U.S. patent application number 16/296852 was filed with the patent office on 2020-01-23 for antibodies with ph dependent antigen binding.
The applicant listed for this patent is Pfizer Inc., RINAT NEUROSCIENCE CORP.. Invention is credited to Jeffrey Raymond CHABOT, Javier Fernando CHAPARRO RIGGERS, Bruce Charles GOMES, Hong LIANG, KapiI MAYAWALA, Jerome Thomas METTETAL, II, Jaume PONS, Arvind RAJPAL, David Louis SHELTON.
Application Number | 20200024366 16/296852 |
Document ID | / |
Family ID | 44483937 |
Filed Date | 2020-01-23 |
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United States Patent
Application |
20200024366 |
Kind Code |
A1 |
PONS; Jaume ; et
al. |
January 23, 2020 |
ANTIBODIES WITH pH DEPENDENT ANTIGEN BINDING
Abstract
The present invention relates to antibodies with pH dependent
binding to its antigen such that the affinity for antigen binding
at physiological pH (i.e., pH 7.4) is greater than at endosomal pH
(i.e., pH 6.0 or 5.5). In other words, the K.sub.D or k.sub.off
ratio at pH 5.5/pH 7.4 or at pH 6.0/pH 7.4 is more than, or ranges
between, 2, 3, 4, 8, 10, 16, 20, 30, 40, or 100 or more. Such pH
dependent antibodies preferentially dissociate from the antigen in
the endosome. This can increase antibody half life, as compared to
antibodies with equivalent K.sub.Ds at pH 7.4 but with no pH
dependent binding, when the antigen is one that undergoes
antigen-mediated clearance (e.g., PCSK9). Antibodies with pH
dependent binding can decrease total antigen half life when the
antigen undergoes reduced clearance when bound to antibody (e.g.,
IL6). Antibodies with pH dependent binding can also prolong the
decrease in antigen which is not antibody-bound. This can be
important when antagonizing a target antigen typically present at
high levels (e.g., IgE, DKK1, C5 and SOST). In addition, such
antibodies can increase antigen half life when the antigen is a
receptor and the receptor has increased clearance when bound to
antibody (e.g., GMCSF receptor).
Inventors: |
PONS; Jaume; (San Francisco,
CA) ; CHABOT; Jeffrey Raymond; (Medford, MA) ;
CHAPARRO RIGGERS; Javier Fernando; (San Mateo, CA) ;
GOMES; Bruce Charles; (Ashburnham, MA) ; LIANG;
Hong; (Hillsborough, CA) ; MAYAWALA; KapiI;
(New Brunswick, NJ) ; METTETAL, II; Jerome Thomas;
(Cambridge, MA) ; RAJPAL; Arvind; (San Francisco,
CA) ; SHELTON; David Louis; (Oakland, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pfizer Inc.
RINAT NEUROSCIENCE CORP. |
New York
South San Francisco |
NY
CA |
US
US |
|
|
Family ID: |
44483937 |
Appl. No.: |
16/296852 |
Filed: |
March 8, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14677983 |
Apr 3, 2015 |
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16296852 |
|
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13045345 |
Mar 10, 2011 |
9029515 |
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14677983 |
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61313102 |
Mar 11, 2010 |
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61447638 |
Feb 28, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/565 20130101;
C07K 2317/70 20130101; C07K 2317/76 20130101; C07K 2317/56
20130101; A61P 3/06 20180101; C07K 16/40 20130101; C07K 2317/732
20130101; C07K 2317/94 20130101; C07K 16/28 20130101; C07K 2317/734
20130101; C07K 2317/92 20130101; C07K 16/4291 20130101; C07K
2319/21 20130101 |
International
Class: |
C07K 16/42 20060101
C07K016/42; C07K 16/40 20060101 C07K016/40; C07K 16/28 20060101
C07K016/28 |
Claims
1-11. (canceled)
12. A method of extending interval dosing and/or decreasing the
therapeutic dose for treating a patient with a therapeutic
antibody, said method comprising administering to the patient a
therapeutically effective amount of the antibody, wherein one or
more amino acid substitutions have been engineered into one or more
complementarity determining regions of said antibody as compared to
the original unsubstituted antibody such that said antibody
specifically binds an antigen with higher affinity at pH 7.4 than
at pH 6.0, wherein the K.sub.D ratio and the k.sub.off ratio at pH
6.0/pH 7.4 at 25.degree. C. is 2, 3, 4, 8, 10, 16, or more, and the
K.sub.D ratio and/or the k.sub.off ratio is higher as compared to
the unsubstituted antibody, wherein the antigen is soluble and not
membrane bound, and wherein said antibody prolongs the
antibody-mediated decrease in antigen that is not antibody-bound as
compared to said unsubstituted antibody.
13-23. (canceled)
24. The method of claim 12, wherein the at least one amino acid
substitution comprises at least one histidine substitution.
25. The method of claim 12, wherein the antigen is not
interleukin-6 receptor.
26. The method of claim 12, wherein said antibody with pH dependent
binding is an antibody drug conjugate, mediates antibody dependent
cell-mediated cytotoxicity (ADCC), and/or mediates
complement-dependent cytotoxicity (CDC).
27. The method of claim 12, wherein pH dependent binding of the
antibody has the K.sub.D ratio and/or k.sub.off ratio at pH 6.0/pH
7.4 and at 25.degree. C. which is 20, 30, 40, 100 or more.
28. The method of claim 27, wherein the antigen is PCSK9, IgE, C5,
or DKK1.
29. The method of claim 28, wherein the antigen is PCSK9 and the pH
dependent binding of the antibody to the antigen at pH 7.4 and at
25.degree. C. has a K.sub.D ranging between 0.01 nM and 100 nM and
a K.sub.D ratio of 10, 30, 100 or more.
30. The method of claim 29, wherein the binding of the antibody to
antigen at pH 7.4 and at 25.degree. C. has a K.sub.D ranging
between 0.1 nM to 10 nM.
31. The method of claim 28, wherein the antigen is C5 and the pH
dependent binding of the antibody to the antigen at pH 7.4 and 25C
has a K.sub.D ranging between 0.1 nM and 100 nM and the k.sub.off
ratio at pH 6.0/pH 7.4 at 25.degree. C. is 10, 30 or more.
32. The method of claim 31, wherein the k.sub.off ratio is 30 or
more.
33. A method of making an antibody with prolonged half-life by
regulating antibody-antigen binding affinity in a pH dependent
manner, said method comprising engineering at least one amino acid
substitution into the complementarity determining region of said
antibody wherein said antibody specifically binds an antigen with
higher affinity at pH 7.4 than at pH 6.0, wherein the K.sub.D ratio
and the k.sub.off ratio at pH 6.0/pH 7.4 and at 25.degree. C. is 2,
3, 4, 8, 10, 16, or more, and wherein said antigen is soluble and
not membrane bound.
34. The method of claim 33, wherein the at least one amino acid
substitution comprises at least one histidine substitution.
35. The method of claim 33, wherein the antigen is not
interleukin-6 receptor.
36. The method of claim 33, wherein the antigen is PCSK9, IgE, C5,
or DKK1.
Description
[0001] This application is a is a continuation of U.S. application
Ser. No. 14/677,983, filed Apr. 3, 2015, which is a divisional of
U.S. application Ser. No. 13/045,345, filed Mar. 10, 2011, now
granted as U.S. Pat. No. 9,029,515, which claims priority, under 35
USC .sctn. 119(e), to U.S. Provisional Application Ser. No.
61/313,102, filed Mar. 11, 2010, and U.S. Provisional Application
Ser. No. 61/447,638, filed Feb. 28, 2011, hereby incorporated by
reference in their entireties.
REFERENCE TO SEQUENCE LISTING
[0002] This application is being filed electronically via EFS-Web
and includes an electronically submitted sequence listing in .txt
format. The .txt file contains a sequence listing entitled
"SequenceListingPC33956C.txt" created on Mar. 7, 2019 and having a
size of 12 KB. The sequence listing contained in this .txt file is
part of the specification and is herein incorporated by reference
in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to antibodies, e.g., full
length antibodies or antigen-binding portions thereof, that have pH
dependent binding such that the K.sub.D and/or k.sub.off ratio at
endosomal pH/physiologic pH (e.g., pH 5.5/pH 7.4 or pH 6.0/pH 7.4)
is 2 or greater.
BACKGROUND OF THE INVENTION
[0004] Monoclonal antibodies (mAbs) have become important
therapeutic options for numerous diseases (Brekke and Sandlie, Nat
Rev Drug Discov 2: 52-62, 2003; Maggon, Curr Med Chem 14:
1978-1987, 2007). Most of the mAbs now on the market are IgG
antibodies. Their relatively long half-life is mediated by FcRn
binding. IgG uptake into the cell occurs via fluid phase
pinocytosis, and the IgG subsequently binds to FcRn in the
acidified environment (pH 6.0) of the endosomal compartment (Lobo
et al., J Pharm Sci 93: 2645-2668, 2004). FcRn-bound IgG is thought
to be protected from degradation by recycling to the cell surface
where the neutral pH facilitates dissociation and release of the
IgG into circulation. Unbound IgG, by contrast, is believed to be
transferred to lysosomes and subsequently degraded (Lencer and
Blumberg, Trends Cell Biol 15: 15: 5-9, 2005).
[0005] Recently, various technologies for optimizing the functional
activity of an IgG antibody by introducing specific substitutions
have been applied in order to reduce dose and/or dose frequency and
improve efficacy and safety (Presta, Curr. Opinion Immunol 20:
460-470, 2008). Generally, optimization of IgG antibodies can be
classified into engineering the Fc constant region, to impact
antibody binding to FcRn, Fc.gamma.R and the complement system, and
engineering the variable region to impact binding affinity.
[0006] Several works describe engineering the constant region to
increase binding to Fc .gamma.-receptors and thus enhance the
effector function of an IgG1 antibody (Stavenhagen et al., Cancer
Res 67: 8882-8890, 2007; Zalevsky et al., Blood 113: 3735-3743,
2009). Substitutions such as S239D/I332E/A330L or
F243L/R292P/Y300L/V305I/P396L into IgG1 have been shown to improve
Fc .gamma.-receptor IIIa binding and exhibit superior
antibody-dependent cellular cytotoxicity (ADCC) activity in vitro
and superior efficacy in vivo compared with wild-type IgG1. Hence,
compared with wild-type antibodies, antibodies with such
substitutions are expected to show superior efficacy at the same
dose or comparable efficacy at a lower dose and/or with lesser
frequency of dosing in human.
[0007] Another method to lower the dose and/or frequency of dosing
is to reduce the elimination of an IgG antibody. The long half-life
of IgG antibodies is reported to be dependent on its binding to
FcRn. Therefore, substitutions that increase the binding affinity
of IgG to FcRn at pH 6.0 while maintaining the pH dependence of the
interaction by engineering the constant region have been
extensively studied (Ghetie et al., Nature Biotech. 15: 637-640,
1997; Hinton et al., JBC 279: 6213-6216, 2004; Dall'Acqua et al., J
Immunol 117: 1129-1138, 2006). Substitutions, such as M428L/N434S,
led to increased half life and an increased pharmacodynamic effect
in the variants (Zalevsky et al., Nature Biotech. 28: 157-159,
2010). Several works have reported successful increase in the
half-life by introducing substitutions such as T250Q/M428L or
M252Y/S254T/T256E to increase binding to FcRn at an acidic pH. In a
non-human primate pharmacokinetic study, T250Q/M428L substitution
to IgG1 showed a half-life of 35 days, a significant increase over
the 14-day half-life of wild-type IgG1 (Hinton et al., J Immunol
176: 346-356, 2006).
[0008] Although substitutions in the constant region are able to
significantly improve the functions of therapeutic IgG antibodies,
substitutions in the strictly conserved constant region have the
risk of immunogenicity in human (Presta, supra, 2008; De Groot and
Martin, Clin Immunol 131: 189-201, 2009) and substitution in the
highly diverse variable region sequence might be less immunogenic.
Reports concerned with the variable region include engineering the
CDR residues to improve binding affinity to the antigen (Rothe et
al., Expert Opin Biol Ther 6: 177-187, 2006; Bostrom et al.,
Methods Mol Biol 525: 353-376, 2009; Thie et al., Methods Mol Biol
525: 309-322, 2009) and engineering the CDR and framework residues
to improve stability (Wom and Pluckthun, J Mol Biol 305: 989-1010,
2001; Ewert et al., Methods 34: 184-199, 2004) and decrease
immunogenicity risk (De Groot and Martin, supra, 2009; Jones et
al., Methods Mol Bio 525: 405-423, xiv, 2009). As reported,
improved affinity to the antigen can be achieved by affinity
maturation using the phage or ribosome display of a randomized
library. Improved stability can be rationally obtained from
sequence- and structure-based rational design. Decreased
immunogenicity risk (deimmunization) can be accomplished by various
humanization methodologies and the removal of T-cell epitopes,
which can be predicted using in silico technologies or determined
by in vitro assays. Additionally, variable regions have been
engineered to lower pl. A longer half life was observed for these
antibodies as compared to wild type antibodies despite comparable
FcRn binding (Igawa et al., PEDS, Advance Access, doi:
10.1093/protein/gzq009, 2010).
[0009] The present invention relates to engineering or selecting
antibodies with pH dependent antigen binding to modify antibody
and/or antigen half life. IgG2 antibody half life can be shortened
if antigen-mediated clearance mechanisms normally degrade the
antibody when bound to the antigen. Similarly, the antigen:antibody
complex can impact the half-life of the antigen, either extending
half-life by protecting the antigen from the typical degradation
processes, or shortening the half-life via antibody-mediated
degradation. The present invention relates to antibodies with
higher affinity for antigen at pH 7.4 as compared to endosomal pH
(i.e., pH 5.5-6.0) such that the K.sub.D ratio at pH 5.5/pH 7.4 or
at pH 6.0/pH 7.4 is 2 or more.
[0010] The invention relates to an antibody with such pH dependent
binding to its antigen, and methods of designing, making and using
such antibodies. Examples of useful antibodies target antigens such
as proprotein convertase subtilisin kexin type 9 (PCSK9), also
known as NARC-1, IgE, dickkopf-related protein 1 (DKK1), Complement
5 (C5), sclerostin (SOST) and GMCSF receptor.
[0011] PCSK9 was identified as a protein with a genetic mutation in
some forms of familial hypercholesterolemia. PCSK9 is synthesized
as a zymogen that undergoes autocatalytic processing at a
particular motif in the endoplasmic reticulum. Population studies
have shown that some PCSK9 mutations are "gain-of-function" and are
found in individuals with autosomal dominant hypercholesterolemia,
while other "loss-of-function" (LOF) mutations are linked with
reduced plasma cholesterol. Morbidity and mortality studies in this
group clearly demonstrated that reducing PCSK9 function
significantly diminished the risk of cardiovascular disease.
SUMMARY OF THE INVENTION
[0012] The present invention relates to antibodies with pH
dependent binding to its antigen such that the affinity for antigen
binding at physiological pH (i.e., pH 7.4) is greater than at
endosomal pH (i.e., pH 6.0 or 5.5). In other words, the K.sub.D or
k.sub.off ratio at pH 5.5/pH 7.4 or at pH 6.0/pH 7.4 is more than,
or ranges between, 2, 3, 4, 8, 10, 16, 20, 30, 40, or 100 or more.
Such pH dependent antibodies preferentially dissociate from the
antigen in the endosome. This can increase antibody half life, as
compared to antibodies with equivalent K.sub.Ds at pH 7.4 but with
no pH dependent binding, when the antigen is one that undergoes
antigen-mediated clearance (e.g., PCSK9). Antibodies with pH
dependent binding can decrease total antigen half life when the
antigen undergoes reduced clearance when bound to antibody (e.g.,
IL6). Antibodies with pH dependent binding can also prolong the
antibody-mediated decrease in antigen which is not antibody-bound.
This can be important when antagonizing a target antigen typically
present at high levels (e.g., IgE, DKK1, C5 and SOST). In addition,
such antibodies can increase antigen half life when the antigen is
a receptor and the receptor has increased clearance when bound to
antibody (e.g., GMCSF receptor). In any embodiment of the invention
described below, the K.sub.D and k.sub.off can be measured at
either 25.degree. C. or 37.degree. C.
[0013] In a preferred embodiment, the antibody with pH dependent
binding which specifically binds an antigen with higher affinity at
pH 7.4 than at pH 6.0, wherein the K.sub.D ratio and/or the
k.sub.off ratio at pH 6.0/pH 7.4 and at 25.degree. C. is more than,
or ranges between, 2, 3, 4, 8, 10, 16, or more, and wherein the
antibody has reduced plasma clearance in vivo when exposed to said
antigen as compared to an antibody without pH dependent binding
that has a similar affinity for the antigen at pH 7.4, but has a
comparable K.sub.D and/or k.sub.off ratio at pH 6.0/pH 7.4 that is
less than 2. Preferably, the antigen is not interleukin-6 receptor
(IL6R), or, preferably, the antibody is not an anti-IL6R antibody
Fv3-m73, Fv4-m73 or H3pI/L73 as disclosed in WO 2010/106812 or WO
2009/041621.
[0014] In another preferred embodiment, the antibody with pH
dependent binding which specifically binds an antigen with higher
affinity at pH 7.4 than at pH 6.0, wherein the K.sub.D ratio and/or
the k.sub.off ratio at pH 6.0/pH 7.4 and at 25.degree. C. is more
than, or ranges between, 2, 3, 4, 8, 10, 16, or more, and wherein
the antigen is both membrane bound and soluble in vivo and wherein
the antibody mediates increased localization to a cell membrane
receptor as compared to an antibody that has a similar affinity for
the antigen at pH 7.4 but has a comparable K.sub.D and/or k.sub.off
ratio at pH 6.0/pH 7.4 that is less than 2. Preferably, the antigen
is not the IL6R or, preferably, the antibody is not an anti-IL6R
antibody Fv3-m73, Fv4-m73 or H3pI/L73 as disclosed in WO
2010/106812 or WO 2009/041621. In another preferred embodiment, the
antigen is a soluble receptor that is a non-signaling decoy. In
still other preferred embodiments, the antibody with pH dependent
binding is an antibody drug conjugate, mediates antibody dependent
cell-mediated cytotoxicity (ADCC), and/or complement-dependent
cytotoxicity (CDC).
[0015] The invention includes an antibody with pH dependent binding
which specifically binds an antigen with higher affinity at pH 7.4
than at pH 6.0, wherein the K.sub.D ratio and/or the k.sub.off
ratio at pH 6.0/pH 7.4 and at 25.degree. C. is more than, or ranges
between, 2, 3, 4, 8, 10, 16, or more, and wherein the decrease in
the in vivo amount of non-antibody bound antigen is prolonged when
exposed to said antibody as compared to an antibody without pH
dependent binding that has a similar affinity for the antigen at pH
7.4, but has a comparable K.sub.D and/or k.sub.off ratio at pH
6.0/pH 7.4 that is less than 2.
[0016] The invention provides an antibody with pH dependent binding
which specifically binds an antigen with higher affinity at pH 7.4
than at pH 6.0, wherein the K.sub.D ratio and/or the k.sub.off
ratio at pH 6.0/pH 7.4 and at 25.degree. C. is more than, or ranges
between, 2, 3, 4, 8, 10, 16, or more, and wherein there is a
decrease in the vivo amount of antibody-bound antigen as compared
to an antibody without pH dependent binding that has a similar
affinity for the antigen at pH 7.4, but has a comparable K.sub.D
and/or k.sub.off ratio at pH 6.0/pH 7.4 that is less than 2. In a
preferred embodiment, the antigen is osteopontin.
[0017] The invention also provides for an agonist antibody with pH
dependent binding which specifically binds an antigen with higher
affinity at pH 7.4 than at pH 6.0, wherein the K.sub.D ratio and/or
the k.sub.off ratio at pH 6.0/pH 7.4 and at 25.degree. C. is more
than, or ranges between, 2, 3, 4, 8, 10, 16, or more, and wherein
the antigen is a receptor and the receptor has reduced clearance in
vivo when exposed to said antibody as compared to an antibody that
has similar affinity for the receptor at pH 7.4, but has a
comparable K.sub.D and/or k.sub.off ratio at pH 6.0/pH 7.4 that is
less than 2. In a preferred embodiment, the receptor is GMCSF
receptor.
[0018] In other preferred embodiments of any of the preceding
antibodies, the K.sub.D ratio or k.sub.off ratio at pH 6.0/pH 7.4
is more than, or ranges between, 20, 30, 40 or 100 or more. In
other preferred embodiments, the preferred K.sub.D ratio or
k.sub.off ratio at pH 6.0/pH 7.4 ranges between 2-3, 2-4, 2-8,
2-10, 2-16, or 2-20 or more, or 3-4, 3-8, 3-10, 3-16 or 3-20, or
4-8, 4-10, 4-16, or 4-20 or more, or 8 to 10, 8-16, 8-20 or more,
10-16, 10-20 or more, or 16-20 or more.
[0019] In other preferred embodiments of the previously described
antibodies, the antibody binding to the antigen at pH 7.4 and at pH
25.degree. C. has a K.sub.D of about 0.01 nM to about 100 nM, or,
more preferably, at about 0.1 nM to about 10 nM.
[0020] In other preferred embodiments of the previously described
antibodies, the binding of the antibody to the antigen at pH 7.4
has a k.sub.off of about 1.times.10E-4 s-1 to about 1.times.10E-1
s-1, more preferably, about 1.times.10E-3 s-1 to about
1.times.10E-1 s-1.
[0021] In another preferred embodiment of the previously described
antibodies, the antigen is PCSK9. In one preferred embodiment, the
anti-PCSK9 antibody is not PCSK9 antibody H1M300N (see
US2010/0166768). In other preferred embodiments, the antigen is
IgE, C5, or DKK1 and, in preferred embodiments, the K.sub.D ranges
between 1.0 nM to about 10 nM or between 1.0 nM to about 100
nM.
[0022] The invention also provides a method of extending interval
dosing and/or decreasing the therapeutic dose for treating a
patient with a therapeutic antibody, said method comprising
administering to the patient a therapeutically effective amount of
the antibody of any of the previously described antibodies of the
invention, wherein said antibody has an extended pharmacodynamic
effect and/or half life as compared to an antibody that has similar
affinity at pH 7.4, but has a K.sub.D ratio and/or k.sub.off ratio
at pH 6.0/7.4 and at 25.degree. C. that is less than 2.
[0023] Also contemplated by the invention is a method of making an
antibody with prolonged half-life and/or pharmacodynamic effect by
regulating antibody binding affinity in a pH dependent manner, said
method comprising selecting for antibody CDR histidine residues or
other residues that optimize the microenvironment affecting pKa,
such that antibody antigen binding has a K.sub.D ratio and/or
k.sub.off ratio at pH 6.0/pH7.4 that is more than, or ranges
between, 2, 3, 4, 8, 10, 16, or more. The invention also
contemplates antibodies made by this method, including antibodies
with 1, 2, 3, 4, 5, or more histidine substitutions in CDR residues
that optimize the microenvironment affecting pKa.
[0024] In a preferred embodiment of the above-described method, the
method further comprises mutagenizing the antibody to achieve
antibody affinity with a K.sub.D at pH 7.4 of at least 100 nM as
measured at 25.degree. C. In another embodiment, the invention
provides for an antibody library enriched for histidines in CDR
residues or other residues that optimize the microenvironment
affecting pKa.
[0025] In other preferred embodiments, the invention provides an
isolated antibody which specifically binds to PCSK9 and comprises a
heavy chain variable region (VH) complementary determining region
one (CDR1), a VH CDR2, and a VH CDR3 from the VH amino acid
sequence shown in SEQ ID NO: 4 or 5 or a variant thereof having
one, two, three or more conservative amino acid substitutions in
CDR1, CDR2, and/or CDR3.
[0026] In a preferred embodiment, the antibody further comprises
the light chain variable region (VL) CDR1, CDR2, and CDR3 of the VL
amino acid sequence shown in SEQ ID NO: 3 or a variant thereof
having one, two, three or more conservative amino acid
substitutions in CDR1, CDR2, and/or CDR3.
[0027] The invention also provides for isolated antibody which
specifically binds to PCSK9 and comprises a heavy chain variable
region (VH) complementary determining region one (CDR1) having the
amino acid sequence shown in SEQ ID NO:6, a VH CDR2 having the
amino acid sequence shown in SEQ ID NO:7, and/or VH CDR3 having the
amino acid sequence shown in SEQ ID NO:8, or a variant thereof
having one or more conservative amino acid substitutions in CDR1,
CDR2, and/or CDR3, as well as an isolated antibody which
specifically binds PCSK9 and comprises a VH CDR1 having the amino
acid sequence shown in SEQ ID NO:6, a VH CDR2 having the amino acid
sequence shown in SEQ ID NO:7, and/or VH CDR3 having the amino acid
sequence shown in SEQ ID NO:9, or a variant thereof having one,
two, three or more conservative amino acid substitutions in CDR1,
CDR2, and/or CDR3.
[0028] In a further embodiment, the invention contemplates an
isolated antibody comprising a light chain variable region (VL)
CDR1 having the amino acid sequence shown in SEQ ID NO:10, a VL
CDR2 having the amino acid sequence shown in SEQ ID NO:11, and/or
VL CDR3 having the amino acid sequence shown in SEQ ID NO:12, or a
variant thereof having one, two, three or more conservative amino
acid substitutions in CDR1, CDR2, and/or CDR3.
[0029] In preferred embodiments of the above, the antibody further
comprises a VL CDR1 having the amino acid sequence shown in SEQ ID
NO:10, a VL CDR2 having the amino acid sequence shown in SEQ ID
NO:11, and/or VL CDR3 having the amino acid sequence shown in SEQ
ID NO:12, or a variant thereof having one, two, three or more
conservative amino acid substitutions in CDR1, CDR2, and/or CDR3,
preferably, the VH region comprises SEQ ID NO: 4 or SEQ ID NO: 5
and the VL region comprises SEQ ID NO: 3, or a variant thereof
having one, two, three or more conservative amino acid
substitutions in SEQ ID NO: 4, SEQ ID NO: 5 and/or SEQ ID NO:
3.
[0030] In another preferred embodiment of the PCSK9 antibodies of
the present invention, the antibody has one or more Fc mutations,
preferably, N434S, N434H, M428L-N434H double mutant, M428L-N434A
double mutant, T250Q-M428L double mutant, and M428L-N434S double
mutant
[0031] In another embodiment, the invention provides for an
antibody or antigen-binding portion thereof, encoded by the
plasmids deposited at the ATCC and having ATCC Accession No.
PTA-10547, or PTA-10548, and/or PTA-10549.
[0032] Also contemplated by the invention are pharmaceutical
compositions comprising a therapeutically effective amount of any
of the above described antibodies, a host cell that recombinantly
produces the antibody of any of the previously described
antibodies, an isolated nucleic acid encoding any of the previously
described antibodies, and an isolated nucleic acid encoding any of
the previously described antibodies.
[0033] Also contemplated by the invention is a method for reducing
a level of LDL-cholesterol in blood of a subject in need thereof,
comprising administering to the subject a therapeutically effective
amount of any of the antibodies of the invention targeting the
PCSK9 antigen.
BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS
[0034] FIG. 1 is a graph showing the increase in antibody
concentration over time as a function of the K.sub.D ratio.
[0035] FIG. 2 is a graph showing the decrease in free ligand
(antigen) over time as a function of the K.sub.D ratio.
[0036] FIG. 3A is a graph showing the effect of changing k.sub.off
on antibody concentration over time. FIG. 3B is a graph showing the
effect of changing k.sub.on on antibody concentration over
time.
[0037] FIG. 4A and FIG. 4B are heatmap displays showing how many
days longer an antibody with pH dependent binding would decrease
serum antigen concentration as a function of K.sub.D (R), serum
half-life of antigen and serum concentration of antigen. R is
equivalent to the K.sub.D ratio at endosomal pH versus physiologic
pH. FIG. 4A shows the heatmaps for K.sub.D at 0.1 nM and 1 nM. FIG.
4B shows the heatmaps for K.sub.D at 10 nM and 100 nM.
[0038] FIG. 5A and FIG. 5B validate the predictability of the pH
dependent antibody modeling. The model successfully predicted the
total antibody concentration for 5A10 (FIG. 5A). FIG. 5B is a graph
demonstrating the time course effect of 5A10 on LDL.
[0039] FIG. 6A and FIG. 6B also validate the predictability of the
pH dependent antibody modeling. The model successfully predicted
the total antibody concentration for 5L1721H23_6L3H3 (6L3H3) (FIG.
6A). FIG. 6B is a graph demonstrating the time course effect of
6L3H3 on LDL. The pH dependent binding antibody 6L3H3 extended the
interval in which LDL was lowered as compared to 5A10.
[0040] FIG. 7A and FIG. 7B show the time course of the total
cholesterol effect when administering various PCSK9 antibodies.
FIG. 7A shows the dose dependent effect of 5A10 on total
cholesterol. FIG. 7B shows the dose dependent effect of pH
dependent antibody 5L1721H23_6H3. This effect is extended as
compared to the effect of 5A10.
[0041] FIG. 8A is a graph which shows that antibodies with pH
dependent binding, 5L1721H23_6H3 and 5L1721H23_6L3H3, have reduced
antibody degradation and an extended half life as compared to
antibodies without pH dependent binding. FIG. 8B is a graph which
demonstrates that the effect shown in FIG. 8A is due to target
mediated degradation. Degradation of antibody in PCSK9 null mice
increased dramatically following injection of PCSK9.
[0042] FIG. 9A and FIG. 9B are graphs which illustrate the effect
of pH sensitive PCSK9 antagonist antibodies and non-pH sensitive
PCSK9 antagonist antibodies on cholesterol levels in monkeys. While
no dramatic change in HDL levels were detected (FIG. 9A), the pH
sensitive antibodies mediated a more prolonged reduction in LDL
levels (FIG. 9B) as compared to non-pH dependent antibody L1L3.
[0043] FIG. 10 is a graph demonstrating that the PCSK9 antibodies
with pH dependent binding had a prolonged half life in vivo as
compared to the non-pH dependent antibodies.
[0044] FIG. 11 is a heatmap showing the general modeling for pH
dependent binding. Such antibodies directed against antigens DKK1,
IgE, or C5, can significantly increase the number of days the
antigen experienced reduced levels of antigen as compared to an
antibody without pH dependent binding.
[0045] FIG. 12 models the time course for antigen concentration
following administration of an antibody with pH dependent binding
directed against antigen IgE.
[0046] FIG. 13 models the time course for antigen concentration
following administration of an antibody with pH dependent binding
directed against antigen DKK1.
[0047] FIG. 14 models the time course for antigen concentration
following administration of an antibody with pH dependent binding
directed against antigen C5.
[0048] FIG. 15 details the trafficking model for antibodies with pH
dependent binding used for modeling.
DETAILED DESCRIPTION OF THE INVENTION
[0049] The present invention relates to antibodies with pH
dependent binding to its antigen such that the affinity for antigen
binding at physiological pH (i.e., pH 7.4) is greater than at
endosomal pH (i.e., pH 6.0 or 5.5). In other words, the K.sub.D or
k.sub.off ratio at pH 5.5/pH 7.4 or at pH 6.0/pH 7.4 is more than,
or ranges between, 2, 3, 4, 8, 10, 16, 20, 30, 40, or 100 or more.
Such pH dependent antibodies preferentially dissociate from the
antigen in the endosome. This can increase antibody half life in
the circulation, as compared to antibodies with equivalent K.sub.Ds
at pH 7.4 but with no pH dependent binding, when the antigen is one
that undergoes antigen-mediated clearance (e.g., PCSK9). Antibodies
with pH dependent binding can decrease the average total antigen
half life when the antigen undergoes reduced clearance when bound
to antibody (e.g., IL6). Antibodies with pH dependent binding can
also prolong the decrease in antigen which is not antibody-bound.
This can be important when antagonizing a target antigen typically
present at high levels (e.g., IgE, DKK1, C5 and SOST). In addition,
such antibodies can increase antigen half life when the antigen is
a receptor and the receptor has increased clearance when bound to
antibody (e.g., GMCSF receptor).
[0050] If the antigen mediates target-mediated degradation, then
using such antibodies with pH dependent binding to achieve
dissociation in the endosome can increase the pharmacodynamic
effect of the antibody, for example, when the antigen undergoes
target-mediated clearance (e.g., PCSK9). The antibody with pH
dependent binding dissociates from the antigen, escapes
antigen-mediated degradation, can recycle out of the cell via FcRn
binding and will have a longer half-life than an antibody with
similar K.sub.D at pH 7.4 but with no pH dependent binding.
[0051] Using such antibodies with pH dependent binding is also
useful therapeutically when the soluble antigen is present at high
concentration (e.g., IgE, C5, DKK1, or SOST). Upon dissociation
from the antigen in the endosome and antigen degradation in the
lysozome, the antibody can recycle into the plasma to bind
additional free antigen, can prolong the decrease in non-antibody
bound antigen, and can decrease the therapeutic dose required, as
compared to an antibody with similar K.sub.D at pH 7.4 but without
pH dependent binding.
[0052] Additionally, using antibodies with pH dependent binding can
be useful when the antigen is present in membrane bound as well as
soluble form, e.g., a receptor, and it is desired to enhance
binding to the membrane bound form. By dissociating from the
soluble form, the antibody has the increased opportunity to re-bind
to the membrane form, increasing antibody in proximity to the cell
membrane. If bound to the membrane form in a divalent manner, the
effective affinity may be higher, or the effective dissociation
rate may be slower, through the effect of avidity.
[0053] This has application for using antibody-drug conjugates
(ADCs) when targeting an antigen present in both membrane-bound and
soluble form. In FcRn-containing cells in the endothelium, soluble
antigen will be cleared with the ADC recycling to the plasma
compartment, allowing for opportunity to bind membrane bound
antigen. With antibodies with pH dependent binding, increased
binding to the membrane bound form, either divalently or
monovalently, will cause increased antibody internalization with
the membrane bound antigen and cell death. If bound to the receptor
in a divalent matter, the avidity may increase the effective
affinity or slow the effective rate of dissociation.
[0054] The mechanism for ADCC and complement dependent cytotoxicity
(CDC) can also be exploited in using antibodies with pH dependent
binding. In FcRn-containing cells in the endothelium, soluble
antigen will be cleared and the ADC recycled to the plasma
compartment, allowing for the opportunity to bind membrane-bound
antigen. Liberating the antibodies from the soluble receptor will
increase the free antibody available that can then bind to membrane
bound antigen and increase cell killing.
General Techniques
[0055] 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.,
1993-1998) 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).
Definitions
[0056] An "antibody" 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 encompasses not
only intact polyclonal or monoclonal antibodies, but also any
antigen binding fragment thereof (i.e., "antigen-binding portion")
or single chain thereof, fusion proteins comprising an antibody,
and any other modified configuration of the immunoglobulin molecule
that comprises an antigen recognition site, including, for example
without limitation, single chain (scFv) and domain antibodies
(e.g., human, camelid, or shark domain antibodies), maxibodies,
minibodies, intrabodies, diabodies, triabodies, tetrabodies, vNAR
and bis-scFv (see e.g., Hollinger and Hudson, Nature Biotech 23:
1126-1136, 2005). An antibody includes an antibody of any class,
such as 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.
[0057] The term "antigen binding portion" of an antibody, as used
herein, refers to one or more fragments of an intact antibody that
retain the ability to specifically bind to a given antigen (e.g.,
target X). Antigen binding functions of an antibody can be
performed by fragments of an intact antibody. Examples of binding
fragments encompassed within the term "antigen binding portion" of
an antibody include Fab, Fab', F(ab').sub.2, an Fd fragment
consisting of the VH and CH1 domains, an Fv fragment consisting of
the VL and VH domains of a single arm of an antibody, a single
domain antibody (dAb) fragment (Ward et al., Nature 341:544-546,
1989), and an isolated complementarity determining region
(CDR).
[0058] As used herein, the "CDRs" may be defined in accordance with
any of Kabat, Chothia, extended, AbM, contact, and/or
conformational definitions. The identity of the amino acid residues
in a particular antibody that make up a CDR can be determined using
methods well known in the art. As used herein, antibody CDRs may be
identified as the hypervariable regions originally defined by Kabat
et al. See, e.g., Kabat et al., 1992, Sequences of Proteins of
Immunological Interest, 5th ed., Public Health Service, NIH,
Washington D.C. The positions of the CDRs may also be identified as
the structural loop structures originally described by Chothia and
others. See, e.g., Chothia et al., Nature 342:877-883, 1989. Other
approaches to CDR identification include the "AbM definition,"
which is a compromise between Kabat and Chothia and is derived
using Oxford Molecular's AbM antibody modeling software (now
Accelrys.RTM.), or the "contact definition" of CDRs based on
observed antigen contacts, set forth in MacCallum et al., J. Mol.
Biol. 262:732-745, 1996. In another approach, referred to herein as
the "conformational definition" of CDRs, the positions of the CDRs
may be identified as the residues that make enthalpic contributions
to antigen binding. See, e.g., Makabe et al., Journal of Biological
Chemistry, 283:1156-1166, 2008. Still other CDR boundary
definitions may not strictly follow one of the above approaches,
but will nonetheless overlap with at least a portion of the Kabat
CDRs, although they may be shortened or lengthened in light of
prediction or experimental findings that particular residues or
groups of residues or even entire CDRs do not significantly impact
antigen binding. As used herein, a CDR may refer to CDRs defined by
any approach known in the art, including combinations of
approaches.
[0059] As used herein, "monoclonal antibody" refers to an antibody
obtained from a population of substantially homogeneous antibodies,
i.e., the individual antibodies comprising the population are
identical except for possible naturally-occurring mutations that
may be present in minor amounts. Monoclonal antibodies are highly
specific, being directed against a single antigenic site.
Furthermore, in contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. The modifier
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies to
be used in accordance with the present invention may be made by the
hybridoma method first described by Kohler and Milstein, 1975,
Nature 256:495, or may be made by recombinant DNA methods such as
described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may
also be isolated from phage libraries generated using the
techniques described in McCafferty et al., 1990, Nature
348:552-554, for example.
[0060] As used herein, "humanized" antibody refers to forms of
non-human (e.g., murine) antibodies that are chimeric
immunoglobulins, immunoglobulin chains, or fragments thereof (such
as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding
subsequences of antibodies) that contain minimal sequence derived
from non-human immunoglobulin. Preferably, 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.
Preferred are antibodies having Fc regions modified as described in
WO 99/58572. Other forms of humanized antibodies have one or more
CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and/or CDR H3) 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.
[0061] As used herein, "human antibody" means an antibody having an
amino acid sequence corresponding to that of an antibody that can
be produced by a human and/or which has been made using any of the
techniques for making human antibodies known to those skilled in
the art or disclosed herein. This definition of a human antibody
includes antibodies comprising at least one human heavy chain
polypeptide or at least one human light chain polypeptide. One such
example is an antibody comprising murine light chain and human
heavy chain polypeptides. Human antibodies can be produced using
various techniques known in the art. In one embodiment, the human
antibody is selected from a phage library, where that phage library
expresses human antibodies (Vaughan et al., 1996, Nature
Biotechnology, 14:309-314; Sheets et al., 1998, Proc. Natl. Acad.
Sci. (USA) 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol.
Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581). Human
antibodies can also be made by immunization of animals into which
human immunoglobulin loci have been transgenically introduced in
place of the endogenous loci, e.g., mice in which the endogenous
immunoglobulin genes have been partially or completely inactivated.
This approach is described in U.S. Pat. Nos. 5,545,807; 5,545,806;
5,569,825; 5,625,126; 5,633,425; and 5,661,016. Alternatively, the
human antibody may be prepared by immortalizing human B lymphocytes
that produce an antibody directed against a target antigen (such B
lymphocytes may be recovered from an individual or may have been
immunized in vitro). See, e.g., Cole et al. Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al.,
1991, J. Immunol., 147 (1):86-95; and U.S. Pat. No. 5,750,373.
[0062] A "variable region" of an antibody refers to the variable
region of the antibody light chain or the variable region of the
antibody heavy chain, either alone or in combination. As known in
the art, the variable regions of the heavy and light chain each
consist of four framework regions (FR) connected by three
complementarity determining regions (CDRs) that contain
hypervariable regions. The CDRs in each chain are held together in
close proximity by the FRs and, with the CDRs from the other chain,
contribute to the formation of the antigen-binding site of
antibodies. There are at least two techniques for determining CDRs:
(1) an approach based on cross-species sequence variability (i.e.,
Kabat et al. Sequences of Proteins of Immunological Interest, (5th
ed., 1991, National Institutes of Health, Bethesda Md.)); and (2)
an approach based on crystallographic studies of antigen-antibody
complexes (Al-lazikani et al, 1997, J. Molec. Biol. 273:927-948).
As used herein, a CDR may refer to CDRs defined by either approach
or by a combination of both approaches.
[0063] As known in the art a "constant region" of an antibody
refers to the constant region of the antibody light chain or the
constant region of the antibody heavy chain, either alone or in
combination.
[0064] As used herein, the term "PCSK9" refers to any form of PCSK9
and variants thereof that retain at least part of the activity of
PCSK9. Unless indicated differently, such as by specific reference
to human PCSK9, PCSK9 includes all mammalian species of native
sequence PCSK9, e.g., human, canine, feline, equine, and bovine.
One exemplary human PCSK9 is found as Uniprot Accession Number
Q8NBP7.
[0065] As used herein, a "PCSK9 antagonist antibody" refers to an
antibody that is able to inhibit PCSK9 biological activity and/or
downstream pathway(s) mediated by PCSK9 signaling, including
PCSK9-mediated down-regulation of the LDLR, and PCSK9-mediated
decrease in LDL blood clearance. A pH dependent PCSK9 antagonist
antibody encompasses antibodies that block, antagonize, suppress or
reduce (to any degree including significantly) PCSK9 biological
activity, including downstream pathways mediated by PCSK9
signaling, such as LDLR interaction and/or elicitation of a
cellular response to PCSK9. For purpose of the present invention,
it will be explicitly understood that the term "PCSK9 antagonist
antibody" encompasses all the previously identified terms, titles,
and functional states and characteristics whereby the PCSK9 itself,
a PCSK9 biological activity (including but not limited to its
ability to mediate any aspect of interaction with the LDLR, down
regulation of LDLR, and decreased blood LDL clearance), or the
consequences of the biological activity, are substantially
nullified, decreased, or neutralized in any meaningful degree. In
some embodiments, a pH dependent PCSK9 antagonist antibody binds
PCSK9 and prevents interaction with the LDLR. Examples of PCSK9
antagonist antibodies are provided herein.
[0066] The terms "polypeptide", "oligopeptide", "peptide" and
"protein" are used interchangeably herein to refer to chains of
amino acids of any length, preferably, relatively short (e.g.,
10-100 amino acids). The chain may be linear or branched, it may
comprise modified amino acids, and/or may be interrupted by
non-amino acids. The terms also encompass an amino acid chain that
has been modified naturally or by intervention; for example,
disulfide bond formation, glycosylation, lipidation, acetylation,
phosphorylation, or any other manipulation or modification, such as
conjugation with a labeling component. Also included within the
definition are, for example, polypeptides containing one or more
analogs of an amino acid (including, for example, unnatural amino
acids, etc.), as well as other modifications known in the art. It
is understood that the polypeptides can occur as single chains or
associated chains.
[0067] As known in the art, "polynucleotide," or "nucleic acid," as
used interchangeably herein, refer to chains of nucleotides of any
length, and include DNA and RNA. The nucleotides can be
deoxyribonucleotides, ribonucleotides, modified nucleotides or
bases, and/or their analogs, or any substrate that can be
incorporated into a chain by DNA or RNA polymerase. A
polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and their analogs. If present, modification
to the nucleotide structure may be imparted before or after
assembly of the chain. The sequence of nucleotides may be
interrupted by non-nucleotide components. A polynucleotide may be
further modified after polymerization, such as by conjugation with
a labeling component. Other types of modifications include, for
example, "caps", substitution of one or more of the naturally
occurring nucleotides with an analog, internucleotide modifications
such as, for example, those with uncharged linkages (e.g., methyl
phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.)
and with charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), those containing pendant moieties, such
as, for example, proteins (e.g., nucleases, toxins, antibodies,
signal peptides, poly-L-lysine, etc.), those with intercalators
(e.g., acridine, psoralen, etc.), those containing chelators (e.g.,
metals, radioactive metals, boron, oxidative metals, etc.), those
containing alkylators, those with modified linkages (e.g., alpha
anomeric nucleic acids, etc.), as well as unmodified forms of the
polynucleotide(s). Further, any of the hydroxyl groups ordinarily
present in the sugars may be replaced, for example, by phosphonate
groups, phosphate groups, protected by standard protecting groups,
or activated to prepare additional linkages to additional
nucleotides, or may be conjugated to solid supports. The 5' and 3'
terminal OH can be phosphorylated or substituted with amines or
organic capping group moieties of from 1 to 20 carbon atoms. Other
hydroxyls may also be derivatized to standard protecting groups.
Polynucleotides can also contain analogous forms of ribose or
deoxyribose sugars that are generally known in the art, including,
for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or
2'-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric
sugars, epimeric sugars such as arabinose, xyloses or lyxoses,
pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs
and abasic nucleoside analogs such as methyl riboside. One or more
phosphodiester linkages may be replaced by alternative linking
groups. These alternative linking groups include, but are not
limited to, embodiments wherein phosphate is replaced by
P(O)S("thioate"), P(S)S ("dithioate"), (O)NR.sub.2 ("amidate"),
P(O)R, P(O)OR', CO or CH.sub.2 ("formacetal"), in which each R or
R' is independently H or substituted or unsubstituted alkyl (1-20
C) optionally containing an ether (--O--) linkage, aryl, alkenyl,
cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a
polynucleotide need be identical. The preceding description applies
to all polynucleotides referred to herein, including RNA and
DNA.
[0068] An antibody "specifically binds" or "preferentially binds"
to a target 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 a PCSK9 epitope is an antibody that binds
this epitope with greater affinity, avidity, more readily, and/or
with greater duration than it binds to other PCSK9 epitopes or
non-PCSK9 epitopes. It is also understood by reading this
definition that, for example, an antibody (or moiety or epitope)
that specifically or preferentially binds to a first target may or
may not specifically or preferentially bind to a second target. 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.
[0069] A "non-signalling decoy" is a soluble receptor isoform or a
binding protein that sequesters ligand from its cognate
receptor(s).
[0070] As used herein, "substantially pure" refers to material
which is at least 50% pure (i.e., free from contaminants), more
preferably, at least 90% pure, more preferably, at least 95% pure,
yet more preferably, at least 98% pure, and most preferably, at
least 99% pure.
[0071] A "host cell" includes an individual cell or cell culture
that can be or has been a recipient for vector(s) for incorporation
of polynucleotide inserts. Host cells include progeny of a single
host cell, and the progeny may not necessarily be completely
identical (in morphology or in genomic DNA complement) to the
original parent cell due to natural, accidental, or deliberate
mutation. A host cell includes cells transfected in vivo with a
polynucleotide(s) of this invention.
[0072] As known in the art, the term "Fc region" is used to define
a C-terminal region of an immunoglobulin heavy chain. The "Fc
region" may be a native sequence Fc region or a variant Fc region.
Although the boundaries of the Fc region of an immunoglobulin heavy
chain might vary, the human IgG heavy chain Fc region is usually
defined to stretch from an amino acid residue at position Cys226,
or from Pro230, to the carboxyl-terminus thereof. The numbering of
the residues in the Fc region is that of the EU index as in Kabat.
Kabat et al., Sequences of Proteins of Immunological Interest, 5th
Ed. Public Health Service, National Institutes of Health, Bethesda,
Md., 1991. The Fc region of an immunoglobulin generally comprises
two constant domains, CH2 and CH3.
[0073] As used in the art, "Fc receptor" and "FcR" describe a
receptor that binds to the Fc region of an antibody. The preferred
FcR is a native sequence human FcR. Moreover, a preferred FcR is
one which binds an IgG antibody (a gamma receptor) and includes
receptors of the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII
subclasses, including allelic variants and alternatively spliced
forms of these receptors. Fc.gamma.RII receptors include
Fc.gamma.RIIA (an "activating receptor") and Fc.gamma.RIIB (an
"inhibiting receptor"), which have similar amino acid sequences
that differ primarily in the cytoplasmic domains thereof. FcRs are
reviewed in Ravetch and Kinet, 1991, Ann. Rev. Immunol., 9:457-92;
Capel et al., 1994, Immunomethods, 4:25-34; and de Haas et al.,
1995, J. Lab. Clin. Med., 126:330-41. "FcR" also includes the
neonatal receptor, FcRn, which is responsible for the transfer of
maternal IgGs to the fetus (Guyer et al., 1976 J. Immunol.,
117:587; and Kim et al., 1994, J. Immunol., 24:249).
[0074] The term "compete", as used herein with regard to an
antibody, means that a first antibody, or an antigen-binding
portion thereof, binds to an epitope in a manner sufficiently
similar to the binding of a second antibody, or an antigen-binding
portion thereof, such that the result of binding of the first
antibody with its cognate epitope is detectably decreased in the
presence of the second antibody compared to the binding of the
first antibody in the absence of the second antibody. The
alternative, where the binding of the second antibody to its
epitope is also detectably decreased in the presence of the first
antibody, can, but need not be the case. That is, a first antibody
can inhibit the binding of a second antibody to its epitope without
that second antibody inhibiting the binding of the first antibody
to its respective epitope. However, where each antibody detectably
inhibits the binding of the other antibody with its cognate epitope
or ligand, whether to the same, greater, or lesser extent, the
antibodies are said to "cross-compete" with each other for binding
of their respective epitope(s). Both competing and cross-competing
antibodies are encompassed by the present invention. Regardless of
the mechanism by which such competition or cross-competition occurs
(e.g., steric hindrance, conformational change, or binding to a
common epitope, or portion thereof), the skilled artisan would
appreciate, based upon the teachings provided herein, that such
competing and/or cross-competing antibodies are encompassed and can
be useful for the methods disclosed herein.
[0075] A "functional Fc region" possesses at least one effector
function of a native sequence Fc region. Exemplary "effector
functions" include C1q binding; CDC; Fc receptor binding;
antibody-dependent cell-mediated cytotoxicity; phagocytosis;
down-regulation of cell surface receptors (e.g., B cell receptor),
etc. Such effector functions generally require the Fc region to be
combined with a binding domain (e.g., an antibody variable domain)
and can be assessed using various assays known in the art for
evaluating such antibody effector functions.
[0076] A "native sequence Fc region" comprises an amino acid
sequence identical to the amino acid sequence of an Fc region found
in nature. A "variant Fc region" comprises an amino acid sequence
which differs from that of a native sequence Fc region by virtue of
at least one amino acid modification, yet retains at least one
effector function of the native sequence Fc region. Preferably, the
variant Fc region has at least one amino acid substitution compared
to a native sequence Fc region or to the Fc region of a parent
polypeptide, e.g., from about one to about ten amino acid
substitutions, and preferably, from about one to about five amino
acid substitutions in a native sequence Fc region or in the Fc
region of the parent polypeptide. The variant Fc region herein will
preferably possess at least about 80% sequence identity with a
native sequence Fc region and/or with an Fc region of a parent
polypeptide, and most preferably, at least about 90% sequence
identity therewith, more preferably, at least about 95%, at least
about 96%, at least about 97%, at least about 98%, at least about
99% sequence identity therewith.
[0077] By "Minimal Anticipated Biological Effect Level (MABEL)" is
meant the minimal anticipated dose level leading to a minimal
biological effect in humans. Safety factors are usually applied for
the calculation for the first dose in man from MABEL. The
calculation of MABEL should utilize all relevant in vitro and in
vivo pharmacokinetic and pharmacodynamic information.
[0078] As used herein, "treatment" and "therapeutically effective"
are approaches for obtaining beneficial or desired clinical
results. For purposes of this invention related to pH dependent
PCSK9 antagonist antibodies, beneficial or desired clinical results
include, but are not limited to, one or more of the following:
enhancement of LDL clearance and reducing incidence or amelioration
of aberrant cholesterol and/or lipoprotein levels resulting from
metabolic and/or eating disorders, or including familial
hypercholesterolemia, atherogenic dyslipidemia, atherosclerosis,
and, more generally, cardiovascular disease (CVD).
[0079] "Reducing incidence" means any of reducing severity (which
can include reducing need for and/or amount of (e.g., exposure to)
other drugs and/or therapies generally used for this condition. As
is understood by those skilled in the art, individuals may vary in
terms of their response to treatment, and, as such, for example, a
"method of reducing incidence" reflects administering the pH
dependent antibody based on a reasonable expectation that such
administration may likely cause such a reduction in incidence in
that particular individual.
[0080] "Ameliorating" means a lessening or improvement of one or
more symptoms after administering a treatment as compared to not
administering a treatment. "Ameliorating" also includes shortening
or reduction in duration of a symptom.
[0081] As used herein, an "effective dosage" or "effective amount"
of drug, compound, or pharmaceutical composition is an amount
sufficient to affect any one or more beneficial or desired results.
For prophylactic use, beneficial or desired results include
eliminating or reducing the risk, lessening the severity, or
delaying the outset of the disease, including biochemical,
histological and/or behavioral symptoms of the disease, its
complications and intermediate pathological phenotypes presenting
during development of the disease. For therapeutic use of a pH
dependent PCSK9 antagonist antibody, beneficial or desired results
include clinical results such as reducing hypercholesterolemia or
one or more symptoms of dyslipidemia, atherosclerosis, CVD, or
coronary heart disease, decreasing the dose of other medications
required to treat the disease, enhancing the effect of another
medication, and/or delaying the progression of the disease of
patients. An effective dosage can be administered in one or more
administrations. For purposes of this invention, an effective
dosage of drug, compound, or pharmaceutical composition is an
amount sufficient to accomplish prophylactic or therapeutic
treatment either directly or indirectly. As is understood in the
clinical context, an effective dosage of a drug, compound, or
pharmaceutical composition may or may not be achieved in
conjunction with another drug, compound, or pharmaceutical
composition. Thus, an "effective dosage" may be considered in the
context of administering one or more therapeutic agents, and a
single agent may be considered to be given in an effective amount
if, in conjunction with one or more other agents, a desirable
result may be or is achieved.
[0082] An "individual" or a "subject" is a mammal, more preferably,
a human. Mammals also include, but are not limited to, farm
animals, sport animals, pets, primates, horses, dogs, cats, mice
and rats.
[0083] As used herein, "vector" means a construct, which is capable
of delivering, and, preferably, expressing, one or more gene(s) or
sequence(s) of interest in a host cell. Examples of vectors
include, but are not limited to, viral vectors, naked DNA or RNA
expression vectors, plasmid, cosmid or phage vectors, DNA or RNA
expression vectors associated with cationic condensing agents, DNA
or RNA expression vectors encapsulated in liposomes, and certain
eukaryotic cells, such as producer cells.
[0084] As used herein, "expression control sequence" means a
nucleic acid sequence that directs transcription of a nucleic acid.
An expression control sequence can be a promoter, such as a
constitutive or an inducible promoter, or an enhancer. The
expression control sequence is operably linked to the nucleic acid
sequence to be transcribed.
[0085] As used herein, "pharmaceutically acceptable carrier" or
"pharmaceutical acceptable excipient" includes any material which,
when combined with an active ingredient, allows the ingredient to
retain biological activity and is non-reactive with the subject's
immune system. Examples include, but are not limited to, any of the
standard pharmaceutical carriers such as a phosphate buffered
saline solution, water, emulsions such as oil/water emulsion, and
various types of wetting agents. Preferred diluents for aerosol or
parenteral administration are phosphate buffered saline (PBS) or
normal (0.9%) saline. Compositions comprising such carriers are
formulated by well known conventional methods (see, for example,
Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed.,
Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science
and Practice of Pharmacy, 20th Ed., Mack Publishing, 2000).
[0086] The term "k.sub.on", as used herein, refers to the rate
constant for association of an antibody to an antigen.
Specifically, the rate constants (k.sub.on and k.sub.off) and
equilibrium dissociation constants are measured using Fab antibody
fragments (i.e., univalent) and antigen.
[0087] The term "k.sub.off", as used herein, refers to the rate
constant for dissociation of an antibody from the antibody/antigen
complex.
[0088] The term "K.sub.D", as used herein, refers to the
equilibrium dissociation constant of an antibody-antigen
interaction.
[0089] Determinations of the association and dissociation rate
constants, k.sub.a and k.sub.d respectively, to determine K.sub.D
and k.sub.off ratios, are made using a surface plasmon
resonance-based biosensor to characterize an analyte/ligand
interaction under conditions where the analyte is monovalent with
respect to binding a ligand that is immobilized at low capacity
onto a sensor surface via a capture reagent. The analysis is
performed using a kinetic titration methodology as described in
Karlsson et al., Anal. Biochem 349, 136-147, 2006. The sensor chip,
capturing reagent, and assay buffer employed for a given assay are
chosen to give stable capture of ligand onto the sensor surface,
minimize non-specific binding of the analyte to the surfaces, and
yield analyte-binding responses that are appropriate for kinetic
analysis, per the recommendations in Myszka, J. Mol. Recognit 12,
279-284, 1999. The analyte-binding responses per analyte/ligand
interaction are double referenced and fit to a 1:1 Langmuir "mass
transport limited model" with k.sub.a, k.sub.d and R.sub.max as
global parameters as described in Myszka & Morton et al.,
Biophys. Chem 64, 127-137 (1997). The equilibrium dissociation
constant, K.sub.D, is deduced from the ratio of the kinetic rate
constants, K.sub.D=k.sub.d/k.sub.a. Such determinations preferably
take place at 25.degree. C. or 37.degree. C.
A. Methods for Preventing or Treating Disorders
[0090] In one aspect regarding pH dependent PCSK9 antagonist
antibodies, the invention provides a method for treating or
preventing hypercholesterolemia, and/or at least one symptom of
dyslipidemia, atherosclerosis, CVD or coronary heart disease, in an
individual comprising administering to the individual an effective
amount of a PH dependent pH dependent PCSK9 antagonist antibody
that antagonizes circulating PCSK9.
[0091] In a further aspect, the invention provides an effective
amount of a pH dependent PCSK9 antagonist antibody that antagonizes
circulating PCSK9 for use in treating or preventing
hypercholesterolemia, and/or at least one symptom of dyslipidemia,
atherosclerosis, CVD or coronary heart disease, in an individual.
The invention further provides the use of an effective amount of a
pH dependent PCSK9 antagonist antibody that antagonizes
extracellular or circulating PCSK9 in the manufacture of a
medicament for treating or preventing hypercholesterolemia, and/or
at least one symptom of dyslipidemia, atherosclerosis, CVD or
coronary heart disease, in an individual.
[0092] Advantageously, therapeutic administration of the antibody
results in lower blood cholesterol and/or lower blood LDL.
Preferably, blood cholesterol and/or blood LDL is at least about
10% or 15% lower than before administration. More preferably, blood
cholesterol and/or blood LDL is at least about 20% lower than
before administration of the antibody. Yet more preferably, blood
cholesterol and/or blood LDL is at least 30% lower than before
administration of the antibody. Advantageously, blood cholesterol
and/or blood LDL is at least 40% lower than before administration
of the antibody. More advantageously, blood cholesterol and/or
blood LDL is at least 50% lower than before administration of the
antibody. Very preferably, blood cholesterol and/or blood LDL is at
least 60% lower than before administration of the antibody. Most
preferably, blood cholesterol and/or blood LDL is at least 70%
lower than before administration of the antibody.
[0093] With respect to all methods described herein, reference to
pH dependent antibodies against any appropriate antigen also
includes compositions comprising one or more additional agents.
These compositions may further comprise suitable excipients, such
as pharmaceutically acceptable excipients including buffers, which
are well known in the art. The present invention can be used alone
or in combination with other conventional methods of treatment.
[0094] The pH dependent antibody can be administered to an
individual via any suitable route. It should be apparent to a
person skilled in the art that the examples described herein are
not intended to be limiting but to be illustrative of the
techniques available. Accordingly, in some embodiments, the pH
dependent antibody is administered to an individual in accord with
known methods, such as intravenous administration, e.g., as a bolus
or by continuous infusion over a period of time, by intramuscular,
intraperitoneal, intracerebrospinal, transdermal, subcutaneous,
intra-articular, sublingually, intrasynovial, via insufflation,
intrathecal, oral, inhalation or topical routes. Administration can
be systemic, e.g., intravenous administration, or localized.
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, pH dependent antibody can be aerosolized using a
fluorocarbon formulation and a metered dose inhaler, or inhaled as
a lyophilized and milled powder.
[0095] In one embodiment, a pH dependent 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 pH dependent antibody or
local delivery catheters, such as infusion catheters, indwelling
catheters, or needle catheters, synthetic grafts, adventitial
wraps, shunts and stents or other implantable devices, site
specific carriers, direct injection, or direct application. See,
e.g., PCT Publ. No. WO 00/53211 and U.S. Pat. No. 5,981,568.
[0096] Various formulations of a pH dependent antibody may be used
for administration. In some embodiments, the pH dependent antibody
may be administered neat. In some embodiments, pH dependent
antibody and a pharmaceutically acceptable excipient may be in
various formulations. Pharmaceutically acceptable excipients are
known in the art, and are relatively inert substances that
facilitate administration of a pharmacologically effective
substance. For example, an excipient can give form or consistency,
or act as a diluent. Suitable excipients include but are not
limited to stabilizing agents, wetting and emulsifying agents,
salts for varying osmolarity, encapsulating agents, buffers, and
skin penetration enhancers. Excipients as well as formulations for
parenteral and nonparenteral drug delivery are set forth in
Remington, The Science and Practice of Pharmacy, 20th Ed., Mack
Publishing (2000).
[0097] These agents can be combined with pharmaceutically
acceptable vehicles such as saline, Ringer's solution, dextrose
solution, and the like. The particular dosage regimen, i.e., dose,
timing and repetition, will depend on the particular individual and
that individual's medical history.
[0098] Antibodies with pH dependent binding can also be
administered via inhalation, as described herein. Generally, for
administration of pH dependent antibodies, an initial candidate
dosage can be about 2 mg/kg. For the purpose of the present
invention, a typical daily dosage might range from about any of
about 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 example, dosage of about 1 mg/kg, about 2.5 mg/kg, about
5 mg/kg, about 10 mg/kg, and about 25 mg/kg may be used. 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, for example, to reduce blood LDL levels. 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, in some embodiments,
dosing from one to four times a week is contemplated. In other
embodiments dosing once a month or once every other month or every
three months is contemplated. The progress of this therapy is
easily monitored by conventional techniques and assays. The dosing
regimen (including the antibody used) can vary over time.
[0099] For the purpose of the present invention, the appropriate
dosage of a pH dependent antibody will depend on the antibody (or
compositions thereof) employed, the type and severity of symptoms
to be treated, whether the agent is administered for preventive or
therapeutic purposes, previous therapy, the patient's clinical
history and response to the agent, the patient's blood antigen
levels, the patient's synthesis and clearance rate for antigen, the
patient's clearance rate for the administered agent, and the
discretion of the attending physician. Typically the clinician will
administer a pH dependent antibody until a dosage is reached that
achieves the desired result. Dose and/or frequency can vary over
course of treatment. 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
symptoms, e.g., hypercholesterolemia. Alternatively, sustained
continuous release formulations of antibodies may be appropriate.
Various formulations and devices for achieving sustained release
are known in the art.
[0100] In one embodiment, dosages for an antagonist antibody may be
determined empirically in individuals who have been given one or
more administration(s) of an antagonist antibody. Individuals are
given incremental dosages of a antibody. To assess efficacy, an
indicator of the disease can be followed.
[0101] Administration of a pH dependent antibody in accordance with
the method in the present invention 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 a pH dependent antibody may be
essentially continuous over a preselected period of time or may be
in a series of spaced doses.
[0102] In some embodiments, more than one antagonist antibody may
be present. At least one, at least two, at least three, at least
four, at least five different, or more antagonist antibodies and/or
peptides can be present. Generally, those antibodies or peptides
may have complementary activities that do not adversely affect each
other. A pH dependent antibody can also be used in conjunction with
other therapeutics. A pH dependent antibody can also be used in
conjunction with other agents that serve to enhance and/or
complement the effectiveness of the agents.
[0103] Acceptable carriers, excipients, or stabilizers are nontoxic
to recipients at the dosages and concentrations employed, and may
comprise buffers such as phosphate, citrate, and other organic
acids; salts such as sodium chloride; antioxidants including
ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens, such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.,
Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
[0104] Liposomes containing the pH dependent antibody are prepared
by methods known in the art, such as described in Epstein, et al.,
1985, Proc. Natl. Acad. Sci. USA 82:3688; Hwang, et al., 1980,
Proc. Natl Acad. Sci. USA 77:4030; 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.
[0105] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacrylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington, The Science and Practice of
Pharmacy, 20th Ed., Mack Publishing (2000).
[0106] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules. 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.
[0107] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by, for example,
filtration through sterile filtration membranes. Therapeutic pH
dependent 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.
[0108] 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 .mu.m, particularly 0.1
and 0.5 .mu.m, and have a pH in the range of 5.5 to 8.0.
[0109] The emulsion compositions can be those prepared by mixing a
pH dependent antibody with Intralipid.TM. or the components thereof
(soybean oil, egg phospholipids, glycerol and water).
[0110] 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. 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.
B. pH Dependent Antibodies
[0111] The antibodies useful in the present invention can encompass
monoclonal antibodies, polyclonal antibodies, antibody fragments
(e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies,
bispecific antibodies, heteroconjugate antibodies, single chain
(ScFv), mutants thereof, fusion proteins comprising an antibody
portion (e.g., a domain antibody), human antibodies, humanized
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. The antibodies may be murine, rat,
human, or any other origin (including chimeric or humanized
antibodies).
[0112] In some embodiments, the pH dependent antibody is a
monoclonal antibody. The pH dependent antibody can also be
humanized. In other embodiments, the antibody is human.
[0113] In some embodiments, the antibody comprises a modified
constant region, such as a constant region that is immunologically
inert, that is, having a reduced potential for provoking an immune
response. In some embodiments, the constant region is modified as
described in Eur. J. Immunol., 1999, 29:2613-2624; PCT Publ. No.
WO99/58572; and/or UK Patent Application No. 9809951.8. The Fc can
be human IgG.sub.2 or human IgG.sub.4. The Fc can be human
IgG.sub.2 containing the mutation A330P331 to S330S331
(IgG.sub.2.DELTA.a), in which the amino acid residues are numbered
with reference to the wild type IgG2 sequence. Eur. J. Immunol.,
1999, 29:2613-2624. In some embodiments, the antibody comprises a
constant region of IgG.sub.4 comprising the following mutations
(Armour et al., 2003, Molecular Immunology 40 585-593):
E233F234L235 to P233V234A235 (IgG.sub.4.DELTA.c), in which the
numbering is with reference to wild type IgG4. In yet another
embodiment, the Fc is human IgG.sub.4 E233F234L235 to P233V234A235
with deletion G236 (IgG.sub.4.DELTA.b). In another embodiment the
Fc is any human IgG.sub.4 Fc (IgG.sub.4, IgG.sub.4.DELTA.b or
IgG.sub.4.DELTA.c) containing hinge stabilizing mutation S228 to
P228 (Aalberse et al., 2002, Immunology 105, 9-19). In another
embodiment, the Fc can be aglycosylated Fc.
[0114] In some embodiments, the constant region is aglycosylated by
mutating the oligosaccharide attachment residue (such as Asn297)
and/or flanking residues that are part of the glycosylation
recognition sequence in the constant region. In some embodiments,
the constant region is aglycosylated for N-linked glycosylation
enzymatically. The constant region may be aglycosylated for
N-linked glycosylation enzymatically or by expression in a
glycosylation deficient host cell.
[0115] One way of determining binding affinity of antibodies to
antigen is by measuring binding affinity of monofunctional Fab
fragments of the antibody. To obtain monofunctional Fab fragments,
an antibody (for example, IgG) can be cleaved with papain or
expressed recombinantly. The affinity of a Fab fragment of an
antibody can be determined by surface plasmon resonance
(Biacore3000.TM. surface plasmon resonance (SPR) system, Biacore,
INC, Piscataway N.J.) equipped with pre-immobilized streptavidin
sensor chips (SA) using HBS-EP running buffer (0.01M HEPES, pH 7.4,
0.15 NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20). Biotinylated
antigen can be diluted into HBS-EP buffer to a concentration of
less than 0.5 .mu.g/mL and injected across the individual chip
channels using variable contact times, to achieve two ranges of
antigen density, either 50-200 response units (RU) for detailed
kinetic studies or 800-1,000 RU for screening assays. Regeneration
studies have shown that 25 mM NaOH in 25% v/v ethanol effectively
removes the bound Fab while keeping the activity of antigen on the
chip for over 200 injections. Typically, serial dilutions (spanning
concentrations of 0.1-10.times. estimated K.sub.D) of purified Fab
samples are injected for 1 min at 100 .mu.L/minute and dissociation
times of up to 2 hours are allowed. The concentrations of the Fab
proteins are determined by ELISA and/or SDS-PAGE electrophoresis
using a Fab of known concentration (as determined by amino acid
analysis) as a standard. Kinetic association rates (k.sub.on) and
dissociation rates (k.sub.off) are obtained simultaneously by
fitting the data globally to a 1:1 Langmuir binding model
(Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods
Enzymology 6. 99-110) using the BIAevaluation program. Equilibrium
dissociation constant (K.sub.D) values are calculated as
k.sub.off/k.sub.on. This protocol is suitable for use in
determining binding affinity of an antibody to any antigen,
including human or another mammalian species (such as mouse, rat,
primate). Binding affinity of an antibody is generally measured at
25.degree. C., but can also be measured at 37.degree. C.
[0116] The antibodies may be made by any method known in the art.
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 human and mouse
antibodies are known in the art and/or are described herein. For pH
dependent antibodies against PCSK9, a currently preferred method of
making the antibodies comprises the immunization of PCSK9 knockout
(PCSK9-/-) animals as disclosed herein.
[0117] 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.
[0118] 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., 1982, In
Vitro, 18:377-381. Available myeloma lines, including but not
limited to X63-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 PCSK9 monoclonal antibodies of the
subject invention. 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).
[0119] Hybridomas that may be used as a source of antibodies
encompass all derivatives, progeny cells of the parent hybridomas
that produce monoclonal antibodies specific for PCSK9, or a portion
thereof.
[0120] 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 human PCSK9, 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 trypsin 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.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R and R.sup.1 are
different alkyl groups, can yield a population of antibodies (e.g.,
monoclonal antibodies).
[0121] Once antibodies are generated or selected, the antibodies
can be optimized for pH dependent binding, for example, as
disclosed in Example 1 herein.
[0122] If desired, the pH dependent antibody (monoclonal or
polyclonal) of interest 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. Production of recombinant
monoclonal antibodies in cell culture can be carried out through
cloning of antibody genes from B cells by means known in the art.
See, e.g., Tiller et al., 2008, J. Immunol. Methods 329, 112; U.S.
Pat. No. 7,314,622.
[0123] In an alternative, the polynucleotide sequence may be used
for genetic manipulation to "humanize" the antibody or to improve
the affinity, or other characteristics of the antibody. For
example, the constant region may be engineered to more nearly
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 antigen and greater efficacy. It will be
apparent to one of skill in the art that one or more polynucleotide
changes can be made to the pH dependent antibody and still maintain
its antigen binding ability.
[0124] There are four general steps to humanize a monoclonal
antibody. These are: (1) determining the nucleotide and predicted
amino acid sequence of the starting antibody light and heavy
variable domains; (2) designing the humanized antibody, i.e.,
deciding which antibody framework region to use during the
humanizing process; (3) the actual humanizing
methodologies/techniques; and (4) the transfection and expression
of the humanized antibody. See, for example, U.S. Pat. Nos.
4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761;
5,693,762; 5,585,089; and 6,180,370.
[0125] A number of "humanized" antibody molecules comprising an
antigen-binding site derived from a non-human immunoglobulin have
been described, including chimeric antibodies having rodent or
modified rodent V regions and their associated CDRs fused to human
constant domains. See, for example, Winter et al., 1991, Nature
349:293-299; Lobuglio et al., 1989, Proc. Nat. Acad. Sci. USA
86:4220-4224; Shaw et al., 1987, J Immunol. 138:4534-4538; and
Brown et al., 1987, Cancer Res. 47:3577-3583. Other references
describe rodent CDRs grafted into a human supporting framework
region (FR) prior to fusion with an appropriate human antibody
constant domain. See, for example, Riechmann et al., 1988, Nature
332:323-327; Verhoeyen et al., 1988, Science 239:1534-1536; and
Jones et al., 1986, Nature 321:522-525. Another reference describes
rodent CDRs supported by recombinantly engineered rodent framework
regions. See, for example, European Patent Publ. No. 0519596. These
"humanized" molecules are designed to minimize unwanted
immunological response toward rodent anti-human antibody molecules
which limits the duration and effectiveness of therapeutic
applications of those moieties in human recipients. For example,
the antibody constant region can be engineered such that it is
immunologically inert (e.g., does not trigger complement lysis).
See, e.g., PCT Publ. No. WO99/58572; UK Patent Application No.
9809951.8. Other methods of humanizing antibodies that may also be
utilized are disclosed by Daugherty et al., 1991, Nucl. Acids Res.
19:2471-2476 and in U.S. Pat. Nos. 6,180,377; 6,054,297; 5,997,867;
5,866,692; 6,210,671; and 6,350,861; and in PCT Publ. No. WO
01/27160.
[0126] In yet another alternative, fully human antibodies may 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 or
more robust immune response may also be used for generation of
humanized or human antibodies. Examples of such technology are
Xenomouse.TM. from Abgenix, Inc. (Fremont, Calif.),
HuMAb-Mouse.RTM. and TC Mouse.TM. from Medarex, Inc. (Princeton,
N.J.), and the VelocImmune.RTM. mouse from Regeneron
Pharmaceuticals, Inc. (Tarrytown, N.Y.).
[0127] In an alternative, antibodies may be made recombinantly and
expressed using any method known in the art. In another
alternative, antibodies may be made recombinantly by phage display
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. 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. According to this technique, antibody V domain
genes are cloned in-frame into either a major or minor coat protein
gene of a filamentous bacteriophage, such as M13 or fd, and
displayed as functional antibody fragments on the surface of the
phage particle. Because the filamentous particle contains a
single-stranded DNA copy of the phage genome, selections based on
the functional properties of the antibody also result in selection
of the gene encoding the antibody exhibiting those properties.
Thus, the phage mimics some of the properties of the B cell. Phage
display can be performed in a variety of formats; see, e.g.,
Johnson, Kevin S. and Chiswell, David J., 1993, Current Opinion in
Structural Biology 3:564-571. Several sources of V-gene segments
can be used for phage display. Clackson et al., 1991, Nature
352:624-628 isolated a diverse array of anti-oxazolone antibodies
from a small random combinatorial library of V genes derived from
the spleens of immunized mice. A repertoire of V genes from
unimmunized human donors can be constructed and antibodies to a
diverse array of antigens (including self-antigens) can be isolated
essentially following the techniques described by Mark et al.,
1991, J. Mol. Biol. 222:581-597, or Griffith et al., 1993, EMBO J.
12:725-734. In a natural immune response, antibody genes accumulate
mutations at a high rate (somatic hypermutation). Some of the
changes introduced will confer higher affinity, and B cells
displaying high-affinity surface immunoglobulin are preferentially
replicated and differentiated during subsequent antigen challenge.
This natural process can be mimicked by employing the technique
known as "chain shuffling." (Marks et al., 1992, Bio/Technol.
10:779-783). In this method, the affinity of "primary" human
antibodies obtained by phage display can be improved by
sequentially replacing the heavy and light chain V region genes
with repertoires of naturally occurring variants (repertoires) of V
domain genes obtained from unimmunized donors. This technique
allows the production of antibodies and antibody fragments with
affinities in the pM-nM range. A strategy for making very large
phage antibody repertoires (also known as "the mother-of-all
libraries") has been described by Waterhouse et al., 1993, Nucl.
Acids Res. 21:2265-2266. Gene shuffling can also be used to derive
human antibodies from rodent antibodies, where the human antibody
has similar affinities and specificities to the starting rodent
antibody. According to this method, which is also referred to as
"epitope imprinting", the heavy or light chain V domain gene of
rodent antibodies obtained by phage display technique is replaced
with a repertoire of human V domain genes, creating rodent-human
chimeras. Selection on antigen results in isolation of human
variable regions capable of restoring a functional antigen-binding
site, i.e., the epitope governs (imprints) the choice of partner.
When the process is repeated in order to replace the remaining
rodent V domain, a human antibody is obtained (see PCT Publ. No. WO
93/06213). Unlike traditional humanization of rodent antibodies by
CDR grafting, this technique provides completely human antibodies,
which have no framework or CDR residues of rodent origin.
[0128] It is apparent that although the above discussion pertains
to humanized antibodies, the general principles discussed are
applicable to customizing antibodies for use, for example, in dogs,
cats, primate, equines and bovines. It is further apparent that one
or more aspects of humanizing an antibody described herein may be
combined, e.g., CDR grafting, framework mutation and CDR
mutation.
[0129] Antibodies may be made recombinantly by first isolating the
antibodies and antibody producing cells from host animals,
obtaining the gene sequence, and using the gene sequence to express
the antibody recombinantly in host cells (e.g., CHO cells). Another
method which may be employed is to express the antibody sequence in
plants (e.g., tobacco) or transgenic milk. Methods for expressing
antibodies recombinantly in plants or milk have been disclosed.
See, for example, Peeters, 2001, et al. Vaccine 19:2756; Lonberg,
N. and D. Huszar, 1995, Int. Rev. Immunol 13:65; and Pollock, et
al., 1999, J Immunol Methods 231:147. Methods for making
derivatives of antibodies, e.g., humanized, single chain, etc. are
known in the art.
[0130] Immunoassays and flow cytometry sorting techniques such as
fluorescence activated cell sorting (FACS) can also be employed to
isolate antibodies that are specific for the desired antigen.
[0131] The antibodies can be bound to many different carriers.
Carriers can be active and/or inert. Examples of well-known
carriers include polypropylene, polystyrene, polyethylene, dextran,
nylon, amylases, glass, natural and modified celluloses,
polyacrylamides, agaroses and magnetite. The nature of the carrier
can be either soluble or insoluble for purposes of the invention.
Those skilled in the art will know of other suitable carriers for
binding antibodies, or will be able to ascertain such, using
routine experimentation. In some embodiments, the carrier comprises
a moiety that targets the myocardium.
[0132] DNA encoding the monoclonal antibodies is 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 expression
vectors (such as expression vectors disclosed in PCT Publ. No. WO
87/04462), 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 Publ. No. WO 87/04462. The DNA also may
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, "chimeric" or
"hybrid" antibodies are prepared that have the binding specificity
of a monoclonal antibody herein.
[0133] pH dependent antibodies and polypeptides derived from
antibodies can be identified or characterized using methods known
in the art, whereby reduction, amelioration, or neutralization of
biological activity is detected and/or measured. In some
embodiments, a pH dependent antibody or polypeptide is identified
by incubating a candidate agent with antigen and monitoring binding
and/or attendant reduction or neutralization of a biological
activity. The binding assay may be performed with purified antigen
polypeptide(s), or with cells naturally expressing, or transfected
to express, to polypeptide(s). In one embodiment, the binding assay
is a competitive binding assay, where the ability of a candidate
antibody to compete with a known antagonist for binding is
evaluated. The assay may be performed in various formats, including
the ELISA format. In other embodiments, a pH dependent antibody is
identified by incubating a candidate agent with the antigen and
monitoring binding and attendant inhibition of LDLR expression
and/or blood cholesterol clearance.
[0134] Following initial identification, the activity of a
candidate pH dependent antibody can be further confirmed and
refined by bioassays that are known to test the targeted biological
activities. Alternatively, bioassays can be used to screen
candidates directly. Some of the methods for identifying and
characterizing antibodies, peptides, or aptamers are described in
detail in the Examples.
[0135] Antibodies with pH dependent binding may be characterized
using methods well known in the art. For example, one method is to
identify the epitope to which it 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 a pH
dependent antibody binds. Epitope mapping is commercially available
from various sources, for example, Pepscan Systems (Edelhertweg 15,
8219 PH Lelystad, The Netherlands). 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. 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 pH dependent antibody binds can be
determined in a systematic screening by using overlapping peptides
derived from the antigen sequence and determining binding by the
antibody. According to the gene fragment expression assays, the
open reading frame encoding the antigen is fragmented either
randomly or by specific genetic constructions and the reactivity of
the expressed fragments of 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 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 antigen
in which various fragments of the polypeptide have been replaced
(swapped) with sequences from antigen from another species, or a
closely related, but antigenically distinct protein (such as
another member of the proprotein convertase family). By assessing
binding of the antibody to the mutant antigen, the importance of
the particular antigen fragment to antibody binding can be
assessed.
[0136] Yet another method which can be used to characterize a pH
dependent antibody is to use competition assays with other
antibodies known to bind to the same antigen to determine if the pH
dependent antibody binds to the same epitope as other antibodies.
Competition assays are well known to those of skill in the art.
[0137] An expression vector can be used to direct expression of a
pH dependent antibody. One skilled in the art is familiar with
administration of expression vectors to obtain expression of an
exogenous protein in vivo. See, e.g., U.S. Pat. Nos. 6,436,908;
6,413,942; and 6,376,471. Administration of expression vectors
includes local or systemic administration, including injection,
oral administration, particle gun or catheterized administration,
and topical administration. In another embodiment, the expression
vector is administered directly to the sympathetic trunk or
ganglion, or into a coronary artery, atrium, ventrical, or
pericardium.
[0138] Targeted delivery of therapeutic compositions containing an
expression vector, or subgenomic polynucleotides can also be used.
Receptor-mediated DNA delivery techniques are described in, for
example, Findeis et al., 1993, Trends Biotechnol. 11:202; Chiou et
al., 1994, Gene Therapeutics: Methods And Applications Of Direct
Gene Transfer (J. A. Wolff, ed.); Wu et al., 1988, J. Biol. Chem.
263:621; Wu et al., 1994, J. Biol. Chem. 269:542; Zenke et al.,
1990, Proc. Natl. Acad. Sci. USA 87:3655; Wu et al., 1991, J. Biol.
Chem. 266:338. Therapeutic compositions containing a polynucleotide
are administered in a range of about 100 ng to about 200 mg of DNA
for local administration in a gene therapy protocol. 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 can also be used during a gene therapy protocol. The
therapeutic polynucleotides and polypeptides can be delivered using
gene delivery vehicles. The gene delivery vehicle can be of viral
or non-viral origin (see generally, Jolly, 1994, Cancer Gene
Therapy 1:51; Kimura, 1994, Human Gene Therapy 5:845; Connelly,
1995, Human Gene Therapy 1:185; and Kaplitt, 1994, Nature Genetics
6:148). Expression of such coding sequences can be induced using
endogenous mammalian or heterologous promoters. Expression of the
coding sequence can be either constitutive or regulated.
[0139] 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 Publ. 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 Publ. 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, 1992,
Hum. Gene Ther. 3:147, can also be employed.
[0140] 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,
1992, Hum. Gene Ther. 3:147); ligand-linked DNA (see, e.g., Wu, J.,
1989, Biol. Chem. 264:16985); eukaryotic cell delivery vehicles
cells (see, e.g., U.S. Pat. No. 5,814,482; PCT Publ. 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 Publ. 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 Publ. Nos. WO 95/13796; WO 94/23697;
WO 91/14445; and EP 0524968. Additional approaches are described in
Philip, 1994, Mol. Cell Biol., 14:2411, and in Woffendin, 1994
Proc. Natl. Acad. Sci. 91:1581.
[0141] This invention encompasses compositions, including
pharmaceutical compositions, comprising antibodies described herein
or made by the methods and having the characteristics described
herein. As used herein, compositions comprise one or more pH
dependent antibody, and/or one or more polynucleotides comprising
sequences encoding one or more these antibodies. These compositions
may further comprise suitable excipients, such as pharmaceutically
acceptable excipients including buffers, which are well known in
the art.
[0142] The invention also provides CDR portions of pH dependent
antibodies (including Chothia and Kabat CDRs). Determination of CDR
regions is well within the skill of the art. It is understood that
in some embodiments, CDRs can be a combination of the Kabat and
Chothia CDR (also termed "combined CDRs" or "extended CDRs"). In
some embodiments, the CDRs are the Kabat CDRs. In other
embodiments, the CDRs are the Chothia CDRs. In other words, in
embodiments with more than one CDR, the CDRs may be any of Kabat,
Chothia, combination CDRs, or combinations thereof.
[0143] The invention also provides methods of making any of these
antibodies or polypeptides. The antibodies of this invention can be
made by procedures known in the art. The polypeptides can be
produced by proteolytic or other degradation of the antibodies, by
recombinant methods (i.e., single or fusion polypeptides) as
described above or by chemical synthesis. Polypeptides of the
antibodies, especially shorter polypeptides up to about 50 amino
acids, are conveniently made by chemical synthesis. Methods of
chemical synthesis are known in the art and are commercially
available. For example, an antibody could be produced by an
automated polypeptide synthesizer employing the solid phase method.
See also, U.S. Pat. Nos. 5,807,715; 4,816,567; and 6,331,415.
[0144] In another alternative, the antibodies and peptides can be
made recombinantly using procedures that are well known in the art.
In one embodiment, a polynucleotide comprises a sequence encoding
the heavy chain and/or the light chain variable regions of antibody
4A5, 5A10, 6F6, 7D4 or L1L3. The sequence encoding the antibody of
interest may be maintained in a vector in a host cell and the host
cell can then be expanded and frozen for future use. Vectors
(including expression vectors) and host cells are further described
herein.
[0145] The invention also encompasses scFv of antibodies of this
invention. Single chain variable region fragments are made by
linking light and/or heavy chain variable regions by using a short
linking peptide. Bird et al., 1988, Science 242:423-426. An example
of a linking peptide is (GGGGS).sub.3 (SEQ ID NO:22), which bridges
approximately 3.5 nm between the carboxy terminus of one variable
region and the amino terminus of the other variable region. Linkers
of other sequences have been designed and used. Bird et al., 1988,
supra. Linkers should be short, flexible polypeptides and
preferably comprised of less than about 20 amino acid residues.
Linkers can in turn be modified for additional functions, such as
attachment of drugs or attachment to solid supports. The single
chain variants can be produced either recombinantly or
synthetically. For synthetic production of scFv, an automated
synthesizer can be used. For recombinant production of scFv, a
suitable plasmid containing polynucleotide that encodes the scFv
can be introduced into a suitable host cell, either eukaryotic,
such as yeast, plant, insect or mammalian cells, or prokaryotic,
such as E. coli. Polynucleotides encoding the scFv of interest can
be made by routine manipulations such as ligation of
polynucleotides. The resultant scFv can be isolated using standard
protein purification techniques known in the art.
[0146] Other forms of single chain antibodies, such as diabodies
are also encompassed. Diabodies are bivalent, bispecific antibodies
in which VH and VL domains are expressed on a single polypeptide
chain, but using a linker that is too short to allow for pairing
between the two domains on the same chain, thereby forcing the
domains to pair with complementary domains of another chain and
creating two antigen binding sites (see e.g., Holliger, P., et al.,
1993, Proc. Natl. Acad Sci. USA 90:6444-6448; Poljak, R. J., et
al., 1994, Structure 2:1121-1123).
[0147] For example, bispecific antibodies, monoclonal antibodies
that have binding specificities for at least two different
antigens, can be prepared using the antibodies disclosed herein.
Methods for making bispecific antibodies are known in the art (see,
e.g., Suresh et al., 1986, Methods in Enzymology 121:210).
Traditionally, the recombinant production of bispecific antibodies
was based on the coexpression of two immunoglobulin heavy
chain-light chain pairs, with the two heavy chains having different
specificities (Millstein and Cuello, 1983, Nature 305,
537-539).
[0148] According to one approach to making bispecific antibodies,
antibody variable domains with the desired binding specificities
(antibody-antigen combining sites) are fused to immunoglobulin
constant domain sequences. The fusion preferably is with an
immunoglobulin heavy chain constant domain, comprising at least
part of the hinge, CH2 and CH3 regions. It is preferred to have the
first heavy chain constant region (CH1), containing the site
necessary for light chain binding, present in at least one of the
fusions. DNAs encoding the immunoglobulin heavy chain fusions and,
if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are cotransfected into a suitable
host organism. This provides for great flexibility in adjusting the
mutual proportions of the three polypeptide fragments in
embodiments when unequal ratios of the three polypeptide chains
used in the construction provide the optimum yields. It is,
however, possible to insert the coding sequences for two or all
three polypeptide chains in one expression vector when the
expression of at least two polypeptide chains in equal ratios
results in high yields or when the ratios are of no particular
significance.
[0149] In one approach, the bispecific antibodies are composed of a
hybrid immunoglobulin heavy chain with a first binding specificity
in one arm, and a hybrid immunoglobulin heavy chain-light chain
pair (providing a second binding specificity) in the other arm.
This asymmetric structure, with an immunoglobulin light chain in
only one half of the bispecific molecule, facilitates the
separation of the desired bispecific compound from unwanted
immunoglobulin chain combinations. This approach is described in
PCT Publ. No. WO 94/04690.
[0150] Heteroconjugate antibodies, comprising two covalently joined
antibodies, are also within the scope of the invention. Such
antibodies have been used to target immune system cells to unwanted
cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection
(PCT Publ. Nos. WO 91/00360 and WO 92/200373; EP 03089).
Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents and techniques
are well known in the art, and are described in U.S. Pat. No.
4,676,980.
[0151] Chimeric or hybrid antibodies also may be prepared in vitro
using known methods of synthetic protein chemistry, including those
involving cross-linking agents. For example, immunotoxins may be
constructed using a disulfide exchange reaction or by forming a
thioether bond. Examples of suitable reagents for this purpose
include iminothiolate and methyl-4-mercaptobutyrimidate.
[0152] Humanized antibody comprising one or more CDRs of antibodies
5A10 or 7D4 or one or more CDRs derived from antibodies 5A10 or 7D4
can be made, for example, using any methods know in the art. For
example, four general steps may be used to humanize a monoclonal
antibody. These are: (1) determining the nucleotide and predicted
amino acid sequence of the starting antibody light and heavy
variable domains; (2) designing the humanized antibody, i.e.,
deciding which antibody framework region to use during the
humanizing process; (3) using the actual humanizing
methodologies/techniques; and (4) transfecting and expressing the
humanized antibody. See, for example, U.S. Pat. Nos. 4,816,567;
5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762;
5,585,089; and 6,180,370.
[0153] In the recombinant humanized antibodies, the Fc portion can
be modified to avoid interaction with Fc.gamma..quadrature.
receptor and the complement and immune systems. The techniques for
preparation of such antibodies are described in WO 99/58572. 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. See, for example,
U.S. Pat. Nos. 5,997,867 and 5,866,692.
[0154] Humanized antibody comprising the light or heavy chain
variable regions or one or more CDRs of an antibody or its
variants, or one or more CDRs derived from the antibody or its
variants, can be made using any methods known in the art.
[0155] Humanized antibodies may be made by any method known in the
art.
[0156] The invention encompasses modifications to the antibodies
and polypeptides of the invention, including functionally
equivalent antibodies which do not significantly affect their
properties and variants which have enhanced or decreased activity
and/or affinity. For example, the amino acid sequence may be
mutated to obtain an antibody with the desired binding affinity to
its antigen. Modification of polypeptides is routine practice in
the art and need not be described in detail herein. Modification of
polypeptides is exemplified in the Examples. Examples of modified
polypeptides include polypeptides with conservative substitutions
of amino acid residues, one or more deletions or additions of amino
acids which do not significantly deleteriously change the
functional activity, or which mature (enhance) the affinity of the
polypeptide for its ligand, or use of chemical analogs.
[0157] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue or the antibody fused to an epitope
tag. Other insertional variants of the antibody molecule include
the fusion to the N- or C-terminus of the antibody of an enzyme or
a polypeptide which increases the half-life of the antibody in the
blood circulation.
[0158] Substitution variants have at least one amino acid residue
in the antibody molecule removed and a different residue inserted
in its place. The sites of greatest interest for substitutional
mutagenesis include the hypervariable regions, but FR alterations
are also contemplated. Conservative substitutions are shown in
Table 1 under the heading of "conservative substitutions." If such
substitutions result in a change in biological activity, then more
substantial changes, denominated "exemplary substitutions", or as
further described below in reference to amino acid classes, may be
introduced and the products screened.
TABLE-US-00001 TABLE 1 Amino Acid Substitutions Original
Conservative Residue Substitutions Exemplary Substitutions Ala (A)
Val Val; Leu; Ile Arg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His;
Asp, Lys; Arg Asp (D) Glu Glu; Asn Cys (C) Ser Ser; Ala Gln (Q) Asn
Asn; Glu Glu (E) Asp Asp; Gln Gly (G) Ala Ala His (H) Arg Asn; Gln;
Lys; Arg Ile (I) Leu Leu; Val; Met; Ala; Phe; Norleucine Leu (L)
Ile Norleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn
Met (M) Leu Leu; Phe; Ile Phe (F) Tyr Leu; Val; Ile; Ala; Tyr Pro
(P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr; Phe
Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile; Leu; Met; Phe; Ala;
Norleucine
[0159] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side-chain properties: (1)
Non-polar: Norleucine, Met, Ala, Val, Leu, Ile; (2) Polar without
charge: Cys, Ser, Thr, Asn, Gln; (3) Acidic (negatively charged):
Asp, Glu; (4) Basic (positively charged): Lys, Arg; (5) Residues
that influence chain orientation: Gly, Pro; and (6) Aromatic: Trp,
Tyr, Phe, His.
[0160] Non-conservative substitutions are made by exchanging a
member of one of these classes for another class.
[0161] Any cysteine residue not involved in maintaining the proper
conformation of the antibody also may be substituted, generally
with serine, to improve the oxidative stability of the molecule and
prevent aberrant cross-linking. Conversely, cysteine bond(s) may be
added to the antibody to improve its stability, particularly where
the antibody is an antibody fragment such as an Fv fragment.
[0162] Amino acid modifications can range from changing or
modifying one or more amino acids to complete redesign of a region,
such as the variable region. Changes in the variable region can
alter binding affinity and/or specificity. In some embodiments, no
more than one to five conservative amino acid substitutions are
made within a CDR domain. In other embodiments, no more than one to
three conservative amino acid substitutions are made within a CDR
domain. In still other embodiments, the CDR domain is CDR H3 and/or
CDR L3.
[0163] Modifications also include glycosylated and nonglycosylated
polypeptides, as well as polypeptides with other post-translational
modifications, such as, for example, glycosylation with different
sugars, acetylation, and phosphorylation. Antibodies are
glycosylated at conserved positions in their constant regions
(Jefferis and Lund, 1997, Chem. Immunol. 65:111-128; Wright and
Morrison, 1997, TibTECH 15:26-32). The oligosaccharide side chains
of the immunoglobulins affect the protein's function (Boyd et al.,
1996, Mol. Immunol. 32:1311-1318; Wittwe and Howard, 1990, Biochem.
29:4175-4180) and the intramolecular interaction between portions
of the glycoprotein, which can affect the conformation and
presented three-dimensional surface of the glycoprotein (Jefferis
and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech.
7:409-416). Oligosaccharides may also serve to target a given
glycoprotein to certain molecules based upon specific recognition
structures. Glycosylation of antibodies has also been reported to
affect ADCC. In particular, CHO cells with tetracycline-regulated
expression of .beta.(1,4)-N-acetylglucosaminyltransferase III
(GnTIII), a glycosyltransferase catalyzing formation of bisecting
GlcNAc, was reported to have improved ADCC activity (Umana et al.,
1999, Nature Biotech. 17:176-180).
[0164] Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine, asparagine-X-threonine, and
asparagine-X-cysteine, where X is any amino acid except proline,
are the recognition sequences for enzymatic attachment of the
carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a polypeptide
creates a potential glycosylation site. O-linked glycosylation
refers to the attachment of one of the sugars
N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid,
most commonly serine or threonine, although 5-hydroxyproline or
5-hydroxylysine may also be used.
[0165] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
[0166] The glycosylation pattern of antibodies may also be altered
without altering the underlying nucleotide sequence. Glycosylation
largely depends on the host cell used to express the antibody.
Since the cell type used for expression of recombinant
glycoproteins, e.g., antibodies, as potential therapeutics is
rarely the native cell, variations in the glycosylation pattern of
the antibodies can be expected (see, e.g., Hse et al., 1997, J.
Biol. Chem. 272:9062-9070).
[0167] In addition to the choice of host cells, factors that affect
glycosylation during recombinant production of antibodies include
growth mode, media formulation, culture density, oxygenation, pH,
purification schemes and the like. Various methods have been
proposed to alter the glycosylation pattern achieved in a
particular host organism including introducing or overexpressing
certain enzymes involved in oligosaccharide production (U.S. Pat.
Nos. 5,047,335; 5,510,261 and 5,278,299). Glycosylation, or certain
types of glycosylation, can be enzymatically removed from the
glycoprotein, for example, using endoglycosidase H (Endo H),
N-glycosidase F, endoglycosidase F1, endoglycosidase F2,
endoglycosidase F3. In addition, the recombinant host cell can be
genetically engineered to be defective in processing certain types
of polysaccharides. These and similar techniques are well known in
the art.
[0168] Other methods of modification include using coupling
techniques known in the art, including, but not limited to,
enzymatic means, oxidative substitution and chelation.
Modifications can be used, for example, for attachment of labels
for immunoassay. Modified polypeptides are made using established
procedures in the art and can be screened using standard assays
known in the art, some of which are described below and in the
Examples.
[0169] In some embodiments of the invention, the antibody comprises
a modified constant region, such as a constant region that is
immunologically inert or partially inert, e.g., does not trigger
complement mediated lysis, does not stimulate ADCC, or does not
activate microglia; or have reduced activities (compared to the
unmodified antibody) in any one or more of the following:
triggering complement mediated lysis, stimulating ADCC, or
activating microglia. Different modifications of the constant
region may be used to achieve optimal level and/or combination of
effector functions. See, for example, Morgan et al., 1995,
Immunology 86:319-324; Lund et al., 1996, J. Immunology 157:4963-9
157:4963-4969; Idusogie et al., 2000, J. Immunology 164:4178-4184;
Tao et al., 1989, J. Immunology 143: 2595-2601; and Jefferis et
al., 1998, Immunological Reviews 163:59-76. In some embodiments,
the constant region is modified as described in Eur. J. Immunol.,
1999, 29:2613-2624; PCT Publ. No. WO99/58572; and/or UK Patent
Application No. 9809951.8. In other embodiments, the antibody
comprises a human heavy chain IgG2 constant region comprising the
following mutations: A330P331 to S330S331 (amino acid numbering
with reference to the wild type IgG2 sequence). Eur. J. Immunol.,
1999, 29:2613-2624. In still other embodiments, the constant region
is aglycosylated for N-linked glycosylation. In some embodiments,
the constant region is aglycosylated for N-linked glycosylation by
mutating the glycosylated amino acid residue or flanking residues
that are part of the N-glycosylation recognition sequence in the
constant region. For example, N-glycosylation site N297 may be
mutated to A, Q, K, or H. See, Tao et al., 1989, J. Immunology 143:
2595-2601; and Jefferis et al., 1998, Immunological Reviews
163:59-76. In some embodiments, the constant region is
aglycosylated for N-linked glycosylation. The constant region may
be aglycosylated for N-linked glycosylation enzymatically (such as
removing carbohydrate by enzyme PNGase), or by expression in a
glycosylation deficient host cell.
[0170] Other antibody modifications include antibodies that have
been modified as described in PCT Publ. No. WO 99/58572. These
antibodies comprise, in addition to a binding domain directed at
the target molecule, an effector domain having an amino acid
sequence substantially homologous to all or part of a constant
domain of a human immunoglobulin heavy chain. These antibodies are
capable of binding the target molecule without triggering
significant complement dependent lysis, or cell-mediated
destruction of the target. In some embodiments, the effector domain
is capable of specifically binding FcRn and/or Fc.gamma.RIIb. These
are typically based on chimeric domains derived from two or more
human immunoglobulin heavy chain CH2 domains. Antibodies modified
in this manner are particularly suitable for use in chronic
antibody therapy, to avoid inflammatory and other adverse reactions
to conventional antibody therapy.
[0171] The invention includes affinity matured embodiments. For
example, affinity matured antibodies can be produced by procedures
known in the art (Marks et al., 1992, Bio/Technology, 10:779-783;
Barbas et al., 1994, Proc Nat. Acad. Sci, USA 91:3809-3813; Schier
et al., 1995, Gene, 169:147-155; Yelton et al., 1995, J. Immunol.,
155:1994-2004; Jackson et al., 1995, J. Immunol., 154(7):3310-9;
Hawkins et al., 1992, J. Mol. Biol., 226:889-896; and PCT Publ. No.
WO2004/058184).
[0172] The following methods may be used for adjusting the affinity
of an antibody and for characterizing a CDR. One way of
characterizing a CDR of an antibody and/or altering (such as
improving) the binding affinity of a polypeptide, such as an
antibody, termed "library scanning mutagenesis". Generally, library
scanning mutagenesis works as follows. One or more amino acid
positions in the CDR are replaced with two or more (such as 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino
acids using art recognized methods. This generates small libraries
of clones (in some embodiments, one for every amino acid position
that is analyzed), each with a complexity of two or more members
(if two or more amino acids are substituted at every position).
Generally, the library also includes a clone comprising the native
(unsubstituted) amino acid. A small number of clones, e.g., about
20-80 clones (depending on the complexity of the library), from
each library are screened for binding affinity to the target
polypeptide (or other binding target), and candidates with
increased, the same, decreased, or no binding are identified.
Methods for determining binding affinity are well-known in the art.
Binding affinity may be determined using Biacore surface plasmon
resonance analysis, which detects differences in binding affinity
of about 2-fold or greater. Biacore is particularly useful when the
starting antibody already binds with a relatively high affinity,
for example a K.sub.D of about 10 nM or lower. Screening using
Biacore surface plasmon resonance is described in the Examples,
herein.
[0173] Binding affinity may be determined using Kinexa Biocensor,
scintillation proximity assays, ELISA, ORIGEN immunoassay (IGEN),
fluorescence quenching, fluorescence transfer, and/or yeast
display. Binding affinity may also be screened using a suitable
bioassay.
[0174] In some embodiments, every amino acid position in a CDR is
replaced (in some embodiments, one at a time) with all 20 natural
amino acids using art recognized mutagenesis methods (some of which
are described herein). This generates small libraries of clones (in
some embodiments, one for every amino acid position that is
analyzed), each with a complexity of 20 members (if all 20 amino
acids are substituted at every position).
[0175] In some embodiments, the library to be screened comprises
substitutions in two or more positions, which may be in the same
CDR or in two or more CDRs. Thus, the library may comprise
substitutions in two or more positions in one CDR. The library may
comprise substitution in two or more positions in two or more CDRs.
The library may comprise substitution in 3, 4, 5, or more
positions, said positions found in two, three, four, five or six
CDRs. The substitution may be prepared using low redundancy codons.
See, e.g., Table 2 of Balint et al., 1993, Gene 137(1):109-18).
[0176] The CDR may be CDRH3 and/or CDRL3. The CDR may be one or
more of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and/or CDRH3. The CDR
may be a Kabat CDR, a Chothia CDR, or an extended CDR.
[0177] Candidates with improved binding may be sequenced, thereby
identifying a CDR substitution mutant which results in improved
affinity (also termed an "improved" substitution). Candidates that
bind may also be sequenced, thereby identifying a CDR substitution
which retains binding.
[0178] Multiple rounds of screening may be conducted. For example,
candidates (each comprising an amino acid substitution at one or
more position of one or more CDR) with improved binding are also
useful for the design of a second library containing at least the
original and substituted amino acid at each improved CDR position
(i.e., amino acid position in the CDR at which a substitution
mutant showed improved binding). Preparation, screening, and
selection of this library is discussed further below.
[0179] Library scanning mutagenesis also provides a means for
characterizing a CDR, in so far as the frequency of clones with
improved binding, the same binding, decreased binding or no binding
also provide information relating to the importance of each amino
acid position for the stability of the antibody-antigen complex.
For example, if a position of the CDR retains binding when changed
to all 20 amino acids, that position is identified as a position
that is unlikely to be required for antigen binding. Conversely, if
a position of CDR retains binding in only a small percentage of
substitutions, that position is identified as a position that is
important to CDR function. Thus, the library scanning mutagenesis
methods generate information regarding positions in the CDRs that
can be changed to many different amino acids (including all 20
amino acids), and positions in the CDRs which cannot be changed or
which can only be changed to a few amino acids.
[0180] Candidates with improved affinity may be combined in a
second library, which includes the improved amino acid, the
original amino acid, and may further include additional
substitutions at that position, depending on the complexity of the
library that is desired, or permitted using the desired screening
or selection method. In addition, if desired, and adjacent amino
acid position can be randomized to at least two or more amino
acids. Randomization of adjacent amino acids may permit additional
conformational flexibility in the mutant CDR, which may, in turn,
permit or facilitate the introduction of a larger number of
improving mutations. The library may also comprise substitution at
positions that did not show improved affinity in the first round of
screening.
[0181] The second library is screened or selected for library
members with improved and/or altered binding affinity using any
method known in the art, including screening using Biacore surface
plasmon resonance analysis, and selection using any method known in
the art for selection, including phage display, yeast display, and
ribosome display.
[0182] The invention also encompasses fusion proteins comprising
one or more fragments or regions from the antibodies or
polypeptides of this invention. In one embodiment, a fusion
polypeptide is provided that comprises at least 10 contiguous amino
acids of a variable light chain region shown in SEQ ID NO: 3 and/or
at least 10 amino acids of a variable heavy chain region shown in
SEQ ID NOs: 4 or 5. In other embodiments, a fusion polypeptide is
provided that comprises at least about 10, at least about 15, at
least about 20, at least about 25, or at least about 30 contiguous
amino acids of the variable light chain region and/or at least
about 10, at least about 15, at least about 20, at least about 25,
or at least about 30 contiguous amino acids of the variable heavy
chain region. In another embodiment, the fusion polypeptide
comprises one or more CDR(s). In still other embodiments, the
fusion polypeptide comprises CDR H3 (VH CDR3) and/or CDR L3 (VL
CDR3). For purposes of this invention, a fusion protein contains
one or more antibodies and another amino acid sequence to which it
is not attached in the native molecule, for example, a heterologous
sequence or a homologous sequence from another region. Exemplary
heterologous sequences include, but are not limited to a "tag" such
as a FLAG tag or a 6His tag. Tags are well known in the art.
[0183] A fusion polypeptide can be created by methods known in the
art, for example, synthetically or recombinantly. Typically, the
fusion proteins of this invention are made by preparing an
expressing a polynucleotide encoding them using recombinant methods
described herein, although they may also be prepared by other means
known in the art, including, for example, chemical synthesis.
[0184] This invention also provides compositions comprising
antibodies or polypeptides conjugated (for example, linked) to an
agent that facilitate coupling to a solid support (such as biotin
or avidin). For simplicity, reference will be made generally to
antibodies with the understanding that these methods apply to any
of the antigen binding and/or antagonist embodiments described
herein. Conjugation generally refers to linking these components as
described herein. The linking (which is generally fixing these
components in proximate association at least for administration)
can be achieved in any number of ways. For example, a direct
reaction between an agent and an antibody is possible when each
possesses a substituent capable of reacting with the other. For
example, a nucleophilic group, such as an amino or sulfhydryl
group, on one may be capable of reacting with a carbonyl-containing
group, such as an anhydride or an acid halide, or with an alkyl
group containing a good leaving group (e.g., a halide) on the
other.
[0185] An antibody or polypeptide of this invention may be linked
to a labeling agent such as a fluorescent molecule, a radioactive
molecule or any others labels known in the art. Labels are known in
the art which generally provide (either directly or indirectly) a
signal.
[0186] The invention also provides compositions (including
pharmaceutical compositions) and kits comprising, as this
disclosure makes clear, any or all of the antibodies and/or
polypeptides described herein.
[0187] The invention also provides isolated polynucleotides
encoding the antibodies and peptides of the invention, and vectors
and host cells comprising the polynucleotide.
[0188] In another aspect, the invention provides polynucleotides
encoding any of the antibodies (including antibody fragments) and
polypeptides described herein, such as antibodies and polypeptides
having impaired effector function. Polynucleotides can be made and
expressed by procedures known in the art.
[0189] In another aspect, the invention provides compositions (such
as pharmaceutical compositions) comprising any of the
polynucleotides of the invention. In some embodiments, the
composition comprises an expression vector comprising a
polynucleotide encoding the antibody as described herein. In other
embodiment, the composition comprises an expression vector
comprising a polynucleotide encoding any of the antibodies or
polypeptides described herein. In still other embodiments, the
composition comprises either or both of the polynucleotides shown
in SEQ ID NO: 23 and SEQ ID NO: 24. Expression vectors, and
administration of polynucleotide compositions are further described
herein.
[0190] In another aspect, the invention provides a method of making
any of the polynucleotides described herein.
[0191] Polynucleotides complementary to any such sequences are also
encompassed by the present invention. Polynucleotides may be
single-stranded (coding or antisense) or double-stranded, and may
be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules
include HnRNA molecules, which contain introns and correspond to a
DNA molecule in a one-to-one manner, and mRNA molecules, which do
not contain introns. Additional coding or non-coding sequences may,
but need not, be present within a polynucleotide of the present
invention, and a polynucleotide may, but need not, be linked to
other molecules and/or support materials.
[0192] Polynucleotides may comprise a native sequence (i.e., an
endogenous sequence that encodes an antibody or a portion thereof)
or may comprise a variant of such a sequence. Polynucleotide
variants contain one or more substitutions, additions, deletions
and/or insertions such that the immunoreactivity of the encoded
polypeptide is not diminished, relative to a native immunoreactive
molecule. The effect on the immunoreactivity of the encoded
polypeptide may generally be assessed as described herein. Variants
preferably exhibit at least about 70% identity, more preferably, at
least about 80% identity, yet more preferably, at least about 90%
identity, and most preferably, at least about 95% identity to a
polynucleotide sequence that encodes a native antibody or a portion
thereof.
[0193] Two polynucleotide or polypeptide sequences are said to be
"identical" if the sequence of nucleotides or amino acids in the
two sequences is the same when aligned for maximum correspondence
as described below. Comparisons between two sequences are typically
performed by comparing the sequences over a comparison window to
identify and compare local regions of sequence similarity. A
"comparison window" as used herein, refers to a segment of at least
about 20 contiguous positions, usually 30 to about 75, or 40 to
about 50, in which a sequence may be compared to a reference
sequence of the same number of contiguous positions after the two
sequences are optimally aligned.
[0194] Optimal alignment of sequences for comparison may be
conducted using the Megalign program in the Lasergene suite of
bioinformatics software (DNASTAR, Inc., Madison, Wis.), using
default parameters. This program embodies several alignment schemes
described in the following references: Dayhoff, M. O., 1978, A
model of evolutionary change in proteins--Matrices for detecting
distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein
Sequence and Structure (National Biomedical Research Foundation,
Washington D.C.), Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990,
Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in
Enzymology vol. 183, (Academic Press, Inc., San Diego, Calif.);
Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E.
W. and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971,
Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol.
4:406-425; Sneath, P. H. A. and Sokal, R. R., 1973, Numerical
Taxonomy the Principles and Practice of Numerical Taxonomy (Freeman
Press, San Francisco, Calif.); Wilbur, W. J. and Lipman, D. J.,
1983, Proc. Natl. Acad. Sci. USA 80:726-730.
[0195] Preferably, the "percentage of sequence identity" is
determined by comparing two optimally aligned sequences over a
window of comparison of at least 20 positions, wherein the portion
of the polynucleotide or polypeptide sequence in the comparison
window may comprise additions or deletions (i.e., gaps) of 20
percent or less, usually 5 to 15 percent, or 10 to 12 percent, as
compared to the reference sequences (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
The percentage is calculated by determining the number of positions
at which the identical nucleic acid bases or amino acid residue
occurs in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the reference sequence (i.e. the window size) and
multiplying the results by 100 to yield the percentage of sequence
identity.
[0196] Variants may also, or alternatively, be substantially
homologous to a native gene, or a portion or complement thereof.
Such polynucleotide variants are capable of hybridizing under
moderately stringent conditions to a naturally occurring DNA
sequence encoding a native antibody (or a complementary
sequence).
[0197] Suitable "moderately stringent conditions" include
prewashing in a solution of 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH
8.0); hybridizing at 50.degree. C.-65.degree. C., 5.times.SSC,
overnight; followed by washing twice at 65.degree. C. for 20
minutes with each of 2.times., 0.5.times. and 0.2.times.SSC
containing 0.1% SDS.
[0198] As used herein, "highly stringent conditions" or "high
stringency conditions" are those that: (1) employ low ionic
strength and high temperature for washing, for example 0.015 M
sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate
at 50.degree. C.; (2) employ during hybridization a denaturing
agent, such as formamide, for example, 50% (v/v) formamide with
0.1% bovine serum albumin/0.1% FicoII/0.1% polyvinylpyrrolidone/50
mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride,
75 mM sodium citrate at 42.degree. C.; or (3) employ 50% formamide,
5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium
phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5.times.Denhardt's
solution, sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and
10% dextran sulfate at 42.degree. C., with washes at 42.degree. C.
in 0.2.times.SSC (sodium chloride/sodium citrate) and 50% formamide
at 55.degree. C., followed by a high-stringency wash consisting of
0.1.times.SSC containing EDTA at 55.degree. C. The skilled artisan
will recognize how to adjust the temperature, ionic strength, etc.
as necessary to accommodate factors such as probe length and the
like.
[0199] It will be appreciated by those of ordinary skill in the art
that, as a result of the degeneracy of the genetic code, there are
many nucleotide sequences that encode a polypeptide as described
herein. Some of these polynucleotides bear minimal homology to the
nucleotide sequence of any native gene. Nonetheless,
polynucleotides that vary due to differences in codon usage are
specifically contemplated by the present invention. Further,
alleles of the genes comprising the polynucleotide sequences
provided herein are within the scope of the present invention.
Alleles are endogenous genes that are altered as a result of one or
more mutations, such as deletions, additions and/or substitutions
of nucleotides. The resulting mRNA and protein may, but need not,
have an altered structure or function. Alleles may be identified
using standard techniques (such as hybridization, amplification
and/or database sequence comparison).
[0200] The polynucleotides of this invention can be obtained using
chemical synthesis, recombinant methods, or PCR. Methods of
chemical polynucleotide synthesis are well known in the art and
need not be described in detail herein. One of skill in the art can
use the sequences provided herein and a commercial DNA synthesizer
to produce a desired DNA sequence.
[0201] For preparing polynucleotides using recombinant methods, a
polynucleotide comprising a desired sequence can be inserted into a
suitable vector, and the vector in turn can be introduced into a
suitable host cell for replication and amplification, as further
discussed herein. Polynucleotides may be inserted into host cells
by any means known in the art. Cells are transformed by introducing
an exogenous polynucleotide by direct uptake, endocytosis,
transfection, F-mating or electroporation. Once introduced, the
exogenous polynucleotide can be maintained within the cell as a
non-integrated vector (such as a plasmid) or integrated into the
host cell genome. The polynucleotide so amplified can be isolated
from the host cell by methods well known within the art. See, e.g.,
Sambrook et al., 1989, supra.
[0202] Alternatively, PCR allows reproduction of DNA sequences. PCR
technology is well known in the art and is described in U.S. Pat.
Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as PCR:
The Polymerase Chain Reaction, Mullis et al., 1994, eds.
(Birkauswer Press, Boston, Mass.).
[0203] RNA can be obtained by using the isolated DNA in an
appropriate vector and inserting it into a suitable host cell. When
the cell replicates and the DNA is transcribed into RNA, the RNA
can then be isolated using methods well known to those of skill in
the art, as set forth in Sambrook et al., 1989, supra, for
example.
[0204] Suitable cloning vectors may be constructed according to
standard techniques, or may be selected from a large number of
cloning vectors available in the art. While the cloning vector
selected may vary according to the host cell intended to be used,
useful cloning vectors will generally have the ability to
self-replicate, may possess a single target for a particular
restriction endonuclease, and/or may carry genes for a marker that
can be used in selecting clones containing the vector. Suitable
examples include plasmids and bacterial viruses, e.g., pUC18,
pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19,
pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors
such as pSA3 and pAT28. These and many other cloning vectors are
available from commercial vendors such as BioRad, Strategene, and
Invitrogen.
[0205] Expression vectors generally are replicable polynucleotide
constructs that contain a polynucleotide according to the
invention. It is implied that an expression vector must be
replicable in the host cells either as episomes or as an integral
part of the chromosomal DNA. Suitable expression vectors include
but are not limited to plasmids, viral vectors, including
adenoviruses, adeno-associated viruses, retroviruses, cosmids, and
expression vector(s) disclosed in PCT Publ. No. WO 87/04462. Vector
components may generally include, but are not limited to, one or
more of the following: a signal sequence; an origin of replication;
one or more marker genes; suitable transcriptional controlling
elements (such as promoters, enhancers and terminator). For
expression (i.e., translation), one or more translational
controlling elements are also usually required, such as ribosome
binding sites, translation initiation sites, and stop codons.
[0206] The vectors containing the polynucleotides of interest can
be introduced into the host cell by any of a number of appropriate
means, including electroporation, transfection employing calcium
chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or
other substances; microprojectile bombardment; lipofection; and
infection (e.g., where the vector is an infectious agent such as
vaccinia virus). The choice of introducing vectors or
polynucleotides will often depend on features of the host cell.
[0207] The invention also provides host cells comprising any of the
polynucleotides described herein. Any host cells capable of
over-expressing heterologous DNAs can be used for the purpose of
isolating the genes encoding the antibody, polypeptide or protein
of interest. Non-limiting examples of mammalian host cells include
but are not limited to COS, HeLa, NSO, and CHO cells. See also PCT
Publ. No. WO 87/04462. Suitable non-mammalian host cells include
prokaryotes (such as E. coli or B. subtillis) and yeast (such as S.
cerevisae, S. pombe; or K. lactis). Preferably, the host cells
express the cDNAs at a level of about 5 fold higher, more
preferably, 10 fold higher, even more preferably, 20 fold higher
than that of the corresponding endogenous antibody or protein of
interest, if present, in the host cells. Screening the host cells
for a specific binding to antigen is effected by an immunoassay or
FACS. A cell overexpressing the antibody or protein of interest can
be identified.
C. Compositions
[0208] The compositions used in the methods of the invention
comprise an effective amount of a pH dependent antibody, or a pH
dependent antibody derived polypeptide, described herein. Examples
of such compositions, as well as how to formulate them, are also
described in an earlier section and below. In one embodiment, the
composition comprises one or more pH dependent antibodies. In other
embodiments, the pH dependent antibody recognizes human PCSK9. In
still other embodiments, the pH dependent antibody is humanized. In
yet other embodiments, the pH dependent antibody comprises a
constant region that does not trigger an unwanted or undesirable
immune response, such as antibody-mediated lysis or ADCC. In other
embodiments, the pH dependent antibody comprises one or more CDR(s)
of the antibody (such as one, two, three, four, five, or, in some
embodiments, all six CDRs). In some embodiments, the pH dependent
antibody is human.
[0209] The composition used in the present invention can further
comprise pharmaceutically acceptable carriers, excipients, or
stabilizers (Remington: The Science and Practice of Pharmacy 20th
Ed., 2000, Lippincott Williams and Wilkins, Ed. K. E. Hoover), in
the form of lyophilized formulations or aqueous solutions.
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations, 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.
[0210] In one embodiment, the antibody is administered in a
formulation as a sterile aqueous solution having a pH that ranges
from about 5.0 to about 6.5 and comprising from about 1 mg/ml to
about 200 mg/ml of antibody, from about 1 millimolar to about 100
millimolar of histidine buffer, from about 0.01 mg/ml to about 10
mg/ml of polysorbate 80, from about 100 millimolar to about 400
millimolar of trehalose, and from about 0.01 millimolar to about
1.0 millimolar of disodium EDTA dihydrate.
[0211] The pH dependent antibody and compositions thereof can also
be used in conjunction with other agents that serve to enhance
and/or complement the effectiveness of the agents.
D. Kits
[0212] The invention also provides kits for use in the instant
methods. Kits of the invention include one or more containers
comprising a pH dependent antibody (such as a humanized antibody)
or peptide described herein and instructions for use in accordance
with any of the methods of the invention described herein.
Generally, these instructions comprise a description of
administration of the pH dependent antibody for the above described
therapeutic treatments.
[0213] In some embodiments, the antibody is a humanized antibody.
In some embodiments, the antibody is human. In other embodiments,
the antibody is a monoclonal antibody. The instructions relating to
the use of a pH dependent 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.
[0214] 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 a pH dependent antibody. The container
(e.g., pre-filled syringe or autoinjector) may further comprise a
second pharmaceutically active agent.
[0215] 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.
Mutations and Modifications
[0216] To express the antibodies of the present invention, DNA
fragments encoding V.sub.H and V.sub.L regions can first be
obtained using any of the methods described above. Various
modifications, e.g., mutations, deletions, and/or additions can
also be introduced into the DNA sequences using standard methods
known to those of skill in the art. For example, mutagenesis can be
carried out using standard methods, such as PCR-mediated
mutagenesis, in which the mutated nucleotides are incorporated into
the PCR primers such that the PCR product contains the desired
mutations or site-directed mutagenesis.
[0217] One type of substitution, for example, that may be made is
to change one or more cysteines in the antibody, which may be
chemically reactive, to another residue, such as, without
limitation, alanine or serine. For example, there can be a
substitution of a non-canonical cysteine. The substitution can be
made in a CDR or framework region of a variable domain or in the
constant domain of an antibody. In some embodiments, the cysteine
is canonical.
[0218] The antibodies may also be modified, e.g., in the variable
domains of the heavy and/or light chains, e.g., to alter a binding
property of the antibody. For example, a mutation may be made in
one or more of the CDR regions to increase or decrease the K.sub.D
of the antibody for antigen, to increase or decrease k.sub.off, or
to alter the binding specificity of the antibody. Techniques in
site-directed mutagenesis are well-known in the art. See, e.g.,
Sambrook et al. and Ausubel et al., supra.
[0219] A modification or mutation may also be made in a framework
region or constant domain to increase the half-life of a pH
dependent. See, e.g., PCT Publ. No. WO 00/09560. A mutation in a
framework region or constant domain can also be made to alter the
immunogenicity of the antibody, to provide a site for covalent or
non-covalent binding to another molecule, or to alter such
properties as complement fixation, FcR binding and
antibody-dependent cell-mediated cytotoxicity. According to the
invention, a single antibody may have mutations in any one or more
of the CDRs or framework regions of the variable domain or in the
constant domain.
[0220] In a process known as "germlining", certain amino acids in
the V.sub.H and V.sub.L sequences can be mutated to match those
found naturally in germline V.sub.H and V.sub.L sequences. In
particular, the amino acid sequences of the framework regions in
the V.sub.H and V.sub.L sequences can be mutated to match the
germline sequences to reduce the risk of immunogenicity when the
antibody is administered. Germline DNA sequences for human V.sub.H
and V.sub.L genes are known in the art (see e.g., the "Vbase" human
germline sequence database; see also Kabat, E. A., et al. (1991)
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publ. No.
91-3242; Tomlinson et al., 1992, J. Mol. Biol. 227:776-798; and Cox
et al., 1994, Eur. J. Immunol. 24:827-836.
[0221] Another type of amino acid substitution that may be made is
to remove potential proteolytic sites in the antibody. Such sites
may occur in a CDR or framework region of a variable domain or in
the constant domain of an antibody. Substitution of cysteine
residues and removal of proteolytic sites may decrease the risk of
heterogeneity in the antibody product and thus increase its
homogeneity. Another type of amino acid substitution eliminates
asparagine-glycine pairs, which form potential deamidation sites,
by altering one or both of the residues. In another example, the
C-terminal lysine of the heavy chain of a pH dependent antibody of
the invention can be cleaved. In various embodiments of the
invention, the heavy and light chains of the antibodies may
optionally include a signal sequence.
[0222] Once DNA fragments encoding the V.sub.H and V.sub.L segments
of the present invention are obtained, these DNA fragments can be
further manipulated by standard recombinant DNA techniques, for
example to convert the variable region genes to full-length
antibody chain genes, to Fab fragment genes, or to a scFv gene. In
these manipulations, a V.sub.L- or V.sub.H-encoding DNA fragment is
operatively linked to another DNA fragment encoding another
protein, such as an antibody constant region or a flexible linker.
The term "operatively linked", as used in this context, is intended
to mean that the two DNA fragments are joined such that the amino
acid sequences encoded by the two DNA fragments remain
in-frame.
[0223] The isolated DNA encoding the V.sub.H region can be
converted to a full-length heavy chain gene by operatively linking
the V.sub.H-encoding DNA to another DNA molecule encoding heavy
chain constant regions (CH1, CH2 and CH3). The sequences of human
heavy chain constant region genes are known in the art (see e.g.,
Kabat, E. A., et al., 1991, Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publ. No. 91-3242) and DNA fragments encompassing
these regions can be obtained by standard PCR amplification. The
heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA,
IgE, IgM or IgD constant region, but most preferably is an IgG1 or
IgG2 constant region. The IgG constant region sequence can be any
of the various alleles or allotypes known to occur among different
individuals, such as Gm(1), Gm(2), Gm(3), and Gm(17). These
allotypes represent naturally occurring amino acid substitution in
the IgG1 constant regions. For a Fab fragment heavy chain gene, the
V.sub.H-encoding DNA can be operatively linked to another DNA
molecule encoding only the heavy chain CH1 constant region. The CH1
heavy chain constant region may be derived from any of the heavy
chain genes.
[0224] The isolated DNA encoding the V.sub.L region can be
converted to a full-length light chain gene (as well as a Fab light
chain gene) by operatively linking the V.sub.L-encoding DNA to
another DNA molecule encoding the light chain constant region, CL.
The sequences of human light chain constant region genes are known
in the art (see e.g., Kabat, E. A., et al., 1991, Sequences of
Proteins of Immunological Interest, Fifth Edition, U.S. Department
of Health and Human Services, NIH Publ. No. 91-3242) and DNA
fragments encompassing these regions can be obtained by standard
PCR amplification. The light chain constant region can be a kappa
or lambda constant region. The kappa constant region may be any of
the various alleles known to occur among different individuals,
such as Inv(1), Inv(2), and Inv(3). The lambda constant region may
be derived from any of the three lambda genes.
[0225] To create a scFv gene, the V.sub.H- and V.sub.L-encoding DNA
fragments are operatively linked to another fragment encoding a
flexible linker, e.g., encoding the amino acid sequence
(Gly4-Ser).sub.3, such that the V.sub.H and V.sub.L sequences can
be expressed as a contiguous single-chain protein, with the V.sub.L
and V.sub.H regions joined by the flexible linker (See e.g., Bird
et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl.
Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature
348:552-554. The single chain antibody may be monovalent, if only a
single V.sub.H and V.sub.L are used, bivalent, if two V.sub.H and
V.sub.L are used, or polyvalent, if more than two V.sub.H and
V.sub.L are used. Bispecific or polyvalent antibodies may be
generated that bind specifically to antigen and to another
molecule.
[0226] In another embodiment, a fusion antibody or immunoadhesin
may be made that comprises all or a portion of an antibody of the
invention linked to another polypeptide. In another embodiment,
only the variable domains of the antibody are linked to the
polypeptide. In another embodiment, the V.sub.H domain of an
antibody is linked to a first polypeptide, while the V.sub.L domain
of an antibody is linked to a second polypeptide that associates
with the first polypeptide in a manner such that the V.sub.H and
V.sub.L domains can interact with one another to form an antigen
binding site. In another preferred embodiment, the V.sub.H domain
is separated from the V.sub.L domain by a linker such that the
V.sub.H and V.sub.L domains can interact with one another. The
V.sub.H-linker-V.sub.L antibody is then linked to the polypeptide
of interest. In addition, fusion antibodies can be created in which
two (or more) single-chain antibodies are linked to one another.
This is useful if one wants to create a divalent or polyvalent
antibody on a single polypeptide chain, or if one wants to create a
bispecific antibody.
[0227] In other embodiments, other modified antibodies may be
prepared using pH dependent antibody encoding nucleic acid
molecules. For instance, "Kappa bodies" (Ill et al., 1997, Protein
Eng. 10:949-57), "Minibodies" (Martin et al., 1994, EMBO J.
13:5303-9), "Diabodies" (Holliger et al., 1993, Proc. Natl. Acad.
Sci. USA 90:6444-6448), or "Janusins" (Traunecker et al., 1991,
EMBO J. 10:3655-3659 and Traunecker et al., 1992, Int. J. Cancer
(Suppl.) 7:51-52) may be prepared using standard molecular
biological techniques following the teachings of the
specification.
[0228] Bispecific antibodies or antigen-binding fragments can be
produced by a variety of methods including fusion of hybridomas or
linking of Fab' fragments. See, e.g., Songsivilai & Lachmann,
1990, Clin. Exp. Immunol. 79:315-321, Kostelny et al., 1992, J.
Immunol. 148:1547-1553. In addition, bispecific antibodies may be
formed as "diabodies" or "Janusins." In some embodiments, the
bispecific antibody binds to two different epitopes of antigen. In
some embodiments, the modified antibodies described above are
prepared using one or more of the variable domains or CDR regions
from a human antibody provided herein.
[0229] Generation of Antigen-Specific Antibodies
[0230] DNA of the heavy and light chains of representative
antibodies of the present invention, 5L1721H23_6L3 and
5L1721H23_6L3H3, were deposited in the American Type Culture
Collection (ATCC) (under the terms of the Budapest Treaty) on Dec.
22, 2009, and were assigned the accession numbers in Table 2. All
restrictions on the availability to the public of the plasmids so
deposited will be irrevocably removed upon the issuance of a patent
from the specification of the present invention. The antibody
references for the heavy and light chains of 5L1721H23_6L3 are
UC-H5H23 and UC-H5L1721-6L3, respectively. The antibody references
for the heavy and light chains of 5L1721H23_6L3H3 are UC-H5H23-6H3
and UC-H5L1721-6L3, respectively.
TABLE-US-00002 TABLE 2 Antibody Reference ATCC Accession No.
UC-H5H23 PTA-10547 UC-H5H23-6H3 PTA-10548 UC-H5L1721-6L3
PTA-10549
EXAMPLES
Example 1: Modeling Antibody pH Dependent Binding to PCSK9
Antigen
[0231] Computer modeling was used to predict whether an antibody
with pH dependent antigen binding could impact antibody half life
the duration of the lowering of the PCSK9 serum concentration. For
the purpose of this modeling the following assumptions were made: 1
uM dose of antibody in blood; 21 day (non pH dependent) antibody
half-life; a simulation run duration of 100 days; for the pH
dependent antibody, a K.sub.on=1e5/M/s, a K.sub.off @ neutral
pH=K.sub.D*K.sub.on, the pH dependent binding is modeled as an
increase in K.sub.off in the acidic endosomes; and K.sub.off at
acidic pH=R*K.sub.off at neutral pH.
[0232] The time rates of change of the species in the model are
specified in terms of model parameters by the differential
equations below. Generally, the model is allowed to proceed with
antibody levels set to zero until it reaches a steady state; at
that time, a bolus of antibody is simulated by resetting [mAb] from
zero to a level reflective of the dose, and then allowing the model
described below to proceed.
[ P ] ' = k P , create - k activeuptake [ P } - k protein , on [
LDLR ] [ P ] + k P , off [ P LDLR ] - k protein , on [ LDL LDLR ] [
P ] + k P , off [ LDL P LDLR ] - 2 k on , neutral [ mAb ] [ P ] + k
off , neutral [ mAb P ] - k on , neutral [ mAb P ] [ P ] + 2 k off
, neutral [ mAb P P ] - k endouptake [ P ] - k endouptake A [ P ]
early V [ LDL ] ' = k L , create - k L , clear [ LDL ] - k protein
, on [ LDLR ] [ LDL ] + k L , off [ LDL LDLR ] - k protein , on [ P
LDLR ] [ LDL ] + k L , off [ LDL P LDLR ] [ LDLR ] ' = k R , create
- k R , clear [ LDLR ] - k protein , on [ LDLR ] [ LDL ] + k L ,
off [ LDL LDLR ] - k protein , on [ LDLR ] [ P ] + k P , off [ P
LDLR ] + k recycle [ LDL LDLR ] in [ mAb ] ' = - 2 k on , neutral [
mAb ] [ P ] + k off , neutral [ mAb P ] - k endouptake [ mAb ] - k
endouptake A [ mAb ] early V + k endouptake ( 1 - A ) F [ mAb ]
late V + .alpha. k endouptake ( 1 - A ) F [ mAb ] active V [ mAb P
] ' = 2 k on , neutral [ mAb ] [ P ] - k off , neutral [ mAb P ] -
k on , neutral [ mAb P ] [ P ] + 2 k off , neutral [ mAb P P ] - k
endouptake [ mAb P ] - k endouptake A [ mAb P ] early V + k
endouptake ( 1 - A ) F [ mAb P ] late V - k activeuptake [ mAb P ]
[ mAb P P ] ' = k on , neutral [ mAb P ] [ P ] - 2 k off , neutral
[ mAb P P ] - k endouptake [ mAb P P ] - k endouptake A [ mAb P P ]
early V + k endouptake ( 1 - A ) F [ mAb P P ] late V - 2 k
activeuptake [ mAb P P ] [ LDL LDLR ] ' = k protein , on [ LDLR ] [
LDL ] - k L , off [ LDL LDLR ] - k protein , on [ LDL LDLR ] [ P ]
+ k P , off [ LDL P LDLR ] - k internalize [ LDL LDLR ] [ P LDLR ]
' = k protein , on [ LDLR ] [ P ] - k P , off [ P LDLR ] - k
protein , on [ P LDLR ] [ LDL ] + k L , off [ LDL P LDLR ] - k
internalize [ P LDLR ] [ LDL P LDLR ] ' = k protein , on [ LDL LDLR
] [ P ] - k P , off [ LDL P LDLR ] + k protein , on [ P LDLR ] [
LDL ] - k L , off [ LDL P LDLR ] - k internalize [ LDL P LDLR ] [ P
LDLR ] i n ' = k internalize [ P LDLR ] - k recycle [ P LDLR ] in [
LDL LDLR ] i n ' = k internalize [ LDL LDLR ] - k recycle [ LDL
LDLR ] in [ LDL P LDLR ] i n ' = k internalize [ LDL P LDLR ] - k
recycle [ LDL P LDLR ] in [ mAb ] early ' = k endouptake [ mAb ] -
k endouptake A [ mAb ] early V early - 2 k on , neutral [ mAb ]
early [ P ] early + k off , neutral [ mAb P ] early - k endouptake
( 1 - A ) [ mAb ] early V early [ P ] early ' = k endouptake [ P ]
- k endouptake A [ P ] early V early - 2 k on , neutral [ mAb ]
early [ P ] early + k off , neutral [ mAb P ] early - k on ,
neutral [ mAb P ] early [ P ] early + 2 k off , neutral [ mAb P P ]
early - k endouptake ( 1 - A ) [ P ] early V early [ mAb P ] early
' = k endouptake [ mAb P ] - k endouptake A [ mAb P ] early V early
+ 2 k on , neutral [ mAb ] early [ P ] early - k off , neutral [
mAb P ] early - k on , neutral [ mAb P ] early [ P ] early + 2 k
off , neutral [ mAb P P ] early - k endouptake ( 1 - A ) [ mAb P ]
early V early [ mAb P P ] early ' = k endouptake [ mAb P P ] - k
endouptake A [ mAb P P ] early V early + k on , neutral [ mAb P ]
early [ P ] early - 2 k off , neutral [ mAb P P ] early - k
endouptake ( 1 - A ) [ mAb P P ] early V early [ mAb ] late ' = k
endouptake ( 1 - A ) [ mAb ] early V late - 2 k on , acidic [ mAb ]
late [ P ] late + k off , acidic [ mAb P ] late - k endouptake ( 1
- A ) ( 1 - F ) [ mAb ] late V late [ P ] late ' = k endouptake ( 1
- A ) [ P ] early V late - 2 k on , acidic [ mAb ] late [ P ] late
+ k off , acidic [ mAb P ] late - k on , acidic [ mAb P P ] late [
P ] late + 2 k off , acidic [ mAb P P ] late - k endouptake ( 1 - A
) [ P ] late V late [ mAb P ] late ' = k endouptake ( 1 - A ) [ mAb
P ] early V late + 2 k on , acidic [ mAb ] late [ P ] late - k off
, acidic [ mAb P ] . late - k on , acidic [ mAb P ] late [ P ] late
+ 2 k off , acidic [ mAb P P ] late - k endouptake ( 1 - A ) F [
mAb P ] late V late - k endouptake ( 1 - A ) ( 1 - F ) [ mAb P ]
late V late [ mAb P P ] late ' = k endouptake ( 1 - A ) [ mAb P P ]
early V late + k on , acidic [ mAb P ] late [ P ] late - 2 k off ,
acidic [ mAb P P ] late - k endouptake ( 1 - A ) F [ mAb P P ] late
V late - k endouptake ( 1 - A ) ( 1 - F ) [ mAb P P ] late V late [
mAb ] active ' = - 2 k on , acidic [ mAb ] active [ P ] active + k
off , acidic [ mAb P ] active - .alpha. k endouptake ( 1 - A ) ( 1
- F ) [ mAb ] active - .alpha. k endouptake ( 1 - A ) F [ mAb ]
active V active [ P ] active ' = - 2 k on , active [ mAb ] active [
P ] active + k off , acidic [ mAb P ] active - k on , acidic [ mAb
P ] active [ P ] active + 2 k off , acidic [ mAb P P ] active -
.alpha. k endouptake ( 1 - A ) [ P ] active [ mAb P ] active ' = k
activeuptake [ mAb P ] V V active + 2 k on , active [ mAb ] active
[ P ] active - k off , acidic [ mAb P ] active - k on , acidic [
mAb P ] active [ P ] active + 2 k off , acidic [ mAb P P ] active -
.alpha. k endouptake ( 1 - A ) [ mAb P ] active [ mAb P P ] active
' = 2 k activeuptake [ mAb P P ] V V active + k on , acidic [ mAb P
] active [ P ] active - 2 k off , acidic [ mAb P P ] active -
.alpha. k endouptake ( 1 - A ) [ mAb P P ] active ##EQU00001##
[0233] The parameters used in the model are described below in
Table 3. In some cases, parameters are derived from other
physiologically-relevant or measured quantities; these are
indicated in the table as well.
TABLE-US-00003 TABLE 3 (N) = non-pH dependent mAb; (AS) = pH
dependent mAb Parameter Description Value V Blood volume 2 mL
V.sub.early Early endosome volume 0.06 mL V.sub.late Late endosome
volume 0.06 mL V.sub.active Active uptake endosome volume 0.3 mL
k.sub.R,create Rate of LDLR synthesis 0.1 nM/hr k.sub.P,create Rate
of PCSK9 synthesis 1.714 nM/hr k.sub.L,create Rate of LDL synthesis
10.44 nM/hr k.sub.activeuptake First order rate of active PCSK9
uptake In(2)/t.sub.activeuptake t.sub.activeuptake Time scale for
active PCSK9 uptake 1 hr k.sub.R,clear First order clearance rate
for LDLR 0.1872 hr.sup.-1 k.sub.L,clear First order clearance rate
for LDL 0.018054 hr.sup.-1 k.sub.protein,on Protein-protein
association rate 100000M.sup.-1 s.sup.-1 k.sub.L,off LDL/LDLR
dissociation rate constant k.sub.protein,on K.sub.d,L K.sub.d,L
LDL/LDLR affinity 10 nM k.sub.P,off PCSK9/LDLR dissociation rate
constant k.sub.protein,on K.sub.d,P K.sub.d,P PCSK9/LDLR affinity
170 nM k.sub.internalize First order LDLR internalization rate
In(2)/t.sub.internalize t.sub.internalize Time scale for LDLR
internalization 5 min k.sub.recycle First order LDLR recycling rate
In(2)/t.sub.recycle t.sub.recycle Time scale for LDLR recycling 10
min k.sub.on,neutral Antibody/PCSK9 association rate (pH 1.01
.times. 10.sup.5M.sup.-1 s.sup.-1 7.4) (N) 6.37 .times.
10.sup.4M.sup.-1 s.sup.-1 (AS) k.sub.off,neutral Antibody/PCSK9
dissociation rate (pH 4.85 .times. 10.sup.-4 s.sup.-1 (N) 7.4) 5.16
.times. 10.sup.-4 s.sup.-1 (AS) k.sub.on,acidic Antibody/PCSK9
association rate (pH 3.73 .times. 10.sup.5M.sup.-1 s.sup.-1 5.5)
(N) 1.6 .times. 10.sup.5M.sup.-1 s.sup.-1 (AS) k.sub.off,acidic
Antibody/PCSK9 dissociation rate (pH 1.94 .times. 10.sup.-3
s.sup.-1 (N) 5.5) 0.0187 s.sup.-1 (AS) k.sub.endouptake Volume rate
of uptake into early 8 .mu.L/min endosomes A Automatic recycling
fraction 0.7 (dimensionless) F FcRn-mediated antibody recycling
0.972 efficiency (dimensionless) .alpha. Increase in active
endosome processing 4 (dimensionless) time (compared to fluid phase
endosomes)
[0234] One depiction of the modeling is shown in FIG. 1. Increasing
the K.sub.D ratio of endosomal pH (i.e., pH 5.5 or 6.0)/physiologic
pH 7.4 from 1 to 2 resulted in an increase in antibody
concentration over time, which was correlated with the value of the
K.sub.D ratio. When the K.sub.D ratio was changed, or k.sub.off or
k.sub.on, the antigen free ligand concentration was changed (FIG.
2) as well as antibody concentration (FIG. 3) in correlation with
the increase K.sub.D ratio (FIG. 2), increased k.sub.off (FIG. 3A)
and decreased k.sub.on (FIG. 3B). These results were not dependent
upon designating whether endosomal pH was 5.5 or 6.0.
[0235] A heatmap display (FIG. 4) was constructed for a general
model of antibodies with pH dependent binding, effectively showing
how many days longer the antibody with pH dependent binding should
decrease the serum concentration of antigen beyond that observed
for a non-pH dependent antibody. Knockdown days are defined by
integral of: 1-(C.sub.serum(t))/(C.sub.serum(t=0)) from day 0 to
day 100, e.g., a 100% reduction in serum concentration for N days
followed by a return to pretreatment level would correspond to N
knockdown days. The heat maps show the increase in knockdown days
for the pH dependent antibodies, as compared to the control
antibodies, based upon the K.sub.D at neutral pH and also the
K.sub.D ratio (R). Effects are seen throughout all K.sub.D values
modeled, particularly between 0.01 and 100 nM, and more
particularly between K.sub.D of 0.1 and 10 nM. Changes in certain
parameters, e.g., dose size, antibody kinetics, duration of
simulation, or other quantities will lead to some differences in
the values shown on the heat maps.
[0236] Modeling predictions for the performance of PCSK9
antibodies, with or without pH dependent binding, correlated well
with actual experimental results. In FIG. 5A, the total antibody
concentration over time after administering antibody 5A10, which
has no pH dependent binding and a K.sub.D ratio of 1.1, is shown.
Counterpart LDL levels are shown in FIG. 5B. In FIG. 6A, the total
antibody concentration over time after administering antibody
5L1721H23_6L3H3, which has pH dependent binding and a K.sub.D ratio
of 14.4, is shown. Counterpart LDL levels are also shown in FIG.
6B. For these models and experiments in FIGS. 5 and 6, a pH of 5.5
was assumed and the experimental K.sub.D values for the antibodies
were calculated at pH 5.5. The pH dependent binding antibody
demonstrated prolonged total antibody concentration and a prolonged
inhibition of LDL levels as compared to 5A10. Further discussion of
the general model for antibodies with pH dependent binding is shown
in Example 4.
Example 2: Generating and Screening Anti-PCSK9 Antibodies with pH
Dependent PCSK9 Binding
[0237] Histidine scan mutagenesis was conducted for all CDRs of
monoclonal antibody h5A10 (also known as 5A10 hu frame). This
antibody originates from mouse monoclonal antibody 5A10 (also known
as 5A10.138), but has human framework regions. The sequence of the
h5A10 antibody used as the starting template is shown below (CDRs
are in bold font):
TABLE-US-00004 Variable Light chain: (SEQ ID NO: 1)
DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYS
ASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSTPRTFGQ GTKLEIK Variable
Heavy chain: (SEQ ID NO: 2)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGE
INPSGGRTNYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARER
PLYAMDYWGQGTTVTVSS
[0238] The antibody h5A10 was cloned into a bacterial expression
vector, which allows the expression of Fabs in the periplasm of E.
coli. Using primers, every CDR position was mutated to histidine,
resulting in 58 single histidine mutants of the h5A10 antibody.
[0239] Antibodies with these single histidine mutants were
expressed in the periplasm of E. coli after induction with IPTG and
purified using a histidine tag on the C-terminus of the CH1. The
affinities of the purified Fabs were measured at pH 5.5 and 7.4
with Biacore. Mutations influencing the K.sub.D ratio of pH 5.5/pH
7.4 were found in four different CDR's (L1, L2, H2 & H3). The
pH dependent change on the K.sub.D ranged between minimally
decreased and significantly decreased.
[0240] Mutations from three CDRs (L1, L2 & H2) increased the
K.sub.D-ratio of pH 5.5/pH 7.4 without destroying the affinity at
pH 7.4, and the antibodies combining the CDRs containing these
mutations were generated. For h5A10 and all mutants, the heavy
chain CDR1 (H1) sequence is GYTFTSYYMH (SEQ ID NO: 6). For 5A10,
single, double, and triple mutants, and 5L1721H23_6H3, the light
chain CDR3 (L3), is QQRYSTPRT (SEQ ID NO: 27). For the other
affinity matured mutants and L1L3-based mutants, L3 is QQRYSLWRT
(SEQ ID NO: 12). The CDR sequences of the antibodies containing
these various combinations of mutations are shown in Table 4 below.
Sequence number identifiers are provided in parentheticals.
TABLE-US-00005 TABLE 4 L1 L2 H2 H3 h 5A10 KASQDVSTAVA(13)
SASYRYT(17) EINPSGGRTNYNEKFKS(19) ERPLYAMDY(8) Single mutants: 5L-6
KASQDHSTAVA(14) 5L1-7 KASQDVHTAVA(10) 5L2-1 HASYRYT(11) 5L2-4
SASHRYT(18) 5H2-3 EIHPSGGRTNYNEKFKS(7) 5H3-5 ERPLHAMDY(21) Double
mutants: 5L17 21 KASQDVHTAVA(10) HASYRYT(11) 5L17 24
KASQDVHTAVA(10) SASHRYT(18) 5L17H23 KASQDVHTAVA(10)
EIHPSGGRTNYNEKFKS(7) 5L21 24 HASHRYT(19) 5L21 H23 HASYRYT(11)
EIHPSGGRTNYNEKFKS(7) 5L24 H23 SASHRYT(18) EIHPSGGRTNYNEKFKS(7)
Triple mutants: 5L1721 KASQDVHTAVA(10) HASYRYT(11)
EIHPSGGRTNYNEKFKS(7) H23 5L1724 KASQDVHTAVA(10) SASHRYT(18)
EIHPSGGRTNYNEKFKS(7) H23 5L1724H35 KASQDVHTAVA(10) SASHRYT(18)
ERPLHAMDY(21) Affinity matured mutants: 5L1721H23_ KASQDVHTAVA(10)
HASYRYT(11) EIHPSGGRTNYNEKFKS(7) ERPLYASDL(9) 6H3 5L1721H23_
KASQDVHTAVA(10) HASYRYT(11) EIHPSGGRTNYNEKFKS(7) 6L3 5L1724H23_
KASQDVHTAVA(10) SASHRYT(18) EIHPSGGRTNYNEKFKS(7) 6L3 5L1721H23_
KASQDVHTAVA(10) HASYRYT(11) EIHPSGGRTNYNEKFKS(7) ERPLYASDL(9) 6L3H3
5111724H23_ KASQDVHTAVA(10) SASHRYT(18) EIHPSGGRTNYNEKFKS(7)
ERPLYASDL(9) 6L3H3 L1L3-based mutants: L1L3 RASQGISSALA(15)
SASYRYT(17) EISPFGGRTNYNEKFKS(20) ERPLYASDL(9) 6L1721H23
RASQGIHSALA(16) HASYRYT(11) EIHPFGGRTNYNEKFKS(7) 6L1721
RASQGIHSALA(16) HASYRYT(11) 6L21H2335 HASYRYT(11)
EIHPFGGRTNYNEKFKS(7)
[0241] No changes were made in the framework sequences for these
antibodies as compared to 5A10.138. For example, the sequences for
the variable heavy chains of 5L1721H23_6L3 and 5L1721H23_6L3H3 are
provided below. The same variable light chain was used in each of
these antibodies and is also provided below. CDRs are highlighted
in bold.
TABLE-US-00006 Variable light chain of 5L1721H23_6L3 and
5L1721H23_6L3H3: (SEQ ID NO: 3)
DIQMTQSPSSLSASVGDRVTITCKASQDVHTAVAWYQQKPGKAPKLLIYH
ASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRTFGQ GTKLEIK Variable
heavy chain 5L1721H23_6L3: (SEQ ID NO: 4)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGE
IHPSGGRTNYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARER
PLYAMDYWGQGTTVTVSS Variable heavy chain 5L1721H23_6L3H3: (SEQ ID
NO: 5) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGE
IHPSGGRTNYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARER
PLYASDLWGQGTTVTVSS Variable light chain (kappa) of 5L1721H23_6L3
and 5L1721H23_6L3H3 (SEQ ID NO: 23)
gatatccagatgacacagtccccatcctccctgtctgcctctgtgggcga
ccgcgtcaccatcacctgcaaggcctctcaggatgtgcatactgctgtag
cctggtatcagcagaagccaggcaaagccccaaaactgctgatctaccat
gcatcctaccgctacactggtgtcccatcacgcttcagtggcagtggctc
tggtacagatttcaccttcaccattagcagcctgcaaccagaagatattg
ccacttattactgccagcaacgttatagtctgtggcgcacgttcggtcaa
ggcaccaagctggagatcaaa Variable heavy chain of 5L1721H23_6L3H3 (SEQ
ID NO: 24) caggtgcagctggtgcagtctggtgctgaggtgaagaagcctggcgcttc
cgtgaaggtttcctgcaaagcatctggttacacctttaccagctactata
tgcactgggtgcgccaagcccctggtcaaggcctggagtggatgggcgag
attcatcctagcggcggtcgtactaactacaatgagaagttcaagagccg
cgtgactatgactcgcgatacctccaccagcactgtctacatggaactga
gctctctgcgctctgaggacactgctgtgtattactgtgcccgcgagcgc
cccctgtatgctagcgacctgtggggccagggtaccacggtcaccgtctc ctca
[0242] These antibodies were expressed and purified for
determination of their K.sub.D at 7.4 and their K.sub.D ratio of
pH5.5/pH 7.4, as measured on a surface plasmon resonance Biacore
3000 biosensor equipped with a research-grade sensor chip using
HBS-EP running buffer (Biacore AB, Uppsala, Sweden--now GE
Healthcare). Rabbit polyclonal anti-Ms IgGs were amine-coupled at
saturating levels onto the chip using a standard
N-hydroxysuccinimide/ethyldimethylaminopropyl carbodiimide
(NHS/EDC) chemistry. The buffer was switched to HBS-EP+1 mg/mL
BSA+1 mg/mL CM-dextran. Full-length PCSK9 IgGs were diluted to
about 15 .mu.g/mL and captured for 1 min at 5 .mu.L/min to give
levels of about 500 RU per flow cell, leaving one blank to serve as
a reference channel. 3.73-302 nM hPCSK9 or 2.54-206 nM mPCSK9 were
injected as a 5-membered 3-fold series for 1 min at 100 .mu.L/min.
Dissociation was monitored for 5 min. The chip was regenerated
after the last injection of each titration with two 30 sec pulses
of 100 mM phosphoric acid. Buffer cycles provided blanks for
double-referencing the data, which were then fit globally to a
simple binding model using Biaevaluation software v.4.1. Affinities
were deduced from the quotient of the kinetic rate constants
(K.sub.D=k.sub.off/k.sub.on). The results for affinity to human
PCSK9 are shown in Table 5, and the results for affinity to mouse
PCSK9 are shown in Table 6. These data show that antibodies can be
designed and selected to have higher affinity for human or mouse
PCSK9 at pH 7.4, and a lower affinity at pH 5.5.
TABLE-US-00007 TABLE 5 Binding affinities for the anti-PCSK9
antibodies binding to human PCSK9 at pH 7.4 and 5.5. huPCS huPCS K9
pH K9 pH 7.4 5.5 pH kon Koff Rmax KD kon Koff Rmax KD 5.5/7.4
(1/Ms) (1/s) (RU) (nM) (1/Ms) (1/s) (RU) (nM) KD 6L1721 3.29E+04
7.08E-05 880 2.2 1.66E+05 8.40E-04 1059 5.1 2.3 5A10.B8 4.38E+04
1.04E-04 1080 2.4 1.31E+05 3.91E-04 1020 3.0 1.3 5L1721H23_6L3H3
4.42E+04 1.72E-04 934 3.9 8.28E+04 4.80E-03 1430 58 14.9 6L1721H23
5.02E+04 2.01E-04 645 4.0 1.10E+05 2.61E-03 1140 24 6 5L1721H23_6L3
3.09E+04 2.14E-04 974 6.9 6.45E+04 0.0166 1320 257 37.2
5L1721H23_6H3 3.74E+04 2.73E-04 461 7.3 1.41E+05 7.72E-03 398 55
7.5 5L1721H23 3.17E+04 3.99E-04 990 13 1.24E+05 0.0179 1030 144
11.1 5L1724H23_6L3H3 2.23E+04 3.42E-04 1020 15 9.55E+04 7.89E-03
1500 83 5.5 5L1724H23_6L3 3.59E+04 7.90E-04 910 22 7.54E+04 0.0407
1050 540 24.5 6L21H2335 5.81E+04 7.33E-03 892 126 6.55E+05 2.38E-01
624 363 2.9 5L1724H23 -- -- 20 -- 2.03E+05 7.93E-03 114 39
5L1724H35 -- -- 40 -- -- -- 0 --
TABLE-US-00008 TABLE 6 Binding affinities for the anti-PCSK9
antibodies binding to mouse PCSK9 at pH 7.4 and 5.5. msPCK9 msPCK9
pH 7.4 pH 5.5 huPSCK9 kon Koff Rmax KD kon Koff Rmax KD pH 5.5/7.4
(1/Ms) (1/s) (RU) (nM) (1/Ms) (1/s) (RU) (nM) KD 6L1721 8.80E+04
2.28E-04 1260 2.6 3.38E+05 1.77E-03 1120 5.2 2.0 5A10_WT 1.01E+05
4.85E-04 1410 4.8 3.73E+05 1.94E-03 1050 5.2 1.1 5L1721H23_6L3H3
6.37E+04 5.16E-04 1630 8.1 1.60E+05 0.0187 1420 117 14.4 6L1721H23
6.48E+04 4.69E-04 1230 7.2 2.04E+05 7.29E-03 1220 36 5.0
5L1721H23_6L3 4.75E+04 6.76E-04 1600 14 1.05E+05 0.0428 1360 408
29.1 5L1721H23_6H3 1.13E+05 8.99E-04 615 8.0 3.07E+05 0.0341 460
111 13.9 5L1721H23 6.12E+04 1.41E-03 1380 23 1.02E+05 0.0588 1100
576 25.0 5L1724H23_6L3H3 6.31E+04 8.27E-04 1690 13 2.11E+05 0.0263
1520 125 9.6 5L1724H23_6L3 5.31E+04 2.18E-03 1440 41 1.60E+05 0.121
1100 756 18.4 6L21H2335 8.99E+04 1.72E-02 1340 191 5.26E+05 0.237
502 451 2.4 5L1724H23 -- -- 40 -- 2.73E+05 2.02E-02 91 74 5L1724H35
4.43E+04 1.53E-03 164 35 -- -- 0 --
[0243] Affinity and kinetic parameters for PCSK9 antibodies h5A10
and 5L1721H23_6L3H3 as well as PCSK9 antibody H1M300N (see
US2010/0166768, e.g., at Table 7). All experiments were performed
on a Biacore 2000 biosensor.
[0244] An anti-human Fc sensor chip was prepared by activating all
flow cells of a Biacore CM4 sensor chip with a 1:1 (v/v) mixture of
400 mM EDC and 100 mM NHS for 7 minutes, at a flow rate of 10
.mu.L/min. An anti-human Fc reagent (Goat F(AB')2 Fragment to Human
IgG Fc, Cappel Catalog #: 55053) was diluted to 60 .mu.g/mL in 10
mM Sodium Acetate pH 5.0 and injected on all flow cells for 7
minutes at 20 .mu.L/min. All flow cells were blocked with 100 mM
ethylenediamine in 150 mM Borate buffer pH 8.5 for 7 minutes at 10
.mu.L/min.
[0245] The kinetics assay was run using a kinetic titration
methodology as described in Karlsson et al., Anal. Biochem 349,
136-147 (2006).
[0246] The same antibody (e.g., 5L1721H23_6L3H3) was captured onto
downstream flow cells (flow cells 2, 3 and 4) at 2 .mu.g/mL at a
flow rate of 10 .mu.L/m in for 30 seconds, 60 seconds and 120
seconds for flow cells 2, 3 and 4 respectively. Flow cell 1 was
used as a reference surface. Following capture of antibodies, PCSK9
was injected at 30 .mu.L/min on all flow cells in a series of
injections from low to high concentration. The top concentration
was 200 nM PCSK9 and the dilution factor was 3-fold. Each PCSK9
injection was two-minutes, the dissociation time after the 200 nM
PCSK9 injection was 20 minutes. A similar set of injections was
performed with running buffer in place of PCSK9 for
double-referencing purposes (double-referencing as described in
Myszka, J. Mol. Recognit 12, 279-284, 1999). After each analysis
cycle all flow cells were regenerated with three 30-second
injections of 75 mM Phosphoric Acid. The sensorgrams from flow
cells 2 and 3 for a given PCSK9/antibody pair were fit globally to
a simple 1:1 Langmuir with mass transport binding model.
[0247] The experiments were performed at pH 6.0 and 7.4 using
sample and running buffers of 10 mM Sodium Phosphate, 150 mM Sodium
Chloride, 0.05% Tween-20, pH 6 and 10 mM Sodium Phosphate, 150 mM
Sodium Chloride, 0.05% Tween-20, pH 7.4, respectively.
TABLE-US-00009 TABLE 7 pH dependent binding ratios pH 6.0 (Sodium
Phosphate) pH 7.4 (Sodium Phosphate) K.sub.D, k.sub.d, k.sub.a
k.sub.d t.sub.1/2 K.sub.D k.sub.a k.sub.d t.sub.1/2 K.sub.D pH
6/K.sub.D, pH 6/k.sub.d, Antibody (M.sup.-1s.sup.-1) (s.sup.-1)
(min) (pM) (M.sup.-1s.sup.-1) (s.sup.-1) (min) (pM) pH 7.4 pH 7.4
5A10_WT 3.31E+05 <6E-5 <193 <181 2.41E+05 .sup. <6E-05
<193 <249 5L1721H_236L3H3 3.07E+05 2.86E-03 4.0 9316 2.42E+05
1.74E-04 66.4 719 13.0 16.4 H1M300N 4.27E+05 3.21E-04 36.0 752
1.67E+05 6.92E-05 166.9 414 1.8 4.6
Example 3: PH Dependent PCSK9 Binding Antibodies have an Extended
Pharmacodynamic Effect on Lowering Cholesterol
A. pH Dependent PCSK9 Binding Antagonist Antibodies Lower Serum
Cholesterol for an Extended Duration in Mice
[0248] To determine if pH dependent PCSK9 binding antagonist
antibodies can lower cholesterol levels in vivo for an extended
duration as compared to non-pH dependent antibodies, the time
course effects of acid sensitive antibodies 5L1721H23_6H3 and
5L1721H23_6L3H3 and non-acid sensitive antibodies h5A10 (only dosed
at 10 mg/kg) and 5A10.138 were tested on serum cholesterol when
injected at 1, 3, or 10 mg/kg into mice. All four antibodies have
similar binding affinities of 5-14 nM at neutral pH (PH 7.4) to
mouse PCSK9. Antibodies 5L1721H23_6H3 and 5L1721H23 6L3H3 have
reduced affinities of 117 nM and 408 nM, respectively, at pH 5.5,
whereas h5A10 and 5A10.138 have K.sub.D at pH 5.5 similar to that
of 7.4 (5.2 nM). Male C57/bl6 mice, 6 to 7 weeks old, were kept on
a 12 hr light/dark cycle, bled to collect approximately 70 .mu.l
serum on day -7. Antagonist PCSK9 antibodies and a control isotype
matching monoclonal antibody that does not bind to any known
mammalian proteins were injected IV into male 7 week old C57/bl6
mice and serum samples were collected on days 5, 12, 19, 26, 61,
and 75 post-injection. All serum samples were analyzed for total
cholesterol, triglyceride, HDL cholesterol on the Ace Alera
instrument (Alfa Wassermann, West Caldwell, N.J.) and LDL
cholesterol levels were calculated using Friedewald equation. FIG.
7 shows a rapid and dose-dependent decrease in total cholesterol
levels following injection of a PCSK9 antagonist antibody. LDL
cholesterol levels in mice were too low to be reliably measured and
calculated. At 10 mg/kg dose, all four antibodies lowered
HDL-cholesterol by 35-40% on days 5 and 12, while 5L1721H23_6H3 and
5L1721H23_6L3H3 injected animals did not recover until day 61,
h5A10 and 5A10.138 injected animals returned to baseline levels on
days 26 and 33, respectively.
B. pH Dependent PCSK9 Binding Antibodies have an Extended Half-Life
in Mice
[0249] Serum concentrations of antibody were determined in the same
study described in example b to determine whether PH sensitive
anti-PCSK9 antibodies resulted in extended antibody half-life.
Normal anti-PCSK9 antibodies such as h5A10 and 5A10.136 have
dose-dependent shorter half life compared to antibodies that binds
to other soluble antigens, due to PCSK9-mediated degradation of
antibody/antigen complex. As shown in FIG. 8A, the pH dependent
binding property reduced antibody degradation and extended the half
life of anti-PCSK9 antibodies 5L1721H23_6H3 and 5L1721H23_6L3H3. To
further demonstrate that the prolonged PK of 5L1721H23_6H3 and
5L1721H23 6L3H3 was a result of diminished PCSK9-mediated clearance
of antibodies, a similar time course study was conducted in PCSK9
knockout mice. The differences in serum antibody concentrations and
the rates of reduction between the PH sensitive and non-sensitive
antibodies were insignificant until 3 mg/kg human PCSK9 was
injected in the mice. Following this injection, the non-PH
sensitive PCSK9 antibodies demonstrated increased degradation as
compared to the PH sensitive antibodies and the negative control
antibody (FIG. 8B). These results indicate that the observed
decreased degradation of pH dependent PCSK9 binding PCSK9
antibodies results from dissociation of the antibody from PCSK9
and, therefore, rescue of the antibody from PCSK9-mediated
degradation.
C. pH Sensitive PCSK9 Antagonist Antibodies Lower Serum Cholesterol
for an Extended Duration in Monkeys
[0250] FIG. 9B illustrates the effect of PH sensitive anti-PCSK9
antagonist antibodies 5L1721H23_6H3 and 5L1721H23_6L3H3 and non
sensitive anti-PCSK9 antibody L1L3 on serum LDL-cholesterol levels
of cynomolgus as percent control. Antibodies (1.5 mg/kg of each)
were administered on day 0 to female cynomolgus monkeys via i.v.
bolus injection. The LDL-cholesterol was reduced to 50% of baseline
by day in all three antibody-treated groups. While the
LDL-cholesterol levels returned to baseline by day 10 following
administration of non-pH sensitive antibody, LDL cholesterol stayed
suppressed until day 21 in monkeys treated with pH sensitive
antibodies. HDL levels remained essentially unchanged as a result
of antibody treatment (FIG. 9A). FIG. 10 demonstrates that the half
life of pH sensitive anti-PCSK9 antibodies were extended as
compared to non-pH sensitive L1L3.
Example 4: General Modeling for Antibodies with pH Dependent
Antigen Binding
[0251] Computer modeling was used to predict whether an antibody
with pH dependent binding to its generic antigen could impact
antibody half life and/or the duration of lowering the antigen's
amount or serum concentration. For purposes of this modeling, the
following assumptions were made: 1) antibody, 1 uM dose of antibody
in blood, 21 day antibody half-life; 2) simulation run for 100
days; antibody binding and pH dependent binding, K.sub.on=1e5/M/s,
K.sub.off @ neutral pH=K.sub.D*K.sub.on; pH dependent binding is
modeled as an increase in K.sub.off in the acidic endosomes,
K.sub.off at acidic pH=R*K.sub.off at neutral pH.
[0252] FIG. 15 details the trafficking model for antibodies with pH
dependent binding used for modeling, and the equations defining the
model are as follows:
d/dt(Normal_Dose)=-k_dist*Normal_Dose
d/dt(mAb)=k_dist*Normal_Dose-(mAb*kon*Normal_antigen)+(kon*KD*mAb_antige-
n-k_blood_endo*mAb/V.sub.Norm+f1*k_blood_endo*mAb_e/V.sub.Norm+(1-f1)*f2*k-
_blood_endo*cmp1x_1e/V.sub.Norm
d/dt(mAb_antigen)=(mAb*kon*Normal_antigen)-(kon*KD*mAb_antigen)-k_blood_-
endo*mAb_antigen/+V.sub.Norm+f1*k_blood_endo*mAb_antigen_e/V.sub.Norm+(1-f-
1)*f2*k_blood_endo*cmp1x_1e/V.sub.Norm
d/dt(Normal_antigen)=(mAb*kon*Normal_antigen)+(kon*KD*mAb_antigen)+1n(2)-
/antigen_halflife*antigen_level-1n(2)/antigen_halflife*Normal_antigen-k_bl-
ood_endo*Normal_antigen/V.sub.Norm+f1*k_blood_endo*Endosomes_antigen_e/V.s-
ub.Norm
d/dt(mAb_e)=k_blood_endo*mAb/V.sub.Endo-f1*k_blood_endo*mAb_e/V.sub.Endo-
-(mAb_e*kon*Endosomes_antigen_e)+(kon*KD*mAb_antigen_e)-(1-f1)*k_blood_end-
o*mAb_e/V.sub.Endo
d/dt(mAb_antigen_e)=k_blood_endo*mAb_antigen/V.sub.Endo-f1*k_blood_endo*-
mAb_antigen_e/V.sub.Endo+(mAb_e*kon*Endosomes_antigen_e)-(kon*KD*mAb_antig-
en_e)-(1-f1)*k_blood_endo*mAb_antigen_e/V.sub.Endo
d/dt(Endosomes_antigen_e)=k_blood_endo*Normal_antigen/V.sub.Endo-f1*k_bl-
ood_endo*Endosomes_antigen_e/V.sub.Endo-(mAb_e*kon*Endosomes_antigen_e)+(k-
on*KD*mAb_antigen_e)-(1-f1)*k_blood_endo*Endosomes_antigen_e/V.sub.Endo
d/dt(mAb_1e)=-(1-f1)*f2*k_blood_endo*mAb_1e/V.sub.LEndo+(1-f1)*k_blood_e-
ndo*mAb_e/V.sub.LEndo-(mAb_1e*kon*Antigen_1e)+(R*kon*KD*cp1x_1e)-(1-f1)*(1-
-f2)*k_blood_endo*mAb_1e/V.sub.LEndo
d/dt(cp1x_1e)=-(1-f1)*f2*k_blood_endo*cp1x_1e/V.sub.LEndo+(1-f1)*k_blood-
_endo*mAb_antigen_e/V.sub.LEndo+(mAb_1e*kon*Antigen_1e)-(R*kon*KD*cp1x_1e)-
-(1-f1)*(1-f2)*k_blood_endo*cp1x_1e/V.sub.Endo
d/dt(Antigen_1e)=+(1-f1)*k_blood_endo*Endosomes_antigen_e/V.sub.LEndo-(m-
Ab_1e*kon*Antigen_1e)+(R*kon*KD*cp1x_1e)-(1-f1)*k_brood_endo*Antigen_1e/V.-
sub.LEndo
[0253] The parameters used in the model are described below in
Table 8.
TABLE-US-00010 TABLE 8 VNorm 5.6 L Volume of blood VEndo 0.06 L
Volume of early endosomes VLEndo 0.06 L Volume of late endosomes
k_dist 0.48/days Rate of transport into blood kon 0.0001/nM/s
mAb-antigen association rate k_blood_endo 11.5 L/day Rate of
endosomal internalization f1 0.7 Fraction recycling from early
endosomes f2 0.95 Fraction mAb recycling from late endosomes
K.sub.D variable Binding affinity [nM] R variable Ratio of affinity
at endosomal vs. neutral pH antigen_halflife variable Half-life of
antigen in blood [days] antigen_level variable Level of antigen in
the blood [nM]
[0254] The heatmaps shown in FIG. 4 show how much more an antibody
with pH dependent binding would impact antigen knockdown beyond
knockdown by an antibody without pH dependent binding. This
knockdown quantity is determined by measuring of the area between
the free antigen curves for the two antibodies over the 100 day
simulation. It can be thought of as how many days longer an
antibody with pH dependent binding would impact antigen knockdown
beyond knockdown by an antibody without pH dependent binding,
though these two metrics do not have to correlate exactly. Lighter
areas on the map reflect longer duration and/or stronger knockdown
of free antigen. The heatmap for FIG. 11 shows how the potential
antibodies with pH dependent antigen binding would impact antigen
knockdown if the antigen was DKK1, IgE or C5. For each of these
antigens, an antibody with pH dependent binding could extend
antigen knockdown.
[0255] FIG. 12 shows more detailed modeling for an antibody with pH
dependent binding to IgE. An extended duration of IgE knockdown
occurs of the antibody has a K.sub.D of about 1 nM or greater and a
K.sub.D ratio at pH 6.0/pH7.4 (R) of 3 or more.
[0256] Similar analysis for the antigen DKK1 is shown in FIG. 13
and for C5 in FIG. 14. For DKK1, an ideal range of K.sub.D is
1.0-100 nM across the K.sub.D ratio range of 3-30. For C5, the
ideal range of K.sub.D is also 1.0-100 nM for the K.sub.D ratio
range of 3-30.
Example 5: Generating and Screening Anti-IgE Antibodies with pH
Dependent IgE Binding
[0257] Histidine substitutions were made at the positions of the
underlined residues in anti-IgE antibody 5.948-H100Y, which has the
following variable light chain (V.sub.L) and variable heavy chain
(V.sub.H) amino acid sequences (CDRs are in bold font).
TABLE-US-00011 VL (SEQ ID NO: 25)
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHRNGYNYLDWYLQKPGQSP
QLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQ TPPATFGGGTKVEIK
VH (SEQ ID NO: 26)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWMG
WMDPNSGNTGYAQKFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCAR
GYYDSDGYYSFSGMDVWGQGTTVTVSS
[0258] The antibodies were tested for IgE binding at 25.degree. C.
For determination of affinities and kinetic constants, all
experiments were performed on a Biacore T200 biosensor.
[0259] A biotin-capture surface was prepared by immobilization of
NeutrAvidin: all flow cells of a Biacore CM4 sensor chip were
activated with a 1:1 (v/v) mixture of 400 mM EDC and 100 mM NHS for
7 minutes, at a flow rate of 10 .mu.L/min. NeutrAvidin (Pierce,
product #: 31000) was diluted to 100 .mu.g/mL in 10 mM Sodium
Acetate pH 4.5 and injected on all flow cells for 7 minutes at 20
.mu.L/min. All flow cells were blocked with 100 mM Ethylenediamine
in 150 mM Borate buffer pH 8.5 for 7 minutes at 10 .mu.L/min.
[0260] The IgE construct (human IgE-Fc (Ch2-Ch3-Ch4), expressed and
purified in-house) was minimally biotinylated by incubating IgE
with an equimolar amount of EZ-Link NHS-LC-LC-biotin (Thermo
Scientific, catalog #21343) for 30 min at room temperature. Free
biotin was removed via buffer exchange.
[0261] All Biacore experiments were performed using two of the four
flow cells (1&2 or 3&4) of the sensor chip in parallel. The
downstream flow cell was used as analysis flow cell while the other
flow cell was used as a reference surface. Thus, biotinylated IgE
(B-IgE) was captured on the analysis flow cell and every purified
Fab fragment was injected over both. The sensorgram from the
reference flow cell was subtracted from that of the analysis flow
cell.
[0262] B-IgE was diluted into running buffer to a final
concentration of 5 ug/mL immediately before capture. At pH 6.0,
B-IgE was injected at 10 uL/min for 60 seconds. At pH 7.4, B-IgE
was injected for 80 seconds to obtain similar capture levels. The
kinetics assay was run using a kinetic titration methodology as
described in Karlsson et al., Anal Biochem 349: 136-147, 2006.
[0263] Following capture of B-IgE, a purified Fab fragment was
injected at 30 .mu.L/min on analysis and reference flow cells in a
series of injections from low to high concentration. The top
concentration of Fab ranged from 30 nM to 200 nM and the dilution
factor was 3-fold. Each Fab dilution was injected for 2 minutes,
followed by a final dissociation time of 30 minutes after the last
injection. A similar set of injections was performed with running
buffer in place of Fab for double-referencing purposes
(double-referencing as described in Myszka, J Mol Recognit 12:
279-284, 1999). The double-referenced sensorgrams were fit globally
to a simple 1:1 Langmuir with mass transport binding model.
[0264] The experiments were performed at pH 6.0 and 7.4 using
sample and running buffers of 10 mM Sodium Phosphate, 150 mM Sodium
Chloride, 0.05% Tween-20, 1 mg/mL BSA, pH 6 and 10 mM Sodium
Phosphate, 150 mM Sodium Chloride, 0.05% Tween-20, 1 mg/mL BSA, pH
7.4, respectively.
[0265] As shown in Table 9 below, histidine substitution in the IgE
antibody 5.948-H100Y successfully resulted in antibodies with a
faster k.sub.off at pH 6.0 and higher K.sub.D and k.sub.off ratios
at pH 6.0/pH 7.4.
TABLE-US-00012 TABLE 9 Effect of histidine substitution on antigen
binding by IgE antibodies pH 6.0 pH 7.4 kd- KD ratio kd KD KD pH
6/kd- pH 6.0/pH ka(M-1S-1) kd (s-1) (min) (M) ka(M-1S-1) kd (s-1)
kd (min) (M) pH 7.4 7.4 H100Y 1.02E+06 1.52E-04 76.00 1.49E-10
1.03E+06 5.01E-05 230.5879 4.87E-11 3.03 3.1 L38H312H100Y 3.29E+05
1.22E-02 0.95 3.71E-08 2.02E+05 1.08E-03 10.69672 5.33E-09 11.30
7.0 H25H100Y 2.98E+05 1.51E-03 7.65 5.08E-09 3.11E+05 1.97E-04
58.64189 6.34E-10 7.66 8.0 H17H25H100Y 2.23E+05 3.64E-03 3.17
1.63E-08 2.22E+05 4.22E-04 27.37548 1.90E-09 8.63 8.6
[0266] The disclosures of all references cited herein are hereby
incorporated by reference herein.
Sequence CWU 1
1
271107PRTHomo sapien 1Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser
Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Tyr Arg Tyr Thr
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr
Tyr Tyr Cys Gln Gln Arg Tyr Ser Thr Pro Arg 85 90 95Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 1052118PRTHomo sapien 2Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Glu Ile Asn Pro Ser Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe
50 55 60Lys Ser Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Glu Arg Pro Leu Tyr Ala Met Asp Tyr Trp
Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser 1153107PRTHomo
sapien 3Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val His
Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45Tyr His Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile
Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln
Gln Arg Tyr Ser Leu Trp Arg 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys 100 1054118PRTHomo sapien 4Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Glu Ile His
Pro Ser Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe 50 55 60Lys Ser Arg
Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Glu Arg Pro Leu Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110Thr Val Thr Val Ser Ser 1155118PRTHomo sapien 5Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25
30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45Gly Glu Ile His Pro Ser Gly Gly Arg Thr Asn Tyr Asn Glu Lys
Phe 50 55 60Lys Ser Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr
Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Glu Arg Pro Leu Tyr Ala Ser Asp Leu
Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser
115610PRTHomo sapien 6Gly Tyr Thr Phe Thr Ser Tyr Tyr Met His1 5
10717PRTHomo sapien 7Glu Ile His Pro Ser Gly Gly Arg Thr Asn Tyr
Asn Glu Lys Phe Lys1 5 10 15Ser89PRTHomo sapien 8Glu Arg Pro Leu
Tyr Ala Met Asp Tyr1 599PRTHomo sapien 9Glu Arg Pro Leu Tyr Ala Ser
Asp Leu1 51011PRTHomo sapien 10Lys Ala Ser Gln Asp Val His Thr Ala
Val Ala1 5 10117PRTHomo sapien 11His Ala Ser Tyr Arg Tyr Thr1
5129PRTHomo sapien 12Gln Gln Arg Tyr Ser Leu Trp Arg Thr1
51311PRTHomo sapien 13Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala1
5 101411PRTHomo sapien 14Lys Ala Ser Gln Asp His Ser Thr Ala Val
Ala1 5 101511PRTHomo sapien 15Arg Ala Ser Gln Gly Ile Ser Ser Ala
Leu Ala1 5 101611PRTHomo sapien 16Arg Ala Ser Gln Gly Ile His Ser
Ala Leu Ala1 5 10177PRTHomo sapien 17Ser Ala Ser Tyr Arg Tyr Thr1
5187PRTHomo sapien 18Ser Ala Ser His Arg Tyr Thr1 51917PRTHomo
sapien 19Glu Ile Asn Pro Ser Gly Gly Arg Thr Asn Tyr Asn Glu Lys
Phe Lys1 5 10 15Ser2017PRTHomo sapien 20Glu Ile Ser Pro Phe Gly Gly
Arg Thr Asn Tyr Asn Glu Lys Phe Lys1 5 10 15Ser219PRTHomo sapien
21Glu Arg Pro Leu His Ala Met Asp Tyr1 52215PRTHomo sapien 22Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10
1523321DNAHomo sapien 23gatatccaga tgacacagtc cccatcctcc ctgtctgcct
ctgtgggcga ccgcgtcacc 60atcacctgca aggcctctca ggatgtgcat actgctgtag
cctggtatca gcagaagcca 120ggcaaagccc caaaactgct gatctaccat
gcatcctacc gctacactgg tgtcccatca 180cgcttcagtg gcagtggctc
tggtacagat ttcaccttca ccattagcag cctgcaacca 240gaagatattg
ccacttatta ctgccagcaa cgttatagtc tgtggcgcac gttcggtcaa
300ggcaccaagc tggagatcaa a 32124354DNAHomo sapien 24caggtgcagc
tggtgcagtc tggtgctgag gtgaagaagc ctggcgcttc cgtgaaggtt 60tcctgcaaag
catctggtta cacctttacc agctactata tgcactgggt gcgccaagcc
120cctggtcaag gcctggagtg gatgggcgag attcatccta gcggcggtcg
tactaactac 180aatgagaagt tcaagagccg cgtgactatg actcgcgata
cctccaccag cactgtctac 240atggaactga gctctctgcg ctctgaggac
actgctgtgt attactgtgc ccgcgagcgc 300cccctgtatg ctagcgacct
gtggggccag ggtaccacgg tcaccgtctc ctca 35425113PRTHomo sapien 25Asp
Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10
15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Arg
20 25 30Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly
Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Met Gln Ala 85 90 95Leu Gln Thr Pro Pro Ala Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile 100 105 110Lys26125PRTHomo sapien 26Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25
30Asp Ile Asn Tr