U.S. patent application number 14/773766 was filed with the patent office on 2016-01-21 for anti-plasma kallikrein antibodies.
This patent application is currently assigned to Dyax Corp.. The applicant listed for this patent is DYAX CORP.. Invention is credited to Stephen R. COMEAU, Jon A. KENNISTON, Andrew NIXON.
Application Number | 20160017055 14/773766 |
Document ID | / |
Family ID | 51581689 |
Filed Date | 2016-01-21 |
United States Patent
Application |
20160017055 |
Kind Code |
A1 |
NIXON; Andrew ; et
al. |
January 21, 2016 |
ANTI-PLASMA KALLIKREIN ANTIBODIES
Abstract
Disclosed herein are antibodies capable of binding to plasma
kallikrein and inhibit its activity. Such antibodies interact with
one or more critical residues in the catalytic domain of the plasma
kallikrein. The antibodies may also contain specific heavy chain
complementarity determining region 3 (CDRs) motifs and optionally
specific residues at certain positions within both the heavy chain
variable region and the light chain variable region.
Inventors: |
NIXON; Andrew; (Hanover,
MA) ; KENNISTON; Jon A.; (Hingham, MA) ;
COMEAU; Stephen R.; (Chelmsford, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DYAX CORP. |
Burlington |
MA |
US |
|
|
Assignee: |
Dyax Corp.
Burlington
MA
|
Family ID: |
51581689 |
Appl. No.: |
14/773766 |
Filed: |
March 14, 2014 |
PCT Filed: |
March 14, 2014 |
PCT NO: |
PCT/US2014/027100 |
371 Date: |
September 9, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61791822 |
Mar 15, 2013 |
|
|
|
Current U.S.
Class: |
424/158.1 ;
530/387.3; 530/389.3 |
Current CPC
Class: |
C07K 2317/92 20130101;
A61P 31/04 20180101; C07K 16/40 20130101; C07K 2317/565 20130101;
A61P 29/00 20180101; A61P 35/00 20180101; A61P 43/00 20180101; C07K
2317/76 20130101; A61P 9/00 20180101; C07K 2317/34 20130101 |
International
Class: |
C07K 16/40 20060101
C07K016/40 |
Claims
1. An isolated antibody that binds human plasma kallikrein (PKal),
wherein the antibody interacts with one or more of amino acid
residues in the human PKal and inhibits its activity by at least
50%, wherein the amino acid residues are selected from the group
consisting of V410, L412, T413, A414, Q415, R416, L418, C419, H434,
C435, F436, D437, G438, L439, W445, Y475, K476, V477, S478, E479,
G480, D483, F524, E527, K528, Y552, D554, Y555, A564, D572, A573,
C574, K575, G576, S578, T596, S597, W598, G599, E600, G601, C602,
A603, R604, Q607, P608, G609, V610, and Y611.
2. The isolated antibody of claim 1, wherein the antibody binds an
epitope of the PKal, the epitope comprising the segment selected
from the group consisting of: (i) V410-C419, (ii) H434-L439, (iii)
Y475-G480, (iv) F524-K528, (v) Y552-Y555, (vi) D572-S578, (vii)
T596-R604, and (viii) Q607-Y611.
3. The isolated antibody of claim 1, wherein the antibody inhibits
the activity of PKal by at least 80%.
4. The isolated antibody of claim 1, wherein the antibody has an
apparent Ki (.sub.Ki,app) lower than about 1 nM.
5. The isolated antibody of claim 4, wherein the antibody has a
K.sub.i,app lower than about 0.1 nM.
6. The isolated antibody of claim 4, wherein the antibody has a
K.sub.i,app lower than about 0.05 nM.
7. The isolated antibody of claim 1, wherein the antibody has a
binding affinity (K.sub.D) for the PKal of less than 10.sup.-6
M.
8. The isolated antibody of claim 1, wherein the antibody
preferentially binds the PKal as relative to a mutant of the PKal
that contains one or more mutations at positions R551, Q553, Y555,
T558, and R560.
9. An isolated antibody that binds human plasma killikrein, wherein
the antibody comprises a heavy chain variable region that comprises
complementarity determining region 1 (HC CDR1), complementarity
determining region 2 (HC CDR2), and complementarity determining
region 3 (HC CDR3), and wherein the HC CDR3 comprises the motif
X.sub.99R.sub.100X.sub.101G.sub.102X.sub.103P.sub.104R.sub.105X.sub.106X.-
sub.107X.sub.108X.sub.109X.sub.110X.sub.111, in which: X.sub.99 is
R or Q, X.sub.101 is T, I, R, S, or P, X.sub.103 is V, I, or L,
X.sub.106 is R or W, X.sub.107 is D or N, X.sub.108 is A, S, D, E,
or V, X.sub.109 is F or L, X.sub.110 is D, E, or N, and X.sub.111
is I, N, M, or S.
10. The isolated antibody of claim 9, wherein the antibody inhibits
the activity of PKal by at least 80%.
11. The isolated antibody of claim 9, wherein the antibody has a
K.sub.i,app lower than about 1 nM.
12. The isolated antibody of claim 11, wherein the antibody has a
K.sub.i,app lower than about 0.1 nM.
13. The isolated antibody of claim 12, wherein the antibody has a
K.sub.i,app lower than about 0.05 nM.
14. The isolated antibody of claim 9, wherein the antibody has a
binding affinity (K.sub.D) for the PKal of less than 10.sup.-6
M.
15. The isolated antibody of claim 9, wherein X.sub.99 is Q and
X.sub.101 is I, R, S, or P.
16. The isolated antibody of claim 9, wherein X.sub.106 is W and
X.sub.111 is N, M, or S.
17. The isolated antibody of claim 9, wherein X.sub.101 is I,
X.sub.108 is E, and X.sub.103 is I or L.
18. The isolated antibody of claim 9, wherein X.sub.101 is I and
X.sub.103 is I or L.
19. The isolated antibody of claim 9, wherein X.sub.103 is I or L
and X.sub.110 is D, E, or N.
20. The isolated antibody of claim 9, wherein the heavy chain
variable region includes H.sub.31 in the HC CDR1.
21. The isolated antibody of claim 9, wherein the heavy chain
variable region includes F.sub.27, F.sub.29, or both in the
framework region 1 (FR1).
22. The isolated antibody of claim 9, further comprising a light
chain variable region that comprises complementarity determining
region 1 (LC CDR1), complementarity determining region 2 (LC CDR2),
and complementarity determining region 3 (LC CDR3).
23. The isolated antibody of claim 22, wherein the LC CDR2 includes
K.sub.50, L.sub.54, E.sub.55, S.sub.56, or a combination
thereof.
24. The isolated antibody of claim 23, wherein the light chain
variable region further includes G.sub.57 in the framework region 3
(FR3).
25. The isolated antibody of claim 22, wherein the light chain
variable includes N.sub.45 in the framework region 2 (FR2).
26. The isolated antibody of claim 1, wherein the antibody is a
full-length antibody or an antigen-binding fragment thereof.
27. The isolated antibody of claim 1, wherein the antibody is a
human antibody or a humanized antibody.
28. The isolated antibody of claim 1, wherein the antibody
preferentially bind an active PKal.
29. A pharmaceutical composition comprising an antibody of claim 1
and a pharmaceutically acceptable carrier.
30. A method for treating a disease associated with plasma
kallikrein, comprising administering to a subject in need thereof
an effective amount of the pharmaceutical composition of claim
29.
31. The method of claim 30, wherein the subject is a human patient
diagnosed with, suspected of having, or at risk for the disease.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the filing date of
U.S. Provisional Application No. 61/791,822, filed Mar. 15, 2013,
the entire contents of which are incorporated by reference
herein.
BACKGROUND OF THE INVENTION
[0002] Plasma kallikrein is a serine protease component of the
contact system and a potential drug target for different
inflammatory, cardiovascular, infectious (sepsis) and oncology
diseases (Sainz I. M. et al., Thromb Haemost 98, 77-83, 2007). The
contact system is activated by either factor XIIa upon exposure to
foreign or negatively charged surfaces or on endothelial cell
surfaces by prolylcarboxypeptidases (Sainz I. M. et al., Thromb
Haemost 98, 77-83, 2007). Activation of the plasma kallikrein
amplifies intrinsic coagulation via its feedback activation of
factor XII and enhances inflammation via the production of the
proinflammatory nonapeptide bradykinin. As the primary kininogenase
in the circulation, plasma kallikrein is largely responsible for
the generation of bradykinin in the vasculature. A genetic
deficiency in the C1-inhibitor protein (C1-INH), the major natural
inhibitor of plasma kallikrein, leads to hereditary angioedema
(HAE). Patients with HAE suffer from acute attacks of painful edema
often precipitated by unknown triggers (Zuraw B. L. et al., N Engl
J Med 359, 1027-1036, 2008).
[0003] Through the use of pharmacological agents or genetic studies
in animal models, the plasma kallikrein-kinin system (plasma KKS)
has been implicated in various diseases. Thus, it is of great
interest to identify agents that inhibit plasma kallikrein
activity, thereby effective in treating diseases associated with
plasma kallikrein.
SUMMARY OF THE INVENTION
[0004] The present invention is based on the determination of
crystal structures of a complex formed by the catalytic domain of
human plasma kallikrein (PKal) and the Fab fragment of DX.sub.2930
(an antibody specifically binds human PKal and effectively inhibits
its activity), and the identification of residues in both plasma
kallirein (PKal) and the antibody that are critical to the
interaction between the two molecules and/or to the inhibition of
the pKal activity.
[0005] Accordingly, the present disclosure features anti-PKal
antibodies capable of inhibiting its activity (e.g., by at least
50%), pharmaceutical compositions comprising such, and uses of the
pharmaceutical compositions for treating diseases and disorders
associated with plasma kallikrein.
[0006] In one aspect, the present disclosure provides an isolated
antibody that binds human plasma kallikrein (PKal), wherein the
antibody interacts with one or more of amino acid residues in the
human PKal and inhibits its activity by at least 50%. The amino
acid residues in the PKal that interact with the antibody can be
V410, L412, T413, A414, Q415, R416, L418, C419, H434, C435, F436,
D437, G438, L439, W445, Y475, K476, V477, S478, E479, G480, D483,
F524, E527, K528, Y552, D554, Y555, A564, D572, A573, C574, K575,
G576, S578, T596, S597, W598, G599, E600, G601, C602, A603, R604,
Q607, P608, G609, V610, and Y611 as indicated in FIG. 2 (boldfaced
and underlined).
[0007] In some examples, the anti-PKal antibody can bind an epitope
of the PKal, the epitope comprising one of the following segments
in PKal (FIG. 2): V410-C419, H434-L439, Y475-G480, F524-K528,
Y552-Y555, D572-S578, T596-R604, or Q607-Y611.
[0008] In other examples, the antibody preferentially binds the
PKal as relative to a mutant of the PKal (e.g., an inactive mutant)
that contains one or more mutations at positions R551, Q553, Y555,
T558, and R560 (e.g., Mutant 2 shown in FIG. 5).
[0009] In another aspect, the present disclosure provides an
isolated antibody that binds human plasma kallikrein, wherein the
antibody comprises a heavy chain variable region that comprises
complementarity determining region 1 (HC CDR1), complementarity
determining region 2 (HC CDR2), and complementarity determining
region 3 (HC CDR3). The HC CDR3 in the antibody comprises the
motif
X.sub.99R.sub.100X.sub.101G.sub.102X.sub.103P.sub.104R.sub.105X.sub.106X.-
sub.107X.sub.108X.sub.109X.sub.110X.sub.111 (SEQ ID NO: 58), in
which X.sub.99 is R or Q, X.sub.101 is T, I, R, S, or P, X.sub.103
is V, I, or L, X.sub.106 is R or W, X.sub.107 is D or N, X.sub.108
is A, S, D, E, or V, X.sub.109 is F or L, X.sub.110 is D, E, or N,
and X.sub.iii is I, N, M, or S.
[0010] In some examples, X.sub.99 can be Q and X.sub.101 can be I,
R, S, or P. In other examples, X.sub.106 can be W and X.sub.iii can
be N, M, or S. Alternatively or in addition, X.sub.101 can be I,
X.sub.108 can be E, and X.sub.103 can be I or L. In yet other
examples, X.sub.101 can be I and X.sub.103 can be I or L, or
X.sub.103 can be I or L and X.sub.110 can be D, E, or N.
[0011] In some embodiments, the heavy chain variable region of the
anti-PKal antibody described herein includes H.sub.31 in the HC
CDR1. Alternatively or in addition, the heavy chain variable region
includes F.sub.27, F.sub.29, or both in the framework region 1
(FR1).
[0012] The anti-PKal antibody described herein can further comprise
a light chain variable region that comprises complementarity
determining region 1 (LC CDR1), complementarity determining region
2 (LC CDR2), and complementarity determining region 3 (LC CDR3). In
some embodiments, the LC CDR2 includes K.sub.50, L.sub.54,
E.sub.55, S.sub.56, or a combination thereof. Alternatively or in
addition, the light chain variable region further includes G.sub.57
in the framework region 3 (FR3). When necessary, the light chain
variable includes N.sub.45 in the framework region 2 (FR2).
[0013] Any of the anti-PKal antibodies described herein can inhibit
the activity of PKal by at least 50% (e.g., at least 80%, 90%, 95%,
or 99%). In some instances, the antibody has an apparent Ki
(.sub.Ki,app) lower than about 1 nM (e.g., lower than about 0.1 nM,
or lower than about 0.05 nM). Alternatively or in addition, the
anti-PKal antibody described herein can have a binding affinity
(K.sub.D) for the PKal of less than 10.sup.-6 M (e.g., less than
10.sup.-7 M, 10.sup.-8 M, or 10.sup.-9 M).
[0014] The anti-PKal antibodies described herein can be a
full-length antibody or an antigen-binding fragment thereof.
Alternatively or in addition, the antibody can be a human antibody
or a humanized antibody.
[0015] Also within the scope of the present disclosure are
pharmaceutical compositions for use in treating various diseases
and disorders associated with plasma kallikrein, or for use in
manufacturing a medicament for treating the diseases and disorders.
The pharmaceutical compositions each comprise one or more anti-PKal
antibodies as described herein and a pharmaceutically acceptable
carrier.
[0016] Further, described herein are methods for treating a disease
associated with plasma kallikrein, comprising administering to a
subject in need thereof an effective amount of the pharmaceutical
composition, which comprises one or more of the anti-PKal
antibodies described herein. In some examples, the subject is a
human patient diagnosed with, suspected of having, or at risk for
the disease.
[0017] The details of one or more embodiments of the invention are
set forth in the description below. Other features or advantages of
the present invention will be apparent from the following drawings
and detailed description of several embodiments, and also from the
appending claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present disclosure, which can be better understood
by reference to one or more of these drawings in combination with
the detailed description of specific embodiments presented
herein.
[0019] FIG. 1 shows the amino acid sequence of the heavy chain
variable region (V.sub.H) and light chain variable region (V.sub.L)
of a parent antibody, M0162-A04, from which DX.sub.2930 was
derived, and their alignment with the corresponding germline
V.sub.H and V.sub.L genes as indicated. Variations in M0162-A04 as
compared to the germline sequences are indicated (boldfaced).
[0020] FIG. 2 shows the amino acid sequence (SEQ ID NO:40) of the
catalytic domain of human plasma kallikrein (residues 391-638 of
the full length human PKal). The boldfaced and underlined residues
refer to those that are involved in the interaction with the Fab
fragment of DX.sub.2930 as identified by the crystal structure
discussed in Example 1 below.
[0021] FIG. 3 is a graph showing the apparent Ki (K.sub.i, app) of
a number of antibody mutants derived from M0162-A04 against human
PKal.
[0022] FIG. 4 is a graph showing the apparent Ki (K.sub.i, app) of
clone X.sub.115-F02 (see Table 1 below) against wild-type PKal and
a number of PKal mutants.
[0023] FIG. 5 shows the amino acid sequences of a number of PKal
mutants (catalytic domain), which were produced in Pichia
cells.
DETAILED DESCRIPTION OF THE INVENTION
[0024] DX-2930 is a fully human IgG derived from parent clone
M0162-A04. The amino acid sequences of the V.sub.H and V.sub.L of
M0162-A04 are shown in FIG. 1. Their alignment with the
corresponding germline VH gene (VH3.sub.--3-23) and VL gene
(VK1_L12) is also shown in FIG. 1. Compared to the HC CDR3 of
M0162-A04, the HC CDR3 of DX-2930 includes the variations of T101I,
I103V, and A108E (see Table 2 below; the HC CDR3 of DX-2930 being
identical to M0199-A08). The Chothia Numbering Scheme is used in
the present disclosure. http://www.bioinf.org.uk/abs/.
[0025] Table 1 below provides structural information of DX-2930,
its parent antibody M0162-A04, and variants thereof. See also
US20120201756 and US20110200611.
TABLE-US-00001 TABLE 1 Structural Properties of DX-2930 and Related
Variants Name Properties M162-A04 This is the parent antibody of
DX-2930 that was discovered in the initial phage display selection
efforts (Ki, app = 2.5 nM). This antibody differs from DX-2930 at 3
critical amino acids in the CDR3 of the heavy chain and the
germlined positions. M199-A08 Fab discovered following the affinity
maturation of M0162-A04 using the Hv-CDR3 spiking method (Ki, app ~
0.06 nM). This antibody shares the same amino acids in the variable
region with DX-2930 but was not germlined and does not contain a Fc
fragment. X115-F02 Fully human IgG, kappa light chain 1 amino acid
in the light chain was mutated to their germline sequence. The DNA
sequence of X115-F02 was optimized for expression in CHO cells
Expressed transiently in 293T cells following subcloning into the
pRH1-CHO vector DX-2930 Fully human IgG, kappa light chain
(X124-G01) 1 amino acid in the light chain and 2 amino acids in the
heavy were mutated to their germline sequence. The DNA sequence of
DX-2930 was optimized for expression in CHO cells and cloned into
the pEh1 vector for stable expression using the glutamate synthase
system. The Fc of DX-2930 was modified to remove the C- terminal
lysine reside, in order to obtain a more homogeneous product.
[0026] Crystal structures (with different resolutions) of a complex
formed between the Fab fragment of DX-2930 and the catalytic domain
of human plasma kallikrein (PKal) was determined. Based on the
structural information provided by the crystal structures, a number
of interacting residues in both the catalytic domain of human PKal
and the antibody (in both V.sub.H and V.sub.L) were identified. The
interacting residues in the PKal are important targets for
developing antibodies capable of inhibiting the PKal activity.
Similarly, the interacting residues in the antibody also provide
important structural information for designing anti-PKal antibodies
with high inhibitory activity.
[0027] Further, affinity maturation analysis was performed to
develop high affinity anti-PKal antibodies, using clone M0162-A04
as the parent. Results obtained from affinity maturation matches
with the structural information provided by the crystal structures.
Based on the structural information and the affinity maturation
results, specific VH and VL motifs/residues were identified for
designing anti-PKal antibodies with high inhibitory activities.
[0028] Accordingly, described herein are antibodies capable of
binding to plasma kallikrein (e.g., human plasma kallikrein; PKal)
and inhibiting its activity, and uses thereof for treating diseases
and disorders associated with plasma kallikrein. Such antibodies
interact with one or more critical residues in the catalytic domain
of the PKal and/or comprise specific motifs/residues in either the
heavy chain variable region (e.g., HC CDR1 or HC CDR3) or the light
chain variable region (e.g., LC CDR2), or both.
Antibodies Binding to PKal
[0029] The present disclosure provides isolated antibodies that
bind PKal, particularly the catalytic domain of the PKal, such as
human PKal. The term "isolated antibody" used herein refers to an
antibody substantially free from naturally associated molecules,
i.e., the naturally associated molecules constituting at most 20%
by dry weight of a preparation containing the antibody. Purity can
be measured by any appropriate method, e.g., column chromatography,
polyacrylamide gel electrophoresis, and HPLC.
[0030] An antibody (interchangeably used in plural form) is an
immunoglobulin molecule capable of specific binding to a target,
such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.,
through at least one antigen recognition site, located in the
variable region of the immunoglobulin molecule. As used herein, the
term "antibody" encompasses not only intact (i.e., full-length)
polyclonal or monoclonal antibodies, but also antigen-binding
fragments thereof (such as Fab, Fab', F(ab').sub.2, Fv), single
chain (scFv), mutants thereof, fusion proteins comprising an
antibody portion, humanized antibodies, chimeric antibodies,
diabodies, linear antibodies, single chain antibodies,
multispecific antibodies (e.g., bispecific antibodies) and any
other modified configuration of the immunoglobulin molecule that
comprises an antigen recognition site of the required specificity,
including glycosylation variants of antibodies, amino acid sequence
variants of antibodies, and covalently modified antibodies. An
antibody includes an antibody of any class, such as IgD, IgE, IgG,
IgA, or IgM (or sub-class thereof), and the antibody need not be of
any particular class. Depending on the antibody amino acid sequence
of the constant domain of its heavy chains, immunoglobulins can be
assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these
may be further divided into subclasses (isotypes), e.g., IgG1,
IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant domains
that correspond to the different classes of immunoglobulins are
called alpha, delta, epsilon, gamma, and mu, respectively. The
subunit structures and three-dimensional configurations of
different classes of immunoglobulins are well known.
[0031] The antibodies described herein are capable of binding to a
PKal, particularly the catalytic domain of a PKal (e.g., human
PKal), thereby inhibiting the activity of PKal. In some instances,
the antibodies described herein can inhibit the activity of PKal by
at least 50%, e.g., 60%, 70%, 80%, 90%, 95%, or higher. The
inhibition constant (Ki) provides a measure of inhibitor potency;
it is the concentration of inhibitor required to reduce enzyme
activity by half and is not dependent on enzyme or substrate
concentrations. The inhibitory activity of an anti-PKal antibody
can be determined by routine methods, such as the method described
in Example 2 below.
[0032] In some examples, the inhibitory activity of an anti-PKal
antibody is determined by the apparent Ki (K.sub.i,app) value. The
K.sub.i,app value of an antibody obtained at different substrate
concentrations by measuring the inhibitory effect of different
concentrations of the antibody on the extent of the reaction (e.g.,
enzyme activity); fitting the change in pseudo-first order rate
constant as a function of inhibitor concentration to the Morrison
equation (Equation 1) yields an estimate of the apparent Ki value.
For a competitive inhibitor, the Ki is obtained from the
y-intercept extracted from a linear regression analysis of a plot
of K.sub.i,app versus substrate concentration.
v = v o - v o ( ( K i , app + I + E ) - ( K i , app + I + E ) 2 - 4
I E 2 E ) Equation 1 ##EQU00001##
[0033] In some examples, the anti-PKal antibodies described herein
have a K.sub.i,app value lower than 1 nM, e.g., 0.5 nM, 0.2 nM, 0.1
nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM,
0.02 nM, 0.01 nM, or lower. The K.sub.i,app value of an antibody
can be estimated following the methods known in the art and
described herein (Example 2).
[0034] The antibodies described herein can be murine, rat, human,
or any other origin (including chimeric or humanized antibodies).
In some examples, the antibody comprises a modified constant
region, such as a constant region that is immunologically inert,
e.g., does not trigger complement mediated lysis, or does not
stimulate antibody-dependent cell mediated cytotoxicity (ADCC).
ADCC activity can be assessed using methods disclosed in U.S. Pat.
No. 5,500,362. In other embodiments, the constant region is
modified as described in Eur. J. Immunol. (1999) 29:2613-2624; PCT
Application No. PCT/GB99/01441; and/or UK Patent Application No.
9809951.8.
[0035] Any of the antibodies described herein can be either
monoclonal or polyclonal. A "monoclonal antibody" refers to a
homogenous antibody population and a "polyclonal antibody" refers
to a heterogeneous antibody population. These two terms do not
limit the source of an antibody or the manner in which it is
made.
[0036] In one example, the antibody used in the methods described
herein is a humanized antibody. Humanized antibodies refer to forms
of non-human (e.g. murine) antibodies that are specific chimeric
immunoglobulins, immunoglobulin chains, or antigen-binding
fragments thereof that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a complementary determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat, or rabbit having the desired
specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore, the
humanized antibody may comprise residues that are found neither in
the recipient antibody nor in the imported CDR or framework
sequences, but are included to further refine and optimize antibody
performance. In general, the humanized antibody will comprise
substantially all of at least one, and typically two, variable
domains, in which all or substantially all of the CDR regions
correspond to those of a non-human immunoglobulin and all or
substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region or domain (Fc), typically that of a human immunoglobulin.
Antibodies may have Fc regions modified as described in WO
99/58572. Other forms of humanized antibodies have one or more CDRs
(one, two, three, four, five, six) which are altered with respect
to the original antibody, which are also termed one or more CDRs
"derived from" one or more CDRs from the original antibody.
Humanized antibodies may also involve affinity maturation.
[0037] In another example, the antibody described herein is a
chimeric antibody, which can include a heavy constant region and a
light constant region from a human antibody. Chimeric antibodies
refer to antibodies having a variable region or part of variable
region from a first species and a constant region from a second
species. Typically, in these chimeric antibodies, the variable
region of both light and heavy chains mimics the variable regions
of antibodies derived from one species of mammals (e.g., a
non-human mammal such as mouse, rabbit, and rat), while the
constant portions are homologous to the sequences in antibodies
derived from another mammal such as human. In some embodiments,
amino acid modifications can be made in the variable region and/or
the constant region.
[0038] In some embodiments, the anti-PKal antibodies described
herein have a suitable binding affinity to a PKal or the catalytic
domain thereof. As used herein, "binding affinity" refers to the
apparent association constant or K.sub.A. The K.sub.A is the
reciprocal of the dissociation constant (K.sub.D). The antibody
described herein may have a binding affinity (K.sub.D) of at least
10.sup.-5, 10.sup.-6, 10.sup.-7, 10.sup.-8 M, 10.sup.-9, 10.sup.-10
M or lower. An increased binding affinity corresponds to a
decreased K.sub.D. Higher affinity binding of an antibody to a
first target relative to a second target can be indicated by a
higher K.sub.A (or a smaller numerical value K.sub.D) for binding
the first target than the K.sub.A (or numerical value K.sub.D) for
binding the second target. In such cases, the antibody has
specificity for the first target (e.g., a protein in a first
conformation or mimic thereof) relative to the second target (e.g.,
the same protein in a second conformation or mimic thereof; or a
second protein). Differences in binding affinity (e.g., for
specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5,
10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or
10.sup.5 fold.
[0039] Binding affinity can be determined by a variety of methods
including equilibrium dialysis, equilibrium binding, gel
filtration, ELISA, surface plasmon resonance, or spectroscopy
(e.g., using a fluorescence assay). Exemplary conditions for
evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH7.4,
150 mM NaCl, 0.005% (v/v) Surfactant P20). These techniques can be
used to measure the concentration of bound binding protein as a
function of target protein concentration. The concentration of
bound binding protein ([Bound]) is related to the concentration of
free target protein ([Free]) and the concentration of binding sites
for the binding protein on the target where (N) is the number of
binding sites per target molecule by the following equation:
[Bound]=[N][Free]/(Kd+[Free])
[0040] It is not always necessary to make an exact determination of
K.sub.A, though, since sometimes it is sufficient to obtain a
quantitative measurement of affinity, e.g., determined using a
method such as ELISA or FACS analysis, is proportional to K.sub.A,
and thus can be used for comparisons, such as determining whether a
higher affinity is, e.g., 2-fold higher, to obtain a qualitative
measurement of affinity, or to obtain an inference of affinity,
e.g., by activity in a functional assay, e.g., an in vitro or in
vivo assay.
Antibodies Targeting Specific Residues in Human Plasma
Kallikrein
[0041] In some embodiments, the anti-PKal antibodies interact with
one or more of the residues (e.g., at least 3, 5, 8, 10, 15, 20,
25, 30, 35, 40, or 45) in the catalytic domain of human PKal,
including V410, L412, T413, A414, Q415, R416, L418, C419, H434,
C435, F436, D437, G438, L439, W445, Y475, K476, V477, 5478, E479,
G480, D483, F524, E527, K528, Y552, D554, Y555, A564, D572, A573,
C574, K575, G576, 5578, T596, 5597, W598, G599, E600, G601, C602,
A603, R604, Q607, P608, G609, V610, and Y611 (numbers based on the
full length prekallikrein amino acid sequence). The positions of
these residues are indicated in FIG. 2 (boldfaced and underlined).
These residues are identified as important to the pKal activity,
according to the crystal structures described in Example 1 below.
In some embodiments, the anti-PKal antibodies interact with one or
more of the residues (e.g., at least 3, 5, 8, 10, 15, 20, or 23) in
the catalytic domain of human PKal, including L418, C419, H434,
C435, D437, G438, L439, Y475, D483, F524, D572, A573, C574, K575,
G576, S578, T596, S597, W598, G599, E600, G601, and C602 (numbers
based on the full length prekallikrein amino acid sequence).
[0042] In some embodiments, the anti-PKal antibodies interact with
one or more of the residues (e.g., at least 3, 5, or 8) in the
catalytic domain of human PKal, including K476, V477, S478, E479,
G480, Y552, D554, and Y555 (numbers based on the full length
prekallikrein amino acid sequence).
[0043] In some embodiments, the anti-PKal antibodies interact with
one or more of the residues (e.g., at least 3, 5, 8, or 10) in the
catalytic domain of human PKal, including V410, L412, T413, A414,
Q415, R416, E527, K528, A603, and R604 (numbers based on the full
length prekallikrein amino acid sequence).
[0044] In some embodiments, the anti-PKal antibodies interact with
one or more of the residues (e.g., at least 3, 5, or 6) in the
catalytic domain of human PKal, including W445, Q607, P608, G609,
V610, and Y611 (numbers based on the full length prekallikrein
amino acid sequence).
[0045] In some embodiments, the anti-PKal antibodies interact with
one or more of the residues (e.g., at least 3, 5, 8, or 9) in the
catalytic domain of human PKal, including F524, D572, A573, C574,
K575, G576, S578, G601, and C602 (numbers based on the full length
prekallikrein amino acid sequence).
[0046] In some embodiments, the anti-PKal antibodies interact with
one or more of the residues (e.g., at least 3, 5, or 8) in the
catalytic domain of human PKal, including L418, C419, H434, C435,
D437, G438, Y475, and D483 (numbers based on the full length
prekallikrein amino acid sequence).
[0047] In some embodiments, the anti-PKal antibodies interact with
one or more of the residues (e.g., at least 3 or 4) in the
catalytic domain of human PKal, including 5597, W598, G599, and
E600 (numbers based on the full length prekallikrein amino acid
sequence).
[0048] Interacting means that the distance between two residues in
a complex formed by two binding partners is lower than a
predetermined value, e.g., <6 .ANG., <4 .ANG., or <2
.ANG.. For example, an interacting residue in one binding partner
can have has at least 1 atom within a given threshold (e.g., <6
.ANG., <4 .ANG., or <2 .ANG.) of at least 1 atom from a
residue of the other binding partner on the complexed structure.
Interacting does not require actual binding. Interacting residues
are suggested as involved in antibody recognition.
[0049] In some embodiments, the antibodies described herein bind
human PKal at an epitope comprising one or more of the residues
listed above. An "epitope" refers to the site on a target compound
that is bound by an antibody such as a Fab or full length antibody.
An epitope can be linear, which is typically 6-15 aa in length.
Alternatively, the epitope can be conformational.
[0050] In some examples, the anti-PKal antibodies described herein
binds an epitope that comprises the following segments: V410-C419,
H434-L439, Y475-G480, F524-K528, Y552-Y555, D572-S578, T596-R604,
or Q607-Y611.
[0051] In some examples, the antibody disclosed herein specifically
binds PKal or an epitope therein. An antibody that "specifically
binds" (used interchangeably herein) to a target or an epitope is a
term well understood in the art, and methods to determine such
specific binding are also well known in the art. A molecule is said
to exhibit "specific binding" if it reacts or associates more
frequently, more rapidly, with greater duration and/or with greater
affinity with a particular target antigen than it does with
alternative targets. An antibody "specifically binds" to a target
antigen if it binds with greater affinity, avidity, more readily,
and/or with greater duration than it binds to other substances. For
example, an antibody that specifically (or preferentially) binds to
human PKal or an epitope therein is an antibody that binds this
target antigen with greater affinity, avidity, more readily, and/or
with greater duration than it binds to other antigens or other
epitopes in the same antigen. It is also understood by reading this
definition that, for example, an antibody that specifically binds
to a first target antigen may or may not specifically or
preferentially bind to a second target antigen. As such, "specific
binding" or "preferential binding" does not necessarily require
(although it can include) exclusive binding. Generally, but not
necessarily, reference to binding means preferential binding.
[0052] In one example, the anti-PKal antibodies described herein
preferentially bind wild-type as compared to a mutant that includes
mutations at one or more of R551, Q553, Y555, T558, and R560, e.g.,
Mutant 2 described in Example 3. Such antibodies may bind wild-type
PKal at a much higher affinity as compared to the mutant (e.g., at
least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold,
500-fold, 1,000-fold higher). Alternatively or in addition, the
antibodies exhibit a much higher inhibitory activity against the
wild-type pKal as relative to the mutant (e.g., at least 2-fold,
5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold
higher).
[0053] In other examples, the anti-PKal antibodies described herein
binds active PKal, including wild-type pKal and functional variant
thereof. The antibody can preferentially bind an active PKal as
relative to its binding to an inactive mutant.
Anti-Plasma Kallikrein Antibodies Having Specific Motifs and/or
Residues
[0054] In some embodiments, the anti-PKal antibody described herein
comprises a V.sub.H and a V.sub.L, each of which comprises three
CDRs flanked by framework regions (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4;
see FIG. 1). The CDR3 of the heavy chain can comprise the motif:
X.sub.99R.sub.100X.sub.101G.sub.102X.sub.103P.sub.104R.sub.105X.sub.106X.-
sub.107X.sub.108X.sub.110X.sub.111, in which X.sub.99 is R or Q,
X.sub.101 is T, I, R, S, or P, X.sub.103 is V, I, or L, X.sub.106
is R or W, X.sub.107 is D or N, X.sub.108 is A, S, D, E, or V,
X.sub.109 is F or L, X.sub.110 is D, E, or N, and X.sub.111 is I,
N, M, or S. In some examples, X.sub.99 is Q and X.sub.101 is I, R,
S, or P. Alternatively or in addition, X.sub.106 is W and X.sub.111
is N, M, or S. In other examples, X.sub.101 is I, X.sub.108 is E,
and X.sub.103 is I or L; or X.sub.101 is I and X.sub.103 is I or L.
In yet other examples, X.sub.103 is I or L and X.sub.110 is D, E,
or N.
[0055] In addition, such an anti-pKal antibody can include one or
more other residues that are identified based on the crystal
structures discussed herein as being involved in interacting with
the catalytic domain of human PKal. These residues can be located
in the V.sub.H or the V.sub.L chain. Examples include E1, V2, F27,
T28, F29, and S30 in the FR1 of the V.sub.H, H.sub.31 in the HC
CDR1; S31 and W32 in the LC CDR1, Y49 in the FR1 of the V.sub.L
chain, K50, T53, L54, and E55, and S56 in LC CDR2, and G57 and V58
the FR3 of the V.sub.L chain.
[0056] The anti-PKal antibodies as described above can use any
germline heavy chain and light chain V genes as the framework.
Heavy chain V genes include, but are not limited to, IGHV1-2,
IGHV1-3, IGHV1-8, IGHV1-18, IGHV1-24, IGHV1-45, IGHV1-46, IGHV1-58,
IGHV1-69, IGHV2-5, IGHV2-26, IGHV2-70, IGHV3-7, IGHV3-9, IGHV3-11,
IGHV3-13, IGHV3-15, IGHV3-20, IGHV3-21, IGHV3-23, IGHV3-30,
IGHV3-33, IGHV3-43, IGHV3-48, IGHV3-49, IGHV3-53, IGHV3-64,
IGHV3-66, IGHV3-72, IGHV3-73, IGHV3-74, IGHV4-4, IGHV4-28,
IGHV4-31, IGHV4-34, IGHV4-39, IGHV4-59, IGHV4-61, IGHV4-B,
IGHV5-51, IGHV6-1, and IGHV7-4-1.
[0057] In some examples, the antibody uses a .kappa. light chain.
Light chain VK genes include, but are not limited to, V genes for
IGKV1-05, IGKV1-06, IGKV1-08, IGKV1-09, IGKV1-12, IGKV1-13,
IGKV1-16, IGKV1-17, IGKV1-27, IGKV1-33, IGKV1-37, IGKV1-39,
IGKV1D-16, IGKV1D-17, IGKV1D-43, IGKV1D-8, IGKV2-24, IGKV2-28,
IGKV2-29, IGKV2-30, IGKV2-40, IGKV2D-26, IGKV2D-29, IGKV2D-30,
IGKV3-11, IGKV3-15, IGKV3-20, IGKV3D-07, IGKV3D-11, IGKV3D-20,
IGKV4-1, IGKV5-2, IGKV6-21, and IGKV6D-41. In other examples, the
antibody uses a .lamda. light chain, e.g., any of IGLV1-IGLV10.
[0058] The antibody also can use any germline heavy J segment
(e.g., heavy chain IGJH1-IGJH6) and light chain J segment (e.g.,
IGJK1, IGJK2, IGJK3, IGJK4, or IGJK5), which can subject to
variations, such as deletions at the C-terminus, N-terminus, or
both.
[0059] Germline antibody gene/segment sequences are well known in
the art. See, e.g., http://www.vbase2.org/vbstat.php.
[0060] In some examples, the anti-PKal antibody described herein
uses VH3.sub.--3-23 and/or VK1_L12 as the framework for the heavy
chain and/or the light chain. It may include substantially similar
HC CDR1, HC CDR2, and/or HC CDR3, and LC CDR1, LC CDR2, and/or LC
CDR3 as those in M0162-A04 (FIG. 1), e.g., containing up to 5, 4,
3, 2, or 1 amino acid residue variations as compared to the
corresponding CDR region in M0162-A04.
[0061] In other examples, the anti-PKal antibody comprises a
V.sub.H chain that includes a V.sub.H CDR1, V.sub.H CDR2, and
V.sub.H CDR3 at least 75% (e.g., 80%, 85%, 90%, 95%, or 98%)
identical to the corresponding V.sub.H CDRs of M0162-A04, and a
V.sub.L chain that includes a V.sub.L CDR1, V.sub.L CDR2, and
V.sub.L CDR3 at least 75% (e.g., 80%, 85%, 90%, 95%, or 98%)
identical to the corresponding V.sub.L CDRs of M0162-A04.
[0062] Alternatively, the anti-PKal antibody comprises a V.sub.H
chain at least 75% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to
the V.sub.H chain (mature or precursor) of M0162-A04 and/or a
V.sub.L chain at least 75% (e.g., 80%, 85%, 90%, 95%, or 98%)
identical to the V.sub.L chain (mature of precursor) of
M0162-A04.
[0063] The "percent identity" of two amino acid sequences is
determined using the algorithm of Karlin and Altschul Proc. Natl.
Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul
Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is
incorporated into the NBLAST and XBLAST programs (version 2.0) of
Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein
searches can be performed with the XBLAST program, score=50,
wordlength=3 to obtain amino acid sequences homologous to the
protein molecules of interest. Where gaps exist between two
sequences, Gapped BLAST can be utilized as described in Altschul et
al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing
BLAST and Gapped BLAST programs, the default parameters of the
respective programs (e.g., XBLAST and NBLAST) can be used.
[0064] In some instances, conservative mutations can be introduced
into the CDRs in M0162-A04, e.g., at positions where the residues
are not likely to be involved in interacting with PKal as
determined based on the crystal structure. As used herein, a
"conservative amino acid substitution" refers to an amino acid
substitution that does not alter the relative charge or size
characteristics of the protein in which the amino acid substitution
is made. Variants can be prepared according to methods for altering
polypeptide sequence known to one of ordinary skill in the art such
as are found in references which compile such methods, e.g.
Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,
Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N. Y., 1989, or Current Protocols in Molecular Biology, F.
M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.
Conservative substitutions of amino acids include substitutions
made amongst amino acids within the following groups: (a) M, I, L,
V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g)
E, D.
[0065] In some embodiments, the anti-PKal antibodies described here
are not those described in US 20110200611, which is incorporated by
reference herein.
[0066] In some embodiments, the anti-PKal antibodies described
herein bind to the same epitope as DX-2930 and/or compete for
binding with DX-2930, with the proviso that the anti-PKal antibody
is not DX-2930. In some embodiments, the anti-Pkal antibodies
described herein bind to the sequence SWGE (SEQ ID NO: 48) and/or
DACKG (SEQ ID NO: 49) in PKal. In some embodiments, the anti-Pkal
antibodies described herein do not bind to the sequence SWGE (SEQ
ID NO: 48) and/or DACKG (SEQ ID NO: 49) in Pkal. In some
embodiments, the anti-Pkal antibodies described herein bind to the
sequence DGL, SEG, TSWGEG (SEQ ID NO: 50) and/or DACKG (SEQ ID NO:
49) in Pkal. In some embodiments, the anti-Pkal antibodies
described herein do not bind to the sequence DGL, SEG, TSWGEG (SEQ
ID NO: 50) and/or DACKG (SEQ ID NO: 49) in Pkal. In some
embodiments, the anti-Pkal antibodies described herein do not bind
to the sequence LVTNEECQKRYQDYKITQQ (SEQ ID NO: 51), WVTGWGFSKEKGEI
(SEQ ID NO: 52), ACKGDSGGPL (SEQ ID NO: 53), SWGDI (SEQ ID NO: 54),
HDIALIKL (SEQ ID NO: 55), TPFSQIKEIIIHQNY (SEQ ID NO: 56), and/or
AHCFDGLPLQDVWRIY (SEQ ID NO: 57).
[0067] In some embodiments, the anti-PKal antibody described herein
binds to an epitope located in the active domain of PKal (the whole
epitope or a portion thereof) and is different from that DX-2930.
The epitope of such an antibody may have overlapping residues with
those of the epitope of DX-2930. Alternatively, there can be no
overlapping residues between the two epitopes.
[0068] The sequences of the full length heavy chain and light chain
of DX-2930 are shown below.
TABLE-US-00002 DX-2930 Heavy Chain Amino Acid Sequence (451 amino
acids) (SEQ ID NO: 46)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGKGLEWVSG
IYSSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAYRR
IGVPRRDEFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G DX-2930 Light
Chain Amino Acid Sequence (213 amino acids) (SEQ ID NO: 47)
DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYK
ASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFGQG
TKVEIKRTVAAPSVFIFRPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
SSPVTKSFNRGEC
[0069] In the above sequences, the constant regions are italicized
and the CDR regions are in boldface and underlined.
Antibody Preparation
[0070] Antibodies capable of binding PKal as described herein can
be made by any method known in the art. See, for example, Harlow
and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory, New York.
[0071] In some embodiments, antibodies specific to a target antigen
(e.g., a human PKal or the catalytic domain thereof) can be made by
the conventional hybridoma technology. The full-length target
antigen or a fragment thereof, optionally coupled to a carrier
protein such as KLH, can be used to immunize a host animal for
generating antibodies binding to that antigen. The route and
schedule of immunization of the host animal are generally in
keeping with established and conventional techniques for antibody
stimulation and production, as further described herein. General
techniques for production of mouse, humanized, and human antibodies
are known in the art and are described herein. It is contemplated
that any mammalian subject including humans or antibody producing
cells therefrom can be manipulated to serve as the basis for
production of mammalian, including human hybridoma cell lines.
Typically, the host animal is inoculated intraperitoneally,
intramuscularly, orally, subcutaneously, intraplantar, and/or
intradermally with an amount of immunogen, including as described
herein.
[0072] Hybridomas can be prepared from the lymphocytes and
immortalized myeloma cells using the general somatic cell
hybridization technique of Kohler, B. and Milstein, C. (1975)
Nature 256:495-497 or as modified by Buck, D. W., et al., In Vitro,
18:377-381 (1982). Available myeloma lines, including but not
limited to X.sub.63-Ag8.653 and those from the Salk Institute, Cell
Distribution Center, San Diego, Calif., USA, may be used in the
hybridization. Generally, the technique involves fusing myeloma
cells and lymphoid cells using a fusogen such as polyethylene
glycol, or by electrical means well known to those skilled in the
art. After the fusion, the cells are separated from the fusion
medium and grown in a selective growth medium, such as
hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate
unhybridized parent cells. Any of the media described herein,
supplemented with or without serum, can be used for culturing
hybridomas that secrete monoclonal antibodies. As another
alternative to the cell fusion technique, EBV immortalized B cells
may be used to produce the anti-PKal monoclonal antibodies
described herein. The hybridomas are expanded and subcloned, if
desired, and supernatants are assayed for anti-immunogen activity
by conventional immunoassay procedures (e.g., radioimmunoassay,
enzyme immunoassay, or fluorescence immunoassay).
[0073] Hybridomas that may be used as source of antibodies
encompass all derivatives, progeny cells of the parent hybridomas
that produce monoclonal antibodies capable of interfering with the
PKal activity. Hybridomas that produce such antibodies may be grown
in vitro or in vivo using known procedures. The monoclonal
antibodies may be isolated from the culture media or body fluids,
by conventional immunoglobulin purification procedures such as
ammonium sulfate precipitation, gel electrophoresis, dialysis,
chromatography, and ultrafiltration, if desired. Undesired activity
if present, can be removed, for example, by running the preparation
over adsorbents made of the immunogen attached to a solid phase and
eluting or releasing the desired antibodies off the immunogen.
Immunization of a host animal with a target antigen or a fragment
containing the target amino acid sequence conjugated to a protein
that is immunogenic in the species to be immunized, e.g., keyhole
limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean
tryp sin inhibitor using a bifunctional or derivatizing agent, for
example maleimidobenzoyl sulfosuccinimide ester (conjugation
through cysteine residues), N-hydroxysuccinimide (through lysine
residues), glutaraldehyde, succinic anhydride, SOCl, or
R1N.dbd.C.dbd.NR, where R and R1 are different alkyl groups, can
yield a population of antibodies (e.g., monoclonal antibodies).
[0074] If desired, an antibody (monoclonal or polyclonal) of
interest (e.g., produced by a hybridoma) may be sequenced and the
polynucleotide sequence may then be cloned into a vector for
expression or propagation. The sequence encoding the antibody of
interest may be maintained in vector in a host cell and the host
cell can then be expanded and frozen for future use. In an
alternative, the polynucleotide sequence may be used for genetic
manipulation to "humanize" the antibody or to improve the affinity
(affinity maturation), or other characteristics of the antibody.
For example, the constant region may be engineered to more resemble
human constant regions to avoid immune response if the antibody is
used in clinical trials and treatments in humans. It may be
desirable to genetically manipulate the antibody sequence to obtain
greater affinity to the target antigen and greater efficacy in
inhibiting the activity of PKal. It will be apparent to one of
skill in the art that one or more polynucleotide changes can be
made to the antibody and still maintain its binding specificity to
the target antigen.
[0075] In other embodiments, fully human antibodies can be obtained
by using commercially available mice that have been engineered to
express specific human immunoglobulin proteins. Transgenic animals
that are designed to produce a more desirable (e.g., fully human
antibodies) or more robust immune response may also be used for
generation of humanized or human antibodies. Examples of such
technology are Xenomouse.TM. from Amgen, Inc. (Fremont, Calif.) and
HuMAb-Mouse.TM. and TC Mouse.TM. from Medarex, Inc. (Princeton,
N.J.). In another alternative, antibodies may be made recombinantly
by phage display or yeast technology. See, for example, U.S. Pat.
Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et
al., (1994) Annu. Rev. Immunol. 12:433-455, and. Alternatively, the
phage display technology (McCafferty et al., (1990) Nature
348:552-553) can be used to produce human antibodies and antibody
fragments in vitro, from immunoglobulin variable (V) domain gene
repertoires from unimmunized donors.
[0076] Antigen-binding fragments of an intact antibody (full-length
antibody) can be prepared via routine methods. For example, F(ab')2
fragments can be produced by pepsin digestion of an antibody
molecule, and Fab fragments that can be generated by reducing the
disulfide bridges of F(ab')2 fragments.
[0077] Genetically engineered antibodies, such as humanized
antibodies, chimeric antibodies, single-chain antibodies, and
bi-specific antibodies, can be produced via, e.g., conventional
recombinant technology. In one example, DNA encoding a monoclonal
antibodies specific to a target antigen can be readily isolated and
sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the monoclonal
antibodies). The hybridoma cells serve as a preferred source of
such DNA. Once isolated, the DNA may be placed into one or more
expression vectors, which are then transfected into host cells such
as E. coli cells, simian COS cells, Chinese hamster ovary (CHO)
cells, or myeloma cells that do not otherwise produce
immunoglobulin protein, to obtain the synthesis of monoclonal
antibodies in the recombinant host cells. See, e.g., PCT
Publication No. WO 87/04462. The DNA can then be modified, for
example, by substituting the coding sequence for human heavy and
light chain constant domains in place of the homologous murine
sequences, Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851,
or by covalently joining to the immunoglobulin coding sequence all
or part of the coding sequence for a non-immunoglobulin
polypeptide. In that manner, genetically engineered antibodies,
such as "chimeric" or "hybrid" antibodies; can be prepared that
have the binding specificity of a target antigen.
[0078] Techniques developed for the production of "chimeric
antibodies" are well known in the art. See, e.g., Morrison et al.
(1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al. (1984)
Nature 312, 604; and Takeda et al. (1984) Nature 314:452.
[0079] Methods for constructing humanized antibodies are also well
known in the art. See, e.g., Queen et al., Proc. Natl. Acad. Sci.
USA, 86:10029-10033 (1989). In one example, variable regions of VH
and VL of a parent non-human antibody are subjected to
three-dimensional molecular modeling analysis following methods
known in the art. Next, framework amino acid residues predicted to
be important for the formation of the correct CDR structures are
identified using the same molecular modeling analysis. In parallel,
human VH and VL chains having amino acid sequences that are
homologous to those of the parent non-human antibody are identified
from any antibody gene database using the parent VH and VL
sequences as search queries. Human VH and VL acceptor genes are
then selected.
[0080] The CDR regions within the selected human acceptor genes can
be replaced with the CDR regions from the parent non-human antibody
or functional variants thereof. When necessary, residues within the
framework regions of the parent chain that are predicted to be
important in interacting with the CDR regions (see above
description) can be used to substitute for the corresponding
residues in the human acceptor genes.
[0081] A single-chain antibody can be prepared via recombinant
technology by linking a nucleotide sequence coding for a heavy
chain variable region and a nucleotide sequence coding for a light
chain variable region. Preferably, a flexible linker is
incorporated between the two variable regions. Alternatively,
techniques described for the production of single chain antibodies
(U.S. Pat. Nos. 4,946,778 and 4,704,692) can be adapted to produce
a phage or yeast scFv library and scFv clones specific to a PKal
can be identified from the library following routine procedures.
Positive clones can be subjected to further screening to identify
those that inhibits PKal activity.
[0082] Antibodies obtained following a method known in the art and
described herein can be characterized using methods well known in
the art. For example, one method is to identify the epitope to
which the antigen binds, or "epitope mapping." There are many
methods known in the art for mapping and characterizing the
location of epitopes on proteins, including solving the crystal
structure of an antibody-antigen complex, competition assays, gene
fragment expression assays, and synthetic peptide-based assays, as
described, for example, in Chapter 11 of Harlow and Lane, Using
Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N. Y., 1999. In an additional example,
epitope mapping can be used to determine the sequence to which an
antibody binds. The epitope can be a linear epitope, i.e.,
contained in a single stretch of amino acids, or a conformational
epitope formed by a three-dimensional interaction of amino acids
that may not necessarily be contained in a single stretch (primary
structure linear sequence). Peptides of varying lengths (e.g., at
least 4-6 amino acids long) can be isolated or synthesized (e.g.,
recombinantly) and used for binding assays with an antibody. In
another example, the epitope to which the antibody binds can be
determined in a systematic screening by using overlapping peptides
derived from the target antigen sequence and determining binding by
the antibody. According to the gene fragment expression assays, the
open reading frame encoding the target antigen is fragmented either
randomly or by specific genetic constructions and the reactivity of
the expressed fragments of the antigen with the antibody to be
tested is determined. The gene fragments may, for example, be
produced by PCR and then transcribed and translated into protein in
vitro, in the presence of radioactive amino acids. The binding of
the antibody to the radioactively labeled antigen fragments is then
determined by immunoprecipitation and gel electrophoresis. Certain
epitopes can also be identified by using large libraries of random
peptide sequences displayed on the surface of phage particles
(phage libraries). Alternatively, a defined library of overlapping
peptide fragments can be tested for binding to the test antibody in
simple binding assays. In an additional example, mutagenesis of an
antigen binding domain, domain swapping experiments and alanine
scanning mutagenesis can be performed to identify residues
required, sufficient, and/or necessary for epitope binding. For
example, domain swapping experiments can be performed using a
mutant of a target antigen in which various fragments of the PKal
polypeptide have been replaced (swapped) with sequences from a
closely related, but antigenically distinct protein (such as
another member of the neurotrophin protein family). By assessing
binding of the antibody to the mutant PKal (e.g., those mutants
described in Example 2 below), the importance of the particular
antigen fragment to antibody binding can be assessed.
[0083] Alternatively, competition assays can be performed using
other antibodies known to bind to the same antigen to determine
whether an antibody binds to the same epitope as the other
antibodies. Competition assays are well known to those of skill in
the art.
[0084] Any of the suitable methods known in the art, e.g., the
epitope mapping methods as described herein, can be applied to
determine whether the anti-PKal antibody binds one or more of the
specific residues/segments in the PKal as described herein.
Further, the interaction of the antibody with one or more of those
defined residues in PKal can be determined by routine technology.
For example, a crystal structure can be determined following the
method disclosed in Example 1 below and the distances between the
residues in PKal and one or more residues in the antibody can be
determined accordingly. Based on such distance, whether a specific
residue in PKal interacts with one or more residues in the antibody
can be determined. Further, suitable methods, such as competition
assays and target mutagenesis assays can be applied to determine
the preferential binding of a candidate anti-PKa1 antibody to the
PKal as compared to another target such as a mutant PKal.
Pharmaceutical Compositions
[0085] One or more of the above-described anti-PKal antibodies can
be mixed with a pharmaceutically acceptable carrier (excipient),
including buffer, to form a pharmaceutical composition for use in
alleviating a disease or disorder that is associated with PKal.
"Acceptable" means that the carrier must be compatible with the
active ingredient of the composition (and preferably, capable of
stabilizing the active ingredient) and not deleterious to the
subject to be treated. Pharmaceutically acceptable excipients
(carriers) including buffers, which are well known in the art. See,
e.g., Remington: The Science and Practice of Pharmacy 20th Ed.
(2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover. In one
example, a pharmaceutical composition described herein contains
more than one anti-PKal antibodies that recognize different
epitopes/residues of the target antigen.
[0086] The pharmaceutical compositions to be used in the present
methods can comprise pharmaceutically acceptable carriers,
excipients, or stabilizers in the form of lyophilized formulations
or aqueous solutions. (Remington: The Science and Practice of
Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E.
Hoover). Acceptable carriers, excipients, or stabilizers are
nontoxic to recipients at the dosages and concentrations used, and
may comprise buffers such as phosphate, citrate, and other organic
acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrans; 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.TM., PLURONICS.TM. or
polyethylene glycol (PEG). Pharmaceutically acceptable excipients
are further described herein.
[0087] In some examples, the pharmaceutical composition described
herein comprises liposomes containing the anti-PKal antibody, which
can be prepared by methods known in the art, such as described in
Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang,
et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat.
Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation
time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful
liposomes can be generated by the reverse phase evaporation method
with a lipid composition comprising phosphatidylcholine,
cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE).
Liposomes are extruded through filters of defined pore size to
yield liposomes with the desired diameter.
[0088] The anti-PKal antibody may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are known in the art, see, e.g., Remington, The Science
and Practice of Pharmacy 20th Ed. Mack Publishing (2000).
[0089] In other examples, the pharmaceutical composition described
herein can be formulated in sustained-release format. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), sucrose
acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.
[0090] The pharmaceutical compositions to be used for in vivo
administration must be sterile. This is readily accomplished by,
for example, filtration through sterile filtration membranes.
Therapeutic antibody compositions are generally placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0091] The pharmaceutical compositions described herein can be in
unit dosage forms such as tablets, pills, capsules, powders,
granules, solutions or suspensions, or suppositories, for oral,
parenteral or rectal administration, or administration by
inhalation or insufflation. For preparing solid compositions such
as tablets, the principal active ingredient can be mixed with a
pharmaceutical carrier, e.g. conventional tableting ingredients
such as corn starch, lactose, sucrose, sorbitol, talc, stearic
acid, magnesium stearate, dicalcium phosphate or gums, and other
pharmaceutical diluents, e.g. water, to form a solid preformulation
composition containing a homogeneous mixture of a compound of the
present invention, or a non-toxic pharmaceutically acceptable salt
thereof. When referring to these preformulation compositions as
homogeneous, it is meant that the active ingredient is dispersed
evenly throughout the composition so that the composition may be
readily subdivided into equally effective unit dosage forms such as
tablets, pills and capsules. This solid preformulation composition
is then subdivided into unit dosage forms of the type described
above containing from 0.1 to about 500 mg of the active ingredient
of the present invention. The tablets or pills of the novel
composition can be coated or otherwise compounded to provide a
dosage form affording the advantage of prolonged action. For
example, the tablet or pill can comprise an inner dosage and an
outer dosage component, the latter being in the form of an envelope
over the former. The two components can be separated by an enteric
layer that serves to resist disintegration in the stomach and
permits the inner component to pass intact into the duodenum or to
be delayed in release. A variety of materials can be used for such
enteric layers or coatings, such materials including a number of
polymeric acids and mixtures of polymeric acids with such materials
as shellac, cetyl alcohol and cellulose acetate. Suitable
surface-active agents include, in particular, non-ionic agents,
such as polyoxyethylenesorbitans (e.g. Tween.TM. 20, 40, 60, 80 or
85) and other sorbitans (e.g. Span.TM. 20, 40, 60, 80 or 85).
Compositions with a surface-active agent will conveniently comprise
between 0.05 and 5% surface-active agent, and can be between 0.1
and 2.5%. It will be appreciated that other ingredients may be
added, for example mannitol or other pharmaceutically acceptable
vehicles, if necessary.
[0092] Suitable emulsions may be prepared using commercially
available fat emulsions, such as Intralipid.TM., Liposyn.TM.,
Infonutrol.TM., Lipofundin.TM. and Lipiphysan.TM.. The active
ingredient may be either dissolved in a pre-mixed emulsion
composition or alternatively it may be dissolved in an oil (e.g.
soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or
almond oil) and an emulsion formed upon mixing with a phospholipid
(e.g. egg phospholipids, soybean phospholipids or soybean lecithin)
and water. It will be appreciated that other ingredients may be
added, for example glycerol or glucose, to adjust the tonicity of
the emulsion. Suitable emulsions will typically contain up to 20%
oil, for example, between 5 and 20%. The fat emulsion can comprise
fat droplets between 0.1 and 1.0 .im, particularly 0.1 and 0.5 .im,
and have a pH in the range of 5.5 to 8.0.
[0093] The emulsion compositions can be those prepared by mixing an
anti-PKal antibody with Intralipid.TM. or the components thereof
(soybean oil, egg phospholipids, glycerol and water).
[0094] Pharmaceutical compositions for inhalation or insufflation
include solutions and suspensions in pharmaceutically acceptable,
aqueous or organic solvents, or mixtures thereof, and powders. The
liquid or solid compositions may contain suitable pharmaceutically
acceptable excipients as set out above. In some embodiments, the
compositions are administered by the oral or nasal respiratory
route for local or systemic effect.
[0095] Compositions in preferably sterile pharmaceutically
acceptable solvents may be nebulised by use of gases. Nebulised
solutions may be breathed directly from the nebulising device or
the nebulising device may be attached to a face mask, tent or
intermittent positive pressure breathing machine. Solution,
suspension or powder compositions may be administered, preferably
orally or nasally, from devices which deliver the formulation in an
appropriate manner.
Use of Anti-PKal Antibodies for Treating Diseases/Disorders
Associated with Plasma Kallikrein
[0096] The anti-PKal antibodies described herein would be effective
in treating a disease or disorder associated the PKal. Examples of
such diseases and conditions which can be treated (or prevented) by
a plasma kallikrein binding protein described herein include:
rheumatoid arthritis, gout, intestinal bowel disease, oral
mucositis, neuropathic pain, inflammatory pain, spinal
stenosis-degenerative spine disease, arterial or venous thrombosis,
post operative ileus, aortic aneurysm, osteoarthritis, vasculitis,
edema, hereditary angioedema, cerebral edema, pulmonary embolism,
stroke, clotting induced by ventricular assistance devices or
stents, head trauma or peri-tumor brain edema, sepsis, acute middle
cerebral artery (MCA) ischemic event (stroke), restenosis (e.g.,
after angioplasty), systemic lupus erythematosis nephritis, burn
injury, and DME. A plasma kallikrein binding protein described
herein can also be used to promote wound healing. A plasma
kallikrein binding protein described herein can also be used as an
oncology treatment by mechanisms that include, but are not limited
to, blocking production of pro-angiogenic bradykinin.
[0097] To practice the method disclosed herein, an effective amount
of the pharmaceutical composition described above can be
administered to a subject (e.g., a human) in need of the treatment
via a suitable route, such as intravenous administration, e.g., as
a bolus or by continuous infusion over a period of time, by
intramuscular, intraperitoneal, intracerobrospinal, subcutaneous,
intra-articular, intrasynovial, intrathecal, oral, inhalation or
topical routes. Commercially available nebulizers for liquid
formulations, including jet nebulizers and ultrasonic nebulizers
are useful for administration. Liquid formulations can be directly
nebulized and lyophilized powder can be nebulized after
reconstitution. Alternatively, anti-PKal antibodies can be
aerosolized using a fluorocarbon formulation and a metered dose
inhaler, or inhaled as a lyophilized and milled powder.
[0098] The subject to be treated by the methods described herein
can be a mammal, more preferably a human. Mammals include, but are
not limited to, farm animals, sport animals, pets, primates,
horses, dogs, cats, mice and rats. A human subject who needs the
treatment may be a human patient having, at risk for, or suspected
of having a disease/disorder associated with PKal, such as those
noted above. A subject having a PKal-associated disease or disorder
can be identified by routine medical examination, e.g., laboratory
tests, organ functional tests, CT scans, or ultrasounds. A subject
suspected of having any of such disease/disorder might show one or
more symptoms of the disease/disorder. A subject at risk for the
disease/disorder can be a subject having one or more of the risk
factors for that disease/disorder.
[0099] "An effective amount" as used herein refers to the amount of
each active agent required to confer therapeutic effect on the
subject, either alone or in combination with one or more other
active agents. Effective amounts vary, as recognized by those
skilled in the art, depending on the particular condition being
treated, the severity of the condition, the individual patient
parameters including age, physical condition, size, gender and
weight, the duration of the treatment, the nature of concurrent
therapy (if any), the specific route of administration and like
factors within the knowledge and expertise of the health
practitioner. These factors are well known to those of ordinary
skill in the art and can be addressed with no more than routine
experimentation. It is generally preferred that a maximum dose of
the individual components or combinations thereof be used, that is,
the highest safe dose according to sound medical judgment. It will
be understood by those of ordinary skill in the art, however, that
a patient may insist upon a lower dose or tolerable dose for
medical reasons, psychological reasons or for virtually any other
reasons.
[0100] Empirical considerations, such as the half-life, generally
will contribute to the determination of the dosage. For example,
antibodies that are compatible with the human immune system, such
as humanized antibodies or fully human antibodies, may be used to
prolong half-life of the antibody and to prevent the antibody being
attacked by the host's immune system. Frequency of administration
may be determined and adjusted over the course of therapy, and is
generally, but not necessarily, based on treatment and/or
suppression and/or amelioration and/or delay of a disease/disorder
associated with PKal. Alternatively, sustained continuous release
formulations of an anti-PKal may be appropriate. Various
formulations and devices for achieving sustained release are known
in the art.
[0101] In one example, dosages for an anti-PKal antibody as
described herein may be determined empirically in individuals who
have been given one or more administration(s) of the antibody.
Individuals are given incremental dosages of the antagonist. To
assess efficacy of the antagonist, an indicator of the
disease/disorder can be followed.
[0102] Generally, for administration of any of the antibodies
described herein, an initial candidate dosage can be about 2 mg/kg.
For the purpose of the present disclosure, a typical daily dosage
might range from about any of 0.1 .mu.g/kg to 3 .mu.g/kg to 30
.mu.g/kg to 300 .mu.g/kg to 3 mg/kg, to 30 mg/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 symptoms occurs or until sufficient therapeutic levels are
achieved to alleviate a disease or disorder associated with PKal,
or a symptom thereof. An exemplary dosing regimen comprises
administering an initial dose of about 2 mg/kg, followed by a
weekly maintenance dose of about 1 mg/kg of the antibody, or
followed by a maintenance dose of about 1 mg/kg every other week.
However, other dosage regimens may be useful, depending on the
pattern of pharmacokinetic decay that the practitioner wishes to
achieve. For example, dosing from one-four times a week is
contemplated. In some embodiments, dosing ranging from about 3
.mu.g/mg to about 2 mg/kg (such as about 3 .mu.g/mg, about 10
.mu.g/mg, about 30 .mu.g/mg, about 100 .mu.g/mg, about 300
.mu.g/mg, about 1 mg/kg, and about 2 mg/kg) may be used. In some
embodiments, dosing frequency is once every week, every 2 weeks,
every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8
weeks, every 9 weeks, or every 10 weeks; or once every month, every
2 months, or every 3 months, or longer. The progress of this
therapy is easily monitored by conventional techniques and assays.
The dosing regimen (including the antibody used) can vary over
time.
[0103] In some embodiments, for an adult patient of normal weight,
doses ranging from about 0.3 to 5.00 mg/kg may be administered. The
particular dosage regimen, i.e., dose, timing and repetition, will
depend on the particular individual and that individual's medical
history, as well as the properties of the individual agents (such
as the half-life of the agent, and other considerations well known
in the art).
[0104] For the purpose of the present disclosure, the appropriate
dosage of an anti-PKal antibody will depend on the specific
antibody (or compositions thereof) employed, the type and severity
of the disease/disorder, whether the antibody is administered for
preventive or therapeutic purposes, previous therapy, the patient's
clinical history and response to the antagonist, and the discretion
of the attending physician. Typically the clinician will administer
an anti-PKal antibody, until a dosage is reached that achieves the
desired result. Administration of an anti-PKal antibody can be
continuous or intermittent, depending, for example, upon the
recipient's physiological condition, whether the purpose of the
administration is therapeutic or prophylactic, and other factors
known to skilled practitioners. The administration of an anti-PKal
antibody may be essentially continuous over a preselected period of
time or may be in a series of spaced dose, e.g., either before,
during, or after developing a disease or disorder associated with
PKal.
[0105] As used herein, the term "treating" refers to the
application or administration of a composition including one or
more active agents to a subject, who has a disease/disorder
associated with PKal, a symptom of the disease/disorder, or a
predisposition toward the disease/disorder, with the purpose to
cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve,
or affect the disorder, the symptom of the disease, or the
predisposition toward the disease/disorder.
[0106] Alleviating a disease/disorder associated with PKal includes
delaying the development or progression of the disease, or reducing
disease severity. Alleviating the disease does not necessarily
require curative results. As used therein, "delaying" the
development of a disease/disorder associated with PKal means to
defer, hinder, slow, retard, stabilize, and/or postpone progression
of the disease. This delay can be of varying lengths of time,
depending on the history of the disease and/or individuals being
treated. A method that "delays" or alleviates the development of a
disease, or delays the onset of the disease, is a method that
reduces probability of developing one or more symptoms of the
disease in a given time frame and/or reduces extent of the symptoms
in a given time frame, when compared to not using the method. Such
comparisons are typically based on clinical studies, using a number
of subjects sufficient to give a statistically significant
result.
[0107] "Development" or "progression" of a disease means initial
manifestations and/or ensuing progression of the disease.
Development of the disease can be detectable and assessed using
standard clinical techniques as well known in the art. However,
development also refers to progression that may be undetectable.
For purpose of this disclosure, development or progression refers
to the biological course of the symptoms. "Development" includes
occurrence, recurrence, and onset. As used herein "onset" or
"occurrence" of a disease/disorder associated with PKal includes
initial onset and/or recurrence.
[0108] In some embodiments, the anti-PKal antibody described herein
is administered to a subject in need of the treatment at an amount
sufficient to inhibit the activity of PKal by at least 20% (e.g.,
30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) in vivo. In other
embodiments, the antibody is administered in an amount effective in
reducing the PKal level by at least 20% (e.g., 30%, 40%, 50%, 60%,
70%, 80%, 90% or greater).
[0109] Conventional methods, known to those of ordinary skill in
the art of medicine, can be used to administer the pharmaceutical
composition to the subject, depending upon the type of disease to
be treated or the site of the disease. This composition can also be
administered via other conventional routes, e.g., administered
orally, parenterally, by inhalation spray, topically, rectally,
nasally, buccally, vaginally or via an implanted reservoir. The
term "parenteral" as used herein includes subcutaneous,
intracutaneous, intravenous, intramuscular, intraarticular,
intraarterial, intrasynovial, intrasternal, intrathecal,
intralesional, and intracranial injection or infusion techniques.
In addition, it can be administered to the subject via injectable
depot routes of administration such as using 1-, 3-, or 6-month
depot injectable or biodegradable materials and methods.
[0110] Injectable compositions may contain various carriers such as
vegetable oils, dimethylacetamide, dimethylformamide, ethyl
lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols
(glycerol, propylene glycol, liquid polyethylene glycol, and the
like). For intravenous injection, water soluble antibodies can be
administered by the drip method, whereby a pharmaceutical
formulation containing the antibody and a physiologically
acceptable excipients is infused. Physiologically acceptable
excipients may include, for example, 5% dextrose, 0.9% saline,
Ringer's solution or other suitable excipients. Intramuscular
preparations, e.g., a sterile formulation of a suitable soluble
salt form of the antibody, can be dissolved and administered in a
pharmaceutical excipient such as Water-for-Injection, 0.9% saline,
or 5% glucose solution.
[0111] In one embodiment, an anti-PKal antibody is administered via
site-specific or targeted local delivery techniques. Examples of
site-specific or targeted local delivery techniques include various
implantable depot sources of the anti-PKal antibody or local
delivery catheters, such as infusion catheters, an indwelling
catheter, or a needle catheter, synthetic grafts, adventitial
wraps, shunts and stents or other implantable devices, site
specific carriers, direct injection, or direct application. See,
e.g., PCT Publication No. WO 00/53211 and U.S. Pat. No.
5,981,568.
[0112] Targeted delivery of therapeutic compositions containing an
antisense polynucleotide, expression vector, or subgenomic
polynucleotides can also be used. Receptor-mediated DNA delivery
techniques are described in, for example, Findeis et al., Trends
Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods
And Applications Of Direct Gene Transfer (J. A. Wolff, ed.) (1994);
Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem.
(1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990)
87:3655; Wu et al., J. Biol. Chem. (1991) 266:338.
[0113] Therapeutic compositions containing a polynucleotide (e.g.,
those encoding the anti-PKal antibodies described herein) are
administered in a range of about 100 ng to about 200 mg of DNA for
local administration in a gene therapy protocol. In some
embodiments, concentration ranges of about 500 ng to about 50 mg,
about 1 .mu.g to about 2 mg, about 5 .mu.g to about 500 .mu.g, and
about 20 .mu.g to about 100 .mu.g of DNA or more can also be used
during a gene therapy protocol.
[0114] The therapeutic polynucleotides and polypeptides described
herein can be delivered using gene delivery vehicles. The gene
delivery vehicle can be of viral or non-viral origin (see
generally, Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human
Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995)
1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of
such coding sequences can be induced using endogenous mammalian or
heterologous promoters and/or enhancers. Expression of the coding
sequence can be either constitutive or regulated.
[0115] Viral-based vectors for delivery of a desired polynucleotide
and expression in a desired cell are well known in the art.
Exemplary viral-based vehicles include, but are not limited to,
recombinant retroviruses (see, e.g., PCT Publication Nos. WO
90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO
93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; GB
Patent No. 2,200,651; and EP Patent No. 0 345 242),
alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki
forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC
VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus
(ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and
adeno-associated virus (AAV) vectors (see, e.g., PCT Publication
Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO
95/11984 and WO 95/00655). Administration of DNA linked to killed
adenovirus as described in Curiel, Hum. Gene Ther. (1992) 3:147 can
also be employed.
[0116] Non-viral delivery vehicles and methods can also be
employed, including, but not limited to, polycationic condensed DNA
linked or unlinked to killed adenovirus alone (see, e.g., Curiel,
Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, e.g., Wu, J.
Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicles
cells (see, e.g., U.S. Pat. No. 5,814,482; PCT Publication Nos. WO
95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic
charge neutralization or fusion with cell membranes. Naked DNA can
also be employed. Exemplary naked DNA introduction methods are
described in PCT Publication No. WO 90/11092 and U.S. Pat. No.
5,580,859. Liposomes that can act as gene delivery vehicles are
described in U.S. Pat. No. 5,422,120; PCT Publication Nos. WO
95/13796; WO 94/23697; WO 91/14445; and EP Patent No. 0524968.
Additional approaches are described in Philip, Mol. Cell. Biol.
(1994) 14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994)
91:1581.
[0117] The particular dosage regimen, i.e., dose, timing and
repetition, used in the method described herein will depend on the
particular subject and that subject's medical history. In some
embodiments, more than one anti-PKal antibodies, or a combination
of an anti-PKal antibody and another suitable therapeutic agent,
may be administered to a subject in need of the treatment. The
antagonist can be the same type or different from each other. The
anti-PKa1 antibody can also be used in conjunction with other
agents that serve to enhance and/or complement the effectiveness of
the agents.
[0118] Treatment efficacy for a disease/disorder associated with
PKal can be assessed by methods well-known in the art.
Kits for Use in Alleviating Diseases/Disorders Associated with
Plasma Kallikrein
[0119] The present disclosure also provides kits for use in
alleviating diseases/disorders associated with plasma kallikrein.
Such kits can include one or more containers comprising an
anti-PKal antibody, e.g., any of those described herein.
[0120] In some embodiments, the kit can comprise instructions for
use in accordance with any of the methods described herein. The
included instructions can comprise a description of administration
of the anti-PKal antibody to treat, delay the onset, or alleviate a
target disease as those described herein. The kit may further
comprise a description of selecting an individual suitable for
treatment based on identifying whether that individual has the
target disease. In still other embodiments, the instructions
comprise a description of administering an antibody to an
individual at risk of the target disease.
[0121] The instructions relating to the use of an anti-PKal
antibody generally include information as to dosage, dosing
schedule, and route of administration for the intended treatment.
The containers may be unit doses, bulk packages (e.g., multi-dose
packages) or sub-unit doses. Instructions supplied in the kits of
the invention are typically written instructions on a label or
package insert (e.g., a paper sheet included in the kit), but
machine-readable instructions (e.g., instructions carried on a
magnetic or optical storage disk) are also acceptable.
[0122] The label or package insert indicates that the composition
is used for treating, delaying the onset and/or alleviating a
disease or disorder associated with PKal. Instructions may be
provided for practicing any of the methods described herein.
[0123] The kits of this invention are in suitable packaging.
Suitable packaging includes, but is not limited to, vials, bottles,
jars, flexible packaging (e.g., sealed Mylar or plastic bags), and
the like. Also contemplated are packages for use in combination
with a specific device, such as an inhaler, nasal administration
device (e.g., an atomizer) or an infusion device such as a
minipump. A kit may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). The container
may also have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is an anti-PKal antibody as those
described herein.
[0124] Kits may optionally provide additional components such as
buffers and interpretive information. Normally, the kit comprises a
container and a label or package insert(s) on or associated with
the container. In some embodiments, the invention provides articles
of manufacture comprising contents of the kits described above.
General Techniques
[0125] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are within the skill of the art.
Such techniques are explained fully in the literature, such as,
Molecular Cloning: A Laboratory Manual, second edition (Sambrook,
et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis
(M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana
Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed.,
1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed.,
1987); Introduction to Cell and Tissue Culture (J. P. Mather and P.
E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory
Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds.,
1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press,
Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.
Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.
Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular
Biology (F. M. Ausubel, et al., eds., 1987); PCR: The Polymerase
Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in
Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in
Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A.
Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997);
Antibodies: a practical approach (D. Catty, ed., IRL Press,
1988-1989); Monoclonal antibodies: a practical approach (P.
Shepherd and C. Dean, eds., Oxford University Press, 2000); Using
antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring
Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.
D. Capra, eds., Harwood Academic Publishers, 1995).
[0126] Without further elaboration, it is believed that one skilled
in the art can, based on the above description, utilize the present
invention to its fullest extent. The following specific embodiments
are, therefore, to be construed as merely illustrative, and not
limitative of the remainder of the disclosure in any way
whatsoever. All publications cited herein are incorporated by
reference for the purposes or subject matter referenced herein.
Example 1
Identification of Critical Residues in the Catalytic Domain of
Human Plasma Kallikrein Based on Crystal Structures of DX-2930-PKal
Complex
[0127] The catalytic domain of human plasma kallikrein (FIG. 2),
fused with a His-tag, was expressed in insect cells and purified
initially by a nickel affinity column. The His-tag was removed from
the plasma kallikrein via trypsin digestion and the free plasma
kallikrein was purified by a benzamidine affinity column, followed
by a SEC column. The purified product was examined on a PAGE gel.
The result indicates that the catalytic domain of human plasma
kallikrein was properly expressed and purified.
[0128] DX-2930 was prepared via routine recombinant technology and
purified. A recombinant Fab fragment of DX-2930 was produced via
routine method and purified.
[0129] The DX-2930 Fab fragment and the catalytic domain of human
plasma kallikrein were mixed at various concentrations under
suitable conditions allowing formation of antibody-PKal complexes.
The complexes thus formed were examined using HPLC to determine the
antibody-PKal ratio in the complexes. Accordingly, the suitable
concentrations of both the antibody and the PKal were identified
for formation of a 1:1 complex.
[0130] The antibody-PKal complex was kept under various conditions
allowing for crystallization. Diffraction analysis was performed on
the crystallized complex. The crystal structures (2.1 .ANG. and 2.4
.ANG.) were determined based on the diffraction statistics.
[0131] According to the crystal structures, residues in the
catalytic domain of human Pkal that are involved in the interaction
with DX-2930 were identified. These residues are indicated
(boldfaced and underlined) in FIG. 2, which provides the amino acid
sequence of the catalytic domain of human PKal (residues 391-638 of
human PKal).
[0132] In addition, residues in DX-2930 that interact with PKal
were also identified based on the crystal structure, including E1,
V2, F27, T28, F29, S30, H31, R100, I101, G102, V103, P104, R105,
R106, D107, G107, K108, and D111 in the heavy chain variable
region, and S31, W32, Y49, K50, T53, L54, E55, S56, G57, and V58 in
the light chain variable region.
[0133] These results indicate that HC CDR3 of DX-2930 is the main
region that interacts with PKal (see FIG. 1) and a couple of
residues in the HC CDR1 and FR1 might also contribute to the
interaction with PKal. In the light chain, the LC CDR2 region was
found to contribute to the interaction.
[0134] Further, the results also indicate that variations at
certain positions with the HC CDR3 region may be allowed. For
example, position 103 requires small hydrophobic residues such as V
or I. As another example, R106 may be replaced with W, and E108 may
be replaced with S or D without substantially affecting the PKal
binding activity. Similarly, D110 might be replaced with E.
Example 2
Affinity Maturation Results Match Structural Information Derived
from Crystal Structure
[0135] The heavy chain variable region, particularly the HC CDR3
region, of antibody M0162-A04 was subject to affinity maturation.
Various mutants having amino acid variations at one or more
positions in the HC CDR3 region were generated and their
K.sub.i,app values were determined following routine methods.
[0136] Briefly, PKal and a Fab at various concentrations are
incubated together for 1 hour at 30.degree. C. A substrate peptide
(cleavable by PKal) is then added to this PKal-Fab mixture. The
rate of substrate peptide cleavage/proteolysis is then measured,
and plotted against the concentrations of the Fab. This plot is
then fit to the Morrison equation, which calculates the K.sub.i,app
value. The results thus obtained are shown in FIG. 3 and Table 2
below:
TABLE-US-00003 TABLE 2 Summary of Hv-CDR3 Affinity Maturation
Results Ki,app SEQ Initial Name Hv CDR3 (nM) ID NO M0162-A04
RRTGIPRRDAFDI 2.5 1 M0199-A11 --R---------- 2 2 M0201-F11
--S---------- 3 3 M0202-A08 -------W----- 2.8 4 M0201-A06
---------V--- 3.8 5 M0202-E03 -----------E- 2 6 M0199-B01
------------N 1.6 7 M0200-B01 ------------S 3.6 8 M0201-H06
----V-------- 0.6 9 M0202-H05 ----V----V--- 0.26 10 M0201-H08
----V-----L-N 0.8 11 M0200-E11 ----V-------N 0.4 12 M0200-H07
----V---N---N 0.4 13 M0202-F06 ----V--W----- 0.33 14 M0200-A10
----V----S--- 0.25 15 M0202-G03 ----V----S-E- 0.4 16 M0202-A12
Q---V----S-N- 0.1 17 M0202-H03 ----V--W-D--- 0.1 18 M0201-A07
----V----E--- 0.1 19 M0202-C02 --P-V-------- 0.6 20 M0202-B04
--S-V-------- 0.2 21 M0202-E06 --R-V----D--- 0.06 22 M0202-A01
--I-V-------- 0.3 23 M0202-D09 --I-V----S--- 0.2 24 M0200-D03
--I-V----S--M 0.1 25 M0202-C09 --I-V----D--- 0.06 26 M0199-A08
--I-V----E--- 0.06 27 X133-B02 --I---------- 2.2 28 X133-D06
--I------E--- 0.33 29 X135-A01 ----A-------- 247.7 30 X133-G05
----S-------- 1405.6 31 X133-F10 ----L-------- 14.7 32 X135-A03
---------E--- 1.1 33
[0137] The affinity maturation results indicate that variations at
certain positions within the HC CDR3 region result in high
affinity/inhibitory anti-PKal antibodies as compared to the parent
M0162-A04 clone. These results match with the structural
information provided in Example 1 above. Note that the HC CDR3
region of clone M0199-A08 is identical to that of DX-2930.
Example 3
Impact of Mutations in Plasma Kallikrein on Antibody Inhibitory
Activity
[0138] The inhibitory activities of mutant X.sub.115-F02 against
various PKal mutants were examined.
[0139] X.sub.115-F02 is an IgG that is the same as DX-2930 except
that it contains a C-terminal lysine residue not present in DX-2930
and was expressed in HEK293T cells rather than CHO cells (Table 1
above). The binding specificity and affinity of X.sub.115-F02 is
the same as DX-2930.
[0140] The wild type and four mutants of plasma kallikrein used in
this study (FIG. 5) are recombinant catalytic domains expressed and
purified from pichia pastoris. Mutant 1 contains the following
mutations in the S3 subsite of the active site: S478A, N481A,
S506A, Y507A) (numbers based on the full length prekallikrein amino
acid sequence). Mutant 2 contains the following mutations in the S
subsite of the active site: R551A, Q553A, Y555A, T558A, R560A.
Mutant 4 contains the following mutations that are distal from the
active site: N396A, S398A, W399A. Mutant 3 was found to be inactive
and therefore was not tested in the activity assay. Mutant 3
contains the following mutations in the S subsite of the active
site: D572A, K575A, D577A.
[0141] The inhibitory activity of X.sub.115-F02 against the
wild-type PKal and the mutants were carried out using the method
described in Example 2 above and the K.sub.i,app values were
determined. As shown in FIG. 4, the mutations in Mutant 1 and 4 did
not significantly affect the potency of X.sub.115-F02 inhibition of
plasma kallikrein. Surprisingly, the mutations in Mutant 2 reduced
the potency approximately 65-fold. These results indicate that
residues R551A, Q553A, Y555A, T558A, R560A and their adjacent
residues might be important to the inhibitory activity of
X.sub.115-F02 (DX-2930).
Other Embodiments
[0142] All of the features disclosed in this specification may be
combined in any combination. Each feature disclosed in this
specification may be replaced by an alternative feature serving the
same, equivalent, or similar purpose. Thus, unless expressly stated
otherwise, each feature disclosed is only an example of a generic
series of equivalent or similar features.
[0143] From the above description, one skilled in the art can
easily ascertain the essential characteristics of the present
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions. Thus, other embodiments
are also within the claims.
Sequence CWU 1
1
58113PRTArtificial sequenceHv-CDR3 1Arg Arg Thr Gly Ile Pro Arg Arg
Asp Ala Phe Asp Ile 1 5 10 213PRTArtificial sequenceHv-CDR3 2Arg
Arg Arg Gly Ile Pro Arg Arg Asp Ala Phe Asp Ile 1 5 10
313PRTArtificial sequenceHv-CDR3 3Arg Arg Ser Gly Ile Pro Arg Arg
Asp Ala Phe Asp Ile 1 5 10 413PRTArtificial sequenceHv-CDR3 4Arg
Arg Thr Gly Ile Pro Arg Trp Asp Ala Phe Asp Ile 1 5 10
513PRTArtificial sequenceHv-CDR3 5Arg Arg Thr Gly Ile Pro Arg Arg
Asp Val Phe Asp Ile 1 5 10 613PRTArtificial sequenceHv-CDR3 6Arg
Arg Thr Gly Ile Pro Arg Arg Asp Ala Phe Glu Ile 1 5 10
713PRTArtificial sequenceHv-CDR3 7Arg Arg Thr Gly Ile Pro Arg Arg
Asp Ala Phe Asp Asn 1 5 10 813PRTArtificial sequenceHv-CDR3 8Arg
Arg Thr Gly Ile Pro Arg Arg Asp Ala Phe Asp Ser 1 5 10
913PRTArtificial sequenceHv-CDR3 9Arg Arg Thr Gly Val Pro Arg Arg
Asp Ala Phe Asp Ile 1 5 10 1013PRTArtificial sequenceHv-CDR3 10Arg
Arg Thr Gly Val Pro Arg Arg Asp Val Phe Asp Ile 1 5 10
1113PRTArtificial sequenceHv-CDR3 11Arg Arg Thr Gly Val Pro Arg Arg
Asp Ala Leu Asp Asn 1 5 10 1213PRTArtificial sequenceHv-CDR3 12Arg
Arg Thr Gly Val Pro Arg Arg Asp Ala Phe Asp Asn 1 5 10
1313PRTArtificial sequenceHv-CDR3 13Arg Arg Thr Gly Val Pro Arg Arg
Asn Ala Phe Asp Asn 1 5 10 1413PRTArtificial sequenceHv-CDR3 14Arg
Arg Thr Gly Val Pro Arg Trp Asp Ala Phe Asp Ile 1 5 10
1513PRTArtificial sequenceHv-CDR3 15Arg Arg Thr Gly Val Pro Arg Arg
Asp Ser Phe Asp Ile 1 5 10 1613PRTArtificial sequenceHv-CDR3 16Arg
Arg Thr Gly Val Pro Arg Arg Asp Ser Phe Glu Ile 1 5 10
1713PRTArtificial sequenceHv-CDR3 17Gln Arg Thr Gly Val Pro Arg Arg
Asp Ser Phe Asn Ile 1 5 10 1813PRTArtificial sequenceHv-CDR3 18Arg
Arg Thr Gly Val Pro Arg Trp Asp Asp Phe Asp Ile 1 5 10
1913PRTArtificial sequenceHv-CDR3 19Arg Arg Thr Gly Val Pro Arg Arg
Asp Glu Phe Asp Ile 1 5 10 2013PRTArtificial sequenceHv-CDR3 20Arg
Arg Pro Gly Val Pro Arg Arg Asp Ala Phe Asp Ile 1 5 10
2113PRTArtificial sequenceHv-CDR3 21Arg Arg Ser Gly Val Pro Arg Arg
Asp Ala Phe Asp Ile 1 5 10 2213PRTArtificial sequenceHv-CDR3 22Arg
Arg Arg Gly Val Pro Arg Arg Asp Asp Phe Asp Ile 1 5 10
2313PRTArtificial sequenceHv-CDR3 23Arg Arg Ile Gly Val Pro Arg Arg
Asp Ala Phe Asp Ile 1 5 10 2413PRTArtificial sequenceHv-CDR3 24Arg
Arg Ile Gly Val Pro Arg Arg Asp Ser Phe Asp Ile 1 5 10
2513PRTArtificial sequenceHv-CDR3 25Arg Arg Ile Gly Val Pro Arg Arg
Asp Ser Phe Asp Met 1 5 10 2613PRTArtificial sequenceHv-CDR3 26Arg
Arg Ile Gly Val Pro Arg Arg Asp Asp Phe Asp Ile 1 5 10
2713PRTArtificial sequenceHv-CDR3 27Arg Arg Ile Gly Val Pro Arg Arg
Asp Glu Phe Asp Ile 1 5 10 2813PRTArtificial sequenceHv-CDR3 28Arg
Arg Ile Gly Ile Pro Arg Arg Asp Ala Phe Asp Ile 1 5 10
2913PRTArtificial sequenceHv-CDR3 29Arg Arg Ile Gly Ile Pro Arg Arg
Asp Glu Phe Asp Ile 1 5 10 3013PRTArtificial sequenceHv-CDR3 30Arg
Arg Thr Gly Ala Pro Arg Arg Asp Ala Phe Asp Ile 1 5 10
3113PRTArtificial sequenceHv-CDR3 31Arg Arg Thr Gly Ser Pro Arg Arg
Asp Ala Phe Asp Ile 1 5 10 3213PRTArtificial sequenceHv-CDR3 32Arg
Arg Thr Gly Leu Pro Arg Arg Asp Ala Phe Asp Ile 1 5 10
3313PRTArtificial sequenceHv-CDR3 33Arg Arg Thr Gly Ile Pro Arg Arg
Asp Glu Phe Asp Ile 1 5 10 34106PRTArtificial
sequence559A-M0162-A04 light chain 34Asp Ile Gln Met Thr Gln Ser
Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 Tyr
Lys Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Thr Tyr
Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
35102PRTArtificial sequence559A-M0162-A04 light chain and germline
light chain comparison 35Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Leu Leu Ile Tyr 35 40 45 Ala Ser Leu Glu
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 50 55 60 Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe 65 70 75 80
Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Tyr Trp Thr Phe Gly Gln Gly 85
90 95 Thr Lys Val Glu Ile Lys 100 36106PRTArtificial
sequenceGermline light chain 36Asp Ile Gln Met Thr Gln Ser Pro Ser
Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala
Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Trp
Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
37122PRTArtificial sequence559A-M0162-A04 heavy chain 37Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr 20 25
30 Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Gly Ile Tyr Ser Ser Gly Gly Ile Thr Val Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Tyr Arg Arg Thr Gly Ile Pro
Arg Arg Asp Ala Phe Asp Ile Trp 100 105 110 Gly Gln Gly Thr Met Val
Thr Val Ser Ser 115 120 38104PRTArtificial sequence559A-M0162-A04
heavy chain and germline comparison 38Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Met 20 25 30 Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ile Ser 35 40 45 Gly
Gly Thr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 50 55
60 Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala
65 70 75 80 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Phe Asp Ile Trp
Gly Gln 85 90 95 Gly Thr Met Val Thr Val Ser Ser 100
39113PRTArtificial sequenceGermline heavy chain 39Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ala Phe Asp Ile Trp Gly Gln
Gly Thr Met Val Thr Val Ser 100 105 110 Ser 40248PRTH. sapiens
40Ile Val Gly Gly Thr Asn Ser Ser Trp Gly Glu Trp Pro Trp Gln Val 1
5 10 15 Ser Leu Gln Val Lys Leu Thr Ala Gln Arg His Leu Cys Gly Gly
Ser 20 25 30 Leu Ile Gly His Gln Trp Val Leu Thr Ala Ala His Cys
Phe Asp Gly 35 40 45 Leu Pro Leu Gln Asp Val Trp Arg Ile Tyr Ser
Gly Ile Leu Asn Leu 50 55 60 Ser Asp Ile Thr Lys Asp Thr Pro Phe
Ser Gln Ile Lys Glu Ile Ile 65 70 75 80 Ile His Gln Asn Tyr Lys Val
Ser Glu Gly Asn His Asp Ile Ala Leu 85 90 95 Ile Lys Leu Gln Ala
Pro Leu Asn Tyr Thr Glu Phe Gln Lys Pro Ile 100 105 110 Ser Leu Pro
Ser Lys Gly Asp Thr Ser Thr Ile Tyr Thr Asn Cys Trp 115 120 125 Val
Thr Gly Trp Gly Phe Ser Lys Glu Lys Gly Glu Ile Gln Asn Ile 130 135
140 Leu Gln Lys Val Asn Ile Pro Leu Val Thr Asn Glu Glu Cys Gln Lys
145 150 155 160 Arg Tyr Gln Asp Tyr Lys Ile Thr Gln Arg Met Val Cys
Ala Gly Tyr 165 170 175 Lys Glu Gly Gly Lys Asp Ala Cys Lys Gly Asp
Ser Gly Gly Pro Leu 180 185 190 Val Cys Lys His Asn Gly Met Trp Arg
Leu Val Gly Ile Thr Ser Trp 195 200 205 Gly Glu Gly Cys Ala Arg Arg
Glu Gln Pro Gly Val Tyr Thr Lys Val 210 215 220 Ala Glu Tyr Met Asp
Trp Ile Leu Glu Lys Thr Gln Ser Ser Asp Gly 225 230 235 240 Lys Ala
Gln Met Gln Ser Pro Ala 245 41248PRTArtificial
sequence(klkb1)-Mut1-forPichia 41Ile Val Gly Gly Thr Asn Ser Ser
Trp Gly Glu Trp Pro Trp Gln Val 1 5 10 15 Ser Leu Gln Val Lys Leu
Thr Ala Gln Arg His Leu Cys Gly Gly Ser 20 25 30 Leu Ile Gly His
Gln Trp Val Leu Thr Ala Ala His Cys Phe Asp Gly 35 40 45 Leu Pro
Leu Gln Asp Val Trp Arg Ile Tyr Ser Gly Ile Leu Asn Leu 50 55 60
Ser Asp Ile Thr Lys Asp Thr Pro Phe Ser Gln Ile Lys Glu Ile Ile 65
70 75 80 Ile His Gln Asn Tyr Lys Val Ala Glu Gly Ala His Asp Ile
Ala Leu 85 90 95 Ile Lys Leu Gln Ala Pro Leu Asn Tyr Thr Glu Phe
Gln Lys Pro Ile 100 105 110 Ser Leu Pro Ala Ala Gly Asp Thr Ser Thr
Ile Tyr Thr Asn Cys Trp 115 120 125 Val Thr Gly Trp Gly Phe Ser Lys
Glu Lys Gly Glu Ile Gln Asn Ile 130 135 140 Leu Gln Lys Val Asn Ile
Pro Leu Val Thr Asn Glu Glu Cys Gln Lys 145 150 155 160 Arg Tyr Gln
Asp Tyr Lys Ile Thr Gln Arg Met Val Cys Ala Gly Tyr 165 170 175 Lys
Glu Gly Gly Lys Asp Ala Cys Lys Gly Asp Ser Gly Gly Pro Leu 180 185
190 Val Cys Lys His Asn Gly Met Trp Arg Leu Val Gly Ile Thr Ser Trp
195 200 205 Gly Glu Gly Cys Ala Arg Arg Glu Gln Pro Gly Val Tyr Thr
Lys Val 210 215 220 Ala Glu Tyr Met Asp Trp Ile Leu Glu Lys Thr Gln
Ser Ser Asp Gly 225 230 235 240 Lys Ala Gln Met Gln Ser Pro Ala 245
42248PRTArtificial sequence(klkb1)-Mut2-forPichia 42Ile Val Gly Gly
Thr Asn Ser Ser Trp Gly Glu Trp Pro Trp Gln Val 1 5 10 15 Ser Leu
Gln Val Lys Leu Thr Ala Gln Arg His Leu Cys Gly Gly Ser 20 25 30
Leu Ile Gly His Gln Trp Val Leu Thr Ala Ala His Cys Phe Asp Gly 35
40 45 Leu Pro Leu Gln Asp Val Trp Arg Ile Tyr Ser Gly Ile Leu Asn
Leu 50 55 60 Ser Asp Ile Thr Lys Asp Thr Pro Phe Ser Gln Ile Lys
Glu Ile Ile 65 70 75 80 Ile His Gln Asn Tyr Lys Val Ser Glu Gly Asn
His Asp Ile Ala Leu 85 90 95 Ile Lys Leu Gln Ala Pro Leu Asn Tyr
Thr Glu Phe Gln Lys Pro Ile 100 105 110 Ser Leu Pro Ser Lys Gly Asp
Thr Ser Thr Ile Tyr Thr Asn Cys Trp 115 120 125 Val Thr Gly Trp Gly
Phe Ser Lys Glu Lys Gly Glu Ile Gln Asn Ile 130 135 140 Leu Gln Lys
Val Asn Ile Pro Leu Val Thr Asn Glu Glu Cys Gln Lys 145 150 155 160
Ala Tyr Ala Asp Ala Lys Ile Ala Gln Ala Met Val Cys Ala Gly Tyr 165
170 175 Lys Glu Gly Gly Lys Asp Ala Cys Lys Gly Asp Ser Gly Gly Pro
Leu 180 185 190 Val Cys Lys His Asn Gly Met Trp Arg Leu Val Gly Ile
Thr Ser Trp 195 200 205 Gly Glu Gly Cys Ala Arg Arg Glu Gln Pro Gly
Val Tyr Thr Lys Val 210 215 220 Ala Glu Tyr Met Asp Trp Ile Leu Glu
Lys Thr Gln Ser Ser Asp Gly 225 230 235 240 Lys Ala Gln Met Gln Ser
Pro Ala 245 43248PRTArtificial sequence(klkb1)-Mut3-forPichia 43Ile
Val Gly Gly Thr Asn Ser Ser Trp Gly Glu Trp Pro Trp Gln Val 1 5 10
15 Ser Leu Gln Val Lys Leu Thr Ala Gln Arg His Leu Cys Gly Gly Ser
20 25 30 Leu Ile Gly His Gln Trp Val Leu Thr Ala Ala His Cys Phe
Asp Gly 35 40 45 Leu Pro Leu Gln Asp Val Trp Arg Ile Tyr Ser Gly
Ile Leu Asn Leu 50 55 60 Ser Asp Ile Thr Lys Asp Thr Pro Phe Ser
Gln Ile Lys Glu Ile Ile 65 70 75 80 Ile His Gln Asn Tyr Lys Val Ser
Glu Gly Asn His Asp Ile Ala Leu 85 90 95 Ile Lys Leu Gln Ala Pro
Leu Asn Tyr Thr Glu Phe Gln Lys Pro Ile 100 105 110 Ser Leu Pro Ser
Lys Gly Asp Thr Ser Thr Ile Tyr Thr Asn Cys Trp 115 120 125 Val Thr
Gly Trp Gly Phe Ser Lys Glu Lys Gly Glu Ile Gln Asn Ile 130 135 140
Leu Gln Lys Val Asn Ile Pro Leu Val Thr Asn Glu Glu Cys Gln Lys 145
150 155 160 Arg Tyr Gln Asp Tyr Lys Ile Thr Gln Arg Met Val Cys Ala
Gly Tyr 165 170 175 Lys Glu Gly Gly Lys Ala Ala Cys Ala Gly Ala Ser
Gly Gly Pro Leu 180 185 190 Val Cys Lys His Asn Gly Met Trp Arg Leu
Val Gly Ile Thr Ser Trp 195 200 205 Gly Glu Gly Cys Ala Arg Arg Glu
Gln Pro Gly Val Tyr Thr Lys Val 210 215 220 Ala Glu Tyr Met
Asp Trp Ile Leu Glu Lys Thr Gln Ser Ser Asp Gly 225 230 235 240 Lys
Ala Gln Met Gln Ser Pro Ala 245 44248PRTArtificial
sequence(klkb1)-Mut4-forPichia 44Ile Val Gly Gly Thr Ala Ser Ala
Ala Gly Glu Trp Pro Trp Gln Val 1 5 10 15 Ser Leu Gln Val Lys Leu
Thr Ala Gln Arg His Leu Cys Gly Gly Ser 20 25 30 Leu Ile Gly His
Gln Trp Val Leu Thr Ala Ala His Cys Phe Asp Gly 35 40 45 Leu Pro
Leu Gln Asp Val Trp Arg Ile Tyr Ser Gly Ile Leu Asn Leu 50 55 60
Ser Asp Ile Thr Lys Asp Thr Pro Phe Ser Gln Ile Lys Glu Ile Ile 65
70 75 80 Ile His Gln Asn Tyr Lys Val Ser Glu Gly Asn His Asp Ile
Ala Leu 85 90 95 Ile Lys Leu Gln Ala Pro Leu Asn Tyr Thr Glu Phe
Gln Lys Pro Ile 100 105 110 Ser Leu Pro Ser Lys Gly Asp Thr Ser Thr
Ile Tyr Thr Asn Cys Trp 115 120 125 Val Thr Gly Trp Gly Phe Ser Lys
Glu Lys Gly Glu Ile Gln Asn Ile 130 135 140 Leu Gln Lys Val Asn Ile
Pro Leu Val Thr Asn Glu Glu Cys Gln Lys 145 150 155 160 Arg Tyr Gln
Asp Tyr Lys Ile Thr Gln Arg Met Val Cys Ala Gly Tyr 165 170 175 Lys
Glu Gly Gly Lys Asp Ala Cys Lys Gly Asp Ser Gly Gly Pro Leu 180 185
190 Val Cys Lys His Asn Gly Met Trp Arg Leu Val Gly Ile Thr Ser Trp
195 200 205 Gly Glu Gly Cys Ala Arg Arg Glu Gln Pro Gly Val Tyr Thr
Lys Val 210 215 220 Ala Glu Tyr Met Asp Trp Ile Leu Glu Lys Thr Gln
Ser Ser Asp Gly 225 230 235 240 Lys Ala Gln Met Gln Ser Pro Ala 245
45248PRTArtificial sequence(klkb1)-parentforPichia 45Ile Val Gly
Gly Thr Asn Ser Ser Trp Gly Glu Trp Pro Trp Gln Val 1 5 10 15 Ser
Leu Gln Val Lys Leu Thr Ala Gln Arg His Leu Cys Gly Gly Ser 20 25
30 Leu Ile Gly His Gln Trp Val Leu Thr Ala Ala His Cys Phe Asp Gly
35 40 45 Leu Pro Leu Gln Asp Val Trp Arg Ile Tyr Ser Gly Ile Leu
Asn Leu 50 55 60 Ser Asp Ile Thr Lys Asp Thr Pro Phe Ser Gln Ile
Lys Glu Ile Ile 65 70 75 80 Ile His Gln Asn Tyr Lys Val Ser Glu Gly
Asn His Asp Ile Ala Leu 85 90 95 Ile Lys Leu Gln Ala Pro Leu Asn
Tyr Thr Glu Phe Gln Lys Pro Ile 100 105 110 Ser Leu Pro Ser Lys Gly
Asp Thr Ser Thr Ile Tyr Thr Asn Cys Trp 115 120 125 Val Thr Gly Trp
Gly Phe Ser Lys Glu Lys Gly Glu Ile Gln Asn Ile 130 135 140 Leu Gln
Lys Val Asn Ile Pro Leu Val Thr Asn Glu Glu Cys Gln Lys 145 150 155
160 Arg Tyr Gln Asp Tyr Lys Ile Thr Gln Arg Met Val Cys Ala Gly Tyr
165 170 175 Lys Glu Gly Gly Lys Asp Ala Cys Lys Gly Asp Ser Gly Gly
Pro Leu 180 185 190 Val Cys Lys His Asn Gly Met Trp Arg Leu Val Gly
Ile Thr Ser Trp 195 200 205 Gly Glu Gly Cys Ala Arg Arg Glu Gln Pro
Gly Val Tyr Thr Lys Val 210 215 220 Ala Glu Tyr Met Asp Trp Ile Leu
Glu Lys Thr Gln Ser Ser Asp Gly 225 230 235 240 Lys Ala Gln Met Gln
Ser Pro Ala 245 46451PRTArtificial sequenceDX-2930 Heavy Chain
46Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His
Tyr 20 25 30 Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Gly Ile Tyr Ser Ser Gly Gly Ile Thr Val
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Tyr Arg Arg Ile
Gly Val Pro Arg Arg Asp Glu Phe Asp Ile Trp 100 105 110 Gly Gln Gly
Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys
Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260
265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385
390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly 450
47213PRTArtificial sequenceAmino acids from Pkal 47Asp Ile Gln Met
Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Lys Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Asn Thr Tyr Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165
170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 484PRTArtificial
sequenceAmino acids from Pkal 48Ser Trp Gly Glu 1 495PRTArtificial
sequenceAmino acids from Pkal 49Asp Ala Cys Lys Gly 1 5
506PRTArtificial sequenceAmino acids from Pkal 50Thr Ser Trp Gly
Glu Gly 1 5 5119PRTArtificial sequenceAmino acids from Pkal 51Leu
Val Thr Asn Glu Glu Cys Gln Lys Arg Tyr Gln Asp Tyr Lys Ile 1 5 10
15 Thr Gln Gln 5214PRTArtificial sequenceAmino acids from Pkal
52Trp Val Thr Gly Trp Gly Phe Ser Lys Glu Lys Gly Glu Ile 1 5 10
5310PRTArtificial sequenceAmino acids from Pkal 53Ala Cys Lys Gly
Asp Ser Gly Gly Pro Leu 1 5 10 545PRTArtificial sequenceAmino acids
from Pkal 54Ser Trp Gly Asp Ile 1 5 558PRTArtificial sequenceAmino
acids from Pkal 55His Asp Ile Ala Leu Ile Lys Leu 1 5
5615PRTArtificial sequenceAmino acids from Pkal 56Thr Pro Phe Ser
Gln Ile Lys Glu Ile Ile Ile His Gln Asn Tyr 1 5 10 15
5716PRTArtificial sequenceAmino acids from Pkal 57Ala His Cys Phe
Asp Gly Leu Pro Leu Gln Asp Val Trp Arg Ile Tyr 1 5 10 15
5813PRTArtificial sequenceHv-CDR3 58Xaa Arg Xaa Gly Xaa Pro Arg Xaa
Xaa Xaa Xaa Xaa Xaa 1 5 10
* * * * *
References