New Polypeptides
20170260238 · 2017-09-14
Assignee
Inventors
- Lars Abrahmsén (Stockholm, SE)
- Caroline Ekblad (Saltsjö-Boo, SE)
- Elin Gunneriusson (Saltsjöbaden, SE)
- Torbjörn Gräslund (Hägersten, SE)
- Johan Seijsing (Stockholm, SE)
- John Löfblom (Huddinge, SE)
- Malin Lindborg (Saltsjö-boo, SE)
- Fredrik Frejd (Stockholm, SE)
- Lindvi Gudmundsdotter (Stockholm, SE)
Cpc classification
A61P1/04
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
A61P7/00
HUMAN NECESSITIES
C07K2319/30
CHEMISTRY; METALLURGY
C07K14/00
CHEMISTRY; METALLURGY
C07K14/70535
CHEMISTRY; METALLURGY
C07K2319/74
CHEMISTRY; METALLURGY
International classification
Abstract
The present disclosure relates to dimers of engineered polypeptides having a binding affinity for the neonatal Fc receptor FcRn, and provides an FcRn binding dimer, comprising a first monomer unit, a second monomer unit and an amino acid linker, wherein said first and second monomer unit search comprises an FcRn binding motif. Said FcRn binding dimer binds FcRn with higher capacity compared to said first monomer unit or second monomer unit alone. The present disclosure also relates to the use of said FcRn binding dimer as an agent for modifying pharmacokinetic and pharmacodynamic properties and as a therapeutic agent.
Claims
1. A neonatal Fc receptor (FcRn) binding dimer, comprising a first monomer unit, a second monomer unit and an amino acid linker, wherein said first and second monomer unit each comprises an FcRn binding motif (BM), which motif consists of the amino acid sequence EX.sub.2 X.sub.3 X.sub.4 AX.sub.6 X.sub.7 EIR WLPNLX.sub.16X.sub.17 X.sub.18 QR X.sub.21 AFIX.sub.25 X.sub.26LX.sub.28 X.sub.29 wherein, independently from each other, X.sub.2 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y; X.sub.3 is selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W and Y; X.sub.4 is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W and Y; X.sub.6 is selected from A, E, F, G, H, I, K, Q, R, S and V; X.sub.7 is selected from A, F, H, K, N, Q, R, S and V; X.sub.16 is selected from N and T; X.sub.17 is selected from F, W and Y; X.sub.18 is selected from A, D, E and N; X.sub.21 is selected from A, S, V and W; X.sub.25 is selected from D, E, G, H, I, K, L, N, Q, R, S, T, V, W and Y; X.sub.26 is selected from K and S; X.sub.28 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y; and X.sub.29 is selected from D and R, and wherein said FcRn binding dimer binds FcRn with a higher binding capacity compared to said first monomer or said second monomer alone.
2. The FcRn binding dimer according to claim 1, wherein the BM of at least one of said first and second monomer units consists of an amino acid sequence selected from i) EX.sub.2 X.sub.3 X.sub.4 AX.sub.6 HEIR WLPNLTX.sub.17 X.sub.18 QR X.sub.21 AFIX.sub.25 KLX.sub.28 D wherein, independently from each other, X.sub.2 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y; X.sub.3 is selected from A, D, E, G, H, K, L, M, N, Q, R, S, T, V and Y; X.sub.4 is selected from A, D, E, F, G, I, K, L, N, Q, R, S, T, V and Y; X.sub.6 is selected from A, G, K, R, S and V; X.sub.17 is selected from F, W and Y; X.sub.18 is selected from A, D, E and N; X.sub.21 is selected from A, S, V and W; X.sub.25 is selected from D, G, H, K, L, N, R, V and W; X.sub.28 is selected from A, D, E, H, K, L, N, Q, R, S, T, W and Y; and ii) an amino acid sequence which has at least 96% identity to a sequence defined by i).
3. The FcRn binding dimer according to claim 1, wherein said first and second monomer units comprise identical BM sequences.
4. The FcRn binding dimer according to claim 1, wherein said first and second monomer units comprise different BM sequences.
5. The FcRn binding dimer according to claim 1, wherein at least one of said first and second monomer units comprises an FcRn binding motif BM corresponding to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO: 1-353.
6. The FcRn binding dimer according to claim 5, wherein at least one of said first and second monomer units comprises an FcRn binding motif BM corresponding to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:75 and SEQ ID NO:77.
7. The FcRn binding dimer according to claim 1, wherein at least one of said first and second monomer units comprises a sequence selected from the group consisting of: xi) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; xv) AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK; xvii) VDAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK; wherein [BM] is an FcRn binding motif as defined in claim 1; and an amino acid sequence which has at least 94% identity to the sequence defined in xi), xv) or xvii).
8. The FcRn binding dimer according to claim 7, wherein at least one of said first and second monomer units comprises a sequence xi) selected from the group consisting of SEQ ID NO:354-357 and SEQ ID NO:360-367.
9. The FcRn binding dimer according to claim 1, wherein at least one of said first and second monomer units comprises a sequence selected from the group consisting of: xxi) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is an FcRn binding motif as defined in claim 1; and xxii) an amino acid sequence which has at least 94% identity to the sequence defined in xxi).
10. The FcRn binding dimer according to claim 9, wherein at least one of said first and second monomer units comprises a sequence xxi) selected from the group consisting of SEQ ID NO:1-353.
11. The FcRn binding dimer according to claim 10, wherein at least one of said first and second monomer units comprises a sequence xxi) selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:75 and SEQ ID NO:77.
12. The FcRn binding dimer according to claim 8, wherein said first and second monomer units comprise SEQ ID NO:365 and SEQ ID NO:365; SEQ ID NO:366 and SEQ ID NO:366; or SEQ ID NO:367 and SEQ ID NO:367, respectively.
13. The FcRn binding dimer according to claim 1, wherein said linker has a general formula selected from
(G.sub.nS.sub.m).sub.p and (S.sub.nG.sub.m).sub.p, wherein, independently, n=1-7, m=0-7, n+m≦8 and p=1-7.
14. The FcRn binding dimer according to claim 1, which is capable of binding to FcRn with at least 2 times higher capacity than the corresponding first monomer unit or second monomer unit alone.
15. A fusion protein or conjugate comprising a first moiety consisting of the FcRn binding dimer according to claim 1; and a second moiety consisting of a polypeptide having a desired biological activity.
16. The FcRn binding dimer according to claim 1, which inhibits binding of IgG to FcRn.
17. The FcRn binding dimer according to claim 1, which binds FcRn such that the ability of the FcRn binding dimer to block IgG binding to FcRn is at least 2 times higher compared to the blocking ability of the corresponding first or second monomer unit alone.
18. A composition comprising the FcRn binding dimer according to claim 1 and at least one pharmaceutically acceptable excipient or carrier.
19. A method of blocking IgG binding to FcRn, the method comprising contacting the FcRn binding dimer according to claim 1 with FcRn under conditions such that the FcRn binding dimer binds to FcRn.
20. A method of treatment, comprising administering the FcRn binding dimer according to claim 1 to a subject in need of treatment of a condition selected from the group consisting of auto-immune conditions, allo-immune conditions, epilepsy and seizures.
21. The method of treatment according to claim 20, wherein the FcRn binding dimer is administered in the form of a composition comprising the FcRn binding dimer and at least one pharmaceutically acceptable excipient or carrier.
22. The fusion protein or conjugate according to claim 15, which inhibits binding of IgG to FcRn.
23. The fusion protein or conjugate according to claim 15 which binds FcRn such that the ability of the FcRn binding dimer to block IgG binding to FcRn is at least 2 times higher compared to the blocking ability of the corresponding first or second monomer unit alone.
24. A composition comprising the fusion protein or conjugate according to claim 15 and at least one pharmaceutically acceptable excipient or carrier.
25. A method of blocking IgG binding to FcRn, the method comprising contacting the fusion protein or conjugate according to claim 15 with FcRn under conditions such that the FcRn binding dimer binds to FcRn.
26. A method of treatment, comprising administering the fusion protein or conjugate according to claim 15 to a subject in need of treatment of a condition selected from the group consisting of auto-immune conditions, allo-immune conditions, epilepsy and seizures.
27. The method of treatment according to claim 26, wherein the fusion protein or conjugate is administered in the form of a composition comprising the fusion protein or conjugate and at least one pharmaceutically acceptable excipient or carrier.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0350]
[0351]
[0352]
[0353]
[0354]
[0355]
[0356]
[0357]
[0358]
[0359]
[0360]
[0361]
[0362]
[0363]
[0364]
[0365]
[0366]
[0367]
EXAMPLES
Summary
[0368] The following Examples disclose the development of novel Z variant molecules targeting the neonatal Fc receptor (FcRn). The Z variants were obtained using phage display technology. The genes encoding FcRn binding polypeptides described herein were sequenced, and the corresponding amino acid sequences are listed in
Example 1
Production of Human αFcRn and Human β2-Microglobulin (B2M)
[0369] In this Example, the extracellular domain (ECD) of human αFcRn (SEQ ID NO:379) in complex with human β2-microglobulin (SEQ ID NO:380) (complex denoted FcRn) and human β2-microglobulin in non-complexed form (denoted B2M) were produced as soluble proteins. Human FcRn and B2M produced in this Example were used for phage selection, ELISA and Biacore assays in Examples 2 and 3.
Materials and Methods
[0370] Construction of Plasmids Containing the Genes for Human αFcRn and Human β2-Microglobulin to be Used for Co-Expression:
[0371] The genes encoding human αFcRn (Genbank BC008734.2) and human β2-microglobulin (B2M) (Genbank BC032589.1) were obtained from OpenBiosystems. Using PCR overlap extension, a gene fragment encoding amino acids 24-290 of human αFcRn (αFcRn.sub.ECD) (SEQ ID NO:379) was amplified to a construct consisting of attB1-site/Kozak sequence followed by a gene encoding: an Ig kappa chain leader sequence, hFcRn.sub.ECD, a GS-linker and a flag tag, followed by an attB2 site. A similar construct was made containing a gene fragment encoding amino acids 21-119 of human B2M (SEQ ID NO:380), except that a His.sub.6 tag replaced the flag tag. The constructs were inserted into the plasmid pDONOR221 (Invitrogen, cat. no. 12536-017) by recombination using the Gateway system (Invitrogen, cat. no. 11789020, Gateway® BP Clonase® II Enzyme mix), according to the manufacturer's recommendations. After verification of correct sequences, the human αFcRn.sub.ECD construct was inserted into 2K7.sub.bsd (Suter et al. (2006) Stem Cells 24:615-623) using multi-site gateway cloning together with the promoter-containing plasmid pENTR-CMV (Tai et al. (2012) PLoS One 7(9):e46269), resulting in the vector 2K7.sub.bsd-CMV-hFcRn.sub.ECD. The human B2M gene construct was similarly inserted into 2K7.sub.neo (Suter et al., supra), giving the vector 2K7.sub.neo-CMV-hB2M.
[0372] Cell Culture, Preparation of Recombinant Lentiviral Vectors and Gene Insertions into SKOV-3 Cell Line:
[0373] The HEK293T and SKOV-3 cell lines were obtained from ATCC. Cells were grown at 37° C. in a humidified incubator in the presence of 5% CO.sub.2. Complete medium for the HEK293T cell line was Dulbeccos modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% Antibiotic Antimycotic Solution (AA) and 1% MEM Non-essential Amino Acid Solution (NEAA). Complete medium for the SKOV-3 cell line was McCoy's 5A medium supplemented with 10% FBS and 1% AA.
[0374] The plasmids 2K7.sub.bsd-CMV-hFcRn.sub.ECD and 2K7.sub.neo-CMV-hB2M were separately co-transfected together with VSV-G envelope and gag/pol packaging plasmid into HEK293T cells using calcium chloride transfection (Zufferey et al. (1997) Nat Biotechnol 15(9):871-5; Jakobsson et al. (2006) J Neurosci Res 84:58-67). HEK293 culture supernatants containing formed lentiviral particles with human αFcRn.sub.ECD and human B2M transgenes, respectively, were cleared from cell debris by centrifugation and filtration. The two types of lentiviral particles were used to sequentially transduce SKOV-3 cells. Successful double integrants containing both the human αFcRn.sub.ECD and the B2M genes were selected for by the addition of blasticidin (Invitrogen) and G418 sulfate (Invitrogen) to culture medium while passaging the cells for two weeks. The resulting, stably transduced SKOV-3 cell line was denoted SKOV-3 hFcRn.sub.ECD/hB2M.
[0375] Expression of Recombinant Human FcRn:
[0376] SKOV-3 cells, co-expressing human αFcRn.sub.ECD and B2M resulting in human FcRn, were expanded and 1.5×10.sup.7 cells were seeded in a HYPERFlask (Corning) in 560 ml complete growth medium. After five days, when the cells had settled and multiplied, the medium was changed to complete growth medium without FBS. After five days, the culture was terminated and the supernatant was collected, passed through a 45 μm filter and frozen at −80° C.
[0377] Purification of Recombinant Human FcRn Using Human IgG Chromatography:
[0378] Protein purification was carried out in an ÄKTA Explorer system (GE Healthcare). Human IgG (Pharmacia), 1 ml in 0.2 M NaHCO.sub.3, 0.5 M NaCl pH 8.3 at a concentration of 10 mg/ml, was coupled to a 1 ml HiTrap NHS-activated HP column (GE Healthcare) according to the manufacturer's instruction. The supernatant containing recombinant human FcRn from SKOV-3 cells was thawed and the pH was adjusted to 5.8 with HCl. The supernatant was subsequently loaded in batches of 100 ml onto the column previously equilibrated with 20 mM Bis-Tris pH 5.8. The column was washed with 20 ml of 20 mM Bis-Tris pH 5.8 and eluted in fractions of 1 ml using 50 mM Tris, pH 8.1. Buffer exchange to PBS (phosphate buffered saline, 10 mM phosphate, 137 mM NaCl, 2.68 mM KCl, pH 7.4) was performed using dialysis.
[0379] SDS-PAGE and Western Blot:
[0380] The purity of the eluted fractions from the protein purification was analyzed by SDS-PAGE and staining with GelCode Blue Stain Reagent (Pierce) and SilverXpress® Silver Staining Kit (Invitrogen). Western blotting was carried out using an Amersham Hybond™-C Extra nitrocellulose membrane (GE Healthcare). The membrane was blocked with 5% non-fat dry milk (Semper) in TBS+T (50 mM Trizma base, 150 mM NaCl, 0.05% Tween-20, pH 8) for 1 hour, then probed with a mixture of rabbit anti-FCGRT polyclonal antibody (Atlas Antibodies) at a concentration of 0.15 μg/ml and rabbit anti-B2M polyclonal antibody (Atlas Antibodies) at a concentration of 0.23 μg/ml in TBS+T. The membrane was subsequently incubated with stabilized goat anti-rabbit antibody conjugated with horse radish peroxidase (Pierce) diluted 1:10,000 in TBS+T. After addition of TMB Substrate (Pierce), an image of the membrane was acquired on Amersham Hyperfilm ECL (GE Healthcare). The Hyperfilm was processed using GBX developer and GBX fixer (Sigma-Aldrich).
[0381] Production of a Non-Complexed Form of Human B2M:
[0382] Human B2M was produced in E. coli. The expression and purification was performed essentially as described in Sandalova et al. (2005) Acta Chryst F61:1090-1093 and Michaëlsson et al. (2001) J Immunol 166:7327-7334. The purified protein, consisting of amino acids 21-119 of human B2M, in urea was subjected to arginine refolding as follows; 0.5 mg of B2M was rapidly added to 2 ml refolding buffer (20 ml 1 M Tris-HCl pH 8.0, 16.87 g L-Arginine (buffered with HCl), 0.8 ml 0.5 M EDTA, 61 mg GSSG, 307 mg GSH and milli-Q water to a final volume of 200 ml, pH 8.0, and supplemented with protease inhibitor (Roche, cat. no. 11 873 580 001)). The refolding procedure was performed at 4° C. during 4 hours. Refolded B2M protein was buffer exchanged to PBS using a PD-10 column (GE Healthcare).
Results
[0383] Construction of Plasmids Containing the Genes for Human αFcRn and Human β2-Microglobulin to be Used for Co-Expression:
[0384] Genes encoding the extracellular domain of the α-chain of human FcRn (αFcRn.sub.ECD) and human B2M were inserted into the lentiviral transfer plasmids 2K7.sub.bsd and 2K7.sub.neo, respectively. In both cases, the inserted gene is under the control of a CMV promoter. The genes were extended so that the resulting proteins would have an Ig kappa chain leader sequence in the N-terminus to target the protein for export through the endoplasmic reticulum to the culture medium (the signal sequence was cleaved upon secretion). In addition, αFcRn.sub.ECD had a C-terminal spacer sequence followed by a FLAG-tag for potential detection. Human B2M had a C-terminal spacer sequence followed by a His.sub.6 tag for potential detection. The spacer sequence was added to enhance accessibility of the tag. The lentiviral transfer plasmids also contained two different antibiotic resistance genes to allow selection of cells where both constructs had been inserted.
[0385] Expression and Purification of Recombinant Human FcRn:
[0386] The genes encoding αFcRn.sub.ECD and B2M were inserted into the genome of SKOV-3 by lentiviruses, and the resulting FcRn protein was secreted into the culture medium. To capture only FcRn having retained pH-dependent IgG binding, affinity chromatography using immobilized IgG was used where the receptor was captured at pH 5.8 and eluted at pH 8.1. Captured protein was eluted in three fractions.
[0387] SDS-PAGE and Western Blot:
[0388] To investigate the presence of two peptide chains (αFcRn.sub.ECD and B2M) of the produced FcRn protein, and to analyze the purity of the eluted material, an SDS-PAGE analysis was performed on the eluted fractions. For the gel stained with GelCode Blue Stain, two bands were detected with molecular weights of 12 and 36 kDa, respectively. This corresponds approximately to the theoretical molecular weights of the non-glycosylated peptide chains of 12 kDa for B2M and 31 kDa for αFcRn.sub.ECD. The αFcRn.sub.ECD part of the protein contains one glycosylation site and it was therefore expected that its molecular mass would be higher than 31 kDa. The gel was also silver stained to increase sensitivity and possibly detect impurities. A band of approximately 66 kDa was detected in the first eluted fraction, which could correspond to BSA (bovine serum albumin) originating from cell attachment. The total amount of protein recovered in fraction 2 and 3 corresponded to 1.4 mg/l culture medium. A western blot analysis on the pooled material was carried out, which showed essentially only the two major bands and in addition a very weak band below 12 kDa which might correspond to a degradation product.
Example 2
Selection and ELISA Binding of FcRn Binding Z Variants
[0389] In this Example, human FcRn was used as target in phage display selections using a phage library of Z variants. Selected clones were DNA sequenced, produced in E. coli periplasmic fractions and assayed against FcRn in ELISA (enzyme-linked immunosorbent assay).
Materials and Methods
[0390] Biotinylation of Target Protein FcRn and of B2M:
[0391] Human FcRn and human B2M, produced as described in Example 1, were biotinylated using No-Weigh EZ-Link Sulfo-NHS-LC-Biotin (Pierce, cat. no. 21327) at a 31×(FcRn) and 10×(B2M) molar excess, respectively, according to the manufacturer's recommendations. The reactions were performed at room temperature (RT) for 30 min. Subsequent buffer exchange to PBS was performed using Slide-a-lyzer dialysis cassettes (FcRn; Pierce, cat. no. 66380, 10,000 MWCO and B2M; Pierce, cat. no. 66333, 3,500 MWCO), according to the manufacturer's instructions.
[0392] Phage Display Selection of FcRn Binding Z Variants:
[0393] A library of random variants of protein Z displayed on bacteriophage, constructed in phagemid pAY02592 essentially as described in Grönwall et al. (2007) J Biotechnol, 128:162-183, was used to select FcRn binding Z variants. In this library, an albumin binding domain (ABD, GA3 of protein G from Streptococcus strain G148) is used as fusion partner to the Z variants. The library is denoted Zlib006Naive.II and has a size of 1.5×10.sup.10 library members (Z variants). E. coli RRIΔM15 cells (Rüther et al., (1982) Nucleic Acids Res 10:5765-5772) from a glycerol stock containing the phagemid library Zlib006Naive.II, were inoculated in 20 l of a defined proline free medium [dipotassium hydrogenphosphate 7 g/l, trisodium citrate dihydrate 1 g/l, uracil 0.02 g/l, YNB (Difco™ Yeast Nitrogen Base w/o amino acids, Becton Dickinson) 6.7 g/l, glucose monohydrate 5.5 g/l, L-alanine 0.3 g/l, L-arginine monohydrochloride 0.24 g/l, L-asparagine monohydrate 0.11 g/l, L-cysteine 0.1 g/l, L-glutamic acid 0.3 g/l, L-glutamine 0.1 g/l, glycine 0.2 g/l, L-histidine 0.05 g/l, L-isoleucine 0.1 g/l, L-leucine 0.1 g/l, L-lysine monohydrochloride 0.25 g/l, L-methionine 0.1 g/l, L-phenylalanine 0.2 g/l, L-serine 0.3 g/l, L-threonine 0.2 g/l, L-tryptophane 0.1 g/l, L-tyrosine 0.05 g/l, L-valine 0.1 g/l], supplemented with 100 μg/ml ampicillin. The cultivations were grown at 37° C. in a fermenter (Belach Bioteknik, BR20). When the cells reached an optical density at 600 nm (OD.sub.600) of 0.75, approximately 2.6 l of the cultivation was infected using a 10× molar excess of M13K07 helper phage (New England Biolabs, cat. no. N0315S). The cells were incubated for 30 minutes, whereupon the fermenter was filled up to 20 l with TSB-YE (Tryptic Soy Broth-Yeast Extract; 30 g/l TSB, 5 g/l yeast extract) supplemented with 100 μM isopropyl-β-D-1-thiogalactopyranoside (IPTG) for induction of expression and with 25 μg/ml kanamycin and 12.5 μg/ml carbenicillin and grown at 30° C. for 22 h. The cells in the cultivation were pelleted by centrifugation at 15,900 g. The phage particles were precipitated from the supernatant twice in PEG/NaCl (polyethylene glycol/sodium chloride), filtered and dissolved in PBS and glycerol as described in Grönwall et al., supra. Phage stocks were stored at −80° C. before use.
[0394] Selections against biotinylated human FcRn were performed in four cycles divided in two different tracks. Phage stock preparation and selection procedure were performed essentially as described for selection against another biotinylated target in WO2009/077175. The amplification of phage between the selection cycles was performed by infecting E. coli RRIΔM15 with phage, then performing cultivation in solution as follows. Eluted phage and 10×excess of M13K07 helper phage compared to bacteria were allowed to simultaneously infect log phase bacteria at 37° C. for 30 min without rotation, followed by 30 min with slow rotation. Prior to infection, bacteria were grown to log phase in the defined proline free medium described above. Infected bacteria were pelleted by centrifugation at 4,300 g for 10 min and resuspended in 200 ml TSB+YE medium supplemented with 0.1 mM IPTG, 25 μg/ml kanamycin and 100 μg/ml ampicillin and cultivated at 30° C. overnight for phage production.
[0395] The selection buffer consisted of 100 mM sodium phosphate and 150 mM sodium chloride adjusted to pH 5.5 with hydrogen chloride and supplemented with 0.1% gelatin and 0.1% Tween-20. At selection, human serum albumin (HSA, Albucult, Novozymes) was added to the selection buffer to a final concentration of 1.5 μM. In order to reduce the amount of background binders, pre-selection was performed by incubation of phage stock with Dynabeads® M-280 Streptavidin (SA-beads, Dynal, cat. no. 112.06) for 1 hour at RT. A second pre-selection was performed during 30 min at RT against human B2M immobilized in immunotubes (Nunc, cat. no. 444474). 5 μg/ml of human B2M in carbonate buffer (Sigma, cat. no. 068K8214) was immobilized in the tube at 7° C. for >1 h. After washing twice with tap water, the tubes were blocked with PBS+0.5% casein (Sigma, cat. no. C8654) for 30 min at RT before use. All tubes and beads used in the selection were pre-blocked with PBS+0.1% gelatin. Selection was performed in solution at RT, followed by capture of target-phage complexes on SA-beads where 1 mg beads per 2.9 μg biotinylated FcRn were used. In cycle 1 of the selections, 100 nM biotinylated FcRn was used and two washes of two min each were performed using selection buffer. An increased stringency, using a lowered target concentration and an increased number of washes, was applied in the subsequent cycles: 50 nM/5 washes, 25 nM/8 washes and 10 nM/12 washes were applied in cycle 2, 3 and 4, respectively. After the washes, bound phage was eluted from the two selection tracks using two different procedures; 1) 500 μl 0.1 M glycine-HCl, pH 2.2, followed by immediate neutralization with 50 μl 1 M Tris-HCl, pH 8.0, and 450 μl PBS, or; 2) 500 μl of 100 mM sodium phosphate and 150 mM sodium chloride, pH 8.0 and neutralization with 500 μl PBS.
[0396] Sequencing:
[0397] PCR fragments were amplified from single colonies using a standard PCR program and the primers AFFI-21 (5′-tgcttccggctcgtatgttgtgtg (SEQ ID NO:385)) and AFFI-22 (5′-cggaaccagagccaccaccgg (SEQ ID NO:386)). Sequencing of amplified fragments was performed using the biotinylated oligonucleotide AFFI-72 (5′-biotin-cggaaccagagccaccaccgg (SEQ ID NO:387)) and a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), used in accordance with the manufacturer's protocol. The sequencing reactions were purified by binding to magnetic streptavidin coated beads (Detach Streptavidin Beads, Nordiag, cat. no. 2012-01) using a Magnatrix 8000 (Magnetic Biosolution), and analyzed on ABI PRISM® 3130xl Genetic Analyzer (PE Applied Biosystems).
[0398] Production of Z Variants for ELISA:
[0399] Sequenced Z variants were produced by inoculating single colonies from the selections into 10 ml TSB-YE medium supplemented with 100 μg/ml ampicillin and 0.1 mM IPTG and incubating for 24 h at 37° C. Cells were pelleted by centrifugation, re-suspended in 2 ml PBST (PBS supplemented with 0.05% Tween-20), frozen at −80° C. and thawed in a water bath, to release the periplasmic fraction of the cells. The freeze-thawing procedure was repeated seven times and cells were then pelleted by centrifugation. The supernatant of the periplasmic extract contained the Z variants as fusions to ABD, expressed as AQHDEALE-[Z#####]-VDYV-[ABD]-YVPG (Grönwall et al., supra). Z##### refers to individual, 58 amino acid residue Z variants.
[0400] ELISA K.sub.D Analysis of Z Variants:
[0401] The binding of Z variants to FcRn was analyzed in ELISA assays. Half-area 96-well ELISA plates were coated with 2 μg/ml of an anti-ABD goat antibody (produced in-house) diluted in coating buffer (50 mM sodium carbonate, pH 9.6) at 4° C. overnight. The antibody solution was poured off and the wells were blocked with 100 μl of PBSC (PBS supplemented with 0.5% casein) for 1.5 h at RT. The blocking solution was discarded and 50 μl periplasmic solution, diluted 1:4, was added to the wells and incubated for 1.5 h at RT under slow shaking. The solutions were poured off and the wells were washed four times with either 0.05% PCT buffer, pH 6.0 (McIlvaines phosphate-citrate buffer, pH 6.0, supplemented with 0.05% Tween-20) or 0.05% PCT buffer, pH 7.4 (McIlvaines phosphate-citrate buffer, pH 7.4, supplemented with 0.05% Tween-20). The target protein, biotinylated human FcRn, was added to the wells in a 1:3 diluted concentration series from 2 μg/ml (45 nM) to 0.3 ng/ml (6.9 μM) diluted in PCC buffer, pH 6.0 or pH 7.4, (McIlvaines phosphate-citrate buffer, pH 6.0 or pH 7.4, supplemented with 0.5% casein), respectively. The plates were incubated for 1.5 h at RT followed by washes as described above. Streptavidin conjugated HRP (Thermo Scientific, cat. no. N100) was diluted 1:30 000 in PCC buffer, pH 6.0 or pH 7.4, respectively, and added to the wells followed by 45 min incubation.
[0402] After washing as described above, 50 μl ImmunoPure TMB substrate (Thermo Scientific, cat. no. 34021) was added to the wells and the plates were treated according to the manufacturer's recommendations. Absorbance was measured at 450 nm using a multi-well plate reader, Victor.sup.3 (Perkin Elmer). A Z variant binding an irrelevant protein was used as negative control and a blank was created by omitting the periplasmic step. A Z variant which bound to FcRn in a pre-experiment (Z07918, SEQ ID NO:1) was used as positive control. Measured values were analyzed using GraphPad Prism 5 (GraphPad Software, Inc.) and non-linear regression in order to determine the affinities (K.sub.D) of the interactions.
[0403] ELISA Specificity Analysis of Z Variants:
[0404] In another ELISA experiment, the specificities of the Z variants were tested by assaying them against 2 μg/ml biotinylated human proteins B2M, PSMA (produced in-house) and IgG (polyclonal, Pharmacia) and against PCC buffer pH 6.0 or pH 7.4, respectively. The assay was performed at pH 6.0 and at pH 7.4, respectively, as described above. The biotinylated proteins or buffer were added to the wells instead of FcRn in the target protein step.
Results
[0405] Phage Display Selection of FcRn Binding Z Variants:
[0406] Individual clones were obtained after four cycles of phage display selections against biotinylated human FcRn.
[0407] Sequencing:
[0408] Sequencing was performed on clones picked at random from selection round four. Each Z variant was given a unique identification number ##### and individual variants are referred to as Z#####. The amino acid sequences of the 58 amino acid residues long Z variants are listed in
[0409] The deduced FcRn binding motifs of these Z variants extend from residue 8 to residue 36 in sequences with SEQ ID NO:1-16 and SEQ ID NO:353 in
[0410] ELISA Assays with Z Variants:
[0411] Sixteen clones were produced as ABD fusion proteins in E. coli. The periplasmic fractions were used in an ELISA against a dilution series of human FcRn. The clones were: Z07909 (SEQ ID NO:13), Z07918 (SEQ ID NO:1), Z07930 (SEQ ID NO:6), Z07960 (SEQ ID NO:4), Z10109 (SEQ ID NO:3), Z10111 (SEQ ID NO:8), Z10127 (SEQ ID NO:12), Z10129 (SEQ ID NO:9), Z10140 (SEQ ID NO:5), Z10141 (SEQ ID NO:10), Z10145 (SEQ ID NO:15), Z10152 (SEQ ID NO:14), Z10156 (SEQ ID NO:11), Z10161 (SEQ ID NO:16), Z10183 (SEQ ID NO:7) and Z10193 (SEQ ID NO:2). K.sub.D values were determined for all variants at pH 6.0 and for three variants at pH 7.4 (Table 2). For thirteen variants, data was not obtained for a K.sub.D analysis at pH 7.4. None of the sixteen variants displayed non-specific binding when assayed against human B2M, IgG or PSMA.
TABLE-US-00002 TABLE 2 ELISA KD analysis of Z-ABD variants in E. coli periplasmic fractions. Z variant SEQ ID NO: KD pH 6.0 (M) KD pH 7.4 (M) Z07909 13 24.5 × 10.sup.−9 n.d. Z07918 1 2.0 × 10.sup.−9 10.9 × 10.sup.−9 Z07930 6 10.4 × 10.sup.−9 n.d. Z07960 4 6.0 × 10.sup.−9 n.d. Z10109 3 3.9 × 10.sup.−9 23.9 × 10.sup.−9 Z10111 8 11.4 × 10.sup.−9 n.d. Z10127 12 21.3 × 10.sup.−9 n.d. Z10129 9 17.6 × 10.sup.−9 n.d. Z10140 5 8.8 × 10.sup.−9 n.d. Z10141 10 21.2 × 10.sup.−9 n.d. Z10145 15 42.0 × 10.sup.−9 n.d. Z10152 14 24.6 × 10.sup.−9 n.d. Z10156 11 21.3 × 10.sup.−9 n.d. Z10161 16 163.0 × 10.sup.−9 n.d. Z10183 7 10.9 × 10.sup.−9 n.d. Z10193 2 2.3 × 10.sup.−9 25.9 × 10.sup.−9 n.d. = not determinable
Example 3
Production and Characterization of FcRn Binding Z Variants
[0412] In this Example, seventeen Z variants were produced in E. coli, purified and assayed against human FcRn in Biacore. A subset of said variants was also assayed against mouse FcRn. Circular dichroism (CD) spectroscopy was performed for a subset of Z variants for investigation of their secondary structure.
Materials and Methods
[0413] Subcloning of Z Variants:
[0414] The DNA of seventeen FcRn binding Z variants (SEQ ID NO:1-16 and SEQ ID NO:353) was amplified from the library vector pAY02592. A subcloning strategy for construction of monomeric Z variant molecules with N-terminal His.sub.6 tag was applied using standard molecular biology techniques (essentially as described in detail in WO2009/077175 for Z variants binding another target). The Z gene fragments were subcloned into the expression vector pAY01448 resulting in the encoded sequence MGSSHHHHHHLQ-[Z#####]-VD.
[0415] In addition, the FcRn binding variant Z07918 (SEQ ID NO:1), but starting with the amino acids AE instead of VD and denoted Z11948 (SEQ ID NO:354), was cloned as homodimeric constructs with two different linkers between the Z variants and followed by a C-terminal His.sub.6 tag. This was performed using conventional molecular biology methods including DNA amplification, restriction with suitable restriction enzymes and ligation of the DNA. The two linkers were obtained from Thermo Fisher Scientific. The Z gene fragments were subcloned into the expression vector (pET-26 origin, Novagen) resulting in the encoded sequence [Z#####]-GT-(G.sub.4S)-PR-[Z#####]-LEHHHHHH and [Z#####]-GT-(G.sub.4S).sub.3-[Z#####]-LEHHHHHH, respectively.
[0416] Cultivation and Purification:
[0417] E. coli BL21(DE3) cells (Novagen) were transformed with plasmids containing the gene fragment of each respective FcRn binding Z variant and cultivated at 37° C. in 800 or 1000 ml of TSB-YE medium supplemented with 50 μg/ml kanamycin. At OD.sub.600=2, IPTG was added to induce expression at a final concentration of 0.17 or 0.2 mM and the culture was incubated at 37° C. for another 5 h. The cells were harvested by centrifugation.
[0418] Approximately 2-5 g of each cell pellet was resuspended in 10-25 ml binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4) supplemented with Benzonase® (Merck, cat. no. 1.01654.0001) to a concentration of 15 U/ml and Lysozyme (Sigma, cat. no. L-7651) to a concentration of 0.5 mg/ml. After cell disruption by three freeze-thawing cycles or sonication, cell debris was removed by centrifugation and each supernatant was applied on a 1 ml His GraviTrap IMAC column (GE Healthcare, cat. no. 11-0033-99). Contaminants were removed by washing with wash buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 or 60 mM imidazole, pH 7.4), and the FcRn binding Z variants were subsequently eluted with elution buffer 1 (20 mM sodium phosphate, 0.5 M sodium chloride, 250 mM imidazole, pH 7.4) or elution buffer 2 (0.1 M acetic acid, 0.5 M sodium chloride, pH 4.5). Purified Z variants were buffer exchanged to PBS using PD-columns (GE Healthcare), according to the manufacturer's protocol. Protein concentrations were determined by measuring the absorbance at 280 nm, using a NanoDrop® ND-1000 spectrophotometer, and using the extinction coefficient of the respective protein. The purity of the FcRn binding Z variants was analyzed by SDS-PAGE stained with Coomassie Blue. The identity of each purified FcRn binding Z variant was confirmed using LC/MS analysis.
[0419] CD Analysis:
[0420] Purified His.sub.6-tagged Z variants were diluted to 0.5 mg/ml in PBS. For each diluted Z variant, a CD spectrum at 250-195 nm or 250-190 nm was obtained at 20° C. In addition, a variable temperature measurement (VTM) was performed to determine the melting temperature (Tm). In the VTM, the absorbance was measured at 221 nm while the temperature was raised from 20 to 90° C., with a temperature slope of 5° C./min. A new CD spectrum was obtained at 20° C. after the heating procedure in order to study the refolding ability of the Z variants. The CD measurements were performed on a Jasco J-810 spectropolarimeter (Jasco Scandinavia AB) using a cell with an optical path-length of 1 mm.
[0421] Biacore Binding and Kinetic Analysis:
[0422] The interaction of FcRn binding His.sub.6-tagged Z variants with human FcRn was analyzed in a Biacore 2000 instrument (GE Healthcare). Human FcRn was immobilized in a flow cell on the carboxylated dextran layer of a CM5 chip surface (GE Healthcare). The immobilization was performed using amine coupling chemistry according to the manufacturer's protocol and using HBS-EP (GE Healthcare) as running buffer. One flow cell surface on the chip was activated and deactivated for use as blank during analyte injections. In the two binding experiments presented below, McIlvaines phosphate-citrate buffer pH 6.0 supplemented with 0.005% Tween-20 (0.005% PCT) was used as running buffer. In all experiments, a flow rate of 50 μl/min was used.
[0423] In one experiment, the dissociation at pH 6.0 was compared to the dissociation at pH 7.4. His.sub.6-tagged Z variants and a human monoclonal IgG1 were diluted in running buffer to a final concentration of 250 nM or 2.5 nM, respectively, and injected over the FcRn chip for 1 minute using the co-inject procedure. The second injection of the co-inject procedure, representing the dissociation phase of the interactions, contained either running buffer (pH 6.0) or 0.005% PCT pH 7.4. The Z variants were allowed to dissociate for 1 minute, except for Z07918 and Z10193, which were allowed to dissociate for 4 minutes, before a surface equilibration during 5 minutes in running buffer. IgG was allowed to dissociate for 4 minutes before equilibration. Buffer injections were performed in a similar way; co-injection of buffer pH 6.0 followed by pH 6.0 or co-injection of buffer pH 6.0 followed by pH 7.4. The results were analyzed in BiaEvaluation software 4.1 (GE Healthcare). Curves of the blank surface were subtracted from the curves of the ligand surface. In addition, curves of buffer injections were subtracted from the Z variant curves and from the IgG curves to adjust for the buffer effects.
[0424] In another experiment, approximate kinetic constants (k.sub.on and k.sub.off) and affinities (K.sub.D) were determined for a subset of His.sub.6-tagged Z variants. Three concentrations of the Z variants were injected for 1 minute followed by dissociation in running buffer for 1 minute. The surfaces were equilibrated with running buffer during 7.5 minutes before the start of next cycle. Injected concentrations were either 675 nM, 225 nM and 75 nM (Z10140, Z10156 and Z10183) or 225 nM, 75 nM and 25 nM (Z07918 and Z10193). Kinetic constants were calculated from the sensorgrams using the Langmuir 1:1 model of BiaEvaluation software 4.1 (GE Healthcare).
[0425] In a separate experiment, the affinity of the interactions of Z variants to hFcRn (SEQ ID NO:379) and mFcRn (SEQ ID NO:384), respectively, was measured at both pH 6.0 and pH 7.4 on a Biacore 3000 instrument (GE Healthcare). hFcRn and mFcRn were produced essentially as described in Example 1 but using mouse 3T3 cells instead of human SKOV-3 cells for production of mFcRn, and immobilized on separate flow cells on a CM5 chip in acetate buffer at pH 4.65. The immobilization level was approximately 1000 RU for both receptors. A reference flow cell was created by activation and deactivation. 0.005% PCT pH 6.0 or 7.4 was used as running buffer and for dilution of the analytes. All analyses were performed at 25° C. The affinity constants for the His.sub.6-tagged Z variants Z07918 (SEQ ID NO:1), Z07960 (SEQ ID NO:4) and Z10193 (SEQ ID NO:2) were determined by injecting a dilution series from 1024 nM to 0.5 nM (pH 6.0) or from 10240 nM to 5 nM (pH 7.4). The affinities were derived using GraphPad Prism 5 software, using a one site binding saturation model.
[0426] AlphaLISA Blocking Assay:
[0427] The potential of Z variants to inhibit binding of IgG to FcRn was analyzed in an AlphaLISA assay with an EnSpire multiplate reader 2300 (Perkin Elmer). Human IgG (Roactemra) was immobilized on AlphaLISA acceptor beads (Perkin Elmer, cat. no. 6772002) according to the manufacturer's recommendations. Stepwise serial dilutions 1:3 of His-tagged Z variants to final concentrations of 250 nM to 38 μM were made in a 384-well plate (Perkin Elmer, cat. no. G6005350) and incubated for 45 min with 10 nM biotinylated human FcRn (Biorbyt, cat. no. orb84388; biotinylated essentially as described in Example 2) in AlphaLISA buffer (Perkin Elmer, cat. no. AL000F) adjusted to pH 6.0 using HCl. IgG-coated Acceptor beads were added to a final concentration of 10 μM and incubated for 45 min. Finally, streptavidin coated Donor beads (Perkin Elmer, cat. no. 6772002) were added to a final concentration of 40 μg/ml and incubated for 30 min. All incubations were performed at RT in the dark. The plate was analyzed in the EnSpire instrument and the IC50 values were calculated using GraphPad Prism 5.
Results
[0428] Cultivation and Purification:
[0429] The seventeen FcRn binding Z variants (SEQ ID NO:1-16 and SEQ ID NO:353), constructed with an N-terminal His.sub.6 tag, were produced in E. coli. The amount of IMAC-purified protein from approximately 2-5 g bacterial pellets, determined spectrophotometrically by measuring the absorbance at 280 nm, ranged from approximately 10 mg to 20 mg for the different FcRn binding Z variants. SDS-PAGE analysis of each final protein preparation showed that these predominantly contained the FcRn binding Z variant. The correct identity and molecular weight of each FcRn binding Z variant was confirmed by HPLC-MS analysis.
[0430] CD Analysis:
[0431] The CD spectra determined for six Z variants showed that each had an α-helical structure at 20° C. This result was also verified in the variable temperature measurements, wherein melting temperatures (Tm) were determined (Table 3). A reversible folding was seen for the six Z variants when overlaying spectra measured before and after heating to 90° C.
TABLE-US-00003 TABLE 3 Melting temperatures for a selection of Z variants. Z variant SEQ ID NO: Tm (°C.) Z07909 13 56 Z07918 1 49 Z07930 6 56 Z07960 4 58 Z10109 3 61 Z10193 2 59
[0432] Biacore Binding and Kinetic Analyses:
[0433] The binding of seventeen His.sub.6-tagged Z variants to human FcRn and the dissociation at different pH were tested in a Biacore instrument by sequentially injecting each of the Z variants at pH 6.0 and either buffer pH 6.0 or pH 7.4 over a chip surface containing FcRn. The ligand immobilization level of the surface was 1668 RU human FcRn. The seventeen Z variants showed binding to FcRn at pH 6.0, and for all variants, faster off-rates were seen at pH 7.4 compared to pH 6.0. The result for IgG was similar, displaying a faster off-rate at pH 7.4. The variants Z07918 and Z10193 showed the slowest dissociation curves. Sensorgrams for a subset of variants and IgG are displayed in
TABLE-US-00004 TABLE 4 Biacore kinetic constants and affinities for hFcRn binding at pH 6.0. Z variant SEQ ID NO: k.sub.on (M.sup.−1s.sup.−1) k.sub.off (s.sup.−1) K.sub.D (M) Z07918 1 1.4 × 10.sup.6 0.022 1.6 × 10.sup.−8 Z10140 5 1.4 × 10.sup.6 0.12 8.6 × 10.sup.−8 Z10156 11 7.6 × 10.sup.5 0.28 3.7 × 10.sup.−7 Z10183 7 1.0 × 10.sup.6 0.13 1.3 × 10.sup.−7 Z10193 2 1.5 × 10.sup.6 0.033 2.2 × 10.sup.−8
[0434] The kinetic constants of five Z variants interacting with FcRn at pH 6.0 were determined (see Table 4). The immobilization level of the surface was 2015 RU human FcRn. For each Z variant, kinetic constants were calculated using a curve set of three injected concentrations.
[0435] Affinity (K.sub.D) constants were also determined for His.sub.6-tagged Z variants Z07918 (SEQ ID NO:1), Z07960 (SEQ ID NO:4) and Z10193 (SEQ ID NO:2) interacting with human and mouse FcRn at pH 6.0 and pH 7.4 (Table 5). For all three variants, K.sub.D values were lower at pH 6.0 compared to pH 7.4.
TABLE-US-00005 TABLE 5 Biacore affinities for hFcRn and mFcRn at pH 6.0 and pH 7.4. SEQ ID K.sub.D (M) hFcRn K.sub.D (M) mFcRn Z variant NO: pH 6.0 pH 7.4 pH 6.0 pH 7.4 Z07918 1 1.2 × 10.sup.−8 >5 × 10.sup.−7 9.0 × 10.sup.−8 >5 × 10.sup.−7 Z07960 4 5.0 × 10.sup.−8 >1 × 10.sup.−6 3.5 × 10.sup.−7 >5 × 10.sup.−6 Z10193 2 1.4 × 10.sup.−8 >5 × 10.sup.−7 9.5 × 10.sup.−8 >5 × 10.sup.−7
TABLE-US-00006 TABLE 6 Calculated IC50 values from AlphaLISA blocking assay. Z variant SEQ ID NO IC50 (M) Z07909 13 4.6 × 10.sup.−8 Z07918 1 2.1 × 10.sup.−9 Z07930 6 4.2 × 10.sup.−8 Z07960 4 4.2 × 10.sup.−8 Z10109 3 5.7 × 10.sup.−8 Z10111 8 4.6 × 10.sup.−8 Z10140 5 5.6 × 10.sup.−8 Z10183 7 3.9 × 10.sup.−8 Z10193 2 1.2 × 10.sup.−8 Z13993 353 1.3 × 10.sup.−7 Z11948-(G.sub.4S)-Z11948 368 3.8 × 10.sup.−10 Z11948-(G.sub.4S).sub.3-Z11948 369 4.1 × 10.sup.−10
[0436] AlphaLISA Blocking Assay:
[0437] The ability of seventeen His.sub.6-tagged monomeric Z variants (SEQ ID NO:1-16 and SEQ ID NO:353) and two dimeric variant, Z11948-G.sub.4S-Z11948 and Z11948-(G.sub.4S).sub.3-Z11948 to inhibit IgG binding to FcRn was tested in an AlphaLISA blocking assay. Serial dilutions of the Z variants were incubated with biotinylated human FcRn and the blocking ability of each respective variant was measured after addition of IgG coated Acceptor beads and subsequently streptavidin coated Donor beads. Inhibition could be measured as a decrease in AlphaLISA counts for positive Z variants. The calculated IC50 values for the ten monomeric variants and the two dimeric variants that were shown to block IgG binding to FcRn in this assay are shown in Table 6.
Example 4
Binding of FcRn Binding Z Variants to Human or Mouse FcRn/eGFP Transfected HeLa Cells
[0438] In this Example, the binding ability of FcRn binding Z variants was investigated. The production of HeLa cells expressing human and murine FcRn-eGFP gene transgene and the use of these cells for flow cytometry analysis with Alexa Fluor@ 647 labeled Z variants is described.
Materials and Methods
[0439] Cloning of FcRn-eGFP and B2M Viral Vectors:
[0440] The genes encoding murine FcRn (mFcRn, Genbank BC003786.1, OpenBiosystems) and murine B2M (mB2M, Genbank BC085164.1, OpenBiosystems) were amplified in a similar way as the genes for human FcRn and human B2M as described in Example 1. Human and murine FcRn and B2M genes were amplified as follows: for hFcRn, the sequence encoding amino acids 1-365 (SEQ ID NO:382) was amplified; for hB2M, the sequence encoding amino acids 21-119 (SEQ ID NO:380) was amplified; for mFcRn, the sequence encoding amino acids 1-369 (SEQ ID NO:383) was amplified; and for mB2M, the sequence encoding amino acids 21-119 (SEQ ID NO:381) was amplified. The vector pHR-cPPT-CMV-EGFP (Jakobsson et al. (2003) J Neurosci Res 73:876-85) and FcRn PCR amplicons (human and murine) were cut using the restriction enzymes BamHI (human) or BclI (murine) and MluI (New England Biolabs, cat. nos. R0136M, R0160L and R0198L, respectively), and ligated using T4 DNA Ligase (New England Biolabs, cat. no. M0202M). The ligation mix was chemically transformed into E. coli RRIΔM15 and spread on ampicillin plates. Colonies were picked and screened with suitable primer pairs. The construct encoding the original signal peptide, human or murine FcRn and eGFP at the cytoplasmic tail were verified by sequencing and denoted pHR-cPPT-CMV-hFcRn-eGFP and pHR-cPPT-CMV-mFcRn-eGFP, respectively.
[0441] The human and murine B2M PCR amplicons were inserted into the plasmid pDONOR221 (Invitrogen, cat. no. 12536-017) by recombination using the Gateway system (Invitrogen, cat. no. 11789020, Gateway® BP Clonase® II Enzyme mix) according to the manufacturer's recommendations. After verification of correct sequences, human or murine B2M was inserted into p2k7_gtc (Suter et al., supra) using a multi-site gateway cloning system (Invitrogen, cat. no. 11791020, Gateway® LR Clonase® II Enzyme mix) together with the promoter containing plasmid pENTR-CMV (Tai et al. supra), resulting in the vectors 2k7.sub.neo-CMV-hB2M and 2k7.sub.neo-CMV-mB2M, respectively.
[0442] Lentiviral Transduction of HeLa Cells:
[0443] The vector pairs 2k7.sub.neo-CMV-hB2M and pHR-cPPT-CMV-hFcRn-eGFP or 2k7.sub.neo-CMV-mB2M and pHR-cPPT-CMV-mFcRn-eGFP were co-transfected together with VSV-G envelope and gag/pol packaging plasmid into HEK293T cells using calcium chloride transfection (Zufferey et al., supra; Jakobsson et al. (2006) supra). HEK293T culture supernatants containing formed lentiviral particles with FcRn and B2M transgenes respectively were used to sequentially transduce HeLa Cervix adenocarcinoma cells (Cell Line Service) at low passage number. The resulting two stably transduced HeLa cell lines are in the following denoted hFcRn-eGFP (transduced with genes for human FcRn-eGFP and hB2M) and mFcRn-eGFP (transduced with genes for mouse FcRn-eGFP and mB2M).
[0444] Alexa Fluor® 647 Labeling of FcRn Binding Z Variants:
[0445] The three His.sub.6-tagged Z variants Z07918, Z07930 and Z07960 were labeled with Alexa Fluor® 647 Carboxylic Acid Succinimidyl Ester (Invitrogen cat. no. A20106). Before labeling, buffer was exchanged to 0.2 M carbonate buffer, pH 8.3, using Vivaspin500 centrifugal filter units (10 kDa MWCO, Vivaproducts cat. no. 512-2838) spun at 10,000 g. The labeling was performed in the Vivaspin500 and 1 μl of Alexa Fluor® 647 Succinimidyl Ester dye (40 μg/μl in DMSO corresponding to 1.3×molar excess) was added to 200 μg/25 μl Z variant. The mixes were incubated at RT in the dark for 40 minutes in a wiggling rota mixer. The reaction mixes were subsequently put on ice for 3.5 hours and free dye was removed by washing with 15×100 μl PBS in the Vivaspin500.
[0446] Immunofluorescence Staining of Human and Mouse FcRn-eGFP Transfected HeLa-Cells with FcRn Binding Z Variants:
[0447] hFcRn-eGFP and mFcRn-eGFP HeLa cells were harvested by trypsination and washed twice in PBS at pH 6.0 before counting. 100,000 cells were pipetted per well of a v-bottomed 96 well plate (Nunc, cat no 277143) and the cells in the plate were subsequently pelleted at 1,700 rpm for 4 min at 4° C. The supernatants were removed and the cells were fixed with 50 μl of 2% formaldehyde (Sigma Aldrich, cat. no. F8775) in PBS at pH 6.0 for 10 min at RT. Cells were thereafter washed with 2×100 μl PBS, pH 6.0, saturated with casein (PBSC), and resuspended in PBSC with 0.1% saponin (AppliChem, cat no A4518.0100) containing 620 nM of Alexa Fluor@ 647 labeled His.sub.6-tagged Z variants; Z07960, Z07930 and Z07918. Transduced HeLa cells, incubated with buffer alone, were used as control. The cells were incubated for 1 h at 8° C. on a shaker in the dark, washed with 2×100 μl PBSC and resuspended in 180 μl of PBS, pH 6.0, containing 1% BSA (fraction V, Merck, cat. no. 1.12018.0100). 10,000 cells/well were analyzed in a Gallios Flow Cytometer (Beckman Coulter) and the data was analyzed using Kaluza software (Beckman Coulter).
Results
[0448] Flow cytometry analysis was utilized to determine whether the FcRn binding Z variants could bind to human and/or mouse FcRn on human or mouse FcRn/eGFP transduced HeLa cells. The experiment was performed at pH 6.0 with Alexa Fluor® 647 labeled Z07960, Z07930 and Z07918 (SEQ ID NO:4, 6 and 1, respectively). Dot plot analysis (y-axis: Alexa Fluor® 647, x-axis: eGFP) showed that the transduced cell population could be divided into FcRn-eGFP negative and positive population (
Example 5
Blocking of IgG Binding to FcRn with the FcRn Binding Z Variant Z07918
[0449] In this Example, the potential competition of FcRn binding Z variants with IgG for binding to FcRn was investigated in a cell based assay. Such binding will result in blocking of the IgG-FcRn interaction.
Materials and Methods
[0450] Blocking of IgG-FcRn Immunofluorescence Staining:
[0451] Human or murine FcRn-eGFP transduced HeLa cells were prepared as described in Example 4. Fixed cells were resuspended in 50 μl of a mix of either 100 nM Alexa Fluor@ 647-conjugated human or mouse IgG (Jackson laboratories, cat. no. 009-600-003 and 015-600-003, respectively) and 1000, 100, 10, 1 or 0 (buffer control) nM His.sub.6-tagged Z07918 diluted in PBS-casein, pH 6.0, containing 0.1% saponin (AppliChem). The cells were incubated for 1 h at 37° C. on a shaker in the dark, washed with 2×100 μl PBS-casein pH 6.0 and re-suspended in 180 μl of PBS, pH 6.0, containing 1% BSA. Data from 10,000 cells/well (except somewhat fewer cells for mouse 100 nM mIgG-Alexa Fluor® 647) were obtained using a Gallios Flow Cytometer (Beckman Coulter) and the data was analyzed using Kaluza software (Beckman Coulter).
Results
[0452] The experiment was performed to determine if the FcRn binding Z variant Z07918 (SEQ ID NO:1) blocks the IgG-FcRn interaction. Human or murine FcRn-eGFP transduced HeLa cells were incubated with human or mouse Alexa Fluor@ 647-conjugated IgG. The binding was blocked with unlabeled Z07918 at different concentrations. Due to the heterogeneous expression of FcRn by the transduced HeLa cells (described in Example 4), the MFI values for Alexa Fluor® 647 in gate N of each sample was subtracted by the corresponding MFI values in gate M (
Example 6
Pharmacokinetic Study of Three FcRn Binding Z Variants
[0453] In this Example, the ability of FcRn binding Z variants to prolong serum half-life of a non-specific Z variant was investigated by a pharmacokinetic study performed in mice.
Materials and Methods
[0454] Subcloning of Z Variants:
[0455] A subset of Z variants (Z07918, Z07960 and Z10193) was submitted to a second subcloning. DNA from the subcloned His.sub.6-tagged variants in Example 3 was used as template. First, PCR amplification using suitable primer pairs was performed to create genes encoding Z variants starting with the amino acids AE instead of VD. The mutated Z variants are listed in
[0456] Cultivation and Purification:
[0457] Z variants in fusion with PP013-Z03638 were produced in E. coli as described in Example 3. Approximately 3 g of each cell pellet was re-suspended in 30 ml TST-buffer (25 mM Tris-HCl, 1 mM EDTA, 200 mM NaCl, 0.05% Tween20, pH 8.0) supplemented with Benzonase® (Merck). After cell disruption by sonication and clarification by centrifugation, each supernatant was applied on a gravity flow column with 5 ml agarose immobilized with an anti-ABD ligand (see WO2014/064237). After washing with TST-buffer and 5 mM NH.sub.4Ac buffer, pH 5.5, the Z variants were eluted with 0.1 M HAc. Acetonitrile (ACN) was added to a final concentration of 10% to the eluted fractions from the anti-ABD agarose affinity chromatography purification step and the samples were loaded on a 3 ml Resource 15RPC column (GE Healthcare), previously equilibrated with RPC solvent A (0.1% trifluoroacetic acid (TFA), 10% ACN, 90% water). After column wash with RPC solvent A, bound protein was eluted with a linear gradient 0-50% RPC solvent B (0.1% TFA, 80% ACN, 20% water) during 60 ml. Fractions containing pure Z variant were identified by SDS-PAGE analysis and pooled. After the RPC purification, the buffer of the pools was exchanged to PBS using a HiPrep 26/10 Desalting column (GE Healthcare). Finally, the Z variants were purified on 1 ml EndoTrap red columns (Hyglos, cat. no. 321063) to ensure low endotoxin content.
[0458] Protein concentrations, purities and the identity of each purified Z variant were analyzed as described in Example 3.
[0459] Biacore Analysis:
[0460] Expressed and purified Z variants fused to PP013-Z03638 were assayed against human FcRn at pH 6.0 essentially as described for the kinetic analysis in Example 3. The Z variants and the negative control Z03638-PP013 were injected at 40 nM, 160 nM and 640 nM during 1 minute followed by dissociation for 2.5 minutes and equilibration for 1 minute. Kinetic constants and affinities were determined for the Z variants using the BiaEvaluation software.
[0461] Pharmacokinetic Study:
[0462] Z11947, Z11946 and Z11948 fused to PP013-Z03638 were administered intravenously (i.v.) to male NMRI mice (Charles River, Germany) at a dose of 92 nmol/kg body weight. Sera from groups of three mice were obtained at 0.08, 6, 18, 78, 120, 168 and 240 hours. The concentration of respective Z variant was determined by ELISA.
[0463] ELISA:
[0464] Half-area 96-well ELISA plates were coated at 4° C. overnight with 50 μl/well of an Z specific goat antibody (produced in-house) diluted to 4 μg/ml in coating buffer (50 mM sodium carbonate, pH 9.6). The antibody solution was poured off and the wells were blocked with 100 μl of PBSC for 1.5 h at RT. The sera were diluted in PBSC containing 1% mouse serum (matrix) from 1:100 to 1:51, 200 in a two-fold dilution series in a dilutions plate. A standard titration for respective Z variant and four quality controls (very low, low, medium and high control) diluted in matrix were included on each plate. 50 μl of the dilutions were transferred per well and the ELISA plates were incubated for 1.5 h at RT. The plates were washed four times with PBST. Bound Z variants were detected with 50 μl/well of rabbit anti-PP013 Ig (produced in-house) diluted to 4 μg/ml in PBSC. The plates were subsequently incubated for 1.5 h at RT followed by washes as described above. HRP conjugated donkey anti-rabbit HRP obtained from Jackson laboratories (cat. no. 711-035-152), diluted 1:20,000 in PBSC, was added and the plates were incubated for 1 hour. After washing as described above, 50 μl of ImmunoPure TMB substrate was added to the wells and the plates were developed according to the manufacturer's recommendations. After 15 minutes of development, the absorbance was measured at 450 nm using a multi-well plate reader (Victor.sup.3). The absorbance values were analyzed using GraphPad Prism 5 to determine the concentrations (cubic-spline curve fit) and area under curve (AUC). The concentrations were then plotted as their natural logarithms against time. The resulting curves followed a two compartment model and the terminal half-life was calculated as In2 divided by the slope based on the last three time points.
Results
[0465] Cultivation and Purification:
[0466] The three FcRn binding Z variants Z11947, Z11946 and Z11948 (SEQ ID NO:356, 355 and 354), constructed as Z#####-PP013-Z03638, and the negative control Z03638-PP013, were produced in E. coli. The amount of purified protein from approximately 3 g bacterial pellets, determined spectrophotometrically by measuring the absorbance at 280 nm, ranged from approximately 10 to 25 mg for the different FcRn binding Z variants. SDS-PAGE analysis of each final protein preparation showed that they predominantly contained respective FcRn binding Z variant. The correct molecular weight of each FcRn binding Z variant was confirmed by LC/MS analysis.
TABLE-US-00007 TABLE 7 Kinetic constants and affinities for FcRn at pH 6.0 of Z variants produced as fusions to PP013-Z03638. Z variant SEQ ID NO: k.sub.on (M.sup.−1s.sup.−1) k.sub.off (s.sup.−1) K.sub.D (M) Z11948 354 7.73 × 10.sup.5 0.047 6.2 × 10.sup.−8 Z11946 355 3.35 × 10.sup.5 0.275 8.2 × 10.sup.−7 Z11947 356 6.54 × 10.sup.5 0.064 9.8 × 10.sup.−8
[0467] Biacore Analysis:
[0468] The binding to FcRn was analyzed for the three PP013-Z03638 fused Z variants. The immobilization level of the surface was 548 RU of human FcRn. The resulting rough kinetic constants and affinities for the target binding at pH 6.0 are displayed in Table 7. Fitted curves are displayed in
[0469] Pharmacokinetic Study:
[0470] The pharmacokinetic profiles of the above-mentioned constructs of Z variants fused to PP013-Z03638 were compared to the negative control Z03638-PP013 in a mouse pharmacokinetic study. In previous work, e.g. as described in PCT application WO2009/016043, it is shown that ABD fusion proteins have a long half-life in serum, caused by ABD binding to serum albumin. In accordance with the previous results, terminal half-life of ABD-fused Z variant molecule (Z03638-PP013) was approximately 43 hours, which is comparable to half-life of mouse albumin (35 hours). The terminal half-lives of the constructs containing FcRn binding Z variant molecule in addition to ABD were two- to three-fold longer (
Example 7
Design and Construction of a Maturation Library of FcRn Binding Z Variants
[0471] In this Example, a maturated library was constructed. The library was used for selections of FcRn binding Z variants. Selections from maturated libraries are usually expected to result in binders with increased affinity (Orlova et al., (2006) Cancer Res 66(8):4339-48). In this study, randomized single stranded linkers were generated using split-pool synthesis enabling incorporation of defined codons in desired positions in the synthesis.
Materials and Methods
[0472] Library Design:
[0473] The library was based on the sixteen sequences of the human FcRn binding Z variants in Table 1 and further described in Examples 2-6. In the new library, 13 variable positions in the Z molecule scaffold were biased towards certain amino acid residues, according to a strategy mainly based on the binding motifs of the Z variants defined in SEQ ID NO:1-16. A DNA linker was generated using split-pool synthesis containing the 147 bp partially randomized helix 1 and 2 of the amino acid sequence: 5′-AA ATA AAT CTC GAG GTA GAT GCC AAA TAC GCC AAA GAA NNN NNN NNN GCG NNN NNN GAG ATC NNN NNN TTA CCT AAC TTA ACC NNN NNN CAA NNN NNN GCC TTC ATC NNN AAA TTA NNN GAT GAC CCA AGC CAG AGC TCA TTA TTT A-3′ (SEQ ID NO:388; randomized codons are illustrated as NNN) flanked by restriction sites XhoI and SacI, was ordered from DNA 2.0 (Menlo Park, Calif., USA). The theoretical distributions of amino acid residues in the new library, including eight variable amino acid positions (9, 10, 11, 13, 14, 24, 32 and 35) and five constant amino acid positions (17, 18, 25, 27 and 28) in the Z molecule scaffold are given in Table 8. The resulting theoretical library size is 5.3×10.sup.8 variants.
TABLE-US-00008 TABLE 8 Design of library for maturation. Amino acid No of position in the Randomization (amino acid amino Z variant abbreviations) acids Proportion 9 A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, Y 16 1/16 10 A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W, Y 17 1/17 11 A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, Y 16 1/16 13 A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W, Y 17 1/17 14 A, F, H (25%), I, K, L, N, Q, R, S, T, V, W, Y 14 3/52, 13/52 (H) 17 R 1 1 18 W 1 1 24 F, Y 2 1/2 25 D 1 1 27 R 1 1 28 V 1 1 32 A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, Y 16 1/16 35 A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, Y 16 1/16
[0474] Library Construction:
[0475] The library was amplified using AmpliTaq Gold polymerase (Applied Biosystems, cat. no. 4311816) during 12 cycles of PCR and pooled products were purified with QIAquick PCR Purification Kit (QIAGEN, cat. no. 28106) according to the supplier's recommendations. The purified pool of randomized library fragments was digested with restriction enzymes XhoI and SacI-HF (New England Biolabs, cat. no. R0146L, and cat. no. R3156M) and concentrated using a PCR Purification Kit. Subsequently, the product was subjected to preparative 2.5% agarose (Nuisieve GTC agarose, Cambrex, Invitrogen) gel electrophoresis and purified using QIAGEN gel extraction Kit (QIAGEN, cat. no. 28706) according to the supplier's recommendations.
[0476] The phagemid vector pAY02592 (essentially as pAffi1 described in Grönwall et al., supra) was restricted with the same enzymes, purified using phenol/chloroform extraction and ethanol precipitation. The restricted fragments and the restricted vector were ligated in a molar ratio of 5:1 with T4 DNA ligase (Fermentas, cat. no. EL0011) for 2 hours at RT, followed by overnight incubation at 4° C. The ligated DNA was recovered by phenol/chloroform extraction and ethanol precipitation, followed by dissolution in 10 mM Tris-HCl, pH 8.5. Thus, the resulting library in vector pAY02592 encoded Z variants, each fused to an albumin binding domain (ABD) derived from streptococcal protein G.
[0477] The ligation reactions (approximately 160 ng DNA/transformation) were electroporated into electrocompetent E. coli ER2738 cells (50 μl, Lucigen, Middleton, Wis., USA). Immediately after electroporation, approximately 1 ml of recovery medium (supplied with the ER2738 cells) was added. The transformed cells were incubated at 37° C. for 60 min. Samples were taken for titration and for determination of the number of transformants. The cells were thereafter pooled and cultivated overnight at 37° C. in 1 l of TSB-YE medium, supplemented with 2% glucose, 10 μg/ml tetracycline and 100 μg/ml ampicillin. The cells were pelleted for 7 min at 4,000 g and resuspended in a PBS/glycerol solution (approximately 40% glycerol). The cells were aliquoted and stored at −80° C. Clones from the library of Z variants were sequenced in order to verify the content and to evaluate the outcome of the constructed library vis-à-vis the library design. Sequencing was performed as described in Example 1 and the amino acid distribution was verified.
[0478] Preparation of Phage Stock:
[0479] Phage stock containing the phagemid library was prepared in a 20 l fermenter (Belach Bioteknik). Cells from a glycerol stock containing the phagemid library were inoculated in 10 l of TSB-YE (Tryptic Soy Broth-Yeast Extract; 30 g/l TSB, 5 g/l yeast extract) supplemented with 1 g/l glucose, 100 mg/l ampicillin and 10 mg/l tetracycline. When the cells reached an optical density at 600 nm (OD600) of 0.6, approximately 1.5 l of the cultivation was infected using a 5×molar excess of M13K07 helper phage. The cells were incubated for 30 min, whereupon the fermenter was filled up to 10 l with complex fermentation medium [2.5 g/l (NH.sub.4).sub.2SO.sub.4; 5.0 g/l yeast extract; 30 g/l tryptone, 2 g/l K.sub.2HPO.sub.4; 3 g/l KH.sub.2PO.sub.4, 1.25 g/l; Na.sub.3C.sub.6H.sub.5O.sub.7.2 H.sub.2O; Breox FMT30 antifoaming agent 0.1 ml/l]. The following components were added: 10 ml carbenicillin 25 mg/ml; 5 ml kanamycin 50 mg/ml; 1 ml 1 M isopropyl-β-D-1-thiogalactopyranoside (IPTG); 17.5 ml/l of 300 g/l MgSO.sub.4, and 5 ml of a trace element solution [35 g/l FeCl.sub.3. 6 H.sub.2O; 10.56 g/l ZnSO.sub.4.7 H.sub.2O; 2.64 g/l CuSO.sub.4.5 H.sub.2O; 13.2 g/l MnSO.sub.4. H.sub.2O; 13.84 g/l CaCl.sub.2.2 H.sub.2O, dissolved in 1.2 M HCl]. A glucose limited fed-batch cultivation was started where a 600 g/l glucose solution was fed to the reactor (3.5 g/h in the start, 37.5 g/h after 20 h and until the end of the cultivation). pH was controlled at pH 7 through the automatic addition of 25% NH.sub.4OH, air was supplemented (5 l/min), and the stirrer was set at 500 rpm. After 24 h of fed-batch cultivation the OD600 was 33.2. The cells in the cultivation were pelleted by centrifugation at 15,900 g. The phage particles were precipitated from the supernatant twice in PEG/NaCl, filtered and dissolved in PBS and glycerol as in Example 2. Phage stocks were stored at −80° C. until use in selection.
Results
[0480] Library Construction:
[0481] The new library was designed based on a set of 16 FcRn binding Z variants with verified binding properties (Example 2-6). The theoretical size of the designed library was 5.3×10.sup.8 Z variants. The actual size of the library, determined by titration after transformation to E. coli ER2738 cells, was 4.5×10.sup.9 transformants.
[0482] The library quality was tested by sequencing of 96 transformants and by comparing their actual sequences with the theoretical design. The contents of the actual library compared to the designed library were shown to be satisfying. A maturated library of potential binders to FcRn was thus successfully constructed.
Example 8
Selection and Screening of Z Variants from a Maturated Library
Materials and Methods
[0483] Phage Display Selection of Matured FcRn Binding Z Variants:
[0484] The target proteins human FcRn (Biorbyt, cat. no. orb84388) and murine FcRn (Biorbyt, cat. no. orb99076) were biotinylated essentially as described in Example 2 using biotin at 10× molar excess. Phage display selections, using the new library of Z variant molecules described in Example 7, were performed in four cycles against human FcRn or murine FcRn essentially as in Example 2 but with the following exceptions. Selection buffers were 0.1% PCTG buffer, pH 5.5 (McIlvaines phosphate-citrate buffer, pH 5.5, supplemented with 0.1% Tween-20 and 0.1% gelatin) or 0.1% PCTG buffer, pH 7.4, (McIlvaines phosphate-citrate buffer, pH 7.4, supplemented with 0.1% Tween-20 and 0.1% gelatin) respectively. Prior to selection, HSA was added to the selection buffers to a final concentration of 1.5 μM. All tubes and beads used in the selection were pre-blocked with either of the two different selections buffers. A pre-selection step, by incubation of phage stock with SA-beads for 45 min, was performed in cycle 1. For capture of phage-target complexes, 1 mg beads per 1.1 μg biotinylated human FcRn or 1.6 μg biotinylated murine FcRn was used. Washes were performed with 0.1% PCT buffer pH 5.5 or pH 7.4 except for tracks 2-1-2-1 and 2-1-2-2 where 0.1% PCT supplemented with 25 nM IgG (Herceptin®) or 10 nM IgG, respectively, was used as outlined in Table 9.
[0485] The five tracks (1-5) in cycle 1 were divided in the second to fourth cycles, resulting in totally seven tracks (1-1 to 5-1) in cycle 2, eleven tracks (1-1-1 to 5-1-1) in cycle 3 and fourteen tracks (1-1-1-1 to 5-1-1-1) in cycle 4. The bound phage particles were eluted as described in Example 2.
[0486] An overview of the selection strategy, describing an increased stringency in subsequent cycles, using a lowered target concentration and an increased number of washes, is shown in Table 9.
[0487] Amplification of Phage Particles:
[0488] Amplification of phage particles between selection cycle 1 and 2 was performed essentially as described in Example 2, with the following exceptions. E. coli ER2738 was used for phage amplification and M13K07 helper phage was used in 5×excess. The amplification of phage particles between the selection cycles 2 and 4 was done by performing infection of bacteria in solution as follows. After infection of log phase E. coli ER2738 with phage particles, TSB supplemented with 2% glucose, 10 μg/ml tetracycline and 100 μg/ml ampicillin was added, followed by incubation with rotation for 30 min at 37° C. Thereafter, the bacteria were infected with M13K07 helper phage in 5×excess. The infected bacteria were pelleted by centrifugation, re-suspended in TSB-YE medium supplemented with 100 μM IPTG, 25 μg/ml kanamycin and 100 μg/ml ampicillin, and grown overnight at 30° C. The overnight cultures were pelleted in a centrifuge, and phage particles in the supernatant were precipitated twice with PEG/NaCl buffer. Finally, the phage particles were re-suspended in selection buffer before entering the next selection cycle.
[0489] In the final selection cycle, log phase bacteria were infected with eluate and diluted before spreading onto TBAB plates (30 g/l tryptose blood agar base, Oxoid cat. no. CM0233B) supplemented with 0.2 g/l ampicillin in order to form single colonies to be used in ELISA screening.
TABLE-US-00009 TABLE 9 Overview of the maturation selection data. Phage stock Target Selection from library or Target conc. Selection Number of Cycle track selection track species (nM) pH Wash pH washes 1 1 Zlib006FcRn.l human 100 7.4 7.4 2 1 2 Zlib006FcRn.l human 100 7.4 5.5 2 1 3 Zlib006FcRn.l human 25 5.5 5.5 4 1 4 Zlib006FcRn.l murine 100 7.4 7.4 2 1 5 Zlib006FcRn.l murine 100 5.5 5.5 2 2 1-1 1 human 50 7.4 7.4 4 2 2-1 2 human 50 7.4 5.5 4 2 2-2 2 human 25 5.5 7.4 6 2 3-1 3 human 5 5.5 7.4 4 2 3-2 3 human 5 5.5 5.5 8 2 4-1 4 murine 50 7.4 5.5 2 2 5-1 5 murine 100 5.5 5.5 2 3 1-1-1 1-1 human 10 7.4 7.4 8 3 1-1-2 1-1 human 5 5.5 7.4 8 3 2-1-1 2-1 human 10 7.4 5.5 8 3 2-1-2 2-1 human 5 7.4 5.5 12 3 2-2-1 2-2 human 10 7.4 5.5 12 3 2-2-2 2-2 human 5 7.4 5.5 15 3 3-1-1 3-1 human 1 5.5 7.4 8 3 3-2-1 3-2 human 0.5 5.5 5.5 12 3 3-2-2 3-2 human 0.25 5.5 5.5 16 3 4-1-1 4-1 murine 10 7.4 5.5 6 3 5-1-1 5-1 murine 5 5.5 5.5 8 4 1-1-1-1 1-1-1 human 1 7.4 7.4 12 4 1-1-1-2 1-1-1 human 0.25 7.4 7.4 15 4 1-1-2-1 1-1-2 human 0.5 7.4 5.5 15 4 1-1-2-2 1-1-2 human 0.1 5.5 7.4 15 4 2-1-1-1 2-1-1 human 1 7.4 5.5 15 4 2-1-1-2 2-1-1 human 0.5 7.4 5.5 15 4 2-1-2-1 2-1-2 human 0.25 7.4 5.5 20 (+IgG) 4 2-1-2-2 2-1-2 human 0.1 7.4 5.5 20 (+IgG) 4 2-2-1-1 2-2-1 and 2-2-2 human 0.5 5.5 7.4 15 4 2-2-2-1 2-2-1 and 2-2-2 human 0.5 7.4 5.5 20 4 3-1-1-1 3-1-1 human 1 5.5 7.4 12 4 3-2-1-1 3-2-1 and 3-2-2 human 0.5 5.5 5.5 16 4 4-1-1-1 4-1-1 murine 1 7.4 5.5 12 4 5-1-1-1 5-1-1 murine 0.5 5.5 5.5 15
[0490] Sequencing of Potential Binders:
[0491] Individual clones from the different selection tracks were picked for sequencing. All clones run in the ELISA screening were sequenced. Amplification of gene fragments and sequence analysis of gene fragments were performed essentially as described in Example 2.
[0492] ELISA Screening of Z Variants:
[0493] Single colonies containing Z variants (expressed as Z variant ABD fusion proteins as described in Example 2) were randomly picked from the selected clones of the FcRn maturated library and grown in 1 ml cultivations essentially as described in Example 2. Preparation of the periplasmic supernatants was performed as in Example 2 with eight freeze thawing cycles and the periplasmic fractions were used undiluted in the ELISA screening. ELISA screenings were performed at both pH 6.0 and pH 7.4 essentially as described in Example 2 using biotinylated human FcRn at a concentration of 2 nM in each well. The periplasmic fraction of the primary FcRn binder Z10193 (SEQ ID NO:2; assayed in above experiments) was used as a positive control. Periplasm containing the ABD moiety only was used as a negative control.
[0494] ELISA K.sub.D Analysis of FcRn Binding Z Variants:
[0495] A selection of FcRn binders was subjected to an analysis of the response against a dilution series of biotinylated human FcRn using ELISA at both pH 6.0 and pH 7.4 as described above. Biotinylated human FcRn was added at a concentration of 30 nM and diluted stepwise 1:3 down to 14 μM. As a background control, all Z variants were also assayed with no target protein added. Periplasm samples containing the primary FcRn binder Z07918 (SEQ ID. NO: 1) was included and analyzed as a positive control. Periplasm containing the ABD moiety only was used as a negative control. Data were analyzed using GraphPad Prism 5 and non-linear regression and K.sub.D values (the half maximal effective concentration) were calculated.
Results
[0496] Phage Display Selection of Maturated FcRn Binding Z Variants:
[0497] Selection was performed in totally 14 parallel tracks containing four cycles each. The different selection tracks differed in target concentration, target type (human FcRn or murine FcRn), selection time, and wash conditions.
[0498] Sequencing of Potential Binders:
[0499] Randomly picked clones were sequenced. Each individual Z variant was given an identification number, Z#####, as described in Example 2. In total, 445 new unique Z variant molecules were identified.
[0500] The amino acid sequences of a subset of the 58 amino acid residues long Z variants are listed in
[0501] ELISA Screening of Z Variants:
[0502] Clones obtained after four selection cycles were produced in 96-well plates and screened for FcRn binding activity using ELISA. All randomly picked clones were analyzed. At pH 6.0, 333 of the 445 unique Z variants were found to give a response of 0.3 AU or higher (corresponding to at least 3× the negative control) against human FcRn at a concentration of 2 nM. At pH 7.4, 278 of the 445 unique Z variants were found to give a response of 0.3 AU or higher (corresponding to at least 3× the negative control) against human FcRn at a concentration of 2 nM. Clones with a positive signal against human FcRn were found in all tracks (including those with murine target) except 1-1-1-1. The negative controls had absorbances of 0.070-0.096 AU (pH 6.0) and 0.060-0.112 AU (pH 7.4), respectively. The average response of the blank controls was 0.070 AU (pH 6.0) and 0.062 (pH 7.4).
[0503] ELISA K.sub.D Analysis of FcRn Binding Z Variants:
[0504] A subset of Z variants was selected based on the result in the ELISA experiment described above (highest ELISA value at pH 6.0 and/or pH 7.4) and subjected to a target titration in ELISA format. Periplasm samples were incubated with a serial dilution of biotinylated human FcRn. A periplasm sample with the primary binder Z07918 (SEQ ID NO:1) was also assayed as a positive control. Obtained values were analyzed and their respective K.sub.D values were calculated (Table 10).
TABLE-US-00010 TABLE 10 Calculated K.sub.D values from ELISA titration analysis of Z-ABD variants from the maturation. K.sub.D K.sub.D Z SEQ pH 6.0 pH 7.4 variant ID NO: (M) (M) Z13573 17 1.1 × 10.sup.−9 3.8 × 10.sup.−9 Z13574 18 1.2 × 10.sup.−9 5.0 × 10.sup.−9 Z13577 19 9.9 × 10.sup.−10 1.4 × 10.sup.−9 Z13578 20 1.0 × 10.sup.−9 2.5 × 10.sup.−9 Z13579 21 1.2 × 10.sup.−9 5.3 × 10.sup.−9 Z13581 22 1.1 × 10.sup.−9 3.3 × 10.sup.−9 Z13583 23 8.0 × 10.sup.−10 1.5 × 10.sup.−9 Z13585 24 1.2 × 10.sup.−9 1.7 × 10.sup.−9 Z13586 25 1.2 × 10.sup.−9 2.3 × 10.sup.−9 Z13587 26 1.4 × 10.sup.−9 6.9 × 10.sup.−9 Z13588 27 1.0 × 10.sup.−9 2.3 × 10.sup.−9 Z13592 28 9.5 × 10.sup.−10 1.8 × 10.sup.−9 Z13594 29 1.3 × 10.sup.−9 6.3 × 10.sup.−9 Z13596 30 1.5 × 10.sup.−9 3.6 × 10.sup.−9 Z13597 31 1.4 × 10.sup.−9 6.0 × 10.sup.−9 Z13598 32 1.1 × 10.sup.−9 1.7 × 10.sup.−9 Z13600 33 1.4 × 10.sup.−9 4.0 × 10.sup.−9 Z13604 43 1.3 × 10.sup.−9 4.1 × 10.sup.−9 Z13605 35 1.3 × 10.sup.−9 3.8 × 10.sup.−9 Z13609 36 1.3 × 10.sup.−9 2.7 × 10.sup.−9 Z13611 37 1.3 × 10.sup.−9 2.5 × 10.sup.−9 Z13612 38 1.2 × 10.sup.−9 8.6 × 10.sup.−9 Z13613 39 1.2 × 10.sup.−9 4.3 × 10.sup.−9 Z13615 40 1.2 × 10.sup.−9 3.1 × 10.sup.−9 Z13616 41 9.6 × 10.sup.−10 1.7 × 10.sup.−9 Z13617 42 1.2 × 10.sup.−9 1.9 × 10.sup.−9 Z13620 43 1.4 × 10.sup.−9 3.3 × 10.sup.−9 Z13621 44 8.6 × 10.sup.−10 1.4 × 10.sup.−9 Z13622 45 1.1 × 10.sup.−9 2.1 × 10.sup.−9 Z13624 46 1.3 × 10.sup.−9 3.4 × 10.sup.−9 Z13625 47 1.3 × 10.sup.−9 2.8 × 10.sup.−9 Z13626 48 1.2 × 10.sup.−9 2.7 × 10.sup.−9 Z13627 49 1.2 × 10.sup.−9 2.9 × 10.sup.−9 Z13628 50 1.3 × 10.sup.−9 5.5 × 10.sup.−9 Z13629 51 1.2 × 10.sup.−9 8.5 × 10.sup.−9 Z13633 52 1.5 × 10.sup.−9 6.2 × 10.sup.−9 Z13634 53 1.1 × 10.sup.−9 2.3 × 10.sup.−9 Z13635 54 1.0 × 10.sup.−9 1.7 × 10.sup.−9 Z13637 55 1.3 × 10.sup.−9 4.8 × 10.sup.−9 Z13638 56 1.2 × 10.sup.−9 2.9 × 10.sup.−9 Z13639 57 1.3 × 10.sup.−9 3.0 × 10.sup.−9 Z13640 58 1.1 × 10.sup.−9 1.9 × 10.sup.−9 Z13641 59 1.1 × 10.sup.−9 1.8 × 10.sup.−9 Z13644 60 1.3 × 10−9 2.8 × 10−9 Z13645 61 1.2 × 10.sup.−9 2.5 × 10.sup.−9 Z13648 62 1.6 × 10.sup.−9 3.3 × 10.sup.−9 Z13651 63 1.2 × 10.sup.−9 2.7 × 10.sup.−9 Z13652 64 1.4 × 10.sup.−9 2.9 × 10.sup.−9 Z13654 65 9.5 × 10.sup.−10 2.9 × 10.sup.−9 Z13655 66 1.1 × 10.sup.−9 2.4 × 10.sup.−9 Z13656 67 1.1 × 10.sup.−9 3.7 × 10.sup.−9 Z13657 68 2.1 × 10.sup.−9 3.9 × 10.sup.−9 Z13659 69 2.2 × 10.sup.−9 3.1 × 10.sup.−9 Z13663 70 9.3 × 10.sup.−10 1.5 × 10.sup.−9 Z13664 71 2.4 × 10.sup.−9 4.2 × 10.sup.−9 Z13667 72 1.2 × 10.sup.−9 2.3 × 10.sup.−9 Z13669 73 9.2 × 10.sup.−10 1.7 × 10.sup.−9 Z13672 74 2.5 × 10.sup.−9 5.6 × 10.sup.−9 Z13674 75 9.2 × 10.sup.−10 1.3 × 10.sup.−9 Z13675 76 9.6 × 10.sup.−10 2.2 × 10.sup.−9 Z13676 77 9.4 × 10.sup.−10 3.1 × 10.sup.−9 Z13678 78 2.0 × 10.sup.−9 3.3 × 10.sup.−9 Z13684 79 1.0 × 10.sup.−9 2.2 × 10.sup.−9 Z13688 80 1.3 × 10.sup.−9 2.1 × 10.sup.−9 Z13691 81 1.8 × 10.sup.−9 2.7 × 10.sup.−9 Z13692 82 1.3 × 10.sup.−9 3.7 × 10.sup.−9 Z13694 83 9.8 × 10.sup.−10 3.6 × 10.sup.−9 Z13695 84 1.8 × 10.sup.−9 5.3 × 10.sup.−9 Z13697 85 1.2 × 10.sup.−9 2.4 × 10.sup.−9 Z13706 86 2.0 × 10.sup.−9 6.4 × 10.sup.−9 Z13708 87 1.9 × 10.sup.−9 4.4 × 10.sup.−9 Z13710 88 1.6 × 10.sup.−9 2.6 × 10.sup.−9 Z13711 89 2.1 × 10.sup.−9 4.9 × 10.sup.−9 Z13714 90 2.1 × 10.sup.−9 6.0 × 10.sup.−9 Z13716 91 1.8 × 10.sup.−9 5.8 × 10.sup.−9 Z13719 92 2.6 × 10.sup.−9 7.3 × 10.sup.−9 Z13720 93 2.5 × 10.sup.−9 4.5 × 10.sup.−7 Z13721 94 1.9 × 10.sup.−9 2.9 × 10.sup.−9 Z13725 95 1.8 × 10.sup.−9 4.9 × 10.sup.−9 Z13727 96 2.1 × 10.sup.−9 5.9 × 10.sup.−9 Z13728 97 2.6 × 10.sup.−9 6.7 × 10.sup.−9 Z13732 98 2.1 × 10.sup.−9 9.4 × 10.sup.−9 Z13735 99 1.6 × 10.sup.−9 9.1 × 10.sup.−9 Z13736 100 1.7 × 10.sup.−9 3.0 × 10.sup.−9 Z13740 101 2.0 × 10.sup.−9 5.0 × 10.sup.−9 Z13742 102 2.4 × 10.sup.−9 7.6 × 10.sup.−9 Z13747 103 1.3 × 10.sup.−9 2.3 × 10.sup.−9 Z13749 104 2.8 × 10.sup.−9 1.2 × 10.sup.−8 Z13750 105 2.7 × 10.sup.−9 8.4 × 10.sup.−9 Z13751 106 2.0 × 10.sup.−9 3.8 × 10.sup.−9 Z13752 107 2.0 × 10.sup.−9 5.8 × 10.sup.−9 Z13758 108 1.9 × 10.sup.−9 6.5 × 10.sup.−9 Z13759 109 2.1 × 10.sup.−9 5.6 × 10.sup.−9 Z13760 110 2.1 × 10.sup.−9 5.8 × 10.sup.−9 Z13761 111 1.9 × 10.sup.−9 3.7 × 10.sup.−9 Z13771 112 1.5 × 10.sup.−9 2.0 × 10.sup.−9 Z13773 113 2.5 × 10.sup.−9 4.9 × 10.sup.−9 Z13776 114 2.2 × 10.sup.−9 5.5 × 10.sup.−9 Z13777 115 2.4 × 10.sup.−9 4.6 × 10.sup.−9 Z13780 116 2.1 × 10.sup.−9 4.0 × 10.sup.−9 Z13782 117 2.2 × 10.sup.−9 4.2 × 10.sup.−9 Z13783 118 1.4 × 10.sup.−9 2.2 × 10.sup.−9 Z13786 119 2.3 × 10.sup.−9 4.7 × 10.sup.−9 Z13792 120 2.0 × 10.sup.−9 2.9 × 10.sup.−9 Z13796 121 2.3 × 10.sup.−9 4.2 × 10.sup.−9 Z13799 122 1.9 × 10.sup.−9 5.6 × 10.sup.−9 Z13806 123 1.6 × 10.sup.−9 3.1 × 10.sup.−9 Z13808 124 2.4 × 10.sup.−9 5.5 × 10.sup.−9 Z13811 125 2.0 × 10.sup.−9 3.1 × 10.sup.−9 Z13812 126 2.3 × 10.sup.−9 1.1 × 10.sup.−8 Z13823 127 2.9 × 10.sup.−9 3.8 × 10.sup.−9 Z13824 128 1.9 × 10.sup.−9 3.8 × 10.sup.−9 Z13838 129 2.6 × 10.sup.−9 5.4 × 10.sup.−9 Z13840 130 2.2 × 10.sup.−9 4.1 × 10.sup.−9 Z13842 131 2.2 × 10.sup.−9 5.5 × 10.sup.−9 Z13845 132 2.6 × 10.sup.−9 4.2 × 10.sup.−9 Z13846 133 2.3 × 10.sup.−9 4.3 × 10.sup.−9 Z13848 134 2.1 × 10.sup.−9 3.1 × 10.sup.−9 Z13849 135 2.1 × 10.sup.−9 3.0 × 10.sup.−9 Z13860 136 2.3 × 10.sup.−9 8.7 × 10.sup.−9 Z13865 137 2.5 × 10.sup.−9 5.6 × 10.sup.−9 Z13866 138 2.0 × 10.sup.−9 2.8 × 10.sup.−9 Z13875 139 2.0 × 10.sup.−9 3.4 × 10.sup.−9 Z13879 140 2.1 × 10.sup.−9 3.0 × 10.sup.−9
Example 9
Production and Characterization of Z Variants from a Maturated Library
[0505] In this Example, twelve Z variants were produced in E. coli, purified and assayed for stability, for binding to FcRn as well as for inhibition of IgG binding to FcRn.
Materials and Methods
[0506] Subcloning of Z Variants into Expression Vectors:
[0507] The DNA of twelve FcRn binding Z variants (Z13577 (SEQ ID NO:19), Z13578 (SEQ ID NO:20), Z13583 (SEQ ID NO:23), Z13592 (SEQ ID NO:28), Z13616 (SEQ ID NO:41), Z13621 (SEQ ID NO:44), Z13654 (SEQ ID NO:65), Z13663 (SEQ ID NO:70), Z13669 (SEQ ID NO:73), Z13674 (SEQ ID NO:75), Z13675 (SEQ ID NO:76) and Z13676 (SEQ ID NO:77)) was amplified from the library vector pAY02592. The subcloning was performed as described in Example 3. The Z gene fragments were subcloned into the expression vector pAY01448 resulting in the encoded sequence MGSSHHHHHHLQ-[Z#####]-VD.
[0508] Production of Z Variants:
[0509] Cultivation and purification of the His.sub.6-tagged Z variants were performed essentially as described in Example 3. In order to obtain higher purity, a reversed phase chromatography (RPC) step was added after the IMAC purification of a second batch of the twelve matured variants and the primary Z variant Z07918. Samples from this batch were used where indicated.
[0510] CD Analysis:
[0511] In order to determine the melting temperatures (Tm) and assess the secondary structure of the Z variants (RPC purified batch), CD analysis was carried out as described in Example 3.
[0512] Biacore Binding and Kinetic Analyses:
[0513] The interaction of FcRn binding His.sub.6-tagged Z variants with human FcRn was analyzed in a Biacore 2000 instrument essentially as described in Example 3. Human FcRn (hFcRn) or cynomolgus FcRn (cFcRn) purchased from Biorbyt (cat. no. orb84388 and orb99075, respectively) were used as target protein. In a first set of experiments, 100 nM of the Z variants was injected at pH 6.0 during 2 min at 30 μl/min over immobilized hFcRn followed by dissociation in buffers of pH 6.0 or pH 7.4 using the co-inject procedure. The dissociation phase was 4 min and the equilibration time between the analyte injections was 30 min. In a second set of experiments, approximate kinetic constants (k.sub.on and k.sub.off) and affinities (K.sub.D) were determined for a subset of Z variants injected at concentrations of 540 nM, 180 nM, 60 nM, 20 nM and 6.7 nM over immobilized hFcRn. As above, the analytes were injected during 2 min at 30 μl/min, the dissociation phase was 4 min and the equilibration time between the analyte injections was 30 min.
[0514] In a third set of experiments, a kinetic analysis of the twelve matured Z variants and the primary Z variant Z07918 (SEQ ID NO:1) (RPC purified batches) binding to hFcRn and cFcRn was performed at pH 6. A concentration series of His.sub.6-tagged Z variants (270, 90, 30 and 10 nM) were injected during 4 min at 30 μl/min over hFcRn and cFcRn, immobilized in different flow cells of a CM5 chip surface. 0.005% PCT pH 6.0 was used as running buffer and for dilutions of the His.sub.6-tagged Z variants. Dissociation in running buffer was allowed for 20 min, followed by surface regeneration by injection of 3×30 second pulses of 0.005% PCT pH 7.4 and equilibration ten minutes before the start of next cycle.
[0515] AlphaLISA Blocking Assay:
[0516] The potential of Z variants to inhibit binding of IgG to FcRn was analyzed in the AlphaLISA assay described in Example 3.
Results
[0517] Production of Z Variants:
[0518] The twelve FcRn binding Z variants constructed with an N-terminal His.sub.6 tag were produced in E. coli. SDS-PAGE analysis of each final protein preparation showed that these predominantly contained the FcRn binding Z variant. The correct identity and molecular weight of each FcRn binding Z variant was confirmed by HPLC-MS analysis.
[0519] CD Analysis:
[0520] Determined melting temperatures are shown in Table 11. Reversible folding was seen for all FcRn binding Z variants when overlaying spectra measured before and after heating to 90° C.
[0521] Biacore Binding and Kinetic Analyses:
[0522] In a first set of experiments, the binding of the twelve Z variants to human FcRn and the dissociation at different pH were tested in a Biacore instrument by sequentially injecting each of the Z variants at pH 6.0 and either buffer pH 6.0 or buffer pH 7.4 over a chip surface containing FcRn. The ligand immobilization level of the surface was 890 RU human FcRn. The twelve Z variants showed binding to FcRn at pH 6.0, and for all variants, faster off-rates were seen at pH 7.4 compared to pH 6.0.
[0523] The kinetic constants of the Z variants Z13577 (SEQ ID NO:19) and Z13621 (SEQ ID NO:44) interacting with FcRn at pH 6.0 were determined in a second set of experiments (see Table 12). Kinetic constants were calculated using curve sets of two or four injected concentrations of Z13577 and Z13621, respectively.
TABLE-US-00011 TABLE 11 Melting temperatures for a set of matured FcRn binding Z variants. Z variant SEQ ID NO: Tm (°C.) Z13577 19 61 Z13578 20 57 Z13583 23 51 Z13592 28 58 Z13616 41 60 Z13621 44 49 Z13654 65 58 Z13663 70 60 Z13669 73 45 Z13674 75 50 Z13675 76 48 Z13676 77 45 Z07918 1 49
TABLE-US-00012 TABLE 12 Biacore kinetic constants and affinities for FcRn binding at pH 6.0. Z variant SEQ ID NO: k.sub.on (M.sup.−1s.sup.−1) k.sub.off (s.sup.−1) K.sub.D (M) Z13577 19 3.0 × 10.sup.5 4.0 × 10.sup.−3 13 × 10.sup.−9 Z13621 44 6.4 × 10.sup.5 3.7 × 10.sup.−3 6 × 10.sup.−9
[0524] In a third set of experiments, the kinetic constants of thirteen His.sub.6-tagged Z variants interacting with human or cynomolgus FcRn at pH 6.0 were determined (Table 13). The FcRn immobilization levels of the chip surfaces were 1196 RU (human) and 788 RU (cynomolgus), respectively. For each Z variant, kinetic constants were calculated using a curve set of four injected concentrations.
TABLE-US-00013 TABLE 13 Biacore kinetic constants and affinities for human and cynomolgus FcRn binding at pH 6.0. hFcRn cFcRn Z SEQ k.sub.on k.sub.off k.sub.on k.sub.off variant ID NO: (M.sup.−1s.sup.−1) (s.sup.−1) K.sub.D (M) (M.sup.−1s.sup.−1) (s.sup.−1) K.sub.D (M) Z13577 19 7.2 × 10.sup.5 2.9 × 10.sup.−3 4.1 × 10.sup.−9 8.2 × 10.sup.5 4.4 × 10.sup.−3 5.4 × 10.sup.−9 Z13578 20 4.1 × 10.sup.5 7.3 × 10.sup.−3 1.8 × 10.sup.−8 5.6 × 10.sup.5 1.1 × 10.sup.−2 2.1 × 10.sup.−8 Z13583 23 4.5 × 10.sup.5 2.6 × 10.sup.−3 5.8 × 10.sup.−9 6.7 × 10.sup.5 4.4 × 10.sup.−3 6.6 × 10.sup.−9 Z13592 28 5.9 × 10.sup.5 7.2 × 10.sup.−3 1.2 × 10.sup.−8 7.5 × 10.sup.5 1.1 × 10.sup.−2 1.5 × 10.sup.−8 Z13616 41 2.9 × 10.sup.5 3.1 × 10.sup.−3 1.0 × 10.sup.−8 4.3 × 10.sup.5 4.8 × 10.sup.−3 1.1 × 10.sup.−8 Z13621 44 4.1 × 10.sup.5 2.8 × 10.sup.−3 6.8 × 10.sup.−9 6.1 × 10.sup.5 4.6 × 10.sup.−3 7.6 × 10.sup.−9 Z13654 65 6.0 × 10.sup.5 9.5 × 10.sup.−3 1.6 × 10.sup.−8 8.4 × 10.sup.5 1.3 × 10.sup.−2 1.5 × 10.sup.−8 Z13663 70 3.9 × 10.sup.5 3.4 × 10.sup.−3 8.7 × 10.sup.−9 5.2 × 10.sup.5 5.3 × 10.sup.−3 1.0 × 10.sup.−8 Z13669 73 5.6 × 10.sup.5 2.8 × 10.sup.−3 4.9 × 10.sup.−9 8.2 × 10.sup.5 4.6 × 10.sup.−3 5.6 × 10.sup.−9 Z13674 75 5.3 × 10.sup.5 3.7 × 10.sup.−3 7.0 × 10.sup.−9 8.3 × 10.sup.5 5.9 × 10.sup.−3 7.1 × 10.sup.−9 Z13675 76 4.9 × 10.sup.5 5.1 × 10.sup.−3 1.0 × 10.sup.−8 7.5 × 10.sup.5 8.0 × 10.sup.−3 1.1 × 10.sup.−8 Z13676 77 6.5 × 10.sup.5 3.6 × 10.sup.−3 5.5 × 10.sup.−9 9.6 × 10.sup.5 5.9 × 10.sup.−3 6.2 × 10.sup.−9 Z07918 1 2.6 × 10.sup.5 4.2 × 10.sup.−3 1.6 × 10.sup.−8 3.8 × 10.sup.5 7.0 × 10.sup.−3 1.9 × 10.sup.−8
TABLE-US-00014 TABLE 14 Calculated IC50 values from AlphaLISA blocking assay. Z variant SEQ ID NO: IC50 (M) Z13577 19 1.2 × 10.sup.−8 Z13578 20 1.2 × 10.sup.−8 Z13583 23 2.7 × 10.sup.−9 Z13592 28 6.4 × 10.sup.−9 Z13616 41 7.4 × 10.sup.−9 Z13621 44 3.2 × 10.sup.−9 Z13654 65 3.5 × 10.sup.−9 Z13663 70 1.1 × 10.sup.−8 Z13669 73 5.2 × 10.sup.−9 Z13674 75 2.5 × 10.sup.−9 Z13675 76 8.2 × 10.sup.−9 Z13676 77 3.9 × 10.sup.−9
[0525] AlphaLISA Blocking Analysis:
[0526] The ability of twelve maturated His.sub.6-tagged monomeric Z variants to inhibit IgG binding to FcRn was tested in an AlphaLISA blocking assay. Serial dilutions of the Z variants were incubated with biotinylated human FcRn and the blocking ability of each respective variant was measured after addition of IgG coated Acceptor beads and subsequently streptavidin coated Donor beads. Inhibition could be measured as a decrease in AlphaLISA counts for positive Z variants. All twelve tested Z variants were shown to block IgG binding to FcRn and the calculated IC50 values are shown in Table 14.
Example 10
Comparison of Blocking Capacity of IgG Binding to FcRn
[0527] In this Example, the IgG blocking capacity of the FcRn binding Z variant His.sub.6-Z07918 (SEQ ID NO:1) was compared to Intravenous immunoglobulin (IVIg) and Subcutaneous immunoglobulin (SCIg) currently used in the treatment of some autoimmune disorders.
Materials and Methods
[0528] Blocking of IG-FcRn Immunofluorescence Staining:
[0529] Human or murine FcRn-eGFP transduced HeLa cells were prepared as described in Example 4. Fixed cells were resuspended in 50 μl of a mix of 50 nM Alexa Fluor® 647-conjugated human IgG (Jackson laboratories, cat. no. 009-600-003) and His.sub.6-tagged Z07918, IVIg (Octagam®, Octapharma) or SCIg (Gammanorm®, Octapharma), respectively, diluted at concentrations of 1000, 100, 10, 1, 0.1 or 0 (buffer control) nM in McIlvanes buffer pH 6.0, containing 2.5% FBS Ultra low IgG (Life Technologies) and 0.1% saponin (AppliChem). The cells were incubated for 1 h at 37° C. in the dark, washed with 2×100 μl McIlvanes, pH 6.0, containing 2.5% FBS Ultra low IgG and re-suspended in 180 μl of McIlvanes, pH 6.0, containing 1% BSA. Data from 10,000 GFP/FcRn positive cells were obtained using a FACS Calibur (Beckman Coulter) and the data was analyzed using Flowing software 2.5.0 (Turku University).
Results
[0530] The experiment was performed to determine if the FcRn binding Z variant His.sub.6-Z07918 (SEQ ID NO:1) blocks the IgG-FcRn interaction and compare the blocking effect to IVIg and SCIg. Human or murine FcRn-eGFP transduced HeLa cells were incubated with human Alexa Fluor@ 647-conjugated IgG. The binding was blocked with unlabeled His.sub.6-Z07918, IVIg or SCIg at different concentrations. The results showed that His.sub.6-Z07918 effectively blocked hIgG binding to hFcRn to a similar extent as IVIg or SCIg (
Example 11
Increased IgG Catabolism by FcRn Binding Z Variants in Mice
[0531] The ability of the FcRn binding Z variant Z07918 to block IgG binding to FcRn in vitro was shown in Example 10. In this example, the blocking ability of the same Z variant was evaluated in vivo. Blocking of IgG-FcRn interactions in vivo will lead to increased IgG catabolism and concomitant reduced levels of IgG (Mezo 2008, supra).
Materials and Methods
[0532] Animal Study:
[0533] The FcRn-binding Z variants Z11948 (SEQ ID NO:354) and Z07918-PP013 (Z07918 (SEQ ID NO:1) identical to Z11948 but with the N-terminus starting with the amino acids VD instead of AE, in fusion with the ABD variant PP013 (SEQ ID NO:377)) or vehicle (PBS buffer), were administered to male NMRI (Charles River), at a dose of 16.3 μmol/kg. The mice were treated with five intravenous injections given at 0, 24, 48, 72 and 96 h. Serum samples were taken at 0, 72, 120 and 168 h (termination of study) and stored at −20° C. The concentration of mouse IgG in serum was quantified by ELISA.
[0534] Mouse IgG ELISA:
[0535] The concentration of mouse IgG in mouse serum samples was analyzed by a mouse IgG ELISA kit (Mabtech 3825-1AD-6) and performed as described by the manufacturer. The concentration of mIgG was calculated from a standard curve provided and GraphPad Prism 5 using a non-linear regression formula. The concentration of IgG in individual mice at 24, 72, 120 and 168 h were related to the level at 0 h and the results are therefore presented as percentage of IgG (0 h).
Results
[0536] The results showed a reduction of mouse IgG concentration in mice treated with FcRn-specific Z variants. Both Z11948 and the ABD-fused variant Z07918-PP013 lowered the concentration of endogenous IgG in mice in vivo. Most pronounced effects were obtained with the ABD-fused variant and after 120 hours. Thus, the results indicates that the FcRn-specific Z variants blocked recycling of IgG resulting in increased IgG catabolism and subsequent lower levels of IgG in mice.
Example 12
In Vitro Transcytosis of FcRn Binding Z Variants
[0537] In this Example, the FcRn binding Z variants are tested for their ability to be transported through epithelial or endothelial cells or recycled by FcRn in vitro. A drug containing a Z variant with the power of transcytosis will facilitate drug uptake after for example oral or pulmonary administration.
Materials and Methods
[0538] Cells, for example T84, MDCK, HeLa, CaCo2, CaLu-1 and/or CaLu-3 cells, with or without endogenous or recombinant expression of FcRn, are grown in respective growth medium on a membrane in a transwell to form a monolayer. The integrity of monolayers can be evaluated by measuring the electrical resistance or adding a probe that is not able to penetrate or being actively transported over the cell monolayer. A defined monolayer of cells is pulsed from the apical or basolateral side with ligand such as FcRn binding Z variants, HSA or IgG in a buffer such as HBSS (Hanks' Balanced Salt Solution, SigmaAldrich, cat. no. H9269) or growth medium at a suitable pH and temperature, and chased with buffers such as HBSS or growth medium at a suitable pH and temperature on the opposite side.
[0539] In a variant of this assay, ligands can be chased with buffers such as HBSS or growth medium at suitable pH and temperature on the same side as administration to measure recycled ligand as well. This can be done in a transwell or in a cell culture dish. Cells are seeded into transwell or cell culture dishes and pulsed with ligands such as FcRn binding Z variants, HSA or IgG. Endocytosed ligands will bind to FcRn and return to the cell surface at the same or opposite side as they were loaded. After pulsing, free ligands are removed by washing the cells with cold buffer. To chase ligands, warm buffer or medium is added to the cells and, after a period in the range from 10 minutes to several hours, the buffer or medium is removed and assayed for the presence of ligands.
[0540] In a variant of this assay, ligands such as FcRn binding Z variants, HSA or IgG can be used to block the binding to FcRn by ligands such as other FcRn binding Z variants, HSA or IgG by administering them at the same time or sequentially to the cells.
[0541] The amount of ligand can be quantified by methods such as ELISA, HPLC-MS, fluorescent dye or radio labeling.
[0542] The results of the experiment described above are expected to show that the FcRn-specific Z variants can be transcytosed and/or recycled in vitro.
Example 13
Binding of Homodimeric FcRn Binding Polypeptides to Human FcRn/eGFP Transfected HeLa Cells
[0543] In this Example, the binding ability of homodimeric FcRn binding polypeptides was investigated and compared to the binding ability of monomeric primary and maturated Z variants. The production of HeLa cells expressing human FcRn-eGFP gene transgene was performed as described in Example 4 and the use of these cells for flow cytometry analysis with Alexa Fluor@ 647 labeled Z variants is described.
Materials and Methods
[0544] Alexa Fluor@ 647 Labeling of FcRn Binding Polypeptides:
[0545] The two homodimeric His.sub.6-tagged polypeptides Z11948-(G.sub.4S).sub.3-Z11948 (SEQ ID NO:369) and Z11948-(G.sub.4S)-Z11948 (SEQ ID NO:368), the primary monomeric His.sub.6-tagged Z variant Z07918 (SEQ ID NO:1) and the maturated monomeric His.sub.6-tagged Z-variants Z13583 (SEQ ID NO:23), Z13621 (SEQ ID NO:44), Z13654 (SEQ ID NO:65) and Z13674 (SEQ ID NO:75) were labeled with Alexa Fluor@ 647 Carboxylic Acid Succinimidyl Ester (Invitrogen cat. no. A20106). Before labeling, the pH in the sample suspensions (in PBS pH 7.4) was adjusted to 8.3 by addition of 10 μl of 0.1 M sodium bicarbonate buffer, pH 8.3, to 90 μl sample suspension. 10 μl of Alexa Fluor® 647 Succinimidyl Ester dye (10 mg/ml in DMSO corresponding to 4×molar excess) was added to 100 μl of each sample suspension. The mixes were incubated at RT in the dark for 1 h in a wiggling rota mixer. The reaction mixes were immediately transferred to dialysis cassettes (3500 MWCO) (Thermo Scientific cat. no. 66333) and free dye was removed by dialysis in PBS pH 7.4.
[0546] Immunofluorescence Staining of Human FcRn-eGFP Transfected HeLa-Cells with FcRn Binding Polypeptides:
[0547] hFcRn-eGFP HeLa cells were harvested by trypsination and washed twice in McIlvanes buffer, pH 6.0 before counting. 100,000 cells were pipetted per well of a v-bottomed 96 well plate (Nunc, cat no 277143) and the cells in the plate were subsequently pelleted at 1,700 rpm for 4 min at 4° C. The supernatants were removed and the cells were fixed with 50 μl of 2% formaldehyde (Sigma Aldrich, cat. no. F8775) in McIlvanes buffer for 10 min at RT. Cells were thereafter washed with 2×100 μl McIlvanes buffer, pH 6.0, containing 2.5% FBS Ultra low IgG (Life Technologies), and resuspended in McIlvanes buffer, pH 6.0, containing 2.5% FBS Ultra low IgG and 0.1% saponin (AppliChem, cat no A4518.0100) containing 640 nM of Alexa Fluor® 647 labeled His.sub.6-tagged polypeptides; Z11948-(G.sub.4S).sub.3-Z11948 and Z11948-(G.sub.4S)-Z11948 and Z07918. Transduced HeLa cells, incubated with buffer alone, were used as control. The cells were incubated for 1 h at 8° C. on a shaker in the dark. The cells were then subjected to two different washing conditions; 2×150 μl McIlvanes buffer, pH 6.0, containing 2.5% FBS ultra low IgG or 2×150 μl PBS, pH 7.4, containing 2.5% FBS Ultra low IgG and a 20 min incubation step in PBS, pH 7.4, containing 2.5% FBS Ultra low IgG. After washing, all samples were re-suspended in 180 μl of McIlvanes, pH 6.0, containing 2.5% FBS Ultra low IgG. Data from 10,000 GFP/FcRn positive cells were obtained using a FACS Calibur (Beckman Coulter) and the data was analyzed using Flowing software 2.5.0 (Turku University).
Results
[0548] Flow cytometry analysis was utilized to determine whether FcRn binding dimers could bind to human FcRn on human FcRn/eGFP transduced HeLa cells and to compare their binding ability to the monomeric FcRn binding Z variants. The analysis was also performed to determine if the pH dependent detachment from the FcRn protein was affected by the dimeric format. The experiment was performed at pH 6.0 with washings at pH 6.0 or pH 7.4 with Alexa Fluor® 647 labeled dimers Z11948-(G.sub.4S).sub.3-Z11948 and Z11948-(G.sub.4S)-Z11948, and monomers Z07918, Z13583, Z13621, Z13654 and Z13674. Z11948 and Z07918 are identical in sequence apart from the first two amino acid residues (AE vs VD). The calculated MFI values are presented in
Example 14
Comparison of Blocking Capacity of IgG Binding to FcRn
[0549] In this Example, the IgG blocking capacity of the FcRn binding dimers Z11948-(G.sub.4S).sub.3-Z11948 and Z11948-(G.sub.4S)-Z11948 was compared to that of monomeric FcRn binding Z variants, as well as to intravenous immunoglobulin (IVIg) and subcutaneous immunoglobulin (SCIg) currently used in the treatment of some autoimmune disorders.
Materials and Methods
[0550] Blocking of IgG-FcRn Immunofluorescence Staining:
[0551] Human FcRn-eGFP transduced HeLa cells were prepared as described in Example 4. Fixed cells were resuspended in 50 μl of a mix of 50 nM Alexa Fluor@ 647-conjugated human IgG (Jackson laboratories, cat. no. 009-600-003) and His.sub.6-tagged Z11948-(G.sub.4S).sub.3-Z11948 (SEQ ID NO:369), Z11948-(G.sub.4S)-Z11948 (SEQ ID NO:368), Z07918 (SEQ ID NO:1), Z13583 (SEQ ID NO:23), Z13621 (SEQ ID NO:44); IVIg (Octagam®, Octapharma) or SCIg (Gammanorm®, Octapharma), respectively, diluted at concentrations of 1000, 100, 10, 1, 0.1 or 0 (buffer control) nM in McIlvanes buffer, pH 6.0, containing 2.5% FBS Ultra low IgG (Life Technologies) and 0.1% saponin (AppliChem). The cells were incubated for 1 h at 37° C. in the dark, washed with 2×100 μl McIlvanes buffer, pH 6.0, containing 2.5% FBS Ultra low IgG and re-suspended in 180 μl of McIlvanes buffer, pH 6.0, containing 1% BSA. Data from 10,000 GFP/FcRn positive cells was obtained using a FACS Calibur (Beckman Coulter) and analyzed using Flowing software 2.5.0 (Turku University).
Results
[0552] The experiment was performed to determine if the FcRn binding dimers Z11948-(G.sub.4S).sub.3-Z11948 and Z11948-(G.sub.4S)-Z11948 block the IgG-FcRn interaction, and compare the blocking effect to that of the monomeric FcRn binding Z variants Z07918, Z13583 and Z13621, as well as IVIg and SCIg. Human FcRn-eGFP transduced HeLa cells were incubated with human Alexa Fluor® 647 conjugated IgG. The binding was blocked with unlabeled Z variants, IVIg or SCIg at different concentrations. The results showed that the FcRn binding dimers have an improved blocking effect in terms of hIgG binding to hFcRn compared to the monomeric Z variant Z07918, IVIg and SCIg (
TABLE-US-00015 TABLE 15 Calculated IC50 values from HeLa cell IgG blocking assay. Designation SEQ ID NO: IC50 (M) Z11948-(G.sub.4S).sub.3-Z11948 369 4.5 × 10.sup.−9 Z11948-(G.sub.4S)-Z11948 368 3.5 × 10.sup.−9 Z13583 23 3.3 × 10.sup.−8 Z13621 44 1.7 × 10.sup.−8 Z07918 1 4.8 × 10.sup.−8 IVIg n.a. 1.7 × 10.sup.−7 SCIg n.a. 1.2 × 10.sup.−7
Example 15
Production of Dimeric FcRn Binding Polypeptides
Materials and Methods
[0553] The Z variants Z17303 (SEQ ID NO:357), Z18632 (SEQ ID NO:365), Z18633 (SEQ ID NO:366) and Z18634 (SEQ ID NO:367) were constructed as dimers in fusion with the albumin binding variant PP013 (SEQ ID NO:377) in the general format [Z#####]-ASGS-PP013-GT-(G.sub.4S)-[Z######]. The resulting polypeptides were denoted ZAZ3824 (SEQ ID NO:373), ZAZ3869 (SEQ ID NO:374), ZAZ3870 (SEQ ID NO:375) and ZAZ3871 (SEQ ID NO:376), respectively. Dimeric polypeptides of the Z variant Z17303 in fusion with PP013 was also constructed with different linkers or C-terminal fusion of PP013, resulting in the polypeptides Z17303-GAP(G.sub.4S).sub.3TS-PP013-GT(G.sub.4S).sub.3PR-Z17303 and Z17303-GAP(G.sub.4S).sub.3TS-Z17303-GT(G.sub.4S).sub.3PR-PP013, denoted ZAZ3715 (SEQ ID NO:371) and ZZA3716 (SEQ ID NO:372), respectively. Furthermore, Z17303 was constructed as a dimer without PP013, but with an N-terminal His.sub.6-tag resulting in the polypeptide GSS-His.sub.6-LQ-Z17303-GT(G.sub.4S).sub.3-Z17303, denoted ZZ3556 (SEQ ID NO:370).
[0554] Cultivation was performed as described in Example 3. Purification of PP013 containing polypeptides was carried out by anti-ABD affinity chromatography and RPC as described in Example 6, whereas purification of His.sub.6-tagged ZZ3556 was performed by IMAC as described in Example 3.
Results
[0555] The seven FcRn binding dimeric polypeptides, constructed either with a His.sub.6-tag or an ABD moiety, were produced in E. coli. The amount of affinity purified protein, determined spectrophotometrically by measuring the absorbance at 280 nm, ranged from 2-18 mg per g bacterial pellet. SDS-PAGE analysis of each final protein preparation showed that these predominantly contained the FcRn binding polypeptide. The correct identity and molecular weight of each FcRn binding polypeptide was confirmed by HPLC-MS analysis.
Example 16
Binding of Homo- and/or Heterodimeric FcRn Binding Polypeptides to Human FcRn/eGFP Transfected HeLa Cells
[0556] In this Example, the binding ability of homo- and/or heterodimeric FcRn binding polypeptides comprising maturated Z variants is investigated. HeLa cells expressing human FcRn-eGFP gene transgene, produced as described in Example 4, are used for flow cytometry analysis with Alexa Fluor® 647 labeled Z variants.
Materials and Methods
[0557] Alexa Fluor@ 647 Labeling of FcRn Binding Z Variants:
[0558] Homo- and/or heterodimeric FcRn binding polypeptides are labeled with Alexa Fluor@ 647 Carboxylic Acid Succinimidyl Ester (Invitrogen cat. no. A20106) as described in Example 13.
[0559] Immunofluorescence Staining of Human FcRn-eGFP Transfected HeLa-Cells with FcRn Binding Polypeptides:
[0560] hFcRn-eGFP HeLa cells are harvested by trypsination and washed twice in McIlvanes buffer, pH 6.0, before counting. 100,000 cells are pipetted per well of a v-bottomed 96 well plate (Nunc, cat no 277143) and the cells in the plate are subsequently pelleted at 1,700 rpm for 4 min at 4° C. The supernatants are removed and the cells are fixed with 50 μl of 2% formaldehyde (Sigma Aldrich, cat. no. F8775) in McIlvanes buffer for 10 min at RT. Cells are thereafter washed with 2×100 μl McIlvanes buffer, pH 6.0, containing 2.5% FBS Ultra low IgG (Life Technologies), and resuspended in McIlvanes buffer, pH 6.0, containing 2.5% FBS Ultra low IgG and 0.1% saponin (AppliChem, cat no A4518.0100) containing 640 nM of Alexa Fluor® 647 labeled His.sub.6-tagged homo- and/or heterodimeric FcRn binding polypeptides and a corresponding monomeric Z variant.
[0561] Examples of formats for homo- and/or heterodimers include Z#####-(G.sub.4S).sub.3-Z##### and Z#####-(G.sub.4S)-Z#####, where Z#### for example is selected from Z13583 (SEQ ID NO:23), Z13621 (SEQ ID NO:44), Z13654 (SEQ ID NO:65) or Z13674 (SEQ ID NO:75), or the same Z variants starting with amino acid residues AE instead of VD, as for example in Z17303 (SEQ ID NO:357), which is identical to Z13621 (SEQ ID NO:44) apart from the N-terminal AE. Cloning may optionally be performed with a C-terminal His.sub.6 tag as described in Example 3 or with an N-terminal His.sub.6 tag as in SEQ ID NO:362.
[0562] Transduced HeLa cells, incubated with buffer alone, are used as control. The cells are incubated for 1 h at 8° C. on a shaker in the dark. The cells are then subjected to two different washing conditions; 2×150 μl McIlvanes buffer, pH 6.0, containing 2.5% FBS Ultra low IgG or 2×150 μl PBS, pH 7.4, containing 2.5% FBS Ultra low IgG and a 20 min incubation step in PBS, pH 7.4, containing 2.5% FBS Ultra low IgG. After washing, all samples are re-suspended in 180 μl of McIlvanes, pH 6.0, containing 2.5% FBS Ultra low IgG. Data from 10,000 GFP/FcRn positive cells are obtained using a FACS Calibur (Beckman Coulter) and the data is analyzed using Flowing software 2.5.0 (Turku University).
Results
[0563] Flow cytometry analysis is used to determine whether the homo- and/or heterodimeric FcRn binding polypeptides comprising maturated Z variants can bind to human FcRn on human FcRn/eGFP transduced HeLa cells and compare the binding ability to the monomeric FcRn binding Z variants or dimeric variants comprising primary Z variants. The analysis is also performed to determine if the pH dependent detachment from the FcRn protein is affected by the dimeric format. The experiments are performed at pH 6.0 with washings at pH 6.0 or pH 7.4 with Alexa Fluor@ 647 labeled FcRn binding Z variants. The results from the experiment are expected to show that homo- and/or heterodimeric formats, as well as the inclusion of maturated Z variants with an improved affinity for FcRn, increase the binding capacity of the FcRn binding polypeptides and that the pH dependent detachment from FcRn is decreased for said polypeptides.
Example 17
pH Dependent Binding of Dimeric Polypeptides to Human FcRn
[0564] In this Example, the capacity of dimeric polypeptides to bind FcRn at different pH values was investigated by ELISA and compared to the binding capacity of a monomeric Z variant.
Materials and Methods
[0565] The capacity of the dimeric polypeptides ZAZ3824 (SEQ ID NO:373), ZAZ3869 (SEQ ID NO:374), ZAZ3870 (SEQ ID NO:375) and ZAZ3871 (SEQ ID NO:376), as well as the monomeric Z variant Z13621 (SEQ ID NO:44), to bind human FcRn at different pH values was tested in an ELISA where all binding and washing steps were performed at either pH 6.0 or pH 7.4. Half-area 96-well ELISA plates were coated at 4° C. overnight with 4 μg/ml of hFcRn (Biorbyt, cat. no. orb84388) diluted in PBS. The plates were washed twice in tap water and the wells were blocked with 100 μl of PBSC (PBS, pH 7.4, supplemented with 1% casein) for 1.5 h at RT. The blocking solution was poured off and the wells subjected to pH 6.0 treatment were washed once with McIlvaines phosphate-citrate buffer, pH 6.0. The different FcRn binding polypeptides were added at a concentration of 100 nM and diluted stepwise 1:10 down to 0.1 μM in either PCC (McIlvaines phosphate-citrate buffer, pH 6.0, supplemented with 1% casein) or PBSC. 50 μl of the dilutions were transferred per well and the ELISA plates were incubated for 1.5 h at RT. The plates were washed four times in either PCT (McIlvaines phosphate-citrate buffer, pH 6.0, supplemented with 0.05% Tween-20) or PBST (PBS, pH 7.4, supplemented with 0.05% Tween-20). Bound polypeptides were detected with 50 μl/well of a Z specific mouse antibody (produced in-house) diluted to 2 μg/ml in either PCC or PBSC. The plates were subsequently incubated for 1.5 h at RT followed by washing as described above. HRP-conjugated goat anti-mouse Ig obtained from DAKO (P0447), diluted 1:5000 in either PCC or PBSC, was added and the plates were incubated at RT for 1 h. After washing (as above), 50 μl of ImmunoPure TMB substrate was added to each well and the plates were developed according to the manufacturer's recommendations. After 30 min of development, the absorbance was measured at 450 nm using a multi-well plate reader (Victor.sup.3) and the EC50 values were calculated using GraphPad Prism 5.
Results
[0566] The analysis was performed to compare the binding potential of monomeric versus dimeric format at different pH, but also to determine whether introduced scaffold mutations (Y5F, N52S and D53E; see further in Examples 24 and 25) would affect the pH dependent binding to FcRn. The experiment was performed in an ELISA format at pH 6.0 or pH 7.4. The results showed that the dimeric format was superior to the monomeric format in binding FcRn regardless of pH (up to 10× and 85× improvement at pH 6.0 and pH 7.4, respectively). The most potent binding, both at pH 6.0 and pH 7.4, was seen for the dimeric polypeptide ZAZ3824, which also showed a similar binding capacity (EC50 value) at pH 6.0 and pH 7.4. The ELISA titration curves are shown in
TABLE-US-00016 TABLE 16 Calculated EC50 values from binding analysis at pH 6.0 and pH 7.4 EC50 (M) Designation SEQ ID NO: pH 6.0 pH 7.4 Z13621 44 9.9 × 10.sup.−10 7.7 × 10.sup.−9 ZAZ3824 373 9.9 × 10.sup.−11 9.1 × 10.sup.−11 ZAZ3869 375 3.0 × 10.sup.−10 7.3 × 10.sup.−10 ZAZ3870 375 2.6 × 10.sup.−10 5.7 × 10.sup.−10 ZAZ3871 376 1.4 × 10.sup.−10 1.8 × 10.sup.−10
Example 18
Comparison of Blocking Capacity of IgG Binding to FcRn
[0567] In this Example, the potential of polypeptides to inhibit binding of IgG to FcRn was analyzed using two different in vitro methods: AlphaLISA and a cell based assay.
Materials and Methods
[0568] AlphaLISA Blocking Assay:
[0569] The capacity of the dimeric FcRn binding polypeptides ZZ3556 (SEQ ID NO:370), ZAZ3715 (SEQ ID NO:371) and ZZA3716 (SEQ ID NO:372) as well as the monomeric Z13621 (SEQ ID NO:44) to block IgG-FcRn interaction was analyzed using an AlphaLISA assay. Human IgG (Roactemra) was immobilized on AlphaLISA acceptor beads (Perkin Elmer, cat. no. 6772002) according to the manufacturer's recommendations. Human FcRn (Biorbyt, cat. no. orb84388) was biotinylated essentially as described in Example 2. Polypeptides were serially diluted 1:3 in AlphaLISA buffer (Perkin Elmer, cat. no. AL000F) pH 6.0 (adjusted using HCl) to final concentrations of 250 nM to 13 pM in a 384-well plate (Perkin Elmer, cat. no. G6005350) and incubated for 45 min with 10 nM biotinylated hFcRn. IgG-coated acceptor beads were added to a final concentration of 10 μg/ml and incubated for 45 min. Finally, streptavidin coated donor beads (Perkin Elmer, cat. no. 6760002) were added to a final concentration of 40 μg/ml and incubated for 30 min. All incubations were performed at RT in the dark. The plate was analyzed in the EnSpire multiplate reader 2300 (Perkin Elmer) and the IC50 values were calculated using GraphPad Prism 5.
[0570] HeLa Cell IgG-FcRn Blocking Assay:
[0571] Human FcRn-eGFP transduced HeLa cells were prepared as described in Example 4. The polypeptides ZZ3556 (SEQ ID NO:370), ZAZ3715 (SEQ ID NO:371), ZZA3716 (SEQ ID NO:372), ZAZ3824 (SEQ ID NO:373), ZAZ3869 (SEQ ID NO:374), ZAZ3870 (SEQ ID NO:375), ZAZ3871 (SEQ ID NO:376), Z13621 (SEQ ID NO:44), Z18632 (SEQ ID NO:365), Z18633 (SEQ ID NO:366) and Z18634 (SEQ ID NO:367), as well as IVIg (Octagam®, Octapharma) or SCIg (Gammanorm®, Octapharma), were each diluted to concentrations of 1000, 100, 10, 1, 0.1 or 0 (buffer control) nM in McIlvanes buffer, pH 6.0, containing 2.5% FBS Ultra low IgG (Life Technologies) and 0.1% saponin (AppliChem) and 50 nM Alexa Fluor@ 647-conjugated human IgG (Jackson laboratories, cat. no. 009-600-003). Fixed cells were resuspended in 100 μl of the mixture and were incubated for 1 h at 37° C. in the dark. Cells were washed and resuspended in McIlvanes buffer, pH 6.0, containing 2.5% FBS Ultra low IgG. Data from 10,000 GFP/FcRn positive cells was obtained using a FACS Calibur (Beckman Coulter), analyzed using Flowing software 2.5.0 (Turku University) and IC50 values were calculated using GraphPad Prism 5.
Results
[0572] AlphaLISA:
[0573] The ability of one monomeric and three dimeric polypeptides to inhibit IgG binding to FcRn was tested in an AlphaLISA blocking assay. The results show that the dimeric polypeptides had better IgG blocking capacity compared to the monomeric format. The two ABD fused dimeric polypeptides, ZAZ3715 and ZZA3716, had IC50 values very similar to that of ZZ3556, which does not contain an ABD moiety. The calculated IC50 values of the AlphaLISA blocking assay are summarized in Table 17.
[0574] HeLa Cell IgG Blocking Assay:
[0575] Human FcRn-eGFP transduced HeLa cells were incubated with human Alexa Fluor 647-conjugated IgG and selected polypeptides to assess the ability of the polypeptides to block IgG-FcRn interactions. Intravenous immunoglobulin (IVIg) and Subcutaneous immunoglobulin (ScIg) currently used in the treatment of some autoimmune disorders, were also included in the test. The experiment was performed at pH 6.0. The results showed that the dimeric format had an improved IgG blocking capacity compared to the monomeric format. ZAZ3715 (longer linkers) was compared to ZAZ3824 (shorter linkers), and the two constructs showed similar IC50 values. There was also no difference between IC50 values obtained for ZAZ3715 and ZZA3716, i.e. dimer constructs with ABD at different positions. All tested polypeptides had superior IgG blocking effect compared to IVIg and SCIg. The calculated IC50 values of the cell blocking assay are summarized in Table 18.
TABLE-US-00017 TABLE 17 Calculated IC50 values showing IgG blocking capacity of polypeptides in AlphaLISA. Designation SEQ ID NO: IC50 (M) Z13621 44 1.0 × 10.sup.−9 ZZ3556 370 1.9 × 10.sup.−10 ZAZ3715 371 1.7 × 10.sup.−10 ZZA3716 372 1.8 × 10.sup.−10
TABLE-US-00018 TABLE 18 Calculated IC50 values showing IgG blocking capacity of polypeptides in a HeLa cell based assay at pH 6.0. Designation SEQ ID NO: IC50 (M) Z13621 44 2.2 × 10.sup.−8 Z18632 365 8.4 × 10.sup.−8 Z18633 366 5.7 × 10.sup.−8 Z18634 367 3.6 × 10.sup.−8 ZZ3556 370 1.9 × 10.sup.−9 ZAZ3715 371 3.6 × 10.sup.−9 ZZA3716 372 3.5 × 10.sup.−9 ZAZ3824 373 3.8 × 10.sup.−9 ZAZ3869 374 4.7 × 10.sup.−9 ZAZ3870 375 5.3 × 10.sup.−9 ZAZ3871 376 4.1 × 10.sup.−9 SCIg — 1.2 × 10.sup.−7 IVIg — 1.5 × 10.sup.−7
Example 19
Comparison of Blocking Capacity of IgG Recycling
[0576] In this Example, the effect of homo- and/or heterodimeric FcRn binding polypeptides on IgG recycling are investigated in FcRn transduced MDCK.2 cells.
Materials and Methods
[0577] Lentiviral Transduction of MDCK.2 Cells:
[0578] The vector pairs 2k7.sub.neo-CMV-hB2M and pHR-cPPT-CMV-hFcRn-eGFP are co-transfected together with VSV-G envelope and gag/pol packaging plasmid into HEK293T cells using calcium chloride transfection (Zufferey et al., supra; Jakobsson et al. (2006) supra). HEK293T culture supernatants containing formed lentiviral particles with FcRn and B2M transgenes respectively are used to sequentially transduce MDCK.2 cells (ATCC cat. no. CRL-2936) at low passage number. The resulting stably transduced MDCK.2 cell lines are denoted hFcRn-eGFP (transduced with genes for human FcRn-eGFP and hB2M).
[0579] Blocking Capacity of IgG Recycling:
[0580] Human FcRn-eGFP transduced MDCK.2 cells are plated at 25 000 cells/well in a 96-well plate and incubated overnight at 37° C., 5% CO.sub.2. The cells are incubated with homo- and/or heterodimeric FcRn binding polypeptides at concentrations ranging from 200 to 0.01 nM in McIlvanes buffer, pH 6.0, containing 2.5% FBS Ultra low IgG before addition of 500 ng/ml Alexa Fluor@ 647-conjugated human IgG (Jackson laboratories, cat. no. 009-600-003) and incubation for one additional hour. The cells are washed McIlvanes buffer, pH 6.0, containing 2.5% FBS Ultra low IgG and then incubated in PBS, pH 7.4, containing 2.5% FBS Ultra low IgG for 2 h in 37° C., 5% CO.sub.2. The supernatants are then analyzed for the presence of the Alexa Fluor® 647-conjugated human IgG in an EnSpire multiplate reader (Perkin Elmer). The inhibition curves are analyzed by non-linear regression using the GraphPad Prism 5 software to determine the IC50 values.
Results
[0581] The results from the experiment are expected to show a dose dependent reduction in IgG recycling through the action of dimeric FcRn binding polypeptides.
Example 20
Increased IgG Catabolism by Dimeric FcRn Binding Polypeptides in FcRn Transgenic Mice
[0582] In this Example, the effect of homodimeric FcRn binding polypeptides on IgG catabolism was investigated in human FcRn transgenic mice on two different occasions.
Materials and Methods
[0583] Animal Studies:
[0584] In the first study, homodimeric FcRn-binding polypeptide ZAZ3715 (SEQ ID NO:371) or ZZA3716 (SEQ ID NO:372), or vehicle (PBS buffer), was administered at 0 hours by i.v. injection to male B6.Cg-Fcgrttml Dcr Tg(FCGRT)32Dcr/DcrJ mice (Jackson Laboratory, stock no. 14565) at a dose of 6 mg/kg. 24 hours prior to polypeptide administration, 500 mg/kg hIgG (Kiovig, Baxter) was administered by i.v. injection. Serum samples were collected at −168, 0, 24, 72, 120 and 144 h (termination of study) and stored at −20° C.
[0585] In the second study, homodimeric FcRn-binding polypeptide ZAZ3869 (SEQ ID NO:374), ZAZ3870 (SEQ ID NO:375) or ZAZ3871 (SEQ ID NO:376), or vehicle (PBS buffer), was administered at 0 hours by i.v. injection to male B6.Cg-Fcgrttml Dcr Tg(FCGRT)32Dcr/DcrJ mice (Jackson Laboratory, stock no. 14565) at a dose of 6 mg/kg. 24 hours prior to polypeptide administration, 500 mg/kg hIgG (Kiovig, Baxter) was administered by i.v. injection. Serum samples were collected at −168, 0, 24, 72, 120 and 144 h (termination of study) and stored at −20° C.
[0586] Human IgG ELISA:
[0587] The concentration of human IgG in mouse serum samples collected from the two studies was analyzed with a human IgG AlphaLISA kit (Perkin Elmer, cat. no. AL205C) as described by the manufacturer. The concentration of hIgG was calculated from a standard curve and GraphPad Prism 5 using a non-linear regression formula.
Results
[0588] The results from the experiments show a reduction in the concentrations of human IgG over time through the action of the FcRn binding polypeptides. There were little or no difference in the reduction of IgG levels between the two groups receiving the polypeptides ZAZ3715 and ZZA3716 (
[0589] Thus, the results from the two experiments indicate that the FcRn-specific polypeptides blocked recycling of IgG, resulting in increased IgG catabolism and subsequent lower levels of IgG in human FcRn transgenic mice.
Example 21
Increased IgG Catabolism by Dimeric FcRn Binding Polypeptides in NMRI Mice
[0590] In this Example, the effect of homodimeric FcRn binding polypeptides on IgG catabolism was investigated in NMRI mice.
Materials and Methods
[0591] Animal Study:
[0592] Homodimeric FcRn-binding polypeptide ZAZ3715 (SEQ ID NO:371) or ZZA3824 (SEQ ID NO:373), or vehicle (PBS buffer), was administered at 0 hours by i.v. injection to female NMRI mice, at a dose of 0.6 or 1.7 μmol/kg. Serum samples were collected at 0, 24, 48 and 72 h (termination of study) and stored at −20° C.
[0593] Mouse IgG ELISA:
[0594] The concentration of mouse IgG in mouse serum samples was analyzed by a mouse IgG ELISA kit (Mabtech, cat.no. 3825-1AD-6) and performed as described by the manufacturer. The concentration of mIgG was calculated from a standard curve and GraphPad Prism 5 using a non-linear regression formula. The concentration of IgG in individual mice at 24, 48 and 72 h were related to the level at 0 h and the results are therefore presented as percentage of IgG (0 h).
[0595] PK ELISA:
[0596] Concentrations of FcRn-binding polypeptides in mouse serum samples were determined by ELISA. In this assay, 96-well half area plates were coated with a mouse anti Z polyclonal antibody (produced in-house) at a concentration of 4 μg/ml in PBS (50 μl/well) and incubated overnight at 4° C. Next, the plates were rinsed twice in tap water and blocked with blocking buffer for 1 hour. An in-house Z variant standard was titrated in a 3-fold dilution series (0.003-300 ng/ml) and diluted serum samples were added to the coated ELISA plates (50 μl/well) and incubated for 1.5 h at RT. The plates were washed 4 times in an automated ELISA washer and 4 μg/ml (50 μl/well) of a goat anti-ABD polyclonal antibody (produced in-house) was added. After incubation for 1 h, the plates were washed and 50 μl of anti-goat IgG-HRP (Jackson, cat. no. 711-035-152) at a concentration of 20 ng/ml was added to each well. After one additional hour of incubation and subsequent washing, the plates were developed with 50 μl TMB per well and the reactions were stopped with 50 μl 2M H.sub.2SO.sub.4. The absorbance at 450 nm was measured in a 96-well plate reader (Victor.sup.3).
Results
[0597] IgG Catabolism:
[0598] The results showed a reduction of mouse IgG concentration in mice treated with the FcRn-specific polypeptides ZAZ3715 and ZAZ3824, respectively (
[0599] Pharmacokinetic Analysis:
[0600] The pharmacokinetic profiles of ZAZ3715 and ZAZ3824 are shown in
Example 22
Increased IgG Catabolism by FcRn Binding Dimers in Cynomolgus Monkeys
[0601] In this Example, the effect of homo- or heterodimeric FcRn binding polypeptides on IgG catabolism is investigated in cynomolgus monkeys.
Materials and Methods
[0602] Animal Study:
[0603] Homo- and/or heterodimeric FcRn binding polypeptides recombinantly fused to PP013 (SEQ ID NO:377) are prepared. Examples of such FcRn binding dimers include SEQ ID NO:371-376 disclosed herein.
[0604] The FcRn binding dimers including an albumin binding domain are then administered to cynomolgus monkeys at a dose of 2, 0.4 and 0.1 μmol/kg. The dimeric polypeptides are injected intravenously at 0 hours. The monkeys are bled 1 h prior to the first administration and daily up to day 21, and then once a week up to day 50. Sera are prepared and stored at −20° C. The concentration of cynomolgus IgG is determined by ELISA.
[0605] Homo- and/or heterodimeric FcRn binding polypeptides without an albumin binding domain are administered to cynomolgus monkeys at a dose of 2, 0.4 and 0.1 μmol/kg. The dimeric polypeptides are administered three times per week for two weeks. The monkeys are bled 1 h prior to the first administration and daily up to day 21, and then once a week up to day 50. Sera are prepared and stored at −20° C. The concentration of cynomolgus IgG is determined by ELISA.
[0606] Cynomolgus IgG ELISA:
[0607] The concentration of cynomolgus IgG in serum samples is determined with a human/cynomolgus IgG ELISA kit (for example Mabtech 3850-1AD-6, which is cross-reactive with both human and cynomolgus IgG) as described by the manufacturer.
Results
[0608] The results from the experiment are expected to show a dose dependent reduction in the concentrations of cynomolgus IgG through the action of FcRn binding dimers, with a more pronounced effect for such dimers that also comprise an albumin binding domain.
Example 23
Pharmacokinetic Study of Dimeric FcRn Binding Polypeptides
[0609] In this Example, the serum half-life of homo- and/or heterodimeric FcRn binding polypeptides is investigated in a pharmacokinetic study performed in mice.
Materials and Methods
[0610] Pharmacokinetic Study:
[0611] FcRn binding polypeptides, as homo- and/or heterodimers alone or recombinantly fused to PP013 (SEQ ID NO:377) are administered intravenously (i.v.) to male NMRI mice (Charles River, Germany) at a dose of 92 nmol/. Sera from groups of three mice are obtained at 0.08, 6, 18, 78, 120, 168 and 240 hours. The concentration of respective polypeptide is determined by ELISA.
[0612] ELISA:
[0613] Half-area 96-well ELISA plates are coated at 4° C. overnight with 50 μl/well of a goat antibody specific for Z variants in general (produced in-house) diluted to 4 μg/ml in coating buffer (50 mM sodium carbonate, pH 9.6). The antibody solution is poured off and the wells are blocked with 100 μl of PBSC for 1.5 h at RT. The sera are diluted in PBSC containing 1% mouse serum (matrix) from 1:100 to 1:51, 200 in a two-fold dilution series in a dilutions plate. A standard titration for respective Z variant polypeptide and four quality controls (very low, low, medium and high control) diluted in matrix are included on each plate. 50 μl of the dilutions are transferred per well and the ELISA plates are incubated for 1.5 h at RT. The plates are washed four times with PBST. Bound Z variant polypeptides are detected with 50 μl/well of rabbit anti-PP013 Ig (produced in-house) or 50 μl/well of mouse anti-Z mAb (produced in-house) diluted to 4 μg/ml in PBSC. The plates are subsequently incubated for 1.5 h at RT followed by washes as described above. HRP conjugated donkey anti-rabbit HRP (Jackson laboratories; cat. no. 711-035-152), diluted 1:20,000 in PBSC, is added and the plates are incubated for 1 h. After washing as described above, 50 μl of ImmunoPure TMB substrate is added to the wells and the plates are developed according to the manufacturer's recommendations. After 15 min of development, the absorbance is measured at 450 nm using a multi-well plate reader (Victor.sup.3). The absorbance values are analyzed using GraphPad Prism 5 to determine the concentrations (cubic-spline curve fit) and area under curve (AUC). The concentrations are then plotted as their natural logarithms against time. The resulting curves are expected to follow a two compartment model and the terminal half-life is calculated as In2 divided by the slope based on the last three time points.
Results
[0614] The results from the experiment described herein are expected to show a two compartment elimination phase with a terminal half-life of approximately 60 min for polypeptides without ABD and approximately 90 hours for polypeptides comprising ABD.
Example 24
Generation, Stability Study and Binding Assessment of Scaffold-Modified FcRn Binding Polypeptides
[0615] The following Example discloses scaffold modified FcRn binding Z variants exhibiting improved stability at elevated temperatures. The Z variants Z17347 (SEQ ID NO:358), with the amino acid substitutions N52S and D53E, and Z17348 (SEQ ID NO:359), with the amino acid substitutions D36R, D37Q, S39E, N52S and D53E, are compared to their parent molecule Z11948 (SEQ ID NO:354) in terms of stability and binding capacity to FcRn.
Materials and Methods
[0616] Generation of Scaffold-Modified Polypeptides:
[0617] Z17347 (SEQ ID NO:358), Z17348 (SEQ ID NO:359) and Z11948 (SEQ ID NO:354) were cloned with an N-terminal 6×Histidine-tag (His.sub.6) and obtained constructs encoded polypeptides in the format MGSSHHHHHHLQ-[Z#####]. Mutations were introduced in the plasmids of the modified Z variants using overlapping oligonucleotide primer pairs encoding the desired amino acid substitutions and by applying established molecular biology techniques. The correct plasmid sequences were verified by DNA sequencing.
[0618] E. coli (strain T7E2) cells (GeneBridge) were transformed with plasmids containing the gene fragments encoding the original and scaffold modified Z variants. The cells were cultivated at 37° C. in TSB-YE medium supplemented with 50 μg/ml kanamycin and protein expression was subsequently induced by addition of IPTG. Pelleted cells were disrupted using a FastPrep®-24 homogenizer (Nordic Biolabs) and cell debris was removed by centrifugation. Each supernatant containing the Z variant as a His.sub.6-tagged protein was purified by immobilized metal ion affinity chromatography (IMAC) using His GraviTrap™ columns (GE Healthcare) according to the manufacturers instructions. Purified Z variants were buffer exchanged to phosphate-buffered saline (PBS; 1.47 mM KH.sub.2PO.sub.4, 8.1 mM Na.sub.2HPO.sub.4, 137 mM NaCl, 2.68 mM KCl, pH 7.4) using PD-10 desalting columns (GE Healthcare). The correct identity of each polypeptide was verified by SDS-PAGE and HPLC-MS.
[0619] Circular Dichroism Spectroscopy Analysis:
[0620] Circular dichroism (CD) analysis was carried out as described in Example 3 to determine the melting temperatures (Tm) and assess potential changes in the secondary structure of the inventive polypeptides as a result of the amino acid substitutions.
[0621] Comparative Stability Study:
[0622] The His.sub.6-tagged Z variants, formulated in PBS pH 7.4, were diluted to 1 mg/ml and 200 μl aliquotes were incubated at 37° C. for 2 weeks. Samples collected prior to and after the stability test were analyzed by SDS-PAGE using 10% Bis-Tris NuPAGE gels (Invitrogen) and by loading 5 μg protein into each well. The stability was assessed by the appearance of new variants after incubation at the elevated temperature and mutated variants were compared to the original polypeptide.
[0623] Binding Assessment of Scaffold-Modified Polypeptides:
[0624] The His.sub.6-tagged Z variants were further assessed in terms of preserved binding capacity to FcRn after introduction of alterations in the scaffold, as well as after having been subjected to the stability test, i.e. incubated at 37° C. for 2 weeks. Comparative kinetic constants (k.sub.on and k.sub.off) and affinities (K.sub.D) were determined using a Biacore 2000 instrument. The target protein human FcRn (Biorbyt, cat. no. orb 84388) was immobilized on the carboxylated dextran layer surface of a CM5 chip (GE Healthcare). The immobilization was performed using amine coupling chemistry according to the manufacturer's protocol and using HBS-EP as running buffer. One flow cell surface on the chip was activated and deactivated for use as blank during analyte injections. The immobilization level of hFcRn on the surface was approximately 750 RU. The Z variants were diluted in running buffer to final concentrations of 3.33, and 30 nM and injected for 3 min, followed by 15 min of dissociation in running buffer. Regeneration by three pulses of HBS-EP followed by 10 min equilibration in running buffer was applied after each analyte injection. Kinetic constants were calculated from the sensorgrams using the Langmuir 1:1 model of the BiaEvaluation software 4.1 (GE Healthcare). Curves of the blank surface were subtracted from the curves of the ligand surfaces and the data from the buffer cycles were subtracted from the data of the test-sample cycles to correct for any drift in signal.
Results
[0625] Circular Dichroism Spectroscopy Analysis:
[0626] The Tm of each respective Z variant as determined from the midpoint of the transition in the CD signal vs. temperature plot is shown in Table 19. The mutated Z variants showed preserved alphahelical structure and refolded reversibly after heating to 90° C.
TABLE-US-00019 TABLE 19 Melting temperatures for original and mutated Z variants. Designation SEQ ID NO Tm (°C.) Original vs modified Z11948 354 48 Original Z17347 358 50 Modified Z17348 359 44 Modified
[0627] Comparative Stability Study:
[0628] The scaffold modified Z variants Z17347 and Z17348 showed an improved stability compared to the original polypeptide Z11948. The second band visible on the gel just above the main band for Z11948 was not visible in samples of Z17347 and Z17348 (
[0629] Binding Assessment of Scaffold-Modified Polypeptides:
[0630] The comparative kinetic constants for the FcRn-binding Z variants are shown in Table 20. The affinity was marginally effected by the amino acid substitutions ND to SE in position 52-53, such as in Z17347 (SEQ ID NO:358), as well as by the substitutions ND to SE in position 52-53 in combination with D36R, D37Q and S39E, such as in Z17348 (SEQ ID NO:359), and functional binders were obtained with K.sub.D in the range of 10.sup.−9 M. The assessed variants also had preserved binding capabilities after 2 weeks incubation at 37° C.
TABLE-US-00020 TABLE 20 Comparative kinetic analysis of original and modified Z variants. Original vs SEQ ID scaffold Test sample NO: modified k.sub.a (Ms.sup.−1) k.sub.d (s.sup.−1) K.sub.D (M)* Z11948 (0) 354 Original 1.60 × 10.sup.6 4.56 × 10.sup.−3 2.9 × 10.sup.−9 Z11948 (2w) 354 Original 3.15 × 10.sup.6 5.75 × 10.sup.−3 1.8 × 10.sup.−9 Z17347 (0) 358 Modified 1.18 × 10.sup.6 7.99 × 10.sup.−3 6.7 × 10.sup.−9 Z17347 (2w) 358 Modified 2.27 × 10.sup.6 8.79 × 10.sup.−3 3.9 × 10.sup.−9 Z17348 (0) 359 Modified 1.82 × 10.sup.6 1.00 × 10.sup.−2 5.5 × 10.sup.−9 Z17348 (2w) 359 Modified 1.28 × 10.sup.6 8.09 × 10.sup.−3 6.3 × 10.sup.−9 *The K.sub.D values should not be regarded as absolute, as these were determined for comparative purposes and only included a limited number of sample concentrations.
Example 25
Generation and Assessment of Additional Scaffold-Modified FcRn Binding Polypeptides
[0631] In this Example, additional variants with the scaffold amino acid substitutions N52S and D53E, and some also with the amino acid substitution Y5F, were analyzed in terms of their stability and binding to FcRn as compared to their respective parent FcRn binding Z variant. The results show that the structure, stability and FcRn binding capacity are retained in the mutated variants.
Materials and Methods
[0632] Generation of Scaffold-Modified Polypeptides:
[0633] The amino acid substitutions N52S and D53E were introduced in the plasmids of His.sub.6-tagged Z13578 (SEQ ID NO:20), Z13583 (SEQ ID NO:23), Z13616 (SEQ ID NO:41), Z13621 (SEQ ID NO:44) and Z13674 (SEQ ID NO:75) using established molecular biology techniques resulting in the respective Z variants Z18614 (SEQ ID NO:360), Z18615 (SEQ ID NO:361), Z18616 (SEQ ID NO:362), Z18617 (SEQ ID NO:363) and Z18618 (SEQ ID NO:364). Additional modifications at position 5, were the tyrosine residue was substituted to phenylalanine, as well as N-terminal modification to start with the amino acid residues AE instead of VD, resulted in the Z variants Z18632 (SEQ ID NO:365), Z18633 (SEQ ID NO:366) and Z18634 (SEQ ID NO:367) having binding motifs (BM) identical to Z13578, Z13616 and Z13621, respectively. Cultivation and purification was performed essentially as described in Example 3 and Example 9. The five Z variants Z18614-Z18618 were purified by IMAC only, whereas the three Z variants Z18632-Z18634 were further purified by RPC.
[0634] CD Analysis:
[0635] CD analysis was carried out as described in Example 3 to determine the melting temperatures (Tm) and assess potential changes in the secondary structure of the mutated Z variants compared to their respective parent Z variant.
[0636] Biacore Binding Analysis:
[0637] A binding analysis at pH 6.0, using a Biacore instrument, was performed essentially as described in Example 3. A concentration series (270, 90, 30, 10 and 3.3 nM) of the His.sub.6-tagged Z variants Z18632, Z18633 and Z18634 and their respective corresponding parent Z variants Z13578, Z13616 and Z13621, were injected during 4 min at 30 μl/min over hFcRn (Biorbyt, cat. no. orb84388) and cFcRn (Biorbyt, cat.no. orb99075), immobilized in different flow cells of a CM5 chip surface. 0.005% PCT pH 6.0 was used as running buffer and for dilutions of the His.sub.6-tagged Z variants. Dissociation in running buffer was allowed for 20 min, followed by surface regeneration by injection of 3×30 second pulses of 0.005% PCT pH 7.4 and equilibration for 15 min before the start of next cycle.
Results
[0638] Cultivation and Purification:
[0639] The FcRn binding Z variants were constructed with an N-terminal His.sub.6-tag and produced in E. coli. SDS-PAGE analysis of each final protein preparation showed that it predominantly contained the Z variant. The correct identity and molecular weight of each FcRn binding Z variant was confirmed by HPLC-MS analysis.
[0640] CD Analysis:
[0641] The Tm of the mutated FcRn binding Z variants were identical, or nearly identical, to the Tm of the respective parent Z variant (Table 21). Furthermore, reversible folding was observed for all seven Z variants by overlaying spectra obtained before and after heating to 90° C.
[0642] Biacore Binding Analysis:
[0643] The binding profiles for interactions with FcRn at pH 6.0 were compared pairwise for three mutated Z variants and their respective parent Z variants; Z18632/Z13578 (SEQ ID NO:365/20), Z18633/Z13616 (SEQ ID NO:366/41) and Z18634/Z13621 (SEQ ID NO:367/44). Overlays of sensorgrams from the 90 nM injections of the Z variants over hFcRn and cFcRn surfaces show that the mutated Z variants retained their ability to bind to human and cynomolgus FcRn (
TABLE-US-00021 TABLE 21 Melting temperatures for scaffold mutated and parent Z variants. Mutated SEQ ID Parent SEQ ID Z variant NO: Tm (°C.) Z variant NO: Tm (°C.) Z18614 360 57 Z13578 20 55 Z18615 361 50 Z13583 23 51 Z18616 362 62 Z13616 41 60 Z18617 363 49 Z13621 44 49 Z18618 364 50 Z13674 75 50 Z18632 365 56 Z13578 20 57 Z18633 366 61 Z13616 41 60 Z18634 367 48 Z13621 44 49
ITEMIZED LISTING OF EMBODIMENTS
[0644] 1. FcRn binding dimer, comprising a first monomer unit, a second monomer unit and an amino acid linker, wherein said first and second monomer unit each comprises an FcRn binding motif (BM), which motif consists of the amino acid sequence
[0645] EX.sub.2 X.sub.3 X.sub.4 AX.sub.6 X.sub.7 EIR WLPNLX.sub.16X.sub.17 X.sub.18 QR X.sub.21 AFIX.sub.25 X.sub.26LX.sub.28 X.sub.29
wherein, independently from each other,
[0646] X.sub.2 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y;
[0647] X.sub.3 is selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W and Y;
[0648] X.sub.4 is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W and Y;
[0649] X.sub.6 is selected from A, E, F, G, H, I, K, Q, R, S and V;
[0650] X.sub.7 is selected from A, F, H, K, N, Q, R, S and V;
[0651] X.sub.16 is selected from N and T;
[0652] X.sub.17 is selected from F, W and Y;
[0653] X.sub.18 is selected from A, D, E and N;
[0654] X.sub.21 is selected from A, S, V and W;
[0655] X.sub.25 is selected from D, E, G, H, I, K, L, N, Q, R, S, T, V, W and Y;
[0656] X.sub.26 is selected from K and S;
[0657] X.sub.28 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y; and
[0658] X.sub.29 is selected from D and R,
and wherein said FcRn binding dimer binds FcRn with a higher binding capacity compared to said first monomer unit or said second monomer unit alone.
[0659] 2. FcRn binding dimer according to item 1, wherein, independently from each other,
[0660] X.sub.2 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y;
[0661] X.sub.3 is selected from A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W and Y;
[0662] X.sub.4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y;
[0663] X.sub.6 is selected from A, E, F, G, H, I, K, Q, R and S;
[0664] X.sub.7 is selected from A, F, H, K, N, Q, R, S and V;
[0665] X.sub.16 is selected from N and T;
[0666] X.sub.17 is selected from F and Y;
[0667] X.sub.18 is D;
[0668] X.sub.21 is V;
[0669] X.sub.25 is selected from D, E, H, I, K, L, N, Q, R, S, T, V, W and Y;
[0670] X.sub.26 is selected from K and S;
[0671] X.sub.28 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V and W; and
[0672] X.sub.29 is selected from D and R.
[0673] 3. FcRn binding dimer according to item 1, wherein the BM of at least one of said first and second monomer units consists of an amino acid sequence selected from [0674] i) EX.sub.2 X.sub.3 X.sub.4 AX.sub.6 HEIR WLPNLTX.sub.17 X.sub.18 QR X.sub.21 AFIX.sub.25 KLX.sub.28 D
wherein, independently from each other,
[0675] X.sub.2 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y;
[0676] X.sub.3 is selected from A, D, E, G, H, K, L, M, N, Q, R, S, T, V and Y;
[0677] X.sub.4 is selected from A, D, E, F, G, I, K, L, N, Q, R, S, T, V and Y;
[0678] X.sub.6 is selected from A, G, K, R, S and V;
[0679] X.sub.17 is selected from F, W and Y;
[0680] X.sub.18 is selected from A, D, E and N;
[0681] X.sub.21 is selected from A, S, V and W;
[0682] X.sub.25 is selected from D, G, H, K, L, N, R, V and W;
[0683] X.sub.28 is selected from A, D, E, H, K, L, N, Q, R, S, T, W and Y;
and [0684] ii) an amino acid sequence which has at least 96% identity to a sequence defined by i).
[0685] 4. FcRn binding dimer according to any one of items 1-3, wherein X.sub.6X.sub.7 is selected from AH and GH in at least one of said first and second monomer units.
[0686] 5. FcRn binding dimer according to item 4, wherein X.sub.6X.sub.7 is AH in at least one of said first and second monomer units.
[0687] 6. FcRn binding dimer according to item 4, wherein X.sub.6X.sub.7 is GH in at least one of said first and second monomer units.
[0688] 7. FcRn binding dimer according to any preceding item, wherein X.sub.17X.sub.18 is selected from FD and YD in at least one of said first and second monomer units.
[0689] 8. FcRn binding dimer according to item 7, wherein X.sub.17X.sub.18 is FD in at least one of said first and second monomer units.
[0690] 9. FcRn binding dimer according to any preceding item, wherein the sequence of the BM of at least one of said first and second monomer units fulfills at least three of the six conditions I-VI: [0691] I. X.sub.6 is selected from A, G, K and S, such as in particular A; [0692] II. X.sub.7 is H; [0693] III. X.sub.17 is selected from F and Y, such as in particular F; [0694] IV. X.sub.18 is D; [0695] V. X.sub.21 is selected from V and W, such as in particular V; [0696] VI. X.sub.25 is selected from H and R, such as in particular H.
[0697] 10. FcRn binding dimer according to item 9, wherein the sequence fulfills at least four of the six conditions I-VI.
[0698] 11. FcRn binding dimer according to item 10, wherein the sequence fulfills at least five of the six conditions I-VI.
[0699] 12. FcRn binding dimer according to item 11, wherein the sequence fulfills all of the six conditions I-VI.
[0700] 13. FcRn binding dimer according to any preceding item, wherein said first and second monomer units comprise identical BM sequences.
[0701] 14. FcRn binding dimer according to any one of items 1-12, wherein said first and second monomer units comprise different BM sequences.
[0702] 15. FcRn binding dimer according to any preceding item, wherein at least one of said first and second monomer units comprises an FcRn binding motif BM corresponding to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-353, such as the group consisting of SEQ ID NO:17-352.
[0703] 16. FcRn binding dimer according to item 15, wherein at least one of said first and second monomer units comprises a BM corresponding to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-15, SEQ ID NO:17-140 and SEQ ID NO:353.
[0704] 17. FcRn binding dimer according to item 16, wherein at least one of said first and second monomer units comprises a BM corresponding to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-2 and SEQ ID NO:17-140, such as the group consisting of SEQ ID NO:17-140.
[0705] 18. FcRn binding dimer according to item 17, wherein at least one of said first and second monomer units comprises a BM corresponding to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-2, SEQ ID NO:17-92, SEQ ID NO:94-103, SEQ ID NO:105-125 and SEQ ID NO:127-140, such as the group consisting of SEQ ID NO:17-92, SEQ ID NO:94-103, SEQ ID NO:105-125 and SEQ ID NO:127-140.
[0706] 19. FcRn binding dimer according to item 16, wherein at least one of said first and second monomer units comprises a BM corresponding to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-8, SEQ ID NO:13, SEQ ID NO:19-20, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:75-77 and SEQ ID NO:353, such as the group consisting of SEQ ID NO:19-20, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:70, SEQ ID NO:73 and SEQ ID NO:75-77.
[0707] 20. FcRn binding dimer according to item 18 or 19, wherein at least one of said first and second monomer units comprises a BM corresponding to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:73 and SEQ ID NO:75-77, such as the group consisting of SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:73 and SEQ ID NO:75-77.
[0708] 21. FcRn binding dimer according to item 20, wherein at least one of said first and second monomer units comprises a BM corresponding to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:75 and SEQ ID NO:77, such as the group consisting of SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:75 and SEQ ID NO:77.
[0709] 22. FcRn binding dimer according to item 21, wherein at least one of said first and second monomer units comprises a BM corresponding to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65 and SEQ ID NO:75, such as the group consisting of SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65 and SEQ ID NO:75.
[0710] 23. FcRn binding dimer according to item 22, wherein at least one of said first and second monomer units comprises a BM corresponding to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:23 and SEQ ID NO:75, such as the group consisting of SEQ ID NO:23 and SEQ ID NO:75.
[0711] 24. FcRn binding dimer according to item 22, wherein at least one of said first and second monomer units comprises a BM corresponding to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:41 and SEQ ID NO:44, such as the group consisting of SEQ ID NO:20 and SEQ ID NO:41; the group consisting of SEQ ID NO:20 and SEQ ID NO:44; or the group consisting of SEQ ID NO:41 and SEQ ID NO:44.
[0712] 25. FcRn binding dimer according to item 22, wherein at least one of said first and second monomer units comprises a BM corresponding to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:23 and SEQ ID NO:44, such as the group consisting of SEQ ID NO:23 and SEQ ID NO:44.
[0713] 26. FcRn binding dimer according to item 24 or 25, wherein at least one of said first and second monomer units comprises a BM corresponding to the sequence from position 8 to position 36 in sequence SEQ ID NO:44.
[0714] 27. FcRn binding dimer according to any preceding item, wherein both said first and second monomer units independently comprise a BM corresponding to the sequence from position 8 to position 36 in a sequence selected as defined in any one of items 15-26.
[0715] 28. FcRn binding dimer according to item 27, wherein both said first and second monomer units independently comprise a BM corresponding to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:44 and SEQ ID NO:75, such as the group consisting of SEQ ID NO:20, SEQ ID NO:41 and SEQ ID NO:44.
[0716] 29. FcRn binding dimer according to item 28, wherein both said first and second monomer units comprise a BM corresponding to the sequence from position 8 to position 36 in SEQ ID NO:44.
[0717] 30. FcRn binding dimer according to any preceding item, wherein said FcRn binding motif BM in at least one of said first and second monomers forms part of a three-helix bundle protein domain.
[0718] 31. FcRn binding dimer according to item 30, wherein said BM essentially forms part of two helices with an interconnecting loop, within said three-helix bundle protein domain.
[0719] 32. FcRn binding dimer according to item 31, wherein said three-helix bundle protein domain is selected from bacterial receptor domains.
[0720] 33. FcRn binding dimer according to item 32, wherein said three-helix bundle protein domain is selected from domains of protein A from Staphylococcus aureus or derivatives thereof.
[0721] 34. FcRn binding dimer according to any preceding item, wherein at least one of said first and second monomer units comprises a binding module (BMod), which module consists of an amino acid sequence selected from: [0722] iii) K-[BM]-DPSQS X.sub.aX.sub.bLLX.sub.c EAKKL X.sub.dX.sub.eX.sub.fQ;
wherein
[0723] [BM] is an FcRn binding motif as defined in any one of items 1-29,
[0724] provided that X.sub.29 is D;
[0725] X.sub.a is selected from A and S;
[0726] X.sub.b is selected from N and E;
[0727] X.sub.c is selected from A, S and C;
[0728] X.sub.d is selected from E, N and S;
[0729] X.sub.e is selected from D, E and S;
[0730] X.sub.f is selected from A and S;
and [0731] iv) an amino acid sequence which has at least 93% identity to a sequence defined by iii).
[0732] 35. FcRn binding dimer according to any one of items 1-33, wherein at least one of said first and second monomer units comprises a binding module (BMod), which module consists of an amino acid sequence selected from: [0733] v) K-[BM]-QPEQS X.sub.aX.sub.bLLX.sub.c EAKKL X.sub.dX.sub.eX.sub.fQ;
wherein
[0734] [BM] is an FcRn binding motif as defined in any one of items 1-29,
[0735] provided that X.sub.29 is R;
[0736] X.sub.a is selected from A and S;
[0737] X.sub.b is selected from N and E;
[0738] X.sub.c is selected from A, S and C;
[0739] X.sub.d is selected from E, N and S;
[0740] X.sub.e is selected from D, E and S;
[0741] X.sub.f is selected from A and S;
and [0742] vi) an amino acid sequence which has at least 93% identity to a sequence defined by v).
[0743] 36. FcRn binding dimer according to item 34, wherein at least one of said first and second monomer units comprises a BMod corresponding to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-353, SEQ ID NO:358 and SEQ ID NO:360-364, such as the group consisting of SEQ ID NO:17-352 and SEQ ID NO:360-364.
[0744] 37. FcRn binding dimer according to item 36, wherein at least one of said first and second monomer units comprises a BMod corresponding to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-15, SEQ ID NO:17-140, SEQ ID NO:353, SEQ ID NO:358 and SEQ ID NO:360-364.
[0745] 38. FcRn binding dimer according to item 37, wherein at least one of said first and second monomer units comprises a BMod corresponding to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-2, SEQ ID NO:17-140, SEQ ID NO:358 and SEQ ID NO:360-364, such as the group consisting of SEQ ID NO:17-140 and SEQ ID NO:360-364.
[0746] 39. FcRn binding dimer according to item 38, wherein at least one of said first and second monomer units comprises a BMod corresponding to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-2, SEQ ID NO:17-92, SEQ ID NO:94-103, SEQ ID NO:105-125, SEQ ID NO:127-140, SEQ ID NO:358 and SEQ ID NO:360-364, such as the group consisting of SEQ ID NO:17-92, SEQ ID NO:94-103, SEQ ID NO:105-125, SEQ ID NO:127-140 and SEQ ID NO:360-364.
[0747] 40. FcRn binding dimer according to item 36, wherein at least one of said first and second monomer units comprises a BMod corresponding to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-8, SEQ ID NO:13, SEQ ID NO:19-20, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:75-77, SEQ ID NO:353, SEQ ID NO:358 and SEQ ID NO:360-364, such as the group consisting of SEQ ID NO:19-20, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:75-77 and SEQ ID NO:360-364.
[0748] 41. FcRn binding dimer according to item 39 or 40, wherein at least one of said first and second monomer units comprises a BMod corresponding to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75-77, SEQ ID NO:358 and SEQ ID NO:360-364, such as the group consisting of SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75-77 and SEQ ID NO:360-364.
[0749] 42. FcRn binding dimer according to item 41, wherein at least one of said first and second monomer units comprises a BMod corresponding to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:358 and SEQ ID NO:360-364, such as the group consisting of SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:75, SEQ ID NO:77 and SEQ ID NO:360-364.
[0750] 43. FcRn binding dimer according to item 42, wherein at least one of said first and second monomer units comprises a BMod corresponding to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:75, SEQ ID NO:358 and SEQ ID NO:360-364, such as the group consisting of SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:75 and SEQ ID NO:360-364.
[0751] 44. FcRn binding dimer according to item 43, wherein at least one of said first and second monomer units comprises a BMod corresponding to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:23, SEQ ID NO:75, SEQ ID NO:358, SEQ ID NO:361 and SEQ ID NO:364, such as the group consisting of SEQ ID NO:23, SEQ ID NO:75, SEQ ID NO:361 and SEQ ID NO:364.
[0752] 45. FcRn binding dimer according to item 43, wherein at least one of said first and second monomer units comprises a BMod corresponding to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:360, SEQ ID NO:362 and SEQ ID NO:363, such as the group consisting of SEQ ID NO:20, SEQ ID NO:41, SEQ ID NO:360 and SEQ ID NO:362; the group consisting of SEQ ID NO:20, SEQ ID NO:44, SEQ ID NO:360 and SEQ ID NO:363; or the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:362 and SEQ ID NO:363.
[0753] 46. FcRn binding dimer according to item 43, wherein at least one of said first and second monomer units comprises a BMod corresponding to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:23, SEQ ID NO:44, SEQ ID NO:358, SEQ ID NO:361 and SEQ ID NO:363, such as the group consisting of SEQ ID NO:23, SEQ ID NO:44, SEQ ID NO:361 and SEQ ID NO:363.
[0754] 47. FcRn binding dimer according to item 46, wherein at least one of said first and second monomer units comprises a BMod corresponding to the sequence from position 7 to position 55 in SEQ ID NO:44.
[0755] 48. FcRn binding dimer according to any one of items 1-13 and 15-47, wherein both said first and second monomer units comprise a BMod corresponding to the sequence from position 7 to position 55 in a sequence selected from the group as defined in any one of items 36-47.
[0756] 49. FcRn binding dimer according to item 48, wherein both said first and second monomer units comprise a BMod corresponding to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:75 and SEQ ID NO:360-364, such as the group consisting of SEQ ID NO:20, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:360, SEQ ID NO:362 and SEQ ID NO:363.
[0757] 50. FcRn binding dimer according to item 49, wherein both said first and second monomer units comprise a BMod corresponding to the sequence from position 7 to position 55 in SEQ ID NO:44.
[0758] 51. FcRn binding dimer according to any preceding item, wherein at least one of said first and second monomer units comprises a sequence selected from the group consisting of: [0759] vii) YAK-[BM]-DPSQS SELLX.sub.c EAKKL NDSQA P;
wherein [BM] is an FcRn binding motif as defined in any one of items 1-29 and X.sub.c is selected from A, S and C; and [0760] viii) an amino acid sequence which has at least 94% identity to a sequence defined by vii).
[0761] 52. FcRn binding dimer according to any one of items 1-50, wherein at least one of said first and second monomer units comprises a sequence selected from the group consisting of: [0762] ix) FAK-[BM]-DPSQS SELLX.sub.c EAKKL SESQA P;
wherein [BM] is an FcRn binding motif as defined in any one of items 1-29 and X.sub.c is selected from A, S and C; and [0763] x) an amino acid sequence which has at least 94% identity to a sequence defined by ix).
[0764] 53. FcRn binding dimer according to any one of items 1-50, wherein at least one of said first and second monomer units comprises a sequence selected from the group consisting of: [0765] xi) FNK-[BM]-DPSQS ANLLX.sub.c EAKKL NDAQA P;
wherein [BM] is an FcRn binding motif as defined in any one of items 1-29 and X.sub.c is selected from A and C; and [0766] xii) an amino acid sequence which has at least 94% identity to a sequence defined by xi).
[0767] 54. FcRn binding dimer according to item 33, wherein at least one of said first and second monomer units comprises a sequence selected from:
[0768] ADNNFNK-[BM]-DPSQSANLLSEAKKLNESQAPK;
[0769] ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK;
[0770] ADNKFNK-[BM]-DPSVSKEILAEAKKLNDAQAPK;
[0771] ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAPK;
[0772] AQHDE-[BM]-DPSQSANVLGEAQKLNDSQAPK;
[0773] VDNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK;
[0774] AEAKYAK-[BM]-DPSESSELLSEAKKLNKSQAPK;
[0775] VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK;
[0776] VDAKYAK-[BM]-DPSQSSELLAEAKKLNDSQAPK;
[0777] AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
[0778] AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAP;
[0779] AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
[0780] AEAKFAK-[BM]-DPSQSSELLSEAKKLN DSQAP;
[0781] AEAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK;
[0782] AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK;
[0783] AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAP;
[0784] AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK;
[0785] AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAP;
[0786] AEAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK;
[0787] AEAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK;
[0788] AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK;
[0789] AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAP;
[0790] AEAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK;
[0791] AEAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK;
[0792] AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK;
[0793] AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAP;
[0794] AEAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK;
[0795] AEAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK;
[0796] VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
[0797] VDAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
[0798] VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK;
[0799] VDAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK;
[0800] VDAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK;
[0801] VDAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK;
[0802] VDAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK;
[0803] VDAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK;
[0804] VDAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK;
[0805] VDAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK;
[0806] VDAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK;
[0807] VDAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK;
[0808] VDAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK;
[0809] VDAKYAK-[BM]-DPSQSSELLAEAKKLN KAQAPK;
[0810] AEAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK; and
[0811] ADAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
wherein [BM] is an FcRn binding motif as defined in any one of items 1-29.
[0812] 55. FcRn binding dimer according to any preceding item, wherein at least one of said first and second monomer units comprises a sequence selected from the group consisting of: [0813] xiii) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
wherein [BM] is an FcRn binding motif as defined in any one of items 1-29; and [0814] xiv) an amino acid sequence which has at least 94% identity to the sequence defined in xiii).
[0815] 56. FcRn binding dimer according to item 55, wherein at least one of said first and second monomer units comprises a sequence xiii) selected from the group consisting of SEQ ID NO:354-357, such as selected from SEQ ID NO:354 and 357.
[0816] 57. FcRn binding dimer according to item 56, wherein at least one of said first and second monomer units comprises a sequence xiii) which is SEQ ID NO:357.
[0817] 58. FcRn binding dimer according to any one of items 1-54, wherein at least one of said first and second monomer units comprises a sequence selected from the group consisting of: [0818] xv) AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK;
wherein [BM] is an FcRn binding motif as defined in any one of items 1-29; and [0819] xvi) an amino acid sequence which has at least 94% identity to the sequence defined in xv).
[0820] 59. FcRn binding dimer according to item 58, wherein at least one of said first and second monomer units comprises a sequence xv) selected from the group consisting of SEQ ID NO:365-367.
[0821] 60. FcRn binding dimer according to any one of items 1-54, wherein at least one of said first and second monomer units comprises a sequence selected from the group consisting of: [0822] xvii) VDAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK;
wherein [BM] is an FcRn binding motif as defined in any one of items 1-29; and [0823] xviii) an amino acid sequence which has at least 94% identity to the sequence defined in xvii).
[0824] 61. FcRn binding dimer according to item 60, wherein at least one of said first and second monomer units comprises a sequence xvii) selected from the group consisting of SEQ ID NO:360-364.
[0825] 62. FcRn binding dimer according to any preceding item, wherein at least one of said first and second monomer units comprises a sequence selected from the group consisting of: [0826] xix) AEAKYAK-[BM]-RQPESSELLSEAKKLSESQAPK;
wherein [BM] is an FcRn binding motif as defined in any one of items 1-29; and [0827] xx) an amino acid sequence which has at least 94% identity to the sequence defined in xix).
[0828] 63. FcRn binding dimer according to item 62, wherein at least one of said first and second monomer units comprises a sequence xix) which is SEQ ID NO:359.
[0829] 64. FcRn binding dimer according to any one of items 1-54, wherein at least one of said first and second monomer units comprises a sequence selected from the group consisting of: [0830] xxi) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
wherein [BM] is an FcRn binding motif as defined in any one of items 1-29; and [0831] xxii) an amino acid sequence which has at least 94% identity to the sequence defined in xxi).
[0832] 65. FcRn binding dimer according to item 64, wherein at least one of said first and second monomer units comprises a sequence xxi) selected from the group consisting of SEQ ID NO:1-353, such as the group consisting of SEQ ID NO:17-352.
[0833] 66. FcRn binding dimer according to item 65, wherein at least one of said first and second monomer units comprises a sequence xxi) selected from the group consisting of SEQ ID NO:1-15, SEQ ID NO:17-140 and SEQ ID NO:353, or comprises a sequence xxi) selected from the group consisting of SEQ ID NO:1-2 and SEQ ID NO:17-140, such as the group consisting of SEQ ID NO:17-140.
[0834] 67. FcRn binding dimer according to item 66, wherein at least one of said first and second monomer units comprises a sequence xxi) selected from the group consisting of SEQ ID NO:1-2, SEQ ID NO:17-92, SEQ ID NO:94-103, SEQ ID NO:105-125 and SEQ ID NO:127-140, such as the group consisting of SEQ ID NO:17-92, SEQ ID NO:94-103, SEQ ID NO:105-125 and SEQ ID NO:127-140.
[0835] 68. FcRn binding dimer according to item 67, wherein at least one of said first and second monomer units comprises a sequence xxi) selected from the group consisting of SEQ ID NO:1-8, SEQ ID NO:13, SEQ ID NO:19-20, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:75-77 and SEQ ID NO:353, such as the group consisting of SEQ ID NO:19-20, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:70, SEQ ID NO:73 and SEQ ID NO:75-77.
[0836] 69. FcRn binding dimer according to item 66 or 68, wherein at least one of said first and second monomer units comprises a sequence xxi) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:73 and SEQ ID NO:75-77, such as the group consisting of SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:73 and SEQ ID NO:75-77.
[0837] 70. FcRn binding dimer according to item 69, wherein at least one of said first and second monomer units comprises a sequence xxi) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:75 and SEQ ID NO:77, such as the group consisting of SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:75 and SEQ ID NO:77.
[0838] 71. FcRn binding dimer according to item 70 wherein at least one of said first and second monomer units comprises a sequence xxi) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65 and SEQ ID NO:75, such as the group consisting of SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65 and SEQ ID NO:75, such as the group consisting of SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:44 and SEQ ID NO:75.
[0839] 72. FcRn binding dimer according to item 71, wherein at least one of said first and second monomer units comprises a sequence xxi) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:23 and SEQ ID NO:75, such as the group consisting of SEQ ID NO:23 and SEQ ID NO:75.
[0840] 73. FcRn binding dimer according to item 71, wherein at least one of said first and second monomer units comprises a sequence xxi) selected from the group consisting of SEQ ID NO:20, SEQ ID NO:41 and SEQ ID NO:44.
[0841] 74. FcRn binding dimer according to item 71, wherein at least one of said first and second monomer units comprises a sequence xxi) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:23 and SEQ ID NO:44, such as the group consisting of SEQ ID NO:23 and SEQ ID NO:44.
[0842] 75. FcRn binding dimer according to item 73 or 74, wherein at least one of said first and second monomer units comprises a sequence xxi) which is SEQ ID NO:44.
[0843] 76. FcRn binding dimer according to any one of items 1-13 and 15-75, wherein both said first and second monomer units correspond to a sequence selected from the group as defined in any one of items 55, 56 and 62-75.
[0844] 77. FcRn binding dimer according to item 76, wherein both said first and second monomer units correspond to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:75, SEQ ID NO:354, SEQ ID NO:357 and SEQ ID NO:360-367, such as the group consisting of SEQ ID NO:20, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:360, SEQ ID NO:362, SEQ ID NO:363, SEQ ID NO:365, SEQ ID NO:366 and SEQ ID NO:367.
[0845] 78. FcRn binding dimer according to item 77, wherein both said first and second monomer units correspond to SEQ ID NO:1 or SEQ ID NO:357.
[0846] 79. FcRn binding dimer according to item 77, wherein both said first and second monomer units correspond to SEQ ID NO:20, SEQ ID NO:360 or SEQ ID NO:365.
[0847] 80. FcRn binding dimer according to item 77, wherein both said first and second monomer units correspond to SEQ ID NO:41, SEQ ID NO:362 or SEQ ID NO:366.
[0848] 81. FcRn binding dimer according to item 77, wherein both said first and second monomer units correspond to SEQ ID NO:44, SEQ ID NO:363 or SEQ ID NO:367.
[0849] 82. FcRn binding dimer according to any preceding item, wherein said linker is selected from the group consisting of flexible amino acid linkers, rigid amino acid linkers and cleavable amino acid linkers.
[0850] 83. FcRn binding dimer according to item 82, wherein said linker is arranged between said first monomer unit and said second monomer unit.
[0851] 84. FcRn binding dimer according to item 82 or 83, wherein said linker is a flexible linker comprising amino acid residues selected from the group consisting of glycine, serine and threonine.
[0852] 85. FcRn binding dimer according to claim 84, wherein said linker has a general formula selected from
(G.sub.nS.sub.m).sub.p and (S.sub.nG.sub.m).sub.p,
wherein, independently,
[0853] n=1-7,
[0854] m=0-7,
[0855] n+m≦8 and
[0856] p=1-7.
[0857] 86. FcRn binding dimer according to claim 85, wherein n=1-5.
[0858] 87. FcRn binding dimer according to any one of claims 85-86, wherein m=0-5.
[0859] 88. FcRn binding dimer according to any one of claims 85-87, wherein p=1-5.
[0860] 89. FcRn binding dimer according to any one of claims 86-88, wherein n=4, m=1 and p=1-4.
[0861] 90. FcRn binding dimer according to claim 89, wherein said linker is (G.sub.4S).sub.3.
[0862] 91. FcRn binding dimer according item 89, wherein said linker is G.sub.4S.
[0863] 92. FcRn binding dimer according to any preceding item, which is capable of binding to FcRn with at least 2 times, such as at least 3 times, such as at least 4 times, such as at least 5 times, such as at least 6 times, such as at least 7 times, such as at least 8 times, such as at least 9 times, such as at least 10 times, such as at least 25 times, such as at least 50 times, such as at least 100 times higher capacity than the corresponding first monomer unit or second monomer unit alone.
[0864] 93. FcRn binding dimer according to item 92, which is capable of binding to FcRn at pH 6.0 with at least 2 times, such as at least 3 times higher capacity than the corresponding first monomer unit or second monomer unit alone.
[0865] 94. FcRn binding dimer according to item 92, which is capable of binding to FcRn at pH 7.4 with at least 2 times, such as at least 3 times, such as at least 4 times, such as at least 5 times, such as at least 6 times, such as at least 7 times, such as at least 8 times, such as at least 9 times, such as at least 10 times higher capacity than the corresponding first monomer unit or second monomer unit alone.
[0866] 95. FcRn binding dimer according to any preceding item, which is capable of binding to FcRn at pH 6.0 such that the K.sub.D value of the interaction is at most 1×10.sup.−7 M, such as at most 1×10.sup.−8 M, such as at most 1×10.sup.−9 M, such as at most 1×10.sup.−10 M, such as at most 1×10.sup.−11 M, such as at most 1×10.sup.−12 M.
[0867] 96. FcRn binding dimer according to any preceding item, wherein the K.sub.D value of the interaction between FcRn binding polypeptide and FcRn at pH 7.4 is higher than the K.sub.D value of said interaction at pH 6.0, such as at least 2 times higher, such as at least 5 times higher, such as at least 10 times higher, such as at least 50 times higher, such as at least 100 times higher than the K.sub.D value of said interaction at pH 6.0.
[0868] 97. FcRn binding dimer according to any preceding item, wherein the K.sub.D value of the interaction between FcRn binding polypeptide and FcRn at pH 7.4 is at least 1×10.sup.−10 M, such as at least 1×10.sup.−9 M, such as at least 1×10.sup.−8M, such as at least 1×10.sup.−7 M, such as at least 1×10.sup.−6 M, such as at least 1×10.sup.−5 M.
[0869] 98. FcRn binding dimer according to any one of items 1-94, wherein the K.sub.D value of said interaction at pH 7.4 is the same as or lower than the K.sub.D value of said interaction at pH 6.0.
[0870] 99. FcRn binding dimer according to any one of items 1-94, wherein the K.sub.D value of said interaction at pH 7.4 is at most 1×10.sup.−7 M, such as at most 1×10.sup.−8 M, such as at most 1×10.sup.−9 M, such as at most 1×10.sup.−10 M, such as at most 1×10.sup.−11 M, such as at most 1×10.sup.−12 M.
[0871] 100. FcRn binding dimer according to any preceding item, wherein at least one of said first and second monomer units comprises at least one additional amino acid at the C-terminal and/or N-terminal end.
[0872] 101. FcRn binding dimer according to item 100, wherein said at least one additional amino acid extension improves or simplifies production, purification, stabilization in vivo or in vitro, coupling or detection of the polypeptide.
[0873] 102. Fusion protein or conjugate comprising [0874] a first moiety consisting of an FcRn binding dimer according to any preceding item; and [0875] a second moiety consisting of a polypeptide having a desired biological activity.
[0876] 103. Fusion protein or conjugate according to item 102, wherein the in vivo half-life of said fusion protein or conjugate is longer than the in vivo half-life of the polypeptide having a desired biological activity per se.
[0877] 104. Fusion protein or conjugate according to any one of items 102-103, wherein said desired biological activity is a therapeutic activity.
[0878] 105. Fusion protein or conjugate according to any one of items 100-102, wherein said desired biological activity is a binding activity to a selected target.
[0879] 106. Fusion protein or conjugate according to item 105, wherein said selected target is albumin.
[0880] 107. Fusion protein or conjugate according to item 106, wherein said albumin binding activity is provided by the albumin binding domain of streptococcal protein G, or a derivative thereof.
[0881] 108. Fusion protein or conjugate according to any one of items 106-107, wherein said albumin binding activity increases in vivo half-life of the fusion protein or conjugate.
[0882] 109. Fusion protein or conjugate according to any one of items 103-104, wherein said desired biological activity is an enzymatic activity.
[0883] 110. Fusion protein or conjugate according to any one of items 103-105, wherein the second moiety having a desired biological activity is a therapeutically active polypeptide.
[0884] 111. Fusion protein or conjugate according to any one of items 103-105 and 109-110, wherein the second moiety having a desired biological activity is selected from the group consisting of enzymes, hormones, growth factors, chemokines and cytokines.
[0885] 112. FcRn binding dimer, fusion protein or conjugate according to any preceding item, which inhibits binding of IgG to FcRn.
[0886] 113. FcRn binding dimer, fusion protein or conjugate according to item 112, which binds FcRn such that the ability of the FcRn binding dimer to block IgG binding to FcRn is at least 2 times higher, such as at least 3 times higher, such as at least 4 times higher, such as at least 5 times higher, such as at least 10 times, such as at least 15 times, such as at least 20 times, such as at least 25 times higher compared to the blocking ability of the corresponding first or second monomer unit alone.
[0887] 114. FcRn binding dimer, fusion protein or conjugate according to item 112 or 113, wherein the K.sub.D value of the interaction between said FcRn binding polypeptide, fusion protein or conjugate and FcRn is lower than the K.sub.D value of the interaction between IgG and FcRn.
[0888] 115. FcRn binding dimer, fusion protein or conjugate according to any preceding item, further comprising a label.
[0889] 116. FcRn binding dimer, fusion protein or conjugate according to item 115, wherein said label is selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radionuclides and radioactive particles.
[0890] 117. FcRn binding dimer, fusion protein or conjugate according to any preceding item, comprising a chelating environment provided by a polyaminopolycarboxylate chelator conjugated to the FcRn binding polypeptide via a thiol group of a cysteine residue or an amine group of a lysine residue.
[0891] 118. FcRn binding dimer, fusion protein or conjugate according to item 117, wherein the polyaminopolycarboxylate chelator is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid or a derivative thereof.
[0892] 119. FcRn binding dimer, fusion protein or conjugate according to item 118, wherein the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid derivative is 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide.
[0893] 120. FcRn binding dimer, fusion protein or conjugate according to item 117, wherein the polyaminopolycarboxylate chelator is 1,4,7-triazacyclononane-1,4,7-triacetic acid or a derivative thereof.
[0894] 121. FcRn binding dimer, fusion protein or conjugate according to item 117, wherein the polyaminopolycarboxylate chelator is diethylenetriaminepentaacetic acid or derivatives thereof.
[0895] 122. A polynucleotide encoding a polypeptide according to any one of items 1-114.
[0896] 123. Expression vector comprising a polynucleotide according to item 122.
[0897] 124. Host cell comprising an expression vector according to item 123.
[0898] 125. Method of producing a polypeptide according to any one of items 1-114, comprising [0899] culturing a host cell according to item 124 under conditions permissive of expression of said polypeptide from said expression vector, and [0900] isolating said polypeptide.
[0901] 126. Composition comprising an FcRn binding dimer, fusion protein or conjugate according to any one of items 1-121 and at least one pharmaceutically acceptable excipient or carrier.
[0902] 127. Composition according to item 126, further comprising at least one additional active agent.
[0903] 128. Composition according to any one of items 126-127, which is adapted for administration by a route selected from the group consisting of oral administration, intranasal administration, pulmonar administration, vaginal administration, rectal administration, intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection and intradermal injection.
[0904] 129. FcRn binding dimer, fusion protein or conjugate according to any one of items 1-121 or composition according to any one of items 126-128 for use as a medicament.
[0905] 130. FcRn binding dimer, fusion protein, conjugate or composition for use according to item 129, wherein said medicament is intended for treatment or prophylaxis of an auto-immune condition.
[0906] 131. FcRn binding dimer, fusion protein, conjugate or composition for use according to item 129, wherein said medicament is intended for treatment or prophylaxis of an allo-immune condition.
[0907] 132. FcRn binding dimer, fusion protein, conjugate or composition for use according to item 129, wherein said medicament is intended for treatment or prophylaxis of a condition selected from the group consisting of epilepsy and seizures.
[0908] 133. Method of treatment or prophylaxis of a subject in need thereof, comprising administering to the subject a therapeutically or prophylactically active amount of an FcRn binding dimer, fusion protein or conjugate according to any one of items 1-121 or composition according to any one of items 126-128.
[0909] 134. Method according to item 133, for treatment or prophylaxis of an auto-immune condition.
[0910] 135. Method according to item 133, for treatment or prophylaxis of an allo-immune condition.
[0911] 136. Method according to item 133, for treatment or prophylaxis of a condition selected from the group consisting of epilepsy and seizures.