ANTIBODIES SPECIFIC FOR IL-17A FUSED TO HYALURONAN BINDING PEPTIDE TAGS
20170291939 · 2017-10-12
Assignee
Inventors
- Franco E. Di Padova (Birsfelden, CH)
- Joy Ghosh (Boston, MA)
- Thomas Huber (Basel, CH)
- Jean-Michel Rene Rondeau (Rixheim, FR)
Cpc classification
C07K2319/35
CHEMISTRY; METALLURGY
C07K2319/20
CHEMISTRY; METALLURGY
C07K2319/33
CHEMISTRY; METALLURGY
C07K2319/30
CHEMISTRY; METALLURGY
C07K2317/33
CHEMISTRY; METALLURGY
C07K2317/94
CHEMISTRY; METALLURGY
C07K2319/70
CHEMISTRY; METALLURGY
A61K47/6845
HUMAN NECESSITIES
A61K39/3955
HUMAN NECESSITIES
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/34
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
International classification
Abstract
The present disclosure relates to antibodies and proteins comprising an antigen-binding portion thereof that specifically bind to the pro-inflammatory cytokine IL-17A and a peptide tag that binds hyaluronan (HA). The disclosure more specifically relates to specific antibodies and proteins that are IL-17A antagonists (inhibit the activities of IL-17A and IL-17AF) and are capable of inhibiting IL-17A induced cytokine production in in vitro assays, and having an inhibitory effect in an antigen-induced arthritis model in vivo. The disclosure further relates to compositions and methods of use for said antibodies and proteins to treat pathological disorders that can be treated by inhibiting IL-17A or IL17AF mediated activity, such as rheumatoid arthritis, psoriasis, systemic lupus erythematosus (SLE), lupus nephritis, chronic obstructive pulmonary disease, asthma or cystic fibrosis or other autoimmune and inflammatory disorders.
Claims
1. An isolated antibody or protein comprising an antigen-binding portion thereof, comprising a CDR amino acid sequence having at least 95% identity to: a) those encoded by SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 3, and a CDR amino acid sequence having at least 64% identity to: b) those encoded by SEQ ID NO: 42, SEQ ID NO: 23 and SEQ ID NO: 11, and wherein said antibody or protein specifically binds to homodimeric IL-17A and heterodimeric IL-17AF, but does not specifically bind to homodimeric IL-17F, and further comprising a peptide tag that binds hyaluronan (HA) selected from the group consisting of: SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, and SEQ ID NO:96.
2. An isolated antibody or protein according to claim 1, wherein the IL-17A, IL-17AF or IL-17F are selected from one or more of cynomolgus monkey, rhesus macaque monkey, marmoset monkey, rat, mouse or human.
3. An isolated antibody or protein according to claim 1, comprising an amino acid sequence having at least 95% identity to: a) SEQ ID NO: 12, and an amino acid sequence having at least 90% identity to: b) SEQ ID NO: 43.
4. An isolated antibody or protein according to claim 1, comprising an amino acid sequence having at least 95% identity to: a) SEQ ID NO: 14, and an amino acid sequence having at least 95% identity to: b) SEQ ID NO: 44.
5. An isolated antibody or protein according to claim 1, comprising a light chain variable region comprising a CDR1, a CDR2, and a CDR3 domain selected from the group consisting of: a) a light chain CDR1 domain of SEQ ID NO: 73, wherein the first variable amino acid is selected from the group consisting of Gly (G) and Val (V); the second variable amino acid is selected from the group consisting of Tyr (Y), Asn (N) and Ile (I); the third variable amino acid is selected from the group consisting of Trp (W) and Ser (S); and the fourth variable amino acid is selected from the group consisting of Glu (E) and Ala (A); b) a light chain CDR2 domain of SEQ ID NO: 74, wherein the variable amino acid is selected from the group consisting of Asn (N) and Gln (Q); and c) a light chain CDR3 domain of SEQ ID NO: 75, wherein the variable amino acid is selected from the group consisting of Asn (N) and Asp (D).
6. An isolated antibody or protein according to claim 1 comprising heavy chain CDRs comprising, in sequence: a) SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 3, and light chain CDRs comprising, in sequence: b) SEQ ID NO: 42, SEQ ID NO: 23 and SEQ ID NO: 11, c) SEQ ID NO: 42, SEQ ID NO: 10 and SEQ ID NO: 11, d) SEQ ID NO: 34, SEQ ID NO: 23 and SEQ ID NO: 11, or e) SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24.
7. An isolated antibody or protein according to claim 1 comprising an immunoglobulin heavy chain comprising a) SEQ ID NO: 12, and an immunoglobulin light chain comprising b) SEQ ID NO: 43, c) SEQ ID NO: 53, d) SEQ ID NO: 35, or e) SEQ ID NO: 25.
8. An isolated antibody or protein according to claim 1 comprising an immunoglobulin heavy chain comprising a) SEQ ID NO: 14, and an immunoglobulin light chain comprising b) SEQ ID NO: 44, c) SEQ ID NO: 54, d) SEQ ID NO: 36, or e) SEQ ID NO: 26.
9. An isolated antibody or protein, which binds to the same epitope as an isolated antibody or protein according to claim 1.
10. An isolated antibody or protein according to claim 9, which binds to an IL-17A epitope which comprises Arg78, Glu80, and Trp90.
11. An isolated antibody or protein according to claim 10, wherein the epitope further comprises any one or more of Tyr85 or Arg124.
12. An isolated antibody or protein, which comprises an antigen recognition surface having epitope recognition characteristics equivalent to an antibody or protein as defined in claim 1.
13. An isolated antibody or protein, which is cross-blocked from binding to IL-17A or IL-17AF by at least one of the antibodies as defined in claim 1.
14. An isolated antibody or protein according to claim 1, wherein the antibody or protein does not specifically bind to: a) any one or more of human IL-17F homodimer, IL-17B homodimer, IL-17C homodimer, IL-17D homodimer, IL-17E homodimer, and/or b) any one or more of cynomolgus monkey IL-17F homodimer, mouse IL-17F homodimer, and/or c) any one or more of other human cytokines selected from the group consisting of IL-1, IL-3, IL-4, IL-6, IL-8, gIFN, TNF alpha, EGF, GMCSF, TGF beta 2, and/or d) any one or more of other mouse cytokines, selected from the group consisting of IL-1b, IL-2, IL-4, IL-6, IL-12, IL18, IL23, IFN or TNF.
15. An isolated antibody or protein according to claim 1, wherein binding to IL-17A a) inhibits or blocks binding between IL-17A and its receptor, and b) reduces or neutralizes IL-17A activity.
16. An isolated antibody or protein according to claim 1, wherein the binding affinity for human IL-17A is below 200 pM or 100 pM as measured by Biacore™.
17. An isolated antibody or protein according to claim 1, which is capable of inhibiting IL-6 secretion, or GRO-alpha secretion when assessed in vitro, preferably using cultured chondrocytes or fibroblasts.
18. An isolated antibody or protein according to claim 1, which is capable of inhibiting knee swelling in an antigen induced arthritis experimental model in vivo.
19. An isolated antibody or protein according to claim 1, which is conjugated to a further active moiety.
20. An isolated antibody or protein according to claim 1, which is a monoclonal antibody or an antigen-binding portion thereof, preferably a chimeric, humanized, or human antibody or portion thereof
21. An isolated antibody or protein according to claim 1, wherein said peptide tag that binds hyaluronan (HA) is selected from the group consisting of SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, and SEQ ID NO:96.
22. The isolated antibody or protein according to claim 1, wherein said peptide tag is linked directly to said antibody or protein.
23. The isolated antibody or protein according to claim 1, wherein said peptide tag is linked indirectly to said antibody or protein via a linker.
24. A pharmaceutical composition comprising an antibody or protein according to claim 1, in combination with one or more pharmaceutically acceptable excipient, diluent or carrier.
25. A pharmaceutical composition according to claim 24, further comprising one or more additional active ingredients.
26.-31. (canceled)
32. A method of treating a pathological disorder mediated by IL-17A or that can be treated by inhibiting IL-6 or GRO-alpha secretion, said method comprising administering an effective amount of an isolated antibody or protein according to claim 1, such that the disorder is alleviated.
33. A method according to claim 32, wherein the disorder is an inflammatory disorder or condition.
34. A method according to claim 33 wherein the disorder is arthritis, rheumatoid arthritis, psoriasis, chronic obstructive pulmonary disease, systemic lupus erythematosus (SLE), lupus nephritis, asthma, multiple sclerosis or cystic fibrosis.
35. An isolated nucleic acid molecule encoding any one of the antibodies or proteins as defined in claim 1.
36. A cloning or expression vector comprising one or more nucleic acid sequences according to claim 35, wherein the vector is suitable for the recombinant production of the encoded antibody or protein.
37. A host cell comprising one or more cloning or expression vectors according to claim 36.
38. An isolated nucleic acid molecule according to claim 35, wherein the nucleic acid molecule is messenger RNA (mRNA),
39. A process for the production of an isolated antibody or protein according to claim 1, comprising culturing a host cell which comprises one or more cloning or expression vectors comprising one or more nucleic acid sequences encoding one or more of said isolated antibodies or proteins, purifying and recovering said antibody or protein.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXAMPLES
[0368] XAB1 is a human IgG1/κ monoclonal antibody. It was generated using standard molecular biological techniques. In brief, the Medarex system was used. Mice were immunized with recombinant human IL-17A. Mice were euthanized by CO.sub.2 inhalation and spleen cells were harvested and fused with a myeloma cell line using PEG 4000. Fused cells were plated into wells with a feeder layer of peritoneal cells. Supernatants were taken from cultured cells and assayed for IL-17A reactive mAbs by ELISA. Clones positive for the production of IL-17A mAbs were selected and plated out.
[0369] The hybridoma responsible for the secretion of XAB1 was identified for further characterization on the basis of initial promising antibody/antigen binding characteristics such as binding affinity for IL-17A, ability to block IL-17A binding to its receptor, and ability to block IL-17A mediated biological effects in in vitro assays.
[0370] The amino acid sequence of XAB1 is SEQ ID NO: 14 (heavy chain) and SEQ ID NO: 15 (light chain). XAB1 was chosen for subsequent affinity maturation.
[0371] As a first step toward structure-guided affinity maturation, the crystal structure of the XAB1 Fab in the free state as well as the corresponding Fv complex with human IL-17A were determined as described below. The analysis of the three-dimensional structure of the XAB1 Fv complex with human IL-17A allowed for a rational affinity maturation process to be carried out alongside, and as an alternative to, a more randomised process. Further details are provided below.
[0372] In addition, X-ray crystallography was used to characterise some of the affinity matured variant antibodies that were generated. Analysis of crystal data from the affinity matured variants allowed for a deeper understanding of the binding behaviour of the variant antibodies and some unexpected properties were discovered as will be described further below.
Example 1. Crystal Structure of the XAB1 Fab in the Free State
[0373] (i) Material and Methods
[0374] Standard molecular biological protocols were used to obtain the XAB1 Fab antibody fragment. In brief, the Fab was cloned and expressed in E. coli W3110 with a C-terminal hexahistidine tag on the heavy-chain. The recombinant protein was purified by Ni-chelate chromatography followed by size-exclusion chromatography on a SPX-75 column in 10 mM TRIS pH 7.4, 25 mM NaCl. The XAB1 Fab was then concentrated by ultra-filtration to 10.4 mg/ml and crystallized.
[0375] Standard crystallization protocols were followed. In brief, crystals were grown at 19° C. in SD2 96 well-plates, using the method of vapour diffusion in sitting drops. The protein stock was mixed 1:1 with a crystallization buffer containing 40% PEG 300, 0.1M sodium phosphate-citrate pH 4.2. Total drop size was 0.4 μl. Prior to X-ray data collection, one crystal was mounted in a nylon cryo-loop and directly flash cooled into liquid nitrogen.
[0376] X-ray data collection and processing was carried out using standard protocols. Briefly, X-ray data to 2.1 Å resolution were collected at the Swiss Light Source, beamline X10SA, with a MAR225 CCD detector, using 1.0000 Å X-ray radiation. In total, 180 images of 1.0° oscillation each were recorded at a crystal-to-detector distance of 190 mm and processed with the HKL2000 software package. The crystal belonged to space group C2 with cell parameters a=51.63 Å, b=132.09 Å, c=77.25 Å, α=90.00°, β=98.88°, γ=90.00° and one XAB1 Fab molecule in the asymmetric unit. R-sym to 2.1 Å resolution was 10.4% and data completeness 99.0%.
[0377] The structure was determined by molecular replacement with the program PHASER. Search models for the V.sub.H/V.sub.L and C.sub.H1/C.sub.L domains were generated from PDB entry 1 HEZ. Iterative model building and refinement were performed with the programs Coot (Crystallographic Object-Oriented Toolkit) and CNX (Crystallography & NMR eXplorer) version 2002, until no further significant improvements could be made to the model. Final R- and R-free for all data were 0.188 and 0.231, respectively. The final refined model showed a root-mean-square deviation (RMSD) from ideal bond lengths and bond angles of 0.004 Å and 0.9°, respectively.
[0378] (ii) Results
[0379] The results of the X-ray refinement of the XAB1 Fab are provided in
[0380] Table 9 and the three-dimensional structure is shown in
TABLE-US-00010 TABLE 9 X-ray refinement of the XAB1 Fab with the program CNX. REMARK 3 REMARK 3 REFINEMENT. REMARK 3 PROGRAM : CNX 2002 REMARK 3 AUTHORS : Brunger, Adams, Clore, Delano, REMARK 3 Gros, Grosse-Kunstleve, Jiang, REMARK 3 Kuszewski, Nilges, Pannu, Read, REMARK 3 Rice, Simonson, Warren REMARK 3 and REMARK 3 Accelrys Inc., REMARK 3 Yip, Dzakula). REMARK 3 REMARK 3 DATA USED IN REFINEMENT. REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) : 2.10 REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) : 33.33 REMARK 3 DATA CUTOFF (SIGMA(F)) : 0.0 REMARK 3 DATA CUTOFF HIGH (ABS(F)) : 19645630.62 REMARK 3 DATA CUTOFF LOW (ABS(F)) : 0.000000 REMARK 3 COMPLETENESS (WORKING+TEST) (%) : 98.2 REMARK 3 NUMBER OF REFLECTIONS : 29298 REMARK 3 REMARK 3 FIT TO DATA USED IN REFINEMENT. REMARK 3 CROSS-VALIDATION METHOD : THROUGHOUT REMARK 3 FREE R VALUE TEST SET SELECTION : RANDOM REMARK 3 R VALUE (WORKING SET) : 0.188 REMARK 3 FREE R VALUE : 0.231 REMARK 3 FREE R VALUE TEST SET SIZE (%) : 4.9 REMARK 3 FREE R VALUE TEST SET COUNT : 1436 REMARK 3 ESTIMATED ERROR OF FREE R VALUE : 0.006 REMARK 3 REMARK 3 FIT IN THE HIGHEST RESOLUTION BIN. REMARK 3 TOTAL NUMBER OF BINS USED : 6 REMARK 3 BIN RESOLUTION RANGE HIGH (A) : 2.10 REMARK 3 BIN RESOLUTION RANGE LOW (A) : 2.23 REMARK 3 BIN COMPLETENESS (WORKING+TEST) (%) : 94.7 REMARK 3 REFLECTIONS IN BIN (WORKING SET) : 4478 REMARK 3 BIN R VALUE (WORKING SET) : 0.201 REMARK 3 BIN FREE R VALUE : 0.241 REMARK 3 BIN FREE R VALUE TEST SET SIZE (%) : 4.5 REMARK 3 BIN FREE R VALUE TEST SET COUNT : 213 REMARK 3 ESTIMATED ERROR OF BIN FREE R VALUE : 0.016 REMARK 3 REMARK 3 NUMBER OF NON-HYDROGEN ATOMS USED IN REFINEMENT. REMARK 3 PROTEIN ATOMS : 3311 REMARK 3 NUCLEIC ACID ATOMS : 0 REMARK 3 HETEROGEN ATOMS : 5 REMARK 3 SOLVENT ATOMS : 313 REMARK 3 REMARK 3 B VALUES. REMARK 3 FROM WILSON PLOT (A**2) : 21.1 REMARK 3 MEAN B VALUE (OVERALL, A**2) : 27.4 REMARK 3 OVERALL ANISOTROPIC B VALUE. REMARK 3 B11 (A**2) : −6.02 REMARK 3 B22 (A**2) : 3.30 REMARK 3 B33 (A**2) : 2.73 REMARK 3 B12 (A**2) : 0.00 REMARK 3 B13 (A**2) : 3.82 REMARK 3 B23 (A**2) : 0.00 REMARK 3 REMARK 3 BULK SOLVENT MODELING. REMARK 3 METHOD USED : FLAT MODEL REMARK 3 KSOL : 0.399279 REMARK 3 BSOL : 54.4727 (A**2) REMARK 3 REMARK 3 ESTIMATED COORDINATE ERROR. REMARK 3 ESD FROM LUZZATI PLOT (A) : 0.21 REMARK 3 ESD FROM SIGMAA (A) : 0.12 REMARK 3 LOW RESOLUTION CUTOFF (A) : 5.00 REMARK 3 REMARK 3 CROSS-VALIDATED ESTIMATED COORDINATE ERROR. REMARK 3 ESD FROM C-V LUZZATI PLOT (A) : 0.29 REMARK 3 ESD FROM C-V SIGMAA (A) : 0.14 REMARK 3 REMARK 3 RMS DEVIATIONS FROM IDEAL VALUES. REMARK 3 BOND LENGTHS (A) : 0.004 REMARK 3 BOND ANGLES (DEGREES) : 0.9 REMARK 3 DIHEDRAL ANGLES (DEGREES) : 21.4 REMARK 3 IMPROPER ANGLES (DEGREES) : 0.58 REMARK 3 REMARK 3 ISOTROPIC THERMAL MODEL : RESTRAINED REMARK 3 REMARK 3 ISOTROPIC THERMAL FACTOR RESTRAINTS. RMS SIGMA REMARK 3 MAIN-CHAIN BOND (A**2) : 1.41 ; 1.50 REMARK 3 MAIN-CHAIN ANGLE (A**2) : 2.21 ; 2.00 REMARK 3 SIDE-CHAIN BOND (A**2) : 2.31 ; 2.00 REMARK 3 SIDE-CHAIN ANGLE (A**2) : 3.44 ; 2.50 REMARK 3 REMARK 3 NCS MODEL : NONE REMARK 3 REMARK 3 NCS RESTRAINTS. RMS SIGMA/WEIGHT REMARK 3 GROUP 1 POSITIONAL (A) : NULL ; NULL REMARK 3 GROUP 1 B-FACTOR (A**2) : NULL ; NULL REMARK 3 REMARK 3 PARAMETER FILE 1 : protein_rep.param REMARK 3 PARAMETER FILE 2 : water_rep.param REMARK 3 TOPOLOGY FILE 1 : protein_no_cter.top REMARK 3 TOPOLOGY FILE 2 : water.top REMARK 3 REMARK 3 OTHER REFINEMENT REMARKS: NULL SSBOND 1 CYS L 23 CYS L 88 SSBOND 2 CYS L 134 CYS L 194 SSBOND 3 CYS H 22 CYS H 96 SSBOND 4 CYS H 143 CYS H 199 CRYST1 51.627 132.089 77.247 90.00 98.88 90.00 C 1 2 1 8 ORIGX1 1.000000 0.000000 0.000000 0.00000 ORIGX2 0.000000 1.000000 0.000000 0.00000 ORIGX3 0.000000 0.000000 1.000000 0.00000 SCALE1 0.019370 0.000000 0.003027 0.00000 SCALE2 0.000000 0.007571 0.000000 0.00000 SCALE3 0.000000 0.000000 0.013103 0.00000
[0381]
Example 2. Crystal Structure of the XAB1 Fv Complex with Human IL-17A: Analysis of the Paratope for Structure-Guided Affinity Maturation
[0382] (i) Material and Methods
[0383] Standard molecular biological protocols were used to obtain the XAB1 Fv antibody fragment. In brief, the Fv was cloned and expressed in E. coli W3110 with a C-terminal hexahistidine tag on the heavy-chain and a C-terminal Strep-tag on the light-chain. The recombinant protein was purified by Ni-chelate chromatography.
[0384] The XAB1 Fv fragment complex with human IL-17A was then prepared using standard methodology. In brief, human IL-17A (1.1 mg) was mixed with an excess of Fv (2.7 mg) and the complex was run on a S100 size-exclusion chromatography, in 10 mM TRIS pH 7.4, 25 mM NaCl. The protein complex was then concentrated by ultra-filtration to 21.2 mg/ml and crystallized.
[0385] Standard crystallization protocols were followed. In brief, crystals were grown at 19° C. in SD2 96 well-plates, using the method of vapour diffusion in sitting drops. The protein stock was mixed 1:1 with a crystallization buffer containing 10% PEG 20,000, 0.1M Bicine pH 9.0, 2.0% (v/v) dioxane. Total drop size was 0.4 μl. Prior to X-ray data collection, one crystal was briefly transferred into a 1:1 mix of the crystallization buffer with 20% PEG 20,000, 30% glycerol, and then flash cooled into liquid nitrogen.
[0386] X-ray data collection and processing was carried out using standard protocols. Briefly, X-ray data to 3.0 Å resolution were collected at the Swiss Light Source, Beamline X10SA, with a MAR225 CCD detector, using 1.0000 Å X-ray radiation. In total, 110 images of 1.0° oscillation each were recorded at a crystal-to-detector distance of 300 mm and processed with the HKL2000 software package. The crystal belonged to space group P2.sub.12.sub.12 with cell parameters a=184.31 Å, b=55.81 Å, c=70.99 Å, α=β=γ=90°. R-sym to 3.0 Å resolution was 11.2% and data completeness 99.9%.
[0387] The structure was determined by molecular replacement with the program PHASER. A search model for the XAB1 Fv was generated from the crystal structure of the XAB1 Fab previously determined (see Example 1). A search model for IL-17A was generated from the published human IL-17F crystal structure (PDB entry 1jpy). Iterative model building and refinement were performed with Coot (Crystallographic Object-Oriented Toolkit) and CNX (Crystallography & NMR eXplorer) version 2002, until no further significant improvements could be made to the model. Final R- and R-free for all data were 0.215 and 0.269, respectively. The final refined model showed a root-mean-square deviation (RMSD) from ideal bond lengths and bond angles of 0.007 Å and 1.0°, respectively.
[0388] (ii) Results
[0389] The molecular replacement calculations revealed a dimeric complex comprising one IL-17A homodimer with two XAB1 Fv fragments bound. The results of the X-ray refinement of the XAB1 Fv complex with human IL-17A are provided in Table 10 and the three-dimensional structure of this complex is shown in
TABLE-US-00011 TABLE 10 X-ray refinement of the XAB1 Fv complex with IL-17A obtained by the program CNX. REMARK 3 REMARK 3 REFINEMENT. REMARK 3 PROGRAM : CNX 2002 REMARK 3 AUTHORS : Brunger, Adams, Clore, Delano, REMARK 3 Gros, Grosse-Kunstleve, Jiang, REMARK 3 Kuszewski, Nilges, Pannu, Read, REMARK 3 Rice, Simonson, Warren REMARK 3 and REMARK 3 Accelrys Inc., REMARK 3 (Badger, Berard, Kumar, Szalma, REMARK 3 Yip, Dzakula). REMARK 3 REMARK 3 DATA USED IN REFINEMENT. REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) : 3.01 REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) : 47.74 REMARK 3 DATA CUTOFF (SIGMA(F)) : 0.0 REMARK 3 DATA CUTOFF HIGH (ABS(F)) : 15276175.80 REMARK 3 DATA CUTOFF LOW (ABS(F)) : 0.000000 REMARK 3 COMPLETENESS (WORKING+TEST) (%) : 99.5 REMARK 3 NUMBER OF REFLECTIONS : 15190 REMARK 3 REMARK 3 FIT TO DATA USED IN REFINEMENT. REMARK 3 CROSS-VALIDATION METHOD : THROUGHOUT REMARK 3 FREE R VALUE TEST SET SELECTION : RANDOM REMARK 3 R VALUE (WORKING SET) : 0.215 REMARK 3 FREE R VALUE : 0.269 REMARK 3 FREE R VALUE TEST SET SIZE (%) : 4.9 REMARK 3 FREE R VALUE TEST SET COUNT : 748 REMARK 3 ESTIMATED ERROR OF FREE R VALUE : 0.010 REMARK 3 REMARK 3 FIT IN THE HIGHEST RESOLUTION BIN. REMARK 3 TOTAL NUMBER OF BINS USED : 6 REMARK 3 BIN RESOLUTION RANGE HIGH (A) : 3.00 REMARK 3 BIN RESOLUTION RANGE LOW (A) : 3.19 REMARK 3 BIN COMPLETENESS (WORKING+TEST) (%) : 94.6 REMARK 3 REFLECTIONS IN BIN (WORKING SET) : 2234 REMARK 3 BIN R VALUE (WORKING SET) : 0.301 REMARK 3 BIN FREE R VALUE : 0.350 REMARK 3 BIN FREE R VALUE TEST SET SIZE (%) : 5.3 REMARK 3 BIN FREE R VALUE TEST SET COUNT : 124 REMARK 3 ESTIMATED ERROR OF BIN FREE R VALUE : 0.031 REMARK 3 REMARK 3 NUMBER OF NON-HYDROGEN ATOMS USED IN REFINEMENT. REMARK 3 PROTEIN ATOMS : 5007 REMARK 3 NUCLEIC ACID ATOMS : 0 REMARK 3 HETEROGEN ATOMS : 0 REMARK 3 SOLVENT ATOMS : 33 REMARK 3 REMARK 3 B VALUES. REMARK 3 FROM WILSON PLOT (A**2) : 54.9 REMARK 3 MEAN B VALUE (OVERALL, A**2) : 44.8 REMARK 3 OVERALL ANISOTROPIC B VALUE. REMARK 3 B11 (A**2) : 5.66 REMARK 3 B22 (A**2) : 0.97 REMARK 3 B33 (A**2) : −6.63 REMARK 3 B12 (A**2) : 0.00 REMARK 3 B13 (A**2) : 0.00 REMARK 3 B23 (A**2) : 0.00 REMARK 3 REMARK 3 BULK SOLVENT MODELING. REMARK 3 METHOD USED : FLAT MODEL REMARK 3 KSOL : 0.313124 REMARK 3 BSOL : 20.608 (A**2) REMARK 3 REMARK 3 ESTIMATED COORDINATE ERROR. REMARK 3 ESD FROM LUZZATI PLOT (A) : 0.33 REMARK 3 ESD FROM SIGMAA (A) : 0.39 REMARK 3 LOW RESOLUTION CUTOFF (A) : 5.00 REMARK 3 REMARK 3 CROSS-VALIDATED ESTIMATED COORDINATE ERROR. REMARK 3 ESD FROM C-V LUZZATI PLOT (A) : 0.44 REMARK 3 ESD FROM C-V SIGMAA (A) : 0.51 REMARK 3 REMARK 3 RMS DEVIATIONS FROM IDEAL VALUES. REMARK 3 BOND LENGTHS (A) : 0.007 REMARK 3 BOND ANGLES (DEGREES) : 1.0 REMARK 3 DIHEDRAL ANGLES (DEGREES) :22.1 REMARK 3 IMPROPER ANGLES (DEGREES) : 0.78 REMARK 3 REMARK 3 ISOTROPIC THERMAL MODEL: RESTRAINED REMARK 3 REMARK 3 ISOTROPIC THERMAL FACTOR RESTRAINTS. RMS SIGMA REMARK 3 MAIN-CHAIN BOND (A**2) : 1.46 ; 1.50 REMARK 3 MAIN-CHAIN ANGLE (A**2) : 2.62 ; 2.00 REMARK 3 SIDE-CHAIN BOND (A**2) : 1.63 ; 2.00 REMARK 3 SIDE-CHAIN ANGLE (A**2) : 2.62 ; 2.50 REMARK 3 REMARK 3 NCS MODEL : NONE REMARK 3 REMARK 3 NCS RESTRAINTS. RMS SIGMA/WEIGHT REMARK 3 GROUP 1 POSITIONAL (A) : NULL ; NULL REMARK 3 GROUP 1 B-FACTOR (A**2) : NULL ; NULL REMARK 3 REMARK 3 PARAMETER FILE 1 : protein_rep.param REMARK 3 PARAMETER FILE 2 : water rep.param REMARK 3 TOPOLOGY FILE 1 : protein_no_cter.top REMARK 3 TOPOLOGY FILE 2 : water.top REMARK 3 REMARK 3 OTHER REFINEMENT REMARKS: NULL SSBOND 1 CYS L 23 CYS L 88 SSBOND 2 CYS H 22 CYS H 96 SSBOND 3 CYS A 23 CYS A 88 SSBOND 4 CYS B 22 CYS B 96 SSBOND 5 CYS C 94 CYS C 144 SSBOND 6 CYS C 99 CYS C 146 SSBOND 7 CYS D 94 CYS D 144 SSBOND 8 CYS D 99 CYS D 146 CRYST1 184.306 55.813 70.991 90.00 90.00 90.00 P21 21 2 24 ORIGX1 1.000000 0.000000 0.000000 0.00000 ORIGX2 0.000000 1.000000 0.000000 0.00000 ORIGX3 0.000000 0.000000 1.000000 0.00000 SCALE1 0.005426 0.000000 0.000000 0.00000 SCALE2 0.000000 0.017917 0.000000 0.00000 SCALE3 0.000000 0.000000 0.014086 0.00000
[0390]
[0391] A detailed analysis of the complex was performed. A careful visual inspection of the crystal structure with the programs Coot and Pymol was carried out, and the amount of protein surface buried at the antibody-antigen interface was calculated with the program AREAIMOL of the CCP4 program suite. Intermolecular contacts were defined using a cut-off distance of 3.9 Å between antibody and antigen atoms. An overview of binding can be summarized as follows. Binding of XAB1 is symmetrical; each Fv fragment binds to an equivalent epitope on the IL17A homodimer.
[0392] The binding of each Fv fragment buried on average 1732A.sup.2 of combined surface, and involved 30 antibody and 25 IL-17A amino acid residues. The contribution to the buried surface of the XAB1 light-chain (around 560 Å.sup.2) was greater than that of the heavy chain (around 275 Å.sup.2). In addition, CDRH2 did not make any direct contacts to IL-17A and appeared to be too far from the protein antigen to provide opportunities for affinity maturation. The CDRH1 contribution appeared to be limited to one amino-acid side-chain only (Tyr32); this CDR was also too far from IL-17A to offer opportunities for affinity maturation through amino acid substitutions. The XAB1 CDRH3 made multiple tight contacts with IL-17A. However, careful inspection of the structure in this region failed to reveal any opportunity for further enhancing these contacts by point mutations; therefore, CDRH3 was deemed unsuitable as a target region for affinity enhancement. In contrast, inspection of the light-chain CDRs showed multiple opportunities for affinity maturation. Among the three light-chain CDRs, CDRL1 was considered most promising, and based on this observation, the inventors proposed to randomize positions 30 to 32 of the light-chain in an attempt to strengthen contacts to IL-17A residues Arg124, Phe133 and Tyr85.
Affinity Maturation by Rational Design
[0393] Based on the results above, it was seen that the XAB1 interface with homodimeric IL-17A was comparatively small and was characterized by a dominant contribution from the light chain, no involvement from CDRH2, and mainly indirect contribution by CDRH1 (i.e. via stabilization of CDRH3). Accordingly, the heavy chain of XAB1 did not appear to offer promising opportunities for affinity maturation.
[0394] In contrast, the XAB1 light chain did offer some opportunities in amino acid residues 30 to 32, with an optional insertion of up to 4 amino acid residues (CDRL1), amino acids 51 to 53 and 56 (CDRL2) and amino acid residues 92 and 93, with an optional insertion of up to 4 amino acid residues.
[0395] The availability of the published crystal structure for homodimeric human IL-17F, and the structure of homodimeric IL-17F in complex with the human receptor IL-17RA allowed for predictions to be made based on the observed structures of crystallized IL-17A and IL-17A in complex with XAB1 (and variants thereof).
[0396] The structural similarities predicted between IL-17F and IL-17A (on the basis of sequence identity and homology) were investigated. IL-17F and IL-17A bore a structural resemblance. The inventors hypothesized that IL-17A would bind to the N-terminal domain of its receptor in the same manner as has been shown for the published IL-17F/IL-17RA complex (Ely L K et al 2009, Nat Immunol. 10:1245-51).
[0397] On the basis of the observed structure and comparison of the known sequences for human IL-17A and IL-17F, along with the sequence of IL-17A derived from other species, a number of additional predictions were made by the inventors:
[0398] It was expected that XAB1 (and antibodies variants derived there from having an improved affinity for the epitope targeted by XAB1) would be highly specific for human IL-17A. It was hypothesized that such antibodies would retain some cross-reactivity with IL-17A from other species (on the basis of the high degree of conserved sequence identity or homology between species). However, on the basis of the available sequence data and structural predictions it was not clear to what extent cross-reactivity with species variants of IL-17A could be expected. Given the lack of structural similarity with other Interleukins, cross-reactivity with such molecules (from humans or other species) was expected to be very unlikely.
[0399] In addition, differences between the sequences of IL-17A and IL-17F (in particular N-terminal region) gave rise to predictions that the anti-IL-17A antibodies of the disclosure would not bind to IL-17F. For example, overlays of the two crystal structures indicated that steric hindrance would prevent binding between these antibodies and IL-17F. Furthermore, an extrapolation to the structure of IL-17AF heterodimers also suggested that such interference, in particular in the N-terminal region, would hamper binding of the antibodies to IL-17AF heterodimers and thereby result in a lack of binding to IL-17AF, i.e. a lack of cross-reactivity by these antibodies for IL-17AF heterodimers.
Example 3. Generation of Affinity Matured Antibody Variants
[0400] Actual affinity maturation of the initial antibody XAB1 focused on the light chain, for reasons discussed above. The work was carried out in three steps: (i) library generation, (ii) library screening, and (iii) candidate characterisation.
[0401] The protein engineering work (i.e. affinity maturation) was carried out in the Fab fragment format for ease of handling. Candidates were formatted back to full IgG after engineering.
[0402] (i) Library Generation
[0403] The DNA sequence encoding the variable domain of the light chain was mutated to create a library of gene variants. Two different approaches (A and B) were used for library generation, providing two separate libraries.
[0404] 1) Method a—Random Mutation by Error Prone PCR:
[0405] The DNA region encoding the variable domain of the light chain of XAB1 was randomly mutated using error prone PCR. In more detail, this region was amplified using the polymerase Mutazyme II, which introduced mutations at a high frequency (for more detail, see the guidance supplied with the GeneMorph II random mutagenesis kit, supplied by Stratagene #200550). However, any suitable random mutation technique or strategy could be used.
[0406] The pool of PCR fragment variants was then cloned by cutting and pasting into the expression vector of XAB1. Essentially, the parent, unmutated sequence was cut out of the expression vector and replaced by a randomly mutagenized sequence which was pasted in its place. Standard molecular biology techniques were used to accomplish this.
[0407] This resulted in a library of expression vector variants comprising a variety of randomly mutagenized variable domain sequences.
[0408] 2) Method B—Mutation by Rational Design:
[0409] Under this approach, the generation of the library was guided by the structural analysis carried out as a precursor to affinity maturation. Specific amino acid residues (in particular in CDR1 of the light chain of XAB1) were targeted based on the epitope and paratope information derived from the crystal structure described above.
[0410] Three amino acid residues, selected on the basis of the crystal structure information, were fully randomised. Standard molecular biology approaches were used for the construction.
[0411] Firstly, a fragment of the variable region encoding the appropriate CDR and a first part of the light chain framework was amplified by PCR, using degenerate oligonucleotides. That is, the oligonucleotides, encoding the CDR were synthesised in such a way as to provide a variety of bases at a defined position or positions. Design of the oligonucleotide enabled randomization of specifically targeted amino acid positions in the CDR by NNK degenerated codons (in which N stands for all 4 bases, A, T, C and G and K for G and T) and allowed all 20 natural amino acids at those positions.
[0412] Following this first step, a second fragment overlapping the first one and encoding the remaining part of the light chain, was also amplified by PCR. Both fragments were then assembled by an “assembly” PCR to generate the complete variable light chain and cloned back into the expression vector in a ‘cut and paste’ manner. Thereby the parental sequence was replaced with a range of rationally mutated sequences, whereby at specific amino acid positions all 20 natural amino acids were represented.
[0413] (ii) Library Screening
[0414] Once libraries comprising sequences encoding XAB1 variants had been generated it was necessary to screen them in order to select those which had superior characteristics to the parental XAB1 sequence, for example higher affinity for IL-17A.
[0415] Two screening techniques were used. Firstly, a high throughput screening was done by “colony filtration screening” (CFS). This assay permitted a convenient screening of large number of clones. It allowed reduction to positive hits prior to ELISA screening, which was useful in particular for the random approach “method A” as the library size was much larger (>10.sup.5) compared to the library size in “method B” (only 8000). ELISA screening is convenient for 10.sup.4 clones or less and gives more quantitative results.
[0416] 1) Colony Filtration Screening (CFS):
[0417] The protocol for CFS was based on Skerra et al. 1991, Anal Biochem 196:151-155. Some adaptations were made.
[0418] E. coli colonies expressing the Fab variant libraries were grown on a filter on top of a Petri dish containing LB agar and glucose. In parallel, a PVDF membrane was coated with the target protein (IL-17A). The coated membrane was placed on the agar plate. The filter with colonies of Fab fragment expressing E. coli was placed on top of the membrane. The Fab fragments expressed by the cells diffused from the colonies and bound the target IL-17A. The Fab fragment thus captured on the PVDF membrane was then detected using a secondary antibody conjugated with alkaline phosphatase for Western staining. The conditions for selecting only variants with improved binding properties were previously established using XAB1 as reference.
[0419] More specifically, after transformation of E. coli cells with the library, the cells were spread on a Durapore™ membrane filter (0.22 μm GV, Millipore®, cat #GVWP09050) placed on a Petri dish containing LB agar+1% glucose+antibiotic of interest. The plates were incubated overnight at 30° C.
[0420] The PVDF membrane (Immobilon-P, Millipore®, cat #IPVH08100) was pre-wet in methanol, washed in PBS and coated with a huIL-17A solution at 1 pg/ml in PBS. The membrane was incubated overnight at room temperature. After coating, the membrane was washed 2 times in Tris buffered saline (TBS)+0.05% Tween (TBST) and blocked two hours at room temperature in 5% milk TBST. Then, the membrane was washed four times in TBST and soaked in 2×YT medium with 1 mM IPTG. This membrane, called the capture membrane was placed onto a LB agar plate with 1 mM IPTG+antibiotic of interest, and was covered with the Durapore membrane with the colonies on top. The resulting sandwich was incubated four hours at 30° C.
[0421] After this incubation, the capture membrane was washed 4 times with TBST and blocked in 5% milk TBST for 1 hour at room temperature. Then, the membrane was washed once with TBST and incubated with a secondary antibody (anti-hu_kappa light chain antibody, alkaline-phosphatase (AP) conjugated, Sigma # A3813, diluted 1:5000 in 2% milk TBST), 1 hour at room temperature. Afterward, the membrane was washed 4 times in TBST, once in TBS and incubated in the substrate solution (SigmaFast BCIP/NBT tablet, 1 tablet in 10 ml H.sub.2O). When the signal reached the expected intensity the membrane was washed with water and allowed to dry.
[0422] After development of the signal on the capture membrane, the colonies giving stronger signal than the parental XAB1 were picked and allowed to proceed to a secondary ELISA screening described below.
[0423] 2) ELISA Screening:
[0424] Following the CFS, ELISA was used to screen the candidates selected by CFS. In brief, for the relative low number of variants identified by error-prone PCR mutagenesis (i.e. library A) the ELISA was performed manually in a 96 well format. In contrast, for the libraries constructed by rational design (method B), a larger number of improved clones needed to be screened at ELISA level to be able to discriminate between their different binding affinity to IL-17A and identify the clones with the highest affinity. An ELISA robot was used for that purpose in a 384 well plate format. However, the ELISA protocol was the same in each case, the only difference being the volumes of reagents.
[0425] a) Cell Cultures:
[0426] Clones were first grown overnight at 30° C., 900 rpm, in 2×YT medium+1% glucose+antibiotic of interest. The plates containing these cultures were called master plates. The next day, aliquots of cultures from the master plates were transferred into expression plates containing 2×YT medium+0.1% glucose+antibiotic of interest. These plates were incubated at 30° C., 900 rpm about 3 hours. Then isopropyl β-D-1-thiogalactopyranoside (IPTG) solution was added to a final concentration of 0.5 mM. The plates were incubated overnight at 30° C., 990 rpm.
[0427] The next day, lysis buffer (2×) Borate buffered saline (BBS) solution (Teknova #60205)+2.5 mg/ml lysosyme+10u/ml Benzonase) was added to the cultures. Plates were incubated 1 hour at room temperature, then 12.5% milk TBST was added for blocking. After 30 min incubation, cells lysates were diluted 1:10 in 2% milk TBST and were transferred into the ELISA plates.
[0428] b) ELISA:
[0429] ELISA plates (Nunc Maxisorp) were coated with a huIL-17A solution at 1 μg/ml during 1 hour. The plates were washed once with TBST and blocked 1 hour with 5% milk TBST. After blocking, plates were washed 3 times with TBST and then, diluted cell lysates were loaded on the plates and incubated 1 hour. Afterward, plates were washed 3 times with TBST and were incubated 1 hour with a secondary antibody AP conjugated.
[0430] The plates were finally washed 3 times with TBST and then incubated with the substrate solution (AttoPhos substrate Set, Roche #11 681 982 001). The whole process was performed at room temperature.
[0431] In addition to the “classic” ELISA described above, modified ELISA were also undertaken for a better discrimination between clones with very high affinity (in the picomolar range) for the target protein. An “off-rate” ELISA and a “competition” ELISA were developed for this purpose, as detailed below.
[0432] c) “Off-Rate” ELISA:
[0433] For this assay, the modification compared to the “classic” ELISA protocol regarded the washing step after the binding step (incubation of cell lysate in ELISA plates). In the “classic” protocol, the plate was washed 3 times with TBST. The washing solution was dispensed and immediately aspirated, without any incubation time. For the “off-rate” ELISA, the plate was washed 6 times during at least 3 hours. This long wash increased the stringency of the assay, and allowed identifying clones with a slow off-rate.
[0434] d) “Competition” ELISA:
[0435] This modified ELISA protocol included an extra step after the binding step. After incubation of cell lysate, the plates were washed 3 times with TBST and then, a solution of the parental XAB1 (200 nM in 2% milk TBST) was incubated overnight at room temperature. This long incubation with an excess of the parental Fab allowed, as in the case of “off-rate” ELISA, to identify clones with slow off-rate, which lead to better discrimination between clones with an affinity in the picomolar range. The rest of the protocol was similar to the “classic” ELISA protocol. The secondary antibody used in this case was an AP conjugated anti-Flag tag antibody, since the Fabs variants from the library had a Flag tag at the C-terminus of the heavy chain but not the parental XAB1 Fab used for the competition.
[0436] (iii) Candidate Characterisation
[0437] The hits identified during the screening were produced on a larger scale for further physicochemical characterisation and to confirm high affinity binding to IL-17A, and/or other advantageous properties in additional assays. These are described below in more detail.
[0438] (iv) Results: Screening and Initial Characterization of Candidates Following Affinity Maturation of XAB1 [0439] 1) Random Mutagenesis Approach (Method A):
[0440] The mutation rate after the error-prone PCR library generation was found to peak at 2 to 3 mutations per gene. Around 3×10.sup.4 clones were screened by colony filter screening and a number of 94 clones were identified as improved and allowed to proceed to binding, off-rate and competition ELISA. ELISA data in combination with sequencing results led to the identification of 6 candidates highlighting 3 potential hot spots for improvement, Gly at position 28 to Val (G28V) in LCDR1; Gly at position 66 to Val (G66V) or Ser (G66S) in framework 3; Asn92 to Asp (N92D) in LCDR3 (data not shown, but positioning is identical to that of XAB2, VL, i.e. SEQ ID NO: 25).
[0441] A stop codon was observed in one of the clones, but was not relevant as the E. coli strain used was an amber suppressor strain allowing read-through. Based on the data obtained, a G28V and G66V mutation appeared to cause the best improvement. A variant of XAB1 was generated by standard molecular biology techniques carrying the two point mutations mentioned. A further variant was cloned having the N92D substitution in addition, in order to test whether the removal of the potential post-translational deamidation site (N92, S93) would be beneficial. More detailed profiling was done on those two variants, in particular of the triple mutant variant referred to as XAB_A2 which finally led to XAB2. In XAB2, amino acids number 1 to 23 according to the Kabat definition constitute framework 1, amino acids number 24 to 34 (Kabat) constitute LCDR1, amino acids number 35 to 49 (Kabat) constitute framework 2, amino acids 50 to 56 (Kabat) constitute LCDR2, amino acids 57 to 88 (Kabat) constitute framework 3, amino acids 89 to 97 (Kabat) constitute LCDR3 and amino acids 98 to 107 (Kabat) constitute framework 4. The same subdivision of other VL sequences according to embodiments of the disclosure also applies.
[0442] Thus, the G66V substitution mentioned above is in a framework region, which is called the outer loop. This framework region is able to contribute to binding in some cases. Based on the available structural information it was retrospectively suggested that this mutation indeed might be able to interact with a region of IL-17A which cannot be resolved from the crystal structure but may be in proximity to the outer loop.
[0443] 2) Rational Mutagenesis Approach (Method B):
[0444] A snapshot of the amino acid distribution at the randomized positions was generated by sequencing of 32 randomly picked members. There was no significant bias, though statistics with this low number of sequences cannot be done. Around 4×10.sup.4 clones were screened which oversampled the theoretical library size of 8000. A high number of hits were identified and 2630 clones were allowed to proceed to ELISA screening. Performing binding, off-rate and competition ELISA, 60 clones with the highest improvements were sequenced. In those 60 clones 22 unique sequences were found, and the result is summarized in Table 11.
TABLE-US-00012 TABLE 11 ELISA of all selected 22 unique candidates. Values are normalized to parental Fab XAB1. The representation indicates how often a certain sequence was found within the 60 hits. The difference in amino acid sequence is given in the three last columns. XAB1 has the amino acids I S A at those positions. ELISA signals determined from crude extract of Fab expression culture from E. coli. Off- Candidate Classic rate Competiton Repre- name ELISA ELISA ELISA sentation % 30 31 32 MB491 2.1 43.0 44.2 5 F F W MB483 3.1 47.1 45.2 2 F W T MB447 3.0 45.5 57.0 5 F W W MB457 2.7 34.7 41.0 5 I W S MB464 2.7 34.9 36.9 7 I Y Q MB432 2.3 44.2 37.3 12 L F A MB454 2.9 34.2 36.6 2 L W A MB444 3.2 48.9 52.4 2 L W E MB456 2.4 45.1 46.7 2 L W H MB440 2.8 52.5 54.0 5 L W Q MB450 2.9 41.5 53.3 5 M W W MB435 2.7 44.7 44.6 2 N W E MB438 2.7 41.5 41.1 7 P Y A MB453 2.7 43.3 46.4 9 V F W MB448 2.9 40.4 51.5 5 V W M MB486 1.9 58.5 64.9 2 W W M MB434 2.4 44.4 39.5 7 W W Y MB458 2.7 33.0 42.1 5 W Y Q MB463 2.7 34.2 31.6 2 Y F E MB468 2.8 43.9 60.0 5 Y W E MB433 2.3 39.7 29.3 2 Y W G MB461 2.9 49.8 62.8 2 Y W T
[0445] Of the 22 unique clones, 6 were selected for 0.5 L scale standard E. coli expression and two step purification by IMAC (Ni-NTA) and SEC. Purified Fabs were then used to confirm the improvement in binding by ELISA.
[0446] ELISA results of selected and purified Fab candidates in comparison to XAB1 are shown in
[0447]
Example 4. Targeting a Potential Post-Translational Deamidation Site
[0448] The inventors hypothesized that the amino acid motif asparagine followed by glycine (NG) or, to lower extend also when followed by serine (NS), may be susceptible to post-translational deamidation. Such motifs are present in L-CDR2 (position 56/57) and L-CDR3 (92/93) of the antibody XAB1. Four IgG variants were generated in order to test whether the NG site could be removed without affecting binding and activity properties. These four point mutation variants were cloned by standard molecular biology procedures and produced by standard transient transfection of HEK cells in 100 ml scale and purified via a protein A column.
[0449] Purified IgG variants were analyzed in an in vitro neutralization assay (e.g. as described in examples 12 and 13) to compare their activity to the parental XAB1 IgG. Results showed that out of these four variants, three had a reduced activity. But the candidate XAB_B12 (mutation N56Q) retained activity compared to the parental XAB1.
TABLE-US-00013 TABLE 12 Overview of sequence modifications to XAB1, and corresponding effect on in vitro neutralization. IC50 (nM) Kabat CDR Hu Hu Residue# L-CDR2 IL-6 IL-8 Kabat# 49 50 51 52 53 54 55 56 57 sec sec IgG Generic variants name XAB1 XAB1 Y D A S S L E N G 4 3 XAB_G57T XAB_A6 Y D A S S L E N T 22 23 XAB_N56Q XAB_B12 Y D A S S L E Q G 2 3 XAB_N56T XAB_B13 Y D A S S L E T G 11 15 XAB_N56S XAB_B14 Y D A S S L E S G 13 17
[0450] Having thus identified the most suitable substitution, it was introduced to the most promising hits identified during the affinity maturation process, resulting in XAB2 (XAB_A2 N56Q), XAB3 (MB468 N56Q), XAB4 (MB435 N56Q). They were produced by standard transient transfection of HEK cells and purified via protein A column along with XAB5 (MB435), which still carried the NG site.
[0451] The NG motif was removed (N56Q) for the XAB2, XAB3, XAB4, but was still present in XAB5. The NS motif in L-CDR3 was removed (N92D) in XAB2, as found during the random affinity maturation approach. Therefore, an optimal set of variants was available to test the susceptibility for deamidation of the potential sites.
[0452] The four purified candidates were diluted in a buffer pH 8 and incubated at 40° C. in order to force the deamidation reaction. Aliquots were taken at several time points to determine the degree of deamidation by cation exchange chromatography (CEX), according to principles well known to a person skilled in the art, and the in vitro neutralization activity by a cell based assay was determined (e.g. as described in examples 12 and 13).
[0453] CEX results showed an increase of acidic variants percentage over time, as expected for any IgG, likely due to post-translational modification sites in the antibody framework, but the extent of increase was higher for XAB5 than for the other candidates, i.e. 72% vs. 46% after one week and 94% vs. 83% after 4 weeks. Finally, in vitro neutralization activity assay results correlated with the CEX results, showing that XAB5 lost activity after 4 weeks incubation during forced deamidation condition. Size-exclusion chromatography-multi angle light scattering methodology (SEC-MALS), well known to a person skilled in the art, was used to monitor the aggregation levels in the samples.
[0454] The data is summarized in Table 13.
TABLE-US-00014 TABLE 13 Analysis by SEC-MALS, in vitro neutralization activity and CEX. M.sup.a) EC.sub.50.sup.b) EC.sub.50.sup.b) Antibody [%] [ng/ml] CEX.sup.c) [%] M.sup.a) [%] [ng/ml] CEX.sup.c) [%] T = 0 weeks T = 1 weeks XAB2 99 45 15 98 n.d. 45 XAB3 99 40 14 98 n.d. 44 XAB5 99 45 18 98 n.d. 72 XAB4 99 48 15 98 n.d. 48 M.sup.a) EC.sub.50.sup.b) NG.sup.d) NS.sup.d) Antibody [%] [ng/ml] CEX.sup.c) [%] sites sites T = 4 weeks XAB2 95 47 85 0 0 XAB3 97 40 81 0 1 XAB5 94 61 94 1 1 XAB4 94 47 84 0 1 .sup.a)monomer by SEC-MALS, .sup.b)inhibition of IL-6 secretion after cell stimulation with 80 ng/ml IL-17, .sup.c)acidic variants by exchange chromatography, .sup.d)number of sites in CDRs (framework region not considered)
[0455] These data indicated successful removal of a potential post-translational deamidation site, which could have had an effect on antibody activity. This is advantageous, since XAB2, XAB3 and XAB4 are therefore likely to achieve a more homogeneous product than XAB1 as no post-translational deamidation can occur during production or storage affecting the antibody activity.
Example 5. X-Ray Analysis of Antibody Variants Derived by Affinity Maturation: XAB2
[0456] In brief, the XAB2 Fv was cloned and expressed in E. coli TGf1—with a C-terminal hexahistidine tag on the heavy-chain and a C-terminal Strep-tag on the light-chain, according to principles well known to a person skilled in the art. The recombinant protein was purified by Ni-chelate chromatography and size-exclusion chromatography (SPX-75).
[0457] The XAB2 Fv fragment complex with human IL-17A was then prepared using standard methodology. In brief, human IL-17A (1.5 mg) was mixed with an excess of XAB2 Fv (3.7 mg) and the complex was run on a S100 size-exclusion chromatography, in 10 mM TRIS pH 7.4, 25 mM NaCl. The protein complex was then concentrated by ultra-filtration to 26.3 mg/ml and crystallized.
[0458] Standard crystallization protocols were followed. In brief, crystals were grown at 19° C. in SD2 96-well plates, using the method of vapour diffusion in sitting drops. The protein stock was mixed 1:1 with a crystallization buffer containing 0.2M calcium acetate, 20% PEG 3,350. Total drop size was 0.4 μl. Prior to X-ray data collection, one crystal was briefly transferred into a 1:1 mix of the crystallization buffer with 30% PEG 3,350, 30% glycerol, and then flash cooled into liquid nitrogen.
[0459] X-ray data collection and processing was carried out using standard protocols. Briefly, X-ray data to 2.0 Å resolution were collected at the Swiss Light Source, beamline X06DA, with a MAR225 CCD detector, using 1.0000 Å X-ray radiation. In total, 360 images of 0.5° oscillation each were recorded at a crystal-to-detector distance of 190 mm and processed with the XDS software package. The crystal belonged to space group P2.sub.12.sub.12 with cell parameters a=184.72 Å, b=55.56 Å, c=71.11 Å, α=β=γ=90°. R-sym to 2.0 Å resolution was 5.2% and data completeness 100.0%.
[0460] As the crystal of the XAB2 Fv complex was highly isomorphous with the crystal of the XAB1 Fv complex (Example 2), the structure of the latter was used as input model for an initial run of crystallographic refinement with the program CNX. Iterative model correction and refinement was performed with Coot (Crystallographic Object-Oriented Toolkit) and CNX (Crystallography & NMR eXplorer) version 2002, until no further significant improvements could be made to the crystallographic model. Final R- and R-free for all data were 0.214 and 0.259, respectively. The final refined model showed a root-mean-square deviation (RMSD) from ideal bond lengths and bond angles of 0.005 Å and 0.9°, respectively.
[0461] Results
[0462] The results of the X-ray refinement of the XAB2 Fv complex with human IL-17A are provided in Table 14 and the three-dimensional structure of this complex is shown in
TABLE-US-00015 TABLE 14 X-ray refinement of the XAB2 Fv complex with IL-17A obtained by the program CNX. REMARK 3 REMARK 3 REFINEMENT. REMARK 3 PROGRAM : CNX 2002 REMARK 3 AUTHORS : Brunger, Adams, Clore, Delano, REMARK 3 Gros, Grosse-Kunstleve, Jiang, REMARK 3 Kuszewski, Nilges, Pannu, Read, REMARK 3 Rice, Simonson, Warren REMARK 3 and REMARK 3 Accelrys Inc., REMARK 3 (Badger, Berard, Kumar, Szalma, REMARK 3 Yip, Dzakula). REMARK 3 REMARK 3 DATA USED IN REFINEMENT. REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) : 2.00 REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) : 71.11 REMARK 3 DATA CUTOFF (SIGMA(F)) : 0.0 REMARK 3 DATA CUTOFF HIGH (ABS(F)) : 2329350.20 REMARK 3 DATA CUTOFF LOW (ABS(F)) : 0.000000 REMARK 3 COMPLETENESS (WORKING+TEST) (%) : 99.8 REMARK 3 NUMBER OF REFLECTIONS : 50409 REMARK 3 REMARK 3 FIT TO DATA USED IN REFINEMENT. REMARK 3 CROSS-VALIDATION METHOD : THROUGHOUT REMARK 3 FREE R VALUE TEST SET SELECTION : RANDOM REMARK 3 R VALUE (WORKING SET) : 0.214 REMARK 3 FREE R VALUE : 0.259 REMARK 3 FREE R VALUE TEST SET SIZE (%) : 5.0 REMARK 3 FREE R VALUE TEST SET COUNT : 2521 REMARK 3 ESTIMATED ERROR OF FREE R VALUE : 0.005 REMARK 3 REMARK 3 FIT IN THE HIGHEST RESOLUTION BIN. REMARK 3 TOTAL NUMBER OF BINS USED : 6 REMARK 3 BIN RESOLUTION RANGE HIGH (A) : 2.00 REMARK 3 BIN RESOLUTION RANGE LOW (A) : 2.13 REMARK 3 BIN COMPLETENESS (WORKING+TEST) (%) :100.0 REMARK 3 REFLECTIONS IN BIN (WORKING SET) : 7858 REMARK 3 BIN R VALUE (WORKING SET) : 0.262 REMARK 3 BIN FREE R VALUE : 0.304 REMARK 3 BIN FREE R VALUE TEST SET SIZE (%) : 5.0 REMARK 3 BIN FREE R VALUE TEST SET COUNT : 414 REMARK 3 ESTIMATED ERROR OF BIN FREE R VALUE : 0.015 REMARK 3 REMARK 3 NUMBER OF NON-HYDROGEN ATOMS USED IN REFINEMENT. REMARK 3 PROTEIN ATOMS : 5055 REMARK 3 NUCLEIC ACID ATOMS : 0 REMARK 3 HETEROGEN ATOMS : 0 REMARK 3 SOLVENT ATOMS : 376 REMARK 3 REMARK 3 B VALUES. REMARK 3 FROM WILSON PLOT (A**2) : 27.8 REMARK 3 MEAN B VALUE (OVERALL, A**2) : 37.3 REMARK 3 OVERALL ANISOTROPIC B VALUE. REMARK 3 B11 (A**2) : −0.85 REMARK 3 B22 (A**2) : 3.93 REMARK 3 B33 (A**2) : −3.08 REMARK 3 B12 (A**2) : 0.00 REMARK 3 B13 (A**2) : 0.00 REMARK 3 B23 (A**2) : 0.00 REMARK 3 REMARK 3 BULK SOLVENT MODELING. REMARK 3 METHOD USED : FLAT MODEL REMARK 3 KSOL : 0.338594 REMARK 3 BSOL : 46.0594 (A**2) REMARK 3 REMARK 3 ESTIMATED COORDINATE ERROR. REMARK 3 ESD FROM LUZZATI PLOT (A) : 0.25 REMARK 3 ESD FROM SIGMAA (A) : 0.19 REMARK 3 LOW RESOLUTION CUTOFF (A) : 5.00 REMARK 3 REMARK 3 CROSS-VALIDATED ESTIMATED COORDINATE ERROR. REMARK 3 ESD FROM C-V LUZZATI PLOT (A) : 0.31 REMARK 3 ESD FROM C-V SIGMAA (A) : 0.22 REMARK 3 REMARK 3 RMS DEVIATIONS FROM IDEAL VALUES. REMARK 3 BOND LENGTHS (A) : 0.005 REMARK 3 BOND ANGLES (DEGREES) : 0.9 REMARK 3 DIHEDRAL ANGLES (DEGREES) : 21.0 REMARK 3 IMPROPER ANGLES (DEGREES) : 0.70 REMARK 3 REMARK 3 ISOTROPIC THERMAL MODEL: RESTRAINED REMARK 3 REMARK 3 ISOTROPIC THERMAL FACTOR RESTRAINTS. RMS SIGMA REMARK 3 MAIN-CHAIN BOND (A**2) : 1.49 ; 1.50 REMARK 3 MAIN-CHAIN ANGLE (A**2) : 2.44 ; 2.00 REMARK 3 SIDE-CHAIN BOND (A**2) : 1.95 ; 2.00 REMARK 3 SIDE-CHAIN ANGLE (A**2) : 2.93 ; 2.50 REMARK 3 REMARK 3 NCS MODEL : NONE REMARK 3 REMARK 3 NCS RESTRAINTS. RMS SIGMA/WEIGHT REMARK 3 GROUP 1 POSITIONAL (A) : NULL ; NULL REMARK 3 GROUP 1 B-FACTOR (A**2) : NULL ; NULL REMARK 3 REMARK 3 PARAMETER FILE 1 : protein_rep.param REMARK 3 PARAMETER FILE 2 : water_rep.param REMARK 3 PARAMETER FILE 3 : ion.param REMARK 3 TOPOLOGY FILE 1 : protein.top REMARK 3 TOPOLOGY FILE 2 : water.top REMARK 3 TOPOLOGY FILE 4 : ion.top REMARK 3 REMARK 3 OTHER REFINEMENT REMARKS: NULL SSBOND 1 CYS L 23 CYS L 88 SSBOND 2 CYS H 22 CYS H 96 SSBOND 3 CYS A 23 CYS A 88 SSBOND 4 CYS B 22 CYS B 96 SSBOND 5 CYS C 94 CYS C 144 SSBOND 6 CYS C 99 CYS C 146 SSBOND 7 CYS D 94 CYS D 144 SSBOND 8 CYS D 99 CYS D 146 CRYST1 184.719 55.558 71.109 90.00 90.00 90.00 P21 21 2 24 ORIGX1 1.000000 0.000000 0.000000 0.00000 ORIGX2 0.000000 1.000000 0.000000 0.00000 ORIGX3 0.000000 0.000000 1.000000 0.00000 SCALE1 0.005414 0.000000 0.000000 0.00000 SCALE2 0.000000 0.017999 0.000000 0.00000 SCALE3 0.000000 0.000000 0.014063 0.00000
[0463]
[0464]
Example 6. X-Ray Analysis of Antibody Variants Derived by Affinity Maturation: XAB5
[0465] The XAB5 Fv was cloned and expressed in E. coli TGf1—with a C-terminal hexahistidine tag on the heavy-chain and a C-terminal Strep-tag on the light-chain. The recombinant protein was purified by Ni-chelate chromatography followed by size-exclusion chromatography on a SPX-75 column, in PBS buffer. LC-MS analysis showed the expected mass for the heavy-chain (13703.4 Da), and the presence of two forms of the light-chain: full-length (115aa; 12627.3 Da; ca. 27%) and with truncated Strep-tag (A1 to Q112; 12222.8 Da; ca. 73%).
[0466] The XAB5 Fv fragment complex with human IL-17A was then prepared using standard methodology. In brief, human IL-17A (1.4 mg) was mixed with an excess of XAB5 Fv (3.4 mg) and the complex was run on a S100 size-exclusion chromatography, in 10 mM TRIS pH 7.4, 25 mM NaCl. The protein complex was then concentrated by ultra-filtration to 16.5 mg/ml and crystallized.
[0467] Standard crystallization protocols were followed. In brief, crystals were grown at 19° C. in SD2 96 well-plates, using the method of vapour diffusion in sitting drops. The protein stock was mixed 1:1 with a crystallization buffer containing 15% PEG 5,000 MME, 0.1M MES pH 6.5, 0.2M ammonium sulfate. Total drop size was 0.4 μl. Prior to X-ray data collection, one crystal was briefly transferred into a 1:1 mix of the crystallization buffer with 20% PEG 5,000 MME, 40% glycerol, and then flash cooled into liquid nitrogen.
[0468] X-ray data collection and processing was carried out using standard protocols. Briefly, X-ray data to 3.1 Å resolution were collected at the Swiss Light Source, beamline X10SA, with a Pilatus detector, using 1.00000 Å X-ray radiation. In total, 720 images of 0.25° oscillation each were recorded at a crystal-to-detector distance of 520 mm and processed with the XDS software package. The crystal belonged to space group C222.sub.1 with cell parameters a=55.37 Å, b=84.08 Å, c=156.35 Å, α=β=γ=90°. R-sym to 3.1 Å resolution was 8.9% and data completeness 99.7%.
[0469] The structure was determined by molecular replacement with the program Phaser, using search models derived from the previously-determined XAB2 Fv complex. Iterative model correction and refinement was performed with Coot (Crystallographic Object-Oriented Toolkit) and CNX (Crystallography & NMR eXplorer) version 2002, until no further significant improvements could be made to the crystallographic model Final R- and R-free for all data were 0.222 and 0.305, respectively. The final refined model showed a root-mean-square deviation (RMSD) from ideal bond lengths and bond angles of 0.008 Å and 1.2°, respectively.
[0470] Results
[0471] The results of the X-ray refinement of the XAB5 Fv complex with human IL-17A are provided in Table 15 and the three-dimensional structure of this complex is shown in
TABLE-US-00016 TABLE 15 X-ray refinement of the XAB5 Fv complex with IL-17A obtained by the program CNX. REMARK 3 REMARK 3 REFINEMENT. REMARK 3 PROGRAM : CNX 2002 REMARK 3 AUTHORS : Brunger, Adams, Clore, Delano, REMARK 3 Gros, Grosse-Kunstleve, Jiang, REMARK 3 Kuszewski, Nilges, Pannu, Read, REMARK 3 Rice, Simonson, Warren REMARK 3 and REMARK 3 Accelrys Inc., REMARK 3 (Badger, Berard, Kumar, Szalma, REMARK 3 Yip, Dzakula). REMARK 3 REMARK 3 DATA USED IN REFINEMENT. REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) : 3.11 REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) : 46.25 REMARK 3 DATA CUTOFF (SIGMA(F)) : 0.0 REMARK 3 DATA CUTOFF HIGH (ABS(F)) : 3778977.84 REMARK 3 DATA CUTOFF LOW (ABS(F)) : 0.000000 REMARK 3 COMPLETENESS (WORKING+TEST) (%) : 99.0 REMARK 3 NUMBER OF REFLECTIONS : 6801 REMARK 3 REMARK 3 FIT TO DATA USED IN REFINEMENT. REMARK 3 CROSS-VALIDATION METHOD : THROUGHOUT REMARK 3 FREE R VALUE TEST SET SELECTION : RANDOM REMARK 3 R VALUE (WORKING SET) : 0.222 REMARK 3 FREE R VALUE : 0.305 REMARK 3 FREE R VALUE TEST SET SIZE (%) : 5.0 REMARK 3 FREE R VALUE TEST SET COUNT : 340 REMARK 3 ESTIMATED ERROR OF FREE R VALUE : 0.017 REMARK 3 REMARK 3 FIT IN THE HIGHEST RESOLUTION BIN. REMARK 3 TOTAL NUMBER OF BINS USED : 6 REMARK 3 BIN RESOLUTION RANGE HIGH (A) : 3.10 REMARK 3 BIN RESOLUTION RANGE LOW (A) : 3.29 REMARK 3 BIN COMPLETENESS (WORKING+TEST) (%) : 89.9 REMARK 3 REFLECTIONS IN BIN (WORKING SET) : 961 REMARK 3 BIN R VALUE (WORKING SET) : 0.293 REMARK 3 BIN FREE R VALUE : 0.403 REMARK 3 BIN FREE R VALUE TEST SET SIZE (%) : 4.9 REMARK 3 BIN FREE R VALUE TEST SET COUNT : 50 REMARK 3 ESTIMATED ERROR OF BIN FREE R VALUE : 0.057 REMARK 3 REMARK 3 NUMBER OF NON-HYDROGEN ATOMS USED IN REFINEMENT. REMARK 3 PROTEIN ATOMS : 2492 REMARK 3 NUCLEIC ACID ATOMS : 0 REMARK 3 HETEROGEN ATOMS : 5 REMARK 3 SOLVENT ATOMS : 4 REMARK 3 REMARK 3 B VALUES. REMARK 3 FROM WILSON PLOT (A**2) : 85.2 REMARK 3 MEAN B VALUE (OVERALL, A**2) : 71.0 REMARK 3 OVERALL ANISOTROPIC B VALUE. REMARK 3 B11 (A**2) : 23.21 REMARK 3 B22 (A**2) : 7.23 REMARK 3 B33 (A**2) : −30.44 REMARK 3 B12 (A**2) : 0.00 REMARK 3 B13 (A**2) : 0.00 REMARK 3 B23 (A**2) : 0.00 REMARK 3 REMARK 3 BULK SOLVENT MODELING. REMARK 3 METHOD USED : FLAT MODEL REMARK 3 KSOL : 0.389339 REMARK 3 BSOL : 59.5295 (A**2) REMARK 3 REMARK 3 ESTIMATED COORDINATE ERROR. REMARK 3 ESD FROM LUZZATI PLOT (A) : 0.35 REMARK 3 ESD FROM SIGMAA (A) : 0.42 REMARK 3 LOW RESOLUTION CUTOFF (A) : 5.00 REMARK 3 REMARK 3 CROSS-VALIDATED ESTIMATED COORDINATE ERROR. REMARK 3 ESD FROM C-V LUZZATI PLOT (A) : 0.51 REMARK 3 ESD FROM C-V SIGMAA (A) : 0.45 REMARK 3 REMARK 3 RMS DEVIATIONS FROM IDEAL VALUES. REMARK 3 BOND LENGTHS (A) : 0.008 REMARK 3 BOND ANGLES (DEGREES) : 1.2 REMARK 3 DIHEDRAL ANGLES (DEGREES) : 23.1 REMARK 3 IMPROPER ANGLES (DEGREES) : 0.73 REMARK 3 REMARK 3 ISOTROPIC THERMAL MODEL: RESTRAINED REMARK 3 REMARK 3 ISOTROPIC THERMAL FACTOR RESTRAINTS. RMS SIGMA REMARK 3 MAIN-CHAIN BOND (A**2) : 1.40 ; 1.50 REMARK 3 MAIN-CHAIN ANGLE (A**2) : 2.49 ; 2.00 REMARK 3 SIDE-CHAIN BOND (A**2) : 1.82 ; 2.00 REMARK 3 SIDE-CHAIN ANGLE (A**2) : 2.93 ; 2.50 REMARK 3 REMARK 3 NCS MODEL : NONE REMARK 3 REMARK 3 NCS RESTRAINTS. RMS SIGMA/WEIGHT REMARK 3 GROUP 1 POSITIONAL (A) : NULL ; NULL REMARK 3 GROUP 1 B-FACTOR (A**2) : NULL ; NULL REMARK 3 REMARK 3 PARAMETER FILE 1 : protein_rep.param REMARK 3 PARAMETER FILE 2 : water_rep.param REMARK 3 PARAMETER FILE 3 : ion.param REMARK 3 TOPOLOGY FILE 1 : protein_no_cter.top REMARK 3 TOPOLOGY FILE 2 : water.top REMARK 3 TOPOLOGY FILE 4 : ion.top REMARK 3 REMARK 3 OTHER REFINEMENT REMARKS: NULL SSBOND 1 CYS S 23 CYS S 88 SSBOND 2 CYS S 22 CYS S 96 SSBOND 3 CYS S 94 CYS S 144 SSBOND 4 CYS S 99 CYS S 146 CRYST1 55.372 84.082 156.350 90.00 90.00 90.00 C 2 2 21 24 ORIGX1 1.000000 0.000000 0.000000 0.00000 ORIGX2 0.000000 1.000000 0.000000 0.00000 ORIGX3 0.000000 0.000000 1.000000 0.00000 SCALE1 0.018060 0.000000 0.000000 0.00000 SCALE2 0.000000 0.011893 0.000000 0.00000 SCALE3 0.000000 0.000000 0.006396 0.00000
[0472]
[0473]
Example 7. X-Ray Analysis of Antibody Variants Derived by Affinity Maturation: XAB4
[0474] The XAB4 Fv was cloned and expressed in E. coli TG1 cells with a C-terminal hexahistidine tag on the heavy-chain and a C-terminal Strep-tag on the light-chain. The recombinant protein was purified by Ni-chelate chromatography.
[0475] The XAB4 Fv fragment complex with human IL-17A was then prepared using standard methodology. In brief, human IL-17A (0.5 mg) was mixed with an excess of XAB4 Fv (1.2 mg) and the complex was run on a SPX-75 size-exclusion chromatography, in 10 mM TRIS pH 7.4, 25 mM NaCl. The protein complex was then concentrated by ultra-filtration to 6.9 mg/ml and crystallized.
[0476] Standard crystallization protocols were followed. In brief, crystals were grown at 19° C. in VDX 24 well-plates, using the method of vapour diffusion in hanging drops. The protein stock was mixed 2:1 with a crystallization buffer containing 15% PEG 5,000 MME, 0.1M MES pH 6.5, 0.2M ammonium sulfate. Total drop size was 3.0 μl. Prior to X-ray data collection, one crystal was briefly transferred into a 1:1 mix of the crystallization buffer with 25% PEG 5,000 MME, 20% glycerol, and then flash cooled into liquid nitrogen.
[0477] X-ray data collection and processing was carried out using standard protocols. Briefly, X-ray data to 3.15 Å resolution were collected at the Swiss Light Source, beamline X10SA, with a Pilatus detector, using 0.99984 Å X-ray radiation. In total, 720 images of 0.25° oscillation each were recorded at a crystal-to-detector distance of 500 mm and processed with the XDS software package. The crystal belonged to space group C222.sub.1 with cell parameters a=55.76 Å, b=87.11 Å, c=156.31 Å, α=β=γ=90°. R-sym to 3.15 Å resolution was 5.5% and data completeness 99.9%.
[0478] As the crystal of the XAB4 Fv complex was nearly isomorphous with the crystal of the XAB5 Fv complex (Example 6), the structure of the latter was used as input model for structure determination by molecular replacement with the program Phaser. Iterative model correction and refinement was performed with Coot (Crystallographic Object-Oriented Toolkit) and Autobuster version 1.11.2 (Buster version 2.11.2), until no further significant improvements could be made to the crystallographic model. Final R- and R-free for all data were 0.197 and 0.253, respectively. The final refined model showed a root-mean-square deviation (RMSD) from ideal bond lengths and bond angles of 0.009 Å and 1.0°, respectively.
[0479] (i) Results
[0480] The results of the X-ray refinement of the XAB4 Fv complex with human IL-17A are provided in Table 16 and the three-dimensional structure of this complex is shown in
TABLE-US-00017 TABLE 16 X-ray refinement of the XAB4 Fv complex with IL-17A obtained by the program Autobuster. REMARK 3 REMARK 3 REFINEMENT. REMARK 3 PROGRAM : BUSTER 2.11.2 REMARK 3 AUTHORS : BRICOGNE,BLANC,BRANDL,FLENSBURG,KELLER, REMARK 3 : PACIOREK,ROVERSI,SHARFF,SMART,VONRHEIN,WOMACK; REMARK 3 : MATTHEWS,TEN EYCK,TRONRUD REMARK 3 REMARK 3 DATA USED IN REFINEMENT. REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) : 3.15 REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) : 78.15 REMARK 3 DATA CUTOFF (SIGMA(F)) : 0.0 REMARK 3 COMPLETENESS FOR RANGE (%) : 99.85 REMARK 3 NUMBER OF REFLECTIONS : 6881 REMARK 3 REMARK 3 FIT TO DATA USED IN REFINEMENT. REMARK 3 CROSS-VALIDATION METHOD : THROUGHOUT REMARK 3 FREE R VALUE TEST SET SELECTION : RANDOM REMARK 3 R VALUE (WORKING+TEST SET) : 0.1998 REMARK 3 R VALUE (WORKING SET) : 0.1972 REMARK 3 FREE R VALUE : 0.2531 REMARK 3 FREE R VALUE TEST SET SIZE (%) : 5.01 REMARK 3 FREE R VALUE TEST SET COUNT : 345 REMARK 3 ESTIMATED ERROR OF FREE R VALUE : NULL REMARK 3 REMARK 3 FIT IN THE HIGHEST RESOLUTION BIN. REMARK 3 TOTAL NUMBER OF BINS USED : 5 REMARK 3 BIN RESOLUTION RANGE HIGH (ANGSTROMS) : 3.15 REMARK 3 BIN RESOLUTION RANGE LOW (ANGSTROMS) : 3.52 REMARK 3 BIN COMPLETENESS (WORKING+TEST) (%) : 99.85 REMARK 3 REFLECTIONS IN BIN (WORKING+TEST SET) : 1916 REMARK 3 BIN R VALUE (WORKING+TEST SET) : 0.2376 REMARK 3 REFLECTIONS IN BIN (WORKING SET) : 1820 REMARK 3 BIN R VALUE (WORKING SET) : 0.2326 REMARK 3 BIN FREE R VALUE : 0.3295 REMARK 3 BIN FREE R VALUE TEST SET SIZE (%) : 5.01 REMARK 3 BIN FREE R VALUE TEST SET COUNT : 96 REMARK 3 ESTIMATED ERROR OF BIN FREE R VALUE : NULL REMARK 3 REMARK 3 NUMBER OF NON-HYDROGEN ATOMS USED IN REFINEMENT. REMARK 3 PROTEIN ATOMS : 2499 REMARK 3 NUCLEIC ACID ATOMS : 0 REMARK 3 HETEROGEN ATOMS : 5 REMARK 3 SOLVENT ATOMS : 0 REMARK 3 REMARK 3 B VALUES. REMARK 3 FROM WILSON PLOT (A**2) : 102.42 REMARK 3 MEAN B VALUE (OVERALL, A**2) : 124.95 REMARK 3 OVERALL ANISOTROPIC B VALUE. REMARK 3 B11 (A**2) : −11.5511 REMARK 3 B22 (A**2) : −28.0012 REMARK 3 B33 (A**2) : 39.5523 REMARK 3 B12 (A**2) : 0.0000 REMARK 3 B13 (A**2) : 0.0000 REMARK 3 B23 (A**2) : 0.0000 REMARK 3 REMARK 3 ESTIMATED COORDINATE ERROR. REMARK 3 ESD FROM LUZZATI PLOT (A) : 0.787 REMARK 3 DPI (BLOW EQ-9) BASED ON FREE R VALUE (A) : 0.474 REMARK 3 REMARK 3 REFERENCES: BLOW, D. (2002) ACTA CRYST D58, 792-797 REMARK 3 REMARK 3 CORRELATION COEFFICIENTS. REMARK 3 CORRELATION COEFFICIENT FO-FC : 0.9113 REMARK 3 CORRELATION COEFFICIENT FO-FC FREE : 0.8848 REMARK 3 REMARK 3 X-RAY WEIGHT: 20.89 REMARK 3 REMARK 3 GEOMETRY FUNCTION. REMARK 3 RESTRAINT LIBRARIES. REMARK 3 NUMBER OF LIBRARIES USED : 8 REMARK 3 LIBRARY 1 : protgeo_eh99.dat (V1.8) 20110121 STANDARD REMARK 3 AMINO ACID DICTIONARY. BONDS AND ANGLES FROM REMARK 3 ENGH AND HUBER EH99. OTHER VALUES BASED ON REMARK 3 PREVIOUS TNT OR TAKEN FROM CCP4. INCLUDES REMARK 3 HYDROGEN ATOMS. REMARK 3 LIBRARY 2 : exoticaa.dat (V1.8) 20100430 COLLECTION OF REMARK 3 NON-STANDARD AMINO ACIDS, MAINLY EH91 WITHOUT REMARK 3 IDEAL DISTANCE INFO REMARK 3 LIBRARY 3: nuclgeo.dat (V1.14) 20091104 REMARK 3 LIBRARY 4: bcorrel.dat (V1.15) 20080423 REMARK 3 LIBRARY 5: contact.dat (V1.20.2.1) 20110510 REMARK 3 LIBRARY 6 : idealdist_contact.dat (V1.7) 20110119 REMARK 3 IDEAL-DISTANCE CONTACT TERM DATA AS USED IN REMARK 3 PROLSQ. VALUES USED HERE ARE BASED ON THE REFMAC REMARK 3 5.5 IMPLEMENTATION. REMARK 3 LIBRARY 7 : restraints for SO4 (SULFATE ION) from cif REMARK 3 dictionary SO4.cif using refmacdict2tnt revision REMARK 3 1.23.2.7; buster common-compounds v 1.0 (05 May REMARK 3 2011) REMARK 3 LIBRARY 8 : assume.dat (V1.10) 20110113 REMARK 3 REMARK 3 NUMBER OF GEOMETRIC FUNCTION TERMS DEFINED: 15 REMARK 3 TERM COUNT WEIGHT FUNCTION. REMARK 3 BOND LENGTHS : 2566 ; 2.00 ; HARMONIC REMARK 3 BOND ANGLES : 3486 ; 2.00 ; HARMONIC REMARK 3 TORSION ANGLES : 860 ; 2.00 ; SINUSOIDAL REMARK 3 TRIGONAL CARBON PLANES : 61 ; 2.00 ; HARMONIC REMARK 3 GENERAL PLANES : 369 ; 5.00 ; HARMONIC REMARK 3 ISOTROPIC THERMAL FACTORS : 2566 ; 20.00 ; HARMONIC REMARK 3 BAD NON-BONDED CONTACTS : NULL ; NULL ; NULL REMARK 3 IMPROPER TORSIONS : NULL ; NULL ; NULL REMARK 3 PSEUDOROTATION ANGLES : NULL ; NULL ; NULL REMARK 3 CHIRAL IMPROPER TORSION : 323 ; 5.00 ; SEMIHARMONIC REMARK 3 SUM OF OCCUPANCIES : NULL ; NULL ; NULL REMARK 3 UTILITY DISTANCES : NULL ; NULL ; NULL REMARK 3 UTILITY ANGLES : NULL ; NULL ; NULL REMARK 3 UTILITY TORSION : NULL ; NULL ; NULL REMARK 3 IDEAL-DIST CONTACT TERM : 2984 ; 4.00 ; SEMIHARMONIC REMARK 3 REMARK 3 RMS DEVIATIONS FROM IDEAL VALUES. REMARK 3 BOND LENGTHS (A) : 0.009 REMARK 3 BOND ANGLES (DEGREES) : 1.00 REMARK 3 PEPTIDE OMEGA TORSION ANGLES (DEGREES) : 4.39 REMARK 3 OTHER TORSION ANGLES (DEGREES) : 18.96 REMARK 3 REMARK 3 SIMILARITY. REMARK 3 NCS. REMARK 3 NCS REPRESENTATION : NONE REMARK 3 TARGET RESTRAINTS. REMARK 3 TARGET REPRESENTATION : LSSR REMARK 3 TARGET STRUCTURE : xab5_il17a_complex_final_buster.pdb REMARK 3 REMARK 3 TLS DETAILS. REMARK 3 NUMBER OF TLS GROUPS : 3 REMARK 3 REMARK 3 TLS GROUP : 1 REMARK 3 SET : { H|* } REMARK 3 ORIGIN FOR THE GROUP (A): 10.9676 −8.7396 −10.1379 REMARK 3 T TENSOR REMARK 3 T11: −0.1266 T22: 0.0257 REMARK 3 T33: −0.2829 T12: −0.3040 REMARK 3 T13: −0.0312 T23: 0.1050 REMARK 3 L TENSOR REMARK 3 L11: 7.4496 L22: 4.4770 REMARK 3 L33: 4.2880 L12: 1.1123 REMARK 3 L13: −1.8044 L23: 3.0307 REMARK 3 S TENSOR REMARK 3 S11: 0.2013 S12: 0.3070 S13: −0.5774 REMARK 3 S21: 0.4752 S22: −0.5377 S23: 0.7096 REMARK 3 S31: 1.0885 S32: −1.0885 S33: 0.3364 REMARK 3 REMARK 3 TLS GROUP : 2 REMARK 3 SET : { I|*} REMARK 3 ORIGIN FOR THE GROUP (A): 22.7365 0.7101 −35.1243 REMARK 3 T TENSOR REMARK 3 T11: −0.1883 T22: 0.1529 REMARK 3 T33: −0.3560 T12: 0.0318 REMARK 3 T13: −0.1985 T23: 0.0144 REMARK 3 L TENSOR REMARK 3 L11: 2.7494 L22: 9.3427 REMARK 3 L33: 3.8648 L12: 0.8073 REMARK 3 L13: −0.6650 L23: −2.0544 REMARK 3 S TENSOR REMARK 3 S11: 0.0485 S12: 0.3188 S13: 0.0579 REMARK 3 S21: 0.0595 S22: 0.1433 S23: 0.7000 REMARK 3 S31: 0.0050 S32: −0.6066 S33: −0.1917 REMARK 3 REMARK 3 TLS GROUP : 3 REMARK 3 SET : { L|* } REMARK 3 ORIGIN FOR THE GROUP (A): 33.2517 −11.1794 −14.2151 REMARK 3 T TENSOR REMARK 3 T11: 0.0667 T22: −0.1645 REMARK 3 T33: −0.2360 T12: 0.1870 REMARK 3 T13: −0.2270 T23: −0.1209 REMARK 3 L TENSOR REMARK 3 L11: 3.3694 L22: 3.7848 REMARK 3 L33: 8.8916 L12: −0.6497 REMARK 3 L13: −2.6132 L23: 0.8234 REMARK 3 S TENSOR REMARK 3 S11: −0.0839 S12: −0.2629 S13: −0.1560 REMARK 3 S21: 0.3804 S22: 0.7574 S23: −0.5378 REMARK 3 S31: 1.0885 S32: 1.0885 S33: −0.6736 REMARK 3 REMARK 3 REFINEMENT NOTES. REMARK 3 NUMBER OF REFINEMENT NOTES : 1 REMARK 3 NOTE 1 : IDEAL-DIST CONTACT TERM CONTACT SETUP. ALL REMARK 3 ATOMS HAVE CCP4 ATOM TYPE FROM LIBRARY REMARK 3 REMARK 3 OTHER REFINEMENT REMARKS: NULL REMARK 3 SSBOND 1 CYS H 22 CYS H 96 1555 1555 2.03 SSBOND 2 CYS I 94 CYS I 144 1555 1555 2.05 SSBOND 3 CYS I 99 CYS I 146 1555 1555 2.04 SSBOND 4 CYS L 23 CYS L 88 1555 1555 2.07 CISPEP 1 TYR I 85 PRO I 86 0 3.67 CISPEP 2 GLU I 125 PRO I 126 0 −9.25 CISPEP 3 PRO I 126 PRO I 127 0 5.92 CISPEP 4 SER L 7 PRO L 8 0 −6.50 CISPEP 5 TYR L 94 PRO L 95 0 −6.52 CRYST1 55.760 87.109 156.306 90.00 90.00 90.00 C 2 2 21
[0481]
[0482]
[0483]
[0484] To summarize, X-ray crystallography analysis confirmed that the variant antibodies selected for further analysis retained their target specificity and bound with high affinity to essentially the same epitope as the parental XAB1 antibody. Tighter binding between each of the variant antibodies and IL-17A was observed, as a result of additional or improved binding contacts (see Table 17 below).
[0485] Further characterisation of the variant antibodies was conducted as described below.
TABLE-US-00018 TABLE 17 X-ray analyses of the IL-17A epitope bound by XAB1, XAB2, XAB4 and XAB5: summary and structure-based, qualitative classification of epitope residues. Epitope residue class XAB1 XAB2 XAB4 XAB5 Very Arg 78, Glu Arg 78, Glu Arg 78, Glu Arg 78, Glu important 80, Trp 90 80, Trp 90 80, Tyr 85, 80, Tyr 85, epitope Trp 90, Trp 90, residues Arg 124 Arg 124 Other Pro 82, Ser Arg 43*, Pro Pro 82, Ser Pro 82, Ser important 87, Val 88, 82, Ser 87, 87, Val 88 87, Val 88 epitope Arg 124 Val 88, Arg residues 124 Additional Val 45*, Leu Pro 42*, Val Val 45*, Leu Val 45*, Leu contributions 49, Ile 51, 45*, Leu 49, 49, Asp 81, 49, Asp 81, Asp 81, Glu Ile 51, Asp Glu 83, Pro Glu 83, Pro 83, Tyr 85, 81, Glu 83, 86, Pro 130, 86, Pro 130, Asn 131, Tyr 85, Asn Phe 133, Lys Phe 133, Lys Lys 137* 131, Lys 137* 137* 137* Little or no Thr 44*, Leu Leu 76, His Arg 43*, Asn Arg 43*, Asn direct 76, His 77, 77, Asn 79, 50, Ser 64, 50, Leu 76, contribution Asn 79, Arg Arg 84, Pro Tyr 67, Leu His 77, Asn 84, Pro 86, 86, Lys 93, 76, His 77, 79, Arg 84, Lys 93, Glu Glu 118*, Asn 79, Arg Glu 118*, Leu 118*, Pro 130, 84, Glu 118*, 122, Asn 131, Pro 130, Phe 133 Leu 122, Asn Leu 135* Phe 133 131, Leu 135* *residue contributed by the second IL-17A subunit.
Example 8. Affinity Measurements and Cross-Reactivity Measured by Biacore™
[0486] Determination of kinetic binding parameters was achieved by surface plasmon resonance measurements using the optical biosensor Biacore™ T200 or T100 (http://www.biacore.com). This technology allows the label-free determination of the microscopic rate constants for binding (k.sub.a) and dissociation (k.sub.d) of a ligand to a receptor. It is therefore especially suited for characterizing the antibody-antigen interactions.
[0487] Indirect binding of antibodies to the Biacore™ chip surface was done via an anti-human Ig antibody (GE Healthcare Bio-Sciences AB; Cat. No. BR-1008-39) 25 μg/ml in immobilization buffer (10 mM Sodium acetate pH 5.0) or through protein A (RepliGen: rPA-50) 20 μg/ml in immobilization buffer (10 mM Sodium acetate pH 5.0 or pH 4.0).
[0488] Antibody was diluted into blank buffer to a final concentration of 1.00 or 1.25 μg/ml.
[0489] Affinity measurements for the determination of dissociation constants of XAB4 or XAB1 was performed for recombinant huIL-17A (SEQ ID NO: 78, e.g. 2-fold increasing concentrations from 0.14 to 8.8 nM), recombinant huIL-17A/F heterodimer (e.g. 2-fold increasing concentrations from 0.13 to 8 nM), recombinant huIL-17F (SEQ ID NO: 77; e.g. 2-fold increasing concentrations from 7.8 to 500 nM) cynomolgus IL-17A (SEQ ID NO: 79; e.g. 2-fold increasing concentrations from 0.63 to 40 nM) rhesus IL-17A (SEQ ID NO: 82; e.g. 2-fold increasing concentrations from 1.6 to 100 nM), marmoset IL-17A (SEQ ID NO: 82; e.g. 2-fold increasing concentrations from 0.63 to 40 nM), recombinant mIL-17A (SEQ ID NO: 83; e.g. 2-fold increasing concentrations from 0.78 to 50 nM), recombinant mIL-17A/F (R&D Systems® Cat#5390-IL; e.g. 2-fold increasing concentrations from 1.25 to 40 nM) rat IL-17A (SEQ ID NO: 85; e.g. 2-fold increasing concentrations from 0.78 to 50 nM), using the indirect coupling/binding method (see above) and surface was regenerated with 10 mM glycine pH 1.75 or MgCl.sub.2 (3 M). One chip surface was coated and reused without significant loss of binding capacity. Ligand concentrations were chosen to start below the K.sub.D and to end at a concentration higher than ten times the K.sub.D.
[0490] Similar but not identical conditions were used to measure affinity of XAB2 and XAB3.
[0491] The kinetic traces were evaluated with the Biacore™ T200 Control Software version 1.0. The full set of these traces with increasing concentrations is taken together and is called a run. Two zero concentration samples (blank runs) were included in each analyte concentration series to allow double-referencing during data evaluation
[0492] Results
[0493] The binding of the anti-IL-17 antibodies XAB4, XAB1, XAB2 and XAB3 to human, cynomolgus monkey, marmoset monkey, rhesus monkey, mouse and rat IL-17A, to human and mouse IL-17A/F heterodimer and to human IL-17F was determined by surface plasmon resonance using the Biacore™ technology.
[0494] The kinetic rate constants for association (k.sub.a) and dissociation (k.sub.d), as well as the dissociation equilibrium constant (K.sub.D) were calculated.
[0495] The affinity data of XAB4 is shown in Table 18, the affinity data of XAB1 is shown in
[0496] Table 19, the affinity data of XAB2 is shown in Table 20, and the affinity data of XAB3 is shown in Table 21. Affinity maturation of XAB1, XAB2 and XAB3 increased the affinity towards human, cynomolgus monkey, mouse and rat IL-17A.
TABLE-US-00019 TABLE 18 Affinity and kinetic rate constants of XAB4 binding. Antigen k.sub.a (1/MS) k.sub.d (1/s) K.sub.D (M) huIL-17A 4.1 ± 0.1E+06 2.3 ± 0.1E−05 5.7 ± 0.0E−12 huIL-17A/F 8.9 ± 0.2E+5 <1.0 ± 0.0E−05* <1.1 ± 0.0E−11* huIL-17F n.d. n.d. n.d. cynoIL-17A 4.1 ± 0.5E+05 1.3 ± 0.0E−05 3.1 ± 0.4E−11 marmIL-17A 1.2 ± 0.0E+06 2.2 ± 0.0E−05 1.8 ± 0.0E−11 rhesIL-17A 3.0 ± 0.1E+05 1.2 ± 0.1E−05 4.0 ± 0.1E−11 mIL-17A 3.8 ± 0.1E+05 6.2 ± 0.3E−05 1.6 ± 0.1E−10 mIL-17A/F 2.421E+05 6.305E−05 2.604 E−10 ratIL-17A 5.5 ± 0.4E+05 4.6 ± 0.9E−05 8.4 ± 1.0E−11 n.d. = not determinable, applied antigen conc. range too low and non-specific binding of antigen to reference flow cell observed at the highest antigen concentrations (500-50 pM). *dissociation rate outside the limits that can be measured by the instrument (k.sub.d < 1 × 10.sup.−5 1/s)
TABLE-US-00020 TABLE 19 Affinity and kinetic rate constants of XAB1 binding. Antigen k.sub.a (1/MS) k.sub.d (1/s) K.sub.D (M) huIL-17A 2.33E+06 9.39E−05 4.03E−11 huIL-17A/F 9.097E+05 0.001342 1.475E−09 huIL-17F n.d. n.d. n.d. cynoIL-17A 2.14E+05 1.13E−04 5.26E−10 rhesIL-17A 8.87E+05 9.97E−05 1.12E−09 mIL-17A 4.05E+05 1.43E−04 3.53E−10 mIL-17A/F 1.8757E+05 9.547E−04 5.093E−09 ratIL-17A 5.44E+05 1.64E−04 3.01E−10 n.d. = not determinable, applied antigen conc. range too low and non-specific binding of antigen to reference flow cell observed at three highest antigen concentrations (500-50 pM).
TABLE-US-00021 TABLE 20 Affinity and kinetic rate constants of XAB2 binding. Antigen k.sub.a (1/MS) k.sub.d (1/s) K.sub.D (M) huIL-17A 4.09E+06 7.12E−05 1.76E−11
TABLE-US-00022 TABLE 21 Affinity and kinetic rate constants of XAB3 binding. Antigen k.sub.a (1/MS) k.sub.d (1/s) K.sub.D (M) huIL-17A 5.48E+06 5.01E−05 9.58E−12 huIL-17A/F 3.37E+06 1.03E−04 3.29E−11 huIL-17F n.d. n.d. n.d. cynoIL-17A 1.21E+06 4.23E−05 3.49E−11 mIL-17A 5.87E+05 1.01E−04 1.74E−10 ratIL-17A 9.05E+05 7.59E−05 8.26E−11 n.d. = not determinable
[0497] The affinities and kinetic rate constants for XAB2, XAB3 and XAB5 are comparable to those observed for XAB4.
Example 9. Binding in ELISA to IL-17A and Other Family Members
[0498] A titration of the antibodies of interest on different antigens was carried out. Briefly, wells of ELISA microtiter plates (Nunc Immuno plates MaxiSorp: Invitrogen, Cat#4-39454A) were coated with 1 μg/ml of recombinant huIL-17A (SEQ ID NO: 76; 1.8 mg/ml), recombinant huIL-17A/F (0.59 mg/ml), recombinant huIL-17F (SEQ ID NO: 77; 1.8 mg/ml)), recombinant huIL-17B (R&D Systems® Cat#1248IB/CF), recombinant huIL-17C (R&D Systems® Cat#1234IL/CF), recombinant huIL-17D (R&D Systems® Cat#1504IL/CF), recombinant huIL-17E (R&D Systems® Cat#1258-IL/CF), recombinant cynoIL-17A (SEQ ID NO: 79; 0.21 mg/ml), recombinant cynoIL-17F (SEQ ID NO: 80; 1.525 mg/ml), recombinant mIL-17A (SEQ ID NO: 83; 2.8 mg/ml), recombinant mIL-17A/F (R&D Systems® Cat#5390-IL), recombinant mIL-17F (SEQ ID NO: 84; 0.2 mg/ml) and recombinant ratIL-17A (SEQ ID NO: 85; 3.8 mg/ml) (100 μl/well) in phosphate buffered saline (PBS) without Ca and Mg (10×; Invitrogen Cat#14200-083) 0.02% NaN.sub.3 (Sigma Cat# S-8032) and incubated overnight at 4° C.
[0499] The following day, microtiter plates were blocked with 300 μl of PBS/2% BSA (fraction V; Roche Cat#10 735 094 001)/0.02% NaN.sub.3 for 1 h at 37° C. Plates were then washed 4 times with PBS/0.05% Tween 20 (Sigma Cat# P7949)/0.02% NaN.sub.3. XAB4 or XAB1 were added at 1 μg/ml in triplicate wells (100 μl/well) for 3 h at room temperature.
[0500] To verify coating of antigens to the plates, control antibodies were used and in particular, a mouse mAb anti-huIL-17F, (Novartis, 5 μg/ml) a goat anti-hu-IL-17B (R&D Systems® Cat# AF1248; 10 μg/ml), a mouse mAb anti-huIL-17C (R&D Systems® Cat# MAB1234; 10 μg/ml), a goat anti-huIL-17D (R&D Systems® Cat# AF1504; 10 μg/ml), a mouse mAb anti hu-IL-17E (R&D Systems® Cat# MAB1258; 10 μg/ml), a mouse anti-mIL-17A or anti-mIL-17A/F (Novartis; 1 μg/ml), and a rat anti-mIL-17F (R&D Systems® Cat# MAB2057; 1 μg/ml;) (100 μl/well in PBS, 0.02% NaN.sub.3 for 3 h at RT).
[0501] Plates were then washed 4 times with PBS/0.05% Tween 20/0.02% NaN.sub.3. Then, an alkaline phosphatase-conjugated goat anti-human IgG antibody (Sigma Cat# A9544) was added to the wells that received test antibody at a dilution of 1/20000 (100 μl/well) for 2 h 30 min at RT. To the wells, that received mouse mAb, an alkaline phosphatase-conjugated goat anti-mouse IgG antibody (Sigma Cat# A7434) was added at a dilution of 1/10000 (100 μl/well) for 2 h 30 min at RT. An alkaline phosphatase conjugated mouse anti goat IgG antibody (Sigma Cat# A8062) was added to the goat antibodies at a dilution of 1/50000 (100 μl/well) for 2 h 30 min at RT. Plates were then washed 4 times and 100 μl of the substrate (p-nitrophenyl phosphate tablets; Sigma; 5 mg Cat# N9389; 20 mg Cat#. N2765) dissolved in diethanolamine buffer pH 9.8, to give a final concentration of 1 mg/ml, were added to each well.
[0502] Plates were read after 30 min in a Spectra Max M5 Microplate Reader (Molecular Devices) using filters of 405 and 490 nm. Values are the means±SEM of triplicate values.
[0503] Results
[0504] These studies show that XAB4 and XAB1 are able to bind human and mouse IL-17A, and human and mouse IL-17A/F. In addition it is shown that XAB4 is able to bind cynomolgus and rat IL-17A. Binding to human, cynomolgus and mouse IL-17F was not detected under these experimental conditions as well as binding to other human family members (IL-17B, IL-17C, IL-17D and IL-17E).
TABLE-US-00023 TABLE 22 Cross-reactivity of XAB4 and XAB1 to human, cynomolgus monkey, mouse and rat IL-17 family members, by ELISA. Control Control XAB1 antibody (1 XAB4 antibody (1 (1 μg/ml) or 10 μg/ml) (1 μg/ml) or 10 μg/ml) O.D values O.D values O.D values O.D values (mean ± (mean ± (mean ± SEM) (mean ± SEM) SEM) SEM) hu IL-17A 2.471 ± 0.0448 1.302 ± 0.0554 hu IL-17A/F 2.137 ± 0.0429 1.222 ± 0.0202 hu IL-17F 0.049 ± 0.0056 0.032 ± 1.913 ± 0.0005 0.0483 hu IL-17B 0.034 ± 0.0007 0.283 ± 0.0066 0.049 ± 1.441 ± 0.0013 0.0283 hu IL-17C 0.036 ± 0.0002 0.290 ± 0.0027 0.032 ± 0.558 ± 0.0002 0.0169 hu IL-17D 0.034 ± 0.0005 0.292 ± 0.0048 0.031 ± 0.867 + 0.0010 0.0372 hu IL-17E 0.035 ± 0.0014 0.833 ± 0.0239 0.033 ± 2.054 ± 0.0003 0.0378 cyno IL-17A 1.926 ± 0.0355 cyno IL-17F 0.085 ± 0.0336 mouse IL- 1.585 ± 0.0428 1.086 ± 0.0119 1.439 ± 3.697 ± 17A 0.0354 0.0602 mouse 2.263 ± 0.0243 1.142 ± 0.0315 1.762 ± 2.084 ± IL-17A/F 0.0097 0.0223 mouse IL- 0.098 ± 0.0060 1.294 ± 0.0134 0.044 ± 1.770 ± 17F 0.0008 0.0302 rat IL-17A 1.772 ± 0.1668
Example 10. Cross-Reactivity to Other Human, Mouse and Rat Interleukins by ELISA
[0505] In another set of experiments the cross-reactivity of antibodies of the disclosure for selected human, mouse or rat cytokines was evaluated.
[0506] Triplicate wells of ELISA microtiter plates (Nunc Immuno plates MaxiSorp: Invitrogen Cat#4-39454A) were coated with 100 μl/well of the following cytokines: recombinant huIL1β (Novartis), recombinant huIL-3 (R&D Systems® Cat#203-IL/CF), recombinant huIL-4 (R&D Systems® Cat#204-IL/CF), recombinant huIL-6 (R&D Systems® Cat#206-IL-1010/CF), recombinant huIL-8 (R&D Systems® Cat#208-IL-010/CF), recombinant huIL-12 (R&D Systems® Cat#219-IL-005/CF), recombinant huIL-13 (Novartis), recombinant huIL-17A (SEQ ID NO: 76), recombinant huIL-17A/F, recombinant huIL-17F (SEQ ID NO: 77), recombinant huIL-1β (MBL Cat# B003-5), recombinant huIL-20 (Novartis), recombinant huIL-23 (R&D Systems® Cat#1290-IL-010/CF), recombinant hulFNγ (Roche), recombinant huTNFα (Novartis), recombinant huEGF (Sigma Cat#E9644.), recombinant huTGFβ2 (Novartis), recombinant mIL-1β (R&D Systems® Cat#401-ML), recombinant mIL-2 (R&D Systems® 402-ML-020/CF), recombinant mIL-6 (R&D Systems® Cat#406-ML-010/CF), recombinant mIL-12 (R&D Systems® Cat#419-ML-010/CF), recombinant mIL-17A (SEQ ID NO: 83), recombinant mIL-17A/F (R&D Systems® Cat#5390-IL), recombinant mIL-17F (R&D Systems® Cat#2057-IL/CF), recombinant mIL-1β (MBL Cat#B004-5), recombinant mIL-23 (R&D Systems® Cat#1887-ML), recombinant mIFN-γ (R&D Systems® Cat#485-MT), recombinant mTNFα (R&D Systems® Cat#410-MT), recombinant rat IL-4 (R&D Systems® Cat#504-RL/CF), recombinant rat IL-6 (R&D Systems® Cat#506-RL-010), recombinant ratIL-12 (R&D Systems® Cat#1760-RL/CF), recombinant ratIL-17A (SEQ ID NO: 85), recombinant ratIL-23 (R&D Systems® Cat#3136-RL-010/CF), recombinant ratTNFα (R&D Systems® Cat#510-RT/CF), at 1 μg/ml with the exception of recombinant mIL-6, recombinant mIL-12 and recombinant mTNFα which were coated at 0.5 μg/ml in phosphate buffered saline (PBS) without Ca and Mg (10×; Invitrogen Cat#14200-083) 0.02% NaN.sub.3 (Sigma Cat# S-8032) and incubated overnight at 4° C.
[0507] The following day, microtiter plates were blocked with 300 μl of PBS/2% BSA (fraction V; Roche Cat#10 735 094 001)/0.02% NaN.sub.3 for 1 h at 37° C. Plates were then washed 4 times with PBS/0.05% Tween 20 (Sigma Cat# P7949)/0.02% NaN.sub.3.
[0508] The antibodies of the disclosure were added at 10 μg/ml (100 μl/well) for 3 h at room temperature. To verify coating of antigens to the plates, 100 μl/well of the following control antibodies were used: a mouse anti-huIL1β (R&D Systems® Cat# MAB601), a mouse anti-huIL-3 (R&D Systems® Cat# MAB603), a mouse anti-huIL4 (R&D Systems® Cat# MAB604), a mouse anti-huIL-6 (R&D Systems® Cat# MAB206), a mouse anti-hu-IL8 (R&D Systems® Cat# MAB208), a mouse anti-huIL-12 (R&D Systems® Cat# MAB219), a mouse anti-huIL-13 (Novartis), a mouse anti-huIL-17A (Novartis), a mouse anti-huIL-17F (Novartis), a mouse anti-huIL-1β (MBL Cat# D043-3), a mouse anti-huIL-20 (Abcam Cat# ab57227), a goat anti-huIL-23 (R&D Systems® Cat# AF1716), a mouse anti-hulFN-γ (R&D Systems® Cat# MAB285), a mouse anti-huTNF-α (R&D Systems® Cat# MAB610), a mouse anti-hu-EGF (R&D Systems® Cat# MAB236), a human anti-huTGFβ2 (Novartis), a rat anti-mIL-1β (R&D Systems® Cat# MAB401), a rat anti-mIL-2 (R&D Systems® Cat# MAB402), a rat anti-mIL-6 (R&D Systems® Cat# MAB406), a rat anti-mIL-12 (R&D Systems® Cat# MAB419), a mouse anti-m/ratIL-17A (Novartis), a rat anti-mIL-17F (R&D Systems® Cat# MAB2057), a rat anti-mIL-1β (MBL Cat# D047-3), a rat anti-mIFN-γ (R&D Systems® Cat# MAB485), a goat anti-mTNFα (R&D Systems® Cat# AF-410-NA), a mouse anti-rat IL-4 (R&D Systems® Cat# MAB504), a goat anti-rat IL-6 (R&D Systems® Cat# AF506), a goat anti-rat IL-12 (R&D Systems® Cat# AF1760), a mouse anti-rat IL-23 (R&D Systems® Cat# MAB3510), a mouse anti-rat TNFα (R&D Systems® Cat# MAB510). They were added at 1 or 5 μg/ml, in PBS, 0.02% NaN.sub.3 for 3 h at RT.
[0509] Plates were then washed 4 times with PBS/0.05% Tween 20/0.02% NaN.sub.3. Then, an alkaline phosphatase-conjugated goat anti-human IgG antibody (Sigma Cat# A9544) was added to the wells with human antibodies at a dilution of 1/20000 (100 μl/well). An alkaline phosphatase-conjugated goat anti-mouse IgG antibody (Sigma Cat# A1047) was added to the wells with mouse antibodies at a dilution of 1/10000 (100 μl/well). An alkaline phosphatase-conjugated rabbit anti-goat IgG antibody (Sigma Cat# A7650) was added to the wells with goat antibodies at a dilution of 1/1000 (100 μl/well) and an alkaline phosphatase-conjugated rabbit anti rat-IgG antibody (Sigma Cat# A6066) was added to the wells with rat antibodies at a dilution of 1/20000 (100 μl/well). The secondary antibodies were incubated for 2 h 30 min at RT. Plates were then washed 4 times and 100 μl of the substrate (p-nitrophenyl phosphate tablets; Sigma; 5 mg Cat#. N9389 or 20 mg Cat# N2765) dissolved in diethanolamine buffer pH 9.8, to give a final concentration of 1 mg/ml, were added to each well.
[0510] Plates were read after 30 min at RT or ON at 4° C. in a Spectra Max M5 Microplate Reader (Molecular Devices) using filters of 405 and 490 nm. Values are the means±SEM of triplicate values.
[0511] Results
[0512] The data obtained show that both XAB4 and XAB1 are highly selective for IL-17A of human, mouse and rat origin and for IL-17A/F of human and mouse origin. In addition, under the conditions tested, the reactivity of XAB1 at 10 μg/ml for human IL-17F (not seen at 1 μg/ml, see above) is not observed with XAB4. Reactivity for the other cytokines tested was not detected.
TABLE-US-00024 TABLE 23 Cross-reactivity of XAB4 and XAB1 to human cytokines by ELISA. Control Control XAB4 antibody XAB1 antibody (10 μg/ml) (5 μg/ml) (10 μg/ml) (1 μg/ml) O.D values O.D values O.D values O.D values (mean ± SEM) (mean ± SEM) (mean ± SEM) (mean ± SEM) IL1β 0.015 ± 0.0075 0.867 ± 0.0107 −0.110 ± 3.071 ± 0.0486 0.0901 IL3 0.167 ± 0.1288 0.732 ± 0.0194 −0.049 ± 2.931 ± 0.0779 0.0738 IL4 0.047 ± 0.0089 0.806 ± 0.0617 0.057 ± 0.0458 2.555 ± 0.1499 IL6 −0.015 ± 1.452 ± 0.2020 −0.044 ± 2.976 ± 0.1025 0.0103 0.0838 IL8 0.018 ± 0.0078 3.130 ± 0.0109 0.058 ± 0.0431 3.153 ± 0.1228 IL12 0.009 ± 0.0058 0.853 ± 0.0496 −0.097 ± 2.964 ± 0.1370 0.1600 IL13 0.019 ± 0.0085 2.639 ± 0.0309 0.125 ± 0.0706 2.639 ± 0.0309 IL17A 3.178 ± 0.0697 3.136 ± 0.0644 2.745 ± 0.0879 2.731 ± 0.0850 IL17A/ 3.100 ± 0.0458 3.024 ± 0.0816 2.644 ± 0.2517 3.024 ± 0.0816 F IL17F 0.035 ± 0.0138 3.114 ± 0.0672 0.613 ± 0.4162 3.185 ± 0.0110 IL18 −0.001 ± 3.313 ± 0.2080 −0.086 ± 3.313 ± 0.2080 0.0234 0.0170 IL20 0.039 ± 0.0117 3.039 ± 0.0671 0.335 ± 0.2442 3.118 ± 0.0252 IL23 −0.022 ± 3.435 ± 0.0878 0.085 ± 0.0678 3.350 ± 0.0886 0.0450 IFN-γ 0.048 ± 0.0676 3.419 ± 0.0404 0.059 ± 0.0511 3.236 ± 0.0312 TNF-α 0.009 ± 0.0197 3.373 ± 0.0550 0.289 ± 0.0318 3.275 ± 0.0440 EGF 0.126 ± 0.0858 3.432 ± 0.1050 0.062 ± 0.0427 3.233 ± 0.1126 TGFβ2 0.018 ± 0.0190 3.397 ± 0.0358 0.146 ± 0.0653 3.246 ± 0.0303 BSA 0.009 ± 0.0194 0.010 ± 0.0192 0.043 ± 0.0033 0.149 ± 0.0558 N.B. the negative values are due to the fact that the blank (O.D. value of wells without specific antibodies) is subtracted.
TABLE-US-00025 TABLE 24 Cross-reactivity of XAB4 and XAB1 to mouse cytokines by ELISA. XAB4 Control antibody XAB1 Control antibody (10 μg/ml) (5 μg/ml) (10 μg/ml) (5 μg/ml) O.D values O.D values O.D values O.D values (mean ± SEM) (mean ± SEM) (mean ± SEM) (mean ± SEM) IL-1β 0.022 ± 0.0057 0.611 ± 0.0665 0.007 ± 0.0123 0.624 ± 0.0455 IL2 0.024 ± 0.0227 3.548 ± 0.1283 0.022 ± 0.0125 3.295 ± 0.0557 IL6 0.031 ± 0.0063 3.291 ± 0.0174 0.038 ± 0.0091 3.340 ± 0.1115 IL12 0.035 ± 0.0110 3.359 ± 0.0094 −0.005 ± 0.0121 3.295 ± 0.0331 IL17A 3.285 ± 0.0445 3.180 ± 0.0702 2.974 ± 0.0281 3.186 ± 0.0505 IL17A/F 3.342 ± 0.1047 3.407 ± 0.1102 3.169 ± 0.0340 3.214 ± 0.0145 IL17F 0.034 ± 0.0122 3.359 ± 0.0247 −0.058 ± 0.0326 3.264 ± 0.0309 IL18 0.054 ± 0.0149 2.650 ± 0.0227 0.022 ± 0.0123 2.572 ± 0.0145 IL23 0.058 ± 0.0139 0.601 ± 0.0314 0.009 ± 0.0007 0.590 ± 0.0378 IFN-γ 0.038 ± 0.0114 2.751 ± 0.0515 0.048 ± 0.0063 2.388 ± 0.2351 TNF-α 0.065 ± 0.0154 3.258 ± 0.1097 0.025 ± 0.0081 3.476 ± 0.0714 BSA 0.015 ± 0.0078 0.035 ± 0.0047 0.015 ± 0.0078 0.035 ± 0.0047 N.B. the negative values are due to the fact that the blank (O.D. value of wells without specific antibodies) is subtracted.
TABLE-US-00026 TABLE 25 Cross-reactivity of XAB4 and XAB1 to rat cytokines by ELISA. XAB4 Control antibody XAB1 Control antibody (10 μg/ml) (5 μg/ml) (10 μg/ml) (5 μg/ml) O.D values O.D values O.D values O.D values (mean ± SEM) (mean ± SEM) (mean ± SEM) (mean ± SEM) IL4 0.026 ± 0.0082 3.168 ± 0.0297 0.017 ± 0.0092 3.324 ± 0.1092 IL6 0.021 ± 0.0028 3.116 ± 0.0318 0.000 ± 0.0141 3.253 ± 0.1078 IL12 0.009 ± 0.0113 3.185 ± 0.0921 −0.007 ± 0.0082 3.310 ± 0.0692 IL17A 3.483 ± 0.0910 3.156 ± 0.0890 1.202 ± 0.0136 3.359 ± 0.0670 IL23 0.023 ± 0.0050 3.380 ± 0.2127 0.011 ± 0.0010 3.199 ± 0.1078 TNF-α 0.020 ± 0.0104 3.346 ± 0.1376 0.003 ± 0.0029 3.159 ± 0.0854 BSA 0.015 ± 0.0078 0.035 ± 0.0047 0.015 ± 0.0078 0.035 ± 0.0047 N.B. the negative values are due to the fact that the blank (O.D. value of wells without specific antibodies) is subtracted.
Example 11. IL-17A-IL-17RA and IL-17A/F-IL-17RA In Vitro Competitive Binding Inhibition Assay
[0513] Human IL-17RA was used from a stock solution (BTP22599: 1.68 mg/ml=46.2 μM). ELISA microtiter plates were coated with human IL-17RA (100 μl/well, 1 μg/ml, ˜27.5 nM) in PBS/0.02% NaN.sub.3 and incubated overnight at room temperature. The following day the plates were blocked with 300 μl of PBS/2% BSA/0.02% NaN.sub.3 for 1 h at 37° C. Then the plates were washed 4 times with PBS/0.05% Tween20/0.02% NaN.sub.3.
[0514] Following this preparation, titration of antibody variants (50 μl, concentrations from 12 nM to 0.12 nM for IL-17A and 1200 nM to 40 nM for IL-17A/F, steps of 3) were pre-incubated with human IL-17A biotin (50 μl at 0.94 nM) or IL-17A/F (50 μl at 31 nM) for 30 minutes at room temperature.
[0515] 100 μl of the mixture were added to the well for 3 hours and 30 minutes at room temperature. After washing with PBS/0.05% Tween20/0.02% NaN.sub.3, four times alkaline phosphatase-conjugated streptavidin was added at a final dilution of 1/10000 (100 μl/well). After 45 minutes at room temperature plates were washed again 4 times with PBS/0.05% Tween20/0.02% NaN.sub.3 and the substrate p-nitrophenylphosphate in diethanolamine buffer pH 9.8 (1 mg/ml), was added (100 μl/well).
[0516] Plates were read after 30 minutes in spectra Max M5 Microplate reader, filters 405 and 490 nm (triplicates). The calculation of the percentage of inhibition and IC.sub.50 for different antibody variants was done using a four parameter logistic model (Excel XIfit; FIT model 205).
[0517] Results
[0518] Data show that both XAB4 and XAB1 are able to block the binding of huIL-17A and huIL-17A/F to the huIL-17RA. The higher affinity of XAB4 for IL-17A and IL-17A/F is reflected in a higher inhibitory capacity. IC.sub.50 values are reported in the table. The higher concentrations needed to block the IL-17A/F-IL-17RA interaction are mostly explained by the fact that about 30 fold higher concentrations of IL-17A/F were used in the assay. The antibody binds to the A subunit of A/F and therefore cannot prevent binding of the F subunit to the IL-17RA. However, binding of F to IL-17RA is rather weak, in the 300 nM range.
TABLE-US-00027 TABLE 26 XAB4 and XAB1 inhibit the binding of huIL-17A and huIL-17A/F to huIL-17RA. XAB4 XAB1 Control Ligand\Receptor IC50 (nM) IC50 (nM) antibody interaction (mean ± SEM) (mean ± SEM) (nM) huIL-17A\huIL-17RA 0.321 ± 0.037 0.830 ± 0.112 >60 huIL-17A/F\huIL-17RA 153.9 ± 18.9 301.3 ± 51.9
Example 12. In Vitro Neutralisation of Human IL-17A and IL-17A/F Activity by Antibody Variants of the Disclosure
[0519] (i) Assay on C20A4C16 Cells (Human Chondrocyte Cell Line)
[0520] C20A4C16, or C-20/A4, clone 6, (Goldring M B, et al 1994, J Clin Invest; 94:2307-16) cells were cultured in RPMI (Gibco Cat#61870-010) supplemented with 10% fetal calf serum ultra-low IgG (Gibco Cat#16250-078; lot 1074403), β-mercapto ethanol (5×10.sup.−5 M final), and Normocin (0.1 mg/ml; InvivoGen Cat# ant-nr-2).
[0521] The cells were detached from plastic using an Accutase solution (PAA Cat# L11-007). Cells were distributed into 96 well microtiter plates at a density of 5×10.sup.3 in 100 μl well in RPMI 1640 (Gibco Cat#61870-010) without fetal calf serum, β-mercaptoethanol (5×10.sup.−5 M final) and Normocin (0.1 mg/ml).
[0522] The C20A4C16 cells were allowed to adhere to the plates overnight. The next morning, different concentrations of recombinant huIL-17A (SEQ ID NO: 76; MW 32000), recombinant huIL-17A/F (MW 32800), recombinant huIL-17F (SEQ ID NO: 77; MW 30000), or control medium in the presence of human TNFα (Novartis; MW 17500) were added in a volume of 50 μl to triplicate wells in the presence of 50 μl of different concentrations of test antibody (XAB4; XAB1), control antibody (Simulect® 1.1% solution, Batch C0011; 831179) or control medium to reach the final volume of 200 μl/well and the final concentration of 0.5% fetal calf serum.
[0523] HuIL-17A (30 pM), huIL-17A/F (300 pM) and huIL-17F (10 nM) were added together with huTNFα (6 pM). XAB4 (MW 150000) was added in a concentration range from 1 to 0.003 nM to neutralize huIL-17A, in a concentration range from 10 to 0.03 nM to neutralize huIL-17A/F and in a concentration range from 3 μM to 30 nM for huIL-17F. XAB1 (MW 150000) was added in a concentration range from 3 to 0.01 nM to neutralize huIL-17A, in a concentration range from 10 to 0.03 nM to neutralize huIL-17A/F and in a concentration range from 3 μM to 30 nM for huIL-17F. Simulect® was added in a concentration range between 3 μM to 100 nM. Culture supernatants were collected after an incubation of 24 h and huIL-6 production was measured by ELISA.
[0524] (ii) Assay on BJ Cells (Human Fibroblasts)
[0525] BJ cells (human skin fibroblasts from ATCC Cat# CRL 2522) were cultured in RPMI (Gibco Cat#61870-010) supplemented with 10% fetal calf serum ultra-low IgG (Gibco Cat#16250-078; lot 1074403), β-mercaptoethanol (5×10.sup.−5 M final) and Normocin (0.1 mg/ml; InvivoGen Cat# ant-nr-2). The cells were detached from plastic using an Accutase solution (PAA Cat# L11-007).
[0526] The cells were distributed into 96 well microtiter plates at a density of 5×10.sup.3 in 100 μl well in RPMI 1640 without fetal calf serum, fl-mercaptoethanol (5×10.sup.−5 M final) and Normocin (0.1 mg/ml). The BJ cells were allowed to adhere to the plates overnight. The next morning, different concentrations of rhuIL-17A (SEQ ID NO: 76; MW 32000), rhuIL-17A/F (MW 32800) and rhuIL-17F (SEQ ID NO: 77; MW 30000), or control medium in the presence of human TNFα (Novartis; MW 17500) were added in a volume of 50 μl to triplicate wells in the presence of 50 μl of different concentrations of test antibody (XAB4; XAB1), control antibody (Simulect® 1.1% solution, Batch # C0011; 831179), or control medium to reach the final volume of 200 μl/well and the final concentration of 2.5% fetal calf serum.
[0527] HuIL-17A (30 pM), huIL-17A/F (300 pM) and huIL-17F (10 nM) were added together with huTNFα (6 pM). XAB4 (MW 150000) was added in a concentration range from 1 to 0.003 nM to neutralize huIL-17A, in a concentration range from 10 to 0.03 nM to neutralize huIL-17A/F and in a concentration range from 3 μM to 30 nM for huIL-17F. XAB1 (MW 150000) was added in a concentration range from 3 to 0.01 nM to neutralize huIL-17A, in a concentration range from 10 to 0.03 nM to neutralize huIL-17A/F and in a concentration range from 3 μM to 30 nM for huIL-17F. Simulect® was added in a concentration range between 3 μM to 100 nM. Culture supernatants were collected after an incubation of 24 h and huIL-6 and huGROα production were measured by ELISA.
[0528] (iii) Detection Assays
[0529] 1) ELISA for Detection of Human IL-6 Production
[0530] ELISA microtiter plates were coated with an anti-human IL-6 mouse Mab (R&D Systems® Cat# MAB206; 100 μl/well at 1 μg/ml) in PBS 0.02% NaN.sub.3 and incubated overnight at +4° C. The following day, microtiter plates were blocked with 300 μl of PBS/2% BSA/0.02% NaN.sub.3 for 3 h at room temperature. Plates were then washed 4 times with PBS/0.05% Tween20/0.02% NaN.sub.3. Culture supernatants of C20A4C16 (final dilution 1:5 for cultures stimulated with huIL-17A plus huTNFα, or 1:2 for cultures stimulated with huTNFα plus huIL-17A/F or IL-17F; 100 μl/well) or BJ cells (final dilution 1:10 for cultures stimulated with huIL-17A plus huTNFα, or 1:5 for cultures stimulated with huTNFα plus huIL-17A/F or IL-17F; 100 μl/well) were added.
[0531] To establish a titration curve, rhuIL-6 (Novartis; 100 μl/well) was titrated from 500 pg/ml to 7.8 pg/ml in 1:2 dilution steps. After an overnight incubation at room temperature, plates were washed 4 times with PBS/0.05% Tween 20/0.02% NaN.sub.3. A biotin-conjugated goat anti-human IL-6 antibody was added (R&D Systems® Cat# BAF206; 30 ng/ml; 100 μl/well). Samples were left to react for 4 h at room temperature. After washing (4 times), alkaline phosphatase-conjugated streptavidin (Jackson Immunoresearch Cat#016-050-084) was added at a final dilution of 1/10000 (100 μl/well).
[0532] After 40 minutes at room temperature, plates were washed again 4 times. P-Nitrophenyl Phosphate substrate tablets (Sigma; 5 mg, Cat# N9389; 20 mg, Cat# N2765) were dissolved in diethanolamine buffer pH 9.8 to give a final concentration of 1 mg/ml. 100 μl were added to each well and the O.D. was read after 1 h in a Spectra Max M5 Microplate Reader (Molecular Devices) using filters of 405 and 490 nm.
[0533] 2) ELISA for Detection of Human GROα Production
[0534] ELISA microtiter plates were coated with an anti-human GROα mouse mAb (R&D Systems® Systems® Cat# MAB275; 100 μl/well at 1.5 μg/ml) in PBS/0.02% NaN.sub.3 and incubated overnight at 4° C. The following day, microtiter plates were blocked with 300 μl of PBS/2% BSA/0.02% NaN.sub.3 for 3 h at room temperature. Plates were then washed 4 times with PBS/0.05% Tween20/0.02% NaN.sub.3. Culture supernatants of BJ cells (final dilution 1:2; 100 μl/well) were added.
[0535] To establish a titration curve, human GROα (R&D Systems® Cat#275-GR/CF; 100 μl/well) was titrated from 2 ng/ml to 0.03 ng/ml in 1:2 dilution steps.) After an overnight incubation at room temperature, plates were washed 4 times with PBS/0.05% Tween 20/0.02% NaN.sub.3.
[0536] A biotin-conjugated goat anti-human GROα antibody was added (R&D Systems® Cat# BAF275; 100 ng/ml; 100 μl/well). Samples were left to react for 4 h at room temperature. After washing (4 times), alkaline phosphatase-conjugated streptavidin (Jackson Immunoresearch Cat#016-050-084) was added at a final dilution of 1/10000 (100 μl/well). After 40 minutes at room temperature, plates were washed again 4 times. P-Nitrophenyl Phosphate substrate tablets (Sigma; 5 mg Cat# N9389; 20 mg, Cat# N2765) were dissolved in diethanolamine buffer pH 9.8 to give a final concentration of 1 mg/ml. 100 μl were added to each well and the O.D. was read after 1 h in a Spectra Max M5 Microplate Reader (Molecular Devices) using filters of 405 and 490 nm.
[0537] 3) Calculations
[0538] Data are reported as Means+/− SEM. Four parameter curve fitting was used for ELISA calculations. IC.sub.50 values for inhibition of IL-6 and GRO-α secretion by antibodies were calculated using XIfit (FIT model 205).
[0539] (iv) Results
[0540] 1) Assay on C20A4C16 Cells (Human Chondrocyte Cell Line)
[0541] Both XAB4 and XAB1 are able to neutralize the induction of huIL-6 secretion by C20A4C16 cells stimulated with rhuIL-17A and rhuIL-17A/F in the presence of rhuTNFα. Control antibody (Simulect®) at 100 nM has no effect. IC.sub.50 values (means±SEM) for XAB4 and XAB1 are reported in
[0542] Table 27. No inhibition on huIL-17F is observed even at Ab concentrations of 3 μM.
TABLE-US-00028 TABLE 27 Inhibitory effects of XAB4 and XAB1 on huIL-6 secretion by C20A4CI6 cells. XAB4 XAB1 IC50 (nM) IC50 (nM) Control (means ± (means ± antibody Stimuli SEM) SEM) (nM) rhuIL-17A (1 nM).sup.a 0.44 ± 0.06 >100 rhuIL-17A/F (3 nM).sup.a 1.30 ± 0.18 >100 rhuIL-17F (30 nM).sup.a >3000 >1000 rhuIL-17A (30 pM) + 0.024 ± 0.004 1.21 ± 0.09 >3000 rhuTNF-α (6 pM).sup.b rhuIL-17A/F (300 pM) + 0.108 ± 0.02 >10 >3000 rhuTNF-α (6 pM).sup.b rhuIL-17F (10 nM) + >3000 >3000 >3000 rhuTNF-α (6 pM).sup.b .sup.aBackground of hu IL-6 production without stimulation (0.13 ± 0.003) is subtracted .sup.bBackground of huIL-6 production in cultures with TNF alone (0.20 ± 0.003) is subtracted
[0543] From these experiments it is evident that the parental XAB1 antibody shares neutralizing activity with its derivatives. The XAB4 variant is also seen to have a higher neutralizing activity than XAB1.
[0544] In an additional experiment, analogous to the experiment described above, all the antibodies XAB1-XAB5 were compared, as seen in
[0545] Table 28. Here it can be seen that the inhibition profiles for XAB2, XAB3 and XAB5 are comparable to those observed for XAB4 and XAB1, especially to XAB4.
TABLE-US-00029 TABLE 28 Table Inhibitory effects of XAB antibodies on huIL-6 secretion by C20A4CI6 cells. XAB1 XAB2 XAB3 XAB4 XAB5 IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM) Means ± Means ± Means ± Means ± Means ± Stimuli SEM SEM SEM SEM SEM rhuIL-17A 0.29 ± 0.72 ± 0.63 ± 0.51 ± 0.55 ± (0.5 nM).sup.a 0.03 0.08 0.15 0.04 0.01 .sup.aBackground of HuIL-6 production without stimuli (0.04 ± 1.13 ng/ml) is subtracted.
[0546] 2) Assay on BJ Cells (Human Fibroblasts)
[0547] Both XAB4 and XAB1 neutralize the induction of huIL-6 and huGROα secretion by BJ cells stimulated with rhuIL-17A and rhuIL-17A/F in the presence of huTNFα. Control antibody (Simulect®) at 100 nM has no effect. IC.sub.50 values for inhibition of IL-6 and hu GROα are reported in
[0548] Table 29 and Table 30. Inhibition on huIL-17F is not observed even at Ab concentrations of 3 μM. From these experiments it is evident that the parental XAB1 antibody shares neutralizing activity with its derivatives.
[0549] The XAB4 variant is also seen to have a higher neutralizing activity than XAB1.
TABLE-US-00030 TABLE 29 Inhibitory effect of XAB4 and XAB1 on huIL-6 secretion by BJ cells. XAB4 XAB1 Control IC50 (nM) IC50 (nM) antibody Stimuli Means ± SEM Means ± SEM (nM) rhuIL-17A (1 nM).sup.a 0.63 ± 0.02 >100 rhuIL-17A/F (3 nM).sup.a 1.68 ± 0.05 >100 rhuIL-17F (30 nM).sup.a >3000 >1000 rhuIL-17A (30 pM) + 0.012 ± 0.002 0.47 ± 0.02 >3000 rhuTNF-α (6 pM).sup.b rhuIL-17A/F (300 pM) + 0.17 ± 0.01 3.83 ± 0.63 >3000 rhuTNF-α (6 pM).sup.b rhuIL-17F (10 nM) + >3000 >3000 >3000 rhuTNF-α (6 pM).sup.b .sup.aBackground of hu IL-6 production without stimuli (0.32 ± 0.002 ng/ml) is subtracted. .sup.bBackground of huIL-6 production in cultures stimulated with TNF alone (0.45 ± 0.02 ng/ml) is subtracted.
TABLE-US-00031 TABLE 30 Inhibitory effect of XAB4 and XAB1 on hu-GRO-alpha secretion by BJ cells. XAB4 XAB1 Control IC50 (nM) IC50 (nM) antibody Stimuli Means ± SEM Means ± SEM (nM) IL-17A (1 nM).sup.a 0.35 ± 0.01 >100 IL-17A/F (3 nM).sup.a 1.11 ± 0.05 >100 IL-17F (30 nM).sup.a >3000 >1000 IL-17A (30 pM) + 0.007 ± 0.0004 0.72 ± 0.12 >3000 TNF-α (6 pM).sup.b IL-17A/F (300 pM) + 0.1 ± 0.01 6.22 ± 0.44 >3000 TNF-α (6 pM).sup.b IL-17F (10 nM) + >3000 >3000 >3000 TNF-α (6 pM).sup.b .sup.aBackground of hu GROα production without stimuli (0.03 ± 0.01 ng/ml) is subtracted. .sup.bBackground of hu GROα production in cultures with TNF alone (0.15 ± 0.008 ng/ml) is subtracted.
[0550] In additional experiments, analogous to the experiments described above, all the antibodies XAB1-XAB5 were compared, as seen in Table 31 and Table 32. Here it can be seen that the inhibition profiles for XAB2, XAB3 and XAB5 are comparable to those observed for XAB4 and XAB1, especially to XAB4.
TABLE-US-00032 TABLE 31 Inhibitory effects of XAB antibodies on huIL-6 secretion by BJ cells. XAB1 XAB2 XAB3 XAB4 XAB5 IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM) Means ± Means ± Means ± Means ± Means ± Stimuli SEM SEM SEM SEM SEM rhuIL-17A 4.97 ± 0.64 ± 0.50 ± 0.55 ± 0.54 ± (0.5 nM).sup.a 0.59 0.22 0.002 0.04 0.02 .sup.aBackground of HuIL-6 production without stimuli (0.15 ± 4.06 ng/ml) is subtracted
TABLE-US-00033 TABLE 32 Inhibitory effects of XAB antibodies on huGROα secretion by BJ cells. XAB1 XAB2 XAB3 XAB4 XAB5 IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM) Means ± Means ± Means ± Means ± Means ± Stimuli SEM SEM SEM SEM SEM rhuIL-17A 1.39 ± 0.40 ± 0.42 ± 0.44 ± 0.46 ± (0.5 nM).sup.a 0.07 0.06 0.01 0.04 0.05 .sup.aBackground of HuGROα production without stimuli (0.03 ± 0.02 ng/ml) is subtracted
Example 13. In Vitro Neutralization of Mouse IL-17A and IL-17A/F Activity by Antibody Variants of the Disclosure
[0551] CMT-93 cells (ATCC CCL-223) were cultured in RPMI (Gibco Cat#61870-010) supplemented with 10% fetal calf serum ultra-low IgG (Gibco Cat#16250-078; lot 1074403), β-mercaptoethanol (5×10.sup.−5 M final) and Normocin (0.1 mg/ml; InvivoGen Cat# ant-nr-2).
[0552] The cells were detached from plastic using an Accutase solution (PAA Cat# L11-007) and distributed into 96 wells microtiter plates at a density of 5×10.sup.3 in 100 μl well in RPMI 1640 without fetal calf serum, β-mercaptoethanol and normocin.
[0553] The cells were allowed to adhere to the plates overnight. The next morning, rmIL-17A (SEQ ID NO: 83, MW 31000) at 1 nM, rmIL-17A/F (R&D Systems® Cat#5390-IL; MW 30400) at 3 nM, rmIL-17F (SEQ ID NO: 84; MW 30000) at 30 nM, rratIL-17A (SEQ ID NO: 85; MW 31000) at 1 nM or control medium were added in a volume of 50 μl to triplicate wells in the presence of 50 μl of different concentrations of test antibodies (XAB4 or XAB1), control antibodies (Simulect® 1.1% solution; C0011, 831179) or control medium to reach the final volume of 200 μl/well and the final concentration of 1% fetal calf serum.
[0554] Culture supernatants were collected after an incubation of 24 h and KC production was measured by ELISA.
[0555] (i) ELISA for Detection of Mouse KC Production
[0556] ELISA microtiter plates were coated with a rat anti-mouse KC MAb (R&D Systems® Cat# MAB453; 100 μl/well at 1 μg/ml) in PBS/0.02% NaN.sub.3 and incubated overnight at 4° C. The following day, microtiter plates were blocked with 300 μl of PBS/2% BSA/0.02% NaN.sub.3 for 3 h at room temperature. Plates were then washed 4 times with PBS/0.05% Tween20/0.02% NaN.sub.3. Culture supernatants of CMT-93 cells (final dilution 1:5; 100 μl/well) were added.
[0557] To establish a titration curve, mouse KC (R&D Systems® #453-KC, 100 μl/well) was titrated from 1 ng/ml to 0.016 ng/ml in 1:2 dilution steps. After an overnight incubation at room temperature, plates were washed 4 times with PBS/0.05% Tween 20/0.02% NaN.sub.3. A biotin-conjugated goat anti-mouse KC antibody (R&D Systems® Cat# BAF453; 100 μl/well) at 0.1 μg/ml was added. Samples were left to react for 4 h at room temperature. After washing (4 times), alkaline phosphatase-conjugated streptavidin (Jackson Immunoresearch Cat#016-050-084) was added at a final dilution of 1/10000 (100 μl/well). After 40 minutes at room temperature, plates were washed again 4 times. P-Nitrophenyl Phosphate substrate tablets (Sigma; 5 mg Cat# N9389; 20 mg Cat# N2765) were dissolved in diethanolamine buffer pH 9.8 to give a final concentration of 1 mg/ml. 100 μl culture supernatants were added to each well and the O.D. was read after 1 h in a Spectra Max M5 Microplate Reader (Molecular Devices) using filters of 405 and 490 nm.
[0558] (ii) Calculations
[0559] Data are reported as Means+/−SEM. Four parameter curve fitting was used for ELISA calculations. IC.sub.50 values for inhibition of KC secretion by antibodies were calculated using XIfit™ (FIT model 205).
[0560] (iii) Results
[0561] Both XAB4 and XAB1 are able to neutralize the induction of mouse KC secretion by CMT-93 cells stimulated with mouse or rat IL-17A and mouse IL-17A/F. Control antibody (Simulect®) has no effect. IC.sub.50 values (means±SEM) for XAB4 and XAB1 are reported in Table 33. Inhibition on huIL-17F is not observed even at Ab concentrations of 10 μM.
TABLE-US-00034 TABLE 33 Inhibitory effect of XAB4 and XAB1 on mouse KC secretion by CMT-93 cells. XAB4 XAB1 IC50 (nM) IC50 (nM) Control Means ± Means ± antibody Stimuli SEM SEM (nM) mIL-17A (1 nM).sup.a 13.8 ± 0.48 539 ± 29.4 >3000 mIL-17A/F (3 nM).sup.a 10.3 ± 1.06 >1000 >3000 mIL-17F (30 nM).sup.a >10000 >10000 >3000 rIL-17A (1 nM).sup.a 6.7 ± 0.84 467 ± 25.1 >3000 .sup.aBackground of KC production without stimuli (0.07 ± 0.001 ng/ml) is subtracted.
[0562] From these experiments it is evident that both the parental XAB1 antibody, as well as its derivates, has neutralizing activity. The XAB4 variant is also seen to have a higher neutralizing activity than XAB1.
[0563] In an additional experiment, analogous to the experiment described above, all the antibodies XAB1-XAB5 were compared, as seen in Table 34. Here it can be seen that the inhibition profiles for XAB2, XAB3 and XAB5 are comparable to those observed for XAB4 and XAB1, especially to XAB4.
TABLE-US-00035 TABLE 34 Inhibitory effects of XAB antibodies on KC secretion by CMT-93 cells. XAB1 XAB2 XAB3 XAB4 XAB5 IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM) Means ± Means ± Means ± Means ± Means ± Stimuli SEM SEM SEM SEM SEM mIL-17A 128 ± 20.9 ± <1 7.0 ± 7.8 ± (0.15 nM).sup.a 14.2 0.96 0.29 0.78 .sup.aBackground of KC production without stimuli (0.19 ± 5.81 ng/ml) is subtracted.
Example 14. Rat Antigen-Induced Arthritis Assay (Rat AIA)
[0564] Female Lewis rats (120-150 g) were sensitized intradermally on the back at two sites to methylated bovine serum albumin (mBSA) homogenized 1:1 with complete Freund's adjuvant on days −21 and −14 (0.1 ml containing 5 mg/ml mBSA). On day 0, the rats were anaesthetized using a 5% isoflurane/air mixture and maintained using isoflurane at 3.5% via a face mask for the intra-articular injections. The right knee received 50 μl of 10 mg/ml mBSA in 5% glucose solution (antigen injected knee), while the left knee received 50 μl of 5% glucose solution alone (vehicle injected knee). The diameters of the left and right knees were then measured using calipers immediately after the intra-articular injections and again on days 2, 4, and 7.
[0565] Treatments were administered by single subcutaneous injection on day-3. The antibody of the disclosure was injected at 0.15, 1.5, 15 and 116 mg/kg. Right knee swelling was calculated as a ratio of left knee swelling, and the R/L knee swelling ratio plotted against time to give Area Under the Curve (AUC) graphs for control and treatment groups. The percentage inhibitions of the individual animals in each treatment group AUCs were calculated vs. the control group AUC (0% inhibition) using an Excel spreadsheet.
[0566] Results
[0567] Results are shown in Table 35. Dose related inhibition of right knee swelling was demonstrated for XAB4 with a calculated ED.sub.50 of 1.68 mg/kg s.c.
TABLE-US-00036 TABLE 35 Effects of single dose treatment with XAB4 on knee swelling from day 0 to day 7 in Lewis rat antigen-induced arthritis. Antibody dose Percentage inhibition of (mg/kg) knee swelling AUC 0.15 18.46 ± 1.61* 1.5 65.76 ± 3.41** 15 71.59 ± 1.27** 116 77.01 ± 1.72** Data points represent the means ± SEM of n = 5 animals. *p < 0.05 and **p < 0.01 ANOVA followed by Dunnett's test vs Control curve.
[0568] Similarly, dose related inhibition of knee swelling was demonstrated for XAB4 in a model using Wistar rats (data not shown), and in a model using mouse antigen-induced arthritis model (data not shown).
Example 15. Angiogenesis Mechanistic Model
[0569] Chambers containing human IL-17A (between 150 and 200 ng), when placed subcutaneously in a mouse, cause new blood vessel growth around the implant. The amount of angiogenesis correlates with the weight of newly formed tissue in this area. Prophylactic treatment with XAB4 at 0.01, 0.03, 0.1, 0.3, 1 and 3 mg/kg inhibited human IL-17 induced angiogenesis. The 5 higher doses all led to a potent and significant inhibition of tissue chamber weight. The 4 higher doses showed no dose dependency, however, the dose of 0.03 mg/kg was less efficacious than doses of 0.1 mg/kg and above.
[0570] This study demonstrates that the potent angiogenic effect of IL-17A can be neutralized with an anti-IL-17A antibody and provides experimental evidence of the effectiveness of XAB4 for human IL-17A in vivo.
Example 16. Experimental Autoimmune Encephalomyelitis (EAE) Model
[0571] The experimental autoimmune encephalomyelitis (EAE) model is a known animal model for multiple sclerosis (reviewed e.g in Constantinescu et al., Br J Pharmacol 2011). It has been shown that inhibition of IL-17 reduces EAE severity in C57Bl/6 mice (Haak S et al 2009, JCI; 119:61-69).
[0572] Female C57Bl/6 mice (aged 9 weeks, Harlan, Germany) were immunized with a 50/50 mixture of recombinant rat myelin oligodendrocyte glycoprotein peptide (MOG.sub.1-125) (generated in-house) and complete Freund's adjuvant (CFA, generated by adding 8 mg/ml Mycobacterium tuberculosis strain H37RA (Difco) to Incomplete Freund's adjuvant (IFA, Sigma). Immunization was performed by subcutaneous injection with 200 μg/animal of MOG.sub.1-125 at the base of tail on day 0. In addition, 200 ng/animal pertussis toxin (PT) was injected intraperitoneally on days 0 and 2.
[0573] Both therapeutic treatment effect and prophylactic treatment effect of XAB4 was tested.
[0574] Therapeutic Treatment
[0575] For the therapeutic treatment 16 mice were used (8 for XAB4 and 8 for control). Treatment was initiated once the animals had a clinical score of at least 2.5 (severe hind limb weakness) for 3 days. After this, 15 mg/kg XAB4 or isotype control antibody was injected subcutaneously each week with a single dose.
[0576] The results are shown in
[0577] Prophylactic Treatment
[0578] For the prophylactic treatment 19 mice were used (10 for XAB4 and 9 for control). Each animal was treated one day prior to immunization with 15 mg/kg XAB4 or isotype control, through a single subcutaneous injection. After this, 15 mg/kg XAB4 or isotype control antibody was injected subcutaneously each week with a single dose.
[0579] The results are shown in
[0580] Thus, it is shown that prophylactic treatment with XAB4 significantly delayed EAE onset and reduced maximal EAE severity.
Example 17. Attenuation of IL17A-Induced Levels of IL6, CXCL1, IL-8, GM-CSF, and CCL2 in Human Astrocytes
[0581] The effects of XAB4 on the levels of IL-6, CXCL1, IL-8, GM-CSF, and CCL2 in astrocytes isolated from the cerebral cortex of the human brain were investigated. Astrocytes release a number of growth factors, cytokines and chemokines that allow them to regulate cellular communication, migration and survival of neuronal, glial and immune cells. The direct communication of astrocytic end-feet with endothelial cells also allows astrocytes to control function of the blood-brain-barrier. Moreover, astrocytes release and uptake neurotransmitters, such as glutamate, at the synaptic cleft that allow them to regulate synaptic transmission and excitoxicity. It is significant that astrocytes form scar pathology after CNS injury, thus having apparent opposing roles in normal physiology and pathophysiology. In disease, astrocytes are suggested to play roles in a range of psychiatric, neurological and neurodegenerative disorders, where their role in neuroinflammation is likely to be important.
[0582] The data showed that co-stimulation with IL-17A and TNFα enhanced the release of IL-6, CXCL1, IL-8, GM-CSF, and CCL2, and that XAB4 inhibited levels of IL-6, CXCL1, IL-8, GM-CSF, and CCL2 in human astrocytes. These data indicate a dominate role for IL-17A in cytokine release from astrocytes and support their use as drug targets for neuroinflammatory diseases. It is noteworthy that the pretreatment of human astrocytes with XAB4 inhibited IL-17A-induced and IL-17A/TNFα-induced, without affecting TNFα-induced, levels of IL-6, CXCL1, IL-8, GM-CSF, and CCL2. Taken together, the data suggested that selective inhibition of IL-17A signaling with XAB4 attenuates the level of pro-inflammatory cytokines in human astrocytes. In disease, astrocytes are suggested to play roles in a range of psychiatric, neurological and neurodegenerative disorders, where their role in neuroinflammation is likely to be important. Novel drugs that alter astrocyte function are thus of potential value, where regulation of astrocyte function may prove therapeutically useful. Consequently, since XAB4 was shown to have an effect on IL-6, CXCL1, IL-8, GM-CSF, and CCL2 production of astrocytes, it can be concluded that XAB4 may be a useful therapeutic agent, such as for treatment of Multiple Sclerosis (MS).
[0583] Materials and Methods
[0584] All cytokines were purchased from R&D Systems. Basiliximab (Novartis, Basel, Switzerland) was used as isotype control. Primary antibodies used were: anti-IL17RA Alexa Fluor 647 (BG/hIL17AR, Biolegend), anti-IL17RC Alexa Fluor 488 (309822, R&D Systems, UK), anti-p65 (Santa Cruz, USA), mouse IgG Alexa Fluor 647 (MOPC-21, Biolegend, UK), mouse IgG Alexa Fluor 488 (133303, R&D System, UK), mouse IgG Biotin (G155-178, BD Biosciences, Switzerland) and rat IgG PE (A95-1, BD Biosciences, Switzerland). Secondary antibodies and dyes used were: biotinylated goat anti-rabbit IgG (BA1000, Vector, UK), streptavidin conjugated Alexa Fluor 488 and Alexa Fluor 633 (S11223 and S2137, Life Technology, USA), goat anti-mouse Alexa Fluor 488 and Alexa Fluor 633 (A1101 and A21050, Life Technology, USA), streptavidin BV421 (405226, Biolegend, UK), Hoechst 34580 (H21486, Life Technology, USA).
[0585] Human astrocytes derived from cerebral cortex were purchased from ScienCell Research Laboratory (USA) (catalogue number 1800). Cells were grown as per provider's instructions. Briefly cells were grown in human astrocyte media (ScienCell catalogue number 1801) supplemented with 1% astrocyte growth supplement (ScienCell catalogue number 1852), 5% fetal calf serum (ScienCell catalogue number 0010) and 1% Penicillin/Streptomycin (ScienCell catalogue number 0503). Cells were maintained in T75 culture flasks at 5% CO.sub.2 and 37° C. with the media changed every three days until 80% confluent. For all treatments, 70,000 cells well plated in 24-well plates, grown for 3 days, serum starved for 2-4 hr, after which astrocytes were treated for 2 hr with XAB4, and thereafter treated for 18-20 hr with recombinant human cytokines as indicated in the figure legends. The cell pellets were used to quantify mRNA levels of cytokines by qPCR and the supernatants were used to quantify the protein levels of cytokines by HTRF (Cisbio, France, used for IL-6, IL-8 & CXCL1) or AlphaLISA (PerkinElmer, USA, used for CCL2 & GM-CSF).
[0586] Measurement of cytokine mRNA was performed by real time-polymerase chain reaction (RT-PCR). Briefly, astrocytes were lysed for 5 min at room temperature by gently shaking in 350 μl lysis buffer (RLT buffer with 1% β-mercaptoethanol) and total RNA was extracted using RNeasy Microkit (74004, Qiagen, Switzerland). The cDNA was synthesized using SuperScript III reverse transcriptase (18080-400, Life Technology, Switzerland). The expression level of each gene was assessed by q-PCR in a Viia7 Real-time PCR machine (Life Technology, Switzerland). Taqman probes were purchased from Life Technology, Switzerland. Each sample was analyzed in triplicate and normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT). Levels of human IL6, IL8, CXCL1 protein (ng/ml) in human astrocyte supernatant (10 μl) were assessed by HTRF (IL6: 62IL6PEC; IL8: 62IL8PEC; CXCL1: 6FGROPEG, Cisbio, France) and the level of human CCL2 protein (ng/ml) in human astrocyte supernatant (5 μl) was assessed by AlphaLISA human CCL2/MCP1 (AL244C, PerkinElmer, USA). All measurements were performed according to manufacturer's instructions.
[0587] Cells suspensions of human astrocytes were obtained from adherent cultures using PBS-5 mM EDTA. For extracellular staining cells were incubated with whole mouse IgG for 10 min at 4° C. in PBS 2% BSA, and then stained with antibodies for 30 min at 4° C. in PBS 2% BSA. For intracellular staining, cells were permeabilized with Cytofix/Cytoperm solution (554714, BD Biosciences, Switzerland) for 20 min at 4° C. before incubating with antibodies for 30 min at 4° C. After filtration through 70 μm strainer, cells were acquired on a BDFortessa (BD Biosciences, Switzerland) and data analyzed using FlowJo software (Tree Star Inc., USA).
[0588] After compound treatment, cells were washed in PBS (Sigma Aldrich, Germany) followed by fixation in ice-cold 100% methanol for 10 min. Cells were washed 3×5 min in sterile PBS then permeabilized by incubation with 0.2% Triton-X-100 (Sigma Aldrich, Germany) in PBS for 5 min at room temperature. Non-reactive sites were blocked overnight at +4° C. with blocking buffer which consisted of 10% normal goat serum (Life Technology, USA) and 2% bovine serum albumin (Sigma Aldrich, Germany) in PBS. The cells were then incubated in primary antibody overnight at 4° C. The primary antibody was removed and the cells washed 3×5 min PBS after which the secondary fluorescent antibody was applied for 2 hr at room temperature. The coverslips were then washed 5×5 min in PBS and counter stained with Hoescht 34580 nuclear stain. The coverslips were finally mounted on microscope slides in VectashieldR mounting medium (Vector, UK) and the edges of the coverslip sealed with nail varnish. The cells were imaged using a Zeiss LSM 510 META confocal laser scanning microscope utilizing an Axiovert 200M inverted microscope (Zeiss Ltd, Germany).
[0589] Results
[0590] Antagonism of TNF-α or IL-17A stimulation, or IL-17A/TNF-α co-stimulation by XAB4 is shown in
[0591]
[0592] Primary human astrocytes were treated with increasing concentrations of XAB4 (0.01 nM, 0.1 nM 1 nM and 10 nM), with or without IL-17A (50 ng/ml), TNF-α (10 ng/ml), IL-1β, IL-17A/TNF-α and IL-1β/TNF-α. All concentrations used are indicated in the figures. The data shown is a representative of two experiments for XAB4 0.01 nM, and of three experiments for XAB4 0.1 nM, 1 nM and 10 nM. Values shown are means±S. E. M.
[0593] As seen in
[0594] As seen in
[0595] As seen in
[0596] As seen in
[0597] As seen in
[0598] Taken together, the data suggested that selective inhibition of IL-17A signaling with XAB4 attenuates the level of pro-inflammatory cytokines in human astrocytes. In disease, astrocytes are suggested to play roles in a range of psychiatric, neurological and neurodegenerative disorders, where their role in neuroinflammation is likely to be important. Since XAB4 was shown to have an effect on IL-6, CXCL1, IL-8, GM-CSF, and CCL2 production of astrocytes, XAB4 may be a useful therapeutic agent, such as for treatment of Multiple Sclerosis (MS).
Example 18: Generation of Additional Proteins and Nucleic Acids Linked to an HA-Binding Peptide Tag
[0599] To test the ability of the HA-binding peptide tags to extend the half-life of proteins or nucleic acids in the eye, the peptide tags of the invention were linked to antibodies, proteins and nucleic acids which bind IL-17A.
Generation of Peptide Tagged Antibodies and Proteins
[0600] Tagged and untagged recombinant antibodies and proteins were expressed by transient transfections of mammalian expression vectors in HEK293 cells and purified using standard affinity resins for example, KappaSelect (Cat #17-5458-01, GE Healthcare Biosciences®) and HisTrap (Cat #17-5255-01, GE Healthcare Biosciences®). Various antibody and protein formats were tested, including: Fabs, IgGs, Fc Traps and proteins. These antibodies and proteins target, for example, IL-17A.
[0601] Fabs linked to single peptide tags were generated as described above by linking the HA-binding tag sequence to the C-terminal of the heavy chain of a Fab using a GSGGG linker (e.g.: SEQ ID NO: 102). To generate peptide tagged IgGs (e.g.: IgG fusions that contain HA-binding tag sequences) the HA-binding tag sequence was fused to the C-terminal of the heavy chain or light chain of an IgG using a GSGGG linker (e.g.: SEQ ID NO: 102). To generate peptide tagged proteins than contain an Fc portion, for example, Fc trap protein linked to an HA-binding tag, the HA-binding tag was linked to the C-terminal of the Fc portion of the protein using a GSGGG linker (e.g.: SEQ ID NO: 102). To generate additional peptide tagged proteins, the HA-binding tag was linked to the C-terminus of the protein of interest using a GSGGG linker (e.g.: SEQ ID NO: 102). In all cases described above, production of candidates entails nucleotide synthesis encoding the amino acid of desired proteins followed by expression and purification using mammalian expression systems described above.
[0602] The peptide tagged antibodies and peptide antigen binding fragments exemplified herein may also be converted and used in alternate antibody formats. For example, peptide tagged IgGs, can be converted to peptide tagged Fabs or peptide tagged scFvs, or vice versa.
TABLE-US-00037 TABLE 36 Example of protein linked to a peptide tag that binds HA. NVS ID Target HA Tag Format Location of HA tag XAB4 IL-17A None Fab None XAB4T IL-17A SEQ ID Fab C-terminus of XAB4 NO: 33 heavy chain
Example 19: Further Characterization of the Peptide Tagged Protein of Example 18
19a: Biacore Affinity Determination
[0603] Affinity of peptide tagged protein and the parental untagged protein were analyzed on Biacore to determine kinetics for their primary targets as described above (ex: IL-17) as well as for HA binding. In order to determine HA kinetics, biotinylated HA was used in a BIOCAP Biacore format in which biotinylated HA is captured and the sample proteins flowed over at various concentrations. Biotinylated target ligands and biotinylated-HA were used in affinity measurements: Biacore Affinity Determination.
Target Kinetics and Affinity Using the Anti-Fab Method:
[0604] For the anti-Fab capture method, the Human Fab Capture® kit from GE® was used (GE 28958325). Refer to the catalog number more detailed information. For this method, HBS-EP+ running buffer (teknova H8022) was used. A CM5 chip (GE®, BR-1005-30) was used and to this the anti-Fab polyclonal was immobilized to achieve approximately 5,000 RU according to the GE® protocol. Refer to the catalog number on the GE® website to get more detailed information. Two flow cells were used for this method. Flow cell 1 served as the reference cell which only contained the immobilized anti-fab reagent and flow cell 2 served as the binding cell which contained both the anti-fab reagent and the protein samples. The protein samples tested in this method were against C5, Factor P and EPO specific. The protein samples were captured at a flow rate of 10 μl/min for a specific contact time in order to achieve an RU signal for an Rmax of 20. Since the protein analytes have strong affinities for their targets, the starting concentrations of the target analytes started at approximately 10 nM and would include 8 serial dilution points. The target analytes were flowed over at 60 ul/min for 240 seconds with short and longer dissociations times greater than 1000 seconds depending on the sample.
TABLE-US-00038 TABLE 37 Binding affinity of peptide tagged molecule. Both HA and protein target binding was measured. NVS ID Ligand ka (1/M*s) kd (1/s) Affinity (M) Temperature XAB4T 17 kDa 4.82E+06 2.19E−01 4.54E−08 25° C. HA-biotin IL17A-biotin 4.51E+06 3.46E−05 7.68E−12 25° C.
[0605] Protein linked with the HA-binding peptide tag exhibited similar HA binding affinity and retained binding to their primary target (Table 37). In fact, the presence of the peptide tag improved the molecule's primary target binding affinity compared to the untagged molecule.
19b: Rabbit Traditional Ocular PK Determination
[0606] Ocular terminal concentrations of antibodies, Fc traps, and proteins linked to an HA-binding peptide tag in rabbit vitreous were compared to their untagged versions using standard methods as described below and shown in Table 38.
[0607] 5 μg/eye (˜105 pmoles) un-tagged antibody and 6.2 ug/eye (˜105 pmoles) of tagged antibody were injected intravitreally into rabbit eyes (N=6 eyes per antibody). Rabbits were sacrificed 21 days after injection and eyes were enucleated. The enucleated eyes were dissected and the vitreous was separated from other tissues and further homogenized mechanically using a TissueLyzer (QIAGEN®). Antibody levels in the vitreous were measured by ELISA or mass spectrometry.
ELISA Method
[0608] The Maxisorp 384 well plates (Nunc 464718) were coated with a Goat Anti-Human IgG (H+L) (Thermo Fisher 31119) in carbonate buffer (Pierce 28382) overnight at 4 C. In between incubations, plates were washed 3 times with TBST (THERMO SCIENTIFIC® 28360) using a BioTek plate washer. The next day, the plates were blocked for 2 hours at room temperature (or overnight at 4 C) with blocking buffer (5% BSA (SIGMA® A4503), 0.1% Tween-20 (SIGMA® P1379), 0.1% Triton X-100 (SIGMA® P234729) in TBS. Samples were diluted in diluent (2% BSA (SIGMA® A4503), 0.1% Tween-20 (SIGMA® P1379), 0.1% Triton X-100 (SIGMA® P234729) in TBS). Samples were incubated on the plate for 1 hour at room temperature with gentle shaking. The detection antibody was a Goat Anti-Human IgG [F(ab′)2]) conjugated to HRP (Thermo Fisher 31414). The detection antibody was added to the plates for 30 minutes at room temperature with gentle shaking. Ultra TMB was added for 15 minutes (Thermo Fisher 34028). The reaction was quenched with 2N sulfuric acid (Ricca 8310-32). The absorbance of the samples was read on the SpectraMax (450-570 nm). To back-calculate Fab recovery levels from eye tissues, a purified standard was used. For the standard, the top concentration used was 200 ng/mL with 2-fold dilutions. Different pairs of antibodies can be used for Fab recovery from rabbit tissues.
Mass Spectrometry Method
Reduction, Alkylation and Digestion:
[0609] 60 uL of vitreous sample in each well was thawed at room temperature for 10 minutes. 150 uL of 8M Urea (FisherScientific®, Cat No. U15-500) in 50 mM Tris-HCl (Fisher Scientific®, BP153-500) was added to each sample well, followed by addition of 4 uL of 2M DTT (SigmaAldrich®, Cat. No. D9779) to a final concentration of 40 mM DTT. The plate was heated at 58 deg C. for 45 minutes to denature the proteins. Subsequently, cool the plate to room temperature, then add 8 uL of 1M Iodoacetamide (SigmaAldrich®, Cat. No. 11149) for a final concentration of 40 mM and incubate at room temperature for 45 minutes in the dark. Dilute final concentration of urea to below 2M by adding 1.3 mL of 50 mM ammonium bicarbonate (Fisher Scientific®, Cat. No. BP2413-500). Add 10 uL of 0.1 ug/uL trypsin (Promega®, Cat. No. V5111) and incubate at 37° C. overnight.
SPE Cleanup and Filtration
[0610] After digestion, formic acid (Fluka, Cat. No. 56302-50ML-F) was added to each sample to a final concentration of 1% (v/v) to quench trypsin digestion. Oasis® MCX plate (Waters, Cat. No. 186000259) was used to clean up the digested sample. The collected sample solution from cleanup was dried down completely using SpeedVac (ThermoFisher Savant). Once the sample was dried, 60 uL of buffer (0.1% formic acid, 1% ACN (Sigma Aldrich, Cat. No. 34998-4L) and 20 pg/uL heavy labeled internal standard (custom made by ThermoFisher) solution was added to each well, and the plate was shaked for 20 minutes. The reconstituted peptide solution was filtered using AcroPrep™ advanced 96-well filter plates for ultrafiltration (Pall Life Sciences, Cat. No. 8164) filter with 10 KDa MWCO.
LC-MS/MS Analysis:
[0611] 5 uL of each filtered samples was loaded to a 300 um×150 mm Symmetry® C18 column (Waters®, Cat. No. 186003498). Separation was achieved by applying a 5 min gradient from 5% B (acetonitrile in 0.1% formic acid) to 20% B with a flow rate of 5 uL/min. Two peptides (HC_T3: GPSVFPLAPSSK and DDA2: TGIIDYGIR), and two transitions for each peptide (HC_T3: 594.19/699.82 and 594.19/847; DDA2: 504.58/623.68 and 504.58/736.84) were monitored for each sample using Waters Xevo TQS mass spectrometer (Waters). For Eylea and Eylea containing constructs, two transitions (560.28/697.76 and 560.28/709.28) from FNWYVDGVEVHNAK were monitored on the same mass spectrometer using the same LC columns and conditions. Drug molecules containing these peptides were quantified using MS signals resulted from these transitions.
Gyrolab Method
Sample Preparation
[0612] Vitreous samples were thawed at room temperature for 10 minutes. 5 uL of vitreous sample was then diluted 1:2 in Rexxip AN Buffer (Gyros AB®, Inc. Cat P0004994) in a 96-well PCR plate (Thermo Scientific® AB-800, 0.2 mL Skirted 96-well PCR plate). Samples were sealed (Gyros AB®, Inc. microplate foil Cat P0003313) and mixed thoroughly in a plate shaker for 1 minute. Ensuring that no bubbles are found in the bottom of the wells, the samples were placed in the Gyrolab™ xP workstation. A 3-step C-A-D method is executed on the Gyrolab™ xP workstation; capture antibody was flowed through the system first, followed by the analyte (samples), and then the detector antibody. The Gyrolab™ xP workstation performs washes of PBS 0.01% Tween20 (Calbiochem®, Inc. Cat 655206) inbetween each step. The standard curve for free Fc drug measurement was prepared in a diluent containing 50% rabbit vitreous (BioReclamation®, LLC. Cat Rabb-Vitreous) in Rexxip AN. The standard was serially diluted 1:6 from 6000 ng/mL to 0.129 ng/mL. The standard curve for Fab drug measurement was prepared in a diluent containing 10% rabbit vitreous (BioReclamation®, LLC. Cat Rabb-Vitreous) in Rexxip AN. The standard was serially diluted 1:6 from 6000 ng/mL to 0.129 ng/m L.
Detection of Fabs
[0613] Total and free purified drug constructs were analyzed in the Gyrolab™ xP workstation using a Bioaffy1000 CD (Gyros AB, Inc. Cat P0004253). Gyros AB
[0614] Free drug was measured by applying 100 ug/mL biotin-labeled VEGF (Novartis) to a column containing streptavidin coated particles. Vitreous samples were applied to the activated columns and detected by capillary action with 25 nM alexafluor-647 labeled goat anti-Human IgG-heavy and light chain antibody (Bethyl Laboratories®, Cat A80-319A). Note that alexafluor labeling was performed using Life Technologies labeling kit (Cat A-20186). The capture reagent was prepared in PBS 0.01% Tween20 and the detector reagent in Rexxip F (Gyros AB®, Inc. P0004825).
[0615] Total drug was measured by applying 100 ug/mL biotin-labeled goat anti-Human IgG-heavy and light chain antibody (Bethyl Laboratories®, Cat A80-319B). Vitreous samples were applied to the activated columns and detected by capillary action with 10 nM alexafluor-647 labeled goat anti-Human IgG-heavy and light chain antibody (Bethyl Laboratories®, Cat A80-319A).
Detection of Fc Proteins Total and free purified drug constructs were analyzed in the Gyrolab™ xP workstation using a Bioaffy1000 CD (Gyros AB, Inc. Cat P0004253). Free drug was measured by applying 100 ug/mL biotin-labeled VEGF (Novartis) to a column containing streptavidin coated particles. Vitreous samples are applied to the activated columns and detected by capillary action with 25 nM alexafluor-647 labeled anti-Human Fc-specific antibody (R10, Novartis). Total drug was measured by applying 25 ug/mL biotin-labeled goat anti-Human IgG-heavy and light chain antibody (Bethyl Laboratories®, Cat A80-319B). Vitreous samples were applied to the activated columns and detected by capillary action with 12.5 nM alexafluor-647 labeled goat anti-Human IgG-heavy and light chain antibody (Bethyl Laboratories, Cat A80-319A).
Detection of DARPins
[0616] Free purified drug constructs were analyzed in the Gyrolab™ xP workstation using a Bioaffy1000 CD (Gyros AB, Inc. Cat P0004253). Free drug was measured by applying 25 ug/mL biotin-labeled VEGF (Novartis) to a column containing streptavidin coated particles. Vitreous samples are applied to the activated columns and detected by capillary action with 6.25 nM alexafluor-647 labeled Penta HIS antibody (Qiagen®, Cat 35370).
TABLE-US-00039 TABLE 38 Terminal vitreal concentrations and calculated 2- point ocular half-life (t½) values. Terminal Terminal vitreal conc. 2-point vitreal conc. 2-point by ELISA ocular t.sub.1/2 by MS ocular t.sub.1/2 NVS ID (ng/ml) (days) (ng/ml) (days) XAB4T 92.5 3.47 466 5.6
[0617] Fusing an HA-binding peptide tag (SEQ ID NO: 33) to antigen binding fragments including XAB4, resulted in higher ocular terminal concentrations of these molecules as compared to untagged Fabs and proteins. These data indicate that the fusion of the HA-binding peptide tag confers improvement in and ocular half-life (t½) independent of the molecule it is fused to. Consequently, fusion of an HA-binding peptide tag appears to universally increase the ocular retention and ocular half-life of molecules administered intravitreally.
Sequence Information
[0618] Sequence data relating to XAB1, XAB2, XAB3, XAB4 and XAB5 is summarized below for ease of reference.
[0619] Table 1 describes the amino acid sequences (SEQ ID NOs) of the full length heavy and light chains of examples XAB1, XAB2, XAB3, XAB4 and XAB5.
[0620] The antibodies XAB1, XAB2, XAB3, XAB4 or XAB5 can be produced using conventional antibody recombinant production and purification processes. For example, the coding sequences as described in Table 3 or Table 4 are cloned into a production vector for recombinant expression in mammalian production cell line.
[0621] Table 2 summarizes the variable heavy (VH) and light chain (VL) amino acid sequence of XAB1, XAB2, XAB3, XAB4 or XAB5, which can be used to generate chimeric antibodies from XAB1, XAB2, XAB3, XAB4 or XAB5.
[0622] Table 5 summarizes the useful CDR sequences of XAB1, XAB2, XAB3, XAB4 and XAB5 (plus consensus sequences) to generate alternative CDR grafted antibodies, wherein the CDR regions from XAB1, XAB2, XAB3, XAB4 and XAB5 are defined according to Kabat definition.
[0623] Table 6 summarizes the useful CDR sequences of XAB1, XAB2, XAB3, XAB4 and
[0624] XAB5 (plus consensus sequences) to generate alternative CDR grafted antibodies, wherein the CDR regions from XAB1, XAB2, XAB3, XAB4 and XAB5 are defined according to Chothia definition.
[0625] All the sequences referred to in this specification (SEQ ID NOs) are found in Table.
Sequence List
[0626] Useful amino acids and nucleotide sequences for practicing the invention are found in Table.
TABLE-US-00040 TABLE 39 Sequence list Sequence Antibody/ Identifier Amino acid sequence Fragment (SEQ ID NO:) or polynucleotide sequence (PN) XAB1, CDRH1 1 GFTFSSY (CHOTHIA) XAB1, CDRH2 2 KQDGSE (CHOTHIA) XAB1, CDRH3 3 DRGSLYY (CHOTHIA) XAB1, CDRL1 4 SQGIISA (CHOTHIA) XAB1, CDRL2 5 DAS (CHOTHIA) XAB1, CDRL3 6 FNSYPL (CHOTHIA) XAB1, CDRH1 7 SYWMS (KABAT) XAB1, CDRH2 8 NIKQDGSEKYYVDSVKG (KABAT) XAB1, CDRH3 3 DRGSLYY (KABAT) XAB1, CDRL1 9 RPSQGIISALA (KABAT) XAB1, CDRL2 10 DASSLEN (KABAT) XAB1, CDRL3 11 QQFNSYPLT (KABAT) XAB1, VH 12 EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMSQVRQ APGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNS LYLQMNSLRAEDTAVYYCARDRGSLYYWGQGTLVTVSS XAB1, VL 13 AIQLTQSPSSLSASVGDRVTITCRPSQGIISALAWYQQK PGKAPKLLIYDASSLENGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQFNSYPLTFGGGTKVEIK XAB1, HEAVY CHAIN 14 EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMSWVRQ APGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNS LYLQMNSLRAEDTAVYYCARDRGSLYYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK XAB1, LIGHT CHAIN 15 AIQLTQSPSSLSASVGDRVTITCRPSQGIISALAWYQQK PGKAPKLLIYDASSLENGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC PN ENCODING 16 GAGGTGCAGCTGGTCGAGTCTGGCGGCGACCTGGTGCAG SEQ ID NO: 12 CCTGGCGGCAGCCTGAGACTGAGCTGCGCCGCCAGCGGC TTCACCTTCAGCAGCTACTGGATGTCCTGGGTCCGCCAG GCCCCTGGCAAAGGCCTCGAATGGGTGGCCAACATCAAG CAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAG GGCCGGTTCACCATCAGCCGGGACAACGCCAAGAACAGC CTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACC GCCGTGTACTACTGCGCCAGGGACCGGGGCAGCCTGTAC TATTGGGGCCAGGGCACCCTGGTCACCGTGTCCAGC PN ENCODING 17 GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCA SEQ ID NO: 13 TCTGTGGGAGACAGAGTCACCATCACTTGCCGGCCAAGT CAGGGCATTATCAGTGCTTTAGCCTGGTATCAGCAGAAA CCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCC AGTTTGGAAAATGGGGTCCCATCAAGGTTCAGCGGCAGT GGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTG CAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTT AATAGTTACCCTCTCACTTTCGGCGGAGGGACCAAGGTG GAGATCAAA PN ENCODING 18 GAGGTGCAGCTGGTCGAGTCTGGCGGCGACCTGGTGCAG SEQ ID NO: 14 CCTGGCGGCAGCCTGAGACTGAGCTGCGCCGCCAGCGGC TTCACCTTCAGCAGCTACTGGATGTCCTGGGTCCGCCAG GCCCCTGGCAAAGGCCTCGAATGGGTGGCCAACATCAAG CAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAG GGCCGGTTCACCATCAGCCGGGACAACGCCAAGAACAGC CTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACC GCCGTGTACTACTGCGCCAGGGACCGGGGCAGCCTGTAC TATTGGGGCCAGGGCACCCTGGTCACCGTGTCCAGCGCT AGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGC AGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGC CTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCC TGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTC CCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCC AGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAG ACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACC AAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAG ACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTG GGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAG GACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGC GTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAG TTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCC AAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTAC AGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGG CTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAG GCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGCC AAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCC CCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTG ACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCC GTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTAC AAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTC TTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGG CAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAG GCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTG TCCCCCGGCAAG PN ENCODING 19 GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCA SEQ ID NO: 15 TCTGTGGGAGACAGAGTCACCATCACTTGCCGGCCAAGT CAGGGCATTATCAGTGCTTTAGCCTGGTATCAGCAGAAA CCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCC AGTTTGGAAAATGGGGTCCCATCAAGGTTCAGCGGCAGT GGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTG CAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTT AATAGTTACCCTCTCACTTTCGGCGGAGGGACCAAGGTG GAGATCAAACGTACGGTGGCCGCTCCCAGCGTGTTCATC TTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCC AGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAG GCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGC GGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAG GACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGC AAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAG GTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGC TTCAACAGGGGCGAGTGC XAB2, CDRH1 1 GFTFSSY (CHOTHIA) XAB2, CDRH2 2 KQDGSE (CHOTHIA) XAB2, CDRH3 3 DRGSLYY (CHOTHIA) XAB2, CDRL1 20 SQVIISA (CHOTHIA) XAB2, CDRL2 5 DAS (CHOTHIA) XAB2, CDRL3 21 FDSYPL (CHOTHIA) XAB2, CDRH1 7 SYWMS (KABAT) XAB2, CDRH2 8 NIKQDGSEKYYVDSVKG (KABAT) XAB2, CDRH3 3 DRGSLYY (KABAT) XAB2, CDRL1 22 RPSQVIISALA (KABAT) XAB2, CDRL2 23 DASSLEQ (KABAT) XAB2, CDRL3 24 QQFDSYPLT (KABAT) XAB2, VH 12 EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMSWVRQ APGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNS LYLQMNSLRAEDTAVYYCARDRGSLYYWGQGTLVTVSS XAB2, VL 25 AIQLTQSPSSLSASVGDRVTITCRPSQVIISALAWYQQK PGKAPKLLIYDASSLEQGVPSRFSGSVSGTDFTLTISSL QPEDFATYYCQQFDSYPLTFGGGTKVEIK XAB2, HEAVY CHAIN 14 EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMSWVRQ APGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNS LYLQMNSLRAEDTAVYYCARDRGSLYYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK XAB2, LIGHT CHAIN 15 AIQLTQSPSSLSASVGDRVTITCRPSQVIISALAWYQQK PGKAPKLLIYDASSLEQGVPSRFSGSVSGTDFTLTISSL QPEDFATYYCQQFDSYPLTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC PN ENCODING 16 GAGGTGCAGCTGGTCGAGTCTGGCGGCGACCTGGTGCAG SEQ ID NO: 12 CCTGGCGGCAGCCTGAGACTGAGCTGCGCCGCCAGCGGC TTCACCTTCAGCAGCTACTGGATGTCCTGGGTCCGCCAG GCCCCTGGCAAAGGCCTCGAATGGGTGGCCAACATCAAG CAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAG GGCCGGTTCACCATCAGCCGGGACAACGCCAAGAACAGC CTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACC GCCGTGTACTACTGCGCCAGGGACCGGGGCAGCCTGTAC TATTGGGGCCAGGGCACCCTGGTCACCGTGTCCAGC PN ENCODING 17 GCCATCCAGCTGACCCAGAGCCCCAGCAGCCTGAGCGCC SEQ ID NO: 25 AGCGTGGGCGACAGAGTGACCATCACCTGTCGGCCCAGC CAGGTCATCATCAGCGCCCTGGCCTGGTATCAGCAGAAG CCTGGCAAGGCCCCCAAGCTGCTGATCTACGACGCCAGC TCCCTGGAACAGGGCGTGCCCAGCCGGTTCAGCGGCAGC GTGTCCGGCACCGACTTCACCCTGACCATCAGCTCCCTG CAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTTC GACAGCTACCCCCTGACCTTCGGCGGAGGCACCAAGGTG GAAATCAAG PN ENCODING 18 GAGGTGCAGCTGGTCGAGTCTGGCGGCGACCTGGTGCAG SEQ ID NO: 14 CCTGGCGGCAGCCTGAGACTGAGCTGCGCCGCCAGCGGC TTCACCTTCAGCAGCTACTGGATGTCCTGGGTCCGCCAG GCCCCTGGCAAAGGCCTCGAATGGGTGGCCAACATCAAG CAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAG GGCCGGTTCACCATCAGCCGGGACAACGCCAAGAACAGC CTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACC GCCGTGTACTACTGCGCCAGGGACCGGGGCAGCCTGTAC TATTGGGGCCAGGGCACCCTGGTCACCGTGTCCAGCGCT AGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGC AGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGC CTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCC TGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTC CCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCC AGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAG ACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACC AAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAG ACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTG GGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAG GACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGC GTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAG TTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCC AAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTAC AGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGG CTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAG GCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGCC AAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCC CCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTG ACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCC GTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTAC AAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTC TTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGG CAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAG GCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTG TCCCCCGGCAAG PN ENCODING 28 GCCATCCAGCTGACCCAGAGCCCCAGCAGCCTGAGCGCC SEQ ID NO: 26 AGCGTGGGCGACAGAGTGACCATCACCTGTCGGCCCAGC CAGGTCATCATCAGCGCCCTGGCCTGGTATCAGCAGAAG CCTGGCAAGGCCCCCAAGCTGCTGATCTACGACGCCAGC TCCCTGGAACAGGGCGTGCCCAGCCGGTTCAGCGGCAGC GTGTCCGGCACCGACTTCACCCTGACCATCAGCTCCCTG CAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTTC GACAGCTACCCCCTGACCTTCGGCGGAGGCACCAAGGTG GAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATC TTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCC AGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAG GCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGC GGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAG GACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGC AAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAG GTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGC TTCAACAGGGGCGAGTGC ALTERNATIVE PN 29 GAGGTGCAGCTGGTGGAATCAGGAGGCGACCTGGTGCAG ENCODING CCTGGCGGCTCACTGAGACTGAGCTGCGCCGCTAGTGGC SEQ ID NO: 12 TTCACCTTTAGTAGCTACTGGATGAGCTGGGTGCGACAG GCCCCTGGCAAGGGACTGGAGTGGGTGGCCAATATTAAG CAGGACGGCTCAGAGAAGTACTACGTGGACTCAGTGAAG GGCCGGTTCACTATTAGCCGGGATAACGCTAAGAATAGC CTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACC GCCGTGTACTACTGCGCTAGAGATAGAGGCTCACTGTAC TACTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCT ALTERNATIVE PN 30 GCTATTCAGCTGACTCAGTCACCTAGTAGCCTGAGCGCT ENCODING AGTGTGGGCGATAGAGTGACTATCACCTGTAGACCTAGT SEQ ID NO: 25 CAGGTGATCATTAGCGCCCTGGCCTGGTATCAGCAGAAG CCCGGCAAGGCCCCTAAGCTGCTGATCTACGACGCTAGT AGTCTGGAACAGGGCGTGCCCTCTAGGTTTAGCGGCTCA GTGTCAGGCACCGACTTCACCCTGACTATTAGTAGCCTG CAGCCCGAGGACTTCGCTACCTACTACTGTCAGCAGTTC GATAGCTACCCCCTGACCTTCGGCGGAGGCACTAAGGTG GAAATCAAG ALTERNATIVE PN 31 GAGGTGCAGCTGGTGGAATCAGGAGGCGACCTGGTGCAG ENCODING CCTGGCGGCTCACTGAGACTGAGCTGCGCCGCTAGTGGC SEQ NO: 14 TTCACCTTTAGTAGCTACTGGATGAGCTGGGTGCGACAG GCCCCTGGCAAGGGACTGGAGTGGGTGGCCAATATTAAG CAGGACGGCTCAGAGAAGTACTACGTGGACTCAGTGAAG GGCCGGTTCACTATTAGCCGGGATAACGCTAAGAATAGC CTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACC GCCGTGTACTACTGCGCTAGAGATAGAGGCTCACTGTAC TACTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCTGCT AGCACCAAGGGCCCAAGTGTCTTTCCCCTGGCCCCCAGC AGCAAGTCCACAAGCGGAGGCACTGCAGCTCTGGGTTGT CTGGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCC TGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTC CCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGC AGCGTCGTGACTGTGCCTAGTTCCAGCCTGGGCACCCAG ACCTATATCTGCAACGTGAACCACAAGCCCAGCAACACC AAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAG ACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTG GGAGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAG GACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGC GTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAG TTCAACTGGTACGTGGACGGGGTGGAGGTGCACAACGCC AAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTAC AGGGTGGTGTCCGTCCTGACAGTGCTGCACCAGGATTGG CTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAG GCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAGGCC AAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCC CCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTG ACCTGTCTGGTGAAGGGCTTCTACCCCAGCGATATCGCC GTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTAC AAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTC TTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGG CAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAG GCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTG AGCCCCGGCAAG ALTERNATIVE PN 32 GCTATTCAGCTGACTCAGTCACCTAGTAGCCTGAGCGCT ENCODING AGTGTGGGCGATAGAGTGACTATCACCTGTAGACCTAGT SEQ ID NO: 26 CAGGTGATCATTAGCGCCCTGGCCTGGTATCAGCAGAAG CCCGGCAAGGCCCCTAAGCTGCTGATCTACGACGCTAGT AGTCTGGAACAGGGCGTGCCCTCTAGGTTTAGCGGCTCA GTGTCAGGCACCGACTTCACCCTGACTATTAGTAGCCTG CAGCCCGAGGACTTCGCTACCTACTACTGTCAGCAGTTC GATAGCTACCCCCTGACCTTCGGCGGAGGCACTAAGGTG GAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATC TTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCC AGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAG GCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGC GGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAG GACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGC AAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAG GTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGC TTCAACAGGGGCGAGTGC XAB3, CDRH1 1 GFTFSSY (CHOTHIA) XAB3, CDRH2 2 KQDGSE (CHOTHIA) XAB3, CDRH3 3 DRGSLYY (CHOTHIA) XAB3, CDRL1 33 SQGIYWE (CHOTHIA) XAB3, CDRL2 5 DAS (CHOTHIA) XAB3, CDRL3 6 FNSYPL (CHOTHIA) XAB3, CDRH1 7 SYWMS (KABAT) XAB3, CDRH2 8 NIKQDGSEKYYVDSVKG (KABAT) XAB3, CDRH3 3 DRGSLYY (KABAT) XAB3, CDRL1 34 RPSQGIYWELA (KABAT) XAB3, CDRL2 23 DASSLEQ (KABAT) XAB3, CDRL3 11 QQFNSYPLT (KABAT) XAB3, VH 12 EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMSWVRQ APGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNS LYLQMNSLRAEDTAVYYCARDRGSLYYWGQGTLVTVSS XAB3, VL 35 AIQLTQSPSSLSASVGDRVTITCRPSQGIYWELAWYQQK PGKAPKLLIYDASSLEQGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQFNSYPLTFGGGTKVEIK XAB3, HEAVY CHAIN 14 EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMSWVRQ APGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNS LYLQMNSLRAEDTAVYYCARDRGSLYYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK XAB3, LIGHT CHAIN 36 AIQLTQSPSSLSASVGDRVTITCRPSQGIYWELAWYQQK PGKAPKLLIYDASSLEQGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC PN ENCODING 16 GAGGTGCAGCTGGTCGAGTCTGGCGGCGACCTGGTGCAG SEQ ID NO: 12 CCTGGCGGCAGCCTGAGACTGAGCTGCGCCGCCAGCGGC TTCACCTTCAGCAGCTACTGGATGTCCTGGGTCCGCCAG GCCCCTGGCAAAGGCCTCGAATGGGTGGCCAACATCAAG CAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAG GGCCGGTTCACCATCAGCCGGGACAACGCCAAGAACAGC CTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACC GCCGTGTACTACTGCGCCAGGGACCGGGGCAGCCTGTAC TATTGGGGCCAGGGCACCCTGGTCACCGTGTCCAGC PN ENCODING 37 GCCATCCAGCTGACCCAGAGCCCCAGCAGCCTGAGCGCC SEQ ID NO: 35 AGCGTGGGCGACAGAGTGACCATCACCTGTCGGCCCAGC CAGGGCATCTACTGGGAGCTGGCCTGGTATCAGCAGAAG CCTGGCAAGGCCCCCAAGCTGCTGATCTACGACGCCAGC TCCCTGGAACAGGGCGTGCCCAGCCGGTTCAGCGGCAGC GGATCCGGCACCGACTTCACCCTGACCATCAGCTCCCTG CAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTTC AACAGCTACCCCCTGACCTTCGGCGGAGGCACCAAGGTG GAAATCAAG PN ENCODING 18 GAGGTGCAGCTGGTCGAGTCTGGCGGCGACCTGGTGCAG SEQ ID NO: 14 CCTGGCGGCAGCCTGAGACTGAGCTGCGCCGCCAGCGGC TTCACCTTCAGCAGCTACTGGATGTCCTGGGTCCGCCAG GCCCCTGGCAAAGGCCTCGAATGGGTGGCCAACATCAAG CAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAG GGCCGGTTCACCATCAGCCGGGACAACGCCAAGAACAGC CTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACC GCCGTGTACTACTGCGCCAGGGACCGGGGCAGCCTGTAC TATTGGGGCCAGGGCACCCTGGTCACCGTGTCCAGCGCT AGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGC AGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGC CTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCC TGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTC CCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCC AGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAG ACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACC AAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAG ACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTG GGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAG GACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGC GTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAG TTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCC AAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTAC AGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGG CTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAG GCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGCC AAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCC CCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTG ACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCC GTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTAC AAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTC TTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGG CAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAG GCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTG TCCCCCGGCAAG PN ENCODING 38 GCCATCCAGCTGACCCAGAGCCCCAGCAGCCTGAGCGCC SEQ ID NO: 36 AGCGTGGGCGACAGAGTGACCATCACCTGTCGGCCCAGC CAGGGCATCTACTGGGAGCTGGCCTGGTATCAGCAGAAG CCTGGCAAGGCCCCCAAGCTGCTGATCTACGACGCCAGC TCCCTGGAACAGGGCGTGCCCAGCCGGTTCAGCGGCAGC GGATCCGGCACCGACTTCACCCTGACCATCAGCTCCCTG CAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTTC AACAGCTACCCCCTGACCTTCGGCGGAGGCACCAAGGTG GAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATC TTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCC AGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAG GCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGC GGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAG GACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGC AAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAG GTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGC TTCAACAGGGGCGAGTGC ALTERNATIVE PN 29 GAGGTGCAGCTGGTGGAATCAGGAGGCGACCTGGTGCAG ENCODING CCTGGCGGCTCACTGAGACTGAGCTGCGCCGCTAGTGGC SEQ ID NO: 12 TTCACCTTTAGTAGCTACTGGATGAGCTGGGTGCGACAG GCCCCTGGCAAGGGACTGGAGTGGGTGGCCAATATTAAG CAGGACGGCTCAGAGAAGTACTACGTGGACTCAGTGAAG GGCCGGTTCACTATTAGCCGGGATAACGCTAAGAATAGC CTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACC GCCGTGTACTACTGCGCTAGAGATAGAGGCTCACTGTAC TACTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCT ALTERNATIVE PN 39 GCTATTCAGCTGACTCAGTCACCTAGTAGCCTGAGCGCT ENCODING AGTGTGGGCGATAGAGTGACTATCACCTGTAGACCTAGC SEQ ID NO: 35 CAGGGAATCTACTGGGAGCTGGCCTGGTATCAGCAGAAG CCCGGCAAGGCCCCTAAGCTGCTGATCTACGACGCTAGT AGTCTGGAACAGGGCGTGCCCTCTAGGTTTAGCGGCTCA GGCTCAGGCACCGACTTCACCCTGACTATTAGTAGCCTG CAGCCCGAGGACTTCGCTACCTACTACTGTCAGCAGTTT AACTCCTACCCCCTGACCTTCGGCGGAGGCACTAAGGTG GAAATCAAG ALTERNATIVE PN 31 GAGGTGCAGCTGGTGGAATCAGGAGGCGACCTGGTGCAG ENCODING CCTGGCGGCTCACTGAGACTGAGCTGCGCCGCTAGTGGC SEQ ID NO: 14 TTCACCTTTAGTAGCTACTGGATGAGCTGGGTGCGACAG GCCCCTGGCAAGGGACTGGAGTGGGTGGCCAATATTAAG CAGGACGGCTCAGAGAAGTACTACGTGGACTCAGTGAAG GGCCGGTTCACTATTAGCCGGGATAACGCTAAGAATAGC CTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACC GCCGTGTACTACTGCGCTAGAGATAGAGGCTCACTGTAC TACTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCTGCT AGCACCAAGGGCCCAAGTGTCTTTCCCCTGGCCCCCAGC AGCAAGTCCACAAGCGGAGGCACTGCAGCTCTGGGTTGT CTGGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCC TGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTC CCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGC AGCGTCGTGACTGTGCCTAGTTCCAGCCTGGGCACCCAG ACCTATATCTGCAACGTGAACCACAAGCCCAGCAACACC AAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAG ACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTG GGAGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAG GACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGC GTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAG TTCAACTGGTACGTGGACGGGGTGGAGGTGCACAACGCC AAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTAC AGGGTGGTGTCCGTCCTGACAGTGCTGCACCAGGATTGG CTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAG GCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAGGCC AAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCC CCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTG ACCTGTCTGGTGAAGGGCTTCTACCCCAGCGATATCGCC GTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTAC AAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTC TTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGG CAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAG GCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTG AGCCCCGGCAAG ALTERNATIVE PN 40 GCTATTCAGCTGACTCAGTCACCTAGTAGCCTGAGCGCT ENCODING AGTGTGGGCGATAGAGTGACTATCACCTGTAGACCTAGC SEQ ID NO: 36 CAGGGAATCTACTGGGAGCTGGCCTGGTATCAGCAGAAG CCCGGCAAGGCCCCTAAGCTGCTGATCTACGACGCTAGT AGTCTGGAACAGGGCGTGCCCTCTAGGTTTAGCGGCTCA GGCTCAGGCACCGACTTCACCCTGACTATTAGTAGCCTG CAGCCCGAGGACTTCGCTACCTACTACTGTCAGCAGTTT AACTCCTACCCCCTGACCTTCGGCGGAGGCACTAAGGTG GAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATC TTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCC AGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAG GCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGC GGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAG GACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGC AAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAG GTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGC TTCAACAGGGGCGAGTGC XAB4, CDRH1 1 GFTFSSY (CHOTHIA) XAB4, CDRH2 2 KQDGSE (CHOTHIA) XAB4, CDRH3 3 DRGSLYY (CHOTHIA) XAB4, CDRL1 41 SQGINWE (CHOTHIA) XAB4, CDRL2 5 DAS (CHOTHIA) XAB4, CDRL3 6 FNSYPL (CHOTHIA) XAB4, CDRH1 7 SYWMS (KABAT) XAB4, CDRH2 8 NIKQDGSEKYYVDSVKG (KABAT) XAB4, CDRH3 3 DRGSLYY (KABAT) XAB4, CDRL1 42 RPSQGINWELA (KABAT) XAB4, CDRL2 23 DASSLEQ (KABAT) XAB4, CDRL3 11 QQFNSYPLT (KABAT) XAB4, VH 12 EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMSWVRQ APGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNS LYLQMNSLRAEDTAVYYCARDRGSLYYWGQGTLVTVSS XAB4, VL 43 AIQLTQSPSSLSASVGDRVTITCRPSQGINWELAWYQQK PGKAPKLLIYDASSLEQGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQFNSYPLTFGGGTKVEIK XAB4, HEAVY CHAIN 14 EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMSQVRQ APGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNS LYLQMNSLRAEDTAVYYCARDRGSLYYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK XAB4, LIGHT CHAIN 44 AIQLTQSPSSLSASVGDRVTITCRPSQGINWELAWYQQK PGKAPKLLIYDASSLEQGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC PN ENCODING 16 GAGGTGCAGCTGGTCGAGTCTGGCGGCGACCTGGTGCAG SEQ ID NO: 12 CCTGGCGGCAGCCTGAGACTGAGCTGCGCCGCCAGCGGC TTCACCTTCAGCAGCTACTGGATGTCCTGGGTCCGCCAG GCCCCTGGCAAAGGCCTCGAATGGGTGGCCAACATCAAG CAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAG GGCCGGTTCACCATCAGCCGGGACAACGCCAAGAACAGC CTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACC GCCGTGTACTACTGCGCCAGGGACCGGGGCAGCCTGTAC TATTGGGGCCAGGGCACCCTGGTCACCGTGTCCAGC PN ENCODING 45 GCCATCCAGCTGACCCAGAGCCCCAGCAGCCTGAGCGCC SEQ ID NO: 43 AGCGTGGGCGACAGAGTGACCATCACCTGTCGGCCCAGC CAGGGCATCAACTGGGAGCTGGCCTGGTATCAGCAGAAG CCTGGCAAGGCCCCCAAGCTGCTGATCTACGACGCCAGC TCCCTGGAACAGGGCGTGCCCAGCCGGTTCAGCGGCAGC GGATCCGGCACCGACTTCACCCTGACCATCAGCTCCCTG CAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTTC AACAGCTACCCCCTGACCTTCGGCGGAGGCACCAAGGTG GAAATCAAG PN ENCODING 18 GAGGTGCAGCTGGTCGAGTCTGGCGGCGACCTGGTGCAG SEQ ID NO: 14 CCTGGCGGCAGCCTGAGACTGAGCTGCGCCGCCAGCGGC TTCACCTTCAGCAGCTACTGGATGTCCTGGGTCCGCCAG GCCCCTGGCAAAGGCCTCGAATGGGTGGCCAACATCAAG CAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAG GGCCGGTTCACCATCAGCCGGGACAACGCCAAGAACAGC CTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACC GCCGTGTACTACTGCGCCAGGGACCGGGGCAGCCTGTAC TATTGGGGCCAGGGCACCCTGGTCACCGTGTCCAGCGCT AGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGC AGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGC CTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCC TGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTC CCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCC AGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAG ACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACC AAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAG ACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTG GGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAG GACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGC GTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAG TTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCC AAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTAC AGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGG CTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAG GCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGCC AAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCC CCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTG ACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCC GTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTAC AAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTC TTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGG CAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAG GCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTG TCCCCCGGCAAG PN ENCODING 46 GCCATCCAGCTGACCCAGAGCCCCAGCAGCCTGAGCGCC SEQ ID NO: 44 AGCGTGGGCGACAGAGTGACCATCACCTGTCGGCCCAGC CAGGGCATCAACTGGGAGCTGGCCTGGTATCAGCAGAAG CCTGGCAAGGCCCCCAAGCTGCTGATCTACGACGCCAGC TCCCTGGAACAGGGCGTGCCCAGCCGGTTCAGCGGCAGC GGATCCGGCACCGACTTCACCCTGACCATCAGCTCCCTG CAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTTC AACAGCTACCCCCTGACCTTCGGCGGAGGCACCAAGGTG GAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATC TTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCC AGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAG GCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGC GGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAG GACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGC AAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAG GTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGC TTCAACAGGGGCGAGTGC ALTERNATIVE PN 29 GAGGTGCAGCTGGTGGAATCAGGAGGCGACCTGGTGCAG ENCODING CCTGGCGGCTCACTGAGACTGAGCTGCGCCGCTAGTGGC SEQ ID NO: 12 TTCACCTTTAGTAGCTACTGGATGAGCTGGGTGCGACAG GCCCCTGGCAAGGGACTGGAGTGGGTGGCCAATATTAAG CAGGACGGCTCAGAGAAGTACTACGTGGACTCAGTGAAG GGCCGGTTCACTATTAGCCGGGATAACGCTAAGAATAGC CTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACC GCCGTGTACTACTGCGCTAGAGATAGAGGCTCACTGTAC TACTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCT ALTERNATIVE PN 47 GCTATTCAGCTGACTCAGTCACCTAGTAGCCTGAGCGCT ENCODING AGTGTGGGCGATAGAGTGACTATCACCTGTAGACCTAGT SEQ ID NO: 43 CAGGGGATTAACTGGGAGCTGGCCTGGTATCAGCAGAAG CCCGGCAAGGCCCCTAAGCTGCTGATCTACGACGCTAGT AGTCTGGAACAGGGCGTGCCCTCTAGGTTTAGCGGCTCA GGCTCAGGCACCGACTTCACCCTGACTATTAGTAGCCTG CAGCCCGAGGACTTCGCTACCTACTACTGTCAGCAGTTT AACTCCTACCCCCTGACCTTCGGCGGAGGCACTAAGGTG GAAATCAAG ALTERNATIVE PN 31 GAGGTGCAGCTGGTGGAATCAGGAGGCGACCTGGTGCAG ENCODING CCTGGCGGCTCACTGAGACTGAGCTGCGCCGCTAGTGGC SEQ ID NO: 14 TTCACCTTTAGTAGCTACTGGATGAGCTGGGTGCGACAG GCCCCTGGCAAGGGACTGGAGTGGGTGGCCAATATTAAG CAGGACGGCTCAGAGAAGTACTACGTGGACTCAGTGAAG GGCCGGTTCACTATTAGCCGGGATAACGCTAAGAATAGC CTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACC GCCGTGTACTACTGCGCTAGAGATAGAGGCTCACTGTAC TACTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCTGCT AGCACCAAGGGCCCAAGTGTCTTTCCCCTGGCCCCCAGC AGCAAGTCCACAAGCGGAGGCACTGCAGCTCTGGGTTGT CTGGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCC TGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTC CCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGC AGCGTCGTGACTGTGCCTAGTTCCAGCCTGGGCACCCAG ACCTATATCTGCAACGTGAACCACAAGCCCAGCAACACC AAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAG ACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTG GGAGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAG GACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGC GTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAG TTCAACTGGTACGTGGACGGGGTGGAGGTGCACAACGCC AAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTAC AGGGTGGTGTCCGTCCTGACAGTGCTGCACCAGGATTGG CTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAG GCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAGGCC AAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCC CCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTG ACCTGTCTGGTGAAGGGCTTCTACCCCAGCGATATCGCC GTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTAC AAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTC TTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGG CAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAG GCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTG AGCCCCGGCAAG ALTERNATIVE PN 48 GCTATTCAGCTGACTCAGTCACCTAGTAGCCTGAGCGCT ENCODING AGTGTGGGCGATAGAGTGACTATCACCTGTAGACCTAGT SEQ ID NO: 44 CAGGGGATTAACTGGGAGCTGGCCTGGTATCAGCAGAAG CCCGGCAAGGCCCCTAAGCTGCTGATCTACGACGCTAGT AGTCTGGAACAGGGCGTGCCCTCTAGGTTTAGCGGCTCA GGCTCAGGCACCGACTTCACCCTGACTATTAGTAGCCTG CAGCCCGAGGACTTCGCTACCTACTACTGTCAGCAGTTT AACTCCTACCCCCTGACCTTCGGCGGAGGCACTAAGGTG GAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATC TTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCC AGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAG GCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGC GGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAG GACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGC AAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAG GTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGC TTCAACAGGGGCGAGTGC SECOND 49 GAGGTGCAGCTGGTGGAATCTGGCGGCGACCTGGTGCAG ALTERNATIVE PN CCTGGCGGCTCTCTGAGACTGTCTTGCGCCGCCTCCGGC ENCODING TTCACCTTCTCCAGCTACTGGATGTCCTGGGTGCGACAG SEQ ID NO: 12 GCCCCTGGCAAGGGACTGGAATGGGTGGCCAACATCAAG CAGGACGGCTCCGAGAAGTACTACGTGGACTCCGTGAAG GGCCGGTTCACCATCTCCCGGGACAACGCCAAGAACTCC CTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACC GCCGTGTACTACTGCGCCAGGGACCGGGGCTCCCTGTAC TATTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCC SECOND 50 GCCATCCAGCTGACCCAGTCCCCCTCCAGCCTGTCTGCC ALTERNATIVE PN TCCGTGGGCGACAGAGTGACCATCACCTGTCGGCCCTCC ENCODING CAGGGCATCAACTGGGAACTGGCCTGGTATCAGCAGAAG SEQ ID NO: 43 CCCGGCAAGGCCCCCAAGCTGCTGATCTACGACGCCAGC TCCCTGGAACAGGGCGTGCCCTCCAGATTCTCCGGCTCT GGCTCCGGCACCGACTTCACCCTGACCATCTCCAGCCTG CAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTTC AACTCCTACCCCCTGACCTTCGGCGGAGGCACCAAGGTG GAAATCAAG SECOND 51 GAGGTGCAGCTGGTGGAATCTGGCGGCGACCTGGTGCAG ALTERNATIVE PN CCTGGCGGCTCTCTGAGACTGTCTTGCGCCGCCTCCGGC ENCODING TTCACCTTCTCCAGCTACTGGATGTCCTGGGTGCGACAG SEQ ID NO: 14 GCCCCTGGCAAGGGACTGGAATGGGTGGCCAACATCAAG CAGGACGGCTCCGAGAAGTACTACGTGGACTCCGTGAAG GGCCGGTTCACCATCTCCCGGGACAACGCCAAGAACTCC CTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACC GCCGTGTACTACTGCGCCAGGGACCGGGGCTCCCTGTAC TATTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCCGCC TCCACCAAGGGCCCAAGCGTGTTCCCCCTGGCCCCCAGC AGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGC CTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCC TGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTC CCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGC AGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAG ACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACC AAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGTGACAAG ACCCACACCTGCCCCCCCTGCCCAGCCCCCGAGCTGCTG GGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAG GACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGT GTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAG TTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCC AAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTAC AGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGG CTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAG GCCCTGCCAGCCCCAATCGAAAAGACCATCAGCAAGGCC AAGGGCCAGCCAAGAGAGCCCCAGGTGTACACCCTGCCA CCCAGCAGGGAGGAGATGACCAAGAACCAGGTGTCCCTG ACCTGTCTGGTGAAGGGCTTCTACCCAAGCGACATCGCC GTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTAC AAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTC TTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGG CAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAG GCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTG TCCCCAGGCAAG SECOND 52 GCCATCCAGCTGACCCAGTCCCCCTCCAGCCTGTCTGCC ALTERNATIVE PN TCCGTGGGCGACAGAGTGACCATCACCTGTCGGCCCTCC ENCODING CAGGGCATCAACTGGGAACTGGCCTGGTATCAGCAGAAG SEQ ID NO: 44 CCCGGCAAGGCCCCCAAGCTGCTGATCTACGACGCCAGC TCCCTGGAACAGGGCGTGCCCTCCAGATTCTCCGGCTCT GGCTCCGGCACCGACTTCACCCTGACCATCTCCAGCCTG CAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTTC AACTCCTACCCCCTGACCTTCGGCGGAGGCACCAAGGTG GAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATC TTCCCCCCAAGCGACGAGCAGCTGAAGAGCGGCACCGCC AGCGTGGTGTGTCTGCTGAACAACTTCTACCCCAGGGAG GCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGC GGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAG GACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGC AAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAG GTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGC TTCAACAGGGGCGAGTGC XAB5, CDRH1 1 GFTFSSY (CHOTHIA) XAB5, CDRH2 2 KQDGSE (CHOTHIA) XAB5, CDRH3 3 DRGSLYY (CHOTHIA) XAB5, CDRL1 41 SQGINWE (CHOTHIA) XAB5, CDRL2 5 DAS (CHOTHIA) XAB5, CDRL3 6 FNSYPL (CHOTHIA) XAB5, CDRH1 7 SYWMS (KABAT) XAB5, CDRH2 8 NIKQDGSEKYYVDSVKG (KABAT) XAB5, CDRH3 3 DRGSLYY (KABAT) XAB5, CDRL1 42 RPSQGINWELA (KABAT) XAB5, CDRL2 10 DASSLEN (KABAT) XAB5, CDRL3 11 QQFNSYPLT (KABAT) XAB5, VH 12 EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMSQVRQ APGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNS LYLQMNSLRAEDTAVYYCARDRGSLYYWGQGTLVTVSS XAB5, VL 53 AIQLTQSPSSLSASVGDRVTITCRPSQGINWELAWYQQK PGKAPKLLIYDASSLENGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQFNSYPLTFGGGTKVEIK XAB5, HEAVY CHAIN 14 EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMSQVRQ APGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNS LYLQMNSLRAEDTAVYYCARDRGSLYYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK XAB5, LIGHT CHAIN 54 AIQLTQSPSSLSASVGDRVTITCRPSQGINWELAWYQQK PGKAPKLLIYDASSLENGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC PN ENCODING 16 GAGGTGCAGCTGGTCGAGTCTGGCGGCGACCTGGTGCAG SEQ ID NO: 12 CCTGGCGGCAGCCTGAGACTGAGCTGCGCCGCCAGCGGC TTCACCTTCAGCAGCTACTGGATGTCCTGGGTCCGCCAG GCCCCTGGCAAAGGCCTCGAATGGGTGGCCAACATCAAG CAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAG GGCCGGTTCACCATCAGCCGGGACAACGCCAAGAACAGC CTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACC GCCGTGTACTACTGCGCCAGGGACCGGGGCAGCCTGTAC TATTGGGGCCAGGGCACCCTGGTCACCGTGTCCAGC PN ENCODING 55 GCCATCCAGCTGACCCAGAGCCCCAGCAGCCTGAGCGCC SEQ ID NO: 53 AGCGTGGGCGACAGAGTGACCATCACCTGTCGGCCCAGC CAGGGCATCAACTGGGAGCTGGCCTGGTATCAGCAGAAG CCTGGCAAGGCCCCCAAGCTGCTGATCTACGACGCCAGC TCCCTGGAAAACGGCGTGCCCAGCCGGTTCAGCGGCAGC GGATCCGGCACCGACTTCACCCTGACCATCAGCTCCCTG CAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTTC AACAGCTACCCCCTGACCTTCGGCGGAGGCACCAAGGTG GAAATCAAG PN ENCODING 18 GAGGTGCAGCTGGTCGAGTCTGGCGGCGACCTGGTGCAG SEQ ID NO: 14 CCTGGCGGCAGCCTGAGACTGAGCTGCGCCGCCAGCGGC TTCACCTTCAGCAGCTACTGGATGTCCTGGGTCCGCCAG GCCCCTGGCAAAGGCCTCGAATGGGTGGCCAACATCAAG CAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAG GGCCGGTTCACCATCAGCCGGGACAACGCCAAGAACAGC CTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACC GCCGTGTACTACTGCGCCAGGGACCGGGGCAGCCTGTAC TATTGGGGCCAGGGCACCCTGGTCACCGTGTCCAGCGCT AGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGC AGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGC CTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCC TGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTC CCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCC AGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAG ACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACC AAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAG ACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTG GGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAG GACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGC GTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAG TTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCC AAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTAC AGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGG CTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAG GCCCTGCCAGCCCCCATCGAAAAGACCATCAGACAAGGC CAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCC CCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCT GACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGC CGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTA CAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTT CTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTG GCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGA GGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCT GTCCCCCGGCAAG PN ENCODING 56 GCCATCCAGCTGACCCAGAGCCCCAGCAGCCTGAGCGCC SEQ ID NO: 54 AGCGTGGGCGACAGAGTGACCATCACCTGTCGGCCCAGC CAGGGCATCAACTGGGAGCTGGCCTGGTATCAGCAGAAG CCTGGCAAGGCCCCCAAGCTGCTGATCTACGACGCCAGC TCCCTGGAAAACGGCGTGCCCAGCCGGTTCAGCGGCAGC GGATCCGGCACCGACTTCACCCTGACCATCAGCTCCCTG CAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTTC AACAGCTACCCCCTGACCTTCGGCGGAGGCACCAAGGTG GAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATC TTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCC AGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAG GCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGC GGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAG GACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGC AAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAG GTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGC TTCAACAGGGGCGAGTGC ALTERNATIVE PN 29 GAGGTGCAGCTGGTGGAATCAGGAGGCGACCTGGTGCAG ENCODING CCTGGCGGCTCACTGAGACTGAGCTGCGCCGCTAGTGGC SEQ NO: 12 TTCACCTTTAGTAGCTACTGGATGAGCTGGGTGCGACAG GCCCCTGGCAAGGGACTGGAGTGGGTGGCCAATATTAAG CAGGACGGCTCAGAGAAGTACTACGTGGACTCAGTGAAG GGCCGGTTCACTATTAGCCGGGATAACGCTAAGAATAGC CTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACC GCCGTGTACTACTGCGCTAGAGATAGAGGCTCACTGTAC TACTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCT ALTERNATIVE PN 57 GCTATTCAGCTGACTCAGTCACCTAGTAGCCTGAGCGCT ENCODING AGTGTGGGCGATAGAGTGACTATCACCTGTAGACCTAGT SEQ ID NO: 53 CAGGGGATTAACTGGGAGCTGGCCTGGTATCAGCAGAAG CCCGGCAAGGCCCCTAAGCTGCTGATCTACGACGCTAGT AGTCTGGAAAACGGCGTGCCCTCTAGGTTTAGCGGCTCA GGCTCAGGCACCGACTTCACCCTGACTATTAGTAGCCTG CAGCCCGAGGACTTCGCTACCTACTACTGTCAGCAGTTT AACTCCTACCCCCTGACCTTCGGCGGAGGCACTAAGGTG GAAATCAAG ALTERNATIVE PN 31 GAGGTGCAGCTGGTGGAATCAGGAGGCGACCTGGTGCAG ENCODING CCTGGCGGCTCACTGAGACTGAGCTGCGCCGCTAGTGGC SEQ ID NO: 14 TTCACCTTTAGTAGCTACTGGATGAGCTGGGTGCGACAG GCCCCTGGCAAGGGACTGGAGTGGGTGGCCAATATTAAG CAGGACGGCTCAGAGAAGTACTACGTGGACTCAGTGAAG GGCCGGTTCACTATTAGCCGGGATAACGCTAAGAATAGC CTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACC GCCGTGTACTACTGCGCTAGAGATAGAGGCTCACTGTAC TACTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCTGCT AGCACCAAGGGCCCAAGTGTCTTTCCCCTGGCCCCCAGC AGCAAGTCCACAAGCGGAGGCACTGCAGCTCTGGGTTGT CTGGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCC TGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTC CCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGC AGCGTCGTGACTGTGCCTAGTTCCAGCCTGGGCACCCAG ACCTATATCTGCAACGTGAACCACAAGCCCAGCAACACC AAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAG ACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTG GGAGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAG GACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGC GTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAG TTCAACTGGTACGTGGACGGGGTGGAGGTGCACAACGCC AAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTAC AGGGTGGTGTCCGTCCTGACAGTGCTGCACCAGGATTGG CTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAG GCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAGGCC AAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCC CCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTG ACCTGTCTGGTGAAGGGCTTCTACCCCAGCGATATCGCC GTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTAC AAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTC TTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGG CAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAG GCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTG AGCCCCGGCAAG ALTERNATIVE PN 58 GCTATTCAGCTGACTCAGTCACCTAGTAGCCTGAGCGCT ENCODING AGTGTGGGCGATAGAGTGACTATCACCTGTAGACCTAGT SEQ ID NO: 54 CAGGGGATTAACTGGGAGCTGGCCTGGTATCAGCAGAAG CCCGGCAAGGCCCCTAAGCTGCTGATCTACGACGCTAGT AGTCTGGAAAACGGCGTGCCCTCTAGGTTTAGCGGCTCA GGCTCAGGCACCGACTTCACCCTGACTATTAGTAGCCTG CAGCCCGAGGACTTCGCTACCTACTACTGTCAGCAGTTT AACTCCTACCCCCTGACCTTCGGCGGAGGCACTAAGGTG GAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATC TTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCC AGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAG GCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGC GGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAG GACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGC AAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAG GTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGC TTCAACAGGGGCGAGTGC LEADER SEQUENCE 59 MEFGLSWVFLVAILEGVHC OF THE HEAVY CHAIN LEADER SEQUENCE 60 MDMRVPAQLLGLLLLWLPGARC OF THE LIGHT CHAIN PN ENCODING 61 ATGGAATTCGGCCTGAGCTGGGTGTTCCTGGTCGCGATT SEQ ID NO: 59 CTGGAAGGCGTGCACTGC PN ENCODING 62 ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTG SEQ ID NO: 60 CTGCTCTGGCTCCCAGGCGCCAGATGT ALTERNATIVE 63 MAWVWTLPFLMAAAQSVQA LEADER SEQUENCE OF THE HEAVY CHAIN ALTERNATIVE 64 MSVLTQVLALLLLWLTGTRC LEADER SEQUENCE OF THE LIGHT CHAIN ALTERNATIVE PN 65 ATGGCCTGGGTGTGGACCCTGCCCTTCCTGATGGCCGCT ENCODING GCTCAGTCAGTGCAGGCC SEQ ID NO: 63 ALTERNATIVE PN 66 ATGAGCGTGCTGACTCAGGTGCTGGCCCTGCTGCTGCTG ENCODING TGGCTGACCGGCACCCGCTGC SEQ ID NO: 64 SECOND 67 MEWSWVFLFFLSVTTGVHS ALTERNATIVE LEADER SEQUENCE OF THE HEAVY CHAIN SECOND 68 MSVPTQVLGLLLLWLTDARC ALTERNATIVE LEADER SEQUENCE OF THE LIGHT CHAIN SECOND 69 ATGGAATGGTCCTGGGTGTTCCTGTTCTTCCTGTCCGTG ALTERNATIVE PN ACCACAGGCGTGCACTCC ENCODING SEQ ID NO: 67 SECOND 70 ATGTCCGTGCCCACACAGGTGCTGGGCCTGCTGCTGCTG ALTERNATIVE PN TGGCTGACCGACGCCAGATGC ENCODING SEQ ID NO: 68 CONSENSUS, 71 SQX.sub.1IX.sub.2X.sub.3X.sub.4 CDRL1 (CHOTHIA) CONSENSUS, 72 FX.sub.1SYPL CDRL3 (CHOTHIA) CONSENSUS, 73 RPSQX.sub.1IX.sub.2X.sub.3X.sub.4LA CDRL1 (KABAT) CONSENSUS, 74 DASSLEX.sub.1 CDRL2 (KABAT) CONSENSUS, 75 QQFX.sub.1SYPLT CDRL3 (KABAT) huIL-17A 76 GITIPRNPGCPNSEDKNFPRTVMVNLNIHNRNTNTNPKR SSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCIN ADGNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILV SVGCTCVTPIVHHVAEFRH huIL-17F 77 MRKIPKVGHTFFQKPESCPPVPGGSMKLDIGIINENQRV SMSRNIESRSTSPWNYTVTWDPNRYPSEVVQAQCRNLGC INAQGKEDISMNSVPIQQETLVVRRKHQGCSVSFQLEKV LVTVGCTCVTPVIHHVQ alternative hulL-17A 78 GPIVKAGITIPRNPGCPNSEDKNFPRTVMVNLNIHNRNT NTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCR HLGCINADGNVDYHMNSVPIQQEILVLRREPPHCPNSFR LEKILVSVGCTCVTPIVHHVA cynoIL-17A 79 GIAIPRNSGCPNSEDKNFPRTVMVNLNIHNRNTSTNPKR SSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCVK ADGNVDYHMNSVPIQQEILVLRREPRHCPNSFRLEKILV SVGCTCVTPIVHHVA cynoIL-17F 80 MRKIPKVGHTFFQKPESCPPVPEGSMKLDTGIINENQRV SMSRNIESRSTSPWNYTVTWDPNRYPSEVVQAQCKHLGC INAQGKEDISMNSVPIQQETLVLRRKHQGCSVSFQLEKV LVTVGCTCVTPVVHHVQ rhesusIL-17A 81 GIAIPRNSGCPNSEDKNFPRTVMVNLNIHNRNTSTSPKR SSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCVK ADGNVDYHMNSVPIQQEILVLRREPRHCPNSFRLEKILV SVGCTCVTPIVHHVA marmosetIL-17A 82 SPQNPGCPNAEDKNFPRTVMVNLNIRNRNTNSKRASDYY NRSSSPWNLHRNEDPERYPSVIWEAKCRHLGCVDADGNV DYHMNSVPIQQEILVLRREPRHCTNSFRLEKMLVSVGCT CVTPIVHHVA mIL-17A 83 MAAIIPQSSACPNTEAKDFLQNVKVNLKVFNSLGAKVSS RRPSDYLNRSTSPWTLHRNEDPDRYPSVIWEAQCRHQRC VNAEGKLDHHMNSVLIQQEILVLKREPESCPFTFRVEKM LVGVGCTCVASIVRQAA mIL-17F 84 APEPEFRHRKNPKAGVPALQKAGNCPPLEDNTVRVDIRI FNQNQGISVPREFQNRSSSPWDYNITRDPHRFPSEIAEA QCRHSGCINAQGQEDSTMNSVAIQQEILVLRREPQGCSN SFRLEKMLLKVGCTCVKPIVHQAA ratIL-17A 85 MAVLIPQSSVCPNAEANNFLQNVKVNLKVINSLSSKASS RRPSDYLNRSTSPWTLSRNEDPDRYPSVIWEAQCRHQRC VNAEGKLDHHMNSVLIQQEILVLKREPEKCPFTFRVEKM LVGVGCTCVSSIVRHAS huIL-17RA 86 NCTVKNSTCLDDSWIHPRNLTPSSPKDLQIQLHFAHTQQ GDLFPVAHIEWTLQTDASILYLEGAELSVLQLNTNERLC VRFEFLSKLRHHHRRWRFTFSHFVVDPDQEYEVTVHHLP KPIPDGDPNHQSKNFLVPDCEHARMKVTTPCMSSGSLWD PNITVETLEAHQLRVSFTLWNESTHYQILLTSFPHMENH SCFEHMHHIPAPRPEEFHQRSNVTLTLRNLKGCCRHQVQ IQPFFSSCLNDCLRHSATVSCPEMPDTPEPIPDYMPLWE FRHDSGGGLNDIFEAQKIEWHE Linker 87 GSGGG Protein tag 1 (HA10) 88 GVYHREARSGKYKLTYAEAKAVCEFEGGHLATYKQLEAA RKIGFHVCAAGWMAKGRVGYPIVKPGPNCGFGKTGIIDY GIRLNRSERWDAYCYNPHAK Protein tag 2 (HA10.1) 89 GVYHREAQSGKYKLTYAEAKAVCEFEGGHLATYKQLEAA RKIGFHVCAAGWMAKGRVGYPIVKPGPNCGFGKTGIIDY GIRLNRSERWDAYCYNPHA Protein tag 3 (HA 10.2) 90 GVYHREAASGKYKLTYAEAKAVCEFEGGHLATYKQLEAA RKIGFHVCAAGWMAKGRVGYPIVKPGPNCGFGKTGIIDY GIRLNRSERWDAYCYNPHA Protein tag 4 91 ACGVYHREAQSGKYKLTYAEAKAVCEFEGGHLATYKQLE (HA 11) CARKIGFHVCAAGWMAKGRVGYPIVKPGPNCGFGKTGII DYGIRLNRSERWDAYCYNPHA Protein tag 5 92 GVYHREAQSGKYKLTYAEAKAVCEFEGGHLCTYKQLEAA (HA 11.1) RKIGFHVCAAGWMAKGRVGYPIVKPGPNCGFGKTGIIDY GIRLNRSERWDAYCCNPHA Protein tag 6 93 GVYHREAISGKYYLTYAEAKAVCEFEGGHLATYKQLLAA (NVS-X) QKIGFHVCAAGWMAKGRVGYPIVKPGPNCGFGKTGIIDY GIRLNRSERWDAYCYNPHA Protein tag 7 94 GVYHREAISGKYYLTYAEAKAVCEFEGGHLATYKQLQAA (NVS-Y) QKIGFHVCAAGWMAKGRVGYPIVKPGPNCGGFKTGIIDY GIRLNRSERWDAYCYNPHA Protein tag 8 95 ACGVYHREAISGKYYLTYAEAKAVCEFEGGHLATYQLLA (NVS-AX) AQKIGFHVCAAGWMAKGRVGYPIVKPGPNCGFGKTGIID YGIRLNRSERWDAYCYNPHA Protein tag 9 96 ACGVYHREAISGKYYLTYAEAKAVCEFEGGHLATYKQLQ (NVS-AY) AAQKIGFHVCAAGWMAKGRVGYPIVKPGPNCGFGKTGII DYGIRLNRSERWDAYCYNPHA DNA of SEQ ID NO: 89 97 GGAGTCTATCACAGAGAGGCTAGATCAGGCAAGTATAAG (HA10) CTGACCTACGCCGAGGCTAAGGCCGTGTGCGAGTTCGAG GGCGGTCACCTGGCTACCTATAAGCAGCTGGAAGCCGCT AGAAAGATCGGCTTTCACGTGTGCGCCGCTGGCTGGATG GCTAAGGGTAGAGTGGGCTACCCTATCGTGAAGCCTGGC CCTAACTGCGGCTTCGGTAAAACCGGAATTATCGACTAC GGGATTAGGCTGAATAGATCAGAGCGCTGGGACGCCTAC TGCTATAACCCTCACGCTAAG DNA of SEQ ID NO: 90 98 GGAGTCTATCACAGAGAGGCTCAGTCAGGCAAGTATAAG (HA10.1) CTGACCTACGCCGAGGCTAAGGCCGTGTGCGAGTTCGAG GGCGGTCACCTGGCTACCTATAAGCAGCTGGAAGCCGCT AGAAAGATCGGCTTTCACGTGTGCGCCGCTGGCTGGATG GCTAAGGGTAGAGTGGGCTACCCTATCGTGAAGCCTGGC CCTAACTGCGGCTTCGGTAAAACCGGAATTATCGACTAC GGGATTAGGCTGAATAGATCAGAGCGCTGGGACGCCTAC TGCTATAACCCTCACGCC DNA of SEQ ID NO: 34 99 GGAGTCTATCACAGAGAGGCTGCTAGCGGTAAATACAAG (HA 10.2) CTGACCTACGCCGAGGCTAAGGCCGTGTGCGAGTTCGAG GGCGGTCACCTGGCTACCTATAAGCAGCTGGAAGCCGCT AGAAAGATCGGCTTTCACGTGTGCGCCGCTGGCTGGATG GCTAAGGGTAGAGTGGGCTACCCTATCGTGAAGCCTGGC CCTAACTGCGGCTTCGGTAAAACCGGAATTATCGACTAC GGGATTAGGCTGAATAGATCAGAGCGCTGGGACGCCTAC TGCTATAACCCTCACGCC DNA of SEQ ID NO: 35 100 GGCGCCTGTGGCGTGTATCACAGGGAGGCCCAGAGCGGC (HA 11) AAGTACAAGCTCACCTACGCCGAGGCCAAGGCCGTGTGC GAATTCGAGGGCGGCCACCTGGCCACCTACAAGCAGCTG GAGTGCGCCAGGAAGATCGGCTTCCACGTGTGTGCCGCC GGCTGGATGGCCAAAGGCAGAGTGGGCTACCCCATCGTG AAACCCGGCCCCAACTGCGGCTTCGGCAAGACAGGCATC ATCGACTACGGCATCAGGCTGAACAGGAGCGAGAGGTGG GACGCCTACTGCTACAACCCCCACGCC DNA of SEQ ID NO: 36 101 GGAGTGTATCACAGAGAGGCCCAGAGCGGCAAGTACAAG (HA 11.1) CTGACCTACGCCGAGGCCAAGGCCGTGTGTGAGTTCGAG GGCGGCCACCTGTGCACCTACAAGCAGCTGGAGGCCGCC AGGAAGATCGGCTTCCACGTGTGTGCCGCCGGCTGGATG GCTAAAGGCAGGGTGGGCTACCCCATTGTGAAGCCCGGC CCCAATTGCGGCTTCGGCAAGACCGGCATCATCGACTAC GGCATCAGGCTGAACAGGAGCGAGAGGTGGGACGCCTAC TGCTGCAACCCCCACGCC Linker 102 GSGGG