ANTI-TENASCIN C ANTIBODIES AND USES THEREOF
20170306005 · 2017-10-26
Inventors
- Kim Suzanne Midwood (Buckinghamshire, GB)
- Philip Antony Bland-Ward (London, GB)
- Nigel Burns (London, GB)
- Patrick John Hextall (Cambridge, GB)
- Susan Rebecca Aungier (Oxford, GB)
Cpc classification
A61P1/04
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
C07K14/78
CHEMISTRY; METALLURGY
A61P17/02
HUMAN NECESSITIES
C07K2319/30
CHEMISTRY; METALLURGY
A61P9/10
HUMAN NECESSITIES
C07K2317/33
CHEMISTRY; METALLURGY
A61K39/3955
HUMAN NECESSITIES
C07K2317/92
CHEMISTRY; METALLURGY
A61P1/16
HUMAN NECESSITIES
A61P17/16
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
C07K2317/76
CHEMISTRY; METALLURGY
A61P37/06
HUMAN NECESSITIES
International classification
Abstract
There is provided antibodies or antigen-binding fragments, derivatives or variants thereof which are capable of binding to the FBG domain of tenascin-C. There are also provided uses of such antibodies or antigen-binding fragments, derivatives or variants thereof, as well as methods of identifying such antibodies.
Claims
1-88. (canceled)
89. An antibody or binding fragment comprising a VH region wherein CDRH1 is SEQ ID NO: 9, CDRH2 is SEQ ID NO: 10, CDRH3 is independently selected from SEQ ID NO: 11, 30, 32, 34, 36, 38, 40, 42, 44, and 46, and a VL region wherein a CDRL1 is SEQ ID NO 5, CDRL2 is SEQ ID NO: 12 and CDRL3 is SEQ ID NO: 14.
90. An antibody or binding fragment according to claim 89, wherein CDRH3 is SEQ ID NO: 11.
91. An antibody or binding fragment according to claim 90, wherein the VH region comprises SEQ ID NO: 12.
92. An antibody or binding fragment according to claim 89, wherein CDH3 is SEQ ID NO: 30.
93. An antibody or binding fragment according to claim 92, wherein the VH region comprises SEQ ID NO: 31.
94. An antibody or binding fragment according to claim 89, wherein CDH3 is SEQ ID NO: 32.
95. An antibody or binding fragment according to claim 94, wherein the VH region comprises SEQ ID NO: 33.
96. An antibody or binding fragment according to claim 89, wherein CDH3 is SEQ ID NO: 34.
97. An antibody or binding fragment according to claim 96, wherein the VH region comprises SEQ ID NO: 35.
98. An antibody or binding fragment according to claim 89, wherein CDH3 is SEQ ID NO: 36.
99. An antibody or binding fragment according to claim 98, wherein the VH region comprises SEQ ID NO: 37.
100. An antibody or binding fragment according to claim 89, wherein CDH3 is SEQ ID NO: 38.
101. An antibody or binding fragment according to claim 100, wherein the VH region comprises SEQ ID NO: 39.
102. An antibody or binding fragment according to claim 89, wherein CDH3 is SEQ ID NO: 40.
103. An antibody or binding fragment according to claim 102, wherein the VH region comprises SEQ ID NO: 41.
104. An antibody or binding fragment according to claim 89, wherein CDH3 is SEQ ID NO: 42.
105. An antibody or binding fragment according to claim 104, wherein the VH region comprises SEQ ID NO: 43.
106. An antibody or binding fragment according to claim 89, wherein CDH3 is SEQ ID NO: 44.
107. An antibody or binding fragment according to claim 106, wherein the VH region comprises SEQ ID NO: 45.
108. An antibody or binding fragment according to claim 89, wherein CDH3 is SEQ ID NO: 46.
109. An antibody or binding fragment according to claim 108, wherein the VH region comprises SEQ ID NO: 47.
110. An antibody or binding fragment according to claim 89, wherein the VL region comprises SEQ ID NO: 15.
111. An antibody or binding fragment according to claim 89, wherein the VL region comprises SEQ ID NO: 125.
112. A composition comprising an antibody or binding fragment of claim 89 and a pharmaceutically acceptable carrier, excipient and/or diluent.
113. A method of treating a chronic inflammatory condition comprising administering to a subject an effective amount of an antibody or binding fragment of according to claim 89.
Description
[0312] Examples embodying an aspect of the invention will now be described with reference to the following figures:
[0313]
[0314]
[0315]
[0316] Polyclonal derived 2nd round output phage were incubated with wells coated with antigen or fusion partner (Fc or Cd4) and bound phage detected with anti-M13 mAb and Europium-labelled anti-mouse antibody. There is enrichment of antigen-specific binders between rounds 1 and 2 of selection and a greater proportion of huFBG binders compared to anti-Fc or -rCd4 phage in the round 2 output populations.
[0317]
[0318] Antibodies were tested at the highest concentration achievable. In a confirmatory assay of purified scFv-Fc clones, 2A3, 2A5, 2611 and 2D12 were identified as effective blockers of signalling evoked by 10 nM Fc-His-huFBG (a). Assay of purified anti-FBG FAbs highlighted a number of additional hits for further analysis including antibodies A12, B12, C2, D7, D8, F3 and G1. In this experiment cells were stimulated with 3 nM Fc-His-huFBG (b).
[0319]
[0320] Binding was detected using anti-kappa or anti-lambda mAb followed by Europium-conjugated anti-mouse mAb.
[0321]
[0322] Traces indicate binding of human, rat and mouse tenascin-C rCd4-FBG and human tenascin-R rCD4-FBG.
[0323]
[0324] Purified antibodies were incubated with THP1-Blue cells in the presence of 3 nM human Fc-His-FBG for 18 h at 37° C. IC50 values for inhibition of Fc-His-FBG evoked SEAP were B12 (1.7 nM), 2A5 (20.6 nM), D8 (7.2 nM), F3 (8.4 nM).
[0325]
[0326] Purified antibodies were incubated with THP1-Blue cells in the presence of 3 nM human Fc-His-FBG for 18 h at 37° C. IC50 values for inhibition of Fc-His-FBG evoked IL-8 release were B12 (6.9 nM), 2A5 (28.5 nM), D8 (14.9 nM), F3 (13.8 nM).
[0327]
[0328] VH CDR3 randomisation was done in three overlapping blocks of 6 residues each (labelled VH 3.1, VH 3.2, and VH 3.3) and the VL CDR3s were randomised in blocks of two (labelled VL 3.1 and VL 3.2). Arrows indicate the positions of stop codons introduced into the template DNA to eliminate parental clones dominating the library. (B) Oligonucleotides used for CDR3 library generation.
[0329]
[0330] (a) Selections on human rCd4-His-FBG using CDR randomised libraries. (b) Hybrid selections on human and mouse FBG using B12 VH and VL CDR 3 randomised libraries.
[0331]
[0332]
[0333] Primary human PBMCs were incubated (37° C., 24 h) in the presence of 200 nM human Fc-His-FBG and test antibody (100 nM or 1 pM) and supernatants were assayed for IL-8 (a) and TNFa (b). All test antibodies blocked evoked cytokine release. Data indicate mean±s.e. mean of results from 3 separate donors.
[0334]
[0335] Specific staining of the synovium was seen with positive control anti-tenascin-antibody (a), and B12 anti-FBG formatted as mouse IgG2a (c). Lower levels of non-specific staining were observed with non-immune isotype control antibodies (b, d).
[0336]
[0337] Recombinant TNC-FBG (Nascient), TNR-FBG or FIBRINOGEN (Kennedy Institute of Rheumatology (KIR)) detected with the following antibodies A) 165_13_C3 IgG4 MAb at 1:20,000 (0.25 ug/ml), overnight at 4° C. B) B12 IgG4 MAb at 1:20,000 (0.25 ug/ml), overnight at 4° C. C) Anti-Tenascin-R antibody (Santa Cruz Biotechnology, sc-9875) at 1:2,000 (0.1 ug/ml) overnight at 4° C. D) Anti-TNC-FBG polyclonal antibody (Midwood group) at 1:500, overnight at 4° C.
[0338]
[0339] Glioma cell lysate (KIR) and tenascin-C(Nascient) detected with A. B12 IgG4 Mab at 1:20,000, overnight at 4° C.; B. IgG4 isotype control (Eureka therapeutics) at 1:4,000, overnight at 4° C.
[0340]
[0341] Recombinant human TLR4 (R&D systems) in PBS (or PBS alone) was bound to a 96-well plate, after blocking the indicated concentrations of Human Fc-His-FBG. was added and detection was carried out by incubation of an anti-human IgG1 MAb (AbD Serotec, clone 2C11) at 1 ug/ml, an anti-mouse HRP conjugated secondary antibody (AbD Serotec, STAR13B) at 1 ug/ml, and TMB substrate. n=4 mean and SEM shown.
[0342]
[0343]
[0344] (A) Recombinant Human tenascin-C FBG (1 uM) or LPS (Enzo) (1 ng/ml) was pre-incubated for 30 min at RT with MAb C3 (1, 0.2, and 0.04 uM) or isotype control MAb (1 uM) before being added in triplicate to Human M2 macrophage cultures. After 24 h supernatants were taken and subjected to IL-8, IL-6 and TNF cytokine ELISA (BD Biosciences). n=3; (B) Recombinant Murine tenascin-C FBG (1 uM) was pre-incubated for 30 min at RT with MAb C3 (1, 0.2 and 0.04 uM) or isotype control MAb (1 uM) before being added in triplicate to Human M2 macrophage cultures. After 24 h supernatants were taken and subjected to cytokine ELISA. n=3 or over, mean and SEM shown; (C) A protein where the Fc portion is mutated to be inactive (Fc-Mut-His-FBG) was used. Other promising anti-TNC-FBG antibodies, B12 and A4 were also tested in this system. Fc-Mut-His-FBG (1 uM) and C3, 160_01_A4 or B12 (1 uM) were pre-incubated for 30 min at RT before being added to human M2 macrophage cultures. After 24 h supernatants were taken and subjected to cytokine ELISA. n=3, mean and SEM shown.
[0345]
[0346] Recombinant Human tenascin-C FBG (1 uM) was pre-incubated with MAb B12 (1, 0.1, 0.01 or 0.001 uM) or isotype control MAb (1 uM) before being added in triplicate to Human M2 macrophage cultures. After 24 h supernatants were taken and subjected to cytokine ELISA.
[0347]
[0348] The potency of the antibody produced at larger scale was compared to that produced by at laboratory scale. Recombinant Human tenascin-C FBG (1 uM) was pre-incubated for 30 min at RT with MAb C3 (1, 0.2 and 0.04 uM) or isotype control MAb (1 uM) before being added in triplicate to Human M2 macrophage cultures. After 24 h supernatants were taken and subjected to cytokine ELISA. n=1, Ico=laboratory scale, Lon=larger scale.
[0349]
[0350] Recombinant Human tenascin-C FBG (1 uM) was pre-incubated for 30 min at RT with MAb C3 or isotype control MAb before being added in triplicate to Human synovial fibroblast cultures from RA patients. After 24 h supernatants were taken and subjected to cytokine ELISA. n=1, mean and SEM shown
[0351]
[0352] TNC levels in synovial fluid from a rat model of collagen induced arthritis. The amount of TNC measured is shown plotted against the corresponding clinical score for each paw.
[0353]
[0354] Vehicle versus 1 mg/kg, 3 mg/kg and 10 mg/kg C3 antibody. Data are presented as Mean±SEM. Statistical significances: #### p<0.0001 when compared to Day 7, ** p<0.01 when compared to the vehicle-treated group.
[0355]
[0356] Vehicle versus 1 mg/kg, 3 mg/kg and 10 mg/kg C3 antibody. Data are presented as Mean±SEM. Statistical significances: ## p<0.01 and #### p<0.0001 when compared to Day 0, * p<0.05 and ** p<0.01 when compared to the vehicle-treated group.
[0357]
[0358] This table details the primers used to generate expression constructs for use in antigen cloning.
EXAMPLE 1—GENERATION OF PURIFIED TENASCIN-C FBG AS ANTIGEN AND ASSAY REAGENTS
[0359] Purified soluble proteins containing the FBG domain of tenascin-C (TNC FBG) were generated for use as antigens in antibody selections and as reagents in subsequent screening and characterisation assays. To enable selection strategies for isolation of antibodies that bind tenascin-C of multiple mammalian species, a range of DNA expression constructs were synthesised, which incorporated the TNC FBG domain of either human [SEQ ID NO: 92], mouse [SEQ ID NO: 93], rat [SEQ ID NO: 94] or dog [SEQ ID NO: 95]. A human tenascin-R FBG [SEQ ID NO: 96] construct was also prepared for identification of antibodies that displayed unwanted binding to this homologue. Constructs were produced as 6His-tagged proteins with either a rat CD4 or human IgG1 Fc tag coupled to either a C- or N-terminal FBG domain as described below.
Protein Expression Constructs
[0360] All synthetic DNA constructs for antigen expression were synthesised and sequence confirmed by Genscript (Piscataway, USA). FBG domains were cloned into the mammalian expression vectors pBIOCAM4 or BIOCAM5, which fuse the expressed domains with either a rat Cd4 (domains 3 and 4) tag (Chapple et al, 2006) or a human IgG1 Fc tag (Falk et al, 2012) respectively. The vectors were modified from the pCMV/myc/ER plasmid (Invitrogen) (Falk et al, 2012), which contains an endoplasmic reticulum (ER) signal sequence derived from the mouse VH chain, for secretion of expressed proteins. For all constructs which resulted in an N-terminal FBG (e.g. FBG-Fc-His or FBG-rCd4-His) the digested PCR products were ligated with NcoI/NotI cut pBIOCAM4 or pBIOCAM5 vectors. For all constructs which resulted in a C-terminal FBG (e.g. Fc-His-FBG or rCd4-His-FBG), digested PCR products were ligated with BamHI/HindIII cut pBIOCAM4 or pBIOCAM5 vectors. The primers used to amplify the FBG domains are listed in
[0361] Protein expression and cell culture Transfection quality plasmid DNA was prepared using the Machery Nagel Nucleobond Xtra Midi kit (740410.50, Fisher Scientific, UK). HEK293F suspension cells and Freestyle media, for antigen and antibody expression, and RPMI media were from Life Technologies (Paisley, UK). Transfection of HEK293F cells was carried out as described previously (Chapple et al, 2006).
Protein Purification and QC
[0362] Protein affinity purification employed either Ni-NTA agarose or immobilised recombinant protein A resin.
[0363] For purification of His-tagged proteins, culture supernatants were mixed with Ni-NTA agarose (1018240, Qiagen, Crawley, UK) for 1 h and the resin transferred to Proteus 1-step midi spin columns (Generon, UK) for centrifugation (200×g, 2 min). Unbound proteins were washed out with phosphate buffered saline (PBS) supplemented with 20 mM imidazole (pH 8). Bound proteins were eluted in fractions through addition of 300 mM imidazole in PBS (pH 8) and column centrifugation (200×g, 2 min). Pooled fractions containing eluted protein were placed in Gebaflex Midi dialysis tubes (Generon D010; molecular weight cut-off 3.5 kDa) and dialysed against PBS.
[0364] Fc-tagged proteins and antibodies expressed as human IgG4 were purified using protein A sepharose (PC-A25, Generon, Maidenhead, UK). Culture supernatants were clarified by centrifugation (2500×g, 15 min) and mixed with protein A sepharose overnight at 4° C. before transfer of the resin to Proteus 1-step midi spin columns (Generon, UK). Columns were centrifuged (200×g, 2 min) and washed with PBS to remove unbound protein. Fc-tagged or IgG4 proteins were eluted in fractions from the protein A with 0.2 M glycine (pH 2.8) into Tris-HCl (pH 8) by centrifugation (200×g, 2 min). Eluted fractions were pooled and dialysed against PBS in Gebaflex Maxi dialysis tubes (Generon D045; molecular weight cut-off 8 kDa).
[0365] Proteins were analysed for purity and concentration by SDS-PAGE (4-12% gel) and spectrophotometry (OD280 using theoretical extinction coefficient). Where purified proteins were used in cell-based assays the endotoxin content was first determined by limulus amoebocyte lysate chromogenic endotoxin assay (Pierce). Proteins were not used if endotoxin levels exceeded 1 endotoxin unit per milligram (i.e. 1 EU/mg).
EXAMPLE 2—ISOLATION OF PRIMARY ANTI-FBG ANTIBODIES
Antibody Phage Display
[0366] Antibodies against tenascin-C FBG domain were isolated using the Iontas Ltd proprietary human antibody phage display library, which was constructed using DNA isolated from 43 human lymphocyte donors. Selections, phage rescues and subcloning into pSANG10 (Martin et al, 2006) were all performed as described previously (Schofield et al, 2007) using techniques that are well known in the art.
[0367] Two rounds of panning selections were performed on immobilised TNC FBG fused to human IgG1 Fc or rCd4 at either the N terminus of the fusion partner (e.g. FBG-Fc, FBG-rCd4) or at the C terminus (Fc-FBG, rCd4-FBG). Phage antibody libraries containing either kappa (κ) or lambda (λ) variable light chains (V.sub.L) were panned separately to facilitate later sub-cloning to Fab expression vectors containing either constant light (C.sub.L) kappa (κ) or lambda (λ) chains.
[0368] Polyclonal phage populations were prepared from the selected populations and were tested in ELISA (polyclonal phage ELISA) using ELISA plates coated with TNC FBG antigen or appropriate fusion partner (Fc or rCd4). After incubation with phage, plates were washed, and bound phage detected using peroxidase-conjugated anti-M13 antibodies.
Confirmation of scFv Binding to Antigen and Cross-Reactivity Assay by ELISA
[0369] Round 2 selection outputs were expressed as individual scFv clones to confirm antigen recognition in ELISA binding assays. Output populations were sub-cloned into the bacterial expression vector pSANG10 (Martin et al, 2006), transformed into E. coli BL21 (DE3), and individual transformants were induced in 96-well plates as described previously (Schofield et al, 2007). E. coli supernatants were collected and assayed for binding of scFv to TNC FBG using DELFIA-based ELISA, using europium-labelled anti-FLAG detection antibodies. Results for initial ELISAs are summarised in Table 1.
TABLE-US-00002 TABLE 1 Monoclonal scFv ELISA. Values indicate number of clones binding to the relevant immobilised selection antigen. FBG Binders ELISA Signal No. Tag (Fluorescence Units; FU) Selection (ID) screened binders ≧1,000 ≧10,000 ≧100,000 λ FBG-rCd4 95 0 0 0 0 (145) λ FBG-Fc (146) 95 3 0 0 0 λ rCd4-FBG 95 0 14 4 0 (147) λ Fc-FBG (148) 95 1 13 5 1 κ FBG-rCd4 95 0 20 8 1 (150) κ FBG-Fc (151) 95 0 2 1 0 κ rCd4-FBG 95 8 12 4 0 (152) κ Fc-FBG (153) 95 8 10 2 0 λ+ κ FBG-rCd4, 95 0 6 3 1 Fc λ+ κ rCD4-FBG, 95 0 2 1 0 Fc Total 79 28 3
[0370] The most successful selections with the A library were based on panning against the antigens rCd4-FBG and Fc-FBG (selections 147 and 148). For the K library, the most successful selections were obtained with the antigens FBG-rCd4 (150), rCd4-FBG (152) and Fc-FBG (153). The 79 positive clones from this ELISA screen were selected for further analysis.
[0371] Cross-reactivity ELISA showed that 67/79 (85%) of anti-human FBG scFv were cross-reactive to mouse TNC FBG. DNA sequence analysis of the anti-FBG scFv indicated excellent sequence diversity. For example, selections 147 and 148 from the V.sub.L λ library contained 92% unique variable heavy (V.sub.H) complementarity determining region 3 (CDR3) sequences, and selections 150, 152 and 153 from the V.sub.L κ library contained 67%, 91% and 100% unique variable V.sub.H CDR3 sequences, respectively.
[0372] A further 1425 clones isolated from the most effective selections were screened by ELISA and this resulted in the identification of an additional 401 scFv with FBG-binding specificity (Table 2). These clones, together with the 79 scFv identified in initial ELISAs were chosen for further evaluation.
TABLE-US-00003 TABLE 2 Focused monoclonal scFv ELISA of the most effective selection outputs. Tag FBG Selection (ID) No. screened Hits (≧5,000 FU) binders binders λ rCd4-FBG (147) 285 66 0 66 λ Fc-FBG (148) 285 60 0 60 κ FBG-rCd4 (150) 285 86 0 86 κ rCd4-FBG (152) 285 144 2 142 κ Fc-FBG (153) 285 94 47 47 Total 1425 450 49 401
[0373] The 1425 clones were further tested in a specificity ELISA in which each scFv was tested for binding to human Tenascin R FBG and also to human, mouse, rat and dog TNC FBG. Clones were ranked according to the ELISA signal obtained for binding to Tenascin C divided by the signal for Tenascin R FBG binding. The top 250 clones with a ratio above 50 were taken for subcloning and further analysis.
EXAMPLE 3—SCREENING OF PRIMARY ANTI-FBG ANTIBODIES IN A FUNCTIONAL ASSAY
[0374] Anti-FBG scFv were reformatted either as bivalent scFv-Fc or as monomeric Fabs for evaluation of their activity as inhibitors of FBG-evoked signalling in a whole cell assay system.
[0375] The top 50 anti-FBG scFv, ranked by primary ELISA signal, for each of the selections 147, 148, 150, 152 and 153 were sub-cloned into the mammalian expression plasmid pBIOCAM5 (Falk et al, 2012) as individual selection populations and expressed by transient transfection in HEK293F cells (Chapple et al, 2006). For Fab expression, pooled A or K scFv variable heavy (V.sub.H) and variable light (V.sub.L) inserts were cloned into a dual promoter Fab expression vector (pFab-dual-κ or pFab-dual-Δ, depending on the light chain germ-line) using a proprietary Iontas Ltd protocol. Culture supernatants were screened for activity in the THP-1 cell assay and selected scFV-Fc and Fab hits were affinity purified for re-assaying and confirmation of inhibitory activity.
THP1-Blue™ Reporter Cell Assay
[0376] Tenascin-C has been shown to elicit the generation of cytokines in inflammatory cells and fibroblasts by interaction of the FBG domain with cellular TLR4 (Midwood et al, 2009). The receptor signalling cascade leading to generation of inflammatory cytokines such as TNFa, IL-8 and IL-6 involves activation of the transcription factor NF-κB. This process can be studied in ‘reporter’ cell lines modified to respond to NF-κB activation with generation of an easily measured protein signal. The THP1-Blue™ reporter cell line (InvivoGen; Toulouse, France) is derived from the human THP-1 monocyte cell line and stably expresses an NF-κB-inducible secreted alkaline phosphatase (SEAP) reporter construct. These cells also constitutively express cell surface TLR4, which enables the signalling activity of TNC FBG fusion proteins to be readily measured using colorimetric or fluorimetric quantitation of SEAP in culture supernatants using medium- to high-throughput assay methods.
[0377] Activity at low FBG concentrations is critical to the success of any screening assay; if the concentrations of FBG required to produce a robust increase in the reporter protein are too high then the expression levels and concentrations of scFv, Fc-ScFv or Fab constructs required to fully inhibit any such signal would be unacceptable for a screen. Fc-FBG produces a robust SEAP signal at low nM levels in this cell assay (CD4-FBG did not produce a response in this concentration range).
[0378] THP1-Blue™ cells were cultured and passaged in supplemented RPMI media according to supplier's protocols (http://www.invivogen.com/PDF/THP1_Blue_NF_kB_TDS.pdf), except that cells were grown in ultra-low attachment T75 flasks. For assays, THP1-Blue™ cells were added to 96-well tissue culture plates (100,000 cells/well) containing Fc-FBG (3 or 10 nM) in RPMI medium in a total volume of 170 μl. Culture supernatants containing expressed scFv-Fc or Fab, or affinity purified antibody in PBS, was added in a volume of 30 μl and cells were incubated for 18 h at 37° C. Supernatants were harvested and assayed for either SEAP using the Attophos AP fluorimetric quantitation system (S1000; Promega) or IL-8 content using the DuoSet ELISA development system (DY208; R&D Systems, UK) according to the supplier's instructions. Data were plotted and curves fitted using Prism software (GraphPad).
[0379] Screening of anti-FBG antibodies as HEK293F culture supernatants highlighted putative inhibitors of Fc-His-FBG evoked signalling in THP1-Blue™ cells of which 9 were confirmed when re-assayed as purified scFv-Fc (
EXAMPLE 4—FUNCTIONAL CHARACTERISATION OF PRIMARY ANTI-FBG ANTIBODIES
ELISA Cross-Reactivity Assays
[0380] The panel of 9 human FBG signalling inhibitors identified in the THP1-Blue™ functional assay was evaluated by ELISA for cross-reactivity to rat, mouse, and dog FBG. Binding to the human tenascin-R FBG homologue was also determined. Assay wells were coated with human, rat, mouse, and dog TNC FBG-rCD4, or human TNR FBG-rCd4 fusion proteins and binding of Fabs was detected using anti-kappa or anti-lambda mAb followed by Europium-conjugated anti-mouse mAb. ELISA results revealed that 4 Fabs displayed good cross-reactivity to other mammalian homologues of human TNC FBG, with lower apparent binding to human TNR FBG (
Fab 2A5 (VH SEQ ID NO: 4; VL SEQ ID NO: 8),
Fab B12 (VH SEQ ID NO: 12; VL SEQ ID NO: 15),
Fab D8 (VH SEQ ID NO: 19; VL SEQ ID NO: 21), and
Fab F3 (VH SEQ ID NO: 25; VL SEQ ID NO: 29).
[0381] Fabs that showed poor species cross-reactivity to TNC-FBG were not considered further.
Determination of Binding Affinity by Surface Plasmon Resonance
[0382] The affinity and association and dissociation kinetics of selected Fabs for binding to the human, rat and mouse TNC FBG, and human TNR FBG were measured by surface plasmon resonance (SPR) at 25° C. Experiments were performed using a BIAcore T100 instrument with CM5 sensor chip according to the protocol provided with the Human Fab Capture Kit (GE, 28-9583-25). Varying concentrations of rCd4-FBG were injected into a flow-cell with immobilised Fab and a reference flow-cell. After reference signal subtraction, the data was fitted to a global 1:1 fit using the T100 BIAevaluation software (
[0383] The calculated kinetic constants are shown in Table 3. The rank order of affinity of Fabs for human TNC FBG was B12 (110 pM)>D8 (8.49 nM)>2A5 (11.4 nM)>F3 (27.4 nM). All Fabs displayed low nanomolar affinity for rodent TNC FBG, and affinities for human TNR FBG were typically greater than 60-fold lower than human TNR FBG.
Inhibitory Potency Assays
[0384] The potency of purified Fabs for neutralisation of huFc-His-FBG activity was determined in the THP1-Blue™ assay, using measures of TLR4-mediated secreted alkaline phosphatase and IL-8 cytokine production. Assays were conducted as described in Example 2, except that purified Fabs were added to assay wells at a range of concentrations (0.3-100 nM) to enable calculation of IC.sub.50 values using Prism software (GraphPad).
TABLE-US-00004 TABLE 3 Anti-FBG Fab binding kinetic data determined by surface plasmon resonance (SPR) spectroscopy. Kinetics K.sub.D K.sub.a K.sub.d Steady Fab FBG (nM) (M.sup.−1s.sup.−1) × 10.sup.5 (s.sup.−1) × 10.sup.−4 State 2A5 Hu TNC 11.4 4.96 56.3 N/A Mu TNC 78.6 4.41 346.5 N/A Hu TNR 757 2.49 1888.4 706 B12 Hu TNC 0.111 26.62 3.0 N/A Mu TNC 13 52.15 675.5 18.7 Rat TNC 7.9 94.59 747.9 N/A Hu TNR 33.9 13.96 472.5 36.1 D8 Hu TNC 8.49 15.41 130.9 N/A Mu TNC 48.4 14.78 716.1 41.2 Hu TNR 1026 5.55 5696.0 913 F3 Hu TNC 27.4 1.26 34.6 N/A Mu TNC 70.6 0.91 64.2 N/A Hu TNR Off rate too rapid to determine 1808 K.sub.D, equilibrium dissociation constant; K.sub.a, association constant; K.sub.d, dissociation constant
[0385] All antibodies displayed concentration-related inhibition of Fc-His-HuFBG-evoked alkaline phosphatase (
EXAMPLE 5—GENERATION AND ISOLATION OF OPTIMISED ANTIBODIES TO huTNC FBG DOMAIN
Affinity Maturation by Targeted CDR Mutagenesis
[0386] Anti-FBG antibodies 2A5, B12, and F3 were selected for affinity maturation. Targeted CDR mutagenesis was carried out by randomising VH and VL CDR3 residues in blocks of 6 amino acids using Kunkel mutagenesis (Fellouse and Sidhu, 2007; Kunkel et al., 1987; Sidhu and Weiss, 2004). Due to the longer VH CDR3s (10-16 residues) for the given clones randomisation was done in three overlapping blocks and the VL CDR3s (9 residues) were randomised in two overlapping blocks (
TABLE-US-00005 TABLE 4 Estimated sizes of the CDR3 randomised libraries Library Sub library Size Combined size 2A5 VH 2A5 VH 3.1 2.0 × 10.sup.9 7.2 × 10.sup.9 2A5 VH 3.2 2.6 × 10.sup.9 2A5 VH 3.3 2.6 × 10.sup.9 2A5 VL 2A5 VL 3.1 4.0 × 10.sup.9 6.5 × 10.sup.9 2A5 VL 3.2 2.5 × 10.sup.9 B12 VH B12 VH 3.1 1.8 × 10.sup.9 6.1 × 10.sup.9 B12 VH 3.2 1.6 × 10.sup.9 B12 VH 3.3 1.7 × 10.sup.9 B12 VL B12 VL 3.1 2.6 × 10.sup.9 7.7 × 10.sup.9 B12 VL 3.2 5.1 × 10.sup.9 F3 VH F3 VH 3.1 6.0 × 10.sup.9 1.6 × 10.sup.9 F3 VH 3.2 4.6 × 10.sup.9 F3 VH 3.3 6.3 × 10.sup.9 F3 VL F3 VL 3.1 2.1 × 10.sup.9 5.7 × 10.sup.9 F3 VL 3.2 3.6 × 10.sup.9
High Stringency Phage Display Selections
[0387] Phage-antibody selections on streptavidin Dynabeads were performed as described previously (Dyson et al, 2011). Multiple rounds of solution-phase selections were carried out on biotinylated rCd4-His-FBG to enrich for affinity improved clones. The optimum antigen concentrations for each round were determined empirically by selecting against a range of antigen concentrations and comparing the output numbers with a no-antigen control. The stringency of selection was increased by reducing the amount of antigen used in each round. No further rounds of selection were carried out after the selection window (the fold difference between phage titres from selection outputs and no antigen control) dropped below 10. Hence, three rounds of selection (
[0388] The selected populations were sub-cloned into the bacterial expression vector pSANG10 (Martin et al, 2006), transformed into E. coli BL21(DE3), and individual transformants picked (46 per selection) for ELISA and HTRF analyses in order to identify clones with improved binding to mouse FBG and human FBG respectively.
TABLE-US-00006 TABLE 5a Selection output titres. Round 1 selections. Phage output titres were determined as described previously (Schofield et al, 2007) Selection Selection CDR3 window window randomised 10 nM 1 nM 0 nM for 10 nM for 1 nM libraries Selection Selection Selection selection selection 2A5 VH 7 × 10.sup.7 2.9 × 10.sup.7 1 × 10.sup.5 700 290 2A5 VL 3 × 10.sup.7 1.7 × 10.sup.7 5 × 10.sup.4 600 340 B12 VH 6 × 10.sup.7 2.6 × 10.sup.7 1 × 10.sup.5 600 260 B12 VL 6 × 10.sup.7 5 × 10.sup.7 2 × 10.sup.5 300 250 F3 VH >1 × 10.sup.8 8 × 10.sup.7 2 × 10.sup.4 5000 4000 F3 VL 5 × 10.sup.7 1.2 × 10.sup.7 9 × 10.sup.4 555 133
TABLE-US-00007 TABLE 5b Selection output titres. Round 2 selections. Phage output titres were determined as described previously (Schofield et al, 2007) Selection window Selection CDR3 for window randomised 200 pM 50 pM 0 nM 200 pM for 50 pM libraries Selection Selection Selection selection selection 2A5 VH 7 × 10.sup.7 3.8 × 10.sup.7 5 × 10.sup.4 1400 760 2A5 VL 1.4 × 10.sup.7 6 × 10.sup.6 1 × 10.sup.4 1400 600 B12 VH 1 × 10.sup.8 6.75 × 10.sup.7 2 × 10.sup.4 5000 3375 B12 VL 1.2 × 10.sup.8 8.1 × 10.sup.7 4 × 10.sup.4 3000 2025 F3 VH 1.1 × 10.sup.8 9.5 × 10.sup.7 4 × 10.sup.4 2750 2375 F3 VL 7 × 10.sup.7 1.2 × 10.sup.7 1.2 × 10.sup.5 583 100 B12 VH 7 × 10.sup.6 2 × 10.sup.4 350 on mu TNC FBG B12 VL 7.5 × 10.sup.6 4 × 10.sup.4 187 on mu TNC FBG
TABLE-US-00008 TABLE 5c Selection output titres. Round 3 selections. Phage output titres were determined as described previously (Schofield et al, 2007) Selection Selection CDR3 window window randomised 5 pM 1 pM 0 nM for 5 pM for 1 pM libraries Selection Selection Selection selection selection 2A5 VH 6 × 10.sup.6 1 × 10.sup.6 <1 × 10.sup.5 60 10 2A5 VL 1.4 × 10.sup.6 <1 × 10.sup.5 2 × 10.sup.5 7 <1 B12 VH 1.5 × 10.sup.7 4 × 10.sup.6 <1 × 10.sup.5 >150 >40 B12 VL 2.7 × 10.sup.7 3.5 × 10.sup.6 <1 × 10.sup.5 >270 >35 F3 VH 3.5 × 10.sup.6 4 × 10.sup.5 <1 × 10.sup.5 >35 >4 F3 VL 6 × 10.sup.5 <1 × 10.sup.5 2 × 10.sup.5 3 <1 Hybrid selections Selection Selection on B12 window window libraries 20 pM 5 pM 0 pM for 20 pM for 5 pM (Hu-mu-hu) Selection Selection Selection selection selection B12 VH 1 × 10.sup.8 7.7 × 10.sup.6 <1 × 10.sup.5 >1000 >77 B12 VL 1.3 × 10.sup.8 1.8 × 10.sup.7 <1 × 10.sup.5 >1300 >78
ELISA Screen
[0389] An anti-FLAG capture ELISA was performed to screen for clones that had an improved affinity for mouse FBG binding compared with the parental antibodies.
[0390] E. coli BL21 (DE3) clones harbouring scFv pSANG10 expression plasmids were induced in 96-well plates with auto-induction media as described previously (Schofield et al, 2007). E. coli supernatants were harvested for ELISA assays. ELISA used the DELFIA (dissociation enhanced lanthanide fluorescent immunoassay) system with Europium-labelled anti-FLAG antibody (Sigma, Aldrich, UK). Black immunosorb plates (Nunc) were coated overnight with anti-FLAG M2 antibody (Sigma, F3165, 5 μg/ml in PBS, 50 μl per well), in wells blocked by the addition of 2% milk powder, PBS (PBS-M, 300 μl per well). Plates were washed three times with PBS-T (PBS, 0.1% Tween-20) and three times with PBS followed by the addition of a 1:2 dilution of 96-well auto-induction culture supernatants containing expressed scFv in PBS-M (50 μl per well). The plates were incubated for 1 h, washed as above and biotinylated mouse or human rCd4-His-FBG (5 μg/ml in PBS-M, 50 μl) added to each well. Plates were incubated for a further 1 h, washed and Strepravidin-Eu added (Perkin Elmer, 1 μg/ml, PBS-M, 50 μl), incubated for 30 min, washed and DELFIA enhancement solution added (50 μl) and plates read on a Perkin Elmer Fusion plate reader (excitation=320 nm, emission 620 nm). The format of the assay is shown in
[0391] In this assay differences in scFv expression level are normalised because the expression levels of scFv in auto-induction cultures saturate the anti-FLAG coated wells. Therefore, the signals obtained in the assay reflect the amount of biotinylated rCd4-His-FBG bound after washing, which will be a function of the off-rate of that clone for mouse or human FBG. ELISA screening of the selection output from the 2A5 and B12 sub-libraries revealed clones with significantly improved binding to mouse TNC FBG.
HTRF Screen
[0392] An HTRF-based competition assay was developed to screen for antibody variants with improved binding to human TNC FBG.
[0393] All samples and reagents were prepared in assay buffer (50 mM NaPO.sub.4, 0.1% BSA, 0.4 M KF, pH 7.0) at 4× the stated concentration. 5 μl of each reagent was subsequently added to low volume 384-well assay plates (Greiner, 784075) to give a final reaction volume of 20 μl. IgG antibodies were labelled using the d2 labelling kit (CisBio, 62D2DPEA) as directed by the manufacturer. Streptavidin europium cryptate (CisBio, 610SAKLA, Lot#25C) was used at a final concentration of 1.8 ng active moiety (SA) per 20 μl reaction as recommended by the manufacturer. Biotinylated rCd4-His-FBG was prepared using EZ-link Sulfo-NHS-LC-Biotin reagent (Thermo Scientific, 21327) the extent of biotinylation was quantified using biotinylation fluorescence quantitation kit (Thermo Scientific, 46610). Where appropriate, supernatants containing scFv (prepared as described above for ELISA assays) were added to the 384-well assay plate at a final dilution of 1/20 (i.e. 1/5 dilution in assay buffer followed by addition of 5 μl diluted sample to the 20 μl FRET assay). The concentrations of d2-labelled 2A5 IgG and B12 IgG used for screening were 15 nM and 1.25 nM respectively. Unless otherwise stated, biotinylated rCd4-His-FBG (biotin:protein ratio=1.8:1) was present at either 2.2 nM (in assays using the 2A5 IgG antibody) or 1 nM (in experiments using B12 IgG). Samples were incubated for approximately 1 h at room temperature and the FRET signal was determined using a BMG Pherastar instrument: excitation=320 nm; emission=620 nm and 665 nm; integration start time=60 μs; integration time=500 μs; 100 flashes per well. For competition assays containing culture supernatant, biotinylated rCd4-His-FBG antigen was pre-incubated with streptavidin europium cryptate for 45 min prior to addition of reagents to the assay plate. All FRET signals are presented as ΔR, where R=(E665/E620×104) and ΔR=(Rsample−Rbackground fluorescence).
[0394] Culture supernatants containing unlabelled scFv clones from affinity selected mutant libraries were tested for inhibition of the interaction between FBG and the fluorophore-labelled parental IgG antibody. When used to screen the 2A5 variants, this approach yielded a high proportion of clones with improved inhibition relative to the parent (92% of VH CDR3 variants and 79% of VL CDR3 variants). In order to distinguish between the clones that fully inhibited the FRET signal, 2A5 variants were subsequently screened for their ability to compete with B12 IgG. This was a more stringent screen given that the affinity of B12 for human FBG is approximately 100-fold stronger than that of 2A5 (dissociation constants for these interactions, determined by surface plasmon resonance at 25° C. were 0.11 nM and 15 nM, respectively). The relative ranking of clones exhibiting FRET signals within the useful range in both assays was broadly unchanged, indicating that they were competing for similar epitopes. Hence, all 2A5 and B12 scFv variants from affinity maturation selections were screened for their ability to inhibit the binding of B12 IgG molecules to human TNC FBG. The parental clones, expressed as scFvs in parallel with the affinity matured clones, were used as benchmarks (Table 6).
[0395] ScFv were sequenced and a panel of clones with unique VH or VL CDR3 sequences was selected for further study in human IgG4 format, based on their binding to mouse and human TNC FBG in the ELISA and HTRF assays, respectively. The chosen variants of antibody 2A5 displayed ≧10-fold improvement in binding to the mouse FBG and an inhibition of ≧90% (VH CDR3 variants) or ≧83% (VL CDR3 variants) in the HTRF assay.
TABLE-US-00009 TABLE 6 HTRF screen for clones with improved affinity for human rCD4-FBG. % inhibition Total by parent CDR3 Selection clones % inhibition of FRET signal scFv Library type tested 0-25% 25-50% 51-75% 76-85% 86-90% 91-95% ≧96% 2A5 B12 2A5 VH 1 pM 46 3 4 4 15 13 7 0 29 90 2A5 VH Off-rate 46 3 0 11 11 17 3 1 29 90 2A5 VL 5 pM 46 3 2 7 19 14 1 0 21 83 2A5 VL Off-rate 46 10 11 10 5 10 0 0 21 83 B12 VH 100 fM .sup. 46 6 2 3 8 5 6 16 19 86 B12 VH Hybrid 46 3 3 5 5 3 9 18 19 86 5 pM
[0396] Variants of antibody B12 showed ≧4-fold improvement for mouse FBG binding, and ≧91% inhibition of HTRF signal. In total, 31 clones fitting these criteria with unique CDR3 sequences were identified (Table 7).
TABLE-US-00010 TABLE 7 Heavy or light chain CDR3 sequences of clones identified with improved binding to mouse and human TNC FBG and chosen for conversion to human IgG format for further study. Library Clone name CDR sequence B12 VH 165_13_B1 VMSSMEDAFDI SEQ ID NO: 30 165_13_B6 GQKGEGDTFDI SEQ ID NO: 32 165_13_D1 GTRGEGDTFDI SEQ ID NO: 34 165_13_C3 SYQSDEDAFDI SEQ ID NO: 36 165_13_D4 GTVGEGDTFDI SEQ ID NO: 38 165_13_A4 DKYPVLDTFDI SEQ ID NO: 40 165_13_B3 ALARGHDTFDI SEQ ID NO: 42 165_13_E1 DISAVMDVPQT SEQ ID NO: 44 180_11_F5 VMRTGLDTFDI SEQ ID NO: 46 2A5 VH 160_01_E3 QRYVWEALTY SEQ ID NO: 48 160_01_D6 AQADPHLFTY SEQ ID NO: 50 160_01_H4 GRFVWEALTY SEQ ID NO: 52 160_01_A4 AQKETLGNAI SEQ ID NO: 54 160_01_F1 AQSPWSGMTY SEQ ID NO: 56 160_01_G2 YTLDNMALTY SEQ ID NO: 58 161_01_F6 AQKENIANRH SEQ ID NO: 60 (160_01_F6) 161_01_A12 AQPTALANTY SEQ ID NO: 62 161_01_C09 AQLPYLAQTY SEQ ID NO: 64 161_01_H10 AQPVWAPGTY SEQ ID NO: 66 161_01_C11 AQKEWLPDVT SEQ ID NO: 68 162_02_D3 AQIHPLGLTY SEQ ID NO: 70 2A5 VL 162_02_C6 QNQYAGPWT SEQ ID NO: 72 162_02_H5 QNQYTGPWT SEQ ID NO: 74 162_02_F3 QNQYRGPWT SEQ ID NO: 76 162_02_C1 LHHYRAPWT SEQ ID NO: 78 162_02_C2 MHHYRAPWT SEQ ID NO: 80 162_02_F4 MHHYRSPWT SEQ ID NO: 82 162_02_C3 MQHYDGPWT SEQ ID NO: 84 162_02_E11 LHHYRSPTWT SEQ ID NO: 86 162_02_E11 LHHYRSPWT SEQ ID NO: 135 163_02_A12 LHHYREPWT SEQ ID NO: 88 163_02_D11 LHHYKSPWT SEQ ID NO: 90
[0397] These are heavy or light chain sequences of antibody clones that bind to human and mouse TNC FBG and thus have potential utility in the methods, uses, compositions and compounds of the present invention. For example, antibodies that bind TNF FBG having these CDR3 sequences may be useful in identifying, inhibiting the function of, detecting and purifying TNC or TNC FBG.
Conversion to IgG4 Format and Determination of Binding Kinetics
[0398] The 31 scFv of interest were sub-cloned into a human IgG4 expression vector for generation of antibodies as human IgG4 with a hinge-stabilising mutation (S241P; Angal et al, 1993). IgG4 antibodies were transiently expressed in HEK-293F cells and culture supernatants were screened using surface plasmon resonance spectroscopy for ranking of their off-rates for binding to human and mouse TNC FBG, and human TNR FBG. Briefly, surface plasmon resonance (SPR) experiments were performed using a BIAcore T100 instrument and followed the protocol according to the Human antibody capture kit protocol (GE, BR-1008-39). For off-rate screening, 10,000 response units (RU) of anti-human Fc IgG (GE, BR-1008-39) was immobilised on flow-cells (FC1 and FC2) of a Series 5 CM5 dextran sensor chip (BR-1005-30) using EDC/NHS cross-linking chemistry according to the amine coupling kit protocol (GE, BR-1000-50). Culture supernatants containing expressed IgG4 were diluted 1:2 with 2×PBS-T and injected into FC2 (flowrate 5 μl/min, 60 s contact time) to enable antibody capture at 25° C. Antibody capture levels ranged from 308 to 1975 RU depending on the expression level of the antibody in the supernatant. A fixed concentration of antigen (15 nM of human and mouse TNC rCd4-His-FBG and 100 nM of human TNR rCd4-His-FBG) was injected with a flow-path via FC 1 (reference flow cell) and FC 2 (antibody capture flow cell), with a flow rate of 30 μl/min, and the association and dissociation phases measured over 1 and 5 min time periods, respectively. Regeneration of the binding surface employed 3M MgCl.sub.2 with 30 s contact time. Off rates were determined by reference cell subtraction and fitting the sensogram experimental data assuming a 1:1 interaction using BIAevaluation software (GE, BR-1005-97). Results of the off-rate screen are summarised in Table 8.
TABLE-US-00011 TABLE 8 Surface plasmon resonance screen for ranking of human IgG4 anti-FBG off-rates kd (s.sup.−1 × 10.sup.−4) for rCD4-His-FBG Human Clone name TNC FBG Mouse TNC FBG Human TNR FBG 165_13_B1 0.015 0.017 390 165_13_B6 0.056 0.069 37 165_13_D1 0.0014 0.039 43 165_13_C3 0.00095 0.033 120 165_13_D4 0.0062 0.037 40 165_13_A4 8.72 79.7 nd 165_13_B3 0.014 300 nd 165_13_E1 0.014 577 nd 180_11_F5 0.26 10000 nd 160_01_E3 0 558.8 nd 160_01_D6 0.105 558.8 nd 160_01_H4 0.16 170.8 nd 160_01_A4 0.067 0.059 110 160_01_F1 0.04 1540000 nd 160_01_G2 0.125 0.139 10 161_01_F6 0.028 17.1 25 (160_01_F6) 161_01_A12 0.013 0.043 42 161_01_C09 0.00117 0.0023 2.9 161_01_H10 0.25 0.019 91 161_01_C11 0.0022 nd nd 162_02_D3 0.0039 0.0106 64 162_02_C6 0.053 2.4 280 162_02_H5 0.00043 1.67 820 162_02_F3 0.00083 3.3 880 162_02_C1 0.00093 16 27000000 162_02_C2 0.115 17 535000 162_02_F4 0.0059 10 151000 162_02_C3 0.0149 20 6350 162_02_E11 0.011 12 10110000 163_02_A12 0.0032 9.4 288000 163_02_D11 0.0032 9.8 22090000 2A5 parent 91 590000 2720 B12 parent 1.5 300 1001
[0399] Clones were ranked according to low off-rate for human and mouse TNC rCd4-His-FBG, and high-off rate for human TNR rCd4-His-FBG. The 3 highest-ranking antibodies from each library were prioritised for more detailed kinetic analysis as purified IgG4. These clones are shown in Tables 9, 10 and 11.
TABLE-US-00012 TABLE 9 Heavy chain CDR3 amino acid sequences of B12 mutants with improved FBG binding off-rate characteristics Clone VH CDR3 B12 parent DISAVPDTFDI SEQ ID NO: 11 165_13_B1 VMSSMEDAFDI SEQ ID NO: 30 165_13_D1 GTRGEGDTFDI SEQ ID NO: 34 165_13_C3 SYQSDEDAFDI SEQ ID NO: 36
TABLE-US-00013 TABLE 10 Heavy chain CDR3 amino acid sequences of 2A5 mutants with improved FBG binding off-rate characteristics Clone VH CDR3 2A5 parent AQKETYALTY SEQ ID NO: 3 160_01_A4 AQKETLGNAI SEQ ID NO: 54 161_01_H10 AQPVWAPGTY SEQ ID NO: 66 162_02_D3 AQIHPLGLTY SEQ ID NO: 70
TABLE-US-00014 TABLE 11 Light chain CDR3 amino acid sequences of 2A5 mutants with improved FBG binding off-rate characteristics. Clone VL CDR3 2A5 parent QQSYSTPWT SEQ ID NO: 7 162_02_F3 QNQYRGPWT SEQ ID NO: 76 163_02_A12 LHHYREPWT SEQ ID NO: 88 163_02_D11 LHHYKSPWT SEQ ID NO: 90
[0400] Detailed kinetic parameters were evaluated for the 9 prioritised IgG4 antibodies. Binding characteristics were determined for interaction with human, rat and dog TNC rCD4-His-FBG, and human TNR rCD4-His-FBG. Kinetic assays followed essentially the same protocols as for the off-rate determinations described above, with some modifications as follows. To improve the accuracy of kinetic parameter determination, anti-human Fc IgG was immobilised at lower levels (2229 RU), resulting in a corresponding reduction in the amount of anti-FBG IgG4 captured. Purified anti-FBG IgG4 was diluted to a concentration of 3.5 nM in PBS, pH 7.4, 0.05% Tween-20 and injected into FC2 at a flow rate of 10 μl/min, 60 s contact time. This typically resulted in an average of 80 RU of antibody captured (range: 55 RU to 90 RU). Antigens were prepared by doubling dilution in PBS, pH 7.4, 0.05% Tween-20 (highest concentration 100 nM except mouse rCD4-His-FBG which was 7 nM). Assays were performed at 37° C. (30 μl/min, 120 s contact time; mouse rCD4-His-FBGFBG 10 μl/min, 60 s contact time), with both the flow cell and injection chamber equilibrated to this temperature. As before, kinetic parameters were determined by reference cell subtraction and fitting the sensogram experimental data assuming a 1:1 interaction using BIAevaluation software (GE, BR-1005-97).
[0401] AH nine antibodies displayed improved binding to mouse TNC FBG domain compared to the non-affinity matured parent clones, and antibodies 165_13_B1, 165_13_C3, and 160_01_A4 exhibited sub-nanomolar K.sub.D values for binding to human TNC FBG, with >70-fold lower affinity to the human TNR FBG analogue (Table 12).
TABLE-US-00015 TABLE 12 Anti-FBG IgG4 binding kinetic data determined by surface plasmon resonance at 37° C. Antibody K.sub.a K.sub.d Par- rCD4-His-FBG K.sub.D (M.sup.−1s.sup.−1) × (s.sup.−1) × IgG4 ent Species Tenascin (nM) 10.sup.4 10.sup.−4 2A5 2A5 Human TNC 23.8 13.6 323 Mouse TNC 123 8.68 106.5 B12 B12 Human TNC 0.24 47.1 11.2 Mouse TNC 4.5 30 13.8 165_13_B1 B12 Human TNC 0.26 72.7 18.8 Mouse TNC 0.96 73.3 7.06 Rat TNC 2.20 31.1 68.4 Dog TNC 2.85 65.5 187 Human TNR 94.4 12.2 1149 165_13_C3 B12 Human TNC 0.072 116 8.3 Mouse TNC 0.46 97.2 4.45 Rat TNC 1.22 38.9 47.3 Dog TNC 1.80 59.7 108 Human TNR 35.8 12.0 431 160_01_A4 2A5 Human TNC 0.21 23.5 5.0 Mouse TNC 1.23 11.8 1.46 Rat TNC 1.49 12.7 18.9 Dog TNC 0.094 19.0 1.8 Human TNR 15.2 2.6 39.9
EXAMPLE 6—INHIBITION OF TNC FBG-EVOKED CYTOKINE PRODUCTION IN PRIMARY HUMAN PBMCs
[0402] The functional FBG neutralising activity of purified IgG4 antibodies 165_13_61, 165_13_C3, and 160_01_A4 was confirmed in an in vitro assay of FBG-evoked cytokine release in primary human PBMCs.
[0403] Peripheral blood mononuclear cell (PBMC) populations were isolated from three healthy human single donor buffy coat preparations by density gradient centrifugation. Assays were carried out in 96-well plates in a final volume of 200 μl, and the endotoxin content of all reagents and test antibodies was confirmed to be within acceptable limits before use, determined using a limulus amoebocyte lysate (LAL) endotoxin quantitation kit (Pierce).
[0404] Freshly isolated PBMC samples (2×10.sup.5 cells/well) were cultured in the presence of test antibodies (100 nM and 1 μM), control isotype antibody (Sigma 14639; 100 nM and 1 μM), dexamethasone (1 μM) or PBS control for 1 h prior to submaximal stimulation with either bacterial lipopolysaccharide (LPS; E. coli 026:B6; 100 ng/mL) or human Fc-His-FBG (200 nM). Control wells, in which LPS or Fc-His-FBG were replaced with an equal volume of PBS, contained test antibodies or dexamethasone. After incubation (24 h, 37° C.), culture supernatants were collected and stored at −80° C. Samples were thawed to room temperature before assay of supernatants for cytokine content. A 25 μl aliquot of each supernatant was diluted with an equal volume of RPMI medium (Life Technologies) and resulting samples were assayed in duplicate for IL-8 and TNFα by Luminex analysis.
[0405] Incubation of PBMCs with 100 ng/mL LPS for 24 h resulted in IL-8 and TNFa production, which was not inhibited by exposure to either control IgG4 antibody or the anti-FBG antibodies. In contrast, IL-8 and TNFa release evoked by Fc-His-FBG was completely blocked by all test antibodies, but not control IgG4, confirming the potent and specific FBG-neutralising activity of the 3 affinity-matured antibodies 165_13_131, 165_13_C3, and 160_01_A4 (
EXAMPLE 7—ANTI-FBG IgG4 BINDING TO CITRULLINATED FBG
[0406] The binding affinity of antibody B12 to citrullinated FBG was determined by surface plasmon resonance (SPR). B12 was expressed as a human IgG4 with the hinge-stabilising S241P mutation using the QMCF expression technology (Icosagen, Estonia) and purified by protein A affinity chromatography (MabSelect Sure; GE Healthcare).
Citrullination of Human TNC FBG
[0407] Purified human His-FBG was citrullinated using either peptidylarginine deiminase 2 (PAD2; MQ-16.201-2.5, Modiquest, NL) or peptidylarginine deiminase 4 (PAD4; MQ-16.203-2.5, Modiquest, NL) according to the supplier's instructions. Briefly, His-FBG was diluted to 1 mg/ml in the supplied deimination buffer (0.1 M Tris-HCl pH 7.5, 10 mM CaCl.sub.2, 5 mM dithiothreitol) and 250 μl mixed with 125 mU of either PAD2 or PAD4 enzyme followed by incubation at 37° C. for 2 h. Citrullination was confirmed by amino acid analysis of the enzymatically-treated samples. Aliquots of His-FBG in deimination buffer were incubated for 2 h at 37° C. in the absence of added PAD enzyme, for use as non-citrullinated control protein. Citrullinated and unmodified His-FBG proteins were used in SPR experiments as described below.
Surface Plasmon Resonance
[0408] SPR experiments were performed on a BIAcore 3000 instrument. Anti-human IgG (GE Healthcare) was covalently coupled to the surface of a CM5 sensor chip using amino coupling chemistry. The amount of the coupled anti-human IgG expressed in RU units varied between 6500-7000 (6.5-7.0 ng/mm.sup.2). B12-hIgG4 (1-13 nM) was attached to the immobilised anti-human IgG in HBS-EP buffer (10 mM Hepes, 0.15 M NaCl, 2.5 mM EDTA and 0.005% Tween-20) at 25° C. Binding of the His-FBG variants to the immobilised B12-hIgG4 was also measured in HBS-EP buffer at 25° C. The flow rate was 5 μl/min in the immobilization experiments and 20 μl/min for kinetic analyses. The sensor chip surface was regenerated using 3 M MgCl.sub.2. Data were analysed using BIAevaluation program 4.1 (GE Healthcare).
[0409] Analysis of B12-IgG4 binding to citrullinated His-FBG revealed that the kinetic parameters were essentially unchanged when compared to values obtained for binding to unmodified His-FBG (Table 13). These results indicate that anti-FBG antibodies of the B12 lineage would be expected to bind both citrullinated and non-citrullinated forms of TNC FBG in therapeutic or diagnostic applications.
TABLE-US-00016 TABLE 13 Kinetic parameters for interaction of B12-hIgG4 with the His-FBG variants. Each kinetic parameter represents the mean ± s.d. of 3 independent determinations. Analyte K.sub.D (M) K.sub.on (M.sup.−1s.sup.−1) K.sub.off (s.sup.−1) His-FBG (1.7 ± 0.3) × 10.sup.−10 (4.1 ± 0.6) × 10.sup.6 (6.8 ± 0.9) × 10.sup.−4 His-FBG + (3.2 ± 0.3) × 10.sup.−10 (3.0 ± 0.4) × 10.sup.6 (9.6 ± 0.8) × 10.sup.−4 PAD2 His-FBG + (3.2 ± 0.7) × 10.sup.−10 (2.6 ± 0.6) × 10.sup.6 (7.8 ± 0.4) × 10.sup.−4 PAD4
EXAMPLE 8—DETECTION OF TNC FBG IN HUMAN RA TISSUE USING IMMUNOHISTOCHEMISTRY
[0410] Immunohistochemistry studies were performed to determine whether anti-FBG antibodies effectively recognise endogenous forms of the human TNC FBG protein in human tissue. Tenascin-C is expressed at sites of chronic inflammation and its localisation within the inflamed synovium of joints from individuals with rheumatoid arthritis has previously been demonstrated by immunohistochemistry using commercially available antibodies (Goh et al, 2010; Salter D M, 1993).
[0411] The B12 antibody was expressed as mouse IgG2a format using the QMCF expression technology (Icosagen, Estonia) and purified by Protein G affinity chromatography followed by Superdex 200 gel filtration. Control mouse IgG1 anti-tenascin-C antibody (Clone 4F10TT; Takara Clontech), which recognises an EGF domain of full-length human tenascin-C was used as a positive control comparator. Mouse IgG1 (Dako X0931) or IgG2a (Dako X0943) against an irrelevant bacterial antigen were used as control primary antibodies to determine the level of non-specific background staining with these isotypes. Frozen sections of human knee joint synovium from donors with confirmed RA diagnosis (Asterand, UK) were equilibrated to room temperature, fixed (10 min) in 1:1 v/v acetone/methanol, and transferred to wash buffer. Immunostaining was performed using a Dako Autostainer with Envision Flex reagents (Dako K8010) according to manufacturer's protocols. Briefly, fixed tissue slides were placed onto the automated stainer and blocked (peroxidase block, 5 min; protein block, 10 min, Dako X0909) before 30 min application of primary antibody (B12 or Clone 4F10TT; 1, 2, or 4 μg/ml). In some controls, slides were not exposed to primary antibody. After washing, HRP-labelled goat anti-mouse secondary antibody was applied (20 min) and slides were washed again, followed by 10 min application of DAB+ Chromogen. Slides were washed, counterstained with haematoxylin and coverslipped for microscopic visualisation of staining.
[0412] In cryosections of RA synovium that were fixed using acetone/methanol, the anti-TNC FBG B12 mouse IgG2a showed a very similar pattern of staining to that obtained with the positive control antibody Clone 4F10TT. Specific immunostaining was observed in the synovium, fibrous capsule, vasculature and within the interstitium. There was no staining within lymphoid aggregates (
EXAMPLE 9—ANTIBODY SEQUENCES
[0413]
TABLE-US-00017 Antibody 2A5 (SEQ ID NO: 1) VH CDR1: ELSMH (SEQ ID NO: 2) VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 3) VH CDR3: AQKETYALTY VH amino acid sequence: (SEQ ID NO: 4)
IgG4 165 13 C3 (Constant Region with Hinge Modification as Described in Angal 1993) [0414] Reference: Angal S1, King D J, Bodmer M W, Turner A, Lawson A D, Roberts G, Pedley B, Adair J R. Mol Immunol. 1993 January; 30(1):105-8.
TABLE-US-00018 QVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSG ISGSGGSTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKSY QSDEDAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
EXAMPLE 10—PROTEIN SEQUENCES
[0415]
TABLE-US-00019 Amino acid sequence of human tenascin-C FBG domain [SEQ ID NO: 92] IGLLYPFPKDCSQAMLNGDTTSGLYTIYLNGDKAEALEVFCDMTSDGGGW IVFLRRKNGRENFYQNWKAYAAGFGDRREEFWLGLDNLNKITAQGQYELR VDLRDHGETAFAVYDKFSVGDAKTRYKLKVEGYSGTAGDSMAYHNGRSFS TFDKDTDSAITNCALSYKGAFWYRNCHRVNLMGRYGDNNHSQGVNWFHWK GHEHSIQFAEMKLRPSNFRNLEGRRKRA Amino acid sequence of mouse tenascin-C FBG domain [SEQ ID NO: 93] IGLLYPFPRDCSQAMLNGDTTSGLYTIYINGDKTQALEVYCDMTSDGGGW IVFLRRKNGREDFYRNWKAYAAGFGDRREEFWLGLDNLSKITAQGQYELR VDLQDHGESAYAVYDRFSVGDAKSRYKLKVEGYSGTAGDSMNYHNGRSFS TYDKDTDSAITNCALSYKGAFVVYKNCHRVNLMGRYGDNNHSQGVNWFHW KGHEYSIQFAEMKLRPSNFRNLEGRRKRA Amino acid sequence of rat tenascin-C FBG domain [SEQ ID NO: 94] IGLLYPFPRDCSQAMLNGDTTSGLYTIYINGDKTQALEVYCDMTSDGGGW IVFLRRKNGREDFYRNWKAYATGFGDRREEFWLGLDNLSKITAQGQYELR VDLQDHGESAYAVYDRFSVGDAKSRYKLKVEGYSGTAGDSMNYHNGRSFS TYDKDTDSAITNCALSYKGAFWYKNCHRVNLMGRYGDNNHSQGVNWFHWK GHEYSIQFAEMKLRPSNFRNLEGRRKRA Amino acid sequence of dog tenascin-C FBG domain [SEQ ID NO: 95] IGLLYPFPRDCSQAMLNGDTTSGLYTIYLNGDKAQALEVYCDMTSDGGGW IVFLRRKNGREDFYRNWKAYAAGFGDRREEFWLGLDNLHKITAQGQYELR VDLRDHGKTAYAVYDRFSVGDAKTRYKLKVEGYSGTAGDSMAYHNGRSFS TFDKDTDSAITNCALSYKGAFWYKNCHRVNLMGRYGDNNHSQGVNWFHWK GHEYSIQFAEMKLRPSNFRNLEGRRKRA Amino acid sequence of human tenascin-R FBG domain [SEQ ID NO: 96] FPHPQDCAQHLMNGDTLSGVYPIFLNGELSQKLQVYCDMTTDGGGWIVFQ RRQNGQTDFFRKWADYRVGFGNVEDEFWLGLDNIHRITSQGRYELRVDMR DGQEAAFASYDRFSVEDSRNLYKLRIGSYNGTAGDSLSYHQGRPFSTEDR DNDVAVTNCAMSYKGAINWYKNCHRTNLNGKYGESRHSQGINVVYHWKGH EFSIPFVEMKMRPYNHRLMAGRKRQSLQF
EXAMPLE 11—GERMLINED SEQUENCES
[0416] Closest germline matches were determined using IMGT/DomainGapAlign: Ehrenmann F., Kaas Q. and Lefranc M. P. Nucleic Acids Res., 38, D301-307 (2010)
[0417] Changes from non-germlined sequences are shown by an underline of the amino acid. The CDRs are shown by the boxed sequences.
TABLE-US-00020 Antibody 2A5 Framework Germlined: VH amino acid sequence: (SEQ ID NO: 112)
EXAMPLE 12—USE OF ANTIBODIES IN WESTERN BLOTTING
[0418] In order to confirm that the monoclonal antibodies IgG4 C3 (165_13_C3 as referred to above) and IgG4 B12 could be successfully used for western blotting, first specificity was tested using purified proteins (
[0419] As shown by the data in
[0420] In this experiment both C3 and B12 showed specificity for TNC-FBG with very little cross-reactivity with either TNR-FBG or Fibrinogen, indicating their suitability for western blotting applications as they show good specificity for TNC-FBG.
[0421] As shown by the data in
[0422] These results indicate that B12 can detect full length TNC as well as breakdown products and/or splice variants of TNC, and shows low cross-reactivity to other proteins present in the cell lysate.
EXAMPLE 13—ACTIVITY OF THE C3 ANTIBODY IN VITRO
[0423] In order to confirm that the monoclonal antibody C3 (165_13_C3) acts by disrupting the binding of TNC-FBG to its receptor TLR4, first an in vitro binding assay was developed for TLR4 and Fc-His-FBG then the effect of pre-incubation of Fc-His-FBG with C3 was determined.
[0424] Recombinant human TLR4 (R&D systems) (1 ug/ml (14.6 nM)) in PBS (or PBS alone) was bound to a 96-well plate. After blocking (10% BSA) the indicated concentrations of Human Fc-His-FBG was added and detection was carried out by incubation of an anti-human IgG1 MAb (AbD Serotec, clone 2C11) at 1 ug/ml, an anti-mouse HRP conjugated secondary antibody (AbD Serotec, STAR13B) at 1 ug/ml, and TMB substrate. The results are shown in
[0425] As shown in
EXAMPLE 14—ANTI-INFLAMMATORY EFFECT OF ANTIBODIES B12, A4 AND C3
[0426] It was confirmed that the anti-TNC-FBG antibodies B12, A4 (160_01_A4) and C3 (165_13_C3) have an anti-inflammatory effect in a biological system. To do this, human monocytes were isolated from peripheral blood (London blood bank) by Ficoll gradient and counter-flow centrifugation. The monocytes were then differentiated with 100 ng/ml M-CSF (Peprotec) for 5 days to produce M2 macrophages.
[0427] As shown by the results in
[0428]
[0429] To confirm that the FBG-induced cytokine release was induced by the FBG rather than the Fc portion of the protein, a protein where the Fc portion is mutated to be inactive (Fc-Mut-FBG) was used, Anti-TNC-FBG antibodies, B12, C3 (165_13_C3) and A4 (160_01_A4) were also tested for activity against this molecule. Fc-Mut-FBG (1 uM) and C3, A4 or B12 (1 uM) were pre-incubated for 30 min at RT before being added to human M2 macrophage cultures. After 24 h supernatants were taken and subjected to cytokine ELISA. n=3, mean and SEM shown. Results are shown in
[0430]
[0431]
[0432] To take the C3 antibody into animal studies, IgG4 B12 165-13-C3 product was cloned, expressed and purified at a leading contract manufacturing organisation using a commercial GS-CHO expression. cDNAs for the heavy and light chain variable regions were optimised for CHO expression and synthesised (with commercial signal sequences) by Life Technologies prior to cloning into the expression vectors. CHO cells were transfected as pools and the highest expressing pool was taken forward into large-scale shake flask production (22L−11×2L in 5L shake flasks.). Proprietary feeds were administered on day 4 and 8 prior to harvesting the culture on day 12. Material was centrifuged prior to depth filtration and filter sterilisation. Approximately a 5.5 fold concentration of material was performed using tangential flow filtration (30 kDa molecular weight cut off) and the resulting concentrate was filter sterilised again prior to MabSelect SuRe purification. The product was eluted and product was neutralised and then concentrated/diafiltered to approximately 11 mg/mL in 20 mM NaOAc, pH 5.5, 150 mM NaCl. Reduced and non-reduced SDS-PAGE analysis together with size exclusion —HPLC showed material that was highly pure and greater than 98% monomer. Endotoxin was less than 0.1 Eu per mg.
[0433] In this experiment the potency of the larger scale antibody batch was compared to the current smaller scale batch. Recombinant Human tenascin-C FBG (1 uM) was pre-incubated for 30 min at RT with MAb C3 (1, 0.2 and 0.04 uM) or isotype control MAb (1 uM) before being added in triplicate to Human M2 macrophage cultures. After 24 h supernatants were taken and subjected to cytokine ELISA. n=1, Ico=laboratory scale Lon=larger scale material. This experiment shows that both batches of antibodies show equal potency in the reduction of FBG-induced cytokine synthesis, i.e. the results are consistent irrespective of production.
EXAMPLE 15—MONOCLONAL ANTIBODY C3 (165_13_C3) REDUCES THE PRODUCTION OF PRO-INFLAMMATORY CYTOKINES BY RA SYNOVIAL FIBROBLASTS STIMULATED WITH HUMAN TNC-FBG
[0434] It has been reported that synovial fibroblasts could be an important source of pro-inflammatory cytokine release in RA (R Bucala et al. (1991) Constitutive Production of Mitogenic and Inflammatory Cytokines by Rheumatoid Synovial Fibroblasts. J. Exp. Med. 173:569-574), it was therefore tested whether the C3 antibody also showed similar effects on FBG-induced cytokine release as in the macrophages.
[0435] Human RA fibroblasts were grown out of donor RA synovial tissue by digestion of the tissue in RPMI (Lonza) containing 0.5 mg/ml Liberase (Roche) and 0.2 mg/ml DNase (Roche) and incubation at 37° C. for 1-1.5 h. The resulting tissue was pipetted through a 200 μm nylon mesh; the material that did not pass through the mesh was put into a petri-dish containing RPMI with 10% FBS (Life technologies) and 1% pen/strep (Life technologies) and incubated at 37° C. for 5 days. After 5 days synovial fibroblasts grow out of the tissue and the remaining tissue was removed from the RA synovial fibroblast (RASF) culture which was subsequently maintained in DMEM (Lonza) containing 10% FBS and 1% pen/strep. For this experiment RASF were plated out at 1×10.sup.4 cells/well. Recombinant Human TNC-FBG (1 uM) was pre-incubated for 30 min at RT with MAb C3 (1, 0.2 and 0.04 uM) or isotype control MAb (1 uM) before being added in triplicate to the synovial fibroblast cultures. After 24 h supernatants were taken and subjected to cytokine ELISA. n=1, mean and SEM shown (see
[0436] These results indicate that C3 acts to reduce FBG induced pro-inflammatory cytokine release (both IL-8 and IL-6) in RA synovial fibroblasts, showing that this is a potential mechanism in multiple cell types found in the inflamed RA joint.
EXAMPLE 16—LEVELS OF TENASCIN-C IN RAT MODEL
[0437] Expression of tenascin-C in both mouse and rat CIA (collagen-induced arthritis) models was confirmed and disease activity shown to correlate with clinical score.
[0438]
EXAMPLE 17—EVALUATION OF C3 ANTIBODY IN A RAT MODEL OF COLLAGEN-INDUCED ARTHRITIS
[0439] IgG4 C3 (165_13_C3) was tested for therapeutic activity in the standard rat collagen induced arthritis model. Adult male Lewis rats were randomly allocated to experimental groups and allowed to acclimatise for one week. On Day 0, animals were administered with 500 μl of a 1 mg/ml emulsion of type II bovine collagen in incomplete Freund's adjuvant (CII/IFA) by intra-dermal injection in the lower back. On Day 7, animals received a second injection of CII/IFA. Injections were performed under gas (isoflurane) anaesthesia. Treatments were administered according to the Administration Schedule shown below in Table 14.
TABLE-US-00021 TABLE 14 Administration Schedule Disease Group Treatment Dose Route Regimen Induction 1 Vehicle (0.9% n/a IV Twice Day 0, NaCl) weekly*, Day 7: 2 Control IgG4 .sup.1 10 mg/kg IV Day 0-End CII/IFA, 3 IgG4 165_13_C3 1 mg/kg IV ID 4 IgG4 165_13_C3 3 mg/kg IV 5 IgG4 165_13_C3 10 mg/kg IV .sup.1 Fully human IgG4 isotype control, preclinical grade, (ET904, Eureka Therapeutics), n/a: not applicable, IV: intra-venous injections, ID: intra-dermal injections, CII/IFA: Type II collagen and Incomplete Freund's Adjuvant emulsion, *Day 0, Day 3, Day 7, Day 10, Day 14, Day 17, Day 21 and Day 24
[0440] From Day 7 until the end of the experiment, animals were scored three times per week for clinical signs of arthritis by an experimenter blind to the treatments. On Day 0, Day 14, Day 21 and Day 28, paw volumes were measured using a plethysmometer by an experimenter blind to the treatments.
Results
Non-Specific Clinical Observations
[0441] From Day 0 until the end of the experiment, animals were checked daily for non-specific clinical signs to include abnormal posture (hunched), abnormal coat condition (piloerection) and abnormal activity levels (reduced or increased activity). One animal in Group 6 (ID #6.9, antibody 10 mg/kg-treated) did not recover from the isoflurane anaesthesia on Day 21. Animals did not show any non-specific clinical signs such as abnormal posture, abnormal coat condition and abnormal activity levels. One animal in Group 1 (ID #1.10, vehicle-treated) was culled on Day 22, prior to the end of the experiment, due to the severity of the clinical signs of arthritis.
Clinical Scores
[0442] From Day 7 until the end of the experiment, animals were scored three times per week for clinical signs of arthritis to include front and hind limb swelling. The experimenter was blind to the treatments. Each limb was scored on a five-point scale: (0) absence of swelling, (1) slight swelling and/or erythema, (2) mild swelling, (3) moderate swelling and (4) severe swelling and/or joint rigidity. A clinical score was calculated for each animal by adding the score of each limb. Data provided in
Paw Volumes
[0443] On Day 0, Day 14, Day 21 and Day 28, hind paw volumes were measured using a plethysmometer (water-displacement device). Measurements were performed under gas (isoflurane) anaesthesia. The experimenter was blind to the treatment. Right and left hind paw volumes from each animal on each experimental day were averaged.
CONCLUSIONS
[0444] The test antibody, IgG4 C3 (165_13_C3), when administered at 3 mg/kg or 10 mg/kg, significantly reduced the severity of the clinical signs.
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Embodiments of the invention will now be described in the following numbered paragraphs:
[0523] 1. An antibody or antigen-binding fragment, derivative or variant thereof which is capable of binding to the FBG domain of tenascin-C, wherein the antibody or antigen-binding fragment, derivative or variant thereof comprises: one or more sequences selected from SEQ ID NOs: 1-8, 48-91 and 112-114; and/or one or more sequences selected from SEQ ID NOs: 5, 9-15, 30-47 and 115-118; and/or one or more sequences selected from SEQ ID NOs: 5, 13, 16-21 and 119-121; and/or one or more sequences selected from SEQ ID NOs: 22-29 and 122-123.
[0524] 2. The antibody or antigen-binding fragment, derivative or variant thereof of paragraph 1 wherein the antibody or antigen-binding fragment, derivative or variant thereof comprises: one or more CDR sequences selected from SEQ ID NOs: 1-3, 5-7, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90 and 114; and/or one or more CDR sequences selected from SEQ ID NOs: 9-11, 5, 13-14, 30, 32, 34, 36, 38, 40, 42, 44, 46, 116 and 118; and/or one or more CDR sequences selected from SEQ ID NOs 16-18, 5, 13, 20 and 121; and/or one or more CDR sequences selected from SEQ ID NOs 22-24 and 26-28.
[0525] 3. The antibody or antigen-binding fragment, derivative or variant thereof of paragraph 1 or 2 wherein the antibody or antigen-binding fragment, derivative or variant thereof comprises: one or more CDR3 sequences selected from SEQ ID NOs: 3, 7, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88 and 90; and/or one or more CDR3 sequences selected from SEQ ID NOs: 11, 14, 30, 32, 34, 36, 38, 40, 42, 44 and 46; and/or one or more CDR3 sequences selected from SEQ ID NOs 18 and 20; and/or one or more CDR3 sequences selected from SEQ ID NOs 24 and 28.
[0526] 4. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in paragraph 3 wherein the antibody or antigen-binding fragment, derivative or variant thereof comprises: one or more CDR3 sequences selected from SEQ ID NOs: 3, 54, 66 and 70; and/or one or more CDR3 sequences selected from SEQ ID NOs: 7, 76, 88 and 90; and/or one or more CDR3 sequences selected from SEQ ID NOs: 11, 30, 34 and 36.
[0527] 5. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in paragraph 1 wherein the antibody or antigen-binding fragment, derivative or variant thereof comprises: a VH CDR3 sequence selected from SEQ ID NOs: 3, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 and 70; a VH CDR3 sequence selected from SEQ ID NOs: 3, 54, 66 and 70; or a VH CDR3 sequence selected from SEQ ID NOs: 3 and 54.
[0528] 6. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in paragraph 1 or 5 wherein the antibody or antigen-binding fragment, derivative or variant thereof comprises: a VL CDR3 sequence selected from SEQ ID NOs: 7, 72, 74, 76, 78, 80, 82, 84, 86, 88 and 90; a VL CDR3 sequence selected from SEQ ID NOs: 7, 76, 88 and 90; or a VL CDR3 sequence of SEQ ID NO 7.
[0529] 7. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in paragraph 1 wherein the antibody or antigen-binding fragment, derivative or variant thereof comprises a VH sequence comprising the sequence of SEQ ID NO: 4 or 112, and wherein the VH sequence comprises a CDR3 sequence which is replaced with: a VH CDR3 sequence selected from SEQ ID NOs: 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 and 70; a VH CDR3 sequence selected from SEQ ID NOs: 54, 66 and 70; or a VH CDR3 sequence of SEQ ID NO: 54.
[0530] 8. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in paragraph 1 or 7 wherein the antibody or antigen-binding fragment, derivative or variant thereof comprises a VL sequence comprising the sequence of SEQ ID NO: 8 or 113, and wherein the VL sequence comprises a CDR3 sequence which is replaced with: a VL CDR3 sequence selected from SEQ ID NOs: 72, 74, 76, 78, 80, 82, 84, 86, 88 and 90; or a VL CDR3 sequence selected from SEQ ID NOs: 76, 88 and 90.
[0531] 9. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in paragraph 1 wherein the antibody or antigen-binding fragment, derivative or variant thereof comprises: a VH CDR3 sequence selected from SEQ ID NOs: 11, 30, 32, 34, 36, 38, 40, 42, 44 and 46; a VH CDR3 sequence selected from SEQ ID NOs: 11, 30, 34 and 36; or a VH CDR3 sequence selected from SEQ ID NOs 11, 30 and 36.
[0532] 10. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in paragraph 1 wherein the antibody or antigen-binding fragment, derivative or variant thereof comprises a VL sequence comprising the sequence of SEQ ID NO: 15 or 117.
[0533] 11. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in paragraph 1 or 10 wherein the antibody or antigen-binding fragment, derivative or variant thereof comprises a VH sequence comprising the sequence of SEQ ID NO: 12 or 115, and wherein the VH sequence comprises a CDR3 sequence which is replaced with: a VH CDR3 sequence selected from SEQ ID NOs: 30, 32, 34, 36, 38, 40, 42, 44 and 46; a VH CDR3 sequence selected from SEQ ID NOs: 30, 34 and 36; or a VH CDR3 sequence selected from SEQ ID NOs 30 and 36.
[0534] 12. The antibody or antigen-binding fragment, derivative or variant thereof of paragraph 1 wherein the antibody or antigen-binding fragment, derivative or variant thereof comprises: a VL CDR3 sequence of SEQ ID NO: 7 and a VH CDR3 sequence selected from SEQ ID NOs: 3 and 48-70; or comprises a VH CDR3 sequence of SEQ ID NO: 3 and a VL CDR3 sequence selected from SEQ ID NOs: 7 and 72-90; or comprises a VL CDR3 sequence of SEQ ID NO: 14 and a VH CDR3 sequence selected from SEQ ID NOs: 11 and 30-46; or comprises a VH CDR3 sequence of SEQ ID NO: 18 and a VL CDR3 sequence of SEQ ID NO: 20; or comprises a VH CDR3 sequence of SEQ ID NO: 24 and a VL CDR3 sequence of SEQ ID NO: 28.
[0535] 13. The antibody or antigen-binding fragment, derivative or variant thereof of paragraph 1 wherein the antibody or antigen-binding fragment, derivative or variant thereof comprises: at least one CDR sequence selected from SEQ ID NOs: 1-3, and 5-7; or at least one CDR sequence selected from SEQ ID NOs: 1, 2, 48 and 5-7; or at least one CDR sequence selected from SEQ ID NOs: 1, 2, 50 and 5-7; or at least one CDR sequence selected from SEQ ID NOs: 1, 2, 52 and 5-7; or at least one CDR sequence selected from SEQ ID NOs: 1, 2, 54 and 5-7; or at least one CDR sequence selected from SEQ ID NOs: 1, 2, 56 and 5-7; or at least one CDR sequence selected from SEQ ID NOs: 1, 2, 58 and 5-7; or at least one CDR sequence selected from SEQ ID NOs: 1, 2, 60 and 5-7; or at least one CDR sequence selected from SEQ ID NOs: 1, 2, 62 and 5-7; or at least one CDR sequence selected from SEQ ID NOs: 1, 2, 64 and 5-7; or at least one CDR sequence selected from SEQ ID NOs: 1, 2, 66 and 5-7; or at least one CDR sequence selected from SEQ ID NOs: 1, 2, 68 and 5-7; or at least one CDR sequence selected from SEQ ID NOs: 1, 2, 70 and 5-7; or at least one CDR sequence selected from SEQ ID NOs: 1-3, 5, 6 and 72; or at least one CDR sequence selected from SEQ ID NOs: 1-3, 5-6 and 74; or at least one CDR sequence selected from SEQ ID NOs: 1-3, 5, 6 and 76; or at least one CDR sequence selected from SEQ ID NOs: 1-3, 5, 6 and 78; or at least one CDR sequence selected from SEQ ID NOs: 1-3, 5, 6 and 80; or at least one CDR sequence selected from SEQ ID NOs: 1-3, 5, 6 and 82; or at least one CDR sequence selected from SEQ ID NOs: 1-3, 5, 6 and 84; or at least one CDR sequence selected from SEQ ID NOs: 1-3, 5, 6 and 86; or at least one CDR sequence selected from SEQ ID NOs: 1-3, 5, 6 and 88; or at least one CDR sequence selected from SEQ ID NOs: 1-3, 5, 6 and 90; or at least one CDR selected from SEQ ID NOs: 1-3, 5, 7 and 114; or at least one CDR sequence selected from SEQ ID NOs: 9-11 and 5, 13 and 14; or at least one CDR sequence selected from SEQ ID NOs: 9, 10, 30, 5, 13 and 14; or at least one CDR sequence selected from SEQ ID NOs: 9, 10, 32, 5, 13 and 14; or at least one CDR sequence selected from SEQ ID NOs: 9, 10, 34, 5, 13 and 14; or at least one CDR sequence selected from SEQ ID NOs: 9, 10, 36, 5, 13 and 14; or at least one CDR sequence selected from SEQ ID NOs: 9, 10, 38, 5, 13 and 14; or at least one CDR sequence selected from SEQ ID NOs: 9, 10, 40, 5, 13 and 14; or at least one CDR sequence selected from SEQ ID NOs: 9, 10, 42, 5, 13 and 14; or at least one CDR sequence selected from SEQ ID NOs: 9, 10, 44, 5, 13 and 14; or at least one CDR sequence selected from SEQ ID NOs: 9, 10, 46, 5, 13 and 14; or at least one CDR selected from SEQ ID NOs: 9, 11, 116, 5, 14 and 118; or at least one CDR sequence selected from SEQ ID NOs: 16-18 and 5, 13 and 20; or at least one CDR sequence selected from SEQ ID NOs: 16-18 and 5, 121 and 20; or at least one CDR sequence selected from SEQ ID NOs: 22-24 and 26-28.
[0536] 14. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in paragraph 3 wherein the antibody or antigen-binding fragment, derivative or variant thereof comprises: a VH CDR3 sequence selected from SEQ ID NOs: 3 and 54; or a VH CDR3 sequence selected from SEQ ID NOs: 11, 30 and 36.
[0537] 15. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in paragraph 1 wherein the antibody or antigen-binding fragment, derivative or variant thereof comprises VH and/or VL sequences comprising: one or more sequences selected from SEQ ID NOs: 4, 8, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 112 and 113; and/or one or more sequences selected from SEQ ID NOs 12, 15, 31, 33, 35, 37, 39, 41, 43, 45, 47, 115 and 117; and/or one or more sequences selected from SEQ ID NOs 19, 21, 119 and 120; and/or one or more sequences selected from SEQ ID NOs 25, 29, 122 and 123.
[0538] 16. The antibody or antigen-binding fragment, derivative or variant thereof of paragraph 15 wherein the VH sequence is selected from SEQ ID NOs: 4, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71 and 112; and/or selected from SEQ ID NOs 12, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 115; and/or selected from SEQ ID NOs: 19 and 119; and/or selected from: SEQ ID NOs 25 and 122.
[0539] 17. The antibody or antigen-binding fragment, derivative or variant thereof of paragraphs 15 or 16 wherein the VL sequence is selected from SEQ ID NOs: 8, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 113; and/or is selected from SEQ ID NOs: 15 and 117; and/or is selected from SEQ ID NOs: 21 and 120; and/or is selected from SEQ ID NOs: 29 and 123.
[0540] 18. The antibody or antigen-binding fragment, derivative or variant thereof of paragraph 1 wherein the antibody or antigen-binding fragment, derivative or variant thereof comprises both a VH and a VL sequence comprising the sequences of a VH and VL sequence pair selected from the sequence pairs: SEQ ID NOs 4 and 8; SEQ ID NOs 49 and 8; SEQ ID NOs 51 and 8; SEQ ID NOs 53 and 8; SEQ ID NOs 55 and 8; SEQ ID NOs 57 and 8; SEQ ID NOs 59 and 8; SEQ ID NOs 61 and 8; SEQ ID NOs 63 and 8; SEQ ID NOs 65 and 8; SEQ ID NOs 67 and 8; SEQ ID NOs 69 and 8; SEQ ID NOs 71 and 8; SEQ ID NOs 112 and 8; SEQ ID NOs 4 and 113; SEQ ID NOs 49 and 113; SEQ ID NOs 51 and 113; SEQ ID NOs 53 and 113; SEQ ID NOs 55 and 113; SEQ ID NOs 57 and 113; SEQ ID NOs 59 and 113; SEQ ID NOs 61 and 113; SEQ ID NOs 63 and 113; SEQ ID NOs 65 and 113; SEQ ID NOs 67 and 113; SEQ ID NOs 69 and 113; SEQ ID NOs 71 and 113; SEQ ID NOs 4 and 73; SEQ ID NOs 4 and 75; SEQ ID NOs 4 and 77; SEQ ID NOs 4 and 79; SEQ ID NOs 4 and 81; SEQ ID NOs 4 and 83; SEQ ID NOs 4 and 85; SEQ ID NOs 4 and 87; SEQ ID NOs 4 and 89; SEQ ID NOs and 4 and 91; SEQ ID NOs 112 and 73; SEQ ID NOs 112 and 75; SEQ ID NOs 112 and 77; SEQ ID NOs 112 and 79; SEQ ID NOs 112 and 81; SEQ ID NOs 112 and 83; SEQ ID NOs 112 and 85; SEQ ID NOs 112 and 87; SEQ ID NOs 112 and 89; SEQ ID NOs and 112 and 91; SEQ ID NOs and 112 and 113; or selected from the sequence pairs: SEQ ID NOs 12 and 15; SEQ ID NOs 31 and 15; SEQ ID NOs 33 and 15; SEQ ID NOs 35 and 15; SEQ ID NOs 37 and 15; SEQ ID NOs 39 and 15; SEQ ID NOs 41 and 15; SEQ ID NOs 43 and 15; SEQ ID NOs 45 and 15; SEQ ID NOs 47 and 15; SEQ ID NOs 115 and 15; SEQ ID NOs 12 and 117; SEQ ID NOs 31 and 117; SEQ ID NOs 33 and 117; SEQ ID NOs 35 and 117; SEQ ID NOs 37 and 117; SEQ ID NOs 39 and 117; SEQ ID NOs 41 and 117; SEQ ID NOs 43 and 117; SEQ ID NOs 45 and 117; SEQ ID NOs 47 and 117; and SEQ ID NOs 115 and 117; or selected from the sequence pairs: SEQ ID NOs 19 and 21; SEQ ID NOs 19 and 120; SEQ ID NOs 119 and 21; and SEQ ID NOs 119 and 120; or selected from the sequence pairs: SEQ ID NOs 25 and 29; SEQ ID NOs 25 and 123; SEQ ID NOs 122 and 29; and SEQ ID NOs 122 and 123.
[0541] 19. The antibody or antigen-binding fragment, derivative or variant thereof of paragraph 1 comprising a VH sequence comprising a sequence selected from SEQ ID NOs: 4, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71 and 112.
[0542] 20. The antibody or antigen-binding fragment, derivative or variant thereof of paragraph 1 or 19 comprising a VL sequence comprising a sequence selected from SEQ ID NOs: 8, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 113.
[0543] 21. The antibody or antigen-binding fragment, derivative or variant thereof of paragraph 1 or 20 comprising a VH sequence comprising the sequence of SEQ ID NO: 4 or 112.
[0544] 22. The antibody or antigen-binding fragment, derivative or variant thereof of paragraph 1 comprising a VH sequence comprising the sequence of SEQ ID NO: 55.
[0545] 23. The antibody or antigen-binding fragment, derivative or variant thereof of paragraph 1 comprising a VH sequence comprising a sequence selected from SEQ ID NOs: 12, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 115.
[0546] 24. The antibody or antigen-binding fragment, derivative or variant thereof of paragraph 1 comprising a VH sequence comprising the sequence of SEQ ID NO: 12 or 115.
[0547] 25. The antibody or antigen-binding fragment, derivative or variant thereof of paragraph 1 comprising a VH sequence comprising the sequence of SEQ ID NO: 31.
[0548] 26. The antibody or antigen-binding fragment, derivative or variant thereof of paragraph 1 comprising a VH sequence comprising the sequence of SEQ ID NO: 37.
[0549] 27. The antibody or antigen-binding fragment, derivative or variant thereof of paragraph 19 or 21-22 additionally comprising a VL sequence comprising the sequence of SEQ ID NO: 8 or 113.
[0550] 28. The antibody or antigen-binding fragment, derivative or variant thereof of any of paragraphs 23-26 additionally comprising a VL sequence comprising the sequence of SEQ ID NO: 15 or 117.
[0551] 29. The antibody or antigen-binding fragment, derivative or variant thereof of paragraph 1 comprising: a VH sequence comprising the sequence of SEQ ID NO: 37; and a VL sequence comprising the sequence of SEQ ID NO: 15.
[0552] 30. An antibody or antigen-binding fragment, derivative or variant thereof according to any of the preceding paragraphs wherein the antibody or antigen-binding fragment, derivative or variant thereof is a polyclonal or a monoclonal antibody or antigen-binding fragment, derivative or variant thereof.
[0553] 31. An antibody or antigen-binding fragment, derivative or variant thereof according to paragraph 30 wherein the antibody or antigen-binding fragment, derivative or variant thereof is selected from the group consisting of Fv fragments, scFv fragments, Fab, single variable domains and domain antibodies.
[0554] 32. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in any previous paragraph wherein the antibody or antigen-binding fragment, derivative or variant thereof is humanised.
[0555] 33. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in any previous paragraph wherein the antibody or antigen-binding fragment, derivative or variant thereof has specificity for tenascin-C or a domain thereof.
[0556] 34. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in any of paragraph 33 wherein the antibody or antigen-binding fragment, derivative or variant thereof has specificity for the FBG domain of tenascin-C.
[0557] 35. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in any of paragraph 34 wherein the antibody or antigen-binding fragment, derivative or variant thereof neutralises the activity of the FBG domain of tenascin-C.
[0558] 36. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in any previous paragraph wherein said tenascin-C is citrullinated tenascin-C.
[0559] 37. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in paragraph 36 wherein the citrullinated tenascin-C is citrullinated at the FBG domain.
[0560] 38. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in paragraph 37 wherein the citrullinated tenascin-C is citrullinated at only the FBG domain.
[0561] 39. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in any previous paragraph wherein the antibody or antigen-binding fragment, derivative or variant thereof is for modulation of a chronic inflammatory response.
[0562] 40. The antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in paragraph 39 wherein the antibody or antigen-binding fragment, derivative or variant thereof modulates the biological activity of tenascin-C.
[0563] 41. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in paragraph 40 wherein the agent modulates the biological activity of tenascin-C by altering the transcription, translation and/or binding properties of tenascin-C.
[0564] 42. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in any previous paragraph wherein the antibody or antigen-binding fragment, derivative or variant thereof down-regulates the biological activity of tenascin-C.
[0565] 43. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in any previous paragraph wherein the antibody or antigen-binding fragment, derivative or variant thereof up-regulates the biological activity of tenascin-C.
[0566] 44. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in any previous paragraph wherein the antibody or antigen-binding fragment, derivative or variant thereof is an inhibitor of transcription, translation and/or the binding properties of tenascin-C.
[0567] 45. An antibody or antigen-binding fragment, derivative or variant thereof as paragraphed in any previous paragraph wherein the antibody or antigen-binding fragment, derivative or variant thereof is a competitive binding inhibitor of tenascin-C.
[0568] 46. A composition comprising an antibody or antigen-binding fragment, derivative or variant thereof as defined in any of paragraphs 1-45 and a pharmaceutically acceptable carrier, excipient and/or diluent.
[0569] 47. A composition as paragraphed in paragraph 46 further comprising at least one other agent.
[0570] 48. A composition as paragraphed in paragraph 47 wherein the at least one other agent is an anti-inflammatory agent, a statin, a biological agent (biologicals), an immunosuppressive agent, a salicylate and/or a microbicidal agent.
[0571] 49. A composition as paragraphed in paragraph 48 wherein the anti-inflammatory agent is selected from the group consisting non-steroidal anti-inflammatories (NSAIDs), corticosteroids, disease-modifying antirheumatic drugs (DMARDs) or immunosuppressants.
[0572] 50. An antibody or antigen-binding fragment, derivative or variant thereof or composition as defined in paragraphs 1-49 for use as a medicament.
[0573] 51. An antibody or antigen-binding fragment, derivative or variant thereof or composition as defined in paragraphs 1-49 for use in the treatment of a chronic inflammatory condition.
[0574] 52. Use of an antibody or antigen-binding fragment, derivative or variant thereof or composition as defined in paragraphs 1-49 in the manufacture of a medicament for the treatment or diagnosis of a chronic inflammatory condition.
[0575] 53. A method of treating a chronic inflammatory condition comprising administering to a subject an effective amount of an antibody or antigen-binding fragment, derivative or variant thereof or composition as defined in paragraphs 1-49.
[0576] 54. An antibody or antigen-binding fragment, derivative or variant thereof or composition as defined in paragraphs 1-49 for use in the diagnosis of a chronic inflammatory condition and/or determination of prognosis of a patient with a chronic inflammatory condition.
[0577] 55. A method of diagnosing a chronic inflammatory condition and/or determination of prognosis of a patient with a chronic inflammatory condition comprising detecting the presence or absence or amount of the FBG domain of tenascin-C using an antibody or antigen-binding fragment, derivative or variant thereof or composition as defined in paragraphs 1-49.
[0578] 56. An antibody or antigen-binding fragment, derivative or variant thereof or method as defined in paragraph 54 or 55 wherein an increase in the amount of the FBG domain of tenascin-C detected is indicative of a chronic inflammatory condition determination and/or of prognosis of a patient with a chronic inflammatory condition.
[0579] 57. The antibody or antigen-binding fragment, derivative or variant thereof or method of paragraph 56 wherein an increase of at least 50% in the amount of FBG domain of tenascin-C detected compared to normal levels is indicative of a chronic inflammatory condition determination and/or prognosis of a patient with a chronic inflammatory condition.
[0580] 58. An antibody or antigen-binding fragment, derivative or variant thereof or composition as defined in paragraphs 1-49 for use in determining the appropriate treatment for an individual, wherein the amount of the FBG domain of tenascin-C detected indicates the appropriate treatment for the individual.
[0581] 59. A method of determining the appropriate treatment for an individual comprising detecting the presence or absence or amount of the FBG domain of tenascin-C using an antibody or antigen-binding fragment, derivative or variant thereof or composition as defined in paragraphs 1-49, wherein the amount of the FBG domain of tenascin-C detected indicates the appropriate treatment for the individual.
[0582] 60. The antibody or antigen-binding fragment, derivative or variant thereof or composition or method of paragraphs 58 or 59 wherein the appropriate treatment comprises the administration of an effective amount of an agent or composition, the agent or composition may be one or more of: an antibody or antigen-binding fragment, derivative or variant thereof or composition as defined in paragraphs 1-49; DMARDS (such as methotrexate); anti-TNF drug; an anti-IL17 therapy; a T-cell co-stimulation modulator (such as Orencia™—abatacept): an interleukin-6 (IL-6) inhibitor (such as Actemra™—tocilizumab); an anti-CD20 antibody (such as Rituxan™—rituxumab; a B cell activating factor (such as anti-BAFF); an inhibitor of janus kinase (JAK) (such as Tofacitinib™); an inhibitor of spleen tyrosine kinase (Syk) (such as Fostamatinib™); antiTNC antibodies or antibodies to citrullinated tenascin-C domains; and/or an agent that modulates the biological activity of citrullinated and/or non-citrullinated tenascin-C.
[0583] 61. The antibody or antigen-binding fragment, derivative or variant thereof or composition method of paragraphs 58-60 wherein the appropriate treatment targets the FBG domain of tenascin-C.
[0584] 62. The antibody or antigen-binding fragment, derivative or variant thereof or composition or method of paragraphs 58-61 wherein the appropriate treatment is the administration of an effective amount of an antibody or antigen-binding fragment, derivative or variant thereof, or composition as defined in paragraphs 1-49.
[0585] 63. The antibody or antigen-binding fragment, derivative or variant thereof or composition or method of paragraphs 58-62 wherein the individual has a chronic inflammatory condition.
[0586] 64. The antibody or antigen-binding fragment, derivative or variant thereof or composition or method of paragraphs 58-63 wherein an increase in the amount of FBG domain of tenascin-C detected indicates the appropriate treatment.
[0587] 65. The antibody or antigen-binding fragment, derivative or variant thereof or composition or method of paragraph 64 wherein an increase in the amount of FBG domain of tenascin-C detected indicates that an increased amount of the appropriate treatment is required.
[0588] 66. The antibody or antigen-binding fragment, derivative or variant thereof or composition or method of paragraphs 64 or 65 wherein the increase in the amount of FBG domain of tenascin-C detected is an increase of at least 50% compared to normal levels of FBG domain of tenascin-C.
[0589] 67. The antibody or antigen-binding fragment, derivative or variant thereof or composition or method of paragraphs 56-66 wherein the amount of FBG domain of tenascin-C is determined by the use of one or more of: immunoassays; spectrometry; western blot; ELISA; immunoprecipitation; slot or dot blot assay; isoelectric focussing; SDS-PAGE; antibody microarray; immunohistological staining; radio immuno assay (RIA), fluoroimmunoassay; and/or an immunoassay using an avidin-biotin and/or streptoavidin-biotin system.
[0590] 68. An antibody or antigen-binding fragment, derivative or variant thereof, composition, use or method as paragraphed in paragraphs 51-67 wherein the chronic inflammatory response is associated with a condition characterised by inappropriate inflammation.
[0591] 69. An antibody or antigen-binding fragment, derivative or variant thereof, composition, use or method as paragraphed in paragraphs 51-67 wherein the chronic inflammatory response is associated with rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases (including Crohn's disease and ulcerative colitis), non-healing wounds, multiple sclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupus erythrematosus (including systemic lupus erythrematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage, psoriasis, ankylosing spondylitis and cardiovascular disease.
[0592] 70. A kit of parts comprising: [0593] (i) an antibody or antigen-binding fragment, derivative or variant thereof or composition as defined in paragraphs 1-49; [0594] (ii) administration means; and [0595] (iii) instructions for their use
[0596] 71. A kit of parts as paragraphed in paragraph 70 optionally comprising [0597] (iv) at least one other agent.
[0598] 72. A kit of parts for use in determining the chronic inflammatory condition status of a subject comprising: [0599] (i) an antibody or antigen-binding fragment, derivative or variant thereof or composition as defined in paragraphs 1-49; and [0600] (ii) instructions for use
[0601] It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present invention and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the following claims.