Dual specificity antibody fusions

Abstract

The present invention provides dual specificity antibody fusion proteins comprising an antibody Fab or Fab′ fragment with specificity for an antigen of interest, said fragment being fused to at least one single domain antibody which has specificity for a second antigen of interest.

Claims

1. An albumin binding antibody, comprising the sequence given in SEQ ID NO: 56 for CDR-H1, the sequence given in SEQ ID NO: 57 for CDR-H2, the sequence given in SEQ ID NO: 58 for CDR-H3, the sequence given in SEQ ID NO: 59 for CDR-L1, the sequence given in SEQ ID NO: 60 for CDR-L2, and the sequence given in SEQ ID NO: 61 for CDR-L3.

2. The albumin binding antibody of claim 1, further comprising a heavy chain VH domain having the sequence given in SEQ ID NO: 52.

3. The albumin binding antibody of claim 2, further comprising a light chain VL domain having the sequence given in SEQ ID NO: 53.

4. The albumin binding antibody of claim 1, wherein the antibody is a whole antibody or a functionally active fragment or derivative thereof.

5. The albumin binding antibody of claim 1, wherein the antibody is a Fab, a modified Fab, a Fab′, a F(ab′)2, Fv, a scFv, a bi, a tri, or a tetra-valent antibody, a Bis-scFv, a diabody, a triabody, a tetrabody, or an epitope-binding fragment thereof.

6. The albumin binding antibody of claim 1, wherein the antibody is multispecific.

7. The albumin binding antibody of claim 1, wherein the antibody is conjugated to an antibody, a protein, or a molecule.

8. The albumin binding antibody of claim 1, wherein the antibody is humanised.

9. The albumin binding antibody of claim 1, wherein the albumin is human serum albumin.

10. The albumin binding antibody of claim 1, wherein the antibody has a binding affinity for albumin of about 2 μM or better.

11. A pharmaceutical composition, comprising the albumin binding antibody of claim 1.

12. The pharmaceutical composition of claim 11, wherein the composition is for the treatment of a disease or disorder; wherein the disease or disorder is an inflammatory disease or disorder, an immune disease or disorder, a fibrotic disorder and/or a cancer; wherein the inflammatory disease or disorder and/or the immune disease or disorder comprise rheumatoid arthritis, psoriatic arthritis, still's disease, Muckle Wells disease, psoriasis, Crohn's disease, ulcerative colitis, SLE (Systemic Lupus Erythematosus), asthma, allergic rhinitis, atopic dermatitis, multiple sclerosis, vasculitis, Type I diabetes mellitus, transplantation and graft-versus-host disease; wherein the fibrotic disorder comprises idiopathic pulmonary fibrosis (IPF), systemic sclerosis (or scleroderma), kidney fibrosis, diabetic nephropathy, IgA nephropathy, hypertension, end-stage renal disease, peritoneal fibrosis, liver cirrhosis, age-related macular degeneration (ARMD), retinopathy, cardiac reactive fibrosis, scarring, keloids, burns, skin ulcers, angioplasty, coronary bypass surgery, arthroplasty and cataract surgery; and wherein the cancer comprises (i) a malignant new growth that arises from epithelium, found in skin or the lining of breast, ovary, prostate, lung, kidney, pancreas, stomach, bladder or bowel and/or (ii) bone, liver, lung or brain cancer.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1: Diagrammatic representation of Fab-dAbs where the dAb is at the C-terminus

(2) FIG. 2: Diagrammatic representation of Fab-didAbs

(3) FIG. 3: SDS PAGE analysis of FabA-dAbL3 (CK-SG.sub.4SE) (1) and FabA-dAbL3 (CK-G[APAPA].sub.2) (2).

(4) FIG. 4A: Western blot analysis of FabA-dAbL3 (CK-SG.sub.4SE) (1) and FabA-dAbL3 (CK-G[APAPA].sub.2) (2).

(5) FIG. 4B: SDS PAGE of FabB-didAbs Lane M=SeeBlue markers Lanes 1 & 2=IgG control Lane 332 FabB Lane 4 =FabB-didAb, -dAbL1 (CK-G.sub.4Sx2) & dAbH1 (CH1-G.sub.4Sx2) Lane 5=FabB-didAb, -dAbL2 (CK-G.sub.4Sx2) & dAbH2 (CH1-G.sub.4Sx2)

(6) FIG. 5A-FIG. 5P: Sequences of domain antibodies dAbH1(FIG. 5A), dAbH2 (FIG. 5C), dAbL1 (FIG. 5B) and dAbL2 (FIG. 5D) and the CDRs (CDRH1 for dAbH1 is FIG. 5E; CDRH2 for dAbH1 is FIG. 5F; CDRH3 for dAbH1 is FIG. 5G; CDRL1 for dAbL1 is FIG. 5H; CDRL2 for dAbL1 is FIG. 5I; CDRL3 for dAbL1 is FIG. 5J; CDRH1 for dAbH2 is FIG. 5K; CDRH2 for dAbH2 is FIG. 5L; CDRH3 for dAbH2 is FIG. 5M; CDRL1 for dAbL2 is FIG. 5N; CDRL2 for dAbL2 is FIG. 5O; and CDRL3 for dAbL2 is FIG. 5P) derived from each of those antibodies.

(7) FIG. 6A-FIG. 6F: FabB-dAb constructs comprising FabB heavy or light chain variable domain fused to a domain antibody. FIG. 6A is FabB-dabH1 (CH1-G.sub.4Sx2); FIG. 6B is FabB-dabH2 (CH1-G.sub.4Sx2); FIG. 6C is FabB-dabL1 (CH1-G.sub.4Sx2); FIG. 6D -FabB-dabL2 (CH1-G.sub.4Sx2); FIG. 6E is FabB-dabL1 (CK-G.sub.4Sx2); FIG. 6F is FabB-dabL2 (CK-G.sub.4Sx2).

(8) FIG. 7: Fab′ A heavy and light chain sequences and FabA heavy chain sequence.

EXPERIMENTAL

Example 1. Production of a dAb Specific for Human Serum Albumin

(9) An in-frame DNA encoded transcription unit encoding a dAb with specificity for human serum albumin was produced using recombinant DNA technology.

(10) Where desired an in-frame DNA encoded transcription unit encoding a dAb with specificity for a recruitment protein can be produced using recombinant DNA technology.

Example 2. Production of Antibody Fragment

(11) For fusion of a dAb to the C-terminus of the light chain, DNA was synthesised encoding a human kappa light chain constant region (with the Km3 allotype of the kappa constant region), a peptide linker and a dAb and cloned as a SacI-PvuII restriction fragment into the UCB-Celltech in-house expression vector pTTOD(Fab) (a derivative of pTTO-1, described in Popplewell et al., Methods Mol. Biol. 2005; 308: 17-30) which contains DNA encoding the human gamma-1 CH1 constant region. This gave rise to a dicistronic gene arrangement consisting of the gene for the humanised light chain fused via a linker to a dAb followed by the gene for the humanised heavy chain Fab fragment, both under the control of the tac promoter. Also encoded is a unique BspE1 site upstream of the Gly4Ser linker, or an AscI site upstream of the Ala-Pro-rich linker.

(12) For fusion of a dAb the C-terminus of the heavy chain, DNA was synthesised encoding a human CH1 fragment (of the γ1 isotype) followed by a linker encoding sequence and a dAb. This was sublcloned as an ApaI-EcoRI restriction fragment into the UCB-Celltech in-house expression vector pTTOD(Fab) (a derivative of pTTO-1, described in Popplewell et al., above) which contains DNA encoding the human gamma-1 CH1 constant region. This gave rise to a dicistronic gene arrangement consisting of the gene for the humanised light chain a non-coding intergenic sequence and followed by a heavy chain fused via a linker to a dAb, both under the control of the tac promoter. The recombinant expression plasmid was transformed into the E. coli strain W3110 in which expression is induced by addition of IPTG. Expression experiments were performed at small scale initially (5 ml culture volumes) with addition of 200 uM IPTG at OD(600 nm) of approx. 0.5, cells were harvested 2 hours post induction and extracted overnight at 30° C. in Tris/EDTA. Clarified extracts were used for affinity analysis by Biacore. Constructs giving promising expression yields and activities were selected for fermentation.

Methods

(13) In the following examples the antibody chain to which the dAb is fused is denoted either as CK or LC for the cKappa light chain and as CH1 or HC for the heavy chain constant domain, CH1.

Construction of FabA-dAb Fusion Plasmids for Expression in E. coli

(14) Fab-dAb fusion proteins were constructed by fusing dAbL3 or dAbH4 to the C-terminus of the constant region of either the light or heavy chain of FabA. A flexible (SGGGGSE (SEQ ID NO:1)) or a rigid (G(APAPA).sub.2 (SEQ ID NO: 34)) linker was used to link the dAb to the cKappa region (SEQ ID NO:75) whereas the linker DKTHTS (SEQ ID NO:2) was used to link the dAb to the CHI region (SEQ ID NO:76). The DNA sequence coding for the constant region-dAb fusion was manufactured synthetically as fragments to enable sub-cloning into the FabA sequence of the in-house pTTOD vector.

(15) Light chain-dAb fusions were constructed by sub-cloning the SacI-ApaI fragment of the synthesized genes, encoding a C-terminal cKappa fused to either dAbL3 or dAbH4 via either a (SGGGGSE (SEQ ID NO:1)) or a rigid (G(APAPA).sub.2 (SEQ ID NO: 34)) linker, into the corresponding sites of a plasmid capable of expressing FabA. Heavy chain-dAb fusions were constructed by sub-cloning the ApaI-EcoRI fragment of the synthesised genes, encoding a C-terminal CHI fused to either dAbL3 or dAbH4 via a DKTHTS linker, into the corresponding sites of a plasmid capable of expressing FabA.

(16) Fab′ A is derived from an IL-1 beta binding antibody, the heavy and light chain sequences of which are provided in SEQ ID NOs:74 and 75 respectively as shown in FIG. 7. In Fab′A where the light chain has a dAb attached, the hinge of the heavy chain was altered to DKTHTS even where no dAb is attached to the heavy chain (SEQ ID NO:76).

(17) FabA comprises the same light chain sequence (SEQ ID NO:75) and a truncated heavy chain sequence which terminates at the interchain cysteine (SEQ ID NO:77). dAbL3 and dAbH4 are light and heavy chain domain antibodies respectively which bind human serum albumin.

Construction of FabA-didAb Fusion Plasmids for Expression in E. coli

(18) FabA-didAb with dAbL3 or dAbH4 on both light and heavy chains were constructed by sub-cloning the ApaI-EcoRI fragment coding for CH1-dAb fusions into the existing Fab-dAb plasmids where the dAb is fused to the light chain via the flexible linker.

Construction of FabB-dAb Fusion Plasmids for Expression in Mammalian Cells

(19) The FabB-dAbs, FabB-dAbH1 (CH1-G.sub.4Sx2), FabB-dAbH2 (CH1-G.sub.4Sx2), FabB-dAbL1 (CH1-G.sub.4Sx2), FabB-dAbL2 (CH1-G.sub.4Sx2) were all assembled by PCR then cloned into a mammalian expression vector under the control of the HCMV-MIE promoter and SV40E polyA sequence. These were paired with a similar vector containing the FabB light chain for expression in mammalian cells (see below).

(20) FabB is derived from an antibody which bids a cell surface co-stimulatory molecule. dAbH1, dAbH2, dAbL1 and dAbL2 were obtained as described in Example 3.

Construction of FabB-dAb Fusion Plasmids for Expression in Mammalian Cells

(21) The FabB-dAbs, FabB-dAbH1 (CH1-G.sub.4Sx2), FabB-dAbH2 (CH1-G.sub.4Sx2), FabB-dAbL1 (CK-G.sub.4Sx2), FabB-dAbL2 (CK-G.sub.4Sx2) were all assembled by PCR then cloned into a mammalian expression vector under the control of the HCMV-MIE promoter and SV40E polyA sequence.

Mammalian Expression of FabB-dAbs and didAbs

(22) HEK293 cells were transfected with the heavy and light chain plasmids using Invitrogen's 293fectin transfection reagent according to the manufacturer's instructions. Briefly, 2 μg heavy chain plasmid +2 μg light chain plasmid was incubated with 10 μl 293fectin+340 μl Optimem media for 20 mins at RT. The mixture was then added to 5×10.sup.6 HEK293 cells in suspension and incubated for 4 days with shaking at 37° C.

Biacore

(23) Binding affinities and kinetic parameters for the interactions of Fab-dAb constructs were determined by surface plasmon resonance (SPR) conducted on a Biacore T100 using CM5 sensor chips and HBS-EP (10mM HEPES (pH7.4), 150 mM NaCl, 3 mM EDTA, 0.05% v/v surfactant P20) running buffer. Fab-dAb samples were captured to the sensor chip surface using either a human F(ab′).sub.2-specific goat Fab (Jackson ImmunoResearch, 109-006-097) or an in-house generated anti human CH1 monoclonal antibody. Covalent immobilisation of the capture antibody was achieved by standard amine coupling chemistry.

(24) Each assay cycle consisted of firstly capturing the Fab-dAb using a 1 min injection, before an association phase consisting of a 3 min injection of antigen, after which dissociation was monitored for 5 min. After each cycle, the capture surface was regenerated with 2×1 min injections of 40 mM HCl followed by 30 s of 5 mM NaOH. The flow rates used were 10 μl/min for capture, 30 μl/min for association and dissociation phases, and 10 μl/min for regeneration.

(25) For kinetic assays, a titration of antigen (for human serum albumin typically 62.5 nM-2 μM, for IL-1β 1.25-40nM) was performed, a blank flow-cell was used for reference subtraction and buffer-blank injections were included to subtract instrument noise and drift.

(26) Kinetic parameters were determined by simultaneous global-fitting of the resulting sensorgrams to a standard 1:1 binding model using Biacore T100 Evaluation software.

(27) In order to test for simultaneous binding, 3 min injections of either separate 5 μM HSA or 100 nM IL-1β, or a mixed solution of 5 μM HSA and 100 nM IL-1β were injected over the captured Fab-dAb.

(28) Fab-dAb Purification from E. coli

Periplasmic Extraction

(29) E. coli pellets containing the Fab-dAbs within the periplasm were re-suspended in original culture volume with 100 mM Tris/HCl, 10 mM EDTA pH 7.4. These suspensions were then incubated at 4° C. for 16 hours at 250 rpm. The re-suspended pellets were centrifuged at 10000×g for 1 hour at 4° C. The supernatants were removed and 0.45 μm filtered.

Protein-G Capture

(30) The Fab-dAbs were captured from the filtered supernatant by Protein-G chromatography. Briefly the supernatants were applied, with a 20 minute residence time, to a Gammabind Plus Sepharose (GE Healthcare) column equilibrated in 20 mM phosphate, 150 mM NaCl pH7.1. The column was washed with 20 mM phosphate, 150 mM NaCl pH7.1 and the bound material eluted with 0.1M glycine/HCl pH2.8. The elution peak was collected and pH adjusted to ˜pH5 with 1M sodium acetate.

(31) The pH adjusted elutions were concentrated and diafiltered into 50 mM sodium acetate pH4.5 using a 10k MWCO membrane.

Ion Exchange

(32) The Fab-dAbs were further purified by cation exchange chromatography at pH4.5 with a NaCl elution gradient. Briefly the diafiltered Protein-G eluates were applied to a Source15S (GE Healthcare) column equilibrated in 50 mM sodium acetate pH4.5. The column was washed with 50 mM sodium acetate pH4.5 and the bound material eluted with a 20 column volume linear gradient from 0 to 1M NaCl in 50 mM sodium acetate pH4.5. Third column volume fractions were collected through out the gradient. The fractions were analysed by A280 and SDS-PAGE and relevant fractions pooled.

Gel Filtration

(33) If required the Fab-dAbs were further purified by gel filtration. Briefly the FabA-dAbL3 (CK-SG.sub.4SE) pooled ion exchange elution fractions were applied to a Superdex200 (GE Healthcare) column equilibrated in 50 mM sodium acetate, 125 mM NaCl pH 5.0 and eluted with an isocratic gradient of 50 mM sodium acetate, 125 mM NaCl pH 5.0. 1/120 column volume fractions were collected through out the gradient. The fractions were analysed by A280 and SDS-PAGE and relevant fractions pooled. For Fab-dAbs that did not undergo gel filtration, the pooled ion exchange elution fractions were concentrated and diafiltered into 50 mM sodium acetate, 125 mM NaCl pH 5.0 using a 10k MWCO membrane.

SDS-Page

(34) Samples were diluted with water where required and then to 10 μl was added 10 μL 2× sample running buffer. For non-reduced samples, 2 μL of 100 mM NEM was added at this point, for reduced samples 2 μL of 10× reducing agent was added. The sample were vortexed, incubated at 85° C. for 5 mins, cooled and centrifuged at 12500 rpm for 30 secs. The prepared samples were loaded on to a 4-20% acrylamine Tris/Glycine SDS gel and run for 100 mins at 125V. The gels were either transferred onto PVDF membranes for Western blotting or stained with Coomassie Blue protein stain.

Western Blotting

(35) Gels were transferred to PVDF membranes in 12 mM Tris, 96 mM glycine pH8.3 for 16 hours at 150 mA. The PVDF membrane was block for 1 hr with 2% Marvel™ in PBS+0.1% Tween20 (Blocking buffer)

Anti-Light Chain

(36) HRP-rabbit anti-human kappa light chains, 1/5000 dilution in blocking buffer for 1 hr.

Anti-Heavy Chain

(37) mouse anti-human heavy chain, 1/7000 dilution in blocking buffer for 1 hr. Followed by HRP-goat anti-mouse, 1/2000 dilution in blocking buffer for 1 hr.

Anti-His Tag

(38) rabbit anti-His6, 1/1000 dilution in blocking buffer for 1 hr. Followed by HRP-goat anti-rabbit IgG, 1/1000 dilution in blocking buffer for 1 hr.

(39) All blots were washed 6 times with 100 ml PBS+0.1% Tween20 for 10 minutes per wash. The blots were developed with either ECL reagent for 1 min before being exposed to Amersham Hyperfilm, or metal enhanced DAB reagent for 20-30 minutes followed by water.

High Temperature Reverse Phase HPLC

(40) Samples (2 μg) were analysed on a 2.1 mm C8 Poroshell column at 80° C., with a flow rate of 2 ml/min and a gradient of 18-38% B over 4 mins.

(41) A=0.1% TFA in H.sub.20

(42) B=0.065% TFA in 80:20 IPA:MeOH

(43) Detection is by absorption at 214 nm.

ELISA

(44) The yields of Fab-dAb were measured using a sandwich ELISA. Briefly, the Fab-dAb was captured with an anti-CH1 antibody then revealed with an anti-kappa-HRP.

Example 3

Generating Anti-Albumin Antibodies

(45) ½ lop rabbits were immunised with recombinant chromapure human serum albumin (purchased from Jackson). Rabbits received 3 immunisations of 100 ug HSA protein sub cutaneously, the first immunisation in complete Freunds adjuvant and subsequent immunisations in incomplete Freunds. Antibodies 1 and 2 which bind human, mouse and rat serum albumin were isolated using the methods described in WO04/051268. Genes for the heavy chain variable domain (VH) and light chain variable domain (VL) of antibodies 1 and 2 were isolated and sequenced following cloning via reverse transcription PCR.

(46) The light chain grafted sequences were sub-cloned into the rabbit light chain expression vector pVRbcK which contains the DNA encoding the rabbit C-Kappa constant region. The heavy chain grafted sequences were sub-cloned into the rabbit heavy chain expression vector pVRbHFab, which contains the DNA encoding the rabbit Fab′ heavy chain constant region. Plasmids were co-transfected into CHO cells and the antibodies produced screened for albumin binding and affinity (Table 1). Transfections of CHO cells were performed using the Lipofectamine™ 2000 procedure according to manufacturer's instructions (InVitrogen, catalogue No. 11668).

Generating Humanised Domain Antibodies dAbL1, dAbH1, dAbL2 and dAbH2

(47) Humanised VL and VH regions were designed using human V-region acceptor frameworks and donor residues in the framework regions. One grafted VL region (L1 (SEQ ID NO:53) and L2 (SEQ ID NO:55)) and one VH region (H1 (SEQ ID NO:52) and H2 (SEQ ID NO:54)) were designed for each of antibodies 1 and 2 and genes were built by oligonucleotide assembly and PCR mutagenesis. The grafted domain antibodies and their CDRs are shown in FIG. 5.

(48) TABLE-US-00003 TABLE 1 Affinities of anti-albumin antibodies as rabbit Fab as humanised IgG Human SA murineSA Human SA nM nM nM Antibody 1 0.31 2.6 0.82 Antibody 2 0.33 12 0.13

Example 4: Analysis of FabB-dAbs Expressed in Mammalian Cells

(49) FabB-dAb constructs were produced as described in the methods and the supernatants from the tranfected HEK293 cells containing the FabB-dAbs were tested directly in BIAcore.

(50) Kinetic analysis was conducted to assess the interaction of HSA with FabB-dAb constructs. These consisted of either dAbL1, dAbH2 or dAbL3 fused to the C-terminus of CH1 of FabB (See FIG. 6). The FabB-dAbL1 has a higher affinity for HSA , K.sub.D=170 nM, than FabB-dAbL3, K.sub.D=392 nM. The FabB-dAbH2 was shown to possess the poorest affinity towards HSA, K.sub.D=1074 nM, see Table 2.

(51) TABLE-US-00004 TABLE 2 k.sub.a k.sub.d Construct (×10.sup.4 M.sup.−1s.sup.−1) (×10.sup.−3 s.sup.−1) K.sub.D (×10.sup.−9M) FabB-dAbL1 (CH1-G.sub.4Sx2) 1.91 ± 0.74 2.18 ± 1.21 170 ± 78 FabB-dAbH2 (CH1-G.sub.4Sx2) 2.66 ± 0.39   29 ± 4.76 1074 ± 42  FabB-dAbL3 (CH1-G.sub.4Sx2) 2.63 ± 0.39 9.87 ± 1.63  392 ± 119

(52) Affinity and kinetic parameters determined for the binding of HSA to FabBs fused to dAbL1, dAbH2 or dAbL3. The data shown are mean values±SEM. (For FabB-dAbL1 and FabB-dAbH2 n=4. For FabB-dAbL3 n=2).

(53) SDS-PAGE and western blotting of the FabB-dAb proteins confirmed that the FabB-dAbs produced were of the expected size.

Example 5: Analysis of FabB-didAbs Expressed in Mammalian Cells

(54) FabB-didAb constructs were produced as described in the methods and the supernatants from the tranfected HEK293 cells containing the didAbs tested directly in BIAcore.

(55) Further analysis was performed using didAb constructs in which single dAbs were fused to both heavy and light C-termini of Fab. Constructs in which the didAb was derived from a natural heavy and light variable domain pairing showed a marked improvement in affinity compared to the single dAb alone (table 2 and 3). The didAb fusion consisting of two identical dAbL1s showed no improvement in affinity over that seen for the single dAbL1 (data not shown).

(56) TABLE-US-00005 TABLE 3 Construct k.sub.a (×10.sup.4 M.sup.−1s.sup.−1) k.sub.d (×10.sup.−3 s.sup.−1) K.sub.D (×10.sup.−9M) FabB-didAb, -dAbL1 1.78 0.16 9 (CK-G.sub.4Sx2) & dAbH1 (CH1-G.sub.4Sx2) FabB-didAb, -dAbL2 0.54 0.21 39 (CK-G.sub.4Sx2) & dAbH2 (CH1-G.sub.4Sx2)

(57) Affinity and kinetic parameters determined for the binding of HSA to FabBs fused to both dAbL1 & dAbH1 or dAbL2 & dAbH2.

(58) SDS-PAGE of the FabB-didAb proteins confirmed that the FabB-didAbs expressed well and were of the expected size (See FIG. 4B). Note this SDS PAGE gel is total protein expressed by the cell.

Example 6

Analysis of Purified FabA-dAbs

(59) Plasmids for expression of the Fab-dAbs, Fab′A-dAbL3 (CK-SG.sub.4SE) Fab′A-dAbL3 (CK-G[APAPA].sub.2) in E. coli were constructed as described in the methods. The Fab-dAbs were expressed into the periplasm of the E. coli and purified to homogeneity as described in the methods. The purity of the Fab-dAbs were assessed by high temperature reverse phase HPLC, SDS-PAGE and Western blotting. The Fab-dAbs were also assessed for antigen binding by Biacore.

High Temperature Reverse Phase HPLC

(60) High temperature reverse phase HPLC as performed as described in the methods gave quantitative analysis of all species contained in FabA-dAbL3 (CK-SG.sub.4SE) and FabA-dAbL3 (CK-G[APAPA].sub.2). The percentage of each species present is shown in table 4.

(61) TABLE-US-00006 TABLE 4 Quantification of species present in Fab-dAb batches Species Fab′A-dAbL3 (CK-SG.sub.4SE) Fab′A-dAbL3 (CK-G[APAPA].sub.2) 1 0.6% 1.8% 2 0.6% 0.0% 3 1.0% 0.3% 4 0.9% 0.8% Fab-dAb 85.5% 92.9% Di 11.5% 4.2% Fab-dAb

SDS-Page

(62) Fab-dAb samples were prepared under non-reduced and reduced conditions and run on a gel as described in the methods. The gel was Coomassie stained. The banding profile of both Fab-dAb samples, Fab′A-dAbL3 (CK-SG.sub.4SE) and Fab′A-dAbL3 (CK-G[APAPA].sub.2), corresponds well to the profile observed by high temperature reverse phase HPLC (FIG. 3).

Western Blot

(63) Fab-dAb samples were subjected to non-reduced SDS-PAGE followed by western blot analysis with anti-light chain and anti-heavy chain antibodies as described in the methods. This confirmed that the dAb was on the light chain of the Fab and that the heavy chain was unmodified in both samples (FIG. 4A). It also demonstrates that all bands detected by coomassie stained, non-reduced SDS PAGE are Fab-dAb related products.

Biacore

(64) Kinetic analysis by SPR as described in the methods was used to assess the binding of human serum albumin to Fab′A-dAbL3 (CK-SG.sub.4SE) and Fab′A-dAbL3 (CK-G[APAPA].sub.2). The results in table 5 demonstrate that both constructs are able to bind human serum albumin with a similar affinity (K.sub.D) of approximately 1 μM.

(65) TABLE-US-00007 TABLE 5 k.sub.d Construct k.sub.a (×10.sup.4 M.sup.−1s.sup.−1) (×10.sup.−2 s.sup.−1) K.sub.D (×10.sup.−9M) Fab′A-dAbL3 (CK-SG.sub.4SE) 3.44 1.42 411 Fab′A-dAbL3 (CK- 9.61 2.85 296 G[APAPA].sub.2)

(66) Further kinetic analysis demonstrated that all the fusion constructs retained the interaction characteristics of the original FabA towards IL-1β, table 6, with only minor differences seen in the kinetic and affinity parameters.

(67) TABLE-US-00008 TABLE 6 k.sub.a k.sub.d Construct (×10.sup.5 M.sup.−1s.sup.−1) (×10.sup.−5 s.sup.−1) K.sub.D (×10.sup.−12M) Fab′A-dAbL3 (CK-SG.sub.4SE) 1.90 4.21 221 Fab′A-dAbL3 (CK- 2.17 3.99 184 G[APAPA].sub.2) Fab′A 2.02 6.46 320

(68) The potential for each construct to bind simultaneously to both human serum albumin and the IL-1β antigen was assessed by capturing each construct to the sensor chip surface, before performing either separate 3 min injections of 5 μM human serum albumin or 100 nM IL-1β, or a mixed solution of both 5 μM human serum albumin and 100 nM IL-1β. For each Fab-dAb construct the response seen for the combined HSA/IL-1β solution was almost identical to the sum of the responses of the independent injections, see table 7. This shows that the Fab-dAbs are capable of simultaneous binding to both IL-1β and human serum albumin, and that binding of either IL-1β or human serum albumin does not inhibit the interaction of the other. The original FabA bound only to IL-1β, with negligible binding to human serum albumin.

(69) TABLE-US-00009 TABLE 7 Construct Analyte Binding (RU) Fab′A-dAbL3 (CK-SG.sub.4SE) HSA + IL-1β 37.6 HSA 13.2 (37.9) IL-1β 24.7 Fab′A-dAbL3 (CK-G[APAPA].sub.2) HSA + IL-1β 61.9 HSA 30.7 (63.6) IL-1β 32.9 Fab′A HSA + IL-1β 30.3 HSA 1.3 (30.0) IL-1β 28.7

(70) The table above shows the binding response (RU) seen for each construct after separate injections of HSA or IL-1β, or injection of premixed HSA and IL-1β. In each case the final concentration was 5 μM for HSA and 100 nM for IL-1β. The sum of the individual HSA and IL-1β responses is shown in parentheses.

Example:7 FabA didAbs

Expression of FabA-didAbs in E. coli

(71) FabA-dAbs and FabA-didAb fusions terminating with a C-terminal HIS6 tag were expressed in Escherichia coli. After periplasmic extraction, dAb fusion proteins were purified via the C-terminal His6 tag. Fab expression was analysed by Western blotting of a non-reduced gel with anti-CH1 and anti-cKappa antibodies. FabA-dAb and FabA-didAb were expressed as full-length proteins and were shown to react to both antibody detection reagents.

Analysis of FabA-didAbs Expressed in E. coli

(72) Further analysis was conducted to characterise the binding of HSA to FabA constructs to which one or more dAbs were fused. Binding assays were performed on a variety of constructs in which dAbL3 or dAbH4 fused to either the light or heavy chain of the FabA (see Table 8 for details of the constructs and summary of the binding data). Although constructs carrying only dAbH4, on either the light or heavy chain, were seen to bind HSA with comparatively poor affinity (≈9 μM and 3 μM respectively), higher affinity binding was observed for constructs carrying dAbL3, either as a single fusion (on either light or heavy chain) or partnered with a second dAb (dAbL3 or dAbH4) on the opposing chain.

(73) TABLE-US-00010 TABLE 8 k.sub.a k.sub.d Construct (×10.sup.4 M.sup.−1s.sup.−1) (×10.sup.−3 s.sup.−1) K.sub.D (×10.sup.−9M) FabA — — nb FabA-dAbL3 (LC-SG.sub.4SE) 4.46 16.2 363 FabA-dAbH4 (LC SG.sub.4SE) — — 9142 FabA-dAbL3 (HC-DKTHTS) 8.24 15.4 187 FabA-dAbH4 (HC-DKTHTS) — — 2866 FabA-didAb, -dAbL3 3.00 15.1 502 (LC-SG.sub.4SE) & -dAbL3 (HC-DKTHTS) FabA-didAb, -dAbL3 4.36 16.3 373 (LC-SG.sub.4SE) & -dAbH4 (HC-DKTHTS)

(74) Affinity and kinetic parameters determined for the binding of HSA to FabAs carrying dAbL3 or dAbH4 on either light chain (LC) or heavy chain (HC) or both as indicated. No binding (nb) of HSA to the original FabA was detected. The interaction kinetics for the binding of HSA to the FabA with (dAbH4 on HC) or (dAbH4 on LC), were too rapid to determine, therefore affinity (K.sub.D) was determined from steady-state binding.