U.S. patent application number 13/406503 was filed with the patent office on 2012-09-20 for monovalent antigen binding proteins.
This patent application is currently assigned to Hoffmann-La Roche Inc.. Invention is credited to Birgit Bossenmaier, Hubert Kettenberger, Christian Klein, Klaus-Peter Kuenkele, Joerg-Thomas Regula, Wolfgang Schaefer, Manfred Schwaiger, Claudio Sustmann.
Application Number | 20120237507 13/406503 |
Document ID | / |
Family ID | 45819191 |
Filed Date | 2012-09-20 |
United States Patent
Application |
20120237507 |
Kind Code |
A1 |
Bossenmaier; Birgit ; et
al. |
September 20, 2012 |
Monovalent Antigen Binding Proteins
Abstract
The present invention relates to monovalent antigen binding
proteins with a CH1-CL domain exchange, methods for their
production, pharmaceutical compositions containing said antibodies,
and uses thereof.
Inventors: |
Bossenmaier; Birgit;
(Seefeld, DE) ; Kettenberger; Hubert; (Muenchen,
DE) ; Klein; Christian; (Bonstetten, CH) ;
Kuenkele; Klaus-Peter; (Marzling, DE) ; Regula;
Joerg-Thomas; (Muenchen, DE) ; Schaefer;
Wolfgang; (Mannheim, DE) ; Schwaiger; Manfred;
(Wang-Bergen, DE) ; Sustmann; Claudio; (Muenchen,
DE) |
Assignee: |
Hoffmann-La Roche Inc.
Nutley
NJ
|
Family ID: |
45819191 |
Appl. No.: |
13/406503 |
Filed: |
February 27, 2012 |
Current U.S.
Class: |
424/133.1 ;
435/320.1; 435/328; 435/69.6; 530/387.3; 536/23.53 |
Current CPC
Class: |
C07K 2317/77 20130101;
C07K 2317/35 20130101; C07K 16/00 20130101; C07K 2317/92 20130101;
A61P 35/00 20180101; C07K 2317/66 20130101; C07K 2317/76 20130101;
C07K 2317/94 20130101; C07K 2317/732 20130101; C07K 2317/64
20130101; C07K 16/2863 20130101 |
Class at
Publication: |
424/133.1 ;
530/387.3; 435/69.6; 536/23.53; 435/320.1; 435/328 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00; C12N 5/10 20060101
C12N005/10; C12N 15/13 20060101 C12N015/13; C12N 15/85 20060101
C12N015/85; C07K 16/18 20060101 C07K016/18; C12P 21/00 20060101
C12P021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 28, 2011 |
EP |
11156321.9 |
Claims
1. A monovalent antigen binding protein comprising a) a modified
heavy chain of an antibody which specifically binds to an antigen,
wherein the VH domain is replaced by the VL domain of said
antibody; and b) a modified heavy chain of said antibody, wherein
the CH1 domain is replaced by the CL domain of said antibody.
2. The monovalent antigen binding protein according to claim 1,
comprising the CH3 domain of the modified heavy chain of the
antibody of a) and the CH3 domain of the modified heavy chain of
the antibody of b) each meet at an interface which comprises an
original interface between the antibody CH3 domains; wherein said
interface is altered to promote the formation of the monovalent
antigen binding protein, wherein the alteration comprises: i) a CH3
domain of one heavy chain is altered, so that within the original
interface the CH3 domain of one heavy chain that meets the original
interface of the CH3 domain of the other heavy chain within the
monovalent antigen binding protein, an amino acid residue is
replaced with an amino acid residue having a larger side chain
volume, thereby generating a protuberance within the interface of
the CH3 domain of one heavy chain which is positionable in a cavity
within the interface of the CH3 domain of the other heavy chain and
ii) the CH3 domain of the other heavy chain is altered, so that
within the original interface of the second CH3 domain that meets
the original interface of the first CH3 domain within the
monovalent antigen binding protein, an amino acid residue is
replaced with an amino acid residue having a smaller side chain
volume, thereby generating a cavity within the interface of the
second CH3 domain within which a protuberance within the interface
of the first CH3 domain is positionable.
3. The monovalent antigen binding protein according to claim 2,
comprising said amino acid residue having a larger side chain
volume is selected from the group consisting of arginine (R),
phenylalanine (F), tyrosine (Y), tryptophan (W), and said amino
acid residue having a smaller side chain volume is selected from
the group consisting of alanine (A), serine (S), threonine (T),
valine (V).
4. The monovalent antigen binding protein according to claim 3,
comprising both CH3 domains are further altered by the introduction
of cysteine (C) as amino acid in the corresponding positions of
each CH3 domain such that a disulfide bridge between both CH3
domains can be formed.
5. The monovalent antigen binding protein according to claims 1 to
4, comprising is of human IgG1 isotype.
6. The monovalent antigen binding protein according to claim 1,
characterized in comprising a) a modified heavy chain comprising
the amino acid sequence of SEQ ID NO:1; and b) a modified heavy
chain comprising the amino acid sequence of SEQ ID NO:2; or a) a
modified heavy chain comprising the amino acid sequence of SEQ ID
NO:3; and b) a modified heavy chain comprising the amino acid
sequence of SEQ ID NO:4; or a) a modified heavy chain comprising
the amino acid sequence of SEQ ID NO:5; and b) a modified heavy
chain comprising the amino acid sequence of SEQ ID NO:6; or a) a
modified heavy chain comprising the amino acid sequence of SEQ ID
NO:7; and b) a modified heavy chain comprising the amino acid
sequence of SEQ ID NO:8; or a) a modified heavy chain comprising
the amino acid sequence of SEQ ID NO:9; and b) a modified heavy
chain comprising the amino acid sequence of SEQ ID NO:10; or a) a
modified heavy chain comprising the amino acid sequence of SEQ ID
NO:11; and b) a modified heavy chain comprising the amino acid
sequence of SEQ ID NO:12.
7. The monovalent antigen binding protein according to claim 1, 2,
3, 4 or 6, comprising the modified heavy chains of a) and b) are of
IgG1 isotype, and the antigen binding protein is afucosylated with
an amount of fucose of 80% or less of the total amount of
oligosaccharides (sugars) at Asn297 is of human IgG1 isotype.
8. A pharmaceutical composition of a monovalent antigen binding
protein according to claims 1 to 7.
9. A pharmaceutical composition comprising a monovalent antigen
binding protein according to claims 1 to 7 and at least one
pharmaceutically acceptable excipient.
10. The monovalent antigen binding protein according to claims 1 to
7 for use in the treatment of cancer.
11. Use of the monovalent antigen binding protein according to
claims 1 to 7 for the manufacture of a medicament for the treatment
of cancer.
12. A method for the treatment of a patient in need of therapy,
characterized by administering to the patient a therapeutically
effective amount of a monovalent antigen binding protein according
to claims 1 to 7.
13. A method for the preparation of a monovalent antigen binding
protein according to claims 1 to 7 comprising the steps of a)
transforming a host cell with vectors comprising nucleic acid
molecules encoding a monovalent antigen binding protein according
to claims 1 to 7, b) culturing the host cell under conditions that
allow synthesis of said monovalent antigen binding protein
molecule; and c) recovering said monovalent antigen binding protein
molecule from said culture.
14. Nucleic acid encoding the monovalent antigen binding protein
according to claims 1 to 7.
15. A vector comprising nucleic acid according to claim 14.
16. A host cell comprising the vector according to claim 15.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims of priority under 35 USC
.sctn.119(a) to European patent application number EP11156321.9,
filed 28 Feb. 2011, the contents of which is incorporated herein by
reference.
SEQUENCE LISTING
[0002] A sequence listing comprising SEQ ID NOS: 1-12 is attached
hereto. Each sequence provided in the sequence listing is
incorporated herein by reference, in its entirety, for all
purposes.
TECHNICAL FIELD
[0003] The present invention relates to monovalent antigen binding
proteins with a CH1-CL domain exchange, methods for their
production, pharmaceutical compositions containing said antibodies,
and uses thereof.
BACKGROUND OF THE INVENTION
[0004] In the last two decades various engineered antibody
derivatives, either mono or -multispecific, either mono- or
multivalent have been developed and evaluated (see e.g. Holliger,
P., et al., Nature Biotech 23 (2005) 1126-1136; Fischer N., and
Leger O., Pathobiology 74 (2007) 3-14).
[0005] For certain antigens as e.g. c-Met monovalent antibodies
have different properties such as lack of agonistic function or
reduced receptor internalization upon antibody binding than their
corresponding bivalent forms and therefore represent attractive
formats for therapeutic use. E.g. WO 2005/063816 refers to
monovalent antibody fragments as therapeutics.
[0006] US 2004/0033561 describes a method for the generation of
monovalent antibodies based on the co-expression of a
VH-CH1-CH2-CH3 antibody chain with a VL-CL-CH2-CH3 antibody chain;
however, a disadvantage of this method is the formation of a
binding inactive homodimer of VL-CL-CH2-CH3 chains as depicted in
FIG. 2. Due the similar molecular weight such homodimeric
by-products are the difficult to separate.
[0007] WO 2007/048037 also refers to monovalent antibodies based on
the co-expression of a VH-CH1-CH2-CH3 antibody chain with a
VL-CL-CH2-CH3 antibody chain, but having a tagging moiety attached
to the heavy chain for easier purification of the heterodimer from
the difficult-to-separate homodimeric by-product.
[0008] WO 2009/089004 describes another possibility to generate a
heterodimeric monovalent antibody using electrostatic steering
effects.
[0009] WO 2010/145792 relates tetravalent bispecific antibodies,
wherein mismatched byproducts of similar weight are reduced
resulting in higher yields of the desiered bispecific antibody.
SUMMARY OF THE INVENTION
[0010] The invention comprises a monovalent antigen binding protein
comprising [0011] a) a modified heavy chain of an antibody which
specifically binds to an antigen, wherein the VH domain is replaced
by the VL domain of said antibody; and [0012] b) a modified heavy
chain of said antibody, wherein the CH1 domain is replaced by the
CL domain of said antibody.
[0013] In one embodiment of the invention the monovalent antigen
binding protein according to the invention is characterized in that
[0014] the CH3 domain of the modified heavy chain of the antibody
of a) and the CH3 domain of the modified heavy chain of the
antibody of b) each meet at an interface which comprises an
original interface between the antibody CH3 domains; [0015] wherein
said interface is altered to promote the formation of the
monovalent antigen binding protein, wherein the alteration is
characterized in that: [0016] i) the CH3 domain of one heavy chain
is altered, [0017] so that within the original interface the CH3
domain of one heavy chain that meets the original interface of the
CH3 domain of the other heavy chain within the monovalent antigen
binding protein, [0018] an amino acid residue is replaced with an
amino acid residue having a larger side chain volume, thereby
generating a protuberance within the interface of the CH3 domain of
one heavy chain which is positionable in a cavity within the
interface of the CH3 domain of the other heavy chain [0019] and
[0020] ii) the CH3 domain of the other heavy chain is altered,
[0021] so that within the original interface of the second CH3
domain that meets the original interface of the first CH3 domain
within the monovalent antigen binding protein, [0022] an amino acid
residue is replaced with an amino acid residue having a smaller
side chain volume, thereby generating a cavity within the interface
of the second CH3 domain within which a protuberance within the
interface of the first CH3 domain is positionable.
[0023] In one embodiment of the invention this monovalent antigen
binding protein according to the invention is characterized in that
[0024] said amino acid residue having a larger side chain volume is
selected from the group consisting of arginine (R), phenylalanine
(F), tyrosine (Y), tryptophan (W), and said amino acid residue
having a smaller side chain volume is selected from the group
consisting of alanine (A), serine (S), threonine (T), valine
(V).
[0025] In one embodiment of the invention this monovalent antigen
binding protein according to the invention is further characterized
in that [0026] both CH3 domains are further altered by the
introduction of cysteine (C) as amino acid in the corresponding
positions of each CH3 domain such that a disulfide bridge between
both CH3 domains can be formed.
[0027] In one embodiment the monovalent antigen binding protein
according to the invention is characterized in that is of human IgG
isotype.
[0028] In one embodiment the monovalent antigen binding protein
according to the invention is characterized in comprising [0029] a)
a modified heavy chain comprising the amino acid sequence of SEQ ID
NO:1; and [0030] b) a modified heavy chain comprising the amino
acid sequence of SEQ ID NO:2; [0031] or [0032] a) a modified heavy
chain comprising the amino acid sequence of SEQ ID NO:3; and [0033]
b) a modified heavy chain comprising the amino acid sequence of SEQ
ID NO:4; [0034] or [0035] a) a modified heavy chain comprising the
amino acid sequence of SEQ ID NO:5; and [0036] b) a modified heavy
chain comprising the amino acid sequence of SEQ ID NO:6; [0037]
or
[0038] a) a modified heavy chain comprising the amino acid sequence
of SEQ ID NO:7; and [0039] b) a modified heavy chain comprising the
amino acid sequence of SEQ ID NO:8; [0040] or [0041] a) a modified
heavy chain comprising the amino acid sequence of SEQ ID NO:9; and
[0042] b) a modified heavy chain comprising the amino acid sequence
of SEQ ID NO:10; [0043] or [0044] a) a modified heavy chain
comprising the amino acid sequence of SEQ ID NO:11; and [0045] b) a
modified heavy chain comprising the amino acid sequence of SEQ ID
NO:12.
[0046] In one aspect of the invention the monovalent antigen
binding protein according to the invention is characterized in that
the modified heavy chains of a) and b) are of IgG1 isotype, and the
antigen binding protein is afucosylated with an the amount of
fucose of 80% or less (preferably of 65% to 5%) of the total amount
of oligosaccharides (sugars) at Asn297.
[0047] The invention further comprises a method for the preparation
of a monovalent antigen binding protein according to the invention
[0048] comprising the steps of [0049] a) transforming a host cell
with vectors comprising nucleic acid molecules encoding [0050] a
monovalent antigen binding protein according to the invention
[0051] b) culturing the host cell under conditions that allow
synthesis of said monovalent antigen binding protein molecule; and
[0052] c) recovering said monovalent antigen binding protein
molecule from said culture.
[0053] The invention further comprises nucleic acid encoding the
monovalent antigen binding protein according to the invention.
[0054] The invention further comprises vectors comprising nucleic
acid encoding the monovalent antigen binding protein according to
the invention.
[0055] The invention further comprises host cell comprising said
vectors.
[0056] The invention further comprises composition, preferably a
pharmaceutical or a diagnostic composition of a monovalent antigen
binding protein according to the invention.
[0057] The invention further comprises pharmaceutical composition
comprising a monovalent antigen binding protein according to the
invention and at least one pharmaceutically acceptable
excipient.
[0058] The invention further comprises method for the treatment of
a patient in need of therapy, characterized by administering to the
patient a therapeutically effective amount of a monovalent antigen
binding protein according to the invention.
[0059] The antigen binding proteins according to the invention are
based on the principle that a VL-CH1-CH2-CH3 and VH-CL-CH2-CH3
chain only forms heterodimers and cannot form a
difficult-to-separate homodimeric by-product of similar structure
and molecular weight. The effect of this modification lays not
primarily in a reduction of by-products, but in that the only
by-product which is formed is changed from a homodimeric by-product
of similar size into a High-Molecular weight tetramer (FIG. 1D).
This High-Molecular weight tetramer then can be easily removed with
SEC or other MW separation techniques.
[0060] The formed dimeric byproduct (FIG. 1D) can be easily
separated due to the different molecular weight (the molecular
weight is approximately doubled) and structure. Therefore the
purification without the introduction of further modifications
(like e.g. genetic introductions of tags) is possible.
[0061] It has further been found that the monovalent antigen
binding proteins according to the invention have valuable
characteristics such as biological or pharmacological activities
(as e.g. ADCC, or antagonistic biological activity as well as lack
of agonistic activities). They can be used e.g. for the treatment
of diseases such as cancer. The monovalent antigen binding proteins
have furthermore highly valuable pharmacokinetic properties (like
e.g. halftime (term t1/2) or AUC).
DESCRIPTION OF THE FIGURES
[0062] FIG. 1 A) Scheme of the monovalent antigen binding protein
according to the invention with CH1-CL domain exchange based on
VL-CH1-CH2-CH3 and VH-CL-CH2-CH3 chains (abbreviated as MoAb). B)
Scheme of a MoAb according to the invention including
knobs-into-holes in the CH3 domains. C) Scheme of the dimeric
bivalent antigen binding protein (MoAb-Dimer that is formed as a
byproduct which can be easily separated due to different structure
and molecular weight).
[0063] FIG. 2 Scheme of A) a monovalent antibody of VL-CL-CH2-CH3
and of VH-CH1-CH2-CH3 chains (described e.g in US 2004/0033561) and
B) the binding inactive difficult-to-separate homodimer byproduct
of VL-CL-CH2-CH3 chains (described e.g in WO 2007/048037).
[0064] FIG. 3 Biochemical characterization of MoAb c-Met (c-Met 5D5
MoAb ("wt")). (A) Protein A purified antigen binding protein was
separated on a Superdex 200 26/60 column. Individual peaks
correspond to MoAb (3), MoAb Dimer (2) and an aggregate fraction
(1). (B) Peak fractions (1, 2, 3) were pooled and subjected to
SDS-PAGE under non-reducing and reducing conditions. Polyacrylamide
gels were stained with Coomassie Blue dye.
[0065] FIG. 4 Biochemical characterization of monovalent MoAb IGF1R
(IGF1R AK18 MoAb ("wt")). (A) Protein A purified antigen binding
protein was separated on an Superdex 200 26/60 column. Individual
peaks correspond to MoAb (2) and MoAb Dimer (1). (B) Peak fractions
(1, 2) were pooled and subjected to SDS-PAGE under non-reducing and
reducing conditions. Polyacrylamide gels were stained with
Coomassie Blue dye. C) The molecular mass of the peaks fractions 1
and 2 was investigated by SEC-MALLS. Peak 2 was identified as
monovalent antigen binding protein MoAb IGF1R.
[0066] FIG. 5 Biochemical characterization of MoAb Her3 (Her3 205
MoAb ("wt")). (A) Protein A purified antibody was separated on an
Superdex 200 26/60 column. Individual peaks correspond to MoAb (3),
MoAb Dimer (2) and an aggregate fraction (1). (B) Peak fractions
(1, 2, 3) were pooled and subjected to SDS-PAGE under non-reducing
and reducing conditions. Polyacrylamide gels were stained with
Coomassie Blue dye.
[0067] FIG. 6 Biochemical characterization of MoAb Her3 with KiH
mutations (Her3 205 MoAb KiH). (A) Protein A purified antigen
binding protein was separated on an Superdex 200 26/60 column. (B)
Peak fraction was pooled and subjected to SDS-PAGE under
non-reducing and reducing conditions. Polyacrylamide gels were
stained with Coomassie Blue dye.
[0068] FIG. 7 Biochemical characterization of MoAb IGF1R with KiH
mutations (IGF1R AK18 MoAb KiH). (A) Protein A purified antibody
was separated on an Superdex 200 26/60 column. Individual peaks
correspond to MoAb (2) and MoAb Dimer (1). (B) Peak fractions (1,
2) were pooled and subjected to SDS-PAGE under non-reducing and
reducing conditions. Polyacrylamide gels were stained with
Coomassie Blue dye.
[0069] FIG. 8 Biochemical characterization of MoAb c-Met with KiH
mutations (c-Met 5D5 MoAb KiH). (A) Protein A purified antibody was
separated on an Superdex 200 26/60 column. (B) Peak fraction was
pooled and subjected to SDS-PAGE under non-reducing and reducing
conditions. Polyacrylamide gels were stained with Coomassie Blue
dye.
[0070] FIG. 9 c-Met receptor phosphorylation assay in A549 cells.
A549 cells were stimulated with HGF in the absence or presence of
c-Met binding antibodies or c-Met 5D5 MoAb ("wt")). Total cell
lysates were subjected to immunoblot analysis. Asterisk marks
phospho-c-Met band in between two unspecific bands.
[0071] FIG. 10 Cellular binding of MoAb c-Met (c-Met 5D5 MoAb
("wt"))) to A549 cells with flow cytrometric analysis. A549 cells
were incubated with a dilution series of the indicated antibodies.
Bound antibodies were visualized with an Fc-binding secondary
fluorophor coupled antibody.
[0072] FIG. 11 Schematic picture of the surface plasmon resonance
assay applied to analyze the binding affinity of the monovalent
antigen binding protein IGF1R AK18 MoAb ("wt").
[0073] FIG. 12 Cellular binding of MoAb IGF-1R (IGF1R AK18 MoAb
("wt")) to A549 cells with flow cytometric analysis. A549 cells
were incubated with a dilution series of the indicated antibodies.
Bound antibodies were visualized with an Fc-binding secondary
fluorophor coupled antibody.
[0074] FIG. 13 ADCC Assay with parent non-glycoengineered (non-ge)
IGF Mab and parent glycoengineered (ge) IGF Mab and
non-glycoengineered monovalent antigen binding protein IGF MoAb
(IGF1R AK18 MoAb ("wt")). Donor derived peripheral blood
mononuclear cells (PBMC) were incubated with prostate cancer cells
(DU145) in the presence of parent non-ge IGF1R Mab (=1), parent ge
IGF Mab (=2) and non-ge monovalent antigen binding protein IGF MoAb
(=3).
[0075] FIG. 14 Internalization of IGF-1R was assessed following
incubation with parent IGF-1R IgG1 antibody and monovalent antigen
binding protein IGF1R MoAb (IGF1R AK18 MoAb ("wt")), the data show
that internalization of IGF-1R is reduced in terms of potency and
absolute internalization when the monovalent antigen binding
protein IGF MoAb (IGF1R AK18 MoAb ("wt")) is bound.
[0076] FIG. 15 IGF-1 induced autophosphorylation of IGF-1R was
assessed following incubation with IGF-1R IgG1 antibody and
monovalent antigen binding protein IGF1R MoAb (IGF1R AK18 MoAb
("wt")), the data show that IGF-1 induced autophoshorylation of
IGF-1R is reduced in terms of potency when the monovalent antigen
binding protein IGF MoAb (IGF1R AK18 MoAb ("wt")) is bound.
[0077] FIG. 16 Aggregation tendency of the monovalent antigen
binding protein IGF1R MoAb (IGF1R AK18 MoAb ("wt")) was assessed by
a DLS timecourse experiment. Over a period of five days, no
measurable increase in the hydrodynamic radius (Rh) of the isolated
monomer fraction (see FIG. 4) could be detected.
[0078] FIG. 17 ESI-MS spectrum of the monovalent antigen binding
protein IGF MoAb (IGF1R AK18 MoAb ("wt")) after deglycosylation and
under non-reducing conditions.
[0079] FIG. 18 ESI-MS spectrum of the IGF-1R monovalent antigen
binding protein IGF1R MoAb (IGF1R AK18 MoAb ("wt")) after
deglycosylation and reduction.
DETAILED DESCRIPTION OF THE INVENTION
[0080] The invention comprises a monovalent antigen binding protein
comprising [0081] a) a modified heavy chain of an antibody which
specifically binds to an antigen, wherein the VH domain is replaced
by the VL domain of said antibody; and [0082] b) a modified heavy
chain of said antibody, wherein the CH1 domain is replaced by the
CL domain of said antibody.
[0083] In one preferred embodiment of the invention the CH3 domains
of said monovalent antigen binding protein according to the
invention can be altered by the "knobs-into-holes" (KiH) technology
which is described in detail with several examples in e.g. WO
96/027011, Ridgway, J. B., et al., Protein Eng. 9 (1996) 617-621;
and Merchant, A. M., et al., Nat Biotechnol 16 (1998) 677-681. In
this method the interaction surfaces of the two CH3 domains are
altered to increase the heterodimerisation of both heavy chains
containing these two CH3 domains. Each of the two CH3 domains (of
the two heavy chains) can be the "knob", while the other is the
"hole". The effect of this modification is that the High-Molecular
weight tetramer by-product, is reduced significantly.
[0084] The introduction of a disulfide bridge further stabilizes
the heterodimers (Merchant, A. M., et al., Nature Biotech 16 (1998)
677-681; Atwell, S., et al. J. Mol. Biol. 270 (1997) 26-35) and
increases the yield.
[0085] Thus in one aspect of the invention said monovalent antigen
binding protein is further characterized in that [0086] the CH3
domain of the heavy chain of the full length antibody of a) and the
CH3 domain of the modified heavy chain of the full length antibody
of b) each meet at an interface which comprises an original
interface between the antibody CH3 domains; [0087] wherein said
interface is altered to promote the formation of the monovalent
antigen binding protein, wherein the alteration is characterized in
that: [0088] i) the CH3 domain of one heavy chain is altered,
[0089] so that within the original interface the CH3 domain of one
heavy chain that meets the original interface of the CH3 domain of
the other heavy chain within the monovalent antigen binding
protein, [0090] an amino acid residue is replaced with an amino
acid residue having a larger side chain volume, thereby generating
a protuberance within the interface of the CH3 domain of one heavy
chain which is positionable in a cavity within the interface of the
CH3 domain of the other heavy chain and [0091] ii) the CH3 domain
of the other heavy chain is altered, [0092] so that within the
original interface of the second CH3 domain that meets the original
interface of the first CH3 domain within the monovalent antigen
binding protein, [0093] an amino acid residue is replaced with an
amino acid residue having a smaller side chain volume, thereby
generating a cavity within the interface of the second CH3 domain
within which a protuberance within the interface of the first CH3
domain is positionable.
[0094] Preferably said amino acid residue having a larger side
chain volume is selected from the group consisting of arginine (R),
phenylalanine (F), tyrosine (Y), tryptophan (W).
[0095] Preferably said amino acid residue having a smaller side
chain volume is selected from the group consisting of alanine (A),
serine (S), threonine (T), valine (V).
[0096] In one aspect of the invention both CH3 domains are further
altered by the introduction of cysteine (C) as amino acid in the
corresponding positions of each CH3 domain such that a disulfide
bridge between both CH3 domains can be formed.
[0097] In one preferred embodiment, said monovalent antigen binding
protein comprises a T366W mutation in the CH3 domain of the "knobs
chain" and T366S, L368A, Y407V mutations in the CH3 domain of the
"hole chain". An additional interchain disulfide bridge between the
CH3 domains can also be used (Merchant, A. M., et al., Nature
Biotech 16 (1998) 677-681) e.g. by introducing a Y349C mutation
into the CH3 domain of the "knobs chain" and a E356C mutation or a
S354C mutation into the CH3 domain of the "hole chain". Thus in a
another preferred embodiment, said monovalent antigen binding
protein comprises Y349C, T366W mutations in one of the two CH3
domains and E356C, T366S, L368A, Y407V mutations in the other of
the two CH3 domains or said monovalent antigen binding protein
comprises Y349C, T366W mutations in one of the two CH3 domains and
S354C, T366S, L368A, Y407V mutations in the other of the two CH3
domains (the additional Y349C mutation in one CH3 domain and the
additional E356C or S354C mutation in the other CH3 domain forming
a interchain disulfide bridge) (numbering always according to EU
index of Kabat). But also other knobs-in-holes technologies as
described by EP 1 870 459 A1, can be used alternatively or
additionally. A preferred example for said monovalent antigen
binding protein are R409D; K370E mutations in the CH3 domain of the
"knobs chain" and D399K; E357K mutations in the CH3 domain of the
"hole chain" (numbering always according to EU index of Kabat).
[0098] In another preferred embodiment said monovalent antigen
binding protein comprises a T366W mutation in the CH3 domain of the
"knobs chain" and T366S, L368A, Y407V mutations in the CH3 domain
of the "hole chain" and additionally R409D; K370E mutations in the
CH3 domain of the "knobs chain" and D399K; E357K mutations in the
CH3 domain of the "hole chain".
[0099] In another preferred embodiment said monovalent antigen
binding protein comprises Y349C, T366W mutations in one of the two
CH3 domains and S354C, T366S, L368A, Y407V mutations in the other
of the two CH3 domains or said monovalent antigen binding protein
comprises Y349C, T366W mutations in one of the two CH3 domains and
S354C, T366S, L368A, Y407V mutations in the other of the two CH3
domains and additionally R409D; K370E mutations in the CH3 domain
of the "knobs chain" and D399K; E357K mutations in the CH3 domain
of the "hole chain".
[0100] In one embodiment the monovalent antigen binding protein
according to the invention is characterized in comprising [0101] a)
a modified heavy chain comprising the amino acid sequence of SEQ ID
NO:1; and [0102] b) a modified heavy chain comprising the amino
acid sequence of SEQ ID NO:2.
[0103] In one embodiment the monovalent antigen binding protein
according to the invention is characterized in comprising [0104] a)
a modified heavy chain comprising the amino acid sequence of SEQ ID
NO:3; and [0105] b) a modified heavy chain comprising the amino
acid sequence of SEQ ID NO:4.
[0106] In one embodiment the monovalent antigen binding protein
according to the invention is characterized in comprising [0107] a)
a modified heavy chain comprising the amino acid sequence of SEQ ID
NO:5; and [0108] b) a modified heavy chain comprising the amino
acid sequence of SEQ ID NO:6.
[0109] In one embodiment the monovalent antigen binding protein
according to the invention is characterized in comprising [0110] a)
a modified heavy chain comprising the amino acid sequence of SEQ ID
NO:7; and [0111] b) a modified heavy chain comprising the amino
acid sequence of SEQ ID NO:8.
[0112] In one embodiment the monovalent antigen binding protein
according to the invention is characterized in comprising [0113] a)
a modified heavy chain comprising the amino acid sequence of SEQ ID
NO:9; and [0114] b) a modified heavy chain comprising the amino
acid sequence of SEQ ID NO:10.
[0115] In one embodiment the monovalent antigen binding protein
according to the invention is characterized in comprising [0116] a)
a modified heavy chain comprising the amino acid sequence of SEQ ID
NO:11; and [0117] b) a modified heavy chain comprising the amino
acid sequence of SEQ ID NO:12.
[0118] The term "antibody" as used herein denotes a full length
antibody consisting of two antibody heavy chains and two antibody
light chains (see FIG. 1). A heavy chain of full length antibody is
a polypeptide consisting in N-terminal to C-terminal direction of
an antibody heavy chain variable domain (VH), an antibody constant
heavy chain domain 1 (CH1), an antibody hinge region (HR), an
antibody heavy chain constant domain 2 (CH2), and an antibody heavy
chain constant domain 3 (CH3), abbreviated as VH-CH1-HR-CH2-CH3;
and optionally an antibody heavy chain constant domain 4 (CH4) in
case of an antibody of the subclass IgE. Preferably the heavy chain
of full length antibody is a polypeptide consisting in N-terminal
to C-terminal direction of VH, CH1, HR, CH2 and CH3. The light
chain of full length antibody is a polypeptide consisting in
N-terminal to C-terminal direction of an antibody light chain
variable domain (VL), and an antibody light chain constant domain
(CL), abbreviated as VL-CL. The antibody light chain constant
domain (CL) can be .kappa. (kappa) or .lamda. (lambda). The
antibody chains are linked together via inter-polypeptide disulfide
bonds between the CL domain and the CH1 domain (i.e. between the
light and heavy chain) and between the hinge regions of the full
length antibody heavy chains. Examples of typical full length
antibodies are natural antibodies like IgG (e.g. IgG 1 and IgG2),
IgM, IgA, IgD, and IgE.) The antibodies according to the invention
can be from a single species e.g. human, or they can be chimerized
or humanized antibodies. The full length antibodies according to
the invention comprise two antigen binding sites each formed by a
pair of VH and VL, which both specifically bind to the same (first)
antigen. From the these full length antibodies the monovalent
antigen binding proteins of the invention are derived by modifying:
a) the first heavy chain of said antibody by replacing the VH
domain by the VL domain of said antibody; and by modifying b) the
second heavy chain of said antibody by replacing the CH1 domain by
the CL domain of said antibody. Thus the resulting monovalent
antigen binding protein comprise two modified heavy chains and no
light chains.
[0119] The C-terminus of the heavy or light chain of said full
length antibody denotes the last amino acid at the C-terminus of
said heavy or light chain.
[0120] The terms "binding site" or "antigen-binding site" as used
herein denotes the region(s) of antigen binding protein according
to the invention to which a ligand (e.g the antigen or antigen
fragment of it) actually binds and which is derived from antibody
molecule or a fragment thereof (e.g. a Fab fragment). The
antigen-binding site according to the invention comprise an
antibody heavy chain variable domains (VH) and an antibody light
chain variable domains (VL).
[0121] The antigen-binding sites (i. the pairs of VH/VL) that
specifically bind to the desired antigen can be derived a) from
known antibodies to the antigen or b) from new antibodies or
antibody fragments obtained by de novo immunization methods using
inter alia either the antigen protein or nucleic acid or fragments
thereof or by phage display.
[0122] An antigen-binding site of a monovalent antigen binding
protein of the invention contains six complementarity determining
regions (CDRs) which contribute in varying degrees to the affinity
of the binding site for antigen. There are three heavy chain
variable domain CDRs (CDRH1, CDRH2 and CDRH3) and three light chain
variable domain CDRs (CDRL1, CDRL2 and CDRL3). The extent of CDR
and framework regions (FRs) is determined by comparison to a
compiled database of amino acid sequences in which those regions
have been defined according to variability among the sequences.
[0123] Antibody specificity refers to selective recognition of the
antibody for a particular epitope of an antigen. Natural
antibodies, for example, are monospecific. Bispecific antibodies
are antibodies which have two different antigen-binding
specificities. The monovalent antigen binding proteins according to
the invention are "monospecific" and specifically bind to an
epitope of the respective antigen.
[0124] The term "valent" as used within the current application
denotes the presence of a specified number of binding sites in an
antibody molecule. A natural antibody for example has two binding
sites and is bivalent. The term "monovalent antigen binding
protein" denotes the a polypeptide containing only one antigen
binding site.
[0125] The full length antibodies of the invention comprise
immunoglobulin constant regions of one or more immunoglobulin
classes. Immunoglobulin classes include IgG, IgM, IgA, IgD, and IgE
class (or isotypes) and, in the case of IgG and IgA, their
subclasses (or subtypes). In a preferred embodiment, an full length
antibody of the invention and thus a monovalent antigen binding
protein of the invention has a constant domain structure of an IgG
class antibody.
[0126] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of a single amino acid composition.
[0127] The term "chimeric antibody" refers to an antibody
comprising a variable region, i.e., binding region, from one source
or species and at least a portion of a constant region derived from
a different source or species, usually prepared by recombinant DNA
techniques. Chimeric antibodies comprising a murine variable region
and a human constant region are preferred. Other preferred forms of
"chimeric antibodies" encompassed by the present invention are
those in which the constant region has been modified or changed
from that of the original antibody to generate the properties
according to the invention, especially in regard to C1q binding
and/or Fc receptor (FcR) binding. Such chimeric antibodies are also
referred to as "class-switched antibodies". Chimeric antibodies are
the product of expressed immunoglobulin genes comprising DNA
segments encoding immunoglobulin variable regions and DNA segments
encoding immunoglobulin constant regions. Methods for producing
chimeric antibodies involve conventional recombinant DNA and gene
transfection techniques are well known in the art. See, e.g.,
Morrison, S. L., et al., Proc. Natl. Acad. Sci. USA 81 (1984)
6851-6855; U.S. Pat. No. 5,202,238 and U.S. Pat. No. 5,204,244.
[0128] The term "humanized antibody" refers to antibodies in which
the framework or "complementarity determining regions" (CDR) have
been modified to comprise the CDR of an immunoglobulin of different
specificity as compared to that of the parent immunoglobulin. In a
preferred embodiment, a murine CDR is grafted into the framework
region of a human antibody to prepare the "humanized antibody."
See, e.g., Riechmann, L., et al., Nature 332 (1988) 323-327; and
Neuberger, M. S., et al., Nature 314 (1985) 268-270. Particularly
preferred CDRs correspond to those representing sequences
recognizing the antigens noted above for chimeric antibodies. 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.
[0129] The term "human antibody", as used herein, is intended to
include antibodies having variable and constant regions derived
from human germ line immunoglobulin sequences. Human antibodies are
well-known in the state of the art (van Dijk, M. A., and van de
Winkel, J. G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human
antibodies can also be produced in transgenic animals (e.g., mice)
that are capable, upon immunization, of producing a full repertoire
or a selection of human antibodies in the absence of endogenous
immunoglobulin production. Transfer of the human germ-line
immunoglobulin gene array in such germ-line mutant mice will result
in the production of human antibodies upon antigen challenge (see,
e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993)
2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258;
Bruggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human
antibodies can also be produced in phage display libraries
(Hoogenboom, H. R., and Winter, G., J. Mol. Biol. 227 (1992)
381-388; Marks, J. D., et al., J. Mol. Biol. 222 (1991) 581-597).
The techniques of Cole et al. and Boerner et al. are also available
for the preparation of human monoclonal antibodies (Cole, et al.,
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77
(1985); and Boerner, P., et al., J. Immunol. 147 (1991) 86-95). As
already 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).
[0130] 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.
[0131] The "variable domain" (variable domain of a light chain
(VL), variable region of a heavy chain (VH) as used herein denotes
each of the pair of light and heavy chains which is involved
directly in binding the antibody to the antigen. The domains of
variable human light and heavy chains have the same general
structure and each domain comprises four framework (FR) regions
whose sequences are widely conserved, connected by three
"hypervariable regions" (or complementarity determining regions,
CDRs). The framework regions adopt a .beta.-sheet conformation and
the CDRs may form loops connecting the .beta.-sheet structure. The
CDRs in each chain are held in their three-dimensional structure by
the framework regions and form together with the CDRs from the
other chain the antigen binding site. The antibody heavy and light
chain CDR3 regions play a particularly important role in the
binding specificity/affinity of the antibodies according to the
invention and therefore provide a further object of the
invention.
[0132] The terms "hypervariable region" or "antigen-binding portion
of an antibody" when used herein refer to the amino acid residues
of an antibody which are responsible for antigen-binding. The
hypervariable region comprises amino acid residues from the
"complementarity 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 chains of an antibody comprise from N- to
C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
CDRs on each chain are separated by such framework amino acids.
Especially, CDR3 of the heavy chain is the region which contributes
most to antigen binding. 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).
[0133] As used herein, the term "binding" or "specifically binding"
refers to the binding of the monovalent antigen binding protein to
an epitope of the antigen in an in vitro assay, preferably in an
plasmon resonance assay (BIAcore, GE-Healthcare Uppsala, Sweden)
with purified wild-type antigen. The affinity of the binding is
defined by the terms ka (rate constant for the association of the
antibody from the antibody/antigen complex), k.sub.D (dissociation
constant), and K.sub.D (k.sub.D/ka). Binding or specifically
binding means a binding affinity (K.sub.D) of 10.sup.-8 mol/l or
less, preferably 10.sup.-9 M to 10.sup.-13 mol/l. Thus, a
monovalent antigen binding protein according to the invention is
specifically binding to each antigen for which it is specific with
a binding affinity (K.sub.D) of 10.sup.-8 mol/l or less, preferably
10.sup.-9 M to 10.sup.-13 mol/l.
[0134] Binding of the monovalent antigen binding protein to the
Fc.gamma.RIII 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), k.sub.D (dissociation constant), and
K.sub.D (k.sub.D/ka).
[0135] The term "epitope" includes any polypeptide determinant
capable of specific binding to a monovalent antigen binding
proteins. 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 a monovalent antigen
binding protein.
[0136] In certain embodiments, an antibody is said to specifically
bind an antigen when it preferentially recognizes its target
antigen in a complex mixture of proteins and/or macromolecules.
[0137] In a further embodiment the monovalent antigen binding
protein according to the invention is characterized in that said
full length antibody is of human IgG1 subclass, or of human IgG1
subclass with the mutations L234A and L235A.
[0138] In a further embodiment the monovalent antigen binding
protein according to the invention is characterized in that said
full length antibody is of human IgG2 subclass.
[0139] In a further embodiment the monovalent antigen binding
protein according to the invention is characterized in that said
full length antibody is of human IgG3 subclass.
[0140] In a further embodiment the monovalent antigen binding
protein according to the invention is characterized in that said
full length antibody is of human IgG4 subclass or, of human IgG4
subclass with the additional mutations S228P and L235E (also named
IgG4 SPLE).
[0141] The term "constant region" as used within the current
applications denotes the sum of the domains of an antibody other
than the variable region. The constant region is not involved
directly in binding of an antigen, but exhibit various effector
functions. Depending on the amino acid sequence of the constant
region of their heavy chains, antibodies are divided in the classes
(also named isotypes): IgA, IgD, IgE, IgG and IgM, and several of
these may be further divided into subclasses (also named isotypes),
such as IgG1, IgG2, IgG3, and IgG4, IgA1 and IgA2. The heavy chain
constant regions that correspond to the different classes of
antibodies are called .alpha., .delta., .epsilon., .gamma., and
.mu., respectively. The light chain constant regions (CL) which can
be found in all five antibody classes are called .kappa. (kappa)
and .lamda. (lambda).
[0142] The term "constant region derived from human origin" as used
in the current application denotes a constant heavy chain region of
a human antibody of the subclass IgG1, IgG2, IgG3, or IgG4 and/or a
constant light chain kappa or lambda region. Such constant regions
are well known in the state of the art and e.g. described by Kabat,
E. A., (see e.g. Johnson, G. and Wu, T. T., Nucleic Acids Res. 28
(2000) 214-218; Kabat, E. A., et al., Proc. Natl. Acad. Sci. USA 72
(1975) 2785-2788).
[0143] While antibodies of the IgG4 subclass show reduced Fc
receptor (Fc.gamma.RIIIa) binding, antibodies of other IgG
subclasses show strong binding. However Pro238, Asp265, Asp270,
Asn297 (loss of Fc carbohydrate), Pro329, Leu234, Leu235, Gly236,
Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435
are residues which, if altered, provide also reduced Fc receptor
binding (Shields, R. L., et al., J. Biol. Chem. 276 (2001)
6591-6604; Lund, J., et al., FASEB J. 9 (1995) 115-119; Morgan, A.,
et al., Immunology 86 (1995) 319-324; EP 0 307 434).
[0144] In one embodiment an antibody according to the invention has
a reduced FcR binding compared to an IgG1 antibody and the full
length parent antibody is in regard to FcR binding of IgG4 subclass
or of IgG1 or IgG2 subclass with a mutation in 5228, L234, L235
and/or D265, and/or contains the PVA236 mutation. In one embodiment
the mutations in the full length parent antibody are S228P, L234A,
L235A, L235E and/or PVA236. In another embodiment the mutations in
the full length parent antibody are in IgG4 S228P and L235E and in
IgG1 L234A and L235A.
[0145] The constant region of an antibody is directly involved in
ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC
(complement-dependent cytotoxicity). Complement activation (CDC) is
initiated by binding of complement factor C1q to the constant
region of most IgG antibody subclasses. Binding of C1q to an
antibody is caused by defined protein-protein interactions at the
so called binding site. Such constant region binding sites are
known in the state of the art and described e.g. by Lukas, T. J.,
et al., J. Immunol. 127 (1981) 2555-2560; Bunkhouse, R. and Cobra,
J. J., Mol. Immunol. 16 (1979) 907-917; Burton, D. R., et al.,
Nature 288 (1980) 338-344; Thomason, J. E., et al., Mol. Immunol.
37 (2000) 995-1004; Idiocies, E. E., et al., J. Immunol. 164 (2000)
4178-4184; Hearer, M., et al., J. Virol. 75 (2001) 12161-12168;
Morgan, A., et al., Immunology 86 (1995) 319-324; and EP 0 307 434.
Such constant region binding sites are, e.g., characterized by the
amino acids L234, L235, D270, N297, E318, K320, K322, P331, and
P329 (numbering according to EU index of Kabat).
[0146] The term "antibody-dependent cellular cytotoxicity (ADCC)"
refers to lysis of human target cells by an antibody according to
the invention in the presence of effector cells. ADCC is measured
preferably by the treatment of a preparation of antigen expressing
cells with an antibody according to the invention in the presence
of effector cells such as freshly isolated PBMC or purified
effector cells from buffy coats, like monocytes or natural killer
(NK) cells or a permanently growing NK cell line.
[0147] Surprisingly it has been found out that an antigen binding
protein according to the invention show improved ADCC properties
compared to its parent full length antibody. These improved ADCC
effects achieved without further modification of the Fc part like
glycoengineering. The term "complement-dependent cytotoxicity
(CDC)" denotes a process initiated by binding of complement factor
C1q to the Fc part of most IgG antibody subclasses. Binding of C1q
to an antibody is caused by defined protein-protein interactions at
the so called binding site. Such Fc part binding sites are known in
the state of the art (see above). Such Fc part binding sites are,
e.g., characterized by the amino acids L234, L235, D270, N297,
E318, K320, K322, P331, and P329 (numbering according to EU index
of Kabat). Antibodies of subclass IgG1, IgG2, and IgG3 usually show
complement activation including C1q and C3 binding, whereas IgG4
does not activate the complement system and does not bind C1q
and/or C3.
[0148] Cell-mediated effector functions of monoclonal antibodies
can be enhanced by engineering their oligosaccharide component as
described in Umana, P., et al., Nature Biotechnol. 17 (1999)
176-180, and U.S. Pat. No. 6,602,684. IgG1 type antibodies, the
most commonly used therapeutic antibodies, are glycoproteins that
have a conserved N-linked glycosylation site at Asn297 in each CH2
domain. The two complex biantennary oligosaccharides attached to
Asn297 are buried between the CH2 domains, forming extensive
contacts with the polypeptide backbone, and their presence is
essential for the antibody to mediate effector functions such as
antibody dependent cellular cytotoxicity (ADCC) (Lifely, M. R., et
al., Glycobiology 5 (1995) 813-822; Jefferis, R., et al., Immunol.
Rev. 163 (1998) 59-76; Wright, A., and Morrison, S., L., Trends
Biotechnol. 15 (1997) 26-32). Umana, P., et al. Nature Biotechnol.
17 (1999) 176-180 and WO 99/54342 showed that overexpression in
Chinese hamster ovary (CHO) cells of
.beta.(1,4)-N-acetylglucosaminyltransferase III ("GnTIII"), a
glycosyltransferase catalyzing the formation of bisected
oligosaccharides, significantly increases the in vitro ADCC
activity of antibodies. Alterations in the composition of the
Asn297 carbohydrate or its elimination affect also binding to
Fc.gamma.R and C1q (Umana, P., et al., Nature Biotechnol. 17 (1999)
176-180; Davies, J., et al., Biotechnol. Bioeng. 74 (2001) 288-294;
Mimura, Y., et al., J. Biol. Chem. 276 (2001) 45539-45547; Radaev,
S., et al., J. Biol. Chem. 276 (2001) 16478-16483; Shields, R., L.,
et al., J. Biol. Chem. 276 (2001) 6591-6604; Shields, R., L., et
al., J. Biol. Chem. 277 (2002) 26733-26740; Simmons, L., C., et
al., J. Immunol. Methods 263 (2002) 133-147).
[0149] In one aspect of the invention the monovalent antigen
binding protein according to the invention is characterized in that
the modified heavy chains of a) and b) are of IgG1 isotype, and the
antigen binding protein is afucosylated with an the amount of
fucose of 80% or less of the total amount of oligosaccharides
(sugars) at Asn297.
[0150] In one embodiment the antigen binding protein is
afucosylated with an the amount of fucose of 65% to 5% of the total
amount of oligosaccharides (sugars) at Asn297.
[0151] The term "afucosylated antigen binding protein" refers to an
antigen binding proteins of IgG1 or IgG3 isotype (preferably of
IgG1 isotype) with an altered pattern of glycosylation in the Fc
region at Asn297 having a reduced level of fucose residues.
Glycosylation of human IgG1 or IgG3 occurs at Asn297 as core
fucosylated bianntennary complex oligosaccharide glycosylation
terminated with up to 2 Gal residues. These structures are
designated as G0, G1 (.alpha.-1,6 or .alpha.-1,3) or G2 glycan
residues, depending from the amount of terminal Gal residues (Raju,
T. S., BioProcess Int. 1 (2003) 44-53). CHO type glycosylation of
antibody Fc parts is e.g. described by Routier, F. H.,
Glycoconjugate J. 14 (1997) 201-207. Antibodies which are
recombinantely expressed in non glycomodified CHO host cells
usually are fucosylated at Asn297 in an amount of at least 85%. It
should be understood that the term an afucosylated antibody as used
herein includes an antibody having no fucose in its glycosylation
pattern. It is commonly known that typical glycosylated residue
position in an antibody is the asparagine at position 297 according
to the EU numbering system ("Asn297").
[0152] Thus an afucosylated antigen binding protein according to
the invention means an antibody of IgG1 or IgG3 isotype (preferably
of IgG1 isotype) wherein the amount of fucose is 80% or less (e.g.
of 80% to 1%) of the total amount of oligosaccharides (sugars) at
Asn297 (which means that at least 20% or more of the
oligosaccharides of the Fc region at Asn297 are afucosylated). In
one embodiment the amount of fucose is 65% or less (e.g. of 65% to
1%), in one embodiment from 65% to 5%, in one embodiment from 40%
to 20% of the oligosaccharides of the Fc region at Asn297.
According to the invention "amount of fucose" means the amount of
said oligosaccharide (fucose) within the oligosaccharide (sugar)
chain at Asn297, related to the sum of all oligosaccharides
(sugars) attached to Asn 297 (e.g. complex, hybrid and high mannose
structures) measured by MALDI-TOF mass spectrometry and calculated
as average value (for a detailed procedure to determine the amount
of fucose, see e.g. WO 2008/077546). Furthermore in one embodiment,
the oligosaccharides of the Fc region are bisected. The
afucosylated antibody according to the invention can be expressed
in a glycomodified host cell engineered to express at least one
nucleic acid encoding a polypeptide having GnTIII activity in an
amount sufficient to partially fucosylate the oligosaccharides in
the Fc region. In one embodiment, the polypeptide having GnTIII
activity is a fusion polypeptide. Alternatively
.alpha.-1,6-fucosyltransferase activity of the host cell can be
decreased or eliminated according to U.S. Pat. No. 6,946,292 to
generate glycomodified host cells. The amount of antibody
fucosylation can be predetermined e.g. either by fermentation
conditions (e.g. fermentation time) or by combination of at least
two antibodies with different fucosylation amount. Such
afucosylated antigen binding proteins and respective
glycoengineering methods are described in WO 2005/044859, WO
2004/065540, WO 2007/031875, Umana, P., et al., Nature Biotechnol.
17 (1999) 176-180, WO 99/154342, WO 2005/018572, WO 2006/116260, WO
2006/114700, WO 2005/011735, WO 2005/027966, WO 97/028267, US
2006/0134709, US 2005/0054048, US 2005/0152894, WO 2003/035835, WO
2000/061739. These glycoengineered antigen binding proteins
according to the invention have an increased ADCC (compared to the
parent antigen binding proteins). Other glycoengineering methods
yielding afucosylated antigen binding proteins according to the
invention are described e.g. in Niwa, R. et al., J. Immunol.
Methods 306 (2005) 151-160; Shinkawa, T., et al., J. Biol. Chem.,
278 (2003) 3466-3473; WO 03/055993 or US 2005/0249722.
[0153] Thus one aspect of the invention is an afucosylated antigen
binding protein according to the invention which of IgG1 isotype or
IgG3 isotype (preferably of IgG1 isotype) with an amount of fucose
of 60% or less (e.g. of 60% to 1%) of the total amount of
oligosaccharides (sugars) at Asn297, for the treatment of cancer
in. In another aspect of the invention is the use of an
afucosylated anti-CD20 antibody of IgG1 or IgG3 isotype (preferably
of IgG1 isotype) specifically binding to CD20 with an amount of
fucose of 60% or less of the total amount of oligosaccharides
(sugars) at Asn297, for the manufacture of a medicament for the
treatment of cancer. In one embodiment the amount of fucose is
between 60% and 20% of the total amount of oligosaccharides
(sugars) at Asn297. In one embodiment the amount of fucose is
between 60% and 40% of the total amount of oligosaccharides
(sugars) at Asn297. In one embodiment the amount of fucose is
between 0% of the total amount of oligosaccharides (sugars) at
Asn297.
[0154] The "EU numbering system" or "EU index (according to Kabat)"
is generally used when referring to a residue or position in an
immunoglobulin heavy chain constant region (e.g., the EU index is
reported in Kabat et al., Sequences of Proteins of Immunological
Interest, 5th ed., Public Health Service, National Institutes of
Health, Bethesda, Md. (1991) expressly incorporated herein by
reference).
[0155] The term "the sugar chains show characteristics of N-linked
glycans attached to Asn297 of an antibody recombinantly expressed
in a CHO cell" denotes that the sugar chain at Asn297 of the full
length parent antibody according to the invention has the same
structure and sugar residue sequence except for the fucose residue
as those of the same antibody expressed in unmodified CHO cells,
e.g. as those reported in WO 2006/103100.
[0156] The term "NGNA" as used within this application denotes the
sugar residue N-glycolylneuraminic acid.
[0157] The antibody according to the invention is produced by
recombinant means. Thus, one aspect of the current invention is a
nucleic acid encoding the antibody according to the invention and a
further aspect is a cell comprising said nucleic acid encoding an
antibody according to the invention. Methods for recombinant
production are widely known in the state of the art and comprise
protein expression in prokaryotic and eukaryotic cells with
subsequent isolation of the antibody and usually purification to a
pharmaceutically acceptable purity. For the expression of the
antibodies as aforementioned in a host cell, nucleic acids encoding
the respective modified light and heavy chains are inserted into
expression vectors by standard methods. Expression is performed in
appropriate prokaryotic or eukaryotic host cells like CHO cells,
NS0 cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells,
yeast, or E. coli cells, and the antibody is recovered from the
cells (supernatant or cells after lysis). General methods for
recombinant production of antibodies are well-known in the state of
the art and described, for example, in the review articles of
Makrides, S. C., Protein Expr. Purif. 17 (1999) 183-202; Geisse,
S., et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R. J.,
Mol. Biotechnol. 16 (2000) 151-161; Werner, R. G., Drug Res. 48
(1998) 870-880.
[0158] The monovalent antigen binding proteins according to the
invention are suitably separated from the culture medium by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography. DNA and RNA
encoding the monoclonal antibodies is readily isolated and
sequenced using conventional procedures. The hybridoma cells can
serve as a source of such DNA and RNA. Once isolated, the DNA may
be inserted into expression vectors, which are then transfected
into host cells such as HEK 293 cells, CHO cells, or myeloma cells
that do not otherwise produce immunoglobulin protein, to obtain the
synthesis of recombinant monoclonal antibodies in the host
cells.
[0159] Amino acid sequence variants (or mutants) of the monovalent
antigen binding protein are prepared by introducing appropriate
nucleotide changes into the antibody DNA, or by nucleotide
synthesis. Such modifications can be performed, however, only in a
very limited range, e.g. as described above. For example, the
modifications do not alter the above mentioned antibody
characteristics such as the IgG isotype and antigen binding, but
may improve the yield of the recombinant production, protein
stability or facilitate the purification.
[0160] The term "host cell" as used in the current application
denotes any kind of cellular system which can be engineered to
generate the antibodies according to the current invention. In one
embodiment HEK293 cells and CHO cells are used as host cells. As
used herein, the expressions "cell," "cell line," and "cell
culture" are used interchangeably and all such designations include
progeny. Thus, the words "transformants" and "transformed cells"
include the primary subject cell and cultures derived therefrom
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.
[0161] Expression in NS0 cells is described by, e.g., Barnes, L.
M., et al., Cytotechnology 32 (2000) 109-123; Barnes, L. M., et
al., Biotech. Bioeng. 73 (2001) 261-270. Transient expression is
described by, e.g., Durocher, Y., et al., Nucl. Acids. Res. 30
(2002) E9. Cloning of variable domains is described by Orlandi, R.,
et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P.,
et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and
Norderhaug, L., et al., J. Immunol. Methods 204 (1997) 77-87. A
preferred transient expression system (HEK 293) is described by
Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30 (1999)
71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996)
191-199.
[0162] The control sequences that are suitable for prokaryotes, for
example, include a promoter, optionally an operator sequence, and a
ribosome binding site. Eukaryotic cells are known to utilize
promoters, enhancers and polyadenylation signals.
[0163] A nucleic acid is "operably linked" when it is placed in a
functional relationship with another nucleic acid sequence. For
example, DNA for a pre-sequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a pre-protein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading frame. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0164] Purification of monovalent antigen binding proteins is
performed in order to eliminate cellular components or other
contaminants, e.g. other cellular nucleic acids or proteins (e.g.
byproducts) by standard techniques, including alkaline/SDS
treatment, CsCl banding, column chromatography, agarose gel
electrophoresis, and others well known in the art (see Ausubel, F.,
et al. (eds.), Current Protocols in Molecular Biology, Greene
Publishing and Wiley Interscience, New York (1987)). Different
methods are well established and widespread used for protein
purification, such as affinity chromatography with microbial
proteins (e.g. protein A or protein G affinity chromatography), ion
exchange chromatography (e.g. cation exchange (carboxymethyl
resins), anion exchange (amino ethyl resins) and mixed-mode
exchange), thiophilic adsorption (e.g. with beta-mercaptoethanol
and other SH ligands), hydrophobic interaction or aromatic
adsorption chromatography (e.g. with phenyl-sepharose,
aza-arenophilic resins, or m-aminophenylboronic acid), metal
chelate affinity chromatography (e.g. with Ni(II)- and
Cu(II)-affinity material), size exclusion chromatography, and
electrophoretical methods (such as gel electrophoresis, capillary
electrophoresis) (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75
(1998) 93-102). An example of a purification is described in
Example 1 and the corresponding FIGS. 3 to 8.
[0165] One aspect of the invention is a pharmaceutical composition
comprising an antibody according to the invention. Another aspect
of the invention is the use of an antibody according to the
invention for the manufacture of a pharmaceutical composition. A
further aspect of the invention is a method for the manufacture of
a pharmaceutical composition comprising an antibody according to
the invention. In another aspect, the present invention provides a
composition, e.g. a pharmaceutical composition, containing an
antibody according to the present invention, formulated together
with a pharmaceutical carrier.
[0166] One embodiment of the invention is the monovalent antigen
binding protein according to the invention for the treatment of
cancer.
[0167] Another aspect of the invention is said pharmaceutical
composition for the treatment of cancer.
[0168] One embodiment of the invention is the monovalent antigen
binding protein according to the invention for use in the treatment
of cancer.
[0169] Another aspect of the invention is the use of an antibody
according to the invention for the manufacture of a medicament for
the treatment of cancer.
[0170] Another aspect of the invention is method of treatment of
patient suffering from cancer by administering an antibody
according to the invention to a patient in the need of such
treatment.
[0171] As used herein, "pharmaceutical carrier" includes any and
all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like that are physiologically compatible. Preferably, the carrier
is suitable for intravenous, intramuscular, subcutaneous,
parenteral, spinal or epidermal administration (e.g. by injection
or infusion).
[0172] A composition of the present invention can be administered
by a variety of methods known in the art. As will be appreciated by
the skilled artisan, the route and/or mode of administration will
vary depending upon the desired results. To administer a compound
of the invention by certain routes of administration, it may be
necessary to coat the compound with, or co-administer the compound
with, a material to prevent its inactivation. For example, the
compound may be administered to a subject in an appropriate
carrier, for example, liposomes, or a diluent. Pharmaceutically
acceptable diluents include saline and aqueous buffer solutions.
Pharmaceutical carriers include sterile aqueous solutions or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. The use of such
media and agents for pharmaceutically active substances is known in
the art.
[0173] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intra-arterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intra-articular, subcapsular,
subarachnoid, intraspinal, epidural and intrasternal injection and
infusion.
[0174] The term cancer as used herein refers to proliferative
diseases, such as lymphomas, lymphocytic leukemias, lung cancer,
non small cell lung (NSCL) cancer, bronchioloalviolar cell lung
cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the
head or neck, cutaneous or intraocular melanoma, uterine cancer,
ovarian cancer, rectal cancer, cancer of the anal region, stomach
cancer, gastric cancer, colon cancer, breast cancer, uterine
cancer, carcinoma of the fallopian tubes, carcinoma of the
endometrium, carcinoma of the cervix, carcinoma of the vagina,
carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus,
cancer of the small intestine, cancer of the endocrine system,
cancer of the thyroid gland, cancer of the parathyroid gland,
cancer of the adrenal gland, sarcoma of soft tissue, cancer of the
urethra, cancer of the penis, prostate cancer, cancer of the
bladder, cancer of the kidney or ureter, renal cell carcinoma,
carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer,
biliary cancer, neoplasms of the central nervous system (CNS),
spinal axis tumors, brain stem glioma, glioblastoma multiforme,
astrocytomas, schwanomas, ependymonas, medulloblastomas,
meningiomas, squamous cell carcinomas, pituitary adenoma and Ewings
sarcoma, including refractory versions of any of the above cancers,
or a combination of one or more of the above cancers.
[0175] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures, supra, and by the inclusion of
various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol, sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin.
[0176] Regardless of the route of administration selected, the
compounds of the present invention, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
present invention, are formulated into pharmaceutically acceptable
dosage forms by conventional methods known to those of skill in the
art.
[0177] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, the route of administration, the time of administration,
the rate of excretion of the particular compound being employed,
the duration of the treatment, other drugs, compounds and/or
materials used in combination with the particular compositions
employed, the age, sex, weight, condition, general health and prior
medical history of the patient being treated, and like factors well
known in the medical arts.
[0178] The composition must be sterile and fluid to the extent that
the composition is deliverable by syringe. In addition to water,
the carrier preferably is an isotonic buffered saline solution.
[0179] Proper fluidity can be maintained, for example, by use of
coating such as lecithin, by maintenance of required particle size
in the case of dispersion and by use of surfactants. In many cases,
it is preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol or sorbitol, and sodium chloride in
the composition.
[0180] As used herein, the expressions "cell," "cell line," and
"cell culture" are used interchangeably and all such designations
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and cultures derived
therefrom 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. Where
distinct designations are intended, it will be clear from the
context.
[0181] The term "transformation" as used herein refers to process
of transfer of a vectors/nucleic acid into a host cell. If cells
without formidable cell wall barriers are used as host cells,
transfection is carried out e.g. by the calcium phosphate
precipitation method as described by Graham and Van der Eh,
Virology 52 (1978) 546. However, other methods for introducing DNA
into cells such as by nuclear injection or by protoplast fusion may
also be used. If prokaryotic cells or cells which contain
substantial cell wall constructions are used, e.g. one method of
transfection is calcium treatment using calcium chloride as
described by Cohen, F. N, et al., PNAS. 69 (1972) 7110.
[0182] As used herein, "expression" refers to the process by which
a nucleic acid is transcribed into mRNA and/or to the process by
which the transcribed mRNA (also referred to as transcript) is
subsequently being translated into peptides, polypeptides, or
proteins. The transcripts and the encoded polypeptides are
collectively referred to as gene product. If the polynucleotide is
derived from genomic DNA, expression in a eukaryotic cell may
include splicing of the mRNA.
[0183] A "vector" is a nucleic acid molecule, in particular
self-replicating, which transfers an inserted nucleic acid molecule
into and/or between host cells. The term includes vectors that
function primarily for insertion of DNA or RNA into a cell (e.g.,
chromosomal integration), replication of vectors that function
primarily for the replication of DNA or RNA, and expression vectors
that function for transcription and/or translation of the DNA or
RNA. Also included are vectors that provide more than one of the
functions as described.
[0184] An "expression vector" is a polynucleotide which, when
introduced into an appropriate host cell, can be transcribed and
translated into a polypeptide. An "expression system" usually
refers to a suitable host cell comprised of an expression vector
that can function to yield a desired expression product.
[0185] The following examples, sequence listing and figures are
provided to aid the understanding of the present invention, the
true scope of which is set forth in the appended claims. It is
understood that modifications can be made in the procedures set
forth without departing from the spirit of the invention.
DESCRIPTION OF THE SEQUENCE LISTING
[0186] SEQ ID NO:1 c-Met 5D5 MoAb ("wt")-modified heavy chain a)
VL-CH1-CH2-CH3
[0187] SEQ ID NO:2 c-Met 5D5 MoAb ("wt")-modified heavy chain b)
VH-CL-CH2-CH3
[0188] SEQ ID NO:3 IGF1R AK18 MoAb ("wt")-modified heavy chain a)
VL-CH1-CH2-CH3
[0189] SEQ ID NO:4 IGF1R AK18 MoAb ("wt")-modified heavy chain b)
VH-CL-CH2-CH3
[0190] SEQ ID NO:5 Her3 205 MoAb ("wt")-modified heavy chain a)
VL-CH1-CH2-CH3
[0191] SEQ ID NO:6 Her3 205 MoAb ("wt")-modified heavy chain b)
VH-CL-CH2-CH3
[0192] SEQ ID NO:7 c-Met 5D5 MoAb KiH modified heavy chain a)
VL-CH1-CH2-CH3 knob T366W, S354C
[0193] SEQ ID NO:8 c-Met 5D5 MoAb KiH modified heavy chain b)
VH-CL-CH2-CH3 hole L368A, Y407V, T366S, Y349C
[0194] SEQ ID NO:9 IGF1R AK18 MoAb KiH modified heavy chain a)
VL-CH1-CH2-CH3 knob T366W, S354C
[0195] SEQ ID NO:10 IGF1R AK18 MoAb KiH modified heavy chain b)
VH-CL-CH2-CH3 hole L368A, Y407V, T366S, Y349C
[0196] SEQ ID NO:11 Her3 205 MoAb KiH modified heavy chain a)
VL-CH1-CH2-CH3 knob T366W, S354C
[0197] SEQ ID NO:12 Her3 205 MoAb KiH modified heavy chain b)
VH-CL-CH2-CH3 hole L368A, Y407V, T366S, Y349C
EXAMPLES
[0198] The present invention is described in further detain in the
following examples which are not in any way intended to limit the
scope of the invention as claimed. All references cited are herein
specifically incorporated by reference for all that is described
therein. The following examples are offered to illustrate, but not
to limit the claimed invention.
Experimental Procedures
A. Materials and Methods
Recombinant DNA Techniques
[0199] 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.
DNA and Protein Sequence Analysis and Sequence Data Management
[0200] 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.
DNA Sequencing
[0201] DNA sequences were determined by double strand sequencing
performed at SequiServe (Vaterstetten, Germany) and Geneart AG
(Regensburg, Germany).
Gene Synthesis
[0202] 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 subcloned gene fragments was
confirmed by DNA sequencing. DNA sequences encoding for the two
antibody chains (VH-CL-CH2-CH3 and VL-CH1-CH2-CH3) were prepared as
whole fragments by gene synthesis with flanking 5'HpaI and 3'NaeI
restriction sites. Gene Segments coding "knobs-into-hole", meaning
one antibody heavy chain carrying a T366W mutation in the CH3
domain as well as a second antibody heavy chain carrying T366S,
L368A and Y407V mutations in the CH3 domain were synthesized with
5'-BclI and 3'-NaeI restriction sites. In a similar manner, DNA
sequences coding "knobs-into-hole" antibody heavy chain carrying
S354C and T366W mutations in the CH3 domain as well as a second
antibody heavy chain carrying Y349C, T366S, L368A and Y407V
mutations were prepared by gene synthesis with flanking BclI and
NaeI restriction sites. All constructs were designed with a 5'-end
DNA sequence coding for a leader peptide, which targets proteins
for secretion in eukaryotic cells.
Construction of the Expression Plasmids
[0203] A Roche expression vector was used for the construction of
all antibody chains. The vector is composed of the following
elements: [0204] an origin of replication, oriP, of Epstein-Barr
virus (EBV), [0205] an origin of replication from the vector pUC18
which allows replication of this plasmid in E. coli [0206] a
beta-lactamase gene which confers ampicillin resistance in E. coli,
[0207] the immediate early enhancer and promoter from the human
cytomegalovirus (HCMV), [0208] the human 1-immunoglobulin
polyadenylation ("poly A") signal sequence, and [0209] unique HpaI,
BclI, and NaeI restriction sites.
[0210] The immunoglobulin genes in the order of VH-CL-CH2-CH3 and
VL-CH1-CH2-CH3 as well as "knobs-into-hole" constructs were
prepared by gene synthesis and cloned into pGA18 (ampR) plasmids as
described. The pG18 (ampR) plasmids carrying the synthesized DNA
segments and the Roche expression vector were digested either with
HpaI and NaeI or with BclI and NaeI restriction enzymes (Roche
Molecular Biochemicals) and subjected to agarose gel
electrophoresis. Purified DNA segments were then ligated to the
isolated Roche expression vector HpaI/NaeI or BclI/NaeI 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.
Transient Expression of Immunoglobulin Variants in HEK293 Cells
[0211] 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). Briefly, suspension
FreeStyle.TM. 293-F cells were cultivated in FreeStyle.TM. 293
Expression medium at 37.degree. C./8% CO.sub.2. Cells were seeded
in fresh medium at a density of 1-2.times.10.sup.6 viable cells/ml
on the day of transfection. DNA-293fectin.TM. complexes were
prepared in Opti-MEM.RTM. I medium (Invitrogen, USA) using 325
.mu.l of 293fectin.TM. (Invitrogen, Germany) and 250 .mu.g of each
plasmid DNA in a 1:1 molar ratio for a 250 ml final transfection
volume. 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.
[0212] Alternatively, antibodies were generated by transient
transfection in HEK293-EBNA cells. Antibodies were expressed by
transient co-transfection of the respective expression plasmids in
adherently growing HEK293-EBNA cells (human embryonic kidney cell
line 293 expressing Epstein-Barr-Virus nuclear antigen; American
type culture collection deposit number ATCC # CRL-10852, Lot. 959
218) cultivated in DMEM (Dulbecco's modified Eagle's medium, Gibco)
supplemented with 10% Ultra Low IgG FCS (fetal calf serum, Gibco),
2 mM L-Glutamine (Gibco), and 250 .mu.g/ml Geneticin (Gibco). For
transfection FuGENE.TM. 6 Transfection Reagent (Roche Molecular
Biochemicals) was used in a ratio of FuGENE.TM. reagent (.mu.l) to
DNA (.mu.g) of 4:1 (ranging from 3:1 to 6:1). Proteins were
expressed from the respective plasmids using an equimolar ratio of
plasmids. Cells were feeded at day 3 with L-Glutamine ad 4 mM,
Glucose [Sigma] and NAA [Gibco]. Bispecific antibody containing
cell culture supernatants were harvested from day 5 to 11 after
transfection by centrifugation and stored at -200 C. 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.
Purification of Antibodies
[0213] 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. Then, the 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 or 26/60 gel
filtration column (GE Healthcare, Sweden) equilibrated with 20 mM
Histidin, 140 mM NaCl, pH 6.0. Fractions containing purified
antibodies with less than 5% high molecular weight aggregates were
pooled and stored as 1.0 mg/ml aliquots at -80.degree. C.
Analysis of Purified Proteins
[0214] 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 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 (4-12%
Tris-Glycine gels). The aggregate content of 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. For stability
analysis, concentrations of 1 mg/ml of purified proteins were
incubated at 4.degree. C. and 40.degree. C. for 7 days and then
evaluated by high-performance SEC (e.g. HP SEC Analysis (Purified
Protein). 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).
Mass Spectrometry and SEC-MALLS
Mass Spectrometry
[0215] The total deglycosylated mass of antibodies was determined
and confirmed via electrospray ionization mass spectrometry
(ESI-MS). Briefly, 100 .mu.g purified antibodies were
deglycosylated with 50 mU N-Glycosidase F (PNGaseF, ProZyme) in 100
mM KH2PO4/K2HPO4, pH 7 at 37.degree. C. for 12-24 h at a protein
concentration of up to 2 mg/ml and subsequently desalted via HPLC
on a Sephadex G25 column (GE Healthcare). The mass of the
respective heavy and light chains was determined by ESI-MS after
deglycosylation and reduction. In brief, 50 .mu.g antibody in 115
.mu.l were incubated with 60 .mu.l 1M TCEP and 50 .mu.l 8 M
Guanidine-hydrochloride subsequently desalted. The total mass and
the mass of the reduced heavy and light chains was determined via
ESI-MS on a Q-Star Elite MS system equipped with a NanoMate source.
The mass range recorded depends on the samples molecular weight. In
general for reduced antibodies the mass range was set from 600-2000
m/z and for non reduced antibodies or bispecific molecules from
1000-3600 m/z.
SEC-MALLS
[0216] SEC-MALLS (size-exclusion chromatography with multi-angle
laser light scattering) was used to determine the approximate
molecular weight of proteins in solution. According to the light
scattering theory, MALLS allows molecular weight estimation of
macromolecules irrespective of their molecular shape or other
presumptions. SEC-MALLS is based on a separation of proteins
according to their size (hydrodynamic radius) via SEC
chromatography, followed by concentration- and scattered
light-sensitive detectors. SEC-MALLS typically gives rise to
molecular weight estimates with an accuracy that allows clear
discrimination between monomers, dimers, trimers etc., provided the
SEC separation is sufficient.
[0217] In this work, the following instrumentation was used: Dionex
Ultimate 3000 HPLC; column: Superose6 10/300 (GE Healthcare);
eluent: 1.times.PBS; flow rate: 0.25 mL/min; detectors: OptiLab REX
(Wyatt Inc., Dembach), MiniDawn Treos (Wyatt Inc., Dembach).
Molecular weights were calculated with the Astra software, version
5.3.2.13. Protein amounts between 50 and 150 .mu.g were loaded on
the column and BSA (Sigma Aldrich) was used as a reference
protein.
Dynamic Light Scattering (DLS) Timecourse
[0218] Samples (30 .mu.L) at a concentration of approx. 1 mg/mL in
20 mM His/HisCl, 140 mM NaCl, pH 6.0, were filtered via a 384-well
filter plate (0.45 .mu.m pore size) into a 384-well optical plate
(Corning) and covered with 20 .mu.L paraffin oil (Sigma). Dynamic
light scattering data were collected repeatedly during a period of
5 days with a DynaPro DLS plate reader (Wyatt) at a constant
temperature of 40.degree. C. Data were processed with Dynamics
V6.10 (Wyatt).
c-Met Phosphorylation Assay
[0219] 5.times.10e5 A549 cells were seeded per well of a 6-well
plate the day prior HGF stimulation in RPMI with 0.5% FCS (fetal
calf serum). The next day, growth medium was replaced for one hour
with RPMI containing 0.2% BSA (bovine serum albumine). 12.5
.mu.g/mL of the bispecific antibody was then added to the medium
and cells were incubated for 15 minutes upon which HGF (R&D,
294-HGN) was added for further 10 minutes in a final concentration
of 25 ng/mL. Cells were washed once with ice cold PBS containing 1
mM sodium vanadate upon which they were placed on ice and lysed in
the cell culture plate with 100 .mu.L lysis buffer (50 mM Tris-Cl
pH7.5, 150 mM NaCl, 1% NP40, 0.5% DOC, aprotinine, 0.5 mM PMSF, 1
mM sodium-vanadate). Cell lysates were transferred to eppendorf
tubes and lysis was allowed to proceed for 30 minutes on ice.
Protein concentration was determined using the BCA method (Pierce).
30-50 .mu.g of the lysate was separated on a 4-12% Bis-Tris NuPage
gel (Invitrogen) and proteins on the gel were transferred to a
nitrocellulose membrane. Membranes were blocked for one hour with
TBS-T containing 5% BSA and developed with a phospho-specific c-Met
antibody directed against Y1349 (Epitomics, 2319-1) according to
the manufacturer's instructions. Immunoblots were reprobed with an
antibody binding to unphosphorylated c-Met (Santa Cruz,
sc-161).
Her3 (ErbB3) Phosphorylation Assay
[0220] 2.times.10e5 MCF7 cells were seeded per well of a 12-well
plate in complete growth medium (RPMI 1640, 10% FCS). Cells were
allowed to grow to 90% confluency within two days. Medium was then
replaced with starvation medium containing 0.5% FCS. The next day
the respective antibodies were supplemented at the indicated
concentrations 1 hour prior addition of 500 ng/mL Heregulin
(R&D). Upon addition of Heregulin cells were cultivated further
10 minutes before the cells were harvested and lysed. Protein
concentration was determined using the BCA method (Pierce). 30-50
.mu.g of the lysate was separated on a 4-12% Bis-Tris NuPage gel
(Invitrogen) and proteins on the gel were transferred to a
nitrocellulose membrane. Membranes were blocked for one hour with
TBS-T containing 5% BSA and developed with a phospho-specific
Her3/ErbB3 antibody specifically recognizing Tyr1289 (4791, Cell
Signaling).
FACS
[0221] A549 were detached and counted. 1.5.times.10e5 cells were
seeded per well of a conical 96-well plate. Cells were spun down
(1500 rpm, 4.degree. C., 5 min) and incubated for 30 min on ice in
50 .mu.L of a dilution series of the respective bispecific antibody
in PBS with 2% FCS (fetal calf serum). Cells were again spun down
and washed once with 200 .mu.L PBS containing 2% FCS followed by a
second incubation of 30 min with 5 .mu.g/mL of Alexa488-coupled
antibody directed against human Fc which was diluted in PBS
containing 2% FCS (Jackson Immunoresearch, 109116098). Cells were
spun down washed twice with 200 .mu.L PBS containing 2% FCS,
resuspended in BD CellFix solution (BD Biosciences) and incubated
for at least 10 min on ice. Mean fluorescence intensity (mfi) of
the cells was determined by flow cytometry (FACS Canto, BD). Mfi
was determined at least in duplicates of two independent stainings
Flow cytometry spectra were further processed using the FlowJo
software (TreeStar). Half-maximal binding was determined using
XLFit 4.0 (IDBS) and the dose response one site model 205.
Surface Plasmon Resonance
[0222] The binding properties of monovalent anti-IGF-1R antibodies
were analyzed by surface plasmon resonance (SPR) technology using a
Biacore instrument (Biacore, GE-Healthcare, Uppsala). This system
is well established for the study of molecule interactions. It
allows a continuous real-time monitoring of ligand/analyte bindings
and thus the determination of association rate constants (ka),
dissociation rate constants (kd), and equilibrium constants (KD) in
various assay settings. SPR-technology is based on the measurement
of the refractive index close to the surface of a gold coated
biosensor chip. Changes in the refractive index indicate mass
changes on the surface caused by the interaction of immobilized
ligand with analyte injected in solution. If molecules bind to
immobilized ligand on the surface the mass increases, in case of
dissociation the mass decreases. For capturing anti-human IgG
antibody was immobilized on the surface of a CM5 biosensorchip
using amine-coupling chemistry. Flow cells were activated with a
1:1 mixture of 0.1 M N-hydroxysuccinimide and 0.1 M
3-(N,N-dimethylamino)propyl-N-ethylcarbodiimide at a flow rate of 5
.mu.l/min. Anti-human IgG antibody was injected in sodium acetate,
pH 5.0 at 10 .mu.g/ml. A reference control flow cell was treated in
the same way but with vehicle buffers only instead of the capturing
antibody. Surfaces were blocked with an injection of 1 M
ethanolamine/HCl pH 8.5. The IGF-1R antibodies were diluted in
HBS-P and injected. All interactions were performed at 25.degree.
C. (standard temperature). The regeneration solution of 3 M
Magnesium chloride was injected for 60 s at 5 .mu.l/min flow to
remove any non-covalently bound protein after each binding cycle.
Signals were detected at a rate of one signal per second. Samples
were injected at increasing concentrations. FIG. 17 depicts the
applied assay format. A low loading density with capturing antibody
density and IGF-1R antibody was chosen to enforce monovalent
binding.
[0223] For affinity measurements, human FcgIIIa was immobilized to
a CM-5 sensor chip by capturing the His-tagged receptor to an
anti-His antibody (Penta-His, Qiagen) which was coupled to the
surface by standard amine-coupling and blocking chemistry on a SPR
instrument (Biacore T100). After FcgRIIIa capturing, 50 nM IGF1R
antibodies were injected at 25.degree. C. at a flow rate of 5
.mu.L/min. The chip was afterwards regenerated with a 60s pulse of
10 mM glycine-HCl, pH 2.0 solution.
Antibody-Dependent Cellular Cytotoxicity Assay (ADCC)
[0224] Determination of antibody mediated effector functions by
anti-IGF-IR antibodies. In order to determine the capacity of the
generated antibodies to elicit immune effector mechanisms
antibody-dependent cell cytotoxicity (ADCC) studies were performed.
To study the effects of the antibodies in ADCC, DU145 IGF-IR
expressing cells (1.times.106 cells/ml) were labeled with 1 .mu.l
per ml BATDA solution (Perkin Elmer) for 25 minutes at 37.degree.
C. in a cell incubator. Afterwards, cells were washed four times
with 10 ml of RPMI-FM/PenStrep and spun down for 10 minutes at
200.times.g. Before the last centrifugation step, cell numbers were
determined and cells diluted to 1.times.10e5 cells/ml in
RPMI-FM/PenStrep medium from the pellet afterwards. The cells were
plated 5,000 per well in a round bottom plate, in a volume of 50
.mu.l. HuMAb antibodies were added at a final concentration ranging
from 25-0.1 .mu.g/ml in a volume of 50 .mu.l cell culture medium to
50 .mu.l cell suspension. Subsequently, 50 .mu.l of effector cells,
freshly isolated PBMC were added at an E:T ratio of 25:1. The
plates were centrifuged for 1 minutes at 200.times.g, followed by
an incubation step of 2 hours at 37.degree. C. After incubation the
cells were spun down for 10 minutes at 200.times.g and 20 .mu.l of
supernatant was harvested and transferred to an Optiplate 96-F
plate. 200 .mu.l of Europium solution (Perkin Elmer, at room
temperature) were added and plates were incubated for 15 minutes on
a shaker table. Fluorescence is quantified in a time-resolved
fluorometer (Victor 3, Perkin Elmer) using the Eu-TDA protocol from
Perkin Elmer. The magnitude of cell lysis by ADCC is expressed as %
of the maximum release of TDA fluorescence enhancer from the target
cells lysed by detergent corrected for spontaneous release of TDA
from the respective target cells.
IGF-1R Internalization Assay
[0225] The binding of antibodies and antigen binding protein
according the invention to the IGF-1R results in internalization
and degradation of the receptor. This process can be monitored by
incubating IGF-1R expressing HT29 CRC cells with IGF-1R targeting
antibodies followed by a quantification of remaining IGF-1R protein
levels in cell lysates by ELISA.
[0226] For this purpose, HT29 cells at 1.5.times.104 cells/well
were incubated in a 96 well MTP in RPMI with 10% FCS over night at
37.degree. C. and 5% CO2 in order to allow attachment of the cells.
Next morning, the medium was aspirated and 100 .mu.l anti IGF-1R
antibody diluted in RPMI+10% FCS was added in concentrations from
10 nM to 2 pM in 1:3 dilution steps. The cells were incubated with
antibody for 18 hours at 37.degree. C. Afterwards, the medium was
again removed and 120 .mu.l MES lysis buffer (25 mM MES pH 6.5+
Complete) were added.
[0227] For ELISA, 96-Well streptavidin coated polystyrene plates
(Nunc) were loaded with 100 .mu.l MAK<hu
IGF-1R.alpha.>hu-1a-IgG-Bi (Ch.10) diluted 1:200 in 3% BSA/PBST
(final concentration 2.4 .mu.g/ml) and incubated under constant
agitation for 1 hour at room temperature. Afterwards, the well
content was removed and each well was washed three times with 200
.mu.l PBST. 100 .mu.l of the cell lysate solution were added per
well, again incubated for 1 hour at room temperature on a plate
shaker, and washed three times with 200 .mu.l PBST. After removal
of the supernatant, 100 .mu.l/well PAK<human
IGF-1R.alpha.>Ra-C20-IgG (Santa Cruz #sc-713) diluted 1:750 in
3% BSA/PBST was added followed by the same incubation and washing
intervals as described above. In order to detect the specific
antibody bound to IGF-1R, 100 .mu.l/well of a polyclonal
horse-radish-peroxidase-coupled rabbit antibody (Cell Signaling
#7074) diluted 1:4000 in 3% BSA/PBST were added. After another
hour, unbound antibody was again removed by washing thoroughly 6
times as described above. For quantification of bound antibody, 100
.mu.l/well 3,3'-5,5'-Tetramethylbenzidin (Roche, BM-Blue
ID.-Nr.11484281) was added and incubated for 30 minutes at room
temperature. The colorigenic reaction is finally stopped by adding
25 .mu.l/well 1M H2SO4 and the light absorption is measured at 450
nm wavelength. Cells not treated with antibody are used as a
control for 0% downregulation, lysis buffer as background
control.
IGF-1R Autophosphorylation Assay (IGF-1 Stimulation)
[0228] Targeting IGF-1R by IGF-1R antibodies results in inhibition
of IGF-1 induced autophosphorylation. We investigated the
inhibition of autophosphorylation of the monovalent IGF-1R antibody
without knobs-into-holes compared to the parental IGF.-1R IgG1
antibody. For this purpose 3T3-IGF-1R cells, a murine fibroblast
cell line overexpressing human IGF-1R, were treated for 10 minutes
with 10 nM recombinant human IGF-1 in the presence of different
concentrations of monovalent and bivalent IGF-1R antibody. After
lysis of the cells, the levels of phosphorylated IGF-1R protein
were determined by a phospho-IGF-1R specific ELISA, combining a
human IGF-1R specific capture antibody and a phospho-Tyrosine
specific detection antibody.
Determination of PK Properties: Single Dose Kinetics in Mice
Methods
Animals:
[0229] NMRI mice, female, fed, 23-32 g body weight at the time
point of compound administration.
Study Protocol:
[0230] For a single i.v. dose of 10 mg/kg the mice were allocated
to 3 groups with 2-3 animals each. Blood samples are taken from
group 1 at 0.5, 168 and 672 hours, from group 2 at 24 and 336 hours
and from group 3 at 48 and 504 hours after dosing.
[0231] Blood samples of about 100 .mu.L were obtained by
retrobulbar puncture. Serum samples of at least 40 .mu.l were
obtained from blood after 1 hour at room temperature by
centrifugation (9300.times.g) at room temperature for 2.5 min.
Serum samples were frozen directly after centrifugation and stored
frozen at -20.degree. C. until analysis.
Analytics:
[0232] The concentrations of the human antibodies in mice serum
were determined with an enzyme linked immunosorbent assay (ELISA)
using 1% mouse serum. Biotinylated monoclonal antibody against
human Fc.gamma. (mAb<hFc.gamma..sub.PAN>IgG-Bi) was bound to
streptavidin coated microtiterplates in the first step. In the next
step serum samples (in various dilutions) and reference standards,
respectively, were added and bound to the immobilized
mAb<hFc.gamma..sub.PAN>IgG-Bi. Then digoxigenylated
monoclonal antibody against human Fc.gamma.
(mAb<hFc.gamma..sub.PAN>IgG-Dig) was added. The human
antibodies were detected via anti-Dig-horseradish-peroxidase
antibody-conjugate. ABTS-solution was used as the substrate for
horseradish-peroxidase. The specificity of the used capture and
detection antibody, which does not cross react with mouse IgG,
enables quantitative determination of human antibodies in mouse
serum samples.
Calculations:
[0233] The pharmacokinetic parameters were calculated by
non-compartmental analysis, using the pharmacokinetic evaluation
program WinNonlin.TM., version 5.2.1.
TABLE-US-00001 TABLE 1 Computed Pharmacokinetic Parameters:
Abbreviations of Pharmacokinetic Pharmacokinetic Parameters
Parameters Units C0 initial concentration .mu.g/mL estimated only
for bolus IV models C0_NORM initial concentration .mu.g/mL/mg/kg
estimated only for bolus IV models, dose-normalized T0 time at
initial concentration h estimated only for bolus IV models TMAX
time of maximum observed h concentration CMAX maximum observed
.mu.g/mL concentration, occurring at TMAX CMAX_NORM Cmax,
dose-normalized .mu.g/mL/mg/kg AUC_0_INF AUC extrapolated h *
.mu.g/mL AUC_0_LST AUC observed h * .mu.g/mL TLAST Time of last
observed h concentration >0 AUC_0_INF_NORM AUC extrapolated,
dose- h * .mu.g/mL/mg/ normalized kg AUC_0_LST_NORM AUC observed,
dose- h * .mu.g/mL/mg/ normalized kg PCT_AUC_EXTRA percentage AUC %
extrapolated CL_TOTAL total clearance mL/min/kg CL_TOTAL_CTG total
clearance categories L, M, H VSS steady state distribution L/kg
volume VSS_CTG steady state distribution L, M, H volume categories
VZ terminal distribution volume L/kg CL/F total clearance after non
IV mL/min/kg routes or after IV route of prodrug VZ/F terminal
distribution volume L/kg after non IV routes or after IV route of
prodrug MRT_INF mean residence time h (extrapolated) MRT_LST mean
residence time h (observed) HALFLIFE_Z terminal halflife h F
bioavailability after non IV % routes or after IV route of
prodrug
[0234] The following pharmacokinetic parameters were used for
assessing the human antibodies: [0235] The initial concentration
estimated for bolus IV models (CO). [0236] The maximum observed
concentration (C.sub.max), occurring at (T.sub.max). [0237] The
time of maximum observed concentration (T.sub.max). [0238] The area
under the concentration/time curve AUC(0-inf) was calculated by
linear trapezoidal rule (with linear interpolation) from time 0 to
infinity. [0239] The apparent terminal half-life (T.sub.1/2) was
derived from the equation: T.sub.1/2=ln 2/.lamda.z. [0240] Total
body clearance (CL) was calculated as Dose/AUC(0-inf). [0241]
Volume of distribution at steady state (Vss), calculated as
MRT(0-inf).times.CL (MRT(0-inf), defined as
AUMC(0-inf)/AUC(0-inf).
B. Examples
Example 1
Generation of Monovalent Antibody
[0242] We designed monovalent antigen binding proteins against
c-Met (SEQ ID NO:1 and SEQ ID NO:2; c-Met 5D5 MoAb ("wt")), IGF-1R
(SEQ ID NO:3 and SEQ ID NO:4.; IGF1R AK18 MoAb ("wt")) and HER3
(SEQ ID NO:5 and SEQ ID NO:6; Her3 205 MoAb ("wt")) based on the
design principle as shown in FIG. 1A. In addition, the same
monovalent antibodies against c-Met (SEQ ID NO:7 and SEQ ID NO:8;
c-Met 5D5 MoAb KiH), IGF-1R (SEQ ID NO:9 and SEQ ID NO:10; IGF1R
AK18 MoAb KiH) and HER3 (SEQ ID NO:11 and SEQ ID NO:12; Her3 205
MoAb KiH) were designed incorporating mutations in the CH3 parts to
support heterodimerization by the knobs-into-holes (KiH) technology
(Merchant, A. M., et al., Nat. Biotechnol. 16 (1998) 677-681). All
monovalent antibodies were transiently expressed in HEK293 cells as
described above, and subsequently purified via Protein A affinity
chromatography followed by size exclusion.
[0243] FIG. 3-5 depict the chromatograms of the size exclusion
chromatography of the three different monovalent antigen binding
proteins without knobs-into-holes as well as the corresponding
SDS-PAGE under non-reducing and reducing conditions.
[0244] The size of the different peaks was confirmed by SEC-MALLS
(FIG. 4C) and the identity of the isolated proteins was confirmed
by mass spectrometry. Taken together these data show that the
CH1-CL crossover allows the easy purification of a pure monovalent
antibody (peak 3 in FIG. 3, peak 2 in FIG. 4, peak 3 in FIG. 5)
without the need to include knobs-into-holes into the Fc proportion
to enforce heterodimerization. This product can be baseline
separated by size exclusion chromatopgraphy from a bivalent,
dimeric form of the antigen binding protein (MoAb-Dimer) as
byproduct as depicted in FIG. 1C that precedes the peak for the
monovalent antigen binding protein. Most of the cysteine bridges in
the bivalent, dimeric construct which crosslink the dimer are not
closed which leads to the observation that under non-reducing
conditions in SDS-PAGE a main product is observed at 100 kDa and
not as would be expected at 200 kDa (peak 2 in FIG. 3, peak 1 in
FIG. 4, peak 2 in FIG. 5). The additional peak (peak 1 in FIG. 3,
peak 1 in FIG. 5) observable for c-Met 5D5 MoAb ("wt") and Her3 205
MoAb ("wt") depict higher molecular weight aggregates. This is in
contrast to the monovalent antibody as described in WO/2007/048037
where the mixture of heterodimeric and homodimeric monovalent
antibody (FIG. 2) cannot be separated by conventional means.
[0245] FIG. 6-5 depict the chromatograms of the size exclusion
chromatography of the three different monovalent antigen binding
proteins with knobs-into-holes as well as the corresponding
SDS-PAGE under non-reducing and reducing conditions.
[0246] By applying this knobs-into-holes technology for
Fc-heterodimerization the relative yields of heterodimeric
monovalent antigen binding protein compared to the bivalent
MoAb-Dimer could be enhanced as shown in FIGS. 6-8.
Example 2
[0247] c-Met Phosphorylation (FIG. 9)
[0248] c-Met has been described as oncogenic receptor tyrosine
kinase which upon deregulation fosters cellular transformation.
Antibodies targeting c-Met have been described in the past.
MetMAb/OA-5D5 (Genentech) is one such antibody inhibiting
ligand-dependent activation of c-Met. As the bivalent antibody is
activatory, it was engineered as one-armed construct in which one
FAb arm was deleted leaving a monovalent antibody. To demonstrate
similar efficacy of OA-5D5 and monovalent antigen binding protein
c-Met MoAb (c-Met 5D5 MoAb ("wt")), A549 cells were incubated with
the respective antibodies in the absence or presence of HGF, the
only known ligand of c-Met. In contrast to the bivalent MetMAb
(MetMAb (biv. Ab)), neither of the antibodies has activatory
potential in the absence of HGF. Furthermore, as to be expected
c-Met MoAb (c-Met 5D5 MoAb ("wt")) is as efficacious in suppressing
ligand-induced receptor phosphorylation as OA-5D5. An unspecific
human IgG control antibody has no influence on HGF-dependent c-Met
receptor phosphorylation.
Example 3
Cellular Binding to C-Met Expressing Cell Lines (FIG. 10)
[0249] Cellular binding of monovalent antigen binding protein c-Met
MoAb (c-Met 5D5 MoAb ("wt")) was demonstrated on A549 cells. A cell
suspension was incubated with a threefold dilution series
(100-0.0003 .mu.g/mL) of the indicated antibodies. Bound antibodies
were visualized with a secondary Alexa488-coupled antibody binding
to the constant region of human immunoglobulin. Fluorescence
intensity of single cells was measured on a FACS Canto (BD
Biosciences) flow cytometer. No differences in binding of c-Met
MoAb and OA-5D5 are observable indicating that the c-Met MoAb
(c-Met 5D5 MoAb ("wt")) efficiently binds to cell surface
c-Met.
[0250] Half-Maximal Binding
OA-5D5: 1.45 nM
[0251] c-Met MoAb 1.57 nM
Example 4
IGF-1R Binding Affinity (FIG. 11)
[0252] IGF-1R extracellular domain binding of the monovalent
antigen binding protein IGF1R MoAb (IGF1R AK18 MoAb ("wt")) was
compared to the binding of the parental <IGF-1R>IgG1 antibody
by surface Plasmon resonance (SPR). FIG. 17 depicts the scheme of
the SPR assay to determine the monovalent affinity. The analysis
(double determination) showed that the IGF-1R binding affinity is
retained in the monovalent antibody.
TABLE-US-00002 k (on) k (off) KD Mab (IGF-1R) 1.74E+06 6.63E-03
3.80E-09 MoAb (IGF-1R) 1.3E+06 2.9E-03 2.16E-09 MoAb (IGF-1R)
2.4E+06 3.3E-03 1.4E-09
Example 5
Cellular Binding to IGF-1R Expressing Cell Lines (FIG. 12)
[0253] Cellular binding of monovalent antigen binding protein IGF1R
MoAb (IGF1R AK18 MoAb ("wt")) was demonstrated on A549 cells. A549
cells in the logarithmic growth phase were detached with accutase
(Sigma) and 2.times.10e5 cells were used for each individual
antibody incubation. MoAb was added in a threefold dilution series
(100-0.0003 .mu.g/mL). Bound antibodies were visualized with a
secondary Alexa488-coupled antibody (5 .mu.g/mL) binding to the
constant region of human immunoglobulin. Dead cells were stained
with 7-AAD (BD) and excluded from the analysis. Fluorescence
intensity of single cells was measured on a FACS Canto (BD
Biosciences) flow cytometer. The data show that there is a
difference in halfmaximal binding to cells due to the fact that the
IGF-1R IgG1 antibody can bind with two arms to IGF-1R on cells and
exhibits an avidity effect whereas the monovalent antibody can only
bind with one arm.
[0254] Half-Maximal Binding
IGF-1R (150 kDa): 0.76 nM IGF-1R MoAb (100 kDa): 5.65 nM
Example 6
ADCC Induction (FIG. 13)
[0255] Donor-derived peripheral blood mononuclear cells (PBMC) can
be used to measure effector cell recruitment by non-glycoengineered
and glycoengineered antibodies to cancer cells. Lysis of cancer
cells correlates with NK cell mediated cytotoxicity and is
proportional to the antibody's ability to recruit NK cells. In this
particular setting, DU145 prostate cancer cells were incubated in a
1:25 ratio (DU145:PBMC) ratio with PBMC in the absence or presence
of the respective antibodies. After 2 hours cellular lysis was
determined using the BATDA/Europium system as described above. The
magnitude of cell lysis by ADCC is expressed as % of the maximum
release of TDA fluorescence enhancer from the target cells lysed by
detergent corrected for spontaneous release of TDA from the
respective target cells. The data show that despite the lower
apparent affinity for IGF-1R on cells the non-glycoengineered
monovalent antigen binding protein IGF1R MoAb (IGF1R AK18 MoAb
("wt")) is superior in inducing ADCC at high concentrations
compared to the non-glycoengineered parent IGF-1R antibody.
Surprisingly, the non-glycoengineered monovalent antigen binding
protein IGF1R MoAb (IGF1R AK18 MoAb ("wt")) is even superior in
inducing ADCC at high concentrations compared to the
glycoengineered parent IGF-1R antibody that shows a drop in the
ADCC assay going to high concentrations. Monovalent IGF-1R antigen
binding proteins (IGF1R AK18 MoAb ("wt")) that mediate reduced
IGF-1R internalization and enhanced ADCC due to reduced
internalization (see below) and double the amount of Fc-parts to
engage FcRIIIa receptors on effector cells may thus represent a
promising approach to target IGF-1R on cancer cells; as
non-glycoengineered or as glycoengineered antibodies.
Example 7
IGF-1R Internalization Assay (FIG. 14)
[0256] Targeting IGF-1R by bivalent parent IGF-1R antibodies
results in internalization of IGF-1R. We investigated the
internalization properties of the monovalent antigen binding
protein IGF MoAb (IGF1R AK18 MoAb ("wt")). The data in FIG. 14 show
that internalization of IGF-1R is reduced in terms of potency and
absolute internalization when the monovalent antigen binding
protein IGF MoAb (IGF1R AK18 MoAb ("wt")) is bound.
[0257] The targeting IGF-1R on tumor cells by bivalent IGF-1R
antibodies results in internalization and lysosomal degradation of
IGF-1R. We investigated the internalization properties of the
monovalent antigen binding protein IGF MoAb (IGF1R AK18 MoAb
("wt")). For this purpose, HT29 colon cancer cells were treated for
18 hours with different concentrations of monovalent antigen
binding protein IGF1R MoAb (IGF1R AK18 MoAb ("wt")) and bivalent
parent IGF-1R antibody. After lysis of the cells, the remaining
levels of IGF-1R protein were determined by IGF-1R specific
ELISA.
[0258] The data in FIG. 20 show that internalization of IGF-1R is
reduced in terms of potency and absolute internalization when the
monovalent antigen binding protein IGF1R MoAb (IGF1R AK18 MoAb
("wt")) is bound. Maximum internalization was reduced from 83%
(IgG1) to 48% (MoAb), the concentration required for halfmax
inhibition increased from 0.027 nM (IgG1) to 1.5 nM (MoAb).
Example 8
IGF-1R Autophosphorylation (IGF-1 Stimulation) (FIG. 15)
[0259] Targeting IGF-1R by IGF-1R antibodies results in inhibition
of IGF-1 induced autophosphorylation. We investigated the
inhibition of autophosphorylation of the monovalent antigen binding
protein IGF MoAb (IGF1R AK18 MoAb ("wt")) compared to the parent
IGF-1R IgG1 antibody. For this purpose 3T3-IGF-1R cells, a murine
fibroblast cell line overexpressing human IGF-1R, were treated for
10 minutes with 10 nM recombinant human IGF-1 in the presence of
different concentrations of monovalent antigen binding protein
IGF1R MoAb (IGF1R AK18 MoAb ("wt")) and bivalent parent IGF-1R
antibody. After lysis of the cells, the levels of phosphorylated
IGF-1R protein were determined by a phospho-IGF-1R specific ELISA,
combining a human IGF-1R specific capture antibody and a
phospho-Tyrosine specific detection antibody.
[0260] The data in FIG. 15 show that the monovalent antigen binding
protein IGF MoAb (IGF1R AK18 MoAb ("wt")) can inhibit IGF-1 induced
autophosphorylation although at a higher concentration due to
monovalent binding on cells (lack of avidity effect due to bivalent
binding). The concentration required for halfmax inhibition
increased from 1.44 nM (IgG1) to 27.9 nM (MoAb). Since the
difference in IC50 values of monovalent and bivalent antibodies is
slightly less pronounced in IGF-1R autophosphorylation (19 fold)
compared to IGF-1R downregulation (59 fold), the reduced impact of
monovalent binding on downregulation cannot solely explained by
reduced affinity to the IGF-1R.
Example 9
Stability of IGF-1R Monovalent Antigen Binding Protein (FIG.
16)
[0261] The stability of the monovalent antigen binding protein
IGF1R MoAb (IGF1R AK18 MoAb ("wt")) was studied by dynamic light
scattering as described above. Briefly, aggregation tendency of the
monovalent antigen binding protein IGF1R MoAb was assessed by a DLS
timecourse experiment at 40.degree. C. Over a period of five days,
no measurable increase in the hydrodynamic radius (Rh) of the
isolated monomer fraction (c.f. FIG. 10) could be detected (FIG.
22).
Example 10
Determination of PK Properties
[0262] Pharmacokinetic properties of the monovalent antibodies
according to the invention were determined in NMRI mice, female,
fed, 23-32 g body weight at the time point of compound
administration mice in a single dose PK study, as described above
(in the methods sections).
[0263] The PK properties are given in the subsequent table and
indicate that the monovalent antigen binding protein IGF MoAb
(IGF1R AK18 MoAb ("wt")) has improved PK properties compared to the
parental <IGF-1R>IgG1 antibody.
TABLE-US-00003 TABLE 2 Summary of PK properties <IGF-1R> IgG1
antibody <IGF1R> MoAb C0 .mu.g/mL 81.9 298.32 Cmax .mu.g/mL
80.7 290.2 Tmax h 0.5 0.5 AUC0-inf h * .mu.g/mL 9349 20159 term
t1/2 h 106.2 148.9 Cl mL/min/kg 0.018 0.0083 Vss L/kg 0.16
0.082
Example 11
ESI-MS Experiment IGF-1R MoAb (FIGS. 17 and 18)
[0264] The monovalent antigen binding protein IGF MoAb (IGF1R AK18
MoAb ("wt")) was transiently expressed and purified via Protein A
affinity and size exclusion chromatography. After preparative SEC
the antibody eluted within two separate peaks (peak 1 and peak 2),
which were collected. Analytical SEC from the fraction 2 (peak 2)
corresponds to a molecular weight of 100 kDa indicating a defined
monomer. SEC-MALS confirmed the initial SEC result and shows for
the fraction 2 (monomer,) a MW of 99.5 kDa. SDS-PAGE analysis of
this fraction under denaturing and reducing conditions shows one
major band with an apparent molecular weight of 50-60 kDa. Under
non reducing conditions fraction 2 (monomer) shows a major band
around a MW of 100 kDa.
Fraction 1=165 mL
Fraction 2=190 mL
[0265] ESI-MS spectra of deglycosylated MoAbs from fraction 2 show
one peak series corresponding to a monomer with a mass of 98151
Da.
TABLE-US-00004 TABLE 3 Summary of MS data from non reducing ESI-MS
measurements from fraction 2. Molecular weight, Fraction monomer
(theor. 98162 Da) Fraction 2 98151 Da
[0266] MS measurements under reducing conditions of fraction 2 show
the correct sequence and expression of the construct. The MS data
from fraction 2 show two different heavy chains with a molecular
weight of 47959 Da and 50211 Da in approximately equal amounts.
TABLE-US-00005 TABLE 4 Summary of MS data from reducing ESI-MS
measurements under reducing conditions from fraction 2. Molecular
weight, heavy Molecular weight, heavy chain 2 Fraction chain 1
(theor. 50226 Da) (theor. 47961 Da) Fraction 2 50211 Da (pyro Glu
at N- 47959 Da term.)
Example 12
Production of Glycoengineered Antigen Binding Proteins
[0267] For the production of the glycoengineered antigen binding
protein, HEK-EBNA cells are transfected, using the calcium
phosphate method, with four plasmids. Two encoding the antibody
chains, one for a fusion GnTIII polypeptide expression (a GnT-III
expression vector), and one for mannosidase II expression (a Golgi
mannosidase II expression vector) at a ratio of 4:4:1:1,
respectively. Cells are grown as adherent monolayer cultures in T
flasks using DMEM culture medium supplemented with 10% FCS, and are
transfected when they are between 50 and 80% confluent. For the
transfection of a T150 flask, 15 million cells are seeded 24 hours
before transfection in 25 ml DMEM culture medium supplemented with
FCS (at 10% V/V final), and cells are placed at 37.degree. C. in an
incubator with a 5% CO.sub.2 atmosphere overnight. For each T150
flask to be transfected, a solution of DNA, CaCl.sub.2 and water is
prepared by mixing 94 .mu.g total plasmid vector DNA divided
equally between the light and heavy chain expression vectors, water
to a final volume of 469 .mu.l and 469 .mu.l of a 1M CaCl.sub.2
solution. To this solution, 938 .mu.l of a 50 mM HEPES, 280 mM
NaCl, 1.5 mM Na.sub.2HPO.sub.4 solution at pH 7.05 are added, mixed
immediately for 10 sec and left to stand at room temperature for 20
sec. The suspension is diluted with 10 ml of DMEM supplemented with
2% FCS, and added to the T150 in place of the existing medium. Then
additional 13 ml of transfection medium are added. The cells are
incubated at 37.degree. C., 5% CO.sub.2 for about 17 to 20 hours,
then medium is replaced with 25 ml DMEM, 10% FCS. The conditioned
culture medium is harvested approx. 7 days post-media exchange by
centrifugation for 15 min at 210.times.g, the solution is sterile
filtered (0.22 um filter) and sodium azide in a final concentration
of 0.01% w/v is added, and kept at 4.degree. C.
[0268] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
Sequence CWU 1
1
131445PRTArtificial SequenceDescription of Artificial Sequence
Synthetic c-Met 5D5 MoAb ("wt") - modified heavy chain a)
VL-CH1-CH2-CH3 polypeptide 1Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ser
Ser Gln Ser Leu Leu Tyr Thr 20 25 30Ser Ser Gln Lys Asn Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45Ala Pro Lys Leu Leu Ile Tyr
Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95Tyr Tyr Ala
Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110Lys
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120
125Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala145 150 155 160Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly 165 170 175Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly 180 185 190Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu225 230 235
240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys 260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360
365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln 405 410 415Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 435 440 4452453PRTArtificial
SequenceDescription of Artificial Sequence Synthetic c-Met 5D5 MoAb
("wt") - modified heavy chain b) VH-CL-CH2-CH3 polypeptide 2Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25
30Trp Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Gly Met Ile Asp Pro Ser Asn Ser Asp Thr Arg Phe Asn Pro Asn
Phe 50 55 60Lys Asp Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr
Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Thr Tyr Arg Ser Tyr Val Thr Pro Leu Asp
Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser
Val Ala Ala Pro Ser Val Phe 115 120 125Ile Phe Pro Pro Ser Asp Glu
Gln Leu Lys Ser Gly Thr Ala Ser Val 130 135 140Val Cys Leu Leu Asn
Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp145 150 155 160Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr 165 170
175Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr
180 185 190Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys
Glu Val 195 200 205Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
Phe Asn Arg Gly 210 215 220Glu Cys Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu225 230 235 240Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295
300Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu305 310 315 320Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala 325 330 335Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro 340 345 350Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr385 390 395 400Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410
415Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
420 425 430Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser 435 440 445Leu Ser Pro Gly Lys 4503440PRTArtificial
SequenceDescription of Artificial Sequence Synthetic IGF1R AK18
MoAb ("wt") - modified heavy chain a) VL-CH1-CH2-CH3 polypeptide
3Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5
10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile 35 40 45Tyr Asp Ala Ser Lys Arg Ala Thr Gly Ile Pro Ala Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Arg Ser Lys Trp Pro Pro 85 90 95Trp Thr Phe Gly Gln Gly Thr Lys Val
Glu Ser Lys Ser Ser Ala Ser 100 105 110Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr 115 120 125Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 130 135 140Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val145 150 155
160His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
165 170 175Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile 180 185 190Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val 195 200 205Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala 210 215 220Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro225 230 235 240Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 245 250 255Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 260 265 270Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 275 280
285Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
290 295 300Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala305 310 315 320Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro 325 330 335Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr 340 345 350Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser 355 360 365Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 370 375 380Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr385 390 395
400Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
405 410 415Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys 420 425 430Ser Leu Ser Leu Ser Pro Gly Lys 435
4404452PRTArtificial SequenceDescription of Artificial Sequence
Synthetic IGF1R AK18 MoAb ("wt") - modified heavy chain b)
VH-CL-CH2-CH3 polypeptide 4Gln Val Glu Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg1 5 10 15Ser Gln Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ile Ile Trp Phe Asp Gly
Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95Ala Arg Glu
Leu Gly Arg Arg Tyr Phe Asp Leu Trp Gly Arg Gly Thr 100 105 110Leu
Val Ser Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile 115 120
125Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
130 135 140Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
Trp Lys145 150 155 160Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu 165 170 175Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu 180 185 190Ser Lys Ala Asp Tyr Glu Lys
His Lys Val Tyr Ala Cys Glu Val Thr 195 200 205His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu 210 215 220Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu225 230 235
240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser 260 265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360
365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly Lys
4505445PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Her3 205 MoAb ("wt") - modified heavy chain a)
VL-CH1-CH2-CH3 polypeptide 5Asp Ile Val Met Thr Gln Ser Pro Asp Ser
Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ser
Ser Gln Ser Val Leu Asn Ser 20 25 30Gly Asn Gln Lys Asn Tyr Leu Thr
Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr
Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu
Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Ser 85 90 95Asp Tyr Ser
Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110Lys
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120
125Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala145 150 155 160Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly 165 170 175Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly 180 185 190Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu225 230 235
240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys 260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360
365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln 405 410 415Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 435 440 4456454PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Her3 205 MoAb
("wt") - modified heavy chain b) VH-CL-CH2-CH3 polypeptide 6Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Arg Ser Ser 20 25
30Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45Gly Trp Ile Tyr Ala Gly Thr Gly Ser Pro Ser Tyr Asn Gln Lys
Leu 50 55 60Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg His Arg Asp Tyr Tyr Ser Asn Ser Leu
Thr Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val
115 120 125Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
Ala Ser 130 135 140Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
Ala Lys Val Gln145 150 155 160Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln Glu Ser Val 165 170 175Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser Ser Thr Leu 180 185 190Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu 195 200 205Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg 210 215 220Gly
Glu Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu225 230
235 240Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp 245 250 255Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp 260 265 270Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly 275 280 285Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn 290 295 300Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp305 310 315 320Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345
350Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
355 360 365Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile 370 375 380Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr385 390 395 400Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys 405 410 415Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445Ser Leu Ser
Pro Gly Lys 4507445PRTArtificial SequenceDescription of Artificial
Sequence Synthetic c-Met 5D5 MoAb KiH modified heavy chain a)
VL-CH1-CH2-CH3 knob T366W, S354C polypeptide 7Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Tyr Thr 20 25 30Ser Ser Gln
Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45Ala Pro
Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75
80Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
85 90 95Tyr Tyr Ala Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile 100 105 110Lys Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro 115 120 125Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val 130 135 140Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala145 150 155 160Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200
205Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys 260 265 270Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315
320Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Cys 340 345 350Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp
Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
4458453PRTArtificial SequenceDescription of Artificial Sequence
Synthetic c-Met 5D5 MoAb KiH modified heavy chain b) VH-CL-CH2-CH3
hole L368A, Y407V, T366S, Y349C polypeptide 8Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Leu His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Met
Ile Asp Pro Ser Asn Ser Asp Thr Arg Phe Asn Pro Asn Phe 50 55 60Lys
Asp Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Thr Tyr Arg Ser Tyr Val Thr Pro Leu Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro
Ser Val Phe 115 120 125Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
Gly Thr Ala Ser Val 130 135 140Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu Ala Lys Val Gln Trp145 150 155 160Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr 165 170 175Glu Gln Asp Ser
Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 180 185 190Leu Ser
Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val 195 200
205Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
210 215 220Glu Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu225 230 235 240Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr 245 250 255Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val 260 265 270Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu305 310 315
320Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
325 330 335Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro 340 345 350Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln 355 360 365Val Ser Leu Ser Cys Ala Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala 370 375 380Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr385 390 395 400Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu 405 410 415Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440
445Leu Ser Pro Gly Lys 4509440PRTArtificial SequenceDescription of
Artificial Sequence Synthetic IGF1R AK18 MoAb KiH modified heavy
chain a) VL-CH1-CH2-CH3 knob T366W, S354C polypeptide 9Glu Ile Val
Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45Tyr Asp Ala Ser Lys Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu
Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Lys
Trp Pro Pro 85 90 95Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ser Lys
Ser Ser Ala Ser 100 105 110Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr 115 120 125Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro 130 135 140Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val145 150 155 160His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 165 170 175Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 180 185
190Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
195 200 205Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala 210 215 220Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro225 230 235 240Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val 245 250 255Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val 260 265 270Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 275 280 285Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 290 295 300Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala305 310
315 320Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro 325 330 335Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp
Glu Leu Thr 340 345 350Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys
Gly Phe Tyr Pro Ser 355 360 365Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr 370 375 380Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr385 390 395 400Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 405 410 415Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 420 425
430Ser Leu Ser Leu Ser Pro Gly Lys 435 44010452PRTArtificial
SequenceDescription of Artificial Sequence Synthetic IGF1R AK18
MoAb KiH modified heavy chain b) VH-CL-CH2-CH3 hole L368A, Y407V,
T366S, Y349C polypeptide 10Gln Val Glu Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg1 5 10 15Ser Gln Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ile Ile Trp Phe Asp Gly
Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95Ala Arg Glu
Leu Gly Arg Arg Tyr Phe Asp Leu Trp Gly Arg Gly Thr 100 105 110Leu
Val Ser Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile 115 120
125Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
130 135 140Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
Trp Lys145 150 155 160Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu 165 170 175Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu 180 185 190Ser Lys Ala Asp Tyr Glu Lys
His Lys Val Tyr Ala Cys Glu Val Thr 195 200 205His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu 210 215 220Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu225 230 235
240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser 260 265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val
Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360
365Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Val Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly Lys
45011445PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Her3 205 MoAb KiH modified heavy chain a) VL-CH1-CH2-CH3
knob T366W, S354C polypeptide 11Asp Ile Val Met Thr Gln Ser Pro Asp
Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys
Ser Ser Gln Ser Val Leu Asn Ser 20 25 30Gly Asn Gln Lys Asn Tyr Leu
Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile
Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser
Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Ser 85 90 95Asp Tyr
Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105
110Lys Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val 130 135 140Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala145 150 155 160Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly 165 170 175Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys 260 265 270Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310
315 320Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Cys 340 345 350Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Trp Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425
430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
44512454PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Her3 205 MoAb KiH modified heavy chain b) VH-CL-CH2-CH3
hole L368A, Y407V, T366S, Y349C polypeptide 12Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Arg Ser Ser 20 25 30Tyr Ile Ser
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp
Ile Tyr Ala Gly Thr Gly Ser Pro Ser Tyr Asn Gln Lys Leu 50 55 60Gln
Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg His Arg Asp Tyr Tyr Ser Asn Ser Leu Thr Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala
Pro Ser Val 115 120 125Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly Thr Ala Ser 130 135 140Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala Lys Val Gln145 150 155 160Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln Glu Ser Val 165 170 175Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu 180 185 190Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu 195 200
205Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
210 215 220Gly Glu Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu225 230 235 240Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp 245 250 255Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp 260 265 270Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp305 310 315
320Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
325 330 335Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu 340 345 350Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn 355 360 365Gln Val Ser Leu Ser Cys Ala Val Lys Gly
Phe Tyr Pro Ser Asp Ile 370 375 380Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr385 390 395 400Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys 405 410 415Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440
445Ser Leu Ser Pro Gly Lys 450135PRTArtificial SequenceDescription
of Artificial Sequence Synthetic 5xHis tag 13His His His His His1
5
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