Polypeptides binding to human complement C5
09808502 · 2017-11-07
Assignee
Inventors
- Charlotta Berghard (Stockholm, SE)
- Magnus Berglund (Vendelso, SE)
- Patrik Strömberg (Sollentuna, SE)
- Malin Lindborg (Saltsjo-Boo, SE)
- Elin Gunneriusson (Saltsjobaden, SE)
- Joachim Feldwisch (Tyreso, SE)
Cpc classification
A61P29/00
HUMAN NECESSITIES
A61K47/6801
HUMAN NECESSITIES
A61P31/00
HUMAN NECESSITIES
A61K38/16
HUMAN NECESSITIES
A61K47/64
HUMAN NECESSITIES
A61P17/02
HUMAN NECESSITIES
A61P7/00
HUMAN NECESSITIES
C07K14/00
CHEMISTRY; METALLURGY
A61K47/68
HUMAN NECESSITIES
A61P43/00
HUMAN NECESSITIES
C07K2319/31
CHEMISTRY; METALLURGY
A61P13/02
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
A61P37/06
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
A61K38/16
HUMAN NECESSITIES
Abstract
The present invention relates to C5 binding polypeptides, comprising a C5 binding motif, BM, which motif consists of an amino acid sequence selected from i) EX.sub.2X.sub.3X.sub.4A X.sub.6X.sub.7EID X.sub.11LPNL X.sub.16X.sub.17X.sub.18QW X.sub.21AFIX.sub.25X.sub.26LX.sub.28D, and ii) an amino acid sequence which has at least 86% identity to the sequence defined in i), wherein the polypeptide binds to C5. The present invention moreover relates to C5 binding polypeptides for use in therapy, such as for use in treatment of a C5 related condition, and to methods of treatments.
Claims
1. A C5 binding polypeptide, comprising a C5 binding motif, BM, which motif consists of an amino acid sequence selected from TABLE-US-00013 i) (SEQ ID NO: 763) EX.sub.2X.sub.3X.sub.4A X.sub.6X.sub.7EID X.sub.11LPNL X.sub.16X.sub.17X.sub.18QW X.sub.21AFIX.sub.25 X.sub.26LX.sub.28D, wherein, independently of each other, X.sub.2 is selected from H, Q, S, T and V; X.sub.3 is selected from I, L, M and V; X.sub.4 is selected from A, D, E, H, K, L, N, Q, R, S, T and Y; X.sub.6 is selected from N and W; X.sub.7 is selected from A, D, E, H, N, Q, R, S and T; X.sub.11 is selected from A, E, G, H, K, L, Q, R, S, T and Y; X.sub.16 is selected from N and T; X.sub.17 is selected from I, L and V; X.sub.18 is selected from A, D, E, H, K, N, Q, R, S and T; X.sub.21 is selected from I, L and V; X.sub.25 is selected from D, E, G, H, N, S and T; X.sub.26 is selected from K and S; X.sub.28 is selected from A, D, E, H, N, Q, S, T and Y; and ii) an amino acid sequence which has at least 86% identity to the sequence defined in i), wherein the polypeptide binds to C5.
2. A C5 binding polypeptide according to claim 1, wherein the amino acid sequence i) fulfills at least four of the following eight conditions I-VIII: I. X.sub.2 is V; II. X.sub.3 is selected from I and L; III. X.sub.6 is IV. X.sub.7 is selected from D and N; V. X.sub.17 is selected from I and L; VI. X.sub.21 is L; VII. X.sub.25 is N; VIII. X.sub.28 is D.
3. The C5 binding polypeptide according to claim 1, wherein the amino acid sequence is selected from any one of SEQ ID NO:1-248.
4. The C5 binding polypeptide according to claim 3, wherein the amino acid sequence is selected from any one of SEQ ID NO:1-12, SEQ ID NO:20, SEQ ID NO:23-24, SEQ ID NO:26-28, SEQ ID NO:32-35, SEQ ID NO:38-39, SEQ ID NO:41, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:56-57, SEQ ID NO:59, SEQ ID NO:66, SEQ ID NO:78-79, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:106, SEQ ID NO:110, SEQ ID NO:119, SEQ ID NO:125, SEQ ID NO:141, SEQ ID NO:151, SEQ ID NO:161, SEQ ID NO:166, SEQ ID NO:187, SEQ ID NO:197, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:215 and SEQ ID NO:243.
5. The C5 binding polypeptide according to claim 4, wherein the amino acid sequence is selected from any one of SEQ ID NO:1-12.
6. The C5 binding polypeptide according to claim 1, in which said C5 binding motif forms part of a three-helix bundle protein domain.
7. A C5 binding polypeptide, which comprises the amino acid sequence: TABLE-US-00014 (SEQ ID NO: 764) K-[BM]-DPSQS X.sub.aX.sub.bLLX.sub.c EAKKL NDX.sub.dQ; or an amino acid sequence which has at least 79% identity to SEQ ID NO: 764, wherein X.sub.a is selected from A and S; X.sub.b is selected from N and E; X.sub.c is selected from A, S and C; X.sub.d is selected from A and S; and wherein [BM] is a C5 binding motif, which motif consists of the amino acid sequence EX.sub.2X.sub.3X.sub.4A X.sub.6X.sub.7EID X.sub.11LPNL X.sub.16X.sub.17X.sub.18QW X.sub.21AFIX.sub.25 X.sub.26LX.sub.28D (SEQ ID NO: 763), or an amino acid sequence which has at least 86% identity to SEQ ID NO:763, wherein independently of each other: X.sub.2 is selected from H, Q, S, T and V; X.sub.3 is selected from I, L, M and V; X.sub.4 is selected from A, D, E, H, K, L, N, Q, R, S, T and Y; X.sub.6 is selected from N and W; X.sub.7 is selected from A, D, E, H, N, Q, R, S and T; X.sub.11 is selected from A, E, G, H, K, L, Q, R, S, T and Y; X.sub.16 is selected from N and T; X.sub.17 is selected from I, L and V; X.sub.18 is selected from A, D, E, H, K, N, Q, R, S and T; X.sub.21 is selected from I, L and V; X.sub.25 is selected from D, E, G, H, N, S and T; X.sub.26 is selected from K and S; X.sub.28 is selected from A, D, E, H, N, Q, S, T and Y; and wherein the [BM] motif binds to C5.
8. The C5 binding polypeptide according to claim 7, wherein the amino acid sequence is selected from any one of SEQ ID NO:249-496.
9. The C5 binding polypeptide according to claim 8, wherein the amino acid sequence is selected from any one of SEQ ID NO:249-260, SEQ ID NO:268, SEQ ID NO:271-272, SEQ ID NO:274-276, SEQ ID NO:280-283, SEQ ID NO:286-287, SEQ ID NO:289, SEQ ID NO:294, SEQ ID NO:297, SEQ ID NO:304-305, SEQ ID NO:307, SEQ ID NO:314, SEQ ID NO:326-327, SEQ ID NO:335, SEQ ID NO:340, SEQ ID NO:354, SEQ ID NO:358, SEQ ID NO:367, SEQ ID NO:373, SEQ ID NO:389, SEQ ID NO:399, SEQ ID NO:409, SEQ ID NO:414, SEQ ID NO:435, SEQ ID NO:445, SEQ ID NO:451, SEQ ID NO:453, SEQ ID NO:463 and SEQ ID NO:491.
10. The C5 binding polypeptide according to claim 9, wherein the amino acid sequence is selected from any one of SEQ ID NO:249-260.
11. The C5 binding polypeptide according to claim 10, wherein the amino acid sequence is selected from any one of SEQ ID NO:497-757.
12. The C5 binding polypeptide according to claim 11, wherein the amino acid sequence is selected from any one of SEQ ID NO:497-508, SEQ ID NO:516, SEQ ID NO:519-520, SEQ ID NO:522-524, SEQ ID NO:528-531, SEQ ID NO:534-535, SEQ ID NO:537, SEQ ID NO:542, SEQ ID NO:545, SEQ ID NO:552-553, SEQ ID NO:555, SEQ ID NO:562, SEQ ID NO:574-575, SEQ ID NO:583, SEQ ID NO:588, SEQ ID NO:602, SEQ ID NO:606, SEQ ID NO:615, SEQ ID NO:621, SEQ ID NO:637, SEQ ID NO:647, SEQ ID NO:657, SEQ ID NO:662, SEQ ID NO:683, SEQ ID NO:693, SEQ ID NO:699, SEQ ID NO:701, SEQ ID NO:711, SEQ ID NO:739 and SEQ ID NO:746-757.
13. The C5 binding polypeptide according to claim 12, wherein the amino acid sequence is selected from any one of SEQ ID NO:497-508 and SEQ ID NO:746-757.
14. The C5 binding polypeptide according to claim 13, wherein the amino acid sequence is selected from any one of SEQ ID NO:497, SEQ ID NO:498, SEQ ID NO:499, SEQ ID NO:500, SEQ ID NO:501, SEQ ID NO:746, SEQ ID NO:747, SEQ ID NO:748, SEQ ID NO:750 and SEQ ID NO:753.
15. The C5 binding polypeptide according to claim 1, which inhibits cleavage of C5.
16. The C5 binding polypeptide according to claim 1, wherein the C5 binding polypeptide binds to C5 such that the K.sub.D value of the interaction is at most 1×10.sup.−6 M.
17. The C5 binding polypeptide according to claim 1, comprising further C terminal and/or N terminal amino acids that improve production, purification, stabilization in vivo or in vitro, coupling, or detection of the polypeptide.
18. The C5 binding polypeptide according to claim 1, wherein the polypeptide is in multimeric form, comprising at least two C5 binding polypeptide monomer units, the amino acid sequences of which may be the same or different.
19. A C5 binding compound, comprising at least one C5 binding polypeptide according to claim 1; at least one albumin binding domain of streptococcal protein G, or a derivative thereof; and at least one linking moiety for linking said at least one albumin binding domain or derivative thereof to the C or N terminal of said at least one C5 binding polypeptide.
20. The C5 binding compound according to claim 19, having a structure selected from [CBP1]-[L1]-[ALBD]; [CBP1]-[CBP2]-[L1]-[ALBD]; [CBP1]-[L1]-[ALBD]-[L2]-[CBP2]; [ALBD]-[L1]-[CBP1]; [ALBD]-[L1]-[CBP1]-[CBP2]; [CBP1]-[L1]-[CBP2]-[L2]-[ALBD]; and [ALBD]-[L1]-[CBP1]-[L2]-[CBP2] wherein, independently of each other, [CBP1] and [CBP2] are C5 binding polypeptides which may be the same or different; [L1] and [L2] are linking moieties which may be the same or different; and [ALBD] is an albumin binding domain of streptococcal protein G, or derivative thereof.
21. The C5 binding compound according to claim 20, wherein the linking moiety is selected from G; GS; [G.sub.2S].sub.n; [G.sub.3S].sub.n (SEQ ID NO:783); [G.sub.4S].sub.n (SEQ ID NO:784); GS[G.sub.4S].sub.n (SEQ ID NO:785); [S.sub.2G].sub.m; [S.sub.3G].sub.m (SEQ ID NO:786); [S.sub.4G].sub.m (SEQ ID NO:787); and VDGS (SEQ ID NO:788); wherein n is 0-7; and wherein m is 0-7.
22. The C5 binding compound according to claim 19, wherein said albumin binding domain is as set out in SEQ ID NO:759.
23. The C5 binding compound according to claim 19, wherein each of said C5 binding polypeptides is independently selected from any one of SEQ ID NO:497-757.
24. A polynucleotide encoding a polypeptide according to claim 1.
25. A polynucleotide encoding a compound according to claim 19.
26. A combination of a C5 binding polypeptide according to claim 1 with a therapeutic agent.
27. A combination of a C5 binding compound according to claim 19 with a therapeutic agent.
28. A method of treatment of a C5 related condition, comprising administering a C5 binding polypeptide according to claim 1, a C5 binding compound according to claim 19, or the combination according to claim 26, to a mammalian subject in need thereof.
29. The method of treatment according to claim 28, in which binding of the C5 binding polypeptide, the C5 binding compound or the combination to C5 inhibits cleavage of C5.
30. The method of treatment according to claim 28, wherein said C5 related condition is selected from inflammatory disease; autoimmune disease; infectious disease; cardiovascular disease; neurodegenerative disorders; cancer; graft injury; wounds; eye disease; kidney disease; pulmonary diseases; hematological diseases; allergic diseases and dermatological diseases.
31. The method of treatment according to claim 30, wherein said C5 related condition is paroxysmal nocturnal hemoglobinuria (PNH).
32. The method of treatment according to claim 28, wherein said C5 binding polypeptide is administered intravenously, subcutaneously, by inhalation, nasally, orally, intravitreally, or topically.
33. The C5 binding polypeptide according to claim 1, wherein the C5 binding polypeptide binds to C5 such that the K.sub.D value of the interaction is at most 1×10.sup.−7M.
34. The C5 binding polypeptide according to claim 1, wherein the C5 binding polypeptide binds to C5 such that the K.sub.D value of the interaction is at most 1×10.sup.−8 M.
35. The C5 binding polypeptide according to claim 1, wherein the C5 binding polypeptide binds to C5 such that the K.sub.D value of the interaction is at most 1×10.sup.−9 M.
Description
BRIEF DESCRIPTION OF THE FIGURES
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EXAMPLES
(17) The following materials were used throughout this work except where otherwise noted. Escherichia coli strain RR1ΔM15 (Rüther, Nucleic Acids Res 10:5765-5772, 1982). Escherichia coli strain XL1-Blue (Agilent Technologies, cat. no. 200268). Human complement protein C5 (hC5). The full 1676 amino acid pro-protein has GenBank accession number: NP_001726 (SEQ ID NO:760) wherein amino acids 19-673 is the beta chain and amino acids 678-1676 is the alpha chain. Human C5 used herein was purchased from Quidel (cat. no. A403) Cynomolgus complement protein C5 (cC5; SEQ ID NO:762). The cC5 sequence was derived from the Cynomolgus (Macaca fascicularis) genomic sequence (www.ebi.ac.uk/ena; Ebeling et al. (2001) Genome Res. 21(10):1746-1756). The coding region of human C5 was retrieved from www.ensembl.org, and the C5 gene was localized to chromosome 15. The region containing the gene is approximately 110 000 bases long and is contained in contigs CAEC01154150 to CAEC01154178. The contigs were manually joined to a single file and used as a genomic context for the sim4 software to align the coding region of human C5 to the raw Cynomolgus genomic material. Cynomolgus C5 used herein was purified in-house from serum using a three-step procedure; PEG6000 precipitation, ion exchange and OmCI affinity chromatography. Rat Complement protein C5 (rC5; GenBank accession number: XP_001079130) Rat C5 used herein was purified in-house from serum using a three-step procedure; PEG6000 precipitation, ion exchange and OmCI affinity chromatography. Human MG7 (hMG7) domain of complement protein C5, corresponding to amino acid residues 822-931 of human C5 (SEQ ID NO:760; Fredslund et al. (2008) Nature Immunology 9: 753-760) produced in-house in Freestyle HEK293 cells. hMG7 binding protein. OmCI (AF2999, Nunn, M. A. et al. supra) from soft tick Ornithodoros moubata OmCI with a His.sub.6 tag in the C-terminus (SEQ ID NO:761) was produced in-house in E. coli strain Origami(DE3) and purified on a HisTrap1 column.
Example 1: Selection and Screening of Complement Protein C5 Binding Polypeptides
(18) Materials and Methods
(19) Biotinylation of Target Protein hC5:
(20) hC5 was biotinylated according to the manufacturer's recommendations at room temperature (RT) for 40 min using No-Weigh EZ-Link Sulfo-NHS-LC-Biotin (Pierce, cat. no. 21327) at a ten times (10×) molar excess. Subsequent buffer exchange to PBS (10 mM phosphate, 137 mM NaCl, 2.68 mM KCl, pH 7.4) was performed using Protein Desalting Spin Columns (Pierce, cat. no. 89849) according to the manufacturer's instructions.
(21) Phage Display Selection of C5-Binding Polypeptides:
(22) Libraries of random variants of protein Z displayed on bacteriophage, constructed in phagemid pAffi1/pAY00065/pAY02947/pAY02592 essentially as described in Grönwall et al. J Biotechnol 2007, 128:162-183), were used to select C5 binding polypeptides. Three different library vectors were used. Two of these utilize an albumin binding domain (ABD, GA3 of protein G from Streptococcus strain G148) as fusion partner to the Z variants generating the libraries Zlib003Naive.I and Zlib006Naive.II. The third library, Zlib004Naive.I utilizes the Taq DNA polymerase binding molecule Z03639 (denoted Z.sub.TaqS1-1 in Gunneriusson et al. Protein Eng 1999, 12:873-878) as fusion partner. The libraries had the following actual sizes: 3×10.sup.9 (Zlib003Naive.I); 1.5×10.sup.10 (Zlib006Naive.II); and 1.4×10.sup.10 (Zlib004Naive.I), the number referring to the amount of variants.
(23) Phage stocks were prepared either in shake flasks (Zlib003Naive.I) as described in Grönwall et al. supra or in a 20 l fermenter (Zlib006Naive.II and Zlib004Naive.I). Cells from a glycerol stock containing the phagemid library Zlib004Naive.I were inoculated in 20 l of TSB-YE (Tryptic Soy Broth-Yeast Extract; 30 g/l TSB, 5 g/l yeast extract) supplemented with 2% glucose and 100 μg/ml ampicillin. Cells 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 (OD) of 0.7-0.8, approximately 2.6 l of the cultivation was infected using a 10× molar excess of M13K07 helper phage (New England Biolabs #N03155). The cells were incubated for 30 minutes, whereupon the fermenter were filled up to 20 l with TSB-YE supplemented with 100 μM IPTG (isopropyl-β-D-1-thiogalactopyranoside, for induction of expression), 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 and the phage particles remaining in the medium were thereafter precipitated 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.
(24) Selections were performed in four cycles against biotinylated hC5. Phage stock preparation, selection procedure and amplification of phage between selection cycles were performed essentially as described in WO 2009/077175. PBS supplemented with 3% bovine serum albumin (BSA) and 0.1% Tween20 was used as selection buffer and the target-phage complexes were directly captured by DYNABEADS M-280 Streptavidin (Dynal, cat. no. 112.06). 1 mg beads per 10 μg complement protein C5 was used. E. coli strain RR1ΔM15 was used for phage amplification. In cycle 1 of the selections, 100 nM hC5 was used and two washes with PBST 0.1% (PBS supplemented with 0.1% Tween-20) were performed. An increased stringency, using a lowered target concentration and an increased number of washes, was applied in the subsequent three cycles. In cycle 2, 3 and 4; 50 or 33 nM hC5, 25 or 11 nM hC5 and 12.5 or 3.7 nM hC5 were used. In cycle 2, 3 and 4; 4, 6 and 8 washes were performed, using PBST 0.1% in all cycles or PBST 0.2%, 0.3% and 0.4% in cycle 2, 3 and 4.
(25) ELISA Screening of Z Variants:
(26) To test if the selected Z variant molecules could indeed interact with human complement protein C5, ELISA assays were performed. The Z variants were produced by inoculating single colonies from the selections into 1 ml TSB-YE medium supplemented with 100 μg/ml ampicillin and 0.1 mM IPTG in deep-well plates (Nunc, cat. no. 278752). The plates were incubated for 18-24 h at 37° C. Cells were pelleted by centrifugation, re-suspended in 300 μl PBST 0.05% and frozen at −80° C. to release the periplasmic fraction of the cells. Frozen samples were thawed in a water bath and cells were pelleted by centrifugation. The periplasmic supernatant contained the Z variants as fusions to an albumin binding domain (GM of protein G from Streptococcus strain G148), expressed as AQHDEALE-[Z#####]-VDYV-[ABD]-YVPG (SEQ ID NO. 767) (Grönwall et al, supra), or to the Taq DNA polymerase binding molecule Z03639, expressed as AQHDEALE-[Z#####]-VDYV-[Z03639]-YVPG (SEQ ID NO. 768). Z##### refers to individual 58 amino acid residues Z variants.
(27) Half-area 96-well ELISA plates (Costar, cat. no. 3690) were coated with 50 μl/well of coating buffer (50 mM sodium carbonate, pH 9.6) containing 4 μg/ml of an antibody specific for Z variants (Affibody, cat. no. 20.1000.01.0005) and incubated over-night at 4° C. The antibody solution was poured off and the wells were blocked with 100 μl of PBSC (PBS supplemented with 0.5% casein (Sigma, cat. no. C8654) for 1-2 h at RT. The blocking solution was discarded and 50 μl periplasmic solution was added to the wells and incubated for 1 h at RT under slow shaking. The supernatants were poured off and the wells were washed 4 times with PBST 0.05%. Then 50 μl of biotinylated complement protein hC5, at a concentration of 5 μg/ml in PBSC, was added to each well. The plates were incubated for 1.5 h at RT followed by washes as described above. Streptavidin-HRP (Horseradish peroxidase; Dako, cat. no. P0397) was diluted 1:10,000 in PBSC, added to the wells which were then incubated for 45 min. 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 of the wells was measured at 450 nm using a multi-well plate reader, Victor.sup.3 (Perkin Elmer).
(28) As positive control, a periplasmic fraction also containing the PSMA binding molecule Z03938 expressed as AQHDEALE-[Z03938]-VDYV-[Z03639]-YVPG (SEQ ID NO. 769) was assayed against 5 μg/ml biotinylated PSMA protein. As negative control; the same periplasmic preparation was assayed against complement protein hC5. Sequencing was performed for the clones with positive absorbance values against hC5.
(29) Sequencing:
(30) PCR fragments were amplified from single colonies using a standard PCR program and the primers AFFI-21 (5′-tgcttccggctcgtatgttgtgtg) (SEQ ID NO. 770) and AFFI-22 (5′-cggaaccagagccaccaccgg) (SEQ ID NO. 771). Sequencing of amplified fragments was performed using the biotinylated oligonucleotide AFFI-72 (5′-biotin-cggaaccagagccaccaccgg) (SEQ ID NO. 772) 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 3100 Genetic Analyzer (PE Applied Biosystems).
(31) Blocking ELISA:
(32) Clones found positive for hC5 in the ELISA screening were subjected to an ELISA blocking assay in order to elucidate if their target binding was affected by the presence of the hC5 binding proteins OmCI and/or hMG7 binding protein. The blocking ELISA was run using Z variants expressed in periplasmic fractions as described in the section for ELISA screening above, but setting up 5 ml cultures in 12 ml round-bottom tubes and using 2 ml PBST 0.05% for pellet dissolution. The ELISA blocking assay was run as the ELISA screening assay, with a protocol modification introduced at the target step; OmCI or hMG7 binding protein were mixed with the target protein before addition to the assay plate. 5 μg/ml biotinylated hC5 was mixed with 5 times or 20 times molar excess of OmCI or hMG7 binding protein, respectively, then incubated 1 h at RT to allow complex formation before addition to the plate. For each clone, a reference (1), a negative control (2) and a background (3) response/signal, respectively, were obtained as follows: at the target step, solely hC5 was added to the Z variants (as in the screening ELISA) (1); the irrelevant protein PSMA (in house produced) was added to complement protein hC5, instead of OmCI or hMG7 binding protein (2); only buffer was added to the Z variants (3).
(33) Results
(34) Phage Display Selection of Complement Protein C5 Binding Polypeptides:
(35) Individual clones were obtained after two-four cycles of phage display selections against biotinylated hC5.
(36) ELISA Screening of Z Variants:
(37) The clones obtained after four cycles of selection were produced in 96-well plates and screened for complement protein C5 binding activity in ELISA. In total, nearly 400 clones were screened. The absorbance measurements showed many clearly hC5 positive clones. The result from a selection of clones is displayed in Table 1; the Z05363 (SEQ ID NO:510) variant is tagged with ABD, whereas the other listed Z variants are tagged with the Taq binding molecule Z03639 as described in the methods section. The PSMA specific molecule Z03938 used as a negative control gave a positive signal for PSMA, whereas no signal was obtained against hC5.
(38) Blocking ELISA:
(39) Clones positive for hC5 were subjected to a blocking assay using the hC5 binding proteins OmCI and hMG7 binding protein. For five clones, the binding signal to complement protein C5 was completely extinguished by the presence of OmCI, reaching the same level as the background (Table 1). One of these clones, namely the Z05363 variant (SEQ ID NO:510), was also tested for its ability to bind hC5 in the presence of hMG7 binding protein. The hMG7 binding protein did not inhibit the binding of Z05363 to hC5.
(40) TABLE-US-00005 TABLE 1 Response in ELISA to target, with or without blocking molecule for a number of Z variants. Z variant SEQ ID NO: # hC5 (OD 450 nm) OmCI-block Z05363 SEQ ID NO: 510 3.143 complete Z05477 SEQ ID NO: 509 2.872 complete Z05483 SEQ ID NO: 511 0.531 complete Z05538 SEQ ID NO: 512 0.099 complete Z05692 SEQ ID NO: 513 0.944 complete
Sequencing:
(41) Sequencing was performed for the clones with positive absorbance values against complement protein C5 in the ELISA screening. Each 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
Example 2: Production and Characterization of Z Variants
(42) Materials and Methods
(43) Subcloning of Z Variants, Protein Expression and Purification:
(44) Five complement protein C5 binding Z variants (Z05363 (SEQ ID NO:510); Z05477 (SEQ ID NO:509); Z05483 (SEQ ID NO:511); Z05538 (SEQ ID NO:512) and Z05692 (SEQ ID NO:513)) were amplified from pAffi1/pAY00065/pAY02947 library vectors. A subcloning strategy for construction of dimeric Affibody molecules with N-terminal His.sub.6 tags was applied using standard molecular biology techniques and as described in detail in WO 2009/077175. The Z gene fragments were subcloned into the expression vector pAY01448 resulting in the encoded sequence MGSSHHHHHHLQ-[Z#####][Z#####]-VD (SEQ ID NO. 773).
(45) The subcloned Z variants were transformed into E. coli BL21(DE3) and expressed in the multifermenter system Greta (Belach Bioteknik). In brief, cultures were grown at 37° C. in 800 ml TSB-YE-medium containing 50 μg/ml kanamycin. At an OD.sub.600 of ˜1, the cultures were induced through the automatic addition of IPTG to a final concentration of 0.05 mM. Cultures were cooled down to approximately 10° C. after 5 h of induction, and harvested by centrifugation (20 min, 15,900 g). Supernatants were discarded and the cell pellets were collected and stored at −20° C. until further use. Expression levels and the degree of solubility were estimated by SDS-PAGE analysis on 4-12% NUPAGE gels (Invitrogen) using Coomassie blue staining.
(46) For Z variants expressed mainly as soluble protein, the cell pellets were resuspended in binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4) with an addition of 1000 U BENZONASE (Merck, cat. no. 1.01654.001) and disrupted by ultrasonication. For each of the Z variants, the sonicated suspension was clarified by centrifugation (40 min, 25,000 g, 4° C.) and the supernatant was loaded onto a 1 ml His GRAVITRAP column (GE Healthcare). The column was washed with wash buffer (20 mM sodium phosphate, 0.5 M NaCl, 60 mM imidazole, pH 7.4), before eluting the Z variants with 3 ml elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 7.4). Z variants which expressed mainly as insoluble protein were purified likewise, but 8 M urea was included in the binding and wash buffer. If required, the Z variants were further purified by reversed phase chromatography (RPC) on 1 ml RESOURCE columns (GE Healthcare) using water including 0.1% TFA (trifluoroacetic acid) as mobile phase and elution with an appropriate gradient (typically 0-50% over 20 column volumes) of acetonitrile including 0.1% TFA.
(47) The buffer was exchanged to PBS using PD-10 columns (GE Healthcare).
(48) Protein Characterization:
(49) The concentration of the purified Z variants was determined by absorbance measurements at 280 nm using theoretical extinction coefficients. The purity was estimated by SDS-PAGE analysis on 4-12% NUPAGE gels (Invitrogen) using Coomassie blue staining. To verify the identity and to determine the molecular weights of purified Z variants, LC/MS-analyses were performed on an Agilent 1100 LC/MSD system (Agilent Technologies).
(50) CD Analysis:
(51) The purified Z variants were diluted to 0.5 mg/ml in PBS. For each diluted Z variant, a CD spectrum was recorded between 250-195 nm at a temperature of 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. The ability of the Z variant to refold was assessed by collecting an additional CD spectrum at 250-195 nm after cooling to 20° C. 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.
(52) Biacore Binding Analysis:
(53) The interactions of the five subcloned His.sub.6-tagged dimeric hC5-binding Z variants with hC5, cC5, rC5, hMG7 and hIgG (Sigma, cat. no. G4386) were analyzed in a Biacore instrument (GE Healthcare). The Z variants were immobilized in different flow cells on the carboxylated dextran layer of several CM5 chip surfaces (GE Healthcare). The immobilization was performed using amine coupling chemistry according to the manufacturer's protocol. One flow cell surface on each chip was activated and deactivated for use as blank during analyte injections. The analytes, diluted in HBS-EP running buffer (GE Healthcare) to a final concentration of 100 nM, were injected at a flow rate of 10 μl/min for 1 min. After 2 min of dissociation, the surfaces were regenerated with one injection of 10 mM HCl. The results were analyzed in BiaEvaluation software (GE Healthcare). Curves of the blank surface were subtracted from the curves of the ligand surfaces.
(54) Results
(55) Subcloning of Z Variants:
(56) Five selected unique clones (Z05477 (SEQ ID NO:509), Z05363 (SEQ ID NO:510), Z05483 (SEQ ID NO:511), Z05538 (SEQ ID NO:512) and Z05692 (SEQ ID NO:513)) were chosen for subcloning as dimers in the expression vector pAY01448 and were subsequently verified by sequencing.
(57) Protein Production:
(58) The histidine-tagged dimeric Z variants yielded acceptable expression levels of soluble gene product. The purity of produced batches was estimated to exceed 90% as assessed by SDS-PAGE analysis. LC/MS analysis verified the correct molecular weight for all Z variant molecules.
(59) CD Analysis:
(60) The melting temperatures (Tm) of the different Z variants were calculated by determining the midpoint of the transition in the CD signal vs. temperature plot. The results for a number of reversibly folding Z variants are summarized in Table 2 below.
(61) TABLE-US-00006 TABLE 2 Melting temperatures for a number of Z variants. SEQ ID NO: # of Tm Z variant monomeric Z variant (° C.) His.sub.6-(Z05477).sub.2 SEQ ID NO: 509 45 His.sub.6-(Z05363).sub.2 SEQ ID NO: 510 35 His.sub.6-(Z05483).sub.2 SEQ ID NO: 511 44 His.sub.6-(Z05538).sub.2 SEQ ID NO: 512 54 His.sub.6-(Z05692).sub.2 SEQ ID NO: 513 52
Biacore Binding Analysis:
(62) The binding of the five subcloned dimeric Z variants to different species of C5 and MG7, a subdomain of hC5, as well as the background binding to IgG was tested in a Biacore instrument by injecting the different proteins over surfaces containing the Z variants. The ligand immobilization levels for the different Z variants on the surfaces were: Z05363: 2080 RU, Z05477: 2180 RU, Z05483: 2010 RU, Z05538: 2570 RU and Z05692: 3270 RU. The different Z variants were tested for binding to different sets of proteins injected at concentrations of 100 nM, see Table 3. The result for the tested Z variants is displayed in the table as a +/− outcome for each protein. As an example of the Biacore binding analysis,
(63) TABLE-US-00007 TABLE 3 Biacore response of different Z variants against C5 from various species and relevant selected background proteins. SEQ ID NO: # of Z variant monomeric Z variant hC5 cC5 rC5 hMG7 hIgG His.sub.6-(Z05477).sub.2 SEQ ID NO: 509 + + + − − His.sub.6-(Z05363).sub.2 SEQ ID NO: 510 + + + − − His.sub.6-(Z05483).sub.2 SEQ ID NO: 511 + + + − − His.sub.6-(Z05538).sub.2 SEQ ID NO: 512 + + + − − His.sub.6-(Z05692).sub.2 SEQ ID NO: 513 + + − − −
Example 3: Design and Construction of a Maturated Library of Complement Protein C5 Binding Z Variants
(64) In this Example, a maturated library was constructed. The library was used for selections of hC5-binding polypeptides. Selections from maturated libraries are usually expected to result in binders with increased affinity (Orlova et al. Cancer Res 2006, 66(8):4339-48). In this study, randomized double stranded linkers were generated by the SLONOMICS technology which enables incorporation of randomized sets of trinucleotide building blocks using ligations and restrictions of the subsequently built up double stranded DNA.
(65) Materials and Methods
(66) Library Design:
(67) The library was based on a selection of sequences of the hC5 binding Z variants described in Examples 1 and 2. In the new library, 13 variable positions in the Z molecule scaffold were biased towards certain amino acid residues, according to a strategy based on the Z variant sequences defined in SEQ ID NO:509-513 (Z05477, Z05363, Z05483, Z05538, Z05692). A SLONOMAX library of double-stranded DNA, 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/GAG NNN NNN NNN GCA/GCC NNN NNN GAG/GAA ATC/ATT NNN NNN TTA/CTG CCT AAC TTA ACC/ACT NNN NNN CAA/CAG TGG NNN GCC/GCG TTC ATC/ATT NNN AAA/AAG TTA/CTG NNN GAT/GAC GAC CCA AGC CAG AGC TCA TTA TTT A-3′ (SEQ ID NO. 774) (randomized codons are illustrated as NNN) flanked with restriction sites XhoI and SacI, was ordered from Sloning BioTechnology GmbH (Pucheim, Germany). The theoretical distributions of amino acid residues in the new library finally including 12 variable Z positions are given in Table 4.
(68) TABLE-US-00008 TABLE 4 Library design. Amino acid position No in the Z of variant Randomization amino Pro- molecule (amino acid abbreviations) acids portion 9 H, Q, S, T, V 5 1/5 10 I, L, V, W 4 1/4 11 A, D, E, H, K, L, N, R, S, T, Y 12 1/12 13 N, Q, W, Y 4 1/4 14 A, D, E, H, I, K, L, N, Q, R, S, T, V, W, Y 15 1/14 17 D, E 2 1/2 18 A, D, E, G, H, I, K, L, Q, R, S, T, V, Y 14 1/14 24 I, L, V 3 1/3 25 A, D, E, H, K, N, Q, R, S, T, Y 11 1/11 28 I, L, V 3 1/3 32 A, D, E, F, G, H, K, L, N, Q, R, S, T, V 14 1/14 35 A, D, E, H, K, N, Q, R, S, T, W, Y 12 1/12
Library Construction:
(69) 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 (New England Biolabs, cat. no. R01460L, and cat. no. R0156L) and purified once more with PCR Purification Kit. Subsequently, the product was purified using preparative gel electrophoresis on a 1% agarose gel.
(70) 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 (New England Biolabs, cat. no. M0202S), 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.
(71) The ligation reactions (approximately 250 ng DNA/transformation) were electroporated into electrocompetent E. coli RR1ΔM15 cells (100 μl). Immediately after electroporation, approximately 1 ml of SOC medium (TSB-YE media, 1% glucose, 50 μM MgCl.sub.2, 50 μM MgSO.sub.4, 50 μM NaCl and 12.5 μM KCl) was added. The transformed cells were incubated at 37° C. for 50 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 71 of TSB-YE medium, supplemented with 2% glucose and 100 μg/ml ampicillin. The cells were pelleted for 15 min at 4,000 g, 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.
(72) Preparation of Phage Stock:
(73) Cells from the glycerol stock containing the C5 phagemid library were inoculated in 20 l of a defined proline free medium (described in Example 1) supplemented with 100 μg/ml ampicillin, and grown at 37° C. in a fermenter (Belach Bioteknik, BR20). All steps were performed as described in Example 1 for the library Zlib006Naive.II. After cultivation, the cells were pelleted by centrifugation at 15,900 g and the phage particles remaining in the medium were thereafter precipitated twice in PEG/NaCl, filtered and dissolved in PBS and glycerol as described in Example 1. Phage stocks were stored at −80° C. until use in selection.
(74) Results
(75) Library Construction:
(76) The new library was designed based on a set of OmCI-blocked C5 binding Z variants with verified binding properties (Example 1 and 2). The theoretical size of the designed library was 6.7×10.sup.9 Z variants. The actual size of the library, determined by titration after transformation to E. coli. RR1ΔM15 cells, was 1.4×10.sup.9 transformants.
(77) The library quality was tested by sequencing of 64 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. The locked position in the designed amino acid sequence (W in position 27) was reflected in the actual sequence in that only the expected amino acid occurred in that position. A maturated library of hC5 binding polypeptides was thus successfully constructed.
Example 4: Selection, Screening and Characterization of Z Variants from a Maturated Library
(78) Materials and Methods
(79) Phage Display Selection of Complement Protein C5 Binding Polypeptides:
(80) The target protein hC5 was biotinylated as described in Example 1. Phage display selections were performed against hC5 essentially as described in Example 1 using the new library of Z variant molecules described in Example 3. E. coli XL1-Blue was used for phage amplification. Selection was initially performed in two parallel tracks. In one track, the time of selection was 2 h, while in the other track, shorter selection times were used: 20 min in the first cycle and 10 min for subsequent cycles 2-4. These two tracks (1 and 2) were further divided in the second cycle, resulting in totally six tracks (1a-c and 2a-c, differing in target concentration and wash conditions). Selection was performed in a total of four cycles. In cycle 1 of the selections, 25 nM complement protein C5 was used and five washes with PBST 0.1% were performed. An increased stringency, using a lowered target concentration and an increased number of washes, was applied in the subsequent three cycles. In cycle 2, 3 and 4; 10, 5 or 2.5 nM complement protein C5, 4, 1 or 0.25 nM complement protein C5 and 1.6, 0.2 or 0.05 nM complement protein C5 were used. In cycle 2, 3 and 4; 10, 15 and 20 washes were performed using PBST 0.1%. In addition, the second last wash was prolonged to 3 h with a 50× excess of non-biotinylated hC5 in the washing solution for two of the tracks (1c and 2c).
(81) Sequencing of Potential Binders:
(82) 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 as described in Example 1.
(83) ELISA Screening of Z Variants:
(84) Single colonies containing Z variants were randomly picked from the selected clones of the complement protein C5 maturated library and grown in 1 ml cultivations as described in Example 1. Periplasmic proteins were released by 8 repeated freeze-thawing cycles. ELISA screenings were performed essentially as described in Example 1 with the following exceptions. Half-area 96-well ELISA plates were coated with 2 μg/ml of an ABD specific goat antibody (in house produced) diluted in coating buffer. Biotinylated hC5 was used at a concentration of 0.15 μg/ml and incubation performed for 1.5-2 h. Streptavidin conjugated HRP was obtained from Thermo Scientific (cat. no. N100). The Z variant Z05363 (SEQ ID NO:510) originating from the primary selections (Example 1) was used as a positive control as well as a negative control omitting hC5.
(85) Selected maturated Z variants were subjected to a second screen against hC5 at a lower concentration and compared to rC5. The assay was essentially performed as described above. hC5 and rC5 was used at a concentration of 0.05 μg/ml and 4 μg/ml, respectively. The Z variant Z05363 (SEQ ID NO:510) was used as a positive control in this experiment as well. As a negative control, a Z variant binding to PDGF-Rβ (Z01977; described in WO 2009/077175) was assayed against biotinylated hC5 or rC5.
(86) In deep sequence analysis of selected Z variants and correlation of amino acids in the 13 randomized positions with measured melting temperatures and IC.sub.50 values for human C5 and mouse C5 in the hemolysis assay (described in Example 6) suggested a favorable Z variant not identified among the 558 sequenced clones. Based on the Z variant Z05998 (SEQ ID No:499), a single amino acid, Ile in position 10 was substituted with Leu using conventional technology for site directed mutagenesis. The new variant is referred to as Z08044 (SEQ ID NO:498). The deduced complement protein C5 binding motif of this Z variant is listed in
(87) Results
(88) Phage Display Selection of Complement Protein C5 Binding Polypeptides:
(89) Selection was performed in totally six parallel tracks containing four cycles each. The different selection tracks differed in target concentration and wash conditions as follows: 1a) 2 h selection time, high concentration, standard wash, 1b) 2 h selection time, low concentration, standard wash, 1c) 2 h selection time, medium concentration, long wash, 2a) 10 min selection time, high concentration, standard wash, 2b) 10 min selection time, low concentration, standard wash, and 2c) 10 min selection time, medium concentration, long wash. For each selection cycle, the target concentration was decreased and the washing conditions were more stringent. All tracks gave in each round sufficient amounts of phage particles in the eluate. Most phage particles were found in tracks 1a and 2a, representing the highest target concentration and mildest wash conditions.
(90) Sequencing:
(91) Randomly picked clones (558) were sequenced. Each individual Z variant was given an identification number, Z#####, as described in Example 1. In total, 242 new unique Z variant molecules were identified. The amino acid sequences of the 58 amino acid residues long Z variants are listed in
(92) ELISA Screening of Z Variants:
(93) Clones obtained after four selection cycles were produced in 96-well plates and screened for hC5-binding activity using ELISA. All randomly picked clones were analyzed. 229 of the 242 unique Z variants were found to give a higher response (0.3-3.1 AU) against hC5 at a concentration of 0.15 μg/ml compared to the positive control clone Z05363 (SEQ ID NO:510; an average absorbance signal of 0.3 AU), obtained from the primary selections (Example 1). Clones from all selection tracks showed positive signals. The negative controls had an absorbance of approximately 0.1 AU.
(94) Z variants were selected based on their performance in the ELISA screen against hC5 and the occurrence frequency. 43 unique Z variants were assayed against a lower concentration of hC5 (0.05 μg/ml) as well as rC5 (4 μg/ml). A positive result against rC5 was obtained for 40 of the tested Z variants, defined as 2× the signal for the negative control (0.4 AU). The results for all the tested Z variants against the lower concentration of hC5 as well as against rC5 are shown in
Example 5: Subcloning, Production and Characterization of a Subset of Complement Protein C5 Binding Z Variants
(95) Materials and Methods
(96) Subcloning of Z Variant Molecules into Expression Vectors:
(97) Based on sequence analysis and the performance in the ELISA against human and rat complement protein C5, 45 clones were selected for subcloning into the expression vector pAY01448. Monomer Z variant fragments were amplified from the phagemid vector pAY02592 and the subcloning into pAY01448 was performed as described in Example 2, resulting in a vector encoding the protein sequence MGSSHHHHHHLQ-[Z#####]-VD (SEQ ID NO. 775).
(98) Protein Expression and Purification:
(99) The 45 Z variants in the His.sub.6-(Z#####) format, were expressed in an automated multifermenter system as described in Example 2 or similarly in a small scale set-up of 100 ml cultures in shaker flasks induced manually with IPTG to a final concentration of 0.4 mM. Purification was performed using 1 ml His GRAVITRAP columns essentially as described in Example 2 or in a smaller scale using 0.1 ml His SpinTrap (GE Healthcare, cat. no. 28-4013-53). Buffer was exchanged to PBS using PD-10 columns or PD SpinTrap G-25 (GE Healthcare, cat. no. 28-9180-04) according to the manufacturer's instructions. The concentration of purified Z variants was determined by absorbance measurements at 280 nm and the purity and identity was assessed by SDS-PAGE and LC/MS as described in Example 2. Samples were aliquoted and stored at −80° C. until further use.
(100) CD Analysis:
(101) The CD analysis for determination of melting temperatures and folding reversibility was performed as described in Example 2.
(102) Results
(103) Protein Expression and Purification:
(104) All 45 subcloned Z variants could be expressed and the in vitro solubility for all purified variants was good. The purity was estimated by LC/MS to exceed 90% for all variants. The correct molecular weights were verified by LC-MS.
(105) CD Analysis:
(106) CD spectrum measurements performed at 20° C. confirmed the α-helical structure of the Z variants at this temperature. An overlay of the spectrums obtained after the variable temperature measurements (heating to 90° C. followed by cooling to 20° C.) on the spectrums obtained before the variable temperature measurement showed that all Z variants fold back completely, or nearly completely, to their α-helical structures after heating to 90° C. (results not shown). The melting temperatures for a set of Z variants were determined from the variable temperature measurements and are shown in Table 5.
(107) TABLE-US-00009 TABLE 5 Melting temperatures of maturated Z variants with a histidine tag fused directly to the amino terminus of SEQ ID NO: 497 and SEQ ID NO: 499-508. Z variant SEQ ID NO: # of Z variant Tm (° C.) His.sub.6-Z06175 SEQ ID NO: 497 44 His.sub.6-Z05998 SEQ ID NO: 499 45 His.sub.6-Z06009 SEQ ID NO: 500 45 His.sub.6-Z06079 SEQ ID NO: 501 46 His.sub.6-Z06126 SEQ ID NO: 502 44 His.sub.6-Z06140 SEQ ID NO: 503 42 His.sub.6-Z06189 SEQ ID NO: 504 47 His.sub.6-Z06214 SEQ ID NO: 505 44 His.sub.6-Z06215 SEQ ID NO: 506 41 His.sub.6-Z06226 SEQ ID NO: 507 44 His.sub.6-Z06018 SEQ ID NO: 508 46
Example 6: In Vitro Characterization of C5 Binding Z Variants
(108) Materials and Methods
(109) Cloning and Protein Production:
(110) DNA encoding a subset of C5 binding Z variants (SEQ ID NO:745-757) where E. coli codon optimized and synthesized by GeneArt, GmbH. The synthetic genes representing the C5 binding Z variants were subcloned and expressed in E. coli. The expression vectors encoding constructs of monomers or dimers of Z variants optionally linked to an albumin binding domain (ABD094, SEQ ID NO:759) are schematically illustrated in
(111) Intracellularly expressed Z variants were purified using conventional chromatography methods. Homogenization and clarification was performed by sonication followed by centrifugation and filtration. Anion exchange chromatography was used as capture step. Further purification was obtained by hydrophobic interaction chromatography. The purifications were executed at acidic conditions (pH 5.5). Polishing and buffer exchange was performed by size exclusion chromatography. Before concentration to final protein content, the endotoxin level was reduced by polymyxin B affinity chromatography. Produced proteins were analyzed by MALDI-TOF MS and on SDS-PAGE.
(112) In addition, recombinantly expressed OmCI protein (SEQ ID NO:761) was used as a reference molecule in the in vitro studies.
(113) Inhibition of Hemolysis:
(114) For studies of classical complement pathway function and inhibition thereof by C5 binding polypeptides, sheep erythrocytes were prepared from fresh sheep whole blood in Alsever's solution (Swedish National Veterinary Institute) and thereafter treated with rabbit anti-sheep erythrocyte antiserum (Sigma) to become antibody sensitized sheep erythrocyte (EA). The whole process was conducted under aseptic conditions. All other reagents were from commercial sources.
(115) The in vitro assay was run in 96-well U-form microtiter plate by consecutive additions of a test protein, a complement serum and EA suspension. The final concentrations of all reagents, in a total reaction volume of 50 μl per well and at pH 7.3-7.4, were: 0.15 mM CaCl.sub.2; 0.5 mM MgCl.sub.2; 3 mM NaN.sub.3; 138 mM NaCl; 0.1% gelatin; 1.8 mM sodium barbital; 3.1 mM barbituric acid; 5 million EA; complement protein C5 serum at suitable dilution, and C5 binding Z variant at desired concentrations. Different species of complement sera were used in the assay to define cross-species potencies of the Z variants. For mouse serum, a C5 depleted human serum (C5D from Quidel cat. no. A501) had to be supplemented in an equal amount.
(116) The Z variants were pre-incubated with the above described complement serum for 20 min on ice prior to starting the reaction by the addition of EA suspension. The hemolytic reaction was allowed to proceed at 37° C. during agitation for 45 min and was then optionally ended by addition of 100 μl ice-cold saline containing 0.02% Tween 20. The cells were centrifuged to the bottom and the upper portion, corresponding to 100 μl supernatant, was transferred to a transparent microplate having half-area and flat-bottom wells. The reaction results were analyzed as optical density using a microtiter plate reader at a wavelength of 415 nm.
(117) On all test occasions, controls, vehicle and OmCI (SEQ ID NO:761), were included in each plate to define the values of uninhibited and fully inhibited reactions, respectively. These values were used to calculate the % inhibition of the complement hemolysis at any given sample concentration. The inhibitory potencies (IC.sub.50 values) of tested Z variants were defined by applying the same assay in the presence of a controlled concentration of human C5 added to C5 depleted serum. For highly potent inhibitors (low nanomolar to sub-nanomolar), a final C5 concentration of the reaction mixture was controlled at 0.1 nM, which was optionally established by using C5 depleted or deficient sera.
(118) In Vitro Kinetics and Affinity of C5 Binding Z Variants to Immobilized hC5:
(119) The binding affinity of a number of C5 binding Z variants (SEQ ID NO:748-757) to hC5 were analyzed using a Biacore T200 instrument (GE Healthcare). Human C5 (A403, Quidel Corporation) was coupled to a CM5 sensor chip (900 RU) using amine coupling chemistry according to the manufacturer's protocol. The coupling was performed by injecting hC5 at a concentration of 7.5 μg/ml in 10 mM Na-acetate buffer pH 5 (GE Healthcare). The reference cell was treated with the same reagents but without injecting human C5.
(120) All experiments were performed in 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Surfactant P20 (HBS-EP buffer, GE Healthcare). For kinetic analyses, the flow rate was 30 μl/min and data were collected at 25° C. Data from the reference cell were subtracted to compensate for bulk refractive index changes. In most cases, an injection of HBS-EP was also included as control so that the sensorgrams were double blanked. The surfaces were regenerated in HBS-EP buffer.
(121) Binding of Z variants to immobilized hC5 was studied with the single cycle kinetics method, in which five concentrations of sample are injected one after the other in the same cycle without regeneration between injections. Kinetic constants were calculated from the sensorgrams using the Langmuir 1:1 or bivalent analyte model of Biacore T200 Evaluation Software version 1.0.
(122) In Vitro Kinetics and Affinity of C5 Binding Z-ABD Molecules to Immobilized hC5:
(123) Binding of Z-ABD molecules (SEQ ID NO:748-757 fused to ABD094 (SEQ ID NO:759) by a GS linker), to immobilized hC5 was evaluated using a Biacore T200 instrument (GE Healthcare).
(124) Z-ABD constructs where Z06175a (SEQ ID NO:753) as a monomer or dimer have been fused to ABD094 (SEQ ID NO:759) either in the N-terminus or the C-terminus via different linkers as specified in
(125) Steady State Binding of C5 Binding Z Variants to C5 Coated ECL Plates:
(126) The affinity of a number of C5 binding constructs comprising Z variants (SEQ ID NO:745, SEQ ID NO:748-757 optionally fused to ABD094 (SEQ ID NO:759) in constructs as specified in
(127) The Z-ABD variant (SEQ ID NO:748 fused to SEQ ID NO:759 by a GS-linker) to be used as tracer was labeled at a molar ratio 1:12 to 1:20 (protein: SULFO-TAG NHS-Ester, Meso Scale Discovery, cat. no. R91AN-1). The labeling reaction was performed on ice for two hours. Unbound SULFO-TAG was removed using a ZEBA spin desalting column (Thermo Scientific, cat no. 89889) and final protein concentration was measured by using Bradford reagent (Bradford, M. M., Anal. Biochem. 72: 248-254, 1976). The affinity (dissociation constant, K.sub.D) of the SULFO-TAG labeled Z-ABD variant was determined by saturation binding analysis of increasing concentrations of the labeled Z-ABD variant to C5 coated electrochemoluminescence wells (ECL, Meso Scale Discovery). The labeled Z-ABD variant was further analyzed by LC/MS in order to determine the distribution of SULFO-TAG molecules on the Z-ABD variant.
(128) Displacement was carried out by coating ECL, Multi-array 96-well high-bind, non-coated (Meso Scale Discovery, cat. no. L15XB) plates with 50 fmol/well hC5 over night at 4° C. Subsequently, non-specific sites were blocked with PBS with 1% Casein for two hours at RT. Different Z variants optionally fused with ABD094 (SEQ ID NO:759) (see
(129) Selectivity of Z-ABD Binding to C5 Over C3, C4 and IgG:
(130) Binding of one Z-ABD variant (SEQ ID NO:748 fused to SEQ ID NO:759 by a GS-linker) to the closely related complement proteins C3 and C4 from human as well as binding to human IgG (since the origin of the Z-domain, Staphylococcal protein A, is an IgG binding protein) was addressed by surface plasmon resonance (SPR) using a Biacore 2000 instrument (GE Healthcare). The Z-ABD construct was immobilized on a CM5 chip (GE-Healthcare) using amine coupling (70 RU). 40 nM and 400 nM of each of human C3 (A401, Quidel), C4 (A402, Quidel) and IgG (12511, Sigma) diluted in HBS-P buffer (GE Healthcare) were injected over the surface. Each injection was followed by a regeneration cycle with 20 mM NaOH injected for 30 s. Human C5 at the same concentrations was run in parallel as a positive control.
(131) Results
(132) Cloning and Protein Production:
(133) Produced protein variants as schematically described in
(134) Inhibition of Hemolysis:
(135) A subset of C5 binding Z variants were assayed for C5 binding activity in vitro and inhibition of hemolysis in sheep erythrocytes. The concentration of Z variant resulting in 50% inhibition of hemolysis (IC.sub.50) or 50% inhibition of tracer binding to human C5 was calculated. Representative concentration-response curves for Z variants shown as SEQ ID NO:745 and SEQ ID NO:748-757 inhibiting hemolysis as described in the methods section are shown in
(136) The parental Z variant Z05477a (SEQ ID NO:745) fused to ABD094 (SEQ ID NO:759) separated by a short GS linker exhibited an IC.sub.50 value of about 100 nM, whereas the tested second-generation C5 binding Z-ABD variants typically inhibited hemolysis with IC.sub.50 values around or below 1 nM. This suggests a more than 100-fold increase in potency for the C5 binding Z variants identified in the maturation selection and subsequent screening.
(137) In
(138) In Vitro Kinetics:
(139) Kinetic studies of binding characteristics for a number of Z variants (SEQ ID NO:748-757) optionally fused to ABD094 (SEQ ID NO:759), to immobilized hC5, as well as to C5 in the presence of human albumin, were performed using the Biacore T200 instrument.
(140) Data for ten different Z variants fused to ABD094 via a GS linker are presented in Table 6.
(141) TABLE-US-00010 TABLE 6 Human C5-binding characteristics for different Z-ABD fusions SEQ ID NO: Construct # of Z variant k.sub.a (1/Ms) k.sub.d (1/s) K.sub.D (M) Z-GS- SEQ ID NO: 748 6.93 × 10.sup.5 9.04 × 10.sup.−4 1.31 × 10.sup.−9 ABD094 SEQ ID NO: 749 6.75 × 10.sup.5 1.23 × 10.sup.−3 1.83 × 10.sup.−9 SEQ ID NO: 750 7.65 × 10.sup.5 1.34 × 10.sup.−3 1.75 × 10.sup.−9 SEQ ID NO: 751 6.90 × 10.sup.5 1.29 × 10.sup.−3 1.87 × 10.sup.−9 SEQ ID NO: 752 7.02 × 10.sup.5 1.81 × 10.sup.−3 2.58 × 10.sup.−9 SEQ ID NO: 753 7.90 × 10.sup.5 1.01 × 10.sup.−3 1.18 × 10.sup.−9 SEQ ID NO: 754 5.00 × 10.sup.5 1.14 × 10.sup.−3 2.28 × 10.sup.−9 SEQ ID NO: 755 6.84 × 10.sup.5 2.08 × 10.sup.−3 3.05 × 10.sup.−9 SEQ ID NO: 756 3.17 × 10.sup.5 6.37 × 10.sup.−3 2.01 × 10.sup.−9 SEQ ID NO:757 4.63 × 10.sup.5 1.08 × 10.sup.−3 2.34 × 10.sup.−9
(142) Binding of the same Z variant (SEQ ID NO:753) but in different constructs; i.e. with/without ABD and different linkers, were also analyzed using Biacore T200. In addition, the effect of albumin on some Z-ABD fusions was also assessed by running the same analysis in the absence and in the presence of human albumin. These data are presented below in Table 7.
(143) TABLE-US-00011 TABLE 7 Human C5-binding characteristics for a Z-ABD fusion variant Z06175a (SEQ ID NO: 753, abbreviated Z) comprised in different constructs. Human Construct albumin k.sub.a (1/Ms) k.sub.d (1/s) K.sub.D (M) Z-GS-ABD094 − 7.37 × 10.sup.5 1.06 × 10.sup.−3 1.43 × 10.sup.−9 Z-GS-ABD094 + 6.74 × 10.sup.5 9.62 × 10.sup.−4 1.43 × 10.sup.−9 Z-Z-GS-ABD094 − 5.93 × 10.sup.5 3.74 × 10.sup.−4 6.30 × 10.sup.−10 Z-Z-GS-ABD094 + 6.02 × 10.sup.5 4.67 × 10.sup.−4 7.76 × 10.sup.−10 Z-GS-ABD094- − 8.69 × 10.sup.5 5.75 × 10.sup.−4 6.62 × 10.sup.−10 GSGGGGSGGGGS-Z (SEQ ID NO. 777) Z-GS-ABD094- + 6.55 × 10.sup.5 3.83 × 10.sup.−4 5.86 × 10.sup.−10 GSGGGGSGGGGS-Z (SEQ ID NO. 777) Z-Z-GSGGGGSGGGGS- − 4.59 × 10.sup.5 6.32 × 10.sup.−4 1.38 × 10.sup.−9 ABD094 (SEQ ID NO. 778) Z-Z-GSGGGGSGGGGS- + 8.32 × 10.sup.5 9.39 × 10.sup.−4 1.13 × 10.sup.−9 ABD094 (SEQ ID NO. 778) Z-GS − 2.42 × 10.sup.6 1.40 × 10.sup.−3 5.79 × 10.sup.−10 Z-GSGGGGSGGGGS- − 3.64 × 10.sup.5 1.37 × 10.sup.−3 3.75 × 10.sup.−9 ABD094 (SEQ ID NO. 779)
(144) Surprisingly small effects could be seen when comparing the affinities of the constructs for hC5 (SEQ ID NO:760) in the presence and absence of albumin. This suggests that simultaneous binding of albumin to the ABD moiety of the constructs does not interfere with C5 interaction.
(145) Steady State Binding of C5 Binding Z Variants to C5 Coated ECL Plates:
(146) Steady state binding of C5 binding constructs composed of different Z variants (SEQ ID NO:745 and 748-757), optionally fused to ABD094 (SEQ ID NO:759) in constructs as specified in
(147) The C5 binding Z-variants (SEQ ID NO:748-757) fused in the carboxy terminus to ABD094 (SEQ ID NO:759) by a GS linker were found to displace 200 pM SULFO-TAG labeled Z-ABD variant with IC.sub.50 values ranging from about 300 pM to 1 nM (
(148) A number of different constructs containing the same C5 binding domain as a monomer, dimer, with or without ABD as well as a few different linkers between the different domains were also tested (
(149) Selectivity:
(150) Selectivity was addressed using SPR analysis and the surface with the immobilized Z-ABD variant (SEQ ID NO:748 fused to SEQ ID NO:759 by a GS-linker) displayed no significant SPR signal when subjected to 40 and 400 nM of the C5 paralogs human C3 and C4 as well as human IgG. As a comparison, 400 nM human C5 elicited an SPR response of about 450 RU showing that the tested Z-ABD variant indeed is selective for C5 over C3, C4 and IgG.
Example 7: Interaction Studies of Z-ABD Variants with HSA, BSA and Serum Album from Rat and Mouse
(151) Materials and Methods
(152) Two different methods, size exclusion chromatography and Biacore, were used to study the interaction between the albumin binding domain ABD094 fused to a C5 binding Z variants.
(153) Size exclusion chromatography (SEC) was employed to study the interaction between Z06175a-GS-ABD094 (SEQ ID NO:753 fused to SEQ ID NO:759 by a GS linker) and HSA. Briefly, equimolar amounts of Z06175a-GS-ABD094 and recombinant HSA (Novozymes) were preincubated in PBS at room temperature for 60 minutes and subsequently run on a Superdex200 column (GE Healthcare) using the SMART system (GE Healthcare). Z06175a-GS-ABD094 and HSA were also run separately as controls.
(154) Binding to immobilized albumin was studied using a Biacore 2000 instrument (GE Healthcare). Recombinant human albumin (RECOMBUMIN, Novozymes) was coupled to a CM5 sensor chip (385 RU) using amine coupling chemistry as described by the manufacturer. The coupling was performed by injecting human albumin in 10 mM Na-acetate buffer pH 4.5 (GE Healthcare). The reference cell was treated with the same reagents but without injecting human albumin Injection of HBS-EP was also included as control so that the sensorgrams were double blanked. Experiments were performed in HBS-EP buffer, 10 mM glycine-HCl pH 2 (GE Healthcare) was used for regeneration, the flow rate was 30 μl/min and data were collected at 25° C. Two different constructs were tested, Z-ABD (Z06175a-GS-ABD094) and Z-ABD-Z (Z06175a-GS-ABD094-GSGGGGSGGGGS-Z06175a) (SEQ ID NO. 780) at three different concentrations; 25 nM, 100 nM and 400 nM. BIAevaluation version 4.1.1 was used for evaluation of sensorgram data. In a similar fashion, binding of Z-ABD (Z06175a-GS-ABD094) to surfaces immobilized with serum albumin from rat (A4538, Sigma), mouse (A3559, Sigma), and cow (BSA, Sigma) was also investigated.
(155) Results
(156) On a SEC column, larger molecules elute faster than small. As seen in
(157) Biacore 2000 data for the analyzed Z-ABD and Z-ABD-Z variants show that the Z-ABD has a faster on-rate than when ABD is flanked by Z-domains on either side (
(158) Z06175a-GS-ABD094 bound to rat serum albumin with very high affinity (KD<100 pM) whereas the interaction with immobilized mouse serum albumin was weaker (KD of about 4 nM) than both with human and rat serum albumin Interaction with bovine serum albumin was not measurable.
(159) These data agree well with published data on an earlier variant of ABD (Jonsson et al. Protein Engineering, Design & Selection 2008, 21: 515-527) and show that the tested Z-ABD variant is strongly bound to serum albumin in human at clinically relevant concentrations as well as in mouse and rat allowing comparisons of pharmacokinetic data between animals and humans.
Example 8: Pharmacokinetic Studies of C5 Binding Z Variant in Rats
(160) Materials and Methods
(161) Rodent in-Life Phase:
(162) The pharmacokinetics of two C5 binding constructs Z-ABD (Z06175a-GS-ABD094; SEQ ID NO:753 fused to SEQ ID NO:759 by a GS linker,
(163) Determination of C5 Binding Z Variant Concentrations in Serum Samples from Animals Using LC/LC/MS/MS:
(164) Serum concentrations of the administrated C5 binding constructs Z-ABD and Z-ABD-Z, as described above, were determined by mass spectrometry (LC/LC/MS/MS). Serum or plasma samples (25 μl) were diluted with 150 μl of a pepsin agarose (7 mg/ml, Sigma, cat. no. P0609) suspended in 1 M ammonium formate buffer pH 3.0 in a 500 μl Eppendorf tube. The tubes were capped and agitated in an Eppendorf thermomixer compact at 37° C. for 20 min Following agitation, 25 μl of an internal standard solution I(.sup.13C.sub.6;.sup.15N)NKLDDDPSQSSEL (SEQ ID NO. 782) (amino acids 31-44 of the SEQ ID NO:746-757) (Thermo Fisher Scientific GmbH), diluted to 0.5 μM in 0.1% trifluoroacetic acid (TFA), was added. Following addition of internal standard, the samples were mixed and filtered through 0.45 μm cellulose spin filters (Grace).
(165) Standard samples for calibration were prepared by weighing 20 μl of protein stock solution with known protein concentration (5-10 mg/ml) followed by dilution with blank plasma from the species to be analyzed. The first stock plasma standard (3 μM) was diluted further down to 0.1 μM.
(166) 40 μl of the samples were injected into a coupled column system followed by tandem mass spectrometry with multiple reaction monitoring (MRM). The first column was an Ascentis RP-Amide column packed with 5 μm particles (2.1×150 mm, Supelco). An enrichment column; a Brownlee newgard column (3.2×15 mm) packed with 7 μm C18 particles, was used to trap the analyte peptide fraction from the first column. The effluent from the first column was diluted with 1 ml/min water pumped by Shimadzu pump into a whirl mixer (Lee Scientific). The last column was a mixed mode reversed phase and cation exchange column (2.1×100 mm) packed with 5 μm particles Primesep 100 (SIELC Inc).
(167) The mobile phases for the first column (RP-Amide) provided on a first liquid chromatograph (Acquity UPLC) were A: 2% acetonitrile, 0.1% acetic acid, 0.1% TFA, and 97.8% water, and B: acetonitrile with 0.1% acetic acid and 0.02% TFA. The flow was 0.5 ml/min and a linear gradient was used for elution. The sample was eluted at isocratic conditions with 100% A for 1 min, followed by 80% A at 7.9 min. At 8.1 min, the column was washed with 100% B for one minute, followed by reconditioning with 100% A. The effluent from the column was connected to a Valco six port valve controlled from the mass spectrometer software.
(168) The trap column (3.2×15 mm) was connected to the six port valve in back flush mode. The mobile phases for the second column, provided on a second liquid chromatograph (Agilent 1100), were A: 80% acetonitrile, 19.9% water, and 0.1% formic acid, and B: 80% acetonitrile, 19% water, 0.5% acetic acid and 0.5% TFA pumped by an Agilent 1100 liquid chromatograph at 0.5 ml/min and eluted with the following gradient: 100% A during the first 5 minutes followed by B gradually being raised from 0 to 40% from 5 to 10 minutes followed by a raise to 100% B during the next 6 seconds (10 to 10.1 minutes). B was kept at 100% until 11.5 minutes followed by a drop to 0% (100% A) during the next 6 seconds (11.5 to 11.6 minute) and kept at 0% B throughout the cycle until stopped at 13 minutes.
(169) The effluent from the last column was connected to a triple quadrupole mass spectrometer (Sciex API 4000) equipped with an electrospray ion source operated in positive ion mode. The MRM transitions were 780.9>814.4 for the analyte and 784.5>821.4 for the internal standard. The declustering potential was optimized at 55 V and the collision energy to 35 V. The effective collision energy was 70 eV since the precursor ion was doubly charged giving a singly charged fragment ion. The peak area ratios between the analyte and internal standard were used for quantification. Linear calibration curves were obtained with a recovery of 85% and a limit of quantification of about 40 nM.
(170) Ex Vivo Hemolysis:
(171) An ex vivo hemolytic assay for complement activation was performed in order to optimally assemble in vivo conditions for the serum samples from the above described in vivo studies. The serum samples were 5× diluted in a total reaction volume of 25 μl/well comprising 5 million antibody sensitized sheep erythrocytes (EA). In general, a portion of 20 μl EA suspension containing all other components (see Example 6) was mixed (agitation 10 minutes) with 5 μl serum sample to initiate the hemolytic activation at 37° C. For mouse serum samples, such as in example 11, however, 1 μl C5D had to be included in the 20 μl EA suspension. The ex vivo assay was performed essentially as described for the in vitro assay of Example 6. Calculations: Evaluation of the pharmacokinetic parameters was based on individual serum concentration data, the mean (±stdev) is reported for each dose group. Levels below lower limit of quantitation (LLOQ) appearing at terminal sampling points were omitted from the pharmacokinetic analysis. Maximum serum concentration, C.sub.max, and time to observed maximum serum concentration, t.sub.max, were obtained directly from the serum concentration data. The pharmacokinetic parameters; area under curve (AUC, AUC.sub.o-∞ and AUC.sub.0-last calculated by the linear trapezoidal method), subcutaneous bioavailability (F, calculated as (AUC.sub.sc/AUC.sub.iv)*(Dose.sub.iv/Dose.sub.sc)), terminal serum half-life (T.sub.1/2,z, calculated as ln 2/λ.sub.z where estimation of terminal slope, λ.sub.z, was based on at least 4 C=f(t) observations), mean residence time (MRT, calculated as AUMC/AUC), serum clearance (CL, calculated as Dose/AUC.sub.0-∞), volume of distribution at steady state (V.sub.ss, calculated as CL*MRT) and volume of distribution at the terminal phase (V.sub.z, calculated as CL/2) were calculated using WinNonlin software version 5.2.1 (Pharsight Corp., USA), Non-Compartmental-Analysis.
(172) Results
(173) The pharmacokinetic data for Z-ABD and Z-ABD-Z following i.v. (250 nmol/kg) and s.c. (500 nmol/kg) administration are summarized in Table 8. Z-ABD was quantifiable in serum up to 10-14 days post dose in the i.v. group and 14 days in the s.c. group whereas Z-ABD-Z was quantifiable in serum up to 10 days post dose in both dose groups (
(174) Correlating the serum concentration of C5 binding polypeptide with the amount of hemolysis in sheep erythrocytes, it was found that full inhibition of hemolysis under the conditions described (e.g. serum dilution 1:5) was obtained by Z-ABD at serum concentrations above 1 μM (
(175) TABLE-US-00012 TABLE 8 Mean (±stdev) pharmacokinetics of Z-ABD and Z-ABD-Z following i.v. and s.c. administration in male Sprague Dawley rats. Z-ABD Z-ABD-Z Administration route i.v. s.c. i.v. s.c. Dose nmol/kg 250 500 250 500 C.sub.max μM 2.8 (0.2) 0.90 (0.10) T.sub.max h 18 (9.8) 17 (12) AUC.sub.0-∞ μM*h 233 (34) 252 (11) 79 (7.5) 64 (1.2) AUC.sub.0-last μM*h 226 (37) 247 (11) 79 (6.9) 63 (1.0) F % 55 (3.1) 41 (2.6) T.sub.1/2, z h 58 (4.6) 57 (4.2) 36 (0.6) 46 (1.2) MRT h 69 (2.6) 80 (4.6) 27 (1.5) 63 (2.6) CL mL/h*kg 1.1 (0.2) 3.2 (0.2) V.sub.ss mL/kg 73 (12) 83 (10) V.sub.z mL/kg 90 (18) 159 (12)
Example 9: Pharmacokinetic Studies of C5 Binding Z Variants in Monkey
(176) Materials and Methods
(177) The study in life phase was performed at Charles River, Nevada (www.criver.com), formulation of administered drug and analysis of serums samples were performed in house. The pharmacokinetics of a Z-ABD variant (Z06175a (SEQ ID NO:753) fused to ABD094 (SEQ ID NO:759) by a GS linker) was investigated in the male Cynomolgus monkey (n=3) following i.v. (intravenous) and s.c. (subcutaneous) administration. Evaluation of the pharmacokinetic parameters was performed according to Example 8, however following i.v. administration the initial serum half-life (T.sub.1/2α) corresponding to the initial slope of the log-linear serum concentration-time curve, intermediate serum half-life (T.sub.1/2β) corresponding to the slope of the log-linear serum concentration-time curve associated with the secondary (intermediate) phase and terminal serum half-life (T.sub.1/2γ) corresponding to the terminal slope of the log-linear serum concentration-time curve was determined. T.sub.1/2 was calculated as ln 2/λ where estimation of the slope, λ, was based on at least 4 C=f(t) observations. The pharmacokinetic data presented for sc administration are compensated for pre-dose levels of Z-ABD while the graph displaying serum concentration versus time after sc administration show the actual serum concentrations determined. The monkeys were 2-4 years old with a body weight of 2.3-3 kg. Each monkey received a single i.v. dose (540 nmol/kg) followed by a single s.c. dose (1635 nmol/kg) three weeks after the i.v. administration. Blood samples were taken at 10 and 30 minutes and 1, 2, 4, 8, 24, 48, 72, 120, 168, 240, 336 and 504 hours post dose following both administrations. The blood samples were allowed to clot for 20-40 minutes in room temperature and then centrifuged at 1500 to 2200 RCF at 2-8° C. for 10-15 minutes before the serum was harvested and frozen. The serum samples were stored at a temperature below −20° C. until analysis.
(178) Serum concentrations of Z-ABD were analyzed by LC/LC/MS/MS as described in Example 8. Serum concentrations determined by LC/LC/MS/MS were also confirmed by a quantitative sandwich enzyme immunoassay technique. A polyclonal antibody specific for the Z compartment of Z-ABD was coated on to a microplate. Unbound polyclonal antibody was washed away and casein was added as blocking agent to reduce unspecific binding to the plastic surface. Samples and standards were diluted in PBS containing 0.5% casein and between 1-5% monkey normal serum. After washing away unbound casein, standards and samples were pipetted to the wells allowing any Z-ABD, presumed mainly to be associated with serum albumin, present in the sample to bind to the immobilized antibody. After washing away any unbound Z-ABD, an HRP labeled polyclonal antibody specific for albumin was added to detect the immobilized Z-ABD-albumin complex by colorometric methods. Unbound polyclonal antibody was washed away and a substrate solution was added to the wells and color develops in proportion to the amount of Z-ABD bound. Evaluation and calculation of pharmacokinetic parameters were performed as described in Example 8.
(179) Ex vivo hemolysis in serum from cynomolgus monkeys dosed with above described Z-ABD variant was monitored using the method described in Examples 6 and 8 with the modification that the monkey serum was diluted only two-fold compared to five-fold for rodent serum.
(180) Results
(181) Data on the mean (±stdev) pharmacokinetics of each dose group are presented. Serum concentrations of Z-ABD were quantifiable at all time points following both i.v. and s.c. administration by LC/LC/MS/MS (
(182) Following s c administration, and corrected for pre-dose serum levels remaining from the i v administration, maximum serum concentrations (mean C.sub.max 21(3) μM) were reached at 8-24 h after dose. The terminal serum half-life was 206 (40) hours and the mean residence time was 250 (68) hours. The subcutaneous bioavailability was estimated to be above 70%.
(183) The pharmacodynamic effect of the injected Z-ABD variant (Z06175a (SEQ ID NO:753) fused to ABD094 (SEQ ID NO:759) by a GS linker) was monitored by hemolysis. The hemolytic effect in cynomolgus monkey was completely suppressed (<20% of pre-dose) for at least seven days after administration of 5 mg/kg Z-ABD i.v. and 15 mg/kg Z-ABD s.c.
Example 10: In Vivo Studies Using Zymosan Induced Peritonitis
(184) Materials and Methods
(185) Administration to Mice:
(186) C57BL/6 female mice received different concentrations of a Z-ABD fusion molecule (Z06175a-GS-ABD094, SEQ ID NO:753 fused to SEQ ID NO:759 by a GS linker) or the positive control OmCI intraperitoneally (i.p.) 1 hour before induction with zymosan, or subcutaneously (s.c.) 18 hours before induction with zymosan.
(187) 0.8 mg/mouse zymosan was administered i.p. 1 hour later orbital blood samples (in serum vials with coagulation activator) were taken under isoflurane anaesthesia. The animals were killed by cervical dislocation. A skin incision was made, and the abdominal muscular wall was visualized. PBS solution (including 2 mM EDTA) was gently injected into the abdominal cavity. The abdomen was massaged and a sample of fluid (1-2 ml) was withdrawn. The samples were transferred to test tubes and stored on wet ice before centrifugation at 600 g for 10 min. Total protein and C5a concentrations in the supernatant were analyzed.
(188) Blood samples were kept in a refrigerator for at least 30 min and centrifugation was thereafter performed at 2000 g. Serum samples were stored in freezer (−70° C.) for later analysis of hemolytic activity and levels of Z06175a-GS-ABD094.
(189) Analysis of Hemolysis Activity in Serum Samples from Animals:
(190) Analysis of hemolysis activity was performed according to the hemolysis assay described in Examples 6 and 7.
(191) Analysis of C5a Concentration in Lavage from Mice Dosed with Zymosan and C5 Binding Z-ABD Fusion Molecules:
(192) For detection of C5a in mouse peritoneal lavage samples, microtiter plates (MaxiSorp, Nunc) were coated overnight at 4° C. with 100 μl/well of anti-05a antibody (cat. no. MAB21501, R&D Systems) at a concentration of 1 μg/ml in 0.05 M sodium carbonate-bicarbonate buffer, pH 9.6 (cat. no. C-3041, Sigma). The plates were washed three times with PBS containing 0.05% Tween 20 (PBST, cat. no. 09-9410-100, Medicago) and blocked with 200 μl/well of 1% BSA (cat. no. A7030, Sigma) in PBST for 1-1.5 hat RT during agitation at 450 rpm. The plate was again washed three times with PBST and then incubated with 100 μl/well of recombinant mouse C5a standard (cat. no. 2150-05, R&D Systems) at various concentrations in PBST with 0.1% BSA or samples for 2 h at RT during agitation at 450 rpm. High concentration samples were also diluted in PBST with 0.1% BSA. The plate was once again washed three times with PBST and then incubated with 100 μl/well of biotinylated anti-C5a antibody (cat. no. BAF2150, R&D Systems) at a concentration of 0.1 μg/ml for 1.5 h at RT while shaking the plate at 450 rpm. Following 3× washing with PBST, the plate was incubated with 100 μl/well of streptavidin-HRP (cat. no. DY998, R&D Systems) at a 200 fold dilution in blocking buffer for 20 min at RT during agitation at 450 rpm. After three final washes, the plate was developed with 100 μl/well TMB substrate (cat. no. T0440, Sigma) and read after 20-30 min at 650 nm using a Spectramax Plus plate reader (Molecular Devices).
(193) A standard curve was constructed by plotting the absorbance at 650 nm for each standard against its concentration (range 0-4000 pg/ml).
(194) Determination of Z Variant Concentration in Serum Samples from Animals Using ECL:
(195) Serum concentrations of administrated C5 binding Z06175a-GS-ABD094 (SEQ ID NO:753 fused to SEQ ID NO:759 by a GS linker) and Z06175a-GS-ABD094-GSGGGGSGGGS-Z06175a (SEQ ID NO: 789) (SEQ ID NO:753 fused to SEQ ID NO:759 by a GS linker, followed by a GS(G.sub.4S).sub.2 (SEQ ID NO: 785) linker and a second SEQ ID NO:753 motif, see
(196) In similarity with Example 6, a Z-ABD variant (Z06009a, SEQ ID NO:748 fused to ABD094, SEQ ID NO:759 Multi-array plates were coated with the goat anti-Affibody molecule IgG (Affibody AB) overnight at 4° C., and subsequently non-specific sites were blocked with PBS with 1% Casein for two hours at RT.
(197) Meanwhile, serum samples were thawed from −70° C. and diluted in PBS with casein in serum from the same animal strain. Standards and controls were diluted in the corresponding buffer. Samples and standards were incubated for three hours at RT while shaking the plate at 300 rpm. Incubation was terminated by washing 3×150 μL ice-cold PBS-Tween20. Immediately after the final wash, 150 μl 2× reading buffer (4× reading buffer T, Meso Scale Discovery cat. no. R92TC-3 diluted 1:1 in ultrapure H.sub.2O) was added to each well and the signal was detected using a plate reader (SECTOR Imager 2400, Meso Scale Discovery).
(198) In an alternative experiment, plates were coated with human C5 (SEQ ID NO:760, 1 pmol/well). Prior to addition to the coated plate, serum samples and standards, diluted in serum or in serum and PBS with casein (all samples and standards were matched to the same serum concentration), were heated to 60° C. for 30 min in order to denature endogenous C5. This alternative experiment provided a method for exclusive detection of C5 binding proteins, whereas the antibody dependent strategy described above can be applied to all proteins binding to that particular antibody.
(199) Results
(200) Analysis of Serum Concentrations of Z-ABD and Hemolysis Activity in Serum Samples from Animals:
(201) The serum concentrations as well as the ability to affect hemolysis in sheep erythrocytes of the Z-ABD fusion molecule (Z06175a-GS-ABD094, SEQ ID NO:753 fused to SEQ ID NO:759 by a GS linker)) was assessed after administration of a low (20 nmol/kg), medium (100 nmol/kg) and high dose (500 nmol/kg). The serum concentrations were relatively linear with dose, and inhibition of hemolysis confirmed that the molecules in serum were active and that the inhibition of hemolysis indeed also was concentration dependent.
(202) Analysis of C5a Concentration in Lavage from Mice Dosed with Zymosan and C5 Binding Z-ABD Fusion Molecules:
(203) The pro-inflammatory molecule zymosan was administered i.p. and in
Example 11: Pharmacokinetic Studies of C5 Binding Protein in Mice Following Intratracheal Administration
(204) Materials and Methods
(205) The pharmacokinetic profile of the C5 binding construct Z06175a-GS-ABD094 (SEQID NO: 753 fused to SEQ ID NO:759 by a GS linker) following intratracheal administration to female C57b1 mice was studied. Temperature, relative humidity and lighting was set to maintain 22±1° C., 55±5% and a 12 h light—12 h dark cycle and diet and water was provided ad libitum. Animals were anesthetized with isoflurane and dosed directly into the lungs using a microspray with 500 nmol/kg Z06175a-GS-ABD094. As much blood as possible was drawn, under anesthesia by isoflurane, from vena cava at 5 min, 30 min, 1 h, 3 h, 7 h, 16 h, 24 h, 48 h and 72 h (three animals/time point) for preparation of serum samples. Serum samples were prepared by collecting blood in tubes and placing the tubes in the fridge for 20 min. Subsequently, the tubes were centrifuged at 4000 rpm for 10 minutes. A minimum of 100 μl serum was prepared from each blood sample. Serum samples were kept at −70° C. prior to analysis. Serum concentrations of Z06175a-GS-ABD094 in each sample was determined by ECL as described in Example 10 and the ability of serum samples to affect hemolysis in sheep erythrocytes was determined as described in Examples 6 and 8.
(206) Results
(207) The serum concentration in each sample and the corresponding ability to affect hemolysis in sheep erythrocytes are described in
Example 12: Pharmacokinetic Studies of C5 Binding Z Variant in Rabbit Eye Following Topical and Intravitreal Administration
(208) Materials and Methods
(209) Rabbit in-Life Phase:
(210) The pharmacokinetics of a Z variant (Z06175a, SEQ ID NO:753 followed by GS (
(211) The study in-life phase and dissection of eyes from dosed animals (pigmented rabbits, 2-2.5 kg) was performed at Iris Pharma, La Gaude, France (www.iris-pharma.com). Animals were housed individually at 20±2° C. at 55±10% relative humidity with access to food and water ad lib.
(212) Animals were divided in three groups: 1) intravitreal administration (50 μl in each eye, n=3, six eyes totally) followed by dissection and serum sampling after one day, 2) intravitreal administration (50 μl in each eye, n=3) followed by dissection and serum sampling after four days and 3) untreated animals (n=5).
(213) Four distinct eye compartments were dissected (aqueous humor, vitreous, neuro-retina and RPE-choroid) and immediately frozen at −80° C. Formulation of administered drug (20.2 mg/ml in 10 mM phosphate buffer, 145 mM NaCl, pH 7.4) and analysis of drug in various eye compartments were performed in house.
(214) Analysis of Z-Variant in Dissected Eye Compartments:
(215) Dissected eye compartments were shipped on dry ice and stored at −80° C. until analysis. The retina and choroid samples were thawed in 10 times (volume/weight) PBS containing 1% human serum albumin in Lysing Matrix D tubes (MP Biomedical) containing ceramic beads and agitated at speed 4 for 2×20 s in a Savant Bio 101 homogenizer. The homogenate was removed from the beads using a pipette and transferred to a 1.5 ml Eppendorf tube and centrifuged at 900 rpm for ten minutes. The aqueous humor and vitreous samples were treated the same way as retina and choroid with the exception that no homogenization was needed. The vitreous samples from groups one and two were diluted 10 times further in the same buffer as above. Five standards were prepared in PBS with HSA (35.8 μM, 3.58 μM, 358 nM, 35.8 nM and 17.9 nM). Subsequently, standards and samples were subjected to pepsin digestion and analysis of the concentration of Z variant in tissue extracts was determined using the LC/LC/MS/MS method described in Example 8.
(216) Results
(217) The concentrations of Z variant after intravitreal administration were high in all compartments after one day (6-200 μM) and, surprisingly, remained high 4 days post-administration (1.5-78 μM). In particular, the concentration of the Z molecule in the vitreous ranged from 118 to 201 μM (average 161 μM, n=6 eyes) one day after injection and remained at 26 to 78 μM (average 46 μM, n=6) four days post-injection, pointing at a T.sub.1/2 of several days. There appears to be an inverse relationship between size and elimination of drugs after intravitreal injection in rabbit eye described by the following examples; Moxifloxacin (MW<0.35 kDa, T.sub.1/2=1.72 h, Mohan et al. Trans Am Ophthalmol Soc 2005, 103:76-83), ESBA105 (MW=26 kDa, T.sub.1/2=25 h, Ottiger et al. Investigative Ophthalmology & Visual Science 2009, 50: 779-786) and Ranibizumab (MW=48 kDa, T.sub.1/2=2.88 days, Bakri et al. American Academy of Ophthalmology 2007, 114:2179-2182). The Z variant tested here had a molecular weight of 7.0 kDa, suggesting that the elimination of the Z molecule was slower than what would be expected for such a small molecule in vitreous.