U.S. patent number 10,385,139 [Application Number 15/897,847] was granted by the patent office on 2019-08-20 for murine, chimeric, humanized or human anti-tnf-alpha antibodies.
This patent grant is currently assigned to IBC Pharmaceuticals, Inc.. The grantee listed for this patent is IBC Pharmaceuticals, Inc.. Invention is credited to Chien-Hsing Chang, David M. Goldenberg, Rongxiu Li.
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United States Patent |
10,385,139 |
Goldenberg , et al. |
August 20, 2019 |
Murine, chimeric, humanized or human anti-TNF-alpha antibodies
Abstract
The present invention concerns compositions and methods of use
of bispecific antibodies comprising at least one anti-TNF-.alpha.
antibody or antigen-binding fragment thereof and at least one
anti-IL-6 antibody or antigen-binding fragment thereof. Preferably,
the bispecific antibody is in the form of a DNL.RTM. complex. The
anti-TNF-.alpha. or anti-IL-6 antibodies may comprise specific CDR
sequences disclosed herein. The compositions and methods are of use
to treat autoimmune disease, immune system dysfunction or
inflammatory disease, as disclosed herein.
Inventors: |
Goldenberg; David M. (Mendham,
NJ), Li; Rongxiu (Parsippany, NJ), Chang; Chien-Hsing
(Downingtown, PA) |
Applicant: |
Name |
City |
State |
Country |
Type |
IBC Pharmaceuticals, Inc. |
Morris Plains |
NJ |
US |
|
|
Assignee: |
IBC Pharmaceuticals, Inc.
(Morris Plains, NJ)
|
Family
ID: |
53005010 |
Appl.
No.: |
15/897,847 |
Filed: |
February 15, 2018 |
Prior Publication Data
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|
|
Document
Identifier |
Publication Date |
|
US 20180186898 A1 |
Jul 5, 2018 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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15620126 |
Jun 12, 2017 |
9932413 |
|
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15206571 |
Jul 11, 2016 |
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14525690 |
Aug 16, 2016 |
9416197 |
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61898798 |
Nov 1, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K
14/46 (20130101); A61K 47/6803 (20170801); A61K
47/6845 (20170801); C07K 16/248 (20130101); C07K
16/468 (20130101); A61K 45/06 (20130101); C07K
16/241 (20130101); A61K 47/6889 (20170801); A61K
47/6801 (20170801); C07K 2317/565 (20130101); C07K
2317/76 (20130101); C07K 2317/92 (20130101); C07K
2319/70 (20130101); C07K 2317/33 (20130101); C07K
2317/60 (20130101); C07K 2317/24 (20130101); A61K
39/3955 (20130101); C07K 2317/31 (20130101); C07K
2317/55 (20130101); C07K 2319/00 (20130101); A61K
2039/505 (20130101) |
Current International
Class: |
C07K
16/46 (20060101); A61K 47/68 (20170101); C07K
16/24 (20060101); C07K 14/46 (20060101); A61K
45/06 (20060101); A61K 39/00 (20060101); A61K
39/395 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
1544466 |
|
Nov 2004 |
|
CN |
|
2000/068248 |
|
Nov 2000 |
|
WO |
|
2005005638 |
|
Jan 2005 |
|
WO |
|
2006063150 |
|
Jun 2006 |
|
WO |
|
2006/107617 |
|
Oct 2006 |
|
WO |
|
2006/107786 |
|
Oct 2006 |
|
WO |
|
2007/046893 |
|
Apr 2007 |
|
WO |
|
20071075270 |
|
Jul 2007 |
|
WO |
|
2009149189 |
|
Dec 2009 |
|
WO |
|
2010003101 |
|
Jan 2010 |
|
WO |
|
2010003108 |
|
Jan 2010 |
|
WO |
|
2011084714 |
|
Jul 2011 |
|
WO |
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2012163521 |
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Dec 2012 |
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WO |
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Other References
Remicade European Medicines Agency, 2007. cited by examiner .
Freise et al, Molecular Immunology 2015; vol. 67, pp. 142-152.
cited by examiner .
Smith, UNM college of pharmacy, vol. 17, lesson 1, introduction to
diagnostic and therapeutic antibodies, 2012,. cited by examiner
.
Abbas et al., Cellular and Molecular Immunology, W.B. Saunders
Comp. 1991, p. 43. cited by applicant .
Alto et al., "Bioinformatic design of A-kinase anchoring protein-in
silico: a potent and selective peptide antagonist of type II
protein kinase A anchoring" Proc. Natl. Acad. Sci USA Apr. 15,
2003; 100(8):4445-50. cited by applicant .
Backer et al., "Self-Assembled "Dock and Lock" System for Linking
Payloads to Targeting Proteins" Bioconjugate Chem., 2006,
17(4):912-919. cited by applicant .
Baillie et al., "Compartmentalisation of phospodiesterases and
protein kinase A: opposites attract" FEBS Letters 2005;
579:3264-3270. cited by applicant .
Banky et al., "Dimerization/Docking Domain of the Type I.alpha.
Regulatory Subunit of cAMP-dependent Protein Kinase" J. Biol. Chem.
273:35048-55, 1998. cited by applicant .
Basu et al., "Structure-Function Engineering of
Interferon-.beta.-1b for Improving Stability, Solubility, Potency,
Immunogenicity, and Pharmacokinetic Properties by Site-Selective
Mono-PEGylation" Bioconjugate Chem. 2006; 17:618-630. cited by
applicant .
Belardelli et al., "Interferon-alpha in tumor immunity and
immunotherapy" Cytokine Growth Factor Rev. 13(2):119-134(2002).
cited by applicant .
Belardelli et al., "International Meeting on Cancer Vaccines: How
Can We Enhance Efficacy of Therapeutic Vaccines?" Cancer Res.
64:6827-6830 (2004). cited by applicant .
Belardelli et al., "The neglected role of type I interferon in the
T-cell response: implications for its clinical use" Immunol. Today
17(8):369-72 (1996). cited by applicant .
Biron et al., "Natural killer cells in antiviral defense: function
and regulation by innate cytokines" Annu. Rev. Immunol. 17:189-220
(1999). cited by applicant .
Brunda et al., "Modulation of Murine Natural Killer Cell Activity
in Vitro and in Vivo by Recombinant Human Interferons" Cancer Res.
44:597-601 (1984). cited by applicant .
Burns-Hamuro et al., "Distinct interaction modes of an AKAP bound
to two regulatory subunit isoforms of protein kinase A revealed by
amide hydrogen/deuterium exchange" Protein Science (2005),
14:2982-2992. cited by applicant .
Carr et al., "Interaction of the Regulatory Subunit (RII) of
cAMP-dependent Protein Kinase with RII-anchoring Proteins Occurs
through an Amphipathic Helix Binding Motif" J. Biol. Chem.
266:14188-92 (1991). cited by applicant .
Carr et al., "Identification of Sperm-specific Proteins That
Interact with A-kinase Anchoring Proteins in a Manner Similar to
the Type II Regulatory Subunit of PKA" J. Biol. Chem.
276(20):17332-17338 (2001). cited by applicant .
Carrero et al., "Lymphocytes are detrimental during the early
innate immune response against Listeria monocytogenes" J. Exp. Med.
203(4):933-940 (2006). cited by applicant .
Chang et al., "The Dock and Lock Method: A Novel Platform
Technology for Building Multivalent, Multifunctional Structures of
Defined Composition with Retained Bioactivity" Clin. Cancer Res.
Sep. 15, 2007;13(18 Suppl), pp. 5586-5591. cited by applicant .
Chmura et al., "Antibodies with infinite affinity" Proc. Natl.
Acad. Sci. USA 98(15):8480-8484 (2001). cited by applicant .
Colledge et al., "AKAPs: from structure to function" Trends Cell
Biol. 6:216-21 (1999). cited by applicant .
Corbin et al., "Regulation of Adenosine
3',5'-Monophosphate-dependent Protein Kinase" J. Biol. Chem.
248:1813-21 (1973). cited by applicant .
Dhalluin et al., "Structural and Biophysical Characterization of
the 40 kDa PEG-Interferon-.alpha.2a and Its Individual Positional
Isomers" Bioconjugate Chem. 2005;16:504-517. cited by applicant
.
Dodart et al., "Immunotherapy for Alzheimer's Disease: will
vaccination work?" Trends Mol. Med. 9(3):85-87 (2003). cited by
applicant .
Doherty et al., "Site-Specific PEGylation of Engineered Cysteine
Analogues of Recombinant Human Granulocyte-Macrophage
Colony-Stimulating Factor" Bioconjugate Chem. 2005;16:1291-1298.
cited by applicant .
Ferrantini et al., "IFN-.alpha.1 Gene Expression into a Metastatic
Murine Adenocarcinoma (TS/A) Results in CD8+ T Cell-Mediated Tumor
Rejection and Development of Antitumor Immunity" J. Immunol.
153:4604-15 (1994). cited by applicant .
Ferrantini et al., "Interferon-.alpha. and cancer: Mechanisms of
action and new perspectives of clinical use" Biochimie 89: 884-893
(2007). cited by applicant .
Foser et al., "Improved biological and transcriptional activity of
monopegylated interferon-.alpha.-2a isomers" The Pharmacogenomics J
3:312-319 (2003). cited by applicant .
Gillies et al., "High-level expression of chimeric antibodies using
adapted cDNA variable region cassettes" J. Immunol. Methods 125
(1989) 191-202. cited by applicant .
Glennie et al., "Mechanisms of killing by anti-CD20 monoclonal
antibodies" Mol. Immunol. 44:3823-3837 (2007). cited by applicant
.
Gold et al., "A Novel Bispecific, Trivalent Antibody Construct for
Targeting Pancreatic Carcinoma", Cancer Res. 68:4819-26, 2008.
cited by applicant .
Gold et al., "Molecular Basis of AKAP Specificity for PKA
Regulatory Subunits" Mol. Cell Nov. 3, 2006;24(3):383-95. cited by
applicant .
Goldenberg et al., "Multifunctional Antibodies by the Dock-and-Lock
Method for Improved Cancer Imaging and Therapy by Pretargeting" J.
Nucl. Med. 49:158-63, 2008. cited by applicant .
Goldenberg et al., "Properties and structure-function relationships
of veltuzumab (hA20), a humanized anti-CD20 monoclonal antibody"
Blood 113:1062-70 (2009). cited by applicant .
Goodson et al., "Site-Directed PEGylation of Recombinant
Interleukin-2 at its Glycosylation Site" Nat. Biotechnology Apr.
1990;8(4):343-6. cited by applicant .
Grace et al., "Site of Pegylation and Polyethylene Glycol Molecule
Size Attenuate Interferon-.alpha. Antiviral and Antiproliferative
Activities through the JAK/STAT Signaling Pathway" J. Biol. Chem.
2005;280(8):6327-6336. cited by applicant .
Grimley et al., "Prolonged STAT1 Activation Related to the Growth
Arrest of Malignant Lymphoma Cells by Interferon-.alpha." Blood
91(8):3017-27 (1998). cited by applicant .
Gutterman et al., "Leukocyte Interferon-Induced Tumor Regression in
Human Metastatic Breast Cancer, Multiple Myeloma, and Malignant
Lymphoma" Ann. Intern. Med. 93(3):399-406 (1980). cited by
applicant .
Gutterman et al., "Cytokine therapeutics: Lessons from interferon
.alpha." Proc. Natl. Acad. Sci. USA 91:1198-205 (1994). cited by
applicant .
Harris et al., "Effect of pegylation on pharmaceuticals" Nat. Rev.
Drug. Discov. 2:214-221 (2003). cited by applicant .
Hausken et al., "Mutational Analysis of the A-Kinase Anchoring
Protein (AKAP)-binding Site on RII" J. Biol. Chem. 271:29016-22
(1996). cited by applicant .
Hodneland et al., Selective immobilization of proteins to
self-assembled monolayers presenting active site-directed capture
ligands, Proc. Natl. Acad. Sci. USA 2002; 99:5048-5052. cited by
applicant .
Huang et al., "Targeting IFN-.alpha. to B Cell Lymphoma by a
Tumor-Specific Antibody Elicits Potent Antitumor Activities" J.
Immunol. 179:6881-88 (2007). cited by applicant .
Hundsrucker et al., "High-affinity AKAP7.delta.-protein kinase A
interaction yields novel protein kinase A-anchoring disruptor
peptides" Biochem. J. (2006) 396, 297-306. cited by applicant .
Kimby et al., "Long-term molecular remissions in patients with
indolent lymphoma treated with rituximab as a single agent or in
combination with interferon alpha-2a: A randomized phase II study
from the Nordic Lymphoma Group" Leuk. Lymphoma 49(1)102-112 (2008).
cited by applicant .
Kinderman et al., "A Dynamic Mechanism for AKAP Binding to RII
Isoforms of cAMP-Dependent Protein Kinase" Mol. Cell 24(3):397-408
(2006). cited by applicant .
Kinstler et al., "Characterization and Stability of N-terminally
PEGylated rhG-CSF" Pharm. Res. 1996;13(7):996-1002. cited by
applicant .
Kramer et al., "Cell and virus sensitivity studies with recombinant
human alpha interferons" J. Interferon. Res. 3(4):425-35 (1983).
cited by applicant .
Le Bon et al., "Type I Interferons Potently Enhance Humoral
Immunity and Can Promote Isotype Switching by Stimulating Dendritic
Cells In Vivo" Immunity 14:461-470 (2001). cited by applicant .
Lee et al., "Solid-Phase PEGylation of Recombinant Interferon
.alpha.-2a for Site-Specific Modification: Process Performance,
Characterization, and in Vitro Bioactivity" Bioconjugate Chem.
2007; 18:1728-34. cited by applicant .
Lohmann et al., "High-affinity binding of the regulatory subunit
(RII) of cAMP-dependent protein kinase to microtubule-associated
and other cellular proteins" Proc. Natl. Acad. Sci. USA 81:6723-27
(1984). cited by applicant .
Luft et al., "Type I IFNs Enhance the Terminal Differentiation of
Dendritic Cells" J. Immunol. 161:1947-1953 (1998). cited by
applicant .
Mason, Anthony J., "Functional Analysis of the Cysteine Residues of
Activin A" Mol. Endocrinol. 8:325-32 (1994). cited by applicant
.
Matarrese et al., "Type I Interferon Gene Transfer Sensitizes
Melanoma Cells to Apoptosis via a Target Activity on Mitochondrial
Function" Am. J. Pathol. 2002, 160(4):1507-1520. cited by applicant
.
Mecchia et al., "Type I consensus interferon (CIFN) gene transfer
into human melanoma cells up-regulates p53 and enhances
cisplatin-induced apoptosis: implications for new therapeutic
strategies with IFN-alpha" Gene Ther. (2000) 7, 167-179. cited by
applicant .
Newlon et al., "A Novel Mechanism of PKA Anchoring Revealed by
Solution Structures of Anchoring Complexes" EMBO J. 2001;
20:1651-1662. cited by applicant .
Newlon et al., "The molecular basis for protein kinase A anchoring
revealed by solution NMR" Nature Struct. Biol. 1999; 3:222-227.
cited by applicant .
Ngo et al., "Computational Complexity, Protein Structure
Prediction, and the Levinthal Paradox", The Protein Folding Problem
and Tertiary Structure Prediction, Ch. 14, pp. 492-495, (Mertz
& Le Grand, Eds.), Birkhauser Boston, 1994. cited by applicant
.
Osborn et al., "Pharmacokinetic and Pharmacodynamic Studies of a
Human Serum Albumin-Interferon-.alpha. Fusion Protein in Cynomolgus
Monkeys" J. Pharmacol. Exp. Ther. 303(2):540-548 (2002). cited by
applicant .
Oyen et al., "Human testis cDNA for the regulatory subunit
RII.alpha. of cAMP-dependent protein kinase encodes an alternate
amino-terminal region" FEBS Letters 246:57-64, 1989. cited by
applicant .
Ozzello et al., "Conjugation of interferon alpha to a humanized
monoclonal antibody (HuBrE-3vI) enhances the selective localization
and antitumor effects of interferon in breast cancer xenografts"
Breast Cancer Res. Treat. 48: 135-147 (1998). cited by applicant
.
Paquette et al., "Interferon-.alpha. and granulocyte-macrophage
colony-stimulating factor differentiate peripheral blood monocytes
into potent antigen-presenting cells" J. Leukoc. Biol. 64:358-367;
1998. cited by applicant .
Pelham et al., "Interferon-.alpha. conjugation to human osteogenic
sarcoma monoclonal antibody 791T/36" Cancer Immunol. Immuother
1983;15(3):210-216. cited by applicant .
Pepinsky et al., "Improved Pharmacokinetic Properties of a
Polyethylene Glycol-Modified Form of Interferon-.beta.-1a with
Preserved in Vitro Bioactivity" Pharmacol. Exp. Ther. 2001;
297(3):1059-1066. cited by applicant .
Pilling et al., "Interferon-.beta. mediates stromal cell rescue of
T cells from apoptosis" Eur. J. Immunol. 29:1041-1050 (1999). cited
by applicant .
Rabjohn et al., "Molecular Cloning and Epitope Analysis of the
Peanut Allergen Ara h 3" J. Clinical Investigation 103(4):535-542
(1999). cited by applicant .
Raefsky et al., "Studies of Interferon as a regulator of
hematopoietic cells proliferation" J. Immunol. 135(4):2507-2512
(1985). cited by applicant .
Rose et al., "Structural basis of dimerization, coactivator
recognition and MODY3 mutations in HNF-1.alpha." Nature Struct.
Biol. 2000; 7:744-748. cited by applicant .
Rosendahl et al., "A Long-Acting, Highly Potent Interferon
.alpha.-2 Conjugate Created Using Site-Specific PEGylation"
Bioconjugate Chem. 2005;16:200-207. cited by applicant .
Rossi et al., "Novel Designs of Multivalent Anti-CD20 Humanized
Antibodies as Improved Lymphoma Therapeutics" Cancer Res.
68:8384-92 (2008). cited by applicant .
Rossi et al., "Stably tethered multifunctional structures of
defined composition made by the dock and lock method for use in
cancer targeting" Proc. Nal Acad. Sci. Epub Apr. 24, 2006, vol.
103, No. 18, pp. 6841-6846. cited by applicant .
Rustandi et al., "The Ca2+-Dependent Interaction of
S100B(.beta..beta.) with a Peptide Derived from p53", Biochemistry
1998; 37: 1951-1960. cited by applicant .
Sabaawy et al., "Enhancement of 5-fluorouracil cytotoxicity on
human colon cancer cells by retrovirus-mediated interferon-.alpha.
gene transfer" Int. J. Oncol. Jun. 1999; 14(6):1143-51. cited by
applicant .
Salles et al., "Rituximab combined with chemotherapy and interferon
in follicular lymphoma patients: results of the GELA-GOELAMS FL2000
study" Blood 2008; 112:4824-4831. cited by applicant .
Santini et al., "Type I Interferon as a Powerful Adjuvant for
Monocyte-derived Dendritic Cell Development and Activity In Vivo
and in Hu-PBL-SCID Mice" J. Exp. Med. 191(10):1777-1788 (2000).
cited by applicant .
Scott et al., "Type II Regulatory Subunit Dimerization Determines
the Subcellular Localization of the cAMP-dependent Protein Kinase"
J. Biol. Chem. 265:21561-66 (1990). cited by applicant .
Scott et al., "Cyclic nucleotide-dependent protein kinases"
Pharmacol. Ther. 1991;50(1):123-45. cited by applicant .
Seffernick et al., "Melamine Deaminase and Atrazine
Chlorohydrolase: 98 Percent Identical but Functionally Different"
J. Bacteriol. 183(8):2405-2410 (2001). cited by applicant .
Sharkey et al., "Improved Therapeutic Results by Pretargeted
Radioimmunotherapy of Non-Hodgkin's Lymphoma with a New
Recombinant, Trivalent, Anti-CD20, Bispecific Antibody" Cancer Res.
68:5282-90 (2008). cited by applicant .
Sharkey et al., "Metastatic Human Colonic Carcinoma: Molecular
Imaging with Pretargeted SPECT and PET in a Mouse Model" Radiology
246:497-507 (2008). cited by applicant .
Sidky et al., "Inhibition of Angiogenesis by Interferons: Effects
on Tumor- and Lymphocyte-induced Vascular Responses" Cancer Res.
47:5155-5161, Oct. 1, 1987. cited by applicant .
Stein et al., "Characterization of a New Humanized Anti-CD20
Monoclonal Antibody, IMMU-106, and Its Use in Combination with the
Humanized Anti-CD22 Antibody, Epratuzumab, for the Therapy of
Non-Hodgkin's Lymphoma" Clin. Cancer Res. vol. 10, 2868-2878, Apr.
15, 2004. cited by applicant .
Stein et al., "Characterization of a humanized IgG4 anti-HLA-DR
monoclonal antibody that lacks effector cell functions but retains
direct antilymphoma activity and increases the potency of
rituximab" Blood 2006;108:2736-2744. cited by applicant .
Stokka et al., "Characterization of A-kinase-anchoring disruption
using a solution-based assay" Biochem. J. (2006) 400, 493-499.
cited by applicant .
Stryer et al., "Levels of Structure in Protein Architecture",
Biochemistry, 3rd Ed., pp. 31-33, W.H. Freeman & Co., New York,
1988. cited by applicant .
Takaoka et al., "Integration of interferon-.alpha./.beta.
signalling to p53 responses in tumour suppression and antiviral
defence" Nature Jul. 31, 2003;424(6948):516-23. cited by applicant
.
Taylor, S., "cAMP-dependent Protein Kinase" J. Biol. Chem.
1989;264(15):8443-8446. cited by applicant .
Walsh et al., "An Adenosine 3', 5'-Monophosphate-dependant Protein
Kinase from Rabbit Skeletal Muscle" J. Biol. Chem.
243(13):3763-3774 (1968). cited by applicant .
Weck et al., "Comparison of the Antiviral Activities of Various
Cloned Human Interferon-.alpha. Subtypes in Mammalian Cell
Cultures" J. Gen. Virol. (1981), 57, 233-237. cited by applicant
.
Winkler et al., "Changing the Antigen Binding Specificity by Single
Point Mutations of an Anti-p24 (HIV-1) Antibody" J. Immunol.
165:4505-14 (2000). cited by applicant .
Witkowski et al., "Conversion of a .beta.-Ketoacyl Synthase to a
Malonyl Decarboxylase by Replacement of the Active-Site Cysteine
with Glutamine" Biochemistry 38(36):11643-50 (1999). cited by
applicant .
Wong et al., "AKAP Signalling Complexes: Focal Points in Space and
Time" Nat. Rev. Mol. Cell Biol. 12:959-70 (2004). cited by
applicant .
Zhu et al., "Inhibition of tumor growth and metastasis by targeting
tumor-associated angiogenesis with antagonists to the receptors of
vascular endothelial growth factor" Invest. New Drugs 17:195-212,
1999. cited by applicant .
Allard et al., "Targeting CD73 enhances the antitumor activity of
anti-PD-1 and anti-CTLA-4 mAbs", Clin Cancer Res. Oct. 15,
2013;19(20):5626-35. cited by applicant .
Almoallim et al., "Anti-Tumor Necrosis Factor-.alpha. Induced
Systemic Lupus Erythematosus", Open Rheumatol J. 2012;6:315-9.
cited by applicant .
Baeuerle et al., "Bispecific T-cell engaging antibodies for cancer
therapy", Cancer Res. Jun. 15, 2009;69(12):4941-4. cited by
applicant .
Cardillo et al., "Targeting both IGF-1R and mTOR synergistically
inhibits growth of renal cell carcinoma in vitro", BMC Cancer. Apr.
1, 2013;13:170. cited by applicant .
Callahan et al., "At the bedside: CTLA-4- and PD-1-blocking
antibodies in cancer immunotherapy", J Leukoc Biol. Jul.
2013;94(1):41-53. cited by applicant .
Chang et al., "A new method to produce monoPEGylated dimeric
cytokines shown with human interferon-.alpha.2b", Bioconjug Chem.
Oct. 21, 2009;20(10):1899-907. cited by applicant .
Chang et al., "A novel class of anti-HIV agents with multiple
copies of enfuvirtide enhances inhibition of viral replication and
cellular transmission in vitro", PLoS One. 2012;7(7):e41235. cited
by applicant .
Chang et al., "Evaluation of a novel hexavalent humanized
anti-IGF-1R antibody and its bivalent parental IgG in diverse
cancer cell lines", PLoS One. 2012;7(8):e44235. cited by applicant
.
Fischer et al., "Combined inhibition of tumor necrosis factor
.alpha. and interleukin-17 as a therapeutic opportunity in
rheumatoid arthritis: development and characterization of a novel
bispecific antibody", Arthritis Rheumatol. Jan. 2015;67(1):51-62.
cited by applicant .
Flieger et al., "A bispecific single-chain antibody directed
against EpCAM/CD3 in combination with the cytokines interferon
alpha and interleukin-2 efficiently retargets T and CD3+CD56+
natural-killer-like T lymphocytes to EpCAM-expressing tumor cells",
Cancer Immunol Immunother. Oct. 2000;49(8):441-8. cited by
applicant .
Goldenberg, DM., "Targeted therapy of cancer with radiolabeled
antibodies", J Nucl Med. May 2002;43(5):693-713. cited by applicant
.
Gleason et al., "Bispecific and trispecific killer cell engagers
directly activate human NK cells through CD16 signaling and induce
cytotoxicity and cytokine production", Mol Cancer Ther. Dec.
2012;11(12):2674-84. cited by applicant .
Hennigan et al., "Interleukin-6 inhibitors in the treatment of
rheumatoid arthritis", Ther Clin Risk Manag. Aug. 2008;4(4):767-75.
cited by applicant .
Hirabara et al., "Clinical efficacy of abatacept, tocilizumab, and
etanercept in Japanese rheumatoid arthritis patients with
inadequate response to anti-TNF monoclonal antibodies", Clin
Rheumatol. Sep. 2014;33(9):1247-54. cited by applicant .
Intlekofer et al., "At the bench: preclinical rationale for CTLA-4
and PD-1 blockade as cancer immunotherapy", J Leukoc Biol. Jul.
2013;94(1):25-39. cited by applicant .
Kipriyanov et al., "Synergistic antitumor effect of bispecific
CD19.times.CD3 and CD19.times.CD16 diabodies in a preclinical model
of non-Hodgkin's lymphoma", J Immunol. Jul. 1, 2002;169(1):137-44.
cited by applicant .
Kyi et al., "Checkpoint blocking antibodies in cancer
immunotherapy", FEBS Lett. Jan. 21, 2014;588(2):368-76. cited by
applicant .
Liu et al., "Trop-2-targeting tetrakis-ranpirnase has potent
antitumor activity against triple-negative breast cancer", Mol
Cancer. Mar. 10, 2014;13:53. cited by applicant .
Lum et al., "Targeted T-cell Therapy in Stage IV Breast Cancer: A
Phase I Clinical Trial", Clin Cancer Res. May 15,
2015;21(10):2305-14. cited by applicant .
Morales-Kastresana et al., "Combined immunostimulatory monoclonal
antibodies extend survival in an aggressive transgenic
hepatocellular carcinoma mouse model", Clin Cancer Res. Nov. 15,
2013;19(22):6151-62. cited by applicant .
Mori et al., "IL-1.beta. and TNF.alpha.-initiated IL-6-STAT3
pathway is critical in mediating inflammatory cytokines and RANKL
expression in inflammatory arthritis", Int Immunol. Nov.
2011;23(11):701-12. cited by applicant .
Oberst et al., "CEA/CD3 bispecific antibody MEDI-565/AMG 211
activation of T cells and subsequent killing of human tumors is
independent of mutations commonly found in colorectal
adenocarcinomas", MAbs. 2014;6(6):1571-84. cited by applicant .
Ofei et al., "Effects of an engineered human anti-TNF-alpha
antibody (CDP571) on insulin sensitivity and glycemic control in
patients with NIDDM", Diabetes. Jul. 1996;45(7):881-5. cited by
applicant .
Peng et al., "The CEA/CD3-bispecific antibody MEDI-565 (MT111)
binds a nonlinear epitope in the full-length but not a short splice
variant of CEA", PLoS One. 2012;7(5):e36412. cited by applicant
.
Podojil et al., "Targeting the B7 family of co-stimulatory
molecules: successes and challenges", BioDrugs. Feb.
2013;27(1):1-13. cited by applicant .
Rossi et al., "Hexavalent bispecific antibodies represent a new
class of anticancer therapeutics: 1. Properties of anti-CD20/CD22
antibodies in lymphoma", Blood. Jun. 11, 2009;113(24):6161-71.
cited by applicant .
Rossi et al., "The dock-and-lock method combines recombinant
engineering with site-specific covalent conjugation to generate
multifunctional structures", Bioconjug Chem. Mar. 21,
2012;23(3):309-23. cited by applicant .
Rossi et al., "Complex and defined biostructures with the
dock-and-lock method", Trends Pharmacol Sci. Sep.
2012;33(9):474-81. cited by applicant .
Rossi et al., "Optimization of multivalent bispecific antibodies
and immunocytokines with improved in vivo properties", Bioconjug
Chem. Jan. 16, 2013;24(1):63-71. cited by applicant .
Rossi et al., "A new class of bispecific antibodies to redirect T
cells for cancer immunotherapy", MAbs. Mar.-Apr. 2014;6(2):381-91.
cited by applicant .
Rossi et al., "A New Platform for Trivalent Bispecific Antibodies
Used for T-Cell Redirected Killing of B-Cell Malignancies", Nov.
15, 2013; Blood: 122 (21). cited by applicant .
Rossi et al., "A novel Trop-2/CD3 trivalent bispecific antibody
effectively redirects T cells to kill target human pancreatic and
gastric cancer cells", Proceedings of the 105th Annual Meeting of
the American Association for Cancer Research; Apr. 5-9, 2014; San
Diego, CA; Cancer Res 2014;74(19 Suppl):Abstract # 2655. cited by
applicant .
Rossi et al., Novel T-cell redirecting trivalent bispecific
antibodies, Proceedings of the 104th Annual Meeting of the American
Association for Cancer Research; Apr. 6-10, 2013; Washington, DC;
Cancer Res 2013;73(8 Suppl):Abstract # 4747. cited by applicant
.
Rossi et al., "Redirected T-cell killing of solid cancers targeted
with an anti-CD3/Trop-2-bispecific antibody is enhanced in
combination with interferon-.alpha.", Mol Cancer Ther. Oct.
2014;13(10):2341-51. cited by applicant .
Sharkey et al., "Improved cancer therapy and molecular imaging with
multivalent, multispecific antibodies", Cancer Biother Radiopharm.
Feb. 2010;25(1):1-12. cited by applicant .
Shubert et al., "A recombinant triplebody with specificity for CD19
and HLA-DR mediates preferential binding to antigen double-positive
cells by dual-targeting", MAbs. Jan.-Feb. 2012;4(1):45-56. cited by
applicant .
Stedman'S Online Medical Dictionary, Feb. 3, 2017; p. 1. cited by
applicant .
Tanaka et al., "Targeting interleukin-6: all the way to treat
autoimmune and inflammatory diseases", Int J Biol Sci.
2012;8(9)1227-36. cited by applicant .
Topalian et al., "Targeting the PD-1/B7-H1(PD-L1) pathway to
activate anti-tumor immunity", Curr Opin Immunol. Apr.
2012;24(2):207-12. cited by applicant .
Vallera et al., "A bispecific recombinant immunotoxin, DT2219,
targeting human CD19 and CD22 receptors in a mouse xenograft model
of B-cell leukemia/lymphoma", Clin Cancer Res. May 15,
2005;11(10):3879-88. cited by applicant .
Wu et al., "Molecular construction and optimization of anti-human
IL-1alpha/beta dual variable domain immunoglobulin (DVD-Ig)
molecules", MAbs. Jul.-Aug. 2009;1(4):339-47. cited by
applicant.
|
Primary Examiner: Bunner; Bridget E
Assistant Examiner: Hamud; Fozia
Attorney, Agent or Firm: Nakashima; Richard A.
Parent Case Text
RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser.
No. 15/620,126, filed Jun. 12, 2017, which was a divisional of U.S.
patent application Ser. No. 15/206,571 (now abandoned), filed Jul.
11, 2016, which was a divisional of U.S. patent application Ser.
No. 14/525,690 (now issued U.S. Pat. No. 9,416,197), filed Oct. 28,
2014, which claimed the benefit under 35 U.S.C. 119(e) of
provisional U.S. Patent Application Ser. No. 61/898,798, filed Nov.
1, 2013.
Claims
What is claimed is:
1. A murine, chimeric, humanized or human anti-TNF-.alpha. antibody
or antigen-binding fragment thereof comprising the heavy chain CDR
sequences CDR1 (GFWN, SEQ ID NO:113), CDR2 (YISYSGRTYYNPSLKS, SEQ
ID NO:114) and CDR3 (DANYVLDY, SEQ ID NO:115) and the light chain
CDR sequences CDR1 (KSSQSLLNSSTQKNYLA, SEQ ID NO:116), CDR2
(FASARES, SEQ ID NO:117) and CDR3 (QQHYRTPFT, SEQ ID NO:118).
2. The anti-TNF-.alpha. antibody or fragment thereof of claim 1,
wherein the antibody allotype is selected from the group consisting
of nG1m1, G1m3, nG1m1,2 and Km3.
3. The anti-TNF-.alpha. antibody or fragment thereof of claim 1,
wherein the antibody or fragment is a naked antibody or
fragment.
4. The anti-TNF-.alpha. antibody or fragment thereof of claim 1,
wherein the antibody is conjugated to an agent selected from the
group consisting of a drug, an anti-angiogenic agent, a
pro-apoptotic agent, an antibiotic, a hormone, a hormone
antagonist, an immunomodulator, a cytokine, a chemokine, a prodrug,
and an enzyme.
5. The anti-TNF-.alpha. antibody or fragment thereof of claim 4,
wherein the drug possesses a pharmaceutical property selected from
the group consisting of antimitotic, antikinase, anti-tyrosine
kinase, alkylating, antimetabolite, antibiotic, alkaloid,
anti-angiogenic, pro-apoptotic agent, and immune modulator.
6. The anti-TNF-.alpha. antibody or fragment thereof of claim 4,
wherein the drug is selected from the group consisting of
5-fluorouracil, aplidin, azaribine, anastrozole, anthracyclines,
bendamustine, bleomycin, bortezomib, bryostatin-1, busulfan,
calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin,
carmustine, celecoxib, chlorambucil, cisplatinum, Cox-2 inhibitors,
irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans,
cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin,
daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX),
pro-2P-DOX, cyano-morpholino doxorubicin, doxorubicin glucuronide,
epirubicin glucuronide, estramustine, epipodophyllotoxin, estrogen
receptor binding agents, etoposide (VP16), etoposide glucuronide,
etoposide phosphate, floxuridine (FUdR), 3',5'-O-dioleoyl-FudR
(FUdR-dO), fludarabine, flutamide, farnesyl-protein transferase
inhibitors, gemcitabine, hydroxyurea, idarubicin, ifosfamide,
L-asparaginase, lenolidamide, leucovorin, lomustine,
mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,
methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane,
navelbine, nitrosourea, plicomycin, procarbazine, paclitaxel,
pentostatin, PSI-341, raloxifene, semustine, streptozocin,
tamoxifen, temazolomide, transplatinum, thalidomide, thioguanine,
thiotepa, teniposide, topotecan, uracil mustard, vinorelbine,
vinblastine, vincristine and vinca alkaloids.
7. The anti-TNF-.alpha. antibody or fragment thereof of claim 4,
wherein the chemokine is selected from the group consisting of
RANTES, MCAF, MIP1-alpha, MIP1-Beta and IP-10.
8. The anti-TNF-.alpha. antibody or fragment thereof of claim 4,
wherein the anti-angiogenic agent is selected from the group
consisting of angiostatin, baculostatin, canstatin, maspin,
anti-VEGF antibody, anti-PlGF peptide, anti-vascular growth factor
antibody, anti-Flk-1 antibody, anti-Flt-1 antibody, anti-Kras
antibody, anti-cMET antibody, anti-MIF (macrophage
migration-inhibitory factor) antibody, laminin peptide, fibronectin
peptide, plasminogen activator inhibitor, tissue metalloproteinase
inhibitor, interferon, interleukin-12, IP-10, Gro- ,
thrombospondin, 2-methoxyoestradiol, proliferin-related protein,
carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate,
angiopoietin-2, interferon-alpha, herbimycin A, PNU145156E, 16K
prolactin fragment, Linomide (roquinimex), thalidomide,
pentoxifylline, genistein, TNP-470, endostatin, paclitaxel,
accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470,
platelet factor 4 and minocycline.
9. The anti-TNF-.alpha. antibody or fragment thereof of claim 4,
wherein the immunomodulator is selected from the group consisting
of a cytokine, a stem cell growth factor, a lymphotoxin, a
hematopoietic factor, a colony stimulating factor (CSF), an
interferon (IFN), erythropoietin, and thrombopoietin.
10. The anti-TNF-.alpha. antibody or fragment thereof of claim 9,
wherein the cytokine is selected from the group consisting of human
growth hormone, N-methionyl human growth hormone, bovine growth
hormone, parathyroid hormone, thyroxine, insulin, proinsulin,
relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroid
stimulating hormone (TSH), luteinizing hormone (LH), hepatic growth
factor, prostaglandin, fibroblast growth factor, prolactin,
placental lactogen, OB protein, tumor necrosis factor-.alpha.,
tumor necrosis factor- , mullerian-inhibiting substance, mouse
gonadotropin-associated peptide, inhibin, activin, vascular
endothelial growth factor, integrin, thrombopoietin (TPO), NGF- ,
platelet-growth factor, transforming growth factor-.alpha.
(TGF-.alpha.), TGF- , insulin-like growth factor-I, insulin-like
growth factor-II, interferon-.alpha., interferon-.beta.,
interferon-.gamma., interferon-.lamda., macrophage-CSF,
interleukin-1 (IL-1), IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,
IL-18, IL-21, IL-25, LIF, FLT-3, angiostatin, thrombospondin,
endostatin, and LT (lymphotoxin).
Description
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been
submitted electronically in ASCII format and is hereby incorporated
by reference in its entirety. Said ASCII copy, created on Oct. 27,
2014, is named IBC139US1 SL.txt and is 58,035 bytes in size.
FIELD OF THE INVENTION
The present invention relates to compositions and methods of use of
complexes comprising at least one anti-TNF-.alpha. antibody or
antigen-binding fragment thereof and at least one anti-IL-6
antibody or antigen-binding fragment thereof. The complex may be a
bispecific or multispecific antibody or fragment thereof.
Preferably, the complex is a DOCK-AND-LOCK.RTM. (DNL.RTM.) complex,
in which the components are joined using the binding affinity
between a DDD (dimerization and docking domain) moiety of human
protein kinase A (PKA) regulatory subunit RI.alpha., RI.beta.,
RII.alpha. or RII.beta., and an AD (anchoring domain) moiety of an
A-kinase anchoring protein (AKAP), wherein a pair of DDD moieties
forms a dimer that binds to a complementary sequence on the AD
moiety. Although the basic DNL.RTM. complex is trimeric, complexes
with other stoichiometries are possible, such as tetrameric,
pentameric or hexameric. The subject complexes are of use to treat
autoimmune disease, inflammatory disease or other conditions in
which TNF-.alpha. and IL-6 play a pathogenic role. In particularly
preferred embodiments, the disease or condition is selected from
the group consisting of systemic lupus erythematosus (SLE),
rheumatoid arthritis, inflammatory bowel disease, type II diabetes,
obesity, atherosclerosis and cachexia related to cancer.
BACKGROUND OF THE INVENTION
TNF-.alpha. and IL-6 are proinflammatory cytokines involved in the
pathogenesis of various autoimmune diseases, such as rheumatoid
arthritis (RA), systemic lupus erythematosus (SLE), inflammatory
bowel disease, and type 2 diabetes. Blocking the biological
activities of TNF-.alpha. has demonstrated clinical benefits in
patients with RA and Crohn's disease, as exemplified by five
antibody- or receptor-based therapeutics currently on the market.
The promise of IL-6 blockade was also reinforced by the regulatory
approval of one anti-IL-6R antibody for treating RA and juvenile
idiopathic arthritis, with additional antibodies targeting either
IL-6R or IL-6 in advanced clinical trials. As reported by Mori et
al. (Int Immunol 2011; 23: 701-12), IL-6 directly activates STAT3,
whereas TNF-.alpha. indirectly activates STAT3 via stimulating the
expression of IL-6, which then activates STAT3 and triggers a
cytokine amplification loop of IL-6, resulting in sustained STAT3
activation and chronic inflammation.
Numerous antibodies against TNF-.alpha. are commercially available
and/or publicly known, including infliximab (Jansenn Biotech,
Inc.), adalimumab (Abbvie, Inc.), certolizumab pegol (UCB, Inc.)
and golimumab (Centocor). Although these therapeutic agents have
significantly improved the treatment of certain autoimmune
diseases, such as rheumatoid arthritis (RA), it has been reported
that about 30% of RA patients treated with TNF inhibitors
(including anti-TNF.alpha. antibodies) show little to no effect of
the therapy, with about two thirds demonstrating moderate to high
disease activity at 1 year after treatment (Hirabara et al., 2014,
Clin Rheumatol 33:1247-54). Further, loss of therapeutic efficacy
is frequently observed with anti-TNF monoclonal antibodies
(adalimumab, infliximab) in patients receiving concomitant low-dose
methotrexate, due to immunogenicity-related issues (Hirabara et al,
2014). A need exists for more effective compositions and methods
for use of anti-TNF antibodies in treating diseases and conditions
related to TNF-.alpha..
Dysregulated IL-6 production has been demonstrated to play a
pathological role in various autoimmune and chronic inflammatory
diseases. Therapies against IL-6 pathways have commonly targeted
the IL-6 receptor (IL-6R), including the anti-IL-6R antibodies
tocilizumab, and sarilumab. Antibodies targeted directly against
IL-6 have also been developed, such as olokizumab (UCB), siltuximab
(Janssen), BMS-943429 (Bristol-Myers Squibb) and sirukumab
(Centocor). The latter have been used against various autoimmune
diseases and cancers. Following regulatory approval of tocilizumab
for rheumatoid arthritis, Castleman's disease and systemic juvenile
idiopathic arthritis, favorable results of off-label use have been
reported in systemic lupus erythematosus, systemic sclerosis,
polymyositis, vasculitis syndrome including giant cell arteritis,
Takayasu arteritis, cryoglobulinemia, glomerulonephritis and
rheumatoid vasculitis (see, e.g., Tanaka & Kishimoto, 2012, Int
J Biol Sci 8:1227-36). While these results are promising, no
antibodies against IL-6 (as opposed to IL-6R) have yet been
approved for human use in any indication.
A need exists in the field for more effective, well-tolerated
therapeutic agents targeted against TNF and IL-6.
SUMMARY OF THE INVENTION
The present invention concerns compositions and methods of use of
bispecific or multispecific antibodies comprising at least one
anti-TNF-.alpha. antibody or antigen-binding fragment thereof and
at least one anti-IL-6 antibody or antigen-binding fragment
thereof. Preferably, the bispecific or multispecific antibody is in
the form of a DNL.RTM. complex, comprising AD and DDD moiety
binding pairs as described below.
The antibodies may be chimeric, humanized or human antibodies. In
certain preferred embodiments, the antibodies are humanized,
comprising the CDR sequences of, e.g., a murine anti-IL-6 or
anti-TNF-.alpha. antibody and the framework (FR) and constant
region sequences from one or more human antibodies. Methods of
antibody humanization are well known in the art, as discussed in
detail below. The antibody can be of various isotypes, preferably
human IgG1, IgG2, IgG3 or IgG4, more preferably comprising human
IgG1 hinge and constant region sequences. More preferably, the
antibody or fragment thereof may be designed or selected to
comprise human constant region sequences that belong to specific
allotypes, which may result in reduced immunogenicity. Preferred
allotypes for administration include a non-G1m1 allotype (nG1m1),
such as G1m3, G1m3,1, G1m3,2 or G1m3,1,2. More preferably, the
allotype is selected from the group consisting of the nG1m1, G1m3,
nG1m1,2 and Km3 allotypes.
Numerous anti-TNF-.alpha. antibodies are commercially available
and/or publicly known, including but not limited to CDP571 (Ofei et
al., 2011, Diabetes 45:881-85); MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI,
M302B and M303 (Thermo Scientific); 3H15L1, D13H3, TN3, 17H1L4,
MP9-20A4, and 68B6A3 L1 (Life Technologies); NBP1-19532, NB600-587,
NBP2-27223, and NBP2-27224, (NOVUS BIOLOGICALS.RTM.); ab9635,
(ABCAM.RTM.); certolizumab pegol (UCB, Brussels, Belgium);
adalimumab (Abbvie); infliximab and golimumab (Centocor). These and
many other known anti-TNF-.alpha. antibodies may be used in the
claimed methods and compositions.
Numerous anti-IL-6 antibodies are commercially available and/or
publicly known, including but not limited to 5IL6, 4HCLC, 4H16L21,
677B6A2, and 20F3 (Thermo Scientific); NBP1-47810, NBP2025275,
NBP1047355, and NBP2021624 (NOVUS BIOLOGICALS.RTM.); olokizumab
(UCB); siltuximab (Janssen); BMS-943429 (Bristol-Myers Squibb); and
sirukumab (Centocor). These and many other known anti-IL-6
antibodies may be used in the claimed methods and compositions.
The subject antibodies may be co-administered with one or more
other therapeutic agents. The therapeutic agents may be conjugated
to the antibodies or administered separately, either before,
concomitantly with or after the antibody. Therapeutic agents of use
for treating immune or inflammatory diseases are preferably
selected from drugs, anti-angiogenic agents, pro-apoptotic agents,
antibiotics, hormones, hormone antagonists, chemokines, prodrugs,
enzymes, immunomodulators, cytokines or other known agents of use
for immune or inflammatory diseases.
Drugs of use may possess a pharmaceutical property selected from
the group consisting of antimitotic, antikinase (e.g.,
anti-tyrosine kinase), alkylating, antimetabolite, antibiotic,
alkaloid, anti-angiogenic, pro-apoptotic agents, immune modulators,
and combinations thereof.
Exemplary drugs of use may include 5-fluorouracil, aplidin,
azaribine, anastrozole, anthracyclines, bendamustine, bleomycin,
bortezomib, bryostatin-1, busulfan, calicheamycin, camptothecin,
carboplatin, 10-hydroxycamptothecin, carmustine, celecoxib,
chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan
(CPT-11), SN-38, carboplatin, cladribine, camptothecans,
cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin,
daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX),
cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin
glucuronide, estramustine, epipodophyllotoxin, estrogen receptor
binding agents, etoposide (VP16), etoposide glucuronide, etoposide
phosphate, floxuridine (FUdR), 3',5'-O-dioleoyl-FudR (FUdR-dO),
fludarabine, flutamide, farnesyl-protein transferase inhibitors,
gemcitabine, hydroxyurea, idarubicin, ifosfamide, L-asparaginase,
lenolidamide, leucovorin, lomustine, mechlorethamine, melphalan,
mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,
mithramycin, mitomycin, mitotane, navelbine, nitrosourea,
plicomycin, procarbazine, paclitaxel, pentostatin, PSI-341,
raloxifene, semustine, streptozocin, tamoxifen, temazolomide (an
aqueous form of DTIC), transplatinum, thalidomide, thioguanine,
thiotepa, teniposide, topotecan, uracil mustard, vinorelbine,
vinblastine, vincristine and vinca alkaloids.
Chemokines of use may include RANTES, MCAF, MIP1-alpha, MIP1-Beta
and IP-10.
In certain embodiments, anti-angiogenic agents, such as
angiostatin, baculostatin, canstatin, maspin, anti-VEGF antibodies,
anti-PlGF peptides and antibodies, anti-vascular growth factor
antibodies, anti-Flk-1 antibodies, anti-Flt-1 antibodies and
peptides, anti-Kras antibodies, anti-cMET antibodies, anti-MIF
(macrophage migration-inhibitory factor) antibodies, laminin
peptides, fibronectin peptides, plasminogen activator inhibitors,
tissue metalloproteinase inhibitors, interferons, interleukin-12,
IP-10, Gro- , thrombospondin, 2-methoxyoestradiol,
proliferin-related protein, carboxiamidotriazole, CM101,
Marimastat, pentosan polysulphate, angiopoietin-2,
interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,
Linomide (roquinimex), thalidomide, pentoxifylline, genistein,
TNP-470, endostatin, paclitaxel, accutin, angiostatin, cidofovir,
vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline
may be of use.
Immunomodulators of use may be selected from a cytokine, a stem
cell growth factor, a lymphotoxin, a hematopoietic factor, a colony
stimulating factor (CSF), an interferon (IFN), erythropoietin,
thrombopoietin and a combination thereof. Specifically useful are
lymphotoxins such as tumor necrosis factor (TNF), hematopoietic
factors, such as interleukin (IL), colony stimulating factor, such
as granulocyte-colony stimulating factor (G-CSF) or granulocyte
macrophage-colony stimulating factor (GM-CSF), interferon, such as
interferons-.alpha., -.beta. or -.gamma., and stem cell growth
factor, such as that designated "S1 factor". Included among the
cytokines are growth hormones such as human growth hormone,
N-methionyl human growth hormone, and bovine growth hormone;
parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;
prorelaxin; glycoprotein hormones such as follicle stimulating
hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing
hormone (LH); hepatic growth factor; prostaglandin, fibroblast
growth factor; prolactin; placental lactogen, OB protein; tumor
necrosis factor-.alpha. and - ; mullerian-inhibiting substance;
mouse gonadotropin-associated peptide; inhibin; activin; vascular
endothelial growth factor; integrin; thrombopoietin (TPO); nerve
growth factors such as NGF- ; platelet-growth factor; transforming
growth factors (TGFs) such as TGF-.alpha. and TGF- ; insulin-like
growth factor-I and -II; erythropoietin (EPO); osteoinductive
factors; interferons such as interferon-.alpha., -.beta., and
-.gamma.; colony stimulating factors (CSFs) such as macrophage-CSF
(M-CSF); interleukins (ILs) such as IL-1, IL-1.alpha., IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,
IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand or
FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis
factor and LT. Lenolidamide is yet another immunomodulator that has
shown activity in controlling certain cancers, such as multiple
myeloma and hematopoietic tumors.
The antibodies or complexes may be used to treat a variety of
diseases or conditions in which TNF-.alpha. and IL-6 play a
pathogenic role, such as autoimmune, immune dysfunction or
inflammatory diseases. Exemplary diseases or conditions may be
selected from the group consisting of rheumatoid arthritis (RA),
systemic lupus erythematosus, type 2 diabetes, Crohn's disease,
Castleman's disease, juvenile idiopathic arthritis, systemic
sclerosis, polymyositis, vasculitis syndrome, Takayasu arteritis,
cryoglobulinemia, glomerulonephritis, rheumatoid vasculitis,
arthritis, sepsis, septic shock, inflammation, non-septic
hyperinflammatory disorder, nephritis, inflammatory bowel disease,
inflammatory liver injury, acute pancreatitis, acute respiratory
distress syndrome, ischemia-reperfusion injury, ischemic stroke,
graft-vs.-host disease and cachexia related to cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Assay for neutralizing anti-IL-6 antibodies. Supernatants
from clones were incubated with human IL-6 at 37.degree. C. for 1
hour, prior to incubation with HT-29 cells. The cells were
incubated with rhIL-6 alone or in combination with serum for 15 min
at 37.degree. C. and phosphorylation of STAT3 was detected by
Western blotting.
FIG. 2A. Titration of neutralizing anti-IL-6 antibodies. The
ability to block IL-6 induced phosphorylation of STAT3 was
determined by Western blot analysis using the indicated
concentrations of the 2-3B2 anti-IL-6 antibody. A substantial
inhibition of IL-6 dependent phosphorylation was seen as low as
0.067 nM antibody.
FIG. 2B. Titration of neutralizing anti-IL-6 antibodies. The
ability to block IL-6 induced phosphorylation of STAT3 was
determined by Western blot analysis using the indicated
concentrations of the 4-4E6 anti-IL-6 antibody. Approximately
equivalent effects on phosphorylation were observed at 0.67 nM
4-4E6 vs. 0.0067 nM 2-34B2 antibody (FIG. 2A).
FIG. 3. Neutralization activity of TNF-.alpha. mediated
cytotoxicity by immunized mouse sera on WEHI 164 cells. Serum from
mouse #3 was the most effective at inhibiting TNF-.alpha. mediated
cytotoxicity.
FIG. 4. Neutralization activity of TNF-.alpha. mediated
cytotoxicity by antibodies from clones 4C9D11 and 4D3B11 in WEHI
164 cells.
FIG. 5. Neutralization activity of TNF-.alpha. mediated
cytotoxicity by antibodies from clones 4C9D11G11 and 4D3B11C4 in
L929 cells.
FIG. 6. Antibody-based neutralization of rhTNF-.alpha.-induced cell
surface expression of ICAM-1 in ECV-304 cells (a derivative of T24
bladder cancer cell line).
FIG. 7. Amino acid sequence of the anti-IL-6 antibody (2-3B2) heavy
chain (VH) sequence (SEQ ID NO:94). The sequence of a homologous
heavy chain of the B34781 antibody (SEQ ID NO:95), obtained from
the NCBI protein sequence database, is shown for comparison.
Putative CDR sequences (underlined) were identified by comparison
with the known sequence of the homologous B34781 antibody.
FIG. 8. Amino acid sequence of the anti-IL-6 antibody (2-3B2) light
chain (VK) sequence (SEQ ID NO:96). The sequence of a homologous
light chain of AAB53778.1 (SEQ ID NO:97), obtained from the NCBI
protein sequence database, is shown for comparison. Putative CDR
sequences (underlined) were identified by comparison with the known
sequence of the homologous AAB53778.1.
FIG. 9. Activity of cIL6/TNF.alpha. DVD construct for neutralizing
IL-6 induced phosphorylation of STAT3 in HT-29 cells, compared to
parent 2-3B2 anti-IL-6 antibody.
FIG. 10. Amino acid sequence of the anti-TNF-.alpha. antibody (4C9)
heavy chain (VH) sequence (SEQ ID NO:98). The sequence of a
homologous heavy chain of the AAS66033.1 antibody (SEQ ID NO:99),
obtained from the NCBI protein sequence database, is shown for
comparison. Putative CDR sequences (underlined) were identified by
comparison with the known sequence of the homologous AAS66033.1
antibody.
FIG. 11. Amino acid sequence of the anti-IL-6 antibody (4C9) light
chain (VK) sequence (SEQ ID NO:100). The sequence of a homologous
heavy chain of AAS66032.1 (SEQ ID NO:101), obtained from the NCBI
protein sequence database, is shown for comparison. Putative CDR
sequences (underlined) were identified by comparison with the known
sequence of the homologous AAS66032.1.
FIG. 12. Schematic illustration of the synthesis of
C.sub.K-AD2-cIgG-anti-TNF-.alpha.-pdHL2.
FIG. 13. Inhibition of IL-6 induced phosphorylation of STAT3 by
cT*-(c6)-(c6) complex compared to Fab-DDD2-cIL-6 protein.
FIG. 14. Inhibition of natural IL-6 induced phosphorylation of
STAT3 by cT*-(c6)-(c6) complex compared to Fab-DDD2-cIL-6
protein.
FIG. 15. Inhibition of rhTNF-.alpha. induced cell death in L929
cells by anti-TNF-.alpha. antibody constructs.
FIG. 16. Inhibition of cell death induced by natural TNF-.alpha. in
L929 cells by anti-TNF-.alpha. antibody constructs.
FIG. 17. Relative affinities of cT*-(c6)-(c6), c-anti-TNF-.alpha.
and c-anti-IL-6 for IL-6 and TNF-.alpha. from different
species.
FIG. 18A. Role of STAT3 in IL-6 and TNF-.alpha. mediated
pathways.
FIG. 18B. Role of STAT3 in IL-6 and TNF-.alpha. mediated disease
processes.
DEFINITIONS
Unless otherwise specified, "a" or "an" means "one or more".
As used herein, the terms "and" and "or" may be used to mean either
the conjunctive or disjunctive. That is, both terms should be
understood as equivalent to "and/or" unless otherwise stated.
A "therapeutic agent" is an atom, molecule, or compound that is
useful in the treatment of a disease. Examples of therapeutic
agents include antibodies, antibody fragments, peptides, drugs,
toxins, enzymes, nucleases, hormones, immunomodulators, antisense
oligonucleotides, small interfering RNA (siRNA), chelators, boron
compounds, photoactive agents, dyes, and radioisotopes.
A "diagnostic agent" is an atom, molecule, or compound that is
useful in diagnosing a disease. Useful diagnostic agents include,
but are not limited to, radioisotopes, dyes (such as with the
biotin-streptavidin complex), contrast agents, fluorescent
compounds or molecules, and enhancing agents (e.g., paramagnetic
ions) for magnetic resonance imaging (MM).
An "antibody" as used herein refers to a full-length (i.e.,
naturally occurring or formed by normal immunoglobulin gene
fragment recombinatorial processes) immunoglobulin molecule (e.g.,
an IgG antibody) or an immunologically active (i.e., specifically
binding) portion of an immunoglobulin molecule, like an antibody
fragment. An "antibody" includes monoclonal, polyclonal,
bispecific, multispecific, murine, chimeric, humanized and human
antibodies.
A "naked antibody" is an antibody or antigen binding fragment
thereof that is not attached to a therapeutic or diagnostic agent.
The Fc portion of an intact naked antibody can provide effector
functions, such as complement fixation and ADCC (see, e.g.,
Markrides, Pharmacol Rev 50:59-87, 1998). Other mechanisms by which
naked antibodies induce cell death may include apoptosis. (Vaswani
and Hamilton, Ann Allergy Asthma Immunol 81: 105-119, 1998.)
An "antibody fragment" is a portion of an intact antibody such as
F(ab').sub.2, F(ab).sub.2, Fab', Fab, Fv, sFv, scFv, dAb and the
like. Regardless of structure, an antibody fragment binds with the
same antigen that is recognized by the full-length antibody. For
example, antibody fragments include isolated fragments consisting
of the variable regions, such as the "Fv" fragments consisting of
the variable regions of the heavy and light chains or recombinant
single chain polypeptide molecules in which light and heavy
variable regions are connected by a peptide linker ("scFv
proteins"). "Single-chain antibodies", often abbreviated as "scFv"
consist of a polypeptide chain that comprises both a V.sub.H and a
V.sub.L domain which interact to form an antigen-binding site. The
V.sub.H and V.sub.L domains are usually linked by a peptide of 1 to
25 amino acid residues. Antibody fragments also include diabodies,
triabodies and single domain antibodies (dAb).
An antibody or antibody complex preparation, or a composition
described herein, is said to be administered in a "therapeutically
effective amount" if the amount administered is physiologically
significant. An agent is physiologically significant if its
presence results in a detectable change in the physiology of a
recipient subject. In particular embodiments, an antibody
preparation is physiologically significant if its presence invokes
an antitumor response or mitigates the signs and symptoms of an
autoimmune disease state. A physiologically significant effect
could also be the evocation of a humoral and/or cellular immune
response in the recipient subject leading to growth inhibition or
death of target cells.
DOCK-AND-LOCK.RTM. (DNL.RTM.)
In preferred embodiments, a bivalent or multivalent antibody is
formed as a DOCK-AND-LOCK.RTM. (DNL.RTM.) complex (see, e.g., U.S.
Pat. Nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143 and 7,666,400,
the Examples section of each of which is incorporated herein by
reference.) Generally, the technique takes advantage of the
specific and high-affinity binding interactions that occur between
a dimerization and docking domain (DDD) sequence of the regulatory
(R) subunits of cAMP-dependent protein kinase (PKA) and an anchor
domain (AD) sequence derived from any of a variety of AKAP proteins
(Baillie et al., FEBS Letters. 2005; 579: 3264. Wong and Scott,
Nat. Rev. Mol. Cell Biol. 2004; 5: 959). The DDD and AD peptides
may be attached to any protein, peptide or other molecule. Because
the DDD sequences spontaneously dimerize and bind to the AD
sequence, the technique allows the formation of complexes between
any selected molecules that may be attached to DDD or AD
sequences.
Although the standard DNL.RTM. complex comprises a trimer with two
DDD-linked molecules attached to one AD-linked molecule, variations
in complex structure allow the formation of dimers, trimers,
tetramers, pentamers, hexamers and other multimers. In some
embodiments, the DNL.RTM. complex may comprise two or more
antibodies, antibody fragments or fusion proteins which bind to the
same antigenic determinant or to two or more different antigens.
The DNL.RTM. complex may also comprise one or more other effectors,
such as proteins, peptides, immunomodulators, cytokines,
interleukins, interferons, binding proteins, peptide ligands,
carrier proteins, toxins, ribonucleases such as onconase,
inhibitory oligonucleotides such as siRNA, antigens or
xenoantigens, polymers such as PEG, enzymes, therapeutic agents,
hormones, cytotoxic agents, anti-angiogenic agents, pro-apoptotic
agents or any other molecule or aggregate.
PKA, which plays a central role in one of the best studied signal
transduction pathways triggered by the binding of the second
messenger cAMP to the R subunits, was first isolated from rabbit
skeletal muscle in 1968 (Walsh et al., J. Biol. Chem. 1968;
243:3763). The structure of the holoenzyme consists of two
catalytic subunits held in an inactive form by the R subunits
(Taylor, J. Biol. Chem. 1989; 264:8443). Isozymes of PKA are found
with two types of R subunits (RI and RII), and each type has
.alpha. and .beta. isoforms (Scott, Pharmacol. Ther. 1991; 50:123).
Thus, the four isoforms of PKA regulatory subunits are RI.alpha.,
RI.beta., RII.alpha. and RII.beta.. The R subunits have been
isolated only as stable dimers and the dimerization domain has been
shown to consist of the first 44 amino-terminal residues of
RII.alpha. (Newlon et al., Nat. Struct. Biol. 1999; 6:222). As
discussed below, similar portions of the amino acid sequences of
other regulatory subunits are involved in dimerization and docking,
each located near the N-terminal end of the regulatory subunit.
Binding of cAMP to the R subunits leads to the release of active
catalytic subunits for a broad spectrum of serine/threonine kinase
activities, which are oriented toward selected substrates through
the compartmentalization of PKA via its docking with AKAPs (Scott
et al., J. Biol. Chem. 1990; 265; 21561)
Since the first AKAP, microtubule-associated protein-2, was
characterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA.
1984; 81:6723), more than 50 AKAPs that localize to various
sub-cellular sites, including plasma membrane, actin cytoskeleton,
nucleus, mitochondria, and endoplasmic reticulum, have been
identified with diverse structures in species ranging from yeast to
humans (Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The
AD of AKAPs for PKA is an amphipathic helix of 14-18 residues (Carr
et al., J. Biol. Chem. 1991; 266:14188). The amino acid sequences
of the AD are quite varied among individual AKAPs, with the binding
affinities reported for RII dimers ranging from 2 to 90 nM (Alto et
al., Proc. Natl. Acad. Sci. USA. 2003; 100:4445). AKAPs will only
bind to dimeric R subunits. For human RII.alpha., the AD binds to a
hydrophobic surface formed by the 23 amino-terminal residues
(Colledge and Scott, Trends Cell Biol. 1999; 6:216). Thus, the
dimerization domain and AKAP binding domain of human RII.alpha. are
both located within the same N-terminal 44 amino acid sequence
(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO
J. 2001; 20:1651), which is termed the DDD herein.
We have developed a platform technology to utilize the DDD of human
PKA regulatory subunit RI.alpha., RI.beta., RII.alpha. or RII.beta.
and the AD of AKAP as an excellent pair of linker modules for
docking any two entities, referred to hereafter as A and B, into a
noncovalent complex, which could be further locked into a DNL.RTM.
complex through the introduction of cysteine residues into both the
DDD and AD at strategic positions to facilitate the formation of
disulfide bonds. The general methodology of the approach is as
follows. Entity A is constructed by linking a DDD sequence to a
precursor of A, resulting in a first component hereafter referred
to as a. Because the DDD sequence would effect the spontaneous
formation of a dimer, A would thus be composed of a.sub.2. Entity B
is constructed by linking an AD sequence to a precursor of B,
resulting in a second component hereafter referred to as b. The
dimeric motif of DDD contained in a.sub.2 will create a docking
site for binding to the AD sequence contained in b, thus
facilitating a ready association of a.sub.2 and b to form a binary,
trimeric complex composed of a.sub.2b. This binding event is made
irreversible with a subsequent reaction to covalently secure the
two entities via disulfide bridges, which occurs very efficiently
based on the principle of effective local concentration because the
initial binding interactions should bring the reactive thiol groups
placed onto both the DDD and AD into proximity (Chmura et al.,
Proc. Natl. Acad. Sci. USA. 2001; 98:8480) to ligate
site-specifically. Using various combinations of linkers, adaptor
modules and precursors, a wide variety of DNL.RTM. constructs of
different stoichiometry may be produced and used (see, e.g., U.S.
Pat. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and
7,666,400.)
By attaching the DDD and AD away from the functional groups of the
two precursors, such site-specific ligations are also expected to
preserve the original activities of the two precursors. This
approach is modular in nature and potentially can be applied to
link, site-specifically and covalently, a wide range of substances,
including peptides, proteins, antibodies, antibody fragments, and
other effector moieties with a wide range of activities. Utilizing
the fusion protein method of constructing AD and DDD conjugated
effectors described in the Examples below, virtually any protein or
peptide may be incorporated into a DNL.RTM. construct. However, the
technique is not limiting and other methods of conjugation may be
utilized.
A variety of methods are known for making fusion proteins,
including nucleic acid synthesis, hybridization and/or
amplification to produce a synthetic double-stranded nucleic acid
encoding a fusion protein of interest. Such double-stranded nucleic
acids may be inserted into expression vectors for fusion protein
production by standard molecular biology techniques (see, e.g.
Sambrook et al., Molecular Cloning, A laboratory manual, 2.sup.nd
Ed, 1989). In such preferred embodiments, the AD and/or DDD moiety
may be attached to either the N-terminal or C-terminal end of an
effector protein or peptide. However, the skilled artisan will
realize that the site of attachment of an AD or DDD moiety to an
effector moiety may vary, depending on the chemical nature of the
effector moiety and the part(s) of the effector moiety involved in
its physiological activity. Site-specific attachment of a variety
of effector moieties may be performed using techniques known in the
art, such as the use of bivalent cross-linking reagents and/or
other chemical conjugation techniques.
Structure-Function Relationships in AD and DDD Moieties
For different types of DNL.RTM. constructs, different AD or DDD
sequences may be utilized. Exemplary DDD and AD sequences are
provided below.
TABLE-US-00001 DDD1 (SEQ ID NO: 1)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2 (SEQ ID NO: 2)
CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1 (SEQ ID NO: 3)
QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 4) CGQIEYLAKQIVDNAIQQAGC
The skilled artisan will realize that DDD1 and DDD2 are based on
the DDD sequence of the human RII.alpha. isoform of protein kinase
A. However, in alternative embodiments, the DDD and AD moieties may
be based on the DDD sequence of the human RI.alpha. form of protein
kinase A and a corresponding AKAP sequence, as exemplified in DDD3,
DDD3C and AD3 below.
TABLE-US-00002 DDD3 (SEQ ID NO: 5)
SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK DDD3C (SEQ ID
NO: 6) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLE KEEAK AD3
(SEQ ID NO: 7) CGFEELAWKIAKMIWSDVFQQGC
In other alternative embodiments, other sequence variants of AD
and/or DDD moieties may be utilized in construction of the DNL.RTM.
complexes. For example, there are only four variants of human PKA
DDD sequences, corresponding to the DDD moieties of PKA RI.alpha.,
RII.alpha., RI.beta. and RII.beta.. The RII.alpha. DDD sequence is
the basis of DDD1 and DDD2 disclosed above. The four human PKA DDD
sequences are shown below. The DDD sequence represents residues
1-44 of RII.alpha., 1-44 of RII.beta., 12-61 of RI.alpha. and 13-66
of RI.beta.. (Note that the sequence of DDD1 is modified slightly
from the human PKA RII.alpha. DDD moiety.)
TABLE-US-00003 PKA RI.alpha. (SEQ ID NO: 8)
SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEK LEKEEAK PKA RI.beta.
(SEQ ID NO: 9) SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKL
EKEENRQILA PKA RII.alpha. (SEQ ID NO: 10)
SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ PKA RII.beta. (SEQ ID
NO: 11) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER
The structure-function relationships of the AD and DDD domains have
been the subject of investigation. (See, e.g., Burns-Hamuro et al.,
2005, Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem
276:17332-38; Alto et al., 2003, Proc Natl Acad Sci USA
100:4445-50; Hundsrucker et al., 2006, Biochem J 396:297-306;
Stokka et al., 2006, Biochem J 400:493-99; Gold et al., 2006, Mol
Cell 24:383-95; Kinderman et al., 2006, Mol Cell 24:397-408, the
entire text of each of which is incorporated herein by
reference.)
For example, Kinderman et al. (2006, Mol Cell 24:397-408) examined
the crystal structure of the AD-DDD binding interaction and
concluded that the human DDD sequence contained a number of
conserved amino acid residues that were important in either dimer
formation or AKAP binding, underlined in SEQ ID NO:1 below. (See
FIG. 1 of Kinderman et al., 2006, incorporated herein by
reference.) The skilled artisan will realize that in designing
sequence variants of the DDD sequence, one would desirably avoid
changing any of the underlined residues, while conservative amino
acid substitutions might be made for residues that are less
critical for dimerization and AKAP binding.
TABLE-US-00004 (SEQ ID NO: 1)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA
As discussed in more detail below, conservative amino acid
substitutions have been characterized for each of the twenty common
L-amino acids. Thus, based on the data of Kinderman (2006) and
conservative amino acid substitutions, potential alternative DDD
sequences based on SEQ ID NO:1 are shown in Table 1. In devising
Table 1, only highly conservative amino acid substitutions were
considered. For example, charged residues were only substituted for
residues of the same charge, residues with small side chains were
substituted with residues of similar size, hydroxyl side chains
were only substituted with other hydroxyls, etc. Because of the
unique effect of proline on amino acid secondary structure, no
other residues were substituted for proline. A limited number of
such potential alternative DDD moiety sequences are shown in SEQ ID
NO:12 to SEQ ID NO:31 below. The skilled artisan will realize that
an almost unlimited number of alternative species within the genus
of DDD moieties can be constructed by standard techniques, for
example using a commercial peptide synthesizer or well known
site-directed mutagenesis techniques. The effect of the amino acid
substitutions on AD moiety binding may also be readily determined
by standard binding assays, for example as disclosed in Alto et al.
(2003, Proc Natl Acad Sci USA 100:4445-50).
TABLE-US-00005 TABLE 1 Conservative Amino Acid Substitutions in
DDD1 (SEQ ID NO: 1). Consensus sequence disclosed as SEQ ID NO: 87.
S H I Q I P P G L T E L L Q G Y T V E V L R T K N A S D N A S D K R
Q Q P P D L V E F A V E Y F T R L R E A R A N N E D L D S K K D L K
L I I I V V V THIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID
NO: 12) SKIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO:
13) SRIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 14)
SHINIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 15)
SHIQIPPALTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 16)
SHIQIPPGLSELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 17)
SHIQIPPGLTDLLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 18)
SHIQIPPGLTELLNGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 19)
SHIQIPPGLTELLQAYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 20)
SHIQIPPGLTELLQGYSVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 21)
SHIQIPPGLTELLQGYTVDVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 22)
SHIQIPPGLTELLQGYTVEVLKQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 23)
SHIQIPPGLTELLQGYTVEVLRNQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 24)
SHIQIPPGLTELLQGYTVEVLRQNPPDLVEFAVEYFTRLREARA (SEQ ID NO: 25)
SHIQIPPGLTELLQGYTVEVLRQQPPELVEFAVEYFTRLREARA (SEQ ID NO: 26)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVDFAVEYFTRLREARA (SEQ ID NO: 27)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFLVEYFTRLREARA (SEQ ID NO: 28)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFIVEYFTRLREARA (SEQ ID NO: 29)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFVVEYFTRLREARA (SEQ ID NO: 30)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVDYFTRLREARA (SEQ ID NO: 31)
Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50) performed a
bioinformatic analysis of the AD sequence of various AKAP proteins
to design an RII selective AD sequence called AKAP-IS (SEQ ID
NO:3), with a binding constant for DDD of 0.4 nM. The AKAP-IS
sequence was designed as a peptide antagonist of AKAP binding to
PKA. Residues in the AKAP-IS sequence where substitutions tended to
decrease binding to DDD are underlined in SEQ ID NO:3 below. The
skilled artisan will realize that in designing sequence variants of
the AD sequence, one would desirably avoid changing any of the
underlined residues, while conservative amino acid substitutions
might be made for residues that are less critical for DDD binding.
Table 2 shows potential conservative amino acid substitutions in
the sequence of AKAP-IS (AD1, SEQ ID NO:3), similar to that shown
for DDD1 (SEQ ID NO:1) in Table 1 above.
A limited number of such potential alternative AD moiety sequences
are shown in SEQ ID NO:32 to SEQ ID NO:49 below. Again, a very
large number of species within the genus of possible AD moiety
sequences could be made, tested and used by the skilled artisan,
based on the data of Alto et al. (2003). It is noted that FIG. 2 of
Alto (2003) shows an even large number of potential amino acid
substitutions that may be made, while retaining binding activity to
DDD moieties, based on actual binding experiments.
TABLE-US-00006 AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA
TABLE-US-00007 TABLE 2 Conservative Amino Acid Substitutions in AD1
(SEQ ID NO: 3). Consensus sequence disclosed as SEQ ID NO: 88. Q I
E Y L A K Q I V D N A I Q Q A N L D F I R N E Q N N L V T V I S V
NIEYLAKQIVDNAIQQA (SEQ ID NO: 32) QLEYLAKQIVDNAIQQA (SEQ ID NO: 33)
QVEYLAKQIVDNAIQQA (SEQ ID NO: 34) QIDYLAKQIVDNAIQQA (SEQ ID NO: 35)
QIEFLAKQIVDNAIQQA (SEQ ID NO: 36) QIETLAKQIVDNAIQQA (SEQ ID NO: 37)
QIESLAKQIVDNAIQQA (SEQ ID NO: 38) QIEYIAKQIVDNAIQQA (SEQ ID NO: 39)
QIEYVAKQIVDNAIQQA (SEQ ID NO: 40) QIEYLARQIVDNAIQQA (SEQ ID NO: 41)
QIEYLAKNIVDNAIQQA (SEQ ID NO: 42) QIEYLAKQIVENAIQQA (SEQ ID NO: 43)
QIEYLAKQIVDQAIQQA (SEQ ID NO: 44) QIEYLAKQIVDNAINQA (SEQ ID NO: 45)
QIEYLAKQIVDNAIQNA (SEQ ID NO: 46) QIEYLAKQIVDNAIQQL (SEQ ID NO: 47)
QIEYLAKQIVDNAIQQI (SEQ ID NO: 48) QIEYLAKQIVDNAIQQV (SEQ ID NO:
49)
Gold et al. (2006, Mol Cell 24:383-95) utilized crystallography and
peptide screening to develop a SuperAKAP-IS sequence (SEQ ID
NO:50), exhibiting a five order of magnitude higher selectivity for
the RII isoform of PKA compared with the RI isoform. Underlined
residues indicate the positions of amino acid substitutions,
relative to the AKAP-IS sequence, which increased binding to the
DDD moiety of RII.alpha.. In this sequence, the N-terminal Q
residue is numbered as residue number 4 and the C-terminal A
residue is residue number 20. Residues where substitutions could be
made to affect the affinity for RII.alpha. were residues 8, 11, 15,
16, 18, 19 and 20 (Gold et al., 2006). It is contemplated that in
certain alternative embodiments, the SuperAKAP-IS sequence may be
substituted for the AKAP-IS AD moiety sequence to prepare DNL.RTM.
constructs. Other alternative sequences that might be substituted
for the AKAP-IS AD sequence are shown in SEQ ID NO:51-53.
Substitutions relative to the AKAP-IS sequence are underlined. It
is anticipated that, as with the AD2 sequence shown in SEQ ID NO:4,
the AD moiety may also include the additional N-terminal residues
cysteine and glycine and C-terminal residues glycine and
cysteine.
TABLE-US-00008 SuperAKAP-IS (SEQ ID NO: 50) QIEYVAKQIVDYAIHQA
Alternative AKAP sequences (SEQ ID NO: 51) QIEYKAKQIVDHAIHQA (SEQ
ID NO: 52) QIEYHAKQIVDHAIHQA (SEQ ID NO: 53) QIEYVAKQIVDHAIHQA
FIG. 2 of Gold et al. disclosed additional DDD-binding sequences
from a variety of AKAP proteins, shown below.
TABLE-US-00009 RII-Specific AKAPs AKAP-KL (SEQ ID NO: 54)
PLEYQAGLLVQNAIQQAI AKAP79 (SEQ ID NO: 55) LLIETASSLVKNAIQLSI
AKAP-Lbc (SEQ ID NO: 56) LIEEAASRIVDAVIEQVK RI-Specific AKAPs
AKAPce (SEQ ID NO: 57) ALYQFADRFSELVISEAL RIAD (SEQ ID NO: 58)
LEQVANQLADQIIKEAT PV38 (SEQ ID NO: 59) FEELAWKIAKMIWSDVF
Dual-Specificity AKAPs AKAP7 (SEQ ID NO: 60) ELVRLSKRLVENAVLKAV
MAP2D (SEQ ID NO: 61) TAEEVSARIVQVVTAEAV DAKAP1 (SEQ ID NO: 62)
QIKQAAFQLISQVILEAT DAKAP2 (SEQ ID NO: 63) LAWKIAKMIVSDVMQQ
Stokka et al. (2006, Biochem J 400:493-99) also developed peptide
competitors of AKAP binding to PKA, shown in SEQ ID NO:64-66. The
peptide antagonists were designated as Ht31 (SEQ ID NO:64), RIAD
(SEQ ID NO:65) and PV-38 (SEQ ID NO:66). The Ht-31 peptide
exhibited a greater affinity for the RII isoform of PKA, while the
RIAD and PV-38 showed higher affinity for RI.
TABLE-US-00010 Ht31 (SEQ ID NO: 64) DLIEEAASRIVDAVIEQVKAAGAY RIAD
(SEQ ID NO: 65) LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 66)
FEELAWKIAKMIWSDVFQQC
Hundsrucker et al. (2006, Biochem J 396:297-306) developed still
other peptide competitors for AKAP binding to PKA, with a binding
constant as low as 0.4 nM to the DDD of the RII form of PKA. The
sequences of various AKAP antagonistic peptides are provided in
Table 1 of Hundsrucker et al., reproduced in Table 3 below. AKAPIS
represents a synthetic RII subunit-binding peptide. All other
peptides are derived from the RII-binding domains of the indicated
AKAPs.
TABLE-US-00011 TABLE 3 AKAP Peptide sequences Peptide Sequence
AKAPIS QIEYLAKQIVDNAIQQA (SEQ ID NO: 3) AKAPIS-P QIEYLAKQIPDNAIQQA
(SEQ ID NO: 67) Ht31 KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 68)
Ht31-P KGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 69)
AKAP7.delta.-wt-pep PEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 70)
AKAP7.delta.-L304T-pep PEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 71)
AKAP7.delta.-L308D-pep PEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 72)
AKAP7.delta.-P-pep PEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 73)
AKAP7.delta.-PP-pep PEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 74)
AKAP7.delta.-L314E-pep PEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 75)
AKAP1-pep EEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 76) AKAP2-pep
LVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 77) AKAP5-pep
QYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 78) AKAP9-pep
LEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 79) AKAP10-pep
NTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 80) AKAP11-pep
VNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 81) AKAP12-pep
NGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 82) AKAP14-pep
TQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 83) Rab32-pep
ETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 84)
Residues that were highly conserved among the AD domains of
different AKAP proteins are indicated below by underlining with
reference to the AKAP IS sequence (SEQ ID NO:3). The residues are
the same as observed by Alto et al. (2003), with the addition of
the C-terminal alanine residue. (See FIG. 4 of Hundsrucker et al.
(2006), incorporated herein by reference.) The sequences of peptide
antagonists with particularly high affinities for the RII DDD
sequence were those of AKAP-IS, AKAP7.delta.-wt-pep,
AKAP7.delta.-L304T-pep and AKAP7.delta.-L308D-pep.
TABLE-US-00012 AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA
Carr et al. (2001, J Biol Chem 276:17332-38) examined the degree of
sequence homology between different AKAP-binding DDD sequences from
human and non-human proteins and identified residues in the DDD
sequences that appeared to be the most highly conserved among
different DDD moieties. These are indicated below by underlining
with reference to the human PKA RII.alpha. DDD sequence of SEQ ID
NO:1. Residues that were particularly conserved are further
indicated by italics. The residues overlap with, but are not
identical to those suggested by Kinderman et al. (2006) to be
important for binding to AKAP proteins. The skilled artisan will
realize that in designing sequence variants of DDD, it would be
most preferred to avoid changing the most conserved residues
(italicized), and it would be preferred to also avoid changing the
conserved residues (underlined), while conservative amino acid
substitutions may be considered for residues that are neither
underlined nor italicized.
TABLE-US-00013 (SEQ ID NO: 1)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA
A modified set of conservative amino acid substitutions for the
DDD1 (SEQ ID NO:1) sequence, based on the data of Carr et al.
(2001) is shown in Table 4. Even with this reduced set of
substituted sequences, there are over 65,000 possible alternative
DDD moiety sequences that may be produced, tested and used by the
skilled artisan without undue experimentation. The skilled artisan
could readily derive such alternative DDD amino acid sequences as
disclosed above for Table 1 and Table 2.
TABLE-US-00014 TABLE 4 Conservative Amino Acid Substitutions in
DDD1 (SEQ ID NO: 1). Consensus sequence disclosed as SEQ ID NO: 89.
S H I Q I P P G L T E L L Q G Y T V E V L R T N S I L A Q Q P P D L
V E F A V E Y F T R L R E A R A N L D S K K L L I I I V V V
The skilled artisan will realize that these and other amino acid
substitutions in the DDD or AD amino acid sequences may be utilized
to produce alternative species within the genus of AD or DDD
moieties, using techniques that are standard in the field and only
routine experimentation.
Amino Acid Substitutions
In alternative embodiments, the disclosed methods and compositions
may involve production and use of proteins or peptides with one or
more substituted amino acid residues. For example, the DDD and/or
AD sequences used to make DNL.RTM. constructs may be modified as
discussed above.
The skilled artisan will be aware that, in general, amino acid
substitutions typically involve the replacement of an amino acid
with another amino acid of relatively similar properties (i.e.,
conservative amino acid substitutions). The properties of the
various amino acids and effect of amino acid substitution on
protein structure and function have been the subject of extensive
study and knowledge in the art.
For example, the hydropathic index of amino acids may be considered
(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The
relative hydropathic character of the amino acid contributes to the
secondary structure of the resultant protein, which in turn defines
the interaction of the protein with other molecules. Each amino
acid has been assigned a hydropathic index on the basis of its
hydrophobicity and charge characteristics (Kyte & Doolittle,
1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);
phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);
alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8);
tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine
(-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5);
asparagine (-3.5); lysine (-3.9); and arginine (-4.5). In making
conservative substitutions, the use of amino acids whose
hydropathic indices are within .+-.2 is preferred, within .+-.1 are
more preferred, and within .+-.0.5 are even more preferred.
Amino acid substitution may also take into account the
hydrophilicity of the amino acid residue (e.g., U.S. Pat. No.
4,554,101). Hydrophilicity values have been assigned to amino acid
residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0);
glutamate (+3.0); serine (+0.3); asparagine (+0.2); glutamine
(+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-0.1);
alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine
(-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine
(-2.3); phenylalanine (-2.5); tryptophan (-3.4). Replacement of
amino acids with others of similar hydrophilicity is preferred.
Other considerations include the size of the amino acid side chain.
For example, it would generally not be preferred to replace an
amino acid with a compact side chain, such as glycine or serine,
with an amino acid with a bulky side chain, e.g., tryptophan or
tyrosine. The effect of various amino acid residues on protein
secondary structure is also a consideration. Through empirical
study, the effect of different amino acid residues on the tendency
of protein domains to adopt an alpha-helical, beta-sheet or reverse
turn secondary structure has been determined and is known in the
art (see, e.g., Chou & Fasman, 1974, Biochemistry, 13:222-245;
1978, Ann. Rev. Biochem., 47: 251-276; 1979, Biophys. J.,
26:367-384).
Based on such considerations and extensive empirical study, tables
of conservative amino acid substitutions have been constructed and
are known in the art. For example: arginine and lysine; glutamate
and aspartate; serine and threonine; glutamine and asparagine; and
valine, leucine and isoleucine. Alternatively: Ala (A) leu, ile,
val; Arg (R) gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp
(D) asn, glu; Cys (C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp;
Gly (G) ala; His (H) asn, gln, lys, arg; Ile (I) val, met, ala,
phe, leu; Leu (L) val, met, ala, phe, ile; Lys (K) gln, asn, arg;
Met (M) phe, ile, leu; Phe (F) leu, val, ile, ala, tyr; Pro (P)
ala; Ser (S), thr; Thr (T) ser; Trp (W) phe, tyr; Tyr (Y) trp, phe,
thr, ser; Val (V) ile, leu, met, phe, ala.
Other considerations for amino acid substitutions include whether
or not the residue is located in the interior of a protein or is
solvent exposed. For interior residues, conservative substitutions
would include: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala;
Ala and Gly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met;
Phe and Tyr; Tyr and Trp. (See, e.g., PROWL website at
rockefeller.edu) For solvent exposed residues, conservative
substitutions would include: Asp and Asn; Asp and Glu; Glu and Gln;
Glu and Ala; Gly and Asn; Ala and Pro; Ala and Gly; Ala and Ser;
Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu; Leu and Ile;
Ile and Val; Phe and Tyr. (Id.) Various matrices have been
constructed to assist in selection of amino acid substitutions,
such as the PAM250 scoring matrix, Dayhoff matrix, Grantham matrix,
McLachlan matrix, Doolittle matrix, Henikoff matrix, Miyata matrix,
Fitch matrix, Jones matrix, Rao matrix, Levin matrix and Risler
matrix (Idem.)
In determining amino acid substitutions, one may also consider the
existence of intermolecular or intramolecular bonds, such as
formation of ionic bonds (salt bridges) between positively charged
residues (e.g., His, Arg, Lys) and negatively charged residues
(e.g., Asp, Glu) or disulfide bonds between nearby cysteine
residues.
Methods of substituting any amino acid for any other amino acid in
an encoded protein sequence are well known and a matter of routine
experimentation for the skilled artisan, for example by the
technique of site-directed mutagenesis or by synthesis and assembly
of oligonucleotides encoding an amino acid substitution and
splicing into an expression vector construct.
Antibodies and Antibody Fragments
Techniques for preparing monoclonal antibodies against virtually
any target antigen, such as IL-6 or TNF-.alpha., are well known in
the art. See, for example, Kohler and Milstein, Nature 256: 495
(1975), and Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY,
VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991). Briefly,
monoclonal antibodies can be obtained by injecting mice with a
composition comprising an antigen, removing the spleen to obtain
B-lymphocytes, fusing the B-lymphocytes with myeloma cells to
produce hybridomas, cloning the hybridomas, selecting positive
clones which produce antibodies to the antigen, culturing the
clones that produce antibodies to the antigen, and isolating the
antibodies from the hybridoma cultures.
MAbs can be isolated and purified from hybridoma cultures by a
variety of well-established techniques. Such isolation techniques
include affinity chromatography with Protein-A SEPHAROSE.RTM.,
size-exclusion chromatography, and ion-exchange chromatography.
See, for example, Coligan at pages 2.7.1-2.7.12 and pages
2.9.1-2.9.3. Also, see Baines et al., "Purification of
Immunoglobulin G (IgG)," in METHODS IN MOLECULAR BIOLOGY, VOL. 10,
pages 79-104 (The Humana Press, Inc. 1992).
After the initial raising of antibodies to the immunogen, the
antibodies can be sequenced and subsequently prepared by
recombinant techniques. Humanization and chimerization of murine
antibodies and antibody fragments are well known to those skilled
in the art. The use of antibody components derived from humanized,
chimeric or human antibodies obviates potential problems associated
with the immunogenicity of murine constant regions.
Chimeric Antibodies
A chimeric antibody is a recombinant protein in which the variable
regions of a human antibody have been replaced by the variable
regions of, for example, a mouse antibody, including the
complementarity-determining regions (CDRs) of the mouse antibody.
Chimeric antibodies exhibit decreased immunogenicity and increased
stability when administered to a subject. General techniques for
cloning murine immunoglobulin variable domains are disclosed, for
example, in Orlandi et al., Proc. Nat'l Acad. Sci. USA 86: 3833
(1989). Techniques for constructing chimeric antibodies are well
known to those of skill in the art. As an example, Leung et al.,
Hybridoma 13:469 (1994), produced an LL2 chimera by combining DNA
sequences encoding the V.sub..kappa. and V.sub.H domains of murine
LL2, an anti-CD22 monoclonal antibody, with respective human
.kappa. and IgG.sub.1 constant region domains.
Humanized Antibodies
Techniques for producing humanized MAbs are well known in the art
(see, e.g., Jones et al., Nature 321: 522 (1986), Riechmann et al.,
Nature 332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988),
Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu,
Crit. Rev. Biotech. 12: 437 (1992), and Singer et al., J. Immun.
150: 2844 (1993)). A chimeric or murine monoclonal antibody may be
humanized by transferring the mouse CDRs from the heavy and light
variable chains of the mouse immunoglobulin into the corresponding
variable domains of a human antibody. The mouse framework regions
(FR) in the chimeric monoclonal antibody are also replaced with
human FR sequences. As simply transferring mouse CDRs into human
FRs often results in a reduction or even loss of antibody affinity,
additional modification might be required in order to restore the
original affinity of the murine antibody. This can be accomplished
by the replacement of one or more human residues in the FR regions
with their murine counterparts to obtain an antibody that possesses
good binding affinity to its epitope. See, for example, Tempest et
al., Biotechnology 9:266 (1991) and Verhoeyen et al., Science 239:
1534 (1988). Generally, those human FR amino acid residues that
differ from their murine counterparts and are located close to or
touching one or more CDR amino acid residues would be candidates
for substitution.
Human Antibodies
Methods for producing fully human antibodies using either
combinatorial approaches or transgenic animals transformed with
human immunoglobulin loci are known in the art (e.g., Mancini et
al., 2004, New Microbiol. 27:315-28; Conrad and Scheller, 2005,
Comb. Chem. High Throughput Screen. 8:117-26; Brekke and Loset,
2003, Curr. Opin. Phamacol. 3:544-50). A fully human antibody also
can be constructed by genetic or chromosomal transfection methods,
as well as phage display technology, all of which are known in the
art. See for example, McCafferty et al., Nature 348:552-553 (1990).
Such fully human antibodies are expected to exhibit even fewer side
effects than chimeric or humanized antibodies and to function in
vivo as essentially endogenous human antibodies. In certain
embodiments, the claimed methods and procedures may utilize human
antibodies produced by such techniques.
In one alternative, the phage display technique may be used to
generate human antibodies (e.g., Dantas-Barbosa et al., 2005,
Genet. Mol. Res. 4:126-40). Human antibodies may be generated from
normal humans or from humans that exhibit a particular disease
state, such as cancer (Dantas-Barbosa et al., 2005). The advantage
to constructing human antibodies from a diseased individual is that
the circulating antibody repertoire may be biased towards
antibodies against disease-associated antigens.
In one non-limiting example of this methodology, Dantas-Barbosa et
al. (2005) constructed a phage display library of human Fab
antibody fragments from osteosarcoma patients. Generally, total RNA
was obtained from circulating blood lymphocytes (Id.). Recombinant
Fab were cloned from the .mu., .gamma. and .kappa. chain antibody
repertoires and inserted into a phage display library (Id.). RNAs
were converted to cDNAs and used to make Fab cDNA libraries using
specific primers against the heavy and light chain immunoglobulin
sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97). Library
construction was performed according to Andris-Widhopf et al.
(2000, In: Phage Display Laboratory Manual, Barbas et al. (eds),
1.sup.st edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. pp. 9.1 to 9.22). The final Fab fragments were
digested with restriction endonucleases and inserted into the
bacteriophage genome to make the phage display library. Such
libraries may be screened by standard phage display methods, as
known in the art (see, e.g., Pasqualini and Ruoslahti, 1996, Nature
380:364-366; Pasqualini, 1999, The Quart. J. Nucl. Med.
43:159-162).
Phage display can be performed in a variety of formats, for their
review, see e.g. Johnson and Chiswell, Current Opinion in
Structural Biology 3:5564-571 (1993). Human antibodies may also be
generated by in vitro activated B cells. See U.S. Pat. Nos.
5,567,610 and 5,229,275, incorporated herein by reference in their
entirety. The skilled artisan will realize that these techniques
are exemplary and any known method for making and screening human
antibodies or antibody fragments may be utilized.
In another alternative, transgenic animals that have been
genetically engineered to produce human antibodies may be used to
generate antibodies against essentially any immunogenic target,
using standard immunization protocols. Methods for obtaining human
antibodies from transgenic mice are disclosed by Green et al.,
Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994),
and Taylor et al., Int. Immun. 6:579 (1994). A non-limiting example
of such a system is the XENOMOUSE.RTM. (e.g., Green et al., 1999,
J. Immunol. Methods 231:11-23) from Abgenix (Fremont, Calif.). In
the XENOMOUSE.RTM. and similar animals, the mouse antibody genes
have been inactivated and replaced by functional human antibody
genes, while the remainder of the mouse immune system remains
intact.
The XENOMOUSE.RTM. was transformed with germline-configured YACs
(yeast artificial chromosomes) that contained portions of the human
IgH and Igkappa loci, including the majority of the variable region
sequences, along accessory genes and regulatory sequences. The
human variable region repertoire may be used to generate antibody
producing B cells, which may be processed into hybridomas by known
techniques. A XENOMOUSE.RTM. immunized with a target antigen will
produce human antibodies by the normal immune response, which may
be harvested and/or produced by standard techniques discussed
above. A variety of strains of XENOMOUSE.RTM. are available, each
of which is capable of producing a different class of antibody.
Transgenically produced human antibodies have been shown to have
therapeutic potential, while retaining the pharmacokinetic
properties of normal human antibodies (Green et al., 1999). The
skilled artisan will realize that the claimed compositions and
methods are not limited to use of the XENOMOUSE.RTM. system but may
utilize any transgenic animal that has been genetically engineered
to produce human antibodies.
Antibody Fragments
Antibody fragments which recognize specific epitopes can be
generated by known techniques. Antibody fragments are antigen
binding portions of an antibody, such as F(ab').sub.2, Fab',
F(ab).sub.2, Fab, Fv, sFv and the like. F(ab').sub.2 fragments can
be produced by pepsin digestion of the antibody molecule and Fab'
fragments can be generated by reducing disulfide bridges of the
F(ab').sub.2 fragments. Alternatively, Fab' expression libraries
can be constructed (Huse et al., 1989, Science, 246:1274-1281) to
allow rapid and easy identification of monoclonal Fab' fragments
with the desired specificity. F(ab).sub.2 fragments may be
generated by papain digestion of an antibody.
A single chain Fv molecule (scFv) comprises a VL domain and a VH
domain. The VL and VH domains associate to form a target binding
site. These two domains are further covalently linked by a peptide
linker (L). Methods for making scFv molecules and designing
suitable peptide linkers are described in U.S. Pat. Nos. 4,704,692,
4,946,778, R. Raag and M. Whitlow, "Single Chain Fvs." FASEB Vol
9:73-80 (1995) and R. E. Bird and B. W. Walker, "Single Chain
Antibody Variable Regions," TIBTECH, Vol 9: 132-137 (1991).
Techniques for producing single domain antibodies are also known in
the art, as disclosed for example in Cossins et al. (2006, Prot
Express Purif 51:253-259), incorporated herein by reference. Single
domain antibodies (VHH) may be obtained, for example, from camels,
alpacas or llamas by standard immunization techniques. (See, e.g.,
Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol
Methods 281:161-75, 2003; Maass et al., J Immunol Methods
324:13-25, 2007). The VHH may have potent antigen-binding capacity
and can interact with novel epitopes that are inaccessible to
conventional VH-VL pairs. (Muyldermans et al., 2001). Alpaca serum
IgG contains about 50% camelid heavy chain only IgG antibodies
(HCAbs) (Maass et al., 2007). Alpacas may be immunized with known
antigens, such as TNF-.alpha., and VHHs can be isolated that bind
to and neutralize the target antigen (Maass et al., 2007). PCR
primers that amplify virtually all alpaca VHH coding sequences have
been identified and may be used to construct alpaca VHH phage
display libraries, which can be used for antibody fragment
isolation by standard biopanning techniques well known in the art
(Maass et al., 2007). In certain embodiments, anti-pancreatic
cancer VHH antibody fragments may be utilized in the claimed
compositions and methods.
An antibody fragment can be prepared by proteolytic hydrolysis of
the full length antibody or by expression in E. coli or another
host of the DNA coding for the fragment. An antibody fragment can
be obtained by pepsin or papain digestion of full length antibodies
by conventional methods. These methods are described, for example,
by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647 and
references contained therein. Also, see Nisonoff et al., Arch
Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119
(1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page 422
(Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and
2.10.-2.10.4.
Known Antibodies
Various embodiments, for example in combination therapy, may
involve the use of antibodies binding to target antigens besides
IL-6 or TNF-.alpha.. A variety of antibodies are commercially
available and/or known in the art. Antibodies of use may be
commercially obtained, for example, from the American Type Culture
Collection (ATCC, Manassas, Va.). A large number of antibodies
against various disease targets, including but not limited to
tumor-associated antigens, have been deposited at the ATCC and/or
have published variable region sequences and are available for use
in the claimed methods and compositions. See, e.g., U.S. Pat. Nos.
7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509;
7,049,060; 7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018;
7,037,498; 7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852;
6,989,241; 6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813;
6,956,107; 6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547;
6,921,645; 6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475;
6,905,681; 6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594;
6,881,405; 6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062;
6,861,511; 6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370;
6,824,780; 6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450;
6,767,711; 6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981;
6,730,307; 6,720,155; 6,716,966; 6,709,653; 6,693,176; 6,692,908;
6,689,607; 6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734;
6,673,344; 6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833;
6,610,294; 6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745;
6,572,856; 6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058;
6,528,625; 6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915;
6,488,930; 6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529;
6,465,173; 6,461,823; 6,458,356; 6,455,044; 6,455,040, 6,451,310;
6,444,206; 6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726;
6,406,694; 6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350;
6,383,759; 6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481;
6,355,444; 6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571;
6,340,459; 6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744;
6,129,914; 6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540;
5,814,440; 5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595;
5,677,136; 5,587,459; 5,443,953, 5,525,338, the Examples section of
each of which is incorporated herein by reference. These are
exemplary only and a wide variety of other antibodies and their
hybridomas are known in the art. The skilled artisan will realize
that antibody sequences or antibody-secreting hybridomas against
almost any disease-associated antigen may be obtained by a simple
search of the ATCC, NCBI and/or USPTO databases for antibodies
against a selected disease-associated target of interest. The
antigen binding domains of the cloned antibodies may be amplified,
excised, ligated into an expression vector, transfected into an
adapted host cell and used for protein production, using standard
techniques well known in the art (see, e.g., U.S. Pat. Nos.
7,531,327; 7,537,930; 7,608,425 and 7,785,880, the Examples section
of each of which is incorporated herein by reference).
Particular antibodies that may be of use for therapy of cancer
within the scope of the claimed methods and compositions include,
but are not limited to, LL1 (anti-CD74), LL2 and RFB4 (anti-CD22),
RS7 (anti-epithelial glycoprotein-1 (EGP-1)), PAM4 and KC4 (both
anti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA, also known
as CD66e), Mu-9 (anti-colon-specific antigen-p), Immu 31 (an
anti-alpha-fetoprotein), TAG-72 (e.g., CC49), Tn, J591 or HuJ591
(anti-PSMA (prostate-specific membrane antigen)), AB-PG1-XG1-026
(anti-PSMA dimer), D2/B (anti-PSMA), G250 (anti-carbonic anhydrase
IX), hL243 (anti-HLA-DR), alemtuzumab (anti-CD52), bevacizumab
(anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33),
ibritumomab tiuxetan (anti-CD20); panitumumab (anti-EGFR);
rituximab (anti-CD20); tositumomab (anti-CD20); GA101 (anti-CD20);
and trastuzumab (anti-ErbB2). Such antibodies are known in the art
(e.g., U.S. Pat. Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744;
6,306,393; 6,653,104; 6,730.300; 6,899,864; 6,926,893; 6,962,702;
7,074,403; 7,230,084; 7,238,785; 7,238,786; 7,256,004; 7,282,567;
7,300,655; 7,312,318; 7,585,491; 7,612,180; 7,642,239; and U.S.
Patent Application Publ. No. 20040202666 (now abandoned);
20050271671; and 20060193865; the Examples section of each
incorporated herein by reference.) Specific known antibodies of use
include hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No.
7,251,164), hA19 (U.S. Pat. No. 7,109,304), hIMMU31 (U.S. Pat. No.
7,300,655), hLL1 (U.S. Pat. No. 7,312,318,), hLL2 (U.S. Pat. No.
7,074,403), hMu-9 (U.S. Pat. No. 7,387,773), hL243 (U.S. Pat. No.
7,612,180), hMN-14 (U.S. Pat. No. 6,676,924), hMN-15 (U.S. Pat. No.
7,541,440), hR1 (U.S. patent application Ser. No. 12/772,645), hRS7
(U.S. Pat. No. 7,238,785), hMN-3 (U.S. Pat. No. 7,541,440),
AB-PG1-XG1-026 (U.S. patent application Ser. No. 11/983,372,
deposited as ATCC PTA-4405 and PTA-4406) and D2/B (WO 2009/130575)
the text of each recited patent or application is incorporated
herein by reference with respect to the Figures and Examples
sections.
Anti-TNF-.alpha. antibodies are known in the art and may be of use
to treat immune diseases, such as autoimmune disease, immune
dysfunction (e.g., graft-versus-host disease, organ transplant
rejection) or diabetes. Known antibodies against TNF-.alpha.
include the human antibody CDP571 (Ofei et al., 2011, Diabetes
45:881-85); murine antibodies MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI,
M302B and M303 (Thermo Scientific, Rockford, Ill.); infliximab
(Centocor, Malvern, Pa.); certolizumab pegol (UCB, Brussels,
Belgium); and adalimumab (Abbott, Abbott Park, Ill.). These and
many other known anti-TNF-.alpha. antibodies may be used in the
claimed methods and compositions. Other antibodies of use for
therapy of immune dysregulatory or autoimmune disease include, but
are not limited to, anti-B-cell antibodies such as veltuzumab,
epratuzumab, milatuzumab or hL243; tocilizumab (anti-IL-6
receptor); basiliximab (anti-CD25); daclizumab (anti-CD25);
efalizumab (anti-CD11a); muromonab-CD3 (anti-CD3 receptor);
anti-CD40L (UCB, Brussels, Belgium); natalizumab (anti-.alpha.4
integrin) and omalizumab (anti-IgE).
Type-2 diabetes may be treated using known antibodies against
B-cell antigens, such as CD22 (epratuzumab), CD74 (milatuzumab),
CD19 (hA19), CD20 (veltuzumab) or HLA-DR (hL243) (see, e.g., Winer
et al., 2011, Nature Med 17:610-18). Anti-CD3 antibodies also have
been proposed for therapy of type 1 diabetes (Cernea et al., 2010,
Diabetes Metab Rev 26:602-05).
Macrophage migration inhibitory factor (MIF) is an important
regulator of innate and adaptive immunity and apoptosis. It has
been reported that CD74 is the endogenous receptor for MIF (Leng et
al., 2003, J Exp Med 197:1467-76). The therapeutic effect of
antagonistic anti-CD74 antibodies on MIF-mediated intracellular
pathways may be of use for treatment of a broad range of disease
states, such as cancers of the bladder, prostate, breast, lung,
colon and chronic lymphocytic leukemia (e.g., Meyer-Siegler et al.,
2004, BMC Cancer 12:34; Shachar & Haran, 2011, Leuk Lymphoma
52:1446-54); autoimmune diseases such as rheumatoid arthritis and
systemic lupus erythematosus (Morand & Leech, 2005, Front
Biosci 10:12-22; Shachar & Haran, 2011, Leuk Lymphoma
52:1446-54); kidney diseases such as renal allograft rejection
(Lan, 2008, Nephron Exp Nephrol. 109:e79-83); and numerous
inflammatory diseases (Meyer-Siegler et al., 2009, Mediators
Inflamm epub Mar. 22, 2009; Takahashi et al., 2009, Respir Res
10:33; Milatuzumab (hLL1) is an exemplary anti-CD74 antibody of
therapeutic use for treatment of MIF-mediated diseases.
Anti-CD3 antibodies have been reported to reduce development and
progression of atherosclerosis (Steffens et al., 2006, Circulation
114:1977-84). Antibodies against oxidized LDL induced a regression
of established atherosclerosis in a mouse model (Ginsberg, 2007, J
Am Coll Cardiol 52:2319-21). Anti-ICAM-1 antibody was shown to
reduce ischemic cell damage after cerebral artery occlusion in rats
(Zhang et al., 1994, Neurology 44:1747-51).
Antibody Allotypes
Immunogenicity of therapeutic antibodies is associated with
increased risk of infusion reactions and decreased duration of
therapeutic response (Baert et al., 2003, N Engl J Med 348:602-08).
The extent to which therapeutic antibodies induce an immune
response in the host may be determined in part by the allotype of
the antibody (Stickler et al., 2011, Genes and Immunity 12:213-21).
Antibody allotype is related to amino acid sequence variations at
specific locations in the constant region sequences of the
antibody. The allotypes of IgG antibodies containing a heavy chain
.gamma.-type constant region are designated as Gm allotypes (1976,
J Immunol 117:1056-59).
For the common IgG1 human antibodies, the most prevalent allotype
is G1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21).
However, the G1m3 allotype also occurs frequently in Caucasians
(Id.). It has been reported that G1m1 antibodies contain allotypic
sequences that tend to induce an immune response when administered
to non-G1m1 (nG1m1) recipients, such as G1m3 patients (Id.).
Non-G1m1 allotype antibodies are not as immunogenic when
administered to G1m1 patients (Id.).
The human G1m1 allotype comprises the amino acids aspartic acid at
Kabat position 356 and leucine at Kabat position 358 in the CH3
sequence of the heavy chain IgG1. The nG1m1 allotype comprises the
amino acids glutamic acid at Kabat position 356 and methionine at
Kabat position 358. Both G1m1 and nG1m1 allotypes comprise a
glutamic acid residue at Kabat position 357 and the allotypes are
sometimes referred to as DEL and EEM allotypes. A non-limiting
example of the heavy chain constant region sequences for G1m1 and
nG1m1 allotype antibodies is shown for the exemplary antibodies
rituximab (SEQ ID NO:85) and veltuzumab (SEQ ID NO:86).
TABLE-US-00015 Rituximab heavy chain variable region sequence (SEQ
ID NO: 85) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Veltuzumab heavy chain variable
region (SEQ ID NO: 86)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP
KSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence
variations characteristic of IgG allotypes and their effect on
immunogenicity. They reported that the G1m3 allotype is
characterized by an arginine residue at Kabat position 214,
compared to a lysine residue at Kabat 214 in the G1m17 allotype.
The nG1m1,2 allotype was characterized by glutamic acid at Kabat
position 356, methionine at Kabat position 358 and alanine at Kabat
position 431. The G1m1,2 allotype was characterized by aspartic
acid at Kabat position 356, leucine at Kabat position 358 and
glycine at Kabat position 431. In addition to heavy chain constant
region sequence variants, Jefferis and Lefranc (2009) reported
allotypic variants in the kappa light chain constant region, with
the Km1 allotype characterized by valine at Kabat position 153 and
leucine at Kabat position 191, the Km1,2 allotype by alanine at
Kabat position 153 and leucine at Kabat position 191, and the Km3
allotype characterized by alanine at Kabat position 153 and valine
at Kabat position 191.
With regard to therapeutic antibodies, veltuzumab and rituximab
are, respectively, humanized and chimeric IgG1 antibodies against
CD20, of use for therapy of a wide variety of hematological
malignancies and/or autoimmune diseases. Table 5 compares the
allotype sequences of rituximab vs. veltuzumab. As shown in Table
5, rituximab (G1m17,1) is a DEL allotype IgG1, with an additional
sequence variation at Kabat position 214 (heavy chain CH1) of
lysine in rituximab vs. arginine in veltuzumab. It has been
reported that veltuzumab is less immunogenic in subjects than
rituximab (see, e.g., Morchhauser et al., 2009, J Clin Oncol
27:3346-53; Goldenberg et al., 2009, Blood 113:1062-70; Robak &
Robak, 2011, BioDrugs 25:13-25), an effect that has been attributed
to the difference between humanized and chimeric antibodies.
However, the difference in allotypes between the EEM and DEL
allotypes likely also accounts for the lower immunogenicity of
veltuzumab.
TABLE-US-00016 TABLE 5 Allotypes of Rituximab vs. Veltuzumab Heavy
chain position and associated allotypes Complete 214 356/358 431
allotype (allotype) (allotype) (allotype) Rituximab G1m17, 1 K 17
D/L 1 A -- Veltuzumab G1m3 R 3 E/M -- A --
In order to reduce the immunogenicity of therapeutic antibodies in
individuals of nG1m1 genotype, it is desirable to select the
allotype of the antibody to correspond to the G1m3 allotype,
characterized by arginine at Kabat 214, and the nG1m1,2
null-allotype, characterized by glutamic acid at Kabat position
356, methionine at Kabat position 358 and alanine at Kabat position
431. Surprisingly, it was found that repeated subcutaneous
administration of G1m3 antibodies over a long period of time did
not result in a significant immune response. In alternative
embodiments, the human IgG4 heavy chain in common with the G1m3
allotype has arginine at Kabat 214, glutamic acid at Kabat 356,
methionine at Kabat 359 and alanine at Kabat 431. Since
immunogenicity appears to relate at least in part to the residues
at those locations, use of the human IgG4 heavy chain constant
region sequence for therapeutic antibodies is also a preferred
embodiment. Combinations of G1m3 IgG1 antibodies with IgG4
antibodies may also be of use for therapeutic administration.
Immunoconjugates
In certain embodiments, the antibodies or complexes may be
conjugated to one or more therapeutic or diagnostic agents. The
therapeutic agents do not need to be the same but can be different,
e.g. a drug and a radioisotope. For example, .sup.131I can be
incorporated into a tyrosine of an antibody or fusion protein and a
drug attached to an epsilon amino group of a lysine residue.
Therapeutic and diagnostic agents also can be attached, for example
to reduced SH groups and/or to carbohydrate side chains. Many
methods for making covalent or non-covalent conjugates of
therapeutic or diagnostic agents with antibodies or fusion proteins
are known in the art and any such known method may be utilized.
A therapeutic or diagnostic agent can be attached at the hinge
region of a reduced antibody component via disulfide bond
formation. Alternatively, such agents can be attached using a
heterobifunctional cross-linker, such as N-succinyl
3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56:
244 (1994). General techniques for such conjugation are well-known
in the art. See, for example, Wong, CHEMISTRY OF PROTEIN
CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis et al.,
"Modification of Antibodies by Chemical Methods," in MONOCLONAL
ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages
187-230 (Wiley-Liss, Inc. 1995); Price, "Production and
Characterization of Synthetic Peptide-Derived Antibodies," in
MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL
APPLICATION, Ritter et al. (eds.), pages 60-84 (Cambridge
University Press 1995). Alternatively, the therapeutic or
diagnostic agent can be conjugated via a carbohydrate moiety in the
Fc region of the antibody. The carbohydrate group can be used to
increase the loading of the same agent that is bound to a thiol
group, or the carbohydrate moiety can be used to bind a different
therapeutic or diagnostic agent.
Methods for conjugating peptides to antibody components via an
antibody carbohydrate moiety are well-known to those of skill in
the art. See, for example, Shih et al., Int. J. Cancer 41: 832
(1988); Shih et al., Int. J. Cancer 46: 1101 (1990); and Shih et
al., U.S. Pat. No. 5,057,313, incorporated herein in their entirety
by reference. The general method involves reacting an antibody
component having an oxidized carbohydrate portion with a carrier
polymer that has at least one free amine function. This reaction
results in an initial Schiff base (imine) linkage, which can be
stabilized by reduction to a secondary amine to form the final
conjugate.
The Fc region may be absent if the antibody used as the antibody
component is an antibody fragment. However, it is possible to
introduce a carbohydrate moiety into the light chain variable
region of a full length antibody or antibody fragment. See, for
example, Leung et al., J. Immunol. 154: 5919 (1995); Hansen et al.,
U.S. Pat. No. 5,443,953 (1995), Leung et al., U.S. Pat. No.
6,254,868, incorporated herein by reference in their entirety. The
engineered carbohydrate moiety is used to attach the therapeutic or
diagnostic agent.
In some embodiments, a chelating agent may be attached to an
antibody, antibody fragment or fusion protein and used to chelate a
therapeutic or diagnostic agent, such as a radionuclide. Exemplary
chelators include but are not limited to DTPA (such as Mx-DTPA),
DOTA, TETA, NETA or NOTA. Methods of conjugation and use of
chelating agents to attach metals or other ligands to proteins are
well known in the art (see, e.g., U.S. Pat. No. 7,563,433, the
Examples section of which is incorporated herein by reference).
In certain embodiments, radioactive metals or paramagnetic ions may
be attached to proteins or peptides by reaction with a reagent
having a long tail, to which may be attached a multiplicity of
chelating groups for binding ions. Such a tail can be a polymer
such as a polylysine, polysaccharide, or other derivatized or
derivatizable chains having pendant groups to which can be bound
chelating groups such as, e.g., ethylenediaminetetraacetic acid
(EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins,
polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and
like groups known to be useful for this purpose.
Chelates may be directly linked to antibodies or peptides, for
example as disclosed in U.S. Pat. No. 4,824,659, incorporated
herein in its entirety by reference. Particularly useful
metal-chelate combinations include 2-benzyl-DTPA and its monomethyl
and cyclohexyl analogs, used with diagnostic isotopes in the
general energy range of 60 to 4,000 keV, such as .sup.125I,
.sup.131I, .sup.123I, .sup.124I, .sup.62Cu, .sup.64Cu, .sup.18F,
.sup.111In, .sup.67Ga, .sup.68Ga, .sup.99mTe, .sup.94mTe, .sup.11C,
.sup.13N, .sup.15O, .sup.76Br, for radioimaging. The same chelates,
when complexed with non-radioactive metals, such as manganese, iron
and gadolinium are useful for MRI. Macrocyclic chelates such as
NOTA, DOTA, and TETA are of use with a variety of metals and
radiometals, most particularly with radionuclides of gallium,
yttrium and copper, respectively. Such metal-chelate complexes can
be made very stable by tailoring the ring size to the metal of
interest. Other ring-type chelates such as macrocyclic polyethers,
which are of interest for stably binding nuclides, such as
.sup.223Ra for RAIT are encompassed.
More recently, methods of .sup.18F-labeling of use in PET scanning
techniques have been disclosed, for example by reaction of F-18
with a metal or other atom, such as aluminum. The .sup.18F--Al
conjugate may be complexed with chelating groups, such as DOTA,
NOTA or NETA that are attached directly to antibodies or used to
label targetable constructs in pre-targeting methods. Such F-18
labeling techniques are disclosed in U.S. Pat. No. 7,563,433, the
Examples section of which is incorporated herein by reference.
Therapeutic Agents
In alternative embodiments, therapeutic agents such as cytotoxic
agents, anti-angiogenic agents, pro-apoptotic agents, antibiotics,
hormones, hormone antagonists, chemokines, drugs, prodrugs, toxins,
enzymes or other agents may be used, either conjugated to the
subject antibody complexes or separately administered before,
simultaneously with, or after the antibody complex. Drugs of use
may possess a pharmaceutical property selected from the group
consisting of antimitotic, kinase inhibitor, Bruton kinase
inhibitor, alkylating, antimetabolite, antibiotic, alkaloid,
anti-angiogenic, pro-apoptotic agents and combinations thereof.
Exemplary drugs of use include, but are not limited to,
5-fluorouracil, afatinib, aplidin, azaribine, anastrozole,
anthracyclines, axitinib, AVL-101, AVL-291, bendamustine,
bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan,
calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin,
carmustine, celecoxib, chlorambucil, cisplatin (CDDP), Cox-2
inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine,
camptothecans, crizotinib, cyclophosphamide, cytarabine,
dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin,
daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX),
cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin
glucuronide, erlotinib, estramustine, epidophyllotoxin, entinostat,
estrogen receptor binding agents, etoposide (VP16), etoposide
glucuronide, etoposide phosphate, exemestane, fingolimod,
floxuridine (FUdR), 3',5'-O-dioleoyl-FudR (FUdR-dO), fludarabine,
flutamide, farnesyl-protein transferase inhibitors, flavopiridol,
fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib,
gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib,
ifosfamide, imatinib, L-asparaginase, lapatinib, lenolidamide,
leucovorin, LFM-A13, lomustine, mechlorethamine, melphalan,
mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,
mithramycin, mitomycin, mitotane, navelbine, neratinib, nilotinib,
nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel,
PCI-32765, pentostatin, PSI-341, raloxifene, semustine, sorafenib,
streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (an
aqueous form of DTIC), transplatinum, thalidomide, thioguanine,
thiotepa, teniposide, topotecan, uracil mustard, vatalanib,
vinorelbine, vinblastine, vincristine, vinca alkaloids and
ZD1839.
Toxins of use may include ricin, abrin, alpha toxin, saporin,
ribonuclease (RNase), e.g., onconase, DNase I, Staphylococcal
enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria
toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.
Chemokines of use may include RANTES, MCAF, MIP1-alpha, MIP1-Beta
and IP-10.
In certain embodiments, anti-angiogenic agents, such as
angiostatin, baculostatin, canstatin, maspin, anti-VEGF antibodies,
anti-PlGF peptides and antibodies, anti-vascular growth factor
antibodies, anti-Flk-1 antibodies, anti-Flt-1 antibodies and
peptides, anti-Kras antibodies, anti-cMET antibodies, anti-MIF
(macrophage migration-inhibitory factor) antibodies, laminin
peptides, fibronectin peptides, plasminogen activator inhibitors,
tissue metalloproteinase inhibitors, interferons, interleukin-12,
IP-10, Gro- , thrombospondin, 2-methoxyoestradiol,
proliferin-related protein, carboxiamidotriazole, CM101,
Marimastat, pentosan polysulphate, angiopoietin-2,
interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,
Linomide (roquinimex), thalidomide, pentoxifylline, genistein,
TNP-470, endostatin, paclitaxel, accutin, angiostatin, cidofovir,
vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline
may be of use.
Immunomodulators of use may be selected from a cytokine, a stem
cell growth factor, a lymphotoxin, a hematopoietic factor, a colony
stimulating factor (CSF), an interferon (IFN), erythropoietin,
thrombopoietin and a combination thereof. Specifically useful are
lymphotoxins such as tumor necrosis factor (TNF), hematopoietic
factors, such as interleukin (IL), colony stimulating factor, such
as granulocyte-colony stimulating factor (G-CSF) or granulocyte
macrophage-colony stimulating factor (GM-CSF), interferon, such as
interferons-.alpha., -.beta. or -.gamma., and stem cell growth
factor, such as that designated "S1 factor". Included among the
cytokines are growth hormones such as human growth hormone,
N-methionyl human growth hormone, and bovine growth hormone;
parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;
prorelaxin; glycoprotein hormones such as follicle stimulating
hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing
hormone (LH); hepatic growth factor; prostaglandin, fibroblast
growth factor; prolactin; placental lactogen, OB protein; tumor
necrosis factor-.alpha. and - ; mullerian-inhibiting substance;
mouse gonadotropin-associated peptide; inhibin; activin; vascular
endothelial growth factor; integrin; thrombopoietin (TPO); nerve
growth factors such as NGF- ; platelet-growth factor; transforming
growth factors (TGFs) such as TGF-.alpha. and TGF- ; insulin-like
growth factor-I and -II; erythropoietin (EPO); osteoinductive
factors; interferons such as interferon-.alpha., -.beta., and
-.gamma.; colony stimulating factors (CSFs) such as macrophage-CSF
(M-CSF); interleukins (ILs) such as IL-1, IL-1.alpha., IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,
IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand or
FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis
factor and LT.
Radionuclides of use include, but are not limited to--.sup.111In,
.sup.177Lu, .sup.212Bi, .sup.213Bi, .sup.211At, .sup.62Cu,
.sup.67Cu, .sup.90Y, .sup.125I, .sup.131I, .sup.32P, .sup.33P,
.sup.47Sc, .sup.111Ag, .sup.67Ga, .sup.142Pr, .sup.153Sm,
.sup.161Tb, .sup.166Dy, .sup.166Ho, .sup.186Re, .sup.188Re,
.sup.189Re, .sup.212Pb, .sup.223Ra, .sup.225Ac, .sup.59Fe,
.sup.75Se, .sup.77As, .sup.89Sr, .sup.99Mo, .sup.105Rh, .sup.109Pd,
.sup.143Pr, .sup.149Pm, .sup.169Er, .sup.194Ir, .sup.198Au,
.sup.199Au, .sup.227Th and .sup.211Pb. The therapeutic radionuclide
preferably has a decay-energy in the range of 20 to 6,000 keV,
preferably in the ranges 60 to 200 keV for an Auger emitter,
100-2,500 keV for a beta emitter, and 4,000-6,000 keV for an alpha
emitter. Maximum decay energies of useful beta-particle-emitting
nuclides are preferably 20-5,000 keV, more preferably 100-4,000
keV, and most preferably 500-2,500 keV. Also preferred are
radionuclides that substantially decay with Auger-emitting
particles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m,
Pt-109, In-111, Sb-119, 1-125, Ho-161, Os-189m and Ir-192. Decay
energies of useful beta-particle-emitting nuclides are preferably
<1,000 keV, more preferably <100 keV, and most preferably
<70 keV. Also preferred are radionuclides that substantially
decay with generation of alpha-particles. Such radionuclides
include, but are not limited to: Dy-152, At-211, Bi-212, Ra-223,
Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213, Th-227 and
Fm-255. Decay energies of useful alpha-particle-emitting
radionuclides are preferably 2,000-10,000 keV, more preferably
3,000-8,000 keV, and most preferably 4,000-7,000 keV. Additional
potential radioisotopes of use include .sup.11C, .sup.13N,
.sup.15O, .sup.75Br, .sup.198Au, .sup.224Ac, .sup.126I, .sup.133I,
.sup.77Br, .sup.113mIn, .sup.95Ru, .sup.97Ru, .sup.103Ru,
.sup.105Ru, .sup.107Hg, .sup.203Hg, .sup.121mTe, .sup.122mTe,
.sup.125mTe, .sup.165Tm, .sup.167Tm, .sup.168Tm, .sup.197Pt,
.sup.109Pd, .sup.105Rh, .sup.142Pr, .sup.143Pr, .sup.161Tb,
.sup.166Ho, .sup.199Au, .sup.57Co, .sup.58Co, .sup.51Cr, .sup.59Fe,
.sup.75Se, .sup.201Tl, .sup.225Ac, .sup.76Br, .sup.169Yb, and the
like. Some useful diagnostic nuclides may include .sup.18F,
.sup.52Fe, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.67Ga, .sup.68Ga,
.sup.86Y, .sup.89Zr, .sup.94Tc, .sup.94mTc, .sup.99mTc, or In.
Radionuclides and other metals may be delivered, for example, using
chelating groups attached to an antibody or conjugate. Macrocyclic
chelates such as NOTA, DOTA, and TETA are of use with a variety of
metals and radiometals, most particularly with radionuclides of
gallium, yttrium and copper, respectively. Such metal-chelate
complexes can be made very stable by tailoring the ring size to the
metal of interest. Other ring-type chelates, such as macrocyclic
polyethers for complexing .sup.223Ra, may be used.
Therapeutic agents may include a photoactive agent or dye.
Fluorescent compositions, such as fluorochrome, and other
chromogens, or dyes, such as porphyrins sensitive to visible light,
have been used to detect and to treat lesions by directing the
suitable light to the lesion. In therapy, this has been termed
photoradiation, phototherapy, or photodynamic therapy. See Joni et
al. (eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES
(Libreria Progetto 1985); van den Bergh, Chem. Britain (1986),
22:430. Moreover, monoclonal antibodies have been coupled with
photoactivated dyes for achieving phototherapy. See Mew et al., J.
Immunol. (1983), 130:1473; idem., Cancer Res. (1985), 45:4380;
Oseroff et al., Proc. Natl. Acad. Sci. USA (1986), 83:8744; idem.,
Photochem. Photobiol. (1987), 46:83; Hasan et al., Prog. Clin.
Biol. Res. (1989), 288:471; Tatsuta et al., Lasers Surg. Med.
(1989), 9:422; Pelegrin et al., Cancer (1991), 67:2529.
Other useful therapeutic agents may comprise oligonucleotides,
especially antisense oligonucleotides that preferably are directed
against oncogenes and oncogene products, such as bcl-2 or p53. A
preferred form of therapeutic oligonucleotide is siRNA.
Diagnostic Agents
Diagnostic agents are preferably selected from the group consisting
of a radionuclide, a radiological contrast agent, a paramagnetic
ion, a metal, a fluorescent label, a chemiluminescent label, an
ultrasound contrast agent and a photoactive agent. Such diagnostic
agents are well known and any such known diagnostic agent may be
used. Non-limiting examples of diagnostic agents may include a
radionuclide such as .sup.110In, .sup.111In, .sup.177Lu, .sup.18F,
.sup.52Fe, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.67Ga, .sup.68Ga,
.sup.86Y, .sup.90Y, .sup.89Zr, .sup.94mTc, .sup.94Tc, .sup.99mTc,
.sup.120I, .sup.123I, .sup.124I, .sup.125I, .sup.131I,
.sup.154-158Gd, .sup.32P, .sup.11C, .sup.13N, .sup.15O, .sup.186Re,
.sup.188Re, .sup.51Mn, .sup.52mMn, .sup.55Co, .sup.72As, .sup.75Br,
.sup.76Br, .sup.82mRb, .sup.83Sr, or other gamma-, beta-, or
positron-emitters. Paramagnetic ions of use may include chromium
(III), manganese (II), iron (III), iron (II), cobalt (II), nickel
(II), copper (II), neodymium (III), samarium (III), ytterbium
(III), gadolinium (III), vanadium (II), terbium (III), dysprosium
(III), holmium (III) or erbium (III). Metal contrast agents may
include lanthanum (III), gold (III), lead (II) or bismuth (III).
Ultrasound contrast agents may comprise liposomes, such as gas
filled liposomes. Radiopaque diagnostic agents may be selected from
compounds, barium compounds, gallium compounds, and thallium
compounds. A wide variety of fluorescent labels are known in the
art, including but not limited to fluorescein isothiocyanate,
rhodamine, phycoerytherin, phycocyanin, allophycocyanin,
o-phthaldehyde and fluorescamine. Chemiluminescent labels of use
may include luminol, isoluminol, an aromatic acridinium ester, an
imidazole, an acridinium salt or an oxalate ester.
Therapeutic Use
In another aspect, the invention relates to a method of treating a
subject, comprising administering a therapeutically effective
amount of an antibody complex as described herein to a subject.
Diseases that may be treated with the antibody complexes described
herein include, but are not limited to immune diseases (e.g., SLE,
RA, juvenile idiopathic arthritis, Crohn's disease, type 2
diabetes, Castleman's disease) or inflammatory diseases (e.g.,
sepsis, septic shock, inflammation, inflammatory bowel disease,
inflammatory liver injury, acute pancreatitis). Such therapeutics
can be given once or repeatedly, depending on the disease state and
tolerability of the conjugate, and can also be used optimally in
combination with other therapeutic modalities, such as
immunomodulator therapy, immunotherapy, chemotherapy, antisense
therapy, interference RNA therapy, gene therapy, and the like. Each
combination will be adapted to patient condition and prior therapy,
and other factors considered by the managing physician.
As used herein, the term "subject" refers to any animal (i.e.,
vertebrates and invertebrates) including, but not limited to
mammals, including humans. It is not intended that the term be
limited to a particular age or sex. Thus, adult and newborn
subjects, as well as fetuses, whether male or female, are
encompassed by the term.
In preferred embodiments, the antibodies that are used in the
treatment of human disease are human or humanized (CDR-grafted)
versions of antibodies; although murine and chimeric versions of
antibodies can be used. Same species IgG molecules are mostly
preferred to minimize immune responses. This is particularly
important when considering repeat treatments. For humans, a human
or humanized IgG antibody is less likely to generate an anti-IgG
immune response from patients.
In another preferred embodiment, diseases that may be treated using
the antibody complexes include, but are not limited to immune
dysregulation disease and related autoimmune diseases, including
Class III autoimmune diseases such as immune-mediated
thrombocytopenias, such as acute idiopathic thrombocytopenic
purpura and chronic idiopathic thrombocytopenic purpura,
dermatomyositis, Sjogren's syndrome, multiple sclerosis, Sydenham's
chorea, myasthenia gravis, systemic lupus erythematosus, lupus
nephritis, rheumatic fever, polyglandular syndromes, bullous
pemphigoid, diabetes mellitus, Henoch-Schonlein purpura,
post-streptococcal nephritis, erythema nodosum, Takayasu's
arteritis, Addison's disease, rheumatoid arthritis, sarcoidosis,
ulcerative colitis, erythema multiforme, IgA nephropathy,
polyarteritis nodosa, ankylosing spondylitis, Goodpasture's
syndrome, thromboangitis obliterans, Sj gren's syndrome, primary
biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis,
scleroderma, chronic active hepatitis, rheumatoid arthritis,
polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris,
Wegener's granulomatosis, membranous nephropathy, amyotrophic
lateral sclerosis, tabes dorsalis, giant cell
arteritis/polymyalgia, pernicious anemia, rapidly progressive
glomerulonephritis and fibrosing alveolitis, and also juvenile
diabetes, as disclosed in U.S. Provisional Application Ser. No.
60/360,259, filed Mar. 1, 2002 (now expired). Antibodies that may
be of use for combination therapy in these diseases include, but
are not limited to, those reactive with HLA-DR antigens, B-cell and
plasma-cell antigens (e.g., CD19, CD20, CD21, CD22, CD23, CD4, CD5,
CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37,
CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7,
MUC1, Ia, HM1.24, and HLA-DR), IL-6, IL-17. Since many of these
autoimmune diseases are affected by autoantibodies made by aberrant
B-cell populations, depletion of these B-cells is a preferred
method of autoimmune disease therapy. In a preferred embodiment,
the anti-B-cell, anti-T-cell, or anti-macrophage or other such
antibodies of use in the co-treatment of patients with autoimmune
diseases also can be conjugated to result in more effective
therapeutics to control the host responses involved in said
autoimmune diseases, and can be given alone or in combination with
other therapeutic agents, such as TNF inhibitors or anti-IL-6R
antibodies and the like.
In a preferred embodiment, a more effective therapeutic agent can
be provided by using multivalent, multispecific antibodies.
Exemplary bivalent and bispecific antibodies are found in U.S. Pat.
Nos. 7,387,772; 7,300,655; 7,238,785; and 7,282,567, the Examples
section of each of which is incorporated herein by reference. These
multivalent or multispecific antibodies are particularly preferred
in the targeting of disease associated cells which express multiple
antigen targets and even multiple epitopes of the same antigen
target, but which often evade antibody targeting and sufficient
binding for immunotherapy because of insufficient expression or
availability of a single antigen target on the cell. By targeting
multiple antigens or epitopes, said antibodies show a higher
binding and residence time on the target, thus affording a higher
saturation with the drug being targeted in this invention.
Formulation and Administration
Suitable routes of administration of the conjugates include,
without limitation, oral, parenteral, rectal, transmucosal,
intestinal administration, intramuscular, subcutaneous,
intramedullary, intrathecal, direct intraventricular, intravenous,
intravitreal, intraperitoneal, intranasal, or intraocular
injections. The preferred routes of administration are parenteral.
Alternatively, one may administer the compound in a local rather
than systemic manner, for example, via injection of the compound
directly into a solid tumor.
Antibody complexes or immunoconjugates can be formulated according
to known methods to prepare pharmaceutically useful compositions,
whereby the antibody complex or immunoconjugate is combined in a
mixture with a pharmaceutically suitable excipient. Sterile
phosphate-buffered saline is one example of a pharmaceutically
suitable excipient. Other suitable excipients are well-known to
those in the art. See, for example, Ansel et al., PHARMACEUTICAL
DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea &
Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL
SCIENCES, 18th Edition (Mack Publishing Company 1990), and revised
editions thereof.
The antibody complex or immunoconjugate can be formulated for
intravenous administration via, for example, bolus injection or
continuous infusion. Preferably, the antibody of the present
invention is infused over a period of less than about 4 hours, and
more preferably, over a period of less than about 3 hours. For
example, the first 25-50 mg could be infused within 30 minutes,
preferably even 15 min, and the remainder infused over the next 2-3
hrs. Formulations for injection can be presented in unit dosage
form, e.g., in ampoules or in multi-dose containers, with an added
preservative. The compositions can take such forms as suspensions,
solutions or emulsions in oily or aqueous vehicles, and can contain
formulatory agents such as suspending, stabilizing and/or
dispersing agents. Alternatively, the active ingredient can be in
powder form for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
Additional pharmaceutical methods may be employed to control the
duration of action of the antibody complex. Control release
preparations can be prepared through the use of polymers to complex
or adsorb the antibody complex. For example, biocompatible polymers
include matrices of poly(ethylene-co-vinyl acetate) and matrices of
a polyanhydride copolymer of a stearic acid dimer and sebacic acid.
Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of
release of an antibody complex or immunoconjugate from such a
matrix depends upon the molecular weight, the amount of antibody
complex or immunoconjugate within the matrix, and the size of
dispersed particles. Saltzman et al., Biophys. J. 55: 163 (1989);
Sherwood et al., supra. Other solid dosage forms are described in
Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY
SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),
REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing
Company 1990), and revised editions thereof.
Generally, the dosage of an administered antibody complex or
immunoconjugate for humans will vary depending upon such factors as
the patient's age, weight, height, sex, general medical condition
and previous medical history. It may be desirable to provide the
recipient with a dosage that is in the range of from about 1 mg/kg
to 25 mg/kg as a single intravenous infusion, although a lower or
higher dosage also may be administered as circumstances dictate. A
dosage of 1-20 mg/kg for a 70 kg patient, for example, is 70-1,400
mg, or 41-824 mg/m.sup.2 for a 1.7-m patient. The dosage may be
repeated as needed, for example, once per week for 4-10 weeks, once
per week for 8 weeks, or once per week for 4 weeks. It may also be
given less frequently, such as every other week for several months,
or monthly or quarterly for many months, as needed in a maintenance
therapy.
Alternatively, an antibody complex or immunoconjugate may be
administered as one dosage every 2 or 3 weeks, repeated for a total
of at least 3 dosages. Or, twice per week for 4-6 weeks. If the
dosage is lowered to approximately 200-300 mg/m.sup.2 (340 mg per
dosage for a 1.7-m patient, or 4.9 mg/kg for a 70 kg patient), it
may be administered once or even twice weekly for 4 to 10 weeks.
Alternatively, the dosage schedule may be decreased, namely every 2
or 3 weeks for 2-3 months. It has been determined, however, that
even higher doses, such as 20 mg/kg once weekly or once every 2-3
weeks can be administered by slow i.v. infusion, for repeated
dosing cycles. The dosing schedule can optionally be repeated at
other intervals and dosage may be given through various parenteral
routes, with appropriate adjustment of the dose and schedule.
Expression Vectors
Still other embodiments may concern DNA sequences comprising a
nucleic acid encoding an antibody, antibody fragment, toxin or
constituent fusion protein of an antibody complex, such as a
DNL.RTM. construct. Fusion proteins may comprise an antibody or
fragment or toxin attached to, for example, an AD or DDD
moiety.
Various embodiments relate to expression vectors comprising the
coding DNA sequences. The vectors may contain sequences encoding
the light and heavy chain constant regions and the hinge region of
a human immunoglobulin to which may be attached chimeric, humanized
or human variable region sequences. The vectors may additionally
contain promoters that express the encoded protein(s) in a selected
host cell, enhancers and signal or leader sequences. Vectors that
are particularly useful are pdHL2 or GS. More preferably, the light
and heavy chain constant regions and hinge region may be from a
human EU myeloma immunoglobulin, where optionally at least one of
the amino acid in the allotype positions is changed to that found
in a different IgG1 allotype, and wherein optionally amino acid 253
of the heavy chain of EU based on the EU number system may be
replaced with alanine. See Edelman et al., Proc. Natl. Acad. Sci
USA 63: 78-85 (1969). In other embodiments, an IgG1 sequence may be
converted to an IgG4 sequence.
The skilled artisan will realize that methods of genetically
engineering expression constructs and insertion into host cells to
express engineered proteins are well known in the art and a matter
of routine experimentation. Host cells and methods of expression of
cloned antibodies or fragments have been described, for example, in
U.S. Pat. Nos. 7,531,327 and 7,537,930, the Examples section of
each incorporated herein by reference.
Kits
Various embodiments may concern kits containing components suitable
for treating or diagnosing diseased tissue in a patient. Exemplary
kits may contain one or more antibody complexes as described
herein. If the composition containing components for administration
is not formulated for delivery via the alimentary canal, such as by
oral delivery, a device capable of delivering the kit components
through some other route may be included. One type of device, for
applications such as parenteral delivery, is a syringe that is used
to inject the composition into the body of a subject. Inhalation
devices may also be used. In certain embodiments, a therapeutic
agent may be provided in the form of a prefilled syringe or
autoinjection pen containing a sterile, liquid formulation or
lyophilized preparation.
The kit components may be packaged together or separated into two
or more containers. In some embodiments, the containers may be
vials that contain sterile, lyophilized formulations of a
composition that are suitable for reconstitution. A kit may also
contain one or more buffers suitable for reconstitution and/or
dilution of other reagents. Other containers that may be used
include, but are not limited to, a pouch, tray, box, tube, or the
like. Kit components may be packaged and maintained sterilely
within the containers. Another component that can be included is
instructions to a person using a kit for its use.
EXAMPLES
In the working Examples below, the DOCK-AND-LOCK.RTM. (DNL)
technology was used to generate the first bispecific antibody
(bsAb) with high potency to neutralize both TNF-.alpha. and IL-6.
This prototype DNL.RTM. construct, designated cT*-(c6)-(c6),
comprises a chimeric anti-TNF-.alpha. IgG linked at the carboxyl
terminus of each light chain to a pair of dimerized Fab's derived
from a chimeric anti-IL-6 antibody, thus featuring a hexavalent
bsAb capable of blocking 2 and 4 molecules of TNF-.alpha. and IL-6,
respectively, as well as a fully functional Fc. As discussed below,
the exemplary anti-TNF-.alpha./anti-IL-6 bispecific antibody showed
potent activity in in vitro assays designed to test efficacy for
immune diseases such as SLE or RA. However, the person of ordinary
skill will realize that the subject complexes of use are not
limited to the specific DNL.RTM. cT*-(c6)-(c6) complex discussed
below, but more generally encompass bispecific antibodies and/or
antigen-binding antibody fragments with at least one binding site
for IL-6 an at least one binding site for TNF-.alpha..
Example 1. Generation of Neutralizing Mouse Anti-Human IL-6
Monoclonal Antibody
The 2-3B2 mouse monoclonal antibody against human IL-6 was produced
using standard immunological techniques, discussed below, that may
be used to make anti-human IL-6 antibodies in general.
Recombinant human IL-6 (rhIL-6) was obtained from ProSpec-Tany
TechnoGene Ltd. (Rehovot, Israel). Multiple mice were initially
immunized with 30 .mu.g rhIL-6 administered i.p., followed by
booster injections of 30 or 10 .mu.g with or without adjuvant,
according to a standard boosting schedule. Animals were tested for
presence of anti-IL-6 antibodies by ELISA assay using rhIL-6 coated
microtiter plates and serial dilutions of serum. Prior to fusion,
the presence of neutralizing anti-IL-6 antibodies was detected by
the ability to block IL-6 stimulated protein phosphorylation (of
STAT3) using Western blotting (data not shown).
Cells secreting neutralizing anti-IL-6 antibodies were fused with
the P3-X63.Ag8.653 myeloma cell line by PEG mediated cell fusion
using standard techniques to generate antibody-secreting hybridomas
cell lines. The 2-3B2, 4-4F5 and 4-4E6 anti-IL-6 clones were
obtained by selection on HAT medium, cloning and subcloning.
Supernatants from isolated clones containing neutralizing anti-IL-6
were detected by the ability to block IL-6 stimulated STAT3 protein
phosphorylation, determined by Western blotting (FIG. 1). Clones
2-3B2, 4-4E6 and control anti-IL-6 MAb206, but not clone 4-4F5,
were able to block rhIL-6 induced phosphorylation (FIG. 1). Size
exclusion HPLC of antibodies purified by protein A column
chromatography demonstrated the presence of homogeneous antibodies,
which was confirmed by SDS-PAGE (data not shown). Isotyping using
an SBA CLONETYPING.TM. system showed that the anti-IL-6 antibodies
were IgG1/.kappa. murine isotypes.
Binding to human IL-6 was determined Western blotting against
rhIL-6 (FIG. 2). Based on the intensity of labeling using identical
concentrations of antibody, it was determined that the 2-3B2 clone
(FIG. 2A) showed higher affinity for human IL-6 than the 4-4E6
clone (FIG. 2B) and 2-3B2 was selected for production of chimeric
and humanized anti-IL-6 antibodies. Serial dilution demonstrated
that the 2-3B2 antibody was about 100-fold more potent than 4-4E6
for inhibiting the IL-6 induced phosphorylation of STAT3 (FIG.
2A-B). Neither antibody bound to murine IL-6 (not shown).
Example 2. Generation of Neutralizing Mouse Anti-Human TNF-.alpha.
Monoclonal Antibody
Monoclonal antibodies against human TNF-.alpha. were prepared using
standard techniques, as discussed in Example 1 above for IL-6. Mice
were immunized with recombinant human TNF-.alpha. obtained from
ProSpec-Tany TechnoGene Ltd. (Rehovot, Israel). Testing of serum
from immunized mice for anti-TNF-.alpha. antibodies was performed
by ELISA (data not shown).
Neutralizing antibodies were also detected by cytotoxicity assay.
Briefly, WEHI 164 cells (mouse fibrosarcoma) were cultured in RPMI
complete media. Cells were plated at a density of 1.times.10.sup.4
cells/well in 75 .mu.L of medium in 96-well plates and kept in a
37.degree. C. incubator overnight before the assay. On the day of
the assay, sera from the immunized mice were diluted 1:25, 1:125,
1:625, 1:3, 125, 1:15,625, and 1:78,125 in RPMI complete medium
containing 8 .mu.g/mL of actinomycin-D and 0.4 ng/mL of
rhTNF-.alpha.. Twenty-five .mu.L of the diluted sera were added to
the cells in the corresponding wells. The addition of the sera to
the cells made the final dilutions of the sera as 1:100, 1:500,
1:2,500, 1:12,500, 1:62,500, and 1:312,500. The final concentration
of actinomycin-D was 2 .mu.g/mL and rhTNF-.alpha. was 0.1 ng/mL.
Plates were incubated in a 37.degree. C./5% CO2 incubator for 20
hours. After this incubation, 20 .mu.L of MTS reagent was added to
all the wells and the absorbance in each well determined in a plate
reader at 490 nm after two hours. As a negative control, serum from
a naive mouse (not immunized) was diluted in a like manner. One set
of wells was incubated with only actinomycin-D and rhTNF-.alpha. to
determine maximum growth inhibition. Another set of cells remained
untreated (cells grown in media lacking actinomycin-D and
rhTNF-.alpha.). Growth inhibition was measured as percent of
untreated control cell growth.
The results of the cytotoxicity assay are shown in FIG. 3. Serum
from each of the inoculated mice showed the ability to neutralize
rhTNF-.alpha. mediated cytotoxicity. Serum from mouse #3 showed the
greatest ability to inhibit rhTNF-.alpha. mediated
cytotoxicity.
Hybridomas were produced from splenocytes of mice showing the
presence of anti-TNF-.alpha. antibodies by PEG fusion, essentially
as discussed above. Selection of fused hybridomas was performed
using HAT medium. Neutralizing clones 4C9 and 4D3 were obtained
from mouse #3. After further subcloning, antibodies were purified
by chromatography on protein G columns. Purified antibodies were
determined to be homogeneous by size separation HPLC and SDS-PAGE
(data not shown). Isotype analysis, performed as discussed above,
showed that 4C9 was IgG1/.kappa. while 4D3 was IgG2a/.kappa..
The ability of anti-TNF-.alpha. antibodies from clones 4C9D11 and
4D3B11 to neutralize TNF-.alpha.-mediated cytotoxicity was
determined (FIG. 4). WEHI 164 cells were seeded at 1.times.10.sup.4
cells/well into 96-well plates and grown in 200 .mu.L of RPMI
complete medium overnight. On the day of the assay, supernatants
from clones were collected and diluted 1:2. A further 1:5 dilution
was made thereafter. Each dilution was made in RPMI complete medium
containing a final concentration of actinomycin-D at 2 .mu.g/mL and
rhTNF-.alpha. at 0.1 ng/mL. Before addition of the diluted
supernatant, the medium in the plate for WEHI 164 cells growth was
removed, and replaced with the diluted supernatant in the
corresponding wells, 100 .mu.L/well. The plate was incubated for 20
hours at 37.degree. C. in a 5% CO2 incubator. After this
incubation, 20 .mu.L of MTS was added to all the wells and the
absorbance in each well determined in a plate reader at 490 nm
after two hours. As a negative control, supernatant from a clone
which stopped producing antibody (ELISA negative) was diluted in a
like manner. One set of wells was incubated with only actinomycin-D
and rhTNF-.alpha. to determine maximum growth inhibition. Another
set of cells remained untreated (cells grown in media lacking
actinomycin-D and rhTNF-.alpha.). Growth inhibition was measured as
percent of untreated control cell growth. The antibody from clone
4D3B11 was more effective at blocking TNF-.alpha. mediated
cytotoxicity in this assay (FIG. 4).
Antibody binding specificity was determined by Western blotting
against rhTNF-.alpha.. Under reducing conditions, 4D3B11C4 and the
anti-TNF-.alpha. antibody REMICADE.RTM. (infliximab) showed no or
weak binding to human TNF-.alpha., with no binding to human
TNF-.beta. or murine TNF-.alpha. (not shown). Under the same
reducing conditions, antibody 4C9D11G11 showed strong binding to
human TNF-.alpha., with no binding to human TNF-.beta. or murine
TNF-.alpha. (not shown).
Neutralization of rhTNF-.alpha. induced cytotoxicity by
anti-TNF-.alpha. antibodies was determined in a different in vitro
system (FIG. 5). L929 cells (mouse fibroblasts) were seeded at
2.times.10.sup.4 cells/well into 96-well plates and grown in 90
.mu.L of MEM medium overnight (10% horse serum complete medium). On
the following day, the purified antibodies were diluted 1:5 in MEM
medium (containing a final concentration of actinomycin-D at 20
.mu.g/mL and rhTNF-.alpha. at 1 ng/mL) for an antibody
concentration range of 10,000 to 3.2 ng/mL. The antibodies were
pre-incubated with rhTNF-.alpha. at RT for one hour. After this
pre-incubation, 10 .mu.L of the diluted antibodies were then added
to the 90 .mu.L cells in the corresponding wells, that made the
final concentration of the antibodies from 1000 to 0.32 ng/mL, with
a final concentration of actinomycin-D and rhTNF-.alpha. at 2
.mu.g/mL and 0.1 ng/mL, respectively. The plate was incubated for
20 hours at 37.degree. C. Following this incubation, 20 .mu.L of
MTS was added to all the wells and the absorbance in each well
determined in a plate reader at 490 nm after two hours. As a
negative control, an anti-hTNF-.alpha. antibody, 4C3
(non-neutralizing), was diluted in a like manner. REMICADE.RTM.,
the commercial anti-TNF-.alpha. antibody was also diluted in a like
manner as a positive control. One set of wells was incubated with
only actinomycin-D and rhTNF-.alpha. to determine maximum growth
inhibition.
Under these conditions, the 4C9D11G11 antibody (EC.sub.50 11.2
ng/mL) was more effective than 4D3B11C4 (EC.sub.50 22.1 ng/mL) at
inhibiting TNF-.alpha.-induced cytotoxicity (FIG. 5). Neither
monoclonal antibody was as effective as REMICADE.RTM. (EC.sub.50
3.6 ng/mL) (FIG. 5).
An assay was performed for antibody based neutralization of
rhTNF-.alpha.-induced cell surface expression of ICAM-1 (FIG. 6).
ECV-304 cells (a derivative of T24, bladder cancer cell line) were
seeded at 2.times.10.sup.5 cells/well into 6-well plates, grown in
10% FBS Medium 199 for 6 hours for attaching. Varying doses of the
mAbs or REMICADE.RTM. (positive control) were mixed with constant
amounts of rhTNF-.alpha. (10 ng/mL). The mixture of the antibodies
and rhTNF-.alpha. was pre-incubated at 37.degree. C. for two hours,
and then pipetted into the appropriate corresponding wells in
duplicate. Cells were then grown for 72 hours in a 37.degree. C.
incubator. After this incubation, supernatant was removed and cells
were trypsinized and transferred to 15 mL tubes. Cells were washed
with cold PBS/0.5% BSA two times, supernatant was removed and the
cell pellets were re-suspended in the residual wash buffer
(.about.100 .mu.L). An aliquot of 25 .mu.L from the cell suspension
from each sample was then transferred to 4 mL flow tubes. Cells
were Fc-blocked by treatment with 1 .mu.g of human IgG for 15 min
at RT and then incubated with fluorescent-conjugated anti-CD54
reagent for 45 min at 4.degree. C. Cells were then washed with 4 mL
of PBS/5% BSA for two times and re-suspended in 400 .mu.L of PBS
and then subjected to flow-cytometric analysis (FACS). One set of
cells remained untreated as background fluorescent control. Another
set of cells treated with only 10 ng/mL of rhTNF-.alpha. served as
the positive control for obtaining maximum fluorescent (i.e.
maximum ICAM-1 up-regulation).
The 4C9 clone again showed higher neutralizing activity than the
4D3 clone and 4C9 was selected for chimerization.
Example 3. Production of Chimeric Anti-IL-6 Antibody from 2-3B2
Hybridoma
Total RNA was extracted from hybridomas 2-3B2 cells by standard
techniques and mRNA was separated from the total RNA fraction. The
mRNA was used as a template for VH and VK cDNA synthesis, using a
QIAGEN.RTM. OneStep RT-PCR kit. Primers used were as shown below
(restriction sites are underlined).
TABLE-US-00017 Vk1 BACK (PNAS 86:3833-3837,1989) (SEQ ID NO: 90)
GACATTCAGCTGACCCAGTCTCCA CK3'-BH: (Biotechniques 15:286-291, 1993)
(SEQ ID NO: 91) GCCGGATCCTCACTGGATGGTGGGAAGATGGATACA VH1 BACK:
(PNAS 86:3833-3837, 1989) AGGTSMARCTGCAGSAGTCWGG (SEQ ID NO: 92, S
= C/G, M = A/C, R = A/G, W = A/T) CH1-C: (Clinical Cancer Res
5:3095s-3100s, 1999) (SEQ ID NO: 93) AGCTGGGAAGGTGTGCAC
The VH and Vk cDNA sequences were cloned into the pGEMT vector for
sequencing by the Sanger dideoxy technique, using an automated DNA
sequencer. The putative VH (SEQ ID NO:94) and Vk (SEQ ID NO:96)
murine amino sequences are shown in FIG. 7 and FIG. 8. The
locations of the heavy and light chain CDR sequences are proposed,
based on homology with the known heavy and light chain antibody
sequences of B34871 (SEQ ID NO:95) and AAB53778.1 (SEQ ID NO:97),
respectively, from the NCBI protein sequence database. The
indicated 2-3B2 heavy chain CDR sequences are CDR1 (GFTFSRFGMH, SEQ
ID NO:107), CDR2 (YIGRGSSTIYYADTVKG, SEQ ID NO:108) and CDR3
(SNWDGAMDY, SEQ ID NO:109). The 2-3B2 light chain CDR sequences are
CDR1 (RASGNIHNFLA, SEQ ID NO:110), CDR2 (NAETLAD, SEQ ID NO:111)
and CDR3 (QHFWSTPWT, SEQ ID NO:112).
The VH and VK sequences from the 2-3B2 anti-IL-6 antibody and the
VH and VK sequences from the 4C9 anti-TNF-.alpha. antibody were
used to make a cIL6/TNF.alpha. DVD (dual variable domain) antibody
construct. (See, e.g., Wu et al., 2009, MAbs 1:339-47.) The
resulting bispecific DVD construct was compared with the parent
2-3B2 anti-IL-6 antibody for the ability to inhibit IL-6 induced
phosphorylation of STAT3 on HT-29 cells (FIG. 9). As shown in FIG.
9, the DVD construct showed the same efficacy as the parent
anti-IL-6 antibody for inhibition of IL-6 mediated
phosphorylation.
The sequences for restriction sites and leader peptides for cloning
into vector pdHL2 were added to the VH and VK sequences of 2-3B2.
The complete sequences were synthesized commercially (GenScript,
Piscataway, N.J.). The 2-3B2-VH-pUC57 and 2-3B2-VK-pUC57 vectors
were produced by incorporating the VH sequence as a XhoI-HindIII
insert and the VK sequence as a XbaI-BamH1 insert into
corresponding sites in pUC57. A vector expressing chimeric 2-3B2
antibody was produced starting with the hA20-pdHL2-IgG vector (see,
e.g., Goldenberg et al., 2002, Blood 100:11 Abstract 2260). The
hA20-VH sequence was replaced with cIL6-VH and the hA20-VK sequence
was replaced with cIL6-VK by restriction enzyme digestion and
ligation. The resulting chimeric 2-3B2 antibody comprised the
murine VH and VK sequences of 2-3B2, attached to human antibody
constant region sequences. After transfection, screening and
antibody purification on a protein A column, a chimeric anti-IL6
clone 1B5 (c-IL6-1B5) was obtained as a homogeneous antibody
preparation, as confirmed by HPLC and SDS-PAGE (not shown). The
final clone is identified as 1B5A9.
Example 4. Production of Chimeric Anti-TNF-.alpha. Antibody from
4C9 Hybridoma
Total RNA was extracted from hybridomas 4C9 cells by standard
techniques and mRNA was separated from the total RNA fraction. The
mRNA was used as a template for VH and VK cDNA synthesis, using a
PHUSION.RTM. High Fidelity PCR kit (Thermo Scientific, Pittsburgh,
Pa.). Primers used were as disclosed in Example 3 above.
The VH and Vk cDNA sequences were cloned into the pGEMT vector for
sequencing by the Sanger dideoxy technique, using an automated DNA
sequencer. The putative VH (SEQ ID NO:98) and Vk (SEQ ID NO:100)
murine amino sequences are shown in FIG. 10 and FIG. 11. The
locations of the heavy and light chain CDR sequences are proposed,
based on homology with the known heavy and light chain antibody
sequences of AAS66033.1 (SEQ ID NO:99) and AAS66032.1 (SEQ ID
NO:101), respectively, from the NCBI protein sequence database. The
indicated 4C9 heavy chain CDR sequences are CDR1 (GFWN, SEQ ID
NO:113), CDR2 (YISYSGRTYYNPSLKS, SEQ ID NO:114) and CDR3 (DANYVLDY,
SEQ ID NO:115). The 4C9 light chain CDR sequences are CDR1
(KSSQSLLNSSTQKNYLA, SEQ ID NO:116), CDR2 (FASARES, SEQ ID NO:117)
and CDR3 (QQHYRTPFT, SEQ ID NO:118).
An optimized DNA sequence encoding the TNF-.alpha. VH, also
comprising a 5' leader sequence and 3' flanking sequence, was
designed as shown in SEQ ID NO:102 below and cloned into pdHL2. The
optimized 4C9-VH sequence is underlined. The DNA sequence was
synthesized by GenScript (Piscataway, N.J.).
TABLE-US-00018 (SEQ ID NO: 102)
CTCGAGCACACAGGACCTCACCATGGGATGGAGCTGTATCATCCTCTTCT
TGGTAGCAACAGCTACAGGTAAGGGGCTCACAGTAGCAGGCTTGAGGTCT
GGACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAG
GTGTCCACTCCGTGCAGCTGCAGGAGAGCGGACCCTCCCTGGTGAAGCCT
AGTCAGACCCTGAGCCTGACATGCTCCGTGACTGGGGACTCTATCACCAG
TGGCTTCTGGAACTGGATTCGGAAGTTCCCAGGAAACAAGTTTGAATACA
TGGGATATATCTCTTACAGTGGGCGCACATACTATAACCCCAGCCTGAAG
TCCAGGCTGTCTATTACAAGAGACACTTCTAAAAACCAGTTTTATCTGCA
GCTGAACAGCGTGACTGCCGAGGATACTGCTACCTACTATTGTGCCAGGG
ACGCTAATTATGTGCTGGATTACTGGGGCCAGGGAACCACACTGACCGTG
AGCTCCGGTGAGTCCTTACAACCTCTCTCTTCTATTCAGCTTAAATAGAT
TTTACTGCATTTGTTGGGGGGGAAATGTGTGTATCTGAATTTCAGGTCAT
GAAGGACTAGGGACACCTTGGGAGTCAGAAAGGGTCATTGGGAAGCTT
An optimized DNA sequence encoding the TNF-.alpha. VK, also
comprising a 5' leader sequence and 3' flanking sequence, was
designed as shown in SEQ ID NO:103 below and cloned into pdHL2. The
optimized 4C9-VK sequence is underlined. The DNA sequence was
synthesized by GenScript (Piscataway, N.J.).
TABLE-US-00019 (SEQ ID NO: 103)
TCTAGACACAGGACCTCACCATGGGATGGAGCTGTATCATCCTCTTCTTG
GTAGCAACAGCTACAGGTAAGGGGCTCACAGTAGCAGGCTTGAGGTCTGG
ACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGT
GTCCACTCCGACATCCAGCTGACCCAGAGCCCCAGCTCCCTGGCTATGTC
CGTGGGACAGAAGGTGACAATGAACTGCAAATCTAGTCAGTCTCTGCTGA
ACAGCTCCACTCAGAAGAATTACCTGGCTTGGTTCCAGCAGAAGCCCGGG
CAGAGTCCTAAACTGCTGGTGTATTTTGCCTCTGCTAGGGAGAGTGGCGT
GCCAGACAGATTCATCGGCAGCGGCAGCGGGACCGATTTTACCCTGACAA
TTTCTAGTGTGCAGGCCGAGGACCTGGCTGATTACTTCTGTCAGCAGCAC
TATCGGACTCCCTTCACCTTTGGCTCCGGAACAAAGCTGGAGATCAAGCG
TGAGTAGAATTTAAACTTTGCTTCCTCAGTTGGATCC
The VH and VK coding sequences were inserted into pUC57 and then
pdHL2 for expression of the chimeric 4C9 antibody as discussed in
Example 3 above. The chimeric 4C9 was produced by transfection of
pdHL2, screening for transfectants and antibody purification on a
protein A column. The selected clone was designated 6A9. The
purified antibody was determined to be homogeneous by HPLC and
SDS-PAGE (not shown). A binding affinity assay for chimeric
anti-TNF-.alpha. showed a dissociation constant (K.sub.D) of 4.13
e.sup.-11 (not shown).
Example 5. Construction of CH1-DDD2-cFab-anti-IL-6-pGSHL
The hLL2-Fab-DDD2-pGSHL#2 plasmid (see, e.g., WO2013181087A2; Rossi
et al., 2009, Blood 113:6161-71; U.S. Patent Publ. Nos.
20130323204, 20140212425) was used as a starting material for
production of a DDD2 conjugated Fab anti-IL-6 antibody fragment.
The hLL2-DDD2 plasmid was digested with XbaI/XhoI and the 6577 bp
vector was isolated. cIL6-pdHL2 (Example 3) was digested with
XbaI/XhoI and the 2604 bp cIL6 coding insert was isolated. The two
were ligated to form the 9182 bp Vk-cIL6-Fab-DDD2-pGSHL vector.
After screening by PstI digestion and electrophoresis, the 9182 bp
vector was digested with XhoI/Hind3/Alkaline phosphatase and an
8536 vector was isolated. The cIL6-pdHL2 vector, comprising a 648
bp cIL6-VH coding insert was digested with XhoI/Hind3. The 648 bp
VH encoding insert was ligated with the 8536 bp vector and VK
insert to generate C.sub.H1-DDD2-cFab-anti-IL-6-pGSHL. The final
construct was then transfected, clones were picked and purified by
Kappa-select (GE Healthcare Life Sciences, Piscataway, N.J.). The
purified antibody product of C.sub.H1-DDD2-cFab-anti-IL-6 appeared
homogeneous on HPLC and SDS-PAGE (not shown). The DDD2-derivatized
cIL6-Fab showed equivalent activity to the underivatized cIL6 or an
hR1-(IL6).sub.4 construct when assayed for inhibition of IL-6
induced STAT3 phosphorylation (not shown).
Example 6. Construction of
C.sub.K-AD2-cIgG-anti-TNF-.alpha.-pdHL2
The Ck-AD2-IgG-hA20-pdHL2 plasmid (see, e.g., WO201262583A1; Chang
et al., 2012, PLoS ONE 7(8): e44235; U.S. Patent Publ. Nos.
20130323204, 20070140966) was used as a starting material for
production of an AD2 conjugated IgG anti-TNF-.alpha. antibody. The
Ck-AD2-hA20 plasmid was digested with BamHI/XhoI to obtain the
Ck-AD2 coding portion. Plasmid cIgG-anti-TNF-.alpha.-pdHL2 (Example
4) was digested with BamHI/XhoI to obtain
.DELTA.C.sub.K-cIgG-anti-TNF-.alpha.-pdHL2. The two were ligated to
form Ck-AD2-cIgG-anti-TNF-.alpha.-pdHL2 (see FIG. 12). The
C.sub.K-AD2-cIgG-anti-TNF-.alpha.-pdHL2 vector was used to
transform DHF.alpha. competent cells. Colonies were picked and
purified by mini-Prep. Plasmid DNA was analyzed by restriction
endonuclease digestion and agarose gel electrophoresis (not shown).
The plasmid DNA was purified by Maxi-Prep and the insert was DNA
sequenced. The DNA sequences encoding cTNF-.alpha.-VH, AD2 and
cTNF-.alpha.-VK are shown in SEQ ID NOs 104-106 below.
TABLE-US-00020 cTNF-.alpha.-VH (SEQ ID NO: 104)
GTGCAGCTGCAGGAGAGCGGACCCTCCCTGGTGAAGCCTAGTCAGACCCT
GAGCCTGACATGCTCCGTGACTGGGGACTCTATCACCAGTGGCTTCTGGA
ACTGGATTCGGAAGTTCCCAGGAAACAAGTTTGAATACATGGGATATATC
TCTTACAGTGGGCGCACATACTATAACCCCAGCCTGAAGTCCAGGCTGTC
TATTACAAGAGACACTTCTAAAAACCAGTTTTATCTGCAGCTGAACAGCG
TGACTGCCGAGGATACTGCTACCTACTATTGTGCCAGGGACGCTAATTAT
GTGCTGGATTACTGGGGCCAGGGAACCACACTGACCGTGAGCTCC AD2 (SEQ ID NO: 105)
TGTGGCCAGATCGAGTACCTGGCCAAGCAGATCGTGGACAACGCCATCCA GCAGGCCGGGTGC
cTNF-.alpha.-VK (SEQ ID NO: 106)
GACATCCAGCTGACCCAGAGCCCCAGCTCCCTGGCTATGTCCGTGGGACA
GAAGGTGACAATGAACTGCAAATCTAGTCAGTCTCTGCTGAACAGCTCCA
CTCAGAAGAATTACCTGGCTTGGTTCCAGCAGAAGCCCGGGCAGAGTCCT
AAACTGCTGGTGTATTTTGCCTCTGCTAGGGAGAGTGGCGTGCCAGACAG
ATTCATCGGCAGCGGCAGCGGGACCGATTTTACCCTGACAATTTCTAGTG
TGCAGGCCGAGGACCTGGCTGATTACTTCTGTCAGCAGCACTATCGGACT
CCCTTCACCTTTGGCTCCGGAACAAAGCTGGAGATCAAGCGTGAGTAGAA
TTTAAACTTTGCT
After transfection, screening, expression and antibody
purification, clone 4A5 encoding C.sub.K-AD2-cIgG-4A5 was
obtained.
Example 7. Construction of cT*-(c6)-(c6) Anti-IL-6/Anti-TNF-.alpha.
Bispecific DNL.RTM. Complex
The Ck-AD2-cIgG-4A5 and C.sub.H1-DDD2-cFab-anti-IL-6 fusion
proteins were used to make a DOCK-AND-LOCK.RTM. (DNL).RTM. complex,
using techniques disclosed herein and in issued U.S. Pat. Nos.
7,550,143; 7,521,056; 7,534,866; 7,527,787; 7,666,400; 7,858,070;
7,871,622; 7,906,121; 7,906,118; 8,163,291; 7,901,680; 7,981,398;
8,003,111; 8,034,352; 8,562,988; 8,211,440; 8,491,914; 8,282,934;
8,246,960; 8,349,332; 8,277,817; 8,158,129; 8,475,794; 8,597,659;
8,481,041; 8,435,540 and 8,551,480, the Examples section of each
incorporated herein by reference.
The intact DNL.RTM. complex was formed by mixing the AD2 and DDD2
components together under reducing conditions and allowing the
complementary sequences on the DDD moiety to form a dimer that
binds to the AD moiety. Twenty five mg of C.sub.K-AD2-cIgG-4A5 was
mixed with 50 mg of C.sub.H1-DDD2-cFab-anti-IL-6. A 1/10 volume of
1M Tris, pH 7.5, 1 mM EDTA, 2 mM reduced glutathione was added to
the reaction and the proteins were reduced overnight at room
temperature. The complexes were then oxidized with 4 mM oxidized
glutathione at room temperature for 3 hours to form disulfide bonds
between the AD2 and DDD2 moieties to stabilize the complex.
Chromatography of the complex on a MABSELECT.TM. column was
performed. After loading, the column was washed with 0.04M PBS, pH
7.4+1 mM oxidized glutathione, followed by a PBS wash and elution
with 0.1M citrate (pH 3.5). The elution volume was 25 ml (2.5 ml of
3M Tris, pH 8.6+22.5 ml of eluate). The concentration measured by
OD280 was 2.3 mg/ml (57.5 mg total).
The product was dialyzed against two 5-L changes of 0.04M PBS, pH
7.4. The final concentration by OD280 was 1.8 mg/ml (52.5 mg
total). Purified complex was analyzed by SE-HPLC, which confirmed
the presence of cT*-(c6)-(c6) as an apparently homogeneous peak
(not shown). The results were confirmed by SDS-PAGE. The activity
of the purified cT*-(c6)-(c6) bispecific antibody complex was then
examined.
The cT*-(c6)-(c6) complex showed greater activity than the
Fab-DDD2-cIL-6 protein for inhibiting IL-6 induced phosphorylation
of STAT3 (FIG. 13). HT-29 cells were seeded at 2.times.10.sup.6
cells/well in 6-well plates, grown overnight. The indicated
antibodies were pre-incubated with hIL6 at 37.degree. C. for 1
hour. Then media containing rhIL-6 alone or in combination with
antibodies was added to the HT-29 cells for 30 min at 37.degree. C.
After the incubation, the supernatant was removed, cells were
washed and lysed.
Two SDS-PAGE gels were run, transferred to nitrocellulose
membranes. Membranes were cut at 60 KDa, the upper portions was
probed with either anti-p-STAT3 or anti-t-STAT3 (FIG. 13). The
lower portions were probed with b-actin for loading control (FIG.
13). This assay showed that TNF-(IL6)-(IL6) neutralized IL-6 with
similar or greater potency compared to anti-IL-6 Fab-DDD2.
FIG. 14 shows that the cT*-(c6)-(c6) complex was able to neutralize
natural IL-6 induced phosphorylation of STAT3 in HT-29 cells. HT-29
cells were seeded at 2.times.10.sup.6 cells/well in 6-well plates
and grown overnight. TNF-(IL6)-(IL6) or chimeric anti-IL6 1B5A9 was
pre-incubated with the supernatant containing 10 ng/mL of natural
IL6 released from collagen Type II stimulated RA patient PBMCs at
37.degree. C. for 1 hour. At the end of the incubation, the
supernatant containing natural IL-6 alone or in combination with
antibodies was added to the HT-29 cells for 30 min at 37.degree. C.
After incubation, the supernatant was removed and cells were washed
and lysed. Two SDS-PAGE gels were run, transferred to
nitrocellulose membranes. Membranes were cut at 60 KDa, the upper
portion was probed with either anti-p-STAT3 or anti-t-STAT3 (FIG.
14). The lower portions were probed with b-actin for loading
control (FIG. 14).
The ability to neutralize TNF-.alpha. induced cell death was also
examined for cT*-(c6)-(c6) compared to other anti-TNF-.alpha.
antibody constructs (FIG. 15). In the presence of 2 .mu.g/mL of
actinomycin-D, recombinant human TNF-.alpha. at 0.1 ng/mL induced
about 70% cell death in L929 cells. As shown, the
TNF-.alpha.-IL6-IL6, chimeric anti-TNF-.alpha. clone 6A9 and
Ck-AD2-cTNF-.alpha.-IgG clone 4A5 were able to neutralize the
activity of rhTNF-.alpha., and inhibit cell death in a
dose-response manner (FIG. 15), just like their parent antibody
4C9.
TNF-.alpha.-(IL6)-(IL6) and Ck-AD2-cTNF-.alpha. were also able to
neutralize cell death of L929 cells induced by natural human
TNF-.alpha. (released from RA PBMCs) (FIG. 16). Upon stimulation by
type II collagen for 5 days, natural human TNF-.alpha. is released
from the cultured PBMCs isolated from a rheumatoid arthritis
patient (S22). In the presence of 2 .mu.g/mL of actinomycin-D,
TNF-.alpha. at 0.1 ng/mL induced about 76% cell death. As shown in
FIG. 16, TNF-.alpha.-IL6-IL6 and Ck-AD2-cTNF-.alpha.-IgG clone 4A5
were able to neutralize the activity of the natural human
TNF-.alpha., and inhibit cell death in a dose-response manner.
The ability of cT*-(c6)-(c6) to bind to IL-6 or TNF-.alpha. from
rat, monkey or human was determined by ELISA. The results are
summarized in FIG. 17, which shows that the affinity of
cT*-(c6)-(c6) for IL-6 or TNF-.alpha. from different species was
approximately the same as the individual antibodies, and that the
antibodies showed approximately similar dissociation constants for
human, Cynomolgus monkey and canine antigens.
As can be seen in FIG. 18, STAT3 plays a central role in both
TNF-.alpha. and IL-6 mediated pathways and disease processes and
inhibition of STAT3 phosphorylation induced by TNF-.alpha. or IL-6
is a reasonable surrogate to determine the efficacy of
anti-TNF-.alpha. or anti-IL-6 antibody complexes as moderators of
such disease processes. Because TNF-.alpha. and IL-6 play
pathogenic roles in the development of a variety of autoimmune,
immune dysfunction or inflammatory diseases, including but not
limited to systemic lupus erythematosus, rheumatoid arthritis,
inflammatory bowel disease, type II diabetes, obesity,
atherosclerosis and cachexia related to cancer, the presence
results show that bispecific anti-IL-6/anti-TNF-.alpha. antibodies,
such as cT*-(c6)-(c6), are of use for treatment of such
TNF-.alpha./IL-6 mediated diseases or conditions.
SEQUENCE LISTINGS
1
118144PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu
Leu Leu Gln Gly Tyr1 5 10 15Thr Val Glu Val Leu Arg Gln Gln Pro Pro
Asp Leu Val Glu Phe Ala 20 25 30Val Glu Tyr Phe Thr Arg Leu Arg Glu
Ala Arg Ala 35 40245PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 2Cys Gly His Ile Gln Ile Pro Pro Gly
Leu Thr Glu Leu Leu Gln Gly1 5 10 15Tyr Thr Val Glu Val Leu Arg Gln
Gln Pro Pro Asp Leu Val Glu Phe 20 25 30Ala Val Glu Tyr Phe Thr Arg
Leu Arg Glu Ala Arg Ala 35 40 45317PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 3Gln
Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn Ala Ile Gln Gln1 5 10
15Ala421PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Cys Gly Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val
Asp Asn Ala Ile1 5 10 15Gln Gln Ala Gly Cys 20550PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
5Ser Leu Arg Glu Cys Glu Leu Tyr Val Gln Lys His Asn Ile Gln Ala1 5
10 15Leu Leu Lys Asp Ser Ile Val Gln Leu Cys Thr Ala Arg Pro Glu
Arg 20 25 30Pro Met Ala Phe Leu Arg Glu Tyr Phe Glu Arg Leu Glu Lys
Glu Glu 35 40 45Ala Lys 50655PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 6Met Ser Cys Gly Gly Ser
Leu Arg Glu Cys Glu Leu Tyr Val Gln Lys1 5 10 15His Asn Ile Gln Ala
Leu Leu Lys Asp Ser Ile Val Gln Leu Cys Thr 20 25 30Ala Arg Pro Glu
Arg Pro Met Ala Phe Leu Arg Glu Tyr Phe Glu Arg 35 40 45Leu Glu Lys
Glu Glu Ala Lys 50 55723PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 7Cys Gly Phe Glu Glu Leu Ala
Trp Lys Ile Ala Lys Met Ile Trp Ser1 5 10 15Asp Val Phe Gln Gln Gly
Cys 20851PRTHomo sapiens 8Ser Leu Arg Glu Cys Glu Leu Tyr Val Gln
Lys His Asn Ile Gln Ala1 5 10 15Leu Leu Lys Asp Val Ser Ile Val Gln
Leu Cys Thr Ala Arg Pro Glu 20 25 30Arg Pro Met Ala Phe Leu Arg Glu
Tyr Phe Glu Lys Leu Glu Lys Glu 35 40 45Glu Ala Lys 50954PRTHomo
sapiens 9Ser Leu Lys Gly Cys Glu Leu Tyr Val Gln Leu His Gly Ile
Gln Gln1 5 10 15Val Leu Lys Asp Cys Ile Val His Leu Cys Ile Ser Lys
Pro Glu Arg 20 25 30Pro Met Lys Phe Leu Arg Glu His Phe Glu Lys Leu
Glu Lys Glu Glu 35 40 45Asn Arg Gln Ile Leu Ala 501044PRTHomo
sapiens 10Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu Gln
Gly Tyr1 5 10 15Thr Val Glu Val Gly Gln Gln Pro Pro Asp Leu Val Asp
Phe Ala Val 20 25 30Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Arg Gln
35 401144PRTHomo sapiens 11Ser Ile Glu Ile Pro Ala Gly Leu Thr Glu
Leu Leu Gln Gly Phe Thr1 5 10 15Val Glu Val Leu Arg His Gln Pro Ala
Asp Leu Leu Glu Phe Ala Leu 20 25 30Gln His Phe Thr Arg Leu Gln Gln
Glu Asn Glu Arg 35 401244PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 12Thr His Ile Gln Ile Pro
Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr1 5 10 15Thr Val Glu Val Leu
Arg Gln Gln Pro Pro Asp Leu Val Glu Phe Ala 20 25 30Val Glu Tyr Phe
Thr Arg Leu Arg Glu Ala Arg Ala 35 401344PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
13Ser Lys Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr1
5 10 15Thr Val Glu Val Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe
Ala 20 25 30Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35
401444PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 14Ser Arg Ile Gln Ile Pro Pro Gly Leu Thr Glu
Leu Leu Gln Gly Tyr1 5 10 15Thr Val Glu Val Leu Arg Gln Gln Pro Pro
Asp Leu Val Glu Phe Ala 20 25 30Val Glu Tyr Phe Thr Arg Leu Arg Glu
Ala Arg Ala 35 401544PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 15Ser His Ile Asn Ile Pro
Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr1 5 10 15Thr Val Glu Val Leu
Arg Gln Gln Pro Pro Asp Leu Val Glu Phe Ala 20 25 30Val Glu Tyr Phe
Thr Arg Leu Arg Glu Ala Arg Ala 35 401644PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
16Ser His Ile Gln Ile Pro Pro Ala Leu Thr Glu Leu Leu Gln Gly Tyr1
5 10 15Thr Val Glu Val Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe
Ala 20 25 30Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35
401744PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 17Ser His Ile Gln Ile Pro Pro Gly Leu Ser Glu
Leu Leu Gln Gly Tyr1 5 10 15Thr Val Glu Val Leu Arg Gln Gln Pro Pro
Asp Leu Val Glu Phe Ala 20 25 30Val Glu Tyr Phe Thr Arg Leu Arg Glu
Ala Arg Ala 35 401844PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 18Ser His Ile Gln Ile Pro
Pro Gly Leu Thr Asp Leu Leu Gln Gly Tyr1 5 10 15Thr Val Glu Val Leu
Arg Gln Gln Pro Pro Asp Leu Val Glu Phe Ala 20 25 30Val Glu Tyr Phe
Thr Arg Leu Arg Glu Ala Arg Ala 35 401944PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
19Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu Asn Gly Tyr1
5 10 15Thr Val Glu Val Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe
Ala 20 25 30Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35
402044PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 20Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu
Leu Leu Gln Ala Tyr1 5 10 15Thr Val Glu Val Leu Arg Gln Gln Pro Pro
Asp Leu Val Glu Phe Ala 20 25 30Val Glu Tyr Phe Thr Arg Leu Arg Glu
Ala Arg Ala 35 402144PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 21Ser His Ile Gln Ile Pro
Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr1 5 10 15Ser Val Glu Val Leu
Arg Gln Gln Pro Pro Asp Leu Val Glu Phe Ala 20 25 30Val Glu Tyr Phe
Thr Arg Leu Arg Glu Ala Arg Ala 35 402244PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
22Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr1
5 10 15Thr Val Asp Val Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe
Ala 20 25 30Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35
402344PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 23Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu
Leu Leu Gln Gly Tyr1 5 10 15Thr Val Glu Val Leu Lys Gln Gln Pro Pro
Asp Leu Val Glu Phe Ala 20 25 30Val Glu Tyr Phe Thr Arg Leu Arg Glu
Ala Arg Ala 35 402444PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 24Ser His Ile Gln Ile Pro
Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr1 5 10 15Thr Val Glu Val Leu
Arg Asn Gln Pro Pro Asp Leu Val Glu Phe Ala 20 25 30Val Glu Tyr Phe
Thr Arg Leu Arg Glu Ala Arg Ala 35 402544PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
25Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr1
5 10 15Thr Val Glu Val Leu Arg Gln Asn Pro Pro Asp Leu Val Glu Phe
Ala 20 25 30Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35
402644PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 26Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu
Leu Leu Gln Gly Tyr1 5 10 15Thr Val Glu Val Leu Arg Gln Gln Pro Pro
Glu Leu Val Glu Phe Ala 20 25 30Val Glu Tyr Phe Thr Arg Leu Arg Glu
Ala Arg Ala 35 402744PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 27Ser His Ile Gln Ile Pro
Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr1 5 10 15Thr Val Glu Val Leu
Arg Gln Gln Pro Pro Asp Leu Val Asp Phe Ala 20 25 30Val Glu Tyr Phe
Thr Arg Leu Arg Glu Ala Arg Ala 35 402844PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
28Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr1
5 10 15Thr Val Glu Val Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe
Leu 20 25 30Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35
402944PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 29Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu
Leu Leu Gln Gly Tyr1 5 10 15Thr Val Glu Val Leu Arg Gln Gln Pro Pro
Asp Leu Val Glu Phe Ile 20 25 30Val Glu Tyr Phe Thr Arg Leu Arg Glu
Ala Arg Ala 35 403044PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 30Ser His Ile Gln Ile Pro
Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr1 5 10 15Thr Val Glu Val Leu
Arg Gln Gln Pro Pro Asp Leu Val Glu Phe Val 20 25 30Val Glu Tyr Phe
Thr Arg Leu Arg Glu Ala Arg Ala 35 403144PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
31Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr1
5 10 15Thr Val Glu Val Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe
Ala 20 25 30Val Asp Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35
403217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 32Asn Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn
Ala Ile Gln Gln1 5 10 15Ala3317PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 33Gln Leu Glu Tyr Leu Ala Lys
Gln Ile Val Asp Asn Ala Ile Gln Gln1 5 10 15Ala3417PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 34Gln
Val Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn Ala Ile Gln Gln1 5 10
15Ala3517PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 35Gln Ile Asp Tyr Leu Ala Lys Gln Ile Val Asp Asn
Ala Ile Gln Gln1 5 10 15Ala3617PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 36Gln Ile Glu Phe Leu Ala Lys
Gln Ile Val Asp Asn Ala Ile Gln Gln1 5 10 15Ala3717PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 37Gln
Ile Glu Thr Leu Ala Lys Gln Ile Val Asp Asn Ala Ile Gln Gln1 5 10
15Ala3817PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 38Gln Ile Glu Ser Leu Ala Lys Gln Ile Val Asp Asn
Ala Ile Gln Gln1 5 10 15Ala3917PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 39Gln Ile Glu Tyr Ile Ala Lys
Gln Ile Val Asp Asn Ala Ile Gln Gln1 5 10 15Ala4017PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 40Gln
Ile Glu Tyr Val Ala Lys Gln Ile Val Asp Asn Ala Ile Gln Gln1 5 10
15Ala4117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 41Gln Ile Glu Tyr Leu Ala Arg Gln Ile Val Asp Asn
Ala Ile Gln Gln1 5 10 15Ala4217PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 42Gln Ile Glu Tyr Leu Ala Lys
Asn Ile Val Asp Asn Ala Ile Gln Gln1 5 10 15Ala4317PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 43Gln
Ile Glu Tyr Leu Ala Lys Gln Ile Val Glu Asn Ala Ile Gln Gln1 5 10
15Ala4417PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 44Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Gln
Ala Ile Gln Gln1 5 10 15Ala4517PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 45Gln Ile Glu Tyr Leu Ala Lys
Gln Ile Val Asp Asn Ala Ile Asn Gln1 5 10 15Ala4617PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 46Gln
Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn Ala Ile Gln Asn1 5 10
15Ala4717PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 47Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn
Ala Ile Gln Gln1 5 10 15Leu4817PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 48Gln Ile Glu Tyr Leu Ala Lys
Gln Ile Val Asp Asn Ala Ile Gln Gln1 5 10 15Ile4917PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 49Gln
Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn Ala Ile Gln Gln1 5 10
15Val5017PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 50Gln Ile Glu Tyr Val Ala Lys Gln Ile Val Asp Tyr
Ala Ile His Gln1 5 10 15Ala5117PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 51Gln Ile Glu Tyr Lys Ala Lys
Gln Ile Val Asp His Ala Ile His Gln1 5 10 15Ala5217PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 52Gln
Ile Glu Tyr His Ala Lys Gln Ile Val Asp His Ala Ile His Gln1 5 10
15Ala5317PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 53Gln Ile Glu Tyr Val Ala Lys Gln Ile Val Asp His
Ala Ile His Gln1 5 10 15Ala5418PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 54Pro Leu Glu Tyr Gln Ala Gly
Leu Leu Val Gln Asn Ala Ile Gln Gln1 5 10 15Ala
Ile5518PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 55Leu Leu Ile Glu Thr Ala Ser Ser Leu Val Lys Asn
Ala Ile Gln Leu1 5 10 15Ser Ile5618PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 56Leu
Ile Glu Glu Ala Ala Ser Arg Ile Val Asp Ala Val Ile Glu Gln1 5 10
15Val Lys5718PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 57Ala Leu Tyr Gln Phe Ala Asp Arg Phe
Ser Glu Leu Val Ile Ser Glu1 5 10 15Ala Leu5817PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 58Leu
Glu Gln Val Ala Asn Gln Leu Ala Asp Gln Ile Ile Lys Glu Ala1 5 10
15Thr5917PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 59Phe Glu Glu Leu Ala Trp Lys Ile Ala Lys Met
Ile
Trp Ser Asp Val1 5 10 15Phe6018PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 60Glu Leu Val Arg Leu Ser Lys
Arg Leu Val Glu Asn Ala Val Leu Lys1 5 10 15Ala
Val6118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 61Thr Ala Glu Glu Val Ser Ala Arg Ile Val Gln Val
Val Thr Ala Glu1 5 10 15Ala Val6218PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 62Gln
Ile Lys Gln Ala Ala Phe Gln Leu Ile Ser Gln Val Ile Leu Glu1 5 10
15Ala Thr6316PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 63Leu Ala Trp Lys Ile Ala Lys Met Ile
Val Ser Asp Val Met Gln Gln1 5 10 156424PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 64Asp
Leu Ile Glu Glu Ala Ala Ser Arg Ile Val Asp Ala Val Ile Glu1 5 10
15Gln Val Lys Ala Ala Gly Ala Tyr 206518PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 65Leu
Glu Gln Tyr Ala Asn Gln Leu Ala Asp Gln Ile Ile Lys Glu Ala1 5 10
15Thr Glu6620PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 66Phe Glu Glu Leu Ala Trp Lys Ile Ala
Lys Met Ile Trp Ser Asp Val1 5 10 15Phe Gln Gln Cys
206717PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 67Gln Ile Glu Tyr Leu Ala Lys Gln Ile Pro Asp Asn
Ala Ile Gln Gln1 5 10 15Ala6825PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 68Lys Gly Ala Asp Leu Ile Glu
Glu Ala Ala Ser Arg Ile Val Asp Ala1 5 10 15Val Ile Glu Gln Val Lys
Ala Ala Gly 20 256925PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 69Lys Gly Ala Asp Leu Ile Glu
Glu Ala Ala Ser Arg Ile Pro Asp Ala1 5 10 15Pro Ile Glu Gln Val Lys
Ala Ala Gly 20 257025PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 70Pro Glu Asp Ala Glu Leu Val
Arg Leu Ser Lys Arg Leu Val Glu Asn1 5 10 15Ala Val Leu Lys Ala Val
Gln Gln Tyr 20 257125PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 71Pro Glu Asp Ala Glu Leu Val
Arg Thr Ser Lys Arg Leu Val Glu Asn1 5 10 15Ala Val Leu Lys Ala Val
Gln Gln Tyr 20 257225PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 72Pro Glu Asp Ala Glu Leu Val
Arg Leu Ser Lys Arg Asp Val Glu Asn1 5 10 15Ala Val Leu Lys Ala Val
Gln Gln Tyr 20 257325PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 73Pro Glu Asp Ala Glu Leu Val
Arg Leu Ser Lys Arg Leu Pro Glu Asn1 5 10 15Ala Val Leu Lys Ala Val
Gln Gln Tyr 20 257425PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 74Pro Glu Asp Ala Glu Leu Val
Arg Leu Ser Lys Arg Leu Pro Glu Asn1 5 10 15Ala Pro Leu Lys Ala Val
Gln Gln Tyr 20 257525PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 75Pro Glu Asp Ala Glu Leu Val
Arg Leu Ser Lys Arg Leu Val Glu Asn1 5 10 15Ala Val Glu Lys Ala Val
Gln Gln Tyr 20 257625PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 76Glu Glu Gly Leu Asp Arg Asn
Glu Glu Ile Lys Arg Ala Ala Phe Gln1 5 10 15Ile Ile Ser Gln Val Ile
Ser Glu Ala 20 257725PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 77Leu Val Asp Asp Pro Leu Glu
Tyr Gln Ala Gly Leu Leu Val Gln Asn1 5 10 15Ala Ile Gln Gln Ala Ile
Ala Glu Gln 20 257825PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 78Gln Tyr Glu Thr Leu Leu Ile
Glu Thr Ala Ser Ser Leu Val Lys Asn1 5 10 15Ala Ile Gln Leu Ser Ile
Glu Gln Leu 20 257925PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 79Leu Glu Lys Gln Tyr Gln Glu
Gln Leu Glu Glu Glu Val Ala Lys Val1 5 10 15Ile Val Ser Met Ser Ile
Ala Phe Ala 20 258025PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 80Asn Thr Asp Glu Ala Gln Glu
Glu Leu Ala Trp Lys Ile Ala Lys Met1 5 10 15Ile Val Ser Asp Ile Met
Gln Gln Ala 20 258125PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 81Val Asn Leu Asp Lys Lys Ala
Val Leu Ala Glu Lys Ile Val Ala Glu1 5 10 15Ala Ile Glu Lys Ala Glu
Arg Glu Leu 20 258225PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 82Asn Gly Ile Leu Glu Leu Glu
Thr Lys Ser Ser Lys Leu Val Gln Asn1 5 10 15Ile Ile Gln Thr Ala Val
Asp Gln Phe 20 258325PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 83Thr Gln Asp Lys Asn Tyr Glu
Asp Glu Leu Thr Gln Val Ala Leu Ala1 5 10 15Leu Val Glu Asp Val Ile
Asn Tyr Ala 20 258425PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 84Glu Thr Ser Ala Lys Asp Asn
Ile Asn Ile Glu Glu Ala Ala Arg Phe1 5 10 15Leu Val Glu Lys Ile Leu
Val Asn His 20 2585330PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 85Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75
80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
33086330PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 86Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235
240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 325 3308744PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptideMOD_RES(1)..(1)Ser or
ThrMOD_RES(2)..(2)His, Lys or ArgMOD_RES(4)..(4)Gln or
AsnMOD_RES(8)..(8)Gly or AlaMOD_RES(10)..(10)Thr or
SerMOD_RES(11)..(11)Glu or AspMOD_RES(14)..(14)Gln or
AsnMOD_RES(15)..(15)Gly or AlaMOD_RES(17)..(17)Thr or
SerMOD_RES(19)..(19)Glu or AspMOD_RES(22)..(22)Arg or
LysMOD_RES(23)..(24)Gln or AsnMOD_RES(27)..(27)Asp or
GluMOD_RES(30)..(30)Glu or AspMOD_RES(32)..(32)Ala, Leu, Ile or
ValMOD_RES(34)..(34)Glu or AspMOD_RES(37)..(37)Thr or
SerMOD_RES(38)..(38)Arg or LysMOD_RES(40)..(40)Arg or
LysMOD_RES(41)..(41)Glu or AspMOD_RES(42)..(42)Ala, Leu, Ile or
ValMOD_RES(43)..(43)Arg or LysMOD_RES(44)..(44)Ala, Leu, Ile or Val
87Xaa Xaa Ile Xaa Ile Pro Pro Xaa Leu Xaa Xaa Leu Leu Xaa Xaa Tyr1
5 10 15Xaa Val Xaa Val Leu Xaa Xaa Xaa Pro Pro Xaa Leu Val Xaa Phe
Xaa 20 25 30Val Xaa Tyr Phe Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa 35
408817PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)Gln or AsnMOD_RES(2)..(2)Ile, Leu
or ValMOD_RES(3)..(3)Glu or AspMOD_RES(4)..(4)Tyr, Phe, Thr or
SerMOD_RES(5)..(5)Leu, Ile or ValMOD_RES(7)..(7)Lys or
ArgMOD_RES(8)..(8)Gln or AsnMOD_RES(11)..(11)Asp or
GluMOD_RES(12)..(12)Asn or GlnMOD_RES(15)..(16)Gln or
AsnMOD_RES(17)..(17)Ala, Leu, Ile or Val 88Xaa Xaa Xaa Xaa Xaa Ala
Xaa Xaa Ile Val Xaa Xaa Ala Ile Xaa Xaa1 5 10
15Xaa8944PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMOD_RES(1)..(1)Ser or ThrMOD_RES(4)..(4)Gln or
AsnMOD_RES(10)..(10)Thr or SerMOD_RES(18)..(18)Val, Ile, Leu or
AlaMOD_RES(23)..(23)Gln or AsnMOD_RES(33)..(33)Val, Ile, Leu or
AlaMOD_RES(34)..(34)Glu or AspMOD_RES(37)..(37)Thr or
SerMOD_RES(38)..(38)Arg or LysMOD_RES(40)..(40)Arg or
LysMOD_RES(42)..(42)Ala, Leu, Ile or ValMOD_RES(44)..(44)Ala, Leu,
Ile or Val 89Xaa His Ile Xaa Ile Pro Pro Gly Leu Xaa Glu Leu Leu
Gln Gly Tyr1 5 10 15Thr Xaa Glu Val Leu Arg Xaa Gln Pro Pro Asp Leu
Val Glu Phe Ala 20 25 30Xaa Xaa Tyr Phe Xaa Xaa Leu Xaa Glu Xaa Arg
Xaa 35 409024DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 90gacattcagc tgacccagtc tcca
249136DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 91gccggatcct cactggatgg tgggaagatg gataca
369222DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 92aggtsmarct gcagsagtcw gg 229318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
93agctgggaag gtgtgcac 1894117PRTMus musculus 94Val Lys Leu Gln Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser1 5 10 15Arg Lys Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Phe Gly 20 25 30Met His Trp
Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val Ala 35 40 45Tyr Ile
Gly Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val Lys 50 55 60Gly
Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe Leu65 70 75
80Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95Arg Ser Asn Trp Asp Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr
Ser 100 105 110Val Thr Val Ser Ser 11595120PRTMus musculus 95Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser1 5 10
15Arg Gln Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly
20 25 30Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val
Ala 35 40 45Tyr Ile Ser Arg Gly Gly Asn Thr Ile Tyr Tyr Ala Asn Thr
Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr
Leu Phe Leu65 70 75 80Gln Met Thr Ser Leu Arg Ser Asp Asp Thr Ala
Met Tyr Tyr Cys Ala 85 90 95Arg Ser His Tyr Tyr Gly Tyr Phe Tyr Ala
Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Thr Leu Thr Val Ser Ser
115 12096123PRTMus musculus 96Asp Ile Gln Leu Thr Gln Ser Pro Ala
Ser Leu Ser Ala Ser Val Gly1 5 10 15Glu Thr Val Thr Ile Thr Cys Arg
Ala Ser Gly Asn Ile His Asn Phe 20 25 30Leu Ala Trp Tyr Gln Gln Lys
Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45Tyr Asn Ala Glu Thr Leu
Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Trp 85 90 95Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105
110Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 115 12097123PRTMus
musculus 97Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser
Val Gly1 5 10 15Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile
His Asn Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro
Gln Leu Leu Val 35 40
45Tyr Asn Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln
Pro65 70 75 80Glu Asp Phe Gly Thr Tyr Tyr Cys His His Phe Trp Ser
Thr Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Val Lys Arg
Ala Asp Ala Ala 100 105 110Pro Thr Val Ser Ile Leu Pro Pro Ser Ser
Glu 115 12098115PRTMus musculus 98Val Gln Leu Gln Glu Ser Gly Pro
Ser Leu Val Lys Pro Ser Gln Thr1 5 10 15Leu Ser Leu Thr Cys Ser Val
Thr Gly Asp Ser Ile Thr Ser Gly Phe 20 25 30Trp Asn Trp Ile Arg Lys
Phe Pro Gly Asn Lys Phe Glu Tyr Met Gly 35 40 45Tyr Ile Ser Tyr Ser
Gly Arg Thr Tyr Tyr Asn Pro Ser Leu Lys Ser 50 55 60Arg Leu Ser Ile
Thr Arg Asp Thr Ser Lys Asn Gln Phe Tyr Leu Gln65 70 75 80Leu Asn
Ser Val Thr Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Arg 85 90 95Asp
Ala Asn Tyr Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105
110Val Ser Ser 11599114PRTMus musculus 99Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser Gln Ser Leu Ser1 5 10 15Leu Thr Cys Ser Val
Ser Gly Tyr Ser Ile Thr Ser Gly Tyr Phe Trp 20 25 30Asn Trp Ile Arg
Gln Phe Ser Gly Asn Lys Leu Glu Trp Met Gly Tyr 35 40 45Ile Ser Tyr
Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu Lys Asn Arg 50 55 60Ile Ser
Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Lys Leu65 70 75
80Asn Ser Val Thr Pro Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Asp
85 90 95Gly Asp Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr
Val 100 105 110Ser Ser100114PRTMus musculus 100Asp Ile Gln Leu Thr
Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly1 5 10 15Gln Lys Val Thr
Met Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30Ser Thr Gln
Lys Asn Tyr Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln 35 40 45Ser Pro
Lys Leu Leu Val Tyr Phe Ala Ser Ala Arg Glu Ser Gly Val 50 55 60Pro
Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75
80Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln
85 90 95His Tyr Arg Thr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu
Ile 100 105 110Lys Arg101109PRTMus musculus 101Leu Thr Gln Ser Pro
Ser Ser Leu Ala Met Ser Val Gly Gln Lys Val1 5 10 15Thr Met Asn Cys
Lys Ser Ser Gln Ser Leu Leu Asn Ser Tyr Thr Gln 20 25 30Lys Asn Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys 35 40 45Leu Leu
Val Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg 50 55 60Phe
Met Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser65 70 75
80Val Gln Thr Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln His Tyr Arg
85 90 95Ile Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100
105102648DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 102ctcgagcaca caggacctca ccatgggatg
gagctgtatc atcctcttct tggtagcaac 60agctacaggt aaggggctca cagtagcagg
cttgaggtct ggacatatat atgggtgaca 120atgacatcca ctttgccttt
ctctccacag gtgtccactc cgtgcagctg caggagagcg 180gaccctccct
ggtgaagcct agtcagaccc tgagcctgac atgctccgtg actggggact
240ctatcaccag tggcttctgg aactggattc ggaagttccc aggaaacaag
tttgaataca 300tgggatatat ctcttacagt gggcgcacat actataaccc
cagcctgaag tccaggctgt 360ctattacaag agacacttct aaaaaccagt
tttatctgca gctgaacagc gtgactgccg 420aggatactgc tacctactat
tgtgccaggg acgctaatta tgtgctggat tactggggcc 480agggaaccac
actgaccgtg agctccggtg agtccttaca acctctctct tctattcagc
540ttaaatagat tttactgcat ttgttggggg ggaaatgtgt gtatctgaat
ttcaggtcat 600gaaggactag ggacaccttg ggagtcagaa agggtcattg ggaagctt
648103537DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 103tctagacaca ggacctcacc atgggatgga
gctgtatcat cctcttcttg gtagcaacag 60ctacaggtaa ggggctcaca gtagcaggct
tgaggtctgg acatatatat gggtgacaat 120gacatccact ttgcctttct
ctccacaggt gtccactccg acatccagct gacccagagc 180cccagctccc
tggctatgtc cgtgggacag aaggtgacaa tgaactgcaa atctagtcag
240tctctgctga acagctccac tcagaagaat tacctggctt ggttccagca
gaagcccggg 300cagagtccta aactgctggt gtattttgcc tctgctaggg
agagtggcgt gccagacaga 360ttcatcggca gcggcagcgg gaccgatttt
accctgacaa tttctagtgt gcaggccgag 420gacctggctg attacttctg
tcagcagcac tatcggactc ccttcacctt tggctccgga 480acaaagctgg
agatcaagcg tgagtagaat ttaaactttg cttcctcagt tggatcc
537104345DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 104gtgcagctgc aggagagcgg accctccctg
gtgaagccta gtcagaccct gagcctgaca 60tgctccgtga ctggggactc tatcaccagt
ggcttctgga actggattcg gaagttccca 120ggaaacaagt ttgaatacat
gggatatatc tcttacagtg ggcgcacata ctataacccc 180agcctgaagt
ccaggctgtc tattacaaga gacacttcta aaaaccagtt ttatctgcag
240ctgaacagcg tgactgccga ggatactgct acctactatt gtgccaggga
cgctaattat 300gtgctggatt actggggcca gggaaccaca ctgaccgtga gctcc
34510563DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 105tgtggccaga tcgagtacct ggccaagcag
atcgtggaca acgccatcca gcaggccggg 60tgc 63106363DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
106gacatccagc tgacccagag ccccagctcc ctggctatgt ccgtgggaca
gaaggtgaca 60atgaactgca aatctagtca gtctctgctg aacagctcca ctcagaagaa
ttacctggct 120tggttccagc agaagcccgg gcagagtcct aaactgctgg
tgtattttgc ctctgctagg 180gagagtggcg tgccagacag attcatcggc
agcggcagcg ggaccgattt taccctgaca 240atttctagtg tgcaggccga
ggacctggct gattacttct gtcagcagca ctatcggact 300cccttcacct
ttggctccgg aacaaagctg gagatcaagc gtgagtagaa tttaaacttt 360gct
36310710PRTMus musculus 107Gly Phe Thr Phe Ser Arg Phe Gly Met His1
5 1010817PRTMus musculus 108Tyr Ile Gly Arg Gly Ser Ser Thr Ile Tyr
Tyr Ala Asp Thr Val Lys1 5 10 15Gly1099PRTMus musculus 109Ser Asn
Trp Asp Gly Ala Met Asp Tyr1 511011PRTMus musculus 110Arg Ala Ser
Gly Asn Ile His Asn Phe Leu Ala1 5 101117PRTMus musculus 111Asn Ala
Glu Thr Leu Ala Asp1 51129PRTMus musculus 112Gln His Phe Trp Ser
Thr Pro Trp Thr1 51134PRTMus musculus 113Gly Phe Trp
Asn111416PRTMus musculus 114Tyr Ile Ser Tyr Ser Gly Arg Thr Tyr Tyr
Asn Pro Ser Leu Lys Ser1 5 10 151158PRTMus musculus 115Asp Ala Asn
Tyr Val Leu Asp Tyr1 511617PRTMus musculus 116Lys Ser Ser Gln Ser
Leu Leu Asn Ser Ser Thr Gln Lys Asn Tyr Leu1 5 10 15Ala1177PRTMus
musculus 117Phe Ala Ser Ala Arg Glu Ser1 51189PRTMus musculus
118Gln Gln His Tyr Arg Thr Pro Phe Thr1 5
* * * * *