U.S. patent application number 17/465092 was filed with the patent office on 2021-12-23 for method for purifying active polypeptides or immunoconjugates.
The applicant listed for this patent is MedImmune Limited. Invention is credited to Alan HUNTER, Thomas LINKE, Hasige SATHISH, Ambarish SHAH, Christopher THOMPSON, William K. WANG.
Application Number | 20210395370 17/465092 |
Document ID | / |
Family ID | 1000005825676 |
Filed Date | 2021-12-23 |
United States Patent
Application |
20210395370 |
Kind Code |
A1 |
LINKE; Thomas ; et
al. |
December 23, 2021 |
METHOD FOR PURIFYING ACTIVE POLYPEPTIDES OR IMMUNOCONJUGATES
Abstract
The present invention provides methods for isolating an active
polypeptide or immunoconjugate by purification of a solution
containing both the active polypeptide or immunoconjugate and an
acidic variant thereof, such as a deamidated variant, using anion
exchange chromatography. The present invention also provides
compositions, formulations, and unit dosage forms comprising the
purified polypeptide or immunoconjugate.
Inventors: |
LINKE; Thomas;
(Gaithersburg, MD) ; WANG; William K.;
(Gaithersburg, MD) ; SHAH; Ambarish;
(Gaithersburg, MD) ; SATHISH; Hasige;
(Gaithersburg, MD) ; HUNTER; Alan; (Gaithersburg,
MD) ; THOMPSON; Christopher; (Gaithersburg,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MedImmune Limited |
Cambridge |
|
GB |
|
|
Family ID: |
1000005825676 |
Appl. No.: |
17/465092 |
Filed: |
September 2, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16786634 |
Feb 10, 2020 |
11136396 |
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17465092 |
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16124441 |
Sep 7, 2018 |
10556955 |
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16786634 |
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15423928 |
Feb 3, 2017 |
10072083 |
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16124441 |
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13813083 |
Apr 8, 2013 |
9580461 |
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PCT/US2011/045524 |
Jul 27, 2011 |
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15423928 |
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61369148 |
Jul 30, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/6849 20170801;
B01D 15/363 20130101; A61K 39/3955 20130101; C07K 2317/56 20130101;
B01D 15/166 20130101; B01J 41/14 20130101; B01J 41/20 20130101;
A61K 47/02 20130101; A61K 47/6829 20170801; C07K 1/18 20130101;
C07K 16/2851 20130101; C07K 2319/35 20130101; A61K 2039/505
20130101; C07K 16/2803 20130101; C07K 2319/55 20130101; C07K
2317/94 20130101; C07K 17/02 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; B01D 15/16 20060101 B01D015/16; B01D 15/36 20060101
B01D015/36; B01J 41/14 20060101 B01J041/14; B01J 41/20 20060101
B01J041/20; A61K 47/68 20060101 A61K047/68; C07K 1/18 20060101
C07K001/18; C07K 17/02 20060101 C07K017/02; A61K 39/395 20060101
A61K039/395; A61K 47/02 20060101 A61K047/02 |
Claims
1. A method of purifying an active immunoconjugate, wherein said
immunoconjugate is deamidated at one or more residues, and wherein
said deamidation results in an inhibition of potency of said
immunoconjugate, the method comprising: (a) contacting the
immunoconjugate with an anion exchange AIEX chromatography matrix;
and (b) eluting the bound immunoconjugate from the AIEX
chromatography matrix with a high salt buffer, thereby separating
the active immunoconjugate from the deamidated variant.
2. A method of producing a purified polypeptide from a solution
comprising the polypeptide and an acidic variant of the
polypeptide, wherein said acidic variant of the polypeptide results
in an inhibition of potency of said polypeptide, the method
comprising: (a) contacting the polypeptide with an anion exchange
(AIEX) chromatography matrix; and (b) eluting the bound polypeptide
from the AIEX chromatography matrix with a high salt buffer,
thereby separating said polypeptide from the acidic variant and
producing a purified polypeptide.
3. A method of producing a purified polypeptide or immunoconjugate
from a solution comprising the polypeptide and an acidic variant of
the polypeptide, the method comprising: (a) producing the
polypeptide or immunoconjugate in a bacterial cell which expresses
the polypeptide or immunoconjugate; (b) isolating inclusion bodies
containing the polypeptide or immunoconjugate from the bacterial
cells; (c) refolding the polypeptide or immunoconjugate isolated
from the inclusion bodies; (d) contacting the composition
containing the polypeptide or immunoconjugate with an AIEX
chromatography matrix; and (b) eluting the bound polypeptide or
immunoconjugate from the AIEX chromatography matrix with a high
salt buffer, thereby purifying the polypeptide or immunoconjugte
from the solution.
3. The method of claim 2 or 3, wherein the acidic variant is a
deamidated variant.
4. The method of any of claims 1-3, wherein the AIEX matrix
contains quaternary amine and tertiary amine ion exchange
groups.
5. The method of claim 4, wherein the AIEX matrix contains a
quaternary amino (Q) group.
6. The method of claim 5, wherein the AIEX matrix is Q
sepharose.
7. The method of any of claims 1-6, wherein the polypeptide is
eluted with a linear or step salt gradient.
8. The method of claim 7, wherein the polypeptide is eluted with a
linear salt gradient.
9. The method of claim 8, wherein the linear salt gradient is from
about 150 mM NaCl in Tris/HCl, pH 8.0 to about 300 mM NaCl in
Tris/HCl, pH 8.0.
10. The method of claim 9, wherein the linear salt gradient is from
about 175 mM NaCl in Tris/HCl, pH 8.0 to about 275 mM NaCl in
Tris/HCl, pH 8.0.
11. The method of claim 9, wherein the linear salt gradient is from
about 192 mM NaCl in Tris/HCl, pH 8.0 to about 245 mM NaCl in
Tris/HCl, pH 8.0.
12. The method of any of claims 1-11, wherein between about 75 to
about 99% of the acidic or deamidated variant is removed.
13. The method of claim 12, wherein about 80% of the acidic or
deamidated variant is removed.
14. The method of claim 12, wherein about 85% of the acidic or
deamidated variant is removed.
15. The method of claim 12, wherein about 90% of the acidic or
deamidated variant is removed.
16. The method of claim 12, wherein about 95% of the acidic or
deamidated variant is removed.
17. The method of claim 12, wherein about 96%, 97%, 98%, or 99% of
the acidic or deamidated variant is removed.
18. The method of any of claims 1-17, wherein the polypeptide or
immunoconjugate comprises an antibody or antigen binding fragment
thereof.
19. The method of claim 18, wherein the antibody or antigen binding
fragment comprises a Fab, a Fab', a F(ab').sub.2, a Fd, a single
chain Fv or scFv, a disulfide linked Fv, a V-NAR domain, an IgNar,
an intrabody, an IgG.DELTA.CH2, a minibody, a F(ab').sub.3, a
tetrabody, a triabody, a diabody, a single-domain antibody, DVD-Ig,
Fcab, mAb.sup.2, a (scFv).sub.2, or a scFv-Fc.
20. The method of claim 18 or 19, wherein the antibody or antigen
binding fragment binds a cell surface receptor.
21. The method of claim 20, wherein the cell surface receptor is
CD22.
22. The method of any of claims 1-21, wherein the polypeptide or
immunoconjugate comprises a toxin.
23. The method of claim 22, wherein the toxin is selected from the
group consisting of: Pseudomonas exotoxin, ricin, abrin, diphtheria
toxin and subunits thereof, as well as botulinum toxins A through F
or variants, or derivatives thereof.
24. The method of claim 22 or 23, wherein the toxin is a
Pseudomonas exotoxin, or variant thereof.
25. The method of claim 24, wherein said Pseudomonas exotoxin, or
variant thereof has an amino acid sequence selected from the group
consisting of SEQ ID NOs:16-22.
26. The method of claim 24, wherein said Pseudomonas exotoxin, or
variant thereof has the amino acid sequence of SEQ ID NO:22.
27. The method of claim 18 or 19, wherein said antibody or antigen
binding fragment thereof comprises a V.sub.H and a V.sub.L
sequence.
28. The method of claim 27, wherein said V.sub.H sequence is
selected from the group consisting of SEQ ID NOs: 6-11.
29. The method of claim 27, wherein said V.sub.L sequence is
selected from the group consisting of SEQ ID NOs: 2, and 12-15.
30. The method of any of claims 1-29, wherein the polypeptide or
immunoconjugate comprises an anti-CD22 antibody or antigen binding
fragment thereof and a PE or variant thereof.
31. The method of claim 30, wherein the immunoconjugate is the
CAT-8015 immunotoxin comprising the V.sub.H-PE38 subunit of SEQ ID
NO:1 and the V.sub.L subunit of SEQ ID NO:2.
32. A composition comprising a purified immunoconjugate having less
than between about 25% and about 1% deamidated species, wherein
said immunoconjugate is purified by the method of any of claims
1-31.
33. The composition of claim 32, wherein less than about 25% of the
deamidated species is present.
34. The composition of claim 32, wherein less than about 20% of the
deamidated species is present.
35. The composition of claim 32, wherein less than about 10% of the
deamidated species is present.
36. The composition of claim 32, wherein less than about 5% of the
deamidated species is present.
37. The composition of claim 32, wherein less than about 3% of the
deamidated species is present.
38. The composition of claim 32, wherein less than about 2% of the
deamidated species is present.
39. The composition of claim 32, wherein less than about 1% of the
deamidated species is present.
40. A pharmaceutical composition comprising the purified
immunoconjugate of any of claims 32-39 and a pharmaceutically
acceptable carrier.
41. A composition comprising a purified immunoconjugate having less
than between about 20% and about 1% deamidated species, wherein
said immunoconjugate comprises an anti-CD22 antibody or antigen
binding fragment thereof and a PE toxin or variant thereof.
42. The composition of claim 41, wherein said immunoconjugate
comprises the V.sub.H-PE38 subunit of SEQ ID NO:1 and the V.sub.L
subunit of SEQ ID NO:2.
43. The composition of claim 41, wherein said purified
immunoconjugate has less than 20% of deamidated species.
44. A unit dosage form of a purified immunoconjugate in the range
of 0.1 mg to 6 mg, wherein said immunoconjugate comprises an
anti-CD22 antibody or antigen binding fragment thereof and a PE
toxin or variant thereof.
45. The unit dosage form of claim 44, wherein said immunoconjugate
comprises the V.sub.H-PE38 subunit of SEQ ID NO:1 and the V.sub.L
subunit of SEQ ID NO:2.
46. A formulation comprising the composition of any of claims 32-43
and at least one excipient selected from the group consisting of
sodium chloride, potassium dihydrogen phosphate, disodium hydrogen
phosphate, and sodium hydroxide and water.
47. A formulation comprising the composition of any of claims 32-43
comprising 1 mg/mL CAT-8015 in 25 mM sodium phosphate, 4% sucrose,
8% glycine, 0.02% polysorbate 80 (PS80), pH 7.4.
48. The formulation of claim any one of claims 41 to 43 that is
further lyophilized.
49. A method of modifying the bioactivity of a polypeptide solution
comprising a polypeptide and a deamidated variant, the method
comprising separating the polypeptide from the deamidated variant
by linear elution AIEX chromatography; and combining the purified
polypeptide and deamidated variant in fixed quantities to obtain
the desired bioactivity of the polypeptide solution.
50. The method of any of claims 24-27, wherein said one or more
deamidated residues are present within the Pseudomonas exotoxin or
variant thereof.
51. The method of claim 47, wherein said immunoconjugate is encoded
by the amino acid sequence of SEQ ID NO:1 and is deamidated at
position 358 of SEQ ID NO:1.
52. The method of claim 3, wherein refolding the polypeptide or
immunoconjugate isolated from the inclusion bodies comprises
solubilization of inclusion bodies at a pH in a range of about pH
9.0 to about pH 10.5.
53. The method of claim 3 or 52, wherein refolding the polypeptide
or immunoconjugate isolated from the inclusion bodies comprises
solubilization of inclusion bodies at a pH of 10.5, a pH of 10.0, a
pH of 9.5, or pH of 9.0.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation of U.S. patent
application Ser. No. 16/124,441, filed Sep. 7, 2018 and issued as
U.S. Pat. No. 10,556,955, which is a continuation of U.S. patent
application Ser. No. 15/423,928, filed Feb. 3, 2017 and issued as
U.S. Pat. No. 10,072,083, which is a continuation of U.S. patent
application Ser. No. 13/813,083, filed Apr. 8, 2013 and issued as
U.S. Pat. No. 9,580,461. U.S. patent application Ser. No.
13/813,083 is a U.S. National Stage application of International
Patent Application No. PCT/US2011/045524, filed Jul. 27, 2011,
which claims the benefit of U.S. Provisional Patent Application No.
61/369,148, filed Jul. 30, 2010. Each of the aforementioned patent
applications is incorporated by reference herein in its
entirety.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
[0002] Incorporated by reference in its entirety herein is a
computer-readable nucleotide/amino acid Sequence Listing submitted
with this application as a text file entitled
"MOXE-300-US-CNT-SequenceListing", created on Jan. 25, 2017, having
a size of 39,577 bytes.
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] The present invention provides methods for purifying an
active polypeptide or immunoconjugate from a solution containing
the polypeptide or immunoconjugate and an acidic variant thereof,
wherein said acidic variant is a deamidated species of said
polypeptide or immunoconjugate. The present invention also provides
formulations containing such purified polypeptides or
immunoconjugates.
Background Art
[0004] The large-scale, economic purification of proteins is a
factor for the biopharmaceutical industry. Therapeutic proteins are
typically produced using prokaryotic or eukaryotic cell lines that
are engineered to express the protein of interest from a
recombinant plasmid containing the gene encoding the protein.
Separation of the desired protein from the mixture of components
fed to the cells and cellular by-products to an adequate purity,
e.g., sufficient for use as a human therapeutic, poses a formidable
challenge to biologics manufacturers for several reasons.
[0005] Manufacturers of protein-based pharmaceutical products must
comply with strict regulatory standards, including extremely
stringent purity requirements. To ensure safety, regulatory
agencies, such as Food and Drug Administration (FDA), require that
protein-based pharmaceutical products are substantially free from
impurities, including both product related contaminants such as
aggregates, fragments and variants of the recombinant protein and
process related contaminants such as host cell proteins, media
components, viruses, DNA and endotoxins. While various protein
purification schemes are available and widely used in the
biopharmaceutical industry, they typically include an
affinity-purification step, such as Protein A purification in the
case of antibodies, in order to reach a pharmaceutically acceptable
degree of purity.
[0006] The development of a purification scheme applicable to a
particular biomolecule or various biomolecules that is scaleable,
controllable, and that strategically employs the use of particular
resins or a combination of resins will allow its integration into
product development at a very early stage in overall drug
development. This approach to the design of a purification scheme
can minimize costly changes to manufacturing processes which may
otherwise be necessary later in drug development or, worse, after
approval. As the process is scaled-up and approaches good
manufacturing practices (GMP) production conditions, additional
inherent complexities arise, including those associated with resin
packing and buffer preparation. The manufacturing process, and its
capacity, can be improved by simplifying the purification scheme,
by eliminating process steps and maximizing throughput and
productivity, while maintaining the integrity and purity of the
molecule that is being purified. Therefore, it would be desirable
and advantageous to start with a simple and efficient process that
can produce a drug substance of high quality and safety.
[0007] One complexity associated with the purification of a drug
product is the maintenance of potency throughout the purification
process. Many factors can contribute to a reduction or inhibition
of potency, including the modification of the drug product during
the development process. Such modification can occur at various
stages of the process, for example, when the protein is being
expressed in the cell, or when a protein that has been isolated
from a cell is subject to various conditions or buffers. The
present invention provides a method for purifying an active
polypeptide or immunoconjugate from a solution containing a
modified variant of the polypeptide or immunoconjugate, where the
presence of this modified variant results in an inhibition in
potency of the final drug product.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention provides a method of purifying a
polypeptide of interest from a solution containing the polypeptide
and an acidic variant, such as a deamidated variant, of the
polypeptide.
[0009] In particular, the present invention provides a method of
purifying an active immunoconjugate, where the immunoconjugate is
deamidated at one or more residues, and wherein the deamidation
results in an inhibition of potency of said immunoconjugate, the
method comprising: (a) contacting the immunoconjugate with an anion
exchange AIEX chromatography matrix; and (b) eluting the bound
immunoconjugate from the AIEX chromatography matrix with a high
salt buffer, thereby separating the active immunoconjugate from the
deamidated variant.
[0010] The invention also provides a method of producing a purified
polypeptide from a solution comprising the polypeptide and an
acidic variant of the polypeptide, where the acidic variant of the
polypeptide results in an inhibition of potency of the polypeptide,
the method comprising: (a) contacting the polypeptide with an anion
exchange (AIEX) chromatography matrix; and (b) eluting the bound
polypeptide from the AIEX chromatography matrix with a high salt
buffer, thereby separating said polypeptide from the acidic variant
and producing a purified polypeptide.
[0011] The invention further provides a method of producing a
purified polypeptide or immunoconjugate from a solution comprising
the polypeptide and an acidic variant of the polypeptide, the
method comprising: (a) producing the polypeptide or immunoconjugate
in a bacterial cell which expresses the polypeptide or
immunoconjugate; (b) isolating inclusion bodies containing the
polypeptide or immunoconjugate from the bacterial cells; (c)
refolding the polypeptide or immunoconjugate isolated from the
inclusion bodies; (d) contacting the composition containing the
polypeptide or immunoconjugate with an AIEX chromatography matrix;
and (e) eluting the bound polypeptide or immunoconjugate from the
AIEX chromatography matrix with a high salt buffer, thereby
purifying the polypeptide or immunoconjugate from the solution.
[0012] In certain embodiments, the acidic variant is a deamidated
variant. In further embodiments, between about 75 to about 99% of
the acidic or deamidated variant is removed during the purification
process, in particular about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%
or 99%.
[0013] The AIEX matrix of the invention contains quaternary amine
or tertiary amine ion exchange groups, a quaternary amino (Q)
group. In certain embodiments, the AIEX matrix is Q sepharose.
[0014] The polypeptide or immunoconjugate of the invention is
eluted with a linear or step salt gradient. In certain embodiments,
the linear salt gradient is from about 150 mM NaCl in Tris/HCl, pH
8.0 to about 300 mM NaCl in Tris/HCl, pH 8.0, from about 175 mM
NaCl in Tris/HCl, pH 8.0 to about 275 mM NaCl in Tris/HCl, pH 8.0,
or from about 192 mM NaCl in Tris/HCl, pH 8.0 to about 245 mM NaCl
in Tris/HCl, pH 8.0.
[0015] In one embodiment, the polypeptide or immunoconjugate of the
invention comprises an antibody or antigen binding fragment
thereof, where the antibody or antigen binding fragment comprises a
Fab, a Fab', a F(ab')2, a Fd, a single chain Fv or scFv, a
disulfide linked Fv, a V NAR domain, an IgNar, an intrabody, an
IgG-.DELTA.CH2, a minibody, a F(ab')3, a tetrabody, a triabody, a
diabody, a single-domain antibody, DVD-Ig, Fcab, mAb2, a (scFv)2,
or a scFv-Fc. In certain embodiments, the antibody or antigen
binding fragment binds a cell surface receptor, such as the cell
surface receptor is CD22. In further embodiments, the antibody or
antigen binding fragment thereof comprises a V.sub.H and V.sub.L
sequence, where the V.sub.H sequence is selected from the group
consisting of SEQ ID NOs: 6-11 and the V.sub.L sequence is selected
from the group consisting of SEQ ID NOs: 2, and 12-15.
[0016] In another embodiment, the polypeptide or immunoconjugate
comprises a toxin, where the toxin is selected from the group
consisting of: Pseudomonas exotoxin, ricin, abrin, diphtheria toxin
and subunits thereof, as well as botulinum toxins A through F or
variants, or derivatives thereof. In certain embodiments, the
Pseudomonas exotoxin, or variant thereof has an amino acid sequence
selected from the group consisting of SEQ ID NOs:16-22. In a
particular embodiment, the immunoconjugate is the CAT-8015
immunotoxin comprising the V.sub.H-PE38 subunit of SEQ ID NO:1 and
the V.sub.L subunit of SEQ ID NO:2.
[0017] The invention also provides a composition comprising a
purified immunoconjugate having less than between about 25% and
about 1% deamidated species, wherein said immunoconjugate is
purified by any of the methods described above. The composition can
have less than about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of
the deamidated species present. In certain embodiments, the
composition is a pharmaceutical composition comprising a purified
polypeptide or immunoconjugate and a pharmaceutically acceptable
carrier.
[0018] The invention also provides a formulation comprising 1 mg/mL
CAT-8015 in 25 mM sodium phosphate, 4% sucrose, 8% glycine, 0.02%
polysorbate 80 (PS80), pH 7.4. In further embodiments, the
formulation is lyophilized.
[0019] The invention also provides a method of modifying the
bioactivity of a polypeptide solution comprising a polypeptide and
a deamidated variant, the method comprising separating the
polypeptide from the deamidated variant by linear elution AIEX
chromatography; and combining the purified polypeptide and
deamidated variant in fixed quantities to obtain the desired
bioactivity of the polypeptide solution.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0020] FIG. 1. A graph depicting an ion exchange chromatography
(IEC) profile of a CAT-8015 reference standard. The pre-peak of
CAT-8015 represents the majority of inactive deamidated, or
iso-deamidated CAT-8015, while the main peak contains the majority
of the active, intact CAT-8015 immunoconjugate.
[0021] FIG. 2. A graph depicting the correlation between the
percent of relative potency of CAT-8015 and the percent of pre-peak
in the sample.
[0022] FIG. 3. A graph depicting an elution profile of bench-scale
purification of CAT-8015 by Q Sepharose HP Chromatography. CAT-8015
was purified using Q Sepharose HP. The majority of active, intact
CAT-8015 resided in fractions D5, D7, and D9 (the main peak
spanning from D3 to D12 as indicated above the peak).
[0023] FIG. 4. An SDS-PAGE analysis of QHP load and eluate pool
samples (bench-scale purification). Lane 1 corresponds to the QHP
load pool; Lane 2 corresponds to the QHP eluate pool; and Lane 3
corresponds to a CAT-8015 reference standard.
[0024] FIG. 5. Large-scale purification of CAT-8015 by Q Sepharose
HP Chromatography. CAT-8015 was purified using Q Sepharose HP. As
shown in the figure and Table 3, the majority of active, intact
CAT-8015 resided in fractions 5, 6, and 7.
[0025] FIG. 6. An SDS-PAGE analysis of QHP load and eluate pool
samples (large-scale purification). Lane 1 corresponds to the QHP
load pool; and Lane 2 corresponds to the QHP eluate pool.
[0026] FIG. 7. A graph depicting percent Pre-Peak in HA Product as
a Function of Solubilization pH as described in Example 6.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The invention provides methods for purifying an active
polypeptide or immunoconjugate from a solution containing the
polypeptide or immunoconjugate and an acidic variant thereof. In
one embodiment, the acidic variant comprises a deamidated form of
the polypeptide or immunoconjugate. In contrast to the expected
elution behavior from an anion exchange column, the bulk of
deamidated variants elute prior to intact polypeptides under salt
gradient elution conditions. Details of the methods are provided
herein.
[0028] The terms "polypeptide," "peptide," "protein," and "protein
fragment" are used interchangeably herein to refer to a polymer of
amino acid residues. The terms apply to amino acid polymers in
which one or more amino acid residue is an artificial chemical
mimetic of a corresponding naturally occurring amino acid, as well
as to naturally occurring amino acid polymers and non-naturally
occurring amino acid polymers.
[0029] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function similarly to the naturally occurring amino
acids. Naturally occurring amino acids are those encoded by the
genetic code, as well as those amino acids that are later modified,
e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine.
Amino acid analogs refers to compounds that have the same basic
chemical structure as a naturally occurring amino acid, e.g., an
alpha carbon that is bound to a hydrogen, a carboxyl group, an
amino group, and an R group, e.g., homoserine, norleucine,
methionine sulfoxide, methionine methyl sulfonium. Such analogs can
have modified R groups (e.g., norleucine) or modified peptide
backbones, but retain the same basic chemical structure as a
naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions
similarly to a naturally occurring amino acid. Negatively charged
amino acids include aspartic acid (or aspartate) and glutamic acid
(or glutamate). Positively charged amino acids include arginine,
histidine, and lysine.
[0030] The "composition" to be purified herein comprises the
polypeptide of interest and one or more impurities. The composition
may be "partially purified" (i.e., having been subjected to one or
more purification steps, such as by non-affinity chromatography
described herein or may be obtained directly from a host cell or
organism producing the polypeptide (e.g., the composition may
comprise harvested cell culture fluid).
[0031] The terms "polypeptide" or "polypeptide of interest" or
"protein of interest" and "target protein" or "protein" are used
interchangeably and refer to a protein or polypeptide such as an
antibody or immunoconjugate (as defined herein) that is to be
purified by a method of the invention from a mixture of proteins
and, other materials such as an acidic variant of the polypeptide
of interest.
[0032] An "acidic variant" is a variant of a polypeptide or
immunoconjugate which is more acidic (e.g., as determined by cation
exchange chromatography) than the polypeptide of interest. An
example of an acidic variant is a deamidated variant.
[0033] Deamidated proteins are those that have had some or all of
the free amide functional groups hydrolyzed to carboxylic acids,
such as conversion of glutamines to glutamic acid. The rate of this
reaction is dependent on the primary sequence, three-dimensional
structure, pH, temperature, buffer type, ionic strength and other
solution properties. Importantly, the deamidation reaction
introduces a negative charge into the molecule. As described
further below, the protein deamidation can have a negative impact
on protein activity.
[0034] As used herein, the terms "antibody" and "immunoglobulin"
are used interchangeably in the broadest sense and include
monoclonal antibodies (e.g., full length or intact monoclonal
antibodies), polyclonal antibodies, multivalent antibodies,
multispecific antibodies (e.g., bispecific antibodies so long as
they exhibit the desired biological activity) and antibody
fragments as described herein. The term "bispecific antibody" is
intended to include any antibody that has two different binding
specificities, i.e., the antibody binds two different epitopes,
which can be located on the same target antigen or, more commonly,
on different target antigens.
[0035] Native antibodies and immunoglobulins 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
between 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 and heavy chain
variable domains (Clothia et al., J. Mol. Biol. 186, 651-66,
(1985)); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82,
4592-4596 (1985)). Five human immunoglobulin classes are defined on
the basis of their heavy chain composition, and are named IgG, IgM,
IgA, IgE, and IgD. The IgG-class and IgA-class antibodies are
further divided into subclasses, namely, IgG1, IgG2, IgG3, and
IgG4, and IgA1 and IgA2. The heavy chains in IgG, IgA, and IgD
antibodies have three constant region domains, that are designated
CH1, CH2, and CH3, and the heavy chains in IgM and IgE antibodies
have four constant region domains, CH1, CH2, CH3, and CH4. Thus,
heavy chains have one variable region and three or four constant
regions Immunoglobulin structure and function are reviewed, for
example, in Harlow et al., Eds., Antibodies: A Laboratory Manual,
Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor
(1988).
[0036] The term "antibody fragment" refers to a portion of an
intact antibody and refers to the antigenic determining variable
regions of an intact antibody. Examples of antibody fragments
include, but are not limited to Fab, Fab', F(ab')2, Fv and single
chain Fv fragments, linear antibodies, single chain antibodies, and
multispecific antibodies formed from antibody fragments.
[0037] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific and bind a single antigen.
Furthermore, in contrast to polyclonal antibody preparations that
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. That an antibody
"selectively binds" or "specifically binds" means that the antibody
reacts or associates more frequently, more rapidly, with greater
duration, with greater affinity, or with some combination of the
above to an epitope than with alternative substances, including
unrelated proteins. "Selectively binds" or "specifically binds"
means, for instance, that an antibody binds to a protein with a
K.sub.D of at least about 0.1 mM, but more usually at least about 1
.mu.M. "Selectively binds" or "specifically binds" means at times
that an antibody binds to a protein at times with a K.sub.D of at
least about 0.1 .mu.M or better, and at other times at least about
0.01 .mu.M or better. Because of the sequence identity between
homologous proteins in different species, specific binding can
include an antibody that recognizes a tumor cell marker protein in
more than one species.
[0038] The 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
(U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad.
Sci. USA 81:6851-6855 (1984)).
[0039] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies can comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the 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 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 the
following review articles and references cited therein: 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).
[0040] A "human antibody" is one that possesses an amino acid
sequence that 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.
[0041] The term "immunoconjugate" or "conjugate" or "immunotoxin"
as used herein refers to a compound or a derivative thereof that is
linked to a cell binding agent (e.g., an anti-CD22 antibody or
fragment thereof) and is defined by a generic formula: C-L-A,
wherein C=cytotoxin, L=linker, and A=cell binding agent (e.g.,
anti-CD22 antibody or antibody fragment) Immunoconjugates can also
be defined by the generic formula in reverse order: A-L-C.
[0042] The term "cytotoxin" or "cytotoxic agent" as used herein
refers to a substance that inhibits or prevents the function of
cells and/or causes destruction of cells. The term is intended to
include radioactive isotopes (e.g., At.sup.211, I.sup.131,
I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153,
Bi.sup.212, P.sup.32 and radioactive isotopes of Lu),
chemotherapeutic agents e.g., methotrexate, adriamycin, vinca
alkaloids (vincristine, vinblastine, etoposide), doxorubicin,
melphalan, mitomycin C, chlorambucil, daunorubicin or other
intercalating agents, enzymes and fragments thereof such as
nucleolytic enzymes, antibiotics, and toxins such as small molecule
toxins or enzymatically active toxins of bacterial, fungal, plant
or animal origin, including fragments and/or variants thereof, and
the various antitumor or anticancer agents disclosed below.
Examples of cytotoxic agents include, but are not limited to,
abrin, ricin, Pseudomonas exotoxin (PE), diphtheria toxin (DT),
botulinum toxin, or modified toxins thereof. For example, PE and DT
are highly toxic compounds that typically bring about death through
liver toxicity. PE and DT, however, can be modified into a form for
use as an immunotoxin by removing the native targeting component of
the toxin (e.g., domain la of PE or the B chain of DT) and
replacing it with a different targeting moiety, such as an
antibody.
[0043] In some embodiments, the toxin is Pseudomonas exotoxin.
Pseudomonas exotoxin A (PEA) is an extremely active monomeric
protein (molecular weight 66 kD), secreted by Pseudomonas
aeruginosa, which inhibits protein synthesis in eukaryotic cells
through the inactivation of elongation factor 2 (EF-2) by
catalyzing its ADP-ribosylation (catalyzing the transfer of the ADP
ribosyl moiety of oxidized NAD onto EF-2).
[0044] The toxin contains three structural domains that act in
concert to cause cytotoxicity. Domain Ia (amino acids 1-252)
mediates cell binding. Domain II (amino acids 253-364) is
responsible for translocation into the cytosol and domain III
(amino acids 400-613) mediates ADP ribosylation of elongation
factor 2, which inactivates the protein and causes cell death. The
function of domain Ib (amino acids 365-399) remains undefined,
although a large part of it, amino acids 365-380, can be deleted
without loss of cytotoxicity. See Siegall et al., J. Biol. Chem.
264: 14256-14261 (1989).
[0045] The Pseudomonas exotoxins (PEs) employed in the present
invention include the native sequence, cytotoxic fragments of the
native sequence, and conservatively modified variants of native PE
and its cytotoxic fragments. Cytotoxic fragments of PE include
those which are cytotoxic with or without subsequent proteolytic or
other processing in the target cell (e.g., as a protein or
pre-protein). Cytotoxic fragments of PE include PE40, PE38, and
PE35. PE40 is a truncated derivative of PE as previously described
in the art. See, Pai et al., Proc. Natl. Acad. Sci. USA, 88:3358-62
(1991); Kondo et al., J. Biol. Chem. 263:9470-9475 (1988). PE38 is
a truncated PE composed of amino acids 253-364 and 381-613 of
native PE. PE35 is a 35 kD carboxyl-terminal fragment of PE
composed of a Met at position 280 followed by amino acids 281-364
and 381-613 of native PE. In one embodiment, the cytotoxic fragment
PE38 is employed. PE38 is a pro-protein which can be activated to
its cytotoxic form upon processing within a cell.
[0046] A "PE immunoconjugate" or "PE immunotoxin" is an
immunoconjugate or immunotoxin comprising an antibody or antigen
binding fragment thereof and a PE toxin or variant thereof.
[0047] By "purifying" a polypeptide or immunoconjugate from a
composition comprising the polypeptide and one or more impurities
is meant increasing the degree of purity of the polypeptide in the
composition by removing (completely or partially) at least one
impurity from the composition. According to the present invention,
purification is performed without the use of an affinity
chromatography step.
[0048] The term "chromatography" refers to the process by which a
solute of interest in a mixture is separated from other solutes in
a mixture as a result of differences in rates at which the
individual solutes of the mixture migrate through a stationary
medium under the influence of a moving phase, or in bind and elute
processes.
[0049] The term "ion-exchange" and "ion-exchange chromatography"
refers to the chromatographic process in which a solute of interest
(such as a protein) in a mixture interacts with a charged compound
linked (such as by covalent attachment) to a solid phase ion
exchange material such that the solute of interest interacts
non-specifically with the charged compound more or less than solute
impurities or contaminants in the mixture. The contaminating
solutes in the mixture elute from a column of the ion exchange
material faster or slower than the solute of interest or are bound
to or excluded from the resin relative to the solute of interest.
"Ion-exchange chromatography" specifically includes cation
exchange, anion exchange, and mixed mode chromatography.
[0050] The phrase "ion exchange material" refers to a solid phase
that is negatively charged (i.e., a cation exchange resin) or
positively charged (i.e., an anion exchange resin). The charge may
be provided by attaching one or more charged ligands to the solid
phase, e.g., by covalent linking. Alternatively, or in addition,
the charge may be an inherent property of the solid phase (e.g., as
is the case for silica, which has an overall negative charge).
[0051] An "anion exchange resin" refers to a solid phase which is
positively charged, thus having one or more positively charged
ligands attached thereto. Any positively charged ligand attached to
the solid phase suitable to form the anionic exchange resin can be
used, such as quaternary amino groups Commercially available anion
exchange resins include DEAE cellulose, Poros PI 20, PI 50, HQ 10,
HQ 20, HQ 50, D 50 from Applied Biosystems, Sartobind Q from
Sartorius, MonoQ, MiniQ, Source 15Q and 30Q, Q, DEAE and ANX
Sepharose Fast Flow, Q Sepharose High Performance, QAE SEPHADEX.TM.
and FAST Q SEPHAROSE.TM. (GE Healthcare), WP PEI, WP DEAM, WP QUAT
from J. T. Baker, Hydrocell DEAE and Hydrocell QA from Biochrom
Labs Inc., UNOsphere Q, Macro-Prep DEAE and Macro-Prep High Q from
Biorad, Ceramic HyperD Q, ceramic HyperD DEAE, Trisacryl M and LS
DEAE, Spherodex LS DEAE, QMA Spherosil LS, QMA Spherosil M and
Mustang Q from Pall Technologies, DOWEX Fine Mesh Strong Base Type
I and Type II Anion Resins and DOWEX MONOSPHER E 77, weak base
anion from Dow Liquid Separations, Intercept Q membrane, Matrex
Cellufine A200, A500, Q500, and Q800, from Millipore, Fractogel EMD
TMAE, Fractogel EMD DEAE and Fractogel EMD DMAE from EMD, Amberlite
weak strong anion exchangers type I and II, DOWEX weak and strong
anion exchangers type I and II, Diaion weak and strong anion
exchangers type I and II, Duolite from Sigma-Aldrich, TSK gel Q and
DEAE 5PW and 5PW-HR, Toyopearl SuperQ-650S, 650M and 650C, QAE-550C
and 650S, DEAE-650M and 650C from Tosoh, QA52, DE23, DE32, DE51,
DE52, DE53, Express-Ion D and Express-Ion Q from Whatman.
[0052] By "solid phase" is meant a non-aqueous matrix to which one
or more charged ligands can adhere. The solid phase may be a
purification column, a discontinuous phase of discrete particles, a
membrane, or filter etc. Examples of materials for forming the
solid phase include polysaccharides (such as agarose and
cellulose); and other mechanically stable matrices such as silica
(e.g., controlled pore glass), poly(styrenedivinyl)benzene,
polyacrylamide, ceramic particles and derivatives of any of the
above.
[0053] The term "specific binding" as used herein, such as to
describe interactions between a molecule of interest and a ligand
bound to a solid phase matrix, refers to the generally reversible
binding of a protein of interest to a ligand through the combined
effects of spatial complementarity of protein and ligand structures
at a binding site coupled with electrostatic forces, hydrogen
bonding, hydrophobic forces, and/or van der Waals forces at the
binding site. The greater the spatial complementarity and the
stronger the other forces at the binding site, the greater will be
the binding specificity of a protein for its respective ligand.
Non-limiting examples of specific binding includes antibody-antigen
binding, enzyme-substrate binding, enzyme-cofactor binding, metal
ion chelation, DNA binding protein-DNA binding, regulatory
protein-protein interactions, and the like.
[0054] The term "non-specific binding" as used herein, such as to
describe interactions between a molecule of interest and a ligand
or other compound bound to a solid phase matrix, refers to binding
of a protein of interest to the ligand or compound on a solid phase
matrix through electrostatic forces, hydrogen bonding, hydrophobic
forces, and/or van der Waals forces at an interaction site, but
lacking structural complementarity that enhances the effects of the
non-structural forces. Examples of non-specific interactions
include, but are not limited to, electrostatic, hydrophobic, and
van der Waals forces as well as hydrogen bonding.
[0055] A "salt" is a compound formed by the interaction of an acid
and a base. A salt useful for the invention include, but are not
limited to acetate (e.g., sodium acetate), citrate (e.g., sodium
citrate), chloride (e.g., sodium chloride), sulphate (e.g., sodium
sulphate), or a potassium salt.
[0056] The term "detergent" refers to ionic and nonionic
surfactants such as polysorbates (e.g., polysorbates 20 or 80);
poloxamers (e.g., poloxamer 188); Triton; sodium dodecyl sulfate
(SDS); sodium lauryl sulfate; sodium octyl glucoside; 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 oleoyl-taurate; and the MONAQUAT.TM. series (Mona
Industries, Inc., Paterson, N.J.). A useful detergent is a
polysorbate, such as polysorbate 20 (TWEEN 20.RTM.) or polysorbate
80 (TWEEN 80.RTM.).
[0057] A "buffer" used in the present invention is a solution that
resists changes in pH by the addition of acid or base by the action
of its acid-base conjugates components. Various buffers can be
employed in a method of the present invention depending on the
desired pH of the buffer and the particular step in the
purification process [see Buffers. A Guide for the Preparation and
Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem
Corporation (1975)]. Non-limiting examples of buffer components
that can be used to control the pH range desirable for a method of
the invention include acetate, citrate, histidine, phosphate,
ammonium buffers such as ammonium acetate, succinate, MES, CHAPS,
MOPS, MOPSO, HEPES, Tris, and the like, as well as combinations of
these TRIS-malic acid-NaOH, maleate, chloroacetate, formate,
benzoate, propionate, pyridine, piperazine, ADA, PIPES, ACES, BES,
TES, tricine, bicine, TAPS, ethanolamine, CHES, CAPS, methylamine,
piperidine, O-boric acid, carbonic acid, lactic acid, butanedioic
acid, diethylmalonic acid, glycylglycine, HEPPS, HEPPSO, imidazole,
phenol, POPSO, succinate, TAPS, amine-based, benzylamine, trimethyl
or dimethyl or ethyl or phenyl amine, ethylenediamine, or
morpholine. Additional components (additives) can be present in a
buffer as needed, e.g., salts can be used to adjust buffer ionic
strength, such as sodium chloride, sodium sulfate and potassium
chloride; and other additives such as amino acids (such as glycine
and histidine), chaotropes (such as urea), alcohols (such as
ethanol, mannitol, glycerol, and benzyl alcohol), detergents (see
supra.), and sugars (such as sucrose, mannitol, maltose, trehalose,
glucose, and fructose). The buffer components and additives, and
the concentrations used, can vary according to the type of
chromatography practiced in the invention.
[0058] The "loading buffer" is that which is used to load the
composition comprising the polypeptide molecule of interest and one
or more impurities onto the ion exchange resin. The loading buffer
has a conductivity and/or pH such that the polypeptide molecule of
interest (and generally one or more impurities) is/are bound to the
ion exchange resin or such that the protein of interest flows
through the column while the impurities bind to the resin.
[0059] The term "wash buffer" when used herein refers to a buffer
used to wash or re-equilibrate the ion exchange resin, prior to
eluting the polypeptide molecule of interest. Conveniently, the
wash buffer and loading buffer may be the same, but this is not
required.
[0060] The "elution buffer" is used to elute the polypeptide of
interest from the solid phase. The conductivity and/or pH of the
elution buffer is/are such that the polypeptide of interest is
eluted from the ion exchange resin.
[0061] The "pI" or "isoelectric point" of a polypeptide refer to
the pH at which the polypeptide's positive charge balances its
negative charge. pI can be calculated from the net charge of the
amino acid residues or sialic acid residues of attached
carbohydrates of the polypeptide or can be determined by
isoelectric focusing.
[0062] By "binding" a molecule to an ion exchange material is meant
exposing the molecule to the ion exchange material under
appropriate conditions (pH/conductivity) such that the molecule is
reversibly immobilized in or on the ion exchange material by virtue
of ionic interactions between the molecule and a charged group or
charged groups of the ion exchange material.
[0063] By "washing" the ion exchange material is meant passing an
appropriate buffer through or over the ion exchange material.
[0064] To "elute" a molecule (e.g., polypeptide or impurity) from
an ion exchange material is meant to remove the molecule therefrom
by altering the ionic strength of the buffer surrounding the ion
exchange material such that the buffer competes with the molecule
for the charged sites on the ion exchange material.
[0065] As used in the present disclosure and claims, the singular
forms "a," "an," and "the" include plural forms unless the context
clearly dictates otherwise.
[0066] It is understood that wherever embodiments are described
herein with the language "comprising," otherwise analogous
embodiments described in terms of "consisting of" and/or
"consisting essentially of" are also provided.
[0067] The term "and/or" as used in a phrase such as "A and/or B"
herein is intended to include both "A and B," "A or B," "A," and
"B." Likewise, the term "and/or" as used in a phrase such as "A, B,
and/or C" is intended to encompass each of the following
embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and
C; A and B; B and C; A (alone); B (alone); and C (alone).
[0068] Pseudomonas Exotoxin and Other Toxins
[0069] Toxins can be employed with antibodies of the present
invention to yield immunotoxins. Exemplary toxins include ricin,
abrin, diphtheria toxin and subunits thereof, as well as botulinum
toxins A through F. These toxins are readily available from
commercial sources (e.g., Sigma Chemical Company, St. Louis, Mo.).
Diphtheria toxin is isolated from Corynebacterium diphtheriae.
Ricin is the lectin RCA60 from Ricinus communis (Castor bean). The
term also references toxic variants thereof. For example, see, U.S.
Pat. Nos. 5,079,163 and 4,689,401. Ricinus communis agglutinin
(RCA) occurs in two forms designated RCA.sub.60 and RCA.sub.120
according to their molecular weights of approximately 65 and 120
kD, respectively (Nicholson & Blaustein, J. Biochem. Biophys.
Acta 266:543 (1972)). The A chain is responsible for inactivating
protein synthesis and killing cells. The B chain binds ricin to
cell-surface galactose residues and facilitates transport of the A
chain into the cytosol (Olsnes, et al., Nature 249:627-631 (1974)
and U.S. Pat. No. 3,060,165).
[0070] Abrin includes toxic lectins from Abrus precatorius. The
toxic principles, abrin a, b, c, and d, have a molecular weight of
from about 63 to 67 kD and are composed of two disulfide-linked
polypeptide chains A and B. The A chain inhibits protein synthesis;
the B-chain (abrin-b) binds to D-galactose residues (see, Funatsu,
et al., Agr. Biol. Chem. 52:1095 (1988); and Olsnes, Methods
Enzymol. 50:330-335 (1978)).
[0071] In preferred embodiments of the present invention, the toxin
is Pseudomonas exotoxin (PE). The Pseudomonas exotoxin (or exotoxin
A) is an exotoxin produced by Pseudomonas aeruginosa. The term
"Pseudomonas exotoxin" as used herein refers to a full-length
native (naturally occurring) PE or a PE that has been modified.
Such modifications may include, but are not limited to, elimination
of domain Ia, various amino acid deletions in domains Ib, II and
III, single amino acid substitutions and the addition of one or
more sequences at the carboxyl terminus such as KDEL (SEQ ID NO:3)
and REDL (SEQ ID NO:4). See Siegall, et al., J. Biol. Chem.
264:14256-14261 (1989). In a preferred embodiment, the cytotoxic
fragment of PE retains at least 50%, preferably 75%, more
preferably at least 90%, and most preferably 95% of the
cytotoxicity of native PE. In a most preferred embodiment, the
cytotoxic fragment is more toxic than native PE.
[0072] Native Pseudomonas exotoxin A (PEA) is an extremely active
monomeric protein (molecular weight 66 kD), secreted by Pseudomonas
aeruginosa, which inhibits protein synthesis in eukaryotic cells.
The native PEA sequence is provided in commonly assigned U.S. Pat.
No. 5,602,095, incorporated herein by reference. The method of
action is inactivation of the ADP-ribosylation of elongation factor
2 (EF-2). The exotoxin contains three structural domains that act
in concert to cause cytotoxicity. Domain Ia (amino acids 1-252)
mediates cell binding. Domain II (amino acids 253-364) is
responsible for translocation into the cytosol and domain III
(amino acids 400-613) mediates ADP ribosylation of elongation
factor 2. The function of domain lb (amino acids 365-399) remains
undefined, although a large part of it, amino acids 365-380, can be
deleted without loss of cytotoxicity. See Siegall, et al., (1989),
supra.
[0073] PE employed in the present invention include the native
sequence, cytotoxic fragments of the native sequence, and
conservatively modified variants of native PE and its cytotoxic
fragments. PE variants useful in the invention are described in
U.S. Pat. No. 7,355,012, and WO 2007/016150 and WO 2009/032954.
Cytotoxic fragments of PE include those which are cytotoxic with or
without subsequent proteolytic or other processing in the target
cell (e.g., as a protein or pre-protein). Cytotoxic fragments of PE
include PE40, PE38, and PE35.
[0074] In preferred embodiments, the PE has been modified to reduce
or eliminate non-specific cell binding, frequently by deleting
domain Ia as taught in U.S. Pat. No. 4,892,827, although this can
also be achieved, for example, by mutating certain residues of
domain Ia. U.S. Pat. No. 5,512,658, for instance, discloses that a
mutated PE in which Domain Ia is present but in which the basic
residues of domain Ia at positions 57, 246, 247, and 249 are
replaced with acidic residues (glutamic acid, or "E")) exhibits
greatly diminished non-specific cytotoxicity. This mutant form of
PE is sometimes referred to as PE4E.
[0075] PE40 is a truncated derivative of PE as previously described
in the art, with a deletion of domain Ia of the native PE molecule.
See, Pai, et al., Proc. Nat'l Acad. Sci. USA 88:3358-62 (1991); and
Kondo, et al., J. Biol. Chem. 263:9470-9475 (1988). PE35 is a 35 kD
carboxyl-terminal fragment of PE in which amino acid residues 1-279
have been deleted and the molecule commences with a met at position
280 followed by amino acids 281-364 and 381-613 of native PE. PE35
and PE40 are disclosed, for example, in U.S. Pat. Nos. 5,602,095
and 4,892,827. PE4E is a form of PE where all of the domains of
native PE are present, but where the basic residues of domain Ia at
positions 57, 246, 247 and 249 are replaced with acidic residues
(glutamine acid, or "E").
[0076] In some preferred embodiments, the cytotoxic fragment PE38
is employed. PE38 is a truncated PE pro-protein composed of amino
acids 253-364 and 381-613 which is activated to its cytotoxic form
upon processing within a cell (see e.g., U.S. Pat. Nos. 5,608,039,
7,355,012, and Pastan et al., Biochim. Biophys. Acta
1333:C.sub.1-C.sub.6 (1997)).
[0077] As noted above, some or all of domain lb may be deleted, and
the remaining portions joined by a linker or directly by a peptide
bond. Some of the amino portion of domain II may be deleted. And,
the C-terminal end may contain the native sequence of residues
609-613 (REDLK) (SEQ ID NO:5), or may contain a variation found to
maintain the ability of the construct to translocate into the
cytosol, such as REDL (SEQ ID NO:4) or KDEL (SEQ ID NO:3), and
repeats of these sequences. See, e.g., U.S. Pat. Nos. 5,854,044;
5,821,238; and 5,602,095 and WO 99/51643. While in preferred
embodiments, the PE is PE4E, PE40, or PE38, any form of PE in which
non-specific cytotoxicity has been eliminated or reduced to levels
in which significant toxicity to non-targeted cells does not occur
can be used in the immunotoxins of the present invention so long as
it remains capable of translocation and EF-2 ribosylation in a
targeted cell.
[0078] Conservatively Modified Variants of PE
[0079] Conservatively modified variants of PE or cytotoxic
fragments thereof have at least 80% sequence similarity, preferably
at least 85% sequence similarity, more preferably at least 90%
sequence similarity, and most preferably at least 95% sequence
similarity at the amino acid level, with the PE of interest, such
as PE38.
[0080] The term "conservatively modified variants" applies to both
amino acid and nucleic acid sequences. With respect to particular
nucleic acid sequences, conservatively modified variants refer to
those nucleic acid sequences which encode identical or essentially
identical amino acid sequences, or if the nucleic acid does not
encode an amino acid sequence, to essentially identical nucleic
acid sequences. Because of the degeneracy of the genetic code, a
large number of functionally identical nucleic acids encode any
given polypeptide. For instance, the codons GCA, GCC, GCG and GCU
all encode the amino acid alanine. Thus, at every position where an
alanine is specified by a codon, the codon can be altered to any of
the corresponding codons described without altering the encoded
polypeptide. Such nucleic acid variations are "silent variations,"
which are one species of conservatively modified variations. Every
nucleic acid sequence herein which encodes a polypeptide also
describes every possible silent variation of the nucleic acid. One
of skill will recognize that each codon in a nucleic acid (except
AUG, which is ordinarily the only codon for methionine) can be
modified to yield a functionally identical molecule. Accordingly,
each silent variation of a nucleic acid which encodes a polypeptide
is implicit in each described sequence.
[0081] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid.
[0082] Pseudomonas exotoxins employed in the invention can be
assayed for the desired level of cytotoxicity by assays well known
to those of skill in the art. Thus, cytotoxic fragments of PE and
conservatively modified variants of such fragments can be readily
assayed for cytotoxicity. A large number of candidate PE molecules
can be assayed simultaneously for cytotoxicity by methods well
known in the art. For example, subgroups of the candidate molecules
can be assayed for cytotoxicity. Positively reacting subgroups of
the candidate molecules can be continually subdivided and reassayed
until the desired cytotoxic fragment(s) is identified. Such methods
allow rapid screening of large numbers of cytotoxic fragments or
conservative variants of PE.
Anti-CD22/PE Immunoconjugates
[0083] In one embodiment, the polypeptide of interest comprises an
antibody that specifically binds CD22. "CD22" refers to a
lineage-restricted B cell antigen belonging to the Ig superfamily.
It is expressed in 60-70% of B cell lymphomas and leukemias and is
not present on the cell surface in early stages of B cell
development or on stem cells. See, e.g., Vaickus et al., Crit. Rev.
Oncol/Hematol. 11:267-297 (1991). In another embodiment, the
polypeptide of interest is an antibody fragment that binds CD22
(e.g., Fab, or scFv).
[0084] As used herein, the term "anti-CD22" in reference to an
antibody, refers to an antibody that specifically binds CD22 and
includes reference to an antibody which is generated against CD22.
In some embodiments, the CD22 is a primate CD22 such as human CD22.
In one embodiment, the antibody is generated against human CD22
synthesized by a non-primate mammal after introduction into the
animal of cDNA which encodes human CD22. In a further embodiment,
the polypeptide of interest is a CD22 antibody immunoconjugate that
comprises the PE38 exotoxin.
[0085] One example of a CD22/PE38 immunoconjugate is CAT-8015
described in International Patent Application Publication Nos. WO
98/41641 and WO2003/27135, U.S. Pat. Nos. 7,541,034, 7,355,012, and
U.S. Publication No. 2007/0189962, all of which are herein
incorporated by reference. CAT-8015 is a recombinant immunotoxin
protein composed of an antibody Fv fragment based on the murine
anti-CD22 antibody RFB4 fused to a truncated form of the
Pseudomonas exotoxin protein, PE38. The anti-CD22 Fv fragment
consists of two domains, a V.sub.L and a V.sub.H, where the latter
was modified to improve binding to the human CD22 target. The
CAT-8015 protein is comprised of two independent polypeptides, the
V.sub.L chain (SEQ ID NO:2), and the V.sub.H chain, fused at the
C-terminus to the PE38 domain (V.sub.H-PE38) (SEQ ID NO:1). Other
V.sub.L and V.sub.H-PE38 sequences useful in this invention are
described in U.S. Pat. Nos. 7,541,034, 7,355,012, and 2007/0189962.
Both domains were designed to each contain engineered cysteine
residues that permit formation of an intermolecular disulfide bond.
This feature increases the stability of the fusion protein.
[0086] The amino acid sequence of the V.sub.H-P38 Subunit (SEQ ID
NO:1) of CAT-8015 is the following:
TABLE-US-00001 (SEQ ID NO: 1)
MEVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKCLEWV
AYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCA
RHSGYGTHWGVLFAYWGQGTLVTVSAKASGGPEGGSLAALTAHQACHLP
LETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALA
SPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGP
ADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEE
RGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYG
YAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVER
LIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNV
GGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK
[0087] The PE38 sequence is shown in bold, and the five amino acid
linker between the V.sub.H domain and the PE38 domain is shown
underlined.
[0088] The amino acid sequence of the V.sub.L Subunit (SEQ ID NO:2)
of CAT-8015 is the following:
TABLE-US-00002 (SEQ ID NO: 2)
MDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLI
YYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWT FGCGTKLEIK
[0089] In further embodiments, the amino acid sequence of the
V.sub.H domain of the immunoconjugate is one of the following:
TABLE-US-00003 (SEQ ID NO: 6)
MEVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKCLEWV
AYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCA
RHSGYGTHWGVLFAYWGQGTLVTVSA (SEQ ID NO: 7)
MEVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKCLEWV
AYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCA
RHSGYGYNWGVLFAYWGQGTLVTVSA (SEQ ID NO: 8)
MEVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKCLEWV
AYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCA
RHSGYGTTWGVLFAYWGQGTLVTVSA (SEQ ID NO: 9)
MEVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKCLEWV
AYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCA
RHSGYGSTYGVLFAYWGQGTLVTVSA (SEQ ID NO: 10)
MEVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKCLEWV
AYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCA
RHSGYGTHWGVLFAYWGQGTLVTVSA (SEQ ID NO: 11)
MEVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKCLEWV
AYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCA
RHSGYGSSYGVLFAYWGQGTLVTVSA
[0090] In additional embodiments, the amino acid sequence of the
V.sub.L domain of the immunoconjugate is one of the following:
TABLE-US-00004 (SEQ ID NO: 12)
MDIQMTQTTSSLSASLGDRVTISCRASQDIARYLNWYQQKPDGTVKLLI
YYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWT FGCGTKLEIK (SEQ
ID NO: 13) MDIQMTQTTSSLSASLGDRVTISCRASQDIHGYLNWYQQKPDGTVKLLI
YYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWT FGCGTKLEIK (SEQ
ID NO: 14) MDIQMTQTTSSLSASLGDRVTISCRASQDIGRYLNWYQQKPDGTVKLLI
YYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWT FGCGTKLEIK (SEQ
ID NO: 15) MDIQMTQTTSSLSASLGDRVTISCRASQDIRGYLNWYQQKPDGTVKLLI
YYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWT FGCGTKLEIK
[0091] In certain other embodiments, the PE toxin of the
immunoconjugate is a PE or variant thereof selected from the
following:
TABLE-US-00005 Native PE (SEQ ID NO: 16)
AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVL
EGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWS
LNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLAR
DATFFVRAHESNEMQPTLAISHAGVSVVMAQTQPRREKRWSEWASGKVL
CLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVIS
HRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRL
VALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLA
AAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDVS
FSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRA
RSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRS
SLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETI
LGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQP GKPPREDLK PE40
(SEQ ID NO: 17) GGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAAR
LSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVR
QGTGNDEAGAANADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLG
DGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVF
GGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVY
VPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRL
ETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYAS QPGKPPREDLK PE38
(SEQ ID NO: 18) GGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAAR
LSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVR
QGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWT
VERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVEGGVRARSQDLDAIWRG
FYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTL
AAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIP
SAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK PE35 (SEQ ID NO: 19)
MWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAI
REQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYP
TGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLE
AAQSIVEGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRN
GALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGP
EEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAIS
ALPDYASQPGKPPREDLK PE-LR (SEQ ID NO: 20)
RHRQPRGWEQLPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERG
YVFVGYHGTFLEAAQSIVEGGVRARSQDLDAIWRGFYIAGDPALAYGYAQ
DQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGH
PLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLD
PSSIPDKEQAISALPDYASQPGKPPREDLK PE-LR-6X (SEQ ID NO: 21)
RHRQPRGWEQLPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEEGG
YVFVGYHGTFLEAAQSIVEGGVRARSQDLDAIWAGFYIAGDPALAYGYAQ
DQEPDAAGRIRNGALLRVYVPRSSLPGFYATSLTLAAPEAAGEVERLIGH
PLPLRLDAITGPEEAGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLD
PSSIPDSEQAISALPDYASQPGKPPREDLK PE-38 (CAT-8015) (SEQ ID NO: 22)
PEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLA
ARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERF
VRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQN
WTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVEGGVRARSQDLDAIW
RGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSL
TLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVV
IPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK
[0092] The PE toxin of the immunoconjugate is fused or conjugated
to either the V.sub.H or V.sub.L domain directly or via a linker at
either the N-terminus or the C-terminus of the V.sub.H or V.sub.L
domain. An example of a linker is described above for CAT-8015 and
corresponds to the amino acid sequence KASGG (SEQ ID NO: 23).
Additional linkers can be readily generated by techniques known in
the art.
[0093] Expression of a PE Immunoconjugate
[0094] The PE immunoconjugate of the present invention is expressed
in cells, such as bacterial cells, and then isolated from inclusion
bodies. The PE immunoconjugate isolated from inclusion bodies is
then further purified using downstream purification steps.
[0095] A variety of host-expression vector systems may be utilized
to express the PE immunoconjugate of the present invention. Such
host-expression systems represent vehicles by which the coding
sequences of interest may be produced and subsequently purified,
but also represent cells which may, when transformed or transfected
with the appropriate nucleotide coding sequences, express an
antibody molecule of the invention in situ. These include but are
not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing antibody coding
sequences; yeast (e.g., Saccharomyces, Pichia) transformed with
recombinant yeast expression vectors containing antibody coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing antibody coding
sequences; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors containing antibody coding sequences; or
mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3 cells)
harboring recombinant expression constructs containing promoters
derived from the genome of mammalian cells (e.g., metallothionein
promoter) or from mammalian viruses (e.g., the adenovirus late
promoter; the vaccinia virus 7.5K promoter).
[0096] DNA encoding each of the V.sub.L and V.sub.H-PE toxin (e.g.,
V.sub.H-PE38) polypeptides is introduced into an expression vector
by techniques well known in the art.
[0097] A "vector" refers to any vehicle for the cloning of and/or
transfer of a nucleic acid into a host cell. A vector may be a
replicon to which another DNA segment may be attached so as to
bring about the replication of the attached segment. A "replicon"
refers to any genetic element (e.g., plasmid, phage, cosmid,
chromosome, virus) that functions as an autonomous unit of DNA
replication in vivo, i.e., capable of replication under its own
control. The term "vector" includes vehicles for introducing the
nucleic acid into a cell in vitro, ex vivo or in vivo. A large
number of vectors known in the art may be used to manipulate
nucleic acids, incorporate response elements and promoters, such as
inducible promoters, into genes, etc. Possible vectors include, for
example, plasmids such as pBR322 or pUC plasmid derivatives, or the
Bluescript vector. For example, the insertion of the DNA fragments
corresponding to response elements and promoters into a suitable
vector can be accomplished by ligating the appropriate DNA
fragments into a chosen vector that has complementary cohesive
termini. Alternatively, the ends of the DNA molecules may be
enzymatically modified or any site may be produced by ligating
nucleotide sequences (linkers) into the DNA termini. Such vectors
may be engineered to contain selectable marker genes that provide
for the selection of cells. Such markers allow identification
and/or selection of host cells that express the proteins encoded by
the marker.
[0098] The term "expression vector" refers to a vector, plasmid or
vehicle designed to enable the expression of an inserted nucleic
acid sequence following transformation into the host. The cloned
gene, i.e., the inserted nucleic acid sequence, e.g., a gene
encoding an anti-CD22 V.sub.H, anti-CD22 V.sub.L, or anti-CD22
V.sub.H or V.sub.L fused to a PE toxin, is usually placed under the
control of control elements such as a promoter, a minimal promoter,
an enhancer, or the like. Initiation control regions or promoters,
which are useful to drive expression of a nucleic acid in the
desired host cell are numerous and familiar to those skilled in the
art. Virtually any promoter capable of driving expression of these
genes can be used in an expression vector, including but not
limited to, viral promoters, bacterial promoters, animal promoters,
mammalian promoters, synthetic promoters, constitutive promoters,
tissue specific promoters, pathogenesis or disease related
promoters, developmental specific promoters, inducible promoters,
light regulated promoters; including, but are not limited to, the
SV40 early (SV40) promoter region, the promoter contained in the 3'
long terminal repeat (LTR) of Rous sarcoma virus (RSV), the E1A or
major late promoter (MLP) of adenoviruses (Ad), the human
cytomegalovirus (HCMV) immediate early promoter, the herpes simplex
virus (HSV) thymidine kinase (TK) promoter, the baculovirus IE1
promoter, the elongation factor 1 alpha (EF1) promoter, the
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, the
phosphoglycerate kinase (PGK) promoter, the ubiquitin C (Ube)
promoter, the albumin promoter, the regulatory sequences of the
mouse metallothionein-L promoter and transcriptional control
regions, the ubiquitous promoters (HPRT, vimentin, .beta.-actin,
tubulin and the like), the promoters of the intermediate filaments
(desmin, neurofilaments, keratin, GFAP, and the like), the
promoters of therapeutic genes (of the MDR, CFTR or factor VIII
type, and the like), pathogenesis or disease related-promoters. In
addition, these expression sequences may be modified by addition of
enhancer or regulatory sequences and the like.
[0099] The term "expression" refers to the biological production of
a product encoded by a coding sequence. In most cases a DNA
sequence, including the coding sequence, is transcribed to form a
messenger-RNA (mRNA). The messenger-RNA is then translated to form
a polypeptide product which has a relevant biological activity.
Also, the process of expression may involve further processing
steps to the RNA product of transcription, such as splicing to
remove introns, and/or post-translational processing of a
polypeptide product.
[0100] The V.sub.L and V.sub.H-PE38 polypeptides are expressed in
cells, e.g., bacterial cells, such as E. coli. The polypeptides are
expressed, e.g., in E. coli cells and isolated from inclusion
bodies. In certain embodiments, the V.sub.L and V.sub.H-PE38
subunits are expressed in different cells. For example, the V.sub.L
is expressed in one cell on a first vector and the V.sub.H-PE38 is
expressed in a different cell on a second vector. Inclusion bodies
from each cell line are recovered and solubilized. In certain
embodiments, the inclusion bodies are solubilized at a pH in a
range of about 9.0 to about 10.5. In further embodiments, the
inclusion bodies are solubilized at a pH of 9.0, at a pH of 9.5, at
a pH of 10.0 or a pH of 10.5. The solubilized V.sub.L and
V.sub.H-PE38 inclusion bodies are combined to form an
immunoconjugate comprising the V.sub.L and V.sub.H-PE38
subunits.
[0101] In other embodiments, the V.sub.L and V.sub.H-PE38 subunits
are expressed in the same cell on different vectors, for example,
the V.sub.L is expressed in one cell on a first vector, and the
V.sub.H-PE38 is expressed in the same cell on a different vector.
Inclusion bodies from the cell are recovered, solubilized and the
V.sub.L and V.sub.H-PE38 subunits combined to form an
immunoconjugate. In certain other embodiments, the V.sub.L and
V.sub.H-PE38 subunits are expressed on the same vector in the same
cell.
[0102] Downstream chromatography steps are utilized to further
purify this immunoconjugate.
Chromatography Conditions
[0103] As appreciated in the art, load, wash, and elution
conditions for use in the chromatography of the invention will
depend on the specific chromatography media/ligands used. The
process of the invention can, of course, be used in combination
with other protein purification methodologies, such as salt
precipitation, affinity chromatography, hydroxyapatite
chromatography, reverse phase liquid chromatography, or any other
commonly used protein purification technique. It is contemplated,
however, that the process of the present invention will eliminate
or significantly reduce the need for other purification steps.
[0104] Anionic exchange chromatography is also performed during
chromatographic separation of the polypeptide of interest. As is
well known in the art, anion exchangers may be based on various
materials with respect to the matrix as well as to the attached
charged groups. For example, the following matrices may be used, in
which the materials mentioned may be more or less cross-linked:
agarose based (such as SEPHAROSE Fast Flow.RTM. (such as
Q-SEPHAROSE FF), and SEPHAROSE High Performance.RTM.; cellulose
based (such as DEAE SEPHACEL.RTM.); silica based and synthetic
polymer based, or resins such as SuperQ-650 (from TOSOH BIOSEP) and
Macro High Q (from BIO-RAD). For the anion exchange resin, the
charged groups which are covalently attached to the matrix may,
e.g., be diethylaminoethyl (DEAE), quaternary aminoethyl (QAE),
and/or quaternary ammonium (Q). In certain embodiments, the resin
is selected from the group including, but not limited to, Q
Sepharose High Performance, Q Sepharose Fast Flow, DEAE Sepharose
Fast Flow, Capto Q, Capto DEAE, Toyopearls SuperQ 650 (M),
Toyopearls SuperQ 650 (S), Toyopearls DEAE 650 (M), Toyopearls DEAE
650 (S), TSKgel SuperQ-5PW (30), TSKgel SuperQ-5PW (20), TSKgel
DEAE-5PW (30), TSKgel DEAE-5PW (20), EMD Chemicals: Fractogel EMD
DEAE (S), Fractogel EMD DEAE (M), Fractogel EMD DMAE (S), Fractogel
EMD DMAE (M), Fractogel EMD TMAE (S), Fractogel EMD TMAE (M), and
Baker Bond XWP500 PolyQuat-35, SPE. In one embodiment of the
present process, the anion exchange resin employed is Q-SEPHAROSE
FP.RTM..
[0105] Although any of these resins may be used for small scale
purification of antibodies, resins of certain size and lower cost
are amenable to manufacturing scale separation. If the size of the
resin is too small, there is considerable back pressure generated
in the system. In addition, the amount of polypeptide that can be
purified is limited. If the resin is costly to make or purchase, it
is not economically feasible/practical for use in large scale
purification.
[0106] Thus, the resin used in the present invention must be of a
certain size to provide efficient scale-up without being
prohibitively expensive. "Manufacturing level purification" means
purification of antibodies from a recombinant preparation on a
scale that meets commercial scale production. The resin used in the
predetermination step should be the same as that used in the final
protocol for manufacturing level purification because one may not
easily predict the variation in conditions necessary to separate
the aggregates if the resin is changed. A particular resin that is
useful in small scale or bench top purification may not be amenable
to large scale purification. Such resins useful for the present
invention include, e.g., Q-SEPHAROSE HP. However, the skilled
artisan would recognize other anion exchange resins useful for
commercial scale production.
[0107] The volume of resin used when packing an anion exchange
chromatography column is reflected by the dimensions of the column,
i.e., the diameter of the column and the height of the resin, and
varies depending on, e.g., the amount of antibody in the applied
solution and the binding capacity of the resin used.
[0108] Before performing an anion exchange chromatography, the
exchange resin may be equilibrated with a buffer. Any of a variety
of buffers are suitable for the equilibration of exchange resin,
e.g., sodium acetate, sodium phosphate, TRIS(hydroxymethyl)
amino-methane, TRIS, phosphate, bis-TRIS, and L-histidine. Persons
skilled in the art will appreciate that numerous other buffers may
be used for the equilibration as long as the pH and conductivity
are about the same as for the applied antibody solution. When
performing the "bind-washout" process, the equilibration buffers
and the wash buffers are the same. When performing the "bind-elute"
process, the elution buffers may be made of one or more buffer
substances to control the pH. The salt used is, e.g., a highly
soluble salt, such as sodium chloride or potassium phosphate, but
any salt may be used that maintains the functionality of the
antibody and allows removal of the antibody monomer from the
resin.
[0109] In performing the "bind-elute" process, the elution of the
antibody monomers from the resin may be performed with a
substantially non-denaturating buffer having a pH and ionic
strength sufficient to efficiently elute the monomeric antibody,
thereby recovering an antibody-containing eluate, while leaving the
aggregates bound to the resin. In this context, efficient elution
means that at least 75%, or at least 80%, or at least 85%, or at
least 90%, or at least 95% of the antibody loaded onto the resin is
recovered. Only about 1.0%, preferably only 0.5%, most preferably
less than 0.1% aggregates remain in the antibody preparation
following ion exchange.
[0110] In one embodiment, the elution is carried out as a step
gradient elution. In another embodiment, the elution is carried out
in a linear gradient.
[0111] Surprisingly, deamidated variants of the immunoconjugate
proteins eluted at higher salt concentration despite the apparent
net increase of negative charge due to deamidation of an asparagine
residue. Therefore, these reduced potency variants were separated
from the more active proteins by the ion exchange chromatography
described herein.
[0112] In certain embodiments of the invention, about 75% to about
99% of the acidic or deamidated variant present within the starting
sample of the polypeptide or immunoconjugate is removed during the
purification process. In other embodiments, at least 75%, 80%, 85%,
90%, 95%, 97%, 98% or 99% of the deamidated variant is removed. The
composition comprising the active polypeptide or immunoconjugate
thus has less than between about 25% and about 1% deamidated
species, for example, less than 25%, less than 20%, less than 15%,
less than 10%, less than 5%, less than 4%, less than 3%, less than
2% or less than 1%.
[0113] Deamidated variants of the invention include
immunoconjugates comprising a PE toxin or variant thereof, wherein
the deamidation occurs at one or more residues within the
immunconjugate, for example, at one or more residues within the PE
toxin or variant thereof. In certain embodiments, deamidation
occurs at 1, 2 3, 4 or 5 residues within the immunoconjugate. In
other embodiments, an immunoconjugate comprising a PE toxin or
variant thereof is deamidated at 1, 2, 3, 4, or 5 residues within
the PE toxin or variant thereof, for example at position 358 of SEQ
ID NO:1, at position 495 of SEQ ID NO: 16, at position 243 of SEQ
ID NO:17, at position 227 of SEQ ID NO:18, at position 200 of SEQ
ID NO:19, at position 212 of SEQ ID NO: 20, at position 212 of SEQ
ID NO: 21 or at position 229 of SEQ ID NO: 22.
[0114] In one embodiment, the salt concentration of the eluting
buffer is sufficiently high to displace the antibody monomers from
the resin without displacing the aggregates. However, it is
contemplated that an increase in pH and a lower salt concentration
can be used to elute the antibody monomers from the resin.
[0115] Any or all chromatographic steps of the present invention
can be carried out by any mechanical means. Chromatography may be
carried out, for example, in a column. The column may be run with
or without pressure and from top to bottom or bottom to top. The
direction of the flow of fluid in the column may be reversed during
the chromatography process. Chromatography may also be carried out
using a batch process in which the solid media is separated from
the liquid used to load, wash, and elute the sample by any suitable
means, including gravity, centrifugation, or filtration.
Chromatography may also be carried out by contacting the sample
with a filter that absorbs or retains some molecules in the sample
more strongly than others. In the following description, the
various embodiments of the present invention are described in the
context of chromatography carried out in a column. It is
understood, however, that use of a column is merely one of several
chromatographic modalities that may be used, and the illustration
of the present invention using a column does not limit the
application of the present invention to column chromatography, as
those skilled in the art may readily apply the teachings to other
modalities as well, such as those using a batch process or
filter.
[0116] A variety of different loading, washing and elution
conditions can be used, as desired. In some embodiments, the
initial loading conditions are adapted such that the protein (e.g.,
antibody) eluted from the initial non-HT is applied directly to the
HT column.
[0117] Elution can be achieved, for example, by changing the salt
conditions in the liquid phase. For example, the salt and/or
conductivity of the liquid phase is increased (linearly or
step-wise) to a point that which the antibody elutes. Exemplary
washing conditions include, e.g., 10 mM phosphate, pH 6.7, with
elution achieved by increasing the salt concentration (step-wise or
in a linear fashion) (e.g., to 10 mM phosphate, 1.5M NaCl, pH 6.7).
All of the various embodiments or options described herein can be
combined in any and all variations.
[0118] Before the sample is applied to the column, the column can
be equilibrated in the buffer or salt that will be used to
chromatograph the protein. As discussed below, chromatography (and
loading of the protein to be purified) can occur in a variety of
buffers or salts including sodium, potassium, ammonium, magnesium,
calcium, chloride, fluoride, acetate, phosphate, and/or citrate
salts and/or Tris buffer. Citrate buffers and salts are preferred
by those skilled in the art for their ease of disposal. Such
buffers or salts can have a pH of at least about 5.5. In some
embodiments, equilibration may take place in a solution comprising
a Tris or a sodium phosphate buffer. In some embodiments,
equilibration takes place at a pH of at least about 5.5.
Equilibration may take place at pHs between about 6.0 and about
8.6, preferably at pHs between about 6.5 and 7.5. Most preferably,
the solution comprises a sodium phosphate buffer at a concentration
of about 25 millimolar and at a pH of about 6.8.
[0119] The protein purification process of the present invention is
applicable to removal of an acidic variant from any protein. Some
proteins specifically contemplated for use with the invention
include antibodies or fragments thereof. Other proteins include,
but are not limited to recombinant fusion proteins comprising one
or more constant antibody immunoglobulin domains, optionally an Fc
portion of an antibody, and a protein of interest.
Formulations
[0120] Formulations of the purified polypeptides or
immunoconjugates are prepared for storage and use by combining a
purified polypeptide or immunoconjugate of the present invention
with a pharmaceutically acceptable vehicle (e.g., carrier,
excipient) (Remington, The Science and Practice of Pharmacy 20th
Edition Mack Publishing, 2000). Suitable pharmaceutically
acceptable vehicles include, but are not limited to, nontoxic
buffers such as phosphate, citrate, and other organic acids; salts
such as sodium chloride; antioxidants including ascorbic acid and
methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium
chloride; hexamethonium chloride; benzalkonium chloride;
benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens, such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight
polypeptides (e.g., less than about 10 amino acid residues);
proteins such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
carbohydrates such as monosaccharides, disaccharides, glucose,
mannose, or dextrins; chelating agents such as EDTA; sugars such as
sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions
such as sodium; metal complexes (e.g., Zn-protein complexes); and
non-ionic surfactants such as TWEEN or polyethylene glycol
(PEG).
[0121] The antibody and/or immunoconjugate compositions of this
invention (i.e., PE linked to an antibody), are particularly useful
for parenteral administration, such as intravenous administration
or administration into a body cavity or lumen of an organ. The
compositions for administration will commonly comprise a solution
of the antibody and/or immunoconjugate dissolved in a
pharmaceutically acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers can be used, e.g., buffered saline
and the like. These solutions are sterile and generally free of
undesirable matter. These compositions may be sterilized by
conventional, well known sterilization techniques. The compositions
may contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions such as pH
adjusting and buffering agents, toxicity adjusting agents and the
like, for example, sodium acetate, sodium chloride, potassium
chloride, calcium chloride, sodium lactate and the like. The
concentration of fusion protein in these formulations can vary
widely, and will be selected primarily based on fluid volumes,
viscosities, body weight and the like in accordance with the
particular mode of administration selected and the patient's
needs.
[0122] Thus, a typical pharmaceutical immunoconjugate composition
for intravenous administration would be at a total treatment of
about 0.3 to about 50 .mu.g/kg per day, in particular 20-50
.mu.g/kg per day with the dosage preferably administered
continuously or allocated at three times per day. Actual methods
for preparing administrable compositions will be known or apparent
to those skilled in the art and are described in more detail in
such publications as Remington's Pharmaceutical Science, 19th ed.,
Mack Publishing Company, Easton, Pa. (1995).
[0123] The composition including the present invention's
immunoconjugate can be administered for therapeutic treatments. In
therapeutic applications, compositions are administered to a
patient suffering from a disease, in an amount sufficient to cure
or at least partially arrest the disease and its complications. An
amount adequate to accomplish this is defined as a "therapeutically
effective dose." Amounts effective for this use will depend upon
the severity of the disease and the general state of the patient's
health.
[0124] Single or multiple administrations of the compositions may
be administered depending on the dosage and frequency as required
and tolerated by the patient. In any event, the composition should
provide a sufficient quantity of the proteins of this invention to
effectively treat the patient. The dosage can be administered three
times a day every other day or continuously every other day for a
cycle of, e.g., 21 days, but may be applied periodically until
either a therapeutic result is achieved or until side effects
warrant discontinuation of therapy. Generally, the dose should be
sufficient to treat or ameliorate symptoms or signs of disease
without producing unacceptable toxicity to the patient. An
effective amount of the compound is that which provides either
subjective relief of a symptom(s) or an objectively identifiable
improvement as noted by the clinician or other qualified
observer.
[0125] In one embodiment, the immunoconjugate is formulated as a
pharmaceutical composition comprising at least one acceptable
excipient. Pharmaceutically acceptable CAT-8015 immunoconjugate
formulations include 0.5 mg/mL to 2.5 mg/mL CAT-8015, usually 1.0
mg/mL, 1.1 mg/mL, 1.2 mg mL, 1.3 mg/mL, 1.4 mg/mL or 1.5 mg/mL in
25 mM sodium phosphate, 4% sucrose, 8% glycine, 0.02% polysorbate
80 (PS80), pH 7.4. In additional embodiments, the sodium phosphate
can be in a range of 20 mM to 100 mM, 25 mM to 50 mM, or 25 mM to
35 mM; the sucrose can be at 2%, 3%, 4%, 5% or 6%; the glycine can
be in the range of 5-10%, usually, 5%, 6%, or 7%; the polysorbate
80 can be in a range from about 0.01% to about 1%, usually 0.01%,
0.02%, 0.03%, 0.04% or 0.05%; with a pH in the range of 6.5 to 8.0,
usually at pH 7.2, 7.3, 7.4, 7.5 or 7.6. Other buffering agents
known to one of ordinary skill in the art can also be utilized.
[0126] In certain embodiments of the invention, the formulation is
lyophilized. The term "lyophilized" refers to any composition or
formulation that is prepared in dry form by rapid freezing and
dehydration, in the frozen state under high vacuum. "Lyophilizing"
or "lyophilization" refers to a process of freezing and drying a
solution. Lyophilized formulations or compositions are often made
ready for use or reconstituted by addition of sterile distilled
water. In certain embodiments, the lyophilized formulation of the
invention is reconstituted into a vial.
[0127] For intravenous administration, a formulation of the
invention, such as a liquid formulation or a formulation
reconstituted from a lyophilized formulation is placed in a vial
where the immunoconjugate in the formulation is present at
concentrations as described above. This formulation is extracted
from the vial and added to an intravenous (IV) bag solution, where
the IV bag contains from about 30 mL to about 100 mL solution,
usually 50 mL, 60 mL, 70 mL or 80 mL. A separate IV bag "protectant
solution" can also be added to the total volume of the IV bag where
the protectant solution contains polysorbate 80 in an amount such
that the polysorbate 80 present in the final IV bag solution is in
a range of 0.001% to about 3% polysorbate 80, usually in the range
of about 0.01% to about 0.1%, and more usually at 0.01%, 0.02%,
0.03%, 0.04% or 0.05%. The protectant solution can be
pre-formulated in a vial such that the polysorbate 80 is at a
concentration of about 0.5% to about 5%, and can be 0.5%, 1.0%,
1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5% or 5.0% The protectant
solution prevents adsorption of the immunoconjugate or drug (e.g.,
CAT-8015) to contact surfaces of the IV bag, thereby preventing or
inhibiting the immunoconjugate or drug from sticking to the IV bag
during administration and allowing the patient to receive the
appropriate dosage of immunoconjugate or drug. The IV bag solution
can be administered by infusion to the patient for various
durations, usually 30 minutes to 1 hour, usually 30 minutes.
[0128] Among various uses of the immunoconjugates and formulations
of the present invention are included a variety of disease
conditions caused by specific human cells that may be eliminated by
the toxic action of the protein. One application for the
immunoconjugates of the invention is for the treatment of B cell
malignancies or malignant B cells expressing CD22. Exemplary B cell
malignancies include chronic B-lymphocytic cells (B-CLL), pediatric
acute lyphocytic leukemia (pALL), follicular lymphoma (FL), diffuse
large B cell lymphoma (DLBCL), Non Hodgkins lymphoma (NHL) and
hairy cell leukemia (HCL).
[0129] All documents, patents, journal articles and other materials
cited in the present application are hereby incorporated by
reference.
[0130] Although the present invention has been fully described in
conjunction with several embodiments thereof with reference to the
accompanying drawings, it is to be understood that various changes
and modifications may be apparent to those skilled in the art. Such
changes and modifications are to be understood as included within
the scope of the present invention as defined by the appended
claims, unless they depart therefrom.
EXAMPLES
Example 1. Expression, Recovery and Inclusion Body Isolation of
CAT-8015
[0131] Fermentation of separate cell lines containing CAT-8015
V.sub.L and CAT-8015 V.sub.H-PE38 expression vectors was performed.
The fermentor was harvested by continuous centrifugation. The
fermentor harvest was passed through a continuous centrifuge at 2
to 8.degree. C. at a rate of 0.5 to 0.8 L per minute and
centrifuged at a speed of approximately 15,000 rpm. After
centrifugation the cell paste was frozen at .ltoreq.-70.degree.
C.
[0132] Following this treatment, the V.sub.H-PE38 and V.sub.L cell
pastes were thawed for 12 to 24 hours at 2 to 8.degree. C. The
cells were lysed to release inclusion bodies containing the V.sub.L
and V.sub.H-PE38 products. The resulting inclusion bodies were
subsequently solubilized and the V.sub.H-PE38 and V.sub.L products
obtained.
[0133] The product was concentrated to approximately 1 mg/mL
(determined by Coomassie total protein assay) using a 30 kDa
ultrafiltration hollow fiber cartridge. The retentate was then
diafiltered with 5 to 6 volumes of 20 mM Tris, 100 mM urea pH 7.4
to achieve a conductivity of 2.5 to 3.0 mS/cm. This product was
stored up to 72 hours at 2 to 8.degree. C.
Example 2. Analytical-Scale Purification of Active CAT-8015 by
Anion Exchange Chromatography with High Performance Resins
[0134] Expression of the V.sub.H-P38 subunit resulted in a
formation of a deamidated variant of the subunit. The deamidation
was found to occur in the PE38 portion of the immunoconjugate.
Deamidation of the V.sub.H-P38 subunit resulted in decreased
potency of the CAT-8015 protein. Surprisingly, the below described
chromatographic conditions were successful in removing the
deamidated variant, thus providing the ability to remove the
inactive species during purification. Since the deamidation
occurred in the PE38 portion of the fusion construct, the
chromatographic conditions can be applied to removal of any
deamidated variant of a PE conjugate.
[0135] CAT-8015 was renatured from isolated inclusion bodies and
subsequently purified by a 4-column process. Table 1 provides an
overview of the renaturation and purification unit operations.
TABLE-US-00006 TABLE 1 CAT-8015 production Step Unit Operation 1
Fermentation 2 Primary Recovery 3 Inclusion Body Isolation 4 Refold
5 Capture Step 6 Intermediate Purification Step I 7 Intermediate
Purification Step II 8 Anion Exchange Chromatography 9 Formulation
(Drug Substance)
[0136] FIG. 1 shows the analytical ion exchange chromatography
(IEC) profile of a sample of a CAT-8015 reference standard. As
shown in the profile, a pre-peak emerges prior to the elution of
the main peak. The individual fractions eluted from the IEC are
assayed for CAT-8015 biological activity relative to a reference
standard using an apoptosis bioassay that measures the ability of
the test sample to induce dose dependent apoptotic death of the
CD22 receptor-expressing Daudi cell line. Once bound to CD22 and
internalized, CAT-8015 induces Daudi cells to undergo apoptosis via
Caspase 3/7 activation that can be measured by Caspase-Glo.TM. 3/7
Assay System. The potency of the test sample is determined by
dividing the 50% effective concentration (EC50) of the Reference
Standard by the EC50 of the test sample. The results of the
apoptosis bioassay demonstrated that the relative potency of
CAT-8015 is correlated with the percentage of pre-peak of CAT-8015
as diagrammed in FIG. 1. FIG. 2 shows the correlation between this
relative potency and the percentage of pre-peak.
[0137] Pre-peak and main peak fractions from multiple IEC analysis
were collected, pooled and subjected to peptide mapping and liquid
chromatography-mass spectrometry/mass spectrometry (LC-MS/MS)
analysis. Results were compared to those obtained from peptide
mapping and LC-MS/MS experiments of purified CAT-8015. The analysis
of purified CAT-8015 drug substance revealed that Asn-358 was
partially deamidated to Asp-358 and iso-Asp-358 (Table 2). Asp-358
and iso-Asp358 were found to be significantly enriched in the
pre-peak fraction whereas the main peak fraction was enriched in
intact CAT-8015 (Table 2). Taken together, the results demonstrate
that deamidation at Asn-358 lead to a loss of relative potency in a
cell based bioassay. The Asp-358 residue is present within the PE
toxin portion of the immunoconjugate thus indicating that an
immunoconjugate containing a PE toxin, or variant thereof, in which
Asp-358 is present will likely be subject to deamidation and
subject to a loss of potency or activity.
TABLE-US-00007 TABLE 2 Distribution of Amino Acid 358 in CAT-8015
Drug Substance, Pre-Peak and Main Peak Fractions based on Peptide
Mapping and LC-MS/MS Analysis. Amino Drug Acid Substance (%)
Pre-Peak (%) Main Peak (%) N358 78.1 3.1 88.9 D358 11.9 44.0 2.2
iso-D358 10.0 52.9 8.9 D358 = deamidated Asn-358; iso-D358 =
iso-deamidated Asn-358; N358 = Asn-358.
[0138] Separation of deamidated CAT-8015 from intact CAT-8015 was
achieved by anion exchange chromatography with strong ion exchange
groups such as Q (quaternary amino) coupled to small diameter
resins such as Source 15 (particle diameter: 15 .mu.m; GE
Healthcare) and Sepharose High Performance (particle diameter: 34
.mu.m; GE Healthcare). The application of small diameter
chromatography resin in biomanufacturing processes is complicated
by the generation of significant back pressures under typical
operating conditions as defined by column geometry, flow rates and
buffer composition. Based on these considerations and the
requirement for a high resolution chromatography step Q Sepharose
High Performance was chosen for the separation of deamidated
CAT-8015 from intact CAT-8015. Chromatography conditions were
developed that achieved high resolution while maintaining
operability at various manufacturing scales.
Example 3. Bench-Scale Purification of CAT-8015
[0139] The column was first pre-equilibrated with 5 column volumes
(CVs) of Buffer C (Pre-Equilibration/Stripping Buffer: 10 mM
Tris/HCl, pH 8.0, 1.0M NaCl) and subsequently equilibrated with 5
CVs Buffer A (Equilibration Buffer: 10 mM Tris/HCl, pH 8.0) at a
linear flow rate of 100 cm/hr. The chromatography resin was Q
Sepharose High Performance (QHP, GE Healthcare) in a Millipore
Vantage column, 2.2 cm.times.19.5 cm, and run on an AKTA Explorer.
The intermediate purification product pool was prepared for loading
onto the high performance anion exchange column by diafiltration
with 10 volumes of Buffer A using a 10 kDa MWCO membrane. The
diafiltered hydrophobic interaction product pool was loaded onto
the QHP column at a linear flow rate of 100 cm/hr, followed by a 2
CV re-equilibration step with Buffer A at the same flow rate.
CAT-8015 was eluted with a 10 CV linear gradient from 35% Buffer B
(Elution Buffer: 10 mM Tris/HCl, pH 8.0, 500 mM NaCl) to 55% Buffer
B at a linear flow rate of 100 cm/hr. Elution of product was
monitored at 280 nm. Fractions were collected and analyzed for %
pre-peak by analytical ion exchange chromatography (IEC). Fractions
containing less than 25% pre-peak were pooled. The QHP pool was
analyzed for % pre-peak by analytical IEC on a strong anion
exchange column Relative potency was measured by an apoptosis
bioassay as described above.
[0140] At pH 8.0, CAT-8015 strongly bound to the anion exchange
resin with no protein detected in the flow-through fraction by
absorbance at A280. After an initial wash step with 175 mM NaCl in
Tris/HCl, pH 8.0, CAT-8015 was eluted from the column with a linear
salt gradient from 35% B (175 mM NaCl in Tris/HCl, pH 8.0) to 55% B
(275 mM NaCl in Tris/HCl, pH 8.0). CAT-8015 eluted from the column
between 39% B (192 mM NaCl in Tris/HCl, pH 8.0) and 49% B (245 mM
NaCl in Tris/HCl, pH 8.0). FIG. 3 shows the QHP chromatography
profile of CAT-8015.
[0141] Fractions were analyzed by analytical IEC. Table 3 shows the
results for fractions eluted between 44.5% B (223 mM NaCl) and
47.2% B (236 mM NaCl).
TABLE-US-00008 TABLE 3 IEC Analysis of Collected QHP Fractions
Fraction No. % Pre-Peak .sup.a % Main Peak C12 41.0 59 D12 34.5
65.5 D11 27.0 73 D9 17.6 82.4 D7 15.9 84.1 D5 18.7 81.3 D3 21.9
78.1 .sup.a Pre-peak contains >90% deamidated CAT-8015.
[0142] Table 3 demonstrates that anion exchange chromatography
operated in a linear salt gradient elution mode is able to separate
deamidated CAT-8015 from intact CAT 8015 in an effective
manner.
[0143] Surprisingly, intact CAT-8015 was eluted at a higher salt
concentration despite the apparent net increase of negative charge
due to deamidation of an asparagine residue. This result is
consistent with the chromatography profile observed by IEC.
CAT-8015-containing samples were injected on an analytical anion
exchange column (PL-SAX, Varian) equilibrated at pH 8.0 with a
Tris/HCl buffer system and eluted by a combination of step and
gradient elution steps (FIG. 1).
[0144] Fractions were combined according to the pooling criteria of
less than 25% pre-peak content. The QHP pool was analyzed for %
pre-peak and relative potency by SDS-PAGE, analytical IEC and
apoptosis bioassay. The SDS-PAGE analysis, as shown in FIG. 4,
demonstrates that QHP load pool and eluate samples contained highly
purified CAT-8015. However, the QHP load pool did not meet target
specification for purity by IEC and bioactivity. Purity and potency
measurements for the QHP load pool as compared to the QHP eluate
pool, as presented in Table 4 below, demonstrate that the anion
exchange chromatography step with QHP resulted in a significant
increase in purity by IEC and relative potency of CAT-8015. The QHP
load pool was generated from Intermediate Purification Step II.
TABLE-US-00009 TABLE 4 CAT-8015 Purity by IEC and Bioactivity
Relative Potency Step % Pre-Peak % Main Peak (%) QHP Load Pool 53.8
46.2 52 QHP Eluate Pool 16.5 83.8 80
[0145] The QHP product pool was subsequently diafiltered into
formulation buffer to generate CAT-8015 drug substance.
[0146] Thus, the manufacture of CAT-8015 drug substance requires
the separation of deamidated CAT-8015 from active CAT-8015. The
capability of anion exchange chromatography with high performance
resins such as QHP to separate deamidated CAT-8015 from intact
CAT-8015 and to increase bioactivity to target specifications is a
pre-requisite for the successful manufacture of CAT-8015 drug
substance.
Example 4. Large-Scale Purification of CAT-8015
[0147] The column was first pre-equilibrated with 5 CVs Buffer C
(Pre-Equilibration/Stripping Buffer: 10 mM Tris/HCl, pH 8.0, 1.0M
NaCl) at a linear flow rate of 66 cm/hr and subsequently
equilibrated with 5 CVs Buffer A at a linear flow rate of 76 cm/hr.
The chromatography resin was Q Sepharose High Performance (QHP, GE
Healthcare), in a BP300, 30 cm.times.22 cm column bed, run on a K
Prime instrument. The intermediate purification product pool was
prepared for loading onto the high performance anion exchange
column by diafiltration with 10 volumes of Buffer A (Equilibration
Buffer: 10 mM Tris/HCl, pH 8.0) using a 10 kDa MWCO membrane. The
diafiltered product pool was loaded onto the QHP column at a linear
flow rate of 64 cm/hr, followed by a 2 CV re-equilibration step
with Buffer A at 76 cm/hr. CAT-8015 was eluted with a 10 CV linear
gradient from 35% Buffer B (Elution Buffer: 10 mM Tris/HCl, pH 8.0,
500 mM NaCl) to 55% Buffer B at a linear flow rate of 76 cm/hr.
Elution of product was monitored at 280 nm. Fractions were
collected and analyzed for % pre-peak by analytical ion exchange
chromatography (IEC). Fractions containing less than 25% pre-peak
were pooled. The QHP pool was analyzed for % pre-peak by analytical
IEC on a strong anion exchange column. Relative potency was
measured by an apoptosis bioassay.
[0148] Large scale anion exchange chromatography of CAT-8015 with
QHP was performed as described above. QHP purification was carried
out at reduced flow rates due to equipment constraints. CAT-8015
eluted from the column at conductivities between 22.3 mS/cm and
26.4 mS/cm. FIG. 5 shows the QHP chromatography profile of CAT-8015
purified according to the method described above.
[0149] Fractions were analyzed by analytical IEC. Table 5 shows the
results for fractions eluted at conductivities between 23.8 and
25.4 mS/cm. Table 5 demonstrates that anion exchange chromatography
operated in a linear salt gradient elution mode was able to
separate deamidated CAT-8015 from intact CAT 8015. Separation of
deamidated CAT-8015 from intact CAT-8015 took place within a
conductivity range of less than 2 mS/cm.
TABLE-US-00010 TABLE 5 IEC Analysis of Collected Fractions %
Pre-peak purity Fraction % Pre-Peak % Main Peak 1 55.1 44.9 2 39.0
61 3 30.1 69.9 4 25.1 74.9 5 18.7 81.3 6 14.7 85.3 7 16.4 83.6
[0150] Fractions 4-7 were combined according to the pooling
criteria of less than 25% pre-peak content. The QHP pool was
analyzed for % pre-peak and relative potency by SDS-PAGE and SEC,
analytical IEC and apoptosis bioassay. The SDS-PAGE analysis, as
shown in FIG. 6, demonstrates that QHP load pool and eluate samples
contained highly purified CAT-8015. However, the QHP load pool did
not meet target specification for purity by SEC, IEC and relative
potency. Purity and potency measurements for the QHP load pool as
compared to the QHP eluate pool, as presented in Tables 6 and 7
below, demonstrate that the anion exchange chromatography step with
QHP resulted in a significant increase in purity by SEC, IEC and
relative potency of CAT-8015. The QHP load pool was generated from
Intermediate Purification Step II.
TABLE-US-00011 TABLE 6 CAT-8015 Purity by SEC Step % Monomer %
Aggregate % Other QHP Load Pool 92.7 0.7 6.6 QHP Eluate Pool 99.0
1.0 0
TABLE-US-00012 TABLE 7 CAT-8015 Purity by IEC and Bioactivity
Relative Potency Step % Pre-Peak % Main Peak (%) QHP Load Pool 50.3
49.7 51 QHP Eluate Pool 17 83 75
[0151] The QHP product pool was subsequently diafiltered into
formulation buffer to generate CAT-8015 drug substance.
[0152] Examples 2-4 demonstrate the capacity of anion exchange
chromatography with resins such as Q Sepharose High Performance to
separate deamidated CAT-8015 from intact CAT-8015 and to increase
its relative potency to meet target specifications (see Tables 4
and 6). Deamidated CAT-8015 differs from intact CAT-8015 by one
additional negative charge. In contrast to the expected elution
behavior from an anion exchange column, the bulk of deamidated
CAT-8015 elutes prior to intact CAT-8015 under salt gradient
elution conditions (see Tables 3 and 5). This unexpected elution
pattern was observed at analytical scale, bench scale, and large
scale anion exchange chromatography. This elution pattern was
unexpected as the linear high salt elution buffer would be expected
to result in a higher negative charge of the variant. Thus,
Examples 2-4 demonstrate that using a linear elution buffer, a
deamidated species can be removed from active immunoconjugates
using anion-exchange chromatography. Separation of deamidated
CAT-8015 from intact CAT-8015 took place within a particular range
of conductivities, underscoring the need for high resolution anion
exchange resins, careful control of elution conditions and
in-process testing of collected fractions.
Example 5. Modifying the Bioactivity of CAT-8015 Formulations
[0153] The potency of CAT-8015 compositions was calibrated by
mixing specific quantities of the deamidated pre-peak with the
active main peak. To obtain compositions comprising a particular
potency of CAT-8015, aliquots of CAT-8015 pre-peak product were
combined with aliquots of CAT-8015 main peak product to achieve a
composition with a particular potency of CAT-8015. By controlling
the level of CAT-8015 potency in the composition, a CAT-8015
formulation was generated for administration of a particular volume
of reconstituted CAT-8015 at a desired dose.
Example 6. Adjusting pH During Solubilization Results in Reduced
Deamidation
[0154] Species as Measured after Capture and Intermediate
Purification Steps
[0155] While deamidated species can be removed from active
immunoconjugates using the purification steps as described above,
levels of deamidated species of CAT-8015 can also be effectively
reduced by adjusting the pH at earlier steps in the purification
process (i.e., the refold step (Step 4 of Table 1 above). The
refold procedure utilized to achieve a lower level of deamidated
species of CAT-8015 includes the following substeps:
[0156] Refold Substep 1: Solubilization, Clarification and
Concentration: V.sub.H-PE38 and V.sub.L inclusion bodies were
thawed for 12-24 hours at room temperature (15-30.degree. C.).
V.sub.H-PE38 and V.sub.L inclusion bodies were combined in a 1:1
molar ratio and adjusted to 15% (w/v) solids by adding 50 mM Tris,
20 mM EDTA, pH 7.4. The inclusion bodies were solubilized by adding
5 kg of inclusion body solubilization buffer (50 mM ethanolamine, 8
M urea, 0.5 M arginine, 2 mM EDTA, 10 mM DTE) for each kg of 15%
(w/v) solids inclusion body suspension. The pH of the inclusion
body solubilization buffer was varied between pH 9.0 and 10.5 in
0.5 pH unit increments. Solubilization was carried out for 2 hours
at room temperature (15-30.degree. C.) with constant stirring.
Solubilized inclusion bodies were clarified by depth filtration
through a series of filters. The clarified filtrate was
concentrated by tangential flow filtration to 5-6 g/L using a 5 kDa
molecular weight cutoff (MWCO) ultra filtration membrane.
[0157] Refold Substep 2: Refold: The refolding of CAT-8015 was
initiated by a 10-fold dilution of the clarified and concentrated
inclusion body filtrate into pre-chilled (2-8.degree. C.) refolding
buffer (50 mM ethanolamine, 1 M arginine, 2 mM EDTA, 0.91 mM
oxidized glutathione, pH 9.5). The refold solution was maintained
at 2-8.degree. C. for 48-72 hours with continuous mixing. The
refold was terminated by bringing the refold solution to room
temperature (15-30.degree. C.) prior to concentration and
diafiltration. The refold solution was concentrated by tangential
flow filtration with a 10 kDa MWCO membrane, and diafiltered with
10 volumes of 20 mM potassium phosphate, pH 7.4. The concentrated
and diafiltered refold solution was filtered through a 0.2 .mu.m
filter (TMAE load).
[0158] As part of the capture step (Step 5 of Table 1 above), the
CAT-8015 preparation obtained from the refold procedure above was
loaded onto a Fractogel TMAE column (EMD Biosciences or equivalent)
equilibrated with 20 mM potassium phosphate, pH 7.4. After loading,
the column was first washed with 20 mM potassium phosphate, pH 7.4,
and then with 20 mM potassium phosphate, 0.1% (w/w) Triton X 100,
pH 7.4, followed by a subsequent wash with 20 mM potassium
phosphate, 100 mM sodium chloride, pH 7.4. The product was eluted
from the column in reverse flow with 20 mM potassium phosphate, 200
mM sodium chloride pH 7.4. The column was stripped with 2 M sodium
chloride, sanitized with 1 N sodium hydroxide and stored in 0.1 N
sodium hydroxide at room temperature.
[0159] As part of the intermediate purification step 1,
hydroxyapatite chromatography was performed. The hydroxyapatite
chromatography step was operated as a flow-through chromatography
step. The product obtained from the capture step above was loaded
directly without any further adjustments onto a ceramic
hydroxyapatite column (Bio-Rad Laboratories or equivalent)
equilibrated with 400 mM potassium phosphate, 200 mM sodium
chloride, pH 7.4, followed by 20 mM potassium phosphate, 200 mM
sodium chloride, pH 7.4. Under the conditions of the
chromatography, the product was collected in the flow-through
fraction (HA product). The column was stripped with 400 mM
potassium phosphate, 200 mM sodium chloride, pH 7.4, sanitized with
1 N sodium hydroxide and stored in 0.1 N sodium hydroxide at room
temperature.
[0160] The percent pre-peak in the HA product from above was
analyzed by high performance anion exchange chromatography. Table 8
and FIG. 7 show the percent pre-peak in HA product as a function of
solubilization pH. As shown in Table 8 and FIG. 7, solubilizing the
V.sub.H-PE38 and V.sub.L inclusion bodies at a lower pH leads to
less deamidated CAT-8015 product. The capability of controlling
CAT-8015 deamidation at an early step in the renaturation and
purification process can increase overall process yield while
maintaining the quality of the final purified drug substance.
TABLE-US-00013 TABLE 8 Percent Pre-Peak in HA Product as a Function
of Solubilization pH Solubilization pH 9.0 9.5 10.0 10.5 Pre Peak
(%) 9.8 14.5 22.1 31.8
Sequence CWU 1
1
231476PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly1 5 10 15Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser
Gly Phe Ala Phe Ser Ile 20 25 30Tyr Asp Met Ser Trp Val Arg Gln Thr
Pro Glu Lys Cys Leu Glu Trp 35 40 45Val Ala Tyr Ile Ser Ser Gly Gly
Gly Thr Thr Tyr Tyr Pro Asp Thr 50 55 60Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Leu65 70 75 80Tyr Leu Gln Met Ser
Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr 85 90 95Cys Ala Arg His
Ser Gly Tyr Gly Thr His Trp Gly Val Leu Phe Ala 100 105 110Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ala Lys Ala Ser Gly 115 120
125Gly Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys
130 135 140His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg
Gly Trp145 150 155 160Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln
Arg Leu Val Ala Leu 165 170 175Tyr Leu Ala Ala Arg Leu Ser Trp Asn
Gln Val Asp Gln Val Ile Arg 180 185 190Asn Ala Leu Ala Ser Pro Gly
Ser Gly Gly Asp Leu Gly Glu Ala Ile 195 200 205Arg Glu Gln Pro Glu
Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala 210 215 220Glu Ser Glu
Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly225 230 235
240Ala Ala Asn Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn
245 250 255Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val
Ser Phe 260 265 270Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg
Leu Leu Gln Ala 275 280 285His Arg Gln Leu Glu Glu Arg Gly Tyr Val
Phe Val Gly Tyr His Gly 290 295 300Thr Phe Leu Glu Ala Ala Gln Ser
Ile Val Phe Gly Gly Val Arg Ala305 310 315 320Arg Ser Gln Asp Leu
Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly 325 330 335Asp Pro Ala
Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala 340 345 350Arg
Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg 355 360
365Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro
370 375 380Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu
Pro Leu385 390 395 400Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu
Gly Gly Arg Leu Glu 405 410 415Thr Ile Leu Gly Trp Pro Leu Ala Glu
Arg Thr Val Val Ile Pro Ser 420 425 430Ala Ile Pro Thr Asp Pro Arg
Asn Val Gly Gly Asp Leu Asp Pro Ser 435 440 445Ser Ile Pro Asp Lys
Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala 450 455 460Ser Gln Pro
Gly Lys Pro Pro Arg Glu Asp Leu Lys465 470 4752108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
2Met Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu1 5
10 15Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser
Asn 20 25 30Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys
Leu Leu 35 40 45Ile Tyr Tyr Thr Ser Ile Leu His Ser Gly Val Pro Ser
Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
Ser Asn Leu Glu65 70 75 80Gln Glu Asp Phe Ala Thr Tyr Phe Cys Gln
Gln Gly Asn Thr Leu Pro 85 90 95Trp Thr Phe Gly Cys Gly Thr Lys Leu
Glu Ile Lys 100 10534PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 3Lys Asp Glu
Leu144PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Arg Glu Asp Leu155PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Arg
Glu Asp Leu Lys1 56124PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 6Met Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly1 5 10 15Gly Ser Leu Lys Leu
Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ile 20 25 30Tyr Asp Met Ser
Trp Val Arg Gln Thr Pro Glu Lys Cys Leu Glu Trp 35 40 45Val Ala Tyr
Ile Ser Ser Gly Gly Gly Thr Thr Tyr Tyr Pro Asp Thr 50 55 60Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu65 70 75
80Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr
85 90 95Cys Ala Arg His Ser Gly Tyr Gly Thr His Trp Gly Val Leu Phe
Ala 100 105 110Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 115
1207124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 7Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly1 5 10 15Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser
Gly Phe Ala Phe Ser Ile 20 25 30Tyr Asp Met Ser Trp Val Arg Gln Thr
Pro Glu Lys Cys Leu Glu Trp 35 40 45Val Ala Tyr Ile Ser Ser Gly Gly
Gly Thr Thr Tyr Tyr Pro Asp Thr 50 55 60Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Leu65 70 75 80Tyr Leu Gln Met Ser
Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr 85 90 95Cys Ala Arg His
Ser Gly Tyr Gly Tyr Asn Trp Gly Val Leu Phe Ala 100 105 110Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ala 115 1208124PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
8Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly1 5
10 15Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser
Ile 20 25 30Tyr Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Cys Leu
Glu Trp 35 40 45Val Ala Tyr Ile Ser Ser Gly Gly Gly Thr Thr Tyr Tyr
Pro Asp Thr 50 55 60Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Thr Leu65 70 75 80Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu
Asp Thr Ala Met Tyr Tyr 85 90 95Cys Ala Arg His Ser Gly Tyr Gly Thr
Thr Trp Gly Val Leu Phe Ala 100 105 110Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ala 115 1209124PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 9Met Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly1 5 10 15Gly Ser Leu Lys Leu
Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ile 20 25 30Tyr Asp Met Ser
Trp Val Arg Gln Thr Pro Glu Lys Cys Leu Glu Trp 35 40 45Val Ala Tyr
Ile Ser Ser Gly Gly Gly Thr Thr Tyr Tyr Pro Asp Thr 50 55 60Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu65 70 75
80Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr
85 90 95Cys Ala Arg His Ser Gly Tyr Gly Ser Thr Tyr Gly Val Leu Phe
Ala 100 105 110Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 115
12010124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 10Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly1 5 10 15Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser
Gly Phe Ala Phe Ser Ile 20 25 30Tyr Asp Met Ser Trp Val Arg Gln Thr
Pro Glu Lys Cys Leu Glu Trp 35 40 45Val Ala Tyr Ile Ser Ser Gly Gly
Gly Thr Thr Tyr Tyr Pro Asp Thr 50 55 60Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Leu65 70 75 80Tyr Leu Gln Met Ser
Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr 85 90 95Cys Ala Arg His
Ser Gly Tyr Gly Thr His Trp Gly Val Leu Phe Ala 100 105 110Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ala 115 12011124PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
11Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly1
5 10 15Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser
Ile 20 25 30Tyr Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Cys Leu
Glu Trp 35 40 45Val Ala Tyr Ile Ser Ser Gly Gly Gly Thr Thr Tyr Tyr
Pro Asp Thr 50 55 60Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Thr Leu65 70 75 80Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu
Asp Thr Ala Met Tyr Tyr 85 90 95Cys Ala Arg His Ser Gly Tyr Gly Ser
Ser Tyr Gly Val Leu Phe Ala 100 105 110Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ala 115 12012108PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 12Met Asp Ile Gln Met
Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu1 5 10 15Gly Asp Arg Val
Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ala Arg 20 25 30Tyr Leu Asn
Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu 35 40 45Ile Tyr
Tyr Thr Ser Ile Leu His Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu65 70 75
80Gln Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro
85 90 95Trp Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys 100
10513108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 13Met Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
Leu Ser Ala Ser Leu1 5 10 15Gly Asp Arg Val Thr Ile Ser Cys Arg Ala
Ser Gln Asp Ile His Gly 20 25 30Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
Asp Gly Thr Val Lys Leu Leu 35 40 45Ile Tyr Tyr Thr Ser Ile Leu His
Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp
Tyr Ser Leu Thr Ile Ser Asn Leu Glu65 70 75 80Gln Glu Asp Phe Ala
Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro 85 90 95Trp Thr Phe Gly
Cys Gly Thr Lys Leu Glu Ile Lys 100 10514108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
14Met Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu1
5 10 15Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Gly
Arg 20 25 30Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys
Leu Leu 35 40 45Ile Tyr Tyr Thr Ser Ile Leu His Ser Gly Val Pro Ser
Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
Ser Asn Leu Glu65 70 75 80Gln Glu Asp Phe Ala Thr Tyr Phe Cys Gln
Gln Gly Asn Thr Leu Pro 85 90 95Trp Thr Phe Gly Cys Gly Thr Lys Leu
Glu Ile Lys 100 10515108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 15Met Asp Ile Gln Met Thr
Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu1 5 10 15Gly Asp Arg Val Thr
Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Gly 20 25 30Tyr Leu Asn Trp
Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu 35 40 45Ile Tyr Tyr
Thr Ser Ile Leu His Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser
Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu65 70 75
80Gln Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro
85 90 95Trp Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys 100
10516597PRTPseudomonas aeruginosa 16Ala Glu Glu Ala Phe Asp Leu Trp
Asn Glu Cys Ala Lys Ala Cys Val1 5 10 15Leu Asp Leu Lys Asp Gly Val
Arg Ser Ser Arg Met Ser Val Asp Pro 20 25 30Ala Ile Ala Asp Thr Asn
Gly Gln Gly Val Leu His Tyr Ser Met Val 35 40 45Leu Glu Gly Gly Asn
Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu 50 55 60Ser Ile Thr Ser
Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu65 70 75 80Pro Asn
Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser 85 90 95Trp
Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn 100 105
110Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His
115 120 125Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu
Ala Lys 130 135 140Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His
Glu Ser Asn Glu145 150 155 160Met Gln Pro Thr Leu Ala Ile Ser His
Ala Gly Val Ser Val Val Met 165 170 175Ala Gln Thr Gln Pro Arg Arg
Glu Lys Arg Trp Ser Glu Trp Ala Ser 180 185 190Gly Lys Val Leu Cys
Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Tyr 195 200 205Leu Ala Gln
Gln Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly Lys Ile 210 215 220Tyr
Arg Val Leu Ala Gly Asn Pro Ala Lys His Asp Leu Asp Ile Lys225 230
235 240Pro Thr Val Ile Ser His Arg Leu His Phe Pro Glu Gly Gly Ser
Leu 245 250 255Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu
Glu Thr Phe 260 265 270Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln
Leu Glu Gln Cys Gly 275 280 285Tyr Pro Val Gln Arg Leu Val Ala Leu
Tyr Leu Ala Ala Arg Leu Ser 290 295 300Trp Asn Gln Val Asp Gln Val
Ile Arg Asn Ala Leu Ala Ser Pro Gly305 310 315 320Ser Gly Gly Asp
Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala 325 330 335Arg Leu
Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg 340 345
350Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Gly Pro Ala Asp
355 360 365Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala
Glu Phe 370 375 380Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg
Gly Thr Gln Asn385 390 395 400Trp Thr Val Glu Arg Leu Leu Gln Ala
His Arg Gln Leu Glu Glu Arg 405 410 415Gly Tyr Val Phe Val Gly Tyr
His Gly Thr Phe Leu Glu Ala Ala Gln 420 425 430Ser Ile Val Phe Gly
Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala 435 440 445Ile Trp Arg
Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly 450 455 460Tyr
Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly465 470
475 480Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe
Tyr 485 490 495Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly
Glu Val Glu 500 505 510Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu
Asp Ala Ile Thr Gly 515 520 525Pro Glu Glu Glu Gly Gly Arg Leu Glu
Thr Ile Leu Gly Trp Pro Leu 530
535 540Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro
Arg545 550 555 560Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro
Asp Lys Glu Gln 565 570 575Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser
Gln Pro Gly Lys Pro Pro 580 585 590Arg Glu Asp Leu Lys
59517361PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 17Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln
Ala Cys His Leu Pro1 5 10 15Leu Glu Thr Phe Thr Arg His Arg Gln Pro
Arg Gly Trp Glu Gln Leu 20 25 30Glu Gln Cys Gly Tyr Pro Val Gln Arg
Leu Val Ala Leu Tyr Leu Ala 35 40 45Ala Arg Leu Ser Trp Asn Gln Val
Asp Gln Val Ile Arg Asn Ala Leu 50 55 60Ala Ser Pro Gly Ser Gly Gly
Asp Leu Gly Glu Ala Ile Arg Glu Gln65 70 75 80Pro Glu Gln Ala Arg
Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu 85 90 95Arg Phe Val Arg
Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn 100 105 110Ala Asp
Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala 115 120
125Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr
130 135 140Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser
Thr Arg145 150 155 160Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu
Gln Ala His Arg Gln 165 170 175Leu Glu Glu Arg Gly Tyr Val Phe Val
Gly Tyr His Gly Thr Phe Leu 180 185 190Glu Ala Ala Gln Ser Ile Val
Phe Gly Gly Val Arg Ala Arg Ser Gln 195 200 205Asp Leu Asp Ala Ile
Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala 210 215 220Leu Ala Tyr
Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg225 230 235
240Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu
245 250 255Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu
Ala Ala 260 265 270Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro
Leu Arg Leu Asp 275 280 285Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly
Arg Leu Glu Thr Ile Leu 290 295 300Gly Trp Pro Leu Ala Glu Arg Thr
Val Val Ile Pro Ser Ala Ile Pro305 310 315 320Thr Asp Pro Arg Asn
Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro 325 330 335Asp Lys Glu
Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro 340 345 350Gly
Lys Pro Pro Arg Glu Asp Leu Lys 355 36018345PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
18Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro1
5 10 15Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln
Leu 20 25 30Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr
Leu Ala 35 40 45Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg
Asn Ala Leu 50 55 60Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala
Ile Arg Glu Gln65 70 75 80Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu
Ala Ala Ala Glu Ser Glu 85 90 95Arg Phe Val Arg Gln Gly Thr Gly Asn
Asp Glu Ala Gly Ala Ala Asn 100 105 110Gly Pro Ala Asp Ser Gly Asp
Ala Leu Leu Glu Arg Asn Tyr Pro Thr 115 120 125Gly Ala Glu Phe Leu
Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg 130 135 140Gly Thr Gln
Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln145 150 155
160Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu
165 170 175Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg
Ser Gln 180 185 190Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala
Gly Asp Pro Ala 195 200 205Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu
Pro Asp Ala Arg Gly Arg 210 215 220Ile Arg Asn Gly Ala Leu Leu Arg
Val Tyr Val Pro Arg Ser Ser Leu225 230 235 240Pro Gly Phe Tyr Arg
Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala 245 250 255Gly Glu Val
Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp 260 265 270Ala
Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu 275 280
285Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro
290 295 300Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser
Ile Pro305 310 315 320Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp
Tyr Ala Ser Gln Pro 325 330 335Gly Lys Pro Pro Arg Glu Asp Leu Lys
340 34519318PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 19Met Trp Glu Gln Leu Glu Gln Cys
Gly Tyr Pro Val Gln Arg Leu Val1 5 10 15Ala Leu Tyr Leu Ala Ala Arg
Leu Ser Trp Asn Gln Val Asp Gln Val 20 25 30Ile Arg Asn Ala Leu Ala
Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu 35 40 45Ala Ile Arg Glu Gln
Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala 50 55 60Ala Ala Glu Ser
Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu65 70 75 80Ala Gly
Ala Ala Asn Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu 85 90 95Arg
Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val 100 105
110Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu
115 120 125Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val
Gly Tyr 130 135 140His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val
Phe Gly Gly Val145 150 155 160Arg Ala Arg Ser Gln Asp Leu Asp Ala
Ile Trp Arg Gly Phe Tyr Ile 165 170 175Ala Gly Asp Pro Ala Leu Ala
Tyr Gly Tyr Ala Gln Asp Gln Glu Pro 180 185 190Asp Ala Arg Gly Arg
Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val 195 200 205Pro Arg Ser
Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala 210 215 220Ala
Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu225 230
235 240Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly
Arg 245 250 255Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr
Val Val Ile 260 265 270Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val
Gly Gly Asp Leu Asp 275 280 285Pro Ser Ser Ile Pro Asp Lys Glu Gln
Ala Ile Ser Ala Leu Pro Asp 290 295 300Tyr Ala Ser Gln Pro Gly Lys
Pro Pro Arg Glu Asp Leu Lys305 310 31520230PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
20Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu Pro Thr Gly Ala Glu1
5 10 15Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr
Gln 20 25 30Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu
Glu Glu 35 40 45Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu
Glu Ala Ala 50 55 60Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser
Gln Asp Leu Asp65 70 75 80Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly
Asp Pro Ala Leu Ala Tyr 85 90 95Gly Tyr Ala Gln Asp Gln Glu Pro Asp
Ala Arg Gly Arg Ile Arg Asn 100 105 110Gly Ala Leu Leu Arg Val Tyr
Val Pro Arg Ser Ser Leu Pro Gly Phe 115 120 125Tyr Arg Thr Ser Leu
Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val 130 135 140Glu Arg Leu
Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr145 150 155
160Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro
165 170 175Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr
Asp Pro 180 185 190Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile
Pro Asp Lys Glu 195 200 205Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala
Ser Gln Pro Gly Lys Pro 210 215 220Pro Arg Glu Asp Leu Lys225
23021230PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 21Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu
Pro Thr Gly Ala Glu1 5 10 15Phe Leu Gly Asp Gly Gly Asp Val Ser Phe
Ser Thr Arg Gly Thr Gln 20 25 30Asn Trp Thr Val Glu Arg Leu Leu Gln
Ala His Arg Gln Leu Glu Glu 35 40 45Gly Gly Tyr Val Phe Val Gly Tyr
His Gly Thr Phe Leu Glu Ala Ala 50 55 60Gln Ser Ile Val Phe Gly Gly
Val Arg Ala Arg Ser Gln Asp Leu Asp65 70 75 80Ala Ile Trp Ala Gly
Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr 85 90 95Gly Tyr Ala Gln
Asp Gln Glu Pro Asp Ala Ala Gly Arg Ile Arg Asn 100 105 110Gly Ala
Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe 115 120
125Tyr Ala Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val
130 135 140Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala
Ile Thr145 150 155 160Gly Pro Glu Glu Ala Gly Gly Arg Leu Glu Thr
Ile Leu Gly Trp Pro 165 170 175Leu Ala Glu Arg Thr Val Val Ile Pro
Ser Ala Ile Pro Thr Asp Pro 180 185 190Arg Asn Val Gly Gly Asp Leu
Asp Pro Ser Ser Ile Pro Asp Ser Glu 195 200 205Gln Ala Ile Ser Ala
Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro 210 215 220Pro Arg Glu
Asp Leu Lys225 23022347PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 22Pro Glu Gly Gly Ser Leu
Ala Ala Leu Thr Ala His Gln Ala Cys His1 5 10 15Leu Pro Leu Glu Thr
Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu 20 25 30Gln Leu Glu Gln
Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr 35 40 45Leu Ala Ala
Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn 50 55 60Ala Leu
Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg65 70 75
80Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu
85 90 95Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly
Ala 100 105 110Ala Asn Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu
Arg Asn Tyr 115 120 125Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly
Asp Val Ser Phe Ser 130 135 140Thr Arg Gly Thr Gln Asn Trp Thr Val
Glu Arg Leu Leu Gln Ala His145 150 155 160Arg Gln Leu Glu Glu Arg
Gly Tyr Val Phe Val Gly Tyr His Gly Thr 165 170 175Phe Leu Glu Ala
Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg 180 185 190Ser Gln
Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp 195 200
205Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg
210 215 220Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro
Arg Ser225 230 235 240Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr
Leu Ala Ala Pro Glu 245 250 255Ala Ala Gly Glu Val Glu Arg Leu Ile
Gly His Pro Leu Pro Leu Arg 260 265 270Leu Asp Ala Ile Thr Gly Pro
Glu Glu Glu Gly Gly Arg Leu Glu Thr 275 280 285Ile Leu Gly Trp Pro
Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala 290 295 300Ile Pro Thr
Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser305 310 315
320Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser
325 330 335Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys 340
345235PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 23Lys Ala Ser Gly Gly1 5
* * * * *