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OA16388A - Anti-IL-23 antibodies. - Google Patents

Anti-IL-23 antibodies. Download PDF

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Publication number
OA16388A
OA16388A OA1201300166 OA16388A OA 16388 A OA16388 A OA 16388A OA 1201300166 OA1201300166 OA 1201300166 OA 16388 A OA16388 A OA 16388A
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OA
OAPI
Prior art keywords
antibody
seq
amino acid
acid sequence
antigen
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OA1201300166
Inventor
Rachel Rebecca Barrett
Keith Canada
Katrina Mary Catron
Rober COPENHAVER
Lee Edward Frego
Ernest Lee Raymond
Sanjaya Singh
Xiangyang Zhu
Original Assignee
Boehringer Ingelheim International Gmbh
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Publication of OA16388A publication Critical patent/OA16388A/en

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Abstract

The present invention relates to anti-IL23p19 binding compounds, in particular new humanized anti-IL-23p19 antibodies and therapeutic and diagnostic methods and compositions for using the same.

Description

Boehringer Ingelheim International GmbH
Anti-IL-23 Antibodies
Technical Field of the Invention
This invention generally relates to anti-IL-23p19 antibodies for diagnostic and therapeutic use. More specifically, humanized anti-IL-23p19 antibodies and methods of use for the treatment of various diseases or disorders are disclosed. Pharmaceutical compositions and kits comprising such compounds are also disclosed.
Background of the Invention
Higher eukaryotes hâve evolved an intricate response to pathogens that is initiated by the innate immune response and followed by the adaptive immune response. Together these two mechanisms not only eradicate pathogens that infect the organism but also establish a long term immunological response against future exposures. Deficiencies in these responses can resuit in increased susceptibility to infections and/or alterations of the adaptive immune response leading to chronic inflammation and autoimmunity. IL-12, a heterodimeric cytokine consisting of a p40 and a p35 protein subunit, has long been considered the hallmark cytokine of the innate immune response with major influence on adaptive immunity. However, data from investigation of this cytokine’s biological rôle led to confusing results. For example, while p40-deficient mice were résistant to Collagen
Induced Arthritis (CIA) and Experimental Autoimmune Encephalomyelitis (EAE), p35deficient mice were susceptible to both and even displayed exacerbated disease. Such conundrums began to be resolved with the discovery in the late 1990’s of a new member of the IL-12 cytokine family with a distinct rôle in the immune response - IL-23. IL-23 is composed of a common subunit (p40) with IL-12 and a unique p19 subunit.
Despite this shared p40 subunit, the rôles for IL-23 and IL-12 are quite different. IL-12 is important for Th 1 responses via promotion of Th1 cell différentiation, prolifération and activation. In contrast, IL-23 supports the development and maintenance of a recently defined set of CD4+ T helper cells termed Th17 cells due to their ability to produce IL-17 and related cytokines. There is mounting evidence that IL-23 is involved in chronic autoimmune inflammation and the modulation of IL-23 activity could provide promising thérapies against autoimmune diseases. y/'
-2There is therefore a need for antagonist molécules against IL-23 with bénéficiai pharmacological properties, which can be used as therapeutic agents to treat diseases, in particular immunological and autoimmune diseases in humans.
Accordingly, one aim of the présent invention is to provide anti-IL-23 antagonist molécules, in particular anti-IL-23 antagonist molécules which hâve high binding affinity to IL-23.
A further aim of the présent invention is to provide anti-IL-23 antagonist molécules, which hâve high specificity for IL-23.
A further aim of the présent invention is to provide anti-IL-23 antagonists, which hâve high blocking activity for the association of IL-23 and its receptor.
A further aim of the présent invention is to provide anti-IL-23 antagonists, which hâve potent cellular activity.
A further aim of the présent invention is to provide anti-IL-23 antagonists, which hâve a favorable bioavailability.
A further aim of the présent invention is to provide anti-IL-23 antagonists, which hâve favorable biophysical properties.
Further aims of the présent invention include combinations of any of the aims set forth above.
Summary of the Invention
The présent invention addresses the above needs and provides antibodies that bind to the p19 subunit of the IL-23 protein. In one aspect, an antibody of the présent invention binds to human IL-23 with high affinity. In another aspect, an antibody of the présent invention inhibits the IL-23 stimulated production of IL-17 from mouse splénocytes. In another aspect, an antibody of the présent invention does not bind to nor antagonize IL12, which is a closely related family member to IL-23.
In one embodiment, the présent invention provides anti-IL-23p19 antibodies that are derived from mouse hybridomas, for example monoclonal antibodies. In one embodiment, the présent invention provides full length anti-IL-23p19 antibodies. In another embodiment, the présent invention provides anti-IL-23p19 humanized antibodies, for example humanized monoclonal anti-IL-23p19 antibodies, for example
-3full length humanized monoclonal anti-IL-23p19 antibodies. In one aspect, a humanized antibody of the présent invention binds to human IL-23 with high affinity. In another aspect, a humanized antibody of the présent invention also binds to cynomolgus IL-23 with high affinity. In a further aspect, a humanized antibody of the présent invention inhibits IL-23-induced STAT3 phosphorylation in DB cells. In another aspect, a humanized antibody of the présent invention antagonizes the action of IL-23 by binding to the p19 subunit of IL-23, for example as measured by the inhibition of cytokines such as IL-17 and IL-22, whose production is stimulated by IL-23. In a further aspect, a humanized antibody of the présent invention has a favorable pharmacokinetic (PK) profile. In a further aspect, a humanized antibody of the présent invention has favorable biophysical properties, such as quality, stability or solubility, for example as defined by the percentage of antibody in monomer form.
Further embodiments encompass DNA molécules encoding antibodies of the présent invention, expression vectors and host cells comprising such DNA molécules, and methods of making antibodies of the présent invention. The présent invention further provides therapeutic uses for the antibodies of the présent invention, in particular against immunological and autoimmune diseases.
In one embodiment, the présent invention further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof comprising a light chain CDR1 (L-CDR1) sequence of SEQ ID NO:1,4, 6, 7, 8, 11, 15, 18, 19, 22, 27 or 30; a light chain CDR2 (L-CDR2) sequence of SEQ ID NO:2, 5, 9,12,16, 20, 23, 25, 28 or 31 ; a light chain CDR3 (LCDR3) sequence of SEQ ID NO:3, 10, 13, 14, 17, 21,24, 26, 29, or 32; a heavy chain CDR1 (H-CDR1 ) sequence of SEQ ID NO:33, 36, 38, 40, 43, 45, 48, 51, 54, 57, 60, 63, 66, 67, 68, 69, 77 or 80; a heavy chain CDR2 (H-CDR2) sequence of SEQ ID NO:34, 39, 41, 46, 49, 52, 55, 58, 61, 64, 70, 72, 73, 75, 78 or 81; and a heavy chain CDR3 (HCDR3) sequence of SEQ ID NO:35, 37, 42, 44, 47, 50, 53, 56, 59, 62, 65, 71, 74, 76, 79 or 82. In one embodiment, the anti-IL-23p19 antibody or antigen-binding fragment thereof comprises a light chain variable région comprising a L-CDR1 listed above, a LCDR2 listed above and a L-CDR3 listed above, and a heavy chain variable région comprising a H-CDR1 listed above, a H-CDR2 listed above and a H-CDR3 listed above.
-4ln one embodiment, the présent invention further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a HCDR3 sequence of SEQ ID NO:1,2, 3, 33, 34, and 35, respectively; or a L-CDR1, a LCDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:4, 5, 3, 36, 34 and 37, respectively; or a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a HCDR2 and a H-CDR3 sequence of SEQ ID NO:1, 2, 3, 38, 39 and 35, respectively; or a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:6, 2, 3, 40, 41 and 42, respectively; or a L-CDR1, a L-CDR2, a L-CDR3, a HCDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:7, 2, 3, 43, 41 and 44, respectively; or a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:8, 9, 10, 45, 46 and 47, respectively; or a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:8, 9, 10, 48, 49 and 50, respectively; or a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:11, 12, 13, 51, 52 and 53, respectively; or a LCDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:7, 2, 14, 54, 55 and 56, respectively; or a L-CDR1, a L-CDR2, a L-CDR3, a HCDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID ΝΟ.Ί 5, 16, 17, 57, 58 and 59, respectively; or a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:18, 16, 17, 60, 61 and 62, respectively; or a L-CDR1, a LCDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:19, 20, 21,63, 66, 67 or 68, 64 and 65, respectively; or a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:22, 23, 24, 69, 70 and 71, respectively; or a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:22, 25, 26, 55, 72 and 71, respectively; or a L-CDR1, a LCDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:8, 9, 10, 45, 73 and 74, respectively; or a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a HCDR2 and a H-CDR3 sequence of SEQ ID NO:27, 28, 29, 45, 75 and 76, respectively; or a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:8, 9, 10, 77, 78 and 79, respectively; or a L-CDR1, a L-CDR2, a L-CDR3, a
-5H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:30, 31, 32, 80, 81 and 82, respectively. In one embodiment, the anti-IL-23p19 antibody or antigen-binding fragment thereof comprises a light chain variable région comprising a L-CDR1, L-CDR2 and LCDR3 combination listed above, and a heavy chain variable région comprising a HCDR1, H-CDR2 and H-CDR3 combination listed above.
In one embodiment, the présent invention further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable région comprising the amino acid sequence of SEQ ID NO:84 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO: 121; or a light chain variable région comprising the amino acid sequence of SEQ ID NO:86 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO:123; or a light chain variable région comprising the amino acid sequence of SEQ ID NO:88 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO:125; or a light chain variable région comprising the amino acid sequence of SEQ ID NO:90 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO:127; or a light chain variable région comprising the amino acid sequence of SEQ ID NO:91 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO:128; or a light chain variable région comprising the amino acid sequence of SEQ ID NO:93 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO: 130; or a light chain variable région comprising the amino acid sequence of SEQ ID NO:95 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO:132; or a light chain variable région comprising the amino acid sequence of SEQ ID NO:97 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO: 134; or a light chain variable région comprising the amino acid sequence of SEQ ID NO:99 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO:136; or a light chain variable région comprising the amino acid sequence of SEQ ID NO:101 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO:138; or a light chain variable région comprising the amino acid sequence of SEQ ID NO: 103 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO:140; or a light chain variable région comprising the amino acid sequence of *
-6SEQ ID NO:105 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO: 142; or a light chain variable région comprising the amino acid sequence of SEQ ID NO:107 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO:144; or a light chain variable région comprising the amino acid sequence of SEQ ID NO:109 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO:146; or a light chain variable région comprising the amino acid sequence of SEQ ID NO:111 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO: 148; or a light chain variable région comprising the amino acid sequence of SEQ ID NO:113 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO:150; or a light chain variable région comprising the amino acid sequence of SEQ ID NO:115 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO:152; or a light chain variable région comprising the amino acid sequence of SEQ ID NO:117 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO:154; or a light chain variable région comprising the amino acid sequence of SEQ ID NO:119 and a heavy chain variable région comprising the amino acid sequence SEQIDNO:156.
In one embodiment, the présent invention further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable région comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 158, 160, 162 and 164 and a heavy chain variable région comprising an amino acid sequence selected from the group consisting of SEQ ID NO:166, 168,170 and 172.
In one embodiment, the présent invention further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof has a Kd for IL-23 of less than 40pM, or a Kd for IL-23 of less than 20pM, or Kd for IL-23 of less than 10pM or KD for IL-23 of less than 1pM.
-7ln a further embodiment, the présent invention provides an anti-IL-23p19 antibody or antigen-binding fragment thereof that binds to human IL-23p19 at an epitope consisting of amino acid residues 108 to 126 and amino acid residues 137 to 151 of SEQ ID NO: 181.
In a further embodiment, the présent invention provides an anti-IL-23p19 antibody or antigen-binding fragment thereof that competitively binds to human IL-23p19 with an antibody of the présent invention. In one enbodiment, the présent invention provides an anti-IL-23p19 antibody or antigen-binding fragment thereof that competitively binds to human IL-23p19 with a humanized monoclonal anti-IL-23p19 antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 174 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 176. In one enbodiment, the présent invention provides an anti-IL-23p19 antibody or antigen-binding fragment thereof that competitively binds to human IL-23p19 with a humanized monoclonal anti-IL-23p19 antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO:174 and a heavy chain comprising the amino acid sequence of SEQ ID NO:178. In one enbodiment, the présent invention provides an anti-IL-23p19 antibody or antigenbinding fragment thereof that competitively binds to human IL-23p19 with a humanized monoclonal anti-IL-23p19 antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO:180 and a heavy chain comprising the amino acid sequence of SEQ ID NO:176. In one enbodiment, the présent invention provides an anti-IL-23p19 antibody or antigen-binding fragment thereof that competitively binds to human IL-23p19 with a humanized monoclonal anti-IL-23p19 antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 180 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 178.
In one embodiment, the anti-IL-23p19 antibody is a humanized antibody. In one embodiment, the anti-IL-23p19 antibody is a monoclonal antibody. In one embodiment, the anti-IL-23p19 antibody is a full length antibody. In one embodiment, the anti-IL23p19 antibody is a humanized monoclonal antibody, for example a full length humanized monocional antibody. In one embodiment the antigen-binding fragment is a
-8Fab, F(ab')2, or single chain Fv fragment. In one embodiment, the antigen-binding fragment comprises a light chain variable région and a heavy chain variable région.
In one embodiment, the présent invention further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:19 (CDR1-L)·, the amino acid sequence of SEQ ID NO:20 (CDR2-L); the amino acid sequence of SEQ ID NO:21 (CDR3-L): the amino acid sequence of SEQ ID NO:63, 66, 67 or 68 (CDR1-H); the amino acid sequence of SEQ ID NO:64 (CDR2-H)’, and the amino acid sequence of SEQ ID NO:65 (CDR3-H).
In one embodiment, the présent invention further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:19 (CDR1-L); the amino acid sequence of SEQ ID NO:20 (CDR2-L)', the amino acid sequence of SEQ ID NO:21 (CDR3-L)'. the amino acid sequence of SEQ ID NO:66 (CDR1-H)', the amino acid sequence of SEQ ID NO:64 (CDR2-H)', and the amino acid sequence of SEQ ID NO:65 (CDR3-H).
In one embodiment, the présent invention further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable région comprising the amino acid sequence of SEQ ID NO:19 (CDR1-L)\ the amino acid sequence of SEQ ID NO:20 (CDR2-L)·, and the amino acid sequence of SEQ ID NO:21 (CDR3-L); and a heavy chain variable région comprising the amino acid sequence of SEQ ID NO: 63, 66, 67 or 68 (CDR1-H)', the amino acid sequence of SEQ ID NO:64 (CDR2-H)·, and the amino acid sequence of SEQ ID NO:65 (CDR3-H).
In one embodiment, the présent invention further provides an anti-IL-23p19 or antigenbinding fragment thereof antibody, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable région comprising the amino acid sequence of m
-9SEQ ID NO:19 (CDR1-L); the amino acid sequence of SEQ ID NO:20 (CDR2-L)·, and the amino acid sequence of SEQ ID NO:21 (CDR3-L)', and a heavy chain variable région comprising the amino acid sequence of SEQ ID NO:66 (CDR1-H); the amino acid sequence of SEQ ID NO:64 (CDR2-H)·, and the amino acid sequence of SEQ ID NO:65 (CDR3-H).
In one embodiment, the présent invention further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-bînding fragment thereof comprises a light chain variable région comprising the amino acid sequence of any one of SEQ ID NO:158, 160, 162 or 164; and a heavy chain variable région comprising the amino acid sequence any one of SEQ ID NO:166, 168, 170 or 172.
In one embodiment, the présent invention further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable région comprising the amino acid sequence of SEQ ID NO:160 and a heavy chain variable région comprising the amino acid sequence SEQIDNO;166.
In one embodiment, the présent invention further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable région comprising the amino acid sequence of SEQ ID NO: 160 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO:168.
In one embodiment, the présent invention further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable région comprising the amino acid sequence of SEQ ID NO:158 and a heavy chain variable région comprising the amino acid sequence SEQID NO:166.
-10ln one embodiment, the présent invention further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable région comprisîng the amino acid sequence of SEQ ID NO: 158 and a heavy chain variable région comprisîng the amino acid sequence SEQIDNO:168.
In one embodiment, the anti-IL-23p19 antibody is a humanized antibody. In one embodiment, the anti-IL-23p19 antibody is a monoclonal antibody. In one embodiment, the anti-IL-23p19 antibody is a full length antibody. In one embodiment, the anti-IL23p19 antibody is a humanized monoclonal anti-IL-23p19 antibody, for example a full length humanized monoclonal antibody. In one embodiment, the antigen-binding fragment is a Fab, F(ab')2, or single chain Fv fragment. In one embodiment, the antigenbinding fragment comprises a light chain variable région and a heavy chain variable région.
In one embodiment, the présent invention further provides an antibody comprisîng the amino acid sequence SEQ ID NO:166 or 168 linked to a human lgG1, lgG2, lgG3, lgG4, IgM, IgA or IgE heavy chain constant région. An antibody comprisîng the amino acid sequence of SEQ ID NO: 166 or 168 linked to a human lgG1 heavy chain constant région. An antibody comprisîng the amino acid sequence of SEQ ID NO:158 or 160 linked to a human kappa or lambda light chain constant région. An antibody comprisîng the amino acid sequence of SEQ ID NO: 158 or 160 linked to a human kappa light chain constant région.
In one embodiment, the présent invention further provides an antibody comprisîng the amino acid sequence of SEQ ID NO:166 or 168 linked to a human lgG1 heavy chain constant région; and the amino acid sequence of SEQ ID NO: 158 or 160 linked to a human kappa light chain constant région.
In one embodiment, the présent invention further provides a humanized monoclonal anti-IL-23p19 antibody comprisîng a light chain variable région comprisîng the amino
-11 acid sequence selected from the group consisting of any one of SEQ ID NO:158, 160, 162 and 164 and a heavy chaîn variable région comprising the amino acid sequence selected from the group consisting of any one of SEQ ID NO:166, 168,170 and 172.
In one embodiment, the présent invention further provides a humanized monoclonal anti-ÎL-23p19 antibody comprising a light chain variable région comprising the amino acid sequence of SEQ ID NO: 160 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO:166.
In one embodiment, the présent invention further provides a humanized monoclonal anti-IL-23p19 antibody comprising a light chain variable région comprising the amino acid sequence of SEQ ID NO:160 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO:168.
In one embodiment, the présent invention further provides a humanized monoclonal anti-IL-23p19 antibody comprising a light chain variable région comprising the amino acid sequence of SEQ ID NO:158 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO:166.
In one embodiment, the présent invention further provides a humanized monoclonal anti-IL-23p19 antibody comprising a light chain variable région comprising the amino acid sequence of SEQ ID NO:158 and a heavy chain variable région comprising the amino acid sequence SEQ ID ΝΟ.Ί68.
In one embodiment, the présent invention further provides a humanized monoclonal anti-IL-23p19 antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO:174 or 180 and a heavy chain comprising the amino acid sequence of SEQ ID NO:176 or 178.
In one embodiment, the présent invention further provides a humanized monoclonal anti-IL-23p19 antibody comprising a light chain comprising the amino acid sequence of \j/
-12SEQ ID NO:174 and a heavy chain comprising the amino acid sequence of SEQ ID NO:176.
In one embodiment, the présent invention further provides a humanized monoclonal anti-IL-23p19 antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO:174 and a heavy chain comprising the amino acid sequence of SEQ ID NO:178.
In one embodiment, the présent invention further provides a humanized monoclonal anti-IL-23p19 antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO:180 and a heavy chain comprising the amino acid sequence of SEQ ID NO:176.
In one embodiment, the présent invention further provides a humanized monoclonal anti-IL-23p19 antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 180 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 178.
In a further embodiment, the présent invention relates to an anti-IL-23p19 antibody or antigen-binding fragment thereof comprising a humanized light chain variable domain comprising the CDRs of SEQ ID NO: 160 and framework régions having an amino acid sequence at least 90% identical to the amino acid sequence of the framework régions of the variable domain light chain amino acid sequence of SEQ ID NO:160 and a humanized heavy chain variable domain comprising the CDRs of SEQ ID NO:166 and framework régions having an amino acid sequence at least 90% identical to the amino acid sequence of the framework régions of the variable domain heavy chain amino acid sequence of SEQ ID NO: 166. In one embodiment, the anti-lL-23p19 antibody is a humanized monoclonal antibody, for example a full length humanized monoclonal antibody.
-13In a further embodiment, the présent invention relates to an anti-IL-23p19 antibody or antigen-binding fragment thereof comprising a humanized light chain variable domain comprising the CDRs of SEQ ID NO:160 and framework régions having an amino acid sequence at least 90% identical to the amino acid sequence of the framework régions of the variable domain light chain amino acid sequence of SEQ ID NO:160 and a humanized heavy chain variable domain comprising the CDRs of SEQ ID NO:168 and framework régions having an amino acid sequence at least 90% identical to the amino acid sequence of the framework régions of the variable domain heavy chain amino acid sequence of SEQ ID NO:168. In one embodiment, the anti-IL-23p19 antibody is a humanized monoclonal antibody, for example a full length humanized monoclonal antibody.
In a further embodiment, the présent invention relates to an anti-IL-23p19 antibody or antigen-binding fragment thereof comprising a humanized light chain variable domain comprising the CDRs of SEQ ID NO: 158 and framework régions having an amino acid sequence at least 90% identical to the amino acid sequence of the framework régions of the variable domain light chain amino acid sequence of SEQ ID NO:158 and a humanized heavy chain variable domain comprising the CDRs of SEQ ID NO:166 and framework régions having an amino acid sequence at least 90% identical to the amino acid sequence of the framework régions of the variable domain heavy chain amino acid sequence of SEQ ID NO:166. In one embodiment, the anti-IL-23p19 antibody is a humanized monoclonal antibody, for example a full length humanized monoclonal antibody.
In a further embodiment, the présent invention relates to an anti-IL-23p19 antibody or antigen-binding fragment thereof comprising a humanized light chain variable domain comprising the CDRs of SEQ ID NO: 158 and framework régions having an amino acid sequence at least 90% identical to the amino acid sequence of the framework régions of the variable domain light chain amino acid sequence of SEQ ID NO: 158 and a humanized heavy chain variable domain comprising the CDRs of SEQ ID NO:168 and framework régions having an amino acid sequence at least 90% identical to the amino
- 14acid sequence of the framework régions of the variable domain heavy chain amino acid sequence of SEQ ID NO: 168. In one embodiment, the anti-IL-23p19 antibody is a humanized monoclonal antibody, for example a full length humanized monoclonal antibody.
In one aspect, a humanized anti-IL-23p19 antibody of the présent invention may be further characterized by a Kd for human IL-23 equal or less than 1 pM. In one aspect, there is no shift in binding on-rate in 50% human sérum.
In one aspect, a humanized anti-IL-23p19 antibody of the présent invention may be further characterized in that it blocks IL-23 binding to human IL-23R/Fc in vitro.
In one aspect, a humanized anti-IL-23p19 antibody of the présent invention may be further characterized in that it does not bind to human IL-12.
In one aspect, a humanized anti-IL-23p19 antibody of the présent invention may be further characterized in that it inhibits human IL-23 induced IL-17 production in mouse splénocytes with ICso’s equal or less than 20 pM.
In one aspect, a humanized anti-IL-23p19 antibody of the présent invention may be further characterized in that it inhibits human IL-23 induced STAT3 phosphorylation in human DB cells with ICso’s equal or less than 40 pM.
In one aspect, a humanized anti-IL-23p19 antibody of the présent invention may be further characterized in that it has no predicted activity in ADCC/CDC.
In one aspect, a humanized anti-IL-23p19 antibody of the présent invention may be further characterized in that it has a Ko equal or less than_1 pM for cynomolgus monkey IL-23.
In one aspect, a humanized anti-IL-23p19 antibody of the présent invention may be further characterized in that it has no cross reactivity to mouse or rat IL-23.
In one aspect, a humanized anti-IL-23p19 antibody of the présent invention may be further characterized in that it inhibits human IL-23 induced IL-17 and IL-22 production in a mouse ear at 80% or greater inhibition of both cytokines at 1 mg/kg.
In one aspect, a humanized anti-IL-23p19 antibody of the présent invention may be further characterized by a melting température of 83°C as determined by differential scanning calorimetry.
-15In one aspect, a humanized anti-IL-23p19 antibody of the présent invention may be further characterized by solubility equal or greater than 100 mg/ml, as measured by UV spectroscopy and monitored by turbidity.
In a further aspect, a humanized anti-IL-23p19 antibody of the présent invention may be further characterized in that it is présent in at least 90% monomer form, or in at least 92% monomer form, or in at least 95% monomer form in a buffer.
In a further aspect, a humanized anti-lL-23p19 antibody of the présent invention may be further characterized in that it remains in at least 90% monomer form, or in at least 92% monomer form, or in at least 95% monomer form in a buffer for one month or for four months.
In one aspect, the humanized anti-IL-23p19 antibody is a humanized monoclonal antibody, for example a full length humanized monoclonal antibody.
Further embodiments encompass a DNA molécule encoding a variable iight chain région above, a DNA molécule encoding a variable heavy chain région above, a DNA molécule encoding a Iight chain région above, or a DNA molécule encoding a heavy chain région above.
Further embodiments encompass an expression vector containing a DNA molécule above. In one embodiment, an expression vector comprises a DNA molécule encoding the constant heavy chain and/or the constant Iight chain, respectively, linked to the DNA molécule encoding the variable heavy chain and/or the variable Iight chain, respectively. Further embodiments encompass a host cell carrying one or more expression vectors above. In one embodiment, a host is a mammalian cell.
Further embodiments encompass a method for producing an antibody or antigenbinding fragment thereof above comprising transfecting a mammalian host cell with one or more of the vectors above, cultivating the host cell and recovering and purifying the antibody or antigen-binding fragment thereof. —
-16Further embodiments encompass a method for producing an antibody or antigenbinding fragment thereof above comprising obtaining a mammalian host cell comprising one or more of the vectors above, and cultivatîng the host cell. In one embodiment, the method further comprises recovering and purifying the antibody or antigen-binding fragment thereof.
In one embodiment, the présent invention further provides an antibody or antigenbinding fragment thereof above for use in medicine. In one embodiment, the use is the treatment of an inflammatory disease, of an autoimmune disease, of a respiratory disease, of a metabolic disorder or of cancer. In one embodiment, the use is for the treatment of psoriasis, inflammatory bowel disease (Crohn’s disease, ulcerative colitis), psoriatic arthritis, multiple sclerosis, rheumatoid arthritis, or ankylosing spondylitis. In one embodiment, the use is for the treatment of psoriasis. In one embodiment, the use is for the treatment of inflammatory bowel disease.
In one embodiment, the présent invention further provides a pharmaceutical composition comprising an antibody molécule or antigen-binding fragment above and a pharmaceutically acceptable carrier.
In one embodiment, the présent invention further provides a method for treating an inflammatory disease, an autoimmune disease, a respiratory disease, a metabolic disorder or cancer comprising administering to a subject in need thereof, for example a patient, an effective amount of an anti-IL-23p19 antibody or antigen-binding fragment thereof above, or a pharmaceutical composition comprising the antibody or antigenbinding fragment thereof. In one embodiment, the antibody or antigen-binding fragment is administered by a parentéral route of administration, or is administered intravenously or subcutaneously. In one embodiment, the antibody or antigen-binding fragment is administered subcutaneously. In one embodiment, the disease is psoriasis, inflammatory bowel disease (Crohn’s disease, ulcerative colitis), psoriatic arthritis, multiple sclerosis, rheumatoid arthritis, or ankylosing spondylitis. In one embodiment, the disease is psoriasis. In one embodiment, the disease is inflammatory bowel disease.
-17In one embodiment, the présent invention further provides a method for inhibiting the binding of IL-23 to the IL-23 receptor on a mammalian cell, comprising administering to the cell an antibody molécule or antigen-binding fragment above, whereby signaling mediated by the IL-23 receptor is inhibited.
In one embodiment, the présent invention further provides a method for treating a subject having an IL-23-associated disorder, comprising administering to the subject an antibody or antigen-binding fragment above, which antibody or antigen-binding fragment binds to human IL-23.
In one embodiment, the présent invention further provides a method for detecting and/or quantifying IL-23 levels in a biological sample by contacting the sample with an antibody or antigen binding fragment above and detecting binding of the antibody or fragment thereof with IL-23p19. This information can be used to diagnose an IL-23-associated disorder. Thus, methods are provided for diagnosing an IL-23-associated disorder or for determining if a subject has an increased risk of developing an IL-23-associated disorder, wherein the method comprises contacting a biological sample from a subject with an antibody or antigen binding fragment above and detecting binding of the antibody or antigen binding fragment to IL-23p19 to détermine the expression or concentration of IL-23.
In one embodiment, the présent invention further provides a method for inhibiting the binding of IL-23 to the IL-23 receptor on a cell, comprising administering to the cell or cellular environment an antibody or antigen-binding fragment above, whereby signaling mediated by the IL-23 receptor is inhibited.
Brief Description of the Figures
Figure 1 : Alignment of mouse and humanized variable régions. Figure 1a: Anti-IL-23p19 6B8 Engineered Vk régions. Figure 1b: Anti-IL-23p19 6ΒΘ Engineered VH régions.
-18The numbering of the amino acids is by the standard Kabat numbering scheme. Regular font = Human; italic/underlined font = Murine; shadowed font = Synthetic; bold/italic/underlined = CDR.
Figure 2: Compétition binding assay of human of IL-23 binding to IL-23R/Fc.
Detailed Description
The p19 subunit of IL-23 (also referred to herein as IL-23p19 and p19 subunit) is a 189 amino acid polypeptide containing a 21 aa leader sequence (Oppmann et al. Immunity 13:715 (2000), SEQ ID NO: 181). The biological activity of the molécule is only detected when it is partnered with the IL-12p40 subunit to form IL-23. IL-23 is predominantly expressed by activated dendritic cells (DCs) and phagocytic cells. The receptor for IL-23 was found to be composed of the IL-12R01 subunit of IL-12 receptor partnered with a unique subunit called IL-23R (Parham et al. J. Immunol. 168:5699 (2002)). Expression of the receptor is detected primarily on memory T cells and NK cells. Thus, expression of this cytokine:receptor pair appears to be restricted to spécifie populations of immune cells. While it was first thought that IL-12 and IL-23 would share many functions, the data has shown the picture to be different. Whereas IL-12 has a prédominant rôle in the production of Th1 cells, IL-23 was found to be critically involved in the production and maintenance of a recently recognized Th cell subset termed Th17 (Kikly et al. Curr. Opin. Immunol. 18:670 (2006), Kastelein et al. Ann. Rev. Immunol. 25:221 (2007)). These cells produce IL-17A, IL-17F, IL-22 and other pro-inflammatory cytokines such as IL-6 and TNF-α. As described below, animal model studies on the rôle of these Th 17 cells show their importance as a driving force in chronic inflammation and autoimmunity.
The présent invention provides antibodies that bind to the p19 subunit of the IL-23, in particular human IL-23p19. The présent invention also relates to humanized antibodies that recognize the p19 subunit of IL-23. In spécifie embodiments, the sequence of these humanized antibodies has been identified based on the sequences of certain lead mouse antibodies.
-19The lead mouse antibodies of the présent invention were derived from mouse hybridomas. The immunization of the mice is carried out using different techniques. For example, antibodies that are spécifie for human IL-23p19 proteins or fragments thereof can be raised against an immunogenic antigen such as an isolated IL-23p19 protein, an isolated IL-23 protein, an isolated hybrid IL-23 protein, and/or a portion thereof of any of the above (including synthetic peptides). For example, a hybrid IL-23 protein comprising a mouse IL-23p40 subunit and a human IL-23p19 subunit is used to immunize mice. Préparation of immunogenic antigens and monoclonal antibody production can be performed using any suitable technique known in the art.
The lead mouse antibodies were selected based on their high affinity to human IL-23. Accordingly, in one aspect, the présent invention provides an antibody that binds to human IL-23 with high affinity. Selected mouse antibodies were humanized to resuit in humanized antibodies. The humanized antibodies of the présent invention bind to human IL-23 with high affinity. Accordingly, in another aspect, the présent invention provides a humanized antibody that binds to human IL-23 with high affinity. Accordingly, in one embodiment, the présent invention provides an anti-IL-23p19 antibody having a KD of less than 40pM. In a further embodiment, the présent invention provides an anti-IL-23p19 antibody having a Kd of less than 20pM. In a further embodiment, the présent invention provides an anti-IL-23p19 antibody having a Ko less than 10pM. In a further embodiment, the présent invention provides an anti-IL-23p19 antibody having a Kd less than 1pM.
In another aspect, an antibody of the présent invention binds to IL-23p19 with high affinity in the absence of human sérum or in the presence of 50% human sérum.
In a further aspect, a humanized antibody of the présent invention also binds to cynomolgus monkey IL-23 with high affinity.
In another aspect, an antibody of the présent invention binds to IL-23, but does not bind to IL-12. In a further aspect, an antibody of the présent invention does not interfère with the biological activity of IL-12, which is a closely related family member to IL-23.
In another aspect, an antibody of the présent invention inhibits the IL-23 stimulated production of IL-17 from mouse splénocytes.
-20In a further aspect, a humanized antibody of the présent invention inhibits IL-23-induced STAT3 phosphorylation in DB cells.
In a further aspect, a humanized antibody of the présent invention antagonizes the action of IL-23 by binding to the p19 subunit of IL-23, as measured by the inhibition of cytokines such as IL-17 and IL-22, whose production is stimulated by IL-23, and detected by the réduction in the levels of these cytokines.
In a further aspect, a humanized antibody of the présent invention has a favorable pharmacokinetic profile (PK) profile, as exemplified by in vivo half life in cynomolgus monkeys,
In a further aspect, a humanized monoclonal anti-IL-23p19 antibody of the présent invention has favorable biophysical properties, for example quality, stability, or solubility. In one aspect, the anti-IL-23p19 antibody is a humanized antibody. In one aspect, the anti-IL-23p19 antibody is a monoclonal antibody. In one aspect, the anti-IL-23p19 antibody is a full length antibody. In one aspect, the anti-IL-23p19 antibody is a humanized monoclonal antibody, for example a full length humanized monoclonal antibody.
An antibody or antigen-binding fragment thereof of the présent invention recognizes spécifie IL-23p19 antigen epitope or IL-23p19 epitope. As used herein these terms refer to a molécule (e.g., a peptide) or a fragment of a molécule capable of immunoreactivity with an anti-IL-23p19 antibody and, for example, include an IL-23p19 antigenic déterminant recognized by the any of the antibodies having a light chain/heavy chain sequence combination of SEQ ID NO:84/121, 86/123, 88/125, 90/127, 91/128, 93/130, 95/132, 97/134, 99/136, 101/138, 103/140, 105/142, 107/144, 109/146, 111/148, 113/150, 115/152, 117/154, 119/156, 160/166, 160/168, 158/166 or 158/168. IL-23p19 antigen epitopes can be included in proteins, protein fragments, peptides or the like. The epitopes are most commonly proteins, short oligopeptides, oligopeptide mimics (i.e., organic compounds that mimic antibody binding properties of the IL-23p19 antigen), or combinations thereof. The minimum size of a peptide or polypeptide epitope for an antibody is thought to be about four to five amino acids. Peptide or polypeptide epitopes contain for example at least seven amino acids or for example at least nine m/
-21 amino acids or for example between about 15 to about 20 amino acids. Since an antibody can recognize an antigenic peptide or polypeptide in its tertiary form, the amino acids comprising an epitope need not be contiguous, and in some cases, may not even be on the same peptide chain. Epitopes may be determined by various techniques known in the art, such as X-ray crystallography, Hydrogen/Deuterium Exchange Mass Spectrometry (HXMS), site-directed mutagenesis, alanine scanning mutagenesis, and peptide screening methods.
The generalized structure of antibodies or immunoglobulin is well known to those of skill in the art. These molécules are heterotetrameric glycoproteins, typically of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains and are typically referred to as full length antibodies. Each light chain is covalently linked to a heavy chain by one disulfide bond to form a heterodimer, and the heterotrameric molécule is formed through a covalent disulfide linkage between the two identical heavy chains of the heterodimers. Although the light and heavy chains are linked together by one disulfide bond, the number of disulfide linkages between the two heavy chains varies by immunoglobulin isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at the aminoterminus a variable domain (Vh), followed by three or four constant domains (Chi, Ch2. Ch3, and Ch4), as well as a hinge région between Cm and Ch2· Each light chain has two domains, an amino-terminal variable domain (VL) and a carboxy-terminal constant domain (CL). The VL domain associâtes non-covalently with the VH domain, whereas the Cl domain is commonly covalently linked to the CHi domain via a disulfide bond. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains (Chothia et al., 1985, J. Mol. Biol. 186:651-663). Variable domains are also referred herein as variable régions.
Certain domains within the variable domains differ extensively between different antibodies i.e., are hypervariable. These hypervariable domains contain residues that are directly involved in the binding and specificity of each particular antibody for its spécifie antigenic déterminant. Hypervariability, both in the light chain and the heavy chain variable domains, is concentrated in three segments known as complementarity vZ
-22determining régions (CDRs) or hypervariable loops (HVLs). CDRs are defined by sequence comparison in Kabat et al., 1991, In: Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., whereas HVLs (also referred herein as CDRs) are structurally defined according to the three-dimensional structure of the variable domain, as described by Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-917. These two methods resuit in slightly different identifications of a CDR. As defined by Kabat, CDR-L1 is positioned at about residues 24-34, CDR-L2, at about residues 50-56, and CDR-L3, at about residues 89-97 in the light chain variable domain; CDR-H1 is positioned at about residues 31-35, CDR-H2 at about residues 50-65, and CDR-H3 at about residues 95-102 in the heavy chain variable domain. The exact residue numbers that encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely détermine which residues comprise a particular CDR given the variable région amino acid sequence of the antibody. The CDR1, CDR2, CDR3 of the heavy and light chains therefore define the unique and functional properties spécifie for a given antibody.
The three CDRs within each of the heavy and light chains are separated by framework régions (FR), which contain sequences that tend to be less variable. From the amino terminus to the carboxy terminus of the heavy and light chain variable domains, the FRs and CDRs are arranged in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The largely β-sheet configuration of the FRs brings the CDRs within each of the chains into close proximity to each other as well as to the CDRs from the other chain. The resulting conformation contributes to the antigen binding site (see Kabat et al., 1991, NIH Publ. No. 91-3242, Vol. I, pages 647-669), although not ail CDR residues are necessarily directly involved in antigen binding.
FR residues and Ig constant domains are not directly involved in antigen binding, but contribute to antigen binding and/or médiate antibody effector function. Some FR residues are thought to hâve a significant effect on antigen binding in at least three ways: by noncovalently binding directly to an epitope, by interacting with one or more CDR residues, and by affecting the interface between the heavy and light chains. The constant domains are not directly involved in antigen binding but médiate various Ig
-23effector functions, such as participation of the antibody in antibody dépendent cellular cytotoxicity (ADCC), complément dépendent cytotoxicity (CDC) and antibody dépendent cellular phagocytosis (ADCP).
The light chains of vertebrate immunoglobulins are assigned to one of two clearly distinct classes, kappa (κ) and lambda (λ), based on the amino acid sequence of the constant domain. By comparison, the heavy chains of mammalian immunoglobulins are assigned to one of five major classes, according to the sequence of the constant domains: IgA, IgD, IgE, IgG, and IgM. IgG and IgA are further divided into subclasses (isotypes), e.g., IgGi, lgG2, lgG3, IgGi, IgAi, and lgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the classes of native immunoglobulins are well known.
The terms, antibody, anti-IL-23p19 antibody, humanized anti-IL-23p19 antibody, humanized anti-IL-23p19 epitope antibody, and variant humanized anti-IL-23p19 epitope antibody specifically encompass monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments such as variable domains and other portions of antibodies that exhibit a desired biological activity, e.g., IL-23p19 binding.The term monoclonal antibody (mAb) refers to an antibody that is highly spécifie, being directed against a single antigenic déterminant, an “epitope”. Therefore, the modifier monoclonal is indicative of antibodies directed to the identical epitope and is not to be construed as requiring production of the antibody by any particular method. It should be understood that monoclonal antibodies can be made by any technique or methodology known in the art; including e.g., the hybridoma method ( Kohler et al., 1975, Nature 256:495), or recombinant DNA methods known in the art (see, e.g., U.S. Pat. No. 4,816,567), or methods of isolation of monoclonal recombinantly produced using phage antibody libraries, using techniques described in Clackson et al., 1991, Nature 352: 624628, and Marks et al., 1991, J. Mol. Biol. 222: 581-597. w--16388
-24The term monomer refers to a homogenous form of an antibody. For example, for a full-length antibody, monomer means a monomeric antibody having two identical heavy chains and two identical light chains.
Chimeric antibodies consist of the heavy and light chain variable régions of an antibody from one species (e.g., a non-human mammal such as a mouse) and the heavy and light chain constant régions of another species (e.g., human) antibody and can be obtained by linking the DNA sequences encoding the variable régions of the antibody from the first species (e.g., mouse) to the DNA sequences for the constant régions of the antibody from the second (e.g. human) species and transforming a host with an expression vector containing the linked sequences to allow it to produce a chimeric antibody. Alternatively, the chimeric antibody also could be one in which one or more régions or domains of the heavy and/or light chain is identical with, homologous to, or a variant of the corresponding sequence in a monoclonal antibody from another immunoglobulin class or isotype, or from a consensus or germline sequence. Chimeric antibodies can include fragments of such antibodies, provided that the antibody fragment exhibits the desired biological activity of its parent antibody, for example binding to the same epitope (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81: 6851-6855).
The terms, antibody fragment, anti-IL-23p19 antibody fragment, anti-IL-23p19 epitope antibody fragment, humanized anti-IL-23p19 antibody fragment, humanized anti-IL-23p19 epitope antibody fragment, variant humanized anti-IL-23p19 epitope antibody fragment refer to a portion of a full length anti-IL-23p19 antibody, in which a variable région or a functional capability is retained, for example, spécifie IL-23p19 epitope binding. Examples of antibody fragments include, but are not limited to, a Fab, Fab', F(ab')2l Fd, Fv, scFv and scFv-Fc fragment, a diabody, a linear antibody, a singlechain antibody, a minibody, a diabody formed from antibody fragments, and multispecific antibodies formed from antibody fragments.
Full length antibodies can be treated with enzymes such as papain or pepsin to generate useful antibody fragments. Papain digestion is used to produces two identical antigen-binding antibody fragments called Fab fragments, each with a single antigen
-25binding site, and a residual Fc fragment. The Fab fragment also contains the constant domain of the light chain and the Chi domain of the heavy chain. Pepsin treatment yields a F(ab')2 fragment that has two antigen-binding sites and is still capable of crosslinking antigen.
Fab' fragments differ from Fab fragments by the presence of additional residues including one or more cysteines from the antibody hinge région at the C-terminus of the Chi domain. F(ab')2 antibody fragments are pairs of Fab' fragments linked by cysteine residues in the hinge région. Other chemical couplings of antibody fragments are also known.
Fv fragment contains a complété antigen-recognition and binding site consîsting of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. In this configuration, the three CDRs of each variable domain interact to define an antigen-biding site on the surface of the Vh-Vl dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody.
A single-chain Fv or scFv antibody fragment is a single chain Fv variant comprising the VH and Vl domains of an antibody where the domains are présent in a single polypeptide chain. The single chain Fv is capable of recognizing and binding antigen. The scFv polypeptide may optionally also contain a polypeptide linker positioned between the Vh and VL domains in order to facilitate formation of a desired threedimensional structure for antigen binding by the scFv (see, e.g., Pluckthun, 1994, In The Pharmacology of monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315).
A diabody refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (V.sub.H) connected to a light chain variable domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L or V.sub.LV.sub.H). Diabodies are described more fully in, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448.
Other recognized antibody fragments include those that comprise a pair of tandem Fd segments (Vh-Chi-Vh-Chi) to form a pair of antigen binding régions. These “linearrv/'
-26antibodies can be bispecific or monospecific as described in, for example, Zapata et al. 1995, Protein Eng. 8(10):1057-1062.
A “humanized antibody” or a “humanized antibody fragment” is a spécifie type of chimeric antibody which includes an immunoglobulin amino acid sequence variant, or fragment thereof, which is capable of binding to a predetermined antigen and which, comprises one or more FRs having substantially the amino acid sequence of a human immunoglobulin and one or more CDRs having substantially the amino acid sequence of a non-human immunoglobulin. This non-human amino acid sequence often referred to as an import sequence is typically taken from an import antibody domain, particularly a variable domain. In general, a humanized antibody includes at least the CDRs or HVLs of a non-human antibody, inserted between the FRs of a human heavy or light chain variable domain. The présent invention describes spécifie humanized anti-IL23p19 antibodies which contain CDRs derived from the mouse monoclonal antibodies or humanized CDRs shown in Tables 3 and 4 inserted between the FRs of human germline sequence heavy and light chain variable domains. It will be understood that certain mouse FR residues may be important to the fonction of the humanized antibodies and therefore certain of the human germline sequence heavy and light chain variable domains residues are modified to be the same as those of the corresponding mouse sequence.
In another aspect, a humanized anti-IL-23p19 antibody comprises substantially ail of at least one, and typically two, variable domains (such as contained, for example, in Fab, Fab', F(ab')2, Fabc, and Fv fragments) in which ail, or substantially ail, of the CDRs correspond to those of a non-human immunoglobulin, and specifically herein, ail of the CDRs are mouse or humanized sequences as detailed in Tables 1 through 4 herein below and ail, or substantially ail, of the FRs are those of a human immunoglobulin consensus or germline sequence. In another aspect, a humanized anti- IL-23p19 antibody also includes at least a portion of an immunoglobulin Fc région, typically that of a human immunoglobulin. Ordinarily, the antibody will contain both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include one
-27or more of the CHi, hinge, Ch2, CH3, and/or CH4 régions of the heavy chain, as appropriate.
A humanized anti-IL-23p19 antibody can be selected from any class of immunoglobuline, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG-i, lgG2, lgG3, lgG4, IgA-r and lgA2. For example, the constant domain can be a complément fixing constant domain where it is desired that the humanized antibody exhibit cytotoxic activity, and the isotype is typically IgG-ι. Where such cytotoxic activity is not désirable, the constant domain may be of another isotype, e.g., lgG2. An alternative humanized anti-IL-23p19 antibody can comprise sequences from more than one immunoglobulin class or isotype, and selecting particular constant domains to optimize desired effector functions is within the ordinary skill in the art. In spécifie embodiments, the présent invention provides antibodies that are lgG1 antibodies and more particularly, are lgG1 antibodies in which there is a knock-out of effector functions.
The FRs and CDRs, or HVLs, of a humanized anti-IL-23p19 antibody need not correspond precisely to the parental sequences. For example, one or more residues in the import CDR, or HVL, or the consensus or germline FR sequence may be altered (e.g., mutagenized) by substitution, insertion or délétion such that the resulting amino acid residue is no longer identical to the original residue in the corresponding position in either parental sequence but the antibody nevertheless retains the function of binding to IL-23p19. Such alteration typically will not be extensive and will be conservative alterations. Usually, at least 75% of the humanized antibody residues will correspond to those of the parental consensus or germline FR and import CDR sequences, more often at least 90%, and most frequently greater than 95%, or greater than 98% or greater than 99%.
Immunoglobulin residues that affect the interface between heavy and light chain variable régions (the VL-VH interface) are those that affect the proximity or orientation of the two chains with respect to one another. Certain residues that may be involved in interchain interactions include Vl residues 34, 36, 38, 44, 46, 87, 89, 91, 96, and 98 and Vh residues 35, 37, 39, 45, 47, 91,93, 95, 100, and 103 (utilizing the numbering System set forth in Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes^/
-28of Health, Bethesda, Md., 1987)). U.S. Pat. No. 6,407,213 also discusses that resîdues such as VL resîdues 43 and 85, and VH resîdues 43 and 60 also may be involved in this interaction. While these resîdues are indicated for human IgG only, they are applicable across species. Important antibody resîdues that are reasonably expected to be involved in interchain interactions are selected for substitution into the consensus sequence.
The terms consensus sequence and consensus antibody refer to an amino acid sequence which comprises the most frequently occurring amino acid residue at each location in ail immunoglobulins of any particular class, isotype, or subunit structure, e.g., a human immunoglobulin variable domain. The consensus sequence may be based on immunoglobulins of a particular species or of many species. A consensus sequence, structure, or antibody is understood to encompass a consensus human sequence as described in certain embodiments, and to refer to an amino acid sequence which comprises the most frequently occurring amino acid resîdues at each location in ail human immunoglobulins of any particular class, isotype, or subunit structure. Thus, the consensus sequence contains an amino acid sequence having at each position an amino acid that is présent in one or more known immunoglobulins, but which may not exactly duplicate the entire amino acid sequence of any single immunoglobulin. The variable région consensus sequence is not obtained from any naturally produced antibody or immunoglobulin. Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., and variants thereof. The FRs of heavy and light chain consensus sequences, and variants thereof, provide useful sequences for the préparation of humanized anti-IL-23p19 antibodies. See, for example, U.S. Pat. Nos. 6,037,454 and 6,054,297.
Human germline sequences are found naturally in the human population. A combination of those germline genes generates antibody diversity. Germline antibody sequences for the light chain of the antibody corne from conserved human germline kappa or lambda v-genes and j-genes. Similarly the heavy chain sequences corne from germline v-, dand j-genes (LeFranc, M-P, and LeFranc, G, “The Immunoglobulin Facts Book Academie Press, 2001).
-29As used herein, variant, anti- IL-23p19 variant, humanized anti- IL-23p19 variant, or variant humanized anti- IL-23p19 each refers to a humanized anti-IL-23p19 antibody having at least a lîght chain variable murine CDR from any of the sequences as shown in Table 1 or a heavy chain murine CDR sequence derived from the murine monoclonal antibody as shown in Table 2. Variants include those having one or more amino acid changes in one or both light chain or heavy chain variable domains, provided that the amino acid change does not substantially impair binding of the antibody to IL-23p19. Exemplary humanized antibodies produced herein include those designated as Antibody A, Antibody B, Antibody C and Antibody D, and the various light chains and heavy chains of the same are shown in SEQ ID Nos:174 and 180, and SEQ ID Nos:176 and 178, respectively.
An isolated antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of the antibody's natural environment are those materials that may interfère with diagnostic or therapeutic uses of the antibody, and can be enzymes, hormones, or other proteinaceous or nonproteinaceous solutés. In one aspect, the antibody will be purified to at least greater than 95% isolation by weight of antibody.
An isolated antibody includes an antibody in situ within recombinant cells in which it is produced, since at least one component of the antibody's natural environment will not be présent. Ordinarily however, an isolated antibody will be prepared by at least one purification step in which the recombinant cellular material is removed.
The term antibody performance refers to factors that contribute to antibody récognition of antigen or the effectiveness of an antibody in vivo. Changes in the amino acid sequence of an antibody can affect antibody properties such as folding, and can influence physical factors such as initial rate of antibody binding to antigen (ka), dissociation constant of the antibody from antigen (kd), affinity constant of the antibody for the antigen (Kd), conformation of the antibody, protein stability, and half life of the antibody.
The term epitope tagged when used herein, refers to an anti-IL-23p19 antibody fused to an epitope tag. An epitope tag is a polypeptide having a sufficient number of amino
-30acids to provide an epitope for antibody production, yet is designed such that it does not interfère with the desired activity of the humanized anti-IL-23p19 antibody. The epitope tag is usually sufficiently unique such that an antibody raised against the epitope tag does not substantially cross-react with other epitopes. Suitable tag polypeptides generally contain at least 6 amino acid residues and usually contain about 8 to 50 amino acid residues, or about 9 to 30 residues. Examples of epitope tags and the antibody that binds the epitope include the flu HA tag polypeptide and its antibody 12CA5 (Field et al., 1988 Mol. Cell. Biol. 8: 2159-2165; c-myc tag and 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., 1985, Mol. Cell. Biol. 5(12):3610-3616; and Herpes simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al. 1990, Protein Engineering 3(6): 547-553). In certain embodiments, the epitope tag is a salvage receptor binding epitope. As used herein, the term salvage receptor binding epitope refers to an epitope of the Fc région of an IgG molécule (such as IgGi, lgG2, lgG3, or lgG4) that is responsible for increasing the in vivo sérum half-life of the IgG molécule.
In some embodiments, the antibodies of the présent invention may be conjugated to a cytotoxic agent. This is any substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (such as I131, I125, Y90, and Re186), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant, or animal origin, and fragments thereof. Such cytotoxic agents can be coupled to the humanized antibodies of the présent invention using standard procedures, and used, for example, to treat a patient indicated for therapy with the antibody.
A chemotherapeutic agent is a chemical compound useful in the treatment of cancer. There are numerous examples of chemotherapeutic agents that could be conjugated with the therapeutic antibodies of the présent invention. Examples of such chemotherapeutic agents include alkylating agents such a thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine;
-31 acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozeiesin, carzelesin, and bizelesin synthetic analogues); cryptophycines (particularly cryptophycin 1 and cryptophycin 8); dolastatin, auristatins, (including analogues monomethylauristatin E and monomethyl-auristatin F); duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine; trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calichemicin gammall and calicheamicin phiH, see for example, Agnew, Chem. Intl. Ed. Engl., 33:183-186; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-Lnorleucine, doxorubicin (Adriamycin™) (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubucin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycine, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such a methotrexate and 5-fluorouracil (5-FU); folie acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adranals such as aminoglutéthimide, mitotane, trilostane; folie acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; democolcine; diaziquone; elfomithine;
-32elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone, mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 212',2’,-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitabronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (Gemzar™); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; Vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine Navelbine™); novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or dérivatives of any of the above. Also included in this définition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and sélective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Nolvadex™), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston™); aromatase inhibitors that inhibit the enzyme aromatase, which régulâtes estrogen production in the adrenal glands, such as, for example, 4(5)imidazoles, aminoglutéthimide, megestrol acetate (Megace™), exemestane, formestane, fadrozole, vorozole (Rivisor™), letrozole (Femara™), and anastrozole (Arimidex™); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or dérivatives of any of the above. Any one or more of these agents may be conjugated to the humanized antibodies of the présent invention to provide a useful therapeutic agent for the treatment of various disorders.
The antibodies also may be conjugated to prodrugs. A prodrug is a precursor or dérivative form of a pharmaceutically active substance that is less cytotoxic to tumor<V
-33cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active form. See, for example, Wilman, 1986, Prodrugs in Cancer Chemotherapy, In Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast and Stella et al., 1985, Prodrugs: A Chemical Approach to Targeted Drug Delivery, In: Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press. Useful prodrugs include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs, and optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5fluorouridine prodrugs that can be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized into a prodrug form include, but are not limited to, those chemotherapeutic agents described above.
For diagnostic as well as therapeutic monitoring purposes, the antibodies of the invention also may be conjugated to a label, either a label alone or a label and an additional second agent (prodrug, chemotherapeutic agent and the like). A label, as distinguished from the other second agents refers to an agent that is a détectable compound or composition and it may be conjugated directly or indirectly to a humanized antibody of the présent invention. The label may itself be détectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is détectable. Labeled humanized anti-IL-23p19 antibody can be prepared and used in various applications including in vitro and in vivo diagnostics.
The antibodies of the présent invention may be formulated as part of a liposomal préparation in order to affect delivery thereof in vivo. A liposome is a small vesicle composed of various types of lipids, phospholipids, and/or surfactant. Liposomes are useful for delivery to a mammal of a compound or formulation, such as a humanized anti-IL-23p19 antibody disclosed herein, optionally, coupled to or in combination with one or more pharmaceutically active agents and/or labels. The components of the
-34liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
Certain aspects of the présent invention related to isolated nucleic acids that encode one or more domains of the humanized antibodies of the présent invention. An isolated nucleic acid molécule is a nucleic acid molécule that is identified and separated from at least one contaminant nucleic acid molécule with which it is ordinarily associated in the natural source of the antibody nucleic acid. An isolated nucleic acid molécule is distinguished from the nucleic acid molécule as it exists in natural cells.
In various aspects of the présent invention one or more domains of the humanized antibodies will be recombinantly expressed. Such recombinant expression may employ one or more control sequences, i.e., polynucleotide sequences necessary for expression of an operably linked coding sequence in a particular host organism. The control sequences suitable for use in prokaryotic cells include, for example, promoter, operator, and ribosome binding site sequences. Eukaryotic control sequences include, but are not limited to, promoters, polyadenylation signais, and enhancers. These control sequences can be utilized for expression and production of humanized anti-IL-23p19 antibody in prokaryotic and eukaryotic host cells.
A nucleic acid sequence is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, a nucleic acid presequence or secretory leader is operably linked to a nucleic acid encoding a polypeptide if it is expressed as a preprotein that participâtes in the sécrétion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, operably linked means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers are optionally contiguous. Linking can be accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers can be used.
As used herein, the expressions cell, cell line, and cell culture are used interchangeably and ail such désignations include the progeny thereof. Thus,
-35transformants and transformed cells include the primary subject cell and cultures derived therefrom without regard for the number of transfers.
The term mammal for purposes of treatment refers to any animal classified as a mammal, including humans, domesticated and farm animais, and zoo, sports, or pet animais, such as dogs, horses, cats, cows, and the like. Preferably, the mammal is human.
A disorder, as used herein, is any condition that would benefit from treatment with a humanized anti-IL-23p19 antibody described herein. This includes chronic and acute disorders or diseases including those pathological conditions that prédisposé the mammal to the disorder in question. Non-limiting examples or disorders to be treated herein include inflammatory, angiogenic, autoimmune and immunologie disorders, respiratory disorders, cancer, hematological malignancies, benign and malignant tumors, leukemias and lymphoid malignancies.
The terms cancer and cancerous refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
As used herein, the term IL-23-associated disorder or IL-23-associated disease refers to a condition in which IL-23 activity contributes to the disease and typically where IL-23 is abnormally expressed. An IL-23-associated disorder includes diseases and disorders of the immune System, such as autoimmune disorders and inflammatory disorders. Such conditions include, but are not limited to, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), scleroderma, Sjogren's syndrome, multiple sclerosis, psoriasis, psoriatic arthritis, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), pulmonary inflammation, asthma, idiopathic thrombocytopénie purara (ITP) and ankylosing spondylitis.
The term intravenous infusion refers to introduction of an agent into the vein of an animal or human patient over a period of time greater than approximately 15 minutes, generally between approximately 30 to 90 minutes.
-36The term intravenous bolus or intravenous push refers to drug administration into a vein of an animal or human such that the body receives the drug in approximately 15 minutes or less, generally 5 minutes or less.
The term subcutaneous administration refers to introduction of an agent under the skin of an animal or human patient, préférable within a pocket between the skin and underlying tissue, by relatively slow, sustained delivery from a drug réceptacle. Pinching or drawing the skin up and away from underlying tissue may create the pocket.
The term subcutaneous infusion refers to introduction of a drug under the skin of an animal or human patient, preferably within a pocket between the skin and underlying tissue, by relatively slow, sustained delivery from a drug réceptacle for a period of time including, but not limited to, 30 minutes or less, or 90 minutes or less. Optionally, the infusion may be made by subcutaneous implantation of a drug delivery pump implanted under the skin of the animal or human patient, wherein the pump delivers a predetermined amount of drug for a predetermined period of time, such as 30 minutes, 90 minutes, or a time period spanning the length of the treatment regimen.
The term subcutaneous bolus refers to drug administration beneath the skin of an animal or human patient, where bolus drug delivery is less than approximately 15 minutes; in another aspect, less than 5 minutes, and in still another aspect, less than 60 seconds. In yet even another aspect, administration is within a pocket between the skin and underlying tissue, where the pocket may be created by pinching or drawing the skin up and away from underlying tissue.
The term therapeutically effective amount is used to refer to an amount of an active agent that relieves or améliorâtes one or more of the symptoms of the disorder being treated. In another aspect, the therapeutically effective amount refers to a target sérum concentration that has been shown to be effective in, for example, slowing disease progression. Efficacy can be measured in conventional ways, depending on the condition to be treated.
The terms treatment and therapy and the like, as used herein, are meant to include therapeutic as well as prophylactic, or suppressive measures for a disease or disorder leading to any clinically désirable or bénéficiai effect, including but not limited toru/^
-37alleviation or relief of one or more symptoms, régression, slowing or cessation of progression of the disease or disorder. Thus, for example, the term treatment includes the administration of an agent prior to or following the onset of a symptom of a disease or disorder thereby preventing or removing one or more signs of the disease or disorder. As another example, the term includes the administration of an agent after clinical manifestation of the disease to combat the symptoms of the disease. Further, administration of an agent after onset and after clinical symptoms hâve developed where administration affects clinical parameters of the disease or disorder, such as the degree of tissue injury or the amount or extent of metastasis, whether or not the treatment leads to amelioration of the disease, comprises treatment or therapy as used herein. Moreover, as long as the compositions of the invention either alone or in combination with another therapeutic agent alleviate or ameliorate at least one symptom of a disorder being treated as compared to that symptom in the absence of use of the humanized anti-IL-23p19 antibody composition, the resuit should be considered an effective treatment of the underlying disorder regardless of whether ali the symptoms of the disorder are alleviated or not.
The term package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
Antibodies
In one aspect, described and disclosed herein are anti-IL-23 antibodies, in particular humanized anti-IL-23p19 antibodies, and compositions and articles of manufacture comprising one or more anti-IL-23 antibody, in particular one or more humanized anti-IL23p19 antibody of the présent invention. Also described are binding agents that include an antigen-binding fragment of an anti-IL-23 antibody, in particular a humanized anti-IL23p19 antibody. The humanized anti-IL-23p19 antibodies and binding agents can inhibit the production of Th17 associated cytokines, which contribute to chronic autoimmune and inflammatory diseases. The humanized anti-IL-23p19 antibodies and binding agents can thus be used in the treatment of a variety of diseases or disorders. A humanized
-38anti-IL-23p19 antibody and an IL-23p19 binding agent each includes at least a portion that specificatly recognizes an IL-23p19 epitope (i.e., an antigen-binding fragment).
In the initial characterization mouse antibodies were selected based on IL-23p19 binding characterization.
Accordingly in one aspect, an antibody of the présent invention has a Kd for IL-23, in particular human IL-23, of less than 100pM. In another aspect, an antibody of the présent invention has a KD of less than 40pM. In another aspect, an antibody of the présent invention has a Kd of less than 20pM. In another aspect, an antibody of the présent invention has a KD of less than 10pM. In another aspect, a monoclonal antibody 10 of the présent invention has a Kd of less than 1 pM.
The selected mouse antibodies hâve the following light chain variable régions and heavy chain variable régions as shown in Table 1 and 2:
Table 1: Anti-IL-23p19 Mouse Leads - VK Sequences
2Dlvk GACATTGTGCTGACCCAATCTCCAGGTTCTTTGGCTGTGTCTCTAGGGCA GAGGGCCACCATATCCTGCAGAACCAGTGAAAGTGTTTATAGTTATGGCC AAAATTTTATACACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTC CTCATCTATCGTGCATCCAACCTGGAATCTGGGATCCCTGCCAGGTTCAG TGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATGAATCCTGTGGAGG CTGATGATGTTGCAACCTATTACTGTCAGCAAACTAATGAGGATCCGTAC ACGTTCGGAGGGGGGACCAAGCTGGAAATAAGA (SEQ ID NO:83)
DIVLTQSPGSLAVSLGQRATISCRTSESVYSYGQNFIHWYQQKPGQPPKL LIYRASNLESGIPARFSGSGSRTDFTLTMNPVEADDVATYYCQQTNEDPY TFGGGTKLEIR (SEQ ID NO:84)
6B8Vk GACATTGTGATGACCCAGTCTCACAAATTCTTGTCCACATCAGTGGGAGA CAGGGTCACCATCACTTGCAAGGCCAGTCGGGATGTGGCTATTGCTGTAG CCTGGTATCAACAGAAACCAGGGCAATCTCCTAAACTACTTCTTTTCTGG GCATCCACCCGACACACTGGGGTCCCTGATCGCTTCACAGGCAGTGGATC TCGGACAGATTTCACTCTCACCATTAGCAATGTGCAGTCTGAAGACTTGG
CAGATTATTTCTGTCACCAATATAGCAGCTATCCATTCACGTTCGGCTCG GGGACAAAGTTGGAAATAAAG (SEQ ID NO:85)
DIVMTQSHKFLSTSVGDRVTITCKASRDVAIAVAWYQQKPGQSPKLLLFW ASTRHTGVPDRFTGSGSRTDFTLTISNVQSEDLADYFCHQYSSYPFTFGS GTKLEIK (SEQ ID NO:86)
9D12-Vk GACATTGCGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTGGGGCA GAGGGCCACCATATCCTGCAGAGCCAGTGAAACTATTAATTTTTATGGCA CTAGTTTTATGCACTGGTACCAGCAGAAACCAGGACAGTCACCCAAACTC CTCATCTATCGTGCATCCAACCTAGAATCTGGGATCCCTGCCAGGTTCAG TGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTAATCCTGTGGAGG CTGATGATGTTGCAACCTATTACTGTCAGCAAACTAATGAGGATCCGTAC ACGTTCGGAGGGGGGACTAAGTTGGAAATAAAA (SEQ ID NO:87)
DIALTQSPASLAVSLGQRATISCRASETINFYGTSFMHWYQQKPGQSPKL LIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQTNEDPY TFGGGTKLEIK (SEQ ID NO:88)
ISCllvk GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGA TCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG GAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAG CTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTT CAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAACAGAGTGG AGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCG TACACGTTCGGAGGGGGGACCCAGCTGGAAATAAAA (SEQ ID NO: 89)
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPK LLIYKVSNRFSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHVP YTFGGGTQLEIK (SEQ ID NO:90)
15Flvk DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQRSHESPRLLIKY ASQSISGIPSRFSGSGSGSDFTLTINSVEPEDVGVYYCQNGHSFPFTFGS GTKLEIK (SEQ ID NO:91)
18D3vk GACATTGTGATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGA
TAGAGTCTCTCTTTCCTGCAGGGCCAGTCAGAGTATTAGCGACTACTTAT ACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAATTT GCTTCCCAATCCATCTCTGGGATCCCCTCCAGGTTCACTGGCAGTGGATC AGGGTCAGATTTCACTCTCAGTATCGACAGTGTGGAACCTGATGATGTTG GAGTCTTTTTCTGTCAAAATGGTCACAGCTTTCCGTTCACGTTCGGAGGG GGGACCAAGCTGGAAATAAAA (SEQ ID NO:92)
DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLYWYQQKSHESPRLLIKF ASQSISGIPSRFTGSGSGSDFTLSIDSVEPDDVGVFFCQNGHSFPFTFGG GTKLEIK (SEQ ID NO:93)
18C4vk GACATTGTGATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGA TAGAGTCTCTCTTTCCTGCAGGGCCAGCCAGAGTATTAGCGAGTACTTAC ACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAATAT GCTTCCCAATCCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATC AGGGTCAGATTTCACTCTCAGTATCAACAGTGTGGAACCTGAAGATGTTG GAGTGTATTACTGTCAAAATGGTCACAGCTTTCCATTCACGTTCGGCTCG GGGACAAAGTTGGAAATAAAA (SEQ ID NO:94)
DIVMTQSPATLSVTPGDRVSLSCRASQSISEYLHWYQQKSHESPRLLIKY ASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPFTFGS GTKLEIK (SEQ ID NO:95)
18E5vk GACATTGTGATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGA TAGAGTCTCTCTTTCCTGCAGGGCCAGCCAGAGTATTAGCGACTACTTAT ACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAATTT GCTTCCCAATCCATCTCTGGGATCCCCTCCAGGTTCACTGGCAGTGGATC AGGGTCAGATTTCACTCTCAGTATCGACAGTGTGGAACCTGATGATGTTG GAGTCTTTTTCTGTCAAAATGGTCACAGCTTTCCGTTCACGTTCGGAGGG GGGACCAAGCTGGAAATAAAA (SEQ ID NO:96)
DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLYWYQQKSHESPRLLIKF ASQSISGIPSRFTGSGSGSDFTLSIDSVEPDDVGVFFCQNGHSFPFTFGG GTKLEIK (SEQ ID NO:97)
20E8vk GACATTGTGATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGA TAGAGTCTCTCTTTCCTGCAGGGCCAGCCAGAGTATTAGCGAGTATTTAC
ACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAATAT GCTTCCCAATCCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATC AGGGTCAGATTTCACTCTCAGTATCAACAGTGTGGAACCTGAAGATGTTG GAGTTTATTACTGTCAAAATGGTCACAGCTTTCCATTCACGTTCGGCTCG GGGACAAAGTTGGAAATAAAA (SEQ ID NO:98)
DIVMTQSPATLSVTPGDRVSLSCRASQSISEYLHWYQQKSHESPRLLIKY ASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPFTFGS GTKLEIK (SEQ ID NO:99)
22E2vk GACATTGTGATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGA TAGAGTCTCTCTCTCCTGCAGGGCCAGCCAGAGTATTAGCGTCTACTTAC ACTGGTATCAACAAAAATCACCTGAGTCTCCAAGGCTTCTCATCAAATAT GCTTCCCAATCCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATC AGGGTCAGATTTCACTCTCAGTATCAACAGTGTGGAACCTGAAGATGTTG GAGTTTATTACTGTCAAAATGGTCACAGCTTTCCATTCACGTTCGGCTCG GGGACAAAGTTGGAAATAAAA (SEQ ID NO:100)
DIVMTQSPATLSVTPGDRVSLSCRASQSISVYLHWYQQKSPESPRLLIKY ASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPFTFGS GTKLEIK (SEQ ID NO:101)
24A54vk GACATTGTGATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAAA TAGAGTCTCTCTTTCCTGCAGGGCCAGCCAGAGTATTAGCGACTACTTAC ACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAATAT GCTTCCCAATCCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATC AGGGTCAAATTTCACTCTCAGTATCAACAGTGTGGAACCTGAAGATGTTG GAGTGTATTATTGTCAAAATGGTCACAGCTTTCCATTCACGTTCGGCTCG GGGACAAAGTTGGAAATAAAA (SEQ ID NO:102)
DIVMTQSPATLSVTPGNRVSLSCRASQSISDYLHWYQQKSHESPRLLIKY ASQSISGIPSRFSGSGSGSNFTLSINSVEPEDVGVYYCQNGHSFPFTFGS GTKLEIK (SEQ ID NO:103)
26F7VX GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTTTCTCTGGGGCA GAGGGCCACCATCTCATGCAGGGCCAGCAAAAGTGTCAGATTCTCTGACT ATTTTTATATGCACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTC
CTCATCTACCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAG TGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGG AGGAGGATGCTGCAACCTATTACTGTCAGAACAGTAGGGAGCTTCCGTAC ACGTTCGGAGGGGGGACCAAGCTGGAGATAAAA (SEQ ID NO:104)
DIVLTQSPASLAVSLGQRATISCRASKSVRFSDYFYMHWYQQKPGQPPKL LIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQNSRELPY TFGGGTKLEIK (SEQ ID NO:105)
27G8vk GACATTGTGTTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCA GAGGGCCACCATCTCATGCAGGGCCAGCAAAAGTGTCAGTACATCTGGCT ATAGTTATATACACTGGTACCAACAGAAACCGGGACAGCCACCCAAATTC CTCATCTATCTTGCATCCAACCTAGATTCTGGGGTCCCTGCCAGGTTCAG TGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGG AGGAGGATGCTGCAACCTATTACTGTCAGCACAGTAGGGAGCTTCCGTAC ACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO:106)
DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYIHWYQQKPGQPPKF LIYLASNLDSGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPY TFGGGTKLEIK (SEQ ID NO:107)
31H9vk GACATTGTGATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGA TAGAGTCTCTCTTTCCTGCAGGGCCAGCCAGAGTATTAGCGACTACTTAC ACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAATAT GCTTCCCAATCCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATC AGGGTCAGATTTCACTCTCAGTATCAACAGTGTGGAACCTGAAGATGTTG GAGTGTATTACTGTCAAAATGGTCACAGCTTTCCGTACACGTTCGGAGGG GGGACCAAGCTGGAAATAAAA (SEQ ID NO:108)
DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKY ASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPYTFGG GTKLEIK (SEQ ID NO:109)
34G3Vk GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGA TCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG GAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAG CTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCCGACAGGTT
CAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGG AGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCG TACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAT (SEQ ID NO:110)
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPK LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVP YTFGGGTKLEIN (SEQ ID NO:111)
34D9Vk GACATTATGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGA CAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGGGTAATGCTGTGG TCTGGTATCAACAAAAACCAGGGCAATCTCCTAAACTACTGATTTACTGG GCATCCACCCGGCACATTGGAGTCCCTGATCGCTTCACAGGCAGTGGATC TGGGACAGATTTCACTCTCACCATTACCAATGTGCAGTCTGAAGACTTGG CAGATTATTTCTGTCAGCAATATAGCAGCTATCTCACGTTCGGTGCTGGG ACCAAGCTGGAGCTGAAA (SEQ ID NO:112)
DIMMTQSHKFMSTSVGDRVSITCKASQDVGNAVVWYQQKPGQSPKLLIYW ASTRHIGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYSSYLTFGAG TKLELK (SEQ ID NO:113)
43F5vk GATGTTGTGATGACCCAATCTCCACTCTCCCTGCCTGTCAGTCTTGGAGA TCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG GAAACACCTATCTACATTGGTACCTGCTGAAGCCAGGCCAGTCTCCAAAG CTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTT CAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGG AGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCG TACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO:114)
DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLLKPGQSPK LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVP YTFGGGTKLEIK (SEQ ID NO:115)
73H10Vk GACATCCAGATGACTCAGTCTCCAGTTTTCCTGTCTGCATCTGTGGGAGA AACTGTCACCATCACATGTCGAGCAAGTGAGAATATTGACAGTTATTTAG
CATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTTTGCT GCACGAAACTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATC AGGCACACAGTATTCTCTCAAGATCAACAGAATGCAGTCTGAAGATGTTG CGAGATACTACTGTCAACATTATTATAGTACTCCATTCACGTTCGGCTCG GGGACAAAGTTGGAAATAGAA {SEQ ID NO:116)
DIQMTQSPVFLSASVGETVTITCRASENIDSYLAWYQQKQGKSPQLLVFA ARNLADGVPSRFSGSGSGTQYSLKINRMQSEDVARYYCQHYYSTPFTFGS GTKLEIE (SEQ ID NO:117)
74H3Vk GACATCCAGATGACTCAGTCGCCAGCTTCCCTGTCTGCATCTGTGGGAGA AACTGTCATCTTCACATGTCGAGCAAGTGAGAATATTGACAGTTATTTAG CATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATGCT GCAACAAACTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATC AGGCACACAGTATTCTCTCAAGATCAACAGCCTGCAGTCTGAAGATGTTG CGAGATATTACTGTCTACATTATTATAGTACTCCATTCACGTTCGGCTCG GGGACAGAGTTGGAAATAAAA (SEQ ID NO:118)
DIQMTQSPASLSASVGETVIFTCRASENIDSYLAWYQQKQGKSPQLLVYA ATNLADGVPSRFSGSGSGTQYSLKINSLQSEDVARYYCLHYYSTPFTFGS GTELEIK (SEQ ID NO:119)
Table 2: Anti-IL-23p19 Mouse Leads - VH Sequences
2Dlvh CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGC CTGTCCATCACATGCACTGTCTCTGGGTTCTCATTAACCACCTATGCTATA AGCTGGGTTCGCCAGTCACCAGGAAAGGGTCTGGAGTGGCTTGGAGTCATA TGGACTGGTGGAGGCACAAAATATAATTCAGCTCTCAAATCCAGACTGAGC ATCAGCAAAGACAACTCCAAGAGTCAAGTTTTCTTAAAAATGAACAGTCTG CAAACTGATGACACAGCCAGGTACTACTGTGCCAGAAAGGACTATAATTAC GGGGGTGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID N0:120)
QVQLKESGPGLVAPSQSLSITCTVSGFSLTTYAISWVRQSPGKGLEWLGVI WTGGGTKYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYCARKDYNY GGAMDYWGQGTSVTVSS (SEQ ID NO:121)
6B8VH CAGGTTCAGCTGCAACAGTCTGACGCTGAGTTGGTGAAACCTGGCACTTCA
GTGAAGACATCCTGCAAAATTTCTGGCAACACCTTCACTGACCAAACTATT CACTGGATGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATT TATCCTAGAGATGATAGTCCTAAGTACAATGAGAACTTCAAGGGCAAGGCC ACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAACAGT CTGACATCTGAGGACTCTGCAGTCTATTTCTGTGCAATCCCAGACAGGTCA GGCTACGCCTGGTTTATTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCT TCA (SEQ ID NO:122)
QVQLQQSDAELVKPGTSVKTSCKISGNTFTDQTIHWMKQRPEQGLEWIGYI YPRDDSPKYNENFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCAIPDRS GYAWFIYWGQGTLVTVSS (SEQ ID NO:123)
9D12VH CAGGTGCAGCTGAAGGAGTCAGGACCTGTCCTGGTGGCGCCCTCACAGAGC CTGTCCATCACATGCACTGTCTCTGGGTTCTCATTAAACAACTTTGCTATA AGTTGGGTTCGTCAGCCACCAGGAAAGGGTCTGGAGTGGCTTGGAGCAATA TGGACTGGTGGAGGCACAAATTATAATTCAGCTCTCAAATCCAGACTGAGC ATCAGCAAAGACAACTCCAAGAGTCAAGTTTTCTTAAAAATGAACAGTCTG CAAACTGATGACACAGCCAGGTATTATTGTGTCAGAAAGGACTATAGTTAC GGGGGTGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO:124)
QVQLKESGPVLVAPSQSLSITCTVSGFSLNNFAISWVRQPPGKGLEWLGAI WTGGGTNYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYCVRKDYSY GGAMDYWGQGTSVTVSS (SEQ ID NO:125)
ISCllvh GAGGTCCAGCTGCAACAGTCTGGACCTGTGCTGGTGAAGCCTGGGGCTTCA GTGAAGATGTCCTGTAAGGCTTCTGGATACACATTCACTGACTACTATATG AACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGTTATT ATTCCTTACAACGGTGGTACTAGCTACAACCAGAAGTTCAAGGGCAAGGCC ACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGGAGCTCAACAGC CTGACATCTGAGGACTCTGCAGTCTATTACTGTGCACGAGATGGTCACCGC TGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO:126)
EVQLQQSGPVLVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGVI
IPYNGGTSYNQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARDGHR WYFDVWGTGTTVTVSS (SEQ ID NO:127)
lSFlvh EVQLQQSGPELVKPGASVKMSCKASGYTFTCCIMHWVKQKPGQGLEWIGYI NPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARRWDE AYWGQGTLVTVSA (SEQ ID NO:128)
18D3vh GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTCAAGCCTGGGGCTTCA GTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTCGCTATCTTATT CACTGGGTGAAACAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATT AATCCTTACAATGATGGTACTAAATACAATGAGAAGTTCAAAGGCAAGGCC ACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAGCAGC CTGACCTCTGAGGACTCTGCGGTCTATTACTGTACCTCTAACTGGGACCTC GACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 129)
EVQLQQSGPELVKPGASVKMSCKASGYTFTRYLIHWVKQKPGQGLEWIGYI NPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCTSNWDL DYWGQGTTLTVSS (SEQ ID NO:130)
18C4vh GAGGTCCAGCTGCAGCAGTCTGGACCTGAAGTGGTAAAGCCTGGGGCTTCA GTGAAGATGTCCTGCAAGGCCTCTGGATACACATTCACTAGTTCTGTTATA CACTGGGTGAAGCAGAAGGCTGGGCAGGGCCTTGAGTGGATTGGATATATC AATCCCTATAATGATGGTACTAAGTACAATGAGAAGTTCAAAGGCAAGGCC ACACTGACTTCAGACAGATCCTCCAGCACAGCCTACATGGAGCTCAGCAGC CTGACCTCTGAGGACTCTGCGGTCTATTACTGTACAAGACGGTTGGACGAG GCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:131)
EVQLQQSG PEVVKPGASVKMSCKASGYT FTSSVIHWVKQKAGQGLEWIGYI NPYNDGTKYNEKFKGKATLTSDRSSSTAYMELSSLTSEDSAVYYCTRRLDE AYWGQGTLVTVSA (SEQ ID NO:132)
18E5vh GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTGCA GTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTCGCTATCTTATT CACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATT
AATCCTTACAATGATGGTACTAAATATAATGAGAAGTTCAAAGGCAAGGCC ACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAGCAGC CTGACCTCTGAGGACTCTGCGGTCTATTACTGTACCTCTAATTGGGACCTC GACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:133)
EVQLQQSGPELVKPGAAVKMSCKASGYTFTRYLIHWVKQKPGQGLEWIGYI NPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCTSNWDL DYWGQGTTLTVSS (SEQ ID NO:134)
20E8vh GAGGTCCAGCTGCAGCAGTCTGGACCTGAACTGGTAAAGCCTGGGGCTTCA GTGAAGATGTCCTGCAAGGCCTCTGGATACACATTCACTAGTTCTGTTATG CACTGGGTGAAGCAGAAGGCTGGGCAGGGCCTTGAGTGGATTGGATATATC AATCCCTATAATGATGGTACTCAGTACAATGAGAAGTTCAAAGGCAAGGCC ACACTGACTTCAGACAAATTTTCCAGCACAGCCTACATGGAGCTCAGCAGC CTGACCTCTGAGGACTCTGCGGTCTATTACTGTACAAGACGGTTGGACGAG GCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:135)
EVQLQQSGPELVKPGASVKMSCKASGYTFTSSVMHWVKQKAGQGLEWIGYI NPYNDGTQYNEKFKGKATLTSDKFSSTAYMELSSLTSEDSAVYYCTRRLDE AYWGQGTLVTVSA (SEQ ID NO:136)
22E2vh GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCA GTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTCTATTATT CACTGGGTGAAGCAGAGGCCTGGGCAGGGCCTTGAGTGGATTGGATATATT AATCCTTACGATGATGTTACTAAGTACAATGAGAAGTTCAAAGGCAAGGCC ACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAGCAGC CTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGACGGTGGGACGAG TCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:137)
EVQLQQSGPELVKPGASVKMSCKASGYTFTSSIIHWVKQRPGQGLEWIGYI NPYDDVTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARRWDE SYWGQGTLVTVSA (SEQ ID NO:138)
24A5vh GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCA
GTGAAGATGTCCTGCAAGGCTTCTGGATACACTTTCACTACCTCTATTATG CACTGGGTGAAACAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATT AATCCTTACGATGATGTTACTAAGTACAATGAAAAGTTCAAAGGCAAGGCC ACATTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAGCAGC CTGACCTCTGAGGACTCTGCAGTCTATTACTGTGTAAGACGGTGGGACGAG GCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:139)
EVQLQQSGPELVKPGASVKMSCKASGYTFTTSIMHWVKQKPGQGLEWIGYI NPYDDVTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCVRRWDE AYWGQGTLVTVSA (SEQ ID NO:140)
26F7VH GAGGTCCAGCTGCAACAATCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCA GTGAAGATATCCTGTAAGGCTTCTGGATACACGTTTACTGACTACTACATG AACTGGGTGAGGCAGAGCCATGGAGAGAGCCTTGAGTGGATTGGAGATTTT AATCATAACAATGATGTTATTACTTACAACCCGAAGTTCAAGGGCAAGGTC ACATTGACTGTAGAGAAGTCTTCCACCACAGCCTACATGGAGCTCCGCAGC CTGTCATCTGAGGACTCTGCAGTCTATTACTGTGCAAGGGGGCTACGAGGC TACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO:141)
EVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVRQSHGESLEWIGDF NHNNDVITYNPKFKGKVTLTVEKSSTTAYMELRSLSSEDSAVYYCARGLRG YYAMDYWGQGTSVTVSS (SEQ ID NO:142)
27G8vh CAGGTTCAGCTGCAACAGTCTGACGCTGAGTTGGTGAAACCTGGAGCTTCA GTGAAGATATCCTGCAAGGTTTCTGGCTACACCTTCACTGACCATACTATT CACTGGATGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATT TATCCTAGAGATGGTTATCCTAAGTTCAATGAGAAGTTCAAGGGCAAGGCC ACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAACAGC CTGACATCTGAGGACTCTGCAGTCTATTTCTGTGCAAGACGGCCCCCTTAC TATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCGCCGTCTCCTCA (SEQ ID NO:143)
QVQLQQSDAELVKPGASVKISCKVSGYTFTDHTIHWMKQRPEQGLEWIGYI YPRDGYPKFNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCARRPPY
YAMDYWGQGTSVAVSS (SEQ ID NO:144)
31H9vh GAGGTCCAACTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCA GTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGGTATCTTATG CACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGTTATATT AATCCTTACAATGATGGTACTAATTACAATGAGAAGTTCAAAGGCAAGGCC ACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAGCAGC CTGACCTCTGAGGACTCTGCGGTCTATTACTGTTCCCTTAACTGGGACTAT GCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:145)
EVQLQQSGPELVKPGASVKMSCKASGYTFTRYLMHWVKQKPGQGLEWIGYI NPYNDGTNYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCSLNWDY AYWGQGTLVTVSA (SEQ ID NO:146)
34G3VH GAGTTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGCGCTTCA GTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGACTACAACATG AACTGGGTGAAGCAGAGCAAAGGAAAGAGCCTTGAGTGGATTGGAGTAATT ATTCCTAACTATGGTTTTACTAGCTACAATCAGAACTTCAAGGGCAAGGCC ACTTTGACTGTAGACCAGTCTTCCAGCACAGCCCACATGCAGCTCAACAGT GTGACATCTGAGGACTCTGCAGTCTATTACTGTGTAAGAGATGGGGGAATA CTCCTCTGGTATCTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCC TCA (SEQ ID NO:147)
EFQLQQSGPELVKPGASVKISCKASGYSFTDYNMNWVKQSKGKSLEWIGVI IPNYGFTSYNQNFKGKATLTVDQSSSTAHMQLNSVTSEDSAVYYCVRDGGI LLWYLDVWGTGTTVTVSS (SEQ ID NO:148)
34D9VH7 GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCA GTGAAGATACCCTGCAAGGCTTCTGGATACACATTCACTGACTACAACATG GACTGGGTGAAGAAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATC AATCCTCACAATGGTGGTACTATCTACAACCAGAAGTTCAAGGGCAAGGCC ACATTGACTGTAGACAAGTCCTCCAGCACAGCCCACATGGAGCTCCGCAGC CTGACATCTGAGGACACTGCAGTCTATTACTGTGCAAGAAATTACTACGGT AGTAGTTACGGCTGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACC GTCTCGTCA (SEQ ID NO:149) f
EVQLQQSGPELVKPGASVKIPCKASGYTFTDYNMDWVKKSHGKSLEWIGDI NPHNGGTIYNQKFKGKATLTVDKSSSTAHMELRSLTSEDTAVYYCARNYYG SSYGWYFDVWGTGTTVTVSS (SEQ ID NO:150)
43F5vh GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCA GTGAAGATTTCCTGCAGGGCTTCTGGTTACTCATTCACTGGCTACTACATG AACTGGGTGAAGCAAAGTCCTGAAAAGAGCCTTGAGTGGATTGGAGAGATT ATTCCTACCACTGGTGGTACTTCCTACAACCAGAAGTTCAAGGCCAAGGCC ACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAAGAGC CTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGAGAGCGGTGGG TTCTACTGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCC TCA (SEQ ID NO:151)
EVQLQQSGPELVKPGASVKISCRASGYSFTGYYMNWVKQSPEKSLEWIGEI IPTTGGTSYNQKFKAKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARESGG FYWYFDVWGTGTTVTVSS (SEQ ID NO:152)
73H10VH GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCA GTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGGTATGTTATG CACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATT AATCCTTACAATGATGTTACTAAGTACAATGAGAAGTTCAAAGGCAAGGCC ACACTGACTTCAGACAGATCCTCCAGCACAGCCTACATGAAACTCAGCAGC CTGACCTCTGAGGACTCTGCGGTCTATTATTGTGCAAGAAACTGGGACGTT CCTTACTGGGGCCAAGGGACTCTGATCACTGTCTCTGCA (SEQ ID NO:153)
EVQLQQSGPELVKPGASVKMSCKASGYTFTRYVMHWVKQKPGQGLEWIGYI NPYNDVTKYNEKFKGKATLTSDRSSSTAYMKLSSLTSEDSAVYYCARNWDV PYWGQGTLITVSA (SEQ ID NO:154)
74H3VH GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCA GTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGGTATCTTATG CACTGGGTGAAGCAGAAGCCTGGACAGGGCCTTGAGTGGATTGGATATATT AATCCTTACAATGATGGTACTAAGTACAATGAGAGGTTCAAAGGCAAGGCC
ACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAGCAGC CTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGAAACTGGGACGTA CCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:155)
EVQLQQSGPELVKPGASVKMSCKASGYTFTRYLMHWVKQKPGQGLEWIGYI NPYNDGTKYNERFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARNWDV PYWGQGTLVTVSA (SEQ ID NO:156)
Human framework sequences were selected for each of the mouse leads based on the framework homology, CDR structure, conserved canonical residues, conserved interface packing residues and other parameters.
The mouse light chain and heavy chain CDRs of the various mouse antibodies are shown in Table 3 and Table 4, respectively. Table 4 also shows three heavy chains CDRs derived from the mouse antibody 6B8 through the humanization process.
Table 3: LIGHT CHAIN CDR sequences
L-CDR1 L-CDR2 L-CDR3
18C4 RASQSISEYLH (SEQ ID NO:1) YASQSIS (SEQ ID NO;2) QNGHSFPFT (SEQ ID NO:3)
18E5 RASQSISDYLY (SEQ ID NO:4) FASQSIS (SEQ ID NO:5) QNGHSFPFT (SEQ ID NO:3)
18D3 RASQSISDYLY (SEQ ID NO:4) FASQSIS (SEQ ID NO:5) QNGHSFPFT (SEQ ID NO:3)
20E8 RASQSISEYLH (SEQID NO:1) YASQSIS (SEQ ID NO:2) QNGHSFPFT (SEQ ID NO:3)
22E2 RASQSISVYLH (SEQID NO:6) YASQSIS (SEQ ID NO:2) QNGHSFPFT (SEQ ID NO:3)
24A5 RASQSISDYLH (SEQ ID NO:7) YASQSIS (SEQ ID NO:2) QNGHSFPFT (SEQ ID NO:3)
15C11 RSSQSLVHSNGNTYLH KVSNRFS SQSTHVPYT
(SEQ ID N0:8) (SEQ ID NO:9) (SEQ ID NO:10)
43F 5 RSSQSLVHSNGNTYLH (SEQ ID N0:8) KVSNRFS (SEQ ID NO:9) SQSTHVPYT (SEQ ID NO:10)
27G8 RASKSVSTSGYSYIH (SEQ ID N0:11) LASNLDS (SEQID NO:12) QHSRELPYT (SEQ ID NO:13)
31H9 RASQSISDYLH (SEQ ID N0:7) YASQSIS (SEQ ID NO:2) QNGHSFPYT (SEQ ID NO:14)
2D1 RTSESVYSYGQNFIH (SEQ ID NO: 15) RASNLES (SEQIDNO:16) QQTNEDPYT (SEQ ID NO:17)
9D12 RASETINFYGTSFMH (SEQ ID NO:18) RASNLES (SEQ ID NO:16) QQTNEDPYT (SEQID NO:17)
6B8 KASRDVAIAVA (SEQ ID NO:19) WASTRHT (SEQ ID NO:20) HQYSSYPFT (SEQ ID NO:21)
73H10 RASENIDSYLA (SEQ ID NO:22) AARNLAD (SEQ ID NO:23) QHYYSTPFT (SEQ ID NO:24)
74H3 RASENIDSYLA (SEQ ID NO:22) AATNLAD (SEQ ID NO:25) LHYYSTPFT (SEQ ID NO:26)
35H8 RSSQSLVHSNGNTYLH (SEQ ID NO:8) KVSNRFS (SEQ ID NO:9) SQSTHVPYT (SEQ ID NO:10)
26F7 RASKSVRFSDYFYMH (SEQ ID NO:27) LASNLES (SEQ ID NO:28) QNSRELPYT (SEQ ID NO:29)
34G3 RSSQSLVHSNGNTYLH (SEQ ID NO:8) KVSNRFS (SEQ ID NO:9) SQSTHVPYT (SEQIDNO:10)
34D9 KASQDVGNAW (SEQ ID NO:30) WASTRHI (SEQ ID NO:31) QQYSSYLT (SEQ ID NO:32)
Table 4: HEAVY CHAIN CDR sequences
H-CDR1 H-CDR2 H-CDR3
18C4 GYTFTSSVIH (SEQ ID NO:33) YINPYNDGTKYNEKFKG (SEQ ID NO:34) RLDEAY (SEQ ID NO:35)
18E5 GYTFTRYLIH (SEQ ID NO:36) YINPYNDGTKYNEKFKG (SEQ ID NO:34) NWDLDY (SEQ ID NO:37)
18D3 GYTFTRYLIH (SEQ ID NO:36) YINPYNDGTKYNEKFKG (SEQ ID NO:34) NWDLDY (SEQ ID NO:37)
20E8 GYTFTSSVMH (SEQ ID NO:38) YINPYNDGTQYNEKFKG (SEQ ID NO:39) RLDEAY (SEQ ID NO:35)
22E2 GYTFTSSIIH (SEQ ID NO:40) YINPYDDVTKYNEKFKG (SEQIDNO:41) RWDESY (SEQ ID NO:42)
24A5 GYTFTTSIMH (SEQ ID NO:43) YINPYDDVTKYNEKFKG (SEQIDNO:41) RWDEAY (SEQ ID NO:44)
15C11 GYTFTDYYMN (SEQ ID NO:45) VIIPYNGGTSYNQKFKG (SEQ ID NO:46) DGHRWYFDV (SEQ ID NO:47)
43F5 GYSFTGYYMN (SEQ ID NO:48) EIIPTTGGTSYNQKFKA (SEQ ID NO:49) ESGGFYWYFDV (SEQ ID NO:50)
27G8 GYTFTDHTIH (SEQ ID N0:51) YIYPRDGYPKFNEKFKG (SEQ ID NO:52) RPPYYAMDY (SEQ ID NO:53)
31H9 GYTFTRYLMH (SEQ ID NO: 54) YINPYNDGTNYNEKFKG (SEQ ID NO:55) NWDYAY (SEQ ID NO:56)
2D1 GFSLTTYAIS (SEQ ID NO:57) VIWTGGGTKYNSALKS (SEQ ID NO:58) KDYNYGGAMDY (SEQ ID NO:59)
9D12 GFSLNNFAIS (SEQ ID NO:60) AIWTGGGTNYNSALKS (SEQ ID NO:61) KDYSYGGAMDY (SEQ ID NO:62)
6B8 GNTFTDQTIH (SEQ ID NO:63) YIYPRDDSPKYNENFKG (SEQ ID NO:64) PDRSGYAWFIY (SEQ ID NO:65)
Hu_6B8-2 GYTFTDQTIH (SEQ ID NO:66) YIYPRDDSPKYNENFKG (SEQ ID NO:64) PDRSGYAWFIY (SEQ ID NO:65)
Hu_6B8-5 GFTFTDQTIH (SEQ ID NO:67) YIYPRDDSPKYNENFKG (SEQ ID NO:64) PDRSGYAWFIY (SEQ ID NO:65)
Hu_6B8- 36/65 GGTFTDQTIH (SEQ ID NO:68) YIYPRDDSPKYNENFKG (SEQ ID NO:64) PDRSGYAWFIY (SEQ ID NO:65)
73H10 GYTFTRYVMH (SEQ ID NO:69) YINPYNDVTKYNEKFKG (SEQ ID NO:70) NWDVPY (SEQ ID NO:71)
74H3 GYTFTRYLMH (SEQ ID NO: 54) YINPYNDGTKYNERFKG (SEQ ID NO:72) NWDVPY (SEQ ID NO:71)
35H8 GYTFTDYYMN VIIPYNGGISYNQKFKG NDYDWYFDV
(SEQ ID NO:45) (SEQ ID NO:73) (SEQ ID NO:74)
26F7 GYTFTDYYMN (SEQ ID NO:45) DFNHNNDVITYNPKFKG (SEQ ID NO:75) GLRGYYAMDY (SEQ ID NO:76)
34G3 GYSFTDYNMN (SEQ ID NO:77) VIIPNYGFTSYNQNFKG (SEQ ID NO:78) DGGILLWYLDV (SEQ ID NO:79)
34D9 GYTFTDYNMD (SEQ ID NO:80) DINPHNGGTIYNQKFKG (SEQ ID NO:81) NYYGSSYGWYFDV (SEQ ID NO:82)
The CDRs listed above in Tables 3 and 4 are defined using the Chothia numbering System (Al-Lazikani et al., (1997) JMB 273,927-948).
Fabs that showed better or equal binding as compared to the chimeric parent Fab were selected for conversion to IgG. 6B8 was converted to an IgGIKO format. IgG 1 KO (knock-out of effector functions) has two mutations in the Fc région, Leu234Ala and Leu235Ala, which reduce effector function such as FcyR and complément binding. The IgG format is described in the literature (see for example Hezareh et al. (2001) Journal of Virology 75: 12161-12168). Example 1 describes the humanization process in further detail. The results of such humanization resulted in humanized antibody sequences. A représentative number of humanized light chain and heavy chain variable régions derived from mouse antibody 6B8 are provided and shown in Tables 5 and 6. An alignment between the humanized light chain and heavy chain variable régions derived from mouse antibody 6B8 and the chain and heavy chain variable régions from mouse antibody 6B8 is shown in Figure 1.
Selected combination of humanized light chain and heavy chain variable régions derived from mouse antibody 6B8 resulted in Antibodies A, B, C and D:
Antibody A: 6B8-lgG1KO-2 with lgK-66 (heavy chain variable région 6B8CVH-02 and light chain variable région 6B8CVK-66);
Antibody B: 6B8-lgG1KO-5 with lgK-66 (heavy chain variable région 6B8CVH-05 and light chain variable région 6B8CVK-66);
Antibody C: 6B8-lgG1KO-2 with lgK-65 (heavy chain variable région 6B8CVH-02 and light chain variable région 6B8CVK-65);
-55Antibody D: 6B8-lgG1KO-5 with lgK-65 (heavy chain variable région 6B8CVH-05 and Iight chain variable région 6B8CVK-65).
Antibodies A, B, C and D hâve the heavy and Iight chain sequences shown in Table 7.
Table 5: Humanized 6B8-VK Sequences
6B8CVK-65 Gacatccagatgacccagagcccaagcagcctgagcgccagcgtggg cgaccgcgtgaccatcacctgcaaggccagccgcgacgtggccatcg ccgtggcctggtaccagcagaagccaggcaaggtgccaaagctgctg ctgttctgggccagcacccgccacaccggcgtgccagaccgcttcag cggcagcggcagcggcaccgacttcaccctgaccatcagcagcctgc agccagaggacctggccgactactactgccaccagtacagcagctac ccattcaccttcggccagggcaccaagctggagatcaag (SEQ ID NO:157)
DIQMTQSPSSLSASVGDRVTITCKASRDVAIAVAWYQQKPGKVPKLL LFWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDLADYYCHQYSSY PFTFGQGTKLEIK (SEQ ID NO:158)
6B8CVK-66 Gacatccagatgacccagagcccaagcagcctgagcgccagcgtggg cgaccgcgtgaccatcacctgcaaggccagccgcgacgtggccatcg ccgtggcctggtaccagcagaagccaggcaaggtgccaaagctgctg atctactgggccagcacccgccacaccggcgtgccaagccgcttcag cggcagcggcagccgcaccgacttcaccctgaccatcagcagcctgc agccagaggacgtggccgactacttctgccaccagtacagcagctac ccattcaccttcggcagcggcaccaagctggagatcaag (SEQ ID NO:159)
DIQMTQSPSSLSASVGDRVTITCKASRDVAIAVAWYQQKPGKVPKLL IYWASTRHTGVPSRFSGSGSRTDFTLTISSLQPEDVADYFCHQYSSY PFTFGSGTKLEIK (SEQ ID NO:160)
6B8CVK-67 Gacatccagatgacccagagcccaagcagcctgagcgccagcgtggg cgaccgcgtgaccatcacctgcaaggccagccgcgacgtggccatcg ccgtggcctggtaccagcagaagccaggcaaggtgccaaagctgctg
ctgtactgggccagcacccgccacaccggcgtgccaagccgcttcag cggcagcggcagccgcaccgacttcaccctgaccatcagcagcctgc agccagaggacgtggccacctactactgccaccagtacagcagctac ccattcaccttcggcagcggcaccaagctggagatcaag (SEQ ID NO:161)
DIQMTQSPSSLSASVGDRVTITCKASRDVAIAVAWYQQKPGKVPKLL LYWASTRHTGVPSRFSGSGSRTDFTLTISSLQPEDVATYYCHQYSSY PFTFGSGTKLEIK (SEQ ID NO:162)
6B8CVK-78 Gacatccagatgacccagagcccaagcagcctgagcgccagcgtggg cgaccgcgtgaccatcacctgcaaggccagccgcgacgtggccatcg ccgtggcctggtaccagcagaagccaggcaaggtgccaaagctgctg ctgttctgggccagcacccgccacaccggcgtgccagaccgcttcag cggcagcggcagccgcaccgacttcaccctgaccatcagcagcctgc agccagaggacctggccgactactactgccaccagtacagcagctac ccattcaccttcggcagcggcaccaagctggagatcaag (SEQ ID NO:163)
DIQMTQSPSSLSASVGDRVTITCKASRDVAIAVAWYQQKPGKVPKLL LFWASTRHTGVPDRFSGSGSRTDFTLTISSLQPEDLADYYCHQYSSY PFTFGSGTKLEIK (SEQ ID NO:164)
Table 6: Humanized 6B8-VH Sequence
6B8CVH-02 Caggtgcagctggtgcagagcggcgccgaggtgaagaagccaggcag cagcgtgaaggtgagctgcaaggccagcggctacaccttcaccgacc agaccatccactggatgcgccaggccccaggccagggcctggagtgg atcggctacatctacccacgcgacgacagcccaaagtacaacgagaa cttcaagggcaaggtcaccatcaccgccgacaagagcaccagcaccg cctacatggagctgagcagcctgcgcagcgaggacaccgccgtgtac tactgcgccatcccagaccgcagcggctacgcctggttcatctactg gggccagggcaccctggtgaccgtgagcagc (SEQ ID NO:165)
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDQTIHWMRQAPGQGLEW
IGYIYPRDDSPKYNENFKGKVTITADKSTSTAYMELSSLRSEDTAVY YCAIPDRSGYAWFIYWGQGTLVTVSS (SEQ ID NO:166)
6B8CVH-05 Caggtgcagctggtgcagagcggcgccgaggtgaagaagccaggcag cagcgtgaaggtgagctgcaaggccagcggcttcaccttcaccgacc agaccatccactgggtgcgccaggccccaggccagggcctggagtgg atgggctacatctacccacgcgacgacagcccaaagtacaacgagaa cttcaagggcaaggtcaccctgaccgccgacaagagcaccagcaccg cctacatggagctgagcagcctgcgcagcgaggacaccgccgtgtac tactgcgccatcccagaccgcagcggctacgcctggttcatctactg gggccagggcaccctggtgaccgtgagcagc (SEQ ID NO:167)
QVQLVQSGAEVKKPGSSVKVSCKASGFTFTDQTIHWVRQAPGQGLEW MGYIYPRDDSPKYNENFKGKVTLTADKSTSTAYMELSSLRSEDTAVY YCAIPDRSGYAWFIYWGQGTLVTVSS (SEQ ID NO:168)
6B8CVH-36 Caggtgcagctggtgcagagcggcgccgaggtgaagaagccaggcag cagcgtgaagaccagctgcaaggccagcggcggcaccttcaccgacc agaccatccactgggtgcgccagcgcccaggccagggcctggagtgg atgggctacatctacccacgcgacgacagcccaaagtacaacgagaa cttcaagggccgcgtcaccatcaccgccgacaagagcaccagcaccg cctacatggagctgagcagcctgcgcagcgaggacaccgccgtgtac tactgcgccatcccagaccgcagcggctacgcctggttcatctactg gggccagggcaccctggtgaccgtgagcagc (SEQ ID NO:169)
QVQLVQSGAEVKKPGSSVKTSCKASGGTFTDQTIHWVRQRPGQGLEW MGYIYPRDDSPKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVY YCAIPDRSGYAWFIYWGQGTLVTVSS (SEQ ID NO:170)
6B8CVH-65 Caggtgcagctggtgcagagcggcgccgaggtgaagaagccaggcag cagcgtgaaggtgagctgcaaggccagcggcggcaccttcaccgacc agaccatccactgggtgcgccaggccccaggccagggcctggagtgg atgggctacatctacccacgcgacgacagcccaaagtacaacgagaa tttcaagggccgcgtcaccctgaccgccgacaagagcaccagcaccg cctacatggagctgagcagcctgcgcagcgaggacaccgccgtgtac ttctgcgcccgcccagaccgcagcggctacgcctggttcatctactg
gggccagggcaccctggtgaccgtgagcagc (SEQ ID NO:171)
QVQLVQSGAEVKKPGSSVKVSCKASGGTFTDQTIHWVRQAPGQGLEW MGYIYPRDDSPKYNENFKGRVTLTADKSTSTAYMELSSLRSEDTAVY FCARPDRSGYAWFIYWGQGTLVTVSS (SEQ ID NO:172)
Table 7: Heavy and Light Chain DNA and Amino Acid Sequences for Antibodies A, B, C, and D
Antibody A IgK light Chain #66 Gacatccagatgacccagagcccaagcagcctgagcg
ccagcqtqqqcqaccqcqtgaccatcacctqcaaqqc
cagccgcgacgtggccatcgccgtggcctggtaccag
cagaagccaggcaaggtgccaaagctgctgatctact
qqqccagcacccgccacaccggcgtgccaaqccqctt
caqcqqcaqcqqcaqccqcaccqacttcaccctqacc
atcagcagcctgcagccagaggacgtggccgactact
tctqccaccaqtacaqcaqctacccattcaccttcqq
caqcgqcaccaaqctggagatcaagcgtactqtqqct gcaccatctgtcttcatcttcccgccatctgatgagc agttgaaatctggaactgcctctgttgtgtgcctgct gaataacttctatcccagagaggccaaagtacagtgg aaggtggataacgccctccaatcgggtaactcccagg agagtgtcacagagcaggacagcaaggacagcaccta cagcctcagcagcaccctgacgctgagcaaagcagac tacgagaaacacaaagtctacgcctgcgaagtcaccc atcagggcctgagctcgcccgtcacaaagagcttcaa caggggagagtgt (SEQ ID NO:173)
DIQMTQSPSSLSASVGDRVTITCKASRDVAIAVAWYQ
QKPGKVPKLLIYWASTRHTGVPSRFSGSGSRTDFTLT
ISSLQPEDVADYFCHQYSSYPFTFGSGTKLEIKRTVA
APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:174)
IgGl KO Heavy Chain #2 Caggtgcagctggtgcagagcggcgccgaqqtqaaga
agccaqgcagcagcgtgaaggtgagctgcaaggccag
cqqctacaccttcaccqaccaqaccatccactqqatq
cqccaqqccccaqqccaqqqcctqqaqtqqatcqqct
acatctacccacgcgacgacagcccaaagtacaacga
qaacttcaaqqqcaaqqtcaccatcaccqccgacaaq
agcaccagcaccgcctacatggagctgagcagcctgc
gcaqcqaqgacaccgccgtgtactactgcgccatccc
agaccgcagcggctacgcctggttcatctactggggc
cagggcaccctggtgaccgtgagcagcgcctccacca
agggcccatcggtcttccccctggcaccctcctccaa gagcacctctgggggcacagcggccctgggctgcctg gtcaaggactacttccccgaaccggtgacggtgtcgt ggaactcaggcgccctgaccagcggcgtgcacacctt cccggctgtcctacagtcctcaggactctactccctc agcagcgtggtgaccgtgccctccagcagcttgggca cccagacctacatctgcaacgtgaatcacaagcccag caacaccaaggtcgacaagagagttgagcccaaatct tgtgacaaaactcacacatgcccaccgtgcccagcac cagaagctgctgggggaccgtcagtcttcctcttccc cccaaaacccaaggacaccctcatgatctcccggacc cctgaggtcacatgcgtcgtggtggacgtgagccacg aagaccctgaggtcaagttcaactggtacgtggacgg cgtggaggtgcataatgccaagacaaagccgcgggag gagcagtacaacagcacgtaccgtgtggtcagcgtcc tcaccgtcctgcaccaggactggctgaatggcaagga gtacaagtgcaaggtctccaacaaagccctcccagcc cccatcgagaaaaccatctccaaagccaaagggcagc
cccgagaaccacaggtgtacaccctgcccccatcccg ggaggagatgaccaagaaccaggtcagcctgacctgc ctggtcaaaggcttctatcccagcgacatcgccgtgg agtgggagagcaatgggcagccggagaacaactacaa gaccacgcctcccgtgctggactccgacggctccttc ttcctctacagcaagctcaccgtggacaagagcaggt ggcagcaggggaacgtcttctcatgctccgtgatgca tgaggctctgcacaaccactacacgcagaagagcctc tccctgtctccgggt {SEQ ID NO:175)
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDQTIHWM
RQAPGQGLEWIGYIYPRDDSPKYNENFKGKVTITADK
STSTAYMELSSLRSEDTAVYYCAIPDRSGYAWFIYWG
QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG (SEQ ID NO:176)
Antibody B IgK light Chain #66 (SEQ ID NO:173)
(SEQ ID NO:174)
IgGlKO Heavy Chain #5 caggtgcagctggtgcagagcggcgccgaggtgaaga
agccaggcagcagcgtgaaggtgaqctgcaaqqccaq
cggcttcaccttcaccgaccagaccatccactgggtg
cgccaggccccaggccagggcctggagtggatgqgct
acatctacccacgcgacgacagcccaaagtacaacqa
gaacttcaagggcaaggtcaccctgaccqccqacaaq
agcaccagcaccgcctacatggagctgagcagcctqc
gcagcgaggacaccgccgtgtactactgcqccatccc
agaccgcaqcggctacgcctqqttcatctactqqqqc
cagggcaccctggtgaccgtgagcagcqcctccacca agggcccatcggtcttccccctggcaccctcctccaa gagcacctctgggggcacagcggccctgggctgcctg gtcaaggactacttccccgaaccggtgacggtgtcgt ggaactcaggcgccctgaccagcggcgtgcacacctt cccggctgtcctacagtcctcaggactctactccctc agcagcgtggtgaccgtgccctccagcagcttgggca cccagacctacatctgcaacgtgaatcacaagcccag caacaccaaggtcgacaagagagttgagcccaaatct tgtgacaaaactcacacatgcccaccgtgcccagcac cagaagctgctgggggaccgtcagtcttcctcttccc cccaaaacccaaggacaccctcatgatctcccggacc cctgaggtcacatgcgtcgtggtggacgtgagccacg aagaccctgaggtcaagttcaactggtacgtggacgg cgtggaggtgcataatgccaagacaaagccgcgggag gagcagtacaacagcacgtaccgtgtggtcagcgtcc tcaccgtcctgcaccaggactggctgaatggcaagga gtacaagtgcaaggtctccaacaaagccctcccagcc cccatcgagaaaaccatctccaaagccaaagggcagc cccgagaaccacaggtgtacaccctgcccccatcccg ggaggagatgaccaagaaccaggtcagcctgacctgc ctggtcaaaggcttctatcccagcgacatcgccgtgg
agtgggagagcaatgggcagccggagaacaactacaa gaccacgcctcccgtgctggactccgacggctccttc ttcctctacagcaagctcaccgtggacaagagcaggt ggcagcaggggaacgtcttctcatgctccgtgatgca tgaggctctgcacaaccactacacgcagaagagcctc tccctgtctccgggt (SEQ ID NO:177)
QVQLVQSGAEVKKPGSSVKVSCKASGFTFTDQTIHWV
RQAPGQGLEWMGYIYPRDDSPKYNENFKGKVTLTADK
STSTAYMELSSLRSEDTAVYYCAIPDRSGYAWFIYWG
QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG (SEQ ID NO:178)
Antibody C IgK light Chain #65 Gacatccagatgacccagagcccaagcagcctgagcg
ccaqcgtgggcgaccgcgtgaccatcacctgcaaggc
cagccgcgacgtggccatcgccgtggcctggtaccag
cagaagccaggcaaggtgccaaagctgctgctgttct
gggccagcacccgccacaccggcgtgccagaccgctt
caqcqgcagcggcagcggcaccgacttcaccctgacc
atcagcagcctgcagccagaggacctggccgactact
actgccaccagtacagcagctacccattcaccttcgg
ccaqqqcaccaaqctggagatcaagcgtactqtqqct gcaccatctgtcttcatcttcccgccatctgatgagc
agttgaaatctggaactgcctctgttgtgtgcctgct gaataacttctatcccagagaggccaaagtacagtgg aaggtggataacgccctccaatcgggtaactcccagg agagtgtcacagagcaggacagcaaggacagcaccta cagcctcagcagcaccctgacgctgagcaaagcagac tacgagaaacacaaagtctacgcctgcgaagtcaccc atcagggcctgagctcgcccgtcacaaagagcttcaa caggggagagtgt (SEQ ID NO:179)
DIQMTQSPSSLSASVGDRVTITCKASRDVAIAVAWYQ
QKPGKVPKLLLFWASTRHTGVPDRFSGSGSGTDFTLT
ISSLQPEDLADYYCHQYSSYPFTFGQGTKLEIKRTVA
APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:180)
IgGlKO Heavy Chain #2 (SEQ ID NO:175)
(SEQ ID NO:176)
Antibody D IgK light Chain #65 (SEQ ID NO:179)
(SEQ ID NO:180)
IgGlKO (SEQ ID NO:177) —
Heavy Chain #5
(SEQ ID NO:178)
Light chains and heavy chain variable régions of Antibodies A, B, C, and D are underlined in Table 7 above.
In one aspect, a humanized anti-IL-23p19 antibody of the présent invention has at least one of the properties below. In a further aspect, a humanized anti-IL-23p19 antibody of the présent invention has any combination of at least two, or at least 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the properties below. In a further aspect, a humanized anti-IL-23p19 antibody of the présent invention has ail the properties below.
• Kd for human IL-23 < 1 pM (no shift in binding on-rate in 50% human sérum) • Blocks IL-23 binding to human IL-23R/Fc in vitro • No binding to human IL-12 • Inhibîts human IL-23 induced IL-17 production in mouse splénocytes with IC5o's < 20 pM • Inhibîts human IL-23 induced STAT3 phosphorylation in human DB cells with IC50’s <
pM • No predicted activity in ADCC/CDC • KD <1 pM for cynomolgus monkey IL-23 • No cross reactivity to mouse or rat IL-23 • Inhibîts human IL-23 induced IL-17 and IL-22 production in mouse ear (>80% inhibition of both cytokines at 1 mg/kg) • Stability 83°C (melting température 83°C as determined by différentiel scanning calorimetry)
Solubility >100 mg/ml (as measured by UV spectroscopy and monitored by turbidity)(Vji<_' • Subcutaneous administration of 1.0 mg/kg in three cynomolgus monkeys shows sustained £ 10 nM exposure for approximately 28 days with a bioavailability of approximately 70%.
By no predicted activity in ADCC/DC, it is meant herein that a humanized anti-IL-23p19 antibody of the présent invention has reduced affinity to Fc receptor and therefore is predicted not to hâve activity in ADCC/CDC.
In one aspect, a humanized anti-IL-23p19 antibody of the présent invention has at least one of the properties below. In a further aspect, a humanized anti-IL-23p19 antibody of the présent invention has any combination of at least two, or at least 3, 4, 5, 6, 7, 8, 9, or 10 of the properties below. In a further aspect, a humanized anti-IL-23p19 antibody of the présent invention has ail the properties below.
• Kd for human IL-23 < 1 pM (no shift in binding on-rate in 50% human sérum) • Blocks IL-23 binding to human IL-23R/Fc in vitro • No binding to human IL-12 • Inhibits human IL-23 induced IL-17 production in mouse splénocytes with IC5o’s < 20 pM • Inhibits human IL-23 induced STAT3 phosphorylation in human DB cells with IC5o's <
pM • No predicted activity in ADCC/CDC • Kd <1 pM for cynomolgus monkey IL-23 • No cross reactivity to mouse or rat IL-23 • Inhibits human IL-23 induced IL-17 and IL-22 production in mouse ear(>80% inhibition of both cytokines at 1 mg/kg) • Stability 83°C (melting température 83°C as determined by differential scanning calorimetry) • Solubility >100 mg/ml (as measured by UV spectroscopy and monitored by turbidity).
In a further aspect, a humanized antibody of the présent invention has at least one of the following binding properties (properties A). In a further aspect, a humanized anti-IL
-6623p19 antibody of the présent invention has any combination of at least two, or at least 3, of the properties below. In a further aspect, a humanized anti-IL-23p19 antibody of the présent invention has al! the properties below.
• KD for human IL-23 < 1 pM (no shift in binding on-rate in 50% human sérum) • No binding to human IL-12 • Kd <1 pM for cynomolgus monkey IL-23 • No cross reactivity to mouse or rat IL-23.
In particular, a humanized antibody of the présent invention has a KD for human IL-23 < 1 pM (no shift in binding on-rate in 50% human sérum) and no binding to human IL-12.
In a further aspect, a humanized antibody of the présent invention has at least one of the following functional properties (properties B). In a further aspect, a humanized antiIL-23p19 antibody of the présent invention has any combination of at least two, or at least 3, of the properties below. In a further aspect, a humanized anti-IL-23p19 antibody of the présent invention has ail the properties below.
• Blocks IL-23 binding to human IL-23R/Fc in vitro • Inhibits human IL-23 induced IL-17 production in mouse splénocytes with IC5o’s < 20 pM • Inhibits human IL-23 induced STAT3 phosphorylation in human DB cells with ICso’s < 40 pM • Inhibits human IL-23 induced IL-17 and IL-22 production in mouse ear (>80% inhibition of both cytokines at 1 mg/kg).
In a further aspect, a humanized antibody of the présent invention has at least one of the following properties (properties C). In a further aspect, a humanized anti-IL-23p19 antibody of the présent invention has any combination of at least two, or at least 3, of the properties below. In a further aspect, a humanized anti-IL-23p19 antibody of the présent invention has ail the properties below.
• No predicted activity in ADCC/CDC • Stability 83QC (melting température 83°C as determined by differential scanning calorimetry) • Solubility >100 mg/ml (as measured by UV spectroscopy and monitored by turbidity) • Subcutaneous administration of 1.0 mg/kg in three cynomolgus monkeys shows sustained > 10 nM exposure for approximately 28 days with a bioavailability of approximately 70%.
In a further aspect, a humanized antibody of the présent invention has at least one of the following properties (properties C). In a further aspect, a humanized anti-IL-23p19 antibody of the présent invention has any combination of at least two of the properties below. In a further aspect, a humanized anti-IL-23p19 antibody of the présent invention has ail the properties below.
• No predicted activity in ADCC/CDC • Stability 83°C (melting température 83°C as determined by differential scanning calorimetry) • Solubility >100 mg/ml (as measured by UV spectroscopy and monitored by turbidity).
In a further aspect, a humanized antibody of the présent invention has at least one property A, at least one property B and at least one property C. In a further aspect, a humanized anti-IL-23p19 antibody of the présent invention has any combination of at least two, or at least 3, of the properties A, B, and C.
In some aspects, the humanized antibody displays blocking activity, whereby it decreases the binding of IL-23 to IL-23 receptor by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, or by at least 95%. The ability of an antibody to block binding of IL-23 to the IL-23 receptor can be measured using compétitive binding assays known in the art. Alternatively, the blocking activity of an antibody can be measured by assessing the biological effects of IL-23, such as the production of IL-17 and IL-22 to détermine if signaiing mediated by the IL-23 receptor is inhibited.
-68In a further aspect, the présent invention provides a humanized anti-IL-23p19 antibody having favorable biophysical properties. In one aspect, a humanized anti-IL-23p19 antibody of the présent invention is présent in at least 90% monomer form, or in at least 92% monomer form, or in at least 95% monomer form in a buffer. In a further aspect, a humanized anti-IL-23p19 antibody of the présent invention remains in at least 90% monomer form, or in at least 92% monomer form, or in at least 95% monomer form in a buffer for one month or for four months.
In one aspect, a humanized antibody of the présent invention is Antibody A, Antibody B, Antibody C or Antibody D. Accordingly, in one embodiment, a humanized antibody of the présent invention comprises the light chain sequence of SEQ ID NO: 174 and the heavy chain sequence of SEQ ID NO:176 (Antibody A). In another embodiment, a humanized antibody of the présent invention comprises the light chain sequence of SEQ ID NO:174 and the heavy chain sequence of SEQ ID NO:178 (Antibody B). In another embodiment, a humanized antibody of the présent invention comprises the light chain sequence of SEQ ID ΝΟ.Ί80 and the heavy chain sequence of SEQ ID NO:176 (Antibody C). In another embodiment, a humanized antibody of the présent invention comprises the light chain sequence of SEQ ID NO:180 and the heavy chain sequence of SEQ ID NO:178 (Antibody D).
In a further embodiment, a humanized antibody of the présent invention consists of the light chain sequence of SEQ ID NO: 174 and the heavy chain sequence of SEQ ID NO:176 (Antibody A). In a further embodiment, a humanized antibody of the présent invention consists of the light chain sequence of SEQ ID NO:174 and the heavy chain sequence of SEQ ID NO:178 (Antibody B). In a further embodiment, a humanized antibody of the présent invention consists of the light chain sequence of SEQ ID NO:180 and the heavy chain sequence of SEQ ID NO:176 (Antibody C). In a further embodiment, a humanized antibody of the présent invention consists of the light chain sequence of SEQ ID NO:180 and the heavy chain sequence of SEQ ID NO:178 (Antibody D).
In some embodiments, the humanized anti-IL-23p19 antibodies, incîuding antigenbinding fragments thereof, such as heavy and light chain variable régions, comprise an amino acid sequence of the residues derived from Antibody A (light chain sequence =
-69SEQ ID NO:174; heavy chain sequence = SEQ ID NO:176), Antibody B (light chain sequence = SEQ ID NO:174; heavy chain sequence = SEQ ID NO:178), Antibody C (light chain sequence = SEQ ID NO:180; heavy chain sequence = SEQ ID NO;176) or Antibody D (light chain sequence = SEQ ID NO:180; heavy chain sequence = SEQ ID NO:178).
In a further embodiment, the présent invention provides an anti-IL-23p19 antibody or an antigen-binding fragment thereof that binds to human IL-23p19 at an epitope consisting of amino acid residues 108 to 126 and amino acid residues 137 to 151 of SEQ ID NO: 181.
In a further embodiment, the présent invention provides an anti-IL-23p19 antibody or antigen-binding fragment thereof that competitively binds to human IL-23p19 with an antibody of the présent invention, for example Antibody A, Antibody B, Antibody C or Antibody D described herein. The ability of an antibody or antigen-binding fragment to competitively bind to IL-23p19 can be measured using compétitive binding assays known in the art.
The humanized anti-IL-23p19 antibodies optionally include spécifie amino acid substitutions in the consensus or germline framework régions. The spécifie substitution of amino acid residues in these framework positions can improve various aspects of antibody performance including binding affinity and/or stability, over that demonstrated in humanized antibodies formed by direct swap of CDRs or HVLs into the human germline framework régions.
In some embodiments, the présent invention describes other monoclonal antibodies with a light chain variable région having the amino acid sequence set forth in of SEQ ID NO; 84, 86, 88, 90, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117 or 119. In some embodiments, the présent invention describes other monoclonal antibodies with a heavy chain variable région having the amino acid sequence set forth in of SEQ ID NO: 121, 123, 125, 127, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154 or 156 (see Tables 1 and 2 above). The CDR sequence of these mouse antibodies are shown in Tables 3 and 4. Placing such CDRs into FRs of the human consensus
-70heavy and light chain variable domains will yield useful humanized antibodies of the présent invention.
In particular, the présent invention provides monoclonal antibodies with the combinations of light chain variable and heavy chain variable régions of SEQ ID NO:84/121, 86/123, 88/125, 90/127, 91/128, 93/130, 95/132, 97/134, 99/136, 101/138, 103/140, 105/142, 107/144, 109/146, 111/148, 113/150, 115/152, 117/154 or 119/156. Such variable régions can be combined with human constant régions.
In some embodiments, the présent invention describes other humanized antibodies with light chain variable région sequences having the amino acid sequence set forth in of SEQ ID NO:158, 160, 162 or 164. In some embodiments, the présent invention describes other humanized antibodies with heavy chain variable région sequences having the amino acid sequence set forth in of SEQ ID NO: 166, 168, 170 or 172 (see Tables 5 and 6 above). The CDR sequences of these antibodies are shown in Tables 3 and 4. In particular, the présent invention provides monoclonal antibodies with the combinations of light chain variable and heavy chain variable régions of SEQ ID NO: 160/166, 160/168, 158/166 or 158/168. Such variable régions can be combined with human constant régions.
In a further embodiment, the présent invention relates to an anti-IL-23p19 antibody or antigen-binding fragment thereof comprising a humanized light chain variable domain comprising the CDRs of SEQ ID NO:160 and framework régions having an amino acid sequence at least 90% identical, at least 93% identical or at least 95% identical to the amino acid sequence of the framework régions of the variable domain light chain amino acid sequence of SEQ ID NO: 160 and a humanized heavy chain variable domain comprising the CDRs of SEQ ID NO:166 and framework régions having an amino acid sequence at least 90% identical, at least 93% identical or at least 95% identical to the amino acid sequence of the framework régions of the variable domain heavy chain amino acid sequence of SEQ ID NO:166. In one embodiment, the anti-IL-23p19 antibody is a humanized monoclonal antibody.
In a further embodiment, the présent invention relates to an anti-IL-23p19 antibody or antigen-binding fragment thereof comprising a humanized light chain variable domain
-71 comprising the CDRs of SEQ ID NO:160 and framework régions having an amino acid sequence at least 90% identical, at least 93% identical or at least 95% identical to the amino acid sequence of the framework régions of the variable domain Iight chain amino acid sequence of SEQ ID NO:160 and a humanized heavy chain variable domain comprising the CDRs of SEQ ID NO:168 and framework régions having an amino acid sequence at least 90% identical, at least 93% identical or at least 95% identical to the amino acid sequence of the framework régions of the variable domain heavy chain amino acid sequence of SEQ ID NO:168. In one embodiment, the anti-IL-23p19 antibody is a humanized monoclonal antibody.
In a further embodiment, the présent invention relates to an anti-IL-23p19 antibody or antigen-binding fragment thereof comprising a humanized Iight chain variable domain comprising the CDRs of SEQ ID NO:158 and framework régions having an amino acid sequence at least 90% identical, at least 93% identical or at least 95% identical to the amino acid sequence of the framework régions of the variable domain Iight chain amino acid sequence of SEQ ID NO:158 and a humanized heavy chain variable domain comprising the CDRs of SEQ ID NO: 166 and framework régions having an amino acid sequence at least 90% identical, at least 93% identical or at least 95% identical to the amino acid sequence of the framework régions of the variable domain heavy chain amino acid sequence of SEQ ID NO: 166. In one embodiment, the anti-IL-23p19 antibody is a humanized monoclonal antibody.
In a further embodiment, the présent invention relates to an anti-IL-23p19 antibody or antigen-binding fragment thereof comprising a humanized Iight chain variable domain comprising the CDRs of SEQ ID NO:158 and framework régions having an amino acid sequence at least 90% identical, at least 93% identical or at least 95% identical to the amino acid sequence of the framework régions of the variable domain Iight chain amino acid sequence of SEQ ID NO:158 and a humanized heavy chain variable domain comprising the CDRs of SEQ ID NO:168 and framework régions having an amino acid sequence at least 90% identical, at least 93% identical or at least 95% identical to the amino acid sequence of the framework régions of the variable domain heavy chain
-72amino acid sequence of SEQ ID NO:168. In one embodiment, the anti-IL-23p19 antibody is a humanized monoclonal antibody.
In some spécifie embodiments, the humanized anti-IL-23p19 antibodies disclosed herein comprise at least a heavy or a light chain variable domain comprising the CDRs or HVLs of the murine monoclonal antibodies or humanized antibodies as shown in Tables 1 through 6 above and the FRs of the human germline heavy and light chain variable domains.
The CDRs of these sequences are shown in Tables 3 and 4. Accordingly, in one aspect, the présent invention provides an anti-IL-23p19 antibody or antigen-binding fragment thereof comprising a light chain CDR1 (L-CDR1) sequence of SEQ ID NO:1,4, 6, 7, 8, 11, 15, 18, 19, 22, 27 or 30; a light chain CDR2 (L-CDR2) sequence of SEQ ID NO:2, 5, 9, 12, 16, 20, 23, 25, 28 or 31; a light chain CDR3 (L-CDR3) sequence of SEQ ID NO:3, 10, 13, 14, 17, 21,24, 26, 29, or 32; a heavy chain CDR1 (H-CDR1) sequence of SEQ ID NO:33, 36, 38, 40, 43, 45, 48, 51, 54, 57, 60, 63, 66, 67, 68, 69, 77 or 80; a heavy chain CDR2 (H-CDR2) sequence of SEQ ID NO:34, 39, 41, 46, 49, 52, 55, 58, 61, 64, 70, 72, 73, 75, 78 or 81; and a heavy chain CDR3 (H-CDR3) sequence of SEQ ID NO:35, 37, 42, 44, 47, 50, 53, 56, 59, 62, 65, 71, 74, 76, 79 or 82. In one aspect, the anti-IL-23p19 antibody or antigen-binding fragment thereof comprises a light chain variable région comprising a L-CDR1 listed above, a L-CDR2 listed above and a LCDR3 listed above, and a heavy chain variable région comprising a H-CDR1 listed above, a H-CDR2 listed above and a H-CDR3 listed above.
In a further aspect, the présent invention provides an anti-IL-23p19 antibody or antigenbinding fragment thereof comprising:
a) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:1,2, 3, 33, 34, and 35, respectively; or
b) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:4, 5, 3, 36, 34 and 37, respectively; or
c) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:1,2, 3, 38, 39 and 35, respectively; or \λ/
d) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:6, 2, 3, 40, 41 and 42, respectively; or
e) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:7, 2, 3, 43, 41 and 44, respectively; or
f) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:8, 9,10, 45, 46 and 47, respectively; or
g) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:8, 9, 10, 48, 49 and 50, respectively; or
h) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:11, 12, 13, 51, 52 and 53, respectively; or
i) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:7, 2, 14, 54, 55 and 56, respectively; or
j) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO: 15, 16, 17, 57, 58 and 59, respectively; or
k) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:18, 16, 17, 60, 61 and 62, respectively; or
l) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:19, 20, 21, 63, 66, 67 or 68, 64 and 65, respectively; or
m) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:22, 23, 24, 69, 70 and 71, respectively; or
n) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:22, 25, 26, 55, 72 and 71, respectively; or
o) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:8, 9, 10, 45, 73 and 74, respectively; or
p) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:27, 28, 29, 45, 75 and 76, respectively; or
q) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:8, 9, 10, 77, 78 and 79, respectively; or
r) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:30, 31, 32, 80, 81 and 82, respectively.
In one aspect, the anti-IL-23p19 antibody or antigen-binding fragment thereof comprises a light chain variable région comprising a L-CDR1, L-CDR2 and L-CDR3 combination listed above, and a heavy chain variable région comprising a H-CDR1, H-CDR2 and HCDR3 combination listed above.
In spécifie embodiments, it is contemplated that chimeric antibodies with switched CDR régions (i.e., for example switching one or two CDRs of one of the mouse antibodies or humanized antibody derived therefrom with the analogous CDR from another mouse antibody or humanized antibody derived therefrom) between these exemplary immunoglobulins may yield useful antibodies.
In certain embodiments, the humanized anti-IL-23p19 antibody is an antibody fragment. Various antibody fragments hâve been generally discussed above and there are techniques that hâve been developed for the production of antibody fragments. Fragments can be derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., 1992, Journal of Biochemical and Biophysical Methods 24:107-117; and Brennan et al., 1985, Science 229:81). Alternatively, the fragments can be produced directly in recombinant host cells. For example, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (see, e.g., Carter et al., 1992, Bio/Technology 10:163-167). By another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. Accordingly, in one aspect, the présent invention provides antibody fragments comprising the CDRs described herein, in particular one of the combinations of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 described herein. In a further aspect, the présent invention provides antibody fragments comprising the variable régions described herein, for example one of the combinations of light chain variable régions and heavy chain variable régions described herein.
Certain embodiments include an F(ab')2 fragment of a humanized anti-IL-23p19 antibody comprise a light chain sequence of any of SEQ ID NO: 174 or 180 in combination with a heavy chain sequence of SEQ ID NO: 176 or 178. Such embodiments can include an intact antibody comprising such an F(ab')2. w—
-75In some embodiments, the antibody or antibody fragment includes a constant région that médiates effector function. The constant région can provide antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC) responses against an IL-23 expressing target cell. The effector domain(s) can be, for example, an Fc région of an Ig molécule.
The effector domain of an antibody can be from any suitable vertebrate animal species and isotypes. The isotypes from different animal species differ in the abilities to médiate effector functions. For example, the ability of human immunoglobulin to médiate CDC and ADCC/ADCP is generally in the order of lgM=lgGis=lgG3>lgG2>lgG4 and lgGi~lgG3>lgG2/lgM/lgG4, respectively. Murine immunoglobulins médiate CDC and ADCC/ADCP generally in the order of murine lgM=lgG3»lgG2b>lgG2a»lgG1 and igG2b>lgG2a>lgG1»lgG3, respectively. In another example, murine lgG2a médiates ADCC while both murine lgG2a and IgM médiate CDC.
Antibody Modifications
The humanized anti-IL-23p19 antibodies and agents can include modifications of the humanized anti-IL-23p19 antibody or antigen-binding fragment thereof. For example, it may be désirable to modify the antibody with respect to effector function, so as to enhance the effectiveness of the antibody in treating cancer. One such modification is the introduction of cysteine residue(s) into the Fc région, thereby allowing interchain disulfide bond formation in this région. The homodimeric antibody thus generated can hâve improved internalization capability and/or increased complement-mediated cell killing and/or antibody-dependent cellular cytotoxicity (ADCC). See, for example, Caron et al., 1992, J. Exp Med. 176:1191-1195; and Shopes, 1992, J. Immunol. 148:29182922. Homodimeric antibodies having enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al., 1993, Cancer Research 53: 2560-2565. Alternative^, an antibody can be engineered to contain dual Fc régions, enhancing complément lysis and ADCC capabilities of the antibody. See Stevenson et al., 1989, Anti-Cancer Drug Design 3: 219-230. --16388
-76Antibodies with improved ability to support ADCC hâve been generated by modifying the glycosylation pattern of their Fc région. This is possible since antibody glycosylation at the asparagine residue, N297, in the CK2 domain is involved in the interaction between IgG and Fcy receptors prerequisite to ADCC. Host cell lines hâve been engineered to express antibodies with altered glycosylation, such as increased bisecting Nacetylglucosamine or reduced fucose. Fucose réduction provides greater enhancement to ADCC actîvity than does increasing the presence of bisecting N-acetylglucosamine. Moreover, enhancement of ADCC by low fucose antibodies is independent of the FcyRI I la V/F polymorphism.
Modifying the amino acid sequence of the Fc région of antibodies is an alternative to glycosylation engineering to enhance ADCC. The binding site on human IgGi for Fcy receptors has been determined by extensive mutational analysis. This led to the génération of humanized IgGi antibodies with Fc mutations that increase the binding affinity for FcyRIIIa and enhance ADCC in vitro. Additionally, Fc variants hâve been obtained with many different permutations of binding properties, e.g., improved binding to spécifie FcyR receptors with unchanged or diminished binding to other FcyR receptors.
Another aspect includes immunoconjugates comprising the humanized antibody or fragments thereof conjugated to a cytotoxic agent such as a chemotherapeutic agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the génération of such immunoconjugates hâve been described above. Enzymatically active toxins and fragments thereof that can be used to form useful immunoconjugates include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, the tricothecenes, and the like. A variety of radionuclides are
-77available for the production of radioconjugated humanized anti-IL-23p19 antibodies. Examples include 212Bi, 131l,131 In, 9°Y, and 186Re.
Conjugates of the humanized anti-IL-23p19 antibody and cytotoxic or chemotherapeutic agent can be made by known methods, using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional dérivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldéhydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium dérivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene
2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., 1987, Science 238:1098. Carbon-14-labeled 1-isothiocyanatobenzyl-3methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. Conjugates also can be formed with a cleavable linker.
The humanized anti-IL-23p19 antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688; Hwang et al., 1980, Proc. Natl. Acad. Sci. USA 77:4030; and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes having enhanced circulation time are disclosed, for example, în U.S. Pat. No. 5,013,556.
Particularly useful liposomes can be generated by the reverse phase évaporation method with a lipid composition comprising phosphatidylcholine, cholestérol and PEGderivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of an antibody disclosed herein can be conjugated to the liposomes as described in Martin et al., 1982, J. Biol. Chem. 257:286-288 via a disulfide interchange reaction. A chemotherapeutic agent (such as doxorubicin) is optionally contained within the liposome. See, e.g., Gabizon et al., 1989, J. National Cancer Inst. 81(19):1484.
-78The antibodies described and disclosed herein can also be used in ADEPT (AntibodyDirected Enzyme Prodrug Therapy) procedures by conjugating the antibody to a prodrug-activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic agent), to an active anti-cancer drug. See, for example, WO 81/01145, WO 88/07378, and U.S. Pat. No. 4,975,278. The enzyme component of the immunoconjugate useful for ADEPT is an enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form. Spécifie enzymes that are useful in ADEPT include, but are not limited to, alkaline phosphatase for converting phosphate-containing prodrugs into free drugs; arylsulfatase for converting sulfate-containing prodrugs into free drugs; cytosine deaminase for converting non-toxic 5-fluorocytosine into the anti-cancer drug,
5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases, and cathepsins (such as cathepsins B and L), for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, for converting prodrugs containing D-amino acid substituents; carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidase for converting glycosylated prodrugs into free drugs; β-lactamase for converting drugs derivatized with β-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies having enzymatic activity (abzymes) can be used to convert the prodrugs into free active drugs (see, for example, Massey, 1987, Nature 328: 457-458). Antibody-abzyme conjugates can be prepared by known methods for delivery of the abzyme to a tumor cell population, for example, by covalently binding the enzyme to the humanized anti-IL23p19antibody/heterobifunctional crosslinking reagents discussed above. Alternatively, fusion proteins comprising at least the antigen binding région of an antibody disclosed herein linked to at least a functionally active portion of an enzyme as described above can be constructed using recombinant DNA techniques (see, e.g., Neuberger et al., 1984, Nature 312:604-608).
In certain embodiments, it may be désirable to use a humanized antî-IL-23p19 antibody fragment, rather than an intact antibody, to increase tissue pénétration, for example. It may be désirable to modify the antibody fragment in order to increase its sérum half life.
-79This can be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment. In one method, the appropriate région of the antibody fragment can be altered (e.g., mutated), or the epitope can be incorporated into a peptide tag that is then fused to the antibody fragment at either end or in the middle, for example, by DNA or peptide synthesis. See, e.g., WO 96/32478.
In other embodiments, covalent modifications of the humanized anti-IL-23p19 antibody are aiso included. Covalent modifications include modification of cysteinyl residues, histidyl residues, lysinyl and amino-terminal residues, arginyl residues, tyrosyl residues, carboxyl side groups (aspartyl or glutamyl), glutaminyl and asparaginyl residues, or seryl, or threonyl residues. Another type of covalent modification involves chemically or enzymatically coupling glycosides to the antibody. Such modifications may be made by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if applicable. Other types of covalent modifications of the antibody can be introduced into the molécule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the amino- or carboxy-terminal residues.
Removal of any carbohydrate moieties présent on the antibody can be accomplished chemically or enzymatically. Chemical deglycosylation is described by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981, Anal. Biochem., 118:131. Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., 1987, Meth. Enzymol 138:350.
Another type of useful covalent modification comprises linking the antibody to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in one or more of U.S. Pat. No. 4,640,835, U.S. Pat. No. 4,496,689, U.S. Pat No. 4,301,144, U.S. Pat. No. 4,670,417, U.S. Pat. No. 4,791,192 and U.S. Pat. No. 4,179,337.
Humanizatîon and Amino Acid Sequence Variants
-80Amino acid sequence variants of the anti-IL-23p19 antibody can be prepared by introducing appropriate nucléotide changes into the anti-IL-23p19 antibody DNA, or by peptide synthesis. Such variants include, for example, délétions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the anti-IL23p19 antibodies of the examples herein. Any combination of délétions, insertions, and substitutions is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter posttranslational processes of the humanized or variant anti-IL-23p19 antibody, such as changing the number or position of glycosylation sites.
A useful method for identification of certain residues or régions of the anti-IL-23p19 antibody that are preferred locations for mutagenesis is called alanine scanning mutagenesis, as described by Cunningham and Wells (Science, 244:1081-1085 (1989)). Here, a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (typically alanine) to affect the interaction of the amino acids with IL-23p19 antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis is conducted at the target codon or région and the expressed antiIL-23p19 antibody variants are screened for the desired activity.
Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an anti-IL-23p19 antibody fused to an epitope tag. Other insertional variants of the anti-IL-23p19 antibody molécule include a fusion to the N- or C-terminus of the anti-IL-23p19 antibody of an enzyme or a polypeptide which increases the sérum half-life of the antibody. —
-81 Another type of variant is an amino acid substitution variant. These variants hâve at least one amino acid residue in the anti-IL-23p19 antibody molécule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include the hypervariable régions, but FR alterations are also 5 contemplated. Conservative substitutions are shown in Table 5 under the heading of preferred substitutions. If such substitutions resuit in a change in biological activity, then more substantial changes, denominated exemplary substitutions, or as further described below in reference to amino acid classes, may be introduced and the products screened.
10 TABLE Θ:
Original Residue Exemplary Substitutions Preferred Substitutions
Ala (A) val; leu; ile val
Arg (R) lys; gin; asn lys
Asn (N) gin; his; asp, lys; arg
15 gin
Asp (D) glu; asn glu
Cys (C) ser; ala ser
Gin (Q) asn; glu asn
Glu (E) asp; gin asp
20 Gly(G) ala ala
His(H) arg; asn; gin; lys;
arg
lle(l) leu; val; met; ala;
phe; norleucine leu
25 Leu (L) ile; norleucine; val;
met; ala; phe île /
Lys (K) arg; gin; asn arg y/
Met (M) leu; phe; ile leu
Phe (F) tyr; leu; val; ile; ala;
tyr
Pro (P) ala ala
Ser (S) thr thr
Thr(T) ser ser
Trp (W) tyr; phe tyr
Tyr (Y) phe;trp; thr; ser
phe
Val (V) leu; ile; met; phe
ala; norleucine; leu
In proteîn chemistry, it is generally accepted that the biological properties of the antibody can be accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molécule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:
(1) hydrophobie: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gin, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
-83Any cysteine residue not involved in maintaining the proper conformation of the humanized or variant anti-IL-23p19 antibody also may be substituted, generally with serine, to improve the oxidative stability of the molécule, prevent aberrant crosslinking, or provide for established points of conjugation to a cytotoxic or cytostatic compound. Conversely, cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).
A type of substitutional variant involves substituting one or more hypervariable région residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further development will hâve improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants is affinity maturation using phage display. Briefly, several hypervariable région sites (e.g., 6-7 sites) are mutated to generate ail possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity). In order to identify candidate hypervariable région sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable région residues contributing significantly to antigen binding. Alternatively, or in addition, it may be bénéficiai to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and human IL-23p19. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.
Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. By altering is meant deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not présent in the antibody. κκ/”
-84In some embodiments, it may be désirable to modify the antibodies of the invention to add glycosylations sites. Glycosylation of antibodies is typically either N-linked or Olinked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagineX-threonine, where X is any amino acid except proline, are the récognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide créâtes a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars Naceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Thus, in order to glycosylate a given protein, e.g., an antibody, the amino acid sequence of the protein is engineered to contain one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).
Nucleic acid molécules encoding amino acid sequence variants of the anti-IL-23p19 antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or préparation by oligonucleotide-mediated (or sitedirected) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the anti-IL-23p19 antibody.
Polynucleotides, Vectors, Host Cells, and Recombinant Methods
Other embodiments encompass isolated polynucleotides that comprise a sequence encoding a humanized anti-IL-23p19 antibody, vectors, and host cells comprising the polynucleotides, and recombinant techniques for production of the humanized antibody. The isolated polynucleotides can encode any desired form of the anti-IL-23p19 antibody including, for example, full length monoclonal antibodies, Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molécules, and multispecific antibodies formed from antibody fragments.
-85Some embodiments include isolated polynucleotides comprising sequences that encode the light chain variable région of an antibody or antibody fragment having the amino acid sequence of any of SEQ ID NO: SEQ ID NO:84, 86, 88, 90, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117 or 119. Exemplary polynucleotide sequences encoding such amino acid sequences are SEQ ID NO:83, 85, 87, 89, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116 and 118. Some embodiments include isolated polynucleotides comprising sequences that encode the heavy chain variable région of an antibody or antibody fragment having the amino acid sequence of SEQ ID NO:121, 123, 125, 127, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154 or 156. Exemplary polynucleotide sequences encoding such amino acid sequences are SEQ ID NO: 120, 122, 124, 126, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153 or 155.
Some embodiments include isolated polynucleotides comprising sequences that encode the light chain variable région of an antibody or antibody fragment having the amino acid sequence of any of SEQ ID NO:158, 160, 162 or 164. Exemplary polynucleotide sequences encoding such amino acid sequences are SEQ ID NO:157, 159, 161 or 163. Some embodiments include isolated polynucleotides comprising sequences that encode the heavy chain variable région of an antibody or antibody fragment having the amino acid sequence of SEQ ID NO: 166, 168, 170 or 172. Exemplary polynucleotide sequences encoding such amino acid sequences are SEQ ID NO: 165, 167, 169 or 171.
Some embodiments include isolated polynucleotides comprising sequences that encode the light chain of an antibody having the amino acid sequence of any of SEQ ID N0:174 or 180. Exemplary polynucleotide sequences encoding such amino acid sequences are SEQ ID NO:173 or 179. Some embodiments include isolated polynucleotides comprising sequences that encode the heavy chain of an antibody having the amino acid sequence of SEQ ID NO:176 or 178. Exemplary polynucleotide sequences encoding such amino acid sequences are SEQ ID NO:175 or 177.
In one aspect, the isolated polynucleotide sequence(s) encodes an antibody or antibody fragment having a light chain and a heavy chain variable région comprising the amino acid sequences of SEQ ID NO:174 and SEQ ID NO:176, respectively; SEQ ID ΝΟ:174ψνζ^'
-86and SEQ ID NO:178, respectively; SEQ ID NO:180 and SEQ ID NO:176, respectively; SEQ ID NO:180 and SEQ ID NO:178, respectively. Exemplary polynucleotide sequences encoding such amino acid sequences are SEQ ID NO: 173 and 175, respectively, SEQ ID NO: 173 and 177, respectively, SEQ ID NO: 179 and 175, respectively, SEQ ID NO: 179 and 177, respectively.
The polynucleotide(s) that comprise a sequence encoding a humanized anti-IL-23p19 antibody or a fragment or chain thereof can be fused to one or more regulatory or control sequence, as known in the art, and can be contained in suitable expression vectors or host cell as known in the art. Each of the polynucleotide molécules encoding the heavy or light chain variable domains can be independently fused to a polynucleotide sequence encoding a constant domain, such as a human constant domain, enablîng the production of intact antibodies. Alternatively, polynucleotides, or portions thereof, can be fused together, providing a template for production of a single chain antibody.
For recombinant production, a polynucleotide encoding the antibody is inserted into a replicable vector for cloning (amplification of the DNA) or for expression. Many suitable vectors for expressing the recombinant antibody are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of réplication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
The humanized anti-IL-23p19 antibodies can also be produced as fusion polypeptides, in which the antibody is fused with a heterologous polypeptide, such as a signal sequence or other polypeptide having a spécifie cleavage site at the amino terminus of the mature protein or polypeptide. The heterologous signal sequence selected is typically one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the humanized anti-IL-23p19 antibody signal sequence, the signal sequence can be substituted by a prokaryotic signal sequence. The signal sequence can be, for example, alkaline phosphatase, penicillinase, lipoprotein, heat-stable enterotoxin II leaders, and the like. For yeast sécrétion, the native signal sequence can be substituted, for example,
-87with a leader sequence obtained from yeast invertase alpha-factor (including Saccharomyces and Kluyveromyces α-factor leaders), acid phosphatase, C. albicans glucoamylase, or the signal described in WO90/13646. In mammalian cells, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, can be used. The DNA for such precursor région is ligated in reading frame to DNA encoding the humanized anti-IL-23p19 antibody.
Expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of réplication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of réplication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2-υ. plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, and BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of réplication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter).
Expression and cloning vectors may contain a gene that encodes a selectable marker to facilitate identification of expression. Typical selectable marker genes encode proteins that confer résistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tétracycline, or alternatively, are complément auxotrophic deficiencies, or in other alternatives supply spécifie nutrients that are not présent in complex media,
e.g., the gene encoding D-alanîne racemase for Bacilli.
One example of a sélection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug résistance and thus survive the sélection regimen. Examples of such dominant sélection use the drugs neomycin, mycophenolic acid, and hygromycin. Common selectable markers for mammalian cells are those that enable the identification of cells competent to take up a nucleic acid encoding a humanized anti-IL-23p19 antibody, such as DHFR (dihydrofolate reductase), thymidine kinase, metallothionein-l yt/'
-88and -Il (such as primate metallothionein genes), adenosine deaminase, ornithine decarboxylase, and the like. Cells transformed with the DH FR sélection gene are first identified by culturing ail of the transformants in a culture medium that contains methotrexate (Mtx), a compétitive antagonist of DHFR. An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line déficient in DHFR activity (e.g., DG44).
Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding anti-IL-23p19 antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3‘phosphotransferase (APH), can be selected by cell growth in medium containing a sélection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See, e.g., U.S. Pat. No. 4,965,199.
Where the recombinant production is performed in a yeast cell as a host cell, the TRP1 gene présent in the yeast plasmid YRp7 (Stinchcomb et al., 1979, Nature 282: 39) can be used as a selectable marker. The TRP1 gene provides a sélection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 (Jones, 1977, Genetics 85:12). The presence of the trp1 lésion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2p-deficient yeast strains such as ATCC 20,622 and 38,626 are complemented by known plasmids bearing the LEU2 gene.
In addition, vectors derived from the 1.6 pm circular plasmid pKD1 can be used for transformation of Kluyveromyces yeasts. Alternatively, an expression System for largescale production of recombinant calf chymosin was reported for K. lactis (Van den Berg, 1990, Bio/Technology 8:135). Stable multi-copy expression vectors for sécrétion of mature recombinant human sérum albumin by industrial strains of Kluyveromyces hâve also been disclosed (Fleer et al., 1991, Bio/Technology 9:968-975).
Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid molécule encoding an anti-IL-23p19 antibody or polypeptide chain thereof. Promoters suitable for use with prokaryotic hosts
-89include phoA promoter, β-lactamase and lactose promoter Systems, alkaline phosphatase, tryptophan (trp) promoter System, and hybrid promoters such as the tac promoter. Other known bacterial promoters are also suitable. Promoters for use in bacterial Systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the humanized anti-IL-23p19 antibody.
Many eukaryotic promoter sequences are known. Virtually ail eukaryotic genes hâve an AT-rich région located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT région where N may be any nucléotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. Ail of these sequences are suitably inserted into eukaryotic expression vectors.
Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
Inducible promoters hâve the additionaî advantage of transcription controlled by growth conditions. These include yeast promoter régions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, dérivative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. Yeast enhancers also are advantageously used with yeast promoters.
Humanized anti-IL-23p19 antibody transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomégalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or
-90an immunoglobulin promoter, or from heat-shock promoters, provided such promoters are compatible with the host cell Systems.
The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of réplication. The immédiate early promoter of the human cytomégalovirus is conveniently obtained as a Hindi II E restriction fragment. A System for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this System is described in U.S. Pat. No. 4,601,978. See also Reyes et al., 1982, Nature 297:598-601, disclosing expression of human p-interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous sarcoma virus long terminal repeat can be used as the promoter.
Another useful element that can be used in a recombinant expression vector is an enhancer sequence, which is used to increase the transcription of a DNA encoding a humanized anti-IL-23p19 antibody by higher eukaryotes. Many enhancer sequences are now known from mammalian genes (e.g., globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, an enhancer from a eukaryotic cell virus is used. Examples include the SV40 enhancer on the late side of the réplication origin (bp 100-270), the cytomégalovirus early promoter enhancer, the polyoma enhancer on the late side of the réplication origin, and adenovirus enhancers. See also Yaniv, 1982, Nature 297:17-18 for a description of enhancing éléments for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5' or 3' to the humanized anti-IL23p19 antibody-encoding sequence, but is preferably located at a site 5' from the promoter.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) can also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated régions of eukaryotic or viral DNAs or cDNAs. These régions contain nucléotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding anti-IL-23p19 antibody. One useful transcription termination component
-91 is the bovine growth hormone polyadenylation région. See WO94/11026 and the expression vector disclosed therein. In some embodiments, humanized anti-IL-23p19 antibodies can be expressed using the CHEF System. (See, e.g., U.S. Pat. No. 5,888,809; the disclosure of which is incorporated by reference herein.)
Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisais, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 P disclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for humanized anti-IL-23p19antibody-encoding vectors. Saccharomyces cerevisiae, or common baker’s yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other généra, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastors (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Pénicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
Suitable host cells for the expression of glycosylated humanized anti-IL-23p19 antibody are derived from multicellular organisme. Examples of invertebrate cells include plant and insect cells, including, e.g., numerous baculoviral strains and variants and^^/^
-92corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori (silk worm). A variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used, particularly for transfection of Spodoptera frugiperda cells.
Plant cell cultures of cotton, corn, potato, soybean, pétunia, tomato, and tobacco can also be utilized as hosts.
In another aspect, expression of humanized anti-IL-23p19 is carried out in vertebrate cells. The propagation of vertebrate cells in culture (tissue culture) has become routine procedure and techniques are widely available. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, (Graham et al., 1977, J. Gen Virol. 36: 59), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells/-DHFR1 (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77: 4216; e.g., DG44), mouse sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TR1 cells (Mather et al., 1982, Annals N.Y. Acad. Sci. 383: 44-68), MRC 5 cells, FS4 cells, and human hepatoma line (Hep G2).
Host cells are transformed with the above-described expression or cloning vectors for humanized anti-IL-23p19 antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
The host cells used to produce a humanized anti-IL-23p19 antibody described herein may be cultured in a variety of media. Commercially available media such as Ham’s F10 (Sigma-Aldrich Co., St. Louis, Mo.), Minimal Essential Medium ((MEM), (Sigma-Aldrich
-93Co.), RPMI-1640 (Sigma-Aldrich Co.), and Dulbecco's Modifîed Eagle's Medium ((DMEM), Sigma-Aldrich Co.) are suitable for culturing the host cells. In addition, any of the media described in one or more of Ham et al., 1979, Meth. Enz. 58: 44, Barnes et al., 1980, Anal. Biochem. 102: 255, U.S. Pat. No. 4,767,704, U.S. Pat. No. 4,657,866, U.S. Pat. No. 4,927,762, U.S. Pat. No. 4,560,655, U.S. Pat. No. 5,122,469, WO 90/103430, and WO 87/00195 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnésium, and phosphate), buffers (such as HEPES), nucléotides (such as adenosine and thymidine), antibiotics (such as gentamicin), trace éléments (defined as inorganic compounds usually présent at final concentrations in the micromolar range), and glucose or an équivalent energy source. Other suppléments may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as température, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, the cells may be disrupted to release protein as a first step. Particulate débris, either host cells or lysed fragments, can be removed, for example, by centrifugation or ultrafiltration. Carter et al., 1992, Bio/Technology 10:163-167 describes a procedure for isolating antibodies that are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 minutes. Cell débris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression Systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants. A variety of methods can be used to isolate the antibody from the host cell. sa/”'
-94The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a typical purification technique. The suitability of protein A as an affinity ligand dépends on the species and isotype of any immunoglobulin Fc domain that is présent in the antibody. Protein A can be used to purify antibodies that are based on human gammal, gamma2, or gamma4 heavy chains (see, e.g., Lindmark et al., 1983 J. Immunol. Meth. 62:1-13). Protein G is recommended for ail mouse isotypes and for human gamma3 (see, e.g., Guss et al., 1986 EMBO J. 5:1567-1575). A matrix to which an affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, éthanol précipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate précipitation are also available depending on the antibody to be recovered.
Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobie interaction chromatography using an elution buffer at a pH between about 2.5-4.5, typically performed at low sait concentrations (e.g., from about 0-0.25M sait).
Also included are nucleic acids that hybridize under low, moderate, and high stringency conditions, as defined herein, to ail or a portion (e.g., the portion encoding the variable région) of the nucléotide sequence represented by isolated polynucleotide sequence(s) that encode an antibody or antibody fragment of the présent invention.The hybridizing portion of the hybridizing nucleic acid is typically at least 15 (e.g., 20, 25, 30 or 50) nucléotides in length. The hybridizing portion of the hybridizing nucleic acid is at least 80%, e.g., at least 90%, at least 95%, or at least 98%, identical to the sequence of a portion or ail of a nucleic acid encoding an anti-IL-23p19 polypeptide (e.g., a heavy
-95chain or light chain variable région), or its complément. Hybridizing nucleic acids of the type described herein can be used, for example, as a cloning probe, a primer, e.g., a PCR primer, or a diagnostic probe.
Some embodiments include isolated polynucleotides including sequences that encode an antibody or antibody fragment having the amino acid sequence of any one of SEQ ID NO: 84, 86, 88, 90, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117 or 119, and that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the polynucleotide sequences of SEQ ID NO:83, 85, 87, 89, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116or118.
Some embodiments include isolated polynucleotides including sequences that encode an antibody or antibody fragment having the amino acid sequence of any one of SEQ ID NO: 158, 160, 162 or 164, and that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the polynucleotide sequences of SEQ ID NO: 157, 159, 161 or 163.
Some embodiments include isolated polynucleotides including sequences that encode an antibody or antibody fragment having the amino acid sequence of any one of SEQ ID NO: 121, 123, 125, 127, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154 or 156, and that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the polynucleotide sequences of SEQ ID NO: 120, 122, 124, 126, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153 or 155.
Some embodiments include isolated polynucleotides including sequences that encode an antibody or antibody fragment having the amino acid sequence of any one of SEQ ID NO: 166, 168, 170 or 172, and that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the polynucleotide sequences of SEQ ID NO: 165, 167, 169 or 171.
Some embodiments include isolated polynucleotides including sequences that encode an antibody or antibody fragment having the light chain variable région amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NO: 84, 86, 88, 90, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117 or 119. Some embodiments
-96include isolated polynucleotides including sequences that encode an antibody or antibody fragment having the light chain variable région amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NO:158, 160, 162 or 164. Some embodiments include isolated polynucleotides including sequences that encode an antibody or antibody fragment having the heavy chain variable région amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NO:121, 123, 125, 127, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154 or 156. Some embodiments include isolated polynucleotides including sequences that encode an antibody or antibody fragment having the heavy chain variable région amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of any of one SEQ ID NO:166, 168, 170 or 172. As used herein, the terms identical or percent identity, in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or hâve a specified percentage of nucléotides or amino acid residues that are the same, when compared and aligned for maximum correspondence. To détermine the percent identity, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucléotides at corresponding amino acid positions or nucléotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucléotide as the corresponding position in the second sequence, then the molécules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total # of positions (e.g., overlapping positions)x100). In some embodiments, the two sequences that are compared are the same length after gaps are introduced within the sequences, as appropriate (e.g., excluding additional sequence extending beyond the sequences being compared). For example, when variable région sequences are compared, the leader and/or constant domain sequences are not considered. For^iz—
-97sequence comparisons between two sequences, a corresponding CDR refers to a
CDR in the same location in both sequences (e.g., CDR-H1 of each sequence).
The détermination of percent identity or percent similarity between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucléotide searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain nucléotide sequences homologous to a nucleic acid encoding a protein of interest. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to protein of interest. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molécules (ld.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. Another preferred, nonlimiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5; and FASTA described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. If ktup=2, similar régions in the two sequences being compared are found by looking at pairs of aligned residues; if ktup=1, single aligned amino acids are examined. ktup can be set to 2 or 1 for protein sequences, or from 1 to 6 for DNA sequences. The default if ktup is not specified is 2 for proteins and 6 m/-''
-98for DNA. Alternatively, protein sequence alignment may be carried oui using the CLUSTAL W algorithm, as described by Higgins et al., 1996, Methods Enzymol. 266:383-402.
Non-Therapeutic Uses
The antibodies described herein are useful as affinity purification agents. In this process, the antibodies are immobilized on a solid phase such a Protein A resin, using methods well known in the art. The immobilized antibody is contacted with a sample containing the IL-23p19 protein (or fragment thereof) to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially ail the material in the sample except the IL-2319 protein, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the IL-23p19 protein from the antibody.
Anti-IL-23p19 antibodies, for example humanized anti-IL-23p19 antibodies, are also useful in diagnostic assays to detect and/or quantify IL-23 protein, for example, detecting IL-23 expression in spécifie cells, tissues, or sérum. The anti-IL-23p19 antibodies can be used diagnostically to, for example, monitor the development or progression of a disease as part of a clinical testing procedure to, e.g., détermine the efficacy of a given treatment and/or prévention regimen. Détection can be facilitated by coupling the anti-IL-23p19 antibody. Examples of détectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron émission tomographies, and nonradioactive paramagnetic métal ions. See, for example, U.S. Patent No. 4,741,900 for métal ions which can be conjugated to antibodies for use as diagnostics according to the présent invention.
The anti-IL-23p19 antibodies can be used in methods for diagnosing an IL-23associated disorder (e.g., a disorder characterized by abnormal expression of IL-23) or to détermine if a subject has an increased risk of developing an IL-23-associated disorder. Such methods include contacting a biological sample from a subject with an IL-23p19 antibody and detecting binding of the antibody to IL-23p19. By biological
-99sample is intended any biological sample obtained from an individual, cell line, tissue culture, or other source of cells potentially expressing IL-23, Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.
In some embodiments, the method can further comprise comparing the level of IL-23 in a patient sample to a control sample (e.g., a subject that does not hâve an IL-23associated disorder) to détermine if the patient has an IL-23-associated disorder or is at risk of developing an IL-23-associated disorder.
It will be advantageous in some embodiments, for example, for diagnostic purposes to label the antibody with a détectable moiety. Numerous détectable labels are available, including radioisotopes, fluorescent labels, enzyme substrate labels and the like. The label may be indirectly conjugated with the antibody using various known techniques. For example, the antibody can be conjugated with biotin and any of the three broad categories of labels mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label with the antibody, the antibody can be conjugated with a small hapten (such as digoxin) and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g., anti-digoxin antibody). Thus, indirect conjugation of the label with the antibody can be achieved.
Exemplary radioisotopes labels include 35S, 14C, 125l, 3H, and 131l. The antibody can be labeled with the radioisotope, using the techniques described in, for exampie, Current Protocols in Immunology, Volumes 1 and 2, 1991, Coligen et al., Ed. Wiley-lnterscience, New York, N.Y., Pubs. Radioactivity can be measured, for example, by scintillation counting.
Exemplary fluorescent labels include labels derived from rare earth chelates (europium chelates) or fluorescein and its dérivatives, rhodamine and its dérivatives, dansyl, Lissamine, phycoerythrin, and Texas Red are available. The fluorescent labels can be conjugated to the antibody via known techniques, such as those disclosed in Current Protocols in Immunology, for example. Fluorescence can be quantified using a fluorimeter.
-100 There are various well-characterized enzyme-substrate labels known in the art (see,
e.g., U.S. Pat. No. 4,275,149 for a review). The enzyme generally catalyzes a chemical alteration of the chromogenic substrate that can be measured using various techniques. For example, alteration may be a color change in a substrate that can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above. The chemiluminescent substrate becomes electronically excited by a chemical reaction and may then émit light that can be measured, using a chemiluminometer, for example, or donates energy to a fluorescent acceptor.
Examples of enzymatic labels include luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocydic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are described, for example, in O'Sullivan et al., 1961, Methods for the Préparation of Enzyme-Antibody Conjugales for use in Enzyme Immunoassay, in Methods in Enzym. (J. Langone & H. Van Vunakis, eds.), Academie press, N.Y., 73: 147-166.
Examples of enzyme-substrate combinations include, for example: Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor such as orthophenylene diamine (OPD) or 3,3',5,5'tetramethyl benzidine hydrochloride (TMB); alkaline phosphatase (AP) with paraNitrophenyl phosphate as chromogenic substrate; and β-D-galactosidase (β-D-Gal) with a chromogenic substrate such as p-nitrophenyl-p-D-galactosidase or fluorogenic substrate 4-methylumbelliferyl-p-D-galactosidase.
Numerous other enzyme-substrate combinations are available to those skilled in the art. For a general review of these, see U.S. Pat. No. 4,275,149 and U.S. Pat. No. 4,318,980.r\J
- 101 In another embodiment, the humanized anti-IL-23p19 antibody is used unlabeled and detected with a labeled antibody that binds the humanized anti-IL-23p19 antibody.
The antibodies described herein may be employed in any known assay method, such as compétitive binding assays, direct and indirect sandwich assays, and immunoprécipitation assays. See, e.g., Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).
The anti-IL-23p19 antibody or antigen binding fragment thereof can be used to inhibit the binding of IL-23 to the IL-23 receptor. Such methods comprise administering an anti-IL-23p19 antibody or antigen binding fragment thereof to a cell (e.g., a mammalian cell) or cellular environment, whereby signaling mediated by the IL-23 receptor is inhibited. These methods can be performed in vitro or in vivo. By “cellular environment is intended the tissue, medium, or extracellular matrix surrounding a cell. The anti-IL23p19 antibody or antigen binding fragment thereof is administered to the cellular environment of a cell in such a manner that the antibody or fragment is capable of binding to IL-23 molécules outside of and surrounding the cell, therefore, preventing the binding of IL-23 to its receptor.
Diagnostic Kits
An anti-IL-23p19 antibody can be used in a diagnostic kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic assay. Where the antibody is labeled with an enzyme, the kit may include substrates and cofactors required by the enzyme such as a substrate precursor that provides the détectable chromophore or fluorophore. In addition, other additives may be included such as stabilizers, buffers (for example a block buffer or lysis buffer), and the like. The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents that substantially optimize the sensitivity of the assay. The reagents may be provided as dry powders, usually lyophilized, including excipients that on dissolution will provide a reagent solution having the appropriate concentration. hT
-102Therapeutic Uses
In another embodiment, a humanized anti-IL-23p19 antibody disclosed herein is useful in the treatment of various disorders associated with the expression of IL-23p19 as described herein. Methods for treating an IL-23 associated disorder comprise administering a therapeutically effective amount of a humanized anti-IL-23p19 antibody to a subject in need thereof.
The humanized anti-IL-23p19 antibody or agent is administered by any suitable means, including parentéral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local immunosuppressive treatment, intralesional administration (including perfusing or otherwise contacting the graft with the antibody before transplantation). The humanized anti-IL-23p19 antibody or agent can be administered, for example, as an infusion or as a bolus. Parentéral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the humanized anti-IL-23p19 antibody is suitably administered by puise infusion, particularly with declining doses of the antibody. In one aspect, the dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
For the prévention or treatment of disease, the appropriate dosage of antibody will dépend on a variety of factors such as the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for préventive or therapeutic purposes, previous therapy, the patient's clinical hîstory and response to the antibody, and the discrétion of the attending physician. The antibody is suitably administered to the patient at one time or over a sériés of treatments.
Depending on the type and severity of the disease, about 1 gg/kg to 20 mg/kg (e.g., 0.115 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several
-103 days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays. An exemplary dosing regimen is that disclosed in WO 94/04188.
The term suppression is used herein in the same context as “amelioration and “alleviation to mean a lessening of one or more characteristics of the disease.
The antibody composition will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for considération in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The therapeutically effective amount of the antibody to be administered will be governed by such considérations, and is the minimum amount necessary to prevent, ameliorate, or treat the disorder associated with IL-23 expression.
The antibody need not be, but is optionally, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents dépends on the amount of humanized anti-IL-23p19 antibody présent in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99% of the heretofore employed dosages.
IL-23-Associated Disorders
The anti-IL-23p19 antibodies or agents are useful for treating or preventing an immunological disorder characterized by abnormal expression of IL-23, e.g., by inappropriate activation of immune cells (e.g., lymphocytes or dendritic cells). Such abnormal expression of IL-23 can be due to, for example, increased IL-23 protein levels. The anti-IL-23p19 antibodies or antigen binding fragments thereof also find use in the treatment or prévention of respiratory disorders, metaboiic disorders, for example diabètes mellitus, and certain cancers. Treatment or prévention of the immunologicaliX>/^
-104disorder, respiratory disorder, metabolic disorder or cancer, according to the methods described herein, is achieved by administering to a subject in need of such treatment or prévention an effective amount of the anti-IL-23p19 antibody or agent, whereby the antibody decreases the activity of IL-23 associated with the disease state.
Immunological diseases that are characterized by inappropriate activation of immune cells and that can be treated or prevented by the methods described herein can be classified, for example, by the type(s) of hypersensitivity reaction(s) that underlie the disorder. These reactions are typically classified into four types: anaphylactic reactions, cytotoxic (cytolytic) reactions, immune complex reactions, or cell-mediated immunity (CMI) reactions (also referred to as delayed-type hypersensitivity (DTH) reactions). (See, e.g., Fundamental Immunology (William E. Paul ed., Raven Press, N.Y., 3rd ed. 1993).) Immunological diseases include inflammatory diseases and autoimmune diseases.
Spécifie examples of such immunological diseases include the following: rheumatoid arthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis, allergie encephalomyelitis), endocrine opthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Grave's disease, glomerulonephritis, autoimmune hepatological disorder, inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), anaphylaxis, allergie reaction, Sjogren's syndrome, type I diabètes mellitus, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, inflammatory myositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anémia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopénie purpura, hemolytic anémia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyl), and telangiectasia), male and female autoimmune infertility, ankylosing spondolytis, ulcerative colitis, mixed connective tissue disease, polyarteritisv</^
-105 nedosa, systemic necrotizing vasculitis, atopie dermatitis, atopie rhinitis, Goodpasture's syndrome, Chagas’ disease, sarcoidosis, rheumatic fever, asthma, récurrent abortion, anti-phospholipid syndrome, farmer's lung, erythema multiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird-fancier’s lung, toxic epidermal necrolysis, Alport's syndrome, alveolitis, allergie alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, Takayasu’s arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki's disease, dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum, psoriasis, psoriatic arthritis, erythroblastosis fetalis, éosinophilie faciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft versus host disease, transplantation rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenstrom’s macroglobulemia, Evan's syndrome, acute respiratory distress syndrome, pulmonary inflammation, osteoporosis, delayed type hypersensitivity and autoimmune gonadal failure.
In some embodiments, the immunological disorder is a T cell-mediated immunological disorder and accordingly, the anti-IL-23p19 antibodies and agents as described herein are also useful for treating or preventing T cell-mediated immunological disorders.
In one aspect, the anti-IL-23p19 antibodies or agents are useful for treating or preventing a respiratory disorder in which IL-23 is abnormally expressed. Treatment or prévention of the respiratory disorder, according to the methods described herein, is achieved by administering to a subject in need of such treatment or prévention an effective amount of the anti-IL-23p19 antibody or agent, whereby the antibody decreases the activity of IL-23 associated with the disease state.These include, but are not limited to: respiratory complaints, obstructive pulmonary diseases of various origins, pulmonary emphysema of various origins, restrictive pulmonary diseases, interstitial pulmonary diseases, interstitial lung disease, cystic fibrosis, bronchitis of various origins, u/-'
-106 bronchiectasis, ARDS (adult respiratory distress syndrome) and ail forms of pulmonary oedema; obstructive pulmonary diseases selected from among COPD (chronic obstructive pulmonary disease), asthma, bronchial asthma, paediatric asthma, severe asthma, acute asthma attacks and chronic bronchitis; pulmonary emphysema which has its origins in COPD (chronic obstructive pulmonary disease) or a 1-protéinase inhibitor deficiency; restrictive pulmonary diseases selected from among allergie alveolitis, restrictive pulmonary diseases triggered by work-related noxious substances, such as asbestosis or silicosis, and restriction caused by lung tumours, such as lymphangiosis carcinomatosa, bronchoalveolar carcinoma and lymphomas; pneumonia caused by infections, such as for example infection by viruses, bacteria, fungi, protozoa, helminths or other pathogens, pneumonitis caused by various factors, such as for example aspiration and left heart insufficiency, radiation-induced pneumonitis or fibrosis, collagénoses, such as for example lupus erythematosus, systemic scleroderma or sarcoidosis, granulomatoses, such as for example Boeck’s disease, idiopathic interstitiel pneumonia or idiopathic pulmonary fibrosis (IPF); mucoviscidosis, bronchitis caused by bacterial or viral infection, allergie bronchitis and toxic bronchitis; bronchiectasis; pulmonary oedema, for example, toxic pulmonary oedema after aspiration or inhalation of toxic substances and foreign substances; rhinitis, arthritis and related arthropathies, psoriasis, myeloid leukemia, multiple sclerosis, Alzheimer's disease, glomerulonephritis, and chronic atopie dermatitis.
In another aspect, the anti-IL-23p19 antibodies and agents as described herein are also useful for treating cancers, in which IL-23 is abnormally expressed.
IL-23-expressing cancers that can be treated by the methods described herein include, for example, leukemia, such as acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia (e.g., myeloblastic, promyelocytic, myelomonocytic, monocytic, or erythroleukemia), chronic leukemia, chronic myelocytic (granulocytic) leukemia, or chronic lymphocytic leukemia; Polycythemia vera; Lymphoma (e.g., Hodgkin's disease or Non-Hodgkin’s disease); multiple myeloma, Waldenstrom's macroglobulinemia; heavy chain disease; solid tumors such sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, ostéogénie sarcoma, osteosarcoma, vt/~
- 107 chordoma, angîosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, rénal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, non small cell lung carcinoma, bladder carcinoma, épithélial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, or esophageal carcinoma).
Pharmaceutical Compositions and Administration Thereof
A composition comprising an IL-23p19 binding agent (e.g., an anti-IL-23p19 antibody) can be administered to a subject having or at risk of having an îmmunological disorder, respiratory disorder or a cancer. The invention further provides for the use of a IL-23p19 binding agent (e.g., an anti-IL-23p19 antibody) in the manufacture of a médicament for prévention or treatment of a cancer, respiratory disorder or îmmunological disorder. The term subject as used herein means any mammalian patient to which an IL23p19binding agent can be administered, including, e.g., humans and non-human mammals, such as primates, rodents, and dogs. Subjects specifically intended for treatment using the methods described herein include humans. The antibodies or agents can be administered either alone or in combination with other compositions in the prévention or treatment of the îmmunological disorder, respiratory disorder or cancer. Such compositions which can be administered in combination with the antibodies or agents include methotrexate (MTX) and immunomodulators, e.g. antibodies or small molécules. \k/
- 108Examples of antibodies for use in such pharmaceutical compositions are those that comprise a humanized antibody or antibody fragment having the light chain variable région amino acid sequence of any of SEQ ID NO: 84, 86, 88, 90, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117 or 119. Examples of antibodies for use in such pharmaceutical compositions are also those that comprise a humanized antibody or antibody fragment having the heavy chain variable région amino acid sequence of any of SEQ ID NO: 121, 123, 125, 127, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154 or 156.
Further examples of antibodies for use in such pharmaceutical compositions are also those that comprise a humanized antibody or antibody fragment having the light chain variable région amino acid sequence of any of SEQ ID NO: 158, 160, 162 or 164. Preferred antibodies for use in such pharmaceutical compositions are also those that comprise a humanized antibody or antibody fragment having the heavy chain variable région amino acid sequence of any of SEQ ID NO:166,168,170 or 172,
Further examples of antibodies for use in such pharmaceutical compositions are also those that comprise a humanized antibody or antibody fragment having the light chain variable région and heavy chain variable région of any of SEQ ID NO: 160 and 166, SEQ ID NO: 160 and 168, SEQ ID NO: 158 and 166 or SEQ ID NO: 158 and 168.
Further examples of antibodies for use in such pharmaceutical compositions are also those that comprise a humanized antibody having the light chain région amino acid sequence of any of SEQ ID NO:174 or 180. Preferred antibodies for use in such pharmaceutical compositions are also those that comprise humanized antibody having the heavy chain variable région amino acid sequence of any of SEQ ID NO:176 or 178.
Further examples of antibodies for use in such pharmaceutical compositions are also those that comprise Antibody A, Antibody B, Antibody C or Antibody D.
Various delivery Systems are known and can be used to administer the IL-23p19 binding agent. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, épidural, and oral routes. The IL23p19 binding agent can be administered, for example by infusion, bolus or injection, and can be administered together with other biologically active agents such as
-109 chemotherapeutic agents. Administration can be systemic or local. In preferred embodiments, the administration is by subcutaneous injection. Formulations for such injections may be prepared in for example prefilled syringes that may be administered once every other week.
In spécifie embodiments, the IL-23p19 binding agent composition is administered by injection, by means of a cathéter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber. Typically, when administering the composition, materials to which the anti-IL-23p19 antibody or agent does not absorb are used.
In other embodiments, the anti-IL-23p19 antibody or agent is delivered in a controlled release System. In one embodiment, a pump may be used (see, e.g., Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used. (See, e.g., Medical Applications of Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fia., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Bail eds., Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. See also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105.) Other controlled release Systems are discussed, for example, in Langer, supra.
An IL-23p19 binding agent (e.g., an anti-IL-23p19 antibody) can be administered as pharmaceutical compositions comprisîng a therapeutically effective amount of the binding agent and one or more pharmaceutically compatible ingrédients.
In typical embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous or subcutaneous administration to human beings. Typically, compositions for administration by injection are solutions in stérile isotonie aqueous buffer. Where necessary, the pharmaceutical can also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the
- 110 ingrédients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent Where the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing stérile pharmaceutical grade water or saline. Where the pharmaceutical is administered by injection, an ampoule of stérile water for injection or saline can be provided so that the ingrédients can be mixed prior to administration.
Further, the pharmaceutical composition can be provided as a pharmaceutical kit comprising (a) a container containing a IL-23p19 binding agent (e.g., an anti-IL-23p19 antibody) in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (e.g., stérile water) for injection. The pharmaceutically acceptable diluent can be used for reconstitution or dilution of the lyophilized anti-IL-23p19 antibody or agent. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
The amount of the IL-23p19 binding agent (e.g., anti-IL-23p19 antibody) that is effective in the treatment or prévention of an immunological disorder or cancer can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The précisé dose to be employed in the formulation will also dépend on the route of administration, and the stage of immunological disorder or cancer, and should be decided according to the judgment of the practitioner and each patients circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test Systems.
Generally, the dosage of an anti-IL-23p19 antibody or IL-23p19 binding agent administered to a patient with an immunological disorder or IL-23p19-expressing cancer is typically about 0.1 mg/kg to about 100 mg/kg of the subject’s body weight. The dosage administered to a subject is about 0.1 mg/kg to about 50 mg/kg, about 1 mg/kg to about 30 mg/kg, about 1 mg/kg to about 20 mg/kg, about 1 mg/kg to about 15 mg/kg, or about 1 mg/kg to about 10 mg/kg of the subject's body weight.
-111 Exemplary doses include, but are not limited to, from 1 ng/kg to 100 mg/kg. In some embodiments, a dose is about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15 mg/kg or about 16 mg/kg. The dose can be administered, for example, daily, once per week (weekly), twice per week, thrice per week, four times per week, five times per week, six times per week, biweekly or monthly, every two months, or every three months. In spécifie embodiments, the dose is about 0.5 mg/kg/week, about 1 mg/kg/week, about 2 mg/kg/week, about 3 mg/kg/week, about 4 mg/kg/week, about 5 mg/kg/week, about 6 mg/kg/week, about 7 mg/kg/week, about 8 mg/kg/week, about 9 mg/kg/week, about 10 mg/kg/week, about 11 mg/kg/week, about 12 mg/kg/week, about 13 mg/kg/week, about 14 mg/kg/week, about 15 mg/kg/week or about 16 mg/kg/week. In some embodiments, the dose ranges from about 1 mg/kg/week to about 15 mg/kg/week.
In some embodiments, the pharmaceutical compositions comprising the IL-23p19 binding agent can further comprise a therapeutic agent, either conjugated or unconjugated to the binding agent. The anti-IL-23p19 antibody or IL-23p19 binding agent can be co-administered in combination with one or more therapeutic agents for the treatment or prévention of immunological disorders or cancers.
Such combination therapy administration can hâve an additive or synergistic effect on disease parameters (e.g., severity of a symptom, the number of symptoms, or frequency of relapse).
With respect to therapeutic regimens for combinatorial administration, in a spécifie embodiment, an anti-IL-23p19 antibody or IL-23p19 binding agent is administered concurrently with a therapeutic agent. In another spécifie embodiment, the therapeutic agent is administered prior or subséquent to administration of the anti-IL-23p19 antibody or IL-23p19 binding agent, by at least an hour and up to several months, for example at least an hour, five hours, 12 hours, a day, a week, a month, or three months, prior or subséquent to administration of the anti-JL-23p19 antibody or IL-23p19 binding agent.
-112Articles of Manufacture
In another aspect, an article of manufacture containing materials useful for the treatment of the disorders described above is included. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition that is effective for treating the condition and may hâve a stérile access port. For example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. The active agent in the composition is the humanized anti-IL-23p19 antibody. The label on or associated with the container indicates that the composition is used for treating the condition of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials désirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
The invention is further described in the following examples, which are not intended to limit the scope of the invention.
Examples
Example 1: Production of Humanized Anti-IL-23p19 Antibodies
Mouse lead antibody 6B8 was converted to a chimeric antibody consisting of the mouse variable domain of 6B8 and a human constant IgGIKO domain. Mouse antibody 6B8 is shown in Tables 1 and 2 herein above. The IgGIKO (knock out) has two replacement mutations (Leu234Ala and Leu235Ala) that eliminate ADCC and CDC activity by reducing effector functions such as FcyR and complément binding. The variable domains of the mouse and chimeric antibodies are identical. Chimeric antibodies are generated to confirm the function of the antibody and to ensure the correct sequence has been obtained. The variable région of the antibody is then humanized through a design and screening process. A library was made where human and mouse residues
-113were varied in such a way that in any given position there could be either a human or mouse residue. Such a library was made for those amino acids that were different between human germline and mouse antibody. Only the clones that retain the function of the parent mouse antibody were selected. Représentative humanized variable 5 régions for antibody 6B8 are shown in Tables 5 and 6.
In this manner, Antibody A, Antibody B, Antibody C and Antibody D were humanized antibodies derived from mouse antibody 6B8 (cloned into a human lgG1-KO (KO=knock-out)/kappa backbone. Antibodies A, B, C and D are shown in Table 7.
Example 2: Binding of antibodies to recombinant IL-23 protein
A) Kinetics and affinity of mouse anti-IL-23p19 antibodies binding to recombinant human IL-23 are shown below (Table 9). Kinetics and binding affinities were measured using the Fortebio Octet (Fortebio, Menlo Park, CA) using material generated from hybridoma following single column purification. Since the Octet is not a fluidics based technology, this method does not provide précisé détermination of off-rate. In some cases, only a estimate of affinity can be obtained.
Table 9
Antibody ka(1/Ms) kd(1/s) KD(pM)
18C4 3.84E+05 2.14E-06 5.57
18E5 3.29E+05 2.61 E-06 7.93
18D3 3.19E+05 2.16E-06 6.78
20 E8 4.21E+05 2.69E-04 638
22 E2 3.46E+05 3.53E-04 1024
24A5 2.02E+05 4.57E-06 22.6
15C11 4.11E+05 1.07E-05 26
43F5 1.72E+05 5.96E-06 34.6
27G8 1.57E+05 4.26E-06 27.2
31H9 2.99E+05 3.45E-06 11.5
2D1 < 1e-6 < 1
-114-
9D12 < 1e-6 < 1
6B8 < 1e-6 < 1
73H10 5.29E+04 5.24E-06 99.2
74H3 3.06E+04 2.09E-06 68.3
35H8
26F7 4.76E+05 1.34E-05 28.1
34G3 9.1ΘΕ+05 3.10E-05 32.8
34D9 3.44E+03 1.87E-06 544
B) Affinities were measured for humanized antibodies derived from mouse antibody
6B8. Kinetic binding data, measured using the ProteON XPR36 (Biorad, Hercules, CA) and globally fit to a 1:1 binding model, demonstrated the interactions with recombinant IL-23 either with orwithout a 21 amino acid linker covalently joining the p19 and p40 subunits to be of high affinity, in the range of 1 pM - 100 pM (Table 10). Antibody 6H12 (disclosed in WO 2007/027714), antibody QF20 (disclosed in WO 2007/024846) and antibody C1273 (disclosed in WO 2007/005955) were also tested.
Table 10
Antibody Human IL-23 with linker Human IL-23 no linker
ka (1/Ms) kd (1/s) KD (PM) ka (1/Ms) kd (1/s) KD (pM)
Mouse 6B8 5.57E+05 1.38E-05 24.5
Antibody A 6.27E+05 < 1e-6 < 1 5.51 E+05 < 1e-6 < 1
Antibody B 3.56E+05 < 1e-6 < 1 5.17E+05 < 1e-6 < 1
Antibody C 3.74E+05 1.19E-05 31.8 4.54E+05 1.65E-05 36.3
Antibody D 3.82E+05 4.07E-05 107 3.66E+05 4.93E-05 135
-115-
C-1273 3.60E+05 5.75E-06 15.8
6H12 4.99E+05 1.07E-04 214
QF20 2.03E+05 5.89E-06 2.91
C) Affinity and kinetic data for the anti-IL-23p19 antibodies binding to cynomologous IL23 were measured on the ProteON XPR36, and globally fit to a 1:1 binding model (Table
11). Antibody 6H12 (disclosed in WO 2007/027714), antibody QF20 (disclosed in WO 2007/024846) and antibody C1273 (disclosed in WO 2007/005955) were also tested.
Table 11
Antibody KD(pM) ka (1/Ms) kd (1/s)
Antibody A < 1 2.95E+06 < 1e-6
Antibody B < 1 2.99E+06 < 1e-6
Antibody C 2.9 3.23E+06 9.36E-06
Antibody D 15.9 2.07E+06 3.29E-05
C-1273 >5,000 n/a n/a
6H12 157 9.91 E+05 1.56E-04
QF20 1.2 3.90E+06 4.78E-06
D) Molecular Selectivity over human IL-12
The anti-IL-23p19 antibodies were also injected over a human IL-12 surface at a concentration of 100 nM. The binding signal for these antibodies measured using the Fortebio Octet is zéro, which indicates that these antibodies selectively bind to human IL-23. The binding of the anti-IL-23p19 antibodies to IL-23 was also analyzed in the presence of 50% human sérum and no significant effect of sérum on binding on-rate was observed demonstrating high specificity.
Example 3: Compétition binding assay of human IL-23 binding to human IL-23R/Fc Human IL-23R-Fc was captured on the biosensor surface and 10 nM of human IL-23 was injected. The sensorgram indicates the spécifie binding between IL-23 and the IL-23 receptor (Figure 2, top trace). Antibodies were then co-injected with 10 nM human IL-23^/^
- 116to assess whether antibody binding to the IL-23 could inhibit the interaction between IL23 and the IL-23 receptor. In this example, if the antibody binds to human IL-23 and is able to inhbit the interaction then reduced or no binding will be observed (Figure 2, bottom trace). In the example shown an équivalent molar concentration of Antibody A was co-injected with 10 nM recombinant human IL-23.
Example 4: Functional Cell Assays, Inhibition of IL-17 production from IL-23 stimulated mouse splénocytes
One functional cell assay for anti-IL-23p19 antibodies measures their ability to inhibit IL23 stimulated IL-17 production from mononuclear cells isolated from mouse spleens. Human recombinant IL-23 protein is capable of stimulating IL-17 release from mouse splénocytes. In addition, a natural source of human IL-23 found in the supernatant of activated human monocytic THP-1 cells can be used to stimulate IL-17 production from mouse mononuclear cells.
Human recombinant IL-23 or natural human IL-23 from activated THP-1 cells was preincubated with titrated anti-IL-23p19 antibodies. The IL-23/antibody combinations were then added to freshly isolated murine splénocytes. Recombinant IL-23 alone was used as a positive control. After two days in culture, cell supernatants were collected and assayed for IL-17 by ELISA (R&D Systems, Minneapolis, MN). Représentative IC50 values for anti-IL-23p19 antibodies are shown below. The tested antibodies are mouse antibodies derived from hybridomas (rows 1-19, see tables 1 and 2), chimeric antibodies (rows 20-23), and Antibodies A to D. Antibody 6H12 (disclosed in WO 2007/027714), antibody QF20 (disclosed in WO 2007/024846) and antibody C1273 (disclosed in WO 2007/005955) were also tested.
Table 12
Antibody IC50 Values (pM), recombinant human IL23 IC50 Values (pM), natural human IL-23
18C4 471 100, 413
18E5 not determined 9, 9, 13
-117-
18D3 234 not determined
20E8 not determined 438, 561
22E2 61, 130 117, 35
24A5 22, 37 85, 31
15C11 126 232
43F5 250, 8000 8000
27G8 235 5000
31H9 960 2000
2D1 not determined 2336,1911,1597
9D12 59 281,138
6B8 13 8,2
73H10 1411 not determined
74H3 1352 not determined
36H8 not determined not determined
26F7 27 2, 8
34G3 336 27, 25
34D9 510 456
Chimeric 18E5 31 8, 36, 10, 9
Chimeric 22E2 100 9, 178
Chimeric 24A5 404 95, 102
Chimeric 6B8 26, 37, 57 5, 2, 6, 3
Antibody A 5, 5, 5, 15 1, 1
Antibody B 13, 30, 54, 42 9, 8
Antibody C 53, 71, 162, 89 16, 32
Antibody D 236, 225, 614, 458 133, 125
6H12 1600, 806, 1300 957, 4400, 1013, 439
QF20 not determined 7, 12
C1273 not determined 93, 44
- 118Example 5: Functional specificity testing against IL-12 in a human activated T cell assay
Anti-IL-23p19 antibodies were tested for functional inhibition of IL-12 in a human activated T cell assay, Human recombinant IL-12 (1 ng/ml) was preincubated with 5 pg/ml anti-IL-23p19 antibodies. The IL-12/antibody combinations were then added to human PHA-derived T cell blasts. Recombinant IL-12 alone was used as a positive control. An anti-IL-12p70 antibody (Bender MedSystems, Vienna, Austria) was used as a control inhibitory antibody. After two days in culture, cell supernatants were collected and assayed for IFN-γ by ELISA (R&D Systems). Samples were tested in triplicate and the average pg/ml of IFN-γ was determined. Results (with standard déviations) are shown in the table below.
Table 13
Antibody Cytokine Stimulation Average pg/ml IFN-γ +/Standard Déviation
no antibody None 87 +/- 7
no antibody 1 ng/ml IL-12 532 +/- 51
chimeric 18E5 1 ng/ml IL-12 511 +/- 3
chimeric 6B8 1 ng/ml IL-12 523 +/- 60
Antibody A 1 ng/ml IL-12 497 +/- 30
Antibody B 1 ng/ml IL-12 537 +/- 2
Antibody C 1 ng/ml IL-12 495 +/- 25
Antibody D 1 ng/ml IL-12 539 +/- 38
anti-IL-12p70 antibody 1 ng/ml IL-12 119 +/- 12
Example 6: Inhibition of IL-23 induced STAT3 phosphorylation in the human cell line DB
- 119 The human cell line DB (ATCC, Manassas, VA) responds to IL-23 stimulation through an endogenous IL-23Rcomplex (IL-23R and IL-12Rp1) and phosphorylâtes STAT3 in an IL-23 dose dépendent manner. An assay was developed for testing anti-IL-23p19 antibody inhibition of IL-23 induced STAT3 phosphorylation. DB cells were plated at 1x10e6 cells/well in a 96 well plate. Antibodies to be tested were serially diluted and preincubated with recombinant human IL-23 (10 ng/ml)for 1 hour at room température.
The antibody/IL-23 mixture was then added to the cells for 30 minutes at 37°C. Cells were harvested by centrifugation at 4°C for 10 minutes and then iysed in ice cold buffer (Cell Signaling Technology, Beverly, MA). A portion of the lysate was run in a phosphoSTAT3 ELISA (Invitrogen). Antibody IC50 values were calculated as percent inhibition of STAT3 phosphorylation compared to control wells without antibody. Représentative IC50 values are shown in the table below.
Table 14
Antibody IC50 (pM)
Antibody A 25, 15, 38, 23, 13, 18
Antibody B 73, 84
Antibody C 132, 80
Antibody D 158
QF20 26, 26, 27
C-1273 163, 438
Example 7: In vivo model of IL-23 induced cytokine production in the mouse ear An in vivo model in the mouse was used. Recombinant human IL-23 is injected into the skin of the mouse ear for 4 consecutive days resulting in epidermal thickening and upregulation of IL-17 and IL-22 protein. Anti-IL-23p19 antibodies were evaluated in this model. A single intraperitoneal injection of 1mg/kg or 5 mg/kg antibody was administered 1 hour prior to the initial IL-23 injection into the skin. Recombinant human IL-23 (with linker) was injected once daily for 3 additional days and tissue was collected for cytokine assessment. Inhibition of cytokine production was demonstrated for the χχΧ-'
-120antibodies. The résulte of three experiments are shown in the table below (exp. 1 : rows
1-7, exp. 2: rows 8-10, exp. 3: rows 11-14).
Table 15
Ear Tissue IL-17 pg/ml Mean +/SEM Ear Tissue IL-17 Percent Inhibition Ear Tissue IL- 22 pg/ml Mean +/SEM Ear Tissue IL-22 Percent Inhibition
0.1% BSA + Citrate Buffer i.p. (Unstimulated Control) 3+/-1 NA 1 +/-0 NA
0.3ug IL-23 + Citrate Buffer i.p. (Vehicle Control) 25 +/- 3 NA 274 +/- 30 NA
0.3ug IL-23 + 1mg/kg Antibody 6B8 7+/-2 81 57 +/-19 80
0.3ug IL-23 + 1mg/kg Antibody A 2+/-1 101 17+/-3 94
0.3ug IL-23 + 1mg/kg Antibody B 5+/-1 93 30 +/- 2 89
0.3ug IL-23 + 1mg/kg Antibody C 11 +/- 1 66 108 +/-12 61
0.3ug IL-23 + 1mg/kg Antibody D 10 +/-1 67 151 +/-12 45
0.1% BSA + Vehicle (Unstimulated Control) 14+/-1 NA 1 +/- 1 NA
0.3ug IL-23 + Vehicle 31 +/- 4 NA 129 +/-29 NA
0.3ug IL-23 + 5mg/kg 24A5 14+/- 1 102 10+/-5 93
0.1% BSA + mIgG (Unstimulated Control) 17+/-1 NA 4+/-1 NA
0.3ug IL-23 + mlgG (Vehicle Control) 30 +/- 2 NA 208 +/- 40 NA
0.3ug IL-23 + 5mg/kg 24A5 16+/-0 109 28 +/- 5 88
-121 -
0.3ug IL-23 + 5mg/kg 21 +/- 2 70 53 +/- 41 80
18E5
Example 8: Pharmacokinetic Studies in Cynomolgus Monkey
Humanized anti-IL-23p19 antibodies were administered by ten minute intravenous infusion at a dose of 1.0 mg/kg to three cynomolgus monkeys. Sérum samples were collected over a 6 week time course and free antibody concentrations were measured using a spécifie ELISA. The sérum concentration-time profiles for the antibodies and the corresponding pharmacokinetic parameters are summarized in the Table 16 below.
Table 16
Antibody CL (ml/d/kg) Vol (ml/kg) AUC (nM»h/ml) Ti/2(days) M RT (days)
Antibody A 5.2 88 32262 12.1 17.2
Antibody B 6.0 87 27030 10.1 14.8
Antibody C 4.7 91 34642 14.1 19,6
Antibody D 3.4 67 47633 12.6 19.8
Example 9: Expression in NSO cells and biophysical data
Transfection of NSO cells and génération of stable pools:
NSO cells were grown in the presence of 1% FBS before transfection. 40x10e6 cells were collected and resuspended in 0.8ml in media containing 2% FBS with 20ug of linearized DNA (heavy chain and light chain expression vectors) and then cells were incubated on ice for approximately 15 min before electroporation of the cells at 750V/25uF (Bio-Rad Gene Puiser Xcell). Cells were recovered with 2% FBS for approximately 48 hours at 37°C and 5% CO2 then plated in 96 well plates at 2x10e5 cells/ml containing G418 and mycophenolic acid for 14-21 days until formation of colonies,
-122Supernatant from 96 well plates with colonies were screened by ELISA. ELISA plates were coated with 1 ug/ml of goat anti-kappa (Southern Biotech, Birmingham, AL) in PBS and diluted supernatant were incubated and then detected with goat anti-human IgG FcHRP (from Jackson ImmunoResearch Laboratories, West Grave, PA). The positive colonies were pooled for scale up. Titers for antibody production were determined by ForteBio using protein A tips according to manufacture protocol. The titers for Antibody A and Antibody D were between 250-350 mg/L, with more than 80% recovery from protein purification, and more than 94% monomer after IEX purification. Proteins were resuspended in a final buffer containing 20mM Sodium Citrate and 115mM NaCI, pH 6.0 and are stable at 4 °C for at least 4 months and with solubility up to 100 mg/ml in this buffer.
Table 17
Protein A Column IEX Column
Titer (mg/L) Yield (mg/L) Recovery Yield (mg/L) Recovery
Antibody A 345 275 80% 221 80%
Antibody D 248 225 90% 175 78%
Table 18
Quality Stability Solubility
AUC fresh (%M) SEC fresh (%M) AUC 1 month (%M) SEC 1 month (%M) AUC 4months (%M) SEC 4months (%M) AUC at 100 mg/ml (%M)
Antibody A 98 99 97 99 96 99 99
Antibody D 94 100 98 100 99 99 97
-123AUC: Analytical Ultracentrifugation as measured by the sédimentation velocity method at concentrations of 0.5 -1 mg/ml; SEC: Size exclusion chromatography; %M: percent monomer.
Example 10: Epitope mapping
Hydrogen/Deuterium Exchange Mass Spectrometry (HXMS) was employed to map the epitope of Antibody A binding to human IL-23p19. This method determined the susceptibility of the amide backbone hydrogens of IL-23p19 to exchange with D2O. The experiment was conducted with IL-23 alone and IL-23 with added Antibody A. Régions of the IL-23p19 sequence showing significant protection from exchange due to binding of Antibody A were thus identified. Resolution of the method is determined by the peptides produced by digestion with pepsin or Protease XVIII, These IL-23p19 derived peptides were identified by additional control experiments with unexchanged samples employing standard accurate mass and HPLC MS/MS technologies.
Recombinant human IL-23 was used. For the protein + antibody sample, 50ul of IL-23 (0.8mg/ml) was incubated with 10ui of Antibody A (12.7mg/ml) for 15 minutes at room température. The final molar ratio Antibody A/IL-23 was 1.2:1.
For the exchange 5ul of IL-23 protein was added to 50ul deuterated buffer (50mM PBS in D2O) and incubated for 100 seconds at room température, 50ul of 2M Urea/0.5M TCEP was added and incubated for 60 seconds at room température. 5ul pepsin or Protease XVIII (4mg/ml in 0.1% formic acid) was added and the sample was immediately cooled to 4 °C.
After 5 minutes 50ul of sample was injected onto a Shimadzu HPLC System (SCL10A controller and two LC10AD pumps) under the following conditions:
Mobile Phase A = 99/1/0.1 (water/acetonitrile/formic acid).
Mobile Phase B = 95/5/0.1 (acetonitrile/water/formic acid).
Flow rate = 100ul/min.
Column = Phenomenex Jupiter C5, 5u, 50x1.0mm.
Mobile phase lines, column, injector loop are in ice baths.
Gradient = Time 0 (3%B), Time 2.2 (3%B), Time 10.1 (90%B), Time 12.0
-124 (90%B), Time 12.1 (3%B).
Mass Spectrometry was carried out as follows:
Mass Spec = Thermo Orbitrap Vélos (0900865).
Methods:
A. Fragmentation (to ID peptides): 12 minute acquisition time (3 minute start delay), full-scan FTMS at 30,000 resolution, seven ion trap data dépendant scans (CID).
B. MS Runs: 12 minute acquisition time (3 minute start delay), full-scan FTMS at 60,000 résolution.
Pepsin and Protease XVIII peptides were identified using fragmentation data and the program Proteome Discoverer (Thermoscientific, Waltham, MA). Identified peptides were visually compared (protein alone vs. protein with antibody présent) using Xcalibur software (Thermoscientific). No significant shifts in exchange were observed for IL-23 alone vs. IL-23 with Antibody A outside of the IL-23p19 région. For the p19 portion of the protein, data was analyzed using the program PepMap (Thermoscientific). This program calculâtes the average mass for exchanged peptides. PepMap results were checked and those peptides that did not yield verified results were calculated with the aid of Microsoft Excel.
The régions of the IL-23 sequence showing significant protection from exchange due to binding of Antibody A were identified as amino acid residues 108 to 126 of SEQ ID NO:181 and amino acid residues 137 to 151 of SEQ ID NO:181.

Claims (27)

Claims: What is Claimed is:
1, wherein the antibody or antigen-binding fragment thereof comprises:
a) a light chain variable région comprising the amino acid sequence of SEQ ID NO:19 (CDR1-L)·, the amino acid sequence of SEQ ID NO:20 (CDR2-L)', and the amino acid sequence of SEQ ID NO:21 (CDR3-L)·, and
b) a heavy chain variable région comprising the amino acid sequence of SEQ ID NO:66 (CDR1-H)·, the amino acid sequence of SEQ ID NO:64 (CDR2-H)·, and the amino acid sequence of SEQ ID NO:65 (CDR3-H).^/~
-126 -
1, wherein the antibody or antigen-binding fragment thereof comprises:
a) a light chain variable région comprising the amino acid sequence of SEQ ID NO:19 (CDR1-L)·, the amino acid sequence of SEQ ID NO:20 (CDR2-L); and the amino acid sequence of SEQ ID NO:21 (CDR3-L)', and
b) a heavy chain variable région comprising the amino acid sequence of SEQ ID NO:63 (CDR1-H); the amino acid sequence of SEQ ID NO:64 (CDR2-H); and the amino acid sequence of SEQ ID NO:65 (CDR3-H).
1) An anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises:
a) a light chain variable région comprising the amino acid sequence of SEQ ID NO:19 (CDR1-L)‘, the amino acid sequence of SEQ ID NO:20 (CDR2-L)·, and the amino acid sequence of SEQ ID NO:21 (CDR3-L); and
b) a heavy chain variable région comprising the amino acid sequence of SEQ ID NO: 63, 66, 67 or 68 (CDR1-H); the amino acid sequence of SEQ ID NO:64 (CDR2-H)·, and the amino acid sequence of SEQ ID NO:65 (CDR3-H).
2) The anti-IL-23p19 antibody or antigen-binding fragment thereof according to claim
3) The anti-IL-23p19 antibody or antigen-binding fragment thereof according to claim
4) The anti-IL-23p19 antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises:
a) a light chain variable région comprising the amino acid sequence of SEQ ID NO:19 (CDR1-L); the amino acid sequence of SEQ ID NO:20 (CDR2-L)', and the amino acid sequence of SEQ ID NO:21 (CDR3-L)·, and
b) a heavy chain variable région comprising the amino acid sequence of SEQ ID NO:67 (CDR1-H)·, the amino acid sequence of SEQ ID NO:64 (CDR2-H)' and the amino acid sequence of SEQ ID NO:65 (CDR3-H).
5) The anti-IL-23p19 antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises:
a) a light chain variable région comprising the amino acid sequence of SEQ ID NO:19 (CDR1-L); the amino acid sequence of SEQ ID NO:20 (CDR2-L); and the amino acid sequence of SEQ ID NO:21 (CDR3-L)·, and
b) a heavy chain variable région comprising the amino acid sequence of SEQ ID NO:68 (CDR1-H)', the amino acid sequence of SEQ ID NO:64 (CDR2-H)‘, and the amino acid sequence of SEQ ID NO:65 (CDR3-H).
6) The anti-IL-23p19 antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable région comprising the amino acid sequence of any one of SEQ ID NO: 158, 160,162 or 164; and a heavy chain variable région comprising the amino acid sequence any one of SEQ ID NO:166,168, 170 or 172.
7) The anti-IL-23p19 antibody or antigen-binding fragment thereof according to claim 6, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable région comprising the amino acid sequence of SEQ ID NO:160 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO:166.
-127 -
8) The anti-IL-23p19 antibody or antigen-binding fragment thereof according to claim 6, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable région comprising the amino acid sequence of SEQ ID NO:160 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO:168.
9) The anti-IL-23p19 antibody or antigen-binding fragment thereof according to claim 6, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable région comprising the amino acid sequence of SEQ ID NO:158 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO:166.
10) The anti-IL-23p19 antibody or antigen-binding fragment thereof according to claim 6, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable région comprising the amino acid sequence of SEQ ID NO:158 and a heavy chain variable région comprising the amino acid sequence SEQ ID NO:168.
11 ) The anti-IL-23p19 antibody or antigen-binding fragment thereof according to claim 6, wherein the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:166 or 168 linked to a human lgG1 heavy chain constant région; and the amino acid sequence of SEQ ID NO:158 or 160 linked to a human kappa light chain constant région.
12) The anti-IL-23p19 antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises: a) a humanized light chain variable domain comprising the CDRs of SEQ ID NO:158 or 160 and framework régions having an amino acid sequence at least 90% identical to the amino acid sequence of the framework régions of the variable domain light chain amino acid sequence of SEQ ID NO:158 or 160; and
-128 -
b) a humanized heavy chain variable domain comprising the CDRs of SEQ ID NO:166 or 168 and framework régions having an amino acid sequence at least 90% identical to the amino acid sequence of the framework régions of the variable domain heavy chain amino acid sequence of SEQ ID NO:166 or 168.
13) The anti-IL-23p19 antibody according to any one of daims 1 to 12, wherein said antibody is a monoclonal antibody.
14) The anti-ÎL-23p19 antibody according to claim 1, wherein the antibody is a monoclonal anti-IL-23p19 antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO:174 or 180 and a heavy chain comprising the amino acid sequence of SEQ ID NO:176 or 178.
15) The monoclonal anti-IL-23p19 antibody according to claim 14, wherein said antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:174 and a heavy chain comprising the amino acid sequence of SEQ ID NO:176.
16) The monoclonal anti-lL-23p19 antibody according to claim 14, wherein said antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:174 and a heavy chain comprising the amino acid sequence of SEQ ID NO:178.
17) The monoclonal anti-IL-23p19 antibody according to claim 14, wherein said antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:180 and a heavy chain comprising the amino acid sequence of SEQ ID NO:176. vZ
-129-
18) The monoclonal anti-IL-23p19 antibody according to claim 14, wherein said antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 180 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 178.
19) An anti-IL-23p19 antibody or antigen-binding fragment thereof that binds to human IL-23p19 at an epitope consisting of amino acid residues 108 to 126 and amino acid residues 137 to 151 of SEQ ID NO: 181.
20) An anti-IL-23p19 antibody or antigen-binding fragment thereof that competitively binds to human IL-23p19 with:
a) a humanized monoclonal anti-IL-23p19 antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 174 and a heavy chain comprising the amino acid sequence of SEQ ID NO:176; or
b) a humanized monoclonal anti-IL-23p19 antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 174 and a heavy chain comprising the amino acid sequence of SEQ ID NO:178; or
c) a humanized monoclonal anti-IL-23p19 antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO:180 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 176; or
d) a humanized monoclonal anti-IL-23p19 antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 180 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 178.
21 ) The anti-lL-23p19 antibody according to any one of daims 19 or 20, wherein said antibody is a monoclonal antibody.
22) An antibody or antigen-binding fragment according to any one of daims 1 to 21 for use in medicine.
-130-
23) The antibody or antigen-binding fragment according to claim 22, wherein the use is the treatment of an inflammatory disease, of an autoimmune disease, of a respiratory disease, of a metabolic disorder or of cancer.
24) The antibody or antigen-binding fragment according to claim 22, wherein the use is for the treatment of psoriasis, inflammatory bowel disease, psoriatic arthritis, multiple sclerosis, rheumatoid arthritis, Crohn’s disease or ankylosing spondylitis.
25) A pharmaceutical composition comprisîng an antibody or antigen-binding fragment according to any one of daims 1 to 21 and a pharmaceutically acceptable carrier.
26) An isolated polynucleotide comprisîng a sequence encoding a light chain variable région of an antibody or antibody fragment having the amino acid sequence of SEQ ID NO:158, 160,162 or 164, or encoding a heavy chain variable région of an antibody or antibody fragment having the amino acid sequence of SEQ ID NO: 166, 168, 170 or 172.
27) The isolated polynucleotide according to claim 26, wherein said polynucleotide comprises a sequence encoding a light chain of an antibody having the amino acid sequence of SEQ ID NO:174 or 180, or encoding a heavy chain of an antibody having the amino acid sequence of SEQ ID NO:176 or 178.
OA1201300166 2010-11-04 2011-11-02 Anti-IL-23 antibodies. OA16388A (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US61/410158 2010-11-04
US61/411953 2010-11-10
US61/412594 2010-11-11
US61/448785 2011-03-03

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OA16388A true OA16388A (en) 2015-10-07

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