U.S. patent application number 14/290563 was filed with the patent office on 2014-12-25 for dual variable domain immunoglobulin and uses thereof.
This patent application is currently assigned to AbbVie Inc.. The applicant listed for this patent is AbbVie Inc.. Invention is credited to Richard W. DIXON, Tariq GHAYUR, Jochen G. SALFELD, Chengbin WU.
Application Number | 20140377805 14/290563 |
Document ID | / |
Family ID | 39107276 |
Filed Date | 2014-12-25 |
United States Patent
Application |
20140377805 |
Kind Code |
A1 |
WU; Chengbin ; et
al. |
December 25, 2014 |
Dual Variable Domain Immunoglobulin and Uses Thereof
Abstract
The present invention relates to engineered multivalent and
multispecific binding proteins, methods of making, and specifically
to their uses in the prevention and/or treatment of acute and
chronic inflammatory and other diseases.
Inventors: |
WU; Chengbin; (Shanghai,
CN) ; GHAYUR; Tariq; (Holliston, MA) ; DIXON;
Richard W.; (Jefferson, MA) ; SALFELD; Jochen G.;
(North Grafton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AbbVie Inc. |
North Chicago |
IL |
US |
|
|
Assignee: |
AbbVie Inc.
North Chicago
IL
|
Family ID: |
39107276 |
Appl. No.: |
14/290563 |
Filed: |
May 29, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13601774 |
Aug 31, 2012 |
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14290563 |
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12459624 |
Jul 2, 2009 |
8258268 |
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13601774 |
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11507050 |
Aug 18, 2006 |
7612181 |
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12459624 |
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60709911 |
Aug 19, 2005 |
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60732892 |
Nov 2, 2005 |
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Current U.S.
Class: |
435/69.6 |
Current CPC
Class: |
A61P 15/00 20180101;
A61P 19/02 20180101; A61P 19/04 20180101; C07K 2317/56 20130101;
A61P 21/00 20180101; A61P 35/02 20180101; A61P 37/06 20180101; A61P
35/00 20180101; A61P 7/06 20180101; A61P 25/06 20180101; Y02A 50/30
20180101; Y02A 50/386 20180101; Y02A 50/412 20180101; Y02A 50/58
20180101; A61P 9/00 20180101; A61P 11/06 20180101; A61P 13/12
20180101; A61P 31/12 20180101; C07K 16/245 20130101; C07K 16/2887
20130101; A61P 29/00 20180101; A61P 17/06 20180101; C07K 16/2809
20130101; A61K 47/6803 20170801; A61P 17/00 20180101; C07K 2317/24
20130101; C07K 2317/51 20130101; C07K 16/241 20130101; A61P 31/00
20180101; C07K 16/2896 20130101; A61P 11/00 20180101; A61P 37/08
20180101; A61K 45/06 20130101; A61K 47/42 20130101; A61P 25/16
20180101; A61P 31/04 20180101; C07K 16/40 20130101; Y02A 50/41
20180101; A61P 1/16 20180101; A61K 39/3955 20130101; A61P 1/00
20180101; A61P 3/10 20180101; C07K 2317/31 20130101; A61P 25/18
20180101; A61K 2039/505 20130101; A61P 25/28 20180101; C07K 16/24
20130101; A61P 21/02 20180101; C07K 16/46 20130101; A61P 31/18
20180101; A61P 41/00 20180101; A61P 19/10 20180101; A61P 23/00
20180101; A61P 25/24 20180101; C07K 16/22 20130101; C07K 16/467
20130101; A61P 25/00 20180101; A61P 25/32 20180101; C07K 2317/522
20130101; C07K 2317/76 20130101; C07K 2317/64 20130101; A61K
51/1093 20130101; A61P 9/10 20180101; C07K 16/468 20130101; C07K
16/244 20130101 |
Class at
Publication: |
435/69.6 |
International
Class: |
C07K 16/46 20060101
C07K016/46 |
Claims
1-65. (canceled)
66. A method of producing a binding protein, comprising culturing a
host cell in culture medium, wherein said host cell comprises: (a)
a first nucleic acid encoding a first polypeptide chain of the
binding protein that comprises formula 1: 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 (b) a second nucleic acid encoding a
second polypeptide chain of the binding protein that comprises
formula 2: VD1-(X1)n-VD2-C, 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 n is 0 or 1; wherein the first nucleic acid is
present on the same or a different expression vector as the second
nucleic acid; wherein the culturing of the host cell is under
conditions sufficient to produce the first and second polypeptide
chains; and wherein one of the first polypeptide chain and one of
the second polypeptide chain form a divalent form of the binding
protein comprising two antigen binding sites, and wherein two of
the first polypeptide chains and two of the second polypeptides
form a tetravalent form of the binding protein comprising four
antigen binding sites.
67. The method of claim 66, wherein 50%-75% of the binding protein
produced is a dual specific tetravalent binding protein.
68. The method of claim 66, wherein 75%-90% of the binding protein
produced is a dual specific tetravalent binding protein.
69. The method of claim 66, wherein 90%-95% of the binding protein
produced is a dual specific tetravalent binding protein.
70-76. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a division of U.S. Ser. No. 13/601,774
filed Aug. 31, 2012, which is a division of U.S. Ser. No.
12/459,624 filed Jul. 2, 2009, now U.S. Pat. No. 8,258,268, which
is a division of U.S. Ser. No. 11/507,050 filed Aug. 18, 2006, now
U.S. Pat. No. 7,612,181, which claims the benefit of priority to
U.S. Provisional Application No. 60/709,911 filed Aug. 19, 2005,
and to U.S. Provisional Application No. 60/732,892 filed Nov. 2,
2005.
FIELD OF THE INVENTION
[0002] The present invention relates to multivalent and
multispecific binding proteins, methods of making, and specifically
to their uses in the prevention and/or treatment of acute and
chronic inflammatory, cancer, and other diseases.
BACKGROUND OF THE INVENTION
[0003] Engineered proteins, such as multispecific antibodies
capable of binding two or more antigens are known in the art. Such
multispecific binding proteins can be generated using cell fusion,
chemical conjugation, or recombinant DNA techniques.
[0004] Bispecific antibodies have been produced using the quadroma
technology (see Milstein, C. and A. C. Cuello, Nature, 1983,
305(5934):537-40) based on the somatic fusion of two different
hybridoma cell lines expressing murine monoclonal antibodies with
the desired specificities of the bispecific antibody. Because of
the random pairing of two different Ig heavy and light chains
within the resulting hybrid-hybridoma (or quadroma) cell line, up
to ten different immunogloblin species are generated of which only
one is the functional bispecific antibody. The presence of
mispaired by-products, and significantly reduced production yields,
means sophisticated purification procedures are required.
[0005] Bispecific antibodies can be produced by chemical
conjugation of two different mAbs (see Staerz, U. D., et al.,
Nature, 1985, 314(6012): 628-31). This approach does not yield
homogeneous preparation. Other approaches have used chemical
conjugation of two different monoclonal antibodies or smaller
antibody fragments (see Brennan, M., et al., Science, 1985,
229(4708):81-3). Another method is the coupling of two parental
antibodies with a hetero-bifunctional crosslinker, but the
resulting preparations of bispecific antibodies suffer from
significant molecular heterogeneity because reaction of the
crosslinker with the parental antibodies is not site-directed. To
obtain more homogeneous preparations of bispecific antibodies two
different Fab fragments have been chemically crosslinked at their
hinge cysteine residues in a site-directed manner (see Glennie, M.
J., et al., J. Immunol, 1987. 139(7): 2367-75). But this method
results in Fab'2 fragments, not full IgG molecule.
[0006] A wide variety of other recombinant bispecific antibody
formats have been developed in the recent past (see Kriangkum, J.,
et al., Biomol Eng, 2001, 18(2):31-40). Amongst them tandem
single-chain Fv molecules and diabodies, and various derivatives
there of, are the most widely used formats for the construction of
recombinant bispecific antibodies. Routinely, construction of these
molecules starts from two single-chain Fv (scFv) fragments that
recognize different antigens (see Economides, A. N., et al., Nature
Med., 2003, 9(1): 47-52). Tandem scFv molecules (taFv) represent a
straightforward format simply connecting the two scFv molecules
with an additional peptide linker The two scFv fragments present in
these tandem scFv molecules form separate folding entities. Various
linkers can be used to connect the two scFv fragments and linkers
with a length of up to 63 residues (see Nakanishi, K., et al., Annu
Rev Immunol, 2001, 19: 423-74). Although the parental scFv
fragments can normally be expressed in soluble form in bacteria, it
is, however, often observed that tandem scFv molecules form
insoluble aggregates in bacteria. Hence, refolding protocols or the
use of mammalian expression systems are routinely applied to
produce soluble tandem scFv molecules. In a recent study, in vivo
expression by transgenic rabbits and cattle of a tandem scFv
directed against CD28 and a melanoma-associated proteoglycan was
reported (see Gracie, J. A., et al., J Clin Invest, 1999,
104(10):1393-401). In this construct, the two scFv molecules were
connected by a CH1 linker and serum concentrations of up to 100
mg/L of the bispecific antibody were found. Various strategies
including variations of the domain order or using middle linkers
with varying length or flexibility were employed to allow soluble
expression in bacteria. A few studies have now reported expression
of soluble tandem scFv molecules in bacteria (see Leung, B. P., et
al., J. Immunol, 2000, 164(12): 6495-502; Ito, A., et al., J.
Immunol, 2003, 170(9):4802-9; Karni, A., et al., J. Neuroimmunol,
2002, 125(1-2):134-40) using either a very short A1a3 linker or
long glycine/serine-rich linkers. In a recent study, phage display
of a tandem scFv repertoire containing randomized middle linkers
with a length of 3 or 6 residues was employed to enrich for those
molecules that are produced in soluble and active form in bacteria.
This approach resulted in the isolation of a preferred tandem scFv
molecule with a 6 amino acid residue linker (see Arndt, M. and J.
Krauss, Methods Mol Biol, 2003, 207:305-21). It is unclear whether
this linker sequence represents a general solution to the soluble
expression of tandem scFv molecules. Nevertheless, this study
demonstrated that phage display of tandem scFv molecules in
combination with directed mutagenesis is a powerful tool to enrich
for these molecules, which can be expressed in bacteria in an
active form.
[0007] Bispecific diabodies (Db) utilize the diabody format for
expression. Diabodies are produced from scFv fragments by reducing
the length of the linker connecting the VH and VL domain to
approximately 5 residues (see Peipp, M. and T. Valerius, Biochem
Soc Trans, 2002, 30(4):507-11). This reduction of linker size
facilitates dimerization of two polypeptide chains by crossover
pairing of the VH and VL domains. Bispecific diabodies are produced
by expressing, two polypeptide chains with, either the structure
VHA-VLB and VHB-VLA (VH-VL configuration), or VLA-VHB and VLB-VHA
(VL-VH configuration) within the same cell. A large variety of
different bispecific diabodies have been produced in the past and
most of them cab be expressed in soluble form in bacteria. However,
a recent comparative study demonstrates that the orientation of the
variable domains can influence expression and formation of active
binding sites (see Mack, M., G. Riethmuller, and P. Kufer, Proc
Natl Acad Sci USA, 1995, 92(15):7021-5). Nevertheless, soluble
expression in bacteria represents an important advantage over
tandem scFv molecules. However, since two different polypeptide
chains are expressed within a single cell inactive homodimers can
be produced together with active heterodimers. This necessitates
the implementation of additional purification steps in order to
obtain homogenous preparations of bispecific diabodies. One
approach to force the generation of bispecific diabodies is the
production of knob-into-hole diabodies (see Holliger, P., T.
Prospero, and G. Winter, Proc Natl Acad Sci USA, 1993, 90(14):
6444-8.18). This was demonstrated for a bispecific diabody directed
against HER2 and CD3. A large knob was introduced in the VH domain
by exchanging Val37 with Phe and Leu45 with Trp and a complementary
hole was produced in the VL domain by mutating Phe98 to Met and
Tyr87 to Ala, either in the anti-HER2 or the anti-CD3 variable
domains. By using this approach the production of bispecific
diabodies could be increased from 72% by the parental diabody to
over 90% by the knob-into-hole diabody. Importantly, production
yields did only slightly decrease as a result of these mutations.
However, a reduction in antigen-binding activity was observed for
several analyzed constructs. Thus, this rather elaborate approach
requires the analysis of various constructs in order to identify
those mutations that produce heterodimeric molecule with unaltered
binding activity. In addition, such approach requires mutational
modification of the immunoglobulin sequence at the constant region,
thus creating non-native and non-natural form of the antibody
sequence, which may result in increased immunogenicity, poor in
vivo stability, as well as undesireable pharmacokinetics.
[0008] Single-chain diabodies (scDb) represent an alternative
strategy to improve the formation of bispecific diabody-like
molecules (see Holliger, P. and G. Winter, Cancer Immunol
Immunother, 1997. 45(3-4): 128-30; Wu, A. M., et al.,
Immunotechnology, 1996, 2(1): 21-36). Bispecific single-chain
diabodies are produced by connecting the two diabody-forming
polypeptide chains with an additional middle linker with a length
of approximately 15 amino acid residues. Consequently, all
molecules with a molecular weight corresponding to monomeric
single-chain diabodies (50-60 kDa) are bispecific. Several studies
have demonstrated that bispecific single chain diabodies are
expressed in bacteria in soluble and active form with the majority
of purified molecules present as monomers (see Holliger, P. and G.
Winter, Cancer Immunol Immunother, 1997, 45(3-4):128-30; Wu, A. M.,
et al., Immunotechnology, 1996, 2(1):21-36; Pluckthun, A. and P.
Pack, Immunotechnology, 1997, 3(2): 83-105; Ridgway, J. B., et al.,
Protein Eng, 1996, 9(7): 617-21). Thus, single-chain diabodies
combine the advantages of tandem scFvs (all monomers are
bispecific) and diabodies (soluble expression in bacteria).
[0009] More recently diabody have been fused to Fc to generate more
Ig-like molecules, named di-diabody (see Lu, D., et al., J Biol
Chem, 2004, 279(4): 2856-65). In addition, multivalent antibody
construct comprising two Fab repeats in the heavy chain of an IgG
and capable of binding four antigen molecules has been described
(see WO 01/77342A1, and Miller, K., et al., J Immunol, 2003,
170(9): 4854-61).
[0010] There is a need in the art for improved multivalent binding
proteins capable of binding two or more antigens. The present
invention provides a novel family of binding proteins capable of
binding two or more antigens with high affinity.
SUMMARY OF THE INVENTION
[0011] This invention pertains to multivalent binding proteins
capable of binding two or more antigens. The present invention
provides a novel family of binding proteins capable of binding two
or more antigens with high affinity.
[0012] In one embodiment the invention provides a binding protein
comprising a polypeptide chain, wherein said polypeptide chain
comprises VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first variable
domain, VD2 is a second variable domain, C is a constant domain, X1
represents an amino acid or polypeptide, X2 represents an Fc region
and n is 0 or 1. In a preferred embodiment the VD1 and VD2 in the
binding protein are heavy chain variable domains. More preferably
the heavy chain variable domain is selected from the group
consisting of a murine heavy chain variable domain, a human heavy
chain variable domain, a CDR grafted heavy chain variable domain,
and a humanized heavy chain variable domain. In a preferred
embodiment VD1 and VD2 are capable of binding the same antigen. In
another embodiment VD1 and VD2 are capable of binding different
antigens. Preferably C is a heavy chain constant domain. More
preferably X1 is a linker with the proviso that X1 is not CH1. Most
preferably X1 is a linker selected from the group consisting of
AKTTPKLEEGEFSEAR (SEQ ID NO:118); AKTTPKLEEGEFSEARV (SEQ ID
NO:119); AKTTPKLGG (SEQ ID NO:120); SAKTTPKLGG (SEQ ID NO:121);
SAKTTP (SEQ ID NO:122); RADAAP (SEQ ID NO:123); RADAAPTVS (SEQ ID
NO:124); RADAAAAGGPGS (SEQ ID NO:125); RADAAAA(G4S)4 (SEQ ID
NO:126); SAKTTPKLEEGEFSEARV (SEQ ID NO:127); ADAAP (SEQ ID NO:40);
ADAAPTVSIFPP (SEQ ID NO:103); TVAAP (SEQ ID NO:44); TVAAPSVFIFPP
(SEQ ID NO:50); QPKAAP (SEQ ID NO:88); QPKAAPSVTLFPP (SEQ ID
NO:92); AKTTPP (SEQ ID NO:38); AKTTPPSVTPLAP (SEQ ID NO:128);
AKTTAP (SEQ ID NO:129); AKTTAPSVYPLAP (SEQ ID NO:99); ASTKGP (SEQ
ID NO:42); ASTKGPSVFPLAP (SEQ ID NO:48), GGGGSGGGGSGGGGS (SEQ ID
NO:130); GENKVEYAPALMALS (SEQ ID NO:131); GPAKELTPLKEAKVS (SEQ ID
NO:132); and GHEAAAVMQVQYPAS (SEQ ID NO:133). Preferably X2 is an
Fc region. More preferably X2 is a variant Fc region.
[0013] In a preferred embodiment the binding protein disclosed
above comprises a polypeptide chain, wherein said polypeptide chain
comprises 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, and X2 is an Fc region.
[0014] In another embodiment VD1 and VD2 in the binding protein are
light chain variable domains. Preferably the light chain variable
domain is selected from the group consisting of a murine light
chain variable domain, a human light chain variable domain, a CDR
grafted light chain variable domain, and a humanized light chain
variable domain. In one embodiment VD1 and VD2 are capable of
binding the same antigen. In another embodiment VD1 and VD2 are
capable of binding different antigens. Preferably C is a light
chain constant domain. More preferably X1 is a linker with the
proviso that X1 is not CL1. Preferably X1 is a linker selected from
the group consisting of AKTTPKLEEGEFSEAR (SEQ ID NO:118);
AKTTPKLEEGEFSEARV (SEQ ID NO:119); AKTTPKLGG (SEQ ID NO:120);
SAKTTPKLGG (SEQ ID NO:121); SAKTTP (SEQ ID NO:122); RADAAP (SEQ ID
NO:123); RADAAPTVS (SEQ ID NO:124); RADAAAAGGPGS (SEQ ID NO:125);
RADAAAA(G4S)4 (SEQ ID NO:126); SAKTTPKLEEGEFSEARV (SEQ ID NO:127);
ADAAP (SEQ ID NO:40); ADAAPTVSIFPP (SEQ ID NO:103); TVAAP (SEQ ID
NO:44); TVAAPSVFIFPP (SEQ ID NO:50); QPKAAP (SEQ ID NO:88);
QPKAAPSVTLFPP (SEQ ID NO:92); AKTTPP (SEQ ID NO:38); AKTTPPSVTPLAP
(SEQ ID NO:128); AKTTAP (SEQ ID NO:129); AKTTAPSVYPLAP (SEQ ID
NO:99); ASTKGP (SEQ ID NO:42); and ASTKGPSVFPLAP (SEQ ID NO:48).
Preferably the binding protein does not comprise X2.
[0015] In a preferred embodiment the binding protein disclosed
above comprises a polypeptide chain, wherein said polypeptide chain
comprises 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.
[0016] In another preferred embodiment the invention provides a
binding protein comprising two polypeptide chains, wherein said
first polypeptide chain comprises 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, and X2 is an Fc region;
and said second polypeptide chain comprises 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. Most preferably the Dual Variable Domain
(DVD) binding protein comprises four polypeptide chains wherein the
first two polypeptide chains comprises VD1-(X1)n-VD2-C-(X2)n,
respectively 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, and X2 is an Fc region; and the second two polypeptide chain
comprises VD1-(X1)n-VD2-C-(X2)n respectively, 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. Such a Dual Variable Domain (DVD) protein has four antigen
binding sites.
[0017] In another preferred embodiment the binding proteins
disclosed above are capable of binding one or more targets.
Preferably the target is selected from the group consisting of
cytokines, cell surface proteins, enzymes and receptors. Preferably
the binding protein is capable of modulating a biological function
of one or more targets. More preferably the binding protein is
capable of neutralizing one or more targets. The binding protein of
the invention is capable of binding cytokines selected from the
group consisting of lymphokines, monokines, and polypeptide
hormones. In a specific embodiment the binding protein is capable
of binding pairs of cytokines selected from the group consisting of
IL-1.alpha. and IL-1.beta.; IL-12 and IL-18, TNF.alpha. and IL-23,
TNF.alpha. and IL-13; TNF and IL-18; TNF and IL-12; TNF and
IL-1beta; TNF and MIF; TNF and IL-17; and TNF and IL-15; TNF and
VEGF; VEGFR and EGFR; IL-13 and IL-9; IL-13 and IL-4; IL-13 and
IL-5; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and
MIF; IL-13 and TGF-.beta.; IL-13 and LHR agonist; IL-13 and CL25;
IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and ADAMS; and TNF.alpha.
and PGE4, IL-13 and PED2, TNF and PEG2. In another embodiment the
binding protein of the invention is capable of binding pairs of
targets selected from the group consisting of CD138 and CD20; CD138
and CD40; CD19 and CD20; CD20 and CD3; CD38 & CD138; CD38 and
CD20; CD38 and CD40; CD40 and CD20; CD-8 and IL-6; CSPGs and RGM A;
CTLA-4 and BTNO2; IGF1 and IGF2; IGF1/2 and Erb2B; IL-12 and TWEAK;
IL-13 and IL-1.beta.; MAG and RGM A; NgR and RGM A; NogoA and RGM
A; OMGp and RGM A; PDL-1 and CTLA-4; RGM A and RGM B; Te38 and
TNF.alpha.; TNF.alpha. and Blys; TNF.alpha. and CD-22; TNF.alpha.
and CTLA-4; TNF.alpha. and GP130; TNF.alpha. and IL-12p40; and
TNF.alpha. and RANK ligand.
[0018] In one embodiment, the binding protein capable of binding
human IL-1.alpha. and human IL-1.beta. comprises a DVD heavy chain
amino acid sequence selected from the group consisting of SEQ ID
NO. 33, SEQ ID NO. 37, SEQ ID NO. 41, SEQ ID NO. 45, SEQ ID NO. 47,
SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, and SEQ
ID NO. 59; and a DVD light chain amino acid sequence selected from
the group consisting of SEQ ID NO. 35, SEQ ID NO. 39, SEQ ID NO.
43, SEQ ID NO. 46, SEQ ID NO. 49, SEQ ID NO. 52, SEQ ID NO. 54, SEQ
ID NO. 56, SEQ ID NO. 58, and SEQ ID NO. 60. In another embodiment,
the binding protein capable of binding murine IL-1.alpha. and
murine IL-1.beta. comprises a DVD heavy chain amino acid sequence
SEQ ID NO. 105, and a DVD light chain amino acid sequence SEQ ID
NO. 109.
[0019] In one embodiment, the binding protein capable of binding
IL-12 and IL-18 comprises a DVD heavy chain amino acid sequence
selected from the group consisting of SEQ ID NO. 83, SEQ ID NO. 90,
SEQ ID NO. 93, SEQ ID NO. 95, and SEQ ID NO. 114; and a DVD light
chain amino acid sequence selected from the group consisting of SEQ
ID NO. 86, SEQ ID NO. 91, SEQ ID NO. 94, SEQ ID NO. 46, SEQ ID NO.
96, and SEQ ID NO. 116.
[0020] In one embodiment the binding protein capable of binding
CD20 and CD3 comprises a DVD heavy chain amino acid sequence is SEQ
ID NO. 97, and a DVD light chain SEQ ID NO. 101.
[0021] In another embodiment the binding protein of the invention
is capable of binding one, two or more cytokines, cytokine-related
proteins, and cytokine receptors selected from the group consisting
of BMP1, BMP2, BMP3B (GDF10), BMP4, BMP6, BMP8, CSF1 (M-CSF), CSF2
(GM-CSF), CSF3 (G-CSF), EPO, FGF1 (aFGF), FGF2 (bFGF), FGF3
(int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF9, FGF10,
FGF11, FGF12, FGF12B, FGF14, FGF16, FGF17, FGF19, FGF20, FGF21,
FGF23, IGF1, IGF2, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNB1,
IFNG, IFNW1, FIL1, FIL1 (EPSILON), FIL1 (ZETA), IL1A, IL1B, IL2,
IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13,
IL14, IL15, IL16, IL17, IL17B, IL18, IL19, IL20, IL22, IL23, IL24,
IL25, IL26, IL27, IL28A, IL28B, IL29, IL30, PDGFA, PDGFB, TGFA,
TGFB1, TGFB2, TGFB3, LTA (TNF-b), LTB, TNF (TNF-.alpha.), TNFSF4
(OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27
ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TNFSF10
(TRAIL), TNFSF11 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April),
TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, FIGF (VEGFD),
VEGF, VEGFB, VEGFC, IL1R1, IL1R2, IL1RL1, IL1RL2, IL2RA, IL2RB,
IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, IL10RA,
IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R,
IL18R1, IL20RA, IL21R, IL22R, IL1HY1, IL1RAP, IL1RAPL1, IL1RAPL2,
IL1RN, IL6ST, IL18BP, IL18RAP, IL22RA2, AIF1, HGF, LEP (leptin),
PTN, and THPO.
[0022] The binding protein of the invention is capable of binding
one or more chemokines, chemokine receptors, and chemokine-related
proteins selected from the group consisting of CCL1 (I-309), CCL2
(MCP-1/MCAF), CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7
(MCP-3), CCL8 (mcp-2), CCL11 (eotaxin), CCL13 (MCP-4), CCL15
(MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19
(MIP-3b), CCL20 (MIP-3a), CCL21 (SLC/exodus-2), CCL22 (MDC/STC-1),
CCL23 (MPIF-1), CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK), CCL26
(eotaxin-3), CCL27 (CTACK/ILC), CCL28, CXCL1 (GRO1), CXCL2 (GRO2),
CXCL3 (GRO3), CXCL5 (ENA-78), CXCL6 (GCP-2), CXCL9 (MIG), CXCL10
(IP 10), CXCL11 (I-TAC), CXCL12 (SDF1), CXCL13, CXCL14, CXCL16, PF4
(CXCL4), PPBP (CXCL7), CX3CL1 (SCYD1), SCYE1, XCL1 (lymphotactin),
XCL2 (SCM-1b), BLR1 (MDR15), CCBP2 (D6/JAB61), CCR1 (CKR1/HM145),
CCR2 (mcp-1RB/RA), CCR3 (CKR3/CMKBR3), CCR4, CCR5 (CMKBR5/ChemR13),
CCR6 (CMKBR6/CKR-L3/STRL22/DRY6), CCR7 (CKR7/EBI1), CCR8
(CMKBR8/TER1/CKR-L1), CCR9 (GPR-9-6), CCRL1 (VSHK1), CCRL2 (L-CCR),
XCR1 (GPR5/CCXCR1), CMKLR1, CMKOR1 (RDC1), CX3CR1 (V28), CXCR4,
GPR2 (CCR10), GPR31, GPR81 (FKSG80), CXCR3 (GPR9/CKR-L2), CXCR6
(TYMSTR/STRL33/Bonzo), HM74, IL8RA (IL8Ra), IL8RB (IL8Rb), LTB4R
(GPR16), TCP10, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6,
CKLFSF7, CKLFSF8, BDNF, C5R1, CSF3, GRCC10 (C10), EPO, FY (DARC),
GDF5, HIF1A, IL8, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREM1,
TREM2, and VHL. The binding protein of the invention is capable of
binding cell surface protein selected from the group consisting of
integrins. The binding protein of the invention is capable of
binding enzyme selected from the group consisting of kinases and
proteases. The binding protein of the invention is capable of
binding receptor selected from the group consisting of lymphokine
receptor, monokine receptor, and polypeptide hormone receptor.
[0023] In a preferred embodiment the binding protein is
multivalent. More preferably the binding protein is multispecific.
The multivalent and or multispecific binding proteins described
above have desirable properties particularly from a therapeutic
standpoint. For instance, the multivalent and or multispecific
binding protein may (1) be internalized (and/or catabolized) faster
than a bivalent antibody by a cell expressing an antigen to which
the antibodies bind; (2) be an agonist antibody; and/or (3) induce
cell death and/or apoptosis of a cell expressing an antigen which
the multivalent antibody is capable of binding to. The "parent
antibody" which provides at least one antigen binding specificity
of the multivalent and or multispecific binding proteins may be one
which is internalized (and/or catabolized) by a cell expressing an
antigen to which the antibody binds; and/or may be an agonist, cell
death-inducing, and/or apoptosis-inducing antibody, and the
multivalent and or multispecific binding protein as described
herein may display improvement(s) in one or more of these
properties. Moreover, the parent antibody may lack any one or more
of these properties, but may be endowed with them when constructed
as a multivalent binding protein as hereindescribed.
[0024] In another embodiment the binding protein of the invention
has an on rate constant (Kon) to one or more targets 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. Preferably, the binding protein of the invention
has an on rate constant (Kon) to one or more targets between
10.sup.2M.sup.-1s.sup.-1 to 10.sup.3M.sup.-1s.sup.-1; between
10.sup.3M.sup.-1s.sup.-1 to 10.sup.4M.sup.-1s.sup.-1; between
10.sup.4M.sup.-1s.sup.-1 to 10.sup.5M.sup.-1s.sup.-1; or between
10.sup.5M.sup.-1s.sup.-1 to 10.sup.6M.sup.-1s.sup.-1, as measured
by surface plasmon resonance.
[0025] In another embodiment the binding protein has an off rate
constant (Koff) for one or more targets 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.
Preferably, the binding protein of the invention has an off rate
constant (Koff) to one or more targets of 10's.sup.-1 to
10.sup.-4s.sup.-1; of 10.sup.-4s.sup.-1 to 10.sup.-5s.sup.-1; or of
10.sup.-5s.sup.-1 to 10's.sup.-1, as measured by surface plasmon
resonance.
[0026] In another embodiment the binding protein has a dissociation
constant (K.sub.D) to one or more targets 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.-11 M; at most
about 10.sup.-12 M; at most about 10.sup.42 M; and at most
10.sup.-13 M. Preferably, the binding protein of the invention has
a dissociation constant (K.sub.D) to IL-12 or IL-23 of 10.sup.-7 M
to 10.sup.-8 M; of 10.sup.-8 M to 10.sup.-9 M; of 10.sup.-9 M to
10.sup.-10 M; of 10.sup.-10 to 10.sup.-11 M; of 10.sup.-11 M to
10.sup.-12 M; or of 10.sup.-12 to M 10.sup.-13M.
[0027] In another embodiment the binding protein described above is
a conjugate further comprising an agent selected from the group
consisting of; an immunoadhension molecule, an imaging agent, a
therapeutic agent, and a cytotoxic agent. Preferably the imaging
agent is selected from the group consisting of a radiolabel, an
enzyme, a fluorescent label, a luminescent label, a bioluminescent
label, a magnetic label, and biotin. More preferably the imaging
agent is a radiolabel 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.
Preferably the therapeutic or cytotoxic agent is 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, a toxin, and an apoptotic
agent.
[0028] In another embodiment the binding protein described above is
a crystallized binding protein and exists as a crystal. Preferably
the crystal is a carrier-free pharmaceutical controlled release
crystal. More preferably the crystallized binding protein has a
greater half life in vivo than the soluble counterpart of said
binding protein. Most preferably the crystallized binding protein
retains biological activity.
[0029] In another embodiment the binding protein described above is
glycosylated. Preferably the glycosylation is a human glycosylation
pattern.
[0030] One aspect of the invention pertains to an isolated nucleic
acid encoding any one of the binding protein disclosed above. A
further embodiment provides a vector comprising the isolated
nucleic acid disclosed above wherein said vector is selected from
the group consisting of pcDNA; pTT (Durocher et al., Nucleic Acids
Research 2002, Vol 30, No. 2); pTT3 (pTT with additional multiple
cloning site; pEFBOS (Mizushima, S. and Nagata, S., (1990) Nucl.
Acids Res. Vol. 18, No. 17); pBV; pJV; pcDNA3.1 TOPO.RTM.; pEF6
TOPO.RTM.; and pBJ.
[0031] In another aspect a host cell is transformed with the vector
disclosed above. Preferably the host cell is a prokaryotic cell.
More preferably the host cell is E. coli. In a related embodiment
the host cell is an eukaryotic cell. Preferably the eukaryotic cell
is selected from the group consisting of protist cell, animal cell,
plant cell and fungal cell. More preferably the host cell is a
mammalian cell including, but not limited to, CHO and COS; or a
fungal cell such as Saccharomyces cerevisiae; or an insect cell
such as Sf9.
[0032] Another aspect of the invention provides a method of
producing a binding protein disclosed above comprising culturing
any one of the host cells also disclosed above in a culture medium
under conditions sufficient to produce the binding protein.
Preferably 50%-75% of the binding protein produced by this method
is a dual specific tetravalent binding protein. More preferably
75%-90% of the binding protein produced by this method is a dual
specific tetravalent binding protein. Most preferably 90%-95% of
the binding protein produced is a dual specific tetravalent binding
protein.
[0033] Another embodiment provides a binding protein produced
according to the method disclosed above.
[0034] One embodiment provides a composition for the release of a
binding protein wherein the composition comprises a formulation
which in turn comprises a crystallized binding protein, as
disclosed above and an ingredient; and at least one polymeric
carrier. Preferably the polymeric carrier is a polymer selected
from 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 polyeaccharides, blends and copolymers
thereof. Preferably the ingredient is selected from the group
consisting of albumin, sucrose, trehalose, lactitol, gelatin,
hydroxypropyl-.beta.-cyclodextrin, methoxypolyethylene glycol and
polyethylene glycol. Another embodiment provides a method for
treating a mammal comprising the step of administering to the
mammal an effective amount of the composition disclosed above.
[0035] The invention also provides a pharmaceutical composition
comprising a binding protein, as disclosed above and a
pharmaceutically acceptable carrier. In a further embodiment the
pharmaceutical composition comprises at least one additional
therapeutic agent for treating a disorder. Preferably the
additional agent is selected from the group consisting of:
Therapeutic agent, imaging agent, cytotoxic agent, angiogenesis
inhibitors (including but not limited to anti-VEGF antibodies or
VEGF-trap); kinase inhibitors (including but not limited to KDR and
TIE-2 inhibitors); co-stimulation molecule blockers (including but
not limited to anti-B7.1, anti-B7.2, CTLA4-Ig, anti-CD20); adhesion
molecule blockers (including but not limited to anti-LFA-1 Abs,
anti-E/L selectin Abs, small molecule inhibitors); anti-cytokine
antibody or functional fragment thereof (including but not limited
to anti-IL-18, anti-TNF, anti-IL-6/cytokine receptor antibodies);
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.
[0036] In another aspect, the invention provides a method for
treating a human subject suffering from a disorder in which the
target, or targets, capable of being bound by the binding protein
disclosed above is detrimental, comprising administering to the
human subject a binding protein disclosed above such that the
activity of the target, or targets in the human subject is
inhibited and treatment is achieved. Preferably the disorder is
selected from the group comprising 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, 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, polyglandular deficiency type II
(Schmidt's syndrome), adult (acute) respiratory distress syndrome,
alopecia, alopecia areata, 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 syndrome, acquired immunodeficiency
related diseases, hepatitis B, hepatitis C, common variable
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 (classic
autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis
(anti-LKM antibody hepatitis), autoimmune mediated hypoglycaemia,
type B insulin resistance with acanthosis nigricans,
hypoparathyroidism, 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, Sjogren'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, cholestasis, idiosyncratic liver
disease, drug-induced hepatitis, non-alcoholic steatohepatitis,
allergy, 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), lung cancer, breast cancer, stomach cancer, bladder cancer,
colon cancer, pancreatic cancer, ovarian cancer, prostate cancer,
rectal cancer, 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, atrial 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-CD 3
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,
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 hemophagocytic 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, hemochromatosis, 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 migraine headache, idiopathic migraine headache,
mitochondrial multisystem disorder, mixed connective tissue
disease, monoclonal gammopathy, multiple myeloma, multiple systems
degenerations (Menzel, Dejerine-Thomas, Shy-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 muscular
atrophies, neutropenic fever, non-Hodgkin's lymphoma, occlusion of
the abdominal aorta and its branches, occlusive arterial disorders,
OKT3 therapy, orchitis/epididymitis, 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, Raynaud's disease, Refsum's disease, regular narrow QRS
tachycardia, renovascular hypertension, reperfusion injury,
restrictive cardiomyopathy, sarcomas, 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, thromboangiitis 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 hemophagocytic
syndrome, Wernicke-Korsakoff syndrome, Wilson's disease, xenograft
rejection of any organ or tissue.
[0037] In another aspect the invention provides a method of
treating a patient suffering from a disorder comprising the step of
administering any one of the binding proteins disclosed above
before, concurrent, or after the administration of a second agent,
as discussed above. In a preferred embodiment the second agent is
selected from the group consisting of 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. monoclonal antibodies, anti-IL-6 monoclonal
antibodies, growth factors, elastase inhibitors,
pyridinyl-imidazole compounds, antibodies or agonists of TNF, LT,
IL-1, IL-2, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-16, IL-18,
IL-23, EMAP-II, GM-CSF, FGF, and PDGF, antibodies of CD2, CD3, CD4,
CD8, CD-19, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their
ligands, methotrexate, cyclosporin, FK506, rapamycin, mycophenolate
mofetil, leflunomide, NSAIDs, ibuprofen, corticosteroids,
prednisolone, phosphodiesterase inhibitors, adenosine agonists,
antithrombotic agents, complement inhibitors, adrenergic agents,
IRAK, NIK, IKK, p38, MAP kinase inhibitors, IL-1.beta. converting
enzyme inhibitors, TNF.alpha. converting enzyme inhibitors, T-cell
signalling inhibitors, metalloproteinase inhibitors, sulfasalazine,
azathioprine, 6-mercaptopurines, angiotensin converting enzyme
inhibitors, soluble cytokine receptors, soluble p55 TNF receptor,
soluble p75 TNF receptor, sIL-1RI, sIL-1RII, sIL-6R,
antiinflammatory cytokines, IL-4, IL-10, IL-11, IL-13 and
TGF.beta..
[0038] In a preferred embodiment the pharmaceutical compositions
disclosed above are administered to the subject by at least one
mode selected from parenteral, subcutaneous, intramuscular,
intravenous, intraarticular, 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.
[0039] One aspect of the invention provides at least one
anti-idiotype antibody to at least one binding protein of the
present invention. The anti-idiotype antibody includes any protein
or peptide containing molecule that comprises at least a portion of
an immunoglobulin molecule such as, but not limited to, at least
one complementarily determining region (CDR) of a heavy or light
chain or a ligand binding portion thereof, a heavy chain or light
chain variable region, a heavy chain or light chain constant
region, a framework region, or; any portion thereof, that can be
incorporated into a binding protein of the present invention.
[0040] In another embodiment the binding proteins of the invention
are capable of binding one or more targets selected from the group
consisting of ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A;
Aggrecan; AGR2; AICDA; AIF1; AIG1; AKAP1; AKAP2; AMH; AMHR2;
ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1
(zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF; BAG1; BAI1; BCL2;
BCL6; BDNF; BLNK; BLR1 (MDR15); B1yS; BMP1; BMP2; BMP3B (GDF10);
BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1 (plectin); BRCA1;
C19orf10 (IL27w); C3; C4A; C5; C5R1; CANT1; CASP1; CASP4; CAV1;
CCBP2 (D6/JAB61); CCL1 (I-309); CCL11 (eotaxin); CCL13 (MCP-4);
CCL15 (MIP-1d); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19
(MIP-3b); CCL2 (MCP-1); MCAF; CCL20 (MIP-3a); CCL21 (MIP-2); SLC;
exodus-2; CCL22 (MDC/STC-1); CCL23 (MPIF-1); CCL24
(MPIF-2/eotaxin-2); CCL25 (TECK); CCL26 (eotaxin-3);
CCL27(CTACK/ILC); CCL28; CCL3 (MIP-1a); CCL4 (MIP-1b); CCL5
(RANTES); CCL7 (MCP-3); CCL8 (mcp-2); CCNA1; CCNA2; CCND1; CCNE1;
CCNE2; CCR1 (CKR1/HM145); CCR2 (mcp-1RB/RA); CCR3 (CKR3/CMKBR3);
CCR4; CCR5 (CMKBR5/ChemRl3); CCR6 (CMKBR6/CKR-L3/STRL22/DRY6); CCR7
(CKR7/EBI1); CCR8 (CMKBR8/TER1/CKR-L1); CCR9 (GPR-9-6); CCRL1
(VSHK1); CCRL2 (L-CCR); CD164; CD19; CD1C; CD20; CD200; CD-22;
CD24; CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD40; CD40L;
CD44; CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CD8; CD80;
CD81; CD83; CD86; CDH1 (E-cadherin); CDH10; CDH12; CDH13; CDH18;
CDH19; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6;
CDK7; CDK9; CDKN1A (p21Wap1/Cip1); CDKN1B (p27Kip1); CDKN1C; CDKN2A
(p16INK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CERT; CHGA; CHGB;
Chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6;
CKLFSF7; CKLFSF8; CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin);
CMKLR1; CMKOR1 (RDC1); CNR1; COL18A1; COL1A1; COL4A3; COL6A1; CR2;
CRP; CSF1 (M-CSF); CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNB1
(b-catenin); CTSB (cathepsin B); CX3CL1 (SCYD1); CX3CR1 (V28);
CXCL1 (GRO1); CXCL10 (IP-10); CXCL11 (I-TAC/IP-9); CXCL12 (SDF1);
CXCL13; CXCL14; CXCL16; CXCL2 (GRO2); CXCL3 (GRO3); CXCL5
(ENA-78/LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9/CKR-L2);
CXCR4; CXCR6 (TYMSTR/STRL33/Bonzo); CYB5; CYC1; CYSLTR16; DAB2IP;
DES; DKFZp451J0118; DNCL1; DPP4; E2F1; ECGF1; EDG1; EFNA1; EFNA3;
EFNB2; EGF; EGFR; ELAC2; ENG; ENOL; ENO2; ENO3; EPHB4; EPO; ERBB2
(Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); FADD; FasL; FASN; FCER1A;
FCER2; FCGR3A; FGF; FGF1 (aFGF); FGF10; FGF11; FGF12; FGF12B;
FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF20;
FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2);
FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FIL1 (EPSILON); FIL1
(ZETA); F1112584; F1125530; FLRT1 (fibronectin); FLT1; FOS; FOSL1
(FRA-1); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-6ST;
GATA3; GDF5; GFI1; GGT1; GM-CSF; GNAS1; GNRH1; GPR2 (CCR10); GPR31;
GPR44; GPR81 (FKSG80); GRCC10 (C10); GRP; GSN (Gelsolin); GSTP1;
HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; HGF; HIF1A; HIP1; histamine
and histamine receptors; HLA-A; HLA-DRA; HM74; HMOX1; HUMCYT2A;
ICEBERG; ICOSL; ID2; IFN-a; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6;
IFNA7; IFNB1; IFNgamma; IFNW1; IGBP1; IGF1; IGF1R; IGF2; IGFBP2;
IGFBP3; IGFBP6; IL-1; IL10; IL10RA; IL10RB; IL11; IL11RA; IL-12;
IL12A; IL12B; IL12RB1; IL12RB2; IL13; IL13RA1; IL13RA2; IL14; IL15;
IL15RA; IL16; IL17; IL17B; IL17C; IL17R; IL18; IL18BP; IL18R1;
IL18RAP; IL19; IL1A; IL1B; IL1F10; IL1F5; IL1F6; IL1F7; IL1F8;
IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1;
IL1RL2 IL1RN; IL2; IL20; IL20RA; IL21R; IL22; IL22R; IL22RA2; IL23;
IL24; IL25; IL26; IL27; IL28A; IL28B; IL29; IL2RA; IL2RB; IL2RG;
IL3; IL30; IL3RA; IL4; IL4R; IL5; IL5RA; IL6; IL6R; IL6ST
(glycoprotein 130); IL7; IL7R; IL8; IL8RA; IL8RB; IL8RB; IL9; IL9R;
ILK; INHA; INHBA; INSL3; INSL4; IRAK1; IRAK2; ITGA1; ITGA2; ITGA3;
ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b 4 integrin); JAG1;
JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GC Box BP); KLF6;
KLK10; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9;
KRT1; KRT19 (Keratin 19); KRT2A; KRTHB6 (hair-specific type II
keratin); LAMAS; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA
(TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp;
MAP2K7 (c-Jun); MDK; MIB1; midkine; MIF; MIP-2; MKI67 (Ki-67);
MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-III); MTSS1; MUC1
(mucin); MYC; MYD88; NCK2; neurocan; NFKB1; NFKB2; NGFB (NGF);
NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1
(NM23A); NOX5; NPPB; NROB1; NROB2; NR1D1; NR1D2; NR1H2; NR1H3;
NR1H4; NR1I2; NR1I3; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2;
NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1;
NRP1; NRP2; NT5E; NTN4; ODZ1; OPRD1; P2RX7; PAP; PART1; PATE; PAWR;
PCA3; PCNA; PDGFA; PDGFB; PECAM1; PF4 (CXCL4); PGF; PGR;
phosphacan; PIAS2; PIK3CG; PLAU (uPA); PLG; PLXDC1; PPBP (CXCL7);
PPID; PR1; PRKCQ; PRKD1; PRL; PROC; PROK2; PSAP; PSCA; PTAFR; PTEN;
PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RARB; RGS1; RGS13; RGS3; RNF110
(ZNF144); ROBO2; S100A2; SCGB1D2 (lipophilin B); SCGB2A1
(mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelial
Monocyte-activating cytokine); SDF2; SERPINA1; SERPINA3; SERPINB5
(maspin); SERPINE1 (PAI-1); SERPINF1; SHBG; SLA2; SLC2A2; SLC33A1;
SLC43A1; SLIT2; SPP1; SPRR1B (Spr1); ST6GAL1; STAB1; STATE; STEAP;
STEAP2; TB4R2; TBX21; TCP10; TDGF1; TEK; TGFA; TGFB1; TGFB1I1;
TGFB2; TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1
(thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-1); TIMP3; tissue
factor; TLR10; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF;
TNF-a; TNFAIP2 (B94); TNFAIP3; TNFRSF11A; TNFRSF1A; TNFRSF1B;
TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9;
TNFSF10 (TRAIL); TNFSF11 (TRANCE); TNFSF12 (APO3L); TNFSF13
(April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF15 (VEGI); TNFSF18;
TNFSF4 (OX40 ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); TNFSF7
(CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-1BB ligand); TOLLIP;
Toll-like receptors; TOP2A (topoisomerase Iia); TP53; TPM1; TPM2;
TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM1; TREM2;
TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC; versican; VHL C5; VLA-4;
XCL1 (lymphotactin); XCL2 (SCM-1b); XCR1 (GPR5/CCXCR1); YY1; and
ZFPM2.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1A is a schematic representation of Dual Variable
Domain (DVD)-Ig constructs and shows the strategy for generation of
a DVD-Ig from two parent antibodies; FIG. 1B, is a schematic
representation of constructs DVD1-Ig, DVD2-Ig, and two chimeric
mono-specific antibodies from hybridoma clones 2D13.E3
(anti-IL-1.alpha.) and 13F5.G5 (anti-IL-1.beta.).
DETAILED DESCRIPTION OF THE INVENTION
[0042] This invention pertains to multivalent and/or multispecific
binding proteins capable of binding two or more antigens.
Specifically, the invention relates to dual variable domain
immunoglobulins (DVD-Ig), and pharmaceutical compositions thereof,
as well as nucleic acids, recombinant expression vectors and host
cells for making such DVD-Igs. Methods of using the DVD-Igs of the
invention to detect specific antigens, either in vitro or in vivo
are also encompassed by the invention.
[0043] 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.
[0044] 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.
[0045] That the present invention may be more readily understood,
select terms are defined below.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] "Biological activity" as used herein, refers to all inherent
biological properties of the antigen. Biological properties include
but are not limited to binding receptor; induction of cell
proliferation, inhibiting cell growth, inductions of other
cytokines, induction of apoptosis, and enzymatic activity.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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; 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. 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: 9266-73.). 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., 0.2000 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.
[0054] 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. 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 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 e.g., 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 e.g.,
Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA
90:6444-6448; Poljak, R. J., 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).
[0055] The term "multivalent binding protein" is used throughout
this specification to denote a binding protein comprising two or
more antigen binding sites. The multivalent binding protein is
preferably engineered to have the 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. DVD binding proteins comprising two
heavy chain DVD polypeptides and two light chain DVD polypeptides
are referred to a DVD Ig. Each half of a DVD Ig comprises a heavy
chain DVD polypeptide, and a light chain DVD polypeptide, and two
antigen binding sites. Each binding site comprises a heavy chain
variable domain and a light chain variable domain with a total of 6
CDRs involved in antigen binding per antigen binding site.
[0056] 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):
537-40), by chemical conjugation of two different mAbs (see Staerz,
U. D., et al., Nature, 1985. 314(6012): 628-31), or by
knob-into-hole or similar approaches which introduces mutations in
the Fc region (see Holliger, P., T. Prospero, and G. Winter, Proc
Natl Acad Sci USA, 1993. 90(14): 6444-8.18), 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 mono-valent
for each antigen it binds to.
[0057] 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 bi-valent for each
antigen it binds to.
[0058] A "functional antigen binding site" of a binding protein is
one which 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.
[0059] The term "cytokine" is a generic term for proteins released
by one cell population, which 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 hormone 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; tumor necrosis factor-alpha
and -beta; mullerian-inhibiting substance; mouse
gonadotropin-associated peptide; inhibin; activin; vascular
endothelial growth factor; integrin; thrombopoietin (TPO); nerve
growth factors such as NGF-alpha; platelet-growth factor;
transforming growth factors (TGFs) such as TGF-alpha and TGF-beta;
insulin-like growth factor-1 and -11; erythropoietin (EPO);
osteoinductive factors; interferons such as interferon-alpha, -beta
and -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-18,
IL-23; a tumor necrosis factor such as TNF-alpha or TNF-beta; 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.
[0060] The term "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, P., et
al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J.,
et al. (1994) Structure 2:1121-1123). Prefered linker include, but
are not limited to, AKTTPKLEEGEFSEAR (SEQ ID NO:118);
AKTTPKLEEGEFSEARV (SEQ ID NO:119); AKTTPKLGG (SEQ ID NO:120);
SAKTTPKLGG (SEQ ID NO:121); SAKTTP (SEQ ID NO:122); RADAAP (SEQ ID
NO:123); RADAAPTVS (SEQ ID NO:124); RADAAAAGGPGS (SEQ ID NO:125);
RADAAAA(G4S)4 (SEQ ID NO:126); SAKTTPKLEEGEFSEARV (SEQ ID NO:127);
ADAAP (SEQ ID NO:40); ADAAPTVSIFPP (SEQ ID NO:103); TVAAP (SEQ ID
NO:44); TVAAPSVFIFPP (SEQ ID NO:50); QPKAAP (SEQ ID NO:88);
QPKAAPSVTLFPP (SEQ ID NO:92); AKTTPP (SEQ ID NO:38); AKTTPPSVTPLAP
(SEQ ID NO:128); AKTTAP (SEQ ID NO:129); AKTTAPSVYPLAP (SEQ ID
NO:99); ASTKGP (SEQ ID NO:42); ASTKGPSVFPLAP (SEQ ID NO:48).
[0061] 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.
[0062] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigen. Furthermore, in contrast to polyclonal antibody
preparations that typically include different antibodies directed
against different determinants (epitopes), each monoclonal antibody
is directed against a single determinant on the antigen. The
modifier "monoclonal" is not to be construed as requiring
production of the antibody by any particular method.
[0063] 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.
[0064] 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.
[0065] An "affinity matured" antibody is one with one or more
alterations in one or more CDRs thereof which result an improvement
in the affinity of the antibody for antigen, compared to a parent
antibody which does not possess those alteration(s). Preferred
affinity matured antibodies will have nanomolar or even picomolar
affinities for the target antigen. Affinity matured antibodies are
produced by procedures known in the art. Marks et al. BioTechnology
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):3310-9 (1995); and Hawkins et al., J. Mol. Biol 226:889-896
(1992).
[0066] The term "chimeric antibody" refers to antibodies which
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.
[0067] The term "CDR-grafted antibody" refers to antibodies which
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.
[0068] The term "humanized antibody" refers to antibodies which
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.
[0069] 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 Acad, 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.
[0070] 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 &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.
[0071] 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.
[0072] 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.
[0073] As used herein, the term "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%, preferably 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. Preferably, 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.
[0074] As used herein, the term "neutralizing" refers to
neutralization of biological activity of a cytokine when a binding
protein specifically binds the cytokine. Preferably the
neutralizing binding protein binds the cytokine and reduces its
biologically activity by at least about 20%, 40%, 60%, 80%, 85% or
more.
[0075] The term "activity" includes activities such as the binding
specificity/affinity of a DVD-Ig for two or more antigens.
[0076] 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.
[0077] 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, U., et al. (1991)
Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol.
Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem.
198:268-277.
[0078] The term "K.sub.on", as used herein, is intended to refer to
the on rate constant for association of an antibody to the antigen
to form the antibody/antigen complex as is known in the art.
[0079] The term "K.sub.off", as used herein, is intended to refer
to the off rate constant for dissociation of an antibody from the
antibody/antigen complex as is known in the art.
[0080] The term "K.sub.d", as used herein, is intended to refer to
the dissociation constant of a particular antibody-antigen
interaction as is known in the art.
[0081] 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. Preferably, the label is a
detectable marker, e.g., incorporation of a radiolabeled amino acid
or attachment to a polypeptide of biotinyl moieties that can be
detected by marked avidin (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); 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, such as gadolinium chelates.
[0082] The term "conjugate" refers to a binding protein, such as an
antibody, 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.
[0083] The terms "crystal", and "crystallized" as used herein,
refer to 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, which 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, R. and Ducruix,
Crystallization of Nucleic Acids and Proteins, a Practical
Approach, 2nd ed., pp. 1-16, Oxford University Press, New York,
N.Y., (1999)."
[0084] The term "polynucleotide" as referred to herein means a
polymeric form of two or more nucleotides, either ribonucleotides
or deoxvnucleotides or a modified form of either type of
nucleotide. The term includes single and double stranded forms of
DNA but preferably is double-stranded DNA.
[0085] The term "isolated polynucleotide" as used herein 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.
[0086] 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.
[0087] 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 which 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.
[0088] "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.
[0089] The term "recombinant host cell" (or simply "host cell"), as
used herein, is intended to refer to a cell into which exogenous
DNA has been introduced. It should be understood that such terms
are intended to refer not only to the particular subject cell, but,
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. Preferably host cells
include prokaryotic and eukaryotic cells selected from any of the
Kingdoms of life. Preferred eukaryotic cells include protist,
fungal, plant and animal cells. Most preferably host cells include
but are not limited to the prokaryotic cell line E. Coli; mammalian
cell lines CHO, HEK 293 and COS; the insect cell line Sf9; and the
fungal cell Saccharomyces cerevisiae.
[0090] 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 (2d ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by
reference for any purpose.
[0091] "Transgenic organism", as known in the art and as used
herein, 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.
[0092] The term "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 a cytokine). 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.
[0093] 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 a
cytokine). 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.
[0094] 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, polypeptides, nucleic
acids, carbohydrates, or any other molecules that bind to the
antigen.
[0095] The term "antagonist" or "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 the antigen. Antagonists
and inhibitors of antigens may include, but are not limited to,
proteins, nucleic acids, carbohydrates, or any other molecules,
which bind to the antigen.
[0096] As used herein, the term "effective amount" refers to the
amount of a therapy which 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).
[0097] 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, mice, rats, monkeys, dogs, rabbits and other
animals. Such substances include, but are not limited to, blood,
serum, urine, synovial fluid, cells, organs, tissues, bone marrow,
lymph nodes and spleen.
I. Generation of DVD Binding Protein
[0098] The invention pertains to Dual Variable Domain binding
proteins capable of binding one or more targets and methods of
making the same. Preferably the binding protein comprises a
polypeptide chain, wherein said polypeptide chain comprises
VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first variable domain, VD2
is a second variable domain, C is a constant domain, X1 represents
an amino acid or polypeptide, X2 represents an Fc region and n is 0
or 1. The binding protein of the invention can be generated using
various techniques. The invention provides expression vectors, host
cell and methods of generating the binding protein.
a. Generation of Parent Monoclonal Antibodies
[0099] The variable domains of the DVD binding protein can be
obtained from parent antibodies, including polyclonal and
monoclonal antibodies capable of binding antigens of interest.
These antibodies may be naturally occurring or may be generated by
recombinant technology.
[0100] 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,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-581
(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.
Hybridomas are selected, cloned and further screened for desirable
characteristics, including robust hybridoma growth, high antibody
production and desirable antibody characteristics, as discussed in
Example 1 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. In a preferred embodiment, the
hybridomas are mouse hybridomas. 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
antibody capable of binding a specific antigen.
[0101] Recombinant monoclonal antibodies are also 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 WO 92/02551 and Babcook
et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848. In this
method, single cells secreting antibodies of interest, e.g.,
lymphocytes derived from an immunized animal, are identified, and,
heavy- and light-chain variable region 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 the antigen of interest. 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 WO 97/29131 and PCT Publication
WO 00/56772.
[0102] Monoclonal antibodies are also produced by immunizing a
non-human animal comprising some, or all, of the human
immunoglobulin locus with an antigen of interest. In a preferred
embodiment, the non-human animal is a XENOMOUSE.RTM. 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 WO
91/10741, published July 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), Green and
Jakobovits J. Exp. Med. 188:483-495 (1998), the disclosures of
which are hereby incorporated by reference.
[0103] In vitro methods also can be used to make the parent
antibodies, 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. (1991)
Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod
Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;
McCafferty et al., Nature (1990) 348:552-554; Griffiths et al.
(1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol
226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.
(1992) Proc. Natl. Acad. Sci. USA, 89:3576-3580; Garrad et al.
(1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc
Acid Res 19:4133-4137; and Barbas et al. (1991) Proc. Natl. Acad.
Sci. USA, 88:7978-7982, US patent application publication
20030186374, and PCT Publication No. WO 97/29131, the contents of
each of which are incorporated herein by reference.
[0104] Parent 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
Brinkman 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
application No. PCT/GB91/01134; PCT publications WO 90/02809; WO
91/10737; WO 92/01047; 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; 5,821,047; 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.
[0105] 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., AJRI 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 240:1038-1040 (1988).
[0106] 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 parent antibodies. 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.
[0107] In another approach the parent antibodies 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 parent
antibodies include those disclosed in Wittrup, et al. U.S. Pat. No.
6,699,658 incorporated herein by reference.
[0108] The antibodies described above can be further modified to
generate CDR grafted and Humanized parent antibodies. CDR-grafted
parent antibodies comprise heavy and light chain variable region
sequences from a human antibody wherein one or more of the CDR
regions of VH and/or VL are replaced with CDR sequences of murine
antibodies capable of binding antigen of interest. 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 such
antibodies are known in the art (see EP 239,400; PCT publication WO
91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089),
veneering or resurfacing (EP 592,106; EP 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 (U.S. Pat. No.
5,565,352).
[0109] Humanized antibodies are antibody molecules from non-human
species antibody that binds the desired antigen having one or more
complementarity determining regions (CDRs) from the non-human
species and framework regions from a human immunoglobulin molecule.
Known human Ig sequences are disclosed, e.g.,
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/Immuno-logy.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/.about.jraats/linksl.html;
www.recab.uni-hd.de/immuno.bme.nwu.edu/;
www.mrc-cpe.cam.ac.uk/imt-doc/pu-blic/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/AHOsem-inar/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/h-umanisation/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.fmolina/Web-pages/Pept/spottech.html;
www.jerini.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.
[0110] Framework 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 (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 (1986); Verhoeyen et al., Science 239:1534
(1988)), Sims et al., J. Immunol. 151: 2296 (1993); Chothia and
Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl.
Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol.
151:2623 (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 WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630,
US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755;
WO90/14443, WO90/14424, WO90/14430, EP 229246, EP 592,106; EP
519,596, EP 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,
5,766886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101,
5,585,089, 5,225,539; 4,816,567, each entirely incorporated herein
by reference, included references cited therein.
[0111] Parent monoclonal antibodies may be selected from various
monoclonal antibodies capable of binding specific targets and well
known in the art. These include, but are not limited to 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
2005/0147610 A1), anti-05, anti-CBL, anti-CD147, anti-gp120,
anti-VLA-4, anti-CD11a, anti-CD18, anti-VEGF, anti-CD40L, anti-Id,
anti-ICAM-1, anti-CXCL13, anti-CD2, anti-EGFR, anti-TGF-beta 2,
anti-E-selectin, anti-Fact VII, anti-Her2/neu, anti-F gp,
anti-CD11/18, anti-CD14, anti-ICAM-3, anti-CD80, anti-CD4,
anti-CD3, anti-CD23, anti-beta2-integrin, anti-alpha4beta7,
anti-CD52, anti-HLA DR, anti-CD22, 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-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-8, anti-IL-9, anti-IL-13, anti-IL-13 receptor, anti-IL-17,
and anti-IL-23 (see Presta LG. 2005 Selection, design, and
engineering of therapeutic antibodies J Allergy Clin Immunol.
116:731-6 and Clark, M., "Antibodies for Therapeutic Applications,"
Department of Pathology, Cambridge University, UK, 15 Oct. 2000,
published online at M. Clark's home page at the website for the
Department of Pathology, Cambridge University.)
[0112] Parent monoclonal antibodies 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.RTM., 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, 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.TM. (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 monoclonal antibody 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, etanercept (ENBREL.RTM.), an anti-TNFalpha
Fc fusion developed by Immunex/Amgen, 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, J695, an anti-IL-12 antibody being developed by
Cambridge Antibody Technology and 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.RTM., an anti-CD4 antibody being
developed by Genmab, HUMAX.TM.-IL15, an anti-IL15 antibody being
developed by Genmab and Amgen, HUMAX.TM.-Inflam, being developed by
Genmab and Medarex, HUMAX.TM.-Cancer, an anti-Heparanase I antibody
being developed by Genmab and Medarex and Oxford GlycoSciences,
HUMAX.TM.-Lymphoma, being developed by Genmab and Amgen,
HUMAX.TM.-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-CD4.RTM., 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/J&J, CNTO 1275, an
anti-cytokine antibody being developed by Centocor/J&J, 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, all of the above-cited references in this
paragraph are expressly incorporated herein by reference.
B. Construction of DVD Molecules:
[0113] The dual variable domain immunoglobulin (DVD-Ig) molecule is
designed such that two different light chain variable domains (VL)
from the two different parent mAbs 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 (FIG. 1A).
[0114] The variable domains can be obtained using recombinant DNA
techniques from a parent antibody generated by any one of the
methods described above. In a preferred embodiment the variable
domain is a murine heavy or light chain variable domain. More
preferably the variable domain is a CDR grafted or a humanized
variable heavy or light chain domain. Most preferably the variable
domain is a human heavy or light chain variable domain.
[0115] 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. Preferably 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 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 molecules may also comprise 2 or more
non-Ig domains.
[0116] The linker sequence may be a single amino acid or a
polypeptide sequence. Preferably the linker sequences are selected
from the group consisting of AKTTPKLEEGEFSEAR (SEQ ID NO:118);
AKTTPKLEEGEFSEARV (SEQ ID NO:119); AKTTPKLGG (SEQ ID NO:120);
SAKTTPKLGG (SEQ ID NO:121); SAKTTP (SEQ ID NO:122); RADAAP (SEQ ID
NO:123); RADAAPTVS (SEQ ID NO:124); RADAAAAGGPGS (SEQ ID NO:125);
RADAAAA(G.sub.4S).sub.4 (SEQ ID NO:126); SAKTTPKLEEGEFSEARV (SEQ ID
NO:127); ADAAP (SEQ ID NO:40); ADAAPTVSIFPP (SEQ ID NO:103); TVAAP
(SEQ ID NO:44); TVAAPSVFIFPP (SEQ ID NO:50); QPKAAP (SEQ ID NO:88);
QPKAAPSVTLFPP (SEQ ID NO:92); AKTTPP (SEQ ID NO:38); AKTTPPSVTPLAP
(SEQ ID NO:128); AKTTAP (SEQ ID NO:129); AKTTAPSVYPLAP (SEQ ID
NO:99); ASTKGP (SEQ ID NO:42); ASTKGPSVFPLAP (SEQ ID NO:48);
GGGGSGGGGSGGGGS (SEQ ID NO:130); GENKVEYAPALMALS (SEQ ID NO:131);
GPAKELTPLKEAKVS (SEQ ID NO:132); and GHEAAAVMQVQYPAS (SEQ ID
NO:133). 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 of the
invention were 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,
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, therefore minimize to a large extent any immunogenicity
potentially arising from the linkers and junctions.
[0117] 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 (e.g., G4S repeats);
hinge region-derived sequences; and other natural sequences from
other proteins.
[0118] In a preferred embodiment a constant domain is linked to the
two linked variable domains using recombinant DNA techniques.
Preferably sequence comprising linked heavy chain variable domains
is linked to a heavy chain constant domain and sequence comprising
linked light chain variable domains is linked to a light chain
constant domain. Preferably the constant domains are human heavy
chain constant domain and human light chain constant domain
respectively. Most preferably 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. Most preferably the Fc region is a human Fc
region. In a preferred embodiment the Fc region includes Fc region
from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.
[0119] 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 targets, and methods of
making the same, is provided in the Examples section below.
C. Production of DVD Proteins
[0120] Binding proteins 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 DVD heavy and DVD 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 proteins of the
invention in either prokaryotic or eukaryotic host cells,
expression of DVD proteins 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 DVD protein.
[0121] 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 DVD proteins are introduced into mammalian host cells, the
DVD proteins are produced by culturing the host cells for a period
of time sufficient to allow for expression of the DVD proteins in
the host cells or, more preferably, secretion of the DVD proteins
into the culture medium in which the host cells are grown. DVD
proteins can be recovered from the culture medium using standard
protein purification methods.
[0122] In a preferred system for recombinant expression of DVD
proteins of the invention, a recombinant expression vector encoding
both the DVD heavy chain and the DVD light chain is introduced into
dhfr-CHO cells by calcium phosphate-mediated transfection. Within
the recombinant expression vector, the DVD 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 heavy and light chains and intact DVD 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 protein from the culture medium.
Still further the invention provides a method of synthesizing a DVD
protein of the invention by culturing a host cell of the invention
in a suitable culture medium until a DVD protein of the invention
is synthesized. The method can further comprise isolating the DVD
protein from the culture medium.
[0123] 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 WO2001/077342(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. Separation of 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.
[0124] 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".
[0125] At least 50%, preferably 75% and more preferably 90% of the
assembled, and expressed dual variable domain immunoglobulin
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".
[0126] The present invention provides a preferred method to express
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.
[0127] The present invention provides a more preferred 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", 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.
[0128] The present invention provides a most preferred 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", 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.
II. Derivatized DVD Binding Proteins:
[0129] One embodiment provides a labeled binding protein wherein
the binding protein 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 binding protein 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 association of the binding protein with
another molecule (such as a streptavidin core region or a
polyhistidine tag).
[0130] Useful detectable agents with which a binding protein 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. A binding protein may also be derivatized with detectable
enzymes, such as alkaline phosphatase, horseradish peroxidase,
glucose oxidase and the like. When a binding protein 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. A binding protein may also be derivatized with biotin,
and detected through indirect measurement of avidin or streptavidin
binding.
[0131] Another embodiment of the invention provides a crystallized
binding protein 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.
[0132] Crystallized binding protein of the invention may be
produced according methods known in the art and as disclosed in WO
02072636, incorporated herein by reference.
[0133] 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. 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 (2005), pp. 11-16). 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. (1993) 30:1361-1367), or result in increased affinity for
the antigen (Wallick, S. C., et al., Exp. Med. (1988)
168:1099-1109; Wright, A., et al., EMBO J. (1991) 10:2717
2723).
[0134] 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.
[0135] 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 WO2003016466A2, and U.S. Pat. Nos. 5,714,350 and
6,350,861, each of which is incorporated herein by reference in its
entirety.
[0136] 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 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. (1999) Nat. Biotech. 17:176-1, as well as, European Patent
No: EP 1,176,195; PCT Publications WO 03/035835; WO 99/54342, each
of which is incorporated herein by reference in its entirety.
[0137] 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.
[0138] 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.
[0139] 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 (U.S patent applications
20040018590 and 20020137134 and PCT publication WO2005100584
A2).
[0140] In addition to the binding proteins, the present invention
is also directed to an anti-idiotypic (anti-Id) antibody 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. The 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.
[0141] 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.
III. Uses of DVD-Ig
[0142] Given their ability to bind to two or more antigens the
binding proteins of the invention can be used to detect the
antigens (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 DVD-Ig 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.
[0143] The binding proteins of the invention preferably are capable
of neutralizing the activity of the antigens both in vitro and in
vivo. Accordingly, such DVD-Igs can be used to inhibit antigen
activity, e.g., in a cell culture containing the antigens, in human
subjects or in other mammalian subjects having the antigens with
which a binding protein of the invention cross-reacts. In another
embodiment, the invention provides a method for reducing antigen
activity in a subject suffering from a disease or disorder in which
the antigen activity is detrimental. A binding protein of the
invention can be administered to a human subject for therapeutic
purposes.
[0144] As used herein, the term "a disorder in which antigen
activity is detrimental" is intended to include diseases and other
disorders in which the presence of the antigen 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 antigen activity is detrimental is
a disorder in which reduction of antigen 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 the antigen in a biological fluid of a subject
suffering from the disorder (e.g., an increase in the concentration
of antigen in serum, plasma, synovial fluid, etc. of the subject).
Non-limiting examples of disorders that can be treated with the
binding proteins of the invention include those disorders discussed
below and in the section pertaining to pharmaceutical compositions
of the antibodies of the invention.
[0145] The DVD-Igs of the invention may bind one antigen or
multiple antigens. Such antigens include, but are not limited to,
the targets listed in the following databases, which databases are
incorporated herein by reference. These target databases include
those listings: 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.ac.jp/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).
[0146] DVD-Igs are useful as therapeutic agents to simultaneously
block two different targets to enhance efficacy/safety and/or
increase patient coverage. Such targets may include soluble targets
(IL-13 and TNF) and cell surface receptor targets (VEGFR and EGFR).
It can also be used to induce redirected cytotoxicity between tumor
cells and T cells (Her2 and CD3) for cancer therapy, or between
autoreactive cell and effector cells for
autoimmune/transplantation, or between any target cell and effector
cell to eliminate disease-causing cells in any given disease.
[0147] In addition, DVD-Ig can be used to trigger receptor
clustering and activation when it is designed to target two
different epitopes on the same receptor. This may have benefit in
making agonistic and antagonistic anti-GPCR therapeutics. In this
case, DVD-Ig can be used to target two different epitopes on one
cell for clustering/signaling (two cell surface molecules) or
signaling (on one molecule). Similarly, a DVD-Ig molecule can be
designed to triger CTLA-4 ligation, and a negative signal by
targeting two different epitopes (or 2 copies of the same epitope)
of CTLA-4 extracellular domain, leading to down regulation of the
immune response. CTLA-4 is a clinically validated target for
therapeutic treatment of a number of immunological disorders.
CTLA-4/B7 interactions negatively regulate T cell activation by
attenuating cell cycle progression, IL-2 production, and
proliferation of T cells following activation, and CTLA-4 (CD152)
engagement can down-regulate T cell activation and promote the
induction of immune tolerance. However, the strategy of attenuating
T cell activation by agonistic antibody engagement of CTLA-4 has
been unsuccessful since CTLA-4 activation requires ligation. The
molecular interaction of CTLA-4/B7 is in "skewed zipper" arrays, as
demonstrated by crystal structural analysis (Stamper 2001 Nature
410:608). However none of the currently available CTLA-4 binding
reagents have ligation properties, including anti-CTLA-4 monoclonal
antibodies. There have been several attempts to address this issue.
In one case, a cell member-bound single chain antibody was
generated, and significantly inhibited allogeneic rejection in mice
(Hwang et al., J. Immunol., 169: 633-637 (2002). In a separate
case, artificial APC surface-linked single-chain antibody to CTLA-4
was generated and demonstrated to attenuate T cell responses
(Griffin 2000 JI 164:4433). In both cases, CTLA-4 ligation was
achieved by closely localized member-bound antibodies in artificial
systems. While these experiments provide proof-of-concept for
immune down-regulation by triggering CTLA-4 negative signaling, the
reagents used in these reports are not suitable for therapeutic
use. To this end, CTLA-4 ligation may be achieved by using a DVD-Ig
molecule, which target two different epitopes (or 2 copies of the
same epitope) of CTLA-4 extracellular domain. The rationale is that
the distance spanning two binding sites of an IgG, approximately
150-170A, is too large for active ligation of CTLA-4 (30-50 .ANG.
between 2 CTLA-4 homodimer). However the distance between the two
binding sites on DVD-Ig (one arm) is much shorter, also in the
range of 30-50 .ANG., allowing proper ligation of CTLA-4.
[0148] Similarly, DVD-Ig can target two different members of a cell
surface receptor complex (e.g., IL-12R alpha and beta).
Furthermore, DVD-Ig can target CR1 and a soluble protein/pathogen
to drive rapid clearance of the target soluble
protein/pathogen.
[0149] 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 an 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 (ABT-578) 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;
Drug/device combinations for local drug therapies and infection
prophylaxis, Wu, Peng; Grainger, David W., Biomaterials (2006),
27(11), 2450-2467; Mediation of the cytokine network in the
implantation of orthopedic devices., Marques, A. P.; Hunt, J. A.;
Reis, Rui L., Biodegradable Systems in Tissue Engineering and
Regenerative Medicine (2005), 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 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 (Aoki 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 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
[0150] 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.
1. Human Autoimmune and Inflammatory Response
[0151] Many proteins have been implicated in general autoimmune and
inflammatory responses, including C5, CCL1 (I-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 (I-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,
TGFB1I1, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, TH1L, TNF,
TNFRSF1A, TNFRSF1B, TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF11A, TNFRSF21,
TNFSF4, TNFSF5, TNFSF6, TNFSF11, VEGF, ZFPM2, and RNF110 (ZNF144).
In one aspect, DVD-Igs capable of binding one or more of the
targets listed above are provided.
2. Asthma
[0152] 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. There
is increasing evidence that IL-13 in mice mimics many of the
features of asthma, including AHR, mucus hypersecretion and airway
fibrosis, independently of eosinophilic inflammation (Finotto et
al., International Immunology (2005), 17(8): 993-1007; Padilla et
al., Journal of Immunology (2005), 174(12): 8097-8105).
[0153] IL-13 has been implicated as having a pivotal role in
causing pathological responses associated with asthma. The
development of anti-IL-13 monoclonal antibody therapy to reduce the
effects of IL-13 in the lung is an exciting new approach that
offers considerable promise as a novel treatment for asthma.
However other mediators of differential immunological pathways are
also involved in asthma pathogenesis, and blocking these mediators,
in addition to IL-13, may offer additional therapeutic benefit.
Such target pairs include, but are not limited to, IL-13 and a
pro-inflammatory cytokine, such as tumor necrosis factor-.alpha.
(TNF-.alpha.). TNF-.alpha. may amplify the inflammatory response in
asthma and may be linked to disease severity (McDonnell, et al.,
Progress in Respiratory Research (2001), 31(New Drugs for Asthma,
Allergy and COPD), 247-250.). This suggests that blocking both
IL-13 and TNF-.alpha. may have beneficial effects, particularly in
severe airway disease. In a preferred embodiment the DVD-Ig of the
invention binds the targets IL-13 and TNF.alpha. and is used for
treating asthma.
[0154] 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).
[0155] Based on the rationale disclosed above and using the same
evaluation model for efficacy and safety other pairs of targets
that DVD-Ig molecules can bind and be useful to treat asthma may be
determined. Preferably such targets include, but are not limited
to, IL-13 and IL-1beta, since IL-1beta is also implicated in
inflammatory response in asthma; IL-13 and cytokines and chemokines
that are involved in inflammation, such as IL-13 and IL-9; IL-13
and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13 and TARC; IL-13
and MDC; IL-13 and MIF; IL-13 and TGF-.beta.; IL-13 and LHR
agonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; and
IL-13 and ADAMS. The present invention also provides DVD-Igs
capable of binding one or more targets involved in asthma selected
from the group consisting of CSF1 (MCSF), CSF2 (GM-CSF), CSF3
(GCSF), FGF2, IFNA1, IFNB1, IFNG, histamine and histamine
receptors, IL1A, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9,
IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17, IL18, IL19,
KITLG, PDGFB, IL2RA, IL4R, IL5RA, IL8RA, IL8RB, IL12RB1, IL12RB2,
IL13RA1, IL13RA2, IL18R1, TSLP, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7,
CCL8, CCL13, CCL17, CCL18, CCL19, CCL20, CCL22, CCL24, CX3CL1,
CXCL1, CXCL2, CXCL3, XCL1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7,
CCR8, CX3CR1, GPR2, XCR1, FOS, GATA3, JAK1, JAK3, STATE, TBX21,
TGFB1, TNFSF6, YY1, CYSLTR1, FCER1A, FCER2, LTB4R, TB4R2, LTBR, and
Chitinase.
3. Rheumatoid Arthritis
[0156] 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. 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 monoclonal antibody
(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
(MRA), CTLA4Ig (abatacept, Genovese Mcet 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. Other cytokines
have been identified and have been shown to be of benefit in animal
models, including interleukin-15, interleukin-17, and
interleukin-18, and clinical trials of these agents are currently
under way. Dual-specific antibody therapy, combining anti-TNF and
another mediator, 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. Blocking other
pairs of targets involved in RA including, but not limited to, TNF
and IL-18; TNF and IL-12; TNF and IL-23; TNF and IL-/beta; TNF and
MIF; TNF and IL-17; and TNF and IL-15 with specific DVD Igs is also
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 DD., Comp Med. (2005) 55(2):114-22).
4. SLE
[0157] 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. The following
targets may be involved in SLE and can potentially be used for
DVD-Ig approach for therapeutic intervention: 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: B1yS, 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-a, and TNF-a are
also contemplated. Combination of targets discussed above will
enhance therapeutic efficacy for SLE which can be tested in a
number of lupus preclinical models (see Peng SL (2004) Methods Mol
Med.; 102:227-72).
5. Multiple Sclerosis
[0158] 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.
[0159] 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 an anti-IL-12 mAb has
beneficial effects in the myelin-induced EAE model in common
marmosets.
[0160] 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.
[0161] One aspect of the invention pertains to DVD Ig molecules
capable of binding one or more, preferably 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, and CCR2. A preferred embodiment includes a
dual-specific anti-IL-12/TWEAK DVD Ig as a therapeutic agent
beneficial for the treatment of MS. Several animal models for
assessing the usefulness of the DVD molecules to treat MS are known
in the art (see Steinman L, et al., (2005) Trends Immunol.
26(11):565-71; Lublin FD., 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 B A,
et al., (2005) J Immunol 175(7):4761-8. 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).
6. Sepsis
[0162] 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-I). 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.
[0163] 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 an
apoptotic mediator, may have added benefit. One aspect of the
invention pertains to DVD Igs capable of binding one or more
targets involved in sepsis, preferably two targets, 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, IL10, IL1B, NFKB1, PROC, TNFRSF1A, CSF3,
IL10, IL1B, IL6, ADORA2A, CCR3, IL10, IL1B, IL1RN, MIF, NFKB1,
PTAFR, TLR2, TLR4, GPR44, HMOX1, 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).
7. Neurological Disorders
7.1. Neurodegenerative Diseases
[0164] Chronic neurodegenerative diseases are usually age-dependent
diseases 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 disease) 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-b peptide (including the A-b
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-b 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-b 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;
Michelle C Janelsins, et al., J Neuroinflammation. 2005; 2: 23;
Soloman B., Curr Alzheimer Res. 2004; 1: 149; Igor Klyubin, et al.,
Nat Med. 2005; 11: 556-61; Arancio 0, 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).
[0165] The DVD-Ig molecules of the invention can bind one or more
targets involved in chronic neurodegenerative diseases such as
Alzheimer's. Such targets include, but are not limited to, any
mediator, soluble or cell surface, implicated in AD pathogenesis
e.g., AGE (S100 A, amphoterin), 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). 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 alpha-synuclein and inflammatory mediators
such as TNF, IL-1, MCP-1 can prove effective therapy for
Parkinson's disease and are contemplated in the invention.
7.2 Neuronal Regeneration and Spinal Cord Injury
[0166] 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., Trends Neurosci. 2003, 26:193; Marco Domeniconi, et
al., J Neurol Sci. 2005, 233:43; Milan Makwana, et al., FEBS J.
2005, 272:2628; Barry J. Dickson, Science. 2002, 298:1959; Felicia
Yu Hsuan Teng, et al., J Neurosci Res. 2005, 79:273; Tara Karnezis,
et al., Nature Neuroscience 2004, 7:736; Gang Xu, et al., J.
Neurochem. 2004, 91:1018).
[0167] In one aspect, DVD-Igs capable of binding 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-a; AB 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-b, 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-b and S100 A. Furthermore, neurite outgrowth
inihibitors 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 an neurite
outgrowth inhibitor molecule alone.
8. Oncological Disorders
[0168] Monoclonal antibody therapy has emerged as an important
therapeutic modality for cancer (von Mehren, M., et al., 2003
Monoclonal antibody therapy for cancer. Annu Rev Med.; 54: 343-69).
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. DVD Igs capable of binding the following pairs of targets
to treat oncological disease are also contemplated: IGF1 and IGF2;
IGF1/2 and Erb2B; VEGFR and EGFR; CD20 and CD3, CD138 and CD20,
CD38 and CD20, CD38 & CD138, CD40 and CD20, CD138 and CD40,
CD38 and CD40. Other target combinations include one or more
members of the EGF/erb-2/erb-3 family. Other targets (one or more)
involved in oncological diseases that DVD Igs may bind include, but
are not limited to those selected from the group consisting of:
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, EGF, FGF10, FGF18, FGF2, FGF4, FGF7, IGF1, IGF1R, IL2,
VEGF, BCL2, CD164, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C,
CDKN3, GNRH1, IGFBP6, IL1A, IL1B, ODZ1, PAWR, PLG, TGFB1I1, AR,
BRCA1, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, E2F1, EGFR, ENOL, 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, NR1I3, NR2F6, NR4A3, ESR1, ESR2, NROB1,
NROB2, NR1D2, NR1H2, NR1H4, NR1I2, 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, DAB2IP, DES,
DNCL1, ELAC2, ENO2, ENO3, FASN, F1112584, F1125530, 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, BAH, 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 (p16INK4a), 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).
IV. Pharmaceutical Composition
[0169] The invention also provides pharmaceutical compositions
comprising a binding protein, of the invention and a
pharmaceutically acceptable carrier. The pharmaceutical
compositions comprising binding proteins 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 binding
proteins of the invention. In another embodiment, the
pharmaceutical composition comprises one or more binding proteins
of the invention and one or more prophylactic or therapeutic agents
other than binding proteins of the invention for treating a
disorder. Preferably, the prophylactic or therapeutic agents 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.
[0170] The binding proteins of the invention can be incorporated
into pharmaceutical compositions suitable for administration to a
subject. Typically, the pharmaceutical composition comprises a
binding protein 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.
[0171] 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, etc. 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,
and 5,290,540; 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, a binding protein 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.). 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.
[0172] 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 a binding protein of the invention of a subject
to prevent, treat, manage, and/or ameliorate a disorder or one or
more symptoms thereof.
[0173] 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.,
Medical Applications of Controlled Release, Langer and Wise (eds.),
CRC Pres., Boca Raton, Fla. (1974); Controlled Drug
Bioavailability, Drug Product Design and Performance, Smolen and
Ball (eds.), Wiley, New York (1984); Langer and Peppas, 1983, J.
Macromol. Sci-Rev. Macromol. Chem. Phys., C23(1):61-126; 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(2-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,
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).
[0174] 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. Int'l. Symp. Control. Rel. Bioact. Mater.
24:853-854; and Lam et al., 1997, "Microencapsulation of
Recombinant Humanized Monoclonal Antibody for Local Delivery,"
Proc. Int'l. Symp. Control Rd. Bioact. Mater. 24:759-760, each of
which is incorporated herein by reference in their entireties.
[0175] 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.
[0176] 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 lignocaine to
ease pain at the site of the injection.
[0177] 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) 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.
[0178] 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
nebuliser, 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.
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).
[0179] 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, and 5,290,540; 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, a binding protein 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.).
[0180] 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.
[0181] 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).
[0182] 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.
[0183] 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 sachette
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.
[0184] 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.
[0185] The binding proteins 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
binding protein. The injectable solution can be composed of either
a liquid or lyophilized dosage form in a flint or amber vial,
ampule 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).
[0186] 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 the binding proteins 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 2006/104968 incorporated herein by reference).
[0187] 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.
[0188] 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.
[0189] The binding proteins 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.
[0190] In certain embodiments, a binding protein 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.
[0191] Supplementary active compounds can also be incorporated into
the compositions. In certain embodiments, a binding protein 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-12 activity is detrimental. For example, a
binding protein 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.
[0192] In certain embodiments, a binding protein 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.
application Ser. No. 09/428,082 and WO 00/24782 (PCT Application
No. WO 99/25044), which are hereby incorporated by reference for
any purpose.
[0193] In a specific embodiment, nucleic acid sequences encoding a
binding protein 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.
[0194] 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, Clin.
Pharm., 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;
Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;
Mulligan, Science 260:926-932 (1993); Morgan and Anderson, 1993,
Ann. Rev. Biochem. 62:191-217; and Robinson, C., Trends
Biotechnol., 11(5): 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, N.Y. (1990).
Detailed description of various methods of gene therapy are
disclosed in US20050042664 A1 which is incorporated herein by
reference.
[0195] The binding proteins of the invention are useful in treating
various diseases wherein the targets that are recognized by the
binding proteins are detrimental. Such diseases include, but are
not limited to, 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 immune
disease associated with organ transplantation, 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, 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, polyglandular
deficiency type II (Schmidt's syndrome), adult (acute) respiratory
distress syndrome, alopecia, alopecia areata, 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 syndrome, acquired
immunodeficiency related diseases, hepatitis B, hepatitis C, common
variable 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 (classic
autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis
(anti-LKM antibody hepatitis), autoimmune mediated hypoglycaemia,
type B insulin resistance with acanthosis nigricans,
hypoparathyroidism, osteoarthrosis, primary sclerosing cholangitis,
psoriasis type 1, psoriasis type 2, idiopathic leucopaenia,
autoimmune neutropaenia, renal disease NOS, glomerulonephritides,
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,
Sjogren'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, cholestasis, idiosyncratic liver
disease, drug-induced hepatitis, non-alcoholic steatohepatitis,
allergy, 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), cancers such as lung, breast, stomach, bladder, colon,
pancreas, ovarian, prostate and rectal cancer, 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, atrial 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,
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 hemophagocytic 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, hemochromatosis, 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 migraine headache, idiopathic migraine headache,
mitochondrial multisystem disorder, mixed connective tissue
disease, monoclonal gammopathy, multiple myeloma, multiple systems
degenerations (Menzel, Dejerine-Thomas, Shy-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 muscular
atrophies, neutropenic fever, non-Hodgkin's lymphoma, occlusion of
the abdominal aorta and its branches, occlusive arterial disorders,
OKT3 therapy, orchitis/epididymitis, 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, Raynaud's disease, Refsum's disease, regular narrow QRS
tachycardia, renovascular hypertension, reperfusion injury,
restrictive cardiomyopathy, sarcomas, 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, thromboangiitis 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 hemophagocytic
syndrome, Wernicke-Korsakoff syndrome, Wilson's disease, xenograft
rejection of any organ or tissue. (see Peritt et al. PCT
publication No. WO 2002/097048 A2, Leonard et al., PCT publication
No. WO 9/524918 A1, and Salfeld et al., PCT publication No. WO
00/56772A1).
[0196] The binding proteins of the invention can be used to treat
humans suffering from autoimmune diseases, in particular those
associated with inflammation, including, rheumatoid arthritis,
spondylitis, allergy, autoimmune diabetes, autoimmune uveitis.
Preferably, the binding proteins of the invention or
antigen-binding portions thereof, are used to treat rheumatoid
arthritis, Crohn's disease, multiple sclerosis, insulin dependent
diabetes mellitus and psoriasis.
[0197] A binding protein of the invention also can be administered
with one or more additional therapeutic agents useful in the
treatment of various diseases. A binding protein of the invention
can be used alone or in combination to treat such diseases.
[0198] It should be understood that the binding proteins 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 which effects the viscosity of the
composition.
[0199] 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.
[0200] Preferred combinations to treat autoimmune and inflammatory
diseases 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 DVD Igs 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 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, IL-23, interferons, EMAP-II, GM-CSF,
FGF, and PDGF. Binding proteins 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).
[0201] 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.RTM. infliximab), CDP 571, and soluble p55
or p75 TNF receptors, derivatives, thereof, (p75TNFR1gG
(ENBREL.RTM. etanercept) 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 include key players of the autoimmune response which
may act parallel to, dependent on or in concert with IL-12
function; especially preferred are IL-18 antagonists including
IL-18 antibodies or soluble IL-18 receptors, or IL-18 binding
proteins. It has been shown that IL-12 and IL-18 have overlapping
but distinct functions and a combination of antagonists to both may
be most effective. 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.
[0202] The binding proteins of the invention may also be combined
with agents, such as methotrexate, 6-MP, azathioprine
sulphasalazine, mesalazine, olsalazine
chloroquinine/hydroxychloroquine, pencillamine, aurothiomalate
(intramuscular and oral), azathioprine, cochicine, 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,
adenosine agonists, antithrombotic agents, complement inhibitors,
adrenergic agents, agents which interfere with signalling by
proinflammatory cytokines such as TNF 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
signalling 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.RTM. etanercept
and p55TNFRIgG (lenercept)), sIL-1RI, sIL-1RII, sIL-6R),
antiinflammatory cytokines (e.g., IL-4, IL-10, IL-11, IL-13 and
TGFI3), 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, SC10-469, 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.
[0203] Nonlimiting additional agents which can also be used in
combination with a binding protein to treat rheumatoid arthritis
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, 5295; 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-2Ra;
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), 5131;
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-1.beta. 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 (CRAB) 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; antivirals; and immune
modulating agents.
[0204] 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: small
molecule inhibitor of KDR (ABT-123), 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; ABT-874; ABT-325
(anti-IL 18); anti-IL 15; BIRB-796; SC10-469; VX-702; AMG-548;
VX-740; roflumilast; IC-485; CDC-801; and mesopram.
[0205] 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. monoclonal antibodies; anti-IL-6 monoclonal
antibodies; 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
signalling 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 signalling 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
TGFI3).
[0206] Preferred 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, D2E7 (PCT
Publication No. WO 97/29131; HUMIRA.RTM. adalimumab), CA2
(REMICADE.RTM. infliximab), CDP 571, TNFR-Ig constructs,
(p75TNFRIgG (ENBREL.RTM. etanercept) and p55TNFRIgG (lenercept))
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.
[0207] Non-limiting examples of therapeutic agents for multiple
sclerosis 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.RTM.; Biogen); interferon-.beta.1b (BETASERON.RTM.;
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.RTM.; 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,
adenosine agonists, antithrombotic agents, complement inhibitors,
adrenergic agents, agents which interfere with signalling 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-1RI, sIL-1RII, sIL-6R) and antiinflammatory cytokines (e.g.,
IL-4, IL-10, IL-13 and TGF.beta.).
[0208] Preferred 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.
[0209] 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, ANTEGREN.RTM. natalizumab--ELAN/Biogen), interferon
gamma antagonists, IL-4 agonists.
[0210] 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.
[0211] 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.
[0212] 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.
[0213] 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.
[0214] 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).
[0215] 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-lp.
[0216] 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.
[0217] 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 (ABT-123),
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.
[0218] 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.
[0219] 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, ABT-578, acetaminophen.
[0220] 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.
[0221] Preferred 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, D2E7 (PCT
Publication No. WO 97/29131; HUMIRA.RTM. adalimumab), CA2
(REMICADE.RTM. infliximab), CDP 571, TNFR-Ig constructs,
(p75TNFRIgG (ENBREL.RTM. etanercept) and p55TNFRIgG
(lenercept)).
[0222] The pharmaceutical compositions of the invention may include
a "therapeutically effective amount" or a "prophylactically
effective amount" of a binding protein 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 binding protein 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
binding protein 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.
[0223] 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.
[0224] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of a binding protein of the
invention is 0.1-20 mg/kg, more preferably 1-10 mg/kg. 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.
[0225] 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. 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
Generation of Dual Variable Domain Immunoglobulin (DVD-Ig)
[0226] The dual variable domain immunoglobulin (DVD-Ig) molecule is
designed such that two different light chain variable domains (VL)
from the two different parent mAbs 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 (FIG. 1A).
Example 1.1
Generation of Murine Monoclonal Antibodies to IL-1.alpha. and
IL-1.beta.
[0227] Monoclonal Antibodies to IL-1.alpha. and IL-1.beta. were
generated as follows using Hybridoma technology well known in the
art.
Example 1.1.a
Immunization of Mice
[0228] Purified recombinant human IL-1.alpha. and murine IL-1.beta.
(R&D Systems) were used as immunogens as well as coating
antigens in titer assays and screening ELISA. Immunizing dosages
ranged from 5.0 to 20.0 .mu.g/mouse/injection for all antigens for
both primary and boost immunizations. IMMUNEASY.TM. adjuvant was
purchased from Qiagen (Waltham, Mass.) and used at Adjuvant/antigen
ratio of 20 ml IMMUNEASY.TM. adjuvant per 10.0 .mu.g antigen. Each
group of animals to be immunized contained 5 IL-1.alpha..beta. KO
mice obtained from Dr. Yoichiro Iwakura (University of Tokyo,
Minato-ku, Tokyo, Japan). The mice were immunized according to
dosing schedule described below. MRC-5 cells were purchased from
ATCC (Manassas, Va.) and used for IL-1 bioassay. Human IL-8 ELISA
kits and control mouse anti-hIL-1.alpha. and .beta. antibodies
(MAB200 and MAB201) were purchased from R&D Systems
(Minneapolis, Minn.).
[0229] Briefly, adjuvant-antigen mixture was prepared by first
gently mixing the adjuvant in a vial using a vortex. The desired
amount of adjuvant was removed from the vial and put into an
autoclaved 1.5 mL microcentrifuge tube. The antigen was prepared in
PBS or saline with concentration ranging from 0.5-1.0 mg/ml. The
calculated amount of antigen was then added to the microcentrifuge
tube with the adjuvant and the solution was mixed by gently
pipetting up and down 5 times. The adjuvant-antigen mixture was
incubated at room temperature for 15 min and then mixed again by
gently pipetting up and down 5 times. The adjuvant-antigen solution
was drawn into the proper syringe for animal injection. A total of
5-20 pg of antigen was injected in a volume of 50-100 .mu.l. Each
animal was immunized, and then boosted 2 to 3 times depending on
the titer Animals with good titers were given a final intravenous
boost before fusion and generation of hybridomas.
Example 1.1.B
Screening Hybridomas
[0230] Hybridomas, generated as described above, were screened and
antibody titer determined using ELISA: Protein antigens were
directly coated on ELISA plates for detecting the specific
antibodies using standard ELISA procedures. Briefly, ELISA plates
were coated with 100 .mu.l of either rhIL-1.alpha. or rhIL-1.beta.
(1.0 .mu.g/ml in PBS) overnight at 4.degree. C. Plates were washed
3 times with 250 .mu.l PBS/0.5% TWEEN.RTM.20 polyethylene-sorbitan
monolaurate and blocked with 200 .mu.l blocking buffer (2% BSA in
PBS with 0.5% Tween.RTM.20 polyethylene-sorbitan monolaurate).
Diluted sera or hybridoma supernatant (100 .mu.l) was added to each
well, and incubated at room temperature for 2 hrs. Plates were then
washed 3 times with PBS/0.5% Tween.RTM.20 polyethylene-sorbitan
monolaurate, HRP-goat anti-murine IgG was used for detection, and
binding ODs were observed at 450 nm. Hybridoma clones producing
antibodies that showed high specific binding activity in the ELISA
were subcloned and purified, and affinity (Biacore) and potency
(MRC-5 bioassay) of the antibodies were characterized as
follows.
Example 1.1.C
Characterization of Murine Monoclonal Antibodies to IL-1.alpha. and
IL-1.beta.
[0231] The following assays were used to characterize the
antibodies produced by the hybridomas described in example
1.1.B.
Example 1.1.C.1
Surface Plasmon Resonance
[0232] Real-time binding interactions between captured antibody
(mouse anti-rmIL1 antibody captured on a biosensor matrix via goat
anti-mouse IgG) and rmIL-1 were measured by surface plasmon
resonance (SPR) using the BIAcore system (Biacore AB, Uppsala,
Sweden) according to manufacturer's instructions and standard
procedures. Briefly, rmIL-1 was diluted in HBS running buffer
(Biacore AB) and 50 .mu.l aliquots were injected through the
immobilized protein matrices at a flow rate of 5 ml/min. The
concentrations of rhIL1 employed were 62.5, 125, 187.5, 250, 375,
500, 750, 1000, 1500 and 2000 nM. To determine the dissociation
constant (off-rate), association constant (on-rate), BIAcore
kinetic evaluation software (version 3.1) was used.
Example 1.1.C.2
Anti-IL-1 Bioassay
[0233] The MRC-5 cell line is a human lung fibroblast cell line
that produces IL-8 in response to human IL-1.alpha. and IL-1.beta.
in a dose-dependent manner (see Dinarello, C. A., K. Muegge, and S.
K. Durum. 2000. Current Protocols in Immunology 6:1). MRC-5 cells
were cultured in 10% FBS complete MEM and grown at 37.degree. C. in
a 5% CO.sub.2 incubator. To determine neutralizing potencies of the
mAbs against recombinant human IL-1.alpha. or IL-1.beta., different
concentrations (0-10 .mu.g/ml) of mAb (50 .mu.l) was added to a
96-well plate and pre-incubated with 50 .mu.l of rhIL-1.alpha. or
rhIL-1b (10-50 pg/ml) for 1 hr at 37.degree. C. The supernatants
were harvested, diluted, and IL-8 concentrations measured by ELISA
using a standard IL-8 ELISA kit (R&D Systems). Antibody potency
was determined by its ability to inhibit IL-8 production by MRC-5
cells.
[0234] Based on Biacore and MRC-5 bioassay, a number of murine
anti-hIL-1.alpha. and anti-hIL-1b antibodies with high affinity and
potency were identified, as shown in Table 1 below:
TABLE-US-00001 TABLE 1 Generation and characterization of murine
anti-hIL-1a/b mAbs. mAb Clone# Specificity K.sub.D (M) IC.sub.50
(M) 3D12.E3 hIL-1.alpha. 1.11E-09 6.70E-10 18F4.2C8 hIL-1.alpha.
5.78E-10 8.90E-11 6H3.1A4.3E11 hIL-1.alpha. 3.54E-10 2.40E-10
13F5.G5 hIL-1.beta. 2.91E-10 6.00E-10 1B12.4H4 hIL-1.beta. 2.13E-10
5.30E-10 6B12.4F6 hIL-1.beta. 5.54E-10 3.20E-10
Example 1.1.D
Cloning and Sequencing of the Murine Monoclonal Antibodies to
IL-1.alpha. and IL-1.beta.
[0235] Cloning and sequencing of the variable heavy (VH) and light
(VL) genes of all anti-IL-1.alpha./b mAbs described in Table 1 and
additional antibodies were carried out after isolation and
purification of the total RNA from the each hybridoma cell line
using TRIZOL.RTM. reagent (Invitrogen) according to the
manufacturer's instructions. Amplification of both VH and VL genes
was carried out using the IgGVH and Ig.kappa.VL oligonucleotides
from the Mouse Ig-Primer Set (Novagen, Madison, Wis.) with One-tube
RT-PCR kit (Qiagen) as suggested by the manufacturer. DNA fragments
resulting from productive amplifications were cloned into
pCR-TOPO.RTM. vector (Invitrogen) according to the manufacturer's
instructions. Multiple VH and VL clones were then sequenced by the
dideoxy chain termination method using an ABI 3000 sequencer
(Applied Biosystems, Foster City, Calif.). The sequences of all mAb
VL and VH genes are shown below in Table 2.
TABLE-US-00002 TABLE 2 Murine monoclonal antibodies capable of
binding human IL-1.alpha. or IL-1.beta. Sequence Sequence Protein
Identifier 12345678901234567890 VH 3D12.E3 SEQ ID NO.: 1
QIQLVQSGPELKKPGETVKI SCKASGYTFRNYGMNWVKQA PGKDLKRMAWINTYTGESTY
ADDFKGRFAFSLETSASTAY LQINNLKNEDTATYFCARGI YYYGSSYAMDYWGQGTSVTV SS
VL 3D12.E3 SEQ ID NO.: 2 NIQMTQTTSSLSASLGDRVT ISCRASQDISNCLNWYQQKP
DGTVKLLIYYTSRLHSGVPS RFSGSGSGTDYSLTISNLEQ EDIATYFCQQGKTLPYAFGG
GTKLEINR VH 18F4.2C8 SEQ ID NO.: 3 EVQLQQSGAELVKPGASVKL
SCTASGLNIKDTYMHWLKQR PEQGLEWIGRIDPANGNAKY DPRFLGKATITADTSSNTAY
LQLSSLTSEDTAVYYCARGD GNFHFDYWGQGTTLTVSS VL 18F4.2C8 SEQ ID NO.: 4
DIVMTQSQRFMSTSVGDRVS VTCKASQNVGTNIAWYQQKP GQSPRALIYSASYRYSGVPD
RFTGSGSGTDFTLTISNVQS VDLAEYFCQQYTRYPLTFGG GTKLEIKR VH 6H3.1A4.3E11
SEQ ID NO.: 5 QVQLQQPGAELVRPGASVKL SCKASGYTFTTYWMNWVKQR
PEQGLEWIGRIDPYDSETLY SQKFKDTAILTVDKSSSTAY MQLSSLTSEDSAVYYCARYG
FDYWGQGTTLTVSS VL 6H3.1A4.3E11 SEQ ID NO.: 6 QIVLTQSPALMSASPGEKVT
MTCSASSSVNYMYWYQQKPR SSPKPWIYLTSNLASGVPAR FSGSGSGTSYSLTISSMEAE
DAATYYCQQWNSNPYTFGGG TKLEMKR VH 13F5.G5 SEQ ID NO.: 7
QVQLQQSGAELVRPGSSVKI SCKASGYAFSSYWMNWVKQR PGQGLEWIGQIYPGDGDTNY
NGKFKGKATLTADKSSSTSY MQLSGLTSEDSAMYFCVRFP TGNDYYAMDYWGQGTSVTVS S VL
13F5.G5 SEQ ID NO.: 8 NIVLTQSPASLAVSLGQRAT ISCRASESVDSYGNSYMHWY
QQKPGQPPKLLIYLASNLES GVPARFSGSGSRTDFTLTID PVEADDAATYYCQQNNEDPF
TFGSGTKLEIKR VH 1B12.4H4 SEQ ID NO.: 9 QVHLKESGPGLVAPSQSLSI
TCTVSGFSLTDYGVSWIRQP PGKGLEWLGLIWGGGDTYYN SPLKSRLSIRKDNSKSQVFL
KMNSLQTDDTAVYYCAKQRT LWGYDLYGMDYWGQGTSVTV SS VL 1B12.4H4 SEQ ID
ETTVTQSPASLSMAIGEKVT NO.: 10 IRCITSTDIDVDMNWYQQKP
GEPPKLLISQGNTLRPGVPS RFSSSGSGTDFVFIIENMLS EDVADYYCLQSDNLPLTFGA
GTKLELKR VH 6B12.4F6 SEQ ID EVQLQQSGPELVKTGTSVKI NO.: 11
SCKASGYSFTGYYMHWVRQS HGKSLEWIGYISCYNGFTSY NPKFKGKATFTVDTSSSTAY
IQFSRLTSEDSAVYYCARSD YYGTNDYWGQGTTLTVSS VL 6B12.4F6 SEQ ID
QIVLTQSPAIMSASPGEKVT NO.: 12 ITCSASSSVSYMHWFQQKPG
ASPKLWIYSTSNLASGVPAR FSGSGSGTSYSLTVSRMEAE DAATYYCQQRSTYPYTFGGG
TKLEIKR
Example 1.2
Generation and Characterization of Murine-Human Chimeric
Antibodies
[0236] All mAbs described above were converted to chimeric (with
human constant region) and expressed, purified, and characterized
to confirm activity and will be used as controls for subsequent
[0237] DVD-Ig analysis. To convert 3D12.E3 into chimeric form,
3D12.E3-VL was PCR amplified using primers P1 and P2; meanwhile
human Ck gene (in pBOS vector generated in-house at ABC) was
amplified using primers P3 and P4. Both PCR reactions were
performed according to standard PCR techniques and procedures. The
two PCR products were gel-purified, and used together as
overlapping template for the subsequent overlapping PCR reaction
using primers P1 and P4 using standard PCR conditions. The final
PCR product, the chimeric light chain 3D12.E3-VL-hCk, was subcloned
into pEF6 TOPO.RTM. mammalian expression vector (Invitrogen) by
TOPO.RTM. vector cloning according to the manufacturer's
instructions. Table 3 shows the PCR primers' sequences:
TABLE-US-00003 TABLE 3 P1: 5' ATG GTG TCC ACA GCT CAG TTC C 3' SEQ
ID NO. 13 P2: 5' GC AGC CAC CGT ACG CCG GTT TAT TTC CAG 3' SEQ ID
NO. 14 P3: 5' CGT ACG GTG GCT GCA CCA TCT GTC 3' SEQ ID NO. 15 P4:
5' TCA ACA CTC TCC CCT GTT GAA GC 3' SEQ ID NO. 16
[0238] To convert 3D12.E3 heavy chain into chimeric form,
3D12.E3-VH was PCR amplified using primers P5 and P6; meanwhile
human C.gamma.1 gene (in pBOS vector generated in-house at ABC) was
amplified using primers P7 and P8. Both PCR reactions were
performed according to standard PCR techniques and procedures. The
two PCR products were gel-purified, and used together as
overlapping template for the subsequent overlapping PCR reaction
using primers P5 and P8 using standard PCR conditions. The final
PCR product, the chimeric light chain 3D12.E3-VH-hC.gamma.1, was
subcloned into pcDNA3.1 TOPO.RTM. mammalian expression vector
(Invitrogen) according to the manufacturer's instructions. Table 4
shows the PCR primers' sequences:
TABLE-US-00004 TABLE 4 P5: 5' ATG GCT TGG GTG TGG ACC TTG C 3' SEQ
ID NO. 17 P6: 5' GGG CCC TTG GTC GAC GCT GAG GAG ACG GTG ACT GAG G
3' SEQ ID NO. 18 P7: 5' GCG TCG ACC AAG GGC CCA TCG GTC TTC C 3'
SEQ ID NO. 19 P8: 5' TC ATT TAC CCG GAG ACA GGG AGA GGC 3' SEQ ID
NO. 20
[0239] Similarly, chimeric 13F5.G5-VH-C.gamma.1 was generated using
primers P21/P22 (for VH) and P7/P8 (for hC.gamma.1) and cloned into
pcDNA3.1 TOPO.RTM. vector, and chimeric 13F5.G5-VL-C.kappa. was
generated using primers P23/P24 (for VL) and P3/P4 (for hCk) and
cloned into pEF6 TOPO.RTM. vector. Table 5 shows the PCR primers'
sequences:
TABLE-US-00005 TABLE 5 P21: 5' ATA GAA TGG AGC TGG GTT TTC CTC 3'
SEQ ID NO. 21 P22: 5' GGG CCC TTG GTC GAC GC TGA GGA GAC GGT GAC
TGA 3' SEQ ID NO. 22 P23: 5' ATG GTC CTC ATG TCC TTG CTG TTC 3' SEQ
ID NO. 23 P24: 5' GC AGC CAC CGT ACG CCG TTT TAT TTC CAG CTT TG 3'
SEQ ID NO. 24
[0240] To express chimeric Abs, 13F5.G5-VL-C.kappa. and
13F5.G5-VH-C.gamma.1 were co-expressed in COS using
LIPOFECTAMINE.TM. transfection reagent (Invitrogen) for 72 hr, and
the medium collected and IgG purified by Protein A chromatography.
Similarly, 13F5.G5-VL-C.kappa. and 13F5.G5-VH-C.gamma.1 were
co-expressed in COS using LIPOFECTAMINE.TM. transfection reagent
(Invitrogen) for 72 hr, and the medium collected and IgG purified
by Protein A chromatography. Both purified chimeric Abs were
characterized by Biacore and MRC-5 bioassay to confirm activity.
The results showed that these chimeric Abs displayed similar
affinity and potency to that of the original murine mAbs.
Example 1.3
Construction, Expression, and Purification of IL-1.alpha./.beta.
Dual Variable Domain Immunoglobulin (DVD-Ig)
[0241] The construct used to generate DVD-Ig capable of binding
hIL-1.alpha. and hIL-1.beta. is illustrated in FIG. 1B. Briefly,
parent mAbs including two high affinity murine Abs,
anti-hIL-1.alpha. (clone 3D12.E3) and anti-hIL-1.beta. (clone
13F5.G5), were obtained by immunizing Balb/c mice with recombinant
IL-1.alpha. protein (rhIL-1.alpha.) and recombinant IL-1.beta.
protein (rhIL-1.beta.), respectively. The VL/VH genes of these two
hybridoma clones were isolated by RT-PCR using the mouse Ig Primer
Kit (Novagen, Madison, Wis.). The VL/VH genes were first converted
into chimeric antibodies (with human constant regions) to confirm
activity and potency. To generate DVD1-Ig, the VH and VL of 13F5.G5
was directly fused to the N-terminus of the VH and VL of 3D12.E3,
respectively (as shown in FIG. 1B). The DVD2-Ig was constructed
similarly, except that it had a linker between the two variable
domains in both the light chain (the linker sequence is ADAAP (SEQ
ID NO:40)) and the heavy chain (the linker sequence is AKTTPP (SEQ
ID NO:38)). These sequences were selected from the N-termini of
murine Ck and CH1 sequences. These linker sequences, selected from
the N-termini of murine Ck and CH1, are natural extension of the
variable domains and exhibit a flexible conformation without
significant secondary structures based on the analysis of several
Fab crystal structures. The detailed procedures of the PCR cloning
is described below:
Example 1.3.A
Molecular Cloning of hIL-La/bDVD1-Ig
[0242] 13F5.G5-VH was PCR amplified using primers P21 and P25;
meanwhile 3D12.E3-VH-hC.gamma.1 was amplified using primers P14 and
P8. Both PCR reactions were performed according to standard PCR
techniques and procedures. The two PCR products were gel-purified,
and used together as overlapping template for the subsequent
overlapping PCR reaction using primers P21 and P8 using standard
PCR conditions. The final PCR product, the DVD1-Ig heavy chain
hIL-1a/bDVD1-VH-hC.gamma.1, was subcloned into pcDNA3.1 TOPO.RTM.
mammalian expression vector (Invitrogen) according to the
manufacturer's instructions. Table 6 shows the PCR primers'
sequences:
TABLE-US-00006 TABLE 6 P14: 5' CAG ATC CAG TTG GTG CAG TCT GG3' SEQ
ID NO. 25 P25: 5' CAC CAA CTG GAT CTG TGA GGA GAC GGT GAC TGA GG 3'
SEQ ID NO. 26
[0243] To generate hIL-1a/bDVD1-Ig light chain, 13F5.G5-VL was PCR
amplified using primers P23 and P26; meanwhile 3D12.E3-VL-hCK was
amplified using primers P16 and P4. Both PCR reactions were
performed according to standard PCR techniques and procedures. The
two PCR products were gel-purified, and used together as
overlapping template for the subsequent overlapping PCR reaction
using primers P23 and P4 using standard PCR conditions. The final
PCR product, the hIL-1a/bDVD1-Ig light chain
hIL-1a/bDVD1-VL-hC.kappa., was subcloned into pEF6 TOPO.RTM.
mammalian expression vector (Invitrogen) according to the
manufacturer's instructions. Table 7 shows the PCR primers'
sequences:
TABLE-US-00007 TABLE 7 P16: 5' AAT ATC CAG ATG ACA CAG ACT ACA TCC
3' SEQ ID NO. 27 P26: 5' GTGT CAT CTG GAT ATT CCG TTT TAT TTC CAG
CTT TG 3' SEQ ID NO. 28
Example 1.3.B
Molecular Cloning of hIL-1a/bDVD2-Ig
[0244] 13F5.G5-VH was PCR amplified using primers P21 and P17;
meanwhile 3D12.E3-VH-hCyl was amplified using primers P18 and P8.
Both PCR reactions were performed according to standard PCR
techniques and procedures. The two PCR products were gel-purified,
and used together as overlapping template for the subsequent
overlapping PCR reaction using primers P21 and P8 using standard
PCR conditions. The final PCR product, the DVD2-Ig heavy chain
hIL-1a/bDVD2-VH-hC.gamma.1, was subcloned into pcDNA3.1 TOPO.RTM.
mammalian expression vector (Invitrogen) according to the
manufacturer's instructions. Table 8 shows the PCR primers'
sequences:
TABLE-US-00008 TABLE 8 P17: 5' TGG GGG TGT CGT TTT GGC TGA GG 3'
SEQ ID NO. 29 P18: 5' GCC AAA ACG ACA CCC CCA CAG ATC CAG TTG GTG
CAG 3' SEQ ID NO. 30
[0245] To generate hIL-1a/bDVD2-Ig light chain, 13F5.G5-VL was PCR
amplified using primers P23 and P19; meanwhile 3D12.E3-VL-hC.kappa.
was amplified using primers P20 and P4. Both PCR reactions were
performed according to standard PCR techniques and procedures. The
two PCR products were gel-purified, and used together as
overlapping template for the subsequent overlapping PCR reaction
using primers P23 and P4 using standard PCR conditions. The final
PCR product, the hIL-1a/bDVD2-Ig light chain
hIL-1a/bDVD2-VL-hC.kappa., was subcloned into pEF6 TOPO.RTM.
mammalian expression vector (Invitrogen) according to the
manufacturer's instructions. Table 9 shows the PCR primers'
sequences:
TABLE-US-00009 TABLE 9 P19: 5' TGG TGC AGC ATC AGC CCG TTT TAT TTC
3' SEQ ID NO. 31 P20: 5' GCT GAT GCT GCA CCA AAT ATC CAG ATG ACA
CAG 3' SEQ ID NO. 32
[0246] The final sequences of hIL-1a/bDVD1-Ig and hIL-1a/bDVD2-Ig
are described in Table 10:
TABLE-US-00010 TABLE 10 Amino acid sequence of
hIL-1.alpha./.beta.DVD1-Ig and hIL-1.alpha./.beta.DVD2-Ig Protein
Sequence Sequence Protein region Identifier 12345678901234567890
DVD HEAVY SEQ ID QVQLQQSGAELVRPGSSVKI VARIABLE NO.: 33
SCKASGYAFSSYWMNWVKQR hIL-1a/bDVD1-Ig PGQGLEWIGQIYPGDGDTNY
NGKFKGKATLTADKSSSTSY MQLSGLTSEDSAMYFCVRFP TGNDYYAMDYWGQGTSVTVS
SQIQLVQSGPELKKPGETVK ISCKASGYTFRNYGMNWVKQ APGKDLKRMAWINTYTGEST
YADDFKGRFAFSLETSASTA YLQINNLKNEDTATYFCARG IYYYGSSYAMDYWGQGTSVT VSS
VH 13F5.G5 SEQ ID QVQLQQSGAELVRPGSSVKI NO.: 7 SCKASGYAFSSYWMNW
VKQRPGQGLEWIGQIYPGDG DTNYNGKFKGKATLTADKSS STSYMQLSGLTSEDSA
MYFCVRFPTGNDYYAMDYWG QGTSVTVSS Linker None 3D12.E3 VH SEQ ID
QIQLVQSGPELKKPGETVKI NO.: 1 SCKASGYTFRNYGMNWVKQA
PGKDLKRMAWINTYTGESTY ADDFKGRFAFSLETSASTAY LQINNLKNEDTATYFCARGI
YYYGSSYAMDYWGQGTSVTV SS CH SEQ ID ASTKGPSVFPLAPSSKSTSG NO.: 34
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD LIGHT SEQ ID NIVLTQSPASLAVSLGQRAT VARIABLE NO.: 35
ISCRASESVDSYGNSYMHWY hIL-1a/bDVD1-Ig QQKPGQPPKLLIYLASNLES
GVPARFSGSGSRTDFTLTID PVEADDAATYYCQQNNEDPF TFGSGTKLEIKRNIQMTQTT
SSLSASLGDRVTISCRASQD ISNCLNWYQQKPDGTVKLLI YYTSRLHSGVPSRFSGSGSG
TDYSLTISNLEQEDIATYFC QQGKTLPYAFGGGTKLEINR R 13F5.G5 VL SEQ ID
NIVLTQSPASLAVSLGQRAT NO.: 8 ISCRASESVDSYGNSYMHWY
QQKPGQPPKLLIYLASNLES GVPARFSGSGSRTDFTLTID PVEADDAATYYCQQNNEDPF
TFGSGTKLEIKR Linker None 3D12.E3 VL SEQ ID NIQMTQTTSSLSASLGDRVT
NO.: 2 ISCRASQDISNCLNWYQQKP DGTVKLLIYYTSRLHSGVPS
RFSGSGSGTDYSLTISNLEQ EDIATYFCQQGKTLPYAFGG GTKLEINR CL SEQ ID
TVAAPSVFIFPPSDEQLKSG NO.: 36 TASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS
FNRGEC DVD HEAVY SEQ ID QVQLQQSGAELVRPGSSVKI VARIABLE NO.: 37
SCKASGYAFSSYWMNWVKQR hIL-1a/bDVD2-Ig PGQGLEWIGQIYPGDGDTNY
NGKFKGKATLTADKSSSTSY MQLSGLTSEDSAMYFCVRFP TGNDYYAMDYWGQGTSVTVS
SAKTTPPQIQLVQSGPELKK PGETVKISCKASGYTFRNYG MNWVKQAPGKDLKRMAWINT
YTGESTYADDFKGRFAFSLE TSASTAYLQINNLKNEDTAT YFCARGIYYYGSSYAMDYWG
QGTSVTVSS 13F5.G5 VH SEQ ID QVQLQQSGAELVRPGSSVKI NO.: 7
SCKASGYAFSSYWMNWVKQR PGQGLEWIGQIYPGDGDTNY NGKFKGKATLTADKSSSTSY
MQLSGLTSEDSAMYFCVRFP TGNDYYAMDYWGQGTSVTVS S Linker SEQ ID AKTTPP
NO.: 38 3D12.E3 VH SEQ ID QIQLVQSGPELKKPGETVKI NO.: 1
SCKASGYTFRNYGMNWVKQA PGKDLKRMAWINTYTGESTY ADDFKGRFAFSLETSASTAY
LQINNLKNEDTATYFCARGI YYYGSSYAMDYWGQGTSVTV SS CH SEQ ID
ASTKGPSVFPLAPSSKSTSG NO.: 34 GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ
ID NIVLTQSPASLAVSLGQRAT VARIABLE HIL- NO.: 39 ISCRASESVDSYGNSYMHWY
1a/bDVD2-Ig QQKPGQPPKLLIYLASNLES GVPARFSGSGSRTDFTLTID
PVEADDAATYYCQQNNEDPF TFGSGTKLEIKRADAAPNIQ MTQTTSSLSASLGDRVTISC
RASQDISNCLNWYQQKPDGT VKLLIYYTSRLHSGVPSRFS GSGSGTDYSLTISNLEQEDI
ATYFCQQGKTLPYAFGGGTK LEINR 13F5.G5 VL SEQ ID NIVLTQSPASLAVSLGQRAT
NO.: 8 ISCRASESVDSYGNSYMHWY QQKPGQPPKLLIYLASNLES
GVPARFSGSGSRTDFTLTID PVEADDAATYYCQQNNEDPF TFGSGTKLEIKR Linker SEQ
ID ADAAP NO.: 40 3D12.E3 VL SEQ ID NIQMTQTTSSLSASLGDRVT NO.: 2
ISCRASQDISNCLNWYQQKP DGTVKLLIYYTSRLHSGVPS RFSGSGSGTDYSLTISNLEQ
EDIATYFCQQGKTLPYAFGG GTKLEINR CL SEQ ID TVAAPSVFIFPPSDEQLKSG NO.:
36 TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKS FNRGEC
Example 1.3.C
Expression and Purification of hIL-1a/bDVD1-Igs
[0247] The heavy and light chain of each construct was subcloned
into pcDNA3.1 TOPO.RTM. and pEF6 TOPO.RTM. vectors (Invitrogen
Inc.), respectively, and sequenced to ensure accuracy. The plasmids
encoding the heavy and light chains of each construct were
transiently expressed using LIPOFECTAMINE.TM. 2000 and
293fectin.TM. transfection reagents, respectively in COS cells as
well as human embryonic kidney 293 cells (American Type Culture
Collection, Manassas, Va.). The cell culture media was harvested 72
hr-post transient transfection and antibodies purified using
protein A chromatography (Pierce, Rockford, Ill.) according to
manufacturer's instructions. The Abs were analyzed by SDS-PAGE and
quantitated by A280 and BCA (Pierce, Rockford, Ill.). Table 11
shows that the expression levels of hIL-1 a/bDVD1-Ig and hIL-1
a/bDVD2-Ig are comparable to that of the chimeric Abs, indicating
that the DVD-Ig can be expressed efficiently in mammalian
cells.
TABLE-US-00011 TABLE 11 Expression and molecular weight analysis of
hIL-1a/bDVD-Ig Expression level (ng/ml) Free- Molecular mass
(Dalton) style Light Heavy Full COS 293 Chain Chain length Mock 0 0
3D12.E3-Ch 2788 3886 23,696 49,914 147,220 13F5.G5-Ch 3260 3562
24,084 49,518 147,204 DVD1-Ig 2988 3300 35,797 64,380 200,346
(35,790) (64,371) (200,521) DVD2-Ig 2433 3486 36,222 64,976 202,354
(36,220) (64,973) (202,573) The molecular mass of the light chain,
heavy chain, and full length of DVD1-Ig and DVD2-Ig determined
experimentally by mass spectrometry are shown in parenthesis.
Example 1.4
Mass Spectrometry and SEC Analysis of hIL-1a/b DVD-Ig
[0248] For measuring molecular weight (MW) of light and heavy
chains of DVD-Ig, 10 uL of DVD-Ig (0.8 ug/uL) was reduced by 1.0 M
DTT solution (5 uL). A PLRP-S, 8u, 4000A, and 1.times.150 mm
protein column (Michrom BioResource, Auburn, Mass.) was used to
separate heavy and light chains of DVD-Ig. Agilent HP1100 Capillary
HPLC (Agilent Technologies Inc., Pala Alto, Calif.) was used with
the mass spectrometer QSTAR.RTM. (Applied Biosystems, Foster City,
Calif.). The valco valve was set at 10 minutes to switch the flow
from waste to MS for desalting sample. Buffer A was 0.02% TFA,
0.08% FA, 0.1% ACN and 99.8% HPLC-H20. Buffer B contained 0.02%
TFA, 0.08% FA, 0.1% HPLC-H2O, and 99.8% ACN. The HPLC flow rate was
50 uL/min, and the sample injection volume was 8.0 mL. The
temperature of the column oven was set at 60.degree. C., and
separation gradient was: 5% B for 5 minutes; 5% B to 65% B for 35
minutes; 65% B to 95% B for another 5 minutes, and 95% B to 5% B
for 5 minutes. TOFMS scan was from 800 to 2500 amu, and cycles were
3600. To determine the MW of full length DVD-Ig, a Protein
MICROTRAP.TM. cartridge (Michrom BioResource, Auburn, Mass.) was
used for desalting the sample. The HPLC gradient was: 5% B for 5
minutes; 5% B to 95% B in 1 minutes; and from 95% B to 5% B in
another 4 minutes. The QSTAR.RTM. TOFMS scan was from 2000 to 3500
amu, and cycles were 899. All MS raw data were analyzed using the
Analyst QS software (Applied Biosystems). For SEC analysis of the
DVD-Ig, purified DVD-Ig and chimeric Abs, in PBS, were applied on a
Superose 6 10/300 G2, 300.times.10 mm column (Amersham Bioscience,
Piscataway, N.J.). An HPLC instrument, Model 10A (Shimadzu,
Columbia, Md.) was used for SEC. All proteins were determined using
UV detection at 280 nm and 214 nm. The elution was isocratic at a
flow rate of 0.5 mL/min. For stability study, samples in the
concentration range of 0.2-0.4 mg/ml in PBS underwent 3 freeze-thaw
cycles between -80.degree. C. and 25.degree. C., or were incubated
at 4.degree. C., 25.degree. C., or 40.degree. C., for 4 weeks and 8
weeks, followed by SEC analysis.
[0249] DVD-Ig and chimeric Abs were purified by protein A
chromatography. The purification yield (3-5 mg/L) was consistent
with hIgG quantification of the expression medium for each protein.
The composition and purity of the purified DVD-Igs and chimeric Abs
were analyzed by SDS-PAGE in both reduced and non-reduced
conditions. In non-reduced condition, each of the four proteins
migrated as a single band. The DVD-Ig proteins showed larger M.W.
than the chimeric Abs, as expected. In non-reducing condition, each
of the four proteins yielded two bands, one heavy chain and one
light chain. Again, the heavy and light chains of the DVD-Igs were
larger in size than that of the chimeric Abs. The SDS-PAGE showed
that each DVD-Ig is expressed as a single species, and the heavy
and light chains are efficiently paired to form an IgG-like
molecule. The sizes of the heavy and light chains as well as the
full-length protein of two DVD-Ig molecules are consistent with
their calculated molecular mass based on amino acid sequences (see
Table 11).
[0250] In order to determine the precise molecular weight of
DVD-Ig, mass spectrometry was employed. As shown in Table I, the
experimentally determined molecular mass of each DVD-Ig, including
the light chain, heavy chain, and the full-length protein, is in
good agreement with the predicted value. To further study the
physical properties of DVD-Ig in solution, size exclusion
chromatography (SEC) was used to analyze each protein. Both
chimeric Abs and DVD2-Ig exhibited a single peak, demonstrating
physical homogeneity as monomeric proteins. The 3D12.E3 chimeric Ab
showed a smaller physical size then 13F5.G5 chimeric Ab, indicating
that 3D12.E3 chimeric Ab adopted a more compact, globular shape.
DVD1-Ig revealed a major peak as well as a shoulder peak on the
right, suggesting that a portion of DVD1-Ig is possibly in an
aggregated form in current buffer condition.
Example 1.5
Analysis of In Vitro Stability of hIL-1a/b DVD-Igs
[0251] The physical stability of DVD-Ig was tested as follows.
Purified antibodies in the concentration range of 0.2-0.4 mg/ml in
PBS underwent 3 freeze-thaw cycles between -80.degree. C. and
25.degree. C., or were incubated at 4.degree. C., 25.degree. C., or
40.degree. C., for 4 weeks and 8 weeks, followed by analysis using
size exclusion chromatography (SEC) analysis (see Table 12).
TABLE-US-00012 TABLE 12 in vitro stability analysis of hIL-1a/b
DVD-Ig by SEC 3D12.E3-Ch 13F5.G5-Ch DVD1-Ig DVD2-Ig Agg Ab Frgm Agg
Ab Frgm Agg Ab Frgm Agg Ab Frgm 3xFreeze- 1.72 98.28 0.00 13.0 87.0
0.0 46.50 53.50 0.00 0.0 100.0 0.0 Thaw 4.degree. C. @ 0.85 99.15
0.00 4.2 95.8 0.0 42.43 56.63 0.94 0.0 100.0 0.0 4 Wks 25.degree.
C. @ 1.29 98.71 0.00 0.0 100.0 0.0 45.66 54.34 0.00 0.0 100.0 0.0 4
Wks 40.degree. C. @ 1.65 98.35 0.00 20.3 78.1 1.6 36.70 59.42 3.88
0.0 100.0 0.0 4 Wks 4.degree. C. @ 5.35 90.33 4.32 2.2 97.8 0.0
38.18 56.91 4.91 0.0 100.0 0.0 8 Wks 25.degree. C. @ 1.11 60.55
38.34 1.4 97.5 1.0 24.42 67.39 8.19 0.0 100.0 0.0 8 Wks 40.degree.
C. @ 4.74 81.47 13.79 34.6 65.4 0.0 20.55 67.16 12.29 0.0 100.0 0.0
8 Wks The degree of aggregation and fragmentation are shown in
percentage, whereas the percentage of Ab represents intact
molecule. Agg: aggregates; Ab: intact antibody; Frgm:
fragments.
[0252] Both chimeric Abs showed minor degrees of aggregation and
fragmentation, normal for a regular IgG molecule. DVD1-Ig showed
some aggregation on SCE after purification. In the stability
analysis, DVD1-Ig also showed aggregations in PBS under different
conditions; however the percentage of aggregated form of DVD1-Ig
did not increase during prolonged storage or at higher
temperatures. The percentage of the fragmented form of DVD1-Ig were
in the normal range, similar to that of the chimeric 3D12.E3 Ab. In
contrast, DVD2-Ig showed exceptional stability. Neither aggregation
nor fragmentation was detected for DVD2-Ig in all conditions
tested, and 100% of DVD2-Ig maintained as intact monomeric
molecule.
Example 1.6
Determination of Antigen Binding Affinity of hIL-1a/bDVD-Igs
[0253] The kinetics of DVD-Ig binding to rhIL1-.alpha. and
rhIL1-.beta. was determined by surface plasmon resonance-based
measurements with a Biacore 3000 instrument (Biacore AB, Uppsala,
Sweden) using 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 AB (Uppsala, Sweden) or otherwise from a
different source as described herein. Approximately, 5000 RU of
goat 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 mg/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-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 ten
injections (using global fit analysis) using the Bioevaluation
4.0.1 software. Purified DVD-Ig samples were diluted in
HEPES-buffered saline for capture across goat anti-human IgG Fc
specific reaction surfaces and injected over reaction matrices at a
flow rate of 5 ml/min. The association and dissociation rate
constants, kon (M-1s-1) and koff (s-1) were determined under a
continuous flow rate of 25 ml/min. Rate constants were derived by
making kinetic binding measurements at ten different antigen
concentrations ranging from 1.25 to 1000 nM. The equilibrium
dissociation constant (M) of the reaction between DVD-Ig and
rhIL1.alpha./.beta. was then calculated from the kinetic rate
constants by the following formula: KD=koff/kon. Aliquots of
rhIL1.alpha./.beta. samples were also simultaneously injected over
a blank reference and reaction CM surface to record and subtract
any nonspecific binding background to eliminate the majority of the
refractive index change and injection noise. Surfaces were
regenerated with two subsequent 25 ml injections of 10 mM Glycine
(pH 1.5) at a flow rate of 5 ml/min. The anti-Fc antibody
immobilized surfaces were completely regenerated and retained their
full capture capacity over twelve cycles. The apparent
stoichiometry of the captured DVD-Ig-rhIL1.alpha./.beta. complex
was calculated under saturating binding conditions (steady-state
equilibrium) using the following formula*
Stoichiometry = rhIL 1 .alpha. / .beta. response ( RU ) DVD
response ( RU ) .times. DVD - Ig MW ) rhIL 1 .alpha. / .beta. MW
##EQU00001##
[0254] The Biacore analysis indicated the chimeric Abs possessed
similar binding kinetics and affinities to IL-1 as the original
hybridoma mAbs, indicating that the correct VL/VH sequences had
been isolated (Table III). The overall binding parameters of the
two DVD-Igs to hIL-1.alpha. were similar, with the affinities of
the DVD-Igs being only 2-3 fold less than that of the chimeric
3D12.E3 Ab. The binding affinity of DVD2-Ig to hIL-1.beta. was
slightly less than the chimeric Ab 13F5.G5, but 3-fold higher than
that of DVD1-Ig. The affinity of the two DVD-Igs to hIL-1 as
compared to the affinity of chimeric Abs to hIL-1 was similar as
indicated by the evaluation of the stoichiometry to IL-1. Both
chimeric Abs, being bivalent monospecific, bound to IL-1.alpha. and
IL-1.beta. on Biocore with a stoichiometry of 1.6 and 1.7,
respectively. This is common for an IgG due to inter-molecular
interference when antibodies are immobilized densely on the Biacore
sense chip resulting in stoichiometry being in the range from 1.5
to 2.0. The stoichiometry of both DVD-Igs for hIL-1.alpha. and
hIL-1.beta. were similar to that of the two chimeric Abs,
indicating that both DVD-Igs possessed bivalent binding capability
to each antigen.
TABLE-US-00013 TABLE 13 Functional characterization of anti-IL-1
DVD-Ig molecule k.sub.on k.sub.off K.sub.d Potency Antigen (M-1
s-1) (s-1) (M) Stoichiometry IC.sub.50 (M) 3D13.E3 hIL-1.alpha.
6.43E+05 7.13E-04 1.11E-09 2.0 6.70E-10 3D12.E3-Ch hIL-1.alpha.
4.12E+05 5.52E-04 1.34E-09 1.6 7.00E-10 DVD1-Ig hIL-1.alpha.
3.70E+04 1.05E-04 2.83E-09 1.8 2.30E-09 DVD2-Ig hIL-1.alpha.
7.35E+04 2.52E-04 3.42E-09 2.0 2.90E-09 13F5.G5 hIL-1.beta.
2.13E+06 6.21E-04 2.91E-10 1.8 6.00E-10 13F5.G5-Ch hIL-1.beta.
1.41E+06 6.54E-04 4.62E-10 1.7 5.30E-10 DVD1-Ig hIL-1.beta.
6.09E+05 1.59E-03 2.60E-09 1.5 3.10E-09 DVD2-Ig hIL-1.beta.
1.19E+06 9.50E-04 7.98E-10 1.8 1.60E-09 Affinity and stoichiometry
were measured by Biacore; Potency (IC.sub.50) was determined by
MRC-5 bioassay.
[0255] In addition, tetravalent dual-specific antigen binding of
DVD-Ig was also analyzed by Biacore (Table 14). DVD-Ig was first
captured via a goat anti-human Fc antibody on the Biacore sensor
chip, and the first antigen was injected and a binding signal
observed. As the DVD-Ig was saturated by the first antigen, the
second antigen was then injected and the second signal observed.
This was done either by first injecting IL-1.beta. then IL-1.alpha.
or by first injecting IL-1.alpha. followed by IL-1.beta. for
DVD2-Ig. In either sequence, a dual-binding activity was detected.
Similar results were obtained for DVD1-Ig. Thus each DVD-Ig was
able to bind both antigens simultaneously as a dual-specific
tetravalent molecule. As shown in Table IV, the stoichiometry of
both DVD-Ig to the first antigen, either hIL-1.alpha. or
hIL-1.beta., were larger than 1.5, similar to that of mono-specific
bivalent binding. Upon the injection of the second antigen, while
DVD-Ig was already occupied by the first antigen, the stoichiometry
of both DVD-Igs to the second antigen (i.e., hIL-1.alpha. or
hIL-1.beta.) was between 1.0 and 1.3. Thus DVD-Ig is able to bind
two IL-1.alpha. and two IL-.beta. molecules. DVD-Ig was first
captured via a goat anti-human Fc antibody on the Biacore sensor
chip, and the first antigen was injected and a binding signal
observed, followed by the injection of the second antigen.
TABLE-US-00014 TABLE 14 Stoichiometry analysis of hIL-1a/b DVD-Ig
in tetravalent dual-specific binding to IL-1.alpha./.beta. Response
Unit Stoichiometry Captured Ab 1st antigen 2nd antigen
hIL-1.alpha.:DVD-Ig hIL-1.beta.:DVD-Ig DVD1-Ig: hIL-1.alpha.:
hIL-1.beta.: 2.3 1.0 932 190 75 DVD1-Ig: hIL-1.beta.: hIL-1.alpha.:
1.1 1.5 1092 141 107 DVD2-Ig: hIL-1.alpha.: hIL-1.beta.: 1.8 1.3
1324 209 137 DVD2-Ig: hIL-1.beta.: hIL-1.alpha.: 1.2 1.6 1184 159
131
Example 1.7
Determination of Functional Homogeneity of DVD-Ig
[0256] Because DVD2-Ig was purified by Protein A chromatography
instead of target-specific affinity chromatography, any potential
misfolded and/or mismatched VL/VH domains, if present, can be
assessed by binding studies against the 2 different antigens. Such
binding analysis was conducted by size exclusion liquid
chromatography (SEC). DVD2-Ig, alone or after a 120-min incubation
period at 37.degree. C. with IL-1.alpha., IL-1.beta., or both
IL-1.alpha. and IL-1.beta., in equal molar ratio, were applied to
the column. Each of the antigens was also run alone as controls.
The SEC results indicated that DVD2-Ig was able to bind IL-1.alpha.
and IL-1.beta. in solution, and such binding resulted in a shift to
the SEC signal indicating an increase in the dynamic size of
DVD2-Ig when it was in complex with either antigen. The shift of
the DVD2-Ig signal was 100%, not partial, suggesting all DVD2-Ig
molecules were able to bind the antigen. In the presence of both
IL-1.alpha. and IL-1.beta., there was a further and complete shift
of the DVD2-Ig signal, indicating all DVD2-Ig molecules were able
to bind both antigens in a uniform fashion. This experiment
demonstrated that DVD-Ig was expressed as a functionally
homogeneous protein. This has significant implications as it
demonstrates that DVD-Ig can be produced as a homogeneous single,
functional species, which differs from all previously described
bi-specific, multi-specific, and multi-valent immunoglobulin-like
and immunoglobulin-derived molecules.
Example 1.8
Determination of Biological Activity of DVD-Ig
[0257] The biological activity of DVD-Ig was measured using MRC-5
bioassay. The MRC-5 cell line is a human lung fibroblast cell line
that produces IL-8 in response to human IL-1.alpha. and IL-1.beta.
in a dose-dependent manner. MRC-5 cells were obtained from ATCC and
cultured in 10% FBS complete MEM at 37.degree. C. in a 5% CO2
incubator. To determine neutralizing activity of the DVD-Ig against
human IL-1.alpha. or IL-1.beta., 50 ul of Ab (1E-7 to 1E-12 M) in
MEM/10% FBS was added to a 96 well plate and pre-incubated with 50
ul of hIL-1.alpha. or hIL-1.beta. (200 pg/ml) for 1 hr at
37.degree. C., 5% CO2. MRC-5 cells at a concentration of 1E5/ml
were then added (100 ul) to all wells and the plates were incubated
overnight at 37.degree. C. in a 5% CO2 incubator. The supernatants
were harvested, and human IL-8 production measured by standard
ELISA (R&D Systems, Minneapolis, Minn.). Neutralizing activity
of the DVD-Ig was determined by its ability to inhibit IL-8
production.
[0258] As shown in Table 13, both DVD-Igs were able to neutralize
hIL-1.alpha. and hIL-1.beta.. Consistent with the binding affinity
to hIL-1 a, the neutralizing activities of DVD1-Ig and DVD2-Ig
against hIL-1.alpha. were also similar, i.e., 3-fold less than that
of the chimeric Abs (see Table III). Consistent with its binding
affinity for hIL-1.beta., the neutralizing activity of DVD2-Ig to
hIL-1.beta. is slightly less than that of the chimeric Ab 13F5.G5,
but 3-fold higher than that of DVD1-Ig. Overall there was no
significant decrease in the biological activities of DVD-Ig
molecules compared to the original mAbs.
[0259] To determine if DVD-Ig was able to inhibit IL-8 production
in the presence of both IL-1.alpha. and IL-1.beta., equal amounts
of hIL-1.alpha. and hIL-1.beta. were added in the same culture
system of MRC-5 assay. Both DVD1-Ig and DVD2-Ig were able to
inhibit IL-8 synthesis by MRC-5 cells in the presence of both
IL-1.alpha. and IL-1.beta., with activities similar to that of
mono-assays where only one cytokine was present (Table 13). In this
assay where both IL-1.alpha. and IL-1.beta. were present, the
dual-inhibition activity of DVD2-Ig (1.2 nM) was higher than that
of DVD1-Ig (2.2 nM).
Example 2
Analysis of Linker Size and Variable Domain Orientation in the
DVD-Ig Molecule
[0260] Additional DVD-Ig molecules with different parent mAb pairs,
as shown in Table 15, were constructed. For each pair of mAbs, four
different DVD-Ig constructs were generated: 2 with a short linker
and 2 with a long linker, each in two different domain
orientations: a-b-C(alpha-beta-constant domain) and
b-a-C(beta-alpha-constant domain). The linker sequences, were
derived from the N-terminal sequence of human Ck or CH1 domain, as
follows:
TABLE-US-00015 Short linker: light chain: TVAAP; (SEQ ID NO: 44)
heavy chain: ASTKGP (SEQ ID NO: 42) Long linker: light chain:
TVAAPSVFIFPP; (SEQ ID NO: 50) heavy chain: ASTKGPSVFPLAP. (SEQ ID
NO: 48)
[0261] All heavy and light chain constructs were subcloned into the
pBOS expression vector, and expressed in COS cells or freestyle 293
cells.
[0262] To construct new DVD clones, the variable domains of the two
mAbs, both light chain and heavy chain, were first jointed in
tandem using overlapping PCR as described for hIL-1abDVD1-Ig and
hIL-1abDVD2-Ig. The jointed pieces were then subcloned in pBOS
vector using homologous recombination. Briefly, vectors were
linearized by restriction digestion (2 ug of pBOS-hCk vector were
digested with FspAI and BsiWI in O+ buffer, and 2 ug of
pBOS-hC.gamma. z,non a vector was digested with FspAI and SaII in
O+ buffer). The digested samples were run on 1% agarose gel and the
backbone fragment purified in 50 ul water. For homologous
recombination and transformation, DH5.alpha. competent cells were
thaw on ice, and mixed with 20-50 ng jointed PCR product and 20-50
ng of linearized vector (in every 50 ul DH5.alpha. cells). The
mixture was mixed gently and incubated on ice for 45 minutes,
followed by heat shock at 42.degree. C. for 1 minute. Then 100 ul
SOC medium were added and incubated at 37.degree. C. for 1 hour.
The transformation culture was inoculated on LB/Agar plates
containing Ampicilin and incubated at 37.degree. C. for 18-20
hours. The bacterial clones were isolated, from which DNA was
purified and subjected to sequencing analysis. The final
sequence-verified clones were co-transfected (matching HV and LC of
the same Ab pair) in COS or 293 cells for Ab expression and
purification, as previously described.
[0263] Characteristics of the purified DVD-Ig proteins are
summarized in Table 16. The left section of the table 16 shows the
specificity, binding affinity, and neutralization potency of the 2
pairs of mAbs used for the construction of the new hIL-1a/bDVD-Ig
molecules. Antibodies 18F4.2C8 and 1B12.4H4 (see example 1.1.D)
were used to construct hIL-1a/bDVD3a-Ig, hIL-1a/bDVD4a-Ig,
hIL-1a/bDVD3b-Ig, and hIL-1a/bDVD4b-Ig. hIL-1a/bDVD3a-Ig and
hIL-1a/bDVD4a-Ig were in a-b-C orientation, with a short and long
linker, respectively. hIL-1a/bDVD3b-Ig and hIL-1a/bDVD4b-Ig were in
b-a-C orientation, with a short and long linker, respectively.
Antibodies 6H3.1A4 and 6B12.4F6 were used to construct
hIL-1a/bDVD5a-Ig, hIL-1a/bDVD6a-Ig, hIL-1a/bDVD5b-Ig, and
hIL-1a/bDVD6b-Ig. hIL-1a/bDVD5a-Ig and hIL-1a/bDVD6a-Ig were in
a-b-C orientation, with a short and long linker, respectively.
hIL-1a/bDVD5b-Ig and hIL-1a/bDVD6b-Ig were in b-a-C orientation,
with a short and long linker, respectively. The molecular cloning
of these additional hIL-1a/bDVD-Igs were performed using the
procedure previously described for hIL-1 a/bDVD1-Ig (see example
1.3), using overlapping PCR procedures. The amino acid sequences of
these additional hIL-1a/bDVD-Igs are disclosed in Table 15.
TABLE-US-00016 TABLE 15 Amino acid sequence of heavy chain and
light chain of six DVD Ig capable of binding IL-1.alpha. and
IL-1.beta.. Protein Sequence Sequence Protein region Identifier
12345678901234567890 DVD HEAVY SEQ ID EVQLQQSGAELVKPGASVKL VARIABLE
NO.: 41 SCTASGLNIKDTYMHWLKQR hIL-1a/b DVD3a-Ig PEQGLEWIGRIDPANGNAKY
DPRFLGKATITADTSSNTAY LQLSSLTSEDTAVYYCARGD GNFHFDYWGQGTTLTVSSAS
TKGPQVHLKESGPGLVAPSQ SLSITCTVSGFSLTDYGVSW IRQPPGKGLEWLGLIWGGGD
TYYNSPLKSRLSIRKDNSKS QVFLKMNSLQTDDTAVYYCA KQRTLWGYDLYGMDYWGQGT
SVTVSS 18F4.2C8 VH SEQ ID EVQLQQSGAELVKPGASVKL NO.: 3
SCTASGLNIKDTYMHWLKQR PEQGLEWIGRIDPANGNAKY DPRFLGKATITADTSSNTAY
LQLSSLTSEDTAVYYCARGD GNFHFDYWGQ GTTLTVSS LINKER SEQ ID ASTKGP NO.:
42 1B12.4H4 VH SEQ ID QVHLKESGPGLVAPSQSLSI NO.: 9
TCTVSGFSLTDYGVSWIRQP PGKGLEWLGLIWGGGDTYYN SPLKSRLSIRKDNSKSQVFL
KMNSLQTDDTAVYYCAKQRT LWGYDLYGMDYWGQGTSVTV SS CH SEQ ID
ASTKGPSVFPLAPSSKSTSG NO.: 34 GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ
ID DIVMTQSQRFMSTSVGDRVS VARIABLE HIL- NO.: 43 VTCKASQNVGTNIAWYQQKP
1a/b DVD3a-Ig GQSPRALIYSASYRYSGVPD RFTGSGSGTDFTLTISNVQS
VDLAEYFCQQYTRYPLTFGG GTKLEIKRTVAAPETTVTQS PASLSMAIGEKVTIRCITST
DIDVDMNWYQQKPGEPPKLL ISQGNTLRPGVPSRFSSSGS GTDFVFIIENMLSEDVADYY
CLQSDNLPLTFGAGTKLELK RR 18F4.2C8 VL SEQ ID DIVMTQSQRFMSTSVGDRVS
NO.: 4 VTCKASQNVGTNIAWYQQKP GQSPRALIYSASYRYSGVPD
RFTGSGSGTDFTLTISNVQS VDLAEYFCQQYTRYPLTFGG GTKLEIKR LINKER SEQ ID
TVAAP NO.: 44 1B12.4H4 VL SEQ ID ETTVTQSPASLSMAIGEKVT NO.: 10
IRCITSTDIDVDMNWYQQKP GEPPKLLISQGNTLRPGVPS RFSSSGSGTDFVFIIENMLS
EDVADYYCLQSDNLPLTFGA GTKLELKR CL SEQ ID TVAAPSVFIFPPSDEQLKSG NO.:
36 TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID QVHLKESGPGLVAPSQSLSI
VARIABLE NO.: 45 TCTVSGFSLTDYGVSWIRQP hIL-1a/b DVD3b-
PGKGLEWLGLIWGGGDTYYN Ig SPLKSRLSIRKDNSKSQVFL KMNSLQTDDTAVYYCAKQRT
LWGYDLYGMDYWGQGTSVTV SSASTKGPEVQLQQSGAELV KPGASVKLSCTASGLNIKDT
YMHWLKQRPEQGLEWIGRID PANGNAKYDPRFLGKATITA DTSSNTAYLQLSSLTSEDTA
VYYCARGDGNFHFDYWGQGT TLTVSS 1B12.4H4 VH SEQ ID QVHLKESGPGLVAPSQSLSI
NO.: 9 TCTVSGFSLTDYGVSWIRQP PGKGLEWLGLIWGGGDTYYN
SPLKSRLSIRKDNSKSQVFL KMNSLQTDDTAVYYCAKQRT LWGYDLYGMDYWGQGTSVTV SS
LINKER SEQ ID ASTKGP NO.: 42 18F4.2C8 VH SEQ ID
EVQLQQSGAELVKPGASVKL NO.: 3 SCTASGLNIKDTYMHWLKQR
PEQGLEWIGRIDPANGNAKY DPRFLGKATITADTSSNTAY LQLSSLTSEDTAVYYCARGD
GNFHFDYWGQGTTLTVSS CH SEQ ID ASTKGPSVFPLAPSSKSTSG NO.: 34
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD LIGHT SEQ ID ETTVTQSPASLSMAIGEKVT VARIABLE HIL- NO.:
46 IRCITSTDIDVDMNWYQQKP 1a/b DVD3b-Ig GEPPKLLISQGNTLRPGVPS
RFSSSGSGTDFVFIIENMLS EDVADYYCLQSDNLPLTFGA GTKLELKRTVAAPDIVMTQS
QRFMSTSVGDRVSVTCKASQ NVGTNIAWYQQKPGQSPRAL IYSASYRYSGVPDRFTGSGS
GTDFTLTISNVQSVDLAEYF CQQYTRYPLTFGGGTKLEIK R 1B12.4H4 VL SEQ ID
ETTVTQSPASLSMAIGEKVT NO.: 10 IRCITSTDIDVDMNWYQQKP
GEPPKLLISQGNTLRPGVPS RFSSSGSGTDFVFIIENMLS EDVADYYCLQSDNLPLTFGA
GTKLELKR LINKER SEQ ID TVAAP NO.: 44 18F4.2C8 VL SEQ ID
DIVMTQSQRFMSTSVGDRVS NO.: 4 VTCKASQNVGTNIAWYQQKP
GQSPRALIYSASYRYSGVPD RFTGSGSGTDFTLTISNVQS VDLAEYFCQQYTRYPLTFGG
GTKLEIKR CL SEQ ID TVAAPSVFIFPPSDEQLKSG NO.: 36
TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID EVQLQQSGAELVKPGASVKL
VARIABLE hIL- NO.: 47 SCTASGLNIKDTYMHWLKQR 1a/1b DVD4a-Ig
PEQGLEWIGRIDPANGNAKY DPRFLGKATITADTSSNTAY LQLSSLTSEDTAVYYCARGD
GNFHFDYWGQGTTLTVSSAS TKGPSVFPLAPQVHLKESGP GLVAPSQSLSITCTVSGFSL
TDYGVSWIRQPPGKGLEWLG LIWGGGDTYYNSPLKSRLSI RKDNSKSQVFLKMNSLQTDD
TAVYYCAKQRTLWGYDLYGM DYWGQGTSVTVSS 18F4.2C8 VH SEQ ID
EVQLQQSGAELVKPGASVKL NO.: 3 SCTASGLNIKDTYMHWLKQR
PEQGLEWIGRIDPANGNAKY DPRFLGKATITADTSSNTAY LQLSSLTSEDTAVYYCARGD
GNFHFDYWGQGTTLTVSS LINKER SEQ ID ASTKGPSVFPLAP NO.: 48 1B12.4H4 VH
SEQ ID QVHLKESGPGLVAPSQSLSI NO.: 9 TCTVSGFSLTDYGVSWIRQP
PGKGLEWLGLIWGGGDTYYN SPLKSRLSIRKDNSKSQVFL KMNSLQTDDTAVYYCAKQRT
LWGYDLYGMDYWGQGTSVTV SS CH SEQ ID ASTKGPSVFPLAPSSKSTSG NO.: 34
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD LIGHT SEQ ID DIVMTQSQRFMSTSVGDRVS VARIABLE HIL- NO.:
49 VTCKASQNVGTNIAWYQQKP 1a/bDVD4a-Ig GQSPRALIYSASYRYSGVPD
RFTGSGSGTDFTLTISNVQS VDLAEYFCQQYTRYPLTFGG GTKLEIKRTVAAPSVFIFPP
ETTVTQSPASLSMAIGEKVT IRCITSTDIDVDMNWYQQKP GEPPKLLISQGNTLRPGVPS
RFSSSGSGTDFVFIIENMLS EDVADYYCLQSDNLPLTFGA GTKLELKR 18F4.2C8 VL SEQ
ID DIVMTQSQRFMSTSVGDRVS NO.: 4 VTCKASQNVGTNIAWYQQKP
GQSPRALIYSASYRYSGVPD RFTGSGSGTDFTLTISNVQS
VDLAEYFCQQYTRYPLTFGG GTKLEIKR LINKER SEQ ID TVAAPSVFIFPP NO.: 50
1B12.4H4 VL SEQ ID ETTVTQSPASLSMAIGEKVT NO.: 10
IRCITSTDIDVDMNWYQQKP GEPPKLLISQGNTLRPGVPS RFSSSGSGTDFVFIIENMLS
EDVADYYCLQSDNLPLTFGA GTKLELKR CL SEQ ID TVAAPSVFIFPPSDEQLKSG NO.:
36 TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID QVHLKESGPGLVAPSQSLSI
VARIABLE hIL- NO.: 51 TCTVSGFSLTDYGVSWIRQP 1a/b DVD4b-Ig
PGKGLEWLGLIWGGGDTYYN SPLKSRLSIRKDNSKSQVFL KMNSLQTDDTAVYYCAKQRT
LWGYDLYGMDYWGQGTSVTV SSASTKGPSVFPLAPEVQLQ QSGAELVKPGASVKLSCTAS
GLNIKDTYMHWLKQRPEQGL EWIGRIDPANGNAKYDPRFL GKATITADTSSNTAYLQLSS
LTSEDTAVYYCARGDGNFHF DYWGQGTTLTVSS 1B12.4H4 VH SEQ ID
QVHLKESGPGLVAPSQSLSI NO.: 9 TCTVSGFSLTDYGVSWIRQP
PGKGLEWLGLIWGGGDTYYN SPLKSRLSIRKDNSKSQVFL KMNSLQTDDTAVYYCAKQRT
LWGYDLYGMDYWGQGTSVTV SS LINKER SEQ ID ASTKGPSVFPLAP NO.: 48
18F4.2C8 VH SEQ ID EVQLQQSGAELVKPGASVKL NO.: 3 SCTASGLNIKDTYMHWLKQR
PEQGLEWIGRIDPANGNAKY DPRFLGKATITADTSSNTAY LQLSSLTSEDTAVYYCARGD
GNFHFDYWGQGTTLTVSS CH SEQ ID ASTKGPSVFPLAPSSKSTSG NO.: 34
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD LIGHT SEQ ID ETTVTQSPASLSMAIGEKVT VARIABLE HIL- NO.:
52 IRCITSTDIDVDMNWYQQKP 1a/1b DVD4b-Ig GEPPKLLISQGNTLRPGVPS
RFSSSGSGTDFVFIIENMLS EDVADYYCLQSDNLPLTFGA GTKLELKRTVAAPSVFIFPP
DIVMTQSQRFMSTSVGDRVS VTCKASQNVGTNIAWYQQKP GQSPRALIYSASYRYSGVPD
RFTGSGSGTDFTLTISNVQS VDLAEYFCQQYTRYPLTFGG GTKLEIKR 1B12.4H4 VL SEQ
ID ETTVTQSPASLSMAIGEKVT NO.: 10 IRCITSTDIDVDMNWYQQKP
GEPPKLLISQGNTLRPGVPS RFSSSGSGTDFVFIIENMLS EDVADYYCLQSDNLPLTFGA
GTKLELKR LINKER SEQ ID TVAAPSVFIFPP NO.: 50 18F4.2C8 VL SEQ ID
DIVMTQSQRFMSTSVGDRVS NO.: 4 VTCKASQNVGTNIAWYQQKP
GQSPRALIYSASYRYSGVPD RFTGSGSGTDFTLTISNVQS VDLAEYFCQQYTRYPLTFGG
GTKLEIKR CL SEQ ID TVAAPSVFIFPPSDEQLKSG NO.: 36
TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID QVQLQQPGAELVRPGASVKL
VARIABLE hIL- NO.: 53 SCKASGYTFTTYWMNWVKQR 1a/1b DVD5a-Ig
PEQGLEWIGRIDPYDSETLY SQKFKDTAILTVDKSSSTAY MQLSSLTSEDSAVYYCARYG
FDYWGQGTTLTVSSASTKGP EVQLQQSGPELVKTGTSVKI SCKASGYSFTGYYMHWVRQS
HGKSLEWIGYISCYNGFTSY NPKFKGKATFTVDTSSSTAY IQFSRLTSEDSAVYYCARSD
YYGTNDYWGQGTTLTVSS 6H3.1A4.3E11 SEQ ID QVQLQQPGAELVRPGASVKL VH NO.:
5 SCKASGYTFTTYWMNWVKQR PEQGLEWIGRIDPYDSETLY SQKFKDTAILTVDKSSSTAY
MQLSSLTSEDSAVYYCARYG FDYWGQGTTLTVSS LINKER SEQ ID ASTKGP NO.: 42
6B12.4F6 VH SEQ ID EVQLQQSGPELVKTGTSVKI NO.: 11
SCKASGYSFTGYYMHWVRQS HGKSLEWIGYISCYNGFTSY NPKFKGKATFTVDTSSSTAY
IQFSRLTSEDSAVYYCARSD YYGTNDYWGQGTTLTVSS CH SEQ ID
ASTKGPSVFPLAPSSKSTSG NO.: 34 GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ
ID QIVLTQSPALMSASPGEKVT VARIABLE HIL- NO.: 54 MTCSASSSVNYMYWYQQKPR
1a/b DVD5a-Ig SSPKPWIYLTSNLASGVPAR FSGSGSGTSYSLTISSMEAE
DAATYYCQQWNSNPYTFGGG TKLEMKRTVAAPQIVLTQSP AIMSASPGEKVTITCSASSS
VSYMHWFQQKPGASPKLWIY STSNLASGVPARFSGSGSGT SYSLTVSRMEAEDAATYYCQ
QRSTYPYTFGGGTKLEIKR 6H3.1A4.3E11 SEQ ID QIVLTQSPALMSASPGEKVT VL
NO.: 6 MTCSASSSVNYMYWYQQKPR SSPKPWIYLTSNLASGVPAR
FSGSGSGTSYSLTISSMEAE DAATYYCQQWNSNPYTFGGG TKLEMKR LINKER SEQ ID
TVAAP NO.: 44 6B12.4F6 VL SEQ ID QIVLTQSPAIMSASPGEKVT NO.: 12
ITCSASSSVSYMHWFQQKPG ASPKLWIYSTSNLASGVPAR FSGSGSGTSYSLTVSRMEAE
DAATYYCQQRSTYPYTFGGG TKLEIKR CL SEQ ID TVAAPSVFIFPPSDEQLKSG NO.: 36
TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID EVQLQQSGPELVKTGTSVKI
VARIABLE hIL- NO.: 55 SCKASGYSFTGYYMHWVRQS 1a/b DVD5b-Ig
HGKSLEWIGYISCYNGFTSY NPKFKGKATFTVDTSSSTAY IQFSRLTSEDSAVYYCARSD
YYGTNDYWGQGTTLTVSSAS TKGPQVQLQQPGAELVRPGA SVKLSCKASGYTFTTYWMNW
VKQRPEQGLEWIGRIDPYDS ETLYSQKFKDTAILTVDKSS STAYMQLSSLTSEDSAVYYC
ARYGFDYWGQGTTLTVSS 6B12.4F6 VH SEQ ID EVQLQQSGPELVKTGTSVKI NO.: 11
SCKASGYSFTGYYMHWVRQS HGKSLEWIGYISCYNGFTSY NPKFKGKATFTVDTSSSTAY
IQFSRLTSEDSAVYYCARSD YYGTNDYWGQGTTLTVSS LINKER SEQ ID ASTKGP NO.:
42 6H3.1A4.3E11 SEQ ID QVQLQQPGAELVRPGASVKL VH NO.: 5
SCKASGYTFTTYWMNWVKQR PEQGLEWIGRIDPYDSETLY SQKFKDTAILTVDKSSSTAY
MQLSSLTSEDSAVYYCARYG FDYWGQGTTLTVSS CH SEQ ID ASTKGPSVFPLAPSSKSTSG
NO.: 34 GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE
MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ ID
QIVLTQSPAIMSASPGEKVT VARIABLE HIL- NO.: 56 ITCSASSSVSYMHWFQQKPG
1a/b DVD5b-Ig ASPKLWIYSTSNLASGVPAR FSGSGSGTSYSLTVSRMEAE
DAATYYCQQRSTYPYTFGGG TKLEIKRTVAAPQIVLTQSP ALMSASPGEKVTMTCSASSS
VNYMYWYQQKPRSSPKPWIY LTSNLASGVPARFSGSGSGT SYSLTISSMEAEDAATYYCQ
QWNSNPYTFGGGTKLEMKR
6B12.4F6 VL SEQ ID QIVLTQSPAIMSASPGEKVT NO.: 12
ITCSASSSVSYMHWFQQKPG ASPKLWIYSTSNLASGVPAR FSGSGSGTSYSLTVSRMEAE
DAATYYCQQRSTYPYTFGGG TKLEIKR LINKER SEQ ID TVAAP NO.: 44
6H3.1A4.3E11 SEQ ID QIVLTQSPALMSASPGEKVT VL NO.: 6
MTCSASSSVNYMYWYQQKPR SSPKPWIYLTSNLASGVPAR FSGSGSGTSYSLTISSMEAE
DAATYYCQQWNSNPYTFGGG TKLEMKR CL SEQ ID TVAAPSVFIFPPSDEQLKSG NO.: 36
TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID QVQLQQPGAELVRPGASVKL
VARIABLE hIL- NO.: 57 SCKASGYTFTTYWMNWVKQR 1a/b DVD6a-Ig
PEQGLEWIGRIDPYDSETLY SQKFKDTAILTVDKSSSTAY MQLSSLTSEDSAVYYCARYG
FDYWGQGTTLTVSSASTKGP SVFPLAPEVQLQQSGPELVK TGTSVKISCKASGYSFTGYY
MHWVRQSHGKSLEWIGYISC YNGFTSYNPKFKGKATFTVD TSSSTAYIQFSRLTSEDSAV
YYCARSDYYGTNDYWGQGTT LTVSS 6H3.1A4.3E11 SEQ ID QVQLQQPGAELVRPGASVKL
VH NO.: 5 SCKASGYTFTTYWMNWVKQR PEQGLEWIGRIDPYDSETLY
SQKFKDTAILTVDKSSSTAY MQLSSLTSEDSAVYYCARYG FDYWGQGTTLTVSS LINKER SEQ
ID ASTKGPSVFPLAP NO.: 48 6B12.4F6 VH SEQ ID EVQLQQSGPELVKTGTSVKI
NO.: 11 SCKASGYSFTGYYMHWVRQS HGKSLEWIGYISCYNGFTSY
NPKFKGKATFTVDTSSSTAY IQFSRLTSEDSAVYYCARSD YYGTNDYWGQGTTLTVSS CH SEQ
ID ASTKGPSVFPLAPSSKSTSG NO.: 34 GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ
ID QIVLTQSPALMSASPGEKVT VARIABLE HIL- NO.: 58 MTCSASSSVNYMYWYQQKPR
1a/b DVD 6a-Ig SSPKPWIYLTSNLASGVPAR FSGSGSGTSYSLTISSMEAE
DAATYYCQQWNSNPYTFGGG TKLEMKRTVAAPSVFIFPPQ IVLTQSPAIMSASPGEKVTI
TCSASSSVSYMHWFQQKPGA SPKLWIYSTSNLASGVPARF SGSGSGTSYSLTVSRMEAED
AATYYCQQRSTYPYTFGGGT KLEIKRR 6H3.1A4.3E11 SEQ ID
QIVLTQSPALMSASPGEKVT VL NO.: 6 MTCSASSSVNYMYWYQQKPR
SSPKPWIYLTSNLASGVPAR FSGSGSGTSYSLTISSMEAE DAATYYCQQWNSNPYTFGGG
TKLEMKR LINKER SEQ ID TVAAPSVFIFPP NO.: 50 6B12.4F6 VL SEQ ID
QIVLTQSPAIMSASPGEKVT NO.: 12 ITCSASSSVSYMHWFQQKPG
ASPKLWIYSTSNLASGVPAR FSGSGSGTSYSLTVSRMEAE DAATYYCQQRSTYPYTFGGG
TKLEIKR CL SEQ ID RTVAAPSVFIFPPSDEQLKS NO.: 36 GTASVVCLLNNFYPREAKVQ
WKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTK
SFNRGEC DVD HEAVY SEQ ID EVQLQQSGPELVKTGTSVKI VARIABLE hIL- NO.: 59
SCKASGYSFTGYYMHWVRQS 1a/b DVD6b-Ig HGKSLEWIGYISCYNGFTSY
NPKFKGKATFTVDTSSSTAY IQFSRLTSEDSAVYYCARSD YYGTNDYWGQGTTLTVSSAS
TKGPSVFPLAPQVQLQQPGA ELVRPGASVKLSCKASGYTF TTYWMNWVKQRPEQGLEWIG
RIDPYDSETLYSQKFKDTAI LTVDKSSSTAYMQLSSLTSE DSAVYYCARYGFDYWGQGTT
LTVSS 6B12.4F6 VH SEQ ID EVQLQQSGPELVKTGTSVKI NO.: 11
SCKASGYSFTGYYMHWVRQS HGKSLEWIGYISCYNGFTSY NPKFKGKATFTVDTSSSTAY
IQFSRLTSEDSAVYYCARSD YYGTNDYWGQGTTLTVSS LINKER SEQ ID ASTKGPSVFPLAP
NO.: 48 6H3.1A4.3E11 SEQ ID QVQLQQPGAELVRPGASVKL VH NO.: 5
SCKASGYTFTTYWMNWVKQR PEQGLEWIGRIDPYDSETLY SQKFKDTAILTVDKSSSTAY
MQLSSLTSEDSAVYYCARYG FDYWGQGTTLTVSS CH SEQ ID ASTKGPSVFPLAPSSKSTSG
NO.: 34 GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE
MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ ID
QIVLTQSPAIMSASPGEKVT VARIABLE HIL- NO.: 60 ITCSASSSVSYMHWFQQKPG
1a/b DVD6b-Ig ASPKLWIYSTSNLASGVPAR FSGSGSGTSYSLTVSRMEAE
DAATYYCQQRSTYPYTFGGG TKLEIKRTVAAPSVFIFPPQ IVLTQSPALMSASPGEKVTM
TCSASSSVNYMYWYQQKPRS SPKPWIYLTSNLASGVPARF SGSGSGTSYSLTISSMEAED
AATYYCQQWNSNPYTFGGGT KLEMKRR 6B12.4F6 VL SEQ ID
QIVLTQSPAIMSASPGEKVT NO.: 12 ITCSASSSVSYMHWFQQKPG
ASPKLWIYSTSNLASGVPAR FSGSGSGTSYSLTVSRMEAE DAATYYCQQRSTYPYTFGGG
TKLEIKR LINKER SEQ ID TVAAPSVFIFPP NO.: 50 6H3.1A4.3E11 SEQ ID
QIVLTQSPALMSASPGEKVT VL NO.: 6 MTCSASSSVNYMYWYQQKPR
SSPKPWIYLTSNLASGVPAR FSGSGSGTSYSLTISSMEAE DAATYYCQQWNSNPYTFGGG
TKLEMKR CL SEQ ID TVAAPSVFIFPPSDEQLKSG NO.: 36 TASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS
FNRGEC
[0264] Characteristics of the new DVD constructs are summarized in
Table 16. Affinity (Kd) and biological activity (IC50) were
determined by Biacore and MRC-5 bioassay, respectively. SDS-PAGE
analysis of all new DVD proteins showed normal migration patterns
in both reduced and non-reduced conditions, similar to a regular
antibody and DVD1/2-Ig.
TABLE-US-00017 TABLE 16 Characterization of new DVD-Ig molecules
derived from new mAb pairs K.sub.d IC.sub.50 Affinity (K.sub.d) M
Potency (IC.sub.50) M mAb Specif. (M) (M) DVD Orient. Linker
IL-1.alpha. IL-1.beta. IL-1.alpha. IL-1.beta. 18F4.2C8
rhIL-1.alpha. 5.95E-10 3.30E-10 DVD3a a-b-C short 8.37E-10 6.37E-08
7.50E-10 NA DVD4a a-b-C long 7.01E-10 9.30E-10 3.50E-10 1.00E-08
1B12.4H4 rhIL-1.beta. 2.61E-10 6.00E-10 DVD3b b-a-C short 1.24E-09
1.90E-10 7.00E-10 4.00E-10 DVD4b b-a-C long 5.60E-10 1.28E-10
3.50E-10 5.00E-10 6H3.1A4 rhIL-1.alpha. 3.54E-10 2.40E-10 DVD5a
a-b-C short 5.08E-10 1.25E-08 2.60E-09 1.90E-08 DVD6a a-b-C long
1.06E-09 2.09E-09 2.30E-09 7.00E-08 6B12.4F6 rhIL-1.beta. 5.54E-10
4.00E-10 DVD5b b-a-C short 1.32E-08 6.71E-10 3.30E-09 2.50E-10
DVD6b b-a-C long 8.20E-10 6.97E-10 1.00E-09 7.50E-10 NA: no
neutralization activity detected.
[0265] The functional characterization of the new DVD molecules
revealed that with either orientation, DVDs with the long linker
performed better than the ones with the short linker in terms of
binding and neutralizing of both antigens. With respect to DVDs
with the long linkers, those with the b-a-C orientation showed good
binding to and neutralization of both antigens, while the DVDs with
an a-b-C orientation showed good binding to and neutralization of
IL-1.alpha. and reduced binding to and neutralization of IL-1.beta.
(e.g., DVD4b vs. DVD4a). The DVD-Ig molecule, DVD4b, bound and
neutralized both IL-1.alpha. and IL-1.beta. with sub-nM and fully
retained the binding and neutralizing characteristics of the parent
mAbs.
Example 3
Generation of DVD-Ig capable of binding IL-12 and IL-18
[0266] DVD-Ig molecules capable of binding IL-12 and IL-18 were
constructed as described above using two parent mAbs, one against
human IL-12p40 (ABT874), and the other against human IL-18
(ABT325). Four different anti-IL12/18 DVD-Ig constructs were
generated: 2 with short linker and 2 with long linker, each in two
different domain orientations: 12-18-C and 18-12-C(Table VI). The
linker sequences, derived from the N-terminal sequence of human
C.sub..lamda./C.sub..kappa. or CH1 domain, were as follows:
[0267] For DVD1218 constructs (ABT874 has a V.sub..lamda.):
TABLE-US-00018 light chain (.lamda.): Short linker: QPKAAP; (SEQ ID
NO: 88) Long linker: QPKAAPSVTLFPP (SEQ ID NO: 92) heavy chain
(.gamma.1): Short linker: ASTKGP; (SEQ ID NO: 42) Long linker:
ASTKGPSVFPLAP (SEQ ID NO: 48)
[0268] For DVD1812 constructs (ABT325 has a V.sub..kappa.):
TABLE-US-00019 light chain (.kappa.): Short linker: TVAAP; (SEQ ID
NO: 44) Long linker: TVAAPSVFIFPP (SEQ ID NO: 50) heavy chain
(.gamma.1): Short linker: ASTKGP; (SEQ ID NO: 42) Long linker:
ASTKGPSVFPLAP. (SEQ ID NO: 48)
[0269] All heavy and light chain constructs were subcloned into the
pBOS expression vector, and expressed in COS cells or freestyle 293
cells, followed by purification by Protein A chromatography. The
purified materials were subjected to SDS-PAGE and SEC, and their
profiles were similar to that of the DVD2-Ig.
[0270] The table 17 below describes the heavy chain and light chain
constructs used to express each anti-IL12/IL18 DVD-Ig protein.
TABLE-US-00020 TABLE 17 Constructs to express anti-IL12/IL18 DVD-Ig
proteins DVD-Ig protein Heavy chain construct Light chain construct
DVD1218SL DVD1218HC-SL DVD1218LC-SL DVD1218LL DVD1218HC-LL
DVD1218LC-LL DVD1812SL DVD1812HC-SL DVD1812LC-SL DVD1812LL
DVD1812HC-LL DVD1812LC-LL
Example 3.1.1
Molecular Cloning of DNA Constructs for DVD1218SL and DVD1218LL
[0271] To generate heavy chain constructs DVD1218HC-LL and
DVD1218HC-SL, VH domain of ABT-874 was PCR amplified using primers
Primer 1 and Primer 2L or Primer 2S respectively; meanwhile VH
domain of ABT-325 was amplified using primers Primer 3L or Primer
3S and Primer 4 respectively. Both PCR reactions were performed
according to standard PCR techniques and procedures. The two PCR
products were gel-purified, and used together as overlapping
template for the subsequent overlapping PCR reaction using primers
Primer 1 and Primer 4 using standard PCR conditions. The
overlapping PCR products were subcloned into Srf I and Sal I double
digested pBOS-hC.gamma.1,z non-a mammalian expression vector
(Abbott) by using standard homologous recombination approach.
[0272] To generate light chain constructs DVD1218LC-LL and
DVD1218LC-SL, VL domain of ABT-874 was PCR amplified using primers
Primer 5 and Primer 6L or Primer 6S respectively; meanwhile VL
domain of ABT-325 was amplified using primers Primer 7L or Primer
7S and Primer 8 respectively. Both PCR reactions were performed
according to standard PCR techniques and procedures. The two PCR
products were gel-purified, and used together as overlapping
template for the subsequent overlapping PCR reaction using primers
Primer 5 and Primer 8 using standard PCR conditions. The
overlapping PCR products were subcloned into Srf I and Not I double
digested pBOS-hCk mammalian expression vector (Abbott) by using
standard homologous recombination approach. The primers used for
these constructions are listed below in table 18:
TABLE-US-00021 TABLE 18 Primer1:
TAGAGATCCCTCGACCTCGAGATCCATTGTGCCCGGGCGCCA SEQ ID NO.: 61
CCATGGAGTTTGGGCTGAGC Primer2- SEQ ID NO.: 62 S:
CACCTCTGGGCCCTTGGTCGACGCTGAAGAGACGGTGACCATTGT Primer2- SEQ ID NO.:
63 L: GGGTGCCAGGGGGAAGACCGATGGGCCCTTGGTCGACGCTGAAGA GACGGTGACCATTGT
Primer3- SEQ ID NO.: 64 S:
TCTTCAGCGTCGACCAAGGGCCCAGAGGTGCAGCTGGTGCAGTCT Primer3- SEQ ID NO.:
65 L: GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCGAGGTG CAGCTGGTGCAGTCT
Primer 4: GTAGTCCTTGACCAGGCAGCC SEQ ID NO.: 66 Primer5:
TAGAGATCCCTCGACCTCGAGATCCATTGTGCCCGGGCGCCA SEQ ID NO.: 67
CCATGACTTGGACCCCACTC Primer 6- SEQ ID NO.: 68 S:
TATTTCGGGGGCAGCCTTGGGCTGACCTAGTACTGTGACCTTGGT Primer6- SEQ ID NO.:
69 L: GGGCGGGAACAGAGTGACCGAGGGGGCAGCCTTGGGCTGACCTA GTACTGTGACCTTGGT
Primer7- SEQ ID NO.: 70 S:
CTAGGTCAGCCCAAGGCTGCCCCCGAAATAGTGATGACGCAGTCT Primer7- SEQ ID NO.:
71 L: CAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCGAAATAG TGATGACGCAGTCT
Primer8: GTCCCAGGTGGGGACCCTCACTCTAGAGTCGCGGCCGCCTA SEQ ID NO.: 72
ACACTCTCCCCTGTTGAA
Similar approach has been used to generate DVD1812SL and DVD1812LL
as described below:
Example 3.1.2
Molecular Cloning of DNA Constructs for DVD1812SL and DVD1812LL
[0273] To generate heavy chain constructs DVD1812HC-LL and
DVD1812HC-SL, VH domain of ABT-325 was PCR amplified using primers
Primer 1 and Primer 9L or Primer 9S respectively; meanwhile VH
domain of ABT-874 was amplified using primers Primer 10L or Primer
10S and Primer 4 respectively. Both PCR reactions were performed
according to standard PCR techniques and procedures. The two PCR
products were gel-purified, and used together as overlapping
template for the subsequent overlapping PCR reaction using primers
Primer 1 and Primer 4 using standard PCR conditions. The
overlapping PCR products were subcloned into Srf I and Sal I double
digested pBOS-hC.gamma.1,z non-a mammalian expression vector
(Abbott) by using standard homologous recombination approach. The
following are primers' sequences:
[0274] To generate light chain constructs DVD1812LC-LL and
DVD1812LC-SL, VL domain of ABT-325 was PCR amplified using primers
Primer 11 and Primer 12L or Primer 12S respectively; meanwhile VL
domain of ABT-874 was amplified using primers Primer 13L or Primer
13S and Primer 14 respectively. Both PCR reactions were performed
according to standard PCR techniques and procedures. The two PCR
products were gel-purified, and used together as overlapping
template for the subsequent overlapping PCR reaction using primers
Primer 11 and Primer 14 using standard PCR conditions. The
overlapping PCR products were subcloned into Srf I and Not I double
digested pBOS-hCk mammalian expression vector (Abbott) by using
standard homologous recombination approach. The primers used for
these constructions are listed below in table 19:
TABLE-US-00022 TABLE 19 Primer 9-S: SEQ ID NO.: 73
CACCTGTGGGCCCTTGGTCGACGCTGAAGAGACGGTGACCATTGT Primer 9-L: SEQ ID
NO.: 74 GGGTGCCAGGGGGAAGACCGATGGGCCCTTGGTCGACGCTGAAGAG
ACGGTGACCATTGT Primer 10- SEQ ID NO.: 75 S:
TCTTCAGCGTCGACCAAGGGCCCACAGGTGCAGCTGGTGGAGTCT Primer 10- SEQ ID
NO.: 76 L: GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCCAGGTG
CAGCTGGTGGAGTCT Primer 11: SEQ ID NO.: 77
TAGAGATCCCTCGACCTCGAGATCCATTGTGCCCGGGCGCCACCATG GAAGCCCCAGCGCAGCTT
Primer 12-S: SEQ ID NO.: 78
AGACTGTGGTGCAGCCACAGTTCGTTTAATCTCCAGTCGTGT Primer 12- SEQ ID NO.:
79 L: TGGCGGGAAGATGAAGACAGATGGTGCAGCCACAGTTCGTTTAAT CTCCAGTCGTGT
Primer 13-S: SEQ ID NO.: 80
AAACGAACTGTGGCTGCACCACAGTCTGTGCTGACTCAGCCC Primer 13- SEQ ID NO.:
81 L: ACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCACAGTCTGTGC TGACTCAGCCC
Primer 14: SEQ ID NO.: 82
GTCCCAGGTGGGGACCCTCACTCTAGAGTCGCGGCCGCTCATGAAC ATTCTGTAGGGGC
[0275] The final DNA sequences for eight heavy and light chain
constructs of anti-IL12/IL-18 DVD-Ig are as shown in table 20:
TABLE-US-00023 TABLE 20 Amino acid sequence of DVD binding proteins
capable of binding IL-12 and IL-18 Protein Sequence Sequence
Protein region Identifier 12345678901234567890 DVD HEAVY SEQ ID
QVQLVESGGGVVQPGRSLRL VARIABLE NO.: 83 SCAASGFTFSSYGMHWVRQA
DVD1218HC-SL PGKGLEWVAFIRYDGSNKYY ADSVKGRFTISRDNSKNTLY
LQMNSLRAEDTAVYYCKTHG SHDNWGQGTMVTVSSASTKG PEVQLVQSGTEVKKPGESLK
ISCKGSGYTVTSYWIGWVRQ MPGKGLEWMGFIYPGDSETR YSPTFQGQVTISADKSFNTA
FLQWSSLKASDTAMYYCARV GSGWYPYTFDIWGQGTMVTV SS ABT-874 VH SEQ ID
QVQLVESGGGVVQPGRSLRL NO.: 84 SCAASGFTFSSYGMHWVRQA
PGKGLEWVAFIRYDGSNKYY ADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCKTHG
SHDNWGQGTMVTVSS LINKER SEQ ID ASTKGP NO.: 42 ABT-325 VH SEQ ID
EVQLVQSGTEVKKPGESLKI NO.: 85 SCKGSGYTVTSYWIGWVRQM
PGKGLEWMGFIYPGDSETRY SPTFQGQVTISADKSFNTAF LQWSSLKASDTAMYYCARVG
SGWYPYTFDIWGQGTMVTVS S CH SEQ ID ASTKGPSVFPLAPSSKSTSG NO.: 34
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD LIGHT SEQ ID MTWTPLLFLTLLLHCTGSLS VARIABLE NO.: 86
QSVLTQPPSVSGAPGQRVTI DVD1218LC-SL SCSGSRSNIGSNTVKWYQQL
PGTAPKLLIYYNDQRPSGVP DRFSGSKSGTSASLAITGLQ AEDEADYYCQSYDRYTHPAL
LFGTGTKVTVLGQPKAAPEI VMTQSPATLSVSPGERATLS CRASESISSNLAWYQQKPGQ
APRLFIYTASTRATDIPARF SGSGSGTEFTLTISSLQSED FAVYYCQQYNNWPSITFGQG
TRLEIKR ABT-874 VL SEQ ID QSVLTQPPSVSGAPGQRVTI NO.: 87
SCSGSRSNIGSNTVKWYQQL PGTAPKLLIYYNDQRPSGVP DRFSGSKSGTSASLAITGLQ
AEDEADYYCQSYDRYTHPAL LFGTGTKVTVLG LINKER SEQ ID QPKAAP NO.: 88
ABT-325 VL SEQ ID EIVMTQSPATLSVSPGERAT NO.: 89 LSCRASESISSNLAWYQQKP
GQAPRLFIYTASTRATDIPA RFSGSGSGTEFTLTISSLQS EDFAVYYCQQYNNWPSITFG
QGTRLEIKR CL SEQ ID TVAAPSVFIFPPSDEQLKSG NO.: 36
TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID QVQLVESGGGVVQPGRSLRL
VARIABLE NO.: 90 SCAASGFTFSSYGMHWVRQA DVD1218HC-LL
PGKGLEWVAFIRYDGSNKYY ADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCKTHG
SHDNWGQGTMVTVSSASTKG PSVFPLAPEVQLVQSGTEVK KPGESLKISCKGSGYTVTSY
WIGWVRQMPGKGLEWMGFTY PGDSETRYSPTFQGQVTISA DKSFNTAFLQWSSLKASDTA
MYYCARVGSGWYPYTFDIWG QGTMVTVSS ABT-874 VH SEQ ID
QVQLVESGGGVVQPGRSLRL NO.: 84 SCAASGFTFSSYGMHWVRQA
PGKGLEWVAFIRYDGSNKYY ADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCKTHG
SHDNWGQGTMVTVSS LINKER SEQ ID ASTKGPSVFPLAP NO.: 48 ABT-325 VH SEQ
ID EVQLVQSGTEVKKPGESLKI NO.: 85 SCKGSGYTVTSYWIGWVRQM
PGKGLEWMGFIYPGDSETRY SPTFQGQVTISADKSFNTAF LQWSSLKASDTAMYYCARVG
SGWYPYTFDIWGQGTMVTVS S CH SEQ ID ASTKGPSVFPLAPSSKSTSG NO.: 34
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD LIGHT SEQ ID QSVLTQPPSVSGAPGQRVTI VARIABLE NO.: 91
SCSGSRSNIGSNTVKWYQQL DVD1218LC-LL PGTAPKLLIYYNDQRPSGVP
DRFSGSKSGTSASLAITGLQ AEDEADYYCQSYDRYTHPAL LFGTGTKVTVLGQPKAAPSV
TLFPPEIVMTQSPATLSVSP GERATLSCRASESISSNLAW YQQKPGQAPRLFIYTASTRA
TDIPARFSGSGSGTEFTLTI SSLQSEDFAVYYCQQYNNWP SITFGQGTRLEIKR ABT-874 VL
SEQ ID QSVLTQPPSVSGAPGQRVTI NO.: 87 SCSGSRSNIGSNTVKWYQQL
PGTAPKLLIYYNDQRPSGVP DRFSGSKSGTSASLAITGLQ AEDEADYYCQSYDRYTHPAL
LFGTGTKVTVLG LINKER SEQ ID QPKAAPSVTLFPP NO.: 92 ABT-325 VL SEQ ID
EIVMTQSPATLSVSPGERAT NO.: 89 LSCRASESISSNLAWYQQKP
GQAPRLFIYTASTRATDIPA RFSGSGSGTEFTLTISSLQS EDFAVYYCQQYNNWPSITFG
QGTRLEIKR CL SEQ ID TVAAPSVFIFPPSDEQLKSG NO.: 36
TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID EVQLVQSGTEVKKPGESLKI
VARIABLE NO.: 93 SCKGSGYTVTSYWIGWVRQM DVD1812HC-SL
PGKGLEWMGFIYPGDSETRY SPTFQGQVTISADKSFNTAF LQWSSLKASDTAMYYCARVG
SGWYPYTFDIWGQGTMVTVS SASTKGPQVQLVESGGGVVQ PGRSLRLSCAASGFTFSSYG
MHWVRQAPGKGLEWVAFIRY DGSNKYYADSVKGRFTISRD NSKNTLYLQMNSLRAEDTAV
YYCKTHGSHDNWGQGTMVTV SS ABT-325 VH SEQ ID EVQLVQSGTEVKKPGESLKI NO.:
85 SCKGSGYTVTSYWIGWVRQM PGKGLEWMGFIYPGDSETRY SPTFQGQVTISADKSFNTAF
LQWSSLKASDTAMYYCARVG SGWYPYTFDIWGQGTMVTVS S LINKER SEQ ID ASTKGP
NO.: 42 ABT-874 VH SEQ ID QVQLVESGGGVVQPGRSLRL NO.: 84
SCAASGFTFSSYGMHWVRQA PGKGLEWVAFIRYDGSNKYY ADSVKGRFTISRDNSKNTLY
LQMNSLRAEDTAVYYCKTHG SHDNWGQGTMVTVSS CH SEQ ID ASTKGPSVFPLAPSSKSTSG
NO.: 34 GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGG
PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE
MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYT QKSLSLSPGK DVD LIGHT SEQ ID
EIVMTQSPATLSVSPGERAT VARIABLE NO.: 94 LSCRASESISSNLAWYQQKP
DVD1812LC-SL GQAPRLFIYTASTRATDIPA RFSGSGSGTEFTLTISSLQS
EDFAVYYCQQYNNWPSITFG QGTRLEIKRTVAAPQSVLTQ PPSVSGAPGQRVTISCSGSR
SNIGSNTVKWYQQLPGTAPK LLIYYNDQRPSGVPDRFSGS KSGTSASLAITGLQAEDEAD
YYCQSYDRYTHPALLFGTGT KVTVLG ABT-325 VL SEQ ID EIVMTQSPATLSVSPGERAT
NO.: 89 LSCRASESISSNLAWYQQKP GQAPRLFIYTASTRATDIPA
RFSGSGSGTEFTLTISSLQS EDFAVYYCQQYNNWPSITFG
QGTRLEIKR LINKER SEQ ID TVAAP NO.: 44 ABT-874 VL SEQ ID
QSVLTQPPSVSGAPGQRVTI NO.: 87 SCSGSRSNIGSNTVKWYQQL
PGTAPKLLIYYNDQRPSGVP DRFSGSKSGTSASLAITGLQ AEDEADYYCQSYDRYTHPAL
LFGTGTKVTVLG CL SEQ ID TVAAPSVFIFPPSDEQLKSG NO.: 36
TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKS FNRGEC DVD HEAVY SEQ ID EVQLVQSGTEVKKPGESLKI
VARIABLE NO.: 95 SCKGSGYTVTSYWIGWVRQM DVD1812HC-LL
PGKGLEWMGFIYPGDSETRY SPTFQGQVTISADKSFNTAF LQWSSLKASDTAMYYCARVG
SGWYPYTFDIWGQGTMVTVS SASTKGPSVFPLAPQVQLVE SGGGVVQPGRSLRLSCAASG
FTFSSYGMHWVRQAPGKGLE WVAFIRYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSL
RAEDTAVYYCKTHGSHDNWG QGTMVTVSS ABT-325 VH SEQ ID
EVQLVQSGTEVKKPGESLKI NO.: 85 SCKGSGYTVTSYWIGWVRQM
PGKGLEWMGFIYPGDSETRY SPTFQGQVTISADKSFNTAF LQWSSLKASDTAMYYCARVG
SGWYPYTFDIWGQGTMVTVS S LINKER SEQ ID ASTKGPSVFPLAP NO.: 48 ABT-875
VH SEQ ID QVQLVESGGGVVQPGRSLRL NO.: 84 SCAASGFTFSSYGMHWVRQA
PGKGLEWVAFIRYDGSNKYY ADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCKTHG
SHDNWGQGTMVTVSS CH SEQ ID ASTKGPSVFPLAPSSKSTSG NO.: 34
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT
QKSLSLSPGK DVD LIGHT SEQ ID EIVMTQSPATLSVSPGERAT VARIABLE NO.: 96
LSCRASESISSNLAWYQQKP DVD1812LC-LL GQAPRLFIYTASTRATDIPA
RFSGSGSGTEFTLTISSLQS EDFAVYYCQQYNNWPSITFG QGTRLEIKRTVAAPSVFIFP
PQSVLTQPPSVSGAPGQRVT ISCSGSRSNIGSNTVKWYQQ LPGTAPKLLIYYNDQRPSGV
PDRFSGSKSGTSASLAITGL QAEDEADYYCQSYDRYTHPA LLFGTGTKVTVLG ABT-325 VL
SEQ ID EIVMTQSPATLSVSPGERAT NO.: 89 LSCRASESISSNLAWYQQKP
GQAPRLFIYTASTRATDIPA RFSGSGSGTEFTLTISSLQS EDFAVYYCQQYNNWPSITFG
QGTRLEIKR LINKER SEQ ID TVAAPSVFIFPP NO.: 50 ABT-874 VL SEQ ID
QSVLTQPPSVSGAPGQRVTI NO.: 87 SCSGSRSNIGSNTVKWYQQL
PGTAPKLLIYYNDQRPSGVP DRFSGSKSGTSASLAITGLQ AEDEADYYCQSYDRYTHPAL
LFGTGTKVTVLG CL SEQ ID TVAAPSVFIFPPSDEQLKSG NO.: 36
TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKS FNRGEC
Example 3.2
Determination of Antigen Binding Affinity of IL-1211L-18 DVD
Igs
[0276] The binding affinity of anti-IL-12/18 DVD-Igs to hIL-12 and
hIL-18 were determined by Biacore (Table 21). The neutralization
activity against IL-18 was determined by KG-1 assay (Konishi, K.,
et al.). Briefly, IL-18 samples (in a final concentration of 2
ng/ml) were pre-incubated with DVD-Ig (in final concentrations
between 0 and 10 mg/ml) at 37.degree. C. for 1 hr, and then added
to KG-1 cells (3.times.10.sup.6/ml) in RPMI medium containing 10
ng/ml hTNF, followed by incubation at 37.degree. C. for 16-20 hr.
The culture supernatants were collected and human IFN-.gamma.
production in each sample was determined by ELISA (R&D
Systems). Inhibition activities of the DVD molecules against IL-18,
presented as IC.sub.50 values, are shown in Table VI. To determine
the inhibition activities of anti-IL-12/18 DVD molecules against
IL-12, an IL-12-induced IFN-.gamma. production assay from activated
PHA blast cells was employed (D'Andrea, A et al.). For production
of human IFN-.gamma., PHA blast cells were incubated for 18 hours
with human IL-12. Sub-maximal stimulation (55-75% of maximum) was
obtained with a human IL-12 concentration of 200 pg/mL.
Supernatants were assayed for IFN-.gamma. using a specific human
IFN-.gamma. ELISA (Endogen, Cambridge, Mass.). Neutralizing IL-12
DVDs interfere with IL-12 induced IFN-.gamma. production. The
neutralization activity of DVD is determined by measuring the DVD
concentration required to inhibit 50% of the IFN-.gamma. production
by human PHA blast cells, as shown in Table 21.
TABLE-US-00024 TABLE 21 Characterization of anti-IL-18/IL-12 DVD-Ig
molecules K.sub.d IC.sub.50 Affinity (K.sub.d, M) Potency
(IC.sub.50, M) MAb Specif. (M) (M) DVD Orient. Linker IL-12 IL-18
IL-12 IL-18 ABT874 hIL-12 6.47E-11 5.0E-12 DVD1218-SL 12-18-C short
3.81E-11 6.22E-10 6.93E-12 1.8E-10 DVD1218-LL 12-18-C long 2.38E-11
6.64E-10 3.04E-12 1.8E-10 ABT325 hIL-18 1.37E-10 3.0E-10 DVD1812-SL
18-12-C short 1.82E-09 1.91E-10 3.66E-10 4.0E-11 DVD1812-LL 18-12-C
long 1.13E-10 1.62E-10 1.18E-10 7.8E-11 Affinity (Kd) was
determined by Biacore and potency (IC50) determined by KG-1
bioassay (IL-18) and PBMC assay (IL-12).
[0277] Table 21 shows the specificity, binding affinity, and
neutralization activity of the 2 fully human mAbs used for the
construction of the anti-IL-12/IL-18 DVD molecules. As shown in the
Table VI, these mAbs have high affinity and neutralization
activity. A summary of the characterization of the anti-IL-18/IL-12
DVD constructs is shown in Table VI. SDS-PAGE analysis of all new
DVD proteins showed normal migration patterns in both reduced and
non-reduced conditions, similar to a regular antibody and
DVD1/2-Ig. SEC analysis indicated all molecules were normal,
exhibiting peaks in the 200 kD region. The Biacore binding data are
consistent with the neutralization activity in the biological
assays.
Example 3.3
Biological activity of anti-IL-1211L-18 DVD-Ig in vivo
[0278] Both IL-12 and IL-18 are required to produce optimal
IFN.gamma. in response to various stimuli. The biological activity
of anti-IL-12/IL-18 DVD-Ig in vivo was determined using the
huPBMC-SCID mouse model. In this model, anti-IL-12 antibody
(ABT-874) anti-IL-18 antibody (ABT-325) or the
anti-IL-12/anti-IL-18 DVD-Ig were injected i.p. or i.v. (250
mg/mouse each) followed by transfer of freshly purified human PBMCs
(huPBMC) i.p. into SCID mice. Fifteen minutes later, mice were
challenged with dried staphylococcus aureus cells (SAC) to induce
human IFN.gamma. production. Animals (n=7-8/group) were sacrificed
18-20 hrs later and serum huIFN.gamma. levels were determined by
ELISA. ABT 874 and ABT-325 inhibited SAC-induced IFN.gamma. by
approximately 70% which represents maximum IFN.gamma. inhibition
with each compound in this model. However, treatment of mice with
ABT-874+ABT-325 and anti-IL-12/anti-IL-18 DVD-Ig inhibited
IFN.gamma. production by almost 100%. These results suggest that
the anti-IL-12/anti-IL-18 DVD-Ig molecule is functionally active in
vivo.
Example 3.4
Pharmacokinetic and Pharmacodynamic Studies of Anti-IL-1211L-18
DVD-Ig
[0279] The overall Pharmacokinetic and pharmacodynamicprofile of
anti-IL-12/IL-18 DVD-Ig was similar to the parent mAbs in mice,
i.e., 73% bioavailability, comparable to regular IgG. Similar
pharmacokinetics, i.e., rapid clearance after day 6-8, was also
observed for other mAbs (e.g., human, rat etc.,) probably due to
anti-human IgG response.
[0280] Male SD rats were dosed with anti-IL-12/IL-18 DVD-Ig at 4
mg/kg either i.v. or s.c. The early part of the PK curves looked
normal and very similar to those of other human antibodies. An
accurate half-life in both groups could not be derived because of
the rapid clearance of DVD-Ig beginning on day 6. The sudden drop
in DVD-Ig concentration after day 6 may be due to the RAHA
response. However, similar profile has also been observed for one
of the parent antibodies (ABT-874) used for construction of this
DVD-Ig in this particular experiment, as well as other
mono-specific human antibodies previously studied. Based on DVD-Ig
concentration up to day 6 in both s.c and i.v. groups,
bioavailability of DVD-Ig was estimated. Two out of three rats
showed 80-95% bioavailability, and the average bioavailability in
the three mice was 73%
Example 3.5
Physical/Chemical Characterization of Anti-IL-12/Anti-IL-18
DVD-Ig
[0281] Results of physical and chemical characterization of 293
cell-derived, protein A purified, anti-IL-12/anti-IL-18 DVD-Ig are
summarized in Table 22.
TABLE-US-00025 TABLE 22 Physical/Chemical Characterization of
anti-IL-12/anti-IL-18 DVD-Ig Parameters Assay/ Tested Methodology
Findings/Comments Affinity (Kd) IL-12 Biacore 38 pM (65 pM for
ABT-874) IL-18 Biacore 622 pM (137 pM for ABT-325) Potency (IC50)
IL-12 PHA-Blast 7 pM (5 pM for ABT874) Assay IL-18 KG-1 Assay 180
pM (300 pM for ABT-325) M.W MS HC: 64130 (theo. 64127) LC: 36072
(theo. 36072) Amino acid Sequencing- All matched sequence MS
Disulfide Peptide All 20 disulfide bonds are matched bonds mapping
Glycosylation Similar to other in-house fully human profile
antibodies-NGA2F and NGA1F observed as the major forms Charge
Cation Homogeneity heterogeneity Exchange (WCX-10) PI cIEF 9.42
(ABT-874: 9.46) Dynamic size DSL 7.69 nM (5.34 nM for ABT-325)
Purity/ SDS-PGE Homogeneity on both reducing (~64 aggregates Kd HC
and ~36 Kd LC bands) and non-reducing (one band) gels SEC One peak
(~100%) observed immediately after protein A purification by SEC
AUC ~16-17% aggregates observed after 2 cycles of freeze-thaw by
AUC Stability SEC ~5% aggregates after 2 freeze-thaw cycles,
(freeze/thaw) increased to ~13% after additional 10 freeze-thaw
cycles. The reason for that is unsolved (process-related, sequence-
specific, or LC lamda/kappa hybrid) PK profile Rat i.v. &
Similar to (or limited by) parental mAbs. s.c. Bioavailability Rat
i.v. vs Average 73%; Overall similar to parental s.c. mAbs
Example 3.6
Generation of an Additional Anti-12/Anti-18 DVD-Ig
(1D4.1-ABT325)
[0282] An additional anti-IL-12/IL-18 DVD-Ig molecule with a
different parent anti-IL-12 mAb (clone#1D4.1), as shown in Table
23, was constructed. The 1D4.1-ABT325 DVD-Ig construct was
generated with a short linker derived from the N-terminal sequence
of human Ck and CH1 domain, as follows: [0283] Short linker: light
chain: TVAAP (SEQ ID NO:44); heavy chain: ASTKGP (SEQ ID
NO:42).
[0284] All heavy and light chain constructs were subcloned into the
pBOS expression vector, expressed in COS cells or freestyle 293
cells, and characterized as described above. 1D4.1-ABT325 DVD-Ig
fully retains the activities of the two original mAbs (Table
24).
TABLE-US-00026 TABLE 23 Amino acid sequence of 1D4.1-ABT325 DVD-Ig
Protein Sequence Sequence Protein region Identifier
12345678901234567890 1D4.1-ABT325 SEQ ID EVTLRESGPALVKPTQTLTL
DVD-Ig HEAVY NO.: 114 TCTFSGFSLSKSVMGVSWIR VARIABLE
QPPGKALEWLAHIYWDDDKY YNPSLKSRLTISKDTSKNQV VLTMTNMDPVDTATYYCARR
GIRSAMDYWGQGTTVTVSSA STKGPEVQLVQSGTEVKKPG ESLKISCKGSGYTVTSYWIG
WVRQMPGKGLEWMGFIYPGD SETRYSPTFQGQVTISADKS FNTAFLQWSSLKASDTAMYY
CARVGSGWYPYTFDIWGQGT MVTVSS 1D4.1 VH SEQ ID EVTLRESGPALVKPTQTLTL
NO.: 115 TCTFSGFSLSKSVMGVSWIR QPPGKALEWLAHIYWDDDKY
YNPSLKSRLTISKDTSKNQV VLTMTNMDPVDTATYYCARR GIRSAMDYWGQGTTVTVSS
LINKER SEQ ID ASTKGP NO.: 42 ABT-325 VH SEQ ID EVQLVQSGTEVKKPGESLKI
NO.: 85 SCKGSGYTVTSYWIGWVRQM PGKGLEWMGFIYPGDSETRY
SPTFQGQVTISADKSFNTAF LQWSSLKASDTAMYYCARVG SGWYPYTFDIWGQGTMVTVS S CH
SEQ ID ASTKGPSVFPLAPSSKSTSG NO.: 34 GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK 1D4.1-ABT325
SEQ ID DIVMTQSPDSLAVSLGERAT DVD-Ig LIGHT NO.: 116
INCKASQSVSNDVAWYQQKP VARIABLE GQPPKLLIYYASNRYTGVPD
RFSGSGSGTDFTLTISSLQA EDVAVYYCQQDYNSPWTFGG GTKVEIKRTVAAPEIVMTQS
PATLSVSPGERATLSCRASE SISSNLAWYQQKPGQAPRLF IYTASTRATDIPARFSGSGS
GTEFTLTISSLQSEDFAVYY CQQYNNWPSITFGQGTRLEI KR 1D4.1 VL SEQ ID
DIVMTQSPDSLAVSLGERAT NO.: 117 INCKASQSVSNDVAWYQQKP
GQPPKLLIYYASNRYTGVPD RFSGSGSGTDFTLTISSLQA EDVAVYYCQQDYNSPWTFGG
GTKVEIKR LINKER SEQ ID TVAAP NO.: 44 ABT-325 VL SEQ ID
EIVMTQSPATLSVSPGERAT NO.: 89 LSCRASESISSNLAWYQQKP
GQAPRLFIYTASTRATDIPA RFSGSGSGTEFTLTISSLQS EDFAVYYCQQYNNWPSITFG
QGTRLEIKR CL SEQ ID TVAAPSVFIFPPSDEQLKSG NO.: 36
TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKS FNRGEC
TABLE-US-00027 TABLE 24 Characterization 1D4.1-ABT325 DVD-Ig
molecule Affinity (K.sub.d, M) Potency (IC.sub.50, M) mAb IL-12
IL-18 IL-12 IL-18 1D4.1 1.20E-10 N/A 4.18E-10 N/A ABT325 N/A
1.91E-10 N/A 6.87E-11 1D4.1-ABT325 DVD-Ig 1.33E-10 1.59E-10
2.17E-10 1.20E-10 Affinity (Kd) was determined by Biacore and
potency (IC50) determined by KG-1 bioassay (IL-18) and PBMC assay
(IL-12).
Example 4
Generation of anti-CD20/anti-CD3 DVD-Ig
[0285] Anti-CD20/anti-CD3 DVD-Igs were generated using murine
anti-human-CD20 (clone 5F1) and anti-human-CD3 (clone OKT3) parent
antibodies. The initial constructs included 2 DVD-Igs with
different domain orientations. The anti-CD3/anti-CD20 DVD-Ig was
constructed in the order of V.sub.CD3-linker-V.sub.CD20-constant,
and anti-CD20/anti-CD3 DVD-Ig was constructed in the order of
V.sub.CD20-linker-V.sub.CD3-constant. However, in a preliminary
cell surface binding study, anti-CD20 binding activity was
diminished in the anti-CD3/anti-CD20 DVD-Ig molecule, even though
the anti-CD3 activity was conserved in this design. In contrast,
both anti-CD3 and anti-CD20 binding activities were fully conserved
in the anti-CD20/anti-CD3 DVD-Ig molecule, indicating this is the
optimal domain orientation for these two mAbs/targets combination
in a DVD-Ig format. Therefore the anti-CD20/anti-CD3 DVD-Ig
construct was chosen for subsequent studies.
[0286] The anti-CD20/anti-CD3 DVD-Ig was generated as chimeric Ig
i.e., the constant region was a human constant region. For binding
analysis, human T cell line Jurkat and B cell line Raji were
blocked with human IgG and then stained with murine anti-hCD3 mAb
OKT3, murine anti-hCD20 mAb 1F5, and anti-CD20/anti-CD3 DVD-Ig.
Cells were then washed and stained with FITC-labeled goat
anti-murine IgG (with no anti-hIgG cross-reactivity). Anti-CD20/CD3
DVD-Ig bound both T and B cells, whereas CD3 and CD20 mAbs bound
only T or B cells, respectively. The amino acid sequence of
CD20/CD3 DVD-Ig is disclosed in Table 25.
TABLE-US-00028 TABLE 25 Amino acid sequence of CD20CD3DVD-Ig
Protein Sequence Sequence Protein region Identifier
12345678901234567890 DVD HEAVY SEQ ID QVQLRQPGAELVKPGASVKM VARIABLE
NO.: 97 SCKASGYTFTSYNMHWVKQT CD20CD3DVD-Ig PGQGLEWIGAIYPGNGDTSY
NQKFKGKATLTADKSSSTAY MQLSSLTSEDSAVYYCARSH YGSNYVDYFDYWGQGTTLTV
SSAKTTAPSVYPLAPQVQLQ QSGAELARPGASVKMSCKAS GYTFTRYTMHWVKQRPGQGL
EWIGYINPSRGYTNYNQKFK DKATLTTDKSSSTAYMQLSS LTSEDSAVYYCARYYDDHYC
LDYWGQGTTLTVSS 5F1 VH SEQ ID QVQLRQPGAELVKPGASVKM NO.: 98
SCKASGYTFTSYNMHWVKQT PGQGLEWIGAIYPGNGDTSY NQKFKGKATLTADKSSSTAY
MQLSSLTSEDSAVYYCARSH YGSNYVDYFDYWGQGTTLTV SS LINKER SEQ ID
AKTTAPSVYPLAP NO.: 99 OKT3 VH SEQ ID QVQLQQSGAELARPGASVKM NO.: 100
SCKASGYTFTRYTMHWVKQR PGQGLEWIGYINPSRGYTNY NQKFKDKATLTTDKSSSTAY
MQLSSLTSEDSAVYYCARYY DDHYCLDYWGQGTTLTVSS CH SEQ ID
ASTKGPSVFPLAPSSKSTSG NO.: 34 GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK CD20CD3DVD-Ig
SEQ ID QIVLSQSPAILSASPGEKVT LIGHT VARIABLE NO.: 101
MTCRASSSLSFMHWYQQKPG SSPKPWIYATSNLASGVPAR FSGSGSGTSYSLTISRVEAE
DAATYFCHQWSSNPLTFGAG TKLELKRADAAPTVSIFPPQ IVLTQSPAIMSASPGEKVTM
TCSASSSVSYMNWYQQKSGT SPKRWIYDTSKLASGVPAHF RGSGSGTSYSLTISGMEAED
AATYYCQQWSSNPFTFGSGT KLEINR 5F1 VL SEQ ID QIVLSQSPAILSASPGEKVT NO.:
102 MTCRASSSLSFMHWYQQKPG SSPKPWIYATSNLASGVPAR FSGSGSGTSYSLTISRVEAE
DAATYFCHQWSSNPLTFGAG TKLELKR LINKER SEQ ID ADAAPTVSIFPP NO.: 103
OKT3 VL SEQ ID QIVLTQSPAIMSASPGEKVT NO.: 104 MTCSASSSVSYMNWYQQKSG
TSPKRWIYDTSKLASGVPAH FRGSGSGTSYSLTISGMEAE DAATYYCQQWSSNPFTFGSG
TKLEINR CL SEQ ID TVAAPSVFIFPPSDEQLKSG NO.: 36 TASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS
FNRGEC
Example 5
Generation of mIL-1.alpha./.beta.DVD-Ig
[0287] To study key issues concerning pharmacokinetics, in vivo
efficacy, tissue penetration, and immunogenicity of DVD-Ig
molecules, mouse-anti-mouse IL-1.alpha./.beta. DVD-Ig molecules
were constructed as described below.
Example 5.1
Construction of mIL-1.alpha./.beta.DVD-Ig
[0288] Mouse-anti-mouse IL-1.alpha./.beta. DVD-Ig molecules were
constructed using two mouse anti-mouse IL-1.alpha./.beta. mAbs
(9H10 and 10G11) generated from IL-1.alpha..beta. double KO mice.
Mouse anti-mouse IL-1.alpha., and mouse anti-mouse IL-1.beta.,
monoclonal antibodies were generated by immunizing
IL-1.alpha./.beta. double KO mice with mouse IL-1.alpha., or mouse
IL-1.beta., respectively. One mouse anti-mouse IL-1.alpha. (Clone
9H10), and one mouse anti-mouse IL-1.beta. mAb (clone 10G11), were
selected and used to generate mIL-1.alpha./.beta. DVD-Ig molecules.
Various linker sizes and different domain orientations were tested.
The final functional mIL-1.alpha./.beta. DVD-Ig molecules was
constructed in an orientation of
V(anti-mIL-1.beta.)-linker-V(anti-mIL-1.beta.)-murine constant
region (C.gamma.2a and CK). The cloning, expression, and
purification procedures were similar to that of the hIL-1a/.beta.
DVD-Ig. The cloning of mIL-1.alpha./.beta. DVD-Ig was carried out
using similar overlapping PCR and homologous recombination as
described for hIL-1.alpha./.beta. DVD3-Ig. The sequences of
mIL-1.alpha./.beta. DVD-Ig are shown below in Table 26:
TABLE-US-00029 TABLE 26 Amino acid sequence of mIL-1.alpha./.beta.
DVD-Ig Protein Sequence Sequence Protein region Identifier
12345678901234567890 mIL-1.alpha./.beta. DVD-Ig SEQ ID
EVQLQQSGPELVKPGTSVKM HEAVY NO.: 105 SCKTSGYTFTSYVMHWVKQK VARIABLE
PGQGLEWIGYIIPYNDNTKY NEKFKGKATLTSDKSSSTAY MELSSLTSEDSAVYYCARRN
EYYGSSFFDYWGQGTTLTVS SAKTTAPSVYPLAPQVILKE SGPGILQPSQTLSLTCSFSG
FSLSTYGTAVNWIRQPSGKG LEWLAQIGSDDRKLYNPFLK SRITLSEDTSNSQVFLKITS
VDTEDSATYYCANGVMEYWG LGTSVTVSS 10G11 VH SEQ ID EVQLQQSGPELVKPGTSVKM
NO.: 106 SCKTSGYTFTSYVMHWVKQK PGQGLEWIGYIIPYNDNTKY
NEKFKGKATLTSDKSSSTAY MELSSLTSEDSAVYYCARRN EYYGSSFFDYWGQGTTLTVS S
LINKER SEQ ID AKTTAPSVYPLAP NO.: 99 9H10 VH SEQ ID
QVILKESGPGILQPSQTLSL NO.: 107 TCSFSGFSLSTYGTAVNWIR
QPSGKGLEWLAQIGSDDRKL YNPFLKSRITLSEDTSNSQV FLKITSVDTEDSATYYCANG
VMEYWGLGTSVTVSS CH SEQ ID AKTTAPSVYPLAPVCGDTTG NO.: 108
SSVTLGCLVKGYFPEPVTLT WNSGSLSSGVHTFPAVLQSD LYTLSSSVTVTSSTWPSQSI
TCNVAHPASSTKVDKKIEPR GPTIKPCPPCKCPAPNLLGG PSVFIFPPKIKDVLMISLSP
IVTCVVVDVSEDDPDVQISW FVNNVEVHTAQTQTHREDYN STLRVVSALPIQHQDWMSGK
EFKCKVNNKDLPAPIERTIS KPKGSVRAPQVYVLPPPEEE MTKKQVTLTCMVTDFMPEDI
YVEWTNNGKTELNYKNTEPV LDSDGSYFMYSKLRVEKKNW VERNSYSCSVVHEGLHNHHT
TKSFSRTPGK mIL-1.alpha./.beta. DVD-Ig SEQ ID DIQMTQSPASLSASVGETVT
LIGHT VARIABLE NO.: 109 ITCRGSGILHNYLVWYQQKQ GKSPQLLVYSAKILADGVPS
RFSGSGSGTQYSLKINSLQP EDFGSYYCQHFWSTPFTFGS GTKLEIKRADAAPTVSIFPP
SIVMTQTPKFLLVSAGDRVT ITCKASQSVNHDVAWYQQMP GQSPKLLIYFASNRYTGVPD
RFTGSGYGTDFTFTISTVQA EDLAVYFCQQDYSSPYTFGG GTKLEIKR 10G11 VL SEQ ID
DIQMTQSPASLSASVGETVT NO.: 110 ITCRGSGILHNYLVWYQQKQ
GKSPQLLVYSAKILADGVPS RFSGSGSGTQYSLKINSLQP EDFGSYYCQHFWSTPFTFGS
GTKLEIKR LINKER SEQ ID ADAAPTVSIFPP NO.: 111 9H10 VL SEQ ID
SIVMTQTPKFLLVSAGDRVT NO.: 112 ITCKASQSVNHDVAWYQQMP
GQSPKLLIYFASNRYTGVPD RFTGSGYGTDFTFTISTVQA EDLAVYFCQQDYSSPYTFGG
GTKLEIKR CL SEQ ID ADAAPTVSIFPPSSEQLTSG NO.: 113
GASVVCFLNNFYPKDINVKW KIDGSERQNGVLNSWTDQDS KDSTYSMSSTLTLTKDEYER
HNSYTCEATHKTSTSPIVKS FNRNEC
[0289] Murine mIL-1.alpha./.beta. DVD-Ig retained affinity/in vitro
potency against both IL-1.alpha. and IL-.beta.. Table 27 shows the
characterization of mAbs 9H10 (anti-mIL-1.alpha.), 10G11
(anti-mIL-1.beta.), and mIL-1.alpha./.beta. DVD-Ig.
TABLE-US-00030 TABLE 27 Characterization of mDVD4-Ig Antigen
K.sub.D (M) IC.sub.50 (M) 9H10 mIL-1.alpha. 1.73E-10 2.00E-10 10G11
mIL-1.beta. 2.30E-10 3.70E-10 mIL-1.alpha./.beta.DVD-Ig
mIL-1.alpha. 7.66E-10 2.00E-09 mIL-1.beta. 6.94E-10 8.00E-10
Example 5.2
In Vivo Activity of mIL-1.alpha./.beta.DVD-Ig in Arthritis
Model
[0290] The therapeutic effects of anti-IL-1alpha, anti-IL-1beta,
combined anti-IL-1-alpha/anti-IL-1beta, and murine
anti-IL-1alpha/beta DVD4-Ig, 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. To neutralize IL-1, mice were
injected every other day with 1-3 mg/kg of anti-IL-1alpha mAb,
anti-IL-1beta mAb, combination of anti-IL-1-alpha/anti-IL-1beta
mAbs, or murine anti-IL-1alpha/beta DVD4-Ig intraperitoneally. Mice
were carefully examined three times a week for the visual
appearance of arthritis in peripheral joints, and scores for
disease activity determined.
[0291] Blockade of IL-1 in the therapeutic mode effectively reduced
the severity of arthritis, with anti-IL-lbeta showing greater
efficacy (24% reduction in mean arthritis score compared to control
group) than anti-IL-1-alpha (10% reduction). An additive effect was
observed between anti-IL-1-alpha and anti-IL-lbeta, with a 40%
reduction in mean arthritis score in mice treated with both
anti-IL-1alpha and anti-IL-lbeta mAbs. Surprisingly, at the same
dose level, the treatment of mDVD-Ig exhibited 47% reduction in
mean arthritis score, demonstrating the in vivo therapeutic
efficacy of mDVD-Ig. Similar efficacy was also observed in the
measurements of joint swelling in this animal model.
[0292] The present invention incorporates by reference in their
entirety techniques well known in the field of molecular biology
and drug delivery. These techniques include, but are not limited
to, techniques described in the following publications: [0293]
Ausubel et al. (eds.), Current Protocols in Molecular Biology, John
Wiley & Sons, NY (1993); [0294] Ausubel, F. M. et al. eds.,
Short Protocols In Molecular Biology (4th Ed. 1999) John Wiley
& Sons, NY. (ISBN 0-471-32938-X). [0295] Controlled Drug
Bioavailability, Drug Product Design and Performance, Smolen and
Ball (eds.), Wiley, New York (1984); [0296] Giege, R. and Ducruix,
Crystallization of Nucleic Acids and Proteins, a Practical
Approach, 2nd ed., Oxford University Press, New York, N.Y., (1999)
pp. 1-16; [0297] 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; [0298] Hammerling, et al., in: Monoclonal Antibodies and
T-Cell Hybridomas 563-581 (Elsevier, N.Y., 1981); [0299] Harlow et
al., Antibodies: A Laboratory Manual, (Cold Spring Harbor
Laboratory Press, 2nd ed. 1988); [0300] Kabat et al., Sequences of
Proteins of Immunological Interest (National Institutes of Health,
Bethesda, Md. (1987) and (1991); [0301] 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; [0302] Kontermann and Dubel eds., Antibody Engineering
(2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).
[0303] Kriegler, Gene Transfer and Expression, A Laboratory Manual,
Stockton Press, N.Y. (1990); [0304] Lu and Weiner eds., Cloning and
Expression Vectors for Gene Function Analysis (2001) BioTechniques
Press. Westborough, Mass. 298 pp. (ISBN 1-881299-21-X). [0305]
Medical Applications of Controlled Release, Langer and Wise (eds.),
CRC Pres., Boca Raton, Fla. (1974); [0306] 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). [0307] Sambrook, J., et al. eds., Molecular
Cloning: A Laboratory Manual (2d Ed. 1989) Cold Spring Harbor
Laboratory Press, N.Y. Vols. 1-3. (ISBN 0-87969-309-6). [0308]
Sustained and Controlled Release Drug Delivery Systems, J. R.
Robinson, ed., Marcel Dekker, Inc., New York, 1978 [0309]
Winnacker, E. L. From Genes To Clones: Introduction To Gene
Technology (1987) VCH Publishers, NY (translated by Horst
Ibelgaufts). 634 pp. (ISBN 0-89573-614-4).
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[0505] Although a number of embodiments and features have been
described above, it will be understood by those skilled in the art
that modifications and variations of the described embodiments and
features may be made without departing from the present disclosure
or the invention as defined in the appended claims. Each of the
publications mentioned herein is incorporated by reference.
Sequence CWU 1
1
1331122PRTMus musculusPEPTIDE(1)..(122)Murine monoclonal antibody
3D12 binding human Il-1a (VH) 1Gln Ile Gln Leu Val Gln Ser Gly Pro
Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Arg Asn Tyr 20 25 30 Gly Met Asn Trp Val
Lys Gln Ala Pro Gly Lys Asp Leu Lys Arg Met 35 40 45 Ala Trp Ile
Asn Thr Tyr Thr Gly Glu Ser Thr Tyr Ala Asp Asp Phe 50 55 60 Lys
Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70
75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe
Cys 85 90 95 Ala Arg Gly Ile Tyr Tyr Tyr Gly Ser Ser Tyr Ala Met
Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115
120 2108PRTMus musculusPEPTIDE(1)..(108)Murine monoclonal antibody
3D12 capable of binding human IL-1a (VL) 2Asn Ile Gln Met Thr Gln
Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr
Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Cys 20 25 30 Leu Asn
Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu
Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Lys Thr
Leu Pro Tyr 85 90 95 Ala Phe Gly Gly Gly Thr Lys Leu Glu Ile Asn
Arg 100 105 3118PRTMus musculusPEPTIDE(1)..(118)Murine monoclonal
antibody 18F4 capable of binding human IL-1a (VH) 3Glu Val Gln Leu
Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val
Lys Leu Ser Cys Thr Ala Ser Gly Leu Asn Ile Lys Asp Thr 20 25 30
Tyr Met His Trp Leu Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35
40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Ala Lys Tyr Asp Pro Arg
Phe 50 55 60 Leu Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn
Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Gly Asn Phe His Phe
Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115
4108PRTMus musculusPEPTIDE(1)..(108)Murine monoclonal antibody 18F4
capable of binding human IL-1a (VL) 4Asp Ile Val Met Thr Gln Ser
Gln Arg Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Val
Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn 20 25 30 Ile Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Ala Leu Ile 35 40 45 Tyr
Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
65 70 75 80 Val Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Thr Arg Tyr
Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 5114PRTMus musculusPEPTIDE(1)..(114)Murine monoclonal
antibody 6H3 capable of binding human IL-1a (VH) 5Gln Val Gln Leu
Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val
Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30
Trp Met Asn Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35
40 45 Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr Leu Tyr Ser Gln Lys
Phe 50 55 60 Lys Asp Thr Ala Ile Leu Thr Val Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Gly Phe Asp Tyr Trp Gly
Gln Gly Thr Thr Leu Thr Val 100 105 110 Ser Ser 6107PRTMus
musculusPEPTIDE(1)..(107)Murine monoclonal antibody 6H3 capable of
binding human IL-1a (VL) 6Gln Ile Val Leu Thr Gln Ser Pro Ala Leu
Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser
Ala Ser Ser Ser Val Asn Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys
Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Leu Thr Ser Asn
Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser
Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Ser Asn Pro Tyr Thr 85
90 95 Phe Gly Gly Gly Thr Lys Leu Glu Met Lys Arg 100 105
7121PRTMus musculusPEPTIDE(1)..(121)Murine monoclonal antibody 13F5
capable of binding human IL-1b (VH) 7Gln Val Gln Leu Gln Gln Ser
Gly Ala Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser
Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn
Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly
Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55
60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ser Tyr
65 70 75 80 Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Met Tyr
Phe Cys 85 90 95 Val Arg Phe Pro Thr Gly Asn Asp Tyr Tyr Ala Met
Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser 115
120 8112PRTMus musculusPEPTIDE(1)..(112)Murine monoclonal antibody
13F5 capable of binding human IL-1b (VL) 8Asn Ile Val Leu Thr Gln
Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr
Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr 20 25 30 Gly Asn
Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50
55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile
Asp 65 70 75 80 Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln
Gln Asn Asn 85 90 95 Glu Asp Pro Phe Thr Phe Gly Ser Gly Thr Lys
Leu Glu Ile Lys Arg 100 105 110 9122PRTMus
musculusPEPTIDE(1)..(122)Murine monoclonal antibody 1B12 capable of
binding human IL-1b (VH) 9Gln Val His Leu Lys Glu Ser Gly Pro Gly
Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val
Ser Gly Phe Ser Leu Thr Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Leu Ile Trp
Gly Gly Gly Asp Thr Tyr Tyr Asn Ser Pro Leu Lys 50 55 60 Ser Arg
Leu Ser Ile Arg Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Val Tyr Tyr Cys Ala 85
90 95 Lys Gln Arg Thr Leu Trp Gly Tyr Asp Leu Tyr Gly Met Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120
10108PRTMousePEPTIDE(1)..(108)Murine monoclonal antibody 1B12
capable of binding human IL-1b (VL) 10Glu Thr Thr Val Thr Gln Ser
Pro Ala Ser Leu Ser Met Ala Ile Gly 1 5 10 15 Glu Lys Val Thr Ile
Arg Cys Ile Thr Ser Thr Asp Ile Asp Val Asp 20 25 30 Met Asn Trp
Tyr Gln Gln Lys Pro Gly Glu Pro Pro Lys Leu Leu Ile 35 40 45 Ser
Gln Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Ser 50 55
60 Ser Gly Ser Gly Thr Asp Phe Val Phe Ile Ile Glu Asn Met Leu Ser
65 70 75 80 Glu Asp Val Ala Asp Tyr Tyr Cys Leu Gln Ser Asp Asn Leu
Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg
100 105 11118PRTMus musculusPEPTIDE(1)..(118)Murine monoclonal
antibody 6B12 capable of binding human IL-1b (VH) 11Glu Val Gln Leu
Gln Gln Ser Gly Pro Glu Leu Val Lys Thr Gly Thr 1 5 10 15 Ser Val
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30
Tyr Met His Trp Val Arg Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35
40 45 Gly Tyr Ile Ser Cys Tyr Asn Gly Phe Thr Ser Tyr Asn Pro Lys
Phe 50 55 60 Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Ile Gln Phe Ser Arg Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Tyr Tyr Gly Thr Asn
Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115
12107PRTMus musculusPEPTIDE(1)..(107)Murine monoclonal antibody
6B12 capable of binding human IL-1b (VL) 12Gln Ile Val Leu Thr Gln
Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr
Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp
Phe Gln Gln Lys Pro Gly Ala Ser Pro Lys Leu Trp Ile Tyr 35 40 45
Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50
55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Val Ser Arg Met Glu Ala
Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Thr Tyr
Pro Tyr Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 1322DNAArtificialPCR primer 13atggtgtcca cagctcagtt cc
221429DNAArtificialPCR primer 14gcagccaccg tacgccggtt tatttccag
291524DNAArtificialPCR Primer 15cgtacggtgg ctgcaccatc tgtc
241623DNAArtificialPCR Primer 16tcaacactct cccctgttga agc
231722DNAArtificialPCR Primer 17atggcttggg tgtggacctt gc
221837DNAArtificialPCR Primer 18gggcccttgg tcgacgctga ggagacggtg
actgagg 371928DNAArtificialPCR Primer 19gcgtcgacca agggcccatc
ggtcttcc 282026DNAArtificialPCR Primer 20tcatttaccc ggagacaggg
agaggc 262124DNAArtificialPCR Primer 21atagaatgga gctgggtttt cctc
242235DNAArtificialPCR Primer 22gggcccttgg tcgacgctga ggagacggtg
actga 352324DNAArtificialPCR Primer 23atggtcctca tgtccttgct gttc
242434DNAArtificialPCR Primer 24gcagccaccg tacgccgttt tatttccagc
tttg 342523DNAArtificialPCR Primer 25cagatccagt tggtgcagtc tgg
232635DNAArtificialPCR Primer 26caccaactgg atctgtgagg agacggtgac
tgagg 352727DNAArtificialPCR Primer 27aatatccaga tgacacagac tacatcc
272836DNAArtificialPCR Primer 28gtgtcatctg gatattccgt tttatttcca
gctttg 362923DNAArtificialPCR Primer 29tgggggtgtc gttttggctg agg
233036DNAArtificialPCR Primer 30gccaaaacga cacccccaca gatccagttg
gtgcag 363127DNAArtificialPCR Primer 31tggtgcagca tcagcccgtt
ttatttc 273233DNAArtificialPCR Primer 32gctgatgctg caccaaatat
ccagatgaca cag 3333243PRTArtificialChimeric mouse/human VH region
33Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser 1
5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser
Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn
Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp
Lys Ser Ser Ser Thr Ser Tyr 65 70 75 80 Met Gln Leu Ser Gly Leu Thr
Ser Glu Asp Ser Ala Met Tyr Phe Cys 85 90 95 Val Arg Phe Pro Thr
Gly Asn Asp Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr
Ser Val Thr Val Ser Ser Gln Ile Gln Leu Val Gln Ser 115 120 125 Gly
Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys 130 135
140 Ala Ser Gly Tyr Thr Phe Arg Asn Tyr Gly Met Asn Trp Val Lys Gln
145 150 155 160 Ala Pro Gly Lys Asp Leu Lys Arg Met Ala Trp Ile Asn
Thr Tyr Thr 165 170 175 Gly Glu Ser Thr Tyr Ala Asp Asp Phe Lys Gly
Arg Phe Ala Phe Ser 180 185 190 Leu Glu Thr Ser Ala Ser Thr Ala Tyr
Leu Gln Ile Asn Asn Leu Lys 195 200 205 Asn Glu Asp Thr Ala Thr Tyr
Phe Cys Ala Arg Gly Ile Tyr Tyr Tyr 210 215 220 Gly Ser Ser Tyr Ala
Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr 225 230 235 240 Val Ser
Ser 34330PRTHomo sapiensPEPTIDE(1)..(330)Sequence of CH region
34Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1
5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315
320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
35221PRTArtificialChimeric mouse/human VL region 35Asn Ile Val Leu
Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg
Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr 20 25 30
Gly Asn Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35
40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro
Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu
Thr Ile Asp 65 70 75 80 Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr
Cys Gln Gln Asn Asn 85 90 95 Glu Asp Pro Phe Thr Phe Gly Ser Gly
Thr Lys Leu Glu Ile Lys Arg 100 105 110 Asn Ile Gln Met Thr Gln Thr
Thr Ser Ser Leu Ser Ala Ser Leu Gly 115 120 125 Asp Arg Val Thr Ile
Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Cys 130 135 140 Leu Asn Trp
Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 145 150 155 160
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 165
170 175 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu
Gln 180 185 190 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Lys Thr
Leu Pro Tyr 195 200 205 Ala Phe Gly Gly Gly Thr Lys Leu Glu Ile Asn
Arg Arg 210 215 220 36106PRTHomo sapiensPEPTIDE(1)..(106)Sequence
of CL region 36Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80 His Lys Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val
Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105
37249PRTArtificialChimeric mouse/human VH region 37Gln Val Gln Leu
Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30
Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys
Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
Thr Ser Tyr 65 70 75 80 Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser
Ala Met Tyr Phe Cys 85 90 95 Val Arg Phe Pro Thr Gly Asn Asp Tyr
Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr Val
Ser Ser Ala Lys Thr Thr Pro Pro Gln 115 120 125 Ile Gln Leu Val Gln
Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr 130 135 140 Val Lys Ile
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Arg Asn Tyr Gly 145 150 155 160
Met Asn Trp Val Lys Gln Ala Pro Gly Lys Asp Leu Lys Arg Met Ala 165
170 175 Trp Ile Asn Thr Tyr Thr Gly Glu Ser Thr Tyr Ala Asp Asp Phe
Lys 180 185 190 Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr
Ala Tyr Leu 195 200 205 Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala
Thr Tyr Phe Cys Ala 210 215 220 Arg Gly Ile Tyr Tyr Tyr Gly Ser Ser
Tyr Ala Met Asp Tyr Trp Gly 225 230 235 240 Gln Gly Thr Ser Val Thr
Val Ser Ser 245 386PRTArtificialLinker peptide 38Ala Lys Thr Thr
Pro Pro 1 5 39225PRTArtificialChimeric mouse/human VL region 39Asn
Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10
15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr
20 25 30 Gly Asn Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln
Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser
Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp
Phe Thr Leu Thr Ile Asp 65 70 75 80 Pro Val Glu Ala Asp Asp Ala Ala
Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95 Glu Asp Pro Phe Thr Phe
Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Ala Asp Ala Ala
Pro Asn Ile Gln Met Thr Gln Thr Thr Ser Ser Leu 115 120 125 Ser Ala
Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln 130 135 140
Asp Ile Ser Asn Cys Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr 145
150 155 160 Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly
Val Pro 165 170 175 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr
Ser Leu Thr Ile 180 185 190 Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr
Tyr Phe Cys Gln Gln Gly 195 200 205 Lys Thr Leu Pro Tyr Ala Phe Gly
Gly Gly Thr Lys Leu Glu Ile Asn 210 215 220 Arg 225
405PRTArtificialLinker peptide 40Ala Asp Ala Ala Pro 1 5
41246PRTArtificialChimeric mouse/human VH region 41Glu Val Gln Leu
Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val
Lys Leu Ser Cys Thr Ala Ser Gly Leu Asn Ile Lys Asp Thr 20 25 30
Tyr Met His Trp Leu Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35
40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Ala Lys Tyr Asp Pro Arg
Phe 50 55 60 Leu Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn
Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Gly Asn Phe His Phe
Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Gln Val His Leu 115 120 125 Lys Glu Ser Gly Pro
Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Ile 130 135 140 Thr Cys Thr
Val Ser Gly Phe Ser Leu Thr Asp Tyr Gly Val Ser Trp 145 150 155 160
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Leu Ile Trp 165
170 175 Gly Gly Gly Asp Thr Tyr Tyr Asn Ser Pro Leu Lys Ser Arg Leu
Ser 180 185 190 Ile Arg Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys
Met Asn Ser 195 200 205 Leu Gln Thr Asp Asp Thr Ala Val Tyr Tyr Cys
Ala Lys Gln Arg Thr 210 215 220 Leu Trp Gly Tyr Asp Leu Tyr Gly Met
Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Ser Val Thr Val Ser Ser
245 426PRTArtificialLinker peptide 42Ala Ser Thr Lys Gly Pro 1 5
43222PRTArtificialChimeric mouse/human VL region 43Asp Ile Val Met
Thr Gln Ser Gln Arg Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg
Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn 20 25 30
Ile Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Ala Leu Ile 35
40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn
Val Gln Ser 65 70 75 80 Val Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr
Thr Arg Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Glu Thr Thr Val Thr Gln
Ser Pro Ala Ser Leu Ser Met Ala Ile 115 120 125 Gly Glu Lys Val Thr
Ile Arg Cys Ile Thr Ser Thr Asp Ile Asp Val 130 135 140 Asp Met Asn
Trp Tyr Gln Gln Lys Pro Gly Glu Pro Pro Lys Leu Leu 145 150 155 160
Ile Ser Gln Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser 165
170 175 Ser Ser Gly Ser Gly Thr Asp Phe Val Phe Ile Ile Glu Asn Met
Leu 180 185 190 Ser Glu Asp Val Ala Asp Tyr Tyr Cys Leu Gln Ser Asp
Asn Leu Pro 195 200 205 Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
Lys Arg Arg 210 215 220 445PRTArtificialLinker peptide 44Thr Val
Ala Ala Pro 1 5 45246PRTArtificialChimeric mouse/human VH region
45Gln Val His Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1
5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp
Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
Glu Trp Leu 35 40 45 Gly Leu Ile Trp Gly Gly Gly Asp Thr Tyr Tyr
Asn Ser Pro Leu Lys 50 55 60 Ser Arg Leu Ser Ile Arg Lys Asp Asn
Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr
Asp Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Gln Arg Thr Leu
Trp Gly Tyr Asp Leu Tyr Gly Met Asp Tyr Trp 100 105 110 Gly Gln Gly
Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Glu
Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 130 135
140 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Leu Asn Ile Lys Asp Thr
145 150 155 160 Tyr Met His Trp Leu Lys Gln Arg Pro Glu Gln Gly Leu
Glu Trp Ile 165 170 175 Gly Arg Ile Asp Pro Ala Asn Gly Asn Ala Lys
Tyr Asp Pro Arg Phe 180 185 190 Leu Gly Lys Ala Thr Ile Thr Ala Asp
Thr Ser Ser Asn Thr Ala Tyr 195 200 205 Leu Gln Leu Ser Ser Leu Thr
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 210 215 220 Ala Arg Gly Asp Gly
Asn Phe His Phe Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Thr Leu
Thr Val Ser Ser 245 46221PRTArtificialChimeric mouse/human VL
region 46Glu Thr Thr Val Thr Gln Ser Pro Ala Ser Leu Ser Met Ala
Ile Gly 1 5 10 15 Glu Lys Val Thr Ile Arg Cys Ile Thr Ser Thr Asp
Ile Asp Val Asp 20 25 30 Met Asn Trp Tyr Gln Gln Lys Pro Gly Glu
Pro Pro Lys Leu Leu Ile 35 40 45 Ser Gln Gly Asn Thr Leu Arg Pro
Gly Val Pro Ser Arg Phe Ser Ser 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Val Phe Ile Ile Glu Asn Met Leu Ser 65 70 75 80 Glu Asp Val Ala
Asp Tyr Tyr Cys Leu Gln Ser Asp Asn Leu Pro Leu 85 90 95 Thr Phe
Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110
Pro Asp Ile Val Met Thr Gln Ser Gln Arg Phe Met Ser Thr Ser Val 115
120 125 Gly Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly
Thr 130 135 140 Asn Ile Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro
Arg Ala Leu 145 150 155 160 Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly
Val Pro Asp Arg Phe Thr 165 170 175 Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Asn Val Gln 180 185 190 Ser Val Asp Leu Ala Glu
Tyr Phe Cys Gln Gln Tyr Thr Arg Tyr Pro 195 200 205 Leu Thr Phe Gly
Gly Gly Thr Lys Leu Glu Ile Lys Arg 210 215 220
47253PRTArtificialChimeric mouse/human VH region 47Glu Val Gln Leu
Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val
Lys Leu Ser Cys Thr Ala Ser Gly Leu Asn Ile Lys Asp Thr 20 25 30
Tyr Met His Trp Leu Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35
40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Ala Lys Tyr Asp Pro Arg
Phe 50 55 60 Leu Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn
Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Gly Asn Phe His Phe
Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Gln Val
His Leu Lys Glu Ser Gly Pro Gly Leu Val Ala 130 135 140 Pro Ser Gln
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu 145 150 155 160
Thr Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu 165
170 175 Glu Trp Leu Gly Leu Ile Trp Gly Gly Gly Asp Thr Tyr Tyr Asn
Ser 180 185 190 Pro Leu Lys Ser Arg Leu Ser Ile Arg Lys Asp Asn Ser
Lys Ser Gln 195 200 205 Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp
Asp Thr Ala Val Tyr 210 215 220 Tyr Cys Ala Lys Gln Arg Thr Leu Trp
Gly Tyr Asp Leu Tyr Gly Met 225 230 235 240 Asp Tyr Trp Gly Gln Gly
Thr Ser Val Thr Val Ser Ser 245 250 4813PRTArtificialLinker peptide
48Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 1 5 10
49228PRTArtificialChimeric mouse/human VL region 49Asp Ile Val Met
Thr Gln Ser Gln Arg Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg
Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn 20 25 30
Ile Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Ala Leu Ile 35
40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn
Val Gln Ser 65 70 75 80 Val Asp Leu Ala Glu Tyr Phe Cys Gln Gln
Tyr
Thr Arg Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro
Pro Glu Thr Thr Val Thr Gln Ser Pro 115 120 125 Ala Ser Leu Ser Met
Ala Ile Gly Glu Lys Val Thr Ile Arg Cys Ile 130 135 140 Thr Ser Thr
Asp Ile Asp Val Asp Met Asn Trp Tyr Gln Gln Lys Pro 145 150 155 160
Gly Glu Pro Pro Lys Leu Leu Ile Ser Gln Gly Asn Thr Leu Arg Pro 165
170 175 Gly Val Pro Ser Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe
Val 180 185 190 Phe Ile Ile Glu Asn Met Leu Ser Glu Asp Val Ala Asp
Tyr Tyr Cys 195 200 205 Leu Gln Ser Asp Asn Leu Pro Leu Thr Phe Gly
Ala Gly Thr Lys Leu 210 215 220 Glu Leu Lys Arg 225
5012PRTArtificialLinker peptide 50Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro 1 5 10 51253PRTArtificialChimeric mouse/human VH
region 51Gln Val His Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro
Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser
Leu Thr Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly
Lys Gly Leu Glu Trp Leu 35 40 45 Gly Leu Ile Trp Gly Gly Gly Asp
Thr Tyr Tyr Asn Ser Pro Leu Lys 50 55 60 Ser Arg Leu Ser Ile Arg
Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser
Leu Gln Thr Asp Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Gln
Arg Thr Leu Trp Gly Tyr Asp Leu Tyr Gly Met Asp Tyr Trp 100 105 110
Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115
120 125 Ser Val Phe Pro Leu Ala Pro Glu Val Gln Leu Gln Gln Ser Gly
Ala 130 135 140 Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys
Thr Ala Ser 145 150 155 160 Gly Leu Asn Ile Lys Asp Thr Tyr Met His
Trp Leu Lys Gln Arg Pro 165 170 175 Glu Gln Gly Leu Glu Trp Ile Gly
Arg Ile Asp Pro Ala Asn Gly Asn 180 185 190 Ala Lys Tyr Asp Pro Arg
Phe Leu Gly Lys Ala Thr Ile Thr Ala Asp 195 200 205 Thr Ser Ser Asn
Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu 210 215 220 Asp Thr
Ala Val Tyr Tyr Cys Ala Arg Gly Asp Gly Asn Phe His Phe 225 230 235
240 Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 245 250
52228PRTArtificialChimeric mouse/human VL region 52Glu Thr Thr Val
Thr Gln Ser Pro Ala Ser Leu Ser Met Ala Ile Gly 1 5 10 15 Glu Lys
Val Thr Ile Arg Cys Ile Thr Ser Thr Asp Ile Asp Val Asp 20 25 30
Met Asn Trp Tyr Gln Gln Lys Pro Gly Glu Pro Pro Lys Leu Leu Ile 35
40 45 Ser Gln Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser
Ser 50 55 60 Ser Gly Ser Gly Thr Asp Phe Val Phe Ile Ile Glu Asn
Met Leu Ser 65 70 75 80 Glu Asp Val Ala Asp Tyr Tyr Cys Leu Gln Ser
Asp Asn Leu Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu
Leu Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro
Pro Asp Ile Val Met Thr Gln Ser Gln 115 120 125 Arg Phe Met Ser Thr
Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys 130 135 140 Ala Ser Gln
Asn Val Gly Thr Asn Ile Ala Trp Tyr Gln Gln Lys Pro 145 150 155 160
Gly Gln Ser Pro Arg Ala Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser 165
170 175 Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe
Thr 180 185 190 Leu Thr Ile Ser Asn Val Gln Ser Val Asp Leu Ala Glu
Tyr Phe Cys 195 200 205 Gln Gln Tyr Thr Arg Tyr Pro Leu Thr Phe Gly
Gly Gly Thr Lys Leu 210 215 220 Glu Ile Lys Arg 225
53238PRTArtificialChimeric mouse/human VH region 53Gln Val Gln Leu
Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val
Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30
Trp Met Asn Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35
40 45 Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr Leu Tyr Ser Gln Lys
Phe 50 55 60 Lys Asp Thr Ala Ile Leu Thr Val Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Gly Phe Asp Tyr Trp Gly
Gln Gly Thr Thr Leu Thr Val 100 105 110 Ser Ser Ala Ser Thr Lys Gly
Pro Glu Val Gln Leu Gln Gln Ser Gly 115 120 125 Pro Glu Leu Val Lys
Thr Gly Thr Ser Val Lys Ile Ser Cys Lys Ala 130 135 140 Ser Gly Tyr
Ser Phe Thr Gly Tyr Tyr Met His Trp Val Arg Gln Ser 145 150 155 160
His Gly Lys Ser Leu Glu Trp Ile Gly Tyr Ile Ser Cys Tyr Asn Gly 165
170 175 Phe Thr Ser Tyr Asn Pro Lys Phe Lys Gly Lys Ala Thr Phe Thr
Val 180 185 190 Asp Thr Ser Ser Ser Thr Ala Tyr Ile Gln Phe Ser Arg
Leu Thr Ser 195 200 205 Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser
Asp Tyr Tyr Gly Thr 210 215 220 Asn Asp Tyr Trp Gly Gln Gly Thr Thr
Leu Thr Val Ser Ser 225 230 235 54219PRTArtificialChimeric
mouse/human VL region 54Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met
Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala
Ser Ser Ser Val Asn Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro
Arg Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Leu Thr Ser Asn Leu
Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly
Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp
Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Ser Asn Pro Tyr Thr 85 90
95 Phe Gly Gly Gly Thr Lys Leu Glu Met Lys Arg Thr Val Ala Ala Pro
100 105 110 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser
Pro Gly 115 120 125 Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser
Val Ser Tyr Met 130 135 140 His Trp Phe Gln Gln Lys Pro Gly Ala Ser
Pro Lys Leu Trp Ile Tyr 145 150 155 160 Ser Thr Ser Asn Leu Ala Ser
Gly Val Pro Ala Arg Phe Ser Gly Ser 165 170 175 Gly Ser Gly Thr Ser
Tyr Ser Leu Thr Val Ser Arg Met Glu Ala Glu 180 185 190 Asp Ala Ala
Thr Tyr Tyr Cys Gln Gln Arg Ser Thr Tyr Pro Tyr Thr 195 200 205 Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 210 215
55238PRTArtificialChimeric mouse/human VH region 55Glu Val Gln Leu
Gln Gln Ser Gly Pro Glu Leu Val Lys Thr Gly Thr 1 5 10 15 Ser Val
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30
Tyr Met His Trp Val Arg Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35
40 45 Gly Tyr Ile Ser Cys Tyr Asn Gly Phe Thr Ser Tyr Asn Pro Lys
Phe 50 55 60 Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Ile Gln Phe Ser Arg Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Tyr Tyr Gly Thr Asn
Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Gln Val Gln Leu 115 120 125 Gln Gln Pro Gly Ala
Glu Leu Val Arg Pro Gly Ala Ser Val Lys Leu 130 135 140 Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Thr Tyr Trp Met Asn Trp 145 150 155 160
Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp 165
170 175 Pro Tyr Asp Ser Glu Thr Leu Tyr Ser Gln Lys Phe Lys Asp Thr
Ala 180 185 190 Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met
Gln Leu Ser 195 200 205 Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr
Cys Ala Arg Tyr Gly 210 215 220 Phe Asp Tyr Trp Gly Gln Gly Thr Thr
Leu Thr Val Ser Ser 225 230 235 56219PRTArtificialChimeric
mouse/human VL region 56Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met
Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala
Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Phe Gln Gln Lys Pro
Gly Ala Ser Pro Lys Leu Trp Ile Tyr 35 40 45 Ser Thr Ser Asn Leu
Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly
Thr Ser Tyr Ser Leu Thr Val Ser Arg Met Glu Ala Glu 65 70 75 80 Asp
Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Thr Tyr Pro Tyr Thr 85 90
95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
100 105 110 Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser
Pro Gly 115 120 125 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser
Val Asn Tyr Met 130 135 140 Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser
Pro Lys Pro Trp Ile Tyr 145 150 155 160 Leu Thr Ser Asn Leu Ala Ser
Gly Val Pro Ala Arg Phe Ser Gly Ser 165 170 175 Gly Ser Gly Thr Ser
Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 180 185 190 Asp Ala Ala
Thr Tyr Tyr Cys Gln Gln Trp Asn Ser Asn Pro Tyr Thr 195 200 205 Phe
Gly Gly Gly Thr Lys Leu Glu Met Lys Arg 210 215
57245PRTArtificialChimeric mouse/human VH region 57Gln Val Gln Leu
Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val
Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30
Trp Met Asn Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35
40 45 Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr Leu Tyr Ser Gln Lys
Phe 50 55 60 Lys Asp Thr Ala Ile Leu Thr Val Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Gly Phe Asp Tyr Trp Gly
Gln Gly Thr Thr Leu Thr Val 100 105 110 Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Glu 115 120 125 Val Gln Leu Gln Gln
Ser Gly Pro Glu Leu Val Lys Thr Gly Thr Ser 130 135 140 Val Lys Ile
Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Tyr 145 150 155 160
Met His Trp Val Arg Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly 165
170 175 Tyr Ile Ser Cys Tyr Asn Gly Phe Thr Ser Tyr Asn Pro Lys Phe
Lys 180 185 190 Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Ser Thr
Ala Tyr Ile 195 200 205 Gln Phe Ser Arg Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys Ala 210 215 220 Arg Ser Asp Tyr Tyr Gly Thr Asn Asp
Tyr Trp Gly Gln Gly Thr Thr 225 230 235 240 Leu Thr Val Ser Ser 245
58227PRTArtificialChimeric mouse/human VL region 58Gln Ile Val Leu
Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys
Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met 20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr 35
40 45 Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly
Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met
Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn
Ser Asn Pro Tyr Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Met
Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro
Gln Ile Val Leu Thr Gln Ser Pro Ala 115 120 125 Ile Met Ser Ala Ser
Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Ala 130 135 140 Ser Ser Ser
Val Ser Tyr Met His Trp Phe Gln Gln Lys Pro Gly Ala 145 150 155 160
Ser Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val 165
170 175 Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu
Thr 180 185 190 Val Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr
Cys Gln Gln 195 200 205 Arg Ser Thr Tyr Pro Tyr Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile 210 215 220 Lys Arg Arg 225
59245PRTArtificialChimeric mouse/human VH region 59Glu Val Gln Leu
Gln Gln Ser Gly Pro Glu Leu Val Lys Thr Gly Thr 1 5 10 15 Ser Val
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30
Tyr Met His Trp Val Arg Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35
40 45 Gly Tyr Ile Ser Cys Tyr Asn Gly Phe Thr Ser Tyr Asn Pro Lys
Phe 50 55 60 Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Ile Gln Phe Ser Arg Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Tyr Tyr Gly Thr Asn
Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Gln Val
Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg 130 135 140 Pro Gly Ala
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe 145 150 155 160
Thr Thr Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Glu Gln Gly Leu 165
170 175 Glu Trp Ile Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr Leu Tyr
Ser 180 185 190 Gln Lys Phe Lys Asp Thr Ala Ile Leu Thr Val Asp Lys
Ser Ser Ser 195 200 205 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser
Glu Asp
Ser Ala Val 210 215 220 Tyr Tyr Cys Ala Arg Tyr Gly Phe Asp Tyr Trp
Gly Gln Gly Thr Thr 225 230 235 240 Leu Thr Val Ser Ser 245
60227PRTArtificialChimeric mouse/human VL region 60Gln Ile Val Leu
Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys
Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30
His Trp Phe Gln Gln Lys Pro Gly Ala Ser Pro Lys Leu Trp Ile Tyr 35
40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly
Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Val Ser Arg Met
Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser
Thr Tyr Pro Tyr Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro
Gln Ile Val Leu Thr Gln Ser Pro Ala 115 120 125 Leu Met Ser Ala Ser
Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala 130 135 140 Ser Ser Ser
Val Asn Tyr Met Tyr Trp Tyr Gln Gln Lys Pro Arg Ser 145 150 155 160
Ser Pro Lys Pro Trp Ile Tyr Leu Thr Ser Asn Leu Ala Ser Gly Val 165
170 175 Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu
Thr 180 185 190 Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr
Cys Gln Gln 195 200 205 Trp Asn Ser Asn Pro Tyr Thr Phe Gly Gly Gly
Thr Lys Leu Glu Met 210 215 220 Lys Arg Arg 225
6162DNAArtificialPCR Primer 61tagagatccc tcgacctcga gatccattgt
gcccgggcgc caccatggag tttgggctga 60gc 626245DNAArtificialPCR Primer
62cacctctggg cccttggtcg acgctgaaga gacggtgacc attgt
456360DNAArtificialPCR Primer 63gggtgccagg gggaagaccg atgggccctt
ggtcgacgct gaagagacgg tgaccattgt 606445DNAArtificialPCR Primer
64tcttcagcgt cgaccaaggg cccagaggtg cagctggtgc agtct
456560DNAArtificialPCR Primer 65gcgtcgacca agggcccatc ggtcttcccc
ctggcacccg aggtgcagct ggtgcagtct 606621DNAArtificialPCR Primer
66gtagtccttg accaggcagc c 216762DNAArtificialPCR Primer
67tagagatccc tcgacctcga gatccattgt gcccgggcgc caccatgact tggaccccac
60tc 626845DNAArtificialPCR Primer 68tatttcgggg gcagccttgg
gctgacctag tactgtgacc ttggt 456960DNAArtificialPCR Primer
69gggcgggaac agagtgaccg agggggcagc cttgggctga cctagtactg tgaccttggt
607045DNAArtificialPCR Primer 70ctaggtcagc ccaaggctgc ccccgaaata
gtgatgacgc agtct 457160DNAArtificialPCR Primer 71cagcccaagg
ctgccccctc ggtcactctg ttcccgcccg aaatagtgat gacgcagtct
607259DNAArtificialPCR Primer 72gtcccaggtg gggaccctca ctctagagtc
gcggccgcct aacactctcc cctgttgaa 597345DNAArtificialPCR Primer
73cacctgtggg cccttggtcg acgctgaaga gacggtgacc attgt
457460DNAArtificialPCR Primer 74gggtgccagg gggaagaccg atgggccctt
ggtcgacgct gaagagacgg tgaccattgt 607545DNAArtificialPCR Primer
75tcttcagcgt cgaccaaggg cccacaggtg cagctggtgg agtct
457660DNAArtificialPCR Primer 76gcgtcgacca agggcccatc ggtcttcccc
ctggcacccc aggtgcagct ggtggagtct 607765DNAArtificialPCR Primer
77tagagatccc tcgacctcga gatccattgt gcccgggcgc caccatggaa gccccagcgc
60agctt 657842DNAArtificialPCR Primer 78agactgtggt gcagccacag
ttcgtttaat ctccagtcgt gt 427957DNAArtificialPCR Primer 79tggcgggaag
atgaagacag atggtgcagc cacagttcgt ttaatctcca gtcgtgt
578042DNAArtificialPCR Primer 80aaacgaactg tggctgcacc acagtctgtg
ctgactcagc cc 428157DNAArtificialPCR Primer 81actgtggctg caccatctgt
cttcatcttc ccgccacagt ctgtgctgac tcagccc 578259DNAArtificialPCR
Primer 82gtcccaggtg gggaccctca ctctagagtc gcggccgctc atgaacattc
tgtaggggc 5983242PRTArtificialantibody VH region 83Gln Val Gln Leu
Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Lys Thr His Gly Ser His Asp Asn Trp
Gly Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys
Gly Pro Glu Val Gln Leu Val Gln Ser 115 120 125 Gly Thr Glu Val Lys
Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys 130 135 140 Gly Ser Gly
Tyr Thr Val Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln 145 150 155 160
Met Pro Gly Lys Gly Leu Glu Trp Met Gly Phe Ile Tyr Pro Gly Asp 165
170 175 Ser Glu Thr Arg Tyr Ser Pro Thr Phe Gln Gly Gln Val Thr Ile
Ser 180 185 190 Ala Asp Lys Ser Phe Asn Thr Ala Phe Leu Gln Trp Ser
Ser Leu Lys 195 200 205 Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg
Val Gly Ser Gly Trp 210 215 220 Tyr Pro Tyr Thr Phe Asp Ile Trp Gly
Gln Gly Thr Met Val Thr Val 225 230 235 240 Ser Ser 84115PRTHomo
sapiensPEPTIDE(1)..(115)Sequence of ABT-874 VH region 84Gln Val Gln
Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Lys Thr His Gly Ser His Asp Asn
Trp Gly Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser 115
85121PRTHomo sapiensPEPTIDE(1)..(121)Sequence of ABT-325 VH region
85Glu Val Gln Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Glu 1
5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Val Thr Ser
Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu
Glu Trp Met 35 40 45 Gly Phe Ile Tyr Pro Gly Asp Ser Glu Thr Arg
Tyr Ser Pro Thr Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp
Lys Ser Phe Asn Thr Ala Phe 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys
Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Val Gly Ser
Gly Trp Tyr Pro Tyr Thr Phe Asp Ile Trp Gly 100 105 110 Gln Gly Thr
Met Val Thr Val Ser Ser 115 120 86247PRTArtificialantibody VL
region 86Met Thr Trp Thr Pro Leu Leu Phe Leu Thr Leu Leu Leu His
Cys Thr 1 5 10 15 Gly Ser Leu Ser Gln Ser Val Leu Thr Gln Pro Pro
Ser Val Ser Gly 20 25 30 Ala Pro Gly Gln Arg Val Thr Ile Ser Cys
Ser Gly Ser Arg Ser Asn 35 40 45 Ile Gly Ser Asn Thr Val Lys Trp
Tyr Gln Gln Leu Pro Gly Thr Ala 50 55 60 Pro Lys Leu Leu Ile Tyr
Tyr Asn Asp Gln Arg Pro Ser Gly Val Pro 65 70 75 80 Asp Arg Phe Ser
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile 85 90 95 Thr Gly
Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr 100 105 110
Asp Arg Tyr Thr His Pro Ala Leu Leu Phe Gly Thr Gly Thr Lys Val 115
120 125 Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Glu Ile Val Met Thr
Gln 130 135 140 Ser Pro Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala
Thr Leu Ser 145 150 155 160 Cys Arg Ala Ser Glu Ser Ile Ser Ser Asn
Leu Ala Trp Tyr Gln Gln 165 170 175 Lys Pro Gly Gln Ala Pro Arg Leu
Phe Ile Tyr Thr Ala Ser Thr Arg 180 185 190 Ala Thr Asp Ile Pro Ala
Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu 195 200 205 Phe Thr Leu Thr
Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr 210 215 220 Tyr Cys
Gln Gln Tyr Asn Asn Trp Pro Ser Ile Thr Phe Gly Gln Gly 225 230 235
240 Thr Arg Leu Glu Ile Lys Arg 245 87112PRTHomo
sapiensPEPTIDE(1)..(112)Sequence of ABT-874 VL region 87Gln Ser Val
Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg
Val Thr Ile Ser Cys Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn 20 25
30 Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45 Ile Tyr Tyr Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg
Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile
Thr Gly Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln
Ser Tyr Asp Arg Tyr Thr 85 90 95 His Pro Ala Leu Leu Phe Gly Thr
Gly Thr Lys Val Thr Val Leu Gly 100 105 110 886PRTArtificiallinker
region 88Gln Pro Lys Ala Ala Pro 1 5 89109PRTHomo
sapiensPEPTIDE(1)..(109)Sequence of ABT-325 VL region 89Glu Ile Val
Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Ile Ser Ser Asn 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Phe Ile
35 40 45 Tyr Thr Ala Ser Thr Arg Ala Thr Asp Ile Pro Ala Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Tyr Asn Asn Trp Pro Ser 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg
Leu Glu Ile Lys Arg 100 105 90249PRTArtificialantibody VH region
90Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Lys Thr His Gly Ser
His Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Glu
Val Gln Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Glu 130 135
140 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Val Thr Ser Tyr
145 150 155 160 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu
Glu Trp Met 165 170 175 Gly Phe Ile Tyr Pro Gly Asp Ser Glu Thr Arg
Tyr Ser Pro Thr Phe 180 185 190 Gln Gly Gln Val Thr Ile Ser Ala Asp
Lys Ser Phe Asn Thr Ala Phe 195 200 205 Leu Gln Trp Ser Ser Leu Lys
Ala Ser Asp Thr Ala Met Tyr Tyr Cys 210 215 220 Ala Arg Val Gly Ser
Gly Trp Tyr Pro Tyr Thr Phe Asp Ile Trp Gly 225 230 235 240 Gln Gly
Thr Met Val Thr Val Ser Ser 245 91234PRTArtificialantibody VL
region 91Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro
Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Arg Ser Asn
Ile Gly Ser Asn 20 25 30 Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly
Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Tyr Asn Asp Gln Arg Pro
Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr
Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln 65 70 75 80 Ala Glu Asp Glu
Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg Tyr Thr 85 90 95 His Pro
Ala Leu Leu Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110
Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Glu Ile Val 115
120 125 Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly Glu Arg
Ala 130 135 140 Thr Leu Ser Cys Arg Ala Ser Glu Ser Ile Ser Ser Asn
Leu Ala Trp 145 150 155 160 Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Phe Ile Tyr Thr Ala 165 170 175 Ser Thr Arg Ala Thr Asp Ile Pro
Ala Arg Phe Ser Gly Ser Gly Ser 180 185 190 Gly Thr Glu Phe Thr Leu
Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe 195 200 205 Ala Val Tyr Tyr
Cys Gln Gln Tyr Asn Asn Trp Pro Ser Ile Thr Phe 210 215 220 Gly Gln
Gly Thr Arg Leu Glu Ile Lys Arg 225 230 9213PRTArtificiallinker
region 92Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro 1 5 10
93242PRTArtificialantibody VH region 93Glu Val Gln Leu Val Gln Ser
Gly Thr Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser
Cys Lys Gly Ser Gly Tyr Thr Val Thr Ser Tyr 20 25 30 Trp Ile Gly
Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly
Phe Ile Tyr Pro Gly Asp Ser Glu Thr Arg Tyr Ser Pro Thr Phe 50 55
60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Phe Asn Thr Ala Phe
65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr
Tyr Cys 85 90 95 Ala Arg Val Gly Ser Gly Trp Tyr Pro Tyr Thr Phe
Asp Ile Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Gln 115 120 125 Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg Ser 130 135 140 Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly 145 150 155 160 Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 165 170 175 Phe
Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 180
185 190 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
Leu 195 200 205 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Lys 210 215 220 Thr His Gly Ser His Asp Asn Trp Gly Gln Gly
Thr Met Val Thr Val 225 230 235 240 Ser Ser
94226PRTArtificialantibody VL region 94Glu Ile Val Met Thr Gln Ser
Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Glu Ser Ile Ser Ser Asn 20 25 30 Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Phe Ile 35 40 45 Tyr
Thr Ala Ser Thr Arg Ala Thr Asp Ile Pro Ala Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp
Pro Ser 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
Arg Thr Val Ala 100 105 110 Ala Pro Gln Ser Val Leu Thr Gln Pro Pro
Ser Val Ser Gly Ala Pro 115 120 125 Gly Gln Arg Val Thr Ile Ser Cys
Ser Gly Ser Arg Ser Asn Ile Gly 130 135 140 Ser Asn Thr Val Lys Trp
Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys 145 150 155 160 Leu Leu Ile
Tyr Tyr Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg 165 170 175 Phe
Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly 180 185
190 Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg
195 200 205 Tyr Thr His Pro Ala Leu Leu Phe Gly Thr Gly Thr Lys Val
Thr Val 210 215 220 Leu Gly 225 95249PRTArtificialantibody VH
region 95Glu Val Gln Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro
Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr
Val Thr Ser Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly
Lys Gly Leu Glu Trp Met 35 40 45 Gly Phe Ile Tyr Pro Gly Asp Ser
Glu Thr Arg Tyr Ser Pro Thr Phe 50 55 60 Gln Gly Gln Val Thr Ile
Ser Ala Asp Lys Ser Phe Asn Thr Ala Phe 65 70 75 80 Leu Gln Trp Ser
Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg
Val Gly Ser Gly Trp Tyr Pro Tyr Thr Phe Asp Ile Trp Gly 100 105 110
Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115
120 125 Val Phe Pro Leu Ala Pro Gln Val Gln Leu Val Glu Ser Gly Gly
Gly 130 135 140 Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly 145 150 155 160 Phe Thr Phe Ser Ser Tyr Gly Met His Trp
Val Arg Gln Ala Pro Gly 165 170 175 Lys Gly Leu Glu Trp Val Ala Phe
Ile Arg Tyr Asp Gly Ser Asn Lys 180 185 190 Tyr Tyr Ala Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 195 200 205 Ser Lys Asn Thr
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 210 215 220 Thr Ala
Val Tyr Tyr Cys Lys Thr His Gly Ser His Asp Asn Trp Gly 225 230 235
240 Gln Gly Thr Met Val Thr Val Ser Ser 245
96233PRTArtificialantibody VL region 96Glu Ile Val Met Thr Gln Ser
Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Glu Ser Ile Ser Ser Asn 20 25 30 Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Phe Ile 35 40 45 Tyr
Thr Ala Ser Thr Arg Ala Thr Asp Ile Pro Ala Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp
Pro Ser 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Gln
Ser Val Leu Thr Gln Pro 115 120 125 Pro Ser Val Ser Gly Ala Pro Gly
Gln Arg Val Thr Ile Ser Cys Ser 130 135 140 Gly Ser Arg Ser Asn Ile
Gly Ser Asn Thr Val Lys Trp Tyr Gln Gln 145 150 155 160 Leu Pro Gly
Thr Ala Pro Lys Leu Leu Ile Tyr Tyr Asn Asp Gln Arg 165 170 175 Pro
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser 180 185
190 Ala Ser Leu Ala Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr
195 200 205 Tyr Cys Gln Ser Tyr Asp Arg Tyr Thr His Pro Ala Leu Leu
Phe Gly 210 215 220 Thr Gly Thr Lys Val Thr Val Leu Gly 225 230
97254PRTArtificialantibody VH region 97Gln Val Gln Leu Arg Gln Pro
Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Met His
Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly
Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55
60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Ser His Tyr Gly Ser Asn Tyr Val Asp Tyr
Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
Ala Lys Thr Thr Ala Pro 115 120 125 Ser Val Tyr Pro Leu Ala Pro Gln
Val Gln Leu Gln Gln Ser Gly Ala 130 135 140 Glu Leu Ala Arg Pro Gly
Ala Ser Val Lys Met Ser Cys Lys Ala Ser 145 150 155 160 Gly Tyr Thr
Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro 165 170 175 Gly
Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr 180 185
190 Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp
195 200 205 Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr
Ser Glu 210 215 220 Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp
Asp His Tyr Cys 225 230 235 240 Leu Asp Tyr Trp Gly Gln Gly Thr Thr
Leu Thr Val Ser Ser 245 250 98122PRTHomo
sapiensPEPTIDE(1)..(122)Sequence of 5F1 VH region 98Gln Val Gln Leu
Arg Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val
Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30
Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys
Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser His Tyr Gly Ser Asn Tyr
Val Asp Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Leu Thr
Val Ser Ser 115 120 9913PRTArtificiallinker region 99Ala Lys Thr
Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro 1 5 10 100119PRTHomo
sapiensPEPTIDE(1)..(119)Sequence of OKT3 VH region 100Gln Val Gln
Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser
Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25
30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln
Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser
Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr
Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser
Ser 115 101226PRTArtificialantibody VL region 101Gln Ile Val Leu
Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys
Val Thr Met Thr Cys Arg Ala Ser Ser Ser Leu Ser Phe Met 20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35
40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly
Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val
Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Phe Cys His Gln Trp Ser
Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu
Lys Arg Ala Asp Ala Ala Pro 100 105 110 Thr Val Ser Ile Phe Pro Pro
Gln Ile Val Leu Thr Gln Ser Pro Ala 115 120 125 Ile Met Ser Ala Ser
Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala 130 135 140 Ser Ser Ser
Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr 145 150 155 160
Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val 165
170 175 Pro Ala His Phe Arg Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu
Thr 180 185 190 Ile Ser Gly Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr
Cys Gln Gln 195 200 205 Trp Ser Ser Asn Pro Phe Thr Phe Gly Ser Gly
Thr Lys Leu Glu Ile 210 215 220 Asn Arg 225 102107PRTHomo
sapiensPEPTIDE(1)..(107)Sequence of 5F1 VL region 102Gln Ile Val
Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu
Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Leu Ser Phe Met 20 25
30 His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser
Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg
Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Phe Cys His Gln Trp
Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu
Leu Lys Arg 100 105 10312PRTArtificiallinker region 103Ala Asp Ala
Ala Pro Thr Val Ser Ile Phe Pro Pro 1 5 10 104107PRTHomo
sapiensPEPTIDE(1)..(107)Sequence of OKT3 VL region 104Gln Ile Val
Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu
Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25
30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg
Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly
Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
Ser Ser Asn Pro Phe Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu
Ile Asn Arg 100 105 105249PRTArtificialantibody VH region 105Glu
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr 1 5 10
15 Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45 Gly Tyr Ile Ile Pro Tyr Asn Asp Asn Thr Lys Tyr
Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys
Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser
Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asn Glu Tyr
Tyr Gly Ser Ser Phe Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr
Leu Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser 115 120 125 Val Tyr
Pro Leu Ala Pro Gln Val Ile Leu Lys Glu Ser Gly Pro Gly 130 135 140
Ile Leu Gln Pro Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly 145
150 155 160 Phe Ser Leu Ser Thr Tyr Gly Thr Ala Val Asn Trp Ile Arg
Gln Pro 165 170 175 Ser Gly Lys Gly Leu Glu Trp Leu Ala Gln Ile Gly
Ser Asp Asp Arg 180 185 190 Lys Leu Tyr Asn Pro Phe Leu Lys Ser Arg
Ile Thr Leu Ser Glu Asp 195 200 205 Thr Ser Asn Ser Gln Val Phe Leu
Lys Ile Thr Ser Val Asp Thr Glu 210 215 220 Asp Ser Ala Thr Tyr Tyr
Cys Ala Asn Gly Val Met Glu Tyr Trp Gly 225 230 235 240 Leu Gly Thr
Ser Val Thr Val Ser Ser 245 106121PRTHomo
sapiensPEPTIDE(1)..(121)Sequence of 10G11 VH region 106Glu Val Gln
Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr 1 5 10 15 Ser
Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25
30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 Gly Tyr Ile Ile Pro Tyr Asn Asp Asn Thr Lys Tyr Asn Glu
Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser
Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asn Glu Tyr Tyr Gly
Ser Ser Phe Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Leu Thr
Val Ser Ser 115 120 107115PRTHomo sapiensPEPTIDE(1)..(115)Sequence
of 9H10 VH region 107Gln Val Ile Leu Lys Glu Ser Gly Pro Gly Ile
Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser
Gly Phe Ser Leu Ser Thr Tyr 20 25 30 Gly Thr Ala Val Asn Trp Ile
Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala Gln Ile
Gly Ser Asp Asp Arg Lys Leu Tyr Asn Pro Phe 50 55 60 Leu Lys Ser
Arg Ile Thr Leu Ser Glu Asp Thr Ser Asn Ser Gln Val 65 70 75 80 Phe
Leu Lys Ile Thr Ser Val Asp Thr Glu Asp Ser Ala Thr Tyr Tyr 85 90
95 Cys Ala Asn Gly Val Met Glu Tyr Trp Gly Leu Gly Thr Ser Val Thr
100 105
110 Val Ser Ser 115 108330PRTMus musculusPEPTIDE(1)..(320)Sequence
of CH region 108Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro
Val Cys Gly 1 5 10 15 Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys
Leu Val Lys Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Leu Thr Trp
Asn Ser Gly Ser Leu Ser Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Asp Leu Tyr Thr Leu 50 55 60 Ser Ser Ser Val Thr
Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile 65 70 75 80 Thr Cys Asn
Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 85 90 95 Ile
Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys 100 105
110 Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro
115 120 125 Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val
Thr Cys 130 135 140 Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val
Gln Ile Ser Trp 145 150 155 160 Phe Val Asn Asn Val Glu Val His Thr
Ala Gln Thr Gln Thr His Arg 165 170 175 Glu Asp Tyr Asn Ser Thr Leu
Arg Val Val Ser Ala Leu Pro Ile Gln 180 185 190 His Gln Asp Trp Met
Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn 195 200 205 Lys Asp Leu
Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly 210 215 220 Ser
Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu 225 230
235 240 Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe
Met 245 250 255 Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys
Thr Glu Leu 260 265 270 Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser
Asp Gly Ser Tyr Phe 275 280 285 Met Tyr Ser Lys Leu Arg Val Glu Lys
Lys Asn Trp Val Glu Arg Asn 290 295 300 Ser Tyr Ser Cys Ser Val Val
His Glu Gly Leu His Asn His His Thr 305 310 315 320 Thr Lys Ser Phe
Ser Arg Thr Pro Gly Lys 325 330 109228PRTArtificialantibody VL
region 109Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Gly Ser Gly Ile
Leu His Asn Tyr 20 25 30 Leu Val Trp Tyr Gln Gln Lys Gln Gly Lys
Ser Pro Gln Leu Leu Val 35 40 45 Tyr Ser Ala Lys Ile Leu Ala Asp
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln
Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly
Ser Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Phe 85 90 95 Thr Phe
Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110
Pro Thr Val Ser Ile Phe Pro Pro Ser Ile Val Met Thr Gln Thr Pro 115
120 125 Lys Phe Leu Leu Val Ser Ala Gly Asp Arg Val Thr Ile Thr Cys
Lys 130 135 140 Ala Ser Gln Ser Val Asn His Asp Val Ala Trp Tyr Gln
Gln Met Pro 145 150 155 160 Gly Gln Ser Pro Lys Leu Leu Ile Tyr Phe
Ala Ser Asn Arg Tyr Thr 165 170 175 Gly Val Pro Asp Arg Phe Thr Gly
Ser Gly Tyr Gly Thr Asp Phe Thr 180 185 190 Phe Thr Ile Ser Thr Val
Gln Ala Glu Asp Leu Ala Val Tyr Phe Cys 195 200 205 Gln Gln Asp Tyr
Ser Ser Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu 210 215 220 Glu Ile
Lys Arg 225 110108PRTHomo sapiensPEPTIDE(1)..(108)Sequence of 10G11
VL region 110Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Gly Ser Gly
Ile Leu His Asn Tyr 20 25 30 Leu Val Trp Tyr Gln Gln Lys Gln Gly
Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Ser Ala Lys Ile Leu Ala
Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe
Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Phe 85 90 95 Thr
Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105
11112PRTArtificiallinker region 111Ala Asp Ala Ala Pro Thr Val Ser
Ile Phe Pro Pro 1 5 10 112108PRTHomo
sapiensPEPTIDE(1)..(108)Sequence of 9H10 VL region 112Ser Ile Val
Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Asn His Asp 20 25
30 Val Ala Trp Tyr Gln Gln Met Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45 Tyr Phe Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe
Thr Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser
Thr Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln
Asp Tyr Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys Arg 100 105 113106PRTMus
musculusPEPTIDE(1)..(106)Sequence of CL region 113Ala Asp Ala Ala
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln 1 5 10 15 Leu Thr
Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr 20 25 30
Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln 35
40 45 Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
Thr 50 55 60 Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu
Tyr Glu Arg 65 70 75 80 His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys
Thr Ser Thr Ser Pro 85 90 95 Ile Val Lys Ser Phe Asn Arg Asn Glu
Cys 100 105 114246PRTArtificialantibody VH region 114Glu Val Thr
Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 10 15 Thr
Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Lys Ser 20 25
30 Val Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu
35 40 45 Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Tyr Tyr Asn
Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser
Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val
Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Arg Gly Ile Arg Ser
Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Glu Val Gln 115 120 125 Leu Val Gln Ser
Gly Thr Glu Val Lys Lys Pro Gly Glu Ser Leu Lys 130 135 140 Ile Ser
Cys Lys Gly Ser Gly Tyr Thr Val Thr Ser Tyr Trp Ile Gly 145 150 155
160 Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Phe Ile
165 170 175 Tyr Pro Gly Asp Ser Glu Thr Arg Tyr Ser Pro Thr Phe Gln
Gly Gln 180 185 190 Val Thr Ile Ser Ala Asp Lys Ser Phe Asn Thr Ala
Phe Leu Gln Trp 195 200 205 Ser Ser Leu Lys Ala Ser Asp Thr Ala Met
Tyr Tyr Cys Ala Arg Val 210 215 220 Gly Ser Gly Trp Tyr Pro Tyr Thr
Phe Asp Ile Trp Gly Gln Gly Thr 225 230 235 240 Met Val Thr Val Ser
Ser 245 115119PRTHomo sapiensPEPTIDE(1)..(119)Sequence of 1D4.1 VH
region 115Glu Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro
Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser
Leu Ser Lys Ser 20 25 30 Val Met Gly Val Ser Trp Ile Arg Gln Pro
Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala His Ile Tyr Trp Asp
Asp Asp Lys Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr
Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met
Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala
Arg Arg Gly Ile Arg Ser Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110
Thr Thr Val Thr Val Ser Ser 115 116222PRTArtificialantibody VL
region 116Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser
Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser
Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr
Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala
Val Tyr Tyr Cys Gln Gln Asp Tyr Asn Ser Pro Trp 85 90 95 Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110
Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro 115
120 125 Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Ile Ser
Ser 130 135 140 Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Phe 145 150 155 160 Ile Tyr Thr Ala Ser Thr Arg Ala Thr Asp
Ile Pro Ala Arg Phe Ser 165 170 175 Gly Ser Gly Ser Gly Thr Glu Phe
Thr Leu Thr Ile Ser Ser Leu Gln 180 185 190 Ser Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro 195 200 205 Ser Ile Thr Phe
Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg 210 215 220 117108PRTHomo
sapiensPEPTIDE(1)..(108)Seqeunce of 1D4.1 VL region 117Asp Ile Val
Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu
Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile
35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
Asp Tyr Asn Ser Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys Arg 100 105 11816PRTArtificiallinker peptide 118Ala Lys
Thr Thr Pro Lys Leu Glu Glu Gly Glu Phe Ser Glu Ala Arg 1 5 10 15
11917PRTArtificiallinker peptide 119Ala Lys Thr Thr Pro Lys Leu Glu
Glu Gly Glu Phe Ser Glu Ala Arg 1 5 10 15 Val
1209PRTArtificiallinker peptide 120Ala Lys Thr Thr Pro Lys Leu Gly
Gly 1 5 12110PRTArtificiallinker peptide 121Ser Ala Lys Thr Thr Pro
Lys Leu Gly Gly 1 5 10 1226PRTArtificiallinker peptide 122Ser Ala
Lys Thr Thr Pro 1 5 1236PRTArtificiallinker peptide 123Arg Ala Asp
Ala Ala Pro 1 5 1249PRTArtificiallinker peptide 124Arg Ala Asp Ala
Ala Pro Thr Val Ser 1 5 12512PRTArtificiallinker peptide 125Arg Ala
Asp Ala Ala Ala Ala Gly Gly Pro Gly Ser 1 5 10
12627PRTArtificiallinker peptide 126Arg Ala Asp Ala Ala Ala Ala Gly
Gly Gly Gly Ser Gly Gly Gly Gly 1 5 10 15 Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser 20 25 12718PRTArtificiallinker peptide 127Ser
Ala Lys Thr Thr Pro Lys Leu Glu Glu Gly Glu Phe Ser Glu Ala 1 5 10
15 Arg Val 12813PRTArtificiallinker peptide 128Ala Lys Thr Thr Pro
Pro Ser Val Thr Pro Leu Ala Pro 1 5 10 1296PRTArtificiallinker
peptide 129Ala Lys Thr Thr Ala Pro 1 5 13015PRTArtificiallinker
peptide 130Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser 1 5 10 15 13115PRTArtificiallinker peptide 131Gly Glu Asn Lys
Val Glu Tyr Ala Pro Ala Leu Met Ala Leu Ser 1 5 10 15
13215PRTArtificiallinker peptide 132Gly Pro Ala Lys Glu Leu Thr Pro
Leu Lys Glu Ala Lys Val Ser 1 5 10 15 13315PRTArtificiallinker
peptide 133Gly His Glu Ala Ala Ala Val Met Gln Val Gln Tyr Pro Ala
Ser 1 5 10 15
* * * * *
References