U.S. patent application number 14/258994 was filed with the patent office on 2014-11-13 for il-17 binding proteins.
This patent application is currently assigned to ABBVIE INC.. The applicant listed for this patent is Shaughn H. Bryant, Anca Clabbers, Chung-Ming Hsieh, Margaret Hugunin, Yuliya Kutskova, Mary R. Leddy, Bradford L. McRae, John E. Memmott, Anwar Murtaza, Jennifer M. Perez, Edit Tarcsa, Craig Wallace, Suju Zhong. Invention is credited to Shaughn H. Bryant, Anca Clabbers, Chung-Ming Hsieh, Margaret Hugunin, Yuliya Kutskova, Mary R. Leddy, Bradford L. McRae, John E. Memmott, Anwar Murtaza, Jennifer M. Perez, Edit Tarcsa, Craig Wallace, Suju Zhong.
Application Number | 20140335564 14/258994 |
Document ID | / |
Family ID | 42710240 |
Filed Date | 2014-11-13 |
United States Patent
Application |
20140335564 |
Kind Code |
A1 |
Hsieh; Chung-Ming ; et
al. |
November 13, 2014 |
IL-17 BINDING PROTEINS
Abstract
Proteins that bind IL-17 and/or IL-17F are described along with
there use in composition and methods for treating, preventing, and
diagnosing IL-17 related diseases and for detecting IL-17 in cells,
tissues, samples, and compositions.
Inventors: |
Hsieh; Chung-Ming; (Newton,
MA) ; Hugunin; Margaret; (North Grafton, MA) ;
Murtaza; Anwar; (Westborough, MA) ; McRae; Bradford
L.; (Northborough, MA) ; Kutskova; Yuliya;
(Northborough, MA) ; Memmott; John E.;
(Framingham, MA) ; Perez; Jennifer M.; (Worcester,
MA) ; Zhong; Suju; (Shrewsbury, MA) ; Tarcsa;
Edit; (Westborough, MA) ; Clabbers; Anca;
(Rutland, MA) ; Wallace; Craig; (Sterling, MA)
; Bryant; Shaughn H.; (Worcester, MA) ; Leddy;
Mary R.; (North Attleboro, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hsieh; Chung-Ming
Hugunin; Margaret
Murtaza; Anwar
McRae; Bradford L.
Kutskova; Yuliya
Memmott; John E.
Perez; Jennifer M.
Zhong; Suju
Tarcsa; Edit
Clabbers; Anca
Wallace; Craig
Bryant; Shaughn H.
Leddy; Mary R. |
Newton
North Grafton
Westborough
Northborough
Northborough
Framingham
Worcester
Shrewsbury
Westborough
Rutland
Sterling
Worcester
North Attleboro |
MA
MA
MA
MA
MA
MA
MA
MA
MA
MA
MA
MA
MA |
US
US
US
US
US
US
US
US
US
US
US
US
US |
|
|
Assignee: |
ABBVIE INC.
North Chicago
IL
|
Family ID: |
42710240 |
Appl. No.: |
14/258994 |
Filed: |
April 22, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12718841 |
Mar 5, 2010 |
8835610 |
|
|
14258994 |
|
|
|
|
61209272 |
Mar 5, 2009 |
|
|
|
Current U.S.
Class: |
435/69.6 ;
435/252.33; 435/254.21; 435/320.1; 435/335; 435/419; 536/23.53 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61P 25/14 20180101; A61P 19/04 20180101; A61P 37/06 20180101; A61K
39/3955 20130101; A61P 3/10 20180101; A61P 25/06 20180101; C07K
16/241 20130101; C07K 2317/21 20130101; A61P 5/14 20180101; C07K
2317/565 20130101; C07K 2317/92 20130101; A61P 27/02 20180101; A61P
21/02 20180101; A61P 1/04 20180101; A61P 7/06 20180101; A61P 37/08
20180101; A61P 31/04 20180101; A61P 31/16 20180101; A61P 33/06
20180101; A61K 2039/507 20130101; A61P 31/12 20180101; C07K 2317/31
20130101; A61P 9/06 20180101; A61P 31/00 20180101; C07K 16/244
20130101; Y02A 50/30 20180101; Y02A 50/386 20180101; A61P 11/00
20180101; C07K 2317/50 20130101; A61P 9/00 20180101; A61P 37/00
20180101; A61P 29/00 20180101; A61P 1/16 20180101; A61P 19/02
20180101; Y02A 50/466 20180101; A61P 7/00 20180101; C07K 2317/64
20130101; A61P 17/00 20180101; A61P 35/00 20180101; A61P 17/14
20180101; A61P 11/02 20180101; A61P 31/10 20180101; C07K 2317/76
20130101; A61P 15/00 20180101; A61P 11/06 20180101; A61P 21/04
20180101; A61P 25/28 20180101; C07K 16/468 20130101; Y02A 50/41
20180101; Y02A 50/412 20180101; A61P 13/00 20180101; A61P 35/02
20180101; A61P 5/18 20180101; A61P 19/00 20180101; A61P 25/00
20180101; A61P 25/16 20180101; A61P 9/10 20180101; A61P 13/12
20180101; A61P 17/02 20180101; A61P 31/14 20180101; A61K 2300/00
20130101; A61P 17/04 20180101; A61P 17/06 20180101 |
Class at
Publication: |
435/69.6 ;
536/23.53; 435/320.1; 435/252.33; 435/335; 435/419; 435/254.21 |
International
Class: |
C07K 16/24 20060101
C07K016/24 |
Claims
1-102. (canceled)
103. An isolated nucleic acid encoding a binding protein amino acid
sequence, wherein the binding protein comprises an antigen binding
domain capable of binding human IL-17, wherein the antigen binding
domain comprises at least one CDR comprising an amino acid sequence
selected from the group consisting of: CDR-H1.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5 (SEQ ID NO:925), wherein;
X.sub.1 is N, A, D, or S; X.sub.2 is Y, F, or L; X.sub.3 is G, D,
or A; X.sub.4 is M or I; and X.sub.5 is H, D, or S; CDR-H2.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16-X.sub.17
(SEQ ID NO:926), wherein; X.sub.1 is V, W, or G; X.sub.2 is I, T,
M, or F; X.sub.3 is S, N, T, or D; X.sub.4 is Y or P; X.sub.5 is D,
N, or I; X.sub.6 is G, S, L, or E; X.sub.7 is S or G; X.sub.8 is N,
T, or E; X.sub.9 is K, T, or A; X.sub.10 is Y, G, N, or V; X.sub.11
is Y or V; X.sub.12 is A; X.sub.13 is D, P, or Q; X.sub.14 is S, K,
or N; X.sub.15 is V or F; X.sub.16 is K, R, or Q; and X.sub.17 is
G; CDR-H3.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16-X.sub.17
(SEQ ID NO:927), wherein; X.sub.1 is V, S, E, or I; X.sub.2 is G,
S, P, or R; X.sub.3 is A, E, N, or P; X.sub.4 is S, D, or W;
X.sub.5 is G, E, F, or L; X.sub.6 is D, G, or W; X.sub.7 is Y, I,
N, or G; X.sub.8 is Y, T, G, or A; X.sub.9 is Y or I; X.sub.10 is
S, G, or Y; X.sub.11 is Y, F, or T; X.sub.12 is G, T, or is not
present; X.sub.13 is L, H, or is not present; X.sub.14 is H or is
not present; X.sub.15 is F or is not present; X.sub.16 is D; and
X.sub.17 is V, N, or Y; CDR-L1.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-X.sub.12-X.sub.13 (SEQ ID NO:928), wherein;
X.sub.1 is S, R, or K; X.sub.2 is G or A; X.sub.3 is S or D;
X.sub.4 is N, K, or Q; X.sub.5 is S or is not present; X.sub.6 is N
or is not present; X.sub.7 is I, L, N, or D; X.sub.8 is G or I;
X.sub.9 is S, N, G, or D; X.sub.10 is H, R, S, or D; X.sub.11 is S,
Y, A, or D; X.sub.12 is V, A, L, or M; and X.sub.13 is N, C, or H;
CDR-L2. X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7
(SEQ ID NO:929), wherein; X.sub.1 is G, Q, Y, or E; X.sub.2 is I,
D, or A; X.sub.3 is G, N, S, or T; X.sub.4 is Q, K, or T; X.sub.5
is R, S, or L; X.sub.6 is P, I, or V; and X.sub.7 is S or P; and
CDR-L3.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11 (SEQ ID NO:930), wherein; X.sub.1 is A, Q, H, or
L; X.sub.2 is T or Q; X.sub.3 is W, S, or H; X.sub.4 is D or T;
X.sub.5 is D or S; X.sub.6 is S, T, L, or F; X.sub.7 is L, T, or P;
X.sub.8 is G, H, or Y; X.sub.9 is G, S, or T; X.sub.10 is Y or is
not present; and X.sub.11 is V or is not present; a CDR-H1, a
CDR-H2, and a CDR-H3 of any variable heavy region (VH) in Table 21,
Table 23, Table 24, or Table 27; and a CDR-L1, a CDR-L2, and a
CDR-L3 of any variable light region (VL) in Table 21, Table 23,
Table 25, or Table 27.
104. A vector comprising an isolated nucleic acid of claim 103.
105. (canceled)
106. A host cell comprising the vector of claim 104.
107-115. (canceled)
116. A method of producing a binding protein that binds human
IL-17, comprising culturing the host cell of claim 106 in a culture
medium under conditions sufficient to produce a binding protein
that binds human IL-17.
117-201. (canceled)
202. An isolated nucleic acid encoding a binding protein amino acid
sequence, wherein the binding protein is capable of binding human
IL-17, and wherein the binding protein comprises: an Ig constant
heavy region having an amino acid sequence selected from the group
consisting of SEQ ID NO:3 and SEQ ID NO: 4; an Ig constant light
region having an amino acid sequence selected from the group
consisting of SEQ ID NO:5 and SEQ ID NO: 6; an Ig variable heavy
region having an amino acid sequence of a VH in Table 21, Table 23,
Table 24, or Table 27; and an Ig variable light region having an
amino acid sequence of a VL in Table 21, Table 23, Table 25, or
Table 27.
203. A vector comprising an isolated nucleic acid of claim 202.
204. A host cell comprising a vector of claim 203.
205. A method of producing a binding protein that binds human
IL-17, comprising culturing the host cell of claim 204 in a culture
medium under conditions sufficient to produce a binding protein
that binds human IL-17.
206. An isolated nucleic acid encoding a binding protein amino acid
sequence, wherein the binding protein is capable of binding human
IL-17, the binding protein comprising: an Ig constant heavy region
having an amino acid sequence of SEQ ID NO:3; an Ig constant light
region having an amino acid sequence of SEQ ID NO:5; an Ig variable
heavy region having an amino acid sequence of a VH in Table 21,
Table 23, Table 24, or Table 27; and an Ig variable light region
having an amino acid sequence of a VL in Table 21, Table 23, Table
25, or Table 27.
207. A vector comprising an isolated nucleic acid of claim 206.
208. A host cell comprising a vector of claim 207.
209. A method of producing a binding protein that binds human
IL-17, comprising culturing the host cell of claim 208 in a culture
medium under conditions sufficient to produce a binding protein
that binds human IL-17.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a non-provisional application claiming
the benefit of U.S. Provisional Application No. 61/209,272, filed
Mar. 5, 2009.
FIELD OF THE INVENTION
[0002] The present invention relates to IL-17 binding proteins, and
specifically to their uses in the prevention and/or treatment of
acute and chronic immunological diseases such as rheumatoid
arthritis, osteoarthritis, psoriasis, multiple sclerosis, and other
autoimmune diseases.
BACKGROUND OF THE INVENTION
[0003] Interleukin-17A (IL-17A, synonymous with IL-17) is a
cytokine produced from the TH17 lineage of T cells. IL-17 was
originally designated "CTL-associated antigen 8" (CTLA-8) when
cloned from a rodent T cell hybridoma and identified as a protein
having amino acid sequence homology with the thirteenth open frame
(ORF-13) of herpesviurs saimiri, a .gamma. herpes virus that causes
T cell lymphoma in monkeys and rabbits (Rouvier et al., J.
Immunol., 150 5445-5556 (1993); Yao et al., Immunity, 3: 811-821
(1995)). The human equivalent of CTLA-8 was later cloned and
designated "IL-17" (Yao et al., J. Immunol., 155(12): 5483-5486
(1995); Fossiez et al., J. Exp. Med., 183(6): 2593-2603 (1996)).
The human gene for IL-17 encodes a 155 amino acid polypeptide
comprising a 19 amino acid signal sequence and a 132 amino acid
mature domain.
[0004] Human IL-17A is a glycoprotein with a M.sub.r of 17,000
daltons (Spriggs et al., J. Clin. Immunol., 17: 366-369 (1997)).
IL-17A may exist as either a homodimer or as a heterodimer
complexed with the homolog IL-17F to form heterodimeric IL-17A/F.
IL-17F (IL-24, ML-1) shares a 55% amino acid identity with IL-17A.
IL-17A and IL-17F also share the same receptor (IL-17R), which is
expressed on a wide variety of cells including vascular endothelial
cells, peripheral T cells, B cells, fibroblast, lung cells,
myelomonocytic cells, and marrow stromal cells (Kolls et al.,
Immunity, 21: 467-476 (2004); Kawaguchi et al., J. Allergy Clin.
Immunol., 114(6): 1267-1273 (2004); Moseley et al., Cytokine Growth
Factor Rev., 14(2): 155-174 (2003)). Additional IL-17 homologs have
been identified (IL-17B, IL-17C, IL-17D, IL-E). These other family
members share less than 30% amino acid identity with IL-17A (Kolls
et al., 2004).
[0005] IL-17A is involved in the induction of proinflammatory
responses and induces or mediates expression of a variety of other
cytokines, factors, and mediators including tissue necrosis
factor-alpha (TNF-.alpha.), IL-6, IL-8, IL-1.beta., granulocyte
colony-stimulating factor (G-CSF), prostaglandin E.sub.2
(PGE.sub.2), IL-10, IL-12, IL-1R antagonist, leukemia inhibitory
factor, and stromelysin (Yao et al., J. Immunol., 155(12):
5483-5486 (1995); Fossiez et al., J. Exp. Med., 183(6): 2593-2603
(1996); Jovanovic et al., J. Immunol., 160: 3513-3521 (1998);
Teunissen et al., J. Investig. Dermatol., 111: 645-649 (1998);
Chabaud et al., J. Immunol., 161: 409-414 (1998)). IL-17 also
induces nitric oxide in chondrocytes and in human osteoarthritis
explants (Shalom-Barak et al., J. Biol. Chem., 273: 27467-27473
(1998); Attur et al., Arthritis Rheum., 40: 1050-1053 (1997)).
[0006] Through its role in T cell mediated autoimmunity, IL-17
induces the release of cytokines, chemokines, and growth factors
(as noted above), is an important local orchestrator of neutrophil
accumulation, and plays a role in cartilage and bone destruction.
There is growing evidence that targeting IL-17 signaling might
prove useful in a variety of autoimmune diseases including
rheumatoid arthritis (RA), psoriasis, Crohn's disease, multiple
sclerosis (MS), psoriatric disease, asthma, and lupus (SLE) (see,
e.g., Aggarwal et al., J. Leukoc. Biol., 71(1): 1-8 (2002);
Lubberts et al., "Treatment with a neutralizing anti-murine
interleukin-17 antibody after the onset of collagen-induced
arthritis reduces joint inflammation, cartilage destruction, and
bone erosion," Arthritis Rheum., 50: 650-659 (2004)).
[0007] The pathogenic role of TNF in arthritis is well established
as TNF-.alpha. antagonists reduce inflammation and limit
progression of cartilage damage and bone erosion in human disease
(van den Berg. "Anti-cytokine therapy in chronic destructive
arthritis," Arthritis Res., 3: 18-26 (2001)). Although TNF
antagonists have revolutionized RA therapy, a significant portion
of patients do not respond adequately to these drugs. Preclinical
studies with TNF-.alpha. and IL-17 point to both independent and
overlapping roles in arthritis pathophysiology. Whereas IL-17 or
TNF-.alpha. alone exert only modest effects on proinflammatory gene
expression, the combination of IL-17 with TNF-.alpha. leads to
strong synergistic responses. This synergy results in upregulation
of cytokines (LeGrand et al., Arthritis Rheum., 44: 2078-2083
(2001)) and proinflammatory chemokines (Chabaud et al., J.
Immunol., 167: 6015-6020 (2001)) and also in the induction of
cartilage and bone destruction (Van Bezooijen et al., Ann. Rheum.
Dis., 61: 870-876 (2002)). Interaction between TNF-.alpha. and
IL-17 has been demonstrated as a predicting factor for joint damage
progression in humans in a two-year prospective study of RA
patients (Kirkham et al., Arthritis Rheum., 54: 1122-1131 (2006)).
In addition, IL-17 mRNA levels correlate poorly with TNF-.alpha.
expression in RA, indicating that IL-17 blockade might complement
TNF-.alpha. antagonists for optimal treatment of RA (Kohno et al.,
Mod. Rheumatol., 18: 15-22 (2008)).
[0008] Although a variety of antibodies to IL-17 have been
described in the nearly two decades of work since the discovery of
this critical proinflammatory cytokine, there remains a need for
improved antibodies that can effectively mediate or neutralize the
activity of IL-17 in the inflammatory response and autoimmune
disorders.
SUMMARY OF THE INVENTION
[0009] This invention pertains to proteins that bind human IL-17
(same as "IL-17A"). Binding proteins of the invention include but
are not limited to antibodies, antigen binding portions thereof,
and multivalent, multispecific binding proteins such as DVD-Ig.TM.
binding proteins that can bind human IL-17 and another target, such
as TNF-.alpha.. The invention also provides methods of making and
using the IL-17 binding proteins described herein as well as
various compositions that may be used in methods of detecting IL-17
in a sample or in methods of treating or preventing a disorder in
an individual that is associated with or suspected to be associated
with IL-17 activity. Binding proteins described herein may bind
human IL-17A homodimer and/or heterodimers of IL-17A and the IL-17F
homolog.
[0010] In one aspect of the invention, there is provided a binding
protein comprising an antigen binding domain capable of binding
human IL-17, said antigen binding domain comprising at least one
CDR comprising an amino acid sequence selected from the group
consisting of: [0011] CDR-H1.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5 (SEQ ID NO:919), wherein;
[0012] X.sub.1 is D or S; [0013] X.sub.2 is Y; [0014] X.sub.3 is E
or G; [0015] X.sub.4 is I, M, V, or F; [0016] X.sub.5 is H; [0017]
CDR-H2.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16-X.sub.17
(SEQ ID NO:920), wherein; [0018] X.sub.1 is V; [0019] X.sub.2 is T,
I, or N; [0020] X.sub.3 is D, H, or W; [0021] X.sub.4 is P or is
not present; [0022] X.sub.5 is E, G, or S; [0023] X.sub.6 is S, N,
or D; [0024] X.sub.7 is G; [0025] X.sub.8 is G or T; [0026] X.sub.9
is T; [0027] X.sub.10 is L, A, T, or F; [0028] X.sub.11 is H or Y;
[0029] X.sub.12 is N; [0030] X.sub.13 is P, Q, or S; [0031]
X.sub.14 is K, A, or N; [0032] X.sub.15 is F or L; [0033] X.sub.16
is D, K, or R; and [0034] X.sub.17 is G, D, or S; [0035] CDR-H3.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-X.sub.12 (SEQ ID NO:921), wherein; [0036] X.sub.1
is Y, F, or D; [0037] X.sub.2 is Y, L, S, or G; [0038] X.sub.3 is
K, T, R, or Y; [0039] X.sub.4 is Y or W; [0040] X.sub.5 is E, D, or
I; [0041] X.sub.6 is S, G, or Y; [0042] X.sub.7 is F, Y, or T;
[0043] X.sub.8 is Y, F, or M; [0044] X.sub.9 is G, T, or is not
present; [0045] X.sub.10 is M or is not present; [0046] X.sub.11 is
D; and [0047] X.sub.12 is Y; [0048] CDR-L1.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X-
.sub.9-X.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16
(SEQ ID NO:922), wherein; [0049] X.sub.1 is S, K, or R; [0050]
X.sub.2 is A or S; [0051] X.sub.3 is S; [0052] X.sub.4 is S or Q;
[0053] X.sub.5 is S or is not present; [0054] X.sub.6 is L or is
not present; [0055] X.sub.7 is V or is not present; [0056] X.sub.8
is H or is not present; [0057] X.sub.9 is S or is not present;
[0058] X.sub.10 is S, N, or is not present; [0059] X.sub.11 is S,
V, or G; [0060] X.sub.12 is I, N, or S; [0061] X.sub.13 is S, N, T,
or I; [0062] X.sub.14 is Y or D; [0063] X.sub.15 is M, V, L, or I;
and [0064] X.sub.16 is C, A, H, Y, or G; [0065] CDR-L2.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7 (SEQ ID
NO:923), wherein; [0066] X.sub.1 is D, Y, K, A, or H; [0067]
X.sub.2 is T, A, or V; [0068] X.sub.3 is S or F; [0069] X.sub.4 is
K, N, or E; [0070] X.sub.5 is L or R; [0071] X.sub.6 is A, Y, or F;
and [0072] X.sub.7 is S or T;
[0073] and [0074] CDR-L3.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9
(SEQ ID NO:924), wherein; [0075] X.sub.1 is Q, S, or H; [0076]
X.sub.2 is Q; [0077] X.sub.3 is R, D, S, or G; [0078] X.sub.4 is S,
Y, or T; [0079] X.sub.5 is S, G, or H; [0080] X.sub.6 is Y, S, V,
or A; [0081] X.sub.7 is P or is not present; [0082] X.sub.8 is W,
Y, or L; and [0083] X.sub.9 is T.
[0084] In an embodiment, a binding protein according to the
invention comprises at least one CDR comprising an amino acid
sequence selected from the group consisting of: [0085] residues
31-35 of SEQ ID NO:26; residues 50-66 of SEQ ID NO:26; residues
99-110 of SEQ ID NO:26; [0086] residues 24-33 of SEQ ID NO:27;
residues 49-55 of SEQ ID NO:27; residues 88-96 of SEQ ID NO:27;
[0087] residues 31-35 of SEQ ID NO:28; residues 50-66 of SEQ ID
NO:28; residues 99-108 of SEQ ID NO:28; [0088] residues 24-34 of
SEQ ID NO:29; residues 50-56 of SEQ ID NO:29; residues 89-97 of SEQ
ID NO:29; [0089] residues 31-35 of SEQ ID NO:30; residues 50-66 of
SEQ ID NO:30; residues 99-108 of SEQ ID NO:30; [0090] residues
24-34 of SEQ ID NO:31; residues 50-56 of SEQ ID NO:31; residues
89-97 of SEQ ID NO:31; [0091] residues 31-35 of SEQ ID NO:32;
residues 50-65 of SEQ ID NO:32; residues 98-109 of SEQ ID NO:32;
[0092] residues 24-39 of SEQ ID NO:33; residues 55-61 of SEQ ID
NO:33; residues 94-101 of SEQ ID NO:33; [0093] residues 31-35 of
SEQ ID NO:34; Residues 50-66 of SEQ ID NO:34; residues 99-110 of
SEQ ID NO:34; [0094] residues 24-33 of SEQ ID NO:35; residues 49-55
of SEQ ID NO:35; residues 88-96 of SEQ ID NO:35; [0095] residues
31-35 of SEQ ID NO:36; residues 50-66 of SEQ ID NO:36; residues
99-108 of SEQ ID NO:36; [0096] residues 24-34 of SEQ ID NO:37;
residues 50-56 of SEQ ID NO:37; residues 89-97 of SEQ ID NO:37;
[0097] residues 31-35 of SEQ ID NO:38; residues 50-65 of SEQ ID
NO:38; residues 98-107 of SEQ ID NO:38; [0098] residues 24-39 of
SEQ ID NO:39; residues 55-61 of SEQ ID NO:39; and residues 94-102
of SEQ ID NO:39.
[0099] In another embodiment, an IL-17 binding protein of the
invention comprises at least 3 CDRs described above.
[0100] In another embodiment, an IL-17 binding protein comprises at
least 3 CDRs selected from a variable domain CDR set consisting
of:
TABLE-US-00001 VH 7D7 CDR Set VH 7D7 CDR-H1 Residues 31-35 of SEQ
ID NO: 26 VH 7D7 CDR-H2 Residues 50-66 of SEQ ID NO: 26 VH 7D7
CDR-H3 Residues 99-110 of SEQ ID NO: 26 VL 7D7 CDR Set VL 7D7
CDR-L1 Residues 24-33 of SEQ ID NO: 27 VL 7D7 CDR-L2 Residues 49-55
of SEQ ID NO: 27 VL 7D7 CDR-L3 Residues 88-96 of SEQ ID NO: 27 VH
6C6 CDR Set VH 6C6 CDR-H1 Residues 31-35 of SEQ ID NO: 28 VH 6C6
CDR-H2 Residues 50-66 of SEQ ID NO: 28 VH 6C6 CDR-H3 Residues
99-108 of SEQ ID NO: 28 VL 6C6 CDR Set VL 6C6 CDR-L1 Residues 24-34
of SEQ ID NO: 29 VL 6C6 CDR-L2 Residues 50-56 of SEQ ID NO: 29 VL
6C6 CDR-L3 Residues 89-97 of SEQ ID NO.: 29 VH 1D8 CDR Set VH 1D8
CDR-H1 Residues 31-35 of SEQ ID NO: 30 VH 1D8 CDR-H2 Residues 50-66
of SEQ ID NO: 30 VH 1D8 CDR-H3 Residues 99-108 of SEQ ID NO: 30 VL
1D8 CDR Set VL 1D8 CDR-L1 Residues 24-34 of SEQ ID NO: 31 VL 1D8
CDR-L2 Residues 50-56 of SEQ ID NO: 31 VL 1D8 CDR-L3 Residues 89-97
of SEQ ID NO: 31 VH 8B12 CDR Set VH 8B12 CDR-H1 Residues 31-35 of
SEQ ID NO: 32 VH 8B12 CDR-H2 Residues 50-65 of SEQ ID NO: 32 VH
8B12 CDR-H3 Residues 98-109 of SEQ ID NO: 32 VL 8B12 CDR Set VL
8B12 CDR-L1 Residues 24-39 of SEQ ID NO: 33 VL 8B12 CDR-L2 Residues
55-61 of SEQ ID NO: 33 VL 8B12 CDR-L3 Residues 94-101 of SEQ ID NO:
33 VH 10F7 CDR Set VH 10F7 CDR-H1 Residues 31-35 of SEQ ID NO: 34
VH 10F7 CDR-H2 Residues 50-66 of SEQ ID NO: 34 VH 10F7 CDR-H3
Residues 99-110 of SEQ ID NO: 34 VL 10F7 CDR Set VL 10F7 CDR-L1
Residues 24-33 of SEQ ID NO: 35 VL 10F7 CDR-L2 Residues 49-55 of
SEQ ID NO: 35 VL 10F7 CDR-L3 Residues 88-96 of SEQ ID NO: 35 VH 5C5
CDR Set VH 5C5 CDR-H1 Residues 31-35 of SEQ ID NO: 36 VH 5C5 CDR-H2
Residues 50-66 of SEQ ID NO: 36 VH 5C5 CDR-H3 Residues 99-108 of
SEQ ID NO: 36 VL 5C5 CDR Set VL 5C5 CDR-L1 Residues 24-34 of SEQ ID
NO: 37 VL 5C5 CDR-L2 Residues 50-56 of SEQ ID NO: 37 VL 5C5 CDR-L3
Residues 89-97 of SEQ ID NO: 37 VH 10G9 CDR Set VH 10G9 CDR-H1
Residues 31-35 of SEQ ID NO: 38 VH 10G9 CDR-H2 Residues 50-65 of
SEQ ID NO: 38 VH 10G9 CDR-H3 Residues 98-107 of SEQ ID NO: 38 VL
10G9 CDR Set VL 10G9 CDR-L1 Residues 24-39 of SEQ ID NO: 39 VL 10G9
CDR-L2 Residues 55-61 of SEQ ID NO: 39 VL 10G9 CDR-L3 Residues
94-102 of SEQ ID NO: 39
[0101] In another embodiment, an IL-17 binding protein may comprise
at least two variable domain CDR sets described above. Preferably,
the two variable domain CDR sets are selected from the group
consisting of:
[0102] VH 7D7 CDR Set and VL 7D7 CDR Set;
[0103] VH 6C6 CDR Set and VL 6C6 CDR Set;
[0104] VH 1D8 CDR Set and VL 1D8 CDR Set;
[0105] VH 8B12 CDR Set and VL 8B12 CDR Set;
[0106] VH 10F7 CDR Set and VL 10F7 CDR Set; and
[0107] VH 5C5 CDR Set and VL 5C5 CDR Set; and
[0108] VH 10G9 CDR Set and VL 10G9 Set.
In other embodiment, a binding protein comprise one or more CDRs
described above further comprises a human acceptor framework.
Preferably, the human framework comprises an amino acid sequence
selected group consisting:
[0109] SEQ ID NO.:7
[0110] SEQ ID NO.:8
[0111] SEQ ID NO.:9
[0112] SEQ ID NO.:10
[0113] SEQ ID NO.:11
[0114] SEQ ID NO.:12
[0115] SEQ ID NO.:13
[0116] SEQ ID NO.:14
[0117] SEQ ID NO.:15
[0118] SEQ ID NO.:16
[0119] SEQ ID NO.:17
[0120] SEQ ID NO.:18
[0121] SEQ ID NO.:19
[0122] SEQ ID NO.:20
[0123] SEQ ID NO.:21
[0124] SEQ ID NO.:22
[0125] SEQ ID NO.:23
[0126] SEQ ID NO.:24 AND
[0127] SEQ ID NO.:25.
[0128] An IL-17 binding protein may comprise a human acceptor
framework comprising at least one Framework Region amino acid
substitution, wherein the amino acid sequence of the framework is
at least 65% identical to the sequence of said human acceptor
framework and comprises at least 70 amino acid residues identical
to said human acceptor framework.
[0129] In another embodiment, an IL-17 binding protein comprises a
human acceptor framework, wherein said acceptor framework comprises
at least one framework region amino acid substitution at a key
residue, said key residue selected from the group consisting of:
[0130] a residue adjacent to a CDR; [0131] a glycosylation site
residue; [0132] a rare residue;
[0133] a residue capable of interacting with human IL-13; [0134] a
residue capable of interacting with a CDR; [0135] a canonical
residue; [0136] a contact residue between heavy chain variable
region and light chain variable region; [0137] a residue within a
Vernier zone; and [0138] a residue in a region that overlaps
between a Chothia-defined variable heavy chain CDR1 and a
Kabat-defined first heavy chain framework.
[0139] In a preferred embodiment, an IL-17A binding protein may
comprise a key residue, wherein said key residue is selected from
the group consisting of: 2H, 4H, 24H, 26H, 27H, 29H, 34H, 35H, 37H,
39H, 44H, 45H, 47H, 48H, 49H, 50H, 51H, 58H, 59H, 60H, 63H, 67H,
69H, 71H, 73H, 76H, 78H, 91H, 93H, 94H, 2L, 4L, 25L, 29L, 27bL,
33L, 34L, 36L, 38L, 43L, 44L, 46L, 47L, 48L, 49L, 55L, 58L, 62L,
64L, 71L, 87L, 89L, 90L, 91L, 94L, 95L (all Kabat numbering). A
preferred subset of these residues for the humanization of IL-17
antibodies consists of 27H, 48H, 67H, 69H, 93H, 36L, 43L, 46L, 47L,
49L, 58L, 71L, and 87L.
[0140] In yet another embodiment, an IL-17 binding protein
according to the invention comprises a consensus human variable
domain that is a consensus human variable domain described
herein.
[0141] In a preferred embodiment, an IL-17 binding protein
comprises at least one variable domain having an amino acid
sequence selected from the group consisting of:
[0142] SEQ ID NO:60
[0143] SEQ ID NO:61
[0144] SEQ ID NO:62
[0145] SEQ ID NO:63
[0146] SEQ ID NO:64
[0147] SEQ ID NO:65
[0148] SEQ ID NO:66
[0149] SEQ ID NO:67
[0150] SEQ ID NO:68
[0151] SEQ ID NO:69
[0152] SEQ ID NO:70
[0153] SEQ ID NO:71
[0154] SEQ ID NO:72
[0155] SEQ ID NO:73
[0156] SEQ ID NO:74
[0157] SEQ ID NO:75
[0158] SEQ ID NO:76
[0159] SEQ ID NO:931
[0160] SEQ ID NO:932 AND
[0161] SEQ ID NO:933.
[0162] More preferably, an IL-17 binding protein described herein
comprises two variable domains, wherein said two variable domains
have amino acid sequences selected from the group consisting
of:
[0163] SEQ ID NO:60 & SEQ ID NO:62,
[0164] SEQ ID NO:60 & SEQ ID NO:63,
[0165] SEQ ID NO:60 & SEQ ID NO:64,
[0166] SEQ ID NO:60 & SEQ ID NO:65,
[0167] SEQ ID NO:60 & SEQ ID NO:66
[0168] SEQ ID NO:60 & SEQ ID NO:67,
[0169] SEQ ID NO:60 & SEQ ID NO:68,
[0170] SEQ ID NO:61 & SEQ ID NO:62,
[0171] SEQ ID NO:61 & SEQ ID NO:63,
[0172] SEQ ID NO:61 & SEQ ID NO:64,
[0173] SEQ ID NO:61 & SEQ ID NO:65,
[0174] SEQ ID NO:61 & SEQ ID NO:66,
[0175] SEQ ID NO:61 & SEQ ID NO:67, AND
[0176] SEQ ID NO:61 & SEQ ID NO:68.
[0177] In another embodiment, a binding protein described herein
comprises at least one variable domain having an amino acid
sequence selected from the group consisting of:
[0178] SEQ ID NO:60
[0179] SEQ ID NO:61
[0180] SEQ ID NO:62
[0181] SEQ ID NO:63
[0182] SEQ ID NO:64
[0183] SEQ ID NO:65
[0184] SEQ ID NO:66
[0185] SEQ ID NO:67
[0186] SEQ ID NO:68
[0187] SEQ ID NO:69
[0188] SEQ ID NO:70
[0189] SEQ ID NO:71
[0190] SEQ ID NO:72
[0191] SEQ ID NO:73
[0192] SEQ ID NO:74
[0193] SEQ ID NO:75
[0194] SEQ ID NO:76
[0195] SEQ ID NO:931
[0196] SEQ ID NO:932 AND
[0197] SEQ ID NO:933.
[0198] In another embodiment, a binding protein of the invention
comprises an antigen binding domain capable of binding human IL-17,
said antigen binding domain comprising at least one CDR comprising
an amino acid sequence selected from the group consisting of:
[0199] CDR-H1. X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5 (SEQ ID
NO:925), wherein; [0200] X.sub.1 is N, A, D, or S; [0201] X.sub.2
is Y, F, or L; [0202] X.sub.3 is G, D, or A; [0203] X.sub.4 is M or
I; and [0204] X.sub.5 is H, D, or S; [0205] CDR-H2.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16-X.sub.17
(SEQ ID NO:926), wherein; [0206] X.sub.1 is V, W, or G; [0207]
X.sub.2 is I, T, M, or F; [0208] X.sub.3 is S, N, T, or D; [0209]
X.sub.4 is Y or P; [0210] X.sub.5 is D, N, or I; [0211] X.sub.6 is
G, S, L, or E; [0212] X.sub.7 is S or G; [0213] X.sub.8 is N, T, or
E; [0214] X.sub.9 is K, T, or A; [0215] X.sub.10 is Y, G, N, or V;
[0216] X.sub.11 is Y or V; [0217] X.sub.12 is A; [0218] X.sub.13 is
D, P, or Q; [0219] X.sub.14 is S, K, or N; [0220] X.sub.15 is V or
F; [0221] X.sub.16 is K, R, or Q; and [0222] X.sub.17 is G; [0223]
CDR-H3.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16-X.sub.17
(SEQ ID NO:927), wherein; [0224] X.sub.1 is V, S, E, or I; [0225]
X.sub.2 is G, S, P, or R; [0226] X.sub.3 is A, E, N, or P; [0227]
X.sub.4 is S, D, or W; [0228] X.sub.5 is G, E, F, or L; [0229]
X.sub.6 is D, G, or W; [0230] X.sub.7 is Y, I, N, or G; [0231]
X.sub.8 is Y, T, G, or A; [0232] X.sub.9 is Y or I; [0233] X.sub.10
is S, G, or Y; [0234] X.sub.11 is Y, F, or T; [0235] X.sub.12 is G,
T, or is not present; [0236] X.sub.13 is L, H, or is not present;
[0237] X.sub.14 is H or is not present; [0238] X.sub.15 is F or is
not present; [0239] X.sub.16 is D; and [0240] X.sub.17 is V, N, or
Y; [0241] CDR-L1.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-X.sub.12-X.sub.13 (SEQ ID NO:928), wherein; [0242]
X.sub.1 is S, R, or K; [0243] X.sub.2 is G or A; [0244] X.sub.3 is
S or D; [0245] X.sub.4 is N, K, or Q; [0246] X.sub.5 is S or is not
present; [0247] X.sub.6 is N or is not present; [0248] X.sub.7 is
I, L, N, or D; [0249] X.sub.8 is G or I; [0250] X.sub.9 is S, N, G,
or D; [0251] X.sub.10 is H, R, S, or D; [0252] X.sub.11 is S, Y, A,
or D; [0253] X.sub.12 is V, A, L, or M; and [0254] X.sub.13 is N,
C, or H; [0255] CDR-L2.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7 (SEQ ID
NO:929), wherein; [0256] X.sub.1 is G, Q, Y, or E; [0257] X.sub.2
is I, D, or A; [0258] X.sub.3 is G, N, S, or T; [0259] X.sub.4 is
Q, K, or T; [0260] X.sub.5 is R, S, or L; [0261] X.sub.6 is P, I,
or V; and [0262] X.sub.7 is S or P;
[0263] and [0264] CDR-L3.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11 (SEQ ID NO:930), wherein; [0265] X.sub.1 is A, Q,
H, or L; [0266] X.sub.2 is T or Q; [0267] X.sub.3 is W, S, or H;
[0268] X.sub.4 is D or T; [0269] X.sub.5 is D or S; [0270] X.sub.6
is S, T, L, or F; [0271] X.sub.7 is L, T, or P; [0272] X.sub.8 is
G, H, or Y; [0273] X.sub.9 is G, S, or T; [0274] X.sub.10 is Y or
is not present; and [0275] X.sub.11 is V or is not present; [0276]
a CDR-H1 amino acid sequence, a CDR-H2 amino acid sequence, and a
CDR-H3 amino acid sequence of any variable heavy region (VH) in
Table 21, Table 23, Table 24, or Table 27; and [0277] a CDR-L1
amino acid sequence, a CDR-L2 amino acid sequence, and a CDR-L3
amino acid sequence of any variable light region (VL) in Table 21,
Table 23, Table 25, or Table 27.
[0278] In a preferred embodiment, at least one CDR of the
above-described IL-17 binding protein comprises an amino acid
sequence selected from the group consisting of: [0279] residues
31-35 of SEQ ID NO:40; residues 50-66 of SEQ ID NO.:40; residues
99-103 of SEQ ID NO.:40; [0280] residues 23-35 of SEQ ID NO:41;
residues 51-57 of SEQ ID NO.:41; residues 90-110 of SEQ ID NO.:41;
[0281] residues 31-35 of SEQ ID NO:42; residues 50-66 of SEQ ID
NO.:42; residues 99-103 of SEQ ID NO.:42; [0282] residues 23-35 of
SEQ ID NO:43; residues 51-57 of SEQ ID NO.:43; residues 90-110 of
SEQ ID NO.:43; [0283] residues 31-35 of SEQ ID NO:44; residues
50-66 of SEQ ID NO.:44; residues 99-101 of SEQ ID NO.:44; [0284]
residues 23-35 of SEQ ID NO:45; residues 51-57 of SEQ ID NO.:45;
residues 90-110 of SEQ ID NO.:45; [0285] residues 31-35 of SEQ ID
NO:46; residues 50-66 of SEQ ID NO.:46; residues 99-115 of SEQ ID
NO.:46; [0286] residues 24-34 of SEQ ID NO:47; residues 50-56 of
SEQ ID NO.:47; residues 89-97 of SEQ ID NO.:47; [0287] residues
31-35 of SEQ ID NO:48; residues 50-66 of SEQ ID NO.:48; residues
99-101 of SEQ ID NO.:48; [0288] residues 24-34 of SEQ ID NO:49;
residues 50-56 of SEQ ID NO.:49; and residues 89-97 of SEQ ID
NO.:49; [0289] an amino acid sequence for a CDR-H1, an amino acid
sequence for a CDR-H2, and an amino acid sequence for a CDR-H3 of a
VH in Table 21, Table 23, Table 24, or Table 27; and [0290] an
amino acid sequence for a CDR-L1, an amino acid sequence for a
CDR-L2, and an amino acid sequence for a CDR-L3 of a VL in Table
21, Table 23, Table 25, or Table 27.
[0291] In another embodiment, the IL-17 binding protein of the
invention comprises at least 3 CDRs described above.
[0292] In another embodiment, an IL-17 binding protein of the
invention comprises an antigen binding domain that comprises a
V.sub.H. More preferably, the V.sub.H comprises an amino acid
sequence selected from the group consisting of: SEQ ID NO:40, SEQ
ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, an amino acid
sequence of a VH in Table 21, an amino acid sequence of a VH in
Table 23, an amino acid sequence of a VH in Table 24, and an amino
acid sequence of a VH in Table 27.
[0293] In another embodiment, the IL-17 binding protein of the
invention comprises an antigen binding domain that comprises a
V.sub.L. Preferably, the V.sub.L comprises an amino acid sequence
selected from the group consisting of SEQ ID NO:41, SEQ ID NO:43,
SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, an amino acid sequence of
a VL in Table 21, an amino acid sequence of a VL in Table 23, an
amino acid sequence of a VL in Table 25, and an amino acid sequence
of a VL in Table 27.
[0294] In a further embodiment, the IL-17 binding protein of the
invention comprises an antigen binding domain comprising a V.sub.H
and a V.sub.L. In a preferred embodiment, the V.sub.H comprises an
amino acid sequence selected from the group consisting of: SEQ ID
NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, an
amino acid sequence of a VH in Table 21, an amino acid sequence of
a VH in Table 23, an amino acid sequence of a VH in Table 24, and
an amino acid sequence of a VH in Table 27.
[0295] More preferably, the V.sub.L of an IL-17 binding protein of
the invention comprises an amino acid sequence of a VL in Table 21,
an amino acid sequence of a VL in Table 23, an amino acid sequence
of a VL in Table 25, or an amino acid sequence of a VL in Table 27
and the V.sub.H comprises an amino acid sequence of a VH in Table
21, an amino acid sequence of a VH in Table 23, an amino acid
sequence of a VH in Table 24, or an amino acid sequence of a VH in
Table 27.
[0296] In another embodiment, an IL-17 binding protein described
herein, further comprises a heavy chain immunoglobulin constant
domain selected from the group consisting of: a human IgM constant
domain; a human IgG1 constant domain; a human IgG2 constant domain;
a human IgG3 constant domain; a human IgG4 constant domain; a human
IgE constant domain and a human IgA constant domain. Preferably,
the heavy chain immunoglobulin constant region is a human IgG1
constant domain. More preferably, the human IgG1 constant domain
comprises amino acid sequence selected from the group consisting of
SEQ ID NO:3 and SEQ ID NO:4.
[0297] In another embodiment, an IL-17 binding protein described
herein comprises a light chain immunoglobulin constant domain is a
human Ig kappa constant domain or a human Ig lambda constant
domain. A preferred human Ig kappa constant domain comprises amino
acid sequence SEQ ID NO:5. A preferred human Ig lambda constant
domain comprises amino acid sequence SEQ ID NO:6.
[0298] In another embodiment, an IL-17 binding protein described
herein is selected from the group consisting of: an immunoglobulin
molecule; an scFv; a monoclonal antibody; a human antibody; a
chimeric antibody; a humanized antibody; a single domain antibody;
a Fab fragment; an Fab' fragment; an F(ab')2; an Fv; and a
disulfide linked Fv. In a preferred embodiment, the IL-17 binding
protein is a human antibody.
[0299] Another aspect of the invention is a binding protein capable
of binding human IL-17, wherein the binding protein comprises:
[0300] an Ig constant heavy region having an amino acid sequence
selected from the group consisting of SEQ ID NO:3 and SEQ ID NO: 4;
[0301] an Ig constant light region having an amino acid sequence
selected from the group consisting of SEQ ID NO:5 and SEQ ID NO: 6;
[0302] an Ig variable heavy region having an amino acid sequence of
a VH in Table 21, an amino acid sequence of a VH in Table 23, an
amino acid sequence of a VH in Table 24, or an amino acid sequence
of a VH in Table 27; and [0303] an Ig variable light region having
an amino acid sequence of a VL in Table 21, an amino acid sequence
of a VL in Table 23, an amino acid sequence of a VL in Table 25, or
an amino acid sequence of a VL in Table 27.
[0304] More preferably, a binding protein according to the
invention is capable of binding human IL-17 and comprises: [0305]
an Ig constant heavy region having an amino acid sequence of SEQ ID
NO:3; [0306] an Ig constant light region having an amino acid
sequence of SEQ ID NO:5; [0307] an Ig variable heavy region having
an amino acid sequence of a VH in Table 21, an amino acid sequence
of a VH in Table 23, an amino acid sequence of a VH in Table 24, or
an amino acid sequence of a VH in Table 27; and [0308] an Ig
variable light region having an amino acid sequence of a VL in
Table 21, an amino acid sequence of a VL in Table 23, an amino acid
sequence of a VL in Table 25, or an amino acid sequence of a VL in
Table 27.
[0309] In another aspect, the invention provides a multivalent,
multispecific DVD-Ig.TM. binding protein comprising a polypeptide
chain, wherein said polypeptide chain comprises
VD1-(X1)n-VD2-C-(X2)n, wherein; [0310] VD1 is a first heavy chain
variable domain; [0311] VD2 is a second heavy chain variable
domain; [0312] C is a heavy chain constant domain; [0313] X1 is a
linker with the proviso that it is not CH1; [0314] X2 is an Fc
region; and [0315] n is 0 or 1;
[0316] wherein the binding protein is capable of binding human
IL-17 and TNF-.alpha.;
[0317] wherein VD1 comprises an amino acid sequence of a variable
heavy region (VH) of an anti-TNF-.alpha. antibody wherein said
amino acid sequence is any of SEQ ID NOs:563, 573, 578, 593, 628,
638, 648, 658, 668, 678, 688, 698, 708, 718, 728, 738, 748, 758,
763, 773, 783, 793, 803, 813, 823, 833, 843, 853, 863, 873, and
883; and
[0318] wherein VD2 comprises the amino acid sequence of a VH region
of anti-IL-17 antibody wherein said amino acid sequence is any of
SEQ ID NOs:565, 575, 580, 595, 630, 640, 650, 660, 670, 680, 690,
700, 710, 720, 730, 740, 750, 760, 765, 775, 795, 805, 815, 825,
835, 845, 855, 865, 875, and 885.
[0319] In an embodiment of the DVD-Ig binding protein described
above, VD1 and VD2 comprise an amino acid sequence of any of SEQ ID
NOs: 562, 572, 577, 592, 627, 637, 647, 657, 667, 677, 687, 697,
707, 717, 727, 737, 747, 757, 762, 772, 782, 792, 802, 812, 822,
832, 842, 852, 862, 872, and 882.
[0320] In another embodiment, the invention provides a multivalent,
multispecific DVD-Ig binding protein comprising a polypeptide
chain, wherein said polypeptide chain comprises
VD1-(X1)n-VD2-C-(X2)n, wherein; [0321] VD1 is a first light chain
variable domain; [0322] VD2 is a second light chain variable
domain; [0323] C is a light chain constant domain; [0324] X1 is a
linker with the proviso that it is not CH1; [0325] X2 does not
comprise an Fc region; and [0326] n is 0 or 1;
[0327] wherein the binding protein is capable of binding human
IL-17 and TNF-.alpha.;
[0328] wherein VD1 comprises an amino acid sequence of a variable
light region (VL) of an anti-TNF-.alpha. antibody wherein said
amino acid sequence is any of SEQ ID NOs:568, 583, 588, 598, 603,
608, 613, 618, 623, 633, 643, 653, 663, 673, 683, 693, 703, 713,
723, 733, 743, 753, 768, 778, 788, 798, 808, 818, 828, 848, 858,
868, and 878; and
[0329] wherein VD2 comprises an amino acid sequence of a VL region
of anti-IL-17 antibody wherein said amino acid sequence is any of
SEQ ID NOs:570, 585, 590, 600, 605, 610, 615, 620, 625, 635, 645,
655, 665, 675, 685, 695, 705, 715, 725, 735, 745, 755, 770, 780,
790, 800, 810, 820, 830, 850, 860, 870, and 880.
[0330] In an embodiment of the DVD-Ig binding protein described
above the VD1 and VD2 light chain variable domains comprise an
amino acid sequence of any of SEQ ID NOs:567, 582, 587, 597, 602,
607, 612, 617, 622, 632, 642, 652, 662, 672, 682, 692, 702, 712,
722, 732, 742, 752, 767, 777, 787, 797, 807, 817, 827, 847, 857,
867, and 877.
[0331] In a preferred embodiment of a multivalent, multispecific
DVD-Ig binding protein described herein, n is 0.
[0332] In another embodiment, the invention provides a multivalent,
multispecific DVD-Ig binding protein comprising first and second
polypeptide chains, wherein said first polypeptide chain comprises
a first VD1-(X1)n-VD2-C-(X2)n, wherein [0333] VD1 is a first heavy
chain variable domain; [0334] VD2 is a second heavy chain variable
domain; [0335] C is a heavy chain constant domain; [0336] X1 is a
linker with the proviso that it is not CH1; and [0337] X2 is an Fc
region; and [0338] wherein said second polypeptide chain comprises
a second VD1-(X1)n-VD2-C-(X2)n, wherein [0339] VD1 is a first light
chain variable domain; [0340] VD2 is a second light chain variable
domain; [0341] C is a light chain constant domain; [0342] X1 is a
linker with the proviso that it is not CH1; [0343] X2 does not
comprise an Fc region; and [0344] n is 0 or 1;
[0345] wherein the binding protein is capable of binding human
IL-17 and TNF-.alpha.;
[0346] wherein said VD1 and VD2 heavy chain variable domains
comprise an amino acid sequence of any of SEQ ID NOs: 562, 572,
577, 592, 627, 637, 647, 657, 667, 677, 687, 697, 707, 717, 727,
737, 747, 757, 762, 772, 782, 792, 802, 812, 822, 832, 842, 852,
862, 872, and 882; and
[0347] wherein said VD1 and VD2 light chain variable domains
comprise an amino acid sequence selected from the group consisting
of SEQ ID NOs:567, 582, 587, 597, 602, 607, 612, 617, 622, 632,
642, 652, 662, 672, 682, 692, 702, 712, 722, 732, 742, 752, 767,
777, 787, 797, 807, 817, 827, 847, 857, 867, and 877.
[0348] In a preferred embodiment of a multivalent, multispecific
DVD-Ig binding protein according to the invention, X1 or X2 is an
amino acid sequence selected from the group consisting of SEQ ID
NOs:888-918.
[0349] In another embodiment, a multivalent, multispecific DVD-Ig
binding protein described herein comprises two first polypeptide
chains and two second polypeptide chains.
[0350] In an embodiment of a multivalent, multispecific DVD-Ig
binding protein described herein, the Fc region is selected from
the group consisting of native sequence Fc region and a variant
sequence Fc region. Preferably, the Fc region is selected from the
group consisting of an Fc region from an IgG1, IgG2, IgG3, IgG4,
IgA, IgM, IgE, and IgD.
[0351] In another embodiment of a multivalent, multispecific DVD-Ig
binding protein described herein comprising a first and second
polypeptide chains, VD1 of the first polypeptide chain and said VD1
of the second polypeptide chain are obtained from the same first
and second parent antibody, respectively, or antigen binding
portion thereof.
[0352] In an embodiment of a TNF-.alpha. and IL-17 binding DVD-Ig
protein described herein, a parental anti-TNF-.alpha. antibody
binds TNF-.alpha. with a potency different from the potency with
which a parental anti-IL-17 antibody binds human IL-17.
[0353] In another embodiment of a TNF-.alpha. and IL-17 binding
DVD-Ig protein described herein, a parental anti-TNF-.alpha.
antibody binds TNF-.alpha. with an affinity different from the
affinity with which said anti-IL-17 antibody binds human IL-17.
[0354] In another embodiment of a TNF-.alpha. and IL-17 binding
DVD-Ig protein described herein an anti-TNF-.alpha. antibody and
said anti-IL-17 antibody are selected from the group consisting of
a human antibody, a CDR grafted antibody, and a humanized
antibody.
[0355] In another embodiment, a TNF-.alpha. and IL-17 binding
DVD-Ig protein described herein possesses at least one desired
property exhibited by said anti-TNF-.alpha. antibody or said
anti-IL-17 antibody. Preferably, the desired property is selected
from one or more antibody parameters. More preferably, the antibody
parameters are selected from the group consisting of antigen
specificity, affinity to antigen, potency, biological function,
epitope recognition, stability, solubility, production efficiency,
immunogenicity, pharmacokinetics, bioavailability, tissue cross
reactivity, and orthologous antigen binding.
[0356] In another embodiment, the invention provides a multivalent,
multispecific DVD-Ig binding protein capable of binding two
antigens comprising four polypeptide chains, wherein two
polypeptide chains comprise VD1-(X1)n-VD2-C-(X2)n, wherein [0357]
VD1 is a first heavy chain variable domain; [0358] VD2 is a second
heavy chain variable domain; [0359] C is a heavy chain constant
domain; [0360] X1 is a linker with the proviso that it is not CH1;
and [0361] X2 is an Fc region; and
[0362] wherein two polypeptide chains comprise
VD1-(X1)n-VD2-C-(X2)n, wherein [0363] VD1 is a first light chain
variable domain; [0364] VD2 is a second light chain variable
domain; [0365] C is a light chain constant domain; [0366] X1 is a
linker with the proviso that it is not CH1; [0367] X2 does not
comprise an Fc region; and [0368] n is 0 or 1;
[0369] wherein the VD1 and VD2 heavy chain variable domains
comprise an amino acid sequence of any of SEQ ID NOs: 562, 572,
577, 592, 627, 637, 647, 657, 667, 677, 687, 697, 707, 717, 727,
737, 747, 757, 762, 772, 782, 792, 802, 812, 822, 832, 842, 852,
862, 872, and 882; and
[0370] wherein the VD1 and VD2 light chain variable domains
comprise an amino acid sequence selected from the group consisting
of SEQ ID NOs:567, 582, 587, 597, 602, 607, 612, 617, 622, 632,
642, 652, 662, 672, 682, 692, 702, 712, 722, 732, 742, 752, 767,
777, 787, 797, 807, 817, 827, 847, 857, 867, and 877.
[0371] In another embodiment, the invention provides method of
producing a multivalent, multispecific DVD-Ig binding protein
described herein, comprising culturing a host cell carrying a
vector comprising a nucleic acid described herein in culture medium
under conditions sufficient to produce the binding protein.
Preferably, 50%-75% of the binding protein produced according the
method is a dual specific tetravalent DVD-Ig binding protein
described herein. More preferably, 75%-90% of the binding protein
produced according to this method is a dual specific tetravalent
binding protein. Even more preferably, 90%-95% of the binding
protein produced is a dual specific tetravalent binding
protein.
[0372] Another embodiment of the invention is a protein produced
according to the described method.
[0373] In another embodiment, the invention provides a
pharmaceutical composition comprising a multivalent, multispecific
DVD-Ig binding protein described herein and a pharmaceutically
acceptable carrier.
[0374] In another embodiment, a pharmaceutical composition
comprising a multivalent, multispecific DVD-Ig binding protein
further comprises at least one additional agent. Preferably, the
additional agent is selected from the group consisting of:
therapeutic agent, imaging agent, cytotoxic agent, angiogenesis
inhibitors; kinase inhibitors; co-stimulation molecule blockers;
adhesion molecule blockers; anti-cytokine antibody or functional
fragment thereof; methotrexate; cyclosporin; rapamycin; FK506;
detectable label or reporter; a TNF antagonist; an antirheumatic; a
muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug
(NSAID), an analgesic, an anesthetic, a sedative, a local
anesthetic, a neuromuscular blocker, an antimicrobial, an
antipsoriatic, a corticosteroid, an anabolic steroid, an
erythropoietin, an immunization, an immunoglobulin, an
immunosuppressive, a growth hormone, a hormone replacement drug, a
radiopharmaceutical, an antidepressant, an antipsychotic, a
stimulant, an asthma medication, a beta agonist, an inhaled
steroid, an epinephrine or analog, a cytokine, and a cytokine
antagonist.
[0375] Another embodiment of the invention provides a method for
treating a subject for a disease or a disorder by administering to
the subject a multivalent, multispecific DVD-Ig binding protein
described herein that binds TNF-.alpha. and IL-17 such that
treatment is achieved.
[0376] The invention also provides a method for generating a
multivalent, multispecific DVD-Ig binding protein described herein
capable of binding TNF-.alpha. and human IL-17 comprising the steps
of: [0377] a) obtaining a first parent antibody or antigen binding
portion thereof, capable of binding TNF-.alpha.; [0378] b)
obtaining a second parent antibody or antigen binding portion
thereof, capable of binding human IL-17; [0379] c) constructing
first and third polypeptide chains comprising
VD1-(X1)n-VD2-C-(X2)n, wherein [0380] VD1 is a first heavy chain
variable domain obtained from said first parent antibody or antigen
binding portion thereof; [0381] VD2 is a second heavy chain
variable domain obtained from said second parent antibody or
antigen binding portion thereof; [0382] C is a heavy chain constant
domain; [0383] X1 is a linker with the proviso that it is not CH1;
[0384] X2 is an Fc region; and [0385] n is 0 or 1; and [0386] d)
constructing second and fourth polypeptide chains comprising
VD1-(X1)n-VD2-C-(X2)n, wherein [0387] VD1 is a first light chain
variable domain obtained from said first parent antibody or antigen
binding portion thereof; [0388] VD2 is a second light chain
variable domain obtained from said second parent antibody or
antigen binding thereof; [0389] C is a light chain constant domain;
[0390] X1 is a linker with the proviso that it is not CH1; [0391]
X2 does not comprise an Fc region; and [0392] n is 0 or 1; and
[0393] e) expressing said first, second, third and fourth
polypeptide chains; such that a DVD-Ig binding protein capable of
binding TNF-.alpha. and human IL-17 is generated, wherein the
binding protein is capable of binding TNF-.alpha. and human
IL-17;
[0394] wherein VD1 and VD2 heavy chain variable domains comprise an
amino acid sequence selected from the group consisting of SEQ ID
NOs: of SEQ ID NOs: 562, 572, 577, 592, 627, 637, 647, 657, 667,
677, 687, 697, 707, 717, 727, 737, 747, 757, 762, 772, 782, 792,
802, 812, 822, 832, 842, 852, 862, 872, and 882; and
[0395] wherein the VD1 and VD2 light chain variable domains
comprise an amino acid sequence selected from the group consisting
of SEQ ID NOs:567, 582, 587, 597, 602, 607, 612, 617, 622, 632,
642, 652, 662, 672, 682, 692, 702, 712, 722, 732, 742, 752, 767,
777, 787, 797, 807, 817, 827, 847, 857, 867, and 877.
[0396] In another embodiment of the method described above, said
first parent antibody or antigen binding portion thereof, and said
second parent antibody or antigen binding portion thereof, are
selected from the group consisting of a human antibody, a CDR
grafted antibody, and a humanized antibody.
[0397] In another embodiment of the method described above, said
first parent antibody or antigen binding portion thereof, and said
second parent antibody or antigen binding portion thereof, are
selected from the group consisting of a Fab fragment, a
F(ab').sub.2 fragment, a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region; a Fd
fragment consisting of the VH and CH1 domains; a Fv fragment
consisting of the VL and VH domains of a single arm of an antibody,
a dAb fragment, an isolated complementarity determining region
(CDR), a single chain antibody, and diabodies.
[0398] In another embodiment of the method, the first parent
antibody or antigen binding portion thereof possesses at least one
desired property exhibited by the DVD-Ig binding protein.
[0399] In another embodiment of the method described above the
second parent antibody or antigen binding portion thereof possesses
at least one desired property exhibited by the DVD-Ig binding
protein.
[0400] Preferably, in the method described above, the Fc region is
selected from the group consisting of a native sequence Fc region
and a variant sequence Fc region. More preferably, the Fc region is
selected from the group consisting of an Fc region from an IgG1,
IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.
[0401] In another embodiment, in a method described above, a
desired property is selected from one or more antibody parameters
of the first parent antibody or antigen binding portion
thereof.
[0402] In another embodiment, in a method described above, a
desired property is selected from one or more antibody parameters
of the second parent antibody.
[0403] Preferably, said antibody parameters are selected from the
group consisting of antigen specificity, affinity to antigen,
potency, biological function, epitope recognition, stability,
solubility, production efficiency, immunogenicity,
pharmacokinetics, bioavailability, tissue cross reactivity, and
orthologous antigen binding.
[0404] In another embodiment of the method described above, the
first parent antibody or antigen binding portion thereof, binds
said first antigen with a different affinity than the affinity with
which said second parent antibody or antigen binding portion
thereof, binds said second antigen.
[0405] In another embodiment, the first parent antibody or antigen
binding portion thereof, binds said first antigen with a different
potency than the potency with which said second parent antibody or
antigen binding portion thereof, binds said second antigen.
[0406] In another embodiment, the invention provides a method for
generating a DVD-Ig binding protein capable of binding TNF-.alpha.
and human IL-17 with desired properties comprising the steps of
[0407] a) obtaining a first parent antibody or antigen binding
portion thereof, capable of binding TNF-.alpha. and possessing at
least one desired property exhibited by the Dual Variable Domain
Immunoglobulin;
[0408] b) obtaining a second parent antibody or antigen binding
portion thereof, capable of binding human IL-17 and possessing at
least one desired property exhibited by the Dual Variable Domain
Immunoglobulin;
[0409] c) constructing first and third polypeptide chains
comprising VD1-(X1)n-VD2-C-(X2)n, wherein; [0410] VD1 is a first
heavy chain variable domain obtained from said first parent
antibody or antigen binding portion thereof; [0411] VD2 is a second
heavy chain variable domain obtained from said second parent
antibody or antigen binding portion thereof; [0412] C is a heavy
chain constant domain; [0413] X1 is a linker with the proviso that
it is not CH1; [0414] X2 is an Fc region; and [0415] n is 0 or
1;
[0416] d) constructing second and fourth polypeptide chains
comprising VD1-(X1)n-VD2-C-(X2)n, wherein; [0417] VD1 is a first
light chain variable domain obtained from said first parent
antibody or antigen binding portion thereof; [0418] VD2 is a second
light chain variable domain obtained from said second parent
antibody or antigen binding portion thereof; [0419] C is a light
chain constant domain; [0420] X1 is a linker with the proviso that
it is not CH1; [0421] X2 does not comprise an Fc region; and [0422]
n is 0 or 1;
[0423] e) expressing said first, second, third, and fourth
polypeptide chains; such that a DVD-Ig binding capable of binding
said TNF-.alpha. and human IL-17 with desired properties is
generated,
[0424] wherein VD1 and VD2 heavy chain variable domains comprise an
amino acid sequence selected from the group consisting of SEQ ID
NOs: of SEQ ID NOs: 562, 572, 577, 592, 627, 637, 647, 657, 667,
677, 687, 697, 707, 717, 727, 737, 747, 757, 762, 772, 782, 792,
802, 812, 822, 832, 842, 852, 862, 872, and 882; and
[0425] wherein the VD1 and VD2 light chain variable domains
comprise an amino acid sequence selected from the group consisting
of SEQ ID NOs:567, 582, 587, 597, 602, 607, 612, 617, 622, 632,
642, 652, 662, 672, 682, 692, 702, 712, 722, 732, 742, 752, 767,
777, 787, 797, 807, 817, 827, 847, 857, 867, and 877.
[0426] In another embodiment, an IL-17 binding protein described
herein binds human IL-17 and is capable of modulating a biological
function of IL-17.
[0427] The invention also provides a neutralizing binding protein,
wherein the neutralizing binding protein comprises an IL-17 binding
protein as described above, and wherein said neutralizing binding
protein is capable of neutralizing IL-17.
[0428] In another embodiment, a neutralizing IL-17 binding protein
according to the invention binds a human IL-17 is selected from the
group consisting of pro-human IL-17; mature-human IL-17, and
truncated-human IL-17.
[0429] Preferably a neutralizing IL-17 binding protein described
herein diminishes the ability of IL-17 to bind to its receptor.
Even more preferably, a neutralizing IL-17 binding protein
diminishes the ability of pro-human IL-17, mature human IL-17, or a
truncated human IL-17 to bind to the IL-17 receptor.
[0430] In another embodiment, a neutralizing IL-17 binding protein
described herein is capable of reducing one or more of IL-17
biological activities selected from the group consisting of: Th1
modulation; Th2 modulation; Nk modulation; neutrophil modulation;
monocyte-macrophage lineage modulation; neutrophil modulation;
eosinophil modulation; B-cells modulation; cytokine modulation;
chemokine modulation; adhesion molecule modulation; and cell
recruitment modulation.
[0431] Preferably, an IL-17 binding protein of the invention has an
on rate constant (K.sub.on) to said target selected from the group
consisting of: at least about 10.sup.2M.sup.-1s.sup.-1; at least
about 10.sup.3M.sup.-1s.sup.-1; at least about
10.sup.4M.sup.-1s.sup.-1; at least about 10.sup.5M.sup.-1s.sup.-1;
and at least about 10.sup.6M.sup.-1s.sup.-1; as measured by surface
plasmon resonance.
[0432] In another embodiment, an IL-17 binding protein has an off
rate constant (K.sub.off) to said target selected from the group
consisting of: at most about 10.sup.-3s.sup.-1; at most about
10.sup.-4s.sup.-1; at most about 10.sup.-5s.sup.-1; and at most
about 10.sup.-6s.sup.-1, as measured by surface plasmon
resonance.
[0433] In another embodiment, an IL-17 binding protein of the
invention has a dissociation constant (K.sub.D) to said target
selected from the group consisting of: at most about 10.sup.-7 M;
at most about 10.sup.-8M; at most about 10.sup.-9 M; at most about
10.sup.-10 M; at most about 10.sup.-11 M; at most about 10.sup.12
M; m and at most 10.sup.-13M.
[0434] Another aspect of the invention provides an IL-17 binding
protein construct that comprises an IL-17 binding protein described
herein and further comprises a linker polypeptide or an
immunoglobulin constant domain. Preferred IL-17 binding protein
constructs of the invention comprise an IL-17 binding protein
selected from the group consisting [0435] an immunoglobulin
molecule, [0436] a monoclonal antibody, [0437] a chimeric antibody,
[0438] a CDR-grafted antibody, [0439] a humanized antibody, [0440]
a Fab, [0441] a Fab', [0442] a F(ab')2, [0443] a Fv, [0444] a
disulfide linked Fv, [0445] a scFv, [0446] a single domain
antibody, [0447] a diabody, [0448] a multispecific antibody, [0449]
a dual specific antibody, [0450] a bispecific antibody, and [0451]
a DVD-Ig.TM..
[0452] In a preferred embodiment, an IL-17 binding protein
construct of the invention comprises a heavy chain immunoglobulin
constant domain selected from the group consisting of: [0453] a
human IgM constant domain, [0454] a human IgG1 constant domain,
[0455] a human IgG2 constant domain, [0456] a human IgG3 constant
domain, [0457] a human IgG4 constant domain, [0458] a human IgE
constant domain, [0459] and [0460] a human IgA constant domain.
[0461] In yet another embodiment, an IL-17 binding protein
construct comprises an immunoglobulin constant domain having an
amino acid sequence selected from the group consisting of: [0462]
SEQ ID NO:3 [0463] SEQ ID NO:4 [0464] SEQ ID NO:5 [0465] and [0466]
SEQ ID NO:6.
[0467] In another embodiment, an IL-17 binding protein construct
described herein has a greater half life in vivo than the soluble
counterpart of said IL-17 binding protein construct.
[0468] Another aspect of the invention provides an IL-17 binding
protein conjugate comprising an IL-17 binding protein construct,
wherein the IL-17 binding protein conjugate further comprises an
agent selected from the group consisting of: an immunoadhesion
molecule, an imaging agent, a therapeutic agent, and a cytotoxic
agent.
[0469] Preferred imaging agents useful in making IL-17 binding
protein conjugates and in other aspects of the invention include,
but are not limited to, a radiolabel, an enzyme, a fluorescent
label, a luminescent label, a bioluminescent label, a magnetic
label, and biotin.
[0470] Preferred radiolabels useful in the invention include, but
are not limited, to those selected from the group consisting of:
.sup.3H, .sup.14C, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In,
.sup.125I, .sup.131I, .sup.177Lu, .sup.166Ho, and .sup.153Sm.
[0471] In another embodiment, an IL-17 binding protein conjugate of
the invention comprises a therapeutic or cytotoxic agent selected
from the group consisting of an anti-metabolite, an alkylating
agent, an antibiotic, a growth factor, a cytokine, an
anti-angiogenic agent, an anti-mitotic agent, an anthracycline,
toxin, and an apoptotic agent.
[0472] In another embodiment, binding proteins described herein
possesses a human glycosylation pattern.
[0473] In another embodiment, an IL-17 binding protein described
herein, including IL-17 binding protein constructs and IL-17
binding protein conjugates, may be in the form of a crystallized
binding protein. Preferred crystalline forms retain at least some
and preferably essentially all of the biologically activity of the
uncrystallized form of an IL-17 binding protein described herein.
Such crystalline forms may also be used as a carrier-free
pharmaceutical controlled release crystallized IL-17 binding
proteins.
[0474] In another embodiment, the invention provides isolated
nucleic acids encoding IL-17 binding proteins, including binding
protein constructs, described herein. Such nucleic acids may be
inserted into a vector for carrying out various genetic analyses
and recombinant techniques for expressing, characterizing, or
improving one or more properties of an IL-17 binding protein
described herein. Preferred vectors for cloning nucleic acids
encoding binding proteins described herein include, but are not
limited, pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, and pBJ.
[0475] The invention also provides a host cell comprising a vector
comprising a nucleic acid encoding a binding protein described
herein. Host cells useful in the invention may be prokaryotic or
eukaryotic. A preferred prokaryotic host cell is Escherichia coli.
Eukaryotic cells useful as host cells in the invention include
protist cell, animal cell, plant cell, and fungal cell.
[0476] A preferred fungal cell is a yeast cell, including
Saccharomyces cerevisiae.
[0477] A preferred animal cell useful as a host cell according to
the invention includes, but is not limited to, a mammalian cell, an
avian cell, and an insect cell. Preferred mammalian cells include
CHO and COS cells. An insect cell useful as a host cell according
to the invention is an insect Sf9 cell.
[0478] A vector may comprise a nucleic acid encoding an IL-17
binding protein described herein in which the nucleic acid is
operably linked to appropriate transcriptional and/or translational
sequences that permit expression of the binding protein in a
particular host cell carrying the vector.
[0479] In another aspect, the invention provides a method of
producing an IL-17 binding protein comprising culturing a host cell
comprising a vector encoding the IL-17 binding protein in culture
medium under conditions sufficient to produce the binding protein
capable of binding IL-17. The protein so produced can be isolated
and used in various compositions and methods described herein.
[0480] Compositions of the invention include a composition for the
release of a binding protein said composition comprising: [0481]
(a) a formulation, wherein said formulation comprises a
crystallized binding protein, described herein, and an ingredient;
and [0482] (b) at least one polymeric carrier.
[0483] Preferred polymeric carriers useful in compositions of the
invention include, without limitation, one or more of the group
consisting of: poly (acrylic acid), poly (cyanoacrylates), poly
(amino acids), poly (anhydrides), poly (depsipeptide), poly
(esters), poly (lactic acid), poly (lactic-co-glycolic acid) or
PLGA, poly (b-hydroxybutryate), poly (caprolactone), poly
(dioxanone); poly (ethylene glycol), poly
((hydroxypropyl)methacrylamide, poly [(organo) phosphazene], poly
(ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone),
maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols,
albumin, alginate, cellulose and cellulose derivatives, collagen,
fibrin, gelatin, hyaluronic acid, oligosaccharides,
glycaminoglycans, sulfated polysaccharides, blends and copolymers
thereof.
[0484] In another aspect, an ingredient of a composition of the
invention is selected from the group consisting of albumin,
sucrose, trehalose, lactitol, gelatin,
hydroxypropyl-.beta.-cyclodextrin, methoxypolyethylene glycol and
polyethylene glycol.
[0485] In yet another embodiment, the invention provides a method
for treating a mammal comprising the step of administering to the
mammal an effective amount of a composition described herein.
[0486] The invention also provides pharmaceutical compositions
comprising an IL-17 binding protein described herein and a
pharmaceutically acceptable carrier. A pharmaceutically acceptable
carrier may also serve as an adjuvant to increase the absorption or
dispersion of the IL-17 binding protein in a composition of the
invention. A preferred adjuvant is hyaluronidase.
[0487] In another embodiment, a pharmaceutical composition further
comprises at least one additional therapeutic agent for treating a
disorder in which IL-17 activity is detrimental.
[0488] In another embodiment, the invention provides a method for
reducing human IL-17 activity comprising contacting human IL-17
with an IL-17 binding protein described herein such that human
IL-17 activity is reduced.
[0489] In another embodiment, a pharmaceutical composition
comprising an IL-17 binding protein described herein comprises at
least one additional agent. Preferably, the additional agent is
selected from the group consisting of: therapeutic agent, imaging
agent, cytotoxic agent, angiogenesis inhibitors; kinase inhibitors;
co-stimulation molecule blockers; adhesion molecule blockers;
anti-cytokine antibody or functional fragment thereof;
methotrexate; cyclosporin; rapamycin; FK506; detectable label or
reporter; a TNF antagonist; an antirheumatic; a muscle relaxant, a
narcotic, a non-steroid anti-inflammatory drug (NSAID), an
analgesic, an anesthetic, a sedative, a local anesthetic, a
neuromuscular blocker, an antimicrobial, an antipsoriatic, a
corticosteroid, an anabolic steroid, an erythropoietin, an
immunization, an immunoglobulin, an immunosuppressive, a growth
hormone, a hormone replacement drug, a radiopharmaceutical, an
antidepressant, an antipsychotic, a stimulant, an asthma
medication, a beta agonist, an inhaled steroid, an epinephrine or
analog, a cytokine, and a cytokine antagonist.
[0490] Another embodiment of the invention provides a method for
treating a subject for a disease or a disorder by administering to
the subject a multivalent, multispecific DVD-Ig binding protein
described herein that binds TNF-.alpha. and IL-17 such that
treatment is achieved.
[0491] In another embodiment, a disorder that may treated by a
method of the invention comprising administering to a subject an
IL-17 binding protein described herein is selected from the group
comprising rheumatoid arthritis, osteoarthritis, juvenile chronic
arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis,
reactive arthritis, spondyloarthropathy, systemic lupus
erythematosus, Crohn's disease, ulcerative colitis, inflammatory
bowel disease, insulin dependent diabetes mellitus, thyroiditis,
asthma, allergic diseases, psoriasis, dermatitis scleroderma, graft
versus host disease, organ transplant rejection, acute or chronic
immune disease associated with organ transplantation, sarcoidosis,
atherosclerosis, disseminated intravascular coagulation, Kawasaki's
disease, Grave's disease, nephrotic syndrome, chronic fatigue
syndrome, Wegener's granulomatosis, Henoch-Schoenlein purpurea,
microscopic vasculitis of the kidneys, chronic active hepatitis,
uveitis, septic shock, toxic shock syndrome, sepsis syndrome,
cachexia, infectious diseases, parasitic diseases, acquired
immunodeficiency syndrome, acute transverse myelitis, Huntington's
chorea, Parkinson's disease, Alzheimer's disease, stroke, primary
biliary cirrhosis, hemolytic anemia, malignancies, heart failure,
myocardial infarction, Addison's disease, sporadic, polyglandular
deficiency type I and polyglandular deficiency type II, Schmidt's
syndrome, adult (acute) respiratory distress syndrome, alopecia,
alopecia greata, seronegative arthropathy, arthropathy, Reiter's
disease, psoriatic arthropathy, ulcerative colitic arthropathy,
enteropathic synovitis, chlamydia, yersinia and salmonella
associated arthropathy, spondyloarthropathy, atheromatous
disease/arteriosclerosis, atopic allergy, autoimmune bullous
disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid,
linear IgA disease, autoimmune haemolytic anaemia, Coombs positive
haemolytic anaemia, acquired pernicious anaemia, juvenile
pernicious anaemia, myalgic encephalitis/Royal Free Disease,
chronic mucocutaneous candidiasis, giant cell arteritis, primary
sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired
Immunodeficiency Disease Syndrome, Acquired Immunodeficiency
Related Diseases, Hepatitis B, Hepatitis C, common varied
immunodeficiency (common variable hypogammaglobulinaemia), dilated
cardiomyopathy, female infertility, ovarian failure, premature
ovarian failure, fibrotic lung disease, cryptogenic fibrosing
alveolitis, post-inflammatory interstitial lung disease,
interstitial pneumonitis, connective tissue disease associated
interstitial lung disease, mixed connective tissue disease
associated lung disease, systemic sclerosis associated interstitial
lung disease, rheumatoid arthritis associated interstitial lung
disease, systemic lupus erythematosus associated lung disease,
dermatomyositis/polymyositis associated lung disease, Sjogren's
disease associated lung disease, ankylosing spondylitis associated
lung disease, vasculitic diffuse lung disease, haemosiderosis
associated lung disease, drug-induced interstitial lung disease,
fibrosis, radiation fibrosis, bronchiolitis obliterans, chronic
eosinophilic pneumonia, lymphocytic infiltrative lung disease,
postinfectious interstitial lung disease, gouty arthritis,
autoimmune hepatitis, type-1 autoimmune hepatitis (classical
autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis
(anti-LKM antibody hepatitis), autoimmune mediated hypoglycaemia,
type B insulin resistance with acanthosis nigricans,
hypoparathyroidism, acute immune disease associated with organ
transplantation, chronic immune disease associated with organ
transplantation, osteoarthrosis, primary sclerosing cholangitis,
psoriasis type 1, psoriasis type 2, idiopathic leucopaenia,
autoimmune neutropaenia, renal disease NOS, glomerulonephritides,
microscopic vasculitis of the kidneys, Lyme disease, discoid lupus
erythematosus, male infertility idiopathic or NOS, sperm
autoimmunity, multiple sclerosis (all subtypes), sympathetic
ophthalmia, pulmonary hypertension secondary to connective tissue
disease, Goodpasture's syndrome, pulmonary manifestation of
polyarteritis nodosa, acute rheumatic fever, rheumatoid
spondylitis, Still's disease, systemic sclerosis, Sjorgren's
syndrome, Takayasu's disease/arteritis, autoimmune
thrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroid
disease, hyperthyroidism, goitrous autoimmune hypothyroidism
(Hashimoto's disease), atrophic autoimmune hypothyroidism, primary
myxoedema, phacogenic uveitis, primary vasculitis, vitiligo acute
liver disease, chronic liver diseases, alcoholic cirrhosis,
alcohol-induced liver injury, choleosatatis, idiosyncratic liver
disease, Drug-Induced hepatitis, Non-alcoholic Steatohepatitis,
allergy and asthma, group B streptococci (GBS) infection, mental
disorders (e.g., depression and schizophrenia), Th2 Type and Th1
Type mediated diseases, acute and chronic pain (different forms of
pain), and cancers such as lung, breast, stomach, bladder, colon,
pancreas, ovarian, prostate and rectal cancer and hematopoietic
malignancies (leukemia and lymphoma) Abetalipoproteinemia,
Acrocyanosis, acute and chronic parasitic or infectious processes,
acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid
leukemia (AML), acute or chronic bacterial infection, acute
pancreatitis, acute renal failure, adenocarcinomas, aerial ectopic
beats, AIDS dementia complex, alcohol-induced hepatitis, allergic
conjunctivitis, allergic contact dermatitis, allergic rhinitis,
allograft rejection, alpha-1-antitrypsin deficiency, amyotrophic
lateral sclerosis, anemia, angina pectoris, anterior horn cell
degeneration, anti-CD3 therapy, antiphospholipid syndrome,
anti-receptor hypersensitivity reactions, aortic and peripheral
aneurysms, aortic dissection, arterial hypertension,
arteriosclerosis, arteriovenous fistula, ataxia, atrial
fibrillation (sustained or paroxysmal), atrial flutter,
atrioventricular block, B cell lymphoma, bone graft rejection, bone
marrow transplant (BMT) rejection, bundle branch block, Burkitt's
lymphoma, Burns, cardiac arrhythmias, cardiac stun syndrome,
cardiac tumors, cardiomyopathy, cardiopulmonary bypass inflammation
response, cartilage transplant rejection, cerebellar cortical
degenerations, cerebellar disorders, chaotic or multifocal atrial
tachycardia, chemotherapy associated disorders, chronic myelocytic
leukemia (CML), chronic alcoholism, chronic inflammatory
pathologies, chronic lymphocytic leukemia (CLL), chronic
obstructive pulmonary disease (COPD), chronic salicylate
intoxication, colorectal carcinoma, congestive heart failure,
conjunctivitis, contact dermatitis, cor pulmonale, coronary artery
disease, Creutzfeldt-Jakob disease, culture negative sepsis, cystic
fibrosis, cytokine therapy associated disorders, Dementia
pugilistica, demyelinating diseases, dengue hemorrhagic fever,
dermatitis, dermatologic conditions, diabetes, diabetes mellitus,
diabetic arteriosclerotic disease, Diffuse Lewy body disease,
dilated congestive cardiomyopathy, disorders of the basal ganglia,
Down's Syndrome in middle age, drug-induced movement disorders
induced by drugs which block CNS dopamine receptors, drug
sensitivity, eczema, encephalomyelitis, endocarditis,
endocrinopathy, epiglottitis, Epstein-Barr virus infection,
erythromelalgia, extrapyramidal and cerebellar disorders, familial
hematophagocytic lymphohistiocytosis, fetal thymus implant
rejection, Friedreich's ataxia, functional peripheral arterial
disorders, fungal sepsis, gas gangrene, gastric ulcer, glomerular
nephritis, graft rejection of any organ or tissue, gram negative
sepsis, gram positive sepsis, granulomas due to intracellular
organisms, hairy cell leukemia, Hallervorden-Spatz disease,
Hashimoto's thyroiditis, hay fever, heart transplant rejection,
hemachromatosis, hemodialysis, hemolytic uremic
syndrome/thrombolytic thrombocytopenic purpura, hemorrhage,
hepatitis (A), His bundle arrhythmias, HIV infection/HIV
neuropathy, Hodgkin's disease, hyperkinetic movement disorders,
hypersensitivity reactions, hypersensitivity pneumonitis,
hypertension, hypokinetic movement disorders,
hypothalamic-pituitary-adrenal axis evaluation, idiopathic
Addison's disease, idiopathic pulmonary fibrosis, antibody mediated
cytotoxicity, Asthenia, infantile spinal muscular atrophy,
inflammation of the aorta, influenza a, ionizing radiation
exposure, iridocyclitis/uveitis/optic neuritis,
ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid
arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma,
kidney transplant rejection, legionella, leishmaniasis, leprosy,
lesions of the corticospinal system, lipedema, liver transplant
rejection, lymphedema, malaria, malignant Lymphoma, malignant
histiocytosis, malignant melanoma, meningitis, meningococcemia,
metabolic/idiopathic, migraine headache, mitochondrial multi-system
disorder, mixed connective tissue disease, monoclonal gammopathy,
multiple myeloma, multiple systems degenerations (Mencel
Dejerine-Thomas Shi-Drager and Machado-Joseph), myasthenia gravis,
mycobacterium avium intracellulare, mycobacterium tuberculosis,
myelodysplastic syndrome, myocardial infarction, myocardial
ischemic disorders, nasopharyngeal carcinoma, neonatal chronic lung
disease, nephritis, nephrosis, neurodegenerative diseases,
neurogenic I muscular atrophies, neutropenic fever, non-Hodgkins
lymphoma, occlusion of the abdominal aorta and its branches,
occlusive arterial disorders, OKT3.RTM. therapy,
orchitis/epidydimitis, orchitis/vasectomy reversal procedures,
organomegaly, osteoporosis, pancreas transplant rejection,
pancreatic carcinoma, paraneoplastic syndrome/hypercalcemia of
malignancy, parathyroid transplant rejection, pelvic inflammatory
disease, perennial rhinitis, pericardial disease, peripheral
atherosclerotic disease, peripheral vascular disorders,
peritonitis, pernicious anemia, pneumocystis carinii pneumonia,
pneumonia, POEMS syndrome (polyneuropathy, organomegaly,
endocrinopathy, monoclonal gammopathy, and skin changes syndrome),
post perfusion syndrome, post pump syndrome, post-MI cardiotomy
syndrome, preeclampsia, Progressive supranucleo Palsy, primary
pulmonary hypertension, radiation therapy, Raynaud's phenomenon and
disease, Raynaud's disease, Refsum's disease, regular narrow QRS
tachycardia, renovascular hypertension, reperfusion injury,
restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea,
Senile Dementia of Lewy body type, seronegative arthropathies,
shock, sickle cell anemia, skin allograft rejection, skin changes
syndrome, small bowel transplant rejection, solid tumors, specific
arrhythmias, spinal ataxia, spinocerebellar degenerations,
streptococcal myositis, structural lesions of the cerebellum,
Subacute sclerosing panencephalitis, Syncope, syphilis of the
cardiovascular system, systemic anaphylaxis, systemic inflammatory
response syndrome, systemic onset juvenile rheumatoid arthritis,
T-cell or FAB ALL, Telangiectasia, thromboangitis obliterans,
thrombocytopenia, toxicity, transplants, trauma/hemorrhage, type
III hypersensitivity reactions, type IV hypersensitivity, unstable
angina, uremia, urosepsis, urticaria, valvular heart diseases,
varicose veins, vasculitis, venous diseases, venous thrombosis,
ventricular fibrillation, viral and fungal infections, viral
encephalitis/aseptic meningitis, viral-associated hemaphagocytic
syndrome, Wernicke-Korsakoff syndrome, Wilson's disease, xenograft
rejection of any organ or tissue, acute coronary syndromes, acute
idiopathic polyneuritis, acute inflammatory demyelinating
polyradiculoneuropathy, acute ischemia, adult Still's disease,
alopecia greata, anaphylaxis, anti-phospholipid antibody syndrome,
aplastic anemia, arteriosclerosis, atopic eczema, atopic
dermatitis, autoimmune dermatitis, autoimmune disorder associated
with streptococcus infection, autoimmune enteropathy, autoimmune
hearing loss, autoimmune lymphoproliferative syndrome (ALPS),
autoimmune myocarditis, autoimmune premature ovarian failure,
blepharitis, bronchiectasis, bullous pemphigoid, cardiovascular
disease, catastrophic antiphospholipid syndrome, celiac disease,
cervical spondylosis, chronic ischemia, cicatricial pemphigoid,
clinically isolated syndrome (cis) with risk for multiple
sclerosis, conjunctivitis, childhood onset psychiatric disorder,
chronic obstructive pulmonary disease (COPD), dacryocystitis,
dermatomyositis, diabetic retinopathy, diabetes mellitus, disk
herniation, disk prolapse, drug induced immune hemolytic anemia,
endocarditis, endometriosis, endophthalmitis, episcleritis,
erythema multiforme, erythema multiforme major, gestational
pemphigoid, Guillain-Barre syndrome (GBS), hay fever, Hughes
syndrome, idiopathic Parkinson's disease, idiopathic interstitial
pneumonia, IgE-mediated allergy, immune hemolytic anemia, inclusion
body myositis, infectious ocular inflammatory disease, inflammatory
demyelinating disease, inflammatory heart disease, inflammatory
kidney disease, IPF/UIP, iritis, keratitis, keratojunctivitis
sicca, Kussmaul disease or Kussmaul-Meier disease, Landry's
paralysis, Langerhan's cell histiocytosis, livedo reticularis,
macular degeneration, microscopic polyangiitis, morbus bechterev,
motor neuron disorders, mucous membrane pemphigoid, multiple organ
failure, myasthenia gravis, myelodysplastic syndrome, myocarditis,
nerve root disorders, neuropathy, non-A non-B hepatitis, optic
neuritis, osteolysis, ovarian cancer, pauciarticular JRA,
peripheral artery occlusive disease (PAOD), peripheral vascular
disease (PVD), peripheral artery, disease (PAD), phlebitis,
polyarteritis nodosa (or periarteritis nodosa), polychondritis,
polymyalgia rheumatica, poliosis, polyarticular JRA, polyendocrine
deficiency syndrome, polymyositis, polymyalgia rheumatica (PMR),
post-pump syndrome, primary Parkinsonism, prostate and rectal
cancer and hematopoietic malignancies (leukemia and lymphoma),
prostatitis, pure red cell aplasia, primary adrenal insufficiency,
recurrent neuromyelitis optica, restenosis, rheumatic heart
disease, sapho (synovitis, acne, pustulosis, hyperostosis, and
osteitis), scleroderma, secondary amyloidosis, shock lung,
scleritis, sciatica, secondary adrenal insufficiency, silicone
associated connective tissue disease, sneddon-wilkinson dermatosis,
spondylitis ankylosans, Stevens-Johnson syndrome (SJS), systemic
inflammatory response syndrome, temporal arteritis, toxoplasmic
retinitis, toxic epidermal necrolysis, transverse myelitis, TRAPS
(tumor necrosis factor receptor, type 1 allergic reaction, type II
diabetes, urticaria, usual interstitial pneumonia (UIP),
vasculitis, vernal conjunctivitis, viral retinitis,
Vogt-Koyanagi-Harada syndrome (VKH syndrome), wet macular
degeneration, wound healing, yersinia and salmonella associated
arthropathy.
[0492] Another embodiment of the invention provides a method for
treating a subject for a disease or a disorder in which IL-17
activity is detrimental by administering to the subject an IL-17
binding protein described herein such that treatment is achieved.
The method can be used to a treat a disorder selected from the
group consisting of respiratory disorders; asthma; allergic and
nonallergic asthma; asthma due to infection; asthma due to
infection with respiratory syncytial virus (RSV); chronic
obstructive pulmonary disease (COPD); other conditions involving
airway inflammation; eosinophilia; fibrosis and excess mucus
production; cystic fibrosis; pulmonary fibrosis; atopic disorders;
atopic dermatitis; urticaria; eczema; allergic rhinitis; and
allergic enterogastritis; inflammatory and/or autoimmune conditions
of the skin; inflammatory and/or autoimmune conditions of
gastrointestinal organs; inflammatory bowel diseases (IBD);
ulcerative colitis; Crohn's disease; inflammatory and/or autoimmune
conditions of the liver; liver cirrhosis; liver fibrosis; liver
fibrosis caused by hepatitis B and/or C virus; scleroderma; tumors
or cancers; hepatocellular carcinoma; glioblastoma; lymphoma;
Hodgkin's lymphoma; viral infections; HTLV-1 infection (e.g., from
HTLV-1); suppression of expression of protective type 1 immune
responses, and suppression of expression of protective type 1
immune responses during vaccination.
[0493] In a further embodiment of the above method, the
administering to the subject is by at least one mode selected from
parenteral, subcutaneous, intramuscular, intravenous,
intra-articular, intrabronchial, intraabdominal, intracapsular,
intracartilaginous, intracavitary, intracelial, intracerebellar,
intracerebroventricular, intracolic, intracervical, intragastric,
intrahepatic, intramyocardial, intraosteal, intrapelvic,
intrapericardiac, intraperitoneal, intrapleural, intraprostatic,
intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,
intrasynovial, intrathoracic, intrauterine, intravesical, bolus,
vaginal, rectal, buccal, sublingual, intranasal, and
transdermal.
[0494] Another aspect of the invention is a method of treating a
patient suffering from a disorder in which IL-17 is detrimental
comprising the step of administering an IL-17A binding protein
described herein before, concurrent, or after the administration of
a second agent, wherein the second agent is selected from the group
consisting of inhaled steroids; beta-agonists; short-acting or
long-acting beta-agonists; antagonists of leukotrienes or
leukotriene receptors; ADVAIR; IgE inhibitors; anti-IgE antibodies;
XOLAIR; phosphodiesterase inhibitors; PDE4 inhibitors; xanthines;
anticholinergic drugs; mast cell-stabilizing agents; Cromolyn; IL-4
inhibitors; IL-5 inhibitors; eotaxin/CCR3 inhibitors; antagonists
of histamine or its receptors including H1, H2, H3, and H4;
antagonists of prostaglandin D or its receptors DP1 and CRTH2; TNF
antagonists; a soluble fragment of a TNF receptor; ENBREL.RTM.; TNF
enzyme antagonists; TNF converting enzyme (TACE) inhibitors;
muscarinic receptor antagonists; TGF-beta antagonists; interferon
gamma; perfenidone; chemotherapeutic agents, methotrexate;
leflunomide; sirolimus (rapamycin) or an analog thereof, CCI-779;
COX2 or cPLA2 inhibitors; NSAIDs; immunomodulators; p38 inhibitors;
TPL-2, MK-2 and NFkB inhibitors; budenoside; epidermal growth
factor; corticosteroids; cyclosporine; sulfasalazine;
aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole;
lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide;
antioxidants; thromboxane inhibitors; IL-1 receptor antagonists;
anti-IL-1.beta. antibodies; anti-IL-6 antibodies; growth factors;
elastase inhibitors; pyridinyl-imidazole compounds; antibodies or
agonists of TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-9, IL-10, IL-11, IL-12, IL-14, IL-15, IL-16, IL-17, IL-18,
IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27,
IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, EMAP-II, GM-CSF, FGF, or
PDGF; antibodies of CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40,
CD45, CD69, CD90 or their ligands; FK506; rapamycin; mycophenolate
mofetil; ibuprofen; prednisolone; phosphodiesterase inhibitors;
adensosine agonists; antithrombotic agents; complement inhibitors;
adrenergic agents; IRAK, NIK, IKK, p38, or MAP kinase inhibitors;
IL-1.beta. converting enzyme inhibitors; TNF-.alpha. converting
enzyme inhibitors; T-cell signaling inhibitors; metalloproteinase
inhibitors; 6-mercaptopurines; angiotensin converting enzyme
inhibitors; soluble cytokine receptors; soluble p55 TNF receptor;
soluble p75 TNF receptor; sIL-1RI; sIL-1RII; sIL-6R;
anti-inflammatory cytokines; IL-4; IL-10; IL-11; and
TGF-.beta..
DETAILED DESCRIPTION OF THE INVENTION
[0495] This invention pertains to IL-17 binding proteins,
including, but not limited to, anti-IL-17 antibodies, or
antigen-binding portions thereof, that bind IL-17 and multivalent,
multispecific binding proteins such as DVD-Ig.TM. that bind IL-17
and another target. Various aspects of the invention relate to
antibodies and antibody fragments, DVD-Ig binding proteins, and
pharmaceutical compositions thereof, as well as nucleic acids,
recombinant expression vectors and host cells for making such IL-17
binding proteins, including antibodies, DVD-Ig binding proteins,
and fragments thereof. Methods of using the IL-17 binding proteins
of the invention to detect human IL-17A homodimer and/or IL-17A/F
heterodimer; to inhibit human IL-17A homodimer and/or IL-17A/F
heterodimer, either in vitro or in vivo; and to regulate gene
expression are also encompassed by the invention.
[0496] The invention also encompasses any binding protein or
antibody capable of competing with an IL-17 binding protein
described herein.
[0497] Unless otherwise defined herein, scientific and technical
terms used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. The meaning and scope of the terms should be clear,
however, in the event of any latent ambiguity, definitions provided
herein take precedent over any dictionary or extrinsic definition.
Further, unless otherwise required by context, singular terms shall
include pluralities and plural terms shall include the singular. In
this application, the use of "or" means "and/or" unless stated
otherwise. Furthermore, the use of the term "including", as well as
other forms, such as "includes" and "included", is not limiting.
Also, terms such as "element" or "component" encompass both
elements and components comprising one unit and elements and
components that comprise more than one subunit unless specifically
stated otherwise.
[0498] Generally, nomenclatures used in connection with, and
techniques of, cell and tissue culture, molecular biology,
immunology, microbiology, genetics and protein and nucleic acid
chemistry and hybridization described herein are those well known
and commonly used in the art. The methods and techniques of the
present invention are generally performed according to conventional
methods well known in the art and as described in various general
and more specific references that are cited and discussed
throughout the present specification unless otherwise indicated.
Enzymatic reactions and purification techniques are performed
according to manufacturer's specifications, as commonly
accomplished in the art or as described herein. The nomenclatures
used in connection with, and the laboratory procedures and
techniques of, analytical chemistry, synthetic organic chemistry,
and medicinal and pharmaceutical chemistry described herein are
those well known and commonly used in the art. Standard techniques
are used for chemical syntheses, chemical analyses, pharmaceutical
preparation, formulation, and delivery, and treatment of
patients.
[0499] That the present invention may be more readily understood,
select terms are defined below.
[0500] The term "polypeptide" as used herein, refers to any
polymeric chain of amino acids. The terms "peptide" and "protein"
are used interchangeably with the term polypeptide and also refer
to a polymeric chain of amino acids. The term "polypeptide"
encompasses native or artificial proteins, protein fragments and
polypeptide analogs of a protein sequence. A polypeptide may be
monomeric or polymeric. Use of "polypeptide" herein is intended to
encompass polypeptide and fragments and variants (including
fragments of variants) thereof, unless otherwise contradicted by
context. For an antigenic polypeptide, a fragment of polypeptide
optionally contains at least one contiguous or nonlinear epitope of
polypeptide. The precise boundaries of the at least one epitope
fragment can be confirmed using ordinary skill in the art. The
fragment comprises at least about 5 contiguous amino acids, such as
at least about 10 contiguous amino acids, at least about 15
contiguous amino acids, or at least about 20 contiguous amino
acids. A variant of polypeptide is as described herein.
[0501] The term "isolated protein" or "isolated polypeptide" is a
protein or polypeptide that by virtue of its origin or source of
derivation is not associated with naturally associated components
that accompany it in its native state; is substantially free of
other proteins from the same species; is expressed by a cell from a
different species; or does not occur in nature. Thus, a polypeptide
that is chemically synthesized or synthesized in a cellular system
different from the cell from which it naturally originates will be
"isolated" from its naturally associated components. A protein may
also be rendered substantially free of naturally associated
components by isolation, using protein purification techniques well
known in the art.
[0502] The term "recovering" as used herein, refers to the process
of rendering a chemical species such as a polypeptide substantially
free of naturally associated components by isolation, e.g., using
protein purification techniques well known in the art.
[0503] The term "human IL-17" (abbreviated herein as "hIL-17"), as
used herein, includes a dimeric cytokine protein. The term includes
a homodimeric protein comprising two 15 kD IL-17A proteins. The
homodimeric protein is referred to as an "IL-17 protein". The term
human "IL-17" is intended to include recombinant human IL-17
(rhIL-17) which can be prepared by standard recombinant expression
methods. The sequence of human IL-17A is shown in Table 1.
[0504] The term "human IL-17A/F", identical to "hIL-17A/F", as used
herein, includes the 15 kD human IL-17A and the 15 kD subunit of
the human cytokine IL-17F. The amino acid sequences of human IL-17A
and IL-17F are shown in Table 1.
TABLE-US-00002 TABLE 1 Sequence of Human IL-17 A and Human IL-17 F.
Pro- Sequence Sequence tein Identifier
12345678901234567890123456789012 Human SEQ ID
GITIPRNPGCPNSEDKNFPRTVMVNLNIHNRN IL-17A NO.: 1
TNTNPKRSSDYYNRSTSPWNLHRNEDPERYPS VIWEAKCRHLGCINADGNVDYHMNSVPIQQEI
LVLRREPPHCPNSFRLEKILVSVGCTCVTPIV HHVA Human SEQ ID
RKIPKVGHTFFQKPESCPPVPGGSMKLDIGII IL-17F NO.: 2
NENQRVSMSRNIESRSTSPWNYTVTWDPNRYP SEVVQAQCRNLGCINAQGKEDISMNSVPIQQE
TLVVRRKHQGCSVSFQLEKVLVTVGCTCVTPV IHHVQ
[0505] "Biological activity" as used herein, refers to all inherent
biological properties of the cytokine. Biological properties of
IL-17A and IL-17A/F include, but are not limited to, binding to an
IL-17 receptor.
[0506] The terms "specific binding" or "specifically binding", as
used herein, in reference to the interaction of an antibody, a
protein, or a peptide with a second chemical species, mean that the
interaction is dependent upon the presence of a particular
structure (e.g., an antigenic determinant or epitope) on the
chemical species; for example, an antibody recognizes and binds to
a specific protein structure rather than to proteins generally. If
an antibody is specific for epitope "A", the presence of a molecule
containing epitope A (or free, unlabeled A), in a reaction
containing labeled "A" and the antibody, will reduce the amount of
labeled A bound to the antibody.
[0507] The term "antibody", as used herein, broadly refers to any
immunoglobulin (Ig) molecule comprised of four polypeptide chains,
two heavy (H) chains and two light (L) chains, or any functional
fragment, mutant, variant, or derivation thereof, which retains the
essential epitope binding features of an Ig molecule. Such mutant,
variant, or derivative antibody formats are known in the art.
Nonlimiting embodiments of which are discussed below.
[0508] In a full-length antibody, each heavy chain is comprised of
a heavy chain variable region (abbreviated herein as HCVR or VH)
and a heavy chain constant region. The heavy chain constant region
is comprised of three domains: CH1, CH2, and CH3. Each light chain
is comprised of a light chain variable region (abbreviated herein
as LCVR or VL) and a light chain constant region. The light chain
constant region is comprised of one domain, CL. The VH and VL
regions can be further subdivided into regions of hypervariability,
termed complementarity determining regions (CDR), interspersed with
regions that are more conserved, termed framework regions (FR).
Each VH and VL is composed of three CDRs and four FRs, arranged
from amino-terminus to carboxy-terminus in the following order:
FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can
be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class
(e.g., IgG 1, IgG2, IgG 3, IgG4, IgA1 and IgA2) or subclass.
[0509] The term "Fc region" is used to define the C-terminal region
of an immunoglobulin heavy chain, which may be generated by papain
digestion of an intact antibody. The Fc region may be a native
sequence Fc region or a variant Fc region. The Fc region of an
immunoglobulin generally comprises two constant domains, a CH2
domain, and a CH3 domain, and optionally comprises a CH4 domain.
Replacements of amino acid residues in the Fc portion to alter
antibody effector function are known in the art (Winter et al.,
U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fc portion of an
antibody mediates several important effector functions, for
example, cytokine induction, ADCC, phagocytosis, complement
dependent cytotoxicity (CDC), and half-life/clearance rate of
antibody and antigen-antibody complexes. In some cases these
effector functions are desirable for therapeutic antibody but in
other cases might be unnecessary or even deleterious, depending on
the therapeutic objectives. Certain human IgG isotypes,
particularly IgG1 and IgG3, mediate ADCC and CDC via binding to
Fc.gamma.Rs and complement C1q, respectively. Neonatal Fc receptors
(FcRn) are the critical components determining the circulating
half-life of antibodies. In still another embodiment at least one
amino acid residue is replaced in the constant region of the
antibody, for example the Fc region of the antibody, such that
effector functions of the antibody are altered. The dimerization of
two identical heavy chains of an immunoglobulin is mediated by the
dimerization of CH3 domains and is stabilized by the disulfide
bonds within the hinge region (Huber et al. Nature, 264: 415-20;
Thies et al 1999 J. Mol. Biol., 293: 67-79.). Mutation of cysteine
residues within the hinge regions to prevent heavy chain-heavy
chain disulfide bonds will destabilize dimeration of CH3 domains.
Residues responsible for CH3 dimerization have been identified
(Dall'Acqua 1998 Biochemistry, 37: 9266-9273.). Therefore, it is
possible to generate a monovalent half-Ig. Interestingly, these
monovalent half Ig molecules have been found in nature for both IgG
and IgA subclasses (Seligman 1978 Ann. Immunol., 129: 855-70;
Biewenga et al 1983 Clin Exp Immunol 51: 395-400). The
stoichiometry of FcRn: Ig Fc region has been determined to be 2:1
(West et al. 2000 Biochemistry, 39: 9698-708), and half Fc is
sufficient for mediating FcRn binding (Kim et al 1994, Eur. J.
Immunol., 24: 542-548.). Mutations to disrupt the dimerization of
CH3 domain may not have greater adverse effect on its FcRn binding
as the residues important for CH3 dimerization are located on the
inner interface of CH3 b sheet structure, whereas the region
responsible for FcRn binding is located on the outside interface of
CH2-CH3 domains. However, the half Ig molecule may have certain
advantage in tissue penetration due to its smaller size than that
of a regular antibody. In one embodiment, at least one amino acid
residue is replaced in the constant region of the binding protein
of the invention, for example the Fc region, such that the
dimerization of the heavy chains is disrupted, resulting in half
DVD Ig molecules. The anti-inflammatory activity of IgG is
completely dependent on sialylation of the N-linked glycan of the
IgG Fc fragment. The precise glycan requirements for
anti-inflammatory activity has been determined, such that an
appropriate IgG1 Fc fragment can be created, thereby generating a
fully recombinant, sialylated IgG1 Fc with greatly enhanced potency
(Anthony, R. M., et al. (2008) Science 320:373-376).
[0510] The term "antigen-binding portion" of an antibody (or simply
"antibody portion"), as used herein, refers to one or more
fragments of an antibody that retain the ability to specifically
bind to an antigen (e.g., hIL-17). It has been shown that the
antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Such antibody embodiments may
also be bispecific, dual specific, or multi-specific formats;
specifically binding to two or more different antigens. Examples of
binding fragments encompassed within the term "antigen-binding
portion" of an antibody include (i) a Fab fragment, a monovalent
fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a
F(ab').sub.2 fragment, a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) a
Fd fragment consisting of the VH and CH1 domains; (iv) a Fv
fragment consisting of the VL and VH domains of a single arm of an
antibody, (v) a dAb fragment (Ward et al., (1989) Nature,
341:544-546, Winter et al., PCT Publication No. WO 90/05144 A1
herein incorporated by reference), which comprises a single
variable domain; and (vi) an isolated complementarity determining
region (CDR). Furthermore, although the two domains of the Fv
fragment, VL and VH, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the VL
and VH regions pair to form monovalent molecules (known as single
chain Fv (scFv); see, for example, Bird et al. (1988) Science, 242:
423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA, 85:
5879-5883). Such single chain antibodies are also intended to be
encompassed within the term "antigen-binding portion" of an
antibody. Other forms of single chain antibodies, such as diabodies
are also encompassed. Diabodies are bivalent, bispecific antibodies
in which VH and VL domains are expressed on a single polypeptide
chain, but using a linker that is too short to allow for pairing
between the two domains on the same chain, thereby forcing the
domains to pair with complementary domains of another chain and
creating two antigen binding sites (see, for example, Holliger, et
al. (1993) Proc. Natl. Acad. Sci. USA, 90: 6444-6448; Poljak et al.
(1994) Structure, 2: 1121-1123). Such antibody binding portions are
known in the art (Kontermann and Dubel eds., Antibody Engineering
(2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5)). In
addition single chain antibodies also include "linear antibodies"
comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which,
together with complementary light chain polypeptides, form a pair
of antigen binding regions (Zapata et al. Protein Eng.
8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870).
[0511] An immunoglobulin constant (C) domain refers to a heavy (CH)
or light (CL) chain constant domain. Murine and human IgG heavy
chain and light chain constant domain amino acid sequences are
known in the art.
[0512] The term "IL-17 binding protein construct" (or "binding
protein construct") as used herein refers to a polypeptide
comprising one or more of the antigen binding portions of the
invention linked to a linker polypeptide or an immunoglobulin
constant domain. Linker polypeptides comprise two or more amino
acid residues joined by peptide bonds and are used to link one or
more antigen binding portions. Such linker polypeptides are well
known in the art (see e.g., Holliger et al. (1993) Proc. Natl.
Acad. Sci. USA, 90: 6444-6448; Poljak et al., (1994) Structure, 2:
1121-1123). An immunoglobulin constant domain refers to a heavy or
light chain constant domain. Human IgG heavy chain and light chain
constant domain amino acid sequences are known in the art and
represented in Table 2.
TABLE-US-00003 TABLE 2 Sequence of Human IgG Heavy Chain Constant
Domain and Light Chain Constant Domain Sequence Sequence Protein
Identifier 12345678901234567890123456789012 Ig gamma-1 SEQ ID NO.:
3 ASTKGPSVFFLAPSSKSTSGGTAALGCLVKDY constant region
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK Ig gamma-1 SEQ ID NO.:
4 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY constant region
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS mutant
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGK Ig Kappa constant SEQ ID NO.: 5
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY region
PREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
VTKSFNRGEC Ig Lambda SEQ ID NO.: 6 QPKAAPSVTLFPPSSEELQANKATLVCLISDF
constant region YPGAVTVAWKADSSPVKAGVETTTPSKQSNNK
YAASSYLSLTPEQWKSHRSYSCQVTHEGSTVE KTVAPTECS
[0513] Still further, an IL-17 binding protein, such as an antibody
or antigen-binding portion thereof, may be part of a larger
immunoadhesion molecule, formed by covalent or noncovalent
association of the antibody or antibody portion with one or more
other proteins or peptides. Examples of such immunoadhesion
molecules include use of the streptavidin core region to make a
tetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) Human
Antibodies and Hybridomas, 6: 93-101) and use of a cysteine
residue, a marker peptide and a C-terminal polyhistidine tag to
make bivalent and biotinylated scFv molecules (Kipriyanov, S. M.,
et al. (1994) Mol. Immunol., 31: 1047-1058). Antibody portions,
such as Fab and F(ab').sub.2 fragments, can be prepared from whole
antibodies using conventional techniques, such as papain or pepsin
digestion, respectively, of whole antibodies. Moreover, antibodies,
antibody portions and immunoadhesion molecules can be obtained
using standard recombinant DNA techniques, as described herein.
[0514] An "isolated antibody", as used herein, is intended to refer
to an antibody that is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that specifically binds hIL-17 is substantially free of
antibodies that specifically bind antigens other than hIL-17). An
isolated antibody that specifically binds hIL-17 may, however, have
cross-reactivity to other antigens, such as IL-17 molecules from
other species. Moreover, an isolated antibody may be substantially
free of other cellular material and/or chemicals.
[0515] The term "monoclonal antibody" or "mAb" as used herein
refers to an antibody obtained from a population of substantially
homogeneous antibodies, i.e., the individual antibodies comprising
the population are identical except for possible naturally
occurring mutations that may be present in minor amounts.
Monoclonal antibodies are highly specific, being directed against a
single antigen. Furthermore, in contrast to polyclonal antibody
preparations that typically include different antibodies directed
against different determinants (epitopes), each mAb is directed
against a single determinant on the antigen. The modifier
"monoclonal" is not to be construed as requiring production of the
antibody by any particular method.
[0516] The term "human antibody", as used herein, is intended to
include antibodies having variable and constant regions derived
from human germline immunoglobulin sequences. The human antibodies
of the invention may include amino acid residues not encoded by
human germline immunoglobulin sequences (e.g., mutations introduced
by random or site-specific mutagenesis in vitro or by somatic
mutation in vivo), for example in the CDRs and in particular CDR3.
However, the term "human antibody", as used herein, is not intended
to include antibodies in which CDR sequences derived from the
germline of another mammalian species, such as a mouse, have been
grafted onto human framework sequences.
[0517] The term "recombinant human antibody", as used herein, is
intended to include all human antibodies that are prepared,
expressed, created or isolated by recombinant means, such as
antibodies expressed using a recombinant expression vector
transfected into a host cell (described further in Section II C,
below), antibodies isolated from a recombinant, combinatorial human
antibody library (Hoogenboom H. R., (1997) TIB Tech., 15: 62-70;
Azzazy H., and Highsmith W. E., (2002) Clin. Biochem., 35: 425-445;
Gavilondo J. V., and Larrick J. W. (2002) BioTechniques, 29:
128-145; Hoogenboom H., and Chames P. (2000) Immunology Today, 21:
371-378), antibodies isolated from an animal (e.g., a mouse) that
is transgenic for human immunoglobulin genes (see, e.g., Taylor, L.
D., et al. (1992) Nucl. Acids Res., 20: 6287-6295; Kellermann S-A.,
and Green L. L. (2002) Current Opinion in Biotechnology, 13:
593-597; Little M. et al (2000) Immunology Today, 21: 364-370) or
antibodies prepared, expressed, created or isolated by any other
means that involves splicing of human immunoglobulin gene sequences
to other DNA sequences. Such recombinant human antibodies have
variable and constant regions derived from human germline
immunoglobulin sequences. In certain embodiments, however, such
recombinant human antibodies are subjected to in vitro mutagenesis
(or, when an animal transgenic for human Ig sequences is used, in
vivo somatic mutagenesis) and thus the amino acid sequences of the
VH and VL regions of the recombinant antibodies are sequences that,
while derived from and related to human germline VH and VL
sequences, may not naturally exist within the human antibody
germline repertoire in vivo.
[0518] The term "chimeric antibody" refers to antibodies that
comprise heavy and light chain variable region sequences from one
species and constant region sequences from another species, such as
antibodies having murine heavy and light chain variable regions
linked to human constant regions.
[0519] The term "CDR-grafted antibody" refers to antibodies that
comprise heavy and light chain variable region sequences from one
species but in which the sequences of one or more of the CDR
regions of VH and/or VL are replaced with CDR sequences of another
species, such as antibodies having murine heavy and light chain
variable regions in which one or more of the murine CDRs (e.g.,
CDR3) has been replaced with human CDR sequences.
[0520] The terms "Kabat numbering", "Kabat definitions", and "Kabat
labeling" are used interchangeably herein. These terms, which are
recognized in the art, refer to a system of numbering amino acid
residues which are more variable (i.e., hypervariable) than other
amino acid residues in the heavy and light chain variable regions
of an antibody, or an antigen binding portion thereof (Kabat et al.
(1971) Ann. NY Aca., Sci., 190: 382-391 and Kabat, E. A., et al.
(1991) Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242). For the heavy chain variable region, the
hypervariable region ranges from amino acid positions 31 to 35 for
CDR1, amino acid positions 50 to 65 for CDR2, and amino acid
positions 95 to 102 for CDR3. For the light chain variable region,
the hypervariable region ranges from amino acid positions 24 to 34
for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid
positions 89 to 97 for CDR3.
[0521] As used herein, the term "CDR" refers to the complementarity
determining region within antibody variable sequences. There are
three CDRs in each of the variable regions of the heavy chain and
the light chain, which are designated CDR1, CDR2 and CDR3, for each
of the variable regions. The term "CDR set" as used herein refers
to a group of three CDRs that occur in a single variable region
capable of binding the antigen. The exact boundaries of these CDRs
have been defined differently according to different systems. The
system described by Kabat (Kabat et al., Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda,
Md. (1987) and (1991)) not only provides an unambiguous residue
numbering system applicable to any variable region of an antibody,
but also provides precise residue boundaries defining the three
CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and
coworkers (Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987) and
Chothia et al., Nature, 342: 877-883 (1989)) found that certain
sub-portions within Kabat CDRs adopt nearly identical peptide
backbone conformations, despite having great diversity at the level
of amino acid sequence. These sub-portions were designated as L1,
L2, and L3 or H1, H2, and H3 where the "L" and the "H" designates
the light chain and the heavy chains regions, respectively. These
regions may be referred to as Chothia CDRs, which have boundaries
that overlap with Kabat CDRs. Other boundaries defining CDRs
overlapping with the Kabat CDRs have been described by Padlan
(FASEB J., 9: 133-139 (1995)) and MacCallum (J. Mol. Biol., 262(5):
732-45 (1996)). Still other CDR boundary definitions may not
strictly follow one of the above systems, but will nonetheless
overlap with the Kabat CDRs, although they may be shortened or
lengthened in light of prediction or experimental findings that
particular residues or groups of residues or even entire CDRs do
not significantly impact antigen binding. The methods used herein
may utilize CDRs defined according to any of these systems,
although preferred embodiments use Kabat or Chothia defined
CDRs.
[0522] As used herein, the term "canonical" residue refers to a
residue in a CDR or framework that defines a particular canonical
CDR structure as defined by Chothia et al. (J. Mol. Biol., 196:
901-907 (1987); Chothia et al., J. Mol. Biol., 227: 799 (1992),
both are incorporated herein by reference). According to Chothia et
al., critical portions of the CDRs of many antibodies have nearly
identical peptide backbone confirmations despite great diversity at
the level of amino acid sequence. Each canonical structure
specifies primarily a set of peptide backbone torsion angles for a
contiguous segment of amino acid residues forming a loop.
[0523] An "affinity matured" antibody is an antibody with one or
more alterations in one or more CDRs thereof which result in an
improvement in the affinity of the antibody for a target antigen,
compared to a parent antibody which does not possess the
alteration(s). Exemplary affinity matured antibodies will have
nanomolar or even picomolar affinities for the target antigen. A
variety of procedures for producing affinity matured antibodies are
known in the art. For example, Marks et al., Bio/Technology, 10:
779-783 (1992) describes affinity maturation by VH and VL domain
shuffling. Random mutagenesis of CDR and/or framework residues is
described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91:
3809-3813 (1994); Schier et al., Gene, 169: 147-155 (1995); Yelton
et al., J. Immunol., 155: 1994-2004 (1995); Jackson et al., J.
Immunol., 154(7): 3310-3319 (1995); Hawkins et al, J. Mol. Biol.,
226: 889-896 (1992). Selective mutation at selective mutagenesis
positions and at contact or hypermutation positions with an
activity enhancing amino acid residue is described in U.S. Pat. No.
6,914,128B1.
[0524] The term "multivalent binding protein" denotes a binding
protein comprising two or more antigen binding sites. A multivalent
binding protein is preferably engineered to have three or more
antigen binding sites, and is generally not a naturally occurring
antibody. The term "multispecific binding protein" refers to a
binding protein capable of binding two or more related or unrelated
targets. "Dual variable domain" ("DVD") binding proteins of the
invention comprise two or more antigen binding sites and are
tetravalent or multivalent binding proteins. DVDs may be
monospecific, i.e., capable of binding one antigen, or
multispecific, i.e., capable of binding two or more antigens. A DVD
binding protein comprising two heavy chain DVD polypeptides and two
light chain DVD polypeptides is referred to as a "DVD
immunoglobulin" or "DVD-Ig". Each half of a DVD-Ig comprises a
heavy chain DVD polypeptide and a light chain DVD polypeptide, and
two or more antigen binding sites. Each binding site comprises a
heavy chain variable domain and a light chain variable domain with
a total of six CDRs involved in antigen binding per antigen binding
site.
[0525] A description of the design, expression, and
characterization of DVD-Ig molecules is provided in PCT Publication
No. WO 2007/024715, U.S. Pat. No. 7,612,181, and Wu et al., Nature
Biotech., 25: 1290-1297 (2007). A preferred example of such DVD-Ig
molecules comprises a heavy chain that comprises the structural
formula VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first heavy chain
variable domain, VD2 is a second heavy chain variable domain, C is
a heavy chain constant domain, X1 is a linker with the proviso that
it is not CH1, X2 is an Fc region, and n is 0 or 1, but preferably
1; and a light chain that comprises the structural formula
VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first light chain variable
domain, VD2 is a second light chain variable domain, C is a light
chain constant domain, X1 is a linker with the proviso that it is
not CH1, and X2 does not comprise an Fc region; and n is 0 or 1,
but preferably 1. Such a DVD-Ig may comprise two such heavy chains
and two such light chains, wherein each chain comprises variable
domains linked in tandem without an intervening constant region
between variable regions, wherein a heavy chain and a light chain
associate to form tandem functional antigen binding sites, and a
pair of heavy and light chains may associate with another pair of
heavy and light chains to form a tetrameric binding protein with
four functional antigen binding sites. In another example, a DVD-Ig
molecule may comprise heavy and light chains that each comprise
three variable domains (VD1, VD2, VD3) linked in tandem without an
intervening constant region between variable domains, wherein a
pair of heavy and light chains may associate to form three antigen
binding sites, and wherein a pair of heavy and light chains may
associate with another pair of heavy and light chains to form a
tetrameric binding protein with six antigen binding sites.
[0526] A DVD-Ig binding protein may bind one or more epitopes of
IL-17, the IL-17A monomer, the IL-17F monomer, and dimers thereof.
A DVD-Ig binding protein may also bind an epitope of IL-17 and an
epitope of a second target antigen other than an IL-17A and/or
IL-17F polypeptide.
[0527] The term "bispecific antibody", as used herein, refers to
full-length antibodies that are generated by quadroma technology
(see Milstein, C. and A. C. Cuello, Nature, 1983. 305(5934): p.
537-40), by chemical conjugation of two different monoclonal
antibodies (see Staerz, U. D., et al., Nature, 1985. 314(6012): p.
628-31), or by knob-into-hole or similar approaches which
introduces mutations in the Fc region (see Holliger et al., Proc.
Natl. Acad. Sci. USA, 1993, 90(14): 6444-6448), resulting in
multiple different immunoglobulin species of which only one is the
functional bispecific antibody. By molecular function, a bispecific
antibody binds one antigen (or epitope) on one of its two binding
arms (one pair of HC/LC), and binds a different antigen (or
epitope) on its second arm (a different pair of HC/LC). By this
definition, a bispecific antibody has two distinct antigen binding
arms (in both specificity and CDR sequences), and is monovalent for
each antigen it binds to.
[0528] The term "dual-specific antibody", as used herein, refers to
full-length antibodies that can bind two different antigens (or
epitopes) in each of its two binding arms (a pair of HC/LC) (see
PCT publication WO 02/02773). Accordingly a dual-specific binding
protein has two identical antigen binding arms, with identical
specificity and identical CDR sequences, and is bivalent for each
antigen to which it binds.
[0529] A "functional antigen binding site" of a binding protein is
one that is capable of binding a target antigen. The antigen
binding affinity of the antigen binding site is not necessarily as
strong as the parent antibody from which the antigen binding site
is derived, but the ability to bind antigen must be measurable
using any one of a variety of methods known for evaluating antibody
binding to an antigen. Moreover, the antigen binding affinity of
each of the antigen binding sites of a multivalent antibody herein
need not be quantitatively the same.
[0530] The term "cytokine" is a generic term for proteins that are
released by one cell population and that act on another cell
population as intercellular mediators. Examples of such cytokines
are lymphokines, monokines, and traditional polypeptide hormones.
Included among the cytokines are growth hormones, such as human
growth hormone, N-methionyl human growth hormone, and bovine growth
hormone; parathyroid hormone; thyroxine; insulin; proinsulin;
relaxin; prorelaxin; glycoprotein hormones, such as follicle
stimulating hormone (FSH), thyroid stimulating hormone (TSH), and
luteinizing hormone (LH); hepatic growth factor; fibroblast growth
factor; prolactin; placental lactogen; a tumor necrosis factor such
as tumor necrosis factor-alpha (TNF-.alpha.) and tumor necrosis
factor-beta (TNF-.beta.); mullerian-inhibiting substance; mouse
gonadotropin-associated peptide; inhibin; activin; vascular
endothelial growth factor; integrin; thrombopoietin (TPO); nerve
growth factors such as NGF-alpha (NGF-.alpha.); platelet-growth
factor; placental growth factor; transforming growth factors (TGFs)
such as TGF-alpha (TGF-.alpha.) and TGF-beta (TGF-.beta.);
insulin-like growth factor-1 and -11; erythropoietin (EPO);
osteoinductive factors; interferons such as interferon-alpha
(IFN-.alpha.), interferon-beta (IFN-.beta.), and interferon-gamma
(IFN-.gamma.); colony stimulating factors (CSFs) such as
macrophage-CSF (M-CSF); granulocyte macrophage-CSF (GM-CSF); and
granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,
IL-13, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-33; and other
polypeptide factors including LIF and kit ligand (KL). As used
herein, the term cytokine includes proteins from natural sources or
from recombinant cell culture and biologically active equivalents
of the native sequence cytokines.
[0531] As used herein, the terms "donor" and "donor antibody" refer
to an antibody providing one or more CDRs. In a preferred
embodiment, the donor antibody is an antibody from a species
different from the antibody from which the framework regions are
obtained or derived. In the context of a humanized antibody, the
term "donor antibody" refers to a non-human antibody providing one
or more CDRs.
[0532] As used herein, the term "framework" or "framework sequence"
refers to the remaining sequences of a variable region minus the
CDRs. Because the exact definition of a CDR sequence can be
determined by different systems, the meaning of a framework
sequence is subject to correspondingly different interpretations.
The six CDRs (CDR-L1, -L2, and -L3 of light chain and CDR-H1, -H2,
and -H3 of heavy chain) also divide the framework regions on the
light chain and the heavy chain into four sub-regions (FR1, FR2,
FR3 and FR4) on each chain, in which CDR1 is positioned between FR1
and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4.
Without specifying the particular sub-regions as FR1, FR2, FR3 or
FR4, a framework region, as referred by others, represents the
combined FR's within the variable region of a single, naturally
occurring immunoglobulin chain. As used herein, a FR represents one
of the four sub-regions, and FRs represents two or more of the four
sub-regions constituting a framework region.
[0533] As used herein, the terms "acceptor" and "acceptor antibody"
refer to the antibody providing or nucleic acid sequence encoding
at least 80%, at least 85%, at least 90%, at least 95%, at least
98%, or 100% of the amino acid sequences of one or more of the
framework regions. In some embodiments, the term "acceptor" refers
to the antibody amino acid providing or nucleic acid sequence
encoding the constant region(s). In yet another embodiment, the
term "acceptor" refers to the antibody amino acid providing or
nucleic acid sequence encoding one or more of the framework regions
and the constant region(s). In a specific embodiment, the term
"acceptor" refers to a human antibody amino acid or nucleic acid
sequence that provides or encodes at least 80%, preferably, at
least 85%, at least 90%, at least 95%, at least 98%, or 100% of the
amino acid sequences of one or more of the framework regions. In
accordance with this embodiment, an acceptor may contain at least
1, at least 2, at least 3, least 4, at least 5, or at least 10
amino acid residues that does (do) not occur at one or more
specific positions of a human antibody. An acceptor framework
region and/or acceptor constant region(s) may be, e.g., derived or
obtained from a germline antibody gene, a mature antibody gene, a
functional antibody (e.g., antibodies well known in the art,
antibodies in development, or antibodies commercially
available).
[0534] Human heavy chain and light chain acceptor sequences are
known in the art. In one embodiment of the invention the human
heavy chain and light chain acceptor sequences are selected from
the sequences listed from V-base (http://vbase.mrc-cpe.cam.ac.uk/)
or from IMGT.RTM., the international ImMunoGeneTics information
System.RTM.(http://imgt.cines.fr/textes/IMGTrepertoire/LocusGenes/).
In another embodiment of the invention the human heavy chain and
light chain acceptor sequences are selected from the sequences
described in Table 3 and Table 4.
TABLE-US-00004 TABLE 3 Heavy Chain Acceptor Sequences SEQ Protein
Sequence ID No. region 12345678901234567890123456789012 7 VH1-69
FR1 QVQLVQSGAEVKKPGSSVKVSCKASGGTFS 8 VH1-69 FR2 WVRQAPGQGLEWMG 9
VH1-69 FR3 RVTITADKSTSTAYMELSSLRSEDTAVYYCAR 10 JH1/JH4/JH5
WGQGTLVTVSS FR4 11 JH3 FR4 WGQGTMVTVSS 12 JH6 FR4 WGQGTTVTVSS
TABLE-US-00005 TABLE 4 Light Chain Acceptor Sequences SEQ ID
Sequence No. Protein region 12345678901234567890123456789012 13
1-17/A30 FR1 DIQMTQSPSSLSASVGDRVTITC 14 1-17/A30 FR2
WYQQKPGKAPKRLIY 15 1-17/A30 FR3 GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC 16
6-21/A26 FR1 EIVLTQSPDFQSVTPKEKVTITC 17 6-21/A26 FR2
WYQQKPDQSPKLLIK 18 6-21/A26 FR3 GVPSRFSGSGSGTDFTLTINSLEAEDAATYYC 13
1-33/O18 FR1 DIQMTQSPSSLSASVGDRVTITC 19 1-33/O18 FR2
WYQQKPGKAPKLLIY 20 1-33/O18 FR3 GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC 21
3-15/L2 FR1 EIVMTQSPATLSVSPGERATLSC 22 3-15/L2 FR2 WYQQKPGQAPRLLIY
23 3-15/L2 FR3 GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC 24 JK2 FR4
FGQGTKLEIKR 25 JK4 FR4 FGGGTKVEIKR
[0535] As used herein, the term "germline antibody gene" or "gene
fragment" refers to an immunoglobulin sequence encoded by
non-lymphoid cells that have not undergone the maturation process
that leads to genetic rearrangement and mutation for expression of
a particular immunoglobulin. (See, e.g., Shapiro et al., Crit. Rev.
Immunol., 22(3): 183-200 (2002); Marchalonis et al., Adv. Exp. Med.
Biol., 484: 13-30 (2001)). One of the advantages provided by
various embodiments of the present invention stems from the
recognition that germline antibody genes are more likely than
mature antibody genes to conserve essential amino acid sequence
structures characteristic of individuals in the species, hence less
likely to be recognized as from a foreign source when used
therapeutically in that species.
[0536] As used herein, the term "key" residues refer to certain
residues within the variable region that have more impact on the
binding specificity and/or affinity of an antibody, in particular a
humanized antibody. A key residue includes, but is not limited to,
one or more of the following: a residue that is adjacent to a CDR,
a potential glycosylation site (can be either N- or O-glycosylation
site), a rare residue, a residue capable of interacting with the
antigen, a residue capable of interacting with a CDR, a canonical
residue, a contact residue between heavy chain variable region and
light chain variable region, a residue within the Vernier zone, and
a residue in the region that overlaps between the Chothia
definition of a variable heavy chain CDR1 and the Kabat definition
of the first heavy chain framework.
[0537] The term "humanized antibody" refers to antibodies that
comprise heavy and light chain variable region sequences from a
non-human species (e.g., a mouse) but in which at least a portion
of the VH and/or VL sequence has been altered to be more
"human-like", i.e., more similar to human germline variable
sequences. One type of humanized antibody is a CDR-grafted
antibody, in which human CDR sequences are introduced into
non-human VH and VL sequences to replace the corresponding nonhuman
CDR sequences. Also "humanized antibody" is an antibody or a
variant, derivative, analog or fragment thereof which
immunospecifically binds to an antigen of interest and which
comprises a framework (FR) region having substantially the amino
acid sequence of a human antibody and a complementary determining
region (CDR) having substantially the amino acid sequence of a
non-human antibody. As used herein, the term "substantially" in the
context of a CDR refers to a CDR having an amino acid sequence at
least 80%, at least 85%, at least 90%, at least 95%, at least 98%
or at least 99% identical to the amino acid sequence of a non-human
antibody CDR. A humanized antibody comprises substantially all of
at least one, and typically two, variable domains (Fab, Fab',
F(ab')2, FabC, Fv) in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin (i.e.,
donor antibody) and all or substantially all of the framework
regions are those of a human immunoglobulin consensus sequence. In
an embodiment, a humanized antibody also comprises at least a
portion of an immunoglobulin constant region (Fc), typically that
of a human immunoglobulin. In some embodiments, a humanized
antibody contains both the light chain as well as at least the
variable domain of a heavy chain. The antibody also may include the
CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some
embodiments, a humanized antibody only contains a humanized light
chain. In some embodiments, a humanized antibody only contains a
humanized heavy chain. In specific embodiments, a humanized
antibody only contains a humanized variable domain of a light chain
and/or humanized heavy chain.
[0538] A humanized antibody may be selected from any class of
immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any
isotype including without limitation IgG1, IgG2, IgG3, and IgG4.
The humanized antibody may comprise sequences from more than one
class or isotype, and particular constant domains may be selected
to optimize desired effector functions using techniques well known
in the art.
[0539] The framework and CDR regions of a humanized antibody need
not correspond precisely to the parental sequences, e.g., the donor
antibody CDR or the consensus framework may be mutagenized by
substitution, insertion and/or deletion of at least one amino acid
residue so that the CDR or framework residue at that site does not
correspond to either the donor antibody or the consensus framework.
In a preferred embodiment, such mutations, however, will not be
extensive. Usually, at least 80%, preferably at least 85%, more
preferably at least 90%, and most preferably at least 95% of the
humanized antibody residues will correspond to those of the
parental FR and CDR sequences. As used herein, the term "consensus
framework" refers to the framework region in the consensus
immunoglobulin sequence. As used herein, the term "consensus
immunoglobulin sequence" refers to the sequence formed from the
most frequently occurring amino acids (or nucleotides) in a family
of related immunoglobulin sequences (see e.g., Winnaker, From Genes
to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a
family of immunoglobulins, each position in the consensus sequence
is occupied by the amino acid occurring most frequently at that
position in the family. If two amino acids occur equally
frequently, either can be included in the consensus sequence.
[0540] With respect to constructing DVD-Ig or other binding protein
molecules, a "linker" is used to denote polypeptides comprising two
or more amino acid residues joined by peptide bonds and are used to
link one or more antigen binding portions. Such linker polypeptides
are well known in the art (see, e.g., Holliger et al. (1993) Proc.
Natl. Acad. Sci. USA, 90: 6444-6448; Poljak et al. (1994)
Structure, 2: 1121-1123). Exemplary linkers include, but are not
limited to, GGGGSG (SEQ ID NO:887), GGSGG (SEQ ID NO:888),
GGGGSGGGGS (SEQ ID NO:889), GGSGGGGSGS (SEQ ID NO:890),
GGSGGGGSGGGGS (SEQ ID NO:891), GGGGSGGGGSGGGG (SEQ ID NO:892),
GGGGSGGGGSGGGGS (SEQ ID NO:893), ASTKGP (SEQ ID NO:894),
ASTKGPSVFPLAP (SEQ ID NO:895), TVAAP (SEQ ID NO:896), TVAAPSVFIFPP
(SEQ ID NO:897), AKTTPKLEEGEFSEAR (SEQ ID NO:898),
AKTTPKLEEGEFSEARV (SEQ ID NO:899), AKTTPKLGG (SEQ ID NO:900),
SAKTTPKLGG (SEQ ID NO:901), SAKTTP (SEQ ID NO:902), RADAAP (SEQ ID
NO:903), RADAAPTVS (SEQ ID NO:904), RADAAAAGGPGS (SEQ ID NO:905),
RADAAAAGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:906), SAKTTPKLEEGEFSEARV
(SEQ ID NO:907), ADAAP (SEQ ID NO:908), ADAAPTVSIFPP (SEQ ID
NO:909), QPKAAP (SEQ ID NO:910), QPKAAPSVTLFPP (SEQ ID NO:911),
AKTTPP (SEQ ID NO:912), AKTTPPSVTPLAP (SEQ ID NO:913), AKTTAP (SEQ
ID NO:914), AKTTAPSVYPLAP (SEQ ID NO:915), GENKVEYAPALMALS (SEQ ID
NO:916), GPAKELTPLKEAKVS (SEQ ID NO:917), and GHEAAAVMQVQYPAS (SEQ
ID NO:918).
[0541] As used herein, "Vernier" zone refers to a subset of
framework residues that may adjust CDR structure and fine-tune the
fit to antigen as described by Foote and Winter (1992, J. Mol.
Biol. 224:487-499, which is incorporated herein by reference).
Vernier zone residues form a layer underlying the CDRs and may
impact on the structure of CDRs and the affinity of the
antibody.
[0542] As used herein, the term "neutralizing" refers to
neutralization of the biological activity of an antigen (e.g., the
cytokine IL-17) when a binding protein specifically binds the
antigen. Preferably, a neutralizing binding protein described
herein binds to hIL-17 and/or hIL-17A/F resulting in the inhibition
of a biological activity of hIL-17 and/or hIL-17A/F. Preferably,
the neutralizing binding protein binds hIL-17 and/or hIL-17A/F and
reduces a biologically activity of hIL-17 and/or hIL-17A/F by at
least about 20%, 40%, 60%, 80%, 85%, or more Inhibition of a
biological activity of hIL-17 and/or hIL-17A/F by a neutralizing
binding protein can be assessed by measuring one or more indicators
of hIL-17 and/or hIL-17A/F biological activity well known in the
art. For example inhibition of human IL-6 secretion by IL-17
induction in HS27 cells.
[0543] The term "activity" includes activities such as the binding
specificity/affinity of an antibody for an antigen, for example, an
anti-hIL-17 antibody that binds to an IL-17 antigen and/or the
neutralizing potency of an antibody, for example, an anti-hIL-17
antibody whose binding to hIL-17 inhibits the biological activity
of hIL-17, for example, inhibition of human IL-6 secretion by IL-17
induction in HS27 cells.
[0544] The term "epitope" includes any polypeptide determinant
capable of specific binding to an immunoglobulin or T-cell
receptor. In certain embodiments, epitope determinants include
chemically active surface groupings of molecules such as amino
acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain
embodiments, may have specific three dimensional structural
characteristics, and/or specific charge characteristics. An epitope
is a region of an antigen that is bound by an antibody. In certain
embodiments, an antibody is said to specifically bind an antigen
when it preferentially recognizes its target antigen in a complex
mixture of proteins and/or macromolecules. Antibodies are said to
"bind to the same epitope" if the antibodies cross-compete (one
prevents the binding or modulating effect of the other). In
addition, structural definitions of epitopes (overlapping, similar,
identical) are informative, but functional definitions are often
more relevant as they encompass structural (binding) and functional
(modulation, competition) parameters.
[0545] The term "surface plasmon resonance", as used herein, refers
to an optical phenomenon that allows for the analysis of real-time
biospecific interactions by detection of alterations in protein
concentrations within a biosensor matrix, for example using the
BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and
Piscataway, N.J.). For further descriptions, see Jonsson, U., et
al. (1993) Ann. Biol. Clin., 51: 19-26; Jonsson et al., (1991)
BioTechniques, 11: 620-627; Johnsson et al., (1995) J. Mol.
Recognit., 8: 125-131; and Johnnson et al. (1991) Anal. Biochem.,
198: 268-277.
[0546] The term "K.sub.on" (also "Kon", "kon"), as used herein, is
intended to refer to the on rate constant for association of a
binding protein (e.g., an antibody) to an antigen to form an
association complex, e.g., antibody/antigen complex, as is known in
the art. The "Kon" also is known by the terms "association rate
constant", or "ka", as used interchangeably herein. This value
indicates the binding rate of an antibody to its target antigen or
the rate of complex formation between an antibody and antigen as is
shown by the equation below:
Antibody ("Ab")+Antigen ("Ag").fwdarw.Ab-Ag.
[0547] The term "K.sub.off" (also "Koff", "koff"), as used herein,
is intended to refer to the off rate constant for dissociation, or
"dissociation rate constant", of a binding protein (e.g., an
antibody) from an association complex (e.g., an antibody/antigen
complex) as is known in the art. This value indicates the
dissociation rate of an antibody from its target antigen or
separation of Ab-Ag complex over time into free antibody and
antigen as shown by the equation below:
Ab+Ag.rarw.Ab-Ag.
[0548] The term "K.sub.D" (also "K.sub.d"), as used herein, is
intended to refer to the "equilibrium dissociation constant", and
refers to the value obtained in a titration measurement at
equilibrium, or by dividing the dissociation rate constant (Koff)
by the association rate constant (Kon). The association rate
constant (Kon), the dissociation rate constant (Koff), and the
equilibrium dissociation constant (K are used to represent the
binding affinity of an antibody to an antigen. Methods for
determining association and dissociation rate constants are well
known in the art. Using fluorescence-based techniques offers high
sensitivity and the ability to examine samples in physiological
buffers at equilibrium. Other experimental approaches and
instruments such as a BIAcore.RTM. (biomolecular interaction
analysis) assay can be used (e.g., instrument available from
BIAcore International AB, a GE Healthcare company, Uppsala,
Sweden). Additionally, a KinExA.RTM. (Kinetic Exclusion Assay)
assay, available from Sapidyne Instruments (Boise, Id.) can also be
used.
[0549] The terms "label" and "detectable label" mean a moiety
attached to a specific binding partner, such as an antibody or an
analyte, e.g., to render the reaction between members of a specific
binding pair, such as an antibody and an analyte, detectable. The
specific binding partner, e.g., antibody or analyte, so labeled is
referred to as "detectably labeled". Thus, the term "labeled
binding protein" as used herein, refers to a protein with a label
incorporated that provides for the identification of the binding
protein. In an embodiment, the label is a detectable marker that
can produce a signal that is detectable by visual or instrumental
means, e.g., incorporation of a radiolabeled amino acid or
attachment to a polypeptide of biotinyl moieties that can be
detected by marked avidin or streptavidin (e.g., streptavidin
containing a fluorescent marker or enzymatic activity that can be
detected by optical or colorimetric methods). Examples of labels
for polypeptides include, but are not limited to, the following:
radioisotopes or radionuclides (e.g., .sup.3H, .sup.14C, .sup.35S,
.sup.90Y, .sup.99Tc, .sup.111In, .sup.125I, .sup.131I, .sup.177Lu,
.sup.166Ho, or .sup.153Sm), chromogens, fluorescent labels (e.g.,
FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g.,
horseradish peroxidase, luciferase, alkaline phosphatase),
chemiluminescent markers, biotinyl groups, predetermined
polypeptide epitopes recognized by a secondary reporter (e.g.,
leucine zipper pair sequences, binding sites for secondary
antibodies, metal binding domains, epitope tags), and magnetic
agents (e.g., gadolinium chelates). Representative examples of
labels commonly employed for immunoassays include moieties that
produce light, e.g., acridinium compounds, and moieties that
produce fluorescence, e.g., fluorescein. Other labels are described
herein. In this regard, the moiety itself may not be detectably
labeled but may become detectable upon reaction with yet another
moiety. Use of the term "detectably labeled" is intended to
encompass the latter type of detectable labeling.
[0550] The term "IL-17 binding protein conjugate" refers to an
IL-17 binding protein described herein chemically linked to a
second chemical moiety, such as a therapeutic or cytotoxic agent.
The term "agent" is used herein to denote a chemical compound, a
mixture of chemical compounds, a biological macromolecule, or an
extract made from biological materials. Preferably the therapeutic
or cytotoxic agents include, but are not limited to, pertussis
toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide,
emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. When employed in the context of an immunoassay, an IL-17
binding protein conjugate may be a detectably labeled antibody,
which is used as the detection antibody.
[0551] The terms "crystal" and "crystallized" as used herein, refer
to a binding protein (e.g., an antibody), or antigen binding
portion thereof, that exists in the form of a crystal. Crystals are
one form of the solid state of matter that is distinct from other
forms such as the amorphous solid state or the liquid crystalline
state. Crystals are composed of regular, repeating,
three-dimensional arrays of atoms, ions, molecules (e.g., proteins
such as antibodies), or molecular assemblies (e.g.,
antigen/antibody complexes). These three-dimensional arrays are
arranged according to specific mathematical relationships that are
well-understood in the field. The fundamental unit, or building
block, that is repeated in a crystal is called the asymmetric unit.
Repetition of the asymmetric unit in an arrangement that conforms
to a given, well-defined crystallographic symmetry provides the
"unit cell" of the crystal. Repetition of the unit cell by regular
translations in all three dimensions provides the crystal. See
Giege et al., Chapter 1, In Crystallization of Nucleic Acids and
Proteins, a Practical Approach, 2nd ed., (Ducruix and Giege, eds.)
(Oxford University Press, New York, 1999) pp. 1-16.
[0552] The term "polynucleotide" means a polymeric form of two or
more nucleotides, either ribonucleotides or deoxynucleotides or a
modified form of either type of nucleotide. The term includes
single and double stranded forms of DNA.
[0553] The term "isolated polynucleotide" shall mean a
polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or
some combination thereof) that, by virtue of its origin, the
"isolated polynucleotide" is not associated with all or a portion
of a polynucleotide with which the "isolated polynucleotide" is
found in nature; is operably linked to a polynucleotide that it is
not linked to in nature; or does not occur in nature as part of a
larger sequence.
[0554] The term "vector", as used herein, is intended to refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid",
which refers to a circular double stranded DNA loop into which
additional DNA segments may be ligated. Another type of vector is a
viral vector, wherein additional DNA segments may be ligated into
the viral genome. Certain vectors are capable of autonomous
replication in a host cell into which they are introduced (e.g.,
bacterial vectors having a bacterial origin of replication and
episomal mammalian vectors). Other vectors (e.g., non-episomal
mammalian vectors) can be integrated into the genome of a host cell
upon introduction into the host cell, and thereby are replicated
along with the host genome. Moreover, certain vectors are capable
of directing the expression of genes to which they are operatively
linked. Such vectors are referred to herein as "recombinant
expression vectors" (or simply, "expression vectors"). In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of plasmids. In the present specification,
"plasmid" and "vector" may be used interchangeably as the plasmid
is the most commonly used form of vector. However, the invention is
intended to include such other forms of expression vectors, such as
viral vectors (e.g., replication defective retroviruses,
adenoviruses and adeno-associated viruses), which serve equivalent
functions.
[0555] The term "operably linked" refers to a juxtaposition wherein
the components described are in a relationship permitting them to
function in their intended manner. A control sequence "operably
linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under conditions
compatible with the control sequences. "Operably linked" sequences
include both expression control sequences that are contiguous with
the gene of interest and expression control sequences that act in
trans or at a distance to control the gene of interest. The term
"expression control sequence" as used herein refers to
polynucleotide sequences that are necessary to effect the
expression and processing of coding sequences to which they are
ligated. Expression control sequences include appropriate
transcription initiation, termination, promoter and enhancer
sequences; efficient RNA processing signals such as splicing and
polyadenylation signals; sequences that stabilize cytoplasmic mRNA;
sequences that enhance translation efficiency (i.e., Kozak
consensus sequence); sequences that enhance protein stability; and
when desired, sequences that enhance protein secretion. The nature
of such control sequences differs depending upon the host organism;
in prokaryotes, such control sequences generally include promoter,
ribosomal binding site, and transcription termination sequence; in
eukaryotes, generally, such control sequences include promoters and
transcription termination sequence. The term "control sequences" is
intended to include components whose presence is essential for
expression and processing, and can also include additional
components whose presence is advantageous, for example, leader
sequences and fusion partner sequences.
[0556] "Transformation", as defined herein, refers to any process
by which exogenous DNA enters a host cell. Transformation may occur
under natural or artificial conditions using various methods well
known in the art. Transformation may rely on any known method for
the insertion of foreign nucleic acid sequences into a prokaryotic
or eukaryotic host cell. The method is selected based on the host
cell being transformed and may include, but is not limited to,
viral infection, electroporation, lipofection, and particle
bombardment. Such "transformed" cells include stably transformed
cells in which the inserted DNA is capable of replication either as
an autonomously replicating plasmid or as part of the host
chromosome. They also include cells which transiently express the
inserted DNA or RNA for limited periods of time.
[0557] The term "recombinant host cell" (or simply "host cell"), is
intended to refer to a cell into which exogenous DNA has been
introduced. In an embodiment, the host cell comprises two or more
(e.g., multiple) nucleic acids encoding antibodies, such as the
host cells described in U.S. Pat. No. 7,262,028, for example. Such
terms are intended to refer not only to the particular subject
cell, but also to the progeny of such a cell. Because certain
modifications may occur in succeeding generations due to either
mutation or environmental influences, such progeny may not, in
fact, be identical to the parent cell, but are still included
within the scope of the term "host cell" as used herein. In an
embodiment, host cells include prokaryotic and eukaryotic cells
selected from any of the Kingdoms of life. In another embodiment,
eukaryotic cells include protist, fungal, plant and animal cells.
In another embodiment, host cells include but are not limited to
the prokaryotic cell line Escherichia coli; mammalian cell lines
CHO, HEK 293, COS, NS0, SP2 and PER.C6; the insect cell line Sf9;
and the fungal cell Saccharomyces cerevisiae.
[0558] Standard techniques may be used for recombinant DNA,
oligonucleotide synthesis, and tissue culture and transformation
(e.g., electroporation, lipofection). Enzymatic reactions and
purification techniques may be performed according to
manufacturer's specifications or as commonly accomplished in the
art or as described herein. The foregoing techniques and procedures
may be generally performed according to conventional methods well
known in the art and as described in various general and more
specific references that are cited and discussed throughout the
present specification. See e.g., Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989).
[0559] "Transgenic organism", as known in the art, refers to an
organism having cells that contain a transgene, wherein the
transgene introduced into the organism (or an ancestor of the
organism) expresses a polypeptide not naturally expressed in the
organism. A "transgene" is a DNA construct, which is stably and
operably integrated into the genome of a cell from which a
transgenic organism develops, directing the expression of an
encoded gene product in one or more cell types or tissues of the
transgenic organism.
[0560] The terms "regulate" and "modulate" are used
interchangeably, and, as used herein, refers to a change or an
alteration in the activity of a molecule of interest (e.g., the
biological activity of hIL-17). Modulation may be an increase or a
decrease in the magnitude of a certain activity or function of the
molecule of interest. Exemplary activities and functions of a
molecule include, but are not limited to, binding characteristics,
enzymatic activity, cell receptor activation, and signal
transduction.
[0561] Correspondingly, the term "modulator," as used herein, is a
compound capable of changing or altering an activity or function of
a molecule of interest (e.g., the biological activity of hIL-17).
For example, a modulator may cause an increase or decrease in the
magnitude of a certain activity or function of a molecule compared
to the magnitude of the activity or function observed in the
absence of the modulator. In certain embodiments, a modulator is an
inhibitor, which decreases the magnitude of at least one activity
or function of a molecule. Exemplary inhibitors include, but are
not limited to, proteins, peptides, antibodies, peptibodies,
carbohydrates or small organic molecules. Peptibodies are
described, e.g., in WO01/83525.
[0562] The term "agonist", as used herein, refers to a modulator
that, when contacted with a molecule of interest, causes an
increase in the magnitude of a certain activity or function of the
molecule compared to the magnitude of the activity or function
observed in the absence of the agonist. Particular agonists of
interest may include, but are not limited to, IL-17 polypeptides,
nucleic acids, carbohydrates, or any other molecule that binds to
hIL-17.
[0563] The terms "antagonist" and "inhibitor", as used herein,
refer to a modulator that, when contacted with a molecule of
interest causes a decrease in the magnitude of a certain activity
or function of the molecule compared to the magnitude of the
activity or function observed in the absence of the antagonist.
Particular antagonists of interest include those that block or
modulate the biological or immunological activity of hIL-17A and/or
hIL-17A/F. Antagonists and inhibitors of hIL-17A and/or hIL-17A/F
may include, but are not limited to, proteins, nucleic acids,
carbohydrates, or any other molecules, which bind to hIL-17A and/or
hIL-17A/F.
[0564] As used herein, the term "effective amount" refers to the
amount of a therapy that is sufficient to reduce or ameliorate the
severity and/or duration of a disorder or one or more symptoms
thereof; prevent the advancement of a disorder; cause regression of
a disorder; prevent the recurrence, development, onset, or
progression of one or more symptoms associated with a disorder;
detect a disorder; or enhance or improve the prophylactic or
therapeutic effect(s) of another therapy (e.g., prophylactic or
therapeutic agent).
[0565] "Patient" and "subject" may be used interchangeably herein
to refer to an animal, such as a mammal, including a primate (for
example, a human, a monkey, and a chimpanzee), a non-primate (for
example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit,
a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a
whale), a bird (e.g., a duck or a goose), and a shark. Preferably,
a patient or subject is a human, such as a human being treated or
assessed for a disease, disorder or condition, a human at risk for
a disease, disorder or condition, a human having a disease,
disorder or condition, and/or human being treated for a disease,
disorder or condition.
[0566] The term "sample", as used herein, is used in its broadest
sense. A "biological sample", as used herein, includes, but is not
limited to, any quantity of a substance from a living thing or
formerly living thing. Such living things include, but are not
limited to, humans, non-human primates, mice, rats, monkeys, dogs,
rabbits and other animals. Such substances include, but are not
limited to, blood (e.g., whole blood), plasma, serum, urine,
amniotic fluid, synovial fluid, endothelial cells, leukocytes,
monocytes, other cells, organs, tissues, bone marrow, lymph nodes
and spleen.
[0567] "Component", "components," and "at least one component,"
refer generally to a capture antibody, a detection or conjugate
antibody, a control, a calibrator, a series of calibrators, a
sensitivity panel, a container, a buffer, a diluent, a salt, an
enzyme, a co-factor for an enzyme, a detection reagent, a
pretreatment reagent/solution, a substrate (e.g., as a solution), a
stop solution, and the like that can be included in a kit for assay
of a test sample, such as a patient urine, serum or plasma sample,
in accordance with the methods described herein and other methods
known in the art. Thus, in the context of the present disclosure,
"at least one component," "component," and "components" can include
a polypeptide or other analyte as above, such as a composition
comprising an analyte such as polypeptide, which is optionally
immobilized on a solid support, such as by binding to an
anti-analyte (e.g., anti-polypeptide) antibody. Some components can
be in solution or lyophilized for reconstitution for use in an
assay.
[0568] "Control" refers to a composition known to not analyte
("negative control") or to contain analyte ("positive control"). A
positive control can comprise a known concentration of analyte.
"Control," "positive control," and "calibrator" may be used
interchangeably herein to refer to a composition comprising a known
concentration of analyte. A "positive control" can be used to
establish assay performance characteristics and is a useful
indicator of the integrity of reagents (e.g., analytes).
[0569] "Predetermined cutoff" and "predetermined level" refer
generally to an assay cutoff value that is used to assess
diagnostic/prognostic/therapeutic efficacy results by comparing the
assay results against the predetermined cutoff/level, where the
predetermined cutoff/level already has been linked or associated
with various clinical parameters (e.g., severity of disease,
progression/nonprogression/improvement, etc.). While the present
disclosure may provide exemplary predetermined levels, it is
well-known that cutoff values may vary depending on the nature of
the immunoassay (e.g., antibodies employed, etc.). It further is
well within the ordinary skill of one in the art to adapt the
disclosure herein for other immunoassays to obtain
immunoassay-specific cutoff values for those other immunoassays
based on this disclosure. Whereas the precise value of the
predetermined cutoff/level may vary between assays, correlations as
described herein (if any) should be generally applicable.
[0570] "Pretreatment reagent," e.g., lysis, precipitation and/or
solubilization reagent, as used in a diagnostic assay as described
herein is one that lyses any cells and/or solubilizes any analyte
that is/are present in a test sample. Pretreatment is not necessary
for all samples, as described further herein. Among other things,
solubilizing the analyte (e.g., polypeptide of interest) may entail
release of the analyte from any endogenous binding proteins present
in the sample. A pretreatment reagent may be homogeneous (not
requiring a separation step) or heterogeneous (requiring a
separation step). With use of a heterogeneous pretreatment reagent
there is removal of any precipitated analyte binding proteins from
the test sample prior to proceeding to the next step of the
assay.
[0571] "Quality control reagents" in the context of immunoassays
and kits described herein, include, but are not limited to,
calibrators, controls, and sensitivity panels. A "calibrator" or
"standard" typically is used (e.g., one or more, such as a
plurality) in order to establish calibration (standard) curves for
interpolation of the concentration of an analyte, such as an
antibody or an analyte. Alternatively, a single calibrator, which
is near a predetermined positive/negative cutoff, can be used.
Multiple calibrators (i.e., more than one calibrator or a varying
amount of calibrator(s)) can be used in conjunction so as to
comprise a "sensitivity panel."
[0572] "Risk" refers to the possibility or probability of a
particular event occurring either presently or at some point in the
future. "Risk stratification" refers to an array of known clinical
risk factors that allows physicians to classify patients into a
low, moderate, high or highest risk of developing a particular
disease, disorder or condition.
[0573] "Specific" and "specificity" in the context of an
interaction between members of a specific binding pair (e.g., an
antigen (or fragment thereof) and an antibody (or antigenically
reactive fragment thereof)) refer to the selective reactivity of
the interaction. The phrase "specifically binds to" and analogous
phrases refer to the ability of antibodies (or antigenically
reactive fragments thereof) to bind specifically to analyte (or a
fragment thereof) and not bind specifically to other entities.
[0574] "Specific binding partner" is a member of a specific binding
pair. A specific binding pair comprises two different molecules,
which specifically bind to each other through chemical or physical
means. Therefore, in addition to antigen and antibody specific
binding pairs of common immunoassays, other specific binding pairs
can include biotin and avidin (or streptavidin), carbohydrates and
lectins, complementary nucleotide sequences, effector and receptor
molecules, cofactors and enzymes, enzyme inhibitors and enzymes,
and the like. Furthermore, specific binding pairs can include
members that are analogs of the original specific binding members,
for example, an analyte-analog Immunoreactive specific binding
members include antigens, antigen fragments, and antibodies,
including monoclonal and polyclonal antibodies as well as
complexes, fragments, and variants (including fragments of
variants) thereof, whether isolated or recombinantly produced.
[0575] "Variant" as used herein means a polypeptide that differs
from a given polypeptide (e.g., IL-17, BNP, NGAL, or HIV
polypeptide, or anti-polypeptide antibody) in amino acid sequence
by the addition (e.g., insertion), deletion, or conservative
substitution of amino acids, but that retains the biological
activity of the given polypeptide (e.g., a variant IL-17 can
compete with anti-IL-17 antibody for binding to IL-17). A
conservative substitution of an amino acid, i.e., replacing an
amino acid with a different amino acid of similar properties (e.g.,
hydrophilicity and degree and distribution of charged regions) is
recognized in the art as typically involving a minor change. These
minor changes can be identified, in part, by considering the
hydropathic index of amino acids, as understood in the art (see,
e.g., Kyte et al., J. Mol. Biol., 157: 105-132 (1982)). The
hydropathic index of an amino acid is based on a consideration of
its hydrophobicity and charge. It is known in the art that amino
acids of similar hydropathic indexes can be substituted and still
retain protein function. In one aspect, amino acids having
hydropathic indexes of .+-.2 are substituted. The hydrophilicity of
amino acids also can be used to reveal substitutions that would
result in proteins retaining biological function. A consideration
of the hydrophilicity of amino acids in the context of a peptide
permits calculation of the greatest local average hydrophilicity of
that peptide, a useful measure that has been reported to correlate
well with antigenicity and immunogenicity (see, e.g., U.S. Pat. No.
4,554,101). Substitution of amino acids having similar
hydrophilicity values can result in peptides retaining biological
activity, for example immunogenicity, as is understood in the art.
In one aspect, substitutions are performed with amino acids having
hydrophilicity values within .+-.2 of each other. Both the
hydrophobicity index and the hydrophilicity value of amino acids
are influenced by the particular side chain of that amino acid.
Consistent with that observation, amino acid substitutions that are
compatible with biological function are understood to depend on the
relative similarity of the amino acids, and particularly the side
chains of those amino acids, as revealed by the hydrophobicity,
hydrophilicity, charge, size, and other properties. "Variant" also
can be used to describe a polypeptide or fragment thereof that has
been differentially processed, such as by proteolysis,
phosphorylation, or other post-translational modification, yet
retains its biological activity or antigen reactivity, e.g., the
ability to bind to IL-17. Use of "variant" herein is intended to
encompass fragments of a variant unless otherwise contradicted by
context.
I. Antibodies that Bind Human IL-17.
[0576] One aspect of the present invention provides isolated murine
monoclonal antibodies, or antigen-binding portions thereof, that
bind to IL-17 with high affinity, a slow off rate and high
neutralizing capacity. A second aspect of the invention provides
chimeric antibodies that bind IL-17. A third aspect of the
invention provides CDR grafted antibodies, or antigen-binding
portions thereof, that bind IL-17. A fourth aspect of the invention
provides humanized antibodies, or antigen-binding portions thereof,
that bind IL-17. A fifth aspect of the invention provides dual
variable domain immunoglobulin (DVD-Ig.TM.) molecules that bind
IL-17 and one other target. Preferably, the antibodies, or portions
thereof, are isolated antibodies. Preferably, the antibodies of the
invention are neutralizing human anti-IL-17A and/or human
anti-IL-17A/F antibodies.
a. Method of Making Anti IL-17 Antibodies
[0577] Anti IL-17 antibodies of the present invention may be made
by any of a number of techniques known in the art.
1. Anti IL-17 Monoclonal Antibodies Using Hybridoma Technology
[0578] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual, 2nd
ed. (Cold Spring Harbor Laboratory Press, 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N.Y., 1981) (said references incorporated by reference
in their entireties). The term "monoclonal antibody" as used herein
is not limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced.
[0579] Methods for producing and screening for specific anti-IL-17
antibodies using hybridoma technology are routine and well known in
the art. In one embodiment, the present invention provides methods
of generating monoclonal antibodies as well as antibodies produced
by the method comprising culturing a hybridoma cell secreting an
antibody of the invention wherein, preferably, the hybridoma is
generated by fusing splenocytes isolated from a mouse immunized
with an antigen of the invention with myeloma cells and then
screening the hybridomas resulting from the fusion for hybridoma
clones that secrete an antibody able to bind a polypeptide of the
invention. Briefly, mice can be immunized with an IL-17 antigen. In
a preferred embodiment, the IL-17 antigen is administered with an
adjuvant to stimulate the immune response. Such adjuvants include
complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides)
or ISCOM (immunostimulating complexes). Such adjuvants may protect
the polypeptide from rapid dispersal by sequestering it in a local
deposit, or they may contain substances that stimulate the host to
secrete factors that are chemotactic for macrophages and other
components of the immune system. Preferably, if a polypeptide is
being administered, the immunization schedule will involve two or
more administrations of the polypeptide, spread out over several
weeks.
[0580] After immunization of an animal with an IL-17 antigen,
antibodies and/or antibody-producing cells may be obtained from the
animal. An anti-IL-17 antibody-containing serum is obtained from
the animal by bleeding or sacrificing the animal. The serum may be
used as it is obtained from the animal, an immunoglobulin fraction
may be obtained from the serum, or the anti-IL-17 antibodies may be
purified from the serum. Serum or immunoglobulins obtained in this
manner are polyclonal, thus having a heterogeneous array of
properties.
[0581] Once an immune response is detected, e.g., antibodies
specific for the antigen IL-17 are detected in the mouse serum, the
mouse spleen is harvested and splenocytes isolated. The splenocytes
are then fused by well-known techniques to any suitable myeloma
cells, for example cells from cell line SP20 available from the
American Type Culture Collection (ATCC, Manassas, Va., US).
Hybridomas are selected and cloned by limited dilution. The
hybridoma clones are then assayed by methods known in the art for
cells that secrete antibodies capable of binding IL-17. Ascites
fluid, which generally contains high levels of antibodies, can be
generated by immunizing mice with positive hybridoma clones.
[0582] In another embodiment, antibody-producing immortalized
hybridomas may be prepared from the immunized animal. After
immunization, the animal is sacrificed and the splenic B cells are
fused to immortalized myeloma cells as is well known in the art.
See, e.g., Harlow and Lane, supra. In a preferred embodiment, the
myeloma cells do not secrete immunoglobulin polypeptides (a
non-secretory cell line). After fusion and antibiotic selection,
the hybridomas are screened using IL-17, or a portion thereof, or a
cell expressing IL-17. In a preferred embodiment, the initial
screening is performed using an enzyme-linked immunoassay (ELISA)
or a radioimmunoassay (RIA), preferably an ELISA. An example of
ELISA screening is provided in PCT Publication No. WO 00/37504,
herein incorporated by reference.
[0583] Anti-IL-17 antibody-producing hybridomas are selected,
cloned, and further screened for desirable characteristics,
including robust hybridoma growth, high antibody production and
desirable antibody characteristics, as discussed further below.
Hybridomas may be cultured and expanded in vivo in syngeneic
animals, in animals that lack an immune system, e.g., nude mice, or
in cell culture in vitro. Methods of selecting, cloning and
expanding hybridomas are well known to those of ordinary skill in
the art.
[0584] In a preferred embodiment, the hybridomas are mouse
hybridomas, as described above. In another preferred embodiment,
the hybridomas are produced in a non-human, non-mouse species such
as rats, sheep, pigs, goats, cattle or horses. In another
embodiment, the hybridomas are human hybridomas, in which a human
non-secretory myeloma is fused with a human cell expressing an
anti-IL-17 antibody.
[0585] Antibody fragments that recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab')2
fragments of the invention may be produced by proteolytic cleavage
of immunoglobulin molecules, using enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain
constant region and the CH1 domain of the heavy chain.
2. Anti-IL-17 Monoclonal Antibodies Using SLAM
[0586] In another aspect of the invention, recombinant antibodies
are generated from single, isolated lymphocytes using a procedure
referred to in the art as the selected lymphocyte antibody method
(SLAM), as described in U.S. Pat. No. 5,627,052; PCT Publication
No. WO 92/02551; and Babcook et al., Proc. Natl. Acad. Sci. USA,
93: 7843-7848 (1996). In this method, single cells secreting
antibodies of interest, e.g., lymphocytes derived from any one of
the immunized animals described in Section 1, are screened using an
antigen-specific hemolytic plaque assay, wherein the antigen IL-17,
a subunit of IL-17, or a fragment thereof, is coupled to sheep red
blood cells using a linker, such as biotin, and used to identify
single cells that secrete antibodies with specificity for IL-17.
Following identification of antibody-secreting cells of interest,
heavy and light chain variable region (VH and VL) cDNAs are rescued
from the cells by reverse transcriptase-PCR, and these variable
regions can then be expressed, in the context of appropriate
immunoglobulin constant regions (e.g., human constant regions), in
mammalian host cells, such as COS or CHO cells. The host cells
transfected with the amplified immunoglobulin sequences, derived
from in vivo selected lymphocytes, can then undergo further
analysis and selection in vitro, for example, by panning the
transfected cells to isolate cells expressing antibodies to IL-17.
The amplified immunoglobulin sequences further can be manipulated
in vitro, such as by in vitro affinity maturation methods such as
those described in PCT Publication No. WO 97/29131 and PCT
Publication No. WO 00/56772.
3. Anti-IL-17 Monoclonal Antibodies Using Transgenic Animals
[0587] In another embodiment of the invention, antibodies are
produced by immunizing a non-human animal comprising some, or all,
of the human immunoglobulin locus with an IL-17 antigen. In a
preferred embodiment, the non-human animal is a XENOMOUSE
transgenic mouse, an engineered mouse strain that comprises large
fragments of the human immunoglobulin loci and is deficient in
mouse antibody production. See, e.g., Green et al., Nature
Genetics, 7: 13-21 (1994) and U.S. Pat. Nos. 5,916,771; 5,939,598;
5,985,615; 5,998,209; 6,075,181; 6,091,001; 6,114,598 and
6,130,364. See also PCT Publication Nos. WO 91/10741, published
Jul. 25, 1991; WO 94/02602, published Feb. 3, 1994; WO 96/34096 and
WO 96/33735, both published Oct. 31, 1996; WO 98/16654, published
Apr. 23, 1998; WO 98/24893, published Jun. 11, 1998; WO 98/50433,
published Nov. 12, 1998; WO 99/45031, published Sep. 10, 1999; WO
99/53049, published Oct. 21, 1999; WO 00/09560, published Feb. 24,
2000; and WO 00/037504, published Jun. 29, 2000. The XENOMOUSE.RTM.
transgenic mouse produces an adult-like human repertoire of fully
human antibodies, and generates antigen-specific human Mabs. The
XENOMOUSE.RTM. transgenic mouse contains approximately 80% of the
human antibody repertoire through introduction of megabase sized,
germline configuration YAC fragments of the human heavy chain loci
and x light chain loci. See, Mendez et al., Nature Genetics,
15:146-156 (1997); and Green and Jakobovits, J. Exp. Med., 188:
483-495 (1998), the disclosures of which are hereby incorporated by
reference.
4. Anti IL-17 Monoclonal Antibodies Using Recombinant Antibody
Libraries
[0588] In vitro methods also can be used to make the antibodies of
the invention, wherein an antibody library is screened to identify
an antibody having the desired binding specificity. Methods for
such screening of recombinant antibody libraries are well known in
the art and include methods described in, for example, Ladner et
al., U.S. Pat. No. 5,223,409; Kang et al., PCT Publication No. WO
92/18619; Dower et al., PCT Publication No. WO 91/17271; Winter et
al., PCT Publication No. WO 92/20791; Markland et al., PCT
Publication No. WO 92/15679; Breitling et al., PCT Publication No.
WO 93/01288; McCafferty et al., PCT Publication No. WO 92/01047;
Garrard et al., PCT Publication No. WO 92/09690; Fuchs et al.,
Bio/Technology, 9: 1369-1372 (1991); Hay et al., Hum. Antibod.
Hybridomas, 3: 81-85 (1992); Huse et al., Science, 246: 1275-1281
(1989); McCafferty et al., Nature, 348: 552-554 (1990); Griffiths
et al., EMBO J., 12: 725-734 (1993); Hawkins et al., J. Mol. Biol.,
226: 889-896 (1992); Clackson et al., Nature, 352: 624-628 (1991);
Gram et al., Proc. Natl. Acad. Sci. USA, 89: 3576-3580 (1992);
Garrard et al., Bio/Technology, 9: 1373-1377 (1991); Hoogenboom et
al., Nucl. Acid Res., 19: 4133-4137 (1991); and Barbas et al.,
Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991); US patent
application publication No. 2003/0186374; and PCT Publication No.
WO 97/29131, the contents of each of which are incorporated herein
by reference.
[0589] The recombinant antibody library may be from a subject
immunized with IL-17A or IL-17F, or a portion of IL-17A or IL-17F.
Alternatively, the recombinant antibody library may be from a naive
subject, i.e., one who has not been immunized with IL-17A or
IL-17F, such as a human antibody library from a human subject who
has not been immunized with human IL-17A or IL-17F. Antibodies of
the invention are selected by screening the recombinant antibody
library with the peptide comprising human IL-17 to thereby select
those antibodies that recognize IL-17. Methods for conducting such
screening and selection are well known in the art, such as
described in the references in the preceding paragraph. To select
antibodies of the invention having particular binding affinities
for hIL-17, such as those that dissociate from human IL-17 with a
particular K.sub.off rate constant, the art-known method of surface
plasmon resonance can be used to select antibodies having the
desired K.sub.off rate constant. To select antibodies of the
invention having a particular neutralizing activity for hIL-17,
such as those with a particular an IC.sub.50, standard methods
known in the art for assessing the inhibition of hIL-17 activity
may be used.
[0590] In one aspect, the invention pertains to an isolated
antibody, or an antigen-binding portion thereof, that binds human
IL-17A and/or human IL-17. Preferably, the antibody is a
neutralizing antibody. In various embodiments, the antibody is a
recombinant antibody or a monoclonal antibody.
[0591] For example, the antibodies of the present invention can
also be generated using various phage display methods known in the
art. In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the
polynucleotide sequences encoding them. In a particular, such phage
can be utilized to display antigen-binding domains expressed from a
repertoire or combinatorial antibody library (e.g., human or
murine). Phage expressing an antigen binding domain that binds the
antigen of interest can be selected or identified with antigen,
e.g., using labeled antigen or antigen bound or captured to a solid
surface or bead. Phage used in these methods are typically
filamentous phage including fd and M13 binding domains expressed
from phage with Fab, Fv, or disulfide stabilized Fv antibody
domains recombinantly fused to either the phage gene III or gene
VIII protein. Examples of phage display methods that can be used to
make the antibodies of the present invention include those
disclosed in Brinkmann et al., J. Immunol. Methods, 182: 41-50
(1995); Ames et al., J. Immunol. Methods, 184: 177-186 (1995);
Kettleborough et al., Eur. J. Immunol., 24: 952-958 (1994); Persic
et al., Gene, 187: 9-18 (1997); Burton et al., Advances in
Immunology, 57:191-280 (1994); PCT Publications Nos. WO 90/02809;
WO 91/10737; WO 92/01047 (PCT/GB91/01134); WO 92/18619; WO
93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426;
5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;
5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743
and 5,969,108; each of which is incorporated herein by reference in
its entirety.
[0592] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies including human antibodies or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and F(ab')2
fragments can also be employed using methods known in the art such
as those disclosed in PCT publication WO 92/22324; Mullinax et al.,
BioTechniques, 12(6): 864-869 (1992); and Sawai et al., Am. J.
Reprod. Immunol., 34: 26-34 (1995); and Better et al., Science,
240: 1041-1043 (1988), (said references incorporated by reference
in their entireties). Examples of techniques which can be used to
produce single-chain Fvs and antibodies include those described in
U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in
Enzymology, 203: 46-88 (1991); Shu et al., Proc. Natl. Acad. Sci.
USA, 90: 7995-7999 (1993); and Skerra et al., Science, 240:
1038-1041 (1988).
[0593] Alternative to screening of recombinant antibody libraries
by phage display, other methodologies known in the art for
screening large combinatorial libraries can be applied to the
identification of dual specificity antibodies of the invention. One
type of alternative expression system is one in which the
recombinant antibody library is expressed as RNA-protein fusions,
as described in PCT Publication No. WO 98/31700 by Szostak and
Roberts, and in Roberts, R. W. and Szostak, J. W. (1997) Proc.
Natl. Acad. Sci. USA, 94: 12297-12302. In this system, a covalent
fusion is created between an mRNA and the peptide or protein that
it encodes by in vitro translation of synthetic mRNAs that carry
puromycin, a peptidyl acceptor antibiotic, at their 3' end. Thus, a
specific mRNA can be enriched from a complex mixture of mRNAs
(e.g., a combinatorial library) based on the properties of the
encoded peptide or protein, e.g., antibody, or portion thereof,
such as binding of the antibody, or portion thereof, to the dual
specificity antigen. Nucleic acid sequences encoding antibodies, or
portions thereof, recovered from screening of such libraries can be
expressed by recombinant means as described above (e.g., in
mammalian host cells) and, moreover, can be subjected to further
affinity maturation by either additional rounds of screening of
mRNA-peptide fusions in which mutations have been introduced into
the originally selected sequence(s), or by other methods for
affinity maturation in vitro of recombinant antibodies, as
described above.
[0594] In another approach the antibodies of the present invention
can also be generated using yeast display methods known in the art.
In yeast display methods, genetic methods are used to tether
antibody domains to the yeast cell wall and display them on the
surface of yeast. In particular, such yeast can be utilized to
display antigen-binding domains expressed from a repertoire or
combinatorial antibody library (e.g., human or murine). Examples of
yeast display methods that can be used to make the antibodies of
the present invention include those disclosed Wittrup et al. in
U.S. Pat. No. 6,699,658, incorporated herein by reference.
B. Production of Recombinant IL-17 Antibodies
[0595] Antibodies of the present invention may be produced by any
of a number of techniques known in the art. For example, expression
from host cells, wherein expression vector(s) encoding the heavy
and light chains is (are) transfected into a host cell by standard
techniques. The various forms of the term "transfection" are
intended to encompass a wide variety of techniques commonly used
for the introduction of exogenous DNA into a prokaryotic or
eukaryotic host cell, e.g., electroporation, calcium-phosphate
precipitation, DEAE-dextran transfection, and the like. Although it
is possible to express the antibodies of the invention in either
prokaryotic or eukaryotic host cells, expression of antibodies in
eukaryotic cells is preferable, and most preferable in mammalian
host cells, because such eukaryotic cells (and in particular
mammalian cells) are more likely than prokaryotic cells to assemble
and secrete a properly folded and immunologically active
antibody.
[0596] Preferred mammalian host cells for expressing the
recombinant antibodies of the invention include Chinese Hamster
Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub
and Chasin, (1980) Proc. Natl. Acad. Sci. USA, 77: 4216-4220, used
with a DHFR selectable marker, e.g., as described in R. J. Kaufman
and P. A. Sharp (1982) Mol. Biol., 159: 601-621), NS0 myeloma
cells, COS cells and SP2 cells. When recombinant expression vectors
encoding antibody genes are introduced into mammalian host cells,
the antibodies are produced by culturing the host cells for a
period of time sufficient to allow for expression of the antibody
in the host cells or, more preferably, secretion of the antibody
into the culture medium in which the host cells are grown.
Antibodies can be recovered from the culture medium using standard
protein purification methods.
[0597] Host cells can also be used to produce functional antibody
fragments, such as Fab fragments or scFv molecules. It will be
understood that variations on the above procedure are within the
scope of the present invention. For example, it may be desirable to
transfect a host cell with DNA encoding functional fragments of
either the light chain and/or the heavy chain of an antibody of
this invention. Recombinant DNA technology may also be used to
remove some, or all, of the DNA encoding either or both of the
light and heavy chains that is not necessary for binding to the
antigens of interest. The molecules expressed from such truncated
DNA molecules are also encompassed by the antibodies of the
invention. In addition, bifunctional antibodies may be produced in
which one heavy and one light chain are an antibody of the
invention and the other heavy and light chain are specific for an
antigen other than the antigens of interest by crosslinking an
antibody of the invention to a second antibody by standard chemical
crosslinking methods.
[0598] In a preferred system for recombinant expression of an
antibody, or antigen-binding portion thereof, of the invention, a
recombinant expression vector encoding both the antibody heavy
chain and the antibody light chain is introduced into dhfr-CHO
cells by calcium phosphate-mediated transfection. Within the
recombinant expression vector, the antibody heavy and light chain
genes are each operatively linked to CMV enhancer/AdMLP promoter
regulatory elements to drive high levels of transcription of the
genes. The recombinant expression vector also carries a DHFR gene,
which allows for selection of CHO cells that have been transfected
with the vector using methotrexate selection/amplification. The
selected transformant host cells are cultured to allow for
expression of the antibody heavy and light chains and intact
antibody is recovered from the culture medium. Standard molecular
biology techniques are used to prepare the recombinant expression
vector, transfect the host cells, select for transformants, culture
the host cells and recover the antibody from the culture medium.
Still further the invention provides a method of synthesizing a
recombinant antibody of the invention by culturing a host cell of
the invention in a suitable culture medium until a recombinant
antibody of the invention is synthesized. The method can further
comprise isolating the recombinant antibody from the culture
medium.
1. Anti hIL-17 Antibodies
[0599] Table 5 is a list of amino acid sequences of VH and VL
regions of preferred murine anti-hIL-17 antibodies of the
invention.
TABLE-US-00006 TABLE 5 List of Amino Acid Sequences of Murine
Anti-hIL-17 Antibody VH and VL Regions SEQ ID Sequence No. Protein
region 123456789012345678901234567890 26 VH 7D7
QVQLQQSGAELVRPGTSVTLSCKASGYRFT DYEIHWIKQTPAQVLEWIGVTDPESGGTLH
NPKFDGKATLTADKSSRTAYMELRSLTSED SAVYYCTRYYKYESFYGMDYWGQGTSVTVS S VH
7D7 CDR- Residues 31-35 DYEIH H1 of SEQ ID NO.: 26 VH 7D7 CDR-
Residues 50-66 VTDPESGGTLHNPKFDG H2 of SEQ ID NO.: 26 VH 7D7 CDR-
Residues 99-110 YYKYESFYGMDY H3 of SEQ ID NO.: 26 27 VL 7D7
QIVLTQSPAIMSAFPGEKVTMTCSASSSIS YMCWYQQKPGTSPKRWICDTSKLASGVPVR
FSGSGSGTSYSLTINSMETEDAATYYCQQR SSYPWTFGGGTKVEIKR VL 7D7 CDR-
Residues 24-33 SASSSISYMC L1 of SEQ ID NO.: 27 VL 7D7 CDR- Residues
49-55 DTSKLAS L2 of SEQ ID NO.: 27 VL 7D7 CDR- Residues 88-96
QQRSSYPWT L3 of SEQ ID NO.: 27 28 VH 6C6
QVQLQQSGAELVRPGASVKLSCKASGYTFS DYEIHWVKQTPVHGLAWIGVIHPGNGGTAY
NQKFKDKATLTADKSSSTAYMELSSLTSED SAVYYCERFLTYEGYFDYWGQGTTLTVSS VH 6C6
CDR- Residues 31-35 DYEIH H1 of SEQ ID NO.: 28 VH 6C6 CDR- Residues
50-66 VIHPGNGGTAYNQKFKD H2 of SEQ ID NO.: 28 VH 6C6 CDR- Residues
99-108 FLTYEGYFDY H3 of SEQ ID NO.: 28 29 VL 6C6
SIVMTQTPKFLLVSAGDRVTITCKASQSVN NDVAWYQHKPGQSPKLLINYASNRYTGVPD
RFTGSGYGTDFTFTISTVQAEDLAIYFCQQ DYGSPYTFGGGTKLEIKR VL 6C6 CDR-
Residues 24-34 KASQSVNNDVA L1 of SEQ ID NO.: 29 VL 6C6 CDR-
Residues 50-56 YASNRYT L2 of SEQ ID NO.: 29 VL 6C6 CDR- Residues
89-97 QQDYGSPYT L3 of SEQ ID NO.: 29 30 VH 1D8
QVQLQQSGAELVRPGASVKLSCKASGYTFS DYEMHWVKQTPVHGLEWIGVIHPGNGGTAY
NQKFRDKATLTADKSSTTAYMELSSLTSED SAVYYCIRFLTYEGYFDYWGQGTTLTVSS VH 1D8
CDR- Residues 31-35 DYEMH H1 of SEQ ID NO.: 30 VH 1D8 CDR- Residues
50-66 VIHPGNGGTAYNQKFRD H2 of SEQ ID NO.: 30 VH 1D8 CDR- Residues
99-108 FLTYEGYFDY H3 of SEQ ID NO.: 30 31 VL 1D8
SIVMTQTPKFLLVSAGDRVTITCKASQSVN NDVAWFQHKPGQSPKLLINYASNRYTGVPD
RFTGSGYGTDFTFTISTVQSEDLAIYFCQQ DYGSPYTFGGGTTLEIKR VL 1D8 CDR-
Residues 24-34 KASQSVNNDVA L1 of SEQ ID NO.: 31 VL 1D8 CDR-
Residues 50-56 YASNRYT L2 of SEQ ID NO.: 31 VL 1D8 CDR- Residues
89-97 QQDYGSPYT L3 of SEQ ID NO.: 31 32 VH 8B12
QVQLKESGPGLVAPSQSLSITCTISGFSLT SYGVHWVRQPPGKGLEWLVVIWSDGTTTYN
SALKSRLSITRDNSKSQVFLKMNSLQTDDT AIYYCARDSTWDYYYTMDYWGQGTPVTVSS VH
8B12 CDR- Residues 31-35 SYGVH H1 of SEQ ID NO.: 32 VH 8B12 CDR-
Residues 50-65 VIWSDGTTTYNSALKS H2 of SEQ ID NO.: 32 VH 8B12 CDR-
Residues 98-109 DSTWDYYYTMDY H3 of SEQ ID NO.: 32 33 VL 8B12
DVVMTQTPLSLPVSLGDQASISCRSSQSLV HSNGNTYLHWYLQKPGQSPKLLIYKVSNRF
SGVPDRFSGSGSGTDFTLKISRVEAEDLGV YFCSQSTHVYTFGGGTKLEIKR VL 8B12 CDR-
Residues 24-39 RSSQSLVHSNGNTYLH L1 of SEQ ID NO.: 33 VL 8B12 CDR-
Residues 55-61 KVSNRFS L2 of SEQ ID NO.: 33 VL 8B12 CDR- Residues
94-101 SQSTHVYT L3 of SEQ ID NO.: 33 34 VH 10F7
QVQLQQSGAELVRPGTSVTLSCKASGYIFT DYEIHWVKQTPVHGLEWIGVNDPESGGTFY
NQKFDGKAELTADKSSSTAYMELRSLTSED SGVYYCTRYYRYESFYGMDYWGQGTSITVS S VH
10F7 CDR- Residues 31-35 DYEIH H1 of SEQ ID NO.: 34 VH 10F7 CDR-
Residues 50-66 VNDPESGGTFYNQKFDG H2 of SEQ ID NO.: 34 VH 10F7 CDR-
Residues 99-110 YYRYESFYGMDY H3 of SEQ ID NO.: 34 35 VL 10F7
QIVLTQSPAIMSASPGEKVTMTCSASSSIS YIYWFQQKPGTSPKRWIYATFELASGVPAR
FSGSGSGTSYSLTISSMEAEDAATYYCHQR SSYPWTFGGGSKLEIKR VL 10F7 CDR-
Residues 24-33 SASSSISYIY L1 of SEQ ID NO.: 35 VL 10F7 CDR-
Residues 49-55 ATFELAS L2 of SEQ ID NO.: 35 VL 10F7 CDR- Residues
88-96 HQRSSYPWT L3 of SEQ ID NO.: 35 36 VH 5C5
QVQLQQSGAELVRPGASVKLSCKALGYTFT DYEFHWVKQTPVHGLEWIGVIHPGNGGTAY
NQNFRDKATLTADKSSSTAYMELSSLTSED SGVYYCTRFLTYEGYFDYWGQGTALTVSS VH 5C5
CDR- Residues 31-35 DYEFH H1 of SEQ ID NO.: 36 VH 5C5 CDR- Residues
50-66 VIHPGNGGTAYNQNFRD H2 of SEQ ID NO.: 36 VH 5C5 CDR- Residues
99-108 FLTYEGYFDY H3 of SEQ ID NO.: 36 37 VL 5C5
NIVMTQTPKFLLVSPGDRVTITCKASQSVS IDVGWFQQKPGQSPKLLIYHASNRYTGVPD
RFTGSGYGTDFTFTVNTVQAEDLAVYFCQQ DYSSPYTFGGGTKLELKR VL 5C5 CDR-
Residues 24-34 KASQSVSIDVG L1 of SEQ ID NO.: 37 VL 5C5 CDR-
Residues 50-56 HASNRYT L2 of SEQ ID NO.: 37 VL 5C5 CDR- Residues
89-97 QQDYSSPYT L3 of SEQ ID NO.: 37 38 VH 10G9
QVQLKESGPALVAPSQSLSFTCTISGFSLS SYGVHWVRQPPGKGLEWLVVIWSDGTTTYN
SALKSRLSISKDNSKSQVFLKMNSLQTDDT AMYYCARDGYYIYTMDYWGQGTSVTVSS VH 10G9
CDR- Residues 31-35 SYGVH H1 of SEQ ID NO.: 38 VH 10G9 CDR-
Residues 50-65 VIWSDGTTTYNSALKS H2 of SEQ ID NO.: 38 VH 10G9 CDR-
Residues 98-107 DGYYIYTMDY H3 of SEQ ID NO.: 38 39 VL 10G9
DVVMTQTPLSLPVSLGDQASISCRSSQSLV HSNGNTYLHWYLQRPGQSPKLLIYKVSNRF
SGVPDRFSGSGSGTDFTLKISRVEAEDLGL YFCSQGTHAPLTFGAGTKLELNR VL 10G9 CDR-
Residues 24-39 RSSQSLVHSNGNTYLH L1 of SEQ ID NO.: 39 VL 10G9 CDR-
Residues 55-61 KVSNRFS L2 of SEQ ID NO.: 39 VL 10G9 CDR- Residues
94-102 SQGTHAPLT L3 of SEQ ID NO.: 39
[0600] Based on an alignment of the amino acid sequences of the
CDRs of the VH and VL regions of the murine anti-hIL-17 antibodies
listed in Table 5, above, the invention provides an IL-17 binding
protein comprising an antigen binding domain capable of binding
human IL-17, said antigen binding domain comprising at least one
CDR comprising an amino acid sequence selected from the group
consisting of: [0601] CDR-H1.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5 (SEQ ID NO:919), wherein;
[0602] X.sub.1 is D or S; [0603] X.sub.2 is Y; [0604] X.sub.3 is E
or G; [0605] X.sub.4 is I, M, V, or F; [0606] X.sub.5 is H; [0607]
CDR-H2.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16-X.sub.17
(SEQ ID NO:920), wherein; [0608] X.sub.1 is V; [0609] X.sub.2 is T,
I, or N; [0610] X.sub.3 is D, H, or W; [0611] X.sub.4 is P or is
not present; [0612] X.sub.5 is E, G, or S; [0613] X.sub.6 is S, N,
or D; [0614] X.sub.7 is G; [0615] X.sub.8 is G or T; [0616] X.sub.9
is T; [0617] X.sub.10 is L, A, T, or F; [0618] X.sub.11 is H or Y;
[0619] X.sub.12 is N; [0620] X.sub.13 is P, Q, or S; [0621]
X.sub.14 is K, A, or N; [0622] X.sub.15 is F or L; [0623] X.sub.16
is D, K, or R; and [0624] X.sub.17 is G, D, or S; [0625] CDR-H3.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-X.sub.12 (SEQ ID NO:921), wherein; [0626] X.sub.1
is Y, F, or D; [0627] X.sub.2 is Y, L, S, or G; [0628] X.sub.3 is
K, T, R, or Y; [0629] X.sub.4 is Y or W; [0630] X.sub.5 is E, D, or
I; [0631] X.sub.6 is S, G, or Y; [0632] X.sub.7 is F, Y, or T;
[0633] X.sub.8 is Y, F, or M; [0634] X.sub.9 is G, T, or is not
present; [0635] X.sub.10 is M or is not present; [0636] X.sub.11 is
D; and [0637] X.sub.12 is Y; [0638] CDR-L1.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X-
.sub.9-X.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16
(SEQ ID NO:922), wherein; [0639] X.sub.1 is S, K, or R; [0640]
X.sub.2 is A or S; [0641] X.sub.3 is S; [0642] X.sub.4 is S or Q;
[0643] X.sub.5 is S or is not present; [0644] X.sub.6 is L or is
not present; [0645] X.sub.7 is V or is not present; [0646] X.sub.8
is H or is not present; [0647] X.sub.9 is S or is not present;
[0648] X.sub.10 is S, N, or is not present; [0649] X.sub.11 is S,
V, or G; [0650] X.sub.12 is I, N, or S; [0651] X.sub.13 is S, N, T,
or I; [0652] X.sub.14 is Y or D; [0653] X.sub.15 is M, V, L, or I;
and [0654] X.sub.16 is C, A, H, Y, or G; [0655] CDR-L2.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7 (SEQ ID
NO:923), wherein; [0656] X.sub.1 is D, Y, K, A, or H; [0657]
X.sub.2 is T, A, or V; [0658] X.sub.3 is S or F; [0659] X.sub.4 is
K, N, or E; [0660] X.sub.5 is L or R; [0661] X.sub.6 is A, Y, or F;
and [0662] X.sub.7 is S or T;
[0663] and [0664] CDR-L3.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9
(SEQ ID NO:924), wherein; [0665] X.sub.1 is Q, S, or H; [0666]
X.sub.2 is Q; [0667] X.sub.3 is R, D, S, or G; [0668] X.sub.4 is S,
Y, or T; [0669] X.sub.5 is S, G, or H; [0670] X.sub.6 is Y, S, V,
or A; [0671] X.sub.7 is P or is not present; [0672] X.sub.8 is W,
Y, or L; and [0673] X.sub.9 is T.
[0674] Table 6 provides a list of amino acid sequences of VH and VL
regions of preferred human anti-hIL-17 antibodies of the
invention.
TABLE-US-00007 TABLE 6 List of Amino Acid Sequences of Human
Anti-hIL-17 Antibody VH and VL Regions SEQ Sequence ID No. Protein
region 123456789012345678901234567890 40 VH IL17-TN-
EVQLLESGGGVVQPGRSLRLSCAASGFIFS L7-G9 NYGMHWVRQAPGKGLEWVAVISYDGSNKYY
ADSVKGRFTISRDNSKNTLYLEMNSLRPED TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS
VH G9 CDR-H1 Residues 31-35 NYGMH of SEQ ID NO.: 40 VH G9 CDR-H2
Residues 50-66 VISYDGSNKYYADSVKG of SEQ ID NO.: 40 VH G9 CDR-H3
Residues 99-103 VGASGDYYYSYGLDV of SEQ ID NO.: 40 41 VL IL17-TN-
QSGLTQPPSASGTPGQTVSISCSGSNSNIG L7-G9 SHSVNWYQQVPGAAPKLLMYGIGQRPSGVP
DRFSVSQSGTSASLAISGLQSEDEADYYCA TWDDSLGGYVFGSGTKVTVLG VL G9 CDR-L1
Residues 23-35 SGSNSNIGSHSVN of SEQ ID NO.: 41 VL G9 CDR-L2
Residues 51-57 GIGQRPS of SEQ ID NO.: 41 VL G9 CDR-L3 Residues
90-110 ATWDDSLGGYV of SEQ ID NO.: 41 42 VH IL17-TN-
EVQLLESGGGVVQPGTFLRLSCAATGFTFS L7-A7 AYGMHWVRQAPGRGLEWVAVTSYDGSNKYY
ADSVKGRFTISRDNSKNTLYLEMNSLRPED TAVYYCAKVGASGDYYYSYGLDVWGQGTMV TVSS
VH A7 CDR-H1 Residues 31-35 AYGMH of SEQ ID NO.: 42 VH A7 CDR-H2
Residues 50-66 VTSYDGSNKYYADSVKG of SEQ ID NO.: 42 VH A7 CDR-H3
Residues 99-103 VGASGDYYYSYGLDV of SEQ ID NO.: 42 43 VL IL17-TN-
QSGLTQPPSASGTPGQTVSISCSGSNSNIG L7-A7 SHSVNWYQQVPGAAPKLLMYGIGQRPSGVP
DRFSVSQSGTSASLAISGLQSEDEADYYCA TWDDSLGGYVFGSGTKVTVLG VL A7 CDR-L1
Residues 23-35 SGSNSNIGSHSVN of SEQ ID NO.: 43 VL A7 CDR-L2
Residues 51-57 GIGQRPS of SEQ ID NO.: 43 VL A7 CDR-L3 Residues
90-110 ATWDDSLGGYV of SEQ ID NO.: 43 44 VH IL17-TN-
EVQLVQSGAEVKKPGASVKVSCKASGYTFS L7-C8 DFDIDWVRQATGLGLEWMGWMNPNSGNTGV
APKFRGRVSMTFNTAIRTAYLELSSLRPDD TAVYFCARSSESEGITIGFDNWGQGTLVTV SS VH
C8 CDR-H1 Residues 31-35 DFDID of SEQ ID NO.: 44 VH C8 CDR-H2
Residues 50-66 WMNPNSGNTGVAPKFRG of SEQ ID NO.: 44 VH C8 CDR-H3
Residues 99-101 SSESEGITIGFDN of SEQ ID NO.: 44 45 VL IL17-TN-
SYELTQPPSVSVSPGQTASIPCSGDKLGNR L7-C8 YACWYKQKPGQPPVLVIYQDNKRPSGISER
YSGSNYGDTATLTITGTQAMDEADYYCQTW DSTTGSYVFGTGTKVTVLG VL C8 CDR-L1
Residues 23-35 SGDKLGNRYAC of SEQ ID NO.: 45 VL C8 CDR-L2 Residues
51-57 QDNKRPS of SEQ ID NO.: 45 VL C8 CDR-L3 Residues 90-110
QTWDSTTGSYV of SEQ ID NO.: 45 46 VH IL17-TN-
EVQLVQSGAEVKKPGESLKISCKASGGSFR K7-B6 SYGISWVRQAPGQGLEWMGGITPILGTANY
AQKFQGRVTITADESTTTAYMELSGLTSDD TAVYYCAREPNDFWNGYYTTHHFDYWGQGT
PVTVSS VH B6 CDR-H1 Residues 31-35 SYGIS of SEQ ID NO.: 46 VH B6
CDR-H2 Residues 50-66 GITPILGTANYAQKFQG of SEQ ID NO.: 46 VH B6
CDR-H3 Residues 99-115 EPNDFWNGYYTTHHFDY of SEQ ID NO.: 46 47 VL
IL17-TN- DVVMTQSPDFQSVTPKEKVTITCRASQNIG K7-B6
SALHWYQQKPDQSPKLLIKYASQSISGVPS RFSGSGSGTDFTLTINGLEAEDAGTYYCHQ
STSLPHTFGQGTKLDIKR VL B6 CDR-L1 Residues 24-34 RASQNIGSALH of SEQ
ID NO.: 47 VL B6 CDR-L2 Residues 50-56 YASQSIS of SEQ ID NO.: 47 VL
B6 CDR-L3 Residues 89-97 HQSTSLPHT of SEQ ID NO.: 47 48 VH IL17-LN-
EVQLVQSGAEVKNPGASVKVSCKVSGSRLS K9-F5 DLAMHWVRQAPGKGPEWMGGFDPDEGETVY
AQNFQGRVSMTEDTSSDTAYMELNSLRSED TAVYYCATIRPWLGGAYYFDNWGQGTLVTV SS VH
F5 CDR-H1 Residues 31-35 DLAMH of SEQ ID NO.: 48 VH F5 CDR-H2
Residues 50-66 GFDPDEGETVYAQNFQG of SEQ ID NO.: 48 VH F5 CDR-H3
Residues 99-101 IRPWLGGAYYFDN of SEQ ID NO.: 48 49 VL IL17-LN-
ETTLTQSPAFMSATPGDKVNISCKASQDID K9-F5 DDMNWYQQKPGEAALFIIQEATTLVPGIPP
RFSGSGYGTDFTLTVNNIQSEDAAYYFCLQ HDSFPYTFGQGTKLEIKR VL F5 CDR-L1
Residues 24-34 KASQDIDDDMN of SEQ ID NO.: 49 VL F5 CDR-L2 Residues
50-56 EATTLVP of SEQ ID NO.: 49 VL F5 CDR-L3 Residues 89-97
LQHDSFPYT of SEQ ID NO.: 49
[0675] Based on an alignment of the amino acid sequences of the
CDRs of the VH and VL regions of the human anti-hIL-17 antibodies
listed in Table 6, above, the invention provides an IL-17 binding
protein comprising an antigen binding domain capable of binding
human IL-17, said antigen binding domain comprising at least one
CDR comprising an amino acid sequence selected from the group
consisting of: [0676] CDR-H1.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5 (SEQ ID NO:925), wherein;
[0677] X.sub.1 is N, A, D, or S; [0678] X.sub.2 is Y, F, or L;
[0679] X.sub.3 is G, D, or A; [0680] X.sub.4 is M or I; and [0681]
X.sub.5 is H, D, or S; [0682] CDR-H2.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16-X.sub.17
(SEQ ID NO:926), wherein; [0683] X.sub.1 is V, W, or G; [0684]
X.sub.2 is I, T, M, or F; [0685] X.sub.3 is 5, N, T, or D; [0686]
X.sub.4 is Y or P; [0687] X.sub.5 is D, N, or I; [0688] X.sub.6 is
G, S, L, or E; [0689] X.sub.7 is S or G; [0690] X.sub.8 is N, T, or
E; [0691] X.sub.9 is K, T, or A; [0692] X.sub.10 is Y, G, N, or V;
[0693] X.sub.11 is Y or V; [0694] X.sub.12 is A; [0695] X.sub.13 is
D, P, or Q; [0696] X.sub.14 is S, K, or N; [0697] X.sub.15 is V or
F; [0698] X.sub.16 is K, R, or Q; and [0699] X.sub.17 is G; [0700]
CDR-H3.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16-X.sub.17
(SEQ ID NO:927), wherein; [0701] X.sub.1 is V, S, E, or I; [0702]
X.sub.2 is G, S, P, or R; [0703] X.sub.3 is A, E, N, or P; [0704]
X.sub.4 is S, D, or W; [0705] X.sub.5 is G, E, F, or L; [0706]
X.sub.6 is D, G, or W; [0707] X.sub.7 is Y, I, N, or G; [0708]
X.sub.8 is Y, T, G, or A; [0709] X.sub.9 is Y or I; [0710] X.sub.10
is S, G, or Y; [0711] X.sub.11 is Y, F, or T; [0712] X.sub.12 is G,
T, or is not present; [0713] X.sub.13 is L, H, or is not present;
[0714] X.sub.14 is H or is not present; [0715] X.sub.15 is F or is
not present; [0716] X.sub.16 is D; and [0717] X.sub.17 is V, N, or
Y; [0718] CDR-L1.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-X.sub.12-X.sub.13 (SEQ ID NO:928), wherein; [0719]
X.sub.1 is S, R, or K; [0720] X.sub.2 is G or A; [0721] X.sub.3 is
S or D; [0722] X.sub.4 is N, K, or Q; [0723] X.sub.5 is S or is not
present; [0724] X.sub.6 is N or is not present; [0725] X.sub.7 is
I, L, N, or D; [0726] X.sub.8 is G or I; [0727] X.sub.9 is S, N, G,
or D; [0728] X.sub.10 is H, R, S, or D; [0729] X.sub.11 is S, Y, A,
or D; [0730] X.sub.12 is V, A, L, or M; and [0731] X.sub.13 is N,
C, or H; [0732] CDR-L2.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7 (SEQ ID
NO:929), wherein; [0733] X.sub.1 is G, Q, Y, or E; [0734] X.sub.2
is I, D, or A; [0735] X.sub.3 is G, N, S, or T; [0736] X.sub.4 is
Q, K, or T; [0737] X.sub.5 is R, S, or L; [0738] X.sub.6 is P, I,
or V; and [0739] X.sub.7 is S or P;
[0740] and [0741] CDR-L3.
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11 (SEQ ID NO:930), wherein; [0742] X.sub.1 is A, Q,
H, or L; [0743] X.sub.2 is T or Q; [0744] X.sub.3 is W, S, or H;
[0745] X.sub.4 is D or T; [0746] X.sub.5 is D or S; [0747] X.sub.6
is S, T, L, or F; [0748] X.sub.7 is L, T, or P; [0749] X.sub.8 is
G, H, or Y; [0750] X.sub.9 is G, S, or T; [0751] X.sub.10 is Y or
is not present; and [0752] X.sub.11 is V or is not present; 2. Anti
hIL-17 Chimeric Antibodies
[0753] A chimeric antibody is a molecule in which different
portions of the antibody are derived from different animal species,
such as antibodies having a variable region derived from a murine
monoclonal antibody and a human immunoglobulin constant region.
Methods for producing chimeric antibodies are known in the art and
discussed in detail in the Examples section. See, e.g., Morrison,
Science, 229: 1202-1207 (1985); Oi et al., BioTechniques, 4:
214-221 (1986); Gillies et al., J. Immunol. Methods, 125: 191-202
(1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which
are incorporated herein by reference in their entireties. In
addition, techniques developed for the production of "chimeric
antibodies" (Morrison et al., Proc. Natl. Acad. Sci. USA, 81:
6851-6855 (1984); Neuberger et al., Nature, 312: 604-608 (1984);
Takeda et al., Nature, 314: 452-454 (1985), which are incorporated
herein by reference in their entireties) by splicing genes from a
mouse antibody molecule of appropriate antigen specificity together
with genes from a human antibody molecule of appropriate biological
activity can be used.
[0754] In one embodiment, the chimeric antibodies of the invention
are produced by replacing the heavy chain constant region of the
murine monoclonal anti human IL-17 antibodies described in section
1 with a human IgG1 constant region.
3. Anti IL-17 CDR-Grafted Antibodies
[0755] CDR-grafted antibodies of the invention comprise heavy and
light chain variable region sequences from a human antibody wherein
one or more of the CDR regions of V.sub.H and/or V.sub.L are
replaced with CDR sequences of the murine antibodies of the
invention. A framework sequence from any human antibody may serve
as the template for CDR grafting. However, straight chain
replacement onto such a framework often leads to some loss of
binding affinity to the antigen. The more homologous a human
antibody is to the original murine antibody, the less likely the
possibility that combining the murine CDRs with the human framework
will introduce distortions in the CDRs that could reduce affinity.
Therefore, it is preferable that the human variable framework that
is chosen to replace the murine variable framework apart from the
CDRs have at least a 65% sequence identity with the murine antibody
variable region framework. It is more preferable that the human and
murine variable regions apart from the CDRs have at least 70%
sequence identify. It is even more preferable that the human and
murine variable regions apart from the CDRs have at least 75%
sequence identity. It is most preferable that the human and murine
variable regions apart from the CDRs have at least 80% sequence
identity. Methods for producing chimeric antibodies are known in
the art and discussed in detail in Example 2.2. (also see EP 0 239
400; PCT Publication No. WO 91/09967; U.S. Pat. Nos. 5,225,539;
5,530,101; and 5,585,089); veneering or resurfacing (see, e.g., EP
0 592 106; EP 0 519 596; Padlan, Molecular Immunology, 28(4/5):
489-498 (1991); Studnicka et al., Protein Engineering, 7(6):
805-814 (1994); Roguska et al., Proc. Natl. Acad. Sci. USA, 91:
969-973 (1994)); and chain shuffling (see, e.g., U.S. Pat. No.
5,565,352).
[0756] In a specific embodiment the invention provides CDR grafted
antibodies with V.sub.H and/or V.sub.L chains as described in Table
7.
TABLE-US-00008 TABLE 7 CDR Grafted Antibodies SEQ Protein Sequence
ID No. region 123456789012345678901234567890 50 h10F7VH.1z
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSD YEIHWVRQAPGQGLEWMGVNDPESGGTFYNQ
KFDGRVTITADKSTSTAYMELSSLRSEDTAV YYCARYYRYESFYGMDYWGQGTTVTVSS 51
h10F7Vk.1z DIQMTQSPSSLSASVGDRVTITCSASSSISY
IYWYQQKPGKAPKRLIYATFELASGVPSRFS GSGSGTEFTLTISSLQPEDFATYYCHQRSSY
PWTFGQGTKLEIKR 52 h10F7Vk.2 EIVLTQSPDFQSVTPKEKVTITCSASSSISY
IYWYQQKPDQSPKLLIKATFELASGVPSRFS GSGSGTDFTLTINSLEAEDAATYYCHQRSSY
PWTFGQGTKLEIKR 53 h10F7Vk.3z DIQMTQSPSSLSASVGDRVTITCSASSSISY
IYWYQQKPGKAPKRLIYATFELASGVPSRFS GSGSGTEFTLTISSLQPEDFATYYCHQRSSY
PWTFGGGTKVEIKR 54 h10F7Vk.4 EIVLTQSPDFQSVTPKEKVTITCSASSSISY
IYWYQQKPDQSPKLLIKATFELASGVPSRFS GSGSGTDFTLTINSLEAEDAATYYCHQRSSY
PWTFGGGTKVEIKR 55 h5C5VH.1z QVQLVQSGAEVKKPGSSVKVSCKASGGTFSD
YEFHWVRQAPGQGLEWMGVIHPGNGGTAYNQ NFRDRVTITADKSTSTAYMELSSLRSEDTAV
YYCARFLTYEGYFDYWGQGTLVTVSS 56 h5C5Vk.1z
DIQMTQSPSSLSASVGDRVTITCKASQSVSI DVGWYQQKPGKAPKLLIYHASNRYTGVPSRF
SGSGSGTDFTFTISSLQPEDIATYYCQQDYS SPYTFGQGTKLEIKR 57 h5C5Vk.2z
DIQMTQSPSSLSASVGDRVTITCKASQSVSI DVGWYQQKPGKAPKLLIYHASNRYTGVPSRF
SGSGSGTDFTFTISSLQPEDIATYYCQQDYS SPYTFGGGTKVEIKR 58 h5C5Vk.3z
EIVMTQSPATLSVSPGERATLSCKASQSVSI DVGWYQQKPGQAPRLLIYHASNRYTGIPARF
SGSGSGTEFTLTISSLQSEDFAVYYCQQDYS SPYTFGQGTKLEIKR 59 h5C5Vk.4z
EIVMTQSPATLSVSPGERATLSCKASQSVSI DVGWYQQKPGQAPRLLIYHASNRYTGIPARF
SGSGSGTEFTLTISSLQSEDFAVYYCQQDYS SPYTFGGGTKVEIKR
4. Anti-hIL-17 Humanized Antibodies
[0757] Humanized antibodies are antibody molecules derived from
non-human species antibody that binds the desired antigen having
one or more complementarity determining regions (CDRs) from the
non-human species antibody and framework regions from a human
immunoglobulin molecule. Known human Ig sequences are disclosed,
e.g., at worldwide web sites:
www.ncbi.nlm.nih.gov/entrez-/query.fcgi;
www.atcc.org/phage/hdb.html; www.sciquest.com/; www.abcam.com/;
www.antibodyresource.com/onlinecomp.html;
www.public.iastate.edu/.about.pedro/research_tools.html;
www.mgen.uni-heidelberg.de/SD/IT/IT.html;
www.whfreeman.com/immunology/CH-05/kuby05.htm;
www.library.thinkquest.org/12429/Immune/Antibody.html;
www.hhmi.org/grants/lectures/1996/vlab/;
www.path.cam.ac.uk/.about.mrc7/m-ikeimages.html;
www.antibodyresource.com/;
mcb.harvard.edu/BioLinks/Immunology.html. www.immunologylink.com/;
pathbox.wustl.edu/.about.hcenter/index.-html;
www.biotech.ufl.edu/.about.hcl/;
www.pebio.com/pa/340913/340913.html-;
www.nal.usda.gov/awic/pubs/antibody/;
www.m.ehime-u.acjp/.about.yasuhito-/Elisa.html;
www.biodesign.com/table.asp;
www.icnet.uk/axp/facs/davies/lin-ks.html;
www.biotech.ufl.edu/.about.fccl/protocol.html;
www.isac-net.org/sites_geo.html;
aximtl.imt.uni-marburg.de/.about.rek/AEP--Start.html;
baserv.uci.kun.nl/.aboutjraats/linksl.html;
www.recab.uni-hd.de/immuno bme.nwu.edu/;
www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html;
www.ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr:8104/;
www.biochem.ucl.ac.uk/.about.martin/abs/index.html;
antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/wwwabgen.html;
www.unizh.ch/.about.honegger/AHOseminar/Slide01.html;
www.cryst.bbk.ac.uk/.about.ubcg07s/; www.nimr
mrc.ac.uk/CC/ccaewg/ccaewg.htm;
www.path.cam.ac.uk/.about.mrc7/humanisation/TAHHP.html;
www.ibt.unam.mx/vir/structure/stat_aim.html;
www.biosci.missouri.edu/smithgp/index.html;
www.cryst.bioc.cam.ac.uk/.abo-ut.finolina/Web-pages/Pept/spottech.html;
wwwjerini.de/fr roducts.htm; www.patents.ibm.com/ibm.html.Kabat et
al., Sequences of Proteins of Immunological Interest, U.S. Dept.
Health (1983), each entirely incorporated herein by reference. Such
imported sequences can be used to reduce immunogenicity or reduce,
enhance or modify binding, affinity, on-rate, off-rate, avidity,
specificity, half-life, or any other suitable characteristic, as
known in the art.
[0758] Framework (FR) residues in the human framework regions may
be substituted with the corresponding residue from the CDR donor
antibody to alter, preferably improve, antigen binding. These
framework substitutions are identified by methods well known in the
art, e.g., by modeling of the interactions of the CDR and framework
residues to identify framework residues important for antigen
binding and sequence comparison to identify unusual framework
residues at particular positions. See, e.g., Queen et al., U.S.
Pat. No. 5,585,089; Riechmann et al., Nature, 332: 323-327 (1988),
which are incorporated herein by reference in their entireties.
Three-dimensional immunoglobulin models are commonly available and
are familiar to those skilled in the art. Computer programs are
available which illustrate and display probable three-dimensional
conformational structures of selected candidate immunoglobulin
sequences. Inspection of these displays permits analysis of the
likely role of the residues in the functioning of the candidate
immunoglobulin sequence, i.e., the analysis of residues that
influence the ability of the candidate immunoglobulin to bind its
antigen. In this way, FR residues can be selected and combined from
the consensus and import sequences so that the desired antibody
characteristic, such as increased affinity for the target
antigen(s), is achieved. In general, the CDR residues are directly
and most substantially involved in influencing antigen binding.
Antibodies can be humanized using a variety of techniques known in
the art, such as but not limited to those described in Jones et
al., Nature, 321:522-525 (1986); Verhoeyen et al., Science,
239:1534-1536 (1988); Sims et al., J. Immunol., 151: 2296-2308
(1993); Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987),
Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285-4289 (1992);
Presta et al., J. Immunol., 151: 2623-2632 (1993); Padlan,
Molecular Immunology, 28(4/5): 489-498 (1991); Studnicka et al.,
Protein Engineering, 7(6): 805-814 (1994); Roguska et al., Proc.
Natl. Acad. Sci. USA, 91: 969-973 (1994); PCT Publication Nos. WO
91/09967; WO 90/14443; WO 90/14424; WO 90/14430; WO 99/06834
(PCT/US98/16280); WO 97/20032 (PCT/US96/18978); WO 92/11272
(PCT/US91/09630); WO 92/03461 (PCT/US91/05939); WO 94/18219
(PCT/US94/01234); WO 92/01047 (PCT/GB91/01134); and WO 93/06213
(PCT/GB92/01755); EP Patent Nos. EP 0 592 106; EP 0 519 596 and EP
0 239 400; U.S. Pat. Nos. 5,565,332; 5,723,323; 5,976,862;
5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886;
5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089;
5,225,539 and 4,816,567, each entirely incorporated herein by
reference, included references cited therein.
5. Anti IL-17 DVD-Ig.TM. Binding Proteins
[0759] Also provided are dual variable domain immunoglobulin
binding proteins (DVD-Igs) that bind one or more epitopes of IL-17,
the IL-17A monomer, the IL-17A dimer, the IL-17F monomer, and the
IL-17A/IL-17F heterodimer A DVD-Ig binding protein may also bind an
epitope of IL-17 and an epitope of a second target antigen other
than an IL-17A and/or IL-17F polypeptide. A preferred embodiment of
such DVD-Ig molecules comprises a heavy chain that comprises the
structural formula VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first
heavy chain variable domain, VD2 is a second heavy chain variable
domain, C is a heavy chain constant domain, X1 is a linker with the
proviso that it is not CH1, X2 is an Fc region, and n is 0 or 1,
and preferably 1; and a light chain that comprises the structural
formula VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first light chain
variable domain, VD2 is a second light chain variable domain, C is
a light chain constant domain, X1 is a linker with the proviso that
it is not CH1, and X2 does not comprise an Fc region; and n is 0 or
1, and preferably 1. Such a DVD-Ig may comprise two such heavy
chains and two such light chains, wherein each chain comprises
variable domains linked in tandem without an intervening constant
region between variable regions, wherein a heavy chain and a light
chain associate to form two tandem antigen binding sites, and a
pair of heavy and light chains may associate with another pair of
heavy and light chains to form a tetrameric binding protein with
four antigen binding sites. In another embodiment, a DVD-Ig
molecule may comprise heavy and light chains that each comprise
three variable domains, e.g., VD1, VD2, VD3, linked in tandem
without an intervening constant region between variable domains,
wherein a pair of heavy and light chains may associate to form
three antigen binding sites, and wherein a pair of heavy and light
chains may associate with another pair of heavy and light chains to
form a tetrameric binding protein with six antigen binding
sites.
[0760] Each variable domain (VD) in a DVD-Ig may be obtained from
one or more "parent" monoclonal antibodies that bind one or more
desired antigens or epitopes, such as IL-17, IL-17F, and/or
non-IL-17 antigens or epitopes (e.g., TNF-.alpha.).
A. Generation of Parent Monoclonal Antibodies
[0761] The variable domains of the DVD-Ig binding protein can be
obtained from parent antibodies, including monoclonal antibodies
(mAb), capable of binding antigens of interest. These antibodies
may be naturally occurring or may be generated by recombinant
technology. It is understood that if an antibody that binds a
desired target antigen or epitope is polyclonal then it is still
necessary to obtain the variable domains of an antigen binding site
of a single antibody from the polyclonal population, i.e., of a
single monoclonal member of the polyclonal population, for use in
generating a DVD-Ig. Monoclonal antibodies may be generated by any
of variety of methods known in the art, including those described
herein (see, sections A.1.-A.4., above).
B. Criteria for Selecting Parent Monoclonal Antibodies
[0762] An embodiment of the invention pertains to selecting parent
antibodies with at least one or more properties desired in the
DVD-Ig molecule. In an embodiment, the desired property is selected
from one or more antibody parameters. In another embodiment, the
antibody parameters are selected from the group consisting of
antigen specificity, affinity to antigen, potency, biological
function, epitope recognition, stability, solubility, production
efficiency, immunogenicity, pharmacokinetics, bioavailability,
tissue cross reactivity, and orthologous antigen binding.
B1. Affinity to Antigen
[0763] The desired affinity of a therapeutic mAb may depend upon
the nature of the antigen, and the desired therapeutic end-point.
In an embodiment, monoclonal antibodies have higher affinities
(Kd=0.01-0.50 pM) when blocking a cytokine-cytokine receptor
interaction as such interaction are usually high affinity
interactions (e.g., <pM-<nM ranges). In such instances, the
mAb affinity for its target should be equal to or better than the
affinity of the cytokine (ligand) for its receptor. On the other
hand, mAb with lesser affinity (>nM range) could be
therapeutically effective, e.g., in clearing circulating
potentially pathogenic proteins e.g., monoclonal antibodies that
bind to, sequester, and clear circulating species of a target
antigen, such as A-.beta. amyloid. In other instances, reducing the
affinity of an existing high affinity mAb by site-directed
mutagenesis or using a mAb with lower affinity for its target could
be used to avoid potential side-effects, e.g., a high affinity mAb
may sequester or neutralize all of its intended target, thereby
completely depleting/eliminating the function(s) of the targeted
protein. In this scenario, a low affinity mAb may
sequester/neutralize a fraction of the target that may be
responsible for the disease symptoms (the pathological or
over-produced levels), thus allowing a fraction of the target to
continue to perform its normal physiological function(s).
Therefore, it may be possible to reduce the Kd to adjust dose
and/or reduce side-effects. The affinity of the parental mAb might
play a role in appropriately targeting cell surface molecules to
achieve desired therapeutic out-come. For example, if a target is
expressed on cancer cells with high density and on normal cells
with low density, a lower affinity mAb will bind a greater number
of targets on tumor cells than normal cells, resulting in tumor
cell elimination via ADCC or CDC, and therefore might have
therapeutically desirable effects. Thus, selecting a mAb with
desired affinity may be relevant for both soluble and surface
targets.
[0764] Signaling through a receptor upon interaction with its
ligand may depend upon the affinity of the receptor-ligand
interaction. Similarly, it is conceivable that the affinity of a
mAb for a surface receptor could determine the nature of
intracellular signaling and whether the mAb may deliver an agonist
or an antagonist signal. The affinity-based nature of mAb-mediated
signaling may have an impact of its side-effect profile. Therefore,
the desired affinity and desired functions of therapeutic
monoclonal antibodies need to be determined carefully by in vitro
and in vivo experimentation.
[0765] The desired Kd of a binding protein (e.g., an antibody) may
be determined experimentally depending on the desired therapeutic
outcome. In an embodiment, parent antibodies with affinity (Kd) for
a particular antigen equal to, or better than, the desired affinity
of the DVD-Ig for the same antigen are selected. The antigen
binding affinity and kinetics are assessed by Biacore or another
similar technique. In one embodiment, each parent antibody has a
dissociation constant (Kd) to its antigen selected from the group
consisting of: at most about 10.sup.-7 M; at most about 10.sup.-8
M; at most about 10.sup.-9 M; at most about 10.sup.-10 M; at most
about 10.sup.-11 M; at most about 10.sup.-12 M; and at most
10.sup.-13 M. First parent antibody from which VD1 is obtained and
second parent antibody from which VD2 is obtained may have similar
or different affinity (K.sub.D) for the respective antigen. Each
parent antibody has an on rate constant (Kon) to the antigen
selected from the group consisting of: at least about
10.sup.2M.sup.-1s.sup.-1; at least about 10.sup.3M.sup.-1s.sup.-1;
at least about 10.sup.4M.sup.-1s.sup.-1; at least about
10.sup.5M.sup.-1s.sup.-1; and at least about
10.sup.6M.sup.-1s.sup.-1, as measured by surface plasmon resonance.
The first parent antibody from which, for example, a VD1 is
obtained and the second parent antibody from which a VD2 is
obtained may have similar or different on rate constant (Kon) for
the respective antigen. In one embodiment, each parent antibody has
an off rate constant (Koff) to the antigen selected from the group
consisting of: at most about 10.sup.-3s.sup.-1; at most about
10.sup.-4s.sup.-1; at most about 10.sup.-5s.sup.-1; and at most
about 10.sup.-6s.sup.-1, as measured by surface plasmon resonance.
The first parent antibody from which VD1 is obtained and the second
parent antibody from which VD2 is obtained may have similar or
different off rate constants (Koff) for the respective antigen.
B2. Potency
[0766] The desired affinity/potency of parental monoclonal
antibodies will depend on the desired therapeutic outcome. For
example, for receptor-ligand (R-L) interactions the affinity (kd)
is equal to or better than the R-L kd (pM range). For simple
clearance of a pathologic circulating proteins, the Kd could be in
low nM range, e.g., clearance of various species of circulating
A-.beta. peptide. In addition, the Kd will also depend on whether
the target expresses multiple copies of the same epitope, e.g., an
mAb targeting conformational epitope in A.beta. oligomers.
[0767] Where VDI and VD2 bind the same antigen, but distinct
epitopes, the DVD-Ig will contain binding sites for the same
antigen, thus increasing avidity and thereby the apparent Kd of the
DVD-Ig. In an embodiment, parent antibodies with equal or lower Kd
than that desired in the DVD-Ig are chosen. The affinity
considerations of a parental mAb may also depend upon whether the
DVD-Ig contains four or more identical antigen binding sites (i.e.,
a DVD-Ig from a single mAb). In this case, the apparent Kd would be
greater than the mAb due to avidity. Such DVD-Igs can be employed
for cross-linking surface receptor, increased neutralization
potency, enhanced clearance of pathological proteins, etc.
[0768] In another embodiment, parent antibodies with neutralization
potency for specific antigen equal to or better than the desired
neutralization potential of the DVD-Ig for the same antigen are
selected. The neutralization potency can be assessed by a
target-dependent bioassay where cells of appropriate type produce a
measurable signal (i.e., proliferation or cytokine production) in
response to target stimulation, and target neutralization by the
mAb can reduce the signal in a dose-dependent manner.
B3. Biological Functions
[0769] Monoclonal antibodies can perform potentially several
functions. Some of these functions are listed in Table 8. These
functions can be assessed by both in vitro assays (e.g., cell-based
and biochemical assays) and in vivo animal models.
TABLE-US-00009 TABLE 8 Some Potential Applications For Therapeutic
Antibodies. Target (Class) Mechanism of Action (target) Soluble
Neutralization of activity (e.g., a cytokine, such IL-17)
(cytokines, other) Enhance clearance (e.g., A.beta. oligomers)
Increase half-life (e.g., GLP 1) Cell Surface Agonist (e.g., GLP1
R, EPO R, etc.) (Receptors, other) Antagonist (e.g., integrins,
etc.) Cytotoxic (CD 20, etc.) Protein deposits Enhance
clearance/degradation (e.g., A.beta. plaques, amyloid deposits)
[0770] MAbs with distinct functions described in the examples
herein and in Table 8 can be selected to achieve desired
therapeutic outcomes. Two or more selected parent monoclonal
antibodies can then be used in DVD-Ig format to achieve two
distinct functions in a single DVD-Ig molecule. For example, a
DVD-Ig can be generated by selecting a parent mAb that neutralizes
function of a specific cytokine, such as IL-17, and selecting a
parent mAb that enhances clearance of a pathological protein.
Similarly, two parent mAbs may be selected that recognize two
different cell surface receptors, one mAb with an agonist function
on one receptor and the other mAb with an antagonist function on a
different receptor. These two selected mAbs, each with a distinct
function, can be used to construct a single DVD-Ig molecule that
will possess the two distinct functions (agonist and antagonist) of
the selected monoclonal antibodies in a single molecule. Similarly,
two antagonistic mAbs to cell surface receptors, each blocking
binding of respective receptor ligands (e.g., EGF and IGF), may be
used in a DVD-Ig format. Conversely, an antagonistic anti-receptor
mAb (e.g., anti-EGFR) and a neutralizing anti-soluble mediator
(e.g., anti-IGF1/2) mAb can be selected to make a DVD-Ig.
B4. Epitope Recognition:
[0771] Different regions of proteins may perform different
functions. For example, specific regions of a cytokine, such as
IL-17, interact with the cytokine receptor to bring about receptor
activation whereas other regions of the protein may be required for
stabilizing the cytokine. In this instance, one may select a mAb
that binds specifically to the receptor interacting region(s) on
the cytokine and thereby block cytokine-receptor interaction. In
some cases, for example certain chemokine receptors that bind
multiple ligands, a mAb that binds to the epitope (region on
chemokine receptor) that interacts with only one ligand can be
selected. In other instances, monoclonal antibodies can bind to
epitopes on a target that are not directly responsible for
physiological functions of the protein, but binding of a mAb to
these regions could either interfere with physiological functions
(steric hindrance) or alter the conformation of the protein such
that the protein cannot function (mAb to receptors with multiple
ligand which alter the receptor conformation such that none of the
ligand can bind). Anti-cytokine monoclonal antibodies that do not
block binding of the cytokine to its receptor, but block signal
transduction have also been identified (e.g., 125-2H, an anti-IL-18
mAb).
[0772] Examples of epitopes and mAb functions include, but are not
limited to, blocking Receptor-Ligand (R-L) interaction
(neutralizing mAb that binds R-interacting site); steric hindrance
resulting in diminished or no R-binding. An antibody can bind the
target at a site other than a receptor binding site, but still
interfere with receptor binding and functions of the target by
inducing conformational change and eliminate function (e.g.,
XOLAIR.RTM. omalizumab, Genetech/Novartis), binding to R but block
signaling (125-2H mAb).
[0773] In an embodiment, the parental mAb needs to target the
appropriate epitope for maximum efficacy. Such epitope should be
conserved in the DVD-Ig. The binding epitope of a mAb can be
determined by several approaches, including co-crystallography,
limited proteolysis of mAb-antigen complex plus mass spectrometric
peptide mapping (Legros V. et al 2000 Protein Sci. 9:1002-10),
phage displayed peptide libraries (O'Connor K H et al 2005 J
Immunol Methods. 299:21-35), as well as mutagenesis (Wu C. et al.
2003 J Immunol 170:5571-7).
B5. Physicochemical and Pharmaceutical Properties:
[0774] Therapeutic treatment with antibodies often requires
administration of high doses, often several mg/kg (due to a low
potency on a mass basis as a consequence of a typically large
molecular weight). In order to accommodate patient compliance and
to adequately address chronic disease therapies and outpatient
treatment, subcutaneous (s.c.) or intramuscular (i.m.)
administration of therapeutic mAbs is desirable. For example, the
maximum desirable volume for s.c. administration is .about.1.0 mL,
and therefore, concentrations of >100 mg/mL are desirable to
limit the number of injections per dose. In an embodiment, the
therapeutic antibody is administered in one dose. The development
of such formulations is constrained, however, by protein-protein
interactions (e.g., aggregation, which potentially increases
immunogenicity risks) and by limitations during processing and
delivery (e.g., viscosity). Consequently, the large quantities
required for clinical efficacy and the associated development
constraints limit full exploitation of the potential of antibody
formulation and s.c. administration in high-dose regimens. It is
apparent that the physicochemical and pharmaceutical properties of
a protein molecule and the protein solution are of utmost
importance, e.g., stability, solubility and viscosity features.
B5.1. Stability
[0775] A "stable" antibody formulation is one in which the antibody
therein essentially retains its physical stability and/or chemical
stability and/or biological activity upon storage. Stability can be
measured at a selected temperature for a selected time period. In
an embodiment, the antibody in the formulation is stable at room
temperature (about 30.degree. C.) or at 40.degree. C. for at least
1 month and/or stable at about 2-8.degree. C. for at least 1 year
for at least 2 years. Furthermore, in an embodiment, the
formulation is stable following freezing (to, e.g., -70.degree. C.)
and thawing of the formulation, hereinafter referred to as a
"freeze/thaw cycle." In another example, a "stable" formulation may
be one wherein less than about 10% and less than about 5% of the
protein is present as an aggregate in the formulation.
[0776] A DVD-Ig stable in vitro at various temperatures for an
extended time period is desirable. One can achieve this by rapid
screening of parental mAbs stable in vitro at elevated temperature,
e.g., at 40.degree. C. for 2-4 weeks, and then assess stability.
During storage at 2-8.degree. C., the protein reveals stability for
at least 12 months, e.g., at least 24 months. Stability (% of
monomeric, intact molecule) can be assessed using various
techniques such as cation exchange chromatography, size exclusion
chromatography, SDS-PAGE, as well as bioactivity testing. For a
more comprehensive list of analytical techniques that may be
employed to analyze covalent and conformational modifications, see,
Jones, A. J. S. (1993) "Analytical methods for the assessment of
protein formulations and delivery systems," In Cleland, J. L.;
Langer, R., editors. Formulation and delivery of peptides and
proteins, 1st edition, (Washington, ACS), pages 22-45; and
Pearlman, R.; Nguyen, T. H. (1990) "Analysis of protein drugs," In
Lee, V. H., editor. Peptide and protein drug delivery, 1st edition
(New York, Marcel Dekker, Inc.) pages 247-301.
[0777] Heterogeneity and aggregate formation: stability of the
antibody may be such that the formulation may reveal less than
about 10%, and, in an embodiment, less than about 5%, in another
embodiment, less than about 2%, or, in an embodiment, within the
range of 0.5% to 1.5% or less in the GMP antibody material that is
present as aggregate. Size exclusion chromatography is a method
that is sensitive, reproducible, and very robust in the detection
of protein aggregates.
[0778] In addition to low aggregate levels, the antibody must, in
an embodiment, be chemically stable. Chemical stability may be
determined by ion exchange chromatography (e.g., cation or anion
exchange chromatography), hydrophobic interaction chromatography,
or other methods such as isoelectric focusing or capillary
electrophoresis. For instance, chemical stability of the antibody
may be such that after storage of at least 12 months at 2-8.degree.
C. the peak representing unmodified antibody in a cation exchange
chromatography may increase not more than 20%, in an embodiment,
not more than 10%, or, in another embodiment, not more than 5% as
compared to the antibody solution prior to storage testing.
[0779] In an embodiment, the parent antibodies display structural
integrity; correct disulfide bond formation, and correct folding:
Chemical instability due to changes in secondary or tertiary
structure of an antibody may impact antibody activity. For
instance, stability as indicated by activity of the antibody may be
such that after storage of at least 12 months at 2-8.degree. C. the
activity of the antibody may decrease not more than 50%, in an
embodiment not more than 30%, or even not more than 10%, or in an
embodiment not more than 5% or 1% as compared to the antibody
solution prior to storage testing. Suitable antigen-binding assays
can be employed to determine antibody activity.
B5.2. Solubility:
[0780] The "solubility" of a mAb correlates with the production of
correctly folded, monomeric IgG. The solubility of the IgG may
therefore be assessed by HPLC. For example, soluble (monomeric) IgG
will give rise to a single peak on the HPLC chromatograph, whereas
insoluble (e.g., multimeric and aggregated) will give rise to a
plurality of peaks. A person skilled in the art will therefore be
able to detect an increase or decrease in solubility of an IgG
using routine HPLC techniques. For a more comprehensive list of
analytical techniques that may be employed to analyze solubility
(see, Jones, A. G. Dep. Chem. Biochem. Eng., Univ. Coll. London,
London, UK. Editor(s): Shamlou, P. Ayazi, Process. Solid-Liquid
Suspensions (1993), 93-117. (Butterworth-Heinemann, Oxford, UK) and
Pearlman, Rodney; Nguyen, Tue H, Advances in Parenteral Sciences
(1990), 4 (Pept. Protein Drug Delivery), 247-301. Solubility of a
therapeutic mAb is critical for formulating to high concentration
often required for adequate dosing. As outlined herein,
solubilities of >100 mg/mL may be required to accommodate
efficient antibody dosing. For instance, antibody solubility may be
not less than about 5 mg/mL in early research phase, in an
embodiment not less than about 25 mg/mL in advanced process science
stages, or in an embodiment not less than about 100 mg/mL, or in an
embodiment not less than about 150 mg/mL. The intrinsic properties
of a protein molecule are important to the physico-chemical
properties of the protein solution, e.g., stability, solubility,
viscosity. However, a person skilled in the art will appreciate
that a broad variety of excipients exist that may be used as
additives to beneficially impact the characteristics of the final
protein formulation. These excipients may include: (i) liquid
solvents, cosolvents (e.g., alcohols such as ethanol); (ii)
buffering agents (e.g., phosphate, acetate, citrate, amino acid
buffers); (iii) sugars or sugar alcohols (e.g., sucrose, trehalose,
fructose, raffinose, mannitol, sorbitol, dextrans); (iv)
surfactants (e.g., polysorbate 20, 40, 60, 80, poloxamers); (v)
isotonicity modifiers (e.g., salts such as NaCl, sugars, sugar
alcohols); and (vi) others (e.g., preservatives, chelating agents,
antioxidants, chelating substances (e.g., EDTA), biodegradable
polymers, carrier molecules (e.g., HSA, PEGs)
[0781] Viscosity is a parameter of high importance with regard to
antibody manufacture and antibody processing (e.g.,
diafiltration/ultrafiltration), fill-finish processes (pumping
aspects, filtration aspects) and delivery aspects (syringeability,
sophisticated device delivery). Low viscosities enable the liquid
solution of the antibody having a higher concentration. This
enables the same dose to be administered in smaller volumes. Small
injection volumes inhere the advantage of lower pain on injection
sensations, and the solutions do not necessarily have to be
isotonic to reduce pain on injection in the patient. The viscosity
of the antibody solution may be such that at shear rates of 100
(1/s) antibody solution viscosity is below 200 mPa s, in an
embodiment below 125 mPa s, in another embodiment below 70 mPa s,
and in yet another embodiment below 25 mPa s or even below 10 mPa
s.
B5.3. Production Efficiency
[0782] The generation of a DVD-Ig that is efficiently expressed in
mammalian cells, such as Chinese hamster ovary cells (CHO), will in
an embodiment require two parental monoclonal antibodies which are
themselves expressed efficiently in mammalian cells. The production
yield from a stable mammalian line (i.e., CHO) should be above
about 0.5 g/L, in an embodiment above about 1 g/L, and in another
embodiment in the range of about 2 to about 5 g/L or more
(Kipriyanov S M, Little M. 1999 Mol Biotechnol. 12:173-201; Carroll
S, Al-Rubeai M. 2004 Expert Opin Biol Ther. 4:1821-9).
[0783] Production of antibodies and Ig fusion proteins in mammalian
cells is influenced by several factors. Engineering of the
expression vector via incorporation of strong promoters, enhancers
and selection markers can maximize transcription of the gene of
interest from an integrated vector copy. The identification of
vector integration sites that are permissive for high levels of
gene transcription can augment protein expression from a vector
(Wurm et al, 2004, Nature Biotechnology, 2004, 22(11): 1393-1398).
Furthermore, levels of production are affected by the ratio of
antibody heavy and light chains and various steps in the process of
protein assembly and secretion (Jiang et al. 2006, Biotechnology
Progress, January-February 2006, vol. 22, no. 1, pp. 313-318).
B6. Immunogenicity
[0784] Administration of a therapeutic mAb may result in certain
incidence of an immune response (i.e., the formation of endogenous
antibodies directed against the therapeutic mAb). Potential
elements that might induce immunogenicity should be analyzed during
selection of the parental monoclonal antibodies, and steps to
reduce such risk can be taken to optimize the parental monoclonal
antibodies prior to DVD-Ig construction. Mouse-derived antibodies
have been found to be highly immunogenic in patients. The
generation of chimeric antibodies comprised of mouse variable and
human constant regions presents a logical next step to reduce the
immunogenicity of therapeutic antibodies (Morrison and Schlom,
1990). Alternatively, immunogenicity can be reduced by transferring
murine CDR sequences into a human antibody framework (reshaping/CDR
grafting/humanization), as described for a therapeutic antibody by
Riechmann et al., 1988. Another method is referred to as
"resurfacing" or "veneering", starting with the rodent variable
light and heavy domains, only surface-accessible framework amino
acids are altered to human ones, while the CDR and buried amino
acids remain from the parental rodent antibody (Roguska et al.,
1996). In another type of humanization, instead of grafting the
entire CDRs, one technique grafts only the "specificity-determining
regions" (SDRs), defined as the subset of CDR residues that are
involved in binding of the antibody to its target (Kashmiri et al.,
2005). This necessitates identification of the SDRs either through
analysis of available three-dimensional structures of
antibody-target complexes or mutational analysis of the antibody
CDR residues to determine which interact with the target.
Alternatively, fully human antibodies may have reduced
immunogenicity compared to murine, chimeric, or humanized
antibodies.
[0785] Another approach to reduce the immunogenicity of therapeutic
antibodies is the elimination of certain specific sequences that
are predicted to be immunogenic. In one approach, after a first
generation biologic has been tested in humans and found to be
unacceptably immunogenic, the B-cell epitopes can be mapped and
then altered to avoid immune detection. Another approach uses
methods to predict and remove potential T-cell epitopes.
Computational methods have been developed to scan and to identify
the peptide sequences of biologic therapeutics with the potential
to bind to MHC proteins (Desmet et al., 2005). Alternatively a
human dendritic cell-based method can be used to identify CD4.sup.+
T-cell epitopes in potential protein allergens (Stickler et al.,
2005; S. L. Morrison and J. Schlom, Important Adv. Oncol. (1990),
pp. 3-18; Riechmann, L., Clark, M., Waldmann, H. and Winter, G.
"Reshaping human antibodies for therapy," Nature (1988) 332:
323-327; Roguska-M-A, Pedersen-J-T, Henry-A-H, Searle-S-M,
Roja-C-M, Avery-B, Hoffee-M, Cook-S, Lambert-J-M, Blattler-W-A,
Rees-A-R, Guild-B-C, "A comparison of two murine mAbs humanized by
CDR-grafting and variable domain resurfacing," Protein Engineering,
(1996), 9: 895-904; Kashmiri-Syed-V-S, De-Pascalis-Roberto,
Gonzales-Noreen-R, Schlom-Jeffrey, "SDR grafting--a new approach to
antibody humanization," Methods (San Diego Calif.), May 2005,
36(1): 25-34; Desmet-Johan, Meersseman-Geert, Boutonnet-Nathalie,
Pletinckx-Jurgen, De-Clercq-Krista, Debulpaep-Maja,
Braeckman-Tessa, Lasters-Ignace, "Anchor profiles of HLA-specific
peptides: analysis by a novel affinity scoring method and
experimental validation," Proteins (2005) 58: 53-69; Stickler-M-M,
Estell-D-A, Harding-F-A., "CD4+ T-cell epitope determination using
unexposed human donor peripheral blood mononuclear cells," J.
Immunother. (2000) 23: 654-60.)
B7. In Vivo Efficacy
[0786] To generate a DVD-Ig molecule with desired in vivo efficacy,
it is important to generate and select mAbs with similarly desired
in vivo efficacy when given in combination. However, in some
instances the DVD-Ig may exhibit in vivo efficacy that cannot be
achieved with the combination of two separate mAbs. For instance, a
DVD-Ig may bring two targets in close proximity leading to an
activity that cannot be achieved with the combination of two
separate mAbs. Additional desirable biological functions are
described herein in section B3. Parent antibodies with
characteristics desirable in the DVD-Ig molecule may be selected
based on factors such as pharmacokinetic half-life (t1/2); tissue
distribution; soluble versus cell surface targets; and target
concentration--soluble/density--surface.
B8. In Vivo Tissue Distribution
[0787] To generate a DVD-Ig molecule with desired in vivo tissue
distribution, in an embodiment, parent mAbs with similar desired in
vivo tissue distribution profile must be selected. Alternatively,
based on the mechanism of the dual-specific targeting strategy, it
may at other times not be required to select parent mAbs with the
similarly desired in vivo tissue distribution when given in
combination. For instance, in the case of a DVD-Ig in which one
binding component targets the DVD-Ig to a specific site thereby
bringing the second binding component to the same target site. For
example, one binding specificity of a DVD-Ig could target pancreas
(islet cells) and the other specificity could bring GLP1 to the
pancreas to induce insulin.
B9. Isotype
[0788] To generate a DVD-Ig molecule with desired properties
including, but not limited to, isotype, effector functions, and the
circulating half-life, parent mAbs are selected that possess
appropriate Fc-effector functions depending on the therapeutic
utility and the desired therapeutic end-point. There are five main
heavy chain classes or isotypes, some of which have several
sub-types and these determine the effector functions of an antibody
molecule. These effector functions reside in the hinge region, CH2,
and CH3 domains of the antibody molecule. However, residues in
other parts of an antibody molecule may have effects on effector
functions as well. The hinge region Fc-effector functions include:
(i) antibody-dependent cellular cytotoxicity (ADCC), (ii)
complement (C1q) binding, activation, and complement-dependent
cytotoxicity (CDC), (iii) phagocytosis/clearance of
antigen-antibody complexes, and (iv) cytokine release in some
instances. These Fc-effector functions of an antibody molecule are
mediated through the interaction of the Fc-region with a set of
class-specific cell surface receptors. Antibodies of the IgG1
isotype are most active while IgG2 and IgG4 having minimal or no
effector functions. The effector functions of the IgG antibodies
are mediated through interactions with three structurally
homologous cellular Fc receptor types (and sub-types) (FcgR1,
FcgRII, and FcgRIII). These effector functions of an IgG1 can be
eliminated by mutating specific amino acid residues in the lower
hinge region (e.g., L234A, L235A) that are required for FcgR and
C1q binding Amino acid residues in the Fc region, in particular the
CH2-CH3 domains, also determine the circulating half-life of the
antibody molecule. This Fc function is mediated through the binding
of the Fc-region to the neonatal Fc receptor (FcRn), which is
responsible for recycling of antibody molecules from the acidic
lysosomes back to the general circulation.
[0789] Whether a mAb should have an active or an inactive isotype
will depend on the desired therapeutic end-point for an antibody.
Some examples of usage of isotypes and desired therapeutic outcome
are listed below: [0790] 1. If the desired end-point is functional
neutralization of a soluble cytokine then an inactive isotype may
be used; [0791] 2. If the desired out-come is clearance of a
pathological protein an active isotype may be used; [0792] 3. If
the desired out-come is clearance of protein aggregates an active
isotype may be used; [0793] 4. If the desired outcome is to
antagonize a surface receptor an inactive isotype is used (Tysabri,
IgG4; OKT3.RTM., mutated IgG1); [0794] 5. If the desired outcome is
to eliminate target cells an active isotype is used (Herceptin,
IgG1 (and with enhanced effector functions); and [0795] 6. If the
desired outcome is to clear proteins from circulation without
entering the CNS an IgM isotype may be used (e.g., clearing
circulating Ab peptide species). The Fc effector functions of a
parental mAb can be determined by various in vitro methods well
known in the art.
[0796] As discussed, the selection of isotype, and thereby the
effector functions will depend upon the desired therapeutic
end-point. In cases where simple neutralization of a circulating
target is desired, for example blocking receptor-ligand
interactions, the effector functions may not be required. In such
instances, isotypes or mutations in the Fc-region of an antibody
that eliminate effector functions are desirable. In other instances
where elimination of target cells is the therapeutic end-point, for
example elimination of tumor cells, isotypes or mutations or
de-fucosylation in the Fc-region that enhance effector functions
are desirable (Presta G L, Adv. Drug Delivery Rev., 58: 640-656,
2006; Satoh M., Iida S., Shitara K., Expert Opinion Biol. Ther., 6:
1161-1173, 2006). Similarly, depending up on the therapeutic
utility, the circulating half-life of an antibody molecule can be
reduced/prolonged by modulating antibody-FcRn interactions by
introducing specific mutations in the Fc region (Dall'Acqua W F,
Kiener P A, Wu H., J. Biol. Chem., 281: 23514-23524 (2006); Petkova
S B., Akilesh S., Sproule T J. et al., Internat. Immunol., 18:
1759-1769 (2006); Vaccaro C., Bawdon R., Wanjie S et al., Proc.
Natl. Acad. Sci. USA, 103: 18709-18714 (2007).
[0797] The published information on the various residues that
influence the different effector functions of a normal therapeutic
mAb may need to be confirmed for a DVD-Ig. It may be possible that
in a DVD-Ig format additional (different) Fc-region residues, other
than those identified for the modulation of monoclonal antibody
effector functions, may be important.
[0798] Overall, the decision as to which Fc-effector functions
(isotype) will be critical in the final DVD-Ig format will depend
up on the disease indication, therapeutic target, desired
therapeutic end-point, and safety considerations. Listed below are
exemplary appropriate heavy chain and light chain constant regions
including, but not limited to: [0799] IgG1--allotype: G1mz [0800]
IgG1 mutant--A234, A235 [0801] IgG2--allotype: G2m(n-) [0802]
Kappa--Km3 [0803] Lambda
[0804] Fc Receptor and C1q Studies:
[0805] The possibility of unwanted antibody-dependent cell-mediated
cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) by
antibody complexing to any overexpressed target on cell membranes
can be abrogated by (for example, L234A, L235A) hinge-region
mutations. These substituted amino acids, present in the IgG1 hinge
region of mAb, are expected to result in diminished binding of mAb
to human Fc receptors (but not FcRn), as FcgR binding is thought to
occur within overlapping sites on the IgG1 hinge region. This
feature of mAb may lead to an improved safety profile over
antibodies containing a wild-type IgG. Binding of mAb to human Fc
receptors can be determined by flow cytometry experiments using
cell lines (e.g., THP-1, K562) and an engineered CHO cell line that
expresses FcgRIIb (or other FcgRs). Compared to IgG1 control
monoclonal antibodies, mAb show reduced binding to FcgRI and
FcgRIIa whereas binding to FcgRIIb is unaffected. The binding and
activation of C1q by antigen/IgG immune complexes triggers the
classical complement cascade with consequent inflammatory and/or
immunoregulatory responses. The C1q binding site on IgGs has been
localized to residues within the IgG hinge region. C1q binding to
increasing concentrations of mAb was assessed by C1q ELISA. The
results demonstrate that mAb is unable to bind to C1q, as expected
when compared to the binding of a wildtype control IgG1. Overall,
the L234A, L235A hinge region mutation abolishes binding of mAb to
FcgRI, FcgRIIa, and C1q, but does not impact the interaction of mAb
with FcgRIIb. These data suggest that in vivo mAb with mutant Fc
will interact normally with the inhibitory FcgRIIb but will likely
fail to interact with the activating FcgRI and FcgRIIa receptors or
C1q.
[0806] Human FcRn Binding:
[0807] The neonatal receptor (FcRn) is responsible for transport of
IgG across the placenta and to control the catabolic half-life of
the IgG molecules. It might be desirable to increase the terminal
half-life of an antibody to improve efficacy, to reduce the dose or
frequency of administration, or to improve localization to the
target. Alternatively, it might be advantageous to do the converse
that is, to decrease the terminal half-life of an antibody to
reduce whole body exposure or to improve the target-to-non-target
binding ratios. Tailoring the interaction between IgG and its
salvage receptor, FcRn, offers a way to increase or decrease the
terminal half-life of IgG. Proteins in the circulation, including
IgG, are taken up in the fluid phase through micropinocytosis by
certain cells, such as those of the vascular endothelia. IgG can
bind FcRn in endosomes under slightly acidic conditions (pH
6.0-6.5) and can recycle to the cell surface, where it is released
under almost neutral conditions (pH 7.0-7.4). Mapping of the
Fc-region-binding site on FcRn80, 16, 17 showed that two histidine
residues that are conserved across species, His310 and His435, are
responsible for the pH dependence of this interaction. Using
phage-display technology, a mouse Fc-region mutation that increases
binding to FcRn and extends the half-life of mouse IgG was
identified (see Victor, G. et al., Nature Biotechnology, 15(7):
637-640 (1997)). Fc-region mutations that increase the binding
affinity of human IgG for FcRn at pH 6.0, but not at pH 7.4, have
also been identified (see, Dall'Acqua et al., J. Immunol., 169(9):
5171-80 (2002)). Moreover, in one case, a similar pH-dependent
increase in binding (up to 27-fold) was also observed for rhesus
FcRn, and this resulted in a twofold increase in serum half-life in
rhesus monkeys compared with the parent IgG (see, Hinton et al., J.
Biol. Chem., 279(8): 6213-6216 (2004)). These findings indicate
that it is feasible to extend the plasma half-life of antibody
therapeutics by tailoring the interaction of the Fc region with
FcRn. Conversely, Fc-region mutations that attenuate interaction
with FcRn can reduce antibody half-life.
B.10. Pharmacokinetics (PK)
[0808] To generate a DVD-Ig molecule with desired pharmacokinetic
profile, in an embodiment, parent mAbs with the similarly desired
pharmacokinetic profile are selected. One consideration is that
immunogenic response to monoclonal antibodies (i.e., "HAHA", human
anti-human antibody response; "HACA", human anti-chimeric antibody
response) further complicates the pharmacokinetics of these
therapeutic agents. In an embodiment, monoclonal antibodies with
minimal or no immunogenicity are used for constructing DVD-Ig
molecules such that the resulting DVD-Igs will also have minimal or
no immunogenicity. Some of the factors that determine the PK of a
mAb include, but are not limited to, intrinsic properties of the
mAb (VH amino acid sequence); immunogenicity; FcRn binding and Fc
functions.
[0809] The PK profile of selected parental monoclonal antibodies
can be easily determined in rodents as the PK profile in rodents
correlates well with (or closely predicts) the PK profile of
monoclonal antibodies in cynomolgus monkey and humans.
[0810] After the parental monoclonal antibodies with desired PK
characteristics (and other desired functional properties as
discussed herein) are selected, the DVD-Ig is constructed. As the
DVD-Ig molecules contain two antigen-binding domains from two
parental monoclonal antibodies, the PK properties of the DVD-Ig are
assessed as well. Therefore, while determining the PK properties of
the DVD-Ig, PK assays may be employed that determine the PK profile
based on functionality of both antigen-binding domains derived from
the 2 parent monoclonal antibodies. The PK profile of a DVD-Ig can
be determined. Additional factors that may impact the PK profile of
DVD-Ig include the antigen-binding domain (CDR) orientation, linker
size, and Fc/FcRn interactions. PK characteristics of parent
antibodies can be evaluated by assessing the following parameters:
absorption, distribution, metabolism and excretion.
[0811] Absorption:
[0812] To date, administration of therapeutic monoclonal antibodies
is via parenteral routes (e.g., intravenous [IV], subcutaneous
[SC], or intramuscular [IM]). Absorption of a mAb into the systemic
circulation following either SC or IM administration from the
interstitial space is primarily through the lymphatic pathway.
Saturable, presystemic, proteolytic degradation may result in
variable absolute bioavailability following extravascular
administration. Usually, increases in absolute bioavailability with
increasing doses of monoclonal antibodies may be observed due to
saturated proteolytic capacity at higher doses. The absorption
process for a mAb is usually quite slow as the lymph fluid drains
slowly into the vascular system, and the duration of absorption may
occur over hours to several days. The absolute bioavailability of
monoclonal antibodies following SC administration generally ranges
from 50% to 100%. In the case of a transport-mediating structure at
the blood-brain barrier (BBB) targeted by the DVD-Ig construct,
circulation times in plasma may be reduced due to enhanced
trans-cellular transport at the blood brain barrier (BBB) into the
CNS compartment, where the DVD-Ig is liberated to enable
interaction via its second antigen recognition site.
[0813] Distribution:
[0814] Following IV administration, monoclonal antibodies usually
follow a biphasic serum (or plasma) concentration-time profile,
beginning with a rapid distribution phase, followed by a slow
elimination phase. In general, a biexponential pharmacokinetic
model best describes this kind of pharmacokinetic profile. The
volume of distribution in the central compartment (Vc) for a mAb is
usually equal to or slightly larger than the plasma volume (2-3
liters). A distinct biphasic pattern in serum (plasma)
concentration versus time profile may not be apparent with other
parenteral routes of administration, such as IM or SC, because the
distribution phase of the serum (plasma) concentration-time curve
is masked by the long absorption portion. Many factors, including
physicochemical properties, site-specific and target-oriented
receptor mediated uptake, binding capacity of tissue, and mAb dose
can influence biodistribution of a mAb. Some of these factors can
contribute to nonlinearity in biodistribution for a mAb.
[0815] Metabolism and Excretion:
[0816] Due to the molecular size, intact monoclonal antibodies are
not excreted into the urine via kidney. They are primarily
inactivated by metabolism (e.g., catabolism). For IgG-based
therapeutic monoclonal antibodies, half-lives typically ranges from
hours or 1-2 days to over 20 days. The elimination of a mAb can be
affected by many factors, including, but not limited to, affinity
for the FcRn receptor, immunogenicity of the mAb, the degree of
glycosylation of the mAb, the susceptibility for the mAb to
proteolysis, and receptor-mediated elimination.
B.11. Tissue Cross-Reactivity Pattern on Human and Tox Species
[0817] Identical staining pattern suggests that potential human
toxicity can be evaluated in tox species. Tox species are those
animal in which unrelated toxicity is studied.
[0818] The individual antibodies are selected to meet two criteria:
(1) tissue staining appropriate for the known expression of the
antibody target and (2) similar staining pattern between human and
tox species tissues from the same organ.
[0819] Criterion 1: Immunizations and/or antibody selections
typically employ recombinant or synthesized antigens (proteins,
carbohydrates or other molecules). Binding to the natural
counterpart and counterscreen against unrelated antigens are often
part of the screening funnel for therapeutic antibodies. However,
screening against a multitude of antigens is often unpractical.
Therefore, tissue cross-reactivity studies with human tissues from
all major organs serve to rule out unwanted binding of the antibody
to any unrelated antigens.
[0820] Criterion 2: Comparative tissue cross reactivity studies
with human and tox species tissues (cynomolgus monkey, dog,
possibly rodents, and others, the same 36 or 37 tissues being
tested as in the human study) help to validate the selection of a
tox species. In the typical tissue cross-reactivity studies on
frozen tissue sections, therapeutic antibodies may demonstrate the
expected binding to the known antigen and/or to a lesser degree
binding to tissues based either on low level interactions
(unspecific binding, low level binding to similar antigens, low
level charge based interactions, etc.). In any case, the most
relevant toxicology animal species is the one with the highest
degree of coincidence of binding to human and animal tissue.
[0821] Tissue cross-reactivity studies follow the appropriate
regulatory guidelines including EC CPMP Guideline III/5271/94
"Production and quality control of mAbs" and the 1997 US FDA/CBER
"Points to Consider in the Manufacture and Testing of Monoclonal
Antibody Products for Human Use". Cryosections (5 .mu.m) of human
tissues obtained at autopsy or biopsy were fixed and dried on
object glass. The peroxidase staining of tissue sections are
performed, using the avidin-biotin system. FDA's Guidance "Points
to Consider in the Manufacture and Testing of Monoclonal Antibody
Products for Human Use". Relevant references include Clarke, J.
(2004), Boon, L. (2002a), Boon, L. (2002b), Ryan, A. (1999).
[0822] Tissue-cross reactivity studies are often done in two
stages, with the first stage including cryosections of 32 tissues
(typically: Adrenal Gland, Gastrointestinal Tract, Prostate,
Bladder, Heart, Skeletal Muscle, Blood Cells, Kidney, Skin, Bone
Marrow, Liver, Spinal Cord, Breast, Lung, Spleen, Cerebellum, Lymph
Node, Testes, Cerebral Cortex, Ovary, Thymus, Colon, Pancreas,
Thyroid, Endothelium, Parathyroid, Ureter, Eye, Pituitary, Uterus,
Fallopian Tube and Placenta) from one human donor. In the second
phase, a full cross reactivity study is performed with up to 38
tissues (including adrenal, blood, blood vessel, bone marrow,
cerebellum, cerebrum, cervix, esophagus, eye, heart, kidney, large
intestine, liver, lung, lymph node, breast mammary gland, ovary,
oviduct, pancreas, parathyroid, peripheral nerve, pituitary,
placenta, prostate, salivary gland, skin, small intestine, spinal
cord, spleen, stomach, striated muscle, testis, thymus, thyroid,
tonsil, ureter, urinary bladder, and uterus) from three unrelated
adults. Studies are done typically at minimally two dose
levels.
[0823] The therapeutic antibody (i.e., test article) and isotype
matched control antibody may be biotinylated for avidin-biotin
complex (ABC) detection; other detection methods may include
tertiary antibody detection for a FITC (or otherwise) labeled test
article, or precomplexing with a labeled anti-human IgG for an
unlabeled test article.
[0824] Briefly, cryosections (about 5 .mu.m) of human tissues
obtained at autopsy or biopsy are fixed and dried on object glass.
The peroxidase staining of tissue sections is performed, using the
avidin-biotin system. First (in case of a precomplexing detection
system), the test article is incubated with the secondary
biotinylated anti-human IgG and developed into immune complex. The
immune complex at the final concentrations of 2 and 10 .mu.g/mL of
test article is added onto tissue sections on object glass and then
the tissue sections were reacted for 30 minutes with a
avidin-biotin-peroxidase kit. Subsequently, DAB
(3,3'-diaminobenzidine), a substrate for the peroxidase reaction,
was applied for 4 minutes for tissue staining. Antigen-Sepharose
beads are used as positive control tissue sections.
[0825] Any specific staining is judged to be either an expected
(e.g., consistent with antigen expression) or unexpected reactivity
based upon known expression of the target antigen in question. Any
staining judged specific is scored for intensity and frequency.
Antigen or serum competition or blocking studies can assist further
in determining whether observed staining is specific or
nonspecific.
[0826] If two selected antibodies are found to meet the selection
criteria--appropriate tissue staining, matching staining between
human and toxicology animal specific tissue--they can be selected
for DVD-Ig generation.
[0827] The tissue cross-reactivity study has to be repeated with
the final DVD-Ig construct, but while these studies follow the same
protocol as outline herein, they are more complex to evaluate
because any binding can come from any of the two parent antibodies,
and any unexplained binding needs to be confirmed with complex
antigen competition studies.
[0828] It is readily apparent that the complex undertaking of
tissue cross-reactivity studies with a multispecific molecule like
a DVD-Ig is greatly simplified if the two parental antibodies are
selected for: (1) lack of unexpected tissue cross-reactivity
findings and (2) appropriate similarity of tissue cross-reactivity
findings between the corresponding human and toxicology animal
species tissues.
B.12. Specificity and Selectivity
[0829] To generate a DVD-Ig molecule with desired specificity and
selectivity, one needs to generate and select parent mAbs with the
similarly desired specificity and selectivity profile.
[0830] Binding studies for specificity and selectivity with a
DVD-Ig can be complex due to the four or more binding sites, two
each for each antigen. Briefly, binding studies using ELISA,
BIAcore, KinExA, or other interaction studies with a DVD-Ig need to
monitor the binding of one, two, or more antigens to the DVD-Ig
molecule. While BIAcore technology can resolve the sequential,
independent binding of multiple antigens, more traditional methods
including ELISA or more modern techniques like KinExA cannot.
Therefore careful characterization of each parent antibody is
critical. After each individual antibody has been characterized for
specificity, confirmation of specificity retention of the
individual binding sites in the DVD-Ig molecule is greatly
simplified.
[0831] It is readily apparent that the complex undertaking of
determining the specificity of a DVD-Ig is greatly simplified if
the two parental antibodies are selected for specificity prior to
being combined into a DVD-Ig.
[0832] Antigen-antibody interaction studies can take many forms,
including many classical protein interaction studies, including
ELISA (enzyme linked immunosorbent assay), mass spectrometry,
chemical cross linking, SEC with light scattering, equilibrium
dialysis, gel permeation, ultrafiltration, gel chromatography,
large-zone analytical SEC, micropreparative ultracentrifugation
(sedimentation equilibrium), spectroscopic methods, titration
microcalorimetry, sedimentation equilibrium (in analytical
ultracentrifuge), sedimentation velocity (in analytical
centrifuge), surface plasmon resonance (including BIAcore).
Relevant references include "Current Protocols in Protein Science,"
John E. Coligan, Ben M. Dunn, David W. Speicher, Paul T, Wingfield
(eds.) Volume 3, chapters 19 and 20, published by John Wiley &
Sons Inc., and references included therein and "Current Protocols
in Immunology," John E. Coligan, Barbara E. Bierer, David H.
Margulies, Ethan M. Shevach, Warren Strober (eds.) published by
John Wiley & Sons Inc and relevant references included
therein.
[0833] Cytokine Release in Whole Blood:
[0834] The interaction of mAb with human blood cells can be
investigated by a cytokine release assay (Wing, M. G., Therapeutic
Immunology (1995), 2(4): 183-190; "Current Protocols in
Pharmacology," S. J. Enna, Michael Williams, John W. Ferkany, Terry
Kenakin, Paul Moser, (eds.) published by John Wiley & Sons Inc;
Madhusudan, S., Clinical Cancer Research (2004), 10(19): 6528-6534;
Cox, J. Methods (2006), 38(4): 274-282; Choi, I., Eur. J. Immunol.,
(2001), 31(1): 94-106). Briefly, various concentrations of mAb are
incubated with human whole blood for 24 hours. The concentration
tested should cover a wide range including final concentrations
mimicking typical blood levels in patients (including but not
limited to 100 ng/ml-100 .mu.g/ml). Following the incubation,
supernatants and cell lysates are analyzed for the presence of
IL-1R.alpha., TNF-.alpha., IL-1b, IL-6 and IL-8. Cytokine
concentration profiles generated for mAb are compared to profiles
produced by a negative human IgG control and a positive LPS or PHA
control. The cytokine profile displayed by mAb from both cell
supernatants and cell lysates are compared to that using control
human IgG. In an embodiment, the monoclonal antibody does not
interact with human blood cells to spontaneously release
inflammatory cytokines.
[0835] Cytokine release studies for a DVD-Ig are complex due to the
four or more binding sites, two each for each antigen. Briefly,
cytokine release studies as described herein measure the effect of
the whole DVD-Ig molecule on whole blood or other cell systems, but
cannot resolve which portion of the molecule causes cytokine
release. Once cytokine release has been detected, the purity of the
DVD-Ig preparation has to be ascertained, because some co-purifying
cellular components can cause cytokine release on their own. If
purity is not the issue, fragmentation of DVD-Ig (including but not
limited to removal of Fc portion, separation of binding sites
etc.), binding site mutagenesis or other methods may need to be
employed to deconvolute any observations. It is readily apparent
that this complex undertaking is greatly simplified if the two
parental antibodies are selected for lack of cytokine release prior
to being combined into a DVD-Ig.
B.13. Cross Reactivity to Other Species for Toxicological
Studies
[0836] In an embodiment, the individual antibodies selected with
sufficient cross-reactivity to appropriate tox species, for
example, cynomolgus monkey. Parental antibodies need to bind to
orthologous species target (i.e. cynomolgus monkey) and elicit
appropriate response (modulation, neutralization, activation). In
an embodiment, the cross-reactivity (affinity/potency) to
orthologous species target should be within 10-fold of the human
target. In practice, the parental antibodies are evaluated for
multiple species, including mouse, rat, dog, monkey (and other
non-human primates), as well as disease model species (i.e. sheep
for asthma model). The acceptable cross-reactivity to tox species
from the parental monoclonal antibodies allows future toxicology
studies of DVD-Ig in the same species. For that reason, the two
parental monoclonal antibodies should have acceptable
cross-reactivity for a common tox species therefore allowing
toxicology studies of DVD-Ig in the same species.
[0837] Parent mAbs may be selected from various mAbs capable of
binding specific targets and well known in the art. These include,
but are not limited to anti-IL-17, anti-IL-17F, anti-TNF antibody
(U.S. Pat. No. 6,258,562), anti-IL-12 and/or anti-IL-12p40 antibody
(U.S. Pat. No. 6,914,128); anti-IL-18 antibody (US patent
application publication No. 2005/0147610 A1), anti-C5, anti-CBL,
anti-CD147, anti-gp120, anti-VLA-4, anti-CD11a, anti-CD18,
anti-VEGF, anti-CD40L, anti CD-40 (e.g., see WO2007124299) anti-Id,
anti-ICAM-1, anti-CXCL13, anti-CD2, anti-EGFR, anti-TGF-beta 2,
anti-HGF, anti-cMet, anti DLL-4, anti-NPR1, anti-PLGF, anti-ErbB3,
anti-E-selectin, anti-Fact VII, anti-Her2/neu, anti-F gp,
anti-CD11/18, anti-CD14, anti-ICAM-3, anti-RON, anti CD-19,
anti-CD80 (e.g., see PCT Publication No. WO 2003/039486), anti-CD4,
anti-CD3, anti-CD23, anti-beta2-integrin, anti-alpha4beta7,
anti-CD52, anti-HLA DR, anti-CD22 (see, e.g., U.S. Pat. No.
5,789,554), anti-CD20, anti-MIF, anti-CD64 (FcR), anti-TCR alpha
beta, anti-CD2, anti-Hep B, anti-CA 125, anti-EpCAM, anti-gp120,
anti-CMV, anti-gpIIbIIIa, anti-IgE, anti-CD25, anti-CD33, anti-HLA,
anti-IGF1,2, anti IGFR, anti-VNRintegrin, anti-IL-1alpha,
anti-IL-1beta, anti-IL-1 receptor, anti-IL-2 receptor, anti-IL-4,
anti-IL-4 receptor, anti-IL5, anti-IL-5 receptor, anti-IL-6,
anti-IL-6R, RANKL, NGF, DKK, alphaVbeta3, anti-IL-8, anti-IL-9,
anti-IL-13, anti-IL-13 receptor, and anti-IL-23; IL-23p19; (see,
Presta, "Selection, design, and engineering of therapeutic
antibodies," J. Allergy Clin. Immunol., 116: 731-736 (2005) and at
worldwide website
http://www.path.cam.ac.uk/.about.mrc7/humanisation/antibodies.html).
[0838] Parent mAbs may also be selected from various therapeutic
antibodies approved for use, in clinical trials, or in development
for clinical use. Such therapeutic antibodies include, but are not
limited to, rituximab (Rituxan.RTM., IDEC/Genentech/Roche) (see for
example U.S. Pat. No. 5,736,137), a chimeric anti-CD20 antibody
approved to treat Non-Hodgkin's lymphoma; HuMax-CD20, an anti-CD20
currently being developed by Genmab, an anti-CD20 antibody
described in U.S. Pat. No. 5,500,362, AME-133 (Applied Molecular
Evolution), hA20 (Immunomedics, Inc.), HumaLYM (Intracel), and
PRO70769 (PCT/US2003/040426, entitled "Immunoglobulin Variants and
Uses Thereof"), trastuzumab (Herceptin.RTM., Genentech) (see for
example U.S. Pat. No. 5,677,171), a humanized anti-Her2/neu
antibody approved to treat breast cancer; pertuzumab (rhuMab-2C4,
Omnitarg.RTM.), currently being developed by Genentech; an
anti-Her2 antibody described in U.S. Pat. No. 4,753,894; cetuximab
(Erbitux.RTM., Imclone) (U.S. Pat. No. 4,943,533; PCT WO 96/40210),
a chimeric anti-EGFR antibody in clinical trials for a variety of
cancers; ABX-EGF (U.S. Pat. No. 6,235,883), currently being
developed by Abgenix-Immunex-Amgen; HuMax-EGFr (U.S. Ser. No.
10/172,317), currently being developed by Genmab; 425, EMD55900,
EMD62000, and EMD72000 (Merck KGaA) (U.S. Pat. No. 5,558,864;
Murthy et al. 1987, Arch Biochem Biophys. 252(2):549-60; Rodeck et
al., 1987, J Cell Biochem. 35(4):315-20; Kettleborough et al.,
1991, Protein Eng. 4(7):773-83); ICR62 (Institute of Cancer
Research) (PCT WO 95/20045; Modjtahedi et al., 1993, J. Cell
Biophys. 1993, 22(1-3):129-46; Modjtahedi et al., 1993, Br J
Cancer. 1993, 67(2):247-53; Modjtahedi et al, 1996, Br J Cancer,
73(2):228-35; Modjtahedi et al, 2003, Int J Cancer, 105(2):273-80);
TheraCIM hR3 (YM Biosciences, Canada and Centro de Immunologia
Molecular, Cuba (U.S. Pat. No. 5,891,996; U.S. Pat. No. 6,506,883;
Mateo et al, 1997, Immunotechnology, 3(1):71-81); mAb-806 (Ludwig
Institute for Cancer Research, Memorial Sloan-Kettering) (Jungbluth
et al. 2003, Proc Natl Acad Sci USA. 100(2):639-44); KSB-102 (KS
Biomedix); MR1-1 (IVAX, National Cancer Institute) (PCT WO
0162931A2); and SC100 (Scancell) (PCT WO 01/88138); alemtuzumab
(Campath.RTM., Millenium), a humanized mAb currently approved for
treatment of B-cell chronic lymphocytic leukemia; muromonab-CD3
(Orthoclone OKT3.RTM.), an anti-CD3 antibody developed by Ortho
Biotech/Johnson & Johnson, ibritumomab tiuxetan (Zevalin.RTM.),
an anti-CD20 antibody developed by IDEC/Schering AG, gemtuzumab
ozogamicin (Mylotarg.RTM.), an anti-CD33 (p67 protein) antibody
developed by Celltech/Wyeth, alefacept (Amevive.RTM.), an
anti-LFA-3 Fc fusion developed by Biogen), abciximab (ReoPro.RTM.),
developed by Centocor/Lilly, basiliximab (Simulect.RTM.), developed
by Novartis, palivizumab (Synagis.RTM.), developed by Medimmune,
infliximab (Remicade.RTM.), an anti-TNFalpha antibody developed by
Centocor, adalimumab (Humira.RTM.), an anti-TNFalpha antibody
developed by Abbott, Humicade.RTM., an anti-TNFalpha antibody
developed by Celltech, golimumab (CNTO-148), a fully human TNF
antibody developed by Centocor, etanercept (Enbrel.RTM.), an p75
TNF receptor Fc fusion developed by Immunex/Amgen, lenercept, an
p55TNF receptor Fc fusion previously developed by Roche, ABX-CBL,
an anti-CD147 antibody being developed by Abgenix, ABX-IL8, an
anti-IL8 antibody being developed by Abgenix, ABX-MA1, an
anti-MUC18 antibody being developed by Abgenix, Pemtumomab
(R1549,90Y-muHMFG1), an anti-MUC1 in development by Antisoma,
Therex (R1550), an anti-MUC1 antibody being developed by Antisoma,
AngioMab (AS1405), being developed by Antisoma, HuBC-1, being
developed by Antisoma, Thioplatin (AS1407) being developed by
Antisoma, Antegren.RTM. (natalizumab), an anti-alpha-4-beta-1
(VLA-4) and alpha-4-beta-7 antibody being developed by Biogen,
VLA-1 mAb, an anti-VLA-1 integrin antibody being developed by
Biogen, LTBR mAb, an anti-lymphotoxin beta receptor (LTBR) antibody
being developed by Biogen, CAT-152, an anti-TGF-.beta.2 antibody
being developed by Cambridge Antibody Technology, ABT 874 (J695),
an anti-IL-12 p40 antibody being developed by Abbott, CAT-192, an
anti-TGF.beta.1 antibody being developed by Cambridge Antibody
Technology and Genzyme, CAT-213, an anti-Eotaxin1 antibody being
developed by Cambridge Antibody Technology, LymphoStat-B.RTM. an
anti-Blys antibody being developed by Cambridge Antibody Technology
and Human Genome Sciences Inc., TRAIL-R1mAb, an anti-TRAIL-R1
antibody being developed by Cambridge Antibody Technology and Human
Genome Sciences, Inc., Avastin.RTM. bevacizumab, rhuMAb-VEGF), an
anti-VEGF antibody being developed by Genentech, an anti-HER
receptor family antibody being developed by Genentech, Anti-Tissue
Factor (ATF), an anti-Tissue Factor antibody being developed by
Genentech, Xolair.RTM. (Omalizumab), an anti-IgE antibody being
developed by Genentech, Raptiva.RTM. (Efalizumab), an anti-CD11a
antibody being developed by Genentech and Xoma, MLN-02 Antibody
(formerly LDP-02), being developed by Genentech and Millenium
Pharmaceuticals, HuMax CD4, an anti-CD4 antibody being developed by
Genmab, HuMax-IL15, an anti-IL15 antibody being developed by Genmab
and Amgen, HuMax-Inflam, being developed by Genmab and Medarex,
HuMax-Cancer, an anti-Heparanase I antibody being developed by
Genmab and Medarex and Oxford GcoSciences, HuMax-Lymphoma, being
developed by Genmab and Amgen, HuMax-TAC, being developed by
Genmab, IDEC-131, and anti-CD40L antibody being developed by IDEC
Pharmaceuticals, IDEC-151 (Clenoliximab), an anti-CD4 antibody
being developed by IDEC Pharmaceuticals, IDEC-114, an anti-CD80
antibody being developed by IDEC Pharmaceuticals, IDEC-152, an
anti-CD23 being developed by IDEC Pharmaceuticals, anti-macrophage
migration factor (MIF) antibodies being developed by IDEC
Pharmaceuticals, BEC2, an anti-idiotypic antibody being developed
by Imclone, IMC-1C11, an anti-KDR antibody being developed by
Imclone, DC101, an anti-flk-1 antibody being developed by Imclone,
anti-VE cadherin antibodies being developed by Imclone,
CEA-Cide.RTM. (labetuzumab), an anti-carcinoembryonic antigen (CEA)
antibody being developed by Immunomedics, LymphoCide.RTM.
(Epratuzumab), an anti-CD22 antibody being developed by
Immunomedics, AFP-Cide, being developed by Immunomedics,
MyelomaCide, being developed by Immunomedics, LkoCide, being
developed by Immunomedics, ProstaCide, being developed by
Immunomedics, MDX-010, an anti-CTLA4 antibody being developed by
Medarex, MDX-060, an anti-CD30 antibody being developed by Medarex,
MDX-070 being developed by Medarex, MDX-018 being developed by
Medarex, Osidem.RTM. (IDM-1), and anti-Her2 antibody being
developed by Medarex and Immuno-Designed Molecules, HuMax.RTM.-CD4,
an anti-CD4 antibody being developed by Medarex and Genmab,
HuMax-IL15, an anti-IL15 antibody being developed by Medarex and
Genmab, CNTO 148, an anti-TNF.alpha. antibody being developed by
Medarex and Centocor/Johnson & Johnson, CNTO 1275, an
anti-cytokine antibody being developed by Centocor/Johnson &
Johnson, MOR101 and MOR102, anti-intercellular adhesion molecule-1
(ICAM-1) (CD54) antibodies being developed by MorphoSys, MOR201, an
anti-fibroblast growth factor receptor 3 (FGFR-3) antibody being
developed by MorphoSys, Nuvion.RTM. (visilizumab), an anti-CD3
antibody being developed by Protein Design Labs, HuZAF.RTM., an
anti-gamma interferon antibody being developed by Protein Design
Labs, Anti-.alpha. 5.beta.1 Integrin, being developed by Protein
Design Labs, anti-IL-12, being developed by Protein Design Labs,
ING-1, an anti-Ep-CAM antibody being developed by Xoma, Xolair.RTM.
(Omalizumab) a humanized anti-IgE antibody developed by Genentech
and Novartis, and MLN01, an anti-Beta2 integrin antibody being
developed by Xoma. In another embodiment, the therapeutics include
KRN330 (Kirin); huA33 antibody (A33, Ludwig Institute for Cancer
Research); CNTO 95 (alpha V integrins, Centocor); MEDI-522 (alpha
V.beta.3 integrin, Medimmune); volociximab (alpha V.beta.1
integrin, Biogen/PDL); Human mAb 216 (B cell glycosolated epitope,
NCI); BiTE MT103 (bispecific CD19.times.CD3, Medimmune);
4G7.times.H22 (Bispecific Bcell.times.FcgammaR1, Medarex/Merck
KGa); rM28 (Bispecific CD28.times.MAPG, US Patent No. EP1444268);
MDX447 (EMD 82633) (Bispecific CD64.times.EGFR, Medarex);
Catumaxomab (removab) (Bispecific EpCAM.times.anti-CD3,
Trion/Fres); Ertumaxomab (bispecific HER2/CD3, Fresenius Biotech);
oregovomab (OvaRex) (CA-125, ViRexx); Rencarex.RTM. (WX G250)
(carbonic anhydrase IX, Wilex); CNTO 888 (CCL2, Centocor); TRC105
(CD105 (endoglin), Tracon); BMS-663513 (CD137 agonist, Brystol
Myers Squibb); MDX-1342 (CD19, Medarex); Siplizumab (MEDI-507)
(CD2, Medimmune); Ofatumumab (Humax-CD20) (CD20, Genmab); Rituximab
(Rituxan) (CD20, Genentech); veltuzumab (hA20) (CD20,
Immunomedics); Epratuzumab (CD22, Amgen); lumiliximab (IDEC 152)
(CD23, Biogen); muromonab-CD3 (CD3, Ortho); HuM291 (CD3 fc
receptor, PDL Biopharma); HeFi-1, CD30, NCI); MDX-060 (CD30,
Medarex); MDX-1401 (CD30, Medarex); SGN-30 (CD30, Seattle
Genentics); SGN-33 (Lintuzumab) (CD33, Seattle Genentics);
Zanolimumab (HuMax-CD4) (CD4, Genmab); HCD122 (CD40, Novartis);
SGN-40 (CD40, Seattle Genentics); Campath1h (Alemtuzumab) (CD52,
Genzyme); MDX-1411 (CD70, Medarex); hLL1 (EPB-1) (CD74.38,
Immunomedics); Galiximab (IDEC-144) (CD80, Biogen); MT293
(TRC093/D93) (cleaved collagen, Tracon); HuLuc63 (CS1, PDL Pharma);
ipilimumab (MDX-010) (CTLA4, Brystol Myers Squibb); Tremelimumab
(Ticilimumab, CP-675,2) (CTLA4, Pfizer); HGS-ETR1 (Mapatumumab)
(DR4TRAIL-R1 agonist, Human Genome Science/Glaxo Smith Kline);
AMG-655 (DR5, Amgen); Apomab (DR5, Genentech); CS-1008 (DR5,
Daiichi Sankyo); HGS-ETR2 (lexatumumab) (DR5TRAIL-R2 agonist, HGS);
Cetuximab (Erbitux) (EGFR, Imclone); IMC-11F8, (EGFR, Imclone);
Nimotuzumab (EGFR, YM Bio); Panitumumab (Vectabix) (EGFR, Amgen);
Zalutumumab (HuMaxEGFr) (EGFR, Genmab); CDX-110 (EGFRvIII, AVANT
Immunotherapeutics); adecatumumab (MT201) (Epcam, Merck);
edrecolomab (Panorex, 17-1A) (Epcam.TM., Glaxo/Centocor); MORAb-003
(folate receptor a, Morphotech); KW-2871 (ganglioside GD3, Kyowa);
MORAb-009 (GP-9, Morphotech); CDX-1307 (MDX-1307) (hCGb, Celldex);
Trastuzumab (Herceptin) (HER2, Celldex); Pertuzumab (rhuMAb 2C4)
(HER2 (DI), Genentech); apolizumab (HLA-DR beta chain, PDL Pharma);
AMG-479 (IGF-1R, Amgen); anti-IGF-1R R1507 (IGF1-R, Roche); CP
751871 (IGF1-R, Pfizer); IMC-A12 (IGF1-R, Imclone); BIIB022
(IGF-1R, Biogen); Mik-beta-1 (IL-2Rb (CD122), Hoffman LaRoche);
CNTO 328 (IL6, Centocor); Anti-KIR (1-7F9) (Killer cell Ig-like
Receptor (KIR), Novo); Hu3S193 (Lewis (y), Wyeth, Ludwig Institute
of Cancer Research); hCBE-11 (LT.beta.R, Biogen); HuHMFG1 (MUC1,
Antisoma/NCI); RAV12 (N-linked carbohydrate epitope, Raven); CAL
(parathyroid hormone-related protein (PTH-rP), University of
California); CT-011 (PD1, CureTech); MDX-1106 (ono-4538) (PD1,
Medarex/Ono); MAb CT-011 (PD1, Curetech); IMC-3G3 (PDGFRa,
Imclone); bavituximab (phosphatidylserine, Peregrine); huJ591
(PSMA, Cornell Research Foundation); muJ591 (PSMA, Cornell Research
Foundation); GC1008 (TGFb (pan) inhibitor (IgG4), Genzyme);
Infliximab (Remicade) (TNFa, Centocor); A27.15 (transferrin
receptor, Salk Institute, INSERN WO 2005/111082); E2.3 (transferrin
receptor, Salk Institute); Bevacizumab (Avastin) (VEGF, Genentech);
HuMV833 (VEGF, Tsukuba Research Lab-WO/2000/034337, University of
Texas); IMC-18F1 (VEGFR1, Imclone); IMC-1121 (VEGFR2, Imclone).
C. Construction of DVD-Ig.TM. Binding Proteins
[0839] A multivalent multispecific dual variable domain
immunoglobulin (DVD-Ig.TM.) binding protein is designed such that
two different light chain variable domains (VL) from two different
parent monoclonal antibodies are linked in tandem directly or via a
short linker by recombinant DNA techniques, followed by the light
chain constant domain. Similarly, the heavy chain comprises two
different heavy chain variable domains (VH) linked in tandem,
followed by the constant domain CH1 and Fc region.
[0840] The variable domains can be obtained using recombinant DNA
techniques from a parent antibody generated by any one of the
methods described herein. In an embodiment, the variable domain is
a murine heavy or light chain variable domain. In another
embodiment, the variable domain is a CDR-grafted or a humanized
variable heavy or light chain domain. In an embodiment, the
variable domain is a human heavy or light chain variable
domain.
[0841] In one embodiment, the first and second variable domains are
linked directly to each other using recombinant DNA techniques. In
another embodiment the variable domains are linked via a linker
sequence. In an embodiment, two variable domains are linked. Three
or more variable domains may also be linked directly or via a
linker sequence. The variable domains may bind the same antigen or
may bind different antigens. DVD-Ig molecules of the invention may
include one immunoglobulin variable domain and one
non-immunoglobulin variable domain such as ligand binding domain of
a receptor, active domain of an enzyme. DVD-Ig molecules may also
comprise two or more non-Ig domains.
[0842] The linker sequence may be a single amino acid or a
polypeptide sequence. In an embodiment, the linker sequences are
selected from the group consisting of GGGGSG (SEQ ID NO:887), GGSGG
(SEQ ID NO:888), GGGGSGGGGS (SEQ ID NO:889), GGSGGGGSGS (SEQ ID
NO:890), GGSGGGGSGGGGS (SEQ ID NO:891), GGGGSGGGGSGGGG (SEQ ID
NO:892), GGGGSGGGGSGGGGS (SEQ ID NO:893), ASTKGP (SEQ ID NO:894),
ASTKGPSVFPLAP (SEQ ID NO:895), TVAAP (SEQ ID NO:896), TVAAPSVFIFPP
(SEQ ID NO:897), AKTTPKLEEGEFSEAR (SEQ ID NO:898),
AKTTPKLEEGEFSEARV (SEQ ID NO:899), AKTTPKLGG (SEQ ID NO:900),
SAKTTPKLGG (SEQ ID NO:901), SAKTTP (SEQ ID NO:902), RADAAP (SEQ ID
NO:903), RADAAPTVS (SEQ ID NO:904), RADAAAAGGPGS (SEQ ID NO:905),
RADAAAAGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:906), SAKTTPKLEEGEFSEARV
(SEQ ID NO:907), ADAAP (SEQ ID NO:908), ADAAPTVSIFPP (SEQ ID
NO:909), QPKAAP (SEQ ID NO:910), QPKAAPSVTLFPP (SEQ ID NO:911),
AKTTPP (SEQ ID NO:912), AKTTPPSVTPLAP (SEQ ID NO:913), AKTTAP (SEQ
ID NO:914), AKTTAPSVYPLAP (SEQ ID NO:915), GENKVEYAPALMALS (SEQ ID
NO:916), GPAKELTPLKEAKVS (SEQ ID NO:917), and GHEAAAVMQVQYPAS (SEQ
ID NO:918). The choice of linker sequences is based on crystal
structure analysis of several Fab molecules. There is a natural
flexible linkage between the variable domain and the CH1/CL
constant domain in Fab or antibody molecular structure. This
natural linkage comprises approximately 10-12 amino acid residues,
contributed by 4-6 residues from C-terminus of V domain and 4-6
residues from the N-terminus of CL/CH1 domain. DVD-Igs described
herein can be generated using N-terminal 5-6 amino acid residues,
or 11-12 amino acid residues, of CL or CH1 as linker in light chain
and heavy chain of DVD-Ig, respectively. The N-terminal residues of
CL or CH1 domains, particularly the first 5-6 amino acid residues,
adopt a loop conformation without strong secondary structures, and
therefore can act as flexible linkers between the two variable
domains. The N-terminal residues of CL or CH1 domains are natural
extension of the variable domains, as they are part of the Ig
sequences, and therefore minimize to a large extent any
immunogenicity potentially arising from the linkers and
junctions.
[0843] Other linker sequences may include any sequence of any
length of CL/CH1 domain but not all residues of CL/CH1 domain; for
example the first 5-12 amino acid residues of the CL/CH1 domains;
the light chain linkers can be from C.kappa. or C.lamda.; and the
heavy chain linkers can be derived from CH1 of any isotypes,
including C.gamma.1, C.gamma.2, C.gamma.3, C.gamma.4, C.alpha.1,
C.alpha.2, C.delta., C.epsilon., and C.mu.. Linker sequences may
also be derived from other proteins such as Ig-like proteins,
(e.g., TCR, FcR, KIR); G/S based sequences; hinge region-derived
sequences; and other natural sequences from other proteins.
[0844] In an embodiment a constant domain is linked to the two
linked variable domains using recombinant DNA techniques. In an
embodiment, a sequence comprising tandemly linked heavy chain
variable domains is linked to a heavy chain constant domain and a
sequence comprising tandemly linked light chain variable domains is
linked to a light chain constant domain. In an embodiment, the
constant domains are human heavy chain constant domain and human
light chain constant domain, respectively. In an embodiment, the
DVD heavy chain is further linked to an Fc region. The Fc region
may be a native sequence Fc region, or a variant Fc region. In
another embodiment, the Fc region is a human Fc region. In another
embodiment the Fc region includes Fc region from IgG1, IgG2, IgG3,
IgG4, IgA, IgM, IgE, or IgD.
[0845] In a most preferred embodiment, two heavy chain DVD
polypeptides and two light chain DVD polypeptides are combined to
form a DVD-Ig molecule. Detailed description of specific DVD-Ig
molecules capable of binding specific target antigens, such as
IL-17, and methods of making the same are provided in the Examples
section below.
D. Production of DVD-Ig Binding Proteins
[0846] DVD-Ig binding proteins of the present invention may be
produced by any of a number of techniques known in the art
including, for example, expression from host cells, wherein
expression vector(s) encoding the DVD-Ig heavy and DVD-Ig light
chains is (are) transfected into a host cell by standard
techniques. The various forms of the term "transfection" are
intended to encompass a wide variety of techniques commonly used
for the introduction of exogenous DNA into a prokaryotic or
eukaryotic host cell, e.g., electroporation, calcium-phosphate
precipitation, DEAE-dextran transfection and the like. Although it
is possible to express the DVD-Ig proteins of the invention in
either prokaryotic or eukaryotic host cells, DVD-Ig proteins are
expressed in eukaryotic cells, for example, mammalian host cells,
because such eukaryotic cells (and in particular mammalian cells)
are more likely than prokaryotic cells to assemble and secrete a
properly folded and immunologically active DVD-Ig protein.
[0847] Exemplary mammalian host cells for expressing the
recombinant antibodies of the invention include Chinese Hamster
Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub
and Chasin, (1980) Proc. Natl. Acad. Sci. USA, 77: 4216-4220, used
with a DHFR selectable marker, e.g., as described in R. J. Kaufman
and P. A. Sharp (1982) Mol. Biol., 159: 601-621), NS0 myeloma
cells, COS cells, SP2 and PER.C6 cells. When recombinant expression
vectors encoding DVD-Ig proteins are introduced into mammalian host
cells, the DVD-Ig proteins are produced by culturing the host cells
for a period of time sufficient to allow for expression of the
DVD-Ig proteins in the host cells or secretion of the DVD proteins
into the culture medium in which the host cells are grown. DVD-Ig
proteins can be recovered from the culture medium using standard
protein purification methods.
[0848] In an exemplary system for recombinant expression of DVD-Ig
proteins of the invention, a recombinant expression vector encoding
both the DVD-Ig heavy chain and the DVD-Ig light chain is
introduced into dhfr-CHO cells by calcium phosphate-mediated
transfection. Within the recombinant expression vector, the DVD-Ig
heavy and light chain genes are each operatively linked to CMV
enhancer/AdMLP promoter regulatory elements to drive high levels of
transcription of the genes. The recombinant expression vector also
carries a DHFR gene, which allows for selection of CHO cells that
have been transfected with the vector using methotrexate
selection/amplification. The selected transformant host cells are
cultured to allow for expression of the DVD-Ig heavy and light
chains and intact DVD-Ig protein is recovered from the culture
medium. Standard molecular biology techniques are used to prepare
the recombinant expression vector, transfect the host cells, select
for transformants, culture the host cells and recover the DVD-Ig
protein from the culture medium. Still further the invention
provides a method of synthesizing a DVD-Ig protein of the invention
by culturing a host cell of the invention in a suitable culture
medium until a DVD-Ig protein of the invention is synthesized. The
method can further comprise isolating the DVD-Ig protein from the
culture medium.
[0849] An important feature of DVD-Ig is that it can be produced
and purified in a similar way as a conventional antibody. The
production of DVD-Ig results in a homogeneous, single major product
with desired dual-specific activity, without any sequence
modification of the constant region or chemical modifications of
any kind. Other previously described methods to generate
"bi-specific", "multi-specific", and "multi-specific multivalent"
full length binding proteins do not lead to a single primary
product but instead lead to the intracellular or secreted
production of a mixture of assembled inactive, mono-specific,
multi-specific, multivalent, full length binding proteins, and
multivalent full length binding proteins with combination of
different binding sites. As an example, based on the design
described by Miller and Presta (PCT Publication No. WO
2001/077342(A1), there are 16 possible combinations of heavy and
light chains. Consequently only 6.25% of protein is likely to be in
the desired active form, and not as a single major product or
single primary product compared to the other 15 possible
combinations. Separation of the desired, fully active forms of the
protein from inactive and partially active forms of the protein
using standard chromatography techniques, typically used in large
scale manufacturing, is yet to be demonstrated.
[0850] Surprisingly, the design of the "dual-specific multivalent
full length binding proteins" of the present invention leads to a
dual variable domain light chain and a dual variable domain heavy
chain which assemble primarily to the desired "dual-specific
multivalent full length binding proteins".
[0851] At least 50%, at least 75%, and at least 90% of the
assembled, and expressed DVD-Ig molecules are the desired
dual-specific tetravalent protein. This aspect of the invention
particularly enhances the commercial utility of the invention.
Therefore, the present invention includes a method to express a
dual variable domain light chain and a dual variable domain heavy
chain in a single cell leading to a single primary product of a
"dual-specific tetravalent full length binding protein".
[0852] The present invention provides a methods of expressing a
dual variable domain light chain and a dual variable domain heavy
chain in a single cell leading to a "primary product" of a
"dual-specific, tetravalent, full length binding protein", where
the "primary product" is more than 50% of all assembled protein,
comprising a dual variable domain light chain and a dual variable
domain heavy chain.
[0853] The present invention provides methods of expressing a dual
variable domain light chain and a dual variable domain heavy chain
in a single cell leading to a single "primary product" of a
"dual-specific, tetravalent, full length binding protein", where
the "primary product" is more than 75% of all assembled protein,
comprising a dual variable domain light chain and a dual variable
domain heavy chain.
[0854] The present invention provides methods of expressing a dual
variable domain light chain and a dual variable domain heavy chain
in a single cell leading to a single "primary product" of a
"dual-specific tetravalent full length binding protein", where the
"primary product" is more than 90% of all assembled protein,
comprising a dual variable domain light chain and a dual variable
domain heavy chain.
6. Production of IL-17 Binding Proteins and Binding
Protein-Producing Cell Lines
[0855] Preferably, LI-17 binding proteins, including anti-IL-17
antibodies, of the present invention exhibit a high capacity to
reduce or to neutralize IL-17 activity, e.g., as assessed by any
one of several in vitro and in vivo assays known in the art.
Preferrably, IL-17 binding proteins of the present invention, also
exhibit a high capacity to reduce or to neutralize IL-17
activity
[0856] In preferred embodiments, a binding protein, or
antigen-binding portion thereof, binds human IL-17, wherein the
binding protein, or antigen-binding portion thereof, dissociates
from human IL-17 with a k.sub.off rate constant of about
0.1s.sup.-1 or less, as determined by surface plasmon resonance, or
which inhibits human IL-17A and/or human IL-17F activity with an
IC.sub.50 of about 1.times.10.sup.-6M or less. Alternatively, the
binding protein, or an antigen-binding portion thereof, may
dissociate from human IL-17 with a k.sub.off rate constant of about
1.times.10.sup.-2 s.sup.-1 or less, as determined by surface
plasmon resonance, or may inhibit human IL-17A and/or human IL-17F
activity with an IC.sub.50 of about 1.times.10.sup.-7M or less.
Alternatively, the binding protein, or an antigen-binding portion
thereof, may dissociate from human IL-17 with a k.sub.off rate
constant of about 1.times.10.sup.-3s.sup.-1 or less, as determined
by surface plasmon resonance, or may inhibit human IL-17A and/or
human IL-17F with an IC.sub.50 of about 1.times.10.sup.-8M or less.
Alternatively, the binding protein, or an antigen-binding portion
thereof, may dissociate from human IL-17 with a k.sub.off rate
constant of about 1.times.10.sup.-4s.sup.-1 or less, as determined
by surface plasmon resonance, or may inhibit human IL-17A and/or
human IL-17F activity with an IC.sub.50 of about 1.times.10.sup.-9M
or less. Alternatively, the binding protein, or an antigen-binding
portion thereof, may dissociate from human IL-17 with a k.sub.off
rate constant of about 1.times.10.sup.-5s.sup.-1 or less, as
determined by surface plasmon resonance, or may inhibit human
IL-17A and/or human IL-17F activity with an IC.sub.50 of about
1.times.10.sup.-10 M or less. Alternatively, the binding protein,
or an antigen-binding portion thereof, may dissociate from human
IL-17 with a k.sub.off rate constant of about
1.times.10.sup.-5s.sup.-1 or less, as determined by surface plasmon
resonance, or may inhibit human IL-17A and/or human IL-17F activity
with an IC.sub.50 of about 1.times.10.sup.-11 M or less.
[0857] In certain embodiments, the binding protein comprises a
heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4,
IgA, IgE, IgM or IgD constant region. Preferably, the heavy chain
constant region is an IgG1 heavy chain constant region or an IgG4
heavy chain constant region. Furthermore, the antibody can comprise
a light chain constant region, either a kappa light chain constant
region or a lambda light chain constant region. Preferably, the
antibody comprises a kappa light chain constant region.
Alternatively, the antibody portion can be, for example, a Fab
fragment or a single chain Fv fragment.
[0858] Replacements of amino acid residues in the Fc portion to
alter antibody effector function are known in the art (Winter et
al., U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fc portion of an
antibody mediates several important effector functions e.g.
cytokine induction, ADCC, phagocytosis, complement dependent
cytotoxicity (CDC) and half-life/clearance rate of antibody and
antigen-antibody complexes. In some cases these effector functions
are desirable for therapeutic antibody but in other cases might be
unnecessary or even deleterious, depending on the therapeutic
objectives. Certain human IgG isotypes, particularly IgG1 and IgG3,
mediate ADCC and CDC via binding to Fc.gamma.Rs and complement C1q,
respectively. Neonatal Fc receptors (FcRn) are the critical
components determining the circulating half-life of antibodies. In
still another embodiment at least one amino acid residue is
replaced in the constant region of the antibody, for example the Fc
region of the antibody, such that effector functions of the
antibody are altered.
[0859] One embodiment provides a labeled binding protein wherein an
antibody or antibody portion of the invention is derivatized or
linked to another functional molecule (e.g., another peptide or
protein). For example, a labeled binding protein of the invention
can be derived by functionally linking an antibody or antibody
portion of the invention (by chemical coupling, genetic fusion,
noncovalent association or otherwise) to one or more other
molecular entities, such as another antibody (e.g., a bispecific
antibody or a diabody), a detectable agent, a cytotoxic agent, a
pharmaceutical agent, and/or a protein or peptide that can mediate
associate of the antibody or antibody portion with another molecule
(such as a streptavidin core region or a polyhistidine tag).
[0860] Useful detectable agents with which a binding protein, such
as an antibody or antibody portion of the invention may be
derivatized include fluorescent compounds. Exemplary fluorescent
detectable agents include fluorescein, fluorescein isothiocyanate,
rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride,
phycoerythrin and the like. An antibody may also be derivatized
with detectable enzymes, such as alkaline phosphatase, horseradish
peroxidase, glucose oxidase and the like. When an antibody is
derivatized with a detectable enzyme, it is detected by adding
additional reagents that the enzyme uses to produce a detectable
reaction product. For example, when the detectable agent
horseradish peroxidase is present, the addition of hydrogen
peroxide and diaminobenzidine leads to a colored reaction product,
which is detectable. An antibody may also be derivatized with
biotin, and detected through indirect measurement of avidin or
streptavidin binding.
[0861] Another embodiment of the invention provides a crystallized
binding protein. Preferably the invention relates to crystals of
whole anti-IL-17 antibodies and fragments thereof as disclosed
herein, and formulations and compositions comprising such crystals.
In one embodiment the crystallized binding protein has a greater
half-life in vivo than the soluble counterpart of the binding
protein. In another embodiment the binding protein retains
biological activity after crystallization.
[0862] Crystallized binding protein of the invention may be
produced according methods known in the art and as disclosed in PCT
Publication No. WO 02072636, incorporated herein by reference.
[0863] Another embodiment of the invention provides a glycosylated
binding protein wherein the antibody or antigen-binding portion
thereof comprises one or more carbohydrate residues. Nascent in
vivo protein production may undergo further processing, known as
post-translational modification. In particular, sugar (glycosyl)
residues may be added enzymatically, a process known as
glycosylation. The resulting proteins bearing covalently linked
oligosaccharide side chains are known as glycosylated proteins or
glycoproteins.
[0864] Naturally occurring antibodies are glycoproteins with one or
more carbohydrate residues in the Fc domain, as well as the
variable domain. Carbohydrate residues in the Fc domain have
important effect on the effector function of the Fc domain, with
minimal effect on antigen binding or half-life of the antibody (R.
Jefferis, Biotechnol. Prog., 21: 11-16 (2005)). In contrast,
glycosylation of the variable domain may have an effect on the
antigen binding activity of the antibody. Glycosylation in the
variable domain may have a negative effect on antibody binding
affinity, likely due to steric hindrance (Co, M. S., et al., Mol.
Immunol., 30: 1361-1367 (1993)), or result in increased affinity
for the antigen (Wallick, S. C., et al., Exp. Med., 168:1099-1109
(1988); Wright, A., et al., EMBO J., 10: 2717-2723 (1991)).
[0865] One aspect of the present invention is directed to
generating glycosylation site mutants in which the O- or N-linked
glycosylation site of the binding protein has been mutated. One
skilled in the art can generate such mutants using standard
well-known technologies. Glycosylation site mutants that retain the
biological activity but have increased or decreased binding
activity are another object of the present invention.
[0866] In still another embodiment, the glycosylation of the
antibody or antigen-binding portion of the invention is modified.
For example, an aglycoslated antibody can be made (i.e., the
antibody lacks glycosylation). Glycosylation can be altered to, for
example, increase the affinity of the antibody for antigen. Such
carbohydrate modifications can be accomplished by, for example,
altering one or more sites of glycosylation within the antibody
sequence. For example, one or more amino acid substitutions can be
made that result in elimination of one or more variable region
glycosylation sites to thereby eliminate glycosylation at that
site. Such aglycosylation may increase the affinity of the antibody
for antigen. Such an approach is described in further detail in PCT
Publication WO 2003/016466A2, and U.S. Pat. Nos. 5,714,350 and
6,350,861.
[0867] Additionally or alternatively, a modified binding protein of
the invention can be made that has an altered type of
glycosylation, such as a hypofucosylated antibody having reduced
amounts of fucosyl residues (see Kanda, Yutaka et al., Journal of
Biotechnology (2007), 130(3), 300-310.) or an antibody having
increased bisecting GlcNAc structures. Such altered glycosylation
patterns have been demonstrated to increase the ADCC ability of
antibodies. Such carbohydrate modifications can be accomplished by,
for example, expressing the antibody in a host cell with altered
glycosylation machinery. Cells with altered glycosylation machinery
have been described in the art and can be used as host cells in
which to express recombinant antibodies of the invention to thereby
produce an antibody with altered glycosylation. See, for example,
Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana
et al., "Engineered glycoforms of an antineuroblastoma IgG1 with
optimized antibody-dependent cellular cytotoxic activity," Nat.
Biotech., 17: 176-180 (1999), as well as, European Patent No: EP
1,176,195; PCT Publication Nos. WO 03/035835 and WO 99/54342.
[0868] Protein glycosylation depends on the amino acid sequence of
the protein of interest, as well as the host cell in which the
protein is expressed. Different organisms may produce different
glycosylation enzymes (e.g., glycosyltransferases and
glycosidases), and have different substrates (nucleotide sugars)
available. Due to such factors, protein glycosylation pattern, and
composition of glycosyl residues, may differ depending on the host
system in which the particular protein is expressed. Glycosyl
residues useful in the invention may include, but are not limited
to, glucose, galactose, mannose, fucose, n-acetylglucosamine and
sialic acid. Preferably the glycosylated binding protein comprises
glycosyl residues such that the glycosylation pattern is human.
[0869] It is known to those skilled in the art that differing
protein glycosylation may result in differing protein
characteristics. For instance, the efficacy of a therapeutic
protein produced in a microorganism host, such as yeast, and
glycosylated utilizing the yeast endogenous pathway may be reduced
compared to that of the same protein expressed in a mammalian cell,
such as a CHO cell line. Such glycoproteins may also be immunogenic
in humans and show reduced half-life in vivo after administration.
Specific receptors in humans and other animals may recognize
specific glycosyl residues and promote the rapid clearance of the
protein from the bloodstream. Other adverse effects may include
changes in protein folding, solubility, susceptibility to
proteases, trafficking, transport, compartmentalization, secretion,
recognition by other proteins or factors, antigenicity, or
allergenicity. Accordingly, a practitioner may prefer a therapeutic
protein with a specific composition and pattern of glycosylation,
for example glycosylation composition and pattern identical, or at
least similar, to that produced in human cells or in the
species-specific cells of the intended subject animal.
[0870] Expressing glycosylated proteins different from that of a
host cell may be achieved by genetically modifying the host cell to
express heterologous glycosylation enzymes. Using techniques known
in the art a practitioner may generate antibodies or
antigen-binding portions thereof exhibiting human protein
glycosylation. For example, yeast strains have been genetically
modified to express non-naturally occurring glycosylation enzymes
such that glycosylated proteins (glycoproteins) produced in these
yeast strains exhibit protein glycosylation identical to that of
animal cells, especially human cells (US patent application
publication Nos. 20040018590 and 20020137134).
[0871] In addition to the binding proteins, the present invention
is also directed to anti-idiotypic (anti-Id) antibodies specific
for such binding proteins of the invention. An anti-Id antibody is
an antibody, which recognizes unique determinants generally
associated with the antigen-binding region of another antibody. The
anti-Id can be prepared by immunizing an animal with the binding
protein or a CDR containing region thereof. The immunized animal
will recognize, and respond to the idiotypic determinants of the
immunizing antibody and produce an anti-Id antibody. It is readily
apparent that it may be easier to generate anti-idiotypic
antibodies to the two or more parent antibodies incorporated into a
DVD-Ig molecule; and confirm binding studies by methods well
recognized in the art (e.g., BIAcore, ELISA) to verify that
anti-idiotypic antibodies specific for the idiotype of each parent
antibody also recognize the idiotype (e.g., antigen binding site)
in the context of the DVD-Ig. The anti-idiotypic antibodies
specific for each of the two or more antigen binding sites of a
DVD-Ig provide ideal reagents to measure DVD-Ig concentrations of a
human DVD-Ig in patient serum. For example, DVD-Ig concentration
assays can be established using a "sandwich assay ELISA format"
with an antibody to a first antigen binding region coated on the
solid phase (e.g., BIAcore chip, ELISA plate, etc.), rinsed with
rinsing buffer, incubation with a serum sample, another rinsing
step, and ultimately incubation with another anti-idiotypic
antibody to the other antigen binding site, itself labeled with an
enzyme for quantitation of the binding reaction. In an embodiment,
for a DVD-Ig with more than two different binding sites,
anti-idiotypic antibodies to the two outermost binding sites (most
distal and proximal from the constant region) will not only help in
determining the DVD-Ig concentration in human serum but also
document the integrity of the molecule in vivo. Each anti-Id
antibody may also be used as an "immunogen" to induce an immune
response in yet another animal, producing a so-called anti-anti-Id
antibody.
[0872] Further, it will be appreciated by one skilled in the art
that a protein of interest may be expressed using a library of host
cells genetically engineered to express various glycosylation
enzymes, such that member host cells of the library produce the
protein of interest with variant glycosylation patterns. A
practitioner may then select and isolate the protein of interest
with particular novel glycosylation patterns. Preferably, the
protein having a particularly selected novel glycosylation pattern
exhibits improved or altered biological properties.
7. Uses of IL-17 Binding Proteins
[0873] Given their ability to bind to human IL-17, the IL-17
binding proteins, or antigen binding portions thereof, of the
invention can be used to detect IL-17A and/or human IL-17F (e.g.,
in a biological sample, such as serum or plasma), using a
conventional immunoassay, such as an enzyme linked immunosorbent
assays (ELISA), an radioimmunoassay (RIA) or tissue
immunohistochemistry. The invention provides a method for detecting
IL-17A and/or human IL-17F in a biological sample comprising
contacting a biological sample with a binding protein, or antigen
binding portion, of the invention and detecting either the binding
protein (or antigen binding portion) bound to IL-17A and/or human
IL-17F or unbound binding protein (or binding portion), to thereby
detect IL-17A and/or human IL-17F in the biological sample. The
binding protein is directly or indirectly labeled with a detectable
substance to facilitate detection of the bound or unbound antibody.
Suitable detectable substances include various enzymes, prosthetic
groups, fluorescent materials, luminescent materials and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase,
or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; and examples of suitable radioactive material include
.sup.3H, .sup.14C, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In,
.sup.125I, .sup.131I, .sup.177Lu, .sup.166Ho, or .sup.153Sm.
[0874] Alternative to labeling the binding protein, human IL-17 can
be assayed in biological fluids by a competition immunoassay
utilizing rhIL-17 standards labeled with a detectable substance and
an unlabeled human IL-17 binding protein. In this assay, the
biological sample, the labeled rhIL-17 standards, and the human
IL-17 binding protein are combined and the amount of labeled
rhIL-17 standard bound to the unlabeled antibody is determined. The
amount of human IL-17 in the biological sample is inversely
proportional to the amount of labeled rhIL-17 standard bound to the
IL-17 binding protein. Similarly, human IL-17 can also be assayed
in biological fluids by a competition immunoassay utilizing rhIL-17
standards labeled with a detectable substance and an unlabeled
human IL-17 binding protein.
[0875] The binding proteins and IL-17 binding portions of the
invention preferably are capable of neutralizing human IL-17A
and/or human IL-17F activity both in vitro and in vivo.
Accordingly, such binding proteins and IL-17 binding portions
thereof of the invention can be used to inhibit hIL-17A and/or
hIL-17F activity, e.g., in a cell culture containing hIL-17A and/or
hIL-17F, in human subjects, or in other mammalian subjects having
IL-17A and/or IL-17F with which an antibody of the invention
cross-reacts. In one embodiment, the invention provides a method
for inhibiting hIL-17A and/or hIL-17F activity comprising
contacting hIL-17A and/or hIL-17F with an IL-17 binding protein or
binding portion thereof of the invention such that hIL-17A and/or
hIL-17F activity is inhibited. For example, in a cell culture
containing, or suspected of containing hIL-17A and/or hIL-17F, an
IL-17 binding protein or binding portion thereof of the invention
can be added to the culture medium to inhibit hIL-17A and/or h
IL-17F activity in the culture.
[0876] In another embodiment, the invention provides a method for
reducing hIL-17A and/or hIL-17F activity in a subject,
advantageously from a subject suffering from a disease or disorder
in which IL-17A or IL-17F activity is detrimental. The invention
provides methods for reducing IL-17A and/or IL-17F activity in a
subject suffering from such a disease or disorder, which method
comprises administering to the subject an antibody or antibody
portion of the invention such that IL-17A and/or IL-17F activity in
the subject is reduced. Preferably, the IL-17A is human IL-17A, the
IL-17F is human IL-17F, and the subject is a human subject.
Alternatively, the subject can be a mammal expressing an IL-17A
and/or IL-17F to which an antibody of the invention is capable of
binding. Still further, the subject can be a mammal into which
IL-17A and/or IL-17F has been introduced (e.g., by administration
of IL-17A and/or IL-17F or by expression of an IL-17A and/or IL-17F
transgene). An IL-17 binding protein of the invention can be
administered to a human subject for therapeutic purposes. Moreover,
a binding protein of the invention can be administered to a
non-human mammal expressing an IL-17A and/or IL-17F with which the
antibody is capable of binding for veterinary purposes or as an
animal model of human disease. Regarding the latter, such animal
models may be useful for evaluating the therapeutic efficacy of
antibodies of the invention (e.g., testing of dosages and time
courses of administration).
[0877] As used herein, the term "a disorder in which IL-17A and/or
IL-17F activity is detrimental" is intended to include diseases and
other disorders in which the presence of IL-17A and/or IL-17F in a
subject suffering from the disorder has been shown to be or is
suspected of being either responsible for the pathophysiology of
the disorder or a factor that contributes to a worsening of the
disorder. Accordingly, a disorder in which IL-17A and/or IL-17F
activity is detrimental is a disorder in which reduction of IL-17A
and/or IL-17F activity is expected to alleviate the symptoms and/or
progression of the disorder. Such disorders may be evidenced, for
example, by an increase in the concentration of IL-17A and/or
IL-17F in a biological fluid of a subject suffering from the
disorder (e.g., an increase in the concentration of IL-17A and/or
IL-17F in serum, plasma, synovial fluid, etc. of the subject),
which can be detected, for example, using an anti-IL-17 antibody as
described above. Non-limiting examples of disorders that can be
treated with the antibodies of the invention include those
disorders discussed in the section below pertaining to
pharmaceutical compositions of the antibodies of the invention.
[0878] The DVD-Igs of the invention may bind IL-17 (e.g., hIL-17 or
hIL-17F) alone or multiple antigens (e.g., hIL-17 and another
non-IL-17 antigen). Thus, a DVD-Ig may block or reduce activity of
huIL-17 and the activity of another target antigen. Such other
target antigens may include soluble targets (e.g., TNF) and cell
surface receptor targets (e.g., VEGFR, EGFR).
[0879] Such other antigens include, but are not limited to, the
targets listed in publically available databases, which databases
include those that are available on the worldwide web and
incorporated herein by reference. These target databases include
those listing:
Therapeutic targets
(http://xin.cz3.nus.edu.sg/group/cjttd/ttd.asp); Cytokines and
cytokine receptors (http://www.cytokinewebfacts.com/,
http://www.copewithcytokines.de/cope.cgi, and
http://cmbi.bjmu.edu.cn/cmbidata/cgf/CGF_Database/cytokine.medic.kumamoto-
-u.acjp/CFC/indexR.html); Chemokines
(http://cytokine.medic.kumamoto-u.ac.jp/CFC/CK/Chemokine.html);
Chemokine receptors and GPCRs
(http://csp.medic.kumamoto-u.ac.jp/CSP/Receptor.html,
http://www.gper.org/7tm/); Olfactory Receptors
(http://senselab.med.yale.edu/senselab/ORDB/default.asp); Receptors
(http://www.iuphar-db.org/iuphar-rd/list/index.htm); Cancer targets
(http://cged.hgc.jp/cgi-bin/input.cgi); Secreted proteins as
potential antibody targets (http://spd.cbi.pku.edu.cn/); Protein
kinases (http://spd.cbi.pku.edu.cn/), and Human CD markers
(http://content.labvelocity.com/tools/6/1226/CD_table_final_locked.pdf)
and (Zola H, 2005 CD molecules 2005: human cell differentiation
molecules Blood, 106:3123-6).
[0880] DVD-Igs are useful as therapeutic agents to simultaneously
block two or more different targets, i.e., hIL-17 (and/or hIL-17F)
and one or more other non-IL-17 target antigens to enhance
efficacy/safety and/or increase patient coverage. Such targets may
include soluble targets (TNF) and cell surface receptor targets
(VEGFR and EGFR).
[0881] Additionally, DVD-Igs of the invention can be employed for
tissue-specific delivery (target a tissue marker and a disease
mediator for enhanced local PK thus higher efficacy and/or lower
toxicity), including intracellular delivery (targeting an
internalizing receptor and a intracellular molecule), delivering to
inside brain (targeting transferrin receptor and a CNS disease
mediator for crossing the blood-brain barrier). DVD-Ig can also
serve as a carrier protein to deliver an antigen to a specific
location via binding to a non-neutralizing epitope of that antigen
and also to increase the half-life of the antigen. Furthermore,
DVD-Ig can be designed to either be physically linked to medical
devices implanted into patients or target these medical devices
(see Burke, Sandra E.; Kuntz, Richard E.; Schwartz, Lewis B.,
Zotarolimus eluting stents. Advanced Drug Delivery Reviews (2006),
58(3), 437-446; Surface coatings for biological activation and
functionalization of medical devices, Hildebrand, H. F.;
Blanchemain, N.; Mayer, G.; Chai, F.; Lefebvre, M.; Boschin, F.,
Surface and Coatings Technology (2006), 200(22-23), 6318-6324; Wu
et al., "Drug/device combinations for local drug therapies and
infection prophylaxis," Biomaterials, 27: 2450-2467 (2006); Marques
et al., "Mediation of the Cytokine Network in the Implantation of
Orthopedic Devices," Chapter 21, In Biodegradable Systems in Tissue
Engineering and Regenerative Medicine, (Reis et al., eds.) (CRC
Press LLC, Boca Raton, 2005) pp. 377-397. Briefly, directing
appropriate types of cell to the site of medical implant may
promote healing and restoring normal tissue function.
Alternatively, inhibition of mediators (including but not limited
to cytokines), released upon device implantation by a DVD-Ig
coupled to or target to a device is also provided. For example,
Stents have been used for years in interventional cardiology to
clear blocked arteries and to improve the flow of blood to the
heart muscle. However, traditional bare metal stents have been
known to cause restenosis (re-narrowing of the artery in a treated
area) in some patients and can lead to blood clots. Recently, an
anti-CD34 antibody coated stent has been described which reduced
restenosis and prevents blood clots from occurring by capturing
endothelial progenitor cells (EPC) circulating throughout the
blood. Endothelial cells are cells that line blood vessels,
allowing blood to flow smoothly. The EPCs adhere to the hard
surface of the stent forming a smooth layer that not only promotes
healing but prevents restenosis and blood clots, complications
previously associated with the use of stents (Aoji et al. 2005 J Am
Coll Cardiol. 45(10):1574-9). In addition to improving outcomes for
patients requiring stents, there are also implications for patients
requiring cardiovascular bypass surgery. For example, a prosthetic
vascular conduit (artificial artery) coated with anti-EPC
antibodies would eliminate the need to use arteries from patients
legs or arms for bypass surgery grafts. This would reduce surgery
and anesthesia times, which in turn will reduce coronary surgery
deaths. DVD-Ig are designed in such a way that it binds to a cell
surface marker (such as CD34) as well as a protein (or an epitope
of any kind, including but not limited to proteins, lipids and
polysaccharides) that has been coated on the implanted device to
facilitate the cell recruitment. Such approaches can also be
applied to other medical implants in general. Alternatively,
DVD-Igs can be coated on medical devices and upon implantation and
releasing all DVDs from the device (or any other need which may
require additional fresh DVD-Ig, including aging and denaturation
of the already loaded DVD-Ig) the device could be reloaded by
systemic administration of fresh DVD-Ig to the patient, where the
DVD-Ig is designed to binds to a target of interest (a cytokine, a
cell surface marker (such as CD34) etc.) with one set of binding
sites and to a target coated on the device (including a protein, an
epitope of any kind, including but not limited to lipids,
polysaccharides and polymers) with the other. This technology has
the advantage of extending the usefulness of coated implants.
A. Use of DVD-Igs in Various Diseases
[0882] DVD-Ig molecules of the invention are also useful as
therapeutic molecules to treat various diseases. Such DVD molecules
may bind one or more targets involved in a specific disease.
Examples of such targets in various diseases are described
below.
Human Autoimmune and Inflammatory Response
[0883] In one aspect, a DVD-Ig binding protein of the invention is
capable of binding human IL-17A and one or more antigens that have
been implicated in general autoimmune and inflammatory responses,
including C5, CCL1 (1-309), CCL11 (eotaxin), CCL13 (mcp-4), CCL15
(MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19, CCL2
(mcp-1), CCL20 (MIP-3a), CCL21 (MIP-2), CCL23 (MPIF-1), CCL24
(MPIF-2/eotaxin-2), CCL25 (TECK), CCL26, CCL3 (MIP-1a), CCL4
(MIP-1b), CCL5 (RANTES), CCL7 (mcp-3), CCL8 (mcp-2), CXCL1, CXCL10
(IP-10), CXCL11 (1-TAC/IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL2,
CXCL3, CXCL5 (ENA-78/LIX), CXCL6 (GCP-2), CXCL9, IL13, IL8, CCL13
(mcp-4), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9,
CX3CR1, IL8RA, XCR1 (CCXCR1), IFNA2, IL10, IL13, IL17C, IL1A, IL1B,
IL1F10, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL22, IL5, IL8, IL9,
LTA, LTB, MIF, SCYE1 (endothelial Monocyte-activating cytokine),
SPP1, TNF, TNFSF5, IFNA2, IL10RA, IL10RB, IL13, IL13RA1, IL5RA,
IL9, IL9R, ABCF1, BCL6, C3, C4A, CEBPB, CRP, ICEBERG, IL1R1, IL1RN,
IL8RB, LTB4R, TOLLIP, FADD, IRAK1, IRAK2, MYD88, NCK2, TNFAIP3,
TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, ACVR1, ACVR1B,
ACVR2, ACVR2B, ACVRL1, CD28, CD3E, CD3G, CD3Z, CD69, CD80, CD86,
CNR1, CTLA4, CYSLTR1, FCER1A, FCER2, FCGR3A, GPR44, HAVCR2, OPRD1,
P2RX7, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, BLR1,
CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL15,
CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24,
CCL25, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9,
CX3CL1, CX3CR1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11,
CXCL12, CXCL13, CXCR4, GPR2, SCYE1, SDF2, XCL1, XCL2, XCR1, AMH,
AMHR2, BMPR1A, BMPR1B, BMPR2, C19orf10 (IL27w), CER1, CSF1, CSF2,
CSF3, DKFZp451J0118, FGF2, GFI1, IFNA1, IFNB1, IFNG, IGF1, IL1A,
IL1B, IL1R1, IL1R2, IL2, IL2RA, IL2RB, IL2RG, IL3, IL4, IL4R, IL5,
IL5RA, IL6, IL6R, IL6ST, IL7, IL8, IL8RA, IL8RB, IL9, IL9R, IL10,
IL10RA, IL10RB, IL11, IL11RA, IL12A, IL12B, IL12RB1, IL12RB2, IL13,
IL13RA1, IL13RA2, IL15, IL15RA, IL16, IL17, IL17R, IL18, IL18R1,
IL19, IL20, KITLG, LEP, LTA, LTB, LTB4R, LTB4R2, LTBR, MIF, NPPB,
PDGFB, TBX21, TDGF1, TGFA, TGFB1, TGFB111, TGFB2, TGFB3, TGFBI,
TGFBR1, TGFBR2, TGFBR3, TH1L, TNF, TNFRSF1A, TNFRSF1B, TNFRSF7,
TNFRSF8, TNFRSF9, TNFRSF11A, TNFRSF21, TNFSF4, TNFSF5, TNFSF6,
TNFSF11, VEGF, ZFPM2, and RNF110 (ZNF144).
Asthma
[0884] Allergic asthma is characterized by the presence of
eosinophilia, goblet cell metaplasia, epithelial cell alterations,
airway hyperreactivity (AHR), and Th2 and Th1 cytokine expression,
as well as elevated serum IgE levels. It is now widely accepted
that airway inflammation is the key factor underlying the
pathogenesis of asthma, involving a complex interplay of
inflammatory cells such as T cells, B cells, eosinophils, mast
cells and macrophages, and of their secreted mediators including
cytokines and chemokines. Corticosteroids are the most important
anti-inflammatory treatment for asthma today, however their
mechanism of action is non-specific and safety concerns exist,
especially in the juvenile patient population. The development of
more specific and targeted therapies is therefore warranted.
[0885] Animal models such as OVA-induced asthma mouse model, where
both inflammation and AHR can be assessed, are known in the art and
may be used to determine the ability of various DVD-Ig molecules to
treat asthma Animal models for studying asthma are disclosed in
Coffman, et al., Journal of Experimental Medicine (2005), 201(12),
1875-1879; Lloyd, et al., Advances in Immunology (2001), 77,
263-295; Boyce et al., Journal of Experimental Medicine (2005),
201(12), 1869-1873; and Snibson, et al., Journal of the British
Society for Allergy and Clinical Immunology (2005), 35(2), 146-52.
In addition to routine safety assessments of these target pairs
specific tests for the degree of immunosuppression may be warranted
and helpful in selecting the best target pairs (see Luster et al.,
Toxicology (1994), 92(1-3), 229-43; Descotes, et al., Developments
in biological standardization (1992), 77 99-102; Hart et al.,
Journal of Allergy and Clinical Immunology (2001), 108(2),
250-257).
[0886] One aspect of the invention pertains to DVD-Ig molecules
capable of binding IL-17 (and/or IL-17F) and one or more, for
example two, targets selected from the group consisting of IL-4,
IL-5, IL-8, IL-9, IL-13, IL-18, IL-5R(.alpha.), TNFSF4,
IL-4R(.alpha.), interferon .alpha., eotaxin, TSLP, PAR-2, PGD2, and
IgE. An embodiment includes a dual-specific anti-IL-17/IL-13 DVD-Ig
as a therapeutic agent beneficial for the treatment of asthma.
Rheumatoid Arthritis (RA)
[0887] Rheumatoid arthritis (RA), a systemic disease, is
characterized by a chronic inflammatory reaction in the synovium of
joints and is associated with degeneration of cartilage and erosion
of juxta-articular bone. Many pro-inflammatory cytokines including
TNF, chemokines, and growth factors are expressed in diseased
joints. Systemic administration of anti-TNF antibody or sTNFR
fusion protein to mouse models of RA was shown to be
anti-inflammatory and joint protective. Various cytokines, included
IL-17 have been implicated in RA. Clinical investigations in which
the activity of TNF in RA patients was blocked with intravenously
administered infliximab (Harriman G, Harper L K, Schaible T F. 1999
Summary of clinical trials in rheumatoid arthritis using
infliximab, an anti-TNFalpha treatment. Ann. Rheum. Dis., 58 Suppl
1: 161-4), a chimeric anti-TNF mAb, has provided evidence that TNF
regulates IL-6, IL-8, MCP-1, and VEGF production, recruitment of
immune and inflammatory cells into joints, angiogenesis, and
reduction of blood levels of matrix metalloproteinases-1 and -3. A
better understanding of the inflammatory pathway in rheumatoid
arthritis has led to identification of other therapeutic targets
involved in rheumatoid arthritis. Promising treatments such as
interleukin-6 antagonists (IL-6 receptor antibody MRA, developed by
Chugai, Roche (see Nishimoto, Norihiro et al., Arthritis &
Rheumatism, (2004), 50(6): 1761-1769), CTLA4Ig (abatacept, Genovese
et al. (2005) "Abatacept for rheumatoid arthritis refractory to
tumor necrosis factor alpha inhibition," N. Engl. J. Med., 353:
1114-23.), and anti-B cell therapy (rituximab, Okamoto H, Kamatani
N. (2004) "Rituximab for rheumatoid arthritis," N. Engl. J. Med.,
351: 1909) have already been tested in randomized controlled trials
over the past year. IL-17 and other cytokines, such as IL-15 and
IL-18, have been identified as playing a role using RA animal
models (therapeutic antibody HuMax-IL.sub.--15, AMG 714 see
Baslund, Bo et al., Arthritis & Rheumatism (2005), 52(9):
2686-2692). Dual-specific antibody therapy, combining anti-TNF and
another mediator, such IL-17, has great potential in enhancing
clinical efficacy and/or patient coverage. For example, blocking
both TNF and VEGF can potentially eradicate inflammation and
angiogenesis, both of which are involved in pathophysiology of RA.
A DVD-Ig binding protein capable of blocking TNF-.alpha. and IL-17
is contemplated. In addition to routine safety assessments of these
target pairs, specific tests for the degree of immunosuppression
may be warranted and helpful in selecting the best target pairs
(see Luster et al., Toxicology (1994), 92(1-3), 229-43; Descotes,
et al., Developments in biological standardization (1992), 77
99-102; Hart et al., Journal of Allergy and Clinical Immunology
(2001), 108(2), 250-257). Whether a DVD-Ig molecule will be useful
for the treatment of rheumatoid arthritis can be assessed using
pre-clinical animal RA models such as the collagen-induced
arthritis mouse model. Other useful models are also well known in
the art (see Brand D D., Comp. Med., (2005) 55(2):114-22). Based on
the cross-reactivity of the parental antibodies for human and mouse
orthologues (e.g., reactivity for human and mouse TNF, human and
mouse IL-15 etc.) validation studies in the mouse CIA model may be
conducted with "matched surrogate antibody" derived DVD-Ig
molecules; briefly, a DVD-Ig based on two (or more) mouse target
specific antibodies may be matched to the extent possible to the
characteristics of the parental human or humanized antibodies used
for human DVD-Ig construction (similar affinity, similar
neutralization potency, similar half-life etc.).
[0888] In an embodiment, a DVD-Ig of the invention that binds
hIL-17 and another non-IL-17 target may also be used to treat other
diseases in which IL-17 plays. Such diseases include, but are not
limited to SLE, multiple sclerosis (MS), sepsis, various
neurological diseases, and cancers (including cervical, breast,
gastric). A more extensive list of diseases and disorders in which
IL-17 plays a role is also provided below.
[0889] An embodiment of the invention pertains to a DVD-Ig
molecules capable of binding huIL-17 and/or hIL-17 F and one or
more targets selected from the group consisting of TNF.alpha.,
IL-12, TWEAK, IL-23, CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4,
TNF.beta., CD45RB, CD200, IFN-.gamma., GM-CSF, FGF, C5, CD52,
sclerostin, and CCR2.
SLE (Lupus)
[0890] The immunopathogenic hallmark of SLE is the polyclonal B
cell activation, which leads to hyperglobulinemia, autoantibody
production and immune complex formation. The fundamental
abnormality appears to be the failure of T cells to suppress the
forbidden B cell clones due to generalized T cell dysregulation. In
addition, B and T-cell interaction is facilitated by several
cytokines such as IL-10 as well as co-stimulatory molecules such as
CD40 and CD40L, B7 and CD28 and CTLA-4, which initiate the second
signal. These interactions together with impaired phagocytic
clearance of immune complexes and apoptotic material, perpetuate
the immune response with resultant tissue injury.
[0891] In one aspect, a DVD-Ig binding protein of the invention is
capable of binding human IL-17A and one or more of the following
antigens that have been implicated in SLE: B cell targeted
therapies: CD-20, CD-22, CD-19, CD28, CD4, CD80, HLA-DRA, IL10,
IL2, IL4, TNFRSF5, TNFRSF6, TNFSF5, TNFSF6, BLR1, HDAC4, HDAC5,
HDAC7A, HDAC9, ICOSL, IGBP1, MS4A1, RGS1, SLA2, CD81, IFNB1, IL10,
TNFRSF5, TNFRSF7, TNFSF5, AICDA, BLNK, GALNAC4S-6ST, HDAC4, HDAC5,
HDAC7A, HDAC9, IL10, IL11, IL4, INHA, INHBA, KLF6, TNFRSF7, CD28,
CD38, CD69, CD80, CD83, CD86, DPP4, FCER2, IL2RA, TNFRSF8, TNFSF7,
CD24, CD37, CD40, CD72, CD74, CD79A, CD79B, CR2, IL1R2, ITGA2,
ITGA3, MS4A1, ST6GAL1, CD1C, CHST10, HLA-A, HLA-DRA, and NT5E.;
co-stimulatory signals: CTLA4 or B7.1/B7.2; inhibition of B cell
survival: BlyS, BAFF; Complement inactivation: C5; Cytokine
modulation: the key principle is that the net biologic response in
any tissue is the result of a balance between local levels of
proinflammatory or anti-inflammatory cytokines (see Sfikakis P P et
al 2005 Curr Opin Rheumatol 17:550-7). SLE is considered to be a
Th-2 driven disease with documented elevations in serum IL-4, IL-6,
IL-10. DVD-Igs capable of binding one or more targets selected from
the group consisting of IL-4, IL-6, IL-10, IFN-.alpha., and
TNF-.alpha. are also contemplated. Combination of targets discussed
herein will enhance therapeutic efficacy for SLE which can be
tested in a number of lupus preclinical models (see, Peng S L
(2004) Methods Mol. Med., 102: 227-72). Based on the
cross-reactivity of the parental antibodies for human and mouse
othologues (e.g., reactivity for human and mouse CD20, human and
mouse Interferon alpha etc.) validation studies in a mouse lupus
model may be conducted with "matched surrogate antibody" derived
DVD-Ig molecules; briefly, a DVD-Ig based two (or more) mouse
target specific antibodies may be matched to the extent possible to
the characteristics of the parental human or humanized antibodies
used for human DVD-Ig construction (similar affinity, similar
neutralization potency, similar half-life etc.).
Multiple Sclerosis (MS)
[0892] Multiple sclerosis (MS) is a complex human autoimmune-type
disease with a predominantly unknown etiology Immunologic
destruction of myelin basic protein (MBP) throughout the nervous
system is the major pathology of multiple sclerosis. MS is a
disease of complex pathologies, which involves infiltration by CD4+
and CD8+ T cells and of response within the central nervous system.
Expression in the CNS of cytokines, reactive nitrogen species and
costimulator molecules have all been described in MS. Of major
consideration are immunological mechanisms that contribute to the
development of autoimmunity. In particular, antigen expression,
cytokine and leukocyte interactions, and regulatory T-cells, which
help balance/modulate other T-cells such as Th1 and Th2 cells, are
important areas for therapeutic target identification.
[0893] IL-12 is a proinflammatory cytokine that is produced by APC
and promotes differentiation of Th1 effector cells. IL-12 is
produced in the developing lesions of patients with MS as well as
in EAE-affected animals. Previously it was shown that interference
in IL-12 pathways effectively prevents EAE in rodents, and that in
vivo neutralization of IL-12p40 using a anti-IL-12 mAb has
beneficial effects in the myelin-induced EAE model in common
marmosets.
[0894] TWEAK is a member of the TNF family, constitutively
expressed in the central nervous system (CNS), with
pro-inflammatory, proliferative or apoptotic effects depending upon
cell types. Its receptor, Fn14, is expressed in CNS by endothelial
cells, reactive astrocytes and neurons. TWEAK and Fn14 mRNA
expression increased in spinal cord during experimental autoimmune
encephalomyelitis (EAE). Anti-TWEAK antibody treatment in myelin
oligodendrocyte glycoprotein (MOG) induced EAE in C57BL/6 mice
resulted in a reduction of disease severity and leukocyte
infiltration when mice were treated after the priming phase.
[0895] One aspect of the invention pertains to DVD-Ig molecules
capable of binding IL-17 (and/or IL-17F) and one or more, for
example two, targets selected from the group consisting of IL-12,
TWEAK, IL-23, CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNF, CD45RB,
CD200, IFNgamma, GM-CSF, FGF, C5, CD52, osteopontin, and CCR2. An
embodiment includes a dual-specific anti-IL-17/TWEAK DVD-Ig as a
therapeutic agent beneficial for the treatment of MS.
[0896] Several animal models for assessing the usefulness of the
DVD-Ig molecules to treat MS are known in the art (see Steinman L,
et al., (2005) Trends Immunol. 26(11):565-71; Lublin F D., et al.,
(1985) Springer Semin Immunopathol. 8(3):197-208; Genain C P, et
al., (1997) J Mol Med. 75(3):187-97; Tuohy V K, et al., (1999) J
Exp Med. 189(7):1033-42; Owens T, et al., (1995) Neurol Clin.
13(1):51-73; and 't Hart et al., J. Immunol., 175(7): 4761-4768
(2005). Based on the cross-reactivity of the parental antibodies
for human and animal species othologues (e.g., reactivity for human
and mouse IL-17, human and mouse TWEAK etc.) validation studies in
the mouse EAE model may be conducted with "matched surrogate
antibody" derived DVD-Ig molecules; briefly, a DVD-Ig based on to
(or more) mouse target specific antibodies may be matched to the
extent possible to the characteristics of the parental human or
humanized antibodies used for human DVD-Ig construction (similar
affinity, similar neutralization potency, similar half-life etc.).
The same concept applies to animal models in other non-rodent
species, where a "matched surrogate antibody" derived DVD-Ig would
be selected for the anticipated pharmacology and possibly safety
studies. In addition to routine safety assessments of these target
pairs specific tests for the degree of immunosuppression may be
warranted and helpful in selecting the best target pairs (see
Luster et al., Toxicology (1994), 92(1-3), 229-43; Descotes, et
al., Developments in biological standardization (1992), 77 99-102;
Jones R. 2000 Rovelizumab (ICOS Corp). IDrugs. 3(4):442-6).
Sepsis
[0897] The pathophysiology of sepsis is initiated by the outer
membrane components of both gram-negative organisms
(lipopolysaccharide [LPS], lipid A, endotoxin) and gram-positive
organisms (lipoteichoic acid, peptidoglycan). These outer membrane
components are able to bind to the CD14 receptor on the surface of
monocytes. By virtue of the recently described toll-like receptors,
a signal is then transmitted to the cell, leading to the eventual
production of the proinflammatory cytokines tumor necrosis
factor-alpha (TNF-alpha) and interleukin-1 (IL-1). Overwhelming
inflammatory and immune responses are essential features of septic
shock and play a central part in the pathogenesis of tissue damage,
multiple organ failure, and death induced by sepsis. Cytokines,
especially tumor necrosis factor (TNF) and interleukin (IL-1), have
been shown to be critical mediators of septic shock. These
cytokines have a direct toxic effect on tissues; they also activate
phospholipase A2. These and other effects lead to increased
concentrations of platelet-activating factor, promotion of nitric
oxide synthase activity, promotion of tissue infiltration by
neutrophils, and promotion of neutrophil activity.
[0898] The treatment of sepsis and septic shock remains a clinical
conundrum, and recent prospective trials with biological response
modifiers (i.e. anti-TNF, anti-MIF) aimed at the inflammatory
response have shown only modest clinical benefit. Recently,
interest has shifted toward therapies aimed at reversing the
accompanying periods of immune suppression. Studies in experimental
animals and critically ill patients have demonstrated that
increased apoptosis of lymphoid organs and some parenchymal tissues
contribute to this immune suppression, anergy, and organ system
dysfunction. During sepsis syndromes, lymphocyte apoptosis can be
triggered by the absence of IL-2 or by the release of
glucocorticoids, granzymes, or the so-called `death` cytokines:
tumor necrosis factor alpha or Fas ligand. Apoptosis proceeds via
auto-activation of cytosolic and/or mitochondrial caspases, which
can be influenced by the pro- and anti-apoptotic members of the
Bcl-2 family. In experimental animals, not only can treatment with
inhibitors of apoptosis prevent lymphoid cell apoptosis; it may
also improve outcome. Although clinical trials with anti-apoptotic
agents remain distant due in large part to technical difficulties
associated with their administration and tissue targeting,
inhibition of lymphocyte apoptosis represents an attractive
therapeutic target for the septic patient. Likewise, a
dual-specific agent targeting both inflammatory mediator and a
apoptotic mediator, may have added benefit. One aspect of the
invention pertains to DVD-Igs capable of binding IL-17 and/or
IL-17F and one or more targets involved in sepsis selected from the
group consisting TNF, IL-1, MIF, IL-6, IL-8, IL-18, IL-12, IL-23,
FasL, LPS, Toll-like receptors, TLR-4, tissue factor, MIP-2,
ADORA2A, CASP1, CASP4, IL-10, IL-1B, NFKB1, PROC, TNFRSF1A, CSF3,
CCR3, IL1RN, MIF, NFKB1, PTAFR, TLR2, TLR4, GPR44, HMOX1, HMG-B1,
midkine, IRAK1, NFKB2, SERPINA1, SERPINE1, and TREM1. The efficacy
of such DVD-Igs for sepsis can be assessed in preclinical animal
models known in the art (see, Buras J A, et al., (2005) Nat. Rev.
Drug Discov., 4(10): 854-65 and Calandra T, et al., (2000) Nat.
Med., 6(2):164-70).
Neurological Disorders and Neurodegenerative Diseases
[0899] Neurodegenerative diseases are either chronic in which case
they are usually age-dependent or acute (e.g., stroke, traumatic
brain injury, spinal cord injury, etc.). They are characterized by
progressive loss of neuronal functions (neuronal cell death,
demyelination), loss of mobility and loss of memory. Emerging
knowledge of the mechanisms underlying chronic neurodegenerative
diseases (e.g., Alzheimer's disease, AD) show a complex etiology
and a variety of factors have been recognized to contribute to
their development and progression e.g., age, glycemic status,
amyloid production and multimerization, accumulation of advanced
glycation-end products (AGE) which bind to their receptor RAGE
(receptor for AGE), increased brain oxidative stress, decreased
cerebral blood flow, neuroinflammation including release of
inflammatory cytokines and chemokines, neuronal dysfunction and
microglial activation. Thus these chronic neurodegenerative
diseases represent a complex interaction between multiple cell
types and mediators. Treatment strategies for such diseases are
limited and mostly constitute either blocking inflammatory
processes with non-specific anti-inflammatory agents (e.g.,
corticosteroids, COX inhibitors) or agents to prevent neuron loss
and/or synaptic functions. These treatments fail to stop disease
progression. Recent studies suggest that more targeted therapies
such as antibodies to soluble A.beta. peptide (including the
A.beta. oligomeric forms) can not only help stop disease
progression but may help maintain memory as well. These preliminary
observations suggest that specific therapies targeting more than
one disease mediator (e.g., A.beta. and a pro-inflammatory cytokine
such as TNF) may provide even better therapeutic efficacy for
chronic neurodegenerative diseases than observed with targeting a
single disease mechanism (e.g., soluble A.beta. alone) (see C. E.
Shepherd, et al, Neurobiol Aging. 2005 Oct. 24; Nelson RB., Curr
Pharm Des. 2005; 11:3335; William L. Klein.; Neurochem Int. 2002;
41:345; Janelsins et al., "Early correlation of microglial
activation with enhanced tumor necrosis factor-alpha and monocyte
chemoattractant protein-I expression specifically within the
entorhinal cortex of triple transgenic Alzheimer's disease mice,"
Journal of Neuroinflammation, 2(23): 1-12 (2005); Soloman B., Curr
Alzheimer Res. 2004; 1:149; Igor Klyubin, et al., Nat Med. 2005;
11:556-61; Arancio O, et al., EMBO Journal (2004) 1-10; Bornemann K
D, et al., Am J Pathol. 2001; 158:63; Deane R, et al., Nat Med.
2003; 9:907-13; and Eliezer Masliah, et al., Neuron. 2005;
46:857).
[0900] The DVD-Ig molecules of the invention can bind IL-17 (and/or
IL-17F) and one or more targets involved in chronic
neurodegenerative diseases such as Alzheimers. Such targets
include, but are not limited to, any mediator, soluble or cell
surface, implicated in AD pathogenesis, e.g., AGE (S100 A,
amphotericin), pro-inflammatory cytokines (e.g., IL-1), chemokines
(e.g., MCP 1), molecules that inhibit nerve regeneration (e.g.,
Nogo, RGM A), molecules that enhance neurite growth (neurotrophins)
and molecules that can mediate transport at the blood brain barrier
(e.g., transferrin receptor, insulin receptor or RAGE). The
efficacy of DVD-Ig molecules can be validated in pre-clinical
animal models such as the transgenic mice that over-express amyloid
precursor protein or RAGE and develop Alzheimer's disease-like
symptoms. In addition, DVD-Ig molecules can be constructed and
tested for efficacy in the animal models and the best therapeutic
DVD-Ig can be selected for testing in human patients. DVD-Ig
molecules can also be employed for treatment of other
neurodegenerative diseases such as Parkinson's disease.
Alpha-Synuclein is involved in Parkinson's pathology. A DVD-Ig
capable of targeting IL-17 (and/or IL-17F) and LINGO-1,
alpha-synuclein, and/or inflammatory mediators such as TNF, IL-1,
MCP-1 can prove effective therapy for Parkinson's disease and are
contemplated in the invention.
Neuronal Regeneration and Spinal Cord Injury
[0901] Despite an increase in knowledge of the pathologic
mechanisms, spinal cord injury (SCI) is still a devastating
condition and represents a medical indication characterized by a
high medical need. Most spinal cord injuries are contusion or
compression injuries and the primary injury is usually followed by
secondary injury mechanisms (inflammatory mediators e.g., cytokines
and chemokines) that worsen the initial injury and result in
significant enlargement of the lesion area, sometimes more than
10-fold. These primary and secondary mechanisms in SCI are very
similar to those in brain injury caused by other means e.g.,
stroke. No satisfying treatment exists and high dose bolus
injection of methylprednisolone (MP) is the only used therapy
within a narrow time window of 8 h post injury. This treatment,
however, is only intended to prevent secondary injury without
causing any significant functional recovery. It is heavily
criticized for the lack of unequivocal efficacy and severe adverse
effects, like immunosuppression with subsequent infections and
severe histopathological muscle alterations. No other drugs,
biologics or small molecules, stimulating the endogenous
regenerative potential are approved, but promising treatment
principles and drug candidates have shown efficacy in animal models
of SCI in recent years. To a large extent the lack of functional
recovery in human SCI is caused by factors inhibiting neurite
growth, at lesion sites, in scar tissue, in myelin as well as on
injury-associated cells. Such factors are the myelin-associated
proteins NogoA, OMgp and MAG, RGM A, the scar-associated CSPG
(Chondroitin Sulfate Proteoglycans) and inhibitory factors on
reactive astrocytes (some semaphorins and ephrins). However, at the
lesion site not only growth inhibitory molecules are found but also
neurite growth stimulating factors like neurotrophins, laminin, L1
and others. This ensemble of neurite growth inhibitory and growth
promoting molecules may explain that blocking single factors, like
NogoA or RGM A, resulted in significant functional recovery in
rodent SCI models, because a reduction of the inhibitory influences
could shift the balance from growth inhibition to growth promotion.
However, recoveries observed with blocking a single neurite
outgrowth inhibitory molecule were not complete. To achieve faster
and more pronounced recoveries either blocking two neurite
outgrowth inhibitory molecules, e.g., Nogo and RGM A, or blocking
an neurite outgrowth inhibitory molecule and enhancing functions of
a neurite outgrowth enhancing molecule, e.g., Nogo and
neurotrophins, or blocking a neurite outgrowth inhibitory molecule,
e.g., Nogo and a pro-inflammatory molecule e.g., TNF, may be
desirable (see McGee A W, et al. (2003) Trends Neurosci., 26: 193;
Marco Domeniconi, et al. (2005) J. Neurol. Sci., 233: 43; Milan
Makwana 1, et al. (2005) FEBS J. 272:2628; Barry J. Dickson (2002)
Science, 298: 1959; Felicia Yu Hsuan Teng, et al. (2005) J.
Neurosci. Res. 79:273; Tara Karnezis, et al. (2004) Nature
Neuroscience, 7: 736; Gang Xu, et al. (2004) J. Neurochem., 91:
1018).
[0902] In one aspect, a DVD-Ig that binds hIL-17 and/or hIL-17F may
also bind one or both of the target pairs such as NgR and RGM A;
NogoA and RGM A; MAG and RGM A; OMGp and RGM A; RGM A and RGM B;
CSPGs and RGM A; aggrecan, midkine, neurocan, versican, phosphacan,
Te38 and TNF-.alpha.; A.beta. globulomer-specific antibodies
combined with antibodies promoting dendrite & axon sprouting
are provided. Dendrite pathology is a very early sign of AD and it
is known that NOGO A restricts dendrite growth. One can combine
such type of ab with any of the SCI-candidate (myelin-proteins) Ab.
Other DVD-Ig targets may include any combination of NgR-p75,
NgR-Troy, NgR-Nogo66 (Nogo), NgR-Lingo, Lingo-Troy, Lingo-p75, MAG
or Omgp. Additionally, targets may also include any mediator,
soluble or cell surface, implicated in inhibition of neurite, e.g.,
Nogo, Ompg, MAG, RGM A, semaphorins, ephrins, soluble A.beta.,
pro-inflammatory cytokines (e.g., IL-1), chemokines (e.g., MIP 1a),
molecules that inhibit nerve regeneration. The efficacy of
anti-nogo/anti-RGM A or similar DVD-Ig molecules can be validated
in pre-clinical animal models of spinal cord injury. In addition,
these DVD-Ig molecules can be constructed and tested for efficacy
in the animal models and the best therapeutic DVD-Ig can be
selected for testing in human patients. In addition, DVD-Ig
molecules can be constructed that target two distinct ligand
binding sites on a single receptor e.g., Nogo receptor which binds
three ligand Nogo, Ompg, and MAG and RAGE that binds A.beta. and
S100 A. Furthermore, neurite outgrowth inhibitors e.g., nogo and
nogo receptor, also play a role in preventing nerve regeneration in
immunological diseases like multiple sclerosis. Inhibition of
nogo-nogo receptor interaction has been shown to enhance recovery
in animal models of multiple sclerosis. Therefore, DVD-Ig molecules
that can block the function of one immune mediator, e.g., a
cytokine like IL-12, and a neurite outgrowth inhibitor molecule,
e.g., Nogo or RGM, may offer faster and greater efficacy than
blocking either an immune or a neurite outgrowth inhibitor molecule
alone.
[0903] In general, antibodies do not cross the blood brain barrier
(BBB) in an efficient and relevant manner. However, in certain
neurologic diseases, e.g., stroke, traumatic brain injury, multiple
sclerosis, etc., the BBB may be compromised and allows for
increased penetration of DVD-Igs and antibodies into the brain. In
other neurological conditions, where BBB leakage is not occurring,
one may employ the targeting of endogenous transport systems,
including carrier-mediated transporters such as glucose and amino
acid carriers and receptor-mediated transcytosis-mediating cell
structures/receptors at the vascular endothelium of the BBB, thus
enabling trans-BBB transport of the DVD-Ig. Structures at the BBB
enabling such transport include but are not limited to the insulin
receptor, transferrin receptor, LRP and RAGE. In addition,
strategies enable the use of DVD-Igs also as shuttles to transport
potential drugs into the CNS including low molecular weight drugs,
nanoparticles and nucleic acids (Coloma M J, et al. (2000) Pharm
Res. 17(3):266-74; Boado R J, et al. (2007) Bioconjug. Chem.
18(2):447-55).
Oncological Disorders
[0904] Monoclonal antibody therapy has emerged as an important
therapeutic modality for cancer (von Mehren et al., Annu. Rev.
Med., 54: 343-69 (2003)). Antibodies may exert antitumor effects by
inducing apoptosis, redirected cytotoxicity, interfering with
ligand-receptor interactions, or preventing the expression of
proteins that are critical to the neoplastic phenotype. In
addition, antibodies can target components of the tumor
microenvironment, perturbing vital structures such as the formation
of tumor-associated vasculature. Antibodies can also target
receptors whose ligands are growth factors, such as the epidermal
growth factor receptor. The antibody thus inhibits natural ligands
that stimulate cell growth from binding to targeted tumor cells.
Alternatively, antibodies may induce an anti-idiotype network,
complement-mediated cytotoxicity, or antibody-dependent cellular
cytotoxicity (ADCC). The use of dual-specific antibody that targets
two separate tumor mediators will likely give additional benefit
compared to a mono-specific therapy.
[0905] In another embodiment, a DVD-Ig that binds hIL-17 (and/or
hIL-17F) of the invention may also be capable of binding another
target involved in oncological diseases including, but not limited
to: IGFR, IGF, VGFR1, PDGFRb, PDGFRa, IGF1,2, ERB3, CDCP, 1BSG2,
ErbB3, CD52, CD20, CD19, CD3, CD4, CD8, BMP6, IL12A, IL1A, IL1B,
IL2, IL24, INHA, TNF, TNFSF10, BMP6, EGF, FGF1, FGF10, FGF11,
FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2, FGF20,
FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GRP,
IGF1, IGF2, IL12A, IL1A, IL1B, IL2, INHA, TGFA, TGFB1, TGFB2,
TGFB3, VEGF, CDK2, FGF10, FGF18, FGF2, FGF4, FGF7, IGF1R, IL2,
BCL2, CD164, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN3,
GNRH1, IGFBP6, IL1A, IL1B, ODZ1, PAWR, PLG, TGFB111, AR, BRCA1,
CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, E2F1, EGFR, ENO1, ERBB2, ESR1,
ESR2, IGFBP3, IGFBP6, IL2, INSL4, MYC, NOX5, NR6A1, PAP, PCNA,
PRKCQ, PRKD1, PRL, TP53, FGF22, FGF23, FGF9, IGFBP3, IL2, INHA,
KLK6, TP53, CHGB, GNRH1, IGF1, IGF2, INHA, INSL3, INSL4, PRL, KLK6,
SHBG, NR1D1, NR1H3, NR113, NR2F6, NR4A3, ESR1, ESR2, NROB1, NROB2,
NR1D2, NR1H2, NR1H4, NR112, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1,
NR2F2, NR3C1, NR3C2, NR4A1, NR4A2, NR5A1, NR5A2, NR6A1, PGR, RARB,
FGF1, FGF2, FGF6, KLK3, KRT1, APOC1, BRCA1, CHGA, CHGB, CLU,
COL1A1, COL6A1, EGF, ERBB2, ERK8, FGF1, FGF10, FGF11, FGF13, FGF14,
FGF16, FGF17, FGF18, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4,
FGF5, FGF6, FGF7, FGF8, FGF9, GNRH1, IGF1, IGF2, IGFBP3, IGFBP6,
IL12A, IL1A, IL1B, IL2, IL24, INHA, INSL3, INSL4, KLK10, KLK12,
KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, MMP2, MMP9,
MSMB, NTN4, ODZ1, PAP, PLAU, PRL, PSAP, SERPINA3, SHBG, TGFA,
TIMP3, CD44, CDH1, CDH10, CDH19, CDH20, CDH7, CDH9, CDH1, CDH10,
CDH13, CDH18, CDH19, CDH20, CDH7, CDH8, CDH9, ROBO2, CD44, ILK,
ITGA1, APC, CD164, COL6A1, MTSS1, PAP, TGFB1I1, AGR2, AIG1, AKAP1,
AKAP2, CANT1, CAV1, CDH12, CLDN3, CLN3, CYB5, CYC1, DAB21P, DES,
DNCL1, ELAC2, ENO2, ENO3, FASN, FLJ12584, FLJ25530, GAGEB1, GAGEC1,
GGT1, GSTP1, HIP1, HUMCYT2A, IL29, K6HF, KAI1, KRT2A, MIB1, PART1,
PATE, PCA3, PIAS2, PIK3CG, PPID, PR1, PSCA, SLC2A2, SLC33A1,
SLC43A1, STEAP, STEAP2, TPM1, TPM2, TRPC6, ANGPT1, ANGPT2, ANPEP,
ECGF1, EREG, FGF1, FGF2, FIGF, FLT1, JAG1, KDR, LAMAS, NRP1, NRP2,
PGF, PLXDC1, STAB1, VEGF, VEGFC, ANGPTL3, BAI1, COL4A3, IL8, LAMAS,
NRP1, NRP2, STAB1, ANGPTL4, PECAM1, PF4, PROK2, SERPINF1, TNFAIP2,
CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5, CXCL6, CXCL9, IFNA1,
IFNB1, IFNG, IL1B, IL6, MDK, EDG1, EFNA1, EFNA3, EFNB2, EGF, EPHB4,
FGFR3, HGF, IGF1, ITGB3, PDGFA, TEK, TGFA, TGFB1, TGFB2, TGFBR1,
CCL2, CDH5, COL18A1, EDG1, ENG, ITGAV, ITGB3, THBS1, THBS2, BAD,
BAG1, BCL2, CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CDH1 (E-cadherin),
CDKN1B (p27Kip1), CDKN2A (p161NK4a), COL6A1, CTNNB1 (b-catenin),
CTSB (cathepsin B), ERBB2 (Her-2), ESR1, ESR2, F3 (TF), FOSL1
(FRA-1), GATA3, GSN (Gelsolin), IGFBP2, IL2RA, IL6, IL6R, IL6ST
(glycoprotein 130), ITGA6 (a6 integrin), JUN, KLK5, KRT19, MAP2K7
(c-Jun), MKI67 (Ki-67), NGFB (NGF), NGFR, NME1 (NM23A), PGR, PLAU
(uPA), PTEN, SERPINB5 (maspin), SERPINE1 (PAI-1), TGFA, THBS1
(thrombospondin-1), TIE (Tie-1), TNFRSF6 (Fas), TNFSF6 (FasL),
TOP2A (topoisomerase Iia), TP53, AZGP1 (zinc-a-glycoprotein), BPAG1
(plectin), CDKN1A (p21Wap1/Cip1), CLDN7 (claudin-7), CLU
(clusterin), ERBB2 (Her-2), FGF1, FLRT1 (fibronectin), GABRP
(GABAa), GNAS1, ID2, ITGA6 (a6 integrin), ITGB4 (b 4 integrin),
KLF5 (GC Box BP), KRT19 (Keratin 19), KRTHB6 (hair-specific type II
keratin), MACMARCKS, MT3 (metallothionectin-III), MUC1 (mucin),
PTGS2 (COX-2), RAC2 (p21Rac2), S100A2, SCGB1D2 (lipophilin B),
SCGB2A1 (mammaglobin 2), SCGB2A2 (mammaglobin 1), SPRR1B (Spr1),
THBS1, THBS2, THBS4, and TNFAIP2 (B94), RON, c-Met, CD64, DLL4,
PLGF, CTLA4, phosphatidylserine, ROBO4, CD80, CD22, CD40, CD23,
CD28, CD55, CD38, CD70, CD74, CD30, CD138, CD56, CD33, CD2, CD137,
DR4, DR5, RANKL, VEGFR2, PDGFR, VEGFR1, MTSP1, MSP, EPHB2, EPHA1,
EPHA2, EpCAM, PGE2, NKG2D, LPA, SIP, APRIL, BCMA, MAPG, FLT3, PDGFR
alpha, PDGFR beta, ROR1, PSMA, PSCA, SCD1, and CD59.
D. Pharmaceutical Composition
[0906] The invention also provides pharmaceutical compositions
comprising an antibody, or antigen-binding portion thereof, of the
invention and a pharmaceutically acceptable carrier. The
pharmaceutical compositions comprising antibodies of the invention
are for use in, but not limited to, diagnosing, detecting, or
monitoring a disorder, in preventing, treating, managing, or
ameliorating of a disorder or one or more symptoms thereof, and/or
in research. In a specific embodiment, a composition comprises one
or more antibodies of the invention. In another embodiment, the
pharmaceutical composition comprises one or more antibodies of the
invention and one or more prophylactic or therapeutic agents other
than antibodies of the invention for treating a disorder in which
IL-17A and/or IL-17F activity is detrimental. In an embodiment, the
prophylactic or therapeutic agents are known to be useful for or
having been or currently being used in the prevention, treatment,
management, or amelioration of a disorder or one or more symptoms
thereof. In accordance with these embodiments, the composition may
further comprise of a carrier, diluent or excipient.
[0907] The antibodies and antibody portions of the invention can be
incorporated into pharmaceutical compositions suitable for
administration to a subject. Typically, the pharmaceutical
composition comprises an antibody or antibody portion of the
invention and a pharmaceutically acceptable carrier. As used
herein, "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like that
are physiologically compatible. Examples of pharmaceutically
acceptable carriers include one or more of water, saline, phosphate
buffered saline, dextrose, glycerol, ethanol and the like, as well
as combinations thereof. In many cases, it will be preferable to
include isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the composition.
Pharmaceutically acceptable carriers may further comprise minor
amounts of auxiliary substances such as wetting or emulsifying
agents, preservatives or buffers, which enhance the shelf life or
effectiveness of the antibody or antibody portion.
[0908] Various delivery systems are known and can be used to
administer one or more antibodies of the invention or the
combination of one or more antibodies of the invention and a
prophylactic agent or therapeutic agent useful for preventing,
managing, treating, or ameliorating a disorder or one or more
symptoms thereof, e.g., encapsulation in liposomes, microparticles,
microcapsules, recombinant cells capable of expressing the antibody
or antibody fragment, receptor-mediated endocytosis (see, e.g., Wu
and Wu, J. Biol. Chem., 262: 4429-4432 (1987)), construction of a
nucleic acid as part of a retroviral or other vector. Methods of
administering a prophylactic or therapeutic agent of the invention
include, but are not limited to, parenteral administration (e.g.,
intradermal, intramuscular, intraperitoneal, intravenous and
subcutaneous), epidural administration, intratumoral
administration, and mucosal administration (e.g., intranasal and
oral routes). In addition, pulmonary administration can be
employed, e.g., by use of an inhaler or nebulizer, and formulation
with an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968;
5,985,320; 5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540;
and 4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572,
WO 97/44013, WO 98/31346, and WO 99/66903, each of which is
incorporated herein by reference their entireties. In one
embodiment, an antibody or antibody portion of the invention,
combination therapy, or a composition of the invention is
administered using Alkermes AIR.RTM. pulmonary drug delivery
technology (Alkermes, Inc., Cambridge, Mass., US). In a specific
embodiment, prophylactic or therapeutic agents of the invention are
administered intramuscularly, intravenously, intratumorally,
orally, intranasally, pulmonary, or subcutaneously. The
prophylactic or therapeutic agents may be administered by any
convenient route, for example by infusion or bolus injection, by
absorption through epithelial or mucocutaneous linings (e.g., oral
mucosa, rectal, and intestinal mucosa, etc.) and may be
administered together with other biologically active agents.
Administration can be systemic or local.
[0909] In an embodiment, specific binding of antibody-coupled
carbon nanotubes (CNTs) to tumor cells in vitro, followed by their
highly specific ablation with near-infrared (NIR) light can be used
to target tumor cells. For example, biotinylated polar lipids can
be used to prepare stable, biocompatible, noncytotoxic CNT
dispersions that are then attached to one or two different
neutralite avidin-derivatized DVD-Igs directed against one or more
tumor antigens (e.g., CD22) (Chakravarty, P. et al. (2008) Proc.
Natl. Acad. Sci. USA, 105:8697-8702).
[0910] In a specific embodiment, it may be desirable to administer
the prophylactic or therapeutic agents of the invention locally to
the area in need of treatment; this may be achieved by, for
example, and not by way of limitation, local infusion, by
injection, or by means of an implant, said implant being of a
porous or non-porous material, including membranes and matrices,
such as sialastic membranes, polymers, fibrous matrices (e.g.,
Tissuel.RTM.), or collagen matrices. In one embodiment, an
effective amount of one or more antibodies of the invention
antagonists is administered locally to the affected area to a
subject to prevent, treat, manage, and/or ameliorate a disorder or
a symptom thereof. In another embodiment, an effective amount of
one or more antibodies of the invention is administered locally to
the affected area in combination with an effective amount of one or
more therapies (e.g., one or more prophylactic or therapeutic
agents) other than an antibody of the invention of a subject to
prevent, treat, manage, and/or ameliorate a disorder or one or more
symptoms thereof.
[0911] In another embodiment, the prophylactic or therapeutic agent
can be delivered in a controlled release or sustained release
system. In one embodiment, a pump may be used to achieve controlled
or sustained release (see Langer, supra; Sefton, 1987, CRC Crit.
Ref Biomed. Eng., 14: 20; 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 to achieve controlled
or sustained release of the therapies of the invention (see, e.g.,
Goodson, J. M., Chapter 6, In Medical Applications of Controlled
Release, Vol. II, Applications and Evaluation, (Langer and Wise,
eds.) (CRC Press, Inc., Boca Raton, 1984), pp. 115-138; Ranger and
Peppas, 1983, J. 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. 7 1:105); U.S.
Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No.
5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCT
Publication No. WO 99/15154; and PCT Publication No. WO 99/20253.
Examples of polymers used in sustained release formulations
include, but are not limited to, poly(-hydroxy ethyl methacrylate),
poly(methyl methacrylate), poly(acrylic acid),
poly(ethylene-co-vinyl acetate), poly(methacrylic acid),
polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),
poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol),
polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and
polyorthoesters. In a preferred embodiment, the polymer used in a
sustained release formulation is inert, free of leachable
impurities, stable on storage, sterile, and biodegradable. In yet
another embodiment, a controlled or sustained release system can be
placed in proximity of the prophylactic or therapeutic target, thus
requiring only a fraction of the systemic dose (see, e.g., Goodson,
in Medical Applications of Controlled Release, supra, vol. 2, pp.
115-138 (1984)).
[0912] Controlled release systems are discussed in the review by
Langer (1990, Science 249:1527-1533). Any technique known to one of
skill in the art can be used to produce sustained release
formulations comprising one or more therapeutic agents of the
invention. See, e.g., U.S. Pat. No. 4,526,938, PCT publication WO
91/05548, PCT publication WO 96/20698, Ning et al., 1996,
"Intratumoral Radioimmunotherapy of a Human Colon Cancer Xenograft
Using a Sustained-Release Gel," Radiotherapy & Oncology, 39:
179-189, Song et al., 1995, "Antibody Mediated Lung Targeting of
Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science
&Technology, 50: 372-397, Cleek et al., 1997, "Biodegradable
Polymeric Carriers for a bFGF Antibody for Cardiovascular
Application," Pro. Intl. Symp. Control. Rel. Bioact. Mater. 24:
853-854, and Lam et al., 1997, "Microencapsulation of Recombinant
Humanized Monoclonal Antibody for Local Delivery," Proc. Intl.
Symp. Control Rel. Bioact. Mater. 24: 759-760, each of which is
incorporated herein by reference in their entireties.
[0913] In a specific embodiment, where the composition of the
invention is a nucleic acid encoding a prophylactic or therapeutic
agent, the nucleic acid can be administered in vivo to promote
expression of its encoded prophylactic or therapeutic agent, by
constructing it as part of an appropriate nucleic acid expression
vector and administering it so that it becomes intracellular, e.g.,
by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by
direct injection, or by use of microparticle bombardment (e.g., a
gene gun; Biolistic, Dupont), or coating with lipids or
cell-surface receptors or transfecting agents, or by administering
it in linkage to a homeobox-like peptide which is known to enter
the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci.
USA, 88: 1864-1868). Alternatively, a nucleic acid can be
introduced intracellularly and incorporated within host cell DNA
for expression by homologous recombination.
[0914] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include, but are not limited
to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral,
intranasal (e.g., inhalation), transdermal (e.g., topical),
transmucosal, and rectal administration. In a specific embodiment,
the composition is formulated in accordance with routine procedures
as a pharmaceutical composition adapted for intravenous,
subcutaneous, intramuscular, oral, intranasal, or topical
administration to human beings. Typically, compositions for
intravenous administration are solutions in sterile isotonic
aqueous buffer. Where necessary, the composition may also include a
solubilizing agent and a local anesthetic, such as lignocamne, to
ease pain at the site of the injection.
[0915] If the compositions of the invention are to be administered
topically, the compositions can be formulated in the form of an
ointment, cream, transdermal patch, lotion, gel, shampoo, spray,
aerosol, solution, emulsion, or other form well-known to one of
skill in the art. See, e.g., Remington's Pharmaceutical Sciences
and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack
Pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage
forms, viscous to semi-solid or solid forms comprising a carrier or
one or more excipients compatible with topical application and
having a dynamic viscosity preferably greater than water are
typically employed. Suitable formulations include, without
limitation, solutions, suspensions, emulsions, creams, ointments,
powders, liniments, salves, and the like, which are, if desired,
sterilized or mixed with auxiliary agents (e.g., preservatives,
stabilizers, wetting agents, buffers, or salts) for influencing
various properties, such as, for example, osmotic pressure. Other
suitable topical dosage forms include sprayable aerosol
preparations wherein the active ingredient, preferably in
combination with a solid or liquid inert carrier, is packaged in a
mixture with a pressurized volatile (e.g., a gaseous propellant,
such as FREON.RTM.) or in a squeeze bottle. Moisturizers or
humectants can also be added to pharmaceutical compositions and
dosage forms if desired. Examples of such additional ingredients
are well known in the art.
[0916] If the method of the invention comprises intranasal
administration of a composition, the composition can be formulated
in an aerosol form, spray, mist or in the form of drops. In
particular, prophylactic or therapeutic agents for use according to
the present invention can be conveniently delivered in the form of
an aerosol spray presentation from pressurized packs or a
nebulizer, with the use of a suitable propellant (e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas).
In the case of a pressurized aerosol the dosage unit may be
determined by providing a valve to deliver a metered amount.
Capsules and cartridges (composed of, e.g., gelatin) for use in an
inhaler or insufflator may be formulated containing a powder mix of
the compound and a suitable powder base such as lactose or
starch.
[0917] If the method of the invention comprises oral
administration, compositions can be formulated orally in the form
of tablets, capsules, cachets, gelcaps, solutions, suspensions, and
the like. Tablets or capsules can be prepared by conventional means
with pharmaceutically acceptable excipients such as binding agents
(e.g., pregelatinised maize starch, polyvinylpyrrolidone, or
hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose, or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc, or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well-known in the art. Liquid preparations for
oral administration may take the form of, but not limited to,
solutions, syrups or suspensions, or they may be presented as a dry
product for constitution with water or other suitable vehicle
before use. Such liquid preparations may be prepared by
conventional means with pharmaceutically acceptable additives such
as suspending agents (e.g., sorbitol syrup, cellulose derivatives,
or hydrogenated edible fats); emulsifying agents (e.g., lecithin or
acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl
alcohol, or fractionated vegetable oils); and preservatives (e.g.,
methyl or propyl-p-hydroxybenzoates or sorbic acid). The
preparations may also contain buffer salts, flavoring, coloring,
and sweetening agents as appropriate. Preparations for oral
administration may be suitably formulated for slow release,
controlled release, or sustained release of a prophylactic or
therapeutic agent(s).
[0918] The method of the invention may comprise pulmonary
administration, e.g., by use of an inhaler or nebulizer, of a
composition formulated with an aerosolizing agent. See, e.g., U.S.
Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064;
5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO
92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903,
each of which is incorporated herein by reference their entireties.
In a specific embodiment, an antibody of the invention, combination
therapy, and/or composition of the invention is administered using
Alkermes AIR.RTM. pulmonary drug delivery technology (Alkermes,
Inc., Cambridge, Mass.).
[0919] The method of the invention may comprise administration of a
composition formulated for parenteral administration by injection
(e.g., by bolus injection or continuous infusion). Formulations for
injection may be presented in unit dosage form (e.g., in ampoules
or in multi-dose containers) with an added preservative. The
compositions may take such forms as suspensions, solutions or
emulsions in oily or aqueous vehicles, and may contain formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the active ingredient may be in powder form for
constitution with a suitable vehicle (e.g., sterile pyrogen-free
water) before use.
[0920] The methods of the invention may additionally comprise of
administration of compositions formulated as depot preparations.
Such long acting formulations may be administered by implantation
(e.g., subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compositions may be formulated
with suitable polymeric or hydrophobic materials (e.g., as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives (e.g., as a sparingly soluble
salt).
[0921] The methods of the invention encompass administration of
compositions formulated as neutral or salt forms. Pharmaceutically
acceptable salts include those formed with anions such as those
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
acids, etc., and those formed with cations such as those derived
from sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0922] Generally, the ingredients of compositions 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 sachet
indicating the quantity of active agent. Where the mode of
administration is infusion, composition can be dispensed with an
infusion bottle containing sterile pharmaceutical grade water or
saline. Where the mode of administration is by injection, an
ampoule of sterile water for injection or saline can be provided so
that the ingredients may be mixed prior to administration.
[0923] In particular, the invention also provides that one or more
of the prophylactic or therapeutic agents, or pharmaceutical
compositions of the invention is packaged in a hermetically sealed
container such as an ampoule or sachette indicating the quantity of
the agent. In one embodiment, one or more of the prophylactic or
therapeutic agents, or pharmaceutical compositions of the invention
is supplied as a dry sterilized lyophilized powder or water free
concentrate in a hermetically sealed container and can be
reconstituted (e.g., with water or saline) to the appropriate
concentration for administration to a subject. Preferably, one or
more of the prophylactic or therapeutic agents or pharmaceutical
compositions of the invention is supplied as a dry sterile
lyophilized powder in a hermetically sealed container at a unit
dosage of at least 5 mg, more preferably at least 10 mg, at least
15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50
mg, at least 75 mg, or at least 100 mg. The lyophilized
prophylactic or therapeutic agents or pharmaceutical compositions
of the invention should be stored at between 2.degree. C. and
8.degree. C. in its original container and the prophylactic or
therapeutic agents, or pharmaceutical compositions of the invention
should be administered within 1 week, preferably within 5 days,
within 72 hours, within 48 hours, within 24 hours, within 12 hours,
within 6 hours, within 5 hours, within 3 hours, or within 1 hour
after being reconstituted. In an alternative embodiment, one or
more of the prophylactic or therapeutic agents or pharmaceutical
compositions of the invention is supplied in liquid form in a
hermetically sealed container indicating the quantity and
concentration of the agent. Preferably, the liquid form of the
administered composition is supplied in a hermetically sealed
container at least 0.25 mg/ml, more preferably at least 0.5 mg/ml,
at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8
mg/ml, at least 10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at
least 50 mg/ml, at least 75 mg/ml or at least 100 mg/ml. The liquid
form should be stored at between 2.degree. C. and 8.degree. C. in
its original container.
[0924] The antibodies and antibody portions of the invention can be
incorporated into a pharmaceutical composition suitable for
parenteral administration. Preferably, the antibody or
antibody-portions will be prepared as an injectable solution
containing 0.1-250 mg/ml antibody. The injectable solution can be
composed of either a liquid or lyophilized dosage form in a flint
or amber vial, ampoule or pre-filled syringe. The buffer can be
L-histidine (1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0
(optimally pH 6.0). Other suitable buffers include but are not
limited to, sodium succinate, sodium citrate, sodium phosphate or
potassium phosphate. Sodium chloride can be used to modify the
toxicity of the solution at a concentration of 0-300 mM (optimally
150 mM for a liquid dosage form). Cryoprotectants can be included
for a lyophilized dosage form, principally 0-10% sucrose (optimally
0.5-1.0%). Other suitable cryoprotectants include trehalose and
lactose. Bulking agents can be included for a lyophilized dosage
form, principally 1-10% mannitol (optimally 2-4%). Stabilizers can
be used in both liquid and lyophilized dosage forms, principally
1-50 mM L-Methionine (optimally 5-10 mM). Other suitable bulking
agents include glycine, arginine, can be included as 0-0.05%
polysorbate-80 (optimally 0.005-0.01%). Additional surfactants
include but are not limited to polysorbate 20 and BRIJ surfactants.
The pharmaceutical composition comprising an antibody or antibody
portion of the invention prepared as an injectable solution for
parenteral administration, can further comprise an agent useful as
an adjuvant, such as those used to increase the absorption, or
dispersion of a therapeutic protein (e.g., antibody). A
particularly useful adjuvant is hyaluronidase (such as Hylenex.RTM.
recombinant human hyaluronidase). Addition of hyaluronidase in the
injectable solution improves human bioavailability following
parenteral administration, particularly subcutaneous
administration. It also allows for greater injection site volumes
(i.e. greater than 1 ml) with less pain and discomfort, and minimum
incidence of injection site reactions (see, WO 2004/078140 and US
patent application publication No. 2006104968).
[0925] The compositions of this invention may be in a variety of
forms. These include, for example, liquid, semi-solid and solid
dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, tablets, pills,
powders, liposomes and suppositories. The preferred form depends on
the intended mode of administration and therapeutic application.
Typical preferred compositions are in the form of injectable or
infusible solutions, such as compositions similar to those used for
passive immunization of humans with other antibodies. The preferred
mode of administration is parenteral (e.g., intravenous,
subcutaneous, intraperitoneal, intramuscular). In a preferred
embodiment, the antibody is administered by intravenous infusion or
injection. In another preferred embodiment, the antibody is
administered by intramuscular or subcutaneous injection.
[0926] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
dispersion, liposome, or other ordered structure suitable to high
drug concentration. Sterile injectable solutions can be prepared by
incorporating the active compound (i.e., antibody or antibody
portion) in the required amount in an appropriate solvent with one
or a combination of ingredients enumerated above, as required,
followed by filtered sterilization. Generally, dispersions are
prepared by incorporating the active compound into a sterile
vehicle that contains a basic dispersion medium and the required
other ingredients from those enumerated above. In the case of
sterile, lyophilized powders for the preparation of sterile
injectable solutions, the preferred methods of preparation are
vacuum drying and spray-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof. The proper fluidity of a
solution can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prolonged
absorption of injectable compositions can be brought about by
including, in the composition, an agent that delays absorption, for
example, monostearate salts and gelatin.
[0927] The antibodies and antibody-portions of the present
invention can be administered by a variety of methods known in the
art, although for many therapeutic applications, the preferred
route/mode of administration is subcutaneous injection, intravenous
injection or infusion. As will be appreciated by the skilled
artisan, the route and/or mode of administration will vary
depending upon the desired results. In certain embodiments, the
active compound may be prepared with a carrier that will protect
the compound against rapid release, such as a controlled release
formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in
the art. See, e.g., Sustained and Controlled Release Drug Delivery
Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,
1978.
[0928] In certain embodiments, an antibody or antibody portion of
the invention may be orally administered, for example, with an
inert diluent or an assimilable edible carrier. The compound (and
other ingredients, if desired) may also be enclosed in a hard or
soft shell gelatin capsule, compressed into tablets, or
incorporated directly into the subject's diet. For oral therapeutic
administration, the compounds may be incorporated with excipients
and used in the form of ingestible tablets, buccal tablets,
troches, capsules, elixirs, suspensions, syrups, wafers, and the
like. To administer a compound of the invention by other than
parenteral administration, it may be necessary to coat the compound
with, or co-administer the compound with, a material to prevent its
inactivation.
[0929] Supplementary active compounds can also be incorporated into
the compositions. In certain embodiments, an antibody or antibody
portion of the invention is coformulated with and/or coadministered
with one or more additional therapeutic agents that are useful for
treating disorders in which IL-17 activity is detrimental. For
example, an anti-hIL-17 antibody or antibody portion of the
invention may be coformulated and/or coadministered with one or
more additional antibodies that bind other targets (e.g.,
antibodies that bind other cytokines or that bind cell surface
molecules). Furthermore, one or more antibodies of the invention
may be used in combination with two or more of the foregoing
therapeutic agents. Such combination therapies may advantageously
utilize lower dosages of the administered therapeutic agents, thus
avoiding possible toxicities or complications associated with the
various monotherapies.
[0930] In certain embodiments, an antibody to IL-17 or fragment
thereof is linked to a half-life extending vehicle known in the
art. Such vehicles include, but are not limited to, the Fc domain,
polyethylene glycol, and dextran. Such vehicles are described,
e.g., in U.S. Ser. No. 09/428,082 and published PCT Publication No.
WO 99/25044, which are hereby incorporated by reference for any
purpose.
[0931] In a specific embodiment, nucleic acid sequences comprising
nucleotide sequences encoding an antibody of the invention or
another prophylactic or therapeutic agent of the invention are
administered to treat, prevent, manage, or ameliorate a disorder or
one or more symptoms thereof by way of gene therapy. Gene therapy
refers to therapy performed by the administration to a subject of
an expressed or expressible nucleic acid. In this embodiment of the
invention, the nucleic acids produce their encoded antibody or
prophylactic or therapeutic agent of the invention that mediates a
prophylactic or therapeutic effect.
[0932] Any of the methods for gene therapy available in the art can
be used according to the present invention. For general reviews of
the methods of gene therapy, see Goldspiel et al., 1993, Clinical
Pharmacy, 12: 488-505; Wu et al., "Delivery systems for gene
therapy," Biotherapy, 3: 87-95 (1991); Tolstoshev, 1993, Ann. Rev.
Pharmacol. Toxicol., 32: 573-596; Mulligan, Science, 260: 926-932
(1993); and Morgan and Anderson, "Human Gene Therapy," Ann. Rev.
Biochem., 62:191-217 (1993); Robinson, C., Trends Biotechnol.,
11:155 (1993). Methods commonly known in the art of recombinant DNA
technology which can be used are described in Ausubel et al.
(eds.), Current Protocols in Molecular Biology, John Wiley
&Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A
Laboratory Manual, Stockton Press, NY (1990). Detailed description
of various methods of gene therapy are disclosed in US application
publication No. US 2005/0042664 A1, which is incorporated herein by
reference.
[0933] IL-17 plays a critical role in the pathology associated with
a variety of diseases involving immune and inflammatory elements.
These diseases include, but are not limited to, Acquired
Immunodeficiency Disease Syndrome; Acquired Immunodeficiency
Related Diseases; acquired pernicious anaemia; Acute coronary
syndromes; acute and chronic pain (different forms of pain); Acute
Idiopathic Polyneuritis; acute immune disease associated with organ
transplantation; acute or chronic immune disease associated with
organ transplantation; Acute Inflammatory Demyelinating
Polyradiculoneuropathy; Acute ischemia; acute liver disease; acute
rheumatic fever; acute transverse myelitis; Addison's disease;
adult (acute) respiratory distress syndrome; Adult Still's Disease;
alcoholic cirrhosis; alcohol-induced liver injury; allergic
diseases; allergy; alopecia; Alopecia greata; Alzheimer's disease;
Anaphylaxis; ankylosing spondylitis; ankylosing spondylitis
associated lung disease; Anti-Phospholipid Antibody Syndrome;
Aplastic anemia; Arteriosclerosis; arthropathy; asthma;
atheromatous disease/arteriosclerosis; atherosclerosis; atopic
allergy; Atopic eczema; Atopic dermatitis; atrophic autoimmune
hypothyroidism; autoimmune bullous disease; Autoimmune dermatitis;
autoimmune diabetes; Autoimmune disorder associated with
Streptococcus infection; Autoimmune Enteropathy; autoimmune
haemolytic anaemia; autoimmune hepatitis; Autoimmune hearing loss;
Autoimmune Lymphoproliferative Syndrome (ALPS); autoimmune mediated
hypoglycaemia; Autoimmune myocarditis; autoimmune neutropenia;
Autoimmune premature ovarian failure; autoimmune thrombocytopenia
(AITP); autoimmune thyroid disease; autoimmune uveitis;
bronchiolitis obliterans; Behcet's disease; Blepharitis;
Bronchiectasis; Bullous pemphigoid; cachexia; Cardiovascular
Disease; Catastrophic Antiphospholipid Syndrome; Celiac Disease;
Cervical Spondylosis; chlamydia; choleosatatis; chronic active
hepatitis; chronic eosinophilic pneumonia; chronic fatigue
syndrome; chronic immune disease associated with organ
transplantation; Chronic ischemia; chronic liver diseases; chronic
mucocutaneous candidiasis; Cicatricial pemphigoid; Clinically
isolated Syndrome (CIS) with Risk for Multiple Sclerosis; common
varied immunodeficiency (common variable hypogammaglobulinaemia);
connective tissue disease associated interstitial lung disease;
Conjunctivitis; Coombs positive haemolytic anaemia; Childhood Onset
Psychiatric Disorder; Chronic obstructive pulmonary disease (COPD);
Crohn's disease; cryptogenic autoimmune hepatitis; cryptogenic
fibrosing alveolitis; Dacryocystitis; depression; dermatitis
scleroderma; dermatomyositis; dermatomyositis/polymyositis
associated lung disease; Diabetic retinopathy; Diabetes mellitus;
dilated cardiomyopathy; discoid lupus erythematosus; Disk
herniation; Disk prolapse; disseminated intravascular coagulation;
Drug-Induced hepatitis; drug-induced interstitial lung disease;
Drug induced immune hemolytic anemia; Endocarditis; Endometriosis;
endophthalmitis; enteropathic synovitis; Episcleritis; Erythema
multiforme; erythema multiforme major; female infertility;
fibrosis; fibrotic lung disease; Gestational pemphigoid; giant cell
arteritis (GCA); glomerulonephritides; goitrous autoimmune
hypothyroidism (Hashimoto's disease); Goodpasture's syndrome; gouty
arthritis; graft versus host disease (GVHD); Grave's disease; group
B streptococci (GBS) infection; Guillain-Barre Syndrome (GBS);
haemosiderosis associated lung disease; Hay Fever; heart failure;
hemolytic anemia; Henoch-Schoenlein purpurea; Hepatitis B;
Hepatitis C; Hughes Syndrome; Huntington's chorea; hyperthyroidism;
hypoparathyroidism; idiopathic leucopaenia; idiopathic
thrombocytopaenia; Idiopathic Parkinson's Disease; idiopathic
interstitial pneumonia; idiosyncratic liver disease; IgE-mediated
Allergy; Immune hemolytic anemiae; Inclusion Body Myositis;
infectious diseases; Infectious ocular inflammatory disease;
inflammatory bowel disease; Inflammatory demyelinating disease;
Inflammatory heart disease; Inflammatory kidney disease; insulin
dependent diabetes mellitus; interstitial pneumonitis; IPF/UIP;
Iritis; juvenile chronic arthritis; juvenile pernicious anaemia;
Juvenile rheumatoid arthritis; Kawasaki's disease; Keratitis;
Keratojunctivitis sicca; Kussmaul disease or Kussmaul-Meier
Disease; Landry's Paralysis; Langerhan's Cell Histiocytosis; linear
IgA disease; Livedo reticularis; Lyme arthritis; lymphocytic
infiltrative lung disease; Macular Degeneration; male infertility
idiopathic or NOS; malignancies; microscopic vasculitis of the
kidneys; Microscopic Polyangiitis; mixed connective tissue disease
associated lung disease; Morbus Bechterev; Motor Neuron Disorders;
Mucous membrane pemphigoid; multiple sclerosis (all subtypes:
primary progressive, secondary progressive, relapsing remitting
etc.); Multiple Organ failure; myalgic encephalitis/Royal Free
Disease; Myasthenia Gravis; Myelodysplastic Syndrome; myocardial
infarction; Myocarditis; nephrotic syndrome; Nerve Root Disorders;
Neuropathy; Non-alcoholic Steatohepatitis; Non-A Non-B Hepatitis;
Optic Neuritis; organ transplant rejection; osteoarthritis;
Osteolysis; Ovarian cancer; ovarian failure; Pancreatitis;
Parasitic diseases; Parkinson's disease; Pauciarticular JRA;
pemphigoid; pemphigus foliaceus; pemphigus vulgaris; peripheral
artery occlusive disease (PAOD); peripheral vascular disease (PVD);
peripheral artery disease (PAD); phacogenic uveitis; Phlebitis;
Polyarteritis nodosa (or periarteritis nodosa); Polychondritis;
Polymyalgia Rheumatica; Poliosis; Polyarticular JRA; Polyendocrine
Deficiency Syndrome; Polymyositis; polyglandular deficiency type I
and polyglandular deficiency type II; polymyalgia rheumatica (PMR);
postinfectious interstitial lung disease; post-inflammatory
interstitial lung disease; Post-Pump Syndrome; premature ovarian
failure; primary biliary cirrhosis; primary myxoedema; primary
parkinsonism; primary sclerosing cholangitis; primary sclerosing
hepatitis; primary vasculitis; prostate and rectal cancer and
hematopoietic malignancies (leukemia and lymphoma); Prostatitis;
psoriasis; psoriasis type 1; psoriasis type 2; psoriatic arthritis;
psoriatic arthropathy; pulmonary hypertension secondary to
connective tissue disease; pulmonary manifestation of polyarteritis
nodosa; Pure red cell aplasia; Primary Adrenal Insufficiency;
radiation fibrosis; reactive arthritis; Reiter's disease; Recurrent
Neuromyelitis Optica; renal disease NOS; Restenosis; rheumatoid
arthritis; rheumatoid arthritis associated interstitial lung
disease; Rheumatic heart disease; SAPHO (synovitis, acne,
pustulosis, hyperostosis, and osteitis); sarcoidosis;
Schizophreniae; Schmidt's syndrome; Scleroderma; Secondary
Amyloidosis; Shock lung; Scleritis; Sciatica; Secondary Adrenal
Insufficiency; sepsis syndrome; septic arthritis; septic shock;
seronegative arthropathy; Silicone associated connective tissue
disease; Sjogren's disease associated lung disease; Sjorgren's
syndrome; Sneddon-Wilkinson Dermatosis; sperm autoimmunity;
spondyloarthropathy; spondylitis ankylosans; Stevens-Johnson
Syndrome (SJS); Still's disease; stroke; sympathetic ophthalmia;
Systemic inflammatory response syndrome; systemic lupus
erythematosus; systemic lupus erythematosus associated lung
disease; systemic sclerosis; systemic sclerosis associated
interstitial lung disease; Takayasu's disease/arteritis; Temporal
arteritis; Th2 Type and Th1 Type mediated diseases; thyroiditis;
toxic shock syndrome; toxoplasmic retinitis; toxic epidermal
necrolysis; Transverse myelitis; TRAPS (Tumor-necrosis factor
receptor type 1 (TNFR)-Associated Periodic Syndrome); type B
insulin resistance with acanthosis nigricans; Type 1 allergic
reaction; type-1 autoimmune hepatitis (classical autoimmune or
lupoid hepatitis); type-2 autoimmune hepatitis (anti-LKM antibody
hepatitis)e; Type II Diabetes; ulcerative colitic arthropathy;
ulcerative colitis; Urticaria; Usual interstitial pneumonia (UIP);
uveitis; vasculitic diffuse lung disease; Vasculitis; Vernal
conjunctivitis; viral retinitis; vitiligo; Vogt-Koyanagi-Harada
syndrome (VKH syndrome); Wegener's granulomatosis; Wet macular
degeneration; Wound healing; yersinia and salmonella associated
arthropathy.
[0934] The antibodies and antibody portions of the invention can be
used to treat humans suffering from autoimmune diseases, in
particular those associated with inflammation, rheumatoid arthritis
(RA), osteoarthritis, psoriasis, multiple sclerosis (MS), and other
autoimmune diseases.
[0935] An antibody or antibody portion of the invention also can be
administered with one or more additional therapeutic agents useful
in the treatment of autoimmune and inflammatory diseases.
[0936] In an embodiment, diseases that can be treated or diagnosed
with the compositions and methods of the invention include, but are
not limited to, primary and metastatic cancers, including
carcinomas of breast, colon, rectum, lung, oropharynx, hypopharynx,
esophagus, stomach, pancreas, liver, gallbladder and bile ducts,
small intestine, urinary tract (including kidney, bladder and
urothelium), female genital tract (including cervix, uterus, and
ovaries as well as choriocarcinoma and gestational trophoblastic
disease), male genital tract (including prostate, seminal vesicles,
testes and germ cell tumors), endocrine glands (including the
thyroid, adrenal, and pituitary glands), and skin, as well as
hemangiomas, melanomas, sarcomas (including those arising from bone
and soft tissues as well as Kaposi's sarcoma), tumors of the brain,
nerves, eyes, and meninges (including astrocytomas, gliomas,
glioblastomas, retinoblastomas, neuromas, neuroblastomas,
Schwannomas, and meningiomas), solid tumors arising from
hematopoietic malignancies such as leukemias, and lymphomas (both
Hodgkin's and non-Hodgkin's lymphomas).
[0937] In another embodiment, an antibody of the invention or
antigen binding portion thereof is used to treat cancer or in the
prevention of metastases from a tumor. Such treatment may involve
administration of the antibody or antigen binding portion thereof
alone or in combination with another therapeutic agent or
treatment, such as radiotherapy and/or a chemotherapeutic
agent.
[0938] The antibodies of the invention, or antigen binding portions
thereof, may be combined with agents that include but are not
limited to, antineoplastic agents, radiotherapy, chemotherapy such
as DNA alkylating agents, cisplatin, carboplatin, anti-tubulin
agents, paclitaxel, docetaxel, taxol, doxorubicin, gemcitabine,
gemzar, anthracyclines, adriamycin, topoisomerase I inhibitors,
topoisomerase II inhibitors, 5-fluorouracil (5-FU), leucovorin,
irinotecan, receptor tyrosine kinase inhibitors (e.g., erlotinib,
gefitinib), COX-2 inhibitors (e.g., celecoxib), kinase inhibitors,
and siRNAs.
[0939] A binding protein of the invention also can be administered
with one or more additional therapeutic agents useful in the
treatment of various diseases.
[0940] Antibodies of the invention, or antigen binding portions
thereof, can be used alone or in combination to treat such
diseases. It should be understood that the antibodies of the
invention or antigen binding portion thereof can be used alone or
in combination with an additional agent, e.g., a therapeutic agent,
said additional agent being selected by the skilled artisan for its
intended purpose. For example, the additional agent can be a
therapeutic agent art-recognized as being useful to treat the
disease or condition being treated by the antibody of the present
invention. The additional agent also can be an agent that imparts a
beneficial attribute to the therapeutic composition, e.g., an agent
that affects the viscosity of the composition.
[0941] It should further be understood that the combinations which
are to be included within this invention are those combinations
useful for their intended purpose. The agents set forth below are
illustrative for purposes and not intended to be limited. The
combinations, which are part of this invention, can be the
antibodies of the present invention and at least one additional
agent selected from the lists below. The combination can also
include more than one additional agent, e.g., two or three
additional agents if the combination is such that the formed
composition can perform its intended function.
[0942] Preferred combinations are non-steroidal anti-inflammatory
drug(s) also referred to as NSAIDS which include drugs like
ibuprofen. Other preferred combinations are corticosteroids
including prednisolone; the well known side-effects of steroid use
can be reduced or even eliminated by tapering the steroid dose
required when treating patients in combination with the anti-IL-17
antibodies of this invention. Non-limiting examples of therapeutic
agents for rheumatoid arthritis with which an antibody or antibody
portion of the invention can be combined include, but are not
limited to, the following: cytokine suppressive anti-inflammatory
drug(s) (CSAIDs); antibodies to or antagonists of other human
cytokines or growth factors, for example, TNF, LT, IL-1, IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, IL-21,
interferons, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of the
invention, or antigen binding portions thereof, can be combined
with antibodies to cell surface molecules such as CD2, CD3, CD4,
CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2),
CD90, CTLA or their ligands including CD154 (gp39 or CD40L).
[0943] Preferred combinations of therapeutic agents may interfere
at different points in the autoimmune and subsequent inflammatory
cascade; preferred examples include TNF antagonists like chimeric,
humanized or human TNF antibodies, D2E7, (PCT Publication No. WO
97/29131), CA2 (Remicade.TM.), CDP 571, and soluble p55 or p75 TNF
receptors, derivatives, thereof, (p75TNFR1gG (Enbrel.TM.) or
p55TNFR1gG (Lenercept), and also TNF.alpha. converting enzyme
(TACE) inhibitors; similarly IL-1 inhibitors
(Interleukin-1-converting enzyme inhibitors, IL-1RA etc.) may be
effective for the same reason. Other preferred combinations include
Interleukin 11. Yet another preferred combination are other key
players of the autoimmune response which may act parallel to,
dependent on or in concert with IL-17 function. Yet another
preferred combination are non-depleting anti-CD4 inhibitors. Yet
other preferred combinations include antagonists of the
co-stimulatory pathway CD80 (B7.1) or CD86 (B7.2) including
antibodies, soluble receptors or antagonistic ligands.
[0944] The antibodies of the invention, or antigen binding portions
thereof, may also be combined with agents, such as methotrexate,
6-MP, azathioprine sulphasalazine, mesalazine, olsalazine
chloroquinine/hydroxychloroquine, pencillamine, aurothiomalate
(intramuscular and oral), azathioprine, colchicine, corticosteroids
(oral, inhaled and local injection), beta-2 adrenoreceptor agonists
(salbutamol, terbutaline, salmeteral), xanthines (theophylline,
aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium
and oxitropium, cyclosporin, FK506, rapamycin, mycophenolate
mofetil, leflunomide, NSAIDs, for example, ibuprofen,
corticosteroids such as prednisolone, phosphodiesterase inhibitors,
adensosine agonists, antithrombotic agents, complement inhibitors,
adrenergic agents, agents which interfere with signaling by
proinflammatory cytokines such as TNF-.alpha. or IL-1 (e.g., IRAK,
NIK, IKK, p38, or MAP kinase inhibitors), IL-1.beta. converting
enzyme inhibitors, TNF.alpha. converting enzyme (TACE) inhibitors,
T-cell signaling inhibitors such as kinase inhibitors,
metalloproteinase inhibitors, sulfasalazine, azathioprine,
6-mercaptopurines, angiotensin converting enzyme inhibitors,
soluble cytokine receptors and derivatives thereof (e.g. soluble
p55 or p75 TNF receptors and the derivatives p75TNFRIgG (Enbrel.TM.
and p55TNFRIgG (Lenercept)), sIL-1RI, sIL-1RII, sIL-6R),
antiinflammatory cytokines (e.g. IL-4, IL-10, IL-11, IL-13 and
TGF.beta.), celecoxib, folic acid, hydroxychloroquine sulfate,
rofecoxib, etanercept, infliximab, naproxen, valdecoxib,
sulfasalazine, methylprednisolone, meloxicam, methylprednisolone
acetate, gold sodium thiomalate, aspirin, triamcinolone acetonide,
propoxyphene napsylate/apap, folate, nabumetone, diclofenac,
piroxicam, etodolac, diclofenac sodium, oxaprozin, oxycodone hcl,
hydrocodone bitartrate/apap, diclofenac sodium/misoprostol,
fentanyl, anakinra, human recombinant, tramadol hcl, salsalate,
sulindac, cyanocobalamin/fa/pyridoxine, acetaminophen, alendronate
sodium, prednisolone, morphine sulfate, lidocaine hydrochloride,
indomethacin, glucosamine sulf/chondroitin, amitriptyline hcl,
sulfadiazine, oxycodone hcl/acetaminophen, olopatadine hcl,
misoprostol, naproxen sodium, omeprazole, cyclophosphamide,
rituximab, IL-1 TRAP, MRA, CTLA4-IG, IL-18 BP, anti-IL-18,
Anti-IL15, BIRB-796, SCIO-469, VX-702, AMG-548, VX-740,
Roflumilast, IC-485, CDC-801, and Mesopram. Preferred combinations
include methotrexate or leflunomide and in moderate or severe
rheumatoid arthritis cases, cyclosporine.
[0945] Non-limiting additional agents which can also be used in
combination with a binding protein to treat rheumatoid arthritis
(RA) include, but are not limited to, the following: non-steroidal
anti-inflammatory drug(s) (NSAIDs); cytokine suppressive
anti-inflammatory drug(s) (CSAIDs); CDP-571/BAY-10-3356 (humanized
anti-TNF.alpha. antibody; Celltech/Bayer); cA2/infliximab (chimeric
anti-TNF.alpha. antibody; Centocor); 75 kdTNFR-IgG/etanercept (75
kD TNF receptor-IgG fusion protein; Immunex; see e.g., Arthritis
& Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol.
44, 235A); 55 kdTNF-IgG (55 kD TNF receptor-IgG fusion protein;
Hoffmann-LaRoche); IDEC-CE9.1/SB 210396 (non-depleting primatized
anti-CD4 antibody; IDEC/SmithKline; see e.g., Arthritis &
Rheumatism (1995) Vol. 38, S185); DAB 486-IL-2 and/or DAB 389-IL-2
(IL-2 fusion proteins; Seragen; see e.g., Arthritis &
Rheumatism (1993) Vol. 36, 1223); Anti-Tac (humanized
anti-IL-2R.alpha.; Protein Design Labs/Roche); IL-4
(anti-inflammatory cytokine; DNAX/Schering); IL-10 (SCH 52000;
recombinant IL-10, anti-inflammatory cytokine; DNAX/Schering);
IL-4; IL-10 and/or IL-4 agonists (e.g., agonist antibodies); IL-1RA
(IL-1 receptor antagonist; Synergen/Amgen); anakinra
(Kineret.RTM./Amgen); TNF-bp/s-TNF (soluble TNF binding protein;
see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9
(supplement), 5284; Amer. J. Physiol.--Heart and Circulatory
Physiology (1995) Vol. 268, pp. 37-42); R973401 (phosphodiesterase
Type IV inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol.
39, No. 9 (supplement), S282); MK-966 (COX-2 Inhibitor; see e.g.,
Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement),
S81); Iloprost (see e.g., Arthritis & Rheumatism (1996) Vol.
39, No. 9 (supplement), S82); methotrexate; thalidomide (see e.g.,
Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement),
5282) and thalidomide-related drugs (e.g., Celgen); leflunomide
(anti-inflammatory and cytokine inhibitor; see e.g., Arthritis
& Rheumatism (1996) Vol. 39, No. 9 (supplement), S131;
Inflammation Research (1996) Vol. 45, pp. 103-107); tranexamic acid
(inhibitor of plasminogen activation; see e.g., Arthritis &
Rheumatism (1996) Vol. 39, No. 9 (supplement), S284); T-614
(cytokine inhibitor; see e.g., Arthritis & Rheumatism (1996)
Vol. 39, No. 9 (supplement), S282); prostaglandin E1 (see e.g.,
Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement),
S282); Tenidap (non-steroidal anti-inflammatory drug; see e.g.,
Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement),
S280); Naproxen (non-steroidal anti-inflammatory drug; see e.g.,
Neuro Report (1996) Vol. 7, pp. 1209-1213); Meloxicam
(non-steroidal anti-inflammatory drug); Ibuprofen (non-steroidal
anti-inflammatory drug); Piroxicam (non-steroidal anti-inflammatory
drug); Diclofenac (non-steroidal anti-inflammatory drug);
Indomethacin (non-steroidal anti-inflammatory drug); Sulfasalazine
(see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9
(supplement), S281); Azathioprine (see e.g., Arthritis &
Rheumatism (1996) Vol. 39, No. 9 (supplement), S281); ICE inhibitor
(inhibitor of the enzyme interleukin-113 converting enzyme); zap-70
and/or lck inhibitor (inhibitor of the tyrosine kinase zap-70 or
lck); VEGF inhibitor and/or VEGF-R inhibitor (inhibitors of
vascular endothelial cell growth factor or vascular endothelial
cell growth factor receptor; inhibitors of angiogenesis);
corticosteroid anti-inflammatory drugs (e.g., SB203580);
TNF-convertase inhibitors; anti-IL-12 antibodies; anti-IL-18
antibodies; interleukin-11 (see e.g., Arthritis & Rheumatism
(1996) Vol. 39, No. 9 (supplement), S296); interleukin-13 (see
e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9
(supplement), S308); interleukin-17 inhibitors (see e.g., Arthritis
& Rheumatism (1996) Vol. 39, No. 9 (supplement), S120); gold;
penicillamine; chloroquine; chlorambucil; hydroxychloroquine;
cyclosporine; cyclophosphamide; total lymphoid irradiation;
anti-thymocyte globulin; anti-CD4 antibodies; CD5-toxins;
orally-administered peptides and collagen; lobenzarit disodium;
Cytokine Regulating Agents (CRAs) HP228 and HP466 (Houghten
Pharmaceuticals, Inc.); ICAM-1 antisense phosphorothioate
oligo-deoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.);
soluble complement receptor 1 (TP10; T Cell Sciences, Inc.);
prednisone; orgotein; glycosaminoglycan polysulphate; minocycline;
anti-IL2R antibodies; marine and botanical lipids (fish and plant
seed fatty acids; see e.g., DeLuca et al. (1995) Rheum. Dis. Clin.
North Am., 21: 759-777); auranofin; phenylbutazone; meclofenamic
acid; flufenamic acid; intravenous immune globulin; zileuton;
azaribine; mycophenolic acid (RS-61443); tacrolimus (FK-506);
sirolimus (rapamycin); amiprilose (therafectin); cladribine
(2-chlorodeoxyadenosine); methotrexate; bcl-2 inhibitors (see
Bruncko, Milan et al., Journal of Medicinal Chemistry (2007),
50(4), 641-662); antivirals and immune modulating agents.
[0946] In one embodiment, the binding protein or antigen-binding
portion thereof, is administered in combination with one of the
following agents for the treatment of rheumatoid arthritis (RA):
small molecule inhibitor of KDR, small molecule inhibitor of Tie-2;
methotrexate; prednisone; celecoxib; folic acid; hydroxychloroquine
sulfate; rofecoxib; etanercept; infliximab; leflunomide; naproxen;
valdecoxib; sulfasalazine; methylprednisolone; ibuprofen;
meloxicam; methylprednisolone acetate; gold sodium thiomalate;
aspirin; azathioprine; triamcinolone acetonide; propoxyphene
napsylate/apap; folate; nabumetone; diclofenac; piroxicam;
etodolac; diclofenac sodium; oxaprozin; oxycodone hcl; hydrocodone
bitartrate/apap; diclofenac sodium/misoprostol; fentanyl; anakinra,
human recombinant; tramadol hcl; salsalate; sulindac;
cyanocobalamin/fa/pyridoxine; acetaminophen; alendronate sodium;
prednisolone; morphine sulfate; lidocaine hydrochloride;
indomethacin; glucosamine sulfate/chondroitin; cyclosporine;
amitriptyline hcl; sulfadiazine; oxycodone hcl/acetaminophen;
olopatadine hcl; misoprostol; naproxen sodium; omeprazole;
mycophenolate mofetil; cyclophosphamide; rituximab; IL-1 TRAP; MRA;
CTLA4-IG; IL-18 BP; IL-12/23; anti-IL 18; anti-IL 15; BIRB-796;
SCIO-469; VX-702; AMG-548; VX-740; Roflumilast; IC-485; CDC-801;
and mesopram.
[0947] Non-limiting examples of therapeutic agents for inflammatory
bowel disease with which a binding protein of the invention can be
combined include the following: budenoside; epidermal growth
factor; corticosteroids; cyclosporin, sulfasalazine;
aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole;
lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide;
antioxidants; thromboxane inhibitors; IL-1 receptor antagonists;
anti-IL-1.beta. mAbs; anti-IL-6 mAbs; growth factors; elastase
inhibitors; pyridinyl-imidazole compounds; antibodies to or
antagonists of other human cytokines or growth factors, for
example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16,
IL-17, IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of the
invention, or antigen binding portions thereof, can be combined
with antibodies to cell surface molecules such as CD2, CD3, CD4,
CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands. The
antibodies of the invention, or antigen binding portions thereof,
may also be combined with agents, such as methotrexate,
cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide,
NSAIDs, for example, ibuprofen, corticosteroids such as
prednisolone, phosphodiesterase inhibitors, adenosine agonists,
antithrombotic agents, complement inhibitors, adrenergic agents,
agents which interfere with signaling by proinflammatory cytokines
such as TNF.alpha. or IL-1 (e.g., IRAK, NIK, IKK, p38 or MAP kinase
inhibitors), IL-1.beta. converting enzyme inhibitors, TNF.alpha.
converting enzyme inhibitors, T-cell signaling inhibitors such as
kinase inhibitors, metalloproteinase inhibitors, sulfasalazine,
azathioprine, 6-mercaptopurines, angiotensin converting enzyme
inhibitors, soluble cytokine receptors and derivatives thereof
(e.g., soluble p55 or p75 TNF receptors, sIL-1RI, sIL-1RII, sIL-6R)
and antiinflammatory cytokines (e.g., IL-4, IL-10, IL-11, IL-13 and
TGF.beta.) and bcl-2 inhibitors.
[0948] Examples of therapeutic agents for Crohn's disease in which
a binding protein can be combined include the following: TNF
antagonists, for example, anti-TNF antibodies, Adalimumab (PCT
Publication No. WO 97/29131; HUMIRA.RTM.), CA2 (REMICADE), CDP 571,
TNFR-Ig constructs, (p75TNFRIgG (ENBREL.RTM.) and p55TNFRIgG
(LENERCEPT.TM.)) inhibitors and PDE4 inhibitors. Antibodies of the
invention, or antigen binding portions thereof, can be combined
with corticosteroids, for example, budenoside and dexamethasone.
Binding proteins of the invention or antigen binding portions
thereof, may also be combined with agents such as sulfasalazine,
5-aminosalicylic acid and olsalazine, and agents which interfere
with synthesis or action of proinflammatory cytokines such as IL-1,
for example, IL-1.beta. converting enzyme inhibitors and IL-1ra.
Antibodies of the invention or antigen binding portion thereof may
also be used with T cell signaling inhibitors, for example,
tyrosine kinase inhibitors 6-mercaptopurines. Binding proteins of
the invention, or antigen binding portions thereof, can be combined
with IL-11. Binding proteins of the invention, or antigen binding
portions thereof, can be combined with mesalamine, prednisone,
azathioprine, mercaptopurine, infliximab, methylprednisolone sodium
succinate, diphenoxylate/atrop sulfate, loperamide hydrochloride,
methotrexate, omeprazole, folate, ciprofloxacin/dextrose-water,
hydrocodone bitartrate/apap, tetracycline hydrochloride,
fluocinonide, metronidazole, thimerosal/boric acid,
cholestyramine/sucrose, ciprofloxacin hydrochloride, hyoscyamine
sulfate, meperidine hydrochloride, midazolam hydrochloride,
oxycodone hcl/acetaminophen, promethazine hydrochloride, sodium
phosphate, sulfamethoxazole/trimethoprim, celecoxib, polycarbophil,
propoxyphene napsylate, hydrocortisone, multivitamins, balsalazide
disodium, codeine phosphate/apap, colesevelam hcl, cyanocobalamin,
folic acid, levofloxacin, methylprednisolone, natalizumab and
interferon-gamma
[0949] Non-limiting examples of therapeutic agents for multiple
sclerosis (MS) with which binding proteins of the invention can be
combined include the following: corticosteroids; prednisolone;
methylprednisolone; azathioprine; cyclophosphamide; cyclosporine;
methotrexate; 4-aminopyridine; tizanidine; interferon-.beta.1a
(AVONEX; Biogen); interferon-.beta.1b (BETASERON; Chiron/Berlex);
interferon .alpha.-n3) (Interferon Sciences/Fujimoto),
interferon-.alpha. (Alfa Wassermann/J&J), interferon
.beta.1A-IF (Serono/Inhale Therapeutics), Peginterferon .alpha. 2b
(Enzon/Schering-Plough), Copolymer 1 (Cop-1; COPAXONE; Teva
Pharmaceutical Industries, Inc.); hyperbaric oxygen; intravenous
immunoglobulin; clabribine; antibodies to or antagonists of other
human cytokines or growth factors and their receptors, for example,
TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-23, IL-15, IL-16, IL-18,
EMAP-II, GM-CSF, FGF, and PDGF. Binding proteins of the invention
can be combined with antibodies to cell surface molecules such as
CD2, CD3, CD4, CD8, CD19, CD20, CD25, CD28, CD30, CD40, CD45, CD69,
CD80, CD86, CD90 or their ligands. Binding proteins of the
invention, may also be combined with agents, such as methotrexate,
cyclosporine, FK506, rapamycin, mycophenolate mofetil, leflunomide,
NSAIDs, for example, ibuprofen, corticosteroids such as
prednisolone, phosphodiesterase inhibitors, adensosine agonists,
antithrombotic agents, complement inhibitors, adrenergic agents,
agents which interfere with signaling by proinflammatory cytokines
such as TNF.alpha. or IL-1 (e.g., IRAK, NIK, IKK, p38 or MAP kinase
inhibitors), IL-1.beta. converting enzyme inhibitors, TACE
inhibitors, T-cell signaling inhibitors such as kinase inhibitors,
metalloproteinase inhibitors, sulfasalazine, azathioprine,
6-mercaptopurines, angiotensin converting enzyme inhibitors,
soluble cytokine receptors and derivatives thereof (e.g., soluble
p55 or p75 TNF receptors, sIL-1R1, sIL-1R11, sIL-6R),
antiinflammatory cytokines (e.g., IL-4, IL-10, IL-13 and TGF.beta.)
and bcl-2 inhibitors.
[0950] Examples of therapeutic agents for multiple sclerosis in
which binding proteins of the invention can be combined to include
interferon-.beta., for example, IFN.beta.1a and IFN.beta.1b;
copaxone, corticosteroids, caspase inhibitors, for example
inhibitors of caspase-1, IL-1 inhibitors, TNF inhibitors, and
antibodies to CD40 ligand and CD80.
[0951] The binding proteins of the invention, may also be combined
with agents, such as alemtuzumab, dronabinol, Unimed, daclizumab,
mitoxantrone, xaliproden hydrochloride, fampridine, glatiramer
acetate, natalizumab, sinnabidol, a-immunokine NNSO3, ABR-215062,
AnergiX.MS, chemokine receptor antagonists, BBR-2778, calagualine,
CPI-1189, LEM (liposome encapsulated mitoxantrone), THC.CBD
(cannabinoid agonist) MBP-8298, mesopram (PDE4 inhibitor), MNA-715,
anti-IL-6 receptor antibody, neurovax, pirfenidone allotrap 1258
(RDP-1258), sTNF-R1, talampanel, teriflunomide, TGF-beta2,
tiplimotide, VLA-4 antagonists (for example, TR-14035, VLA4
Ultrahaler, Antegran-ELAN/Biogen), interferon gamma antagonists,
IL-4 agonists.
[0952] Non-limiting examples of therapeutic agents for Angina with
which binding proteins of the invention can be combined include the
following: aspirin, nitroglycerin, isosorbide mononitrate,
metoprolol succinate, atenolol, metoprolol tartrate, amlodipine
besylate, diltiazem hydrochloride, isosorbide dinitrate,
clopidogrel bisulfate, nifedipine, atorvastatin calcium, potassium
chloride, furosemide, simvastatin, verapamil hcl, digoxin,
propranolol hydrochloride, carvedilol, lisinopril, spironolactone,
hydrochlorothiazide, enalapril maleate, nadolol, ramipril,
enoxaparin sodium, heparin sodium, valsartan, sotalol
hydrochloride, fenofibrate, ezetimibe, bumetanide, losartan
potassium, lisinopril/hydrochlorothiazide, felodipine, captopril,
bisoprolol fumarate.
[0953] Non-limiting examples of therapeutic agents for Ankylosing
Spondylitis with which binding proteins of the invention can be
combined include the following: ibuprofen, diclofenac and
misoprostol, naproxen, meloxicam, indomethacin, diclofenac,
celecoxib, rofecoxib, Sulfasalazine, Methotrexate, azathioprine,
minocyclin, prednisone, etanercept, infliximab.
[0954] Non-limiting examples of therapeutic agents for Asthma with
which binding proteins of the invention can be combined include the
following: albuterol, salmeterol/fluticasone, montelukast sodium,
fluticasone propionate, budesonide, prednisone, salmeterol
xinafoate, levalbuterol hcl, albuterol sulfate/ipratropium,
prednisolone sodium phosphate, triamcinolone acetonide,
beclomethasone dipropionate, ipratropium bromide, azithromycin,
pirbuterol acetate, prednisolone, theophylline anhydrous,
methylprednisolone sodium succinate, clarithromycin, zafirlukast,
formoterol fumarate, influenza virus vaccine, methylprednisolone,
amoxicillin trihydrate, flunisolide, allergy injection, cromolyn
sodium, fexofenadine hydrochloride, flunisolide/menthol,
amoxicillin/clavulanate, levofloxacin, inhaler assist device,
guaifenesin, dexamethasone sodium phosphate, moxifloxacin hcl,
doxycycline hyclate, guaifenesin/d-methorphan,
p-ephedrine/cod/chlorphenir, gatifloxacin, cetirizine
hydrochloride, mometasone furoate, salmeterol xinafoate,
benzonatate, cephalexin, pe/hydrocodone/chlorphenir, cetirizine
hcl/pseudoephed, phenylephrine/cod/promethazine,
codeine/promethazine, cefprozil, dexamethasone,
guaifenesin/pseudoephedrine, chlorpheniramine/hydrocodone,
nedocromil sodium, terbutaline sulfate, epinephrine,
methylprednisolone, metaproterenol sulfate.
[0955] Non-limiting examples of therapeutic agents for COPD with
which binding proteins of the invention can be combined include the
following: albuterol sulfate/ipratropium, ipratropium bromide,
salmeterol/fluticasone, albuterol, salmeterol xinafoate,
fluticasone propionate, prednisone, theophylline anhydrous,
methylprednisolone sodium succinate, montelukast sodium,
budesonide, formoterol fumarate, triamcinolone acetonide,
levofloxacin, guaifenesin, azithromycin, beclomethasone
dipropionate, levalbuterol hcl, flunisolide, ceftriaxone sodium,
amoxicillin trihydrate, gatifloxacin, zafirlukast,
amoxicillin/clavulanate, flunisolide/menthol,
chlorpheniramine/hydrocodone, metaproterenol sulfate,
methylprednisolone, mometasone furoate,
p-ephedrine/cod/chlorphenir, pirbuterol acetate,
p-ephedrine/loratadine, terbutaline sulfate, tiotropium bromide,
(R,R)-formoterol, TgAAT, Cilomilast, Roflumilast.
[0956] Non-limiting examples of therapeutic agents for HCV with
which binding proteins of the invention can be combined include the
following: Interferon-alpha-2a, Interferon-alpha-2b,
Interferon-alpha con1, Interferon-alpha-n1, Pegylated
interferon-alpha-2a, Pegylated interferon-alpha-2b, ribavirin,
Peginterferon alfa-2b+ribavirin, Ursodeoxycholic Acid, Glycyrrhizic
Acid, Thymalfasin, Maxamine, VX-497 and any compounds that are used
to treat HCV through intervention with the following targets: HCV
polymerase, HCV protease, HCV helicase, HCV IRES (internal ribosome
entry site).
[0957] Non-limiting examples of therapeutic agents for Idiopathic
Pulmonary Fibrosis with which binding proteins of the invention can
be combined include the following: prednisone, azathioprine,
albuterol, colchicine, albuterol sulfate, digoxin, gamma
interferon, methylprednisolone sod succ, lorazepam, furosemide,
lisinopril, nitroglycerin, spironolactone, cyclophosphamide,
ipratropium bromide, actinomycin d, alteplase, fluticasone
propionate, levofloxacin, metaproterenol sulfate, morphine sulfate,
oxycodone hcl, potassium chloride, triamcinolone acetonide,
tacrolimus anhydrous, calcium, interferon-alpha, methotrexate,
mycophenolate mofetil, Interferon-gamma-1.beta..
[0958] Non-limiting examples of therapeutic agents for Myocardial
Infarction with which binding proteins of the invention can be
combined include the following: aspirin, nitroglycerin, metoprolol
tartrate, enoxaparin sodium, heparin sodium, clopidogrel bisulfate,
carvedilol, atenolol, morphine sulfate, metoprolol succinate,
warfarin sodium, lisinopril, isosorbide mononitrate, digoxin,
furosemide, simvastatin, ramipril, tenecteplase, enalapril maleate,
torsemide, retavase, losartan potassium, quinapril hcl/mag carb,
bumetanide, alteplase, enalaprilat, amiodarone hydrochloride,
tirofiban hcl m-hydrate, diltiazem hydrochloride, captopril,
irbesartan, valsartan, propranolol hydrochloride, fosinopril
sodium, lidocaine hydrochloride, eptifibatide, cefazolin sodium,
atropine sulfate, aminocaproic acid, spironolactone, interferon,
sotalol hydrochloride, potassium chloride, docusate sodium,
dobutamine hcl, alprazolam, pravastatin sodium, atorvastatin
calcium, midazolam hydrochloride, meperidine hydrochloride,
isosorbide dinitrate, epinephrine, dopamine hydrochloride,
bivalirudin, rosuvastatin, ezetimibe/simvastatin, avasimibe,
cariporide.
[0959] Non-limiting examples of therapeutic agents for Psoriasis
with which binding proteins of the invention can be combined
include the following: small molecule inhibitor of KDR, small
molecule inhibitor of Tie-2, calcipotriene, clobetasol propionate,
triamcinolone acetonide, halobetasol propionate, tazarotene,
methotrexate, fluocinonide, betamethasone diprop augmented,
fluocinolone acetonide, acitretin, tar shampoo, betamethasone
valerate, mometasone furoate, ketoconazole, pramoxine/fluocinolone,
hydrocortisone valerate, flurandrenolide, urea, betamethasone,
clobetasol propionate/emoll, fluticasone propionate, azithromycin,
hydrocortisone, moisturizing formula, folic acid, desonide,
pimecrolimus, coal tar, diflorasone diacetate, etanercept folate,
lactic acid, methoxsalen, hc/bismuth subgal/znox/resor,
methylprednisolone acetate, prednisone, sunscreen, halcinonide,
salicylic acid, anthralin, clocortolone pivalate, coal extract,
coal tar/salicylic acid, coal tar/salicylic acid/sulfur,
desoximetasone, diazepam, emollient, fluocinonide/emollient,
mineral oil/castor oil/na lact, mineral oil/peanut oil,
petroleum/isopropyl myristate, psoralen, salicylic acid,
soap/tribromsalan, thimerosal/boric acid, celecoxib, infliximab,
cyclosporine, alefacept, efalizumab, tacrolimus, pimecrolimus,
PUVA, UVB, sulfasalazine.
[0960] Non-limiting examples of therapeutic agents for Psoriatic
Arthritis with which binding proteins of the invention can be
combined include the following: methotrexate, etanercept,
rofecoxib, celecoxib, folic acid, sulfasalazine, naproxen,
leflunomide, methylprednisolone acetate, indomethacin,
hydroxychloroquine sulfate, prednisone, sulindac, betamethasone
diprop augmented, infliximab, methotrexate, folate, triamcinolone
acetonide, diclofenac, dimethylsulfoxide, piroxicam, diclofenac
sodium, ketoprofen, meloxicam, methylprednisolone, nabumetone,
tolmetin sodium, calcipotriene, cyclosporine, diclofenac
sodium/misoprostol, fluocinonide, glucosamine sulfate, gold sodium
thiomalate, hydrocodone bitartrate/apap, ibuprofen, risedronate
sodium, sulfadiazine, thioguanine, valdecoxib, alefacept,
efalizumab and bcl-2 inhibitors.
[0961] Non-limiting examples of therapeutic agents for Restenosis
with which binding proteins of the invention can be combined
include the following: sirolimus, paclitaxel, everolimus,
tacrolimus, Zotarolimus, acetaminophen.
[0962] Non-limiting examples of therapeutic agents for Sciatica
with which binding proteins of the invention can be combined
include the following: hydrocodone bitartrate/apap, rofecoxib,
cyclobenzaprine hcl, methylprednisolone, naproxen, ibuprofen,
oxycodone hcl/acetaminophen, celecoxib, valdecoxib,
methylprednisolone acetate, prednisone, codeine phosphate/apap,
tramadol hcl/acetaminophen, metaxalone, meloxicam, methocarbamol,
lidocaine hydrochloride, diclofenac sodium, gabapentin,
dexamethasone, carisoprodol, ketorolac tromethamine, indomethacin,
acetaminophen, diazepam, nabumetone, oxycodone hcl, tizanidine hcl,
diclofenac sodium/misoprostol, propoxyphene napsylate/apap,
asa/oxycod/oxycodone ter, ibuprofen/hydrocodone bit, tramadol hcl,
etodolac, propoxyphene hcl, amitriptyline hcl, carisoprodol/codeine
phos/asa, morphine sulfate, multivitamins, naproxen sodium,
orphenadrine citrate, temazepam.
[0963] Examples of therapeutic agents for SLE (Lupus) in which
binding proteins of the invention can be combined include the
following: NSAIDS, for example, diclofenac, naproxen, ibuprofen,
piroxicam, indomethacin; COX2 inhibitors, for example, Celecoxib,
rofecoxib, valdecoxib; anti-malarials, for example,
hydroxychloroquine; Steroids, for example, prednisone,
prednisolone, budenoside, dexamethasone; Cytotoxics, for example,
azathioprine, cyclophosphamide, mycophenolate mofetil,
methotrexate; inhibitors of PDE4 or purine synthesis inhibitor, for
example Cellcept. Binding proteins of the invention, may also be
combined with agents such as sulfasalazine, 5-aminosalicylic acid,
olsalazine, Imuran and agents which interfere with synthesis,
production or action of proinflammatory cytokines such as IL-1, for
example, caspase inhibitors like IL-1.beta. converting enzyme
inhibitors and IL-1ra. Binding proteins of the invention may also
be used with T cell signaling inhibitors, for example, tyrosine
kinase inhibitors; or molecules that target T cell activation
molecules, for example, CTLA-4-IgG or anti-B7 family antibodies,
anti-PD-1 family antibodies. Binding proteins of the invention, can
be combined with IL-11 or anti-cytokine antibodies, for example,
fonotolizumab (anti-IFNg antibody), or anti-receptor receptor
antibodies, for example, anti-IL-6 receptor antibody and antibodies
to B-cell surface molecules. Antibodies of the invention or antigen
binding portion thereof may also be used with LJP 394 (abetimus),
agents that deplete or inactivate B-cells, for example, Rituximab
(anti-CD20 antibody), lymphostat-B (anti-BlyS antibody), TNF
antagonists, for example, anti-TNF antibodies, Adalimumab (PCT
Publication No. WO 97/29131; HUMIRA.RTM.), CA2 (REMICADE.RTM.), CDP
571, TNFR-Ig constructs, (p75TNFRIgG (ENBREL.RTM.) and p55TNFRIgG
(LENERCEPT.RTM.)) and bcl-2 inhibitors, because bcl-2
overexpression in transgenic mice has been demonstrated to cause a
lupus like phenotype (see Marquina, Regina et al., Journal of
Immunology (2004), 172(11), 7177-7185), therefore inhibition is
expected to have therapeutic effects.
[0964] The pharmaceutical compositions of the invention may include
a "therapeutically effective amount" or a "prophylactically
effective amount" of an antibody or antibody portion of the
invention. A "therapeutically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired therapeutic result. A therapeutically effective amount
of the antibody or antibody portion may be determined by a person
skilled in the art and may vary according to factors such as the
disease state, age, sex, and weight of the individual, and the
ability of the antibody or antibody portion to elicit a desired
response in the individual. A therapeutically effective amount is
also one in which any toxic or detrimental effects of the antibody,
or antibody portion, are outweighed by the therapeutically
beneficial effects. A "prophylactically effective amount" refers to
an amount effective, at dosages and for periods of time necessary,
to achieve the desired prophylactic result. Typically, since a
prophylactic dose is used in subjects prior to or at an earlier
stage of disease, the prophylactically effective amount will be
less than the therapeutically effective amount.
[0965] Dosage regimens may be adjusted to provide the optimum
desired response (e.g., a therapeutic or prophylactic response).
For example, a single bolus may be administered, several divided
doses may be administered over time or the dose may be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation. It is especially advantageous to
formulate parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the mammalian subjects to be treated; each unit
containing a predetermined quantity of active compound calculated
to produce the desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for the dosage
unit forms of the invention are dictated by and directly dependent
on (a) the unique characteristics of the active compound and the
particular therapeutic or prophylactic effect to be achieved, and
(b) the limitations inherent in the art of compounding such an
active compound for the treatment of sensitivity in
individuals.
[0966] It is to be noted that dosage values may vary with the type
and severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that dosage
ranges set forth herein are exemplary only and are not intended to
limit the scope or practice of the claimed composition.
Diagnostics
[0967] The disclosure herein also provides diagnostic applications.
This is further elucidated below. Antibodies that bind IL-17 and/or
IL-17F of the invention may be employed in any of a variety of
formats to detect IL-17 and/or IL-17F in vivo, in vitro, or ex vivo
(i.e., in cells or tissues that have been obtained from a living
individual, subjected to a procedure, then returned to the
individual). DVD-Igs of the invention offer the further advantage
of being capable of binding to an epitope of IL-17 as well as other
antigens or epitopes in various diagnostic and detection assay
formats.
I. Method of Assay
[0968] The present disclosure also provides a method for
determining the presence, amount or concentration of an IL-17, or a
fragment thereof, ("analyte") in a test sample using at least one
anti-IL-17 binding protein or antigen binding portion thereof,
including a DVD-Ig, as described herein. Any suitable assay as is
known in the art can be used in the method. Examples include, but
are not limited to, immunoassay, such as sandwich immunoassay
(e.g., monoclonal, polyclonal and/or DVD-Ig sandwich immunoassays
or any variation thereof (e.g., monoclonal/DVD-Ig,
DVD-Ig/polyclonal, etc.), including radioisotope detection
(radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay
(EIA) or enzyme-linked immunosorbent assay (ELISA) (e.g.,
Quantikine ELISA assays, R&D Systems, Minneapolis, Minn.))),
competitive inhibition immunoassay (e.g., forward and reverse),
fluorescence polarization immunoassay (FPIA), enzyme multiplied
immunoassay technique (EMIT), bioluminescence resonance energy
transfer (BRET), and homogeneous chemiluminescent assay, etc. In a
SELDI-based immunoassay, a capture reagent that specifically binds
an analyte (or a fragment thereof) of interest is attached to the
surface of a mass spectrometry probe, such as a pre-activated
protein chip array. The analyte (or a fragment thereof) is then
specifically captured on the biochip, and the captured analyte (or
a fragment thereof) is detected by mass spectrometry.
Alternatively, the analyte (or a fragment thereof) can be eluted
from the capture reagent and detected by traditional MALDI
(matrix-assisted laser desorption/ionization) or by SELDI. A
chemiluminescent microparticle immunoassay, in particular one
employing the ARCHITECT.RTM. automated analyzer (Abbott
Laboratories, Abbott Park, Ill.), is an example of a preferred
immunoassay.
[0969] Methods well-known in the art for collecting, handling and
processing urine, blood, serum and plasma, and other body fluids,
are used in the practice of the present disclosure, for instance,
when anti-IL-17 binding protein as described herein is employed as
an immunodiagnostic reagent and/or in an analyte immunoassay kit.
The test sample can comprise further moieties in addition to the
analyte of interest, such as antibodies, antigens, haptens,
hormones, drugs, enzymes, receptors, proteins, peptides,
polypeptides, oligonucleotides and/or polynucleotides. For example,
the sample can be a whole blood sample obtained from a subject. It
can be necessary or desired that a test sample, particularly whole
blood, be treated prior to immunoassay as described herein, e.g.,
with a pretreatment reagent. Even in cases where pretreatment is
not necessary (e.g., most urine samples), pretreatment optionally
can be done (e.g., as part of a regimen on a commercial
platform).
[0970] The pretreatment reagent can be any reagent appropriate for
use with the immunoassay and kits of the invention. The
pretreatment optionally comprises: (a) one or more solvents (e.g.,
methanol and ethylene glycol) and optionally, salt, (b) one or more
solvents and salt, and optionally, detergent, (c) detergent, or (d)
detergent and salt. Pretreatment reagents are known in the art, and
such pretreatment can be employed, e.g., as used for assays on
Abbott TDx, AxSYM.RTM., and ARCHITECT.RTM. analyzers (Abbott
Laboratories, Abbott Park, Ill.), as described in the literature
(see, e.g., Yatscoff et al., Abbott TDx Monoclonal Antibody Assay
Evaluated for Measuring Cyclosporine in Whole Blood, Clin. Chem.
36: 1969-1973 (1990), and Wallemacq et al., Evaluation of the New
AxSYM Cyclosporine Assay: Comparison with TDx Monoclonal Whole
Blood and EMIT Cyclosporine Assays, Clin. Chem. 45: 432-435
(1999)), and/or as commercially available. Additionally,
pretreatment can be done as described in Abbott's U.S. Pat. No.
5,135,875; European Patent Publication No. 0 471 293; PCT
Publication No. WO 2008/082984; and US Patent Application
Publication No. 2008/0020401 (incorporated by reference in its
entirety for its teachings regarding pretreatment). The
pretreatment reagent can be a heterogeneous agent or a homogeneous
agent.
[0971] With use of a heterogeneous pretreatment reagent, the
pretreatment reagent precipitates analyte binding protein (e.g.,
protein that can bind to an analyte or a fragment thereof) present
in the sample. Such a pretreatment step comprises removing any
analyte binding protein by separating from the precipitated analyte
binding protein the supernatant of the mixture formed by addition
of the pretreatment agent to sample. In such an assay, the
supernatant of the mixture absent any binding protein is used in
the assay, proceeding directly to the antibody capture step.
[0972] With use of a homogeneous pretreatment reagent there is no
such separation step. The entire mixture of test sample and
pretreatment reagent are contacted with a labeled specific binding
partner for analyte (or a fragment thereof), such as a labeled
anti-analyte antibody (or an antigenically reactive fragment
thereof). The pretreatment reagent employed for such an assay
typically is diluted in the pretreated test sample mixture, either
before or during capture by the first specific binding partner.
Despite such dilution, a certain amount of the pretreatment reagent
is still present (or remains) in the test sample mixture during
capture. According to the invention, a preferred labeled specific
binding partner can be a DVD-Ig (or a fragment, a variant, or a
fragment of a variant thereof).
[0973] In a heterogeneous format, after the test sample is obtained
from a subject, a first mixture is prepared. The mixture contains
the test sample being assessed for an analyte (or a fragment
thereof) and a first specific binding partner, wherein the first
specific binding partner and any analyte contained in the test
sample form a first specific binding partner-analyte complex.
Preferably, the first specific binding partner is an anti-analyte
antibody or a fragment thereof. The first specific binding partner
can be a DVD-Ig (or a fragment, a variant, or a fragment of a
variant thereof) as described herein. The order in which the test
sample and the first specific binding partner are added to form the
mixture is not critical. Preferably, the first specific binding
partner is immobilized on a solid phase. The solid phase used in
the immunoassay (for the first specific binding partner and,
optionally, the second specific binding partner) can be any solid
phase known in the art, such as, but not limited to, a magnetic
particle, a bead, a test tube, a microtiter plate, a cuvette, a
membrane, a scaffolding molecule, a film, a filter paper, a disc
and a chip.
[0974] After the mixture containing the first specific binding
partner-analyte complex is formed, any unbound analyte is removed
from the complex using any technique known in the art. For example,
the unbound analyte can be removed by washing. Desirably, however,
the first specific binding partner is present in excess of any
analyte present in the test sample, such that all analyte that is
present in the test sample is bound by the first specific binding
partner.
[0975] After any unbound analyte is removed, a second specific
binding partner is added to the mixture to form a first specific
binding partner-analyte-second specific binding partner complex.
The second specific binding partner is preferably an anti-analyte
antibody that binds to an epitope on analyte that differs from the
epitope on analyte bound by the first specific binding partner.
Moreover, also preferably, the second specific binding partner is
labeled with or contains a detectable label as described above. The
second specific binding partner can be a DVD-Ig (or a fragment, a
variant, or a fragment of a variant thereof) as described
herein.
[0976] Any suitable detectable label as is known in the art can be
used. For example, the detectable label can be a radioactive label
(such as .sup.3H, .sup.125I, .sup.35S, .sup.14C, .sup.32P, and
.sup.33P), an enzymatic label (such as horseradish peroxidase,
alkaline peroxidase, glucose 6-phosphate dehydrogenase, and the
like), a chemiluminescent label (such as acridinium esters,
thioesters, or sulfonamides; luminol, isoluminol, phenanthridinium
esters, and the like), a fluorescent label (such as fluorescein
(e.g., 5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein,
5(6)-carboxyfluorescein, 6-hexachloro-fluorescein,
6-tetrachlorofluorescein, fluorescein isothiocyanate, and the
like)), rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots
(e.g., zinc sulfide-capped cadmium selenide), a thermometric label,
or an immuno-polymerase chain reaction label. An introduction to
labels, labeling procedures and detection of labels is found in
Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd
ed., Springer Verlag, N.Y. (1997), and in Haugland, Handbook of
Fluorescent Probes and Research Chemicals (1996), which is a
combined handbook and catalogue published by Molecular Probes,
Inc., Eugene, Oreg. A fluorescent label can be used in FPIA (see,
e.g., U.S. Pat. Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093,
and 5,352,803). An acridinium compound can be used as a detectable
label in a homogeneous or heterogeneous chemiluminescent assay
(see, e.g., Adamczyk et al., Bioorg. Med. Chem. Lett. 16: 1324-1328
(2006); Adamczyk et al., Bioorg. Med. Chem. Lett. 4: 2313-2317
(2004); Adamczyk et al., Biorg. Med. Chem. Lett. 14: 3917-3921
(2004); and Adamczyk et al., Org. Lett. 5: 3779-3782 (2003)).
[0977] A preferred acridinium compound is an
acridinium-9-carboxamide. Methods for preparing acridinium
9-carboxamides are described in Mattingly, J. Biolumin. Chemilumin.
6: 107-114 (1991); Adamczyk et al., J. Org. Chem., 63: 5636-5639
(1998); Adamczyk et al., Tetrahedron, 55: 10899-10914 (1999);
Adamczyk et al., Org. Lett., 1: 779-781 (1999); Adamczyk et al.,
Bioconjugate Chem., 11: 714-724 (2000); Mattingly et al., In
Luminescence Biotechnology: Instruments and Applications; Dyke, K.
V. Ed.; CRC Press: Boca Raton, pp. 77-105 (2002); Adamczyk et al.,
Org. Lett., 5: 3779-3782 (2003); and U.S. Pat. Nos. 5,468,646,
5,543,524 and 5,783,699. Another preferred acridinium compound is
an acridinium-9-carboxylate aryl ester. An example of an
acridinium-9-carboxylate aryl ester is
10-methyl-9-(phenoxycarbonyllacridinium fluorosulfonate (available
from Cayman Chemical, Ann Arbor, Mich.). Methods for preparing
acridinium 9-carboxylate aryl esters are described in McCapra et
al., Photochem. Photobiol., 4: 1111-21 (1965); Razavi et al.,
Luminescence, 15: 245-249 (2000); Razavi et al., Luminescence, 15:
239-244 (2000); and U.S. Pat. No. 5,241,070. Further details
regarding acridinium-9-carboxylate aryl ester and its use are set
forth in US 2008-0248493.
[0978] Chemiluminescent assays (e.g., using acridinium as described
above or other chemiluminescent agents) can be performed in
accordance with the methods described in Adamczyk et al., Anal.
Chim. Acta, 579(1): 61-67 (2006). While any suitable assay format
can be used, a microplate chemiluminometer (Mithras LB-940,
Berthold Technologies U.S.A., LLC, Oak Ridge, Tenn.) enables the
assay of multiple samples of small volumes rapidly.
[0979] The order in which the test sample and the specific binding
partner(s) are added to form the mixture for chemiluminescent assay
is not critical. If the first specific binding partner is
detectably labeled with a chemiluminescent agent such as an
acridinium compound, detectably labeled first specific binding
partner-analyte complexes form. Alternatively, if a second specific
binding partner is used and the second specific binding partner is
detectably labeled with a chemiluminescent agent such as an
acridinium compound, detectably labeled first specific binding
partner-analyte-second specific binding partner complexes form. Any
unbound specific binding partner, whether labeled or unlabeled, can
be removed from the mixture using any technique known in the art,
such as washing.
[0980] Hydrogen peroxide can be generated in situ in the mixture or
provided or supplied to the mixture (e.g., the source of the
hydrogen peroxide being one or more buffers or other solutions that
are known to contain hydrogen peroxide) before, simultaneously
with, or after the addition of an above-described acridinium
compound. Hydrogen peroxide can be generated in situ in a number of
ways such as would be apparent to one skilled in the art.
[0981] Upon the simultaneous or subsequent addition of at least one
basic solution to the sample, a detectable signal, namely, a
chemiluminescent signal, indicative of the presence of analyte is
generated. The basic solution contains at least one base and has a
pH greater than or equal to 10, preferably, greater than or equal
to 12. Examples of basic solutions include, but are not limited to,
sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium
hydroxide, magnesium hydroxide, sodium carbonate, sodium
bicarbonate, calcium hydroxide, calcium carbonate, and calcium
bicarbonate. The amount of basic solution added to the sample
depends on the concentration of the basic solution. Based on the
concentration of the basic solution used, one skilled in the art
can easily determine the amount of basic solution to add to the
sample.
[0982] The chemiluminescent signal that is generated can be
detected using routine techniques known to those skilled in the
art. Based on the intensity of the signal generated, the amount of
analyte in the sample can be quantified. Specifically, the amount
of analyte in the sample is proportional to the intensity of the
signal generated. The amount of analyte present can be quantified
by comparing the amount of light generated to a standard curve for
analyte or by comparison to a reference standard. The standard
curve can be generated using serial dilutions or solutions of known
concentrations of analyte by mass spectroscopy, gravimetric
methods, and other techniques known in the art. While the above is
described with emphasis on use of an acridinium compound as the
chemiluminescent agent, one of ordinary skill in the art can
readily adapt this description for use of other chemiluminescent
agents.
[0983] Analyte immunoassays generally can be conducted using any
format known in the art, such as, but not limited to, a sandwich
format. Specifically, in one immunoassay format, at least two
antibodies are employed to separate and quantify analyte, such as
human analyte, or a fragment thereof in a sample. More
specifically, the at least two antibodies bind to different
epitopes on an analyte (or a fragment thereof) forming an immune
complex, which is referred to as a "sandwich." Generally, in the
immunoassays one or more antibodies can be used to capture the
analyte (or a fragment thereof) in the test sample (these
antibodies are frequently referred to as a "capture" antibody or
"capture" antibodies) and one or more antibodies can be used to
bind a detectable (namely, quantifiable) label to the sandwich
(these antibodies are frequently referred to as the "detection
antibody," the "detection antibodies," the "conjugate," or the
"conjugates"). Thus, in the context of a sandwich immunoassay
format, a DVD-Ig (or a fragment, a variant, or a fragment of a
variant thereof) as described herein can be used as a capture
antibody, a detection antibody, or both. For example, one DVD-Ig
having a domain that can bind a first epitope on an analyte (or a
fragment thereof) can be used as a capture antibody and/or another
DVD-Ig having a domain that can bind a second epitope on an analyte
(or a fragment thereof) can be used as a detection antibody. In
this regard, a DVD-Ig having a first domain that can bind a first
epitope on an analyte (or a fragment thereof) and a second domain
that can bind a second epitope on an analyte (or a fragment
thereof) can be used as a capture antibody and/or a detection
antibody. Alternatively, one DVD-Ig having a first domain that can
bind an epitope on a first analyte (or a fragment thereof) and a
second domain that can bind an epitope on a second analyte (or a
fragment thereof) can be used as a capture antibody and/or a
detection antibody to detect, and optionally quantify, two or more
analytes. In the event that an analyte can be present in a sample
in more than one form, such as a monomeric form and a
dimeric/multimeric form, which can be homomeric or heteromeric, one
DVD-Ig having a domain that can bind an epitope that is only
exposed on the monomeric form and another DVD-Ig having a domain
that can bind an epitope on a different part of a
dimeric/multimeric form can be used as capture antibodies and/or
detection antibodies, thereby enabling the detection, and optional
quantification, of different forms of a given analyte. Furthermore,
employing DVD-Igs with differential affinities within a single
DVD-Ig and/or between DVD-Igs can provide an avidity advantage. In
the context of immunoassays as described herein, it generally may
be helpful or desired to incorporate one or more linkers within the
structure of a DVD-Ig. When present, optimally the linker should be
of sufficient length and structural flexibility to enable binding
of an epitope by the inner domains as well as binding of another
epitope by the outer domains. In this regard, if a DVD-Ig can bind
two different analytes and one analyte is larger than the other,
desirably the larger analyte is bound by the outer domains.
[0984] Generally speaking, a sample being tested for (for example,
suspected of containing) an IL-17 protein (or a fragment thereof)
can be contacted with at least one capture antibody (or antibodies)
and at least one detection antibody (which can be a second
detection antibody or a third detection antibody or even a
successively numbered antibody, e.g., as where the capture and/or
detection antibody comprise multiple antibodies) either
simultaneously or sequentially and in any order. For example, the
test sample can be first contacted with at least one capture
antibody and then (sequentially) with at least one detection
antibody. Alternatively, the test sample can be first contacted
with at least one detection antibody and then (sequentially) with
at least one capture antibody. In yet another alternative, the test
sample can be contacted simultaneously with a capture antibody and
a detection antibody.
[0985] In the sandwich assay format, a sample suspected of
containing IL-17 (or a fragment thereof) is first brought into
contact with at least one first capture binding protein (e.g.,
IL-17 antibody) under conditions that allow the formation of a
first binding protein/IL-17 complex. If more than one capture
binding protein is used, a first capture binding protein/IL-17
complex comprising two or more capture binding proteins forms. In a
sandwich assay, the binding proteins, i.e., preferably, the at
least one capture binding protein, are used in molar excess amounts
of the maximum amount of IL-17 analyte (or a fragment thereof)
expected in the test sample. For example, from about 5 .mu.g to
about 1 mg of antibody per mL of buffer (e.g., microparticle
coating buffer) can be used.
[0986] Competitive inhibition immunoassays, which are often used to
measure small analytes because binding by only one antibody is
required, comprise sequential and classic formats. In a sequential
competitive inhibition immunoassay a capture binding protein to
IL-17 is coated onto a well of a microtiter plate or other solid
support. When the sample containing the IL-17 is added to the well,
the IL-17 binds to the capture binding protein. After washing, a
known amount of labeled (e.g., biotin or horseradish peroxidase
(HRP)) IL-17 is added to the well. A substrate for an enzymatic
label is necessary to generate a signal. An example of a suitable
substrate for HRP is 3,3',5,5'-tetramethylbenzidine (TMB). After
washing, the signal generated by the labeled analyte is measured
and is inversely proportional to the amount of IL-17 in the sample.
In a classic competitive inhibition immunoassay, a binding protein
to IL-17 is coated onto a solid support (e.g., a well of a
microtiter plate). However, unlike the sequential competitive
inhibition immunoassay, the sample and the labeled IL-17 are added
to the well at the same time. Any IL-17 in the sample competes with
labeled IL-17 for binding to the capture binding protein. After
washing, the signal generated by the labeled IL-17 is measured and
is inversely proportional to the amount of IL-17 in the sample.
[0987] Optionally, prior to contacting the test sample with the at
least one capture binding protein (for example, the first capture
antibody), the at least one capture binding protein can be bound to
a solid support, which facilitates the separation of the first
binding protein/IL-17 (or a fragment thereof) complex from the test
sample. The substrate to which the capture binding protein is bound
can be any suitable solid support or solid phase that facilitates
separation of the capture antibody-analyte complex from the
sample.
[0988] Examples include a well of a plate, such as a microtiter
plate, a test tube, a porous gel (e.g., silica gel, agarose,
dextran, or gelatin), a polymeric film (e.g., polyacrylamide),
beads (e.g., polystyrene beads or magnetic beads), a strip of a
filter/membrane (e.g., nitrocellulose or nylon), microparticles
(e.g., latex particles, magnetizable microparticles (e.g.,
microparticles having ferric oxide or chromium oxide cores and
homo- or hetero-polymeric coats and radii of about 1-10 microns).
The substrate can comprise a suitable porous material with a
suitable surface affinity to bind antigens and sufficient porosity
to allow access by detection antibodies. A microporous material is
generally preferred, although a gelatinous material in a hydrated
state can be used. Such porous substrates are preferably in the
form of sheets having a thickness of about 0.01 to about 0.5 mm,
preferably about 0.1 mm. While the pore size may vary quite a bit,
preferably the pore size is from about 0.025 to about 15 microns,
more preferably from about 0.15 to about 15 microns. The surface of
such substrates can be activated by chemical processes that cause
covalent linkage of an antibody to the substrate. Irreversible
binding, generally by adsorption through hydrophobic forces, of the
antigen or the antibody to the substrate results; alternatively, a
chemical coupling agent or other means can be used to bind
covalently the antibody to the substrate, provided that such
binding does not interfere with the ability of the antibody to bind
to analyte. Alternatively, the antibody can be bound with
microparticles, which have been previously coated with streptavidin
(e.g., DYNAL.RTM. Magnetic Beads, Invitrogen, Carlsbad, Calif.) or
biotin (e.g., using Power-Bind.TM.-SA-MP streptavidin-coated
microparticles (Seradyn, Indianapolis, Ind.)) or
anti-species-specific monoclonal antibodies. If necessary, the
substrate can be derivatized to allow reactivity with various
functional groups on the antibody. Such derivatization requires the
use of certain coupling agents, examples of which include, but are
not limited to, maleic anhydride, N-hydroxysuccinimide, and
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. If desired, one or
more capture reagents, such as antibodies (or fragments thereof),
each of which is specific for analyte(s) can be attached to solid
phases in different physical or addressable locations (e.g., such
as in a biochip configuration (see, e.g., U.S. Pat. No. 6,225,047;
PCT Publication No. WO 99/51773; U.S. Pat. No. 6,329,209; PCT
Publication No. WO 00/56934; and U.S. Pat. No. 5,242,828). If the
capture reagent is attached to a mass spectrometry probe as the
solid support, the amount of analyte bound to the probe can be
detected by laser desorption ionization mass spectrometry.
Alternatively, a single column can be packed with different beads,
which are derivatized with the one or more capture reagents,
thereby capturing the analyte in a single place (see,
antibody-derivatized, bead-based technologies, e.g., the xMAP
technology of Luminex (Austin, Tex.)).
[0989] After the test sample being assayed for analyte (or a
fragment thereof) is brought into contact with the at least one
capture antibody (for example, the first capture antibody), the
mixture is incubated in order to allow for the formation of a first
antibody (or multiple antibody)-analyte (or a fragment thereof)
complex. The incubation can be carried out at a pH of from about
4.5 to about 10.0, at a temperature of from about 2.degree. C. to
about 45.degree. C., and for a period from at least about one (1)
minute to about eighteen (18) hours, preferably from about 1 to
about 24 minutes, most preferably for about 4 to about 18 minutes.
The immunoassay described herein can be conducted in one step
(meaning the test sample, at least one capture antibody and at
least one detection antibody are all added sequentially or
simultaneously to a reaction vessel) or in more than one step, such
as two steps, three steps, etc.
[0990] After formation of the (first or multiple) capture
antibody/analyte (or a fragment thereof) complex, the complex is
then contacted with at least one detection antibody under
conditions which allow for the formation of a (first or multiple)
capture antibody/analyte (or a fragment thereof)/second detection
antibody complex). While captioned for clarity as the "second"
antibody (e.g., second detection antibody), in fact, where multiple
antibodies are used for capture and/or detection, the at least one
detection antibody can be the second, third, fourth, etc.
antibodies used in the immunoassay. If the capture antibody/analyte
(or a fragment thereof) complex is contacted with more than one
detection antibody, then a (first or multiple) capture
antibody/analyte (or a fragment thereof)/(multiple) detection
antibody complex is formed. As with the capture antibody (e.g., the
first capture antibody), when the at least one (e.g., second and
any subsequent) detection antibody is brought into contact with the
capture antibody/analyte (or a fragment thereof) complex, a period
of incubation under conditions similar to those described above is
required for the formation of the (first or multiple) capture
antibody/analyte (or a fragment thereof)/(second or multiple)
detection antibody complex. Preferably, at least one detection
antibody contains a detectable label. The detectable label can be
bound to the at least one detection antibody (e.g., the second
detection antibody) prior to, simultaneously with, or after the
formation of the (first or multiple) capture antibody/analyte (or a
fragment thereof)/(second or multiple) detection antibody complex.
Any detectable label known in the art can be used (see discussion
above, including of the Polak and Van Noorden (1997) and Haugland
(1996) references).
[0991] The detectable label can be bound to the antibodies either
directly or through a coupling agent. An example of a coupling
agent that can be used is EDAC (1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide, hydrochloride), which is commercially available from
Sigma-Aldrich, St. Louis, Mo. Other coupling agents that can be
used are known in the art. Methods for binding a detectable label
to an antibody are known in the art. Additionally, many detectable
labels can be purchased or synthesized that already contain end
groups that facilitate the coupling of the detectable label to the
antibody, such as CPSP-Acridinium Ester (i.e.,
9-[N-tosyl-N-(3-carboxypropyl)]-10-(3-sulfopropyl)acridinium
carboxamide) or SPSP-Acridinium Ester (i.e.,
N10-(3-sulfopropyl)-N-(3-sulfopropyl)-acridinium-9-carboxamide).
[0992] The (first or multiple) capture antibody/analyte/(second or
multiple) detection antibody complex can be, but does not have to
be, separated from the remainder of the test sample prior to
quantification of the label. For example, if the at least one
capture antibody (e.g., the first capture antibody) is bound to a
solid support, such as a well or a bead, separation can be
accomplished by removing the fluid (of the test sample) from
contact with the solid support. Alternatively, if the at least
first capture antibody is bound to a solid support, it can be
simultaneously contacted with the analyte-containing sample and the
at least one second detection antibody to form a first (multiple)
antibody/analyte/second (multiple) antibody complex, followed by
removal of the fluid (test sample) from contact with the solid
support. If the at least one first capture antibody is not bound to
a solid support, then the (first or multiple) capture
antibody/analyte/(second or multiple) detection antibody complex
does not have to be removed from the test sample for quantification
of the amount of the label.
[0993] After formation of the labeled capture
antibody/analyte/detection antibody complex (e.g., the first
capture antibody/analyte/second detection antibody complex), the
amount of label in the complex is quantified using techniques known
in the art. For example, if an enzymatic label is used, the labeled
complex is reacted with a substrate for the label that gives a
quantifiable reaction such as the development of color. If the
label is a radioactive label, the label is quantified using
appropriate means, such as a scintillation counter. If the label is
a fluorescent label, the label is quantified by stimulating the
label with a light of one color (which is known as the "excitation
wavelength") and detecting another color (which is known as the
"emission wavelength") that is emitted by the label in response to
the stimulation. If the label is a chemiluminescent label, the
label is quantified by detecting the light emitted either visually
or by using luminometers, x-ray film, high speed photographic film,
a CCD camera, etc. Once the amount of the label in the complex has
been quantified, the concentration of analyte or a fragment thereof
in the test sample is determined by appropriate means, such as by
use of a standard curve that has been generated using serial
dilutions of analyte or a fragment thereof of known concentration.
Other than using serial dilutions of analyte or a fragment thereof,
the standard curve can be generated gravimetrically, by mass
spectroscopy and by other techniques known in the art.
[0994] In a chemiluminescent microparticle assay employing the
ARCHITECT.RTM. analyzer, the conjugate diluent pH should be about
6.0+/-0.2, the microparticle coating buffer should be maintained at
about room temperature (i.e., at from about 17 to about 27
.delta.C), the microparticle coating buffer pH should be about
6.5+/-0.2, and the microparticle diluent pH should be about
7.8+/-0.2. Solids preferably are less than about 0.2%, such as less
than about 0.15%, less than about 0.14%, less than about 0.13%,
less than about 0.12%, or less than about 0.11%, such as about
0.10%.
[0995] FPIAs are based on competitive binding immunoassay
principles. A fluorescently labeled compound, when excited by a
linearly polarized light, will emit fluorescence having a degree of
polarization inversely proportional to its rate of rotation. When a
fluorescently labeled tracer-antibody complex is excited by a
linearly polarized light, the emitted light remains highly
polarized because the fluorophore is constrained from rotating
between the time light is absorbed and the time light is emitted.
When a "free" tracer compound (i.e., a compound that is not bound
to an antibody) is excited by linearly polarized light, its
rotation is much faster than the corresponding tracer-antibody
conjugate produced in a competitive binding immunoassay. FPIAs are
advantageous over RIAs inasmuch as there are no radioactive
substances requiring special handling and disposal. In addition,
FPIAs are homogeneous assays that can be easily and rapidly
performed.
[0996] In view of the above, a method of determining the presence,
amount, or concentration of analyte (or a fragment thereof) in a
test sample is provided. The method comprises assaying the test
sample for an analyte (or a fragment thereof) by an assay (i)
employing (i') at least one of an antibody, a fragment of an
antibody that can bind to an analyte, a variant of an antibody that
can bind to an analyte, a fragment of a variant of an antibody that
can bind to an analyte, and a DVD-Ig (or a fragment, a variant, or
a fragment of a variant thereof) that can bind to an analyte, and
(ii') at least one detectable label and (ii) comprising comparing a
signal generated by the detectable label as a direct or indirect
indication of the presence, amount or concentration of analyte (or
a fragment thereof) in the test sample to a signal generated as a
direct or indirect indication of the presence, amount or
concentration of analyte (or a fragment thereof) in a control or
calibrator. The calibrator is optionally part of a series of
calibrators, in which each of the calibrators differs from the
other calibrators by the concentration of analyte.
[0997] The method can comprise (i) contacting the test sample with
at least one first specific binding partner for analyte (or a
fragment thereof) selected from the group consisting of an
antibody, a fragment of an antibody that can bind to an analyte, a
variant of an antibody that can bind to an analyte, a fragment of a
variant of an antibody that can bind to an analyte, and a DVD-Ig
(or a fragment, a variant, or a fragment of a variant thereof) that
can bind to an analyte so as to form a first specific binding
partner/analyte (or fragment thereof) complex, (ii) contacting the
first specific binding partner/analyte (or fragment thereof)
complex with at least one second specific binding partner for
analyte (or fragment thereof) selected from the group consisting of
a detectably labeled anti-analyte antibody, a detectably labeled
fragment of an anti-analyte antibody that can bind to analyte, a
detectably labeled variant of an anti-analyte antibody that can
bind to analyte, a detectably labeled fragment of a variant of an
anti-analyte antibody that can bind to analyte, and a detectably
labeled DVD-Ig (or a fragment, a variant, or a fragment of a
variant thereof) so as to form a first specific binding
partner/analyte (or fragment thereof)/second specific binding
partner complex, and (iii) determining the presence, amount or
concentration of analyte in the test sample by detecting or
measuring the signal generated by the detectable label in the first
specific binding partner/analyte (or fragment thereof)/second
specific binding partner complex formed in (ii). A method in which
at least one first specific binding partner for analyte (or a
fragment thereof) and/or at least one second specific binding
partner for analyte (or a fragment thereof) is a DVD-Ig (or a
fragment, a variant, or a fragment of a variant thereof) as
described herein can be preferred.
[0998] Alternatively, the method can comprise contacting the test
sample with at least one first specific binding partner for an
IL-17 analyte (or a fragment thereof) selected from the group
consisting of an antibody, a fragment of an antibody that can bind
to an analyte, a variant of an antibody that can bind to an
analyte, a fragment of a variant of an antibody that can bind to an
analyte, and a DVD-Ig (or a fragment, a variant, or a fragment of a
variant thereof) and simultaneously or sequentially, in either
order, contacting the test sample with at least one second specific
binding partner, which can compete with analyte (or a fragment
thereof) for binding to the at least one first specific binding
partner and which is selected from the group consisting of a
detectably labeled analyte, a detectably labeled fragment of
analyte that can bind to the first specific binding partner, a
detectably labeled variant of analyte that can bind to the first
specific binding partner, and a detectably labeled fragment of a
variant of analyte that can bind to the first specific binding
partner. Any IL-17 (or a fragment thereof) present in the test
sample and the at least one second specific binding partner compete
with each other to form a first specific binding partner/analyte
(or fragment thereof) complex and a first specific binding
partner/second specific binding partner complex, respectively. The
method further comprises determining the presence, amount or
concentration of analyte in the test sample by detecting or
measuring the signal generated by the detectable label in the first
specific binding partner/second specific binding partner complex
formed in (ii), wherein the signal generated by the detectable
label in the first specific binding partner/second specific binding
partner complex is inversely proportional to the amount or
concentration of analyte in the test sample.
[0999] The above methods can further comprise diagnosing,
prognosticating, or assessing the efficacy of a
therapeutic/prophylactic treatment of a patient from whom the test
sample was obtained. If the method further comprises assessing the
efficacy of a therapeutic/prophylactic treatment of the patient
from whom the test sample was obtained, the method optionally
further comprises modifying the therapeutic/prophylactic treatment
of the patient as needed to improve efficacy. The method can be
adapted for use in an automated system or a semi-automated
system.
[1000] With regard to the methods of assay (and kit therefor), it
may be possible to employ commercially available anti-analyte
antibodies or methods for production of anti-analyte as described
in the literature. Commercial supplies of various antibodies
include, but are not limited to, Santa Cruz Biotechnology Inc.
(Santa Cruz, Calif.), GenWay Biotech, Inc. (San Diego, Calif.), and
R&D Systems (RDS; Minneapolis, Minn.).
[1001] Generally, a predetermined level can be employed as a
benchmark against which to assess results obtained upon assaying a
test sample for analyte or a fragment thereof, e.g., for detecting
disease or risk of disease. Generally, in making such a comparison,
the predetermined level is obtained by running a particular assay a
sufficient number of times and under appropriate conditions such
that a linkage or association of analyte presence, amount or
concentration with a particular stage or endpoint of a disease,
disorder or condition or with particular clinical indicia can be
made. Typically, the predetermined level is obtained with assays of
reference subjects (or populations of subjects). The analyte
measured can include fragments thereof, degradation products
thereof, and/or enzymatic cleavage products thereof.
[1002] In particular, with respect to a predetermined level as
employed for monitoring disease progression and/or treatment, the
amount or concentration of analyte or a fragment thereof may be
"unchanged," "favorable" (or "favorably altered"), or "unfavorable"
(or "unfavorably altered"). "Elevated" or "increased" refers to an
amount or a concentration in a test sample that is higher than a
typical or normal level or range (e.g., predetermined level), or is
higher than another reference level or range (e.g., earlier or
baseline sample). The term "lowered" or "reduced" refers to an
amount or a concentration in a test sample that is lower than a
typical or normal level or range (e.g., predetermined level), or is
lower than another reference level or range (e.g., earlier or
baseline sample). The term "altered" refers to an amount or a
concentration in a sample that is altered (increased or decreased)
over a typical or normal level or range (e.g., predetermined
level), or over another reference level or range (e.g., earlier or
baseline sample).
[1003] The typical or normal level or range for analyte is defined
in accordance with standard practice. Because the levels of analyte
in some instances will be very low, a so-called altered level or
alteration can be considered to have occurred when there is any net
change as compared to the typical or normal level or range, or
reference level or range, that cannot be explained by experimental
error or sample variation. Thus, the level measured in a particular
sample will be compared with the level or range of levels
determined in similar samples from a so-called normal subject. In
this context, a "normal subject" is an individual with no
detectable disease, for example, and a "normal" (sometimes termed
"control") patient or population is/are one(s) that exhibit(s) no
detectable disease, respectively, for example. Furthermore, given
that analyte is not routinely found at a high level in the majority
of the human population, a "normal subject" can be considered an
individual with no substantial detectable increased or elevated
amount or concentration of analyte, and a "normal" (sometimes
termed "control") patient or population is/are one(s) that
exhibit(s) no substantial detectable increased or elevated amount
or concentration of analyte. An "apparently normal subject" is one
in which analyte has not yet been or currently is being assessed.
The level of an analyte is said to be "elevated" when the analyte
is normally undetectable (e.g., the normal level is zero, or within
a range of from about 25 to about 75 percentiles of normal
populations), but is detected in a test sample, as well as when the
analyte is present in the test sample at a higher than normal
level. Thus, inter alia, the disclosure provides a method of
screening for a subject having, or at risk of having, a particular
disease, disorder, or condition. The method of assay can also
involve the assay of other markers and the like.
[1004] Accordingly, the methods described herein also can be used
to determine whether or not a subject has or is at risk of
developing a given disease, disorder or condition. Specifically,
such a method can comprise the steps of:
[1005] (a) determining the concentration or amount in a test sample
from a subject of IL-17 (or a fragment thereof) (e.g., using the
methods described herein, or methods known in the art); and
[1006] (b) comparing the concentration or amount of IL-17 (or a
fragment thereof) determined in step (a) with a predetermined
level, wherein, if the concentration or amount of analyte
determined in step (a) is favorable with respect to a predetermined
level, then the subject is determined not to have or be at risk for
a given disease, disorder or condition. However, if the
concentration or amount of IL-17 determined in step (a) is
unfavorable with respect to the predetermined level, then the
subject is determined to have or be at risk for a given disease,
disorder or condition.
[1007] Additionally, provided herein is method of monitoring the
progression of disease in a subject. Optimally the method
comprising the steps of:
[1008] (a) determining the concentration or amount in a test sample
from a subject of IL-17;
[1009] (b) determining the concentration or amount in a later test
sample from the subject of IL-17; and
[1010] (c) comparing the concentration or amount of analyte as
determined in step (b) with the concentration or amount of IL-17
determined in step (a), wherein if the concentration or amount
determined in step (b) is unchanged or is unfavorable when compared
to the concentration or amount of IL-17 determined in step (a),
then the disease in the subject is determined to have continued,
progressed or worsened. By comparison, if the concentration or
amount of IL-17 as determined in step (b) is favorable when
compared to the concentration or amount of IL-17 as determined in
step (a), then the disease in the subject is determined to have
discontinued, regressed or improved.
[1011] Optionally, the method further comprises comparing the
concentration or amount of IL-17 analyte as determined in step (b),
for example, with a predetermined level. Further, optionally the
method comprises treating the subject with one or more
pharmaceutical compositions for a period of time if the comparison
shows that the concentration or amount of analyte as determined in
step (b), for example, is unfavorably altered with respect to the
predetermined level.
[1012] Still further, the methods can be used to monitor treatment
in a subject receiving treatment with one or more pharmaceutical
compositions. Specifically, such methods involve providing a first
test sample from a subject before the subject has been administered
one or more pharmaceutical compositions. Next, the concentration or
amount in a first test sample from a subject of IL-17 is determined
(e.g., using the methods described herein or as known in the art).
After the concentration or amount of IL-17 is determined,
optionally the concentration or amount of IL-17 is then compared
with a predetermined level. If the concentration or amount of IL-17
as determined in the first test sample is lower than the
predetermined level, then the subject is not treated with one or
more pharmaceutical compositions. However, if the concentration or
amount of IL-17 as determined in the first test sample is higher
than the predetermined level, then the subject is treated with one
or more pharmaceutical compositions for a period of time. The
period of time that the subject is treated with the one or more
pharmaceutical compositions can be determined by one skilled in the
art (for example, the period of time can be from about seven (7)
days to about two years, preferably from about fourteen (14) days
to about one (1) year).
[1013] During the course of treatment with the one or more
pharmaceutical compositions, second and subsequent test samples are
then obtained from the subject. The number of test samples and the
time in which said test samples are obtained from the subject are
not critical. For example, a second test sample could be obtained
seven (7) days after the subject is first administered the one or
more pharmaceutical compositions, a third test sample could be
obtained two (2) weeks after the subject is first administered the
one or more pharmaceutical compositions, a fourth test sample could
be obtained three (3) weeks after the subject is first administered
the one or more pharmaceutical compositions, a fifth test sample
could be obtained four (4) weeks after the subject is first
administered the one or more pharmaceutical compositions, etc.
[1014] After each second or subsequent test sample is obtained from
the subject, the concentration or amount of IL-17 analyte is
determined in the second or subsequent test sample is determined
(e.g., using the methods described herein or as known in the art).
The concentration or amount of IL-17 as determined in each of the
second and subsequent test samples is then compared with the
concentration or amount of analyte as determined in the first test
sample (e.g., the test sample that was originally optionally
compared to the predetermined level). If the concentration or
amount of IL-17 as determined in step (c) is favorable when
compared to the concentration or amount of analyte as determined in
step (a), then the disease in the subject is determined to have
discontinued, regressed or improved, and the subject should
continue to be administered the one or pharmaceutical compositions
of step (b). However, if the concentration or amount determined in
step (c) is unchanged or is unfavorable when compared to the
concentration or amount of analyte as determined in step (a), then
the disease in the subject is determined to have continued,
progressed or worsened, and the subject should be treated with a
higher concentration of the one or more pharmaceutical compositions
administered to the subject in step (b) or the subject should be
treated with one or more pharmaceutical compositions that are
different from the one or more pharmaceutical compositions
administered to the subject in step (b). Specifically, the subject
can be treated with one or more pharmaceutical compositions that
are different from the one or more pharmaceutical compositions that
the subject had previously received to decrease or lower said
subject's analyte level.
[1015] Generally, for assays in which repeat testing may be done
(e.g., monitoring disease progression and/or response to
treatment), a second or subsequent test sample is obtained at a
period in time after the first test sample has been obtained from
the subject. Specifically, a second test sample from the subject
can be obtained minutes, hours, days, weeks or years after the
first test sample has been obtained from the subject. For example,
the second test sample can be obtained from the subject at a time
period of about 1 minute, about 5 minutes, about 10 minutes, about
15 minutes, about 30 minutes, about 45 minutes, about 60 minutes,
about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6
hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours,
about 11 hours, about 12 hours, about 13 hours, about 14 hours,
about 15 hours, about 16 hours, about 17 hours, about 18 hours,
about 19 hours, about 20 hours, about 21 hours, about 22 hours,
about 23 hours, about 24 hours, about 2 days, about 3 days, about 4
days, about 5 days, about 6 days, about 7 days, about 2 weeks,
about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7
weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11
weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15
weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19
weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23
weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27
weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31
weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35
weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 39
weeks, about 40 weeks, about 41 weeks, about 42 weeks, about 43
weeks, about 44 weeks, about 45 weeks, about 46 weeks, about 47
weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51
weeks, about 52 weeks, about 1.5 years, about 2 years, about 2.5
years, about 3.0 years, about 3.5 years, about 4.0 years, about 4.5
years, about 5.0 years, about 5.5. years, about 6.0 years, about
6.5 years, about 7.0 years, about 7.5 years, about 8.0 years, about
8.5 years, about 9.0 years, about 9.5 years or about 10.0 years
after the first test sample from the subject is obtained.
[1016] When used to monitor disease progression, the above assay
can be used to monitor the progression of disease in subjects
suffering from acute conditions. Acute conditions, also known as
critical care conditions, refer to acute, life-threatening diseases
or other critical medical conditions involving, for example, the
cardiovascular system or excretory system. Typically, critical care
conditions refer to those conditions requiring acute medical
intervention in a hospital-based setting (including, but not
limited to, the emergency room, intensive care unit, trauma center,
or other emergent care setting) or administration by a paramedic or
other field-based medical personnel. For critical care conditions,
repeat monitoring is generally done within a shorter time frame,
namely, minutes, hours or days (e.g., about 1 minute, about 5
minutes, about 10 minutes, about 15 minutes, about 30 minutes,
about 45 minutes, about 60 minutes, about 2 hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 10 hours, about 11 hours, about 12
hours, about 13 hours, about 14 hours, about 15 hours, about 16
hours, about 17 hours, about 18 hours, about 19 hours, about 20
hours, about 21 hours, about 22 hours, about 23 hours, about 24
hours, about 2 days, about 3 days, about 4 days, about 5 days,
about 6 days or about 7 days), and the initial assay likewise is
generally done within a shorter timeframe, e.g., about minutes,
hours or days of the onset of the disease or condition.
[1017] The assays also can be used to monitor the progression of
disease in subjects suffering from chronic or non-acute conditions.
Non-critical care or, non-acute conditions, refers to conditions
other than acute, life-threatening disease or other critical
medical conditions involving, for example, the cardiovascular
system and/or excretory system. Typically, non-acute conditions
include those of longer-term or chronic duration. For non-acute
conditions, repeat monitoring generally is done with a longer
timeframe, e.g., hours, days, weeks, months or years (e.g., about 1
hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours,
about 6 hours, about 7 hours, about 8 hours, about 9 hours, about
10 hours, about 11 hours, about 12 hours, about 13 hours, about 14
hours, about 15 hours, about 16 hours, about 17 hours, about 18
hours, about 19 hours, about 20 hours, about 21 hours, about 22
hours, about 23 hours, about 24 hours, about 2 days, about 3 days,
about 4 days, about 5 days, about 6 days, about 7 days, about 2
weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks,
about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about
11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15
weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19
weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23
weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27
weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31
weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35
weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 39
weeks, about 40 weeks, about 41 weeks, about 42 weeks, about 43
weeks, about 44 weeks, about 45 weeks, about 46 weeks, about 47
weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51
weeks, about 52 weeks, about 1.5 years, about 2 years, about 2.5
years, about 3.0 years, about 3.5 years, about 4.0 years, about 4.5
years, about 5.0 years, about 5.5. years, about 6.0 years, about
6.5 years, about 7.0 years, about 7.5 years, about 8.0 years, about
8.5 years, about 9.0 years, about 9.5 years or about 10.0 years),
and the initial assay likewise generally is done within a longer
time frame, e.g., about hours, days, months or years of the onset
of the disease or condition.
[1018] Furthermore, the above assays can be performed using a first
test sample obtained from a subject where the first test sample is
obtained from one source, such as urine, serum or plasma.
Optionally, the above assays can then be repeated using a second
test sample obtained from the subject where the second test sample
is obtained from another source. For example, if the first test
sample was obtained from urine, the second test sample can be
obtained from serum or plasma. The results obtained from the assays
using the first test sample and the second test sample can be
compared. The comparison can be used to assess the status of a
disease or condition in the subject.
[1019] Moreover, the present disclosure also relates to methods of
determining whether a subject predisposed to or suffering from a
given disease, disorder or condition will benefit from treatment.
In particular, the disclosure relates to analyte companion
diagnostic methods and products. Thus, the method of "monitoring
the treatment of disease in a subject" as described herein further
optimally also can encompass selecting or identifying candidates
for therapy.
[1020] Thus, in particular embodiments, the disclosure also
provides a method of determining whether a subject having, or at
risk for, a given disease, disorder or condition is a candidate for
therapy. Generally, the subject is one who has experienced some
symptom of a given disease, disorder or condition or who has
actually been diagnosed as having, or being at risk for, a given
disease, disorder or condition, and/or who demonstrates an
unfavorable concentration or amount of analyte or a fragment
thereof, as described herein.
[1021] The method optionally comprises an assay as described
herein, where IL-17 is assessed before and following treatment of a
subject with one or more pharmaceutical compositions (e.g.,
particularly with a pharmaceutical related to a mechanism of action
involving analyte), with immunosuppressive therapy, or by
immunoabsorption therapy, or where analyte is assessed following
such treatment and the concentration or the amount of analyte is
compared against a predetermined level. An unfavorable
concentration of amount of IL-17 observed following treatment
confirms that the subject will not benefit from receiving further
or continued treatment, whereas a favorable concentration or amount
of analyte observed following treatment confirms that the subject
will benefit from receiving further or continued treatment. This
confirmation assists with management of clinical studies, and
provision of improved patient care.
[1022] It goes without saying that, while certain embodiments
herein are advantageous when employed to assess a given disease,
disorder or condition as discussed herein, the assays and kits can
be employed to assess analyte in other diseases, disorders and
conditions. The method of assay can also involve the assay of other
markers and the like.
[1023] The method of assay also can be used to identify a compound
that ameliorates a given disease, disorder or condition. For
example, a cell that expresses analyte can be contacted with a
candidate compound. The level of expression of analyte in the cell
contacted with the compound can be compared to that in a control
cell using the method of assay described herein.
II. Kits
[1024] A kit for assaying a test sample for the presence, amount or
concentration of an analyte (or a fragment thereof) in a test
sample is also provided. The kit comprises at least one component
for assaying the test sample for IL-17 (or a fragment thereof) and
instructions for assaying the test sample for the analyte (or a
fragment thereof). The at least one component for assaying the test
sample for the analyte (or a fragment thereof) can include a
composition comprising an anti-IL-17 binding protein, such as a
monoclonal antibody or DVD-Ig (or a fragment, a variant, or a
fragment of a variant thereof), as described herein and which is
optionally immobilized on a solid phase.
[1025] The kit can comprise at least one component for assaying the
test sample for an IL-17 analyte by immunoassay, e.g.,
chemiluminescent microparticle immunoassay, and instructions for
assaying the test sample for an IL-17 analyte by immunoassay, e.g.,
chemiluminescent microparticle immunoassay. For example, the kit
can comprise at least one specific binding partner for IL-17, such
as an anti-IL-17 monoclonal/polyclonal antibody (or a fragment
thereof that can bind to the IL-17 analyte, a variant thereof that
can bind to the analyte, or a fragment of a variant that can bind
to the analyte) or an anti-IL-17 DVD-Ig (or a fragment, a variant,
or a fragment of a variant thereof), either of which can be
detectably labeled. Alternatively or additionally, the kit can
comprise detectably labeled IL-17 analyte (or a fragment thereof
that can bind to an anti-analyte, monoclonal/polyclonal antibody or
an anti-analyte DVD-Ig (or a fragment, a variant, or a fragment of
a variant thereof)), which can compete with any analyte in a test
sample for binding to an anti-analyte monoclonal/polyclonal
antibody (or a fragment thereof that can bind to the analyte, a
variant thereof that can bind to the analyte, or a fragment of a
variant that can bind to the analyte) or an anti-analyte DVD-Ig (or
a fragment, a variant, or a fragment of a variant thereof), either
of which can be immobilized on a solid support. The kit can
comprise a calibrator or control, e.g., isolated or purified
analyte. The kit can comprise at least one container (e.g., tube,
microtiter plates or strips, which can be already coated with a
first specific binding partner, for example) for conducting the
assay, and/or a buffer, such as an assay buffer or a wash buffer,
either one of which can be provided as a concentrated solution, a
substrate solution for the detectable label (e.g., an enzymatic
label), or a stop solution. Preferably, the kit comprises all
components, i.e., reagents, standards, buffers, diluents, etc.,
which are necessary to perform the assay. The instructions can be
in paper form or computer-readable form, such as a disk, CD, DVD,
or the like.
[1026] Any binding protein, such as an anti-IL-17 binding protein
or an anti-analyte DVD-Ig, or tracer can incorporate a detectable
label as described herein, such as a fluorophore, a radioactive
moiety, an enzyme, a biotin/avidin label, a chromophore, a
chemiluminescent label, or the like, or the kit can include
reagents for carrying out detectable labeling. The antibodies,
calibrators and/or controls can be provided in separate containers
or pre-dispensed into an appropriate assay format, for example,
into microtiter plates.
[1027] Optionally, the kit includes quality control components (for
example, sensitivity panels, calibrators, and positive controls).
Preparation of quality control reagents is well-known in the art
and is described on insert sheets for a variety of immunodiagnostic
products. Sensitivity panel members optionally are used to
establish assay performance characteristics, and further optionally
are useful indicators of the integrity of the immunoassay kit
reagents, and the standardization of assays.
[1028] The kit can also optionally include other reagents required
to conduct a diagnostic assay or facilitate quality control
evaluations, such as buffers, salts, enzymes, enzyme co-factors,
enzyme substrates, detection reagents, and the like. Other
components, such as buffers and solutions for the isolation and/or
treatment of a test sample (e.g., pretreatment reagents), also can
be included in the kit. The kit can additionally include one or
more other controls. One or more of the components of the kit can
be lyophilized, in which case the kit can further comprise reagents
suitable for the reconstitution of the lyophilized components.
[1029] The various components of the kit optionally are provided in
suitable containers as necessary, e.g., a microtiter plate. The kit
can further include containers for holding or storing a sample
(e.g., a container or cartridge for a urine sample). Where
appropriate, the kit optionally also can contain reaction vessels,
mixing vessels, and other components that facilitate the
preparation of reagents or the test sample. The kit can also
include one or more instruments for assisting with obtaining a test
sample, such as a syringe, pipette, forceps, measured spoon, or the
like.
[1030] If the detectable label is at least one acridinium compound,
the kit can comprise at least one acridinium-9-carboxamide, at
least one acridinium-9-carboxylate aryl ester, or any combination
thereof. If the detectable label is at least one acridinium
compound, the kit also can comprise a source of hydrogen peroxide,
such as a buffer, a solution, and/or at least one basic solution.
If desired, the kit can contain a solid phase, such as a magnetic
particle, bead, test tube, microtiter plate, cuvette, membrane,
scaffolding molecule, film, filter paper, disc or chip.
III. Adaptation of Kit and Method
[1031] The kit (or components thereof), as well as the method of
determining the presence, amount or concentration of an analyte in
a test sample by an assay, such as an immunoassay as described
herein, can be adapted for use in a variety of automated and
semi-automated systems (including those wherein the solid phase
comprises a microparticle), as described, e.g., in U.S. Pat. Nos.
5,089,424 and 5,006,309, and as commercially marketed, e.g., by
Abbott Laboratories (Abbott Park, Ill.) as ARCHITECT.RTM..
[1032] Some of the differences between an automated or
semi-automated system as compared to a non-automated system (e.g.,
ELISA) include the substrate to which the first specific binding
partner (e.g., an anti-analyte, monoclonal/polyclonal antibody (or
a fragment thereof, a variant thereof, or a fragment of a variant
thereof) or an anti-analyte DVD-Ig (or a fragment thereof, a
variant thereof, or a fragment of a variant thereof) is attached;
either way, sandwich formation and analyte reactivity can be
impacted), and the length and timing of the capture, detection
and/or any optional wash steps. Whereas a non-automated format,
such as an ELISA, may require a relatively longer incubation time
with sample and capture reagent (e.g., about 2 hours), an automated
or semi-automated format (e.g., ARCHITECT.RTM., Abbott
Laboratories) may have a relatively shorter incubation time (e.g.,
approximately 18 minutes for ARCHITECT.RTM.). Similarly, whereas a
non-automated format, such as an ELISA, may incubate a detection
antibody, such as the conjugate reagent, for a relatively longer
incubation time (e.g., about 2 hours), an automated or
semi-automated format (e.g., ARCHITECT.RTM.) may have a relatively
shorter incubation time (e.g., approximately 4 minutes for the
ARCHITECT.RTM.).
[1033] Other platforms available from Abbott Laboratories include,
but are not limited to, AxSYM.RTM., IMx.RTM. (see, e.g., U.S. Pat.
No. 5,294,404), PRISM.RTM., EIA (bead), and Quantum.TM. II, as well
as other platforms. Additionally, the assays, kits and kit
components can be employed in other formats, for example, on
electrochemical or other hand-held or point-of-care assay systems.
The present disclosure is, for example, applicable to the
commercial Abbott Point of Care (i-STAT.RTM., Abbott Laboratories)
electrochemical immunoassay system that performs sandwich
immunoassays Immunosensors and their methods of manufacture and
operation in single-use test devices are described, for example in,
U.S. Pat. No. 5,063,081, US Patent Application Publication No.
2003/0170881, US Patent Application Publication No. 2004/0018577,
US Patent Application Publication No. 2005/0054078, and US Patent
Application Publication No. 2006/0160164.
[1034] In particular, with regard to the adaptation of an analyte
assay to the I-STAT.RTM. system, the following configuration is
preferred. A microfabricated silicon chip is manufactured with a
pair of gold amperometric working electrodes and a silver-silver
chloride reference electrode. On one of the working electrodes,
polystyrene beads (0.2 mm diameter) with immobilized anti-analyte,
monoclonal/polyclonal antibody (or a fragment thereof, a variant
thereof, or a fragment of a variant thereof) or anti-analyte DVD-Ig
(or a fragment thereof, a variant thereof, or a fragment of a
variant thereof), are adhered to a polymer coating of patterned
polyvinyl alcohol over the electrode. This chip is assembled into
an I-STAT.RTM. cartridge with a fluidics format suitable for
immunoassay. On a portion of the wall of the sample-holding chamber
of the cartridge there is a layer comprising a specific binding
partner for an analyte, such as an anti-analyte,
monoclonal/polyclonal antibody (or a fragment thereof, a variant
thereof, or a fragment of a variant thereof that can bind the
analyte) or an anti-analyte DVD-Ig (or a fragment thereof, a
variant thereof, or a fragment of a variant thereof that can bind
the analyte), either of which can be detectably labeled. Within the
fluid pouch of the cartridge is an aqueous reagent that includes
p-aminophenol phosphate.
[1035] In operation, a sample suspected of containing an analyte is
added to the holding chamber of the test cartridge, and the
cartridge is inserted into the I-STAT.RTM. reader. After the
specific binding partner for an analyte has dissolved into the
sample, a pump element within the cartridge forces the sample into
a conduit containing the chip. Here it is oscillated to promote
formation of the sandwich. In the penultimate step of the assay,
fluid is forced out of the pouch and into the conduit to wash the
sample off the chip and into a waste chamber. In the final step of
the assay, the alkaline phosphatase label reacts with p-aminophenol
phosphate to cleave the phosphate group and permit the liberated
p-aminophenol to be electrochemically oxidized at the working
electrode. Based on the measured current, the reader is able to
calculate the amount of analyte in the sample by means of an
embedded algorithm and factory-determined calibration curve.
[1036] It further goes without saying that the methods and kits as
described herein necessarily encompass other reagents and methods
for carrying out the immunoassay. For instance, encompassed are
various buffers such as are known in the art and/or which can be
readily prepared or optimized to be employed, e.g., for washing, as
a conjugate diluent, microparticle diluent, and/or as a calibrator
diluent. An exemplary conjugate diluent is ARCHITECT.RTM. conjugate
diluent employed in certain kits (Abbott Laboratories, Abbott Park,
Ill.) and containing 2-(N-morpholino)ethanesulfonic acid (MES), a
salt, a protein blocker, an antimicrobial agent, and a detergent.
An exemplary calibrator diluent is ARCHITECT.RTM. human calibrator
diluent employed in certain kits (Abbott Laboratories, Abbott Park,
Ill.), which comprises a buffer containing MES, other salt, a
protein blocker, and an antimicrobial agent. Additionally, as
described in U.S. patent application Ser. No. 12/650,241 (see, also
PCT/US2009/069846), improved signal generation may be obtained,
e.g., in an I-Stat cartridge format, using a nucleic acid sequence
linked to the signal antibody as a signal amplifier.
[1037] It will be readily apparent to those skilled in the art that
other suitable modifications and adaptations of the methods of the
invention described herein are obvious and may be made using
suitable equivalents without departing from the scope of the
invention or the embodiments disclosed herein.
[1038] Having now described the present invention in detail, the
same will be more clearly understood by reference to the following
examples, which are included for purposes of illustration only and
are not intended to be limiting of the invention.
EXAMPLES
Example 1
Murine, Chimeric, and Humanized Anti-Human IL-17 Antibodies
1.1: Construction and Expression of Murine Monoclonal and
Recombinant Chimeric Anti Human IL-17 Antibodies
[1039] Murine monoclonal antibodies to human IL-17 (hIL-17) were
obtained by standard methods. Balb/c and A/J mice, 4-6 weeks of
age, were immunized and boosted subcutaneously with recombinant
human IL-17 Animals were injected every three weeks, beginning with
a primary injection of 15 .mu.g in complete Freund's adjuvant and
injection boosts of 15 .mu.g in Incomplete Freund's Adjuvant. Mice
selected for fusion were injected intravenously with IL-17 in
saline, four days prior to fusion. Spleens from immunized animals
were removed and single cell suspensions were prepared. SP2/0
myeloma cells were harvested from culture and washed. Spleen cells
and tumor cells were mixed at a ratio of 5: I and fused using 50%
PEG 3000 using standard techniques (Kohler and Milstein, Nature,
256: 495-497 (1975)). Fused cells were seeded in 96 well plates in
selective media, at a density of 2.5.times.10.sup.5 spleen cells
per well. Fusions were incubated at 37.degree. C. for 7-10 days.
When macroscopic colonies were observed, supernatants were removed
and tested by ELISA. Cells that show binding to human and cyno
IL-17 proteins were subcloned and antibody proteins purified.
Murine anti hIL-17 monoclonal antibodies 10F7, 5C5, 6C6, 7D7, 1D8,
8B12, and 10G9 were isolated and the amino acid sequences of the
variable heavy (VH) and variable light (VL) chains determined See,
Table 5.
[1040] The DNA encoding the heavy chain constant region of murine
anti-human IL-17 monoclonal antibodies 10F7, 5C5, 6C6, 7D7, 1D8,
8B12, and 10G9 was replaced by a cDNA fragment encoding the human
IgG1 constant region containing 2 hinge-region amino acid mutations
by homologous recombination in bacteria. These mutations were a
leucine to alanine change at position 234 (EU numbering) and a
leucine to alanine change at position 235 (Lund et al., J.
Immunol., 147: 2657 (1991)). The light chain constant region of
each of these antibodies was replaced by a human kappa constant
region. Full-length chimeric antibodies were transiently expressed
in HEK293 cells by co-transfection of chimeric heavy and light
chain cDNAs ligated into the pHybE expression plasmid. Cell
supernatants containing recombinant chimeric antibody were purified
by Protein A Sepharose chromatography and bound antibody was eluted
by addition of acid buffer. Antibodies were neutralized and
dialyzed into PBS.
[1041] The purified chimeric anti-human IL-17 monoclonal antibodies
were then tested for their ability to bind the hIL-17 protein by
ELISA to confirm antigen binding. See, Example 1.5.1, below.
1.2: Construction of CDR Grafted and Humanized Anti Human IL-17
Antibodies
[1042] By applying standard methods well known in the art, the CDR
sequences of VH and VL chains of monoclonal antibody 10F7 and 5C5
(see Table 5, above) were grafted into different human heavy and
light chain acceptor sequences.
[1043] Based on sequence VH and VL alignments with the VH and VL
sequences of monoclonal antibody 10F7 of the present invention the
following known human sequences are selected:
[1044] a) VH1-69 (IGHV1-69) and the joining sequences hJH6 for
constructing heavy chain acceptor sequences
[1045] b) 1-17/A30 and 6-21/A26 as well as hJK2 and hJK4 for
constructing light chain acceptor sequences
[1046] By grafting the corresponding VH and VL CDRs of 10F7 into
said acceptor sequences, the CDR-grafted, humanized, and modified
VH and VL sequences were prepared.
[1047] Based on sequence VH and VL alignments with the VH and VL
sequences of monoclonal antibody 5C5 of the present invention the
following known human sequences are selected:
[1048] c) VH1-69 (IGHV1-69) and the joining sequences hJH1, hJH3,
hJH4, hJH5, and hJH6 for constructing heavy chain acceptor
sequences
[1049] d) 1-33/O18 and 3-15/L2 as well as hJK2 and hJK4 for
constructing light chain acceptor sequences
[1050] By grafting the corresponding VH and VL CDRs of 5C5 into
said acceptor sequences, the CDR-grafted, humanized, and modified
VH and VL sequences were prepared (see, Table 7, above).
1.3: Construction of Framework Back Mutations in CDR-Grafted
Antibodies
[1051] To generate humanized antibody framework back mutations,
mutations were introduced into the CDR-grafted antibody sequences
by de novo synthesis of the variable domain and/or using mutagenic
primers and PCR, and methods well known in the art. Different
combinations of back mutations and other mutations are constructed
for each of the CDR-grafts as follows. Residue numbers for these
mutations are based on the Kabat numbering system. [1052] For heavy
chains h10F7VH.1z, one or more of the following Vernier and VH/VL
interfacing residues were back mutated as follows: M48.fwdarw.I,
V67.fwdarw.A, I69.fwdarw.L, and A93.fwdarw.T. [1053] Additional
mutations include the following: Q1.fwdarw.E, G27.fwdarw.Y, and
S30.fwdarw.T. [1054] For light chain h10F7Vk.1z and 3z one or more
of the following Vernier and VH/VL interfacing residues were back
mutated as follows: D1.fwdarw.Q, Q3.fwdarw.V, M4.fwdarw.L,
Y36.fwdarw.F, A43.fwdarw.S, L47.fwdarw.W, and F71.fwdarw.Y. [1055]
Additional mutation include the following: E70.fwdarw.D,
D1.fwdarw.E [1056] For light chain h10F7Vk.2 and 4 one or more of
the following Vernier and VH/VL interfacing residues were back
mutated as follows: E1.fwdarw.Q, Y36.fwdarw.F, L46.fwdarw.R,
L47.fwdarw.W, K49.fwdarw.Y, and F71.fwdarw.Y [1057] For heavy
chains h5C5VH.1z, one or more of the following Vernier and VH/VL
interfacing residues were back mutated as follows: M48.fwdarw.I,
V67.fwdarw.A, I69.fwdarw.L, A93.fwdarw.T [1058] Additional
mutations include the following: Q1.fwdarw.E, S16.fwdarw.A,
G27.fwdarw.Y, and S30.fwdarw.T [1059] For light chain h5C5Vk.1z and
2z one or more of the following Vernier and VH/VL interfacing
residues were back mutated as follows: D1.fwdarw.N, Q3.fwdarw.V,
Y36.fwdarw.F, A43.fwdarw.S, and Y87.fwdarw.F. [1060] Additional
mutation include the following: S7.fwdarw.T, I83.fwdarw.F [1061]
For light chain h5C5Vk.3z and 4z one or more of the following
Vernier and VH/VL interfacing residues were back mutated as
follows: E1.fwdarw.N, Y36.fwdarw.F, A43.fwdarw.S, I58.fwdarw.V,
Y87.fwdarw.F. [1062] Additional mutation include the following:
S7.fwdarw.T, E70.fwdarw.D, and S80.fwdarw.P
1.4: Generation of Humanized Anti-hIL-17 Antibodies Containing
Framework Back Mutations in CDR-Grafted Antibodies
[1063] The following humanized variable regions of the murine
monoclonal antibody 10F7 were cloned into IgG expression vectors
for functional characterization.
TABLE-US-00010 TABLE 9 Sequences of humanized variable regions. SEQ
ID Sequence NO: Protein region 123456789012345678901234567890 60
h10F7VH.1 EVQLVQSGAEVKKPGSSVKVSCKASGYTFTD
YEIHWVRQAPGQGLEWMGVNDPESGGTFYNQ KFDGRVTITADKSTSTAYMELSSLRSEDTAV
YYCARYYRYESFYGMDYWGQGTTVTVSS 61 h10F7VH.1a
EVQLVQSGAEVKKPGSSVKVSCKASGYTFTD YEIHWVRQAPGQGLEWIGVNDPESGGTFYNQ
KFDGRATLTADKSTSTAYMELSSLRSEDTAV YYCTRYYRYESFYGMDYWGQGTTVTVSS 62
h10F7Vk.1 DIQMTQSPSSLSASVGDRVTITCSASSSISY
IYWYQQKPGKAPKRLIYATFELASGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCHQRSSY
PWTFGQGTKLEIKR 63 h10F7Vk.1a DIQLTQSPSSLSASVGDRVTITCSASSSISY
IYWFQQKPGKSPKRWIYATFELASGVPSRFS GSGSGTDYTLTISSLQPEDFATYYCHQRSSY
PWTFGQGTKLEIKR 64 h10F7Vk.1b QIVLTQSPSSLSASVGDRVTITCSASSSISY
IYWFQQKPGKSPKRWIYATFELASGVPSRFS GSGSGTDYTLTISSLQPEDFATYYCHQRSSY
PWTFGQGTKLEIKR 65 h10F7Vk.1c EIVLTQSPSSLSASVGDRVTITCSASSSISY
IYWFQQKPGKSPKRWIYATFELASGVPSRFS GSGSGTDYTLTISSLQPEDFATYYCHQRSSY
PWTFGQGTKLEIKR 66 h10F7Vk.2 EIVLTQSPDFQSVTPKEKVTITCSASSSISY
IYWYQQKPDQSPKLLIKATFELASGVPSRFS GSGSGTDFTLTINSLEAEDAATYYCHQRSSY
PWTFGQGTKLEIKR 67 h10F7Vk.2a EIVLTQSPDFQSVTPKEKVTITCSASSSISY
IYWFQQKPDQSPKRWIYATFELASGVPSRFS GSGSGTDYTLTINSLEAEDAATYYCHQRSSY
PWTFGQGTKLEIKR 68 h10F7Vk.2b QIVLTQSPDFQSVTPKEKVTITCSASSSISY
IYWFQQKPDQSPKRWIYATFELASGVPSRFS GSGSGTDYTLTINSLEAEDAATYYCHQRSSY
PWTFGQGTKLEIKR
[1064] The different combinations of humanized 10F7 antibody based
on the VH and VL shuffling are listed in the table below.
TABLE-US-00011 TABLE 10 10F7 h10F7 h10F7 h10F7 h10F7 h10F7 h10F7
h10F7 Vk Vk.1 Vk.1a Vk.1b Vk.1c Vk.2 Vk.2a Vk.2b 10F7VH 4 9
h10F7VH.1 18 2 5 12 14 7 10 16 h10F7VH.1a 1 3 6 13 15 8 11 17 VH.1a
contains 4 back-mutations (M48I, V67A, I69L, A93T) Vk.1a contains 4
back-mutations (M4L, Y36F, A43S, L47W) Vk.1b contains two
additional back-mutations (D1Q and Q3V) Vk.1c changed the
back-mutation D1Q to D1E to avoid pyroglutamate Vk.2a contains 4
back-mutations (Y36F, L46R, L47W, K49Y) Vk.2b contains one
additional back-mutations (E1Q)
[1065] The following humanized variable regions of the murine
monoclonal antibody 5C5 were cloned into IgG expression vectors for
functional characterization.
TABLE-US-00012 TABLE 11 SEQ Protein Sequence ID No. region
123456789012345678901234567890 69 h5C5VH.1
EVQLVQSGAEVKKPGASVKVSCKASGYTFTD YEFHWVRQAPGQGLEWMGVIHPGNGGTAYNQ
NFRDRVTITADKSTSTAYMELSSLRSEDTAV YYCARFLTYEGYFDYWGQGTLVTVSS 70
h5C5VH.1a EVQLVQSGAEVKKPGASVKVSCKASGGTFTD
YEFHWVRQAPGQGLEWIGVIHPGNGGTAYNQ NFRDRATLTADKSTSTAYMELSSLRSEDTAV
YYCTRFLTYEGYFDYWGQGTLVTVSS 931 h5C5VH.1b
EVQLVQSGAEVKKPGASVKVSCKASGYTFTD YEFHWVRQAPGQGLEWIGVIHPGNGGTAYNQ
NFRDRATLTADKSTSTAYMELSSLRSEDTAV YYCTRFLTYEGYFDYWGQGTLVTVSS 71
h5C5Vk.1 DIQMTQSPSSLSASVGDRVTITCKASQSVSI
DVGWYQQKPGKAPKLLIYHASNRYTGVPSRF SGSGSGTDFTFTISSLQPEDFATYYCQQDYS
SPYTFGQGTKLEIKR 72 h5C5Vk.1a DIQMTQSPSSLSASVGDRVTITCKASQSVSI
DVGWFQQKPGKSPKLLIYHASNRYTGVPSRF SGSGSGTDFTFTISSLQPEDFATYFCQQDYS
SPYTFGQGTKLEIKR 73 h5C5Vk.1b NIVMTQTPSSLSASVGDRVTITCKASQSVSI
DVGWFQQKPGKSPKLLIYHASNRYTGVPSRF SGSGSGTDFTFTISSLQPEDFATYFCQQDYS
SPYTFGQGTKLEIKR 932 h5C5Vk.1c NIVMTQSPSSLSASVGDRVTITCKASQSVSI
DVGWFQQKPGKSPKLLIYHASNRYTGVPSRF SGSGSGTDFTFTISSLQPEDFATYFCQQDYS
SPYTFGQGTKLEIKR 74 h5C5Vk.3 EIVMTQSPATLSVSPGERATLSCKASQSVSI
DVGWYQQKPGQAPRLLIYHASNRYTGIPARF SGSGSGTDFTLTISSLQPEDFAVYYCQQDYS
SPYTFGQGTKLEIKR 75 h5C5Vk.3a EIVMTQSPATLSVSPGERATLSCKASQSVSI
DVGWFQQKPGQSPRLLIYHASNRYTGVPARF SGSGSGTDFTLTISSLQPEDFAVYFCQQDYS
SPYTFGQGTKLEIKR 76 h5C5Vk.3b NIVMTQTPATLSVSPGERATLSCKASQSVSI
DVGWFQQKPGQSPRLLIYHASNRYTGVPARF SGSGSGTDFTLTISSLQPEDFAVYFCQQDYS
SPYTFGQGTKLEIKR 933 h5C5Vk.3c NIVMTQSPATLSVSPGERATLSCKASQSVSI
DVGWFQQKPGQSPRLLIYHASNRYTGVPARF SGSGSGTDFTLTISSLQPEDFAVYFCQQDYS
SPYTFGQGTKLEIKR
TABLE-US-00013 TABLE 12 5C5 h5C5 h5C5 h5C5 h5C5 h5C5 h5C5 Vk Vk.1
Vk.1a Vk.1c Vk.3 Vk.3a Vk.3c 5C5VH 1 4 7 16 10 13 19 h5C5VH.1 2 5 8
17 11 14 20 h5C5VH.1b 3 6 9 18 12 15 21 VH.1b contains 4
back-mutations (M48I, V67A, I69L, A93T) Vk.1a contains 3
back-mutations (Y36F, A43S, Y87F) Vk.1c contains two additional
back-mutations (D1N, Q3V) Vk.3a contains 4 back-mutations (Y36F,
A43S, I58V, Y87F) Vk.3c contains one additional back-mutation
(E1N)
1.5: Functional Characterization of Mouse and Humanized IL-17
Antibodies
1.5.1: IL-17 Enzyme-Linked Immunosorbent Assay Protocol (ELISA)
[1066] The following protocol is used to characterize the binding
of IL-17 antibodies to human IL-17 by enzyme-linked immunosorbent
assay (ELISA). [1067] 1. Coat ELISA plate with 50 .mu.l per well of
Goat anti Mouse IgG-Fc at 2 ug/ml, overnight at 4.degree. C.
(Jackson cat#115-005-164). [1068] 2. Wash plate 3.times.PBS/Tween.
[1069] 3. Add 50 ul Mab diluted to 1 .mu.g/ml in PBS/0.1% BSA to
appropriate wells. Incubate 1 hr at room temperature (RT). [1070]
4. Wash plate 3.times. with PBS/Tween. [1071] 5. Add 50 .mu.l of
serial diluted biotin-IL17 (human IL-17, IL-17A/F, cyno IL-17) to
appropriate wells. Incubate 1 hr at room temperature (RT). [1072]
6. Wash plate 3.times. with PBS/Tween. [1073] 7. Add 50 .mu.l of
Streptavidin (Thermo Scientific cat#21126) diluted 1:10,000 in
PBS/0.1% BSA. Incubate 1 hr at RT. [1074] 8. Wash plate 3.times.
with PBS/Tween. [1075] 9. Add 50 .mu.l of TMB (Zymed cat#002023),
allow reaction to proceed for 1 minute. [1076] 10. Stop reaction
with 50 .mu.l 2N H.sub.2SO.sub.4. [1077] 11. Read absorbance at 450
nm.
TABLE-US-00014 [1077] TABLE 13 Binding of mouse antibodies to human
IL-17 by ELISA EC 50 nMoles Mab Hu A/A Cyno A/A Hu A/F ML109-12H9
1.60 2.10 2.21 ML109-8A9 0.76 0.64 0.73 ML109-6C6 0.56 0.44 0.63
ML109-10G9 0.83 1.10 1.17 ML109-1D8 0.59 0.37 0.54 ML109-8B12 1.01
1.50 1.13 ML109-7D7 0.93 0.62 0.56 ML109-10F7 0.54 0.67 0.75
ML109-5C5 0.76 0.78 0.74 ML107-14F1 0.46 0.95 0.88
TABLE-US-00015 TABLE 14 Binding of humanized antibodies to human
IL-17 by ELISA EC50 in hIL17A Antibody Name ELISA (nM) IL17-h10F7.1
1.7 IL17-h10F7.2 >9398 IL17-h10F7.3 0.05 IL17-h10F7.4 0.04
IL17-h10F7.5 0.06 IL17-h10F7.6 0.05 IL17-h10F7.7 0.05 IL17-h10F7.8
3.9 IL17-h10F7.9 0.04 IL17-h10F7.10 0.04 IL17-h10F7.11 0.10
IL17-h10F7.12 0.14 IL17-h10F7.13 0.10 IL17-h10F7.14 0.13
IL17-h10F7.15 0.14 IL17-h10F7.16 0.09 IL17-h10F7.17 0.10
IL17-h5C5.1 1 IL17-h5C5.2 1.2 IL17-h5C5.3 1.37 IL17-h5C5.5 0.05
IL17-h5C5.6 0.11 IL17-h5C5.7 0.03 IL17-h5C5.8 0.07 IL17-h5C5.9 0.03
IL17-h5C5.10 0.05 IL17-h5C5.11 0.03 IL17-h5C5.12 0.23 IL17-h5C5.13
0.03 IL17-h5C5.14 0.03 IL17-h5C5.15 0.02 IL17-h5C5.16 0.02
IL17-h5C5.17 0.03 IL17-h5C5.18 0.03 IL17-h5C5.19 0.03 IL17-h5C5.20
0.03 IL17-h5C5.21 0.03
1.5.2: Assay for IL-17a and IL-17a/F Induced IL-6 Secretion in
Primary Human Foreskin Fibroblasts HS27
[1078] The human HS27 cell line (ATCC Accession # CRL-1634)
secretes IL-6 in response to IL-17. The IL-17-induced IL-6
secretion is inhibited by neutralizing anti-IL-17 antibodies (See,
e.g., J. Immunol., 155: 5483-5486 (1995); Cytokine, 9: 794-800
(1997)).
[1079] HS27 cells were maintained in assay medium: DMEM high
glucose medium (Gibco #11965) with 10% fetal bovine serum
(Gibco#26140), 4 mM L-glutamine, 1 mM sodium pyruvate, penicillin G
(100 U/500 ml) and streptomycin (100 .mu.g/500 ml). Cells were
grown in T150 flasks until they were about 80-90% confluent the day
of the assay. Human IL-17A (R&D Systems, #317-IL/CF), or
cynomolgous monkey (cyno) IL-17A (generated at Abbott) was
reconstituted in sterile PBS without Ca.sup.2+ and Mg.sup.2+ stored
frozen, freshly thawed for use and diluted to 240 pM (4.times.) or
4 nM(4.times.) for IL-17A/F in assay medium. Serial dilutions of
antibodies were made in a separate plate (4.times. concentrations),
mixed with equal volume of 240 pM (4.times.) of huIL-17 or
cynoIL-17A or 4 nM (4.times.) huIL-17A/F and incubated at
37.degree. C. for 1 hr. HS27 cells (typically about 20,000 cells in
50 .mu.l assay medium) were added to each well of a 96-well
flat-bottom tissue culture plate (Costar #3599), followed by
addition of 50 .mu.l of the pre-incubated antibody plus IL-17 mix.
The final concentration of human and cynoIL-17A was 60 pM. The
final concentration of human IL-17A/F was 1 nM. Cells were
incubated for about 24 hrs at 37.degree. C. The media supernatants
were then collected. The level of IL-17 neutralization was measured
by determination of IL-6 amounts in supernatant using a commercial
Meso Scale Discovery kit (cat# L411AKB-1) according to
manufacturer's instruction. IC50 values were obtained using
logarithm of antibody versus IL-6 amount variable slope fit.
1.5.3: Assay for IL-17 and TNF-.alpha. Induced IL-6 Secretion from
Murine Embryonic Fibroblast Cell Line (NIH3T3)
[1080] The murine NIH3T3 cell line (ATCC Accession # CRL-1658)
secretes IL-6 in response to murine, rat, or rabbit IL-17A and
murine TNF.alpha. (R&D Systems, Cat#410-MT). The IL-17 induced
IL-6 secretion is inhibited by neutralizing anti-IL-17
antibodies.
[1081] NIH3T3 cells were maintained in assay medium: DMEM
(Invitrogen Cat#11965-092) with 10% fetal bovine serum
(Gibco#26140-079), 1% Non Essential Amino Acids, 2 mM L-glutamine,
1 mM sodium pyruvate, penicillin G (100 U/500 ml), and streptomycin
(100 .mu.g/500 ml). Cells were grown in T150 flasks until they were
about 80-90% confluent the day of the assay. Rat IL17A (Prospec
bio, Cat# CYT-542) was reconstituted in sterile PBS, without Ca2+
and Mg2+, with 0.1% BSA, aliquoted and stored frozen at 100
.mu.g/mL. Rabbit IL17A (Abbott, A-1239293.0) was aliquoted and
stored frozen at 260 .mu.g/mL. Murine TNF-.alpha. was reconstituted
in 0.1% BSA/PBS without Ca.sup.2+ and Mg.sup.2+ at a concentration
of 10 .mu.g/mL, aliquoted, and stored frozen. Freshly thawed IL-17
antibodies were diluted to 200 .mu.g/ml (4.times.) in assay medium.
Serial dilutions of antibodies were made in a separate plate
(4.times. concentrations), mixed with equal volume of 40 ng/ml
(4.times.) murine or rat IL-17A or 100 ng/mL rabbit IL-17A, and
incubated at 37.degree. C. for 1 hr.
[1082] NIH3T3 cells (typically about 400,000 cells in 50 .mu.l
assay medium) were added to each well of a 96-well flat-bottom
tissue culture plate (Costar #3599), followed by addition of 50
.mu.l of the pre-incubated antibody plus IL-17 mix. Mu TNF-.alpha.
at 5.5 ng/mL (10.times.) was added in 11 .mu.l of media to each
well. The final concentration of IL-17A was 10 ng/ml for murine and
rat and 25 ng/mL for rabbit. The final concentration for mu
TNF.alpha. was 0.55 ng/mL. Cells were incubated for about 24 hrs at
37.degree. C. The media supernatants were then collected. The level
of IL-17 neutralization was measured by determination of IL-6
amounts in supernatant using a commercial Meso Scale Discovery kit
(cat# K112AKA-4) according to manufacturer's instruction. IC50
values were obtained using logarithm of antibody versus IL-6 amount
variable slope fit.
1.5.4: Affinity Measurement of IL-17 Antibodies by Surface Plasmon
Resonance
[1083] The binding of antibodies to purified recombinant human,
cyno, rat, mouse, rabbit IL-17 and human IL-17A/F were determined
by surface plasmon resonance-based measurements with a Biacore.RTM.
3000 instrument (Biacore.RTM. AB, Uppsala, Sweden) using running
HBS-EP (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, and 0.005%
surfactant P20) at 25.degree. C. All chemicals were obtained from
Biacore.RTM. AB (Uppsala, Sweden) or otherwise from a different
source as described in the text. Approximately 5000 RU of goat
anti-mouse or anti-human IgG (Fc.gamma.) fragment specific
polyclonal antibody (Pierce Biotechnology Inc, Rockford, Ill.)
diluted in 10 mM sodium acetate (pH 4.5) was directly immobilized
across a CM5 research grade biosensor chip using a standard amine
coupling kit according to manufacturer's instructions and
procedures at 25 .mu.g/ml. Unreacted moieties on the biosensor
surface were blocked with ethanolamine Modified carboxymethyl
dextran surface in flowcell 2 and 4 was used as a reaction surface.
Unmodified carboxymethyl dextran without goat anti-mouse or
anti-human IgG in flow cell 1 and 3 was used as the reference
surface. For kinetic analysis, rate equations derived from the 1:1
Langmuir binding model were fitted simultaneously to association
and dissociation phases of all eight injections (using global fit
analysis) with the use of Biaevaluation 4.0.1 software. Purified
antibodies were diluted in HEPES-buffered saline for capture across
goat anti-mouse or anti-human IgG specific reaction surfaces.
Antibodies to be captured as a ligand (25 .mu.g/ml) were injected
over reaction matrices at a flow rate of 5 .mu.l/min. The
association and dissociation rate constants, kon (unit M-1s-1) and
koff (unit s-1) were determined under a continuous flow rate of 25
.mu.l/min. Rate constants were derived by making kinetic binding
measurements at ten different antigen concentrations ranging from
10 nM to 200 nM. The equilibrium dissociation constant (unit M) of
the reaction between antibodies and recombinant purified IL-17
antigens was then calculated from the kinetic rate constants by the
following formula: K.sub.D=koff/kon. Binding was recorded as a
function of time and kinetic rate constants are calculated. In this
assay, on-rates as fast as 10.sup.6 M.sup.-1s.sup.-1 and off-rates
as slow as 10.sup.-6 s.sup.-1 can be measured.
1.5.5: Neutralizing Potency of Mouse Anti-Human IL-17
Antibodies
[1084] Potency of anti-IL-17 antibodies was assessed using
IL-17-driven IL-6 production in HS27 cells for human and cyno
antigens or IL-17 (according to the assay described above, Example
1.5.2.). The table below summarizes potencies to human IL-17A.
TABLE-US-00016 TABLE 15 Hybridoma-derived Potency (pM) anti-IL-17
MAb Hu IL-17A 1D8.4B10 62 10G9.3B11 131 6C6.3B4 36 12H9.3B5 937
8A9.5C7 415 8B12.4H2 137 7D7.3C5 81 5C5.3A8 23 10F7.3D1 54
14F1.5F4.3A7 450
1.5.6: Neutralizing Potency of Humanized IL-17 Antibodies of the
10F7 Lineage
[1085] Potency of the humanized 10F7 antibodies was assessed using
IL-17-driven IL-6 production in HS27 cells for human and cyno
antigens (assay described above, Example 1.5.2) or IL-17 and
TNF-.alpha.-driven IL-6 production in NIH3T3 cells (assay described
above, Example 1.5.3) for rat, mouse, and rabbit antigens. The
table below summarizes potencies to human, cyno and rat IL-17A.
Only active humanized antibodies were tested for their
potencies.
TABLE-US-00017 TABLE 16 Potency (pM) Humanized Hu Cyno Rat
Anti-IL-17 MAb IL-17A IL-17A IL-17A h10F7.4 117 ND ND h10F7.6 264
313 ND h10F7.9 293 ND ND h10F7.11 326 622 ND h10F7.13 242 429 ND
h10F7.15 309 499 ND h10F7.17 510 293 9118 m10F7hIgG1 40 79 ND Note:
m10FhIgG1 is a chimeric antibody containing the murine 10F7
variable domains. ND: not determined.
1.5.7: Neutralizing Potency of Humanized IL-17 Antibodies of the
5C5 Lineage
[1086] Potency of the humanized 5C5 antibodies was assessed using
IL-17-driven IL-6 production in HS27 cells for human and cyno
antigens (assay described above, Example 1.5.2) or IL-17 and
TNF-.alpha.-driven IL-6 production in NIH3T3 cells for rat, mouse
and rabbit antigens (assay described above, Example 1.5.3). The
table below summarizes potencies to human, cyno, and rat IL-17A.
Only active humanized antibodies were tested for their
potencies.
TABLE-US-00018 TABLE 17 Humanized Potency (pM) Anti-IL-17 Hu Cyno
Rat MAb IL-17A IL-17A IL-17A h5C5.2 6 11 ND h5C5.3 5 12 2519 h5C5.8
17 20 13080 h5C5.9 14 19 5678 h5C5.11 115 90 ND h5C5.14 18 16 ND
h5C5.15 13 12 ND h5C5.19 11 9 ND h5C5.20 12 13 ND h5C5.21 9 9 ND
m5C5.hIgG1 11 ND 4911 Note: m5C5.hIgG1 is a chimeric antibody
containing the murine 5C5 variable domains. ND: not determined
1.5.8: Affinity Measurement of IL-17 Antibodies by Surface Plasmon
Resonance
[1087] The binding of antibodies to purified recombinant human
(HuIL-17A), cyno (Cyno IL-17A), rat (RatIL-17A), mouse (MuIL-17A),
rabbit IL-17 (RabIL-17A), and human IL-17A/F (HuIL-17A/F) were
determined by surface plasmon resonance-based measurements with a
Biacore.RTM. 3000 instrument (Biacore.RTM. AB, Uppsala, Sweden)
using running HBS-EP (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA,
and 0.005% surfactant P20) at 25.degree. C. as described above
(Example 1.5.4). The tables below show the affinity measurements
for selected mouse monoclonal and humanized anti-human IL-17A
antibodies.
TABLE-US-00019 TABLE 18 IL-17 Antigen Mouse Antibody HuIL-17A
HuIL-17A/F Cyno IL-17A RatIL-17A MuIL-17A RabIL-17A 10F7 (M)
6.26E-11 6.73E-10 6.84E-11 1.97E-10 2.21E-10 ND Kon (1/Ms) 2.54E+06
1.02E+06 2.12E+06 3.17E+06 1.60E+06 Koff (1/s) 1.59E-04 6.86E-04
1.45E-04 6.23E-04 3.54E-04 6C6 (M) 1.87E-11 1.89E-10 4.25E-11
1.42E-10 3.57E-10 ND Kon (1/Ms) 3.63E+06 1.94E+06 2.73E+06 4.95E+06
2.44E+06 Koff (1/s) 6.78E-05 3.66E-04 1.16E-04 7.04E-04
8.70E-04
TABLE-US-00020 TABLE 19 Humanized IL-17 Antigen Antibody HuIL-17A
HuIL-17A/F Cyno IL-17A RatIL-17A MuIL-17A RabIL-17A h5C5.8 (VH.1-
3.93E-10 1.28E-09 2.17E-10 1.42E-08 1.86E-09 ND Vk.1a) (M) Kon
(1/Ms) 1.13E+06 9.48E+05 1.04E+06 7.74E+05 2.85E+05 Koff (1/s)
4.44E-04 1.21E-03 2.26E-04 1.10E-02 5.31E-04 h5C5.9 (VH.1b-
4.40E-10 1.21E-09 2.28E-10 8.48E-09 2.09E-09 ND Vk.1a) (M) Kon
(1/Ms) 1.02E+06 9.50E+05 1.05E+06 1.13E+06 2.79E+05 Koff (1/s)
4.49E-04 1.15E-03 2.39E-04 9.58E-03 5.82E-04 h5C5.14 (VH.1-
3.63E-10 1.11E-09 2.45E-10 5.56E-09 1.86E-09 ND Vk.3a) (M) Kon
(1/Ms) 1.30E+06 1.07E+06 9.34E+05 7.16E+05 2.66E+05 Koff (1/s)
4.72E-04 1.19E-03 2.29E-04 3.98E-03 4.94E-04 h5C5.15 (VH.1b-
4.58E-10 1.43E-09 3.58E-10 5.80E-09 2.83E-09 ND Vk.3a) (M) Kon
(1/Ms) 9.54E+05 7.81E+05 6.03E+05 6.21E+05 1.97E+05 Koff (1/s)
4.37E-04 1.12E-03 2.16E-04 3.60E-03 5.57E-04 h5C5.18 (VH.1b-
5.21E-10 1.31E-09 4.37E-10 9.15E-09 2.91E-09 ND Vk.1c) (M) Kon
(1/Ms) 9.47E+05 7.72E+05 5.81E+05 4.60E+05 1.72E+05 Koff (1/s)
4.93E-04 1.01E-03 2.54E-04 4.21E-03 5.00E-04 h5C5.21 (VH.1b-
7.29E-10 1.39E-09 4.06E-10 5.97E-09 3.06E-09 ND Vk.3c) (M) Kon
(1/Ms) 5.42E+05 8.01E+05 5.91E+05 4.02E+05 1.71E+05 Koff (1/s)
3.95E-04 1.11E-03 2.40E-04 2.40E-03 5.24E-04 h10F7.6 (VH.1a-
3.02E-10 1.14E-09 2.17E-10 9.19E-10 9.80E-10 ND Vk.1a) (M) Kon
(1/Ms) 6.12E+05 6.09E+05 7.11E+05 4.94E+05 3.92E+05 Koff (1/s)
1.85E-04 6.96E-04 1.54E-04 4.54E-04 3.84E-04 h10F7.11 (VH.1a-
5.24E-10 2.11E-09 7.73E-10 3.77E-09 2.21E-09 ND Vk.2a) (M) Kon
(1/Ms) 2.71E+05 4.08E+05 4.54E+05 1.77E+05 2.36E+05 Koff (1/s)
1.42E-04 8.60E-04 3.51E-04 6.67E-04 5.22E-04 ND: Not
determined.
Example 2
Fully Human Anti-IL-17 Antibodies
[1088] 2.1 Generation of Affinity-Matured Fully Human IL-17
Antibodies from IL17-TN-L7-G9 and from IL17-K7-B6.
[1089] Fully human anti hIL-17 monoclonal antibodies IL17-TN-L7-G9,
IL-17-TN-L7-A7, IL-TN-L7-C8, IL17-TN-K7-B6, and IL17-LN-K9-F5 were
isolated by PROfusion mRNA display technology from human antibody
libraries by their ability to bind human IL-17 proteins. The amino
acid sequences of the variable heavy (VH) and variable light (VL)
chains were determined from DNA sequencing. See, Table 6.
[1090] The IL17-TN-L7-G9 human antibody to human IL-17 was
subsequently affinity matured by PROfusion mRNA display technology.
One light chain library was constructed to contain limited
mutagenesis at the following residues: CDRL1: 26, 26, 30, 31, 32,
34; CDRL2: 50, 51, 52, 53; CDRL3: 89, 90, 94, 95a, 95b, 96 (Kabat
numbering). This library also contained G3V framework germline
back-mutations as well as binary diversities at position 48(I/M),
64(G/V), 66(K/Q), and 100(S/T) to allow for framework germ-lining
during library selections. Two heavy chain libraries were made to
contain limited mutagenesis in CDRH1 and CDRH2 at residues 30, 31,
32, 50, 52, 52a, 55, 56, 57, 58, and 60 (Kabat numbering) or in
CDRH3 at residues 95-100, 100a-100g, and 102. All three libraries
were selected separately for the ability to bind IL-17 in the
presence of decreasing concentrations of biotinylated human IL-17,
cyno IL-17, or human IL-17A/F and recovered by magnetic
streptavidin particles (Invitrogen). All mutated CDR sequences were
then recombined into one library and the recombined library is
subjected to more stringent selection conditions before individual
antibodies are identified.
[1091] The table below provides a list of amino acid sequences of
VH and VL of the fully human G9 antibody which were subjected to
the affinity maturation selection protocol. Amino acid residues of
individual CDRs of each VH and VL sequence are indicated in
bold.
TABLE-US-00021 TABLE 20 List of Amino Acid Sequences of VH and VL
regions of fully human IL17-TN-L7-G9. SEQ ID Protein Sequence No.
region 123456789012345678901234567890 77 VH G9
EVQLLESGGGVVQPGRSLRLSCAASGFIFS NYGMHWVRQAPGKGLEWVAVISYDGSNKYY
ADSVKGRFTISRDNSKNTLYLEMNSLRPED TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS
VH G9 Residues 31-35 NYGMN CDR-H1 of SEQ-ID NO: 77 VH G9 Residues
50-66 VISYDGSNKYYADSVKG CDR-H2 of SEQ-ID NO: 77 VH G9 Residues
99-113 VGASGDYYYSYGLDV CDR-H3 of SEQ-ID NO: 77 78 VL G9
QSVLTQPPSASGTPGQTVSISCSGSNSNIG SHSVNWYQQVPGAAPKLLMYGIGQRPSGVP
DRFSVSQSGTSASLAISGLQSEDEADYYCA TWDDSLGGYVFGSGTKVTVLG VL G9 Residues
23-35 SGSNSNIGSHSVN CDR-L1 of SEQ-ID NO: 78 VL G9 Residues 51-57
GIGQRPS CDR-L2 of SEQ-ID NO: 78 VL G9 Residues 90-100 ATWDDSLGGYV
CDR-L3 of SEQ-ID NO: 78
In Table 20, the amino acid sequence for VL G9 (SEQ ID NO:78)
contains the G3V framework germline backmutation, mentioned above,
of the VL (SEQ ID NO:41) of fully human IL17-TN-L7-G9 shown in
Table 6.
[1092] The table below provides a list of amino acid sequences of
VH and VL regions of affinity matured fully human IL-17 antibodies
derived from IL17-TN-L7-G9. Amino acid residues of individual CDRs
of each VH and VL sequence are indicated in bold.
TABLE-US-00022 TABLE 21 List of amino acid sequences of affinity
matured G9 VH/VL variants SEQ ID Sequence NO: Protein region
123456789012345678901234567890 79 G9 VH #1 Same as Parental
EVQLLESGGGVVQPGRSLRLSCAASGFIFS NYGMHWVRQAPGKGLEWVAVISYDGSNKYY
ADSVKGRFTISRDNSKNTLYLEMNSLRPED TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS
80 G9 VL #1 QSVLTQPPSASGTPGQTVSISCSGRQSNIGR
HYVDWYQQVPGAAPKLLMYYDSIRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGSGTKVTVL 81 G9 VH #2 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISYDGSNAYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 82 G9 VL #2 Same as VL #1
QSVLTQPPSASGTPGQTVSISCSGRQSNIGR HYVDWYQQVPGAAPKLLMYYDSIRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGSGTKVTVL 83 G9 VH #3
EVQLLESGGGVVQPGRSLRLSCAASGFIFGN YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
SVKGRFTISRDNSKNTLYLEMNSPRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 84
G9 VL #3 QSVLTQPPSASGTPGQTVSISCSGRQSNIGR
HYVDWYEQVPGAAPKLLMYYDSIRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCGMWD
DSLAGYVFGSGTKVTVL 85 G9 VH #4 EVQLLESGGGVVQPGRSLRLSCAASGFIFRN
YGMHWVRQAPGKGLEWVAVIAYDGSRQYYSD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDLWGQGTTVTVSS 86 G9 VL #4
QSVLTQPPSASGTPGQTVSISCSGRQSNIGR HYVDWYQQVPGAAPKLLMYYDSIRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCGMWD DSLAGYVFGSGTKVTVL 87 G9 VH #5
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN HGMHWVRQAPGKGLEWVAVIASDGSNKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGRGTTVTVSS 88
G9 VL #5 Same as VL #1 QSVLTQPPSASGTPGQTVSISCSGRQSNIGR
HYVDWYQQVPGAAPKLLMYYDSIRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGSGTKVTVL 89 G9 VH #6 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD SVKGRFTISKDNSKNTLHLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 90 G9 VL #6
QSVLTQPPSASGTPGQTVSISCSGRQSNIGR HYVDWYQQVPGAAPKLLMYYDSIRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGTGTKVTVL 100 G9 VH #7 Same
as Parental EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 101 G9 VL #7 Same as VL #6
QSVLTQPPSASGTPGQTVSISCSGRQSNIGR HYVDWYQQVPGAAPKLLMYYDSIRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGTGTKVTVL 102 G9 VH #8
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPKDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 103
G9 VL #8 Same as VL #6 QSVLTQPPSASGTPGQTVSISCSGRQSNIGR
HYVDWYQQVPGAAPKLLMYYDSIRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGTGTKVTVL 104 G9 VH #9 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISFDGSNKYYPD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 105 G9 VL #9
QSVLTQPPSASGTPGQTVSISCSGRQSNIGR HYVDWYQQVPGAAPKLLMYYDSIRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVLGTGTKVTVL 106 G9 VH #10
EVQLLESGGGVVQPGRSLRLSCAASGFIFTN YGMHWVRQAPGKGLEWVAVISFDGSNRYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 107
G9 VL #10 Same as VL #6 QSVLTQPPSASGTPGQTVSISCSGRQSNIGR
HYVDWYQQVPGAAPKLLMYYDSIRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGTGTKVTVL 108 G9 VH #11 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISYDGRNKYYED SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 109 G9 VL #11
QSVLTQPPSASGTPGQTVSISCSGRQSNIGR HYVDWYQQVPGAAPKLLMYYDSIRPSGVPDR
FSGSQSGTSASLVISGLQSEDEADYYCATWD DSLGGYVFGTGTKVTVL 110 G9 VH #12
EVQLLESGGGVVQPGRSLRLSCAASGFIFRN YGMHWVRQAPGKGLEWVAVISYDGRNKYYTD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 111
G9 VL #12 Same as VL #1 QSVLTQPPSASGTPGQTVSISCSGRQSNIGR
HYVDWYQQVPGAAPKLLMYYDSIRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGSGTKVTVL 112 G9 VH #13 EVQLLESGGGVVQPGRSLRLSCAASGFIFRN
YGMHWVRQAPGKGLEWVAVISYDGTNTYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 113 G9 VL #13 Same as VL #6
QSVLTQPPSASGTPGQTVSISCSGRQSNIGR HYVDWYQQVPGAAPKLLMYYDSIRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGTGTKVTVL 114 G9 VH #14
EVQLLESGGGVVQPGRSLRLSCAASGFIFRN YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 115
G9 VL #14 Same as VL #6 QSVLTQPPSASGTPGQTVSISCSGRQSNIGR
HYVDWYQQVPGAAPKLLMYYDSIRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGTGTKVTVL 116 G9 VH #15 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISNDGSNTYYAD SVKGRFTISRDNSENTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 117 G9 VL #15
QSVLTQPPSASGTPGQTVSISCSGRQSNIGR HYVDWYQQVPGAAPKLLMYYDSIRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DWLAGYVFGTGTKVTVL 118 G9 VH #16
Same as Parental EVQLLESGGGVVQPGRSLRLSCAASGFIFS
NYGMHWVRQAPGKGLEWVAVISYDGSNKYY ADSVKGRFTISRDNSKNTLYLEMNSLRPED
TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS 119 G9 VL #16
QSVLTQPPSASGTPGQTVSISCSGRQSNIGR HYVDWYQQVPGAAPKLLMYYDSIRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DFLAGYVFGTGTKVTVL 120 G9 VH #17
Same as Parental EVQLLESGGGVVQPGRSLRLSCAASGFIFS
NYGMHWVRQAPGKGLEWVAVISYDGSNKYY ADSVKGRFTISRDNSKNTLYLEMNSLRPED
TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS 121 G9 VL #17
QSVLSQPPSASGTPGQTVSISCSGTNSNIGR HAVDWYQQVPGAAPKLLMYYDSIRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGTGTKVTVL 122 G9 VH #18
EVQLLESGGGVVQPGRSLRLSCAASGFIFRN NGMHWVRQAPGKGLEWVAVISRDGSNKYYAD
SVKGRFTISRDNSKNTLYLEMNRLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 123
G9 VL #18 QSVLTQPPSASGTPGQTVSISCSGRQSNIGN
HYVDWYQQVPGAAPKLLIYGDVIRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DWLAGYVFGTGTKVTVL 124 G9 VH #19 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISYDGSNSYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 125 G9 VL #19 Same as VL #18
EVQLLESGGGVVQPGRSLRLASCAASGFIFRN NGMHWVRQAPGKGLEWVAVISRDGSNKYYAD
SVKGRFTISRDNSKNTLYLEMNRLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 126
G9 VH #20 EVQLLESGGGVVQPGRSLRLSCAASGFIFSD
YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 127 G9 VL #20
QSVLTQPPSASGTPGQTVSISCSGRQSNIGN HYVDWYQQVPGAAPKLLIYGDVIRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DWLAGYVFGSGTKVTVL 128 G9 VH #21
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVISNDGSNKYYSD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 129
G9 VL #21 QSGLTQPPSASGTPGQTVSISCSGSNSNIGR
HPVDWYQQVPGAAPKLLIYYDDQRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGTGTKVTVL 130 G9 VH #22 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISNDGGNKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 131 G9 VL #22
QSVLTQPPSASGTPGQTVSISCSGSNSNIGR HPVDWYQQVPGAAPKLLIYYDDQRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGTGTKVTVL 132 G9 VH #23
Same as VH #14 EVQLLESGGGVVQPGRSLRLSCAASGFIFRN
YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS
133 G9 VL #23 Same as VL #22 QSVLTQPPSASGTPGQTVSISCSGSNSNIGR
HPVDWYQQVPGAAPKLLIYYDDQRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGTGTKVTVL 134 G9 VH #24 EVQLLESGGGVVQPGRSLRLSCAASGFIFGN
YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 135 G9 VL #24 Same as VL #22
QSVLTQPPSASGTPGQTVSISCSGSNSNIGR HPVDWYQQVPGAAPKLLIYYDDQRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGTGTKVTVL 136 G9 VH #25
EVQLLESGGGVVQPGRSLRLSCAASGFIFNN HGMHWVRQAPGKGLEWVAVISYDGWNKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 137
G9 VL #25 Same as VL #22 QSVLTQPPSASGTPGQTVSISCSGSNSNIGR
HPVDWYQQVPGAAPKLLIYYDDQRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGTGTKVTVL 138 G9 VH #26 EVQLLESGGGVVQPGRSLRLSCGASGFIFRN
YGMHWVRQAPGKGLEWVAVISYDGTSKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 139 G9 VL #26
QSVLTQPPSASGTPGQTVSISCSGSNSNIGR HPVDWYQQVPGAAPKLLIYYDDQRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGSGTKVTVL 140 G9 VH #27
EVQLLESGGGVVQPGRSLRLSCAASGFIFPN YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 141
G9 VL #27 Same as VL #22 QSVLTQPPSASGTPGQTVSISCSGSNSNIGR
HPVDWYQQVPGAAPKLLIYYDDQRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGTGTKVTVL 142 G9 VH #28 Same as Parental
EVQLLESGGGVVQPGRSLRLSCAASGFIFS NYGMHWVRQAPGKGLEWVAVISYDGSNKYY
ADSVKGRFTISRDNSKNTLYLEMNSLRPED TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS
143 G9 VL #28 QSVLTQPPSASGTPGQTVSISCSGRPSNIGS
HAVDWYQQVPGAAPKLLMYYTDLRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DHLAGYVFGTGTKVTVL 144 G9 VH #29 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISCDGRNKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGNYYYSYGLDVWGQGTTVTVSS 145 G9 VL #29
QSVLTQPPSASGTPGQTVSISCSGRNSNIGY HTVDWYQQVPGAAPKLLMYYSVQRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCASWD DSLGGYVFGTGTKVTVL 146 G9 VH #30
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVISYDGSNRYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 147
G9 VL #30 QSVLTQPPSASGTPGQTVSISCSGRNSNIGN
HPVDWYQQVPGAAPKLLMYYTGIRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCASWD
DRLGGYVFGTGTKVTVL 148 G9 VH #31 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVIAYDGSNKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 149 G9 VL #31
QSVLTQPPSASGTPGQTVSISCSGGKSNIGS HPVDWYQQVPGAAPKLLMYYVGMRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLAGYVFGTGTKVTVL 150 G9 VH #32
Same as Parental EVQLLESGGGVVQPGRSLRLSCAASGFIFS
NYGMHWVRQAPGKGLEWVAVISYDGSNKYY ADSVKGRFTISRDNSKNTLYLEMNSLRPED
TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS 151 G9 VL #32
QSVLTQPPSASGTPGQTVSISCSGRNSNIGN HYVDWYQQVPGAAPKLLIYGTGSRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DWLAEYVFGTGTKVTVL 152 G9 VH #33
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN NGMHWVRQAPGKGLEWVAVISSDGSHGYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 153
G9 VL #33 QSVLTQPPSASGTPGQTVSISCSGRMSNIGY
HSVDWYQQVPGAAPKLLIYGISQRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DWLAGYVFGTGTKVTVL 154 G9 VH #34 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
NGMHWVRQAPGKGLEWVAVISYDGSNKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 155 G9 VL #34
QSVLTQPPSASGTPGQTVSISCSGRSSNIGN HSVDWYQQVPGAAPKLLMYGIGQRPSGVPDR
FSVSQSGTSASLAISGLQSEDEADYYCATWD DWLGGYVFGTGTKVTVL 156 G9 VH #35
EVQLLESGGGVVQPGRSLRLSCAASGFIFNN HGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEYTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 157
G9 VL #35 QSVLTQPPSASGTPGQTVSISCSGRSSNIGC
HAVDWYQQVPGAAPKLLMYGISERPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DRLAAYVFGTGTKVTVL 158 G9 VH #36 Same as Parental
EVQLLESGGGVVQPGRSLRLSCAASGFIFS NYGMHWVRQAPGKGLEWVAVISYDGSNKYY
ADSVKGRFTISRDNSKNTLYLEMNSLRPED TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS
159 G9 VL #36 QSVLTQPPSASGTPGQTVSISCSGRISNIGN
HAVDWYQQVPGAAPKLLMYGIGQRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DWLAGYVFGSGTKVTVL 160 G9 VH #37 EVQLLESGGGVVQPGRSLRLSCAASGFIFSY
HGMHWVRQAPGKGLEWVAVISYDGSNKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 161 G9 VL #37
QSVLTQPPSASGTPGQTVSISCSGRNSNIGN HSVYWYQQVPGAAPKLLMYESDQRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DWLGVYVFGSGTKVTVL 162 G9 VH #38
Same as Parental EVQLLESGGGVVQPGRSLRLSCAASGFIFS
NYGMHWVRQAPGKGLEWVAVISYDGSNKYY ADSVKGRFTISRDNSKNTLYLEMNSLRPED
TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS 163 G9 VL #38
QSVLTQPPSASGTPGQTVSISCSGRRSNIGY HSVDWYQQVPGAAPKLLMYGDDQRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DWLGGYVFGTGTKVTVL 164 G9 VH #39
EVQLLESGGGVVQPGRSLRLSCAASGFIFNN YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 165
G9 VL #39 QSVLTQPPSASGTPGQTVSISCSGRNSNIGN
HSVDWYQQVPGAAPKLLMYWSGHRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DRLGGYVFGTGTKVTVL 166 G9 VH #40 EVQLLESGGGVVQPGRSLRLSCAASGFIFRN
YGMHWVRQAPGKGLEWVAVISRDGSGKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 167 G9 VL #40
QSVLTQPPSASGTPGQTVSISCSGTNSNIGN HSVDWYQQVPGAAPKLLMYGVGERPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DRLGGYVFGTGTKVTVL 168 G9 VH #41
Same as VH #39 EVQLLESGGGVVQPGRSLRLSCAASGFIFNN
YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 169 G9 VL #41
QSVLTQPPSASGTPGQTVSISCSGRNSNIGS HSVDWYQQVPGAAPKLLMYYNGQRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DWLGGYVFGSGTKVTVL 170 G9 VH #42
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVISYDGSKQYYAD
SVKGRFTISRDNSKDTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 171
G9 VL #42 QSVLTQPPSASGTPGQTVSISCSGRNSNIGS
HSVDWYQQVPGAAPKLLMYYNGHRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DWLGGYVFGTGTKVTVL 172 G9 VH #43 EVQLLESGGGVVQPGRSLRLSCAASGFIFGH
YGMHWVRQAPGKGLEWVAVISYDGRNQYYVD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 173 G9 VL #43
QSVLTQPPSASGTPGQTVSISCSGRTSNIGR HFVDWYQQVPGAAPKLLMYYGDMRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGSGTKVTVL 174 G9 VH #44
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVISYDGTNKYYRD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGALGDYYYTYGLDVWGQGTTVTVSS 175
G9 VL #44 QSVLTQPPSASGTPGQTVSISCSGSKSNIGR
HSVDWYQQVPGAAPKLLMYYSDMRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGTGTKVTVL 176 G9 VH #45 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISYDGSRGYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 177 G9 VL #45
QSVLTQPPSASGTPGQTVSISCSGSRSNIGR HCVDWYQQVPGAAPKLLIYFDDLRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DRLAGYVFGSGTKVTVL 178 G9 VH #46
EVQLLESGGGMVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVIAYDGSRKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 179
G9 VL #46 QSVLTQPPSASGTPGQTVSISCSGSHSNIGR
HSVDWYQQVPGAAPKLLMYFTDYRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DLLAGYVFGTGTKVTVL 180 G9 VH #47 EVQLLESGGGVVQPGRSLRLSCAASGFIFNN
YGMHWVRQAPGKGLEWVAVISMDGSRKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 181 G9 VL #47
QSVLTQPPSASGTPGQTVSISCSGSPSNIGR HYVHWYQQVPGAAPKLLMYFTDQRPSGVPDR
FSGSQSGTSASLAISGLQSEDETDYYCATWD DSLGGYVFGSGTKVTVL 182 G9 VH #48
EVQLLESGGGVVQPGRSLRLSCAASGFIFTN YGMHWVRQAPGKGLEWVAVISSDGSNKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 183
G9 VL #48 QSVLTQPPSASGTPGQTVSISCSGSNSNIGS
HYVDWYQQVPGAAPKLLMYYGGYRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGTGTKVTVL 184 G9 VH #49 EVQLLESGGGVVQPGRSLRLSCAASGFIFRD
YGMHWVRQAPGKGLEWVAVISNDGWNKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 185 G9 VL #49
QSVLTQPPSASGTPGQTVSISCSGSKSNIGS HAVDWYQQVPGAAPKLLMYYGGYRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGSGTKVTVL 186 G9 VH #50
Same as Parental EVQLLESGGGVVQPGRSLRLSCAASGFIFS
NYGMHWVRQAPGKGLEWVAVISYDGSNKYY ADSVKGRFTISRDNSKNTLYLEMNSLRPED
TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS 187 G9 VL #50
QSVLTQPPSASGTPGQTVSISCSGSNSNIGG HYVDWYQQVPGAAPKLLMYYTGYRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGAYVFGTGTKVTVL 188 G9 VH #51
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
SVKGRFTISRDNPKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 189
G9 VL #51 QSVLTQPPSASGTPGQTVSISCSGTNSNIGS
HSVDWYQQVPGAAPKLLMYYTGYRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGSGTKVTVL 190 G9 VH #52 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISSDGSNKYYSD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 191 G9 VL #52
QSVLTQPPSASRTPGQTVSISCSGNNSNIGT HYVDWYQQVPGAAPKLLMYYTGYRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGSGTKVTVL 192 G9 VH #53
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYRAKVGASGDYYYSYGLDVWGQGTTVTVSS 193
G9 VL #53 QSVLTQPPSASGTPGQTVSISCSGTNSNIGR
HYVDWYQQVPGAAPKLLMYFGGYRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGSGTKVTVL 194 G9 VH #54 Same as Parental
EVQLLESGGGVVQPGRSLRLSCAASGFIFS NYGMHWVRQAPGKGLEWVAVISYDGSNKYY
ADSVKGRFTISRDNSKNTLYLEMNSLRPED TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS
195 G9 VL #54 QSVLTQPPSASGTPGQTVSISCSGSHSNIGR
HPVDWYQQVPGAAPKLLMYYGGYRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGSGTKVTVL 196 G9 VH #55 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGMEWVAVISYDGSNKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDAWGQGTTVTVSS 197 G9 VL #55
QSVLTQPPSASGTPGQTVSISCSGSHSNIGS HPVDWYQQVPGAAPKLLMYYSVYRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGSGTKVTVL 198 G9 VH #56
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVIAYDGRNKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 199
G9 VL #56 QSVLTQPPSASGTPGQTVSISCSGNHSNIGN
HFVDWYQQVPGAAPKLLMYYSWYRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGSGTKVTVL 200 G9 VH #57 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKLGASGDYYYSYGLDVWGQGTTVTVSS 201 G9 VL #57
QSVLTQPPSASGTPGQTVSISCSGSHSNIGR HYVDWYQQVPGAAPKLLMYYTGERPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGTGTKVTVL 202 G9 VH #58
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVISSDGSNKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKLGASGPYYYTYGLDVWGQGTTVTVSS 203
G9 VL #58 QSVLTQPPSASGTPGQTVSISCSGSNSNIGR
HTVDWYQQVPGAAPKLLMYYTGMRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DKLGGYDFGSGTKVTVL 204 G9 VH #59 EVQLLESGGGVVQPGRSLRLSCAASGFIFSD
YGMHWVRQAPGKGLEWVAVISYDGSRKYYED SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 205 G9 VL #59
QSVLTQPPSASGTPGQTVSISCSGTKSNIGR HYVDWYQQVPGAAPKLLMYWSGERPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGTGTKVTVL 206 G9 VH #60
EVQLLESGGGVVQPGRSLRLSCAASGFIFSD YGMHWVRQAPGKGLEWVAVIAFDGSRKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 207
G9 VL #60 QSVLTQPPSASGTPGQTVSISCSGRPSNIGR
HSVDWYQQVPGAAPKLLMYYFGERPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGTGTKVTVL 208 G9 VH #61 Same as Parental
EVQLLESGGGVVQPGRSLRLSCAASGFIFS NYGMHWVRQAPGKGLEWVAVISYDGSNKYY
ADSVKGRFTISRDNSKNTLYLEMNSLRPED TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS
209 G9 VL #61 QSVLTQPPSASGTPGQTVSISCSGYDSNIGR
HSVDWYQQVPGAAPKLLMYYYGYRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DLLGGYVFGTGTKVTVL 210 G9 VH #62 Same as Parental
EVQLLESGGGVVQPGRSLRLSCAASGFIFS NYGMHWVRQAPGKGLEWVAVISYDGSNKYY
ADSVKGRFTISRDNSKNTLYLEMNSLRPED TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS
211 G9 VL #62 QSVLTQPPSASGTPGQTVSISCSGTISNIGR
HSVDWYQQVPGAAPKLLMYYNGHRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGTGTKVTVL 212 G9 VH #63 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISRDGSHKYYAD SVKGRFTISRGNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 213 G9 VL #63
QSVLTQPPSASGTPGQTVSISCSGRYSNIGR HAVDWYQQVPGAAPKLLMYYIGHRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGSGTKVTVL 214 G9 VH #64
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVISNDGHGKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 215
G9 VL #64 QSVLTQPPSASGTPGQTVSISCSGSNSNIGR
HAVDWYQQVPGAAPKLLMYYIGERPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGSGTKVTVL 216 G9 VH #65 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
NGMHWVRQAPGKGLEWVAVISYDGSKKYYAD SVKGRFTISRDNSYNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDFWGQGTTVTVSS 217 G9 VL #65
QSVLTQPPSASGTPGQTVSISCSGTTSNIGR HAVDWYQQVPGAAPKLLMYYNGWRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGSGTKVTVL 218 G9 VH #66
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVISFDGSRKYYVD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKLGASGDYYYSYGLDVWGQGTTVTVSS 219
G9 VL #66 QSVLTQPPSASGTPGQTVSISCSGKNSNIGN
HYVHWYQQVPGAAPKLLMYDFGQRPSGVPDR FSVSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGTGTKVTVL 220 G9 VH #67 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISSDGSKNYYVD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 221 G9 VL #67
QSVLTQPPSASGTPGQTVSISCSGTNSNIGN HFVHWYQQVPGAAPKLLMYDFSQRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DWLVGYVFGTGTKVTVL 222 G9 VH #68
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAQGKGLEWVAVISYDGSREYYGD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYCLDVWGQGTTVTVSS 223
G9 VL #68 QSMLTQPPSASGTPGQTVSISCSGRHSNIGS
HEVHWYQQVPGAAPKLLMYDFGQRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DWLGGYVFGTGTKVTVL 224 G9 VH #69 EVQLLESGGGVVQPGRSLRLSCAASGFIFSD
YGMHWVRQAPGKGLEWVAVISYDGCNQYYPD SVKQRFTISRDNSYNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 225 G9 VL #69
QSGLTQPPSASGTPGQTVSISCSGRTSNIGN HSVHWYQQVPGAAPKLLMYGFGVRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DWLGSYVFGTGTKVTVL 226 G9 VH #70
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVISNDGSNKYYTD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 227
G9 VL #70 QSVLTQPPSASGTPGQTVSISCSGKNSNIGR
HSVHWYQQVPGAAPKLLMYWFGTRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DRLGGYVFGTGTKVTVL 228 G9 VH #71 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISNDGSNKYYLD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 229 G9 VL #71
QSVLTQPPSASGTPGQTVSISCSGRNSNIGY
HPVHWYQQVPGAAPKLLIYDFGLRPSGVPDR FSGSQSGTSASLSISGLQSEDEADYYCATWD
DWLGGYVFGTGTKVTVL 230 G9 VH #72 EVQLLESGGGVVQPGRSLRLSCAASGFIFTN
YGMHWVRQAPGKGLEWVAVISHDGCNNYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGAYYYSYGLDVWGQGTTVTVSS 231 G9 VL #72
QSVLTQPPSASGTPGQTVSISCSGRNSNIGR HAVDWYQQVPGAAPKLLMYGIGQRPSGVPDR
FSVSQSGTSASLAISGLQSEDEADYYCATWD DWLGGYVFGTGTKVTVL 232 G9 VH #73
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVISYDGSNKYYGD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 233
G9 VL #73 QSVLTQPPSASGTPGQTVSISCSGTNSNIGS
HAVDWYQQVPGAAPKLLMYGIDQRPSGVPDR FSVSQSGTSASLAISGLQSEDEADYYCATWD
DWLAGYVFGTGTKVTVL 234 G9 VH #74 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISYDGTNKYYSD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 235 G9 VL #74
QSVLTQPPSASGTPGQTVSISCSGRNSNIGS HSVHWYQQVPGAAPKLLMYGIGERPSGVPDR
FSVSQSGTSASLAISGLQSEDEADYYCATWD DWLAGYVFGTGTKVTVL 236 G9 VH #75
Same as Parental EVQLLESGGGVVQPGRSLRLSCAASGFIFS
NYGMHWVRQAPGKGLEWVAVISYDGSNKYY ADSVKGRFTISRDNSKNTLYLEMNSLRPED
TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS 237 G9 VL #75
QSVLTQPPSASGTPGQTVSISCSGRNSNIGS HYVHWYQQVPGAAPKLLMYDFGLRPSGVPDR
FSVSQSGTSASLAISGLQSEDEADYYCATWD DMLGGYVFGTGTKVTVL 238 G9 VH #76
Same as Parental EVQLLESGGGVVQPGRSLRLSCAASGFIFS
NYGMHWVRQAPGKGLEWVAVISYDGSNKYY ADSVKGRFTISRDNSKNTLYLEMNSLRPED
TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS 239 G9 VL #76
QSVLTQPPTASGTPGQTVSISCSGRNSNIGN HSVHWYQQVPGAAPKLLMYDIGFRPSGVPDR
FSVSQSGTSASLAISGLQSEDEADYYCATWD DWLGGYVFGTGTKVTVL 240 G9 VH #77
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVISYDGSNKYYSD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 241
G9 VL #77 QSVLTQPPSASGTPGQTVSISCSGSHSNIGR
HPVDWYQQVPGAAPKLLMYDFGQRPSGVPDR FSVSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGTGTKVTVL 242 G9 VH #78 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISCDGSNKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 243 G9 VL #78
QSVLTQPPSASGTPGQTVSISCSGTNSNIGS HSVHWYQQVPGAAPKLLMYWFGERPSGVPDR
FSGSPSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGSGTKVTVL 244 G9 VH #79
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGGSGEYYYSPGLDVWGQGTTVTVSS 245
G9 VL #79 QSVLTQPPSASGTPGQTVSISCSGSNSNIGS
RSVTWYQQVPGAAPKLLMYWFGHRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLAEYVFGTGTKVTVL 246 G9 VH #80 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISYDGSKQYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 247 G9 VL #80
QSVLTQPPSASGTPGQTVSISCSGSNSNIGY HSVHWYQQVPGAAPKLLMYYFGNRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGTGTKVTVL 248 G9 VH #81
EVQLLESGGGVVQPGRSLRLSCAASGFIFRN YGMHWVRQAPGKGLEWVAVISYDGSNTYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGAYGDYYYSYGLDVWGQGTTVTVSS 249
G9 VL #81 QSVLTQPPSASGTPGQTVSISCSGTNSNIGS
HSVHWYQQVPGAAPKLLIYDFGDRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DRLGGYVFGTGTKVTVL 250 G9 VH #82 EVQLLESGGGVVQPGRSLRLSCAASGFIFRN
YGMHWVRQAPGKGLEWVAVISYDGRRNYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKLGAYGPYYYSYGLDVWGQGTMVTVSS 251 G9 VL #82
QSVLTQPPSASGTPGQTVSISCSGGNSNIGC HSVHWYQQVPGAAPKLLMYGMGYRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGSGTKVTVL 252 G9 VH #83
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVISYDGGNTYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGANGDYYYSYGLDVWGQGTTVTVSS 253
G9 VL #83 QSVLTQPPSASGTPGQTVSISCSGTNSNIGN
HAVNWYQQVPGAAPKLLMYGLGMRPSGVPDR FSDSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGTGTKVTVL 254 G9 VH #84 Same as VH #31
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVIAYDGSNKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 255
G9 VL #84 QSVLTQPPSASGTPGQTVSISCSGHNSNIGS
HTVNWYQQVPGAAPKLLMYEIGYRPSGVPDR FSVSQSGTSASLAISGLQSEDEADYYCATWD
DWLADYVFGSGTKVTVL 256 G9 VH #85 EVQLLESGGGVVQPGRSLRLSCAASGFIFRN
YGMHWVRQAPGKGLEWVAVIAYDGSRKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 257 G9 VL #85
QSVLTQPPSASGTPGQTVSISCSGSNSNIGS HDVTWYQQVPGAAPKLLMYDMWERPSGVPDR
FSVSQSGTSASLAISGLQSEDEADYYCATWD DWLGGYVFGSGTKVTVL 258 G9 VH #86
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN QGMHWVRQAPGKGLEWVAVIAYDGSKKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 259
G9 VL #86 QSVLTQPPSASGTPGQTVSISCSGSNSNIGS
HFVHWYQQVPGAAPKLLMYEYGWRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCAIWD
DRLVGYVFGSGTKVTVL 260 G9 VH #87 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVTVISYDGSNKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGAYGDYYYSYGLDGWGQGTTVTVSS 261 G9 VL #87
QSVLTQPPSASGTPGQTVSISCSGRQSNIGS HYVHWYQQVPGAAPKLLMYGIGERPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DRLVGYVFGSGTKVTVL 262 G9 VH #88
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN FGMHWVRQAPGKGLEWVAVISYDGSRKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGAYGDYYYSYGLDVWGQGTTVTVSS 263
G9 VL #88 QSVLTQPPSASGTPGQTVSISCSGSNSNIGV
HSVHWYQQVPGAAPKLLMYGIGQRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DRLAGYVFGTGTKVTVL 264 G9 VH #89 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISYDGRNKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDEWGQGTTVTVSS 265 G9 VL #89
QSVLTQPPSASGTPGQTVSISCSGRNSNIGN HYVHWYQQVPGAAPKLLMYSIGLRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DRLAGYVFGTGTKVTVL 266 G9 VH #90
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVIAADGRKKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGAWGDYYYTYGLDSWGQGTTVTVSS 267
G9 VL #90 QSVLTQPPSASGTPGQTVSISCSGRYSNIGN
HSVHWYRQVPGAAPKLLMYGIDYRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DWLGGYVFGTGTKVTVL 268 G9 VH #91 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVIGYDGYRKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 269 G9 VL #91
QSVLTQPPSASRTPGQTVPISCSGRHSNIGN HSVHWYQQVPGAAPKLLMYGIGERPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DWLAGYVFGTGTKVT 270 G9 VH #92
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVIAYDGRKKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 271
G9 VL #92 QSVLTQPPSASGTPGQTVSISCSGRNSNIGN
HSVHWYQQVPGAAPKLLMYGTGHRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCASWD
DGLAGYVFGSGTKVTVL 272 G9 VH #93 Same as Parental
EVQLLESGGGVVQPGRSLRLSCAASGFIFS NYGMHWVRQAPGKGLEWVAVISYDGSNKYY
ADSVKGRFTISRDNSKNTLYLEMNSLRPED TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS
273 G9 VL #93 QSVLTQPPSASGTPGQTVSISCSGRKSNIGS
HSVHWYQQVPGAAPKLLMYGYGERPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCAMWD
DWLGGYVFGTGTKVTVL 274 G9 VH #94 Same as Parental
EVQLLESGGGVVQPGRSLRLSCAASGFIFS NYGMHWVRQAPGKGLEWVAVISYDGSNKYY
ADSVKGRFTISRDNSKNTLYLEMNSLRPED TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS
275 G9 VL #94 QSVLTQPPSASGTPGQTVSISCSGSISNIGS
HSVHWYQQVPGAAPKLLMYGLGYRPSGVPDR FSVSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGTGTKVTVL 276 G9 VH #95 EVQLLESGGGVVQPGRSLRLSCAASGFIFNN
NGMHWVRQAPGKGLEWVAVISKDGRNKYYGD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGAYGDYYYSYGLDVWGQGTTVTVSS 277 G9 VL #95
QSVLTQPPSASGTPGQTVSISCSGSKSNIGS HYVHWYQQVPGAAPKLLMYGICQRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGTGTKVTVL 278 G9 VH #96 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISRDGRRKYYSD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGALGDYYYSYGLDVWGQGTTVTVSS 279 G9 VL #96
QSVLTQPPSASGTPGQTVSISCSGSNSNIGR HYVHWYQQVPGAAPKLLMYGIGQRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGSGTKVTVL 280 G9 VH #97
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVISNDGTNKYYTD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 281
G9 VL #97 QSVLTQPPSASGTPGQTVSISCSGSKSNIGR
HFVHWYQQVPGAAPKLLMYANGQRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGSGTKVTVL 282 G9 VH #98 EVQLLESGGGVVQPGRSLRLSCAASGFIFRN
NGMHWVRQAPGKGLEWVAVISYDGRNKYYED SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSFGLDVWGQGTTVTVSS 283 G9 VL #98
QSVLTQPPSASGTPGQTVSISCSGSISNIGR HAVHWYQQVPGAAPKLLMYYFGERPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGSGTKVTVL 284 G9 VH #99
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEHTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 285
G9 VL #99 QSVLTQPPSASGTPGQTVSISCSGRNSNIGR
HHVHWYQQVPGAAPKLLMYGIGYRPSGIPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGAYVFGSGTKVTVL 286 G9 VH #100 Same as Parental
EVQLLESGGGVVQPGRSLRLSCAASGFIFS NYGMHWVRQAPGKGLEWVAVISYDGSNKYY
ADSVKGRFTISRDNSKNTLYLEMNSLRPED TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS
287 G9 VL #100 QSVLTQPPSASGTPGQTVSISCSGNRSNIGH
HSVHWYQQVPGAAPKLLMYGISERPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGSGTKVTVL 288 G9 VH #101 Same as Parental
EVQLLESGGGVVQPGRSLRLSCAASGFIFS NYGMHWVRQAPGKGLEWVAVISYDGSNKYY
ADSVKGRFTISRDNSKNTLYLEMNSLRPED TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS
289 G9 VL #101 QSVLTQPPSASGTPGQTVSISCSGITSNIGR
HSVHWYQQVPGAAPKLLMYGICQRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGSGTKVTVL 290 G9 VH #102 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISYDGANRYYSD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 291 G9 VL #102
QSVLTQPPSASGTPGQTVSISCSGRNSNIGR HSVHWYQQVPGAAPKLLMYWYGQRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGSGTKVTVL 292 G9 VH #103
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVISYDGSNTYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 293
G9 VL #103 QSVLTQPPSASGTPGQTVSISCSGYNSNIGR
HYVHWYQQVPGAAPKLLMYSNGLRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGSGTKVTVL 294 G9 VH #104 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISYDGSNKYYVD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGHGTTVTVSS 295 G9 VL #104
QSVLTQPPSASGTPGQTVSISCSGINSNIGR HYVNWYQQVPGAAPKLLIYADGWRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGAYVFGSGTKVTVL 296 G9 VH #105
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVISYDGGNQYYVD
SVKGRFTISRDNSKNTLYLEMNSLARPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS
297 G9 VL #105 QSVLTQPPSASGTPGQTVSISCSGSNSNIGR
HYVHWYQQVPGAAPKLLMYFNGVRPSGVPDR FSGSQSGTSASLTISGLQSEDEADYYCATWD
DWLADYVFGSGTKVTVL 298 G9 VH #106 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISYDGHNQYYQD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 299 G9 VL #106
QSVLTQPPSASGTPGQTVSISCSGANSNIGR HYVHWYQQVPGAAPKLLIYYDGMRPSGVPDR
FSGSQSGTSASLAISGPQSEDEADYYCATWD DRLVGYVFGTGTKVTVL 300 G9 VH #107
EVQLLESGGGVVQPGRSLRLSCAASGFIFRN YGMHWVRQAPGKGLEWVAVISHDGSNKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 301
G9 VL #107 QSVLTQPPSASGTPGQTVSISCSGINSNIGY
HYVHWYQQVPGAGPKLLIYGSGERPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLVGYVFGTGTKVTVL 302 G9 VH #108 Same as Parental
EVQLLESGGGVVQPGRSLRLSCAASGFIFS NYGMHWVRQAPGKGLEWVAVISYDGSNKYY
ADSVKGRFTISRDNSKNTLYLEMNSLRPED TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS
303 G9 VL #108 QSVLTQPPSASGTPGQTVSISCSGHNSNIGY
HYVHWYQQVPGAAPKLLIYGDGWRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCGTWD
DWLGGYVFGTGTKVTVL 304 G9 VH #109 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVHQAPGKGLEWVAVISHDGSKQYYAD SLKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKLGASGDYYYSYGLDYWGQGTTVTVSS 305 G9 VL #109
QSVLTQPPSASGTPGQTVSISCSGHNSNIGY HYVHWYQQVPGAAPKLLIYGDGWRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGSGTKVTVL 306 G9 VH #110
EVQLLESGGGVVQPGRSLRLSCAASGFIFGN YGMHWVRQAPGKGLEWVAVISYDGSNKYYGD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGAYGDYYYSYGLDVWGQGTTVTVSS 307
G9 VL #110 QSVLTQPPSASGTPGQTVSISCSGRQSNIGR
HSVDWYQQVPGAAPKLIIYGIGQRPSGVPDR FSGSQSGTSASLTISGLQSEDEADYYCATWD
DSLGGYVFGTGTKVTVL 308 G9 VH #111 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGAYGDYYYSYGLDFWGQGTTVTVSS 309 G9 VL #111
QSVLTQPPSASGTPGQTVSISCSGSNSNIGR HYVHWYQQVPGAAPKLLIYGVGQRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLAGGYVFGSGTKVTVL 310 G9 VH #112
EVQLLESGGGVVQPGSSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
SVKGRFTISRDNSKNSLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 311
G9 VL #112 QSVLTQPPSASGTPGQTASISCSGSNSNIGR
HYVDWYQQVPGAAPKLLIYGGGQRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGSGTKVTVL 312 G9 VH #113 EVQLLESGGGVVQPGRSLRLSCAASGFIFTN
YGMHWVRQAPGKGLEWVAVISHDGSNKYYAD SVKGRFTIPRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 313 G9 VL #113
QSVLTQPPSASGTPGQTVSISCSGRNSNIGR HSVHWYQQVPGAAPKLLIYDVGQRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DWLAGYVFGSGTKVTVL 314 G9 VH #114
EVQLLESGGGVVQPGRSLRLSCAASGFIFRN YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
SVKGRFTISRDNSKNTLYLEMNSLKPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 315
G9 VL #114 QSVLTQPPSASGTPGQTVSISCSGSNSNIGR
HSVHWYQQVPGAAPKLLIYGDGQRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DRLGGYVFGSGTKVTVL 316 G9 VH #115 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGAFGDYYYSYGLDVWGQGTTVTVSS 317 G9 VL #115
QSVLTQPPSASGTPGQTVSISCSGNKSNIGR HYVHWYQQVPGAAPKLLMYYDGQRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCGTWD DRLGGYVFGTGTKVTVL 318 G9 VH #116
EVQLLESGGGVVQPGRSLRLSCAASGFLFSN YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGAFGDYYYSYGLDVWGQGTTVTVSS 319
G9 VL #116 QSVLTQPPSASGTPGQTVSISCSGSNSNIGM
HYVHWYQQVPGAAPKLLMYYSGLRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGTGTKVTVL 320 G9 VH #117 EVQLLESGGGVVQPGRSPRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 321 G9 VL #117
QSVLTQPPSASGTPGQTVSISCSGSRSNIGR HYVHWYQQVPGAAPKLLMYDYFYRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGTGTKVTVL 322 G9 VH #118
Same as VH #73 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISYDGSNKYYGD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 323 G9 VL #118
QSVLTQPPSASGTPGQTVSISCSGSNSNIGN HYVHWYQQVPGAAPKLLIYDFGYRPSGVPDR
FSVSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGSGTKVTVL 324 G9 VH #119
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVISHDGWSKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDAWGQGTTVTVSS 325
G9 VL #119 QSVLTQPPSASGTPGQTVSISCSGNYSNIGT
HYVHWYQQVPGAAPKLLIYDFGHRPSGVPDR FSVSQSGTSASLAISGLQSEDEADYYCATWD
DRLAGYVFGSGTKVTVL 326 G9 VH #120 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDLWGQGTTVTVSS 327
G9 VL #120 QSVLTQPPSASGTPGQTVSISCSGSYSNIGS
HYVHWYQQVPGAAPKLLMYDFGERPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGSGTKVTVL 328 G9 VH #121 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISYDGGNAYYAD SVKGRFTISRDNSENTLYLEMNSLRPEDTAV
YYCAKLGASGDYYYSYGLDVWGQGTTVTVSS 329 G9 VL #121
QSVLTQPPSASGTPGQTVSISCSGSNSNIGS HYVHWYQQVPGAAPKLLIYWGGLRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCALWD DRLGGYVFGTGTKVTVL 330 G9 VH #122
EVQLLESGGGVVQPGRSLRLSCAASGFIFSN YGMHWVRQAPGKGLEWVAVISYDGGYKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKLSLSGDYYYSYGFDGWGQGTTVTVSS 331
G9 VL #122 QSVLTQPPSASGTPGQTVSISCSGSKSNIGS
HYVHWYQQVPGAAPKLLMYDFGQRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCGTWD
DWLGGYVFGTGTKVTVL 332 G9 VH #123 EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISYDGRNKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGAKGDYYYSYGLDLWGQGTTVTVSS 333 G9 VL #123
QSVLTQPPSASGTPGQTVSISCSGNRSNIGS HYVHWYQQVPGAAPKLLIYSMGQRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGTGTKVTVL 334 G9 VH #124
Same as Parental EVQLLESGGGVVQPGRSLRLSCAASGFIFS
NYGMHWVRQAPGKGLEWVAVISYDGSNKYY ADSVKGRFTISRDNSKNTLYLEMNSLRPED
TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS 335 G9 VL #124
QSVLTQPPSASGTPGQTVSISCSGSHSNIGR HPVDWYQQVPGAAPKLLMYYGGYRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DRLVGYVFGSGTKVTVL 336 G9 VH #125
Same as VH #14 EVQLLESGGGVVQPGRSLRLASCAASGFIFRN
YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 337 G9 VL #125 Same as VL #26
QSVLTQPPSASGTPGQTVSISCSGSNSNIGR HPVDWYQQVPGAAPKLLIYYDDQRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCATWD DSLGGYVFGSGTKVTVL
[1093] The sequences of the individual CDRs of the VH and VL
regions of the affinity matured fully human IL-17 antibodies from
IL17-TN-L7-G9 in the above table can be aligned to provide
consensus CDR sequences such as those in the table below.
TABLE-US-00023 TABLE 22 CDR Sequence Consensus region Identifier
Sequence CDR- SEQ ID X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 H1
NO.: 338 N Y G M H D N Y H H Q F CDR- SEQ ID X.sub.1 X.sub.2
X.sub.3 X.sub.4 X.sub.5 X.sub.6 X.sub.7 X.sub.8 X.sub.9 X.sub.10
X.sub.11 X.sub.12 X.sub.13 X.sub.14 X.sub.15 X.sub.16 X.sub.17 H2
NO.: 339 I S Y D G S N K Y Y A D S V K G A N R R Q S L G S T K T V
H G S R G R W H N T F H G A E C C Y G P M Y S R K A E Q A L CDR-
SEQ ID X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6 X.sub.7
X.sub.8 X.sub.9 X.sub.10 X.sub.11 X.sub.12 X.sub.13 X.sub.14
X.sub.15 H3 NO.: 340 V G A S G D Y Y Y S Y G L D V L S L Y P T P F
L G L N F Y F E G W A F N A K S E CDR-L1 SEQ ID X.sub.1 X.sub.2
X.sub.3 X.sub.4 X.sub.5 X.sub.6 X.sub.7 X.sub.8 X.sub.9 X.sub.10
X.sub.11 X.sub.12 X.sub.13 NO.: 341 S G R N S N I G R H Y V D S Q S
R S H T K N P N N H Y A T H R T F Y I Y C T Y T V H K I M E G P H D
A S G C M D CDR-L2 SEQ ID X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5
X.sub.6 X.sub.7 NO.: 342 Y D G Q R P S G I S I D F D Y W T V E F S
W L S N C H E G F W A Y M V V M T L S N F D CDR-L3 SEQ ID X.sub.1
X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6 X.sub.7 X.sub.8 X.sub.9
X.sub.10 X.sub.11 NO.: 343 A T W D D S L G G Y V G S W A A D M R V
E L M D I L V K S H G F
[1094] The sequence data for CDR-H1 of the VH regions of the
affinity matured fully human IL-17 antibodies from IL17-TN-L7-G9
also revealed that the first amino acid (X.sub.1 position) of
CDR-H1 is preferably preceded by an amino acid selected from S, R,
N, T, G, and P.
[1095] Considering the aligned CDRs from affinity matured human
IL-17 antibodies (Table 22) and from the isolated fully human IL-17
antibodies (Table 6) leads to a more comprehensive set of consensus
CDR sequences. See, Summary of Invention, supra.
[1096] The following were converted into IgG for further
characterization.
TABLE-US-00024 TABLE 23 SEQ ID Sequence No: Protein region
123456789012345678901234567890 344 G9-1VH Same as Parental
EVQLLESGGGVVQPGRSLRLSCAASGFIFS NYGMHWVRQAPGKGLEWVAVISYDGSNKYY
ADSVKGRFTISRDNSKNTLYLEMNSLRPED TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS
345 G9-1VL QSVLTQPPSASGTPGQTVSISCSGSHSNIGR
HPVDWYQQVPGAAPKLLMYYGGYRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DRLVGYVFGSGTKVTVLG 346 G9-3VH Same as Parental
EVQLLESGGGVVQPGRSLRLSCAASGFIFS NYGMHWVRQAPGKGLEWVAVISYDGSNKYY
ADSVKGRFTISRDNSKNTLYLEMNSLRPED TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS
347 G9-3VL QSVLTQPPSASGTPGQTVSISCSGRNSNIGS
HYVHWYQQVPGAAPKLLMYDFGLRPSGVPDR FSVSQSGTSASLAISGLQSEDEADYYCATWD
DMLGGYVFGTGTKVTVLG 348 G9-4VL QSVLTQPPSASGTPGQTVSISCSGRQSNIGR
HYVDWYQQVPGAAPKLLMYYDSIRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGSGTKVTVLG 349 G9-M3VH Same as Parental
EVQLLESGGGVVQPGRSLRLSCAASGFIFS NYGMHWVRQAPGKGLEWVAVISYDGSNKYY
ADSVKGRFTISRDNSKNTLYLEMNSLRPED TAVYYCAKVGASGDYYYSYGLDVWGQGTTV TVSS
350 G9-M2VL QSVLTQPPSASGTPGQTVSISCSGHNSNIGY
HYVHWYQQVPGAAPKLLIYGDGWRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCGTWD
DWLGGYVFGTGTKVTVLG G9-1,3,4 and M2 have an identical VH sequence as
the parental IL17-TN-L7-G9 351 G9-2VH
EVQLLESGGGVVQPGRSLRLSCAASGFIFRN YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 352
G9-2VL QSVLTQPPSASGTPGQTVSISCSGSNSNIGR
HPVDWYQQVPGAAPKLLIYYDDQRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DSLGGYVFGSGTKVTVLG 353 G9-5VH EVQLLESGGGVVQPGRSLRLSCAASGFIFRN
YGMHWVRQAPGKGLEWVAVIAYDGSRQYYSD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDLWGQGTTVTVSS 354 G9-5VL
QSVLTQPPSASGTPGQTVSISCSGRQSNIGR HYVDWYQQVPGAAPKLLMYYDSIRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCGMWD DSLAGYVFGSGTKVTVLG 355 G9-6VH
EVQLLESGGGVVQPGRSLRLSCAASGFIFSD YGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDVWGQGTTVTVSS 356
G9-6VL QSVLTQPPSASGTPGQTVSISCSGRQSNIGN
HYVDWYQQVPGAAPKLLIYGDVIRPSGVPDR FSGSQSGTSASLAISGLQSEDEADYYCATWD
DWLAGYVFGSGTKVTVLG 357 G9-M1VH EVQLLESGGGVVQPGRSLRLSCAASGFIFSN
YGMHWVRQAPGKGLEWVAVISYDGGYKYYAD SVKGRFTISRDNSKNTLYLEMNSLRPEDTAV
YYCAKLSLSGDYYYSYGFDGWGQGTTVTVSS 358 G9-M1VL
QSVLTQPPSASGTPGQTVSISCSGSKSNIGS HYVHWYQQVPGAAPKLLMYDFGQRPSGVPDR
FSGSQSGTSASLAISGLQSEDEADYYCGTWD DWLGGYVFGTGTKVTVLG
[1097] Similarly, affinity maturation selection methods were also
employed to generate affinity matured fully human IL-17 antibodies
from IL-17-TN-K7-B6. (The amino acid sequences of the VH and VL
regions of the fully human IL-17-TN-K7-B6 antibody are shown in
Table 6 above.) One light chain library is constructed to contain
limited mutagenesis at the following residues: CDRL1: 28, 30, 31,
32; CDRL2: 50, 53, 55; CDRL3: 91, 92, 93, 96, 97 (Kabat numbering).
This library also contains V2I, M4L, and L104V framework germline
back-mutations as well as binary diversities at position 77 (S/G),
84(G/A), and 100(P/Q) to allow for framework germ-lining during
library selections. Two heavy chain libraries are made to contain
limited mutagenesis in CDRH1 and CDRH2 at residues 30, 31, 33, 34,
35, 52a, 53, 54, 55, 56, 57, and 58 (Kabat numbering) or in CDRH3
at residues 95-100, 100a-100i, and 102. The heavy chain libraries
also contain a P108T framework germline back-mutation in addition
to binary diversities at residues 16(S/E), 18(V/L), 20(V/I), and
73(K/E) to allow for framework germ-lining during library
selections. All three libraries are selected separately by
decreasing concentrations of human IL-17, cyno IL-17, or human
IL-17A/F. All mutated CDR sequences are then recombined into one
library and the recombined library is subjected to more strigent
selection conditions before individual antibodies are
identified.
[1098] The table below provides a list of amino acid sequences of
VH regions of affinity matured fully human IL-17 antibodies derived
from IL-17-TN-K7-B6 Amino acid residues of individual CDRs of each
VH sequence are indicated in bold.
TABLE-US-00025 TABLE 24 List of amino acid sequences of affinity
matured B6 VH variants Clone SEQ ID NO: VH J313M2S3#11 359
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITTFFGITDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYATHDFDYWGQGTTVTVSS J313M2S3#45 360
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITNFFGWTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYATHDFDYWGQGTTVTVSS J313M2S3#61 361
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITNFFGAVDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYATHDFDYWGQGTTVTVSS J313M2S3#14 362
EVQLVQSGAEVKKPGESLKISCKASGGSFVSYGICWVRQAPGQ
GLEWMGGITNFFGTTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYATHDFDYWGQGTTVTVSS J313M2S3#26 363
EVQLVQSGAEVKKPGESLKISCKASGGSFSSYGIVWVRQAPGQ
GLEWMGGITTFFGATDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYATHDFDYWGQGTTVTVSS J313M2S3#86 364
EVQLVQSGAEVKKPGESLKISCKASGGSFSSYGIVWVRQAPGQ
GLEWMGGITTFFGATDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYATHDFDIWGQGTTVTVSS J313M2S3#82 365
EVQLVQSGAEVKKPGESLKISCKASGGSFSSYGIVWVRQAPGQ
GLEWMGGITTFFGATDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYDTHDFDSWGQGTTVTVSS J313M2S3#59 366
EVQLVQSGAEVKKPGESLKVSCKASGGSFKSYGISWVRQAPGQ
GLEWMGGITIFFGTVDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYNTHDFDYWGQGTTVTVSS J313M2S3#90 367
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITHFFGTVDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYDTHDLDYWGQGTTVTVSS J313M2S3#16 368
EVQLVQSGAEVKMPGSSLKISCKASGGSFRGYGISWVRQAPGQ
GLEWMGGITPFFGWADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYDTHHFDYWGQGTTVTVSS J313M2S3#24 369
EVQLVQSGAEVKMPGSSLKISCKASGGSFRGYGISWVRQAPGQ
GLEWMGGITPFFGWADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYDTHDFDSWGQGTTVTVSS J313M2S3#42 370
EVQLVQSGAEVKKPGESLKISCKASGGSFKGYGISWVRQAPGQ
GLEWMGGITPFFGWTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYATHDFDYWGQGTTVTVSS J313M2S3#78 371
EVQLVQSGAEVKKPGESLKISCKASGGSFKGYGISWVRQAPGQ
GLEWMGGITPFFGWTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYDTHHFDYWGQGTTVTVSS J313M2S3#18 372
EVQLVQSGAEVKKPGESLKVSCKASGGRFRTYGISWVRQAPGQ
GLEWMGGITNFFGWTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARVPNEFWNGYYATDYFDNWGQGTTVTVSS J313M2S3#30 373
EVQLVQSGAEVKKPGESLKVSCKASGGRFRTYGISWVRQAPGQ
GLEWMGGITNFFGWTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNEFWNGYYTTHDFDSWGQGTTVTVSS J313M2S3#12 374
EVQLVQSGAEVKKPGESLKISCKASGGSFISYGISWVRQAPGQ
GLEWMGGITHFFGSTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYATHDFDYWGQGTTVTVSS J313M2S3#13 375
EVQLVQSGAEVKKPGESLKISCKASGGSFISYGISWVRQAPGQ
GLEWMGGITHFFGSTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARVPNEFWNGYYATQDFDYWGQGTTVTVSS J313M2S3# 2 376
EVQLVQSGAEVKKPGESLKISCKASGGSFISYGISWVRQAPGQ
GLEWMGGITHFFGSTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARVPNEFWNGYYATDYFDYWGQGTTVTVSS J313M2S3#46 377
EVQLVQSGAEVKKPGESLKISCKASGGSFISYGISWVRQAPGQ
GLEWMGGITHFFGSTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNEFWNGYYTTHDFDSWGQGTTVTVSS J313M2S3#28 378
EVQLVQSGAEVKKPGSSLKISCKASGGSFLSYGISWVRQAPGL
GLEWMGGITLFFGTVDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNEFWNGYYSTHDFDYWGQGTTVTVSS J313M2S3#74 379
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITLFFGTVDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNEFWNGYYATHDFDYWGQGTTVTVSS J313M2S3#34 380
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITLFFGISDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNEFWNGYYTTHDFDSWGQGTTVTVSS J313M2S3#69 381
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITNFFGAVDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNEFWNGYYTTHDFDAWGQGTTVTVSS J313M2S3#85 382
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITNFFGMEDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNEFWNGYYSTHDFDYWGQGTTVTVSS J313M2S3#15 383
EVQLVQSGAEVKKPGESLKISCKASGGSFSGYGTSWVRQAPGQ
GLEWMGGITHFFGAVDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYATHDFDYWGQGTTVTVSS J313M2S3#31 384
EVQLVQSGAEVKKPGESLKISCKASGGSFSGYGTSWVRQAPGQ
GLEWMGGITHFFGAVDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPHEFWNGYYATHDFDYWGQGTTVTVSS J313M2S3#25 385
EVQLVQSGAEVKKPGESLKISCKASGGSFSGYGTSWVRQAPGQ
GLEWMGGITHFFGAVDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYDTHDFDSWGQGTTVTVSS J313M2S3#49 386
EVQLVQSGAEVKKPGESVKVSCKASGGSFSGYGTSWVRQAPGQ
GLEWMGGITHFFGAVDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNEFWNGYYSTHDFDYWGQGTTVTVSS J313M2S3# 6 387
EVQLVQSGAEVKKPGESVKVSCKASGGSFSGYGISWVRQAPGQ
GLEWMGGITNFFGIVDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYATHDFDYWGQGTTVTVSS J313M2S3#75 388
EVQLVQSGAEVKKPGESVKVSCKASGGSFSGYGISWVRQAPGQ
GLEWMGGITNFFGIVDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNEFWNGYYATHDFDYWGQGTTVTVSS J313M2S3#17 389
EVQLVQSGAEVKKPGSSVKVSCKASGGSFGGYGIGWVRQAPGQ
GLEWMGGITPFFGFADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYSTHDFDSWGQGTTVTVSS J313M2S3#79 390
EVQLVQSGAEVKKPGSSVKVSCKASGGSFSSYGICWVRQAPGQ
GLEWMGGVTTFFGFADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYATHDFDYWGQGTTVTVSS J313M2S3#94 391
EVQLVQSGAEVKKPGSSVKVSCKASGGSFTSYGISWVRQAPGQ
GLEWVGGITTFFGVADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPHEFWNGYYATHDFDYWGQGTTVTVSS J313M2S3#37 392
EVQLVQSGAEVKKPGESVKVSCKASGGSFRGYGISWVRQAPGQ
GLEWMGGITHFFGMTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYATHDFDYWGQGTTVTVSS J313M2S3#81 393
EVQLVQSGAEVKKPGESVKVSCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITTFFGTTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYATHDFDYWGQGTTVTVSS J313M2S3#77 394
EVQLVQSGAEVKKPGESVKVSCKASGGSFRSYGIGWVRQAPGQ
GLEWMGGITNFFGVEDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYATHDFDYWGQGTTVTVSS J313M2S3#54 395
EVQLVQSGAEVKKPGESVKISCKASGGSFLSYGISWVRQAPGQ
GLEWMGGITHFFGISDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYATHDFDYWGQGTTVTVSS J313M2S3#89 396
EVQLVQSGAEVKKPGESVKISCKASGGSFPSYGISWVRQAPGQ
GLEWMGGITNFFGIRDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNEFWNGYYATHDFDYWGQGTTVTVSS J313M2S3#32 397
EVQLVQSGAEVKKPGESVKISCKASGGSFSSYGISWVRQAPGQ
GLEWMGGITVFFGTADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARVPNEFWNGYYATQDFDYWGQGTTVTVSS J313M2S3#55 398
EVQLVQSGAEVKKPGSSVKISCKASGGSFSSYGISWVRQAPGQ
GLEWMGGITLFFGTTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYDTHDFDSWGQGTTVTVSS J313M2S3#27 399
EVQLVQSGAEVKKPGSSVKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITHFFGMVDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNEFWNGYYATHDFDYWGQGTTVTVSS J313M2S3#50 400
EVQLVQSGAEVKKPGSSVKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITHFFGMVDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNEFWNGYYSTHDFDYWGQGTTVTVSS J313M2S3#39 401
EVQLVQSGAEVKKPGSSVKISCKASGGSFTSYGISWVRQAPGQ
GLEWMGGITNFFGSTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNEFWNGYYSTHDFDYWGQGTTVTVSS J313M2S3#91 402
EVQLVQSGAEVKKPGSSVKISCKASGGSFTSYGISWVRQAPGQ
GLEWMGGITNFFGSTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNEFWNGYFATNDFDYWGQGTTVTVSS J313M2S3# 5 403
EVQLVQSGAEVKKPGESVKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITHFFGITDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYDTHDFDSWGQGTTVTVSS J313M2S3#47 404
EVQLVQSGAEVKKPGESVKVSCKASGGSFISYGISWVRQAPGQ
GLEWMGGITHFFGTVDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNEFWNGYYTTHDFDSWGQGTTVTVSS J313M2S3#72 405
EVQLVQSGAEVKKPGESVKVSCKASGGSFISYGFSWVRQAPGQ
GLEWMGGITVFFGHVYYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNEFWNGYYSTHDFDYWGQGTTVTVSS J313M2S3# 1 406
EVQLVQSGAEVKKPGSSVKVSCKASGGSFISYGYNWVRQAPGQ
GLEWMGGITPFFGTADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARESSEFWNGYYCTQDFDLWGQGTTVTVSS J313M2S3#44 407
EVQLVQSGAEVKKPGSSVKVSCKASGGSFLSYGFSWVRQAPGQ
GLEWMGGITPFFGLADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARESSEFWNGYYCTQDFDLWGQGTTVTVSS J313M2S3# 3 408
EVQLVQSGAEVKKPGESLKISCKASGGSFTTYGMNWVRQAPGQ
GLEWMGGITPFFGWADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARESSEFWNGYYCTQDFDLWGQGTTVTVSS J313M2S3#29 409
EVQLVQSGAEVKKPGESLKISCKASGGSFSSYGFGWVRQAPGQ
GLEWMGGITPFFGIADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARESSEFWNGYYVTNDFDYWGQGTTVTVSS J299M2S3#10 410
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITPILGTANYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARESNEFWNGYYPTLDLDSWGQGTTVTVSS J299M2S3#23 411
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITPILGTANYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARESNEFWNGYYPTQDYDFWGQGTTVTVSS J299M2S3#20 412
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITPILGTANYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARESSAFWNGYYNTNDLDYWGQGTTVTVSS J299M2S3#35 413
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITPILGTANYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARESTEFWNGYYNTNDYDNWGQGTTVTVSS J299M2S3#16 414
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITPILGTANYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARESNDFWNGYYTTDDFDYWGQGTTVTVSS J299M2S3#18 415
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITPILGTANYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARESNDFWNGYYTTDDLDYWGQGTTVTVSS J299M2S3#27 416
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITPILGTANYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARESNDFWNGYYTTDDFDCWGQGTTVTVSS J299M2S3#15 417
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITPILGTANYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARVPNEFWNGYFPTQDFDYWGQGTTVTVSS J299M2S3#17 418
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITPILGTANYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARETNEFWNGYFPTQDFDYWGQGTTVTVSS J299M2S3#43 419
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITPILGTANYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNEFWNGYFPTQDFDYWGQGTTVTVSS J299M2S3#32 420
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITPILGTANYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNEFWNGYFPTLDFDCWGQGTTVTVSS J299M2S3#42 421
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITPILGTANYAQKFQDRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNEFWNGYFATNDFDYWGQGTTVTVSS J299M2S3#11 422
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITPILGTANYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYATDNFDYWGQGTTVTVSS J299M2S3#13 423
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITPILGTANYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYSTDHFDCWGQGTTVTVSS J299M2S3#14 424
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITPILGTANYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARVPNDFWNGYFATDYFDDWGQGTTVTVSS J299M2S3#21 425
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITPILGTANYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYFATDHFDFWGQGTTVTVSS J299M2S3#28 426
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITPILGTANYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYDTDHFDYWGQGTTVTVSS J299M2S3#2 427
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITPILGTANYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCARDPNEFWNGYYATHDFDHWGQGTTVTVSS J313M2S3#23 428
EVQLVQSGAEVKKPGSSVKISCKASGGSFGSYGISWVRQAPGQ
GLEWMGGITPFFGWADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNEFWNGYYSTDHFDYWGQGTTITVSS J294M2S3#27 429
EVQLVQSGAEVKKPGESVKISCKASGGSFPSYGISWVRQAPGQ
GLEWMGGITFFFGAADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J313M2S3#62 430
EVQLVQSGAEVKKPGESVKISCKASGGSFISYGTSWVRQAPGQ
GLEWMGGITHFFGTTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#1 431
EVQLVQSGAEVKKPGESVKISCKASGGSFISYCTSWVRQAPGQ
GLEWMGGITHFFGSADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#39 432
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYSTSWVRQAPGQ
GLEWMGGITHFFGTADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J313M2S3#65 433
EVQLVQSGAEVKKPGESLKISCKASGGSFISYGISWVRQAPGQ
GLEWMGGITHFFGSTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#14 434
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITHFFGTTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J299M2S3#24 435
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITHFFLTTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#42 436
EVQLVQSGAEVKKPGESLKVSCKASGGSFTSYGISWVRQAPGQ
GLEWMGGITHMFATTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#5 437
EVQLVQSGAEVKKPGESLKISCKASGGSFISYGISWVRQAPGQ
GLEWMGGITPFLGATDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#19 438
EVQLVQSGAEVKKPGSSLKISCKASGGSFRSYGISWVRQAPGQ
GLEWMGGITPFFGFIDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#44 439
EVQLVQSGAEVKKPGESLKISCKASGGSFRSYVISWVRQAPGQ
GLEWMGGITPFFGTVDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#12 440
EVQLVQSGAEVKKPGESLKISCKASGGSFIVYGFSWVRQAPGQ
GLEWMGGITPFLGTVDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#2 441
EVQLVQSGAEVKKPGESLKVSCKASGGSFLTYGFSWVRQAPGQ
GLEWMGGITTFLGTVNYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#23 442
EVQLVQSGAEVKKPGESLKISCKASGGSFTSYVFSWVRQAPGQ
GLEWMGGITPLLGISDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#34 443
EVQLVQSGAEVKKPGESLKISCKASGGSFTSYGVSWVRQAPGQ
GLEWMGGITTILGTSDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#47 444
EVQLVQSGAEVKKPGESLKISCKASGGSFISYGFSWVRQAPGQ
GLEWMGGITPIFGTGDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#15 445
EVQLVQSGAEVKKPGESVKISCKASGGSFWSYDFSWVRQAPGQ
GLEWMGGITTFFESADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#25 446
EVQLVQSGAEVKKPGSSLKISCKASGGSFTSYDFSWVRQAPGQ
GLEWMGGITTFFGLADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#21 447
EVQLVQSGAEVKKPGESLKISCKASGGSFSGYDFSWVRQAPGQ
GLEWMGGITPFFGSAYYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#24 448
EVQLVQSGAEVKKPGESLKISCKASGGSFSSYVFSWVRQAPGQ
GLEWMGGITPFFGIADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J313M2S3#88 449
EVQLVQSGAEVKKPGSSVKISCKASGGSFTSYGISWVRQAPGQ
GLEWMGGITNFFGSTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#13 450
EVQLVQSGAEVKKPGSSVKVSCKASGGSFGSYVISWVRQAPGQ
GLEWMGGITPFFGSTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J313M2S3#93 451
EVQLVQSGAEVKKPGSSVKVSCKASGGSFSSYGISWVRQAPGQ
GLEWMGGITHFFGTTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#28 452
EVQLVQSGAEVKKPGSSVKVSCKASGGSFRSYIISWVRQAPGQ
GLEWMGGITPFFGTADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#16 453
EVQLVQSGAEVKKPGSSLKVSCKASGGSFISYGYSWVRQAPGQ
GLEWMGGITTFFGTTDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#29 454
EVQLVQSGAEVKKPGSSVKVSCKASGGSFISYGYNWVRQAPGQ
GLEWMGGITPFFGTADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#11 455
EVQLVQSGAEVKKPGSSVKVSCKASGGSFLSYGFSWVRQAPGQ
GLEWMGGITPFFGFADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#37 456
EVQLVQSGAEVKKPGESVKVSCKASGGSFSSYVFSWVRQAPGQ
GLEWMGGITPFFGTADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#10 457
EVQLVQSGAEVKKPGSSVKVSCKASGGSFISYDYSWVRQAPGQ
GLEWMGGITPFFGIVNYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#8 458
EVQLVQSGAEVKKPGESVKVSCKASGGSFGSYDYSWVRQAPGQ
GLEWMGGITTFFGASTYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#30 459
EVQLVQSGAEVKKPGSSVKISCKASGGSFHSYDYSWVRQAPGQ
GLEWMGGITSFFGTEDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#33 460
EVQLVQSGAEVKKPGSSVKISCKASGGSFTGYVYSWVRQAPGQ
GLEWMGGITPFFGTADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#17 461
EVQLVQSGAEVKKPGESVKVSCKASGGSFTSYDFSWVRQAPGQ
GLEWMGGITPFLGTAIYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#20 462
EVQLVQSGAEVKKPGESVKISCKASGGSFSSYDFSWVRQAPGQ
GLEWMGGITPILGTENYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#38 463
EVQLVQSGAEVKKPGESVKISCKASGGSFSSYDYSWVRQAPGQ
GLEWMGGITPFLGMSSYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#36 464
EVQLVQSGAEVKKPGESLKISCKASGGSFTRYDFSWVRQAPGQ
GLEWMGGITPFLGTSNYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#6 465
EVQLVQSGAEVKKPGSSLKISCKASGGSFISYDFSWVRQAPGQ
GLEWMGGITPFLGFSNYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#18 466
EVQLVQSGAEVKKPGSSVKISCKASGGSFSSYDFSWVRQAPGQ
GLEWMGGITPFLGTADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#35 467
EVQLVQSGAEVKKPGSSVKISCKASGGSFSSYDFSWVRQAPGQ
GLEWMGGITTFLGTVVYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#32 468
EVQLVQSGAEVKKPGSSVKISCKASGGSFSSYVVSWVRQAPGQ
GLEWMGGITPFLGTVDYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#22 469
EVQLVQSGAEVKKPGSSVKISCKASGGSFISYGFHWVRQAPGQ
GLEWMGGITPFFGTDNYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#41 470
EVQLVQSGAEVKKPGESVKISCKASGGSFISYGFSWVRQAPGQ
GLEWMGGITPFFGLGTYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#31 471
EVQLVQSGAEVKKPGESVKISCKASGGSFLSYDFSWVRQAPGQ
GLEWMGGITPFFGAANYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS J294M2S3#48 472
EVQLVQSGAEVKKPGESVKISCKASGGSFLSYGFSWVRQAPGQ
GLEWMGGITPFFGTADYAQKFQGRVTITADESTTTAYMELSGL
TSDDTAVYYCAREPNDFWNGYYTTHHFDYWGQGTTVTVSS
[1099] The table below provides a list of amino acid sequences of
VL regions of affinity matured fully human IL-17 antibodies derived
from IL-17-TN-K7-B6 Amino acid residues of individual CDRs of each
VL sequence are indicated in bold.
TABLE-US-00026 TABLE 25 Clone SEQ ID NO: Light Chain Variable
Region (VL) J313M2S3#11 473 EIVLTQSPDFQSVTPKEKVTITCRASQIIGSELHWYQ
QKPDQSPKLLIKYASHSISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSYDLPHTFGQGTKVDIK J313M2S3#88 474
EIVLTQPPDFQSVTPKEKVTITCRASQDIGSELHWYQ
QKPDQSPKLLIKYASHSISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSYDLPYTFGQGTKVDIK J313M2S3#32 475
EIVLTQSPDFQSVTPKEKVTITCRASQDIGSELHWYQ
QKPDQSPKLLIKYASHSISGVPSRFSGSGSGTDFTLT
INSLEAEDAGTYYCHQSYWLPNTFGQGTKVDIK J313M2S3#77 476
EIVLTQSPDFQSVTPKEKVTITCRASQDIGSELHWYQ
QKPDQSPKLLIKYASHSGSGVPSRFSGSGSGTDFTLT
INSLEAEDAATYYCHQSYNLPITFGQGTKVDIK J313M2S3#28 477
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSELHWYQ
QKPDQSPKLLIKYASWSQSGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSYDLPYTFGQGTKVDIK J313M2S3#12 478
EIVLTQSPDFQSVTLKEKVTITCRASQDIGSELHWYQ
QKPDQSPKLLIKYASNSISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSMSLPHTFGQGTKVDIK J294M2S3#10 479
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSALHWYQ
QKPDQSPKLLIKYASQSISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSTSLPHTFGQGTKVDIK J294M2S3#21 480
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSALHWYQ
QKPDQSPKLLIKYASQSISGVPPRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSTSLPHTFGQGTKVDIK J294M2S3#35 481
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSALHWYQ
QKPDQSPKLLIKYASQSISGVPSRFSGSGSGADFTLT
INGLEAEDAGTYYCHQSTSLPHTFGQGTKVDIK J299M2S3#17 482
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSALHWYQ
QKPDQSPKLLIKYASQSISGVPSRFGGSGSGTDFTLT
INGLEAEDAGTYYCHQSTSLPHTFGQGTKVDIK J299M2S3#42 483
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSALHWYQ
QKPDQSPKLLVKYASQSISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSTSLPHTFGQGTKVDIK J294M2S3#44 484
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSALHWYQ
QKPDQPPKLLIKYASQSISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSTSLPHTFGQGTKVDIK J294M2S3#16 485
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSALHWYQ
QKPGQSPKLLIKYASQSISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSTSLPHTFGQGTKVDIK J299M2S3#11 486
EIVLTQSPDFQSATPKEKVTITCRASQNIGSALHWYQ
QKPDQSPKLLIKYASQSISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQGTSLPHTFGQGTKVDIK J299M2S3#13 487
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSALHWYQ
QKPDQSPKLLIKYASQSISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQGTSLPHTFGQGTKVDIK J299M2S3#14 488
EIVLTQSPDFQSVTPKEKVTITCRASQDIGSALHWYQ
QKPDQSPKLLIKYASQSISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSTSLPHTFGQGTKVDIK J313M2S3#16 489
EIVLTQSPDFQSVTPKEKVTITCRASQDIGSALHWYQ
QKPDQSPKLLIKYASHSVSGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSDILPHTFGPGTKVDIK J313M2S3#50 490
EIVLTQSPDFQSVTPKGKVTITCRASQDIGSALHWYQ
QKPDQSPKLLIKYASYSVSGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSDILPHTFGPGTKVDIK J313M2S3#69 491
EIVLTQSPDFQSVTPKEKVTITCRASQDIGAALHWYQ
QKPDQSPKLLIKYASHSNPGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSDILPQTFGQGTKVDIK J313M2S3#39 492
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSSLHWYQ
QKPDQSPKLLIKYASHSISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSDFLPHTFGQGTKVDIK J313M2S3#85 493
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSSLHWYQ
QKPDQSPKLLIKYASYPISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSDILPHTFGQGTKVDIK J313M2S3#94 494
EIVLTQSPDFRSVTPKEKVTITCRASQNIGSALHWYQ
QKPDQSPKLLIKYASHSISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSDDLPYTFGQGTKVDIK J313M2S3#2 495
EIVLTQSPDFQSVTPKEKVTITCRASQDIGSELHWYQ
QKPDQSPKLLIKYASHSNSGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSSWLPHTFGPGTKVDIK J313M2S3#86 496
EIVLTQSPDFQSVTPKEKVTITCRASQDIGSELHWYQ
QKPDQSPKLLIKYASESMSGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSS#LPHSFGPGTKVDIK J313M2S3#89 497
EIVLTQSPDFQSVTPKEKVTITCRASQDIGYELHWYQ
QKPDQSPKLLIKYASHSISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSSELPHTFGRGTKVDIK J313M2S3#91 498
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSELHWYQ
QKPGQSPKLLIKYASYSSSGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSSCLPHTFGQGTKVGIK J313M2S3#13 499
EIVLTQSPDFQSVTPKEKVTITCRASQEIGFALHWYQ
QKPGQSPKLLIKYASHSISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQTEELPYTFGPGTKVDIK J313M2S3#15 500
EIVLTQSPDFQSVTPKEKVTITCRASQNIGAELHWYQ
QKPDQSPKLLIKYASHSISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQTDRLPYSFGPGTKVDIK J313M2S3#78 501
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSELHWYQ
QKPDQSPKLLIKYASHSISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQTFSLPYTFGPGTKVDIK J313M2S3#17 502
DIVLTQSPDFQSVTPKEKVTITCRASQDIGSELHWYQ
QKPDQPPKLLIKYASHSTSGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQTDSLPYTFGPGTKVDIK J313M2S3#93 503
EIVLTQSPDFQSVTPKEKVTITCRASQDIGSELHWYQ
QKPDQSPKLLIKYASHSVSGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQTSDLPYTFGPGTKVDIK J313M2S3#49 504
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSELHWYQ
QKPDQSPKLLIKYASHSMSGVPSRFSGSGSGTDFTLT
INGLEAEDAATYYCHQTNSLPYTFGPGTKVDIK J313M2S3#9 505
EIVLTQSPDFQSVTPKEKVTITCRASQDIGSGLHWYQ
QKPDQSPKLLIKYASESMSGVPSRFSGSGSGTDFTLT
INGLEAEDAATYYCHQTDWLPYTFGPGTKVDIK J313M2S3#3 506
EIVLTQSPDFQSVTPREKVTITCRASQDIGSDLHWYQ
QKPDQSPKLLIKYASHSISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQTNSLPYTFGQGTKVDIK J313M2S3#37 507
EIVLTQSPDFQSVTPKEKVTITCRASQDIDSDLHWYQ
QKPDQSPKLLIKYASHSISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQTFNLPYTFGPGTKVDIK J313M2S3#44 508
EIVLTQSPDFQSVTPKEKVTITCRASQDIGSDLHWYQ
QKPDQSPKLLIKYASNSISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQTAYLPQTFGPGTIVDIK J313M2S3#42 509
EIVLTQSPDFQSVTPKEKVTITCRASQDIGSDLHWYQ
QKPDQSPKLLIKYASYSTSGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQTDGLPYTFGQGTKVDIK J313M2S3#62 510
EIVLTQSPDFQSVTPKEKVTITCRASQDIGSELHWYQ
QKPDQSPKLLIKYASESISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQTDSLPYTFGQGTKVDIK J313M2S3#65 511
EIVLTQSPDFQSVTPKEKVTITCRASQDIGSELHWYQ
QKPDQSPKLLIKYASHSASGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQTTLLPYTFGQGTKVDIK J313M2S3#23 512
EIVLTQSPDFQSVTPKGKVTITCRASQDIGSELHWYQ
QKPDQSPKLLIKYASHSGSGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQGFSLPHTFGQGTKVDIK J313M2S3#74 513
EVVLTQSPDFQSVTPKEKVTITCRASQDIGSELHWYQ
QKPDQSPKLLIKYASESGSGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSFCLPYTFGQGTKVDIK J313M2S3#26 514
EIVLTQSPDFQSVTPKEKVTITCRASQDIGSELHWYQ
QKPDQSPKLLIKYASSSTSGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSTSLPYTFGQGTKVDIK J313M2S3#54 515
EIVLTQSPDFQSVTPKEKVTITCRASQDIGSELHWYQ
QKPDQSPKLLIKYASSSLSGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSDYLPYTFGQGTIVDIK J313M2S3#1 516
EIVLTQSPDFQSVTPKEKVTITCRASQDIGEALHWYQ
QKPDQSPKLLIKYASHSISGVPSRFSGSGSGTDFTLT
INGLEAEDAATYYCHQSDWLPYTFGQGTKVDIK J313M2S3#18 517
EIVLTQSPDFQSVTPKEKVTITCRASQEIGASLHWYQ
QKPDQSPKLLIKYASHSASGVPSRFSGSGSGTDFTLT
INSLEAEDAATYYCHQSDGLPYTFGQGTKVDIK J313M2S3#55 518
EIVLTQSPDFQSVTPKEKVTITCRASQDIGGELHWYQ
QKPDQSPKLLIKYASHSISGVPSRFSGSGSGTDFTLT
INGLEAEDAATYYCHQSNDLPYTFGQGTKVDIK J313M2S3#59 519
EIVLTQSPDFQSVTPKEKVTITCRASQDIETELHWYQ
QKPDQSPKLLIKYASHSISGVPSRFSGSGSGTDFTLT
INGLEAEDAATYYCHQTYGLPYTFGQGTKVDIK J313M2S3#24 520
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSDLHWYQ
QKPDQSPKLLIKYASHSISGVPSRFSGSGSGTDFTLT
INGLEAEDAATYYCHQSMGLPYTFGQGTKVDIK J313M2S3#25 521
EIVLTQSPDFQSVTPKEKVTITCRASQNIGYELHWYQ
QKPDQSPKLLIKYASHSISGVPSRFSGSGSGTDFTLT
INGLEAEDAATYYCHQSFTLPYTFGPGTKVDIK J313M2S3#47 522
EIVLTQSPDFQSVTPKEKVTITCRASQNIGTELHWYQ
QKPDQSPKLLIKYASHCISGVPSRFSGSGSGTDFTLT
INGLEAEDAATYYCHQSESLPHTFGQGTKVDIK J313M2S3#5 523
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSELHWYQ
QKPDQSPKLLIKYASHSISGVPSRFSGSGSGTDFTLT
INGLEAEDAATYYCHQSDTLPHTFGQGTKVDIK J313M2S3#14 524
EIVLTQSPDFQSVTPKEKVTITCRASQDIGSELHWYQ
QKPDQSPKLLIKYASESVSGVPSRFSGSGSGTDFTLT
INGLEAEDAATYYCHQSTNLPYTFGQGTKVDIK J313M2S3#34 525
EIVLTQSPDFQSVTPKEKVTITCRASQDIGSELHWYQ
QKPDQSPKLLIKYASHSVSGVPSRFSGSGSGTDFTLT
INGLEAEDAATYYCHQSSILPHTFGQGTKVDIK J313M2S3#45 526
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSELHWYQ
QKPDQSPKLLIKYASHSVSGVPSRFSGSGSGTDFTLT
INGLEAEDAATYYCHQSTWLPYTFGQGTKVDIK J313M2S3#90 527
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSELHWYQ
QKPDQSPKLLIKYASHSVSGVPSRFSGSGSGTDFTLT
INGLEAEDAATYYCHQSAFLPYIFGQGTKVDIK J313M2S3#27 528
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSELHWYQ
QKPDQSPKLLIKYASYSVSGVPSRFSGSGSGTDFTLT
INGLEAEDAATYYCHQSISLPQTFGQGTKVDIK J313M2S3#33 529
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSELHWYQ
QKPDQSPKLLIKYASHSTSGVPSRFSGSGSGTDFTLT
INGLEAEDAATYYCHQSASLPITFGQGTKVDIK J313M2S3#29 530
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSGLHWYQ
QKPDQSPKLLIKYASYSISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSAYLPYTFGPGTKVDIK J313M2S3#81 531
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSELHWYQ
QKPDQSPKLLIKYASHSISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSAVLPDTFGPGTKVDIK J313M2S3#31 532
EIVLTQSPDFQSVAPKEKVTITCRASQNIGSELHWYQ
QKPDQSPKLLIKYASSSISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSSDLPYTFGPGTKVDIK J313M2S3#72 533
EIVLTQSPDFQSVTPKEKVTITCRASQNIDSELHWYQ
QKPDQSPKLLIKYASESISGVPSRFSGSGSGTDFALT
INGLEAEDAGTYYCHQSFSLPYTFGPGTKVDIK J313M2S3#75 534
EIVLTQSPDFQSVTPKEKVTITCRASQNIDSELHWYQ
QKPDQSPKLLIKYASESISGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSAFLPYTFGQGTKVDIK J313M2S3#6 535
EIVLTQSPDFQSVTPKEKVTITCRASQNIDSDLHWYQ
QKPDQSPKLLIKYASHSVSGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSYSLPFNFGPGTKVDIK J313M2S3#79 536
EIVLTQSPDFQSVTPKEKVTITCRASQNIGSELHWYQ
QKPDQSPKLLIKYASHSVSGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSYMLPYSFGPGTKVDIK J313M2S3#82 537
EIVLTQSPDFQSVTPKEKVTITCRASQNIGEELHWYQ
QKPDQSPKLLIKYASSSPSGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSFSLPVTFGPGTKVDIK J313M2S3#30 538
EIVLTQSPDFQSVAPKEKVTITCRASQNIGSELHWYQ
QKPDQSPKLLIKYASHSVSGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSISLPYTFGQGTKVDIK J313M2S3#46 539
EIVLTQSPDFQSVTPKEKVTITCRASQAIGSELHWYQ
QKPDQSPKLLIKYASYSVSGVPSRFSGSGSGTDFTLT
INGLEAEDAGTYYCHQSASLPYTFGQGTKVDIK
[1100] The sequences of the individual CDRs of the VH and VL
regions of the affinity matured fully human IL-17 antibodies from
IL17-TN-K7-B6 in the above tables can be aligned to provide
consensus CDR sequences such as those in the table below.
TABLE-US-00027 TABLE 26 CDR Sequence Consensus region Identifier
Sequence CDR- SEQ ID X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 H1
NO.: 540 S Y G I S G D F G T V T C V S Y N R A M V C V H I CDR- SEQ
ID X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6 X.sub.7 X.sub.8
X.sub.9 X.sub.10 X.sub.11 X.sub.12 X.sub.13 X.sub.14 X.sub.15
X.sub.16 X.sub.17 H2 NO.: 541 G I T P F F G T A D Y A Q K F Q G V H
I L M I T N D N M L S V Y T V E W S S L S A A E T I L F D V V L P I
S M I F V G H R CDR- SEQ ID X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5
X.sub.6 X.sub.7 X.sub.8 X.sub.9 X.sub.10 X.sub.11 X.sub.12 X.sub.13
X.sub.14 X.sub.15 X.sub.16 X.sub.17 H3 NO.: 542 E P N E F W N G Y Y
T T H D F D Y D S S D F A D H L S V T T A S Q Y Y L H D N N N P L F
N C C I V H A D CDR-L1 SEQ ID X.sub.1 X.sub.2 X.sub.3 X.sub.4
X.sub.5 X.sub.6 X.sub.7 X.sub.8 X.sub.9 X.sub.10 X.sub.11 NO.: 543
R A S Q N I G S A L H W V D D Y E E E A D A E S I F G T G CDR-L2
SEQ ID X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6 X.sub.7 NO.:
544 Y A S H S I S Q P V P Y C T E N N A S M W L G S F P Q CDR-L3
SEQ ID X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6 X.sub.7
X.sub.8 X.sub.9 NO.: 545 H Q S T S L P H T T D D Y S G A N Q A Y G
F I S I I N F T V L N F D I Y N E W M C E R V L M
[1101] The sequence data for CDR-H1 of the VH regions of the
affinity matured fully human IL-17 antibodies from IL17-TN-K7-B6
also revealed that the first amino acid (X.sub.1 position) of
CDR-H1 is preferably preceded by an amino acid selected from R, S,
I, T, G, L, K, V, P, W, and H.
[1102] Considering the aligned CDRs from affinity matured human
IL-17 antibodies (Tables 22 and 26) and from the isolated fully
human IL-17 antibodies (Table 6) leads to a more comprehensive set
of consensus CDR sequences. See, Summary of Invention, supra.
[1103] The following were converted into IgG for further
characterization.
TABLE-US-00028 TABLE 27 SEQ Sequence ID No: Protein region
123456789012345678901234567890 546 pJP243
EVQLVQSGAEVKKPGESVKISCKASGGSFRS B6.1 #5VH
YGISWVRQAPGQGLEWMGGITHFFGITDYAQ (B6-5VH)
KFQGRVTITADESTTTAYMELSGLTSDDTAV YYCAREPNDFWNGYYDTHDFDSWGQGTTVTV SS
pJP243 CDR-H1 Residues 31-35 SYGIS B6.1 #5VH of SEQ ID NO.: 546
pJP243 CDR-H2 Residues 50-66 GITHFFGITDYAQKFQG B6.1 #5VH of SEQ ID
NO.: 546 pJP243 CDR-H3 Residues 99-115 EPNDFWNGYYDTHDFDS B6.1 #5VH
of SEQ ID NO.: 546 547 pJP244 EIVLTQSPDFQSVTPKEKVTITCRASQNIGS B6.1
#5VK ELHWYQQKPDQSPKLLIKYASHSISGVPSRF (B6-5Vk)
SGSGSGTDFTLTINGLEAEDAATYYCHQSDT LPHTFGQGTKVDIK pJP244 CDR-L1
Residues 24-34 RASQNIGSELH B6.1 #5VK of SEQ ID NO.: 547 pJP244
CDR-L2 Residues 50-56 YASHSIS B6.1 #5VK of SEQ ID NO.: 547 pJP244
CDR-L3 Residues 84-97 ATYYCHQSDTLPHT B6.1 #5VK of SEQ ID NO.: 547
548 pJP245 EVQLVQSGAEVKKPGESLKISCKASGGSFSG B6.1
YGTSWVRQAPGQGLEWMGGITHFFGAVDYAQ #15VH
KFQGRVTITADESTTTAYMELSGLTSDDTAV (B6-15VH)
YYCARDPNEFWNGYYATHDFDYWGQGTTVTV SS pJP245 CDR-H1 Residues 31-35
GYGTS B6.1 of SEQ ID #15VH NO.: 548 pJP245 CDR-H2 Residues 50-66
GITHFFGAVDYAQKFQG B6.1 of SEQ ID #15VH NO.: 548 pJP245 CDR-H3
Residues 99-115 DPNEFWNGYYATHDFDY B6.1 of SEQ #15VH ID NO.: 548 549
pJP246 EIVLTQSPDFQSVTPKEKVTITCRASQNIGA B6.1
ELHWYQQKPDQSPKLLIKYASHSISGVPSRF #15Vk
SGSGSGTDFTLTINGLEAEDAGTYYCHQTDR (B6-15Vk) LPYSFGPGTKVDIK pJP246
CDR-L1 Residues 24-34 RASQNIGAELH B6.1 of SEQ ID #15Vk NO.: 549
pJP246 CDR-L2 Residues 50-56 YASHSIS B6.1 of SEQ ID #15Vk NO.: 549
pJP246 CDR-L3 Residues 89-97 HQTDRLPYS B6.1 of SEQ ID #15Vk NO.:
549 550 pJP247 EVQLVQSGAEVKMPGSSLKISCKASGGSFRG B6.1
YGISWVRQAPGQGLEWMGGITPFFGWADYAQ #16VH
KFQGRVTITADESTTTAYMELSGLTSDDTAV (B6-16VH)
YYCARDPNEFWNGYYDTHHFDYWGQGTTVTV SS pJP247 CDR-H1 Residues 31-35
GYGIS B6.1 of SEQ ID #16VH NO.: 550 pJP247 CDR-H2 Residues 50-66
GITPFFGWADYAQKFQG B6.1 of SEQ ID #16VH NO.: 550 pJP247 CDR-H3
Residues 99-115 DPNEFWNGYYDTHHFDY B6.1 of SEQ #16VH ID NO.: 550 551
pJP248 EIVLTQSPDFQSVTPKEKVTITCRASQDIGS B6.1
ALHWYQQKPDQSPKLLIKYASHSVSGVPSRF #16Vk
SGSGSGTDFTLTINGLEAEDAGTYYCHQSDI (B6-16Vk) LPHTFGPGTKVDIK pJP248
CDR-L1 Residues 24-34 RASQDIGSALH B6.1 of SEQ ID #16Vk NO.: 551
pJP248 CDR-L2 Residues 50-56 YASHSVS B6.1 of SEQ ID #16Vk NO.: 551
pJP248 CDR-L3 Residues 89-97 HQSDILPHT B6.1 of SEQ ID #16Vk NO.:
551 552 pJP249 EVQLVQSGAEVKKPGSSVKVSCKASGGSFGG B6.1
YGIGWVRQAPGQGLEWMGGITPFFGFADYAQ #17VH
KFQGRVTITADESTTTAYMELSGLTSDDTAV (B6-17VH)
YYCARDPNEFWNGYYSTHDFDSWGQGTTVTV SS pJP247 CDR-H1 Residues 31-35
GYGIG B6.1 of SEQ ID #16VH NO.: 552 pJP247 CDR-H2 Residues 50-66
GITPFFGFADYAQKFQG B6.1 of SEQ ID #16VH NO.: 552 pJP247 CDR-H3
Residues 99-115 DPNEFWNGYYSTHDFDS B6.1 of SEQ #16VH ID NO.: 552 553
pJP252 EIVLTQSPDFQSVTPKEKVTITCRASQDIGS B6.1
ELHWYQQKPDQPPKLLIKYASHSTSGVPSRF #17Vk
SGSGSGTDFTLTINGLEAEDAGTYYCHQTDS (B6-17Vk) LPYTFGPGTKVDIK pJP252
CDR-L1 Residues 24-34 RASQDIGSELH B6.1 of SEQ ID #17Vk NO.: 553
pJP252 CDR-L2 Residues 50-56 YASHSTS B6.1 of SEQ ID #17Vk NO.: 553
pJP252 CDR-L3 Residues 89-97 HQTDSLPYT B6.1 of SEQ ID #17Vk NO.:
553 554 pJP251 EVQLVQSGAEVKKPGESLKVSCKASGGRFRT B6.1
YGISWVRQAPGQGLEWMGGITNFFGWTDYAQ #18VH
KFQGRVTITADESTTTAYMELSGLTSDDTAV (B6-18VH)
YYCARVPNEFWNGYYATDYFDNWGQGTTVTV SS pJP251 CDR-H1 Residues 31-35
TYGIS B6.1 of SEQ ID #18VH NO.: 554 pJP251 CDR-H2 Residues 50-66
GITNFFGWTDYAQKFQG B6.1 of SEQ ID #18VH NO.: 554 pJP251 CDR-H3
Residues 99-115 VPNEFWNGYYATDYFDN B6.1 of SEQ #18VH ID NO.: 554 555
pJP250 EIVLTQSPDFQSVTPKEKVTITCRASQEIGA B6.1
SLHWYQQKPDQSPKLLIKYASHSASGVPSRF #18Vk
SGSGSGTDFTLTINSLEAEDAATYYCHQSDG (B6-18Vk) LPYTFGQGTKVDIK pJP250
CDR-L1 Residues 24-34 RASQEIGASLH B6.1 of SEQ ID #18Vk NO.: 555
pJP250 CDR-L2 Residues 50-56 YASHSAS B6.1 of SEQ ID #18Vk NO.: 555
pJP250 CDR-L3 Residues 89-97 HQSDGLPYT B6.1 of SEQ ID #18Vk NO.:
555 556 pJP253 EVQLVQSGAEVKKPGESLKISCKASGGSFIS B6.1
YGISWVRQAPGQGLEWMGGITHFFGSTDYAQ #46VH
KFQGRVTITADESTTTAYMELSGLTSDDTAV (B6-46VH)
YYCAREPNEFWNGYYTTHDFDSWGQGTTVTV SS pJP253 CDR-H1 Residues 31-35
SYGIS B6.1 of SEQ ID #46VH NO.: 556 pJP253 CDR-H2 Residues 50-66
GITHFFGSTDYAQKFQG B6.1 of SEQ ID #46VH NO.: 556 pJP253 CDR-H3
Residues 99-115 EPNEFWNGYYTTHDFDS B6.1 of SEQ #46VH ID NO.: 556 557
pJP254 EIVLTQSPDFQSVTPKEKVTITCRASQAIGS B6.1
ELHWYQQKPDQSPKLLIKYASYSVSGVPSRF #46Vk
SGSGSGTDFTLTINGLEAEDAGTYYCHQSAS (B6-46Vk) LPYTFGQGTKVDIK pJP254
CDR-L1 Residues 24-34 RASQAIGSELH B6.1 of SEQ ID #46Vk NO.: 557
pJP254 CDR-L2 Residues 50-56 YASYSVS B6.1 of SEQ ID #46Vk NO.: 557
pJP254 CDR-L3 Residues 89-97 HQSASLPYT B6.1 of SEQ ID #46Vk NO.:
557 558 pJP255 EVQLVQSGAEVKKPGSSVKVSCKASGGSFLS B6.1
YGFSWVRQAPGQGLEWMGGITPFFGLADYAQ #44VH
KFQGRVTITADESTTTAYMELSGLTSDDTAV (B6-44VH)
YYCARESSEFWNGYYCTQDFDLWGQGTTVTV SS pJP255 CDR-H1 Residues 31-35
SYGFS B6.1 of SEQ ID #44VH NO.: 558 pJP255 CDR-H2 Residues 50-66
GITPFFGLADYAQKFQG B6.1 of SEQ ID #44VH NO.: 558 pJP255 CDR-H3
Residues 99-115 ESSEFWNGYYCTQDFDL B6.1 of SEQ #44VH ID NO.: 559 559
pJP256 EIVLTQSPDFQSVTPKEKVTITCRASQDIGS B6.1
DLHWYQQKPDQSPKLLIKYASNSISGVPSRF #44Vk
SGSGSGTDFTLTINGLEAEDAGTYYCHQTAY (B6-44Vk) LPQTFGPGTKVDIK pJP256
CDR-L1 Residues 24-34 RASQDIGSDLH B6.1 of SEQ ID #44Vk NO.: 559
pJP256 CDR-L2 Residues 50-56 YASNSIS B6.1 of SEQ ID #44Vk NO.: 559
pJP256 CDR-L3 Residues 89-97 HQTAYLPQT B6.1 of SEQ ID #44Vk NO.:
559 560 B6.11 VH EVQLVQSGAEVKKPGSSVKVSCKASGGSFLS (pairs
YGFSWVRQAPGQGLEWMGGITPFFGFADYAQ with
KFQGRVTITADESTTTAYMELSGLTSDDTAV parental
YYCAREPNDFWNGYYTTHHFDYWGQGTTVTV VL, SEQ SS ID NO: 47) B6.11 VH
CDR-H1 Residues 31-35 SYGFS of SEQ ID NO.: 560 B6.11 VH CDR-H2
Residues 50-66 GITPFFGFADYAQKFQG of SEQ ID NO.: 560 B6.11 VH CDR-H3
Residues 99-115 EPNDFWNGYYTTHHFDY of SEQ ID NO.: 560 561 B6.29 VH
EVQLVQSGAEVKKPGSSVKVSCKASGGSFIS (pairs
YGYNWVRQAPGQGLEWMGGITPFFGTADYAQ with
KFQGRVTITADESTTTAYMELSGLTSDDTAV parental
YYCAREPNDFWNGYYTTHHFDYWGQGTTVTV VL, SEQ SS ID NO: 47) B6.29 VH
CDR-H1 Residues 31-35 SYGYN of SEQ ID NO.: 561 B6.29 VH CDR-H2
Residues 50-66 GITPFFGTADYAQKFQG of SEQ ID NO.: 561 B6.29 VH CDR-H3
Residues 99-115 EPNDFWNGYYTTHHFDY of SEQ ID NO.: 561
2.2: Functional Characterization of Human IL-17 Antibodies
2.2.1: IL-17 Enzyme-Linked Immunosorbent Assay Protocol
[1104] IL-17 binding by the human antibodies was assessed by ELISA
(assay described above, Example 1.5.1). Results are shown in the
table below.
TABLE-US-00029 TABLE 28 Binding of human antibodies to human IL-17
by ELISA MAb EC50 in hIL-17 ELISA (pM) IL-17-TN-K7-B6 447
IL-17-TN-L7-C8 517 IL-17-TN-L7-A7 131 IL-17-TN-L7-G9 165
IL-17-LN-K9-F5 7445 IL17-G9-1 130 IL17-G9-2 180 IL17-G9-3 50
IL17-G9-4 60 IL17-G9-5 120 IL17-G9-6 120 IL17-G9-M1 80 IL17-G9-M2
80 IL17-B6-5 220 IL17-B6-15 130 IL17-B6-16 160 IL17-B6-17 130
IL17-B6-18 160 IL17-B6-46 140 IL17-B6-11 58 IL17-B6-29 90
2.2.2: Neutralizing Potency of Human IL-17 Antibodies
[1105] Potency of several fully human antibodies identified by
PROfusion mRNA display technology from human antibody libraries was
assessed using the IL-17-driven IL-6 production in HS27 cells
(assay described above, Example 1.5.2). The table below summarizes
potencies to human IL-17A. It should be noted that the final
concentration of human IL-17A in these assays was 0.3 nM.
TABLE-US-00030 TABLE 29 Potency (nM) Anti-IL-17 Hu Hu Cyno MAb
IL-17A IL-17A/F IL-17A A7 1.8 1.8 12 B6 4.5 8.1 18 C8 4.5 2.6 18 G9
0.8 2.0 12
2.2.3: Neutralizing Potency of Affinity Matured Human
Antibodies
[1106] Potency of fully human antibodies derived from affinity
maturation of IL17-TN-L7-G9 and IL17-TN-K7-B6 was assessed using
IL-17-driven IL-6 production in HS27 cells for human and cyno
antigens (assay described above, Example 1.5.2). For murine, rat,
or rabbit IL-17A neutralization assay the IL-17 and TNF-induced
IL-6 secretion from murine embryonic fibroblast cell line NIH3T3
was used (assay described above, Example 1.5.3). The table below
summarizes the neutralizing potencies.
TABLE-US-00031 TABLE 30 Potency (pM) Anti-IL-17 Hu Hu Cyno Rat
Mouse Rabbit MAb IL-17A IL-17A/F IL-17A IL-17A IL-17A IL-17A B6-5
18 134 12 73 64 320 B6-11 20 71 33 6100 NI ND B6-15 24 162 10 17 ND
ND B6-16 26 114 13 167 ND ND B6-17 15 137 11 111 69 232 B6-29 134
ND ND ND ND ND B6-44 120 288 65 ND ND ND B6-46 59 ND ND ND ND ND
G9-1 27 99 604 NI NI ND G9-2 24 147 157 NI NI NI G9-3 334 ND ND ND
ND ND G9-4 38 119 247 NI NI ND G9-5 310 ND ND ND ND ND G9-6 884 ND
ND ND ND ND G9-M1 88 ND ND NI NI ND G9-M2 38 44 105 NI NI ND NI: no
inhibition. ND: not determined
2.2.3: Affinity Measurement of IL-17 Antibodies by Surface Plasmon
Resonance
[1107] The binding of human anti-IL-17 antibodies to purified
recombinant human (HuIL-17A), cyno (Cyno IL-17A), rat (RatIL-17A),
mouse (MuIL-17A), rabbit IL-17 (RabIL-17A), and human IL-17A/F
(HuIL-17A/F) was determined by surface plasmon resonance-based
measurements with a Biacore.RTM. 3000 instrument (Biacore.RTM. AB,
Uppsala, Sweden) using running HBS-EP (10 mM HEPES [pH 7.4], 150 mM
NaCl, 3 mM EDTA, and 0.005% surfactant P20) at 25.degree. C. as
described above (Example 1.5.4). The table below shows the affinity
measurements for selected human anti-human IL-17A antibodies.
TABLE-US-00032 TABLE 31 Affinity measure of anti-human IL-17
antibodies. IL-17 Antigen Human antibody HuIL-17A HuIL-17A/F Cyno
IL-17A RatIL-17A MuIL-17A RabIL-17A G9 (M) 2.80E-10 9.07E-10
2.07E-09 ND ND ND Kon (1/Ms) 3.09E+05 1.07E+05 9.60E+04 Koff (1/s)
8.65E-05 9.70E-05 1.99E-04 G9-1 (M) 2.57E-11 3.13E-12 2.96E-12 ND
ND ND Kon (1/Ms) 4.08E+05 3.20E+05 3.38E+05 Koff (1/s) 1.05E-05
1.00E-06 1.00E-06 G9-2 (M) <4.33E-12 4.37E-10 <2.70E-12 ND ND
ND Kon (1/Ms) 2.31E+05 1.98E+05 3.71E+05 Koff (1/s) <1.00E-6
8.66E-05 <1.00E-6 G9-4 (M) 4.69E-12 4.24E-12 3.77E-12 ND ND ND
Kon (1/Ms) 2.13E+05 2.36E+05 2.65E+05 Koff (1/s) 1.00E-06 1.00E-06
1.00E-06 G9-M1 (M) 3.22E-12 2.44E-10 2.65E-12 ND ND ND Kon (1/Ms)
3.11E+05 1.62E+05 3.77E+05 Koff (1/s) 1.00E-06 3.95E-05 1.00E-06
G9-M2 (M) 8.26E-12 1.03E-11 <2.45E-12 ND ND ND Kon (1/Ms)
2.81E+05 1.37E+05 4.08E+05 Koff (1/s) 2.32E-06 1.41E-06 <1.00E-6
B6 (M) 8.40E-10 2.19E-08 9.92E-10 ND ND ND Kon (1/Ms) 1.75E+04
7.98E+03 2.53E+04 Koff (1/s) 1.47E-05 1.75E-04 2.51E-05 B6-5 (M)
3.18E-12 2.22E-12 5.39E-12 9.05E-12 3.95E-11 3.17E-12 Kon (1/Ms)
7.20E+06 1.33E+07 1.21E+07 6.12E+06 2.58E+06 7.03E+06 Koff (1/s)
2.29E-05 2.95E-05 6.52E-05 5.54E-05 1.02E-04 2.23E-05 B6-15 (M)
7.30E-12 6.47E-12 4.01E-12 ND ND ND Kon (1/Ms) 8.25E+06 8.82E+06
1.37E+07 Koff (1/s) 6.02E-05 5.71E-05 5.50E-05 B6-16 (M) 7.94E-12
1.64E-11 4.83E-12 ND ND ND Kon (1/Ms) 3.78E+06 4.00E+06 9.27E+06
Koff (1/s) 3.00E-05 6.57E-05 4.48E-05 B6-17 (M) 7.85E-13 1.70E-12
1.03E-12 9.64E-12 1.13E-10 3.51E-12 Kon (1/Ms) 3.95E+06 7.23E+06
1.02E+07 6.13E+06 1.49E+06 9.58E+06 Koff (1/s) 3.10E-06 1.23E-05
1.05E-05 5.91E-05 1.69E-04 3.36E-05 ND: Not determined.
2.2.4: Pharmacokinetic Analysis of IL-17 Antibodies in Rat
[1108] Pharmacokinetic studies of human anti-human IL-17 antibodies
were carried out in Sprague Dawley rats. Male rats were dosed
intravenously with a single dose of 4 mg/kg of antibody proteins
and serum samples were analyzed using antigen capture based
chemiluminescent MSD (Meso Scale Discovery) method. Pharmacokinetic
parameters were calculated by non-compartmental analysis using
WinNonlin.
2.2.4.1: Preparation of Rat Serum
[1109] Surgically altered (jugular vein cannulated, JVC) and
regular male Sprague-Dawley Rats (approximately seven weeks old,
weighing 240-390 grams) were purchased from Charles River
Laboratories (Wilmington, Mass.). The animals were housed in rooms
maintained at constant temperature and humidity under 12 h
light/dark cycle, fed with normal rodent chow and were allowed food
and water ad libitum. Hydration and clinical conditions of the
animals were monitored daily.
[1110] Blood samples were collected (0.2 mL) at various timepoints,
allowed to clot for 30 minutes at room temperature, and centrifuged
for 8 minutes at 13,200 rpm. Then, serum was transferred to
eppendorf tubes and stored frozen at -80.degree. C.
2.2.4.2: MSD Assay Used to Quantify IL-17 Antibody in PK Serum
Samples
[1111] MSD streptavidin plates (Meso Scale Discovery) were washed
with phosphate buffered saline containing 0.05% Tween-20 (diluted
from 10.times.PBS, Abbott Bioresearch Center, Media Room,
Worcester, Mass. and Tween-20, Sigma, St. Louis, Mo.). Plates were
blocked with 150 .mu.L/well blocking solution (MSD Block, Meso
Scale Discovery, diluted to 3% final concentration in PBS) for 1
hour, covered, with shaking (600 rpm) at room temperature.
[1112] Prior to analysis, rat serum samples were thawed on ice,
mixed gently, and centrifuged at 14,000 rpm for 3 minutes at
4.degree. C. in an eppendorf centrifuge. Standard curve and control
samples were prepared in rat serum. Study samples, standard curve
samples, blanks, and quality control samples were incubated in
solution in a separate 2 mL deep well 96-well plate (Corning,
Corning, N.Y.) 1:1:1=V:V:V with biotinylated human IL-17(0.1 ug/mL
in assay buffer) and sulfo-tagged goat anti-human IgG (Meso Scale
Discovery, 1 .mu.g/mL in assay buffer) for 1 hour at room
temperature. The samples were then transferred to the MSD plates
and incubated for an additional hour with shaking (600 rpm) at room
temperature. The MSD plates were washed and developed with 2.times.
Read Buffer (Meso Scale Discovery). Chemiluminiscence was measured
within ten minutes on the MSD Sector Imager 6000.
[1113] Standard curves were analyzed using four-parameter logistic
fit and sample concentrations were calculated by XLfit4 software
version 2.2.1 Build 16, (Microsoft Corporation, Redmond, Wash.).
Pharmacokinetic parameters were calculated for each animal using
Winonlin software version 5.0.1 (Pharsight Corporation, Mountain
View, Calif.) by noncompartmental analysis.
[1114] The pharmacokinetic profile for selected human anti-human
IL-17A antibodies is shown in the table below.
TABLE-US-00033 TABLE 32 Pharmacokinetic profile of human IL-17
antibodies. T1/2 Vss CI mAb (day) (mL/kg) (ml/hr/kg) IL-17-B6-17
11.9 66 0.18 IL17-B6-5 11.8 68 0.18 IL-17-G9-2 11.1 130 0.37
2.3: Therapeutic Efficacy of IL-17 Antibodies
2.3.1: Neutralizing Potency of IL-17 Antibodies in Acute IL-17
Induced KC Model
[1115] Two of the fully human antibodies to human IL-17 were
evaluated in the acute in vivo rhIL17-induced KC model. Female
BALB/cJ mice were pre-dosed with antibodies intra-peritoneally
(i.p.), and 18 hrs later they were injected i.p. with 3 .mu.g
rhIL17 in a 500 .mu.L volume. After 1 hour, the mice were
sacrificed, and the levels of KC were assessed by MesoScale. ED50
values for % inhibition of KC were determined. As shown in Table W,
Both B6-5 and B6-17 fully neutralized IL-17 induced KC production
with ED50s of 7.5 and 17.1 mg/kg respectively.
TABLE-US-00034 TABLE 33 ED50 of anti-IL-17 antibodies in
recombinant human IL-17 induced KC model. Anti-IL-17 MAb ED50
(mg/kg) B6-5 7.5 B6-17 17.1
Example 3
Generation of TNF/IL-17 DVD-Ig.TM. Molecules
3.1: Construction of TNF/IL-17 DVD-Ig DNA Constructs
[1116] Anti-TNF antibody variable domains (D2E7) are combined with
multiple IL-17 antibody variable domains by overlapping PCR
amplification with intervening linker DNA sequences. The amplified
PCR products are subcloned into expression vectors suitable for
transient expression in HEK293 cells and the open reading frame
regions are confirmed by sequencing before DVD-Ig expression.
3.2: Expression and Production of TNF/IL17 DVD-Ig Binding
Proteins
[1117] After DNA confirmation by sequencing, all DVD-Ig DNA
constructs were expanded in E. coli and DNA are purified using
Qiagen Hispeed Maxi Prep (CAT#12662, QIAGEN). DVD-Ig DNA was
transfected into log phase 293E cells (0.5.times.10.sup.6/ml,
viability >95%) by mixing PEI and DNA @ 2:1 ratio with 0.2
.mu.g/ml heavy chain DNA and 0.3 .mu.g/ml light chain DNA. DNA:PEI
complex was formed at room temperature in TC hood for fifteen
minutes before adding to 293E cells. Twenty four later, 0.5% TN1
was added to 293E cells. At day five, supernatant was collected for
human IgG1 titer measurement. Cell supernatant was harvested at day
seven and filtered through 0.2 .mu.M PES filter. Supernatant was
purified by using Protein A Sepharose Affinity Chromatography
according to manufacturer's instruction. Purified DVD-Igs were
eluted off the column by 0.1 M glycine (pH 2.99) and dialyzed into
15 mM histidine buffer (pH 6.0) immediately. The binding proteins
were quantitated by A280 and analyzed by mass spectrometry and
SEC.
3.3: Sequences of TNF/IL-17 DVD-12 Constructs
[1118] Amino acid sequence of heavy chain and light chain of DVD-Ig
proteins capable of binding human TNF and hIL-17 were determined.
The amino acid sequences of variable heavy chains, variable light
chains, and constant regions of TNF/IL-17 DVD-Ig binding proteins
are shown in the table below.
TABLE-US-00035 TABLE 34 Sequences of variable and constant regions
of TNF/IL-17 DVD-Ig binding proteins. Protein Sequence Protein
region Sequence Identifier 12345678901234567890 DVD HEAVY SEQ ID
NO.: 562 EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA
D2-GS6-B6 DVD PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS SGGGGSGEVQLVQSGAEVKK
PGESLKISCKASGGSFRSYG ISWVRQAPGQGLEWMGGITP ILGTANYAQKFQGRVTITAD
ESTTTAYMELSGLTSDDTAV YYCAREPNDFWNGYYTTHHF DYWGQGTPVTVSS D2 VH SEQ
ID NO.: 563 EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA
PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS S LINKER SEQ ID NO.: 564 GGGGSG B6-VH SEQ ID
NO.: 565 EVQLVQSGAEVKKPGESLKI SCKASGGSFRSYGISWVRQA
PGQGLEWMGGITPILGTANY AQKFQGRVTITADESTTTAY MELSGLTSDDTAVYYCAREP
NDFWNGYYTTHHFDYWGQGT PVTVSS CH SEQ ID NO.: 566 ASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD LIGHT SEQ ID NO.: 567 DIQMTQSPSSLSASVGDRVT VARIABLE
ITCRASQGIRNYLAWYQQKP E7-SL-B6 DVD GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKRGGSGGDVVMTQS
PDFQSVTPKEKVTITCRASQ NIGSALHWYQQKPDQSPKLL IKYASQSISGVPSRFSGSGS
GTDFTLTINGLEAEDAGTYY CHQSTSLPHTFGQGTKLDIK R E7 VL SEQ ID NO.: 568
DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID
NO.: 569 GGSGG B6 VL SEQ ID NO.: 570 DVVMTQSPDFQSVTPKEKVT
ITCRASQNIGSALHWYQQKP DQSPKLLIKYASQSISGVPS RFSGSGSGTDFTLTINGLEA
EDAGTYYCHQSTSLPHTFGQ GTKLDIKR CL SEQ ID NO.: 571
TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID
NO.: 572 EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA
D2-SL-B6 DVD PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS SASTKGPEVQLVQSGAEVKK
PGESLKISCKASGGSFRSYG ISWVRQAPGQGLEWMGGITP ILGTANYAQKFQGRVTITAD
ESTTTAYMELSGLTSDDTAV YYCAREPNDFWNGYYTTHHF DYWGQGTPVTVSS D2 VH SEQ
ID NO.: 573 EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA
PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS S LINKER SEQ ID NO.: 574 ASTKGP B6-VH SEQ ID
NO.: 575 EVQLVQSGAEVKKPGESLKI SCKASGGSFRSYGISWVRQA
PGQGLEWMGGITPILGTANY AQKFQGRVTITADESTTTAY MELSGLTSDDTAVYYCAREP
NDFWNGYYTTHHFDYWGQGT PVTVSS CH SEQ ID NO.: 576 ASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD HEAVY SEQ ID NO.: 577 EVQLVESGGGLVQPGRSLRL VARIABLE
SCAASGFTFDDYAMHWVRQA D2-GS6-G9-DVD PGKGLEWVSAITWNSGHIDY
ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS
SGGGGSGEEVQLLESGGGVV QPGRSLRLSCAASGFIFSNY GMHWVRQAPGKGLEWVAVIS
YDGSNKYYADSVKGRFTISR DNSKNTLYLEMNSLRPEDTA VYYCAKVGASGDYYYSYGLD
VWGQGTTVTVSS D2 VH SEQ ID NO.: 578 EVQLVESGGGLVQPGRSLRL
SCAASGFTFDDYAMHWVRQA PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS S LINKER SEQ ID NO.: 579
GGGGSG G9 VH SEQ ID NO.: 580 EVQLLESGGGVVQPGRSLRL
SCAASGFIFSNYGMHWVRQA PGKGLEWVAVISYDGSNKYY ADSVKGRFTISRDNSKNTLY
LEMNSLRPEDTAVYYCAKVG ASGDYYYSYGLDVWGQGTTV TVSS CH SEQ ID NO.: 581
ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ ID NO.: 582
DIQMTQSPSSLSASVGDRVT VARIABLE ITCRASQGIRNYLAWYQQKP E7-GS6-G9 DVD
GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ
GTKVEIKRGGSGGQSVLTQP PSASGTPGQTVSISCSGSNS NIGSHSVNWYQQVPGAAPKL
LMYGIGQRPSGVPDRFSVSQ SGTSASLAISGLQSEDEADY YCATWDDSLGGYVFGSGTKV
TVLGQPKAAPSVTLFPPSSE ELQANKATLVCLISDFYPGA VTVAWKADSSPVKAGVETTT
PSKQSNNKYAASSYLSLTPE QWKSHRSYSCQVTHEGSTVE KTVAPTECS E7 VL SEQ ID
NO.: 583 DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP
GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ
GTKVEIKR LINKER SEQ ID NO.: 584 GGSGG G9 VL SEQ ID NO.: 585
QSVLTQPPSASGTPGQTVSI SCSGSNSNIGSHSVNWYQQV PGAAPKLLMYGIGQRPSGVP
DRFSVSQSGTSASLAISGLQ SEDEADYYCATWDDSLGGYV FGSGTKVTVLG CL SEQ ID
NO.: 586 TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS
FNRGEC DVD LIGHT SEQ ID NO.: 587 DIQMTQSPSSLSASVGDRVT VARIABLE
ITCRASQGIRNYLAWYQQKP E7-SL-G9 DVD GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKRTVAAPQSVLTQP
PSASGTPGQTVSISCSGSNS NIGSHSVNWYQQVPGAAPKL LMYGIGQRPSGVPDRFSVSQ
SGTSASLAISGLQSEDEADY YCATWDDSLGGYVFGSGTKV TVLG E7 VL SEQ ID NO.:
588 DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP
GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID
NO.: 589 TVAAP G9 VL SEQ ID NO.: 590 QSVLTQPPSASGTPGQTVSI
SCSGSNSNIGSHSVNWYQQV PGAAPKLLMYGIGQRPSGVP DRFSVSQSGTSASLAISGLQ
SEDEADYYCATWDDSLGGYV FGSGTKVTVLG CL SEQ ID NO.: 591
TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID
NO.: 592 EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA
D2-SL-G9 DVD PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS SASTKGPEVQLLESGGGVVQ
PGRSLRLSCAASGFIFSNYG MHWVRQAPGKGLEWVAVISY DGSNKYYADSVKGRFTISRD
NSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDV WGQGTTVTVSS D2 VH SEQ ID
NO.: 593 EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA
PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS S LINKER SEQ ID NO.: 594 ASTKGP G9 VH SEQ ID
NO.: 595 EVQLLESGGGVVQPGRSLRL SCAASGFIFSNYGMHWVRQA
PGKGLEWVAVISYDGSNKYY ADSVKGRFTISRDNSKNTLY LEMNSLRPEDTAVYYCAKVG
ASGDYYYSYGLDVWGQGTTV TVSS CH SEQ ID NO.: 596 ASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD LIGHT SEQ ID NO.: 597 DIQMTQSPSSLSASVGDRVT VARIABLE
ITCRASQGIRNYLAWYQQKP E7-GS6-G9-1 DVD GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKRGGSGGQSVLTQP
PSASGTPGQTVSISCSGSHS NIGRHPVDWYQQVPGAAPKL LMYYGGYRPSGVPDRFSGSQ
SGTSASLAISGLQSEDEADY YCATWDDRLVGYVFGSGTKV TVLG E7 VL SEQ ID NO.:
598 DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID
NO.: 599 GGSGG G9-1 VL SEQ ID NO.: 600 QSVLTQPPSASGTPGQTVSI
SCSGSHSNIGRHPVDWYQQV PGAAPKLLMYYGGYRPSGVP DRFSGSQSGTSASLAISGLQ
SEDEADYYCATWDDRLVGYV FGSGTKVTVLG CL SEQ ID NO.: 601
TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD LIGHT SEQ ID
NO.: 602 DIQMTQSPSSLSASVGDRVT VARIABLE ITCRASQGIRNYLAWYQQKP
E7-SL-G9-1 DVD GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP
EDVATYYCQRYNRAPYTFGQ GTKVEIKRTVAAPQSVLTQP PSASGTPGQTVSISCSGSHS
NIGRHPVDWYQQVPGAAPKL LMYYGGYRPSGVPDRFSGSQ SGTSASLAISGLQSEDEADY
YCATWDDRLVGYVFGSGTKV TVLG E7 VL SEQ ID NO.: 603
DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID
NO.: 604 TVAAP G9-1 VL SEQ ID NO.: 605 QSVLTQPPSASGTPGQTVS
ISCSGSHSNIGRHPVDWYQ QVPGAAPKLLMYYGGYRPS GVPDRFSGSQSGTSASLAI
SGLQSEDEADYYCATWDDR LVGYVFGSGTKVTVLG CL SEQ ID NO.: 606
TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD LIGHT SEQ ID
NO.: 607 DIQMTQSPSSLSASVGDRVT VARIABLE ITCRASQGIRNYLAWYQQKP
E7-GS6-G9-4 DVD GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP
EDVATYYCQRYNRAPYTFGQ GTKVEIKRGGSGGQSVLTQP PSASGTPGQTVSISCSGRQS
NIGRHYVDWYQQVPGAAPKL LMYYDSIRPSGVPDRFSGSQ SGTSASLAISGLQSEDEADY
YCATWDDSLGGYVFGSGTKV TVLG E7 VL SEQ ID NO.: 608
DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID
NO.: 609 GGSGG G9-4 VL SEQ ID NO.: 610 QSVLTQPPSASGTPGQTVSI
SCSGRQSNIGRHYVDWYQQV PGAAPKLLMYYDSIRPSGVP DRFSGSQSGTSASLAISGLQ
SEDEADYYCATWDDSLGGYV FGSGTKVTVLG CL SEQ ID NO.: 611
TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD LIGHT SEQ ID
NO.: 612 DIQMTQSPSSLSASVGDRVT VARIABLE ITCRASQGIRNYLAWYQQKP
E7-SL-G9-4 DVD GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP
EDVATYYCQRYNRAPYTFGQ GTKVEIKRTVAAPQSVLTQP PSASGTPGQTVSISCSGRQS
NIGRHYVDWYQQVPGAAPKL LMYYDSIRPSGVPDRFSGSQ SGTSASLAISGLQSEDEADY
YCATWDDSLGGYVFGSGTKV TVLG E7 VL SEQ ID NO.: 613
DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID
NO.: 614 TVAAP G9-4 VL SEQ ID NO.: 615 QSVLTQPPSASGTPGQTVSI
SCSGRQSNIGRHYVDWYQQV PGAAPKLLMYYDSIRPSGVP DRFSGSQSGTSASLAISGLQ
SEDEADYYCATWDDSLGGYV FGSGTKVTVLG CL SEQ ID NO.: 616
TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD LIGHT SEQ ID
NO.: 617 DIQMTQSPSSLSASVGDRVT VARIABLE ITCRASQGIRNYLAWYQQKP
E7-GS6-G9-M2 GKAPKLLIYAASTLQSGVPS DVD RFSGSGSGTDFTLTISSLQP
EDVATYYCQRYNRAPYTFGQ GTKVEIKRGGSGGQSVLTQP PSASGTPGQTVSISCSGHNS
NIGYHYVHWYQQVPGAAPKL LIYGDGWRPSGVPDRFSGSQ SGTSASLAISGLQSEDEADY
YCGTWDDWLGGYVFGTGTKV TVLG E7 VL SEQ ID NO.: 618
DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID
NO.: 619 GGSGG G9-M2 VL SEQ ID NO.: 620 QSVLTQPPSASGTPGQTVSI
SCSGHNSNIGYHYVHWYQQV PGAAPKLLIYGDGWRPSGVP DRFSGSQSGTSASLAISGLQ
SEDEADYYCGTWDDWLGGYV FGTGTKVTVLG CL SEQ ID NO.: 621
TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD LIGHT SEQ ID
NO.: 622 DIQMTQSPSSLSASVGDRVT
VARIABLE ITCRASQGIRNYLAWYQQKP E7-SL-G9-M2 DVD GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKRTVAAPQSVLTQP
PSASGTPGQTVSISCSGHNS NIGYHYVHWYQQVPGAAPKL LIYGDGWRPSGVPDRFSGSQ
SGTSASLAISGLQSEDEADY YCGTWDDWLGGYVFGTGTKV TVLG E7 VL SEQ ID NO.:
623 DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID
NO.: 624 TVAAP G9-M2 VL SEQ ID NO.: 625 QSVLTQPPSASGTPGQTVSI
SCSGHNSNIGYHYVHWYQQV PGAAPKLLIYGDGWRPSGVP DRFSGSQSGTSASLAISGLQ
SEDEADYYCGTWDDWLGGYV FGTGTKVTVLG CL SEQ ID NO.: 626
TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID
NO.: 627 EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA
D2-GS10-G9-2 DVD PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS SGGGGSGGGGSEVQLLESGG
GVVQPGRSLRLSCAASGFIF RNYGMHWVRQAPGKGLEWVA VISYDGSNKYYADSVKGRFT
ISRDNSKNTLYLEMNSLRPE DTAVYYCAKVGASGDYYYSY GLDVWGQGTTVTVSS D2 VH SEQ
ID NO.: 628 EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA
PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS S LINKER SEQ ID NO.: 629 GGGGSGGGGS G9-2 VH
SEQ ID NO.: 630 EVQLLESGGGVVQPGRSLRL SCAASGFIFRNYGMHWVRQA
PGKGLEWVAVISYDGSNKYY ADSVKGRFTISRDNSKNTLY LEMNSLRPEDTAVYYCAKVG
ASGDYYYSYGLDVWGQGTTV TVSS CH SEQ ID NO.: 631 ASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD LIGHT SEQ ID NO.: 632 DIQMTQSPSSLSASVGDRVT VARIABLE
ITCRASQGIRNYLAWYQQKP E7-GS10-G9-2 DVD GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKRGGSGGGGSGSQS
VLTQPPSASGTPGQTVSISC SGSNSNIGRHPVDWYQQVPG AAPKLLIYYDDQRPSGVPDR
FSGSQSGTSASLAISGLQSE DEADYYCATWDDSLGGYVFG SGTKVTVLG E7 VL SEQ ID
NO.: 633 DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP
GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ
GTKVEIKR LINKER SEQ ID NO.: 634 GGSGGGGSGS G9-2 VL SEQ ID NO.: 635
QSVLTQPPSASGTPGQTVSI SCSGSNSNIGRHPVDWYQQV PGAAPKLLIYYDDQRPSGVP
DRFSGSQSGTSASLAISGLQ SEDEADYYCATWDDSLGGYV FGSGTKVTVLG CL SEQ ID
NO.: 636 TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS
FNRGEC DVD HEAVY SEQ ID NO.: 637 EVQLVESGGGLVQPGRSLRL VARIABLE
SCAASGFTFDDYAMHWVRQA D2-GS6-G9-2 DVD PGKGLEWVSAITWNSGHIDY
ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS
SGGGGSGEVQLLESGGGVVQ PGRSLRLSCAASGFIFRNYG MHWVRQAPGKGLEWVAVISY
DGSNKYYADSVKGRFTISRD NSKNTLYLEMNSLRPEDTAV YYCAKVGASGDYYYSYGLDV
WGQGTTVTVSS D2 VH SEQ ID NO.: 638 EVQLVESGGGLVQPGRSLRL
SCAASGFTFDDYAMHWVRQA PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS S LINKER SEQ ID NO.: 639
GGGGSG G9-2 VH SEQ ID NO.: 640 EVQLLESGGGVVQPGRSLRL
SCAASGFIFRNYGMHWVRQA PGKGLEWVAVISYDGSNKYY ADSVKGRFTISRDNSKNTLY
LEMNSLRPEDTAVYYCAKVG ASGDYYYSYGLDVWGQGTTV TVSS CH SEQ ID NO.: 641
ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ ID NO.: 642
DIQMTQSPSSLSASVGDRVT VARIABLE ITCRASQGIRNYLAWYQQKP E7-GS6-G9-2 DVD
GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ
GTKVEIKRGGSGGQSVLTQP PSASGTPGQTVSISCSGSNS NIGRHPVDWYQQVPGAAPKL
LIYYDDQRPSGVPDRFSGSQ SGTSASLAISGLQSEDEADY YCATWDDSLGGYVFGSGTKV TVLG
E7 VL SEQ ID NO.: 643 DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP
GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ
GTKVEIKR LINKER SEQ ID NO.: 644 GGSGG G9-2 VL SEQ ID NO.: 645
QSVLTQPPSASGTPGQTVSI SCSGSNSNIGRHPVDWYQQV PGAAPKLLIYYDDQRPSGVP
DRFSGSQSGTSASLAISGLQ SEDEADYYCATWDDSLGGYV FGSGTKVTVLG CL SEQ ID
NO.: 646 TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS
FNRGEC DVD HEAVY SEQ ID NO.: 647 EVQLVESGGGLVQPGRSLRL VARIABLE
SCAASGFTFDDYAMHWVRQA D2-GS14-G9-2 DVD PGKGLEWVSAITWNSGHIDY
ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS
SGGGGSGGGGSGGGGEVQLL ESGGGVVQPGRSLRLSCAAS GFIFRNYGMHWVRQAPGKGL
EWVAVISYDGSNKYYADSVK GRFTISRDNSKNTLYLEMNS LRPEDTAVYYCAKVGASGDY
YYSYGLDVWGQGTTVTVSS D2 VH SEQ ID NO.: 648 EVQLVESGGGLVQPGRSLRL
SCAASGFTFDDYAMHWVRQA PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS S LINKER SEQ ID NO.: 649
GGGGSGGGGSGGGG G9-2 VH SEQ ID NO.: 650 EVQLLESGGGVVQPGRSLRL
SCAASGFIFRNYGMHWVRQA PGKGLEWVAVISYDGSNKYY ADSVKGRFTISRDNSKNTLY
LEMNSLRPEDTAVYYCAKVG ASGDYYYSYGLDVWGQGTTV TVSS CH SEQ ID NO.: 651
ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ ID NO.: 652
DIQMTQSPSSLSASVGDRVT VARIABLE ITCRASQGIRNYLAWYQQKP E7-GS14-G9-2 DVD
GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ
GTKVEIKRGGSGGGGSGGGG SQSVLTQPPSASGTPGQTVS ISCSGSNSNIGRHPVDWYQQ
VPGAAPKLLIYYDDQRPSGV PDRFSGSQSGTSASLAISGL QSEDEADYYCATWDDSLGGY
VFGSGTKVTVLG E7 VL SEQ ID NO.: 653 DIQMTQSPSSLSASVGDRVT
ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP
EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID NO.: 654 GGSGGGGSGGGGS
G9-2VL SEQ ID NO.: 655 QSVLTQPPSASGTPGQTVSI SCSGSNSNIGRHPVDWYQQV
PGAAPKLLIYYDDQRPSGVP DRFSGSQSGTSASLAISGLQ SEDEADYYCATWDDSLGGYV
FGSGTKVTVLG CL SEQ ID NO.: 656 TVAAPSVFIFPPSDEQLKSG
TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID NO.: 657
EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA D2-SL-G9-2 DVD
PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS SASTKGPEVQLLESGGGVVQ PGRSLRLSCAASGFIFRNYG
MHWVRQAPGKGLEWVAVISY DGSNKYYADSVKGRFTISRD NSKNTLYLEMNSLRPEDTAV
YYCAKVGASGDYYYSYGLDV WGQGTTVTVSS D2 VH SEQ ID NO.: 658
EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA PGKGLEWVSAITWNSGHIDY
ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS S
LINKER SEQ ID NO. 659 ASTKGP G9-2 VH SEQ ID NO.: 660
EVQLLESGGGVVQPGRSLRL SCAASGFIFRNYGMHWVRQA PGKGLEWVAVISYDGSNKYY
ADSVKGRFTISRDNSKNTLY LEMNSLRPEDTAVYYCAKVG ASGDYYYSYGLDVWGQGTTV TVSS
CH SEQ ID NO.: 661 ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ
ID NO.: 662 DIQMTQSPSSLSASVGDRVT VARIABLE ITCRASQGIRNYLAWYQQKP
E7-SL-G9-2 DVD GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP
EDVATYYCQRYNRAPYTFGQ GTKVEIKRTVAAPQSVLTQP PSASGTPGQTVSISCSGSNS
NIGRHPVDWYQQVPGAAPKL LIYYDDQRPSGVPDRFSGSQ SGTSASLAISGLQSEDEADY
YCATWDDSLGGYVFGSGTKV TVLG E7 VL SEQ ID NO.: 663
DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID
NO.: 664 TVAAP G9-2 VL SEQ ID NO.: 665 QSVLTQPPSASGTPGQTVSI
SCSGSNSNIGRHPVDWYQQV PGAAPKLLIYYDDQRPSGVP DRFSGSQSGTSASLAISGLQ
SEDEADYYCATWDDSLGGYV FGSGTKVTVLG CL SEQ ID NO.: 666
TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID
NO.: 667 EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA
D2-GS10-B6-17 DVD PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS SGGGGSGGGGSEVQLVQSGA
EVKKPGSSVKVSCKASGGSF GGYGIGWVRQAPGQGLEWMG GITPFFGFADYAQKFQGRVT
ITADESTTTAYMELSGLTSD DTAVYYCARDPNEFWNGYYS THDFDSWGQGTTVTVSS D2 VH
SEQ ID NO.: 668 EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA
PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS S LINKER SEQ ID NO.: 669 GGGGSGGGGS B6-17 VH
SEQ ID NO.: 670 EVQLVQSGAEVKKPGSSVKV SCKASGGSFGGYGIGWVRQA
PGQGLEWMGGITPFFGFADY AQKFQGRVTITADESTTTAY MELSGLTSDDTAVYYCARDP
NEFWNGYYSTHDFDSWGQGT TVTVSS CH SEQ ID NO.: 671 ASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD LIGHT SEQ ID NO.: 672 DIQMTQSPSSLSASVGDRVT VARIABLE
ITCRASQGIRNYLAWYQQKP E7-GS10-B6-17 GKAPKLLIYAASTLQSGVPS DVD
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKRGGSGGGGSGSEI
VLTQSPDFQSVTPKEKVTIT CRASQDIGSELHWYQQKPDQ PPKLLIKYASHSTSGVPSRF
SGSGSGTDFTLTINGLEAED AGTYYCHQTDSLPYTFGPGT KVDIKR E7 VL SEQ ID NO.:
673 DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID
NO.: 674 GGSGGGGSGS B6-17 VL SEQ ID NO.: 675 EIVLTQSPDFQSVTPKEKVT
ITCRASQDIGSELHWYQQKP DQPPKLLIKYASHSTSGVPS RFSGSGSGTDFTLTINGLEA
EDAGTYYCHQTDSLPYTFGP GTKVDIKR CL SEQ ID NO.: 676
TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID
NO.: 677 EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA
D2-GS6-B6-17 DVD PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS SGGGGSGEVQLVQSGAEVKK
PGSSVKVSCKASGGSFGGYG IGWVRQAPGQGLEWMGGITP FFGFADYAQKFQGRVTITAD
ESTTTAYMELSGLTSDDTAV YYCARDPNEFWNGYYSTHDF DSWGQGTTVTVSS D2 VH SEQ
ID NO.: 678 EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA
PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS S LINKER SEQ ID NO.: 679 GGGGSG B6-17 VH SEQ
ID NO.: 680 EVQLVQSGAEVKKPGSSVKV SCKASGGSFGGYGIGWVRQA
PGQGLEWMGGITPFFGFADY AQKFQGRVTITADESTTTAY MELSGLTSDDTAVYYCARDP
NEFWNGYYSTHDFDSWGQGT TVTVSS CH SEQ ID NO.: 681 ASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ
ID NO.: 682 DIQMTQSPSSLSASVGDRVT VARIABLE ITCRASQGIRNYLAWYQQKP
E7-GS6-B6-17 DVD GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP
EDVATYYCQRYNRAPYTFGQ GTKVEIKRGGSGGEIVLTQS PDFQSVTPKEKVTITCRASQ
DIGSELHWYQQKPDQPPKLL IKYASHSTSGVPSRFSGSGS GTDFTLTINGLEAEDAGTYY
CHQTDSLPYTFGPGTKVDIK R E7 VL SEQ ID NO.: 683 DIQMTQSPSSLSASVGDRVT
ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP
EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID NO.: 684 GGSGG B6-17 VL
SEQ ID NO.: 685 EIVLTQSPDFQSVTPKEKVT ITCRASQDIGSELHWYQQKP
DQPPKLLIKYASHSTSGVPS RFSGSGSGTDFTLTINGLEA EDAGTYYCHQTDSLPYTFGP
GTKVDIKR CL SEQ ID NO.: 686 TVAAPSVFIFPPSDEQLKSG
TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID NO.: 687
EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA D2-GS14-B6-17
DVD PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS SGGGGSGGGGSGGGGEVQLV QSGAEVKKPGSSVKVSCKAS
GGSFGGYGIGWVRQAPGQGL EWMGGITPFFGFADYAQKFQ GRVTITADESTTTAYMELSG
LTSDDTAVYYCARDPNEFWN GYYSTHDFDSWGQGTTVTVS S D2 VH SEQ ID NO.: 688
EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA PGKGLEWVSAITWNSGHIDY
ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS S
LINKER SEQ ID NO.: 689 GGGGSGGGGSGGGG B6-17 VH SEQ ID NO.: 690
EVQLVQSGAEVKKPGSSVKV SCKASGGSFGGYGIGWVRQA PGQGLEWMGGITPFFGFADY
AQKFQGRVTITADESTTTAY MELSGLTSDDTAVYYCARDP NEFWNGYYSTHDFDSWGQGT
TVTVSS CH SEQ ID NO.: 691 ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ
ID NO.: 692 DIQMTQSPSSLSASVGDRVT VARIABLE ITCRASQGIRNYLAWYQQKP
E7-GS14-B6-17 GKAPKLLIYAASTLQSGVPS DVD RFSGSGSGTDFTLTISSLQP
EDVATYYCQRYNRAPYTFGQ GTKVEIKRGGSGGGGSGGGG SEIVLTQSPDFQSVTPKEKV
TITCRASQDIGSELHWYQQK PDQPPKLLIKYASHSTSGVP SRFSGSGSGTDFTLTINGLE
AEDAGTYYCHQTDSLPYTFG PGTKVDIKR E7 VL SEQ ID NO.: 693
DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID
NO.: 694 GGSGGGGSGGGGS B6-17 VL SEQ ID NO.: 695
EIVLTQSPDFQSVTPKEKVT ITCRASQDIGSELHWYQQKP DQPPKLLIKYASHSTSGVPS
RFSGSGSGTDFTLTINGLEA EDAGTYYCHQTDSLPYTFGP GTKVDIKR CL SEQ ID NO.:
696 TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID
NO.: 697 EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA
D2-SL-B6-17 DVD PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS SASTKGPEVQLVQSGAEVKK
PGSSVKVSCKASGGSFGGYG IGWVRQAPGQGLEWMGGITP FFGFADYAQKFQGRVTITAD
ESTTTAYMELSGLTSDDTAV YYCARDPNEFWNGYYSTHDF DSWGQGTTVTVSS D2 VH SEQ
ID NO.: 698 EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA
PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS S LINKER SEQ ID NO.: 699 ASTKGP B6-17 VH SEQ
ID NO.: 700 EVQLVQSGAEVKKPGSSVKV SCKASGGSFGGYGIGWVRQA
PGQGLEWMGGITPFFGFADY AQKFQGRVTITADESTTTAY MELSGLTSDDTAVYYCARDP
NEFWNGYYSTHDFDSWGQGT TVTVSS CH SEQ ID NO.: 701 ASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD LIGHT SEQ ID NO.: 702 DIQMTQSPSSLSASVGDRVT VARIABLE
ITCRASQGIRNYLAWYQQKP E7-SL-B6-17 DVD GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKRTVAAPEIVLTQS
PDFQSVTPKEKVTITCRASQ DIGSELHWYQQKPDQPPKLL IKYASHSTSGVPSRFSGSGS
GTDFTLTINGLEAEDAGTYY CHQTDSLPYTFGPGTKVDIK R E7 VL SEQ ID NO.: 703
DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID
NO.: 704 TVAAP B6-17 VL SEQ ID NO.: 705 EIVLTQSPDFQSVTPKEKVT
ITCRASQDIGSELHWYQQKP DQPPKLLIKYASHSTSGVPS RFSGSGSGTDFTLTINGLEA
EDAGTYYCHQTDSLPYTFGP GTKVDIKR CL SEQ ID NO.: 706
TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID
NO.: 707 EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA
D2-LL-B6-17 DVD PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS SASTKGPSVFPLAPEVQLVQ
SGAEVKKPGSSVKVSCKASG GSFGGYGIGWVRQAPGQGLE WMGGITPFFGFADYAQKFQG
RVTITADESTTTAYMELSGL TSDDTAVYYCARDPNEFWNG YYSTHDFDSWGQGTTVTVSS D2
VH SEQ ID NO.: 708 EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA
PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS S LINKER SEQ ID NO.: 709 ASTKGPSVFPLAP B6-17
VH SEQ ID NO.: 710 EVQLVQSGAEVKKPGSSVKV SCKASGGSFGGYGIGWVRQA
PGQGLEWMGGITPFFGFADY AQKFQGRVTITADESTTTAY MELSGLTSDDTAVYYCARDP
NEFWNGYYSTHDFDSWGQGT TVTVSS CH SEQ ID NO.: 711 ASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD LIGHT SEQ ID NO.: 712 DIQMTQSPSSLSASVGDRVT VARIABLE
ITCRASQGIRNYLAWYQQKP E7-LL-B6-17 DVD GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKRTVAAPSVFIFPP
EIVLTQSPDFQSVTPKEKVT ITCRASQDIGSELHWYQQKP DQPPKLLIKYASHSTSGVPS
RFSGSGSGTDFTLTINGLEA EDAGTYYCHQTDSLPYTFGP GTKVDIKR E7 VL SEQ ID
NO.: 713 DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP
GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ
GTKVEIKR LINKER SEQ ID NO.: 714 TVAAPSVFIFPP B6-17 VL SEQ ID NO.:
715 EIVLTQSPDFQSVTPKEKVT ITCRASQDIGSELHWYQQKP DQPPKLLIKYASHSTSGVPS
RFSGSGSGTDFTLTINGLEA EDAGTYYCHQTDSLPYTFGP GTKVDIKR CL SEQ ID NO.:
716 TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID
NO.: 717 EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA
D2-GS10-B6-5 DVD PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS SGGGGSGGGGSEVQLVQSGA
EVKKPGESVKISCKASGGSF RSYGISWVRQAPGQGLEWMG GITHFFGITDYAQKFQGRVT
ITADESTTTAYMELSGLTSD DTAVYYCAREPNDFWNGYYD THDFDSWGQGTTVTVSS D2 VH
SEQ ID NO.: 718 EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA
PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS S LINKER SEQ ID NO.: 719 GGGGSGGGGS B6-5 VH
SEQ ID NO.: 720 EVQLVQSGAEVKKPGESVKI SCKASGGSFRSYGISWVRQA
PGQGLEWMGGITHFFGITDY AQKFQGRVTITADESTTTAY MELSGLTSDDTAVYYCAREP
NDFWNGYYDTHDFDSWGQGT TVTVSS CH SEQ ID NO.: 721 ASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD LIGHT SEQ ID NO.: 722 DIQMTQSPSSLSASVGDRVT VARIABLE
E7- ITCRASQGIRNYLAWYQQKP GS10-B6-5 DVD GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKRGGSGGGGSGSEI
VLTQSPDFQSVTPKEKVTIT CRASQNIGSELHWYQQKPDQ SPKLLIKYASHSISGVPSRF
SGSGSGTDFTLTINGLEAED AATYYCHQSDTLPHTFGQGT KVDIKR E7 VL SEQ ID NO.:
723 DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID
NO.: 724 GGSGGGGSGS B6-5 VL SEQ ID NO.: 725 EIVLTQSPDFQSVTPKEKVT
ITCRASQNIGSELHWYQQKP DQSPKLLIKYASHSISGVPS RFSGSGSGTDFTLTINGLEA
EDAATYYCHQSDTLPHTFGQ GTKVDIKR CL SEQ ID NO.: 726
TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID
NO.: 727 EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA
D2-GS6-B6-5 DVD PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS SGGGGSGEVQLVQSGAEVKK
PGESVKISCKASGGSFRSYG ISWVRQAPGQGLEWMGGITH FFGITDYAQKFQGRVTITAD
ESTTTAYMELSGLTSDDTAV YYCAREPNDFWNGYYDTHDF DSWGQGTTVTVSS D2 VH SEQ
ID NO.: 728 EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA
PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS S LINKER SEQ ID NO.: 729 GGGGSG B6-5 VH SEQ ID
NO.: 730 EVQLVQSGAEVKKPGESVKI SCKASGGSFRSYGISWVRQA
PGQGLEWMGGITHFFGITDY AQKFQGRVTITADESTTTAY MELSGLTSDDTAVYYCAREP
NDFWNGYYDTHDFDSWGQGT TVTVSS CH SEQ ID NO.: 731 ASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD LIGHT SEQ ID NO.: 732 DIQMTQSPSSLSASVGDRVT VARIABLE
ITCRASQGIRNYLAWYQQKP E7-SL-B6-5 DVD GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKRTVAAPEIVLTQS
PDFQSVTPKEKVTITCRASQ NIGSELHWYQQKPDQSPKLL IKYASHSISGVPSRFSGSGS
GTDFTLTINGLEAEDAATYY CHQSDTLPHTFGQGTKVDIK R E7 VL SEQ ID NO.: 733
DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID
NO.: 734 TVAAP B6-5 VL SEQ ID NO.: 735 EIVLTQSPDFQSVTPKEKVT
ITCRASQNIGSELHWYQQKP DQSPKLLIKYASHSISGVPS RFSGSGSGTDFTLTINGLEA
EDAATYYCHQSDTLPHTFGQ GTKVDIKR CL SEQ ID NO.: 736
TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID
NO.: 737 EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA
D2-SL-B6-15 DVD PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS SASTKGPEVQLVQSGAEVKK
PGESLKISCKASGGSFSGYG TSWVRQAPGQGLEWMGGITH FFGAVDYAQKFQGRVTITAD
ESTTTAYMELSGLTSDDTAV YYCARDPNEFWNGYYATHDF DYWGQGTTVTVSS D2 VH SEQ
ID NO.: 738 EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA
PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS S
LINKER SEQ ID NO.: 739 ASTKGP B6-15 VH SEQ ID NO.: 740
EVQLVQSGAEVKKPGESLKI SCKASGGSFSGYGTSWVRQA PGQGLEWMGGITHFFGAVDY
AQKFQGRVTITADESTTTAY MELSGLTSDDTAVYYCARDP NEFWNGYYATHDFDYWGQGT
TVTVSS CH SEQ ID NO.: 741 ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ
ID NO.: 742 DIQMTQSPSSLSASVGDRVT VARIABLE ITCRASQGIRNYLAWYQQKP
E7-GS6-B6-15DVD GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP
EDVATYYCQRYNRAPYTFGQ GTKVEIKRGGSGGEIVLTQS PDFQSVTPKEKVTITCRASQ
NIGAELHWYQQKPDQSPKLL IKYASHSISGVPSRFSGSGS GTDFTLTINGLEAEDAGTYY
CHQTDRLPYSFGPGTKVDIK R E7 VL SEQ ID NO.: 743 DIQMTQSPSSLSASVGDRVT
ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP
EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID NO.: 744 GGSGG B6-15 VL
SEQ ID NO.: 745 EIVLTQSPDFQSVTPKEKVT ITCRASQNIGAELHWYQQKP
DQSPKLLIKYASHSISGVPS RFSGSGSGTDFTLTINGLEA EDAGTYYCHQTDRLPYSFGP
GTKVDIKR CL SEQ ID NO.: 746 TVAAPSVFIFPPSDEQLKSG
TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID NO.: 747
EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA D2-GS10-h5C5Vh1
PGKGLEWVSAITWNSGHIDY DVD ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS SGGGGSGGGGSEVQLVQSGA EVKKPGASVKVSCKASGYTF
TDYEFHWVRQAPGQGLEWMG VIHPGNGGTAYNQNFRDRVT ITADKSTSTAYMELSSLRSE
DTAVYYCARFLTYEGYFDYW GQGTLVTVSS D2 VH SEQ ID NO.: 748
EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA PGKGLEWVSAITWNSGHIDY
ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS S
LINKER SEQ ID NO.: 749 GGGGSGGGGS H5C5 Vh1VH SEQ ID NO.: 750
EVQLVQSGAEVKKPGASVKV SCKASGYTFTDYEFHWVRQA PGQGLEWMGVIHPGNGGTAY
NQNFRDRVTITADKSTSTAY MELSSLRSEDTAVYYCARFL TYEGYFDYWGQGTLVTVSS CH
SEQ ID NO.: 751 ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ
ID NO.: 752 DIQMTQSPSSLSASVGDRVT VARIABLE ITCRASQGIRNYLAWYQQKP
E7-GS10-h5C5Vk3a GKAPKLLIYAASTLQSGVPS DVD RFSGSGSGTDFTLTISSLQP
EDVATYYCQRYNRAPYTFGQ GTKVEIKRGGSGGGGSGSEI VMTQSPATLSVSPGERATLS
CKASQSVSIDVGWFQQKPGQ SPRLLIYHASNRYTGVPARF SGSGSGTDFTLTISSLQPED
FAVYFCQQDYSSPYTFGQGT KLEIKR E7 VL SEQ ID NO.: 753
DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID
NO.: 754 GGSGGGGSGS H5C5 Vk3a VL SEQ ID NO.: 755
EIVMTQSPATLSVSPGERAT LSCKASQSVSIDVGWFQQKP GQSPRLLIYHASNRYTGVPA
RFSGSGSGTDFTLTISSLQP EDFAVYFCQQDYSSPYTFGQ GTKLEIKR CL SEQ ID NO.:
756 TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID
NO.: 757 EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA
D2-GS10-6C6 DVD PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS SGGGGSGGGGSQVQLQQSGA
ELVRPGASVKLSCKASGYTF SDYEIHWVKQTPVHGLAWIG VIHPGNGGTAYNQKFKDKAT
LTADKSSSTAYMELSSLTSE DSAVYYCERFLTYEGYFDYW GQGTTLTVSS D2 VH SEQ ID
NO.: 758 EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA
PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS S LINKER SEQ ID NO.: 759 GGGGSGGGGS 6C6VH SEQ
ID NO.: 760 QVQLQQSGAELVRPGASVKL SCKASGYTFSDYEIHWVKQT
PVHGLAWIGVIHPGNGGTAY NQKFKDKATLTADKSSSTAY MELSSLTSEDSAVYYCERFL
TYEGYFDYWGQGTTLTVSS CH SEQ ID NO.: 761 ASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD HEAVY SEQ ID NO.: 762 EVQLVESGGGLVQPGRSLRL VARIABLE
SCAASGFTFDDYAMHWVRQA D2-GS10-h10F7Vh1a PGKGLEWVSAITWNSGHIDY DVD
ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS
SGGGGSGGGGSEVQLVQSGA EVKKPGSSVKVSCKASGYTF TDYEIHWVRQAPGQGLEWMG
VNDPESGGTFYNQKFDGRVT ITADKSTSTAYMELSSLRSE DTAVYYCARYYRYESFYGMD
YWGQGTTVTVSS D2 VH SEQ ID NO.: 763 EVQLVESGGGLVQPGRSLRL
SCAASGFTFDDYAMHWVRQA PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS S LINKER SEQ ID NO.: 764
GGGGSGGGGS H10F7Vh1aVH SEQ ID NO.: 765 EVQLVQSGAEVKKPGSSVKV
SCKASGYTFTDYEIHWVRQA PGQGLEWMGVNDPESGGTFY NQKFDGRVTITADKSTSTAY
MELSSLRSEDTAVYYCARYY RYESFYGMDYWGQGTTVTVS S CH SEQ ID NO.: 766
ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ ID NO.: 767
DIQMTQSPSSLSASVGDRVT VARIABLE ITCRASQGIRNYLAWYQQKP
E7-GS10-h10F7Vk1a GKAPKLLIYAASTLQSGVPS
DVD RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKRGGSGGGGSGSDI
QLTQSPSSLSASVGDRVTIT CSASSSISYIYWFQQKPGKS PKRWIYATFELASGVPSRFS
GSGSGTDYTLTISSLQPEDF ATYYCHQRSSYPWTFGQGTK LEIKR E7 VL SEQ ID NO.:
768 DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID
NO.: 769 GGSGGGGSGS H10F7 Vk1a VL SEQ ID NO.: 770
DIQLTQSPSSLSASVGDRVT ITCSASSSISYIYWFQQKPG KSPKRWIYATFELASGVPSR
FSGSGSGTDYTLTISSLQPE DFATYYCHQRSSYPWTFGQG TKLEIKR CL SEQ ID NO.:
771 TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID
NO.: 772 EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA
D2-GS6-h5C5Vh1 PGKGLEWVSAITWNSGHIDY DVD ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS SGGGGSGEVQLVQSGAEVKK
PGASVKVSCKASGYTFTDYE FHWVRQAPGQGLEWMGVIHP GNGGTAYNQNFRDRVTITAD
KSTSTAYMELSSLRSEDTAV YYCARFLTYEGYFDYWGQGT LVTVSS D2 VH SEQ ID NO.:
773 EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA PGKGLEWVSAITWNSGHIDY
ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS S
LINKER SEQ ID NO.: 774 GGGGSG H5C5 Vh1VH SEQ ID NO.: 775
EVQLVQSGAEVKKPGASVKV SCKASGYTFTDYEFHWVRQA PGQGLEWMGVIHPGNGGTAY
NQNFRDRVTITADKSTSTAY MELSSLRSEDTAVYYCARFL TYEGYFDYWGQGTLVTVSS CH
SEQ ID NO.: 776 ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ
ID NO.: 777 DIQMTQSPSSLSASVGDRVT VARIABLE ITCRASQGIRNYLAWYQQKP
E7-SL-h5C5Vk1a GKAPKLLIYAASTLQSGVPS DVD RFSGSGSGTDFTLTISSLQP
EDVATYYCQRYNRAPYTFGQ GTKVEIKRTVAAPDIQMTQS PSSLSASVGDRVTITCKASQ
SVSIDVGWFQQKPGKSPKLL IYHASNRYTGVPSRFSGSGS GTDFTFTISSLQPEDFATYF
CQQDYSSPYTFGQGTKLEIK R E7 VL SEQ ID NO.: 778 DIQMTQSPSSLSASVGDRVT
ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP
EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID NO.: 779 TVAAP H5C5
Vk1a VL SEQ ID NO.: 780 DIQMTQSPSSLSASVGDRVT ITCKASQSVSIDVGWFQQKP
GKSPKLLIYHASNRYTGVPS RFSGSGSGTDFTFTISSLQP EDFATYFCQQDYSSPYTFGQ
GTKLEIKR CL SEQ ID NO.: 781 TVAAPSVFIFPPSDEQLKSG
TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID NO.: 782
EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA D2-GS6-h5C5Vh1b
PGKGLEWVSAITWNSGHIDY DVD ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS SGGGGSGEVQLVQSGAEVKK PGASVKVSCKASGYTFTDYE
FHWVRQAPGQGLEWIGVIHP GNGGTAYNQNFRDRATLTAD KSTSTAYMELSSLRSEDTAV
YYCTRFLTYEGYFDYWGQGT LVTVSS D2 VH SEQ ID NO.: 783
EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA PGKGLEWVSAITWNSGHIDY
ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS S
LINKER SEQ ID NO.: 784 GGGGSG H5C5 Vh1bVH SEQ ID NO.: 785
EVQLVQSGAEVKKPGASVKV SCKASGYTFTDYEFHWVRQA PGQGLEWIGVIHPGNGGTAY
NQNFRDRATLTADKSTSTAY MELSSLRSEDTAVYYCTRFL TYEGYFDYWGQGTLVTVSS CH
SEQ ID NO.: 786 ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ
ID NO.: 787 DIQMTQSPSSLSASVGDRVT VARIABLE ITCRASQGIRNYLAWYQQKP
E7-SL-h5C5Vk3a GKAPKLLIYAASTLQSGVPS DVD RFSGSGSGTDFTLTISSLQP
EDVATYYCQRYNRAPYTFGQ GTKVEIKRTVAAPEIVMTQS PATLSVSPGERATLSCKASQ
SVSIDVGWFQQKPGQSPRLL IYHASNRYTGVPARFSGSGS GTDFTLTISSLQPEDFAVYF
CQQDYSSPYTFGQGTKLEIK R E7 VL SEQ ID NO.: 788 DIQMTQSPSSLSASVGDRVT
ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP
EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID NO.: 789 TVAAP H5C5
Vk3a VL SEQ ID NO.: 790 EIVMTQSPATLSVSPGERAT LSCKASQSVSIDVGWFQQKP
GQSPRLLIYHASNRYTGVPA RFSGSGSGTDFTLTISSLQP EDFAVYFCQQDYSSPYTFGQ
GTKLEIKR CL SEQ ID NO.: 791 TVAAPSVFIFPPSDEQLKSG
TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID NO.: 792
EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA D2-GS6-10F7 DVD
PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS SGGGGSGQVQLQQSGAELVR PGTSVTLSCKASGYIFTDYE
IHWVKQTPVHGLEWIGVNDP ESGGTFYNQKFDGKAELTAD KSSSTAYMELRSLTSEDSGV
YYCTRYYRYESFYGMDYWGQ GTSITVSS D2 VH SEQ ID NO.: 793
EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA PGKGLEWVSAITWNSGHIDY
ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS S
LINKER SEQ ID NO.: 794 GGGGSG 10F7VH SEQ ID NO.: 795
QVQLQQSGAELVRPGTSVTL SCKASGYIFTDYEIHWVKQT PVHGLEWIGVNDPESGGTFY
NQKFDGKAELTADKSSSTAY MELRSLTSEDSGVYYCTRYY RYESFYGMDYWGQGTSITVS S CH
SEQ ID NO.: 796 ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD LIGHT SEQ ID NO.: 797 DIQMTQSPSSLSASVGDRVT VARIABLE
ITCRASQGIRNYLAWYQQKP E7-SL-10F7 DVD GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKRTVAAPQIVLTQS
PAIMSASPGEKVTMTCSASS SISYIYWFQQKPGTSPKRWI YATFELASGVPARFSGSGSG
TSYSLTISSMEAEDAATYYC HQRSSYPWTFGGGSKLEIKR E7 VL SEQ ID NO.: 798
DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID
NO.: 799 TVAAP 10F7 VL SEQ ID NO.: 800 QIVLTQSPAIMSASPGEKVT
MTCSASSSISYIYWFQQKPG TSPKRWIYATFELASGVPAR FSGSGSGTSYSLTISSMEAE
DAATYYCHQRSSYPWTFGGG SKLEIKR CL SEQ ID NO.: 801
TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID
NO.: 802 EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA
D2-GS6-5C5 DVD PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS SGGGGSGQVQLQQSGAELVR
PGASVKLSCKALGYTFTDYE FHWVKQTPVHGLEWIGVIHP GNGGTAYNQNFRDKATLTAD
KSSSTAYMELSSLTSEDSGV YYCTRFLTYEGYFDYWGQGT ALTVSS D2 VH SEQ ID NO.:
803 EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA PGKGLEWVSAITWNSGHIDY
ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS S
LINKER SEQ ID NO.: 804 GGGGSG 5C5 VH SEQ ID NO.: 805
QVQLQQSGAELVRPGASVKL SCKALGYTFTDYEFHWVKQT PVHGLEWIGVIHPGNGGTAY
NQNFRDKATLTADKSSSTAY MELSSLTSEDSGVYYCTRFL TYEGYFDYWGQGTALTVSS CH
SEQ ID NO.: 806 ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ
ID NO.: 807 DIQMTQSPSSLSASVGDRVT VARIABLE ITCRASQGIRNYLAWYQQKP
E7-SL-5C5 DVD GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP
EDVATYYCQRYNRAPYTFGQ GTKVEIKRTVAAPNIVMTQT PKFLLVSPGDRVTITCKASQ
SVSIDVGWFQQKPGQSPKLL IYHASNRYTGVPDRFTGSGY GTDFTFTVNTVQAEDLAVYF
CQQDYSSPYTFGGGTKLELK R E7 VL SEQ ID NO.: 808 DIQMTQSPSSLSASVGDRVT
ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP
EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID NO.: 809 TVAAP 5C5 VL
SEQ ID NO.: 810 NIVMTQTPKFLLVSPGDRVT ITCKASQSVSIDVGWFQQKP
GQSPKLLIYHASNRYTGVPD RFTGSGYGTDFTFTVNTVQA EDLAVYFCQQDYSSPYTFGG
GTKLELKR CL SEQ ID NO.: 811 TVAAPSVFIFPPSDEQLKSG
TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID NO.: 812
EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA D2-GS6-1D8 DVD
PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS SGGGGSGQVQLQQSGAELVR PGASVKLSCKASGYTFSDYE
MHWVKQTPVHGLEWIGVIHP GNGGTAYNQKFRDKATLTAD KSSTTAYMELSSLTSEDSAV
YYCIRFLTYEGYFDYWGQGT TLTVSS D2 VH SEQ ID NO.: 813
EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA PGKGLEWVSAITWNSGHIDY
ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS S
LINKER SEQ ID NO.: 814 GGGGSG 1D8VH SEQ ID NO.: 815
QVQLQQSGAELVRPGASVKL SCKASGYTFSDYEMHWVKQT PVHGLEWIGVIHPGNGGTAY
NQKFRDKATLTADKSSTTAY MELSSLTSEDSAVYYCIRFL TYEGYFDYWGQGTTLTVSS CH
SEQ ID NO.: 816 ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ
ID NO.: 817 DIQMTQSPSSLSASVGDRVT VARIABLE ITCRASQGIRNYLAWYQQKP
E7-SL-1D8 DVD GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP
EDVATYYCQRYNRAPYTFGQ GTKVEIKRTVAAPSIVMTQT PKFLLVSAGDRVTITCKASQ
SVNNDVAWFQHKPGQSPKLL INYASNRYTGVPDRFTGSGY GTDFTFTISTVQSEDLAIYF
CQQDYGSPYTFGGGTTLEIK R E7 VL SEQ ID NO.: 818 DIQMTQSPSSLSASVGDRVT
ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP
EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID NO.: 819 TVAAP 1D8 VL
SEQ ID NO.: 820 SIVMTQTPKFLLVSAGDRVT ITCKASQSVNNDVAWFQHKP
GQSPKLLINYASNRYTGVPD RFTGSGYGTDFTFTISTVQS EDLAIYFCQQDYGSPYTFGG
GTTLEIKR CL SEQ ID NO.: 821 TVAAPSVFIFPPSDEQLKSG
TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID NO.: 822
EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA D2-GS6-6C6 DVD
PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS SGGGGSGQVQLQQSGAELVR PGASVKLSCKASGYTFSDYE
IHWVKQTPVHGLAWIGVIHP GNGGTAYNQKFKDKATLTAD KSSSTAYMELSSLTSEDSAV
YYCERFLTYEGYFDYWGQGT TLTVSS D2 VH SEQ ID NO.: 823
EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA PGKGLEWVSAITWNSGHIDY
ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS S
LINKER SEQ ID NO.: 824 GGGGSG 6C6VH SEQ ID NO.: 825
QVQLQQSGAELVRPGASVKL SCKASGYTFSDYEIHWVKQT PVHGLAWIGVIHPGNGGTAY
NQKFKDKATLTADKSSSTAY MELSSLTSEDSAVYYCERFL TYEGYFDYWGQGTTLTVSS CH
SEQ ID NO.: 826 ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD LIGHT SEQ ID NO.: 827 DIQMTQSPSSLSASVGDRVT VARIABLE
ITCRASQGIRNYLAWYQQKP E7-SL-6C6 DVD GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKRTVAAPSIVMTQT
PKFLLVSAGDRVTITCKASQ SVNNDVAWYQHKPGQSPKLL INYASNRYTGVPDRFTGSGY
GTDFTFTISTVQAEDLAIYF CQQDYGSPYTFGGGTKLEIK R E7 VL SEQ ID NO.: 828
DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID
NO.: 829 TVAAP 6C6VL SEQ ID NO.: 830 SIVMTQTPKFLLVSAGDRVT
ITCKASQSVNNDVAWYQHKP GQSPKLLINYASNRYTGVPD RFTGSGYGTDFTFTISTVQA
EDLAIYFCQQDYGSPYTFGG GTKLEIKR CL SEQ ID NO.: 831
TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID
NO.: 832 EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA
D2-GS6-8B12 DVD PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS SGGGGSGQVQLKESGPGLVA
PSQSLSITCTISGFSLTSYG VHWVRQPPGKGLEWLVVIWS DGTTTYNSALKSRLSITRDN
SKSQVFLKMNSLQTDDTAIY YCARDSTWDYYYTMDYWGQG TPVTVSS D2 VH SEQ ID NO.:
833 EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA PGKGLEWVSAITWNSGHIDY
ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS S
LINKER SEQ ID NO.: 834 GGGGSG 8B12VH SEQ ID NO.: 835
QVQLKESGPGLVAPSQSLSI TCTISGFSLTSYGVHWVRQP PGKGLEWLVVIWSDGTTTYN
SALKSRLSITRDNSKSQVFL KMNSLQTDDTAIYYCARDST WDYYYTMDYWGQGTPVTVSS CH
SEQ ID NO.: 836 ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ
ID NO.: 837 DIQMTQSPSSLSASVGDRVT VARIABLE ITCRASQGIRNYLAWYQQKP
E7-SL-8B12 DVD GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP
EDVATYYCQRYNRAPYTFGQ GTKVEIKRTVAAPDVVMTQT PLSLPVSLGDQASISCRSSQ
SLVHSNGNTYLHWYLQKPGQ SPKLLIYKVSNRFSGVPDRF SGSGSGTDFTLKISRVEAED
LGVYFCSQSTHVYTFGGGTK LEIKR E7 VL SEQ ID NO.: 838
DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID
NO.: 839 TVAAP 8B12 VL SEQ ID NO.: 840 DVVMTQTPLSLPVSLGDQAS
ISCRSSQSLVHSNGNTYLHW YLQKPGQSPKLLIYKVSNRF SGVPDRFSGSGSGTDFTLKI
SRVEAEDLGVYFCSQSTHVY TFGGGTKLEIKR CL SEQ ID NO.: 841
TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID
NO.: 842 EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA
D2-GS10-B6-11 DVD PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS SGGGGSGGGGSEVQLVQSGA
EVKKPGSSVKVSCKASGGSF LSYGFSWVRQAPGQGLEWMG GITPFFGFADYAQKFQGRVT
ITADESTTTAYMELSGLTSD DTAVYYCAREPNDFWNGYYT THHFDYWGQGTTVTVSS D2 VH
SEQ ID NO.: 843 EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA
PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS S LINKER SEQ ID NO.: 844 GGGGSGGGGS B6-11VH
SEQ ID NO.: 845 EVQLVQSGAEVKKPGSSVKV SCKASGGSFLSYGFSWVRQA
PGQGLEWMGGITPFFGFADY AQKFQGRVTITADESTTTAY MELSGLTSDDTAVYYCAREP
NDFWNGYYTTHHFDYWGQGT TVTVSS CH SEQ ID NO.: 846 ASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD LIGHT SEQ ID NO.: 847 DIQMTQSPSSLSASVGDRVT VARIABLE
ITCRASQGIRNYLAWYQQKP E7-GS10-B6.1 DVD GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKRGGSGGGGSGSEI
VLTQSPDFQSVTPKEKVTIT CRASQNIGSALHWYQQKPDQ SPKLLIKYASQSISGVPSRF
SGSGSGTDFTLTINGLEAED AGTYYCHQSTSLPHTFGQGT KVDIKR E7 VL SEQ ID NO.:
848 DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID
NO.: 849 GGSGGGGSGS B6.1 VL SEQ ID NO.: 850 EIVLTQSPDFQSVTPKEKVT
ITCRASQNIGSALHWYQQKP DQSPKLLIKYASQSISGVPS RFSGSGSGTDFTLTINGLEA
EDAGTYYCHQSTSLPHTFGQ GTKVDIKR CL SEQ ID NO.: 851
TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID
NO.: 852 EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA
D2-LL-B6-11 DVD PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS SASTKGPSVFPLAPEVQLVQ
SGAEVKKPGSSVKVSCKASG GSFLSYGFSWVRQAPGQGLE WMGGITPFFGFADYAQKFQG
RVTITADESTTTAYMELSGL TSDDTAVYYCAREPNDFWNG YYTTHHFDYWGQGTTVTVSS D2
VH SEQ ID NO.: 853 EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA
PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS S LINKER SEQ ID NO.: 854 ASTKGPSVFPLAP B6-11VH
SEQ ID NO.: 855 EVQLVQSGAEVKKPGSSVKV SCKASGGSFLSYGFSWVRQA
PGQGLEWMGGITPFFGFADY AQKFQGRVTITADESTTTAY MELSGLTSDDTAVYYCAREP
NDFWNGYYTTHHFDYWGQGT TVTVSS CH SEQ ID NO.: 856 ASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD LIGHT SEQ ID NO.: 857 DIQMTQSPSSLSASVGDRVT VARIABLE
ITCRASQGIRNYLAWYQQKP E7-LL-B6.1 DVD GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKRTVAAPSVFIFPP
EIVLTQSPDFQSVTPKEKVT ITCRASQNIGSALHWYQQKP DQSPKLLIKYASQSISGVPS
RFSGSGSGTDFTLTINGLEA EDAGTYYCHQSTSLPHTFGQ GTKVDIKR E7 VL SEQ ID
NO.: 858 DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP
GKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ
GTKVEIKR LINKER SEQ ID NO.: 859 TVAAPSVFIFPP B6.1 VL SEQ ID NO.:
860 EIVLTQSPDFQSVTPKEKVT ITCRASQNIGSALHWYQQKP DQSPKLLIKYASQSISGVPS
RFSGSGSGTDFTLTINGLEA EDAGTYYCHQSTSLPHTFGQ GTKVDIKR CL SEQ ID NO.:
861 TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID
NO.: 862 EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA
D2-SL-B6-11 DVD PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS STVAAPEVQLVQSGAEVKKP
GSSVKVSCKASGGSFLSYGF SWVRQAPGQGLEWMGGITPF FGFADYAQKFQGRVTITADE
STTTAYMELSGLTSDDTAVY YCAREPNDFWNGYYTTHHFD YWGQGTTVTVSS D2 VH SEQ ID
NO.: 863 EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA
PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS S LINKER SEQ ID NO.: 864 TVAAP B6-11VH SEQ ID
NO.: 865 EVQLVQSGAEVKKPGSSVKV SCKASGGSFLSYGFSWVRQA
PGQGLEWMGGITPFFGFADY AQKFQGRVTITADESTTTAY MELSGLTSDDTAVYYCAREP
NDFWNGYYTTHHFDYWGQGT TVTVSS CH SEQ ID NO.: 866 ASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD LIGHT SEQ ID NO.: 867 DIQMTQSPSSLSASVGDRVT VARIABLE
ITCRASQGIRNYLAWYQQKP E7-GS6-B6.1 DVD GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKRGGSGGEIVLTQS
PDFQSVTPKEKVTITCRASQ NIGSALHWYQQKPDQSPKLL IKYASQSISGVPSRFSGSGS
GTDFTLTINGLEAEDAGTYY CHQSTSLPHTFGQGTKVDIK R E7 VL SEQ ID NO.: 868
DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID
NO.: 869 GGSGG B6.1VL SEQ ID NO.: 870 EIVLTQSPDFQSVTPKEKVT
ITCRASQNIGSALHWYQQKP DQSPKLLIKYASQSISGVPS RFSGSGSGTDFTLTINGLEA
EDAGTYYCHQSTSLPHTFGQ GTKVDIKR CL SEQ ID NO.: 871
TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID
NO.: 872 EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA
D2-SL-B6-16DVD PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS STVAAPEVQLVQSGAEVKMP
GSSLKISCKASGGSFRGYGI SWVRQAPGQGLEWMGGITPF FGWADYAQKFQGRVTITADE
STTTAYMELSGLTSDDTAVY YCARDPNEFWNGYYDTHHFD YWGQGTTVTVSS D2 VH SEQ ID
NO.: 873 EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA
PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS S LINKER SEQ ID NO.: 874 TVAAP B6-16VH SEQ ID
NO.: 875 EVQLVQSGAEVKMPGSSLKI SCKASGGSFRGYGISWVRQA
PGQGLEWMGGITPFFGWADY AQKFQGRVTITADESTTTAY MELSGLTSDDTAVYYCARDP
NEFWNGYYDTHHFDYWGQGT TVTVSS CH SEQ ID NO.: 876 ASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD LIGHT SEQ ID NO.: 877 DIQMTQSPSSLSASVGDRVT VARIABLE
ITCRASQGIRNYLAWYQQKP E7-GS6-B6-16 DVD GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKRGGSGGEIVLTQS
PDFQSVTPKEKVTITCRASQ DIGSALHWYQQKPDQSPKLL IKYASHSVSGVPSRFSGSGS
GTDFTLTINGLEAEDAGTYY CHQSDILPHTFGPGTKVDIK R E7 VL SEQ ID NO.: 878
DIQMTQSPSSLSASVGDRVT ITCRASQGIRNYLAWYQQKP GKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQP EDVATYYCQRYNRAPYTFGQ GTKVEIKR LINKER SEQ ID
NO.: 879 GGSGG B6-16VL SEQ ID NO.: 880 EIVLTQSPDFQSVTPKEKVT
ITCRASQDIGSALHWYQQKP DQSPKLLIKYASHSVSGVPS RFSGSGSGTDFTLTINGLEA
EDAGTYYCHQSDILPHTFGP GTKVDIKR CL SEQ ID NO.: 881
TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID
NO.: 882 EVQLVESGGGLVQPGRSLRL VARIABLE SCAASGFTFDDYAMHWVRQA
D2-GS6-B6-16DVD PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCAKVS YLSTASSLDYWGQGTLVTVS SGGGGSGEVQLVQSGAEVKM
PGSSLKISCKASGGSFRGYG ISWVRQAPGQGLEWMGGITP FFGWADYAQKFQGRVTITAD
ESTTTAYMELSGLTSDDTAV YYCARDPNEFWNGYYDTHHF DYWGQGTTVTVSS D2 VH SEQ
ID NO.: 883 EVQLVESGGGLVQPGRSLRL SCAASGFTFDDYAMHWVRQA
PGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVS
YLSTASSLDYWGQGTLVTVS S LINKER SEQ ID NO.: 884 GGGGSG B6-16VH SEQ ID
NO.: 885 EVQLVQSGAEVKMPGSSLKI SCKASGGSFRGYGISWVRQA
PGQGLEWMGGITPFFGWADY AQKFQGRVTITADESTTTAY MELSGLTSDDTAVYYCARDP
NEFWNGYYDTHHFDYWGQGT TVTVSS
CH SEQ ID NO.: 886 ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK
3.4: DVD-Ig Heavy and Light Chain Combinations
[1119] The following table lists the heavy and light chain
sequences used for the expression of different TNF/IL-17 DVD-Ig
binding proteins.
TABLE-US-00036 TABLE 35 DVD-Ig Name Heavy chain construct Light
chain construct D2E7-B6.2 DVD-Ig D2-GS6-B6 DVD HC E7-SL-B6 DVD LC
D2E7-B6.4DVD-Ig D2-SL-B6 DVD HC E7-SL-B6 DVD LC D2E7-G9.1 DVD-Ig
D2-GS6-G9 DVD HC E7-GS6-G9 DVD LC D2E7-G9.2DVD-Ig D2-GS6-G9 DVD HC
E7-SL-G9 DVD LC D2E7-G9.3DVD-Ig D2-SL-G9 DVD HC E7-GS6-G9 DVD LC
D2E7-G9.4 DVD-Ig D2-SL-G9 DVD HC E7-SL-G9 DVD LC D2E7-B6-5.2 DVD-Ig
D2-GS6-B6-5 DVD HC E7-SL-B6-5 DVD LC D2E7-B6-5.8 DVD-Ig
D2-GS10-B6-5 DVD HC E7-GS10-B6-5 DVD LC D2E7-B6-11.3 DVD-Ig
D2-SL-B6-11 DVD HC E7-GS6-B6.1 DVD LC D2E7-B6-11.5 DVD-Ig
D2-LL-B6-11 DVD HC E7-LL-B6.1 DVD LC D2E7-B6-11.8 DVD-Ig
D2-GS10-B6-11 DVD HC E7-GS10-B6.1 DVD LC D2E7-B6-15.3 DVD-Ig
D2-SL-B6-15 DVD HC E7-GS6-B6-15 DVD LC D2E7-B6-16.1 DVD-Ig
D2-GS6-B6-16 DVD HC E7-GS6-B6-16 DVD LC D2E7-B6-16.3 DVD-Ig
D2-SL-B6-16 DVD HC E7-GS6-B6-16 DVD LC D2E7-B6-17.1 DVD-Ig
D2-GS6-B6-17 DVD HC E7-GS6-B6-17 DVD LC D2E7-B6-17.2 DVD-Ig
D2-GS6-B6-17 DVD HC E7-SL-B6-17 DVD LC D2E7-B6-17.3 DVD-Ig
D2-SL-B6-17 DVD HC E7-GS6-B6-17 DVD LC D2E7-B6-17.4 DVD-Ig
D2-SL-B6-17 DVD HC E7-SL-B6-17 DVD LC D2E7-B6-17.5 DVD-Ig
D2-LL-B6-17 DVD HC E7-LL-B6-17 DVD LC D2E7-B6-17.6 DVD-Ig
D2-SL-B6-17 DVD HC E7-LL-B6-17 DVD LC D2E7-B6-17.7 DVD-Ig
D2-LL-B6-17 DVD HC E7-SL-B6-17 DVD LC D2E7-B6-17.8 DVD-Ig
D2-GS10-B6-17 DVD HC E7-GS10-B6-17 DVD LC D2E7-B6-17.9 DVD-Ig
D2-GS14-B6-17 DVD HC E7-GS14-B6-17 DVD LC D2E7-G9-1.1 DVD-Ig
D2-GS6-G9 DVD HC E7-GS6-G9-1 DVD LC D2E7-G9-1.2 DVD-Ig D2-GS6-G9
DVD HC E7-SL-G9-1 DVD LC D2E7-G9-1.3 DVD-Ig D2-SL-G9 DVD HC
E7-GS6-G9-1 DVD LC D2E7-G9-1.4 DVD-Ig D2-SL-G9 DVD HC E7-SL-G9-1
DVD LC D2E7-G9-2.1 DVD-Ig D2-GS6-G9-2 DVD HC E7-GS6-G9-2 DVD LC
D2E7-G9-2.2 DVD-Ig D2-GS6-G9-2 DVD HC E7-SL-G9-2 DVD LC D2E7-G9-2.3
DVD-Ig D2-SL-G9-2 DVD HC E7-GS6-G9-2 DVD LC D2E7-G9-2.4 DVD-Ig
D2-SL-G9-2 DVD HC E7-SL-G9-2 DVD LC D2E7-G9-2.8 DVD-Ig D2-GS10-G9-2
DVD HC E7-GS10-G9-2 DVD LC D2E7-G9-2.9 DVD-Ig D2-GS14-G9-2 DVD HC
E7-GS14-G9-2 DVD LC D2E7-G9-4.1 DVD-Ig D2-GS6-G9 DVD HC E7-GS6-G9-4
DVD LC D2E7-G9-4.2 DVD-Ig D2-GS6-G9 DVD HC E7-SL-G9-4 DVD LC
D2E7-G9-4.3 DVD-Ig D2-SL-G9 DVD HC E7-GS6-G9-4 DVD LC D2E7-G9-4.4
DVD-Ig D2-SL-G9 DVD HC E7-SL-G9-4 DVD LC D2E7-G9-M2.1 DVD-Ig
D2-GS6-G9 DVD HC E7-GS6-G9-M2 DVD LC D2E7-G9-M2.2 DVD-Ig D2-GS6-G9
DVD HC E7-SL-G9-M2 DVD LC D2E7-G9-M2.3 DVD-Ig D2-SL-G9 DVD HC
E7-GS6-G9-M2 DVD LC D2E7-G9-M2.4 DVD-Ig D2-SL-G9 DVD HC E7-SL-G9-M2
DVD LC D2E7-h5C5.8 DVD-Ig D2-GS6-h5C5Vh1 DVD HC E7-SL-h5C5Vk1a DVD
LC D2E7-h5C5.9DVD-Ig D2-GS6-h5C5Vh1b DVD HC E7-SL-h5C5Vk1a DVD LC
D2E7-h5C5.15 DVD-Ig D2-GS6-h5C5Vh1b DVD HC E7-SL-h5C5Vk3a DVD LC
D2E7-h5C5DVD.8-Ig D2-GS10-h5C5.Vh1 DVD HC E7-GS10-h5C5Vk3a DVD LC
D2E7-10F7 DVD-Ig D2-GS6-10F7 DVD HC E7-SL-10F7 DVD LC D2E7-h10F7.8
DVD-Ig D2-GS10-h10F7Vh1a DVD HC E7-GS10-h10F7Vk1a DVD LC
D2E7-1D8DVD-Ig D2-GS6-1D8 DVD HC E7-SL-1D8 DVD LC D2E7-5C5 DVD-Ig
D2-GS6-5C5 DVD HC E7-SL-5C5 DVD LC D2E7-8B12 DVD-Ig D2-GS6-8B12 DVD
HC E7-SL-8B12 DVD LC D2E7-6C6 DVD-Ig D2-GS6-6C6 DVD HC E7-SL-6C6
DVD LC D2E7-6C6.8 DVD-Ig D2-GS10-6C6 DVD HC E7-GS10-h5C5Vk3a DVD
LC
3.5: Functional Characterization of TNF/IL-17 DVD-Ig Proteins
3.5.1: IL-17 Enzyme-Linked Immunosorbent Assay Protocol
[1120] IL-17 binding by TNF/IL-17 DVD-Ig binding proteins was
assessed by ELISA (assay described above, Example 1.5.1). Results
are shown in the table below.
TABLE-US-00037 TABLE 36 Binding of TNF/IL-17 DVD-Ig proteins to
human IL-17 by ELISA DVD-Ig Name EC50 in hIL-17 ELISA (pM)
D2E7-B6.2 DVD-Ig 540 D2E7-B6.4DVD-Ig 20000 D2E7-G9.1 DVD-Ig 234
D2E7-G9.2DVD-Ig 201 D2E7-G9.3DVD-Ig 167 D2E7-G9.4 DVD-Ig 163
D2E7-B6-5.2 DVD-Ig 167 D2E7-B6-5.8 DVD-Ig 150 D2E7-B6-11.3 DVD-Ig
140 D2E7-B6-11.5 DVD-Ig 110 D2E7-B6-11.8 DVD-Ig 130 D2E7-B6-15.3
DVD-Ig 2000 D2E7-B6-16.1 DVD-Ig 363 D2E7-B6-16.3 DVD-Ig 8000
D2E7-B6-17.1 DVD-Ig 210 D2E7-B6-17.2 DVD-Ig 180 D2E7-B6-17.3 DVD-Ig
153 D2E7-B6-17.4 DVD-Ig 171 D2E7-B6-17.5 DVD-Ig >500
D2E7-B6-17.6 DVD-Ig 150 D2E7-B6-17.7 DVD-Ig 150 D2E7-B6-17.8 DVD-Ig
70 D2E7-B6-17.9 DVD-Ig 50 D2E7-G9-1.1 DVD-Ig 190 D2E7-G9-1.2 DVD-Ig
170 D2E7-G9-1.3 DVD-Ig 130 D2E7-G9-1.4 DVD-Ig 1000 D2E7-G9-2.1
DVD-Ig 230 D2E7-G9-2.2 DVD-Ig 200 D2E7-G9-2.3 DVD-Ig 190
D2E7-G9-2.4 DVD-Ig 160 D2E7-G9-2.8 DVD-Ig 153 D2E7-G9-2.9 DVD-Ig
171 D2E7-G9-4.1 DVD-Ig 270 D2E7-G9-4.2 DVD-Ig 200 D2E7-G9-4.3
DVD-Ig 190 D2E7-G9-4.4 DVD-Ig 200 D2E7-G9-M2.1 DVD-Ig 200
D2E7-G9-M2.2 DVD-Ig 190 D2E7-G9-M2.3 DVD-Ig 160 D2E7-G9-M2.4 DVD-Ig
200 D2E7-h5C5.8 DVD-Ig 180 D2E7-h5C5.9DVD-Ig 142 D2E7-h5C5.15
DVD-Ig 211 D2E7-h10F7.8 DVD-Ig 182 D2E7-h5C5DVD.8-Ig 1416
D2E7-6C6.8 DVD-Ig 528 D2E7-10F7 DVD-Ig 238 D2E7-1D8DVD-Ig 3300
D2E7-5C5 DVD-Ig 500 D2E7-8B12 DVD-Ig 300 D2E7-6C6 DVD-Ig 1500
3.5.2: IL-17 and S1P-Induced IL-6 Secretion from Murine Embryonic
Fibroblast Cell Line (NIH3T3)
[1121] The murine NIH3T3 cell line (ATCC #CRL-1658) secretes IL-6
in response to murine, rat or rabbit IL-17 and S1P (Cayman
Chemical, Cat#62570). The IL-17 induced IL-6 secretion is inhibited
by neutralizing anti-IL-17 antibodies.
[1122] NIH3T3 cells were maintained in assay medium: DMEM
(Invitrogen Cat#11965-092) with 10% fetal bovine serum
(Gibco#26140-079), 1% Non Essential Amino Acids, 2 mM L-glutamine,
1 mM sodium pyruvate, penicillin G (100 U/500 ml) and streptomycin
(100 .mu.g/500 ml). Cells were grown in T150 flasks until they were
about 80-90% confluent the day of the assay.
[1123] Murine IL17A HIS (Abbott, A-1229793.0) was diluted to 40
.mu.g/mL in 0.1% BSA/PBS without Ca.sup.2+ and Mg.sup.2+,
aliquoted, and stored frozen. Rat IL17A (Prospec bio, Cat#CYT-542)
was reconstituted in sterile PBS, without Ca.sup.2+ and Mg.sup.2+,
with 0.1% BSA, aliquoted and stored frozen at 100 .mu.g/mL. Rabbit
IL17A (Abbott, A-1239293.0) was aliquoted and stored frozen at 260
.mu.g/mL. S1p was reconstituted in 0.3 M NaOH at 10.54 mM,
aliquoted, and stored frozen. Freshly thawed IL-17 antibodies were
diluted to 200 .mu.g/ml (4.times.) in assay medium. Serial
dilutions of antibodies were made in a separate plate (4.times.
concentrations), mixed with equal volume of 40 ng/ml (4.times.)
murine or rat IL-17A or 100 ng/mL rabbit IL17A, and incubated at
37.degree. C. for 1 hr.
[1124] NIH3T3 cells (typically about 400,000 cells in 50 .mu.l
assay medium) were added to each well of a 96-well flat-bottom
tissue culture plate (Costar #3599), followed by addition of 50
.mu.l of the pre-incubated antibody plus IL-17 mix. S1P at 100
.mu.M (10.times.) was added in 11 .mu.l of media to each well. The
final concentration of IL-17A was 10 ng/ml for murine and rat and
25 ng/mL for rabbit. The final concentration for S1P was 10 .mu.M.
Cells were incubated for about 24 hrs at 37.degree. C. The media
supernatants were then collected. The level of IL-17 neutralization
was measured by determination of IL-6 amounts in supernatant using
a commercial Meso Scale Discovery kit (cat# K112AKA-4) according to
manufacturer's instruction. IC50 values were obtained using
logarithm of antibody versus IL-6 amount variable slope fit.
3.5.3: IL-17 Neutralization Potency of TNF/IL-17 DVD-12
Molecules
[1125] Multiple DVD-Ig molecules having iterative changes in the
linkers (short versus long linker and different sequences)
connecting the outer D2E7 variable domains to the inner B6-17
variable domains were generated for functional characterization
(B6-5.2, B6-5.8, B6-11.3, B6-11.5, B6-11.8, etc.). Potency of
DVD-Ig molecules was assessed using IL-17 or IL-17A/F driven human
IL-6 production in HS27 cells for human and cyno antigens (assay
described above, Example 1.5.2) or IL-17 plus S1P driven mouse IL-6
production in NIH3T3 cells for rat, mouse, and rabbit antigens
(assay described above, Example 3.5.2). The table below summarizes
potencies to human IL-17A and A/F and cyno, rat, mouse and rabbit
IL-17A.
TABLE-US-00038 TABLE 37 Potency (nM) Cyno Mouse Rabbit TNF/IL-17
DVD-Ig Hu IL-17A Hu IL-17A/F IL-17A Rat IL-17A IL-17A IL-17A
D2E7-B6.2 DVD-Ig 3.2 ND ND ND ND ND D2E7-B6.4DVD-Ig 85.9 ND ND ND
ND ND D2E7-G9.1 DVD-Ig 2.5 ND ND ND ND ND D2E7-G9.2DVD-Ig 5.6 ND ND
ND ND ND D2E7-G9.3DVD-Ig 1.6 ND ND ND ND ND D2E7-G9.4 DVD-Ig 1.8 ND
ND ND ND ND D2E7-B6-5.2 DVD-Ig 1.42 ND ND ND ND 0.32 D2E7-B6-5.8
DVD-Ig 0.064 0.233 0.13 5.2 8 11 D2E7-B6-11.3 DVD-Ig 0.519 3.400
0.622 >20 NI ND D2E7-B6-11.5 DVD-Ig 0.349 1.760 0.535 >20 NI
ND D2E7-B6-11.8 DVD-Ig 0.547 1.780 1.148 ND ND 0.23 D2E7-B6-16.1
DVD-Ig 0.758 ND 2.711 ND NI ND D2E7-B6-17.1 DVD-Ig 0.335 ND 0.913
>20 NI NI D2E7-B6-17.2 DVD-Ig 0.282 ND 0.509 ND NI ND
D2E7-B6-17.3 DVD-Ig 0.188 0.488 0.588 >20 NI ND D2E7-B6-17.4
DVD-Ig 0.453 0.545 1.465 >20 NI >20 D2E7-B6-17.6 DVD-Ig >1
ND ND ND ND ND D2E7-B6-17.7 DVD-Ig >1 ND ND ND ND ND
D2E7-B6-17.8 DVD-Ig 0.035 0.210 0.082 5.1 11 6.8 D2E7-B6-17.9
DVD-Ig 0.027 0.167 0.060 ND >20 ND D2E7-G9-1.1 DVD-Ig 0.882 ND
2.350 ND ND ND D2E7-G9-1.2 DVD-Ig 3.004 ND 11.070 ND ND ND
D2E7-G9-1.3 DVD-Ig 0.507 ND 4.276 ND ND ND D2E7-G9-1.4 DVD-Ig 0.828
ND 4.168 ND ND ND D2E7-G9-2.1 DVD-Ig 0.407 ND 1.710 ND ND ND
D2E7-G9-2.2 DVD-Ig 0.482 ND 2.405 ND ND ND D2E7-G9-2.3 DVD-Ig 0.254
0.384 1.381 ND ND ND D2E7-G9-2.4 DVD-Ig 0.227 0.696 1.476 ND ND ND
D2E7-G9-2.8 DVD-Ig 0.160 0.435 0.798 NI ND ND D2E7-G9-M2.1 DVD-Ig
0.660 ND 4.530 ND ND ND D2E7-G9-M2.2 DVD-Ig 1.789 ND 4.989 ND ND ND
D2E7-G9-M2.3 DVD-Ig 1.256 ND 3.060 ND ND ND D2E7-G9-M2.4 DVD-Ig
1.312 ND 1.903 ND ND ND D2E7-h5C5.8 DVD-Ig 0.726 ND ND ND ND ND
D2E7-h5C5.9DVD-Ig 0.896 ND ND ND ND ND D2E7-h5C5.15 DVD-Ig 0.708 ND
ND ND ND ND D2E7-10F7 DVD-Ig 7.2 ND 45 ND ND ND D2E7-1D8DVD-Ig 19
ND ND ND ND ND D2E7-5C5 DVD-Ig 2.6 ND 17 ND ND ND D2E7-8B12 DVD-Ig
14 ND ND ND ND ND D2E7-6C6 DVD-Ig 3.9 ND ND ND ND ND NI: no
inhibition. ND: not determined.
3.5.4: TNF Neutralization Potency of TNF/IL-17 DVD-Ig Molecules
3.5.4.1: L929 Bioassay for Measuring TNF Neutralization Potency
[1126] Human TNF Lot No. 1277249 (1.85 mg/mL) was prepared at
Abbott Bioresearch Center (Worcester, Mass., US) and received from
the Biologics Pharmacy. Mouse TNF Lot 1420095 (1.0 mg/mL) was
prepared at Abbott Bioresearch Center and received from the
Biologics Pharmacy. Rat TNF Lot 1436667 (3.0 mg/mL) was prepared at
Abbott Bioresearch Center and received from the Biologics Pharmacy.
Rabbit TNF was purchased from R&D Systems (cat#5670-TG-025).
Rhesus/Macaque TNF (rhTNF) was purchased from R&D Systems
(cat#1070-RM-025). Actinomycin D (catalog# A1410) was purchased
from Sigma Aldrich and resuspended at a stock concentration of 10
mg/mL in DMSO.
[1127] Assay Media: 10% FBS (Hyclone#SH30070.03), Gibco reagents:
RPMI 1640 (#21870), 2 mM L-glutamine (#25030), 50 units/mL
penicillin/50 .mu.g/mL streptomycin (#15140), 0.1 mM MEM
non-essential amino acids (#11140) and 5.5.times.10.sup.-5 M
2-mercaptoethanol (#21985-023).
[1128] L929 cells were grown to a semi-confluent density and
harvested using 0.05% tryspin (Gibco#25300). The cells were washed
with PBS, counted, and resuspended at 1E6 cells/mL in assay media
containing 4 .mu.g/mL actinomycin D. The cells were seeded in a
96-well plate (Costar#3599) at a volume of 50 .mu.L and 5E4
cells/well. Wells received 50 .mu.L of assay media, bringing the
volume to 100 .mu.L.
[1129] A test sample was prepared as follows. The DVD-Ig.TM. and
control IgG were diluted to a 4.times. concentration in assay media
and serial 1:3 dilutions were performed. TNF species were diluted
to the following concentrations in assay media: 400 pg/mL huTNF,
200 pg/mL muTNF, 600 pg/mL ratTNF, and 100 pg/mL rabTNF. Antibody
sample (200 .mu.L) was added to the TNF (200 .mu.L) in a 1:2
dilution scheme and allowed to incubate for 0.5 h at room
temperature.
[1130] To measure huTNF neutralization potency of DVD-Ig in this
assay, the DVD-Ig/TNF solution was added to the plated cells at 100
.mu.L for a final concentration of DVD-Ig at 375 nM-0.019 nM
DVD-Ig. The final concentration of TNF was as follows: 100 pg/mL
huTNF, 50 pg/mL muTNF, 150 pg/mL ratTNF, and 25 pg/mL rabTNF. The
plates were incubated for 20 h at 37.degree. C., 5% CO.sub.2. To
quantitate viability, 100 .mu.L was removed from the wells and 10
.mu.L of WST-1 reagent (Roche cat#11644807001) was added. Plates
were incubated under assay conditions for 3.5 h, centrifuged at
500.times.g, and 75 .mu.L of supernatant transferred to an ELISA
plate (Costar cat#3369). The plates were read at OD 420-600 nm on a
Spectromax 190 ELISA plate reader. The neutralization potency of
selected TNF/IL-17 DVD-Ig binding proteins is shown in the table
below.
TABLE-US-00039 TABLE 38 TNF neutralization potency IC50 (nM) Human
Mouse Rat Rabbit Cyno/Rhesus TNF TNF TNF TNF TNF D2E7-B6-5.8 DVD-Ig
0.015 >50 NI NI 0.011 D2E7-B6-17.8 DVD- 0.019 >50 NI NI 0.016
Ig D2E7-G9-2.8 DVD-Ig 0.012 3.037 NI NI 0.012
3.5.5: Affinity Measurement
[1131] The binding of TNF/IL-17 DVD-Igs to purified recombinant
human, cyno, rat, mouse, rabbit IL-17 and human IL-17A/F as well as
to human and cyno/rhesus TNF were determined using surface plasmon
resonance as described above (Example 1.5.4)
TABLE-US-00040 TABLE 39 Affinity measurements for TNF/IL-17 DVD-Ig
Cyno/ Human Human Cyno Rat Mouse Rabbit Human Rhesus IL-17A
IL-17A/F IL-17A IL-17A IL-17A IL-17A TNF TNF D2E7-10F7 (M) 1.17E-09
2.16E-09 8.49E-10 3.55E-09 3.90E-09 ND ND ND Kon (1/Ms) 1.50E+05
1.70E+05 2.18E+05 9.88E+04 6.07E+04 Koff (1/s) 1.75E-04 3.67E-04
1.85E-04 3.51E-04 2.37E-04 D2E7-6C6 (M) 1.06E-09 1.78E-09 1.78E-09
1.21E-08 1.45E-08 ND ND ND Kon (1/Ms) 2.02E+05 1.31E+05 1.12E+05
6.71E+04 6.42E+04 Koff (1/s) 2.15E-04 2.33E-04 1.99E-04 8.11E-04
9.31E-04 D2E7-SL-G9 (M) 8.38E-10 ND ND ND ND ND 5.11E-11 ND Kon
(1/Ms) 7.40E+04 3.15E+06 Koff (1/s) 6.20E-05 1.61E-04 D2E7-GS-G9
(M) 1.35E-10 ND ND ND ND ND 2.63E-11 ND Kon (1/Ms) 7.16E+04
2.37E+06 Koff (1/s) 9.64E-06 6.24E-05 D2E7-B6.2 (M) 4.02E-10 ND ND
ND ND ND 6.09E-11 ND Kon (1/Ms) 4.92E+04 2.66E+06 Koff (1/s)
1.98E-05 1.62E-04 D2E7-B6-17.8 (M) 4.54E-11 5.19E-11 7.12E-11
3.59E-10 5.83E-10 2.42E-10 1.11E-11 1.36E-11 Kon (1/Ms) 2.80E+05
1.88E+05 1.70E+05 4.60E+04 3.36E+04 7.30E+04 3.15E+06 2.02E+06 Koff
(1/s) 1.27E-05 9.75E-06 1.21E-05 1.65E-05 1.96E-05 1.77E-05
3.49E-05 2.74E-05 D2E7-B6-5.8 (M) 3.37E-11 5.10E-11 1.67E-10
1.40E-09 2.51E-09 3.50E-09 9.93E-12 1.45E-11 Kon (1/Ms) 1.67E+05
3.63E+05 1.74E+05 7.27E+04 2.31E+04 7.69E+04 3.04E+06 2.06E+06 Koff
(1/s) 5.63E-06 1.85E-05 2.90E-05 1.02E-04 5.80E-05 2.69E-04
3.02E-05 2.99E-05 D2E7-G9-2.8 (M) <3.3E-11 4.24E-10 <3.2E-11
ND ND ND 3.50E-11 7.97E-12 Kon (1/Ms) 3.00E+04 4.50E+04 3.09E+04
2.14E+06 1.58E+06 Koff (1/s) <1.0E-06 1.91E-05 <1.0E-06
7.50E-05 1.26E-05 ND: not determined
3.5.6: Pharmacokinetic Analysis of TNF/IL-17 DVD-Ig in Rat
[1132] Pharmacokinetic studies with TNF/IL-17 DVD-Ig were carried
out in Sprague Dawley rats essentially as described above (Example
2.2.4). Male rats were dosed intravenously or subcutaneously with a
single dose of 4 mg/kg of TNF/IL-17 DVD-Ig, and serum samples were
analyzed using antigen capture based chemiluminescent MSD (Meso
Scale Discovery) method.
[1133] Pharmacokinetic parameters were calculated by
non-compartmental analysis using WinNonlin. The MSD assay format
was essentially the same except that the biotinylated human TNF was
used in place of biotinylated human IL-17 for sample
incubation.
[1134] The pharmacokinetic profile of TNF/IL-17 DVD-Ig is shown in
the tables below.
TABLE-US-00041 TABLE 40 Subcutaneous injection D2E7-IL-17-B6-17.8
DVD hIgG1/K T1/2 (day) Cmax (.mu.g/mL) Tmax (day) % F 13.2 30.1 3.0
84
TABLE-US-00042 TABLE 41 Intravenous injection T1/2 Vss Cl DVD-Ig
(day) (mL/kg) (ml/hr/kg) D2E7-B6-17.8 DVD-Ig 12.9 97 0.25
D2E7-B6-5.8 DVD-Ig 9.9 77 0.23 D2E7-G9-2.8 DVD-Ig 8.9 100 0.36
3.6: Therapeutic Efficacy of TNF/IL-17 DVD-Ig and Combined TNF and
IL-17 Antibodies
3.6.1: Neutralizing Potency of TNF/IL-17 DVD-Ig in Acute IL-17
Induced KC Model
[1135] The ability of the TNF/IL-17 DVD-Ig binding proteins to
neutralize cytokines in vivo was demonstrated using a mouse model
in which recombinant human IL-17 induced the murine chemokine KC.
Female BALB/cJ mice were pre-dosed with antibodies
intra-peritoneally (i.p.), and 18 hrs later they were injected i.p.
with 3 .mu.g rhIL-17 in a 500 .mu.L volume. After 1 hour, the mice
were sacrificed, and the levels of KC were assessed by MesoScale.
ED50 values for % inhibition of KC were determined and shown in the
table below.
TABLE-US-00043 TABLE 42 ED50 (mg/kg) TNF/IL-17 DVD-Ig rh
IL17-induced KC D2E7-B6-5.8 DVD-Ig 7.6 D2E7-B6-17.8 DVD-Ig 5.2
D2E7-G9-2.8 DVD-Ig 17.2
3.6.2: Neutralizing Potency of TNF/IL-17 DVD-Ig in
rhTNF/D-Galactosamine-Induced Lethality Mouse Model
[1136] The TNF arms of the TNF/IL17 DVD-Ig molecules were also
evaluated in the rhTNF/D-galactosamine-induced lethality model.
Female C57BL6/N mice were pre-dosed with binding protein (i.p.) and
18 hr later were challenged (i.p.) with 0.1 .mu.g rh TNF and 20 mg
D-galactosamine in 500 .mu.L 0.9% sodium chloride and monitored for
survival over 48 hours. ED50 values for percent survival were
calculated. As shown in the table below, three distinct
anti-IL-17/TNF DVD-Ig constructs were tested in these models and
all three constructs fully neutralized human IL-17 and human TNF
induced bioactivity.
TABLE-US-00044 TABLE 43 ED50 (mg/kg) TNF/IL-17 DVD-Ig rh
TNF/Dgal-induced Lethality D2E7-B6-5.8 DVD-Ig 0.009 D2E7-B6-17.8
DVD-Ig 0.004 D2E7-G9-2.8 DVD-Ig 0.028
Example 4
Therapeutic Efficacy of IL-17 Antibodies in Mouse Collagen-Induced
Arthritis Model
[1137] The therapeutic effects of anti-IL-17 were evaluated in a
collagen-induced arthritis mouse model well known in the art.
Briefly, male DBA-1 mice were immunized with bovine type II
collagen in CFA at the base of the tail. The mice were boosted with
zymosan intraperitoneally (i.p) at day 21. After disease onset at
day 24-27, mice were selected and divided into separate groups of
10 mice each. The mean arthritis score of the control group, and
anti-cytokine groups was comparable at the start of treatment. Mice
were injected (i.p.) every other day with anti-IL-17 mAb MAB421 (12
mg/kg, R&D Systems, Inc., Minneapolis, Minn., US). Mice were
carefully examined three times a week for the visual appearance of
arthritis in peripheral joints, and scores of disease activity were
determined Statistical differences were determined by Student's
t-test and p values of <0.05 were considered significant.
[1138] Treatment of mice with either anti-IL-17 antibody reduced
mean arthritic score (MAS) by 43%, as shown in the table below.
TABLE-US-00045 TABLE 44 Inhibition of arthritic score by anti-IL-17
antibody Antibody Dose % inhibition Specificity treatment (mg/kg)
MAS Anti-IL-17 MAB421 12 43
Example 5
Therapeutic Efficacy of IL-17 Antibodies in Mouse Experimental
Autoimmune Encephalomyelitis (EAE) Mode
[1139] SJL/J mice were immunized using the following protocol for
induction of active experimental autoimmune encephalomyelitis
(EAE), a model of human multiple sclerosis (MS): stock solutions of
2 mg/ml PLP.sub.139-151 in PBS and 4 mg/ml M. tuberculosis H37Ra
(heat killed and desiccated) in incomplete Freund's adjuvant were
combined at 1:1 ratio to give a 1 mg/ml PLP.sub.139-151 emulsion.
Mice were immunized (s.c.) with 0.1 ml of emulsion spread over
three sites on the dorsal flank. Mice also receive 60 ng pertussis
toxin (i.p.) on of immunization. Mice were dosed (s.c.) with either
mouse IgG or anti-mouse IL-17 antibody (MAB421, R&D Systems) on
days 7, 14, and 21 post immunization Animals were followed for
clinical signs of disease using the following scoring system:
0--normal; 1--loss of tail tone (flaccid); 2--impaired righting
reflex, irregular gait; 3--partial hind limb paresis; 4--complete
hind limb paralysis; 5--moribund/dead. Animals were euthanized on
day 28. The data from this experiment are summarized in the table
below. The anti-IL-17 antibody significantly inhibited the clinical
course of disease at the 1.5 mg/kg dose tested. The mean maximal
score and the mean cumulative score were also affected by this
treatment. Anti-IL-17 mAb treatment also significantly inhibited
the frequency of disease relapses. These data are consistent will
an important role for IL-17 in tissue-specific autoimmune responses
in the CNS.
TABLE-US-00046 TABLE 45 PBS MAB421 Incidence 15/15 13/15 Mean Onset
Day* 10.9 10.3 (+/-1.5) (+/-1.0) Mean Maximal Score* 3 2.4 (+/-0.7)
(+/-1.0) Mean Cumulative Score* 36.3 21 (onset to conclusion)
(+/-16.7) (+/-21.5) Avg Daily Score* 1.3 0.7* (+/-0.6) (+/-0.8)
Relapse Rate 9/15 3/13 *p < 0.05 (students t test vs. PBS
Example 6
Therapeutic Efficacy of Combined TNF and IL-17 Antibodies in Mouse
Collagen-Induced Arthritis Model
[1140] The therapeutic effects of anti-IL-17, anti-TNF, and
combined anti-IL-17/anti-TNF were evaluated in a collagen-induced
arthritis mouse model well known in the art. Briefly, male DBA-1
mice were immunized with bovine type II collagen in CFA at the base
of the tail. The mice were boosted with zymosan intraperitoneally
(i.p) at day 21. After disease onset at (day 24-27), mice were
selected and divided into separate groups of 12 mice each. The mean
arthritis score of the control group, and anti-cytokine groups was
comparable at the start of treatment. Mice were injected with
anti-IL-17 mAb MAB421 (12 mg/kg), anti-TNF antibody 8C11 (12
mg/kg), or a combination of anti-IL-17/anti-TNF mAbs (12 mg/kg
each) i.p, two times a week. Mice were monitored daily for the
first week and three times a week subsequently for the visual
appearance of arthritis in peripheral joints, and scores of disease
activity were determined. Statistical differences were determined
by non-parametric Mann-Whitney test and p values of <0.05 were
considered significant.
[1141] Treatment of mice with either anti-IL-17 or anti-TNF
antibodies alone reduced mean arthritic score (MAS) by 43 and 36%
respectively, as shown in the table below. Treatment of mice with
both anti-IL-17 and anti-TNF monoclonal antibodies further reduced
MAS scores to 60% (p<0.05), demonstrating superior efficacy
compared to treatment of either monoclonal antibody alone.
TABLE-US-00047 TABLE 46 Inhibition of arthritic score by anti-IL-17
and TNF antibodies Dose % inhibition Specificity Antibody treatment
(mg/kg) MAS Anti-IL-17 MAB421 12 43 Anti-TNF 8C11 12 36 Anti-IL-17
+ TNF MAB421 + 8C11 24 60 (12 each)
INCORPORATION BY REFERENCE
[1142] The present invention incorporates by reference in their
entirety techniques well known in the field of molecular biology,
drug delivery, immunology, molecular biology and cell biology.
These techniques include, but are not limited to, techniques
described in the following publications: Ausubel et al. (eds.),
Current Protocols in Molecular Biology, John Wiley & Sons, NY
(1993); Ausubel, F. M. et al. eds., Short Protocols In Molecular
Biology (4th Ed. 1999) John Wiley & Sons, NY. (ISBN
0-471-32938-X). Controlled Drug Bioavailability Drug Product Design
and Performance, Smolen and Ball (eds.), Wiley, New York (1984);
Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids
and Proteins, a Practical Approach, 2nd ed., pp. 20 1-16, Oxford
University Press, New York, N.Y., (1999); Goodson, in Medical
Applications of Controlled Release, vol. 2, pp. 115-138 (1984);
Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas
563-681 (Elsevier, N.Y., 1981; Harlow et al., Antibodies: A
Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988); Kabat et al., Sequences of Proteins of Immunological
Interest (National Institutes of Health, Bethesda, Md. (1987) and
(1991); Kabat, E. A., et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242; Kontermann and
Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York.
790 pp. (ISBN 3-540-41354-5); Kriegler, Gene Transfer and
Expression, A Laboratory Manual, Stockton Press, NY (1990); Lu and
Weiner eds., Cloning and Expression Vectors for Gene Function
Analysis (2001) BioTechniques Press. Westborough, Mass. 298 pp.
(ISBN 1-881299-21-X), Medical Applications of Controlled Release,
Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Old, R.
W. & S. B. Primrose, Principles of Gene Manipulation: An
Introduction To Genetic Engineering (3d Ed. 1985) Blackwell
Scientific Publications, Boston. Studies in Microbiology; V.2:409
pp. (ISBN 0-632-01318-4); Sambrook, J. et al. eds., Molecular
Cloning: A Laboratory Manual (2d Ed. 1989) Cold Spring Harbor
Laboratory Press, NY. Vols. 1-3 (ISBN 0-87969-309-6); Sustained and
Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,
Marcel Dekker, Inc., New York, 1978; Winnacker, E. L. From Genes To
Clones: Introduction To Gene Technology (1987) VCH Publishers, N.Y.
(translated by Horst Ibelgaufts). 634 pp. (ISBN 0-89573-614-4).
[1143] Further, the contents of all cited references (including
literature references, patents, patent applications, and websites)
that maybe cited throughout this application are hereby expressly
incorporated by reference in their entirety for any purpose, as are
the references cited therein.
EQUIVALENTS
[1144] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting of the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes that come within the meaning and range of equivalency of
the claims are therefore intended to be embraced herein.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140335564A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140335564A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References