U.S. patent application number 15/845970 was filed with the patent office on 2018-10-11 for trispecific antibodies specific for her2 and a blood brain barrier receptor and methods of use.
This patent application is currently assigned to Hoffmann-La Roche Inc.. The applicant listed for this patent is Hoffmann-La Roche Inc.. Invention is credited to Christian KLEIN, Julia KRUEGER, Ekkehard MOESSNER, Jens NIEWOEHNER.
Application Number | 20180291110 15/845970 |
Document ID | / |
Family ID | 53489884 |
Filed Date | 2018-10-11 |
United States Patent
Application |
20180291110 |
Kind Code |
A1 |
KLEIN; Christian ; et
al. |
October 11, 2018 |
TRISPECIFIC ANTIBODIES SPECIFIC FOR HER2 AND A BLOOD BRAIN BARRIER
RECEPTOR AND METHODS OF USE
Abstract
The present invention relates to trispecific antibodies binding
to HER2 and a blood-brain barrier receptor (BBB-R), methods for
their production, pharmaceutical compositions containing said
antibodies, and uses thereof.
Inventors: |
KLEIN; Christian;
(Bonstetten, CH) ; KRUEGER; Julia; (Wuerzburg,
DE) ; MOESSNER; Ekkehard; (Kreuzlingen, CH) ;
NIEWOEHNER; Jens; (Muenchen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc. |
Little Falls |
NJ |
US |
|
|
Assignee: |
Hoffmann-La Roche Inc.
Little Falls
NJ
|
Family ID: |
53489884 |
Appl. No.: |
15/845970 |
Filed: |
December 18, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/EP2016/064124 |
Jun 20, 2016 |
|
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15845970 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/18 20130101;
C07K 2317/31 20130101; C07K 2317/73 20130101; C07K 16/32 20130101;
C07K 2317/33 20130101; C07K 2317/51 20130101; C07K 2317/94
20130101; C07K 2317/515 20130101; C07K 16/2881 20130101; C07K
2317/41 20130101; A61P 35/00 20180101; C07K 2317/92 20130101; C07K
2317/734 20130101; C07K 2317/60 20130101; C07K 2317/24 20130101;
C07K 2317/732 20130101; A61K 2039/507 20130101; C07K 2317/34
20130101; A61K 2039/505 20130101; C07K 2317/622 20130101 |
International
Class: |
C07K 16/32 20060101
C07K016/32; C07K 16/18 20060101 C07K016/18 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 24, 2015 |
EP |
15173640.2 |
Claims
1. A trispecific antibody specifically binding to HER2 and a
blood-brain barrier receptor (BBB-R), comprising a first monovalent
antigen binding site specific for extracellular domain II of HER2
and a second monovalent antigen binding site specific for
extracellular domain IV of HER2, and a third monovalent antigen
binding site specific for a BBB-R.
2. The trispecific antibody of claim 1, wherein the BBB-R of the
third antigen binding site is selected from the group consisting of
transferrin receptor (TfR), insulin receptor, insulin-like growth
factor receptor (IGF receptor), low density lipoprotein
receptor-related protein 8 (LRP8), low density lipoprotein
receptor-related protein 1 (LRP1), and heparin-binding epidermal
growth factor-like growth factor (HB-EGF).
3. The trispecific antibody of claim 1 wherein the third antigen
binding site is specific for the transferrin receptor.
4. The trispecific antibody of claim 3 wherein the third antigen
binding site specifically binds to an epitope in the transferrin
receptor comprised within the amino acid sequence of SEQ ID NO:
202, 203 and/or 204.
5. The trispecific antibody of claim 1, further comprising a first
Fab molecule capable of specific binding to extracellular domain II
of HER2 and a second Fab molecule capable of specific binding to
extracellular domain IV of HER2, wherein the sequence of the
variable light chain of the first Fab molecule is identical to the
sequence of the variable light chain of the second Fab
molecule.
6. The trispecific antibody of claim 5, further comprising (a) a
first heavy chain comprising a heavy chain CDR1 comprising an amino
acid sequence selected from the group consisting of SEQ ID NO: 55,
SEQ ID NO: 58, and SEQ ID NO: 14; a heavy chain CDR 2 comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO: SEQ ID NO: 15, and SEQ ID NO: 60; a heavy chain CDR 3
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 56, SEQ ID NO: 59, and SEQ ID NO: 16; and
(b) a second heavy chain comprising a heavy chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 20, a heavy chain CDR2
comprising the amino acid sequence of SEQ ID NO: 29, a heavy chain
CDR3 comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 30 and SEQ ID NO: 79; and (c) a first and
a second light chain, wherein the variable light chains of the
first and second light chain each comprise a light chain CDR1
comprising the amino acid sequence of SEQ ID NO: 89, a light chain
CDR2 comprising the amino acid sequence of SEQ ID NO: 90, and a
light chain CDR3 comprising the amino acid sequence of SEQ ID NO:
19.
7. The trispecific antibody of claim 5, further comprising two
variable light chains comprising the amino acid sequence of SEQ ID
NO: 54, a first heavy chain comprising a variable heavy chain
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 64, SEQ ID NO: 70, and SEQ ID NO: 68; and
a second heavy chain comprising a variable heavy chain comprising
an amino acid sequence selected from the group consisting of SEQ ID
NO: 92 and SEQ ID NO: 117.
8. The trispecific antibody of claim 1, further comprising a first
Fab molecule capable of specific binding to extracellular domain II
of HER2 and a second Fab molecule capable of specific binding to
extracellular domain IV of HER2, wherein either the variable
regions or the constant regions of the heavy and light chain of at
least one Fab fragment are exchanged.
9. The trispecific antibody of claim 8, wherein the first Fab
molecule comprises a heavy chain CDR1 comprising the amino acid
sequence of SEQ ID NO: 14, a heavy chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 15, a heavy chain CDR3 comprising the
amino acid sequence of SEQ ID NO: 16; a light chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 11, a light chain CDR2
comprising the amino acid sequence of SEQ ID NO: 12, and a light
chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13;
wherein the second Fab molecule comprises a heavy chain CDR1
comprising the amino acid sequence of SEQ ID NO: 20, a heavy chain
CDR2 comprising the amino acid sequence of SEQ ID NO: 108, a heavy
chain CDR3 comprising the amino acid sequence of SEQ ID NO: 79, a
light chain CDR1 comprising the amino acid sequence of SEQ ID NO:
107, a light chain CDR2 comprising the amino acid sequence of SEQ
ID NO: 18, and a light chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 19.
10. The trispecific antibody of claim 8, wherein the first Fab
molecule comprises a heavy chain CDR1 comprising the amino acid
sequence of SEQ ID NO: 14, a heavy chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 15, a heavy chain CDR3 comprising the
amino acid sequence of SEQ ID NO: 16, a light chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 11, a light chain CDR2
comprising the amino acid sequence of SEQ ID NO: 12, and a light
chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13; and
wherein the second Fab molecule comprises a heavy chain CDR1
comprising the amino acid sequence of SEQ ID NO: 20, a heavy chain
CDR2 comprising the amino acid sequence of SEQ ID NO: 29, a heavy
chain CDR3 comprising an amino acid sequence selected from the
group consisting of SEQ ID NO: 79, SEQ ID NO: 78, SEQ ID NO: 80,
SEQ ID NO: 87, SEQ ID NO: 88; a light chain CDR1 comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO: 104, SEQ ID NO: 103, and SEQ ID NO: 158; a light chain CDR2
comprising the amino acid sequence of SEQ ID NO: 18; and a light
chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19.
11. The trispecific antibody of claim 8, wherein the first Fab
molecule comprises a variable heavy chain comprising the amino acid
sequence of SEQ ID NO: 22 and a variable light chain comprising the
amino acid sequence of SEQ ID NO: 24, and wherein the second Fab
molecule comprises the amino acid sequence of SEQ ID NO: 105 and a
light chain variable region comprising the amino acid sequence of
SEQ ID NO: 106.
12. The trispecific antibody of claim 1, wherein the third antigen
binding site specific for a BBB-R is a scFv or a scFab.
13. The trispecific antibody of claim 12, wherein the scFv or scFab
is connected to the N-terminus of an IgG molecule comprising the
first and second antigen binding site.
14. The trispecific antibody of claim 12, wherein the scFv or scFab
is connected to the C-terminus of the first or second subunit of
the Fc domain.
15. The trispecific antibody of claim 1, wherein the third antigen
binding site comprises a heavy chain CDR1 comprising the amino acid
sequence of SEQ ID NO: 186, a heavy chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 187, a heavy chain CDR3 comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO: 188, SEQ ID NO: 206 and SEQ ID NO: 174; a light chain CDR1
comprising the amino acid sequence of SEQ ID NO: 189, a light chain
CDR2 comprising the amino acid sequence of SEQ ID NO: 190, and a
light chain CDR3 comprising the amino acid sequence of SEQ ID
NO:191.
16. The trispecific antibody of claim 1, wherein the third antigen
binding site comprises a heavy chain CDR1 comprising the amino acid
sequence of SEQ ID NO: 172, a heavy chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 173, a heavy chain CDR3 comprising the
amino acid sequence of SEQ ID NO: 174; a light chain CDR1
comprising the amino acid sequence of SEQ ID NO: 175, a light chain
CDR2 comprising the amino acid sequence of SEQ ID NO: 176, and a
light chain CDR3 comprising the amino acid sequence of SEQ ID
NO:177.
17. The trispecific antibody of claim 1, wherein the third antigen
binding site comprises a variable heavy chain comprising the amino
acid sequence of SEQ ID NO: 178 and a variable light chain
comprising the amino acid sequence of SEQ ID NO: 179, or a
humanized version thereof.
18. The trispecific antibody of claim 1, wherein the third antigen
binding site comprises a variable heavy chain comprising the amino
acid sequence of SEQ ID NO: 192 or SEQ ID NO: 205 and a variable
light chain comprising the amino acid sequence of SEQ ID NO:
193.
19. The trispecific antibody of claim 1, wherein the third antigen
binding site comprises the heavy chain CDR1 of SEQ ID NO: 180, the
heavy chain CDR2 of SEQ ID NO: 181, the heavy chain CDR3 of SEQ ID
NO: 182; the light chain CDR1 of SEQ ID NO: 183, the light chain
CDR2 of SEQ ID NO: 184, and the light chain CDR3 of SEQ ID NO:
185.
20. The trispecific antibody of claim 1, wherein the third antigen
binding site comprises a variable heavy chain comprising the amino
acid sequence of SEQ ID NO: 166 and a variable light chain
comprising the amino acid sequence of SEQ ID NO: 167, or a
humanized version thereof.
21. The trispecific antibody of claim 1, wherein the third antigen
binding site comprises a variable heavy chain comprising an amino
acid sequence selected from the group consisting of SEQ ID NO: 92,
SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ
ID NO: 199, and SEQ ID NO: 200, and a variable light chain
comprising an amino acid sequence selected from the group of SEQ ID
NO: 201, SEQ ID NO: 207, SEQ ID NO: 208, and SEQ ID NO:209.
22. A pharmaceutical composition comprising the bispecific antibody
of claim 1.
23. (canceled)
24. (canceled)
25. (canceled)
26. (canceled)
27. (canceled)
28. (canceled)
29. A nucleic acid sequence comprising a sequence encoding a heavy
chain of the trispecific antibody of claim 1.
30. A nucleic acid sequence comprising a sequence encoding a light
chain of the trispecific antibody of claim 1.
31. An expression vector comprising a nucleic acid sequence of
claim 29.
32. A prokaryotic or eukaryotic host cell comprising a vector
according to claim 31.
33. A method of producing an antibody comprising culturing the host
cell of claim 32 so that the antibody is produced.
34. (canceled)
35. A method of treating an individual having cancer comprising
administering to the individual an effective amount of the
trispecific antibody of claim 1.
36. The method of claim 35, wherein the wherein the cancer is a
HER2-positive cancer with brain metastases.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of International
Application PCT/EP2016/064124, filed Jun. 20, 2016, which claims
the benefit of priority of European Patent Application No.
15173640.2 filed Jun. 24, 2015, the disclosures of both which
are-incorporated herein by reference in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Dec. 12, 2017 is named P32940US_ST25.txt and is 337,315 bytes in
size.
FIELD OF THE INVENTION
[0003] The present invention relates to trispecific antibodies
binding to HER2 and a blood-brain barrier receptor (BBB-R), methods
for their production, pharmaceutical compositions containing said
antibodies, and uses thereof.
BACKGROUND
[0004] Antibodies specific for tumor-associated antigens are a
valuable approach in cancer therapy because they mediate selective
destruction of tumor cells, while leaving healthy cells and tissues
undamaged. Treatment of cancers affecting the central nervous
system (CNS) with large molecules such as antibodies remains
challenging as brain penetration is severely limited by the largely
impermeable blood-brain barrier (BBB). Past studies have shown that
a very small percentage (approximately 0.1%) of an IgG circulating
in the bloodstream crosses through the BBB into the CNS
(Felgenhauer, Klin. Wschr. 52: 1158-1164 (1974)), where the CNS
concentration of the antibody may be insufficient to permit a
robust effect. Among the many strategies to overcome this obstacle
is to utilize transcytosis trafficking pathways of endogenous
receptors expressed at the brain capillary endothelium. Recombinant
proteins such as monoclonal antibodies have been designed against
these receptors to enable receptor-mediated delivery of large
molecules to the brain. However, strategies to maximize brain
uptake while minimizing reverse transcytosis back to the blood, and
the extent of accumulation after therapeutic dosing, remain
unexplored. Furthermore, whether antibodies that cross the BBB are
pharmacodynamically functional is unknown.
[0005] Members of the ErbB family of receptor tyrosine kinases are
important mediators of cell growth, differentiation and survival.
The receptor family includes four distinct members, including
epidermal growth factor receptor (EGFR or ErbB1), HER2 (ErbB2 or
p185''e''), HER3 (ErbB3) and HER4 (ErbB4 or tyro2). HER2 is a
transmembrane surface-bound receptor tyrosine kinase and is
normally involved in the signal transduction pathways leading to
cell growth and differentiation. HER2 is a promising target for
treatment of breast cancer as it was found to be overexpressed in
about one-quarter of breast cancer patients (Bange et al, 2001,
Nature Medicine 7:548).
[0006] The murine monoclonal antibody 4D5 is targeting HER2
specifically in HER2 overexpressing cancer cells, while having no
effect on cells expressing physiological levels of HER2. The
humanized (4D5) monoclonal antibody (hu4D5) is commercially known
as the drug Herceptin.RTM. (trastuzumab, rhuMAb HER2, U.S. Pat. No.
5,821,337), which gained FDA marketing approval in late 1998.
[0007] Herceptin was the first monoclonal antibody developed for
the treatment of HER2-positive breast cancer and has increased
survival times for patients so that they are now the same as for
patients with HER2-negative breast cancer. Before Herceptin
treatment, shorter survival outcomes were expected for patients
diagnosed with HER2-positive breast cancer, compared to patients
with HER2-negative disease. In the CLEOPATRA study, PERJETA in
combination with Herceptin and chemotherapy has shown the extension
of survival times for patients with this aggressive disease even
further than Herceptin.
[0008] Pertuzumab (PERJETA.TM., rhuMab 2C4, U.S. Pat. No.
7,862,817) is a humanized monoclonal antibody, which is designed
specifically to prevent the HER2 receptor from pairing (dimerising)
with other HER receptors (EGFR/HER1, HER3 and HER4) on the surface
of cells, a process that is believed to play a role in tumor growth
and survival. The combination of PERJETA, Herceptin and
chemotherapy is thought to provide a more comprehensive blockade of
HER signaling pathways. PERJETA is approved in combination with
Herceptin (trastuzumab) and docetaxel in adult patients with
HER2-positive metastatic or locally recurrent unresectable breast
cancer and gained FDA approval for neoadjuvant breast cancer
treatment in September 2013. Pertuzumab binds to domain II of HER2,
essential for dimerization, while Ttrastuzumab binds to
extracellular domain IV of HER2.
[0009] Li et al (Cancer Research. 2013) describe bispecific,
bivalent antibodies to ErbB2 that overcome trastuzumab resistance.
The bispecific, bivalent antibodies described therein are based on
the native Trastuzumab and Pertuzumab sequences.
[0010] Central nervous system (CNS) metastases are observed in up
to half of patients with HER2-positive metastatic breast cancer
(MBC), with incidence likely to continue to rise due to longer
survival through improved systemic treatments. Drugs that
specifically block HER2 to stop the growth of cancer cells are
called HER2-targeted therapies. Examples of these drugs include
trastuzumab (Herceptin), lapatinib (Tykerb), pertuzumab (Perjeta),
and ado-trastuzumab emtansine (Kadcyla), commonly referred to as
T-DM1. Some of these drugs may be used together with chemotherapy.
Unfortunately, these drugs are not usually able to reach the brain
as easily as they can reach the rest of the body, with lapatinib
being a possible exception. Therefore, when cancer spreads to the
brain it is usually treated with surgery and/or radiation therapy.
While radiotherapy-based approaches can be effective, there are
potential short- and long-term toxicities, and patients frequently
progress. CNS response to existing systemic therapies has been
generally poor, and there is a high unmet need with no approved
treatment for CNS metastases in HER2-positive MBC.
[0011] Surprisingly the inventors of the present application found
that optimizing the native Trastuzumab and Pertuzumab sequences and
combining these optimized variants with a third binder targeting a
blood brain barrier receptor (BBBR) results in a functional
molecule with improved properties compared to the combination of
the monospecific antibodies rhuMab 2C4 and hu 4D5. Hence the new
trispecific format combines the superior characteristics of the
bispecific HER2 antibodies known in the art with the advantages of
a classical monospecific HER2 antibody and specific CNS targeting:
The novel trispecific antibodies of the present invention are
monovalent for the two different HER2 epitopes, resulting in the
same avidity effect as the bivalent parental antibodies and
specifically target the CNS via a BBBR binding moiety. In contrast,
tetravalent antibodies may differ in their avidity for HER2 on
cells. The avidity effect of the novel trispecific HER2 antibodies
may result in a superior safety window on cell types with low HER2
expression such as in normal tissues or cardiac tissues where
inhibition of HER2 and/or ADCC may not be desired.
[0012] In one aspect of the invention a trispecific antibody
binding to HER2 and a blood-brain barrier receptor (BBB-R) is
provided, comprising an IgG molecule wherein one of the Fab
fragments is replaced by a crossover Fab fragment. Crossover Fab
fragments are Fab fragments wherein either the variable regions or
the constant regions of the heavy and light chain are exchanged.
Bispecific antibody formats comprising crossover Fab fragments have
been described, for example, in WO2009080252, WO2009080253,
WO2009080251, WO2009080254, WO2010/136172, WO2010/145792 and
WO2013/026831. The native Trastuzumab sequences have been optimized
in their CDRs to improve the stability of the antibody CDRs against
spontaneous chemical modification, the resulting sequences
framework-grafted to avoid mispairing, resulting in highly potent
trispecific antibodies that specifically bind to HER2; finally they
can be produced with high yield and only low percentage of side
products comparable to the conventional parental Her2 antibodies.
Chain mispairing of light chains resulting from the fact that both
pertuzumab and trastuzumab are based on a comparable framework
region has been overcome by grafting the CDRs on a completely novel
antibody framework.
[0013] In another aspect of the invention trispecific antibodies
binding to HER2 and a blood-brain barrier receptor (BBB-R) are
provided wherein the two binding moieties for HER2 comprise
identical light chains based on a consensus of the parental
trastuzumab and pertuzumab light chains and the corresponding
pertuzumab heavy chain has been remodeled. The use of this
so-called `common light chain` principle, i.e. combining two
binders that share one light chain but still have separate
specificities, prevents light chain mispairing and in this
particular case retains the epitope specificity of the parental
antibodies. As a consequence, there are less side products during
production, facilitating the homogenous preparation of the
trispecific antigen binding molecules at high yields. Surprisingly
the inventors of the present invention found that the bispecific
HER2 antibodies in the monovalent common light chain format have an
increased affinity to the pertuzumab epitope, and show superior
inhibitory effects on cell proliferation and induction of cell
dependent cytotoxicity (CDC) as compared to the combination of the
parental antibodies. Complement dependent cytotoxicity (CDC) is
very important for the optimal therapeutic monoclonal antibodies
(mAb) function and is totally conserved even after a chemotherapy
treatment. However, this activity is generated by some antibodies
but not all of them.
SUMMARY
[0014] The present invention relates to a trispecific antibody
specifically binding to HER2 and a blood-brain barrier receptor
(BBB-R), comprising a first monovalent antigen binding site
specific for extracellular domain II of HER2 and a second
monovalent antigen binding site specific for extracellular domain
IV of HER2, and a third monovalent antigen binding site specific
for a BBB-R. In one aspect the BBB-R of the third antigen binding
site is selected from the group consisting of transferrin receptor
(TfR), insulin receptor, insulin-like growth factor receptor (IGF
receptor), low density lipoprotein receptor-related protein 8
(LRP8), low density lipoprotein receptor-related protein 1 (LRP1),
and heparin-binding epidermal growth factor-like growth factor
(HB-EGF). In one aspect the third antigen binding site is specific
for the transferrin receptor. In one aspect the third antigen
binding site specifically binds to an epitope in the transferrin
receptor comprised within the amino acid sequence of SEQ ID NO:
202, 203 and/or 204.
[0015] In one aspect the trispecific antibody specifically binding
to HER2 and a blood-brain barrier receptor (BBB-R), comprises a
first Fab molecule capable of specific binding to extracellular
domain II of HER2 and a second Fab molecule capable of specific
binding to extracellular domain IV of HER2, wherein the sequence of
the variable light chain of the first Fab molecule is identical to
the sequence of the variable light chain of the second Fab
molecule. In one aspect the trispecific antibody specifically
binding to HER2 and a blood-brain barrier receptor (BBB-R)
comprises (a) a first heavy chain comprising a heavy chain CDR1
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 55, SEQ ID NO: 58 and SEQ ID NO: 14; a
heavy chain CDR 2 comprising an amino acid sequence selected from
the group consisting of SEQ ID NO: 77; SEQ ID NO: 15 and SEQ ID NO:
60 and a heavy chain CDR 3 comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 56 or SEQ ID NO:
59 and SEQ ID NO: 16, and (b) a second heavy chain comprising a
heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:
20, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID
NO: 29 and a heavy chain CDR3 comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 30 and SEQ ID NO:
79; and (c) a first and a second light chain, wherein the variable
light chains of the first and second light chain comprise the CDRs
comprising the amino acid sequence of SEQ ID NO: 89, SEQ ID NO: 90
and SEQ ID NO: 19. In one aspect the trispecific antibody
specifically binding to HER2 and a blood-brain barrier receptor
(BBB-R) comprises two variable light chains comprising an amino
acid sequence of SEQ ID NO: 54, a first heavy chain comprising a
variable heavy chain comprising an amino acid sequence selected
from the group consisting of SEQ ID NO: 64, SEQ ID NO: 70 and SEQ
ID NO: 68, and a second heavy chain comprising a variable heavy
chain comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 92 and SEQ ID NO: 117. In one aspect the
trispecific antibody specifically binding to HER2 and a blood-brain
barrier receptor (BBB-R) comprises a first Fab molecule capable of
specific binding to extracellular domain II of HER2 and a second
Fab molecule capable of specific binding to extracellular domain IV
of HER2, wherein either the variable regions or the constant
regions of the heavy and light chain of at least one Fab fragment
are exchanged. In one aspect the trispecific antibody specifically
binding to HER2 and a blood-brain barrier receptor (BBB-R)
comprises a first Fab molecule comprising a heavy chain CDR1
comprising the amino acid sequence of SEQ ID NO: 14, a heavy chain
CDR2 comprising the amino acid sequence of SEQ ID NO: 15 and a
heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:
16; and a light chain CDR1 comprising the amino acid sequence of
SEQ ID NO: 11; a light chain CDR2 comprising the amino acid
sequence of SEQ ID NO: 12 and a light chain CDR3 comprising the
amino acid sequence of SEQ ID NO: 13, and a second Fab molecule
comprising a heavy chain CDR1 comprising the amino acid sequence of
SEQ ID NO: 20; a heavy chain CDR2 comprising the amino acid
sequence of SEQ ID NO: 108; a heavy chain CDR3 comprising the amino
acid sequence of SEQ ID NO: 79; and a light chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 107, a light chain CDR2
comprising the amino acid sequence of SEQ ID NO: 18 and a light
chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19. In
one aspect the trispecific antibody specifically binding to HER2
and a blood-brain barrier receptor (BBB-R) comprises a first Fab
molecule comprising a heavy chain CDR1 comprising the amino acid
sequence of SEQ ID NO: 14, a heavy chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 15 and a heavy chain CDR3 comprising
the amino acid sequence of SEQ ID NO: 16; and a light chain CDR1
comprising the amino acid sequence of SEQ ID NO: 11; a light chain
CDR2 comprising the amino acid sequence of SEQ ID NO: 12 and a
light chain CDR3 comprising the amino acid sequence of SEQ ID NO:
13, and a second Fab molecule comprising a heavy chain CDR1
comprising the amino acid sequence of SEQ ID NO: 20, a heavy chain
CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and a
heavy chain CDR3 comprising an amino acid sequence selected from
the group consisting of SEQ ID NO: 79, SEQ ID NO: 78, SEQ ID NO:
80, SEQ ID NO: 87, SEQ ID NO: 88; and a light chain CDR1 comprising
an amino acid sequence selected from the group consisting of SEQ ID
NO: 104, SEQ ID NO: 103 and SEQ ID NO: 158; a light chain CDR2
comprising the amino acid sequence of SEQ ID NO: 18 and a light
chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19. In
one aspect the trispecific antibody specifically binding to HER2
and a blood-brain barrier receptor (BBB-R) comprises a first Fab
molecule comprising a variable heavy chain comprising an amino acid
sequence of SEQ ID NO: 22 and a variable light chain comprising an
amino acid sequence of SEQ ID NO: 24 and wherein a second Fab
molecule comprising an amino acid sequence of SEQ ID NO: 105 and a
light chain variable region comprising an amino acid sequence of
SEQ ID NO: 106. In one aspect the trispecific antibody specifically
binding to HER2 and a blood-brain barrier receptor (BBB-R)
comprises a third antigen binding site specific for a BBB-R
selected from a scFv or a scFab. In one aspect the scFv or scFab is
connected to the N-terminus of an IgG molecule comprising the first
and second antigen binding site. In one aspect the scFv or scFab is
connected to the C-terminus of the first or second subunit of the
Fc domain. In one aspect the trispecific antibody specifically
binding to HER2 and a blood-brain barrier receptor (BBB-R)
comprises a third antigen binding site comprising a 1. a heavy
chain CDR1 comprising the amino acid sequence of SEQ ID NO: 186, a
heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:
187 and a heavy chain CDR3 comprising the amino acid sequence
selected from the group of SEQ ID NO: 188, SEQ ID NO: 206 or SEQ ID
NO: 174; and a light chain CDR1 comprising the amino acid sequence
of SEQ ID NO: 189, a light chain CDR2 comprising the amino acid
sequence of SEQ ID NO: 190 and a light chain CDR3 comprising the
amino acid sequence of SEQ ID NO:191. In one aspect the trispecific
antibody specifically binding to HER2 and a blood-brain barrier
receptor (BBB-R) comprises a third antigen binding site comprising
a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:
172, a heavy chain CDR2 comprising the amino acid sequence of SEQ
ID NO: 173 and a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 174; and a light chain CDR1 comprising the
amino acid sequence of SEQ ID NO: 175, a light chain CDR2
comprising the amino acid sequence of SEQ ID NO: 176 and a light
chain CDR3 comprising the amino acid sequence of SEQ ID NO:177.
[0016] In one aspect the trispecific antibody specifically binding
to HER2 and a blood-brain barrier receptor (BBB-R) comprises a
third antigen binding site comprising a variable heavy chain
comprising an amino acid sequence of SEQ ID NO: 178 and a variable
light chain comprising an amino acid sequence of SEQ ID NO: 179 or
a humanized version thereof. In one aspect the trispecific antibody
specifically binding to HER2 and a blood-brain barrier receptor
(BBB-R) comprises a third antigen binding site comprising 1. a
variable heavy chain comprising an amino acid sequence of SEQ ID
NO: 192 or SEQ ID NO: 205 and a variable light chain comprising an
amino acid sequence of SEQ ID NO: 193.
[0017] In one aspect the trispecific antibody specifically binding
to HER2 and a blood-brain barrier receptor (BBB-R) comprises a
third antigen binding site comprising a heavy chain CDR1 of SEQ ID
NO: 180, a heavy chain CDR2 of SEQ ID NO: 181 and a heavy chain
CDR3 of SEQ ID NO: 182; and a light chain CDR1 of SEQ ID NO: 183, a
light chain CDR2 of SEQ ID NO: 184 and a light chain CDR3 of SEQ ID
NO: 185. In one aspect the trispecific antibody specifically
binding to HER2 and a blood-brain barrier receptor (BBB-R)
comprises a third antigen binding site comprising a variable heavy
chain comprising an amino acid sequence of SEQ ID NO: 166 and a
variable light chain comprising an amino acid sequence of SEQ ID
NO: 167 or a humanized version thereof. In one aspect the
trispecific antibody specifically binding to HER2 and a blood-brain
barrier receptor (BBB-R) comprises a third antigen binding site
comprising a variable heavy chain comprising an amino acid sequence
of SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197,
SEQ ID NO: 198, SEQ ID NO: 199, or SEQ ID NO: 200, and a variable
light chain comprising an amino acid sequence selected from the
group of SEQ ID NO: 201, SEQ ID NO: 207, SEQ ID NO: 208, or SEQ ID
NO:209.
[0018] In one aspect the trispecific antibody specifically binding
to HER2 and a blood-brain barrier receptor (BBB-R) comprises a
third antigen binding site comprising sequence alterations in the
CDRs resulting in a reduced affinity.
[0019] In a second object the present invention relates to a
pharmaceutical composition comprising a trispecific antibody of the
present invention.
[0020] In a third object the present invention relates to a
trispecific antibody of the present invention for the treatment of
cancer. In another embodiment, use of the trispecific antibody as a
medicament is provided. Preferably said use is for the treatment of
cancer. In one aspect said cancer is a HER2-positive cancer with
brain metastases.
[0021] In further objects the present invention relates to a
nucleic acid sequence comprising a sequence encoding a heavy chain
of a trispecific antibody of the present invention, a nucleic acid
sequence comprising a sequence encoding a light chain of a
trispecific 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
[0022] FIGS. 1A-D: Schematic drawing of Trastuzumab and Pertuzumab
bispecific antibodies in a 2+2 IgG-scFv format. The antibodies are
bivalent for each antigen binding site and are able to bind two
different paratopes in the ErbB2/HER2 receptor
(antigen1=trastuzumab specificity, i.e. extracellular domain IV of
HER2; antigen2=pertuzumab specificity extracellular domain II of
HER2) (A): The single chain Fv (scFv) is fused C-terminally to the
heavy chain in the order VH-VL (TvAB12, SEQ ID NOs 123 and 124).
(B): The single chain Fv (scFv) is fused N-terminally to the light
chain in the order VL-VH (TvAB13, SEQ ID NOs 125 and 126). (C) The
single chain Fv (scFv) is fused C-terminally to the light chain in
the order VL-VH (TvAB16: SEQ ID NOs 127 and 128, TvAB20: SEQ ID NOs
131 and 132). (D): The single chain Fv (scFv) is fused C-terminally
to the heavy chain in the order VL-VH (TvAB17: SEQ ID NOs 129 and
130).
[0023] FIGS. 2A-B: Purification of Trastuzumab and Pertuzumab
bispecific antibodies in a 2+2 IgG-scFv format. (A): Size-exclusion
purification of TvAb12 (SEQ ID NOs 123 and 124) on a 26/60 Superdex
200 column. (B): SDS-Page analysis of main peak fraction
originating from size-exclusion chromatography (NR=non-reducing,
R=reducing conditions).
[0024] FIGS. 3A-B: Purification of Trastuzumab and Pertuzumab
bispecific antibodies in a 2+2 IgG-scFv format. (A): Size-exclusion
purification of TvAb16 (SEQ ID NOs 127 and 128) on a 26/60 Superdex
200 column. (B): SDS-Page analysis of main peak fraction
originating from size-exclusion chromatography (NR=non-reducing,
R=reducing conditions).
[0025] FIGS. 4A-B: Purification of Trastuzumab and Pertuzumab
bispecific antibodies in a 2+2 IgG-scFv format. (A): Size-exclusion
purification of TvAb20 (SEQ ID NOs 131 and 132) on a 26/60 Superdex
200 column. Main product peak marked with "1". (B) SDS-Page
analysis of main peak fraction originating from size-exclusion
chromatography (NR=non-reducing, R=reducing conditions).
[0026] FIGS. 5A-B: Off-rates of Trastuzumab variants as determined
by SPR method (ProteOn instrument) after incubating the samples for
1, 2, or 3 months at 40.degree. in buffer 40 mM Histidin, 150 mM
NaCl, pH5.0. The off rates of the variant does not change over the
investigated time period. "602": D98E mutation in heavy chain and
T31V mutation in light chain.
[0027] FIGS. 6A-C: Off-rates of Trastuzumab variants as determined
by SPR method (ProteOn instrument) after incubating the samples for
1, 2, or 3 months at 40.degree. C. in 40 mM Histidin, 150 mM NaCl,
at different pH. The off rates of the N30S variant were very slow,
and therefore contain a high degree of uncertainty. "602": D98E
mutation in heavy chain and T31V mutation in light chain, "N30T":
D98E mutation in heavy chain and N30T mutation in light chain,
"N30S": D98E mutation in heavy chain and N30S mutation in light
chain. (A): pH5.0. (B): pH6.0, (C): pH7.4.
[0028] FIGS. 7A-L: Binding of Trastuzumab and Trastuzumab
stabilization variants after stress to KPL-4 cells. Trastuzumab and
3 different stabilized Trastuzumab variants were incubated for one,
two and three month in buffer with different pH values at
40.degree. C. The stressed antibodies were tested compared to the
antibody at time point zero for binding to KPL-4 cells by flow
cytometry. "602": D98E mutation in heavy chain and T31V mutation in
light chain, "N30T": D98E mutation in heavy chain and N30T mutation
in light chain, "N30S": D98E mutation in heavy chain and N30S
mutation in light chain.
[0029] FIGS. 8A-F: Binding of Trastuzumab and Trastuzumab
stabilization variants after stress to KPL-4 cells. Trastuzumab and
the 2 stabilization variants GA602 (D98E mutation in heavy chain
and T31V mutation in light chain) and GA603 (D98E mutation in heavy
chain and T31V mutation in light chain and FcRN mutation T307Q und
N434A) were incubated for one, two and three month in buffer 1 (40
mM Histidin 150 mM NaCl, pH5.0) or buffer 2 (2. 40 mM Histidin 150
mM NaCl, pH6.0) at 40.degree. C. The stressed antibodies were
tested compared to the antibody at time point zero for binding to
KPL-4 cells by flow cytometry.
[0030] FIGS. 9A-F: ADCC induction with Trastuzumab, GA602 and GA603
after stress on KPL-4 cells. Trastuzumab and the 2 stabilization
variants GA602 (D98E mutation in heavy chain and T31V mutation in
light chain) and GA603 (D98E mutation in heavy chain and T31V
mutation in light chain and FcRN mutation T307Q und N434A) were
incubated for one, two and three month in buffer 1 (40 mM Histidin
150 mM NaCl, pH 5.0) or buffer 2 (2. 40 mM Histidin 150 mM NaCl,
pH6.0) at 40.degree. C. The stressed antibodies were tested
compared to the antibody at time point zero for ADCC induction
after 4 h on KPL-4 cells.
[0031] FIGS. 10A-B: Schematic drawing of Trastuzumab and Pertuzumab
bispecific antibodies in a 1+1 format. (A): single chain Fab
(scFab) based molecules (B): cross-over Fab (xFab) based
molecules.
[0032] FIGS. 11A-B: Purification of CrossMab-XPer (SEQ ID NOs 109,
110, 96, 86). (A): SDS-PAGE showing the purified antibody molecule
under reduced and non-reduced conditions. (B): HP-SEC analysis of
purified CrossMab-XPer.
[0033] FIGS. 12A-B: Q-TOF mass spectrometry comparison of the
spectra of CrossMab-XTra (top, SEQ ID NOs 119, 120, 121,122) and
CrossMab-CDRG (bottom, SEQ ID NOs 109, 110, 111, 112) estimating
the integrity and purity of the antibody molecules.
[0034] FIGS. 13A-B: Proliferation inhibition by non-glycoengineered
HER2 CrossMab (SEQ ID NOs 119, 120, 121,122) after 5 days of
incubation as measured in an AlamarBlue.RTM. assay. (A) BT474 cells
(B) N87 cells.
[0035] FIGS. 14A-C: ADCC induced by different HER2 specific
antibodies using (A) KPL-4, (B) T47D and (C) Calu-3 as target cells
(E:T=25:1, effectors human PBMCs, incubation time 4 h). "HER2
crossmab wt": SEQ ID NOs 119, 120, 121,122, non glycoengineered;
"HER2 crossmab g2": SEQ ID NOs 119, 120, 121,122,
glycoengineered.
[0036] FIGS. 15A-C: Antitumor activity of different anti-Her2
antibodies in the Calu3 non-small cell lung cancer xenograft
(Experiment: BispecHer2_PZ_Calu3_001). SCID beige mice with Calu3
xenograft tumors were treated i.p. once weekly at the indicated
dosages for 7 weeks. Xolair a humanized IgG1 antibody targeting
human IgE was used as a control. Statistical analysis based on
medians at endpoint (day 85) reveals that compared to Xolair the
bispecific HER2 antibodies suppressed tumor growth by 87.5% (s.);
OmniE (SEQ ID NOs 145, 146) by 43.7% (n.s.); Crossmab 003 (SEQ ID
NOs 119, 120, 121, 122, non glycoengineered) by 92.1% (s.); TvAb12
(SEQ ID NOs 123 and 124) by 59.8% (n.s.) and TvAb20 (SEQ ID NOs 131
and 132) by 12.6% (n.s.). Tumor growth curves are depicted as
mean+/-SEM (n=8 in each group).
[0037] FIGS. 16A-C: Antitumor activity of different anti-Her2
antibodies in the KPL-4 breast cancer xenograft (Experiment:
Bispec.Her2_PZ_KPL-4_002). SCID beige mice with KPL-4 xenograft
tumors were treated i.p. once weekly at the indicated dosages for 5
weeks. Xolair a humanized IgG1 antibody targeting human IgE was
used as a control. Statistical analysis based on medians at
endpoint (day 59) reveals that compared to Xolair the bispecific
HER2 antibodies suppressed tumor growth by 120.8% (s.); Crossmab
003 (SEQ ID NOs 119, 120, 121,122, non glycoengineered) by 120.6%
(s.); TvAb12 (SEQ ID NOs 123 and 124) by 70.1% (s.); TvAb20 (SEQ ID
NOs 131 and 132) by 83.4% (s.). OmniE (SEQ ID NOs 145, 146) had no
significant effect on tumor growth. Tumor growth curves are
depicted as mean+/-SEM (n=9 in each group).
[0038] FIGS. 17A-C: Antitumor activity of anti-Her2_005 crossmab
antibody (SEQ ID NOs 119, 120, 121,122, non glycoengineered) in the
KPL-4 breast cancer xenograft (Experiment:
Bispec.Her2_PZ_KPL-4_003). SCID beige mice with KPL-4 xenograft
tumors were treated i.p. once weekly with escalating dosages of the
crossmab ranging from 1 to 20 mg/kg for 5 weeks. Xolair a humanized
IgG1 antibody targeting human IgE was used as a control.
Statistical analysis based on medians at endpoint (day 70) reveals
that compared to Xolair the bispecific HER2 antibodies suppressed
tumor growth by 121.8% (s.); The Her2 crossmab 005 suppressed tumor
growth at a dosage of 1 mg/kg by 25.1% (n.s.); at 5 mg/kg by 112.3%
(s.); at 10 mg/kg by 109.5% (s.) and by 20 mg/kg by 121.8% (s.).
Tumor growth curves are depicted as mean+/-SEM (n=10 in each
group).
[0039] FIGS. 18A-C: Antitumor activity of different anti-Her2
antibodies in the KPL-4 breast cancer xenograft (Experiment:
Bispec.Her2_PZ_KPL-4_009). SCID beige mice with KPL-4 xenograft
tumors were treated i.p. once weekly with the different compounds
for 4 weeks. Xolair a humanized IgG1 antibody targeting human IgE
was used as a control. Statistical analysis based on medians at
endpoint (day 70) reveals that compared to Xolair the bispecific
HER2 antibodies (each dosed at 5 mg/kg) suppressed tumor growth by
83.2% (s.) and both given at a dosage of 10 mg/kg each by 109.5%
(s.). TvAb 16 (SEQ ID NOs 127 and 128) given at two different
dosages (5 mg/kg and 10 mg/kg) had no significant anti-tumoral
effect. TvAb20 (SEQ ID NOs 131 and 132), at a dosage of 5 mg/kg,
suppressed tumor growth by 75.3% (s.) and at a dosage of 10 mg/kg
by 59.8% (n.s.). Tumor growth curves are depicted as mean+/-SEM
(n=10 in each group).
[0040] FIGS. 19A-F: SPR analysis of initial Pertuzumab/Trastuzumab
hybrid light chains. SPR-based kinetic analyses of Pertuzumab,
Trastuzumab, and sequence combinations with the initial Pertuzumab
hybrid LCs harboring amino acid residues of the Trastuzumab LCDR3
region. Smooth lines represent a global fit of the data to a 1:1
interaction model. PertuzumabTrasL3: SEQ ID No: 26, PertuzumabTras
Y91H: SEQ ID No: 28.
[0041] FIGS. 20A-B: SPR analysis of the Pertuzumab and Trastuzumab
HCs in combination with the newly identified common light chain
Pertuzumab (Tras.L3)(QM), SEQ ID No: 54. Shown is the binding of
both antibodies to Her2 at different concentrations. Smooth lines
represent a global fit of the data to a 1:1 interaction model.
[0042] FIGS. 21A-F: Characterization of the affinity-matured
Pertuzumab clones identified by phage display. SPR analysis of the
identified affinity-matured clones. Shown is the binding of
bacterial Fabs to Her2 at different concentrations. Smooth lines
represent a global fit of the data to a 1:1 interaction model. B2:
SEQ ID No: 66, D1: SEQ ID No: 62, E1: SEQ ID No: 68, C8: SEQ ID No:
72, G2: SEQ ID No: 70, A1: SEQ ID No: 74.
[0043] FIG. 22: Schematic drawing of the bi-specific HER2
antibodies with a common light chain.
[0044] FIGS. 23A-F: Purification and analytical characterization of
the bi-specific HER2 antibodies with a common light chain. The
purification method involved an affinity step (protein A) followed
by size exclusion chromatography (Superdex 200, GE Healthcare). The
final product was analyzed and characterized by analytical size
exclusion chromatography (Superdex 200 column). (A)(B): comprising
D1der (SEQ ID NO: 64), (C)(D): comprising G2 (SEQ ID NO: 70),
(E)(F): comprising E1 (SEQ ID NO: 68).
[0045] FIGS. 24A-D: SPR analysis of the Her2 knock-out variants.
Shown are the sensograms of Trastuzumab and Pertuzumab binding to
both knock-out variants. Smooth lines represent a global fit of the
data to a 1:1 interaction model.
[0046] FIG. 25: Binding of bi-specific HER2 antibodies with a
common light chain clone variants to KPL-4 cells. KPL-4 cells were
stained with increasing concentrations of the indicated antibodies.
The antibodies were detected with a FITC labeled anti-human
secondary and the fluorescence was determined by flow cytometry.
"Herceptarg CLC D1-der": SEQ ID NOs 64, 54, 92, "Herceptarg CLC
G2/2": SEQ ID NOs 70, 54, 92, "Herceptarg CLC E1/1": SEQ ID NOs 68,
54, 92; "GA 604": SEQ ID NOs 109, 110, 111, 112.
[0047] FIGS. 26A-F: Proliferation inhibition of BT474, N87, and
SkBr3 cells with bi-specific HER2 antibodies with common light
chain clone variants. BT474 (A)(B), N87 (C)(D), and SkBr3
(E)(F)cells were treated with the three different Herceptarg
variants. As controls Trastuzumab, Pertuzumab and the combination
of both were included. After 5 days, proliferation inhibition was
determined with CellTiter Glo. "Herceptarg CLC D1-der": SEQ ID NOs
64, 54, 92, "Herceptarg CLC G2/2": SEQ ID NOs 70, 54, 92,
"Herceptarg CLC E1/1": SEQ ID NOs 68, 54, 92; "GA 604": SEQ ID NOs
109, 110, 111, 112.
[0048] FIGS. 27A-D: Killing of KPL-4 cells and MDA-MB 231 with
bi-specific HER2 antibodies with common light chain variants.
(A)(B), Antibody dependent killing of KPL-4 cells with PBMCs (E:T
25:1) or was determined by measuring LDH release after 4 h. (C)(D)
Antibody dependent killing of MDA-MB 231 cells with PBMCs (E:T 5:1)
was determined by measuring LDH release after 24 h. "Herceptarg CLC
D1-der": SEQ ID NOs 64, 54, 92, "Herceptarg CLC G2/2": SEQ ID NOs
70, 54, 92, "Herceptarg CLC E1/1": SEQ ID NOs 68, 54, 92; "GA 604":
SEQ ID NOs 109, 110, 111, 112.
[0049] FIG. 28: Proliferation inhibition of BT474 cells with
bi-specific HER2 antibodies with common light chain clone variants.
BT474 cells were treated with the different Herceptarg variants. As
controls Trastuzumab, Pertuzumab and the combination of both were
included. After 6 days, proliferation inhibition was determined
with CellTiter Glo. "Herceptarg CLC D1-der wt": SEQ ID NOs 64, 54,
92, Herceptarg CLC D1-der G2'': SEQ ID NOs 64, 54, 92
(glycoengineered variant) "Herceptarg CrossMab": SEQ ID NOs 109,
110, 111, 112.
[0050] FIG. 29: C1q binding of Her2 antibodies on BT-474 cells.
BT474 cells were incubated with the three Herceptarg variants. As
controls Trastuzumab, Pertuzumab and the combination of both were
included. "Herceptarg CLC D1-der wt": SEQ ID NOs 64, 54, 92,
Herceptarg CLC D1-der G2'': SEQ ID NOs 64, 54, 92 (glycoengineered
variant) "Herceptarg CrossMab": SEQ ID NOs 109,110,111,112.
[0051] FIG. 30: CDC activation on BT-474 cells (LDH release). BT474
cells were incubated with the three Herceptarg variants. As
controls Trastuzumab, Pertuzumab and the combination of both were
included. "Herceptarg CLC D1-der wt": SEQ ID NOs 64, 54, 92,
Herceptarg CLC D1-der G2'': SEQ ID NOs 64, 54, 92 (glycoengineered
variant) "Herceptarg CrossMab": SEQ ID NOs 109, 110, 111, 112.
[0052] FIGS. 31A-B: CDC mediated killing of BT-474 cells (ACEA).
BT474 cells were incubated with the three Herceptarg variants. As
controls Trastuzumab, Pertuzumab and the combination of both were
included. "Herceptarg CLC D1-der wt": SEQ ID NOs 64, 54, 92,
Herceptarg CLC D1-der G2'': SEQ ID NOs 64, 54, 92 (glycoengineered
variant) "Herceptarg CrossMab": SEQ ID NOs 109, 110, 111, 112.
[0053] FIG. 32: In vivo activity of bispecific antibodies. Tumor
volume in mouse xenograft models after treatment with different
Her2 bispecific molecules (10 mg/kg) was compared to treatment with
Trastuzumab, Pertuzumab and the combination of both. "Herceptarg":
SEQ ID NOs 64, 54, 92. "Control": Xolair, a non Her2 binding
antibody.
[0054] FIG. 33: FACS binding of trispecific antibodies specific for
HER2 and transferrin receptor to HER2+BT474.M1 cells.
"Herceptarg-8D3" (SEQ ID NOs.: 165, 163, 161) and "Herceptarg
(LALA)-8D3" (SEQ ID NOs.: 168, 169, 161) are trispecific, trivalent
antibodies specific for extracellular domains II and IV of HER2 and
the transferrin receptor. Positive control: "Herceptarg-LALA" (SEQ
ID NOs: 170, 169, 161) and "Herceptarg" (SEQ ID NOs.: 171, 163,
161) are bispecific, bivalent antibodies specific for extracellular
domains II and IV of HER2. Negative control "pTAU-8D3" is a
bispecific antibody specific for the transferrin receptor and pTau
(SEQ ID. NOs: 210, 211, 212).
[0055] FIG. 34: FACS binding of trispecific antibodies specific for
HER2 and transferrin receptor to Transferrin receptor expressing
(TfR+) BAF3 cells. "Herceptarg-8D3" (SEQ ID NOs.: 165, 163, 161)
and "Herceptarg (LALA)-8D3" (SEQ ID NOs.: 168, 169, 161) are
trispecific, trivalent antibodies specific for extracellular
domains II and IV of HER2 and the transferrin receptor. Positive
control: "Herceptarg-LALA" (SEQ ID NOs: 170, 169, 161) and
"Herceptarg" (SEQ ID NOs.: 171, 163, 161) are bispecific, bivalent
antibodies specific for extracellular domains II and IV of HER2.
Positive control "pTAU-8D3" is a bispecific antibody specific for
the transferrin receptor and pTau (SEQ ID. NOs: 210, 211, 212).
[0056] FIG. 35: Proliferation inhibition assay of trispecific
antibodies specific for HER2 and transferrin receptor.
Herceptarg-8D3'' (SEQ ID NOs.: 165, 163, 161) and "Herceptarg
(LALA)-8D3" (SEQ ID NOs.: 168, 169, 161) are trispecific, trivalent
antibodies specific for extracellular domains II and IV of HER2 and
the transferrin receptor. Positive control: "Herceptarg-LALA" (SEQ
ID NOs: 170, 169, 161) and "Herceptarg" (SEQ ID NOs.: 171, 163,
161) are bispecific, bivalent antibodies specific for extracellular
domains II and IV of HER2. Negative control "pTAU-8D3" is a
bispecific antibody specific for the transferrin receptor and pTau
(SEQ ID. NOs: 210, 211, 212).
[0057] FIG. 36: Herceptarg(LALA)+/-scFab(8D3) penetration from
vascular border into tumor tissue. Mean antibody signal in tumor
tissue as a function of distance from the nearest tumor vessel
shows that the brain shuttle (BS) increases penetration of
Herceptarg into the brain tumor.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
I. Definitions
[0058] Throughout the disclosure, the terms "ErbB2", "ErbB2
receptor", "c-Erb-B2", and "HER2" are used interchangeably, and,
unless otherwise indicated, refer to a native sequence ErbB2 human
polypeptide, or a functional derivative thereof. "ber2", "erbB2"
and "c-erb-B2" refer to the corresponding human gene.
[0059] Herein, "HER2 extracellular domain" or "HER2 ECD" refers to
a domain of HER2 that is outside of a cell, either anchored to a
cell membrane, or in circulation, including fragments thereof. The
amino acid sequence of HER2 is shown in FIG. 1 of WO2013055874. In
one embodiment, the extracellular domain of HER2 may comprise four
domains: "Domain I" (amino acid residues from about 1-195; SEQ ID
NO: 1 of WO2013055874), "Domain H" (amino acid residues from about
196-319; SEQ ID NO:2 of WO2013055874), "Domain III" (amino acid
residues from about 320-488: SEQ ID NO:3 of WO2013055874), and
"Domain IV" (amino acid residues from about 489-630; SEQ ID NO:4 of
WO2013055874) (residue numbering without signal peptide). See
Garrett et al. Mol. Cell. 11: 495-505 (2003), Cho et al. Nature
All: 756-760 (2003), Franklin et al. Cancer Cell 5:317-328 (2004),
and Plowman et al. Proc. Natl. Acad. Sci. 90:1746-1750 (1993), as
well as FIG. 6 in WO2013055874.
[0060] "Antigen binding site specific for extracellular domain II
of HER2" refers to the epitope of Pertuzumab (which is also known
as recombinant humanized monoclonal antibody 2C4 (rhuMAb 2C4)),
also depicted as "epitope 2C4". The "epitope 2C4" is the region in
the extracellular domain of HER2 to which the murine antibody 2C4
and Pertuzumab bind (see e.g. WO2013055874). In order to screen for
antibodies which bind essentially to the 2C4 epitope, a routine
cross-blocking assay such as that described in Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988), can be performed. Preferably the antibody blocks
2C4's binding to HER2 by about 50% or more. Alternatively, epitope
mapping can be performed to assess whether the antibody binds
essentially to the 2C4 epitope of HER2. Epitope 2C4 comprises
residues from Domain II (SEQ ID NO: 2 of WO2013055874) in the
extracellular domain of HER2. 2C4 and Pertuzumab binds to the
extracellular domain of HER2 at the junction of domains I, II and
III (SEQ ID NOs: 1, 2, and 3 of WO2013055874, respectively).
Franklin et al. Cancer Cell 5:317-328 (2004).
[0061] "Antigen binding site specific for extracellular domain IV
of HER2" refers to the epitope of Trastuzumab, also depicted as
"epitope 4D5". The "epitope 4D5" is the region in the extracellular
domain of HER2 to which the antibody 4D5 (ATCC CRL 10463) and
Trastuzumab bind. This epitope is close to the transmembrane domain
of HER2, and within Domain IV of HER2 (SEQ ID NO: 4 of
WO2013055874). To screen for antibodies which bind essentially to
the 4D5 epitope, a routine cross-blocking assay such as that
described in Antibodies, A Laboratory Manual, Cold Spring Harbor
Laboratory, Ed Harlow and David Lane (1988), can be performed.
Alternatively, epitope mapping can be performed to assess whether
the antibody binds essentially to the 4D5 epitope of HER2 {e.g. any
one or more residues in the region from about residue 529 to about
residue 625, inclusive of the HER2 ECD, residue numbering including
signal peptide).
[0062] The "blood-brain barrier" or "BBB" refers to the
physiological barrier between the peripheral circulation and the
brain and spinal cord which is formed by tight junctions within the
brain capillary endothelial plasma membranes, creating a tight
barrier that restricts the transport of molecules into the brain,
even very small molecules such as urea (60 Daltons). The BBB within
the brain, the blood-spinal cord barrier within the spinal cord,
and the blood-retinal barrier within the retina are contiguous
capillary barriers within the CNS, and are herein collectively
referred to an the blood-brain barrier or BBB. The BBB also
encompasses the blood-CSF barrier (choroid plexus) where the
barrier is comprised of ependymal cells rather than capillary
endothelial cells.
[0063] The term "blood brain barrier receptor" or "BBB-R" refers to
an extracellular membrane-linked receptor protein expressed on
brain endothelial cells which is capable of transporting molecules
across the BBB and can be used to transport exogenous administrated
molecules. Examples of BBB-R herein include: transferrin receptor
(TfR), insulin receptor, insulin-like growth factor receptor
(IGF-R), low density lipoprotein receptors including without
limitation low density lipoprotein receptor-related protein 1
(LRP1) and low density lipoprotein receptor-related protein 8
(LRP8), and heparin-binding epidermal growth factor-like growth
factor (HB-EGF). In one specific embodiment the BBB-R is a
transferrin receptor, preferably a human transferrin receptor
and/or a cynomolgous monkey transferrin receptor. The term
"transferrin receptor" or "TfR" refers to a transmembrane
glycoprotein (with a molecular weight of about 180,000) composed of
two disulphide-bonded sub-units (each of apparent molecular weight
of about 90,000) involved in iron uptake in vertebrates. In one
embodiment, the TfR herein is human TfR comprising the amino acid
sequence as in Schneider et al. Nature 311: 675-678 (1984). The TfR
mediates receptor-mediated transcytosis (RMT)
[0064] 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.
[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] 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.
[0067] An "affinity reduced" 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 a reduction in the
affinity of the antibody for antigen. Reduced affinity is
advantageous for the exposure of the trispecific antibody
specifically binding to HER2 and a blood-brain barrier receptor
(BBB-R) in the brain.
[0068] The terms "a bispecific HER2 antibody" and "a bispecific
antibody that specifically binds to HER2" are used interchangeably
and refer to a bispecific antibody that is capable of binding HER2
on both extracellular domains II and IV, respectively, with
sufficient affinity such that the antibody is useful as a
diagnostic and/or therapeutic agent in targeting cells expressing
HER2. In one embodiment, the extent of binding of a bispecific
antibody that specifically binds to HER2 on both extracellular
domains II and IV to an unrelated, non-HER2 protein is less than
about 10% of the binding of the antibody to HER2 as measured, e.g.,
by a Enzyme-linked immunosorbent assay (ELISA), surface plasmon
resonance (SPR) based assays (e.g. Biacore) or flow cytometry
(FACS). In certain embodiments, a bispecific antibody that
specifically binds to HER2 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.-13M).
[0069] The terms "a trispecific antibody binding to HER2 and a
blood-brain barrier receptor (BBB-R)" and "a trispecific antibody
that specifically binds to HER2 and BBB-R" are used interchangeably
and refer to a trispecific antibody that is capable of binding HER2
on both extracellular domains II and IV and a BBB-R, respectively,
with sufficient affinity such that the antibody is useful as a
diagnostic and/or therapeutic agent in targeting CNS cells
expressing HER2. In one embodiment, the extent of binding of a
trispecific antibody that specifically binds to HER2 on both
extracellular domains II and IV and a BBB-R to an unrelated,
non-HER2 or non BBB-R protein is less than about 10% of the binding
of the antibody to HER2 or a BBB-R as measured, e.g., by a
Enzyme-linked immunosorbent assay (ELISA), surface plasmon
resonance (SPR) based assays (e.g. Biacore) or flow cytometry
(FACS). In certain embodiments, the antigen binding site
specifically binding to a BBB-R of the trispecific antibody has an
off-rate of 0.07 to 0.005 l/s for the BBB-R, preferably as
determined by SPR.
[0070] 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., trispecific antibodies), and antibody fragments
so long as they exhibit the desired antigen-binding activity.
[0071] 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',
Fab'-SH, F(ab').sub.2; diabodies, cross-Fab fragments; 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.
[0072] 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. In one embodiment the
trispecific antibodies of the invention comprise at least one Fab
fragment, wherein either the variable regions or the constant
regions of the heavy and light chain are exchanged. Due to the
exchange of either the variable regions or the constant regions,
said 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 trispecific 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).
[0073] A "single chain Fab fragment" or "scFab" is a polypeptide
consisting of an antibody heavy chain variable domain (VH), an
antibody constant domain 1 (CH1), an antibody light chain variable
domain (VL), an antibody light chain constant domain (CL) and a
linker, wherein said antibody domains and said linker have one of
the following orders in N-terminal to C-terminal direction: a)
VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1
or d) VL-CH1-linker-VH-CL; and wherein said linker is a polypeptide
of at least 30 amino acids, preferably between 32 and 50 amino
acids. Said single chain Fab fragments a) VH-CH1-linker-VL-CL, b)
VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 and d)
VL-CH1-linker-VH-CL, are stabilized via the natural disulfide bond
between the CL domain and the CH1 domain. In addition, these single
chain Fab molecules might be further stabilized by generation of
interchain disulfide bonds via insertion of cysteine residues (e.g.
position 44 in the variable heavy chain and position 100 in the
variable light chain according to Kabat numbering). 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.
[0074] By "fused" or "connected" is meant that the components (e.g.
a Fab molecule and an Fc domain subunit) are linked by peptide
bonds, either directly or via one or more peptide linkers.
[0075] The term "linker" as used herein refers to a peptide linker
and is preferably 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.
[0076] The term "immunoglobulin molecule" refers to a protein
having the structure of a naturally occurring antibody. For
example, immunoglobulins of the IgG class are heterotetrameric
glycoproteins of about 150,000 daltons, composed of two light
chains and two heavy chains that are disulfide-bonded. From N- to
C-terminus, each heavy chain has a variable region (VH), also
called a variable heavy domain or a heavy chain variable domain,
followed by three constant domains (CHL CH2, and CH3), also called
a heavy chain constant region. Similarly, from N- to C-terminus,
each light chain has a variable region (VL), also called a variable
light domain or a light chain variable domain, followed by a
constant light (CL) domain, also called a light chain constant
region. The heavy chain of an immunoglobulin may be assigned to one
of five types, called .alpha. (IgA), .delta. (IgD), .epsilon.
(IgE), .gamma. (IgG), or .mu. (IgM), some of which may be further
divided into subtypes, e.g. .gamma..sub.1 (IgG.sub.1),
.gamma..sub.2 (IgG.sub.2), .gamma..sub.3 (IgG.sub.3), .gamma..sub.4
(IgG.sub.4), .alpha..sub.1 (IgA.sub.1) and .alpha..sub.2
(IgA.sub.2). The light chain of an immunoglobulin may be assigned
to one of two types, called kappa (.kappa.) and lambda (.lamda.),
based on the amino acid sequence of its constant domain. An
immunoglobulin essentially consists of two Fab molecules and an Fc
domain, linked via the immunoglobulin hinge region.
[0077] 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.
[0078] The term "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).
[0079] 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 rabbit 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.
[0080] 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.
[0081] "Effector functions" refer to those biological activities
attributable to the Fc region of an antibody, which vary with the
antibody isotype. Examples of antibody effector functions include:
C1q binding and complement dependent cytotoxicity (CDC); Fc
receptor binding; antibody-dependent cell-mediated cytotoxicity
(ADCC); antibody-dependent cellular phagocytosis (ADCP), cytokine
secretion, immune complex-mediated antigen uptake by antigen
presenting cells; down regulation of cell surface receptors (e.g. B
cell receptor); and B cell activation.
[0082] As used herein, the terms "engineer, engineered,
engineering", are considered to include any manipulation of the
peptide backbone or the post-translational modifications of a
naturally occurring or recombinant polypeptide or fragment thereof.
Engineering includes modifications of the amino acid sequence, of
the glycosylation pattern, or of the side chain group of individual
amino acids, as well as combinations of these approaches.
[0083] The term "amino acid mutation" as used herein is meant to
encompass amino acid substitutions, deletions, insertions, and
modifications. Any combination of substitution, deletion,
insertion, and modification can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics, e.g., reduced binding to an Fc receptor, or
increased association with another peptide. Amino acid sequence
deletions and insertions include amino- and/or carboxy-terminal
deletions and insertions of amino acids. Particular amino acid
mutations are amino acid substitutions. For the purpose of altering
e.g. the binding characteristics of an Fc region, non-conservative
amino acid substitutions, i.e. replacing one amino acid with
another amino acid having different structural and/or chemical
properties, are particularly preferred. Amino acid substitutions
include replacement by non-naturally occurring amino acids or by
naturally occurring amino acid derivatives of the twenty standard
amino acids (e.g. 4-hydroxyproline, 3-methylhistidine, ornithine,
homoserine, 5-hydroxylysine). Amino acid mutations can be generated
using genetic or chemical methods well known in the art. Genetic
methods may include site-directed mutagenesis, PCR, gene synthesis
and the like. It is contemplated that methods of altering the side
chain group of an amino acid by methods other than genetic
engineering, such as chemical modification, may also be useful.
Various designations may be used herein to indicate the same amino
acid mutation. For example, a substitution from proline at position
329 of the Fc domain to glycine can be indicated as 329G, G329,
G.sub.329, P329G, or Pro329Gly.
[0084] 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.
[0085] The term "Fc domain" or "Fc region" herein is used to define
a C-terminal region of an immunoglobulin heavy chain that contains
at least a portion of the constant region. The term includes native
sequence Fc regions and variant Fc regions. Although the boundaries
of the Fc region of an IgG heavy chain might vary slightly, the
human IgG heavy chain Fc region is usually defined to extend from
Cys226, or from Pro230, to the carboxyl-terminus of the heavy
chain. However, the C-terminal lysine (Lys447) of the Fc region may
or may not be present. Unless otherwise specified herein, numbering
of amino acid residues in the Fc region or constant region is
according to the EU numbering system, also called the EU index, as
described in Kabat et al., Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md., 1991. A "subunit" of an Fc domain as used
herein refers to one of the two polypeptides forming the dimeric Fc
domain, i.e. a polypeptide comprising C-terminal constant regions
of an immunoglobulin heavy chain, capable of stable
self-association. For example, a subunit of an IgG Fc domain
comprises an IgG CH2 ("first subunit") and an IgG CH3 ("second
subunit") constant domain.
[0086] A "modification promoting the association of the first and
the second subunit of the Fc domain" is a manipulation of the
peptide backbone or the post-translational modifications of an Fc
domain subunit that reduces or prevents the association of a
polypeptide comprising the Fc domain subunit with an identical
polypeptide to form a homodimer. A modification promoting
association as used herein particularly includes separate
modifications made to each of the two Fc domain subunits desired to
associate (i.e. the first and the second subunit of the Fc domain),
wherein the modifications are complementary to each other so as to
promote association of the two Fc domain subunits. For example, a
modification promoting association may alter the structure or
charge of one or both of the Fc domain subunits so as to make their
association sterically or electrostatically favorable,
respectively. Thus, (hetero)dimerization occurs between a
polypeptide comprising the first Fc domain subunit and a
polypeptide comprising the second Fc domain subunit, which might be
non-identical in the sense that further components fused to each of
the subunits (e.g. antigen binding moieties) are not the same. In
some embodiments the modification promoting association comprises
an amino acid mutation in the Fc domain, specifically an amino acid
substitution. In a particular embodiment, the modification
promoting association comprises a separate amino acid mutation,
specifically an amino acid substitution, in each of the two
subunits of the Fc domain.
[0087] "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.
[0088] The terms "full length antibody," "intact antibody," and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a native
antibody structure or having heavy chains that contain an Fc region
as defined herein.
[0089] 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.
[0090] 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. As also mentioned for chimeric and
humanized antibodies according to the invention the term "human
antibody" as used herein also comprises such antibodies which are
modified in the constant region to generate the properties
according to the invention, especially in regard to C1q binding
and/or FcR binding, e.g. by "class switching" i.e. change or
mutation of Fc parts (e.g. from IgG1 to IgG4 and/or IgG1/IgG4
mutation.)
[0091] 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.
[0092] 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.
[0093] 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.
[0094] 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).) Hypervariable regions (HVRs) are also referred to as
complementarity determining regions (CDRs), and these terms 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 A 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 A 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 A 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 A refers to the
CDRs as defined by Oxford Molecular's "AbM" antibody modeling
software.
[0095] 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.
[0096] 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.
[0097] An "immunoconjugate" is an antibody conjugated to one or
more heterologous molecule(s), including but not limited to a
cytotoxic agent.
[0098] 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.
[0099] 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).
[0100] 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.
[0101] "Isolated nucleic acid encoding a trispecific antibody that
specifically binds HER2 and a BBB-R" 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.
[0102] 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.
[0103] 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.
[0104] "Native antibodies" refer to naturally occurring
immunoglobulin molecules with varying structures. For example,
native IgG antibodies are heterotetrameric glycoproteins of about
150,000 daltons, composed of two identical light chains and two
identical heavy chains that are disulfide-bonded. From N- to
C-terminus, each heavy chain has a variable region (VH), also
called a variable heavy domain or a heavy chain variable domain,
followed by three constant domains (CHL CH2, and CH3). Similarly,
from N- to C-terminus, each light chain has a variable region (VL),
also called a variable light domain or a light chain variable
domain, followed by a constant light (CL) domain. The light chain
of an antibody may be assigned to one of two types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequence of
its constant domain.
[0105] 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.
[0106] "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
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.
[0107] "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.
[0108] 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.
[0109] 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.
[0110] 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.
[0111] 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.
[0112] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth.
[0113] A cancer cell with "HER receptor overexpression or
amplification" is one which has significantly higher levels of a
HER receptor protein or gene compared to a noncancerous cell of the
same tissue type. Such overexpression may be caused by gene
amplification or by increased transcription or translation. HER
receptor overexpression or amplification may be determined in a
diagnostic or prognostic assay by evaluating increased levels of
the HER protein present on the surface of a cell {e.g. via an
immunohistochemistry assay; IHC). Alternatively, or additionally,
one may measure levels of HER-encoding nucleic acid in the cell,
e.g. via in situ hybridization (ISH), including fluorescent in situ
hybridization (FISH; see WO98/45479 published October, 1998) and
chromogenic in situ hybridization (CISH; see, e.g. Tanner et al.,
Am. J. Pathol. 157(5): 1467-1472 (2000); Bella et al., J. Clin.
Oncol. 26: (May 20 suppl; abstr 22147) (2008)), southern blotting,
or polymerase chain reaction (PCR) techniques, such as quantitative
real time PCR (qRT-PCR). One may also study HER receptor
overexpression or amplification by measuring shed antigen {e.g.,
HER extracellular domain) in a biological fluid such as serum (see,
e.g., U.S. Pat. No. 4,933,294 issued Jun. 12, 1990; WO91/05264
published Apr. 18, 1991; U.S. Pat. No. 5,401,638 issued Mar. 28,
1995; and Sias et al. J. Immunol. Methods 132: 73-80 (1990)). Aside
from the above assays, various in vivo assays are available to the
skilled practitioner. For example, one may expose cells within the
body of the patient to an antibody which is optionally labeled with
a detectable label, e.g. a radioactive isotope, and binding of the
antibody to cells in the patient can be evaluated, e.g. by external
scanning for radioactivity or by analyzing a biopsy taken from a
patient previously exposed to the antibody.
[0114] A "HER2-positive" cancer comprises cancer cells which have
higher than normal levels of HER2. Examples of HER2-positive cancer
include HER2-positive breast cancer, HER2-positive gastric cancer
and HER2-positive ovarian cancer. Optionally, HER2-positive cancer
has an immunohistochemistry (IHC) score of 2+ or 3+ and/or an in
situ hybridization (ISH) amplification ratio >2.0.
[0115] "Metastatic" cancer refers to cancer which has spread from
one part of the body (e.g. the breast) to another part of the body.
"HER2-positive cancer with brain metastases" refers to
HER2-positive cancer which has spread from one part of the body
(e.g. the breast) to the central nervous system (CNS). 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).
[0116] The term "antigen-binding site of an antibody" when used
herein refer 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".
[0117] Antibody specificity refers to selective recognition of the
antibody for a particular epitope of an antigen. Natural
antibodies, for example, are monospecific. 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.
[0118] "Bispecific antibodies" according to the disclosure are
antibodies which have two different antigen-binding specificities.
Antibodies of the present invention are specific for two different
epitopes of HER2, i.e. the extracellular domains II and IV of HER2.
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.
[0119] "Trispecific antibodies" according to the disclosure are
antibodies which have three different antigen-binding
specificities. Antibodies of the present invention are specific for
two different epitopes of HER2, i.e. the extracellular domains II
and IV of HER2 and a BBB-R. The term "trispecific" antibody as used
herein denotes an antibody that has at least three binding sites
each of which bind to different epitopes.
[0120] Trispecific antibodies may also be used to localize
cytotoxic agents to cells which express HER2 and/or a BBB-R.
Trispecific antibodies can be prepared as full length antibodies or
antibody fragments.
[0121] 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 trispecific antibodies according to the invention are
at least "trivalent" and may be "multivalent" (e.g. "tetravalent"
or "hexavalent").
[0122] Antibodies of the present invention have three binding sites
and are trispecific. That is, the antibodies may be trispecific
even in cases where there are more than three binding sites (i.e.
that the antibody is multivalent). Trispecific antibodies of the
invention include, for example, multivalent single chain
antibodies, diabodies and triabodies, as well as antibodies having
the constant domain structure of full length antibodies to which
further antigen-binding sites (e.g., single chain Fv, a VH domain
and/or a VL domain, Fab, or (Fab)2) are linked via one or more
peptide-linkers. The antibodies can be full length from a single
species, or be chimerized or humanized.
[0123] 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."
[0124] The term "amino acid" as used within this application
denotes the group of naturally occurring carboxy a-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).
[0125] 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.
[0126] "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.
[0127] As used herein, the term "binding" or "specifically binding"
refers to the binding of the antibody to an epitope of the antigen
in an in-vitro assay, preferably in a surface plasmon resonance
assay (SPR, BIAcore, GE-Healthcare Uppsala, Sweden). The affinity
of the binding is defined by the terms ka (rate constant for the
association of the antibody from the antibody/antigen complex), kD
(dissociation constant), and KD (kD/ka). Binding or specifically
binding means a binding affinity (KD) of 10-8 mol/l or less,
preferably 10-9 M to 10-13 mol/l.
[0128] Binding of the antibody to HER2 or a BBB-R can be
investigated by a BIAcore assay (GE-Healthcare Uppsala, Sweden).
The affinity of the binding is defined by the terms ka (rate
constant for the association of the antibody from the
antibody/antigen complex), kD (dissociation constant), and KD
(kD/ka)
[0129] 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.
[0130] As used herein, the terms "engineer, engineered,
engineering," particularly with the prefix "glyco-," as well as the
term "glycosylation engineering" are considered to include any
manipulation of the glycosylation pattern of a naturally occurring
or recombinant polypeptide or fragment thereof. Glycosylation
engineering includes metabolic engineering of the glycosylation
machinery of a cell, including genetic manipulations of the
oligosaccharide synthesis pathways to achieve altered glycosylation
of glycoproteins expressed in cells. Furthermore, glycosylation
engineering includes the effects of mutations and cell environment
on glycosylation. In one embodiment, the glycosylation engineering
is an alteration in glycosyltransferase activity. In a particular
embodiment, the engineering results in altered
glucosaminyltransferase activity and/or fucosyltransferase
activity.
II. Compositions and Methods
[0131] In one aspect, the invention is based on trispecific
antibodies specifically binding to HER2 and a BBB-R. Antibodies of
the invention are useful, e.g., for the treatment or diagnosis of
cancer, in particular a HER2-positive cancer with brain metastases.
In one embodiment the trispecific antibody induces
complement-dependent cytotoxicity (CDC) to a higher degree than the
combination of Pertuzumab or Trastuzumab. In one such embodiment
the complement dependent cytotoxicity of the trispecific antibody
is determined by a LDH assay or a complement assay and compared to
the complement dependent cytotoxicity of the combination of
Pertuzumab and Trastuzumab as determined by the same assay. In one
embodiment the complement dependent cytotoxicity is determined in
vitro on cancer cells, preferably on breast cancer cells.
A. Exemplary BBB-R Binders Useful in the Trispecific Antibodies
Specifically Binding to HER2 and a BBB-R
[0132] In one aspect of the invention, a trispecific antibody
specifically binding to HER2 and a BBB-R is provided, wherein the
antibody comprises a first monovalent antigen binding site that
specifically binds the extracellular domain II of HER2, a second
monovalent antigen binding site that specifically binds the
extracellular domain IV of HER2 and a third monovalent antigen
binding site specifically binding to a BBB-R.
[0133] In one embodiment the BBB-R of the third antigen binding
site is selected from the group consisting of transferrin receptor
(TfR), insulin receptor, insulin-like growth factor receptor (IGF
receptor), low density lipoprotein receptor-related protein 8
(LRP8), low density lipoprotein receptor-related protein 1 (LRP1),
and heparin-binding epidermal growth factor-like growth factor
(HB-EGF). In one preferred embodiment the third antigen binding
site is specific for the transferrin receptor, preferably the human
transferrin receptor.
[0134] In one embodiment, the invention provides a trispecific
antibody that specifically binds to extracellular domains II and IV
of HER2 and to a BBB-R, comprising an antigen binding site that
specifically binds to an epitope in the transferrin receptor
comprised within the amino acid sequence of SEQ ID NO: 202, 203 or
204.
[0135] In one embodiment, the invention provides a trispecific
antibody that specifically binds to extracellular domains II and IV
of HER2 and to a BBB-R comprising
[0136] a heavy chain comprising
(a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 172; (b) a heavy chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 173; (c) a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 174;
[0137] and a light chain comprising
(a) a light chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 175; (b) a light chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 176; (c) a light chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 177.
[0138] In one embodiment, the invention provides a trispecific
antibody that specifically binds to extracellular domains II and IV
of HER2 and to a BBB-R comprising a variable light chain comprising
an amino acid sequence of SEQ ID NO: 178, a heavy chain comprising
a variable heavy chain comprising an amino acid sequence of SEQ ID
NO: 179
[0139] In one embodiment, the invention provides a trispecific
antibody that specifically binds to extracellular domains II and IV
of HER2 and to a BBB-R comprising a humanized light chain variable
domain derived from the variable light chain comprising an amino
acid sequence of SEQ ID NO: 178, a humanized heavy chain variable
domain derived from the heavy chain comprising a variable heavy
chain comprising an amino acid sequence of SEQ ID NO: 179.
[0140] In one embodiment, the invention provides a trispecific
antibody that specifically binds to extracellular domains II and IV
of HER2 and to a BBB-R comprising
[0141] a heavy chain comprising
(a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 180; (b) a heavy chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 181; (c) a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 182;
[0142] and a light chain comprising
(a) a light chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 183; (b) a light chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 184; (c) a light chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 185.
[0143] In one embodiment, the invention provides a trispecific
antibody that specifically binds to extracellular domains II and IV
of HER2 and to a BBB-R comprising a variable light chain comprising
an amino acid sequence of SEQ ID NO: 166, a heavy chain comprising
a variable heavy chain comprising an amino acid sequence of SEQ ID
NO: 167
[0144] In one embodiment, the invention provides a trispecific
antibody that specifically binds to extracellular domains II and IV
of HER2 and to a BBB-R comprising a humanized light chain variable
domain derived from the variable light chain comprising an amino
acid sequence of SEQ ID NO: 166, a humanized heavy chain variable
domain derived from the heavy chain comprising a variable heavy
chain comprising an amino acid sequence of SEQ ID NO: 167.
[0145] In one embodiment the monovalent antigen binding site
specifically binding to the human Transferrin receptor is further
optimized for improved brain exposure. Towards this end, the
affinity of the antigen binding site specifically binding to the
human Transferrin receptor is reduced by introducing certain
mutations into its amino acid sequence.
[0146] The equilibrium dissociation constants (KD) are commonly
used to describe molecular interactions. It is used as a measure of
two molecules' interaction strength (e.g. affinity) with each
other. Thus, the KD value is a measure for the strength of a
bimolecular interaction. However, the KD value as such does not
describe the kinetics of the molecular interaction, i.e. from the
KD value it cannot be deduced on the one hand how quickly the two
molecules bind to each other (association rate constant or "on
rate") and on the other hand how quickly the molecules dissociate
(dissociation rate constant or "off-rate"). Characterizing
bimolecular interactions only by their KD value neglects the fact
that an identical KD value can be made up by extremely different
(differing orders of magnitude) on and off-rates as the KD value is
the ratio thereof. The on and off-rates are important for
characterizing the binding behavior of molecules. The off-rate is
especially important because it characterizes the binding duration
of e.g. an antibody to its antigen. A long off-rate correlates to a
slow dissociation of the formed complex whereas a short off-rate
correlates to a quick dissociation.
[0147] In order to have a long-lasting (i.e. less frequent dosing
requiring) or a tailor-made (e.g. depending on the surrounding
conditions) interaction the off-rates have to be determined
experimentally. This is even more important as it is next to
impossible to predict the off-rate. Additionally the correlation
between off-rate and binding affinity is poor as outlined above.
For example, due to the fact that the KD value is the ratio of on
and off-rate even weak binders can stay bound long to their target
whereas tight binders can dissociate rapidly.
[0148] Herein are disclosed novel humanized and affinity reduced
anti-transferrin receptor binders that have an off-rate for binding
to the human transferrin receptor that is within a certain range in
order to ensure proper BBB shuttling. It has been found that this
range is defined at the one end by the off-rate of the murine
anti-transferrin receptor antibody 128.1 (WO93/10819) determined by
surface plasmon resonance for the cynomolgus transferrin receptor
and at the other end by 5% of that off-rate (i.e. a 20-times slower
dissociation). In one embodiment the monovalent antigen binding
site specifically binding to the human Transferrin receptor (huTfR)
and to the cynomolgus transferrin receptor (cyTfR) has an off-rate
determined by surface plasmon resonance for the cynomolgus
transferrin receptor between 0.1 l/s and 0.005 l/s.
[0149] In one embodiment the off-rate is determined at 500, 250,
125, 62.5, 31.25, 15.625 and 0 nM.
[0150] In one embodiment the off-rate is determined using a surface
plasmon resonance chip with a biotin surface and a running buffer
of 1.times.PBS supplemented with 250 mM sodium chloride at a flow
rate of 10 .mu.L/min. In one embodiment the association is
monitored for 180 seconds and the dissociation is monitored for 600
seconds. In one embodiment the off-rate is determined on a BIAcore
T200. In one embodiment the off-rate is between 0.08 l/s and 0.008
l/s. In one embodiment of all aspects the off-rate is determined at
25.degree. C.
[0151] In one embodiment, the invention provides a trispecific
antibody that specifically binds to extracellular domains II and IV
of HER2 and to a BBB-R comprising a humanized, affinity reduced
light chain variable domain derived from the variable light chain
comprising an amino acid sequence of SEQ ID NO: 178, a humanized,
affinity reduced heavy chain variable domain derived from the heavy
chain comprising a variable heavy chain comprising an amino acid
sequence of SEQ ID NO: 179.
[0152] In one embodiment, the invention provides a trispecific
antibody that specifically binds to extracellular domains II and IV
of HER2 and to a BBB-R comprising a humanized, affinity reduced
light chain variable domain derived from the variable light chain
comprising an amino acid sequence of SEQ ID NO: 166, a humanized,
affinity reduced heavy chain variable domain derived from the heavy
chain comprising a variable heavy chain comprising an amino acid
sequence of SEQ ID NO: 167.
[0153] In one embodiment, the invention provides a trispecific
antibody that specifically binds to extracellular domains II and IV
of HER2 and to a BBB-R comprising a heavy chain comprising
(a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 186; (b) a heavy chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 187; (c) a heavy chain CDR3 comprising the amino acid
sequence selected from the group of SEQ ID NO: 188, SEQ ID NO: 206
or SEQ ID NO: 174;
[0154] and a light chain comprising
(a) a light chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 189; (b) a light chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 190; (c) a light chain CDR3 of SEQ ID NO: 191.
[0155] In one embodiment, the invention provides a trispecific
antibody that specifically binds to extracellular domains II and IV
of HER2 and to a BBB-R comprising a variable light chain comprising
an amino acid sequence of SEQ ID NO: 192, a heavy chain comprising
a variable heavy chain comprising an amino acid sequence of SEQ ID
NO: 193 or SEQ ID NO: 205.
[0156] In one embodiment, the invention provides a trispecific
antibody that specifically binds to extracellular domains II and IV
of HER2 and to a BBB-R comprising a variable heavy chain comprising
an amino acid sequence selected from the group of SEQ ID NO: 194,
SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ
ID NO: 199, or SEQ ID NO: 200, and a variable light chain
comprising an amino acid sequence selected from the group of SEQ ID
NO: 201, SEQ ID NO: 207, SEQ ID NO: 208, or SEQ ID NO:209.
[0157] In one embodiment, the BBB-R antigen binding site comprises
one single chain Fab (scFab) which is connected to a full length
IgG antibody comprising the antigen binding sites specific for
extracellular domains II and IV of HER2, wherein the scFab is
connected either directly or by a linker to the C-terminal end of
the Fc part of one of the heavy chains of the IgG antibody. In
another embodiment the scFab is connected to the N-terminus of the
IgG antibody, e.g. to the N-terminus of the variable light chain or
heavy chain. In one embodiment the scFab is connected to the
N-terminus of the IgG antibody with a linker.
[0158] In one embodiment, the BBB-R antigen binding site comprises
one single chain Fv (scFv) which is connected to a full length IgG
antibody comprising the antigen binding sites specific for
extracellular domains II and IV of HER2, wherein the scFv is
connected either directly or by a linker to the C-terminal end of
the Fc part of one of the heavy chains of the IgG antibody. In
another embodiment the scFv is connected to the N-terminus of the
IgG antibody, e.g. to the N-terminus of the variable light chain or
heavy chain. In one embodiment the scFv is connected to the
N-terminus of the IgG antibody with a linker.
[0159] In one embodiment, the BBB-R antigen binding site comprises
one crossover Fab fragment which is connected to a full length IgG
antibody comprising the antigen binding sites specific for
extracellular domains II and IV of HER2, wherein the crossover Fab
fragment is connected either directly or by a linker to the
C-terminal end of the Fc part of one of the heavy chains of the IgG
antibody. In another embodiment the crossover Fab fragment is
connected to the N-terminus of the IgG antibody, e.g. to the
N-terminus of the variable light chain or heavy chain. In one
embodiment the crossover Fab fragment is connected to the
N-terminus of the IgG antibody with a linker. Crossover Fab
fragments are Fab fragments wherein either the variable regions or
the constant regions of the heavy and light chain are exchanged.
Bispecific antibody formats comprising crossover Fab fragments have
been described, for example, in WO2009080252, WO2009080253,
WO2009080251, WO2009080254, WO2010/136172, WO2010/145792 and
WO2013/026831.
[0160] In one embodiment, the BBB-R antigen binding site comprises
one Fab fragment which is connected to a full length IgG antibody
comprising the antigen binding sites specific for extracellular
domains II and IV of HER2, wherein the Fab fragment is connected
either directly or by a linker to the C-terminal end of the Fc part
of one of the heavy chains of the IgG antibody. In another
embodiment the Fab fragment is connected to the N-terminus of the
IgG antibody, e.g. to the N-terminus of the variable light chain or
heavy chain. In one embodiment the Fab fragment is connected to the
N-terminus of the IgG antibody with a linker.
[0161] The term "linker" denotes a chemical linker or a peptidic
linker that covalently connects BBB-R antigen binding site to the
full length IgG antibody comprising the antigen binding sites
specific for extracellular domains II and IV of HER2.
[0162] Single chain peptidic linkers, comprising of from one to
twenty amino acid residues joined by peptide bonds, can be used. In
certain embodiments, the amino acids are selected from the twenty
naturally-occurring amino acids. In certain other embodiments, one
or more of the amino acids are selected from glycine, alanine,
proline, asparagine, glutamine and lysine. In other embodiments,
the linker is a chemical linker. In certain embodiments, the linker
is a single chain peptidic linker with an amino acid sequence with
a length of at least 25 amino acid residues, in one preferred
embodiment with a length of 32 to 50 amino acid residues. In one
embodiment the peptidic linker is a (G.times.S)n linker with
G=glycine, S=serine, (x=3, n=8, 9 or 10) or (x=4 and n=6, 7 or 8),
in one embodiment with x=4, n=6 or 7, in one preferred embodiment
with x=4, n=7. In one embodiment the linker is (G4S)4. In one
embodiment the linker is (G4S)6G2.
[0163] Conjugation may be performed using a variety of chemical
linkers. For example, the BBB-R antigen binding site may be
connected to the full length IgG antibody comprising the antigen
binding sites specific for extracellular domains II and IV of HER2
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-mal eimidomethyl) 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). The linker may be a "cleavable
linker" facilitating release of the full length IgG antibody
comprising the antigen binding sites specific for extracellular
domains II and IV of HER2 upon delivery to the brain. For example,
an acid-labile linker, peptidase-sensitive linker, photolabile
linker, dimethyl linker or disulfide-containing linker (Chari et
al, Cancer Res. 52 (1992) 127-131; U.S. Pat. No. 5,208,020) may be
used.
[0164] Covalent conjugation can either be direct or via a linker.
In certain embodiments, direct conjugation is by construction of a
polypeptide fusion (i.e. by genetic fusion). In certain
embodiments, direct conjugation is by formation of a covalent bond
between a reactive group on one of the two portions of the BBB-R
antigen binding site and a corresponding group or acceptor on the
full length IgG antibody comprising the antigen binding sites
specific for extracellular domains II and IV of HER2. In certain
embodiments, direct conjugation is by modification (i.e. genetic
modification) of one of the two molecules to be conjugated to
include a reactive group (as non-limiting examples, a sulfhydryl
group or a carboxyl group) that forms a covalent attachment to the
other molecule to be conjugated under appropriate conditions.
Conjugation may also be performed using a variety of linkers. For
example, the BBB-R antigen binding site and the full length IgG
antibody comprising the antigen binding sites specific for
extracellular domains II and IV of HER2 may be conjugated 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). Peptidic linkers, comprised of
from one to twenty amino acid residues joined by peptide bonds, may
also be used. In certain such embodiments, the amino acid residues
are selected from the twenty naturally-occurring amino acids. In
certain other such embodiments, one or more of the amino acid
residues are selected from glycine, alanine, proline, asparagine,
glutamine and lysine. The linker may be a "cleavable linker"
facilitating release of the full length IgG antibody comprising the
antigen binding sites specific for extracellular domains II and IV
of HER2 upon delivery to the brain. For example, an acid-labile
linker, peptidase-sensitive linker, photolabile linker, dimethyl
linker or disulfide-containing linker (Chari et al, Cancer Res. 52
(1992) 127-131; U.S. Pat. No. 5,208,020) may be used. The above
disclosed antigen binding site specifically binding to a BBB-R as
outlined in this section are part of trispecific antibodies
specifically binding to HER2 and a BBR-R. The antigen binding sites
for HER2 as well as the antibody formats of the HER2 binders are
described in sections II B and II C below.
B. Exemplary Trispecific Antibodies Specifically Binding to HER2
and a BBB-R with a Common Light Chain
[0165] In one aspect of the invention, a trispecific antibody
specifically binding to HER2 and a BBB-R is provided, wherein the
antibody comprises a first monovalent antigen binding site that
specifically binds the extracellular domain II of HER2, a second
monovalent antigen binding site that specifically binds the
extracellular domain IV of HER2 and a third monovalent antigen
binding site specifically binding to a BBB-R as outlined in section
II A above. The inventors of the present invention surprisingly
generated a trispecific antibody wherein both binding moieties
(i.e. antigen binding sites) that specifically bind to HER2 share a
common light chain that retains the efficacy of the parent
monospecific antibodies in terms of inhibition of tumor cell
proliferation and has an increased affinity to the extracellular
domain II of HER2. The generation of a trispecific molecule with a
common light chain that retains the binding properties of both of
the parent antibodies is not straight-forward as the common CDRs of
the hybrid light chain have to retain the binding specificity for
both the extracellular domains II and IV of HER2. The use of this
so-called `common light chain` principle, i.e. combining two
binders that share one light chain but still have separate
specificities, prevents light chain mispairing and in this
particular case retains the epitope specificity of the parental
antibodies. As a consequence, there are less side products during
production, facilitating the homogenous preparation of HER2
trispecific antigen binding molecules at high yields. The heavy
chain of Pertuzumab was further optimized, resulting in a more
potent molecule in terms of affinity to the extracellular domain II
of HER2. In addition, the Trastuzumab heavy chain has been
stabilized by introducing certain mutations into the CDRs. The
resulting molecule is superior to the parent Pertuzumab and
Trastuzumab monospecific antibodies, plus targets the CNS through
its BBB-R binding moiety. The trispecific HER2 antibodies in the
monovalent common light chain format have an increased affinity to
the pertuzumab epitope, and show superior inhibitory effects on
cell proliferation as compared to the combination of the parental
antibodies.
[0166] In one embodiment a trispecific antibody is provided,
comprising a first Fab molecule capable of specific binding to
extracellular domain II of HER2 and a second Fab molecule capable
of specific binding to extracellular domain IV of HER2, wherein the
sequence of the variable light chain of the first Fab molecule is
identical to the sequence of the variable light chain of the second
Fab molecule (i.e. the first and the second Fab molecule comprise a
common light chain); and a third monovalent antigen binding site
specifically binding to a BBB-R as outlined in section II A
above.
[0167] In one embodiment, the invention provides a trispecific
antibody that specifically binds to extracellular domains II and IV
of HER2 and to a BBB-R comprising a first heavy chain
comprising
(a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 55; (b) a heavy chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 77; (c) a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 56;
[0168] and a second heavy chain comprising
(a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 20; (b) a heavy chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 29; (c) a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 30;
[0169] and a first and a second light chain comprising
(a) a light chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 89; (b) a light chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 90; (c) a light chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 19.
[0170] In one embodiment, the invention provides a trispecific
antibody that specifically binds to extracellular domains II and IV
of HER2 and to a BBB-R comprising a first heavy chain
comprising
(a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 14; (b) a heavy chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 60; (c) a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 16;
[0171] and a second heavy chain comprising
(a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 20; (b) a heavy chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 29; (c) a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 30;
[0172] and a first and a second light chain comprising
(a) a light chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 89; (b) a light chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 90; (c) a light chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 19.
[0173] In one embodiment, the invention provides a trispecific
antibody that specifically binds to extracellular domains II and IV
of HER2 and to a BBB-R comprising a first heavy chain
comprising
(a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 58; (b) a heavy chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 15; (c) a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 59;
[0174] and a second heavy chain comprising
(a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 20; (b) a heavy chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 29; (c) a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 30;
[0175] and a first and a second light chain comprising
(a) a light chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 89; (b) a light chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 90; (c) a light chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 19.
[0176] In one embodiment the second heavy chain of any of the
embodiments above bears at least one modification in the amino acid
sequence that confers higher chemical stability to the CDRs,
resulting in retained binding to HER2 under stress conditions.
Modifications useful herein are e.g. D98E, D98N, D98T, G99A or
G99S. Surprisingly the inventors found that some modifications of
the CDRs did not only improve the stability of the molecule but
also improved the binding affinity to HER2.
[0177] Hence in one embodiment, the invention provides a tri
specific antibody that specifically binds to extracellular domains
II and IV of HER2 and to a BBB-R comprising
[0178] a first heavy chain comprising
(a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 55; (b) a heavy chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 77; (c) a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 56;
[0179] and a second heavy chain comprising
(a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 20; (b) a heavy chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 29; (c) a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 79;
[0180] and a first and a second light chain comprising
(a) a light chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 89; (b) a light chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 90; (c) a light chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 19.
[0181] In one embodiment, the invention provides a trispecific
antibody that specifically binds to extracellular domains II and IV
of HER2 and to a BBB-R comprising
[0182] a first heavy chain comprising
(a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 14; (b) a heavy chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 60; (c) a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 16;
[0183] and a second heavy chain comprising
(a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 20; (b) a heavy chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 29; (c) a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 79;
[0184] and a first and a second light chain comprising
(a) a light chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 89; (b) a light chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 90; (c) a light chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 19.
[0185] In one embodiment, the invention provides a trispecific
antibody that specifically binds to extracellular domains II and IV
of HER2 and to a BBB-R comprising
[0186] a first heavy chain comprising
(a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 58; (b) a heavy chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 15; (c) a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 59;
[0187] and a second heavy chain comprising
(a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 20; (b) a heavy chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 29; (c) a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 79;
[0188] and a first and a second light chain comprising
(a) a light chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 89; (b) a light chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 90; (c) a light chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 19.
[0189] In one embodiment, the trispecific antibody comprises two
variable light chains comprising an amino acid sequence of SEQ ID
NO 54 (i.e. a common light chain), a first heavy chain comprising a
variable heavy chain comprising an amino acid sequence of SEQ ID
NO: 64, and a second heavy chain comprising a variable heavy chain
comprising an amino acid sequence of SEQ ID NO 92 and a third
monovalent antigen binding site specifically binding to a BBB-R as
outlined in section II A above.
[0190] In one embodiment, the trispecific antibody comprises two
variable light chains comprising an amino acid sequence of SEQ ID
NO 54 (i.e. a common light chain), a first heavy chain comprising a
variable heavy chain comprising an amino acid sequence of SEQ ID
NO: 70, and a second heavy chain comprising a variable heavy chain
comprising an amino acid sequence of SEQ ID NO 92 and a third
monovalent antigen binding site specifically binding to a BBB-R as
outlined in section II A above.
[0191] In one embodiment, the trispecific antibody comprises two
variable light chains comprising an amino acid sequence of SEQ ID
NO 54 (i.e. a common light chain), a first heavy chain comprising a
variable heavy chain comprising an amino acid sequence of SEQ ID
NO: 68, and a second heavy chain comprising a variable heavy chain
comprising an amino acid sequence of SEQ ID NO: 92 and a third
monovalent antigen binding site specifically binding to a BBB-R as
outlined in section II A above.
[0192] In one embodiment the second heavy chain of any of the
embodiments above bears at least one modification in the amino acid
sequence that confers stability to the CDRs and the binding to the
target, e.g. D98E, D98N, D98T, G99A or G99S.
[0193] Hence in one embodiment, the trispecific antibody comprises
two variable light chains comprising an amino acid sequence of SEQ
ID NO: 54 (i.e. a common light chain), a first heavy chain
comprising a variable heavy chain comprising an amino acid sequence
of SEQ ID NO: 64, and a second heavy chain comprising a variable
heavy chain comprising an amino acid sequence of SEQ ID NO: 117 and
a third monovalent antigen binding site specifically binding to a
BBB-R as outlined in section II A above.
[0194] In one embodiment, the trispecific antibody comprises two
variable light chains comprising an amino acid sequence of SEQ ID
NO:54 (i.e. a common light chain), a first heavy chain comprising a
variable heavy chain comprising an amino acid sequence of SEQ ID
NO: 70, and a second heavy chain comprising a variable heavy chain
comprising an amino acid sequence of SEQ ID NO: 117 and a third
monovalent antigen binding site specifically binding to a BBB-R as
outlined in section II A above.
[0195] In one embodiment, the trispecific antibody comprises two
variable light chains comprising an amino acid sequence of SEQ ID
NO:54 (i.e. a common light chain), a first heavy chain comprising a
variable heavy chain comprising an amino acid sequence of SEQ ID
NO: 68, and a second heavy chain comprising a variable heavy chain
comprising an amino acid sequence of SEQ ID NO: 117 and a third
monovalent antigen binding site specifically binding to a BBB-R as
outlined in section II A above.
[0196] In one embodiment, the trispecific antibody of the invention
comprises a first heavy chain constant region comprising the amino
acid sequence of SEQ ID NO: 114.
[0197] In one embodiment, the trispecific antibody of the invention
comprises a second heavy chain constant region comprising the amino
acid sequence of SEQ ID NO: 115.
[0198] In another embodiment the trispecific antibody of the
invention comprises a first light chain constant region comprising
the amino acid sequence of SEQ ID NO: 113.
[0199] In another embodiment the trispecific antibody of the
invention comprises a second light chain constant region comprising
the amino acid sequence of SEQ ID NO: 116.
[0200] In one embodiment a trispecific antibody is provided
comprising SEQ ID NOs: 165, 163 and 161.
[0201] In one embodiment a trispecific antibody is provided
comprising SEQ ID NOs: 168, 169 and 161.
[0202] In one embodiment the trispecific antibody of any of the
above embodiments comprises a Fc domain modification that promotes
heterodimerization as outlined in section D below.
C. Exemplary Trispecific Antibodies Specifically Binding to HER2
and a BBB-R Comprising a Crossover Fab Fragment
[0203] In one embodiment of the invention, a trispecific antibody
specifically binding to HER2 and a BBB-R is provided, wherein the
antibody comprises a first monovalent antigen binding site that
specifically binds to the extracellular domain II of HER2, a second
monovalent antigen binding site that specifically binds to the
extracellular domain IV of HER2 and a third monovalent antigen
binding site specifically binding to a BBB-R as outlined in section
II A above. The inventors of the present invention generated a
second trispecific antibody format wherein one of the binding
moieties (i.e. antigen binding sites) for HER2 is a crossover Fab
fragment. In one aspect of the invention a trispecific antibody is
provided, comprising an IgG molecule, wherein one of the Fab
fragments is replaced by a crossover Fab fragment. Crossover Fab
fragments are Fab fragments wherein either the variable regions or
the constant regions of the heavy and light chain are exchanged.
Bispecific antibody formats comprising crossover Fab fragments have
been described, for example, in WO2009080252, WO2009080253,
WO2009080251, WO2009080254, WO2010/136172, WO2010/145792 and
WO2013/026831. The native Trastuzumab sequence has been optimized
by introducing modifications into the CDRs of both the variable
heavy chain and the variable light chain to improve stability and
affinity, the resulting sequences framework-grafted to avoid
mispairing of the light chains in the trispecific molecule,
resulting in highly potent trispecific antibodies that target HER2
and a BBB-R that can be produced with high yield and only low
percentage of side products. In addition it shows superior
inhibition of tumor cell proliferation as compared to the
combination of the respective parental antibodies.
[0204] In one embodiment, the invention provides a trispecific
antibody specifically binding to HER2 and a BBB-R comprising
a first antigen binding site specific for extracellular domain II
of HER2, comprising (a) a heavy chain CDR1 comprising the amino
acid sequence of SEQ ID NO: 14; (b) a heavy chain CDR2 comprising
the amino acid sequence of SEQ ID NO: 15; (c) a heavy chain CDR3
comprising the amino acid sequence of SEQ ID NO: 16; (d) a light
chain CDR1 comprising the amino acid sequence of SEQ ID NO: 11; (e)
a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:
12; (f) a light chain CDR3 comprising the amino acid sequence of
SEQ ID NO: 13;
[0205] And a second antigen binding site specific for extracellular
domain IV of HER2 comprising
(a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 20; (b) a heavy chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 108; (c) a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 79; (d) a light chain CDR1 comprising the
amino acid sequence of SEQ ID NO: 107; (e) a light chain CDR2
comprising the amino acid sequence of SEQ ID NO: 18; (f) a light
chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19;
[0206] In one embodiment, the trispecific antibody comprises a
first antigen binding site specific for the extracellular domain II
of HER2 comprising a variable heavy chain comprising an amino acid
sequence of SEQ ID NO: 22 and a variable light chain comprising an
amino acid sequence of SEQ ID NO: 24; and a second antigen binding
site specific for the extracellular domain IV of HER2, comprising a
heavy chain variable region comprising an amino acid sequence of
SEQ ID NO: 105 and a light chain variable region comprising an
amino acid sequence of SEQ ID NO: 106, and a third monovalent
antigen binding site specifically binding to a BBB-R as outlined in
section II A above.
[0207] In one embodiment, the invention provides a trispecific
antibody that specifically binds to HER2 and a BBB-R comprising
[0208] a first antigen binding site specific for extracellular
domain II of HER2 comprising
[0209] (a) a heavy chain CDR1 comprising the amino acid sequence of
SEQ ID NO: 14;
[0210] (b) a heavy chain CDR2 comprising the amino acid sequence of
SEQ ID NO: 15;
[0211] (c) a heavy chain CDR3 comprising the amino acid sequence of
SEQ ID NO: 16;
[0212] (d) a light chain CDR1 comprising the amino acid sequence of
SEQ ID NO: 11;
[0213] (e) a light chain CDR2 comprising the amino acid sequence of
SEQ ID NO: 12;
[0214] (f) a light chain CDR3 comprising the amino acid sequence of
SEQ ID NO: 13.
[0215] and a second antigen binding site specific for extracellular
domain IV of HER2, comprising
[0216] (a) a heavy chain CDR1 comprising the amino acid sequence of
SEQ ID NO: 20;
[0217] (b) a heavy chain CDR2 comprising the amino acid sequence of
SEQ ID NO: 29;
[0218] (c) a heavy chain CDR3 comprising the amino acid sequence of
SEQ ID NO: 79;
[0219] (d) a light chain CDR1 comprising the amino acid sequence of
SEQ ID NO: 104;
[0220] (e) a light chain CDR2 comprising the amino acid sequence of
SEQ ID NO: 18;
[0221] (f) a light chain CDR3 comprising the amino acid sequence of
SEQ ID NO: 19.
[0222] In one embodiment, the trispecific antibody comprises a
first antigen binding site specific for the extracellular domain II
of HER2 comprising a variable heavy chain comprising an amino acid
sequence of SEQ ID NO: 22 and a variable light chain comprising an
amino acid sequence of SEQ ID NO: 24; and a second antigen binding
site specific for the extracellular domain IV of
[0223] HER2, comprising a heavy chain variable region comprising an
amino acid sequence of SEQ ID NO: 117 and a light chain variable
region comprising an amino acid sequence of SEQ ID NO: 118, and a
third monovalent antigen binding site specifically binding to a
BBB-R as outlined in section II A above.
[0224] In one embodiment, the invention provides a trispecific
antibody that specifically binds to HER2 and a BBB-R comprising
[0225] a first antigen binding site specific for extracellular
domain II of HER2 comprising
[0226] (a) a heavy chain CDR1 comprising the amino acid sequence of
SEQ ID NO: 14;
[0227] (b) a heavy chain CDR2 comprising the amino acid sequence of
SEQ ID NO: 15;
[0228] (c) a heavy chain CDR3 comprising the amino acid sequence of
SEQ ID NO: 16;
[0229] (d) a light chain CDR1 comprising the amino acid sequence of
SEQ ID NO: 11;
[0230] (e) a light chain CDR2 comprising the amino acid sequence of
SEQ ID NO: 12;
[0231] (f) a light chain CDR3 comprising the amino acid sequence of
SEQ ID NO: 13.
and a second antigen binding site specific for extracellular domain
IV of HER2, comprising
[0232] (a) a heavy chain CDR1 comprising the amino acid sequence of
SEQ ID NO: 20;
[0233] (b) a heavy chain CDR2 comprising the amino acid sequence of
SEQ ID NO: 29;
[0234] (c) a heavy chain CDR3 comprising the amino acid sequence
selected from the group consisting of SEQ ID NO: 79, SEQ ID NO: 78,
SEQ ID NO: 80, SEQ ID NO: 87, SEQ ID NO: 88;
[0235] (d) a light chain CDR1 comprising the amino acid sequence
selected from the group consisting of SEQ ID NO: 104, SEQ ID NO:
103 and SEQ ID NO: 158;
[0236] (e) a light chain CDR2 comprising the amino acid sequence of
SEQ ID NO: 18;
[0237] (f) a light chain CDR3 comprising the amino acid sequence of
SEQ ID NO: 19;
[0238] In one embodiment, the trispecific antibody of the invention
comprises a first heavy chain constant region comprising the amino
acid sequence of SEQ ID NO: 114.
[0239] In one embodiment, the trispecific antibody of the invention
comprises a first heavy chain constant region comprising the amino
acid sequence of SEQ ID NO: 114, wherein the C-terminal Lysine has
been removed.
[0240] In one embodiment, the trispecific antibody of the invention
comprises a second heavy chain constant region comprising the amino
acid sequence of SEQ ID NO: 115.
[0241] In one embodiment, the trispecific antibody of the invention
comprises a second heavy chain constant region comprising the amino
acid sequence of SEQ ID NO: 115, wherein the C-terminal Lysine has
been removed.
[0242] In another embodiment the trispecific antibody of the
invention comprises a first light chain constant region comprising
the amino acid sequence of SEQ ID NO: 113.
[0243] In another embodiment the trispecific antibody of the
invention comprises a second light chain constant region comprising
the amino acid sequence of SEQ ID NO: 116.
[0244] In one embodiment a trispecific antibody is provided
comprising SEQ ID NOs: 109, 110, 111 and 112.
[0245] In one embodiment a trispecific antibody that specifically
binds to HER2 and a BBB-R according to any of the above embodiments
comprises
[0246] an Fc domain,
[0247] one Fab fragment comprising a first antigen binding site
specific for the extracellular domain II of HER2,
[0248] and one Fab fragment comprising a second antigen binding
site specific for the extracellular domain IV of HER2,
wherein either the variable regions or the constant regions of the
heavy and light chain of at least one Fab fragment are exchanged
and a third monovalent antigen binding site specifically binding to
a BBB-R as outlined in section II A above.
[0249] The triispecific antibodies described in this section are
monovalent for the extracellular domain II of HER2 and monovalent
for the extracellular domain IV of HER2. Due to the exchange of
either the variable regions or the constant regions, the Fab
fragment above is also referred to as "cross-Fab fragment" or "xFab
fragment" or "crossover Fab fragment".
[0250] In one embodiment a trispecific antibody that specifically
binds to HER2 and a BBB-R according to any of the above embodiments
comprises
[0251] an Fc domain,
[0252] one Fab fragment comprising an antigen binding site specific
for the extracellular domain II of HER2, wherein either the
variable regions or the constant regions of the heavy and light
chain are exchanged.
[0253] and one Fab fragment comprising an antigen binding site
specific for the extracellular domain IV of HER2, and
a third monovalent antigen binding site specifically binding to a
BBB-R as outlined in section II A above.
[0254] In one embodiment a tri specific antibody that specifically
binds to HER2 and a BBB-R according to any of the above embodiments
comprises
[0255] an Fc domain,
[0256] one Fab fragment comprising an antigen binding site specific
for the extracellular domain II of HER2,
[0257] and one Fab fragment comprising an antigen binding site
specific for the extracellular domain IV of HER2, wherein either
the variable regions or the constant regions of the heavy and light
chain are exchanged, and
a third monovalent antigen binding site specifically binding to a
BBB-R as outlined in section II A above.
[0258] In one embodiment a tri specific antibody that specifically
binds to HER2 and a BBB-R according to any of the above embodiments
comprises
[0259] an Fc domain,
[0260] one Fab fragment comprising an antigen binding site specific
for the extracellular domain II of HER2,
[0261] and one Fab fragment comprising an antigen binding site
specific for the extracellular domain IV of HER2, wherein the
variable regions of the heavy and light chain of the Fab fragment
are exchanged;
and a third monovalent antigen binding site specifically binding to
a BBB-R as outlined in section II A above.
[0262] In one embodiment a trispecific antibody that specifically
binds to HER2 and a BBB-R according to any of the above embodiments
comprises
[0263] an Fc domain,
[0264] one Fab fragments comprising an antigen binding site
specific for the extracellular domain II of HER2,
[0265] and one Fab fragment comprising an antigen binding site
specific for the extracellular domain IV of HER2, wherein the
constant regions of the heavy and light chain are exchanged, and a
third monovalent antigen binding site specifically binding to a
BBB-R as outlined in section II A above.
[0266] In one embodiment said trispecific antibody that
specifically binds to HER2 and a BBB-R according to any of the
above embodiments comprises an Fc domain to which two Fab fragments
are fused to the N-terminus, wherein either the variable regions or
the constant regions of the heavy and light chain of at least one
Fab fragment are exchanged and a third monovalent antigen binding
site specifically binding to a BBB-R as outlined in section II A
above. In one embodiment the two Fab fragments are fused to the
N-terminus of the Fc domain through an immunoglobulin hinge region.
In one embodiment, the immunoglobulin hinge region is a human IgG1
hinge region. In one embodiment the Fab fragment comprising an
antigen binding site specific for the extracellular domain II of
HER2, the Fab fragment comprising an antigen binding site specific
for the extracellular domain IV of HER2 and the Fc domain are part
of an immunoglobulin molecule. In a particular embodiment the
immunoglobulin molecule is an IgG class immunoglobulin. In an even
more particular embodiment the immunoglobulin is an IgG1 subclass
immunoglobulin. In another embodiment the immunoglobulin is an IgG4
subclass immunoglobulin. In a further particular embodiment the
immunoglobulin is a human immunoglobulin. In other embodiments the
immunoglobulin is a chimeric immunoglobulin or a humanized
immunoglobulin.
[0267] In one embodiment a trispecific antibody that specifically
binds to HER2 and a BBB-R according to any of the above embodiments
comprises an Immunoglobulin G (IgG) molecule with one binding site
specific for the extracellular domain II of HER2 and one binding
site specific for the extracellular domain IV of HER2, wherein
either the variable regions or the constant regions of the heavy
and light chain of one arm (Fab fragment) of the IgG molecule are
exchanged, and a third monovalent antigen binding site specifically
binding to a BBB-R as outlined in section II A above.
[0268] In one embodiment a trispecific antibody that specifically
binds to HER2 and a BBB-R according to any of the above embodiments
comprises an Immunoglobulin G (IgG) molecule with one binding site
specific for the extracellular domain II of HER2 and one binding
site specific for the extracellular domain IV of HER2, wherein the
variable regions of the heavy and light chain of one arm (Fab
fragment) of the IgG molecule are exchanged, and a third monovalent
antigen binding site specifically binding to a BBB-R as outlined in
section II A above. In one embodiment the variable regions of the
heavy and light chain of the one arm (Fab fragment) of the IgG
molecule which comprises the binding site specific for the
extracellular domain IV of HER2 are exchanged.
[0269] In one embodiment a trispecific antibody that specifically
binds to HER2 and a BBB-R according to any of the above embodiments
comprises an Immunoglobulin G (IgG) molecule with one binding site
specific for the extracellular domain II of HER2 and one binding
site specific for the extracellular domain IV of HER2, wherein the
constant regions of the heavy and light chain of one arm (Fab
fragment) of the IgG molecule are exchanged, and a third monovalent
antigen binding site specifically binding to a BBB-R as outlined in
section II A above. In one embodiment the constant regions of the
heavy and light chain of the one arm (Fab fragment) of the IgG
molecule which comprises the binding site specific for the
extracellular domain IV of HER2 are exchanged.
[0270] In one embodiment a trispecific antibody that specifically
binds to HER2 and a BBB-R according to any of the above embodiments
comprises an Immunoglobulin G (IgG) molecule with one binding site
specific for the extracellular domain II of HER2 and one binding
site specific for the extracellular domain IV of HER2, wherein the
complete VH-CH1 and VL-CL domains of one arm (Fab fragment) of the
IgG molecule are exchanged; and a third monovalent antigen binding
site specifically binding to a BBB-R as outlined in section II A
above.
[0271] This means that at least one of the Fab fragments is fused
to the N-terminus of the Fc domain via the light chain (VLCL). In
one embodiment the other Fab fragment is fused to the N-terminus of
the Fc domain via the heavy chain (VHCH1). In one embodiment both
Fab fragments are fused to the N-terminus of the Fc domain through
an immunoglobulin hinge region.
[0272] In one embodiment the trispecific antibody of any of the
above embodiments comprises a Fc domain modification that promotes
heterodimerization as outlined in section D below.
D. Fc Domain Modifications Promoting Heterodimerization
[0273] The trispecific antibodies binding to HER2 and a blood-brain
barrier receptor (BBB-R) of the invention comprise different
antigen binding moieties, fused to one or the other of the two
subunits of the Fc domain, thus the two subunits of the Fc domain
are typically comprised in two non-identical polypeptide chains.
Recombinant co-expression of these polypeptides and subsequent
dimerization leads to several possible combinations of the two
polypeptides. To improve the yield and purity of the antibodies of
the invention in recombinant production, it will thus be
advantageous to introduce in the Fc domain of the trispecific
antibodies of the invention a modification promoting the
association of the desired polypeptides.
[0274] Accordingly, in particular embodiments the Fc domain of the
trispecific antibodies of the invention comprises a modification
promoting the association of the first and the second subunit of
the Fc domain. The site of most extensive protein-protein
interaction between the two subunits of a human IgG Fc domain is in
the CH3 domain of the Fc domain. Thus, in one embodiment said
modification is in the CH3 domain of the Fc domain.
[0275] In a specific embodiment said modification is a so-called
"knob-into-hole" modification, comprising a "knob" modification in
one of the two subunits of the Fc domain and a "hole" modification
in the other one of the two subunits of the Fc domain.
[0276] The knob-into-hole technology is described e.g. in U.S. Pat.
No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9,
617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).
Generally, the method involves introducing a protuberance ("knob")
at the interface of a first polypeptide and a corresponding cavity
("hole") in the interface of a second polypeptide, such that the
protuberance can be positioned in the cavity so as to promote
heterodimer formation and hinder homodimer formation. Protuberances
are constructed by replacing small amino acid side chains from the
interface of the first polypeptide with larger side chains (e.g.
tyrosine or tryptophan). Compensatory cavities of identical or
similar size to the protuberances are created in the interface of
the second polypeptide by replacing large amino acid side chains
with smaller ones (e.g. alanine or threonine).
[0277] Accordingly, in a particular embodiment, in the CH3 domain
of the first subunit of the Fc domain of the trispecific antibodies
of the invention an amino acid residue is replaced with an amino
acid residue having a larger side chain volume, thereby generating
a protuberance within the CH3 domain of the first subunit which is
positionable in a cavity within the CH3 domain of the second
subunit, and in the CH3 domain of the second subunit of the Fc
domain an amino acid residue is replaced with an amino acid residue
having a smaller side chain volume, thereby generating a cavity
within the CH3 domain of the second subunit within which the
protuberance within the CH3 domain of the first subunit is
positionable.
[0278] The protuberance and cavity can be made by altering the
nucleic acid encoding the polypeptides, e.g. by site-specific
mutagenesis, or by peptide synthesis.
In a specific embodiment, in the CH3 domain of the first subunit of
the Fc domain the threonine residue at position 366 is replaced
with a tryptophan residue (T366W), and in the CH3 domain of the
second subunit of the Fc domain the tyrosine residue at position
407 is replaced with a valine residue (Y407V). In one embodiment,
in the second subunit of the Fc domain additionally the threonine
residue at position 366 is replaced with a serine residue (T366S)
and the leucine residue at position 368 is replaced with an alanine
residue (L368A).
[0279] In yet a further embodiment, in the first subunit of the Fc
domain additionally the serine residue at position 354 is replaced
with a cysteine residue (S354C), and in the second subunit of the
Fc domain additionally the tyrosine residue at position 349 is
replaced by a cysteine residue (Y349C). Introduction of these two
cysteine residues results in formation of a disulfide bridge
between the two subunits of the Fc domain, further stabilizing the
dimer (Carter, J Immunol Methods 248, 7-15 (2001)).
[0280] In an alternative embodiment a modification promoting
association of the first and the second subunit of the Fc domain
comprises a modification mediating electrostatic steering effects,
e.g. as described in PCT publication WO 2009/089004. Generally,
this method involves replacement of one or more amino acid residues
at the interface of the two Fc domain subunits by charged amino
acid residues so that homodimer formation becomes electrostatically
unfavorable but heterodimerization electrostatically favorable.
[0281] In one embodiment a trispecific antibodies binding to HER2
and a blood-brain barrier receptor (BBB-R) according to any of the
above embodiments comprises an Immunoglobulin G (IgG) molecule
comprising a first antigen binding site specific for extracellular
domain II of HER2 and a second antigen binding site specific for
extracellular domain IV of HER2 and a monovalent third antigen
binding site specific for a BBB-R, wherein the Fc part of the first
heavy chain comprises a first dimerization module and the Fc part
of the second heavy chain comprises a second dimerization module
allowing a heterodimerization of the two heavy chains of the IgG
molecule.
[0282] In a further preferred embodiment, the first dimerization
module comprises knobs and the second dimerization module comprises
holes according to the knobs into holes strategy (see Carter P.;
Ridgway J. B. B.; Presta L. G.: Immunotechnology, Volume 2, Number
1, February 1996, pp. 73-73(1)).
E. Nucleic Acid Sequences, Vectors and Methods of
[0283] The invention further provides isolated polynucleotides
encoding trispecific antibodies binding to HER2 and a blood-brain
barrier receptor (BBB-R) as described herein or a fragment thereof.
The polynucleotides encoding trispecific antibodies of the
invention may be expressed as a single polynucleotide that encodes
the entire trispecific antigen binding molecule or as multiple
(e.g., two or more) polynucleotides that are co-expressed.
Polypeptides encoded by polynucleotides that are co-expressed may
associate through, e.g., disulfide bonds or other means to form a
functional trispecific antibody. For example, the light chain
portion of a Fab fragment may be encoded by a separate
polynucleotide from the portion of the trispecific antibody
comprising the heavy chain portion of the Fab fragment, an Fc
domain subunit and optionally (part of) another Fab fragment. When
co-expressed, the heavy chain polypeptides will associate with the
light chain polypeptides to form the Fab fragment. In another
example, the portion of the trispecific antibody provided therein
comprising one of the two Fc domain subunits and optionally (part
of) one or more Fab fragments could be encoded by a separate
polynucleotide from the portion of the trispecific antibody
provided therein comprising the other of the two Fc domain subunits
and optionally (part of) a Fab fragment. When co-expressed, the Fc
domain subunits will associate to form the Fc domain.
[0284] In one embodiment, the present invention is directed to an
isolated polynucleotide encoding a trispecific antibody of the
invention or a fragment thereof, wherein the polynucleotide
comprises a sequence that encodes a first variable heavy chain
sequence as shown in SEQ ID NOs 63, 67 and 69. In one embodiment,
the present invention is directed to an isolated polynucleotide
encoding a trispecific antibody of the invention or a fragment
thereof, wherein the polynucleotide comprises a sequence that
encodes a second variable heavy chain sequence as shown in SEQ ID
NOs 91 and 133.
[0285] In one embodiment, the present invention is directed to an
isolated polynucleotide encoding a trispecific antibody of the
invention or a fragment thereof, wherein the polynucleotide
comprises a sequence that encodes a variable light chain sequence
as shown in SEQ ID NO: 53.
[0286] In another embodiment, the present invention is directed to
an isolated polynucleotide encoding a trispecific antibody or
fragment thereof, wherein the polynucleotide comprises a sequence
that encodes a polypeptide sequence as shown in SEQ ID NOs 83, 85,
91, 93, 95, 97, 99, 101, 63, 67, 69, 53, 21 and 23.
[0287] In another embodiment, the invention is directed to an
isolated polynucleotide encoding a trispecific antibody of the
invention or a fragment thereof, wherein the polynucleotide
comprises a sequence that encodes a first variable heavy chain
sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,
or 99% identical to an amino acid sequence in SEQ ID NOs 63, 67 and
69.
[0288] In another embodiment, the invention is directed to an
isolated polynucleotide encoding a trispecific antibody of the
invention or a fragment thereof, wherein the polynucleotide
comprises a sequence that encodes a second variable heavy chain
sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,
or 99% identical to an amino acid sequence in SEQ ID NOs 91 and
133.
[0289] In another embodiment, the invention is directed to an
isolated polynucleotide encoding a trispecific antibody of the
invention or a fragment thereof, wherein the polynucleotide
comprises a sequence that encodes a variable light chain sequence
that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
identical to an amino acid sequence in SEQ ID NO: 53.
[0290] In certain embodiments the polynucleotide or nucleic acid is
DNA. In other embodiments, a polynucleotide of the present
invention is RNA, for example, in the form of messenger RNA (mRNA).
RNA of the present invention may be single stranded or double
stranded.
[0291] In further objects the present invention relates to 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.
F. Fc Domain Modifications Reducing Fc Receptor Binding and/or
Effector Function
[0292] In one aspect a trispecific antibodies binding to HER2 and a
blood-brain barrier receptor (BBB-R) according to any of the above
embodiments comprises an Immunoglobulin G (IgG) molecule wherein
the Fc part is modified. The modified Fc part has a reduced binding
affinity for the Fc.gamma. receptors compared to a wildtype Fc
part.
[0293] The Fc domain of the trispecific antibodies of the invention
consists of a pair of polypeptide chains comprising heavy chain
domains of an immunoglobulin molecule. For example, the Fc domain
of an immunoglobulin G (IgG) molecule is a dimer, each subunit of
which comprises the CH2 and CH3 IgG heavy chain constant domains.
The two subunits of the Fc domain are capable of stable association
with each other.
[0294] In one embodiment according the invention the Fc domain of
the trispecific antibodies of the invention is an IgG Fc domain. In
a particular embodiment the Fc domain is an IgG.sub.1 Fc domain. In
another embodiment the Fc domain is an IgG.sub.4 Fc domain. In a
more specific embodiment, the Fc domain is an IgG.sub.4 Fc domain
comprising an amino acid substitution at position S228 (Kabat
numbering), particularly the amino acid substitution S228P. In a
more specific embodiment, the Fc domain is an IgG.sub.4 Fc domain
comprising amino acid substitutions L235E and S228P and P329G. This
amino acid substitution reduces in vivo Fab arm exchange of
IgG.sub.4 antibodies (see Stubenrauch et al., Drug Metabolism and
Disposition 38, 84-91 (2010)). In a further particular embodiment
the Fc domain is human.
[0295] The Fc domain confers favorable pharmacokinetic properties
to the trispecific antibodies of the invention, including a long
serum half-life which contributes to good accumulation in the
target tissue and a favorable tissue-blood distribution ratio. At
the same time it may, however, lead to undesirable targeting of the
trispecific antibodies of the invention to cells expressing Fc
receptors rather than to the preferred antigen-bearing cells.
Accordingly, in particular embodiments the Fc domain of the
trispecific antibodies of the invention exhibits reduced binding
affinity to an Fc receptor and/or reduced effector function, as
compared to a native IgG.sub.1 Fc domain. In one such embodiment
the Fc domain (or the trispecific antibodies of the invention
comprising said Fc domain) exhibits less than 50%, preferably less
than 20%, more preferably less than 10% and most preferably less
than 5% of the binding affinity to an Fc receptor, as compared to a
native IgG.sub.1 Fc domain (or a trispecific antibodies of the
invention comprising a native IgG.sub.1 Fc domain), and/or less
than 50%, preferably less than 20%, more preferably less than 10%
and most preferably less than 5% of the effector function, as
compared to a native IgG.sub.1 Fc domain domain (or a trispecific
antibodies of the invention comprising a native IgG.sub.1 Fc
domain). In one embodiment, the Fc domain (or the trispecific
antibodies of the invention comprising said Fc domain) does not
substantially bind to an Fc receptor and/or induce effector
function. In a particular embodiment the Fc receptor is an
Fc.gamma. receptor. In one embodiment the Fc receptor is a human Fc
receptor. In one embodiment the Fc receptor is an activating Fc
receptor. In a specific embodiment the Fc receptor is an activating
human Fc.gamma. receptor, more specifically human Fc.gamma.RIIIa,
Fc.gamma.RI or Fc.gamma.RIIa, most specifically human
Fc.gamma.RIIIa. In one embodiment the Fc receptor is an inhibitory
Fc receptor. In a specific embodiment the Fc receptor is an
inhibitory human Fc.gamma. receptor, more specifically human
FcgRIIB In one embodiment the effector function is one or more of
CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment
the effector function is ADCC. In one embodiment the Fc domain
domain exhibits substantially similar binding affinity to neonatal
Fc receptor (FcRn), as compared to a native IgG.sub.1 Fc domain
domain. Substantially similar binding to FcRn is achieved when the
Fc domain (or the trispecific antibodies of the invention
comprising said Fc domain) exhibits greater than about 70%,
particularly greater than about 80%, more particularly greater than
about 90% of the binding affinity of a native IgG.sub.1 Fc domain
(or the trispecific antibodies of the invention comprising a native
IgG.sub.1 Fc domain) to FcRn.
[0296] In certain embodiments the Fc domain is engineered to have
reduced binding affinity to an Fc receptor and/or reduced effector
function, as compared to a non-engineered Fc domain. In particular
embodiments, the Fc domain of the trispecific antibodies of the
invention comprises one or more amino acid mutation that reduces
the binding affinity of the Fc domain to an Fc receptor and/or
effector function. Typically, the same one or more amino acid
mutation is present in each of the two subunits of the Fc domain.
In one embodiment the amino acid mutation reduces the binding
affinity of the Fc domain to an Fc receptor. In one embodiment the
amino acid mutation reduces the binding affinity of the Fc domain
to an Fc receptor by at least 2-fold, at least 5-fold, or at least
10-fold. In embodiments where there is more than one amino acid
mutation that reduces the binding affinity of the Fc domain to the
Fc receptor, the combination of these amino acid mutations may
reduce the binding affinity of the Fc domain to an Fc receptor by
at least 10-fold, at least 20-fold, or even at least 50-fold. In
one embodiment the trispecific antibodies of the invention
comprising an engineered Fc domain exhibits less than 20%,
particularly less than 10%, more particularly less than 5% of the
binding affinity to an Fc receptor as compared to a trispecific
antibodies of the invention comprising a non-engineered Fc domain.
In a particular embodiment the Fc receptor is an Fc.gamma.
receptor. In some embodiments the Fc receptor is a human Fc
receptor. In one embodiment the Fc receptor is an inhibitory Fc
receptor. In a specific embodiment the Fc receptor is an inhibitory
human Fc.gamma. receptor, more specifically human FcgRIIB In some
embodiments the Fc receptor is an activating Fc receptor. In a
specific embodiment the Fc receptor is an activating human
Fc.gamma. receptor, more specifically human Fc.gamma.RIIIa,
Fc.gamma.RI or Fc.gamma.RIIa, most specifically human
Fc.gamma.RIIIa. Preferably, binding to each of these receptors is
reduced. In some embodiments binding affinity to a complement
component, specifically binding affinity to C1q, is also reduced.
In one embodiment binding affinity to neonatal Fc receptor (FcRn)
is not reduced. Substantially similar binding to FcRn, i.e.
preservation of the binding affinity of the Fc domain to said
receptor, is achieved when the Fc domain (or the trispecific
antibodies of the invention comprising said Fc domain) exhibits
greater than about 70% of the binding affinity of a non-engineered
form of the Fc domain (or the trispecific antibodies of the
invention comprising said non-engineered form of the Fc domain) to
FcRn. The Fc domain, or the trispecific antibodies of the invention
of the invention comprising said Fc domain, may exhibit greater
than about 80% and even greater than about 90% of such affinity. In
certain embodiments the Fc domain of the trispecific antibodies of
the invention is engineered to have reduced effector function, as
compared to a non-engineered Fc domain. The reduced effector
function can include, but is not limited to, one or more of the
following: reduced complement dependent cytotoxicity (CDC), reduced
antibody-dependent cell-mediated cytotoxicity (ADCC), reduced
antibody-dependent cellular phagocytosis (ADCP), reduced cytokine
secretion, reduced immune complex-mediated antigen uptake by
antigen-presenting cells, reduced binding to NK cells, reduced
binding to macrophages, reduced binding to monocytes, reduced
binding to polymorphonuclear cells, reduced direct signaling
inducing apoptosis, reduced dendritic cell maturation, or reduced T
cell priming. In one embodiment the reduced effector function is
one or more of reduced CDC, reduced ADCC, reduced ADCP, and reduced
cytokine secretion. In a particular embodiment the reduced effector
function is reduced ADCC. In one embodiment the reduced ADCC is
less than 20% of the ADCC induced by a non-engineered Fc domain (or
a trispecific antibody of the invention comprising a non-engineered
Fc domain).
[0297] In one embodiment the amino acid mutation that reduces the
binding affinity of the Fc domain to an Fc receptor and/or effector
function is an amino acid substitution. In one embodiment the Fc
domain comprises an amino acid substitution at a position of E233,
L234, L235, N297, P331 and P329. In a more specific embodiment the
Fc domain comprises an amino acid substitution at a position of
L234, L235 and P329. In some embodiments the Fc domain comprises
the amino acid substitutions L234A and L235A. In one such
embodiment, the Fc domain is an IgG.sub.1 Fc domain, particularly a
human IgG.sub.1 Fc domain. In one embodiment the Fc domain
comprises an amino acid substitution at position P329. In a more
specific embodiment the amino acid substitution is P329A or P329G,
particularly P329G. In one embodiment the Fc domain comprises an
amino acid substitution at position P329 and a further amino acid
substitution at a position selected from E233, L234, L235, N297 and
P331. In a more specific embodiment the further amino acid
substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S.
In particular embodiments the Fc domain comprises amino acid
substitutions at positions P329, L234 and L235. In more particular
embodiments the Fc domain comprises the amino acid mutations L234A,
L235A and P329G ("P329G LALA"). In one such embodiment, the Fc
domain is an IgG.sub.1 Fc domain, particularly a human IgG.sub.1 Fc
domain. The "P329G LALA" combination of amino acid substitutions
almost completely abolishes Fc.gamma. receptor binding of a human
IgG.sub.1 Fc domain, as described in PCT patent application no.
PCT/EP2012/055393, incorporated herein by reference in its
entirety. PCT/EP2012/055393 also describes methods of preparing
such mutant Fc domains and methods for determining its properties
such as Fc receptor binding or effector functions.
[0298] IgG.sub.4 antibodies exhibit reduced binding affinity to Fc
receptors and reduced effector functions as compared to IgG.sub.1
antibodies. Hence, in some embodiments the Fc domain of the
trispecific antibodies of the invention is an IgG.sub.4 Fc domain,
particularly a human IgG.sub.4 Fc domain. In one embodiment the
IgG.sub.4 Fc domain comprises amino acid substitutions at position
S228, specifically the amino acid substitution S228P. To further
reduce its binding affinity to an Fc receptor and/or its effector
function, in one embodiment the IgG.sub.4 Fc domain comprises an
amino acid substitution at position L235, specifically the amino
acid substitution L235E. In another embodiment, the IgG.sub.4 Fc
domain comprises an amino acid substitution at position P329,
specifically the amino acid substitution P329G. In a particular
embodiment, the IgG.sub.4 Fc domain comprises amino acid
substitutions at positions 5228, L235 and P329, specifically amino
acid substitutions S228P, L235E and P329G. Such IgG.sub.4 Fc domain
mutants and their Fc.gamma. receptor binding properties are
described in PCT patent application no. PCT/EP2012/055393,
incorporated herein by reference in its entirety.
[0299] In a particular embodiment the Fc domain exhibiting reduced
binding affinity to an Fc receptor and/or reduced effector
function, as compared to a native IgG.sub.1 Fc domain, is a human
IgG.sub.1 Fc domain comprising the amino acid substitutions L234A,
L235A and optionally P329G, or a human IgG.sub.4 Fc domain
comprising the amino acid substitutions S228P, L235E and optionally
P329G.
[0300] In certain embodiments N-glycosylation of the Fc domain has
been eliminated. In one such embodiment the Fc domain comprises an
amino acid mutation at position N297, particularly an amino acid
substitution replacing asparagine by alanine (N297A) or aspartic
acid (N297D). In addition to the Fc domains described hereinabove
and in PCT patent application no. PCT/EP2012/055393, Fc domains
with reduced Fc receptor binding and/or effector function also
include those with substitution of one or more of Fc domain
residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No.
6,737,056). Such Fc mutants include Fc mutants with substitutions
at two or more of amino acid positions 265, 269, 270, 297 and 327,
including the so-called "DANA" Fc mutant with substitution of
residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).
[0301] Mutant Fc domains can be prepared by amino acid deletion,
substitution, insertion or modification using genetic or chemical
methods well known in the art. Genetic methods may include
site-specific mutagenesis of the encoding DNA sequence, PCR, gene
synthesis, and the like. The correct nucleotide changes can be
verified for example by sequencing.
[0302] Binding to Fc receptors can be easily determined e.g. by
ELISA, or by Surface Plasmon Resonance (SPR) using standard
instrumentation such as a BIAcore instrument (GE Healthcare), and
Fc receptors such as may be obtained by recombinant expression. A
suitable such binding assay is described herein. Alternatively,
binding affinity of Fc domains or cell activating trispecific
antigen binding molecules comprising an Fc domain for Fc receptors
may be evaluated using cell lines known to express particular Fc
receptors, such as human NK cells expressing Fc.gamma.IIIa
receptor.
[0303] Effector function of an Fc domain, or trispecific antibodies
of the invention comprising an Fc domain, can be measured by
methods known in the art. A suitable assay for measuring ADCC is
described herein. Other examples of in vitro assays to assess ADCC
activity of a molecule of interest are described in U.S. Pat. No.
5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063
(1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502
(1985); U.S. Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166,
1351-1361 (1987). Alternatively, non-radioactive assays methods may
be employed (see, for example, ACTI.TM. non-radioactive
cytotoxicity assay for flow cytometry (CellTechnology, Inc.
Mountain View, Calif.); and CytoTox 96.RTM. non-radioactive
cytotoxicity assay (Promega, Madison, Wis.)). Useful effector cells
for such assays include peripheral blood mononuclear cells (PBMC)
and Natural Killer (NK) cells. Alternatively, or additionally, ADCC
activity of the molecule of interest may be assessed in vivo, e.g.
in a animal model such as that disclosed in Clynes et al., Proc
Natl Acad Sci USA 95, 652-656 (1998).
[0304] In some embodiments, binding of the Fc domain to a
complement component, specifically to C1q, is reduced. Accordingly,
in some embodiments wherein the Fc domain is engineered to have
reduced effector function, said reduced effector function includes
reduced CDC. C1q binding assays may be carried out to determine
whether the trispecific antibodies of the invention is able to bind
C1q and hence has CDC activity. See e.g., C1q and C3c binding ELISA
in WO 2006/029879 and WO 2005/100402. To assess complement
activation, a CDC assay may be performed (see, for example,
Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et
al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103,
2738-2743 (2004)).
[0305] The following section describes preferred embodiments of the
trispecific antibodies of the invention comprising Fc domain
modifications reducing Fc receptor binding and/or effector
function.
G. Antibody Variants
[0306] In certain embodiments, amino acid sequence variants of the
trispecific antibodies provided herein are contemplated, in
addition to those described above. For example, it may be desirable
to improve the binding affinity and/or other biological properties
of the trispecific antibody. Amino acid sequence variants of a
trispecific antibody may be prepared by introducing appropriate
modifications into the nucleotide sequence encoding the trispecific
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.
1. Substitution, Insertion, and Deletion Variants
[0307] 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 B under the heading of
"conservative substitutions." More substantial changes are provided
in Table B 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, decreased
immunogenicity, or improved ADCC or CDC.
TABLE-US-00002 TABLE B 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:
[0308] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0309] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0310] (3) acidic: Asp, Glu;
[0311] (4) basic: His, Lys, Arg;
[0312] (5) residues that influence chain orientation: Gly, Pro;
[0313] (6) aromatic: Trp, Tyr, Phe.
[0314] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0315] 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).
[0316] 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.
[0317] 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.
[0318] 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.
[0319] 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.
2. Glycosylation Variants
[0320] In certain embodiments, a trispecific antibody provided
herein is altered to increase or decrease the extent to which the
antibody is glycosylated. Addition or deletion of glycosylation
sites to an antibody may be conveniently accomplished by altering
the amino acid sequence such that one or more glycosylation sites
is created or removed.
[0321] Where the trispecific antibody comprises an Fc region, the
carbohydrate attached thereto may be altered. Native antibodies
produced by mammalian cells typically comprise a branched,
biantennary oligosaccharide that is generally attached by an
N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g.,
Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may
include various carbohydrates, e.g., mannose, N-acetyl glucosamine
(GlcNAc), galactose, and sialic acid, as well as a fucose attached
to a GlcNAc in the "stem" of the biantennary oligosaccharide
structure. In some embodiments, modifications of the
oligosaccharide in a trispecific antibody of the invention may be
made in order to create antibody variants with certain improved
properties.
[0322] In one embodiment, trispecific antibody variants are
provided having a carbohydrate structure that lacks fucose attached
(directly or indirectly) to an Fc region. For example, the amount
of fucose in such antibody may be from 1% to 80%, from 1% to 65%,
from 5% to 65% or from 20% to 40%. The amount of fucose is
determined by calculating the average amount of fucose within the
sugar chain at Asn297, relative to the sum of all glycostructures
attached to Asn 297 (e. g. complex, hybrid and high mannose
structures) as measured by MALDI-TOF mass spectrometry, as
described in WO 2008/077546, for example. Asn297 refers to the
asparagine residue located at about position 297 in the Fc region
(Eu numbering of Fc region residues); however, Asn297 may also be
located about .+-.3 amino acids upstream or downstream of position
297, i.e., between positions 294 and 300, due to minor sequence
variations in antibodies. Such fucosylation variants may have
improved ADCC function. See, e.g., US Patent Publication Nos. US
2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co.,
Ltd). Examples of publications related to "defucosylated" or
"fucose-deficient" antibody variants include: US 2003/0157108; WO
2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US
2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US
2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO
2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol.
Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng.
87: 614 (2004). Examples of cell lines capable of producing
defucosylated antibodies include Lec13 CHO cells deficient in
protein fucosylation (Ripka et al. Arch. Biochem. Biophys.
249:533-545 (1986); US Pat Appl No US 2003/0157108 A1, Presta, L;
and WO 2004/056312 A1, Adams et al., especially at Example 11), and
knockout cell lines, such as alpha-1,6-fucosyltransferase gene,
FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech.
Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng.,
94(4):680-688 (2006); and WO2003/085107).
[0323] Trispecific antibodies variants are further provided with
bisected oligosaccharides, e.g., in which a biantennary
oligosaccharide attached to the Fc region of the trispecific
antibody is bisected by GlcNAc. Such trispecific antibody variants
may have reduced fucosylation and/or improved ADCC function.
Examples of such antibody variants are described, e.g., in WO
2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et
al.); and US 2005/0123546 (Umana et al.). Antibody variants with at
least one galactose residue in the oligosaccharide attached to the
Fc region are also provided. Such antibody variants may have
improved CDC function. Such antibody variants are described, e.g.,
in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO
1999/22764 (Raju, S.).
3. Cysteine Engineered Antibody Variants
[0324] In certain embodiments, it may be desirable to create
cysteine engineered trispecific antibodies, e.g., "thioMAbs," in
which one or more residues of a trispecific antibody are
substituted with cysteine residues. In particular embodiments, the
substituted residues occur at accessible sites of the trispecific
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. In certain embodiments, any one or more of the
following residues may be substituted with cysteine: V205 (Kabat
numbering) of the light chain; A118 (EU numbering) of the heavy
chain; and 5400 (EU numbering) of the heavy chain Fc region.
Cysteine engineered antibodies may be generated as described, e.g.,
in U.S. Pat. No. 7,521,541.
H. Recombinant Methods and Compositions
[0325] Trispecific 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 trispecific
antibodies (or fragments), 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 trispecific 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 trispecific
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
trispecific 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.
promoters 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).
[0326] 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 trispecific
antibody is desired, DNA encoding a signal sequence may be placed
upstream of the nucleic acid encoding a trispecific 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.
[0327] DNA encoding a short protein sequence that could be used to
facilitate later purification (e.g. a histidine tag) or assist in
labeling the trispecific antibody may be included within or at the
ends of the trispecific antibody (fragment) encoding
polynucleotide.
[0328] 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 trispecific 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
trispecific antibodies of the invention or fragments thereof. Host
cells suitable for replicating and for supporting expression of
trispecific 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 trispecific 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., YO, NSO, Sp20 cell).
[0329] 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.
[0330] In one embodiment, a method of producing a trispecific
antibody according to the invention is provided, wherein the method
comprises culturing a host cell comprising a polynucleotide
encoding the trispecific antibody, as provided herein, under
conditions suitable for expression of the trispecific antibody, and
recovering the trispecific antibody from the host cell (or host
cell culture medium).
[0331] The components of the trispecific antibody are genetically
fused to each other. Trispecific antibodies 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 trispecific 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.
[0332] In certain embodiments the Fab fragments forming part of the
trispecific antibody 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).
[0333] Any animal species of antibody, antibody fragment, antigen
binding domain or variable region can be used in the trispecific
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 trispecific 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.
[0334] In certain embodiments, the Fab fragments useful in 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 trispecific 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. 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 is incubated in a solution comprising a first
labeled antibody that binds to the antigen 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.).
[0335] Trispecific 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
trispecific antibody binds. For example, for affinity
chromatography purification of trispecific antibodies 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 trispecific
antibody essentially as described in the Examples. The purity of
the trispecific antibody can be determined by any of a variety of
well known analytical methods including gel electrophoresis, high
pressure liquid chromatography, and the like.
I. Assays
[0336] Trispecific antibodies binding to HER2 and a blood-brain
barrier receptor (BBB-R) 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.
1. Affinity Assays
[0337] The affinity of the trispecific antibodies binding to HER2
and a blood-brain barrier receptor (BBB-R) 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 trispecific antibody provided therein to
HER2 and/or a BBB-R may be evaluated using cell lines expressing
the particular receptor or target antigen, for example by flow
cytometry (FACS). A specific illustrative and exemplary embodiment
for measuring binding affinity is described in the following and in
the Examples below.
[0338] According to one embodiment, K.sub.D is measured by surface
plasmon resonance using a BIACORE.RTM. T100 machine (GE Healthcare)
at 25.degree. C.
[0339] To analyze the interaction between the Fc-portion and Fc
receptors, His-tagged recombinant Fc-receptor is captured by an
anti-Penta His antibody (Qiagen) immobilized on CM5 chips and the
trispecific constructs are used as analytes. Briefly,
carboxymethylated dextran biosensor chips (CM5, GE Healthcare) are
activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the
supplier's instructions. Anti Penta-His antibody is diluted with 10
mM sodium acetate, pH 5.0, to 40 .mu.g/ml before injection at a
flow rate of 5 .mu.l/min to achieve approximately 6500 response
units (RU) of coupled protein. Following the injection of the
ligand, 1 M ethanolamine is injected to block unreacted groups.
Subsequently the Fc-receptor is captured for 60 s at 4 or 10 nM.
For kinetic measurements, four-fold serial dilutions of the
trispecific construct (range between 500 nM and 4000 nM) are
injected in HBS-EP (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM
EDTA, 0.05% Surfactant P20, pH 7.4) at 25.degree. C. at a flow rate
of 30 .mu.l/min for 120 s.
[0340] To determine the affinity to the target antigen, trispecific
constructs are captured by an anti human Fab specific antibody (GE
Healthcare) that is immobilized on an activated CM5-sensor chip
surface as described for the anti Penta-His antibody. The final
amount of coupled protein is approximately 12000 RU. The
trispecific constructs are captured for 90 s at 300 nM. The target
antigens are passed through the flow cells for 180 s at a
concentration range from 250 to 1000 nM with a flowrate of 30
.mu.l/min. The dissociation is monitored for 180 s. Bulk refractive
index differences are corrected for by subtracting the response
obtained on reference flow cell. The steady state response was used
to derive the dissociation constant K.sub.D by non-linear curve
fitting of the Langmuir binding isotherm. Association rates
(k.sub.on) and dissociation rates (k.sub.off) are calculated using
a simple one-to-one Langmuir binding model (BIACORE.RTM. T100
Evaluation Software version 1.1.1) by simultaneously fitting the
association and dissociation sensorgrams. The equilibrium
dissociation constant (K.sub.D) is calculated as the ratio
k.sub.off/k.sub.on. See, e.g., Chen et al., J Mol Biol 293, 865-881
(1999).
2. Binding Assays and Other Assays
[0341] In one aspect, a trispecific antibody of the invention is
tested for its antigen binding activity, e.g., by known methods
such as ELISA, Western blot, etc.
[0342] In another aspect, competition assays may be used to
identify an antibody that competes with a specific anti-HER2 for
binding to HER2. 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-HER2 antibody. In another
aspect, competition assays may be used to identify an antibody that
competes with a specific anti-BBB-R for binding to BBB-R. 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-BBB-R 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.). Further methods are
described in the example section.
3. Activity Assays
[0343] In one aspect, assays are provided for identifying
trispecific antibodies specific for HER2 and a BBB-R thereof having
biological activity. Biological activity may include, e.g., DNA
fragmentation, induction of apoptosis and lysis of targeted cells.
Antibodies having such biological activity in vivo and/or in vitro
are also provided. In one embodiment said activity is induction of
complement-dependent cytotoxicity (CDC). In one embodiment said
trispecific antibodies induce CDC to a higher degree than
pertuzumab or trastuzumab alone. In one embodiment said trispecific
antibodies induce CDC to at least an about 10 times higher degree,
an about 20 times higher degree or an about 30 times higher degree
than pertuzumab or trastuzumab alone. In another embodiment said
trispecific antibodies induce CDC to a higher degree than the
combination of pertuzumab or trastuzumab. In another embodiment
said trispecific antibodies induce CDC to about a 30%, 40%, 50% or
60%, or at least a 30% to 70%, or at least a 40% to 60% higher
degree than the combination of pertuzumab or trastuzumab. The
complement-dependent cytotoxicity (CDC) assay can be performed with
non heat-treated serum or commercially available complement
fractions (see e.g. Lazar, G. A. et al. Engineered antibody Fc
variants with enhanced effector function. Proc. Natl Acad. Sci. USA
103, 4005-4010 (2006)). Target cell killing can be assessed by
several cell viability reagents such as Alamar Blue (Lazar, G. A.
et al. Engineered antibody Fc variants with enhanced effector
function. Proc. Natl Acad. Sci. USA 103, 4005-4010 (2006),
Idusogie, E. E. et al. Engineered antibodies with increased
activity to recruit complement. J. Immunol. 166, 2571-2575 (2001)),
CellTiter-Glo (see e.g. Zhao, X. et al. Targeting C-type
lectin-like molecule-1 for antibody-mediated immunotherapy in acute
myeloid leukemia. Haematologica 95, 71-78 (2009)), LDH release (see
e.g. Konishi, E., Kitai, Y. & Kondo, T. Utilization of
complement-dependent cytotoxicity to measure low levels of
antibodies: application to nonstructural protein 1 in a model of
Japanese encephalitis virus. Clin. Vaccine Immunol. 15, 88-94
(2008) and the examples disclosed herein) or calcein-AM release. In
some embodiments said degree of induction of CDC is determined by a
LDH release assay or a complement assay measuring binding of
complement protein C1q to the antibodies of the invention bound to
a cellular antigen. The CDC induction of the trispecific antibody
is then compared to the CDC induction of either Pertuzumab or
Trastuzumab alone, or the combination of Pertuzumab and
Trastuzumab, with all values for CDC induction being assayed in the
same assay, with the same cell line and the same respective
antibody concentration. If performed in a microtiterplate, the
capability of the trispecific antibodies and the controls (either
Pertuzumab or Trastuzumab alone, or the combination of Pertuzumab
and Trastuzumab) to induce CDC are preferably measured in the same
microtiterplate using the same assay. Exemplary assays are
disclosed, e.g. in example 18 or 19. In one embodiment said
induction of CDC is determined on cancer cells, e.g. breast cancer
cells.
[0344] In certain embodiments, a trispecific 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.
Assays for measuring ADCC or CDC are also described in WO
2004/065540 (see Example 1 therein), the entire content of which is
incorporated herein by reference.
J. Pharmaceutical Formulations
[0345] Pharmaceutical formulations of a trispecific antibody
specific for HER2 and a BBB-R as described herein are prepared by
mixing such trispecific 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.
[0346] 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.
[0347] 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.
[0348] 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).
[0349] 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.
[0350] 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.
K. Therapeutic Methods and Compositions
[0351] Any of the trispecific antibodies specific for HER2 and a
BBB-R provided herein may be used in therapeutic methods.
[0352] In one aspect, a trispecific antibody specific for HER2 and
a BBB-R for use as a medicament is provided. In further aspects, a
trispecific antibody specific for HER2 and a BBB-R for use in
treating cancer is provided. In certain embodiments, a trispecific
antibody specific for HER2 and a BBB-R for use in a method of
treatment is provided. In certain embodiments, the invention
provides a trispecific antibody specific for HER2 and a BBB-R for
use in a method of treating an individual having cancer comprising
administering to the individual an effective amount of the
trispecific antibody specific for HER2 and a BBB-R. 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.
[0353] In a further aspect, the invention provides for the use of a
trispecific antibody specific for HER2 and a BBB-R in the
manufacture or preparation of a medicament. In one embodiment, the
medicament is for treatment of cancer. In one embodiment said
cancer is HER2-positive cancer with brain metastases. 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.
[0354] 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 trispecific antibody specific for HER2 and a BBB-R. 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.
[0355] In a further aspect, the invention provides pharmaceutical
formulations comprising any of the trispecific antibodies specific
for HER2 and a BBB-R provided herein, e.g., for use in any of the
above therapeutic methods. In one embodiment, a pharmaceutical
formulation comprises any of the trispecific antibodies specific
for HER2 and a BBB-R provided herein and a pharmaceutically
acceptable carrier. In another embodiment, a pharmaceutical
formulation comprises any of the trispecific antibodies specific
for HER2 and a BBB-R provided herein and at least one additional
therapeutic agent, e.g., as described below.
[0356] A trispecific antibody of the invention 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.
[0357] Trispecific 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 trispecific
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.
[0358] For the prevention or treatment of disease, the appropriate
dosage of a trispecific antibody of the invention will depend on
the type of disease to be treated, the type of antibody, the
severity and course of the disease, whether the trispecific
antibody is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical history and response to
the trispecific antibody, and the discretion of the attending
physician. The trispecific 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 the trispecific 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 trispecific 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 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 trispecific 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.
[0359] 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 trispecific antibody
specific for HER2 and a BBB-R of the invention.
L. Articles of Manufacture
[0360] 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 trispecific 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 trispecific 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.
[0361] 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 trispecific antibody specific for HER2
and a BBB-R of the invention.
M. Immunoconjugates
[0362] The invention also provides immunoconjugates comprising an
trispecific antibody specific for HER2 and a BBB-R 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.
[0363] 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.
[0364] In another embodiment, an immunoconjugate comprises an
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.
[0365] In another embodiment, an immunoconjugate comprises an
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.
[0366] Conjugates of an 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.
[0367] 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).
III. Examples
[0368] 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.
[0369] 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: Materials and Methods
[0370] Unless stated otherwise the following general methods have
been applied:
[0371] Recombinant DNA Techniques
[0372] Standard methods were used to manipulate DNA as described in
Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The
molecular biological reagents were used according to the
manufacturer's instructions.
[0373] DNA and Protein Sequence Analysis and Sequence Data
Management
[0374] General information regarding the nucleotide sequences of
human immunoglobulins light and heavy chains is given in: Kabat,
E., A., et al., (1991) Sequences of Proteins of Immunological
Interest, Fifth Ed., NIH Publication No 91-3242. Amino acids of
antibody chains are numbered according to EU numbering (Edelman, G.
M., et al., PNAS 63 (1969) 78-85; Kabat, E. A., et al., (1991)
Sequences of Proteins of Immunological Interest, Fifth Ed., NIH
Publication No 91-3242). The GCG's (Genetics Computer Group,
Madison, Wis.) software package version 10.2 and Infomax's Vector
NTI Advance suite version 8.0 was used for sequence creation,
mapping, analysis, annotation and illustration.
[0375] DNA Sequencing
[0376] DNA sequences were determined by double strand sequencing
performed at SequiServe (Vaterstetten, Germany) and Geneart AG
(Regensburg, Germany).
Example 2: Generation of Trastuzumab and Pertuzumab Bispecific
Antibodies in a 2+2 IgG-scFv Format
[0377] Gene Synthesis
[0378] Desired gene segments were prepared by Geneart AG
(Regensburg, Germany) from synthetic oligonucleotides and PCR
products by automated gene synthesis. The gene segments which are
flanked by singular restriction endonuclease cleavage sites were
cloned into pGA18 (ampR) plasmids. The plasmid DNA was purified
from transformed bacteria and concentration determined by UV
spectroscopy. The DNA sequence of the subcloned gene fragments was
confirmed by DNA sequencing.
[0379] Construction of the Expression Plasmids
[0380] The following expression vector was used for the
construction of all heavy and light chain encoding expression
plasmids. The vector is composed of the following elements: [0381]
a hygromycin resistance gene as a selection marker, [0382] an
origin of replication, oriP, of Epstein-Barr virus (EBV), [0383] an
origin of replication from the vector pUC18 which allows
replication of this plasmid in E. coli [0384] a beta-lactamase gene
which confers ampicillin resistance in E. coli, [0385] the
immediate early enhancer and promoter from the human
cytomegalovirus (HCMV), [0386] the human 1-immunoglobulin
polyadenylation ("poly A") signal sequence, and
[0387] The immunoglobulin genes comprising the heavy or light chain
were prepared by gene synthesis and cloned into pGA18 (ampR)
plasmids as described above. Variable heavy chain constructs were
constructed by directional cloning using unique restriction sites.
Variable light chain constructs were ordered as gene synthesis
comprising VL and CL and constructed by directional cloning using
unique restriction sites. The final expression vectors were
transformed into E. coli cells, expression plasmid DNA was isolated
(Miniprep) and subjected to restriction enzyme analysis and DNA
sequencing. Correct clones were grown in 150 ml LB-Amp medium,
again plasmid DNA was isolated (Maxiprep) and sequence integrity
confirmed by DNA sequencing.
[0388] Transient Expression of Immunoglobulin Variants in HEK293
Cells
[0389] Recombinant immunoglobulin variants were expressed by
transient transfection of human embryonic kidney 293-F cells using
the FreeStyle.TM. 293 Expression System according to the
manufacturer's instruction (Invitrogen, USA). For small scale test
expressions 30 ml of 0.5.times.10.sup.6 HEK293F cells/ml were
seeded one day prior to transfection. The next day, plasmid DNA (1
DNA per ml culture volume) was mixed with 1.2 ml Opti-MEM.RTM. I
Reduced Serum Medium (Invitrogen, Carlsbad, Calif., USA) followed
by addition of 40 .mu.l of 293Fectin.TM. Transfection Reagent
(Invitrogen, Carlsbad, Calif., USA). The mixture was incubated for
15 min at room temperature and added drop wise to the cells. One
day post-transfection each flask was fed with 300 .mu.l L-Glutamine
(200 mM, Sigma-Aldrich, Steinheim, Germany) and 600 .mu.l feed7
containing L-asparagine, amino acids, trace elements,
ammonium-Fe(III) citrate, ethanolamine, trace elements, D-glucose,
FreeStyle medium without RPMI. Three days post-transfection cell
concentration, viability and glucose concentration in the medium
were determined using an automated cell viability analyzer
(Vi-CELL.TM. XR, Beckman Coulter, Fullerton, Calif., USA) and a
glucose meter (Accu-CHEK.RTM. Sensor comfort, Roche Diagnostics
GmbH, Mannheim, Germany). In addition each flask was fed with 300
.mu.l of L-glutamine, 300 .mu.l non-essential amino acids solution
(PAN.TM. Biotech, Aidenbach, Germany), 300 .mu.l sodium pyruvate
(100 mM, Gibco, Invitrogen), 1.2 ml feed7 and ad 5 g/L glucose
(D-(+)-Glucose solution 45%, Sigma). Finally, six days
post-transfection antibodies were harvested by centrifugation at
3500 rpm in a X3R Multifuge (Heraeus, Buckinghamshire, England) for
15 min at ambient temperature, the supernatant was sterile filtered
through a Steriflip filter unit (0.22 mm Millipore Express PLUS PES
membrane, Millipore, Bedford, Mass.) and stored at -20.degree. C.
until further use. Large scale transfections up to 5 L were scaled
linearly.
[0390] Purification of Bispecific and Control Antibodies
[0391] Bispecific antibodies were purified from cell culture
supernatants by affinity chromatography using Protein
A-Sepharose.TM. (GE Healthcare, Sweden) and Superdex200 size
exclusion chromatography. Briefly, sterile filtered cell culture
supernatants were applied on a HiTrap ProteinA HP (5 ml) column
equilibrated with PBS buffer (10 mM Na.sub.2HPO.sub.4, 1 mM
KH.sub.2PO.sub.4, 137 mM NaCl and 2.7 mM KCl, pH 7.4). Unbound
proteins were washed out with equilibration buffer. Antibody and
antibody variants were eluted with 0.1 M citrate buffer, pH 2.8,
and the protein containing fractions were neutralized with 0.1 ml 1
M Tris, pH 8.5. Eluted protein fractions were pooled, concentrated
with an Amicon Ultra centrifugal filter device (MWCO: 30 K,
Millipore) to a volume of 3 ml and loaded on a Superdex200 HiLoad
120 ml 16/60 gel filtration column (GE Healthcare, Sweden)
equilibrated with 20 mM Histidin, 140 mM NaCl, pH 6.0. Fractions
containing purified bispecific and control antibodies with less
than 5% high molecular weight aggregates were pooled and stored as
1.0 mg/ml aliquots at -80.degree. C.
[0392] Protein Quantification
[0393] Proteins were quantified by affinity chromatography using
the automated Ultimate 3000 system (Dionex, Idstein, Germany) with
a pre-packed Poros.RTM. A protein A column (Applied Biosystems,
Foster City, Calif., USA). All samples were loaded in buffer A (0.2
M Na.sub.2HPO.sub.4.[2H.sub.2O], pH 7.4) and eluted in buffer B
(0.1 M citric acid, 0.2 M NaCl, pH 2.5). In order to determine the
protein concentration an extinction coefficient of 1.62 was used
for all samples.
[0394] Analysis of Purified Proteins
[0395] The protein concentration of purified protein samples was
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 bispecific and
control antibodies were analyzed by SDS-PAGE in the presence and
absence of a reducing agent (5 mM 1,4-dithiotreitol) and staining
with Coomassie brilliant blue. The NuPAGE.RTM. Pre-Cast gel system
(Invitrogen, USA) was used according to the manufacturer's
instruction (4-20% Tris-Glycine gels). The aggregate content of
bispecific and control antibody samples was analyzed by
high-performance SEC using a Superdex 200 analytical size-exclusion
column (GE Healthcare, Sweden) in 200 mM KH.sub.2PO.sub.4, 250 mM
KCl, pH 7.0 running buffer at 25.degree. C. 25 .mu.g protein were
injected on the column at a flow rate of 0.5 ml/min and eluted
isocratic over 50 minutes. Integrity of the amino acid backbone of
reduced bispecific antibody light and heavy chains was verified by
NanoElectrospray Q-TOF mass spectrometry after removal of N-glycans
by enzymatic treatment with Peptide-N-Glycosidase F (Roche
Molecular Biochemicals).
[0396] Analytical HPLC
[0397] Antibodies were analyzed using a Agilent HPLC 1100 (Agilent
Technologies, Palo Alto, Calif., USA) with a TSK-GEL G3000SW gel
filtration column (7.5 mm ID.times.30 cm, TosoHaas Corp.,
Montgomeryville, Pa., USA). 18 .mu.l of the eluted proteins were
loaded onto the column in Buffer A (0.05 M
K.sub.2HPO.sub.4/KH.sub.2PO.sub.4 in 300 mM NaCl, pH 7.5) and
separated based on size.
[0398] Reducing and Non-Reducing SDS-PAGE
[0399] 7 .mu.l of the eluted proteins were mixed with 2.times.
sample buffer (NuPAGE.RTM. LDS Sample buffer, Invitrogen, Carlsbad,
Calif., USA) and another 7 .mu.l were mixed with 2.times. sample
buffer containing 10% reducing agent (NuPAGE.RTM. Sample Reducing
Agent, Invitrogen, Carlsbad, Calif., USA). Samples were heated to
70.degree. for 10 min and loaded onto a pre-cast NuPAGE.RTM. 4-12%
BisTris Gel (Invitrogen, Carlsbad, Calif., USA). The gel was run
for 45 min at 200V and 125 mA. Afterwards the gel was washed three
times with Millipore water and stained with SimplyBlue.TM.
SafeStain (Invitrogen, Carlsbad, Calif., USA). The gel was
destained overnight in Millipore water. FIGS. 1A-D depict
schematically the different variants of Trastuzumab and Pertuzumab
bispecific antibodies in a 2+2 IgG-scFv format. All bispecific
antibodies are bivalent for each antigen binding site and bind two
different paratopes in the ErbB2/HER2 receptor
(antigen1=trastuzumab specificity; antigen2=pertuzumab
specificity). All bispecific antibodies in a 2+2 IgG-scFv format
described herein are non frame-work grafted, non-CDR optimized, not
glycoengineered and do not bear any mutation in the Fc part.
[0400] FIGS. 2A-B, 3A-B and 4A-B show exemplary size-exclusion
purification graphs, SDS-PAGE analysis and analytical HPLC of
variants of Trastuzumab and Pertuzumab bispecific antibodies in a
2+2 IgG-scFv format. No data shown for TvAB17, TvAB13 and variants
Herceptin-scFv_A to E; all variants of Trastuzumab and Pertuzumab
bispecific antibodies in a 2+2 IgG-scFv format were produced with
the same quality.
TABLE-US-00003 TABLE 1a Sequences of Trastuzumab and Pertuzumab
bispecific antibodies in a 2 + 2 IgG-scFv format SEQ ID NO Name
Sequence TvAB12_2431_TrastuzumabHCscFvOmnitarg(HC_LC) 123 Light
chain
diqmtqspsslsasvgdrvtitcrasqdvntavawyqqkpgkapklliysasflysgvpsrfsgs
(kappa)
rsgtdftltisslqpedfatyycqqhyttpptfgqgtkveikrtvaapsvfifppsdeqlksgta-
svv [Trastuzumb,
cllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevt
1016] hqglsspvtksfnrgec 124 Heavy
evqlvesggglvqpggslrlscaasgfnikdtyihwvrqapgkglewvariyptngytryadsv
chain
kgrftisadtskntaylqmnslraedtavyycsrwggdgfyamdywgqgtivtvssastkgps
[Trastuzumb +
vfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvps
scFv
sslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmis
Omnitarg,
rtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlng
RB40]
keykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiave
wesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslsl
spgkggggsggggsggggsevqlvesggglvqpggslrlscaasgftftdytmdwvrqapgk
clewvadvnpnsggsiynqrfkgrftlsvdrskntlylqmnslraedtavyycarnlgpsfyfdy
wgqgtivtvssggggsggggsggggsggggsdiqmtqspsslsasvgdrvtitckasqdvsig
vawyqqkpgkapklliysasyrytgvpsrfsgsgsgtdifitisslqpedfatyycqqyyiypytf
gcgtkveik TvAB13
[TvAb13_1330scFvTrastuzumab(LC_HC)OmnitargLC_IntronA_cDNA] 125
Light chain
diqmtqspsslsasvgdrvtitcrasqdvntavawyqqkpgkapklliysasflysgvpsrfsgs
[scFv
rsgtdftltisslqpedfatyycqqhyttpptfgqgtkveikggggsggggsggggsevqlvesg
Trastuzumab +
gglvqpggslrlscaasgfnikdtyihwvrqapgkglewvariyptngytryadsvkgrftisad
Omnitarg,
tskntaylqmnslraedtavyycsrwggdgfyamdywgqgtivtvssggggsggggsgggg RB34]
sdiqmtqspsslsasvgdrvtitckasqdvsigvawyqqkpgkapklliysasyrytgvpsrfsg
sgsgtdftltisslqpedfatyycqqyyiypytfgqgtkveikrtvaapsvfifppsdeqlksgtas
vvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyace
vthqglsspvtksfnrgec 126 Heavy
evqlvesggglvqpggslrlscaasgftftdytmdwvrqapgkglewvadvnpnsggsiynqr
chain
fkgrftlsvdrskntlylqmnslraedtavyycarnlgpsfyfdywgqgtivtvssastkgpsvfp
(Omnitarg,
lapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslg
RB33)
tqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpe
vtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkey
kckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesn
gqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk
TvAB16 [TVAb16_2330TrastuzumabLCscFvOmnitarg(LC_HC)] 127 Light
chain
diqmtqspsslsasvgdrvtitcrasqdvntavawyqqkpgkapklliysasflysgvpsrfsgs
[Trastuzumb +
rsgtdftltisslqpedfatyycqqhyttpptfgqgtkveikrtvaapsvfifppsdeqlksgtasvv
scFvOmnitarg,
cllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevt
RB35]
hqglsspvtksfnrgecggggsggggsggggsdiqmtqspsslsasvgdrvtitckasqdvsig
vawyqqkpgkapklliysasyrytgvpsrfsgsgsgtdifitisslqpedfatyycqqyyiypytf
gqgtkveikggggsggggsggggsevqlvesggglvqpggslrlscaasgftftdytmdwvrq
apgkglewvadvnpnsggsiynqrfkgrftlsvdrskntlylqmnslraedtavyycarnlgps
fyfdywgqgtivtvss 128 Heavy
evqlvesggglvqpggslrlscaasgfnikdtyihwvrqapgkglewvariyptngytryadsv
chain
kgrftisadtskntaylqmnslraedtavyycsrwggdgfyamdywgqgtivtvssastkgps
[Trastuzumb,
vfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvps
1036]
sslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmis
rtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlng
keykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiave
wesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslsl
spgk TvAB17 [TvAb17_2431_TrastuzumabHCscFvOmnitarg(LC_HC)] 129
Light chain
diqmtqspsslsasvgdrvtitcrasqdvntavawyqqkpgkapklliysasflysgvpsrfsgs
(kappa)
rsgtdftltisslqpedfatyycqqhyttpptfgqgtkveikrtvaapsvfifppsdeqlksgta-
svv [Trastuzumb,
cllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevt
1016+ hqglsspvtksfnrgec 130 Heavy
evqlvesggglvqpggslrlscaasgfnikdtyihwvrqapgkglewvariyptngytryadsv
chain
kgrftisadtskntaylqmnslraedtavyycsrwggdgfyamdywgqgtivtvssastkgps
[Trastuzumb +
vfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvps
scFvOmnitarg,
sslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmis
RB43]
rtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlng
keykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiave
wesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslsl
spgkggggsggggsggggsdiqmtqspsslsasvgdrvtitckasqdvsigvawyqqkpgka
pklliysasyrytgvpsrfsgsgsgtdftltisslqpedfatyycqqyyiypytfgcgtkveikggg
gsggggsggggsggggsevqlvesggglvqpggslrlscaasgftftdytmdwvrqapgkcle
wvadvnpnsggsiynqrfkgrftlsvdrskntlylqmnslraedtavyycarnlgpsfyfdywg
qgtivtvss TvAB20 [TVAb20_4441TrastuzumabLCscFvOmnitarg(LC_HC)] 131
Light chain
diqmtqspsslsasvgdrvtitcrasqdvntavawyqqkpgkapklliysasflysgvpsrfsgs
[Trastuzumb +
rsgtdftltisslqpedfatyycqqhyttpptfgqgtkveikrtvaapsvfifppsdeqlksgtasvv
scFv
cllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevt
Omnitarg,
hqglsspvtksfnrgecggggsggggsggggsggggsdiqmtqspsslsasvgdrvtitckas- q
RB61]
dvsigvawyqqkpgkapklliysasyrytgvpsrfsgsgsgtdifitisslqpedfatyycqqyyi
ypytfgcgtkveikggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgftft
dytmdwvrqapgkclewvadvnpnsggsiynqrfkgrftlsvdrskntlylqmnslraedtav
yycarnlgpsfyfdywgqgtivtvss 132 Heavy
evqlvesggglvqpggslrlscaasgfnikdtyihwvrqapgkglewvariyptngytryadsv
chain
kgrftisadtskntaylqmnslraedtavyycsrwggdgfyamdywgqgtivtvssastkgps
[Trastuzumb,
vfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvps
1036]
sslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmis
rtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlng
keykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiave
wesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslsl
spgk Herceptarg 2 + 2 OmniE 145 Heavy chain
evqlvesggglvqpggslrlscaasgftftdytmdwvrqapgkglewvadvnpnsggsiynqrfkgrftls
with scFv
vdrskntlylqmnslraedtavyycarnlgpsfyfdywgqgtivtvssastkgpsvfplapss-
kstsggtaaI Trastuzumab
gclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkv
stabilized
epkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvwdvshedpevkfnwyvdgvevh
with
naktkpreeqynstyrwsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlpps-
rde disulphide
ltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmh
bonding
ealhnhytqkslslspgkggggsggggsevqlvesggglvqpggslrlscaasgfnikdtyihwv-
rqapgk
glewvariyptngytryadsvkgrftisadtskntaylqmnslraedtavyycsrwggegfyamdywgcg
tivtvssggggsggggsggggsdiqmtqspsslsasvgdrvtitcrasqdvnvavawyqqkpgkcpklliy
sasflysgvpsrfsgsrsgtdftltisslqpedfatyycqqhyttpptfgqgtlweik 146
Pertuzumab
diqmtqspsslsasvgdrvtitckasqdvsigvawyqqkpgkapklliysasyrytgvpsrfsgsgsgtdftl-
t light chain
isslqpedfatyycqqyyiypytfgqgtkveikrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvq-
w
kvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec
TABLE-US-00004 TABLE 1b) Variants of
TvAB12_2431_TrastuzumabHCscFvOmnitarg(HC_LC) with a stabilizing
disulphide bond in the scFv part to reduce aggregation levels Name
of molecule Disulphide position (VH-VL) Trastuzumab_scFv_WT WT
Trastuzumab_scFv_A 44-100 Trastuzumab_scFv_B 45-98
Trastuzumab_scFv_C 101-46 Trastuzumab_scFv_D 103-44
Trastuzumab_scFv_E 105-43
Example 3: Proliferation Inhibition Assay with Trastuzumab and
Pertuzumab Bispecific Antibodies in a 2+2 IgG-scFv Format
[0401] Cell Lines
[0402] MDA-MB 175 VII cells were maintained in DMEM/F12 medium
(Gibco) supplemented with 10% fetal calf serum and 2 mL
L-glutamine. Propagation of cell lines followed standard cell
culture protocols.
[0403] The ability of the bispecific antibodies to inhibit
proliferation was assessed in the cell line MDA-MB-175 VII.
MDA-MB-175 VII were cultured in DMEM/F12 medium (Gibco)
supplemented with 10% fetal calf serum, 2 mM L-glutamine. Cells in
the logarithmic growth phase were detached, counted and
2.times.10e4 cells were seeded in 100 .mu.L medium per well of a
96-well cell culture plate. Cells were maintained overnight in the
incubator and the following day 100 .mu.L of the respective
antibodies diluted in medium were added in form of a dilution
series to the cells. After a total incubation time of 6 days cell
growth was assessed in an Alamar Blue (Invitrogen) assay. The assay
was performed as recommended by the manufacturer.
[0404] Table 2 shows the potency of selected bispecific antibodies
in the proliferation assay.
TABLE-US-00005 Antibody SEQ ID NO: EC50 [nM] Herceptarg WT 1.84
Herceptarg A 1.89 Herceptarg B 2.66 Herceptarg C 2.56 Herceptarg D
1.63 Herceptarg E 1.75 TvAb12 123/124 4.90 CrossMab 119/ 4.75
120/121/122 Pertuzumab 2.11 Pertuzumab + Herceptin 1.70 Herceptin
2.92
Example 4: Herceptarg Stabilisation
[0405] The aspartate isomerization site at position 98 of the heavy
chain and the asparagine deamidation site at position 30 of the
light chain are stability hotspots of trastuzumab. Those two
positions affect the stability and integrity of the antigen binding
capacity of the antibody. This problem was overcome by introducing
the lyophilized formulations using either sodium succinate or
histidine buffer. In order to increase stability and storage
half-life we intended to replace those known sources of instability
by amino acids, or amino acid stretches that should have a higher
intrinsic stability. We tested herefore the replacement of Asp98 by
Glu in the heavy chain and the replacement of Asn30 by Ser, as well
Thr31 by Val in the light chain. Those mutations were abbreviated
D98E, N30S, and T31V. T31V does not directly influence the
deamidation of N30, but it was assumed that the residue adjacent on
the C-terminal side of an Asparagine would influence the stability
properties of a polypeptide chain.
[0406] The antibody samples were incubated over a period of either
1, 2, or 3 months at 40.degree. C. in one of the three buffers: 40
mM Histidin, 150 mM NaCl, pH5.0, 40 mM Histidin, 150 mM NaCl,
pH6.0, or 40 mM Histidin, 150 mM NaCL, pH7.4. The protein
concentration during this period was always 1 mg/ml. After the
indicated time points, samples were taken and shock frozen in
liquid nitrogen, and then kept at -80.degree. C. until further
analysis. This analysis was carried out on a ProteOn XPR36
instrument (BioRad). Approximately 700 RU of Her2, respectively,
were immobilized on 2 channels of a GLM chip using amine coupling
(vertical orientation). Trastuzumab variants were measured in
duplicates at 6 different analyte concentrations (100, 50, 25,
12.5, 6.25, 0 nM) by injections in horizontal orientation at 100
.mu.l/min. Association rate were recorded for 180s, the
dissociation rate for 600s. Regeneration was performed by two
pulses of 10 mM glycine pH 1.5 and 50 mM NaOH for 60s at 150
.mu.L/min (horizontal orientation). In total four different
Trastuzumab variants were measured. In the first experiment (Tables
3 and 4), only the unmodified Trastuzumab and variant 602 (D98E of
heavy chain and T31V of light chain) were tested. In the second
experiment the unmodified Trastuzumab, variant 602 (D98E of heavy
chain and T31V of light chain) and variants VH:D98E/VL: N30S
VH:D98E/VL: N30T were tested (Table 5, 6, 7).
[0407] Results:
[0408] All variants show the same affinity towards recombinant Her2
antigen as the parental Trastuzumab molecule. After exposure to pH5
or pH6 at 40.degree. C., Trastuzumab lost affinity by a factor
.about.5, mainly driven by increasing the off-rate and keeping the
on-rate unchanged. Variant 602 showed almost undistinguishable
affinities before and after pH stress. Variant N30S had a higher
affinity from the beginning compared to the parental Trastuzumab,
which stayed approximately constant during the stress conditions.
The variants D98E (VH) together with either N30S, N30T, or T31V
(VL) were used in the further experiments. Results are shown in
FIGS. 5A-B and 6A-C and the tables below.
TABLE-US-00006 TABLE 3 Kinetic affinity parameters of Trastuzumab
variants as determined by SPR method (ProteOn instrument) after
incubating the samples for 1, 2, or 3 months at 40.degree. in 40 mM
Histidin, 150 mM NaCl, pH 5.0 buffer. Each measurement was done in
duplicate and both experimental values are shown. Tested were the
resynthesized Trastuzumab (wt) and compared to the D98E T31V
variant in the heavy and light chain, respectively (named clone
602). t0 t1 t2 pH 5 ka kd KD ka kd KD ka wt 1 2.4E+05 1.1E-04
4.3E-10 3.3E+05 1.6E-04 5.0E-10 2.9E+05 2 2.4E+05 4.4E-05 1.8E-10
2.8E+05 1.9E-04 7.0E-10 2.8E+05 602 1 2.6E+05 1.2E-04 4.6E-10
2.7E+05 2.2E-04 8.0E-10 2.9E+05 2 2.3E+05 1.3E-04 5.4E-10 2.8E+05
1.4E-04 5.2E-10 2.9E+05 t2 t3 pH 5 kd KD ka kd KD wt 1 2.6E-04
9.2E-10 2.8E+05 3.4E-04 1.2E-09 2 2.8E-04 9.9E-10 2.9E+05 3.6E-04
1.3E-09 602 1 1.7E-04 5.8E-10 3.0E+05 1.7E-04 5.8E-10 2 1.6E-04
5.3E-10 3.1E+05 1.7E-04 5.5E-10
TABLE-US-00007 TABLE 4 Kinetic affinity parameters of Trastuzumab
variants similar to table 3. Here the samples are incubated at
40.degree. in 40 mM Histidin, 150 mM NaCl, pH 6.0 for the same time
intervals as before. t0 t1 t2 pH 6 ka kd KD ka kd KD ka wt 1
2.8E+05 9.5E-05 3.4E-10 3.0E+05 1.5E-04 5.1E-10 2.5E+05 2 2.8E+05
8.6E-05 3.1E-10 2.8E+05 2.1E-04 7.7E-10 2.6E+05 602 1 2.9E+05
1.4E-04 4.8E-10 3.0E+05 1.6E-04 5.2E-10 3.0E+05 2 2.8E+05 1.4E-04
5.0E-10 2.9E+05 1.5E-04 5.1E-10 2.9E+05 t2 t3 pH 6 kd KD ka kd KD
wt 1 3.1E-04 1.2E-09 2.3E+05 4.0E-04 1.7E-09 2 3.3E-04 1.3E-09
2.4E+05 4.1E-04 1.8E-09 602 1 1.6E-04 5.3E-10 3.0E+05 1.8E-04
5.9E-10 2 1.6E-04 5.5E-10 2.9E+05 1.7E-04 5.7E-10
TABLE-US-00008 TABLE 5 Kinetic affinity parameters of Trastuzumab
variants similar to table 3. Here the samples are incubated at
40.degree. in 40 mM Histidin, 150 mM NaCl, pH 5.0 for the same time
intervals as before. Samples included are the resynthesized
Trastuzumab (wt), the D98E T31V variant in the heavy and light
chain, respectively (named clone 602. This was produced in CHO
instead of HEK). Also the light chain variants N30S, and N30T (both
have the D98E heavy chain variant) t0 t1 t2 pH 5 ka kd KD ka kd KD
ka 602 1 2.20E+05 1.40E-04 6.34E-10 2.32E+05 9.14E-05 3.95E-10
2.32E+05 CHO 2 2.32E+05 2.13E-04 9.21E-10 2.10E+05 1.54E-04
7.35E-10 2.37E+05 N30T 1 2.19E+05 2.11E-04 9.64E-10 2.21E+05
1.46E-04 6.61E-10 2.20E+05 2 2.18E+05 2.61E-04 1.20E-09 2.05E+05
1.85E-04 9.02E-10 2.01E+05 N30S * 1 2.59E+05 2.59E-19 1.00E-24
2.54E+05 1.93E-05 7.58E-11 2.49E+05 2 2.44E+05 2.36E-17 9.68E-23
2.42E+05 3.47E-05 1.43E-10 2.37E+05 wt 1 2.51E+05 1.60E-04 6.39E-10
2.30E+05 3.62E-04 1.58E-09 2.38E+05 2 2.29E+05 1.93E-04 8.43E-10
2.13E+05 2.66E-04 1.25E-09 2.26E+05 t2 t3 pH 5 kd KD ka kd KD 602 1
2.36E-04 1.02E-09 2.01E+05 9.67E-05 4.81E-10 CHO 2 2.35E-04
9.89E-10 2.08E+05 1.30E-04 6.25E-10 N30T 1 1.77E-04 8.05E-10
2.31E+05 2.87E-04 1.24E-09 2 2.02E-04 1.00E-09 2.27E+05 2.82E-04
1.24E-09 N30S * 1 1.16E-16 4.64E-22 2.54E+05 4.11E-18 1.62E-23 2
3.71E-16 1.57E-21 2.37E+05 6.06E-17 2.55E-22 wt 1 6.47E-04 2.72E-09
2.33E+05 7.72E-04 3.32E-09 2 6.88E-04 3.05E-09 2.18E+05 7.91E-04
3.62E-09 * Due to slow dissociation rates, the off-rates and the
dissociation constant contain a high degree of uncertainty.
TABLE-US-00009 TABLE 6 Kinetic affinity parameters of Trastuzumab
variants similar to table 3. Here the samples are incubated at
40.degree. in 40 mM Histidin, 150 mM NaCl, pH 6.0 for the same time
intervals as before. Samples included are the resynthesized
Trastuzumab (wt), the D98E T31V variant in the heavy and light
chain, respectively (named clone 602. This was produced in CHO
instead of HEK). Also the light chain variants N30S, and N30T (both
have the D98E heavy chain variant) t0 t1 t2 pH 6 ka kd KD ka kd KD
ka 602 1 2.07E+05 9.33E-05 4.51E-10 2.30E+05 1.37E-04 5.94E-10
2.19E+05 CHO 2 2.16E+05 1.97E-04 9.12E-10 2.16E+05 2.02E-04
9.32E-10 2.07E+05 N30T 1 2.24E+05 2.45E-04 1.09E-09 2.03E+05
1.24E-04 6.10E-10 2.11E+05 2 2.31E+05 2.71E-04 1.17E-09 1.93E+05
1.57E-04 8.13E-10 2.05E+05 N30S * 1 2.56E+05 1.18E-17 4.62E-23
2.65E+05 2.77E-05 1.04E-10 2.38E+05 2 2.48E+05 8.14E-06 3.28E-11
2.51E+05 4.23E-05 1.68E-10 2.25E+05 wt 1 2.23E+05 7.43E-05 3.33E-10
2.22E+05 4.60E-04 2.07E-09 2.31E+05 2 2.23E+05 7.98E-05 3.59E-10
2.21E+05 4.03E-04 1.82E-09 2.20E+05 t2 t3 pH 6 kd KD ka kd KD 602 1
1.29E-04 5.87E-10 2.14E+05 1.50E-04 7.01E-10 CHO 2 1.52E-04
7.31E-10 2.13E+05 1.60E-04 7.50E-10 N30T 1 2.27E-04 1.08E-09
2.08E+05 2.01E-04 9.67E-10 2 2.55E-04 1.24E-09 2.03E+05 1.98E-04
9.75E-10 N30S * 1 8.97E-19 3.77E-24 2.36E+05 1.84E-05 7.80E-11 2
4.30E-17 1.91E-22 2.33E+05 3.41E-05 1.46E-10 wt 1 9.24E-04 4.00E-09
1.89E+05 9.91E-04 5.24E-09 2 9.31E-04 4.24E-09 1.78E+05 9.95E-04
5.58E-09 * Due to slow dissociation rates, the off-rates and the
dissociation constant contain a high degree of uncertainty.
TABLE-US-00010 TABLE 7 Kinetic affinity parameters of Trastuzumab
variants similar to table 3. Here the samples are incubated at
40.degree. in 40 mM Histidin, 150 mM NaCl, pH 7.4 for the same time
intervals as before. Samples included are the resynthesized
Trastuzumab (wt), the D98E T3IV variant in the heavy and light
chain, respectively (named clone 602. This was produced in CHO
instead of HEK). Also the light chain variants N30S, and N30T (both
have the D98E heavy chain variant) t0 t1 t2 pH 7.4 ka kd KD ka kd
KD ka 602 1 2.15E+05 1.23E-04 5.71E-10 2.32E+05 1.80E-04 7.78E-10
1.97E+05 CHO 2 2.17E+05 2.08E-04 9.55E-10 2.19E+05 2.31E-04
1.06E-09 1.82E+05 N30T 1 2.46E+05 2.19E-04 8.87E-10 2.15E+05
1.86E-04 8.65E-10 2.12E+05 2 2.20E+05 2.30E-04 1.04E-09 2.00E+05
2.03E-04 1.02E-09 1.98E+05 N30S 1 2.58E+05 1.35E-06 5.25E-12
2.60E+05 2.55E-05 9.82E-11 2.43E+05 2 2.36E+05 2.24E-05 9.46E-11
2.49E+05 6.31E-18 2.53E-23 2.27E+05 wt 1 2.25E+05 7.49E-05 3.33E-10
1.99E+05 8.80E-04 4.43E-09 1.64E+05 2 2.12E+05 9.85E-05 4.65E-10
2.00E+05 8.78E-04 4.40E-09 1.67E+05 t2 t3 pH 7.4 kd KD ka kd KD 602
1 1.05E-04 5.34E-10 2.02E+05 2.24E-04 1.11E-09 CHO 2 1.33E-04
7.30E-10 2.00E+05 2.14E-04 1.07E-09 N30T 1 2.64E-04 1.25E-09
1.94E+05 1.41E-04 7.26E-10 2 2.84E-04 1.44E-09 1.73E+05 1.54E-04
8.87E-10 N30S 1 7.28E-21 2.99E-26 2.37E+05 6.30E-05 2.66E-10 2
1.24E-05 5.46E-11 2.21E+05 6.55E-05 2.97E-10 wt 1 1.35E-03 8.27E-09
1.55E+05 1.79E-03 1.15E-08 2 1.29E-03 7.72E-09 1.45E+05 1.63E-03
1.12E-08 * Due to slow dissociation rates, the off-rates and the
dissociation constant contain a high degree of uncertainty.
Example 5: Binding of Trastuzumab and Trastuzumab Stabilization
Variants after Stress to KPL-4 Cells
[0409] Binding
[0410] KPL-4 cells were harvested and resuspended in FACS buffer.
0.2 Mio cells were seeded into a 96 well round bottom plate. The
plate was centrifuged at 400 g for 3 min to pellet the cells. The
supernatant was removed and the cells were resuspended in 40 .mu.l
of the diluted antibodies. The plate was incubated for 30 min at
4.degree. C. to allow binding of the antibodies. To remove unbound
antibodies the cells were centrifuged again and washed twice with
FACS buffer. To detect the antibodies the cells were resuspended in
12 .mu.l diluted secondary goat anti-human Fc specific FITC-labeled
secondary antibody (Jackson ImmunoResearch #109-096-098) and
incubated again for 30 min at 4.degree. C. Afterwards the cells
were washed twice with FACS buffer, resuspended in 200 .mu.l FACS
buffer and the fluorescence was measured with BD Cantoll.
[0411] ADCC
[0412] Target cells were harvested, washed, stained with calcein
(Invitrogen), resuspended in AIM V.RTM. medium (Life Technologies),
and plated at a concentration of 3.times.10.sup.4 cells/well. The
respective antibody dilutions were added in triplicates to the
cells and incubated for 10 min before addition of the effector
cells (peripheral blood mononuclear effector cells [PBMCs]).
Effector (E) and target (T) cells were then incubated for the
indicated time at 37.degree. C. at the indicated E:T ratio
(triplicates for all samples). After incubation the cells were
washed once with PBS and then lysed with borate buffer. Calcein
retention was measured in a Wallac Victor3 1420 Multilabel Counter.
ADCC was calculated using the following formula:
Percentage A D C C = ( [ sample release - spontaneous release
maximal release - spontaneous release ] ) .times. 100.
##EQU00001##
[0413] Spontaneous release, corresponding to target cells incubated
with effector cells without antibody, was defined as 0%
cytotoxicity, and maximal release (target cells lysed with 1%
Triton X-100) was defined as 100% cytotoxicity. The average
percentage of ADCC and standard deviations of the triplicates of
each experiment were calculated.
[0414] Results are shown in FIGS. 7A-L, 8A-F, and 9A-F.
Example 6: Generation of Herceptarg CrossMab and Framework Grafting
on Novel LC06 Based Framework to Achieve Less Mispairing
[0415] Gene Synthesis
[0416] Desired gene segments were prepared by Geneart AG
(Regensburg, Germany) from synthetic oligonucleotides and PCR
products by automated gene synthesis. The gene segments encoding
heavy or light chains with C-terminal attachment of scFv antibody
fragments, "knobs-into-hole" antibody heavy chains carrying S354C
and T366W mutations and "knobs-into-hole" heavy chains carrying
Y349C, T366S, L368A and Y407V mutations in the CH3 domain in
combination with unmodified VH domains, crossed C kappa domains or
scFab antibody fragments as well as unmodified antibody light
chains or CH1 domain exchanged light chains are flanked by singular
restriction endonuclease cleavage sites (BamHI-XbaI, BamHI-XmnI or
BamHI-KpnI) and were cloned into pGA18 (ampR) plasmids. The plasmid
DNA was purified from transformed bacteria and concentration
determined by UV spectroscopy. The DNA sequence of the subcloned
gene fragments was confirmed by DNA sequencing. All constructs were
designed with a 5'-end DNA sequence coding for a leader peptide
(MGWSCIILFLVATATGVHS), which targets proteins for secretion in
eukaryotic cells.
[0417] Construction of the Expression Plasmids
[0418] The expression vector that was used for the construction of
all "knobs-into-hole" heavy chain as well as antibody light chain
encoding expression plasmids comprises the following elements:
[0419] a hygromycin resistance gene as a selection marker, [0420]
an origin of replication, oriP, of Epstein-Barr virus (EBV), [0421]
an origin of replication from the vector pUC18 which allows
replication of this plasmid in E. coli [0422] a beta-lactamase gene
which confers ampicillin resistance in E. coli, [0423] the
immediate early enhancer and promoter from the human
cytomegalovirus (HCMV), [0424] the human 1-immunoglobulin
polyadenylation ("poly A") signal sequence, and [0425] unique BamHI
and XbaI restriction sites.
[0426] The immunoglobulin genes comprising heavy or light chains
with C-terminal attachment of scFv antibody fragments,
"knobs-into-hole" heavy chains with unmodified VH domains, crossed
C kappa domains or scFab fragments as well as unmodified light
chains or CH1 domain exchanged light chains were prepared by gene
synthesis and cloned into pGA18 (ampR) plasmids as described. The
pG18 (ampR) plasmids carrying the synthesized DNA segments and the
expression vector were digested with BamHI and XbaI, BamHI and XmnI
or BamHI and KpnI restriction enzymes (Roche Molecular
Biochemicals) and subjected to agarose gel electrophoresis.
Purified heavy or light chains with C-terminal attachment of scFv
antibody fragments, "knobs-into-hole" heavy and unmodified or
domain exchanged light chain encoding DNA segments were then
ligated to the isolated expression vector BamHI/XbaI, BamHI/XmnI or
BamHI/KpnI fragment resulting in the final expression vectors. The
final expression vectors were transformed into E. coli cells,
expression plasmid DNA was isolated (Miniprep) and subjected to
restriction enzyme analysis and DNA sequencing. Correct clones were
grown in 150 ml LB-Amp medium, again plasmid DNA was isolated
(Maxiprep) and sequence integrity confirmed by DNA sequencing.
[0427] Transient Expression of Bispecific Antibodies in HEK293
Cells
[0428] Recombinant bispecific antibodies were expressed by
transient transfection of human embryonic kidney 293-F cells using
the FreeStyle.TM. 293 Expression System according to the
manufacturer's instruction (Invitrogen, USA). Briefly, suspension
FreeStyle.TM. 293-F cells were cultivated in FreeStyle.TM. 293
Expression medium at 37.degree. C./8% CO.sub.2 and the cells were
seeded in fresh medium at a density of 1.times.10.sup.6 viable
cells/ml one day before transfection. For transfection, DNA was
prepared in 10 ml Dulbecco's PBS (PAA, Austria) using 162.5 .mu.l
of 293-Free.TM. Transfection Reagent (Merck, USA) and 125 .mu.g of
heavy with N-terminal attachment of scFab encoding DNA in a plasmid
ratio of 1:1 with "Knobs-into-hole" heavy chain 1 and 2 and light
chain plasmid DNA in a 1:1:1 molar ratio in 250 ml final
transfection volume. For transfection of Cross Mabs, a plasmid
ratio of 1:1:1:1, 1:1:1:2, 1:1:1:4, 1:1:1:8 of "Knobs-into-hole"
heavy chain 1:unmodified light chain:C kappa domain exchanged
"Knobs-into-hole" heavy chain 2: CH1 domain exchanged light chain
was prepared. The CH1-VL light chain plasmid ration was used at
1.times., 2.times., 4.times. and 8.times. molar ratios to assess
the optimization of chain pairing using a combination of CE-SDS and
Q-TOF spectrometry. Antibody containing cell culture supernatants
were harvested 7 days after transfection by centrifugation at 14000
g for 30 minutes and filtered through a sterile filter (0.22
.mu.m). Supernatants were stored at -20.degree. C. until
purification. The sequences of the resulting antibodies are shown
below in table 8.
[0429] Preparation of the Glycoengineered Derivatives of Bispecific
<Her2GlyMab> Antibodies
[0430] Glycoengineered derivatives of bispecific <Her2GlyMab>
antibodies were produced by co-transfecting HEK293-EBNA cells with
the mammalian antibody heavy and light chain expression vectors
using a calcium phosphate-transfection approach. Exponentially
growing HEK293-EBNA cells were transfected by the calcium phosphate
method. For the production of the glycoengineered antibody, the
cells were co-transfected with a plasmid for a fusion GnTIII
polypeptide expression and a second plasmid for mannosidase II
expression, respectively. Plasmid ratios of bispecific antibodies
were added as described in the material and methods section above.
Cells were grown as adherent monolayer cultures in T flasks using
DMEM culture medium supplemented with 10% FCS, and were transfected
when they were between 50 and 80% confluent. For the transfection
of a T75 flask, 7.5 (to 8) million cells were seeded 24 hours
before transfection in ca 14 ml DMEM culture medium supplemented
with FCS (at 10% V/V final), (eventually 250 neomycin,) and cells
were placed at 37.degree. C. in an incubator with a 5% CO2
atmosphere overnight. For each T75 flask to be transfected, a
solution of DNA, CaCl2 and water was prepared by mixing 47 .mu.g
total plasmid vector DNA, 235 .mu.l of a 1M CaCl2 solution, and
adding water to a final volume of 469 .mu.l. To this solution, 469
.mu.l of a 50 mM HEPES, 280 mM NaCl, 1.5 mM Na2HPO4 solution at pH
7.05 were added, mixed immediately for 10 sec and left to stand at
room temperature for 20 sec. The suspension was diluted with ca. 12
ml of DMEM supplemented with 2% FCS, and added to the T75 in place
of the existing medium. The cells were incubated at 37.degree. C.,
5% CO2 for about 17 to 20 hours, then medium was replaced with ca.
12 ml DMEM, 10% FCS. The conditioned culture medium was harvested 5
to 7 days post-transfection centrifuged for 5 min at 210-300*g,
sterile filtered through a 0.22 .mu.m filter (or alternatively
centrifuged for 5 min at 1200 rpm, followed by a second
centrifugation for 10 min at 4000 rpm) and kept at 4.degree. C.
[0431] Glycoengineered antibodies were purified and formulated as
described above for the non-glycoengineered antibodies. The
oligosaccharides attached to the Fc region of the antibodies were
analysed as described below to determine the amount of fucose.
[0432] Framework Grafting
[0433] Rationale: Similar framework of trastuzumab and pertuzumab
allows mis-pairing of light chains: Both Trastuzumab and Pertuzumab
have a V.sub.HIII V.sub.LkI framework and therefore the light chain
interface affinity to both heavy chains is identical.
[0434] In order to avoid mispairing of light chains in the crossMab
Herceptarg bispecific antibody, the Trastuzumab framework of
"CrossMabXPer Her2GlyMab" was exchanged with the framework of the
non-related antibody LC06. Trastuzumab is related to germlines
hVH3_66 and hVK1D_39 whereas the antibody LC06 corresponds to
germlines hVbase_VH1_1 germline and hVL_3, respectively. Pertuzumab
is related to hVH3_23 and hVK1D_13 showing a very similar framework
system compared to Trastuzumab.
[0435] Both germline acceptor frameworks of LC06 are different from
the Trastuzumab framework, especially, the LC06 lambda light chain,
compared to the Trastzumab kappa light chain. The antibody Fab
crystal structure of Trastuzumab has been superimposed and the
compatibility of the framewords have been structurally evaluated.
CDRI, II and III of the Trastuzumab light chain were grafted onto
the new Lambda framework of LC06. Mutations included were D98E and
N30S, the N30S was used in favour of T31V because of the increase
in affinity to the Her2 extracellular domain with the N30S
modification. It was thought that any reduction of the Kd caused by
the CDR grafting could be compensated by the use of the N30S
mutation. Some accommodations in the acceptor frameworks were
required in order to get the CDRs in their biological active
conformation; for example A(LC06 lambda) at the Kabat position 71
of the light chain has been backmutated to F(Trastuzumab kappa).
The original VHIII-VLkI (Kappa I family) framework of Pertuzumab
was maintained. The resulting bispecific antibody is depicted as
"CrossMab-CDRG Her2GlyMab", with sequences as shown in Table 8
below.
TABLE-US-00011 TABLE 8 Sequences of Herceptarg CrossMab bispecific
antibodies. Construct SEQ ID NO Sequence OAscFab1 Her2GlyMab scFab
133
diqmtqspsslsasvgdrvtitcrasqdynyavawyqqkpgkapklliysasflysgypsrfsg
Trastuzumab
srsgtdftltisslqpedfatyycqqhyttpptfgqgtkveikrtvaapsvfifppsdeqlksgtas
heavy chain 1
vvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyac
evthqglsspvtksfnrgecggggsggggsggggsggggsggggsggggsggevqlvesgg
glvqpggslrlscaasgfnikdtyihwyrqapgkglewvariyptngytryadsvkgrftisadt
skntaylqmnslraedtavyycsrwggegfyamdywgqgtlytyssastkgpsvfplapssk
stsggtaalgclykdyfpepytyswnsgaltsgyhtfpaylqssglyslssyytypssslgtqtyi
cnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcv
vvdvshedpevkfnwyydgvevhnaktkpreeqynstyryvsyltylhqdwlngkeykck
vsnkalpapiektiskakgqprepqvytlppcrdeltknqvslwclvkgfypsdiavewesng
qpennykttppyldsdgsfflyskltvdksrwqqgnyfscsvmhealhnhytqks1s1spgk
Pertuzumab 134
evqlvesggglvqpggslrlscaasgftftdytmdwyrqapgkglewvadynpnsggsiyn heavy
chain 2
qrfkgrftlsydrskntlylqmnslraedtavyycarnlgpsfyfdywgqgtlytyssastkgps
vfplapsskstsggtaalgclykdyfpepytyswnsgaltsgyhtfpaylqssglyslssyytyp
ssslgtqtyicnynhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlm
isrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwl
ngkeykckvsnkalpapiektiskakgqprepqvctlppsrdeltknqvslscavkgfypsdi
avewesngqpennykttppvldsdgsfflvskltvdksrwqqgnvfscsvmhealhnhytq
kslslspgk Pertuzumab 135
diqmtqspsslsasvgdrvtitckasqdvsigvawyqqkpgkapklliysasyrytgvpsrfsg
lght chain 1
sgsgtdftltisslqpedfatyycqqyyiypytfgqgtkveikrtvaapsvfifppsdeqlksgtas
vvellnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyac
evthqglsspvtksfnrgec OAscFab2 Her2GlyMab Pertuzumab 136
evqlvesggglvqpggslrlscaasgftftdytmdwvrqapgkglewvadvnpnsggsiyn heavy
chain 1
qrfkgrftlsvdrskntlylqmnslraedtavyycarnlgpsfyfdywgqgtivtvssastkgps
vfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvp
ssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlm
isrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwl
ngkeykckvsnkalpapiektiskakgqprepqvctlppsrdeltknqvslscavkgfypsdi
avewesngqpennykttppvldsdgsfflvskltvdksrwqqgnvfscsvmhealhnhytq
kslslspgk Pertuzumab 137
diqmtqspsslsasvgdrvtitckasqdvsigvawyqqkpgkapklliysasyrytgvpsrfsg
light chain 1
sgsgtdftltisslqpedfatyycqqyyiypytfgqgtkveikrtvaapsvfifppsdeqlksgtas
vvellnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyac
evthqglsspvtksfnrgec scFab 138
evqlvesggglvqpggslrlscaasgfnikdtyihwvrqapgkglewvariyptngytryads
Trastuzumab
vkgrftisadtskntaylqmnslraedtavyycsrwggegfyamdywgqgtivtvssastkg
heavy chain 2
psvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvt
vpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdt
lmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlhq
dwlngkeykckvsnkalpapiektiskakgqprepqvytlpperdeltknqvslwclvkgfy
psdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhn
hytqkslslspgkggggsggggsggggsggggsggggsggggsggdiqmtqspsslsasvg
drvtitcrasqdvnvavawyqqkpgkapklliysasflysgvpsrfsgsrsgtdifitisslqped
fatyycqqhyttpptfgqgtkveikrtvaapsvfifppsdeqlksgtasvvellnnfypreakvq
wkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnr
gee OAscFabPer1 Her2GlyMab scFab 139
evqlvesggglvqpggslrlscaasgftftdytmdwvrqapgkglewvadvnpnsggsiyn
Pertuzumab
qrfkgrftlsvdrskntlylqmnslraedtavyycarnlgpsfyfdywgqgtivtvssastkgps
heavy chain 1
vfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvp
ssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlm
isrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwl
ngkeykckvsnkalpapiektiskakgqprepqvctlppsrdeltknqvslscavkgfypsdi
avewesngqpennykttppvldsdgsfflvskltvdksrwqqgnvfscsvmhealhnhytq
kslslspgkggggsggggsggggsggggsggggsggggsggdiqmtqspsslsasvgdrvt
itckasqdvsigvawyqqkpgkapklliysasyrytgvpsrfsgsgsgtdifitisslqpedfaty
ycqqyyiypytfgqgtkveikrtvaapsvfifppsdeqlksgtasvvellnnfypreakvqwk
vdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec
Trastuzumab 140
evqlvesggglvqpggslrlscaasgfnikdtyihwvrqapgkglewvariyptngytryads
heavy chain 2
vkgrftisadtskntaylqmnslraedtavyycsrwggegfyamdywgqgtivtvssastkg
psvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvt
vpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdt
lmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhq
dwlngkeykckvsnkalpapiektiskakgqprepqvytlpperdeltknqvslwclvkgfy
psdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhn
hytqkslslspgk Trastuzumab 141
diqmtqspsslsasvgdrvtitcrasqdvnvavawyqqkpgkapklliysasflysgvpsrfsg
light chain 2
srsgtdftltisslqpedfatyycqqhyttpptfgqgtkveikrtvaapsvfifppsdeqlksgtas
vvellnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyac
evthqglsspvtksfnrge OAscFabPer2 Her2GlyMab Trastuzumab 142
evqlvesggglvqpggslrlscaasgfnikdtyihwvrqapgkglewvariyptngytryads
heavy chain 1
vkgrftisadtskntaylqmnslraedtavyycsrwggegfyamdywgqgtivtvssastkg
psvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvt
vpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdt
lmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhq
dwlngkeykckvsnkalpapiektiskakgqprepqvytlpperdeltknqvslwclvkgfy
psdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhn
hytqkslslspgk Trastuzumab 143
diqmtqspsslsasvgdrvtitcrasqdvnvavawyqqkpgkapklliysasflysgvpsrfsg
light chain 1
srsgtdftltisslqpedfatyycqqhyttpptfgqgtkveikrtvaapsvfifppsdeqlksgtas
vvellnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyac
evthqglsspvtksfnrgec scFab 144
evqlvesggglyqpggslrlscaasgftftdytmdwyrqapgkglewvadynpnsggsiyn
Pertuzumab
qrfkgrftlsydrskntlylqmnslraedtavyycarnlgpsfyfdywgqgtlytyssastkgps
heavy chain 2
vfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvp
ssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlm
isrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwl
ngkeykckvsnkalpapiektiskakgqprepqvctlppsrdeltknqvslscavkgfypsdi
avewesngqpennykttppvldsdgsfflvskltvdksrwqqgnvfscsvmhealhnhytq
kslslspgkggggsggggsggggsggggsggggsggggsggdiqmtqspsslsasvgdrvt
itckasqdvsigvawyqqkpgkapklliysasyrytgvpsrfsgsgsgtdifitisslqpedfaty
ycqqyyiypytfgqgtkveikrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwk
vdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec
Cros sMab-XPer Her2GlyMab XPertuzumab 109
evqlvesggglvqpggslrlscaasgftftdytmdwvrqapgkglewvadvnpnsggsiyn heavy
chain
qrfkgrftlsvdrskntlylqmnslraedtavyycamlgpsfyfdywgqgtivtvssasvaaps
vfifppsdeqlksgtasvvellnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsst
itlskadyekhkvyacevthqglsspvtksfnrgecdkthtcppcpapellggpsvflfppkpk
dtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlh
qdwlngkeykckvsnkalpapiektiskakgqprepqvytlppcrdeltknqvslwclvkgf
ypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealh
nhytqkslslspgk XPertuzumab 110
diqmtqspsslsasvgdrvtitckasqdvsigvawyqqkpgkapklliysasyrytgvpsrfsg
light chain
sgsgtdftltisslqpedfatyycqqyyiypytfgqgtkveikssastkgpsvfplapsskstsggt
aalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnh
kpsntkvdkkvepksc Trastuzumab 96
evqlvesggglvqpggslrlscaasgfnikdtyihwvrqapgkglewvariyptngytryads
heavy chain
vkgrftisadtskntaylqmnslraedtavyycsrwggegfyamdywgqgtivtvssastkg
(VH.sub.D98E CH1)
psvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvt
vpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdt
lmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhq
dwlngkeykckvsnkalpapiektiskakgqprepqvctlppsrdeltknqvslscavkgfyp
sdiavewesngqpennykttppvldsdgsfflvskltvdksrwqqgnvfscsvmhealhnh
ytqkslslspgk Trastuzumab 86
diqmtqspsslsasvgdrvtitcrasqdvnvavawyqqkpgkapklliysasflysgvpsrfsg
light chain
srsgtdftltisslqpedfatyycqqhyttpptfgqgtkveikrtvaapsvfifppsdeqlksgtas
(VL.sub.T31V CL)
vvellnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyac
evthqglsspvtksfnrgec CrossMab-XTra Her2GlyMab Pertuzumab 119
evqlvesggglvqpggslrlscaasgftftdytmdwvrqapgkglewvadvnpnsggsiyn heavy
chain
qrfkgrftlsvdrskntlylqmnslraedtavyycarnlgpsfyfdywgqgtivtvssasvaaps
vfifppsdeqlksgtasvvellnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsst
itlskadyekhkvyacevthqglsspvtksfnrgecdkthtcppcpapellggpsvflfppkpk
dtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlh
qdwlngkeykckvsnkalpapiektiskakgqprepqvctlppsrdeltknqvslscavkgfy
psdiavewesngqpennykttppvldsdgsfflvskltvdksrwqqgnvfscsvmhealhn
hytqkslslspgk Pertuzumab 120
diqmtqspsslsasvgdrvtitckasqdvsigvawyqqkpgkapklliysasyrytgvpsrfsg
light chain
sgsgtdftltisslqpedfatyycqqyyiypytfgqgtkveikrtvaapsvfifppsdeqlksgtas
vvellnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyac
evthqglsspvtksfnrgec XTrastuzumab 121
evqlvesggglvqpggslrlscaasgfnikdtyihwvrqapgkglewvariyptngytryads
heavy chain
vkgrftisadtskntaylqmnslraedtavyycsrwggegfyamdywgqgtivtvssastkg
psvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvt
vpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdt
lmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhq
dwlngkeykckvsnkalpapiektiskakgqprepqvytlppcrdeltknqvslwclvkgfy
psdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhn
hytqkslslspgk XTrastuzuma 122
diqmtqspsslsasvgdrvtitcrasqdvnvavawyqqkpgkapklliysasflysgvpsrfsg
light chain
srsgtdftltisslqpedfatyycqqhyttpptfgqgtkveikssastkgpsvfplapsskstsggta
algclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhk
psntkvdkkvepksc Optimized Trastuzumab sequences: CrossMab-XTra
Her2GlyMab and CrossMab-XPer Her2GlyMab Trastuzumab 117
evqlvesggglvqpggslrlscaasgfnikdtyihwvrqapgkglewvariyptngytryads VH
(D98E) vkgrftisadtskntaylqmnslraedtavyycsrwggegfyamdywgqgtivtvss
Trastuzumab 115
astkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglysl
CH1 ssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkth Trastuzumab 118
diqmtqspsslsasvgdrvtitcrasqdvnvavawyqqkpgkapklliysasflysgvpsrfsg VL
(T31V) srsgtdftltisslqpedfatyycqqhyttpptfgqgtkveikr Trastuzumab 116
tvaapsvfifppsdeqlksgtasvvellnnfypreakvqwkvdnalqsgnsqesvteqdskds CL
tyslsstltlskadyekhkvyacevthqglsspvtksfnrgec Trastuzumab 20
gfnikdtyih VH CDR1 Trastuzumab 29 riyptngytryadsvkg VH CDR2
Trastuzumab 79 wggegfyamdy VH CDR3 Trastuzumab 104 rasqdvnvava VL
CDR1 Trastuzumab 18 sasflys VL CDR2 Trastuzumab 19 qqhyttppt VL
CDR3
CrossMab-CDRG Her2GlyMab XPertuzumab 109
evqlvesggglvqpggslrlscaasgftftdytmdwvrqapgkglewvadvnpnsggsiyn heavy
chain
qrfkgrftlsvdrskntlylqmnslraedtavyycarnlgpsfyfdywgqgtivtvssasvaaps
(VHCL)
vfifppsdeqlksgtasvvellnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsst
itlskadyekhkvyacevthqglsspvtksfnrgecdkthtcppcpapellggpsvflfppkpk
dtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlh
qdwlngkeykckvsnkalpapiektiskakgqprepqvytlpperdeltknqvslwclvkgf
ypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealh
nhytqkslslspgk Pertuzumab 110
diqmtqspsslsasvgdrvtitckasqdvsigvawyqqkpgkapklliysasyrytgvpsrfsg
light chain
sgsgtdftltisslqpedfatyycqqyyiypytfgqgtkveikssastkgpsvfplapsskstsggt
(VLCH1)
aalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnh
kpsntkvdkkvepksc Trastuzumab 111
qvqlvqsgaevkkpgasvkvsckasgfnikdtyihwvrqapgqglewmgriyptngytrya CDRG
heavy
qkfqgrvtmtrdtsistaymelsrlrsddtavyycsrwggegfyamdywgqgtmvtvssast
chain
kgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssv
(VHCH1)
vtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpk
dtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlh
qdwlngkeykckvsnkalpapiektiskakgqprepqvctlppsrdeltknqvslscavkgfy
psdiavewesngqpennykttppvldsdgsfflvskltvdksrwqqgnvfscsvmhealhn
hytqks1s1spgk Trastuzumab 112
diqltqppsysvapgqtaritcgasqdvstavawyqqkpgqapvlvvysasflysgipsrfsgs
CDRG light
rsgtdftltisrveagdeadyycqqhyttpptfgtgtkvtvlrtvaapsvfifppsdeqlksgtasv
chain (VLCL)
vellnnfypreakvqw1cvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyace
vthqglsspvtksfnrgec Trastuzumab 105
evqlvqsgaevkkpgasvkvsckasgfnikdtyihwvrqapgqglewmgriyptngytrya CDRG
VH qkfqgrvtmtrdtsistaymelsrlrsddtavyycsrwggegfyamdywgqgtmvtvss
(D98E, CDRG) Trastuzumab 115
astkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglysl
CH1 ssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkth Trastuzumab 106
diqltqppsysvapgqtaritcgasqdvstavawyqqkpgqapvlvvysasflysgipsrfsgs
CDRG VL rsgtdftltisrveagdeadyycqqhyttpptfgtgtkvtvlr (N30T, CDRG)
Trastuzumab 116
tvaapsvfifppsdeqlksgtasvvellnnfypreakvqwkvdnalqsgnsqesvteqdskds CL
tyslsstltlskadyekhkvyacevthqglsspvtksfnrgec Trastuzumab 20
gfnikdtyih CDRG VH CDR1 Trastuzumab 108 riyptngytryaqkfqg CDRG VH
CDR2 Trastuzumab 79 wggegfyamdy CDRG VH CDR3 Trastuzumab 107
gasqdvstava CDRG VL CDR1 Trastuzumab 18 sasflys CDRG VL CDR2
Trastuzumab 19 qqhyttppt CDRG VL CDR3 Pertuzumab sequences in
CrossMab-CDRG Her2GlyMab, CrossMab-XTra Her2GlyMab and
CrossMab-XPer Her2GlyMab: see Pertuzumab wt (parent) sequences
Table 32
[0436] Purification of Bispecific Antibodies
[0437] Bispecific antibodies were purified from cell culture
supernatants by affinity chromatography using
MabSelectSure-Sepharose.TM. (GE Healthcare, Sweden) and Superdex
200 size exclusion (GE Healthcare, Sweden) chromatography. Briefly,
sterile filtered cell culture supernatants were captured on a
MabSelect SuRe resin equilibrated with PBS buffer (10 mM
Na.sub.2HPO.sub.4, 1 mM KH.sub.2PO.sub.4, 137 mM NaCl and 2.7 mM
KCl, pH 7.4), washed with equilibration buffer and eluted with 25
mM sodium citrate at pH 3.0. The eluted protein fractions were
pooled, neutralized with 2M Tris, pH 9.0 and further purified by
size exclusion chromatography using a Superdex 200 26/60 GL (GE
Healthcare, Sweden) column equilibrated with 20 mM histidine, 140
mM NaCl, pH 6.0. Size exclusion chromatography fractions were
analysed by CE-SDS (Caliper Life Science, USA) and bispecific
antibody containing fractions were pooled and stored at -80.degree.
C.
[0438] Analysis of Purified Proteins
[0439] The protein concentration of purified protein samples was
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, antibody integrity and molecular
weight of bispecific and control antibodies were analyzed by CE-SDS
using microfluidic Labchip technology (Caliper Life Science, USA).
5 .mu.l of protein solution was prepared for CE-SDS analysis using
the HT Protein Express Reagent Kit according manufacturer's
instructions and analysed on LabChip GXII system using a HT Protein
Express Chip. Data were analyzed using LabChip GX Software version
3.0.618.0. The aggregate content of bispecific and control antibody
samples was analyzed by high-performance SEC using a Superdex 200
analytical size-exclusion column (GE Healthcare, Sweden) in 200 mM
KH.sub.2PO.sub.4, 250 mM KCl, pH 7.0 running buffer at 25.degree.
C. 25 .mu.g protein were injected on the column at a flow rate of
0.5 ml/min and eluted isocratic over 50 minutes.
[0440] Mass Spectrometry
[0441] The integrity of the amino acid backbone of reduced
bispecific antibody light and heavy chains was verified by
NanoElectrospray Q-TOF mass spectrometry after removal of N-glycans
by enzymatic treatment with Peptide-N-Glycosidase F (Roche
Molecular Biochemicals). The amount of heavy and light chain
mis-pairing was also quantified.
[0442] Surface Plasmon Resonance
[0443] Instrument: BIAacore T100 (GE Healthcare) [0444] Software:
Biacore T100 Control, Version 2.02/2.03 [0445] Biacore T100
Evaluation, Version 2.02/2.03 [0446] Biacore B3000 (Biacore) [0447]
Software: Biacore B3000 Control, Version 4.1.2 [0448]
BIAEvaluation, Version 4.1.1
[0449] Assayformat Chip: CM5-Chip
[0450] Kinetic constants and resulting affinities of
<Her2GlyMab>-molecules were measured for both "Trastuzumab"-
and "Pertuzumab"-functionalities respectively. These two
functionalities were distinguished via pre-complexation of Her2
with either amine coupled parental MAb "Trastuzumab" (FC1/2) or
"Pertuzumab" FC3/4). Complex formation of parental MAb and Her2
commensed after injection of Her2 ECD.
[0451] As a consequence of pre-complexed parental MAb
"Trastuzumab"/Her2 all "Trastuzumab"-binding sites are saturated,
but all "Pertuzumab"-binding sites are available and vice versa.
Finally the binding of the "<Her2GlyMab>"-molecules to be
analyzed was measured via injections using increasing
concentrations with each cycle. Association and dissociation
observed were calculated with a Langmuir 1:1 binding model. To
minimize dissociation of Her2 during the measurements, the kinetic
constants were measured at T=25.degree. C.
[0452] Amine Coupling of Capture Molecules
[0453] Standard amine coupling on flow cells 1 to 4 according to
the manufacturer's instructions: CM5 Chip, T=25.degree. C., running
buffer: HBS-N buffer, activation by mixture of EDC/NHS, aimed at
800 RU; the parental Abs "Trastuzumab" or "Pertuzumab" were diluted
in coupling buffer sodium acetate, pH 4.5, c=2-3 .mu.g/mL; finally
remaining activated carboxyl groups were blocked by injection of 1
M Ethanolamine.
[0454] Chip surface on flow cell 1 and 3 (with either amine coupled
parental mAb "Trastuzumab" or "Pertuzumab") were used as reference
control surface for correction of possible buffer-effects or
non-specific binding.
[0455] Kinetic Characterization of <Her2GlyMab> Molecules at
25.degree. C.
[0456] Running buffer: PBS
[0457] All samples were diluted with running buffer+1 mg/mL
BSA.
[0458] Capturing of HER2 ECD on flow cells 2 and 4: c=100 nM, flow
5 .mu.l/min, time 120 sec. Analyte samples: A classical
concentration series of the <Her2GlyMab>-molecules were
analyzed at five concentrations (c=300, 100, 33.33, 11.11 and 3.7
nM.) at a flow rate of SOW/min was injected. Singles for each
concentration, one as a duplicate; association time: 180 sec.,
dissociation time: 900 sec.
[0459] Final regeneration was performed after each cycle using 10
mM Glycin pH 2.5 for amine coupled Mab "Trastuzumab" and 25 mM NaOH
for amine coupled Mab "Pertuzumab", contact time each 60 sec, flow
rate 30 .mu.l/min.
[0460] Kinetic parameters were calculated by using double
referencing (control reference: binding of analyte to Mabs
Trastuzumab and Pertuzumab respectively; Flow Cell: 1 respectively
3), concentration "0" used as the blank. Calculations were
performed with model `Langmuir binding 1:1, RI (refractive
index)=0.
[0461] Results: Expression & Purification Bispecific, Bivalent
<Her2GlyMab> Antibody Molecules
[0462] According the procedures described in the materials and
methods above, the bispecific, bivalent <Her2GlyMab> antibody
molecules OAscFab1, OAscFab2, OAscFabPer1, OAscFabPer2,
CrossMab-XPer, CrossMab-XTra and CrossMab-CDRG were expressed and
purified. In each molecule the VH and VL of part are based on
optimized Trastuzumab sequences with mutations T31V or N30T in the
variable light chain and mutation D98E in the heavy chain and the
Pertuzumab parent sequence respectively. The schematic structure of
the antibodies is shown in FIGS. 10A-B. The sequences are shown in
Table 8 above.
[0463] Expression of <Her2GlyMab> antibody molecules OAscFab1
Her2 GlyMab, OAscFab2 Her2 GlyMab, OAscFabPer1 Her2 GlyMab,
OAscFabPer2 Her2 GlyMab (all glycoengineered, Knobs-into-holes,
with the Trastuzumab scFab bearing D98E and T31V mutations),
CrossMab-XPer Her2 GlyMab, CrossMab-XTra Her2 GlyMab (both
glycoengineered, Knobs-into-holes, with the Trastuzumab cross-Fab
bearing D98E and T31V mutations), and CrossMab-CDRG Her2 GlyMab
(glycoengineered, Knobs-into-holes, CDR grafted, with the
Trastuzumab cross-Fab bearing D98E and N30S mutations) was
confirmed by western blotting and HP-SEC (FIGS. 11A-B).
Purification of OAscFab1, OAscFab2, OAscFabPer1, OAscFabPer2,
CrossMab-XPer, CrossMab XTra and CrossMab-CDRG led to the yields
shown in Table 9. All OAscFab constructs showed less than 90%
monomer post purification, the reduced purity of these molecules
could not be increased by optimization of the plasmid ratios in
expression (data not shown). However, optimization of the plasmid
chain ratios in expression proved to increase the monomeric
fraction present post purification of the CrossMab antibodies as
described below.
TABLE-US-00012 TABLE 9 Her2GlyMab Purification - Analysis of the
percentage aggregation post protein A and SEC purification using
HP-SEC and the respective protein yields calculated by UV
spectroscopy A280 of the glycoengineered constructs. Purity
Expression Purity post yield post post Expression protein A protein
A SEC yield post Name (%) (mg/L) (%) SEC (mg/L) OAscFab1 48.4 144
90 31.6 (SEQ ID NOs 133, 134, 135) OAscFab2 41 42.9 93.6 12.6 (SEQ
ID NOs 136, 137, 138) OAscFabPer1 41.6 43.4 88.8 4.6 (SEQ ID NOs
139, 140, 141) OAscFabPer2 38.2 34.2 86.7 6.6 (SEQ ID NOs 142, 143,
144) CrossMab-XTra 65.1 28.4 73 10.6 (SEQ ID NOs 119, 120, 121,
122) CrossMab-XPer 76.4 30.9 85 17.9 (SEQ ID NOs 109, 110, 96, 86)
CrossMab- 73 31.5 95 11.9 CDRG (SEQ ID NOs 109, 110, 111, 112)
Example 7: Expression & Purification Bispecific, Bivalent
<Her2GlyMab> Antibody Molecules Optimization of the Plasmid
Ratios Used in Expression
[0464] CrossMab-XTra
[0465] According the procedures described in the materials and
methods above, the bispecific, bivalent <Her2GlyMab> antibody
molecule CrossMab-XTra, was expressed with molar plasmid ratios of
1:1:1:1, 1:1:1:2, 1:1:1:4 and 1:1:1:8 and purified. Expression of
CrossMab-XTra was confirmed by Western blot. After Protein A
purification of cell culture supernatants the construct showed at a
1:1:1:1 equimolar plasmid ratio approximately 73% of bispecific
antibody with the expected molecular weight of approximately 148
kDa as detected by Q-TOF MS; 11% containing 2.times. Pertuzumab
light chains paired with Pertuzumab and XTrastuzumab heavy chains;
9% intact XTrastuzumab antibodies (both heavy and light chains
originating from XTrastuzumab) with the formation of the heavy
chain hole-hole association; 4% XTrastuzumab heavy chain hole-hole
association combined with Pertuzumab light chains only; and 3%
XTrastuzumab heavy chain hole-hole association with 1.times.
XTrastuzumab light chain and 1.times. Pertuzumab light chain.
[0466] The 1:1:1:2 plasmid ratio where the molar ratio of the
crossed Trastuzumab light chain (XHerLC) was expressed 2-fold,
showed approximately 81% of bispecific antibody with the expected
molecular weight of approximately 148 kDa as detected by Q-TOF MS;
1% containing 2.times. Pertuzumab light chains paired with
Pertuzumab and XTrastuzumab heavy chains; 16% intact XTrastuzumab
antibodies (both heavy and light chains originating from
XTrastuzumab) with the formation of the heavy chain hole-hole
association; XTrastuzumab heavy chain hole-hole association
combined with Pertuzumab light chains only were not detected; and
1% XTrastuzumab heavy chain hole-hole association with 1.times.
XTrastuzumab light chain and 1.times. Pertuzumab light chain.
[0467] The 1:1:1:4 plasmid ratio where the molar ratio of the
crossed Trastuzumab light chain was expressed 4-fold, showed
approximately 64% of bispecific antibody with the expected
molecular weight of approximately 148 kDa as detected by Q-TOF MS;
2.times. Pertuzumab light chains paired with Pertuzumab and
XTrastuzumab heavy chains was not detected, however, 12% 2.times.
XTrastuzumab light chains paired with Pertuzumab and XTrastuzumab
heavy chains was detected for the first time at this ratio; 24%
intact XTrastuzumab antibodies (both heavy and light chains
originating from XTrastuzumab) with the formation of the heavy
chain hole-hole association; XTrastuzumab heavy chain hole-hole
association combined with Pertuzumab light chains only were not
detected; XTrastuzumab heavy chain hole-hole association with
1.times. XTrastuzumab light chain and 1.times. Pertuzumab light
chain were not detected.
[0468] The 1:1:1:8 plasmid ratio where the molar ratio of the
crossed Trastuzumab light chain was expressed 8-fold, showed
approximately 45% of bispecific antibody with the expected
molecular weight of approximately 148 kDa as detected by Q-TOF MS;
2.times. Pertuzumab light chains paired with Pertuzumab and
XTrastuzumab heavy chains was not detected, however, 28% 2.times.
XTrastuzumab light chains paired with Pertuzumab and XTrastuzumab
heavy chains was detected for the first time at this ratio; 27%
intact XTrastuzumab antibodies (both heavy and light chains
originating from XTrastuzumab) with the formation of the heavy
chain hole-hole association; XTrastuzumab heavy chain hole-hole
association combined with Pertuzumab light chains only were not
detected; XTrastuzumab heavy chain hole-hole association with
1.times. XTrastuzumab light chain and 1.times. Pertuzumab light
chain were not detected.
TABLE-US-00013 TABLE 10 CrossMab-XTra Her2GlyMab - Q-TOF analysis
and quantification of the by-product profile of the Her2GlyMab
antibody constructs comparing the optimization plasmid titration of
the crossed Trastuzumab light chain (XHer LC). CrossMab- CrossMab-
CrossMab- CrossMab- Detected XTra XTra XTra XTra protein Her2GlyMab
Her2GlyMab Her2GlyMab Her2GlyMab species in MS 1:1:1:1 1:1:1:2
1:1:1:4 1:1:1:8 1 x XHer HC; N.D. N.D. ~12% ~28% 1 x Per HC; 2 x
XHer LC 2 x XHer HC; ~9% ~16% ~24% ~27% 2 x XHer LC CrossMab- ~73%
~81% ~64% ~45% XTra Her2GlyMab (100%) 2 x XHer HC; ~3% ~1% N.D.
N.D. 1 x XHer LC; 1 x Per HC 1 x XHer HC; ~11% ~1% N.D. N.D. 1 x
Per HC; 2 x Per LC 2 x XHer HC; ~4% N.D. N.D. N.D. 2 x Per LC
N.D.--Not Detected
[0469] CrossMab-XPer
[0470] After Protein A purification of cell culture supernatants
the construct showed at a 1:1:1:1 equimolar plasmid ratio
approximately 85% of bispecific antibody with the expected
molecular weight of approximately 148 kDa as detected by Q-TOF MS;
2% containing 2.times. XPertuzumab light chains paired with
XPertuzumab and Trastuzumab heavy chains; 1% intact Trastuzumab
antibodies (both heavy and light chains originating from
Trastuzumab) with the formation of the heavy chain hole-hole
association; 12% 2.times. Trastuzumab light chains paired with
XPertuzumab and Trastuzumab heavy chains. No further species were
detected.
[0471] The 1:1:1:2 plasmid ratio where the molar ratio of the
crossed Pertuzumab light chain (XPerLC) was expressed 2-fold,
showed approximately 89% of bispecific antibody with the expected
molecular weight of approximately 148 kDa as detected by Q-TOF MS;
7% containing 2.times. XPertuzumab light chains paired with
XPertuzumab and Trastuzumab heavy chains; intact Trastuzumab
antibodies (both heavy and light chains originating from
Trastuzumab) with the formation of the heavy chain hole-hole
association were not detected; 4% 2.times. Trastuzumab light chains
paired with XPertuzumab and Trastuzumab heavy chains.
[0472] The 1:1:1:4 plasmid ratio where the molar ratio of the
crossed Pertuzumab light chain (XPerLC) was expressed 4-fold,
showed approximately 74% of bispecific antibody with the expected
molecular weight of approximately 148 kDa as detected by Q-TOF MS;
25% containing 2.times. XPertuzumab light chains paired with
XPertuzumab and Trastuzumab heavy chains; intact Trastuzumab
antibodies (both heavy and light chains originating from
Trastuzumab) with the formation of the heavy chain hole-hole
association were not detected; 1% 2.times. Trastuzumab light chains
paired with XPertuzumab and Trastuzumab heavy chains.
[0473] The 1:1:1:8 plasmid ratio where the molar ratio of the
crossed Pertuzumab light chain (XPerLC) was expressed 8-fold,
showed approximately 52% of bispecific antibody with the expected
molecular weight of approximately 148 kDa as detected by Q-TOF MS;
48% containing 2.times. XPertuzumab light chains paired with
XPertuzumab and Trastuzumab heavy chains; intact Trastuzumab
antibodies (both heavy and light chains originating from
Trastuzumab) with the formation of the heavy chain hole-hole
association were not detected; 2.times. Trastuzumab light chains
paired with XPertuzumab and Trastuzumab heavy chains were not
detected.
[0474] CrossMab-CDRG
[0475] After Protein A purification of cell culture supernatants
the construct showed at a 1:1:1:1 equimolar plasmid ratio
approximately 95% of bispecific antibody with the expected
molecular weight of approximately 148 kDa as detected by Q-TOF MS
and 5% containing 2.times. XPertuzumab light chains paired with
XPertuzumab and Trastuzumab (HerCDRG) heavy chains. No further
species were detected, therefore, further optimization of the
plamid ratios was not performed. The MS spectra performed on the
antibodies CrossMab-XHer and CrossMab-CDRG to compare the byproduct
profile can be seen in FIGS. 12A-B.
TABLE-US-00014 TABLE 11 CrossMab-XPer Her2GlyMab-Q-TOF analysis and
quantification of the by-product profile of the Her2GlyMab antibody
constructs comparing the optimization plasmid titration of the
crossed Trastuzumab light chain (XPer LC). CrossMab- CrossMab-
CrossMab- CrossMab- Detected XPer XPer XPer XPer protein Her2GlyMab
Her2GlyMab Her2GlyMab Her2GlyMab species in MS 1:1:1:1 1:1:1:2
1:1:1:4 1:1:1:8 1 x Her HC; 1 x ~2% ~7% ~25% ~48% XPer HC; 2 x XPer
LC 2 x Her HC; 2 x ~1% n.d. n.d. n.d. Her LC CrossMab-XPer ~85%
~89% ~74% ~52% Her2GlyMab (100%) 1 x Her HC; 1 x ~12% ~4% ~1% n.d.
XPer HC; 2 x Her LC N.D.--Not Detected.
TABLE-US-00015 TABLE 12 CrossMab-CDRG Her2GlyMab-Q-TOF analysis and
quantification of the by-product profile of the Her2GlyMab antibody
construct detecting the presence of mispaired antibody heavy and
light chains CrossMab- CDRG Her2GlyMab Detected protein species in
MS 1:1:1:1 2 x XPer HC; 2 x XPer LC N.D. 2 x HerCDRG HC; 2 x
HerCDRG LC N.D. CrossMab-CDRG Her2GlyMab (100%) ~95% 1 x HerCDRG
HC; 1 x XPer HC; 2 x XPer LC ~5% N.D.--Not Detected
Example 8: Simultaneous Binding of Bispecific Antibodies to Both
Antigens
[0476] The binding of the bispecific antibody was analyzed via
BIAcore as described above.
[0477] In separate assay format samples (CrossMabXPer as well as
for OAscFab1 and OAscFab2) were proven to be functional for
Trastuzumab as well as Pertuzumab specificity. CrossMabXPer showed
kinetic constants and resulting affinities in the same order of
magnitude as the positive controls. Except for a slightly reduced
k.sub.a-rate constant for the Trastuzumab mediated binding OAscFab1
and OAscFab2 showed kinetic constants and resulting affinities in
the same order of magnitude as the positive controls i.e the
parental Mabs.
[0478] Partly bivalent binding of the positive controls--depending
on the ligand density on the CM5-Chip may cause the variation of
the dissociation rate constants in the two experiments depicted in
this example.
TABLE-US-00016 TABLE 13 SPR analysis of the Her2GlyMab affinities -
The association and dissociation rates of the antibodies were
measure using a BIAcoreT100 with a CM5-Chip at 25.degree. C. T =
25.degree. C. analyzed k.sub.a k.sub.d t(1/2) K.sub.D Experiment
function analyte [M.sup.-1 s.sup.-1] [s.sup.-1] [min] [M] UJ2530
"Trastuzumab" Trastuzumab 3.9E+05 8.7E-05 132.5 2.2E-10 UJ2530
"Trastuzumab" Pertuzumab no binding as expected UJ2530
"Trastuzumab" CrossMabXPer 1.0E+05 7.6E-05 151.9 7.4E-10 UJ2530
"Pertuzumab" Trastuzumab no binding as expected UJ2530 "Pertuzumab"
Pertuzumab 4.7E+05 9.8E-05 118.1 2.1E-10 UJ2530 "Pertuzumab"
CrossMabXPer 2.0E+05 1.8E-04 66.0 8.7E-10 UJ2530_b "Trastuzumab"
Trastuzumab 7.0E+05 3.0E-05 382.9 4.3E-11 UJ2530_b "Trastuzumab"
Pertuzumab no binding as expected UJ2530_b "Trastuzumab" OAscFab1
1.6E+04 8.7E-05 133.5 5.4E-09 UJ2530_b "Trastuzumab" OAscFab2
1.8E+04 1.1E-04 100.6 6.2E-09 UJ2530_b "Pertuzumab" Trastuzumab no
binding as expected UJ2530_b "Pertuzumab" Pertuzumab 2.4E+05
2.6E-04 44.8 1.1E-09 UJ2530_b "Pertuzumab" OAscFab1 1.2E+05 2.6E-04
44.3 2.2E-09 UJ2530_b "Pertuzumab" OAscFab2 1.7E+05 2.6E-04 44.8
1.5E-09
Example 9: In Vitro Evaluation of 1+1 Herceptarg CrossMAb and
Glyocoengineered Herceptarg Crossmab
[0479] Proliferation Inhibition Assay
[0480] AlamarBlue.RTM. (Invitrogen) was used for the measurement of
the metabolic activity and proliferation of (A) BT474 and (B) N87
cells after a 5 day incubation in presence of HER2 CrossMab
(CrossMab-XTra Her2GlyMab, SEQ ID NOs 119, 120, 121, 122),
Trastuzumab, Pertuzumab or the combination of
Trastuzumab/Pertuzumab. The bioreduction of the dye reduces the
amount of the oxidized form (blue) and concomitantly increases the
fluorescent intermediate (red).
[0481] Target cells were harvested, washed, resuspended in RPMI
1640 (Gibco)+10% FCS+1% GlutaMAX.TM. (Gibco) and plated at a
concentration of 1.times.10.sup.4 cells/well. Cells were incubated
for 3 hours in the cell incubator before respective antibody
dilutions were added. Plates were gently shaked and incubated for 5
days in the cell incubator.
[0482] 25 .mu.l/well of Alamar Blue were added to the plate and
incubated for 7 h in the incubator.
[0483] Absorbance was monitored at 584 nm and 612 nm in a Wallac
Victor3 1420 Multilabel Counter. For calculation of the percentage
of proliferation inhibition, non-treated controls samples were
included in the assay and defined as 100% proliferation. The
average percentage of proliferation inhibition of the triplicates
of each experiment was calculated.
[0484] Results are shown in FIGS. 13A-B.
[0485] ADCC Assay
[0486] ADCC mediated by HER2 CrossMab (CrossMab-XTra Her2GlyMab,
SEQ ID NOs 119, 120, 121, 122), Trastuzumab, Pertuzumab or the
combo of Trastuzumab/Pertuzumab was assessed on KPL-4 (A), T47D (B)
and Calu-3 (C) cells.
[0487] Target cells were harvested, washed, resuspended in AIM
V.RTM. medium (Life Technologies), and plated at a concentration of
3.times.10.sup.4 cells/well. The respective antibody dilutions were
added in triplicates to the cells and incubated for 10 min before
addition of the effector cells (peripheral blood mononuclear
effector cells [PBMCs]). Effector (E) and target (T) cells were
then incubated for 4 h at 37.degree. C. at an E:T ratio of 25:1
(triplicates for all samples). Lactate dehydrogenase (LDH) release
was measured using the LDH Cytotoxicity Detection Kit (Roche
Applied Science). ADCC was calculated using the following
formula:
Percentage A D C C = ( [ sample release - spontaneous release
maximal release - spontaneous release ] ) .times. 100.
##EQU00002##
[0488] Spontaneous release, corresponding to target cells incubated
with effector cells without antibody, was defined as 0%
cytotoxicity, with maximal release (target cells lysed with 1%
Triton X-100) defined as 100% cytotoxicity. The average percentage
of ADCC and standard deviations of the triplicates of each
experiment were calculated. Results are shown in FIGS. 14A-C.
Example 10: In Vivo Characterization of HER2 CrossMab: Effect of
Bispecific Antibodies Targeting HER2 on Tumor Growth in Calu3 Lung
Cancer and KPL4 Breast Cancer Xenograft
[0489] In Vitro Cultured Cells--Calu3
[0490] This human lung adenocarcinoma cancer cell line has been
established from a human caucasian male with lung cancer. Cells
were obtained from Chugai Pharmaceuticals Co., Ltd. and passaged in
house for working cell bank. Tumor cells are routinely cultured in
RPMI medium (PAN Biotech, Germany) supplemented with 10% fetal
bovine serum glutamine (PAN Biotech, Germany) at 37.degree. C. in a
water-saturated atmosphere at 5% CO2. Culture passage is performed
with trypsin/EDTA 1.times. (PAN) splitting twice/week. Cell passage
P6 is used for in vivo study.
[0491] In Vitro Cultured Cells--KPL-4
[0492] This human breast cancer cell line has been established from
the malignant pleural effusion of a breast cancer patient with an
inflammatory skin metastasis. Cells have been provided by Professor
J. Kurebayashi (Kawasaki Medical School, Kurashiki, Japan). Tumor
cells are routinely cultured in DMEM medium (PAN Biotech, Germany)
supplemented with 10% fetal bovine serum (PAN Biotech, Germany) and
2 mM L-glutamine (PAN Biotech, Germany) at 37.degree. C. in a
water-saturated atmosphere at 5% CO2. Culture passage is performed
with trypsin/EDTA 1.times. (PAN) splitting twice/week. Cell passage
P6 is used for in vivo study.
[0493] Animals
[0494] Female SCID beige (C.B.-17) mice; age 10-12 weeks; body
weight 18-20 g (Charles River Germany, Sulzfeld) or female BALB/C
nu/nu mice; age 8-10 weeks; body weight >20 g (Bomholtgard,
Denmark) are maintained under specific-pathogen-free condition with
daily cycles of 12 h light/12 h darkness according to international
guidelines (GV-Solas; Felasa; TierschG). After arrival animals are
housed in the quarantine part of the animal facility for one week
to get accustomed to new environment and for observation.
Continuous health monitoring is carried out on regular basis. Diet
food (Alltromin) and water (acidified pH 2.5-3) are provided ad
libitum. The experimental study was reviewed and approved by local
government; registration no. 55.2-1-54-2531.2-3-08 Scid-beige
orthotop rodent breast cancer model and 211-2531.2-16/00. 1.2.2
subcutan tumor model.
[0495] Tumor Cell Injection
[0496] At the day of injection tumor cells are harvested
(trypsin-EDTA) from culture flasks (Greiner TriFlask) and
transferred into 50 ml culture medium, washed once and resuspended
in PBS. After an additional washing step with PBS and filtration
(cell strainer; Falcon O 100 .mu.m) the final cell titer is
adjusted to 1.5.times.108/ml. Tumor cell suspension is carefully
mixed with transfer pipette to avoid cell aggregation. Anesthesia
is performed using a Stephens inhalation unit for small animals
with preincubation chamber (plexiglas), individual mouse nose-mask
(silicon) and not flammable or explosive anesthesia compound
Isoflurane (Pharmacia-Upjohn, Germany) in a closed circulation
system. Two days before injection, coat of the SCID beige mice are
shaved. For subcutaneous injection of Calu3 cells, skin of
anaesthetized animals is carefully lifted up with an anatomic
forceps and 100 .mu.l cell suspension (=5.0.times.10e6 cells) is
injected subcutaneously in the right flank of the animals. Cell
suspension is filled into a 1.0 ml tuberculin syringe (Braun,
Melsungen) using a wide injection needle (0.45.times.25 mm). KPL-4
cells (3.times.10e6 cells) are injected orthotopically in a volume
of 20 .mu.l into the right penultimate inguinal mammary fat pad of
each anesthetized mouse. For the orthotopic implantation, the cell
suspension is injected through the skin under the nipple using a
using a Hamilton microliter syringe and a 30G.times.1/2''
needle.
[0497] Monitoring
[0498] Animals are controlled daily for detection of clinical
symptoms of adverse effects. For monitoring throughout the
experiment the body weight of the animals is documented two times
weekly and the tumor volume is measured by caliper twice weekly.
Tumor volume was calculated according to NCI protocol (Tumor
weight=1/2ab.sup.2, where "a" and "b" are the long and the short
diameters of the tumor, respectively). Termination criteria were
the critical tumor mass (up to 1.7 g or O>1.5 cm), body weight
loss more than 20% from baseline, tumor ulceration or poor general
condition of the animals. Study exclusion criteria for the animals
are described and approved in the corresponding
"Tierversuchsanzeige".
[0499] Treatment of Animals
[0500] Mice were randomized for tumor volume, for KPL-4 a mean of
80 mm.sup.3, for Calu3 a mean of 100 mm.sup.3. Mice were treated
once weekly with a volume of 10 ml/kg intra peritoneal. For
combination treatment Trastuzumab was given first and Pertuzumab
was given 24 hrs thereafter. Results are shown in Tables 14 to 17
and FIGS. 15A-C, 16A-C, 17A-C, and 18A-C.
TABLE-US-00017 TABLE 14 BispecHer2_Pz_Calu3_001 (FIGS. 15A-C):
CrossMAb_003 non-ge: non glycoengineered CrossMab-XTra Her2GlyMab
(SEQ ID NOs 119, 120, 121, 122), negative control: anti-IgE
antibody (Omalizumab), Herceptarg 2 + 2 OmniE: Pertuzumab antibody
with the Trastuzumab scFV added onto the c-terminus of the heavy
chains. The trastuzumab scFv contains a stabilizing disulphide bond
between VH 105-VL 43 (SEQ ID NOs: 145, 146), TvAb12 and TvAb20:
scFv 2 + 2 HER2 bispecific antibodies, see example 2. Cumulative No
of Dose Route/Mode of No of dose Group animals Compound (mg/kg)
administration treatments (mg/kg) 1 8 negative control 10 i.p. once
weekly 7 70 2 8 Trastuzumab + 10 i.p. once weekly 7 70 Pertuzumab
10 i.p. once weekly 7 70 3 8 Herceptarg 2 + 13.5 i.p. once weekly 7
94.5 2 OmniE 4 8 CrossMAb_003 20 i.p. once weekly 7 140 non-ge 5 8
TvAb12 11.3 i.p. once weekly 7 79.1 6 8 TvAb20 11.4 i.p. once
weekly 7 79.8
TABLE-US-00018 TABLE 15 BispecHER2_PZ_KPL-4_002 (FIGS. 16A-C):
CrossMAb_003 non-ge: non glycoengineered CrossMab-XTra Her2GlyMab
(SEQ ID NOs 119, 120, 121, 122), negative control: anti-IgE
antibody (Omalizumab), Herceptarg 2 + 2 OmniE: Pertuzumab antibody
with the Trastuzumab scFV added onto the c-terminal of the heavy
chains. The trastuzumab scFv contains a stabilizing disulphide bond
between VH 105-VL 43 (SEQ ID NOs: 145, 146), TvAb12 and TvAb20:
scFv 2 + 2 HER2 bispecific antibodies, see example 2. Cumulative No
of Dose Route/Mode of No of dose Group animals Compound (mg/kg)
administration treatments (mg/kg) 1 9 Negative control 10 i.p. once
weekly 5 50 2 9 Trastuzumab + 10 i.p. once weekly 5 50 Pertuzumab
10 i.p. once weekly 50 3 9 Herceptarg 2 + 13.5 i.p. once weekly 5
67.5 2 OmniE 4 9 CrossMAb_003 20 i.p. once weekly 5 100 non-ge 5 9
TvAb12 11.3 i.p. once weekly 5 56.5 6 9 TvAb20 11.42 i.p. once
weekly 5 57.1
TABLE-US-00019 TABLE 16 Bispec.HER2_PZ_KPL-4_003 (FIGS. 17A-C):
CrossMAb_005 non glycoengineered CrossMab-XTra Her2GlyMab (SEQ ID
NOs 119, 120, 121, 122), negative control: anti-IgE antibody
(Omalizumab). Cumulative No of Dose Route/Mode of No of dose Group
animals Compound (mg/kg) administration treatments (mg/kg) 1 10
Negative control 10 i.p. once weekly 5 50 2 10 Trastuzumab + 10
i.p. once weekly 5 50 Pertuzumab 10 i.p. once weekly 50 3 10
Her2_Crossmab_005 20 i.p. once weekly 5 100 4 10 Her2_Crossmab_005
10 i.p. once weekly 5 50 5 10 Her2_Crossmab_005 5 i.p. once weekly
5 25 6 10 Her2_Crossmab_005 1 i.p. once weekly 5 5
TABLE-US-00020 TABLE 17 Exploratory_PZ_KPL-4_009 (FIGS. 18A-C):
negative control: anti-IgE antibody (Omalizumab), TvAb16 and
TvAb20: scFv 2 + 2 HER2 bispecific antibodies, see example 2.
Cumulative No of Dose Route/Mode of No of dose Group animals
Compound (mg/kg) administration treatments (mg/kg) 1 10 Negative 10
i.p. once weekly 4 40 control 2 10 Trastuzumab + 10 i.p. once
weekly 4 40 Pertuzumab 10 i.p. once weekly 4 40 3 10 Trastuzumab +
5 i.p. once weekly 4 20 Pertuzumab 5 i.p. once weekly 4 20 4 10
TvAb16 10 i.p. once weekly 4 40 5 10 TvAb16 5 i.p. once weekly 4 20
6 10 TvAb20 10 i.p. once weekly 4 40 7 10 TvAb20 5 i.p. once weekly
4 20
Example 11: Generation of a Common Light Chain for Trastuzumab and
Pertuzumab
[0501] Gene Synthesis
[0502] Desired gene segments, where required, were either generated
by PCR using appropriate templates or were synthesized at Geneart
AG (Regensburg, Germany) from synthetic oligonucleotides and PCR
products by automated gene synthesis. In cases where no exact gene
sequence was available, oligonucleotide primers were designed based
on sequences from closest homologues and the genes were isolated by
RT-PCR from RNA originating from the appropriate tissue. The gene
segments flanked by singular restriction endonuclease cleavage
sites were cloned into standard cloning/sequencing vectors. The
plasmid DNA was purified from transformed bacteria and
concentration determined by UV spectroscopy. The DNA sequence of
the subcloned gene fragments was confirmed by DNA sequencing. Gene
segments were designed with suitable restriction sites to allow
subcloning into the respective expression vectors. All constructs
were designed with a 5'-end DNA sequence coding for a leader
peptide which targets proteins for secretion in eukaryotic cells.
SEQ ID NOs: 155, 156, and 157 give exemplary leader peptides.
[0503] Cloning of Antigen Expression Vectors
[0504] A DNA fragment encoding amino acids 1 to 629 of matured
Tyrosine kinase-type cell surface receptor HER2 (Her2, Uniprot:
P04626) was cloned in frame into a mammalian recipient vector
containing an N-terminal leader sequence. In addition, the
construct contains a C-terminal avi-tag allowing specific
biotinylation during co-expression with Bir A biotin ligase and a
His-tag used for purification by immobilized-metal affinity
chromatography (IMAC) (SEQ ID NOs 1 and 2).
[0505] The antigen expression is generally driven by an MPSV
promoter and transcription is terminated by a synthetic polyA
signal sequence located downstream of the CDS. In addition to the
expression cassette, each vector contains an EBV oriP sequence for
autonomous replication in EBV-EBNA expressing cell lines.
[0506] Production and Purification of Antigens and Antibodies
[0507] Both antigens and antibodies were transiently transfected
into HEK 293 cells, stably expressing the EBV-derived protein EBNA.
A simultaneously co-transfected plasmid encoding biotin ligase Bir
A allowed avi tag-specific biotinlylation in vivo. The proteins
were then purified using a protein A column followed by gel
filtration.
[0508] Design of Trastuzumab/Pertuzumab Common Light Chains
[0509] For the generation of a common light chain (CLC) for
Trastuzumab and Pertuzumab, the individual light chains (LC) were
analyzed and compared. Sequence analysis revealed that both
variable domains originate from the same germline sequence. Given
that the Trastuzumab LCDR3 in general and residue H91 in particular
interact specifically with the Trastuzumab-specific epitope on
Her2, the first attempt to create a Trastuzumab/Pertuzumab CLC was
done as follows: Either the complete LCDR3 region or only residue
H91 of Trastuzumab substituted the corresponding positions in the
Pertuzumab LC. As a result, a hybrid LC construct was created
encoding Pertuzumab-derived LCDR1 and 2 but harboring
Trastuzumab-derived LCDR3 amino acid residues. The resulting CLCs,
named either "Pertuzumab (Tras.L3) LC" (DNA sequence of variable
domains listed as SEQ ID NO: 25) or "Pertuzumab (Tras.Y91H) LC"
(SEQ ID NO: 27), were co-expressed with either the Trastuzumab or
the Pertuzumab HC. The resulting four antibodies (protein sequence
of variable domains listed as SEQ ID NOs: 22 and 26, "Pertuzumab
HC" x "Pertuzumab (Tras.L3) LC"; SEQ ID NO: 92 and 26, "Trastuzumab
HC" x "Pertuzumab (Tras.L3) LC"; SEQ ID NOs: 22 and 28, "Pertuzumab
HC" x "Pertuzumab (Trast.Y91H) LC"; SEQ ID NO: 92 and 28,
"Trastuzumab HC" x "Pertuzumab (Tras.Y91H) LC") were purified from
mammalian-derived cell culture supernatant and binding to Her2 was
measured and compared with the respective parental antibodies (SEQ
ID NOs: 22 and 24, "Pertuzumab HC" x "Pertuzumab LC"; SEQ ID NO: 92
and 82, "Trastuzumab HC" x "Trastuzumab LC") by SPR.
[0510] Affinity-Determination by SPR Using BioRad's ProteOn XPR36
Biosensor
[0511] The Affinity (K.sub.D) of the new antibody chain
combinations was measured by surface plasmon resonance using a
ProteOn XPR36 instrument (Biorad) at 25.degree. C. In a first step,
6500 RU of anti-human IgG (Sigma 12136, polyclonal goat antibody)
recognizing hu IgG (Fc-specific) was immobilized on all 6 channels
of a GLM chip by Amine coupling(NaAcetate pH4, 30 .mu.l/min, 300s)
(vertical orientation).
[0512] Each antibody was diluted with PBST (10 mM phosphate, 150 mM
sodium chloride pH 7.4, 0.005% Tween 20) to 2 .mu.g/ml, and then
injected for 60s at 30 .mu.l/minute to achieve immobilization
levels of about 400 response units (RU) in vertical orientation.
Injection of Her2: For one-shot kinetics measurements, injection
direction was changed to horizontal orientation, three-fold
dilution series of purified Her2 (varying concentration ranges
between 300 and 3.7 nM) were injected simultaneously at 100
.mu.l/min along separate channels 1-5, with association times of
180s, and dissociation times of 600s. Buffer (PBST) was injected
along the sixth channel to provide an "in-line" blank for
referencing. Regeneration was performed by two pulses of 10 mM
glycine pH 1.5 and 50 mM NaOH for 30s at 100 .mu.l/min (horizontal
orientation). Association rate constants (k.sub.on) and
dissociation rate constants (k.sub.off) were calculated using a
simple one-to-one Langmuir binding model in ProteOn Manager v3.1
software by simultaneously fitting the association and dissociation
sensorgrams. The equilibrium dissociation constant (K.sub.D) was
calculated as the ratio k.sub.off/k.sub.on.
[0513] As expected, both control antibodies Trastuzumab and
Pertuzumab recognized Her2 with their known affinities in the low
nanomolar or sub-nanomolar range. However, while the affinity of
Pertuzumab HC in combination with the newly designed "Pertuzumab
(Tras.Y91H) LC" was slightly reduced, the same light chain in
combination with Trastuzumab HC did not yield any detectable
binding. This indicates that a single Trastuzumab-derived point
mutation (Y91H) in the Pertuzumab LC is not sufficient translate
binding to Her2 in this chain combination. This is in contrast to
the second CLC variant "Pertuzumab LC (Trast. L3)" which resulted
in weak binding when combined with Trastuzumab HC, while binding
was reduced but clearly visible when co-expressed with the
Pertuzumab HC. A summary of the kinetic and thermodynamic
measurements is given in FIGS. 19A-F and Table 18. Based on this
finding, the CLC "Pertuzumab LC (Trast. L3)" was further modified
in order to restore Her2 binding in combination with Trastuzumab HC
while affecting the affinity as little as possible when
co-expressed with Pertuzumab HC.
TABLE-US-00021 TABLE 18 Kinetic and thermodynamic parameters of
Trastuzumab, Pertuzumab, and hybrid molecules thereof Clone ka
[1/Ms] kd [1/s] KD [M] Trastuzumab 1.01E+05 2.24E-04 2.21E-09
Pertuzumab 8.08E+04 6.69E-05 1.47E-10 Pertuzumab HC 6.47E+04
1.73E-03 2.67E-08 Pertuzumab (Tras.L3) LC Pertuzumab HC 8.99E+04
1.98E-04 2.21E-09 Pertuzumab(Tras.Y91H) LC Trastuzumab HC 4.19E+04
0.05 1.09E-06 Pertuzumab(Tras.L3) LC Trastuzumab HC N/A N/A N/A
Pertuzumab(Tras.Y91H) LC
Example 12: Affinity-Improvement of the Common Light Chain
[0514] Introduction and Characterization of Additional
Trastuzumab-Specific LCDR Residues into the Pertuzumab (Tras.L3)
LC
[0515] Based on the results of the previous SPR measurement,
binding of the designed CLC
[0516] "Pertuzumab (Trast. L3) LC" in combination with the
Pertuzumab HC led to a reduced but still well detectable binding.
In contrast, co-expression of the CLC with Trastuzumab HC resulted
in a very weak affinity in the micromolar range and was
significantly reduced compared to the parental Trastuzumab
antibody.
[0517] In order to further evolve the generation of a CLC and
improve the binding in combination with the Trastuzumab HC,
additional Trastuzumab-specific amino acid of the LCDR1, 2, and 3
as well in framework 3 region that are specific for Trastuzumab
were individually introduced into the sequence of the previously
designed "Pertuzumab (Trast.L3) LC" (DNA sequence of variable
domains listed as SEQ ID NO: 31, 33, 35, 37, 39, 41, 43, 45, 47,
49, and 51). The resulting constructs (protein sequence of variable
domains listed as SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, 46, 48,
50, and 52) were co-expressed with the Trastuzumab HC (SEQ ID NO:
92) or the Pertuzumab HC (SEQ ID NO: 22) and purified from
mammalian-derived cell culture supernatant. Binding to Her2 was
measured and compared with the respective parental antibodies.
[0518] Affinity-Determination of the "Pertuzumab (Trast.L3) LC"
Variants by SPR
[0519] The Affinity (K.sub.D) of the new antibody chain
combinations was measured by surface plasmon resonance using a
ProteOn XPR36 instrument (Biorad) and was performed as described
before. A summary of the kinetic and thermodynamic measurements is
given in table 19.
[0520] Interestingly, none of the additional single mutations in
the "Pertuzumab (Tras.L3) LC" variants significantly affected the
affinity when combined with the Pertuzumab and the affinity was
comparable to "Pertuzumab HC"x"Pertuzumab (Tras.L3) LC". However,
combination of the "Pertuzumab (Tras.L3) LC" variants with the
Trastuzumab HC resulted in significant differences. While back
mutations to the Pertuzumab sequence in the LCDR3 region (P94Y and
T96Y) (protein sequence of variable domains listed as SEQ ID NOs:
50 and 52) completely abolished binding to Her2, introduction of
Pertuzumab-specific mutations (SEQ ID NOs: 32, 34, 36, 38, 40, 42,
44, 46, and 48) yielded an equal or improved affinity compared to
the initial combination "Trastuzumab HC" x "Pertuzumab (Tras.L3)
LC". In particular, introduction of the mutations I31T, G32A, Y53F,
and G66R led to the most significant improvement in binding.
[0521] Simultaneous Introduction of Most Significant
Trastuzumab-Specific Residues into the "Pertuzumab (Tras.L3)
LC"
[0522] Based on the binding measurement described above,
introduction of the 4 most relevant Trastuzumab-specific mutations
into the "Pertuzumab (Tras.L3) LC" was combined. The protein
sequences of the variable domains containing either the individual
mutations are listed as SEQ ID NOs: 36, 40, 42, and 48.) The chain
containing the "quadruple mutation" was named "Pertuzumab (Tras.L3)
(QM) LC" (SEQ ID NOs: 54).
[0523] Affinity-Determination of the "Pertuzumab (Trast.L3)(QM) LC"
by SPR
[0524] In order to characterize the new "Pertuzumab (Tras.L3) (QM)
LC" and to determine the binding affinity when combined with either
Pertuzumab HC or Trastuzumab HC, a further SPR experiment was
performed. The Affinity (K.sub.D) of the new antibody chain
combinations was measured using a ProteOn XPR36 instrument (Biorad)
and was performed as described before. A summary of the kinetic and
thermodynamic measurements is given in FIGS. 20A-B and Table
20.
[0525] Similar to the individual mutations that were introduced and
characterized before, the affinity of the antibody comprising the
Pertuzumab HC and the "Pertuzumab (Tras.L3) (QM) LC" did not
significantly decrease compared to the initial chain combination
"Pertuzumab HC" x "Pertuzumab (Tras.L3) LC". For both chain
combinations, the affinity was in the range of 26 to 34 nM.
Interestingly, the combination of the "Trastuzumab HC" with the
"Pertuzumab (Tras.L3) (QM) LC" leads to a very strong improvement
of the affinity to Her2. Furthermore, its affinity of 200 pM is
even better than the measured affinity of the parental antibody
Trastuzumab (2.2 nM). Given that the combination of "Pertuzumab
(Tras.L3) (QM) LC" with Trastuzumab HC fully restores (or even
exceeds) binding to Her2 and considering that the combination with
the Pertuzumab HC diminishes but not abrogates binding to Her2, it
was apparent to use the "Pertuzumab (Tras.L3) (QM) LC" as a CLC for
both Her2 specificities and restore binding to the
Pertuzumab-specific epitope by affinity-maturation of the
Pertuzumab HC.
TABLE-US-00022 TABLE 19 Kinetic and thermodynamic parameters of
antibodies comprising either a Trastuzumab or Pertuzumab HC in
combination of additional CLC hybrid molecules Pertuzumab HC
Trastuzumab HC Clone ka [1/Ms] kd [1/s] KD [M] ka [1/Ms] kd [1/s]
KD [M] Tras.L3 4.57E+05 2.11E-03 4.62E-09 3.77E+05 2.52E-02
6.68E-08 (K24R) Tras.L3 5.30E+05 1.80E-03 3.40E-09 3.96E+05
2.56E-03 6.46E-08 (S30N) Tras.L3 4.31E+05 1.75E-03 4.06E-09
4.73E+05 3.41E-03 7.21E-09 (I31T) Tras.L3 4.40E+05 1.92E-03
4.35E-09 4.13E+05 1.16E-02 2.80E-08 (I31V) Tras.L3 5.11E+05
1.87E-03 3.66E-09 6.75E+05 2.68E-03 3.97E-09 (G32A) Tras.L3
3.16E+05 2.54E-03 8.03E-09 3.81E+05 1.71E-02 4.49E-08 (Y53F)
Tras.L3 4.64E+05 2.26E-03 4.87E-09 4.16E+05 2.97E-02 7.15E-08
(R54L) Tras.L3 4.43E+05 2.45E-03 5.52E-09 4.70E+05 2.32E+02
4.94E-08 (T56S) Tras.L3 3.58E+05 2.47E-03 6.91E-09 5.14E+05
5.58E-03 1.09E-08 (G66R) Tras.L3 8.05E+05 6.30E-04 7.82E-10 N/A N/A
N/A (T94Y) Tras.L3 3.44E+05 9.70E-04 2.82E-09 N/A N/A N/A (P96Y)
Tras.L3 4.22E+05 2.09E-03 4.96E-09 3.25E+05 2.36E-02 7.25E-08
TABLE-US-00023 TABLE 20 Kinetic and thermodynamic parameters of
antibodies comprising either a Trastuzumab or Pertuzumab HC and the
final CLC Clone ka [1/Ms] kd [1/s] KD [M] Pertuzumab HC 9.46E+04
3.24E-03 3.42E-08 Pertuzumab (Tras.L3)(QM) LC Trastuzumab HC
2.69E+05 5.42E-05 2.02E-10 Pertuzumab (Tras.L3)(QM) LC
Example 13: Affinity Maturation of the Pertuzumab Heavy Chain
[0526] Generation of Pertuzumab-Based 111/113 and 112 Affinity
Maturation Libraries
[0527] Generation of affinity-matured Pertutzumab-derived heavy
chains was carried out by phage display using standard protocols
(Silacci et al, 2005).
[0528] For the generation of Pertuzumab-derived HCs with improved
affinity when jointly expressed with "Pertuzumab (Tras.L3) (QM) LC"
a maturation library randomized in CDR1 and 3 or in CDR2 was
generated. The sequence of the Pertuzumab HC (SEQ ID NO: 22) and of
the "Pertuzumab (Tras.L3) (QM) LC" (SEQ ID NO: 54) was cloned into
a phagemid and used as a template for the randomization. For the
generation of the Pertuzumab HC affinity maturation library
randomized in CDR1 and 3, three fragments were assembled by
"splicing by overlapping extension" (SOE) PCR and cloned into the
phage vector. The following primer combinations were used to
generate the library fragments: fragment 1 (LMB3 (SEQ ID NO: 147)
and AM_omni_H1_TN-ba (SEQ ID NO: 148), fragment 2 (RJH108
(omni_3'H1_fo) (SEQ ID NO: 149) and RJH109 (omni_5'H3_re) (SEQ ID
NO: 150), and fragment 3 (AM_omni_H3_TN_fo (SEQ ID NO: 151) and
RJH99 (SEQ ID NO: 152). After assembly of sufficient amounts of
full length randomized fragment, it was digested with MunI/NheI
alongside with identically treated acceptor phagemid vector. bug of
Fab library insert were ligated with 24 ug of phagemid vector.
Purified ligations were used for 60 transformations resulting in
6.times.10 exp9 transformants. Phagemid particles displaying the
Pertuzumab affinity maturation library were rescued and purified by
PEG/NaCl purification to be used for selections.
[0529] The generation of the CDR2-randomized Pertuzumab HC affinity
maturation library was done similarly, but only 2 fragments were
generated, assembled and cloned into the phagemid using the same
restriction enzymes as before. The following primer combinations
were used to generate the library fragments: fragment 1 (LMB3 (SEQ
ID NO: 147) and RJH110 (omni_5'H2_ba) (SEQ ID NO: 153) and fragment
2 (AM_omni_h2_TN_fo (SEQ ID NO: 154) and RJH99 (SEQ ID NO:152).
Purified ligations were used for 60 transformations resulting in
4.times.10 exp9 transformants. Phagemid particles displaying the
Pertuzumab affinity maturation library were rescued and purified by
PEG/NaCl purification to be used for selections.
TABLE-US-00024 TABLE 21 a) Primer combinations for the generation
of the CDR1 and 3-randomized Pertuzumab affinity-maturation library
Pertuzumab HC affinity maturation (CDR1 and 3) fragment 5'Primer
3'Primer PCR1 LMB3 AM_omni_H1_TN-ba PCR2 RJH108 (omni _3'H1_fo)
RJH109 (omni _5'H3_re) PCR3 AM_omni_H3_TN_fo RJH99
TABLE-US-00025 TABLE 21 b) Primer sequences for the generation of
the CDR1 and 3-randomized Pertuzumab affinity-maturation library
Pertuzumab HC affinity maturation (CDR1 and 3) SEQ ID Name Sequence
147 LMB3 CAGGAAACAGCTATGACCATGATTAC 148 AM_omni_
CCGGTGCCTGACGAACCCAATCCAT 4 3 2 1 H1_TN-ba
AAAGGTAAAACCGCTTGCTGCACAGCTC 1 T = 60%, S/G/R/N/D = 20% (4% each),
rest = 20% (1.7% each) 2 D = 60%, S/N/T/A/R/E/Q/G = 30% (3.8%
each), rest = 10% (1.1% each) 3 Y = 60%, F/S/H/N/D/T = 30% (5.0%
each), rest = 10% (0.9%) 4 T = 60%, A/G/V/S/P/D/N = 30% (4.3%
each), rest = 10% (1.0%) 149 RJH108
ATGGATTGGGTTCGTCAGGCACCGGGTAAAGG (omni_3' H1_fo) 150 RJH109
ATTACGTGCACAATAATACACTGCGGTATCCTC (omni_5' H3_re) 151 AM_omni
TACCGCAGTGTATTATTGTGCACGT 4a 5 5a 6 7 TTC H3_TN_fo 8
TTTGATTATTGGGGTCAGGGCACCCTGGTTAC 4a N = 60%,
G/D/E/Q/V/S/A/P/R/L/T/Y = 40% (3.3% each) 5 L = 60%,
G/Y/S/A/D/T/R/P/V/N/W/F/I/E = 40% (2.9% each) 5a G = 60%,
Y/S/A/D/T/R/P/L/V/N/W/F/I/E = 40% (2.9% each) 6 P = 60%,
G/Y/S/A/D/T/R/L/V/N/W/F/I/E = 40% (2.9% each) 7 S = 60%,
G/Y/P/A/D/T/R/L/V/N/W/F/I/E = 40% (2.9% each) 8 Y = 60%,
G/A/P/W/S/D/T/F/R/K/H = 40% (3.6% each) 152 RJH99
GGCTGAGACTCCTCAAGAGAAGGATTAG
TABLE-US-00026 TABLE 22 a) Primer combinations for the generation
of the CDR2-randomized Pertuzumab affinity-maturation library
Pertuzumab HC affinity maturation (CDR2) fragment 5'Primer 3'Primer
PCR1 LMB3 RJH110(omni_5'H2_ba)
TABLE-US-00027 TABLE 22 b) Primer combinations for the generation
of the CDR2-randomized Pertuzumab affinity-maturation library
Pertuzumab HC affinity maturation (CDR2) SEQ ID Name Sequence 153
RJH110 ATTAACATCTGCAACCCATTCCAGACCTTTAC (omni_5' H2_ba) 154
AM_omni_ GGTCTGGAATGGGTTGCAGATGTTAAT 9 10 11 12 h2_TN_fo GGT 13 ATT
14 AC 15 CGTTTTAAAGGTCGTTTTACCCTGAG 9 P = 60%, G/A/S/T/D/N/F/Y =
30% (3.8% each), rest = 10% (1.0% each) 10 N = 60%, S/D/G/T/R/A =
30% (5.0% each), rest = 10% (0.8%) 11 S = 60%, G = 10%, rest = 30%
(1.8% each)
[0530] Selection of Affinity Matured Pertuzumab HC-Derived
Clones
[0531] Selections against the extracellular domain (ECD) of human
Her2 were carried out using HEK293-expressed protein. The antigen
was enzymatically biotinylated by co-expression of the biotin
ligase Bir A via an N-terminal avi-tag. Panning rounds were
performed in solution according to the following pattern: 1.
binding of .about.10.sup.12 phagemid particles to 10 nM
biotinylated Her2 ECD for 0.5 h in a total volume of 1 ml, 2.
capture of biotinylated Her2 ECD and specifically bound phage
particles by addition of 5.4.times.10.sup.7 streptavidin-coated
magnetic beads for 10 min, 3. washing of beads using 5.times.1 ml
PBS/Tween20 and 5.times.1 ml PBS, 4. elution of phage particles by
addition of 1 ml 100 mM TEA for 10 min and neutralization by adding
500 .mu.l M Tris/HCl pH 7.4, 5. re-infection of exponentially
growing E. coli TG1 bacteria, and 6.infection with helperphage
VCSM13 and subsequent PEG/NaCl precipitation of phagemid particles
to be used in subsequent selection rounds. Selections were carried
out over 3 rounds using decreasing (from 20.times.10.sup.-9M to
1.times.10.sup.-9M) antigen concentrations. In round 2 and 3,
capture of antigen:phage complexes was performed using neutravidin
plates instead of streptavidin beads. In addition, neutravidin
plates were washed for 3 h in 2 l PBS. Specific binders were
identified by ELISA as follows: 100 .mu.l of 30 nM biotinylated
Her2 ECD per well were coated on neutravidin plates. Fab-containing
bacterial supernatants were added and binding Fabs were detected
via their Flag-tags by using an anti-Flag/HRP secondary antibody.
ELISA-positive clones were bacterially expressed as soluble Fab
fragments in 96-well format and supernatants were subjected to a
kinetic screening experiment by SPR-analysis using Proteon
XPR36.
[0532] Affinity-Determination of Affinity-Matured Pertuzumab HC
Variants by SPR
[0533] The Affinity (K.sub.D) of the new Pertuzumab HC variants was
measured by surface plasmon resonance. In a first step, 7000 RU of
polyclonal anti-human Fab antibody were immobilized on all 6
channels of a GLM chip by Amine coupling (NaAcetate pH4.5, 30
.mu.l/min, 300s) (vertical orientation).
[0534] Each antibody-containing bacterial supernatant was filtered
and 3-fold diluted with PBS, and then injected for 180s at 30
.mu.l/minute to achieve immobilization levels of between 100 and
400 response units (RU) in vertical orientation. Injection of Her2:
For one-shot kinetics measurements, injection direction was changed
to horizontal orientation, two-fold dilution series of purified
Her2 (varying concentration ranges between 100 and 6.25 nM) were
injected simultaneously at 100 .mu.l/min along separate channels
1-5, with association times of 180s, and dissociation times of
1000s. Buffer (PBST) was injected along the sixth channel to
provide an "in-line" blank for referencing. Regeneration was
performed by two pulses of 10 mM glycine pH 1.5 and 50 mM NaOH for
30s at 100 .mu.l/min (horizontal orientation). Association rate
constants (k.sub.on) and dissociation rate constants (k.sub.off)
were calculated using a simple one-to-one Langmuir binding model in
ProteOn Manager v3.1 software by simultaneously fitting the
association and dissociation sensorgrams. The equilibrium
dissociation constant (K.sub.D) was calculated as the ratio
k.sub.off/k.sub.on. Clones expressing Fabs with the highest
affinity constants were identified and the heavy chains of the
corresponding phagemids were sequenced. The thermodynamic
measurement of the most affine Pertuzumab HC variants (protein
sequence of variable domains listed as SEQ ID NOs: 62, 66, 68, 70,
72, and 74) is summarized in Table 23 and FIGS. 21A-F.
TABLE-US-00028 TABLE 23 Affinity of selected affinity matured
Pertuzumab clone variants in combination with the common light
chain (CLC) Pertuzumab heavy chain affinity (nM) Pertuzumab 34 Aff
mat. clone D1 1 Aff mat. clone B2 1 Aff mat. clone E1 0.5 Aff mat.
clone G2 1 Aff mat. clone C8 3 Aff mat. clone A1 1
Example 14: Characterization of the Trastuzumab/Pertuzumab
Bispecific Antibodies
[0535] Generation of the Trastuzumab/Pertuzumab Bi-Specific
Anti-Her Antibodies with a CLC
[0536] In the following step, the affinity-matured Pertuzumab HC as
well as the Trastuzumab HC were expressed in combination with the
CLC named "Pertuzumab (Tras.L3) (QM) LC" in a bispecific antibody
format. For the generation of such bispecific antibodies with a
CLC, heterodimerization of the 2 different HCs was achieved by
application of the knob-into-hole technology. Variable domains of
the Pertuzumab affinity-matured clones E1 and G2 (protein sequence
of variable domains listed as SEQ ID NOs: 68 and 70) as well as a
clone "D1-derived" (D1-der) sequence (SEQ ID NOs: 64) were cloned
into a human IgG1 HC containing the "hole" mutations in domain CH3.
A schematic overview of the bispecific antibody with a CLC is shown
in FIG. 22. Affinity-maturation clone "D1-der" combines the CDR1
and 3 mutations of clone D1 (SEQ ID NOs: 62) with additional CDR2
mutations found in other selected clones. The variable domain of
the Tratuzumab HC (SEQ ID NO: 92) was cloned into a human IgG1 HC
harboring the "knob" mutations in domain CH3. The resulting
constructs, called "Herceptarg" constructs, were co-expressed and
purified from mammalian-derived cell culture supernatant. A summary
of the analytical data for all three bi-specific antibodies is
shown clones in FIGS. 23A-F and Table 24.
TABLE-US-00029 TABLE 24 Production of HER2 antibody with common
light chain yield/liter Antibody (mg/I) % Monomer Herceptarg CLC G2
41.3 100 Herceptarg CLC E1 72.1 96 Herceptarg CLC D1- 20.1 100
der.
[0537] Generation of Her2 Knock-Out Antigen Variants
[0538] In order to analyze and characterize binding of the
Herceptarg bi-specific antibodies to each of both epitopes on Her2,
Her2 knock-out variants were designed. In these variants, either of
the two specific epitopes was deleted by mutation of the amino
acids that interact with the respective antibody chains.
[0539] For expression and purification, the respective DNA
fragments were fused in frame to an N-terminal leader sequence and
a C-terminal human IgG1 Fc coding fragment serving as solubility-
and purification tag. An avi-tag at the C-terminal end of the Fc
fragment allowed in vivo biotinylation. In order to express the
antigen in a monomeric form, these Fc chains contained the "knob"
mutations (SEQ ID NOs: 5 and 6, Her2 ECD-Fc(knob); SEQ ID NOs: 7
and 8, Her2 ECD (Pertuzumab KO)-Fc(knob); SEQ ID NOs: 9 and 10,
Her2 ECD (Trastuzumab KO)-Fc(knob)) and was co-expressed in
combination with an "Fc-hole" counterpart (SEQ ID NOs: 3 and
4).
[0540] Affinity-Determination of the Herceptarg Bi-Specific
Antibodies by SPR
[0541] The Affinity (K.sub.D) of the new bi-specific antibodies to
each of their epitopes in her2 was measured by SPR using a ProteOn
XPR36 instrument (Biorad) at 25.degree. C. In a first step, 11000
RU of a polyclonal goat anti-human IgG (Sigma 12136,) recognizing
human IgG (Fc-specific) was immobilized on all 6 channels of a GLM
chip by Amine coupling(NaAcetate pH4, 30 .mu.l/min, 300s) (vertical
orientation).
[0542] Each antibody was diluted with PBST (10 mM phosphate, 150 mM
sodium chloride pH 7.4, 0.005% Tween 20) to 2 .mu.g/ml, and then
injected for 60s at 30 .mu.l/minute to achieve immobilization
levels of about 400 response units (RU) in vertical orientation.
Injection of Her2: For one-shot kinetics measurements, injection
direction was changed to horizontal orientation, two-fold dilution
series of purified monovalent Her2-Fc protein constructs (varying
concentration ranges between 100 and 6.25 nM) were injected
simultaneously at 100 .mu.l/min along separate channels 1-5, with
association times of 180s, and dissociation times of 600s. Buffer
(PBST) was injected along the sixth channel to provide an "in-line"
blank for referencing. Regeneration was performed by two pulses of
10 mM glycine pH 1.5 and 50 mM NaOH for 30s at 100 .mu.l/min
(horizontal orientation). Association rate constants (k.sub.on) and
dissociation rate constants (k.sub.off) were calculated using a
simple one-to-one Langmuir binding model in ProteOn Manager v3.1
software by simultaneously fitting the association and dissociation
sensorgrams. The equilibrium dissociation constant (K.sub.D) was
calculated as the ratio k.sub.off/k.sub.on.
[0543] All antibodies including Trastuzumab, Pertuzumab as well as
three bi-specific Herceptarg constructs comprising the
affinity-matured Pertuzumab HCs, the Trastuzumab HC, and the CLC
"Pertuzumab (Trast.L3) (QM)" were tested for binding to both Her2
knock-out variants. As expected, both Trastuzumab and Pertuzumab
bind to their respective Her2 epitope with the excepted affinity.
Binding to their corresponding knock-out variant was abolished
(FIGS. 24A-D). These results demonstrate that the use of Her2
knock-out epitope variants allows dissecting the binding of the
bispecific antibodies and analyzing the individual affinity of both
specificities. Determination of the individual K.sub.D values of
each Herceptarg clone variant revealed a constant binding affinity
to the Trastuzumab epitope in the expected range of 0.6 to 1.8 nM.
In contrast, binding to the Pertuzumab epitope depends on the
affinity-matured Pertuzumab HC. Among the three Herceptarg clone
variants, clone "D1-der" was found to have the highest affinity
(0.16 nM). A summary of the Thermodynamic data is shown in table
25. In summary, this experiment confirms that we were able to
generate a bi-specific antibody with a CLC and specific for the
epitopes Trastuzumab and Pertuzumab.
[0544] Binding Analysis of the Herceptarg Clone Variants to KPL-4
Cells
[0545] KPL-4 cells were harvested and resuspended in FACS buffer.
0.2 Mio cells were seeded into a 96 well round bottom plate. The
plate was centrifuged at 400 g for 3 min to pellet the cells. The
supernatant was removed and the cells were resuspended in 40 .mu.l
of the diluted antibodies. The plate was incubated for 30 min at
4.degree. C. to allow binding of the antibodies. To remove unbound
antibodies the cells were centrifuged again and washed twice with
FACS buffer. To detect the antibodies the cells were resuspended in
12 .mu.l diluted secondary goat anti-human Fc specific FITC-labeled
secondary antibody (Jackson ImmunoResearch #109-096-098) and
incubated again for 30 min at 4.degree. C. Afterwards the cells
were washed twice with FACS buffer, resuspended in 200 .mu.l FACS
buffer and the fluorescence was measured with BD CantoII. Results
are shown in FIG. 25.
TABLE-US-00030 TABLE 25 Affinity of selected bi-specific antibodies
affinity matured Pertuzumab clone variants in combination with the
CLC KD (nM) Trastuzumab KD (nM) Pertuzumab clone Epitope Epitope
Trastuzumab 2.17 N/A Pertuzumab N/A 0.62 Herceptarg E1 0.72 1.32
Herceptarg G2 1.82 9.65 Herceptarg D1-derived 0.6 0.16 Herceptarg
crossmab 0.8 0.84
TABLE-US-00031 TABLE 26 Proliferation inhibition of various cell
types (IC50 values with confidence interval) cell Trastuzumab +
Herceptarg Herceptarg Herceptarg line Trastuzumab Pertuzumab
Pertuzumab CLC E1/1 CLC D1-der CLC G2/2 GA604 BT474 110.8 205.4
222.3 114.1 78.38 89.99 125.4 (96.7-127.0) (156-270.7)
(176.5-280.1) (106-122.7) (69.03-89).sup. (82.3-98.4) (113.6-138.4)
N87 83.13 205.8 312.9 136.5 92.7 119.1 109.6 (59.7-115.7)
(126.3-335) (156.5-625.6) (126.6-147) (80.7-106.5) (107.6-132).sup.
(98.3-122.2) SkBr3 73.73 nd 55.63 69.01 47.54 92.59 59.84
(43.54-124.8) (25.97-119.2) (55.78-85.4) (26.61-84.9) (70.1-122.3)
(50.2-71.4)
[0546] Proliferation Inhibition Mediated by the Herceptarg Binding
Variants
[0547] Target cells were harvested, washed, resuspended in RPMI
1640 (Gibco)+10% FCS+1% GlutaMAX.TM. (Gibco) and plated at a
concentration of 5.times.10.sup.3 cells/well. Cells were incubated
for 4 hours in the cell incubator before respective antibody
dilutions were added. Plates were gently shaked and incubated for 5
days in the cell incubator. The plates were equilibrated to room
temperature and 100 .mu.l/well of the freshly prepared CellTiter
Glo (Promega) substrate were added to each well. Luminescence was
measured in a Wallac Victor3 1420 Multilabel Counter. Results are
shown in Table 26 and FIGS. 26A-F.
[0548] Herceptarg Clones-Mediated Antibody-Dependent Cell-Mediated
Cytotoxicity (ADCC)
[0549] Target cells were harvested, washed, resuspended in AIM
V.RTM. medium (Life Technologies), and plated at a concentration of
3.times.10.sup.4 cells/well. The respective antibody dilutions were
added in triplicates to the cells and incubated for 10 min before
addition of the effector cells (peripheral blood mononuclear
effector cells [PBMCs]). Effector (E) and target (T) cells were
then incubated for the indicated time at 37.degree. C. at the
indicated E:T ratio (triplicates for all samples). Lactate
dehydrogenase (LDH) release was measured using the LDH Cytotoxicity
Detection Kit (Roche Applied Science). ADCC was calculated using
the following formula:
Percentage A D C C = ( [ sample release - spontaneous release
maximal release - spontaneous release ] ) .times. 100.
##EQU00003##
[0550] Spontaneous release, corresponding to target cells incubated
with effector cells without antibody, was defined as 0%
cytotoxicity, and maximal release (target cells lysed with 1%
Triton X-100) was defined as 100% cytotoxicity. The average
percentage of ADCC and standard deviations of the triplicates of
each experiment were calculated. Results are shown in FIGS.
27A-D.
TABLE-US-00032 TABLE 27 Leader Peptides SEQ ID Protein sequence 155
MDWTWRILFLVAAATGAHS 156 MDMRVPAQLLGLLLLWFPGARC 157
MGWSCIILFLVATATGVHS
Example 15: Proliferation Assay
[0551] 1.times.10.sup.4 BT-474 cells/well were cultured in RPMI/10%
FCS in a 96-well flat bottom plate. After 24 hrs growth medium was
removed and titrated amounts of indicated antibodies were added
(premixed in culture medium) to a final volume of 100 .mu.l. To
determine the number of viable cells in culture, the CellTiter-Glo
Luminescent Cell Viability Assay was performed by quantifying the
present ATP levels as an indicator of metabolically active cells.
Thus, after six days of culture, 100 .mu.l CellTiter-Glo Reaction
Mix (Promega, cat.no. #G7571) was added to the cells, shook for 2
min before 75 .mu.l of the lysate was transferred to a separate
96-well flat bottom titer plate (Costar, cat.no. #3917). After
additional mixing, luminescence was assed according to the
manufacturer's instructions using a Tecan Infinite Reader.
[0552] Results are shown in Table 28 and FIG. 28.
[0553] In the proliferation assay it was shown that the bispecific
Her2 antibodies inhibited proliferation of BT-474 cells more
potently than Pertuzumab or Trastuzumab alone or in combination.
The following bispecific Her2 antibodies were tested: Herceptarg
CLC D1-der wt": SEQ ID NOs 64, 54, 92, Herceptarg CLC D1-der G2":
SEQ ID NOs 64, 54, 92 (glycoengineered variant) "Herceptarg
CrossMab": SEQ ID NOs 109, 110, 111, 112.
TABLE-US-00033 TABLE 28 IC50 BT474 proliferation assay Antibody
treatment IC50 Proliferation [nM] Trastuzumab n.d. Pertuzumab n.d.
Trastuzumab + Pertuzumab 6.20 Herceptarg CLC D1-der. wt 3.31
Herceptarg CLC D1-der. G2 3.93 Herceptarg CrossMab 4.75
Example 16: C1q FACS Binding Assay
[0554] 3.times.10.sup.5 BT-474 cells were incubated with 10
.mu.g/ml of indicated antibody on ice. The following bispecific
Her2 antibodies were tested: Herceptarg CLC D1-der wt": SEQ ID NOs
64, 54, 92, Herceptarg CLC D1-der G2": SEQ ID NOs 64, 54, 92
(glycoengineered variant) "Herceptarg CrossMab": SEQ ID NOs 109,
110, 111, 112.
[0555] After 30 min, 10 .mu.g/ml C1q (Sigma, C1740) was added and
additionally incubated for 20 min. After washing, cells were
counterstained with commercial PE-labeled anti-C1q antibody
(Cedarlane, CL7611PE-SP). After further incubation (30 min, ice),
cells were washed twice and analyzed on a FACS Canto II.
[0556] Results are shown in FIG. 29 and Table 29. This C1q assay
illustrates the binding of recombinant complement factor C1q to
different Her antibodies on BT-474 cells. It was shown that the
highest C1q binding resulted upon treatment with the combination of
Trastuzumab and Pertuzumab, followed by the two CLC bispecific Her2
antibodies. Treatment with the Crossmab resulted only in a slightly
elevated C1q binding.
TABLE-US-00034 TABLE 29 Clq binding assay PE-signal
antibody/antibodies (geomean) trastuzumab 282 pertuzumab 344
combination of trastuzumab and pertuzumab 2157 bispecific anti-HER2
antibody, common light chain 1439 bispecific anti-HER2 antibody,
common light chain, 1036 glycoengineered bispecific anti-HER2
antibody, CrossMab format 489
Example 17: LDH Assay with Baby Rabbit Complement (BRC)
[0557] CHO-K1 Nxre19 cells (IL15R transfected CHO-K1) were seeded
at 10,000 cells/well on 96-well flat bottom cell culture plates
(NUNC, 100 .mu.L/well) and cultivated overnight. IL15-Fc fusion
polypeptide was added (25 .mu.L/well in 5-fold end-concentration)
and incubated for one hour. Thereafter, one vial of Baby Rabbit
complement (Cedarlane, Cat. No. CL3441) was reconstituted with 1 mL
of Aqua bidest. The complement solution was diluted with medium and
25 .mu.L added to the wells. After four hours the plates were
centrifuged at 200 g and 100 .mu.L/well were transferred to another
96-well flat bottom plate. Thereafter 100 .mu.L of LDH reaction mix
(Cytotoxicity Detection Kit, Roche Diagnostic GmbH, Mannheim,
Germany) was added. After an incubation time of 20 min. at
37.degree. C. optical density (OD) was measured at 492/690 nm on a
Tecan Sunrise reader.
TABLE-US-00035 TABLE 30 LDH assay with BRC signal [OD] sample BRC
1/40 BRC 1/30 9000 ng/ml IL15-Fc-fusion with HUC 11.3 12.3 3000
ng/ml IL15-Fc-fusion with HUC 12.3 17.0 1000 ng/ml IL15-Fc-fusion
with HUC 10.2 13.6 333.3 ng/ml IL15-Fc-fusion with HUC 7.8 12.2
111.1 ng/ml IL15-Fc-fusion with HUC 8.3 13.0 37.04 ng/ml
IL15-Fc-fusion with HUC 14.9 19.7 12.35 ng/ml IL15-Fc-fusion with
HUC 43.2 53.0 4.12 ng/ml IL15-Fc-fusion with HUC 41.5 63.8 0 ng/ml
IL15-Fc-fusion with HUC 42.4 48.4
[0558] It can be seen that BRC has a low background toxicity and
shows dose dependent complement toxicity.
Example 18: CDC (Complement Dependent Cytotoxicity) Activation on
BT-474 Cells (LDH Release)
[0559] 1.times.10.sup.4 cells/well were incubated with 10 .mu.g/ml
of the indicated antibodies for 30 min at 37.degree. C. in 150
.mu.l. The following bispecific Her2 antibodies were tested:
Herceptarg CLC D1-der wt": SEQ ID NOs 64, 54, 92, Herceptarg CLC
D1-der G2": SEQ ID NOs 64, 54, 92 (glycoengineered variant)
"Herceptarg CrossMab": SEQ ID NOs 109, 110, 111, 112.
[0560] Then 50 .mu.l Baby Rabbit Complement (Cedarlane, cat. no.
CL3441, batch no. 6312) was added and incubated for further 2 hrs.
Then, the s/n was transferred and mixed with 50 .mu.l LDH Reaction
Mix (Roche) and, after a further incubation of 15 min, extinktion
(Ex.) at 490/620 nm was analyzed on a Tecan Sunrise Reader. The
specific antibody dependent toxicity (mean+/-SD of n=4) on BT-474
cell was calculated as follows: (Ex. sample-Ex. spontanous
lysis/Ex. maximal lysis-spontanous lysis).times.100.
[0561] Results are shown in FIG. 30.
[0562] This CDC assay shows the release of LDH as a marker for
dying/dead cells upon treatment of different anti-Her2 antibodies
(formats, combination) in the presence of baby rabbit complement.
Here, the combination of Trastuzumab and Pertuzumab resulted in a
significant induction of CDC, whereas the parental antibodies alone
did not. Surprisingly, both CLC Herceptarg variants provoked even
superior CDC effects, whereas the Herceptarg Crossmab treatment
results in a CDC reaction less effective than the combination of
the parental antibodies.
Example 19: CDC (Complement Dependent Cytotoxicity)-Mediated
Killing of BT-474 Cells (ACEA)
[0563] 1.times.10.sup.4 BT-474 cells/well were seeded on 96-well
E-Plates (ACEA Biosciences Inc.) and grown overnight in an
Xcelligence device. Growth medium was removed and cells were washed
once with serum-free AIM-V medium (Gibco). 50 .mu.l/well AIM-V
medium and 50 .mu.l antibody in AIM-V (3-fold end concentration)
were added and incubated for 20 min. 50 .mu.l Baby Rabbit
Complement (Cedarlane) was added and CellIndex (CI; as
representative for the viability of the cells) was measured every 5
minutes (see curve). The following bispecific Her2 antibodies were
tested: Herceptarg CLC D1-der wt": SEQ ID NOs 64, 54, 92,
Herceptarg CLC D1-der G2": SEQ ID NOs 64, 54, 92 (glycoengineered
variant) "Herceptarg CrossMab": SEQ ID NOs 109, 110, 111, 112.
[0564] Specific CDC was calculated according following formula,
whereas CI is the normalized cell index:
% C D C = CI Complement control - CI sample CI Complement control
.times. 100 ##EQU00004##
[0565] At two representative time points (1 hr and 2 hrs after
starting the reaction, specific lysis (=CDC-induced cell death) was
calculated and shown in the diagram (mean+/SEM of n=4).
[0566] Results are shown in FIGS. 31A-B and Table 31. This CDC
assay illustrates a change in the cell index as a marker for
dying/dead cells upon treatment with different anti-Her2 antibodies
(formats, combination) in the presence of baby rabbit complement:
Here, the combination of Trastuzumab and Pertuzumab resulted in a
significant induction of CDC, whereas the parental antibodies alone
did not. Surprisingly, both CLC Herceptarg variants provoked even
superior CDC effects, whereas the Herceptarg Crossmab treatment
results in a CDC reaction less effective than the combination of
the parental antibodies.
[0567] One possible reason for the superiority of Herceptarg CLC
D1-der may be the slightly higher affinity to the Trastuzumab
epitope as well as the significantly higher affinity to the
Pertuzumab epitope (see also Table 25).
TABLE-US-00036 TABLE 31 CDC (complement dependent
cytotoxicity)--mediated killing of BT-474 cells (ACEA) specific
lysis [% cell index ACEA] antibody/antibodies 1 hour 2 hours
trastuzumab -3.5 .+-. 0.6 -6.5 .+-. 0.8 pertuzumab -5.3 .+-. 1.0
-8.3 .+-. 2.1 combination of trastuzumab and 20.9 .+-. 6.7 26.3
.+-. 7.0 pertuzumab bispecific anti-HER2 antibody, common 31.8 .+-.
3.4 38.9 .+-. 3.7 light chain (D1 der) bispecific anti-HER2
antibody, common 28.8 .+-. 2.6 35.8 .+-. 2.6 light chain,
glycoengineered (D1 der) bispecific anti-HER2 antibody, 12.9 .+-.
1.4 22.7 .+-. 1.6 CrossMab format
Example 20: Mouse Xenograft Studies
[0568] Cell Line KPL4
[0569] This human breast cancer cell line has been established from
the malignant pleural effusion of a breast cancer patient with an
inflammatory skin metastasis. Cells have been provided by Professor
J. Kurebayashi (Kawasaki Medical School, Kurashiki, Japan). Tumor
cells were routinely cultured in DMEM medium (PAN Biotech, Germany)
supplemented with 10% fetal bovine serum (PAN Biotech, Germany) and
2 mM L-glutamine (PAN Biotech, Germany) at 37.degree. C. in a
water-saturated atmosphere at 5% CO2. Culture passage was performed
with trypsin/EDTA 1.times. (PAN) splitting twice/week. Cell passage
P6 was used for in vivo study.
[0570] Mice
[0571] Female SCID beige (C.B.-17) mice; age 10-12 weeks; body
weight 18-20 g (Charles River Germany, Sulzfeld); body weight
>20 g are maintained under specific-pathogen-free condition with
daily cycles of 12 h light/12 h darkness according to international
guidelines (GV-Solas; Felasa; TierschG). After arrival animals were
housed in the quarantine part of the animal facility for one week
to get accustomed to new environment and for observation.
Continuous health monitoring was carried out on regular basis. Diet
food (Alltromin) and water were provided ad libitum. The
experimental study was reviewed and approved by local
government.
[0572] Tumor Cell Injection
[0573] At the day of injection tumor cells were harvested
(trypsin-EDTA) from culture flasks (Greiner TriFlask) and
transferred into 50 ml culture medium, washed once and resuspended
in PBS. After an additional washing step with PBS and filtration
(cell strainer; Falcon O 100 .mu.m) the final cell titer was
adjusted to 1.5.times.10e8/ml. Tumor cell suspension was carefully
mixed with transfer pipette to avoid cell aggregation. Anesthesia
was performed using a Stephens inhalation unit for small animals
with preincubation chamber (plexiglas), individual mouse nose-mask
(silicon) and not flammable or explosive anesthesia compound
Isoflurane (Pharmacia-Upjohn, Germany) in a closed circulation
system. Two days before injection, coat of the SCID beige mice were
shaved and KPL-4 cells (3.times.10e6 cells) were injected
orthotopically in a volume of 20 .mu.l (using a Hamilton microliter
syringe and a 30G.times.1/2'' needle) into the right penultimate
inguinal mammary fat pad of each anesthetized mouse. The cell
suspension was injected through the skin under the nipple.
[0574] Monitoring
[0575] Animals were controlled daily for detection of clinical
symptoms of adverse effects. For monitoring throughout the
experiment the body weight of the animals was documented two times
weekly and the tumor volume was measured by caliper twice weekly.
Tumor volume was calculated according to NCI protocol (Tumor
weight=1/2ab2, where "a" and "b" are the long and the short
diameters of the tumor, respectively). Termination criteria were
the critical tumor mass (up to 1.7 g or O>1.5 cm), body weight
loss more than 20% from baseline, tumor ulceration or poor general
condition of the animals. Study exclusion criteria for the animals
are described and approved in the corresponding
"Tierversuchsanzeige".
[0576] Treatment
[0577] Mice were randomized for tumor volume of 80 mm.sup.3 and
subsequently treated once weekly with a volume of 10 ml/kg intra
peritoneal. For combination treatment Herceptin was given first and
Perjeta was given 24 hrs thereafter.
[0578] The following bispecific Her2 antibodies was tested:
Herceptarg CLC-D1-der: SEQ ID NOs 64, 54, 92. Results are shown in
FIG. 32.
TABLE-US-00037 No of Dose Route/Mode of Group animals Compound
(mg/kg) administration 1 9 Control (Xolair) 10 i.p. once weekly 2 9
Herceptin 10 i.p. once weekly 3 9 Perjeta 10 i.p. once weekly 4 9
Herceptin plus 10 plus 10 i.p. once weekly Perjeta 5 8 Herceptarg
10 i.p. once weekly CLC-D1-der
TABLE-US-00038 TABLE 32 Parent sequences of Pertuzumab and
Trastuzumab SEQ ID NO Name Sequence Pertuzumab wt (parent)
sequences 22 Pertuzumab
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEW wt VH
VADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVY (10289)
YCARNLGPSFYFDYWGQGTLVTVSS 21 Pertuzumab
GAGGTGCAGCTGGTCGAGTCTGGCGGCGGACTGGTGCAGCCTGGCGG wt VH
CAGCCTGAGACTGAGCTGCGCCGCCAGCGGCTTCACCTTCACCGACT (10289) DNA
ACACCATGGACTGGGTGCGGCAGGCCCCTGGCAAGGGCCTGGAATGG
GTGGCCGACGTGAACCCCAACAGCGGCGGCAGCATCTACAACCAGCG
GTTCAAGGGCCGGTTCACCCTGAGCGTGGACAGAAGCAAGAACACCC
TGTACCTCCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTAC
TACTGCGCCCGGAACCTGGGCCCCAGCTTCTACTTCGACTACTGGGG
CCAGGGCACCCTGGTGACCGTGAGCAGCGCTAGCACCAAGGGCCCAT
CGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACA
GCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGAC
GGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC
CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC 14 Pertuzumab GFTFTDYTMD
wt VH CDR1 15 Pertuzumab DVNPNSGGSIYNQRFKG wt VH CDR2 16 Pertuzumab
NLGPSFYFDY wt VH CDR3 114 Pertuzumab
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT wt CH1
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKV 24 Pertuzumab
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLL wt VL
IYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIY (10290)
PYTFGQGTKVEIK 23 Pertuzumab
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGG wt VL
CGACAGAGTGACCATCACCTGCAAGGCCAGCCAGGACGTGTCCATCG (10290) DNA
GCGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTG
ATCTACAGCGCCAGCTACCGGTACACAGGCGTGCCCAGCCGGTTCAG
CGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGC
AGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACTACATCTAC
CCCTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG 11 Pertuzumab KASQDVSIGVA
wt VL CDR1 12 Pertuzumab SASYRYT wt VL CDR2 13 Pertuzumab QQYYIYPYT
wt VL CDR3 113 Pertuzumab
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL wt CL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC
Trastuzumab wt (parent) sequences 82 Trastuzumab
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLL VL (4245)
IYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTT PPTFGQGTKVEIK 81
Trastuzumab GACATCCAGATGACCCAGAGCCCAAGCTCTCTGTCTGCCTCTGTGGG VL
(4245) DNA CGACAGAGTGACCATCACCTGCAGAGCCAGCCAGGACGTGAACACAG
CCGTGGCCTGGTATCAGCAGAAGCCAGGCAAGGCCCCAAAGCTGCTG
ATCTACAGCGCCAGCTTCCTGTACAGCGGCGTGCCAAGCAGATTCAG
CGGCAGCAGAAGCGGCACAGACTTCACCCTGACCATCAGCAGCCTGC
AGCCAGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACC
CCACCAACCTTCGGACAGGGCACCAAGGTGGAGATCAAG 17 Trastuzumab RASQDVNTAVA
wt VL CDR1 18 Trastuzumab SASFLYS wt VL CDR2 19 Trastuzumab
QQHYTTPPT wt VL CDR3 116 Trastuzumab
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL wt CL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 92
Trastuzumab EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW VH
VARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVY (11345)
YCSRWGGDGFYAMDYWGQGTLVTVSS 91 Trastuzumab
GAAGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGG VH
CAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTAACATAAAGGACA (11345) DNA
CATACATCCACTGGGTGCGCCAAGCACCTGGGAAGGGTCTCGAGTGG
GTGGCTCGGATTTACCCAACAAATGGCTACACCAGGTATGCGGATAG
CGTGAAAGGCCGTITTACCATTICAGCTGATACTICGAAGAACACCG
CCTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTAT
TATTGCTCGCGTTGGGGAGGAGACGGGTTCTATGCTATGGATTACTG
GGGCCAAGGCACCCTGGTGACGGTTAGCTCA 20 Trastuzumab GFNIKDTYIH wt VH
CDR1 29 Trastuzumab RIYPTNGYTRYADSVKG wt VH CDR2 30 Trastuzumab
WGGDGFYAMDY wt VH CDR3 115 Trastuzumab
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT wt CH1
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKV
TABLE-US-00039 TABLE 33 Sequences of antibodies with common light
chain SEQ ID NO Name Sequence Pertuzumab/ Trastuzumab hybrid light
chains 26 Pertuzumab
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLL VL (Trast.
IYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTT L3) PPTFGQGTKVEIK
(10403) 25 Pertuzumab
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGG VL (Trast.
CGACAGAGTGACCATCACATGCAAGGCCAGCCAGGACGTGTCCATCG L3)
GCGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTG (10403)DNA
ATCTACAGCGCCAGCTACCGGTACACCGGCGTGCCCAGCAGATTCAG
CGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGC
AGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACC
CCCCCCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG 28 Pertuzumab
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLL VL (Trast.
IYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYIY H91) PYTFGQGTKVEIK
(10404) 27 Pertuzumab
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGG VL (Trast.
CGACAGAGTGACCATCACCTGCAAGGCCAGCCAGGACGTGTCCATCG H91)
GCGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTG (10404)DNA
ATCTACAGCGCCAGCTACCGGTACACAGGCGTGCCCAGCCGGTTCAG
CGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGC
AGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACATCTAC
CCCTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG 32 Pertuzumab
DIQMTQSPSSLSASVGDRVTITCRASQDVSIGVAWYQQKPGKAPKLL VL
IYSASYRYTGVPSRFSGSGSGTDTTLTISSLQPEDFATYYCQQHYTT (Tras.L3)
PPTFGQGTKVEIK K24R (10949) 31 Pertuzumab
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGG VL
CGACAGAGTGACCATCACATGCCGGGCCAGCCAGGACGTGTCCATCG (Tras.L3)
GCGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTG K24R
ATCTACAGCGCCAGCTACCGGTACACCGGCGTGCCCAGCAGATTCAG (10949)DNA
CGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGC
AGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACC
CCCCCCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG 34 Pertuzumab
DIQMTQSPSSLSASVGDRVTITCKASQDVNIGVAWYQQKPGKAPKLL VL
IYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTT (Tras.L3)
PPTFGQGTKVEIK S30N (10950) 33 Pertuzumab
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGG VL(Tras.L3)
CGACAGAGTGACCATCACATGCAAGGCCAGCCAGGACGTGAACATCG S30N
GCGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTG (10950)DNA
ATCTACAGCGCCAGCTACCGGTACACCGGCGTGCCCAGCAGATTCAG
CGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGC
AGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACC
CCCCCCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG 36 Pertuzumab
DIQMTQSPSSLSASVGDRVTITCKASQDVSTGVAWYQQKPGKAPKLL VL
IYSASYRYTGVPSRFSGSGSGTDFTLTISSTQPEDFATYYCQQHYTT (Tras.L3)
PPTFGQGTKVEIK I31T (10951) 35 Pertuzumab
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGG VL
CGACAGAGTGACCATCACATGCAAGGCCAGCCAGGACGTGTCCACCG (Tras.L3)
GCGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTG I31T
ATCTACAGCGCCAGCTACCGGTACACCGGCGTGCCCAGCAGATTCAG (10951)DNA
CGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGC
AGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACC
CCCCCCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG 38 Pertuzumab
DIQMTQSPSSLSASVGDRVTITCKASQDVSVGVAWYQQKPGKAPKLL VL
IYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTT (Tras.L3)
PPTFGQGTKVEIK I31V (10952) 37 Pertuzumab
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGG VL
CGACAGAGTGACCATCACATGCAAGGCCAGCCAGGACGTGTCCGTCG (Tras.L3)
GCGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTG I31V
ATCTACAGCGCCAGCTACCGGTACACCGGCGTGCCCAGCAGATTCAG (10952)DNA
CGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGC
AGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACC
CCCCCCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG 40 Pertuzumab
DIQMTQSPSSLSASVGDRVTITCKASQDVSIAVAWYQQKPGKAPKLL VL
IYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTT (Tras.L3)
PPTFGQGTKVEIK G32A (10953) 39 Pertuzumab
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGG VL
CGACAGAGTGACCATCACATGCAAGGCCAGCCAGGACGTGTCCATCG (Tras.L3)
CCGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTG G32A
ATCTACAGCGCCAGCTACCGGTACACCGGCGTGCCCAGCAGATTCAG (10953)DNA
CGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGC
AGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACC
CCCCCCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG 42 Pertuzumab
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLL VL
IYSASFRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTT (Tras.L3)
PPTFGQGTKVEIK Y53F (10954) 41 Pertuzumab
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGG VL(Tras.L3)
CGACAGAGTGACCATCACATGCAAGGCCAGCCAGGACGTGTCCATCG Y53F
GCGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTG (10954) DNA
ATCTACAGCGCCAGCTTCCGGTACACCGGCGTGCCCAGCAGATTCAG
CGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGC
AGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACC
CCCCCCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG 44 Pertuzumab
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLL VL (Tras.L3)
IYSASYLYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTT R54L PPTFGQGTKVEIK
(10955) 43 Pertuzumab
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGG (Tras.L3)
CGACAGAGTGACCATCACATGCAAGGCCAGCCAGGACGTGTCCATCG R54L
GCGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTG (10955) DNA
ATCTACAGCGCCAGCTACCTGTACACCGGCGTGCCCAGCAGATTCAG
CGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGC
AGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACC
CCCCCCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG 46 Pertuzumab
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLL (Tras.L3)
IYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTT T56S PPTFGQGTKVEIK
(10956) 45 Pertuzumab
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGG (Tras.L3)
CGACAGAGTGACCATCACATGCAAGGCCAGCCAGGACGTGTCCATCG T56S
GCGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTG (10956) DNA
ATCTACAGCGCCAGCTACCGGTACAGCGGCGTGCCCAGCAGATTCAG
CGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGC
AGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACC
CCCCCCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG 48 Pertuzumab
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLL (Tras.L3)
IYSASYRYTGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTT G66R PPTFGQGTKVEIK
(10957) 47 Pertuzumab
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGG (Tras.L3)
CGACAGAGTGACCATCACATGCAAGGCCAGCCAGGACGTGTCCATCG G66R
GCGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTG (10957) DNA
ATCTACAGCGCCAGCTACCGGTACACCGGCGTGCCCAGCAGATTCAG
CGGCAGCCGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGC
AGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACC
CCCCCCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG 50 Pertuzumab
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLL (Tras.L3)
IYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTY T94Y PPTFGQGTKVEIK
(10958) 49 Pertuzumab
ACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGC (Tras.L3)
GACAGAGTGACCATCACATGCAAGGCCAGCCAGGACGTGTCCATCGG T94Y
CGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGA (10958) DNA
TCTACAGCGCCAGCTACCGGTACACCGGCGTGCCCAGCAGATTCAGC
GGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCA
GCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCTACC
CCCCCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG 52 Pertuzumab
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLL (Tras.L3)
IYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTT P96Y PYTFGQGTKVEIK
(10959) 51 Pertuzumab
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGG (Tras.L3)
CGACAGAGTGACCATCACATGCAAGGCCAGCCAGGACGTGTCCATCG P96Y
GCGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTG (10959) DNA
ATCTACAGCGCCAGCTACCGGTACACCGGCGTGCCCAGCAGATTCAG
CGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGC
AGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACC
CCCTACACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG 54 Pertuzumab
DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLL (Tras.L3)
IYSASFRYTGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTT (QM) PPTFGQGTKVEIK
(11055) 53 Pertuzumab
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCGTGGG (Tras.L3)
CGACAGAGTGACCATCACATGCAAGGCCAGCCAGGACGTGTCCACAG (QM)
CCGTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCCAAGCTGCTG (11055)DNA
ATCTACAGCGCCAGCTTCCGGTACACCGGCGTGCCCAGCAGATTCAG
CGGCAGCAGATCCGGCACCGACTTCACCCTGACCATCAGCTCCCTGC
AGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACC
CCCCCCACATTTGGCCAGGGCACCAAGGTGGAAATCAAG 89 Pertuzumab KASQDVSTAVA
(Tras.L3) (QM)-CDR1 90 Pertuzumab SASFRYT (Tras.L3) (QM)-CDR2 19
Pertuzumab QQHYTTPPT (Tras.L3) (QM)-CDR3 Pertuzumab/Trastuzumab
hybrid light chain affinity matured VH clones 62 Pertuzumab
EVQLVESGGGLVQPGGSLRLSCAASGFTFNDYTMDWVRQAPGKGLEW aff.mat.
VADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVY clone D1
YCARNLGPFFYFDYWGQGTLVTVSS 61 Pertuzumab
GAGGTGCAATTGGTTGAAAGCGGTGGTGGTCTGGTTCAGCCTGGTGG aff.mat.
TAGCCTGCGTCTGAGCTGTGCAGCAAGCGGTTTTACCTTTAACGATT clone D1
ATACCATGGATTGGGTTCGTCAGGCACCGGGTAAAGGTCTGGAATGG DNA
GTTGCAGATGTTAATCCGAATAGCGGTGGTAGCATTTATAACCAGCG
TTTTAAAGGTCGTTTTACCCTGAGCGTTGATCGTAGCAAAAATACCC
TGTATCTGCAAATGAATAGTCTGCGTGCAGAGGATACCGCAGTGTAT
TATTGTGCACGTAACCTGGGTCCGTTCTTCTACTTTGATTATTGGGG
TCAGGGCACCCTGGTTACCGTTAGCAGC 55 Pertuzumab GFTFNDYTMD aff.mat.
clone D1- VH CDR1 15 Pertuzumab DVNPNSGGSIYNQRFKG aff.mat. clone
D1- VH CDR2 56 Pertuzumab NLGPFFYFDY aff.mat. clone D1- VH CDR3 64
Pertuzumab EVQLVESGGGLVQPGGSLRLSCAASGFTFNDYTMDWVRQAPGKGLEW aff.mat.
VADVNPNSGGSIVNRRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVY clone D1-
YCARNLGPFFYFDYWGQGTLVTVSS derived 63 Pertuzumab
GAGGTGCAATTGGTTGAAAGCGGTGGTGGTCTGGTTCAGCCTGGTGG aff.mat.
TAGCCTGCGTCTGAGCTGTGCAGCAAGCGGTTTTACCTTTAACGATT clone D1-
ATACCATGGATTGGGTTCGTCAGGCACCGGGTAAAGGTCTGGAATGG derived,
GTTGCAGATGTTAATCCGAATAGCGGTGGTAGCATTGTTAACCGTCG DNA
TTTTAAAGGTCGTTTTACCCTGAGCGTTGATCGTAGCAAAAATACCC
TGTATCTGCAAATGAATAGTCTGCGTGCAGAGGATACCGCAGTGTAT
TATTGTGCACGTAACCTGGGTCCGTTCTTCTACTTTGATTATTGGGG
TCAGGGCACCCTGGTTACCGTTAGCAGC
55 Pertuzumab GFTFNDYTMD aff.mat. clone D1- derived VH CDR1 77
Pertuzumab DVNPNSGGSIVNRRFKG aff.mat. clone D1- derived VH CDR2 56
Pertuzumab NLGPFFYFDY aff.mat. clone D1- derived VH CDR3 66
Pertuzumab EVQLVESGGGLVQPGGSLRLSCAASGFTFNDYTMDWFRQAPGKGLEW aff.mat.
VADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVY clone B2
YCARNLGPNFYFDYWGQGTLVTVSS 65 Pertuzumab
GAGGTGCAATTGGTTGAAAGCGGTGGTGGTCTGGTTCAGCCTGGTGG aff.mat.
TAGCCTGCGTCTGAGCTGTGCAGCAAGCGGTTTTACCTTTAACGATT clone B2,
ATACCATGGATTGGTTTCGTCAGGCACCGGGTAAAGGTCTGGAATGG DNA
GTTGCAGATGTTAATCCGAATAGCGGTGGTAGCATTTATAACCAGCG
TTTTAAAGGTCGTTTTACCCTGAGCGTTGATCGTAGCAAAAATACCC
TGTATCTGCAAATGAATAGTCTGCGTGCAGAGGATACCGCAGTGTAT
TATTGTGCACGTAATCTGGGTCCGAACTTCTACTTTGATTATTGGGG
TCAGGGCACCCTGGTTACCGTTAGCAGC 55 Pertuzumab GFTFNDYTMD aff.mat.
clone B2- VH CDR1 15 Pertuzumab DVNPNSGGSIYNQRFKG aff.mat. clone
B2- VH CDR2 57 Pertuzumab NLGPNFYFDY aff.mat. clone B2- VH CDR3 68
Pertuzumab EVQLVESGGGLVQPGGSLRLSCAASGFTFADYTMDWVRQAPGKGLEW aff.mat.
VADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVY clone E1
YCARNLGPWFYFDYWGQGTLVTVSS 67 Pertuzumab
GAGGTGCAATTGGTTGAAAGCGGTGGTGGTCTGGTTCAGCCTGGTGG aff.mat.
TAGCCTGCGTCTGAGCTGTGCAGCAAGCGGTTTTACCTTTGCAGATT clone E1,
ATACCATGGATTGGGTTCGTCAGGCACCGGGTAAAGGTCTGGAATGG DNA
GTTGCAGATGTTAATCCGAATAGCGGTGGTAGCATTTATAACCAGCG
TTTTAAAGGTCGTTTTACCCTGAGCGTTGATCGTAGCAAAAATACCC
TGTATCTGCAAATGAATAGTCTGCGTGCAGAGGATACCGCAGTGTAT
TATTGTGCACGTAATCTGGGTCCGTGGTTCTACTTTGATTATTGGGG
TCAGGGCACCCTGGTTACCGTTAGCAGC 58 Pertuzumab GFTFADYTMD aff.mat.
clone E1- VH CDR1 15 Pertuzumab DVNPNSGGSIYNQRFKG aff.mat. clone
E1- VH CDR2 59 Pertuzumab NLGPWFYFDY aff.mat. clone E1- VH CDR3 70
Pertuzumab EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEW aff.mat.
VADVNPNSGGYIVNRRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVY clone G2
YCARNLGPSFYFDYWGQGTLVTVSS 69 Pertuzumab
GAGGTGCAATTGGTTGAAAGCGGTGGTGGTCTGGTTCAGCCTGGTGG aff.mat.
TAGCCTGCGTCTGAGCTGTGCAGCAAGCGGTTTTACCTTTACCGATT clone G2,
ACACAATGGATTGGGTTCGTCAGGCACCGGGTAAAGGTCTGGAATGG DNA
GTTGCAGATGTTAATCCGAACTCTGGTGGTTACATTGTTAACCGTCG
TTTTAAAGGTCGTTTTACCCTGAGCGTTGATCGTAGCAAAAATACCC
TGTATCTGCAAATGAATAGTCTGCGTGCAGAGGATACCGCAGTGTAT
TATTGTGCACGTAATCTGGGTCCGAGCTTCTATTTTGATTATTGGGG
TCAGGGCACCCTGGTTACCGTTAGCAGC 14 Pertuzumab GFTFTDYTMD aff.mat.
clone G2- VH CDR1 60 Pertuzumab DVNPNSGGYIVNRRFKG aff.mat. clone
G2- VH CDR2 16 Pertuzumab NLGPSFYFDY aff.mat. clone G2- VH CDR3 72
Pertuzumab EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEW aff.mat.
VADVNPNSGGSIMNRRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVY clone C8
YCARNLGPSFYFDYWGQGTLVTVSS 71 Pertuzumab
GAGGTGCAATTGGTTGAAAGCGGTGGTGGTCTGGTTCAGCCTGGTGG aff.mat.
TAGCCTGCGTCTGAGCTGTGCAGCAAGCGGTTTTACCTTTACCGATT clone C8,
ACACAATGGATTGGGTTCGTCAGGCACCGGGTAAAGGTCTGGAATGG DNA
GTTGCAGATGTTAATCCGAACTCTGGTGGTTCTATTATGAACCGTCG
TTTTAAAGGTCGTTTTACCCTGAGCGTTGATCGTAGCAAAAATACCC
TGTATCTGCAAATGAATAGTCTGCGTGCAGAGGATACCGCAGTGTAT
TATTGTGCACGTAATCTGGGTCCGAGCTTCTATTTTGATTATTGGGG
TCAGGGCACCCTGGTTACCGTTAGCAGC 14 Pertuzumab GFTFTDYTMD aff.mat.
clone C8- VH CDR1 75 Pertuzumab DVNPNSGGSIMNRRFKG aff.mat. clone
C8- VH CDR2 16 Pertuzumab NLGPSFYFDY aff.mat. clone C8- VH CDR3 74
Pertuzumab EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEW aff.mat.
VADVNPNSGGSIVNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVY clone A1
YCARNLGPWFYFDYWGQGTLVTVSS 73 Pertuzumab
GAGGTGCAATTGGTTGAAAGCGGTGGTGGTCTGGTTCAGCCTGGTGG aff.mat.
TAGCCTGCGTCTGAGCTGTGCAGCAAGCGGTTTTACCTTTACCGATT clone A1,
ACACAATGGATTGGGTTCGTCAGGCACCGGGTAAAGGTCTGGAATGG DNA
GTTGCAGATGTTATCCGAACTCTGGTGGTTCTATTGTTAACCAGCG
TTTTAAAGGTCGTTTTACCCTGAGCGTTGATCGTAGCAAAAATACCC
TGTATCTGCAAATGAATAGTCTGCGTGCAGAGGATACCGCAGTGTAT
TATTGTGCACGTAATCTGGGTCCGTGGTTCTACTTTGATTATTGGGG
TCAGGGCACCCTGGTTACCGTTAGCAGC 14 Pertuzumab GFTFTDYTMD aff.mat.
clone A1- VH CDR1 76 Pertuzumab DVNPNSGGSIVNQRFKG aff.mat. clone
A1- VH CDR2 59 Pertuzumab NLGPWFYFDY aff.mat. clone A1- VH CDR3
Trastuzumab Stabilization Variants 84 Trastuzumab
DIQMTQSPSSLSASVGDRVTITCRASQDVNAAVAWYQQKPGKAPKLL VL T31A
IYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTT (6641)
PPTFGQGTKVEIK 83 Trastuzumab
GATATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGG VL T31A
AGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACGTGAACGCCG (6641)
CTGTAGCGTGGTACCAGCAGAAACCAGGTAAGGCACCGAAGCTATTA
ATTTATAGTGCGAGCTTCCTGTACAGTGGGGTCCCGTCGCGTTTTAG
CGGCTCTCGATCCGGCACGGATTTTACCCTGACCATTAGCAGCCTGC
AGCCTGAAGACTTTGCGACATATTATTGCCAACAGCACTACACAACT
CCTCCCACCTITGGCCAGGGTACGAAAGTTGAAATTAA 103 Trastuzumab RASQDVNAAVA
VL T31A (6641)CDR1 18 Trastuzumab SASFLYS VL T31A (6641)CDR2 19
Trastuzumab QQHYTTPPT VL T31A (6641)CDR3 86 Trastuzumab
DIQMTQSPSSLSASVGDRVTITCRASQDVNVAVAWYQQKPGKAPKLL VL T31V
IYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTT (6642)
PPTFGQGTKVEIK 85 Trastuzumab
GATATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGG VL T31V
AGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACGTGAACGTGG (6642)DNA
CTGTAGCGTGGTACCAGCAGAAACCAGGTAAGGCACCGAAGCTATTA
ATTTATAGTGCGAGCTTCCTGTACAGTGGGGTCCCGTCGCGTTTTAG
CGGCTCTCGATCCGGCACGGATTTTACCCTGACCATTAGCAGCCTGC
AGCCTGAAGACTTTGCGACATATTATTGCCAACAGCACTACACAACT
CCTCCCACCTTTGGCCAGGGTACGAAAGTTGAAATTAAAG 104 Trastuzumab
RASQDVNVAVA VL T31V (6642)CDR1 18 Trastuzumab SASFLYS VL T31V
(6642)CDR2 19 Trastuzumab QQHYTTPPT VL T31V (6642)CDR3 158
Trastuzumab RASQDVSTAVA VL N30S CDR1 94 Trastuzumab
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW VH (D98N)
VARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVY (6636)
YCSRWGGNGFYAMDYWGQGTLVTVSS 93 Trastuzumab
GAAGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGG VH (D98N)
CAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTAACATAAAGGACA (6636) DNA
CATACATCCACTGGGTGCGCCAAGCACCTGGGAAGGGTCTCGAGTGG
GTGGCTCGGATTTACCCAACAAATGGCTACACCAGGTATGCGGATAG
CGTGAAAGGCCGTTTTACCATTTCAGCTGATACTTCGAAGAACACCG
CCTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTAT
TATTGCTCGCGTTGGGGAGGAAACGGGTTCTATGCTATGGATTACTG
GGGCCAAGGCACCCTGGTGACGGTTAGCTCA 20 Trastuzumab GFNIKDTYIH VH (D98N)
(6636) CDR1 29 Trastuzumab RIYPTNGYTRYADSVKG VH (D98N) (6636) CDR2
78 Trastuzumab WGGNGFYAMDY VH (D98N) (6636) CDR3 96 Trastuzumab
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW VH (D98E)
VARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVY (6637)
YCSRWGGEGFYAMDYWGQGTLVTVSS 95 Trastuzumab
GAAGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGG VH (D98E)
CAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTAACATAAAGGACA (6637) DNA
CATACATCCACTGGGTGCGCCAAGCACCTGGGAAGGGTCTCGAGTGG
GTGGCTCGGATTTACCCAACAAATGGCTACACCAGGTATGCGGATAG
CGTGAAAGGCCGTTTTACCATTTCAGCTGATACTTCGAAGAACACCG
CCTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTAT
TATTGCTCGCGTTGGGGAGGAGAGGGGTTCTATGCTATGGATTACTG
GGGCCAAGGCACCCTGGTGACGGTTAGCTCA 20 Trastuzumab GFNIKDTYIH
VH (D98E) (6637) CDR1 29 Trastuzumab RIYPTNGYTRYADSVKG VH (D98E)
(6637) CDR2 79 Trastuzumab WGGEGFYAMDY VH (D98E) (6637) CDR3 98
Trastuzumab EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW VH
(D98T) VARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVY (6638)
YCSRWGGTGFYAMDYWGQGTLVTVSS 97 Trastuzumab
GAAGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGG VH (D98T)
CAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTAACATAAAGGACA (6638) DNA
CATACATCCACTGGGTGCGCCAAGCACCTGGGAAGGGTCTCGAGTGG
GTGGCTCGGATTTACCCAACAAATGGCTACACCAGGTATGCGGATAG
CGTGAAAGGCCGTTTTACCATTTCAGCTGATACTTCGAAGAACACCG
CCTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTAT
TATTGCTCGCGTTGGGGAGGAACCGGGTTCTATGCTATGGATTACTG
GGGCCAAGGCACCCTGGTGACGGTTAGCTCA 20 Trastuzumab GFNIKDTYIH VH (D98T)
(6638) CDR1 29 Trastuzumab RIYPTNGYTRYADSVKG VH (D98T) (6638) CDR2
80 Trastuzumab WGGTGFYAMDY VH (D98T) (6638) CDR3 100 Trastuzumab
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW VH (G99A)
VARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVY (6639)
YCSRWGGDAFYAMDYWGQGTLVTVSS 99 Trastuzumab
GAAGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGG VH (G99A)
CAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTAACATAAAGGACA (6639) DNA
CATACATCCACTGGGTGCGCCAAGCACCTGGGAAGGGTCTCGAGTGG
GTGGCTCGGATTTACCCAACAAATGGCTACACCAGGTATGCGGATAG
CGTGAAAGGCCGTTTTACCATTTCAGCTGATACTTCGAAGAACACCG
CCTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTAT
TATTGCTCGCGTTGGGGAGGAGACGCCTTCTATGCTATGGATTACTG
GGGCCAAGGCACCCTGGTGACGGTTAGCTCA 20 Trastuzumab GFNIKDTYIII VH
(G99A) (6639) CDR1 29 Trastuzumab RIYPTNGYTRYADSVKG VH (G99A)
(6639) CDR2 87 Trastuzumab WGGDAFYAMDY VH (G99A) (6639) CDR3 102
Trastuzumab EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW VH
(G99S) VARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVY (6640)
YCSRWGGDSFYAMDYWGQGTLVTVSS 101 Trastuzumab
GAAGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGG VH (G99S)
CAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTAACATAAAGGACA (6640) DNA
CATACATCCACTGGGTGCGCCAAGCACCTGGGAAGGGTCTCGAGTGG
GTGGCTCGGATTTACCCAACAAATGGCTACACCAGGTATGCGGATAG
CGTGAAAGGCCGTTTTACCATTTCAGCTGATACTTCGAAGAACACCG
CCTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTAT
TATTGCTCGCGTTGGGGAGGAGACAGCTTCTATGCTATGGATTACTG
GGGCCAAGGCACCCTGGTGACGGTTAGCTCA 20 Trastuzumab GFNIKDTYIII VH
(G99S) (6640) CDR1 29 Trastuzumab RIYPTNGYTRYADSVKG VH (G99S)
(6640) CDR2 88 Trastuzumab WGGDSFYAMDY VH (G99S) (6640) CDR3
TABLE-US-00040 TABLE 34 Antigens SEQ ID NO Name Sequence 2 Her2 ECD
MGWSCIILFLVATATGVHSTQVCTGTDMKLRLPASPETHLDMLRHLY
QGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQ
RLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRS
LTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRA
CHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHE
QCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNP
EGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCE
KCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPE
SFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQ
NLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTH
LCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHC
WGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPEC
QPQNGSVTCFGLEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIW
KFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTVDEQLYFQG
GSGLNDIFEAQKIEWHEARAHHHHHH 1 Her2 ECD
ACCCAAGTGTGCACCGGCACAGACATGAAGCTGCGGCTCCCTGCCAG DNA
TCCCGAGACCCACCTGGACATGCTCCGCCACCTCTACCAGGGCTGCC
AGGTGGTGCAGGGAAACCTGGAACTCACCTACCTGCCCACCAATGCC
AGCCTGTCCTTCCTGCAGGATATCCAGGAGGTGCAGGGCTACGTGCT
CATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGA
TTGTGCGAGGCACCCAGCTCTTTGAGGACAACTATGCCCTGGCCGTG
CTAGACAATGGAGACCCGCTGAACAATACCACCCCTGTCACAGGGGC
CTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACAGAGA
TCTTGAAAGGAGGGGTCTTGATCCAGCGGAACCCCCAGCTCTGCTAC
CAGGACACGATTTTGTGGAAGGACATCTTCCACAAGAACAACCAGCT
GGCTCTCACACTGATAGACACCAACCGCTCTCGGGCCTGCCACCCCT
GTTCTCCGATGTGTAAGGGCTCCCGCTGCTGGGGAGAGAGTTCTGAG
GATTGTCAGAGCCTGACGCGCACTGTCTGTGCCGGTGGCTGTGCCCG
CTGCAAGGGGCCACTGCCCACTGACTGCTGCCATGAGCAGTGTGCTG
CCGGCTGCACGGGCCCCAAGCACTCTGACTGCCTGGCCTGCCTCCAC
TTCAACCACAGTGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCAC
CTACAACACAGACACGTTTGAGTCCATGCCCAATCCCGAGGGCCGGT
ATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTACAACTACCTT
TCTACGGACGTGGGATCCTGCACCCTCGTCTGCCCCCTGCACAACCA
AGAGGTGACAGCAGAGGATGGAACACAGCGGTGTGAGAAGTGCAGCA
AGCCCTGTGCCCGAGTGTGCTATGGTCTGGGCATGGAGCACTTGCGA
GAGGTGAGGGCAGTTACCAGTGCCAATATCCAGGAGTTTGCTGGCTG
CAAGAAGATCTTTGGGAGCCTGGCATTTCTGCCGGAGAGCTTTGATG
GGGACCCAGCCTCCAACACTGCCCCGCTCCAGCCAGAGCAGCTCCAA
GTGTTTGAGACTCTGGAAGAGATCACAGGTTACCTATACATCTCAGC
ATGGCCGGACAGCCTGCCTGACCTCAGCGTCTTCCAGAACCTGCAAG
TAATCCGGGGACGAATTCTGCACAATGGCGCCTACTCGCTGACCCTG
CAAGGGCTGGGCATCAGCTGGCTGGGGCTGCGCTCACTGAGGGAACT
GGGCAGTGGACTGGCCCTCATCCACCATAACACCCACCTCTGCTTCG
TGCACACGGTGCCCTGGGACCAGCTCTTTCGGAACCCGCACCAAGCT
CTGCTCCACACTGCCAACCGGCCAGAGGACGAGTGTGTGGGCGAGGG
CCTGGCCTGCCACCAGCTGTGCGCCCGAGGGCACTGCTGGGGTCCAG
GGCCCACCCAGTGTGTCAACTGCAGCCAGTTCCTTCGGGGCCAGGAG
TGCGTGGAGGAATGCCGAGTACTGCAGGGGCTCCCCAGGGAGTATGT
GAATGCCAGGCACTGTTTGCCGTGCCACCCTGAGTGTCAGCCCCAGA
ATGGCTCAGTGACCTGTTTTGGACTGGAGGCTGACCAGTGTGTGGCC
TGTGCCCACTATAAGGACCCTCCCTTCTGCGTGGCCCGCTGCCCCAG
CGGTGTGAAACCTGACCTCTCCTACATGCCCATCTGGAAGTTTCCAG
ATGAGGAGGGCGCATGCCAGCCTTGCCCCATCAACTGCACCCACTCC
TGTGTGGACCTGGATGACAAGGGCTGCCCCGCCGAGCAGAGAGCCAG
CCCTCTGACGGTCGACGAACAGTTATATTTTCAGGGCGGCTCAGGCC
TGAACGACATCTTCGAGGCCCAGAAGATCGAGTGGCACGAGGCTCGA
GCTCACCACCATCACCATCAC 4 Fc (hole)
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKN
QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
KLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 3 Fc (hole)
GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGG DNA
GGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA
TGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC
CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGA
GGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA
CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG
AATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGC
CCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC
CACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAAC
CAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACAT
CGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGA
CCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGC
AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTC
ATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGA
GCCTCTCCCTGTCTCCGGGTAAA 6 Her2 ECD-
TQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNA Fc (knob)
SLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAV
LDNGDPLNNTTPVTGASPGGLRELQLRSLIEILKGGVLIQRNPQLCY
QDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSE
DCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLH
FNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYL
STDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLR
EVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQ
VFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTL
QGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQA
LLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQE
CVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGLEADQCVA
CAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHS
CVDLDDKGCPAEQRASPLTVDGGSPTPPTPGGGSADKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
ALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE 5 Her2 ECD-
ACCCAAGTGTGCACCGGCACAGACATGAAGCTGCGGCTCCCTGCCAG Fc (knob)
TCCCGAGACCCACCTGGACATGCTCCGCCACCTCTACCAGGGCTGCC DNA
AGGTGGTGCAGGGAAACCTGGAACTCACCTACCTGCCCACCAATGCC
AGCCTGTCCTTCCTGCAGGATATCCAGGAGGTGCAGGGCTACGTGCT
CATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGA
TTGTGCGAGGCACCCAGCTCTTTGAGGACAACTATGCCCTGGCCGTG
CTAGACAATGGAGACCCGCTGAACAATACCACCCCTGTCACAGGGGC
CTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACAGAGA
TCTTGAAAGGAGGGGTCTTGATCCAGCGGAACCCCCAGCTCTGCTAC
CAGGACACGATTTTGTGGAAGGACATCTTCCACAAGAACAACCAGCT
GGCTCTCACACTGATAGACACCAACCGCTCTCGGGCCTGCCACCCCT
GTTCTCCGATGTGTAAGGGCTCCCGCTGCTGGGGAGAGAGTTCTGAG
GATTGTCAGAGCCTGACGCGCACTGTCTGTGCCGGTGGCTGTGCCCG
CTGCAAGGGGCCACTGCCCACTGACTGCTGCCATGAGCAGTGTGCTG
CCGGCTGCACGGGCCCCAAGCACTCTGACTGCCTGGCCTGCCTCCAC
TTCAACCACAGTGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCAC
CTACAACACAGACACGTTTGAGTCCATGCCCAATCCCGAGGGCCGGT
ATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTACAACTACCTT
TCTACGGACGTGGGATCCTGCACCCTCGTCTGCCCCCTGCACAACCA
AGAGGTGACAGCAGAGGATGGAACACAGCGGTGTGAGAAGTGCAGCA
AGCCCTGTGCCCGAGTGTGCTATGGTCTGGGCATGGAGCACTTGCGA
GAGGTGAGGGCAGTTACCAGTGCCAATATCCAGGAGTTTGCTGGCTG
CAAGAAGATCTTTGGGAGCCTGGCATTTCTGCCGGAGAGCTTTGATG
GGGACCCAGCCTCCAACACTGCCCCGCTCCAGCCAGAGCAGCTCCAA
GTGTTTGAGACTCTGGAAGAGATCACAGGTTACCTATACATCTCAGC
ATGGCCGGACAGCCTGCCTGACCTCAGCGTCTTCCAGAACCTGCAAG
TAATCCGGGGACGAATTCTGCACAATGGCGCCTACTCGCTGACCCTG
CAAGGGCTGGGCATCAGCTGGCTGGGGCTGCGCTCACTGAGGGAACT
GGGCAGTGGACTGGCCCTCATCCACCATAACACCCACCTCTGCTTCG
TGCACACGGTGCCCTGGGACCAGCTCTTTCGGAACCCGCACCAAGCT
CTGCTCCACACTGCCAACCGGCCAGAGGACGAGTGTGTGGGCGAGGG
CCTGGCCTGCCACCAGCTGTGCGCCCGAGGGCACTGCTGGGGTCCAG
GGCCCACCCAGTGTGTCAACTGCAGCCAGTTCCTTCGGGGCCAGGAG
TGCGTGGAGGAATGCCGAGTACTGCAGGGGCTCCCCAGGGAGTATGT
GAATGCCAGGCACTGTTTGCCGTGCCACCCTGAGTGTCAGCCCCAGA
ATGGCTCAGTGACCTGTTTTGGACTGGAGGCTGACCAGTGTGTGGCC
TGTGCCCACTATAAGGACCCTCCCTTCTGCGTGGCCCGCTGCCCCAG
CGGTGTGAAACCTGACCTCTCCTACATGCCCATCTGGAAGTTTCCAG
ATGAGGAGGGCGCATGCCAGCCTTGCCCCATCAACTGCACCCACTCC
TGTGTGGACCTGGATGACAAGGGCTGCCCCGCCGAGCAGAGAGCCAG
CCCTCTGACGGTCGACGGTGGTAGTCCGACACCTCCGACACCCGGGG
GTGGTTCTGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCT
GAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA
GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGG
TGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG
GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA
GTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC
AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA
GCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCA
GCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGC
TGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTAT
CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAA
CACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCT
TCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG
AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA
CACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCTGA
ACGACATCTTCGAGGCCCAGAAGATTGAATGGCACGAG 8 Her2
TQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNA ECD
SLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAV (pertuzumab
LDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCY KO)-Fc
QDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSE (knob)
DCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLH
FNHSGICELHCPALVTYNTDTRESMPNPEGRYRFGASCVTACPYNYL
STDRGSCTLVCPLANQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLR
EVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQ
VFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTL
QGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQA
LLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQE
CVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGLEADQCVA
CAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHS
CVDLDDKGCPAEQRASPLTVDGGSPTPPTPGGGSADKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
ALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE 7 Her2
ACCCAAGTGTGCACCGGCACAGACATGAAGCTGCGGCTCCCTGCCAG ECD
TCCCGAGACCCACCTGGACATGCTCCGCCACCTCTACCAGGGCTGCC (pertuzumab
AGGTGGTGCAGGGAAACCTGGAACTCACCTACCTGCCCACCAATGCC KO)-Fc
AGCCTGTCCTTCCTGCAGGATATCCAGGAGGTGCAGGGCTACGTGCT (knob) DNA
CATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGA
TTGTGCGAGGCACCCAGCTCTTTGAGGACAACTATGCCCTGGCCGTG
CTAGACAATGGAGACCCGCTGAACAATACCACCCCTGTCACAGGGGC
CTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACAGAGA
TCTTGAAAGGAGGGGTCTTGATCCAGCGGAACCCCCAGCTCTGCTAC
CAGGACACGATTTTGTGGAAGGACATCTTCCACAAGAACAACCAGCT
GGCTCTCACACTGATAGACACCAACCGCTCTCGGGCCTGCCACCCCT
GTTCTCCGATGTGTAAGGGCTCCCGCTGCTGGGGAGAGAGTTCTGAG
GATTGTCAGAGCCTGACGCGCACTGTCTGTGCCGGTGGCTGTGCCCG
CTGCAAGGGGCCACTGCCCACTGACTGCTGCCATGAGCAGTGTGCTG
CCGGCTGCACGGGCCCCAAGCACTCTGACTGCCTGGCCTGCCTCCAC
TTCAACCACAGTGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCAC
CTACAACACAGACACGCGGGAGTCCATGCCCAATCCCGAGGGCCGGT
ATAGATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTACAACTACCTT
TCTACGGACCGGGGATCCTGCACCCTCGTCTGCCCCCTGGCCAACCA
AGAGGTGACAGCAGAGGATGGAACACAGCGGTGTGAGAAGTGCAGCA
AGCCCTGTGCCCGAGTGTGCTATGGTCTGGGCATGGAGCACTTGCGA
GAGGTGAGGGCAGTTACCAGTGCCAATATCCAGGAGTTTGCTGGCTG
CAAGAAGATCTTTGGGAGCCTGGCATTTCTGCCGGAGAGCTTTGATG
GGGACCCAGCCTCCAACACTGCCCCGCTCCAGCCAGAGCAGCTCCAA
GTGTTTGAGACTCTGGAAGAGATCACAGGTTACCTATACATCTCAGC
ATGGCCGGACAGCCTGCCTGACCTCAGCGTCTTCCAGAACCTGCAAG
TAATCCGGGGACGAATTCTGCACAATGGCGCCTACTCGCTGACCCTG
CAAGGGCTGGGCATCAGCTGGCTGGGGCTGCGCTCACTGAGGGAACT
GGGCAGTGGACTGGCCCTCATCCACCATAACACCCACCTCTGCTTCG
TGCACACGGTGCCCTGGGACCAGCTCTTTCGGAACCCGCACCAAGCT
CTGCTCCACACTGCCAACCGGCCAGAGGACGAGTGTGTGGGCGAGGG
CCTGGCCTGCCACCAGCTGTGCGCCCGAGGGCACTGCTGGGGTCCAG
GGCCCACCCAGTGTGTCAACTGCAGCCAGTTCCTTCGGGGCCAGGAG
TGCGTGGAGGAATGCCGAGTACTGCAGGGGCTCCCCAGGGAGTATGT
GAATGCCAGGCACTGTTTGCCGTGCCACCCTGAGTGTCAGCCCCAGA
ATGGCTCAGTGACCTGTTTTGGACTGGAGGCTGACCAGTGTGTGGCC
TGTGCCCACTATAAGGACCCTCCCTTCTGCGTGGCCCGCTGCCCCAG
CGGTGTGAAACCTGACCTCTCCTACATGCCCATCTGGAAGTTTCCAG
ATGAGGAGGGCGCATGCCAGCCTTGCCCCATCAACTGCACCCACTCC
TGTGTGGACCTGGATGACAAGGGCTGCCCCGCCGAGCAGAGAGCCAG
CCCTCTGACGGTCGACGGTGGTAGTCCGACACCTCCGACACCCGGGG
GTGGTTCTGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCT
GAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA
GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGG
TGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG
GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA
GTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC
AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA
GCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCA
GCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGC
TGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTAT
CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAA
CAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCT
TCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG
AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA
CACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCTGA
ACGACATCTTCGAGGCCCAGAAGATTGAATGGCACGAG 10 Her2
TQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNA ECD
SLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAV (trastuzumab
LDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCY KO)-Fc
QDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSE (knob)
DCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLH
FNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYL
STDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLR
EVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQ
VFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTL
QGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQA
LLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQE
CVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGLEARQCVA
CAHYKDRRCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSC
VDLDDKGCPAEQRASPLTVDGGSPTPPTPGGGSADKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA
LPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE 9 Her2
ACCCAAGTGTGCACCGGCACAGACATGAAGCTGCGGCTCCCTGCCAG ECD
TCCCGAGACCCACCTGGACATGCTCCGCCACCTCTACCAGGGCTGCC (trastuzumab
AGGTGGTGCAGGGAAACCTGGAACTCACCTACCTGCCCACCAATGCC KO)-Fc
AGCCTGTCCTTCCTGCAGGATATCCAGGAGGTGCAGGGCTACGTGCT (knob)
CATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGA DNA
TTGTGCGAGGCACCCAGCTCTTTGAGGACAACTATGCCCTGGCCGTG
CTAGACAATGGAGACCCGCTGAACAATACCACCCCTGTCACAGGGGC
CTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACAGAGA
TCTTGAAAGGAGGGGTCTTGATCCAGCGGAACCCCCAGCTCTGCTAC
CAGGACACGATTTTGTGGAAGGACATCTTCCACAAGAACAACCAGCT
GGCTCTCACACTGATAGACACCAACCGCTCTCGGGCCTGCCACCCCT
GTTCTCCGATGTGTAAGGGCTCCCGCTGCTGGGGAGAGAGTTCTGAG
GATTGTCAGAGCCTGACGCGCACTGTCTGTGCCGGTGGCTGTGCCCG
CTGCAAGGGGCCACTGCCCACTGACTGCTGCCATGAGCAGTGTGCTG
CCGGCTGCACGGGCCCCAAGCACTCTGACTGCCTGGCCTGCCTCCAC
TTCAACCACAGTGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCAC
CTACAACACAGACACGTTTGAGTCCATGCCCAATCCCGAGGGCCGGT
ATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTACAACTACCTT
TCTACGGACGTGGGATCCTGCACCCTCGTCTGCCCCCTGCACAACCA
AGAGGTGACAGCAGAGGATGGAACACAGCGGTGTGAGAAGTGCAGCA
AGCCCTGTGCCCGAGTGTGCTATGGTCTGGGCATGGAGCACTTGCGA
GAGGTGAGGGCAGTTACCAGTGCCAATATCCAGGAGTTTGCTGGCTG
CAAGAAGATCTTTGGGAGCCTGGCATTTCTGCCGGAGAGCTTTGATG
GGGACCCAGCCTCCAACACTGCCCCGCTCCAGCCAGAGCAGCTCCAA
GTGTTTGAGACTCTGGAAGAGATCACAGGTTACCTATACATCTCAGC
ATGGCCGGACAGCCTGCCTGACCTCAGCGTCTTCCAGAACCTGCAAG
TAATCCGGGGACGAATTCTGCACAATGGCGCCTACTCGCTGACCCTG
CAAGGGCTGGGCATCAGCTGGCTGGGGCTGCGCTCACTGAGGGAACT
GGGCAGTGGACTGGCCCTCATCCACCATAACACCCACCTCTGCTTCG
TGCACACGGTGCCCTGGGACCAGCTCTTTCGGAACCCGCACCAAGCT
CTGCTCCACACTGCCAACCGGCCAGAGGACGAGTGTGTGGGCGAGGG
CCTGGCCTGCCACCAGCTGTGCGCCCGAGGGCACTGCTGGGGTCCAG
GGCCCACCCAGTGTGTCAACTGCAGCCAGTTCCTTCGGGGCCAGGAG
TGCGTGGAGGAATGCCGAGTACTGCAGGGGCTCCCCAGGGAGTATGT
GAATGCCAGGCACTGTTTGCCGTGCCACCCTGAGTGTCAGCCCCAGA
ATGGCTCAGTGACCTGTTTTGGACTGGAGGCTCGGCAGTGTGTGGCC
TGTGCCCACTATAAGGACAGACGGTGCGTGGCCCGCTGCCCCAGCGG
TGTGAAACCTGACCTCTCCTACATGCCCATCTGGAAGTTTCCAGATG
AGGAGGGCGCATGCCAGCCTTGCCCCATCAACTGCACCCACTCCTGT
GTGGACCTGGATGACAAGGGCTGCCCCGCCGAGCAGAGAGCCAGCCC
TCTGACGGTCGACGGTGGTAGTCCGACACCTCCGACACCCGGGGGTG
GTTCTGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAA
CTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA
CACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGG
ACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC
GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGG
ACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC
CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCC
CCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGA
CCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCC
AGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAA
CTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC
TCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC
GTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCTGAACG
ACATCTTCGAGGCCCAGAAGATTGAATGGCACGAG
TABLE-US-00041 TABLE 35 Full-length antibody sequences of common
light chain antibody "D1 der" SEQ ID NO Name Sequence 159
Trastuzumab EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW
VHCH1-Fc VARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVY KNOB
YCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
ALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK 160 Trastuzumab
GAAGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCC VHCH1- Fc
TGCGTCTGAGCTGCGCGGCCTCCGGATTTAACATAAAGGACACATACATCCA KNOB DNA
CTGGGTGCGCCAAGCACCTGGGAAGGGTCTCGAGTGGGTGGCTCGGATTTAC
CCAACAAATGGCTACACCAGGTATGCGGATAGCGTGAAAGGCCGTTTTACCA
TTTCAGCTGATACTTCGAAGAACACCGCCTATCTGCAAATGAACAGCCTGCG
TGCGGAAGATACGGCCGTGTATTATTGCTCGCGTTGGGGAGGAGACGGGTTC
TATGCTATGGATTACTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCAGCTA
GCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTC
TGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCG
GTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC
CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGT
GCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAG
CCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAA
CTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGT
CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCT
GAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGT
TCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG
GGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTG
CACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAG
CCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG
AGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAAC
CAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCG
TGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC
CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGAC
AAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGG
CTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 161 Common
DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSAS light chain
FRYTGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKV VLCL
EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC 162
Common GACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGTG
light chain
ACCATCACATGCAAGGCCAGCCAGGACGTGTCCACAGCCGTGGCCTGGTATCAGCAG VLCL -
DNA AAGCCTGGCAAGGCCCCCAAGCTGCTGATCTACAGCGCCAGCTTCCGGTACACCGGC
GTGCCCAGCAGATTCAGCGGCAGCAGATCCGGCACCGACTTCACCCTGACCATCAGC
TCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACCCCC
CCCACATTTGGCCAGGGCACCAAGGTGGAAATCAAGCGTACGGTGGCTGCACCATCT
GTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTG
TGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAAC
GCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAA
GTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTC
AACAGGGGAGAGTGT 163 Pertuzumab
EVQLVESGGGLVQPGGSLRLSCAASGFTFNDYTMDWVRQAPGKGLEWVADVN VHCH1 Fc
PNSGGSIVNRRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPFF hole
YFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP
SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE
VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQ
VSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 164 Pertuzumab
GAAGTTCAGCTGGTTGAAAGCGGTGGTGGTCTGGTTCAGCCTGGTGGTAGCC VHCH1 Fc
TGCGTCTGAGCTGTGCAGCAAGCGGTTTTACCTTTAACGATTATACCATGGA hole DNA
TTGGGTTCGTCAGGCACCGGGTAAAGGTCTGGAATGGGTTGCAGATGTTAAT
CCGAATAGCGGTGGTAGCATTGTTAACCGTCGTTTTAAAGGTCGTTTTACCC
TGAGCGTTGATCGTAGCAAAAATACCCTGTATCTGCAAATGAATAGTCTGCG
TGCAGAGGATACCGCAGTGTATTATTGTGCACGTAACCTGGGTCCGTTCTTC
TACTTTGATTATTGGGGTCAGGGCACCCTGGTTACCGTTAGCAGCGCTAGCA
CCAAGGGCCCAAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGG
CGGAACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTG
ACAGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTTCCAG
CCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTCACAGTGCC
TAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCC
AGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAGACCC
ACACCTGTCCCCCTTGTCCTGCCCCTGAGCTGCTGGGCGGACCCAGCGTGTT
CCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAA
GTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCA
ATTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCCGGGA
GGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCAC
CAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTCTCCAACAAGGCCC
TGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGA
ACCCCAGGTGTGCACCCTGCCCCCCAGCAGAGATGAGCTGACCAAGAACCAG
GTGTCCCTGAGCTGTGCCGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGG
AGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGT
GCTGGACAGCGACGGCAGCTTCTTCCTGGTGTCCAAACTGACCGTGGACAAG
AGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCC
TGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCAAG
Example 21: Generation of Trivalent HER2 (Extracellular Domain
IV)-HER2 (Extracellular Domain II)-Transferrin Specific
Antibodies
[0579] Generation of the Expression Plasmids
[0580] Desired proteins were expressed by transient transfection of
human embryonic kidney cells (HEK 293). For the expression of a
desired gene/protein (e.g. antibody-Fab multimeric protein) a
transcription unit comprising the following functional elements was
used: [0581] The immediate early enhancer and promoter from the
human cytomegalovirus (P-CMV) including intron A, [0582] a murine
immunoglobulin heavy chain signal sequence (SS), [0583] a
gene/protein to be expressed (e.g. full length antibody heavy
chain), and [0584] the bovine growth hormone polyadenylation
sequence (BGH pA).
[0585] Besides the expression unit/cassette including the desired
gene to be expressed the basic/standard mammalian expression
plasmid contains: [0586] An origin of replication from the vector
pUC18 which allows replication of this plasmid in E. coli, and
[0587] a beta-lactamase gene which confers ampicillin resistance in
E. coli
[0588] Expression Plasmids Coding for the Following
Antibody+/-scFab Proteins were Constructed:
[0589] 1. BBB-0580 Trivalent Herceptarg-scFab(8D3)
[0590] Heavy chain knob (Herceptin(D98E)(IgG1) knob
SS-(G4S)4-VL-Ck-(G4S)6-GG-VH-CH1) (Seq. 21832/SEQ ID NO.: 165)
[0591] Composition of the knob Herceptin(D98E)-scFab(8D3) heavy
chain fusion protein: [0592] Herceptin(D89E) human IgG1 heavy chain
without C-terminal Lys containing CH3 knob mutation T366W and the
S354C mutation for the formation of an additional disulfide bridge
[0593] Glycine-Serine-linker [0594] Variable light chain domain
(VL) variant of the mouse 8D3 anti-transferrin antibody (Boado et
al., 2009) [0595] Human C-kappa light chain [0596]
Glycine-Serine-linker [0597] Variable heavy chain domain (VL)
variant of the mouse 8D3 anti-transferrin antibody (Boado et al.,
2009) [0598] Human IgG1 CH1 heavy chain domain
[0599] Heavy chain hole (Pertuzumab(affmat.)(IgG1) hole SS (Seq. ID
NO.: 163)
[0600] Composition of the hole Pertuzumab(aff mat.) heavy chain
protein: [0601] Pertuzumab(affmat.) human IgG1 heavy chain
containing the CH3 hole mutations T366S, Y407V and L368A and the
Y349C mutation for the formation of an additional disulfide
bridge
[0602] Light chain (CLC-Herc combo) (clone D1-derSEQ ID NO.: 161)
[0603] See example 14
[0604] 2. BBB-0581 Trivalent Herceptarg(LALA)-scFab(8D3)
[0605] Heavy chain knob (Herceptin(D98E)(IgG1) LALA-PG knob
SS-(G4S)4-VL-Ck-(G4S)6-GG-VH-CH1) (Seq. 21834, SEQ ID. NO.:
168)
[0606] Composition of the knob Herceptin(D98E)-scFab(8D3) heavy
chain fusion protein: [0607] Herceptin(D89E) human IgG1 heavy chain
without C-terminal Lys containing: [0608] CH2 mutations L234A,
L235A and P329G to impair Fc gamma receptor-mediated interaction
with immune effector cells as well as complement activation [0609]
CH3 knob mutation T366W and the S354C mutation for the formation of
an additional disulfide bridge [0610] Glycine-Serine-linker [0611]
Variable light chain domain (VL) variant of the mouse 8D3
anti-transferrin antibody (Boado et al., 2009), SEQ ID NO.: 178
[0612] Human C-kappa light chain [0613] Glycine-Serine-linker
[0614] Variable heavy chain domain (VL) variant of the mouse 8D3
anti-transferrin antibody (Boado et al., 2009), SEQ ID NO.: 179
[0615] Human IgG1 CH1 heavy chain domain
[0616] Heavy chain hole (Pertuzumab(affmat.)(IgG1) LALA-PG hole SS
(Seq. 21836, SEQ ID NO.: 169)
[0617] Composition of the hole Pertuzumab(aff mat.) heavy chain
protein: [0618] Pertuzumab(affmat.) human IgG1 heavy chain
containing: [0619] CH2 mutations L234A, L235A and P329G to impair
Fc gamma receptor-mediated interaction with immune effector cells
as well as complement activation [0620] CH3 hole mutations T366S,
L368A and Y407V and the Y349C mutation for the formation of an
additional disulfide bridge [0621] CH3 hole mutations H435R and
Y436F to avoid protein purification of heavy chain hole-hole
dimers
[0622] Light chain (CLC-Herc combo) (clone D1-derSEQ ID NO.: 161)
[0623] See example 14
[0624] 3. BBB-0582 Bivalent Herceptarg(LALA) (Control Molecule
without BBB-R Binder)
[0625] Heavy chain knob (Herceptin(D98E)(IgG1) LALA-PG (Seq. 21835,
SEQ ID.NO.: 170)
[0626] Composition of the knob Herceptin(D98E) heavy chain protein:
[0627] Herceptin(D89E) human IgG1 heavy chain containing: [0628]
CH2 mutations L234A, L235A and P329G to impair Fc gamma
receptor-mediated interaction with immune effector cells as well as
complement activation [0629] CH3 knob mutation T366W and the S354C
mutation for the formation of an additional disulfide bridge
[0630] Heavy Chain Hole (Pertuzumab(Affmat.)(IgG1) LALA-PG Hole SS
(Seq. 21836 SEQ ID.NO.: 163)
[0631] Composition of the hole Pertuzumab(aff mat.) heavy chain
protein: [0632] Pertuzumab(affmat.) human IgG1 heavy chain
containing: [0633] CH2 mutations L234A, L235A and P329G to impair
Fc gamma receptor-mediated interaction with immune effector cells
as well as complement activation [0634] CH3 hole mutations T366S,
L368A and Y407V and the Y349C mutation for the formation of an
additional disulfide bridge [0635] CH3 hole mutations H435R and
Y436F to avoid protein purification of hole-hole dimers
[0636] Light chain (CLC-Herc combo) (clone D1-derSEQ ID NO.: 161)
[0637] See example 14
[0638] 4. Herceptarg (Control Molecule without BBB-R Binder)
[0639] Heavy chain knob (Herceptin(D98E)(IgG1) (SEQ ID NO.:
171)
[0640] Composition of the knob Herceptin(D98E) heavy chain protein:
[0641] Herceptin(D89E) human IgG1 heavy chain containing CH3 knob
mutation T366W and the S354C mutation for the formation of an
additional disulfide bridge
[0642] Heavy chain hole (Pertuzumab(aff.mat.)(IgG1) hole SS (Seq.
21836 SEQ ID.NO.: 163)
[0643] Composition of the hole Pertuzumab(aff mat.) heavy chain
protein: [0644] Pertuzumab(aff mat.) human IgG1 heavy chain
containing CH3 hole mutations T366S, L368A and Y407V and the Y349C
mutation for the formation of an additional disulfide bridge
[0645] Light chain (CLC-Herc combo) (clone D1-derSEQ ID NO.: 161)
[0646] See example 14
[0647] Sequences are shown in tables 36 and 37.
[0648] Ref Boado R J, Zhang Y, Wang Y, Pardridge W M. Engineering
and expression of a chimeric transferrin receptor monoclonal
antibody for blood-brain barrier delivery in the mouse. Biotechnol
Bioeng. 2009; 102(4):1251-8.
Example 22: Purification of Trivalent HER2 (Extracellular Domain
IV)-HER2 (Extracellular Domain II)-Transferrin Specific Antibodies
and Control Antibodies without BBB-R Binder
[0649] The antibody chains were generated by transient transfection
of HEK293 cells (human embryonic kidney cell line 293-derived)
cultivated in F17 Medium (Invitrogen Corp.). For transfection
293-Free Transfection Reagent (Merck, Millipore) was used. The
antibody chains were expressed from three different plasmids,
coding for the trivalent Herceptarg-scFab(8D3) knob and hole heavy
chains and the Herceptarg light chain or the bivalent Herceptarg
knob and hole heavy chains and the Herceptarg light chain,
respectively. For transfection the three plasmids were used at
plasmid ratios of 2:1:1 (LC: HC knob: HC hole) for trivalent
Herceptarg-scFab(8D3), of 1:1:2 (LC: HC knob: HC hole) for
trivalent Herceptarg(LALA)-scFab(8D3) and bivalent Herceptarg(LALA)
and of 2:1:1(LC: HC knob: HC hole) for bivalent Herceptarg.
Transfections were performed as specified in the manufacturer's
instructions. Antibody-containing cell culture supernatants were
harvested seven days after transfection. Supernatants were stored
frozen until purification.
[0650] Proteins were purified from filtered cell culture
supernatants. Supernatants were applied to a protein A Sepharose
column (GE Healthcare) and washed with PBS pH 7.4. Elution of
antibodies was achieved with 100 mM Citarate buffer at pH 3.0
followed by immediate neutralization of the sample to pH 6.5.
Aggregated protein and other byproducts were separated from
monomeric antibodies by size exclusion chromatography (SEC;
Superdex 200; GE Healthcare) in 20 mM histidine, 140 mM NaCl, pH
6.0. Every single fraction was analyzed on analytical SEC (MabPac
SEC-1, Thermo Scientific) and on a chip-based capillary
electrophoresis system (CE-SDS, LabChipGX, Caliper) for the
quantification of incompletely assembled molecules and other
byproducts. Monomeric antibody fractions without byproducts were
pooled. After concentration using a MILLIPOREAmicon Ultra (30
molecular weight cut off) centrifugal concentrator the protein was
stored at -80.degree. C. Analytical characterization of the
endproduct was done by UV protein determination, CE-SDS, size
exclusion chromatography, mass spectrometry and also by endotoxin
determination.
Example 23: Binding Analysis of Herceptarg-scFab(8D3) and Control
Antibodies to BT474-M1 and BA/F3 Cells
[0651] HER2+BT474-M1 and TfR+BA/F3 cells were used as target cells
for binding analysis of Herceptarg-scFab(8D3) and Herceptarg
control molecules. BT474-M1 cells were maintained in RPMI 1640
supplemented with 10% fetal calf serum and 2 mL L-glutamine and
BA/F3 cells were maintained in RPMI 1640, 10% FCS, 2 mM L-glutamine
and 10 ng/ml rmIL-3. Propagation of cell lines followed standard
cell culture protocols.
[0652] BT474-M1 and BA/F3 cells were harvested and resuspended in
FACS buffer. 0.2 Mio cells were seeded into a 96 well round bottom
plate. The plate was centrifuged at 350 g for 3 min to pellet the
cells. The supernatant was removed and the cells were resuspended
in 120 .mu.l of the diluted antibodies. The plate was incubated for
1h at 4.degree. C. to allow binding of the antibodies. To remove
unbound antibodies the cells were centrifuged again and washed
twice with FACS buffer. To detect the antibodies the cells were
resuspended in 100 .mu.l diluted goat anti-human Fc.gamma. specific
PE-labeled secondary antibody (Jackson ImmunoResearch #109-116-170)
and incubated again for 30 min at 4.degree. C. Afterwards the cells
were washed twice with FACS buffer, resuspended in 50 FACS buffer
and the fluorescence was measured with BD Canto II. Results are
shown in FIGS. 33 and 34. All Herceptarg antibodies specifically
bind to HER2+BT474-M1 cells and the Herceptarg-scFab(8D3) fusion
proteins bind to TfR+BA/F3 cells.
Example 24: Proliferation Inhibition Assay with Trivalent HER2
(Extracellular Domain IV)-HER2 (Extracellular Domain
II)-Transferrin Specific Antibodies and Control Antibodies without
BBB-R Binder on HER2+BT474-M1 Cells
[0653] The ability of the Herceptarg molecules to inhibit
proliferation was assessed in the cell line BT474-M1. Cells in the
logarithmic growth phase were detached, counted and
4.times.10.sup.3 cells were seeded in 60 .mu.L medium per well of a
96-well cell culture plate. Cells were maintained overnight in the
incubator and the following day 60 .mu.L of the respective
antibodies diluted in medium were added in form of a dilution
series to the cells. After a total incubation time of 5 days cell
growth was assessed by CellTiter-Glo.RTM. Luminescent Cell
Viability Assay. The assay was performed as recommended by the
manufacturer.
[0654] FIG. 35 shows inhibition of BT474-M1 cell proliferation
after incubation with Herceptarg+/-scFab(8D3) molecules.
TABLE-US-00042 TABLE 36 BBB-R binders SEQ ID NO Description
Sequence 172 2/99 CDR-H1 FSLSSY 173 2/99 CDR-H2 YIWSGGSTDYASWA 174
2/99 CDR-H3 RYGTSYPDYGDANGFDP 175 2/99 CDR-L1 QASQSISSYLS 176 2/99
CDR-L2 RAS 177 2/99 CDR-L3 CYSSSNVDN 178 2/99 VH
QSMEESGGRLVTPGTPLTLTCTVSGFSLSSYAMSWVRQAPGKGLEWIGYIWSG
GSTDYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARRYGTSYPDYGDA
NGFDPWGPGTLVTVSS 179 2/99 VL
AYDMTQTPASVEVAVGGTVTIKCQASQSISSYLSWYQQKPGQRPKLLIYRAST
LASGVSSRFKGSGSGTQFTLTISGVECADAATYYCQQCYSSSNVDNTFGGGTE VVVKR 180
4/94 CDR-H1 GFNIKDT 181 4/94 CDR-H2 RIDPANGDTKCDPKFQ 182 4/94
CDR-H3 YLYPYYFD 183 4/94 CDR-L1 SESVDTY 184 4/94 CDR-L2 GAS 185
4/94 CDR-L3 TYNYPL 166 4/94 VH
EVQLQQSGAVLVKPGASVKLSCPASGFNIKDTYIHWVIQRPEQGLEWIGRIDP
ANGDTKCDPKFQVKATITADTSSNTAYLQLSSLTSEDTAVYFCVRDYLYPYYF DFWGQGTTLTVSS
167 4/94 VL KIVMTQSPKSMSMSVGERVTLNCRASESVDTYVSWYQQKPEQSPELLIYGAS
NRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYYCGQTYNYPLTFGAGTKLE LKR 186
Humanized SYAMS 2/99 CDR-H1 187 Humanized YIWSGGSTDY ASWAKS 2/99
CDR-H2 188 Humanized RYGTSYPDYGDASGFDP 2/99 CDR-H3 206 Humanized
RYGTSYPDYGDAQGFDP 2/99 CDR-H3 variant 189 Humanized RASQSISSYL A
2/99 CDR-L1 190 Humanized RASTLAS 2/99 CDR-L2 191 Humanized
QQNYASSNVD NT 2/99 CDR-L3 192 Humanized QSMQESGPGL VKPSQTLSLT
CTVSGFSLSS YAMSWIRQHP 2/99 VH GKGLEWIGYIWSGGSTDYASWAKSRVTISK
TSTTVSLKLS SVTAADTAVY YCARRYGTSYPDYGDASGFDPWGQGTLVTV SS 205
Humanized QSMQESGPGL VKPSQTLSLT CTVSGFSLSS YAMSWIRQHP GKGLEWIGYI
2/99 VH WSGGSTDYAS WAKSRVTISK TSTTVSLKLS SVTAADTAVY variant
YCARRYGTSYPDYGDAQGFDPWGQGTLVTVSS 193 Humanized AIQLTQSPSS
LSASVGDRVT ITCRASQSIS SYLAWYQQKP GKAPKLLIYR 2/99 VL ASTLASGVPS
RFSGSGSGTD FTLTISSLQP EDFATYYCQQ NYASSNVDNT FGGGTKVEI 194 Humanized
QVQLVQSGAE VKKPGASVKV SCKASGFNIK DTYIHWVIQA PGQGLEWMGR 2/99 VH
IDPANGDTKS DPKFQVRVTI TADTSTSTVY MELSSLRSED TAVYYCVRDY variant 1
LYPYYFDFWG QGTTVTVSS 195 Humanized QVQLVQSGAE VKKPGASVKV SCKASGFNIK
DTYIHWVIQA PGQGLEWMGR 2/99 VH IDPANGDTKS APKFQVRVTI TADTSTSTVY
MELSSLRSED TAVYYCVRDY variant 2 LYPYYFDFWG QGTTVTVSS 196 Humanized
QVQLVQSGAE VKKPGASVKV SCKASGFNIK DTYIHWVIQA PGQGLEWMGR 2/99 VH
IDPANGDTKS variant 3 DPKFQGRVTI TADTSTSTVY MELSSLRSED TAVYYCVRDY
LYPYYFDFWG QGTTVTVSS 197 Humanized QVQLVQSGAE VKKPGSSVKV SCKASGFNIK
DTYIHWVIQA PGQGLEWMGR 2/99 VH IDPANGDTKS DPKFQGRVTI TADESTSTAY
MELSSLRSED variant 4 TAVYYCVRDY LYPYYFDFWG QGTTVTVSS 198 Humanized
QVQLVQSGAE VKKPGSSVKV SCKASGFNIK DTYIHWVIQR PGQGLEWMGR 2/99 VH
IDPANGDTKS variant 5 DPKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCVRDY
LYPYYFDFWG QGTTVTVSS 199 Humanized QVQLQESGPG LVKPSETLSL TCTVSGFNIK
DTYIHWVIQR PGKGLEWIGR 2/99 VH IDPANGDTKS DPSLQSRVTI SADTSKNQAS
LKLSSVTAAD variant 6 TAVYYCVRDY LYPYYFDFWG QGTTVTVSS 200 Humanized
EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVIQA PGKGLEWVGR 2/99 VH
IDPANGDTKS variant 7 DPSVQVRATI SADTSKNTAY LQMNSLRAED TAVYYCVRDY
LYPYYFDFWG QGTTVTVSS 201 Humanized KIQMTQSPSS LSASVGDRVT ITCRASESVD
TYVSWYQQKP GKAPKLLIYG 2/99 VL ASNRYTGVPS RFSGSGSGTD FTLTISSLQP
EDFADYYCGQ TYNYPLTFGQ variant 1 GTKLEI 207 Humanized KIVLTQSPGT
LSLSPGERAT LSCRASESVD TYVSWYQQKP GQAPRLLIYG 2/99 VL ASNRYTGVPD
variant 2 RFSGSGSGTD FTLTISRLEP EDFADYYCGQ TYNYPLTFGQ GTKLEI 208
Humanized EIVLTQSPGT LSLSPGERAT LSCRASESVD TYVSWYQQKP GQAPRLLIYG
2/99 VL ASNRYTGVPD variant 3 RFSGSGSGTD FTLTISRLEP EDFADYYCGQ
TYNYPLTFGQ GTKLEI 209 Humanized KIVLTQSPGT LSLSPGERAT LSCRASESVD
TYVSWYQQKP GQAPRLLIYG 2/99 VL ASNRYTGIPD variant 4 RFSGSGSGTD
FTLTISRLEP EDFADYYCGQ TYNYPLTFGQ GTKLEI
TABLE-US-00043 TABLE 37 Trivalent HER2 (extracellular domain IV)-
HER2 (extracellular domain II)- Transferrin specific antibodies and
control antibodies without BBB-R binder SEQ ID NO Description
Sequence BBB-0580/Herceptarg-scFab(8D3) 165 Her-knob-
evqlvesggglvqpggslrlscaasgfnikdtyihwvrqapgkglewvariyptngytry
scFab(8D3)
adsvkgrftisadtskntaylqmnslraedtavyycsrwggegfyamdywgqgtlvtvss Heavy
astkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssgl
chain yslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsv
21832 flfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynst
yrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppcrdelt
knqvslwclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwq
qgnvfscsvmhealhnhytqkslslspgggsggggsggggsggggsdiqmtqspasls
asleeivtitcqasqdignwlawyqqkpgkspqlliygatsladgvpsrfsgsrsgtqfslki
srvqvedigiyyclqayntpwtfgggtkveikrtvaapsvfifppsdeqlksgtasvvclln
nfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevt
hqglsspvtksfnrgecggggsggggsggggsggggsggggsggggsggevqlvesgg
glvqpgnsltlscvasgftfsnygmhwirqapkkglewiamiyydsskmnyadtvkgr
ftisrdnskntlylemnslrsedtamyycavptshyvvdvwgqgvsvtvssastkgpsvf
plapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtv
pssslgtqtyicnvnhkpsntkvdkkvepksc 163 Per-hole
evqlvesggglvqpggslrlscaasgftfndytmdwvrqapgkglewvadvnpnsggsi Heavy
vnrrfkgrftlsvdrskntlylqmnslraedtavyycarnlgpffyfdywgqgtlvtvssast
chain
kgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglysl
21833 ssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflf
ppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyr
vvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvctlppsrdeltkn
qvslscavkgfypsdiavewesngqpennykttppvldsdgsfflvskltvdksrwqqg
nvfscsvmhealhnhytqkslslspgk 161 Her/Per
diqmtqspsslsasvgdrvtitckasqdvstavawyqqkpgkapklliysasfrytgvpsr CLC
fsgsrsgtdftltisslqpedfatyycqqhyttpptfgqgtkveik 21831
rtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqds
kdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec
BBB-0581/Herceptarg(LALA-PG)-scFab(8D3) 168 Her-knob-
evqlvesggglvqpggslrlscaasgfnikdtyihwvrqapgkglewvariyptngytry
LALA-PG-
adsvkgrftisadtskntaylqmnslraedtavyycsrwggegfyamdywgqgtlvtvss
scFab(8D3)
astkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssgl
Heavy yslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapeaaggps
chain vflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqyn
21834 styrvvsvltvlhqdwlngkeykckvsnkalgapiektiskakgqprepqvytlppcrdel
tknqvslwclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrw
qqgnvfscsvmhealhnhytqkslslspgggsggggsggggsggggsdiqmtqspasl
sasleeivtitcqasqdignwlawyqqkpgkspqlliygatsladgvpsrfsgsrsgtqfslk
isrvqvedigiyyclqayntpwtfgggtkveikrtvaapsvfifppsdeqlksgtasvvcll
nnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacev
thqglsspvtksfnrgecggggsggggsggggsggggsggggsggggsggevqlvesg
gglvqpgnsltlscvasgftfsnygmhwirqapkkglewiamiyydsskmnyadtvkg
rftisrdnskntlylemnslrsedtamyycavptshyvvdvwgqgvsvtvssastkgpsv
fplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtv
pssslgtqtyicnvnhkpsntkvdkkvepksc 169 Per-hole
Evqlvesggglvqpggslrlscaasgftfndytmdwvrqapgkglewvadvnpnsggs LALA-PG
ivnrrfkgrftlsvdrskntlylqmnslraedtavyycarnlgpffyfdywgqgtlvtvssas
Heavy
tkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglys
chain lssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapeaaggpsvf
21836 lfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynst
yrvvsvltvlhqdwlngkeykckvsnkalgapiektiskakgqprepqvctlppsrdeltk
nqvslscavkgfypsdiavewesngqpennykttppvldsdgsfflvskltvdksrwqq
gnvfscsvmhealhnrftqkslslspgk 161 Her/Per
diqmtqspsslsasvgdrvtitckasqdvstavawyqqkpgkapklliysasfrytgvpsr CLC
fsgsrsgtdftltisslqpedfatyycqqhyttpptfgqgtkveik 21831
rtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqds
kdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec
BBB-0582/Herceptarg-LALA-PG (control molecule without BBB-R binder)
170 Her-knob-
evqlvesggglvqpggslrlscaasgfnikdtyihwvrqapgkglewvariyptngytry
LALA-PG
adsvkgrftisadtskntaylqmnslraedtavyycsrwggegfyamdywgqgtlvtvss Heavy
astkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssgl
Chain yslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapeaaggps
21835 vflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqyn
styrvvsvltvlhqdwlngkeykckvsnkalgapiektiskakgqprepqvytlppcrdel
tknqvslwclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrw
qqgnvfscsvmhealhnhytqkslslspgk 169 Per-hole
Evqlvesggglvqpggslrlscaasgftfndytmdwvrqapgkglewvadvnpnsggs LALA-PG
ivnrrfkgrftlsvdrskntlylqmnslraedtavyycarnlgpffyfdywgqgtlvtvssas
Heavy
tkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglys
chain lssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapeaaggpsvf
21836 lfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynst
yrvvsvltvlhqdwlngkeykckvsnkalgapiektiskakgqprepqvctlppsrdeltk
nqvslscavkgfypsdiavewesngqpennykttppvldsdgsfflvskltvdksrwqq
gnvfscsvmhealhnrftqkslslspgk 161 Her/Per
diqmtqspsslsasvgdrvtitckasqdvstavawyqqkpgkapklliysasfrytgvpsr CLC
fsgsrsgtdftltisslqpedfatyycqqhyttpptfgqgtkveik 21831
rtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqds
kdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec Herceptarg (control
molecule without BBB-R binder) 171 pETR13573
evqlvesggglvqpggslrlscaasgfnikdtyihwvrqapgkglewvariyptngytry
Her-knob
adsvkgrftisadtskntaylqmnslraedtavyycsrwggegfyamdywgqgtlvtvss heavy
astkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssgl
chain yslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsv
flfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynst
yrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppcrdelt
knqvslwclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwq
qgnvfscsvmhealhnhytqkslslspgk 163 Per-hole
evqlvesggglvqpggslrlscaasgftfndytmdwvrqapgkglewvadvnpnsggsi Heavy
vnrrfkgrftlsvdrskntlylqmnslraedtavyycarnlgpffyfdywgqgtlvtvssast
chain
kgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglysl
21833 ssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflf
ppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyr
vvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvctlppsrdeltkn
qvslscavkgfypsdiavewesngqpennykttppvldsdgsfflvskltvdksrwqqg
nvfscsvmhealhnhytqkslslspgk 161 Her/Per
diqmtqspsslsasvgdrvtitckasqdvstavawyqqkpgkapklliysasfrytgvpsr CLC
fsgsrsgtdftltisslqpedfatyycqqhyttpptfgqgtkveik 21831
rtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqds
kdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec
TABLE-US-00044 TABLE 38 Sequence of pTAU-BBB-R control molecule SEQ
ID NO Description Sequence 210 8936_pTau_Rb
Aqvltqttspvsaavgstvtiscqssqsvrtnklawfqqkpgqppkrliysastldfgvpsrf
Mab86_VL_hu
sasgsgtqftltisdvqcddaatyyclgyfdcsiadcvafgggtevvvkrtvaapsvfifpps
Igkappa
deqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlsk
adyekhkvyacevthqglsspvtksfnrgec 211 8975-pTau-
Qsveesggrlvtpgtpltltctvsgfslssnainwvrqapgkglewigyiaysgntyyasw Rb86-
akgrftiskasttvdlkmtsptaedtgtyfcgksniwgpgtlvtvslastkgpsvfplapssk
hugamma1-SS-
stsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtq
hole tyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrt
pevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwl
ngkeykckvsnkalpapiektiskakgqprepqvctlppsrdeltknqvslscavkgfyp
sdiavewesngqpennykttppvldsdgsfflvskltvdksrwqqgnvfscsvmhealh
nhytqkslslspgk 212 8976-pTau-
qsveesggrlvtpgtpltltctvsgfslssnainwvrqapgkglewigyiavsgntyyaswa
Rb86-
kgrftiskasttvdlkmtsptaedtgtyfcgksniwgpgtivtvslastkgpsvfplapsskst
hugamma1-SS-
sggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqty
knob-mTfR-
icnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpe
8D3-scFab
vtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlng
keykckvsnkalpapiektiskakgqprepqvytlppcrdeltknqvslwclvkgfypsd
iavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnh
ytqkslslspgggsggggsggggsggggsdiqmtqspaslsasleeivtitcqasqdignw
lawyqqkpgkspqlliygatsladgvpsrfsgsrsgtqfslkisrvqvedigiyyclqaynt
pwtfgggtkveikrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnal
qsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgecgg
ggsggggsggggsggggsggggsggggsggevqlvesggglvqpgnsltlscvasgftf
snygmhwirqapkkglewiamiyydsskmnyadtvkgrftisrdnskntlylemnslr
sedtamyycavptshyvvdvwgqgvsvtvssastkgpsvfplapsskstsggtaalgclv
kdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsn
tkvdkkvepksc
Example 25: Characterization of Monovalent Anti TfR Antibodies
[0655] Identification of Human and Cynomolgus TfR-Binding
Antibodies by Cell ELISA
[0656] To screen rabbit B-cell or mouse hybridoma supernatants for
antibodies recognizing human and cynomolgus TfR, a cell ELISA using
stably transfected CHO-K1 cells way employed. Stable transfectants
were obtained by transfecting CHO-K1 cells with expression plasmids
containing expression cassettes for the human or cynomolgus TfR as
well as for neomycin-phosphotransferase. After transfection, cells
were diluted in growth medium containing 500 .mu.g/mL G418 (Life
Technologies). After appearance of growing clones, cells were
detached, stained with MEM-75 (Abcam) or 13E4 (Life Technologies)
and PE-labeled secondary antibodies for human or cynomolgus TfR,
and highly fluorescent cells sorted as single cells into
96-well-plate wells (FACS Aria). After 7 days of growth, clones
were again checked for TfR expression and best expressing clones
selected for cell ELISA experiments.
[0657] Briefly, 15,000 cells were seeded per well of a 384-well
plate and incubated for 18 h at 37.degree. C., 5% CO2. Supernatant
was removed using an automated washer (BIOTEK), and 30 .mu.L of
antibody-containing supernatant added to each well, followed by 24
.mu.L of growth medium. After 2 hours of incubation, wells were
emptied and 30 .mu.L of 0.05% glutaraldehyde in PBS added for 45
min. at RT. After 3 washes with PBS/0.025% Tween20 (PBST), 30 .mu.L
of anti-rabbit-HRP or anti-mouse-HRP (Southern Biotech) diluted
1:5000 in Blocking buffer was added and plates incubated for 1 hour
at RT. Wells were washed 6 times with PBST and signal was generated
using 30 .mu.L of TMB per well and absorbance measured at 450
nm.
[0658] Cloning and Expression of Anti-TfR Antibodies [0659]
Recombinant DNA techniques
[0660] Standard methods were used to manipulate DNA as described in
Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The
molecular biological reagents were used according to the
manufacturer's instructions. [0661] Gene and oligonucleotide
synthesis
[0662] Desired gene segments were prepared by chemical synthesis at
Geneart GmbH (Regensburg, Germany). The synthesized gene fragments
were cloned into an E. coli plasmid for propagation/amplification.
The DNA sequences of subcloned gene fragments were verified by DNA
sequencing. Alternatively, short synthetic DNA fragments were
assembled by annealing chemically synthesized oligonucleotides or
via PCR. The respective oligonucleotides were prepared by metabion
GmbH (Planegg-Martinsried, Germany).
[0663] PCR Amplification of V-Domains
[0664] Total RNA was prepared from B-cells lysate (resuspended in
RLT buffer--Qiagen--Cat. N.degree. 79216) using the NucleoSpin 8/96
RNA kit (Macherey & Nagel; 740709.4, 740698) according to
manufacturer's protocol. RNA was eluted with 60 .mu.L RNAse free
water. 6 .mu.L of RNA was used to generate cDNA by reverse
transcriptase reaction using the Superscript III First-Strand
Synthesis SuperMix (Invitrogen 18080-400) and an oligo-dT-primer
according to the manufacturer's instructions. All steps were
performed on a Hamilton ML Star System. 4 .mu.L of cDNA were used
to amplify the immunoglobulin heavy and light chain variable
regions (VH and VL) with the AccuPrime SuperMix (Invitrogen
12344-040) in a final volume of 50 .mu.L using the primers rbHC.up
and rbHC.do for the heavy chain, rbLC.up and rbLC.do for the light
chain of Wild Type Rabbit B cells and BcPCR_FHLC_leader.fw and
BcPCR_huCkappa.rev for the light chain of transgenic rabbit B-cells
(see Table below). All forward primers were specific for the signal
peptide (of respectively VH and VL) whereas the reverse primers
were specific for the constant regions (of respectively VH and VL).
The PCR conditions for the RbVH+RbVL were as follows: Hot start at
94.degree. C. for 5 min.; 35 cycles of 20 sec. at 94.degree. C., 20
sec. at 70.degree. C., 45 sec. at 68.degree. C., and a final
extension at 68.degree. C. for 7 min. The PCR conditions for the
HuVL were as follows: Hot start at 94.degree. C. for 5 min.; 40
cycles of 20 sec. at 94.degree. C., 20 sec. at 52.degree. C., 45
sec. at 68.degree. C., and a final extension at 68.degree. C. for 7
min.
TABLE-US-00045 TABLE 39 rbHC.up
AAGCTTGCCACCATGGAGACTGGGCTGCGCTGGCTTC (SEQ ID NO: 213) rbHCf.do
CCATTGGTGAGGGTGCCCGAG (SEQ ID NO: 214) rbLC.up
AAGCTTGCCACCATGGACAYGAGGGCCCCCACTC (SEQ ID NO: 215) rbLC.do
CAGAGTRCTGCTGAGGTTGTAGGTAC (SEQ ID NO: 216) BcPCR_FHLC_
ATGGACATGAGGGTCCCCGC leader.fw (SEQ ID NO: 217) BcPCR_
GATTTCAACTGCTCATCAGATGGC huCkappa.rev (SEQ ID NO: 218)
[0665] 8 .mu.L of 50 .mu.L PCR solution were loaded on a 48 E-Gel
2% (Invitrogen G8008-02). Positive PCR reactions were cleaned using
the NucleoSpin Extract II kit (Macherey & Nagel; 740609250)
according to manufacturer's protocol and eluted in 50 .mu.L elution
buffer. All cleaning steps were performed on a Hamilton ML Starlet
System.
[0666] Recombinant Expression of Rabbit Monoclonal Bivalent
Antibodies
[0667] For recombinant expression of rabbit monoclonal bivalent
antibodies, PCR-products coding for VH or VL were cloned as cDNA
into expression vectors by the overhang cloning method (RS Haun et
al., BioTechniques (1992) 13, 515-518; M Z Li et al., Nature
Methods (2007) 4, 251-256). The expression vectors contained an
expression cassette consisting of a 5' CMV promoter including
intron A, and a 3' BGH poly adenylation sequence. In addition to
the expression cassette, the plasmids contained a pUC18-derived
origin of replication and a beta-lactamase gene conferring
ampicillin resistance for plasmid amplification in E. coli. Three
variants of the basic plasmid were used: one plasmid containing the
rabbit IgG constant region designed to accept the VH regions while
two additional plasmids containing rabbit or human kappa LC
constant region to accept the VL regions.
[0668] Linearized expression plasmids coding for the kappa or gamma
constant region and VL/VH inserts were amplified by PCR using
overlapping primers.
[0669] Purified PCR products were incubated with T4 DNA-polymerase
which generated single-strand overhangs. The reaction was stopped
by dCTP addition.
[0670] In the next step, plasmid and insert were combined and
incubated with recA which induced site specific recombination. The
recombined plasmids were transformed into E. coli. The next day the
grown colonies were picked and tested for correct recombined
plasmid by plasmid preparation, restriction analysis and
DNA-sequencing.
[0671] For antibody expression, the isolated HC and LC plasmids
were transiently co-transfected into HEK293 cells and the
supernatants were harvested after 1 week.
[0672] Generation of vectors for the expression of rabbit
monoclonal monovalent antibodies
[0673] For recombinant expression of selected candidates as
monoclonal monovalent antibodies rabbit constant regions of all VH
chains were converted into human constant regions enclosing the
knob-mutation in the CH3 segment. For VL chains derived from rabbit
wild-type B-cells, rabbit C kappa constant regions were converted
into human. 4 .mu.L of cDNA of the selected candidates were used to
amplify the immunoglobulin heavy and light chain variable regions
with the AccuPrime SuperMix (Invitrogen 12344-040) in a final
volume of 50 .mu.L with forward primers specific for the signal
peptide and reverse primers specific for the CDR3-J region with (at
the 3' end) overlap sequence (20 bp) homologous to the human
constant regions (respectively of VH and VL). The PCR conditions
for the VH and VL chain amplification were as follows: Hot start at
94.degree. C. for 5 min.; 35 cycles of 20 sec. at 94.degree. C., 20
sec. at 68.degree. C., 45 sec. at 68.degree. C., and a final
extension at 68.degree. C. for 7 min.
[0674] PCR-products coding for VH or VL were cloned as cDNA into
expression vectors by the overhang cloning method (RS Haun et al.,
BioTechniques (1992) 13, 515-518; M Z Li et al., Nature Methods
(2007) 4, 251-256). The expression vectors contained an expression
cassette consisting of a 5' CMV promoter including intron A, and a
3' BGH poly adenylation sequence. In addition to the expression
cassette, the plasmids contained a pUC18-derived origin of
replication and a beta-lactamase gene conferring ampicillin
resistance for plasmid amplification in E. coli. Two variants of
the basic plasmid were used: one plasmid containing the human IgG
constant region designed to accept the new amplified VH chain and a
second plasmid containing the human kappa LC constant region to
accept the VL chain.
[0675] Linearized expression plasmids coding for the kappa or gamma
constant region and VL/VH inserts were amplified by PCR using
overlapping primers.
[0676] Purified PCR products were incubated with T4 DNA-polymerase
which generated single-strand overhangs. The reaction was stopped
by dCTP addition.
[0677] In the next step, plasmid and insert were combined and
incubated with recA which induced site specific recombination. The
recombined plasmids were transformed into E. coli. The next day the
grown colonies were picked and tested for correct recombined
plasmid by plasmid preparation, restriction analysis and
DNA-sequencing.
[0678] Transient Expression of the Monovalent Anti-TfR
Antibodies
[0679] The antibodies were generated in vivo in transiently
transfected HEK293 cells (human embryonic kidney cell line
293-derived) cultivated in F17 Medium (Invitrogen Corp.). For
transfection "293-Free" Transfection Reagent (Novagen) was used.
Antibodies and antibody-based modified molecules as described above
were expressed from individual expression plasmids. Transfections
were performed as specified in the manufacturer's instructions.
Recombinant protein-containing cell culture supernatants were
harvested three to seven days after transfection. Supernatants were
stored at reduced temperature (e.g. -80.degree. C.) until
purification. General information regarding the recombinant
expression of human immunoglobulins in e.g. HEK293 cells is given
in: Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.
[0680] hCMEC/D3 Cell Culture for Transcytosis Assays
[0681] Medium and supplements for hCMEC/D3 (Weksler, B. B. et al.,
FASEB J. 19 (2005), 1872-1874) were obtained from Lonza. hCMEC/D3
cells (passages 26-29) were cultured to confluence on
collagen-coated coverslips (microscopy) or flasks in EBM2 medium
containing 2.5 FBS, a quarter of the supplied growth factors and
fully complemented with supplied hydrocortisone, gentamycin and
ascorbic acid.
[0682] For all transcytosis assays, high density pore (1.times.108
pores/cm2) PET membrane filter inserts (0.4 .mu.m, 12 mm diameter)
were used in 12-well cell culture plates. Optimum media volumes
were calculated to be 400 .mu.L and 1600 .mu.L for apical and
basolateral chambers, respectively. Apical chambers of filter
inserts were coated with rat tail collagen I (7.5 .mu.g/cm2)
followed by fibronectin (5 .mu.g/mL), each incubation lasting for 1
hour at RT. hCMEC/D3 cells were grown to confluent monolayers
(approx. 2.times.105 cells/cm2) for 10-12 days in EMB2 medium.
[0683] Transcytosis Assay of Monovalent Transferrin Receptor
Specific Antibodies
[0684] The entire assay was performed in serum-free EBM2 medium.
Filter inserts with cells were incubated apically with monovalent
antibodies (concentration: 2.67 .mu.g/mL) for 1 hour at 37.degree.
C. following which the entire apical and basolateral media were
collected. From these values, paracellular flux was calculated. The
monolayers were washed at RT in serum-free medium apically (400
.mu.L) and basolaterally (1600 .mu.L) 3.times.3-5 min. each. All
the washes were collected to monitor efficiency of removal of the
unbound antibody. Pre-warmed medium was added to the apical chamber
and the filters transferred to a fresh 12 well plate (blocked
overnight with PBS containing 1% BSA) containing 1600 .mu.L
pre-warmed medium. At this point, cells on filters were lysed in
500 .mu.L RIPA buffer in order to determine specific antibody
uptake. The remaining filters were incubated at 37.degree. C. and
samples collected at various time points to determine apical and/or
basolateral release of antibody. The content of antibody in the
samples was quantified using a highly sensitive IgG ELISA. For each
time point, data were generated from three filter cell
cultures.
[0685] Sensitive IgG ELISA after Transcytosis Assay
[0686] The entire procedure was performed at RT using an automated
washer for the wash steps. A 384-well plate was coated with 30
.mu.L/well of 1 .mu.g/mL anti-human/mouse-IgG, Fc -specific in PBS
for 2 hours followed by 1 hour incubation in blocking buffer PBS
containing 1% BSA or 1% CroteinC for human and mouse IgG assays,
respectively). Serially diluted samples from the transcytosis assay
and standard concentrations of the antibody used in the
transcytosis assay were added to the plate and incubated for 2
hours. After four washes, 30 .mu.L/well of 50 ng/mL
anti-human/mouse-F(ab)2-Biotin in blocking buffer was added and
incubated for a further 2 hours. Following 6 washes, 30 .mu.L/well
of 50 ng/mL (huIgG assay) or 100 ng/mL (mIgG assay)
Poly-HRP40-Streptavidin (Fitzgerald; in PBS containing 1% BSA and
0.05% Tween-20) was added and incubated for 30 min. After 4 washes,
immune complexes were detected by addition of 30 .mu.L/well of BM
Chemiluminescence Substrate (Roche). The luminescence signal was
measured using a luminescence plate reader and concentration
calculated using the fitted standard curve. The sensitivity of the
assay ranged from 10 pg/mL to 10 ng/mL.
[0687] Rabbit anti-transferrin antibody clone 2/99 showed
properties comparable to that of the anti-transferrin receptor
antibody 128.1. This can be seen from the following Table.
TABLE-US-00046 TABLE 40 Characterization of antibody 2/99 (SEQ ID
NO. 178/179) total total sum transcytosis total total sum loading
basolateral apical transported loading transcytosis basolateral
apical transported % of mAb % of mAb % of mAb % of mAb origin [pg]
% basolateral [pg] [pg] [pg] 128.1 128.1 128.1 128.1 mAb 2226 34
757 1229 1986 100 100 100 100 128.1 clone- 2773 36 998 1346 2344
125 132 110 118 2/99 max. EC50 max. geo. BIAcore [ng/mL] geo. EC50
mean ratio ratio BIAcore BIAcore BIAcore t1/2 FACS mean [ng/mL]
Cyno EC50 max off-rate t1/2 off-rate Cyno ratio hTfR- hTfR- FACS
TfR- Cyno/ Cyno/ huTfR huTfR cyTfR TfR t1/2 origin CHO CHO cyTfR
CHO human human [1/s] [min] [1/s] [min] human/Cyno mAb 96 78200 314
52100 3.3 0.6 6.06E-04 19 5.47E-02 0 90.2 128.1 clone- 275 55600
241 52000 0.9 1.0 6.16E-04 19 2.77E-04 42 0.4 2/99
[0688] The mouse anti-human transferrin-receptor antibody 128.1 (WO
93/10819) was taken as reference. The transferrin receptor binder
2/99 shows a transcytosis loading of 705 pg, whereof after 4 hours
170 pg (=24% of loading) can be found in the basolateral
compartment and 294 pg (=42% of loading) can be found in the apical
compartment.
[0689] The transferrin receptor binder 4/94 shows a transcytosis
loading of 3510 pg, whereof after 4 hours 748 pg (=21% of loading)
can be found in the basolateral compartment and 1503 pg (=43% of
loading) can be found in the apical compartment.
Example 26: Epitope Mapping by Cell ELISA of CHO Cells Transfected
with hTfR Mutants
[0690] In order to be able determine the epitope regions on human
transferrin receptor (hTfR), mutations were introduced into the
hTfR sequence at positions, where a cluster of surface-exposed
amino acids had different amino acids in the aligned mouse TfR
sequence (see Table below), following the rationale that in spite
of the significant homology between human and mouse TfR (77%
identity), no antibodies directed to the extracellular part are
known which show good cross-reactivity between both orthologous.
Cloning of plasmids with the corresponding mutations is described
above. To map binding of human TfR binders to those epitopes,
CHO-K1 cells were transiently transfected with the described
plasmids and antibody binding measured in a cell ELISA. Briefly,
104 cells were plated per well of a 96-well plate the day before
experiment in normal growth medium (RPMI/10% FCS). The other day,
medium was changed to OPTI-MEM Serum-Reduced Medium (Gibco), and 10
.mu.L of a mixture of 1200 .mu.L OPTI-MEM, 12 .mu.g plasmid DNA and
12 .mu.L XtremeGENE transfection reagent (Roche) were added to the
wells after 30 minutes of pre-incubation. Cells were incubated for
2 days at 37.degree. C./7.5% CO2, then medium was removed and TfR
antibodies added at concentrations between 1 nM and 100 nM in
growth medium, followed by 2 h incubation at 4.degree. C.
Afterwards, antibody solutions were replaced by 0.05%
glutaraldehyde in PBS and cells fixed for 15 min. at RT, then
washed twice with PBS and incubated with HRP-conjugated
anti-human-Fc secondary antibody (BioRad; 1:2000 in ELISA Blocking
Reagent (Roche)) for 1.5 hours at RT. Signal was generated after 3
washes with PBS using 50 .mu.L of TMB per well and absorbance
measured at 450 nm.
TABLE-US-00047 TABLE 41 Plasmid # mutations in hTfR 10188 -- 18909
Thr518Asp/Gln520Lys/Phe521 Ser/Gln524Arg 18910 Arg325Gln 18911
Ser355Ala/Asp356Arg/Lys358Asn/Thr359Ile 18912
Asp204Gln/Lys205Ser/Arg208Asn 18913
Lys574Gly/Glu575Ala/Ile577Thr/Glu578Gln 18914
Ala196I1e/Gln197Gly/Asn198Gln/Ser199Asn/
Val200Met/Ile201Va1/I1e202Thr/Val203Ile/
Asp204Val/Lys205Gln/Asn206Ser/Gly207Asn/
Arg208Gly/Leu209Asn/Val210Leu/Tyr211Asp/ Leu212Pro 18974
Asp245G1u/Tyr247Ser/Thr248Tyr/Pro249Ser
Example 27: Surface Plasmon Resonance-Based Binding Assay for Human
TfR-Antibody Interaction
[0691] The binding experiment were carried out on a BIAcore B 4000
(GE Healthcare) equipped with C1 sensorchip (GE Healthcare, cat.no.
BR1005-35) pre-treated with anti-human Fab antibody (GE Healthcare,
cat.no 28-9583-25) using a standard amine coupling chemistry
procedure accordingly to the vendor's manual.
[0692] For kinetic measurements the sample antibody was immobilized
applying a contact time of 60 seconds and a flow rate of 10
.mu.L/min in phosphate buffer saline pH 7.4, 0.05% Tween 20 at
25.degree. C. Recombinant His6-tagged human transferrin receptor
(R&D systems, cat.no 2474-TR-050) was applied in increasing
concentrations and the signal monitored over the time. An average
time span of 150 seconds of association time and 600 seconds of
dissociation time at 30 .mu.L/min flow rate was recorded. Data were
fit using a 1:1 binding model (Langmuir isotherm).
TABLE-US-00048 TABLE 42 Sequences of the epitopes of human
transferrin receptor (hTfR) SEQ ID NO Sequence 202 IGQNMVTIVQSNGNL
20 203 NMVTIVQSNGNLDPV 204 QSNGNLDPVESPEGY
Example 28: Surface Plasmon Resonance-Based Binding Assay for Human
TfR-Antibody Interaction
[0693] The binding experiment were carried out on a BIAcore B 4000
(GE Healthcare) equipped with C1 sensor chip (GE Healthcare,
cat.no. BR1005-35) pre-treated with anti-human Fab antibody (GE
Healthcare, cat.no 28-9583-25) using a standard amine coupling
chemistry procedure accordingly to the vendor's manual.
[0694] For kinetic measurements the sample antibody was immobilized
applying a contact time of 60 seconds and a flow rate of 10
.mu.L/min in phosphate buffer saline pH 7.4, 0.05 Tween 20 at
25.degree. C. Recombinant His6-tagged human transferrin receptor
(R&D systems, cat.no 2474-TR-050) was applied in increasing
concentrations and the signal monitored over the time. An average
time span of 150 seconds of association time and 600 seconds of
dissociation time at 30 .mu.L/min flow rate was recorded. Data were
fit using a 1:1 binding model (Langmuir isotherm).
Example 29: Humanization of the VH and VL Domains of Murine and
Rabbit Anti-Transferrin Receptor Antibody
[0695] The non-human anti-transferrin receptor antibodies were
humanized as follows: Based on the characterization of encoding
sequences and amino acid sequences that comprise the VH and VL
domains of a the non-human anti-transferrin receptor antibodies of
the IgG1 class with kappa light chain, a corresponding humanized
anti-transferrin receptor antibody was generated by CDR grafting an
backward/forward mutations based on the human germline framework
VH4_3 and VK1_10 combination for clone 299.
[0696] The humanized antibodies of clone 2/99 as reported herein
were not available by applying standard humanization techniques. It
was required to introduce non-standard mutations in the amino acid
sequence in order to obtain a humanized antibody with transferrin
receptor binding off-rates within the intended range. This is
especially important as the antibodies as reported herein are being
developed for crossing the human blood-brain-barrier to shuttle the
HER bispecific antibody as the therapeutic payload into the
brain.
[0697] It has been found that in order to obtain a suitable and
developable humanized antibody two cysteine amino acid residues in
the light chain of the parental rabbit antibody had to be replaced
by a proline and an asparagine amino acid residue, respectively. In
addition to be within the given off-rate range a serine residue
present in the middle of the rabbit CDRL3 had to be replaced by an
alanine residue.
[0698] Is has further been found that it is advantageous to change
three amino acid residues in the heavy chain at positions 65, 100 g
and 105 (numbering according to Kabat).
[0699] All numbering as used herein is based on the Kabat variable
domain numbering scheme.
[0700] Anti-transferrin receptor antibodies that specifically bind
to human transferrin receptor (huTfR) and cynomolgus transferrin
receptor (cyTfR) were obtained.
Example 30: Method to Determine Human/Cynomolgus TfR Receptor
Affinity
[0701] In this example the method for the determination of the
human transferrin receptor affinities for the comparison of the
dissociation behavior is outlined.
[0702] For all analysis the Biotin CAPture Kit from GE Healthcare
(Instruction 28-9242-34 AB) was used. First the chip was rehydrated
by docking it in the BIAcore T200 instrument. After that, the chip
was left on standby with running buffer overnight. For surface
preparation the Biotin CAPture Reagent was diluted 1:100 in running
buffer (1.times.PBS, supplemented with 0.25 M NaCl). This solution
was injected on Flow Cells 1 to 4 for 360 sec with a flow rate of 2
.mu.L/min. Next the sensor surface was conditioned with three
one-minute injections of regeneration solution provided in the
Biotin CAPture Kit. This has to be done for the docking procedure
or for the first time or after storage. A 100 nM human or
cynomolgus, respectively, mono-biotinylated transferrin receptor
solution should be injected on Flow Cell 2 for 30 sec at a flow
rate of 10 .mu.L/min. For affinity determination injections with
six concentrations (500, 250, 125, 62.5, 31.25, 15.625 and 0 nM)
were applied. They were injected on the "hu-TfR-flow cell" (e.g.
Flow Cell 2 as prepared as outlined above) with an injection time
of 180 sec (association) and a flow rate of 10 .mu.L/min. After
dissociation phase of 600 sec the surface was regenerated according
to the manufacturer's instructions with the regeneration solution
provided in the Biotin CAPture Kit and the next cycle was carried
out.
[0703] The kinetic data was evaluated using the BIAcore T200
evaluation software. Especially the dissociation rate constants of
the different human transferrin receptor binders were taken into
account after the application of the 1:1 Langmuir binding
model.
Example 31: B4000 Relative Ranking of Human/Cynomolgus Transferrin
Receptor Dissociation
[0704] A CAP sensor chip (provided in the Biotin CAPture Kit,
series S #28-9202-34 GE) was mounted into a BIAcore B4000 system,
normalized and addressed in a hydrodynamic manner, according to the
manufacturer's instructions. In the first cycle the CAP reagent (as
provided in the Kit) was addressed to spot 1, 2, 4 and 5 with a
flow rate of 10 .mu.L/min for 300 sec. The human transferrin
receptor capture took place in spot 1 (human transferrin
receptor-biotinylated) and spot 5 (cynomolgus transferrin
receptor-biotinylated) with a flow rate of 10 .mu.L/min and a
contact time of 30 sec. The receptors were diluted to a
concentration of 50 nM with running buffer (1.times.PBS #28995084,
GE Healthcare, supplemented with 0.25 M NaCl). The antibodies were
injected into all flow cells in a concentration series of 100 nM,
50 nM, 25 nM and 0 nM for 180 sec at a flow speed of 30 .mu.L/min.
Dissociation time was set to 300 sec. Regeneration of the whole
complex from the CAP chip has been performed utilizing the
regeneration solution provided in the Biotin CAPture Kit (120 sec
with a flow rate of 10 .mu.L/min). To control the active protein
concentration a second cycle was performed in spot 5 utilizing
biotinylated protein A (#P2165-2MG, Sigma) with a flow rate of 10
.mu.L/min and a contact time of 30 sec. Spot 1 was kept empty in
this control cycle. The antibodies and the regeneration were
handled like in cycle 1. Relevant kinetic data was calculated using
the BIAcore B4000 evaluation software. The dissociation from the
human transferrin receptor has been determined applying the 1:1
dissociation fit.
[0705] Results
[0706] The mouse anti-human transferrin-receptor antibody 128.1 (WO
93/10819) was taken as reference. The new humanized
anti-transferrin receptor antibodies that specifically bind to
human transferrin receptor (huTfR) and cynomolgus transferrin
receptor (cyTfR) had an off-rate for the human transferrin receptor
that was equal to or less than (i.e. at most) that of the
anti-transferrin receptor antibody 128.1 for the cynomolgus
transferrin receptor, whereby the off-rates are determined by
surface plasmon resonance and bound with an off-rate for the human
transferrin receptor that was between and including 0.1 l/s and
0.005 l/s.
[0707] In the following Table the off-rates of humanization
variants of the rabbit light chain variable domain of clone 2/99 in
combination with humanization variants of the rabbit heavy chain
variable domain of clone 2/99 are shown. Binding partner was human
transferrin receptor (determined at 25.degree. C.).
TABLE-US-00049 TABLE 43 VH VL 0 (rb) 1 2 5 6 7 8 9 11 12 .sup. 0
(rb) 4.34E-04 9.08E-04 8.06E-04 7.72E-04 6.63E-04 5.15E-04 4.06E-04
9.01E-04 9.05E-04 9.21E-04 1 5.69E-03 1.00E-03 1.00E-03 1.00E-03
1.00E-03 7.52E-03 3.19E-03 6.94E-03 1.00E-03 1.00E-03 2 1.25E-03
2.86E-03 2.75E-03 2.41E-03 1.87E-03 1.31E-03 1.01E-03 3.99E-03
3.85E-03 6.35E-03 3 1.32E-03 4.31E-03 3.84E-03 3.16E-03 2.82E-03
1.45E-03 1.00E-03 4.17E-03 5.65E-03 5.86E-03 4 1.36E-03 2.56E-03
2.63E-03 2.38E-03 1.87E-03 1.25E-03 7.88E-04 2.70E-03 3.88E-03
3.11E-03 5 1.94E-03 2.71E-03 2.62E-03 2.53E-03 1.66E-03 1.35E-03
1.07E-03 3.50E-03 4.56E-03 5.82E-03 6 1.90E-03 5.38E-03 5.55E-03
4.64E-03 3.06E-03 1.97E-03 1.40E-03 6.83E-03 6.71E-03 7.05E-03 7
4.63E-03 7.33E-03 7.50E-03 6.97E-03 5.63E-03 3.66E-03 2.31E-03
7.61E-03 7.81E-03 7.71E-03 8 1.39E-03 4.85E-03 3.94E-03 3.78E-03
3.01E-03 1.72E-03 1.16E-03 5.23E-03 5.52E-03 5.31E-03 9- 1.41E-03
2.46E-03 2.21E-03 2.03E-03 1.41E-03 1.21E-03 1.01E-03 2.52E-03
2.42E-03 2.19E-03 NYA (SEQ ID NO 193) 10 1.88E-03 6.77E-03 6.49E-03
6.53E-03 4.55E-03 2.64E-03 1.73E-03 7.19E-03 7.16E-03 7.79E-03 12
5.41E-03 7.05E-03 8.14E-03 1.00E-03 7.78E-03 7.75E-03 6.72E-03
1.00E-03 7.87E-03 1.00E-03 14 1.78E-03 2.99E-03 2.44E-03 2.33E-03
2.20E-03 1.53E-03 1.04E-03 3.32E-03 3.51E-03 5.46E-03 15 6.63E-03
6.69E-03 6.38E-03 6.37E-03 4.21E-03 2.73E-03 1.81E-03 7.39E-03
7.09E-03 7.76E-03 17 1.49E-03 7.56E-03 7.12E-03 7.45E-03 7.17E-03
1.87E-03 1.12E-03 4.25E-03 7.55E-03 7.27E-03 VH 23- VL 13 15 16 17
18 19 20 21 22 DANG .sup. 0 (rb) 4.82E-04 7.63E-04 6.53E-04
4.13E-04 1.09E-03 1.00E-03 1.11E-03 5.65E-04 5.06E-04 3.38E-04 1
1.00E-03 7.72E-03 7.71E-03 4.33E-03 1.00E-03 1.00E-03 1.00E-03
7.71E-03 5.78E-03 2.80E-03 2 2.46E-03 2.16E-03 1.97E-03 1.05E-03
4.89E-03 7.69E-03 5.25E-03 1.38E-03 1.26E-03 7.15E-04 3 2.77E-03
2.07E-03 1.73E-03 8.43E-04 6.65E-03 1.00E-03 7.15E-03 1.94E-03
1.43E-03 7.83E-04 4 1.30E-03 1.34E-03 1.27E-03 7.23E-04 3.35E-03
1.00E-03 4.36E-03 1.46E-03 1.18E-03 7.61E-04 5 2.18E-03 2.14E-03
2.23E-03 1.23E-03 3.49E-03 1.00E-03 3.52E-03 1.37E-03 1.41E-03
8.80E-04 6 3.65E-03 3.50E-03 3.39E-03 1.71E-03 6.74E-03 1.00E-03
6.06E-03 2.07E-03 2.14E-03 1.16E-03 7 6.68E-03 5.43E-03 5.25E-03
2.33E-03 7.66E-03 1.00E-03 7.14E-03 2.38E-03 3.37E-03 1.55E-03 8
2.47E-03 2.09E-03 1.97E-03 1.11E-03 6.77E-03 6.71E-03 6.58E-03
1.74E-03 1.65E-03 9.41E-04 9- 1.39E-03 1.42E-03 1.36E-03 9.34E-04
2.21E-03 6.17E-03 1.89E-03 1.13E-03 1.26E-03 7.69E-04 NYA (SEQ ID
NO 193) 10 5.89E-03 3.99E-03 4.24E-03 1.88E-03 7.46E-03 1.00E-03
7.05E-03 2.11E-03 2.09E-03 1.20E-03 12 7.85E-03 7.64E-03 7.54E-03
2.84E-03 1.00E-03 1.00E-03 1.00E-03 7.54E-03 6.44E-03 1.87E-03 14
2.22E-03 1.94E-03 1.75E-03 1.05E-03 3.00E-03 7.96E- 03 2.39E-03
1.03E-03 1.12E-03 7.33E-04 15 7.11E-03 6.03E-03 4.77E-03 1.56E-03
7.58E-03 1.00E-03 7.85E-03 1.92E-03 1.79E-03 9.86E-04 17 3.39E-03
1.69E-03 1.66E-03 9.88E-04 5.30E-03 1.00E-03 4.57E-03 9.97E-04
9.38E-04 6.94E-04
[0708] The combination of VH23-DANG with VL09-NYA (SEQ ID NO 193)
was chosen as starting point for further engineering to develop a
binding site that is reflecting the properties of the antibody
128.1 with respect to the binding to human transferrin receptor
more closely.
[0709] In the following Table the off-rates of different exemplary
variants of VH23-DANG and VL9-NYA (SEQ ID NO 193) as well as
different other variable domain humanization variants for the human
transferrin receptor are shown in comparison (determined according
to Example 31 at 25.degree. C.).
TABLE-US-00050 TABLE 44 VK9- NYA (SEQ VH567- ID NO VK9- VK9- VK9- P
. . . NY VK12- VK17- VK15- VK2- VK6- 193) SYA GYS CYS A HYS TYS AYS
SYS SYS VH23- 8.83E- 3.96E- 4.62E-03 1.53E- 2.29E- 6.97E- 5.22E-
1.32E- DANG 03 03 02 03 03 03 02 VH23- 3.95E- 4.84E- 3.73E- 2.33E-
DASG 02 04 03 03 (SEQ ID NO 192) VH23- 1.49E- 7.55E- 3.75E- 1.51E-
DAQG 02 04 03 03 (SEQ ID NO 205) VH9- 1.82E- 6.52E- 4.02E- DANG 03
03 02 VH9- 1.25E- DAQG 03 VH7- 3.08E- DANG 02 reference: 128.1 =
7.78E-02 (determined for the cynomolgus transferrin receptor).
[0710] In the following Table the kinetic data of different
exemplary variants of VH23 and VL9 are shown in comparison
(determined according to Example 30).
TABLE-US-00051 TABLE 45 BIAcore Assay @ 25.degree. C. TfR kd
[s.sup.-1] ka [s.sup.-1M.sup.-1] kD [M] mAb 128.1 cynomolgus
7.33E-02 5.41E+05 1.36E-07 VH23-DASG/VL09-NYA human 3.95E-02
8.83E+04 4.47E-07 (SEQ ID NO 192/193) VH23-DAQG/VL09-NYA human
1.37E-02 1.21E+05 1.13E-07 (SEQ ID NO 205/193) VH23-DANG/VL09-NYA
human 8.83E-03 1.55E+05 5.72E-08
[0711] In the following Table the off-rates of humanization
variants of the murine light chain variable domain of clone 4/94 in
combination with humanization variants of the murine heavy chain
variable domain of clone 4/94 are shown. Binding partner was human
transferrin receptor.
TABLE-US-00052 TABLE 46 VH 1 2 3 4 (SEQ ID (SEQ ID (SEQ ID (SEQ ID
VL 0 (mu) NO 201) NO 207) NO 208) NO 209) 0 (mu) 1.36E-04 1.37E-04
1.56E-04 1.48E-04 1.77E-04 1 3.70E-04 4.10E-04 4.54E-04 4.76E-04
4.48E-04 (SEQ ID NO 194) 2 3.64E-04 3.99E-04 4.26E-04 4.15E-04
4.38E-04 (SEQ ID NO 195) 3 3.39E-04 3.86E-04 4.30E-04 4.34E-04
4.52E-04 (SEQ ID NO 196) 4 5.42E-04 6.57E-04 7.03E-04 6.83E-04
7.05E-04 (SEQ ID NO 197) 5 5.44E-04 6.84E-04 7.17E-04 7.17E-04
7.28E-04 (SEQ ID NO 198) 6 3.81E-04 4.86E-04 5.27E-04 5.50E-04
5.52E-04 (SEQ ID NO 199) 7 2.32E-04 2.74E-04 2.99E-04 3.06E-04
3.26E-04 (SEQ ID NO 200)
Example 32: In Vivo Characterization of Herceptarg-scFab(8D3)
[0712] A Her2-expressing intracranial tumor xenograft model was
used to compare the brain tumor penetration of Herceptarg-brain
shuttle to Herceptarg alone by 3D fluorescence ultramicroscopy.
[0713] Cell Lines
[0714] The human breast cancer cell line BT474 M1 was maintained in
RPMI 1640 supplemented with 10% fetal calf serum and 2 mL
L-glutamine. Propagation of cell lines followed standard cell
culture protocols.
[0715] Intracranial Implantation of Tumor Cells in Mice
[0716] Female severe combined immunodeficient hairless outbred
(SHO) mice (weight 20 to 27 g) were obtained from Charles River and
were 7 to 9 weeks of age at initiation of experiments. Mice were
anesthetized with i.p. injection of 100 mg/kg ketamine and 10 mg/kg
xylazine. Scalp was disinfected and removed over the whole right
hemisphere of the brain to expose the cranium. The periosteum was
removed by a bone scraper. Next, a 0.7-mm burr hole in diameter was
drilled 2 mm right of midline and 2 mm anterior to bregma with a
dental drill. Mice were then placed in a small animal stereotactic
frame and BT-474 M1 cell suspension (5.times.10.sup.5 cells in 3
.mu.l) was injected using a Hamilton syringe with a 26s-gauge
needle to a depth of 3 mm into the brain tissue. After injection,
needle was slowly withdrawn and the hole was sealed with Cyano
veneer tissue glue. All animal studies were approved by the local
government (file number 55.2-1-54-2532.0-76-14, Government of Upper
Bavaria, Germany).
[0717] Application of Substances
[0718] 30 days after tumor cell implantation mice received an i.v.
injection of Herceptarg(LALA)-scFab(8D3)-Alexa Fluor 750 (5 mg/kg,
n=7) or Herceptarg(LALA)-Alexa Fluor 750 (3.75 mg/kg; n=5) 6 h
before dissection. Five minutes before necropsy, mice were injected
i.v. with 100 .mu.g lectin-Alexa Fluor 750 solution to allow
visualization of blood vessels ex vivo. Substances were
systemically given via the tail vein in a volume of 150 .mu.l.
[0719] Necropsy, Fixation and Optical Clearance of Brains
[0720] Mice were sacrificed by cervical dislocation. Brains were
explanted and transferred to 10% buffered formalin for about 12
hours at room temperature in the dark and thereafter were
dehydrated in a graded tetrahydrofuran series (50%, 70%, 80%, 100%
for 90 minutes each, 100% over night and 100% for 90 minutes).
Afterwards brains were cleared in dibenzyl ether for 2 days until
the specimen became optically transparent.
[0721] 3D Imaging of Brains by Ultramicroscopy
[0722] The optical transparent samples were placed in a commercial
ultramicroscope, equipped with an Imager 3QE camera (both LaVision
Biotec), a 2 My PLAPO 2VC objective lens (Olympus) and a
supercontinuum white light laser (SuperK EXTREME 80 mHz VIS; NKT
Photonics). The samples were scanned with a standard magnification
of 0.63 in 5.1 .mu.m thick virtual sections with filter settings
appropriate for Alexa Fluor 647 (excitation range 655/15 nm,
emission range 680/30 nm) and Alexa Fluor 750 (excitation range
747/33 nm, emission range 786/22 nm) signal detection. Tiff raw
data were converted to dicom files and visualized using the OsiriX
software (Pixmeo). A 3D brain tumor region of interest (ROI) was
defined according to the dimensions of chaotic vessel structures
seen by lectin-Alexa Fluor 750 staining. The determined 3D ROI was
imported to 3D-reconstructed mab-Alexa Fluor 750 data files to read
out mean antibody fluorescent signals within the brain tumors.
[0723] The distinct fluorescent signal intensities of mab-Alexa
Fluor 750 at different distances from nearest lectin-Alexa Fluor
647-stained blood vessels were quantified. This was done with a set
of custom developed image analysis algorithms implemented as a
"Definiens Cognition Network Technology" rule set (Definiens
AG).
[0724] Statistical analysis was performed by GraphPad Prism
software (GraphPad Software, Inc.) using 2-way ANOVA plus Sidak's
multiple comparison test (FIG. 36). The significance levels are
indicated by asterisks (**p<0.01 and ****P<0.0001). All data
are presented as means.+-.standard error of the mean.
Sequence CWU 1
1
21811995DNAArtificial sequenceHer2 ECD DNA 1acccaagtgt gcaccggcac
agacatgaag ctgcggctcc ctgccagtcc cgagacccac 60ctggacatgc tccgccacct
ctaccagggc tgccaggtgg tgcagggaaa cctggaactc 120acctacctgc
ccaccaatgc cagcctgtcc ttcctgcagg atatccagga ggtgcagggc
180tacgtgctca tcgctcacaa ccaagtgagg caggtcccac tgcagaggct
gcggattgtg 240cgaggcaccc agctctttga ggacaactat gccctggccg
tgctagacaa tggagacccg 300ctgaacaata ccacccctgt cacaggggcc
tccccaggag gcctgcggga gctgcagctt 360cgaagcctca cagagatctt
gaaaggaggg gtcttgatcc agcggaaccc ccagctctgc 420taccaggaca
cgattttgtg gaaggacatc ttccacaaga acaaccagct ggctctcaca
480ctgatagaca ccaaccgctc tcgggcctgc cacccctgtt ctccgatgtg
taagggctcc 540cgctgctggg gagagagttc tgaggattgt cagagcctga
cgcgcactgt ctgtgccggt 600ggctgtgccc gctgcaaggg gccactgccc
actgactgct gccatgagca gtgtgctgcc 660ggctgcacgg gccccaagca
ctctgactgc ctggcctgcc tccacttcaa ccacagtggc 720atctgtgagc
tgcactgccc agccctggtc acctacaaca cagacacgtt tgagtccatg
780cccaatcccg agggccggta tacattcggc gccagctgtg tgactgcctg
tccctacaac 840tacctttcta cggacgtggg atcctgcacc ctcgtctgcc
ccctgcacaa ccaagaggtg 900acagcagagg atggaacaca gcggtgtgag
aagtgcagca agccctgtgc ccgagtgtgc 960tatggtctgg gcatggagca
cttgcgagag gtgagggcag ttaccagtgc caatatccag 1020gagtttgctg
gctgcaagaa gatctttggg agcctggcat ttctgccgga gagctttgat
1080ggggacccag cctccaacac tgccccgctc cagccagagc agctccaagt
gtttgagact 1140ctggaagaga tcacaggtta cctatacatc tcagcatggc
cggacagcct gcctgacctc 1200agcgtcttcc agaacctgca agtaatccgg
ggacgaattc tgcacaatgg cgcctactcg 1260ctgaccctgc aagggctggg
catcagctgg ctggggctgc gctcactgag ggaactgggc 1320agtggactgg
ccctcatcca ccataacacc cacctctgct tcgtgcacac ggtgccctgg
1380gaccagctct ttcggaaccc gcaccaagct ctgctccaca ctgccaaccg
gccagaggac 1440gagtgtgtgg gcgagggcct ggcctgccac cagctgtgcg
cccgagggca ctgctggggt 1500ccagggccca cccagtgtgt caactgcagc
cagttccttc ggggccagga gtgcgtggag 1560gaatgccgag tactgcaggg
gctccccagg gagtatgtga atgccaggca ctgtttgccg 1620tgccaccctg
agtgtcagcc ccagaatggc tcagtgacct gttttggact ggaggctgac
1680cagtgtgtgg cctgtgccca ctataaggac cctcccttct gcgtggcccg
ctgccccagc 1740ggtgtgaaac ctgacctctc ctacatgccc atctggaagt
ttccagatga ggagggcgca 1800tgccagcctt gccccatcaa ctgcacccac
tcctgtgtgg acctggatga caagggctgc 1860cccgccgagc agagagccag
ccctctgacg gtcgacgaac agttatattt tcagggcggc 1920tcaggcctga
acgacatctt cgaggcccag aagatcgagt ggcacgaggc tcgagctcac
1980caccatcacc atcac 19952684PRTArtificial sequenceHer2 ECD 2Met
Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10
15 Val His Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu
20 25 30 Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His Leu
Tyr Gln 35 40 45 Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr
Tyr Leu Pro Thr 50 55 60 Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile
Gln Glu Val Gln Gly Tyr 65 70 75 80 Val Leu Ile Ala His Asn Gln Val
Arg Gln Val Pro Leu Gln Arg Leu 85 90 95 Arg Ile Val Arg Gly Thr
Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala 100 105 110 Val Leu Asp Asn
Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr Gly 115 120 125 Ala Ser
Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu 130 135 140
Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr 145
150 155 160 Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn
Gln Leu 165 170 175 Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala
Cys His Pro Cys 180 185 190 Ser Pro Met Cys Lys Gly Ser Arg Cys Trp
Gly Glu Ser Ser Glu Asp 195 200 205 Cys Gln Ser Leu Thr Arg Thr Val
Cys Ala Gly Gly Cys Ala Arg Cys 210 215 220 Lys Gly Pro Leu Pro Thr
Asp Cys Cys His Glu Gln Cys Ala Ala Gly 225 230 235 240 Cys Thr Gly
Pro Lys His Ser Asp Cys Leu Ala Cys Leu His Phe Asn 245 250 255 His
Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr Asn 260 265
270 Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe
275 280 285 Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser
Thr Asp 290 295 300 Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn
Gln Glu Val Thr 305 310 315 320 Ala Glu Asp Gly Thr Gln Arg Cys Glu
Lys Cys Ser Lys Pro Cys Ala 325 330 335 Arg Val Cys Tyr Gly Leu Gly
Met Glu His Leu Arg Glu Val Arg Ala 340 345 350 Val Thr Ser Ala Asn
Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe 355 360 365 Gly Ser Leu
Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser 370 375 380 Asn
Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu 385 390
395 400 Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser
Leu 405 410 415 Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg
Gly Arg Ile 420 425 430 Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln
Gly Leu Gly Ile Ser 435 440 445 Trp Leu Gly Leu Arg Ser Leu Arg Glu
Leu Gly Ser Gly Leu Ala Leu 450 455 460 Ile His His Asn Thr His Leu
Cys Phe Val His Thr Val Pro Trp Asp 465 470 475 480 Gln Leu Phe Arg
Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg 485 490 495 Pro Glu
Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His Gln Leu Cys 500 505 510
Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys Val Asn Cys 515
520 525 Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys Arg Val
Leu 530 535 540 Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys
Leu Pro Cys 545 550 555 560 His Pro Glu Cys Gln Pro Gln Asn Gly Ser
Val Thr Cys Phe Gly Leu 565 570 575 Glu Ala Asp Gln Cys Val Ala Cys
Ala His Tyr Lys Asp Pro Pro Phe 580 585 590 Cys Val Ala Arg Cys Pro
Ser Gly Val Lys Pro Asp Leu Ser Tyr Met 595 600 605 Pro Ile Trp Lys
Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys Pro 610 615 620 Ile Asn
Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys Gly Cys Pro 625 630 635
640 Ala Glu Gln Arg Ala Ser Pro Leu Thr Val Asp Glu Gln Leu Tyr Phe
645 650 655 Gln Gly Gly Ser Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys
Ile Glu 660 665 670 Trp His Glu Ala Arg Ala His His His His His His
675 680 3681DNAArtificial sequenceFc (hole) DNA 3gacaaaactc
acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60ttcctcttcc
ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca
120tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg
gtacgtggac 180ggcgtggagg tgcataatgc caagacaaag ccgcgggagg
agcagtacaa cagcacgtac 240cgtgtggtca gcgtcctcac cgtcctgcac
caggactggc tgaatggcaa ggagtacaag 300tgcaaggtct ccaacaaagc
cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360gggcagcccc
gagaaccaca ggtgtgcacc ctgcccccat cccgggatga gctgaccaag
420aaccaggtca gcctctcgtg cgcagtcaaa ggcttctatc ccagcgacat
cgccgtggag 480tgggagagca atgggcagcc ggagaacaac tacaagacca
cgcctcccgt gctggactcc 540gacggctcct tcttcctcgt gagcaagctc
accgtggaca agagcaggtg gcagcagggg 600aacgtcttct catgctccgt
gatgcatgag gctctgcaca accgcttcac gcagaagagc 660ctctccctgt
ctccgggtaa a 6814227PRTArtificial sequenceFc (hole) 4Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25
30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser
Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155
160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu
Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn Arg Phe Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225
52670DNAArtificial sequenceHer2 ECD-Fc(knob) DNA 5acccaagtgt
gcaccggcac agacatgaag ctgcggctcc ctgccagtcc cgagacccac 60ctggacatgc
tccgccacct ctaccagggc tgccaggtgg tgcagggaaa cctggaactc
120acctacctgc ccaccaatgc cagcctgtcc ttcctgcagg atatccagga
ggtgcagggc 180tacgtgctca tcgctcacaa ccaagtgagg caggtcccac
tgcagaggct gcggattgtg 240cgaggcaccc agctctttga ggacaactat
gccctggccg tgctagacaa tggagacccg 300ctgaacaata ccacccctgt
cacaggggcc tccccaggag gcctgcggga gctgcagctt 360cgaagcctca
cagagatctt gaaaggaggg gtcttgatcc agcggaaccc ccagctctgc
420taccaggaca cgattttgtg gaaggacatc ttccacaaga acaaccagct
ggctctcaca 480ctgatagaca ccaaccgctc tcgggcctgc cacccctgtt
ctccgatgtg taagggctcc 540cgctgctggg gagagagttc tgaggattgt
cagagcctga cgcgcactgt ctgtgccggt 600ggctgtgccc gctgcaaggg
gccactgccc actgactgct gccatgagca gtgtgctgcc 660ggctgcacgg
gccccaagca ctctgactgc ctggcctgcc tccacttcaa ccacagtggc
720atctgtgagc tgcactgccc agccctggtc acctacaaca cagacacgtt
tgagtccatg 780cccaatcccg agggccggta tacattcggc gccagctgtg
tgactgcctg tccctacaac 840tacctttcta cggacgtggg atcctgcacc
ctcgtctgcc ccctgcacaa ccaagaggtg 900acagcagagg atggaacaca
gcggtgtgag aagtgcagca agccctgtgc ccgagtgtgc 960tatggtctgg
gcatggagca cttgcgagag gtgagggcag ttaccagtgc caatatccag
1020gagtttgctg gctgcaagaa gatctttggg agcctggcat ttctgccgga
gagctttgat 1080ggggacccag cctccaacac tgccccgctc cagccagagc
agctccaagt gtttgagact 1140ctggaagaga tcacaggtta cctatacatc
tcagcatggc cggacagcct gcctgacctc 1200agcgtcttcc agaacctgca
agtaatccgg ggacgaattc tgcacaatgg cgcctactcg 1260ctgaccctgc
aagggctggg catcagctgg ctggggctgc gctcactgag ggaactgggc
1320agtggactgg ccctcatcca ccataacacc cacctctgct tcgtgcacac
ggtgccctgg 1380gaccagctct ttcggaaccc gcaccaagct ctgctccaca
ctgccaaccg gccagaggac 1440gagtgtgtgg gcgagggcct ggcctgccac
cagctgtgcg cccgagggca ctgctggggt 1500ccagggccca cccagtgtgt
caactgcagc cagttccttc ggggccagga gtgcgtggag 1560gaatgccgag
tactgcaggg gctccccagg gagtatgtga atgccaggca ctgtttgccg
1620tgccaccctg agtgtcagcc ccagaatggc tcagtgacct gttttggact
ggaggctgac 1680cagtgtgtgg cctgtgccca ctataaggac cctcccttct
gcgtggcccg ctgccccagc 1740ggtgtgaaac ctgacctctc ctacatgccc
atctggaagt ttccagatga ggagggcgca 1800tgccagcctt gccccatcaa
ctgcacccac tcctgtgtgg acctggatga caagggctgc 1860cccgccgagc
agagagccag ccctctgacg gtcgacggtg gtagtccgac acctccgaca
1920cccgggggtg gttctgcaga caaaactcac acatgcccac cgtgcccagc
acctgaactc 1980ctggggggac cgtcagtctt cctcttcccc ccaaaaccca
aggacaccct catgatctcc 2040cggacccctg aggtcacatg cgtggtggtg
gacgtgagcc acgaagaccc tgaggtcaag 2100ttcaactggt acgtggacgg
cgtggaggtg cataatgcca agacaaagcc gcgggaggag 2160cagtacaaca
gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg
2220aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc
catcgagaaa 2280accatctcca aagccaaagg gcagccccga gaaccacagg
tgtacaccct gcccccatgc 2340cgggatgagc tgaccaagaa ccaggtcagc
ctgtggtgcc tggtcaaagg cttctatccc 2400agcgacatcg ccgtggagtg
ggagagcaat gggcagccgg agaacaacta caagaccacg 2460cctcccgtgc
tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag
2520agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc
tctgcacaac 2580cactacacgc agaagagcct ctccctgtct ccgggtaaat
ccggaggcct gaacgacatc 2640ttcgaggccc agaagattga atggcacgag
26706890PRTArtificial sequenceHer2 ECD-Fc(knob) 6Thr Gln Val Cys
Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser 1 5 10 15 Pro Glu
Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln 20 25 30
Val Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser 35
40 45 Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu
Ile 50 55 60 Ala His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu
Arg Ile Val 65 70 75 80 Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala
Leu Ala Val Leu Asp 85 90 95 Asn Gly Asp Pro Leu Asn Asn Thr Thr
Pro Val Thr Gly Ala Ser Pro 100 105 110 Gly Gly Leu Arg Glu Leu Gln
Leu Arg Ser Leu Thr Glu Ile Leu Lys 115 120 125 Gly Gly Val Leu Ile
Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr 130 135 140 Ile Leu Trp
Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu Thr 145 150 155 160
Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro Met 165
170 175 Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln
Ser 180 185 190 Leu Thr Arg Thr Val Cys Ala Gly Gly Cys Ala Arg Cys
Lys Gly Pro 195 200 205 Leu Pro Thr Asp Cys Cys His Glu Gln Cys Ala
Ala Gly Cys Thr Gly 210 215 220 Pro Lys His Ser Asp Cys Leu Ala Cys
Leu His Phe Asn His Ser Gly 225 230 235 240 Ile Cys Glu Leu His Cys
Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr 245 250 255 Phe Glu Ser Met
Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser 260 265 270 Cys Val
Thr Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser 275 280 285
Cys Thr Leu Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp 290
295 300 Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val
Cys 305 310 315 320 Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val Arg
Ala Val Thr Ser 325 330 335 Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys
Lys Ile Phe Gly Ser Leu 340 345 350 Ala Phe Leu Pro Glu Ser Phe Asp
Gly Asp Pro Ala Ser Asn Thr Ala 355 360 365 Pro Leu Gln Pro Glu Gln
Leu Gln Val Phe Glu Thr Leu Glu Glu Ile 370 375 380 Thr Gly Tyr Leu
Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp Leu 385 390 395 400 Ser
Val Phe Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His Asn 405 410
415 Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu Gly
420 425 430 Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile
His His 435 440 445 Asn Thr His Leu Cys Phe Val His Thr Val Pro Trp
Asp Gln Leu Phe 450 455 460 Arg Asn Pro His Gln Ala Leu Leu His Thr
Ala Asn Arg Pro Glu Asp 465 470 475 480 Glu Cys Val Gly Glu Gly Leu
Ala Cys His Gln Leu Cys Ala Arg Gly 485 490 495 His Cys Trp Gly Pro
Gly Pro Thr Gln Cys Val Asn Cys Ser Gln Phe 500 505 510 Leu Arg Gly
Gln Glu Cys Val Glu Glu Cys Arg Val Leu Gln Gly Leu 515 520 525
Pro Arg Glu Tyr Val Asn Ala Arg His Cys Leu Pro Cys His Pro Glu 530
535 540 Cys Gln Pro Gln Asn Gly Ser Val Thr Cys Phe Gly Leu Glu Ala
Asp 545 550 555 560 Gln Cys Val Ala Cys Ala His Tyr Lys Asp Pro Pro
Phe Cys Val Ala 565 570 575 Arg Cys Pro Ser Gly Val Lys Pro Asp Leu
Ser Tyr Met Pro Ile Trp 580 585 590 Lys Phe Pro Asp Glu Glu Gly Ala
Cys Gln Pro Cys Pro Ile Asn Cys 595 600 605 Thr His Ser Cys Val Asp
Leu Asp Asp Lys Gly Cys Pro Ala Glu Gln 610 615 620 Arg Ala Ser Pro
Leu Thr Val Asp Gly Gly Ser Pro Thr Pro Pro Thr 625 630 635 640 Pro
Gly Gly Gly Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro 645 650
655 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
660 665 670 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val 675 680 685 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr 690 695 700 Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu 705 710 715 720 Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His 725 730 735 Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 740 745 750 Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 755 760 765 Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu 770 775
780 Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
785 790 795 800 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn 805 810 815 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu 820 825 830 Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val 835 840 845 Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln 850 855 860 Lys Ser Leu Ser Leu
Ser Pro Gly Lys Ser Gly Gly Leu Asn Asp Ile 865 870 875 880 Phe Glu
Ala Gln Lys Ile Glu Trp His Glu 885 890 72670DNAArtificial
sequenceHer2 ECD(pertuzumab KO)-Fc(knob)DNA 7acccaagtgt gcaccggcac
agacatgaag ctgcggctcc ctgccagtcc cgagacccac 60ctggacatgc tccgccacct
ctaccagggc tgccaggtgg tgcagggaaa cctggaactc 120acctacctgc
ccaccaatgc cagcctgtcc ttcctgcagg atatccagga ggtgcagggc
180tacgtgctca tcgctcacaa ccaagtgagg caggtcccac tgcagaggct
gcggattgtg 240cgaggcaccc agctctttga ggacaactat gccctggccg
tgctagacaa tggagacccg 300ctgaacaata ccacccctgt cacaggggcc
tccccaggag gcctgcggga gctgcagctt 360cgaagcctca cagagatctt
gaaaggaggg gtcttgatcc agcggaaccc ccagctctgc 420taccaggaca
cgattttgtg gaaggacatc ttccacaaga acaaccagct ggctctcaca
480ctgatagaca ccaaccgctc tcgggcctgc cacccctgtt ctccgatgtg
taagggctcc 540cgctgctggg gagagagttc tgaggattgt cagagcctga
cgcgcactgt ctgtgccggt 600ggctgtgccc gctgcaaggg gccactgccc
actgactgct gccatgagca gtgtgctgcc 660ggctgcacgg gccccaagca
ctctgactgc ctggcctgcc tccacttcaa ccacagtggc 720atctgtgagc
tgcactgccc agccctggtc acctacaaca cagacacgcg ggagtccatg
780cccaatcccg agggccggta tagattcggc gccagctgtg tgactgcctg
tccctacaac 840tacctttcta cggaccgggg atcctgcacc ctcgtctgcc
ccctggccaa ccaagaggtg 900acagcagagg atggaacaca gcggtgtgag
aagtgcagca agccctgtgc ccgagtgtgc 960tatggtctgg gcatggagca
cttgcgagag gtgagggcag ttaccagtgc caatatccag 1020gagtttgctg
gctgcaagaa gatctttggg agcctggcat ttctgccgga gagctttgat
1080ggggacccag cctccaacac tgccccgctc cagccagagc agctccaagt
gtttgagact 1140ctggaagaga tcacaggtta cctatacatc tcagcatggc
cggacagcct gcctgacctc 1200agcgtcttcc agaacctgca agtaatccgg
ggacgaattc tgcacaatgg cgcctactcg 1260ctgaccctgc aagggctggg
catcagctgg ctggggctgc gctcactgag ggaactgggc 1320agtggactgg
ccctcatcca ccataacacc cacctctgct tcgtgcacac ggtgccctgg
1380gaccagctct ttcggaaccc gcaccaagct ctgctccaca ctgccaaccg
gccagaggac 1440gagtgtgtgg gcgagggcct ggcctgccac cagctgtgcg
cccgagggca ctgctggggt 1500ccagggccca cccagtgtgt caactgcagc
cagttccttc ggggccagga gtgcgtggag 1560gaatgccgag tactgcaggg
gctccccagg gagtatgtga atgccaggca ctgtttgccg 1620tgccaccctg
agtgtcagcc ccagaatggc tcagtgacct gttttggact ggaggctgac
1680cagtgtgtgg cctgtgccca ctataaggac cctcccttct gcgtggcccg
ctgccccagc 1740ggtgtgaaac ctgacctctc ctacatgccc atctggaagt
ttccagatga ggagggcgca 1800tgccagcctt gccccatcaa ctgcacccac
tcctgtgtgg acctggatga caagggctgc 1860cccgccgagc agagagccag
ccctctgacg gtcgacggtg gtagtccgac acctccgaca 1920cccgggggtg
gttctgcaga caaaactcac acatgcccac cgtgcccagc acctgaactc
1980ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct
catgatctcc 2040cggacccctg aggtcacatg cgtggtggtg gacgtgagcc
acgaagaccc tgaggtcaag 2100ttcaactggt acgtggacgg cgtggaggtg
cataatgcca agacaaagcc gcgggaggag 2160cagtacaaca gcacgtaccg
tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 2220aatggcaagg
agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa
2280accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct
gcccccatgc 2340cgggatgagc tgaccaagaa ccaggtcagc ctgtggtgcc
tggtcaaagg cttctatccc 2400agcgacatcg ccgtggagtg ggagagcaat
gggcagccgg agaacaacta caagaccacg 2460cctcccgtgc tggactccga
cggctccttc ttcctctaca gcaagctcac cgtggacaag 2520agcaggtggc
agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac
2580cactacacgc agaagagcct ctccctgtct ccgggtaaat ccggaggcct
gaacgacatc 2640ttcgaggccc agaagattga atggcacgag
26708890PRTArtificial sequenceHer2 ECD(pertuzumab KO)-Fc(knob) 8Thr
Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser 1 5 10
15 Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln
20 25 30 Val Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn
Ala Ser 35 40 45 Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly
Tyr Val Leu Ile 50 55 60 Ala His Asn Gln Val Arg Gln Val Pro Leu
Gln Arg Leu Arg Ile Val 65 70 75 80 Arg Gly Thr Gln Leu Phe Glu Asp
Asn Tyr Ala Leu Ala Val Leu Asp 85 90 95 Asn Gly Asp Pro Leu Asn
Asn Thr Thr Pro Val Thr Gly Ala Ser Pro 100 105 110 Gly Gly Leu Arg
Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu Lys 115 120 125 Gly Gly
Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr 130 135 140
Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu Thr 145
150 155 160 Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser
Pro Met 165 170 175 Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu
Asp Cys Gln Ser 180 185 190 Leu Thr Arg Thr Val Cys Ala Gly Gly Cys
Ala Arg Cys Lys Gly Pro 195 200 205 Leu Pro Thr Asp Cys Cys His Glu
Gln Cys Ala Ala Gly Cys Thr Gly 210 215 220 Pro Lys His Ser Asp Cys
Leu Ala Cys Leu His Phe Asn His Ser Gly 225 230 235 240 Ile Cys Glu
Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr 245 250 255 Arg
Glu Ser Met Pro Asn Pro Glu Gly Arg Tyr Arg Phe Gly Ala Ser 260 265
270 Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Arg Gly Ser
275 280 285 Cys Thr Leu Val Cys Pro Leu Ala Asn Gln Glu Val Thr Ala
Glu Asp 290 295 300 Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys Pro Cys
Ala Arg Val Cys 305 310 315 320 Tyr Gly Leu Gly Met Glu His Leu Arg
Glu Val Arg Ala Val Thr Ser 325 330 335 Ala Asn Ile Gln Glu Phe Ala
Gly Cys Lys Lys Ile Phe Gly Ser Leu 340 345 350 Ala Phe Leu Pro Glu
Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr Ala 355 360 365 Pro Leu Gln
Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu Ile 370 375 380 Thr
Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp Leu 385 390
395 400 Ser Val Phe Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His
Asn 405 410 415 Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser
Trp Leu Gly 420 425 430 Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu
Ala Leu Ile His His 435 440 445 Asn Thr His Leu Cys Phe Val His Thr
Val Pro Trp Asp Gln Leu Phe 450 455 460 Arg Asn Pro His Gln Ala Leu
Leu His Thr Ala Asn Arg Pro Glu Asp 465 470 475 480 Glu Cys Val Gly
Glu Gly Leu Ala Cys His Gln Leu Cys Ala Arg Gly 485 490 495 His Cys
Trp Gly Pro Gly Pro Thr Gln Cys Val Asn Cys Ser Gln Phe 500 505 510
Leu Arg Gly Gln Glu Cys Val Glu Glu Cys Arg Val Leu Gln Gly Leu 515
520 525 Pro Arg Glu Tyr Val Asn Ala Arg His Cys Leu Pro Cys His Pro
Glu 530 535 540 Cys Gln Pro Gln Asn Gly Ser Val Thr Cys Phe Gly Leu
Glu Ala Asp 545 550 555 560 Gln Cys Val Ala Cys Ala His Tyr Lys Asp
Pro Pro Phe Cys Val Ala 565 570 575 Arg Cys Pro Ser Gly Val Lys Pro
Asp Leu Ser Tyr Met Pro Ile Trp 580 585 590 Lys Phe Pro Asp Glu Glu
Gly Ala Cys Gln Pro Cys Pro Ile Asn Cys 595 600 605 Thr His Ser Cys
Val Asp Leu Asp Asp Lys Gly Cys Pro Ala Glu Gln 610 615 620 Arg Ala
Ser Pro Leu Thr Val Asp Gly Gly Ser Pro Thr Pro Pro Thr 625 630 635
640 Pro Gly Gly Gly Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro
645 650 655 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys 660 665 670 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val 675 680 685 Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr 690 695 700 Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu 705 710 715 720 Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His 725 730 735 Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 740 745 750 Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 755 760
765 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu
770 775 780 Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe
Tyr Pro 785 790 795 800 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn 805 810 815 Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu 820 825 830 Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val 835 840 845 Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln 850 855 860 Lys Ser Leu
Ser Leu Ser Pro Gly Lys Ser Gly Gly Leu Asn Asp Ile 865 870 875 880
Phe Glu Ala Gln Lys Ile Glu Trp His Glu 885 890 92667DNAArtificial
sequenceHer2 ECD(trastuzumab KO)-Fc(knob)DNA 9acccaagtgt gcaccggcac
agacatgaag ctgcggctcc ctgccagtcc cgagacccac 60ctggacatgc tccgccacct
ctaccagggc tgccaggtgg tgcagggaaa cctggaactc 120acctacctgc
ccaccaatgc cagcctgtcc ttcctgcagg atatccagga ggtgcagggc
180tacgtgctca tcgctcacaa ccaagtgagg caggtcccac tgcagaggct
gcggattgtg 240cgaggcaccc agctctttga ggacaactat gccctggccg
tgctagacaa tggagacccg 300ctgaacaata ccacccctgt cacaggggcc
tccccaggag gcctgcggga gctgcagctt 360cgaagcctca cagagatctt
gaaaggaggg gtcttgatcc agcggaaccc ccagctctgc 420taccaggaca
cgattttgtg gaaggacatc ttccacaaga acaaccagct ggctctcaca
480ctgatagaca ccaaccgctc tcgggcctgc cacccctgtt ctccgatgtg
taagggctcc 540cgctgctggg gagagagttc tgaggattgt cagagcctga
cgcgcactgt ctgtgccggt 600ggctgtgccc gctgcaaggg gccactgccc
actgactgct gccatgagca gtgtgctgcc 660ggctgcacgg gccccaagca
ctctgactgc ctggcctgcc tccacttcaa ccacagtggc 720atctgtgagc
tgcactgccc agccctggtc acctacaaca cagacacgtt tgagtccatg
780cccaatcccg agggccggta tacattcggc gccagctgtg tgactgcctg
tccctacaac 840tacctttcta cggacgtggg atcctgcacc ctcgtctgcc
ccctgcacaa ccaagaggtg 900acagcagagg atggaacaca gcggtgtgag
aagtgcagca agccctgtgc ccgagtgtgc 960tatggtctgg gcatggagca
cttgcgagag gtgagggcag ttaccagtgc caatatccag 1020gagtttgctg
gctgcaagaa gatctttggg agcctggcat ttctgccgga gagctttgat
1080ggggacccag cctccaacac tgccccgctc cagccagagc agctccaagt
gtttgagact 1140ctggaagaga tcacaggtta cctatacatc tcagcatggc
cggacagcct gcctgacctc 1200agcgtcttcc agaacctgca agtaatccgg
ggacgaattc tgcacaatgg cgcctactcg 1260ctgaccctgc aagggctggg
catcagctgg ctggggctgc gctcactgag ggaactgggc 1320agtggactgg
ccctcatcca ccataacacc cacctctgct tcgtgcacac ggtgccctgg
1380gaccagctct ttcggaaccc gcaccaagct ctgctccaca ctgccaaccg
gccagaggac 1440gagtgtgtgg gcgagggcct ggcctgccac cagctgtgcg
cccgagggca ctgctggggt 1500ccagggccca cccagtgtgt caactgcagc
cagttccttc ggggccagga gtgcgtggag 1560gaatgccgag tactgcaggg
gctccccagg gagtatgtga atgccaggca ctgtttgccg 1620tgccaccctg
agtgtcagcc ccagaatggc tcagtgacct gttttggact ggaggctcgg
1680cagtgtgtgg cctgtgccca ctataaggac agacggtgcg tggcccgctg
ccccagcggt 1740gtgaaacctg acctctccta catgcccatc tggaagtttc
cagatgagga gggcgcatgc 1800cagccttgcc ccatcaactg cacccactcc
tgtgtggacc tggatgacaa gggctgcccc 1860gccgagcaga gagccagccc
tctgacggtc gacggtggta gtccgacacc tccgacaccc 1920gggggtggtt
ctgcagacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg
1980gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat
gatctcccgg 2040acccctgagg tcacatgcgt ggtggtggac gtgagccacg
aagaccctga ggtcaagttc 2100aactggtacg tggacggcgt ggaggtgcat
aatgccaaga caaagccgcg ggaggagcag 2160tacaacagca cgtaccgtgt
ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 2220ggcaaggagt
acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc
2280atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc
cccatgccgg 2340gatgagctga ccaagaacca ggtcagcctg tggtgcctgg
tcaaaggctt ctatcccagc 2400gacatcgccg tggagtggga gagcaatggg
cagccggaga acaactacaa gaccacgcct 2460cccgtgctgg actccgacgg
ctccttcttc ctctacagca agctcaccgt ggacaagagc 2520aggtggcagc
aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac
2580tacacgcaga agagcctctc cctgtctccg ggtaaatccg gaggcctgaa
cgacatcttc 2640gaggcccaga agattgaatg gcacgag 266710889PRTArtificial
sequenceHer2 ECD(trastuzumab KO)-Fc(knob) 10Thr Gln Val Cys Thr Gly
Thr Asp Met Lys Leu Arg Leu Pro Ala Ser 1 5 10 15 Pro Glu Thr His
Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln 20 25 30 Val Val
Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser 35 40 45
Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu Ile 50
55 60 Ala His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile
Val 65 70 75 80 Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala
Val Leu Asp 85 90 95 Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val
Thr Gly Ala Ser Pro 100 105 110 Gly Gly Leu Arg Glu Leu Gln Leu Arg
Ser Leu Thr Glu Ile Leu Lys 115 120 125 Gly Gly Val Leu Ile Gln Arg
Asn Pro Gln Leu Cys Tyr Gln Asp Thr 130 135 140 Ile Leu Trp Lys Asp
Ile Phe His Lys Asn Asn Gln Leu Ala Leu Thr 145 150 155 160 Leu Ile
Asp Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro Met 165 170 175
Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser 180
185
190 Leu Thr Arg Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro
195 200 205 Leu Pro Thr Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys
Thr Gly 210 215 220 Pro Lys His Ser Asp Cys Leu Ala Cys Leu His Phe
Asn His Ser Gly 225 230 235 240 Ile Cys Glu Leu His Cys Pro Ala Leu
Val Thr Tyr Asn Thr Asp Thr 245 250 255 Phe Glu Ser Met Pro Asn Pro
Glu Gly Arg Tyr Thr Phe Gly Ala Ser 260 265 270 Cys Val Thr Ala Cys
Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser 275 280 285 Cys Thr Leu
Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp 290 295 300 Gly
Thr Gln Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val Cys 305 310
315 320 Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr
Ser 325 330 335 Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe
Gly Ser Leu 340 345 350 Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro
Ala Ser Asn Thr Ala 355 360 365 Pro Leu Gln Pro Glu Gln Leu Gln Val
Phe Glu Thr Leu Glu Glu Ile 370 375 380 Thr Gly Tyr Leu Tyr Ile Ser
Ala Trp Pro Asp Ser Leu Pro Asp Leu 385 390 395 400 Ser Val Phe Gln
Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His Asn 405 410 415 Gly Ala
Tyr Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu Gly 420 425 430
Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His His 435
440 445 Asn Thr His Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu
Phe 450 455 460 Arg Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg
Pro Glu Asp 465 470 475 480 Glu Cys Val Gly Glu Gly Leu Ala Cys His
Gln Leu Cys Ala Arg Gly 485 490 495 His Cys Trp Gly Pro Gly Pro Thr
Gln Cys Val Asn Cys Ser Gln Phe 500 505 510 Leu Arg Gly Gln Glu Cys
Val Glu Glu Cys Arg Val Leu Gln Gly Leu 515 520 525 Pro Arg Glu Tyr
Val Asn Ala Arg His Cys Leu Pro Cys His Pro Glu 530 535 540 Cys Gln
Pro Gln Asn Gly Ser Val Thr Cys Phe Gly Leu Glu Ala Arg 545 550 555
560 Gln Cys Val Ala Cys Ala His Tyr Lys Asp Arg Arg Cys Val Ala Arg
565 570 575 Cys Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr Met Pro Ile
Trp Lys 580 585 590 Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys Pro
Ile Asn Cys Thr 595 600 605 His Ser Cys Val Asp Leu Asp Asp Lys Gly
Cys Pro Ala Glu Gln Arg 610 615 620 Ala Ser Pro Leu Thr Val Asp Gly
Gly Ser Pro Thr Pro Pro Thr Pro 625 630 635 640 Gly Gly Gly Ser Ala
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 645 650 655 Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 660 665 670 Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 675 680
685 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
690 695 700 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln 705 710 715 720 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln 725 730 735 Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala 740 745 750 Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro 755 760 765 Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr 770 775 780 Lys Asn Gln
Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser 785 790 795 800
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 805
810 815 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr 820 825 830 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe 835 840 845 Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys 850 855 860 Ser Leu Ser Leu Ser Pro Gly Lys Ser
Gly Gly Leu Asn Asp Ile Phe 865 870 875 880 Glu Ala Gln Lys Ile Glu
Trp His Glu 885 1111PRTArtificial sequencePertuzumab wt VL CDR1
11Lys Ala Ser Gln Asp Val Ser Ile Gly Val Ala 1 5 10
127PRTArtificial sequencePertuzumab wt VL CDR2 12Ser Ala Ser Tyr
Arg Tyr Thr 1 5 139PRTArtificial sequencePertuzumab wt VL CDR3
13Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr 1 5 1410PRTArtificial
sequencePertuzumab wt VH CDR1 14Gly Phe Thr Phe Thr Asp Tyr Thr Met
Asp 1 5 10 1517PRTArtificial sequencePertuzumab wt VH CDR2 15Asp
Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe Lys 1 5 10
15 Gly 1610PRTArtificial sequencePertuzumab wt VH CDR3 16Asn Leu
Gly Pro Ser Phe Tyr Phe Asp Tyr 1 5 10 1711PRTArtificial
sequenceTrastuzumab wt VL CDR1 17Arg Ala Ser Gln Asp Val Asn Thr
Ala Val Ala 1 5 10 187PRTArtificial sequenceTrastuzumab VL CDR2
18Ser Ala Ser Phe Leu Tyr Ser 1 5 199PRTArtificial
sequenceTrastuzumab VL CDR3 19Gln Gln His Tyr Thr Thr Pro Pro Thr 1
5 2010PRTArtificial sequenceTrastuzumab VH CDR1 20Gly Phe Asn Ile
Lys Asp Thr Tyr Ile His 1 5 10 21558DNAArtificial
sequencePertuzumab wt VH (10289) DNA 21gaggtgcagc tggtcgagtc
tggcggcgga ctggtgcagc ctggcggcag cctgagactg 60agctgcgccg ccagcggctt
caccttcacc gactacacca tggactgggt gcggcaggcc 120cctggcaagg
gcctggaatg ggtggccgac gtgaacccca acagcggcgg cagcatctac
180aaccagcggt tcaagggccg gttcaccctg agcgtggaca gaagcaagaa
caccctgtac 240ctccagatga acagcctgcg ggccgaggac accgccgtgt
actactgcgc ccggaacctg 300ggccccagct tctacttcga ctactggggc
cagggcaccc tggtgaccgt gagcagcgct 360agcaccaagg gcccatcggt
cttccccctg gcaccctcct ccaagagcac ctctgggggc 420acagcggccc
tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg
480aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca
gtcctcagga 540ctctactccc tcagcagc 55822119PRTArtificial
sequencePertuzumab wt VH (10289) 22Glu 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 Phe Thr Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp
Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60
Lys Gly Arg Phe Thr Leu Ser Val Asp Arg 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 Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
23321DNAArtificial sequencePertuzumab wt VL (10290) DNA
23gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc
60atcacctgca aggccagcca ggacgtgtcc atcggcgtgg cctggtatca gcagaagccc
120ggcaaggccc ccaagctgct gatctacagc gccagctacc ggtacacagg
cgtgcccagc 180cggttcagcg gcagcggctc cggcaccgac ttcaccctga
ccatcagcag cctgcagccc 240gaggacttcg ccacctacta ctgccagcag
tactacatct acccctacac cttcggccag 300ggcaccaagg tggagatcaa g
32124107PRTArtificial sequencePertuzumab wt VL (10290) 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 Lys Ala Ser Gln Asp Val Ser Ile Gly 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 Tyr Thr 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 Tyr Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 100 105 25321DNAArtificial sequencePertuzumab VL
(Trast. L3) (10403) DNA 25gacatccaga tgacccagag ccccagcagc
ctgagcgcca gcgtgggcga cagagtgacc 60atcacatgca aggccagcca ggacgtgtcc
atcggcgtgg cctggtatca gcagaagccc 120ggcaaggccc ccaagctgct
gatctacagc gccagctacc ggtacaccgg cgtgcccagc 180agattcagcg
gcagcggctc cggcaccgac ttcaccctga ccatcagcag cctgcagccc
240gaggacttcg ccacctacta ctgccagcag cactacacca ccccccccac
cttcggccag 300ggcaccaagg tggaaatcaa g 32126107PRTArtificial
sequencePertuzumab VL (Trast. L3) (10403) 26Asp 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 Gln Asp Val Ser Ile Gly 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 Tyr Thr 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 His Tyr Thr
Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 27321DNAArtificial sequencePertuzumab VL (Trast. H91)
(10404) DNA 27gacatccaga tgacccagag ccccagcagc ctgagcgcca
gcgtgggcga cagagtgacc 60atcacctgca aggccagcca ggacgtgtcc atcggcgtgg
cctggtatca gcagaagccc 120ggcaaggccc ccaagctgct gatctacagc
gccagctacc ggtacacagg cgtgcccagc 180cggttcagcg gcagcggctc
cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240gaggacttcg
ccacctacta ctgccagcag cactacatct acccctacac cttcggccag
300ggcaccaagg tggagatcaa g 32128107PRTArtificial sequencePertuzumab
VL (Trast. H91) (10404) 28Asp 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 Gln Asp Val Ser Ile Gly 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 Tyr Thr 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 His Tyr Ile Tyr Pro Tyr 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
2917PRTArtificial sequenceTrastuzumab VH CDR2 29Arg Ile Tyr Pro Thr
Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
3011PRTArtificial sequenceTrastuzumab wt VH CDR3 30Trp Gly Gly Asp
Gly Phe Tyr Ala Met Asp Tyr 1 5 10 31321DNAArtificial
sequencePertuzumab VL (Tras.L3) K24R (10949) DNA 31gacatccaga
tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc 60atcacatgcc
gggccagcca ggacgtgtcc atcggcgtgg cctggtatca gcagaagccc
120ggcaaggccc ccaagctgct gatctacagc gccagctacc ggtacaccgg
cgtgcccagc 180agattcagcg gcagcggctc cggcaccgac ttcaccctga
ccatcagcag cctgcagccc 240gaggacttcg ccacctacta ctgccagcag
cactacacca ccccccccac cttcggccag 300ggcaccaagg tggaaatcaa g
32132107PRTArtificial sequencePertuzumab VL (Tras.L3) K24R (10949)
32Asp 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 Val Ser Ile
Gly 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 Tyr Thr 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 His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 33321DNAArtificial
sequencePertuzumab VL(Tras.L3) S30N (10950) DNA 33gacatccaga
tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc 60atcacatgca
aggccagcca ggacgtgaac atcggcgtgg cctggtatca gcagaagccc
120ggcaaggccc ccaagctgct gatctacagc gccagctacc ggtacaccgg
cgtgcccagc 180agattcagcg gcagcggctc cggcaccgac ttcaccctga
ccatcagcag cctgcagccc 240gaggacttcg ccacctacta ctgccagcag
cactacacca ccccccccac cttcggccag 300ggcaccaagg tggaaatcaa g
32134107PRTArtificial sequencePertuzumab VL (Tras.L3) S30N (10950)
34Asp 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 Gln Asp Val Asn Ile
Gly 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 Tyr Thr 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 His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 35321DNAArtificial
sequencePertuzumab VL (Tras.L3) I31T (10951) DNA 35gacatccaga
tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc 60atcacatgca
aggccagcca ggacgtgtcc accggcgtgg cctggtatca gcagaagccc
120ggcaaggccc ccaagctgct gatctacagc gccagctacc ggtacaccgg
cgtgcccagc 180agattcagcg gcagcggctc cggcaccgac ttcaccctga
ccatcagcag cctgcagccc 240gaggacttcg ccacctacta ctgccagcag
cactacacca ccccccccac cttcggccag 300ggcaccaagg tggaaatcaa g
32136107PRTArtificial sequencePertuzumab VL (Tras.L3) I31T (10951)
36Asp 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 Gln Asp Val Ser Thr
Gly 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 Tyr Thr 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 His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 37321DNAArtificial
sequencePertuzumab VL (Tras.L3) I31V (10952) DNA 37gacatccaga
tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc 60atcacatgca
aggccagcca ggacgtgtcc gtcggcgtgg cctggtatca gcagaagccc
120ggcaaggccc ccaagctgct gatctacagc gccagctacc ggtacaccgg
cgtgcccagc 180agattcagcg gcagcggctc cggcaccgac ttcaccctga
ccatcagcag cctgcagccc 240gaggacttcg ccacctacta ctgccagcag
cactacacca ccccccccac cttcggccag 300ggcaccaagg tggaaatcaa g
32138107PRTArtificial sequencePertuzumab VL (Tras.L3) I31V (10952)
38Asp 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 Gln Asp Val Ser Val Gly 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 Tyr Thr 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 His Tyr Thr Thr
Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 39321DNAArtificial sequencePertuzumab VL (Tras.L3) G32A (10953)
DNA 39gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga
cagagtgacc 60atcacatgca aggccagcca ggacgtgtcc atcgccgtgg cctggtatca
gcagaagccc 120ggcaaggccc ccaagctgct gatctacagc gccagctacc
ggtacaccgg cgtgcccagc 180agattcagcg gcagcggctc cggcaccgac
ttcaccctga ccatcagcag cctgcagccc 240gaggacttcg ccacctacta
ctgccagcag cactacacca ccccccccac cttcggccag 300ggcaccaagg
tggaaatcaa g 32140107PRTArtificial sequencePertuzumab VL (Tras.L3)
G32A (10953) 40Asp 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 Gln
Asp Val Ser Ile Ala 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 Tyr
Thr 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 His Tyr Thr Thr Pro Pro 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 41321DNAArtificial
sequencePertuzumab VL (Tras.L3) Y53F (10954) DNA 41gacatccaga
tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc 60atcacatgca
aggccagcca ggacgtgtcc atcggcgtgg cctggtatca gcagaagccc
120ggcaaggccc ccaagctgct gatctacagc gccagcttcc ggtacaccgg
cgtgcccagc 180agattcagcg gcagcggctc cggcaccgac ttcaccctga
ccatcagcag cctgcagccc 240gaggacttcg ccacctacta ctgccagcag
cactacacca ccccccccac cttcggccag 300ggcaccaagg tggaaatcaa g
32142107PRTArtificial sequencePertuzumab VL (Tras.L3) Y53F (10954)
42Asp 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 Gln Asp Val Ser Ile
Gly 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 Phe Arg Tyr Thr 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 His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 43321DNAArtificial
sequencePertuzumab (Tras.L3) R54L (10955) DNA 43gacatccaga
tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc 60atcacatgca
aggccagcca ggacgtgtcc atcggcgtgg cctggtatca gcagaagccc
120ggcaaggccc ccaagctgct gatctacagc gccagctacc tgtacaccgg
cgtgcccagc 180agattcagcg gcagcggctc cggcaccgac ttcaccctga
ccatcagcag cctgcagccc 240gaggacttcg ccacctacta ctgccagcag
cactacacca ccccccccac cttcggccag 300ggcaccaagg tggaaatcaa g
32144107PRTArtificial sequencePertuzumab VL (Tras.L3) R54L (10955)
44Asp 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 Gln Asp Val Ser Ile
Gly 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 Leu Tyr Thr 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 His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 45321DNAArtificial
sequencePertuzumab (Tras.L3) T56S (10956) DNA 45gacatccaga
tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc 60atcacatgca
aggccagcca ggacgtgtcc atcggcgtgg cctggtatca gcagaagccc
120ggcaaggccc ccaagctgct gatctacagc gccagctacc ggtacagcgg
cgtgcccagc 180agattcagcg gcagcggctc cggcaccgac ttcaccctga
ccatcagcag cctgcagccc 240gaggacttcg ccacctacta ctgccagcag
cactacacca ccccccccac cttcggccag 300ggcaccaagg tggaaatcaa g
32146107PRTArtificial sequencePertuzumab (Tras.L3) T56S (10956)
46Asp 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 Gln Asp Val Ser Ile
Gly 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 Tyr 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 His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 47321DNAArtificial
sequencePertuzumab (Tras.L3) G66R (10957) DNA 47gacatccaga
tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc 60atcacatgca
aggccagcca ggacgtgtcc atcggcgtgg cctggtatca gcagaagccc
120ggcaaggccc ccaagctgct gatctacagc gccagctacc ggtacaccgg
cgtgcccagc 180agattcagcg gcagccgctc cggcaccgac ttcaccctga
ccatcagcag cctgcagccc 240gaggacttcg ccacctacta ctgccagcag
cactacacca ccccccccac cttcggccag 300ggcaccaagg tggaaatcaa g
32148107PRTArtificial sequencePertuzumab (Tras.L3) G66R (10957)
48Asp 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 Gln Asp Val Ser Ile
Gly 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 Tyr Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Arg 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 His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 49320DNAArtificial
sequencePertuzumab (Tras.L3) T94Y (10958) DNA 49acatccagat
gacccagagc cccagcagcc tgagcgccag cgtgggcgac agagtgacca 60tcacatgcaa
ggccagccag gacgtgtcca tcggcgtggc ctggtatcag cagaagcccg
120gcaaggcccc caagctgctg atctacagcg ccagctaccg gtacaccggc
gtgcccagca 180gattcagcgg cagcggctcc ggcaccgact tcaccctgac
catcagcagc ctgcagcccg 240aggacttcgc cacctactac tgccagcagc
actacaccta cccccccacc ttcggccagg 300gcaccaaggt ggaaatcaag
32050107PRTArtificial sequencePertuzumab (Tras.L3) T94Y (10958)
50Asp 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 Gln Asp Val Ser Ile
Gly 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 Tyr Thr 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 His Tyr Thr Tyr Pro Pro 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 51321DNAArtificial
sequencePertuzumab (Tras.L3) P96Y (10959) DNA 51gacatccaga
tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc 60atcacatgca
aggccagcca ggacgtgtcc atcggcgtgg cctggtatca gcagaagccc
120ggcaaggccc ccaagctgct gatctacagc gccagctacc ggtacaccgg
cgtgcccagc 180agattcagcg gcagcggctc cggcaccgac ttcaccctga
ccatcagcag cctgcagccc 240gaggacttcg ccacctacta ctgccagcag
cactacacca ccccctacac cttcggccag 300ggcaccaagg tggaaatcaa g
32152107PRTArtificial sequencePertuzumab (Tras.L3) P96Y (10959)
52Asp 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 Gln Asp Val Ser Ile
Gly 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 Tyr Thr 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 His Tyr Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 53321DNAArtificial
sequencePertuzumab (Tras.L3) (QM) (11055) DNA 53gacatccaga
tgacccagag ccccagcagc ctgtctgcca gcgtgggcga cagagtgacc 60atcacatgca
aggccagcca ggacgtgtcc acagccgtgg cctggtatca gcagaagcct
120ggcaaggccc ccaagctgct gatctacagc gccagcttcc ggtacaccgg
cgtgcccagc 180agattcagcg gcagcagatc cggcaccgac ttcaccctga
ccatcagctc cctgcagccc 240gaggacttcg ccacctacta ctgccagcag
cactacacca ccccccccac atttggccag 300ggcaccaagg tggaaatcaa g
32154107PRTArtificial sequencePertuzumab (Tras.L3) (QM) (11055)
54Asp 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 Gln Asp Val Ser Thr
Ala 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 Phe Arg Tyr Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Arg 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 His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 5510PRTArtificial
sequencePertuzumab aff.mat. clone D1- VH CDR1 55Gly Phe Thr Phe Asn
Asp Tyr Thr Met Asp 1 5 10 5610PRTArtificial sequencePertuzumab
aff.mat. clone D1- VH CDR3 56Asn Leu Gly Pro Phe Phe Tyr Phe Asp
Tyr 1 5 10 5710PRTArtificial sequencePertuzumab aff.mat. clone B2-
VH CDR3 57Asn Leu Gly Pro Asn Phe Tyr Phe Asp Tyr 1 5 10
5810PRTArtificial sequencePertuzumab aff.mat. clone E1- VH CDR1
58Gly Phe Thr Phe Ala Asp Tyr Thr Met Asp 1 5 10 5910PRTArtificial
sequencePertuzumab aff.mat. clone E1- VH CDR3 59Asn Leu Gly Pro Trp
Phe Tyr Phe Asp Tyr 1 5 10 6017PRTArtificial sequencePertuzumab
aff.mat. clone G2- VH CDR2 60Asp Val Asn Pro Asn Ser Gly Gly Tyr
Ile Val Asn Arg Arg Phe Lys 1 5 10 15 Gly 61357DNAArtificial
sequencePertuzumab aff.mat. clone D1 DNA 61gaggtgcaat tggttgaaag
cggtggtggt ctggttcagc ctggtggtag cctgcgtctg 60agctgtgcag caagcggttt
tacctttaac gattatacca tggattgggt tcgtcaggca 120ccgggtaaag
gtctggaatg ggttgcagat gttaatccga atagcggtgg tagcatttat
180aaccagcgtt ttaaaggtcg ttttaccctg agcgttgatc gtagcaaaaa
taccctgtat 240ctgcaaatga atagtctgcg tgcagaggat accgcagtgt
attattgtgc acgtaacctg 300ggtccgttct tctactttga ttattggggt
cagggcaccc tggttaccgt tagcagc 35762119PRTArtificial
sequencePertuzumab aff.mat. clone D1 62Glu 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 Phe Thr Phe Asn Asp Tyr 20 25 30 Thr Met Asp
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55
60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg 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 Arg Asn Leu Gly Pro Phe Phe Tyr Phe Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
63357DNAArtificial sequencePertuzumab aff.mat. clone D1-derived,
DNA 63gaggtgcaat tggttgaaag cggtggtggt ctggttcagc ctggtggtag
cctgcgtctg 60agctgtgcag caagcggttt tacctttaac gattatacca tggattgggt
tcgtcaggca 120ccgggtaaag gtctggaatg ggttgcagat gttaatccga
atagcggtgg tagcattgtt 180aaccgtcgtt ttaaaggtcg ttttaccctg
agcgttgatc gtagcaaaaa taccctgtat 240ctgcaaatga atagtctgcg
tgcagaggat accgcagtgt attattgtgc acgtaacctg 300ggtccgttct
tctactttga ttattggggt cagggcaccc tggttaccgt tagcagc
35764119PRTArtificial sequencePertuzumab aff.mat. clone D1-derived
64Glu 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 Phe Thr Phe Asn Asp
Tyr 20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile
Val Asn Arg Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp
Arg 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 Arg Asn Leu Gly
Pro Phe Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 65357DNAArtificial sequencePertuzumab aff.mat.
clone B2, DNA 65gaggtgcaat tggttgaaag cggtggtggt ctggttcagc
ctggtggtag cctgcgtctg 60agctgtgcag caagcggttt tacctttaac gattatacca
tggattggtt tcgtcaggca 120ccgggtaaag gtctggaatg ggttgcagat
gttaatccga atagcggtgg tagcatttat 180aaccagcgtt ttaaaggtcg
ttttaccctg agcgttgatc gtagcaaaaa taccctgtat 240ctgcaaatga
atagtctgcg tgcagaggat accgcagtgt attattgtgc acgtaatctg
300ggtccgaact tctactttga ttattggggt cagggcaccc tggttaccgt tagcagc
35766119PRTArtificial sequencePertuzumab aff.mat. clone B2 66Glu
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 Phe Thr Phe Asn Asp Tyr
20 25 30 Thr Met Asp Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr
Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg
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 Arg Asn Leu Gly Pro
Asn Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser 115 67357DNAArtificial sequencePertuzumab aff.mat.
clone E1, DNA 67gaggtgcaat tggttgaaag cggtggtggt ctggttcagc
ctggtggtag cctgcgtctg 60agctgtgcag caagcggttt tacctttgca gattatacca
tggattgggt tcgtcaggca 120ccgggtaaag gtctggaatg ggttgcagat
gttaatccga atagcggtgg tagcatttat 180aaccagcgtt ttaaaggtcg
ttttaccctg agcgttgatc gtagcaaaaa taccctgtat 240ctgcaaatga
atagtctgcg tgcagaggat accgcagtgt attattgtgc acgtaatctg
300ggtccgtggt tctactttga ttattggggt cagggcaccc tggttaccgt tagcagc
35768119PRTArtificial sequencePertuzumab aff.mat. clone E1 68Glu
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 Phe Thr Phe Ala Asp
Tyr 20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile
Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp
Arg 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 Arg Asn Leu Gly
Pro Trp Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 69357DNAArtificial sequencePertuzumab aff.mat.
clone G2, DNA 69gaggtgcaat tggttgaaag cggtggtggt ctggttcagc
ctggtggtag cctgcgtctg 60agctgtgcag caagcggttt tacctttacc gattacacaa
tggattgggt tcgtcaggca 120ccgggtaaag gtctggaatg ggttgcagat
gttaatccga actctggtgg ttacattgtt 180aaccgtcgtt ttaaaggtcg
ttttaccctg agcgttgatc gtagcaaaaa taccctgtat 240ctgcaaatga
atagtctgcg tgcagaggat accgcagtgt attattgtgc acgtaatctg
300ggtccgagct tctattttga ttattggggt cagggcaccc tggttaccgt tagcagc
35770119PRTArtificial sequencePertuzumab aff.mat. clone G2 70Glu
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 Phe Thr Phe Thr Asp Tyr
20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Tyr Ile Val
Asn Arg Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg
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 Arg Asn Leu Gly Pro
Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser 115 71357DNAArtificial sequencePertuzumab aff.mat.
clone C8, DNA 71gaggtgcaat tggttgaaag cggtggtggt ctggttcagc
ctggtggtag cctgcgtctg 60agctgtgcag caagcggttt tacctttacc gattacacaa
tggattgggt tcgtcaggca 120ccgggtaaag gtctggaatg ggttgcagat
gttaatccga actctggtgg ttctattatg 180aaccgtcgtt ttaaaggtcg
ttttaccctg agcgttgatc gtagcaaaaa taccctgtat 240ctgcaaatga
atagtctgcg tgcagaggat accgcagtgt attattgtgc acgtaatctg
300ggtccgagct tctattttga ttattggggt cagggcaccc tggttaccgt tagcagc
35772119PRTArtificial sequencePertuzumab aff.mat. clone C8 72Glu
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 Phe Thr Phe Thr Asp Tyr
20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Met
Asn Arg Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg
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 Arg Asn Leu Gly Pro
Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser 115 73357DNAArtificial sequencePertuzumab aff.mat.
clone A1 DNA 73gaggtgcaat tggttgaaag cggtggtggt ctggttcagc
ctggtggtag cctgcgtctg 60agctgtgcag caagcggttt tacctttacc gattacacaa
tggattgggt tcgtcaggca 120ccgggtaaag gtctggaatg ggttgcagat
gttaatccga actctggtgg ttctattgtt 180aaccagcgtt ttaaaggtcg
ttttaccctg agcgttgatc gtagcaaaaa taccctgtat 240ctgcaaatga
atagtctgcg tgcagaggat accgcagtgt attattgtgc acgtaatctg
300ggtccgtggt tctactttga ttattggggt cagggcaccc tggttaccgt tagcagc
35774119PRTArtificial sequencePertuzumab aff.mat. clone A1 74Glu
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 Phe Thr Phe Thr Asp Tyr
20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Val
Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg
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 Arg Asn Leu Gly Pro
Trp Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser 115 7517PRTArtificial sequencePertuzumab aff.mat. clone
C8- VH CDR2 75Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Met Asn Arg
Arg Phe Lys 1 5 10 15 Gly 7617PRTArtificial sequencePertuzumab
aff.mat. clone A1- VH CDR2 76Asp Val Asn Pro Asn Ser Gly Gly Ser
Ile Val Asn Gln Arg Phe Lys 1 5 10 15 Gly 7717PRTArtificial
sequencePertuzumab aff.mat. clone D1-derived VH CDR2 77Asp Val Asn
Pro Asn Ser Gly Gly Ser Ile Val Asn Arg Arg Phe Lys 1 5 10 15 Gly
7811PRTArtificial sequenceTrastuzumab VH (D98N) (6636) CDR3 78Trp
Gly Gly Asn Gly Phe Tyr Ala Met Asp Tyr 1 5 10 7911PRTArtificial
sequenceTrastuzumab VH CDR3 79Trp Gly Gly Glu Gly Phe Tyr Ala Met
Asp Tyr 1 5 10 8011PRTArtificial sequenceTrastuzumab VH (D98T)
(6638) CDR3 80Trp Gly Gly Thr Gly Phe Tyr Ala Met Asp Tyr 1 5 10
81321DNAArtificial sequenceTrastuzumab VL (4245) DNA 81gacatccaga
tgacccagag cccaagctct ctgtctgcct ctgtgggcga cagagtgacc 60atcacctgca
gagccagcca ggacgtgaac acagccgtgg cctggtatca gcagaagcca
120ggcaaggccc caaagctgct gatctacagc gccagcttcc tgtacagcgg
cgtgccaagc 180agattcagcg gcagcagaag cggcacagac ttcaccctga
ccatcagcag cctgcagcca 240gaggacttcg ccacctacta ctgccagcag
cactacacca ccccaccaac cttcggacag 300ggcaccaagg tggagatcaa g
32182107PRTArtificial sequenceTrastuzumab VL (4245) 82Asp 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 Val Asn Thr Ala 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 Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Arg 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
His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 105 83321DNAArtificial sequenceTrastuzumab VL T31A
(6641) DNA 83gatatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
cagagtcacc 60atcacttgcc gggcaagtca ggacgtgaac gccgctgtag cgtggtacca
gcagaaacca 120ggtaaggcac cgaagctatt aatttatagt gcgagcttcc
tgtacagtgg ggtcccgtcg 180cgttttagcg gctctcgatc cggcacggat
tttaccctga ccattagcag cctgcagcct 240gaagactttg cgacatatta
ttgccaacag cactacacaa ctcctcccac ctttggccag 300ggtacgaaag
ttgaaattaa a 32184107PRTArtificial sequenceTrastuzumab VL T31A
(6641) 84Asp 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
Val Asn Ala Ala 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 Phe Leu Tyr Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg 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 His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 85322DNAArtificial
sequenceTrastuzumab VL T31V (6642) DNA 85gatatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca
ggacgtgaac gtggctgtag cgtggtacca gcagaaacca 120ggtaaggcac
cgaagctatt aatttatagt gcgagcttcc tgtacagtgg ggtcccgtcg
180cgttttagcg gctctcgatc cggcacggat tttaccctga ccattagcag
cctgcagcct 240gaagactttg cgacatatta ttgccaacag cactacacaa
ctcctcccac ctttggccag 300ggtacgaaag ttgaaattaa ag
32286107PRTArtificial sequenceTrastuzumab VL T31V (6642) 86Asp 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 Val Asn Val Ala 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 Phe Leu Tyr Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Arg 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 His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 100 105 8711PRTArtificial sequenceTrastuzumab VH
(G99A) (6639) CDR3 87Trp Gly Gly Asp Ala Phe Tyr Ala Met Asp Tyr 1
5 10 8811PRTArtificial sequenceTrastuzumab VH (G99S) (6640) CDR3
88Trp Gly Gly Asp Ser Phe Tyr Ala Met Asp Tyr 1 5 10
8911PRTArtificial sequencePertuzumab (Tras.L3) (QM)-CDR1 89Lys Ala
Ser Gln Asp Val Ser Thr Ala Val Ala 1 5 10 907PRTArtificial
sequencePertuzumab (Tras.L3) (QM)-CDR2 90Ser Ala Ser Phe Arg Tyr
Thr 1 5 91360DNAArtificial sequenceTrastuzumab VH (11345) DNA
91gaagtgcaat tggtggaaag cggcggcggc ctggtgcaac cgggcggcag cctgcgtctg
60agctgcgcgg cctccggatt taacataaag gacacataca tccactgggt gcgccaagca
120cctgggaagg gtctcgagtg ggtggctcgg atttacccaa caaatggcta
caccaggtat 180gcggatagcg tgaaaggccg ttttaccatt tcagctgata
cttcgaagaa caccgcctat 240ctgcaaatga acagcctgcg tgcggaagat
acggccgtgt attattgctc gcgttgggga 300ggagacgggt tctatgctat
ggattactgg ggccaaggca ccctggtgac ggttagctca 36092120PRTArtificial
sequenceTrastuzumab VH (11345) 92Glu 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 Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg
Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr 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 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp
Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
93360DNAArtificial sequenceTrastuzumab VH (D98N) (6636) DNA
93gaagtgcaat tggtggaaag cggcggcggc ctggtgcaac cgggcggcag cctgcgtctg
60agctgcgcgg cctccggatt taacataaag gacacataca tccactgggt gcgccaagca
120cctgggaagg gtctcgagtg ggtggctcgg atttacccaa caaatggcta
caccaggtat 180gcggatagcg tgaaaggccg ttttaccatt tcagctgata
cttcgaagaa caccgcctat 240ctgcaaatga acagcctgcg tgcggaagat
acggccgtgt attattgctc gcgttgggga 300ggaaacgggt tctatgctat
ggattactgg ggccaaggca ccctggtgac ggttagctca 36094120PRTArtificial
sequenceTrastuzumab VH (D98N) (6636) 94Glu 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 Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr 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 Ser Arg Trp Gly Gly Asn Gly Phe Tyr Ala Met Asp
Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
95360DNAArtificial sequenceTrastuzumab VH (D98E) (6637) DNA
95gaagtgcaat tggtggaaag cggcggcggc ctggtgcaac cgggcggcag cctgcgtctg
60agctgcgcgg cctccggatt taacataaag gacacataca tccactgggt gcgccaagca
120cctgggaagg gtctcgagtg ggtggctcgg atttacccaa caaatggcta
caccaggtat 180gcggatagcg tgaaaggccg ttttaccatt tcagctgata
cttcgaagaa caccgcctat 240ctgcaaatga acagcctgcg tgcggaagat
acggccgtgt attattgctc gcgttgggga 300ggagaggggt tctatgctat
ggattactgg ggccaaggca ccctggtgac ggttagctca 36096120PRTArtificial
sequenceTrastuzumab VH (D98E) (6637) 96Glu 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 Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr 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 Ser Arg Trp Gly Gly Glu Gly Phe Tyr Ala Met Asp
Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
97360DNAArtificial sequenceTrastuzumab VH (D98T) (6638) DNA
97gaagtgcaat tggtggaaag cggcggcggc ctggtgcaac cgggcggcag cctgcgtctg
60agctgcgcgg cctccggatt taacataaag gacacataca tccactgggt gcgccaagca
120cctgggaagg gtctcgagtg ggtggctcgg atttacccaa caaatggcta
caccaggtat 180gcggatagcg tgaaaggccg ttttaccatt tcagctgata
cttcgaagaa caccgcctat 240ctgcaaatga acagcctgcg tgcggaagat
acggccgtgt attattgctc gcgttgggga 300ggaaccgggt tctatgctat
ggattactgg ggccaaggca ccctggtgac ggttagctca 36098120PRTArtificial
sequenceTrastuzumab VH (D98T) (6638) 98Glu 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 Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr 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 Ser Arg Trp Gly Gly Thr Gly Phe Tyr Ala Met Asp
Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
99360DNAArtificial sequenceTrastuzumab VH (G99A) (6639) DNA
99gaagtgcaat tggtggaaag cggcggcggc ctggtgcaac cgggcggcag cctgcgtctg
60agctgcgcgg cctccggatt taacataaag gacacataca tccactgggt gcgccaagca
120cctgggaagg gtctcgagtg ggtggctcgg atttacccaa caaatggcta
caccaggtat 180gcggatagcg tgaaaggccg ttttaccatt tcagctgata
cttcgaagaa caccgcctat 240ctgcaaatga acagcctgcg tgcggaagat
acggccgtgt attattgctc gcgttgggga 300ggagacgcct tctatgctat
ggattactgg ggccaaggca ccctggtgac ggttagctca 360100120PRTArtificial
sequenceTrastuzumab VH (G99A) (6639) 100Glu 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 Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr
Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala
Asp Thr 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 Ser Arg Trp Gly
Gly Asp Ala Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr
Leu Val Thr Val Ser Ser 115 120 101360DNAArtificial
sequenceTrastuzumab VH (G99S) (6640) DNA 101gaagtgcaat tggtggaaag
cggcggcggc ctggtgcaac cgggcggcag cctgcgtctg 60agctgcgcgg cctccggatt
taacataaag gacacataca tccactgggt gcgccaagca 120cctgggaagg
gtctcgagtg ggtggctcgg atttacccaa caaatggcta caccaggtat
180gcggatagcg tgaaaggccg ttttaccatt tcagctgata cttcgaagaa
caccgcctat 240ctgcaaatga acagcctgcg tgcggaagat acggccgtgt
attattgctc gcgttgggga 300ggagacagct tctatgctat ggattactgg
ggccaaggca ccctggtgac ggttagctca 360102120PRTArtificial
sequenceTrastuzumab VH (G99S) (6640) 102Glu 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 Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr 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 Ser Arg Trp Gly Gly Asp Ser Phe Tyr Ala Met Asp
Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
10311PRTArtificial sequenceTrastuzumab VL T31A (6641) CDR1 103Arg
Ala Ser Gln Asp Val Asn Ala Ala Val Ala 1 5 10 10411PRTArtificial
sequenceTrastuzumab VL CDR1 104Arg Ala Ser Gln Asp Val Asn Val Ala
Val Ala 1 5 10 105120PRTArtificial sequenceTrastuzumab CDRG VH
(D98E, CDRG) 105Glu 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 Phe
Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Tyr Pro Thr Asn
Gly Tyr Thr Arg Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr
Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu
Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser
Arg Trp Gly Gly Glu Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105
110 Gly Thr Met Val Thr Val Ser Ser 115 120 106107PRTArtificial
sequenceTrastuzumab CDRG VL (N30T, CDRG) 106Asp Ile Gln Leu Thr Gln
Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile
Thr Cys Gly Ala Ser Gln Asp Val Ser Thr Ala Val 20 25 30 Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45
Ser Ala Ser Phe Leu Tyr Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser 50
55 60 Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Val Glu Ala
Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Gln His Tyr Thr Thr
Pro Pro Thr 85 90 95 Phe Gly Thr Gly Thr Lys Val Thr Val Leu Arg
100 105 10711PRTArtificial sequenceTrastuzumab CDRG VL CDR1 107Gly
Ala Ser Gln Asp Val Ser Thr Ala Val Ala 1 5 10 10817PRTArtificial
sequenceTrastuzumab CDRG VH CDR2 108Arg Ile Tyr Pro Thr Asn Gly Tyr
Thr Arg Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 109453PRTArtificial
sequenceXPertuzumab heavy chain 109Glu 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 Phe Thr Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp
Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60
Lys Gly Arg Phe Thr Leu Ser Val Asp Arg 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 Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Val
Ala Ala Pro Ser Val Phe 115 120 125 Ile Phe Pro Pro Ser Asp Glu Gln
Leu Lys Ser Gly Thr Ala Ser Val 130 135 140 Val Cys Leu Leu Asn Asn
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp 145 150 155 160 Lys Val Asp
Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr 165 170 175 Glu
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 180 185
190 Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val
195 200 205 Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn
Arg Gly 210 215 220 Glu Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310
315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu
Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Trp Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435
440 445 Leu Ser Pro Gly Lys 450 110212PRTArtificial
sequenceXPertuzumab light chain 110Asp 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 Gln Asp Val Ser Ile Gly 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 Tyr Thr 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 Tyr Ile Tyr
Pro Tyr 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 111450PRTArtificial
sequenceTrastuzumab CDRG heavy chain (VHCH1) 111Gln 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 Phe Asn Ile Lys Asp Thr 20 25 30 Tyr
Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Gln Lys Phe
50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Glu Gly Phe Tyr Ala
Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170
175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295
300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Cys 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu 355 360 365 Ser Cys Ala Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp
Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp 405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420
425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro 435 440 445 Gly Lys 450 112213PRTArtificial sequenceTrastuzumab
CDRG light chain (VLCL) 112Asp Ile Gln Leu Thr Gln Pro Pro Ser Val
Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Ala
Ser Gln Asp Val Ser Thr Ala Val 20 25 30 Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Ser Ala Ser Phe
Leu Tyr Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser 50 55 60 Arg Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr 85
90 95 Phe Gly Thr Gly Thr Lys Val Thr Val Leu Arg Thr Val Ala Ala
Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205
Asn Arg Gly Glu Cys 210 113107PRTArtificial sequencePertuzumab wt
CL 113Arg 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 11498PRTArtificial
sequencePertuzumab wt CH1 114Ala 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 115107PRTArtificial sequenceTrastuzumab CH1
115Ala 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 Lys Thr His 100 105 116106PRTArtificial
sequenceTrastuzumab CL 116Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln 1 5
10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80 His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 100 105 117120PRTArtificial sequenceTrastuzumab
VH (D98E) 117Glu 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 Phe
Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn
Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr 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 Ser
Arg Trp Gly Gly Glu Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105
110 Gly Thr Leu Val Thr Val Ser Ser 115 120 118108PRTArtificial
sequenceTrastuzumab VL (T31V) 118Asp 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 Val Asn Val Ala 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 Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Arg 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 His Tyr Thr Thr
Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 119453PRTArtificial sequencePertuzumab heavy chain 119Glu
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 Phe Thr Phe Thr Asp Tyr
20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr
Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg
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 Arg Asn Leu Gly Pro
Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe 115 120 125 Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 130 135 140
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp 145
150 155 160 Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
Val Thr 165 170 175 Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr 180 185 190 Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala Cys Glu Val 195 200 205 Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser Phe Asn Arg Gly 210 215 220 Glu Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265
270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Cys Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu
Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390
395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys
Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450
120214PRTArtificial sequencePertuzumab light chain 120Asp 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 Gln Asp Val Ser Ile Gly 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 Tyr Thr 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 Tyr Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
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
121450PRTArtificial sequenceXTrastuzumab heavy chain 121Glu 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 Phe Asn Ile Lys Asp Thr 20 25
30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr 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 Ser Arg Trp Gly Gly Glu Gly Phe
Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155
160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280
285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Cys Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Trp Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405
410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro 435 440 445 Gly Lys 450 122212PRTArtificial
sequenceXTrastuzumab light chain 122Asp 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 Val Asn Val Ala 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 Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Arg 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 His Tyr Thr Thr
Pro Pro 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 123214PRTArtificial sequenceLight
chain (kappa) [Trastuzumab, 1016] 123Asp 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 Val Asn Thr Ala 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 Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Arg 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 His Tyr Thr Thr
Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val 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 124711PRTArtificial
sequenceHeavy chain [Trastuzumab + scFv Omnitarg, RB40] 124Glu 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 Phe Asn Ile Lys Asp Thr 20
25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr 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 Ser Arg Trp Gly Gly Asp Gly
Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150
155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275
280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455 460 Ser
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 465 470
475 480 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr
Asp 485 490 495 Tyr Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Cys
Leu Glu Trp 500 505 510 Val Ala Asp Val Asn Pro Asn Ser Gly Gly Ser
Ile Tyr Asn Gln Arg 515 520 525 Phe Lys Gly Arg Phe Thr Leu Ser Val
Asp Arg Ser Lys Asn Thr Leu 530 535 540 Tyr Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr 545 550 555 560 Cys Ala Arg Asn
Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln 565 570 575 Gly Thr
Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 580 585 590
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met 595
600 605 Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val
Thr 610 615 620 Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly Val
Ala Trp Tyr 625 630 635 640 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile Tyr Ser Ala Ser 645 650 655 Tyr Arg Tyr Thr Gly Val Pro Ser
Arg Phe Ser Gly Ser Gly Ser Gly 660 665 670 Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 675 680 685 Thr Tyr Tyr Cys
Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly Cys 690 695 700 Gly Thr
Lys Val Glu Ile Lys 705 710 125471PRTArtificial sequenceLight chain
[scFv Trastuzumab + Omnitarg, RB34] 125Asp 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 Val Asn Thr Ala 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 Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Arg 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 His Tyr Thr Thr
Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val 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 Val Glu 115 120 125 Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly Ser Leu Arg Leu Ser Cys 130 135 140 Ala Ala Ser Gly Phe Asn
Ile Lys Asp Thr Tyr Ile His Trp Val Arg 145 150 155 160 Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr 165 170 175 Asn
Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185
190 Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu
195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly
Gly Asp 210 215 220 Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val 225 230 235 240 Ser Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly 245 250 255 Ser Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val 260 265 270 Gly Asp Arg Val Thr
Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile 275 280 285 Gly Val Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 290 295 300 Ile
Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser 305 310
315 320 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln 325 330 335 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr
Ile Tyr Pro 340 345 350 Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala 355 360 365 Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser 370 375 380 Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu 385 390 395 400 Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 405 410 415 Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 420 425 430
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 435
440 445 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys 450 455 460 Ser Phe Asn Arg Gly Glu Cys 465 470
126449PRTArtificial sequenceHeavy chain (Omnitarg, RB33) 126Glu 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 Phe Thr Phe Thr Asp Tyr 20
25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn
Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg 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 Arg Asn Leu Gly Pro Ser
Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150
155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275
280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395
400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly 435 440 445 Lys 127470PRTArtificial sequenceLight
chain [Trastuzumab + scFvOmnitarg, RB35] 127Asp 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 Val Asn Thr Ala 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 Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Arg 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 His Tyr Thr
Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val 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 Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser 210 215 220 Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu 225 230 235 240 Ser Ala Ser Val Gly Asp Arg Val
Thr Ile Thr Cys Lys Ala Ser Gln 245 250 255 Asp Val Ser Ile Gly Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala 260 265 270 Pro Lys Leu Leu
Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro 275 280 285 Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 290 295 300
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr 305
310 315 320 Tyr Ile Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 325 330 335 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Glu 340 345 350 Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly Ser 355 360 365 Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Thr Asp Tyr Thr 370 375 380 Met Asp Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 385 390 395 400 Asp Val Asn
Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe Lys 405 410 415 Gly
Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr Leu 420 425
430 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
435 440 445 Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln
Gly Thr 450 455 460 Leu Val Thr Val Ser Ser 465 470
128450PRTArtificial sequenceHeavy chain [Trastuzumab, 1036] 128Glu
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 Phe Asn Ile Lys Asp Thr
20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr
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 Ser Arg Trp Gly Gly Asp
Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145
150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265
270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390
395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 129214PRTArtificial
sequenceLight chain (kappa) [Trastuzumab, 1016] 129Asp 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 Val Asn Thr Ala 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 Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Arg 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 His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly
Gln Gly Thr Lys Val 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 130711PRTArtificial sequenceHeavy chain [Trastuzumab +
scFvOmnitarg, RB43] 130Glu 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 Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro
Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Ala Asp Thr 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 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215
220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340
345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455 460
Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 465
470 475 480 Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val
Ser Ile 485 490 495 Gly Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu 500 505 510 Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly
Val Pro Ser Arg Phe Ser 515 520 525 Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln 530 535 540 Pro Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro 545 550 555 560 Tyr Thr Phe
Gly Cys Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly 565 570 575 Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 580 585
590 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
595 600 605 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr
Asp Tyr 610 615 620 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Cys
Leu Glu Trp Val 625 630 635 640 Ala Asp Val Asn Pro Asn Ser Gly Gly
Ser Ile Tyr Asn Gln Arg Phe 645 650 655 Lys Gly Arg Phe Thr Leu Ser
Val Asp Arg Ser Lys Asn Thr Leu Tyr 660 665 670 Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 675 680 685 Ala Arg Asn
Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 690 695 700 Thr
Leu Val Thr Val Ser Ser 705 710 131480PRTArtificial sequenceLight
chain [Trastuzumab + scFv Omnitarg, RB61] 131Asp 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 Val Asn Thr Ala 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 Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Arg 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 His Tyr
Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val 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 Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser 210 215 220 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Asp Ile Gln Met Thr Gln 225 230 235 240 Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly Asp Arg Val Thr Ile Thr 245 250 255 Cys Lys Ala Ser Gln
Asp Val Ser Ile Gly Val Ala Trp Tyr Gln Gln 260 265 270 Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg 275 280 285 Tyr
Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 290 295
300 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
305 310 315 320 Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly
Cys Gly Thr 325 330 335 Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly 340 345 350 Gly Gly Gly Ser Gly Gly Gly Gly Ser
Glu Val Gln Leu Val Glu Ser 355 360 365 Gly Gly Gly Leu Val Gln Pro
Gly Gly Ser Leu Arg Leu Ser Cys Ala 370 375 380 Ala Ser Gly Phe Thr
Phe Thr Asp Tyr Thr Met Asp Trp Val Arg Gln 385 390 395 400 Ala Pro
Gly Lys Cys Leu Glu Trp Val Ala Asp Val Asn Pro Asn Ser 405 410 415
Gly Gly Ser Ile Tyr Asn Gln Arg Phe Lys Gly Arg Phe Thr Leu Ser 420
425 430 Val Asp Arg Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
Arg 435 440 445 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asn Leu
Gly Pro Ser 450 455 460 Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 465 470 475 480 132450PRTArtificial
sequenceHeavy chain [Trastuzumab, 1036] 132Glu 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 Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr 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 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met
Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180
185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305
310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425
430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445 Gly Lys 450 133696PRTArtificial sequencescFab
Trastuzumab heavy chain 1 133Asp 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 Val Asn Val Ala 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 Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Arg 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 His Tyr Thr Thr Pro
Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser 210 215 220 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly 225 230 235 240 Gly Gly Gly Ser Gly Gly Glu Val Gln Leu
Val Glu Ser Gly Gly Gly 245 250 255 Leu Val Gln Pro Gly Gly Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly 260 265 270 Phe Asn Ile Lys Asp Thr
Tyr Ile His Trp Val Arg Gln Ala Pro Gly 275 280 285 Lys Gly Leu Glu
Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr 290 295 300 Arg Tyr
Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr 305 310 315
320 Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
325 330 335 Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Glu Gly Phe
Tyr Ala 340 345 350 Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser 355 360 365 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr 370 375 380 Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro 385 390 395 400 Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 405 410 415 His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 420 425 430 Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 435 440
445 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
450 455 460 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala 465 470 475 480 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro 485 490 495 Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 500 505 510
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 515
520 525 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln 530 535 540 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln 545 550 555 560 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala 565 570 575 Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro 580 585 590 Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr 595 600 605 Lys Asn Gln Val
Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser 610 615 620 Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 625 630 635
640 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
645 650 655 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe 660 665 670 Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys 675 680 685 Ser Leu Ser Leu Ser Pro Gly Lys 690 695
134449PRTArtificial sequencePertuzumab heavy chain 2 134Glu 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 Phe Thr Phe Thr Asp Tyr 20 25
30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln
Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg 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 Arg Asn Leu Gly Pro Ser Phe
Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155
160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280
285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Cys Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Ser 355 360 365 Cys Ala Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys 405
410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly 435 440 445 Lys 135214PRTArtificial sequencePertuzumab
light chain 1 135Asp 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
Gln Asp Val Ser Ile Gly 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
Tyr Thr 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 Tyr Ile Tyr Pro Tyr 85 90 95
Thr Phe Gly Gln Gly Thr Lys Val 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 136449PRTArtificial sequencePertuzumab heavy
chain 1 136Glu 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 Phe Thr
Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly
Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu
Ser Val Asp Arg 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 Arg
Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115
120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235
240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr 340 345 350 Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser 355 360
365 Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys
Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys
137214PRTArtificial sequencePertuzumab light chain 1 137Asp 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 Gln Asp Val Ser Ile Gly 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 Tyr Thr 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 Tyr Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
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
138696PRTArtificial sequencescFab Trastuzumab heavy chain 2 138Glu
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 Phe Asn Ile Lys Asp Thr
20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr
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 Ser Arg Trp Gly Gly Glu
Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145
150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265
270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Cys
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Trp Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390
395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro 435 440 445 Gly Lys Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly 450 455 460 Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser 465 470 475 480 Gly Gly Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 485 490 495 Val Gly
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn 500 505 510
Val Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 515
520 525 Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg
Phe 530 535 540 Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu 545 550 555 560 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln His Tyr Thr Thr 565 570 575 Pro Pro Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg Thr Val 580 585 590 Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 595 600 605 Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 610 615 620 Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 625 630 635
640 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
645 650 655 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys 660 665 670 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr 675 680 685 Lys Ser Phe Asn Arg Gly Glu Cys 690 695
139695PRTArtificial sequencescFab Pertuzumab heavy chain 1 139Glu
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 Phe Thr Phe Thr Asp Tyr
20 25 30 Thr Met
Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50
55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg 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 Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp
Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180
185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305
310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Cys Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser Leu Ser 355 360 365 Cys Ala Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp
Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425
430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser 450 455 460 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly 465 470 475 480 Gly Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val 485 490 495 Gly Asp Arg Val Thr Ile Thr
Cys Lys Ala Ser Gln Asp Val Ser Ile 500 505 510 Gly Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 515 520 525 Ile Tyr Ser
Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser 530 535 540 Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 545 550
555 560 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr
Pro 565 570 575 Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
Thr Val Ala 580 585 590 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser 595 600 605 Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu 610 615 620 Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser 625 630 635 640 Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 645 650 655 Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 660 665 670
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 675
680 685 Ser Phe Asn Arg Gly Glu Cys 690 695 140450PRTArtificial
sequenceTrastuzumab heavy chain 2 140Glu 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 Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr 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 Ser Arg Trp Gly Gly Glu Gly Phe Tyr Ala Met Asp
Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185
190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310
315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr 340 345 350 Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser Leu 355 360 365 Trp Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435
440 445 Gly Lys 450 141213PRTArtificial sequenceTrastuzumab light
chain 2 141Asp 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
Val Asn Val Ala 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 Phe Leu Tyr Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg 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 His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val 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 210 142450PRTArtificial sequenceTrastuzumab heavy chain 1
142Glu 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 Phe Asn Ile Lys
Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr
Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala
Asp Thr 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 Ser Arg Trp Gly
Gly Glu Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130
135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250
255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro
Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Trp
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375
380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450
143214PRTArtificial sequenceTrastuzumab light chain 1 143Asp 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 Val Asn Val Ala 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 Phe Leu Tyr Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Arg 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 His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys
Val 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
144695PRTArtificial sequencescFab Pertuzumab heavy chain 2 144Glu
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 Phe Thr Phe Thr Asp Tyr
20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr
Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg
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 Arg Asn Leu Gly Pro
Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145
150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265
270 Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr 340
345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Ser 355 360 365 Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu
Val Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 450 455 460
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 465
470 475 480 Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val 485 490 495 Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln
Asp Val Ser Ile 500 505 510 Gly Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu 515 520 525 Ile Tyr Ser Ala Ser Tyr Arg Tyr
Thr Gly Val Pro Ser Arg Phe Ser 530 535 540 Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 545 550 555 560 Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro 565 570 575 Tyr
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 580 585
590 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
595 600 605 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu 610 615 620 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser Gly Asn Ser 625 630 635 640 Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr Tyr Ser Leu 645 650 655 Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys His Lys Val 660 665 670 Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 675 680 685 Ser Phe Asn
Arg Gly Glu Cys 690 695 145701PRTArtificial sequenceHeavy chain
with scFv Trastuzumab stabilized with disulphide bonding 145Glu 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 Phe Thr Phe Thr Asp Tyr 20
25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn
Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg 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 Arg Asn Leu Gly Pro Ser
Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150
155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275
280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395
400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly 435 440 445 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Glu Val Gln Leu Val 450 455 460 Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly Ser Leu Arg Leu Ser 465 470 475 480 Cys Ala Ala Ser Gly
Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val 485 490 495 Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro 500 505 510 Thr
Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 515 520
525 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser
530 535 540 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp
Gly Gly 545 550 555 560 Glu Gly Phe Tyr Ala Met Asp Tyr Trp Gly Cys
Gly Thr Leu Val Thr 565 570 575 Val Ser Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly 580 585 590 Gly Ser Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser 595 600 605 Val Gly Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn 610 615 620 Val Ala Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Cys Pro Lys Leu 625 630 635 640
Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe 645
650 655 Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu 660 665 670 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
Tyr Thr Thr 675 680 685 Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 690 695 700 146214PRTArtificial sequencePertuzumab light
chain 146Asp 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 Gln Asp
Val Ser Ile Gly 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 Tyr Thr
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 Tyr Ile Tyr Pro Tyr 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val 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 14726DNAArtificial sequenceLMB3 147caggaaacag
ctatgaccat gattac 2614857DNAArtificial
sequenceAM_omni_H1_TN-bamisc_feature(26)..(26)T=60%, S/G/R/N/D=20%
(4% each), rest=20% (1.7% each)misc_feature(27)..(27)D=60%,
S/N/T/A/R/E/Q/G=30% (3.8% each), rest=10% (1.1%
each)misc_feature(28)..(28)Y=60%, F/S/H/N/D/T=30% (5.0% each),
rest=10% (0.9%)misc_feature(29)..(29)T=60%, A/G/V/S/P/D/N=30% (4.3%
each), rest=10% (1.0%) 148ccggtgcctg acgaacccaa tccatnnnna
aaggtaaaac cgcttgctgc acagctc 5714932DNAArtificial sequenceRJH108
(omni_3'H1_fo) 149atggattggg ttcgtcaggc accgggtaaa gg
3215033DNAArtificial sequenceRJH109 (omni_5'H3_re) 150attacgtgca
caataataca ctgcggtatc ctc 3315166DNAArtificial
sequenceAM_omni_H3_TN_fomisc_feature(26)..(26)N=60%,
G/D/E/Q/V/S/A/P/R/L/T/Y=40% (3.3% each)misc_feature(27)..(27)L=60%,
G/Y/S/A/D/T/R/P/V/N/W/F/I/E=40% (2.9%
each)misc_feature(28)..(28)G=60%, Y/S/A/D/T/R/P/L/V/N/W/F/I/E=40%
(2.9% each)misc_feature(29)..(29)P=60%,
G/Y/S/A/D/T/R/L/V/N/W/F/I/E=40% (2.9%
each)misc_feature(30)..(30)S=60%, G/Y/P/A/D/T/R/L/V/N/W/F/I/E=40%
(2.9% each)misc_feature(34)..(34)Y=60%, G/A/P/W/S/D/T/F/R/K/H=40%
(3.6% each) 151taccgcagtg tattattgtg cacgtnnnnn ttcntttgat
tattggggtc agggcaccct 60ggttac 6615228DNAArtificial sequenceRJH99
152ggctgagact cctcaagaga aggattag 2815332DNAArtificial
sequenceRJH110(omni_5'H2_ba) 153attaacatct gcaacccatt ccagaccttt ac
3215469DNAArtificial
sequenceAM_omni_h2_TN_fomisc_feature(28)..(28)P=60%,
G/A/S/T/D/N/F/Y=30% (3.8% each), rest=10% (1.0%
each)misc_feature(29)..(29)N=60%, S/D/G/T/R/A=30% (5.0% each),
rest=10% (0.8%)misc_feature(30)..(30)S=60%, G=10%, rest=30% (1.8%
each)misc_feature(31)..(31)misc_feature(31)..(31)n is a, c, g, or
tmisc_feature(35)..(35)misc_feature(35)..(35)n is a, c, g, or
tmisc_feature(39)..(39)misc_feature(39)..(39)misc_feature(39)..(39)n
is a, c, g, or tmisc_feature(43)..(43)n is a, c, g, or t
154ggtctggaat gggttgcaga tgttaatnnn nggtnattna acncgtttta
aaggtcgttt 60taccctgag 6915519PRTArtificial sequenceLeader Peptide
1 155Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr
Gly 1 5 10 15 Ala His Ser 15622PRTArtificial sequenceLeader Peptide
2 156Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu
Trp 1 5 10 15 Phe Pro Gly Ala Arg Cys 20 15719PRTArtificial
sequenceLeader Peptide 3 157Met Gly Trp Ser Cys Ile Ile Leu Phe Leu
Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser 15811PRTArtificial
sequenceTrastuzumab VL N30S CDR1 158Arg Ala Ser Gln Asp Val Ser Thr
Ala Val Ala 1 5 10 159450PRTArtificial sequenceTrastuzumab VHCH1-
Fc KNOB 159Glu 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 Phe Asn
Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly
Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Ala Asp Thr 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 Ser Arg
Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115
120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235
240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr
Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360
365 Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450
1601350PRTArtificial sequenceTrastuzumab VHCH1- Fc KNOB DNA 160Gly
Ala Ala Gly Thr Gly Cys Ala Ala Thr Thr Gly Gly Thr Gly Gly 1 5 10
15 Ala Ala Ala Gly Cys Gly Gly Cys Gly Gly Cys Gly Gly Cys Cys Thr
20 25 30 Gly Gly Thr Gly Cys Ala Ala Cys Cys Gly Gly Gly Cys Gly
Gly Cys 35 40 45 Ala Gly Cys Cys Thr Gly Cys Gly Thr Cys Thr Gly
Ala Gly Cys Thr 50
55 60 Gly Cys Gly Cys Gly Gly Cys Cys Thr Cys Cys Gly Gly Ala Thr
Thr 65 70 75 80 Thr Ala Ala Cys Ala Thr Ala Ala Ala Gly Gly Ala Cys
Ala Cys Ala 85 90 95 Thr Ala Cys Ala Thr Cys Cys Ala Cys Thr Gly
Gly Gly Thr Gly Cys 100 105 110 Gly Cys Cys Ala Ala Gly Cys Ala Cys
Cys Thr Gly Gly Gly Ala Ala 115 120 125 Gly Gly Gly Thr Cys Thr Cys
Gly Ala Gly Thr Gly Gly Gly Thr Gly 130 135 140 Gly Cys Thr Cys Gly
Gly Ala Thr Thr Thr Ala Cys Cys Cys Ala Ala 145 150 155 160 Cys Ala
Ala Ala Thr Gly Gly Cys Thr Ala Cys Ala Cys Cys Ala Gly 165 170 175
Gly Thr Ala Thr Gly Cys Gly Gly Ala Thr Ala Gly Cys Gly Thr Gly 180
185 190 Ala Ala Ala Gly Gly Cys Cys Gly Thr Thr Thr Thr Ala Cys Cys
Ala 195 200 205 Thr Thr Thr Cys Ala Gly Cys Thr Gly Ala Thr Ala Cys
Thr Thr Cys 210 215 220 Gly Ala Ala Gly Ala Ala Cys Ala Cys Cys Gly
Cys Cys Thr Ala Thr 225 230 235 240 Cys Thr Gly Cys Ala Ala Ala Thr
Gly Ala Ala Cys Ala Gly Cys Cys 245 250 255 Thr Gly Cys Gly Thr Gly
Cys Gly Gly Ala Ala Gly Ala Thr Ala Cys 260 265 270 Gly Gly Cys Cys
Gly Thr Gly Thr Ala Thr Thr Ala Thr Thr Gly Cys 275 280 285 Thr Cys
Gly Cys Gly Thr Thr Gly Gly Gly Gly Ala Gly Gly Ala Gly 290 295 300
Ala Cys Gly Gly Gly Thr Thr Cys Thr Ala Thr Gly Cys Thr Ala Thr 305
310 315 320 Gly Gly Ala Thr Thr Ala Cys Thr Gly Gly Gly Gly Cys Cys
Ala Ala 325 330 335 Gly Gly Cys Ala Cys Cys Cys Thr Gly Gly Thr Gly
Ala Cys Gly Gly 340 345 350 Thr Thr Ala Gly Cys Thr Cys Ala Gly Cys
Thr Ala Gly Cys Ala Cys 355 360 365 Cys Ala Ala Gly Gly Gly Cys Cys
Cys Ala Thr Cys Gly Gly Thr Cys 370 375 380 Thr Thr Cys Cys Cys Cys
Cys Thr Gly Gly Cys Ala Cys Cys Cys Thr 385 390 395 400 Cys Cys Thr
Cys Cys Ala Ala Gly Ala Gly Cys Ala Cys Cys Thr Cys 405 410 415 Thr
Gly Gly Gly Gly Gly Cys Ala Cys Ala Gly Cys Gly Gly Cys Cys 420 425
430 Cys Thr Gly Gly Gly Cys Thr Gly Cys Cys Thr Gly Gly Thr Cys Ala
435 440 445 Ala Gly Gly Ala Cys Thr Ala Cys Thr Thr Cys Cys Cys Cys
Gly Ala 450 455 460 Ala Cys Cys Gly Gly Thr Gly Ala Cys Gly Gly Thr
Gly Thr Cys Gly 465 470 475 480 Thr Gly Gly Ala Ala Cys Thr Cys Ala
Gly Gly Cys Gly Cys Cys Cys 485 490 495 Thr Gly Ala Cys Cys Ala Gly
Cys Gly Gly Cys Gly Thr Gly Cys Ala 500 505 510 Cys Ala Cys Cys Thr
Thr Cys Cys Cys Gly Gly Cys Thr Gly Thr Cys 515 520 525 Cys Thr Ala
Cys Ala Gly Thr Cys Cys Thr Cys Ala Gly Gly Ala Cys 530 535 540 Thr
Cys Thr Ala Cys Thr Cys Cys Cys Thr Cys Ala Gly Cys Ala Gly 545 550
555 560 Cys Gly Thr Gly Gly Thr Gly Ala Cys Cys Gly Thr Gly Cys Cys
Cys 565 570 575 Thr Cys Cys Ala Gly Cys Ala Gly Cys Thr Thr Gly Gly
Gly Cys Ala 580 585 590 Cys Cys Cys Ala Gly Ala Cys Cys Thr Ala Cys
Ala Thr Cys Thr Gly 595 600 605 Cys Ala Ala Cys Gly Thr Gly Ala Ala
Thr Cys Ala Cys Ala Ala Gly 610 615 620 Cys Cys Cys Ala Gly Cys Ala
Ala Cys Ala Cys Cys Ala Ala Gly Gly 625 630 635 640 Thr Gly Gly Ala
Cys Ala Ala Gly Ala Ala Ala Gly Thr Thr Gly Ala 645 650 655 Gly Cys
Cys Cys Ala Ala Ala Thr Cys Thr Thr Gly Thr Gly Ala Cys 660 665 670
Ala Ala Ala Ala Cys Thr Cys Ala Cys Ala Cys Ala Thr Gly Cys Cys 675
680 685 Cys Ala Cys Cys Gly Thr Gly Cys Cys Cys Ala Gly Cys Ala Cys
Cys 690 695 700 Thr Gly Ala Ala Cys Thr Cys Cys Thr Gly Gly Gly Gly
Gly Gly Ala 705 710 715 720 Cys Cys Gly Thr Cys Ala Gly Thr Cys Thr
Thr Cys Cys Thr Cys Thr 725 730 735 Thr Cys Cys Cys Cys Cys Cys Ala
Ala Ala Ala Cys Cys Cys Ala Ala 740 745 750 Gly Gly Ala Cys Ala Cys
Cys Cys Thr Cys Ala Thr Gly Ala Thr Cys 755 760 765 Thr Cys Cys Cys
Gly Gly Ala Cys Cys Cys Cys Thr Gly Ala Gly Gly 770 775 780 Thr Cys
Ala Cys Ala Thr Gly Cys Gly Thr Gly Gly Thr Gly Gly Thr 785 790 795
800 Gly Gly Ala Cys Gly Thr Gly Ala Gly Cys Cys Ala Cys Gly Ala Ala
805 810 815 Gly Ala Cys Cys Cys Thr Gly Ala Gly Gly Thr Cys Ala Ala
Gly Thr 820 825 830 Thr Cys Ala Ala Cys Thr Gly Gly Thr Ala Cys Gly
Thr Gly Gly Ala 835 840 845 Cys Gly Gly Cys Gly Thr Gly Gly Ala Gly
Gly Thr Gly Cys Ala Thr 850 855 860 Ala Ala Thr Gly Cys Cys Ala Ala
Gly Ala Cys Ala Ala Ala Gly Cys 865 870 875 880 Cys Gly Cys Gly Gly
Gly Ala Gly Gly Ala Gly Cys Ala Gly Thr Ala 885 890 895 Cys Ala Ala
Cys Ala Gly Cys Ala Cys Gly Thr Ala Cys Cys Gly Thr 900 905 910 Gly
Thr Gly Gly Thr Cys Ala Gly Cys Gly Thr Cys Cys Thr Cys Ala 915 920
925 Cys Cys Gly Thr Cys Cys Thr Gly Cys Ala Cys Cys Ala Gly Gly Ala
930 935 940 Cys Thr Gly Gly Cys Thr Gly Ala Ala Thr Gly Gly Cys Ala
Ala Gly 945 950 955 960 Gly Ala Gly Thr Ala Cys Ala Ala Gly Thr Gly
Cys Ala Ala Gly Gly 965 970 975 Thr Cys Thr Cys Cys Ala Ala Cys Ala
Ala Ala Gly Cys Cys Cys Thr 980 985 990 Cys Cys Cys Ala Gly Cys Cys
Cys Cys Cys Ala Thr Cys Gly Ala Gly 995 1000 1005 Ala Ala Ala Ala
Cys Cys Ala Thr Cys Thr Cys Cys Ala Ala Ala 1010 1015 1020 Gly Cys
Cys Ala Ala Ala Gly Gly Gly Cys Ala Gly Cys Cys Cys 1025 1030 1035
Cys Gly Ala Gly Ala Ala Cys Cys Ala Cys Ala Gly Gly Thr Gly 1040
1045 1050 Thr Ala Cys Ala Cys Cys Cys Thr Gly Cys Cys Cys Cys Cys
Ala 1055 1060 1065 Thr Gly Cys Cys Gly Gly Gly Ala Thr Gly Ala Gly
Cys Thr Gly 1070 1075 1080 Ala Cys Cys Ala Ala Gly Ala Ala Cys Cys
Ala Gly Gly Thr Cys 1085 1090 1095 Ala Gly Cys Cys Thr Gly Thr Gly
Gly Thr Gly Cys Cys Thr Gly 1100 1105 1110 Gly Thr Cys Ala Ala Ala
Gly Gly Cys Thr Thr Cys Thr Ala Thr 1115 1120 1125 Cys Cys Cys Ala
Gly Cys Gly Ala Cys Ala Thr Cys Gly Cys Cys 1130 1135 1140 Gly Thr
Gly Gly Ala Gly Thr Gly Gly Gly Ala Gly Ala Gly Cys 1145 1150 1155
Ala Ala Thr Gly Gly Gly Cys Ala Gly Cys Cys Gly Gly Ala Gly 1160
1165 1170 Ala Ala Cys Ala Ala Cys Thr Ala Cys Ala Ala Gly Ala Cys
Cys 1175 1180 1185 Ala Cys Gly Cys Cys Thr Cys Cys Cys Gly Thr Gly
Cys Thr Gly 1190 1195 1200 Gly Ala Cys Thr Cys Cys Gly Ala Cys Gly
Gly Cys Thr Cys Cys 1205 1210 1215 Thr Thr Cys Thr Thr Cys Cys Thr
Cys Thr Ala Cys Ala Gly Cys 1220 1225 1230 Ala Ala Gly Cys Thr Cys
Ala Cys Cys Gly Thr Gly Gly Ala Cys 1235 1240 1245 Ala Ala Gly Ala
Gly Cys Ala Gly Gly Thr Gly Gly Cys Ala Gly 1250 1255 1260 Cys Ala
Gly Gly Gly Gly Ala Ala Cys Gly Thr Cys Thr Thr Cys 1265 1270 1275
Thr Cys Ala Thr Gly Cys Thr Cys Cys Gly Thr Gly Ala Thr Gly 1280
1285 1290 Cys Ala Thr Gly Ala Gly Gly Cys Thr Cys Thr Gly Cys Ala
Cys 1295 1300 1305 Ala Ala Cys Cys Ala Cys Thr Ala Cys Ala Cys Gly
Cys Ala Gly 1310 1315 1320 Ala Ala Gly Ala Gly Cys Cys Thr Cys Thr
Cys Cys Cys Thr Gly 1325 1330 1335 Thr Cys Thr Cys Cys Gly Gly Gly
Thr Ala Ala Ala 1340 1345 1350 161214PRTArtificial sequenceCommon
light chain VLCL 161Asp 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
Gln Asp Val Ser Thr Ala 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 Phe Arg
Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg 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 His Tyr Thr Thr Pro Pro 85 90 95
Thr Phe Gly Gln Gly Thr Lys Val 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 162642PRTArtificial sequenceCommon light chain
VLCL - DNA 162Gly Ala Cys Ala Thr Cys Cys Ala Gly Ala Thr Gly Ala
Cys Cys Cys 1 5 10 15 Ala Gly Ala Gly Cys Cys Cys Cys Ala Gly Cys
Ala Gly Cys Cys Thr 20 25 30 Gly Thr Cys Thr Gly Cys Cys Ala Gly
Cys Gly Thr Gly Gly Gly Cys 35 40 45 Gly Ala Cys Ala Gly Ala Gly
Thr Gly Ala Cys Cys Ala Thr Cys Ala 50 55 60 Cys Ala Thr Gly Cys
Ala Ala Gly Gly Cys Cys Ala Gly Cys Cys Ala 65 70 75 80 Gly Gly Ala
Cys Gly Thr Gly Thr Cys Cys Ala Cys Ala Gly Cys Cys 85 90 95 Gly
Thr Gly Gly Cys Cys Thr Gly Gly Thr Ala Thr Cys Ala Gly Cys 100 105
110 Ala Gly Ala Ala Gly Cys Cys Thr Gly Gly Cys Ala Ala Gly Gly Cys
115 120 125 Cys Cys Cys Cys Ala Ala Gly Cys Thr Gly Cys Thr Gly Ala
Thr Cys 130 135 140 Thr Ala Cys Ala Gly Cys Gly Cys Cys Ala Gly Cys
Thr Thr Cys Cys 145 150 155 160 Gly Gly Thr Ala Cys Ala Cys Cys Gly
Gly Cys Gly Thr Gly Cys Cys 165 170 175 Cys Ala Gly Cys Ala Gly Ala
Thr Thr Cys Ala Gly Cys Gly Gly Cys 180 185 190 Ala Gly Cys Ala Gly
Ala Thr Cys Cys Gly Gly Cys Ala Cys Cys Gly 195 200 205 Ala Cys Thr
Thr Cys Ala Cys Cys Cys Thr Gly Ala Cys Cys Ala Thr 210 215 220 Cys
Ala Gly Cys Thr Cys Cys Cys Thr Gly Cys Ala Gly Cys Cys Cys 225 230
235 240 Gly Ala Gly Gly Ala Cys Thr Thr Cys Gly Cys Cys Ala Cys Cys
Thr 245 250 255 Ala Cys Thr Ala Cys Thr Gly Cys Cys Ala Gly Cys Ala
Gly Cys Ala 260 265 270 Cys Thr Ala Cys Ala Cys Cys Ala Cys Cys Cys
Cys Cys Cys Cys Cys 275 280 285 Ala Cys Ala Thr Thr Thr Gly Gly Cys
Cys Ala Gly Gly Gly Cys Ala 290 295 300 Cys Cys Ala Ala Gly Gly Thr
Gly Gly Ala Ala Ala Thr Cys Ala Ala 305 310 315 320 Gly Cys Gly Thr
Ala Cys Gly Gly Thr Gly Gly Cys Thr Gly Cys Ala 325 330 335 Cys Cys
Ala Thr Cys Thr Gly Thr Cys Thr Thr Cys Ala Thr Cys Thr 340 345 350
Thr Cys Cys Cys Gly Cys Cys Ala Thr Cys Thr Gly Ala Thr Gly Ala 355
360 365 Gly Cys Ala Gly Thr Thr Gly Ala Ala Ala Thr Cys Thr Gly Gly
Ala 370 375 380 Ala Cys Thr Gly Cys Cys Thr Cys Thr Gly Thr Thr Gly
Thr Gly Thr 385 390 395 400 Gly Cys Cys Thr Gly Cys Thr Gly Ala Ala
Thr Ala Ala Cys Thr Thr 405 410 415 Cys Thr Ala Thr Cys Cys Cys Ala
Gly Ala Gly Ala Gly Gly Cys Cys 420 425 430 Ala Ala Ala Gly Thr Ala
Cys Ala Gly Thr Gly Gly Ala Ala Gly Gly 435 440 445 Thr Gly Gly Ala
Thr Ala Ala Cys Gly Cys Cys Cys Thr Cys Cys Ala 450 455 460 Ala Thr
Cys Gly Gly Gly Thr Ala Ala Cys Thr Cys Cys Cys Ala Gly 465 470 475
480 Gly Ala Gly Ala Gly Thr Gly Thr Cys Ala Cys Ala Gly Ala Gly Cys
485 490 495 Ala Gly Gly Ala Cys Ala Gly Cys Ala Ala Gly Gly Ala Cys
Ala Gly 500 505 510 Cys Ala Cys Cys Thr Ala Cys Ala Gly Cys Cys Thr
Cys Ala Gly Cys 515 520 525 Ala Gly Cys Ala Cys Cys Cys Thr Gly Ala
Cys Gly Cys Thr Gly Ala 530 535 540 Gly Cys Ala Ala Ala Gly Cys Ala
Gly Ala Cys Thr Ala Cys Gly Ala 545 550 555 560 Gly Ala Ala Ala Cys
Ala Cys Ala Ala Ala Gly Thr Cys Thr Ala Cys 565 570 575 Gly Cys Cys
Thr Gly Cys Gly Ala Ala Gly Thr Cys Ala Cys Cys Cys 580 585 590 Ala
Thr Cys Ala Gly Gly Gly Cys Cys Thr Gly Ala Gly Cys Thr Cys 595 600
605 Gly Cys Cys Cys Gly Thr Cys Ala Cys Ala Ala Ala Gly Ala Gly Cys
610 615 620 Thr Thr Cys Ala Ala Cys Ala Gly Gly Gly Gly Ala Gly Ala
Gly Thr 625 630 635 640 Gly Thr 163449PRTArtificial
sequencePertuzumab VHCH1 Fc hole 163Glu 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 Phe Thr Phe Asn Asp Tyr 20 25 30 Thr Met Asp Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp
Val Asn Pro Asn Ser Gly Gly Ser Ile Val Asn Arg Arg Phe 50 55 60
Lys Gly Arg Phe Thr Leu Ser Val Asp Arg 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 Arg Asn Leu Gly Pro Phe Phe Tyr Phe Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val
Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225
230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr 340 345
350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser
355 360 365 Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Val
Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys
1641347DNAArtificial sequencePertuzumab VHCH1 Fc hole DNA
164gaagttcagc tggttgaaag cggtggtggt ctggttcagc ctggtggtag
cctgcgtctg 60agctgtgcag caagcggttt tacctttaac gattatacca tggattgggt
tcgtcaggca 120ccgggtaaag gtctggaatg ggttgcagat gttaatccga
atagcggtgg tagcattgtt 180aaccgtcgtt ttaaaggtcg ttttaccctg
agcgttgatc gtagcaaaaa taccctgtat 240ctgcaaatga atagtctgcg
tgcagaggat accgcagtgt attattgtgc acgtaacctg 300ggtccgttct
tctactttga ttattggggt cagggcaccc tggttaccgt tagcagcgct
360agcaccaagg gcccaagcgt gttccctctg gcccccagca gcaagagcac
aagcggcgga 420acagccgccc tgggctgcct ggtcaaggac tacttccccg
agcccgtgac agtgtcctgg 480aacagcggag ccctgaccag cggcgtgcac
acctttccag ccgtgctgca gagcagcggc 540ctgtacagcc tgagcagcgt
ggtcacagtg cctagcagca gcctgggcac ccagacctac 600atctgcaacg
tgaaccacaa gcccagcaac accaaggtgg acaagaaggt ggagcccaag
660agctgcgaca agacccacac ctgtccccct tgtcctgccc ctgagctgct
gggcggaccc 720agcgtgttcc tgttcccccc aaagcccaag gacaccctga
tgatcagccg gacccccgaa 780gtgacctgcg tggtggtgga cgtgtcccac
gaggaccctg aagtgaagtt caattggtac 840gtggacggcg tggaggtgca
caatgccaag accaagcccc gggaggaaca gtacaacagc 900acctaccggg
tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaagag
960tacaagtgca aggtctccaa caaggccctg cctgccccca tcgagaaaac
catcagcaag 1020gccaagggcc agcccagaga accccaggtg tgcaccctgc
cccccagcag agatgagctg 1080accaagaacc aggtgtccct gagctgtgcc
gtcaagggct tctaccccag cgatatcgcc 1140gtggagtggg agagcaacgg
ccagcctgag aacaactaca agaccacccc ccctgtgctg 1200gacagcgacg
gcagcttctt cctggtgtcc aaactgaccg tggacaagag ccggtggcag
1260cagggcaacg tgttcagctg cagcgtgatg cacgaggccc tgcacaacca
ctacacccag 1320aagtccctga gcctgagccc cggcaag
1347165934PRTArtificial sequenceHer-knob-scFab(8D3) Heavy chain
(21832) 165Glu 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 Phe Asn
Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly
Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Ala Asp Thr 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 Ser Arg
Trp Gly Gly Glu Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115
120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235
240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr
Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360
365 Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 450 455 460 Gly Gly Ser
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala 465 470 475 480
Ser Leu Glu Glu Ile Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile 485
490 495 Gly Asn Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro
Gln 500 505 510 Leu Leu Ile Tyr Gly Ala Thr Ser Leu Ala Asp Gly Val
Pro Ser Arg 515 520 525 Phe Ser Gly Ser Arg Ser Gly Thr Gln Phe Ser
Leu Lys Ile Ser Arg 530 535 540 Val Gln Val Glu Asp Ile Gly Ile Tyr
Tyr Cys Leu Gln Ala Tyr Asn 545 550 555 560 Thr Pro Trp Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 565 570 575 Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 580 585 590 Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 595 600 605
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 610
615 620 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr 625 630 635 640 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His 645 650 655 Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val 660 665 670 Thr Lys Ser Phe Asn Arg Gly Glu
Cys Gly Gly Gly Gly Ser Gly Gly 675 680 685 Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 690 695 700 Gly Ser Gly Gly
Gly Gly Ser Gly Gly Glu Val Gln Leu Val Glu Ser 705 710 715 720 Gly
Gly Gly Leu Val Gln Pro Gly Asn Ser Leu Thr Leu Ser Cys Val 725 730
735 Ala Ser Gly Phe Thr Phe Ser Asn Tyr Gly Met His Trp Ile Arg Gln
740 745 750 Ala Pro Lys Lys Gly Leu Glu Trp Ile Ala Met Ile Tyr Tyr
Asp Ser 755 760 765 Ser Lys Met Asn Tyr Ala Asp Thr Val Lys Gly Arg
Phe Thr Ile Ser 770 775 780 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
Glu Met Asn Ser Leu Arg 785 790 795 800 Ser Glu Asp Thr Ala Met Tyr
Tyr Cys Ala Val Pro Thr Ser His Tyr 805 810 815 Val Val Asp Val Trp
Gly Gln Gly Val Ser Val Thr Val Ser Ser Ala 820 825 830 Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 835 840 845 Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 850 855
860 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
865 870 875 880 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu 885 890 895 Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr 900 905 910 Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys 915 920 925 Val Glu Pro Lys Ser Cys 930
166119PRTArtificial sequence4/94 VH 166Glu Val Gln Leu Gln Gln Ser
Gly Ala Val Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser
Cys Pro Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His
Trp Val Ile Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly
Arg Ile Asp Pro Ala Asn Gly Asp Thr Lys Cys Asp Pro Lys Phe 50 55
60 Gln Val Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr
Phe Cys 85 90 95 Val Arg Asp Tyr Leu Tyr Pro Tyr Tyr Phe Asp Phe
Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115
167108PRTArtificial sequence4/94 VL 167Lys Ile Val Met Thr Gln Ser
Pro Lys Ser Met Ser Met Ser Val Gly 1 5 10 15 Glu Arg Val Thr Leu
Asn Cys Arg Ala Ser Glu Ser Val Asp Thr Tyr 20 25 30 Val Ser Trp
Tyr Gln Gln Lys Pro Glu Gln Ser Pro Glu Leu Leu Ile 35 40 45 Tyr
Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55
60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala
65 70 75 80 Glu Asp Leu Ala Asp Tyr Tyr Cys Gly Gln Thr Tyr Asn Tyr
Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg
100 105 168934PRTArtificial sequenceHer-knob-LALA-PG-scFab(8D3)
Heavy chain (21834) 168Glu 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 Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro
Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Ala Asp Thr 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 Ser Arg Trp Gly Gly Glu Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215
220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly
225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu 325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340
345 350 Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu 355 360 365 Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 450 455 460
Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala 465
470 475 480 Ser Leu Glu Glu Ile Val Thr Ile Thr Cys Gln Ala Ser Gln
Asp Ile 485 490 495 Gly Asn Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ser Pro Gln 500 505 510 Leu Leu Ile Tyr Gly Ala Thr Ser Leu Ala
Asp Gly Val Pro Ser Arg 515 520 525 Phe Ser Gly Ser Arg Ser Gly Thr
Gln Phe Ser Leu Lys Ile Ser Arg 530 535 540 Val Gln Val Glu Asp Ile
Gly Ile Tyr Tyr Cys Leu Gln Ala Tyr Asn 545 550 555 560 Thr Pro Trp
Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 565 570 575 Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 580 585 590
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 595
600 605 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly 610 615 620 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr 625 630 635 640 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His 645 650 655 Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val 660 665 670 Thr Lys Ser Phe Asn Arg
Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly 675 680 685 Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 690 695 700 Gly Ser
Gly Gly Gly Gly Ser Gly Gly Glu Val Gln Leu Val Glu Ser 705 710 715
720 Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu Thr Leu Ser Cys Val
725 730 735 Ala Ser Gly Phe Thr Phe Ser Asn Tyr Gly Met His Trp Ile
Arg Gln 740 745 750 Ala Pro Lys Lys Gly Leu Glu Trp Ile Ala Met Ile
Tyr Tyr Asp Ser 755 760 765 Ser Lys Met Asn Tyr Ala Asp Thr Val Lys
Gly Arg Phe Thr Ile Ser 770 775 780 Arg Asp Asn Ser Lys Asn Thr Leu
Tyr Leu Glu Met Asn Ser Leu Arg 785 790 795 800 Ser Glu Asp Thr Ala
Met Tyr Tyr Cys Ala Val Pro Thr Ser His Tyr 805 810 815 Val Val Asp
Val Trp Gly Gln Gly Val Ser Val Thr Val Ser Ser Ala 820 825 830 Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 835 840
845 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
850 855 860 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly 865 870 875 880 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu 885 890 895 Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr 900 905 910 Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys 915 920 925 Val Glu Pro Lys Ser
Cys 930 169449PRTArtificial sequencePer-hole LALA-PG Heavy chain
(21836) 169Glu 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 Phe Thr
Phe Asn Asp Tyr 20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly
Gly Ser Ile Val Asn Arg Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu
Ser Val Asp Arg 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 Arg
Asn Leu Gly Pro Phe Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115
120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 225 230 235
240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr 340 345 350 Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser 355 360
365 Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys
Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn Arg Phe Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys
170450PRTArtificial sequenceHer-knob-LALA-PG Heavy Chain (21835)
170Glu 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 Phe Asn Ile Lys
Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr
Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala
Asp Thr 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 Ser Arg Trp Gly
Gly Glu Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130
135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 225 230 235 240 Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250
255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Gly Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro
Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Trp
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375
380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450
171450PRTArtificial sequencepETR13573Her-knob heavy chain 171Glu
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 Phe Asn Ile Lys Asp Thr
20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr
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 Ser Arg Trp Gly Gly Glu
Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145
150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265
270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Cys
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Trp Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390
395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 1726PRTArtificial
sequence2/99 CDR-H1 172Phe Ser Leu Ser Ser Tyr 1 5
17314PRTArtificial sequence2/99 CDR-H2 173Tyr Ile Trp Ser Gly Gly
Ser Thr Asp Tyr Ala Ser Trp Ala 1 5 10 17417PRTArtificial
sequence2/99 CDR-H3 174Arg Tyr Gly Thr Ser Tyr Pro Asp Tyr Gly Asp
Ala Asn Gly Phe Asp 1 5 10 15 Pro 17511PRTArtificial sequence2/99
CDR-L1 175Gln Ala Ser Gln Ser Ile Ser Ser Tyr Leu Ser 1 5 10
1763PRTArtificial sequence2/99 CDR-L2 176Arg Ala Ser 1
1779PRTArtificial sequence2/99 CDR-L3 177Cys Tyr Ser Ser Ser Asn
Val Asp Asn 1 5 178122PRTArtificial sequence2/99 VH 178Gln Ser Met
Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu
Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Ala 20 25
30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly
35 40 45 Tyr Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Ser Trp Ala
Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp
Leu Lys Ile Thr 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr
Phe Cys Ala Arg Arg Tyr 85 90 95 Gly Thr Ser Tyr Pro Asp Tyr Gly
Asp Ala Asn Gly Phe Asp Pro Trp 100 105 110 Gly Pro Gly Thr Leu Val
Thr Val Ser Ser 115 120 179111PRTArtificial sequence2/99 VL 179Ala
Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10
15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Ser Tyr
20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu
Leu Ile 35 40 45 Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Ser Ser
Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr
Ile Ser Gly Val Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys
Gln Gln Cys Tyr Ser Ser Ser Asn 85 90 95 Val Asp Asn Thr Phe Gly
Gly Gly Thr Glu Val Val Val Lys Arg 100 105 110 1807PRTArtificial
sequence4/94 CDR-H1 180Gly Phe Asn Ile Lys Asp Thr 1 5
18116PRTArtificial sequence4/94 CDR-H2 181Arg Ile Asp Pro Ala Asn
Gly Asp Thr Lys Cys Asp Pro Lys Phe Gln 1 5 10 15 1828PRTArtificial
sequence4/94 CDR-H3 182Tyr Leu Tyr Pro Tyr Tyr Phe Asp 1 5
1837PRTArtificial sequence4/94 CDR-L1 183Ser Glu Ser Val Asp Thr
Tyr 1 5 1843PRTArtificial sequence4/94 CDR-L2 184Gly Ala Ser 1
1856PRTArtificial sequence4/94 CDR-L3 185Thr Tyr Asn Tyr Pro Leu 1
5 1865PRTArtificial sequenceHumanized 2/99 CDR-H1 186Ser Tyr Ala
Met Ser 1 5 18716PRTArtificial sequenceHumanized 2/99 CDR-H2 187Tyr
Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Ser Trp Ala Lys Ser 1 5 10
15 18817PRTArtificial sequenceHumanized 2/99 CDR-H3 188Arg Tyr Gly
Thr Ser Tyr Pro Asp Tyr Gly Asp Ala Ser Gly Phe Asp 1 5 10 15 Pro
18911PRTArtificial sequenceHumanized 2/99 CDR-L1 189Arg Ala Ser Gln
Ser Ile Ser Ser Tyr Leu Ala 1 5 10 1907PRTArtificial
sequenceHumanized 2/99 CDR-L2 190Arg Ala Ser Thr Leu Ala Ser 1 5
19112PRTArtificial sequenceHumanized 2/99 CDR-L3 191Gln Gln Asn Tyr
Ala Ser Ser Asn Val Asp Asn Thr 1 5 10 192122PRTArtificial
sequenceHumanized 2/99 VH 192Gln Ser Met Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln Thr 1 5 10 15 Leu Ser Leu Thr Cys Thr Val
Ser Gly Phe Ser Leu Ser Ser Tyr Ala 20
25 30 Met Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile
Gly 35 40 45 Tyr Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Ser Trp
Ala Lys Ser 50 55 60 Arg Val Thr Ile Ser Lys Thr Ser Thr Thr Val
Ser Leu Lys Leu Ser 65 70 75 80 Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Arg Tyr 85 90 95 Gly Thr Ser Tyr Pro Asp Tyr
Gly Asp Ala Ser Gly Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 120 193109PRTArtificial sequenceHumanized
2/99 VL 193Ala Ile Gln Leu 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 Ser
Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Arg Ala Ser Thr Leu Ala 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 Asn Tyr Ala Ser Ser Asn 85 90 95 Val Asp
Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105
194119PRTArtificial sequenceHumanized 2/99 VH variant 1 194Gln 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 Phe Asn Ile Lys Asp Thr 20
25 30 Tyr Ile His Trp Val Ile Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asp Thr Lys Ser Asp
Pro Lys Phe 50 55 60 Gln Val Arg Val Thr Ile Thr Ala Asp Thr Ser
Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Asp Tyr Leu Tyr Pro
Tyr Tyr Phe Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val
Ser Ser 115 195119PRTArtificial sequenceHumanized 2/99 VH variant 2
195Gln 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 Phe Asn Ile Lys
Asp Thr 20 25 30 Tyr Ile His Trp Val Ile Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asp Thr
Lys Ser Ala Pro Lys Phe 50 55 60 Gln Val Arg Val Thr Ile Thr Ala
Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Asp Tyr
Leu Tyr Pro Tyr Tyr Phe Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr
Val Thr Val Ser Ser 115 196119PRTArtificial sequenceHumanized 2/99
VH variant 3 196Gln 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 Phe
Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Ile Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn
Gly Asp Thr Lys Ser Asp Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr
Ile Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val
Arg Asp Tyr Leu Tyr Pro Tyr Tyr Phe Asp Phe Trp Gly Gln Gly 100 105
110 Thr Thr Val Thr Val Ser Ser 115 197119PRTArtificial
sequenceHumanized 2/99 VH variant 4 197Gln 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 Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His
Trp Val Ile Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly
Arg Ile Asp Pro Ala Asn Gly Asp Thr Lys Ser Asp Pro Lys Phe 50 55
60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu 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 Val Arg Asp Tyr Leu Tyr Pro Tyr Tyr Phe Asp Phe
Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115
198119PRTArtificial sequenceHumanized 2/99 VH variant 5 198Gln 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 Asn Ile Lys Asp Thr 20
25 30 Tyr Ile His Trp Val Ile Gln Arg Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asp Thr Lys Ser Asp
Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu 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 Val Arg Asp Tyr Leu Tyr Pro
Tyr Tyr Phe Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val
Ser Ser 115 199119PRTArtificial sequenceHumanized 2/99 VH variant 6
199Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Asn Ile Lys
Asp Thr 20 25 30 Tyr Ile His Trp Val Ile Gln Arg Pro Gly Lys Gly
Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asp Thr
Lys Ser Asp Pro Ser Leu 50 55 60 Gln Ser Arg Val Thr Ile Ser Ala
Asp Thr Ser Lys Asn Gln Ala Ser 65 70 75 80 Leu Lys Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Asp Tyr
Leu Tyr Pro Tyr Tyr Phe Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr
Val Thr Val Ser Ser 115 200119PRTArtificial sequenceHumanized 2/99
VH variant 7 200Glu 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 Phe
Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Ile Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Asp Pro Ala Asn
Gly Asp Thr Lys Ser Asp Pro Ser Val 50 55 60 Gln Val Arg Ala Thr
Ile Ser Ala Asp Thr 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 Val
Arg Asp Tyr Leu Tyr Pro Tyr Tyr Phe Asp Phe Trp Gly Gln Gly 100 105
110 Thr Thr Val Thr Val Ser Ser 115 201106PRTArtificial
sequenceHumanized 2/99 VL variant 1 201Lys 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 Thr Tyr 20 25 30 Val Ser Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Gly Ala Ser Asn Arg Tyr Thr 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 Asp Tyr Tyr Cys Gly Gln Thr Tyr Asn Tyr
Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105
20215PRTArtificial sequenceHuman transferring receptor (hTFR)
epitope 1 202Ile Gly Gln Asn Met Val Thr Ile Val Gln Ser Asn Gly
Asn Leu 1 5 10 15 20315PRTArtificial sequenceHuman transferring
receptor (hTFR) epitope 2 203Asn Met Val Thr Ile Val Gln Ser Asn
Gly Asn Leu Asp Pro Val 1 5 10 15 20415PRTArtificial sequenceHuman
transferring receptor (hTFR) epitope 3 204Gln Ser Asn Gly Asn Leu
Asp Pro Val Glu Ser Pro Glu Gly Tyr 1 5 10 15 205122PRTArtificial
sequenceHumanized 2/99 VH variant 205Gln Ser Met Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln Thr 1 5 10 15 Leu Ser Leu Thr Cys
Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Ala 20 25 30 Met Ser Trp
Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Tyr
Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Ser Trp Ala Lys Ser 50 55
60 Arg Val Thr Ile Ser Lys Thr Ser Thr Thr Val Ser Leu Lys Leu Ser
65 70 75 80 Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg
Arg Tyr 85 90 95 Gly Thr Ser Tyr Pro Asp Tyr Gly Asp Ala Gln Gly
Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 20617PRTArtificial sequenceHumanized 2/99 CDR-H3 variant
206Arg Tyr Gly Thr Ser Tyr Pro Asp Tyr Gly Asp Ala Gln Gly Phe Asp
1 5 10 15 Pro 207106PRTArtificial sequenceHumanized 2/99 VL variant
2 207Lys 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 Glu Ser Val
Asp Thr Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly
Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Asp
Tyr Tyr Cys Gly Gln Thr Tyr Asn Tyr Pro Leu 85 90 95 Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile 100 105 208106PRTArtificial
sequenceHumanized 2/99 VL variant 3 208Glu 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 Glu Ser Val Asp Thr Tyr 20 25 30 Val Ser Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr
Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro
65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Gly Gln Thr Tyr Asn Tyr
Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105
209106PRTArtificial sequenceHumanized 2/99 VL variant 4 209Lys 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 Glu Ser Val Asp Thr Tyr 20
25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Ile Pro Asp Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Gly
Gln Thr Tyr Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile 100 105 210219PRTArtificial
sequence8936_pTau_RbMab86_VL_huIgkappa 210Ala Gln Val Leu Thr Gln
Thr Thr Ser Pro Val Ser Ala Ala Val Gly 1 5 10 15 Ser Thr Val Thr
Ile Ser Cys Gln Ser Ser Gln Ser Val Arg Thr Asn 20 25 30 Lys Leu
Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45
Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50
55 60 Ala Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val
Gln 65 70 75 80 Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe
Asp Cys Ser 85 90 95 Ile Ala Asp Cys Val Ala Phe Gly Gly Gly Thr
Glu Val Val Val Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180
185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
211438PRTArtificial sequence8975-pTau-Rb86-hugamma1-SS-hole 211Gln
Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10
15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Asn Ala
20 25 30 Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Ile Gly 35 40 45 Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Ser
Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Ala Ser Thr Thr
Val Asp Leu Lys Met Thr 65 70 75 80 Ser Pro Thr Ala Glu Asp Thr Gly
Thr Tyr Phe Cys Gly Lys Ser Asn 85 90 95 Ile Trp Gly Pro Gly Thr
Leu Val Thr Val Ser Leu Ala Ser Thr Lys 100 105 110 Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 115 120 125 Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 130 135 140
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 145
150 155 160 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val 165 170 175 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn 180 185 190 Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro 195 200 205 Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu 210 215 220 Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 225 230 235 240 Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 245 250 255 Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 260
265 270 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn 275 280 285 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp 290 295 300 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro 305 310 315 320 Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu 325 330 335 Pro Gln Val Cys Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 340 345 350 Gln Val Ser Leu
Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile 355 360 365 Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 370 375 380
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys 385
390 395 400 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys 405 410 415 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu 420 425 430 Ser Leu Ser Pro Gly Lys 435
212922PRTArtificial
sequence8976-pTau-Rb86-hugamma1-SS-knob-mTfR-8D3-scFab 212Gln Ser
Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15
Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Asn Ala 20
25 30 Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
Gly 35 40 45 Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Ser Trp
Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Ala Ser Thr Thr Val
Asp Leu Lys Met Thr 65 70 75 80 Ser Pro Thr Ala Glu Asp Thr Gly Thr
Tyr Phe Cys Gly Lys Ser Asn 85 90 95 Ile Trp Gly Pro Gly Thr Leu
Val Thr Val Ser Leu Ala Ser Thr Lys 100 105 110 Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 115 120 125 Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 130 135 140 Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 145 150
155 160 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val 165 170 175 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn 180 185 190 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro 195 200 205 Lys Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu 210 215 220 Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp 225 230 235 240 Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 245 250 255 Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 260 265 270
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 275
280 285 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp 290 295 300 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro 305 310 315 320 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu 325 330 335 Pro Gln Val Tyr Thr Leu Pro Pro
Cys Arg Asp Glu Leu Thr Lys Asn 340 345 350 Gln Val Ser Leu Trp Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 355 360 365 Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 370 375 380 Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 385 390 395
400 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
405 410 415 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu 420 425 430 Ser Leu Ser Pro Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly 435 440 445 Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln
Met Thr Gln Ser Pro Ala 450 455 460 Ser Leu Ser Ala Ser Leu Glu Glu
Ile Val Thr Ile Thr Cys Gln Ala 465 470 475 480 Ser Gln Asp Ile Gly
Asn Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly 485 490 495 Lys Ser Pro
Gln Leu Leu Ile Tyr Gly Ala Thr Ser Leu Ala Asp Gly 500 505 510 Val
Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Gln Phe Ser Leu 515 520
525 Lys Ile Ser Arg Val Gln Val Glu Asp Ile Gly Ile Tyr Tyr Cys Leu
530 535 540 Gln Ala Tyr Asn Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys
Val Glu 545 550 555 560 Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser 565 570 575 Asp Glu Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn 580 585 590 Asn Phe Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala 595 600 605 Leu Gln Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys 610 615 620 Asp Ser Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp 625 630 635 640
Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu 645
650 655 Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly
Gly 660 665 670 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly 675 680 685 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Glu Val Gln 690 695 700 Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Asn Ser Leu Thr 705 710 715 720 Leu Ser Cys Val Ala Ser
Gly Phe Thr Phe Ser Asn Tyr Gly Met His 725 730 735 Trp Ile Arg Gln
Ala Pro Lys Lys Gly Leu Glu Trp Ile Ala Met Ile 740 745 750 Tyr Tyr
Asp Ser Ser Lys Met Asn Tyr Ala Asp Thr Val Lys Gly Arg 755 760 765
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Glu Met 770
775 780 Asn Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Val
Pro 785 790 795 800 Thr Ser His Tyr Val Val Asp Val Trp Gly Gln Gly
Val Ser Val Thr 805 810 815 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro 820 825 830 Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val 835 840 845 Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala 850 855 860 Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 865 870 875 880 Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 885 890
895 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
900 905 910 Val Asp Lys Lys Val Glu Pro Lys Ser Cys 915 920
21337DNAArtificial sequencerbHC.up 213aagcttgcca ccatggagac
tgggctgcgc tggcttc 3721421DNAArtificial sequencerbHCf.do
214ccattggtga gggtgcccga g 2121534DNAArtificial sequencerbLC.up
215aagcttgcca ccatggacay gagggccccc actc 3421626DNAArtificial
sequencerbLC.do 216cagagtrctg ctgaggttgt aggtac
2621720DNAArtificial sequenceBcPCR_FHLC_leader.fw 217atggacatga
gggtccccgc 2021824DNAArtificial sequenceBcPCR_huCkappa.rev
218gatttcaact gctcatcaga tggc 24
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