U.S. patent application number 16/237363 was filed with the patent office on 2019-06-27 for antibodies selective for cells presenting erbb2 at high density.
This patent application is currently assigned to National Research Council of Canada. The applicant listed for this patent is National Research Council of Canada. Invention is credited to Jason Baardsnes, Myriam Banville, Bruno Gaillet, Renald Gilbert, Maria L. Jaramillo, Maureen D. O'Connor-McCourt, Traian Sulea, Ilia Alexandre Tikhomirov.
Application Number | 20190194349 16/237363 |
Document ID | / |
Family ID | 46206496 |
Filed Date | 2019-06-27 |
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United States Patent
Application |
20190194349 |
Kind Code |
A1 |
Tikhomirov; Ilia Alexandre ;
et al. |
June 27, 2019 |
ANTIBODIES SELECTIVE FOR CELLS PRESENTING ERBB2 AT HIGH DENSITY
Abstract
An erbB2 antibody is provided that binds preferentially to
disease cells having an erbB2 density greater than a normal erbB2
density. The erbB2 antibody comprises a heavy chain and a light
chain. Each chain has a constant region and a variable region. Each
variable region comprises framework regions and complementarity
determining regions (CDRs), wherein the CDRs have an amino acid
sequence set forth below: For the heavy chain: CDR1 GFNIKDTYIH (SEQ
ID No. 1) CDR2 RIYPTNGY.sup.57TR.sup.59YADSVKG (SEQ ID No. 2) CDR3
WGGDGFYAMDY (SEQ ID No. 3). For the light chain: CDR1
RASQDVN.sup.30TAVA (SEQ ID No. 4) CDR2 SASF.sup.53LYS (SEQ ID No.
5) CDR3 QQHY.sup.92TTPPT (SEQ ID NO. 6). At least one of Y57, R59,
N30, F53, and Y92 is substituted by an amino acid that confers on
said antibody a reduced erbB2 binding affinity (Kd) that is in the
range from 0.1 nM to 100 nM. The substitution is other than N30A,
F53N, Y92A and Y92F when there is a single substitution in the
antibody light chain.
Inventors: |
Tikhomirov; Ilia Alexandre;
(Toronto, CA) ; Jaramillo; Maria L.;
(Beaconsfield, CA) ; O'Connor-McCourt; Maureen D.;
(Beaconsfield, CA) ; Sulea; Traian; (Kirkland,
CA) ; Gilbert; Renald; (Montreal, CA) ;
Gaillet; Bruno; (Sainte Julienne, CA) ; Baardsnes;
Jason; (Montreal, CA) ; Banville; Myriam;
(Laval, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
National Research Council of Canada |
Ottawa |
|
CA |
|
|
Assignee: |
National Research Council of
Canada
|
Family ID: |
46206496 |
Appl. No.: |
16/237363 |
Filed: |
December 31, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13992051 |
Jun 19, 2013 |
10208129 |
|
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PCT/CA2011/050747 |
Dec 2, 2011 |
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16237363 |
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61419983 |
Dec 6, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/32 20130101;
C07K 2317/24 20130101; C07K 2317/565 20130101; A61P 35/00 20180101;
C07K 2317/92 20130101 |
International
Class: |
C07K 16/32 20060101
C07K016/32 |
Claims
1-22. (canceled)
23. An antibody or a bivalent fragment thereof that binds
preferentially to disease cells having an erbB2 density greater
than a normal erbB2 density, the antibody comprising: a heavy chain
(HC) comprising a complementarity determining region (CDR)1
comprising the sequence GFNIKDTYIH (SEQ ID NO: 1); a CDR2
comprising the sequence RIYPTNGY.sup.57TR.sup.59YADSVKG (SEQ ID NO:
2); and a CDR3 comprising the sequence WGGDGFYAMDY (SEQ ID NO: 3);
and a light chain (LC) comprising a CDR1 comprising the sequence
RASQDVN.sup.30TAVA (SEQ ID NO: 4); a CDR2 comprising the sequence
SASF.sup.53LYS (SEQ ID NO: 5); and a CDR3 comprising the sequence
QQHY.sup.92TTPPT (SEQ ID NO. 6), wherein the heavy chain and the
light chain contain substitutions consisting of HC Y57A, LC F53N,
and LC Y92A.
24. An antibody according to claim 23, the antibody having the
framework region sequences of trastuzumab.
25. An antibody according to claim 23, the antibody having the
framework region sequences and constant region sequences of
trastuzumab.
26. A bivalent fragment of an antibody according to claim 23.
27. A conjugate comprising a cytotoxin or a detectable label and,
conjugated thereto, an antibody or bivalent fragment thereof as
defined according to claim 23.
28. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and an antibody in an amount useful to control
the growth of cells presenting erbB2 at a density greater than the
normal erbB2 density, in a subject in need thereof, wherein the
antibody is an antibody or bivalent fragment thereof as defined
according to claim 23.
29. A method of treating a subject presenting with disease cells
having an erbB2 density greater than normal, comprising treating
the subject with a pharmaceutical composition comprising the
antibody as defined according to claim 23.
30. The method according to claim 29, wherein the disease cells are
cancer cells.
31. The method according to claim 30, wherein the cancer cells are
breast cancer cells.
32. An antibody or a bivalent fragment thereof comprising a heavy
chain and a light chain, each chain having a constant region and a
variable region, each variable region comprising framework regions
and complementarity determining regions (CDRs), wherein: the heavy
chain (HC) comprises a CDR1 comprising the sequence GFNIKDTYIH (SEQ
ID NO: 1); a CDR2 comprising the sequence
RIYPTNGY.sup.57TR.sup.59YADSVKG (SEQ ID NO: 2); and a CDR3
comprising the sequence WGGDGFYAMDY (SEQ ID NO: 3); and the light
chain (LC) comprises a CDR1 comprising the sequence
RASQDVN.sup.30TAVA (SEQ ID NO: 4); a CDR2 comprising the sequence
SASF.sup.53LYS (SEQ ID NO: 5); and a CDR3 comprising the sequence
QQHY.sup.92TTPPT (SEQ ID NO. 6), wherein the heavy chain and the
light chain contain substitutions consisting of HC Y57A, LC F53N,
and LC Y92A, and wherein the antibody further comprises the
framework region sequences of trastuzumab.
33. The antibody of according to claim 32, wherein the antibody
further comprises the constant region sequence of trastuzumab.
34. A pharmaceutical composition comprising the antibody or
bivalent fragment of claim 32.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/992,051, filed on Jun. 19, 2013, which is a
national stage application of PCT International Application No.
PCT/CA2011/050747, filed Dec. 2, 2011, which claims priority to
U.S. Provisional Application Ser. No. 61/419,983, filed Dec. 6,
2010, each of which is incorporated by reference herein in its
entirety.
FIELD OF THE INVENTION
[0002] This invention relates to antibodies having therapeutic and
diagnostic utility. More particularly, the present invention
relates to antibodies that bind selectively to cells that present
erbB2 at abnormally high density. The antibodies are useful
therapeutically and diagnostically in the fields of oncology and
other diseases.
BACKGROUND TO THE INVENTION
[0003] Drugs for the treatment of cancer and other diseases have a
so-called "therapeutic window". In the case of cancer, the
therapeutic window defines the drug dosage that can kill cancer
cells preferentially to normal cells, thereby establishing a safety
range for the use of the drug. The therapeutic window for
conventional chemotherapeutics is narrow with, in many cases,
significant adverse effects coinciding with marginal slowing of
tumour growth. Targeted treatments that spare normal cells are
urgently needed.
[0004] Therapeutic antibodies form a newer class of cancer
therapies that specifically target an antigen presented on the
surface of cancer cells. When the target surface protein is unique
to the cancer cell, adverse antibody effects on normal cells can be
avoided. However, for the majority of antigens, target expression
is not restricted completely to tumour cells, with some normal
cells also expressing the antigen. In these cases, the antibody may
have an effect on normal cells as well as tumor cells, leading to
"on-target, off-tissue" adverse events. In the case of the ErbB2
antigen, because it is present on the surface of normal cells in
cardiac tissue as well as on breast cancer cells, the clinical use
of erbB2-targeting therapeutics is associated with adverse events
that include cardiac toxicity. The incidence and degree of adverse
events is considerably increased when given in combination with
chemotherapeutics, particularly anthracyclines.
[0005] Considering the efficacy of anti-erbB2 therapies in treating
patients that overexpress erbB2, the risk associated with cardiac
toxicity is currently considered acceptable when managed properly.
The risk of anti-erbB2 therapy-associated cardiac toxicity can be
reduced by avoiding co-administration with anthracyclines, or by
administering anti-erbB2 therapy and chemotherapeutics
consecutively.
[0006] Currently, routine LVEF monitoring is performed to assess
cardiac function in patients that are prescribed anti-erbB2
therapies, especially when given in combination with
anthracyclines. This is a time-consuming and expensive process
which requires patient compliance.
[0007] Efforts to improve upon erbB2 antibodies are aimed at
generating antibodies having even greater affinity for the target
antigen. For instance, U.S. Pat. No. 7,435,797 issued Oct. 14, 2008
describes a variety of trastuzumab analogs in which amino acid
substitution is used to further increase target affinity.
Substitutions are made at sites within different complementarity
determining regions of trastuzumab.
[0008] It would be desirable to provide an erbB2 antibody that is
useful to treat subjects presenting with erbB2 over-expressing
disease cells, while avoiding significant interaction with cardiac
and other normal cells that also present the erbB2 antigen.
[0009] It is an object of the present invention to provide
therapeutic antibodies, and fragments and conjugates thereof that
bind effectively to a given target only when that target is
presented at a relatively higher density characteristic of a
disease state.
[0010] It is a further object of the present invention to provide
such antibodies, fragments and conjugates in pharmaceutical
compositions, particularly for therapeutic and diagnostic use.
[0011] It is a further object of the present invention to provide a
method useful, in a subject in need thereof, to control the growth
of disease cells that present erbB2 at a density greater than
normal erbB2 density, while avoiding or minimizing adverse effects
on normal cells.
SUMMARY OF THE INVENTION
[0012] In one aspect, the present invention provides an isolated,
erbB2 antibody or bivalent fragment thereof that binds
preferentially to target cells that present erbB2 at a density
above a normal erbB2 density. Cells that present erbB2 at a density
greater than normal erbB2 density are disease cells, including
cancer cells such as breast cancer cells, that over-express the
her-2 gene, and manifest on their surface a greater number of erbB2
proteins than cells that express the her-2 gene at normal
levels.
[0013] The antibodies of the present invention, and their bivalent
fragments, display a preference for binding to disease cells having
the higher erbB2 density, and show reduced and desirably minimal or
negligible binding to normal cells having a normal erbB2
density.
[0014] The present antibodies and their bivalent binding fragments
thus are well suited for use in reducing or eradicating high
density erbB2 disease cells while minimizing or avoiding effects on
normal cells, thereby reducing adverse events in subjects receiving
erbB2 antibody therapy.
[0015] In one aspect, the erbB2 antibody comprises a heavy chain
and a light chain, each chain having a constant region and a
variable region, each variable region comprising framework regions
and complementarity determining regions (CDRs), wherein the CDRs
have an amino acid sequence set forth below:
TABLE-US-00001 For the heavy chain: (SEQ ID No. 1) CDR1 GFNIKDTYIH
(SEQ ID No. 2) CDR2 RIYPTNGY.sup.57TR.sup.59YADSVKG (SEQ ID No. 3)
CDR3 WGGDGFYAMDY For the light chain: (SEQ ID No. 4) CDR1
RASQDVN.sup.30TAVA (SEQ ID No. 5) CDR2 SASF.sup.53LYS (SEQ ID No.
6) CDR3 QQHY.sup.92TTPPT
wherein at least one of Y57, R59, N30, F53, and Y92 is replaced by
a substituting amino acid that reduces the erbB2 binding affinity
of said antibody. In embodiments, the substituting amino acid(s)
are selected to confer on the antibody a binding affinity (KD) for
erbB2 that is about 10 fold or more weaker than the erbB2 binding
affinity of trastuzumab.
[0016] In embodiments, the present invention provides an ErbB2
antibody comprising a heavy chain and a light chain, each chain
having a constant region and a variable region, wherein the light
chain variable region comprises the sequence of SEQ ID No. 7 and
the heavy chain variable region comprises the sequence of SEQ ID
No. 8, wherein at least one of Y57, R59, N30, F53, and Y92 is
replaced by a substituting amino acid that reduces the erbB2
binding affinity of said antibody.
[0017] In other embodiments, the substituting amino acid is
selected to reduce erbB2 binding affinity of the antibody or
bivalent fragment to a level that substantially eliminates binding
to cells presenting erbB2 at a normal erbB2 density, and retains
effective binding at targeted disease cells that present erbB2 at a
greater density relative to normal cell erbB2 density.
[0018] In still other embodiments, the antibody or bivalent
fragment is a variant of trastuzumab having one or more
substitutions at the residues identified herein.
[0019] In another of its aspects, the present invention provides
conjugates, i.e., immunoconjugates, comprising an antibody or
bivalent fragment thereof according to the present invention and,
conjugated therewith, an agent useful to treat or diagnose cells
presenting erbB2 at a density characteristic of disease cells.
[0020] In a further aspect, the present invention provides
medically useful compositions comprising an antibody, bivalent
fragment thereof or immunoconjugate thereof according to the
present invention, in combination with a medically acceptable
carrier, such as a pharmaceutically acceptable carrier or a
diagnostically useful carrier.
[0021] In a related aspect, the present invention provides a method
for treating a subject having disease cells that present erbB2 at a
density greater than the erbB2 density on normal cells, comprising
the step of administering to the subject an effective amount of an
antibody, bivalent fragment thereof, or an immunoconjugate of the
present invention.
[0022] Subjects so treated will manifest adverse events that are
fewer in number and/or severity given the reduced affinity of the
present antibodies for normal cells and tissue.
[0023] These and other aspects of the present invention are now
described in greater detail with reference to the accompanying
drawings, in which:
REFERENCE TO THE FIGURES
[0024] FIG. 1 shows the in vitro effect of substitution on antibody
affinity for erbB2;
[0025] FIG. 2A is a graph showing binding of antibodies to cell
surface erbB2 present on SkBr3 tumour cells at 0.1 and 1 ug/ml mAb,
and compared to wt mAb (2-1 wt, set arbitrarily to 100%);
[0026] FIG. 2B is a graph showing binding of antibodies to cell
surface erbB2 present on BT474 tumour cells at 0.1 and 1 ug/ml mAb,
and compared to wt mAb (2-1 wt, set arbitrarily to 100%);
[0027] FIG. 2C is a graph showing binding of antibodies to cell
surface erbB2 present on human cardiac myocytes (HCM) at 0.1 and 1
ug/ml mAb, and compared to wt mAb (2-1 wt, set arbitrarily to
100%); and
[0028] FIG. 3 is a graph representing binding selectivity of
antibodies. The ratio of antibody binding to SKBr3 cells or BT474
cells (overexpressing erbB2) divided by their respective binding to
normal HCM (at 0.1 mg/ml) was calculated and compared to that seen
with 2-1 wild type antibody (set arbitrarily to 1).
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
[0029] As used herein, the term "erbB2" refers to any protein that
comprises the expressed and processed product of the her-2 gene,
wherein the protein is designated as UniProtKB/Swiss-Prot P04626-1,
including antibody-binding variants thereof.
[0030] The present invention relates to erbB2 antibodies and
bivalent fragments thereof that display a preference for binding to
disease cells presenting erbB2 at a density greater than normal
cells. On cells that present erbB2, the normal density of erbB2 is
generally less than about 10,000 erbB2 molecules per cell, and is
usually less than about 1,000 erbB2 molecules per cell.
ErbB2-presenting disease cells, on the other hand, present erbB2 at
a density generally greater than 10,000 erbB2 molecules per cell,
and usually greater than about 100,000 erbB2 molecules per cell.
Generally, the erbB2 density is thus about 10.sup.3 or less on
normal cells, and about 10.sup.5 or more on disease cells. The
actual number of erbB2 molecules on any given cell can be
determined by established methods, including the antibody based
radiolabeled binding or flow cytometry binding to live cells herein
exemplified. The binding avidity of the present antibodies is
greater for the higher erbB2 density disease cells than for the
lower erbB2 density normal cells. This greater avidity is revealed
conveniently using techniques established for determining affinity
constants for antibody-target interactions, also as exemplified
herein.
[0031] In embodiments, the present erbB2 antibodies have a binding
affinity for erbB2 that is about 10 fold or more weaker than the
erbB2 binding affinity of trastuzumab. Desirably, the binding
affinity of the antibody for erbB2 is about 15-fold, 20-fold,
25-fold, and preferably 30-fold or more weaker than the erbB2
binding affinity of trastuzumab. In absolute terms, and given an
erbB2 binding affinity of about 0.03 nM for trastuzumab, the
present antibodies incorporate amino acid substitution(s) that
reduce their erbB2 binding affinity to about 0.1 nM and weaker,
e.g., to an erbB2 binding affinity that is in the range from 0.1 nM
to 100 nM, more desirably 0.5 nM to 100 nM, such as 0.7 nM to 100
nM, or 1 nM to 100 nM, or 1 nM to 75 nM, or 1 nM to 50 nM, or 1 nM
to 25 nM, or 1 nM to 10 nM, or 1 nM to 5 nM.
[0032] In embodiments, the antibody is an intact antibody
comprising features common to all natural antibodies, and thus
comprises a heavy chain and a light chain, each chain having a
constant region and a variable region, each variable region
comprising framework regions (FRs) and complementarity determining
regions (CDRs). In the alternative, the antibody is provided as a
bivalent fragment, i.e., an antibody fragment comprising both
"arms" of an intact antibody, joined through a linker that can be
represented by the hinge region of the antibody or any equivalent.
Such bivalent fragments include F(ab).sub.2 fragments and any other
bivalent fragment that retains preference for high density erbB2.
In particular embodiments, the bivalent fragment is a F(ab').sub.2
fragment, generated for instance by papain-based digestion of the
parent antibody using standard procedures for digestion and
subsequent fragment isolation. In the alternative, the bivalent
fragment can be a so-called single chain Fv (scFv), consisting of
the variable light and variable heavy antibody domains joined by an
amino acid linker, or a bivalent form of a so-called diabody
prepared using a 5 amino acid linker such as SGGGG between the
light and heavy chain variable domains and a C-terminal cysteine
modification to GGC to give a final diabody product as
VL-SGGG-VH-GGC. Still other bivalent fragments can be prepared by
coupling the light and heavy chain variable domains through
thioether linkages such as bis-maleimidomethyl ether (BMME),
N,N'-p-phenylene dimaleimide (PDM and N,N'-bismaleimidohexane BMH),
to stabilize the F(ab')2 fragments.
[0033] In the intact antibody or bivalent fragment, the CDRs
comprise or consist of the following amino acid sequences:
TABLE-US-00002 For the heavy chain: (SEQ ID No. 1) CDR 1 GFNIKDTYIH
(SEQ ID No. 2) CDR2 RIYPTNGY.sup.57TR.sup.59YADSVKG (SEQ ID No. 3)
CDR3 WGGDGFYAMDY For the light chain: (SEQ ID No. 4) CDR 1
RASQDVN.sup.30TAVA (SEQ ID No. 5) CDR2 SASF.sup.53LYS (SEQ ID No.
6) CDR3 QQHY.sup.92TTPPT
wherein at least one of Y57, R59, N30, F53, and Y92 is replaced by
a substituting amino acid that reduces the erbB2 binding affinity
of said antibody or bivalent fragment.
[0034] The substituting amino acids are most suitably genetically
encoded amino acids that are selected desirably, but not
essentially, from an amino acid class that is different from the
amino acid class to which the parent amino acid belongs. For
instance, in the case of Y57, suitable substituting amino acids are
those that are not polar/neutral/large amino acids. The selection
process can be conducted by applying computer aided tools that
couple saturation virtual mutagenesis engines with algorithms for
in silico scoring of binding affinities and/or association rates.
Amino acid selections can also be made based on the following Table
1:
TABLE-US-00003 Polarity Charge Amino Acid 3 letter 1 letter (side
chain) (pH 7.4) Size* Alanine Ala A nonpolar neutral tiny Arginine
Arg R polar positive large Asparagine Asn N polar neutral small
Aspartic acid Asp D polar negative small Cysteine Cys C nonpolar
neutral small Glutamic acid Glu E polar negative small Glutamine
Gln Q polar neutral small Glycine Gly G nonpolar neutral tiny
Histidine His H polar neutral (90%) large Isoleucine Ile I nonpolar
neutral large Leucine Leu L nonpolar neutral large Lysine Lys K
polar positive large Methionine Met M nonpolar neutral large
Phenylalanine Phe F nonpolar neutral large Proline Pro P non-polar
neutral small Serine Ser S polar neutral tiny Threonine Thr T polar
neutral small Tryptophan Trp W nonpolar neutral bulky Tyrosine Tyr
Y polar neutral large Valine Val V nonpolar neutral small *based on
volume in A.sup.3, where 50-100 is tiny, 100-150 is small, 150-200
is large and >200 is bulky
[0035] In embodiments, the heavy chain variable region of the
antibody or bivalent fragment incorporates at least one
substitution at Y57 or R59. In other embodiments, the heavy chain
variable region incorporates substitutions at both Y57 and R59. In
an alternative embodiment, the heavy chain variable region is wild
type and incorporates no such substitutions, provided there is at
least one substitution and optionally two substitutions in the
light chain variable region.
[0036] In embodiments, Y57 is replaced by a substituting amino acid
having a side chain that is nonpolar and/or a side chain that is
non-neutral and/or a side chain that is not large. Desirably, Y57
is replaced by an amino acid selected from A, C, G, I, L, M, F, W
and V; preferably from A, G, I, L and V; and more preferably from
A, V, I and L. In a specific embodiment, Y57 is replaced by A57,
thus yielding the substitution designated Y57A.
[0037] In other embodiments, R59 is replaced by a substituting
amino acid having a side chain that is nonpolar and/or is charge
neutral or negative and/or is not large. Desirably, R59 is replaced
by an amino acid having a side chain that is charge neutral or
negative, as well as polar, as well as small, and is selected
desirably from D and E. In a specific embodiment, R59 is replaced
by E59, thus yielding the substitution designated R59E.
[0038] In embodiments, the light chain variable region of the
antibody or bivalent fragment incorporates at least one
substitution at N30 or at F53 or at Y92. In other embodiments, when
the heavy chain comprises at least one substitution, then the light
chain variable region comprises at least two such substitutions, or
all three such substitutions. In a specific embodiment, the light
chain variable region incorporates substitutions at both F53 and
Y92. In another specific embodiment, the light chain variable
region incorporates substitution only at N30, only at F53 or only
at Y92. In an alternative embodiment, the light chain variable
region is wild type and incorporates no such substitutions,
provided there is at least one substitution in the heavy chain
variable region.
[0039] When substituted, N30 is replaced by a substituting amino
acid having a side chain that is either nonpolar and/or is negative
or positive in charge and/or may not be small. In embodiments, N30
is substituted by an amino acid that is not S, and is selected from
R, D, E, or K. In a preferred embodiment, N30 is substituted by D,
yielding the substitution designated N30D.
[0040] When substituted, F53 is replaced by a substituting amino
acid having a side chain that is either polar and/or is charge
positive or negative and/or is not large. In embodiments, F53 is
replaced by R, N, D, E, Q, H, K, S, T or Y. In particular
embodiments, F53 is replaced by N, Q, H, S, T or Y. In a preferred
embodiment, F53 is replaced by N, yielding the substitution
designated F53N.
[0041] When substituted, Y92 is replaced by a substituting amino
acid having a side chain that is nonpolar and/or a side chain that
is non-neutral and/or is not large. Desirably, Y92 is replaced by
an amino acid selected from A, C, G, I, L, M, F, W and V;
preferably from A, G, I, L and V; and more preferably from A, V, I
and L. In a specific embodiment, Y92 is replaced by A92, thus
yielding the substitution designated Y92A.
[0042] The antibody or bivalent fragment thereof comprises at least
one substitution at a location noted above. The at least one
substitution can occur in either the light chain variable region or
the heavy chain variable region. In embodiments, and in the case
where there is a single substitution that is only within the
antibody light chain, that substitution is other than N30A, F53N,
Y92A and Y92F.
[0043] In other embodiments, the antibody or binding fragment
thereof comprises at least two such substitutions, either both in
the light chain variable region, both in the heavy chain variable
region, or at least one substitution in each of the light and heavy
chain variable regions. In specific embodiments, the light chain
variable region and the heavy chain variable region incorporate
substitutions as follows:
TABLE-US-00004 TABLE 2 Heavy chain Light Chain Wild type Y57A R59E
Wild type -- wt, Y57A wt, R59E N30D N30D, wt N30D, Y57A N30D, R59E
F53N F53N, wt F53N, Y57A F53N, R59E Y92A Y92A, wt Y92A, Y57A Y92A,
R59E F53N&Y92A F53N&Y92A, F53N&Y92A, F53N&Y92A, wt
Y57A R59E
[0044] In preferred embodiments, the erbB2 antibody incorporates
(1) a wild type heavy chain and light chain substitutions of both
F53 and Y92, such as F53N and Y92A, or (2) a heavy chain
substituted at Y57, such as Y57A, and a light chain substituted at
N30, such as N30D.
[0045] In addition to the recited three CDRs present in each of the
light and heavy chain variable regions, the heavy and light chains
of the intact antibody comprise four intervening framework regions
that present the CDRs in a conformation suitable for erbB2 binding,
and constant regions that confer antibody effector function. The
CDRs can be integrated into any suitable acceptor antibody, by
grafting the present CDRs into the acceptor antibody, in accordance
with practices and techniques well established for the production
of chimeric, humanized and human antibodies.
[0046] Particularly suitable acceptor antibodies are antibodies
already known to have erbB2 binding affinity. Such donor antibodies
are most desirably of human origin, but they can also derive from
acceptor antibodies of non-human origin, including mouse, rat,
rabbit, goat, sheep, primate and the like. It will be appreciated
that human antibody acceptor sequences different from those
exemplified herein can be identified and used to accommodate the
presently desired CDRs. This is achieved by modeling the structure
of a preferred antibody using for instance the Swiss-Model
[http://swissmodel.expasy.org/repository] or similar software and
selecting, from among the numerous human antibody sequences
available in public databases, a human acceptor antibody sequence
that, with CDR sequences altered as herein preferred, approximates
the same structural conformation as the preferred antibodies. In
embodiments, the acceptor antibodies, and the resulting present
antibodies, are of the IgG1 isotype, but they may also be IgG2 or
IgG4. Moreover, the isotype of the antibody, as dictated by the
constant region, can be manipulated to alter or eliminate the
effector function of the resulting antibody. That is, the constant
region of the present antibodies is either wild type human antibody
constant region, or a variant thereof that incorporates amino acid
modifications, i.e., amino acid additions, substitutions or
deletions that alter the effector function of the constant region,
such as to enhance serum half-life, reduce complement fixation,
reduce antigen-dependent cellular cytotoxicity and improve antibody
stability.
[0047] The number of amino acid modifications in the constant
region is usually not more than 20, such as 1-10 e.g., 1-5
modifications, including conservative amino acid substitutions.
[0048] In embodiments, the half life of the antibody is improved by
incorporating one more amino acid modification, usually in the form
of amino acid substitutions, for instance at residue 252, e.g., to
introduce Thr, at residue 254, e.g., to introduce Ser, and/or at
residue 256 e.g., to introduce Phe. Still other modifications can
be made to improve half-life, such as by altering the CH1 or CL
region to introduce a salvage receptor motif, such as that found in
the two loops of a CH2 domain of an Fc region of an IgG. Such
alterations are described for instance in U.S. Pat. Nos. 5,869,046
and 6,121,022.
[0049] Altered C1q binding, or reduced complement dependent
cytotoxicity, can be introduced by altering constant region amino
acids at locations 329, 331 and 322, as described in U.S. Pat. No.
6,194,551. The ability of the antibody to fix complement can
further be altered by introducing substitutions at positions 231
and 239 of the constant region, as described in WO94/029351.
[0050] The framework regions of the light and heavy chains of the
present antibodies and fragments also desirably have the sequence
of a human antibody variable region, but incorporating the CDRs
herein specified. In embodiments, the heavy chain variable region
is human IgG4 in origin. In specific embodiments, the heavy chain
variable region is that of human IgG, such as the human IgG1
antibody variant having the sequence designated Genbank gi 2414502.
Alternatively, and preferably, the heavy chain variable region is
that of human IgG4 antibody species designated Genbank gi
2414502.
[0051] The framework regions of the heavy and light chains of the
present antibodies may also incorporate amino acid modifications,
i.e., amino acid deletions, additions or substitutions, which
further improve upon the properties of the antibody or fragment, in
accordance with techniques established for antibody humanization.
Such framework modifications can be modeled on the framework
regions of antibody sequences provided in public databases, and on
framework regions of antibodies known to bind erbB2, such as those
antibodies referenced in the background section hereof. Preferred
framework substitutions are those which yield antibodies having a
greater preference for binding erbB2 at the higher density
associated with disease cells, relative to normal cells.
[0052] Framework modifications can also be made to reduce
immunogenicity of the antibody or to reduce or remove T cell
epitopes that reside therein, as described for instance by Carr et
al in US2003/0153043.
[0053] In accordance with embodiments of the present invention, the
heavy and light chain variable regions are modeled on the antibody
trastuzumab, and comprise a light chain variable region of SEQ ID
No.7, and/or a heavy chain variable region having SEQ ID No.8, as
follows:
TABLE-US-00005 Light chain variable region (VL): [SEQ ID No. 7]
DIQMTQSPSSLSASVGDRVTITCRASQDVN.sup.30TAVAWYQQKPGKAPKLL
IYSASF.sup.53LYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHY.sup.92T
TPPTFGQGTKVEIK, wherein N30, F53, and Y92 are as defined
hereinabove; Heavy chain variable region (VH) [SEQ ID No. 8]
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWV
ARTYPTNGY.sup.57TR.sup.59YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVY
YCSRWGGDGFYAMDYWGQGTLVTVSS; wherein Y57 and R59 are as defined
hereinabove.
[0054] In more specific and preferred embodiments, the entire light
and heavy chains of the intact antibody are set out below as SEQ ID
Nos. 9 and 10, respectively:
TABLE-US-00006 Entire Light chain [SEQ ID No. 9]
DIQMTQSPSSLSASVGDRVTITCRASQDVN.sup.30TAVAWYQQKPGKAPKLLI
YSASF.sup.53LYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHY.sup.52TT
PPTEGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE
AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
ACEVTHQGLSSPVTKSFNRGEC; wherein N30, F53, and Y92 are as defined
hereinabove; Entire Heavy chain [SEQ ID No. 10]
EVQLVESGGGLVQPGGSLRLSCAASGENIKDTYIHWVRQAPGKGLEWVA
RIYPTNGY.sup.57TR.sup.59YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYY
CSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA
LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS
SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDTPPPCPRCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK;
wherein Y57 and R59 are as defined hereinabove.
[0055] As noted, final selection of an antibody or binding fragment
is made based on the binding preference displayed by the desired
antibody or bivalent fragment for cells that present erbB2 at a
density greater than normal. The target cells are thus disease
cells presenting greater than normal erbB2 density, as a hallmark.
Screening can be performed in vitro, as exemplified herein, using
as reference cells a first disease cell known from analysis to
present erbB2 at a density greater than normal, such as the human
breast adenocarcinoma cell line SkBr3 ATCC HTB-30 (.about.2.5M
ErbB2/cell) or the human breast ductal carcinoma cell line BT474
ATCC HTB-20 (.about.3M Her2/cell) and a second, normal cell known
from analysis to present erbB2 at a normal density, such as normal
human cardiac myocytes (.about.20,000 ErbB2/cell). The choice of
cardiac myocytes as the reference, normal cell is prudent, given
that marketed ErbB2 antibodies, such as trastuzumab, are known to
elicit cardiac side effects through their interaction with these
cells. Any other human cell line that presents erbB2 at normal
density can be used, in the alternative.
[0056] The cell-based assay can use flow cytometry with appropriate
erbB2 antibody and labeled secondary antibody to report and measure
binding affinity and avidity, as exemplified herein. In the
alternative, selection of the desired antibody can be performed
based on absolute binding affinities obtained for instance using
surface plasmon resonance, also as exemplified herein.
[0057] For purposes of identifying disease cells that can be
targeted by the present erbB2 antibodies and bivalent fragments,
the commercial test known as HerceptTest.RTM. can conveniently be
used. This is a semi-quantitative immunohistochemical assay for
determination of her-2 protein overexpression in breast cancer
tissues. Positive or negative results aid in the classification of
abnormal cells/tissues and provide a basis for treatment with erbB2
antibody.
[0058] The antibodies and binding fragments thus are useful for
both diagnostic purposes, including sample testing and in vivo
imaging, and for therapeutic purposes to treat diseases in which
erbB2 density is increased on disease cells.
[0059] For either purpose, the antibody or binding fragment can be
conjugated to an appropriate agent, to form an immunoconjugate.
Agents appropriate for treating disease include cytotoxic agents
include chemotherapeutics and radiotherapeutics. For diagnostic
purposes, appropriate agents are detectable labels that include
radioisotopes, for whole body imaging, and radioisotopes, enzymes,
fluorescent labels and the like for sample testing.
[0060] For therapy, the cytotoxin may be conjugated with the
antibody or bivalent binding fragment through non-covalent
interaction, but more desirably, are coupled by covalent linkage
either directly or, more preferably, through a suitable linker. In
a preferred embodiment, the conjugate comprises a cytotoxin and an
antibody. Immunoconjugates of the antibody and cytotoxin are made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate, iminothiolane,
bifunctional derivatives of imidoesters such as dimethyl
adipimidate HCL, active esters such as disuccinimidyl suberate,
aldehydes such as glutaraldehyde, bis-azido compounds such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates such as
toluene 2,6-diisocyanate, and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). Carbon-14-labeled
1-isothiocyanobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is a chelating agent suitable for conjugation of radio
nucleotide to the antibody.
[0061] The cytotoxin component of the immunoconjugate can be a
chemotherapeutic agent, a toxin such as an enzymatically active
toxin of bacterial, fungal, plant or animal origin, or fragments
thereof, or a small molecule toxin, or a radioactive isotope such
as .sup.212Bi, .sup.131I, .sup.131In, .sup.111In, .sup.90Y, and
.sup.186Re, or any other agent that acts to inhibit the growth or
proliferation of a cancer cell.
[0062] Chemotherapeutic agents useful in the generation of such
immunoconjugates include adriamycin, doxorubicin, epirubicin,
5-fluoroouracil, cytosine arabinoside ("Ara-C"), cyclophosphamide,
thiotepa, busulfan, cytoxin, taxoids, e.g. paclitaxel, and
docetaxel, toxotere, methotraxate, cisplatin, melphalan,
vinblastine, bleomycin, etoposide, ifosgamide, mitomycin C,
mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide,
daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins,
esperamicins, 5-FU, 6-thioguanine, 6-mercaptopurine, actinomycin D,
VP-16, chlorambucil, melphalan, and other related nitrogen
mustards. Also included are hormonal agents that act to regulate or
inhibit hormone action on tumors such as tamoxifen and onapristone.
Toxins and fragments thereof which can be used include diphtheria A
chain, nonbonding active fragments of diphtheria toxin, cholera
toxin, botulinus toxin, exotoxin A chain (from Pseudomonas
aeruginosa), ricin A chain, abrin A chain, modeccin A chain,
alpha-sarcin, Aleurites fordii proteins, dianthin proteins,
phytolaca Americana proteins (PAPI, PAPII, and PAP-S), momordica
charantia inhibitor, curcin, crotin, sapaonaria, officinalis
inhibitor, gelonin, saporin, mitogellin, restrictocin, phenomycin,
enomycin, and the tricothcenes. Small molecule toxins include, for
example, calicheamicins, maytansinoids, palytoxin and CC1065.
[0063] Pharmaceutical Compositions
[0064] Therapeutic formulations of the antibody, bivalent fragment
or the conjugate are prepared for storage by mixing the antibody or
conjugate having the desired degree of purity with optional
pharmaceutically acceptable carriers, excipients or stabilizers
(Remington's Pharmaceutical Sciences, 16.sup.th edition, Osol, A.
Ed. [1980]), in the form of lyophilized formulations or aqueous
solutions. Acceptable carriers, excipients, or stabilizers are
nontoxic to recipients at the dosages and concentrations employed,
and include buffers such as phosphate, citrate, and other organic
acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins such as serum, albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagines, histidine, arginine or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN, PLURONICS or polyethylene
glycol (PEG).
[0065] The active ingredients to be used for in vivo administration
must be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0066] Sustained-release preparations may be prepared. Suitable
examples of sustained-release include semipermeable matrices of
solid hydrophobic polymers containing the antibody, which matrices
are in the form of shapes articles, e.g., films or microcapsules.
Examples of sustained-release matrices include polyesters,
hydrogels (for example, poly (2-hydroxyethyl-methacrylate),
polyactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,
degradable lactic acid-glycolic acid copolymers such as injectable
microspheres composed of lactic acid-glycolic acid copolymer and
leuprolide acetate, and poly-D-(-)-3-hydroxybutyric acid. While
polymers such as ethylene-vinyl acetate and lactic acid-glycolic
acid enable release of molecules for over 100 days, certain
hydrogels release proteins for shorter time periods. When
encapsulated antibodies remain in the body for a long time, they
may denature or aggregate as a result of exposure to moisture at
37.degree. C., resulting in a loss of biological activity and
possible changes in immunogenicity. Rational strategies can be
devised for stabilization depending on the mechanism involved. For
example, if the aggregation mechanism is discovered to be
intermolecular S--S bond formation through thio-disulfide
interchange, stabilization may be achieved by modifying sulfhydryl
residues, lyophilizing from acidic solutions, controlling moisture
content, using appropriate additives, and developing specific
polymer matrix compositions.
[0067] Pharmaceutical Combinations
[0068] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0069] Other therapeutic regimens may be combined with the
administration of the anti-cancer agents, e.g., antibodies or
conjugates, of the instant invention. For example, the patient to
be treated with such anti-cancer agents may also receive radiation
therapy, such as external beam radiation. Alternatively, or in
addition, a chemotherapeutic agent may be administered to the
patient. Preparation and dosing schedules for such chemotherapeutic
agents may be used according to manufacturers' instructions or as
determined empirically by the skilled practitioner. Preparation and
dosing schedules for such chemotherapy are also described in
Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins,
Baltimore, Md. (1992). The chemotherapeutic agent may precede, or
follow administration or the anti-tumor agent, e.g., antibody, or
may be given simultaneously therewith. The antibody may be combined
with an anti-estrogen compound such as tamoxifen or an
anti-progesterone such as onapristone (see, EP 616812) in dosages
known for such molecules.
[0070] It may be desirable to also administer antibodies or
conjugates against other tumor associated antigens, such as
antibodies which bind to the EGFR, ErbB3, ErbB4, or vascular
endothelial factor (VEGF). Alternatively, or in addition, two or
more antibodies binding that same or two or more different antigens
disclosed herein may be co-administered to the patient. Sometimes
it may be beneficial to also administer one or more cytokines to
the patient. In a preferred embodiment, the antibodies herein are
co-administered with a growth inhibitory agent. For example, the
growth inhibitory agent may be administered first, followed by an
antibody of the present invention. However, simultaneous
administration or administration of the antibody of the present
invention first is also contemplated. Suitable dosages for the
growth inhibitory agent are those presently used and may be lowered
due to combined action (synergy) of the growth inhibitory agent and
the antibody herein.
[0071] Kits
[0072] In another embodiment of the invention, an article of
manufacture containing materials useful for the diagnosis or
treatment of the disorders described herein is provided. The
article of manufacture comprises a container and a label. Suitable
containers include, for example, bottles, vials, syringes, and test
tubes. The containers may be formed from a variety of materials
such as glass or plastic. The container holds a composition which
is effective for treating the condition and may have a sterile
access port (for example the container may be an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle). The label on, or associated with, the container
indicates that the composition is used for treating a cancer
condition. The article of manufacture may further compromise a
second container compromising a pharmaceutically-acceptable buffer,
such as phosphate-buffered saline, Ringer's solution and dextrose
solution. It may further include other matters desirable from a
commercial and use standpoint, including other buffers, diluents,
filters, needles, syringes, and package inserts with instructions
for use.
[0073] Dosing and Administration
[0074] An anti-cancer therapeutic according to the invention may be
administered with a pharmaceutically-acceptable diluent, carrier,
or excipient, in unit dosage form. Any appropriate route of
administration can be employed, for example, parenteral,
intravenous, subcutaneous, intramuscular, intracranial,
intraorbital, ophthalmic, intraventricular, intracapsular,
intraspinal, intracisternal, intraperitoneal, intranasal, aerosol,
or oral administration.
[0075] For the treatment of subjects presenting with cancer cells
presenting erbB2 at greater density than normal cells, the
appropriate dosage of an anti-tumor agent, e.g., an antibody,
fragment or conjugate, will depend on the type of disease to be
treated, as defined above, the severity and course of the disease,
whether the agent is administered for preventative or therapeutic
purposes, previous therapy, the patients clinical history and
response to the agent, and the discretion of the attending
physician. The agent is suitably administered to the patient at one
time or over a series of treatments.
[0076] For example, depending on the type and severity of the
disease, about 1 .mu.g/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of
antibody or conjugate is a candidate dosage for administration to
the patient, whether, for example, by one or more separate
administrations, or by continuous infusion. A typical daily dosage
might range from about 1 .mu.g/kg to 100 mg/kg or more, depending
on the factors mentioned above. For repeated administrations over
several days or longer, depending on the condition, the treatment
is sustained until a desired suppression of disease symptoms
occurs. However, other dosage regimens may be useful. The progress
of this therapy is easily monitored by conventional techniques and
assays.
[0077] It will thus be appreciated that an effective amount of the
antibody, fragment or immunoconjugate is an amount effective alone
or as part of a treatment regimen that retards or inhibits the
growth or proliferation of disease cells presenting with higher
than normal erbB2 density.
[0078] In embodiments, the present antibodies are administered by
intravenous infusion, such as at an initial dose of 4 mg/kg over 90
minutes, then 2 mg/kg over 30 minutes, once weekly for 52 weeks,
with follow up as required.
[0079] The antibody and bivalent fragments are useful in the
treatment of a variety of cancers, to inhibit the growth or
proliferation of cancer cells and tumours comprising them,
including hematopoietic cell cancers and solid tumours. Conditions
or disorders to be treated include benign or malignant tumors
(e.g., renal, liver, kidney, bladder, breast, gastric, ovarian,
colorectal, prostate, pancreatic, lung, vulva, and thyroid);
hepatic carcinomas; sarcomas; glioblastomas; and various head and
neck tumors; leukemias and lymphoid malignancies. In particular
embodiments, the antibody or bivalent fragment are used in the
treatment of such cancer cells that express high density erbB2, as
determined by the screening assays herein described. In particular
embodiments, the cancer cells are erbB2-presenting breast cancer
cells.
[0080] It will be appreciated that subjects who could benefit from
the present method include mammals including humans as well as
livestock, and pets.
[0081] Screening for High Density erbB2 Cancer Cells
[0082] Antibodies and bivalent fragments thereof that bind
selectively to the target antigen, e.g. erbB2, are used, in
accordance with an aspect of the invention, to screen cancer cells
to detect those which present the erbB2 antigen at high density. In
a preferred embodiment, screening is applied to a sample of cancer
cells taken from a subject that is a candidate for erbB2 antibody
therapy. Subjects testing positive for cancer cells that present
the erbB2 antigen at high density can then be scheduled for therapy
with the present antibody or fragment, or an immunoconjugate
thereof. Standard techniques, combined with the antibodies or other
binding agents herein described can be used to screen cancer
cells.
[0083] Desirably, the antibodies incorporate a detectable label.
The label may be detectable by itself. (e.g., radio-isotope labels
or fluorescent labels) or, in the case of an enzymatic label, may
catalyze chemical alteration of a substrate compound or composition
which is detectable. Radionuclides that can serve as detectable
labels include, for example, I-131, I-123, I-125, Y-90, Re-188,
Re-186, At-211, Cu-67, Bi-212, and Pd-109.
[0084] In situ detection of the binding to cancer cells bearing
high density erbB2 can be performed, using the present antibody or
fragment, by immunofluorescence or immunoelectron microscopy. For
this purpose, a histological specimen is removed from the patient,
and a labeled antibody is applied to it, preferably by overlaying
the antibody on a biological sample. This procedure also allows for
distribution of the erbB2 antigen to be examined within biopsied
tumour tissue, to reveal only those sites at which the antigen is
presented at a density higher than normal. It will be apparent for
those skilled in the art that a wide variety of histological
methods are readily available for in situ detection.
[0085] More particularly, erbB2 antibodies or binding fragments may
be used to monitor the presence or absence of antibody reactivity
in a biological sample (e.g., a tissue biopsy, a cell, or fluid)
using standard detection assays. Immunological assays may involve
direct detection, and are particularly suited for screening large
amounts of samples for the presence of erbB2 positive cancer cells.
For example, antibodies may be used in any standard immunoassay
format (e.g., ELISA, Western blot, immunoprecipitation, flow
cytometry or RIA assay) to measure complex formation. Any
appropriate label which may be directly or indirectly visualized
may be utilized in these detection assays including, without
limitation, any radioactive, fluorescent, chromogenic (e.g.,
alkaline phosphatase or horseradish peroxidase), or
chemiluminescent label, or hapten (for example, digoxigenin or
biotin) which may be visualized using a labeled, hapten-specific
antibody or other binding partner (e.g., avidin). Exemplary
immunoassays are described, e.g., in Ausubel et al., supra, Harlow
and Lane, Antibodies: A Laboratory Approach, Cold Spring Harbor
Laboratory, New York (1988), and Moynagh and Schimmel, Nature
400:105, 1999. For example, using the antibodies described herein,
high density erbB2 is readily detected at the cell surface using
standard flow cytometry methods. Samples found to contain increased
levels of labeled complex compared to appropriate control samples
are taken as indicating the presence of high density erbB2, and are
thus indicative of a cancer or other disease amenable to treatment
with the present antibodies.
[0086] The present antibody is produced suitably by recombinant DNA
means, as exemplified herein. For production, there is provided a
DNA molecule that encodes the heavy chain of the present antibody,
and a DNA molecule that encodes the light chain thereof. The DNA
further encodes any suitable signal peptide suitable for expression
of a secretable chain precursor that enables proper externalization
with folding and disulfide formation to elaborate the desired
antibody as a secreted, dimerized and processed protein. To this
end, the present invention provides, in one embodiment, a
polynucleotide comprising a sequence that encodes the variable
region of the light chain of a presently preferred erbB2 antibody,
as set out in SEQ ID No. 9, supra. Also provided, in another
embodiment, is a polynucleotide comprising a sequence that encodes
the variable region of the heavy chain of a presently preferred
erbB2 antibody, as set out in SEQ ID No. 10, supra.
[0087] In more specific embodiments, the present invention provides
a polynucleotide that encodes the entire light chain (SEQ ID No.
11) and the entire heavy chain (SEQ ID No.12) of a preferred erbB2
antibody of the present invention. These sequences are provided at
the end of this disclosure.
[0088] It will be appreciated that polynucleotide equivalents also
can be used, in which synonymous codons are replaced within the
sequences provided, to produce the present antibodies.
[0089] In embodiments, there are also provided vectors that
comprise polynucleotides that encode the heavy chain or the
variable region thereof and that encode the light chain or the
variable region thereof. To express the antibodies, the
polynucleotides are incorporated operably within expression
vectors, i.e., operatively linked to transcriptional and
translational control sequences. Expression vectors include
plasmids, retroviruses, cosmids, and the like. The expression
vector and expression control sequences are chosen to be compatible
with the expression host cell used. The antibody light chain gene
and the antibody heavy gene can be inserted into separate vectors.
In a preferred embodiment, both genes are inserted into the same
expression vector. The antibody genes are inserted into the
expression vector by standard methods (e.g., ligation of
complementary restriction sites on the antibody gene fragment and
vector, or blunt end ligation if no restriction sites are
present).
[0090] A convenient vector is one that encodes a functionally
complete human CH or CL immunoglobulin sequence, with appropriate
restriction sites engineered so that any VH or VL sequence can be
easily inserted and expressed, as described above. In such vectors,
splicing usually occurs between the splice donor site in the
inserted J region, and the splice acceptor site preceding the human
C region, and also at the splice regions that occur within the
human CH exons. Polyadenylation and transcription termination occur
at native chromosomal sites downstream of the coding regions. The
recombinant expression vector can also encode a signal peptide that
facilitates secretion of the antibody chain from a host cell. The
antibody chain gene may be cloned into the vector such that the
signal peptide is linked in-frame to the amino terminus of the
antibody chain gene. The signal peptide can be an immunoglobulin
signal peptide or a heterologous signal peptide (i.e., a signal
peptide from a non-immunoglobulin protein).
[0091] Polynucleotides encoding the heavy chain and/or the light
chain, and vectors comprising these can be used for transformation
of a suitable mammalian host cell. Methods for introduction of
heterologous polynucleotides into mammalian calls include
dextran-mediated transfection, calcium phosphate precipitation,
polybrene-mediated transfection, protoplast fusion,
electroporation, encapsulation of the polynucleotide(s) in
liposomes, biolistic injection and direct microinjection of the DNA
into nuclei. In addition, polynucleotides may be introduced into
mammalian cells by viral vectors. Mammalian cell lines useful as
hosts for expression of the antibody-encoding polynucleotides
include many immortalized cell lines available from the American
Type Culture Collection (ATCC). These include, inter alia, Chine
hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster
kidney (BHK) cells, monkey kidney cells (COS, human hepatocellular
carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and a number
of other cell lines. Mammalian host cells include human, mouse,
rat, dog, monkey, pig, goat, bovine, horse, and hamster cells. Cell
lines of particular preference are selected through determining
which cell lines have high expression levels. Other cell lines that
may be used are insect cell lines, such as S19 cells, amphibian
cells, bacterial cells, plant cells and fungal cells. When
recombinant expression vectors encoding the heavy chain or
antigen-binding portion thereof are introduced into mammalian host
cells, the antibodies are produced by culturing the host cells for
a period of time sufficient to allow for expression of the antibody
in the host cells or, more preferably, secretion of the antibody
into the culture medium in which the host cells are grown.
Antibodies can be recovered from the culture medium using standard
protein purification methods.
[0092] It is likely that antibodies expressed by different cell
lines or in transgenic animals will have different glycosylation
from each other. However, all antibodies encoded by the
polynucleotides provided herein, or comprising the amino acid
sequences provided herein are part of the instant invention.
[0093] Embodiments are now described in the following examples.
EXAMPLES
[0094] The structure of trastuzumab bound to erbB2 [Ref TS1] was
used as starting point for mutant design. Mutations were introduced
only in the CDR regions of the light and heavy chain. First,
single-point mutations were generated and evaluated
computationally. Virtual mutagenesis was carried out with optional
conformational relaxation upon mutation by means of conformational
sampling algorithms, such as Monte Carlo minimization [Ref TS2].
Prediction of antigen-antibody relative binding affinities between
parent and mutant antibodies was carried out with binding affinity
scoring functions, such as the solvated interaction energy (SIE)
function [Ref TS3]. Prediction of relative antigen-antibody
association rates (k.sub.on) between parent and mutant antibodies
was carried out with methods that evaluate long-range electrostatic
interactions, such as HyPARE [Ref TS4]. Candidate single-point
mutants were the assembled into multiple-point mutants and
re-scored for relative binding affinity.
[0095] Multiple-point mutants were generated by combining
single-point mutants between light and heavy chains to achieve the
targeted change in affinity. A requirement was to use as few
single-point mutants as possible and to maximize the number of
generated assembled antibodies. Another desirable feature was to
generate a pool of mutants with reduced affinities due to either
increased dissociation rates (k.sub.-off) or to decreased
association rates (k.sub.-on). Among suitable candidate
single-point mutations, those targeting distinct locations within
the antibody-antigen interface, preferably at its periphery, were
given higher priority.
Preparation of Plasmids
[0096] All the cDNAs encoding the heavy and light chains of the
antibodies were ordered from GeneArt (Regensburg Germany). The
cDNAs were removed from the plasmid provided by GeneArt by
digestion with HindIII and cloned into the HindIII site of plasmid
pKCR5 previously dephosphorylated with calf intestinal phosphatase
(NEB) to prevent recircularization. In pKCR5, transcription of the
cDNA is under the control of the strong CR5 promoter, part of the
cumate gene switch. The plasmid pKCR5 is available from the
Biotechnology Research Institute, Montreal, Canada and is described
by Mullick et al, BMC Biotechnol., 2006, 6:43. This 3.9 kb plasmid
incorporates a HindIII in proper contex with the CR5 promoter and a
rabbit b-globin polyA, together with a B-lactamase gene for
selection, and colE1 and f1 origins of replication. For
transfection of CHO cells, all plasmids were isolated from large
culture of E. coli using the Plasmid Maxi kit (Qiagen Inc,
Mississauga, ON) according to the manufacturer's recommendation.
Briefly, 3 ml of LB medium containing 100 .mu.g/ml ampicillin were
inoculated with a single colony of E. coli and grown for 6 h at
37.degree. C. with vigorous shaking (250 RPM). This preculture was
then used to inoculate 250 ml of LB medium containing 100 .mu.g/ml
ampicillin. The culture was incubated overnight at 37.degree. C.
with vigorous shaking (250 RPM). The bacteria were pelleted by
centrifugation at 6000.times.g, for 15 min, at 4.degree. C. and the
plasmid was isolated using the protocols, buffers and columns
provided by the kit. The pure plasmids was resuspended in sterile
50 mM TRIS, pH 8 and quantified by measuring the optical density at
260 nm.
Cell Line (CHO-cTA; Clone 5F1) and Growth Conditions
[0097] The CHO-cTA cell line (Gaillet, B. et al, Biotechnol. Prog.
23:200-209; Mullick, A., et al. BMC Biotechnol. 2006, 6:43.) used
for transient transfection is a Chinese Hamster Ovary cell line
(CHO) adapted to grow in suspension and in protein-free medium. The
cell line stably expresses the cumate transactivator (cTA) which
activates transcription by binding to the CR5 promoter. The CHO-cTA
are maintained in CD-CHO medium (Invitrogen, CDCHO 10743),
supplemented with 4 mM glutamine, 50 .mu.g/mL and dextran sulfate
(Amersham Pharmacia Biotech) at 37.degree. C. under an atmosphere
of 5% CO.sub.2. When the cells reach a concentration of
1.0.times.10.sup.6 cells/ml (on average three times a week) they
are passaged by diluting them to a concentration of
5.0.times.10.sup.4 cells/ml using fresh medium.
Transient Transfection of CHO-cTA
[0098] Before transfection, the cells were washed with PBS and
resuspended at a concentration of 2.5.times.10.sup.6 cell/ml in
growth medium without dextran sulfate for 3 hrs in suspension
culture. 50 ml of cells were transfected by adding slowly 2.5 ml of
a CDCHO medium supplemented with 1 .mu.g/ml of plasmid and 5
.mu.g/ml. polyethylenimine (PEI Max; Polysciences). After 2 hrs,
the cells were transferred at 30.degree. C. The next days, 50
.mu.g/mL of dextran sulfate was added to the cells and they were
incubated at 30.degree. C. for a total of 4 days. The supernatant
was clarified by centrifugation and filtered through a 0.22 .mu.M
filter and transferred at -80.degree. C. until further
analysis.
Polyacrylamide Gel Electrophoresis (SDS-PAGE)
[0099] Known amounts of supernatant were resuspended into an equal
volume of Laemmli 2.times. and heated at 95.degree. C. for 5 min
and chilled on ice. The samples were then separated on a
polyacrylamide Novex 10% Tris-Glycine gel (Invitrogen Canada Inc.,
Burlington, ON). A standard curve was made by adding known amount
of purified human IgG. The gel was then stained using a solution of
Coomassie Fluor.TM.-Orange (Molecular Probes, Eugene Oreg.)
according to the manufacturer's recommendations. The signal was
visualized and quantified using the Typhoon Scanner.
Western Blot Analysis
[0100] Known amounts of supernatant were separated on a SDS-PAGE as
described above and then transferred onto a Hybond-N nitrocellulose
membrane (Amersham Bioscience Corp., Baie d'Urfee, QC) for 1 h at
275 mA. The membrane was blocked for 1 h in 0.15% Tween 20, 5%
skimmed milk in PBS and incubated for 1 h with an anti-human IgG
conjugated to Cy5 (Jackson, Cat#109-176-099). The signal was
revealed by scanning with the Typhoon Trio+ (Amersham Biosciences,
GE Healthcare).
ELISA
[0101] 96 wells/plates were coated with 50 .mu.l of AffiniPure Goat
Anti-Human IgG, (H+L) (Jackson Immuno Research) and incubated
overnight at 4.degree. C. The wells were washed with PBS and
incubated for 30 min at 37.degree. C. with 100 .mu.l of 1% BSA in
PBS at 37.degree. C. 25 .mu.l of samples diluted with 1% BSA in PBS
were added to the wells, which were incubated for 2 hrs at
37.degree. C. The wells were washed with 0.05% Tween 20 in PBS and
incubated with an alkaline Phosphatase-conjugated AffiniPure Goat
Anti-Human IgG (H+L) (Jackson Immuno Research) for 1 hr at
37.degree. C. The wells were washed with 0.05% Tween 20 in PBS,
followed by PBS. The signal was revealed by incubation with PNPP
for 30 min at 37.degree. C. The signal intensity was measure at 405
nm. A standard curve was made using known amount of purified
antibody (IgG1, kappa from myeloma plasma (Athens Research
Technology).
Purification of Antibody
[0102] The supernatant was concentrated with a Amicon Ultra
(Ultracel-50K) at 1500 rpm to a volume of 500 .mu.l. The wild type
and mutant antibodies were purified using the Nab spin kit Protein
A mini column (Thermo Scientific) according to the manufacture's
recommendations. The purified antibodies were then desalted and
resuspended in PBS using the desalting column PD-10 (GE
Healthcare). The antibodies were then concentrated by
centrifugation on an Amica Ultra 100,000 MWCO membrane. The
purified antibodies were quantified by reading the optical density
at 280 nm using the Nanodrop spectrophotometer. The purified
antibodies were kept frozen at -20.degree. C. in 50% glycerol.
In Vitro Binding by Surface Plasmon Resonance
[0103] Kinetic and affinity analysis was carried out using a BioRad
Proteon surface plasmon resonance instrument. The running buffer
for all steps was 10 mM HEPES, 150 mM NaCl, 3.5 mM EDTA and 0.05%
Tween20 at pH 7.4. An antibody capture sensorchip was prepared by
injecting 6.5 ug/mL of anti-mouse Fc (Jackson Immunochemicals Inc.)
in 10 mM sodium acetate pH 4.5 at flow rate 25 uL/minute over a GLM
sensorchip (BioRad Inc.) that had previously been activated with a
1/10 dilution of sNHS/EDC (BioRad Inc.) until the surface was
saturated (approximately 5000 RUs). This procedure was carried out
in the analyte direction to ensure all of the interspots for
referencing have immobilized anti-mouse Fc. Wild-type trastuzumab
and variants was captured in the ligand direction by injecting 100
uL of 4% culture supernatants or purified samples in running buffer
at flow rate of 25 uL/min until 400 to 800 resonance units have
been captured. This was immediately followed by two pulses of
running buffer in the analyte direction, 50 uL each at flow rate
100 uL/min to stabilize the baseline. Next, the simultaneous
injection of 100 uL of five ErbB2 (eBiosciences Inc.)
concentrations (3-fold dilutions of 30 nM or 20 nM ErbB2) and
buffer blank at a flow rate of 50 uL/min with a 600 s dissociation
was carried out to analyse the ErbB2--antibody interaction. Kinetic
rate constants (on- and off-rates) and affinity constants were
generated from the aligned and double referenced sensorgrams with
the Langmuir binding model using BioRad Proteon Manager software
v3.2.
Cell Culture
[0104] The SkBr3 and BT474 cell lines were obtained from ATCC. Cell
lines were maintained in DMEM (source) containing 10% fetal bovine
serum (Gibco). Primary adult human cardiac myocytes (HCM) were
obtained from ScienCell (Catalog #6200) and cultured using
manufacturer's recommended Cardiac Myocyte Medium. Generally cells
were passaged once or twice a week and used within 4-6 weeks for
all experiments.
Detection of Antibody Binding to Surface erbB2 Level by Flow
Cytometry
[0105] Prior to analysis, cells were plated such that they were not
more than 80% confluent on the day of analysis. Tumor cells
overexpressing her-2(SkBr3, .about.2.5M Her2/cell or BT474,
.about.3M Her2/cell) or normal (human cardiac myocytes,
.about.20,000 Her2/cell) were washed in PBS and harvested by the
addition of cell dissociation buffer (Sigma.). A cell suspension
containing 2.5.times.10.sup.5 in 500 .mu.l corresponding cell
culture media) was incubated with various concentrations (0.01-100
ug/ml) of anti-HER2 antibodies for 2 h at 4.degree. C. (to prevent
internalization). Following 1 wash with cell culture media, primary
antibody was incubated with 2 ug Dylight 488 conjugated AffiniPure
goat anti-human IgG Alexa 488 secondary antibody (Jackson
ImmunoResearch 109-487-003) in 100 ul of media for 1 h at 4.degree.
C. Cells were then pelleted and stored on ice until ready to
analyzed by flow cytometry. Prior to analysis, cell pellets were
resuspended in 300-500 ul media and filtered through a 50 um nylon
mesh filter to remove cell aggregates. Flow cytometry analyses were
performed on 10,000 viable cells gated on forward scattering, side
scattering parameters and propidium iodide dye exclusion using a BD
LSRII flow Cytometer (Becton-Dickinson Biosciences, CA, USA) and a
standard filter set using BD FACSDiva.TM. acquisition software,
according to manufacturer's instructions.
[0106] Specific antibody binding was calculated as the mean
fluorescent intensity of binding to each antibody after background
level subtraction of the mean fluorescent intensity of binding in
the absence of primary antibody (but containing detection
antibody). For all experiments, specific antibody binding was
compared relative to that of the wild type version of trastuzumab
that was produced and purified in the same manner (HC/LC). To
examine the binding selectivity of antibodies, the value of
antibody binding to tumor (overexpressing ErbB2) was divided by the
binding observed with normal human cardiac myocyte cells. This
parameter, named the ratio of binding, was calculated and compared
to that seen with wild type antibody (named 2-1 wt, set arbitrarily
to 1). A commercial source of trastuzumab (Roche) was used as a
benchmark for comparison purposes.
Results:
1. Production of ErbB2 Antibodies.
[0107] Eight cDNAs corresponding to the coding sequence of the
ErbB2 antibodies (Table above) were synthesized (GeneArt). All the
cDNAs were cloned into the HindIII site of pKCR5, an expression
vector regulated by the cumate-switch (pKCR5 vector).
[0108] For each antibody, 50 ml of CHOcTA (expressing the cumate
transactivator, cTA) were transfected with various combinations of
heavy and light chain. Four days after transfection the supernatant
was analyzed by SDS-PAGE (not shown), with quantifications made by
Western Blot (Table 4) and by ELISA (Table 3).
TABLE-US-00007 TABLE 3 Quantification Quantification Heavy and
light chains Western blot ELISA used (Heavy_Light) (mg/L) (mg/L)
1_2 67.02 86.46 2_3 107.27 96.79 2_4 87.69 58.08 2_5 54.36 95.51
2_6 26.21 22.99 7_1 66.04 76.44 7_3 80.14 81.98 7_4 60.72 118.02
7_5 34.49 52.12 7_6 50.10 46.45 8_1 81.49 66.98 8_3 43.78 46.45 8_4
48.22 45.98 8_5 48.77 61.47 8_6 16.62 29.56 K- 0 0 Where, for the
light chain, and for the heavy chain 1 = wild type light chain 2 =
wild type 3 = F53N 4 = Y92A 5 = F53N, Y92A 6 = N30D 7 = Y57A 8 =
R59E
[0109] The wild type and 9 mutants were purified by chromatography
using protein A. The purified proteins were quantified by
OD.sub.280 (Nanodrop) see Table 4. The purified antibodies were
analyzed by non-denaturing and denaturing SDS-PAGE.
TABLE-US-00008 TABLE 4 Quantification of the purified antibodies by
OD.sub.280 (Nanodrop) Purified Concentration mutants (ug/ml) 1_2
Wild type 580 2_3 wt-HC + m1-Lc 500 2_4 wt-HC + m2-LC 370 2_5 wt-HC
+ m3-LC 450 2_6 wt-HC + -m4-LC 270 7_1 m5-HC + wt-LC 500 7_3 m5-HC
+ m1-LC 540 7_4 m5-HC + m2-LC 520 7_5 m5-HC + m3-LC 380 7_6 m5-HC +
m4-LC 310
[0110] Binding Affinity Determination by SPR
[0111] The 2- and 7-series trastuzumab variants had detectible
activity at the 20 nM ErbB2 concentrations used. While the 2-series
fit quite well to a Langmuir binding model (1:1), the 7-series
variants showed complexity in their kinetics as seen by deviations
from ideal behaviour in the dissociation phase. This is especially
evident with variant 7-5 which shows a complex biphasic off-rate
that fit poorly to the Langmuir binding model. The affinity of this
variant would likely be lower than the 0.3 nM indicated in Table 5,
as the modeled fit uses the slow dissociation phase only. Series 8,
those with the heavy chain substitution R59E, did not show any
detectible binding activity indicating this position is critical
for binding activity of trastuzumab. The wild-type version (2-1)
had identical binding behaviour to the commercial trastuzumab.
TABLE-US-00009 TABLE 5 Kinetic parameters of ErbB2 - mAb variant
binding modeled to a 1:1 binding fit ka 1/Ms kd 1/s KD Chi2 RU
Variant 7-1 7.07E+05 1.72E-04 2.43E-10 3.38 Variant 7-3 6.70E+05
9.24E-04 1.38E-09 14.92 Variant 7-4 6.01E+05 1.10E-03 1.83E-09 25.9
Variant 7-5 8.12E+05 2.11E-04 2.59E-10 5.48 Variant 7-6 5.76E+05
2.91E-03 5.05E-09 3.68 Variant 2-3 8.46E+05 1.17E-04 1.39E-10 3.76
Variant 2-4 7.47E+05 2.95E-04 3.95E-10 3.55 Variant 2-5 7.19E+05
1.12E-03 1.56E-09 11.91 Variant 2-6 6.21E+05 4.30E-04 6.92E-10 3.7
Trastuzumab 8.82E+05 2.92E-05 3.31E-11 5.84 Wild-type 2-1 8.56E+05
1.66E-05 1.94E-11 3.99
[0112] Experimental testing of the designed Trastuzumab mutants
using the SPR technique showed that the erbB2 binding affinity is
weakened over 100-fold for 1 mutant, between 10-100-fold for 5
mutants, and below 10-fold for another 3 mutants, relative to the
parent Trastuzumab antibody (see also FIG. 1).
Evaluation of Antibody Binding to Tumor and Normal Cell Lines
[0113] Binding of the antibodies to various cell lines was also
determined by indirect flow cytometry as described in the Materials
and Methods. As shown in FIG. 3, these results demonstrate weaker
binding of 2-5 and 7-6 antibody mutants to HCM cells compared to
tumor cells (relative to binding observed for wt control and
commercial benchmark). Also as shown in FIG. 4, this result clearly
shows that some of the antibody mutants exhibit better binding to
tumor relative to normal HEK cells (e.g. 2-5 exhibits 10-12.times.,
7-6 exhibits 6-8.times. more binding to tumor than normal). The
pattern of binding specificity was similar amongst the tumor cell
lines analyzed (SKBr3 or BT474) suggesting that the selectivity of
binding is universally high for all tumor cells presenting erbB2 at
a density greater than the normal erbB2 density (.about.3 million
receptors per cell or more).
[0114] Polynucleotides encoding the various mutant antibody chains
are provided below. Substituted codons are italicized and in bold
font, and HindIII sites are italicized:
TABLE-US-00010 Light chain: WT [SEQ ID No. 11]
GGTACCAAGCTTGCCACCATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGG
ATCTCTGGCGCCTACGGCGACATCCAGATGACCCAGTCCCCCTCCTCCCTGTCTGCCTCC
GTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCCCAGGATGTGAACACCGCCGTGGCC
TGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTCCGCCTCCTTCCTG
TACTCCGGCGTGCCCTCCCGGTTCTCCGGCTCCAGATCCGGCACCGACTTCACCCTGACC
ATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACC
CCTCCAACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCTCCTTCC
GTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCTGTCGTCTGC
CTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTG
CAGTCCGGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACTCTACCTACTCC
CTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGC
GAAGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGGGCGAGTGC
TGATGAAAGCTTGAGCTC >m1; Light chain: F53N (changed TTC for AAC)
[SEQ ID No. 12]
GGTACCAAGCTTGCCACCATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGG
ATCTCTGGCGCCTACGGCGACATCCAGATGACCCAGTCCCCCTCCTCCCTGTCTGCCTCC
GTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCCCAGGATGTGAACACCGCCGTGGCC
TGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTCCGCCTCC CTG
TACTCCGGCGTGCCCTCCCGGTTCTCCGGCTCCAGATCCGGCACCGACTTCACCCTGACC
ATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACC
CCTCCAACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCTCCTTCC
GTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCTGTCGTCTGC
CTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTG
CAGTCCGGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACTCTACCTACTCC
CTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGC
GAAGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGGGCGAGTGC
TGATGAAAGCTTGAGCTC >m2; Light chain: Y92A (changed TAC for GCC)
[SEQ ID No. 13]
GGTACCAAGCTTGCCACCATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGG
ATCTCTGGCGCCTACGGCGACATCCAGATGACCCAGTCCCCCTCCTCCCTGTCTGCCTCC
GTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCCCAGGATGTGAACACCGCCGTGGCC
TGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTCCGCCTCCTTCCTG
TACTCCGGCGTGCCCTCCCGGTTCTCCGGCTCCAGATCCGGCACCGACTTCACCCTGACC
ATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCAC ACCACC
CCTCCAACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCTCCTTCC
GTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCTGTCGTCTGC
CTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTG
CAGTCCGGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACTCTACCTACTCC
CTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGC
GAAGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGGGCGAGTGC
TGATGAAAGCTTGAGCTC >m3; Light chain: F53N, Y92A (changed TTC for
AAC and TAC for GCC) [SEQ ID No. 14]
GGTACCAAGCTTGCCACCATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGG
ATCTCTGGCGCCTACGGCGACATCCAGATGACCCAGTCCCCCTCCTCCCTGTCTGCCTCC
GTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCCCAGGATGTGAACACCGCCGTGGCC
TGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTCCGCCTCC CTG
TACTCCGGCGTGCCCTCCCGGTTCTCCGGCTCCAGATCCGGCACCGACTTCACCCTGACC
ATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCAC ACCACC
CCTCCAACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCTCCTTCC
GTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCTGTCGTCTGC
CTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTG
CAGTCCGGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACTCTACCTACTCC
CTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGC
GAAGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGGGCGAGTGC
TGATGAAAGCTTGAGCTC >m4; Light chain: N30D (changed AAC for GAC)
[SEQ ID No. 15]
GGTACCAAGCTTGCCACCATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGG
ATCTCTGGCGCCTACGGCGACATCCAGATGACCCAGTCCCCCTCCTCCCTGTCTGCCTCC
GTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCCCAGGATGTG ACCGCCGTGGCC
TGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTCCGCCTCCTTCCTG
TACTCCGGCGTGCCCTCCCGGTTCTCCGGCTCCAGATCCGGCACCGACTTCACCCTGACC
ATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACC
CCTCCAACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCTCCTTCC
GTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCTGTCGTCTGC
CTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTG
CAGTCCGGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACTCTACCTACTCC
CTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGC
GAAGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGGGCGAGTGC
TGATGAAAGCTTGAGCTC Heavy chain: WT [SEQ ID No. 16]
TTAATTAAGCTTGCCACCATGGACTGGACCTGGCGGATCCTGTTTCTGGTGGCCGCTGCT
ACCGGCACACACGCCGAGGTGCAGCTGGTGGAGTCTGGCGGAGGACTGGTGCAGCCTGGC
GGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCAACATCAAGGACACCTACATCCAC
TGGGTCCGGCAGGCTCCAGGCAAGGGACTGGAATGGGTGGCCCGGATCTACCCTACCAAC
GGCTACACCAGATACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCCGCCGACACCTCC
AAGAACACCGCCTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTAC
TGCTCCAGATGGGGAGGCGACGGCTTCTACGCCATGGACTACTGGGGCCAGGGCACCCTG
GTCACTGTGTCCTCTGCCTCCACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTCCAGC
AAGTCTACCTCTGGCGGCACCGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAG
CCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCC
GTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCTCTAGC
CTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGAC
AAGAAGGTGGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCCTCCATGCCCTGCCCCT
GAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCTCCAAAGCCTAAGGACACCCTGATG
ATCTCCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGATCCTGAA
GTGAAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGG
GAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGAC
TGGCTGAACGGCAAAGAGTATAAGTGCAAAGTCTCCAACAAGGCCCTGCCTGCCCCTATC
GAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGGGAACCTCAGGTGTACACCCTGCCT
CCCAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAGGGCTTC
TACCCTTCCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAG
ACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAACTGACCGTG
GACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTG
CACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCTGGCAAGTGATGAAAGCTTGGC GCGCC
>m5; Heavy chain: Y57A (Changed TAC for GCC) [SEQ ID No. 17]
TTAATTAAGCTTGCCACCATGGACTGGACCTGGCGGATCCTGTTTCTGGTGGCCGCTGCT
ACCGGCACACACGCCGAGGTGCAGCTGGTGGAGTCTGGCGGAGGACTGGTGCAGCCTGGC
GGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCAACATCAAGGACACCTACATCCAC
TGGGTCCGGCAGGCTCCAGGCAAGGGACTGGAATGGGTGGCCCGGATCTACCCTACCAAC GGC
ACCAGATACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCCGCCGACACCTCC
AAGAACACCGCCTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTAC
TGCTCCAGATGGGGAGGCGACGGCTTCTACGCCATGGACTACTGGGGCCAGGGCACCCTG
GTCACTGTGTCCTCTGCCTCCACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTCCAGC
AAGTCTACCTCTGGCGGCACCGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAG
CCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCC
GTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCTCTAGC
CTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGAC
AAGAAGGTGGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCCTCCATGCCCTGCCCCT
GAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCTCCAAAGCCTAAGGACACCCTGATG
ATCTCCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGATCCTGAA
GTGAAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGG
GAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGAC
TGGCTGAACGGCAAAGAGTATAAGTGCAAAGTCTCCAACAAGGCCCTGCCTGCCCCTATC
GAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGGGAACCTCAGGTGTACACCCTGCCT
CCCAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAGGGCTTC
TACCCTTCCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAG
ACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAACTGACCGTG
GACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTG
CACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCTGGCAAGTGATGAAAGCTTGGC GCGCC
>m6; Heavy chain: R59E (changed AGA for GAG) [SEQ ID No. 18]
TTAATTAAGCTTGCCACCATGGACTGGACCTGGCGGATCCTGTTTCTGGTGGCCGCTGCT
ACCGGCACACACGCCGAGGTGCAGCTGGTGGAGTCTGGCGGAGGACTGGTGCAGCCTGGC
GGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCAACATCAAGGACACCTACATCCAC
TGGGTCCGGCAGGCTCCAGGCAAGGGACTGGAATGGGTGGCCCGGATCTACCCTACCAAC
GGCTACACC TACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCCGCCGACACCTCC
AAGAACACCGCCTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTAC
TGCTCCAGATGGGGAGGCGACGGCTTCTACGCCATGGACTACTGGGGCCAGGGCACCCTG
GTCACTGTGTCCTCTGCCTCCACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTCCAGC
AAGTCTACCTCTGGCGGCACCGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAG
CCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCC
GTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCTCTAGC
CTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGAC
AAGAAGGTGGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCCTCCATGCCCTGCCCCT
GAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCTCCAAAGCCTAAGGACACCCTGATG
ATCTCCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGATCCTGAA
GTGAAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGG
GAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGAC
TGGCTGAACGGCAAAGAGTATAAGTGCAAAGTCTCCAACAAGGCCCTGCCTGCCCCTATC
GAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGGGAACCTCAGGTGTACACCCTGCCT
CCCAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAGGGCTTC
TACCCTTCCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAG
ACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAACTGACCGTG
GACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTG
CACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCTGGCAAGTGATGAAAGCTTGGC
GCGCC
[0115] Amino acid sequences constituting the antibody mutant chains
are provided below. The signal peptide is indicated using lower
case letters and is not included in the residue numbering. Mutated
positions are italicized and in bold font.
TABLE-US-00011 Anti-HER2 Light chain wild-type [SEQ ID No. 19]
.........|.........|.........|.........|.........|.........|
mvlqtqvfislllwisgaygDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKP
GKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Anti-HER2
Heavy chain wild-type [SEQ ID No. 20]
.........|.........|.........|.........|.........|.........|
mdwtwrilflvaaatgthaEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVARTYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGG
DGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mutant sequences
>m1; Light chain: F53N [SEQ ID No. 21]
mvlqtqvfislllwisgaygDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKP
GKAPKLLIYSAS LYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >m2;
Light chain: Y92A [SEQ ID No. 22]
mvlqtqvfislllwisgaygDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKP
GKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQH TTPPTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >m3;
Light chain: F53N, Y92A [SEQ ID No. 23]
mvlqtqvfislllwisgaygDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKP
GKAPKLLIYSAS LYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQH TTPPTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >m4;
Light chain: N30D [SEQ ID No. 24]
mvlqtqvfislllwisgaygDIQMTQSPSSLSASVGDRVTITCRASQDV TAVAWYQQKP
GKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >m5;
Heavy chain: Y57A [SEQ ID No. 25]
mdwtwrilflvaaatgthaEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVARTYPTNG TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGG
DGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >m6; Heavy
chain: R59E [SEQ ID No. 26]
mdwtwrilflvaaatgthaEVQLVESGGGLVQPGGSLRLSCAASGENIKDTYTHWVRQAP
GKGLEWVARIYPTNGYT YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGG
DGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
REFERENCES
[0116] TS1. Cho, H. S., Mason, K., Ramyar, K. X., Stanley, A. M.,
Gabelli, S. B., Denney, D. W., and Leahy, D. J. (2003) Structure of
the extracellular region of HER2 alone and in complex with the
Herceptin Fab, Nature 421, 756-760. [0117] TS2. Li, Z. and
Scheraga, H. A. (1987) Monte Carlo-minimization approach to the
multiple-minima problem in protein folding, Proc. Nat. Acad. Sci.
U.S.A. 84, 6611-6615. [0118] TS3. Naim, M., Bhat, S., Rankin, K.
N., Dennis, S., Chowdhury, S. F., Siddiqi, I., Drabik, P., Sulea,
T., Bayly, C. I., Jakalian, A., and Purisima, E. O. (2007) Solvated
Interaction Energy (SIE) for Scoring Protein-Ligand Binding
Affinities. 1. Exploring the Parameter Space, J. Chem. Inf. Model.
47, 122-133. [0119] TS4. Selzer, T., Albeck, S., and Schreiber, G.
(2000) Rational design of faster associating and tighter binding
protein complexes, Nat. Struct. Mol. Biol. 7, 537-541.
Sequence CWU 1
1
26110PRTArtificial SequenceHeavy chain CDR1 1Gly Phe Asn Ile Lys
Asp Thr Tyr Ile His1 5 10217PRTArtificial SequenceHeavy chain
CDR2MISC_FEATURE(8)..(8)Xaa is Tyr or an amino acid having a side
chain that is nonpolar and/or a side chain that is non-neutral
and/or a side chain that is not largeMISC_FEATURE(10)..(10)Xaa is
Arg or an amino acid having a side chain that is nonpolar and/or a
side chain that is non-neutral and/or a side chain that is not
large 2Arg Ile Tyr Pro Thr Asn Gly Xaa Thr Xaa Tyr Ala Asp Ser Val
Lys1 5 10 15Gly311PRTArtificial SequenceHeavy chain CDR3 3Trp Gly
Gly Asp Gly Phe Tyr Ala Met Asp Tyr1 5 10411PRTArtificial
SequenceLight chain CDR1MISC_FEATURE(7)..(7)Xaa is Asn or an amino
acid having a side chain that is either nonpolar and/or is negative
or positive in charge and/or may not be small 4Arg Ala Ser Gln Asp
Val Xaa Thr Ala Val Ala1 5 1057PRTArtificial SequenceLight chain
CDR2MISC_FEATURE(4)..(4)Xaa is Phe or an amino acid having a side
chain that is either polar and/or is charge positive or negative
and/or is not large 5Ser Ala Ser Xaa Leu Tyr Ser1 569PRTArtificial
SequenceLight chain CDR3MISC_FEATURE(4)..(4)Xaa is Tyr or an amino
acid having a side chain that is nonpolar and/or a side chain that
is non-neutral and/or is not large. 6Gln Gln His Xaa Thr Thr Pro
Pro Thr1 57107PRTArtificial SequenceLight chain variable
regionMISC_FEATURE(30)..(30)Xaa is Asn or an amino acid having a
side chain that is either nonpolar and/or is negative or positive
in charge and/or may not be small.MISC_FEATURE(53)..(53)Xaa is Phe
or an amino acid having a side chain that is either polar and/or is
charge positive or negative and/or is not
large.MISC_FEATURE(92)..(92)Xaa is Tyr or an amino acid having a
side chain that is nonpolar and/or a side chain that is non-neutral
and/or is not large. 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 Arg Ala Ser
Gln Asp Val Xaa Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Xaa Leu Tyr Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Arg Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln His Xaa Thr Thr Pro Pro 85 90 95Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 1058120PRTArtificial SequenceHeavy
chain variable regionMISC_FEATURE(57)..(57)Xaa is Tyr or an amino
acid having a side chain that is nonpolar and/or a side chain that
is non-neutral and/or a side chain that is not
large.MISC_FEATURE(59)..(59)Xaa is Arg or an amino acid having a
side chain that is nonpolar and/or is charge neutral or negative
and/or is not large. 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
Phe Asn Ile Lys Asp Thr 20 25 30Tyr Ile His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Tyr Pro Thr Asn Gly
Xaa Thr Xaa Tyr Ala Asp Ser Val 50 55 60Lys Gly 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 95Ser Arg Trp Gly
Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr
Leu Val Thr Val Ser Ser 115 1209214PRTArtificial SequenceLight
chainMISC_FEATURE(30)..(30)Xaa is Asn or an amino acid having a
side chain that is either nonpolar and/or is negative or positive
in charge and/or may not be small.MISC_FEATURE(53)..(53)Xaa is Phe
or an amino acid having a side chain that is either polar and/or is
charge positive or negative and/or is not
large.MISC_FEATURE(92)..(92)Xaa is Tyr or an amino acid having a
side chain that is either nonpolar and/or a side chain that is
non-neutral and/or is not large. 9Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Val Xaa Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Xaa
Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Arg Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln His Xaa Thr Thr Pro Pro 85 90 95Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg
Gly Glu Cys 21010450PRTArtificial SequenceHeavy
chainMISC_FEATURE(57)..(57)Xaa is Tyr or an amino acid having a
side chain that is nonpolar and/or a side chain that is non-neutral
and/or a side chain that is not large.MISC_FEATURE(59)..(59)Xaa is
Arg or an amino acid having a side chain that is nonpolar and/or is
charge neutral or negative and/or is not large. 10Glu 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 Phe Asn Ile Lys Asp Thr 20 25 30Tyr Ile
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala
Arg Ile Tyr Pro Thr Asn Gly Xaa Thr Xaa Tyr Ala Asp Ser Val 50 55
60Lys Gly 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 95Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala Pro Glu Leu Leu
Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315
320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445Gly Lys 45011738DNAArtificial SequenceLight chain 11ggtaccaagc
ttgccaccat ggtgctgcag acccaggtgt tcatctccct gctgctgtgg 60atctctggcg
cctacggcga catccagatg acccagtccc cctcctccct gtctgcctcc
120gtgggcgaca gagtgaccat cacctgtcgg gcctcccagg atgtgaacac
cgccgtggcc 180tggtatcagc agaagcctgg caaggcccct aagctgctga
tctactccgc ctccttcctg 240tactccggcg tgccctcccg gttctccggc
tccagatccg gcaccgactt caccctgacc 300atctccagcc tgcagcctga
ggacttcgcc acctactact gccagcagca ctacaccacc 360cctccaacct
tcggccaggg caccaaggtg gagatcaagc ggaccgtggc cgctccttcc
420gtgttcatct tcccaccttc cgacgagcag ctgaagtccg gcaccgcctc
tgtcgtctgc 480ctgctgaaca acttctaccc tcgggaggcc aaggtgcagt
ggaaggtgga caacgccctg 540cagtccggca actcccagga atccgtcacc
gagcaggact ccaaggactc tacctactcc 600ctgtcctcca ccctgaccct
gtccaaggcc gactacgaga agcacaaggt gtacgcctgc 660gaagtgaccc
accagggcct gtccagccct gtgaccaagt ccttcaaccg gggcgagtgc
720tgatgaaagc ttgagctc 73812738DNAArtificial SequenceLight chain
F53N 12ggtaccaagc ttgccaccat ggtgctgcag acccaggtgt tcatctccct
gctgctgtgg 60atctctggcg cctacggcga catccagatg acccagtccc cctcctccct
gtctgcctcc 120gtgggcgaca gagtgaccat cacctgtcgg gcctcccagg
atgtgaacac cgccgtggcc 180tggtatcagc agaagcctgg caaggcccct
aagctgctga tctactccgc ctccaacctg 240tactccggcg tgccctcccg
gttctccggc tccagatccg gcaccgactt caccctgacc 300atctccagcc
tgcagcctga ggacttcgcc acctactact gccagcagca ctacaccacc
360cctccaacct tcggccaggg caccaaggtg gagatcaagc ggaccgtggc
cgctccttcc 420gtgttcatct tcccaccttc cgacgagcag ctgaagtccg
gcaccgcctc tgtcgtctgc 480ctgctgaaca acttctaccc tcgggaggcc
aaggtgcagt ggaaggtgga caacgccctg 540cagtccggca actcccagga
atccgtcacc gagcaggact ccaaggactc tacctactcc 600ctgtcctcca
ccctgaccct gtccaaggcc gactacgaga agcacaaggt gtacgcctgc
660gaagtgaccc accagggcct gtccagccct gtgaccaagt ccttcaaccg
gggcgagtgc 720tgatgaaagc ttgagctc 73813738DNAArtificial
SequenceLight chain Y92A 13ggtaccaagc ttgccaccat ggtgctgcag
acccaggtgt tcatctccct gctgctgtgg 60atctctggcg cctacggcga catccagatg
acccagtccc cctcctccct gtctgcctcc 120gtgggcgaca gagtgaccat
cacctgtcgg gcctcccagg atgtgaacac cgccgtggcc 180tggtatcagc
agaagcctgg caaggcccct aagctgctga tctactccgc ctccttcctg
240tactccggcg tgccctcccg gttctccggc tccagatccg gcaccgactt
caccctgacc 300atctccagcc tgcagcctga ggacttcgcc acctactact
gccagcagca cgccaccacc 360cctccaacct tcggccaggg caccaaggtg
gagatcaagc ggaccgtggc cgctccttcc 420gtgttcatct tcccaccttc
cgacgagcag ctgaagtccg gcaccgcctc tgtcgtctgc 480ctgctgaaca
acttctaccc tcgggaggcc aaggtgcagt ggaaggtgga caacgccctg
540cagtccggca actcccagga atccgtcacc gagcaggact ccaaggactc
tacctactcc 600ctgtcctcca ccctgaccct gtccaaggcc gactacgaga
agcacaaggt gtacgcctgc 660gaagtgaccc accagggcct gtccagccct
gtgaccaagt ccttcaaccg gggcgagtgc 720tgatgaaagc ttgagctc
73814738DNAArtificial SequenceLight chain, F53N, Y92A 14ggtaccaagc
ttgccaccat ggtgctgcag acccaggtgt tcatctccct gctgctgtgg 60atctctggcg
cctacggcga catccagatg acccagtccc cctcctccct gtctgcctcc
120gtgggcgaca gagtgaccat cacctgtcgg gcctcccagg atgtgaacac
cgccgtggcc 180tggtatcagc agaagcctgg caaggcccct aagctgctga
tctactccgc ctccaacctg 240tactccggcg tgccctcccg gttctccggc
tccagatccg gcaccgactt caccctgacc 300atctccagcc tgcagcctga
ggacttcgcc acctactact gccagcagca cgccaccacc 360cctccaacct
tcggccaggg caccaaggtg gagatcaagc ggaccgtggc cgctccttcc
420gtgttcatct tcccaccttc cgacgagcag ctgaagtccg gcaccgcctc
tgtcgtctgc 480ctgctgaaca acttctaccc tcgggaggcc aaggtgcagt
ggaaggtgga caacgccctg 540cagtccggca actcccagga atccgtcacc
gagcaggact ccaaggactc tacctactcc 600ctgtcctcca ccctgaccct
gtccaaggcc gactacgaga agcacaaggt gtacgcctgc 660gaagtgaccc
accagggcct gtccagccct gtgaccaagt ccttcaaccg gggcgagtgc
720tgatgaaagc ttgagctc 73815738DNAArtificial SequenceLight chain
N30D 15ggtaccaagc ttgccaccat ggtgctgcag acccaggtgt tcatctccct
gctgctgtgg 60atctctggcg cctacggcga catccagatg acccagtccc cctcctccct
gtctgcctcc 120gtgggcgaca gagtgaccat cacctgtcgg gcctcccagg
atgtggacac cgccgtggcc 180tggtatcagc agaagcctgg caaggcccct
aagctgctga tctactccgc ctccttcctg 240tactccggcg tgccctcccg
gttctccggc tccagatccg gcaccgactt caccctgacc 300atctccagcc
tgcagcctga ggacttcgcc acctactact gccagcagca ctacaccacc
360cctccaacct tcggccaggg caccaaggtg gagatcaagc ggaccgtggc
cgctccttcc 420gtgttcatct tcccaccttc cgacgagcag ctgaagtccg
gcaccgcctc tgtcgtctgc 480ctgctgaaca acttctaccc tcgggaggcc
aaggtgcagt ggaaggtgga caacgccctg 540cagtccggca actcccagga
atccgtcacc gagcaggact ccaaggactc tacctactcc 600ctgtcctcca
ccctgaccct gtccaaggcc gactacgaga agcacaaggt gtacgcctgc
660gaagtgaccc accagggcct gtccagccct gtgaccaagt ccttcaaccg
gggcgagtgc 720tgatgaaagc ttgagctc 738161445DNAArtificial
SequenceHeavy chain 16ttaattaagc ttgccaccat ggactggacc tggcggatcc
tgtttctggt ggccgctgct 60accggcacac acgccgaggt gcagctggtg gagtctggcg
gaggactggt gcagcctggc 120ggctccctga gactgtcttg cgccgcctcc
ggcttcaaca tcaaggacac ctacatccac 180tgggtccggc aggctccagg
caagggactg gaatgggtgg cccggatcta ccctaccaac 240ggctacacca
gatacgccga ctccgtgaag ggccggttca ccatctccgc cgacacctcc
300aagaacaccg cctacctgca gatgaactcc ctgagggccg aggacaccgc
cgtgtactac 360tgctccagat ggggaggcga cggcttctac gccatggact
actggggcca gggcaccctg 420gtcactgtgt cctctgcctc caccaagggc
ccttccgtgt tccctctggc cccttccagc 480aagtctacct ctggcggcac
cgctgctctg ggctgcctgg tcaaggacta cttccctgag 540cctgtgaccg
tgtcctggaa ctctggcgcc ctgacctccg gcgtgcacac cttccctgcc
600gtgctgcagt cctccggcct gtactccctg tcctccgtcg tgaccgtgcc
ttcctctagc 660ctgggcaccc agacctacat ctgcaacgtg aaccacaagc
cttccaacac caaggtggac 720aagaaggtgg agcctaagtc ctgcgacaag
acccacacct gtcctccatg ccctgcccct 780gagctgctgg gcggaccctc
cgtgttcctg ttccctccaa agcctaagga caccctgatg 840atctcccgga
cccctgaagt gacctgcgtg gtggtggacg tgtcccacga ggatcctgaa
900gtgaagttca attggtacgt ggacggcgtg gaggtgcaca acgccaagac
caagcctcgg 960gaggaacagt acaactccac ctaccgggtg gtgtccgtgc
tgaccgtgct gcaccaggac 1020tggctgaacg gcaaagagta taagtgcaaa
gtctccaaca aggccctgcc tgcccctatc 1080gaaaagacca tctccaaggc
caagggccag cctcgggaac ctcaggtgta caccctgcct 1140cccagcaggg
acgagctgac caagaaccag gtgtccctga cctgtctggt caagggcttc
1200tacccttccg atatcgccgt ggagtgggag tctaacggcc agcctgagaa
caactacaag 1260accacccctc ctgtgctgga ctccgacggc tccttcttcc
tgtactccaa actgaccgtg 1320gacaagtccc ggtggcagca gggcaacgtg
ttctcctgct ccgtgatgca cgaggccctg 1380cacaaccact acacccagaa
gtccctgtcc ctgtctcctg gcaagtgatg aaagcttggc 1440gcgcc
1445171445DNAArtificial SequenceHeavy chain Y57A 17ttaattaagc
ttgccaccat ggactggacc tggcggatcc tgtttctggt ggccgctgct 60accggcacac
acgccgaggt gcagctggtg gagtctggcg gaggactggt gcagcctggc
120ggctccctga gactgtcttg cgccgcctcc ggcttcaaca tcaaggacac
ctacatccac 180tgggtccggc aggctccagg caagggactg gaatgggtgg
cccggatcta ccctaccaac 240ggcgccacca gatacgccga ctccgtgaag
ggccggttca ccatctccgc cgacacctcc 300aagaacaccg cctacctgca
gatgaactcc ctgagggccg aggacaccgc cgtgtactac 360tgctccagat
ggggaggcga cggcttctac gccatggact actggggcca gggcaccctg
420gtcactgtgt cctctgcctc caccaagggc ccttccgtgt tccctctggc
cccttccagc 480aagtctacct ctggcggcac cgctgctctg ggctgcctgg
tcaaggacta cttccctgag 540cctgtgaccg tgtcctggaa ctctggcgcc
ctgacctccg gcgtgcacac cttccctgcc 600gtgctgcagt cctccggcct
gtactccctg tcctccgtcg tgaccgtgcc ttcctctagc 660ctgggcaccc
agacctacat ctgcaacgtg aaccacaagc cttccaacac caaggtggac
720aagaaggtgg agcctaagtc ctgcgacaag acccacacct gtcctccatg
ccctgcccct 780gagctgctgg gcggaccctc cgtgttcctg ttccctccaa
agcctaagga caccctgatg 840atctcccgga cccctgaagt gacctgcgtg
gtggtggacg tgtcccacga ggatcctgaa 900gtgaagttca attggtacgt
ggacggcgtg gaggtgcaca acgccaagac caagcctcgg 960gaggaacagt
acaactccac ctaccgggtg gtgtccgtgc tgaccgtgct gcaccaggac
1020tggctgaacg gcaaagagta taagtgcaaa gtctccaaca aggccctgcc
tgcccctatc 1080gaaaagacca tctccaaggc caagggccag cctcgggaac
ctcaggtgta caccctgcct 1140cccagcaggg acgagctgac caagaaccag
gtgtccctga cctgtctggt caagggcttc 1200tacccttccg atatcgccgt
ggagtgggag tctaacggcc agcctgagaa caactacaag 1260accacccctc
ctgtgctgga ctccgacggc tccttcttcc tgtactccaa actgaccgtg
1320gacaagtccc ggtggcagca gggcaacgtg ttctcctgct ccgtgatgca
cgaggccctg 1380cacaaccact acacccagaa gtccctgtcc ctgtctcctg
gcaagtgatg aaagcttggc 1440gcgcc 1445181445DNAArtificial
SequenceHeavy chain R59E 18ttaattaagc ttgccaccat ggactggacc
tggcggatcc tgtttctggt ggccgctgct 60accggcacac acgccgaggt gcagctggtg
gagtctggcg gaggactggt gcagcctggc 120ggctccctga gactgtcttg
cgccgcctcc ggcttcaaca tcaaggacac ctacatccac 180tgggtccggc
aggctccagg caagggactg gaatgggtgg cccggatcta ccctaccaac
240ggctacaccg agtacgccga ctccgtgaag ggccggttca ccatctccgc
cgacacctcc 300aagaacaccg cctacctgca gatgaactcc ctgagggccg
aggacaccgc cgtgtactac 360tgctccagat ggggaggcga cggcttctac
gccatggact actggggcca gggcaccctg 420gtcactgtgt cctctgcctc
caccaagggc ccttccgtgt tccctctggc cccttccagc 480aagtctacct
ctggcggcac cgctgctctg ggctgcctgg tcaaggacta cttccctgag
540cctgtgaccg tgtcctggaa ctctggcgcc ctgacctccg gcgtgcacac
cttccctgcc 600gtgctgcagt cctccggcct gtactccctg tcctccgtcg
tgaccgtgcc ttcctctagc 660ctgggcaccc agacctacat ctgcaacgtg
aaccacaagc cttccaacac caaggtggac 720aagaaggtgg agcctaagtc
ctgcgacaag acccacacct gtcctccatg ccctgcccct 780gagctgctgg
gcggaccctc cgtgttcctg ttccctccaa agcctaagga caccctgatg
840atctcccgga cccctgaagt gacctgcgtg gtggtggacg tgtcccacga
ggatcctgaa 900gtgaagttca attggtacgt ggacggcgtg gaggtgcaca
acgccaagac caagcctcgg 960gaggaacagt acaactccac ctaccgggtg
gtgtccgtgc tgaccgtgct gcaccaggac 1020tggctgaacg gcaaagagta
taagtgcaaa gtctccaaca aggccctgcc tgcccctatc 1080gaaaagacca
tctccaaggc caagggccag cctcgggaac ctcaggtgta caccctgcct
1140cccagcaggg acgagctgac caagaaccag gtgtccctga cctgtctggt
caagggcttc 1200tacccttccg atatcgccgt ggagtgggag tctaacggcc
agcctgagaa caactacaag 1260accacccctc ctgtgctgga ctccgacggc
tccttcttcc tgtactccaa actgaccgtg 1320gacaagtccc ggtggcagca
gggcaacgtg ttctcctgct ccgtgatgca cgaggccctg 1380cacaaccact
acacccagaa gtccctgtcc ctgtctcctg gcaagtgatg aaagcttggc 1440gcgcc
144519234PRTArtificial SequenceAnti-HER2 light chain 19Met Val Leu
Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser1 5 10 15Gly Ala
Tyr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30Ala
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp 35 40
45Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
50 55 60Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro
Ser65 70 75 80Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser 85 90 95Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln His Tyr 100 105 110Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg 115 120 125Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr145 150 155 160Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185
190Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
195 200 205His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro 210 215 220Val Thr Lys Ser Phe Asn Arg Gly Glu Cys225
23020469PRTArtificial SequenceAnti-HER2 heavy chain 20Met Asp Trp
Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly1 5 10 15Thr His
Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile 35 40
45Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr
Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr
Ser Lys Asn 85 90 95Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly
Phe Tyr Ala Met Asp Tyr 115 120 125Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly145 150 155 160Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185
190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val 210 215 220Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310
315 320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425
430Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
435 440 445Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser 450 455 460Leu Ser Pro Gly Lys46521234PRTArtificial
SequenceLight chain F53N 21Met Val Leu Gln Thr Gln Val Phe Ile Ser
Leu Leu Leu Trp Ile Ser1 5 10 15Gly Ala Tyr Gly Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser 20 25 30Ala Ser Val Gly Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Asp 35 40 45Val Asn Thr Ala Val Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60Lys Leu Leu Ile Tyr Ser
Ala Ser Asn Leu Tyr Ser Gly Val Pro Ser65 70 75 80Arg Phe Ser Gly
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95Ser Leu Gln
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr 100 105 110Thr
Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 115 120
125Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
130 135 140Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr145 150 155 160Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln Ser 165 170 175Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr 180 185 190Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys225 23022234PRTArtificial SequenceLight
chain Y92A 22Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu
Trp Ile Ser1 5 10 15Gly Ala Tyr Gly Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser 20 25 30Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp 35 40 45Val Asn Thr Ala Val Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro 50 55 60Lys Leu Leu Ile Tyr Ser Ala Ser Phe
Leu Tyr Ser Gly Val Pro Ser65 70 75 80Arg Phe Ser Gly Ser Arg Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95Ser Leu Gln Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln His Ala 100 105 110Thr Thr Pro Pro
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 115 120 125Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135
140Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr145 150 155 160Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser 165 170 175Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr 180 185 190Tyr Ser Leu Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205His Lys Val Tyr Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220Val Thr Lys Ser
Phe Asn Arg Gly Glu Cys225 23023234PRTArtificial SequenceLight
chain, F53N, Y92A 23Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu
Leu Trp Ile Ser1 5 10 15Gly Ala Tyr Gly Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser 20 25 30Ala Ser Val Gly Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Asp 35 40 45Val Asn Thr Ala Val Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro 50 55 60Lys Leu Leu Ile Tyr Ser Ala Ser
Asn Leu Tyr Ser Gly Val Pro Ser65 70 75 80Arg Phe Ser Gly Ser Arg
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95Ser Leu Gln Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Ala 100 105 110Thr Thr Pro
Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 115 120 125Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135
140Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr145 150 155 160Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser 165 170 175Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr 180 185 190Tyr Ser Leu Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205His Lys Val Tyr Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220Val Thr Lys Ser
Phe Asn Arg Gly Glu Cys225 23024234PRTArtificial SequenceLight
chain N30D 24Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu
Trp Ile Ser1 5 10 15Gly Ala Tyr Gly Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser 20 25 30Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp 35 40 45Val Asp Thr Ala Val Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro 50 55 60Lys Leu Leu Ile Tyr Ser Ala Ser Phe
Leu Tyr Ser Gly Val Pro Ser65 70 75 80Arg Phe Ser Gly Ser Arg Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95Ser Leu Gln Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr 100 105 110Thr Thr Pro Pro
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 115 120 125Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135
140Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr145 150 155 160Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser 165 170 175Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr 180 185 190Tyr Ser Leu Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205His Lys Val Tyr Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220Val Thr Lys Ser
Phe Asn Arg Gly Glu Cys225 23025469PRTArtificial SequenceHeavy
chain Y57A 25Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala
Ala Thr Gly1 5 10 15Thr His Ala Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Asn Ile 35 40 45Lys Asp Thr Tyr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Arg Ile Tyr Pro Thr
Asn Gly Ala Thr Arg Tyr Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe
Thr Ile Ser Ala Asp Thr Ser Lys Asn 85 90 95Thr Ala Tyr Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ser
Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr 115 120 125Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135
140Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly145 150 155 160Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val 165 170 175Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe 180 185 190Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val 195 200 205Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220Asn His Lys Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys225 230 235 240Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250
255Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
260 265 270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val 275 280 285Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val 290 295 300Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser305 310 315 320Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345 350Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 370 375
380Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala385 390 395 400Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr 405 410 415Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu 420 425 430Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser 435 440 445Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460Leu Ser Pro Gly
Lys46526469PRTArtificial SequenceHeavy chain R59E 26Met Asp Trp Thr
Trp
Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly1 5 10 15Thr His Ala Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Gly Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile 35 40 45Lys Asp
Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu
Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Glu Tyr Ala65 70 75
80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn
85 90 95Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val 100 105 110Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala
Met Asp Tyr 115 120 125Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly145 150 155 160Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200
205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
210 215 220Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val 275 280 285Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315
320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440
445Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
450 455 460Leu Ser Pro Gly Lys4651/30
* * * * *
References