U.S. patent application number 15/596867 was filed with the patent office on 2017-12-07 for immunoglobulins with reduced aggregation.
This patent application is currently assigned to Novartis AG. The applicant listed for this patent is Massachusetts Institute of Technology, Novartis AG. Invention is credited to Naresh CHENNAMSETTY, Bernhard HELK, Veysel KAYSER, Bernhardt TROUT, Vladimir VOYNOV.
Application Number | 20170349650 15/596867 |
Document ID | / |
Family ID | 41165439 |
Filed Date | 2017-12-07 |
United States Patent
Application |
20170349650 |
Kind Code |
A1 |
CHENNAMSETTY; Naresh ; et
al. |
December 7, 2017 |
IMMUNOGLOBULINS WITH REDUCED AGGREGATION
Abstract
The present disclosure relates to immunoglobulins with reduced
aggregation and compositions, methods of generating such
immunoglobulins with computational tools and methods of using such
immunoglobulins particularly in the treatment and prevention of
disease.
Inventors: |
CHENNAMSETTY; Naresh;
(Cambridge, MA) ; HELK; Bernhard; (Basel, CH)
; KAYSER; Veysel; (Cambridge, MA) ; TROUT;
Bernhardt; (Cambridge, MA) ; VOYNOV; Vladimir;
(Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novartis AG
Massachusetts Institute of Technology |
Basel
Cambridge |
MA |
CH
US |
|
|
Assignee: |
Novartis AG
Basel
MA
Massachusetts Institute of Technology
Cambridge
|
Family ID: |
41165439 |
Appl. No.: |
15/596867 |
Filed: |
May 16, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14265149 |
Apr 29, 2014 |
9676841 |
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15596867 |
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13000351 |
Sep 20, 2011 |
8747848 |
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PCT/US2009/047948 |
Jun 19, 2009 |
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14265149 |
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61151368 |
Feb 10, 2009 |
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61074466 |
Jun 20, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/02 20180101;
A61P 1/00 20180101; A61P 25/28 20180101; A61P 27/06 20180101; A61P
31/00 20180101; A61P 37/02 20180101; A61P 9/04 20180101; A61P 43/00
20180101; A61P 9/14 20180101; A61P 19/08 20180101; A61P 35/00
20180101; C07K 2317/565 20130101; A61P 3/02 20180101; A61P 31/18
20180101; A61P 31/04 20180101; C07K 2317/55 20130101; A61P 27/02
20180101; C07K 16/18 20130101; C07K 2317/50 20130101; A61P 25/00
20180101; A61P 1/02 20180101; A61P 31/12 20180101; A61P 29/00
20180101; A61P 37/00 20180101; A61P 9/00 20180101; A61P 19/04
20180101; A61P 19/10 20180101; C07K 16/1063 20130101; A61P 17/06
20180101; C07K 2317/52 20130101; C07K 2317/92 20130101; C07K
2317/21 20130101; C07K 2299/00 20130101; A61K 39/39591 20130101;
A61P 25/02 20180101; A61P 17/00 20180101; A61P 17/02 20180101; A61P
19/02 20180101; A61P 1/04 20180101; A61P 7/06 20180101; A61P 3/10
20180101; A61P 19/06 20180101; A61P 37/06 20180101; C07K 2317/41
20130101; A61P 1/16 20180101; A61P 35/04 20180101; A61P 3/14
20180101 |
International
Class: |
C07K 16/18 20060101
C07K016/18; A61K 39/395 20060101 A61K039/395; C07K 16/10 20060101
C07K016/10 |
Claims
1: A modified immunoglobulin formulation comprising a modified or
isolated immunoglobulin having reduced propensity for aggregation
comprising at least one aggregation reducing mutation at a residue
in a conserved domain of the immunoglobulin that (i) has a
Spatial-Aggregation-Propensity (5 .ANG. radius sphere) of at least
0.15, or (ii) has an Spatial-Aggregation-Propensity (5 .ANG. radius
sphere) of greater than 0.0 and is within 5 .ANG. of a residue
having a Spatial-Aggregation-Propensity (5 .ANG. radius sphere) of
at least 0.15, wherein the at least one aggregation reducing
mutation is a substitution with an amino acid residue that lowers
the Spatial-Aggregation-Propensity (5 .ANG. radius sphere) of the
residue as compared to the unmutated immunoglobulin and the
propensity for aggregation that is reduced is aggregation between
immunoglobulin molecules in a concentrated, liquid solution,
wherein the at least one aggregation reducing mutation is not at
residue 234(hinge) or 235(hinge), wherein the modified or isolated
immunoglobulin is at a concentration of at least 75 mg/mL, wherein
at least eighty percent of the modified or isolated immunoglobulin
is non-aggregated monomer, and wherein the modified or isolated
immunoglobulin comprises a substitution at 253(C.sub.H2).
2: The modified immunoglobulin formulation of claim 1, further
comprising at least one additional aggregation reducing mutation
residue selected from the group consisting of residues from an
aggregation motif 1: 174(C.sub.H1), 175(C.sub.H1), and
181(C.sub.H1); an aggregation motif 2: 226(hinge), 227(hinge),
228(hinge), 229(hinge), 230(hinge), 231(hinge), and 232(hinge); an
aggregation motif 3: 234(hinge) and 235(hinge); an aggregation
motif 4: 252(C.sub.H2); an aggregation motif 5: 282(C.sub.H2); an
aggregation motif 6: 291(C.sub.H2); an aggregation motif 7:
296(C.sub.H2); an aggregation motif 8: 308(C.sub.H2) and
309(C.sub.H2); an aggregation motif 9: 328(C.sub.H2),
329(C.sub.H2), 330(C.sub.H2), and 331(C.sub.H2); an aggregation
motif 10: 395(C.sub.H3), 396(C.sub.H3), 397(C.sub.H3),
398(C.sub.H3), and 404(C.sub.H3); an aggregation motif 11:
443(C.sub.H3); an aggregation motif 12: 110(C.sub.L) and
111(C.sub.L); an aggregation motif 13: 153(C.sub.L) and
154(C.sub.L); and an aggregation motif 14: 201(C.sub.L).
3: The modified immunoglobulin formulation of claim 2, wherein the
at least one aggregation reducing mutation residue is (i) selected
from the group consisting of residues from an aggregation motif 1:
175(C.sub.H1); an aggregation motif 2: 227(hinge), 228(hinge), and
230(hinge); an aggregation motif 3: 234(hinge) and 235(hinge); an
aggregation motif 5: 282(C.sub.H2); an aggregation motif 6:
291(C.sub.H2); an aggregation motif 7: 296(C.sub.H2); an
aggregation motif 8: 309(C.sub.H2); an aggregation motif 9:
329(C.sub.H2) and 330(C.sub.H2); an aggregation motif 10:
395(C.sub.H3) and 398(C.sub.H3); an aggregation motif 11:
443(C.sub.H3); an aggregation motif 12: 110(C.sub.L); an
aggregation motif 13: 154(C.sub.L); and an aggregation motif 14:
201(C.sub.L).
4: A modified immunoglobulin formulation comprising a modified or
isolated immunoglobulin having reduced propensity for aggregation
comprising at least one aggregation reducing mutation, wherein the
at least one aggregation reducing mutation is not at residue
234(hinge) or 235(hinge), and wherein the modified or isolated
immunoglobulin comprises at least one aggregation reducing mutation
at residue 253(C.sub.H2), wherein residue 253 is mutated to a
lysine, wherein the modified or isolated immunoglobulin is at a
concentration of at least 75 mg/mL, and wherein at least eighty
percent of the modified or isolated immunoglobulin is
non-aggregated monomer.
5: The modified immunoglobulin formulation of claim 1, wherein the
aggregation reducing mutation is a substitution with an amino acid
residue that is less hydrophobic than the residue in the unmodified
immunoglobulin.
6: The modified immunoglobulin formulation of claim 1, wherein the
immunoglobulin further comprises a binding affinity for a target
antigen and the binding affinity for the target antigen is at least
seventy percent of the binding affinity of the unmutated
immunoglobulin for the target antigen.
7. (canceled)
8: The modified immunoglobulin formulation of claim 1, wherein at
least eighty-five percent of the modified or isolated
immunoglobulin is non-aggregated monomer.
9: The modified immunoglobulin formulation of claim 1, further
comprising a pharmaceutically acceptable excipient.
10: The modified immunoglobulin formulation of claim 1, wherein the
immunoglobulin formulation shows at least five percent less
aggregate after twenty four hours of accelerated aggregation as
compared to the unmutated immunoglobulin under the same
conditions.
11: The modified immunoglobulin formulation of claim 1, wherein the
immunoglobulin formulation is substantially free of any additive
that reduces aggregation of immunoglobulins.
12: An isolated or recombinant polynucleotide encoding the
immunoglobulin of claim 1.
13: A vector comprising the polynucleotide of claim 12, optionally
comprising an inducible promoter operably linked to the
polynucleotide.
14: A host cell comprising the vector of claim 13.
15: A method of producing an immunoglobulin with a reduced
aggregation propensity comprising: (a) providing a culture medium
comprising the host cell of claim 14; and (b) placing the culture
medium in conditions under which the immunoglobulin is expressed;
and (c) optionally, isolating the immunoglobulin.
16: A modified immunoglobulin formulation comprising a modified or
isolated immunoglobulin having reduced propensity for aggregation
comprising at least one aggregation reducing mutation at a residue
in a conserved domain of the immunoglobulin that (i) has a
Spatial-Aggregation-Propensity (5 .ANG. radius sphere) of at least
0.15, or (ii) has an Spatial-Aggregation-Propensity (5 .ANG. radius
sphere) of greater than 0.0 and is within 5 .ANG. of a residue
having a Spatial-Aggregation-Propensity (5 .ANG. radius sphere) of
at least 0.15, wherein the aggregation reducing mutation is a
substitution with an amino acid residue that lowers the
Spatial-Aggregation-Propensity (5 .ANG. radius sphere) of the
residue as compared to the unmutated immunoglobulin and the
propensity for aggregation that is reduced is aggregation between
immunoglobulin molecules in a concentrated, liquid solution, and a
second aggregation reducing mutation at a residue that (i) has a
Spatial-Aggregation-Propensity (5 .ANG. radius sphere) of at least
0.15, or (ii) has an Spatial-Aggregation-Propensity (5 .ANG. radius
sphere) of greater than 0.0 and is within 5 .ANG. of a residue
having a Spatial-Aggregation-Propensity (5 .ANG. radius sphere) of
at least 0.15, wherein the second aggregation reducing mutation is
a substitution with an amino acid residue that lowers the
Spatial-Aggregation-Propensity (5 .ANG. radius sphere) of the
residue as compared to the unmutated immunoglobulin, wherein the
aggregation reducing mutation and the second aggregation reducing
mutation are in different aggregation motifs, wherein the modified
or isolated immunoglobulin is at a concentration of at least 75
mg/mL, wherein at least eighty percent of the modified or isolated
immunoglobulin is non aggregated monomer, and wherein the modified
or isolated immunoglobulin comprises a substitution at
253(C.sub.H2).
17: The modified immunoglobulin formulation of claim 16, wherein
the aggregation reducing mutation is a substitution with an amino
acid residue that is less hydrophobic than the residue in the
unmodified immunoglobulin.
18: The modified immunoglobulin formulation of claim 16, wherein
the immunoglobulin further comprises a binding affinity for a
target antigen and the binding affinity for the target antigen is
at least seventy percent of the binding affinity of the unmutated
immunoglobulin for the target antigen.
19-21. (canceled)
22: The modified immunoglobulin formulation of claim 1, wherein the
immunoglobulin formulation is free of free histidine, saccharides,
and polyols.
23: The modified immunoglobulin formulation of claim 1, wherein
residue 253(C.sub.H2) is mutated to a lysine.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. patent application
Ser. No. 14/265,149, filed Apr. 29,2014; which is a Divisional of
U.S. patent application Ser. No. 13/000,351, internationally filed
Jun. 19, 2009, now issued as U.S. Pat. No. 8,747,848; which is a
U.S. National Phase application under 35 U.S.C. .sctn.371 of
International Application No. PCT/US2009/047948, filed Jun. 19,
2009; which claims the benefit of U.S. Provisional Patent
Application Nos. 61/074,466, filed Jun. 20, 2008, and 61/151,368,
filed Feb. 10, 2009; all of which are hereby incorporated by
reference in the present disclosure in their entirety.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
619672000111SeqList.txt, date recorded: May 11, 2017, size: 13
KB).
FIELD OF THE INVENTION
[0003] The present disclosure relates to improved immunoglobulins
having reduced aggregation.
BACKGROUND
[0004] Understanding and controlling protein stability has been a
coveted endeavor to Biologists, Chemists, and Engineers. The first
link between amino acid substitution and disease (Ingram. Nature.
1957, 180(4581):326-8.) offered a new and essential perspective on
protein stability in health and disease. The recent tremendous
increase of protein-based pharmaceuticals, particularly
immunoglobulin based pharmaceuticals, has created a new challenge.
Therapeutic proteins are stored in liquid for several months at
very high concentrations. The percent of non-monomeric species
increases with time. As aggregates form, not only the efficacy of
the product decreases, but side effects such as immunological
response upon administration may occur. Assuring stability of
protein pharmaceuticals for the shelf-life of the product is
imperative.
[0005] Because of their potential in the cure of various diseases,
antibodies currently constitute the most rapidly growing class of
human therapeutics (Carter. Nature Reviews Immunology. 2006, 6(5),
343). Since 2001, their market has been growing at an average
yearly growth rate of 35%, the highest rate among all categories of
biotech drugs (S. Aggarwal. Nature. BioTech. 20 2007, 25 (10)
1097).
[0006] Therapeutic immunoglobulins are prepared and stored in
aqueous solutions at high concentrations, as required for the
disease treatment. However, these immunoglobulins are
thermodynamically unstable under these conditions and degrade due
to aggregation. The aggregation in turn leads to a decrease in
antibody activity making the drug ineffective and can even generate
an immunological response. Thus, there is an urgent need to
generate therapeutic immunoglobulins that are less prone to
aggregation.
[0007] Numerous existing approaches for preventing immunoglobulin
aggregation employ the use of additives in protein formulations.
This is different from the direct approach described herein where
immunoglobulin itself is modified based on the aggregation prone
regions predicted from molecular simulations. Additives commonly
used in antibody stabilization are salts of nitrogen-containing
bases, such as arginine, guanidine, or imidazole (EP0025275). Other
suitable additives for stabilization are polyethers (EPA0018609),
glycerin, albumin and dextran sulfate (U.S. Pat. No. 4,808,705),
detergents and surfactants such as polysorbate based surfactants
(Publication DE2652636, and Publication GB2175906 (UK Pat. Appl.
No. GB8514349)), chaperones such as GroEL (Mendoza. Biotechnol.
Tech. 1991, (10) 535-540), citrate buffer (WO9322335) or chelating
agents (WO9225509). Although these additives enable proteins to be
stabilized to some degree in solution, they suffer from certain
disadvantages such as the necessity of additional processing steps
for additive removal.
[0008] Optimized immunoglobulin variants have been generated to
improve other characteristics such as binding of the Fc receptor.
By way of example, a genus of two hundred and sixteen antibody
variants were generated (including L234 and L235 mutant species)
and tested for the effect upon binding to Fc.gamma.RIIIa and
Fc.gamma.RIIb as disclosed in US Pat. Publ. 2004/0132101 (Lazar et
al.). However, Lazar et al. did not test any of the antibody
variants for their propensity for aggregation.
[0009] Thus, there is a need for improved immunoglobulin
compositions, such as antibody therapeutics, that are directly
stabilized without the use of additives.
SUMMARY
[0010] Described herein are improved immunoglobulins which exhibit
reduced aggregation and/or enhanced stability that meet this
need.
[0011] Thus one aspect includes modified and/or isolated
immunoglobulins that have a reduced propensity for aggregation
comprising at least one aggregation reducing mutation at a residue
in a conserved domain of the immunoglobulin that (i) has a
Spatial-Aggregation-Propensity (5 .ANG. radius sphere) of at least
0.15, or (ii) has an Spatial-Aggregation-Propensity (5 .ANG. radius
sphere) of greater than 0.0 and is within 5 .ANG. of a residue
having a Spatial-Aggregation-Propensity (5 .ANG. radius sphere) of
at least 0.15, wherein the at least one aggregation reducing
mutation is a substitution with an amino acid residue that lowers
the Spatial-Aggregation-Propensity (5 .ANG. radius sphere) of the
residue as compared to the unmutated immunoglobulin and the
propensity for aggregation that is reduced is aggregation between
immunoglobulin molecules in a concentrated, liquid solution. In
certain embodiments, the at least one aggregation reducing mutation
is not at a residue corresponding to Kabat residue 234(hinge) or
235(hinge) in IgG1 based upon alignment with the IgG1 sequence. In
certain embodiments that may be combined with the preceding
embodiments, the immunoglobulin has a second aggregation reducing
mutation at a residue that (i) has a Spatial-Aggregation-Propensity
(5 .ANG. radius sphere) of at least 0.15, or (ii) has an
Spatial-Aggregation-Propensity (5 .ANG. radius sphere) of greater
than 0.0 and is within 5 .ANG. of a residue having a
Spatial-Aggregation-Propensity (5 .ANG. radius sphere) of at least
0.15, wherein the second aggregation reducing mutation is a
substitution with an amino acid residue that is a substitution with
an amino acid residue that lowers the
Spatial-Aggregation-Propensity (5 .ANG. radius sphere) of the
residue as compared to the unmutated immunoglobulin. In certain
embodiments that may be combined with the preceding embodiments
having a second aggregation reducing mutation, the aggregation
reducing mutation and the second aggregation reducing mutation are
at least 5 .ANG., at least 10 .ANG., at least 15 .ANG., or at least
20 .ANG. apart. In certain embodiments that may be combined with
the preceding embodiments having a second aggregation reducing
mutation, the aggregation reducing mutation and the second
aggregation reducing mutation are in different aggregation motifs.
In certain embodiments that may be combined with any of the
preceding embodiments, the aggregation reducing mutation is a
substitution with an amino acid residue that is less hydrophobic
than the residue in the unmodified immunoglobulin. In certain
embodiments that may be combined with the preceding embodiments,
the aggregation reducing mutation is a substitution with an amino
acid residue selected from the group consisting of lysine,
arginine, histidine, glutamate, aspartate, glutamine, and
asparagine. In certain embodiments that may be combined with the
preceding embodiments, the aggregation reducing mutation is a
substitution with an amino acid residue selected from the group
consisting of lysine, arginine, and histidine. In certain
embodiments that may be combined with the preceding embodiments,
the aggregation reducing mutation is a substitution with a lysine
residue. In certain embodiments that may be combined with the
preceding embodiments, the Spatial-Aggregation-Propensity (5 .ANG.
radius sphere) is calculated using the Black Mould hydrophobicity
scale normalized so that glycine equals 0. In certain embodiments
that may be combined with the preceding embodiments, the
immunoglobulin is an IgG1, an IgG2, an IgG3, or an IgG4. In certain
embodiments that may be combined with the preceding embodiments,
the immunoglobulin is an IgG1. In certain embodiments that may be
combined with the preceding embodiments, the immunoglobulin has a
human C.sub.H1 domain. In certain embodiments that may be combined
with the preceding embodiments, the immunoglobulin has a human
C.sub.H2 domain. In certain embodiments that may be combined with
the preceding embodiments, the immunoglobulin has a human C.sub.H3
domain. In certain embodiments that may be combined with the
preceding embodiments, the immunoglobulin has a human C.sub.L
domain. In certain embodiments that may be combined with the
preceding embodiments, the immunoglobulin has a binding affinity
for a target antigen and the binding affinity for the target
antigen is at least seventy percent, at least eighty percent, at
least ninety percent at least one hundred percent, or at least one
hundred five percent of the binding affinity of the unmutated
immunoglobulin for the target antigen. In certain embodiments that
may be combined with the preceding embodiments, the concentrated,
liquid solution is at a concentration of at least 10 mg/ml, at
least 20 mg/ml, at least 30 mg/ml, at least 40 mg/ml, at least 50
mg/ml, at least 75 mg/ml, at least 100 mg/ml, at least 125 mg/ml,
or at least 150 mg/ml.
[0012] Another aspect includes a modified or isolated
immunoglobulin that has a reduced propensity (or aggregation
comprising at least one aggregation reducing mutation at a residue
selected from the group consisting of residues from an aggregation
motif 1: 174(C.sub.H1), 175(C.sub.H1), and 181(C.sub.H1); an
aggregation motif 2: 226(hinge), 227(hinge), 228(hinge),
229(hinge), 230(hinge), 231(hinge), and 232(hinge); an aggregation
motif 3: 234(hinge) and 235(hinge); an aggregation motif 4:
252(C.sub.H2), and 253(C.sub.H2); an aggregation motif 5:
282(C.sub.H2); an aggregation motif 6: 291(C.sub.H2); an
aggregation motif 7: 296(C.sub.H2); an aggregation motif 8:
308(C.sub.H2) and 309(C.sub.H2); an aggregation motif 9:
328(C.sub.H2), 329(C.sub.H2), 330(C.sub.H2), and 331(C.sub.H2); an
aggregation motif 10: 395(C.sub.H3), 396(C.sub.H3), 397(C.sub.H3),
398(C.sub.H3), and 404(C.sub.H3); an aggregation motif 11:
443(C.sub.H3); an aggregation motif 12: 110(C.sub.L) and
111(C.sub.L); an aggregation motif 13: 153(C.sub.L) and
154(C.sub.L); and an aggregation motif 14: 201(C.sub.L), wherein
the at least one aggregation reducing mutation is a substitution
with an amino acid residue that is less hydrophobic than the
residue in the unmodified immunoglobulin and the propensity for
aggregation that is reduced is aggregation between immunoglobulin
molecules in a concentrated, liquid solution; and wherein the
residue numbers are the corresponding Kabat residue numbers in IgG1
based upon alignment with the IgG1 sequence. In certain
embodiments, the at least one aggregation reducing mutation residue
is selected from the group consisting of residues from an
aggregation motif 1: 175(C.sub.H1), an aggregation motif 2:
227(hinge), 228(hinge), 230(hinge); an aggregation motif 3:
234(hinge) and 235(hinge); an aggregation motif 4: 253(C.sub.H2);
an aggregation motif 5: 282(C.sub.H2); an aggregation motif 6:
291(C.sub.H2); an aggregation motif 7: 296(C.sub.H2); an
aggregation motif 8: 309(C.sub.H2); an aggregation motif 9:
329(C.sub.H2) and 330(C.sub.H2); an aggregation motif 10:
395(C.sub.H3) and 398(C.sub.H3); an aggregation motif 11:
443(C.sub.H3); an aggregation motif 12: 110(C.sub.L); an
aggregation motif 13: 154(C.sub.L); and an aggregation motif 14:
201(C.sub.L). In certain embodiments that may be combined with the
preceding embodiments, the aggregation reducing mutation is not
residue 234(hinge) or 235(hinge). In certain embodiments that may
be combined with the preceding embodiments, the aggregation
reducing mutation residue is 234(hinge), 235(hinge), 253(C.sub.H2),
or 309(C.sub.H2). In certain embodiments that may be combined with
the preceding embodiments, the aggregation reducing mutation
residue is 253(C.sub.H2) or 309(C.sub.H2). In certain embodiments
that may be combined with any of the preceding embodiments, the
immunoglobulin has a second aggregation reducing mutation at a
hydrophobic residue that (i) has a Spatial-Aggregation-Propensity
of at least 0.15, or (ii) is within 5 .ANG. of a residue having a
Spatial-Aggregation-Propensity of at least 0.15, wherein the at
least one aggregation reducing mutation is a substitution with an
amino acid residue that is less hydrophobic than the residue in the
unmodified immunoglobulin. In certain embodiments that may be
combined with the preceding embodiments having a second aggregation
reducing mutation, the aggregation reducing mutation and the second
aggregation reducing imitation are at least 5 .ANG., at least 10
.ANG., at least 15 .ANG., or at least 20 .ANG. apart. In certain
embodiments that may be combined with any of the preceding
embodiments having a second aggregation reducing mutation, the
aggregation reducing imitation and the second aggregation reducing
mutation are in different aggregation motifs. In certain
embodiments that may be combined with any of the preceding
embodiments, the immunoglobulin has at least fourteen aggregation
reducing mutations wherein each aggregation reducing mutation is
selected from a different aggregation motif. In certain embodiments
that may be combined with any of the preceding embodiments, the
aggregation reducing mutation is substitution with an amino acid
residue selected from the group consisting of lysine, arginine,
histidine, glutamate, aspartate, glutamine, and asparagine. In
certain embodiments that may be combined with any of the preceding
embodiments, the aggregation reducing mutation is substitution with
an amino acid residue selected from the group consisting of lysine,
arginine, and histidine. In certain embodiments that may be
combined with any of the preceding embodiments, the aggregation
reducing mutation is substitution with a lysine residue. In certain
embodiments that may be combined with the preceding embodiments,
the Spatial-Aggregation-Propensity (5 .ANG. radius sphere) is
calculated using the Black Mould hydrophobicity scale normalized so
that glycine equals 0. In certain embodiments that may be combined
with any of the preceding embodiments, the immunoglobulin is an
IgG1, an IgG2, an IgG3, or an IgG4. In certain embodiments that may
be combined with any of the preceding embodiments, the
immunoglobulin comprises an IgG1. In certain embodiments that may
be combined with any of the preceding embodiments, the
immunoglobulin has a human C.sub.H1 domain. In certain embodiments
that may be combined with any of the preceding embodiments, the
immunoglobulin has a human C.sub.H2 domain. In certain embodiments
that may be combined with any of the preceding embodiments, the
immunoglobulin has a human C.sub.H3 domain. In certain embodiments
that may be combined with any of the preceding embodiments, the
immunoglobulin has a human C.sub.L domain. In certain embodiments
that may be combined with any of the preceding embodiments, the
immunoglobulin has a binding affinity for a target antigen and the
binding affinity for the target antigen is at least seventy
percent, at least eighty percent, at least ninety percent, at least
one hundred percent, or at least one hundred five percent of the
binding affinity of the unmutated immunoglobulin for the target
antigen. In certain embodiments that may be combined with the
preceding embodiments, the concentrated, liquid solution is at a
concentration of at least 10 mg/ml, at least 20 mg/ml, at least 30
mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least 75 mg/ml, at
least 100 mg/ml, at least 125 mg/ml, or at least 150 mg/ml.
[0013] Another aspect includes modified immunoglobulin formulations
that can be made up of immunoglobulin of either of the preceding
aspects and any and all combinations of the preceding embodiments
at a concentration of at least 10 mg/ml, at least 20 mg/ml, at
least 30 mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least 75
mg/ml, at least 100 mg/ml, at least 125 mg/ml, or at least 150
mg/ml. In certain embodiments, the immunoglobulin is at a
concentration of greater than the concentration at which the
unmutated immunoglobulin aggregates with itself in a concentrated,
liquid solution under the same conditions. In certain embodiments
that may be combined with the preceding embodiments, at least
eighty percent, at least eighty-five percent, at least ninety
percent, at least ninety-five percent, at least ninety-six percent,
at least ninety-seven percent, at least ninety-eight percent, or at
least ninety-nine percent of the modified immunoglobulin is
non-aggregated monomer. In certain embodiments that may be combined
with any of the preceding embodiments, the formulation includes a
pharmaceutically acceptable excipient. In certain embodiments that
may be combined with any of the preceding embodiments, the
immunoglobulin formulation shows at least five percent, at least
ten percent, at least fifteen percent, at least twenty percent, at
least twenty-five percent, at least thirty percent, at least
thirty-five percent, at least forty percent, or at least fifty
percent less aggregate after twenty four hours of accelerated
aggregation as compared to the unmutated immunoglobulin under the
same conditions. In certain embodiments that may be combined with
the preceding embodiments, the aggregation is measured by SEC-HPLC.
In certain embodiments that may be combined with any of the
preceding embodiments, the immunoglobulin formulation is
substantially free of any additive that reduces aggregation of
immunoglobulins. In certain embodiments that may be combined with
any of the preceding embodiments, the immunoglobulin formulation is
substantially free of free histidine, saccharides and polyols.
[0014] Yet another aspect includes isolated or recombinant
polynucleotides that encode immunoglobulin of either of the
preceding modified immunoglobulin aspects and any and all
combinations of the preceding embodiments. In certain embodiments,
the polynucleotide is in a vector. In certain embodiments, the
vector is an expression vector. In certain embodiments that may be
combined with the preceding embodiments, an inducible promoter is
operably linked to the polynucleotide. Another aspect includes host
cells with the vector of either of the preceding embodiments. In
certain embodiments, the host cells are capable of expressing the
immunoglobulin encoded by the polynucleotide.
[0015] Another aspect includes methods of producing an
immunoglobulin with a reduced aggregation propensity comprising
providing a culture medium comprising the host cell of the
preceding aspect and placing the culture medium in conditions under
which the immunoglobulin is expressed. In certain embodiments, the
methods include an additional step of isolating the immunoglobulin
expressed.
[0016] Another aspect includes methods for reducing the aggregation
propensity of an immunoglobulin in a highly concentrated
pharmaceutical formulation comprising providing an immunoglobulin
that is prone to aggregation; substituting a residue in a conserved
domain of the immunoglobulin that (i) has a
Spatial-Aggregation-Propensity of at least 0.15, or (ii) has an
Spatial-Aggregation-Propensity (5 .ANG. radius sphere) of greater
than 0.0 and is within 5 .ANG. of a residue having a
Spatial-Aggregation-Propensity of at least 0.15, with an amino acid
residue that lowers the Spatial-Aggregation-Propensity (5 .ANG.
radius sphere), and generating a highly concentrated, liquid
formulation of the modified immunoglobulin wherein the modified
immunoglobulin concentration is at least 20 mg/ml, at least 30
mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least 75 mg/ml, at
least 100 mg/ml, at least 125 mg/ml, or at least 150 mg/ml, and
wherein the aggregation propensity that is reduced is aggregation
between immunoglobulin molecules in a concentrated, liquid
solution.
[0017] Another aspect includes uses of either of the preceding
modified immunoglobulin aspects and any and all combinations of the
preceding embodiments in the preparation of a medicament comprising
a highly concentrated liquid formulation wherein the modified
immunoglobulin concentration is at least 20 mg/ml, at least 30
mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least 75 mg/ml, at
least 100 mg/ml, at least 125 mg/ml, or at least 150 mg/ml. In
certain embodiments, the use of the medicament is for the treatment
of antoimmune diseases, immunological diseases, infectious
diseases, inflammatory diseases, neurological diseases, and
oncological and neoplastic diseases including cancer. In certain
embodiments, the use of the medicament is for the treatment of
congestive heart failure (CHF), vasculitis, rosacea, acne, eczema,
myocarditis and other conditions of the myocardium, systemic lupus
erythematosus, diabetes, spondylopathies, synovial fibroblasts, and
bone marrow stroma; bone loss; Paget's disease, osteoclastoma;
breast cancer; disuse osteopenia; malnutrition, periodontal
disease, Gaucher's disease, Langerhans' cell histiocytosis, spinal
cord injury, acute septic arthritis, osteomalacia, Cushing's
syndrome, monoostotic fibrous dysplasia, polyostotic fibrous
dysplasia, periodontal reconstruction, and bone fractures;
sarcoidosis; osteolytic bone cancers, breast cancer, lung cancer,
kidney cancer and rectal cancer; bone metastasis, bone pain
management, and humoral malignant hypercalcemia, ankylosing
spondylitisa and other spondyloarthropathies; transplantation
rejection, viral infections, hematologic neoplasias and
neoplastic-like conditions for example, Hodgkin's lymphoma;
non-Hodgkin's lymphomas (Burkitt's lymphoma, small lymphocytic
lymphoma/chronic lymphocytic leukemia, mycosis fungoides, mantle
cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma,
marginal zone lymphoma, hairy cell leukemia and lymphoplamacytic
leukemia), tumors of lymphocyte precursor cells, including B-cell
acute lymphoblastic leukemia/lymphoma, and T-cell acute
lymphoblastic leukemia/lymphoma, thymoma, tumors of the mature T
and NK cells, including peripheral T-cell leukemias, adult T-cell
leukemia/T-cell lymphomas and large granular lymphocytic leukemia,
Langerhans cell histocytosis, myeloid neoplasias such as acute
myelogenous leukemias, including AML with maturation, AML without
differentiation, acute promyelocytic leukemia, acute myelomonocytic
leukemia, and acute monocytic leukemias, myelodysplastic syndromes,
and chronic myeloproliferative disorders, including chronic
myelogenous leukemia, tumors of the central nervous system, e.g.,
brain tumors (glioma, neuroblastoma, astrocytoma, medulloblastoma,
ependymoma, and retinoblastoma), solid tumor (nasopharyngeal
cancer, basal cell carcinoma, pancreatic cancer, cancer of the bile
duct, Kaposi's sarcoma, testicular cancer, uterine, vaginal or
cervical cancer, ovarian cancer, primary liver cancer or
endometrial cancer, and tumors of the vascular system (angiosarcoma
and hemangiopericytoma), osteoporosis, hepatitis, HIV, AIDS,
spondylarthritis, rheumatoid arthritis, inflammatory bowel diseases
(IBD), sepsis and septic shock, Crohn's disease, psoriasis,
schleraderma, graft versus host disease (GVHD), allogenic graft
rejection, hematologic malignancies, such as multiple myeloma (MM),
myelodyspastic syndrome (MDS) and acute myelogenous leukemia (AML),
inflammation associated with tumors, peripheral nerve injury or
demyelinating diseases. In certain embodiments, the use of the
medicament is for the treatment or plaque psoriasis, ulcerative
colitis, non-Hodgkin's lymphoma, breast cancer, colorectal cancer,
juvenile idiopathic arthritis, macular degeneration, respiratory
syncytial virus, Crohn's disease, rheumatoid arthritis, psoriatic
arthritis, ankylosing spondylitis, osteoporosis, treatment-induced
bone loss, bone metastases, multiple myeloma, Alzheimer's disease,
glaucoma, and multiple sclerosis. In certain embodiments that may
be combined with any of the preceding embodiments, the use of the
medicament further comprises a pharmaceutically acceptable
excipient. In certain embodiments that may be combined with any of
the preceding embodiments, the immunoglobulin in the medicament
shows at least five percent, at least ten percent, at least fifteen
percent, at least twenty percent, at least twenty-five percent, at
least thirty percent, at least thirty-five percent, at least forty
percent, or at least fifty percent less aggregate after twenty four
hours of accelerated aggregation as compared to the unmutated
immunoglobulin under the same conditions. In certain embodiments,
the aggregation is measured by SEC-HPLC. In certain embodiments
that may be combined with any of the preceding embodiments, the
medicament is substantially free of any additive that reduces
aggregation of immunoglobulins. In certain embodiments that may be
combined with any of the preceding embodiments, the medicament is
substantially free of free histidine, saccharides and polyols.
[0018] Another aspect includes uses of either of the preceding
modified immunoglobulin aspects and any and all combinations of the
preceding embodiments as a non-aggregating pharmaceutical active
ingredient.
[0019] Another aspect includes pharmaceutical compositions that
include an immunoglobulin of either of the preceding aspects and
any and all combinations of the preceding embodiments and a
pharmaceutically acceptable excipient. In certain embodiments, the
immunoglobulin is at least a concentration of at least 10 mg/ml, at
least 20 mg/ml, at least 30 mg/ml, at least 40 mg/ml, at least 50
mg/ml, at least 75 mg/ml, at least 100 mg/ml, at least 125 mg/ml,
or at least 150 mg/ml. In certain embodiments, the immunoglobulin
is at a concentration of greater than the concentration at which
the unmutated immunoglobulin aggregates with itself in a
concentrated, liquid solution under the same conditions. In certain
embodiments that may be combined with the preceding embodiments, at
least eighty percent, at least eighty-five percent, at least ninety
percent, at least ninety-five percent, at least ninety-six percent,
at least ninety-seven percent, at least ninety-eight percent, or at
least ninety-nine percent of the modified immunoglobulin is
non-aggregated monomer. In certain embodiments that may be combined
with any of the preceding embodiments, the immunoglobulin
formulations shows at least five percent, at least ten percent, at
least fifteen percent, at least twenty percent, at least
twenty-five percent, at least thirty percent, at least thirty-five
percent, at least forty percent, or at least fifty percent less
aggregate after twenty four hours of accelerated aggregation as
compared to the unmutated immunoglobulin under the same conditions.
In certain embodiments that may be combined with the preceding
embodiments, the aggregation is measured by SEC-HPLC. In certain
embodiments that may be combined with any of the preceding
embodiments, the immunoglobulin formulation is substantially free
of any additive that reduces aggregation of immunoglobulins. In
certain embodiments that may be combined with any of the preceding
embodiments, the immunoglobulin formulation is substantially free
of free histidine, saccharides and polyols.
[0020] Another aspect includes a modified or isolated
immunoglobulin that has a reduced propensity for aggregation
comprising at least one aggregation reducing mutation at a residue
selected from the group consisting of 235(hinge), 241(C.sub.H2),
243(C.sub.H2), 282(C.sub.H2), and 309(C.sub.H2), wherein if residue
235 is selected, it is mutated to a glutamate or a serine, if
residue 282 is selected it is mutated to a lysine, and if residue
309 is selected, it is mutated to a lysine, and wherein the at
least one aggregation reducing mutation is a substitution with an
amino acid residue that is less hydrophobic that then residue in
the unmodified immunoglobulin and the propensity for aggregation
that is reduced is aggregation between immunoglobulin molecules in
a concentrated, liquid solution; and wherein the residue numbers
are the corresponding Kabat residue numbers in IgG1 based upon
alignment with the IgG1 sequence. In certain embodiments, the at
least one aggregation reducing mutation is a mutation of residue
241 to serine, and the modified or isolated immunoglobulin further
comprises a second aggregation reducing mutation of residue 243 to
serine. In certain embodiments, the at least one aggregation
reducing mutation is a mutation of residue 241 to tyrosine, and the
modified or isolated immunoglobulin further comprises a second
aggregation reducing mutation of residue 243 to tyrosine. In
certain embodiments, the at least one aggregation reducing mutation
is a mutation of residue 282 to lysine, and the modified or
isolated immunoglobulin further comprises a second and a third
aggregation reducing mutation, wherein the second aggregation
reducing mutation is a mutation of residue 235 to lysine and the
third aggregation reducing mutation is a mutation of residue 309 to
lysine. In certain embodiments, the immunoglobulin has a second
aggregation reducing mutation at a hydrophobic residue, wherein the
at least one aggregation reducing mutation is a substitution with
an amino acid residue that is less hydrophobic than the residue in
the unmodified immunoglobulin. In certain embodiments that may be
combined with the preceding embodiments having a second aggregation
reducing mutation, the second aggregation reducing mutation (i) has
a Spatial-Aggregation-Propensity of at least 0.15, or (ii) is
within 5 .ANG. or a residue having a Spatial-Aggregation-Propensity
of at least 0.15. In certain embodiments that may be combined with
the preceding embodiments having a second aggregation reducing
mutation, the immunoglobulin has a third aggregation reducing
mutation that (i) has a Spatial-Aggregation-Propensity of at least
0.15, or (ii) is within 5 .ANG. of a residue having a
Spatial-Aggregation-Propensity of at least 0.15, wherein the third
aggregation reducing mutation is a substitution with an amino acid
residue that is less hydrophobic than the residue in the unmodified
immunoglobulin. In certain embodiments that may be combined with
the preceding embodiments having a second aggregation reducing
mutation, the aggregation reducing mutation and the second
aggregation reducing mutation are at least 5 .ANG., at least 10
.ANG., at least 15 .ANG., or at least 20 .ANG. apart. In certain
embodiments that may be combined with any of the preceding
embodiments having a second aggregation reducing mutation, the
aggregation reducing mutation and the second aggregation reducing
mutation are in different aggregation motifs. In certain
embodiments that may be combined with any of the preceding
embodiments, the immunoglobulin has at least fourteen aggregation
reducing mutations wherein each aggregation reducing mutation is
selected from a different aggregation motif. In certain embodiments
that may be combined with any of the preceding embodiments, the
aggregation reducing mutation is substitution with an amino acid
residue selected from the group consisting of lysine, arginine,
histidine, glutamate, aspartate, glutamine, asparagine, tyrosine,
and serine. In certain embodiments that may be combined with any of
the preceding embodiments, the aggregation reducing mutation is
substitution with an amino acid residue selected from the group
consisting of lysine, serine, glutamate, and tyrosine. In certain
embodiments that may be combined with the preceding embodiments,
the Spatial-Aggregation-Propensity (5 .ANG. radius sphere) is
calculated using the Black Mould hydrophobicity scale normalized so
that glycine equals 0. In certain embodiments that may be combined
with any of the preceding embodiments, the immunoglobulin is an
IgG1, an IgG2, an IgG3, or an IgG4. In certain embodiments that may
be combined with any of the preceding embodiments, the
immunoglobulin comprises an IgG1. In certain embodiments that may
be combined with any of the preceding embodiments, the
immunoglobulin has a human C.sub.H1 domain. In certain embodiments
that may be combined with any of the preceding embodiments, the
immunoglobulin has a human C.sub.H2 domain. In certain embodiments
that may be combined with any of the preceding embodiments, the
immunoglobulin has a human C.sub.H3 domain. In certain embodiments
that may be combined with any of the preceding embodiments, the
immunoglobulin has a human C.sub.L domain. In certain embodiments
that may be combined with any of the preceding embodiments, the
immunoglobulin has a binding affinity for a target antigen and the
binding affinity for the target antigen is at least seventy
percent, at least eighty percent, at least ninety percent, at least
one hundred percent, at least one hundred five percent of the
binding affinity of the unmutated immunoglobulin for the target
antigen. In certain embodiments that may be combined with the
preceding embodiments, the concentrated, liquid solution is at a
concentration of at least 10 mg/ml, at least 20 mg/ml, at least 30
mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least 75 mg/ml, at
least 100 mg/ml, at least 125 mg/ml, or at least 150 mg/ml.
[0021] Another aspect includes modified immunoglobulin formulations
that can be made up of immunoglobulin of either of the preceding
aspects and any and all combinations of the preceding embodiments
at a concentration of at least 10 mg/ml, at least 20 mg/ml, at
least 30 mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least 75
mg/ml, at least 100 mg/ml, at least 125 mg/ml, or at least 150
mg/ml. In certain embodiments, the immunoglobulin is at a
concentration of greater than the concentration at which the
unmutated immunoglobulin aggregates with itself in a concentrated,
liquid solution under the same conditions. In certain embodiments
that may be combined with the preceding embodiments, at least
eighty percent, at least eighty-five percent, at least ninety
percent, at least ninety-five percent, at least ninety-six percent,
at least ninety-seven percent, at least ninety-eight percent, or at
least ninety-nine percent of the modified immunoglobulin is
non-aggregated monomer. In certain embodiments that may be combined
with any of the preceding embodiments, the formulation includes a
pharmaceutically acceptable excipient. In certain embodiments that
may be combined with any of the preceding embodiments, the
immunoglobulin formulation shows at least five percent, at least
ten percent, at least fifteen percent, at least twenty percent, at
least twenty-five percent, at least thirty percent, at least
thirty-five percent, at least forty percent, or at least fifty
percent less aggregate after twenty four hours of accelerated
aggregation as compared to the unmutated immunoglobulin under the
same conditions. In certain embodiments that may be combined with
the preceding embodiments, the aggregation is measured by SEC-HPLC.
In certain embodiments that may be combined with any of the
preceding embodiments, the immunoglobulin formulation is
substantially free of any additive that reduces aggregation of
immunoglobulins. In certain embodiments that may be combined with
any of the preceding embodiments, the immunoglobulin formulation is
substantially free of free histidine, saccharides and polyols.
[0022] Yet another aspect includes isolated or recombinant
polynucleotides that encode immunoglobulin of either of the
preceding modified immunoglobulin aspects and any and all
combinations of the preceding embodiments. In certain embodiments,
the polynucleotide is in a vector. In certain embodiments, the
vector is an expression vector. In certain embodiments that may be
combined with the preceding embodiments, an inducible promoter is
operably linked to the polynucleotide. Another aspect includes host
cells with the vector of either of the preceding embodiments. In
certain embodiments, the host cells are capable of expressing the
immunoglobulin encoded by the polynucleotide.
[0023] Another aspect includes methods of producing an
immunoglobulin with a reduced aggregation propensity comprising
providing a culture medium comprising the host cell of the
preceding aspect and placing the culture medium in conditions under
which the immunoglobulin is expressed. In certain embodiments, the
methods include an additional step of isolating the immunoglobulin
expressed.
[0024] Another aspect includes uses of either of the preceding
modified immunoglobulin aspects and any and all combinations of the
preceding embodiments in the preparation of a medicament comprising
a highly concentrated liquid formulation wherein the modified
immunoglobulin concentration is at least 20 mg/ml, at least 30
mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least 75 mg/ml, at
least 100 mg/ml, at least 125 mg/ml, or at least 150 mg/ml. In
certain embodiments, the use of the medicament is for the treatment
of autoimmune diseases, immunological diseases, infectious
diseases, inflammatory diseases, neurological diseases, and
oncological and neoplastic diseases including cancer. In certain
embodiments, the use of the medicament is for the treatment of
congestive heart failure (CHF), vasculitis, rosacea, acne, eczema,
myocarditis and other conditions of the myocardium, systemic lupus
erythematosus, diabetes, spondylopathies, synovial fibroblasts, and
bone marrow stroma; bone loss; Paget's disease, osteoclastoma;
breast cancer; disuse osteopenia; malnutrition, periodontal
disease, Gaucher's disease, Langerhans' cell histiocytosis, spinal
cord injury, acute septic arthritis, osteomalacia, Cushing's
syndrome, monoostotic fibrous dysplasia, polyostotic fibrous
dysplasia, periodontal reconstruction, and bone fractures;
sarcoidosis; osteolytic bone cancers, breast cancer, lung cancer,
kidney cancer and rectal cancer; bone metastasis, bone pain
management, and humoral malignant hypercalcemia, ankylosing
spondylitisa and other spondyloarthropathies; transplantation
rejection, viral infections, hematologic neoplasias and
neoplastic-like conditions for example, Hodgkin's lymphoma;
non-Hodgkin's lymphomas (Burkitt's lymphoma, small lymphocytic
lymphoma/chronic lymphocytic leukemia, mycosis fungoides, mantle
cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma,
marginal zone lymphoma, hairy cell leukemia and lymphoplamacytic
leukemia), tumors of lymphocyte precursor cells, including B-cell
acute lymphoblastic leukemia/lymphoma, and T-cell acute
lymphoblastic leukemia/lymphoma, thymoma, tumors of the mature T
and NK cells, including peripheral T-cell leukemias, adult T-cell
leukemia/T-cell lymphomas and large granular lymphocytic leukemia,
Langerhans cell histocytosis, myeloid neoplasias such as acute
myelogenous leukemias, including AML with maturation, AML without
differentiation, acute promyelocytic leukemia, acute myelomonocytic
leukemia, and acute monocytic leukemias, myelodysplastic syndromes,
and chronic myeloproliferative disorders, including chronic
myelogenous leukemia, tumors of the central nervous system, e.g.,
brain tumors (glioma, neuroblastoma, astrocytoma, medulloblastoma,
ependymoma, and retinoblastoma), solid tumor (nasopharyngeal
cancer, basal cell carcinoma, pancreatic cancer, cancer of the bile
duct, Kaposi's sarcoma, testicular cancer, uterine, vaginal or
cervical cancer, ovarian cancer, primary liver cancer or
endometrial cancer, and tumors of the vascular system (angiosarcoma
and hemangiopericytoma), osteoporosis, hepatitis, HIV, AIDS,
spondylarthritis, rheumatoid arthritis, inflammatory bowel diseases
(IBD), sepsis and septic shock, Crohn's disease, psoriasis,
schleraderma, graft versus host disease (GVHD), allogenic graft
rejection, hematologic malignancies, such as multiple myeloma (MM),
myelodyspastic syndrome (MDS) and acute myelogenous leukemia (AML),
inflammation associated with tumors, peripheral nerve injury or
demyelinating diseases. In certain embodiments, the use of the
medicament is for the treatment or plaque psoriasis, ulcerative
colitis, non-Hodgkin's lymphoma, breast cancer, colorectal cancer,
juvenile idiopathic arthritis, macular degeneration, respiratory
syncytial virus, Crohn's disease, rheumatoid arthritis, psoriatic
arthritis, ankylosing spondylitis, osteoporosis, treatment-induced
bone loss, bone metastases, multiple myeloma, Alzheimer's disease,
glaucoma, and multiple sclerosis. In certain embodiments that may
be combined with any of the preceding embodiments, the use of the
medicament further comprises a pharmaceutically acceptable
excipient. In certain embodiments that may be combined with any of
the preceding embodiments, the immunoglobulin in the medicament
shows at least five percent, at least ten percent, at least fifteen
percent, at least twenty percent, at least twenty-five percent, at
least thirty percent, at least thirty-five percent, at least forty
percent, or at least fifty percent less aggregate after twenty four
hours of accelerated aggregation as compared to the unmutated
immunoglobulin under the same conditions. In certain embodiments,
the aggregation is measured by SEC-HPLC. In certain embodiments
that may be combined with any of the preceding embodiments, the
medicament is substantially free of any additive that reduces
aggregation of immunoglobulins. In certain embodiments that may be
combined with any of the preceding embodiments, the medicament is
substantially free of free histidine, saccharides and polyols.
[0025] Another aspect includes uses of either of the preceding
modified immunoglobulin aspects and any and all combinations of the
preceding embodiments as a non-aggregating pharmaceutical active
ingredient.
[0026] Another aspect includes pharmaceutical compositions that
include an immunoglobulin of either of the preceding aspects and
any and all combinations of the preceding embodiments and a
pharmaceutically acceptable excipient. In certain embodiments, the
immunoglobulin is at a concentration of at least 10 mg/ml, at least
20 mg/ml, at least 30 mg/ml, at least 40 mg/ml, at least 50 mg/ml,
at least 75 mg/ml, at least 100 mg/ml, at least 125 mg/ml, or at
least 150 mg/ml. In certain embodiments, the immunoglobulin is at a
concentration of greater than the concentration at which the
unmutated immunoglobulin aggregates with itself in a concentrated,
liquid solution under the same conditions. In certain embodiments
that may be combined with the preceding embodiments, at least
eighty percent, at least eighty-five percent, at least ninety
percent, at least ninety-five percent, at least ninety-six percent,
at least ninety-seven percent, at least ninety-eight percent, or at
least ninety-nine percent of the modified immunoglobulin is
non-aggregated monomer. In certain embodiments that may be combined
with any of the preceding embodiments, the immunoglobulin
formulation shows at least five percent, at least ten percent, at
least fifteen percent, at least twenty percent, at least
twenty-five percent, at least thirty percent, at least thirty-five
percent, at least forty percent, or at least fifty percent less
aggregate after twenty four hours of accelerated aggregation as
compared to the unmutated immunoglobulin under the same conditions.
In certain embodiments that may be combined with the preceding
embodiments, the aggregation is measured by SEC-HPLC. In certain
embodiments that may be combined with any of the preceding
embodiments, the immunoglobulin formulation is substantially free
of any additive that reduces aggregation of immunoglobulins. In
certain embodiments that may be combined with any of the preceding
embodiments, the immunoglobulin formulation is substantially free
of free histidine, saccharides, and polyols.
[0027] Additional aspects and embodiments of the invention may be
found throughout the specification.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0028] The present disclosure relates to improved immunoglobulins,
particularly human antibodies, that have reduced aggregation. In
certain embodiments, the immunoglobulins of other disclosure are
modified at specific hydrophobic residues within the constant
regions of the heavy or light chains of the immunoglobulin. The
disclosure provided modified immunoglobulins, methods of making
such immunoglobulins, immunoconjugates and multivalent or
multispecific molecules comprising such immunoglobulins and
pharmaceutical compositions containing the immunoglobulins,
immunoconjugates or bispecific molecules of the disclosure.
DEFINITIONS
[0029] The term "antibody" is referred to herein includes whole
antibodies and any antigen binding fragment (i.e.,
"antibody-binding portion") or single chains thereof. A naturally
occurring "antibody" is a glycoprotein comprising at least two
heavy (H) chains and two light (L) chains inter-connected by
disulfide bonds. Each heavy chain is comprised of a heavy chain
variable region (abbreviated herein as V.sub.H) and a heavy chain
constant region. The heavy chain constant region is comprised of
three domains, C.sub.H1, C.sub.H2 and C.sub.H3. Each light chain is
comprised of a light chain variable region (abbreviated herein as
V.sub.L) and a light chain constant region. The light chain
constant region is comprised of one domain, C.sub.L. The V.sub.H
and V.sub.L regions can be further subdivided into regions of
hypervariability, termed complementarity determining regions (CDR),
interspersed with regions that are more conserved, termed framework
regions (FR). Each V.sub.H and V.sub.L is composed of three CDRs
and four FRs arranged from amino-terminus to carboxy-terminus in
the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The
variable regions of the heavy and light chains contain a binding
domain that interacts with an antigen. The constant regions of the
antibodies may mediate the binding of the immunoglobulin to host
tissues or factors, including various cells of the immune system
(e.g., effector cells) and the first component (Clq) of the
classical complement system.
[0030] The term "antigen-binding portion" of an antibody (or simply
"antigen portion"), as used herein, refers to full length or one or
more fragments of an antibody that retain the ability to
specifically bind to an antigen and at least a portion of the
constant region of the heavy or light chain, it has been shown that
the antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Examples of binding fragments
encompassed within the term "antigen-binding portion" of an
antibody include a Fab fragment, a monovalent fragment consisting
of the V.sub.L, V.sub.H, C.sub.L and C.sub.H1 domains; a F(a)2
fragment, a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region; a Fd fragment consisting
of the V.sub.H and C.sub.H1 domains; and a Fv fragment consisting
of the V.sub.L and V.sub.H domains of a single arm of an
antibody.
[0031] Furthermore, although the two domains of the Fv fragment,
V.sub.L and V.sub.H, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the
V.sub.L and V.sub.H regions pair to form monovalent molecules
(known as single chain Fv (scFv); see e.g., Bird et al., 1988
Science 2442:423-426; and Huston et al., 1988 Proc. Natl. Acad.
Sci. 85:5879-5883). Such single chain antibodies are also intended
to be encompassed within the term "antigen-binding region" of an
antibody. These antibody fragments are obtained using conventional
techniques known to those of skill in the art, and the fragments
are screened for utility in the same manner as are intact
antibodies.
[0032] An "isolated" antibody or immunoglobulin, as used herein,
refers to an antibody or immunoglobulin that is substantially free
of other components to which such antibodies or immunoglobulin, are
naturally found. Moreover, an isolated antibody or immunoglobulin
may be substantially free of other cellular material and/or
chemicals.
[0033] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
composition typically displays a single binding specificity and
affinity for a particular epitope.
[0034] The term "human antibody", as used herein, is intended to
include antibodies having variable regions in which both the
framework and CDR regions are derived from sequences of human
origin. Furthermore, if the antibody contains a constant region,
the constant region also is derived from such human sequences,
e.g., human germline sequences, or mutated versions of human
germline sequences or antibody containing consensus framework
sequences derived from human framework sequences analysis as
described in Knappik, et al. (2000. J Mol Biol 296, 57-86).
[0035] The human antibodies of the disclosure may include amino
acid residues not encoded by human sequences (e.g., mutations
introduced by random or site-specific mutagenesis in vitro or by
somatic mutation in vivo). However the term "human antibody", as
used herein, is not intended, to include antibodies in which CDR
sequences derived from the germline of another mammalian species,
such as a mouse, have been grafted onto human framework
sequences.
[0036] The term "human domain", as used herein, is intended to
include immunoglobulin constant region domains derived from
sequences of human origin, e.g., human germline sequences, or
mutated versions of human germline sequences or antibody containing
consensus framework sequences derived from human framework
sequences analysis as described in Knappik, et al. (2000. J Mol
Biol 296, 57-86).
[0037] The term "recombinant human antibody", as used herein,
includes all human antibodies that are prepared, expressed, created
or isolated by recombinant means, such as antibodies isolated from
an animal (e.g., a mouse) that is transgenic or transchromosomal
for human immunoglobulin genes of a hybridoma prepared therefrom,
antibodies isolated from a host cell transformed to express the
human antibody, e.g., from a transfectoma, antibodies isolated from
a recombinant, combinatorial human antibody library, and antibodies
prepared, expressed, created or isolated by any other means that
involve splicing of all or a portion of a human immunoglobulin
gene, sequences to other DNA sequences. Such recombinant human
antibodies have variable regions in which the framework and CDR
regions are derived from human germline immunoglobulin sequences.
In certain embodiments, however, such recombinant human antibodies
can be subjected to in vitro mutagenesis (or, when an animal
transgenic for human Ig sequences is used, in vivo somatic
mutagenesis) and thus the amine acid sequences of the V.sub.H and
V.sub.L regions of the recombinant antibodies are sequences that,
while derived from and related to human germline V.sub.H and
V.sub.L sequences, may not naturally exist within the human
antibody germline repertoire in vivo.
[0038] A "chimeric antibody" is an antibody molecule in which (a)
the constant region, or a portion thereof, is altered, replaced or
exchanged so that the antigen binding site (variable region) is
linked to a constant region of a different or altered class,
effector function and/or species, or an entirely different molecule
which confers new properties to the chimeric antibody, e.g., an
enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the
variable region, or a portion thereof, is altered, replaced or
exchanged with a variable region having a different or altered
antigen specificity. For example, a mouse antibody can be modified
by replacing its constant region with the constant region from a
human immunoglobulin comprising a modification as disclosed herein.
Due to the replacement with a human constant region, the chimeric
antibody can retain its specificity while having reduced
antigenicity in human and reduced aggregation overall as compared
to the original mouse antibody or a chimeric antibody without the
modification as disclosed herein.
[0039] A "humanized" antibody is an antibody that retains the
reactivity of a non-human antibody while being less immunogenic in
humans. This can be achieved, for instance, by retaining the
non-human CDR regions and replacing the remaining parts of the
antibody with their human counterparts (i.e., the constant region
as well as the framework portions of the variable region). See,
e.g., Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6835,
1984; Morrison and Oi, Adv. Immunol., 44:65-92, 1988; Verhoeyen et
al., Science, 239:1534-1536, 1988; Padlan, Molec. Immun.,
28:489-498, 1991; and Padlan, Molec. Immun., 31:169-217, 1994.
Other examples of human engineering technology include, but are not
limited to Xoma technology disclosed in U.S. Pat. No.
5,766,886.
[0040] The term "Humaneering" as used herein infers to a method for
converting non-human antibodies into engineered human antibodies
(See e.g., KalBios' Humaneering.TM. technology).
[0041] As used herein, "isotype"refers to any antibody class (e.g.,
IgM, IgE, IgG such as IgG1 or IgG2) that is provided by the heavy
chain constant region genes that have the aggregation prone motifs
disclosed herein (and therefore are amenable to the modifications
disclosed herein that reduce aggregation).
[0042] As used herein, the term "affinity" refers to the strength
of interaction between antibody and antigen at single antigenic
sites. Within each antigenic site, the variable region of the
antibody "arm" interacts through weak non-covalent forces with
antigen at numerous sites; the more interactions, the stronger the
affinity. The modifications disclosed herein preferably do not
reduce the affinity of the immunoglobulin or antibodies disclosed
herein or the affinity is reduced less than thirty percent, less
than twenty percent, less than ten percent, or less than five
percent. As used herein, when determining whether the modifications
disclosed herein reduce affinity the comparison is made between the
immunoglobulin or antibody with the modification and the same
immunoglobulin lacking the modification but including any unrelated
mutations. By way of example, a humanized antibody with an L234K
mutation as disclosed herein would be compared to the humanized
antibody with the exact same sequence except for the wild type
L234.
[0043] As used herein, the term "subject" includes any human or
nonhuman animal.
[0044] The term "nonhuman animal" includes all vertebrates, e.g.,
mammals and non-mammals, such as nonhuman primates, sheep, dogs,
cats, horses, cows, chickens, amphibians, reptiles, etc.
[0045] As used herein, the term "optimized" means that a nucleotide
sequence has been altered to encode an amino acid sequence using
codons that are preferred in the production cell or organism,
generally a eukaryotic cell, for example, a cell of Pichia, a
Chinese Hamster Ovary cell (CHO) or a human cell. The optimized
nucleotide sequence is engineered to retain completely or as much
as possible the amino acid sequence originally encoded by the
starting nucleotide sequence, which is also known as the "parental"
sequence. Optimized expression of these sequences in other
eukaryotic cells is also envisioned herein. The amino acid
sequences encoded by optimized nucleotide sequences are also
referred to as optimized.
[0046] The term "epitope" means a protein determinant capable of
specific binding to an antibody. Epitopes usually consist of
chemically active surface groupings of molecules such as amino
acids or sugar side chains and usually have specific three
dimensional structural characteristics, as well as specific charge
characteristics. Conformational and nonconformational epitopes are
distinguished in that the binding to the former but not the latter
is lost in the presence of denaturing solvents.
[0047] The term "conservatively modified variant" applies to both
amino acid and nucleic acid sequences. With respect to particular
nucleic acid sequences, conservatively modified variants refers to
those nucleic acids which encode identical or essentially identical
amino acid sequences, or where the nucleic acid does not encode an
amino acid sequence, to essentially identical sequences. Because of
the degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid that encodes a polypeptide is implicit in each described
sequence.
[0048] For polypeptide sequences, "conservatively modified
variants" include individual substitutions, deletions or additions
to a polypeptide sequence which results in the substitution of an
amino acid with a chemically similar amino acid. Conservative
substitution tables providing functionally similar amino acids are
well known in the art. Such conservatively modified variants are in
addition to and do not exclude polymorphic variants, interspecies
homologs, and alleles of the disclosure. The following eight groups
contain amino acids that are conservative substitutions for one
another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D),
Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine
(R), Lysine (K); 5) Isoleucine (I), Leucine (L); Methionine (M),
Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7)
Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M)
(see, e.g., Creighton, Proteins (1984)).
[0049] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same. Two sequences
are "substantially identical" if two sequences have a specified
percentage of amino acid residues or nucleotides that are the same
(i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%,
or 99% identity over a specified region, or, when not specified,
over the entire sequence), when compared and aligned for maximum
correspondence over a comparison window, or designated region as
measured using one of the following sequence comparison algorithms
or by manual alignment and visual inspection. Optionally, the
identity exists over a region that is at least about 50 nucleotides
(or 10 amino acids) in length, or more preferably over a region
that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or
more amino acids) in length.
[0050] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters. When comparing two sequences for identity, it
is not necessary that the sequences be contiguous, but any gap
would carry with it a penalty that would reduce the overall percent
identity. For blastn, the default parameters are Gap opening
penalty=5 and Gap extension penalty=2. For blastp, the default
parameters are Gap opening penalty=11 and Gap extension
penalty=1.
[0051] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions
including, but not limited to from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 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.
Methods of alignment of sequences for comparison are well known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith and
Waterman (1970) Adv. App. Math. 2:482c, by the homology alignment
algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970, by
the search for similarity method of Pearson and Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized implementation
of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the
Wisconsin Genetics Software Package, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by manual alignment and visual
inspection (see, e.g., Brent et al., Current Protocols in Molecular
Biology, John Wiley & Sons, Inc. (ringbou ed., 2003)).
[0052] Two examples of algorithms that are suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al., Nuc.
Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol.
215:403-410, 1990, respectively. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information. This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some position-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits acts as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) or 10, M=5, N=-4 and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength of 3, and expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci.
USA 89:10915, 1989) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands.
[0053] The BLAST algorithm also performs a statistical analysis of
the similarity between twos sequences (see, e.g., Karlin and
Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0054] Other than percentage of sequence identity noted above,
another indication that two nucleic acid sequences or polypeptides
are substantially identical is that the polypeptide encoded by the
first nucleic acid is immunologically cross reactive with the
antibodies raised against the polypeptide encoded by the second
nucleic acid, as described below. Thus, a polypeptide is typically
substantially identical to a second polypeptide, for example, where
the two peptides differ only by conservative substitutions. Another
indication that two nucleic acid sequences are substantially
identical is that the two molecules or their complements hybridize
to each other under stringent conditions, as described below. Yet
another indication that two nucleic acid sequences are
substantially identical is that the same primers can be used to
amplify the sequence.
[0055] The term "operably linked" refers to a functional
relationship between two or more polylnucleotide (e.g., DNA)
segments. Typically, it refers to the functional relationship of a
transcriptional regulatory sequence to a transcribed sequence. For
example, a promoter or enhancer sequence is operably linked to a
coding sequence if it stimulates or modulates transcription of the
coding sequence in am appropriate host cell or other expression
system. Generally, promoter transcriptional regulatory sequences
that are operably linked to a transcribed sequence are physically
contiguous to the transcribed sequence, i.e., they are cis-acting.
However, some transcriptional regulatory sequences, such as
enhancers, need not be physically contiguous or located in close
proximity to the coding sequences whose transcription they
enhance.
[0056] The term "vector" is intended to refer to a polynucleotide
molecular capable of transporting another polynucleotide to which
it has been linked. One type of vector is a "plasmid", which refers
to a circular double stranded DNA loop into which additional DNA
segments may be ligated. Another type of vector is a viral vector,
wherein additional DNA segments may be heated into the viral
genome. Certain vectors are capable of autonomous replication in a
host cell into which they are introduced (e.g., bacterial vectors
having a bacterial origin of replication and episomal mammalian
vectors). Other vectors (e.g., non-episomal mammalian vectors) can
be integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively linked. Such
vectors are referred to herein as "recombinant expression vectors"
(or simply, "expression vectors"). In general, expression vectors
of utility in recombinant DNA techniques are often in the form of
plasmids. In the present speculation, "plasmid" and "vector" may be
used interchangeably as the plasmid is the most commonly used form
of vector. However, the disclosure is intended to include such
other forms of expression vectors such as viral vectors (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[0057] The term "recombinant host cell" (or simply "host cell")
refers to a cell in which a recombinant expression vector has been
introduced. It should be understood that such terms are intended to
refer not only to the particular subject cell but to the progeny of
such a cell. Because certain modifications may occur in succeeding
generation due to either mutation or environmental influences, such
progeny may not, in fact, be identical to the parent cell, but are
still included within the scope of the term "host cell" as used
herein.
[0058] The term "target antigen" refers to the antigen against
which the parent immunoglobulin was raised or otherwise generated
(e.g., by phage display).
[0059] The term "unmutated immunoglobulin" refers to the
immunoglobulin which does not comprise the at least one aggregation
reducing mutations. As used herein, the unmutated immunoglobulin
may be a hypothetical construct for the purposes of comparison of
the aggregation propensity or the binding affinity of the
immunoglobulin with and without the aggregation reducing mutations.
By way of example, a murine antibody that includes humanizing
mutations as well as aggregation reducing mutations is not the
unmutated immunoglobulin. The unmutated immunoglobulin would be the
affinity with the humanizing mutations, but without the aggregation
reducing mutations. Where a mutation is intended to serve more than
one purpose including aggregation reduction, the unmutated
immunoglobulin does not include such mutation.
[0060] The term "aggregation motif" refers to a set of residues
grouped together based upon the following process. First, residues
having an SAP (5 .ANG. radius) of greater than 0.15 are identified.
Then all residues within 5 .ANG. of each residue having an SAP (5
.ANG. radius) of greater than 0.15 are identified. A motif is then
the residue with an SAP (5 .ANG. radius) of greater than 0.15 and
all residues with an SAP (5 .ANG. radius) of greater than 0.0
within 5 .ANG. of the residue with an SAP (5 .ANG. radius) of
greater than 0.15. Any such motifs having at least one residue in
common are merged into a larger motif reiteratively until there are
no remaining motifs which have a residue in common. These remaining
motifs or sets of residues constitute aggregation motifs. Table 2
below sets out the aggregation motifs for the IgG constant
domains.
[0061] It is accordingly an object of the present invention to
provide a modified or isolated immunoglobulin that has a reduced
propensity for aggregation comprising at least one aggregation
reducing mutation at a residue selected from the group consisting
of residues from and an aggregation motif 1: 174(C.sub.H1),
175(C.sub.H1), and 181(C.sub.H1); an aggregation motif 2:
226(hinge), 227(hinge), 228(hinge), 229(hinge), 230(hinge),
231(hinge), and 232(hinge); an aggregation motif 3: 234(hinge) and
235(hinge); an aggregation motif 4: 252(C.sub.H2), and
253(C.sub.H2); an aggregation motif 5: 282(C.sub.H2); an
aggregation motif 6: 291(C.sub.H2); an aggregation motif 7:
296(C.sub.H2); an aggregation motif 8: 308(C.sub.H2) and
309(C.sub.H2); an aggregation motif 9: 328(C.sub.H2),
329(C.sub.H2), 330(C.sub.H2), and 331(C.sub.H2); an aggregation
motif 10: 395(C.sub.H3), 396(C.sub.H3), 397(C.sub.H3),
398(C.sub.H3), and 404(C.sub.H3); an aggregation motif 11:
443(C.sub.H3); an aggregation motif 12: 110(C.sub.L) and
111(C.sub.L); an aggregation motif 13: 153(C.sub.L) and
154(C.sub.L); and an aggregation motif 14: 201(C.sub.L), where the
at least one aggregation reducing mutation is a substitution with
an amino acid residue that is less hydrophobic than the residue in
the unmodified immunoglobulin and the propensity for aggregation
that is reduced is aggregation between immunoglobulin molecules in
a concentrated, liquid solution; and wherein the residue numbers
are the corresponding Kabat residue numbers in IgG1 based upon
alignment with the IgG1 sequence. In certain embodiments, the at
least one aggregation reducing mutation residue is selected from
the group consisting of residues from an aggregation motif 1:
175(C.sub.H1); an aggregation motif 2: 227(hinge), 228(hinge), and
230(hinge); an aggregation motif 3: 234(hinge) and 235(hinge); an
aggregation motif 4: 253(C.sub.H2); an aggregation motif 5:
282(C.sub.H2); an aggregation motif 6: 291(C.sub.H2); an
aggregation motif 7: 296(C.sub.H2); an aggregation motif 8:
309(C.sub.H2); an aggregation motif 9: 329(C.sub.H2) and
330(C.sub.H2); an aggregation motif 10: 395(C.sub.H3) and
398(C.sub.H3); an aggregation motif 11: 443(C.sub.H3); an
aggregation motif 12: 110(C.sub.L); an aggregation motif 13:
154(C.sub.L); and an aggregation motif 14: 201(C.sub.L). In certain
embodiments that may be combined with the preceding embodiments,
the aggregation reducing mutation is not residue 243(hinge) or
235(hinge). In certain embodiments that may be combined with the
preceding embodiments, the aggregation reducing mutation residue is
234(hinge), 235(hinge), 253(CH.sub.H2), or 309(C.sub.H2). In
certain embodiments that may be combined with the preceding
embodiments, the aggregation reducing mutation residue is
253(C.sub.H2) or 309(C.sub.H2). In certain embodiments that may be
combined with nay of the preceding embodiments, the immunoglobulin
has a second aggregation reducing mutation at a hydrophobic residue
that (i) has a Spatial-Aggregation-Propensity of at least 0.15, or
(ii) is within 5 .ANG. of a residue having a
Spatial-Aggregation-Propensity of at least 0.15, wherein the at
least one aggregation reducing mutation is a substitution with an
amino acid residue that is less hydrophobic than the residue in the
unmodified immunoglobulin. In certain embodiments that may be
combined with any of the preceding embodiments having a second
aggregation reducing mutation, the aggregation reducing mutation
and the second aggregation reducing mutation are at least 5 .ANG.,
at least 10 .ANG., at least 15 .ANG.A, or at least 20 .ANG. apart.
In certain embodiments that may be combined with any of the
preceding embodiments having a second aggregation reducing
mutation, the aggregation reducing mutation and the second
aggregation reducing mutation are in different aggregation motifs.
In certain embodiments that may be combined with any of the
preceding embodiments, the immunoglobulin has at least fourteen
aggregation reducing mutations wherein each aggregation reducing
mutation is selected from a different aggregation motif. In certain
embodiments that may be combined with any of the preceding
embodiments, the aggregation reducing mutation is substitution with
an amino acid residue selected from the group consisting of lysine,
arginine, histidine, glutamate, aspartate, glutamine, and
asparagine. In certain embodiments that may be combined with any of
the preceding embodiments, the aggregation reducing mutation is
substitution with an amino acid residue selected from the group
consisting of lysine, arginine, and histidine. In certain
embodiments that may be combined with any of the preceding
embodiments, the aggregation reducing mutation is substitution with
a lysine residue. In certain embodiments that may be combined with
the preceding embodiments, the Spatial-Aggregation-Propensity
(5< radius sphere) is calculated using the Black Mould
hydrophobicity scale normalized so that glycine equals 0. In
certain embodiments that may be combined with any of the preceding
embodiments, the immunoglobulin is an IgG1, an IgG2, an IgG3, or an
IgG4. In certain embodiments that may be combined with any of the
preceding embodiment, the immunoglobulin comprises an IgG1. In
certain embodiments that may be combined with any of the preceding
embodiments, the immunoglobulin has a human C.sub.H1 domain. In
certain embodiment that may be combined with any of the preceding
embodiments, the immunoglobulin has a human C.sub.H2 domain. In
certain embodiments that may be combined with any of the preceding
embodiments, the immunoglobulin has a human C.sub.H3 domain. In
certain embodiments that may be combined with any of the preceding
embodiments, the immunoglobulin has a human C.sub.L domain. In
certain embodiments that may be combined with any of the preceding
embodiments, the immunoglobulin has a binding affinity for a large
target antigen and the binding affinity for the target antigen is
at least seventy percent, at least eighty percent, at least ninety
percent, at least one hundred percent, or at least one hundred five
percent of the binding affinity of the unmutated immunoglobulin for
the target antigen. In certain embodiments that may be combined
with the preceding embodiments, the concentrated, liquid solution
is at a concentration of at least 10 mg/ml, at least 20 mg/ml, at
least 30 mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least 75
mg/ml, at least 100 mg/ml, at least 125 mg/ml, or at least 150
mg/ml.
[0062] It is accordingly a further object of the invention to
provide a modified or isolated immunoglobulin that has a reduced
propensity for aggregation comprising at least one aggregation
reducing mutation at a residue selected from the group consisting
of 235(hinge), 241(C.sub.H2), 243(C.sub.H2), 282(C.sub.H2), and
309(C.sub.H2), wherein if residue 235 is selected, it is mutated to
a glutamate or a serine, if residue 282 is selected it is mutated
to a lysine, and if residue 309 is selected, it is mutated to a
lysine, and wherein the at least one aggregation reducing mutation
is a substitution with an amino acid residue that is less
hydrophobic than the residue in the unmodified immunoglobulin and
the propensity for aggregation that is reduced is aggregation
between immunoglobulin molecules in a concentrated, liquid
solution; and wherein the residue numbers are the corresponding
Kabat residue numbers in IgG1 based upon alignment with the IgG1
sequence. In certain embodiments, the at least one aggregation
reducing mutation is a mutation of residue 241 to serine, and the
modified or isolated immunoglobulin further comprises a second
aggregation reducing mutation of residue 243 to serine. In certain
embodiments, the at least one aggregation reducing mutation is a
mutation of residue 241 to tyrosine, and the modified or isolated
immunoglobulin further comprises a second aggregation reducing
mutation of residue 243 to tyrosine. In certain embodiments, the at
least one aggregation reducing mutation is a mutation of residue
282 to lysine, and the modified or isolated immunoglobulin further
comprises a second and a third aggregation reducing mutation,
wherein the second aggregation reducing mutation is a mutation of
residue 235 to lysine and the third aggregation reducing mutation
is a mutation of residue 309 to lysine. In certain embodiments, the
immunoglobulin has a second aggregation reducing mutation at a
hydrophobic residue, wherein the at least one aggregation reducing
mutation is a substitution with an amino acid residue that is less
hydrophobic than the residue in the unmodified immunoglobulin. In
certain embodiments that may be combined with the preceding
embodiments having a second aggregation reducing mutation, the
second aggregation reducing mutation (i) has a
Spatial-Aggregation-Propensity of at least 0.15, or (ii) is within
5 .ANG. or a residue having a Spatial-Aggregation-Propensity of at
least 0.15. In certain embodiments that may be combined with the
preceding embodiments having a second aggregation reducing
mutation, the immunoglobulin has a third aggregation reducing
mutation that (i) has a Spatial-Aggregation-Propensity of at least
0.15, or (ii) is within 5 .ANG. of a residue having a
Spatial-Aggregation-Propensity of at least 0.15, wherein the third
aggregation reducing mutation is a substitution with an amino acid
residue that is less hydrophobic than the residue in the unmodified
immunoglobulin. In certain embodiments that may be combined with
the preceding embodiments having a second aggregation reducing
mutation, the aggregation reducing mutation and the second
aggregation reducing mutation are at least 5 .ANG., at least 10
.ANG., at least 15 .ANG., or at least 20 .ANG. apart. In certain
embodiments that may be combined with any of the preceding
embodiments having a second aggregation reducing mutation, the
aggregation reducing mutation and the second aggregation reducing
mutation are in different aggregation motifs. In certain
embodiments that may be combined with any of the preceding
embodiments, the immunoglobulin has at least fourteen aggregation
reducing mutations wherein each aggregation reducing mutation is
selected from a different aggregation motif. In certain embodiments
that may be combined with any of the preceding embodiments, the
aggregation reducing mutation is substitution with an amino acid
residue selected from the group consisting of lysine, arginine,
histidine, glutamate, aspartate, glutamine, asparagine, tyrosine,
and serine. In certain embodiments that may be combined with any of
the preceding embodiments, the aggregation reducing mutation is
substitution with an amino acid residue selected from the group
consisting of lysine, serine, glutamate, and histidine. In certain
embodiments that may be combined with the preceding embodiments,
the Spatial-Aggregation-Propensity (5 .ANG. radius sphere) is
calculated using the Black Mould hydrophobicity scale normalized so
that glycine equals 0. In certain embodiments that may be combined
with any of the preceding embodiments, the immunoglobulin is an
IgG1, an IgG2, an IgG3, or an IgG4. In certain embodiments that may
be combined with any of the preceding embodiments, the
immunoglobulin comprises an IgG1. In certain embodiments that may
be combined with any of the preceding embodiments, the
immunoglobulin has a human C.sub.H1 domain. In certain embodiments
that may be combined with any of the preceding embodiments, the
immunoglobulin has a human C.sub.H2 domain. In certain embodiments
that may be combined with any of the preceding embodiments, the
immunoglobulin has a human C.sub.H3 domain. In certain embodiments
that may be combined with any of the preceding embodiments, the
immunoglobulin has a human C.sub.L domain. In certain embodiments
that may be combined with any of the preceding embodiments, the
immunoglobulin has a binding affinity for a target antigen and the
binding affinity for the target antigen is at least seventy
percent, at least eighty percent, at least ninety percent, at least
one hundred percent, or at least one hundred five percent of the
binding affinity of the unmutated immunoglobulin for the target
antigen. In certain embodiments that may be combined with the
preceding embodiments, the concentrated, liquid solution is at a
concentration of at least 10 mg/ml, at least 20 mg/ml, at least 30
mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least 75 mg/ml, at
least 100 mg/ml, at least 125 mg/ml, or at least 150 mg/ml.
[0063] Where immunoglobulin residues are referred to by number
herein, the residue number refers to the Kabat number of the
corresponding residue in the IgG1 molecule when the immunoglobulin
sequence of interest is aligned to the human IgG1 immunoglobulin.
By way of reference, the human IgG1, IgG2, IgG3 and IgG4 constant
domains are aligned:
TABLE-US-00001 C.sub.H1 domain: IgG1 (SEQ ID NO: 1) IgG2 (SEQ ID
NO: 2) IgG4 (SEQ ID NO: 3) IgG3 (SEQ ID NO: 4) ...A.. loop
....B.... loop..C... C'loop..D. 120 130 140 150 160 170 | | | | | |
IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF IgG2
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF IgG4
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF IgG3
ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF .. loop
...E..... loop. ...F... loop ..G....join 180 190 200 210 220 | | |
| | IgG1 PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC IgG2
PAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC IgG4
PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG IgG3
PAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVEPKTP Hinge: IgG1 (SEQ
ID NO: 5) IgG2 (SEQ ID NO: 6) IgG4 (SEQ ID NO: 7) IgG3 (SEQ ID NO:
8) upper middle lower 230 | IgG1 -DKTHT ---------------- CPPCP
APELLGG IgG2 -VE--- ---------------- CPPCP AP-PVAG IgG4 -PP---
---------------- CPSCP APEFLGG IgG3 LGTTHT CPRCPEPK******** CPRCP
APELLGG C.sub.H2 domain: IgG1 (SEQ ID NO: 9) IgG2 (SEQ ID NO: 10)
IgG4 (SEQ ID NO: 11) IgG3 (SEQ ID NO: 12) ..A.. loop ....B.... loop
..C.. C'loop ...D 240 250 260 270 280 290 | | | | | | IgG1
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP IgG2
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKP IgG4
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP IgG3
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKP ... loop
....E... .loop. ...F.....loop ..G... joinC3 300 310 320 330 340 | |
| | | IgG1 REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
IgG2 REEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPRE IgG4
REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE IgG3
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPRE C.sub.H3
domain: IgG1 (SEQ ID NO: 13) IgG2 (SEQ ID NO: 14) IgG4 (SEQ ID NO:
15) IgG3 (SEQ ID NO: 16) ..A.. loop ....B.... loop
..C...C'loop..D.... 350 360 370 380 390 400 | | | | | | IgG1
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS IgG2
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS IgG4
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS IgG3
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYKTTPPVLDS loop
....E... .loop. ...F... loop ....G.... 410 420 430 440 | | | | IgG1
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK IgG2
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK IgG4
DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK IgG3
DGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNHFTQKSLSLSPGK
[0064] Spatial-Aggregation-Propensity
[0065] The invention herein refers to methods for identifying
aggregation prone regions on a protein surface and for preventing
or reducing aggregation of a protein. The invention may be applied
to generate immunoglobulin with reduced aggregation propensity,
i.e., the immunoglobulin in concentrated solution remains primarily
in monomeric form rather than higher order aggregated multimer. The
methods herein represent an advancement in the ability of
computational methods to identity protein regions which may be
modified to reduce the propensity of a protein from aggregating. In
particular, the methods are based, at least in part, on the
calculation of the SAA (Solvent Accessible Area), which is known in
the art for characterizing the surface of a protein. SAA gives the
surface area of each amino acid or protein structure that is in
contact with the solvent. SAA may be typically calculated by
computing the locus of the center of a probe sphere as it rolls
over the protein surface, i.e., the surface of a protein structural
model. The probe sphere has the same radius as that of a water
molecule, R=1.4 .ANG.. Alternative methods of calculating SAA,
described below, are known in the art and are compatible with the
methods described herein. Although SAA is quite useful to
characterize the protein surface, it was not found to be adequate
to characterize the hydrophobic patches on the protein surface that
are potentially aggregation prone because of the following
shortcomings,
1. SAA doesn't distinguish between hydrophobic and hydrophilic
regions 2. SAA is not directly proportional to a residue's
hydrophobicity (for example, MET has more surface area than LEU but
is less hydrophobic) 3. SAA doesn't indicate whether several
hydrophobic residues are close-by and thus could enhance the
hydrophobicity of a certain region. These residues could be
close-by either in primary sequence or in the tertiary structure
even though they are far in primary sequence. Either way, they
could enhance the hydrophobicity of a certain patch on the antibody
surface.
[0066] One measure which is described herein, the Effective-SAA, is
generated by calculating the hydrophobicity of the fraction of the
amino acid which is exposed according to the formula below:
Effective - SAA = SAA SAA fully exposed .times. Residue
hydrophobicity ##EQU00001##
[0067] A further embodiment of the effective-SAA further comprises
summing the Effective-SAA over at least two, at least three, at
least four, at least five or at least six (e.g., two, three, four,
five, six, etc.) amino acid residues which are adjacent in the
primary protein sequence. Although the Effective-SAA represents an
improvement over the basis SAA, it nevertheless lacks the ability
to fully account for the structure of the folded protein and for
the fact that amino acids which are not adjacent in the protein
sequence may be in proximity to one another in the folded
secondary, tertiary, or quaternary structure of a protein. Such
protein folds may form aggregation prone regions which do not
appear in the primary structure alone, or which may only be
detected by more robustly analyzing the folded protein
structure.
[0068] The present invention provides a new, more advanced measure,
called the Spatial-Aggregation-Propensity, which will highlight the
effective hydrophobicity of a certain patch or region on the
protein surface. The Spatial-Aggregation-Propensity is calculated
for defined spatial regions on or near the atoms of a protein
structural model.
[0069] In this context, a "defined spatial region" is a
three-dimensional space or volume chosen to capture a local
physical structure and/or chemical environment on or near the
protein structure. In a particularly preferred embodiment the
Spatial-Aggregation-Propensity is calculated for spherical regions
with radius R centered on atoms in a protein (e.g., atoms in a
protein structural model). The Spatial-Aggregation-Propensity may
also be calculated for spherical regions with radius R centered on
chemical bonds, or positioned in space near the structural model.
Accordingly, in another embodiment the SAP may be calculated for a
defined spatial region centered near at atom, e.g., centered on a
point in space which is between 1-10 .ANG., 1-5 .ANG. or 1-2 .ANG.
from the center of a particular atom or chemical bond.
[0070] In certain embodiments, the chosen radius R is between 1
.ANG. and 50 .ANG.. In particular embodiments the chosen radius is
at least 1 .ANG., at least 3 .ANG., at least 4 .ANG., at least 5
.ANG., at least 6 .ANG., at least 7 .ANG., at least 8 .ANG., at
least 9 .ANG., at least 10 .ANG., at least 11 .ANG., at least 12
.ANG., at least 15 .ANG., at least 20 .ANG., at least 25 .ANG., or
at least 30 .ANG.. In certain embodiments, the chosen radius is
between 5 .ANG. and 15 .ANG., between 5 .ANG. and 12 .ANG., or
between 5 .ANG. and 10 .ANG.. In specific embodiments the chosen
radius is 5 .ANG. or 10 .ANG..
[0071] In other embodiments, the region for which the
Spatial-Aggregation-Propensity is calculated is not spherical. The
possible shape of the region may further comprise a cube, a
cylinder, a cone, an elliptical spheroid, a pyramid, a hemisphere,
or any other shape which may be used to enclose a portion of space.
In such embodiments, the size of the region may be chosen using
measures other than radius, e.g., the distance from the corner of
the shape to a face or vertex.
[0072] In a certain embodiment, the SAP may be used to select
residues in a protein, particularly an antibody or immunoglobulin,
which may be substituted, thus increasing the protein's stability.
In previous studies two main approaches to stabilize a protein in
vitro have been to (1) engineer the protein sequence itself and (2)
include additives in the liquid formulation. Both approaches have
been investigated and significant results have been obtained. The
first approach has relied on screening extensive libraries of
random variants in silico of experimentally. In the second
approach, high-throughput screening for stabilizing additives, as
well as rational design of additives permits identification of
optimal formulations for a therapeutic protein.
[0073] The present invention is expected to streamline the process
of stability enhancement by identifying existing hot-spots for
aggregation computationally, and analyzing variants with
substitutions at those sites experimentally.
[0074] Thus, in general terms, a method for calculating the
Spatial-Aggregation-Propensity for a particular atom its a protein
comprises (a) identifying one or more toms in a structural model,
representing the protein, wherein the one or more atoms are within
a defined spatial region centered on or near the particular atom;
(b) calculating, for each or the one or more atoms in the defined
spatial region, a ratio of the solvent accessible area (SAA) of the
atoms to the SAA of atoms in an identical residue which is fully
exposed; (c) multiplying each ratio by the atom hydrophobicity of
the one or more atoms; and (d) summing the products of step (c);
whereby the sum is the SAP for the particular atom.
[0075] In a related embodiment, the SAP may be calculated according
to a different method comprising (a) identifying one or more amino
acid residues in a structural model representing the protein,
wherein the one or more amino acid residues have at least one atom
within a defined spatial region centered on or near the particular
atom; (b) calculating, for each of the identical one or more amino
acid residues, a ratio of the solvent accessible area (SAA) of
atoms in the amino acid to the SAA of atoms in an identical residue
which is fully exposed; (c) multiplying each ratio by the
hydrophobicity of the one or more amino acid residues as determined
by an amino acid hydrophobicity scale; and (d) summing the products
of step (c); whereby the sum is the SAP for the particular atom. In
preferred embodiments, the structural model is processed prior to
step (a) by allowing the structural model to interact with solvent
in a molecular dynamics simulation. When an amino acid is
identified as having at least one atom within the defined spatial
region, the at least one atom may be required to be exclusively an
atom in an amino acid side chain. Alternatively it may be an atom
required to be a main chain atom.
[0076] In other embodiments, this method may further comprise
optionally conducting a molecular dynamics simulation prior to step
(a) and repeating steps (a)-(d), each time conducting a further
molecular dynamics simulation at a plurality of time steps, thereby
producing multiple sums as in step (d), and calculating the average
of the sums; whereby the calculated average is the SAP for the
particular atom.
[0077] One of skill in the art will appreciate that an embodiment
of the present invention which employs the average of values
calculated over a molecular dynamics simulation will be more
computationally intensive. Such an embodiment will also, in some
cases, provide a more precise or highly resolved map of the
Spatial-Aggregation-Propensity. However, experiments disclosed
herein have shown that the method is still highly accurate when the
molecular dynamics averaging is not employed. In one preferred
embodiment, Spatial-Aggregation-Propensity values may be calculated
for all protein structures in a database, e.g., the Protein Data
Bank (PDB), thereby swiftly identifying hydrophobic residues and
patches on all known protein structures. This method allows rapid
screening of large sets of proteins to identify potential
aggregation prone regions and/or protein interaction sites.
[0078] In a preferred application, the
Spatial-Aggregation-Propensity is described.
[0079] In the following formula:
SAP.sub.atom=.SIGMA..sub.Simulation Average(.SIGMA..sub.atoms
within R of atom((SAA-R/SAA-fe)*atom-hb)
[0080] wherein:
1) SAA-R is SAA of side chain atoms within radius R which is
computed at each simulation snapshot. SAA is preferably calculated
in the simulation model by computing the locus of the center of a
probe sphere as it rolls over the protein surface. The probe sphere
has the same radius as that of a water molecule, R=1.4 A. One of
skill in the art will appreciate that other methods of computing
the SAA would be compatible with the methods described here to
calculate SAP. For example, the SAA may be calculated on only amino
acid side chain atoms. The SAA may also be calculated on only amino
acid main chain atoms (i.e., those atoms of the peptide backbone
and associated hydrogens). Alternatively, the SAA may be calculated
on only amino acid main chain atoms with the exclusion of
associated hydrogens; 2) SAA-fe is SAA of side chain of fully
exposed residue (say for amino acid `X`) which is obtained, in a
preferred embodiment, by calculating the SAA of side chains of the
middle residue in the fully extended conformation of tripeptide
`Ala-X-Ala`; and 3) atom-hb is Atom Hydrophobicity which is
obtained as described above using the hydrophobicity scale of Black
and Mould (Black and Mould, Anal. Biochem. 1991, 193, 72-82). The
scale is normalized such that Glycine has a hydrophobicity of zero.
Therefore, amino acids that are more hydrophobic than Glycine are
positive and less hydrophobic than Glycine are negative on the
hydrophobic scale.
[0081] A residue which is "fully exposed" is a residue, X, in the
fully extended conformation of that tripeptide Ala-X-Ala. One of
skill in the art will appreciate that this arrangement is designed
such that a calculation of SAA on such a residue, X, will yield the
maximum solvent accessible area available. Accordingly, it is
contemplated that other residues besides alanine may be used in the
calculation without wholly disrupting or altering the results.
[0082] As described above, the methods of the present invention may
be applied to any protein structural model including an X-ray
structure using the same formula as above.
[0083] Similarly, if the X-ray structure is not available, the same
Spatial-Aggregation-Propensity parameter can be applied to the
structure generated through homology modeling, and the SAP
parameter may be calculated using the same formula as above.
[0084] In certain embodiments the Spatial-Aggregation-Propensity is
calculated for all atoms in a protein structural model. In some
embodiments, the atomistic Spatial-Aggregation-Propensity values
may be averaged over each individual protein residue, or over small
groups of residues.
[0085] Uses of the SAP Methodology
[0086] In one aspect, the present invention may be used as
described above to identity hydrophobic amino acid residues,
regions or patches in a protein. Without wanting to be held to
specific threshold values, atoms or amino acid residues having a
Spatial-Aggregation-Propensity >0 are considered to be
hydrophobic, or to be in an aggregation prone region. Depending on
the type of protein, the particular structure, and the solvent in
which it exists, it may be desirable to identify atoms or residues
using a cutoff which is slightly below zero, e.g., by choosing
atoms or residues which have a Spatial-Aggregation-Propensity of
greater than -0.1, -0.15, -0.2, etc. Alternatively, it may be
desirable to employ a more stringent cutoff, e.g., 0, 0.05, 1.0,
0.15, 0.2, etc., in order to choose the strongest hydrophobic
atoms, residues, or patches. In addition, as the algorithm gives
higher numbers to residues at the center of a patch, residues
within 3 A, 4 A, 5 A, 7.5 A, or 10 A of the residue meeting the
cutoff can also be selected for mutation to less hydrophobic
residues to reduce aggregation. In another embodiment it may be
advantageous simply to select atoms or residues having
Spatial-Aggregation-Propensity which is larger than atoms or
residues which are nearby either sequentially (i.e., along the
protein sequence) or, in a preferred embodiment, spatially (i.e.,
in the three-dimensional structure). One preferred method for
selecting atoms or residues in a hydrophobic patch is to map the
calculated Spatial-Aggregation-Propensity values, e.g., using a
color coding or numerical coding, onto the protein structural model
from which they were derived, thus visualizing differences in the
Spatial-Aggregation-Propensity across the protein surface and hence
allowing easy selection of hydrophobic patches or residues. In a
particularly preferred embodiment, the calculations for
Spatial-Aggregation-Propensity are carried out separately using two
values chosen for the radius, one of higher resolution, e.g., 5 A,
and one of lower resolution, e.g., 10 A. In such an embodiment
larger or broader hydrophobic patches may be seen on the protein
structure with the lower resolution map. Once hydrophobic patches
of interested are selected on the low resolution map, those patches
may be viewed its greater detail in the higher resolution map which
may, in some embodiments, allow one of skill in the art to more
easily or more accurately choose residues to mutate or modify. For
example, when viewing a hydrophobic patch in the higher resolution
map, it may be desirable to select for mutation the residue which
has the highest SAP score or is the most hydrophobic (e.g., the
most hydrophobic residue in the patch according to the scale of
Black and Mould, Anal. Biochem. 1991, 193, 72-82).
[0087] In a specific embodiment a method to identity an aggregation
prone region on a protein comprise (a) mapping onto the structural
model the SAP as calculated according to any of the methods
described herein for atoms in the protein; and (b) identifying a
region within in the protein having a plurality of atoms having a
SAP>0; wherein the aggregation prone region comprises the amino
acids comprising said plurality of atoms. In such an embodiment the
SAP may be calculated for all the atoms in a protein or a portion
of the atoms. It is contemplated that one may only calculate the
SAP for particular residues or groups of residues which are of
interest.
[0088] In a similar embodiment, it may be informative to plot the
SAP scores of the atoms (or the SAP score as averaged over amino
acid residues). Such a plot showing the SAP score along the atoms
or residues of a protein allows the easy identification of peaks,
may indicate candidates for replacement. In a particularly
preferred embodiment the SAP scores along the atoms or residues in
the protein are plotted in a graph and the Area Under the Curve
(AUC) is calculated for peaks in the graph. In such an embodiment,
peaks with a larger AUC represent larger or more hydrophobic
aggregation prone regions. In particular embodiments it will be
desirable to select for replacement one or more residues which are
identified as existing in a peak, or, more preferably, in a peak
with a large AUC.
[0089] In particular embodiments the present invention may be used
to make an immunoglobulin variant which exhibits a reduced
propensity for aggregation by replacing at least one amino acid
residue within an aggregation prone region in the immunoglobulin
identified by any of the methods described herein with an amino
acid residue which is more hydrophilic then the residue which is
being replaced, such that the propensity for aggregation of the
variant is reduced. As used herein, when amino acid residues are
referred to as "more" or "less" hydrophilic or hydrophobic, it will
be appreciated by the skilled artisan that this signifies more or
less hydrophobic as compared to another amino acid according to a
measure of hydrophobicity (hydrophilicity) known in the art, e.g.,
the hydrophobicity scale of Black and Mould.
[0090] In a similar embodiment the present invention may be used to
make an immunoglobulin variant which exhibits a reduced propensity
for aggregation by generating a plurality of immunoglobulin
variants by replacing, in each variant at least one residue within
an aggregation prone region in the immunoglobulin, wherein the
aggregation prone region is identified using SAP scores calculated
according any method described herein, wherein one or different
residues or different combinations of residues are replaced in each
variant, and wherein the at least one residue is replaced with a
residue which is more hydrophilic; and (b) selecting an
immunoglobulin variant prepared as in (a) which exhibits a reduced
propensity for aggregation.
[0091] In addition, an amino acid residue in an aggregation prone
region may be deleted rather than replaced. In some immunoglobulins
where multiple amino acid residues are selected for replacement,
some residues may be replaced while others are deleted.
[0092] In further embodiments multiple aggregation prone regions or
residues may be identified in an initial immunoglobulin by the
methods described above (e.g., by using a
Spatial-Aggregation-Propensity cutoff above which residues are
selected). Subsequently, a plurality of immunoglobulin variants may
be generated by replacing in said initial immunoglobulin one or
more selected amino acid residues (or one or more residues falling
in selected patch) with amino acid residues which are more
hydrophilic, such that a plurality of immunoglobulin variants are
created representing a variety of different amino acid
substitutions. This population may then be screened to select one
or more immunoglobulin variants which have a reduced propensity for
aggregation. One of skill in the art will appreciate that multiple
aggregation prone regions may be identified, and that one or more
substitutions and/or deletions may be made in one or more
aggregation prone regions. The relative hydrophobicity of the amino
acids may be determined by the hydrophobicity scale of Black and
Mould as described above. In specific embodiments, an amino acid to
be replaced is selected from the group comprising or consisting of
Phe, Leu, Ile, Tyr, Trp, Val, Met, Pro, Cys, Ala, or Gly. In
related embodiments, the more hydrophilic amino acid which will be
substituted into the immunoglobulin will be chosen from the group
comprising or consisting of Thr, Ser, Lys, Gln, Asn, His, Glu, Asp,
and Arg.
[0093] It is accordingly an object of the present invention to
provide modified and/or isolated immunoglobulins that have a
reduced propensity for aggregation comprising at least one
aggregation reducing mutation at a residue in a conserved domain of
the immunoglobulin that (i) has a Spatial-Aggregation-Propensity (5
.ANG. radius sphere) of at least 0.15, or (ii) has an
Spatial-Aggregation-Propensity (5 .ANG. radius sphere) of greater
than 0.0 and is within 5 .ANG. of a residue having a
Spatial-Aggregation-Propensity (5 .ANG. radius sphere) of at least
0.15, wherein the at least one aggregation reducing mutation is a
substitution with an amino acid residue that lowers the
Spatial-Aggregation-Propensity (5 .ANG. radius sphere) of the
residue as compared to the unmutated immunoglobulin and the
propensity for aggregation that is reduced is aggregation between
immunoglobulin molecules in a concentrated, liquid solution. In
certain embodiments, the at least one aggregation reducing mutation
is not at a residue corresponding to Kabat residue 234(hinge) or
235(hinge) in IgG1 based upon alignment with the IgG1 sequence. In
certain embodiments that may be combined with the preceding
embodiments, the immunoglobulin has a second aggregation reducing
mutation at a residue that (i) has a Spatial-Aggregation-Propensity
(5 .ANG. radius sphere) of at least 0.15, or (ii) has an
Spatial-Aggregation-Propensity (5 .ANG. radius sphere) of greater
than 0.0 and is within 5 .ANG. of a residue having a
Spatial-Aggregation-Propensity (5 .ANG. radius sphere) of at least
0.15, wherein the second aggregation reducing mutation is a
substitution with an amino acid residue that is a substitution with
an amino acid residue that lowers the
Spatial-Aggregation-Propensity (5 .ANG. radius sphere) of the
residue as compared to the unmutated immunoglobulin. In certain
embodiments that may be combined with the preceding embodiments
having a second aggregation reducing mutation, the aggregation
reducing mutation and the second aggregation reducing mutation are
at least 5 .ANG., at least 10 .ANG., at least 15 .ANG., or at least
20 .ANG. apart. In certain embodiments that may be combined with
the preceding embodiments having a second aggregation reducing
mutation, the aggregation reducing mutation and the second
aggregation reducing mutation are in different aggregation motifs.
In certain embodiments that may be combined with any of the
preceding embodiments, the aggregation reducing mutation is a
substitution with an amino acid residue that is less hydrophobic
than the residue in the unmodified immunoglobulin. In certain
embodiments that may be combined with the preceding embodiments,
the aggregation reducing mutation is a substitution with an amino
acid residue selected from the group consisting of lysine,
arginine, histidine, glutamate, aspartate, glutamine, and
asparagine. In certain embodiments that may be combined with the
preceding embodiments, the aggregation reducing mutation is a
substitution with an amino acid residue selected from the group
consisting of lysine, arginine, and histidine. In certain
embodiments that may be combined with the preceding embodiments,
the aggregation reducing mutation is a substitution with a lysine
residue. In certain embodiments that may be combined with the
preceding embodiments, the Spatial-Aggregation-Propensity (5 .ANG.
radius sphere) is calculated using the Black Mould hydrophobicity
scale normalized so that glycine equals 0. In certain embodiments
that may be combined with the preceding embodiments, the
immunoglobulin is an IgG1, an IgG2, an IgG3, or an IgG4. In certain
embodiments that may be combined with the preceding embodiments,
the immunoglobulin is an IgG1. In certain embodiments that may be
combined with the preceding embodiments, the immunoglobulin has a
human C.sub.H1 domain. In certain embodiments that may be combined
with the preceding embodiments, the immunoglobulin has a human
C.sub.H2 domain. In certain embodiments that may be combined with
the preceding embodiments, the immunoglobulin has a human C.sub.H3
domain. In certain embodiments that may the combined with the
preceding embodiments, the immunoglobulin fuss a human C.sub.L
domain. In certain embodiments that may be combined with the
preceding embodiments, the immunoglobulin has a binding affinity
for a target antigen and the binding affinity for the target
antigen is at least seventy percent, at least eighty percent, at
least ninety percent, at least one hundred percent, or at least one
hundred five percent of the binding affinity of the unmutation
immunoglobulin for the target antigen. In certain embodiments that
may be combined with the preceding embodiments, the concentrated,
liquid solution is at a concentration of at least 10 mg/ml, at
least 20 mg/ml, at least 30 mg/ml, at least 40 mg/ml, at least 50
mg/ml, at least 75 mg/ml, at least 100 mg/ml, at least 125 mg/ml,
at least 150 mg/ml.
[0094] Immunoglobulin variants may be made by any method known in
the art including site directed mutagenesis and other recombinant
DNA technology, e.g., see U.S. Pat. Nos. 5,284,760; 5,556,747;
5,789,166; 6,878,531, 5,932,419; and, 6,391,548.
[0095] In particular embodiments the present invention may be used
to make an immunoglobulin variant which exhibits a reduced
propensity for aggregation by replacing at least one amino acid
residue within an aggregation prone region in the immunoglobulin
identified by any of the methods described herein with a natural
amino acid residue, a modified amino acid residue, an unusual amino
acid residue, an unnatural acid residue, or an amino acid analog or
derivative which is more hydrophilic then the residue which is
being replaced, such that the propensity for aggregation of the
variant is reduced.
[0096] The synthesis of unnatural amino acids is known to those of
skill in the art, and is further described, e.g., in U.S. Patent
Publication No. 2003-0082575. In general, any method known in the
art to synthesize or incorporate unnatural, modified, or unusual
amino acids into protein may be employed including, but not limited
to those methods described or referenced in the publications Liao
J. Biotechnol Prog. 2007 January-February; 23(1):28-31; Rajesh and
Iqbal. Curr Pharm Biotechnol. 2006 August; 7(4):247-59; Cardillo et
al. Mini Rev Med Chem. 2006 March; 6(3): 293-304; Wang et al. Annu
Rev Biophys Biomol Struct. 2006; 35-225-49; Chakraborty et al.,
Glycoconj J. 2005 March; 22(3):83-93. As a further example, the
Ambrx ReCODE.TM. technology may be employed to develop and
incorporate unnatural amino acids, or unusual amino acids into
proteins as indicated by the methods described herein.
[0097] Immunoglobulin variants according to the invention can
exhibit enhanced or improved stability as determined, for example,
by accelerated stability studies. Exemplary accelerated stability
studies include, but are not limited to, studies featuring
increased storage temperatures. A decrease in the formation of
aggregates observed for a immunoglobulin variant as compared to the
wild type or initial protein indicates an increased stability.
Stability of immunoglobulin variants may also be tested by
measuring the change in the melting temperature transition of a
variant as compared to the wild type or initial immunoglobulin. In
such an embodiment, increased stability would be evident as an
increase in the melting temperature transition in the variant.
Additional methods for measuring protein aggregation are described
in U.S. patent application Ser. No. 10/176,809.
[0098] It is accordingly an object of the present invention to
provide isolated or recombinant polynucleotides that encode
modified immunoglobulins as discussed in paragraphs [0059], [0060],
or [0089] and any and all combinations of their embodiments. In
certain embodiments, the polynucleotide is in a vector. In certain
embodiments, the vector is an expression vector, in certain
embodiments that may be combined with the preceding embodiments, an
inducible promoter is operably linked to the polynucleotide.
Another aspect includes host cells with the vector of either of the
preceding embodiments. In certain embodiments, the host cells are
capable of expressing the immunoglobulin encoded by the
polynucleotide.
[0099] It is accordingly an object of the present invention to
provide methods of producing an immunoglobulin with a reduced
aggregation propensity comprising providing a culture medium
comprising the host cell of the preceding paragraph and placing the
culture medium in conditions under which the immunoglobulin is
expressed. In certain embodiments, the methods include an
additional step of isolating the immunoglobulin expressed.
[0100] In another aspect of the invention the calculated
Spatial-Aggregation-Propensity may be used to identify
protein-protein interaction sites on the surface of a protein
structure. It is known in the art that protein interactions sites
often contain hydrophobic residues or hydrophobic patches. It is
expected that the methods described herein will be useful in
locating binding sites by identifying hydrophobic patches. Such
hydrophobic patches will then be candidates for protein-protein or
protein-ligand recognition sites.
[0101] In some embodiments, the invention further relates to
computer code for determining SAP according to the methods of the
invention. In other embodiments, the invention relates to a
computer, a supercomputer, or cluster of computers dedicated to
performing the methods of the invention. In yet other aspect, the
invention provides a web-based, server based, or internet based
service for determining aggregation prone regions on a protein, the
service comprising accepting data about a protein (e.g., a protein
structural model) from a user (e.g., over the internet) or
retrieving such data from a database such that the service provider
can generate, retrieve, or access a static structure of the
protein, optionally including molecular dynamics modeling of the
protein to provide a dynamic structure of the protein, determining
SAP for atoms or residues of the protein based on the static or
dynamic structure so generated, and returning the SAP data, for
example, as a structural model mapped with said SAP data by the
service provider, to a user. In some embodiments, the user is a
person. In other embodiments the user is a computer system or
automated computes algorithm.
[0102] In some embodiments the present invention proves an SAP
calculation system comprising: a web server for providing a web
service for calculating SAP to a user terminal through the
Internet; a database for storing general information on the
calculation method, amino acid hydrophobicity, etc., and a
calculation server for performing the SAP calculated based on
information in the database and information provided or transmitted
through the internet by the user.
[0103] In some embodiments, the web server and the calculation
server are the same computer system. In some embodiments the
computer system is a supercomputer, cluster computer, or a single
workstation or server. In a related embodiment the web server of
the SAP calculation system further comprises a controller for
controlling the entire operation, a network connection unit for
connection to the Internet, and a web service unit for providing a
web service for calculating SAP to the user terminal connected
through the Internet.
[0104] In addition, embodiments of the present invention further
relate to computer storage products with a computer readable medium
that contain program code for performing various
computer-implemented operations, e.g., calculating the SAP for a
structural model, calculating SAA, calculating effective-SAA,
manipulating structural models, implementing molecular dynamics
simulations, organizing and storing relevant data, performing other
operations described herein. The computer-readable medium is any
data storage device that can store data which can thereafter be
read by a computer system. Examples of computer-readable media
include, but are not limited to hard disks, floppy disks, flash
drives, optical discs (e.g., CDs, DVDs, HD-DVDs, Blu-Ray discs,
etc.) and specially configured hardware devices such as application
specific integrated circuits (ASICs) or programmable logic devices
(PLDs). The computer-readable medium can also be distributed as a
data signal embodied in a carrier wave over a network of coupled
computer systems so that the computer readable code is stored and
executed in a distributed fashion. It will be appreciated by those
skilled in the art that the above described hardware and software
elements are of standard design and construction. The computer,
internet, server, and service related embodiments described above
may further apply to the SAA and the effective-SAA as well as
SAP.
[0105] Pharmaceutical Compositions Containing Immunoglobulins and
Immunoglobulin Variants
[0106] In another aspect, the present invention provides a
composition, e.g., a pharmaceutical composition, containing one or
more immunoglobulin variants produced by the methods of the
invention, formulated together with a pharmaceutically acceptable
carrier. Pharmaceutical compositions of the invention also can be
administered in combination therapy, i.e., combined with other
agents. For example, the combination therapy can include an
immunoglobulin of the present invention combined with at least one
other anti-cancer agent.
[0107] As used, herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal, agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
Preferably, the carrier is suitable for intravenous, intramuscular,
subcutaneous, parenteral, spinal or epidermal administration (e.g.,
by injection or infusion). Depending on the route of
administration, the active compound, i.e., the immunoglobulin or
variant thereof of the invention, may the coated in a material to
protect the compound from the action of acids and other natural
conditions that may inactivate the compound.
[0108] The pharmaceutical compositions of the Invention may include
one or more pharmaceutically acceptable salts. A "pharmaceutically
acceptable salt" refers to a salt that retains the desired
biological activity of the parent compound and does not impart any
undesired toxicological effects (see e.g., Berge, S. M., et al.
(1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid
addition salts and base addition salts. Acid addition salts include
those derived from nontoxic inorganic acids, such as hydrochloric,
nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous
and the like, as well as from nontoxic organic acids such as
aliphatic mono- and dicarboxylic acids, phenylsubstituted alkanoic
acids, hydroxy alkanoic acids, aromatic acids, aliphatic and
aromatic sulfonic acids and the like. Base addition salts include
those derived front alkaline earth metals, such as sodium,
potassium, magnesium, calcium and the like, as well as from
nontoxic organic amines, such as N,N'-dibenzylethylenediamine,
N-methyglucamine, chloroprocaine, choline, diethanolamine,
ethylenediamine, procaine and the like.
[0109] A pharmaceutical composition of the invention also may
include a pharmaceutically acceptable antioxidant. Examples of
pharmaceutically acceptable antioxidants include: (1) water soluble
antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium
bisulfate, sodium metabisulfate, sodium sulfite and the like; (2)
oil-soluble antioxidants, such as ascorbyl palmitate, butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin,
propyl gallate, alpha-tocopherol, and the like; and (3) metal
chelating agents, such as citric acid, ethylenediamine tetraacetic
acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the
like.
[0110] Examples of suitable aqueous and nonaqueous carriers that
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0111] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures, and by the inclusion of various
antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injection pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin.
[0112] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. The use
of such media and agents for pharmaceutically active substances is
known in the art. Except insofar as any conventional media or agent
is incompatible with the active compound, use thereof in the
pharmaceutical compositions of the invention is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0113] Exemplary formulations comprise at least one immunoglobulin
variant of the invention and can comprise lower concentrations of
stabilizing (or disaggregation) agents which can, in addition to
the methods disclosed herein, be used to prevent or diminish
aggregation of an immunoglobulin. Accordingly, conventional methods
used to prevent aggregation may be employed in the development of
pharmaceutical compositions containing immunoglobulin variants
produced by the methods of the present invention. For example, a
variety of stabilizing or disaggregating compounds may be included
in pharmaceutical compositions of the invention depending on their
intended use and their biological toxicity. Such stabilizing
compounds may include, for example, cyclodextrin and its
derivatives (U.S. Pat. No. 5,930,969, alkylglycoside compositions
(U.S. patent application Ser. No. 11/474,049), the use of chaperone
molecules (e.g., LEA (Goyal et al., Biochem J. 2005, 388(Pt
1):151-7; the methods of U.S. Pat. No. 5,688,651), betaine
compounds (Xiao, Burn, Tolbert, Bioconjug Chem. 2008 May 23),
surfactants (e.g., Pluronic F127, Pluronic F68, Tween 20 (Wei et
al. International Journal of Pharmaceutics. 2007, 338(1-2):
125-132)), and the methods described in U.S. Pat. Nos. 5,696,090,
5,688,651, and 6,420,122.
[0114] In addition, proteins, and in particular antibodies, are
stabilized in formulations using combinations of different classes
of excipients, e.g., (1) disaccarides (e.g. Saccharose, Trehalose)
or polyols (e.g. Sorbitol, Mannitol) act as stabilizers by
preferential exclusion and are also able to act as cryoprotectants
during lyophilization, (2) surfactants (e.g. Polysorbat 80,
Polysorbat 20) act by minimizing interactions of proteins on
interfaces like liquid/ice, liquid/material-surface and/or
liquid/air interfaces and (3) buffers (e.g. phosphate-, citrate-,
histidine) help to control and maintain formulation pH.
Accordingly, such disaccharides polyols, surfactants and buffers
may be used in addition to the methods of the present invention to
further stabilize immunoglobulins and prevent their
aggregation.
[0115] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, it will be
preferably to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, monostearate salts and gelatin.
[0116] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredient from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying (lyophilization) that yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0117] The amount of active ingredient which can be combined with a
carrier material to produce a single dosage form will vary
depending upon the subject being treated, and the particular mode
of administration. The amount of active ingredient which can be
combined with a carrier material to produce a single dosage form
will generally be that amount of the composition which produces a
therapeutic effect. Generally, out of one hundred percent, this
amount will range from about 0.01 percent to about ninety-nine
percent of active ingredient, preferably from about 0.1 percent to
about 70 percent, most preferably from about 1 percent to about 30
percent of active ingredient in combination with a pharmaceutically
acceptable carrier.
[0118] It is accordingly an object of the present invention to
provide modified immunoglobulin formulations that can be made upon
of modified immunoglobulins as discussed in paragraphs [0059],
[0060], or [0089] and any and all combinations of their embodiments
at a concentration of at least 10 mg/ml, at least 20 mg/ml, at
least 30 mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least 75
mg/ml, at least 100 mg/ml, at least 125 mg/ml, or at least 150
mg/ml. In certain embodiments, the immunoglobulin is at a
concentration of greater than the concentration at which the
unmutated immunoglobulin aggregates with itself in a concentrated,
liquid solution under the same conditions. In certain embodiments
that may be combined with the preceding embodiments, at least
eighty percent, at least eighty-five percent, at least ninety
percent, at least ninety-five percent, at least ninety-six percent,
at least ninety-seven percent, at least ninety-eight percent, or at
least ninety-nine percent of the modified immunoglobulin is
non-aggregated monomer. In certain embodiment that may be combined
with any of the preceding embodiments, the formulation includes a
pharmaceutically acceptable excipient. In certain embodiments that
may be combined with any of the preceding embodiments, the
immunoglobulin formulation shows at least five percent, at least
ten percent, at least fifteen percent, at least twenty percent, at
least twenty-five percent, at least thirty percent, at least
thirty-five percent, at least forty percent, or at least fifty
percent less aggregate after twenty four hours of accelerated
aggregation as compared to the unmutated immunoglobulin under the
same conditions. In certain embodiments that may be combined with
the preceding embodiments, the aggregation is measured by SEC-HPLC.
In certain embodiments that may be combined with any of the
preceding embodiments, the immunoglobulin formulation is
substantially free of any additive that reduces aggregation of
immunoglobulins. In certain embodiments that may be combined with
any of the preceding embodiments, the immunoglobulin formulation is
substantially free of free histidine, saccharides and polyols.
[0119] It is accordingly an object of the present invention to
provide uses of the modified immunoglobulins as discussed in
paragraphs [0059], [0060], or [0089] and any and all combinations
of their embodiments as a non-aggregating pharmaceutical active
ingredient.
[0120] It is accordingly so object of the present invention to
provide pharmaceutical compositions that include a modified
immunoglobulin as discussed in paragraphs [0059], [0060], or [0089]
and any and all combinations of their embodiments and a
pharmaceutically acceptable excipient. In certain embodiments, the
immunoglobulin is at a concentration of at least 10 mg/ml, at least
20 mg/ml, at least 30 mg/ml, at least 40 mg/ml, at least 50 mg/ml,
at least 75 mg/ml, at least 100 mg/ml, at least 125 mg/ml, or at
least 150 mg/ml. In certain embodiments, the immunoglobulin is at a
concentration of greater than the concentration at which the
unmutated immunoglobulin aggregates with itself in a concentrated,
liquid solution under the same conditions. In certain embodiments
that may be combined with the preceding embodiments, at least
eighty percent, at least eighty five percent, at least ninety
percent, at least ninety-five percent, at least ninety-six percent,
at least ninety-seven percent, at least ninety-eight percent, or at
least ninety-nine percent of the modified immunoglobulin is
non-aggregated monomer. In certain embodiments that may be combined
with any of the preceding embodiments, the immunoglobulin
formulation shows at least five percent, at least ten percent, at
least fifteen percent, at least twenty percent at least twenty-five
percent, at least thirty percent, at least thirty-five percent, at
least forty percent, or at least fifty percent less aggregate after
twenty four hours of accelerated aggregation as compared to the
unmutated immunoglobulin under the same conditions. In certain
embodiments that may be combined with the preceding embodiments,
the aggregation is measured by SEC-HPLC. In certain embodiments
that may be combined with any of the preceding embodiments, the
immunoglobulin formulation is substantially free of any additive
that reduces aggregation of immunoglobulins. In certain embodiments
that may be combined with any of the preceding embodiments, the
immunoglobulin formulation is substantially free of free histidine,
saccharides and polyols.
[0121] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0122] For administration of the immunoglobulin, the dosage ranges
from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg,
of the host body weight. For example dosages can be 0.3 mg/kg body
weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body
weight or 10 mg/kg body weight or within the range of 1-10 mg/kg.
An exemplary treatment regime emails administration once per week,
once every two weeks, once every three weeks, once even four weeks,
once a month, once every 3 months or once every three to 6 months.
Preferred dosage regimens for an immunoglobulin of the invention
include 1 mg/kg body weight or 3 mg/kg body weight via intravenous
administration, with the antibody being given using one of the
following dosing schedules: (i) every four weeks for six dosages,
then every three months; (ii) every three weeks; (iii) 3 mg/kg body
weight once followed by 1 mg/kg body weight every three weeks.
[0123] Alternatively an immunoglobulin of the invention can be
administered as a sustained release formulation, in which case less
frequent administration is required. Dosage and frequency vary
depending on the half-life of the administered substance in the
patient. In general, human antibodies show the longest half life,
followed by humanized antibodies, chimeric antibodies, and nonhuman
antibodies. The dosage and frequency of administration can vary
depending on whether din treatment is prophylactic or therapeutic.
In prophylactic applications, a relatively low dosage is
administered at relatively infrequent intervals over a long period
of time. Some patients continue to receive treatment for the rest
of their lives. In therapeutic applications, a relatively high
dosage at relatively short intervals is sometimes returned until
progression of the disease is reduced or terminated, and preferably
until the patient shows partial or compete amelioration of symptoms
of disease. Thereafter, the patient can be administered a
prophylactic regime.
[0124] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, or the ester, salt or amide thereof, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0125] A "therapeutically effective dosage" of immunoglobulin of
the invention preferably results in a decrease in severity of
disease symptoms, an increase in frequency and duration of disease
symptom-free periods, or a prevention of impairment or disability
due to the disease affliction. For example, for the treatment of
tumors, a "therapeutically effective dosage" preferably inhibits
cell growth or tumor growth by least about 20%, more preferably by
at least about 40%, even more preferably by at least about 60%, but
still more preferably by at least about 80% relative to untreated
subjects. The ability of a compound to inhibit tumor growth can be
evaluated in an animal model system predictive of efficacy in human
tumors. Alternatively, this property of a composition can be
evaluated by examining the ability of the compound to inhibit, such
inhibition in vitro by assays known to the skilled practitioner. A
therapeutically effective amount of a therapeutic compound can
decrease tumor size, or otherwise ameliorate symptoms in a subject.
One of ordinary skill in the art would be able to determine such
amounts based on such factors as the subject's size, the severity
of the subject'symptoms, and the particular composition or route of
administration selected.
[0126] A composition of the present invention can be administered
via one or more routes of administration using one or more of a
variety of methods known in the art. As will be appreciated by the
skilled artisan, the route and/or mode of administration will vary
depending upon the desired results. Preferred routes of
administration for binding moieties of the invention include
intravenous, intramuscular, intradermal, intraperitoneal,
subcutaneous, spinal or other parenteral routes of administration,
for example by injection or infusion. The phase "parenteral
administration" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, interdermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal, epidural and intrasternal injection and
infusion.
[0127] Alternatively, an immunoglobulin of the invention can be
administered via a nonparenteral route, such as a topical,
epidermal or mucosal route of administration, for example,
intranasally, orally, vaginally, rectally, sublingually or
topically.
[0128] The active compounds can be prepared with carriers that will
protect the compound against rapid release, such as a controlled
release formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in
the art. See, e.g., Sustained and Controlled Release Drug Delivery
Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,
1978.
[0129] Therapeutic compositions can be administered with medical
devices known in the art. For example, in a preferred embodiment, a
therapeutic composition of the invention can be administered with a
needleless hypodermic injection device, such as the devices
disclosed in U.S. Pat. No. 5,399,163; 5,383,851; 5,312,335;
5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of
well-known implants and modules useful in the present invention
include U.S. Pat. No. 4,487,603, which discloses an implantable
micro-infusion pump for dispensing medication at a controlled rate;
U.S. Pat. No. 4,486,194, which discloses a therapeutic device for
administering medicants through the skin; U.S. Pat. No. 4,447,233,
which discloses a medication infusion pump for delivering a
medication at a precise infusion rate; U.S. Pat. No. 4,447,224,
which discloses a variable flow implantable infusion apparatus for
continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses
an osmotic drug delivery system having multi-chamber compartments;
and U.S. Pat. No. 4,475,196, which discloses an osmotic drug
delivery system.
[0130] It is accordingly an object of the present invention to
provide methods for reducing the aggregation propensity of an
immunoglobulin in a highly concentrated pharmaceutical formulation
comprising providing an immunoglobulin that is prone to
aggregation; substituting a residue in a conserved domain of the
immunoglobulin that (i) has a Spatial-Aggregation-Propensity of at
least 0.15, or (ii) has an Spatial-Aggregation-Propensity (5 .ANG.
radius sphere) of greater than 0.0 and is within 5 .ANG. of a
residue having a Spatial-Aggregation-Propensity of at least 0.15,
with an amino acid residue that lowers the
Spatial-Aggregation-Propensity (5 < radius sphere), and
generating a highly concentrated, liquid formulation of the
modified immunoglobulin wherein the modified immunoglobulin
concentration is at least 20 mg/ml, at least 30 mg/ml, at least 40
mg/ml, at least 50 mg/ml, at least 75 mg/ml, at least 100 mg/ml, at
least 125 mg/ml, or at least 150 mg/ml, wherein the aggregation
propensity that is reduced is aggregation between immunoglobulin
molecules in a concentrated, liquid solution.
[0131] It is accordingly an object of the present invention to
provide uses of the modified immunoglobulins as discussed in
paragraphs [0059], [0060], or [0089] and any and all combinations
of their embodiments in the preparation of a medicament comprising
a highly concentrated liquid formulation wherein the modified
immunoglobulin concentration is at least 20 mg/ml, at least 30
mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least 75 mg/ml, at
least 100 mg/ml, at least 125 mg/ml, or at least 150 mg/ml. In
certain embodiments, the use of the medicament is for the treatment
of autoimmune diseases, immunological diseases, infectious
diseases, inflammatory diseases, neurological diseases, and
oncological and neoplastic diseases including cancer. In certain
embodiments, the use of the medicament is for the treatment of
congestive heart failure (CHF), vasculitis, rosacea, acne, eczema,
myocarditis and other conditions of the myocardium, systemic lupus
erythematosus, diabetes, spondylopathies, synovial fibroblasts, and
bone marrow stroma; bone loss; Paget's disease, osteoclastoma;
breast cancer; disuse osteopenia; malnutrition, periodontal
disease, Gaucher's disease, Langerhans' cell histiocytosis, spinal
cord injury, acute septic arthritis, osteomalacia, Cushing's
syndrome, monoostotic fibrous dysplasia, polyostotic fibrous
dysplasia, periodontal reconstruction, and bone fractures;
sarcoidosis; osteolytic bone cancers, breast cancer, lung cancer,
kidney cancer and rectal cancer; bone metastasis, bone pain
management, and humoral malignant hypercalcemia, ankylosing
spondylitisa and other spondyloarthropathies; transplantation
rejection, viral infections, hematologic neoplasias and
neoplastic-like conditions for example, Hodgkin's lymphoma;
non-Hodgkin's lymphomas (Burkitt's lymphoma, small lymphocytic
lymphoma/chronic lymphocytic leukemia, mycosis fungoides, mantle
cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma,
marginal zone lymphoma, hairy cell leukemia and lymphoplamacytic
leukemia), tumors of lymphocyte precursor cells, including B-cell
acute lymphoblastic leukemia/lymphoma, and T-cell acute
lymphoblastic leukemia/lymphoma, thymoma, tumors of the mature T
and NK cells, including peripheral T-cell leukemias, adult T-cell
leukemia/T-cell lymphomas and large granular lymphocytic leukemia,
Langerhans cell histocytosis, myeloid neoplasias such as acute
myelogenous leukemias, including AML with maturation, AML without
differentiation, acute promyelocytic leukemia, acute myelomonocytic
leukemia, and acute monocytic leukemias, myelodysplastic syndromes,
and chronic myeloproliferative disorders, including chronic
myelogenous leukemia, tumors of the central nervous system, e.g.,
brain tumors (glioma, neuroblastoma, astrocytoma, medulloblastoma,
ependymoma, and retinoblastoma), solid tumor (nasopharyngeal
cancer, basal cell carcinoma, pancreatic cancer, cancer of the bile
duct, Kaposi's sarcoma, testicular cancer, uterine, vaginal or
cervical cancer, ovarian cancer, primary liver cancer or
endometrial cancer, and tumors of the vascular system (angiosarcoma
and hemangiopericytoma), osteoporosis, hepatitis, HIV, AIDS,
spondylarthritis, rheumatoid arthritis, inflammatory bowel diseases
(IBD), sepsis and septic shock, Crohn's disease, psoriasis,
schleraderma, graft versus host disease (GVHD), allogenic graft
rejection, hematologic malignancies, such as multiple myeloma (MM),
myelodyspastic syndrome (MDS) and acute myelogenous leukemia (AML),
inflammation associated with tumors, peripheral nerve injury or
demyelinating diseases. In certain embodiments, the use of the
medicament is for the treatment or plaque psoriasis, ulcerative
colitis, non-Hodgkin's lymphoma, breast cancer, colorectal cancer,
juvenile idiopathic arthritis, macular degeneration, respiratory
syncytial virus, Crohn's disease, rheumatoid arthritis, psoriatic
arthritis, ankylosing spondylitis, osteoporosis, treatment-induced
bone loss, bone metastases, multiple myeloma, Alzheimer's disease,
glaucoma, and multiple sclerosis. In certain embodiments that may
be combined with any of the preceding embodiments, the use of the
medicament further comprises a pharmaceutically acceptable
excipient. In certain embodiments that may be combined with any of
the preceding embodiments, the immunoglobulin in the medicament
shows at least five percent, at least ten percent, at least fifteen
percent, at least twenty percent, at least twenty-five percent, at
least thirty percent, at least thirty-five percent, at least forty
percent, or at least fifty percent less aggregate after twenty four
hours of accelerated aggregation as compared to the unmutated
immunoglobulin under the same conditions. In certain embodiments,
the aggregation is measured by SEC-HPLC. In certain embodiments
that may be combined with any of the preceding embodiments, the
medicament is substantially free of any additive that reduces
aggregation of immunoglobulins. In certain embodiments that may be
combined with any of the preceding embodiments, the medicament is
substantially free of free histidine, saccharides and polyols.
EXAMPLES
[0132] Molecular simulation techniques for predicting aggregation
prone regions and studying the mechanism of aggregation have mostly
employed comparatively simple simulation methods (Ma and Nussinov.
Curr. Opin. Chem. Biol. 2006, 10, 445-452; Cellmer, et al., TRENDS
in Biotechnology 2007, 25(6), 254) unlike the detailed atomistic
models which may be employed in the present invention. The least
detailed of the simulation models employed was the lattice model,
which was used in numerous studies of protein aggregation (Harrison
et al. J. MoL. Biol. 1999, 286, 593-606; Dima and Thirumalai.
Protein Sci. 2002, 11, 1036-1049; Leonhard et al. Protein Sci.
2004, 13, 358-369; Patro and Przybycien. Biophys. J. 1994,
1274-1289; Patro and Przybycien. Biophys. J. 1996, 70, 2888-2902;
Broglia et al. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12930-12933;
Istrail et al. Comput. Biol. 1999, 6, 143-162; Giugliarelli et al.
Chem. Phys. 2000, 113, 5072-5077; Bratko et al. J. Chem. Phys.
2001, 114, 561-569; Bratko and Blanch J. Chem. Phys. 2003, 118,
5185-5194; Combe and Frenkel Chem. Phys. 2003, 118, 9015-9022; Toma
and Toma. Biomacromolecules 2000, 1, 232-238; Gupta et al. Protein
Sci. 1998, 7, 2642-2652; and Nguyen and Hall Biotechnol. Bioeng.
2002, 80, 823-834). Here each residue is represented as a bead
occupying a single site on a three dimensional lattice. Because of
its simplicity, the lattice model is less computationally demanding
and has been used to simulate large systems for long time scales.
Although these lattice models provide insight into the basic
physics underlying protein aggregation, they do not accurately
represent the secondary and tertiary structure, and cannot
adequately account for different atomistic level interactions such
as hydrogen bonding.
[0133] A more detailed model compared to the lattice model is the
intermediate resolution model in which a few atoms are usually
combined into a single bead, and pseudo-bonds are sometimes
introduced to maintain the backbone bond angles and isomerization
states (Smith and Hall, Mol. Biol. 2001, 312, 187-202; Smith and
Hall. Proteins: Struct., Funct., Genet. 2001, 44, 344-360; Smith
and Hall. Proteins: Struct., Funct., Genet. 2001, 44, 376-391;
Nguyen, et al., Protein Sci. 2004, 13, 2909-2924; Nguyen and Hall,
Proc. Natl. Acad. Sci. U.S.A., 2004, 101(46), 16180-16185; Nguyen
and Hall. J. Am. Chem. Soc., 2006, 128, 1890-1901; Jang, et al.,
Biophys. J. 2004, 86, 31-49; Jang, et al., Protein Sci. 2004, 13,
40-53). This model was successfully used to simulate the formation
of fibrils from systems containing between 12 and 96 polyalanine
peptides (16-residue each) starting from a random state (Nguyen and
Hall, Proc. Natl. Acad. Sci. U.S.A., 2004, 101(46), 16180-16185;
Nguyen and Hall, J. Am. Chem. Soc., 2006, 128, 1890-1901).
Dokholyan and co-workers applied such a model to study the
formation of fibrillar .beta.-sheet structures by eight model Src
SH3 domain proteins (Ding, et al., Mol. Biol. 2002, 324, 851-857)
or by 28 model A .beta. (1-40) peptides (Peng. et al., Phys. Rev.
E: Stat. Ph. Interdiscip. Top. 2004, 69, 41908-41914.).
[0134] Unlike simpler models, atomistic models include all the
atomistic details such as hydrogen bonding and are thus more
accurate than the lattice or the intermediate resolution models.
Such atomistic models have been used either with an explicitly
solvent, or with an implicit solvent where the solvent is treated
as a continuum. The explicit model is more accurate than the
implicit model, but is also more computationally demanding. Such an
atomistic model with implicit solvent was used to study the early
stages of aggregation of the heptapeptide GNNQQNY (SEQ ID NO: 17),
which is a part of the yeast protein Sup35 (Gsponer, et al., Proc.
Natl. Acad. Sci. U.S.A. 2003, 100, 5154-5159.). A similar model was
used for the aggregation of Ab16-22 amyloid peptide (KLVFFAE (SEQ
ID NO: 18)) into antiparallel .beta. Sheets (Klimov and Thirumalai,
Structure 2003, 11, 295-307). Dokholynha and coworkers (Khare, et
al., Proteins, 2005, 61, 617-632.) used an explicit atomistic model
to investigate the ordered aggregation propensity along the
sequence of the enzyme Cu, Zn superoxide dismutase (SOD1). They
have decomposed the SOD1 sequence into overlapping heptapeptides
and performed a large number of explicit water molecular dynamics
simulations (each of 0.5 ns) of monomeric, dimeric and tetrameric
segments. With this they identified the amyloidogenic regions in
the SOD1 sequence to be: the two termini, the .beta.-strands 4 and
7, and the two crossover loops.
[0135] A similar molecular dynamics simulation protocol was
developed to obtain structural information on ordered
.beta.-aggregation of amyloidogenic polypeptides (Cecchini et al.,
J Mol Biol. 2006, 357, 1306-1321.). The procedure is based on the
decomposition of a polypeptide chain into overlapping segments and
equilibrium molecular dynamics (MD) simulations of a small number
of copies of each segment. The .beta.-aggregation propensity along
the sequence of the Alzheimer's A.beta. (1-42) peptide was found to
be highly heterogeneous with a maximum at the segment
V.sub.12HHQKLVFFAE.sub.22 (SEQ ID NO: 19) and minima at four
turn-like dipeptides. Using this technique, the predicted change in
the aggregation propensity of a double-point mutant of the
N-terminal domain of the yeast prion Ure2p was verified in vitro
using the thioflavin T binding assay. Such a procedure to decompose
the polypeptide chain into overlapping segments would be extremely
challenging for systems such as antibodies because of their huge
size. Even at atomistic simulation of a single full antibody in
explicit solvent is very computationally demanding because of the
huge size of an antibody. Therefore, there does not appear to be
full antibody atomistic simulation in the literature.
[0136] However, there have been atomistic simulations of small
parts of the antibody, mostly for the Fab fragment (Noon, et al.,
PNAS, 2002, 99, 6466; Sinha and Smith-Gill, Cell Biochemistry and
Biophysics. 2005, 43, 253). In the work disclosed herein, atomistic
simulations of a full antibody molecular with an explicitly solvent
were performed. Based on these simulations, the aggregation prone
regions on the antibody were identified using the
`Spatial-Aggregation-Propensity` parameter described herein. These
aggregation prone regions were then mutated to design antibodies
with enhanced stability. The Examples described herein refer to
particular, non-limiting embodiments of the invention.
Example 1: Molecular Dynamics Simulation Methodology
[0137] Molecular dynamics simulations were performed for a full
antibody using an all atom model. The initial structure for
simulation for the full antibody was obtained from the X-ray
structures of individual Fab and Fc fragments. The X-ray structure
of a proof-of-concept (POC) Fab fragment selected for modeling onto
the X-ray structure of Fc obtained front the IgG1 antibody 1HZH
(Saphire et al., Science, 2001, 293, 1155). 1HZH was chosen since
the X-ray structure is known for the full antibody and since the Fc
structure is the same for all of the IgG1 class of antibodies. The
structure of a full POC antibody was then obtained by aligning the
Fab and Fc fragments using the 1HZH structure as a model template.
In order to align the fragments at the correct distance and
orientation, the RMSD (Root Mean Square Deviation) was minimized
between the common CYS residues of the fragments and the full
antibody template (1HZH). The CYS residues were chosen because each
antibody sub-domain (C.sub.H1, C.sub.H3 etc.) contains disulphide
bond, and thus CYS residues are broadly distributed across the
whole antibody structure. The resulting full antibody structure was
then used to perform explicitly atom simulations for 30 ns. A G0
glycosylation was used for the simulations since this is the most
common glycosylation pattern observed in antibodies.
[0138] The CHARMM simulation package (Brooks et al. J. Comput.
Chem., 1983, 4, 187) was used for set-up and analysis, and the NAMD
package (Phillips et al. Journal of Computational Chemistry, 2005,
26, 1781) for performing simulations. the CHARMM fully atomistic
force field (MacKerell et al. J. Phys Chem. B. 1998, 102, 3586) was
used for the protein and TIP3P (Jorgensen et al. J. Chem. Phys.,
1983, 79, 926) solvent model for water. The simulations were
performed at 298K and 1 atm in the NPT ensemble. The parameters for
the sugar groups involved in glycosylation of the Fc fragment were
derived to be consistent with the CHARMM force field, following
from the CSFF force field (Kuttel et al. J. Comput. Chem., 2002,
23, 1236). The protonation states of Histidine residues at pH-7
were chosen based on the spatial proximity of electro-negative
groups. The full antibody was solvated in an orthorhombic box since
this minimizes the number of water molecules required and thus
minimizes the computational time. Periodic boundary conditions were
used in all 3 directions. A water solvation shell of 8 .ANG. was
used in each direction of the orthorhombic box. The resulting total
system size was 202130 atoms. Sufficient ions were added to
neutralize the total charge of the system. The charge neutrality is
required by the Ewald summation technique employed to calculate the
contribution of electrostatic interactions in the system.
[0139] After the antibody was solvated, the energy was initially
minimized with SD (Steepest Descents) by fixing the protein to
allow the water to relax around the protein. Then the restraints
were removed and the structure was further minimized with SD and
ABNR (Adopted Basis Hewton-Raphson). The system was then slowly
heated to room temperature with 5.degree. C. increment every 0.5 ps
using a lfs time step. The system was then equilibrated for Ins
before computing properties of interest from the simulation. The
configurations were saved every 0.1 ps during the simulation for
further statistical analysis.
Example 2: Calculation of the Spatial Aggregation Propensity
(SAP)
[0140] In order to overcome the shortcomings of SAA, a new
parameter was defined called `Spatial-Aggregation-Propensity` as
described above.
[0141] In this example, the `Spatial-Aggregation-Propensity` was
calculated for spherical regions with radius R centered on every
atom in the antibody described in Example 1. The value of
Spatial-Aggregation-Propensity was thus evaluated with a 30 ns
simulation average for the Fc-fragment of the antibody for two
different radii of patches (R=5 .ANG., 10 .ANG.) (One of skill in
the art will appreciate various time steps for simulation may be
chosen according to the computation resources available and the
desired resolution of the result). In both cases it was noticed
that the majority of values were negative, indicating that most
exposed regions are hydrophilic. This was as expected since most of
the exposed protein surface is usually hydrophilic. It was also
observed that there are a few regions with positive peaks for
Spatial-Aggregation-Propensity indicating high exposed
hydrophobicity. Going from lower radii of patches (5 .ANG.) to the
higher radii (10 .ANG.) eliminates some peaks, whereas some other
peaks are enhanced. Some peaks were eliminated because in these
regions a small hydrophobic patch (with less than 5 .ANG. radius)
is surrounded by hydrophilic patches; thus, averaging over 10 .ANG.
leads to an effective decrease in hydrophobicity for the region.
Whereas in some other regions the Spatial-Aggregation-Propensity at
R=10 .ANG. is enhanced because of hydrophobic patches surrounding a
similar hydrophobic patch.
[0142] Above, the Spatial-Aggregation-Propensity was calculated as
an average during the 30 ns simulation run. The results calculated
using the simulation were then compared to the
Spatial-Aggregation-Propensity of just the X-ray structure, without
molecular simulation. The Spatial-Aggregation-Propensity (X-ray)
thus was calculated for R=5 .ANG. and for R=10 .ANG.. The
Spatial-Aggregation-Propensity (X-ray) was similar to that of the
simulation-averaged value, having peaks in the same locations but
with differences in the magnitude of the peaks. The differences
were higher with the larger radius of patch, R=10 .ANG.. This is
probably because the differences are additive when looking at
larges patch sizes. These differences arise due to the changing
surface exposure of the residues in the dynamic simulation run.
Nevertheless, this comparison shows that a good initial estimate of
Spatial-Aggregation-Propensity, especially for low radius of patch
R, can be obtained from the X-ray structure itself.
[0143] The Spatial-Aggregation-Propensity values from the
simulation for R=5 .ANG. and 10 .ANG. were mapped onto the antibody
structure. In both cases, the antibody surface was colored
according to the values of the Spatial-Aggregation-Propensity. At
both the radii used in the calculation of Spatial
Aggregation-Propensity (5 .ANG. and 10 .ANG.) is was observed that
the surface is predominantly hydrophilic. This is again as expected
since most of the protein surface is usually hydrophilic. However a
few hydrophobic regions were noticeable. The contrast between the
hydrophobic and hydrophilic regions is more prominent at the higher
radii of patch used in the calculation of SAP. R=10 .ANG.. Certain
of the identified hydrophobic regions have excellent correlation
with regions of the antibody known to interact with other proteins.
One patch around residues 234 and 235 in the hinge region is where
the Fc-receptor interacts. A second patch around residue 253
corresponds to the region in the Fc fragment where protein A and
protein G interact. A significant hydrophobic patch was observed at
the end of the Fab fragment corresponds to the region where the
antibody binds to antigens. Plots of Spatial-Aggregation-Propensity
for R=5 .ANG. and 10 .ANG. respectively, wherein the same
correlation of peaks with interacting regions may be observed. The
protein interaction sites were obtained from X-ray structure of
protein complexes, PDB entries 1T89, 1FC2, and 1FCC (Radaev, J.
Biol. Chem. 2001, 276(19) 16469; Deisenhofer et al. Hoppe-Seyler's
Z Physiol Chem. 1978. 359, 975-985; Deisenhofer, J. Biochemistry.
1981, 20, 2361-2370; Sauer-Eriksson et al. Structure. 1995, 3,
265). The hydrophobic interactions correlate very well with the
Spatial-Aggregation-Propensity positive peaks and the hydrophilic
interactions correlate well with the Spatial-Aggregation-Propensity
negative peaks. Therefore, the Spatial-Aggregation-Propensity
parameter can be used to predict the binding sites of proteins as
well. In the few exceptions in which residues with low
Spatial-Aggregation-Propensity (i.e. close to zero, either positive
or negative) also interact, it was observed that the interactions
are actually with the atoms of the main backbone chain itself,
instead of with the side chains.
[0144] Apart from the hydrophobic patches already shown to interact
with other proteins discussed above, additional hydrophobic patches
on the antibody surface (regions 4 to 6) were identified. Region-5
at the bottom of Fc was significantly hydrophobic, but it is
somewhat buried inside, with hydrophilic region on its borders.
Similarly regions 4 and 6 are hydrophobic and solvent exposed, but
they are facing into the interior of the antibody. Regions 4 and 6
could still be potentially involved in interactions with other
proteins if they are exposed due to significant conformational
changes or unfolding of the antibody. All of the hydrophobic
patches (regions 1 to 6) could also be observed at the smaller
patch radius (R=5 .ANG.), although with less contrast compared to
the higher patch radius (R=10 .ANG.).
[0145] The Spatial-Aggregation-Propensity (X-ray) values which are
based on just the X-ray structure were also mapped onto the
antibody surface, to compare them with the simulation averaged
values. Comparing Spatial-Aggregation-Propensity calculated either
through simulation or using just the X-ray structure showed that
the hydrophobic regions identified were quite similar. There are of
course some differences, such as the intensity of the Protein A
interaction patches. Nevertheless, this comparison demonstrates
that Spatial-Aggregation-Propensity (X-ray) based on just the X-ray
structure can be used to obtain a good description of the
distribution of hydrophobic patches on the surface. This is
important since the atomistic simulation of a full antibody is
computationally demanding. For proteins lacking an X-ray structural
model, the same Spatial-Aggregation-Propensity parameter can be
applied to the structure generated through homology modeling or
ab-initio structure prediction. The homology structure was observed
to be very similar to the X-ray structure, and its
Spatial-Aggregation-Propensity values are also similar to the X-ray
structure.
[0146] Thus Spatial-Aggregation-Propensity identifies the
hydrophobic patches on the surface of the antibody. These patches
could be natively exposed or exposed due to dynamic fluctuations or
partial unfolding of the antibody. Some of these hydrophobic
patches also correlate well with regions interacting with other
proteins. In order to test if these hydrophobic patches predicted
by Spatial-Aggregation-Propensity are involved in aggregation as
well, mutations in these specific regions were performed to change
the hydrophobic residues into hydrophilic residues. The resulting
antibodies showed less aggregation behavior and improved stability.
Apart from identifying aggregation prone residues, it was also
observed that the SAP method correctly identifies the regions of
the antibody prone to binding with other proteins. Therefore, the
method could be broadly applied to all proteins to identify the
aggregation prone regions or binding regions with other
proteins.
Example 3: Selection of Antibody Sites for Stability
Engineering
[0147] The residues identified as having high
Spatial-Aggregation-Propensities (and therefore being at the center
of aggregation prone motifs identified by the inventors) are set
forth to Table 1. Given that these are at the center of the motifs,
these residues as well as those residues within 5 .ANG. (or 10
.ANG. if a 10 .ANG. window is used in the calculation) may be
modified to less hydrophobic residues to reduce aggregation and/or
increase stability. Residues were identified for a human IgG1
antibody (with kappa light chain), and the corresponding residues
in the different IgG classes are shown in Table 1.
TABLE-US-00002 TABLE 1 The aggregation prone motifs in various
domains for all IgG class of antibodies. The differences between
IgGs are underlined. Aggregation prone motifs Residue Residue names
Domain Number IgG1 IgG2 IgG3 IgG4 C.sub.H1 175 L L L L Hinge 234 L
P L F 235 L V L L C.sub.H2 253 I I I I 282 V V V V 291 P P P P 296
Y F Y F 309 L V L L 328 L L L L 329 P P P P 330 A A A S C.sub.H3
398 L L L L 443 L L L L Kappa Lambda C.sub.L 110 V K 154 L P 201 L
--
[0148] Table 1 shows that the motifs are mostly conserved between
different IgGs with a few differences. However, most of the
differences are from a hydrophobic amino acid to another
hydrophobic amino acid. Therefore, the hydrophobicity of the motif
remains intact even with these differences and therefore the other
classes with hydrophobic residues at the same position are also
aggregation prone motifs. There are a few exceptions to this
(A330S, V110K and the deletion of L201) which are not aggregation
prone motifs. Apart from these exceptions, the motifs identified
here have similar exposed hydrophobicity and higher SAP values for
all the IgG class of antibodies.
[0149] Table 2 shows hydrophobic residues organized into
aggregation prone motifs.
TABLE-US-00003 TABLE 2 Fourteen motifs of the constant region of
the IgG1 molecule. Residues with (SAP5 .ANG. > 0) within 5 .ANG.
of (SAP.sub.5.ANG. > 0.15) (Aggregation prone motif Domain
SAP.sub.5.ANG. > 0.15 number) C.sub.H1 175 LEU 174 VAL 1
(119-224) 175 LEU 1 181 TYR 1 Hinge 227 PRO 226 CYS 2 (221-237) 228
PRO 227 PRO 2 229 CYS 228 PRO 2 230 PRO 229 CYS 2 231 ALA 230 PRO 2
232 PRO 231 ALA 2 234 LEU 232 PRO 2 235 LEU 234 LEU 3 235 LEU 3
C.sub.H2 253 ILE 252 MET 4 (238-345) 282 VAL 253 ILE 4 291 PRO 282
VAL 5 296 TYR 291 PRO 6 309 LEU 296 TYR 7 329 PRO 308 VAL 8 330 ALA
309 LEU 8 328 LEU 9 329 PRO 9 330 ALA 9 331 PRO 9 C.sub.H3 395 PRO
395 PRO 10 (346-447) 398 LEU 396 PRO 10 443 LEU 397 VAL 10 398 LEU
10 404 PHE 10 443 LEU 11 C.sub.L 110 VAL 110 VAL 12 (110-214) 154
LEU 111 ALA 12 201 LEU 153 ALA 13 154 LEU 13 201 LEU 14
Example 4--Selection of Antibody Sites for Stability
Engineering
[0150] In order to demonstrate that the aggregation prone motifs
identified by their SAP are involved in aggregation and/or
instability, mutations were generated in the identified regions to
change the hydrophobic residues into hydrophilic residues. Here the
selected residues were all changed to lysine. In general, the amino
acids which form the general motifs can be replaced by amino acids
which are more hydrophilic in the Black and Mould scale, in
particular by Thr, Ser, Lys, Gln, Asn, His, Glu, Asp, and Arg. The
selected regions were as follows: A1 (L235K), A2 (I253K), A3
(L309K), A4 (L309K, L235K), and A5 (L234K, L235K). The resulting
mutant antibodies showed less aggregation behaviour and improved
stability as described in Example 6.
Example 5--Expression and Purification of Antibody Variants
[0151] The selected residues discussed in Example 4 above were
mutated, and the resulting antibody variants were expressed and
purified. Vectors that carry the light chain or the heavy chain
genes of the human IgG1 Antibody A were obtained by subcloning the
genes from proprietary vectors (Novartis) into a gWIZ vector
(Genlantis), optimized for high expression from transient
transfection. Antibody variants were generated following the
Stratagene protocol for site-directed mutagenesis. All constructs
were confirmed by DNA sequencing. Plasmid DNA at the mg scale was
purified from bacterial cultures with DNA Maxi Prep columns
(Invitrogen). The manufacturer's protocols were followed for growth
and transient transfection of FreeStyle HEK 293 cells (Invitrogen).
In brief, for transection of 1 L culture, 1 mg total DNA (0.5 mg of
the HC and LC vectors each) was incubated with 20 ml OptiPro
solution for 15 minutes; at the same time 2 mg of the transfection
reagent polyethyleneimine (PEI (Polysciences) at 1 mg/ml) was
incubated with 20 ml OptiPro solution for 15 minutes. The PEI
solution was then added to the DNA solution, mixed by swirling, and
incubated for another 15 minutes. Aliquots of 20 ml PEI/DNA mix
were added to 500 ml cell cultures at 1.0.times.10.sup.6 cells/ml.
Transfected cells were incubated in a CO.sub.2 incubator at
37.degree. C. for 7-9 days.
[0152] Antibody wild type and variants were purified from the
tissue culture supernatant on a Protein A column (GE Healthcare)
with the use of an FPLC AKTA Purifier system (GE Healthcare).
Antibodies were eluted from the column with 50 mM citrate buffer pH
3.5, and equilibrated to pH 6.6-7.0 with 1M Tris-HCl pH 9.0. This
eluate was passed over a Q Sepharose column (GE Healthcare) to
remove negatively charged impurities. At pH 7.0 and below, the
antibodies are positively charged and remain in the flow-through,
while negatively charged impurities bind to the positively charged
matrix of the Q Sepharose column. The solution with purified
antibody was concentrated with 30K MWCO filters (Millipore, VWR)
and buffer exchanged with 20 mM His buffer pH 6.5 to a final
concentration of 150 mg/ml.
[0153] As a quality control, aliquots of the purified and
concentrated samples were analyzed by SDS-PAGE under non-reducing
and reducing conditions. Protein aliquots of 4 .mu.g per sample
were incubated in denaturing buffer without or with DTT and
resolved on a 10% polyacrylamide gel (Pierce). Variant A1 was
compared to wild type also by circular dichroism wherein the
spectra were essentially identical showing that the two proteins
had essentially the same degree of secondary structure.
Example 6--Biophysical Characterization of Antibody Variants
[0154] The stability of the antibody variants was analyzed with
three different analytical methods.
[0155] Turbidity Assay
[0156] A turbidity assay was carried out at 65.degree. C. for up to
4 hours. Antibody A and variants were at a concentration of 150
mg/ml in 20 mM His, pH 6.5, and diluted 15-fold in 15 mM potassium
phosphate buffer, pH 6.5 to 10 mg/ml for turbidity assessment. In
addition to the qualitative observations, turbidity was quantified
after further diluting the samples to 1 mg/ml and recording the
absorbance values at 320 mm as shown in Table 3.
TABLE-US-00004 TABLE 3 Turbidity assay comparison of Antibody A
wild type and variants. Variant 0 HRS 1 HR 2 HRS 4 HRS WT 0.02 0.06
0.27* *** A1 0.01 0.03 0.04 0.19* A2 0.01 0.04 0.07 ** A3 0.01 0.03
0.05 ** A4 0.01 0.04 0.04 0.13* A5 0.01 0.04 0.09 0.14* Samples at
150 mg/ml were incubated at 65.degree. C. for up to 4 hours.
Asterisks indicates the state of the solution at 10 mg/ml, or if
the sample had gelified, as follows: *denotes a liquid, turbid upon
dilution; ** denotes gel, clear upon dilution; and *** denotes a
gel, turbid upon dilution. Values without asterisks were liquid,
clear upon dilution. The numbers represent absorbance at 320 nm
after further dilution of the samples to 1 mg/ml.
[0157] Size Exclusion--High Performance Liquid Chromatography
(SEC-HPLC)
[0158] As a second and preferred assay, SEC-HPLC was used to
determine monomer loss over time in accelerated aggregation
experiments. Antibody A wild type and variants were incubated in a
thermal cycler (BioRad) at 150 mg/ml at 58.degree. C. for up to 24
hours. For each time point, sample aliquots of 2 .mu.l were diluted
15-fold in 15 mM potassium phosphate buffer, pH 6.5 to a final
concentration of 10 mg/ml. Monomers were resolved from
non-monomeric species by SEC-HPLC on a TSKgel Super SW3000 column
(TOSOH Bioscience), maintained at 22.degree. C., with a mobile
phase of 150 mM potassium phosphate, pH 6.5, at a flow rate of 0.2
ml/min. Percent monomer was calculated as the area of the monomeric
peak divided by the total area of all peaks detected at 280 mm.
[0159] Differential Scanning Microcalorimetry
[0160] Third, the thermodynamic stability of antibody A wild type
and variants was compared by Differential Scanning
Micro-calorimetry (DSC, Microcal). MAbs have characteristic DSC
thermograms with three melting transitions, if not overlapping:
Fab, C.sub.H2, and C.sub.H3 (Ionescu et al., J Pharm Sci, v. 97,
1414, 2008; Mimura et al., J Biol Chem, v. 276, 45539, 2001). At
the experimental conditions used here, antibody A Fab has a melting
transition at 77.degree. C. The C.sub.H2 and C.sub.H3 melting
temperatures are at 73.degree. C. and 83.degree. C. respectively.
Thus, in antibody A, C.sub.H2 is the antibody domain with the
lowest melting temperature.
[0161] Antibody A wild type and variants A1-A15 were analyzed at a
concentration of 2 mg/ml in 20 mM His pH 6.5 buffer and a heating
rate of 1.0 degree per minute. The sample data was analyzed by
subtraction of the reference data, normalization to the protein
concentration and DSC cell volume, and interpolation of a cubic
baseline. The peaks were deconvoluted by non-2-state fit using
Microcal Origin 5.0 software. A comparison of the thermograms
showed an increase of the C.sub.H2 melting transition in the
variants compared to wild type by 1 to 3 degrees, with the
difference most pronounced for the double Variants A4 and A5 (table
4 below).
TABLE-US-00005 TABLE 4 T.sub.m1 is the melting transition for the
C.sub.H2 domain. T.sub.m2 is the melting transition for the Fab
domain. T.sub.m3 is the melting transition for the C.sub.H3 domain.
T.sub.m1 T.sub.m2 T.sub.m3 MAb (.degree. C.) (.degree. C.)
(.degree. C.) WT 73.5 77.3 83.6 A1 76.0 77.8 83.5 A2 75.0 77.5 83.4
A3 75.5 77.6 83.4 A4 76.2 77.7 83.1 A5 76.3 77.9 83.3
Example 7--Summary
[0162] The results from the turbidity, SEC-HPLC and DSC experiments
of antibody A wild type and variants are summarized in Table 5.
TABLE-US-00006 TABLE 5 Summary of results for antibody A wild type
and variants. Relative Stability Based on Variant Mutation Domain
Turbidity HPLC DSC WT na na ++ ++ ++ A1 L235K C.sub.H2 ++++ +++ +++
lower hinge A2 I253K C.sub.H2-C.sub.H3 +++ +++ +++ junction A3
L309K C.sub.H2 +++ +++ +++ A4 L235K L309K C.sub.H2 ++++ ++++ ++++
A5 L234K L235K C.sub.H2 ++++ +++ ++++ Legend: + least stable; ++
stable as WT; +++ more stable; ++++ most stable.
[0163] Each of the three single mutants A1, A2, and A3 showed
improved stability by each of the three analytical methods. In the
turbidity assay, dilution of antibody A wt sample stressed at
65.degree. C. for 2 hrs resulted in clouding of the solution, while
the solutions for all variants remained clear. SEC-HPLC results of
samples stressed at 58.degree. C. for 24 hrs indicated monomer
increase from 91% for wild type to 93-95% for the variants. As the
initial monomer population was 99%, the non-monomeric species in
the variants decreased up to a half compared to wild type. DSC
analysis showed an increase of the melting transition for C.sub.H2
(the domain with the lowest melting transition in antibody A) from
73.degree. C. for wild type to 75-76.degree. C. for the
variants.
[0164] Substituting additional high-SAP residues in Variant A1
further improved stability, as evidenced by the Turbidity results
and the DSC thermograms for Variants A4 and A5. The SEC-HPLC
results showed an improvement over Variant A1 only for Variant A4
(96% monomer after 24-hrs stress) and not for Variant A5 (93%
monomer after 24-hrs stress, as Variant A1).
[0165] By way of confirmation, similar mutations were generated in
a second antibody with the addition of mutation of residues in the
CDR regions of the antibody. All but one of the mutations tested
improved the stability and/or reduced aggregation. Mutations at one
residue in a CDR region did not perform as predicted, however, this
may have been because this variant did not express well which may
have been due to a defected in folding therefore had a greater
degree of aggregation than the wild-type even before the
accelerated aggregation analysis. Thus, all mutations tested in
framework and conserved regions produced the predicted results
thereby proving that the SAP algorithm is robust with the sole
possible exception of the mutations which are unable to fold
properly. However, given that the mutations are all to surface
exposed residues and involve substitution with more hydrophilic
residues, such folding issues are expected to be rare.
Example 8--Stability Analysis of Additional Antibody Variants
[0166] Additional variants were designed and analyzed for improved
stability in the first and second antibody. Sites for mutation were
based on SAP predictions. The mutations in each variant are listed
in Table 6.
TABLE-US-00007 TABLE 6 Position of mutated sites in additional
variants. Starting Variant Antibody Rationale Mutation Domain A6
Ab-1 SAP L235S Hinge A7 Ab-1 SAP V282K C.sub.H2 A8 Ab-1 SAP V282K
Hinge Var A4 L235K L309K C.sub.H2 B6 Ab-2 SAP L235E C.sub.H2
[0167] SEC-HPLC was used to determine monomer loss over time in
accelerated aggregation experiments. For the first antibody, wild
type and variant antibodies were incubated in a thermal cycler
(BioRad) at 150 mg/ml at 58.degree. C. for up to 24 hours. For the
second antibody, wild type and variant antibodies were incubated in
a thermal cycler (BioRad) at 60 mg/ml at 52.degree. C. for up to 36
hours. For each time point, sample aliquots of 2 .mu.l were diluted
15-fold in 1.5 mM potassium phosphate buffer, pH 6.5 to a final
concentration of 1.0 mg/ml. Monomers were resolved from
non-monomeric species by SEC-HPLC on a TSKgel Super SW3000 column
(TOSOH Bioscience), maintained at 22.degree. C., with a mobile
phase of 150 mM potassium phosphate, pH 6.5, at a flow rate of 0.2
ml/min. Percent monomer was calculated as the area of the monomeric
peak divided by the total area of all peaks detected at 280 nm.
[0168] Variant A6 showed an increase from 95.5% for wild-type to
96% monomer at 12 hours and an increase from 91% for wild-type to
92% monomer at 24 hours. Variant A7 showed an increase 96.5% for
wild-type to 97.5% monomer at 12 hours and an increase from 91% for
wild-type to 94% monomer at 24 hours. Variant A8 showed an increase
from 96.5% for wild-type to 98.5% monomer at 12 hours and an
increase from 91% for wild-type to 97% monomer at 24 hours. Variant
B6 showed no significant difference in percentage monomer at 12
hours, showed an increase from 97.5% for wild-type to 98% monomer
at 24 hours, and an increase from 96% for wild-type to 97% monomer
at 36 hours.
Example 9--The Role of Protein-Carbohydrate Interactions in SAP and
Stability
[0169] In this example we determined the effect of glycosylation of
an antibody on SAP values. The SAP was determined at R=5 .ANG. for
the full antibody both with and without glycosylation. The SAP for
antibody with glycosylation was determined from 30 ns molecular
dynamics simulation of the full antibody with G0-glycosylation. The
SAP for antibody without glycosylation was determined with the same
simulation, but where we removed the glycosylation during SAP
analysis. The high SAP regions are the most aggregation prone
regions. It was observed that the removal of glycosylation
significantly increased the SAP in the regions covered by
glycosylation, especially for residues F241 and F243. Therefore,
removal or displacement of glycosylation led to a significant
increase in the aggregation prone regions. These regions could
either directly cause aggregation or lower the free energy barrier
for unfolding, making the non-glycosylated form less-stable
compared to the glycosylated form.
[0170] To further explore the role of protein-carbohydrate
interactions experimentally, two antibody mutants, F241S F243S
(Variant S) and F241Y F243Y (Variant FY), were generated. Variant S
has phenylalanine residues known to interact with the carbohydrate
moiety replaced with polar serine residues that have smaller and
non-aromatic side chains (Deisenhofer, Biochem, 1981, 20, 2361-70;
Krapp et al., J Mol Biol, 2003, 979-89). In Variant FY, the same
phenylalanine residues are replaced by Tyr residues, which have
been suggested to have higher sugar interface propensity (Taroni et
al., Protein Eng, 2000, 13, 89-98). At the same time, other
hydrophobic residues, for example Val264, remain unmodified in this
region. Both wild type and Variant FY had very little if any
sialylation of their carbohydrates, while nearly 50% of the
molecules of Variant FS had at least one sialic acid residue.
[0171] The stability of wild type and Variants FS and FY was
compared in accelerated aggregation experiments and by different
scanning micro-calorimetry (DSC). Samples at 150 mg/ml were induced
to aggregate at 58.degree. C. for up to 36 hrs, and monomer levels
were resolved and quantified by size-exclusion high performance
liquid chromatography (SEC-HPLC). Wild-type monomer levels
gradually decreased from 100 to 96, 91, and 87% for 0, 12, 24, and
36 hrs time points. In comparison, variant FY had reduced monomer
levels by 1-3% in the earlier time points but remained at 88% at 36
hrs, within statistical error of wild type. Variant FS was
significantly less stable at this temperature showing a monomer
decrease from 99% to 39% at 12 hrs, and to 20% at 24 hrs. The
36-hrs samples were not run on SEC-HPLC because of the presence of
abundant visible aggregates.
[0172] DSC results also differentiated between the variants and
wild type. The melting temperature (Tm) of the C.sub.H2 domain
decreased from 73.degree. C. for wild type to 59.degree. C. for
Variant FS. Minor differences in Variant FS, not greater than
1.degree. C., were observed for the melting transitions of Fab and
C.sub.H3 as well. Although the C.sub.H2 melting transition shoulder
of Variant FY overlapped that of wild type, the software fitting
indicated lowering of the C.sub.H2 Tm to 71.degree. C., while the
other two Tm's remain unchanged.
[0173] A number of additional experiments were carried out to
compare Variant FS to wild type and to better understand the
observed decrease in stability. Variant FS retained the same
.beta.-sheet-rich structure as wild type. The variant had different
mobility patterns compared to wild type on reducing as sell as
native gel electrophoresis. Protease treatment experiments were
also carried out to compare protein surface exposure in Variant FS
and wild type. Digestion of the antibodies with Glu-C was more
efficient (more small fragments) for Variant FS than wild type;
that efficiency was largely equalized in the variant and wild type
deglycosylated counterparts, although some differences persisted.
Furthermore, Variant FS retained full FcRn and partial Fc.gamma.RIa
binding function but lost binding to Fc.gamma.RII and Fc.gamma.RIII
receptors.
Sequence CWU 1
1
191101PRTHomo sapiens 1Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Ser Leu Ser 50 55 60 Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile65 70 75 80 Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val 85 90
95 Glu Pro Lys Ser Cys 100 2101PRTHomo sapiens 2Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser
Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Ser Leu Ser
50 55 60 Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr
Tyr Thr65 70 75 80 Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys Thr Val 85 90 95 Glu Arg Lys Cys Cys 100 3101PRTHomo
sapiens 3Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys
Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Ser Leu Ser 50 55 60 Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr65 70 75 80 Cys Asn Val Asp
His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val 85 90 95 Glu Ser
Lys Tyr Gly 100 4101PRTHomo sapiens 4Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Ser Leu Ser 50 55
60 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Thr65 70 75 80 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg Val 85 90 95 Glu Pro Lys Thr Pro 100 517PRTHomo sapiens
5Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1
5 10 15 Gly613PRTHomo sapiens 6Val Glu Cys Pro Pro Cys Pro Ala Pro
Pro Val Ala Gly 1 5 10 714PRTHomo sapiens 7Pro Pro Cys Pro Ser Cys
Pro Ala Pro Glu Phe Leu Gly Gly 1 5 10 826PRTHomo sapiens 8Leu Gly
Thr Thr His Thr Cys Pro Arg Cys Pro Glu Pro Lys Cys Pro 1 5 10 15
Arg Cys Pro Ala Pro Glu Leu Leu Gly Gly 20 25 9106PRTHomo sapiens
9Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 1
5 10 15 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu 20 25 30 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His 35 40 45 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Tyr Val Val 50 55 60 Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr65 70 75 80 Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr 85 90 95 Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu 100 105 10108PRTHomo sapiens 10Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 1 5 10 15 Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 20 25 30
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 35
40 45 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
Arg 50 55 60 Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu
Asn Gly Lys65 70 75 80 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ala Pro Ile Glu 85 90 95 Lys Thr Ile Ser Lys Thr Lys Gly Gln
Pro Arg Glu 100 105 11106PRTHomo sapiens 11Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 1 5 10 15 Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 20 25 30 Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 35 40 45
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Tyr Val Val 50
55 60 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr65 70 75 80 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr 85 90 95 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 100
105 12106PRTHomo sapiens 12Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile 1 5 10 15 Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu 20 25 30 Asp Pro Glu Val Gln Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His 35 40 45 Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Tyr Val Val 50 55 60 Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr65 70 75 80
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 85
90 95 Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 100 105 13100PRTHomo
sapiens 13Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn 1 5 10 15 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile 20 25 30 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr 35 40 45 Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Lys Lys Leu Thr 50 55 60 Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val65 70 75 80 Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 85 90 95 Ser Pro
Gly Lys 100 14100PRTHomo sapiens 14Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn 1 5 10 15 Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 20 25 30 Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 35 40 45 Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Lys Lys Leu Thr 50 55 60
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val65
70 75 80 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu 85 90 95 Ser Pro Gly Lys 100 15100PRTHomo sapiens 15Pro Gln
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn 1 5 10 15
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 20
25 30 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr 35 40 45 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Lys
Arg Leu Thr 50 55 60 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser Cys Ser Val65 70 75 80 Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu 85 90 95 Ser Leu Gly Lys 100
16100PRTHomo sapiens 16Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn 1 5 10 15 Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile 20 25 30 Ala Val Glu Trp Glu Ser Ser
Gly Gln Pro Glu Asn Asn Tyr Lys Thr 35 40 45 Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Lys Lys Leu Thr 50 55 60 Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Ile Phe Ser Cys Ser Val65 70 75 80 Met
His Glu Ala Leu His Asn His Phe Thr Gln Lys Ser Leu Ser Leu 85 90
95 Ser Pro Gly Lys 100 177PRTSaccharomyces cerevisiae 17Gly Asn Asn
Gln Gln Asn Tyr1 5 187PRTHomo sapiens 18Lys Leu Val Phe Phe Ala
Glu1 5 1911PRTHomo sapiens 19Val His His Gln Lys Leu Val Phe Phe
Ala Glu 1 5 10
* * * * *