MULTI-SPECIFIC ANTIBODIES
20230242677 · 2023-08-03
Inventors
- Emily Mary Cairistine Barry (Slough, Berkshire, GB)
- Emma Dave (Slough, Berkshire, GB)
- Sam Philip Heywood (Slough, Berkshire, GB)
Cpc classification
C07K1/22
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
C12N15/63
CHEMISTRY; METALLURGY
C07K16/00
CHEMISTRY; METALLURGY
C07K2317/94
CHEMISTRY; METALLURGY
International classification
C12N15/63
CHEMISTRY; METALLURGY
Abstract
Multi-specific Antibodies The present disclosure relates to a multi-specific antibody comprising or consisting of: a) a polypeptide chain of formula (I): VH-CH1-(CH2)-(CH3)-(X)-(V1); and b) a polypeptide chain of formula (II): (V3)-(Z) -VL-C.sub.L-(Y)-(V2) wherein the polypeptide chain of formula (II) comprises at least one dsscFv, dsFv, scFv, VH or VHH, and wherein the polypeptide chain of formula (I) comprises a protein A binding domain and wherein the polypeptide chain of formula (II) does not bind protein A. The disclosure also provides polynucleotide sequences encoding said multi-specific antibody, vectors comprising the polynucleotides and host cells comprising said vectors and/or polynucleotide sequences. The disclosure also provides pharmaceutical formulations comprising same, for example for use in treatment. There is also provided a method of expressing a multi-specific antibody of the present disclosure from a host cell.
Claims
1. A multi-specific antibody, comprising: a polypeptide chain of formula (I): ##STR00017## a polypeptide chain of formula (II): ##STR00018## wherein: VH represents a heavy chain variable domain; CH1 represents domain 1 of a heavy chain constant region; CH2 represents domain 2 of a heavy chain constant region; CH3 represents domain 3 of a heavy chain constant region; X represents a bond or linker; V1 represents a dsscFv, a dsFv, a scFv, a VH, a VH or a VHH; V3 represents a dsscFv, a dsFv, a scFv, a VH, a VH or a VHH; Z represents a bond or linker; VL represents a light chain variable domain; CL represents a domain from a light chain constant region, such as Ckappa; Y represents a bond or linker; V2 represents a dsscFv, a dsFv, a scFv, a VH, a VH or a VHH; p represents 0 or 1 ; q represents 0 or 1 ; r represents 0 or 1 ; s represents 0 or 1 ; t represents 0 or 1 ; wherein when p is 0, X is absent and when q is 0, Y is absent and when r is 0, Z is absent; and wherein when q is 0, r is 1 and when r is 0, q is 1 ; and wherein the polypeptide chain of formula (II) comprises at least one dsscFv, dsFv, scFv, VH or VHH; and wherein the polypeptide chain of formula (I) comprises a protein A binding domain; and wherein the polypeptide chain of formula (II) does not bind protein A.
2. A multi-specific antibody according to claim 1, wherein the polypeptide chain of formula (I) comprises one, two or three protein A binding domains.
3. A multi-specific antibody according to claim 1, wherein a protein A binding domain is present in VH and/or CH2-CH3 and/or V1.
4. A multi-specific antibody according to claim 1, wherein the polypeptide chain of formula (I) comprises only one protein A binding domain which is present in VH or V1.
5. A multi-specific antibody according to claim 4, wherein the polypeptide chain of formula (I) comprises only one protein A binding domain which is present in VH.
6. A multi-specific antibody according to claim 4, wherein the polypeptide chain of formula (I) comprises only one protein A binding domain which is present in V1.
7. A multi-specific antibody according to claim 1, wherein the protein A binding domain(s) comprise(s) or consist(s) of a VH3 domain or variant thereof which binds protein A.
8. A multi-specific antibody according to claim 1, wherein V2 and/or V3 do/does not comprise a VH3 domain.
9. A multi-specific antibody according to claim 1, wherein V2 and/or V3, comprise(s) a VH3 domain or variant thereof which does not bind protein A.
10. A multi-specific antibody according to claim 1, wherein p is 1.
11. A multi-specific antibody according to claim 1, wherein q is 1.
12. A multi-specific antibody according to claim 1, wherein r is 1.
13. A multi-specific antibody according to claim 1, wherein q is 0 and r is 1.
14. A multi-specific antibody according claim 1, wherein s is 1, t is 1, p is 0, q is 1, r is 0 and wherein V2 is a dsscFv or dsFv.
15. A multi-specific antibody according to claim 1, wherein s is 0 and t is 0, p is 1, q is 1, r is 0, and wherein V1 and V2 both represent a dsscFv.
16. A multi-specific antibody according to claim 1, wherein V1 binds albumin and comprises a VH3 of sequence SEQ ID NO: 78.
17. A multi-specific antibody according to claim 1, wherein X and/or Y and/or Z is a peptide linker.
18. A multi-specific antibody according to claim 1, wherein V1 and/or V2 and/or V3 are/is a dsscFv or a dsFv, and wherein the light chain and heavy chain variable domains of V1 and/or the light chain and heavy chain variable domains of V2 and/or the light chain and heavy chain variable domains of V3 are linked by a disulfide bond between two engineered cysteine residues, wherein the position of the pair of cysteine residues is selected from the group comprising or consisting of: VH37 and VL95, VH44 and VL100, VH44 and VL105, VH45 and VL87, VH100 and VL50, VH1 00b and VL49, VH98 and VL46, VH101 and VL46, VH1 05 and VL43 and VH106 and VL57 (numbering according to Kabat), wherein the VH and VL values are independently within a given V1 or V2 or V3.
19. A polynucleotide encoding a multi-specific antibody defined in claim 1.
20. A vector comprising a polynucleotide defined in claim 19.
21. A host cell comprising a polynucleotide of claim 19.
22. A host cell comprising at least two vectors, each vector comprising a polynucleotide encoding a different polypeptide chain of a multi-specific antibody defined in claim 1.
23. A pharmaceutical composition comprising a multi-specific antibody according to claim 1 and at least one excipient.
24. (canceled)
25. A method of treating a patient in need thereof, the method comprising administering a therapeutically effective amount of a multi-specific antibody according claim 1.
26. A method of producing a multi-specific antibody comprising a polypeptide chain of formula (I) and a polypeptide chain of formula (II) as defined in claim 1, said method comprising: a) Expressing a polypeptide chain of formula (I) and a polypeptide chain of formula (II) in a host cell, wherein the polypeptide chain of formula (II) is in excess over the polypeptide chain of formula (I); b) Recovering the composition of polypeptides expressed at step a), said composition comprising a multi-specific antibody and a LC dimer of formula (II-II); and c) Purifying the multi-specific antibody, wherein when s is 1 and t is 1, said multi-specific antibody is purified as a dimer with two heavy chains of formula (I) and two associated light chains of formula (II) and, wherein when s is 0 and t is 0, said multi-specific antibody is purified as a dimer with one heavy chain of formula (I) and one associated light chain of formula (II); and, wherein the polypeptide chain of formula (II) comprises at least one dsscFv, dsFv, scFv, VH or VHH; and, wherein the polypeptide chain of formula (I) comprises a protein A binding domain; and, wherein the polypeptide chain of formula (II) does not bind protein A; and, wherein step c) comprises subjecting the composition of polypeptides recovered at step b), optionally following at least one purification step, to a Protein A affinity chromatography column.
27. A method of purifying a multi-specific antibody comprising a polypeptide chain of formula (I) and a polypeptide chain of formula (II) as defined in claim 1, said method comprising: a) Obtaining a composition of polypeptide chains of formula (I) and polypeptide chains of formula (II) said composition comprising a multi-specific antibody, wherein when s is 1 and t is 1, the multi-specific antibody is a dimer with two heavy chains of formula (I) and two associated light chains of formula (II) and; when s is 0 and t is 0, the multi-specific antibody is a dimer with one heavy chain of formula (I) and one associated light chain of formula (II); and a dimer of two light chains of formula (II-II), associated together (LC dimer); and, wherein the polypeptide chain of formula (II) comprises at least one dsscFv, dsFv, scFv, VH or VHH; and, wherein the polypeptide chain of formula (I) comprises a protein A binding domain; and, wherein the polypeptide chain of formula (II) does not bind protein A; b) Loading the composition obtained in step a), onto a protein A affinity column, such that the multi-specific antibody is retained on the column whilst the LC dimer does not bind to the column; c) Washing the protein A affinity column; d) Eluting the multi--specific antibody; and, e) Recovering the multi-specific antibody.
Description
BRIEF DESCRIPTION OF THE FIGURES
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[0408]
EXAMPLES
Example 1: Production of an Improved Multi-Specific Antibody Format of the Invention, Example of A Fab-2xdsscFv (TrYbe)
Gene Design and Expression in CHO-S XE Cell Line
[0409] TrYbe antibody was designed with an anti-Antigen#1 (or “Ag#1”) V-region fixed in the Fab position; the anti-albumin(Antigen#2, or “Ag#2” in the following example) V-region (645gL4gH5) and Antigen#3 (or “Ag#3”) V-region (VH1) were reformatted into disulfide-stabilised scFv in the HL orientation (dsHL) and linked to the C-termini of the respective heavy and light chain constant regions via a 11 -amino acid glycine-serine rich linkers. The resulting antibody is referred to as Trybe 03. The sequences of anti-albumin 645 antibody are shown in
[0410] The light chain and heavy chain genes were independently cloned into proprietary mammalian expression vectors for transient expression under the control of a hCMV promoter. Equal ratios of both plasmids were transfected into the CHO-S XE cell line (UCB) using the commercial ExpiCHO expifectamine transient expression kit (Thermo Scientific). The cultures were incubated in Corning roller bottles with vented caps at 37° C., 8.0% CO.sub.2, 190 rpm. After 18-22 h, the cultures were fed with the appropriate volumes of CHO enhancer and feeds for the HiTiter method as provided by the manufacturer. Cultures were reincubated at 32° C., 8.0% CO.sub.2, 190 rpm for an additional 10 to 12 days. The supernatant was harvested by centrifugation at 4000 rpm for 1 h at 4° C. prior to filter-sterilization through a 0.45 .Math.m followed by a 0.2 .Math.m filter. Expression titres were quantified by Protein G HPLC using a 1 ml GE HiTrap Protein G column (GE Healthcare) and Fab standards produced in-house. The expression titre was 160 mg/L.
Purification of TrYbe 03 Using A Protein A Affinity Chromatography
[0411] The TrYbe 03 was purified by native protein A capture step followed by a preparative size exclusion polishing step. Clarified supernatants from standard transient CHO expression were loaded onto a MabSelect (GE Healthcare) column giving a 5 min contact time and washed with binding buffer (20 mM Hepes pH7.4 + 150 mM NaCl). Bound material was eluted with a 0.1 M sodium citrate pH3.1 step elution and neutralised with 2 M Tris/HCl pH8.5 and quantified by absorbance at 280 nm.
[0412] Size exclusion chromatography (SE-UPLC) was used to determine the purity status of the eluted product. The antibody (~2 .Math.g) was loaded on to a BEH200, 200 Å, 1.7 .Math.m, 4.6 mm ID x 300 mm column (Waters ACQUITY) and developed with an isocratic gradient of 0.2 M phosphate pH 7 at 0.35 mL/min. Continuous detection was by absorbance at 280 nm and multi-channel fluorescence (FLR) detector (Waters). The eluted TrYbe 03 antibody was found to be 72 % monomer.
[0413] The neutralised samples were concentrated using Amicon Ultra-15 concentrator (10 kDa molecular weight cut off membrane) and centrifugation at 4000xg in a swing out rotor. Concentrated samples were applied to a XK16/60 Superdex200 column (GE Healthcare) equilibrated in PBS, pH7.4 and developed with an isocratic gradient of PBS, pH7.4 at 1 ml/min. Fractions were collected and analysed by size exclusion chromatography on a BEH200, 200 Å, 1.7 .Math.m, 4.6 mm ID x 300 mm column (Aquity) and developed with an isocratic gradient of 0.2 M phosphate pH 7 at 0.35 mL/min, with detection by absorbance at 280 nm and multi-channel fluorescence (FLR) detector (Waters). Selected monomer fractions were pooled, 0.22 .Math.m sterile filtered and final samples were assayed for concentration by A280 Scanning on DropSense96 (Trinean). Endotoxin level was less than 1.0EU/mg as assessed by Charles River’s EndoSafe® Portable Test System with Limulus Amebocyte Lysate (LAL) test cartridges.
Analysis by Size Exclusion Chromatography
[0414] Monomer status of the final TrYbe 03 was determined by size exclusion chromatography on a BEH200, 200 Å, 1.7 .Math.m, 4.6 mm ID x 300 mm column (Aquity) and developed with an isocratic gradient of 0.2 M phosphate pH 7 at 0.35 mL/min, with detection by absorbance at 280 nm and multi-channel fluorescence (FLR) detector (Waters). The final TrYbe 03 antibody was found to be >99 % monomeric. (
SDS-PAGE Analysis
[0415] For analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) samples were prepared by adding 4 x Novex NuPAGE LDS sample buffer (Life Technologies) and either 10X NuPAGE sample reducing agent (Life Technologies) or 100 mM N-ethylmaleimide (Sigma-Aldrich) to ~ 5 .Math.g purified protein, and were heated to 100° C. for 3 min. The samples were loaded onto a 10 well Novex 4-20% Tris-glycine 1.0 mm SDS-polyacrylamide gel (Life Technologies) and separated at a constant voltage of 225 V for 40 min in Tris-glycine SDS running buffer (Life Technologies). Novex Mark12 wide-range protein standards (Life Technologies) were used as standards. The gel was stained with Coomassie Quick Stain (Generon) and destained in distilled water.
[0416] On non-reducing SDS-PAGE the TrYbe (lane 1), theoretical molecular weight (MW) of ~100 kDa, migrated to ~ 120 kDa (
[0417] The present inventors have observed that Trybe 03 had improved properties over the multi-specific antibodies of the prior art, in particular in that it maximised the amount of proteins of interest (i-e the correct multi-specific antibody) obtained after a one-step purification on a protein A chromatography column. Indeed, previously, the inventors detected appended light chains unpaired with their corresponding heavy chains, co-purified with the multi-specific antibody of interest and which had a propensity to form dimers of appended light chains (appended LC dimers), which needed to be purified away by an additional capture step. Unexpectedly, after the protein A purification step, no light chain or LC dimer was detected as a by-product of the production process of TrYbe 03 and only the desired multi-specific antibody was eluted from the protein A column. In addition, the multi-specific antibody was highly monomeric.
[0418] The inventors made the hypothesis that the isolation and removal of the appended LC dimers occurred concurrently with the purification of Trybe 03.
[0419] To confirm this hypothesis, additional experiments, with alternative multi-specific antibody formats, were performed and are described in the following examples.
Example 2: Production of Alternative Antibody Formats for Further Analysis in Examples 3 to 6
[0420] The constructs as illustrated in
TABLE-US-00008 Antibody construct Description WITTRUP 01 Ag#1 hg1FL, Ag#1 Fab LC- Ag#2 dsscFv HL WITTRUP 02 Ag#1 hg1FL, Ag#1 Fab LC-Ag#4 dsscFv HL TRYBE 03 Ag#1 Fab, Ag#2 dsscFv HL (HC), Ag#3 dsscFv HL (LC) (VH1) TRYBE 04 Ag#1 Fab, Ag#2 dsscFv HL (HC), Ag#3 dsscFv HL (LC) (VH3) TRYBE 05 Ag#1 Fab, Ag#3 dsscFv HL (HC) (VH1), Ag#2 dsscFv HL (LC) TRYBE 06 Ag#1 Fab, Ag#3 dsscFv HL (HC) (VH3), Ag#2 dsscFv HL (LC)
[0421] In the following examples, 645 gH5gL4 dsscFv(HL), i-e Ag#2 dsscFv HL, is termed dsscFv 1.
[0422] Ag#3 dsscFv HL (VH1), comprising a VH1 domain, is termed dsscFv 3B,
[0423] Ag#3 dsscFv HL (VH3), comprising a VH3 domain, is termed dsscFv 3A.
[0424] Ag#4 dsscFv HL is termed dsscFv 2.
Transient Expression
[0425] Heavy and light chain antibody genes were independently cloned into proprietary mammalian expression vectors for transient expression under the control of a hCMV-mie promoter. Plasmids were transfected into a proprietary CHO-SXE cell line using the commercial ExpiCHO expifectamine transient expression kit (Thermo Scientific). The cultures were incubated in Corning roller bottles with vented caps at 37° C., 8.0% CO.sub.2, 190 rpm. After 18-22 h, the cultures were fed with the appropriate volumes of CHO enhancer and feeds for the HiTiter method as provided by the manufacturer. Cultures were then incubated at 32° C., 8.0% CO.sub.2, 190 rpm for an additional 10 to 12 days. The supernatant was harvested by centrifugation at 4000 rpm for 1 h at 4° C. prior to filter-sterilization through a 0.45 .Math.m followed by a 0.2 .Math.m filter.
[0426] Expression titres were quantified by Protein A HPLC and Protein L HPLC using either a 1 ml HiTrap Protein A column or a 1 ml HiTrap Protein L column (GE Healthcare). Columns were equilibrated in a phosphate buffer, 10 .Math.l of sample was injected, column was washed, and an acidic step elution was used to elute the antibody. Concentrations were calculated using the elution peak area for each sample compared to a standard curve generated using in-house purified Fab standards with appropriate molar extinction co-efficient correction.
[0427] Protein L ligand binds via the VL domain, i-e the light chain of antibodies. Protein A binds the CH2/CH3 interface of the Fc and a selection of human VH domains comprising a protein A binding domain.
Expression of Light Chain Plasmids Only
[0428] For expression of the light chains appended with a disulphide stabilised single chain Fv (LC-dsscFv), only the light chain plasmids were transfected, expressed and quantified by the above method. Table 1a lists the titres for these expressed light chain dimers as quantified by both Protein A and Protein L HPLC assays.
[0429] The quantification of LC-dsscFv-1 supernatant gave equivalent results in the Protein L and Protein A assays. In contrast, the LC-dsscFv-2 and LC-dsscFv-3B supernatants were quantifiable by Protein L but the Protein A assay was below the level of quantification. The quantification of the LC-dsscFv-3A expression gave a value of the Protein-A assay of about a third of the Protein L assay.
TABLE-US-00009 Quantification of expressed Light Chain Dimer by Protein A and Protein L HPLC assay. LOQ = Limit of quantification. Description of Light Chain dsscFv (Light Chain Dimer) Protein A mg/L Protein L mg/L dsscFv-1 221.2 220.2 dsscFv-2 <LOQ 250.5 dsscFv-3B <LOQ 155.3 dsscFv-3A 43.9 120.5
TABLE-US-00010 Strength of Light Chain binding to Protein A. Binding strengths have been categorised as strong (++), weak (+), none (-). Antibody Name Description of Light Chain dsscFv Protein A Binding Wittrup 01 dsscFv-1 ++ TrYbe 05 TrYbe 06 Wittrup 02 dsscFv-2 - TrYbe 03 dsscFv-3B - TrYbe 04 dsscFv-3A +
[0430] As shown in Table 1b, LC-dsscFv-1 contains a dsscFv which binds Protein A, explaining why the calculated Protein L and Protein A titres were equivalent (Table 2a). At the contrary, LC-dsscFv-2 and LC-dsscFv-3B were only quantifiable by the Protein L assay and not the Protein A assay and it was confirmed that they do not comprise a protein A binding domain. It was observed that LC-dsscFv-3A contained a dsscFv that binds Protein A weakly, therefore the concentration calculated was only a third of the concentration from the Protein L assay.
[0431] Therefore, the results show that dsscFv-1 and dsscFv-3A comprise a protein A binding domain. In particular, dsscFv-3A comprises a VH3 domain which is able to bind protein A.
[0432] At the contrary, dsscFv-2 and dsscFv-3B do not bind protein A. In particular, dsscFv-3B comprises a VH1 domain which is unable to bind protein A.
Co-Expression of Heavy Chain and Light Chain Plasmids
[0433] For the expression of antibody constructs, equal ratios of heavy and light chain plasmids were co-transfected and expressed by the above method. These antibodies share the same Fab region and isotype.
[0434] To ensure that the test supernatants studied in the following Examples (3, 4, 5 and 6) contained excess light chain, the corresponding light chain only supernatant was added to the antibody supernatant. The resulting test supernatants were quantified by Protein A and Protein L HPLC assays (Table 2a).
[0435] The quantification of Wittrup 01, TrYbe 05 and TrYbe 06 test supernatants gave equivalent results in both Protein A and Protein L assays. For Wittrup 02, TrYbe 03 and TrYbe 04 the concentration determined by Protein A assay was approximately half of that determined by the Protein L assay.
[0436] Wittrup 01, TrYbe 05 and TrYbe 06 share the same light chain, as described in Table 1b and Table 2b, this light chain has a Protein A binding dsscFv, so the calculated Protein L and Protein A titres were equivalent as both the antibody and light chain dimer can bind in both assays. The Protein A assay can be used to determine the concentration of Wittrup 02 and TrYbe 03 as the antibody can bind Protein A, however both have a non-protein A binding dsscFv on the light chain meaning that respective light chain dimers can only be quantified by the Protein L assay, thus accounting for the 2-fold difference between the two assays. TrYbe 04 has a weak Protein A binding dsscFv on the light chain, therefore only some of the light chain dimer binds and the concentration calculated was only half of the concentration from the Protein L assay.
TABLE-US-00011 Quantification of test material by Protein A and Protein L HPLC assay. Samples prepared by spiking light chain only supernatant into the respective antibody supernatants. Sample Name Chain Description of appended dsscFv Protein A mg/L Protein L mg/L Wittrup 01 Heavy - 47.0 66.9 Light dsscFv-1 Wittrup 02 Heavy - 97.2 180.2 Light dsscFv-2 TrYbe 03 Heavy dsscFv-1 95.8 153.4 Light dsscFv-3B TrYbe 04 Heavy dsscFv-1 129.6 219.1 Light dsscFv-3A TrYbe 05 Heavy dsscFv-3B 137.6 143.0 Light dsscFv-1 TrYbe 06 Heavy dsscFv-3A 280.9 296.2 Light dsscFv-1
TABLE-US-00012 Strength of Light Chain binding to Protein A. Binding strengths have been categorised as strong (++), weak (+), none (-). All heavy chains are described as strong binders as they bind through the common Fab (Wittrup & TrYbe) or through the Fc (Wittrup only). Sample Name Chain Description of appended scFv Protein A Binding Wittrup 01 Heavy - ++ Light dsscFv-1 ++ Wittrup 02 Heavy - ++ Light dsscFv-2 - TrYbe 03 Heavy dsscFv-1 ++ Light dsscFv-3B - TrYbe 04 Heavy dsscFv-1 ++ Light dsscFv-3A + TrYbe 05 Heavy dsscFv-3B - Light dsscFv-1 ++ TrYbe 06 Heavy dsscFv-3A + Light dsscFv-1 ++
Example 3: Protein A Purification of Wittrup Antibody Formats; Selecting the dsscFv Variable Region With Appropriate Protein A Binding Properties
[0437] The test supernatants for both Wittrup molecules were prepared as described in Example 2, and contain both antibody and light chain dimer. These Wittrup antibodies share the same IgG component (Fc and Fab) but each has a different dsscFv appended to the light chain. Wittrup 01 has a Protein A binding dsscFv appended to the light chain whereas Wittrup 02 has a non-Protein A binding dsscFv appended to the light chain.
[0438] As shown in Example 2, the Wittrup 01 and Wittrup 02 test supernatants were quantified by Protein A and Protein L HPLC assays (Table 3a). Wittrup 01 gave approximately equivalent results in both assays, whereas for Wittrup 02 the Protein A assay was only half of the Protein L assay. Wittrup 01 has a Protein A binding dsscFv appended to the light chain (Table 3b), so the titres calculated by Protein L and Protein A are equivalent as both ligands can detect light chain dimers. The Protein A assay result for Wittrup 02, which has a non-protein A binding dsscFv appended to the light chain (Table 3b), is significantly lower than the Protein L assay as only the antibody can bind Protein A whereas both Wittrup antibody and light chain dimer can bind Protein L.
TABLE-US-00013 Quantification of test material by Protein A and Protein L HPLC assay. Samples prepared by spiking light chain only supernatant into the respective antibody supernatants. Sample Name Chain Description of appended scFv Protein A mg/L Protein L mg/L Wittrup 01 Heavy - 47.0 66.9 Light dsscFv-1 Wittrup 02 Heavy - 97.2 180.2 Light dsscFv-2
TABLE-US-00014 Strength of Light Chain binding to Protein A. Binding strengths have been categorised as strong (++), weak (+), none (-). Sample Name Chain Description of appended scFv Protein A Binding Wittrup 01 Heavy - ++ Light dsscFv-1 ++ Wittrup 02 Heavy - ++ Light dsscFv-2 -
Protein A Purification
[0439] The test supernatants were loaded onto a MabSelect (GE Healthcare) column with a 15 min contact time and washed with binding buffer (200 mM glycine, pH7.5). The flow through was collected and 0.22 .Math.m sterile filtered. Bound material was eluted with a 0.1 M sodium citrate pH3.2 step elution, the elution peak was collected, neutralised with 2 M Tris-HCl pH8.5 and the purified protein was quantified by absorbance at 280 nm. To confirm that the protein was completely eluted from the column a second elution with 0.1 M Citrate pH2.1 was performed.
Protein L Purification
[0440] The flow throughs from the Protein A purifications were loaded onto a Protein L (GE Healthcare) column with a 10 min contact time and washed with binding buffer (200 mM glycine, pH7.5). The flow through was collected and 0.22 .Math.m sterile filtered. Bound material was eluted with a 0.1 M Glycine/HCl pHy step elution, the elution peak was collected, neutralised with 2 M Tris-HCl pH8.5 and the purified protein was quantified by absorbance at 280 nm. To confirm that the protein was completely eluted from the column a second elution with 0.1 M Citrate pH2.1 was performed.
SDS-PAGE
[0441] For analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) samples were prepared by adding 4 x Novex NuPAGE LDS sample buffer (Life Technologies) and either 10X NuPAGE sample reducing agent (Life Technologies) or 100 mM N-ethylmaleimide (Sigma-Aldrich), and were heated to 100° C. for 3 min. The samples were loaded onto a 15 well Novex 4-20% Tris-glycine 1.0 mm SDS-polyacrylamide gel (Life Technologies) and separated at a constant voltage of 225 V for 40 min in Tris-glycine SDS running buffer (made in-house). Novex Mark12 wide-range protein standards (Life Technologies) were used as molecular weight markers. The gel was stained with Coomassie Quick Stain (Generon) and destained in distilled water.
Results
[0442] To evaluate sequential Protein A and Protein L purifications, reduced (
[0443] Wittrup 01 has a Protein A binding dsscFv appended to the light chain. In the reduced Protein A eluate (lane 1B) there is one band as the heavy and light chains are similar in size and therefore co-migrate to the same position. In the Protein L eluate (lane 1D) there are no detectable bands. This indicates that the light chain dimer was co-purified with the Wittrup 01 antibody during the Protein A purification. In contrast, Wittrup 02 has a non-Protein A binding dsscFv appended to the light chain. The Protein A eluate (lane 2B) looks comparable to the Wittrup 01 Protein A eluate but in the Protein L eluate there is a light chain band present indicating that the light chain dimer was not captured in the Protein A purification but flowed through the column and was subsequently captured in the Protein L purification.
[0444] On the non-reduced gel for Wittrup 01, there are bands for the Wittrup antibody and the light chain dimer in the Protein A eluate (lane 1B). There are also additional bands present in this lane due to incomplete formation of the natural interchain disulphide (ds) bond between the CH1 and C.sub.K in a portion of the molecules. The Protein L eluate (lane 1D) has no detectable bands again showing that the light chain dimer co-purified with the Wittrup 01 in the Protein A purification. For Wittrup 02, there is a Wittrup band in the Protein A eluate (lane 2B) as well as the additional bands due to incomplete disulphide formation. The light chain dimer band can be seen in both the Protein L load and the Protein L Eluate (lane 2C, lane 2D) but not in the Protein A eluate. This further indicates that only the Wittrup 02 antibody was captured in the Protein A purification and that the light chain dimer flowed through the column and was subsequently captured in the Protein L purification.
[0445] In summary, the presence of a dsscFv able to bind Protein A appended to the light chain in the Wittrup antibody resulted in the co-purification of the light chain dimers, which could be avoided by selecting a dsscFv unable to bind protein A appended to the light chain of the Wittrup format. Therefore, the inventors provided an improved multi-specific antibody wherein the light chain may be selected or engineered to be a non-Protein A binder.
Example 4: Protein A Purification of TrYbe Antibody Formats, With Different Variable Region Grafting; Framework Selection for Appropriate Protein A Binding Properties of the Light Chain Appended DsscFv
[0446] The test supernatants for both TrYbe 03 and 04 molecules were prepared as described in Example 2 and contain both antibody and light chain dimer. These TrYbes share the same Fab and the same Protein A binding dsscFv appended to the heavy chain. The light chain appended dsscFvs are derived from the same parent variable region but in TrYbe 03 the CDRs were grafted onto a non-Protein A binding framework (VH1 domain) whereas in TrYbe 04 the CDRs were grafted onto a Protein A binding framework (VH3 domain).
[0447] The TrYbe 03 and TrYbe 04 test supernatants were quantified by Protein A and Protein L HPLC assays (Table 4a) and in both cases the Protein A assay is lower than the Protein L assay.
[0448] The concentration of TrYbe 03 as determined by Protein A assay is about half of the Protein L assay, as this TrYbe has a non-Protein A binding dsscFv on the light chain (Table 4b) only the TrYbe antibody can bind Protein A whereas both the TrYbe 03 and light chain dimer can be quantified by the Protein L assay. TrYbe 04 has a weak Protein A binding dsscFv on the light chain (Table 4b), all the TrYbe and light chain dimer can bind to the Protein L assay, but the Protein A assay binds all the TrYbe and only a proportion of the light chain dimer. Therefore, it is not possible to accurately quantify the total light chain dimer and TrYbe by Protein A in this situation.
TABLE-US-00015 Quantification of test supernatants by Protein A and Protein L HPLC assay. Samples prepared by spiking light chain only supernatant into the respective antibody supernatants. Sample Name Chain Description of appended scFv Protein A mg/L Protein L mg/L TrYbe 03 Heavy dsscFv-1 95.8 153.4 Light dsscFv-3B TrYbe 04 Heavy dsscFv-1 129.6 219.1 Light dsscFv-3A
TABLE-US-00016 Strength of Light Chain binding to Protein A. Binding strengths have been categorised as strong (++), weak (+), none (-). Sample Name Chain Description of appended scFv Protein A Binding TrYbe 03 Heavy dsscFv-1 ++ Light dsscFv-3B - TrYbe 04 Heavy dsscFv-1 ++ Light dsscFv-3A +
Protein A and Protein L Purification Steps, and SDS PAGE Analysis Were Done As Described Above in Example 3
Densitometry
[0449] A Densitometrical analysis was performed on the reduced SDS-PAGE using ImageQuant image analysis software (GE Healthcare). Analysis is displayed as a percentage relative to the density of the heavy chain band.
Results
[0450] To evaluate the sequential Protein A and Protein L purifications, reduced (
[0451] TrYbe 03 has a non-Protein A binding dsscFv appended to the light chain. On the reduced gel in the Protein A eluate (lane 3B), there are two bands corresponding to the heavy and light chains; and in the Protein L eluate (lane 3D) only the light chain band is present. Densitometrical analysis showed the ratio of heavy and light chains present in the protein A eluate is equal. Therefore, only TrYbe 03 was captured by the Protein A purification and that the light chain dimer flowed through the column and was subsequently captured by the Protein L purification. In contrast, TrYbe 04 has a Protein A binding dsscFv appended to the light chain. On the reduced gel, in the Protein A eluate (lane 4B) there is a more intense light chain and less intense heavy chain. Densitometry (
[0452] On the non-reduced gel, for TrYbe 03 there is a TrYbe band in the Protein A eluate (lane 3B) and a light chain dimer band in the Protein L eluate (lane 3D), they are similar in size, so the bands migrate to the same position. There are also heavy and light chain bands in the Protein A eluate and a light chain band in the Protein L eluate, this is due to the incomplete formation of the natural interchain disulphide (ds) bond between the CH1 and C.sub.K in a small proportion of the molecules. This is also evident in the Protein L eluate (lane 3E) as there is non-ds bonded light chain present. Again, these observations indicate that only the TrYbe 03 antibody was captured by the Protein A purification and that the light chain dimer flowed through the column and was subsequently captured by the Protein L purification. For TrYbe 04, in the Protein A eluate (lane 4B) the TrYbe and light chain dimer bands co-migrate to the same position as they are similar in size. There are also heavy and light chain bands present due to incomplete interchain ds bond formation and there is more non-ds bonded light chain as the ds bond formation between two CK is less efficient than for the CH1/C.sub.K pairing. Again, there are no bands in the Protein L eluate (lane 4D) indicating the light chain dimer was co-purified with the TrYbe 04 during the Protein A purification.
[0453] In summary, the presence of a Protein A binding graft of this dsscFv on the light chain resulted in the co-purification of light chain dimer with TrYbe. The same dsscFv was grafted onto a non-Protein A binding framework, then light chain dimer was not captured and only the TrYbe was purified by the Protein A chromatography.
[0454] Therefore, the inventors provided an improved multi-specific antibody wherein the VH framework of the dsscFv appended to the light chain was selected to be a non-Protein A binder. In the present case, a VH1 was selected for its inability to bind protein A. It will be understood by the skilled person that the same results can be obtained by selecting frameworks that do not bind protein A, for example a VH1, a VH2, a VH4, a VH5, a VH6, a naturally occurring VH3 unable to bind protein A, or a variant of a naturally occurring VH3 able to bind protein A, comprising at least one mutation abolishing its ability to bind protein A.
Example 5: Protein A Purification of TrYbe Antibody Formats, With Alternate DsscFv Positioning for Appropriate Protein A Binding Properties of the Light Chain Appended DsscFv
[0455] The test supernatants for both TrYbe 03 and TrYbe 05 molecules were prepared as described in Example 2 and contain both antibody and light chain dimer. These TrYbe share the same Fab and the same pair of dsscFvs but the dsscFvs were appended onto opposite Fab chains. In TrYbe 03 the Protein A binding dsscFv is appended to the heavy chain and the non-Protein A binding dsscFv is appended to the light chain. Alternatively, in TrYbe 05 the Protein A binding dsscFv is appended to the light chain and the non-Protein A binding dsscFv is appended to the heavy chain.
[0456] The TrYbe 03 and TrYbe 05 test supernatants were quantified by Protein A and Protein L HPLC (Table 5a). For TrYbe 03 the Protein A assay is significantly lower than the Protein L assay whereas TrYbe 05 gives equivalent results in both assays.
[0457] The Protein A assay result for TrYbe 03 is significantly lower than the Protein L assay as this TrYbe has a non-Protein A binding dsscFv on the light chain (Table 5b) meaning that only the TrYbe antibody can bind Protein A, whereas both the TrYbe and light chain dimer can bind the Protein L assay. TrYbe 05 has a Protein A binding dsscFv appended to the light chain (Table 5b), so the calculated Protein L and Protein A titres are equivalent as both assays can bind TrYbe and light chain dimers.
TABLE-US-00017 Quantification of test supernatants by Protein A and Protein L HPLC assay. Samples prepared by spiking light chain only supernatant into the respective antibody supernatants. Sample Name Chain Description of appended scFv Protein A mg/L Protein L mg/L TrYbe 03 Heavy dsscFv-1 95.8 153.4 Light dsscFv-3B TrYbe 05 Heavy dsscFv-3B 137.6 143.0 Light dsscFv-1
TABLE-US-00018 Strength of Light Chain binding to Protein A. Binding strengths have been categorised as strong (++), weak (+), none (-). Sample Name Chain Description of appended scFv Protein A Binding TrYbe 03 Heavy dsscFv-1 ++ Light dsscFv-3B – TrYbe 05 Heavy dsscFv-3B – Light dsscFv-1 ++
Protein A and Protein L Purification Steps Were Performed as Described Above. SDS PAGE and Densitometrical Analyses Were Also Performed As Described Above
Results
[0458] To evaluate the sequential Protein A and Protein L purifications, reduced (
[0459] TrYbe 03 has the non-Protein A binding dsscFv appended to the light chain. On the reduced gel, in the Protein A eluate (lane 3B), there are two bands corresponding to the heavy and light chains, and in the Protein L eluate (lane 3D) only the light chain band is present. Densitometrical analysis shows the ratio of heavy and light chains present in the protein A eluate is equal. Therefore, only the TrYbe 03 was captured in the Protein A purification and the light chain dimer flowed through the column and was subsequently captured in the Protein L purification. In contrast, TrYbe 05 has the Protein A binding dsscFv appended to the light chain. In the reduced Protein A eluate (lane 5B) there is 40% more light chain than heavy chain present, and in the Protein L eluate (lane 5D) there are no detectable bands. This indicates that the light chain dimer was co-purified with the TrYbe 05 during the Protein A purification.
[0460] On the non-reduced gel, for TrYbe 03 there is a TrYbe band in the Protein A eluate (lane 3B) and a light chain dimer band in the Protein L eluate (lane 3D), they are similar in size, so the bands migrate to the same position. There are also heavy and light chain bands in the Protein A eluate and a light chain band in the Protein L eluate. These are due to the incomplete formation of the natural interchain disulphide (ds) bond between the CH1 and C.sub.K in a small proportion of the molecules, or the corresponding C.sub.K/C.sub.K interchain disulphide in the light chain dimer. Again, these results indicate that only TrYbe was captured in the Protein A purification and that the light chain dimer flowed through the column and was subsequently captured in the Protein L purification. In the TrYbe 05 Protein A eluate (lane 5B) the TrYbe and light chain dimer bands co-migrate to the same position as they are very similar in size. Again, heavy and light chains due to non-formation of interchain disulphide binds are present as in lane 3B. In addition, there are also no detectable bands in the Protein L eluate (lane 5D) further indication that the light chain dimer was co-purified with the TrYbe during the Protein A purification.
[0461] In summary, the arrangement of the TrYbe molecule such that a Protein A binding dsscFv was appended to the light chain and a non-Protein A binding dsscFv was appended to the heavy chain resulted in the co-purification of both light chain dimer and TrYbe. By reversing this design and swapping the two dsscFvs such that the Protein A binding dsscFv was on the heavy chain and the non-Protein A binding dsscFv was on the light chain, the inventors showed that it was possible to purify only the TrYbe by Protein A affinity chromatography with the light chain dimer flowing through the column.
Example 6: Protein A Purification of TrYbe Antibody Formats, With Inappropriate ScFv Selection for Protein A Binding Properties of the Light Chain Appended ScFv
[0462] The test supernatants for both TrYbe molecules were prepared as described in Example 1 and contain both antibody and light chain dimer. These TrYbes share the same Fab and the same pair of dsscFvs but the dsscFvs were appended onto the opposite Fab chains. Both dsscFvs bind Protein A but with different strengths. In TrYbe 04, the weaker Protein A binding dsscFv is appended to light chain and the strong Protein A binding dsscFv is appended to the heavy chain. Alternatively, in TrYbe 06, the weaker Protein A binding dsscFv is appended to the heavy chain and the strong Protein A binding dsscFv is appended to the light chain.
[0463] The TrYbe 04 and TrYbe 06 test supernatants were quantified by Protein A and Protein L HPLC (Table 6a). For TrYbe 06, the Protein A and Protein L assays gives equivalent results, whereas for TrYbe 04 the Protein A assay is lower than the Protein L assay.
[0464] TrYbe 06 has a strong Protein A binding dsscFv appended to the light chain (Table 7b), so the concentrations calculated for both the Protein L and Protein A assays are equivalent as both TrYbe and the light chain dimer can bind to both assays. TrYbe 04 has a weak Protein A binding dsscFv on the light chain (Table 6b), therefore all the TrYbe and only a proportion of the light chain dimer will bind to the Protein A assay. In contrast both TrYbe and light chain dimer bind fully to the Protein L assay. It is therefore not possible to fully quantify all the light chain dimer present in this test supernatant using the Protein A assay.
TABLE-US-00019 Quantification of test supernatants by Protein A and Protein L HPLC assay. Samples prepared by spiking light chain only supernatant into the respective antibody supernatants. Sample Name Chain Description of appended scFv Protein A mg/L Protein L mg/L TrYbe 04 Heavy dsscFv-1 129.6 219.1 Light dsscFv-3A TrYbe 06 Heavy dsscFv-3A 280.9 296.2 Light dsscFv-1
TABLE-US-00020 Strength of Light Chain binding to Protein A. Binding strengths have been categorised as strong (++), weak (+), none (-). Sample Name Chain Description of appended scFv Protein A Binding TrYbe 04 Heavy dsscFv-1 ++ Light dsscFv-3A + TrYbe 06 Heavy dsscFv-3A + Light dsscFv-1 ++
Protein A and Protein L Purification Steps Were Performed as Described Above. SDS PAGE and Densitometrical Analyses Were Also Performed As Described Above
Results
[0465] To evaluate the sequential Protein A and Protein L purifications, reduced (
[0466] TrYbe 04 has the weaker Protein A binding dsscFv appended to the light chain. On the reduced gel, in the Protein A eluate (lane 4B) there is a more intense light chain and less intense heavy chain. Densitometry showed there to be three times more light chain than heavy chain present. In the Protein L eluate (lane 4D) there are no bands. This indicates that the light chain dimer has co-purified with the TrYbe 04 during the Protein A purification. TrYbe 06 has a strong Protein A binding dsscFv appended to the light chain. In the reduced Protein A eluate (lane 6B) there is one band for both the heavy and light chain as in this example the bands co-migrate. There are no detectable bands in in the protein L eluate (lane 6D). As for TrYbe 06 this suggests that the light chain dimer has co-purified with the TrYbe during the Protein A purification.
[0467] For TrYbe 04, in the non-reduced Protein A eluate (lane 4B) the TrYbe and light chain dimer bands co-migrate to the same position as they are similar in size. There are also heavy and light chain bands due to incomplete interchain ds bond formation. There is more light chain due to the presence of light chain dimer and because the interchain disulphide bond formation between two C.sub.K is less efficient than for the CH1/C.sub.K pairing.
[0468] Like TrYbe 04, the Protein A eluate (lane 6B) for TrYbe 06, in the non-reduced gel, contains the TrYbe and light chain dimer however in this case there are two bands as they migrate slightly differently. There are also heavy and light chain bands but in contrast to the reduced gel they co-migrate so only one band is evident. As before, there are no bands in the Protein L eluate for either TrYbe 04 or TrYbe 06 (lane 4D, lane 6D) indicating the light chain dimer was co-purified with the TrYbe during the Protein A purifications.
[0469] In Summary, the presence of a Protein A binding dsscFv appended to the light chain resulted in co-purification of light chain dimer with TrYbe. This co-purification occured even when the light chain appended dsscFv was only a weak binder of Protein A. Therefore, the inventors showed the importance to completely abolish the ability of the antibody LC to bind protein A.
Example 7: Protein-A Interaction Assay
[0470] A new method has been developed to qualitatively test antibody fragments for Protein-A binding through an interaction assay.
[0471] The assay consists of four key stages: load, wash, elution, re-equilibration. A 100 .Math.l 2.1 x 30 mm POROS™ A 20 .Math.m Column (Thermo Fisher Scientific, Waltham, MA) was equilibrated in running buffer (PBS pH 7.4). 50 .Math.l of 1 mg/ml of test molecules or control molecules were loaded onto the column at 0.2 ml/min using an Agilent 1100 high-performance liquid chromatography (HPLC) system (Palo Alto, CA). Then, the column was washed slowly over 60 column volumes with a running buffer, such as PBS pH 7.4 for 30 minutes before applying an acidic step elution with 0.1 M Glycine-HCl pH 2.7 at 2.0 ml/min, for 2 minutes to remove any residual strong binders. Finally, the column was re-equilibrated in the running buffer (e.g. 50 CV PBS pH 7.4 at a flow-rate of 2.0 ml/min and a further 10 CV at 0.2 ml/min) in preparation for the next injection. Absorbance was read at 280 nm (A280).
Test Molecules
[0472] Test molecules must be monovalent and monomeric, in this case purified BYbe (Fab-dsscFv) molecules with a murine Fab (which does not bind protein A) and dsscFv test V-regions appended to the heavy chain (HC) were used. dsscFv-1, dsscFv-2, dsscFv-3A, dsscFv-3B correspond to the dsscFv molecules used in the previous examples. In addition, dsscFv-4 was used, which comprises VH and VL regions corresponding to those of the hFab-4 binding fragment known to be a strong binder.
Control Molecules
[0473] Control molecules have been used to ensure that the results were accurate. hFab-1 is a human Fab known to be a moderate binder. hFab-4 is a human Fab known to be a strong binder. mu Fab is a murine Fab and does not bind protein A. IgG bind Protein A strongly so an irrelevant IgG was used as control. Finally, human serum albumin (HSA) was used as a negative control.
Results
[0474] The retention times are presented in Table 7. In this Protein A Interaction assay, Protein A non-binders can be defined where the main peak elutes in the Flow Through and therefore has a retention time which is inferior to 0.9 minutes. Peak retention times for weak to strong Protein A binders will range from 1-30 minutes respectively. It can also be expected that for stronger binders the peak shape will broaden as the molecule tumbles down the column. Strong binders may remain bound until the acidic elution step, where a peak at 31 minutes can be observed.
[0475] IgG’s bind Protein A strongly and so the IgG control was only eluted from the column during the acidic step of the assay and so the main peak retention time was 31 minutes. In contrast, the HSA negative control flew straight through the column and thus the main peak has a retention time of 0.7 minutes. The mu Fab used in the Fab-dsscFv test molecules has a main peak retention time <0.9 minutes, therefore we were confident that binding of the test molecules to Protein A occurred only through the dsscFv appended to the heavy chain of the Fab.
[0476] For all Protein A binding V-regions the retention time of the main peak was > 1 minute. The dsscFv-3A was previously described as a weak Protein A binder and has the shortest retention time at only 1.8 minutes.
[0477] Other Protein A binding V-regions (dsscFv-1, dsscFv-4) had later retention times indicating they are stronger binders than dsscFv-3A.
[0478] For Protein A non-binding V-regions (dsscFv-2, dsscFv-3B, dsscFv-mul) the Fab-dsscFv flew straight through the column and the retention time of the main peak is <0.9 minutes.
TABLE-US-00021 Retention times obtained observed in the Protein A interaction assay Assay step: FT Wash Elution Binding Strength: None Weaker > > > > > > Stronger Strong Retention time: 0-1 min 1-30 min 31 min mu Fab, dsscFv-1 3.584 mu Fab, dsscFv-2 0.797 mu Fab, dsscFv-3A 1.818 mu Fab, dsscFv-3B 0.781 mu Fab, dsscFv-4 30.073 mu Fab, ds scFv-mu1 (negative control) 0.835 hFab-1 (moderate binder control) 5.342 hFab-4 (strong binder control) 30.107 mu Fab (non-binder control) 0.798 hu IgG (positive control) 31.275
Example 8: Biacore Assay
[0479] In order to confirm the ability of an antibody construct comprising either natural or engineered Variable regions to bind protein A, the binding can be measured by Surface Plasmon resonance (SPR), in particular using Biacore.
[0480] SPR is a commonly used technology for detailed and quantitative studies of protein-protein interactions. It is often used to determine their equilibrium and kinetic parameters (Hashimoto, 2000). A Biacore method has been established to quantitatively assess the binding of antibody test molecules (such as BYbes) to Protein A. A BIAcore™ T200 instrument (GE Healthcare) was used to carry out the SPR experiments.
[0481] Binding to two forms of native Protein A was assessed: a commercially sourced Protein A purified from S.aureus (Sigma Aldrich), and a recombinant purified form (prepared in-house). Each were immobilised by standard amine coupling chemistry to a CM5 sensor chip surface (GE Healthcare) to a level of approximately 400RU. After which the binding of the test molecules was assessed by titrating each over the chip surface using a 60 s injection at 30 .Math.l/min. HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05 % Polysorbate 20) used as both sample dilute and running buffer, Between each injection, the surface was regenerated using a 60 s injection (at 10 .Math.1/min) injection of 10 mM glycine pH 1.7. Each sample was titrated over a 10-point concentration series in 3-fold dilutions from the highest concentration achievable dependent on the stock concentration (90, 30 or 10 .Math.M) with a 0 nM blank injection was included for each sample to subtracted instrument noise and drift.
[0482] Mouse Fab samples fused to dsscFv sequences were selected as described in the previous example. In addition, the Mu Fab, dsscFv-mu1 was used as a negative control, comprising negative control mouse sequences with known absence of protein A binding.
Results
[0483] Tables 8a and 8b, and
[0484] Non-binding of a test molecule can be confirmed by demonstrating a lack of titratable binding response up to a concentration of 10 .Math.M, with a binding response (at 10 .Math.M) that is no greater than 2-fold higher than the response observed for the negative control at 10 .Math.M.
TABLE-US-00022 Binding of Fab-dsscFv Molecules to Commercial Purified Secreted Protein A Binding (RU) Concentration (M) Mu Fab, dsscFv-1 Mu Fab, dsscFv-2 Mu Fab, dsscFv-4 Mu Fab, dsscFv-3B Mu Fab, dsscFv-3A Mu Fab, dsscFv-mu1 (Negative Control) 9.00E-05 183.5 3.00E-05 128.2 8.6 6.4 90.4 8.5 1.00E-05 79.2 2.7 326.3 2.2 51.8 2.9 3.33E-06 39.2 0.9 257.2 0.7 28.3 0.8 1.11E-06 15.8 0.3 176.8 0.3 14.1 0.3 3.70E-07 5.4 0.1 100.6 0.1 6.0 0.0 1.23E-07 1.7 0.1 45.9 0.1 2.3 -0.1 4.12E-08 0.4 0.1 18.0 0.2 0.9 0.0 1.37E-08 0.2 0.1 6.5 0.2 0.2 -0.1 4.57E-09 0.1 0.1 2.3 0.1 0.0 0.1 1.52E-09 0.0 0.9 0.1 0.0 0.1 5.10E-10 0.5
TABLE-US-00023 Binding of Fab-dsscFv Molecules to Purified Recombinant Protein A Binding (RU) Concentration (M) Mu Fab, dsscFv-1 Mu Fab, dsscFv-2 Mu Fab, dsscFv-4 Mu Fab, dsscFv-3B Mu Fab, dsscFv-3A Mu Fab, dsscFv-mu1 (Negative Control) 9.00E-05 726.4 3.00E-05 449.7 6.8 4.8 343.5 6.0 1.00E-05 255.3 2.3 1524.4 1.9 205.3 2.2 3.33E-06 122.8 0.8 1112.0 0.6 116.8 0.7 1.11E-06 49.2 0.2 679.2 0.2 58.3 0.3 3.70E-07 17.6 0.0 343.7 0.0 24.9 0.1 1.23E-07 6.0 0.0 145.1 0.0 9.3 0.1 4.12E-08 1.9 0.0 54.6 0.0 3.2 0.1 1.37E-08 0.6 0.0 19.0 0.0 1.1 0.1 4.57E-09 0.3 -0.1 6.3 0.1 0.4 0.0 1.52E-09 0.0 2.0 0.0 0.1 0.0 5.10E-10 0.6