Albumin binding antibodies and binding fragments thereof

Abstract

A serum albumin binding antibody or fragment thereof comprising a heavy chain variable domain having the sequence given in SEQ ID NO: 1 or SEQ ID NO:2 and/or comprising a light chain variable domain having the sequence given in SEQ ID NO:3 or SEQ ID NO:4, in particular comprising a heavy chain variable domain and a light chain variable domain having the sequence given in SEQ ID NO: 1 and SEQ ID NO:3 or a heavy chain variable domain and a light chain variable domain having the sequence given in SEQ ID NO: 2 and SEQ ID NO:4. The disclosure also extends to polynucleotides encoding the antibodies or fragments, vectors comprising same and host cells capable of expressing the polynucleotides. The disclosure further includes pharmaceutical compositions comprising the antibodies or fragments and therapeutic used of any one of the same.

Claims

1. A serum albumin binding antibody or fragment thereof comprising a heavy chain variable domain having the sequence given in SEQ ID NO: 1 or SEQ ID NO:2.

2. A serum albumin binding antibody or fragment thereof comprising a light chain variable domain having the sequence given in SEQ ID NO:3 or SEQ ID NO:4.

3. A serum albumin binding antibody or fragment thereof comprising a heavy chain variable domain having the sequence given in SEQ ID NO: 1 and a light chain variable domain having the sequence given in SEQ ID NO:3.

4. A serum albumin binding antibody or fragment thereof comprising a heavy chain variable domain having the sequence given in SEQ ID NO:2 and a light chain variable domain having the sequence given SEQ ID NO:4.

5. A serum albumin binding antibody or fragment according to any one of claims 1 to 4, where the antibody or fragment is selected from the group consisting of a Fab, modified Fab, Fab′, F(ab′)2, Fv, scFv, bi, tri or tetra-valent antibody, Bis-scFv, diabody, triabody, tribody, DVD-Ig and BiTE.

6. A bispecific antibody fusion protein comprising: a heavy chain comprising, in sequence from the N-terminal, a first heavy chain variable domain (VH1), a CH1 domain and a second heavy chain variable domain (VH2), a light chain comprising, in sequence from the N-terminal, a first light chain variable domain (VL1), a CL domain and a second light chain variable domain (VL2), wherein said heavy and light chains are aligned such that VH1 and VL1 form a first antigen binding site and VH2 and VL2 form a second antigen binding site, wherein the antigen bound by the second antigen binding site is human serum albumin and wherein the second heavy chain variable domain (VH2) has the sequence given in SEQ ID NO:1 and the second light chain variable domain (VL2) has the sequence given in SEQ ID NO: 3 or the second heavy chain variable domain (VH2) has the sequence given in SEQ ID NO:2 and the second light chain variable domain (VL2) has the sequence given in SEQ ID NO: 4, and the second heavy chain variable domain (VH2) and second light chain variable domain (VL2) are optionally linked by a disulphide bond.

7. A polynucleotide encoding an antibody or fragment as defined in any one of claim 3, 4, or 6.

8. A vector comprising a polynucleotide as defined in claim 7.

9. A host cell comprising a vector according to claim 8.

10. A process of producing an antibody or fragment comprising expressing same from a host cell as defined in claim 9, and isolating said antibody or fragment.

11. A pharmaceutical formulation comprising an antibody or fragment as defined in any one of claim 3, 4, or 6.

Description

LIST OF FIGURES

(1) FIG. 1A: Diagrammatic representation of a Fab-Fv

(2) FIG. 1B: Diagrammatic representation of a Fab-dsFv

(3) FIGS. 2 to 5: Sequences of the present invention

(4) FIG. 6: Shows binding of AlexaFluor 488 labelled A26 Fab-dsFv to activated human CD4.sup.+OX40.sup.+T cells

(5) FIG. 7: Shows ug/ml of antibody constructs produced by transient expression in HEK293 cells

(6) FIG. 8 Shows SDS-PAGE of Fab disulphide stabilised scFv.

(7) FIG. 9 Shows tabulated data relating to the binding affinity to human serum albumin of various constructs

(8) FIG. 10 Shows tabulated data of affinity Fab binding antigen of various constructs

(9) FIG. 11 Shows ug/ml of antibody constructs produced by transient expression in CHO cells

(10) FIG. 12 Shows SDS-PAGE analysis of various constructs

(11) FIG. 13 Shows thermostablity data for various constructs expressed in CHO cells.

DNA MANIPULATIONS AND GENERAL METHODS

(12) Competent E. coli strains were used for transformations and routine culture growth. DNA restriction and modification enzymes were obtained from Roche Diagnostics Ltd. and New England Biolabs. Plasmid preparations were performed using Maxi Plasmid purification kits (QIAGEN, catalogue No. 12165). DNA sequencing reactions were performed using the ABI Prism Big Dye terminator sequencing kit (catalogue No. 4304149) and run on an ABI 3100 automated sequencer (Applied Biosystems). Data was analysed using the program Sequencher (Genecodes). Oligonucleotides were obtained from Sigma or Invitrogen. Genes encoding initial V-region sequences were constructed by an automated synthesis approach by DNA2.0, and modified to generate the grafted versions by oligonucleotide directed mutagenesis. The concentration of Fab-Fv was determined by a Protein-G based HPLC method.

EXAMPLE 1

Generation and Analysis of Different Humanisation Grafts of 645 in A26Fab-645dsFv

(13) We have previously described the Fab-dsFv antibody format (FIG. 1B) and a humanised anti-albumin antibody known as ‘645gH1gL1’ in WO2010/035012. We have also previously described the generation of a humanised antagonistic anti-OX40 antibody known as ‘A26’ in WO2010096418. Here we describe the generation of a new improved humanised graft of antibody ‘645’ known as 645dsgH5gL4 and the generation of a Fab-dsFv antibody molecule incorporating that graft in the Fv component and the ‘A26’ variable regions in the Fab component.

(14) The sequences of 645gH1 and gL1 are given in FIGS. 3 (a) and (b), SEQ ID NOs 9 and 10.

Construction of A26Fab-645dsFv(gH1gL1) and A26Fab-645dsFv(gH5gL4) Plasmids

(15) The total coding region of A26Fab-645dsFv(gL1) light chain (SEQ ID NO:12) was cloned into a UCB mammalian expression vector under the control of the HCMV-MIE promoter and SV40E polyA sequence. The light chain variable region of 645dsFv(gL1) (SEQ ID NO:10) was mutated to 645dsFv(gL4) (SEQ ID NO:4) by an overlapping PCR method. The total coding region of A26Fab-645dsFv(gH1) heavy chain (SEQ ID NO:11) was cloned into a UCB mammalian expression vector under the control of the HCMV-MIE promoter and SV40E polyA sequence. The heavy chain variable region of 645dsFv(gH1) (SEQ ID NO:9) was mutated to 645dsFv(gH5) (SEQ ID NO:2) by an overlapping PCR method. The constructs were verified by sequencing.

Mammalian Expression of A26Fab-645dsFv(gH1gL1) and A26Fab-645dsFv(gH5gL4)

(16) HEK293 cells were transfected with the heavy and light chain plasmids using Invitrogen's 293fectin transfection reagent according to the manufacturer's instructions. Briefly, 25 μg heavy chain plasmid and 25 μg light chain plasmid were incubated with 100 μl 293fectin and 1700 μl Optipro media for 20 mins at RT. The mixture was then added to 50×10.sup.6 HEK293 cells in 50 ml suspension and incubated for 6 days with shaking at 37° C. After 6 days the supernatant was collected by centrifugation at 1500×g for 10 minutes to remove the cells and then 0.22 μm sterile filtered.

Protein-G Purification of A26Fab-645dsFv(gH1gL1) and A26Fab-645dsFv(gH5gL4)

(17) The ˜50 ml of 0.22 μm filtered supernatants were concentrated to ˜2 ml using Amicon Ultra-15 concentrators with a 10 kDa molecular weight cut off membrane and centrifugation at 4000×g in a swing out rotor. 1.8 ml of concentrated supernatant was applied at 1 ml/min to a 1 ml Gammabind Plus Sepharose (GE Healthcare) column equilibrated in 20 mM phosphate, 40 mM NaCl pH7.4. The column was washed with 20 mM phosphate, 40 mM NaCl pH7.4 and the bound material eluted with 0.1M glycine/HCl pH2.7. The elution peak was collected and pH adjusted to ˜pH7 with 2M Tris/HCl pH8.5. The pH adjusted elution was concentrated and diafiltered into 20 mM phosphate, 150 mM NaCl pH7.4 using Amicon Ultra-15 concentrators with a 10 kDa molecular weight cut off membrane and centrifugation at 4000×g in a swing out rotor, to a final volume of ˜0.3 ml.

Size Exclusion Analysis A26Fab-645dsFv(gH1gL1) and A26Fab-645dsFv(gH5gL4)

(18) Protein-G purified samples were analysed by size exclusion HPLC. The samples were separated on a Superdex 200 10/300 GL Tricorn column (GE Healthcare) developed with an isocratic gradient of PBS pH7.4 at 1 ml/min. Peak detection was at 280 nm and apparent molecular weight was calculated by comparison to a standard curve of known molecular weight proteins verses elution volume. Changing the humanisation graft of the 645dsFv from gH1gL1 to gH5gL4 resulted in an increase in the percentage monomer of the expressed A26Fab-645dsFv from 59% to 71% an increase of 12%, without any change in the thermal stability of the dsFv (data not shown) or in the affinity of binding of the dsFv to HSA (data not shown).

EXAMPLE 2

2.1 BIAcore Kinetics for A26 Fab-dsFv (645gH5gL4) Binding OX40

(19) In this and all subsequent examples the A26 Fab-dsFv 645gH5gL4 had the heavy chain sequence given in SEQ ID NO:7 (FIG. 2 (g)) and the light chain sequence given in SEQ ID NO:8 (FIG. 2(h)) i.e. the heavy chain contained the G4S, G4T, G4S linker given in SEQ ID NO:5, FIG. 2 (e).

(20) BIA (Biamolecular Interaction Analysis) was performed using a BIAcore T200 (GE Healthcare). Affinipure F(ab′).sub.2 Fragment goat anti-human IgG, F(ab′).sub.2 fragment specific (Jackson ImmunoResearch) was immobilised on a CM5 Sensor Chip via amine coupling chemistry to a capture level of ≈5000 response units (RUs). HBS-EP buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% Surfactant P20, GE Healthcare) was used as the running buffer with a flow rate of 10 μL/min. A 10 μL injection of A26 Fab′ at 0.5 μg/mL or A26Fab-dsFv at 1 μg/mL was used for capture by the immobilised anti-human IgG-F(ab′).sub.2. Human OX40 was titrated over the captured A26 at various concentrations (25 nM to 1.5625 nM) at a flow rate of 30 μL/min. The surface was regenerated by 2×10 μL injection of 50 mM HCl, followed by a 5 μL injection of 5 mM NaOH at a flowrate of 10 μL/min. Background subtraction binding curves were analysed using the T200evaluation software (version 1.0) following standard procedures. Kinetic parameters were determined from the fitting algorithm.

(21) TABLE-US-00003 Sample ka (1/Ms) kd (1/s) KD (M) KD (pM) Fab′ 2.18 ± 0.38E+05 1.00E−05 4.68E−11 46.8 Fab-Fv 2.55 ± 0.35E+05 1.04E−05 4.12E−11 41.2 Average of 4 determinations

2.2. BIAcore Kinetics for A26 Fab-dsFv (645gH5gL4) Binding Albumin

(22) BIA (Biamolecular Interaction Analysis) was performed using a BIAcore T200 (GE Healthcare). Affinipure F(ab′).sub.2 Fragment goat anti-human IgG, F(ab′).sub.2 fragment specific (Jackson ImmunoResearch) was immobilised on a CM5 Sensor Chip via amine coupling chemistry to a capture level of ≈5000 response units (RUs). HBS-EP buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% Surfactant P20, GE Healthcare) was used as the running buffer with a flow rate of 10 μl/min. A 10 μL injection of Fab-Fv at 0.75 μg/mL was used for capture by the immobilised anti-human IgG-F(ab′).sub.2. Human Serum Albumin (HSA), Mouse Serum albumin (MSA) and Cynomolgus Serum Albumin (CSA) was titrated over the captured Fab-Fv at various concentrations (50 nM to 6.25 nM) at a flow rate of 30 μL/min. The surface was regenerated by 2×10 μL injection of 50 mM HCl, followed by a 5 μL injection of 5 mM NaOH at a flowrate of 10 μL/min. Background subtraction binding curves were analysed using the T200evaluation software (version 1.0) following standard procedures. Kinetic parameters were determined from the fitting algorithm.

(23) TABLE-US-00004 Sample ka (1/Ms) kd (1/s) KD (M) KD (nM) HSA 5.84E+04 1.63E−04 2.93E−09 2.93 MSA 8.86E+04 3.68E−04 4.16E−09 4.16 CSA  7.1E+04 1.89E−04 2.66E−09 2.66 Average of 3 determinations

2.3 Demonstration of A26 Fab-dsFv(645gH5gL4) binding OX40 and Albumin Simultaneously

(24) The simultaneous binding of human OX40 and Human Serum Albumin to A26 Fab-dsFv was assessed. The A26 Fab-dsFv construct was captured to the sensor chip surface as stated in the method for Biacore kinetics for binding A26 Fab-dsFv albumin. 50 nM HAS, 25 nM OX40 or a mixed solution with final concentration of 50 nM HSA and 25 nM OX40 were titrated separately over the captured A26 Fab-dsFv. The binding response for the combined HSA/OX40 solution was equivalent to the sum of the responses of the independent injections. This confirms that the Fab-dsFv is capable of simultaneous binding to both human OX40 and HSA.

(25) TABLE-US-00005 Sample Analyte Binding (RU) A26 Fab-Fv hOX40 25 HSA  9 hOX40 + HSA 35 (34)

2.4 Cell-Based Affinity of A26 Fab-dsFv (645gH5gL4)

(26) Methods:

(27) A26 Fab-Fv Binding to Human Activated CD4.sup.+OX40.sup.+T Cells.

(28) PBMC were isolated by separation on a Ficoll gradient and activated with 4 μg/mL PHA-L for 3 days at 37° C., 5% CO.sub.2, 100% humidity. CD4.sup.+ T cells were isolated by negative selection using magnetic beads (CD4.sup.+ T cell Isolation Kit II for Human; Miltenyi Biotec). Approximately 1×10.sup.5 cells were incubated in the presence of antibody in either Facs buffer (PBS/0.2% BSA/0.09% NaN3) or Facs buffer supplemented with 5% HSA at 4° C. The final concentration of the antibody ranged from 48 nM-0.0005 nM)). The cells were washed in PBS prior to analysis by flow cytometry using a FACScalibur (Becton Dickinson).Two titration data sets were produced in both buffer conditions, one with A26 Fab-dsFv and the second with an irrelevant control Fab-Fv to determine non-specific binding. The number of moles of bound antibody were calculated by using interpolated values from a standard curve generated by use of beads comprised of differing but known amounts of fluorescent dye. Geometric mean fluorescence values were determined in the flow cytometric analyses of cells and beads. Non-specific binding was subtracted from the A26 Fab-dsFv values and the specific binding curve thus generated analysed by non-linear regression using a one-site binding equation (Graphpad Prism®) to determine the K.sub.D. To determine the affinity of A26 Fab-dsFv for cell surface expressed antigen, saturation binding experiments were performed using activated CD4.sup.+OX40.sup.+ T cells, and Alexa Fluor 488-labelled A26 Fab-dsFv. Specific binding of antibody to receptor at equilibrium across a range of antibody concentrations was used to determine K.sub.D, assuming that only a very small fraction of antibody was bound to receptor at any point on the binding curve.

(29) Equilibrium binding is described using the following equation:

(30) ##STR00001##

(31) The rate of association of antibody with receptor=k.sub.on×[Receptor.sub.free]×[Antibody free]

(32) The rate of dissociation of receptor-antibody complex=k.sub.off×[Receptor−Antibody]

(33) At equilibrium, the association and dissociation rates are equal and an equation can be derived which describes the binding isotherm; on a semi-log plot the binding is sigmoidal. The K.sub.D is defined by k.sub.off/k.sub.on and can be calculated from the binding curve as the concentration at which half-maximal binding occurs.

(34) Binding of AlexaFluor488-labelled A26 Fab-Fv to activated human CD4.sup.+OX40.sup.+ T cells was measured by flow cytometry across a 5-log concentration range.

(35) A representative binding curve for A26 Fab-Fv is shown in FIG. 4.

(36) The mean K.sub.D value obtained on activated cells from 5 different donors is 145 pM.

EXAMPLE 3

Expression of 645gL4gH5 as a scFv

(37) Plasmid Construction

(38) The scFv were expressed from one of two closely related UCB modified mammalian expression plasmids; pVKΔPvuII was used for cloning and expression of scFv in the HL orientation, whilst pKHΔEcoRV was used for cloning and expression of scFv in the LH orientation. All scFv were designed to contain a 20 amino acid linker peptide, (GGGGS).sub.4 (SEQ ID NO:17) and a C-terminal 10×His tag. The scFv acceptor plasmids 362HL and 240LH encode unique restriction sites at the FW1-FW4 borders of vH (PvuII and XhoI) and vL (EcoRV and BsiWI) enabling the restriction cloning of subsequent scFv variable regions in a two step ligation. Genes encoding 645gH5 vH and 645gL4 vL were synthesised by DNA2.0, with cysteine wobbles at Kabat positions vH44 and vL100 for generation of disulphide-stabilised (ds) scFv. These V-region genes were cloned into acceptor scFv plasmids using PvuII and XhoI (vH) or EcoRV and BsiWI (vL) and successful ligation was verified by DNA sequencing.

(39) Expression and Purification

(40) HEK293F cells (50 ml cultures at 10.sup.6 cells/ml) were transfected with 50 μg plasmid DNA and cultured at 37° C. in FreeStyle™ media. Supernatants were harvested 6 days post-transfection and scFv were purified by batch Ni.sup.2+-NTA purification. Purified protein was concentrated and buffer exchanged into PBS for subsequent biophysical characterisation.

(41) Thermostability Assay

(42) Thermofluor assay was performed to assess the thermal stabilities of purified molecules. Purified proteins (0.1 mg/ml) were mixed with SYPRO® Orange dye (Invitrogen), and the mixture dispensed in quadruplicate into a 384 PCR optical well plate. Samples were analysed on a 7900HT Fast Real-Time PCR System (Agilent Technologies) over a temperature range from 20° C. to 99° C., with a ramp rate of 1.1° C./min. Fluorescence intensity changes per well were plotted against temperature and the inflection points of the resulting slopes were used to generate the T.sub.m.

(43) Size Exclusion HPLC

(44) Purified proteins (10 μg and 50 μg) were analysed by size exclusion HPLC on a Superdex 200 10/300 GL Tricorn Column (GE Healthcare). An isocratic gradient of PBS pH7.4 was used at a flow rate of 1 ml/min, with UV detection at 214 nm and 280 nm.

(45) Results Summary

(46) 645gH5gL4 HLds gave 97% monomer and a Tm in ° C. of 75.6.

(47) 645gH5gL4 HL gave 86% monomer and a Tm in ° C. of 75.6.

EXAMPLE 4

Construction of FabA-dsscFv Fusions

(48) Plasmids for Expression in Mammalian Cells.

(49) A single chain Fv (scFv) was constructed by linking the light and heavy chain variable region domains of a human serum albumin binding antibody (SEQ ID: 1 and 3 or 2 and 4) via a flexible linker (SEQ ID: 17) in the HL orientation. Point mutations were introduced into the DNA sequences at selected residues in the framework region of both the heavy chain and the light chain of the Fv. The mutations were introduced to create an interchain disulphide bond between the heavy and light chains of the Fv were heavy chain G44C and light chain G100C to form a disulphide linked-scFv (dsscFv). FabA-dsscFv fusion proteins were constructed by fusing a dsscFv to the C-terminus of the constant region of either the light region (with the Km3 allotype of the kappa constant region), or heavy chain of FabA (human gamma-1 CH1 constant region, γ1 isotype). A flexible (SEQ ID NO: 18 and 5) linker was used to link the scFv to the cKappa region (SEQ ID NO: 19) or CHI region (SEQ ID NO: 20), respectively. The FabA-dsscFv (CL-dsscFv), FabA-dsscFv (CH1-dsscFv), FabA light chain and FabA heavy chain were manufactured chemically and then cloned into mammalian expression vectors under the control of the HCMV-MIE promoter and SV40E polyA sequence.

(50) Various data for these constructs is shown in FIGS. 7 to 13. Thermo stability data for the constructs gave a Tm for each of around 82° C.

(51) Comprising in the context of the present specification is intended to meaning including.

(52) Where technically appropriate embodiments of the invention may be combined. Embodiments are described herein as comprising certain features/elements. The disclosure also extends to separate embodiments consisting or consisting essentially of said features/elements.

(53) It will of course be understood that the present invention has been described by way of example only, is in no way meant to be limiting, and that modifications of detail can be made within the scope of the claims hereinafter. Preferred features of each embodiment of the invention are as for each of the other embodiments mutatis mutandis. All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.