constant region comprising the amino acid
sequence of SEQ ID NO: 21 or SEQ ID. NO. 37.
[0153] In yet another specific embodiment, a bispecific antibody of
the invention comprises a light chain and a heavy chain of a second
Fab fragment specifically binding to CD3, said heavy chain
comprising a heavy chain constant region comprising the amino acid
sequence of SEQ ID NO: 22; and said light chain comprising a light
chain constant region comprising the amino acid sequence of SEQ ID
NO: 21.
[0154] In yet another specific embodiment, a bispecific antibody of
the invention comprises a light chain and a heavy chain of a second
Fab fragment specifically binding to CD3, said heavy chain
comprising a heavy chain constant region comprising the amino acid
sequence of SEQ ID NO: 22; and said light chain comprising a light
chain constant region comprising the amino acid sequence of SEQ ID
NO: 21, and a light chain and a heavy chain of a first Fab fragment
specific for a Tumor Antigen (TA) comprising one or more amino acid
sequences as defined in any of the embodiments described
herein.
[0155] In one embodiment the bispecific antibody of the invention
comprises a light chain and a heavy chain of a second Fab fragment
specifically binding to CD3, comprising a variable light chain of
SEQ ID NO: 19 and a variable heavy chain of SEQ ID NO: 20, and a
heavy chain constant region comprising the amino acid sequence of
SEQ ID NO: 22, and a light chain constant region comprising the
amino acid sequence of SEQ ID NO: 21.
[0156] In one embodiment the bispecific antibody of the invention
comprises a light chain and a heavy chain of a second Fab fragment
specifically binding to CD3, comprising a variable light chain of
SEQ ID NO: 19 and a variable heavy chain of SEQ ID NO: 20, and a
heavy chain constant region comprising the amino acid sequence of
SEQ ID NO: 22, and a light chain constant region comprising the
amino acid sequence of SEQ ID NO: 21, and a light chain and a heavy
chain of a first Fab fragment specific for a Tumor Antigen (TA)
comprising one or more amino acid sequences as defined in any of
the embodiments described herein.
[0157] In one embodiment the bispecific antibody of the invention
comprises a light chain and a heavy chain of a second Fab fragment
specifically binding to CD3, comprising a variable light chain of
SEQ ID NO: 35 and a variable heavy chain of SEQ ID NO: 36, and a
heavy chain constant region comprising the amino acid sequence of
SEQ ID NO: 38, and a light chain constant region comprising the
amino acid sequence of SEQ ID NO: 37.
[0158] In one embodiment the bispecific antibody of the invention
comprises a light chain and a heavy chain of a second Fab fragment
specifically binding to CD3, comprising a variable light chain of
SEQ ID NO: 35 and a variable heavy chain of SEQ ID NO: 36, and a
heavy chain constant region comprising the amino acid sequence of
SEQ ID NO: 38, and a light chain constant region comprising the
amino acid sequence of SEQ ID NO: 37, and a light chain and a heavy
chain of a first Fab fragment specific for a Tumor Antigen (TA)
comprising one or more amino acid sequences as defined in any of
the embodiments described herein.
[0159] In one embodiment the bispecific antibody of the invention
comprises a light chain and a heavy chain of a second Fab fragment
specifically binding to CD3, comprising a variable light chain of
SEQ ID NO: 157 and a variable heavy chain of SEQ ID NO: 158, and a
heavy chain constant region comprising the amino acid sequence of
SEQ ID NO: 22, and a light chain constant region comprising the
amino acid sequence of SEQ ID NO: 21.
[0160] In one embodiment the bispecific antibody of the invention
comprises a light chain and a heavy chain of a second Fab fragment
specifically binding to CD3, comprising a variable light chain of
SEQ ID NO: 157 or an affinity matured variant thereof and a
variable heavy chain of SEQ ID NO: 158 or an affinity matured
variant thereof, and a heavy chain constant region comprising the
amino acid sequence of SEQ ID NO: 22, and a light chain constant
region comprising the amino acid sequence of SEQ ID NO: 21.
Affinity matured variants in this embodiment means that
independently 1, 2, 3 or 4 amino acids of SEQ ID. NO. 158 and/or
SEQ ID. NO. 157 are exchanged.
[0161] In one embodiment the bispecific antibody of the invention
comprises a light chain and a heavy chain of a second Fab fragment
specifically binding to CD3, comprising a variable light chain of
SEQ ID NO: 157 and a variable heavy chain of SEQ ID NO: 158, and a
heavy chain constant region comprising the amino acid sequence of
SEQ ID NO: 22, and a light chain constant region comprising the
amino acid sequence of SEQ ID NO: 21, and a light chain and a heavy
chain of a first Fab fragment specific for a Tumor Antigen (TA)
comprising one or more amino acid sequences as defined in any of
the embodiments described herein.
[0162] In one embodiment the bispecific antibody of the invention
comprises a light chain and a heavy chain of a second Fab fragment
specifically binding to CD3, comprising a variable light chain of
SEQ ID NO: 157 or an affinity matured variant thereof and a
variable heavy chain of SEQ ID NO: 158 or an affinity matured
variant thereof, and a heavy chain constant region comprising the
amino acid sequence of SEQ ID NO: 22, and a light chain constant
region comprising the amino acid sequence of SEQ ID NO: 21 and a
heavy chain of a first Fab fragment specific for a Tumor Antigen
(TA) comprising one or more amino acid sequences as defined in any
of the embodiments described herein. Affinity matured variants in
this embodiment means that independently 1, 2, 3 or 4 amino acids
of SEQ ID. NO. 158 and/or SEQ ID. NO. 157 are exchanged.
[0163] In one embodiment the Tumor Antigen is selected from the
group of Melanoma-associated Chondroitin Sulfate Proteoglycan
(MCSP), Epidermal Growth Factor Receptor (EGFR), Carcinoembryonic
Antigen (CEA), Fibroblast Activation Protein (FAP) and CD33. In one
preferred embodiment the Tumor Antigen is MCSP.
[0164] In one embodiment the T cell activating bispecific antibody
comprises at least one antigen binding site that is specific for
Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP). In
another embodiment the T cell activating bispecific antibody
comprises at least one, typically two or more antigen binding
moieties that can compete with monoclonal antibody M4-3 ML2
(Sequence ID 161 and 162) or an affinity matured variant thereof
for binding to an epitope of MCSP.
[0165] In one embodiment the bispecific antibody comprises a light
chain and a heavy chain of a first Fab fragment specifically
binding to MCSP, wherein the variable heavy chain comprises a CDR1
of SEQ ID. NO. 4, a CDR2 of SEQ ID. NO. 5, a CDR3 of SEQ ID. NO. 6;
and the variable light chain comprises a CDR1 of SEQ ID. NO. 1, a
CDR2 of SEQ ID. NO. 2, and a CDR3 of SEQ ID. NO. 3.
[0166] In one embodiment the bispecific antibody comprises a light
chain and a heavy chain of a first Fab fragment specifically
binding to MCSP, wherein the heavy chain variable region sequence
that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or
100% identical to SEQ ID. NO. 14; and a light chain variable region
is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID. NO. 13.
[0167] In one embodiment the bispecific antibody comprises a light
chain and a heavy chain of a first Fab fragment specifically
binding to MCSP, wherein the heavy chain variable region sequence
that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or
100% identical to SEQ ID. NO. 161; and a light chain variable
region is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or
100% identical to SEQ ID. NO. 162.
[0168] In one embodiment the bispecific antibody comprises a light
chain and a heavy chain of a first Fab fragment specifically
binding to MCSP, wherein the heavy chain variable region sequence
is an affinity matured variant of SEQ ID. NO. 161 and wherein the
light chain variable region sequence is an affinity matured variant
of SEQ ID. NO. 162. Affinity matured variants in this embodiment
means that independently 1, 2, 3 or 4 amino acids of SEQ ID. NO.
161 and/or SEQ ID. NO. 162 are exchanged.
[0169] In one embodiment, the bispecific antibody comprises a light
chain and a heavy chain of a first Fab fragment specifically
binding to MCSP, wherein said heavy chain comprises a constant
region comprising the amino acid sequence of SEQ ID NO: 16. In one
embodiment, the bispecific antibody comprises a light chain and a
heavy chain of a first Fab fragment specifically binding to MCSP,
wherein said heavy chain comprises a constant region comprising the
amino acid sequence of SEQ ID NO: 16, and a light chain and a heavy
chain of a second Fab fragment specific for CD3 comprising one or
more amino acid sequences as defined in any of the embodiments
described herein.
[0170] In one embodiment, the bispecific antibody comprises a light
chain and a heavy chain of a first Fab fragment specifically
binding to MCSP, wherein said light chain comprises a constant
region comprising the amino acid sequence of SEQ ID NO: 15. In one
embodiment, the bispecific antibody comprises a light chain and a
heavy chain of a second antibody specifically binding to MCSP,
wherein said light chain comprises a constant region comprising the
amino acid sequence of SEQ ID NO: 15, and a light chain and a heavy
chain of a second Fab fragment specific for CD3 comprising one or
more amino acid sequences as defined in any of the embodiments
described herein.
[0171] In one embodiment the bispecific antibody comprises a light
chain and a heavy chain of a first Fab fragment specifically
binding to MCSP, wherein the heavy chain constant region comprises
an amino acid sequence of SEQ ID NO: 16; and a light chain constant
region comprising an amino acid sequence of SEQ ID NO: 15.
[0172] In one embodiment the bispecific antibody comprises a light
chain and a heavy chain of a first Fab fragment specifically
binding to MCSP, wherein the heavy chain variable region comprises
an amino acid sequence of SEQ ID NO: 14; and a light chain variable
region comprising an amino acid sequence of SEQ ID NO: 13, and
wherein the heavy chain constant region comprises an amino acid
sequence of SEQ ID NO: 16; and a light chain constant region
comprising an amino acid sequence of SEQ ID NO:15.
[0173] In a further embodiment, the bispecific antibody of the
invention comprises a light chain and a heavy chain of a second Fab
fragment specifically binding to CD3, comprising a variable light
chain of SEQ ID NO: 19 and a variable heavy chain of SEQ ID NO: 20;
and a light chain and a heavy chain of a first Fab fragment
specific for MCSP, comprising a variable light chain of SEQ ID NO:
13 and a variable heavy chain of SEQ ID NO: 14.
[0174] In one embodiment the bispecific antibody comprises a light
chain and a heavy chain of a first Fab fragment specifically
binding to MCSP, wherein the heavy chain variable region comprises
an amino acid sequence of SEQ ID NO: 161; and a light chain
variable region comprising an amino acid sequence of SEQ ID NO:
162, and wherein the heavy chain constant region comprises an amino
acid sequence of SEQ ID NO: 16; and a light chain constant region
comprising an amino acid sequence of SEQ ID NO:15.
[0175] In a further embodiment, the bispecific antibody of the
invention comprises a light chain and a heavy chain of a second Fab
fragment specifically binding to CD3, comprising a variable light
chain of SEQ ID NO: 19 and a variable heavy chain of SEQ ID NO: 20;
and a light chain and a heavy chain of a first Fab fragment
specific for MCSP, comprising a variable light chain of SEQ ID NO:
161 and a variable heavy chain of SEQ ID NO: 162.
[0176] In one embodiment the bispecific antibody comprises a light
chain and a heavy chain of a first Fab fragment specifically
binding to MCSP, wherein the heavy chain variable region comprises
an amino acid sequence of SEQ ID NO: 161 or an affinity matured
variant thereof; and a light chain variable region comprising an
amino acid sequence of SEQ ID NO: 162 or an affinity matured
variant thereof, and wherein the heavy chain constant region
comprises an amino acid sequence of SEQ ID NO: 16; and a light
chain constant region comprising an amino acid sequence of SEQ ID
NO:15. Affinity matured variants in this embodiment means that
independently 1, 2, 3 or 4 amino acids of SEQ ID. NO. 161 and/or
SEQ ID. NO. 162 are exchanged.
[0177] In a further embodiment, the bispecific antibody of the
invention comprises a light chain and a heavy chain of a second Fab
fragment specifically binding to CD3, comprising a variable light
chain of SEQ ID NO: 19 and a variable heavy chain of SEQ ID NO: 20;
and a light chain and a heavy chain of a first Fab fragment
specific for MCSP, comprising a variable light chain of SEQ ID NO:
161 or an affinity matured variant thereof and a variable heavy
chain of SEQ ID NO: 162 or an affinity matured variant thereof.
Affinity matured variants in this embodiment means that
independently 1, 2, 3 or 4 amino acids of SEQ ID. NO. 161 and/or
SEQ ID. NO. 162 are exchanged.
[0178] In a further embodiment, the bispecific antibody of the
invention comprises a light chain and a heavy chain of a second Fab
fragment specifically binding to CD3, comprising a variable light
chain of SEQ ID NO: 158 and a variable heavy chain of SEQ ID NO:
157; and a light chain and a heavy chain of a first Fab fragment
specific for MCSP, comprising a variable light chain of SEQ ID NO:
161 and a variable heavy chain of SEQ ID NO: 162.
[0179] In a further embodiment, the bispecific antibody of the
invention comprises a light chain and a heavy chain of a second Fab
fragment specifically binding to CD3, comprising a variable light
chain of SEQ ID NO: 158 or an affinity matured variant thereof and
a variable heavy chain of SEQ ID NO: 157 or an affinity matured
variant thereof and a light chain and a heavy chain of a first Fab
fragment specific for MCSP, comprising a variable light chain of
SEQ ID NO: 161 or an affinity matured variant thereof and a
variable heavy chain of SEQ ID NO: 162 or an affinity matured
variant thereof. Affinity matured variants in this embodiment means
that independently 1, 2, 3 or 4 amino acids of one or more of SEQ
ID. NO. 157, SEQ ID. NO. 158, SEQ ID. NO. 161 and/or SEQ ID. NO.
162 are exchanged.
[0180] In one embodiment the bispecific antibody comprises a third
Fab fragment, comprising a light chain and a heavy chain
specifically binding to MCSP, wherein the variable heavy chain
comprises a CDR1 of SEQ ID. NO. 4, a CDR2 of SEQ ID. NO. 5, a CDR3
of SEQ ID. NO. 6; and the variable light chain comprises a CDR1 of
SEQ ID. NO. 1, a CDR2 of SEQ ID. NO. 2, and a CDR3 of SEQ ID. NO.
3.
[0181] In one embodiment the bispecific antibody comprises a third
Fab fragment, comprising a a light chain and a heavy chain
specifically binding to MCSP, wherein the heavy chain variable
region sequence that is at least about 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99% or 100% identical to SEQ ID. NO. 14; and a light
chain variable region is at least about 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99% or 100% identical to SEQ ID. NO. 13.
[0182] In one embodiment the bispecific antibody comprises a third
Fab fragment, comprising a a light chain and a heavy chain
specifically binding to MCSP, wherein the heavy chain variable
region sequence that is at least about 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99% or 100% identical to SEQ ID. NO. 161; and a light
chain variable region is at least about 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99% or 100% identical to SEQ ID. NO. 162.
[0183] In one embodiment the bispecific antibody comprises a third
Fab fragment, comprising a a light chain and a heavy chain
specifically binding to MCSP, wherein the heavy chain variable
region sequence is an affinity matured variant of SEQ ID. NO. 161
and wherein the light chain variable region sequence is an affinity
matured variant of SEQ ID. NO. 162. Affinity matured variants in
this embodiment means that independently 1, 2, 3 or 4 amino acids
of SEQ ID. NO. 161 and/or SEQ ID. NO. 162 are exchanged.
[0184] In one embodiment the bispecific antibody comprises a third
Fab fragment, comprising a light chain and a heavy chain
specifically binding to MCSP, wherein said heavy chain comprises a
constant region comprising the amino acid sequence of SEQ ID NO:
16. In one embodiment the bispecific antibody comprises a third Fab
fragment, comprising a light chain and a heavy chain specifically
binding to MCSP, wherein said heavy chain comprises a constant
region comprising the amino acid sequence of SEQ ID NO: 16, and a
light chain and a heavy chain of a second Fab fragment specific for
CD3 comprising one or more amino acid sequences as defined in any
of the embodiments described herein, and a a light chain and a
heavy chain of a first Fab fragment specific for MCSP comprising
one or more amino acid sequences as defined in any of the
embodiments described herein.
[0185] In one embodiment the bispecific antibody comprises a third
Fab fragment, comprising a light chain and a heavy chain
specifically binding to MCSP, wherein said light chain comprises a
constant region comprising the amino acid sequence of SEQ ID NO:
15. In one embodiment, the bispecific antibody comprises a light
chain and a heavy chain of a second antibody specifically binding
to MCSP, wherein said light chain comprises a constant region
comprising the amino acid sequence of SEQ ID NO: 15, and a light
chain and a heavy chain of a second Fab fragment specific for CD3,
and a light chain and a heavy chain of a first Fab fragment
specific for MCSP comprising one or more amino acid sequences as
defined in any of the embodiments described herein.
[0186] In one embodiment the bispecific antibody comprises a third
Fab fragment, comprising a light chain and a heavy chain
specifically binding to MCSP, wherein the heavy chain constant
region comprises an amino acid sequence of SEQ ID NO: 16; and a
light chain constant region comprising an amino acid sequence of
SEQ ID NO: 15.
[0187] In one embodiment the bispecific antibody comprises a third
Fab fragment, comprising a light chain and a heavy chain
specifically binding to MCSP, wherein the heavy chain variable
region comprises an amino acid sequence of SEQ ID NO: 14; and a
light chain variable region comprising an amino acid sequence of
SEQ ID NO: 13, and wherein the heavy chain constant region
comprises an amino acid sequence of SEQ ID NO: 16; and a light
chain constant region comprising an amino acid sequence of SEQ ID
NO:15.
[0188] In one embodiment the bispecific antibody comprises a third
Fab fragment, comprising a light chain and a heavy chain
specifically binding to MCSP, wherein the heavy chain variable
region comprises an amino acid sequence of SEQ ID NO: 161; and a
light chain variable region comprising an amino acid sequence of
SEQ ID NO: 162, and wherein the heavy chain constant region
comprises an amino acid sequence of SEQ ID NO: 16; and a light
chain constant region comprising an amino acid sequence of SEQ ID
NO:15.
[0189] In one embodiment the bispecific antibody comprises a third
Fab fragment, comprising a light chain and a heavy chain
specifically binding to MCSP, wherein the heavy chain variable
region sequence is an affinity matured variant of SEQ ID. NO. 161
and wherein the light chain variable region sequence is an affinity
matured variant of SEQ ID. NO. 162, and wherein the heavy chain
constant region comprises an amino acid sequence of SEQ ID NO: 16;
and a light chain constant region comprising an amino acid sequence
of SEQ ID NO:15. Affinity matured variants in this embodiment means
that independently 1, 2, 3 or 4 amino acids of SEQ ID. NO. 161
and/or SEQ ID. NO. 162 are exchanged.
[0190] In a further embodiment, the bispecific antibody of the
invention comprises a light chain and a heavy chain of a second Fab
fragment specifically binding to CD3, comprising a variable light
chain of SEQ ID NO: 19 and a variable heavy chain of SEQ ID NO: 20;
and a light chain and a heavy chain of a first Fab fragment
specific for MCSP, comprising a variable light chain of SEQ ID NO:
13 and a variable heavy chain of SEQ ID NO: 14, and a light chain
and a heavy chain of a third Fab fragment specific for MCSP,
comprising a variable light chain of SEQ ID NO: 13 and a variable
heavy chain of SEQ ID NO: 14.
[0191] In a further embodiment, the bispecific antibody of the
invention comprises a light chain and a heavy chain of a second Fab
fragment specifically binding to CD3, comprising a variable light
chain of SEQ ID NO: 19 and a variable heavy chain of SEQ ID NO: 20;
and a light chain and a heavy chain of a first Fab fragment
specific for MCSP, comprising a variable light chain of SEQ ID NO:
162 and a variable heavy chain of SEQ ID NO: 161, and a light chain
and a heavy chain of a third Fab fragment specific for MCSP,
comprising a variable light chain of SEQ ID NO: 162 and a variable
heavy chain of SEQ ID NO: 161.
[0192] In a further embodiment, the bispecific antibody of the
invention comprises a light chain and a heavy chain of a second Fab
fragment specifically binding to CD3, comprising a variable light
chain of SEQ ID NO: 19 and a variable heavy chain of SEQ ID NO: 20;
and a light chain and a heavy chain of a first Fab fragment
specific for MCSP, comprising a variable light chain of SEQ ID NO:
162 or an affinity matured variant thereof and a variable heavy
chain of SEQ ID NO: 161 or an affinity matured variant thereof, and
a light chain and a heavy chain of a third Fab fragment specific
for MCSP, comprising a variable light chain of SEQ ID NO: 162 or an
affinity matured variant thereof and a variable heavy chain of SEQ
ID NO: 161 or an affinity matured variant thereof. Affinity matured
variants in this embodiment means that independently 1, 2, 3 or 4
amino acids of SEQ ID. NO. 161 and/or SEQ ID. NO. 162 are
exchanged.
[0193] In a further embodiment, the bispecific antibody of the
invention comprises a light chain and a heavy chain of a second Fab
fragment specifically binding to CD3, comprising a variable light
chain of SEQ ID NO: 157 and a variable heavy chain of SEQ ID NO:
158; and a light chain and a heavy chain of a first Fab fragment
specific for MCSP, comprising a variable light chain of SEQ ID NO:
162 and a variable heavy chain of SEQ ID NO: 161, and a light chain
and a heavy chain of a third Fab fragment specific for MCSP,
comprising a variable light chain of SEQ ID NO: 162 and a variable
heavy chain of SEQ ID NO: 161.
[0194] In a further embodiment, the bispecific antibody of the
invention comprises a light chain and a heavy chain of a second Fab
fragment specifically binding to CD3, comprising a variable light
chain of SEQ ID NO: 157 and a variable heavy chain of SEQ ID NO:
158; and a light chain and a heavy chain of a first Fab fragment
specific for MCSP, comprising a variable light chain of SEQ ID NO:
162 or an affinity matured variant thereof and a variable heavy
chain of SEQ ID NO: 161 or an affinity matured variant thereof, and
a light chain and a heavy chain of a third Fab fragment specific
for MCSP, comprising a variable light chain of SEQ ID NO: 162 or an
affinity matured variant thereof and a variable heavy chain of SEQ
ID NO: 161 or an affinity matured variant thereof. Affinity matured
variants in this embodiment means that independently 1, 2, 3 or 4
amino acids of SEQ ID. NO. 161 and/or SEQ ID. NO. 162 are
exchanged.
[0195] In yet another embodiment said bispecific antibody comprises
one or more amino acid sequences selected from the group of SEQ ID
NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO. 41 and SEQ ID NO.
43.
[0196] In one embodiment said bispecific antibody comprises SEQ ID
NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27.
[0197] In one embodiment the T cell activating bispecific antibody
comprises at least one antigen binding site that is specific for
Epidermal Growth Factor Receptor (EGFR). In another embodiment the
T cell activating bispecific antibody comprises at least one,
typically two or more antigen binding moieties that can compete
with monoclonal antibody GA201 for binding to an epitope of EGFR.
See PCT publication WO 2006/082515, incorporated herein by
reference in its entirety. In one embodiment, the antigen binding
site that is specific for EGFR comprises the heavy chain CDR1 of
SEQ ID NO: 68, the heavy chain CDR2 of SEQ ID NO: 69, the heavy
chain CDR3 of SEQ ID NO: 70, the light chain CDR1 of SEQ ID NO: 71,
the light chain CDR2 of SEQ ID NO: 72, and the light chain CDR3 of
SEQ ID NO: 73. In a further embodiment, the antigen binding site
that is specific for EGFR comprises a heavy chain variable region
sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99% or 100% identical to SEQ ID NO: 74 and a light chain variable
region sequence that is at least about 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99% or 100% identical to SEQ ID NO: 75, or variants
thereof that retain functionality.
[0198] In a further embodiment, the bispecific antibody comprises a
first Fab fragment comprising an antigen binding site that is
specific for EGFR comprising a heavy chain variable region sequence
that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or
100% identical to SEQ ID NO: 74 and a light chain variable region
sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99% or 100% identical to SEQ ID NO: 75, or variants thereof that
retain functionality, and a light chain and a heavy chain of a
second Fab fragment specific for CD3 comprising one or more amino
acid sequences as defined in any of the embodiments described
herein.
[0199] In a further embodiment, the bispecific antibody comprises a
first and a third Fab fragment comprising an antigen binding site
that is specific for EGFR comprising a heavy chain variable region
sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99% or 100% identical to SEQ ID NO: 74 and a light chain variable
region sequence that is at least about 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99% or 100% identical to SEQ ID NO: 75, or variants
thereof that retain functionality, and a light chain and a heavy
chain of a second Fab fragment specific for CD3 comprising one or
more amino acid sequences as defined in any of the embodiments
described herein.
[0200] In one embodiment the T cell activating bispecific antibody
comprises at least one antigen binding site that is specific for
Fibroblast Activation Protein (FAP). In another embodiment the T
cell activating bispecific antibody comprises at least one,
typically two or more antigen binding moieties that can compete
with monoclonal antibody 3F2 for binding to an epitope of FAP. See
European patent application no. EP10172842.6, incorporated herein
by reference in its entirety. In one embodiment, the antigen
binding site that is specific for FAP comprises the heavy chain
CDR1 of SEQ ID NO: 76, the heavy chain CDR2 of SEQ ID NO: 77, the
heavy chain CDR3 of SEQ ID NO: 78, the light chain CDR1 of SEQ ID
NO: 79, the light chain CDR2 of SEQ ID NO: 80, and the light chain
CDR3 of SEQ ID NO: 81. In a further embodiment, the antigen binding
site that is specific for FAP comprises a heavy chain variable
region sequence that is at least about 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99% or 100% identical to SEQ ID NO: 82 and a light chain
variable region sequence that is at least about 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 83, or variants
thereof that retain functionality.
[0201] In a further embodiment, the bispecific antibody comprises a
first Fab fragment comprising an antigen binding site that is
specific for FAP comprising a heavy chain variable region sequence
that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or
100% identical to SEQ ID NO: 82 and a light chain variable region
sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99% or 100% identical to SEQ ID NO: 83, or variants thereof that
retain functionality, and a light chain and a heavy chain of a
second Fab fragment specific for CD3 comprising one or more amino
acid sequences as defined in any of the embodiments described
herein.
[0202] In a further embodiment, the bispecific antibody comprises a
first and a third Fab fragment comprising an antigen binding site
that is specific for FAP comprising a heavy chain variable region
sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99% or 100% identical to SEQ ID NO: 82 and a light chain variable
region sequence that is at least about 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99% or 100% identical to SEQ ID NO: 83, or variants
thereof that retain functionality, and a light chain and a heavy
chain of a second Fab fragment specific for CD3 comprising one or
more amino acid sequences as defined in any of the embodiments
described herein.
[0203] In one embodiment the T cell activating bispecific antibody
comprises at least one antigen binding site that is specific for
Carcinoembryonic Antigen (CEA). In another embodiment the T cell
activating bispecific antibody comprises at least one, typically
two or more antigen binding moieties that can compete with
monoclonal antibody CH1A1A for binding to an epitope of CEA. In one
embodiment the T cell activating bispecific antibody comprises at
least one, typically two or more antigen binding moieties that can
compete with monoclonal antibody CH1A1A clone 98/99
(CH1A1.sub.(98/99)) for binding to an epitope of CEA. See PCT
patent application number PCT/EP2010/062527, incorporated herein by
reference in its entirety. In one embodiment, the antigen binding
site that is specific for CEA comprises the heavy chain CDR1 of SEQ
ID NO: 84, the heavy chain CDR2 of SEQ ID NO: 85, the heavy chain
CDR3 of SEQ ID NO: 86, the light chain CDR1 of SEQ ID NO: 87, the
light chain CDR2 of SEQ ID NO: 88, and the light chain CDR3 of SEQ
ID NO: 89. In a further embodiment, the antigen binding site that
is specific for CEA comprises a heavy chain variable region
sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99% or 100% identical to SEQ ID NO: 90 or SEQ ID NO: 159 and a
light chain variable region sequence that is at least about 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
91 or SEQ ID NO: 160, or variants thereof that retain
functionality.
[0204] In one embodiment the bispecific antibody comprises a light
chain and a heavy chain of a first Fab fragment specifically
binding to CEA, wherein the heavy chain variable region comprises
an affinity matured variant of SEQ ID NO: 159 or thereof; and a
light chain variable region comprising an affinity matured variant
of SEQ ID NO: 160. Affinity matured variants in this embodiment
means that independently 1, 2, 3 or 4 amino acids of SEQ ID. NO.
159 and/or SEQ ID. NO. 160 are exchanged.
[0205] In a further embodiment, the bispecific antibody comprises a
first Fab fragment comprising an antigen binding site that is
specific for CEA comprising a heavy chain variable region sequence
that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or
100% identical to SEQ ID NO: 90 and a light chain variable region
sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99% or 100% identical to SEQ ID NO: 91, or variants thereof that
retain functionality, and a light chain and a heavy chain of a
second Fab fragment specific for CD3 comprising one or more amino
acid sequences as defined in any of the embodiments described
herein.
[0206] In a further embodiment, the bispecific antibody comprises a
first Fab fragment comprising an antigen binding site that is
specific for CEA comprising a heavy chain variable region sequence
that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or
100% identical to SEQ ID NO: 159 and a light chain variable region
sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99% or 100% identical to SEQ ID NO: 160, or variants thereof that
retain functionality, and a light chain and a heavy chain of a
second Fab fragment specific for CD3 comprising one or more amino
acid sequences as defined in any of the embodiments described
herein.
[0207] In one embodiment the bispecific antibody comprises a light
chain and a heavy chain of a first Fab fragment specifically
binding to CEA, wherein the heavy chain variable region comprises
an affinity matured variant of SEQ ID NO: 159; and a light chain
variable region comprising an affinity matured variant of SEQ ID
NO: 160 and a light chain and a heavy chain of a second Fab
fragment specific for CD3 comprising one or more amino acid
sequences as defined in any of the embodiments described herein.
Affinity matured variants in this embodiment means that
independently 1, 2, 3 or 4 amino acids of SEQ ID. NO. 159 and/or
SEQ ID. NO. 160 are exchanged.
[0208] In a further embodiment, the bispecific antibody comprises a
first and a third Fab fragment comprising an antigen binding site
that is specific for CEA comprising a heavy chain variable region
sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99% or 100% identical to SEQ ID NO: 90 or SEQ ID NO: 159 and a
light chain variable region sequence that is at least about 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
91 or SEQ ID NO:160, or variants thereof that retain functionality,
and a light chain and a heavy chain of a second Fab fragment
specific for CD3 comprising one or more amino acid sequences as
defined in any of the embodiments described herein. Affinity
matured variants in this embodiment means that independently 1, 2,
3 or 4 amino acids of SEQ ID. NO. 159 and/or SEQ ID. NO. 160 are
exchanged.
[0209] In a further embodiment, the bispecific antibody comprises a
first and a third Fab fragment comprising an antigen binding site
that is specific for CEA wherein the heavy chain variable region
comprises an affinity matured variant of SEQ ID NO: 159; and the
light chain variable region comprising an affinity matured variant
of SEQ ID NO: 160. Affinity matured variants in this embodiment
means that independently 1, 2, 3 or 4 amino acids of SEQ ID. NO.
159 and/or SEQ ID. NO. 160 are exchanged.
[0210] In one embodiment the T cell activating bispecific antibody
comprises at least one antigen binding site that is specific for
CD33. In one embodiment, the antigen binding site that is specific
for CD33 comprises the heavy chain CDR1 of SEQ ID NO: 92, the heavy
chain CDR2 of SEQ ID NO: 93, the heavy chain CDR3 of SEQ ID NO: 94,
the light chain CDR1 of SEQ ID NO: 95, the light chain CDR2 of SEQ
ID NO: 96, and the light chain CDR3 of SEQ ID NO: 97. In a further
embodiment, the antigen binding site that is specific for CD33
comprises a heavy chain variable region sequence that is at least
about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to
SEQ ID NO: 98 and a light chain variable region sequence that is at
least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID NO: 99, or variants thereof that retain
functionality.
[0211] In a further embodiment, the bispecific antibody comprises a
first Fab fragment comprising an antigen binding site that is
specific for CD33 comprising a heavy chain variable region sequence
that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or
100% identical to SEQ ID NO: 98 and a light chain variable region
sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99% or 100% identical to SEQ ID NO: 99, or variants thereof that
retain functionality, and a light chain and a heavy chain of a
second Fab fragment specific for CD3 comprising one or more amino
acid sequences as defined in any of the embodiments described
herein.
[0212] In a specific embodiment the T cell activating bispecific
antibody comprises a polypeptide sequence encoded by a
polynucleotide sequence that is at least about 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or 100% identical to a sequence selected from
the group of SEQ ID NO: 100, SEQ ID NO: 101 and SEQ ID NO: 102.
[0213] In one embodiment the T cell activating bispecific antibody
comprises a polypeptide sequence encoded by a polynucleotide
sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99% or 100% identical to a sequence selected from the group of SEQ
ID NO: 151, SEQ ID NO. 152 and SEQ ID NO. 153.
[0214] In yet another embodiment said bispecific antibody comprises
one or more amino acid sequences selected from the group of SEQ ID
NO: 100, SEQ ID NO: 101, SEQ ID NO: 151, SEQ ID NO. 152 and SEQ ID
NO. 153.
[0215] In one embodiment of the invention the bispecific antibody
is a humanized antibody, as detailed below.
[0216] In another embodiment of the invention the bispecific
antibody is a human antibody, as detailed below.
[0217] In a second object the present invention relates to a
pharmaceutical composition comprising a bispecific antibody of the
present invention.
[0218] In a third object the present invention relates to a
bispecific antibody of the present invention for the treatment of
cancer. In another embodiment, use of the bispecific antibody as a
medicament is provided. Preferably said use is for the treatment of
cancer.
[0219] In further objects the present invention relates to a
nucleic acid sequence comprising a sequence encoding a heavy chain
of a bispecific antibody of the present invention, a nucleic acid
sequence comprising a sequence encoding a light chain of a
bispecific antibody of the present invention, an expression vector
comprising a nucleic acid sequence of the present invention and to
a prokaryotic or eukaryotic host cell comprising a vector of the
present invention. In addition a method of producing an antibody
comprising culturing the host cell so that the antibody is produced
is provided.
[0220] In a specific embodiment the T cell activating bispecific
antibody comprises a polypeptide sequence encoded by a
polynucleotide sequence that is at least about 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or 100% identical to a sequence selected from
the group of SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID
NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51,
SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID
NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60,
SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID
NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67.
[0221] In a specific embodiment the T cell activating bispecific
antibody comprises a polypeptide sequence encoded by a
polynucleotide sequence that is at least about 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or 100% identical to a sequence selected from
the group of SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID
NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112.
[0222] In a specific embodiment the T cell activating bispecific
antibody comprises a polypeptide sequence encoded by a
polynucleotide sequence that is at least about 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or 100% identical to a sequence selected from
the group of SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID
NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, and SEQ ID
NO: 120.
[0223] In a specific embodiment the T cell activating bispecific
antibody comprises a polypeptide sequence encoded by a
polynucleotide sequence that is at least about 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or 100% identical to a sequence selected from
the group of SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID
NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, and SEQ ID
NO: 128.
[0224] In a specific embodiment the T cell activating bispecific
antibody comprises a polypeptide sequence encoded by a
polynucleotide sequence that is at least about 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or 100% identical to a sequence selected from
the group of SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID
NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, and SEQ ID
NO: 136.
[0225] In a specific embodiment the T cell activating bispecific
antibody comprises a polypeptide sequence encoded by a
polynucleotide sequence that is at least about 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or 100% identical to a sequence selected from
the group of SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 137, SEQ ID
NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO:
142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 154, SEQ ID NO: 155
and SEQ ID NO: 156.
[0226] In a further aspect, a bispecific antibody according to any
of the above embodiments may incorporate any of the features,
singly or in combination, as described in Sections 1-5 below:
[0227] 1. Antibody Affinity
[0228] The affinity of the T cell activating bispecific antibody
for a target antigen can be determined in accordance with the
methods set forth in the Examples by surface plasmon resonance
(SPR), using standard instrumentation such as a BIAcore instrument
(GE Healthcare), and receptors or target proteins such as may be
obtained by recombinant expression. Alternatively, binding of T
cell activating bispecific antibodies for different receptors or
target antigens may be evaluated using cell lines expressing the
particular receptor or target antigen, for example by flow
cytometry (FACS).
[0229] In certain embodiments, a bispecific antibody provided
herein has a dissociation constant (KD) of .ltoreq.1 .mu.M,
.ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM,
.ltoreq.0.01 nM, or .ltoreq.0.001 nM (e.g. 10.sup.-8M or less, e.g.
from 10.sup.-8M to 10.sup.-13M, e.g., from 10.sup.-9M to 10.sup.-13
M).
[0230] According to one embodiment, KD is measured using surface
plasmon resonance assays using a BIACORE.RTM.-2000 or a
BIACORE.RTM.-3000 (BIAcore, Inc., Piscataway, N.J.) at 25.degree.
C. with immobilized antigen CM5 chips at .about.10 response units
(RU). Briefly, carboxymethylated dextran biosensor chips (CM5,
BIACORE, Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
to 5 .mu.g/ml (.about.0.2 .mu.M) before injection at a flow rate of
5 .mu.l/minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of antigen, 1 M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% polysorbate 20 (TWEEN-20.TM.)
surfactant (PBST) at 25.degree. C. at a flow rate of approximately
25 .mu.l/min. Association rates (ka or k.sub.on) and dissociation
rates (kd or k.sub.off) are calculated using a simple one-to-one
Langmuir binding model (BIACORE.RTM. Evaluation Software version
3.2) by simultaneously fitting the association and dissociation
sensorgrams. The equilibrium dissociation constant (KD) is
calculated as the ratio k.sub.off/k.sub.on. See, e.g., Chen et al.,
J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10.sup.6
M.sup.-1 s.sup.-1 by the surface plasmon resonance assay above,
then the on-rate can be determined by using a fluorescent quenching
technique that measures the increase or decrease in fluorescence
emission intensity (excitation=295 nm; emission=340 nm, 16 nm
band-pass) at 25.degree. C. of a 20 nM anti-antigen antibody (Fab
form) in PBS, pH 7.2, in the presence of increasing concentrations
of antigen as measured in a spectrometer, such as a stop-flow
equipped spectrophometer (Aviv Instruments) or a 8000-series
SLM-AMINCO.TM. spectrophotometer (ThermoSpectronic) with a stirred
cuvette.
[0231] 2. Chimeric and Humanized Antibodies
[0232] In certain embodiments, a bispecific antibody provided
herein is a chimeric antibody. Certain chimeric antibodies are
described, e.g., in U.S. Pat. No. 4,816,567; and Morrison et al.,
Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one example, a
chimeric antibody comprises a non-human variable region (e.g., a
variable region derived from a mouse, rat, hamster, rabbit, or
non-human primate, such as a monkey) and a human constant region.
In a further example, a chimeric antibody is a "class switched"
antibody in which the class or subclass has been changed from that
of the parent antibody. Chimeric antibodies include antigen-binding
fragments thereof.
[0233] In certain embodiments, a chimeric antibody is a humanized
antibody. Typically, a non-human antibody is humanized to reduce
immunogenicity to humans, while retaining the specificity and
affinity of the parental non-human antibody. Generally, a humanized
antibody comprises one or more variable domains in which HVRs,
e.g., CDRs, (or portions thereof) are derived from a non-human
antibody, and FRs (or portions thereof) are derived from human
antibody sequences. A humanized antibody optionally will also
comprise at least a portion of a human constant region. In some
embodiments, some FR residues in a humanized antibody are
substituted with corresponding residues from a non-human antibody
(e.g., the antibody from which the HVR residues are derived), e.g.,
to restore or improve antibody specificity or affinity.
[0234] Humanized antibodies and methods of making them are
reviewed, e.g., in Almagro and Fransson, Front. Biosci.
13:1619-1633 (2008), and are further described, e.g., in Riechmann
et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad.
Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,
7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods
36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol.
Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua
et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and
Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J.
Cancer, 83:252-260 (2000) (describing the "guided selection"
approach to FR shuffling).
[0235] Human framework regions that may be used for humanization
include but are not limited to: framework regions selected using
the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296
(1993)); framework regions derived from the consensus sequence of
human antibodies of a particular subgroup of light or heavy chain
variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci.
USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623
(1993)); human mature (somatically mutated) framework regions or
human germline framework regions (see, e.g., Almagro and Fransson,
Front. Biosci. 13:1619-1633 (2008)); and framework regions derived
from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem.
272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.
271:22611-22618 (1996)).
[0236] 3. Human Antibodies
[0237] In certain embodiments, a bispecific antibody provided
herein is a human antibody. Human antibodies can be produced using
various techniques known in the art. Human antibodies are described
generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:
368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459
(2008).
[0238] Human antibodies may be prepared by administering an
immunogen to a transgenic animal that has been modified to produce
intact human antibodies or intact antibodies with human variable
regions in response to antigenic challenge. Such animals typically
contain all or a portion of the human immunoglobulin loci, which
replace the endogenous immunoglobulin loci, or which are present
extrachromosomally or integrated randomly into the animal's
chromosomes. In such transgenic mice, the endogenous immunoglobulin
loci have generally been inactivated. For review of methods for
obtaining human antibodies from transgenic animals, see Lonberg,
Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Pat. Nos.
6,075,181 and 6,150,584 describing XENOMOUSE.TM. technology; U.S.
Pat. No. 5,770,429 describing HUMAB.RTM. technology; U.S. Pat. No.
7,041,870 describing K-M MOUSE.RTM. technology, and U.S. Patent
Application Publication No. US 2007/0061900, describing
VELOCIMOUSE.RTM. technology). Human variable regions from intact
antibodies generated by such animals may be further modified, e.g.,
by combining with a different human constant region.
[0239] Human antibodies can also be made by hybridoma-based
methods. Human myeloma and mouse-human heteromyeloma cell lines for
the production of human monoclonal antibodies have been described.
(See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, pp.
51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.
Immunol., 147: 86 (1991).) Human antibodies generated via human
B-cell hybridoma technology are also described in Li et al., Proc.
Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods
include those described, for example, in U.S. Pat. No. 7,189,826
(describing production of monoclonal human IgM antibodies from
hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268
(2006) (describing human-human hybridomas). Human hybridoma
technology (Trioma technology) is also described in Vollmers and
Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and
Vollmers and Brandlein, Methods and Findings in Experimental and
Clinical Pharmacology, 27(3):185-91 (2005).
[0240] Human antibodies may also be generated by isolating Fv clone
variable domain sequences selected from human-derived phage display
libraries. Such variable domain sequences may then be combined with
a desired human constant domain. Techniques for selecting human
antibodies from antibody libraries are described below.
[0241] 4. Library-Derived Antibodies
[0242] Bispecific antibodies of the invention may be isolated by
screening combinatorial libraries for antibodies with the desired
activity or activities. For example, a variety of methods are known
in the art for generating phage display libraries and screening
such libraries for antibodies possessing the desired binding
characteristics. Such methods are reviewed, e.g., in Hoogenboom et
al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed.,
Human Press, Totowa, N.J., 2001) and further described, e.g., in
the McCafferty et al., Nature 348:552-554; Clackson et al., Nature
352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597
(1992); Marks and Bradbury, in Methods in Molecular Biology
248:161-175 (Lo, ed., Human Press, Totowa, N.J., 2003); Sidhu et
al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol.
Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci.
USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol.
Methods 284(1-2): 119-132 (2004).
[0243] In certain phage display methods, repertoires of VH and VL
genes are separately cloned by polymerase chain reaction (PCR) and
recombined randomly in phage libraries, which can then be screened
for antigen-binding phage as described in Winter et al., Ann. Rev.
Immunol., 12: 433-455 (1994). Phage typically display antibody
fragments, either as single-chain Fv (scFv) fragments or as Fab
fragments. Libraries from immunized sources provide high-affinity
antibodies to the immunogen without the requirement of constructing
hybridomas. Alternatively, the naive repertoire can be cloned
(e.g., from human) to provide a single source of antibodies to a
wide range of non-self and also self antigens without any
immunization as described by Griffiths et al., EMBO J, 12: 725-734
(1993). Finally, naive libraries can also be made synthetically by
cloning unrearranged V-gene segments from stem cells, and using PCR
primers containing random sequence to encode the highly variable
CDR3 regions and to accomplish rearrangement in vitro, as described
by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
Patent publications describing human antibody phage libraries
include, for example: U.S. Pat. No. 5,750,373, and US Patent
Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000,
2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and
2009/0002360.
[0244] Antibodies or antibody fragments isolated from human
antibody libraries are considered human antibodies or human
antibody fragments herein.
[0245] 5. Antibody Variants
[0246] In certain embodiments, amino acid sequence variants of the
bispecific antibodies provided herein are contemplated. For
example, it may be desirable to improve the binding affinity and/or
other biological properties of the bispecific antibody. Amino acid
sequence variants of a bispecific antibody may be prepared by
introducing appropriate modifications into the nucleotide sequence
encoding the bispecific antibody, or by peptide synthesis. Such
modifications include, for example, deletions from, and/or
insertions into and/or substitutions of residues within the amino
acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics, e.g., antigen-binding.
[0247] a) Substitution, Insertion, and Deletion Variants
[0248] In certain embodiments, antibody variants having one or more
amino acid substitutions are provided. Sites of interest for
substitutional mutagenesis include the HVRs and FRs. Conservative
substitutions are shown in Table 1 under the heading of
"conservative substitutions." More substantial changes are provided
in Table 1 under the heading of "exemplary substitutions," and as
further described below in reference to amino acid side chain
classes. Amino acid substitutions may be introduced into an
antibody of interest and the products screened for a desired
activity, e.g., retained/improved antigen binding or decreased
immunogenicity.
TABLE-US-00002 TABLE 1 Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp
Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;
Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met;
Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
Amino acids may be grouped according to common side-chain
properties:
[0249] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0250] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0251] (3) acidic: Asp, Glu;
[0252] (4) basic: His, Lys, Arg;
[0253] (5) residues that influence chain orientation: Gly, Pro;
[0254] (6) aromatic: Tip, Tyr, Phe.
[0255] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0256] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g. a
humanized or human antibody). Generally, the resulting variant(s)
selected for further study will have modifications (e.g.,
improvements) in certain biological properties (e.g., increased
affinity, reduced immunogenicity) relative to the parent antibody
and/or will have substantially retained certain biological
properties of the parent antibody. An exemplary substitutional
variant is an affinity matured antibody, which may be conveniently
generated, e.g., using phage display-based affinity maturation
techniques such as those described herein. Briefly, one or more HVR
residues are mutated and the variant antibodies displayed on phage
and screened for a particular biological activity (e.g. binding
affinity).
[0257] Alterations (e.g., substitutions) may be made in HVRs, e.g.,
to improve antibody affinity. Such alterations may be made in HVR
"hotspots," i.e., residues encoded by codons that undergo mutation
at high frequency during the somatic maturation process (see, e.g.,
Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or SDRs
(a-CDRs), with the resulting variant VH or VL being tested for
binding affinity. Affinity maturation by constructing and
reselecting from secondary libraries has been described, e.g., in
Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien
et al., ed., Human Press, Totowa, N.J., (2001).) In some
embodiments of affinity maturation, diversity is introduced into
the variable genes chosen for maturation by any of a variety of
methods (e.g., error-prone PCR, chain shuffling, or
oligonucleotide-directed mutagenesis). A secondary library is then
created. The library is then screened to identify any antibody
variants with the desired affinity. Another method to introduce
diversity involves HVR-directed approaches, in which several HVR
residues (e.g., 4-6 residues at a time) are randomized. HVR
residues involved in antigen binding may be specifically
identified, e.g., using alanine scanning mutagenesis or modeling.
CDR-H3 and CDR-L3 in particular are often targeted.
[0258] In certain embodiments, substitutions, insertions, or
deletions may occur within one or more HVRs so long as such
alterations do not substantially reduce the ability of the antibody
to bind antigen. For example, conservative alterations (e.g.,
conservative substitutions as provided herein) that do not
substantially reduce binding affinity may be made in HVRs. Such
alterations may be outside of HVR "hotspots" or SDRs. In certain
embodiments of the variant VH and VL sequences provided above, each
HVR either is unaltered, or contains no more than one, two or three
amino acid substitutions.
[0259] A useful method for identification of residues or regions of
an antibody that may be targeted for mutagenesis is called "alanine
scanning mutagenesis" as described by Cunningham and Wells (1989)
Science, 244:1081-1085. In this method, a residue or group of
target residues (e.g., charged residues such as arg, asp, his, lys,
and glu) are identified and replaced by a neutral or negatively
charged amino acid (e.g., alanine or polyalanine) to determine
whether the interaction of the antibody with antigen is affected.
Further substitutions may be introduced at the amino acid locations
demonstrating functional sensitivity to the initial substitutions.
Alternatively, or additionally, a crystal structure of an
antigen-antibody complex to identify contact points between the
antibody and antigen. Such contact residues and neighboring
residues may be targeted or eliminated as candidates for
substitution. Variants may be screened to determine whether they
contain the desired properties.
[0260] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue. Other insertional variants of the
antibody molecule include the fusion to the N- or C-terminus of the
antibody to an enzyme (e.g. for ADEPT) or a polypeptide which
increases the serum half-life of the antibody.
[0261] of Fc domain variants.
[0262] b) Cysteine Engineered Antibody Variants
[0263] In certain embodiments, it may be desirable to create
cysteine engineered bispecific antibodies, e.g., "thioMAbs," in
which one or more residues of a bispecific antibody are substituted
with cysteine residues. In particular embodiments, the substituted
residues occur at accessible sites of the bispecific antibody. By
substituting those residues with cysteine, reactive thiol groups
are thereby positioned at accessible sites of the antibody and may
be used to conjugate the antibody to other moieties, such as drug
moieties or linker-drug moieties, to create an immunoconjugate, as
described further herein. In certain embodiments, any one or more
of the following residues may be substituted with cysteine: V205
(Kabat numbering) of the light chain and A118 (EU numbering) of the
heavy chain. Cysteine engineered antibodies may be generated as
described, e.g., in U.S. Pat. No. 7,521,541.
[0264] c) Antibody Derivatives
[0265] In certain embodiments, a bispecific antibody provided
herein may be further modified to contain additional
nonproteinaceous moieties that are known in the art and readily
available. The moieties suitable for derivatization of the
bispecific antibody include but are not limited to water soluble
polymers. Non-limiting examples of water soluble polymers include,
but are not limited to, polyethylene glycol (PEG), copolymers of
ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,
polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,
poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer,
polyaminoacids (either homopolymers or random copolymers), and
dextran or poly(n-vinyl pyrrolidone)polyethylene glycol,
propropylene glycol homopolymers, prolypropylene oxide/ethylene
oxide co-polymers, polyoxyethylated polyols (e.g., glycerol),
polyvinyl alcohol, and mixtures thereof. Polyethylene glycol
propionaldehyde may have advantages in manufacturing due to its
stability in water. The polymer may be of any molecular weight, and
may be branched or unbranched. The number of polymers attached to
the antibody may vary, and if more than one polymer are attached,
they can be the same or different molecules. In general, the number
and/or type of polymers used for derivatization can be determined
based on considerations including, but not limited to, the
particular properties or functions of the antibody to be improved,
whether the antibody derivative will be used in a therapy under
defined conditions, etc.
[0266] In another embodiment, conjugates of a bispecific antibody
and nonproteinaceous moiety that may be selectively heated by
exposure to radiation are provided. In one embodiment, the
nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc.
Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be
of any wavelength, and includes, but is not limited to, wavelengths
that do not harm ordinary cells, but which heat the
nonproteinaceous moiety to a temperature at which cells proximal to
the antibody-nonproteinaceous moiety are killed.
[0267] B. Recombinant Methods and Compositions
[0268] T cell activating bispecific antibodies of the invention may
be obtained, for example, by solid-state peptide synthesis (e.g.
Merrifield solid phase synthesis) or recombinant production. For
recombinant production one or more polynucleotide encoding the T
cell activating bispecific antibody (fragment), e.g., as described
above, is isolated and inserted into one or more vectors for
further cloning and/or expression in a host cell. Such
polynucleotide may be readily isolated and sequenced using
conventional procedures. In one embodiment a vector, preferably an
expression vector, comprising one or more of the polynucleotides of
the invention is provided. Methods which are well known to those
skilled in the art can be used to construct expression vectors
containing the coding sequence of a T cell activating bispecific
antibody (fragment) along with appropriate
transcriptional/translational control signals. These methods
include in vitro recombinant DNA techniques, synthetic techniques
and in vivo recombination/genetic recombination. See, for example,
the techniques described in Maniatis et al., MOLECULAR CLONING: A
LABORATORY MANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); and
Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene
Publishing Associates and Wiley Interscience, N.Y (1989). The
expression vector can be part of a plasmid, virus, or may be a
nucleic acid fragment. The expression vector includes an expression
cassette into which the polynucleotide encoding the T cell
activating bispecific antibody (fragment) (i.e. the coding region)
is cloned in operable association with a promoter and/or other
transcription or translation control elements. As used herein, a
"coding region" is a portion of nucleic acid which consists of
codons translated into amino acids. Although a "stop codon" (TAG,
TGA, or TAA) is not translated into an amino acid, it may be
considered to be part of a coding region, if present, but any
flanking sequences, for example promoters, ribosome binding sites,
transcriptional terminators, introns, 5' and 3' untranslated
regions, and the like, are not part of a coding region. Two or more
coding regions can be present in a single polynucleotide construct,
e.g. on a single vector, or in separate polynucleotide constructs,
e.g. on separate (different) vectors. Furthermore, any vector may
contain a single coding region, or may comprise two or more coding
regions, e.g. a vector of the present invention may encode one or
more polypeptides, which are post- or co-translationally separated
into the final proteins via proteolytic cleavage. In addition, a
vector, polynucleotide, or nucleic acid of the invention may encode
heterologous coding regions, either fused or unfused to a
polynucleotide encoding the T cell activating bispecific antibody
(fragment) of the invention, or variant or derivative thereof.
Heterologous coding regions include without limitation specialized
elements or motifs, such as a secretory signal peptide or a
heterologous functional domain. An operable association is when a
coding region for a gene product, e.g. a polypeptide, is associated
with one or more regulatory sequences in such a way as to place
expression of the gene product under the influence or control of
the regulatory sequence(s). Two DNA fragments (such as a
polypeptide coding region and a promoter associated therewith) are
"operably associated" if induction of promoter function results in
the transcription of mRNA encoding the desired gene product and if
the nature of the linkage between the two DNA fragments does not
interfere with the ability of the expression regulatory sequences
to direct the expression of the gene product or interfere with the
ability of the DNA template to be transcribed. Thus, a promoter
region would be operably associated with a nucleic acid encoding a
polypeptide if the promoter was capable of effecting transcription
of that nucleic acid. The promoter may be a cell-specific promoter
that directs substantial transcription of the DNA only in
predetermined cells. Other transcription control elements, besides
a promoter, for example enhancers, operators, repressors, and
transcription termination signals, can be operably associated with
the polynucleotide to direct cell-specific transcription. Suitable
promoters and other transcription control regions are disclosed
herein. A variety of transcription control regions are known to
those skilled in the art. These include, without limitation,
transcription control regions, which function in vertebrate cells,
such as, but not limited to, promoter and enhancer segments from
cytomegaloviruses (e.g. the immediate early promoter, in
conjunction with intron-A), simian virus 40 (e.g. the early
promoter), and retroviruses (such as, e.g. Rous sarcoma virus).
Other transcription control regions include those derived from
vertebrate genes such as actin, heat shock protein, bovine growth
hormone and rabbit a-globin, as well as other sequences capable of
controlling gene expression in eukaryotic cells. Additional
suitable transcription control regions include tissue-specific
promoters and enhancers as well as inducible promoters (e.g.
promoter inducible tetracyclins). Similarly, a variety of
translation control elements are known to those of ordinary skill
in the art. These include, but are not limited to ribosome binding
sites, translation initiation and termination codons, and elements
derived from viral systems (particularly an internal ribosome entry
site, or IRES, also referred to as a CITE sequence). The expression
cassette may also include other features such as an origin of
replication, and/or chromosome integration elements such as
retroviral long terminal repeats (LTRs), or adeno-associated viral
(AAV) inverted terminal repeats (ITRs).
Polynucleotide and nucleic acid coding regions of the present
invention may be associated with additional coding regions which
encode secretory or signal peptides, which direct the secretion of
a polypeptide encoded by a polynucleotide of the present invention.
For example, if secretion of the T cell activating bispecific
antigen binding molecule is desired, DNA encoding a signal sequence
may be placed upstream of the nucleic acid encoding a T cell
activating bispecific antibody of the invention or a fragment
thereof. According to the signal hypothesis, proteins secreted by
mammalian cells have a signal peptide or secretory leader sequence
which is cleaved from the mature protein once export of the growing
protein chain across the rough endoplasmic reticulum has been
initiated. Those of ordinary skill in the art are aware that
polypeptides secreted by vertebrate cells generally have a signal
peptide fused to the N-terminus of the polypeptide, which is
cleaved from the translated polypeptide to produce a secreted or
"mature" form of the polypeptide. In certain embodiments, the
native signal peptide, e.g. an immunoglobulin heavy chain or light
chain signal peptide is used, or a functional derivative of that
sequence that retains the ability to direct the secretion of the
polypeptide that is operably associated with it. Alternatively, a
heterologous mammalian signal peptide, or a functional derivative
thereof, may be used. For example, the wild-type leader sequence
may be substituted with the leader sequence of human tissue
plasminogen activator (TPA) or mouse .beta.-glucuronidase. DNA
encoding a short protein sequence that could be used to facilitate
later purification (e.g. a histidine tag) or assist in labeling the
T cell activating bispecific antibody may be included within or at
the ends of the T cell activating bispecific antibody (fragment)
encoding polynucleotide. In a further embodiment, a host cell
comprising one or more polynucleotides of the invention is
provided. In certain embodiments a host cell comprising one or more
vectors of the invention is provided. The polynucleotides and
vectors may incorporate any of the features, singly or in
combination, described herein in relation to polynucleotides and
vectors, respectively. In one such embodiment a host cell comprises
(e.g. has been transformed or transfected with) a vector comprising
a polynucleotide that encodes (part of) a T cell activating
bispecific antibody of the invention. As used herein, the term
"host cell" refers to any kind of cellular system which can be
engineered to generate the T cell activating bispecific antibodies
of the invention or fragments thereof. Host cells suitable for
replicating and for supporting expression of T cell activating
bispecific antibodies are well known in the art. Such cells may be
transfected or transduced as appropriate with the particular
expression vector and large quantities of vector containing cells
can be grown for seeding large scale fermenters to obtain
sufficient quantities of the T cell activating bispecific antibody
for clinical applications. Suitable host cells include prokaryotic
microorganisms, such as E. coli, or various eukaryotic cells, such
as Chinese hamster ovary cells (CHO), insect cells, or the like.
For example, polypeptides may be produced in bacteria in particular
when glycosylation is not needed. After expression, the polypeptide
may be isolated from the bacterial cell paste in a soluble fraction
and can be further purified. In addition to prokaryotes, eukaryotic
microbes such as filamentous fungi or yeast are suitable cloning or
expression hosts for polypeptide-encoding vectors, including fungi
and yeast strains whose glycosylation pathways have been
"humanized", resulting in the production of a polypeptide with a
partially or fully human glycosylation pattern. See Gerngross, Nat
Biotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24,
210-215 (2006). Suitable host cells for the expression of
(glycosylated) polypeptides are also derived from multicellular
organisms (invertebrates and vertebrates). Examples of invertebrate
cells include plant and insect cells. Numerous baculoviral strains
have been identified which may be used in conjunction with insect
cells, particularly for transfection of Spodoptera frugiperda
cells. Plant cell cultures can also be utilized as hosts. See e.g.
U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and
6,417,429 (describing PLANTIBODIES.TM. technology for producing
antibodies in transgenic plants). Vertebrate cells may also be used
as hosts. For example, mammalian cell lines that are adapted to
grow in suspension may be useful. Other examples of useful
mammalian host cell lines are monkey kidney CV1 line transformed by
SV40 (COS-7); human embryonic kidney line (293 or 293T cells as
described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)), baby
hamster kidney cells (BHK), mouse sertoli cells (TM4 cells as
described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkey
kidney cells (CV1), African green monkey kidney cells (VERO-76),
human cervical carcinoma cells (HELA), canine kidney cells (MDCK),
buffalo rat liver cells (BRL 3A), human lung cells (W138), human
liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI
cells (as described, e.g., in Mather et al., Annals N.Y. Acad Sci
383, 44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful
mammalian host cell lines include Chinese hamster ovary (CHO)
cells, including dhfr.sup.- CHO cells (Urlaub et al., Proc Natl
Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO,
NS0, P3X63 and Sp2/0. For a review of certain mammalian host cell
lines suitable for protein production, see, e.g., Yazaki and Wu,
Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana
Press, Totowa, N.J.), pp. 255-268 (2003). Host cells include
cultured cells, e.g., mammalian cultured cells, yeast cells, insect
cells, bacterial cells and plant cells, to name only a few, but
also cells comprised within a transgenic animal, transgenic plant
or cultured plant or animal tissue. In one embodiment, the host
cell is a eukaryotic cell, preferably a mammalian cell, such as a
Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK)
cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). Standard
technologies are known in the art to express foreign genes in these
systems. Cells expressing a polypeptide comprising either the heavy
or the light chain of an antigen binding domain such as an
antibody, may be engineered so as to also express the other of the
antibody chains such that the expressed product is an antibody that
has both a heavy and a light chain. In one embodiment, a method of
producing a T cell activating bispecific antibody according to the
invention is provided, wherein the method comprises culturing a
host cell comprising a polynucleotide encoding the T cell
activating bispecific antibody, as provided herein, under
conditions suitable for expression of the T cell activating
bispecific antigen binding molecule, and recovering the T cell
activating bispecific antibody from the host cell (or host cell
culture medium). The components of the T cell activating bispecific
antibody are genetically fused to each other. T cell activating
bispecific antibody can be designed such that its components are
fused directly to each other or indirectly through a linker
sequence. The composition and length of the linker may be
determined in accordance with methods well known in the art and may
be tested for efficacy. Examples of linker sequences between
different components of T cell activating bispecific antibodies are
found in the sequences provided herein. Additional sequences may
also be included to incorporate a cleavage site to separate the
individual components of the fusion if desired, for example an
endopeptidase recognition sequence. In certain embodiments the one
or more antigen binding moieties of the T cell activating
bispecific antibodies comprise at least an antibody variable region
capable of binding an antigenic determinant. Variable regions can
form part of and be derived from naturally or non-naturally
occurring antibodies and fragments thereof. Methods to produce
polyclonal antibodies and monoclonal antibodies are well known in
the art (see e.g. Harlow and Lane, "Antibodies, a laboratory
manual", Cold Spring Harbor Laboratory, 1988). Non-naturally
occurring antibodies can be constructed using solid phase-peptide
synthesis, can be produced recombinantly (e.g. as described in U.S.
Pat. No. 4,186,567) or can be obtained, for example, by screening
combinatorial libraries comprising variable heavy chains and
variable light chains (see e.g. U.S. Pat. No. 5,969,108 to
McCafferty). Any animal species of antibody, antibody fragment,
antigen binding domain or variable region can be used in the T cell
activating bispecific antibodies of the invention. Non-limiting
antibodies, antibody fragments, antigen binding domains or variable
regions useful in the present invention can be of murine, primate,
or human origin. If the T cell activating antibody is intended for
human use, a chimeric form of antibody may be used wherein the
constant regions of the antibody are from a human. A humanized or
fully human form of the antibody can also be prepared in accordance
with methods well known in the art (see e.g. U.S. Pat. No.
5,565,332 to Winter). Humanization may be achieved by various
methods including, but not limited to (a) grafting the non-human
(e.g., donor antibody) CDRs onto human (e.g. recipient antibody)
framework and constant regions with or without retention of
critical framework residues (e.g. those that are important for
retaining good antigen binding affinity or antibody functions), (b)
grafting only the non-human specificity-determining regions (SDRs
or a-CDRs; the residues critical for the antibody-antigen
interaction) onto human framework and constant regions, or (c)
transplanting the entire non-human variable domains, but "cloaking"
them with a human-like section by replacement of surface residues.
Humanized antibodies and methods of making them are reviewed, e.g.,
in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), and are
further described, e.g., in Riechmann et al., Nature 332, 323-329
(1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033
(1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and
7,087,409; Jones et al., Nature 321, 522-525 (1986); Morrison et
al., Proc Natl Acad Sci 81, 6851-6855 (1984); Morrison and Oi, Adv
Immunol 44, 65-92 (1988); Verhoeyen et al., Science 239, 1534-1536
(1988); Padlan, Molec Immun 31(3), 169-217 (1994); Kashmiri et al.,
Methods 36, 25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,
Mol Immunol 28, 489-498 (1991) (describing "resurfacing");
Dall'Acqua et al., Methods 36, 43-60 (2005) (describing "FR
shuffling"); and Osbourn et al., Methods 36, 61-68 (2005) and
Klimka et al., Br J Cancer 83, 252-260 (2000) (describing the
"guided selection" approach to FR shuffling). Human antibodies and
human variable regions can be produced using various techniques
known in the art. Human antibodies are described generally in van
Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) and
Lonberg, Curr Opin Immunol 20, 450-459 (2008). Human variable
regions can form part of and be derived from human monoclonal
antibodies made by the hybridoma method (see e.g. Monoclonal
Antibody Production Techniques and Applications, pp. 51-63 (Marcel
Dekker, Inc., New York, 1987)). Human antibodies and human variable
regions may also be prepared by administering an immunogen to a
transgenic animal that has been modified to produce intact human
antibodies or intact antibodies with human variable regions in
response to antigenic challenge (see e.g. Lonberg, Nat Biotech 23,
1117-1125 (2005). Human antibodies and human variable regions may
also be generated by isolating Fv clone variable region sequences
selected from human-derived phage display libraries (see e.g.,
Hoogenboom et al. in Methods in Molecular Biology 178, 1-37
(O'Brien et al., ed., Human Press, Totowa, N.J., 2001); and
McCafferty et al., Nature 348, 552-554; Clackson et al., Nature
352, 624-628 (1991)). Phage typically display antibody fragments,
either as single-chain Fv (scFv) fragments or as Fab fragments. In
certain embodiments, the bispecific antibodies of the present
invention are engineered to have enhanced binding affinity
according to, for example, the methods disclosed in U.S. Pat. Appl.
Publ. No. 2004/0132066, the entire contents of which are hereby
incorporated by reference. The ability of the T cell activating
bispecific antibody of the invention to bind to a specific
antigenic determinant can be measured either through an
enzyme-linked immunosorbent assay (ELISA) or other techniques
familiar to one of skill in the art, e.g. surface plasmon resonance
technique (analyzed on a BIACORE T100 system) (Liljeblad, et al.,
Glyco J 17, 323-329 (2000)), and traditional binding assays
(Heeley, Endocr Res 28, 217-229 (2002)). Competition assays may be
used to identify an antibody, antibody fragment, antigen binding
domain or variable domain that competes with a reference antibody
for binding to a particular antigen, e.g. an antibody that competes
with the V9 antibody for binding to CD3. In certain embodiments,
such a competing antibody binds to the same epitope (e.g. a linear
or a conformational epitope) that is bound by the reference
antibody. Detailed exemplary methods for mapping an epitope to
which an antibody binds are provided in Morris (1996) "Epitope
Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana
Press, Totowa, N.J.). In an exemplary competition assay,
immobilized antigen (e.g. CD3) is incubated in a solution
comprising a first labeled antibody that binds to the antigen (e.g.
V9 antibody) and a second unlabeled antibody that is being tested
for its ability to compete with the first antibody for binding to
the antigen. The second antibody may be present in a hybridoma
supernatant. As a control, immobilized antigen is incubated in a
solution comprising the first labeled antibody but not the second
unlabeled antibody. After incubation under conditions permissive
for binding of the first antibody to the antigen, excess unbound
antibody is removed, and the amount of label associated with
immobilized antigen is measured. If the amount of label associated
with immobilized antigen is substantially reduced in the test
sample relative to the control sample, then that indicates that the
second antibody is competing with the first antibody for binding to
the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory
Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y.). T cell activating bispecific antibodies prepared as
described herein may be purified by art-known techniques such as
high performance liquid chromatography, ion exchange
chromatography, gel electrophoresis, affinity chromatography, size
exclusion chromatography, and the like. The actual conditions used
to purify a particular protein will depend, in part, on factors
such as net charge, hydrophobicity, hydrophilicity etc., and will
be apparent to those having skill in the art. For affinity
chromatography purification an antibody, ligand, receptor or
antigen can be used to which the T cell activating bispecific
antibody binds. For example, for affinity chromatography
purification of T cell activating bispecific antibody of the
invention, a matrix with protein A or protein G may be used.
Sequential Protein A or G affinity chromatography and size
exclusion chromatography can be used to isolate a T cell activating
bispecific antibody essentially as described in the Examples. The
purity of the T cell activating bispecific antibodies can be
determined by any of a variety of well known analytical methods
including gel electrophoresis, high pressure liquid chromatography,
and the like.
[0269] C. Assays
[0270] Bispecific antibodies provided herein may be identified,
screened for, or characterized for their physical/chemical
properties and/or biological activities by various assays known in
the art.
[0271] 1. Binding Assays and Other Assays
[0272] In one aspect, a bispecific antibody of the invention is
tested for its antigen binding activity, e.g., by known methods
such as ELISA, Western blot, etc.
[0273] In another aspect, competition assays may be used to
identify an antibody that competes with a specific anti-TA antibody
or an antibody specific for aT-cell activating antigen for binding
to the Tumor Antigen (TA) or a T-cell activating antigen
respectively. In certain embodiments, such a competing antibody
binds to the same epitope (e.g., a linear or a conformational
epitope) that is bound by a specific anti-TA antibody or an
antibody specific for aT-cell activating antigen. Detailed
exemplary methods for mapping an epitope to which an antibody binds
are provided in Morris (1996) "Epitope Mapping Protocols," in
Methods in Molecular Biology vol. 66 (Humana Press, Totowa,
N.J.).
[0274] 2. Activity Assays
[0275] In one aspect, assays are provided for identifying
bispecific antibodies that bind to a T-cell activating antigen and
a Tumor Antigen (TA) thereof having biological activity. Biological
activity may include, e.g., lysis of targeted cells or induction of
apoptosis. Antibodies having such biological activity in vivo
and/or in vitro are also provided.
[0276] In certain embodiments, a bispecific antibody of the
invention is tested for such biological activity. Assays for
detecting cell lysis (e.g. by measurement of LDH release) or
apoptosis (e.g. using the TUNEL assay) are well known in the
art.
[0277] D. Immunoconjugates
[0278] The invention also provides immunoconjugates comprising a
bispecific antibody that binds to a T-cell activating antigen and a
Tumor Antigen (TA) herein conjugated to one or more cytotoxic
agents, such as chemotherapeutic agents or drugs, growth inhibitory
agents, toxins (e.g., protein toxins, enzymatically active toxins
of bacterial, fungal, plant, or animal origin, or fragments
thereof), or radioactive isotopes.
[0279] In one embodiment, an immunoconjugate is an antibody-drug
conjugate (ADC) in which an antibody is conjugated to one or more
drugs, including but not limited to a maytansinoid (see U.S. Pat.
Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an
auristatin such as monomethylauristatin drug moieties DE and DF
(MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and
7,498,298); a dolastatin; a calicheamicin or derivative thereof
(see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285,
5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al.,
Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res.
58:2925-2928 (1998)); an anthracycline such as daunomycin or
doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523
(2006); Jeffrey et al., Bioorganic & Med. Chem. Letters
16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005);
Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000);
Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532
(2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S.
Pat. No. 6,630,579); methotrexate; vindesine; a taxane such as
docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a
trichothecene; and CC1065.
[0280] In another embodiment, an immunoconjugate comprises a
bispecific antibody as described herein conjugated to an
enzymatically active toxin or fragment thereof, including but not
limited to diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin, and the
tricothecenes.
[0281] In another embodiment, an immunoconjugate comprises a
bispecific antibody as described herein conjugated to a radioactive
atom to form a radioconjugate. A variety of radioactive isotopes
are available for the production of radioconjugates. Examples
include At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186,
Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32, Pb.sup.212 and
radioactive isotopes of Lu. When the radioconjugate is used for
detection, it may comprise a radioactive atom for scintigraphic
studies, for example tc99m or I123, or a spin label for nuclear
magnetic resonance (NMR) imaging (also known as magnetic resonance
imaging, mri), such as iodine-123 again, iodine-131, indium-111,
fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,
manganese or iron.
[0282] Conjugates of a bispecific antibody and cytotoxic agent may
be made using a variety of bifunctional protein coupling agents
such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a "cleavable linker" facilitating release of a cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No.
5,208,020) may be used.
[0283] The immunuoconjugates or ADCs herein expressly contemplate,
but are not limited to such conjugates prepared with cross-linker
reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS,
LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,
sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and
sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which
are commercially available (e.g., from Pierce Biotechnology, Inc.,
Rockford, Ill., U.S.A).
[0284] E. Methods and Compositions for Diagnostics and
Detection
[0285] In certain embodiments, any of the bispecific antibodies
that bind to a T-cell activating antigen and a Tumor Antigen (TA)
provided herein is useful for detecting the presence of a T-cell
activating antigen and/or a Tumor Antigen (TA) in a biological
sample. The term "detecting" as used herein encompasses
quantitative or qualitative detection. In certain embodiments, a
biological sample comprises a cell or tissue.
[0286] In one embodiment, a bispecific antibody that binds to a
T-cell activating antigen and a Tumor Antigen (TA) for use in a
method of diagnosis or detection is provided. In a further aspect,
a method of detecting the presence of a T-cell activating antigen 3
and/or a Tumor Antigen (TA) in a biological sample is provided. In
certain embodiments, the method comprises contacting the biological
sample with a bispecific antibody that binds to a T-cell activating
antigen and a Tumor Antigen (TA) as described herein under
conditions permissive for binding of the bispecific antibody that
binds to a T-cell activating antigen and a Tumor Antigen (TA) to a
T-cell activating antigen and/or a Tumor Antigen (TA), and
detecting whether a complex is formed between the bispecific
antibody that binds to a T-cell activating antigen and a Tumor
Antigen (TA) and a T-cell activating antigen and/or a Tumor Antigen
(TA). Such method may be an in vitro or in vivo method. In one
embodiment, a bispecific antibody that binds to a T-cell activating
antigen and a Tumor Antigen (TA) is used to select subjects
eligible for therapy with a bispecific antibody that binds to a
T-cell activating antigen and a Tumor Antigen (TA), e.g. where a
Tumor Antigen (TA) is a biomarker for selection of patients.
[0287] Exemplary disorders that may be diagnosed using an antibody
of the invention include cancer.
[0288] In certain embodiments, labeled bispecific antibodies that
bind to a T-cell activating antigen and a Tumor Antigen (TA) are
provided. Labels include, but are not limited to, labels or
moieties that are detected directly (such as fluorescent,
chromophoric, electron-dense, chemiluminescent, and radioactive
labels), as well as moieties, such as enzymes or ligands, that are
detected indirectly, e.g., through an enzymatic reaction or
molecular interaction. Exemplary labels include, but are not
limited to, the radioisotopes .sup.32P, .sup.14C, .sup.125I,
.sup.3H, and .sup.131I, fluorophores such as rare earth chelates or
fluorescein and its derivatives, rhodamine and its derivatives,
dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and
bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),
alkaline phosphatase, .beta.-galactosidase, glucoamylase, lysozyme,
saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as
uricase and xanthine oxidase, coupled with an enzyme that employs
hydrogen peroxide to oxidize a dye precursor such as HRP,
lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,
bacteriophage labels, stable free radicals, and the like.
[0289] F. Pharmaceutical Formulations
[0290] Pharmaceutical formulations of a bispecific antibody that
binds to a T-cell activating antigen and a Tumor Antigen (TA) as
described herein are prepared by mixing such bispecific antibody
having the desired degree of purity with one or more optional
pharmaceutically acceptable carriers (Remington's Pharmaceutical
Sciences 16th edition, Osol, A. Ed. (1980)), in the form of
lyophilized formulations or aqueous solutions. Pharmaceutically
acceptable carriers are generally nontoxic to recipients at the
dosages and concentrations employed, and include, but are not
limited to: buffers such as phosphate, citrate, and other organic
acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride; benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as polyethylene glycol (PEG). Exemplary
pharmaceutically acceptable carriers herein further include
insterstitial drug dispersion agents such as soluble neutral-active
hyaluronidase glycoproteins (sHASEGP), for example, human soluble
PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX.RTM.,
Baxter International, Inc.). Certain exemplary sHASEGPs and methods
of use, including rHuPH20, are described in US Patent Publication
Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is
combined with one or more additional glycosaminoglycanases such as
chondroitinases.
[0291] Exemplary lyophilized antibody formulations are described in
U.S. Pat. No. 6,267,958. Aqueous antibody formulations include
those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the
latter formulations including a histidine-acetate buffer.
[0292] The formulation herein may also contain more than one active
ingredients as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. Such active ingredients are suitably
present in combination in amounts that are effective for the
purpose intended.
[0293] Active ingredients may be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0294] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules.
[0295] The formulations to be used for in vivo administration are
generally sterile. Sterility may be readily accomplished, e.g., by
filtration through sterile filtration membranes.
[0296] G. Therapeutic Methods and Compositions
[0297] Any of the bispecific antibodies that bind to a T-cell
activating antigen and a Tumor Antigen (TA) provided herein may be
used in therapeutic methods.
[0298] In one aspect, a bispecific antibody that binds to a T-cell
activating antigen and a Tumor Antigen (TA) for use as a medicament
is provided. In further aspects, a bispecific antibody that binds
to a T-cell activating antigen and a Tumor Antigen (TA) use in
treating cancer is provided. In certain embodiments, a bispecific
antibody that binds to a T-cell activating antigen and a Tumor
Antigen (TA) for use in a method of treatment is provided. In
certain embodiments, the invention provides a bispecific antibody
that binds to a T-cell activating antigen and a Tumor Antigen (TA)
for use in a method of treating an individual having cancer
comprising administering to the individual an effective amount of
the bispecific antibody that binds to a T-cell activating antigen
and a Tumor Antigen (TA). In one such embodiment, the method
further comprises administering to the individual an effective
amount of at least one additional therapeutic agent, e.g., as
described below. An "individual" according to any of the above
embodiments is preferably a human.
[0299] In a further aspect, the invention provides for the use of a
bispecific antibody that binds to a T-cell activating antigen and a
Tumor Antigen (TA) in the manufacture or preparation of a
medicament. In one embodiment, the medicament is for treatment of
cancer. In a further embodiment, the medicament is for use in a
method of treating cancer comprising administering to an individual
having cancer an effective amount of the medicament. In one such
embodiment, the method further comprises administering to the
individual an effective amount of at least one additional
therapeutic agent, e.g., as described below. An "individual"
according to any of the above embodiments may be a human.
[0300] In a further aspect, the invention provides a method for
treating cancer. In one embodiment, the method comprises
administering to an individual having cancer an effective amount of
a bispecific antibody that binds to a T-cell activating antigen and
a Tumor Antigen (TA). In one such embodiment, the method further
comprises administering to the individual an effective amount of at
least one additional therapeutic agent, as described below. An
"individual" according to any of the above embodiments may be a
human.
[0301] In a further aspect, the invention provides pharmaceutical
formulations comprising any of the bispecific antibodies that bind
to a T-cell activating antigen and a Tumor Antigen (TA) provided
herein, e.g., for use in any of the above therapeutic methods. In
one embodiment, a pharmaceutical formulation comprises any of the
bispecific antibody that binds to a T-cell activating antigen and a
Tumor Antigen (TA) provided herein and a pharmaceutically
acceptable carrier. In another embodiment, a pharmaceutical
formulation comprises any of the bispecific antibodies that bind to
a T-cell activating antigen and a Tumor Antigen (TA) provided
herein and at least one additional therapeutic agent, e.g., as
described below.
[0302] The bispecific antibodies of the invention can be used
either alone or in combination with other agents in a therapy. For
instance, a bispecific antibody of the invention may be
co-administered with at least one additional therapeutic agent.
[0303] Such combination therapies noted above encompass combined
administration (where two or more therapeutic agents are included
in the same or separate formulations), and separate administration,
in which case, administration of the antibody of the invention can
occur prior to, simultaneously, and/or following, administration of
the additional therapeutic agent and/or adjuvant. Bispecific
antibodies of the invention can also be used in combination with
radiation therapy.
[0304] A bispecific antibody of the invention (and any additional
therapeutic agent) can be administered by any suitable means,
including parenteral, intrapulmonary, and intranasal, and, if
desired for local treatment, intralesional administration.
Parenteral infusions include intramuscular, intravenous,
intraarterial, intraperitoneal, or subcutaneous administration.
Dosing can be by any suitable route, e.g. by injections, such as
intravenous or subcutaneous injections, depending in part on
whether the administration is brief or chronic. Various dosing
schedules including but not limited to single or multiple
administrations over various time-points, bolus administration, and
pulse infusion are contemplated herein.
[0305] Bispecific antibodies of the invention would be formulated,
dosed, and administered in a fashion consistent with good medical
practice. Factors for consideration in this context include the
particular disorder being treated, the particular mammal being
treated, the clinical condition of the individual patient, the
cause of the disorder, the site of delivery of the agent, the
method of administration, the scheduling of administration, and
other factors known to medical practitioners. The bispecific
antibody need not be, but is optionally formulated with one or more
agents currently used to prevent or treat the disorder in question.
The effective amount of such other agents depends on the amount of
antibody present in the formulation, the type of disorder or
treatment, and other factors discussed above. These are generally
used in the same dosages and with administration routes as
described herein, or about from 1 to 99% of the dosages described
herein, or in any dosage and by any route that is
empirically/clinically determined to be appropriate.
[0306] For the prevention or treatment of disease, the appropriate
dosage of a bispecific antibody of the invention (when used alone
or in combination with one or more other additional therapeutic
agents) will depend on the type of disease to be treated, the type
of antibody, the severity and course of the disease, whether the
bispecific antibody is administered for preventive or therapeutic
purposes, previous therapy, the patient's clinical history and
response to the bispecific antibody, and the discretion of the
attending physician. The antibody is suitably administered to the
patient at one time or over a series of treatments. Depending on
the type and severity of the disease, about 1 .mu.g/kg to 15 mg/kg
(e.g. 0.1 mg/kg-10 mg/kg) of bispecific antibody can be an initial
candidate dosage for administration to the patient, whether, for
example, by one or more separate administrations, or by continuous
infusion. One typical daily dosage might range from about 1
.mu.g/kg to 100 mg/kg or more, depending on the factors mentioned
above. For repeated administrations over several days or longer,
depending on the condition, the treatment would generally be
sustained until a desired suppression of disease symptoms occurs.
One exemplary dosage of the bispecific antibody would be in the
range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more
doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any
combination thereof) may be administered to the patient. Such doses
may be administered intermittently, e.g. every week or every three
weeks (e.g. such that the patient receives from about two to about
twenty, or e.g. about six doses of the bispecific antibody). An
initial higher loading dose, followed by one or more lower doses
may be administered. However, other dosage regimens may be useful.
The progress of this therapy is easily monitored by conventional
techniques and assays.
[0307] It is understood that any of the above formulations or
therapeutic methods may be carried out using an immunoconjugate of
the invention in place of or in addition to a bispecific antibody
that binds to a T-cell activating antigen and a Tumor Antigen
(TA).
[0308] H. Articles of Manufacture
[0309] In another aspect of the invention, an article of
manufacture containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
IV solution bags, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds a
composition which is by itself or combined with another composition
effective for treating, preventing and/or diagnosing the condition
and may have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is a bispecific antibody of the invention.
The label or package insert indicates that the composition is used
for treating the condition of choice. Moreover, the article of
manufacture may comprise (a) a first container with a composition
contained therein, wherein the composition comprises a bispecific
antibody of the invention; and (b) a second container with a
composition contained therein, wherein the composition comprises a
further cytotoxic or otherwise therapeutic agent. The article of
manufacture in this embodiment of the invention may further
comprise a package insert indicating that the compositions can be
used to treat a particular condition. Alternatively, or
additionally, the article of manufacture may further comprise a
second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0310] It is understood that any of the above articles of
manufacture may include an immunoconjugate of the invention in
place of or in addition to a bispecific antibody that binds to a
T-cell activating antigen and a Tumor Antigen (TA).
III. Examples
[0311] The following are examples of methods and compositions of
the invention. It is understood that various other embodiments may
be practiced, given the general description provided above.
[0312] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, the descriptions and examples should not be
construed as limiting the scope of the invention. The disclosures
of all patent and scientific literature cited herein are expressly
incorporated in their entirety by reference.
Example 1
Preparation of Fab (MCSP)-CrossFab(CD3)
[0313] The resulting variable region of heavy and light chain DNA
sequences have been subcloned in frame with either the constant
heavy chain or the constant light chain pre-inserted into the
respective recipient mammalian expression vector. The antibody
expression is driven by an MPSV promoter and carries a synthetic
polyA signal sequence at the 3' end of the CDS. In addition each
vector contains an EBV OriP sequence.
[0314] The molecule is produced by co-transfecting HEK293-EBNA
cells with the mammalian expression vectors using a calcium
phosphate-transfection. Exponentially growing HEK293-EBNA cells are
transfected by the calcium phosphate method. Alternatively,
HEK293-EBNA cells growing in suspension are transfected by
polyethylenimine. The cells are transfected with the corresponding
expression vectors in a 1:1:1 ratio ("vector CH1-VH-CK-VH": "vector
light chain": "vector light chain CH1-VL").
[0315] For transfection using calcium phosphate cells are grown as
adherent monolayer cultures in T-flasks using DMEM culture medium
supplemented with 10% (v/v) FCS, and are transfected when they are
between 50 and 80% confluent. For the transfection of a T150 flask,
15 million cells are seeded 24 hours before transfection in 25 ml
DMEM culture medium supplemented with FCS (at 10% v/v final), and
cells are placed at 37.degree. C. in an incubator with a 5% CO2
atmosphere overnight. For each T150 flask to be transfected, a
solution of DNA, CaCl2 and water is prepared by mixing 94 .mu.g
total plasmid vector DNA divided in the corresponding ratio, water
to a final volume of 469 .mu.l and 469 .mu.l of a 1 M CaCl2
solution. To this solution, 938 .mu.l of a 50 mM HEPES, 280 mM
NaCl, 1.5 mM Na2HPO4 solution at pH 7.05 are added, mixed
immediately for 10 s and left to stand at room temperature for 20
s. The suspension is diluted with 10 ml of DMEM supplemented with
2% (v/v) FCS, and added to the T150 in place of the existing
medium. Then additional 13 ml of transfection medium are added. The
cells are incubated at 37.degree. C., 5% CO2 for about 17 to 20
hours, then medium is replaced with 25 ml DMEM, 10% FCS. The
conditioned culture medium is harvested approx. 7 days post-media
exchange by centrifugation for 15 min at 210.times.g, the solution
is sterile filtered (0.22 .mu.m filter) and sodium azide in a final
concentration of 0.01% (w/v) is added, and kept at 4.degree. C.
[0316] For transfection using polyethylenimine HEK293 EBNA cells
are cultivated in suspension serum free in CD CHO culture medium.
For the production in 500 ml shake flask 400 million HEK293 EBNA
cells are seeded 24 hours before transfection. For transfection
cells are centrifuged for 5 min by 210.times.g, supernatant is
replaced by pre-warmed 20 ml CD CHO medium. Expression vectors are
mixed in 20 ml CD CHO medium to a final amount of 200 .mu.g DNA.
After addition of 540 .mu.l PEI solution is vortexed for 15 s and
subsequently incubated for 10 min at room temperature. Afterwards
cells are mixed with the DNA/PEI solution, transferred to a 500 ml
shake flask and incubated for 3 hours by 37.degree. C. in an
incubator with a 5% CO2 atmosphere. After incubation time 160 ml
F17 medium is added and cell are cultivated for 24 hours. One day
after transfection 1 mM valporic acid and 7% Feed 1 (Lonza) is
added. After 7 days cultivation supernatant is collected for
purification by centrifugation for 15 min at 210.times.g, the
solution is sterile filtered (0.22 .mu.m filter) and sodium azide
in a final concentration of 0.01% w/v is added, and kept at
4.degree. C.
[0317] The secreted protein is purified from cell culture
supernatants by affinity chromatography using Protein A and Protein
G affinity chromatography, followed by a size exclusion
chromatographic step. For affinity chromatography supernatant is
loaded on a HiTrap Protein A HP column (CV=5 ml, GE Healthcare)
coupled to a HiTrap Protein G HP column (CV=5 ml, GE Healthcare)
each column equilibrated with 30 ml 20 mM sodium phosphate, 20 mM
sodium citrate, pH 7.5. Unbound protein is removed by washing both
columns with 6 column volume 20 mM sodium phosphate, 20 mM sodium
citrate, pH 7.5. Subsequently an additional wash step is necessary
to wash only the HiTrap Protein G HP column using at least 8 column
volume 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5. The
target protein is eluted from HiTrap Protein G HP column using a
step gradient with 7 column volume 8.8 mM formic acid, pH 3.0.
Protein solution is neutralized by adding 1/10 of 0.5 M sodium
phosphate, pH 8.0. Target protein is concentrated and filtrated
prior loading on a HiLoad Superdex 200 column (GE Healthcare)
equilibrated with 25 mM potassium phosphate, 125 mM sodium
chloride, 100 mM glycine solution of pH 6.7.
[0318] The protein concentration of purified protein samples is
determined by measuring the optical density (OD) at 280 nm, using
the molar extinction coefficient calculated on the basis of the
amino acid sequence. Purity and molecular weight of antibodies are
analyzed by SDS-PAGE in the presence and absence of a reducing
agent (5 mM 1,4-dithiotreitol) and staining with Coomassie
(SimpleBlue.TM. SafeStain from Invitrogen). The NuPAGE.RTM.
Pre-Cast gel system (Invitrogen, USA) is used according to the
manufacturer's instruction (4-12% Tris-Acetate gels or 4-12%
Bis-Tris). The aggregate content of antibody samples is analyzed
using a Superdex 200 10/300GL analytical size-exclusion column (GE
Healthcare, Sweden) in 2 mM MOPS, 150 mM NaCl, 0.02% (w/v) NaN3, pH
7.3 running buffer at 25.degree. C.
Analysis of production and purification of an exemplary
Fab-Crossfab molecule (consisting of three chains:
VHCH1(MCSP)-VLCH1(CD3.sub.V9)=SEQ ID NO:25, VLCL(MCSP)=SEQ ID NO:17
and VHCL(CD3.sub.V9)=SEQ ID NO:23; with an orientation as depicted
in FIG. 1 a)) is shown in FIGS. 2 and 3. This molecule is further
referred to as Fab (MCSP)-Crossfab (CD3) or hu Fab (MCSP)-Crossfab
(CD3).
Example 2
Preparation of Fab (MCSP)-Fab (MCSP)-CrossFab(CD3) and Fab
(MCSP)-CrossFab(CD3)-Fab (MCSP)
[0319] The resulting variable region of heavy and light chain DNA
sequences have been subcloned in frame with either the constant
heavy chain or the constant light chain pre-inserted into the
respective recipient mammalian expression vector. The antibody
expression is driven by an MPSV promoter and carries a synthetic
polyA signal sequence at the 3' end of the CDS. In addition each
vector contains an EBV OriP sequence.
[0320] The molecule is produced by co-transfecting HEK293-EBNA
cells with the mammalian expression vectors using a calcium
phosphate-transfection. Exponentially growing HEK293-EBNA cells are
transfected by the calcium phosphate method. Alternatively,
HEK293-EBNA cells growing in suspension are transfected by
polyethylenimine. The cells are transfected with the corresponding
expression vectors in a 1:2:1 ratio ("vector CH1-VH-CH1-VH-CK-VH":
"vector light chain": "vector light chain CH1-VL").
[0321] For transfection using calcium phosphate cells are grown as
adherent monolayer cultures in T-flasks using DMEM culture medium
supplemented with 10% (v/v) FCS, and are transfected when they are
between 50 and 80% confluent. For the transfection of a T150 flask,
15 million cells are seeded 24 hours before transfection in 25 ml
DMEM culture medium supplemented with FCS (at 10% v/v final), and
cells are placed at 37.degree. C. in an incubator with a 5% CO2
atmosphere overnight. For each T150 flask to be transfected, a
solution of DNA, CaCl2 and water is prepared by mixing 94 .mu.g
total plasmid vector DNA divided in the corresponding ratio, water
to a final volume of 469 .mu.l and 469 .mu.l of a 1 M CaCl2
solution. To this solution, 938 .mu.l of a 50 mM HEPES, 280 mM
NaCl, 1.5 mM Na2HPO4 solution at pH 7.05 are added, mixed
immediately for 10 s and left to stand at room temperature for 20
s. The suspension is diluted with 10 ml of DMEM supplemented with
2% (v/v) FCS, and added to the T150 in place of the existing
medium. Then additional 13 ml of transfection medium are added. The
cells are incubated at 37.degree. C., 5% CO2 for about 17 to 20
hours, then medium is replaced with 25 ml DMEM, 10% FCS. The
conditioned culture medium is harvested approx. 7 days post-media
exchange by centrifugation for 15 min at 210.times.g, the solution
is sterile filtered (0.22 .mu.m filter) and sodium azide in a final
concentration of 0.01% (w/v) is added, and kept at 4.degree. C. For
transfection using polyethylenimine HEK293 EBNA cells are
cultivated in suspension serum free in CD CHO culture medium. For
the production in 500 ml shake flask 400 million HEK293 EBNA cells
are seeded 24 hours before transfection. For transfection cells are
centrifuged for 5 min by 210.times.g, supernatant is replaced by
pre-warmed 20 ml CD CHO medium. Expression vectors are mixed in 20
ml CD CHO medium to a final amount of 200 .mu.g DNA. After addition
of 540 .mu.l PEI solution is vortexed for 15 s and subsequently
incubated for 10 min at room temperature. Afterwards cells are
mixed with the DNA/PEI solution, transferred to a 500 ml shake
flask and incubated for 3 hours by 37.degree. C. in an incubator
with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium
is added and cell are cultivated for 24 hours. One day after
transfection 1 mM valporic acid and 7% Feed 1 (Lonza) is added.
After 7 days cultivation supernatant is collected for purification
by centrifugation for 15 min at 210.times.g, the solution is
sterile filtered (0.22 .mu.m filter) and sodium azide in a final
concentration of 0.01% w/v is added and kept at 4.degree. C.
[0322] The secreted protein is purified from cell culture
supernatants by affinity chromatography using Protein A and Protein
G affinity chromatography, followed by a size exclusion
chromatographic step.
[0323] For affinity chromatography supernatant is loaded on a
HiTrap Protein A HP column (CV=5 ml, GE Healthcare) coupled to a
HiTrap Protein G HP column (CV=5 ml, GE Healthcare) each column
equilibrated with 30 ml 20 mM sodium phosphate, 20 mM sodium
citrate, pH 7.5. Unbound protein is removed by washing both columns
with 6 column volume 20 mM sodium phosphate, 20 mM sodium citrate,
pH 7.5. Subsequently an additional wash step is necessary to wash
only the HiTrap Protein G HP column using at least 8 column volume
20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5. The target
protein is eluted from HiTrap Protein G HP column using a step
gradient with 7 column volume 8.8 mM formic acid, pH 3.0. Protein
solution is neutralized by adding 1/10 of 0.5 M sodium phosphate,
pH 8.0. Target protein is concentrated and filtrated prior loading
on a HiLoad Superdex 200 column (GE Healthcare) equilibrated with
25 mM potassium phosphate, 125 mM sodium chloride, 100 mM glycine
solution of pH 6.7.
[0324] The protein concentration of purified protein samples is
determined by measuring the optical density (OD) at 280 nm, using
the molar extinction coefficient calculated on the basis of the
amino acid sequence. Purity and molecular weight of antibodies are
analyzed by SDS-PAGE in the presence and absence of a reducing
agent (5 mM 1,4-dithiotreitol) and staining with Coomassie
(SimpleBlue.TM. SafeStain from Invitrogen). The NuPAGE.RTM.
Pre-Cast gel system (Invitrogen, USA) is used according to the
manufacturer's instruction (4-12% Tris-Acetate gels or 4-12%
Bis-Tris). The aggregate content of antibody samples is analyzed
using a Superdex 200 10/300GL analytical size-exclusion column (GE
Healthcare, Sweden) in 2 mM MOPS, 150 mM NaCl, 0.02% (w/v)
NaN.sub.3, pH 7.3 running buffer at 25.degree. C. and compared with
prior art antibody fragment (scFv)2 (results see table below).
TABLE-US-00003 Aggregate after 1.sup.st Yield purification HMW LMW
Monomer Construct [mg/1] step [%] [%] [%] [%] (scFv)2 3.84 80 0 0
100 Fab-Crossfab 7.85 13.8 0 0 100 (Fab)2-Crossfab 7.8 3.6 0 0 100
Fab-Crossfab-Fab 5.3 1.7 0.4 0 99.56 HMW = High Molecular Weight;
LMW = Low Molecular Weight
Analysis of production and purification of an exemplary
Fab-Fab-Crossfab molecule (consisting of four chains:
VHCH1(MCSP)-VHCH1(MCSP)-VLCH1(CD3.sub.V9)=SEQ ID NO:26, 2
VLCL(MCSP) chains=SEQ ID NO:17 and one VHCL(CD3.sub.V9) chain=SEQ
ID NO:23; with an orientation as depicted in FIG. 1c)) is shown in
FIGS. 4 and 5. This molecule is further referred to as Fab
(MCSP)-Fab (MCSP)-Crossfab (CD3) or hu Fab (MCSP)-Fab
(MCSP)-Crossfab (CD3). Analysis of production and purification of
an exemplary Fab-Crossfab-Fab molecule (consisting of four chains:
VHCH1(MCSP)-VLCH1(CD3.sub.V9)-VHCH1(MCSP)=SEQ ID NO:27, 2
VLCL(MCSP) chains=SEQ ID NO:17 and one VHCL(CD3.sub.V9) chain=SEQ
ID NO:23; with an orientation as depicted in FIG. 1 e)) is shown in
FIGS. 6 and 7. This molecule is further referred to as Fab
(MCSP)-Fab (MCSP)-Crossfab (CD3) or hu Fab (MCSP)-Fab
(MCSP)-Crossfab (CD3). Analysis of production and purification of
an exemplary Crossfab-Fab-Fab molecule (consisting of four chains:
VLCH1(CD3.sub.2C11)-VHCH1(MCSP)-VHCH1(MCSP)=SEQ ID NO:42, 2
VLCL(MCSP) chains=SEQ ID NO:17 and one VHCL(CD3.sub.2C11) chain=SEQ
ID NO:43; with an orientation as depicted in FIG. 1 d)) is shown in
FIGS. 8 and 9. This molecule is further referred to as murine
Crossfab (CD3)-Fab (MCSP)-Fab (MCSP).
Example 3
Preparation of Fab(CD33)-CrossFab (CD3)
[0325] The resulting variable region of heavy and light chain DNA
sequences have been subcloned in frame with either the constant
heavy chain or the constant light chain pre-inserted into the
respective recipient mammalian expression vector. The antibody
expression is driven by an MPSV promoter and carries a synthetic
polyA signal sequence at the 3' end of the CDS. In addition each
vector contains an EBV OriP sequence.
[0326] The molecule is produced by co-transfecting HEK293-EBNA
cells with the mammalian expression vectors using a calcium
phosphate-transfection. Exponentially growing HEK293-EBNA cells are
transfected by the calcium phosphate method. Alternatively,
HEK293-EBNA cells growing in suspension are transfected by
polyethylenimine. The cells are transfected with the corresponding
expression vectors in a 1:1:1 ratio ("vector CH1-VH-CK-VH": "vector
light chain": "vector light chain CH1-VL").
[0327] For transfection using calcium phosphate cells are grown as
adherent monolayer cultures in T-flasks using DMEM culture medium
supplemented with 10% (v/v) FCS, and are transfected when they are
between 50 and 80% confluent. For the transfection of a T150 flask,
15 million cells are seeded 24 hours before transfection in 25 ml
DMEM culture medium supplemented with FCS (at 10% v/v final), and
cells are placed at 37.degree. C. in an incubator with a 5% CO2
atmosphere overnight. For each T150 flask to be transfected, a
solution of DNA, CaCl2 and water is prepared by mixing 94 .mu.g
total plasmid vector DNA divided in the corresponding ratio, water
to a final volume of 469 .mu.l and 469 .mu.l of a 1 M CaCl2
solution. To this solution, 938 .mu.l of a 50 mM HEPES, 280 mM
NaCl, 1.5 mM Na2HPO4 solution at pH 7.05 are added, mixed
immediately for 10 s and left to stand at room temperature for 20
s. The suspension is diluted with 10 ml of DMEM supplemented with
2% (v/v) FCS, and added to the T150 in place of the existing
medium. Then additional 13 ml of transfection medium are added. The
cells are incubated at 37.degree. C., 5% CO2 for about 17 to 20
hours, then medium is replaced with 25 ml DMEM, 10% FCS. The
conditioned culture medium is harvested approx. 7 days post-media
exchange by centrifugation for 15 min at 210.times.g, the solution
is sterile filtered (0.22 .mu.m filter) and sodium azide in a final
concentration of 0.01% (w/v) is added, and kept at 4.degree. C.
[0328] For transfection using polyethylenimine HEK293 EBNA cells
are cultivated in suspension serum free in CD CHO culture medium.
For the production in 500 ml shake flask 400 million HEK293 EBNA
cells are seeded 24 hours before transfection. For transfection
cells are centrifuged for 5 min by 210.times.g, supernatant is
replaced by pre-warmed 20 ml CD CHO medium. Expression vectors are
mixed in 20 ml CD CHO medium to a final amount of 200 .mu.g DNA.
After addition of 540 .mu.l PEI solution is vortexed for 15 s and
subsequently incubated for 10 min at room temperature. Afterwards
cells are mixed with the DNA/PEI solution, transferred to a 500 ml
shake flask and incubated for 3 hours by 37.degree. C. in an
incubator with a 5% CO2 atmosphere. After incubation time 160 ml
F17 medium is added and cell are cultivated for 24 hours. One day
after transfection 1 mM valporic acid and 7% Feed 1 (LONZA) is
added. After 7 days cultivation supernatant is collected for
purification by centrifugation for 15 min at 210.times.g, the
solution is sterile filtered (0.22 .mu.m filter) and sodium azide
in a final concentration of 0.01% w/v is added, and kept at
4.degree. C.
[0329] The secreted protein is purified from cell culture
supernatants by affinity chromatography using Protein A and
ProteinG affinity chromatography, followed by a size exclusion
chromatographic step.
[0330] For affinity chromatography supernatant is loaded on a
HiTrap ProteinA HP column (CV=5 mL, GE Healthcare) coupled to a
HiTrap ProteinG HP column (CV=5 mL, GE Healthcare) each column
equilibrated with 30 ml 20 mM sodium phosphate, 20 mM sodium
citrate, pH 7.5. Unbound protein is removed by washing both columns
with 6 column volume 20 mM sodium phosphate, 20 mM sodium citrate,
pH 7.5. Subsequently an additional wash step is necessary to wash
only the HiTrap ProteinG HP column using at least 8 column volume
20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5. The target
protein is eluted from HiTrap ProteinG HP column using a step
gradient with 7 column volume 8.8 mM formic acid, pH 3.0. Protein
solution is neutralized by adding 1/10 of 0.5M sodium phosphate, pH
8.0. Target protein is concentrated and filtrated prior loading on
a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 25
mM potassium phosphate, 125 mM sodium chloride, 100 mM glycine
solution of pH 6.7.
[0331] The protein concentration of purified protein samples is
determined by measuring the optical density (OD) at 280 nm, using
the molar extinction coefficient calculated on the basis of the
amino acid sequence. Purity and molecular weight of antibodies are
analyzed by SDS-PAGE in the presence and absence of a reducing
agent (5 mM 1,4-dithiotreitol) and staining with Coomassie
(SimpleBlue.TM. SafeStain from Invitrogen). The NuPAGE.RTM.
Pre-Cast gel system (Invitrogen, USA) is used according to the
manufacturer's instruction (4-12% Tris-Acetate gels or 4-12%
Bis-Tris). The aggregate content of antibody samples is analyzed
using a Superdex 200 10/300GL analytical size-exclusion column (GE
Healthcare, Sweden) in 2 mM MOPS, 150 mM NaCl, 0.02% (w/v) NaN3, pH
7.3 running buffer at 25.degree. C.
[0332] Analysis of production and purification of an exemplary
Fab-Crossfab molecule (consisting of three chains:
VHCH1(CD33)-VLCH1(CD3.sub.V9)=SEQ ID NO:102, VLCL(CD33)=SEQ ID
NO:100 and VHCL(CD3.sub.V9)=SEQ ID NO:23 or SEQ ID NO:101; with an
orientation as depicted in FIG. 1a)) is shown in FIGS. 17 and 18.
This molecule is further referred to as Fab(CD33)-CrossFab (CD3) or
hu Fab(CD33)-CrossFab (CD3).
Example 4
Preparation of the Reference Molecule (scFv)2
[0333] Cloning and Production
[0334] The resulting variable region of heavy and light chain DNA
sequences have been subcloned in frame into the respective
recipient mammalian expression vector. The antibody expression is
driven by an MPSV promoter and carries a synthetic polyA signal
sequence at the 3' end of the CDS. In addition each vector contains
an EBV OriP sequence.
[0335] The molecule is produced by transfecting HEK293-EBNA cells
with the mammalian expression vector using polyethylenimine. HEK293
EBNA cells are cultivated in suspension serum free in CD CHO
culture medium. For the production in 500 ml shake flask 400
million HEK293 EBNA cells are seeded 24 hours before transfection.
For transfection cells are centrifuged for 5 min by 210.times.g,
supernatant is replaced by pre-warmed 20 ml CD CHO medium.
Expression vectors are mixed in 20 ml CD CHO medium to a final
amount of 200 .mu.g DNA. After addition of 540 .mu.l PEI solution
is vortexed for 15 s and subsequently incubated for 10 min at room
temperature. Afterwards cells are mixed with the DNA/PEI solution,
transferred to a 500 ml shake flask and incubated for 3 hours by
37.degree. C. in an incubator with a 5% CO2 atmosphere. After
incubation time 160 ml F17 medium is added and cell are cultivated
for 24 hours. One day after transfection 1 mM valporic acid and 7%
Feed 1 (LONZA) are added. After 7 days cultivation supernatant is
collected for purification by centrifugation for 15 min at
210.times.g, the solution is sterile filtered (0.22 .mu.m filter)
and sodium azide in a final concentration of 0.01% w/v is added,
and kept at 4.degree. C.
[0336] Purification of (scFv).sub.2 (Anti MCSP/Anti huCD3)
[0337] The secreted protein is purified from cell culture
supernatants by affinity chromatography using Immobilized Metal Ion
Affinity Chromatography (IMAC), followed by a size exclusion
chromatographic step.
[0338] Prior first purification step disturbing components from the
supernatant are removed by diafiltration using the tangential flow
filtration system Sarcojet (Sartorius) equipped with a 5.000 MWCO
membrane (Sartocon Slice Cassette, Hydrosart; Sartorius).
Supernatant is concentrated to 210 ml and subsequently diluted in 1
l 20 mM sodium phosphate, 500 mM sodium chloride, pH 6.5. The
protein solution is concentrated again to 210 ml. This process is
repeated twice to ensure a complete buffer exchange.
[0339] For affinity chromatography retentate of the diafiltration
process is loaded on a NiNTA Superflow Cartridge (CV=5 mL, Qiagen)
equilibrated with 25 ml 20 mM sodium phosphate, 500 mM sodium
chloride, 15 mM imidazole, pH 6.5. Unbound protein is removed by
washing with at least 2 column volume 20 mM sodium phosphate, 500
mM sodium chloride, 15 mM imidazole, pH 6.5 followed by an
additional wash step using 3 column volume 20 mM sodium phosphate,
500 mM sodium chloride, 62.5 mM imidazole, pH 6.5. Target protein
is eluted in 2 column volume 20 mM sodium phosphate, 500 mM sodium
chloride, 125 mM imidazole, pH 6.5. Column is washed subsequently
with 20 mM sodium phosphate, 500 mM sodium chloride, 250 mM
imidazole, pH 6.5.
[0340] Target protein is concentrated prior loading on a HiLoad
Superdex 75 column (GE Healthcare) equilibrated with 25 mM
KH.sub.2PO.sub.4, 125 mM NaCl, 200 mM Arginine, pH 6.7. Yields,
aggregate content after the first purification step and final
monomer content is shown in the table above. Comparison of the
aggregate content after the first purification step indicates the
superior stability of the Fab-Crossfab construct in contrast to the
(scFv)2.
[0341] Characterization of (scFv)2
[0342] The protein concentration of purified protein samples is
determined by measuring the optical density (OD) at 280 nm, using
the molar extinction coefficient calculated on the basis of the
amino acid sequence. Purity and molecular weight of antibodies are
analyzed by SDS-PAGE in the presence and absence of a reducing
agent (5 mM 1,4-dithiotreitol) and staining with Coomassie
(SimpleBlue.TM. SafeStain from Invitrogen). The NuPAGE.RTM.
Pre-Cast gel system (Invitrogen, USA) is used according to the
manufacturer's instruction (4-12% Tris-Acetate gels or 4-12%
Bis-Tris). The aggregate content of antibody samples is analyzed
using a Superdex 75 10/300GL analytical size-exclusion column (GE
Healthcare, Sweden) in 2 mM MOPS, 150 mM NaCl, 0.02% (w/v) NaN3, pH
7.3 running buffer at 25.degree. C.
[0343] A schematic drawing of the (scFv)2 molecule is shown in FIG.
21.
[0344] Analysis of production and purification of an exemplary
(scFv)2 molecule (antiMCSP/anti huCD3; consisting two single chain
Fvs: VL-VH (MCSP) and VH-VL (CD3.sub.V9)=SEQ ID NO:149; is shown in
FIGS. 22 and 23. This molecule is further referred to as (scFv)2
(antiMC SP/anti huCD3e).
Example 5
Isolation of Primary Human Pan T Cells from PBMCs
[0345] Peripheral blood mononuclar cells (PBMCs) were prepared by
Histopaque density centrifugation from enriched lymphocyte
preparations (buffy coats) obtained from local blood banks or from
fresh blood from healthy human donors.
[0346] T-cell enrichment from PBMCs was performed using the Pan T
Cell Isolation Kit II (Miltenyi Biotec #130-091-156), according to
the manufacturer's instructions. Briefly, the cell pellets were
diluted in 40 .mu.l cold buffer per 10 Mio cells (PBS with 0.5%
BSA, 2 mM EDTA--sterile filtered) and incubated with 10 .mu.l
Biotin-Antibody Cocktail per 10 Mio cells for 10 min at 4.degree.
C.
[0347] 30 .mu.l cold buffer and 20 .mu.l Anti-Biotin magnetic beads
per 10 Mio cells were added, and the mixture incubated for another
15 min at 4.degree. C.
[0348] Cells were washed by adding 10-20.times. of labeling volume
and a subsequent centrifugation step at 300 g for 10 min. Up to 100
Mio cells were resuspended in 500 .mu.l buffer.
[0349] Magnetic separation of unlabeled human pan T cells was
performed using LS columns (Miltenyi Biotec #130-042-401) according
to the manufacturer's instructions. The resulting T cell population
was counted automatically (ViCell) and stored in AIM-V medium at
37.degree. C., 5% CO2 in the incubator until assay start (not
longer than 24 h).
Example 6
Isolation of Murine Pan T Cells from Splenocytes
[0350] Spleens were isolated from C57BL/6 mice, transferred into a
GentleMACS C-tube (Miltenyi Biotech #130-093-237) containing MACS
buffer (PBS+0.5% BSA+2 mM EDTA) and dissociated with the GentleMACS
Dissociator to obtain single-cell suspensions according to the
manufacturers' instructions.
[0351] The cell suspension was passed through a pre-separation
filter to get rid-off remaining undissociated tissue particles.
After centrifugation at 400 g for 4 minutes at 4.degree. C., ACK
Lysis Buffer was added to to lyse red blood cells (incubation for 5
minutes at room temperature). The remaining cells were washed with
MACS buffer twice, counted and used for the isolation of murine pan
T cells. The negative (magnetic) selection was performed using the
Pan T Cell Isolation Kit from Miltenyi Biotec (#130-090-861),
following the manufacturers' instructions. The resulting T cell
population was counted automatically (ViCell) and used immediately
for further assays.
Example 7
Re-Directed T Cell Cytotoxicity Mediated by Cross-Linked Bispecific
Constructs Targeting CD3 on T Cells and MCSP on Tumor Cells (LDH
Release Assay)
[0352] Bispecific constructs targeting CD3 on human, or mouse T
cells and human on tumor cells, are analyzed by a LDH release assay
regarding their potential to induce T cell-mediated apoptosis of
target cells.
[0353] Briefly, target cells (human Colo-38, human MDA-MB-435,
human melanoma MV-3 or murine B16/F10-huMCSP Fluc 2 clone 48 cells,
all expressing human MCSP) are harvested with Cell Dissociation
Buffer (MCSP is trypsin-sensitive) or trypsin (and then plated the
day before), washed and resuspendend in the appropriate cell
culture medium (see detailed description of the different figures).
20 000-30 000 cells per well are plated in a round-bottom
96-well-plate and the respective antibody dilution was added as
indicated (triplicates). Effector cells were added to obtain a
final E:T ratio of 5:1 (for human pan T cells), 10:1 (for human
PBMCs).
[0354] In addition, 1-10 .mu.g/ml PHA-M (Sigma #L8902), a mixture
of isolectins, isolated from Phaseolus vulgaris, was used as a
mitogenic stimulus to induce human or cynomolgus T cell activation.
For murine T cells, a 5% solution of "rat T-Stim with ConA" (BD
#354115) was used as a positive control for T cell activation.
[0355] For normalization, maximal lysis of the target cells (=100%)
is achieved by incubation of the target cells with a final
concentration of 1% Triton-X-100. Minimal lysis (=0%) refers to
target cells co-incubated with effector cells, but without any
construct or antibody.
[0356] After an overnight incubation of at least 18 h at 37.degree.
C., 5% CO2, LDH release of apoptotic/necrotic target cells into the
supernatant is measured with the LDH detection kit (Roche Applied
Science, #11 644 793 001), according to the manufacturer's
instructions.
[0357] LDH Release Assay with Fab (MCSP)-Crossfab (CD3) and Fab
(MCSP)-Fab (MCSP)-Crossfab (CD3) Bispecific Constructs
[0358] Purified Fab (MCSP)-Crossfab (CD3), Fab (MCSP)-Fab
(MCSP)-Crossfab (CD3) and the (scFv)2 (antiMCSP/anti huCD3e)
reference molecule were analyzed for their potential to induce T
cell-mediated apoptosis in tumor target cells upon crosslinkage of
the construct via binding of both targeting moieties to the
respective antigens on cells. Briefly, huMCSP-expressing MDA-MB-435
human melanoma target cells are harvested with Cell Dissociation
Buffer, washed and resuspendend in AIM-V medium (Invitrogen
#12055-091). 30 000 cells per well were plated in a round-bottom
96-well-plate and the respective antibody dilution was added at the
indicated concentrations. All constructs and controls were adjusted
to the same molarity.
[0359] Human pan T effector cells were added to obtain a final E:T
ratio of 5:1. As a positive control for the activation of human pan
T cells, 1 .mu.g/ml PHA-M (Sigma #L8902) was used. For
normalization, maximal lysis of the target cells (=100%) was
determined by incubation of the target cells with a final
concentration of 1% Triton-X-100. Minimal lysis (=0%) refers to
target cells co-incubated with effector cells, but without any
construct or antibody.
[0360] After an overnight incubation of 20 h at 37.degree. C., 5%
CO2, LDH release of apoptotic/necrotic target cells into the
supernatant was measured with the LDH detection kit (Roche Applied
Science, #11 644 793 001), according to the manufacturer's
instructions.
[0361] As depicted in FIG. 10, the constructs with bivalent
MCSP-targeting show comparable cytotoxic activity compared to the
(scFv)2 (antiMCSP/anti huCD3e) construct, whereas the Fab
(MCSP)-Crossfab (CD3) construct with monovalent MCSP binding is
clearly less potent.
[0362] LDH Release Assay with Fab (MCSP)-Fab (MCSP)-Crossfab (CD3)
Bispecific Construct with MDA-MB-435 Human Melanoma Target
Cells
[0363] Purified Fab (MCSP)-Fab (MCSP)-Crossfab (CD3) and the
(scFv)2 (antiMCSP/anti huCD3e) reference molecule were analyzed for
their potential to induce T cell-mediated apoptosis in tumor target
cells upon crosslinkage of the construct via binding of both
targeting moieties to the respective antigens on cells.
[0364] Briefly, huMCSP-expressing MDA-MB-435 human melanoma target
cells are harvested with Cell Dissociation Buffer, washed and
resuspendend in AIM-V medium (Invitrogen #12055-091). 30 000 cells
per well were plated in a round-bottom 96-well-plate and the
respective antibody dilution was added at the indicated
concentrations. All constructs and controls were adjusted to the
same molarity.
[0365] Human pan T effector cells were added to obtain a final E:T
ratio of 5:1. As a positive control for the activation of human pan
T cells, 5 .mu.g/ml PHA-M (Sigma #L8902) was used. For
normalization, maximal lysis of the target cells (=100%) was
determined by incubation of the target cells with a final
concentration of 1% Triton-X-100. Minimal lysis (=0%) refers to
target cells co-incubated with effector cells, but without any
construct or antibody.
[0366] After an overnight incubation of 21 h at 37.degree. C., 5%
CO2, LDH release of apoptotic/necrotic target cells into the
supernatant was measured with the LDH detection kit (Roche Applied
Science, #11 644 793 001), according to the manufacturer's
instructions.
[0367] As depicted in FIG. 11, the Fab (MCSP)-Fab (MCSP)-Crossfab
(CD3) induces apoptosis in target cells at least comparably good as
the (scFv)2 (antiMCSP/anti huCD3e) molecule.
[0368] LDH Release Assay with Fab (MCSP)-Fab (MCSP)-Crossfab (CD3)
Bispecific Construct with MV-3 Human Melanoma Target Cells
[0369] Purified Fab (MCSP)-Fab (MCSP)-Crossfab (CD3) and the
(scFv)2 (antiMCSP/anti huCD3e) molecule were analyzed for their
potential to induce T cell-mediated apoptosis in tumor target cells
upon crosslinkage of the construct via binding of both targeting
moieties to the respective antigens on cells.
[0370] Briefly, huMCSP-expressing MV-3 human melanoma target cells
are harvested with trypsin on the day before the LDH release assay
was started. Cell were washed and resuspendend in the appropriate
cell culture medium. 30 000 cells per well were plated in a
round-bottom 96-well-plate. The next day, the supernatant was
discarded and 100 .mu.l/well AIM-V medium (Invitrogen #12055-091),
as well as the respective antibody dilution were added at the
indicated concentrations. All constructs and controls were adjusted
to the same molarity.
[0371] Human PBMC effector cells were added to obtain a final E:T
ratio of 10:1. As a positive control for the activation of human
pan T cells, 5 .mu.g/ml PHA-M (Sigma #L8902) was used. For
normalization, maximal lysis of the target cells (=100%) was
determined by incubation of the target cells with a final
concentration of 1% Triton-X-100. Minimal lysis (=0%) refers to
target cells co-incubated with effector cells, but without any
construct or antibody.
[0372] After an overnight incubation of 26 h at 37.degree. C., 5%
CO2, LDH release of apoptotic/necrotic target cells into the
supernatant was measured with the LDH detection kit (Roche Applied
Science, #11 644 793 001), according to the manufacturer's
instructions.
[0373] As depicted in FIG. 12, the Fab (MCSP)-Fab (MCSP)-Crossfab
(CD3) induces apoptosis in target cells at least comparably good as
the (scFv)2 (antiMCSP/anti huCD3e) molecule.
[0374] LDH Release Assay with Fab (MCSP)-Crossfab (CD3) Bispecific
Construct with MV-3 Human Melanoma Target Cells
[0375] An LDH release assay was performed as outlined above. FIG.
19 shows killing of huMCSP-positive MV-3 tumor cells upon
co-culture with human PBMCs (E:T ratio=10:1), treated with Fab
(MCSP)-Crossfab (CD3), respective the (scFv)2 (antiMCSP/anti
huCD3e) reference molecule for .about.24 hours.
[0376] LDH Release Assay with Murine Crossfab (CD3)-Fab (MCSP)-Fab
(MCSP) Bispecific Construct
[0377] Purified with murine Crossfab (CD3)-Fab (MCSP)-Fab (MCSP),
targeting murine CD3, as well as human MCSP, was analyzed for its
potential to induce T cell-mediated apoptosis in tumor target cells
upon crosslinkage of the construct via binding of both targeting
moieties to the respective antigens on cells.
[0378] Briefly, huMCSP-expressing B16/F10-huMCSP Fluc2 clone 48
tumor target cells are harvested with Cell Dissociation Buffer,
washed and resuspendend in RPMI1640 medium, including 1.times.NEAA,
10 mM Hepes, 50 .mu.m 2-b-ME and 1 mM sodium pyruvate.
[0379] 20 000 cells per well were plated in a round-bottom
96-well-plate and the respective antibody dilution was added at the
indicated concentrations. The bispecific construct and the
different IgG controls were adjusted to the same molarity. As an
additional control for the activation of murine T cells "T Cell
Stim with ConA" (BD #354115) was used, diluted 1:160 with assay
medium.
[0380] Murine pan T effector cells, isolated from splenocytes
(C57BL/6 mice) were added to obtain a final E:T ratio of 10:1. For
normalization, maximal lysis of the target cells (=100%) was
determined by incubation of the target cells with a final
concentration of 1% Triton-X-100. Minimal lysis (=0%) refers to
target cells co-incubated with effector cells, but without any
construct or antibody.
[0381] After an incubation for 70 h at 37.degree. C., 5% CO2, LDH
release of apoptotic/necrotic target cells into the supernatant was
measured with the LDH detection kit (Roche Applied Science, #11 644
793 001), according to the manufacturer's instructions.
[0382] As depicted in FIG. 13, the bispecific construct induces
concentration-dependent LDH release from target cells, comparable
to the positive control with "T Cell Stim with ConA".
[0383] LDH Release Assay with Murine Crossfab (CD3)-Fab (MCSP)-Fab
(MCSP) Bispecific Construct
[0384] Purified murine Crossfab (CD3)-Fab (MCSP)-Fab (MCSP),
targeting murine CD3, as well as human MCSP, was analyzed for its
potential to induce T cell-mediated apoptosis in tumor target cells
upon crosslinkage of the construct via binding of both targeting
moieties to the respective antigens on cells.
[0385] Briefly, huMCSP-expressing B16/F10-huMCSP Fluc2 clone 48
tumor target cells are harvested with Cell Dissociation Buffer,
washed and resuspendend in RPMI1640 medium, including 1.times.NEAA,
10 mM Hepes, 50 .mu.M 2-b-ME and 1 mM sodium pyruvate.
[0386] 20 000 cells per well were plated in a round-bottom
96-well-plate and the respective antibody dilution was added to
obtain a final concentration of 50 nM. The bispecific construct and
the different IgG controls were adjusted to the same molarity.
[0387] Murine pan T effector cells, isolated from splenocytes
(C57BL/6 mice) were added to obtain a final E:T ratio of 10:1. To
assess the level of hyperactivation of murine T cells in the
absence of target cells, control wells with 50 nM bispecific
construct and T cells were plated accordingly.
[0388] For normalization, maximal lysis of the target cells (=100%)
was determined by incubation of the target cells with a final
concentration of 1% Triton-X-100. Minimal lysis (=0%) refers to
target cells co-incubated with effector cells, but without any
construct or antibody.
[0389] After an incubation for 70 h at 37.degree. C., 5% CO2, LDH
release of apoptotic/necrotic target cells into the supernatant was
measured with the LDH detection kit (Roche Applied Science, #11 644
793 001), according to the manufacturer's instructions.
[0390] As depicted in FIG. 14, the bispecific construct induces
strong LDH release from target cells. In the absence of target
cells, there is only a slight increase of LDH (reflecting
hyperactivation of T cells) compared to untreated murine T cells,
co-incubated with target cells. None of the control IgGs induces
LDH release of target cells.
Example 8
Cytokine Release Assay (CBA Analysis)
[0391] To assess the de novo secretion of different cytokines upon
T cell activation with CD3-bispecific constructs in the presence or
absence of target cells, human PBMCs were isolated from Buffy Coats
and 0.3 Mio cells per well were plated into a round-bottom 96-well
plate. Alternatively, 280 .mu.l whole blood from a healthy donor
were plated per well of a deep-well 96-well plate.
[0392] Tumor target cells (e.g. MDA-MB-435 cells for
CD3-MCSP-bispecific constructs) were added to obtain a final
E/T-ratio of 10:1. Bispecific constructs and controls were added as
indicated. After an incubation of up to 24 h at 37.degree. C., 5%
CO2, the assay plate was centrifuged for 5 min at 350 g and the
supernatant was transferred into a new deep-well 96-well-plate for
the subsequent analysis.
[0393] The CBA analysis was performed according to manufacturers'
instructions for FACS Cantoll, using the combination of the
following CBA Flex Sets: human granzyme B (BD 560304), human
IFN-.gamma. Flex Set (BD 558269), human TNF Flex Set (BD 558273),
human IL-10 Flex Set (BD 558274), human IL-6 Flex Set (BD 558276),
human IL-4 Flex Set (BD 558272).
[0394] Cytokine Release Assay with MCSP-CD3 Bispecific
Constructs
[0395] The following purified bispecific constructs targeting human
MCSP and human CD3 were analyzed for their ability to induce T
cell-mediated de novo secretion of cytokines in the presence (A, B)
versus absence (C, D) of tumor target cells: "Fab (MCSP)-Fab
(MCSP)-Crossfab (CD3) and the (scFv)2 (antiMCSP/anti huCD3e)
reference molecule.
[0396] Briefly, 280 .mu.l whole blood from a healthy donor were
plated per well of a deep-well 96-well plate. 30 000 Colo-38 tumor
target cells, expressing human MCSP, as well as the different
bispecific constructs and IgG controls were added were added at 1
nM final concentration. The cells were incubated for 24 h at
37.degree. C., 5% CO2 and then centrifuged for 5 min at
350.times.g. The supernatant was transferred into a new deep-well
96-well-plate for the subsequent analysis.
[0397] The CBA analysis was performed according to manufacturers'
instructions for FACS Cantoll, using the combination of the
following CBA Flex Sets: human granzyme B (BD 560304), human
IFN-.gamma. Flex Set (BD 558269), human TNF Flex Set (BD 558273),
human IL-10 Flex Set (BD 558274), human IL-6 Flex Set (BD 558276),
human IL-4 Flex Set (BD 558272).
[0398] FIG. 15 depicts different cytokine levels, that were
measured in the supernatant of whole blood after treatment with 1
nM of different CD3-MCSP bispecific constructs (Fab (MCSP)-Fab
(MCSP)-Crossfab (CD3) and the (scFv)2 (antiMCSP/anti huCD3e)) in
the presence (A, B) or absence (C,D) of Colo-38 tumor cells for 24
hours. 280 .mu.l whole blood were plated per well of a 96-well
plate and 30 000 Colo-38 cells added, as indicated.
[0399] The main cytokine that was secreted upon activation of T
cells in the presence of Colo-38 tumor cells, is IL-6, followed by
IFNgamma. In addition, also the levels of granzyme B increased
enormously upon activation of T cells in the presence of target
cells. In general, the (scFv)2 (antiMCSP/anti huCD3e) construct
elevated the levels of TNF and IFNgamma, as well as granzyme B in
the presence of target cells (A and B) a bit more compared to the
other bispecific construct.
[0400] There was no significant secretion of Th2 cytokines (IL-10
and IL-4) upon activation of T cells by the bispecific constructs
in the presence (or absence) of target cells.
[0401] In this assay, there was also a weak secretion of IFNgamma,
induced by the Fab (MCSP)-Fab (MCSP)-Crossfab (CD3) construct in
the absence of target cells.
[0402] Cytokine Release Assay with MCSP-murineCD3 Bispecific
Constructs
[0403] The purified huMCSP-muCD3-targeting bispecific molecule as
murine Crossfab (CD3)-Fab (MCSP)-Fab (MCSP) was tested by flow
cytometry for its potential to up-regulate the late activation
marker CD25 on CD8+ T cells in the presence of human
MCSP-expressing tumor cells.
[0404] Briefly, MCSP-positive B16/F10-huMCSP Fluc2 clone 48 tumor
cells were harvested with Cell Dissociation buffer, counted and
checked for viability. Cells were adjusted to 0.3.times.10.sup.6
(viable) cells per ml in RPMI1640 medium (including 1.times.NEAA,
10 mM Hepes, 50 .mu.m 2-b-ME, 1 mM sodium pyruvate), 100 .mu.l of
this cell suspension were pipetted per well into a round-bottom
96-well plate (as indicated). 50 .mu.l of the (diluted) bispecific
construct was added to the cell-containing wells to obtain a final
concentration of 50 nM. Human murine T effector cells were isolated
from splenocytes (C57BL/6 mice) and adjusted to 3.times.10.sup.6
(viable) cells per ml in AIM-V medium. 50 .mu.l of this cell
suspension was added per well of the assay plate (see above) to
obtain a final E:T ratio of 10:1. To analyze, if the bispecific
construct is able to activate T cells only in the presence of
target cells, expressing huMCSP, wells were included that contained
50 nM of the respective bispecific molecule, as well as T effector,
but no target cells.
[0405] After incubation for 70 hours at 37.degree. C., 5% CO2,
cells were centrifuged (5 min, 350.times.g) and washed twice with
150 .mu.l/well PBS, including 0.1% BSA.
[0406] Surface staining for CD8a (rat IgG2a; clone 53-6.7;
BioLegend #100712) and CD25 (rat IgG2b; clone 3C7; BD #553075) was
performed according to the suppliers' suggestions. Cells were
washed twice with 150 .mu.l/well PBS, including 0.1% BSA and fixed
for 15 min at 4.degree. C., using 100 .mu.l/well fixation buffer
(BD ##554655).
[0407] After centrifugation, the samples were resuspended in 200
.mu.l/well PBS, 0.1% BSA and analyzed using a FACS Cantoll machine
(Software FACS Diva).
[0408] FIG. 16 shows that the as murine Crossfab (CD3)-Fab
(MCSP)-Fab (MCSP) construct induces up-regulation of CD25 in the
presence of target cells only.
Example 9
Expression of Surface Activation Markers on Primary Human T Cells
Upon Engagement of Bispecific Constructs
[0409] To check for specific activation of T cells upon binding of
CD3 bispecific constructs exclusively in the presence of tumor
target cells, primary human PBMCs (isolated as described above)
were incubated with the indicated concentrations of bispecific
constructs for at least 24 h in the presence or absence of tumor
antigen-positive target cells.
[0410] Briefly, 0.3 million primary human PBMCs were plated per
well of a flat-bottom 96-well plate, containing the huMCSP-positive
target cells (MV-3 tumor cells) or medium. The final effector to
target cell (E:T) ratio was 10:1. The cells were incubated with the
indicated concentration of the CD3-MCSP bispecific constructs (Fab
(MCSP)-Crossfab (CD3); designated as "1+1 non-Fc", and the (scFv)2
(antiMCSP/anti huCD3e) reference molecule (designated as"(scFv)2")
for the indicated incubation times at 37.degree. C., 5% CO2. The
effector cells were stained for CD8, and the early activation
marker CD69 or the late activation marker CD25 and analyzed by FACS
Cantoll.
[0411] FIG. 20 shows the result of this experiment.
[0412] While there are shown and described presently preferred
embodiments of the invention, it is to be distinctly understood
that the invention is not limited thereto but may be otherwise
variously embodied and practiced within the scope of the following
claims.
Sequences
[0413] While there are shown and described presently preferred
embodiments of the invention, it is to be distinctly understood
that the invention is not limited thereto but may be otherwise
variously embodied and practiced within the scope of the following
claims. Legend: GA201=EGFR binder, 3F2=FAP binder, CH1A1A=CEA
binder.
[0414] Protein Sequences
TABLE-US-00004 SEQ ID. NO. Description Sequence 1 CDR1 VL MCSP
SASQGIRNYLN 2 CDR2 VL MCSP YTSSLHS 3 CDR3 VL MCSP QQYSKLPWT 4 CDR1
VH MCSP GYSITSGYYWN 5 CDR2 VH MCSP YITYDGSNNYNPSLKN 6 CDR3 VH MCSP
FDY 7 CDR1 VL CD3.sub.(V9) RASQDIRNYLN 8 CDR2 VL CD3.sub.(V9)
YTSRLES 9 CDR3 VL CD3.sub.(V9) QQGNTLPWT 10 CDR1 VH CD3.sub.(V9)
GYTMN 11 CDR2 VH CD3.sub.(V9) LINPYKGVSTYNQKFKD 12 CDR3 VH
SGYYGDSDWYFDV CD3.sub.(V9) 29 CDR1 VL GSSTGAVTSGYYPN CD3.sub.(H2C)
30 CDR2 VL GTKFLAP CD3.sub.(H2C) 31 CDR3 VL ALWYSNRWV CD3.sub.(H2C)
32 CDR1 VH GFTFNKYAMN CD3.sub.(H2C) 33 CDR2 VH RIRSKYNNYATYYADSVKD
CD3.sub.(H2C) 34 CDR3 VH HGNFGNSYISYWAY CD3.sub.(H2C) 13 VL MCSP
DIVLTQSPSSLSASLGDRVTISCSASQGIRNYLNWY
QQRPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYS
LTISNLEPEDIATYYCQQYSKLPWTFGGGTKLEIK 14 VH MCSP
EVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWN
WIRQFPGNKLEWMGYITYDGSNNYNPSLKNRISITR
DTSKNQFFLKLNSVTTEDTATYYCADFDYWGQGTTL TVSS 15 CL MCSP
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV
QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA
DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 16 CH1 MCSP
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCD
17 LIGHT CHAIN DIVLTQSPSSLSASLGDRVTISCSASQGIRNYLNWYQQR MCSP
PDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLE
PEDIATYYCQQYSKLPWTFGGGTKLEIKRTVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ
SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC 18
HEAVY CHAIN EVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIR MCSP
QFPGNKLEWMGYITYDGSNNYNPSLKNRISITRDTSKN
QFFLKLNSVTTEDTATYYCADFDYWGQGTTLTVSSAST
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCD 19
VL CD3.sub.(V9) QSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKA
PKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFA TYYCQQGNTLPWTFGQGTKVEIK
20 VH CD3.sub.(V9) EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWV
RQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKS
KNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDV WGQGTLVTVSS 21 CL CD3.sub.(V9)
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA
KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS
KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 22 CH CD3.sub.(V9)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSC
23 LIGHT CHAIN EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWV CD3.sub.(V9)
RQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKS (VHCL)
KNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDV
WGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST
YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 24 HEAVY CHAIN
SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV CD3.sub.(V9)(VLCH1)
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSC
35 VL CD3.sub.(H2C) QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPN
WVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGK
AALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTV L 36 VH CD3.sub.(H2C)
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW
VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI
SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSS 37 CL
CD3.sub.(H2C) VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD
YEKHKVYACEVTHQGLSSPVTKSFNRGEC 38 CH1 CD3.sub.(H2C)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSC
39 LIGHT CHAIN EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWV
CD3.sub.(H2C)(VHCL) RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRD
DSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYW
AYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVV
CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD
STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 40 HEAVY CHAIN
SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV CD3.sub.(H2C)(VLCH1)
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSC
25 FAB (MCSP)- EVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIR
XFAB(CD3.sub.(V9)) QFPGNKLEWMGYITYDGSNNYNPSLKNRISITRDTSKN
(VH-CH1-VL- QFFLKLNSVTTEDTATYYCADFDYWGQGTTLTVSSAST CH1)
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSC 26
FAB(MCSP)- EVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIR FAB(MCSP)-
QFPGNKLEWMGYITYDGSNNYNPSLKNRISITRDTSKN XFAB(CD3.sub.(V9))
QFFLKLNSVTTEDTATYYCADFDYWGQGTTLTVSSAST (VH-CH1-VH-
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW CH1-VL-CH1)
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQ
LQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFP
GNKLEWMGYITYDGSNNYNPSLKNRISITRDTSKNQFFL
KLNSVTTEDTATYYCADFDYWGQGTTLTVSSASTKGPS
VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKKVEPKSCDGGGGSGGGGSSSASTKGPS
VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSC 27
FAB(MCSP)- EVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIR
XFAB(CD3.sub.(V9))- QFPGNKLEWMGYITYDGSNNYNPSLKNRISITRDTSKN
FAB(MCSP) QFFLKLNSVTTEDTATYYCADFDYWGQGTTLTVSSAST (VH-CH1-VL-
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW CH1-VH-CH1)
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCGGGGSGGGGSEVQL
QESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPG
NKLEWMGYITYDGSNNYNPSLKNRISITRDTSKNQFFLK
LNSVTTEDTATYYCADFDYWGQGTTLTVSSASTKGPSV
FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSC 28 LINKER
1 GGGGSGGGGS 41 FAB(MCSP)- EVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIR
FAB(MCSP)- QFPGNKLEWMGYITYDGSNNYNPSLKNRISITRDTSKN
XFAB(CD3.sub.(H2C)) QFFLKLNSVTTEDTATYYCADFDYWGQGTTLTVSSAST
(VH-CH1-VH- KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW CH1-VL-CH1)
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQ
LQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFP
GNKLEWMGYITYDGSNNYNPSLKNRISITRDTSKNQFFL
KLNSVTTEDTATYYCADFDYWGQGTTLTVSSASTKGPS
VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKKVEPKSCDGGGGSGGGGSSSASTKGPS
VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSC 42
Murine LIGHT EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWV CHAIN
CD3.sub.(2C11) RQAPGRGLESVAYITSSSINIKYADAVKGRFTVSRDNAK (VHCL)
NLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMV
TVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE
AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Murine
DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQ XFAB(CD3.sub.(2C11))-
KPGKAPKLLIYYTNKLADGVPSRFSGSGSGRDSSFTISSL FAB(MCSP)-
ESEDIGSYYCQQYYNYPWTFGPGTKLEIKSSASTKGPSV FAB(MCSP)
FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT (VL-CH1-VH-
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN CH1-VH-CH1)
HKPSNTKVDKKVEPKSCGGGGSGGGGSEVQLQESGPG
LVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEW
MGYITYDGSNNYNPSLKNRISITRDTSKNQFFLKLNSVT
TEDTATYYCADFDYWGQGTTLTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS
NTKVDKKVEPKSCDGGGGSGGGGSEVQLQESGPGLVK PSQSLSLTCSVTGYSITSGY
YWNWIRQFPGNKLEWMGYITYDGSNNYNPSLKNRISIT
RDTSKNQFFLKLNSVTTEDTATYYCADFDYWGQGTTL
TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS
SLGTQTYICNVNHKPSNTKVDKKVEPKSCD 68 GA201 CDR1 VH DYKIH 69 GA201 CDR2
VH YFNPNSGYSTYAQKFQG 70 GA201 CDR3 VH LSPGGYYVMDA 71 GA201 CDR1 VL
RASQGINNYLN 72 GA201 CDR2 VL NTNNLQT 73 GA201 CDR3 VL LQHNSFPT 74
GA201 VH QVQLVQSGAEVKKPGSSVKVSCKASGFTFTDYKIHWV
RQAPGQGLEWMGYFNPNSGYSTYAQKFQGRVTITADK
STSTAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWG QGTTVTVSS 75 GA201 VL
DIQMTQSPSSLSASVGDRVTITCRASQGINNYLNWYQQ
KPGKAPKRLIYNTNNLQTGVPSRFSGSGSGTEFTLTISSL
QPEDFATYYCLQHNSFPTFGQGTKLEIK 76 3F2 CDR1 VH SYAMS 77 3F2 CDR2 VH
AISGSGGSTYYADSVK
78 3F2 CDR3 VH YCAKGWFG 79 3F2 CDR1 VL RASQSVTSSYL 80 3F2 CDR2 VL
NVGSRRA 81 3F2 CDR3 VL CQQGIMLPP 82 3F2 VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR
QAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSK
NTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTL VTVSS 83 3F2 VL
EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQ
KPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRL
EPEDFAVYYCQQGIMLPPTFGQGTKVEIK 84 CH1A1A CDR1 EFGMN VH 85 CH1A1A
CDR2 WINTKTGEATYVEEFKG VH 86 CH1A1A CDR3 WDFAYYVEAMDY VH 87 CH1A1A
CDR1 KASAAVGTYVA VL 88 CH1A1A CDR2 SASYRKR VL 89 CH1A1A CDR3
HQYYTYPLFT VL 90 CH1A1A VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWV
RQAPGQGLEWMGWINTKTGEATYVEFKGRVTFTTDT
STSTAYMELRSLRSDDTAVYYCARWDFAYYVEAMDY WGQGTTVTVSS 91 CH1A1A VL
DIQMTQSPSSLSASVGDRVTITCKASAAVGTYVAWYQQ
KPGKAPKLLIYSASYRKRGVPSRFSGSGSGTDFTLTISSL
QPEDFATYYCHQYYTYPLFTFGQGTKLEIK 92 Anti-/CD33 CDR1 GYTITDSNIH VH 93
Anti-CD33 CDR2 YIYPYNGGTDYNQ VH 94 Anti-CD33 CDR3 GNPWLAY VH 95
Anti-CD33 CDR1 RASESLDNYGIRFLT VL 96 Anti-CD33 CDR1 AASNQGS VL 97
Anti-CD33 CDR1 QQTKEVPWS VL 98 Anti-CD33 VH
EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVR
QAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPT
NTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVT VSS 99 Anti-CD33 VL
DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWF
QQKPGKAPKLLLMYAASNQGSGVPSRFSGSGSGTEFTLTI
SSLQPDDFATYYCQQTKEVPWSFGQGTKVEVK 100 Light Chain
DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNW antiCD33
FQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLT
ISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIKRTVAA
PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC
101 Light Chain EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYT CD3.sub.(V9)
MNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTI (VH-CL)
SVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSD
WYFDVWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGT
ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC 102 Fab(CD33)-
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHW XFab(CD3.sub.(V9))
VRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADE (VH-CH1-VL-
STNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTL CH1)
VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS
SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGG
GGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWY
QQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTIS
SLQPEDFATYYCQQGNTLPWTFGQGTKVEIKSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVL 145 Linker 2
EPKSCGGGGSGGGGS 146 Linker 3 EPKSCDGGGGSGGGGS 147 Linker 4
GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGG 148 Linker 5
SGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSG 149 (scFv)2
DIVLTQSPSSLSASLGDRVTISCSASQGIRNYLNWYQQR antiMCSP/anti
PDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLE huCD3e
PEDIATYYCQQYSKLPWTFGGGTKLEIKGGGGSGGGGS (MCSP(VL-VH)-
GGGGSEVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYY CD3.sub.(V9)(VH-VL))
WNWIRQFPGNKLEWMGYITYDGSNNYNPSLKNRISITR
DTSKNQFFLKLNSVTTEDTATYYCADFDYWGQGTTLT
VSSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFT
GYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKD
RFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYG
DSDWYFDVWGQGTLVTVSSVEGGSGGSGGSGGSGGV
DDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQ
QKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISS
LQPEDFATYYCQQGNTLPWTFGQGTKVEIKHHHHHH 151 Light Chain
DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWF antiCD33.sub.(Myelotarg)
QQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTI
SSLQPDDFATYYCQQTKEVPWSFGQGTKVEVKRTVAA
PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC
152 Light Chain DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQ CD3.sub.(V9)
KPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSL (VL-CH1)
QPEDFATYYCQQGNTLPWTFGQGTKVEIKSSASTKGPS
VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSC 153 Fab
EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVR (CD33.sub.(Myelotarg))-
QAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPT XFab(CD3.sub.(V9))
NTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVT (VH-CH1-VH-
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP CL)
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGG
GSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMN
WVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISV
DKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWY
FDVWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASV
VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC 157 CD3(CH2527)VL
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWV
QEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTI
TGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL 158 CD3(CH2527)VH
EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWV
RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRD
DSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWF AYWGQGTLVTVSA 159 CEA(CH1A1A
QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWV (98/99))VH
RQAPGQGLEWMGWINTKTGEATYVEEFKGRVTFTTDT
STSTAYMELRSLRSDDTAVYYCARWDFAYYVEAMDY WGQGTTVTVSS 160 CEA(CH1A1A
DIQMTQSPSSLSASVGDRVTITCKASAAVGTYVAWYQQ (98/99))VL
KPGKAPKLLIYSASYRKRGVPSRFSGSGSGTDFTLTISSL
QPEDFATYYCHQYYTYPLFTFGQGTKLEIK 161 MCSP(M4-3 ML2)
QVQLQESGPGLVKPSQTLSLTCTVSGGSITSGYYWNWI VH
RQHPGKGLEWIGYITYDGSNNYNPSLKSRVTISRDTSKN
QFSLKLSSVTAADTAVYYCADFDYWGQGTLVTVSS 162 MCSP(M4-3 ML2)
DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLNWYQQ VL
KPGKAPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSL
QPEDFATYYCQQYSKLPWTFGQGTKVEIK
[0415] DNA Sequences
TABLE-US-00005 SEQ ID. NO. Description Sequence 44 VL MCSP
GATATTGTGCTCACACAGTCTCCATCCTCCCTGTCTGCC
TCTCTGGGAGACAGAGTCACCATCAGTTGCAGTGCAAG
TCAGGGCATTAGAAATTATTTAAACTGGTATCAGCAGA
GACCAGATGGAACTGTTAAACTCCTGATCTATTACACAT
CAAGTTTACACTCAGGAGTCCCATCAAGGTTCAGTGGC
AGTGGGTCTGGGACAGATTATTCTCTCACCATCAGCAAC
CTGGAACCTGAAGATATTGCCACTTACTATTGTCAGCAG
TATAGTAAGCTTCCTTGGACGTTCGGTGGAGGCACCAA GCTGGAAATCAAA 45 VH MCSP
GAGGTGCAGCTGCAGGAATCTGGCCCTGGCCTGGTCAA
GCCAAGCCAGAGTCTGAGCCTGACCTGCAGCGTGACCG
GCTACAGCATTACCAGCGGCTACTACTGGAACTGGATT
CGGCAGTTCCCCGGCAATAAGCTGGAATGGATGGGCTA
CATCACCTACGACGGCAGCAACAACTACAACCCCAGCC
TGAAGAACCGGATCAGCATCACCCGGGACACCAGCAAG
AACCAGTTCTTCCTGAAGCTGAACAGCGTGACCACCGA
GGACACCGCCACATACTATTGCGCCGACTTCGACTACTG
GGGCCAGGGCACCACCCTGACCGTGTCCAGC 46 CL MCSP
CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCA
TCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTG
TGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGT
ACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACT
CCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAG
CACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAG
CAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTC
ACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTT CAACAGGGGAGAGTGTTAG 47 CH1
MCSP GCCAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCC
CAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTG
GGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGAC
AGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGC
ACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACT
CCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTG
GGCACCCAGACCTACATCTGCAACGTGAACCACAAGCC
CAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAG AGCTGTGAT 48 LIGHT CHAIN
ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGCCTCCTG MCSP
CTGCTCTGGTTCCCAGGTGCCAGGTGTGATATTGTGCTC
ACACAGTCTCCATCCTCCCTGTCTGCCTCTCTGGGAGAC
AGAGTCACCATCAGTTGCAGTGCAAGTCAGGGCATTAG
AAATTATTTAAACTGGTATCAGCAGAGACCAGATGGAA
CTGTTAAACTCCTGATCTATTACACATCAAGTTTACACT
CAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGG
ACAGATTATTCTCTCACCATCAGCAACCTGGAACCTGAA
GATATTGCCACTTACTATTGTCAGCAGTATAGTAAGCTT
CCTTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAA
ACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCC
ATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGT
GTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG
TACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAAC
TCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACA
GCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAA
GCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGT
CACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCT TCAACAGGGGAGAGTGTTAG 49
HEAVY ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACA CHAIN MCSP
GCTACCGGTGTGCATTCGGAGGTGCAGCTGCAGGAATC
TGGCCCTGGCCTGGTCAAGCCAAGCCAGAGTCTGAGCC
TGACCTGCAGCGTGACCGGCTACAGCATTACCAGCGGC
TACTACTGGAACTGGATTCGGCAGTTCCCCGGCAATAA
GCTGGAATGGATGGGCTACATCACCTACGACGGCAGCA
ACAACTACAACCCCAGCCTGAAGAACCGGATCAGCATC
ACCCGGGACACCAGCAAGAACCAGTTCTTCCTGAAGCT
GAACAGCGTGACCACCGAGGACACCGCCACATACTATT
GCGCCGACTTCGACTACTGGGGCCAGGGCACCACCCTG
ACCGTGTCCAGCGCCAGCACAAAGGGCCCTAGCGTGTT
CCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAA
CAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCG
AGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACA
AGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGAGCAG
CGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAG
CAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGA
ACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGT GGAGCCCAAGAGCTGTGAT 50 VL
CD3.sub.(V9) GACATCCAGATGACCCAGAGCCCCTCTAGCCTGAGCGC
CAGCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCA
GCCAGGACATCAGAAACTACCTGAACTGGTATCAGCAG
AAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACTACAC
CTCTAGACTGGAAAGCGGCGTGCCCAGCCGGTTTAGCG
GCAGCGGCTCCGGCACCGACTACACCCTGACCATCAGC
AGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCA
GCAGGGCAACACACTCCCCTGGACCTTCGGCCAGGGCA CCAAGGTGGAGATCAAGTCCAGC 51
VH CD3.sub.(V9) GAGGTGCAGCTGGTCGAGAGCGGAGGCGGCCTGGTGCA
GCCTGGCGGCAGCCTGAGACTGAGCTGCGCCGCCAGCG
GCTACAGCTTCACCGGCTACACCATGAACTGGGTCCGG
CAGGCACCTGGCAAGGGACTGGAATGGGTGGCCCTGAT
CAACCCCTACAAGGGCGTGAGCACCTACAACCAGAAGT
TCAAGGACCGGTTCACCATCAGCGTGGACAAGAGCAAG
AACACCGCCTATCTGCAGATGAACAGCCTGCGGGCCGA
GGACACCGCCGTGTACTACTGCGCCAGAAGCGGCTACT
ACGGCGACAGCGACTGGTACTTCGACGTGTGGGGCCAG GGCACCCTCGTGACCGTGTCTAGC 52
CL CD3.sub.(V9) GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATC
TGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTG
CCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC
AGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCC
CAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCA
CCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCA
GACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCAC
CCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCA ACAGGGGAGAGTGTTGA 53 CH
CD3.sub.(V9) ACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCA
GCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTCGGC
TGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTG
TCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACAC
CTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCT
GTCCAGCGTGGTCACCGTGCCCTCCAGCAGCCTGGGCA
CCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGC
AATACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCT GCTGA 54 LIGHT CHAIN
ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACA CD3.sub.(V9)
GCTACCGGTGTGCATTCCGAGGTGCAGCTGGTCGAGAG (VHCL)
CGGAGGCGGCCTGGTGCAGCCTGGCGGCAGCCTGAGAC
TGAGCTGCGCCGCCAGCGGCTACAGCTTCACCGGCTAC
ACCATGAACTGGGTCCGGCAGGCACCTGGCAAGGGACT
GGAATGGGTGGCCCTGATCAACCCCTACAAGGGCGTGA
GCACCTACAACCAGAAGTTCAAGGACCGGTTCACCATC
AGCGTGGACAAGAGCAAGAACACCGCCTATCTGCAGAT
GAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACT
GCGCCAGAAGCGGCTACTACGGCGACAGCGACTGGTAC
TTCGACGTGTGGGGCCAGGGCACCCTCGTGACCGTGTCT
AGCGCTAGCGTGGCTGCACCATCTGTCTTCATCTTCCCG
CCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTT
GTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTA
ACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGA
CAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCA
AAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA
GTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAG CTTCAACAGGGGAGAGTGTTGA 55
HEAVY GACATCCAGATGACCCAGAGCCCCTCTA CHAIN CD3.sub.(V9)
GCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACC (VLCH1)
TGTCGGGCCAGCCAGGACATCAGAAACTACCTGAACTG
GTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGA
TCTACTACACCTCTAGACTGGAAAGCGGCGTGCCCAGC
CGGTTTAGCGGCAGCGGCTCCGGCACCGACTACACCCT
GACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCT
ACTACTGCCAGCAGGGCAACACACTCCCCTGGACCTTC
GGCCAGGGCACCAAGGTGGAGATCAAGTCCAGCGCTA
GCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGC
AGCAAGAGCACCAGCGGCGGCACAGCCGCCCTCGGCTG
CCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTC
CTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCT
TCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTG
TCCAGCGTGGTCACCGTGCCCTCCAGCAGCCTGGGCAC
CCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCA
ATACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTG CTGA 56 VL CD3.sub.(H2C)
CAGACCGTGGTGACACAGGAACCCAGCCTGACCGTCTC
CCCTGGCGGCACCGTGACCCTGACCTGTGGAAGCAGCA
CAGGCGCCGTGACCAGCGGCTACTACCCCAACTGGGTG
CAGCAGAAGCCCGGCCAGGCCCCTAGAGGACTGATCGG
CGGCACCAAGTTTCTGGCCCCTGGCACCCCCGCCAGATT
CTCTGGCTCTCTGCTGGGCGGCAAGGCCGCCCTGACACT
GTCTGGCGTGCAGCCTGAGGACGAGGCCGAGTACTACT
GCGCCCTGTGGTACAGCAACAGATGGGTGTTCGGCGGA GGCACCAAGCTGACCGTGCTGAGCAGC
57 VH CD3.sub.(H2C) GAGGTGCAGCTGGTGGAAAGCGGCGGAGGACTGGTGC
AGCCTGGCGGAAGCCTGAAGCTGTCTTGCGCCGCCAGC
GGCTTCACCTTCAACAAATACGCCATGAACTGGGTGCG
CCAGGCCCCTGGCAAGGGACTGGAATGGGTGGCCCGGA
TCAGAAGCAAGTACAACAACTACGCCACCTACTACGCC
GACAGCGTGAAGGACCGGTTCACCATCAGCCGGGACGA
CAGCAAGAACACCGCCTACCTGCAGATGAACAACCTGA
AAACCGAGGACACCGCCGTGTACTACTGCGTGCGGCAC
GGCAACTTCGGCAACAGCTACATCAGCTACTGGGCCTA
CTGGGGACAGGGCACCCTGGTGACAGTGTCCAGC 58 CL CD3.sub.(H2C)
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGAT
GAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTG
CTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTG
GAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG
AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTA
CAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACT
ACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCAT
CAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAG GGGAGAGTGTTGA 59 CH1
CD3.sub.(H2C) ACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGC
AAGAGCACCAGCGGCGGCACAGCCGCCCTCGGCTGCCT
GGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTG
GAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCC
CCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCC
AGCGTGGTCACCGTGCCCTCCAGCAGCCTGGGCACCCA
GACCTACATCTGCAACGTGAACCACAAGCCCAGCAATA
CCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCTG A 60 LIGHT CHAIN
ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACA CD3.sub.(H2C)
GCTACCGGTGTGCATTCCGAGGTGCAGCTGGTGGAAAG (VHCL)
CGGCGGAGGACTGGTGCAGCCTGGCGGAAGCCTGAAGC
TGTCTTGCGCCGCCAGCGGCTTCACCTTCAACAAATACG
CCATGAACTGGGTGCGCCAGGCCCCTGGCAAGGGACTG
GAATGGGTGGCCCGGATCAGAAGCAAGTACAACAACTA
CGCCACCTACTACGCCGACAGCGTGAAGGACCGGTTCA
CCATCAGCCGGGACGACAGCAAGAACACCGCCTACCTG
CAGATGAACAACCTGAAAACCGAGGACACCGCCGTGTA
CTACTGCGTGCGGCACGGCAACTTCGGCAACAGCTACA
TCAGCTACTGGGCCTACTGGGGACAGGGCACCCTGGTG
ACAGTGTCCAGCGCTAGCGTGGCTGCACCATCTGTCTT
CATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAAC
TGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAG
AGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCC
AATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGAC
AGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACG
CCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTC
ACAAAGAGCTTCAACAGGGGAGAGTGTTGA 61 HEAVY
CAGACCGTGGTGACACAGGAACCCAGCCTGACCGTCTC CHAIN
CCCTGGCGGCACCGTGACCCTGACCTGTGGAAGCAGCA CD3.sub.(H2C)
CAGGCGCCGTGACCAGCGGCTACTACCCCAACTGGGTG (VLCH1)
CAGCAGAAGCCCGGCCAGGCCCCTAGAGGACTGATCGG
CGGCACCAAGTTTCTGGCCCCTGGCACCCCCGCCAGATT
CTCTGGCTCTCTGCTGGGCGGCAAGGCCGCCCTGACACT
GTCTGGCGTGCAGCCTGAGGACGAGGCCGAGTACTACT
GCGCCCTGTGGTACAGCAACAGATGGGTGTTCGGCGGA
GGCACCAAGCTGACCGTGCTGAGCAGCGCTAGCACCA
AGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAG
AGCACCAGCGGCGGCACAGCCGCCCTCGGCTGCCTGGT
CAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGA
ACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCC
GCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCCAG
CGTGGTCACCGTGCCCTCCAGCAGCCTGGGCACCCAGA
CCTACATCTGCAACGTGAACCACAAGCCCAGCAATACC
AAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCTGA 62 FAB(MCSP)-
ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAG XFAB(CD3).sub.(V9)
CAACAGCTACCGGTGTGCATTCGGAGGTGCAGCTGCAG (VH-CH1-VL-
GAAAGCGGCCCTGGCCTGGTGAAACCCAGCCAGAGCCT
CH1) GAGCCTGACCTGCAGCGTGACCGGCTACAGCATCACCA
GCGGCTACTACTGGAACTGGATCAGACAGTTCCCCGGC
AACAAGCTGGAATGGATGGGCTACATCACCTACGACGG
CAGCAACAACTACAACCCCAGCCTGAAGAACAGAATCA
GCATCACCCGGGACACCAGCAAGAACCAGTTCTTCCTG
AAGCTGAACAGCGTGACCACCGAGGACACCGCCACCTA
CTACTGCGCCGACTTCGACTACTGGGGCCAGGGCACCA
CCCTGACCGTGTCCTCCGCTAGCACCAAGGGACCCAGC
GTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGG
CGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACT
TCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCC
TGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAG
AGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACAGT
GCCCAGCAGCAGCCTGGGAACCCAGACCTACATCTGCA
ACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAA
GAAGGTGGAACCCAAGAGCTGCGATGGCGGAGGAGGC
TCCGGAGGCGGAGGCTCTGATATCCAGATGACCCAGAG
CCCCAGCTCTCTGAGCGCCAGCGTGGGCGACAGAGTGA
CCATCACCTGTCGGGCCAGCCAGGACATCAGAAACTAC
CTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAA
GCTGCTGATCTACTACACCAGCAGACTGGAAAGCGGCG
TGCCCTCCAGATTTTCCGGCAGCGGCTCCGGCACCGACT
ACACCCTGACCATCAGCAGCCTGCAGCCCGAGGATTTC
GCCACATATTACTGCCAGCAGGGCAATACCCTGCCCTG
GACCTTCGGACAGGGCACAAAAGTGGAAATCAAG 63 FAB(MCSP)-
ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACA FAB(MCSP)-
GCTACCGGTGTGCATTCGGAGGTGCAGCTGCAGGAATC XFAB(CD3).sub.(V9)
TGGCCCTGGCCTGGTCAAGCCAAGCCAGAGTCTGAGCC (VH-CH1-VH-
TGACCTGCAGCGTGACCGGCTACAGCATTACCAGCGGC CH1-VL-CH1)
TACTACTGGAACTGGATTCGGCAGTTCCCCGGCAATAA
GCTGGAATGGATGGGCTACATCACCTACGACGGCAGCA
ACAACTACAACCCCAGCCTGAAGAACCGGATCAGCATC
ACCCGGGACACCAGCAAGAACCAGTTCTTCCTGAAGCT
GAACAGCGTGACCACCGAGGACACCGCCACATACTATT
GCGCCGACTTCGACTACTGGGGCCAGGGCACCACCCTG
ACCGTGTCCAGCGCCAGCACAAAGGGCCCTAGCGTGTT
CCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAA
CAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCG
AGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACA
AGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGAGCAG
CGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAG
CAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGA
ACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGT
GGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGA
GGCGGTGGCTCCGAGGTGCAGCTGCAGGAATCTGGCCC
TGGCCTGGTCAAGCCAAGCCAGAGTCTGAGCCTGACCT
GCAGCGTGACCGGCTACAGCATTACCAGCGGCTACTAC
TGGAACTGGATTCGGCAGTTCCCCGGCAATAAGCTGGA
ATGGATGGGCTACATCACCTACGACGGCAGCAACAACT
ACAACCCCAGCCTGAAGAACCGGATCAGCATCACCCGG GACACCAGC
AAGAACCAGTTCTTCCTGAAGCTGAACAGCGTGACCAC
CGAGGACACCGCCACATACTATTGCGCCGACTTCGACT
ACTGGGGCCAGGGCACCACCCTGACCGTGTCCAGCGCC
AGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAG
CAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCT
GCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTG
TCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACAC
CTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCT
GAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCA
CCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGC
AACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCT
GTGATGGCGGAGGAGGGTCCGGCGGCGGTGGATCCGAC
ATCCAGATGACCCAGAGCCCCTCTAGCCTGAGCGCCAG
CGTGGGCGACAGAGTGACCATCACCTGTCGGGCCAGCC
AGGACATCAGAAACTACCTGAACTGGTATCAGCAGAAG
CCCGGCAAGGCCCCCAAGCTGCTGATCTACTACACCTCT
AGACTGGAAAGCGGCGTGCCCAGCCGGTTTAGCGGCAG
CGGCTCCGGCACCGACTACACCCTGACCATCAGCAGCC
TGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAG
GGCAACACACTCCCCTGGACCTTCGGCCAGGGCACCAA
GGTGGAGATCAAGTCCAGCGCTAGCACCAAGGGCCCCT
CCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGC
GGCGGCACAGCCGCCCTCGGCTGCCTGGTCAAGGACTA
CTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAG
CCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGC
AGAGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTCACC
GTGCCCTCCAGCAGCCTGGGCACCCAGACCTACATCTG
CAACGTGAACCACAAGCCCAGCAATACCAAGGTGGACA AGAAGGTGGAGCCCAAGAGCTGCTGA
64 FAB(MCSP)- ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACA XFAB
GCTACCGGTGTGCATTCGGAGGTGCAGCTGCAGGAAAG (CD3.sub.(V9))-
CGGCCCTGGCCTGGTGAAACCCAGCCAGAGCCTGAGCC FAB(MCSP)
TGACCTGCAGCGTGACCGGCTACAGCATCACCAGCGGC (VH-CH1-VL-
TACTACTGGAACTGGATCAGACAGTTCCCCGGCAACAA CH1-VH-
GCTGGAATGGATGGGCTACATCACCTACGACGGCAGCA CH1)
ACAACTACAACCCCAGCCTGAAGAACAGAATCAGCATC
ACCCGGGACACCAGCAAGAACCAGTTCTTCCTGAAGCT
GAACAGCGTGACCACCGAGGACACCGCCACCTACTACT
GCGCCGACTTCGACTACTGGGGCCAGGGCACCACCCTG
ACCGTGTCCTCCGCTAGCACCAAGGGACCCAGCGTGTT
CCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAA
CAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCG
AGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCA
GCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGC
GGCCTGTACTCCCTGAGCAGCGTGGTGACAGTGCCCAG
CAGCAGCCTGGGAACCCAGACCTACATCTGCAACGTGA
ACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGT
GGAACCCAAGAGCTGCGATGGCGGAGGAGGCTCCGGA
GGCGGAGGCTCTGATATCCAGATGACCCAGAGCCCCAG
CTCTCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCA
CCTGTCGGGCCAGCCAGGACATCAGAAACTACCTGAAC
TGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCT
GATCTACTACACCAGCAGACTGGAAAGCGGCGTGCCCT
CCAGATTTTCCGGCAGCGGCTCCGGCACCGACTACACC
CTGACCATCAGCAGCCTGCAGCCCGAGGATTTCGCCAC
ATATTACTGCCAGCAGGGCAATACCCTGCCCTGGACCTT
CGGACAGGGCACAAAAGTGGAAATCAAGAGCAGCGCT
TCCACCAAAGGCCCTTCCGTGTTTCCTCTGGCTCCTAGC
TCCAAGTCCACCTCTGGAGGCACCGCTGCTCTCGGATGC
CTCGTGAAGGATTATTTTCCTGAGCCTGTGACAGTGTCC
TGGAATAGCGGAGCACTGACCTCTGGAGTGCATACTTT
CCCCGCTGTGCTGCAGTCCTCTGGACTGTACAGCCTGAG
CAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCC
AGACCTACATCTGCAACGTGAACCACAAGCCCAGCAAC
ACCAAGGTGGACAAGAAGGTGGAACCCAAGTCTTGTGG
CGGAGGCGGATCCGGCGGAGGGGGATCTGAGGTGCAG
CTGCAGGAAAGCGGCCCTGGCCTGGTGAAACCCAGCCA
GAGCCTGAGCCTGACCTGCAGCGTGACCGGCTACAGCA
TCACCAGCGGCTACTACTGGAACTGGATCAGACAGTTC
CCCGGCAACAAGCTGGAATGGATGGGCTACATCACCTA
CGACGGCAGCAACAACTACAACCCCAGCCTGAAGAACA
GAATCAGCATCACCCGGGACACCAGCAAGAACCAGTTC
TTCCTGAAGCTGAACAGCGTGACCACCGAGGACACCGC
CACCTACTACTGCGCCGACTTCGACTACTGGGGCCAGG
GCACCACCCTGACCGTGTCCTCCGCCTCTACCAAGGGCC
CCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACA
TCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGA
CTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGG
CGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCT
GCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCAC
CGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTG
TAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATA AGAAAGTCGAGCCCAAGAGCTGCTGA
65 FAB(MCSP)- ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACA FAB(MCSP)-
GCTACCGGTGTGCATTCGGAGGTGCAGCTGCAGGAATC XFAB
TGGCCCTGGCCTGGTCAAGCCAAGCCAGAGTCTGAGCC (CD3.sub.(H2C))
TGACCTGCAGCGTGACCGGCTACAGCATTACCAGCGGC (VH-CH1-VH-
TACTACTGGAACTGGATTCGGCAGTTCCCCGGCAATAA CH1-VL-CH1)
GCTGGAATGGATGGGCTACATCACCTACGACGGCAGCA
ACAACTACAACCCCAGCCTGAAGAACCGGATCAGCATC
ACCCGGGACACCAGCAAGAACCAGTTCTTCCTGAAGCT
GAACAGCGTGACCACCGAGGACACCGCCACATACTATT
GCGCCGACTTCGACTACTGGGGCCAGGGCACCACCCTG
ACCGTGTCCAGCGCCAGCACAAAGGGCCCTAGCGTGTT
CCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAA
CAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCG
AGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACA
AGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGAGCAG
CGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAG
CAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGA
ACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGT
GGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGA
GGCGGTGGCTCCGAGGTGCAGCTGCAGGAATCTGGCCC
TGGCCTGGTCAAGCCAAGCCAGAGTCTGAGCCTGACCT
GCAGCGTGACCGGCTACAGCATTACCAGCGGCTACTAC
TGGAACTGGATTCGGCAGTTCCCCGGCAATAAGCTGGA
ATGGATGGGCTACATCACCTACGACGGCAGCAACAACT
ACAACCCCAGCCTGAAGAACCGGATCAGCATCACCCGG
GACACCAGCAAGAACCAGTTCTTCCTGAAGCTGAACAG
CGTGACCACCGAGGACACCGCCACATACTATTGCGCCG
ACTTCGACTACTGGGGCCAGGGCACCACCCTGACCGTG
TCCAGCGCCAGCACAAAGGGCCCTAGCGTGTTCCCTCT
GGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCC
GCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCC
GTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGG
CGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCT
GTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCA
GCCTGGGCACCCAGACCTACATCTGCAACGTGAACCAC
AAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGC
CCAAGAGCTGTGATGGCGGAGGAGGGTCCGGCGGCGGT
GGATCCCAGACCGTGGTGACACAGGAACCCAGCCTGAC
CGTCTCCCCTGGCGGCACCGTGACCCTGACCTGTGGAA
GCAGCACAGGCGCCGTGACCAGCGGCTACTACCCCAAC
TGGGTGCAGCAGAAGCCCGGCCAGGCCCCTAGAGGACT
GATCGGCGGCACCAAGTTTCTGGCCCCTGGCACCCCCG
CCAGATTCTCTGGCTCTCTGCTGGGCGGCAAGGCCGCCC
TGACACTGTCTGGCGTGCAGCCTGAGGACGAGGCCGAG
TACTACTGCGCCCTGTGGTACAGCAACAGATGGGTGTTC
GGCGGAGGCACCAAGCTGACCGTGCTGAGCAGCGCTA
GCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGC
AGCAAGAGCACCAGCGGCGGCACAGCCGCCCTCGGCTG
CCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTC
CTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCT
TCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTG
TCCAGCGTGGTCACCGTGCCCTCCAGCAGCCTGGGCAC
CCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCA
ATACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTG CTGA 66 Murine LIGHT
ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACA CHAIN
GCTACCGGTGTGCATTCCGAGGTGCAGCTGGTGGAAAG CD3.sub.(2C11)
CGGCGGAGGCCTGGTGCAGCCCGGCAAGAGCCTGAAGC (VHCL)
TGAGCTGCGAGGCCAGCGGCTTCACCTTCAGCGGCTAC
GGCATGCACTGGGTGAGACAGGCCCCTGGCAGAGGACT
GGAAAGCGTGGCCTACATCACCAGCAGCAGCATCAACA
TTAAGTACGCCGACGCCGTGAAGGGCCGGTTCACCGTG
TCCAGGGATAACGCCAAGAACCTGCTGTTCCTGCAGAT
GAACATCCTGAAGTCCGAGGACACCGCTATGTATTACT
GCGCCAGATTCGACTGGGACAAGAACTACTGGGGCCAG
GGCACCATGGTCACAGTGTCTAGCGCTAGCGTGGCTGC
ACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTT
GAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAA
CTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGG
ATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTC
ACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCA
GCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAA
CACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT
GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GTTGA 67 Murine
ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACA XFAB
GCTACCGGTGTGCATTCCGACATCCAGATGACCCAGAG (CD3.sub.(2C11))-
CCCCAGCAGCCTGCCTGCCAGCCTGGGCGACAGAGTGA FAB(MCSP)-
CCATCAACTGCCAGGCCAGCCAGGACATCAGCAACTAC FAB(MCSP)
CTGAACTGGTATCAGCAGAAGCCTGGCAAGGCCCCCAA (VL-CH1-VH-
GCTGCTGATCTACTACACCAACAAGCTGGCCGACGGCG CH1-VH-
TGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCAGAGAC CH1)
AGCAGCTTCACCATCTCCAGCCTGGAAAGCGAGGACAT
CGGCAGCTACTACTGCCAGCAGTACTACAACTACCCCT
GGACCTTCGGCCCTGGCACCAAGCTGGAAATCAAGAGC
AGCGCTTCCACCAAAGGCCCTTCCGTGTTTCCTCTGGCT
CCTAGCTCCAAGTCCACCTCTGGAGGCACCGCTGCTCTC
GGATGCCTCGTGAAGGATTATTTTCCTGAGCCTGTGACA
GTGTCCTGGAATAGCGGAGCACTGACCTCTGGAGTGCA
TACTTTCCCCGCTGTGCTGCAGTCCTCTGGACTGTACAG
CCTGAGCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGG
GCACCCAGACCTACATCTGCAACGTGAACCACAAGCCC
AGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGT
CTTGTGGCGGAGGCGGATCCGGCGGAGGAGGGTCCGAG
GTGCAGCTGCAGGAATCTGGCCCTGGCCTGGTCAAGCC
AAGCCAGAGTCTGAGCCTGACCTGCAGCGTGACCGGCT
ACAGCATTACCAGCGGCTACTACTGGAACTGGATTCGG
CAGTTCCCCGGCAATAAGCTGGAATGGATGGGCTACAT
CACCTACGACGGCAGCAACAACTACAACCCCAGCCTGA
AGAACCGGATCAGCATCACCCGGGACACCAGCAAGAAC
CAGTTCTTCCTGAAGCTGAACAGCGTGACCACCGAGGA
CACCGCCACATACTATTGCGCCGACTTCGACTACTGGGG
CCAGGGCACCACCCTGACCGTGTCCAGCGCCAGCACAA
AGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAG
AGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGT
GAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGA
ACAGCGGAGCCCTGACAAGCGGCGTGCACACCTTCCCT
GCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAG
CGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGA
CCTACATCTGCAACGTGAACCACAAGCCCAGCAACACC
AAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATG
GCGGAGGAGGGTCCGGAGGCGGTGGCTCCGAGGTGCA
GCTGCAGGAATCTGGCCCTGGCCTGGTCAAGCCAAGCC
AGAGTCTGAGCCTGACCTGCAGCGTGACCGGCTACAGC
ATTACCAGCGGCTACTACTGGAACTGGATTCGGCAGTTC
CCCGGCAATAAGCTGGAATGGATGGGCTACATCACCTA
CGACGGCAGCAACAACTACAACCCCAGCCTGAAGAACC
GGATCAGCATCACCCGGGACACCAGCAAGAACCAGTTC
TTCCTGAAGCTGAACAGCGTGACCACCGAGGACACCGC
CACATACTATTGCGCCGACTTCGACTACTGGGGCCAGG
GCACCACCCTGACCGTGTCCAGCGCCAGCACAAAGGGC
CCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCAC
AAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGG
ACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGC
GGAGCCCTGACAAGCGGCGTGCACACCTTCCCTGCCGT
GCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGG
TCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTAC
ATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGT
GGACAAGAAGGTGGAGCCCAAGAGCTGTGATTGA 104 Light Chain
ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACA antiCD33
GCTACCGGTGTGCATTCCGACATCCAGATGACCCAGAG
CCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGA
CCATCACCTGTCGGGCCAGCGAGAGCGTGGACAACTAC
GGCATCAGCTTCATGAACTGGTTCCAGCAGAAGCCCGG
CAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAATC
AGGGCAGCGGCGTGCCCAGCAGATTCAGCGGCTCTGGC
AGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCA
GCCCGACGACTTCGCCACCTACTACTGCCAGCAGAGCA
AAGAGGTGCCCTGGACCTTCGGCCAGGGCACCAAGGTG
GAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATC
TTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCC
TCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAG
GCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATC
GGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA
AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTG
AGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTG
CGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAA AGAGCTTCAACAGGGGAGAGTGTTAG
105 Light Chain ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACA
(CD3).sub.(V9) GCTACCGGTGTGCATTCCGAGGTGCAGCTGGTCGAGAG (VH-CL)
CGGAGGCGGCCTGGTGCAGCCTGGCGGCAGCCTGAGAC
TGAGCTGCGCCGCCAGCGGCTACAGCTTCACCGGCTAC
ACCATGAACTGGGTCCGGCAGGCACCTGGCAAGGGACT
GGAATGGGTGGCCCTGATCAACCCCTACAAGGGCGTGA
GCACCTACAACCAGAAGTTCAAGGACCGGTTCACCATC
AGCGTGGACAAGAGCAAGAACACCGCCTATCTGCAGAT
GAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACT
GCGCCAGAAGCGGCTACTACGGCGACAGCGACTGGTAC
TTCGACGTGTGGGGCCAGGGCACCCTCGTGACCGTGTCT
AGCGCTAGCGTGGCTGCACCATCTGTCTTCATCTTCCCG
CCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTT
GTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTA
ACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGA
CAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCA
AAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA
GTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAG CTTCAACAGGGGAGAGTGTTGA 106
Fab(CD33)- ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACA CrossFab
GCTACCGGTGTGCATTCCCAGGTGCAGCTGGTGCAGTCT (CD3.sub.(V9))
GGCGCCGAAGTGAAGAAACCCGGCAGCAGCGTGAAGG (VH-CH1-VL-
TGTCCTGCAAGGCCAGCGGCTACACCTTCACCGACTAC CH1)
AACATGCACTGGGTCCGCCAGGCCCCAGGCCAGGGACT
GGAATGGATCGGCTACATCTACCCCTACAACGGCGGCA
CCGGCTACAACCAGAAGTTCAAGAGCAAGGCCACCATC
ACCGCCGACGAGAGCACCAACACCGCCTACATGGAACT
GAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTACT
GCGCCAGAGGCAGACCCGCCATGGACTACTGGGGCCAG
GGCACCCTGGTGACAGTGTCCAGCGCCAGCACAAAGGG
CCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCA
CAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAG
GACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAG
CGGAGCCCTGACAAGCGGCGTGCACACCTTCCCTGCCG
TGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTG
GTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTA
CATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAG
TGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGG
AGGAGGGTCCGGAGGCGGTGGATCCGACATCCAGATGA
CCCAGAGCCCCTCTAGCCTGAGCGCCAGCGTGGGCGAC
AGAGTGACCATCACCTGTCGGGCCAGCCAGGACATCAG
AAACTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGG
CCCCCAAGCTGCTGATCTACTACACCTCTAGACTGGAAA
GCGGCGTGCCCAGCCGGTTTAGCGGCAGCGGCTCCGGC
ACCGACTACACCCTGACCATCAGCAGCCTGCAGCCCGA
GGACTTCGCCACCTACTACTGCCAGCAGGGCAACACAC
TCCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATC
AAGTCCAGCGCTAGCACCAAGGGCCCCTCCGTGTTCCC
CCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAG
CCGCCCTCGGCTGCCTGGTCAAGGACTACTTCCCCGAGC
CCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCC
GGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGG
CCTGTACAGCCTGTCCAGCGTGGTCACCGTGCCCTCCAG
CAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACC
ACAAGCCCAGCAATACCAAGGTGGACAAGAAGGTGGA GCCCAAGAGCTGCTGA 107 MCSP
CDR1 GGCTACTCCATCACCAGTGGTTATTACTGGAAC VH 108 MCSP CDR2
TACATAACCTACGACGGTAGCAATAACTACAACCCATC VH TCTCAAAAAT 109 MCSP CDR3
TTTGACTAC VH 110 MCSP CDR1 AGTGCAAGTCAGGGCATTAGAAATTATTTAAAC VL 111
MCSP CDR2 TACACATCAAGTTTACACTCA VL 112 MCSP
CAGCAGTATAGTAAGCTTCCTTGGACG CDR3VL 113 GA201 CDR1 GACTACAAGATACAC
VH 114 GA201 CDR2 TATTTCAACCCTAACAGCGGTTATAGTACCTACGCACAG VH
AAGTTCCAGGGC 115 GA201 CDR3 CTATCCCCAGGCGGTTACTATGTTATGGATGCC VH
116 GA201 CDR1 CGGGCAAGTCAGGGCATTAACAATTACTTAAAT VL 117 GA201 CDR2
AATACCAACAACTTGCAGACA VL 118 GA201 CDR3 TTGCAGCATAATAGTTTTCCCACG VL
119 GA201 VH CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAA
GCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCTGG
TTTCACATTCACTGACTACAAGATACACTGGGTGCGACA
GGCCCCTGGACAAGGGCTCGAGTGGATGGGATATTTCA
ACCCTAACAGCGGTTATAGTACCTACGCACAGAAGTTC
CAGGGCAGGGTCACCATTACCGCGGACAAATCCACGAG
CACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGG
ACACGGCCGTGTATTACTGTGCGAGACTATCCCCAGGC
GGTTACTATGTTATGGATGCCTGGGGCCAAGGGACCAC CGTGACCGTCTCCTCA 120 GA201
VL GATATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCA
TCTGTCGGAGACCGGGTCACCATCACCTGCCGGGCAAG
TCAGGGCATTAACAATTACTTAAATTGGTACCAGCAGA
AGCCAGGGAAAGCCCCTAAGCGCCTGATCTATAATACC
AACAACTTGCAGACAGGCGTCCCATCAAGGTTCAGCGG
CAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCA
GCCTGCAGCCTGAAGATTTTGCCACCTATTACTGCTTGC
AGCATAATAGTTTTCCCACGTTTGGCCAGGGCACCAAG CTCGAGATCAAG 121 3F2 CDR1 VH
AGCTACGCCATGAGC 122 3F2 CDR2 VH
GCCATCTCCGGCAGCGGAGGCAGCACCTACTACGCCGA CAGCGTGAAG 123 3F2 CDR3 VH
TATTGCGCCAAGGGATGGTTCGGC 124 3F2 CDR1 VL
AGAGCCAGCCAGAGCGTGACCAGCAGCTACCTG 125 3F2 CDR2 VL
AACGTGGGCAGCAGACGGGCC 126 3F2 CDR3 VL TGCCAGCAGGGCATCATGCTGCCCCCC
127 3F2 VH GAGGTGCAGCTGCTGGAATCTGGAGGCGGCCTGGTGCA
GCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCG
GCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGAC
AGGCTCCTGGCAAGGGACTGGAATGGGTGTCCGCCATC
TCCGGCAGCGGAGGCAGCACCTACTACGCCGACAGCGT
GAAGGGCCGGTTCACCATCAGCAGAGACAACAGCAAG
AACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGA
GGATACCGCCGTGTATTATTGCGCCAAGGGATGGTTCG
GCGGCTTCAACTACTGGGGCCAGGGAACCCTGGTGACA GTGTCCAGC 128 3F2 VL
GAGATCGTGCTGACCCAGTCTCCCGGCACCCTGAGCCT
GAGCCCTGGCGAGAGAGCCACCCTGAGCTGCAGAGCCA
GCCAGAGCGTGACCAGCAGCTACCTGGCCTGGTATCAG
CAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCAACGT
GGGCAGCAGACGGGCCACCGGCATCCCCGATAGATTCA
GCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATC
AGCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTG
CCAGCAGGGCATCATGCTGCCCCCCACCTTCGGCCAGG GCACCAAGGTGGAAATCAAG 129
CH1A1A CDR1 GAGTTCGGCATGAAC VH 130 CH1A1A CDR2
TGGATCAACACCAAGACCGGCGAGGCCACCTACGTGGA VH AGAGTTCAAGGGC 131 CH1A1A
CDR3 TGGGACTTCGCCTATTACGTGGAAGCCATGGACTAC VH 132 CH1A1A CDR1
AAGGCCAGTGCGGCTGTGGGTACGTATGTTGCG VL 133 CH1A1A CDR2
TCGGCATCCTACCGCAAAAGG VL 134 CH1A1A CDR3
CACCAATATTACACCTATCCTCTATTCACG VL 135 CH1A1A VH
CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAA
ACCTGGAGCTAGTGTGAAGGTGTCCTGCAAGGCCAGCG
GCTACACCTTCACCGAGTTCGGCATGAACTGGGTCCGA
CAGGCTCCAGGCCAGGGCCTCGAATGGATGGGCTGGAT
CAACACCAAGACCGGCGAGGCCACCTACGTGGAAGAGT
TCAAGGGCAGAGTGACCTTCACCACGGACACCAGCACC
AGCACCGCCTACATGGAACTGCGGAGCCTGAGAAGCGA
CGACACCGCCGTGTACTACTGCGCCAGATGGGACTTCG
CCTATTACGTGGAAGCCATGGACTACTGGGGCCAGGGC ACCACCGTGACCGTGTCTAGC 136
CH1A1A VL GATATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCA
TCTGTGGGAGACAGAGTCACCATCACTTGCAAGGCCAG
TGCGGCTGTGGGTACGTATGTTGCGTGGTATCAGCAGA
AACCAGGGAAAGCACCTAAGCTCCTGATCTATTCGGCA
TCCTACCGCAAAAGGGGAGTCCCATCAAGGTTCAGTGG
CAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCA
GTCTGCAACCTGAAGATTTCGCAACTTACTACTGTCACC
AATATTACACCTATCCTCTATTCACGTTTGGCCAGGGCA CCAAGCTCGAGATCAAG 137
Anti-CD33 GGCTACACCATCACCGACAGCAACATCCAC CDR1 VH 138 Anti-CD33
TACATCTACCCCTACAACGGCGGCACCGACTACAACCA CDR2 VH G 139 Anti-CD33
GGCAACCCCTGGCTGGCCTAT CDR3 VH 140 Anti-CD33
CGGGCCAGCGAGAGCCTGGACAACTACGGCATCCGGTT CDR1 VL TCTGACC 141
Anti-CD33 GCCGCCAGCAACCAGGGCAGC CDR2 VL 142 Anti-CD33
CAGCAGACCAAAGAGGTGCCCTGGTCC CDR3 VL 143 Anti-CD33 VH
GAAGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAA
ACCCGGCAGCAGCGTGAAGGTGTCCTGCAAGGCCAGCG
GCTACACCATCACCGACAGCAACATCCACTGGGTCCGA
CAGGCCCCTGGGCAGAGCCTGGAATGGATCGGCTACAT
CTACCCCTACAACGGCGGCACCGACTACAACCAGAAGT
TCAAGAACCGGGCCACCCTGACCGTGGACAACCCCACC
AACACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGA
GGACACCGCCTTCTACTACTGCGTGAACGGCAACCCCT
GGCTGGCCTATTGGGGCCAGGGAACCCTGGTCACCGTG TCTAGC 144 Anti-CD33 VL
GACATCCAGCTGACCCAGAGCCCCAGCACCCTGTCTGC
CAGCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCA
GCGAGAGCCTGGACAACTACGGCATCCGGTTTCTGACC
TGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCT
GATGTACGCCGCCAGCAACCAGGGCAGCGGCGTGCCAA
GCAGATTCAGCGGCAGCGGCTCCGGCACCGAGTTCACC
CTGACCATCAGCAGCCTGCAGCCCGACGACTTCGCCAC
CTACTACTGCCAGCAGACCAAAGAGGTGCCCTGGTCCT
TCGGCCAGGGCACCAAGGTGGAAGTGAAG 150 (scFv)2
ATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCCACA antiMCSP/anti
GCCACCGGTGTGCATTCCGACATCGTGCTGACCCAGAG huCD3
CCCCAGCAGCCTGAGCGCCAGCCTGGGCGACAGAGTGA (LC007(VL-
CCATCAGCTGCAGCGCCTCCCAGGGCATCAGAAACTAC VH)-V9(VH-
CTGAACTGGTATCAGCAGCGGCCCGACGGCACCGTGAA VL))
GCTGCTGATCTACTACACCAGCTCCCTGCACAGCGGCGT
GCCCAGCAGATTTTCAGGCAGCGGCAGCGGCACTGACT
ACAGCCTGACCATCTCCAACCTGGAACCCGAGGACATT
GCCACCTACTACTGCCAGCAGTACAGCAAGCTGCCCTG
GACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGGGCG
GAGGCGGATCCGGCGGAGGTGGAAGTGGCGGCGGAGG
CTCTGAGGTGCAATTGCAGGAAAGCGGCCCTGGCCTGG
TGAAACCCAGCCAGAGCCTGAGCCTGACCTGCAGCGTG
ACCGGCTACTCCATCACCAGCGGCTACTACTGGAACTG
GATCAGACAGTTCCCCGGAAACAAGCTGGAATGGATGG
GCTACATCACCTACGACGGCAGCAACAACTACAACCCC
AGCCTGAAGAACCGGATCAGCATCACCCGGGACACCAG
CAAGAACCAGTTCTTCCTGAAGCTGAACAGCGTGACCA
CCGAGGATACCGCCACCTATTACTGTGCCGACTTCGACT
ACTGGGGCCAGGGCACCACCCTGACCGTGTCATCCGGT
GGCGGCGGATCCGAAGTGCAGCTGGTGGAGTCTGGCGG
TGGACTGGTGCAGCCAGGCGGCTCCCTGAGACTGAGCT
GCGCCGCCTCCGGCTACAGCTTCACCGGCTACACCATG
AATTGGGTCCGCCAGGCCCCTGGAAAGGGACTGGAATG
GGTGGCCCTGATCAACCCCTACAAGGGCGTGAGCACCT
ACAACCAGAAGTTCAAGGACCGGTTCACCATCAGCGTG
GACAAGAGCAAGAACACAGCCTACCTGCAGATGAACTC
CCTGAGAGCCGAGGATACCGCCGTGTATTACTGTGCCC
GCAGCGGCTACTACGGCGACTCCGACTGGTACTTCGAC
GTGTGGGGGCAGGGAACCCTGGTCACCGTGTCCAGCGT
GGAAGGCGGCAGCGGAGGATCTGGCGGCTCTGGCGGA
AGCGGCGGAGTGGACGATATCCAGATGACACAGTCCCC
CAGCTCCCTGAGCGCCAGCGTGGGCGACAGAGTGACCA
TCACCTGTCGGGCCAGCCAGGACATCCGGAATTATCTC
AATTGGTATCAGCAGAAACCTGGCAAAGCTCCTAAACT
GCTGATCTACTACACCTCCCGGCTGGAAAGCGGCGTGC
CCAGCAGATTTTCCGGCAGCGGGAGCGGCACCGATTAC
ACACTGACCATCAGCAGCCTGCAGCCCGAGGACTTTGC
CACCTACTATTGCCAGCAGGGCAACACCCTGCCCTGGA
CCTTTGGGCAGGGCACAAAGGTGGAGATCAAGCACCAC CACCATCACCACTGA 154 Light
Chain ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACA
antiCD33.sub.(Myelotarg) GCTACCGGTGTGCATTCCGACATCCAGCTGACCCAGAG
CCCCTCCACACTCTCTGCCTCAGTGGGCGATAGGGTCAC
CATTACTTGCAGAGCTAGCGAGTCCCTGGACAACTACG
GAATCCGCTTCCTTACATGGTTTCAGCAGAAGCCTGGAA
AAGCACCAAAGCTGCTCATGTATGCCGCTTCTAATCAA
GGCAGTGGTGTGCCCAGCCGGTTCTCCGGGTCTGGCTCA
GGAACCGAATTTACTCTGACCATTAGCTCCTTGCAGCCT
GATGACTTCGCAACATACTATTGTCAGCAGACCAAGGA
GGTCCCATGGTCTTTTGGTCAAGGCACAAAAGTGGAGG
TTAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCC
CGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTG
TTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCA
AAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGT
AACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGG
ACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC
AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGA
AGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGA GCTTCAACAGGGGAGAGTGTTAG 155
Light Chain ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACA CD3.sub.(V9)
GCTACCGGTGTGCATTCCGATATTCAGATGACCCAGAG (VL-CH1)
CCCCAGCTCTCTGAGCGCCAGCGTGGGCGACAGAGTGA
CCATCACCTGTCGGGCCAGCCAGGACATCAGAAACTAC
CTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAA
GCTGCTGATCTACTACACCAGCAGACTGGAAAGCGGCG
TGCCCTCCAGATTTTCCGGCAGCGGCTCCGGCACCGACT
ACACCCTGACCATCAGCAGCCTGCAGCCCGAGGATTTC
GCCACATATTACTGCCAGCAGGGCAATACCCTGCCCTG
GACCTTCGGACAGGGCACAAAAGTGGAAATCAAGAGC
AGCGCTTCCACCAAAGGCCCTTCCGTGTTTCCTCTGGCT
CCTAGCTCCAAGTCCACCTCTGGAGGCACCGCTGCTCTC
GGATGCCTCGTGAAGGATTATTTTCCTGAGCCTGTGACA
GTGTCCTGGAATAGCGGAGCACTGACCTCTGGAGTGCA
TACTTTCCCCGCTGTGCTGCAGTCCTCTGGACTGTACAG
CCTGAGCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGG
GCACCCAGACCTACATCTGCAACGTGAACCACAAGCCC
AGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGT CTTGTTGA 156 Fab
ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACA (CD33.sub.(Myelotarg))-
GCTACCGGTGTGCATTCCGAGGTGCAGCTGGTGCAGTCT XFab(CD3.sub.(V9))
GGCGCCGAAGTGAAGAAACCCGGCAGCAGCGTGAAGG (VH-CH1-
TGTCCTGCAAGGCCAGCGGCTACACCATCACCGACAGC VH-CL)
AACATCCACTGGGTGCGCCAGGCCCCTGGCCAGTCTCT
GGAATGGATCGGCTACATCTACCCCTACAACGGCGGCA
CCGACTACAACCAGAAGTTCAAGAACCGGGCCACCCTG
ACCGTGGACAACCCCACCAATACCGCCTACATGGAACT
GAGCAGCCTGCGGAGCGAGGACACCGCCTTCTACTACT
GCGTGAACGGCAACCCCTGGCTGGCCTATTGGGGCCAG
GGAACACTCGTGACCGTGTCCAGCGCTAGCACCAAGGG
CCCTAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCAC
CTCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGG
ACTACTTTCCCGAGCCCGTGACAGTGTCCTGGAACTCTG
GCGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTG
CTGCAGTCTAGCGGCCTGTACAGCCTGAGCAGCGTCGT
GACTGTGCCCAGCAGCAGCCTGGGAACCCAGACCTACA
TCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTG
GACAAGAAGGTGGAACCCAAGAGCTGCGACGGCGGAG
GCGGATCCGGGGGAGGGGGATCTGAAGTGCAGCTGGTG
GAAAGCGGCGGAGGCCTGGTGCAGCCTGGGGGATCTCT
GAGACTGAGCTGTGCCGCCTCCGGCTACAGCTTCACCG
GCTACACAATGAATTGGGTGCGGCAGGCTCCCGGCAAG
GGCCTGGAATGGGTGGCCCTGATCAACCCTTACAAGGG
CGTGTCCACCTATAATCAGAAGTTTAAGGACCGCTTCAC
CATCAGCGTGGACAAGTCCAAGAACACCGCCTACCTGC
AGATGAACTCCCTGCGGGCCGAGGATACAGCCGTGTAC
TACTGTGCCAGAAGCGGCTACTACGGCGACAGCGACTG
GTACTTCGACGTGTGGGGACAGGGCACCCTGGTGACCG
TGTCTAGTGCCTCTGTGGCCGCTCCCAGCGTGTTCATCT
TCCCACCTAGCGACGAGCAGCTGAAGTCCGGCACCGCT
TCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAG
GCCAAGGTGCAGTGGAAAGTGGACAATGCCCTGCAGAG
CGGCAACAGCCAGGAAAGCGTGACCGAGCAGGACAGC
AAGGACTCCACCTACAGCCTGTCCAGCACCCTGACACT
GAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCT
GCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACC
AAGAGCTTCAACCGGGGCGAGTGCTGA
[0416] While there are shown and described presently preferred
embodiments of the invention, it is to be distinctly understood
that the invention is not limited thereto but may be otherwise
variously embodied and practiced within the scope of the following
claims.
Sequence CWU 1
1
162111PRTArtificial SequenceCDR1 VL MCSP 1Ser Ala Ser Gln Gly Ile
Arg Asn Tyr Leu Asn 1 5 10 27PRTArtificial SequenceCDR2 VL MCSP
2Tyr Thr Ser Ser Leu His Ser 1 5 39PRTArtificial SequenceCDR3 VL
MCSP 3Gln Gln Tyr Ser Lys Leu Pro Trp Thr 1 5 411PRTArtificial
SequenceCDR1 VH MCSP 4Gly Tyr Ser Ile Thr Ser Gly Tyr Tyr Trp Asn 1
5 10 516PRTArtificial SequenceCDR2 VH MCSP 5Tyr Ile Thr Tyr Asp Gly
Ser Asn Asn Tyr Asn Pro Ser Leu Lys Asn 1 5 10 15 63PRTArtificial
SequenceCDR3 VH MCSP 6Phe Asp Tyr 1 711PRTArtificial SequenceCDR1
VL CD3 7Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn 1 5 10
87PRTArtificial SequenceCDR2 VL CD3 8Tyr Thr Ser Arg Leu Glu Ser 1
5 99PRTArtificial SequenceCDR3 VL CD3 9Gln Gln Gly Asn Thr Leu Pro
Trp Thr 1 5 105PRTArtificial SequenceCDR1 VH CD3 10Gly Tyr Thr Met
Asn 1 5 1117PRTArtificial SequenceCDR2 VH CD3 11Leu Ile Asn Pro Tyr
Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp
1213PRTArtificial SequenceCDR3 VH CD3 12Ser Gly Tyr Tyr Gly Asp Ser
Asp Trp Tyr Phe Asp Val 1 5 10 13107PRTArtificial SequenceVL MCSP
13Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1
5 10 15 Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Arg Asn
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Arg Pro Asp Gly Thr Val Lys
Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu
Thr Ile Ser Asn Leu Glu Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr
Cys Gln Gln Tyr Ser Lys Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys 100 105 14112PRTArtificial SequenceVH MCSP
14Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1
5 10 15 Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser
Gly 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys
Leu Glu Trp 35 40 45 Met Gly Tyr Ile Thr Tyr Asp Gly Ser Asn Asn
Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile Ser Ile Thr Arg Asp
Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr
Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Asp Phe Asp Tyr
Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 100 105 110
15107PRTArtificial SequenceCL MCSP 15Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55
60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100
105 16104PRTArtificial SequenceCH1 MCSP 16Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50
55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp 100
17214PRTArtificial SequenceLIGHT CHAIN MCSP 17Asp Ile Val Leu Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val
Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Arg Asn Tyr 20 25 30 Leu
Asn Trp Tyr Gln Gln Arg Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40
45 Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu
Glu Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser
Lys Leu Pro Trp 85 90 95 Thr Phe Gly Gly 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 18216PRTArtificial
SequenceHEAVY CHAIN MCSP 18Glu Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ser Val
Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Tyr Trp Asn Trp Ile
Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile
Thr Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn
Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80
Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85
90 95 Ala Asp Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser
Ser 100 105 110 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 115 120 125 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 130 135 140 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 145 150 155 160 Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170 175 Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 180 185 190 Tyr Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 195 200 205
Lys Val Glu Pro Lys Ser Cys Asp 210 215 19107PRTArtificial
SequenceVL CD3 (V9) 19Asp 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 Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg
Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
20122PRTArtificial SequenceVH CD3 (V9) 20Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe 50
55 60 Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp
Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 21105PRTArtificial SequenceCL CD3 (V9) 21Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 1 5 10 15 Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 20 25
30 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
35 40 45 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr 50 55 60 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His 65 70 75 80 Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val 85 90 95 Thr Lys Ser Phe Asn Arg Gly Glu
Cys 100 105 22103PRTArtificial SequenceCH1 CD3 (V9) 22Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys 100
23229PRTArtificial SequenceLight chain CD3 (VHCL) (V9) 23Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20
25 30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn
Gln Lys Phe 50 55 60 Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser
Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Tyr Tyr Gly
Asp Ser Asp Trp Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro 115 120 125 Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 130 135 140 Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 145 150
155 160 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
Glu 165 170 175 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Leu Ser Ser 180 185 190 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys Val Tyr Ala 195 200 205 Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser Phe 210 215 220 Asn Arg Gly Glu Cys 225
24212PRTArtificial SequenceHeavy Chain CD3 (VLCH1) V9 24Asp 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 Gln Asp Ile Arg Asn Tyr 20 25
30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Ser Ser Ala Ser Thr 100 105 110 Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 115 120 125 Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 130 135 140 Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 145 150 155
160 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
165 170 175 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys 180 185 190 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu 195 200 205 Pro Lys Ser Cys 210 25438PRTArtificial
SequenceFAB (MCSP)-XFAB (CD3) 25Glu Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ser
Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Tyr Trp Asn Trp
Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr
Ile Thr Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys
Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70
75 80 Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr
Cys 85 90 95 Ala Asp Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr
Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170 175 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 180 185 190
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 195
200 205 Lys Val Glu Pro Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly
Gly 210 215 220 Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser 225 230 235 240 Val Gly Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Asp Ile Arg 245 250 255 Asn Tyr Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu 260 265 270 Leu Ile Tyr Tyr Thr Ser
Arg Leu Glu Ser Gly Val Pro Ser Arg Phe 275 280 285 Ser Gly Ser Gly
Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu 290 295 300 Gln Pro
Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Gly Asn Thr Leu 305 310 315 320 Pro Trp Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Ser Ser Ala 325 330 335 Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 340 345 350 Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 355 360
365 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
370 375 380 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu 385 390 395 400 Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr 405 410 415 Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys 420 425 430 Val Glu Pro Lys Ser Cys 435
26664PRTArtificial SequenceFAB (MCSP)-FAB (MCSP)-XFAB (CD3) 26Glu
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10
15 Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Gly
20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu
Glu Trp 35 40 45 Met Gly Tyr Ile Thr Tyr Asp Gly Ser Asn Asn Tyr
Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr
Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Thr
Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Asp Phe Asp Tyr Trp
Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 100 105 110 Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145
150 155 160 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 165 170 175 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 180 185 190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 195 200 205 Lys Val Glu Pro Lys Ser Cys Asp
Gly Gly Gly Gly Ser Gly Gly Gly 210 215 220 Gly Ser Glu Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro 225 230 235 240 Ser Gln Ser
Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr 245 250 255 Ser
Gly Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu 260 265
270 Glu Trp Met Gly Tyr Ile Thr Tyr Asp Gly Ser Asn Asn Tyr Asn Pro
275 280 285 Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys
Asn Gln 290 295 300 Phe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp
Thr Ala Thr Tyr 305 310 315 320 Tyr Cys Ala Asp Phe Asp Tyr Trp Gly
Gln Gly Thr Thr Leu Thr Val 325 330 335 Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser 340 345 350 Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 355 360 365 Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 370 375 380 Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 385 390
395 400 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr 405 410 415 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val 420 425 430 Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Gly
Gly Gly Gly Ser Gly 435 440 445 Gly Gly Gly Ser Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser 450 455 460 Ala Ser Val Gly Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Asp 465 470 475 480 Ile Arg Asn Tyr
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 485 490 495 Lys Leu
Leu Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser 500 505 510
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser 515
520 525 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly
Asn 530 535 540 Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Ser 545 550 555 560 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser 565 570 575 Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp 580 585 590 Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 595 600 605 Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 610 615 620 Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 625 630 635
640 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
645 650 655 Lys Lys Val Glu Pro Lys Ser Cys 660 27663PRTArtificial
SequenceFAB (MCSP)-XFAB (CD3)-FAB (MCSP) 27Glu Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu
Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Tyr
Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45
Met Gly Tyr Ile Thr Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50
55 60 Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe
Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr
Tyr Tyr Cys 85 90 95 Ala Asp Phe Asp Tyr Trp Gly Gln Gly Thr Thr
Leu Thr Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170 175
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 180
185 190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys 195 200 205 Lys Val Glu Pro Lys Ser Cys Asp Gly Gly Gly Gly Ser
Gly Gly Gly 210 215 220 Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser 225 230 235 240 Val Gly Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Asp Ile Arg 245 250 255 Asn Tyr Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 260 265 270 Leu Ile Tyr Tyr
Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe 275 280 285 Ser Gly
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu 290 295 300
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu 305
310 315 320 Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ser
Ser Ala 325 330 335 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser 340 345 350 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe 355 360 365 Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly 370 375 380 Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 385 390 395 400 Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 405 410 415 Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 420 425
430 Val Glu Pro Lys Ser Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
435 440 445 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Gln 450 455 460 Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser
Ile Thr Ser Gly 465 470 475 480 Tyr Tyr Trp Asn Trp Ile Arg Gln Phe
Pro Gly Asn Lys Leu Glu Trp 485 490 495 Met Gly Tyr Ile Thr Tyr Asp
Gly Ser Asn Asn Tyr Asn Pro Ser Leu 500 505 510 Lys Asn Arg Ile Ser
Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 515 520 525 Leu Lys Leu
Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 530 535 540 Ala
Asp Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 545 550
555 560 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
Lys 565 570 575 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 580 585 590 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser 595 600 605 Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser 610 615 620 Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr 625 630 635 640 Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 645 650 655 Lys Val
Glu Pro Lys Ser Cys 660 2810PRTArtificial SequenceLinker Sequence
28Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 2914PRTArtificial
SequenceCDR1 VL CD3 (H2C) 29Gly Ser Ser Thr Gly Ala Val Thr Ser Gly
Tyr Tyr Pro Asn 1 5 10 307PRTArtificial SequenceCDR2 VL CD3 (H2C)
30Gly Thr Lys Phe Leu Ala Pro 1 5 319PRTArtificial SequenceCDR3 VL
CD3 (H2C) 31Ala Leu Trp Tyr Ser Asn Arg Trp Val 1 5
3210PRTArtificial SequenceCDR1 VH CD3 (H2C) 32Gly Phe Thr Phe Asn
Lys Tyr Ala Met Asn 1 5 10 3319PRTArtificial SequenceCDR2 VH CD3
(H2C) 33Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
Ser 1 5 10 15 Val Lys Asp 3414PRTArtificial SequenceCDR3 VH CD3
(H2C) 34His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp Ala Tyr 1 5
10 35109PRTArtificial SequenceVL CD3 (H2C) 35Gln Thr Val Val Thr
Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr
Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly 20 25 30 Tyr
Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40
45 Leu Ile Gly Gly Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe
50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser
Gly Val 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu
Trp Tyr Ser Asn 85 90 95 Arg Trp Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu 100 105 36125PRTArtificial SequenceVH CD3 (H2C) 36Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr
20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr
Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg
Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu
Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly
Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp 100 105 110 Ala Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 37105PRTArtificial
SequenceCL CD3 (H2C) 37Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu 1 5 10 15 Lys Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro 20 25 30 Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly 35 40 45 Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 50 55 60 Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 65 70 75 80 Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 85 90
95 Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 38103PRTArtificial
SequenceCH1 CD3 (H2C) 38Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95 Lys Val Glu Pro Lys Ser Cys 100 39232PRTArtificial SequenceLIGHT
CHAIN CD3 (VHCL) 39Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Asn Lys Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys
Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp
Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr
Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95
Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp 100
105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Val 115 120 125 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu Lys 130 135 140 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro Arg 145 150 155 160 Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn 165 170 175 Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 180 185 190 Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 195 200 205 Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 210 215 220
Lys Ser Phe Asn Arg Gly Glu Cys 225 230 40214PRTArtificial
SequenceHEAVY CHAIN CD3 (VLCH1) 40Gln Thr Val Val Thr
Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr
Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly 20 25 30 Tyr
Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40
45 Leu Ile Gly Gly Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe
50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser
Gly Val 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu
Trp Tyr Ser Asn 85 90 95 Arg Trp Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Ser Ser Ala 100 105 110 Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser 115 120 125 Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 130 135 140 Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 145 150 155 160 Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 165 170
175 Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
180 185 190 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys 195 200 205 Val Glu Pro Lys Ser Cys 210 41666PRTArtificial
SequenceFAB(MCSP)-FAB(MCSP)-XFAB(CD3) 41Glu Val Gln Leu Gln Glu Ser
Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr
Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Tyr Trp
Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met
Gly Tyr Ile Thr Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55
60 Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80 Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Asp Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu
Thr Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170 175 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 180 185
190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
195 200 205 Lys Val Glu Pro Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly
Gly Gly 210 215 220 Gly Ser Glu Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro 225 230 235 240 Ser Gln Ser Leu Ser Leu Thr Cys Ser
Val Thr Gly Tyr Ser Ile Thr 245 250 255 Ser Gly Tyr Tyr Trp Asn Trp
Ile Arg Gln Phe Pro Gly Asn Lys Leu 260 265 270 Glu Trp Met Gly Tyr
Ile Thr Tyr Asp Gly Ser Asn Asn Tyr Asn Pro 275 280 285 Ser Leu Lys
Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln 290 295 300 Phe
Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr 305 310
315 320 Tyr Cys Ala Asp Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr
Val 325 330 335 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser 340 345 350 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys 355 360 365 Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu 370 375 380 Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu 385 390 395 400 Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 405 410 415 Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 420 425 430
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly 435
440 445 Gly Gly Gly Ser Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr
Val 450 455 460 Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser
Thr Gly Ala 465 470 475 480 Val Thr Ser Gly Tyr Tyr Pro Asn Trp Val
Gln Gln Lys Pro Gly Gln 485 490 495 Ala Pro Arg Gly Leu Ile Gly Gly
Thr Lys Phe Leu Ala Pro Gly Thr 500 505 510 Pro Ala Arg Phe Ser Gly
Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr 515 520 525 Leu Ser Gly Val
Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu 530 535 540 Trp Tyr
Ser Asn Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val 545 550 555
560 Leu Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
565 570 575 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val 580 585 590 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala 595 600 605 Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly 610 615 620 Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly 625 630 635 640 Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 645 650 655 Val Asp Lys
Lys Val Glu Pro Lys Ser Cys 660 665 42223PRTArtificial
SequenceLIGHT CHAIN CD3 42Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Lys Leu Ser Cys Glu Ala
Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Arg Gly Leu Glu Ser Val 35 40 45 Ala Tyr Ile Thr
Ser Ser Ser Ile Asn Ile Lys Tyr Ala Asp Ala Val 50 55 60 Lys Gly
Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Leu Leu Phe 65 70 75 80
Leu Gln Met Asn Ile Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85
90 95 Ala Arg Phe Asp Trp Asp Lys Asn Tyr Trp Gly Gln Gly Thr Met
Val 100 105 110 Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe
Ile Phe Pro 115 120 125 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu 130 135 140 Leu Asn Asn Phe Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp 145 150 155 160 Asn Ala Leu Gln Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 165 170 175 Ser Lys Asp Ser
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 180 185 190 Ala Asp
Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 195 200 205
Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
220 43664PRTMus musculus 43Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Pro Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Asn Cys Gln
Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Asn
Lys Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Arg Asp Ser Ser Phe Thr Ile Ser Ser Leu Glu Ser 65 70 75 80
Glu Asp Ile Gly Ser Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro Trp 85
90 95 Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Ser
Thr 100 105 110 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser 115 120 125 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu 130 135 140 Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His 145 150 155 160 Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 165 170 175 Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 180 185 190 Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 195 200 205
Pro Lys Ser Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val 210
215 220 Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Ser
Leu 225 230 235 240 Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr
Ser Gly Tyr Tyr 245 250 255 Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn
Lys Leu Glu Trp Met Gly 260 265 270 Tyr Ile Thr Tyr Asp Gly Ser Asn
Asn Tyr Asn Pro Ser Leu Lys Asn 275 280 285 Arg Ile Ser Ile Thr Arg
Asp Thr Ser Lys Asn Gln Phe Phe Leu Lys 290 295 300 Leu Asn Ser Val
Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Asp 305 310 315 320 Phe
Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser 325 330
335 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
340 345 350 Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro 355 360 365 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val 370 375 380 His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser 385 390 395 400 Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile 405 410 415 Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 420 425 430 Glu Pro Lys
Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 435 440 445 Glu
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 450 455
460 Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Gly
465 470 475 480 Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys
Leu Glu Trp 485 490 495 Met Gly Tyr Ile Thr Tyr Asp Gly Ser Asn Asn
Tyr Asn Pro Ser Leu 500 505 510 Lys Asn Arg Ile Ser Ile Thr Arg Asp
Thr Ser Lys Asn Gln Phe Phe 515 520 525 Leu Lys Leu Asn Ser Val Thr
Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 530 535 540 Ala Asp Phe Asp Tyr
Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 545 550 555 560 Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 565 570 575
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 580
585 590 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 595 600 605 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 610 615 620 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr 625 630 635 640 Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 645 650 655 Lys Val Glu Pro Lys Ser
Cys Asp 660 44321DNAArtificial SequenceVL MCSP DNA 44gatattgtgc
tcacacagtc tccatcctcc ctgtctgcct ctctgggaga cagagtcacc 60atcagttgca
gtgcaagtca gggcattaga aattatttaa actggtatca gcagagacca
120gatggaactg ttaaactcct gatctattac acatcaagtt tacactcagg
agtcccatca 180aggttcagtg gcagtgggtc tgggacagat tattctctca
ccatcagcaa cctggaacct 240gaagatattg ccacttacta ttgtcagcag
tatagtaagc ttccttggac gttcggtgga 300ggcaccaagc tggaaatcaa a
32145336DNAArtificial SequenceVH MCSP DNA 45gaggtgcagc tgcaggaatc
tggccctggc ctggtcaagc caagccagag tctgagcctg 60acctgcagcg tgaccggcta
cagcattacc agcggctact actggaactg gattcggcag 120ttccccggca
ataagctgga atggatgggc tacatcacct acgacggcag caacaactac
180aaccccagcc tgaagaaccg gatcagcatc acccgggaca ccagcaagaa
ccagttcttc 240ctgaagctga acagcgtgac caccgaggac accgccacat
actattgcgc cgacttcgac 300tactggggcc agggcaccac cctgaccgtg tccagc
33646324DNAArtificial SequenceCL MCSP DNA 46cgtacggtgg ctgcaccatc
tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60ggaactgcct ctgttgtgtg
cctgctgaat aacttctatc ccagagaggc caaagtacag 120tggaaggtgg
ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac
180agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc
agactacgag 240aaacacaaag tctacgcctg cgaagtcacc catcagggcc
tgagctcgcc cgtcacaaag 300agcttcaaca ggggagagtg ttag
32447312DNAArtificial SequenceCH1 MCSP DNA 47gccagcacaa agggccctag
cgtgttccct ctggccccca gcagcaagag cacaagcggc 60ggaacagccg ccctgggctg
cctcgtgaag gactacttcc ccgagcccgt gacagtgtct 120tggaacagcg
gagccctgac aagcggcgtg cacaccttcc ctgccgtgct gcagagcagc
180ggcctgtact ccctgagcag cgtggtcacc gtgcctagca gcagcctggg
cacccagacc 240tacatctgca acgtgaacca caagcccagc aacaccaaag
tggacaagaa ggtggagccc 300aagagctgtg at 31248711DNAArtificial
SequenceLIGHT CHAIN MCSP DNA 48atggacatga gggtccccgc tcagctcctg
ggcctcctgc tgctctggtt cccaggtgcc 60aggtgtgata ttgtgctcac acagtctcca
tcctccctgt ctgcctctct gggagacaga 120gtcaccatca gttgcagtgc
aagtcagggc attagaaatt atttaaactg gtatcagcag 180agaccagatg
gaactgttaa actcctgatc tattacacat caagtttaca ctcaggagtc
240ccatcaaggt tcagtggcag tgggtctggg acagattatt ctctcaccat
cagcaacctg 300gaacctgaag atattgccac ttactattgt cagcagtata
gtaagcttcc ttggacgttc 360ggtggaggca ccaagctgga aatcaaacgt
acggtggctg caccatctgt cttcatcttc 420ccgccatctg atgagcagtt
gaaatctgga actgcctctg ttgtgtgcct gctgaataac 480ttctatccca
gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac
540tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct
cagcagcacc 600ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct
acgcctgcga agtcacccat 660cagggcctga gctcgcccgt cacaaagagc
ttcaacaggg gagagtgtta g 71149705DNAArtificial SequenceHEAVY CHAIN
MCSP DNA 49atgggatgga gctgtatcat cctcttcttg gtagcaacag ctaccggtgt
gcattcggag 60gtgcagctgc aggaatctgg ccctggcctg gtcaagccaa gccagagtct
gagcctgacc 120tgcagcgtga ccggctacag cattaccagc ggctactact
ggaactggat tcggcagttc 180cccggcaata agctggaatg gatgggctac
atcacctacg acggcagcaa caactacaac 240cccagcctga agaaccggat
cagcatcacc cgggacacca gcaagaacca gttcttcctg 300aagctgaaca
gcgtgaccac cgaggacacc gccacatact attgcgccga cttcgactac
360tggggccagg gcaccaccct gaccgtgtcc agcgccagca caaagggccc
tagcgtgttc 420cctctggccc ccagcagcaa gagcacaagc ggcggaacag
ccgccctggg ctgcctcgtg 480aaggactact tccccgagcc cgtgacagtg
tcttggaaca gcggagccct gacaagcggc 540gtgcacacct tccctgccgt
gctgcagagc agcggcctgt actccctgag cagcgtggtc 600accgtgccta
gcagcagcct gggcacccag acctacatct gcaacgtgaa ccacaagccc
660agcaacacca aagtggacaa gaaggtggag cccaagagct gtgat
70550327DNAArtificial SequenceVL CD3 DNA 50gacatccaga tgacccagag
cccctctagc ctgagcgcca gcgtgggcga cagagtgacc 60atcacctgtc gggccagcca
ggacatcaga aactacctga actggtatca gcagaagccc 120ggcaaggccc
ccaagctgct gatctactac acctctagac tggaaagcgg cgtgcccagc
180cggtttagcg gcagcggctc cggcaccgac tacaccctga ccatcagcag
cctgcagccc 240gaggacttcg ccacctacta
ctgccagcag ggcaacacac tcccctggac cttcggccag 300ggcaccaagg
tggagatcaa gtccagc 32751366DNAArtificial SequenceVH CD3 DNA
51gaggtgcagc tggtcgagag cggaggcggc ctggtgcagc ctggcggcag cctgagactg
60agctgcgccg ccagcggcta cagcttcacc ggctacacca tgaactgggt ccggcaggca
120cctggcaagg gactggaatg ggtggccctg atcaacccct acaagggcgt
gagcacctac 180aaccagaagt tcaaggaccg gttcaccatc agcgtggaca
agagcaagaa caccgcctat 240ctgcagatga acagcctgcg ggccgaggac
accgccgtgt actactgcgc cagaagcggc 300tactacggcg acagcgactg
gtacttcgac gtgtggggcc agggcaccct cgtgaccgtg 360tctagc
36652318DNAArtificial SequenceCL CD3 DNA 52gtggctgcac catctgtctt
catcttcccg ccatctgatg agcagttgaa atctggaact 60gcctctgttg tgtgcctgct
gaataacttc tatcccagag aggccaaagt acagtggaag 120gtggataacg
ccctccaatc gggtaactcc caggagagtg tcacagagca ggacagcaag
180gacagcacct acagcctcag cagcaccctg acgctgagca aagcagacta
cgagaaacac 240aaagtctacg cctgcgaagt cacccatcag ggcctgagct
cgcccgtcac aaagagcttc 300aacaggggag agtgttga 31853306DNAArtificial
SequenceCH1 CD3 DNA 53accaagggcc cctccgtgtt ccccctggcc cccagcagca
agagcaccag cggcggcaca 60gccgccctcg gctgcctggt caaggactac ttccccgagc
ccgtgaccgt gtcctggaac 120agcggagccc tgacctccgg cgtgcacacc
ttccccgccg tgctgcagag cagcggcctg 180tacagcctgt ccagcgtggt
caccgtgccc tccagcagcc tgggcaccca gacctacatc 240tgcaacgtga
accacaagcc cagcaatacc aaggtggaca agaaggtgga gcccaagagc 300tgctga
30654747DNAArtificial SequenceLIGHT CHAIN CD3 (VHCL) DNA
54atgggatgga gctgtatcat cctcttcttg gtagcaacag ctaccggtgt gcattccgag
60gtgcagctgg tcgagagcgg aggcggcctg gtgcagcctg gcggcagcct gagactgagc
120tgcgccgcca gcggctacag cttcaccggc tacaccatga actgggtccg
gcaggcacct 180ggcaagggac tggaatgggt ggccctgatc aacccctaca
agggcgtgag cacctacaac 240cagaagttca aggaccggtt caccatcagc
gtggacaaga gcaagaacac cgcctatctg 300cagatgaaca gcctgcgggc
cgaggacacc gccgtgtact actgcgccag aagcggctac 360tacggcgaca
gcgactggta cttcgacgtg tggggccagg gcaccctcgt gaccgtgtct
420agcgctagcg tggctgcacc atctgtcttc atcttcccgc catctgatga
gcagttgaaa 480tctggaactg cctctgttgt gtgcctgctg aataacttct
atcccagaga ggccaaagta 540cagtggaagg tggataacgc cctccaatcg
ggtaactccc aggagagtgt cacagagcag 600gacagcaagg acagcaccta
cagcctcagc agcaccctga cgctgagcaa agcagactac 660gagaaacaca
aagtctacgc ctgcgaagtc acccatcagg gcctgagctc gcccgtcaca
720aagagcttca acaggggaga gtgttga 74755639DNAArtificial
SequenceHEAVY CHAIN (VLCH1) CD3 DNA 55gacatccaga tgacccagag
cccctctagc ctgagcgcca gcgtgggcga cagagtgacc 60atcacctgtc gggccagcca
ggacatcaga aactacctga actggtatca gcagaagccc 120ggcaaggccc
ccaagctgct gatctactac acctctagac tggaaagcgg cgtgcccagc
180cggtttagcg gcagcggctc cggcaccgac tacaccctga ccatcagcag
cctgcagccc 240gaggacttcg ccacctacta ctgccagcag ggcaacacac
tcccctggac cttcggccag 300ggcaccaagg tggagatcaa gtccagcgct
agcaccaagg gcccctccgt gttccccctg 360gcccccagca gcaagagcac
cagcggcggc acagccgccc tcggctgcct ggtcaaggac 420tacttccccg
agcccgtgac cgtgtcctgg aacagcggag ccctgacctc cggcgtgcac
480accttccccg ccgtgctgca gagcagcggc ctgtacagcc tgtccagcgt
ggtcaccgtg 540ccctccagca gcctgggcac ccagacctac atctgcaacg
tgaaccacaa gcccagcaat 600accaaggtgg acaagaaggt ggagcccaag agctgctga
63956333DNAArtificial SequenceVL CD3 DNA 56cagaccgtgg tgacacagga
acccagcctg accgtctccc ctggcggcac cgtgaccctg 60acctgtggaa gcagcacagg
cgccgtgacc agcggctact accccaactg ggtgcagcag 120aagcccggcc
aggcccctag aggactgatc ggcggcacca agtttctggc ccctggcacc
180cccgccagat tctctggctc tctgctgggc ggcaaggccg ccctgacact
gtctggcgtg 240cagcctgagg acgaggccga gtactactgc gccctgtggt
acagcaacag atgggtgttc 300ggcggaggca ccaagctgac cgtgctgagc agc
33357375DNAArtificial SequenceVH CD3 DNA 57gaggtgcagc tggtggaaag
cggcggagga ctggtgcagc ctggcggaag cctgaagctg 60tcttgcgccg ccagcggctt
caccttcaac aaatacgcca tgaactgggt gcgccaggcc 120cctggcaagg
gactggaatg ggtggcccgg atcagaagca agtacaacaa ctacgccacc
180tactacgccg acagcgtgaa ggaccggttc accatcagcc gggacgacag
caagaacacc 240gcctacctgc agatgaacaa cctgaaaacc gaggacaccg
ccgtgtacta ctgcgtgcgg 300cacggcaact tcggcaacag ctacatcagc
tactgggcct actggggaca gggcaccctg 360gtgacagtgt ccagc
37558318PRTArtificial SequenceCL CD3 DNA 58Gly Thr Gly Gly Cys Thr
Gly Cys Ala Cys Cys Ala Thr Cys Thr Gly 1 5 10 15 Thr Cys Thr Thr
Cys Ala Thr Cys Thr Thr Cys Cys Cys Gly Cys Cys 20 25 30 Ala Thr
Cys Thr Gly Ala Thr Gly Ala Gly Cys Ala Gly Thr Thr Gly 35 40 45
Ala Ala Ala Thr Cys Thr Gly Gly Ala Ala Cys Thr Gly Cys Cys Thr 50
55 60 Cys Thr Gly Thr Thr Gly Thr Gly Thr Gly Cys Cys Thr Gly Cys
Thr 65 70 75 80 Gly Ala Ala Thr Ala Ala Cys Thr Thr Cys Thr Ala Thr
Cys Cys Cys 85 90 95 Ala Gly Ala Gly Ala Gly Gly Cys Cys Ala Ala
Ala Gly Thr Ala Cys 100 105 110 Ala Gly Thr Gly Gly Ala Ala Gly Gly
Thr Gly Gly Ala Thr Ala Ala 115 120 125 Cys Gly Cys Cys Cys Thr Cys
Cys Ala Ala Thr Cys Gly Gly Gly Thr 130 135 140 Ala Ala Cys Thr Cys
Cys Cys Ala Gly Gly Ala Gly Ala Gly Thr Gly 145 150 155 160 Thr Cys
Ala Cys Ala Gly Ala Gly Cys Ala Gly Gly Ala Cys Ala Gly 165 170 175
Cys Ala Ala Gly Gly Ala Cys Ala Gly Cys Ala Cys Cys Thr Ala Cys 180
185 190 Ala Gly Cys Cys Thr Cys Ala Gly Cys Ala Gly Cys Ala Cys Cys
Cys 195 200 205 Thr Gly Ala Cys Gly Cys Thr Gly Ala Gly Cys Ala Ala
Ala Gly Cys 210 215 220 Ala Gly Ala Cys Thr Ala Cys Gly Ala Gly Ala
Ala Ala Cys Ala Cys 225 230 235 240 Ala Ala Ala Gly Thr Cys Thr Ala
Cys Gly Cys Cys Thr Gly Cys Gly 245 250 255 Ala Ala Gly Thr Cys Ala
Cys Cys Cys Ala Thr Cys Ala Gly Gly Gly 260 265 270 Cys Cys Thr Gly
Ala Gly Cys Thr Cys Gly Cys Cys Cys Gly Thr Cys 275 280 285 Ala Cys
Ala Ala Ala Gly Ala Gly Cys Thr Thr Cys Ala Ala Cys Ala 290 295 300
Gly Gly Gly Gly Ala Gly Ala Gly Thr Gly Thr Thr Gly Ala 305 310 315
59306DNAArtificial SequenceCH1 CD3 DNA 59accaagggcc cctccgtgtt
ccccctggcc cccagcagca agagcaccag cggcggcaca 60gccgccctcg gctgcctggt
caaggactac ttccccgagc ccgtgaccgt gtcctggaac 120agcggagccc
tgacctccgg cgtgcacacc ttccccgccg tgctgcagag cagcggcctg
180tacagcctgt ccagcgtggt caccgtgccc tccagcagcc tgggcaccca
gacctacatc 240tgcaacgtga accacaagcc cagcaatacc aaggtggaca
agaaggtgga gcccaagagc 300tgctga 30660756DNAArtificial SequenceLIGHT
CHAIN CD3 (VHCL) DNA 60atgggatgga gctgtatcat cctcttcttg gtagcaacag
ctaccggtgt gcattccgag 60gtgcagctgg tggaaagcgg cggaggactg gtgcagcctg
gcggaagcct gaagctgtct 120tgcgccgcca gcggcttcac cttcaacaaa
tacgccatga actgggtgcg ccaggcccct 180ggcaagggac tggaatgggt
ggcccggatc agaagcaagt acaacaacta cgccacctac 240tacgccgaca
gcgtgaagga ccggttcacc atcagccggg acgacagcaa gaacaccgcc
300tacctgcaga tgaacaacct gaaaaccgag gacaccgccg tgtactactg
cgtgcggcac 360ggcaacttcg gcaacagcta catcagctac tgggcctact
ggggacaggg caccctggtg 420acagtgtcca gcgctagcgt ggctgcacca
tctgtcttca tcttcccgcc atctgatgag 480cagttgaaat ctggaactgc
ctctgttgtg tgcctgctga ataacttcta tcccagagag 540gccaaagtac
agtggaaggt ggataacgcc ctccaatcgg gtaactccca ggagagtgtc
600acagagcagg acagcaagga cagcacctac agcctcagca gcaccctgac
gctgagcaaa 660gcagactacg agaaacacaa agtctacgcc tgcgaagtca
cccatcaggg cctgagctcg 720cccgtcacaa agagcttcaa caggggagag tgttga
75661645DNAArtificial SequenceHEAVY CHAIN CD3 (VLCH1) DNA
61cagaccgtgg tgacacagga acccagcctg accgtctccc ctggcggcac cgtgaccctg
60acctgtggaa gcagcacagg cgccgtgacc agcggctact accccaactg ggtgcagcag
120aagcccggcc aggcccctag aggactgatc ggcggcacca agtttctggc
ccctggcacc 180cccgccagat tctctggctc tctgctgggc ggcaaggccg
ccctgacact gtctggcgtg 240cagcctgagg acgaggccga gtactactgc
gccctgtggt acagcaacag atgggtgttc 300ggcggaggca ccaagctgac
cgtgctgagc agcgctagca ccaagggccc ctccgtgttc 360cccctggccc
ccagcagcaa gagcaccagc ggcggcacag ccgccctcgg ctgcctggtc
420aaggactact tccccgagcc cgtgaccgtg tcctggaaca gcggagccct
gacctccggc 480gtgcacacct tccccgccgt gctgcagagc agcggcctgt
acagcctgtc cagcgtggtc 540accgtgccct ccagcagcct gggcacccag
acctacatct gcaacgtgaa ccacaagccc 600agcaatacca aggtggacaa
gaaggtggag cccaagagct gctga 645621056DNAArtificial
SequenceFAB(MCSP)-XFAB (CD3) DNA 62atgggatgga gctgtatcat cctcttcttg
gtagcaacag ctaccggtgt gcattcggag 60gtgcagctgc aggaaagcgg ccctggcctg
gtgaaaccca gccagagcct gagcctgacc 120tgcagcgtga ccggctacag
catcaccagc ggctactact ggaactggat cagacagttc 180cccggcaaca
agctggaatg gatgggctac atcacctacg acggcagcaa caactacaac
240cccagcctga agaacagaat cagcatcacc cgggacacca gcaagaacca
gttcttcctg 300aagctgaaca gcgtgaccac cgaggacacc gccacctact
actgcgccga cttcgactac 360tggggccagg gcaccaccct gaccgtgtcc
tccgctagca ccaagggacc cagcgtgttc 420cccctggcac ccagcagcaa
gagcacatct ggcggaacag ccgctctggg ctgtctggtg 480aaagactact
tccccgagcc cgtgaccgtg tcttggaact ctggcgccct gaccagcggc
540gtgcacacct ttccagccgt gctgcagagc agcggcctgt actccctgag
cagcgtggtg 600acagtgccca gcagcagcct gggaacccag acctacatct
gcaacgtgaa ccacaagccc 660agcaacacca aggtggacaa gaaggtggaa
cccaagagct gcgatggcgg aggaggctcc 720ggaggcggag gctctgatat
ccagatgacc cagagcccca gctctctgag cgccagcgtg 780ggcgacagag
tgaccatcac ctgtcgggcc agccaggaca tcagaaacta cctgaactgg
840tatcagcaga agcccggcaa ggcccccaag ctgctgatct actacaccag
cagactggaa 900agcggcgtgc cctccagatt ttccggcagc ggctccggca
ccgactacac cctgaccatc 960agcagcctgc agcccgagga tttcgccaca
tattactgcc agcagggcaa taccctgccc 1020tggaccttcg gacagggcac
aaaagtggaa atcaag 1056632052DNAArtificial
SequenceFAB(MCSP)-FAB(MCSP)-XFAB (CD3) DNA 63atgggatgga gctgtatcat
cctcttcttg gtagcaacag ctaccggtgt gcattcggag 60gtgcagctgc aggaatctgg
ccctggcctg gtcaagccaa gccagagtct gagcctgacc 120tgcagcgtga
ccggctacag cattaccagc ggctactact ggaactggat tcggcagttc
180cccggcaata agctggaatg gatgggctac atcacctacg acggcagcaa
caactacaac 240cccagcctga agaaccggat cagcatcacc cgggacacca
gcaagaacca gttcttcctg 300aagctgaaca gcgtgaccac cgaggacacc
gccacatact attgcgccga cttcgactac 360tggggccagg gcaccaccct
gaccgtgtcc agcgccagca caaagggccc tagcgtgttc 420cctctggccc
ccagcagcaa gagcacaagc ggcggaacag ccgccctggg ctgcctcgtg
480aaggactact tccccgagcc cgtgacagtg tcttggaaca gcggagccct
gacaagcggc 540gtgcacacct tccctgccgt gctgcagagc agcggcctgt
actccctgag cagcgtggtc 600accgtgccta gcagcagcct gggcacccag
acctacatct gcaacgtgaa ccacaagccc 660agcaacacca aagtggacaa
gaaggtggag cccaagagct gtgatggcgg aggagggtcc 720ggaggcggtg
gctccgaggt gcagctgcag gaatctggcc ctggcctggt caagccaagc
780cagagtctga gcctgacctg cagcgtgacc ggctacagca ttaccagcgg
ctactactgg 840aactggattc ggcagttccc cggcaataag ctggaatgga
tgggctacat cacctacgac 900ggcagcaaca actacaaccc cagcctgaag
aaccggatca gcatcacccg ggacaccagc 960aagaaccagt tcttcctgaa
gctgaacagc gtgaccaccg aggacaccgc cacatactat 1020tgcgccgact
tcgactactg gggccagggc accaccctga ccgtgtccag cgccagcaca
1080aagggcccta gcgtgttccc tctggccccc agcagcaaga gcacaagcgg
cggaacagcc 1140gccctgggct gcctcgtgaa ggactacttc cccgagcccg
tgacagtgtc ttggaacagc 1200ggagccctga caagcggcgt gcacaccttc
cctgccgtgc tgcagagcag cggcctgtac 1260tccctgagca gcgtggtcac
cgtgcctagc agcagcctgg gcacccagac ctacatctgc 1320aacgtgaacc
acaagcccag caacaccaaa gtggacaaga aggtggagcc caagagctgt
1380gatggcggag gagggtccgg cggcggtgga tccgacatcc agatgaccca
gagcccctct 1440agcctgagcg ccagcgtggg cgacagagtg accatcacct
gtcgggccag ccaggacatc 1500agaaactacc tgaactggta tcagcagaag
cccggcaagg cccccaagct gctgatctac 1560tacacctcta gactggaaag
cggcgtgccc agccggttta gcggcagcgg ctccggcacc 1620gactacaccc
tgaccatcag cagcctgcag cccgaggact tcgccaccta ctactgccag
1680cagggcaaca cactcccctg gaccttcggc cagggcacca aggtggagat
caagtccagc 1740gctagcacca agggcccctc cgtgttcccc ctggccccca
gcagcaagag caccagcggc 1800ggcacagccg ccctcggctg cctggtcaag
gactacttcc ccgagcccgt gaccgtgtcc 1860tggaacagcg gagccctgac
ctccggcgtg cacaccttcc ccgccgtgct gcagagcagc 1920ggcctgtaca
gcctgtccag cgtggtcacc gtgccctcca gcagcctggg cacccagacc
1980tacatctgca acgtgaacca caagcccagc aataccaagg tggacaagaa
ggtggagccc 2040aagagctgct ga 2052642049DNAArtificial
SequenceFAB(MCSP)-XFAB (CD3)-FAB(MCSP) 64atgggatgga gctgtatcat
cctcttcttg gtagcaacag ctaccggtgt gcattcggag 60gtgcagctgc aggaaagcgg
ccctggcctg gtgaaaccca gccagagcct gagcctgacc 120tgcagcgtga
ccggctacag catcaccagc ggctactact ggaactggat cagacagttc
180cccggcaaca agctggaatg gatgggctac atcacctacg acggcagcaa
caactacaac 240cccagcctga agaacagaat cagcatcacc cgggacacca
gcaagaacca gttcttcctg 300aagctgaaca gcgtgaccac cgaggacacc
gccacctact actgcgccga cttcgactac 360tggggccagg gcaccaccct
gaccgtgtcc tccgctagca ccaagggacc cagcgtgttc 420cccctggcac
ccagcagcaa gagcacatct ggcggaacag ccgctctggg ctgtctggtg
480aaagactact tccccgagcc cgtgaccgtg tcttggaact ctggcgccct
gaccagcggc 540gtgcacacct ttccagccgt gctgcagagc agcggcctgt
actccctgag cagcgtggtg 600acagtgccca gcagcagcct gggaacccag
acctacatct gcaacgtgaa ccacaagccc 660agcaacacca aggtggacaa
gaaggtggaa cccaagagct gcgatggcgg aggaggctcc 720ggaggcggag
gctctgatat ccagatgacc cagagcccca gctctctgag cgccagcgtg
780ggcgacagag tgaccatcac ctgtcgggcc agccaggaca tcagaaacta
cctgaactgg 840tatcagcaga agcccggcaa ggcccccaag ctgctgatct
actacaccag cagactggaa 900agcggcgtgc cctccagatt ttccggcagc
ggctccggca ccgactacac cctgaccatc 960agcagcctgc agcccgagga
tttcgccaca tattactgcc agcagggcaa taccctgccc 1020tggaccttcg
gacagggcac aaaagtggaa atcaagagca gcgcttccac caaaggccct
1080tccgtgtttc ctctggctcc tagctccaag tccacctctg gaggcaccgc
tgctctcgga 1140tgcctcgtga aggattattt tcctgagcct gtgacagtgt
cctggaatag cggagcactg 1200acctctggag tgcatacttt ccccgctgtg
ctgcagtcct ctggactgta cagcctgagc 1260agcgtggtga cagtgcccag
cagcagcctg ggcacccaga cctacatctg caacgtgaac 1320cacaagccca
gcaacaccaa ggtggacaag aaggtggaac ccaagtcttg tggcggaggc
1380ggatccggcg gagggggatc tgaggtgcag ctgcaggaaa gcggccctgg
cctggtgaaa 1440cccagccaga gcctgagcct gacctgcagc gtgaccggct
acagcatcac cagcggctac 1500tactggaact ggatcagaca gttccccggc
aacaagctgg aatggatggg ctacatcacc 1560tacgacggca gcaacaacta
caaccccagc ctgaagaaca gaatcagcat cacccgggac 1620accagcaaga
accagttctt cctgaagctg aacagcgtga ccaccgagga caccgccacc
1680tactactgcg ccgacttcga ctactggggc cagggcacca ccctgaccgt
gtcctccgcc 1740tctaccaagg gccccagcgt gttccccctg gcacccagca
gcaagagcac atctggcgga 1800acagccgctc tgggctgtct ggtgaaagac
tacttccccg agcccgtgac cgtgtcttgg 1860aactctggcg ccctgaccag
cggcgtgcac acctttccag ccgtgctgca gagcagcggc 1920ctgtactccc
tgtcctccgt ggtcaccgtg ccctctagct ccctgggaac acagacatat
1980atctgtaatg tcaatcacaa gccttccaac accaaagtcg ataagaaagt
cgagcccaag 2040agctgctga 2049652058DNAArtificial SequenceFAB
(MCSP)-FAB(MCSP)-XFAB (CD3) DNA 65atgggatgga gctgtatcat cctcttcttg
gtagcaacag ctaccggtgt gcattcggag 60gtgcagctgc aggaatctgg ccctggcctg
gtcaagccaa gccagagtct gagcctgacc 120tgcagcgtga ccggctacag
cattaccagc ggctactact ggaactggat tcggcagttc 180cccggcaata
agctggaatg gatgggctac atcacctacg acggcagcaa caactacaac
240cccagcctga agaaccggat cagcatcacc cgggacacca gcaagaacca
gttcttcctg 300aagctgaaca gcgtgaccac cgaggacacc gccacatact
attgcgccga cttcgactac 360tggggccagg gcaccaccct gaccgtgtcc
agcgccagca caaagggccc tagcgtgttc 420cctctggccc ccagcagcaa
gagcacaagc ggcggaacag ccgccctggg ctgcctcgtg 480aaggactact
tccccgagcc cgtgacagtg tcttggaaca gcggagccct gacaagcggc
540gtgcacacct tccctgccgt gctgcagagc agcggcctgt actccctgag
cagcgtggtc 600accgtgccta gcagcagcct gggcacccag acctacatct
gcaacgtgaa ccacaagccc 660agcaacacca aagtggacaa gaaggtggag
cccaagagct gtgatggcgg aggagggtcc 720ggaggcggtg gctccgaggt
gcagctgcag gaatctggcc ctggcctggt caagccaagc 780cagagtctga
gcctgacctg cagcgtgacc ggctacagca ttaccagcgg ctactactgg
840aactggattc ggcagttccc cggcaataag ctggaatgga tgggctacat
cacctacgac 900ggcagcaaca actacaaccc cagcctgaag aaccggatca
gcatcacccg ggacaccagc 960aagaaccagt tcttcctgaa gctgaacagc
gtgaccaccg aggacaccgc cacatactat 1020tgcgccgact tcgactactg
gggccagggc accaccctga ccgtgtccag cgccagcaca 1080aagggcccta
gcgtgttccc tctggccccc agcagcaaga gcacaagcgg cggaacagcc
1140gccctgggct gcctcgtgaa ggactacttc cccgagcccg tgacagtgtc
ttggaacagc 1200ggagccctga caagcggcgt gcacaccttc cctgccgtgc
tgcagagcag cggcctgtac 1260tccctgagca gcgtggtcac cgtgcctagc
agcagcctgg gcacccagac ctacatctgc 1320aacgtgaacc acaagcccag
caacaccaaa gtggacaaga aggtggagcc caagagctgt 1380gatggcggag
gagggtccgg cggcggtgga tcccagaccg tggtgacaca ggaacccagc
1440ctgaccgtct cccctggcgg caccgtgacc ctgacctgtg gaagcagcac
aggcgccgtg 1500accagcggct actaccccaa ctgggtgcag cagaagcccg
gccaggcccc tagaggactg 1560atcggcggca ccaagtttct ggcccctggc
acccccgcca gattctctgg ctctctgctg 1620ggcggcaagg ccgccctgac
actgtctggc gtgcagcctg aggacgaggc cgagtactac 1680tgcgccctgt
ggtacagcaa cagatgggtg ttcggcggag gcaccaagct gaccgtgctg
1740agcagcgcta gcaccaaggg cccctccgtg ttccccctgg cccccagcag
caagagcacc 1800agcggcggca cagccgccct cggctgcctg gtcaaggact
acttccccga gcccgtgacc 1860gtgtcctgga acagcggagc cctgacctcc
ggcgtgcaca ccttccccgc cgtgctgcag 1920agcagcggcc tgtacagcct
gtccagcgtg gtcaccgtgc cctccagcag cctgggcacc 1980cagacctaca
tctgcaacgt gaaccacaag cccagcaata ccaaggtgga caagaaggtg
2040gagcccaaga gctgctga 205866729DNAArtificial SequenceLIGHT CHAIN
CD3 (VHCL) DNA 66atgggatgga gctgtatcat cctcttcttg gtagcaacag
ctaccggtgt gcattccgag 60gtgcagctgg tggaaagcgg cggaggcctg gtgcagcccg
gcaagagcct gaagctgagc 120tgcgaggcca gcggcttcac cttcagcggc
tacggcatgc actgggtgag acaggcccct 180ggcagaggac tggaaagcgt
ggcctacatc accagcagca gcatcaacat taagtacgcc 240gacgccgtga
agggccggtt caccgtgtcc agggataacg ccaagaacct gctgttcctg
300cagatgaaca tcctgaagtc cgaggacacc gctatgtatt actgcgccag
attcgactgg 360gacaagaact actggggcca gggcaccatg gtcacagtgt
ctagcgctag cgtggctgca 420ccatctgtct tcatcttccc gccatctgat
gagcagttga aatctggaac tgcctctgtt 480gtgtgcctgc tgaataactt
ctatcccaga gaggccaaag tacagtggaa ggtggataac 540gccctccaat
cgggtaactc ccaggagagt gtcacagagc aggacagcaa ggacagcacc
600tacagcctca gcagcaccct gacgctgagc aaagcagact acgagaaaca
caaagtctac 660gcctgcgaag tcacccatca gggcctgagc tcgcccgtca
caaagagctt caacagggga 720gagtgttga 729672052DNAArtificial
SequenceXFAB (CD3)-FAB(MCSP)-FAB(MCSP) DNA 67atgggatgga gctgtatcat
cctcttcttg gtagcaacag ctaccggtgt gcattccgac 60atccagatga cccagagccc
cagcagcctg cctgccagcc tgggcgacag agtgaccatc 120aactgccagg
ccagccagga catcagcaac tacctgaact ggtatcagca gaagcctggc
180aaggccccca agctgctgat ctactacacc aacaagctgg ccgacggcgt
gcccagcaga 240ttcagcggca gcggctccgg cagagacagc agcttcacca
tctccagcct ggaaagcgag 300gacatcggca gctactactg ccagcagtac
tacaactacc cctggacctt cggccctggc 360accaagctgg aaatcaagag
cagcgcttcc accaaaggcc cttccgtgtt tcctctggct 420cctagctcca
agtccacctc tggaggcacc gctgctctcg gatgcctcgt gaaggattat
480tttcctgagc ctgtgacagt gtcctggaat agcggagcac tgacctctgg
agtgcatact 540ttccccgctg tgctgcagtc ctctggactg tacagcctga
gcagcgtggt gacagtgccc 600agcagcagcc tgggcaccca gacctacatc
tgcaacgtga accacaagcc cagcaacacc 660aaggtggaca agaaggtgga
acccaagtct tgtggcggag gcggatccgg cggaggaggg 720tccgaggtgc
agctgcagga atctggccct ggcctggtca agccaagcca gagtctgagc
780ctgacctgca gcgtgaccgg ctacagcatt accagcggct actactggaa
ctggattcgg 840cagttccccg gcaataagct ggaatggatg ggctacatca
cctacgacgg cagcaacaac 900tacaacccca gcctgaagaa ccggatcagc
atcacccggg acaccagcaa gaaccagttc 960ttcctgaagc tgaacagcgt
gaccaccgag gacaccgcca catactattg cgccgacttc 1020gactactggg
gccagggcac caccctgacc gtgtccagcg ccagcacaaa gggccctagc
1080gtgttccctc tggcccccag cagcaagagc acaagcggcg gaacagccgc
cctgggctgc 1140ctcgtgaagg actacttccc cgagcccgtg acagtgtctt
ggaacagcgg agccctgaca 1200agcggcgtgc acaccttccc tgccgtgctg
cagagcagcg gcctgtactc cctgagcagc 1260gtggtcaccg tgcctagcag
cagcctgggc acccagacct acatctgcaa cgtgaaccac 1320aagcccagca
acaccaaagt ggacaagaag gtggagccca agagctgtga tggcggagga
1380gggtccggag gcggtggctc cgaggtgcag ctgcaggaat ctggccctgg
cctggtcaag 1440ccaagccaga gtctgagcct gacctgcagc gtgaccggct
acagcattac cagcggctac 1500tactggaact ggattcggca gttccccggc
aataagctgg aatggatggg ctacatcacc 1560tacgacggca gcaacaacta
caaccccagc ctgaagaacc ggatcagcat cacccgggac 1620accagcaaga
accagttctt cctgaagctg aacagcgtga ccaccgagga caccgccaca
1680tactattgcg ccgacttcga ctactggggc cagggcacca ccctgaccgt
gtccagcgcc 1740agcacaaagg gccctagcgt gttccctctg gcccccagca
gcaagagcac aagcggcgga 1800acagccgccc tgggctgcct cgtgaaggac
tacttccccg agcccgtgac agtgtcttgg 1860aacagcggag ccctgacaag
cggcgtgcac accttccctg ccgtgctgca gagcagcggc 1920ctgtactccc
tgagcagcgt ggtcaccgtg cctagcagca gcctgggcac ccagacctac
1980atctgcaacg tgaaccacaa gcccagcaac accaaagtgg acaagaaggt
ggagcccaag 2040agctgtgatt ga 2052685PRTArtificial SequenceGA201
HCDR1 68Asp Tyr Lys Ile His 1 5 6917PRTArtificial SequenceGA201
HCDR2 69Tyr Phe Asn Pro Asn Ser Gly Tyr Ser Thr Tyr Ala Gln Lys Phe
Gln 1 5 10 15 Gly 7011PRTArtificial SequenceGA201 HCDR3 70Leu Ser
Pro Gly Gly Tyr Tyr Val Met Asp Ala 1 5 10 7111PRTArtificial
SequenceGA201 LCDR1 71Arg Ala Ser Gln Gly Ile Asn Asn Tyr Leu Asn 1
5 10 727PRTArtificial SequenceGA201 LCDR2 72Asn Thr Asn Asn Leu Gln
Thr 1 5 738PRTArtificial SequenceGA201 LCDR3 73Leu Gln His Asn Ser
Phe Pro Thr 1 5 74120PRTArtificial SequenceGA201 VH 74Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25
30 Lys Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45 Gly Tyr Phe Asn Pro Asn Ser Gly Tyr Ser Thr Tyr Ala Gln
Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr
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 Leu Ser Pro Gly Gly Tyr
Tyr Val Met Asp Ala Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val
Ser Ser 115 120 75106PRTArtificial SequenceGA201 VL 75Asp 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 Gln Gly Ile Asn Asn Tyr 20 25
30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile
35 40 45 Tyr Asn Thr Asn Asn Leu Gln Thr Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln
His Asn Ser Phe Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 105 765PRTArtificial Sequence3F2 HCDR1 76Ser Tyr Ala
Met Ser 1 5 7716PRTArtificial Sequence3F2 HCDR2 77Ala Ile Ser Gly
Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15
788PRTArtificial Sequence3F2 HCDR3 78Tyr Cys Ala Lys Gly Trp Phe
Gly 1 5 7911PRTArtificial Sequence3F2 LCDR1 79Arg Ala Ser Gln Ser
Val Thr Ser Ser Tyr Leu 1 5 10 807PRTArtificial Sequence3F2 LCDR2
80Asn Val Gly Ser Arg Arg Ala 1 5 819PRTArtificial Sequence3F2
LCDR3 81Cys Gln Gln Gly Ile Met Leu Pro Pro 1 5 82117PRTArtificial
Sequence3F2 VH 82Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser
Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp Gly Gln Gly Thr Leu 100
105 110 Val Thr Val Ser Ser 115 83108PRTArtificial Sequence3F2 VL
83Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1
5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ser
Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu 35 40 45 Ile Asn Val Gly Ser Arg Arg Ala Thr Gly Ile
Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr
Tyr Cys Gln Gln Gly Ile Met Leu Pro 85 90 95 Pro Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105 845PRTArtificial SequenceCH1A1A
HCDR1 84Glu Phe Gly Met Asn 1 5 8517PRTArtificial SequenceCH1A1A
HCDR2 85Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe
Lys 1 5 10 15 Gly 8612PRTArtificial SequenceCH1A1A HCDR3 86Trp Asp
Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr 1 5 10 8711PRTArtificial
SequenceCH1A1A LCDR1 87Lys Ala Ser Ala Ala Val Gly Thr Tyr Val Ala
1 5 10 887PRTArtificial SequenceCH1A1A LCDR2 88Ser Ala Ser Tyr Arg
Lys Arg 1 5 8910PRTArtificial SequenceCH1A1A LCDR3 89His Gln Tyr
Tyr Thr Tyr Pro Leu Phe Thr 1 5 10 90121PRTArtificial
SequenceCH1A1A VH 90Gln 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 Thr Glu Phe 20 25 30 Gly Met Asn Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Lys
Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe 50 55 60 Lys Gly Arg Val
Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu
Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly 100
105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
91108PRTArtificial SequenceCH1A1A VL 91Asp 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 Lys Ala Ser Ala Ala Val Gly Thr Tyr 20 25 30 Val Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Ser Ala Ser Tyr Arg Lys Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Thr Tyr
Pro Leu 85 90 95 Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 9210PRTArtificial SequenceAnti-CD33 HCDR1 92Gly Tyr Thr Ile
Thr Asp Ser Asn Ile His 1 5 10 9313PRTArtificial SequenceAnti-CD33
HCDR2 93Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln 1 5 10
947PRTArtificial SequenceAnti-CD33 HCDR3 94Gly Asn Pro Trp Leu Ala
Tyr 1 5 9515PRTArtificial SequenceAnti-CD33 LCDR1 95Arg Ala Ser Glu
Ser Leu Asp Asn Tyr Gly Ile Arg Phe Leu Thr 1 5 10 15
967PRTArtificial SequenceAnti-CD33 LCDR2 96Ala Ala Ser Asn Gln Gly
Ser 1 5 979PRTArtificial SequenceAnti-CD33 LCDR3 97Gln Gln Thr Lys
Glu Val Pro Trp Ser 1 5 98116PRTArtificial SequenceAnti-CD33 VH
98Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Ile Thr Asp
Ser 20 25 30 Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu
Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Asp
Tyr Asn Gln Lys Phe 50 55 60 Lys Asn Arg Ala Thr Leu Thr Val Asp
Asn Pro Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Phe Tyr Tyr Cys 85 90 95 Val Asn Gly Asn Pro
Trp Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser
Ser 115 99111PRTArtificial SequenceAnti-CD33 VL 99Asp Ile Gln Leu
Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Leu Asp Asn Tyr 20 25 30
Gly Ile Arg Phe Leu Thr Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro 35
40 45 Lys Leu Leu Met Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro
Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu
Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Thr Lys 85 90 95 Glu Val Pro Trp Ser Phe Gly Gln Gly
Thr Lys Val Glu Val Lys 100 105 110 100218PRTArtificial
SequenceLight Chain antiCD33 100Asp 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 Ser Val Asp Asn Tyr 20 25 30 Gly Ile Ser Phe Met
Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu
Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser 50 55 60 Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70
75 80 Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser
Lys 85 90 95 Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195
200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
101229PRTArtificial SequenceLight Chain V9 (VH-CL) 101Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25
30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln
Lys Phe 50 55 60 Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys
Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Tyr Tyr Gly Asp
Ser Asp Trp Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Val Ala Ala Pro 115 120 125 Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 130 135 140 Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 145 150 155
160 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
165 170 175 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser Ser 180 185 190 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr Ala 195 200 205 Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser Phe 210 215 220 Asn Arg Gly Glu Cys 225
102396PRTArtificial SequenceCD33 Fab-Crossfab (VH-CH1-VL-CH1)
102Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20
25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45 Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly Tyr Asn
Gln Lys Phe 50 55 60 Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser
Thr Asn 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 Gly Arg Pro 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 Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser 225 230 235 240 Leu Ser Ala Ser
Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 245 250 255 Gln Asp
Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys 260 265 270
Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val 275
280 285 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu
Thr 290 295 300 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln 305 310 315 320 Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile 325 330 335 Lys Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro 340 345 350 Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 355 360 365 Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 370 375 380 Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu 385 390 395 103105PRTHomo
sapiens 103Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro
Tyr Lys 1 5 10 15 Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys
Pro Gln Tyr Pro 20 25 30 Gly Ser Glu Ile Leu Trp Gln His Asn Asp
Lys Asn Ile Gly Gly Asp 35 40 45 Glu Asp Asp Lys Asn Ile Gly Ser
Asp Glu Asp His Leu Ser Leu Lys 50 55 60 Glu Phe Ser Glu Leu Glu
Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg 65 70 75 80 Gly Ser Lys Pro
Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg 85 90 95 Val Cys
Glu Asn Cys Met Glu Met Asp 100 105 104714DNAArtificial
SequenceLight Chain antiCD33 DNA 104atgggatgga gctgtatcat
cctcttcttg gtagcaacag ctaccggtgt gcattccgac 60atccagatga cccagagccc
cagcagcctg agcgccagcg tgggcgacag agtgaccatc 120acctgtcggg
ccagcgagag cgtggacaac tacggcatca gcttcatgaa ctggttccag
180cagaagcccg gcaaggcccc caagctgctg atctacgccg ccagcaatca
gggcagcggc 240gtgcccagca gattcagcgg ctctggcagc ggcaccgact
tcaccctgac catcagcagc 300ctgcagcccg acgacttcgc cacctactac
tgccagcaga gcaaagaggt gccctggacc 360ttcggccagg gcaccaaggt
ggaaatcaag cgtacggtgg ctgcaccatc tgtcttcatc 420ttcccgccat
ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat
480aacttctatc ccagagaggc caaagtacag tggaaggtgg ataacgccct
ccaatcgggt 540aactcccagg agagtgtcac agagcaggac agcaaggaca
gcacctacag cctcagcagc 600accctgacgc tgagcaaagc agactacgag
aaacacaaag tctacgcctg cgaagtcacc 660catcagggcc tgagctcgcc
cgtcacaaag agcttcaaca ggggagagtg ttag 714105747DNAArtificial
SequenceLight Chain V9 (VH-CL) DNA 105atgggatgga gctgtatcat
cctcttcttg gtagcaacag ctaccggtgt gcattccgag 60gtgcagctgg tcgagagcgg
aggcggcctg gtgcagcctg gcggcagcct gagactgagc 120tgcgccgcca
gcggctacag cttcaccggc tacaccatga actgggtccg gcaggcacct
180ggcaagggac tggaatgggt ggccctgatc aacccctaca agggcgtgag
cacctacaac 240cagaagttca aggaccggtt caccatcagc gtggacaaga
gcaagaacac cgcctatctg 300cagatgaaca gcctgcgggc cgaggacacc
gccgtgtact actgcgccag aagcggctac 360tacggcgaca gcgactggta
cttcgacgtg tggggccagg gcaccctcgt gaccgtgtct 420agcgctagcg
tggctgcacc atctgtcttc atcttcccgc catctgatga gcagttgaaa
480tctggaactg cctctgttgt gtgcctgctg aataacttct atcccagaga
ggccaaagta 540cagtggaagg tggataacgc cctccaatcg ggtaactccc
aggagagtgt cacagagcag 600gacagcaagg acagcaccta cagcctcagc
agcaccctga cgctgagcaa agcagactac 660gagaaacaca aagtctacgc
ctgcgaagtc acccatcagg gcctgagctc gcccgtcaca 720aagagcttca
acaggggaga gtgttga 7471061386DNAArtificial SequenceCD33
Fab-Crossfab (VH-CH1-VL-CH1) DNA 106atgggatgga gctgtatcat
cctcttcttg gtagcaacag ctaccggtgt gcattcccag 60gtgcagctgg tgcagtctgg
cgccgaagtg aagaaacccg gcagcagcgt gaaggtgtcc 120tgcaaggcca
gcggctacac cttcaccgac tacaacatgc actgggtccg ccaggcccca
180ggccagggac tggaatggat cggctacatc tacccctaca acggcggcac
cggctacaac 240cagaagttca agagcaaggc caccatcacc gccgacgaga
gcaccaacac cgcctacatg 300gaactgagca gcctgcggag cgaggacacc
gccgtgtact actgcgccag aggcagaccc 360gccatggact actggggcca
gggcaccctg gtgacagtgt ccagcgccag cacaaagggc 420cctagcgtgt
tccctctggc ccccagcagc aagagcacaa gcggcggaac agccgccctg
480ggctgcctcg tgaaggacta cttccccgag cccgtgacag tgtcttggaa
cagcggagcc 540ctgacaagcg gcgtgcacac cttccctgcc gtgctgcaga
gcagcggcct gtactccctg 600agcagcgtgg tcaccgtgcc tagcagcagc
ctgggcaccc agacctacat ctgcaacgtg 660aaccacaagc ccagcaacac
caaagtggac aagaaggtgg agcccaagag ctgtgatggc 720ggaggagggt
ccggaggcgg tggatccgac atccagatga cccagagccc ctctagcctg
780agcgccagcg tgggcgacag agtgaccatc acctgtcggg ccagccagga
catcagaaac 840tacctgaact ggtatcagca gaagcccggc aaggccccca
agctgctgat ctactacacc 900tctagactgg aaagcggcgt gcccagccgg
tttagcggca gcggctccgg caccgactac 960accctgacca tcagcagcct
gcagcccgag gacttcgcca cctactactg ccagcagggc 1020aacacactcc
cctggacctt cggccagggc accaaggtgg agatcaagtc cagcgctagc
1080accaagggcc cctccgtgtt ccccctggcc cccagcagca agagcaccag
cggcggcaca 1140gccgccctcg gctgcctggt caaggactac ttccccgagc
ccgtgaccgt gtcctggaac 1200agcggagccc tgacctccgg cgtgcacacc
ttccccgccg tgctgcagag cagcggcctg 1260tacagcctgt ccagcgtggt
caccgtgccc tccagcagcc tgggcaccca gacctacatc 1320tgcaacgtga
accacaagcc cagcaatacc aaggtggaca agaaggtgga gcccaagagc 1380tgctga
138610733DNAArtificial SequenceMCSP CDR1 VH DNA 107ggctactcca
tcaccagtgg ttattactgg aac 3310848DNAArtificial SequenceMCSP CDR2 VH
108tacataacct acgacggtag caataactac aacccatctc tcaaaaat
481099DNAArtificial SequenceMCSP CDR3 VH 109tttgactac
911033DNAArtificial SequenceMCSP CDR1 VL DNA 110agtgcaagtc
agggcattag aaattattta aac 3311121DNAArtificial SequenceMCSP CDR2 VL
111tacacatcaa gtttacactc a 2111227DNAArtificial SequenceMCSP CDR3
VL 112cagcagtata gtaagcttcc ttggacg 2711315DNAArtificial
SequenceGA201 CDR1 VH 113gactacaaga tacac 1511451DNAArtificial
SequenceGA201 CDR2 VH 114tatttcaacc ctaacagcgg ttatagtacc
tacgcacaga agttccaggg c 5111533DNAArtificial SequenceGA201 CDR3 VH
115ctatccccag gcggttacta tgttatggat gcc 3311633DNAArtificial
SequenceGA201 CDR1 VL 116cgggcaagtc agggcattaa caattactta aat
3311721DNAArtificial SequenceGA201 CDR2 VL 117aataccaaca acttgcagac
a 2111824DNAArtificial SequenceGA201 CDR3 VL 118ttgcagcata
atagttttcc cacg 24119360DNAArtificial SequenceGA201 VH
119caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc
ggtgaaggtc 60tcctgcaagg cctctggttt cacattcact gactacaaga tacactgggt
gcgacaggcc 120cctggacaag ggctcgagtg gatgggatat ttcaacccta
acagcggtta tagtacctac 180gcacagaagt tccagggcag ggtcaccatt
accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag
atctgaggac acggccgtgt attactgtgc gagactatcc 300ccaggcggtt
actatgttat ggatgcctgg ggccaaggga ccaccgtgac cgtctcctca
360120318DNAArtificial SequenceGA201 VL 120gatatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtcggaga ccgggtcacc 60atcacctgcc gggcaagtca
gggcattaac aattacttaa attggtacca gcagaagcca 120gggaaagccc
ctaagcgcct gatctataat accaacaact tgcagacagg cgtcccatca
180aggttcagcg gcagtggatc cgggacagaa ttcactctca ccatcagcag
cctgcagcct 240gaagattttg ccacctatta ctgcttgcag cataatagtt
ttcccacgtt tggccagggc 300accaagctcg agatcaag 31812115DNAArtificial
Sequence3F2 CDR1 VH 121agctacgcca tgagc 1512248DNAArtificial
Sequence3F2 CDR2 VH 122gccatctccg gcagcggagg cagcacctac tacgccgaca
gcgtgaag 4812324DNAArtificial Sequence3F2 CDR3 VH 123tattgcgcca
agggatggtt cggc 2412433DNAArtificial Sequence3F2 CDR1 VL
124agagccagcc agagcgtgac cagcagctac ctg 3312521DNAArtificial
Sequence3F2 CDR2 VL 125aacgtgggca gcagacgggc c 2112627DNAArtificial
Sequence3F2 CDR3 VL 126tgccagcagg gcatcatgct gcccccc
27127351DNAArtificial Sequence3F2 VH 127gaggtgcagc tgctggaatc
tggaggcggc ctggtgcagc ctggcggcag cctgagactg 60tcttgcgccg ccagcggctt
caccttcagc agctacgcca tgagctgggt ccgacaggct 120cctggcaagg
gactggaatg ggtgtccgcc atctccggca gcggaggcag cacctactac
180gccgacagcg tgaagggccg gttcaccatc agcagagaca acagcaagaa
caccctgtac 240ctgcagatga acagcctgcg ggccgaggat accgccgtgt
attattgcgc caagggatgg 300ttcggcggct tcaactactg gggccaggga
accctggtga cagtgtccag c 351128324DNAArtificial Sequence3F2 VL
128gagatcgtgc tgacccagtc tcccggcacc ctgagcctga gccctggcga
gagagccacc 60ctgagctgca gagccagcca gagcgtgacc agcagctacc tggcctggta
tcagcagaag 120cccggccagg cccccagact gctgatcaac gtgggcagca
gacgggccac cggcatcccc 180gatagattca gcggcagcgg ctccggcacc
gacttcaccc tgaccatcag ccggctggaa 240cccgaggact tcgccgtgta
ctactgccag cagggcatca tgctgccccc caccttcggc 300cagggcacca
aggtggaaat caag 32412915DNAArtificial SequenceCH1A1A CDR1 VH
129gagttcggca tgaac 1513051DNAArtificial SequenceCH1A1A CDR2 VH
130tggatcaaca ccaagaccgg cgaggccacc tacgtggaag agttcaaggg c
5113136DNAArtificial SequenceCH1A1A CDR3 VH 131tgggacttcg
cctattacgt ggaagccatg gactac 3613233DNAArtificial SequenceCH1A1A
CDR1 VL 132aaggccagtg cggctgtggg tacgtatgtt gcg
3313321DNAArtificial SequenceCH1A1A CDR2 VL 133tcggcatcct
accgcaaaag g 2113430DNAArtificial SequenceCH1A1A CDR3 VL
134caccaatatt acacctatcc tctattcacg 30135363DNAArtificial
SequenceCH1A1A VH 135caggtgcagc tggtgcagtc tggcgccgaa gtgaagaaac
ctggagctag tgtgaaggtg 60tcctgcaagg ccagcggcta caccttcacc gagttcggca
tgaactgggt ccgacaggct 120ccaggccagg gcctcgaatg gatgggctgg
atcaacacca agaccggcga ggccacctac 180gtggaagagt tcaagggcag
agtgaccttc accacggaca ccagcaccag caccgcctac 240atggaactgc
ggagcctgag aagcgacgac accgccgtgt actactgcgc cagatgggac
300ttcgcctatt acgtggaagc catggactac tggggccagg gcaccaccgt
gaccgtgtct 360agc 363136324DNAArtificial SequenceCH1A1A VL
136gatatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtgggaga
cagagtcacc 60atcacttgca aggccagtgc ggctgtgggt acgtatgttg cgtggtatca
gcagaaacca 120gggaaagcac ctaagctcct gatctattcg gcatcctacc
gcaaaagggg agtcccatca 180aggttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagatttcg caacttacta
ctgtcaccaa tattacacct atcctctatt cacgtttggc 300cagggcacca
agctcgagat caag 32413730DNAArtificial SequenceAnti-CD33 CDR1 VH
137ggctacacca tcaccgacag caacatccac 3013839DNAArtificial
SequenceAnti-CD33 CDR2 VH 138tacatctacc cctacaacgg cggcaccgac
tacaaccag 3913921DNAArtificial SequenceAnti-CD33 CDR3 VH
139ggcaacccct ggctggccta t 2114045DNAArtificial SequenceAnti-CD33
CDR1 VL 140cgggccagcg agagcctgga caactacggc atccggtttc tgacc
4514121DNAArtificial SequenceAnti-CD33 CDR2 VL 141gccgccagca
accagggcag c 2114227DNAArtificial SequenceAnti-CD33 CDR3 VL
142cagcagacca aagaggtgcc ctggtcc 27143348DNAArtificial
SequenceAnti-CD33 VH 143gaagtgcagc tggtgcagtc tggcgccgaa gtgaagaaac
ccggcagcag cgtgaaggtg 60tcctgcaagg ccagcggcta caccatcacc gacagcaaca
tccactgggt ccgacaggcc 120cctgggcaga gcctggaatg gatcggctac
atctacccct acaacggcgg caccgactac 180aaccagaagt tcaagaaccg
ggccaccctg accgtggaca accccaccaa caccgcctac 240atggaactga
gcagcctgcg gagcgaggac accgccttct actactgcgt gaacggcaac
300ccctggctgg cctattgggg ccagggaacc ctggtcaccg tgtctagc
348144333DNAArtificial SequenceAnti-CD33 VL 144gacatccagc
tgacccagag ccccagcacc ctgtctgcca gcgtgggcga cagagtgacc 60atcacctgtc
gggccagcga gagcctggac aactacggca tccggtttct gacctggttc
120cagcagaagc ccggcaaggc ccccaagctg ctgatgtacg ccgccagcaa
ccagggcagc 180ggcgtgccaa gcagattcag cggcagcggc tccggcaccg
agttcaccct gaccatcagc 240agcctgcagc ccgacgactt cgccacctac
tactgccagc agaccaaaga ggtgccctgg 300tccttcggcc agggcaccaa
ggtggaagtg aag 33314515PRTArtificial SequenceLinker 2 145Glu Pro
Lys Ser Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15
14616PRTArtificial SequenceLinker 3 146Glu Pro Lys Ser Cys Asp Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 14732PRTArtificial
SequenceLinker 4 147Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly 20 25 30 14834PRTArtificial SequenceLinker
5 148Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu
Gly 1 5 10 15 Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser
Gly Gly Gly 20 25 30 Ser Gly 149492PRTArtificial Sequence(scFV)2
CD3-MCSP 149Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly
Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Arg Pro Asp Gly
Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Ser Leu His Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro 65 70 75 80 Glu Asp Ile Ala
Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Trp 85 90 95 Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Glu 115
120 125 Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr
Cys 130 135 140 Ser Val Thr Gly Tyr Ser Ile Thr Ser Gly Tyr Tyr Trp
Asn Trp Ile 145 150 155 160 Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp
Met Gly Tyr Ile Thr Tyr 165 170 175 Asp Gly Ser Asn Asn Tyr Asn Pro
Ser Leu Lys Asn Arg Ile Ser Ile 180 185 190 Thr Arg Asp Thr Ser Lys
Asn Gln Phe Phe Leu Lys Leu Asn Ser Val 195 200 205 Thr Thr Glu Asp
Thr Ala Thr Tyr Tyr Cys Ala Asp Phe Asp Tyr Trp 210 215 220 Gly Gln
Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Glu 225 230 235
240 Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly Ser 245 250 255 Leu Arg Leu Ser Cys Ala Ala Ser
Gly Tyr Ser Phe Thr Gly Tyr Thr 260 265 270 Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 275 280 285 Leu Ile Asn Pro
Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys 290 295 300 Asp Arg
Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu 305 310 315
320 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
325 330 335 Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val
Trp Gly 340 345 350 Gln Gly Thr Leu Val Thr Val Ser Ser Val Glu Gly
Gly Ser Gly Gly 355 360 365 Ser Gly Gly Ser Gly Gly Ser Gly Gly Val
Asp Asp Ile Gln Met Thr 370 375 380 Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly Asp Arg Val Thr Ile 385 390 395 400 Thr Cys Arg Ala Ser
Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln 405 410 415 Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg 420 425 430 Leu
Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 435 440
445 Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
450 455 460 Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly
Gln Gly 465 470 475 480 Thr Lys Val Glu Ile Lys His His His His His
His 485 490 1501536DNAArtificial Sequence(scFv)2 MCSP-CD3 DNA
150atgggctggt cctgcatcat cctgtttctg gtggccacag ccaccggtgt
gcattccgac 60atcgtgctga cccagagccc cagcagcctg agcgccagcc tgggcgacag
agtgaccatc 120agctgcagcg cctcccaggg catcagaaac tacctgaact
ggtatcagca gcggcccgac 180ggcaccgtga agctgctgat ctactacacc
agctccctgc acagcggcgt gcccagcaga 240ttttcaggca gcggcagcgg
cactgactac agcctgacca tctccaacct ggaacccgag 300gacattgcca
cctactactg ccagcagtac agcaagctgc cctggacctt cggcggaggc
360accaagctgg aaatcaaggg cggaggcgga tccggcggag gtggaagtgg
cggcggaggc 420tctgaggtgc aattgcagga aagcggccct ggcctggtga
aacccagcca gagcctgagc 480ctgacctgca gcgtgaccgg ctactccatc
accagcggct actactggaa ctggatcaga 540cagttccccg gaaacaagct
ggaatggatg ggctacatca cctacgacgg cagcaacaac 600tacaacccca
gcctgaagaa ccggatcagc atcacccggg acaccagcaa gaaccagttc
660ttcctgaagc tgaacagcgt gaccaccgag gataccgcca cctattactg
tgccgacttc 720gactactggg gccagggcac caccctgacc gtgtcatccg
gtggcggcgg atccgaagtg 780cagctggtgg agtctggcgg tggactggtg
cagccaggcg gctccctgag actgagctgc 840gccgcctccg gctacagctt
caccggctac accatgaatt gggtccgcca ggcccctgga 900aagggactgg
aatgggtggc cctgatcaac ccctacaagg gcgtgagcac ctacaaccag
960aagttcaagg accggttcac catcagcgtg gacaagagca agaacacagc
ctacctgcag 1020atgaactccc tgagagccga ggataccgcc gtgtattact
gtgcccgcag cggctactac 1080ggcgactccg actggtactt cgacgtgtgg
gggcagggaa ccctggtcac cgtgtccagc 1140gtggaaggcg gcagcggagg
atctggcggc tctggcggaa gcggcggagt ggacgatatc 1200cagatgacac
agtcccccag ctccctgagc gccagcgtgg gcgacagagt gaccatcacc
1260tgtcgggcca gccaggacat ccggaattat ctcaattggt atcagcagaa
acctggcaaa 1320gctcctaaac tgctgatcta ctacacctcc cggctggaaa
gcggcgtgcc cagcagattt 1380tccggcagcg ggagcggcac cgattacaca
ctgaccatca gcagcctgca gcccgaggac 1440tttgccacct actattgcca
gcagggcaac accctgccct ggacctttgg gcagggcaca 1500aaggtggaga
tcaagcacca ccaccatcac cactga 1536151218PRTArtificial SequenceLight
Chain VL CD33 (Myelotarg) 151Asp Ile Gln Leu Thr Gln Ser Pro Ser
Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Glu Ser Leu Asp Asn Tyr 20 25 30 Gly Ile Arg Phe Leu
Thr Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu
Met Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser 50 55 60 Arg
Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser 65 70
75 80 Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr
Lys 85 90 95 Glu Val Pro Trp Ser Phe Gly Gln Gly Thr Lys Val Glu
Val Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195
200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
152212PRTArtificial SequenceLight Chain V9 (VL-CH1) 152Asp 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 Gln Asp Ile Arg Asn Tyr 20 25
30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Ser Ser Ala Ser Thr 100 105 110 Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 115 120 125 Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 130 135 140 Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 145 150 155
160 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
165 170 175 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys 180 185 190 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu 195 200 205 Pro Lys Ser Cys 210 153459PRTArtificial
SequenceFab-Crossfab (VH-CH1(CD33 Myelotarg) -VH-CL [V9]) 153Glu
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Ile Thr Asp Ser
20 25 30 Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu
Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Asp Tyr
Asn Gln Lys Phe 50 55 60 Lys Asn Arg Ala Thr Leu Thr Val Asp Asn
Pro Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Phe Tyr Tyr Cys 85 90 95 Val Asn Gly Asn Pro Trp
Leu Ala 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 Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser
Glu Val Gln Leu Val Glu Ser Gly Gly Gly 225 230 235 240 Leu Val Gln
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 245 250 255 Tyr
Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly 260 265
270 Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser
275 280 285 Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Val
Asp Lys 290 295 300 Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp 305 310 315 320 Thr Ala Val Tyr Tyr Cys Ala Arg Ser
Gly Tyr Tyr Gly Asp Ser Asp 325 330 335 Trp Tyr Phe Asp Val Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 340 345 350 Ala Ser Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 355 360 365 Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 370 375 380 Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 385 390
395 400 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser 405 410 415 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu 420 425 430 Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser 435 440 445 Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 450 455 154714DNAArtificial SequenceLight Chain
VL(Myelotarg) DNA 154atgggatgga gctgtatcat cctcttcttg gtagcaacag
ctaccggtgt gcattccgac 60atccagctga cccagagccc ctccacactc tctgcctcag
tgggcgatag ggtcaccatt 120acttgcagag ctagcgagtc cctggacaac
tacggaatcc gcttccttac atggtttcag 180cagaagcctg gaaaagcacc
aaagctgctc atgtatgccg cttctaatca aggcagtggt 240gtgcccagcc
ggttctccgg gtctggctca ggaaccgaat ttactctgac cattagctcc
300ttgcagcctg atgacttcgc aacatactat tgtcagcaga ccaaggaggt
cccatggtct 360tttggtcaag gcacaaaagt ggaggttaag cgtacggtgg
ctgcaccatc tgtcttcatc 420ttcccgccat ctgatgagca gttgaaatct
ggaactgcct ctgttgtgtg cctgctgaat 480aacttctatc ccagagaggc
caaagtacag tggaaggtgg ataacgccct ccaatcgggt 540aactcccagg
agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc
600accctgacgc tgagcaaagc agactacgag aaacacaaag tctacgcctg
cgaagtcacc 660catcagggcc tgagctcgcc cgtcacaaag agcttcaaca
ggggagagtg ttag 714155696DNAArtificial SequenceLight Chain V9
(VL-CH1) DNA 155atgggatgga gctgtatcat cctcttcttg gtagcaacag
ctaccggtgt gcattccgat 60attcagatga cccagagccc cagctctctg agcgccagcg
tgggcgacag agtgaccatc 120acctgtcggg ccagccagga catcagaaac
tacctgaact ggtatcagca gaagcccggc 180aaggccccca agctgctgat
ctactacacc agcagactgg aaagcggcgt gccctccaga 240ttttccggca
gcggctccgg caccgactac accctgacca tcagcagcct gcagcccgag
300gatttcgcca catattactg ccagcagggc aataccctgc cctggacctt
cggacagggc 360acaaaagtgg aaatcaagag cagcgcttcc accaaaggcc
cttccgtgtt tcctctggct 420cctagctcca agtccacctc tggaggcacc
gctgctctcg gatgcctcgt gaaggattat 480tttcctgagc ctgtgacagt
gtcctggaat agcggagcac tgacctctgg agtgcatact 540ttccccgctg
tgctgcagtc ctctggactg tacagcctga gcagcgtggt gacagtgccc
600agcagcagcc tgggcaccca gacctacatc tgcaacgtga accacaagcc
cagcaacacc 660aaggtggaca agaaggtgga acccaagtct tgttga
6961561437DNAArtificial SequenceFab-Crossfab (VH-CH1(CD33
Myelotarg)-VH-CL [V9]) 156atgggatgga gctgtatcat cctcttcttg
gtagcaacag ctaccggtgt gcattccgag 60gtgcagctgg tgcagtctgg cgccgaagtg
aagaaacccg gcagcagcgt gaaggtgtcc 120tgcaaggcca gcggctacac
catcaccgac agcaacatcc actgggtgcg ccaggcccct 180ggccagtctc
tggaatggat cggctacatc tacccctaca acggcggcac cgactacaac
240cagaagttca agaaccgggc caccctgacc gtggacaacc ccaccaatac
cgcctacatg 300gaactgagca gcctgcggag cgaggacacc gccttctact
actgcgtgaa cggcaacccc 360tggctggcct attggggcca gggaacactc
gtgaccgtgt ccagcgctag caccaagggc 420cctagcgtgt tccctctggc
ccctagcagc aagagcacct ctggcggaac agccgccctg 480ggctgcctcg
tgaaggacta ctttcccgag cccgtgacag tgtcctggaa ctctggcgcc
540ctgacaagcg gcgtgcacac ctttccagcc gtgctgcagt ctagcggcct
gtacagcctg 600agcagcgtcg tgactgtgcc cagcagcagc ctgggaaccc
agacctacat ctgcaacgtg 660aaccacaagc ccagcaacac caaggtggac
aagaaggtgg aacccaagag ctgcgacggc 720ggaggcggat ccgggggagg
gggatctgaa gtgcagctgg tggaaagcgg cggaggcctg 780gtgcagcctg
ggggatctct gagactgagc tgtgccgcct ccggctacag cttcaccggc
840tacacaatga attgggtgcg gcaggctccc ggcaagggcc tggaatgggt
ggccctgatc 900aacccttaca agggcgtgtc cacctataat cagaagttta
aggaccgctt caccatcagc 960gtggacaagt ccaagaacac cgcctacctg
cagatgaact ccctgcgggc cgaggataca 1020gccgtgtact actgtgccag
aagcggctac tacggcgaca gcgactggta cttcgacgtg 1080tggggacagg
gcaccctggt gaccgtgtct agtgcctctg tggccgctcc cagcgtgttc
1140atcttcccac ctagcgacga gcagctgaag tccggcaccg cttctgtcgt
gtgcctgctg 1200aacaacttct acccccgcga ggccaaggtg cagtggaaag
tggacaatgc cctgcagagc 1260ggcaacagcc aggaaagcgt gaccgagcag
gacagcaagg actccaccta cagcctgtcc 1320agcaccctga cactgagcaa
ggccgactac gagaagcaca aggtgtacgc ctgcgaagtg 1380acccaccagg
gcctgtctag ccccgtgacc aagagcttca accggggcga gtgctga
1437157109PRTArtificial SequenceCD3 VL 157Gln Ala Val Val Thr Gln
Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu 1 5 10 15 Thr Val Thr Leu
Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr
Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly 35 40 45
Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50
55 60 Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly
Ala 65 70 75 80 Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp
Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 158125PRTArtificial SequenceCD3 VH 158Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser
Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25
30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr
Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp
Ser Gln Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr
Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe
Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ala 115 120 125 159121PRTArtificial
SequenceAnti-CEA VH 159Gln 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 Thr Glu Phe 20 25 30 Gly Met Asn Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr
Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe 50 55 60 Lys Gly Arg
Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly
100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
160108PRTArtificial SequenceAnti-CEA VL 160Asp 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 Lys Ala Ser Ala Ala Val Gly Thr Tyr 20 25 30 Val Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Lys Arg Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys His
Gln Tyr Tyr Thr Tyr Pro Leu 85 90 95 Phe Thr Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys 100 105 161112PRTArtificial SequenceAnti-MCSP
VH 161Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser
Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile
Thr Ser Gly 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln His Pro Gly
Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Thr Tyr Asp Gly Ser
Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser
Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asp Phe
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110
162107PRTArtificial SequenceAnti-MCSP VL 162Asp 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 Gln Gly Ile Arg Asn Tyr 20 25 30 Leu Asn
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys
Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
* * * * *