U.S. patent application number 15/385788 was filed with the patent office on 2017-07-13 for formulations with reduced oxidation.
The applicant listed for this patent is Genentech, Inc.. Invention is credited to Sreedhara ALAVATTAM, Parbir GREWAL, Mary MALLANEY.
Application Number | 20170196837 15/385788 |
Document ID | / |
Family ID | 51729183 |
Filed Date | 2017-07-13 |
United States Patent
Application |
20170196837 |
Kind Code |
A1 |
ALAVATTAM; Sreedhara ; et
al. |
July 13, 2017 |
FORMULATIONS WITH REDUCED OXIDATION
Abstract
The invention provides formulations comprising a protein in
combination with a compound that prevents oxidation of the protein.
The invention also provides methods for making such formulations
and methods of using such formulations. The invention further
provides methods of screening for compounds that prevent oxidation
of a protein in a protein composition and methods of preventing
oxidation of a protein in a formulation.
Inventors: |
ALAVATTAM; Sreedhara; (South
San Francisco, CA) ; MALLANEY; Mary; (South San
Francisco, CA) ; GREWAL; Parbir; (South San
Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
51729183 |
Appl. No.: |
15/385788 |
Filed: |
December 20, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14724568 |
May 28, 2015 |
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15385788 |
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14207911 |
Mar 13, 2014 |
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14724568 |
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61780852 |
Mar 13, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/55 20130101;
C07K 2317/52 20130101; A61K 39/39533 20130101; A61K 31/405
20130101; C07K 16/241 20130101; C07K 16/2878 20130101; A61K
2039/505 20130101; G01N 33/6854 20130101; C12Q 1/28 20130101; A61K
39/39591 20130101 |
International
Class: |
A61K 31/405 20060101
A61K031/405; C12Q 1/28 20060101 C12Q001/28; A61K 39/395 20060101
A61K039/395 |
Claims
1. A liquid formulation comprising a protein and a compound which
prevents oxidation of the protein in the liquid formulation,
wherein the compound is of formula: ##STR00021## wherein R.sup.2 is
selected from hydrogen, hydroxyl, --COOH, and --CH.sub.2COOH;
R.sup.3 is selected from hydrogen, hydroxyl, --COOH,
--CH.sub.2COOH, and --CH.sub.2CHR.sup.3a(NH.sub.2); wherein
R.sup.3a is COOH or hydrogen; R.sup.4, R.sup.5, R.sup.6, and
R.sup.7 are independently selected from hydrogen and hydroxyl;
provided that one of R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6,
and R.sup.7 is hydroxyl; or a pharmaceutically acceptable salt
thereof.
2. The formulation of claim 1, wherein the compound is a compound
of formula: ##STR00022## wherein R.sup.2 and R.sup.3 are
independently selected from hydrogen, hydroxyl, --COOH, and
--CH.sub.2COOH; and R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are
independently selected from hydrogen and hydroxyl; provided that
one of R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 is
hydroxyl; or a pharmaceutically acceptable salt thereof.
3. The formulation of claim 1, wherein the compound is a compound
of formula: ##STR00023## wherein R.sup.3a is COOH or hydrogen;
R.sup.2, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are independently
selected from hydrogen and hydroxyl, provided that one of R.sup.2,
R.sup.4, R.sup.5, R.sup.6, and R.sup.7 is hydroxyl; or a
pharmaceutically acceptable salt thereof.
4. The formulation of claim 1, wherein R.sup.4, R.sup.5 or R.sup.7
is hydroxyl.
5. The formulation of claim 1, wherein the compound is selected
from the group consisting of 5-hydroxy-tryptophan, 5-hydroxy
indole, 7-hydroxy indole, and serotonin.
6. The formulation of claim 1 which is a pharmaceutical formulation
suitable for administration to a subject.
7. The formulation of claim 1 which is aqueous.
8. The formulation of claim 1, wherein the compound in the
formulation is from about 0.3 mM to about 1 mM.
9. The formulation of claim 1, wherein the compound prevents
oxidation of tryptophan, cysteine, histidine, tyrosine, and/or
methionine in the protein.
10. The formulation of claim 1, wherein the compound prevents
oxidation of the protein by a reactive oxygen species.
11. The formulation of claim 10, wherein the reactive oxygen
species is selected from the group consisting of singlet oxygen,
hydrogen peroxide, a hydroxyl radical, and an alkyl peroxide.
12. The formulation of claim 1, wherein the protein is susceptible
to oxidation.
13. The formulation of claim 1, wherein tryptophan in the protein
is susceptible to oxidation.
14. The formulation of claim 1, wherein the protein is an
antibody.
15. The formulation of claim 14, wherein the antibody is a
polyclonal antibody, a monoclonal antibody, a humanized antibody, a
human antibody, a chimeric antibody, or antibody fragment.
16. The formulation of claim 1, wherein the protein concentration
in the formulation is about 1 mg/mL to about 250 mg/mL.
17. The formulation of claim 1, which further comprises one or more
excipients selected from the group consisting of a stabilizer, a
buffer, a surfactant, and a tonicity agent.
18. The formulation of claim 6, wherein the formulation has a pH of
about 4.5 to about 7.0.
19. A method of making a protein formulation comprising adding an
amount of a compound that prevents oxidation of a protein to the
protein formulation, wherein the compound is of formula:
##STR00024## wherein R.sup.2 is selected from hydrogen, hydroxyl,
--COOH, and --CH.sub.2COOH; R.sup.3 is selected from hydrogen,
hydroxyl, --COOH, --CH.sub.2COOH, and
--CH.sub.2CHR.sup.3a(NH.sub.2); wherein R.sup.3a is COOH or
hydrogen; R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are independently
selected from hydrogen and hydroxyl; provided that one of R.sup.2,
R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 is hydroxyl; or a
pharmaceutically acceptable salt thereof.
20. A method of preventing oxidation of a protein in a protein
formulation comprising adding an amount of a compound that prevents
oxidation of the protein to the formulation, wherein the compound
is of formula: ##STR00025## wherein R.sup.2 is selected from
hydrogen, hydroxyl, --COOH, and --CH.sub.2COOH; R.sup.3 is selected
from hydrogen, hydroxyl, --COOH, --CH.sub.2COOH, and
--CH.sub.2CHR.sup.3a(NH.sub.2); wherein R.sup.3a is COOH or
hydrogen; R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are independently
selected from hydrogen and hydroxyl; provided that one of R.sup.2,
R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 is hydroxyl; or a
pharmaceutically acceptable salt thereof.
21. The method of claim 19, wherein the compound is a compound of
formula: ##STR00026## wherein R.sup.2 and R.sup.3 are independently
selected from hydrogen, hydroxyl, --COOH, and --CH.sub.2COOH; and
R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are independently selected
from hydrogen and hydroxyl; provided that one of R.sup.2, R.sup.3,
R.sup.4, R.sup.5, R.sup.6, and R.sup.7 is hydroxyl; or a
pharmaceutically acceptable salt thereof.
22. The method of claim 19, wherein the compound is a compound of
formula: ##STR00027## wherein R.sup.3a is COOH or hydrogen;
R.sup.2, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are independently
selected from hydrogen and hydroxyl, provided that one of R.sup.2,
R.sup.4, R.sup.5, R.sup.6, and R.sup.7 is hydroxyl; or a
pharmaceutically acceptable salt thereof.
23. The method of claim 19, wherein R.sup.4, R.sup.5 or R.sup.7 is
hydroxyl.
24. The method of claim 19, wherein the compound is selected from
the group consisting of 5-hydroxy-tryptophan, 5-hydroxy indole,
7-hydroxy indole, and serotonin.
25. The method of claim 19 which is a pharmaceutical formulation
suitable for administration to a subject.
26. The method of claim 19 which is aqueous.
27. The method of claim 19, wherein the compound in the formulation
is from about 0.3 mM to about 1 mM.
28. The method of claim 19, wherein the compound prevents oxidation
of tryptophan, cysteine, histidine, tyrosine, and/or methionine in
the protein.
29. The method of claim 19, wherein the compound prevents oxidation
of the protein by a reactive oxygen species.
30. The method of claim 29, wherein the reactive oxygen species is
selected from the group consisting of singlet oxygen, hydrogen
peroxide, a hydroxyl radical, and an alkyl peroxide.
31. The method of claim 19, wherein the protein is susceptible to
oxidation.
32. The method of claim 19, wherein tryptophan in the protein is
susceptible to oxidation.
33. The method of claim 19, wherein the protein is an antibody.
34. The method of claim 33, wherein the antibody is a polyclonal
antibody, a monoclonal antibody, a humanized antibody, a human
antibody, a chimeric antibody, or antibody fragment.
35. The method of claim 19, wherein the protein concentration in
the formulation is about 1 mg/mL to about 250 mg/mL.
36. The method of claim 19, wherein the formulation further
comprises one or more excipients selected from the group consisting
of a stabilizer, a buffer, a surfactant, and a tonicity agent.
37. The method of claim 26, wherein the formulation has a pH of
about 4.5 to about 7.0.
38. A method of screening a compound that prevents oxidation of a
protein in a protein composition, comprising selecting a compound
that has lower oxidation potential and less photosensitivity as
compared to L-tryptophan, and testing the effect of the selected
compound on preventing oxidation of the protein.
39. The method of claim 38, wherein the photosensitivity is
measured based on the amount of H.sub.2O.sub.2 produced by the
compound upon light exposure.
40. The method of claim 39, wherein the compound that produces less
than about 10% of the amount of H.sub.2O.sub.2 produced by
L-tryptophan is selected.
41. The method of claim 38, wherein the oxidation potential is
measured by cyclic voltammetry.
42. The method of claim 38, wherein the selected compound is tested
for the effect on preventing oxidation of the protein by reactive
oxygen species generated by 2,2'-azobis(2-amidinopropane)
dihydrochloride (AAPH), light, and/or a Fenton reagent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 14/724,568, filed May 28, 2015, which
is a continuation of U.S. patent application Ser. No. 14/207,911,
filed Mar. 13, 2014, which claims the benefit of U.S. Provisional
Application No. 61/780,852, filed Mar. 13, 2013, the contents of
each of which are hereby incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0002] This invention relates to formulations comprising a protein
and further comprising a compound that prevents oxidation of said
protein, methods for producing and using the formulations as well
as methods of screening for compounds that prevent protein
oxidation in protein compositions.
BACKGROUND OF THE INVENTION
[0003] Oxidative degradation of amino acid residues is a commonly
observed phenomenon in protein pharmaceuticals. A number of amino
acid residues are susceptible to oxidation, particularly methionine
(Met), cysteine (Cys), histidine (His), tryptophan (Trp), and
tyrosine (Tyr) (Li et al., Biotechnology and Bioengineering
48:490-500 (1995)). Oxidation is typically observed when the
protein is exposed to hydrogen peroxide, light, metal ions or a
combination of these during various processing steps (Li et al.,
Biotechnology and Bioengineering 48:490-500 (1995)). In particular,
proteins exposed to light (Wei, et al., Analytical Chemistry
79(7):2797-2805 (2007)), AAPH or Fenton reagents (Ji et al., J
Pharm Sci 98(12):4485-500 (2009)) have shown increased levels of
oxidation on tryptophan residues, whereas those exposed to hydrogen
peroxide have typically shown only methionine oxidation (Ji et al.,
J Pharm Sci 98(12):4485-500 (2009)). Light exposure can result in
protein oxidation through the formation of reactive oxygen species
(ROS) including singlet oxygen, hydrogen peroxide and superoxide
(Li et al., Biotechnology and Bioengineering 48:490-500 (1995);
Wei, et al., Analytical Chemistry 79(7):2797-2805 (2007); Ji et
al., J Pharm Sci 98(12):4485-500 (2009); Frokjaer et al., Nat Rev
Drug Discov 4(4):298-306 (2005)), whereas protein oxidation
typically occurs via hydroxyl radicals in the Fenton mediated
reaction (Prousek et al., Pure and Applied Chemistry
79(12):2325-2338 (2007)) and via alkoxyl peroxides in the AAPH
mediated reaction (Werber et al., J Pharm Sci 100(8):3307-15
(2011)). Oxidation of tryptophan leads to a myriad of oxidation
products, including hydroxytryptophan, kynurenine, and
N-formylkynurenine, and has the potential to impact safety and
efficacy (Li et al., Biotechnology and Bioengineering 48:490-500
(1995); Ji et al., J Pharm Sci 98(12):4485-500 (2009); Frokjaer et
al., Nat Rev Drug Discov 4(4):298-306 (2005)). Oxidation of a
particular tryptophan residue in the heavy chain complementarity
determining region (CDR) of a monoclonal antibody that correlated
to loss of biological function has been reported (Wei, et al.,
Analytical Chemistry 79(7):2797-2805 (2007)). Trp oxidation
mediated by a histidine coordinated metal ion has recently been
reported for a Fab molecule (Lam et al., Pharm Res 28(10):2543-55
(2011)). Autoxidation of polysorbate 20 in the Fab formulation,
leading to the generation of various peroxides, has also been
invoked in the same report. Autoxidation-induced generation of
these peroxides can also lead to methionine oxidation in the
protein during long-term storage of the drug product since Met
residues in proteins have been suggested to act as internal
antioxidants (Levine et al., Proceedings of the National Academy of
Sciences of the United States of America 93(26):15036-15040 (1996))
and are easily oxidized by peroxides. Oxidation of amino acid
residues has the potential to impact the biological activity of the
protein. This may be especially true for monoclonal antibodies
(mAbs). Methionine oxidation at Met254 and Met430 in an IgG1 mAb
potentially impacts serum half-life in transgenic mice (Wang et
al., Molecular Immunology 48(6-7):860-866 (2011)) and also impacts
binding of human IgG1 to FcRn and Fc-gamma receptors
(Bertolotti-Ciarlet et al., Molecular Immunology 46(8-9) 1878-82
(2009)).
[0004] The stability of proteins, especially in liquid state, needs
to be evaluated during drug product manufacturing and storage. The
development of pharmaceutical formulations sometimes includes
addition of antioxidants to prevent oxidation of the active
ingredient. Addition of L-methionine to formulations has resulted
in reduction of methionine residue oxidation in proteins and
peptides (Ji et al., J Pharm Sci 98(12):4485-500 (2009); Lam et
al., Journal of Pharmaceutical Sciences 86(11):1250-1255 (1997))
Likewise, addition of L-tryptophan has been shown to reduce
oxidation of tryptophan residues (Ji et al., J Pharm Sci
98(12):4485-500 (2009); Lam et al., Pharm Res 28(10):2543-55
(2011)). L-Trp, however, possesses strong absorbance in the UV
region (260-290 nm) making it a primary target during
photo-oxidation (Creed, D., Photochemistry and Photobiology
39(4):537-562 (1984)). Trp has been hypothesized as an endogenous
photosensitizer enhancing the oxygen dependent photo-oxidation of
tyrosine (Babu et al., Indian J Biochem Biophys 29(3):296-8 (1992))
and other amino acids (Bent et al., Journal of the American
Chemical Society 97(10):2612-2619 (1975)). It has been demonstrated
that L-Trp can generate hydrogen peroxide when exposed to light and
that L-Trp under UV light produces hydrogen peroxide via the
superoxide anion (McCormick et al., Science 191(4226):468-9 (1976);
Wentworth et al., Science 293(5536):1806-11 (2001); McCormick et
al., Journal of the American Chemical Society 100:312-313 (1978)).
Additionally, tryptophan is known to produce singlet oxygen upon
exposure to light (Davies, M. J., Biochem Biophys Res Commun
305(3):761-70 (2003)). Similar to the protein oxidation induced by
autoxidation of polysorbate 20, it is possible that protein
oxidation can occur upon ROS generation by other excipients in the
protein formulation (e.g. L-Trp) under normal handling
conditions.
[0005] It is apparent from recent studies that the addition of
standard excipients, such as L-Trp and polysorbates, to protein
compositions that are meant to stabilize the protein can result in
unexpected and undesired consequences such as ROS-induced oxidation
of the protein. Therefore, there remains a need for the
identification of alternative excipients for use in protein
compositions and the development of such compositions.
BRIEF SUMMARY OF THE INVENTION
[0006] Provided herein are formulations comprising a protein and a
compound that prevents oxidation of the protein in the formulation,
methods of making the formulations, and methods of screening
compounds that prevent oxidation of a protein in a protein
formulation.
[0007] In one aspect, provided herein is a formulation comprising a
protein and a compound which prevents oxidation of the protein in
the formulation, wherein the compound is of formula:
##STR00001## [0008] wherein R.sup.2 is selected from hydrogen,
hydroxyl, --COOH, and --CH.sub.2COOH; [0009] R.sup.3 is selected
from hydrogen, hydroxyl, --COOH, --CH.sub.2COOH, and
--CH.sub.2CHR.sup.3a(NH.sub.2); wherein R.sup.3a is COOH or
hydrogen; [0010] R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are
independently selected from hydrogen and hydroxyl; [0011] provided
that one of R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, and
R.sup.7 is hydroxyl; [0012] or a pharmaceutically acceptable salt
thereof.
[0013] In some embodiments, the compound is a compound of
formula:
##STR00002## [0014] wherein R.sup.2 and R.sup.3 are independently
selected from hydrogen, hydroxyl, --COOH, and --CH.sub.2COOH; and
[0015] R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are independently
selected from hydrogen and hydroxyl; [0016] provided that one of
R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 is
hydroxyl; [0017] or a pharmaceutically acceptable salt thereof.
[0018] In some embodiments, the compound is a compound of
formula:
##STR00003## [0019] wherein R.sup.3a is COOH or hydrogen; [0020]
R.sup.2, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are independently
selected from hydrogen and hydroxyl, provided that one of R.sup.2,
R.sup.4, R.sup.5, R.sup.6, and R.sup.7 is hydroxyl; [0021] or a
pharmaceutically acceptable salt thereof.
[0022] In some embodiments, R.sup.4, R.sup.5 or R.sup.7 in any of
the formula above is hydroxyl. In some embodiments, the compound is
selected from the group consisting of 5-hydroxy-tryptophan,
5-hydroxy indole, 7-hydroxy indole, and serotonin.
[0023] In some embodiments, the formulation is a liquid
formulation. In some embodiments, the formulation is a
pharmaceutical formulation suitable for administration to a
subject. In some embodiments, the formulation is aqueous.
[0024] In some embodiments, the compound in the formulation is from
about 0.3 mM to about 1 mM. In some embodiments, the compound
prevents oxidation of tryptophan, cysteine, histidine, tyrosine,
and/or methionine in the protein. In some embodiments, the compound
prevents oxidation of the protein by a reactive oxygen species. In
some embodiments, the reactive oxygen species is selected from the
group consisting of singlet oxygen, hydrogen peroxide, a hydroxyl
radical, and an alkyl peroxide.
[0025] In some embodiments, the protein is susceptible to
oxidation. In some embodiments, a tryptophan amino acid in the
protein is susceptible to oxidation. In some embodiments, the
protein is an antibody (e.g., a polyclonal antibody, a monoclonal
antibody, a humanized antibody, a human antibody, a chimeric
antibody, or antibody fragment). In some embodiments, the protein
concentration in the formulation is about 1 mg/mL to about 250
mg/mL.
[0026] In some embodiments, the formulation further comprises one
or more excipients selected from the group consisting of a
stabilizer, a buffer, a surfactant, and a tonicity agent. In some
embodiments, the formulation has a pH of about 4.5 to about
7.0.
[0027] In another aspect, provided herein is a method of making a
protein formulation (such as a liquid formulation) comprising
adding an amount of a compound that prevents oxidation of a protein
to the formulation, wherein the compound is of formula:
##STR00004## [0028] wherein R.sup.2 is selected from hydrogen,
hydroxyl, --COOH, and --CH.sub.2COOH; [0029] R.sup.3 is selected
from hydrogen, hydroxyl, --COOH, --CH.sub.2COOH, and
--CH.sub.2CHR.sup.3a(NH.sub.2); wherein R.sup.3a is COOH or
hydrogen; [0030] R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are
independently selected from hydrogen and hydroxyl; [0031] provided
that one of R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, and
R.sup.7 is hydroxyl; [0032] or a pharmaceutically acceptable salt
thereof.
[0033] In another aspect, provided herein is a method of preventing
oxidation of a protein in a protein formulation (such as a liquid
formulation) comprising adding an amount of a compound that
prevents oxidation of a protein to the formulation, wherein the
compound is of formula:
##STR00005## [0034] wherein R.sup.2 is selected from hydrogen,
hydroxyl, --COOH, and --CH.sub.2COOH; [0035] R.sup.3 is selected
from hydrogen, hydroxyl, --COOH, --CH.sub.2COOH, and
--CH.sub.2CHR.sup.3a(NH.sub.2); wherein R.sup.3a is COOH or
hydrogen; [0036] R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are
independently selected from hydrogen and hydroxyl; [0037] provided
that one of R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, and
R.sup.7 is hydroxyl; [0038] or a pharmaceutically acceptable salt
thereof.
[0039] In some embodiments of the methods described herein, the
compound is a compound of formula:
##STR00006## [0040] wherein R.sup.2 and R.sup.3 are independently
selected from hydrogen, hydroxyl, --COOH, and --CH.sub.2COOH; and
[0041] R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are independently
selected from hydrogen and hydroxyl; [0042] provided that one of
R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 is
hydroxyl; [0043] or a pharmaceutically acceptable salt thereof.
[0044] In some embodiments of the methods described herein, the
compound is a compound of formula:
##STR00007## [0045] wherein R.sup.3a is COOH or hydrogen; [0046]
R.sup.2, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are independently
selected from hydrogen and hydroxyl, provided that one of R.sup.2,
R.sup.4, R.sup.5, R.sup.6, and R.sup.7 is hydroxyl; [0047] or a
pharmaceutically acceptable salt thereof.
[0048] In some embodiments, R.sup.4, R.sup.5 or R.sup.7 in any of
the formula above is hydroxyl. In some embodiments, the compound is
selected from the group consisting of 5-hydroxy-tryptophan,
5-hydroxy indole, 7-hydroxy indole, and serotonin.
[0049] In some embodiments, the formulation is a liquid
formulation. In some embodiments, the formulation is a
pharmaceutical formulation suitable for administration to a
subject. In some embodiments, the formulation is aqueous. In some
embodiments, the compound in the formulation is from about 0.3 mM
to about 1 mM.
[0050] In some embodiments, the compound prevents oxidation of
tryptophan, cysteine, histidine, tyrosine, and/or methionine in the
protein. In some embodiments, the compound prevents oxidation of
the protein by a reactive oxygen species. In some embodiments, the
reactive oxygen species is selected from the group consisting of
singlet oxygen, hydrogen peroxide, a hydroxyl radical, and an alkyl
peroxide.
[0051] In some embodiments, the protein is susceptible to
oxidation. In some embodiments, a tryptophan amino acid in the
protein is susceptible to oxidation. In some embodiments, the
protein is an antibody (e.g., a polyclonal antibody, a monoclonal
antibody, a humanized antibody, a human antibody, a chimeric
antibody, or antibody fragment). In some embodiments, the protein
concentration in the formulation is about 1 mg/mL to about 250
mg/mL.
[0052] In some embodiments, the formulation further comprises one
or more excipients selected from the group consisting of a
stabilizer, a buffer, a surfactant, and a tonicity agent. In some
embodiments, the formulation has a pH of about 4.5 to about
7.0.
[0053] In another aspect, provided herein is a method of screening
a compound that prevents oxidation of a protein in a protein
composition, comprising selecting a compound that has lower
oxidation potential and less photosensitivity as compared to
L-tryptophan, and testing the effect of the selected compound on
preventing oxidation of the protein.
[0054] In some embodiments, the photosensitivity is measured based
on the amount of H.sub.2O.sub.2 produced by the compound upon light
exposure. In some embodiments, the compound that produces less than
about 10% of the amount of H.sub.2O.sub.2 produced by L-tryptophan
is selected. In some embodiments, the oxidation potential is
measured by cyclic voltammetry. In some embodiments, the selected
compound is tested for the effect on preventing oxidation of the
protein by reactive oxygen species generated by
2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), light, and/or
a Fenton reagent.
[0055] It is to be understood that one, some, or all of the
properties of the various embodiments described herein may be
combined to form other embodiments of the present invention. These
and other aspects of the invention will become apparent to one of
skill in the art. These and other embodiments of the invention are
further described by the detailed description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0056] FIG. 1 is a series of graphs demonstrating the oxidation of
A) Fab in mAb1, and B) Fc in mAb1 after eight hours of light
exposure at 250 W/m.sup.2. mAb1 was present at 5 mg/mL in 20 mM
histidine acetate, 250 mM trehalose, 0.02% polysorbate 20. All
vials were placed in the lightbox except the mAb1 Ref Mat. Foil
CTRL vials were covered in foil before placement in the lightbox.
Three separate experimental vials were averaged for each sample,
except "10 mM Met, 1 mM Trp" (*) was the average of two
experimental vials, and mAb1 Ref Mat was one experimental vial with
three independent injections on the HPLC. Error bars represent one
standard deviation.
[0057] FIG. 2 is a graph showing dose dependent H.sub.2O.sub.2
production by L-Trp. Diamonds indicate L-Trp alone; Triangles
indicate L-Trp+SOD; Circles and Squares indicate
L-Trp+NaN.sub.3.+-.SOD. All studies were performed in 20 mM L-His
HCl, pH 5.5.
[0058] FIG. 3 is a series of graphs demonstrating A) Hydrogen
peroxide (H.sub.2O.sub.2) production in 50 mg/mL mAb1 formulations
containing 3.2 mM L-Trp when exposed to ambient light conditions
for 1, 3 and 7 days and B) Percent (%) Fab oxidation in mAb1
formulations containing 3.2 mM L-Trp after 10 days of exposure to
ambient light conditions.
[0059] FIG. 4 is a series of graphs showing hydrogen peroxide
generation by tryptophan derivatives and indole derivatives under
light stress for 4 hours at 250 W/m.sup.2. A) Screening of
tryptophan derivatives (1 mM) for hydrogen peroxide (.mu.M)
generation in a 20 mM HisAc pH5.5 formulation. B) Screening of
indole derivatives (1 mM) for hydrogen peroxide (.mu.M) generation
in a 20 mM HisAc pH5.5 formulation.
[0060] FIG. 5 is a graph showing the effect of NaN.sub.3 on
H.sub.2O.sub.2 production by various Trp derivatives upon light
exposure. Data is shown as a ratio with respect to peroxide
generated by L-Trp.
[0061] FIG. 6 is a graph showing the correlation between oxidation
potential and light-induced peroxide formation. The boxed region
shows candidate antioxidant compounds.
[0062] FIG. 7 is a series of graphs showing the oxidation of A) Fab
in mAb1, and B) Fc in mAb1 after AAPH incubation. All samples were
incubated with AAPH except mAb1 Ref Mat and No AAPH. All samples
were incubated at 40.degree. C. except mAb1 Ref Mat. Data shown are
the average of three experimental samples .+-.1SD, except mAb1 Ref
Mat which is the average of six HPLC injections without error
bars.
[0063] FIG. 8 is series of graphs showing the oxidation of A) Fab
in mAb1, and B) Fc in mAb1 after sixteen hours of light exposure at
250 W/m.sup.2. All vials were placed in the lightbox except the
mAb1 Ref Mat. Foil CTRL vials were covered in foil before placement
in the lightbox. Three separate experimental vials were averaged
for each sample, except L-tryptophanamide (*) was the average of
two experimental vials and mAb1 Ref Mat was one vial with three
independent injections on the HPLC. Error bars represent one
standard deviation.
DETAILED DESCRIPTION
I. Definitions
[0064] Before describing the invention in detail, it is to be
understood that this invention is not limited to particular
compositions or biological systems, which can, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to be limiting.
[0065] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of the active ingredient to be effective, and which
contains no additional components which are unacceptably toxic to a
subject to which the formulation would be administered. Such
formulations are sterile.
[0066] A "sterile" formulation is aseptic or free or essentially
free from all living microorganisms and their spores.
[0067] A "stable" formulation is one in which the protein therein
essentially retains its physical stability and/or chemical
stability and/or biological activity upon storage. Preferably, the
formulation essentially retains its physical and chemical
stability, as well as its biological activity upon storage. The
storage period is generally selected based on the intended
shelf-life of the formulation. Various analytical techniques for
measuring protein stability are available in the art and are
reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee
Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones,
A. Adv. Drug Delivery Rev. 10: 29-90 (1993), for example. Stability
can be measured at a selected amount of light exposure and/or
temperature for a selected time period. Stability can be evaluated
qualitatively and/or quantitatively in a variety of different ways,
including evaluation of aggregate formation (for example using size
exclusion chromatography, by measuring turbidity, and/or by visual
inspection); evaluation of ROS formation (for example by using a
light stress assay or a 2,2'-Azobis(2-Amidinopropane)
Dihydrochloride (AAPH) stress assay); oxidation of specific amino
acid residues of the protein (for example a Trp residue and/or a
Met residue of a monoclonal antibody); by assessing charge
heterogeneity using cation exchange chromatography, image capillary
isoelectric focusing (icIEF) or capillary zone electrophoresis;
amino-terminal or carboxy-terminal sequence analysis; mass
spectrometric analysis; SDS-PAGE analysis to compare reduced and
intact antibody; peptide map (for example tryptic or LYS-C)
analysis; evaluating biological activity or target binding function
of the protein (e.g., antigen binding function of an antibody);
etc. Instability may involve any one or more of: aggregation,
deamidation (e.g. Asn deamidation), oxidation (e.g. Met oxidation
and/or Trp oxidation), isomerization (e.g. Asp isomeriation),
clipping/hydrolysis/fragmentation (e.g. hinge region
fragmentation), succinimide formation, unpaired cysteine(s),
N-terminal extension, C-terminal processing, glycosylation
differences, etc.
[0068] A protein "retains its physical stability" in a
pharmaceutical formulation if it shows no signs or very little of
aggregation, precipitation and/or denaturation upon visual
examination of color and/or clarity, or as measured by UV light
scattering or by size exclusion chromatography.
[0069] A protein "retains its chemical stability" in a
pharmaceutical formulation, if the chemical stability at a given
time is such that the protein is considered to still retain its
biological activity as defined below. Chemical stability can be
assessed by detecting and quantifying chemically altered forms of
the protein. Chemical alteration may involve protein oxidation
which can be evaluated using tryptic peptide mapping, reverse-phase
high-performance liquid chromatography (HPLC) and liquid
chromatography-mass spectrometry (LC/MS), for example. Other types
of chemical alteration include charge alteration of the protein
which can be evaluated by ion-exchange chromatography or icIEF, for
example.
[0070] A protein "retains its biological activity" in a
pharmaceutical formulation, if the biological activity of the
protein at a given time is within about 10% (within the errors of
the assay) of the biological activity exhibited at the time the
pharmaceutical formulation was prepared as determined for example
in an antigen binding assay for a monoclonal antibody.
[0071] As used herein, "biological activity" of a protein refers to
the ability of the protein to bind its target, for example the
ability of a monoclonal antibody to bind to an antigen. It can
further include a biological response which can be measured in
vitro or in vivo. Such activity may be antagonistic or
agonistic.
[0072] A protein which is "susceptible to oxidation" is one
comprising one or more residue(s) that has been found to be prone
to oxidation such as, but not limited to, methionine (Met),
cysteine (Cys), histidine (His), tryptophan (Trp), and tyrosine
(Tyr). For example, a tryptophan amino acid in the Fab portion of a
monoclonal antibody or a methionine amino acid in the Fc portion of
a monoclonal antibody may be susceptible to oxidation.
[0073] By "isotonic" is meant that the formulation of interest has
essentially the same osmotic pressure as human blood. Isotonic
formulations will generally have an osmotic pressure from about 250
to 350 mOsm. Isotonicity can be measured using a vapor pressure or
ice-freezing type osmometer, for example.
[0074] As used herein, "buffer" refers to a buffered solution that
resists changes in pH by the action of its acid-base conjugate
components. The buffer of this invention preferably has a pH in the
range from about 4.5 to about 8.0. For example, histidine acetate
is an example of a buffer that will control the pH in this
range.
[0075] A "preservative" is a compound which can be optionally
included in the formulation to essentially reduce bacterial action
therein, thus facilitating the production of a multi-use
formulation, for example. Examples of potential preservatives
include octadecyldimethylbenzyl ammonium chloride, hexamethonium
chloride, benzalkonium chloride (a mixture of
alkylbenzyldimethylammonium chlorides in which the alkyl groups are
long-chain compounds), and benzethonium chloride. Other types of
preservatives include aromatic alcohols such as phenol, butyl and
benzyl alcohol, alkyl parabens such as methyl or propyl paraben,
catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. In
one embodiment, the preservative herein is benzyl alcohol.
[0076] As used herein, a "surfactant" refers to a surface-active
agent, preferably a nonionic surfactant. Examples of surfactants
herein include polysorbate (for example, polysorbate 20 and,
polysorbate 80); poloxamer (e.g. poloxamer 188); Triton; sodium
dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl
glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine;
lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-,
myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-,
linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-betaine (e.g. lauroamidopropyl);
myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or
disodium methyl oleyl-taurate; and the MONAQUAT.TM. series (Mona
Industries, Inc., Paterson, N.J.); polyethyl glycol, polypropyl
glycol, and copolymers of ethylene and propylene glycol (e.g.
Pluronics, PF68 etc.); etc. In one embodiment, the surfactant
herein is polysorbate 20.
[0077] "Pharmaceutically acceptable" excipients or carriers as used
herein include pharmaceutically acceptable carriers, stabilizers,
buffers, acids, bases, sugars, preservatives, surfactants, tonicity
agents, and the like, which are well known in the art (Remington:
The Science and Practice of Pharmacy, 22.sup.nd Ed., Pharmaceutical
Press, 2012). Examples of pharmaceutically acceptable excipients
include buffers such as phosphate, citrate, acetate, and other
organic acids; antioxidants including ascorbic acid, L-tryptophan
and methionine; low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; metal complexes such as
Zn-protein complexes; chelating agents such as EDTA; sugar alcohols
such as mannitol or sorbitol; salt-forming counterions such as
sodium; and/or nonionic surfactants such as polysorbate, poloxamer,
polyethylene glycol (PEG), and PLURONICS.TM.. "Pharmaceutically
acceptable" excipients or carriers are those which can reasonably
be administered to a subject to provide an effective dose of the
active ingredient employed and that are nontoxic to the subject
being exposed thereto at the dosages and concentrations
employed.
[0078] The protein which is formulated is preferably essentially
pure and desirably essentially homogeneous (e.g., free from
contaminating proteins etc.). "Essentially pure" protein means a
composition comprising at least about 90% by weight of the protein
(e.g., monoclonal antibody), based on total weight of the
composition, preferably at least about 95% by weight. "Essentially
homogeneous" protein means a composition comprising at least about
99% by weight of the protein (e.g., monoclonal antibody), based on
total weight of the composition.
[0079] The terms "protein" "polypeptide" and "peptide" are used
interchangeably herein to refer to polymers of amino acids of any
length. The polymer may be linear or branched, it may comprise
modified amino acids, and it may be interrupted by non-amino acids.
The terms also encompass an amino acid polymer that has been
modified naturally or by intervention; for example, disulfide bond
formation, glycosylation, lipidation, acetylation, phosphorylation,
or any other manipulation or modification, such as conjugation with
a labeling component. Also included within the definition are, for
example, proteins containing one or more analogs of an amino acid
(including, for example, unnatural amino acids, etc.), as well as
other modifications known in the art. Examples of proteins
encompassed within the definition herein include mammalian
proteins, such as, e.g., renin; a growth hormone, including human
growth hormone and bovine growth hormone; growth hormone releasing
factor; parathyroid hormone; thyroid stimulating hormone;
lipoproteins; alpha-1-antitrypsin; insulin A-chain; insulin
B-chain; proinsulin; follicle stimulating hormone; calcitonin;
luteinizing hormone; glucagon; leptin; clotting factors such as
factor VIIIC, factor IX, tissue factor, and von Willebrands factor;
anti-clotting factors such as Protein C; atrial natriuretic factor;
lung surfactant; a plasminogen activator, such as urokinase or
human urine or tissue-type plasminogen activator (t-PA); bombesin;
thrombin; hemopoietic growth factor; tumor necrosis factor-alpha
and -beta; a tumor necrosis factor receptor such as death receptor
5 and CD120; TNF-related apoptosis-inducing ligand (TRAIL); B-cell
maturation antigen (BCMA); B-lymphocyte stimulator (BLyS); a
proliferation-inducing ligand (APRIL); enkephalinase; RANTES
(regulated on activation normally T-cell expressed and secreted);
human macrophage inflammatory protein (MIP-1-alpha); a serum
albumin such as human serum albumin; Muellerian-inhibiting
substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse
gonadotropin-associated peptide; a microbial protein, such as
beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associated
antigen (CTLA), such as CTLA-4; inhibin; activin; platelet-derived
endothelial cell growth factor (PD-ECGF); a vascular endothelial
growth factor family protein (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D,
and P1GF); a platelet-derived growth factor (PDGF) family protein
(e.g., PDGF-A, PDGF-B, PDGF-C, PDGF-D, and dimers thereof);
fibroblast growth factor (FGF) family such as aFGF, bFGF, FGF4, and
FGF9; epidermal growth factor (EGF); receptors for hormones or
growth factors such as a VEGF receptor(s) (e.g., VEGFR1, VEGFR2,
and VEGFR3), epidermal growth factor (EGF) receptor(s) (e.g.,
ErbB1, ErbB2, ErbB3, and ErbB4 receptor), platelet-derived growth
factor (PDGF) receptor(s) (e.g., PDGFR-.alpha. and PDGFR-.beta.),
and fibroblast growth factor receptor(s); TIE ligands
(Angiopoietins, ANGPT1, ANGPT2); Angiopoietin receptor such as TIE1
and TIE2; protein A or D; rheumatoid factors; a neurotrophic factor
such as bone-derived neurotrophic factor (BDNF), neurotrophin-3,
-4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor
such as NGF-b; transforming growth factor (TGF) such as TGF-alpha
and TGF-beta, including TGF-.beta.1, TGF-.beta.2, TGF-.beta.3,
TGF-.beta.4, or TGF-.beta.5; insulin-like growth factor-I and -II
(IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like
growth factor binding proteins (IGFBPs); CD proteins such as CD3,
CD4, CD8, CD19 and CD20; erythropoietin; osteoinductive factors;
immunotoxins; a bone morphogenetic protein (BMP); a chemokine such
as CXCL12 and CXCR4; an interferon such as interferon-alpha, -beta,
and -gamma; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF,
and G-CSF; a cytokine such as interleukins (ILs), e.g., IL-1 to
IL-10; midkine; superoxide dismutase; T-cell receptors; surface
membrane proteins; decay accelerating factor; viral antigen such
as, for example, a portion of the AIDS envelope; transport
proteins; homing receptors; addressing; regulatory proteins;
integrins such as CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4 and
VCAM; ephrins; Bv8; Delta-like ligand 4 (DLL4); Del-1; BMP9; BMP10;
Follistatin; Hepatocyte growth factor (HGF)/scatter factor (SF);
Alk1; Robo4; ESM1; Perlecan; EGF-like domain, multiple 7 (EGFL7);
CTGF and members of its family; thrombospondins such as
thrombospondin1 and thrombospondin2; collagens such as collagen IV
and collagen XVIII; neuropilins such as NRP1 and NRP2; Pleiotrophin
(PTN); Progranulin; Proliferin; Notch proteins such as Notch1 and
Notch4; semaphorins such as Sema3A, Sema3C, and Sema3F; a tumor
associated antigen such as CA125 (ovarian cancer antigen);
immunoadhesins; and fragments and/or variants of any of the
above-listed proteins as well as antibodies, including antibody
fragments, binding to one or more protein, including, for example,
any of the above-listed proteins.
[0080] The term "antibody" herein is used in the broadest sense and
specifically covers monoclonal antibodies (including full length
monoclonal antibodies), polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired biological activity.
[0081] An "isolated" protein (e.g., an isolated antibody) is one
which has been identified and separated and/or recovered from a
component of its natural environment. Contaminant components of its
natural environment are materials which would interfere with
research, diagnostic or therapeutic uses for the protein, and may
include enzymes, hormones, and other proteinaceous or
nonproteinaceous solutes. Isolated protein includes the protein in
situ within recombinant cells since at least one component of the
protein's natural environment will not be present. Ordinarily,
however, isolated protein will be prepared by at least one
purification step.
[0082] "Native antibodies" are usually heterotetrameric
glycoproteins of about 150,000 daltons, composed of two identical
light (L) chains and two identical heavy (H) chains. Each light
chain is linked to a heavy chain by one covalent disulfide bond,
while the number of disulfide linkages varies among the heavy
chains of different immunoglobulin isotypes. Each heavy and light
chain also has regularly spaced intrachain disulfide bridges. Each
heavy chain has at one end a variable domain (V.sub.H) followed by
a number of constant domains. Each light chain has a variable
domain at one end (V.sub.L) and a constant domain at its other end;
the constant domain of the light chain is aligned with the first
constant domain of the heavy chain, and the light chain variable
domain is aligned with the variable domain of the heavy chain.
Particular amino acid residues are believed to form an interface
between the light chain and heavy chain variable domains.
[0083] The term "constant domain" refers to the portion of an
immunoglobulin molecule having a more conserved amino acid sequence
relative to the other portion of the immunoglobulin, the variable
domain, which contains the antigen binding site. The constant
domain contains the C.sub.H1, C.sub.H2 and C.sub.H3 domains
(collectively, CH) of the heavy chain and the CHL (or CL) domain of
the light chain.
[0084] The "variable region" or "variable domain" of an antibody
refers to the amino-terminal domains of the heavy or light chain of
the antibody. The variable domain of the heavy chain may be
referred to as "V.sub.H." The variable domain of the light chain
may be referred to as "V.sub.L." These domains are generally the
most variable parts of an antibody and contain the antigen-binding
sites.
[0085] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
hypervariable regions (HVRs) both in the light-chain and the
heavy-chain variable domains. The more highly conserved portions of
variable domains are called the framework regions (FR). The
variable domains of native heavy and light chains each comprise
four FR regions, largely adopting a beta-sheet configuration,
connected by three HVRs, which form loops connecting, and in some
cases forming part of, the beta-sheet structure. The HVRs in each
chain are held together in close proximity by the FR regions and,
with the HVRs from the other chain, contribute to the formation of
the antigen-binding site of antibodies (see Kabat et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, National
Institute of Health, Bethesda, Md. (1991)). The constant domains
are not involved directly in the binding of an antibody to an
antigen, but exhibit various effector functions, such as
participation of the antibody in antibody-dependent cellular
toxicity.
[0086] The "light chains" of antibodies (immunoglobulins) from any
mammalian species can be assigned to one of two clearly distinct
types, called kappa (".kappa.") and lambda (".lamda."), based on
the amino acid sequences of their constant domains.
[0087] The term IgG "isotype" or "subclass" as used herein is meant
any of the subclasses of immunoglobulins defined by the chemical
and antigenic characteristics of their constant regions. Depending
on the amino acid sequences of the constant domains of their heavy
chains, antibodies (immunoglobulins) can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, and several of these may be further divided into
subclasses (isotypes), e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3,
IgG.sub.4, IgA.sub.1, and IgA.sub.2. The heavy chain constant
domains that correspond to the different classes of immunoglobulins
are called .alpha., .gamma., .epsilon., .gamma., and .mu.,
respectively. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well
known and described generally in, for example, Abbas et al.
Cellular and Mol. Immunology, 4th ed., W.B. Saunders, Co., 2000. An
antibody may be part of a larger fusion molecule, formed by
covalent or non-covalent association of the antibody with one or
more other proteins or peptides.
[0088] The terms "full length antibody," "intact antibody" and
"whole antibody" are used herein interchangeably to refer to an
antibody in its substantially intact form, not antibody fragments
as defined below. The terms particularly refer to an antibody with
heavy chains that contain an Fc region.
[0089] "Antibody fragments" comprise a portion of an intact
antibody, preferably comprising the antigen binding region thereof.
Examples of antibody fragments include Fab, Fab', F(ab').sub.2, and
Fv fragments; diabodies; linear antibodies; single-chain antibody
molecules; and multispecific antibodies formed from antibody
fragments.
[0090] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-combining
sites and is still capable of cross-linking antigen. The Fab
fragment contains the heavy- and light-chain variable domains and
also contains the constant domain of the light chain and the first
constant domain (CH1) of the heavy chain. Fab' fragments differ
from Fab fragments by the addition of a few residues at the carboxy
terminus of the heavy chain CH1 domain including one or more
cysteines from the antibody hinge region. Fab'-SH is the
designation herein for Fab' in which the cysteine residue(s) of the
constant domains bear a free thiol group. F(ab').sub.2 antibody
fragments originally were produced as pairs of Fab' fragments which
have hinge cysteines between them. Other chemical couplings of
antibody fragments are also known.
[0091] "Fv" is the minimum antibody fragment which contains a
complete antigen-binding site. In one embodiment, a two-chain Fv
species consists of a dimer of one heavy- and one light-chain
variable domain in tight, non-covalent association. In a
single-chain Fv (scFv) species, one heavy- and one light-chain
variable domain can be covalently linked by a flexible peptide
linker such that the light and heavy chains can associate in a
"dimeric" structure analogous to that in a two-chain Fv species. It
is in this configuration that the three HVRs of each variable
domain interact to define an antigen-binding site on the surface of
the VH-VL dimer. Collectively, the six HVRs confer antigen-binding
specificity to the antibody. However, even a single variable domain
(or half of an Fv comprising only three HVRs specific for an
antigen) has the ability to recognize and bind antigen, although at
a lower affinity than the entire binding site.
[0092] "Single-chain Fv" or "scFv" antibody fragments comprise the
VH and VL domains of antibody, wherein these domains are present in
a single polypeptide chain. Generally, the scFv polypeptide further
comprises a polypeptide linker between the VH and VL domains which
enables the scFv to form the desired structure for antigen binding.
For a review of scFv, see, e.g., Pluckthun, in The Pharmacology of
Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
Springer-Verlag, New York, pp. 269-315, 1994.
[0093] The term "diabodies" refers to antibody fragments with two
antigen-binding sites, which fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) in the same polypeptide chain (VH-VL). By using a linker that
is too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
Diabodies may be bivalent or bispecific. Diabodies are described
more fully in, for example, EP 404,097; WO 1993/01161; Hudson et
al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl.
Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are
also described in Hudson et al., Nat. Med. 9:129-134 (2003).
[0094] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, e.g., the individual antibodies comprising the
population are identical except for possible mutations, e.g.,
naturally occurring mutations, that may be present in minor
amounts. Thus, the modifier "monoclonal" indicates the character of
the antibody as not being a mixture of discrete antibodies. In
certain embodiments, such a monoclonal antibody typically includes
an antibody comprising a polypeptide sequence that binds a target,
wherein the target-binding polypeptide sequence was obtained by a
process that includes the selection of a single target binding
polypeptide sequence from a plurality of polypeptide sequences. For
example, the selection process can be the selection of a unique
clone from a plurality of clones, such as a pool of hybridoma
clones, phage clones, or recombinant DNA clones. It should be
understood that a selected target binding sequence can be further
altered, for example, to improve affinity for the target, to
humanize the target binding sequence, to improve its production in
cell culture, to reduce its immunogenicity in vivo, to create a
multispecific antibody, etc., and that an antibody comprising the
altered target binding sequence is also a monoclonal antibody of
this invention. In contrast to polyclonal antibody preparations,
which typically include different antibodies directed against
different determinants (epitopes), each monoclonal antibody of a
monoclonal antibody preparation is directed against a single
determinant on an antigen. In addition to their specificity,
monoclonal antibody preparations are advantageous in that they are
typically uncontaminated by other immunoglobulins.
[0095] The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the
invention may be made by a variety of techniques, including, for
example, the hybridoma method (e.g., Kohler and Milstein, Nature,
256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995),
Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor
Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal
Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)),
recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567),
phage-display technologies (see, e.g., Clackson et al., Nature,
352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597
(1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et
al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl.
Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J.
Immunol. Methods 284(1-2): 119-132 (2004), and technologies for
producing human or human-like antibodies in animals that have parts
or all of the human immunoglobulin loci or genes encoding human
immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096;
WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad.
Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258
(1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat.
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and
U.S. Pat. No. 5,661,016; Marks et al., Bio/Technology 10: 779-783
(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison,
Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14:
845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and
Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).
[0096] The monoclonal antibodies herein specifically include
"chimeric" antibodies in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc.
Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies
include PRIMATTZED.RTM. antibodies wherein the antigen-binding
region of the antibody is derived from an antibody produced by,
e.g., immunizing macaque monkeys with the antigen of interest.
[0097] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. In one embodiment, a humanized antibody
is a human immunoglobulin (recipient antibody) in which residues
from a HVR of the recipient are replaced by residues from a HVR of
a non-human species (donor antibody) such as mouse, rat, rabbit, or
nonhuman primate having the desired specificity, affinity, and/or
capacity. In some instances, FR residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Furthermore, humanized antibodies may comprise residues that are
not found in the recipient antibody or in the donor antibody. These
modifications may be made to further refine antibody performance.
In general, a humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin, and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally will also comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see, e.g., Jones et al.,
Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329
(1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See
also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma &
Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions
23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433
(1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.
[0098] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human and/or has been made using any of the techniques for making
human antibodies as disclosed herein. This definition of a human
antibody specifically excludes a humanized antibody comprising
non-human antigen-binding residues. Human antibodies can be
produced using various techniques known in the art, including
phage-display libraries. Hoogenboom and Winter, J. Mol. Biol.,
227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also
available for the preparation of human monoclonal antibodies are
methods described in Cole et al., Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol.,
147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr.
Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be
prepared by administering the antigen to a transgenic animal that
has been modified to produce such antibodies in response to
antigenic challenge, but whose endogenous loci have been disabled,
e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and
6,150,584 regarding XENOMOUSE.TM. technology). See also, for
example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562
(2006) regarding human antibodies generated via a human B-cell
hybridoma technology.
[0099] The term "hypervariable region," "HVR," or "HV," when used
herein refers to the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops. Generally, antibodies comprise six HVRs; three in the VH
(H1, H2, H3), and three in the VL (L1, L2, L3). In native
antibodies, H3 and L3 display the most diversity of the six HVRs,
and H3 in particular is believed to play a unique role in
conferring fine specificity to antibodies. See, e.g., Xu et al.,
Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular
Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003).
Indeed, naturally occurring camelid antibodies consisting of a
heavy chain only are functional and stable in the absence of light
chain. See, e.g., Hamers-Casterman et al., Nature 363:446-448
(1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).
[0100] A number of HVR delineations are in use and are encompassed
herein. The Kabat Complementarity Determining Regions (CDRs) are
based on sequence variability and are the most commonly used (Kabat
et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991)). Chothia refers instead to the location of the structural
loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM
HVRs represent a compromise between the Kabat HVRs and Chothia
structural loops, and are used by Oxford Molecular's AbM antibody
modeling software. The "contact" HVRs are based on an analysis of
the available complex crystal structures. The residues from each of
these HVRs are noted below.
TABLE-US-00001 Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34
L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97
L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B
(Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia
Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102
H96-H101 H93-H101
[0101] HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34
(L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and
26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3)
in the VH. The variable domain residues are numbered according to
Kabat et al., supra, for each of these definitions.
[0102] "Framework" or "FR" residues are those variable domain
residues other than the HVR residues as herein defined.
[0103] The term "variable domain residue numbering as in Kabat" or
"amino acid position numbering as in Kabat," and variations
thereof, refers to the numbering system used for heavy chain
variable domains or light chain variable domains of the compilation
of antibodies in Kabat et al., supra. Using this numbering system,
the actual linear amino acid sequence may contain fewer or
additional amino acids corresponding to a shortening of, or
insertion into, a FR or HVR of the variable domain. For example, a
heavy chain variable domain may include a single amino acid insert
(residue 52a according to Kabat) after residue 52 of H2 and
inserted residues (e.g. residues 82a, 82b, and 82c, etc. according
to Kabat) after heavy chain FR residue 82. The Kabat numbering of
residues may be determined for a given antibody by alignment at
regions of homology of the sequence of the antibody with a
"standard" Kabat numbered sequence
[0104] The Kabat numbering system is generally used when referring
to a residue in the variable domain (approximately residues 1-107
of the light chain and residues 1-113 of the heavy chain) (e.g.,
Kabat et al., Sequences of Immunological Interest. 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.
(1991)). The "EU numbering system" or "EU index" is generally used
when referring to a residue in an immunoglobulin heavy chain
constant region (e.g., the EU index reported in Kabat et al.,
supra). The "EU index as in Kabat" refers to the residue numbering
of the human IgG1 EU antibody.
[0105] The expression "linear antibodies" refers to the antibodies
described in Zapata et al. (1995 Protein Eng, 8(10):1057-1062).
Briefly, these antibodies comprise a pair of tandem Fd segments
(VH--CH1-VH--CH1) which, together with complementary light chain
polypeptides, form a pair of antigen binding regions. Linear
antibodies can be bispecific or monospecific.
[0106] The term "about" as used herein refers to an acceptable
error range for the respective value as determined by one of
ordinary skill in the art, which will depend in part how the value
is measured or determined, i.e., the limitations of the measurement
system. For example, "about" can mean within 1 or more than 1
standard deviations, per the practice in the art. A reference to
"about" a value or parameter herein includes and describes
embodiments that are directed to that value or parameter per se.
For example, a description referring to "about X" includes
description of "X".
[0107] As used in this specification and the appended claims, the
singular forms "a", "an" and "the" include plural referents unless
the content clearly dictates otherwise. Thus, for example,
reference to "a compound" optionally includes a combination of two
or more such compounds, and the like.
[0108] It is understood that aspects and embodiments of the
invention described herein include "comprising," "consisting," and
"consisting essentially of" aspects and embodiments.
II. Protein Formulations and Preparation
[0109] The invention herein relates to formulations comprising a
protein and a compound which prevents oxidation of the protein in
the formulation, the compound is of formula:
##STR00008## [0110] wherein R.sup.2 is selected from hydrogen,
hydroxyl, --COOH, and --CH.sub.2COOH; [0111] R.sup.3 is selected
from hydrogen, hydroxyl, --COOH, --CH.sub.2COOH, and
--CH.sub.2CHR.sup.3a(NH.sub.2); wherein R.sup.3a is COOH or
hydrogen; R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are independently
selected from hydrogen and hydroxyl; provided that one of R.sup.2,
R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 is hydroxyl; or a
pharmaceutically acceptable salt thereof.
[0112] In some embodiments, the compound is a compound of
formula:
##STR00009## [0113] wherein R.sup.2 and R.sup.3 are independently
selected from hydrogen, hydroxyl, --COOH, and --CH.sub.2COOH; and
R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are independently selected
from hydrogen and hydroxyl; provided that one of R.sup.2, R.sup.3,
R.sup.4, R.sup.5, R.sup.6, and R.sup.7 is hydroxyl; or a
pharmaceutically acceptable salt thereof.
[0114] In some embodiments, the compound is a compound of
formula:
##STR00010## [0115] wherein R.sup.3a is COOH or hydrogen; R.sup.2,
R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are independently selected
from hydrogen and hydroxyl, provided that one of R.sup.2, R.sup.4,
R.sup.5, R.sup.6, and R.sup.7 is hydroxyl; or a pharmaceutically
acceptable salt thereof.
[0116] In some embodiments, R.sup.4, R.sup.5 or R.sup.7 in any of
the formula above is hydroxyl. In a further embodiment, the
compound is selected from the group consisting of
5-hydroxy-tryptophan, 5-hydroxy indole, 7-hydroxy indole, and
serotonin. In some embodiments, the formulation is a liquid
formulation. In some embodiments, the compound in the formulation
is from about 0.3 mM to about 10 mM, or up to the highest
concentration that the compound is soluble in the formulation. In
some embodiments, the compound in the formulation is about 0.3 mM
to about 1 mM. In some embodiments, the compound prevents oxidation
of one or more amino acids in the protein selected from group
consisting of tryptophan, cysteine, histidine, tyrosine, and/or
methionine. In some embodiments, the compound prevents oxidation of
the protein by a reactive oxygen species (ROS). In a further
embodiment, the reactive oxygen species is selected from the group
consisting of a singlet oxygen, a superoxide (O.sub.2--), an
alkoxyl radical, a peroxyl radical, a hydrogen peroxide
(H.sub.2O.sub.2), a dihydrogen trioxide (H.sub.2O.sub.3), a
hydrotrioxy radical (HO.sub.3.cndot.), ozone (O.sub.3), a hydroxyl
radical, and an alkyl peroxide. In some embodiments, a protein
described herein is susceptible to oxidation. In some embodiments,
methionine, cysteine, histidine, tryptophan, and/or tyrosine in the
protein is susceptible to oxidation. In some embodiments,
tryptophan and/or methionine in the protein is susceptible to
oxidation. For example, a tryptophan amino acid in the Fab portion
of a monoclonal antibody and/or a methionine amino acid in the Fc
portion of a monoclonal antibody can be susceptible to oxidation.
In some embodiments, the protein is a therapeutic protein. In some
of the embodiments herein, the protein is an antibody. In some
embodiments, the antibody is a polyclonal antibody, a monoclonal
antibody, a humanized antibody, a human antibody, a chimeric
antibody, or an antibody fragment. In a further embodiment, the
compound prevents oxidation of one or more amino acids in the Fab
portion of an antibody. In another further embodiment, the compound
prevents oxidation of one or more amino acids in the Fc portion of
an antibody. In some embodiments, the formulation provided herein
is a pharmaceutical formulation suitable for administration to a
subject. As used herein a "subject" or an "individual" for purposes
of treatment or administration refers to any animal classified as a
mammal, including humans, domestic and farm animals, and zoo,
sports, or pet animals, such as dogs, horses, cats, cows, etc.
Preferably, the mammal is human. In some embodiments, the
formulation is aqueous. In some embodiments herein, the protein
(e.g., the antibody) concentration in the formulation is about 1
mg/mL to about 250 mg/mL. In some embodiments, the formulation
further one or more excipients selected from the group consisting
of a stabilizer, a buffer, a surfactant, and a tonicity agent. For
example, a formulation of the invention can comprise a monoclonal
antibody, a compound as provided herein which prevents oxidation of
the protein (e.g., 5-hydroxy indole), and a buffer that maintains
the pH of the formulation to a desirable level. In some
embodiments, a formulation provided herein has a pH of about 4.5 to
about 7.0.
[0117] Proteins and antibodies in the formulation may be prepared
using methods known in the art. The antibody (e.g., full length
antibodies, antibody fragments and multispecific antibodies) in the
formulation is prepared using techniques available in the art,
non-limiting exemplary methods of which are described in more
detail in the following sections. The methods herein can be adapted
by one of skill in the art for the preparation of formulations
comprising other proteins such as peptide-based inhibitors. See
Molecular Cloning: A Laboratory Manual (Sambrook et al., 4.sup.th
ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
2012); Current Protocols in Molecular Biology (F. M. Ausubel, et
al. eds., 2003); Short Protocols in Molecular Biology (Ausubel et
al., eds., J. Wiley and Sons, 2002); Current Protocols in Protein
Science, (Horswill et al., 2006); Antibodies, A Laboratory Manual
(Harlow and Lane, eds., 1988); Culture of Animal Cells: A Manual of
Basic Technique and Specialized Applications (R. I. Freshney,
6.sup.th ed., J. Wiley and Sons, 2010) for generally well
understood and commonly employed techniques and procedures for the
production of therapeutic proteins, which are all incorporated
herein by reference in their entirety.
[0118] A. Antibody Preparation
[0119] The antibody in the formulations provided herein is directed
against an antigen of interest. Preferably, the antigen is a
biologically important polypeptide and administration of the
antibody to a mammal suffering from a disorder can result in a
therapeutic benefit in that mammal. However, antibodies directed
against nonpolypeptide antigens are also contemplated.
[0120] Where the antigen is a polypeptide, it may be a
transmembrane molecule (e.g. receptor) or ligand such as a growth
factor. Exemplary antigens include molecules such as vascular
endothelial growth factor (VEGF); CD20; ox-LDL; ox-ApoB100; renin;
a growth hormone, including human growth hormone and bovine growth
hormone; growth hormone releasing factor; parathyroid hormone;
thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin;
insulin A-chain; insulin B-chain; proinsulin; follicle stimulating
hormone; calcitonin; luteinizing hormone; glucagon; clotting
factors such as factor VIIIC, factor IX, tissue factor, and von
Willebrands factor; anti-clotting factors such as Protein C; atrial
natriuretic factor; lung surfactant; a plasminogen activator, such
as urokinase or human urine or tissue-type plasminogen activator
(t-PA); bombesin; thrombin; hemopoietic growth factor; a tumor
necrosis factor receptor such as death receptor 5 and CD120; tumor
necrosis factor-alpha and -beta; enkephalinase; RANTES (regulated
on activation normally T-cell expressed and secreted); human
macrophage inflammatory protein (MIP-1-alpha); a serum albumin such
as human serum albumin; Muellerian-inhibiting substance; relaxin
A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated
peptide; a microbial protein, such as beta-lactamase; DNase; IgE; a
cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4;
inhibin; activin; receptors for hormones or growth factors; protein
A or D; rheumatoid factors; a neurotrophic factor such as
bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or
-6 (NT-3, NT4, NT-5, or NT-6), or a nerve growth factor such as
NGF-.beta.; platelet-derived growth factor (PDGF); fibroblast
growth factor such as aFGF and bFGF; epidermal growth factor (EGF);
transforming growth factor (TGF) such as TGF-alpha and TGF-beta,
including TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, TGF-.beta.4, or
TGF-.beta.5; insulin-like growth factor-I and -II (IGF-I and
IGF-II); des (1-3)-IGF-I (brain IGF-I), insulin-like growth factor
binding proteins; CD proteins such as CD3, CD4, CD8, CD19 and CD20;
erythropoietin; osteoinductive factors; immunotoxins; a bone
morphogenetic protein (BMP); an interferon such as
interferon-alpha, -beta, and -gamma; colony stimulating factors
(CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g.,
IL-1 to IL-10; superoxide dismutase; T-cell receptors; surface
membrane proteins; decay accelerating factor; viral antigen such
as, for example, a portion of the AIDS envelope; transport
proteins; homing receptors; addressins; regulatory proteins;
integrns such as CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4 and
VCAM; a tumor associated antigen such as HER2, HER3 or HER4
receptor; and fragments of any of the above-listed
polypeptides.
[0121] (i) Antigen Preparation
[0122] Soluble antigens or fragments thereof, optionally conjugated
to other molecules, can be used as immunogens for generating
antibodies. For transmembrane molecules, such as receptors,
fragments of these (e.g. the extracellular domain of a receptor)
can be used as the immunogen. Alternatively, cells expressing the
transmembrane molecule can be used as the immunogen. Such cells can
be derived from a natural source (e.g. cancer cell lines) or may be
cells which have been transformed by recombinant techniques to
express the transmembrane molecule. Other antigens and forms
thereof useful for preparing antibodies will be apparent to those
in the art.
[0123] (ii) Certain Antibody-Based Methods
[0124] Polyclonal antibodies are preferably raised in animals by
multiple subcutaneous (sc) or intraperitoneal (ip) injections of
the relevant antigen and an adjuvant. It may be useful to conjugate
the relevant antigen to a protein that is immunogenic in the
species to be immunized, e.g., keyhole limpet hemocyanin, serum
albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a
bifunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R
and R.sup.1 are different alkyl groups.
[0125] Animals are immunized against the antigen, immunogenic
conjugates, or derivatives by combining, e.g., 100 .mu.g or 5 .mu.g
of the protein or conjugate (for rabbits or mice, respectively)
with 3 volumes of Freund's complete adjuvant and injecting the
solution intradermally at multiple sites. One month later the
animals are boosted with 1/5 to 1/10 the original amount of peptide
or conjugate in Freund's complete adjuvant by subcutaneous
injection at multiple sites. Seven to 14 days later the animals are
bled and the serum is assayed for antibody titer. Animals are
boosted until the titer plateaus. Preferably, the animal is boosted
with the conjugate of the same antigen, but conjugated to a
different protein and/or through a different cross-linking reagent.
Conjugates also can be made in recombinant cell culture as protein
fusions. Also, aggregating agents such as alum are suitably used to
enhance the immune response.
[0126] Monoclonal antibodies of interest can be made using the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), and further described, e.g., in Hongo et al., Hybridoma, 14
(3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory
Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);
Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas
563-681 (Elsevier, N.Y., 1981), and Ni, Xiandai Mianyixue,
26(4):265-268 (2006) regarding human-human hybridomas. Additional
methods include those described, for example, in U.S. Pat. No.
7,189,826 regarding production of monoclonal human natural IgM
antibodies from hybridoma cell lines. Human hybridoma technology
(Trioma technology) is described in Vollmers and Brandlein,
Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and
Brandlein, Methods and Findings in Experimental and Clinical
Pharmacology, 27(3):185-91 (2005).
[0127] For various other hybridoma techniques, see, e.g., US
2006/258841; US 2006/183887 (fully human antibodies), US
2006/059575; US 2005/287149; US 2005/100546; US 2005/026229; and
U.S. Pat. Nos. 7,078,492 and 7,153,507. An exemplary protocol for
producing monoclonal antibodies using the hybridoma method is
described as follows. In one embodiment, a mouse or other
appropriate host animal, such as a hamster, is immunized to elicit
lymphocytes that produce or are capable of producing antibodies
that will specifically bind to the protein used for immunization.
Antibodies are raised in animals by multiple subcutaneous (sc) or
intraperitoneal (ip) injections of a polypeptide of interest or a
fragment thereof, and an adjuvant, such as monophosphoryl lipid A
(MPL)/trehalose dicrynomycolate (TDM) (Ribi Immunochem. Research,
Inc., Hamilton, Mont.). A polypeptide of interest (e.g., antigen)
or a fragment thereof may be prepared using methods well known in
the art, such as recombinant methods, some of which are further
described herein. Serum from immunized animals is assayed for
anti-antigen antibodies, and booster immunizations are optionally
administered. Lymphocytes from animals producing anti-antigen
antibodies are isolated. Alternatively, lymphocytes may be
immunized in vitro.
[0128] Lymphocytes are then fused with myeloma cells using a
suitable fusing agent, such as polyethylene glycol, to form a
hybridoma cell. See, e.g., Goding, Monoclonal Antibodies:
Principles and Practice, pp. 59-103 (Academic Press, 1986). Myeloma
cells may be used that fuse efficiently, support stable high-level
production of antibody by the selected antibody-producing cells,
and are sensitive to a medium such as HAT medium. Exemplary myeloma
cells include, but are not limited to, murine myeloma lines, such
as those derived from MOPC-21 and MPC-11 mouse tumors available
from the Salk Institute Cell Distribution Center, San Diego, Calif.
USA, and SP-2 or X63-Ag8-653 cells available from the American Type
Culture Collection, Rockville, Md. USA. Human myeloma and
mouse-human heteromyeloma cell lines also have been described for
the production of human monoclonal antibodies (Kozbor, J. Immunol.,
133:3001 (1984); Brodeur et al., Monoclonal Antibody Production
Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New
York, 1987)).
[0129] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium, e.g., a medium that contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
Preferably, serum-free hybridoma cell culture methods are used to
reduce use of animal-derived serum such as fetal bovine serum, as
described, for example, in Even et al., Trends in Biotechnology,
24(3), 105-108 (2006).
[0130] Oligopeptides as tools for improving productivity of
hybridoma cell cultures are described in Franek, Trends in
Monoclonal Antibody Research, 111-122 (2005). Specifically,
standard culture media are enriched with certain amino acids
(alanine, serine, asparagine, proline), or with protein hydrolyzate
fractions, and apoptosis may be significantly suppressed by
synthetic oligopeptides, constituted of three to six amino acid
residues. The peptides are present at millimolar or higher
concentrations.
[0131] Culture medium in which hybridoma cells are growing may be
assayed for production of monoclonal antibodies that bind to an
antibody described herein. The binding specificity of monoclonal
antibodies produced by hybridoma cells may be determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoadsorbent assay
(ELISA). The binding affinity of the monoclonal antibody can be
determined, for example, by Scatchard analysis. See, e.g., Munson
et al., Anal. Biochem., 107:220 (1980).
[0132] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods. See, e.g., Goding, supra. Suitable culture media
for this purpose include, for example, D-MEM or RPMI-1640 medium.
In addition, hybridoma cells may be grown in vivo as ascites tumors
in an animal. Monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography. One
procedure for isolation of proteins from hybridoma cells is
described in US 2005/176122 and U.S. Pat. No. 6,919,436. The method
includes using minimal salts, such as lyotropic salts, in the
binding process and preferably also using small amounts of organic
solvents in the elution process.
[0133] (iii) Certain Library Screening Methods
[0134] Antibodies described herein can be made by using
combinatorial libraries to screen for antibodies with the desired
activity or activities. For example, a variety of methods are known
in the art for generating phage display libraries and screening
such libraries for antibodies possessing the desired binding
characteristics. Such methods are described generally in Hoogenboom
et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al.,
ed., Human Press, Totowa, N.J., 2001). For example, one method of
generating antibodies of interest is through the use of a phage
antibody library as described in Lee et al., J. Mol. Biol. (2004),
340(5):1073-93.
[0135] In principle, synthetic antibody clones are selected by
screening phage libraries containing phage that display various
fragments of antibody variable region (Fv) fused to phage coat
protein. Such phage libraries are panned by affinity chromatography
against the desired antigen. Clones expressing Fv fragments capable
of binding to the desired antigen are adsorbed to the antigen and
thus separated from the non-binding clones in the library. The
binding clones are then eluted from the antigen, and can be further
enriched by additional cycles of antigen adsorption/elution. Any of
the antibodies can be obtained by designing a suitable antigen
screening procedure to select for the phage clone of interest
followed by construction of a full length antibody clone using the
Fv sequences from the phage clone of interest and suitable constant
region (Fc) sequences described in Kabat et al., Sequences of
Proteins of Immunological Interest, Fifth Edition, NIH Publication
91-3242, Bethesda Md. (1991), vols. 1-3.
[0136] In certain embodiments, the antigen-binding domain of an
antibody is formed from two variable (V) regions of about 110 amino
acids, one each from the light (VL) and heavy (VH) chains, that
both present three hypervariable loops (HVRs) or
complementarity-determining regions (CDRs). Variable domains can be
displayed functionally on phage, either as single-chain Fv (scFv)
fragments, in which VH and VL are covalently linked through a
short, flexible peptide, or as Fab fragments, in which they are
each fused to a constant domain and interact non-covalently, as
described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).
As used herein, scFv encoding phage clones and Fab encoding phage
clones are collectively referred to as "Fv phage clones" or "Fv
clones."
[0137] Repertoires of VH and VL genes can be separately cloned by
polymerase chain reaction (PCR) and recombined randomly in phage
libraries, which can then be searched for antigen-binding clones as
described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).
Libraries from immunized sources provide high-affinity antibodies
to the immunogen without the requirement of constructing
hybridomas. Alternatively, the naive repertoire can be cloned to
provide a single source of human antibodies to a wide range of
non-self and also self antigens without any immunization as
described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally,
naive libraries can also be made synthetically by cloning the
unrearranged V-gene segments from stem cells, and using PCR primers
containing random sequence to encode the highly variable CDR3
regions and to accomplish rearrangement in vitro as described by
Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
[0138] In certain embodiments, filamentous phage is used to display
antibody fragments by fusion to the minor coat protein pIII. The
antibody fragments can be displayed as single chain Fv fragments,
in which VH and VL domains are connected on the same polypeptide
chain by a flexible polypeptide spacer, e.g. as described by Marks
et al., J. Mol. Biol., 222: 581-597 (1991), or as Fab fragments, in
which one chain is fused to pIII and the other is secreted into the
bacterial host cell periplasm where assembly of a Fab-coat protein
structure which becomes displayed on the phage surface by
displacing some of the wild type coat proteins, e.g. as described
in Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991).
[0139] In general, nucleic acids encoding antibody gene fragments
are obtained from immune cells harvested from humans or animals. If
a library biased in favor of anti-antigen clones is desired, the
subject is immunized with antigen to generate an antibody response,
and spleen cells and/or circulating B cells other peripheral blood
lymphocytes (PBLs) are recovered for library construction. In one
embodiment, a human antibody gene fragment library biased in favor
of anti-antigen clones is obtained by generating an anti-antigen
antibody response in transgenic mice carrying a functional human
immunoglobulin gene array (and lacking a functional endogenous
antibody production system) such that antigen immunization gives
rise to B cells producing human antibodies against antigen. The
generation of human antibody-producing transgenic mice is described
below.
[0140] Additional enrichment for anti-antigen reactive cell
populations can be obtained by using a suitable screening procedure
to isolate B cells expressing antigen-specific membrane bound
antibody, e.g., by cell separation using antigen affinity
chromatography or adsorption of cells to fluorochrome-labeled
antigen followed by flow-activated cell sorting (FACS).
[0141] Alternatively, the use of spleen cells and/or B cells or
other PBLs from an unimmunized donor provides a better
representation of the possible antibody repertoire, and also
permits the construction of an antibody library using any animal
(human or non-human) species in which antigen is not antigenic. For
libraries incorporating in vitro antibody gene construction, stem
cells are harvested from the subject to provide nucleic acids
encoding unrearranged antibody gene segments. The immune cells of
interest can be obtained from a variety of animal species, such as
human, mouse, rat, lagomorpha, luprine, canine, feline, porcine,
bovine, equine, and avian species, etc.
[0142] Nucleic acid encoding antibody variable gene segments
(including VH and VL segments) are recovered from the cells of
interest and amplified. In the case of rearranged VH and VL gene
libraries, the desired DNA can be obtained by isolating genomic DNA
or mRNA from lymphocytes followed by polymerase chain reaction
(PCR) with primers matching the 5' and 3' ends of rearranged VH and
VL genes as described in Orlandi et al., Proc. Natl. Acad. Sci.
(USA), 86: 3833-3837 (1989), thereby making diverse V gene
repertoires for expression. The V genes can be amplified from cDNA
and genomic DNA, with back primers at the 5' end of the exon
encoding the mature V-domain and forward primers based within the
J-segment as described in Orlandi et al. (1989) and in Ward et al.,
Nature, 341: 544-546 (1989). However, for amplifying from cDNA,
back primers can also be based in the leader exon as described in
Jones et al., Biotechnol., 9: 88-89 (1991), and forward primers
within the constant region as described in Sastry et al., Proc.
Natl. Acad. Sci. (USA), 86: 5728-5732 (1989). To maximize
complementarity, degeneracy can be incorporated in the primers as
described in Orlandi et al. (1989) or Sastry et al. (1989). In
certain embodiments, library diversity is maximized by using PCR
primers targeted to each V-gene family in order to amplify all
available VH and VL arrangements present in the immune cell nucleic
acid sample, e.g. as described in the method of Marks et al., J.
Mol. Biol., 222: 581-597 (1991) or as described in the method of
Orum et al., Nucleic Acids Res., 21: 4491-4498 (1993). For cloning
of the amplified DNA into expression vectors, rare restriction
sites can be introduced within the PCR primer as a tag at one end
as described in Orlandi et al. (1989), or by further PCR
amplification with a tagged primer as described in Clackson et al.,
Nature, 352: 624-628 (1991).
[0143] Repertoires of synthetically rearranged V genes can be
derived in vitro from V gene segments. Most of the human VH-gene
segments have been cloned and sequenced (reported in Tomlinson et
al., J. Mol. Biol., 227: 776-798 (1992)), and mapped (reported in
Matsuda et al., Nature Genet., 3: 88-94 (1993); these cloned
segments (including all the major conformations of the H1 and H2
loop) can be used to generate diverse VH gene repertoires with PCR
primers encoding H3 loops of diverse sequence and length as
described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388
(1992). VH repertoires can also be made with all the sequence
diversity focused in a long H3 loop of a single length as described
in Barbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992).
Human V.kappa. and V.lamda. segments have been cloned and sequenced
(reported in Williams and Winter, Eur. J. Immunol., 23: 1456-1461
(1993)) and can be used to make synthetic light chain repertoires.
Synthetic V gene repertoires, based on a range of VH and VL folds,
and L3 and H3 lengths, will encode antibodies of considerable
structural diversity. Following amplification of V-gene encoding
DNAs, germline V-gene segments can be rearranged in vitro according
to the methods of Hoogenboom and Winter, J. Mol. Biol., 227:
381-388 (1992).
[0144] Repertoires of antibody fragments can be constructed by
combining VH and VL gene repertoires together in several ways. Each
repertoire can be created in different vectors, and the vectors
recombined in vitro, e.g., as described in Hogrefe et al., Gene,
128: 119-126 (1993), or in vivo by combinatorial infection, e.g.,
the loxP system described in Waterhouse et al., Nucl. Acids Res.,
21: 2265-2266 (1993). The in vivo recombination approach exploits
the two-chain nature of Fab fragments to overcome the limit on
library size imposed by E. coli transformation efficiency. Naive VH
and VL repertoires are cloned separately, one into a phagemid and
the other into a phage vector. The two libraries are then combined
by phage infection of phagemid-containing bacteria so that each
cell contains a different combination and the library size is
limited only by the number of cells present (about 10.sup.12
clones). Both vectors contain in vivo recombination signals so that
the VH and VL genes are recombined onto a single replicon and are
co-packaged into phage virions. These huge libraries provide large
numbers of diverse antibodies of good affinity (K.sub.d.sup.-1 of
about 10.sup.-8 M).
[0145] Alternatively, the repertoires may be cloned sequentially
into the same vector, e.g. as described in Barbas et al., Proc.
Natl. Acad. Sci. USA, 88: 7978-7982 (1991), or assembled together
by PCR and then cloned, e.g. as described in Clackson et al.,
Nature, 352: 624-628 (1991). PCR assembly can also be used to join
VH and VL DNAs with DNA encoding a flexible peptide spacer to form
single chain Fv (scFv) repertoires. In yet another technique, "in
cell PCR assembly" is used to combine VH and VL genes within
lymphocytes by PCR and then clone repertoires of linked genes as
described in Embleton et al., Nucl. Acids Res., 20: 3831-3837
(1992).
[0146] The antibodies produced by naive libraries (either natural
or synthetic) can be of moderate affinity (K.sub.d.sup.-1 of about
10.sup.6 to 10.sup.7 M.sup.-1), but affinity maturation can also be
mimicked in vitro by constructing and reselecting from secondary
libraries as described in Winter et al. (1994), supra. For example,
mutation can be introduced at random in vitro by using error-prone
polymerase (reported in Leung et al., Technique 1: 11-15 (1989)) in
the method of Hawkins et al., J. Mol. Biol., 226: 889-896 (1992) or
in the method of Gram et al., Proc. Natl. Acad. Sci USA, 89:
3576-3580 (1992). Additionally, affinity maturation can be
performed by randomly mutating one or more CDRs, e.g. using PCR
with primers carrying random sequence spanning the CDR of interest,
in selected individual Fv clones and screening for higher affinity
clones. WO 9607754 (published 14 Mar. 1996) described a method for
inducing mutagenesis in a complementarity determining region of an
immunoglobulin light chain to create a library of light chain
genes. Another effective approach is to recombine the VH or VL
domains selected by phage display with repertoires of naturally
occurring V domain variants obtained from unimmunized donors and
screen for higher affinity in several rounds of chain reshuffling
as described in Marks et al., Biotechnol., 10: 779-783 (1992). This
technique allows the production of antibodies and antibody
fragments with affinities of about 10.sup.-9 M or less.
[0147] Screening of the libraries can be accomplished by various
techniques known in the art. For example, antigen can be used to
coat the wells of adsorption plates, expressed on host cells
affixed to adsorption plates or used in cell sorting, or conjugated
to biotin for capture with streptavidin-coated beads, or used in
any other method for panning phage display libraries.
[0148] The phage library samples are contacted with immobilized
antigen under conditions suitable for binding at least a portion of
the phage particles with the adsorbent. Normally, the conditions,
including pH, ionic strength, temperature and the like are selected
to mimic physiological conditions. The phages bound to the solid
phase are washed and then eluted by acid, e.g. as described in
Barbas et al., Proc. Natl. Acad. Sci USA, 88: 7978-7982 (1991), or
by alkali, e.g. as described in Marks et al., J. Mol. Biol., 222:
581-597 (1991), or by antigen competition, e.g. in a procedure
similar to the antigen competition method of Clackson et al.,
Nature, 352: 624-628 (1991). Phages can be enriched 20-1,000-fold
in a single round of selection. Moreover, the enriched phages can
be grown in bacterial culture and subjected to further rounds of
selection.
[0149] The efficiency of selection depends on many factors,
including the kinetics of dissociation during washing, and whether
multiple antibody fragments on a single phage can simultaneously
engage with antigen. Antibodies with fast dissociation kinetics
(and weak binding affinities) can be retained by use of short
washes, multivalent phage display and high coating density of
antigen in solid phase. The high density not only stabilizes the
phage through multivalent interactions, but favors rebinding of
phage that has dissociated. The selection of antibodies with slow
dissociation kinetics (and good binding affinities) can be promoted
by use of long washes and monovalent phage display as described in
Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and a
low coating density of antigen as described in Marks et al.,
Biotechnol., 10: 779-783 (1992).
[0150] It is possible to select between phage antibodies of
different affinities, even with affinities that differ slightly,
for antigen. However, random mutation of a selected antibody (e.g.
as performed in some affinity maturation techniques) is likely to
give rise to many mutants, most binding to antigen, and a few with
higher affinity. With limiting antigen, rare high affinity phage
could be competed out. To retain all higher affinity mutants,
phages can be incubated with excess biotinylated antigen, but with
the biotinylated antigen at a concentration of lower molarity than
the target molar affinity constant for antigen. The high
affinity-binding phages can then be captured by streptavidin-coated
paramagnetic beads. Such "equilibrium capture" allows the
antibodies to be selected according to their affinities of binding,
with sensitivity that permits isolation of mutant clones with as
little as two-fold higher affinity from a great excess of phages
with lower affinity. Conditions used in washing phages bound to a
solid phase can also be manipulated to discriminate on the basis of
dissociation kinetics.
[0151] Anti-antigen clones may be selected based on activity. In
certain embodiments, the invention provides anti-antigen antibodies
that bind to living cells that naturally express antigen or bind to
free floating antigen or antigen attached to other cellular
structures. Fv clones corresponding to such anti-antigen antibodies
can be selected by (1) isolating anti-antigen clones from a phage
library as described above, and optionally amplifying the isolated
population of phage clones by growing up the population in a
suitable bacterial host; (2) selecting antigen and a second protein
against which blocking and non-blocking activity, respectively, is
desired; (3) adsorbing the anti-antigen phage clones to immobilized
antigen; (4) using an excess of the second protein to elute any
undesired clones that recognize antigen-binding determinants which
overlap or are shared with the binding determinants of the second
protein; and (5) eluting the clones which remain adsorbed following
step (4). Optionally, clones with the desired blocking/non-blocking
properties can be further enriched by repeating the selection
procedures described herein one or more times.
[0152] DNA encoding hybridoma-derived monoclonal antibodies or
phage display Fv clones is readily isolated and sequenced using
conventional procedures (e.g. by using oligonucleotide primers
designed to specifically amplify the heavy and light chain coding
regions of interest from hybridoma or phage DNA template). Once
isolated, the DNA can be placed into expression vectors, which are
then transfected into host cells such as E. coli cells, simian COS
cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do
not otherwise produce immunoglobulin protein, to obtain the
synthesis of the desired monoclonal antibodies in the recombinant
host cells. Review articles on recombinant expression in bacteria
of antibody-encoding DNA include Skerra et al., Curr. Opinion in
Immunol., 5: 256 (1993) and Pluckthun, Immunol. Revs, 130: 151
(1992).
[0153] DNA encoding the Fv clones can be combined with known DNA
sequences encoding heavy chain and/or light chain constant regions
(e.g. the appropriate DNA sequences can be obtained from Kabat et
al., supra) to form clones encoding full or partial length heavy
and/or light chains. It will be appreciated that constant regions
of any isotype can be used for this purpose, including IgG, IgM,
IgA, IgD, and IgE constant regions, and that such constant regions
can be obtained from any human or animal species. An Fv clone
derived from the variable domain DNA of one animal (such as human)
species and then fused to constant region DNA of another animal
species to form coding sequence(s) for "hybrid," full length heavy
chain and/or light chain is included in the definition of
"chimeric" and "hybrid" antibody as used herein. In certain
embodiments, an Fv clone derived from human variable DNA is fused
to human constant region DNA to form coding sequence(s) for full-
or partial-length human heavy and/or light chains.
[0154] DNA encoding anti-antigen antibody derived from a hybridoma
can also be modified, for example, by substituting the coding
sequence for human heavy- and light-chain constant domains in place
of homologous murine sequences derived from the hybridoma clone
(e.g. as in the method of Morrison et al., Proc. Natl. Acad. Sci.
USA, 81: 6851-6855 (1984)). DNA encoding a hybridoma- or Fv
clone-derived antibody or fragment can be further modified by
covalently joining to the immunoglobulin coding sequence all or
part of the coding sequence for a non-immunoglobulin polypeptide.
In this manner, "chimeric" or "hybrid" antibodies are prepared that
have the binding specificity of the Fv clone or hybridoma
clone-derived antibodies.
[0155] (iv) Humanized and Human Antibodies
[0156] Various methods for humanizing non-human antibodies are
known in the art. For example, a humanized antibody has one or more
amino acid residues introduced into it from a source which is
non-human. These non-human amino acid residues are often referred
to as "import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0157] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework (FR) for the
humanized antibody (Sims et al., J. Immunol., 151:2296 (1993);
Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses
a particular framework derived from the consensus sequence of all
human antibodies of a particular subgroup of light or heavy chains.
The same framework may be used for several different humanized
antibodies (Carter et al., Proc. Natl. Acad Sci. USA, 89:4285
(1992); Presta et al., J. Immunol., 151:2623 (1993)).
[0158] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to one
embodiment of the method, humanized antibodies are prepared by a
process of analysis of the parental sequences and various
conceptual humanized products using three-dimensional models of the
parental and humanized sequences. Three-dimensional immunoglobulin
models are commonly available and are familiar to those skilled in
the art. Computer programs are available which illustrate and
display probable three-dimensional conformational structures of
selected candidate immunoglobulin sequences. Inspection of these
displays permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
[0159] Human antibodies in the formulations and compositions
described herein can be constructed by combining Fv clone variable
domain sequence(s) selected from human-derived phage display
libraries with known human constant domain sequence(s) as described
above. Alternatively, human monoclonal antibodies can be made by
the hybridoma method. Human myeloma and mouse-human heteromyeloma
cell lines for the production of human monoclonal antibodies have
been described, for example, by Kozbor J. Immunol., 133: 3001
(1984); Brodeur et al., Monoclonal Antibody Production Techniques
and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987);
and Boerner et al., J. Immunol., 147: 86 (1991).
[0160] It is possible to produce transgenic animals (e.g., mice)
that are capable, upon immunization, of producing a full repertoire
of human antibodies in the absence of endogenous immunoglobulin
production. For example, it has been described that the homozygous
deletion of the antibody heavy-chain joining region (J.sub.H) gene
in chimeric and germ-line mutant mice results in complete
inhibition of endogenous antibody production. Transfer of the human
germ-line immunoglobulin gene array in such germ-line mutant mice
will result in the production of human antibodies upon antigen
challenge. See, e.g., Jakobovits et al, Proc. Natl. Acad. Sci. USA,
90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993);
Bruggermann et al., Year in Immuno., 7:33 (1993); and Duchosal et
al. Nature 355:258 (1992).
[0161] Gene shuffling can also be used to derive human antibodies
from non-human, e.g. rodent, antibodies, where the human antibody
has similar affinities and specificities to the starting non-human
antibody. According to this method, which is also called "epitope
imprinting", either the heavy or light chain variable region of a
non-human antibody fragment obtained by phage display techniques as
described herein is replaced with a repertoire of human V domain
genes, creating a population of non-human chain/human chain scFv or
Fab chimeras. Selection with antigen results in isolation of a
non-human chain/human chain chimeric scFv or Fab wherein the human
chain restores the antigen binding site destroyed upon removal of
the corresponding non-human chain in the primary phage display
clone, i.e. the epitope governs (imprints) the choice of the human
chain partner. When the process is repeated in order to replace the
remaining non-human chain, a human antibody is obtained (see PCT WO
93/06213 published Apr. 1, 1993). Unlike traditional humanization
of non-human antibodies by CDR grafting, this technique provides
completely human antibodies, which have no FR or CDR residues of
non-human origin.
[0162] (v) Antibody Fragments
[0163] Antibody fragments may be generated by traditional means,
such as enzymatic digestion, or by recombinant techniques. In
certain circumstances there are advantages of using antibody
fragments, rather than whole antibodies. The smaller size of the
fragments allows for rapid clearance, and may lead to improved
access to solid tumors. For a review of certain antibody fragments,
see Hudson et al. (2003) Nat. Med. 9:129-134.
[0164] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
Fab, Fv and ScFv antibody fragments can all be expressed in and
secreted from E. coli, thus allowing the facile production of large
amounts of these fragments. Antibody fragments can be isolated from
the antibody phage libraries discussed above. Alternatively,
Fab'-SH fragments can be directly recovered from E. coli and
chemically coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab').sub.2 fragments can be isolated directly from recombinant
host cell culture. Fab and F(ab').sub.2 fragment with increased in
vivo half-life comprising salvage receptor binding epitope residues
are described in U.S. Pat. No. 5,869,046. Other techniques for the
production of antibody fragments will be apparent to the skilled
practitioner. In certain embodiments, an antibody is a single chain
Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and
5,587,458. Fv and scFv are the only species with intact combining
sites that are devoid of constant regions; thus, they may be
suitable for reduced nonspecific binding during in vivo use. scFv
fusion proteins may be constructed to yield fusion of an effector
protein at either the amino or the carboxy terminus of an scFv. See
Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment
may also be a "linear antibody", e.g., as described in U.S. Pat.
No. 5,641,870, for example. Such linear antibodies may be
monospecific or bispecific.
[0165] (vi) Multispecific Antibodies
[0166] Multispecific antibodies have binding specificities for at
least two different epitopes, where the epitopes are usually from
different antigens. While such molecules normally will only bind
two different epitopes (i.e. bispecific antibodies, BsAbs),
antibodies with additional specificities such as trispecific
antibodies are encompassed by this expression when used herein.
Bispecific antibodies can be prepared as full length antibodies or
antibody fragments (e.g. F(ab').sub.2 bispecific antibodies).
[0167] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the coexpression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Millstein et al., Nature, 305:537-539 (1983)). Because of the
random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829, and in Traunecker et al., EMBO J., 10:3655-3659
(1991).
[0168] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences. The
fusion preferably is with an immunoglobulin heavy chain constant
domain, comprising at least part of the hinge, CH2, and CH3
regions. It is typical to have the first heavy-chain constant
region (CH1) containing the site necessary for light chain binding,
present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host organism. This
provides for great flexibility in adjusting the mutual proportions
of the three polypeptide fragments in embodiments when unequal
ratios of the three polypeptide chains used in the construction
provide the optimum yields. It is, however, possible to insert the
coding sequences for two or all three polypeptide chains in one
expression vector when the expression of at least two polypeptide
chains in equal ratios results in high yields or when the ratios
are of no particular significance.
[0169] In one embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only one half of the
bispecific molecule provides for a facile way of separation. This
approach is disclosed in WO 94/04690. For further details of
generating bispecific antibodies see, for example, Suresh et al.,
Methods in Enzymology, 121:210 (1986).
[0170] According to another approach described in WO96/27011, the
interface between a pair of antibody molecules can be engineered to
maximize the percentage of heterodimers which are recovered from
recombinant cell culture. One interface comprises at least a part
of the C.sub.H 3 domain of an antibody constant domain. In this
method, one or more small amino acid side chains from the interface
of the first antibody molecule are replaced with larger side chains
(e.g. tyrosine or tryptophan). Compensatory "cavities" of identical
or similar size to the large side chain(s) are created on the
interface of the second antibody molecule by replacing large amino
acid side chains with smaller ones (e.g. alanine or threonine).
This provides a mechanism for increasing the yield of the
heterodimer over other unwanted end-products such as
homodimers.
[0171] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/200373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0172] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science, 229: 81 (1985) describe a
procedure wherein intact antibodies are proteolytically cleaved to
generate F(ab').sub.2 fragments. These fragments are reduced in the
presence of the dithiol complexing agent sodium arsenite to
stabilize vicinal dithiols and prevent intermolecular disulfide
formation. The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0173] Recent progress has facilitated the direct recovery of
Fab'-SH fragments from E. coli, which can be chemically coupled to
form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:
217-225 (1992) describe the production of a fully humanized
bispecific antibody F(ab').sub.2 molecule. Each Fab' fragment was
separately secreted from E. coli and subjected to directed chemical
coupling in vitro to form the bispecific antibody.
[0174] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (V.sub.L) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
V.sub.H and V.sub.L domains of one fragment are forced to pair with
the complementary V.sub.L and V.sub.H domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See Gruber et al, J. Immunol,
152:5368 (1994).
[0175] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tuft et al. J.
Immunol. 147: 60 (1991).
[0176] (vii) Single-Domain Antibodies
[0177] In some embodiments, an antibody is a single-domain
antibody. A single-domain antibody is a single polypeptide chain
comprising all or a portion of the heavy chain variable domain or
all or a portion of the light chain variable domain of an antibody.
In certain embodiments, a single-domain antibody is a human
single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g.,
U.S. Pat. No. 6,248,516 B1). In one embodiment, a single-domain
antibody consists of all or a portion of the heavy chain variable
domain of an antibody.
[0178] (viii) Antibody Variants
[0179] In some embodiments, amino acid sequence modification(s) of
the antibodies described herein are contemplated. For example, it
may be desirable to improve the binding affinity and/or other
biological properties of the antibody. Amino acid sequence variants
of the antibody may be prepared by introducing appropriate changes
into the nucleotide sequence encoding the antibody, or by peptide
synthesis. Such modifications include, for example, deletions from,
and/or insertions into and/or substitutions of, residues within the
amino acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics. The amino acid alterations may be introduced in
the subject antibody amino acid sequence at the time that sequence
is made.
[0180] (ix) Antibody Derivatives
[0181] The antibodies in the formulations and compositions of the
invention can be further modified to contain additional
nonproteinaceous moieties that are known in the art and readily
available. In certain embodiments, the moieties suitable for
derivatization of the antibody are water soluble polymers.
Non-limiting examples of water soluble polymers include, but are
not limited to, polyethylene glycol (PEG), copolymers of ethylene
glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl
alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,
poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer,
polyaminoacids (either homopolymers or random copolymers), and
dextran or poly(n-vinyl pyrrolidone)polyethylene glycol,
propropylene glycol homopolymers, prolypropylene oxide/ethylene
oxide co-polymers, polyoxyethylated polyols (e.g., glycerol),
polyvinyl alcohol, and mixtures thereof. Polyethylene glycol
propionaldehyde may have advantages in manufacturing due to its
stability in water. The polymer may be of any molecular weight, and
may be branched or unbranched. The number of polymers attached to
the antibody may vary, and if more than one polymer are attached,
they can be the same or different molecules. In general, the number
and/or type of polymers used for derivatization can be determined
based on considerations including, but not limited to, the
particular properties or functions of the antibody to be improved,
whether the antibody derivative will be used in a therapy under
defined conditions, etc.
[0182] (x) Vectors, Host Cells, and Recombinant Methods
[0183] Antibodies may also be produced using recombinant methods.
For recombinant production of an anti-antigen antibody, nucleic
acid encoding the antibody is isolated and inserted into a
replicable vector for further cloning (amplification of the DNA) or
for expression. DNA encoding the antibody may be readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the antibody). Many
vectors are available. The vector components generally include, but
are not limited to, one or more of the following: a signal
sequence, an origin of replication, one or more marker genes, an
enhancer element, a promoter, and a transcription termination
sequence.
[0184] (a) Signal Sequence Component
[0185] An antibody in the formulations and compositions described
herein may be produced recombinantly not only directly, but also as
a fusion polypeptide with a heterologous polypeptide, which is
preferably a signal sequence or other polypeptide having a specific
cleavage site at the N-terminus of the mature protein or
polypeptide. The heterologous signal sequence selected preferably
is one that is recognized and processed (e.g., cleaved by a signal
peptidase) by the host cell. For prokaryotic host cells that do not
recognize and process a native antibody signal sequence, the signal
sequence is substituted by a prokaryotic signal sequence selected,
for example, from the group of the alkaline phosphatase,
penicillinase, lpp, or heat-stable enterotoxin II leaders. For
yeast secretion the native signal sequence may be substituted by,
e.g., the yeast invertase leader, a factor leader (including
Saccharomyces and Kluyveromyces a-factor leaders), or acid
phosphatase leader, the C. albicans glucoamylase leader, or the
signal described in WO 90/13646. In mammalian cell expression,
mammalian signal sequences as well as viral secretory leaders, for
example, the herpes simplex gD signal, are available.
[0186] (b) Origin of Replication
[0187] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Generally, in cloning vectors this sequence is
one that enables the vector to replicate independently of the host
chromosomal DNA, and includes origins of replication or
autonomously replicating sequences. Such sequences are well known
for a variety of bacteria, yeast, and viruses. The origin of
replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria, the plasmid origin is suitable for yeast,
and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)
are useful for cloning vectors in mammalian cells. Generally, the
origin of replication component is not needed for mammalian
expression vectors (the SV40 origin may typically be used only
because it contains the early promoter.
[0188] (c) Selection Gene Component
[0189] Expression and cloning vectors may contain a selection gene,
also termed a selectable marker. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, or (c) supply critical
nutrients not available from complex media, e.g., the gene encoding
D-alanine racemase for Bacilli.
[0190] One example of a selection scheme utilizes a drug to arrest
growth of a host cell. Those cells that are successfully
transformed with a heterologous gene produce a protein conferring
drug resistance and thus survive the selection regimen. Examples of
such dominant selection use the drugs neomycin, mycophenolic acid
and hygromycin.
[0191] Another example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up antibody-encoding nucleic acid, such as DHFR, glutamine
synthetase (GS), thymidine kinase, metallothionein-I and -II,
preferably primate metallothionein genes, adenosine deaminase,
ornithine decarboxylase, etc.
[0192] For example, cells transformed with the DHFR gene are
identified by culturing the transformants in a culture medium
containing methotrexate (Mtx), a competitive antagonist of DHFR.
Under these conditions, the DHFR gene is amplified along with any
other co-transformed nucleic acid. A Chinese hamster ovary (CHO)
cell line deficient in endogenous DHFR activity (e.g., ATCC
CRL-9096) may be used.
[0193] Alternatively, cells transformed with the GS gene are
identified by culturing the transformants in a culture medium
containing L-methionine sulfoximine (Msx), an inhibitor of GS.
Under these conditions, the GS gene is amplified along with any
other co-transformed nucleic acid. The GS selection/amplification
system may be used in combination with the DHFR
selection/amplification system described above.
[0194] Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with DNA
sequences encoding an antibody of interest, wild-type DHFR gene,
and another selectable marker such as aminoglycoside
3'-phosphotransferase (APH) can be selected by cell growth in
medium containing a selection agent for the selectable marker such
as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or
G418. See U.S. Pat. No. 4,965,199.
[0195] A suitable selection gene for use in yeast is the trp1 gene
present in the yeast plasmid YRp7 (Stinchcomb et al., Nature,
282:39 (1979)). The trp1 gene provides a selection marker for a
mutant strain of yeast lacking the ability to grow in tryptophan,
for example, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12
(1977). The presence of the trp1 lesion in the yeast host cell
genome then provides an effective environment for detecting
transformation by growth in the absence of tryptophan. Similarly,
Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are
complemented by known plasmids bearing the Leu2 gene.
[0196] In addition, vectors derived from the 1.6 .mu.m circular
plasmid pKD1 can be used for transformation of Kluyveromyces
yeasts. Alternatively, an expression system for large-scale
production of recombinant calf chymosin was reported for K. lactis.
Van den Berg, Bio/Technology, 8:135 (1990). Stable multi-copy
expression vectors for secretion of mature recombinant human serum
albumin by industrial strains of Kluyveromyces have also been
disclosed. Fleer et al., Bio/Technology, 9:968-975 (1991).
[0197] (d) Promoter Component
[0198] Expression and cloning vectors generally contain a promoter
that is recognized by the host organism and is operably linked to
nucleic acid encoding an antibody. Promoters suitable for use with
prokaryotic hosts include the phoA promoter, .beta.-lactamase and
lactose promoter systems, alkaline phosphatase promoter, a
tryptophan (trp) promoter system, and hybrid promoters such as the
tac promoter. However, other known bacterial promoters are
suitable. Promoters for use in bacterial systems also will contain
a Shine-Dalgarno (S.D.) sequence operably linked to the DNA
encoding an antibody.
[0199] Promoter sequences are known for eukaryotes. Virtually all
eukaryotic genes have an AT-rich region located approximately 25 to
30 bases upstream from the site where transcription is initiated.
Another sequence found 70 to 80 bases upstream from the start of
transcription of many genes is a CNCAAT region where N may be any
nucleotide. At the 3' end of most eukaryotic genes is an AATAAA
sequence that may be the signal for addition of the poly A tail to
the 3' end of the coding sequence. All of these sequences are
suitably inserted into eukaryotic expression vectors.
[0200] Examples of suitable promoter sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase or other
glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate
dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0201] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP
73,657. Yeast enhancers also are advantageously used with yeast
promoters.
[0202] Antibody transcription from vectors in mammalian host cells
can be controlled, for example, by promoters obtained from the
genomes of viruses such as polyoma virus, fowlpox virus, adenovirus
(such as Adenovirus 2), bovine papilloma virus, avian sarcoma
virus, cytomegalovirus, a retrovirus, hepatitis-B virus, Simian
Virus 40 (SV40), or from heterologous mammalian promoters, e.g.,
the actin promoter or an immunoglobulin promoter, from heat-shock
promoters, provided such promoters are compatible with the host
cell systems.
[0203] The early and late promoters of the SV40 virus are
conveniently obtained as an SV40 restriction fragment that also
contains the SV40 viral origin of replication. The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment. A system for expressing DNA in
mammalian hosts using the bovine papilloma virus as a vector is
disclosed in U.S. Pat. No. 4,419,446. A modification of this system
is described in U.S. Pat. No. 4,601,978. See also Reyes et al.,
Nature 297:598-601 (1982) on expression of human .beta.-interferon
cDNA in mouse cells under the control of a thymidine kinase
promoter from herpes simplex virus. Alternatively, the Rous Sarcoma
Virus long terminal repeat can be used as the promoter.
[0204] (e) Enhancer Element Component
[0205] Transcription of a DNA encoding an antibody by higher
eukaryotes is often increased by inserting an enhancer sequence
into the vector. Many enhancer sequences are now known from
mammalian genes (globin, elastase, albumin, .alpha.-fetoprotein,
and insulin). Typically, however, one will use an enhancer from a
eukaryotic cell virus. Examples include the SV40 enhancer on the
late side of the replication origin (bp 100-270), the
cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late side of the replication origin, and adenovirus enhancers.
See also Yaniv, Nature 297:17-18 (1982) on enhancing elements for
activation of eukaryotic promoters. The enhancer may be spliced
into the vector at a position 5' or 3' to the antibody-encoding
sequence, but is preferably located at a site 5' from the
promoter.
[0206] (f) Transcription Termination Component
[0207] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3',
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding
antibody. One useful transcription termination component is the
bovine growth hormone polyadenylation region. See WO94/11026 and
the expression vector disclosed therein.
[0208] (g) Selection and Transformation of Host Cells
[0209] Suitable host cells for cloning or expressing the DNA in the
vectors herein are the prokaryote, yeast, or higher eukaryote cells
described above. Suitable prokaryotes for this purpose include
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as Escherichia, e.g., E. coli,
Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. One preferred E. coli cloning host is E. coli 294
(ATCC 31,446), although other strains such as E. coli B, E. coli
X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.
These examples are illustrative rather than limiting.
[0210] Full length antibody, antibody fusion proteins, and antibody
fragments can be produced in bacteria, in particular when
glycosylation and Fc effector function are not needed, such as when
the therapeutic antibody is conjugated to a cytotoxic agent (e.g.,
a toxin) that by itself shows effectiveness in tumor cell
destruction. Full length antibodies have greater half-life in
circulation. Production in E. coli is faster and more cost
efficient. For expression of antibody fragments and polypeptides in
bacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et. al.), U.S.
Pat. No. 5,789,199 (Joly et al.), U.S. Pat. No. 5,840,523 (Simmons
et al.), which describes translation initiation region (TIR) and
signal sequences for optimizing expression and secretion. See also
Charlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed.,
Humana Press, Totowa, N.J., 2003), pp. 245-254, describing
expression of antibody fragments in E. coli. After expression, the
antibody may be isolated from the E. coli cell paste in a soluble
fraction and can be purified through, e.g., a protein A or G column
depending on the isotype. Final purification can be carried out
similar to the process for purifying antibody expressed e.g., in
CHO cells.
[0211] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for antibody-encoding vectors. Saccharomyces cerevisiae, or common
baker's yeast, is the most commonly used among lower eukaryotic
host microorganisms. However, a number of other genera, species,
and strains are commonly available and useful herein, such as
Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K.
lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.
wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum
(ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP
402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia
(EP 244,234); Neurospora crassa; Schwanniomyces such as
Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,
Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such
as A. nidulans and A. niger. For a review discussing the use of
yeasts and filamentous fungi for the production of therapeutic
proteins, see, e.g., Gerngross, Nat. Biotech. 22:1409-1414
(2004).
[0212] Certain fungi and yeast strains may be selected in which
glycosylation pathways have been "humanized," resulting in the
production of an antibody with a partially or fully human
glycosylation pattern. See, e.g., Li et al., Nat. Biotech.
24:210-215 (2006) (describing humanization of the glycosylation
pathway in Pichia pastoris); and Gerngross et al., supra.
[0213] Suitable host cells for the expression of glycosylated
antibody are also derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains and
variants and corresponding permissive insect host cells from hosts
such as Spodoptera frugiperda (caterpillar), Aedes aegypti
(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster
(fruitfly), and Bombyx mori have been identified. A variety of
viral strains for transfection are publicly available, e.g., the
L-1 variant of Autographa californica NPV and the Bm-5 strain of
Bombyx mori NPV, and such viruses may be used as the virus herein
according to the invention, particularly for transfection of
Spodoptera frugiperda cells.
[0214] Plant cell cultures of cotton, corn, potato, soybean,
petunia, tomato, duckweed (Leninaceae), alfalfa (M. truncatula),
and tobacco can also be utilized as hosts. See, e.g., U.S. Pat.
Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429
(describing PLANTIBODIES.TM. technology for producing antibodies in
transgenic plants).
[0215] Vertebrate cells may be used as hosts, and propagation of
vertebrate cells in culture (tissue culture) has become a routine
procedure. Examples of useful mammalian host cell lines are monkey
kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human
embryonic kidney line (293 or 293 cells subcloned for growth in
suspension culture, Graham et al., J. Gen Virol. 36:59 (1977));
baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells
(TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells
(CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC
CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2);
canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells
(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75);
human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT
060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.
Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human
hepatoma line (Hep G2). Other useful mammalian host cell lines
include Chinese hamster ovary (CHO) cells, including DHFR.sup.- CHO
cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980));
and myeloma cell lines such as NSO and Sp2/0. For a review of
certain mammalian host cell lines suitable for antibody production,
see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248
(B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp.
255-268.
[0216] Host cells are transformed with the above-described
expression or cloning vectors for antibody production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.
[0217] (b) Culturing the Host Cells
[0218] The host cells used to produce an antibody may be cultured
in a variety of media. Commercially available media such as Ham's
F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640
(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are
suitable for culturing the host cells. In addition, any of the
media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et
al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704;
4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO
87/00195; or U.S. Pat. Re. 30,985 may be used as culture media for
the host cells. Any of these media may be supplemented as necessary
with hormones and/or other growth factors (such as insulin,
transferrin, or epidermal growth factor), salts (such as sodium
chloride, calcium, magnesium, and phosphate), buffers (such as
HEPES), nucleotides (such as adenosine and thymidine), antibiotics
(such as GENTAMYCIN.TM. drug), trace elements (defined as inorganic
compounds usually present at final concentrations in the micromolar
range), and glucose or an equivalent energy source. Any other
necessary supplements may also be included at appropriate
concentrations that would be known to those skilled in the art. The
culture conditions, such as temperature, pH, and the like, are
those previously used with the host cell selected for expression,
and will be apparent to the ordinarily skilled artisan.
[0219] (xi) Purification of Antibody
[0220] When using recombinant techniques, the antibody can be
produced intracellularly, in the periplasmic space, or directly
secreted into the medium. If the antibody is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed fragments, are removed, for example, by
centrifugation or ultrafiltration. Carter et al., Bio/Technology
10:163-167 (1992) describe a procedure for isolating antibodies
which are secreted to the periplasmic space of E. coli. Briefly,
cell paste is thawed in the presence of sodium acetate (pH 3.5),
EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
Cell debris can be removed by centrifugation. Where the antibody is
secreted into the medium, supernatants from such expression systems
are generally first concentrated using a commercially available
protein concentration filter, for example, an Amicon or Millipore
Pellicon ultrafiltration unit. A protease inhibitor such as PMSF
may be included in any of the foregoing steps to inhibit
proteolysis and antibiotics may be included to prevent the growth
of adventitious contaminants.
[0221] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography,
hydrophobic interaction chromatography, gel electrophoresis,
dialysis, and affinity chromatography, with affinity chromatography
being among one of the typically preferred purification steps. The
suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human .gamma.1, .gamma.2, or .gamma.4 heavy chains
(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human .gamma.3 (Guss et
al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity
ligand is attached is most often agarose, but other matrices are
available. Mechanically stable matrices such as controlled pore
glass or poly(styrenedivinyl)benzene allow for faster flow rates
and shorter processing times than can be achieved with agarose.
Where the antibody comprises a C.sub.H3 domain, the Bakerbond
ABX.TM. resin (J. T. Baker, Phillipsburg, N.J.) is useful for
purification. Other techniques for protein purification such as
fractionation on an ion-exchange column, ethanol precipitation,
Reverse Phase HPLC, chromatography on silica, chromatography on
heparin SEPHAROSE.TM. chromatography on an anion or cation exchange
resin (such as a polyaspartic acid column), chromatofocusing,
SDS-PAGE, and ammonium sulfate precipitation are also available
depending on the antibody to be recovered.
[0222] In general, various methodologies for preparing antibodies
for use in research, testing, and clinical are well-established in
the art, consistent with the above-described methodologies and/or
as deemed appropriate by one skilled in the art for a particular
antibody of interest.
[0223] B. Selecting Biologically Active Antibodies
[0224] Antibodies produced as described above may be subjected to
one or more "biological activity" assays to select an antibody with
beneficial properties from a therapeutic perspective. The antibody
may be screened for its ability to bind the antigen against which
it was raised. For example, for an anti-DR5 antibody (e.g.,
drozitumab), the antigen binding properties of the antibody can be
evaluated in an assay that detects the ability to bind to a death
receptor 5 (DR5).
[0225] In another embodiment, the affinity of the antibody may be
determined by saturation binding; ELISA; and/or competition assays
(e.g. RIA's), for example.
[0226] Also, the antibody may be subjected to other biological
activity assays, e.g., in order to evaluate its effectiveness as a
therapeutic. Such assays are known in the art and depend on the
target antigen and intended use for the antibody.
[0227] To screen for antibodies which bind to a particular epitope
on the antigen of interest, a routine cross-blocking assay such as
that described in Antibodies, A Laboratory Manual, Cold Spring
Harbor Laboratory, Ed Harlow and David Lane (1988), can be
performed. Alternatively, epitope mapping, e.g. as described in
Champe et al., J. Biol. Chem. 270:1388-1394 (1995), can be
performed to determine whether the antibody binds an epitope of
interest.
[0228] C. Preparation of the Formulations
[0229] Provided herein are methods of preparing a formulation
comprising a protein and a compound which prevents oxidation of the
protein in the formulation. The formulation may be prepared by
mixing the protein having the desired degree of purity with a
compound which prevents oxidation of the protein in the formulation
(such as a liquid formulation). In certain embodiments, the protein
to be formulated has not been subjected to prior lyophilization and
the formulation of interest herein is an aqueous formulation. In
some embodiments, the protein is a therapeutic protein. In certain
embodiments, the protein is an antibody. In further embodiments,
the antibody is a polyclonal antibody, a monoclonal antibody, a
humanized antibody, a human antibody, a chimeric antibody, or
antibody fragment. In certain embodiments, the antibody is a full
length antibody. In one embodiment, the antibody in the formulation
is an antibody fragment, such as an F(ab').sub.2, in which case
problems that may not occur for the full length antibody (such as
clipping of the antibody to Fab) may need to be addressed. The
therapeutically effective amount of protein present in the
formulation is determined by taking into account the desired dose
volumes and mode(s) of administration, for example. From about 1
mg/mL to about 250 mg/mL, from about 10 mg/mL to about 250 mg/mL,
from about 15 mg/mL to about 225 mg/mL, from about 20 mg/mL to
about 200 mg/mL, from about 25 mg/mL to about 175 mg/mL, from about
25 mg/mL to about 150 mg/mL, from about 25 mg/mL to about 100
mg/mL, from about 30 mg/mL to about 100 mg/mL or from about 45
mg/mL to about 55 mg/mL is an exemplary protein concentration in
the formulation. In some embodiments, the protein described herein
is susceptible to oxidation. In some embodiments, one or more of
the amino acids selected from the group consisting of methionine,
cysteine, histidine, tryptophan, and tyrosine in the protein is
susceptible to oxidation. In some embodiments, tryptophan in the
protein is susceptible to oxidation. In some embodiments,
methionine in the protein is susceptible to oxidation. In some
embodiments, an antibody provided herein is susceptible to
oxidation in the Fab portion and/or the Fc portion of the antibody.
In some embodiments, an antibody provided herein is susceptible to
oxidation at a tryptophan amino acid in the Fab portion of the
antibody. In a further embodiment, the tryptophan amino acid
susceptible to oxidation is in a CDR of the antibody. In some
embodiments, an antibody provided herein is susceptible to
oxidation at a methionine amino acid in the Fc portion of the
antibody.
[0230] The formulations provided herein comprise a protein and a
compound which prevents oxidation of the protein in the
formulation, wherein the compound is of formula:
##STR00011## [0231] wherein R.sup.2 is selected from hydrogen,
hydroxyl, --COOH, and --CH.sub.2COOH; [0232] R.sup.3 is selected
from hydrogen, hydroxyl, --COOH, --CH.sub.2COOH, and
--CH.sub.2CHR.sup.3a(NH.sub.2); wherein R.sup.3a is COOH or
hydrogen; R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are independently
selected from hydrogen and hydroxyl; provided that one of R.sup.2,
R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 is hydroxyl; or a
pharmaceutically acceptable salt thereof.
[0233] In some embodiments, the compound is a compound of
formula:
##STR00012## [0234] wherein R.sup.2 and R.sup.3 are independently
selected from hydrogen, hydroxyl, --COOH, and --CH.sub.2COOH; and
R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are independently selected
from hydrogen and hydroxyl; provided that one of R.sup.2, R.sup.3,
R.sup.4, R.sup.5, R.sup.6, and R.sup.7 is hydroxyl; or a
pharmaceutically acceptable salt thereof.
[0235] In some embodiments, the compound is a compound of
formula:
##STR00013## [0236] wherein R.sup.3a is COOH or hydrogen; R.sup.2,
R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are independently selected
from hydrogen and hydroxyl, provided that one of R.sup.2, R.sup.4,
R.sup.5, R.sup.6, and R.sup.7 is hydroxyl; or a pharmaceutically
acceptable salt thereof.
[0237] In some embodiments, R.sup.4, R.sup.5 or R.sup.7 in any of
the formula above is hydroxyl. In a further embodiment, the
compound is selected from the group consisting of
5-hydroxy-tryptophan, 5-hydroxy indole, 7-hydroxy indole, and
serotonin. In a further embodiment, the compound is selected from
the group consisting of 4-hydroxy indole, 5-hydroxy indole-3-acetic
acid, and 7-hydroxy indole-2-carboxylic acid. In some embodiments,
the formulation is a liquid formulation. In some embodiments, the
compound in the formulation is at a concentration from about 0.3 mM
to about 10 mM, or up to the highest concentration that the
compound is soluble in the formulation. In certain embodiments, the
compound in the formulation is at a concentration from about 0.3 mM
to about 9 mM, from about 0.3 mM to about 8 mM, from about 0.3 mM
to about 7 mM, from about 0.3 mM to about 6 mM, from about 0.3 mM
to about 5 mM, from about 0.3 mM to about 4 mM, from about 0.3 mM
to about 3 mM, from about 0.3 mM to about 2 mM, from about 0.5 mM
to about 2 mM, from about 0.6 mM to about 1.5 mM, or from about 0.8
mM to about 1.25 mM. In some embodiments, the compound in the
formulation is about 1 mM. In some embodiments, the compound
prevents oxidation of one or more amino acids in the protein. In
some embodiments, the compound prevents oxidation of one or more
amino acids in the protein selected from group consisting of
tryptophan, methionine, tyrosine, histidine, and/or cysteine. In
some embodiments, the compound prevents oxidation of the protein by
a reactive oxygen species (ROS). In a further embodiment, the
reactive oxygen species is selected from the group consisting of a
singlet oxygen, a superoxide (O.sub.2--), an alkoxyl radical, a
peroxyl radical, a hydrogen peroxide (H.sub.2O.sub.2), a dihydrogen
trioxide (H.sub.2O.sub.3), a hydrotrioxy radical (HO.sub.3.cndot.),
ozone (O.sub.3), a hydroxyl radical, and an alkyl peroxide. In a
further embodiment, the compound prevents oxidation of one or more
amino acids in the Fab portion of an antibody. In another further
embodiment, the compound prevents oxidation of one or more amino
acids in the Fc portion of an antibody.
[0238] In some embodiments, the formulation (such as a liquid
formulation) further comprises one or more excipients selected from
the group consisting of a stabilizer, a buffer, a surfactant, and a
tonicity agent. In some embodiments, the formulation is prepared in
a pH-buffered solution. The buffer of this invention has a pH in
the range from about 4.5 to about 7.0. In certain embodiments the
pH is in the range from pH 4.5 to 6.5, in the range from pH 4.5 to
6.0, in the range from pH 4.5 to 5.5, in the range from pH 4.5 to
5.0, in the range from pH 5.0 to 7.0, in the range from pH 5.5 to
7.0, in the range from pH 5.7 to 6.8, in the range from pH 5.8 to
6.5, in the range from pH 5.9 to 6.5, in the range from pH 6.0 to
6.5, or in the range from pH 6.2 to 6.5. In certain embodiments of
the invention, the formulation has a pH of 6.2 or about 6.2. In
certain embodiments of the invention, the formulation has a pH of
6.0 or about 6.0. Examples of buffers that will control the pH
within this range include organic and inorganic acids and salts
thereof. For example, acetate (e.g., histidine acetate, arginine
acetate, sodium acetate), succinate (e.g., histidine succinate.
arginine succinate, sodium succinate), gluconate, phosphate,
fumarate, oxalate, lactate, citrate, and combinations thereof. The
buffer concentration can be from about 1 mM to about 600 mM,
depending, for example, on the buffer and the desired isotonicity
of the formulation. In certain embodiments, the formulation
comprises a histidine buffer (e.g., in the concentration front
about 5 mM to 100 mM). Examples of histidine buffers include
histidine chloride, histidine acetate, histidine phosphate,
histidine sulfate, histidine succinate, etc. In certain
embodiments, the formulation comprises histidine and arginine
(e.g., histidine chloride-arginine chloride, histidine
acetate-arginine acetate, histidine phosphate-arginine phosphate,
histidine sulfate-arginine sulfate, histidine succinate-arginine
succinate, etc.). In certain embodiments, the formulation comprises
histidine in the concentration from about 5 mM to 100 mM and the
arginine is in the concentration of 50 mM to 500 mM. In one
embodiment, the formulation comprises histidine acetate (e.g.,
about 20 mM)-arginine acetate (e.g., about 150 mM). In certain
embodiments, the formulation comprises histidine succinate (e.g.,
about 20 mM)-arginine succinate (e.g., about 150 mM). In certain
embodiments, histidine in the formulation from about 10 mM, to
about, 35 mM, about 10 mM to about 30 mM, about 10 mM to about 25
mM, about 10 mM to about 20 mM, about 10 mM to about 15 mM, about
15 mM to about 35 mM, about 20 mM to about 35 mM, about 20 mM to
about 30 mM or about 20 mM to about 25 mM. In further embodiments,
the arginine in the formulation is from about 50 mM to about 500 mM
(e.g., about 100 mM, about 150 mM, or about 200 mM).
[0239] The formulation (such as a liquid formulation) of the
invention can further comprise a saccharide, such as a disaccharide
(e.g., trehalose or sucrose). A "saccharide" as used herein
includes the general composition (CH.sub.2O)n and derivatives
thereof, including monosaccharides, disaccharides, trisaccharides,
polysaccharides, sugar alcohols, reducing sugars, nonreducing
sugars, etc. Examples of saccharides herein include glucose,
sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin,
dextran, erythritol, glycerol, arabitol, sylitol, sorbitol,
mannitol, mellibiose, melezitose, raffinose, mannotriose,
stachyose, maltose, lactulose, maltulose, glucitol, maltitol,
lactitol, iso-maltulose, etc.
[0240] A surfactant can optionally be added to the formulation
(such as a liquid formulation). Exemplary surfactants include
nonionic surfactants such as polysorbates (e.g. polysorbates 20,
80, etc.) or poloxamers (e.g. poloxamer 188, etc.). The amount of
surfactant added is such that it reduces aggregation of the
formulated antibody and/or minimizes the formation of particulates
in the formulation and/or reduces adsorption. For example, the
surfactant may be present in the formulation in an amount from
about 0.001% to about 0.5%, from about 0.005% to about 0.2%, from
about 0.01% to about 0.1%, or from about 0.02% to about 0.06%, or
about 0.03% to about 0.05%. In certain embodiments, the surfactant
is present in the formulation in an amount of 0.04% or about 0.04%.
In certain embodiments, the surfactant is present in the
formulation in an amount of 0.02% or about 0.02%. In one
embodiment, the formulation does not comprise a surfactant.
[0241] In one embodiment, the formulation contains the
above-identified agents (e.g., antibody, buffer, saccharide, and/or
surfactant) and is essentially free of one or more preservatives,
such as benzyl alcohol, phenol, m-cresol, chlorobutanol and
benzethonium Cl. In another embodiment, a preservative may be
included in the formulation, particularly where the formulation is
a multidose formulation. The concentration of preservative may be
in the range from about 0.1% to about 2%, preferably from about
0.5% to about 1%. One or more other pharmaceutically acceptable
carriers, excipients or stabilizers such as those described in
Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980) may be included in the formulation provided that they do not
adversely affect the desired characteristics of the formulation.
Exemplary pharmaceutically acceptable excipients herein further
include insterstitial drug dispersion agents such as soluble
neutral-active hyaluronidase glycoproteins (sHASEGP), for example,
human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20
(HYLENEX.RTM., Baxter International, Inc.). Certain exemplary
sHASEGPs and methods of use, including rHuPH20, are described in US
Patent Publication Nos. 2005/0260186 and 2006/0104968. In one
aspect, a sHASEGP is combined with one or more additional
glycosaminoglycanases such as chondroitinases.
[0242] The formulation may further comprise metal ion chelators.
Metal ion chelators are well known by those of skill in the art and
include, but are not necessarily limited to aminopolycarboxylates,
EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene
glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid), NTA
(nitrilotriacetic acid), EDDS (ethylene diamine disuccinate), PDTA
(1,3-propylenediaminetetraacetic acid), DTPA
(diethylenetriaminepentaacetic acid), ADA (beta-alaninediacetic
acid), MGCA (methylglycinediacetic acid), etc. Additionally, some
embodiments herein comprise phosphonates/phosphonic acid
chelators.
[0243] Tonicity agents are present to adjust or maintain the
tonicity of liquid in a composition. When used with large, charged
biomolecules such as proteins and antibodies, they may also serve
as "stabilizers" because they can interact with the charged groups
of the amino acid side chains, thereby lessening the potential for
inter- and intra-molecular interactions. Tonicity agents can be
present in any amount between 0.1% to 25% by weight, or more
preferably between 1% to 5% by weight, taking into account the
relative amounts of the other ingredients. Preferred tonicity
agents include polyhydric sugar alcohols, preferably trihydric or
higher sugar alcohols, such as glycerin, erythritol, arabitol,
xylitol, sorbitol and mannitol.
[0244] The formulation herein may also contain more than one
protein or a small molecule drug as necessary for the particular
indication being treated, preferably those with complementary
activities that do not adversely affect the other protein. For
example, where the antibody is anti-DR5 (e.g., drozitumab), it may
be combined with another agent (e.g., a chemotherapeutic agent, and
anti-neoplastic agent).
[0245] In some embodiments, the formulation is for in vivo
administration. In some embodiments, the formulation is sterile.
The formulation may be rendered sterile by filtration through
sterile filtration membranes. The therapeutic formulations herein
generally are placed into a container having a sterile access port,
for example, an intravenous solution bag or vial having a stopper
pierceable by a hypodermic injection needle. The route of
administration is in accordance with known and accepted methods,
such as by single or multiple bolus or infusion over a long period
of time in a suitable manner, e.g., injection or infusion by
subcutaneous, intravenous, intraperitoneal, intramuscular,
intraarterial, intralesional or intraarticular routes, topical
administration, inhalation or by sustained release or
extended-release means.
[0246] The formulation of the invention may be stored in liquid or
non-liquid formulation (e.g., lyophilized). The lyophilized
formulation may be reconstituted before administration. In some
embodiments, the concentrations of proteins, compounds and other
excipients described herein refer to concentrations in
reconstituted formulations. In some embodiments, the formulation is
stable upon storage. In some embodiments, the protein in the liquid
formulation is stable upon storage at about 0 to 5.degree. C. for
at least about 12 months, at least about 18 months, at least about
21 months, or at least about 24 months (or at least about 52
weeks). In some embodiments, the physical stability, chemical
stability, or biological activity of the protein in the formulation
is evaluated or measured. Any methods known the art may be used to
evaluate the stability and biological activity. In some
embodiments, the stability is measured by oxidation of the protein
in the formulation (such as a liquid formulation) after storage.
Stability can be tested by evaluating physical stability, chemical
stability, and/or biological activity of the antibody in the
formulation around the time of formulation as well as following
storage. Physical and/or stability can be evaluated qualitatively
and/or quantitatively in a variety of different ways, including
evaluation of aggregate formation (for example using size exclusion
chromatography, by measuring turbidity, and/or by visual
inspection); by assessing charge heterogeneity using cation
exchange chromatography or capillary zone electrophoresis;
amino-terminal or carboxy-terminal sequence analysis; mass
spectrometric analysis; SDS-PAGE analysis to compare reduced and
intact antibody; peptide map (for example tryptic or LYS-C)
analysis; evaluating biological activity or antigen binding
function of the antibody; etc. Instability may result in
aggregation, deamidation (e.g. Asn deamidation), oxidation (e.g.
Trp oxidation), isomerization (e.g. Asp isomeriation),
clipping/hydrolysis/fragmentation (e.g. hinge region
fragmentation), succinimide formation, unpaired cysteine(s),
N-terminal extension, C-terminal processing, glycosylation
differences, etc. In some embodiments, the oxidation in a protein
is determined using one or more of RP-HPLC, LC/MS, or tryptic
peptide mapping. In some embodiments, the oxidation in an antibody
is determined as a percentage using one or more of RP-HPLC, LC/MS,
or tryptic peptide mapping and the formula of:
% Fab Oxidation = 100 Oxidized Fab Peak Area Fab Peak Area +
Oxidized Fab Peak Area ##EQU00001## % Fc Oxidation = 100 Oxidized
Fc Peak Area Fc Peak Area + Oxidized Fc Peak Area
##EQU00001.2##
[0247] The formulations to be used for in vivo administration
should be sterile. This is readily accomplished by filtration
through sterile filtration membranes, prior to, or following,
preparation of the formulation.
[0248] Also provided herein are methods of making a protein
formulation or preventing oxidation of a protein in a protein
formulation comprising adding an amount of a compound that prevents
oxidation of a protein to the protein formulation, wherein the
compound is of formula:
##STR00014## [0249] wherein R.sup.2 is selected from hydrogen,
hydroxyl, --COOH, and --CH.sub.2COOH; [0250] R.sup.3 is selected
from hydrogen, hydroxyl, --COOH, --CH.sub.2COOH, and
--CH.sub.2CHR.sup.3a(NH.sub.2); wherein R.sup.3a is COOH or
hydrogen; R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are independently
selected from hydrogen and hydroxyl; provided that one of R.sup.2,
R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 is hydroxyl; or a
pharmaceutically acceptable salt thereof.
[0251] In some embodiments, the compound is a compound of
formula:
##STR00015## [0252] wherein R.sup.2 and R.sup.3 are independently
selected from hydrogen, hydroxyl, --COOH, and --CH.sub.2COOH; and
R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are independently selected
from hydrogen and hydroxyl; provided that one of R.sup.2, R.sup.3,
R.sup.4, R.sup.5, R.sup.6, and R.sup.7 is hydroxyl; or a
pharmaceutically acceptable salt thereof.
[0253] In some embodiments, the compound is a compound of
formula:
##STR00016## [0254] wherein R.sup.3a is COOH or hydrogen; R.sup.2,
R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are independently selected
from hydrogen and hydroxyl, provided that one of R.sup.2, R.sup.4,
R.sup.5, R.sup.6, and R.sup.7 is hydroxyl; or a pharmaceutically
acceptable salt thereof.
[0255] In some embodiments, R.sup.4, R.sup.5 or R.sup.7 is
hydroxyl. In some embodiments, the compound is selected from the
group consisting of 5-hydroxy-tryptophan, 5-hydroxy indole,
7-hydroxy indole, and serotonin. In certain embodiments, the
formulation comprises an antibody. The amount of the compound that
prevents oxidation of the protein as provided herein is from about
0.3 mM to about 10 mM or any of the amounts disclosed herein.
III. Administration of Protein Formulations
[0256] The formulation (such as a liquid formulation) is
administered to a mammal in need of treatment with the protein
(e.g., an antibody), preferably a human, in accord with known
methods, such as intravenous administration as a bolus or by
continuous infusion over a period of time, by intramuscular,
intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,
intrasynovial, intrathecal, oral, topical, or inhalation routes. In
one embodiment, the liquid formulation is administered to the
mammal by intravenous administration. For such purposes, the
formulation may be injected using a syringe or via an IV line, for
example. In one embodiment, the liquid formulation is administered
to the mammal by subcutaneous administration.
[0257] The appropriate dosage ("therapeutically effective amount")
of the protein will depend, for example, on the condition to be
treated, the severity and course of the condition, whether the
protein is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical history and response to
the protein, the type of protein used, and the discretion of the
attending physician. The protein is suitably administered to the
patient at one time or over a series of treatments and may be
administered to the patient at any time from diagnosis onwards. The
protein may be administered as the sole treatment or in conjunction
with other drugs or therapies useful in treating the condition in
question. As used herein the term "treatment" refers to both
therapeutic treatment and prophylactic or preventative measures.
Those in need of treatment include those already with the disorder
as well as those in which the disorder is to be prevented. As used
herein a "disorder" is any condition that would benefit from
treatment including, but not limited to, chronic and acute
disorders or diseases including those pathological conditions which
predispose the mammal to the disorder in question.
[0258] In a pharmacological sense, in the context of the invention,
a "therapeutically effective amount" of a protein (e.g., an
antibody) refers to an amount effective in the prevention or
treatment of a disorder for the treatment of which the antibody is
effective. As a general proposition, the therapeutically effective
amount of the protein administered will be in the range of about
0.1 to about 50 mg/kg of patient body weight whether by one or more
administrations, with the typical range of protein used being about
0.3 to about 20 mg/kg, preferably about 0.3 to about 15 mg/kg,
administered daily, for example. However, other dosage regimens may
be useful. For example, a protein can be administered at a dose of
about 100 or 400 mg every 1, 2, 3, or 4 weeks or is administered a
dose of about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5,
6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 15.0, or 20.0 mg/kg
every 1, 2, 3, or 4 weeks. The dose may be administered as a single
dose or as multiple doses (e.g., 2 or 3 doses), such as infusions.
The progress of this therapy is easily monitored by conventional
techniques.
IV. Methods of Screening for Compounds for the Prevention of
Protein Oxidation
[0259] Also provided herein are methods of screening a compound
that prevents oxidation of a protein in a protein composition. In
some embodiments, the method comprises selecting a compound that
has lower oxidation potential and less photosensitivity as compared
to L-tryptophan, and testing the effect of the selected compound on
preventing oxidation of the protein. In some embodiments, the
photosensitivity is measured based on the amount of H.sub.2O.sub.2
produced by the compound upon light exposure. For example, a liquid
composition comprising the compound can be exposed to 250 W/m.sup.2
light for a certain amount of time and the resulting H.sub.2O.sub.2
formation is quantified. A compound with less photosensitivity
produces less H.sub.2O.sub.2 upon exposure to a certain amount of
light than a compound that has a higher photosensitivity upon
exposure to the same amount of light. In some embodiments, the
compound that produces less than about 10%, less than about 15%,
less than about 20%, less than about 25% of the amount of
H.sub.2O.sub.2 is selected. H.sub.2O.sub.2 can be produced by
oxidation of amino acid residues in a protein that are susceptible
to oxidation. In some embodiments, the oxidation potential is
measured by cyclic voltammetry.
[0260] In some embodiments, the selected compound is tested for the
effect on preventing oxidation of the protein by reactive oxygen
species generated by 2,2'-azobis(2-amidinopropane) dihydrochloride
(AAPH), light, and/or a Fenton reagent. In any of the embodiments
herein, a method described in the Examples may be used for
screening a compound that prevents oxidation of a protein in a
protein composition.
V. Articles of Manufacture
[0261] In another embodiment of the invention, an article of
manufacture is provided comprising a container which holds the
formulation of the invention and optionally provides instructions
for its use. Suitable containers include, for example, bottles,
vials and syringes. The container may be formed from a variety of
materials such as glass or plastic. An exemplary container is a
3-20 cc single use glass vial. Alternatively, for a multidose
formulation, the container may be 3-100 cc glass vial. The
container holds the formulation and the label on, or associated
with, the container may indicate directions for use. The article of
manufacture may further include other materials desirable from a
commercial and user standpoint, including other buffers, diluents,
filters, needles, syringes, and package inserts with instructions
for use.
[0262] The specification is considered to be sufficient to enable
one skilled in the art to practice the invention. Various
modifications of the invention in addition to those shown and
described herein will become apparent to those skilled in the art
from the foregoing description and fall within the scope of the
appended claims. All publications, patents, and patent applications
cited herein are hereby incorporated by reference in their entirety
for all purposes.
EXAMPLES
[0263] The invention will be more fully understood by reference to
the following examples. They should not, however, be construed as
limiting the scope of the invention. It is understood that the
examples and embodiments described herein are for illustrative
purposes only and that various modifications or changes in light
thereof will be suggested to persons skilled in the art and are to
be included within the spirit and purview of this application and
scope of the appended claims.
Example 1: The Antioxidant L-Trp Produces ROS that Oxidize
Monoclonal Antibodies in Protein Formulations
[0264] Monoclonal antibodies have been shown to produce ROS through
the antibody catalyzed water oxidation pathway (ACWOP) wherein
antibodies potentially catalyze a reaction between water and
singlet oxygen generating hydrogen peroxide (Wentworth et al.,
Science 293(5536):1806-11 (2001); Wentworth et al., Proc Natl Acad
Sci USA 97(20):10930-5 (2000)). In the ACWOP, a variety of ROS,
including superoxide anion, dihydrogen trioxide, ozone, and even
hydrotrioxy radical are generated in the pathway toward production
of hydrogen peroxide (Zhu et al., Proc Natl Acad Sci USA
101(8):2247-52 (2004)). It has been shown that surface exposed
tryptophans in a monoclonal anti-DR5 antibody, drozitumab (CAS
number 912628-39-8), also referred to herein as mAb1, act as
substrate (.sup.1O.sub.2 and O.sub.2.sup.-) generators that
facilitate ACWOP even under mild light conditions in a time and
concentration dependent manner (Sreedhara et al., Mol.
Pharmaceutics (2013)). It was demonstrated that mAb1 was
particularly susceptible to oxidation during storage under
pharmaceutically relevant conditions (Sreedhara et al., Mol.
Pharmaceutics (2013)). Oxidation was shown to be site specific and
localized to Trp53 (W53) on the heavy chain CDR (Fab) as evaluated
by tryptic peptide mapping. Additionally, a reverse-phase HPLC
assay was used to measure the total oxidation in the HC Fab and Fc
regions of mAb1 via a papain digestion, DTT reduction, and
reverse-phase separation. Peaks from RP-HPLC were identified using
LC/MS and showed a strong correlation with results of the tryptic
peptide map, indicating that the RP method could be used as a
surrogate for detection of W53 (i.e. % Fab) oxidation. In the RP
papain digest method, Fab and Fc oxidation peaks eluted before
their respective main peaks, allowing the quantification of % Fab
and % Fc oxidation in relation to their total peak areas. The study
further showed that hydrogen peroxide could serve as a surrogate
for a number of ROS, including superoxide and singlet oxygen.
[0265] To determine if limited light exposure can be used as an
accelerated stress model to study protein oxidation, the same human
monoclonal IgG1 antibody (mAb1) was used to screen and evaluate
potential antioxidants. L-tryptophan (L-Trp), an antioxidant used
in protein formulations, has been recently shown to be
photosensitive (Igarashi et al., Anal Sci 23(8):943-8 (2007)) and
to have the ability to produce H.sub.2O.sub.2 upon light exposure.
The sensitivity of mAb1 to L-Trp under light stress was evaluated,
with and without the addition of L-methionine (L-Met) as a
potential antioxidant. mAb1 was expressed in Chinese Hamster Ovary
(CHO) cells and purified by a series of chromatography methods
including affinity purification by protein A chromatography and
ion-exchange chromatography. mAb1 was prepared at 5 mg/mL in a
formulation of 20 mM histidine acetate, 250 mM trehalose and 0.02%
polysorbate 20 in a glass vial and with 1 mM L-Trp and various
concentrations of L-Met, ranging from 10 mM to 100 mM, and exposed
to eight hours of light at 250 W/m.sup.2 in an Atlas SunTest
CPS+Xenon Test Instrument (Chicago, Ill.). Control vials were
wrapped in aluminum foil and treated similarly. After light
exposure, solutions were prepared for analysis by reverse-phase
HPLC. For RP-HPLC, mAb1 solution from the stress study was prepared
to 1.1 mg/mL in 0.1 M Tris, 4.4 mM EDTA, and 1.1 mM cysteine. 150
.mu.L of 0.1 mg/mL papain was added to 1.35 mL of the mAb1 solution
before incubation at 37.degree. C. for two hours. Following
incubation, 900 .mu.L of the solution was combined with 100 .mu.L
of 1 M dithiothreitol (DTT) and incubated for another thirty
minutes at 37.degree. C. Samples were then run on an Agilent, Inc.
1100/1200 HPLC system (Santa Clara, Calif.) equipped with UV
detection at 280 nm in conjunction with a Varian, Inc. Pursuit 3
.mu.m, 2 mm ID.times.250 mm diphenyl column (Palo Alto, Calif.).
Mobile Phase A was 0.1% TFA in water. Mobile Phase B was 0.1% TFA
in acetonitrile. The mobile phase gradient increased linearly from
34% B at 0 minutes to 43% B at 50.0 minutes, then to 95% B at 50.1
minutes. The gradient remained at 95% B until 60.1 minutes, and
then decreased linearly from 95% B to 34% B between 60.1 and 60.2
minutes. The gradient remained at 34% B until the end of the cycle
at 80.2 minutes. The column temperature was 65.degree. C., total
flow rate was 0.2 mL/min, and injection volume of each sample was 6
.mu.L. Chromatograms were then integrated for quantification of
oxidation.
[0266] Analysis of the light exposure effects of L-Trp and L-Met on
mAb1 Fab oxidation showed that the mAb1 reference material (no
light exposure) and the foil control had about 2% Fab oxidation
(FIG. 1A). Since the foil control and the reference material showed
the same level of Fab oxidation, it was unlikely that heat alone is
causing oxidation of the Fab. When mAb1 was exposed to light ("No
Excipient" sample), the Fab oxidation doubled to 4%. With the
addition of 1 mM L-Trp, the Fab oxidation increased to almost 9%,
suggesting that free L-Trp was generating ROS under light exposure
that may have resulted in oxidation of W53 on the Fab. Further
addition of 10, 25, 50, and 100 mM L-Met to formulation containing
1 mM L-Trp appeared to reduce Fab oxidation slightly, but even 100
molar excess of L-Met dis not reduce Fab oxidation to the level of
the foil control (FIG. 1A).
[0267] Oxidation in the Fc region of mAb1 has been shown to be
predominately of Met residues Met 254 and Met 430 (Sreedhara et
al., Mol. Pharmaceutics (2013)). Analysis of the light exposure
effects of L-Trp and L-Met on mAb1 Fc oxidation showed that the
mAb1 reference material and foil control had about 8% Fc oxidation
even before exposure to light (FIG. 1B). Exposure to light resulted
in only a minor increase in Fc oxidation ("No Excipient") for mAb1
in formulation buffer. However, incubation with 1 mM L-Trp resulted
in over 20% oxidation at these Met sites in the Fc region as seen
by the RP-HPLC assay. Addition of various concentrations of L-Met
(10, 25, 50 and 100 mM) to formulations containing 1 mM L-Trp
reduced the amount of Fc oxidation, although even 100 mM L-Met dis
not reduce Fc oxidation to the level of the controls (FIG. 1B).
[0268] It was previously reported that L-Trp produced
H.sub.2O.sub.2 via superoxide ion and in a sub-stoichiometric
fashion while antibodies under similar conditions were producing
catalytic amounts (Wentworth et al., Science 293(5536):1806-11
(2001); McCormick et al., Journal of the American Chemical Society
100:312-313 (1978)). To test the susceptibility of free L-Trp under
pharmaceutically relevant conditions, such as under both ICH and
ambient light conditions, formulations comprising 0.32 mM to 7.5 mM
of L-Trp were exposed for 3 hours at 250 W/m.sup.2 UV light and
about 150 k lux visible light. Samples were taken and analyzed
immediately via the Amplex assay to detect the amount of
H.sub.2O.sub.2 generated under these conditions. A large quantity
of H.sub.2O.sub.2 was generated by free L-Trp upon light exposure
in a concentration dependent manner (FIG. 2). This H.sub.2O.sub.2
generation was reduced greatly in the presence of 50 mM sodium
azide, a known quencher of singlet oxygen (FIG. 2). When L-Trp was
incubated with a combination of 50 mM NaN.sub.3 and 150 U
superoxide dismutase (SOD) or SOD alone, significant amounts of
H.sub.2O.sub.2 were still detected in the samples not containing
NaN.sub.3. This indicated that, in addition to singlet oxygen,
superoxide ion was also generated upon photo-irradiation that was
converted to H.sub.2O.sub.2 by SOD.
[0269] While confirming the photosensitivity of free L-Trp under
ICH light conditions, the effect of ambient light that was
typically seen in laboratories was studied. Measurements using a
DLM1 digital light meter in various labs indicated an average of
300 lux on a lab benchtop (with white fluorescent lighting), an
average of 3000 lux in a laminar flow hood (with white fluorescent
lighting) and about 10000 lux for a windowsill exposed to southeast
sunlight. Under these conditions, L-Trp in formulation buffers
containing 50 mg/mL mAb1 produced hydrogen peroxide in the
micromolar range as detected using the Amplex assay (FIG. 3A).
Peroxide production increased with both luminosity (300, 3000, and
10000 lux) and time (1, 3, and 7 days). The protein samples were
further analyzed using the mAb1 specific RP-HPLC assay and showed
increased heavy chain Fab oxidation corresponding to oxidation in
W53 with increased luminosity (FIG. 3B). At the same time, % Fc
oxidation in mAb1 under these conditions increased from 5 to 40%
between 300 and 10000 lux, respectively. These levels of light
exposure and time were determined to be pharmaceutically relevant
for drug substance handling under ambient light and temperature
before fill/finish operations and potentially while inspecting drug
product vials. These results supported that L-Trp is photosensitive
and that it produces several reactive oxygen species, including
singlet oxygen, superoxide and H.sub.2O.sub.2 that can be
detrimental to mAb product quality and that care should be taken
while handling and storing L-Trp containing buffers.
Example 2: Screening of Candidate Antioxidant Compounds
[0270] Tryptophan (Trp) is an electron rich amino acid that
undergoes oxidative and electrophilic addition reactions in the
presence of ROS such as hydroxyl radicals and singlet oxygen. Any
potential antioxidant to protect Trp oxidation in proteins should
have similar if not superior reactivity towards these ROS. A series
of compounds that were either based on the L-Trp structure or have
been reported to have antioxidant properties were evaluated.
Compounds screened for antioxidant ability in this study included
derivatives of tryptophan, indole, aromatic acids such as salicylic
acid and anthranilic acid, and some vitamins. The chemical
structures of the various compounds used were based on (A)
Tryptophan derivatives (B) Kynurenine (C) Indole derivatives and
(D) Aromatic acid derivatives:
(A) Tryptophan Derivatives
##STR00017##
TABLE-US-00002 [0271] Name R X A L-Tryptophan COOH H H
5-Hydroxy-Tryptophan COOH OH H 5-Methoxy-Tryptophan COOH OCH.sub.3
H 5-Amino-Tryptophan COOH NH.sub.2 H 5-Fluoro-Tryptophan COOH F H
N-Acetyl-Tryptophan COOH H CH.sub.3C(O) Tryptamine H H H
Tryptophanamide CONH.sub.2 H H Serotonin H OH H Melatonin H
OCH.sub.3 CH.sub.3C(O)
(B) Kynurenine
##STR00018##
[0272] (C) Indole Derivatives
##STR00019##
TABLE-US-00003 [0273] Name Y.sub.2 Y.sub.3 Y.sub.4 Y.sub.5 Y.sub.7
Indole H H H H H Indole-3-Acetic Acid H CH.sub.2COOH H H H
4-Hydroxy Indole H H OH H H 5-Hydroxy Indole H H H OH H 5-Hydroxy
Indole-3- H CH.sub.2COOH H OH H Acetic Acid 7-Hydroxy Indole H H H
H OH 7-Hydroxy Indole-2- COOH H H H OH Carboxylic Acid
(D) Aromatic Acid Derivatives
##STR00020##
TABLE-US-00004 [0274] Name Z.sub.1 Z.sub.2 Salicylic Acid OH H
5-Hydroxy Salicylic Acid OH OH Anthranilic Acid NH.sub.2 H
5-Hydroxy Anthranilic Acid NH.sub.2 OH
[0275] Candidate Antioxidant Compounds Obtained from a
Photosensitivity Screening Assay.
[0276] While L-Trp may have been an effective antioxidant under
certain circumstances, its photosensitivity may limit its utility
during normal processing without special precautions. Hence the
photosensitivity of the above molecules was investigated and rated
for their H.sub.2O.sub.2 generation capability with respect to
L-Trp. As a screening tool, antioxidant candidates were exposed to
light for four hours at 250 W/m.sup.2 and the resulting
H.sub.2O.sub.2 formation was quantified by the Amplexassay.
Specifically, antioxidants were prepared to 1 mM in 20 mM histidine
acetate buffer at pH 5.5. The 1 mM antioxidant solutions were
aliquoted into glass vials (2 mL/glass vial) and exposed to four
hours of light at 250 W/m.sup.2 in an Atlas SunTest CPS+Xenon Test
Instrument (Chicago, Ill.). Total UV dose was 90 watt-hours/square
meter and total visible dose was 0.22 million lux hours over the
4-hour period. Control vials were wrapped in aluminum foil and
treated similarly. The amount of hydrogen peroxide generated after
exposure to light was measured using the Amplex.RTM. Ultra Red
Assay (Invitrogen, Carlsbad, Calif.) following the manufacturer's
recommended procedure. On addition of horseradish peroxidase (HRP),
the dye reacted 1:1 stoichiometrically with H.sub.2O.sub.2,
resulting in the production of fluorescent oxidation product
resorufin. In this study, fluorescence readings were obtained using
a Spectra Max M2 Micro-plate Reader (Molecular Devices, Sunnyvale,
Calif.) with excitation and emission set at 560 nm and 590 nm,
respectively. Final H.sub.2O.sub.2 concentrations were determined
using a standard curve ranging from 0 .mu.m to 20 .mu.m.
[0277] Analysis of hydrogen peroxide (H.sub.2O.sub.2) generation by
tryptophan derivatives upon light exposure showed that under
similar conditions of light (corresponding to 0.22 million lux
hours over a 4-hour period) and buffer (20 mM L-His-acetate, pH
5.5), 5-hydroxy-L-tryptophan produced about one tenth of the
H.sub.2O.sub.2, while kynurenine produced about one fifth of the
H.sub.2O.sub.2, when compared to L-Trp (FIG. 4A). Other tryptophan
derivatives produced anywhere between 30% and 105% of the
H.sub.2O.sub.2 produced by L-Trp. In comparison to L-Trp, Trolox (a
water soluble Vitamin E derivative) produced 123 times more
H.sub.2O.sub.2, and pyridoxine (Vitamin B6) produced 5 times more
H.sub.2O.sub.2 (Table 1). Indole, which has a basic structure like
L-Trp, behaved similarly to L-Trp, but indole-3-acetic acid
produced twice as much H.sub.2O.sub.2 (FIG. 4B). The hydroxy
derivatives of indole behaved like 5-OH-L-tryptophan in that they
produced negligible amounts of H.sub.2O.sub.2 upon light exposure.
Several biochemically relevant derivatives of L-Trp, namely
tryptamine, serotonin and melatonin were also tested. Tryptamine
produced about half as much H.sub.2O.sub.2 as L-Trp (FIG. 4A).
Interestingly, serotonin (5-hydroxytryptamine) behaved much like
the 5-OH derivatives of indole and tryptophan, producing very
little H.sub.2O.sub.2 upon light exposure, while melatonin
(N-acetyl-5-methoxytryptamine) produced less than a third of the
H.sub.2O.sub.2 produced by L-Trp (Table 1).
TABLE-US-00005 TABLE 1 Hydrogen Peroxide Production Ratio between
Tested Compounds and L-Trp (H.sub.2O.sub.2 produced by
Compound)/(H.sub.2O.sub.2 Compound produced by L-Trp) L-Trp 1
L-Trpamide 0.43 N-Acetyl-L-Trp 0.31 N-Acetyl-L-Trpamide 0.34
5-Fluoro-L-Trp 0.71 5-Hydroxy-L-Trp 0.09 5-Methoxy-DL-Trp 1.05
5-Amino-DL-Trp 0.29 L-Kynurenine 0.20 Trolox 122.75 Pyridoxine 5.16
Indole 0.95 Indole-3-Acetic Acid 2.40 4-Hydroxyindole 0.00
5-Hydroxyindole -0.08 5-Hydroxyindole-3-Acetic Acid 0.11
7-Hydroxyindole -0.03 7-Hydroxyindole-2-Carboxylic Acid 0.15
Tryptamine 0.53 Serotonin (5-Hydroxytryptamine) 0.03 Melatonin
(N-Acetyl-5-Methoxytryptamine) 0.28 Salicylic Acid 0.03
5-Hydroxysalicylic Acid 0.84 Anthranilic Acid 2.50
5-Hydroxyanthranilic Acid 0.44
[0278] In order to understand the ROS formed during
photo-irradiation, several of the Trp derivatives in the presence
of 50 mM NaN.sub.3, a known singlet oxygen quencher, were tested
under light exposure as described above. All the compounds tested
showed a substantial decrease in the amount of hydrogen peroxide
generated under these conditions, indicating that singlet oxygen
was a major ROS created upon photo-irradiation of Trp and its
derivatives (FIG. 5).
[0279] Other aromatic compounds such as salicylic acid and
derivatives were also tested based on their reported antioxidant
properties (Baltazar et al., Curr Med Chem 18(21):3252-64 (2011)).
Salicylic acid produced very little H.sub.2O.sub.2 upon light
exposure while its 5-OH derivative behaved like L-Trp (Table 1). On
the other hand, anthranilic acid produced twice as much
H.sub.2O.sub.2 as L-Trp but 5-OH-anthranilic acid produced half as
much H.sub.2O.sub.2 compared to L-Trp (Table 1).
[0280] Candidate Antioxidant Compounds Obtained from a CV Screening
Assay.
[0281] Based on the results from the photosensitivity screening
assay, compounds with aromatic ring substitutions appeared to
impact the amount of hydrogen peroxide generated. Since the goal
was preferential oxidation of the excipient rather than the protein
drug, excipients that had low oxidation potentials may have served
as effective antioxidants. The oxidation/reduction characteristics
of the compounds were investigated. Several compounds, including
L-Trp and derivatives, were evaluated for the protection of Trp
oxidation in proteins using cyclic voltammetry (CV) and rank
ordered based on their oxidation potentials (Table 2).
Specifically, the candidate antioxidants were dissolved in
deionized water and then added to a 0.2 M lithium perchlorate
electrolyte solution. Solutions were characterized with an EG&G
Princeton Applied Research Model 264A Polarograph/Voltammeter with
a Model 616 RDE Glassy Carbon Electrode as working electrode.
Solutions were scanned from -0.10 V to +1.50 V at a scan rate of
either 100 or 500 mV/sec. The analytical cell was purged for four
minutes with nitrogen before scanning of each antioxidant solution.
The input was a linear scan of the potential of a working
electrode, and the output was measurement of the resulting current.
As the potential was scanned (linearly increased or decreased),
electrochemically active species in the CV cell underwent oxidation
and reduction reactions at the surface of the working electrode
that resulted in a current which was continuously measured. Redox
reactions were characterized by sharp increases or decreases in
current (peaks). The potential at which an oxidation reaction
occurred was referred to as the anodic peak potential (or oxidation
potential), and the potential at which a reduction occurred was
referred to as is the cathodic peak (or reduction) potential.
[0282] The oxidation potentials of the excipients in this study
ranged from 0.410 to 1.080 V vs Ag/AgCl (Table 2). Under these
conditions, L-Trp had an irreversible oxidation potential of 0.938
V vs Ag/AgCl. Nine compounds were found to have a lower oxidation
potential than L-Trp, including all of the 5-OH compounds which had
oxidation potentials between 0.535 and 0.600 V vs Ag/AgCl. Of all
the compounds tested, 5-amino-DL-tryptophan had the lowest
oxidation potential at 0.410 V, while the N-acetyl compounds
(0.730-0.880 V), and 5-methoxy-DL-tryptophan (0.890 V) were also
below L-Trp. Seven compounds had higher oxidation potential than
L-Trp (Table 2). These were indole-3-acetic acid,
5-fluoro-L-tryptophan, tryptamine, L-tryptophanamide, L-kynurenine,
5-nitro-DL-tryptophan, and salicylic acid. Salicylic acid had the
highest oxidation potential in this study (1.080 V vs Ag/AgCl). All
the tested compounds showed non-reversible CV indicating that once
oxidized, the species did not tend to receive electrons and
probably could not be involved in further electrochemical
reactions.
TABLE-US-00006 TABLE 2 Oxidation Potentials of Excipients Oxidation
Potential Compound (V vs Ag/AgCl) 5-amino-DL-tryptophan 0.410
5-hydroxyindole-3-acetic acid 0.535 5-hydroxy-L-tryptophan 0.565
5-hydroxyindole 0.580 Serotonin HCl (5-hydroxytryptamine HCl) 0.600
Melatonin (N-acetyl-5-methoxytryptamine) 0.730
N-acetyl-L-tryptophan 0.875 N-acetyl-L-tryptophanamide 0.880
5-methoxy-DL-tryptophan 0.890 L-tryptophan 0.938 Indole-3-acetic
acid 0.948 5-fluoro-L-tryptophan 0.965 Tryptamine HCl 1.010
L-tryptophanamide 1.015 L-kynurenine 1.040 5-nitro-DL-tryptophan
1.055 Salicylic acid 1.080 Oxidation (anodic peak) potentials were
measured using cyclic voltammetry with a glassy carbon working
electrode in 0.2M lithium perchlorate.
[0283] A correlation was determined between oxidation potential and
light-induced H.sub.2O.sub.2 generation for 16 compounds that had
oxidation potentials above and below the oxidation potential of
L-Trp, and H.sub.2O.sub.2 production levels above and below that of
L-Trp (FIG. 6). Since indole and tryptophan behaved similarly in
H.sub.2O.sub.2 production under light exposure, it was possible
that substitutions on the C.sub.3 position of the 5 membered ring
did not affect this property. However, tryptamine with a
--CH.sub.2CH.sub.2NH.sub.2 substitution and indole-3-acetic acid
with a --CH.sub.2COOH substitution at the C.sub.3 position produced
two times less and two times more H.sub.2O.sub.2, respectively,
than L-Trp. These data indicated that the C.sub.3 substitutions
played a role in photo-activation and peroxide generation. The
C.sub.3 substitutions did not affect the oxidation potentials of
the molecules, whereas indole per se had significantly lower
oxidation potential than L-Trp under these experimental conditions.
Substitutions at the C.sub.5 of the 6-membered aromatic ring
behaved quite predictably. In general, compounds with electron
donating groups such as --NH.sub.2 and --OH had lower oxidation
potentials than their parent compounds and also showed low levels
of H.sub.2O.sub.2 production upon photo-activation (e.g.
5-amino-DL-tryptophan, 5-hydroxyindole-3-acetic acid,
5-hydroxy-L-tryptophan, 5-hydroxyindole, serotonin). Similarly,
compounds with high oxidation potential produced more
H.sub.2O.sub.2 (5-methoxy-DL-tryptophan, L-Trp, indole-3-acetic
acid, 5-fluoro-L-tryptophan) under these conditions. There were
exceptions to this correlation; some compounds had high oxidation
potential but did not produce much H.sub.2O.sub.2 (e.g. salicylic
acid and L-kynurenine) indicating that there were potentially other
mechanisms that played an important role for these six membered
aromatic compounds that may not have been observed with compounds
containing the indole backbone of L-Trp. The area of interest was
the quadrant which contained compounds with lower oxidation
potential and lower H.sub.2O.sub.2 production upon light exposure
than L-Trp (FIG. 6, dashed box). Compounds with these two qualities
were considered as new candidate antioxidants because they could
(1) oxidize faster than Trp on the protein and (2) produce very
little H.sub.2O.sub.2 during long term storage and/or ambient
processing during drug product production and therefore could
protect the protein from further oxidation under these
conditions.
Example 3: Candidate Antioxidant Compounds Reduced Oxidation of
Monoclonal Antibody Formulations
[0284] Compounds that, compared to L-Trp, produced less
H.sub.2O.sub.2 upon light treatment as well as those with lower
oxidation potentials than L-Trp were chosen for evaluation of for
their possible antioxidant properties using AAPH, light, and Fenton
reaction as oxidative stress models (Table 3). mAb1 was used as a
model protein to evaluate the effectiveness of select candidate
antioxidants to protect against Trp oxidation by the different
oxidation stress models. Each stress model produced oxidation
through a different mechanism and therefore each was valuable in
the assessment of biopharmaceutical stability. AAPH, or
2,2'-Azobis(2-Amidinopropane) Dihydrochloride, is used as a stress
model to mimic alkyl peroxides potentially generated from
formulation excipients such as degraded polysorbate. Decomposition
of AAPH generates alkyl, alkoxyl, and alkyl peroxyl radicals that
have been shown to oxidize amino acid residues in proteins,
including methionine, tyrosine, and tryptophan residues (Ji et al.,
J Pharm Sci 98(12):4485-500 (2009); Chao et al., Proc Natl Acad Sci
USA 94(7):2969-74 (1997)). Similarly, controlled light could be
used as a stress model to mimic ambient light exposure that drugs
may experience during processing and storage. Light-induced
oxidation of biopharmaceuticals was shown to proceed through a
singlet oxygen (.sup.1O.sub.2) and/or superoxide anion
(O.sub.2.sup.-) mechanism (Sreedhara et al., Mol. Pharmaceutics
(2013)). The Fenton reaction is also commonly used as an oxidative
stress model. This mixture of H.sub.2O.sub.2 and Fe ions generates
oxidation through a metal catalyzed, hydroxyl radical mechanism
(Prousek et al., Pure and Applied Chemistry 79(12):2325-2338
(2007)), and is used to model metal residue from stainless steel
surfaces used in drug manufacturing and storage.
TABLE-US-00007 TABLE 3 Oxidation Stress Models Stress Model
Mechanism Purpose AAPH Alkyl peroxides, alkyl Mimic alkyl peroxides
from radical catalyzed degraded polysorbate Light Singlet oxygen
(.sup.1O.sub.2), Mimic ambient light superoxide anion
(O.sub.2.sup.-), exposure during H.sub.2O.sub.2 processing and
storage Fenton Hydroxyl radical, Mimic metal residue from
(H.sub.2O.sub.2 + Fe) metal catalyzed stainless steel surfaces
[0285] Tryptophan (W53) oxidation on mAb1 was thoroughly
characterized previously using a RP-HPLC and LC-MS method
(Sreedhara et al., Mol. Pharmaceutics (2013)). Briefly, mAb1 was
digested with papain to generate Heavy Chain (HC) Fab, HC Fc, and
Light Chain fragments. The fragments were reduced with DTT, and
then separated and identified via Liquid Chromatography-Mass
Spectrometry (LC-MS). Oxidized versions of the HC Fab and HC Fc
were found to elute earlier than their native counterparts.
Comparison of area integrated under the oxidized and native peaks
was used to quantify HC Fab and Fc oxidation. In addition, LC-MS/MS
peptide maps (by trypsin digestion and by Lys-C digestion) showed
that oxidation of the HC Fab was primarily of a Trp residue, W53,
while oxidation of the HC Fc was attributed predominantly to
oxidation of two Met residues, M254 and M430. By using the papain
digest RP-HPLC method in the present study it was possible to
investigate Trp residue oxidation by quantifying HC Fab oxidation,
and Met residue oxidation by quantifying HC Fc oxidation.
[0286] % Fab oxidation and % Fc oxidation were calculated as
follows:
% Fab Oxidation = 100 Oxidized Fab Peak Area Fab Peak Area +
Oxidized Fab Peak Area ##EQU00002## % Fc Oxidation = 100 Oxidized
Fc Peak Area Fc Peak Area + Oxidized Fc Peak Area
##EQU00002.2##
[0287] For the mAb1 stress study, mAb1 was prepared to 5 mg/mL in a
formulation of 20 mM histidine acetate, 250 mM trehalose, and 0.02%
Polysorbate 20. Antioxidants were added at 1 mM. Glass vials
containing these formulations were exposed to 250 W/m.sup.2 light
in an Atlas SunTest CPS+Xenon Test Instrument (Chicago, Ill.).
Control vials were wrapped in aluminum foil and treated similarly.
After light exposure, solutions were prepared for analysis by
reverse-phase HPLC as described above.
[0288] For the mAb1 AAPH stress study, mAb1 was prepared to 4 mg/mL
in a formulation of 20 mM histidine acetate, 250 mM trehalose, and
0.02% Polysorbate 20. Antioxidants were added at 1 mM. 200 .mu.L of
10 mM AAPH was added to 2 mL of each mAb1 solution and then
incubated at 40.degree. C. for 24 hours. After incubation, each
solution was buffer exchanged with formulation buffer (20 mM
histidine acetate, 250 mM trehalose, and 0.02% Polysorbate 20)
using a PD-10 column so that the final mAb1 concentration was 2.3
mg/mL. After buffer exchange, each solution was prepared for
analysis by reverse-phase HPLC as described above.
[0289] For the mAb1 Fenton stress study, mAb1 was prepared to 3
mg/mL in a formulation of 20 mM histidine hydrochloride pH 6.0.
Antioxidants were added at a final concentration of 1 mM. A final
concentration of 0.2 mM FeCl.sub.3 and 10 ppm H.sub.2O.sub.2 were
added to each mAb1 solution and then incubated at 40.degree. C. for
3 hours. After incubation, each reaction was quenched by addition
of 100 mM L-Met and then prepared for analysis by reverse-phase
HPLC as described above.
[0290] It was determined that incubation of mAb1 with AAPH for 24
hours at 40.degree. C. resulted in 27% Fab (Trp residue) oxidation
(FIG. 7A) and 47% Fc (Met residue) oxidation (FIG. 7B). Seven
excipients that had been previously screened using light stress and
cyclic voltammetry were incubated with mAb1 under the AAPH
conditions to evaluate antioxidant capabilities. Six of the seven
compounds were found to significantly reduce AAPH-induced Fab
oxidation (FIG. 7A). All six of these compounds contained the
indole backbone. Moreover, all the hydroxy derivatives tested
(5-hydroxy-L-Trp, 5-hydroxyindole, 7-hydroxyindole, and serotonin)
reduced Fab oxidation to close to control levels (about 2%).
Meanwhile, salicylic acid had almost no effect on Fab oxidation
under AAPH stress. None of the excipients appeared to impact the
level of AAPH-induced Fc oxidation (FIG. 7B).
[0291] For the light stress study, mAb1 was exposed to 16 hours of
light at 250 W/m.sup.2 while testing the aforementioned seven
excipients (FIG. 8). Exposure of mAb1 to light ("No Excipient")
increased Fab oxidation 3.5 times over the control level ("mAb1 Ref
Mat", FIG. 8A). It was previously shown that L-Trp could protect
against Trp oxidation in the model protein Parathyroid Hormone
(PTH) (Ji et al., J Pharm Sci 98(12):4485-500 (2009)). However,
this study found that addition of 1 mM L-Trp to mAb1 increased the
Fab oxidation over 11-fold, probably through the production of ROS
such as singlet oxygen by light-exposed L-Trp (FIG. 2). Addition of
the hydroxy compounds (5-hydroxy-L-Trp, 5-hydroxyindole,
7-hydroxyindole, and serotonin) protected against light-induced Fab
oxidation, reducing Fab oxidation to near control levels (FIG. 8A).
On the other hand, salicylic acid performed similarly to
L-Tryptophanamide, increasing Fab oxidation 8-fold over the control
level. Similar results were observed for Fc oxidation under light
stress (FIG. 8B). Light exposure of mAb1 resulted in a 40% increase
in Fc oxidation over the control level, whereas addition of L-Trp
increased Fc oxidation to 7 times the control level. Compared to
the control (no excipient), L-Tryptophanamide and salicylic acid
also resulted in more Fc oxidation. The hydroxy compounds produced
similar Fc oxidation as the no excipient control potentially
because they produce much fewer ROS than L-Trp under light
exposure. The light screening and NaN.sub.3 study results in
Example 2 showed a good correlation between the amount of
H.sub.2O.sub.2 generated by an excipient and Fc Met oxidation of
mAb1.
[0292] The Fenton reaction, using a mixture of H.sub.2O.sub.2 and
Fe ions, generates oxidation through a metal catalyzed, hydroxyl
radical reaction (Prousek et al., Pure and Applied Chemistry
79(12):2325-2338 (2007)). This reaction generated Fab, i.e.
tryptophan, oxidation in mAb1. The reaction was also carried out in
the presence of select antioxidants that were useful against both
AAPH and light induced oxidation as reported above. Data related to
the antioxidant properties against Fenton mediated reaction were
analyzed using the RP-HPLC assay as described above. The Fenton
reaction used 10 ppm of H.sub.2O.sub.2 and 0.2 mM of Fe(III). The
reaction was incubated at 40.degree. C. for 3 hours, quenched with
100 mM L-Met and analyzed using RP-HPLC after papain digest. All
samples were the average of three separate vials, and mAb1 control
(Ref Mat) was one vial with five independent injections on the
HPLC. Under the conditions tested, the Fenton reaction produced
about four times the oxidation in the Fab region of mAb1 over the
control. Most of the antioxidants tested, except salicylic acid,
showed similar hydroxyl radical quenching properties to L-Trp,
which protected the Fab oxidation by about 25% with respect to the
no excipient case. In regards to protection against Fc oxidation,
the tested excipients (other than salicylic acid) performed
slightly better than L-Trp.
* * * * *