Binding domain or antibody specific to a human serum albumin (HSA)

Abstract

The present disclosure relates to a method of modulating the half-life of a binding domain specific to a serum carrier protein by mutating the sequence and a modulated binding domain specific to a serum carrier protein.

Claims

1. A binding domain comprising a variable heavy (VH) domain and variable light (VL) domain specific to a serum carrier protein, wherein the VH and VL sequences are selected from combinations of SEQ ID NOs: 3 and 9, SEQ ID NOs: 4 and 8, SEQ ID NOs: 4 and 9, and SEQ ID NOs: 5 and 9.

2. The binding domain according to claim 1, wherein the serum carrier protein is human serum albumin.

3. The binding domain according to claim 2, wherein the binding domain binds to domain II of human serum albumin.

4. The binding domain according to claim 1, wherein the VL and VH sequences are SEQ ID NO: 9 and SEQ ID NO: 3, respectively.

5. The binding domain according to claim 1, wherein the VL and VH sequences are SEQ ID NO: 8 and SEQ ID NO: 4 respectively.

6. The binding domain according to claim 1, wherein the VL and VH sequences are SEQ ID NO: 9 and SEQ ID NO: 5 respectively.

7. The binding domain according to claim 1, wherein the VL and VH sequences are SEQ ID NO: 9 and SEQ ID NO: 4 respectively.

8. The binding domain according to claim 1, wherein the binding domain is humanized.

9. The binding domain according to claim 8, wherein the humanized binding domain comprises a human IgG framework in the VH and/or VL.

10. A pharmaceutical composition comprising the binding domain according to claim 1.

11. An antibody molecule comprising the binding domain according to claim 1.

12. A pharmaceutical composition comprising the antibody molecule according to claim 11.

Description

EXAMPLES

(1) FIG. 1. Humanization and affinity reduction of antibody CA645. The heavy and light chain sequences of antibody CA645 (SEQ ID NO:1 and SEQ ID NO:6) are aligned with human germline acceptor framework sequences VH3 1-3 3-23/JH4 and Vκ1 2-1-(1) L5/Jκ4. Rabbit residues are in red, human residues are in black and CDRs are in blue (J-region CDR residues are shown but acceptor V-region CDRs are not). The grafted VH (gH) fSEQ ID NO:2 and SEQ ID NO:3) and VL (gL) (SEQ ID NO:7 and SEQ ID NO:8) sequences are shown below their corresponding human acceptor germline frameworks. Framework sequence differences between the rabbit and human framework sequences are shown with asterisks. Rabbit framework residues retained in the humanized grafts are highlighted in bold.

(2) FIG. 2. Binding of FcRn to HSA and MSA in the presence or absence of CA645 gL4gH5 Fab. Binding to HSA in absence of Fab (red circle), binding to HSA in presence of Fab (red triangle), binding to MSA in absence of Fab (blue square), binding to MSA in presence of Fab (blue triangle).

(3) FIG. 3. (A) Crystal structure of CA645 gL4gH5 Fab in complex with HSA (B) Superimposition of CA645 Fab-HSA with the crystal structure of FcRn-HSA complex, PDB code 4N0F. FcRn is composed of heavy chain, shown in green, and common β2-microglobulin (β2M), shown in orange (C) Superimposition of CA645 Fab-HSA with the crystal structures of HSA in complex with myristic acid, PDB code 1BJ5, shown in red, ibuprofen, PDB code 2BXG, shown in blue, and warfarin, PDB code 2BXD, shown in magenta. The seven fatty acid (FA) binding sites in albumin are also labelled.

(4) FIG. 4. Superimposition of CA645-HSA with RbSA. Close up views of regions around albumin residues at positions (A) 364, (B) 320 and (C) 358. CA645 heavy chain shown in blue; CA645 light chain shown in silver; HSA shown in wheat; RbSA shown in pink. Clashes are defined as two heavy atoms from different residues being within 2 Å of each other and are denoted by a black circle.

(5) FIG. 5. Pharmacokinetics. CA645 Fab grafts were intravenously injected into mice at 10 mg/kg and serum concentrations of the Fabs were determined at various time points by ELISA. Data were normalized considering maximal concentration at the first time point.

(6) FIG. 6. Percentage of free CA645 Fab in blood versus affinity for MSA. % free Fab calculated for K.sub.D range of 1-10.sup.6 nM (blue diamonds) using solution of mass action quadratic equation..sup.45% free Fab for grafts gL4gH5, gL5gH5, gL4gH37 and gL5gH47 with affinities for MSA of 2.2, 316, 1146 and 62400 nM, respectively, are shown as red squares.

(7) FIG. 7. Sequences of the disclosure

(8) Table 1. Activity profiles of anti-human serum albumin (HSA) antibodies. Fluorescent microvolume assay technology (FMAT) screening of secreted anti-HSA antibodies in B cell supernatants for binding to 100 ng/ml HSA in the presence or absence of 25 μM albumin binding compounds (warfarin, ibuprofen, myristic acid, and copper chloride) and for binding to 100 ng/ml rat serum albumin (RSA). FL=fluorescence intensity. Equilibrium binding constants (K.sub.D) of anti-HSA rabbit Fab fragments for human and mouse serum albumin (MSA), and of equivalent humanized IgG antibodies for HSA, MSA and RSA determined by surface plasmon resonance (SPR).

(9) Table 2. Affinity of CA645 gL4gH5 Fab for serum albumin from different species. Association (k.sub.a) and dissociation (k.sub.d) rate constants and equilibrium binding constants (K.sub.D) determined by SPR.

(10) Table 3. X-ray data collection and refinement statistics. Values in parentheses are for highest-resolution shell.

(11) Table 4. Binding kinetics and pharmacokinetics of CA645 Fab grafts. Association (k.sub.a) and dissociation (k.sub.d) rate constants and equilibrium binding constants (K.sub.D) determined by SPR. 3 mice (M1-3)/group were dosed intravenously at 10 mg/kg with each CA645 graft. Mean and standard deviation (SD) of each group is shown. *measured by steady state.

(12) Table 5 Shows affinity for various grafts.

(13) Table 6. Affinity of CA645 gL4gH5 Fab for HSA over pH range 5.0-7.0.

(14) Table 7. (A) (B) (C) Binding kinetics of CA645 Fab grafts. Association (k.sub.a) and dissociation (k.sub.d) rate constants and equilibrium binding constants (K.sub.D) determined by SPR.

(15) Antibody Discovery

(16) Two Half Lop rabbits were immunised subcutaneously with 200 μg HSA (Jackson ImmunoResearch). Complete Freund's adjuvant (Sigma Aldrich) was co-administered with the first dose and subsequent doses included incomplete Freund's adjuvant. B cells were harvested from the rabbit sera and cultured for 7 days to induce clonal expansion and antibody secretion. Fluorescence microvolume assay technology (FMAT) was used to screen the supernatants for binding to HSA..sup.20-22 The supernatants were mixed with streptavidin beads (Bangs Laboratories, Inc) coated with biotinylated goat anti-rabbit Fc and Alexa Fluor 647 Chrompure Human Albumin (Jackson ImmunoResearch). Plates were read on an Applied Biosystems 8200 Cellular Detection System. The 48 wells with the highest fluorescence intensity (FL) signal were transferred to a single master plate and the screening repeated as before but with two additional screens. In one screen, Alexa Fluor 647 Chrompure Human Albumin was pre-incubated for 1 hour with a 25 μM solution of albumin binders; warfarin, ibuprofen, myristic acid, and copper chloride (all individually sourced from Sigma Aldrich). In the second screen, HSA was replaced with rat serum albumin (Sigma Aldrich) that had been labelled using Alexa Fluor 647® monoclonal antibody labelling kit (Molecular Probes).

(17) Individual HSA specific B-cells were isolated by fluorescent foci method..sup.20-22 B cells from positive wells were mixed with streptavidin beads (Bangs Laboratories, Inc) coated with biotinylated-HSA (Jackson ImmunoResearch) and goat anti-rabbit Fc fragment fluorescein isothiocynate conjugate (Chemicon). Following 1 hour incubation at 37° C., antigen-specific B cells could be identified due to the presence of a fluorescent halo surrounding that B cell. An Olympus IX70 microscope and an Eppendorf micromanipulator were used to identify and transfer the individual B cells to PCR tubes. The heavy and light chain immunoglobulin variable (V) region genes of single cells were amplified by RT-PCR and cloned into UCB mammalian expression vectors containing rabbit heavy C.sub.H1 and rabbit light Cκ regions, respectively. Following transient expression in HEK293 cells, anti-HSA recombinant Fabs were further screened in SPR binding assays against HSA and MSA.

(18) Humanization

(19) Albumin specific antibodies were humanized in silico by grafting the CDRs from antibody V-regions onto the Vκ1 and V.sub.H3 human germline antibody V-region frameworks. The CDR's grafted from the donor to the acceptor sequence were as defined by Kabat et al.,.sup.32 with the exception of CDR-H1 (residues 26-35) where the combined definitions of Kabat et al., and loop structure was used..sup.23 Where a framework residue differed between the donor rabbit sequence and the acceptor human sequence in a position that was considered to be important for retention of antigen binding, then the donor residue was included in the initial conservative graft..sup.21 The conservative graft genes were chemically synthesized by Entelechon, GmbH. Heavy chain graft genes (gH1) were cloned into two UCB expression vectors, one containing human γ1C.sub.H1 domain and another containing the full human γ1 constant region. Light chain graft genes (gL1) were cloned into a UCB expression containing human kappa constant region (Km3 allotype). These constructs were subsequently modified by oligonucleotide-directed mutagenesis to create a number of different variants of both the heavy and light chain grafts. Heavy and light chain vectors were co-transfected into HEK293 cells and the recombinant Fab or IgG molecules screened using a SPR binding assay to measure affinity for HSA, MSA, RSA, CSA, RbSA and BSA.

(20) Antibody Expression

(21) Antibodies were transiently expressed in either HEK-293 cells using 293Fectin lipid transfection (Life Technologies, catalog #12347-019, according to the manufacturer's instructions) or CHO-S XE cells, a CHO-K1 derived cell line,.sup.33 using electroporation. HEK-293 cells were used for small scale expression (<100 ml) to prepare antibodies for SPR analysis. CHO-S XE cells were used for large scale expression (1 litre) to prepare antibodies for crystallography and in vivo pharmacokinetic studies.

(22) Protein Purification

(23) Affinity chromatography was used to purify Fab protein from culture supernatants. Supernatants were passed over a HiTrap Protein G column (GE Healthcare) at a flow rate that gave a column contact time of 25 min. Following a washing step with PBS pH 7.4, the bound material was eluted with 0.1 M glycine pH 2.7 and neutralized with 2 m Tris-HCl (pH 8.5). Fractions containing Fab were pooled, quantified by absorbance at 280 nm, and concentrated using Amicon Ultra centrifugal filters (Merck Millipore). To isolate the monomeric fraction, size exclusion chromatography over a HiLoad 16/60, Superdex 200 column (GE Healthcare) equilibrated with PBS, pH 7.4, was used. Fractions containing monomeric Fab were pooled, quantified, concentrated and stored at 4° C.

(24) Surface Plasmon Resonance

(25) The binding affinities and kinetic parameters for the interactions of antibodies were determined by surface plasmon resonance (SPR) conducted on either a Biacore T200 or Biacore 3000 using CMS sensor chips (GE Healthcare Bio-Sciences AB) and HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% v/v P20, pH7.4) running buffer. For analysis at pH 7.0, 6.0, 5.5 and 5.0, a running buffer of 40 mM citric acid, 80 mM sodium phosphate 50 mM NaCl, 3 mM EDTA, 0.05% v/v P20 was used. The required pH was achieved by altering the ratio of citric acid to sodium phosphate. All experiments were performed at 25° C. The antibody samples were captured to the sensor chip surface using either a human F(ab′).sub.2-specific or human Fc-specific goat Fab (Jackson ImmunoResearch). Covalent immobilisation of the capture antibody was achieved by standard amine coupling chemistry to a level of 6000-7000 response units (RU).

(26) Human (Jackson ImmunoResearch, catalog #009-000-051), mouse (Sigma Aldrich, catalog #A3559), rat (Sigma Aldrich, catalog #A6414), rabbit (Sigma Aldrich, catalog #A0764), bovine (Sigma Aldrich, catalog #05470) and cynomolgus (Equitech-Bio, #CMSA-0050) albumin were titrated over the captured antibody at various concentrations from 50 nM to 500 μM. Each assay cycle consisted of firstly capturing the antibody sample using a 1 min injection, before an association phase consisting of a 3 min injection of albumin, after which dissociation was monitored. After each cycle, the capture surface was regenerated with two 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 both the association and dissociation phases, and 10 μl/min for regeneration. A blank flow-cell was used for reference subtraction and buffer-blank injections were included to subtract instrument noise and drift. Kinetic parameters were determined by simultaneous global-fitting of the resulting sensorgrams to a standard 1:1 binding model using Biacore T200 Evaluation software v2.0.1 and BIAEvaluation software v4.1.1, with the exception of CA645 gL5gH47 which was fitted in prism using steady state affinity model.

(27) To measure the effect of CA645 Fab on the binding potency of FcRn to albumin by SPR, a Biacore3000 instrument was used with a CMS chip prepared by immobilisation of HSA and MSA on separate flow cells to levels of 270 RU and 247 RU respectively. FcRn samples were prepared over the range 50 nM to 50 μM in running buffer, (100 mM MES, 150 mM NaCl, 0.05% v/v P20, pH 5.5) and they also contained either zero or 100 nM CA645 Fab. Each assay cycle was run at a flow rate of 10 μl/min and consisted of either a 5 min injection of 100 nM CA645 Fab to pre-saturate immobilised albumin, followed by a 5 min injection of one of the above FcRn solutions prepared in the presence of CA645 Fab, or a 5 min injection of running buffer followed by a 5 min injection of one of the above FcRn solutions in the absence of CA645 Fab. In either case a third 5 min injection followed immediately at the end of the second injection, using the ‘coinject’ mode, comprising respectively, buffer or 100 nM CA645 Fab. A blank flow-cell was used for reference subtraction and blank cycles, where FcRn was replaced with buffer, were included to subtract drift and noise. Cycle regeneration was as above. Blank corrected plateau binding levels of FcRn were plotted in Prism and fitted to a steady state model.

(28) Binding kinetics of wild type and mutant CA645 Fabs at pH 5.5 were also investigated in reverse format on the Biacore3000 using the immobilised albumin chip. In this case cycles were run where Fab solutions over the range 5 to 5000 nM were injected with 5 min association and dissociation phases. Buffer blank cycles were also included to correct for drift.

(29) Crystallography

(30) To prepare the complex, purified CA645 Fab and fatty acid-free HSA (Sigma Aldrich, catalog #A3782) were mixed in a molar ratio of 1:1 and incubated overnight at 4° C. Both CA645 Fab and the complex were purified by size exclusion chromatography over a HiLoad 16/60, Superdex 200 column (GE Healthcare) equilibrated with 50 mM NaCl, 25 mM Tris, 5% (v/v) glycerol. Fractions containing either CA645 Fab or the complex were pooled and concentrated to 10 mg/ml and 70 mg/ml, respectively. Conditions suitable for crystal growth were identified by the sitting drop vapour diffusion method using commercially available crystallization screens (Qiagen).

(31) To generate diffraction quality crystals, hanging drop vapour diffusion method was used where 1 μl of protein solution was mixed with 1 μl of reservoir solution. For CA645 Fab, the reservoir contained 500 μl 2 M DL-Malic acid. Crystals were harvested and flash frozen in liquid nitrogen without additional cryoprotectant. Diffraction data to 2.68 Å was collected from a single crystal on the 104 beamline at Diamond Light Source, Oxford, UK and processed using MOSFLM and SCALA..sup.34-36 The structure of CA645 Fab was solved by molecular replacement with Phaser,.sup.37 using coordinates of an in-house Fab structure as a search model. For the complex, the reservoir contained 500 μl 0.1 M Citric acid pH 4.4, 0.1 M di-Sodium hydrogen phosphate, 38% v/v Ethanol and 5% v/v Polyethylene glycol 1000 (PEG1000). The crystals were cryoprotected by multiple additions to the drop of 1 μl reservoir buffer containing 25% (v/v) PEG1000, until the concentration of PEG1000 in the drop reached 20%. To minimise crystal stress, each addition was spaced at least 1 hour apart. Crystals were harvested and flash frozen in liquid nitrogen. Diffraction data to 3.58 Å was collected from a single crystal on the 102 beamline at Diamond Light Source, Oxford, UK and processed using XDS..sup.38 The structure of the complex was solved by molecular replacement with Phaser using coordinates of CA645 Fab structure and HSA (PDB code 4G03).sup.39 as search models.

(32) Both initial structures were refined with iterative cycles of simulated annealing, energy minimisation and manual rebuilding using CNS.sup.40,41 and COOT..sup.42 Due to the rather low resolution of the complex, the model was constrained during refinement by using the DEN function of CNS. Model geometry was validated using Molprobity..sup.43 Molecular visualisations were generated with Pymol..sup.44 Data collection and refinement statistics are summarised in Table 3.

(33) Accession Codes

(34) Coordinates and structure factors of CA645 Fab and the CA645 Fab-HSA complex have been deposited in the Protein Data Bank (PDB) with accession codes, X and Y, respectively.

(35) Mouse Pharmacokinetics

(36) Three male BALB/c mice were dosed intravenously at 10 mg/kg with the antibody. Serial blood samples were collected from the tail venipuncture at several time points up to 100 hr post dose. To obtain sera, blood samples were centrifuged for 5 min at 10,000 rpm at room temperature and analysed for the antibody concentration by ELISA. An antibody against the Fab antigen and an anti-human kappa chain-Horseradish peroxidase conjugate (Stratech) was used as the capture and secondary antibody, respectively. A purified sample of the Fab antigen was used as the standard. Plates were developed using TMB peroxidase solution (Sigma-Aldrich) and read at 450 nm (reference at 630 nm). Pharmacokinetic parameters were calculated from the final dataset using Phoenix WinNonlin 6.2 software.

(37) Calculation of Free CA645 Fab

(38) To determine the concentration of unbound Fab (molecular weight=47907 Da), in 2 ml of blood, of a 20 g mouse, following a dose at 10 mg/kg the following equation was used:.sup.31
Free Fab concentration x=(−b+SQRT(b.sup.2−4ac))/2a
Where: b=(−(K.sub.A*[Tab])+1+(K.sub.A*[Tag])) a=K.sub.A=1/K.sub.D c=(−[Tab]) K.sub.D=affinity of Fab K.sub.A=equilibrium constant of association [Tab]=concentration of Fab (2087 nM) [Tag]=concentration of albumin (600 μM)
To calculate percentage of free Fab % Free Fab=([free Fab]/[Tab])*100
Results
Generation and Characterization of a mAb to Serum Albumin Across Species

(39) To generate a panel of anti-HSA antibodies with cross species reactivity, two rabbits were immunized with HSA. B cells were harvested from the sera, cultured, stimulated to secrete IgG and screened using fluorescent microvolume assay technology (FMAT) to identify antigen-specific wells..sup.20-22 Further FMAT screens assessed binding to RSA and binding to HSA in the presence or absence of known albumin binding compounds; warfarin, ibuprofen, myristic acid, and copper chloride. Data for the five top-ranked antibodies is shown in Table 1. The fluorescence intensity signal for binding to HSA was lowest for CA645. However, CA645 did retain 80% of binding activity in the presence of the compounds whereas CA646, CA647, CA648 and CA649 retained only 40%. The levels of binding to HSA and RSA were most closely matched for CA645 and CA646, being within 5-fold. In contrast, the levels of binding to RSA by CA647, CA648 and CA649 were 9 to 18-fold lower than for HSA.

(40) To recover the heavy and light chain variable regions of the five antibodies, fluorescent foci method was used to isolate single B cells and then RT-PCR was performed. The variable regions were cloned into expression vectors containing rabbit heavy C.sub.H1 and light chain constant regions and then the DNA sequenced. This revealed that the antibody sequences were unique with the exception of CA645 and CA646 which had identical heavy chain sequences. Given CA645 and CA646 must bind to the same epitope through the heavy chain it is unclear why CA646 binding was more affected by the presence of ligands. Also determined from the sequencing was that the complementarity determining regions (CDRs) of all of the antibodies lacked histidine residues. This was important for further progression of these antibodies since histidine residues protonate at acidic pH and this can potentially disrupt antigen binding.

(41) Following transfection of HEK293 cells, the recombinant Fab molecules were analysed by surface plasmon resonance (SPR) for affinity for HSA and mouse serum albumin (MSA). CA645 and CA646 both exhibited the strongest affinities for HSA, at 0.31 nM and 0.14 nM, respectively, and for MSA at 2.6 nM and 1.6 nM, respectively (Table 1). Furthermore, their affinities for HSA and MSA were the most closely matched of all of the antibodies. This was in line with the B cell supernatant screening data against HSA and RSA.

(42) Humanization and Selection of Lead Candidate

(43) All five antibodies were humanized by grafting the CDRs onto human Vκ1 and VH3 frameworks and back-mutating framework residues in positions considered important for retention of binding activity..sup.23 The humanization scheme for CA645 is shown in FIG. 1. Of note is that the framework three regions (residues 66-94) of the rabbit donor heavy chains were shorter than that of the human acceptor framework sequences. CA647 and CA649 were shorter by one residue whereas CA645 and CA646 (identical sequences) and CA648 were shorter by two residues. In all cases, the gap was retained in the initial conservative gL1gH1 graft.

(44) The conservative grafts were expressed as human IgG1 antibodies and analysed by SPR for binding to HSA, MSA and RSA. The humanized IgGs displayed the same trend in binding to HSA and MSA as observed with the recombinant parental rabbit Fabs (Table 1). The affinities for HSA of CA647, CA648 and CA649 were similar to those of CA645 and CA646 but they showed a 6- to 10-fold reduction in affinity for MSA by comparison. The affinities for RSA of CA648 and CA649 also showed a 5- to 10-fold reduction in comparison with CA645 and CA646. CA646 exhibited marginally stronger affinities for HSA, MSA and RSA than CA645 but the transient expression yields were 4-fold lower at 35 mg/ml compared with 161 mg/ml (Table 1). Based on the near maximal retention of binding to HSA in the presence of known albumin binders, the consistent binding activity for albumin across multiple species and good yields in transient expression, CA645 was selected as our lead candidate for further progression. Further graft variants of CA645 gL IgH1 were generated by replacing rabbit donor residues with human acceptor residues and filling the gap in framework three of the heavy chain with the equivalent human residues. The graft variants were assessed on affinity for HSA and transient yield expression (data not shown). The final graft pairing selected was gL4 and gH5 (FIG. 1).

(45) The affinities of CA645 gL4gH5 Fab for HSA, MSA, RSA and rabbit serum albumin (RbSA) were shown to be 4.6, 7.1, 54 and 162 nM, respectively (Table 2). Significantly for utility of CA645 gL4gH5 Fab in cynomolgus monkey toxicology studies and disease models, the affinity for cynomolgus serum albumin (CSA) was very similar to that of HSA at 3.3 nM. In addition, CA645 Fab failed to bind to bovine serum albumin.

(46) To determine whether CA645 gL4gH5 Fab is likely to remain bound to albumin in the acidic environment of the early endosome and be recycled to the cell surface, the affinity was measured at pH 5.0-7.0 (Table S1). The affinities of CA645 gL4gH5 Fab for HSA at pH 5.0, pH 5.5, pH 6.0, and pH 7.0 were 7.1, 10.7, 12.5 and 13.3 nM indicating that binding is largely unaffected within this physiologically relevant pH range.

(47) To determine whether HSA can bind to FcRn in the presence of CA645 gL4gH5 Fab, SPR was used. The kinetic assays were conducted at pH5.5 to ensure optimal binding by FcRn to albumin. HSA or MSA was bound directly onto the sensor chip, then either CA645 gL4gH5 Fab, to saturate CA645 albumin binding sites, or running buffer was injected into the flow cell. This was followed by an injection of FcRn plus CA645 Fab, or FcRn alone. CA645 Fab was included in the co-injection with FcRn to maintain CA645 binding site saturation. FIG. 2 shows the levels of FcRn binding to both HSA and MSA following subtraction of the signals for CA645. Binding by FcRn to both albumins was unaffected by the presence of CA645 gL4gH5 Fab.

(48) Crystallography

(49) To identify where CA645 binds to HSA, the crystal structure of CA645 Fab-HSA complex was determined. The CA645 Fab-HSA complex protein preparation was concentrated to 70 mg/ml,.sup.24 and crystallized using ethanol and PEG1000 as precipitants. To aid solving the structure of the complex with molecular replacement, we also determined the structure of unbound CA645 Fab. We observed single copies of both the Fab and Fab-HSA complex in the asymmetric units of their respective crystals (Table 3). The structure of free Fab was refined to 2.68 Å with a final R.sub.work value of 21.14% and R.sub.free value of 25.13%. The structure of the complex was refined to 3.6 Å with a final R.sub.work value of 21.38% and R.sub.free value of 25.23%.

(50) The crystal structure of the CA645 Fab-HSA complex showed that CA645 binds to domain II of HSA (FIG. 3A). Superimposition of the crystal structure of FcRn in complex with HSA (PDB code 4N0F),.sup.25 showed that CA645 does not block binding of HSA to FcRn (FIG. 3B). HSA contains seven fatty acid (FA) binding sites. Sites FA7 and FA3/FA4 are the two main drug binding sites..sup.26 Drugs also bind at sites FA1, FA5 and FA6 but with weaker affinity. Metal ion binding sites are located between domains I and II and at a site at the N-terminus..sup.27 Superimposition of the complex with the crystal structures of HSA in complex with warfarin (PDB code 2BXD),.sup.28 ibuprofen (PDB code 2BXG).sup.28 and myristic acid (PDB code 1BJ5).sup.29 showed that CA645 binds close to site FA6 and does not occlude the main drug (FA7 and FA3/FA4), fatty acid or metal ion binding sites (FIG. 3C).

(51) The binding kinetics of CA645 gL4gH5 Fab to HSA in comparison with those for MSA, CSA, RSA and RbSA (Table.2) may be explained by close visual inspection of the crystal structure. The epitope on HSA is formed by residues F206, G207, R209, C316, K317, AEAKD 320-324, K351, E354, E358, K359, C361, A362 and A364. The affinities of CA645 for CSA (3.3 nM) and MSA (7.1 nM) are very similar to the affinity for HSA (4.6 nM). This is likely due to the presence in CSA and MSA of the same residues that form the epitope in HSA. RSA shares all of these residues except for position 364 which is glycine. Position 364 is located at the tip of a short loop (positions 362-365) that links two α-helices (positions 366-398 and 342-361) together (FIG. 4A). This short loop is bound by CDR's 1 and 2 of the CA645 heavy chain. The affinity of CA645 for RSA is approximately 10-fold lower than for HSA. It is possible that the absence of the alanine side chain increases the flexibility of the loop, compared with that of HSA, and alters the binding kinetics.

(52) RbSA shares all of the HSA epitope residues except positions 320, 358 and 364. Superimposition of the crystal structure of RbSA (PDB code 3V09).sup.30 showed clear clashes with CA645 Fab at positions 320 and 358, and a potential clash at position 364. In RbSA, position 364 is aspartic acid and whilst there was no clear clash, this position is a contact residue and therefore likely to influence binding by CA645. In HSA, position 320 is alanine and it forms a hydrophobic interaction with F58 of CDRH2 (FIG. 4B). In RbSA, position 320 is glutamic acid and it clashes with CDRH2 residues W52 and F58. Residue E358 in HSA forms a hydrogen bond network with S100 and T100a of CDRH3 (FIG. 4C). Position 358 in RbSA is lysine and it clashes with Y99 of CDRH3. The weaker affinity of CA645 for RbSA compared with HSA is entirely due to an 18-fold reduction in the association rate (Table.2). This is likely to be caused by the presence in RbSA of the larger side chains at positions 320 and 358, and possibly 364.

(53) Pharmacokinetics of Reduced Affinity Variants

(54) To investigate the correlation between the half-life of CA645 and its affinity for albumin, we generated a panel of mutants of CA645 gL4gH5 Fab with a broad range of reduced affinities and then analysed their pharmacokinetic properties in mice. The mutations were designed using the crystal structure of the CA645 gL4gH5 Fab-HSA complex as a guide. Oligonucleotide-directed mutagenesis was used to generate twenty variants across six residue positions of the heavy chain and twenty seven variants across six residue positions of the light chain (Tables 4, S2A, S2B and S2C). BALB/c mice were dosed by a single intravenous injection at 10 mg/kg with CA645 gL4gH5 Fab and a subset of four of the Fab variants, gL5gH5, gL4gH37, gL5gH37 and gL5gH47. Blood sera were sampled over 103 hours and the level of Fab quantified by ELISA.

(55) CA645 gL5gH37 showed no detectable binding to HSA by SPR and was cleared rapidly with a serum half-life of only 0.48±0.06 h (Table 4). This is in line with the short half-life (0.7 h) of an anti-TNF Fab observed in rats..sup.19 In contrast gL4gH5 exhibited a significantly extended half-life of 84±4.6 h. The variant with the weakest affinity for which there was no difference in pharmacokinetic profile from gL4gH5 was gL5gH5 (FIG. 5). gL5gH5 contained a single mutation in the light chain, W30A, and its affinity was 453 nM. This affinity was 368-fold weaker than that of gL4gH5 (1.23 nM) but its half-life (96.7±20.4 h) was equivalent to that of gL4gH5. A change in the pharmacokinetic profile was observed for gL4gH37. It has a single mutation in the heavy chain, F58E, and its affinity was 955 nM. This affinity was 776-fold lower than gL4gH5 but the half-life still extended to 61±16.8 h. gL5gH47 contained one mutation in the light chain, W30A, and one mutation in the heavy chain, T100aS, and had an affinity of 52 μM, as measured by steady state SPR. This affinity was 42,276-fold weaker than that of gL4gH5, and yet the pharmacokinetic profile did not differ dramatically from gL4gH37 and the half-life increased to 26.3±3.1 h.

(56) The mutants were designed and selected on the basis of affinity for HSA but the pharmacokinetic model was murine. Therefore, to confirm that the affinities of the mutants for HSA reflected their affinities for albumin in a mouse, SPR was repeated (Table.5). The affinities of gL4gH5 and gL5gH5 were 1.8 and 254 nM for HSA, and similarly 2.2 and 316 nM for MSA. These data were in line with the previously determined affinities of gL4gH5 and gL5gH5 for HSA of 1.23 and 453 nM, respectively.

(57) Using a solution of the mass action quadratic equation, we can estimate the percentage of free Fab in the blood for each of the variants..sup.31 If we assume the concentration of MSA (65.9 kDa) is 44 g/L and the volume of the blood of a 20 g mouse is 2 ml, then for a dose at 10 mg/kg, the concentration of CA645 Fab (47.9 kDa) will be 2087 nM. FIG. 6 shows a graph of the percentage of free Fab versus affinity for albumin in the range 1-10.sup.6 nM. With an affinity for MSA of 2.2 nM, just 0.0003% of gL4gH5 Fab is predicted to be unbound in blood. The affinity for MSA of gL5gH5 Fab is 316 nM. It has a pharmacokinetic profile and half-life that matches that of gL4gH5, and is calculated to have a similarly low level of free Fab at 0.05%. We were unable to measure the affinities of gL4gH37 and gL5gH47 Fabs for MSA. However, as the affinities of gL4gH5 and gL5gH5 were both 1.2-fold weaker for MSA than for HSA (Table 5), it is reasonable to predict that the affinities of gL4gH37 and gL5gH47 will be proportionately 1.2-fold weaker. Therefore, with predicted affinities for MSA of 1146 nM and 62.4 μM, it is calculated that 0.17% of gL4gH37 and 8.57% of gL5gH47, respectively, are potentially free in blood.

(58) TABLE-US-00001 TABLE 1 Binding of various antibodies generated FMAT SPR B cell sup Rabbit Fab Humanized IgG HSA + HSA MSA HSA MSA RSA mAb HSA compounds RSA K.sub.D ×10.sup.−9 K.sub.D ×10.sup.−9 K.sub.D ×10.sup.−9 K.sub.D ×10.sup.−9 K.sub.D ×10.sup.−9 Yield CA no. (FL) (FL) (FL) (M) (M) (M) (M) (M) (mg/ml) 645 272 220 114 0.31 2.6 0.82 2.9 7.9 161 646 2310 964 484 0.14 1.6 0.57 1.7 4.5 35 647 1213 520 69 0.60 36.0 1.30 26 10 23 648 1048 465 72 0.33 12.0 0.13 23 54 312 649 1338 534 142 0.54 13.0 0.32 17 44 188

(59) TABLE-US-00002 TABLE 2 Binding Kinetics of CA645 gL4Gh5 Fab to HAS, MSA, CSA, RSA and RbSA k.sub.a × 10.sup.4 k.sub.d × 10.sup.−4 K.sub.D × 10.sup.−9 Albumin (1/Ms) (1/s) (M) Human 9.0 4.1 4.6 Mouse 4.8 3.4 7.1 Rat 2.4 13 54 Cynomolgus 10 3.5 3.3 Rabbit 0.2 2.9 162 Bovine — — No binding

(60) TABLE-US-00003 TABLE 3 X-ray data Fab_645 Fab_645-HSA Data collection Space group P 3.sub.1 2 1 P 3.sub.1 2 1 Cell dimensions a, b, c (Å) 111.21, 111.21, 217.68, 217.68, 89.20 78.68 α, β, γ (°) 90.00, 90.00, 90.00, 90.00, 120.00  120.00   Resolution (Å) 30.0-2.68 (2.82-2.32) * 30.0-3.58 (3.79-3.58) * R.sub.merge 0.117 (0.357) 0.157 (0.612) R.sub.meas 0.120 (0.365) 0.108 (0.439) CC.sub.1/2 99.7 (98.2) 99.5 (78.5) I/σI 23.0 (10.1) 7.57 (1.64) Completeness (%) 99.5 (99.4) 93.9 (86.1) Redundancy 21.5 (22.2) 2.4 (1.9) Refinement Resolution (Å) 30.00-2.68 30.00-3.6 No. reflections 389, 935 110, 160 R.sub.work/R.sub.free 0.2114/0.2513 0.2138/0.2523 No. atoms Protein 3311 (excluding H) 7870 (ex H) Water 37   — B-factors Protein 31.55 115.14   Water 24.24 — R.m.s. deviations Bond lengths (Å)  0.007 0.010 Bond angles (°)  1.447 1.446

(61) TABLE-US-00004 TABLE 4 mutation SPR CA645 Light Heavy k.sub.a ×10.sup.5 k.sub.d ×10.sup.−4 K.sub.D ×10.sup.−9 Half-life (h) Grafts chain chain (1/Ms) (1/s) (M) M1 M2 M3 Mean SD gL4gH5 — — 1.39 1.72 1.23 85 88 79 84 4.6 gL5gH5 W30A — 1.26 571 453 120 82 88 96.7 20.4 gL4gH37 — F58E 0.61 583 955 55 48 80 61 16.8 gL5gH47 W30A T100aS — — 52 μM* 27 23 29 26.3 3.1 gL5gH37 W30A F58E — — NB 0.54 0.42 0.47 0.48 0.06

(62) TABLE-US-00005 TABLE 5 CA645 Albumin k.sub.a × 10.sup.4 k.sub.d × 10.sup.−4 K.sub.D × 10.sup.−9 Grafts species (1/Ms) (1/s) (M) gL4gH5 HSA 22 4.0 1.8 gL4gH5 MSA 31 6.8 2.2 gL5gH5 HSA 8.5 220 254 gL5gH5 MSA 1.2 38 316

(63) TABLE-US-00006 TABLE 6 KD pH (nM) 5.0 7.1 5.5 10.7 6.0 12.5 7.0 13.3

(64) TABLE-US-00007 TABLE 7A Light Heavy CA645 chain chain k.sub.a × 10.sup.4 k.sub.d × 10.sup.−4 K.sub.D × 10.sup.−9 Grafts mutation mutation (1/Ms) (1/s) (M) gL4gH5 — — 5.75 1.23 2.14 gL10gH5 S28A — 13.80 1.72 1.03 gL12gH5 S28D — 5.54 1.04 1.88 gL13gH5 S28I — 5.76 1.08 1.88 gL14gH5 S28L — 5.77 0.87 1.51 gL27gH5 F32Y — 6.21 5.86 9.44 gL34gH5 S93T — 5.58 1.90 3.41 gL35gH5 S93V — 5.21 2.90 5.57 gL4gH43 — G98E 5.29 3.91 7.39 gL4gH44 — G98L 5.35 2.26 4.22 gL4gH45 — G98V 5.38 4.44 8.25 gL4gH27 — A53G 4.83 0.84 1.73 gL4gH28 — A53V 4.85 5.15 10.62 gL4gH29 — A53S 4.77 3.80 7.97 gL4gH30 — A53T 4.38 10.2 23.17 gL4gH38 — F58Y 4.75 5.81 12.23 gL4gH39 — G98I 5.35 7.50 14.01 gL4gH40 — G98T 5.56 3.56 6.40 gL4gH41 — G98D 5.37 3.51 6.54 gL4gH42 — G98Q 5.31 2.22 4.17 gL4gH5 — — 5.60 1.40 2.51

(65) TABLE-US-00008 TABLE 7B Light Heavy CA645 chain chain k.sub.a × 10.sup.4 k.sub.d × 10.sup.−4 K.sub.D × 10.sup.−9 Grafts mutation mutation (1/Ms) (1/s) (M) gL4gH5 — — 5.91 1.61 2.72 gL15gH5 S31R — 6.01 2.77 4.62 gL16gH5 S31W — 5.53 1.69 3.06 gL17gH5 S31N — 5.88 3.23 5.50 gL18gH5 S31I — 5.32 8.23 15.47 gL19gH5 S31D — 5.20 2.60 5.00 gL20gH5 S31Q — 5.55 7.94 14.30 gL21gH5 S31E — 5.10 3.23 6.34 gL22gH5 S31H — 5.57 4.78 8.58 gL23gH5 S31L — 5.49 8.14 14.82 gL24gH5 S31V — 5.47 8.83 16.14 gL25gH5 S31F — 5.62 2.17 3.85 gL26gH5 S31Y — 6.01 2.48 4.13 gL4gH31 — S54V 4.00 1.67 4.18 gL4gH32 — S54I 3.86 1.69 4.38 gL4gH33 — S54L 3.89 2.40 6.18 gL4gH34 — S54Q 3.85 4.22 10.97 gL4gH35 — S54E 2.65 3.99 15.06 gL4gH5 — — 5.86 1.58 2.70

(66) TABLE-US-00009 TABLE 7C Light Heavy CA645 chain chain k.sub.a × 10.sup.4 k.sub.d × 10.sup.−4 K.sub.D × 10.sup.−9 Grafts mutation mutation (1/Ms) (1/s) (M) gL4gH5 — — 5.62 1.67 2.97 gL11gH5 S28N — 5.64 2.49 4.42 gL28gH5 S67L — 5.90 1.09 1.85 gL29gH5 S67V — 5.77 1.03 1.78 gL30gH5 S67I — 5.84 0.94 1.61 gL31gH5 S67T — 5.72 1.42 2.49 gL32gH5 S67Q — 5.86 1.43 2.44 gL33gH5 S67E — 5.40 1.79 3.32 gL4gH46 — V96Y 5.64 1.84 3.26 gL4gH47 — T100aS 6.16 34.10 55.39 gL4gH5 — — 5.57 2.11 3.80

REFERENCES

(67) 1. Kontermann R E. Strategies for extended serum half-life of protein therapeutics. Curr Opin Biotechnol 2011; 22: 868-876. 2. Sleep D, Cameron J, Evans L R. Albumin as a versatile platform for drug half-life extension. Biochim Biophys Acta 2013; 1830: 5526-5534. 3. Peters T Jr. All About Albumin: Biochemistry, Genetics, and Medical Applications 1996; San Diego, Calif.: Academic Press. 4. Chaudhury C, Mehnaz S, Robinson J M, Hayton W L, Pearl D K, Roopenian D C, Anderson C L. The major histocompatibility complex-related Fc receptor for IgG (FcRn) binds albumin and prolongs its lifespan. J Exp Med 2003; 197: 315-322. 5. Junghans R P, Anderson C L. The protection receptor for IgG catabolism is the beta2-microglobulin-containing neonatal intestinal receptor. Proc Natl Acad Sci USA 1996; 93: 5512-5516. 6. Müller D, Karle A, Meissburger B, Höfig I, Stork R, Kontermann R E. Improved pharmacokinetics of recombinant bispecific antibody molecules by fusion to human serum albumin. J Biol Chem 2007; 282: 12650-12660. 7. Elsadek B, Kratz F. Impact of albumin on drug delivery—New applications on the horizon. J Control Release 2012; 157: 4-28. 8. Sorensen A R, Stidsen C E, Ribel U, Nishimura E, Stuns J, Jonassen I, Bouman S D, Kurtzhals P, Brand C L. Insulin detemir is a fully efficacious, low affinity agonist at the insulin receptor. Diabetes Obes Metab 2010; 12: 655-673. 9. Trüssel S, Dumelin C, Frey K, Villa A, Buller F, Neri D. New strategy for the extension of the serum half-life of antibody fragments. Bioconjug Chem 2009; 20: 2286-2292. 10. Dennis M S, Zhang M, Meng Y G, Kadkhodayan M, Kirchhofer D, Combs D, Damico L A. Albumin binding as a general strategy for improving the pharmacokinetics of proteins. J Biol Chem 2002; 277: 35035-35043. 11. Nguyen A, Reyes A E 2nd, Zhang M, McDonald P, Wong W L, Damico L A, Dennis M S. The pharmacokinetics of an albumin-binding Fab (AB.Fab) can be modulated as a function of affinity for albumin. Protein Eng Des Sel 2006; 9: 291-297. 12. Hopp J, Hornig N, Zettlitz K A, Schwarz A, Fuss N, Müller D, Kontermann R E. The effects of affinity and valency of an albumin-binding domain (ABD) on the half-life of a single-chain diabody-ABD fusion protein. Protein Eng Des Sel 2010; 23: 827-834. 13. Andersen J T, Pehrson, R, Tolmachev V, Daba, M B, Abrahmsén L, Ekblad C. Extending half-life by indirect targeting of the neonatal Fc receptor (FcRn) using a minimal albumin binding domain. J Biol Chem 2011; 286: 5234-5241. 14. Tijink B M, Laeremans T, Budde M, Stigter-van Walsum M, Dreier T, de Haard H J, Leemans C R, van Dongen GAImproved tumor targeting of anti-epidermal growth factor receptor Nanobodies through albumin binding: taking advantage of modular Nanobody technology. Mol Cancer Ther 2008; 8: 2288-2297. 15. Van Roy M, Ververken C, Beirnaert E, Hoefman S, Kolkman J, Vierboom M, Breedveld E, Hart B, Poelmans S, Bontinck L, Hemeryck A, Jacobs S, Baumeister J, Ulrichts H. The preclinical pharmacology of the high affinity anti-IL-6R Nanobody® ALX-0061 supports its clinical development in rheumatoid arthritis. Arthritis Research & Therapy 2015. 16. Holt L J, Basran A, Jones K, Chorlton J, Jespers L S, Brewis N D, Tomlinson I M. Anti-serum albumin domain antibodies for extending the half-lives of short lived drugs. Protein Eng Des Sel 2008; 21: 283-288. 17. O'Connor-Semmes R L, Lin J, Hodge R J, Andrews S, Chism J, Choudhury A. Nunez D J. GSK2374697, a novel albumin-binding domain antibody (AlbudAb), extends systemic exposure of exendin-4: first study in humans-PK/PD and safety. Clin Pharmacol Ther 2014; 96: 704-712. 18. Flanagan R J, Jones A L. Fab antibody fragments. Drug Safety 2004; 27: 1115-1133. 19. Smith B J, Popplewell A, Athwal, D, Chapman A P, Heywood S, West S M, Carrington B, Nesbitt A, Lawson A D, Antoniw P, Eddelston A, Suitters A. Prolonged in vivo residence times of antibody fragments associated with albumin. Bioconjug Chem 2001; 12: 750-756. 20. Lightwood D J, Carrington B, Henry A J, McKnight, A J, Crook K, Cromie K, Lawson A D. Antibody generation through B cell panning on antigen followed by in situ culture and direct RT-PCR on cells harvested en masse from antigen-positive wells. J Immunol Methods 2006; 316: 133-143. 21. Tickle S, Adams R, Brown D, Griffiths M, Lightwood D J, Lawson A D. High-throughput screening for high affinity antibodies. J Lab Auto 2009; 14(5): 303-307. 22. Clargo A M, Hudson A R, Ndlovu W, Wootton R J, Cremin L A, O'Dowd V L, Nowosad C R, Starkie D O, Shaw S P, Compson J E, White D P, MacKenzie B, Snowden J R, Newnham L E, Wright M, Stephens P E, Griffiths M R, Lawson A D, Lightwood D J. The rapid generation of recombinant functional monoclonal antibodies from individual, antigen-specific bone marrow-derived plasma cells isolated using a novel fluorescence-based method. MAbs 2014; 6: 143-159. 23. Adair J R, Athwal D S, Emtage J S. Humanised Antibodies International Patent Publication. 1991; WO91/09967. 24. Curry S. Lessons from the crystallographic analysis of molecule binding to Human Serum Albumin. Drug Metab Pharmacokinet 2009; 24(4): 342-357. 25. Oganesyan V, Damschroder M M, Cook, K E, Li, Q, Gao C, Wu H, Dall'acqua W F. Structural insights into neonatal Fc receptor-based recycling mechanisms. J Biol Chem 2014; 289: 7812-7824. 26. Ascenzi, P. & Fasano, M. Allostery in a monomeric protein: The case of human serum albumin. Biophys Chem 2010; 148: 16-22. 27. Bal W, Sokolowska M, Kurowska E, Faller P. Binding of transition metal ions to albumin: Sites, affinities and rates. Biochim Et Biophys Act 2013; 1830 (12): 5444-5445. 28. Ghuman J, Zunszain P A, Petitpas I, Bhattacharya A A, Otagiri M, Curry S. Structural basis of the drug-binding specificity of human serum albumin. J Mol Biol 2005; 353: 38-52. 29. Curry S, Mandelkow H, Brick P, Franks N. Crystal structure of human serum albumin complexed with fatty acid reveals an asymmetric distribution of binding sites. Nat Struct Biol 1998; 5: 827-835 30. Majorek K A, Porebski P J, Dayal A, Zimmerman M D, Jablonska K, Stewart A J, Chruszcz M, Minor W. Structural and immunologic characterization of bovine, horse, and rabbit serum albumins. Mol Immunol 2012; 52: 174-182 31. Steward M W, Steensgard J. Antibody affinity: thermodynamic aspects and biological significance 1983. CRC Press. 32. Kabat E A, Wu T T, Perry H M, Gottesman K S, Foeller C. Sequences of proteins of immunological interest, 1991; 5th edit Public Health Service, National Institutes of Health, Bethesda, Md. 33. Cain K, Peters S, Hailu H, Sweeney B, Stephens P, Heads J, Sarkar K, Ventom A, Page C, Dickson A. A CHO cell line engineered to express XBP1 and ERO1-Lα has increased levels of transient protein expression. Biotechnol Prog 2013; 29: 697-706. 34. Leslie A G W. Acta Cryst 2006; D62: 48-57 35. Leslie A G W, Powell H R. Evolving Methods for Macromolecular Crystallography 2007; 245: 41-51; ISBN 978-1-4020-6314-5 36. Evans P. Acta Cryst 2006; D62: 72-82 37. McCoy A J, Grosse-Kunstleve R W, Adams P D, Winn M D, Storoni L C, Read R J. Phaser crystallographic software. J Appl Cryst 2007; 40: 658-674 38. Kabsch W. XDS. Acta Cryst 2010; D66: 125-132. 39. Cao H. L, Yin D C. High-resolution Crystal Structural Variance Analysis between Recombinant and Wild-type Human Serum Albumin. To be published. DOI:10.2210/pdb4g03/pdb 40. Brünger A T, Adams P D, Clore G M, DeLano W L, Gros P, Grosse-Kunstleve R W, Jiang J S, Kuszewski J, Nilges M, Pannu N S, Read R J, Rice L M, Simonson T, Warren G L. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 1998; 54 (Pt 5): 905-21 41. Brünger A T. Version 1.2 of the Crystallography and NMR system. Nature Protocols 2007; 2: 2728-2733 42. Emsley P, Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 2004; D60: 2126-32 43. Chen V B, Arendall III W B, Headd J J, Keedy D A, Immormino R M, Kapral G J, Murray L W, Richardson J S, Richardson D C. Acta Crystallogr D Biol Crystallogr 2010; D66: 12-21 44. DeLano W L The PyMOL Molecular Graphics System 2002; DeLano Scientific LLC, San Carlos, Calif.