Antibody and use in diagnosis and therapy of arthropathies

Abstract

The present invention provides a composition comprising an antibody or fragment thereof against oxidized Collagen II (CII) in which the antibody or fragment thereof is conjugated to a pharmaceutically active moiety. The invention also provides a composition comprising an antibody or fragment thereof against oxidized Collagen II (Gil) and a detectable label. The invention further provides the use of such compositions in medicine, in particular for the treatment of an arthropathy, and in methods of diagnosis.

Claims

1. A composition comprising an antibody or fragment thereof against oxidised collagen II (CII) in which the antibody or fragment thereof is conjugated to a pharmaceutically active moiety, wherein the antibody or fragment thereof comprises CDR sequences in the variable heavy (VH) chains and variable light (VL) chains as follows: CDRH2: SIDDSGATTYYADSVKG (SEQ ID NO: 14), CDRH3: NYSSFDY (SEQ ID NO: 48), CDRL2: YASSLQS (SEQ ID NO: 73), and CDRL3: QQAANYPTT (SEQ ID NO: 100), wherein the CDRH1 and CDRL1 sequences are the same as those of scFv 1-11E.

2. A composition as claimed in claim 1, in which the antibody is a polyclonal antibody or a monoclonal antibody.

3. A composition as claimed in claim 1, in which the antibody fragment is a Fab, scFv, single domain (dAb) antibody, diabody, minibody, or scFv-Fc fragment.

4. A composition as claimed in claim 1, in which the antibody is the scFv 1-11E.

5. A composition as claimed in claim 1, in which the composition comprises a proteolytic cleavage site between the antibody or fragment thereof and the pharmaceutically active moiety.

6. A composition as claimed in claim 5, in which the proteolytic cleavage site is a matrix metalloproteinase (MMP) cleavage site, a serine protease cleavage site, or a site cleavable by a parasitic protease derived from a pathogenic organism.

7. A composition as claimed in claim 6, in which the proteolytic cleavage site is a MMP cleavage site.

8. A composition as claimed in claim 7, in which the MMP cleavage site is one or more of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9 or MMP10 as shown in FIG. 5.

9. A composition as claimed in claim 1, in which the pharmaceutically active moiety is an antibody or a fragment thereof, a growth factor, a differentiation factor, a cytokine molecule, an interferon, a bone morphogenetic protein (BMP); a chemokine, a MCP (monocyte chemotactic protein), a cytokine inhibitor; a cytokine receptor, a free-radical scavenging enzyme or a toxin.

10. A composition as claimed in claim 9, in which the pharmaceutically active moiety is an interferon.

11. A composition as claimed in claim 10, in which the pharmaceutically active moiety is interferon beta (IFN-β).

12. A composition as claimed in claim 1, which composition comprises the scFv 1-11E, a MMP cleavage site and IFN-β.

13. A composition as claimed in claim 9, in which the pharmaceutically active moiety is a TNF receptor (TNFR) antibody fusion protein.

14. A composition as claimed in claim 13, in which the pharmaceutically active moiety is TNFR2-Fc.

15. A composition as claimed in claim 14, which composition comprises the scFv 1-11E, a MMP cleavage site and TNFR2-Fc.

16. A composition as claimed in claim 1, in which the pharmaceutically active moiety is a glycosaminoglycan molecule, chondroitin, a non-steroidal anti-inflammatory drug (NSAID), a steroid, sodium hyaluronate or hyaluronic acid, colchicine or hydroxychloroquine.

17. A composition comprising an antibody or fragment thereof against oxidised collagen II (CII) and a detectable label, wherein the antibody or fragment thereof comprises CDR sequences in the variable heavy (VH) chains and variable light (VL) chains as follows: CDRH2: SIDDSGATTYYADSVKG (SEQ ID NO: 14) CDRH3: NYSSFDY (SEQ ID NO: 48) CDRL2: YASSLQS (SEQ ID NO: 73) CDRL3: QQAANYPTT (SEQ ID NO: 100), wherein the CDRH1 and CDRL1 sequences are the same as those of scFv 1-11E.

18. A composition as claimed in claim 17, in which the detectable label is a radionuclide or a dye.

19. A composition as claimed in claim 18, in which the detectable label is a dye.

20. A composition as claimed in claim 9, in which the pharmaceutically active moiety is IL-10.

21. A composition as claimed in claim 20, which composition comprises the scFv 1-11E, a MMP cleavage site, and IL-10.

Description

(1) The invention will now be described by way of reference to the following Examples which are present for the purposes of illustration only and are not to be construed as being limiting on the present invention. Reference is also made in the Examples to the following drawings in which:

(2) FIG. 1 shows representative ELISA of unique scFvs

(3) FIG. 2 shows Western Blotting with scFv 1-11E probe

(4) FIG. 3 shows specific binding to damaged human cartilage tissue by anti-ROS-modified CII scFv in patients with OA in photographs (A) to (H).

(5) FIG. 4 shows the localisation of scFv 1-11E in inflamed paw.

(6) FIG. 5 shows the sequences of the protein cleavage sites of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9 and MMP10.

(7) FIG. 6 shows specific binding to damaged human cartilage tissue by anti-ROS-modified CII scFv in patients with OA.

(8) FIG. 7 shows specific binding to damaged human cartilage tissue by anti-ROS-modified CII scFv in patients with RA.

(9) FIG. 8 shows: (A) and (B) histological staining of the right paw; (C) and (D) 1-11E staining of cartilage in the paw; (E) staining with a non-relevant scFv; all in a mouse RA model.

(10) FIG. 9 shows staining of a joint surface injury in a mouse OA model.

(11) FIG. 10 shows the construction of (A) an IFN-β/1-11E fusion protein; and (B) a TNFR2Fc/scFv fusion protein.

(12) FIG. 11 shows the protocol used for expression of the IFN-β/1-11E fusion protein and the TNFR2Fc/scFv fusion protein.

(13) FIG. 12 is a Western blot of the IFN-β/1-11E fusion protein.

(14) FIG. 13 is a Western blot of (A) and (B) TNFR2Fc/1-11E fusion proteins; (C) a TNFR2Fc/C7 fusion protein

EXAMPLE 1: PREPARATION AND MODIFICATION OF CII

(15) CII was prepared from bovine cartilage as in Miller (Miller, Biochemistry 11(26): 4903-4909, 1972) and subsequently exposed to reactive oxygen generating systems as previously described (Nissim A, 2005). Briefly, CII was modified with (.OH), HOCl (Hawkins CL, 2001; Hawkins CL, 2002), (ONOO.sup.−), or 2M ribose by ON incubation at 37° C. Bovine serum albumin (BSA, Sigma) was also modified as above and was used as control antigen.

EXAMPLE 2: SELECTION OF ANTI-MODIFIED CII SCFV FROM PHAGE-DISPLAY LIBRARY

(16) Phage display antibody technology (Winter G et al, Annu. Rev. Immunol. 12: 433-455, 1994) was used to raise a single chain fragment variable (scFv) that binds only to CII that has been post-translationally modified by free radicals.

(17) A human semi-synthetic scFv library constructed from a single human framework for V.sub.H (DP-47 and JH4) and V.sub.L (DPK9 and JK1) was employed, in which diversity was incorporated in CDR3 and CDR2 (de Wildt R. M et al, Nat. Biotechnol. 18(9): 989-994, 2000). To select for phage binding to modified CII and not to native non-modified CII, subtractive selection was performed using native non-modified CII for subtraction. HOCl modified CII was used as a target for panning as binding to HOCl modified CII was strongest in RA sera (Nissim A, 2005). Glycated CII was used in parallel. Briefly, immunotubes (Nunc-Immuno Tubes, Maxi-Sorp, Nunc, Denmark) were coated with 10 μg/ml CII in phosphate-buffered saline (PBS). After blocking with 2% marvel in PBS (MPBS) coated tubes were exposed for 2 hours to 10.sup.13 transforming units (tu) of the phage library in 2% MPBS. Unbound phage were then transferred to a second immunotube previously coated with HOCl or ribose-modified CII for a further 2 hours incubation at room temperature. Modified CII-bound phage were then used to infect E. coli TG-1 and rescued by helper phage as described (Harrison J. L, 1996). The panning process was repeated three times and E coli TG-1 was infected with the final phage eluted after the third round and individual ampicillin-resistant colonies (phage clones) were selected for further analysis.

EXAMPLE 3: SCREENING AND SEQUENCING OF MODIFIED CII-SPECIFIC PHAGE CLONES

(18) Screening for positive anti-modified CII phage clones was first performed by enzyme-linked immunosorbent assay (ELISA), as previously described (Harrison J. L, 1996). Microtiter plate (Nunc, Paisley, UK) wells were coated with 10 μg/ml native or modified CII and incubated with 100 μl phage suspension for 90 minutes. In addition, native and modified BSA were used as negative control. After removal of the supernatants, the amount of bound phage was determined using peroxidase-labeled anti-M13 antibodies (GE Healthcare Little Chalfont, Buckinghamshire) and developed by using 100 mM 3,3′5,5′ tetramethylbenzidine (TMB) as substrate. The reaction was monitored in an ELISA reader at 450 nm with a reference wavelength of 650 nm using GENios plate reader (TECAN, Theale Court, Reading UK) and Magellan software (TECAN, Theale Court, Reading UK)

(19) The entire scFv DNA fragment of each modified CII bound phage clone was sequenced using the primers LMB-3 (5′-C AGGAAACAGCTATGAC) (SEQ ID NO: 127) and Fd-Seq (5′-GAATTTTCTGTATGAGG) (SEQ ID NO: 128). Sequences were analyzed using Chromas (Technelysium Pty Ltd) and VBASE (http://vbase.mrc-cpe.cam.ac.uk), to identify unique scFv sequences as shown in Table 3.

(20) TABLE-US-00003 TABLE 3 Clone Antigen CDRH2 CDRH3 CDRL2 CDRL3 3-11A HOCl-CII DISSTGSYTAYADSVKG GAGSFDY AASALQS QQSSSTPTT (SEQ ID NO: 1) (SEQ ID (SEQ ID (SEQ ID NO: NO: 39) NO: 61) 86) 6-6E HOCl-CII AISAAGTATAYADSVKG GYDTFDY AASSLQS QQNYGYPNT (SEQ ID NO: 2) (SEQ ID (SEQ ID (SEQ ID NO: NO: 40) NO: 62) 87) 1-7G Ribose-CII SISNSGSYTDYADSVKG GYGSFDY AASTLQS QQANSSPDT (SEQ ID NO: 3) (SEQ ID (SEQ ID (SEQ ID NO: NO: 41) NO: 63) 88) 3-7B HOCl-CII SINNYGSNTAYADSVKG GYSSFDY AASYLQS QQTSSSPDT (SEQ ID NO: 4) (SEQ ID (SEQ ID (SEQ ID NO: NO: 42) NO: 64) 89) 6-9D HOCl-CII SINNYGSNTAYADSVKG GYSSFDY AASYLQS QQTSSSPDT (SEQ ID NO: 4) (SEQ ID (SEQ ID (SEQ ID NO: NO: 42) NO: 64) 89) 1-1C Ribose-CII SISYTGNSTDYADSVKG GYTAFDY YASYLQS QQADSTPTT (SEQ ID NO: 5) (SEQ ID (SEQ ID (SEQ ID NO: NO: 43) NO: 65) 90) 1-8D Ribose-CII SISYTGNSTDYADSVKG GYTAFDY YASYLQS QQADSTPTT (SEQ ID NO: 5) (SEQ ID (SEQ ID (SEQ ID NO: NO: 43) NO: 65) 90) 1-3G Ribose-CII SISYTGNSTDYADSVKG GYTAFDY YASYLQS QQADSTPTT (SEQ ID NO: 5) (SEQ ID (SEQ ID (SEQ ID NO: NO: 43) NO: 65) 90) 4-12C Ribose-CII SISYTGNSTDYADSVKG GYTAFDY YASYLQS QQADSTPTT (SEQ ID NO: 5) (SEQ ID (SEQ ID (SEQ ID NO: NO: 43) NO: 65) 90) 6-3E HOCl-CII SISYTGNSTDYASVKG GYTAFDY YASYLQS QQADSTPTT (SEQ ID NO: 5) (SEQ ID (SEQ ID (SEQ ID NO: NO: 43) NO: 65) 90) 6-9A HOCl-CII NIATDGTTTYYADSVKG NSTYFDY SASTLQS QQAATSPTT (SEQ ID NO: 6) (SEQ ID (SEQ ID (SEQ ID NO: NO: 44) NO: 66) 91) 1-12A Ribose-CII SISNSGTNTDYADSVKG NYASFDY YASYLQS QQGSASPST (SEQ ID NO: 7) (SEQ ID (SEQ ID (SEQ ID NO: NO: 45) NO: 65) 92) 4-6A Ribose-CII SISNSGTNTDYADSVKG NYASFDY YASYLQS QQGSASPST (SEQ ID NO: 7) (SEQ ID (SEQ ID (SEQ ID NO: NO: 45) NO: 65) 92) 4-8A Ribose-CII SISNSGTNTDYADSVKG NYASFDY YASYLQS QQGSASPST (SEQ ID NO: 7) (SEQ ID (SEQ ID (SEQ ID NO: NO: 45) NO: 65) 92) 4-9F Ribose-CII SISNSGTNTDYADSVKG NYASFDY YASYLQS QQGSASPST (SEQ ID NO: 7) (SEQ ID (SEQ ID (SEQ ID NO: NO: 45) NO: 65) 92) 4-4H Ribose-CII SISNSGTNTDYADSVKG NYASFDY YASYLQS QQGSASPST (SEQ ID NO: 7) (SEQ ID (SEQ ID (SEQ ID NO: NO: 45) NO: 65) 92) 3-3A HOCl-CII SISNSGTNTDYADSVKG NYASFDY YASYLQS QQGSASPST (SEQ ID NO: 7) (SEQ ID (SEQ ID (SEQ ID NO: NO: 45) NO: 65) 92) 3-6F HOCl-CII SISNSGTNTDYADSVKG NYASFDY YASYLQS QQGSASPST (SEQ ID NO: 7) (SEQ ID (SEQ ID (SEQ ID NO: NO: 45) NO: 65) 92) 6-10H HOCl-CII SISYTGDSTYYADSVKG NYSAFDY YASYLQS QQADSTPTT (SEQ ID NO: 8) (SEQ ID (SEQ ID (SEQ ID NO: NO: 46) NO: 65) 90) 12E Unknown SINDSGITTYYADSVKG NYSAFDY AASDLQS QQSDSAPTT (SEQ ID NO: 9) (SEQ ID (SEQ ID (SEQ ID NO: NO: 46) NO: 67) 93) 3-5G HOCl-CII SIDSAGASTYYADSVKG NYSAFDY NASSLQS QQSDTYPST (SEQ ID NO: 10) (SEQ ID (SEQ ID (SEQ ID NO: NO: 46) NO: 68) 94) 3-4D HOCl-CII SISYTGDSTYYADSVKG NYSAFDY TASNLQS QQSYASPTT (SEQ ID NO: 8) (SEQ ID (SEQ ID (SEQ ID NO: NO: 46) NO: 69) 95) 3-5D HOCl-CII SISYTGDSTYYADSVKG NYSAFDY TASNLQS QQSYASPTT (SEQ ID NO: 8) (SEQ ID (SEQ ID (SEQ ID NO: NO: 46) NO: 69) 95) 6-4E HOCl-CII SISYTGDSTYYADSVKG NYSAFDY TASNLQS QQTGSYPTT (SEQ ID NO: 8) (SEQ ID (SEQ ID (SEQ ID NO: NO: 46) NO: 69) 96) 3-6B HOCl-CII SINATGYGTYYADSVKG NYSDFDY SASALQS QQGDSYPTT (SEQ ID NO: 11) (SEQ ID (SEQ ID (SEQ ID NO: NO: 47) NO: 70) 97) 3-6G HOCl-CII SINSNGTDTYYADSVKG NYSDFDY TASALQS QQGYGAPTT (SEQ ID NO: 12) (SEQ ID (SEQ ID (SEQ ID NO: NO: 47) NO: 71) 98) 4-11F Ribose-CII SISATGSSTYYADSVKG NYSDFDY SASDLQS QQSSYTPTT (SEQ ID NO: 13) (SEQ ID (SEQ ID (SEQ ID NO: NO: 47) NO: 72) 99) 6-7H HOCl-CII SISATGSSTYYADSVKG NYSDFDY SASDLQS QQSSYTPTT (SEQ ID NO: 13) (SEQ ID (SEQ ID (SEQ ID NO: NO: 47) NO: 72) 99) 1-11E Ribose-CII SIDDSGATTYYADSVKG NYSSFDY YASSLQS QQAANYPTT (SEQ ID NO: 14) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 73) 100) 1-2F Ribose-CII SIDDSGATTYYADSVKG NYSSFDY YASSLQS QQAANYPTT (SEQ ID NO: 14) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 73) 100) 1-6H Ribose-CII SIDDSGATTYYADSVKG NYSSFDY YASSLQS QQAANYPTT (SEQ ID NO: 14) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 73) 100) 3-8D HOCl-II SIDDSGATTYYADSVKG NYSSFDY YASSLQS QQAANYPTT (SEQ ID NO: 14) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 73) 100) 1-4D Ribose-CII SIASTGDSTYYADSVKG NYSSFDY SASALQS QQASNYPTT (SEQ ID NO: 15) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 70) 101) 4-2F Ribose-CII SIASTGDSTYYADSVKG NYSSFDY SASALQS QQASNYPTT (SEQ ID NO: 15) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 70) 101) 3-3B HOCl-CII SIASTGDSTYYADSVKG NYSSFDY SASALQS QQASNYPTT (SEQ ID NO: 15) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 70) 101) 3-5C HOC1-CII SIASTGDSTYYADSVKG NYSSFDY SASALQS QQASNYPTT ) (SEQ ID NO: 15 (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 70) 101) 6-9C HOCl-CII SIASTGDSTYYADSVKG NYSSFDY SASALQS QQASNYPTT (SEQ ID NO: 15) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 70) 101) 4G Unknown SIASTGDSTYYADSVKG NYSSFDY SASALQS QQASNYPTT (SEQ ID NO: 15) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 70) 101) 3-12F HOCl-CII SISTNGSSTYYADSVKG NYSSFDY DASGLQS QQGDTSPTT (SEQ ID NO: 16) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 74) 102) 3-4G HOCl-CII SISTNGSSTYYADSVKG NYSSFDY DASGLQS QQGDTSPTT (SEQ ID NO: 16) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 74) 102) 6-11F HOCl-CII SISTNGSSTYYADSVKG NYSSFDY DASGLQS QQGDTSPTT (SEQ ID NO: 16) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 74) 102) 6-11H HOCl-CII SISTNGSSTYYADSVKG NYSSFDY DASGLQS QQGDTSPTT (SEQ ID NO: 16) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 74) 102) 3-2C HOCl-CII SIDTTGTTTYFADSVKG NYSSFDY SASYLQS QQGYSAPTT (SEQ ID NO: 17) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 75) 103) 5B Unknown TISYSGNNTYYADSVKG NYSSFDY TASSLQS QQGYTSPTT (SEQ ID NO: 18) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 76) 104) 6-10G HOCl-CII SIDAGGNGTYYADSVKG NYSSFDY TASNLQS QQNNYYPTT (SEQ ID NO: 19) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 77) 105) 1-4H Ribose-CII SIDAGGNGTYYADSVKG NYSSFDY YASSLQS QQSDAYPTT (SEQ ID NO: 19) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 73) 106) 4-5A HOCl-CII SIDAGGNGTYYADSVKG NYSSFDY YASSLQS QQSDAYPTT (SEQ ID NO: 19) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 73) 106) 4-1B HOCl-CII SIDAGGNGTYYADSVKG NYSSFDY YASSLQS QQSDAYPTT (SEQ ID NO: 19) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 73) 106) 4-12D HOCl-CII SIDAGGNGTYYADSVKG NYSSFDY YASSLQS QQSDAYPTT (SEQ ID NO: 19) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 73) 106) 6-4B HOCl-CII SIDSAGNATYYADSVKG NYSSFDY AASTLQS TSNYPTTQQ (SEQ ID NO: 20) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 78) 107) 1-2E Ribose-CII SITDSGDTTYYADSVKG NYSTFDY SASSLQS QQSNATPTT (SEQ ID NO: 21) (SEQ ID (SEQ ID (SEQ ID NO: NO: 49) NO: 79) 108) 1-7F Ribose-CII SITDSGDTTYYADSVKG NYSTFDY SASSLQS QQSNATPTT (SEQ ID NO: 21) (SEQ ID (SEQ ID (SEQ ID NO: NO: 49) NO: 79) 108) 1-10F Ribose-CII SITDSGDTTYYADSVKG NYSTFDY SASSLQS QQSNATPTT (SEQ ID NO: 21) (SEQ ID (SEQ ID (SEQ ID NO: NO: 49) NO: 79) 108) 1-9G Ribose-CII SITDSGDTTYYADSVKG NYSTFDY SASSLQS QQSNATPTT (SEQ ID NO: 21) (SEQ ID (SEQ ID (SEQ ID NO: NO: 49) NO: 79) 108) 4-1C Ribose-CII SITDSGDTTYYADSVKG NYSTFDY SASSLQS QQSNATPTT (SEQ ID NO: 21) (SEQ ID (SEQ ID (SEQ ID NO: NO: 49) NO: 79) 108) 6-7G HOCl-CII SIATTGDNTYYADSVKG NYSYFDY TASTLQS QQAAGNPTT (SEQ ID NO: 22) (SEQ ID (SEQ ID (SEQ ID NO: NO: 50) NO: 80) 109) 3-7H HOCl-CII AINAYGGSTYYADSVKG NYSYFDY AASSLQS QQGSDYPTT (SEQ ID NO: 23) (SEQ ID (SEQ ID (SEQ ID NO: NO: 50) NO: 62) 110) 6-1F HOCl-CII AINAYGGSTYYADSVKG NYSYFDY AASSLQS QQGSDYPTT (SEQ ID NO: 23) (SEQ ID (SEQ ID (SEQ ID NO: NO: 50) NO: 62) 110) 6-3B HOCl-CII SIATTGTSTTYADSVKG NYSYFDY TASSLQS QQGSTAPTT (SEQ ID NO: 24) (SEQ ID (SEQ ID (SEQ ID NO: NO: 50) NO: 76) 111) 4H Unknown SIATTGTSTTYADSVKG NYSYFDY TASSLQS QQGSTAPTT (SEQ ID NO: 24) (SEQ ID (SEQ ID (SEQ ID NO: NO: 50) NO: 76) 111) 3-9A HOCl-CII TIDTAGSYTDYADSVKG NYSYFDY GASTLQS QQSTASPST (SEQ ID NO: 25) (SEQ ID (SEQ ID (SEQ ID NO: NO: 50) NO: 81) 112) 6-10D HOCl-CII SISNNGSSTYYADSVKG NYSYFDY AASNLQS QQTSSYPTT (SEQ ID NO: 26) (SEQ ID (SEQ ID (SEQ ID NO: NO: 50) NO: 82) 113) 3-5H HOCl-CII SIAYGGAGTDYADSVKG NYTAFDY AASYLQS QQGAGSPST (SEQ ID NO: 27) (SEQ ID (SEQ ID (SEQ ID NO: NO: 51) NO: 64) 114) 3-2F HOCl-CII AIANTGSATNYADSVKG NYTAFDY DASTLQS QQRNTSPTT (SEQ ID NO: 28) (SEQ ID (SEQ ID (SEQ ID NO: NO: 51) NO: 83) 115) 1-6G Ribose-CII SISTAGTYTDYADSVKG NYTDFDY SASYLQS QQSNTSPAT (SEQ ID NO: 29) (SEQ ID (SEQ ID (SEQ ID NO: NO: 52) NO: 75) 116) 3-11H HOCl-CII SISTAGTYTDYADSVKG NYTDFDY SASYLQS QQSNTSPAT (SEQ ID NO: 29) (SEQ ID (SEQ ID (SEQ ID NO: NO: 52) NO: 75) 116) 6-9F HOCl-CII SINDTGYTTYYADSVKG NYTYFDY TASTLQS QQAYTAPTT (SEQ ID NO: 30) (SEQ ID (SEQ ID (SEQ ID NO: NO: 53) NO: 80) 117) 3-9D HOCl-CII SIASSGTTTYYADSVKG SYADFDY AASNLQS QQADTYPTT (SEQ ID NO: 31) (SEQ ID (SEQ ID (SEQ ID NO: NO: 54) NO: 82) 118) 4-3H Ribose-CII TITSTGAATAYADSVKG SYATFDY AASYLQS QQAANSPDT (SEQ ID NO: 32) (SEQ ID (SEQ ID (SEQ ID NO: NO: 55) NO: 64) 119) 3-3E HOCl-CII AIDGTGYGTAYADSVKG SYDTFDY GASSLQS QQTSDYPNT (SEQ ID NO: 33) (SEQ ID (SEQ ID (SEQ ID NO: NO: 56) NO: 84) 120) 3-10C HOCl-CII SIANAGTATYYADSVKG SYSNFDY SASTLQS QQASTSPTT (SEQ ID NO: 34) (SEQ ID (SEQ ID (SEQ ID NO: NO: 57) NO: 66) 121) 3-11E HOCl-CII SIDSAGDSTYYADSVKG SYSYFDY TASYLQS QQASDYPTT (SEQ ID NO: 35) (SEQ ID (SEQ ID (SEQ ID NO: NO: 58) NO: 85) 122) 6-8C HOCl-CII SISSSGDTTYYADSVKG SYSYFDY TASTLQS QQSSSNPTT (SEQ ID NO: 36) (SEQ ID (SEQ ID (SEQ ID NO: NO: 58) NO: 80) 123) 6-11D HOCl-CII SIDTGGSYTDYADSVKG SYTTFDY SASYLQS QQGSNSPTT (SEQ ID NO: 37) (SEQ ID (SEQ ID (SEQ ID NO: NO: 59) NO: 75) 124) 4-5H Ribose-CII SIDTGGSYTDYADSVKG SYTTFDY SASYLQS QQGSNSPTT (SEQ ID NO: 37) (SEQ ID (SEQ ID (SEQ ID NO: NO: 59) NO: 75) 124) 6-5F HOCl-CII SIDTGGSYTDYADSVKG SYTTFDY SASYLQS QQGSNSPTT (SEQ ID NO: 37) (SEQ ID (SEQ ID (SEQ ID NO: NO: 59) NO: 75) 124) 6-7F HOCl-CII SIDTGGSYTDYADSVKG SYTTFDY SASYLQS QQGSNSPTT (SEQ ID NO: 37) (SEQ ID (SEQ ID (SEQ ID NO: NO: 59) NO: 75) 124) 1-10D Ribose-CII SIDASGANTAYADSVKG TYGTFDY SASYLQS QQSATTPDT (SEQ ID NO: 38) (SEQ ID (SEQ ID (SEQ ID NO: NO: 60) NO: 75) 125)

EXAMPLE 4: PRODUCTION AND PURIFICATION OF ANTI-MODIFIED CII-SCFV

(21) The reactive phage clones obtained from E coli TG-1 bacteria were used to infect E coli HB2151 non-suppressor bacterial strain to obtain soluble scFv. After overnight induction with 1 mM IPTG at 30° C., the antibody fragments, derived from the V.sub.H3 family, were harvested from the supernatant and periplasmic space as described (Harrison J. L, 1996) and purified on a protein A affinity column (GE Healthcare Ltd, Little Chalfont, Buckinghamshire). Binding of purified scFv to modified CII was first analyzed by ELISA as above except that mouse anti-myc tag antibody (Santa Cruz Biotechnology, INC, Wembley, UK) followed by anti-mouse-HRP conjugate (Sigma, Dorset, UK) were used to probe bound scFv.

(22) Anti-Modified CII scFv Raised by Phage Display Human Antibody Library

(23) After three rounds of subtractive selection 82 phage clones specific to either glycated CII or HOCl modified CII were selected out of which 42 clones had unique sequences. 15 representative clones with different binding patterns but with good expression were then studied for further analysis (FIG. 1 and Table 1). As shown in FIG. 1, out of these 15 clones there were 9 clones with stronger binding to modified CII, 3 clones bound to all forms of CII and and 3 clones had no binding reactivity to any form of CII. Three scFvs that have different binding characteristics were then further studied: Clone 1-11E binds to modified CII (glycated, HOCl and to some extent to peroxynitrated CII), clone 6-11D binds to both native and modified CII and clone 12E that does not bind to any form of CII. None of these scFv bound to native or free radical modified BSA, or to collagen type III (data not shown).

EXAMPLE 5: WESTERN BLOTTING

(24) Western blot using scFv as probe and modified or native CII as target antigens was done as described (Nissim A, 2005). Briefly, modified and native CII (2 μg of each) were run on a 7.5% denaturing SDS gel and electroblotted into a nitrocellulose membrane. After blocking with 2% MPBS, membranes were incubated with 10 μg/ml purified scFv in 2% MPBS for 2 hr at room temperature, followed by incubation with mouse anti-myc tag (Santa Cruz Biotechnology, INC, Wembley, UK) and then with anti-mouse-HRP (Sigma, Dorset). Membranes were washed three times with 0.1% Tween PBS (5 min each) and three times with PBS (5 min each) before development with ECL (GE Healthcare Ltd. Little Chalfont, Buckinghamshire).

(25) Comparative Analysis of Human RA Serum and scFv Binding to CII by Western Blotting

(26) 1-11E binds several CII fragments between 50 and 150 kDa as well as to a band >250 kDa which resulted from CII cross linking due to the ROS reactivity (FIG. 2 lane 2-5). 1-11E also binds to native CII corresponding to a band below 150 KDa (FIG. 2 lane 1). Binding to native CII in Western blotting was also seen in sera from RA patients that did not bind to native CII in ELISA but only to ROS modified CII in ELISA (for example sera 33 (Nissim A, 2005)).

EXAMPLE 6: IMMUNOHISTOCHEMISTRY OF HUMAN OA AND RA CARTILAGE USING SELECTED ANTI-MODIFIED CII SCFV

(27) One osteochondral sample was obtained from the femoral condyle of a patient (female, 63 years old) undergoing prosthetic knee replacement for OA. One sample of normal human cartilage was obtained post-mortem from a preserved area of a knee with unicompartimental OA undergoing joint replacement (female, 54 years old). In both cases, cartilage was fixed overnight at 4° C. in 4% paraformaldehyde, decalcified for 15 days in 0.5M EDTA at 4° C., washed in PBS, and embedded in paraffin according to standard protocols. Safranin 0 staining was performed according to standard protocols (Rosenberg, 1971). All samples were obtained in accordance with institutional policies and regulations.

(28) For immunostaining, 5 mm thick sections were cut, deparaffinized and hydrated according to standard protocols. After endogenous peroxidase quenching in 0.5% hydrogen peroxide for 15 min antigen retrieval was done by 45 min incubation of slides with 3 mg/ml pepsin (Zymed, Chandlers Ford, Hampshire, UK) at 37° C. followed by two washes with PBS. Endogenous avidin activity was blocked using a commercially available kit (Vector Laboratories, Orton Southgate, Peterborough, UK) according to the manufacturer's instructions. This was followed by 30 min blocking with 0.5% BSA Immunostaining was performed using the selected scFv (10 μg/ml and 1 μg/ml) as well as control commercial mouse anti-CII antibodies (diluted 1:100 and 1:1000 dilution; Chemicon International, Chandlers Ford, Hampshire, UK) and polyclonal anti-CII antibodies (diluted 1:100, 1:1000) from collagen induced arthritis (CIA) mice. ScFv or control antibodies were added to the slide in blocking buffer (0.5% BSA in PBS plus 0.05% sodium azide) and left overnight at 4° C. When scFv were used for probing, next day slides were washed with PBS for 2 minutes and incubated for 30 minutes with anti-myc tag mouse antibodies to bind to the myc tag incorporated at the carboxy terminal end of the scFv (diluted 1:200, Santa Cruz Biotechnology Inc, Wembley, UK). After two washes as above anti-mouse biotinylated antibodies were added (Vector kit PK-6102) followed by two washes with PBS and development with DAB substrate (DAKO, Ely, Cambridgeshire, UK) and nuclear counterstaining with Mayer's haematoxylin. Slides were finally dehydrated and mounted with DPX mount (BDH, London, UK)

(29) Specific Binding to Damaged Human Cartilage Tissue by Anti-ROS-Modified CII scFv

(30) The cartilage extracellular matrix is a complex structure where several molecules interact to form a structural and functional unit. There is therefore the chance that the tertiary and quaternary structure of collagens in the intact tissue may alter the specificity of binding of the phage antibodies that had been selected in vitro. To determine binding specificity in the intact tissue, the capacity of anti-ROS-modified CII scFv to bind to CII within the matrix complex structure and to present immunoreactivity with damaged OA cartilage as opposed to normal cartilage was tested. 1-11E stained the extracellular matrix of cartilage tissue that displayed marked features of OA (FIG. 3A, B) with mostly pericellular staining (FIG. 3 B), with severe damage of the extracellular matrix determined with reduced staining with safranin 0 (FIG. 3 C). No staining by scFv 1-11E was detected when using histologically normal cartilage from normal cartilage (FIG. 3 D, E). By contrast, polyclonal antibodies from CIA mice bound the OA cartilage in both the damaged and non-damaged regions strongly (FIG. 3 F) and weakly stained with safranin 0 (FIG. 3 C). A commercial anti-CII mAb did not stain the damaged cartilage areas stained by 1-11E on an adjacent section (FIG. 3 G) but intensely stained a histologically normal cartilage (FIG. 3 H), suggesting that the epitope recognised by the commercial antibody is lost in the OA section.

(31) FIG. 6 also shows staining of OA cartilage. Although synovial inflammation in OA is not as extensive as in RA and inflammatory cells are not significant in numbers, low grade synovitis is nearly a constant feature in OA. Abnormal mechanical force appears to stimulate chrondocytes to produce some of the same inflammatory mediators and ROS as the infiltrating leukocytes present in inflamed RA joints, leading to post-translational modifications of CII in OA. FIG. 6 confirms the results shown in FIG. 3 and shows that staining of OA cartilage section is only pericellular, around the chrondocytes.

(32) A further sample was obtained from a patient (female, 47 years old) undergoing total right knee replacement for RA. Fixing and staining protocols were as described above.

(33) FIG. 7 shows staining of the RA cartilage. In RA, infiltrating inflammatory cells consume increased amounts of oxygen, resulting in the generation of reactive oxygen species (ROS), which may cause excessive degradation of the extracellular matrix leading to cartilage destruction and chemical post-translational modification of CII by ROS. FIG. 7 shows uniteral staining of RA cartilage across the section. This is due to the high influx of immune cells that produce high levels of ROS.

EXAMPLE 7: CONSTRUCTION AND EXPRESSION OF DIABODY

(34) Out of the unique scFv assessed for specific binding to modified CII as well as best expression in bacteria, the most promising scFv, 1-11E of 25 kDa, was engineered to a larger fragment of 55 KDa. The linker between the V.sub.H and V.sub.L was shortened by digesting the phagemid vector with XhoI and SalI and relegation. This results in bivalent diabody, a superior molecule with an increased half life (Hudson, 2005) built from two scFv. Expression and screening of diabody binders was done as above. Molecular weight profile of the resulted expressed diabody was analyzed by gel filtration.

EXAMPLE 8: INDUCING ARTHRITIS IN THE ANIMAL MODELS

(35) Male C3H mice (age 17-19 weeks) were used. 100 mg of dessicated non-viable T.B. strain H37RA (Difco 231141) was added to 30 ml of incomplete Freunds adjuvant (IFA, Difco 263910) to form complete Freunds adjuvant (CFA). An equal volume of CFA was added to a 2 mg/ml solution (in PBS) of methylated BSA (mBSA) (Sigma A1009). The mixture was then emulsified on ice using an Ultra-Turrax T25 homogeniser at 13500-20500 rpm until a fluffy milky consistency was obtained. Mice were anaesthetised with Hypnorm, and 100 μl of 1 mg/ml (i.e. 100 μg) mBSA in CFA was injected over 2-3 separate sites intradermally. 1 week later, the immunisation was repeated as previously, except that no bacteria were added (i.e. IFA/mBSA). Two weeks after the 2nd immunisation, mice were anaesthetised with nitrous/oxygen and halothane, and inflammation was induced by injecting 50 μl of 1 mg/ml (i.e. 50 μg) mBSA in PBS into the animals' left hind paw. As a control, 50 μl PBS was injected into the right hind paw. Inflammation was measured using calipers to measure the paw thickness. Swelling was seen only in the right paws from 24 hours, and persisted until 1 week later. 2 weeks later, the swelling had totally subsided.

EXAMPLE 9: IMAGING OF ANTI-ROS MODIFIED CII LOCALIZATION

(36) 50 μg of 1-11E diabody was radiolabelled with 20 MBq of sodium [I-125] iodide (GE Healthcare, Amersham, UK) using the iodogen method (Perbio Science, Cramlingham, UK) and diluted in PBS to a final volume of 240 μl. Radiochemical purity was determined by thin-layer chromatography on silica gel (ITLC, Pall Corporation, Portsmouth, UK) using 85% methanol as mobile phase. A volume of 100 μl of the labeled diabody was injected intravenously via the tail vein into two arthritis-bearing C3H mice 24 hours after injection of the mBSA. Four and 22 hours later the mice were anaesthetized by ip injection of Ketamine/Xylazine. The mice were imaged on a NanoSPECT/CT scanner (Bioscan Inc, Washington, USA) using a four-detector/36×1.4 mm pinhole configuration. 30-50,000 counts were acquired for the SPECT study over 20-50 minutes.

(37) Imaging of 1-11E Localisation into the Inflamed Paw

(38) SPECT and CT images from the NanoSPECT/CT camera were fused and displayed using PMOD software. FIG. 4 shows a representative image acquired 22 hours after injection of the radiotracer. Increased uptake of radioactivity is clearly seen in the rear left (inflamed) paw relative to the right (normal paw).

EXAMPLE 10: STAINING OF CARTILAGE IN MOUSE RA MODEL

(39) Staining of cartilage was observed in the mouse mBSA model described in Example 8 above, except that C57BL mice were used.

(40) Mice were sensitised with mBSA (100 μg) in CFA intradermally at the base of the tail, and challenged either intra-articularly (both knees) or intra-plantarly (right, saline left) with 500 μg mBSA in saline 14 days later.

(41) Staining of cartilage is shown in FIG. 8.

(42) Paw:

(43) 12 hours post challenge with mBSA, the right paw was grossly inflamed in the subplantar region (seen by haematoxylin and eosin (H&E) staining), as shown in FIGS. 8A and 8B. The cartilage in the mBSA paws is uniformly and strongly stained by 1-11E, as shown in FIGS. 8C and 8D. The left paw injected by saline had no subplantar inflammation. Some cartilage within the left paw joints was stained heterogeneously, perhaps associated with spontaneous osteoarthritis.

EXAMPLE 11: STAINING OF JOINT IN MOUSE OA MODEL

(44) Staining was observed in mice with joint surface injury.

(45) Seven week old C57BL/6 male mice were utilized for these experiments (Dell'Accio F et al, Arthritis Res Ther. 2006; 8(5):R139). The mice were anesthetized and subjected to medial para-patellar arthrotomy. The patellar groove was exposed by lateral patellar dislocation. A longitudinal full thickness injury was made in the patellar groove using a custom made device in which the length of a 26G needle was limited by a glass bead (injured knee). The patellar dislocation was then reduced and the joint capsule and the skin sutured in separate layers. The animals were killed after 4 weeks and the knees dissected for histological and histochemical analysis.

(46) Staining methods are as set out in Example 6 above, except that rabbit anti-myc followed by anti-rabbit-HRP were used to avoid cross-reactivity with mouse antibody in the tissue.

(47) As shown in FIG. 9, there is strong staining at the site of the injury.

EXAMPLE 12: PRODUCTION OF FUSION PROTEINS: 1-11E WITH ANTI-INFLAMMATORY CYTOKINES

(48) Cloning of IFN-Beta/1-11E

(49) pFastBac1.AH was created from pFastBac1 (Invitrogen) by cutting out BamHI/HindIII fragment containing multiple cloning sites (MCS), and replacing with a linker to give another MCS of BamHI-KpnI-HindIII-ApaI.

(50) Mouse interferon b (mIFNb) was cloned into the HindIII-EcoRI sites, followed by a MMP cleavage site and 1-11E which were cloned into the NotI and ApaI sites as shown in FIG. 10A. The MMP cleavage site can be cleaved by MPP-1 and MMP-3.

(51) Mouse interferon-beta was amplified with the following primers:

(52) TABLE-US-00004 forward: mIFNBHindFOR (SEQ ID NO: 129) 5′ gct aag ctt atg aac aac agg tgg atT 3′    HindIII     Start                 * reverse: mIFNBEcoRIREV (SEQ ID NO: 130) 5′ CGC GAA TTC GTT TTG GAA GTT TCT GGT 3′

(53) 1-11E was amplified with the following primers:

(54) TABLE-US-00005 forward: NotI1-11Efor: (SEQ ID NO: 131) 5′cag GC GGC CGC a ATG GCC GAG GTG CAG CTG 3′       NotI       * Start reverse: 1-11EApaRev (SEQ ID NO: 132) 5 ′CTTGGGCCCTCAATGGTGGTGGTGATGGTGTCTAGACCGTTT GATTTCCACCTT 3′

(55) 1-11E was amplified with NotI/ApaI ends to include a histidine (His) tag and then cloned into FastBac1.AH mIFN-b/MMP/SP/His and cut with Not/Apa to liberate SP/His.

(56) The mIFN-beta/His construct was cloned by amplifying mIFN-b with HindIII/ApaI with the following primers:

(57) TABLE-US-00006 forward (this primer is the same as the primer used for cloning IFN-b/MMP/1-11E/His): mIFNBHindFOR (SEQ ID NO: 129) 5′ gct aag ctt atg aac aac agg tgg atT 3′    HindIII     Start                 * reverse primer: mIFN-bApaRev (SEQ ID NO: 133) 5′ CTTGGGCCCTCAATGGTGGTGGTGATGGTGTCTAGAGTTTTGGA AGTTTCTGGT 3′

(58) These constructs were transformed into DH10Bac cells from Invitrogen and the sequence was confirmed as follows:

(59) TABLE-US-00007 IFN-beta/MMP/1-11E/His (50.4 kDa) (SEQ ID NO: 134) MNNRWILHAAFLLCFSTTALSINYKQLQLQERTNIRKCQELLEQLNGKIN  50 LTYRADFKIPMEMTEKMQKSYTAFAIQEMLQNVFLVFRNNFSSTGWNETI 100 VVRLLDELHQQTVFLKTVLEEKQEERLTWEMSSTALHLKSYYWRVQRYLK 150 LMKYNSYAWMVVRAEIFRNFLIIRRLTRNFQNEFGGGGSPLGLWAGGGSA 200 AAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE 250 WVSSIDDSGATTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC 300 AKNYSSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSAS 350 VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYYASSLQSGVPSRFSG 400 SGSGTDFTLTISSLQPEDFATYYCQQAANYPTTFGQGTKVEIKRDIHHHH 450 HH*

(60) Within this sequence, the IFN-beta portion is from amino acids 1 to 182 as follows:

(61) TABLE-US-00008 (SEQ ID NO: 135) MNNRWILHAAFLLCFSTTALSINYKQLQLQERTNIRKCQELLEQLNGKIN  50 LTYRADFKIPMEMTEKMQKSYTAFAIQEMLQNVFLVFRNNFSSTGWNETI 100 VVRLLDELHQQTVFLKTVLEEKQEERLTWEMSSTALHLKSYYWRVQRYLK 150 LMKYNSYAWMVVRAEIFRNFLIIRRLTRNFQN

(62) The MMP linker portion is from amino acids 183 to 202 as follows:

(63) TABLE-US-00009 (SEQ ID NO: 136) EFGGGGSPLGLWAGGGSA 200 AA

(64) The 1-11E portion is from amino acids 203 to 446 as follows:

(65) TABLE-US-00010 (SEQ ID NO: 137) MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE 250 WVSSIDDSGATTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC 300 AKNYSSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSAS 350 VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYYASSLQSGVPSRFSG 400 SGSGTDFTLTISSLQPEDFATYYCQQAANYPTTFGQGTKVEIKRDI

(66) The His tag is from amino acids 448 to 502 as follows:

(67) TABLE-US-00011 (SEQ ID NO: 138) HHHH 450 HH*

(68) IFN-Beta/his (23.2 kDa)

(69) TABLE-US-00012 (SEQ ID NO: 139) MNNRWILHAAFLLCFSTTALSINYKQLQLQERTNIRKCQELLEQLNGKIN 50 LTYRADFKIPMEMTEKMQKSYTAFAIQEMLQNVFLVFRNNFSSTGWNETI 100 VVRLLDELHQQTVFLKTVLEEKQEERLTWEMSSTALHLKSYYWRVQRYLK 150 LMKYNSYAWMVVRAEIFRNFLIIRRLTRNFQNDIHHHHHH*

(70) Within this sequence, the IFN-beta portion is from amino acids 1 to 184 as follows:

(71) TABLE-US-00013 (SEQ ID NO: 140) MNNRWILHAAFLLCFSTTALSINYKQLQLQERTNIRKCQELLEQLNGKIN 50 LTYRADFKIPMEMTEKMQKSYTAFAIQEMLQNVFLVFRNNFSSTGWNETI 100 VVRLLDELHQQTVFLKTVLEEKQEERLTWEMSSTALHLKSYYWRVQRYLK 150 LMKYNSYAWMVVRAEIFRNFLIIRRLTRNFQNDI

(72) The His tag is from amino acids 185 to 190 as follows:

(73) TABLE-US-00014 (SEQ ID NO: 138) HHHHHH*

(74) The protocol for expression of the constructs is shown in FIG. 11.

(75) Briefly, the constructs were transformed into competent DH10Bac cells (Invitrogen) to generate bacmid vectors. Recombinant bacmid vectors were confirmed by blue-white screening and PCR according to Invitrogen instructions. Bacmid DNA was transfected into Sf9 insect cells using cellfectin according to Invitrogen instructions.

(76) Baculovirus (P1) was harvested from the supernatant of transfected cells, and used to infect fresh Sf9 cells to amplify the virus stocks. P3 virus was used to infect High 5 insect cells for 72 hours, and the supernatant was collected and run on an SDS-PAGE gel. Recombinant proteins were detected by Western blot using anti-tetra-His antibody (Qiagen) and anti-mouse HRP (Sigma).

(77) The test expression of the fusion constructs is shown in FIG. 12.

(78) Fusion Proteins: 1-11E/C7 with TNFR2-Fc

(79) Cloning of TNFR2-Fc/1-11E and TNFR2-Fc/C7

(80) pFastBac1.AH was created from pFastBac1 (Invitrogen) by cutting out BamHI/HindIII fragment containing multiple cloning sites (MCS), and replacing with a linker to give another MCS of BamHI-KpnI-HindIII-ApaI.

(81) TNFR2Fc was cloned into the HindIII-EcoRI sites, followed by a MMP cleavage site and scFv (1-11E or C7) which were cloned into the NotI and ApaI sites as shown in FIG. 10B.

(82) Mouse TNFR2-Fc was amplified with the following primers:

(83) TABLE-US-00015 forward primer: (SEQ ID NO: 141) 5′ GCT aag ctt ATG GCG CCC GCC GCC CTC 3′ reverse primer: (SEQ ID NO: 142) 5′ CTTGAATTCTTTACCCAGAGACCGGGA 3′

(84) 1-11E was amplified with the same primers as above for the INFb.

(85) The sequence of TNFR2Fc/MMP/1-11E is as follows:

(86) TABLE-US-00016 (SEQ ID NO: 143) MAPAALWVALVFELQLWATGHTVPAQVVLTPYKPEPGYECQISQEYYDRK AQMCCAKCPPGQYVKHFCNKTSDTVCADCEASMYTQVWNQFRTCLSCSSS CTTDQVEIRACTKQQNRVCACEAGRYCALKTHSGSCRQCMRLSKCGPGFG VASSRAPNGNVLCKACAPGTFSDTTSSTDVCRPHRICSILAIPGNASTDA VCAPESDGSPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTC VVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQ DWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKK EFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLR VQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK---EFGGGGSPLGL WAGGGSAAA---MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSW VRQAPGKGLEWVSSIDDSGATTYYADSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCAKNYSSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQM TQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYYASSL QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAANYPTTFGQGTKV EIKRDIHHHHHH

(87) Of this sequence, the TNFR2Fc portion is as follows:

(88) TABLE-US-00017 (SEQ ID NO: 144) MAPAALWVALVFELQLWATGHTVPAQVVLTPYKPEPGYECQISQEYYDRK 50 AQMCCAKCPPGQYVKHFCNKTSDTVCADCEASMYTQVWNQFRTCLSCSSS 100 CTTDQVEIRACTKQQNRVCACEAGRYCALKTHSGSCRQCMRLSKCGPGFG 150 VASSRAPNGNVLCKACAPGTFSDTTSSTDVCRPHRICSILAIPGNASTDA 200 VCAPESDGSPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTC 250 VVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQ 300 DWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKK 350 EFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLR 400 VQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK*

(89) The MMP linker portion is as follows:

(90) TABLE-US-00018 (SEQ ID NO: 136) EFGGGGSPLGLWAGGGSAAA

(91) The 1-11E portion is as follows:

(92) TABLE-US-00019 (SEQ ID NO: 137) MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE WVSSIDDSGATTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AKNYSSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSAS VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYYASSLQSGVPSRFSG SGSGTDFTLTISSLQPEDFATYYCQQAANYPTTFGQGTKVEIKRDI

(93) The His tag is as follows:

(94) TABLE-US-00020 (SEQ ID NO: 138) HHHHHH*

(95) As a negative control a non specific scFv was developed that binds to Hen Egg Lysosyme (HEL). Clone C7 was the best expressed and was taken forward for TNFR2Fc fusion as done for 1-11E.

(96) Sequence of TNFR2Fc/MMP/C7:

(97) TABLE-US-00021 (SEQ ID NO: 147) MAPAALWVALVFELQLWATGHTVPAQVVLTPYKPEPGYECQISQEYYDRK AQMCCAKCPPGQYVKHFCNKTSDTVCADCEASMYTQVWNQFRTCLSCSSS CTTDQVEIRACTKQQNRVCACEAGRYCALKTHSGSCRQCMRLSKCGPGFG VASSRAPNGNVLCKACAPGTFSDTTSSTDVCRPHRICSILAIPGNASTDA VCAPESDGSPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTC VVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQ DWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKK EFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLR VQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGKEFGGGGSPLGLWAG GGSAAAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPG KGLEWVSTISYAGASTAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCAKTSTSFDYWGQGTLVTVSTDIQMTQSPSSLSASVGDRVTITCRAS QSISSYLNWYQQKPGKAPKLLIYNASYLQSGVPSRFSGSGSGTDFTLTIS SLQPEDFATYYCQQAYAGPYTFGQGTKVEIKRDIHHHHHH*

(98) Of this sequence, the TNFR2Fc portion is as follows:

(99) TABLE-US-00022 (SEQ ID NO: 148) MAPAALWVALVFELQLWATGHTVPAQVVLTPYKPEPGYECQISQEYYDRK 50 AQMCCAKCPPGQYVKHFCNKTSDTVCADCEASMYTQVWNQFRTCLSCSSS 100 CTTDQVEIRACTKQQNRVCACEAGRYCALKTHSGSCRQCMRLSKCGPGFG 150 VASSRAPNGNVLCKACAPGTFSDTTSSTDVCRPHRICSILAIPGNASTDA 200 VCAPESDGSPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTC 250 VVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQ 300 DWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKK 350 EFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLR 400 VQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK*

(100) The MMP linker portion is as follows:

(101) TABLE-US-00023 (SEQ ID NO: 149) EFGGGGSPLGLWAGGGSAAA

(102) The C7 portion is as follows:

(103) TABLE-US-00024 (SEQ ID NO: 150) MAEVQLLESGGGLVQPGGSLRLSCAASGFT FSSYAMSWVRQAPGKGLEWVSTISYAGASTAYADSVKGRFTISRDNSKNT LYLQMNSLRAEDTAVYYCAKTSTSFDYWGQGTLVTVSTDIQMTQSPSSLS ASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYNASYLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQAYAGPYTFGQGTKVEIKRDI

(104) The His tag is as follows:

(105) TABLE-US-00025 (SEQ ID NO: 138) HHHHHH*

(106) The protocol for expression of the constructs is shown in FIG. 11 and is as described above for the IFN-beta/1-11E fusion protein.

(107) Infected Hi-5 cells were grown for 3 days at 27° C. After 3 days, different 100, 50, 25 and 12 microliter aliquots of cell supernatant were taken for Western blot analysis. Fusion protein was probed with anti-His tag antibodies. As shown in FIG. 13 the apparent molecular weight of the TNFR2Fc fusion proteins is slightly above 75 kDa which reflects the predicted 50 kDa TNFR2Fc plus 30 kDa scFv.