BACKGROUND OF THE INVENTION

The bradykinin B1 receptor has been implicated in pathogenesis of inflammatory disease and chronic pain. By modulating tissue inflammation and renal fibrosis, the B1 receptor has also been associated with pathogenesis of acute kidney injury as well as chronic kidney diseases which are the main causes of end-stage renal failure.

In humans, the major agonists of the bradykinin B1 receptor are the kinins. Kinins are bioactive peptides produced from the proteolytic cleavage of kininogen proteins. The major kinin agonists of bradykinin B1 receptor are the decapeptide Kallidin, and the nonapeptide des-Arg10-Kallidin (formed by the proteolytic cleavage the c-terminal arginine form Kallidin). Therefore, agents that can inhibit the binding of Kallidin and des-Arg10-Kallidin to the bradykinin B1 receptor have the potential to treat or prevent bradykinin B1 receptor-mediated pathologies.

Accordingly, there is a need in the art for novel agents that inhibit the binding of Kallidin and des-Arg10-Kallidin to the bradykinin B1 receptor for use in the treatment of bradykinin B1 receptor-mediated human pathologies.

SUMMARY OF THE INVENTION

The present invention provides antibodies, or antigen binding fragments thereof, that specifically bind Kallidin and des-Arg10-Kallidin and prevent binding to the bradykinin B1 receptor. Such antibodies are particularly useful for treating Kallidin and des-Arg10-Kallidin-associated diseases or disorders (e.g., pain or fibrosis). The invention also provides pharmaceutical compositions, as well as nucleic acids encoding anti-Kallidin and des-Arg10-Kallidin antibodies, recombinant expression vectors and host cells for making such antibodies, or fragments thereof. Methods of using antibodies, or fragments thereof, of the invention to detect Kallidin and des-Arg10-Kallidin or to modulate Kallidin and des-Arg10-Kallidin activity, either in vitro or in vivo, are also encompassed by the invention. The invention also provides methods of making antibodies that specifically bind to des-Arg9- Bradykinin and des-Arg10-Kallidin-like peptide.

Accordingly, in one aspect the invention provides an isolated monoclonal antibody or antigen binding fragment thereof that:

a) specifically binds to Kallidin or des-Arg10-Kallidin but not to Bradykinin or des-Arg9-Bradykinin;

b) specifically binds to Kallidin or des-Arg10-Kallidin with a KD of less than 1 x1 0"10 M;

c) specifically binds to Kallidin or des-Arg10-Kallidin with a K0ff of less than 1 x1 04 s"1 ; or d) specifically binds to Kallidin or des-Arg10-Kallidin and inhibits binding to the bradykinin B1 receptor.

In one embodiment, the antibody or antigen binding fragment thereof binds to the N-terminal Lysine residue of Kallidin or des-Arg10-Kallidin.

In another embodiment, the antibody or antigen binding fragment thereof inhibits the binding of Kallidin or des-Arg10-Kallidin to a bradykinin-1 receptor.

In another embodiment, the antibody or antigen binding fragment thereof binds specifically to mouse Kallidin-like peptide (KLP).

In another embodiment, the antibody or antigen binding fragment thereof comprises a heavy chain variable domain comprising an HCDR3 amino acid sequence selected from the group consisting of:

a) SEQ ID NO: 7 [Χ, Υ Χζ X3D X4HAM X5Y], wherein

X2 is R, D, A, V, L, I, M, F, Y or W,

X3 is Y, F, W or H,

X4 is D, E or Y, and,

X5 is D or E;

b) SEQ ID NO: 63 [X1 EYDGX2YX3X4LDX5], wherein

X! is W or F,

X2 is N or no amino acid;

X3 is Y or S,

X4 is D or P, and

X5 is F or Y;

c) SEQ ID NO: 13;

d) SEQ ID NO: 32;

e) SEQ ID NO: 40;

f) SEQ ID NO: 47; and

g) SEQ ID NO: 55.

In another embodiment, the antibody or antigen binding fragment thereof comprises an HCDR2 amino acid sequence selected from the group consisting of:

Furthermore, in accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook et al., 1989") ; DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985) ; Oligonucleotide Synthesis (M.J. Gait ed. 1984) ; Nucleic Acid Hybridization [B.D. Hames & S.J.Higgins eds. (1985)]; Transcription And Translation [B.D. Hames & S.J. Higgins, eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1 986)] ; B. Perbal, A Practical Guide To Molecular Cloning (1984) ; F.M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

Example 1 : Hybridoma Production: Immunization of Mice with Kallidin Peptide Conjugated to KLH and Antibody Generation against Human BKR1 Ligands

The objective was to develop cross-reactive antibodies against Kallidin (KD; SEQ ID NO:1 ) and des-arg— Kallidin (DAKD; SEQ ID NO:2) that would inhibit these ligands binding to the human BKR1 . Generally, immunization of mice with KLH conjugated KD through additional cysteines on either the C- or N- terminus of the peptide was used to obtain mouse splenocytes for fusion with mouse myeloma cell lines as a fusion partner to produce the hybridomas.

Briefly, the immunization protocol was as follows: BALB/c Mice (8-20 week-old na'ive female) were immunized intraperitoneally with a mixture of even amounts of KLH-KD and KD-KLH in phosphate buffered saline (PBS) as an antigen total of 100 ug per mouse mixed at 1 :1 ratio of Sigma Adjuvant System (Sigma cat #6322) in a total volume of 200 μΙ per mouse (day 0). On day 21 , mice were boosted with a mixture of even amounts of KLH-KD and KD-KLH in PBS as an antigen total of 50 ug per mouse mixed at 1 :1 ratio of Sigma Adjuvant System (Sigma cat #6322) in a total volume of 200 μΙ per mouse. On day 30, blood samples were harvested for KD specific antibody titer evaluation. On day 51 , mice were boosted for fusion with a mixture of even amounts of KLH-KD and KD-KLH in PBS as an antigen total of 50 ug per mouse mixed at 1 :1 ratio of Sigma Adjuvant System (Sigma cat #6322) in a total volume of 200 μΙ per mouse. At day 55 mice were sacrificed by C02 chamber, blood was collected through the cardiac puncture and spleen was harvested for hybridoma production.

Hybridomas were made by fusing mouse myeloma cells that are deficient in adenosine phosphoribosyltransferase (APRT) with spleen cells from mice immunized with specific antigens. A selection system using HAT (hypoxanthine, azaserine, and thymidine) medium eliminates all but the fusion cells that are APRT+. Successful hybridomas must also retain the immunoglobulin (Igh) heavy chain, one of the immunoglobulin light chain loci and secrete functional antibody.

Hybridoma Production Medium (IMDM) was made by combining the following: 500ml Iscove's Modified Dulbecco's Medium (HyClone SH30259.01 ), 50ml fetal bovine serum (HyClone

SH30070.03), 5ml L-glutamine (Gibco Invitrogen cat # 25030), 5ml non-essential amino acids (Gibco Invitrogen cat # 1 1 140050), 5m! sodium pyruvate (Gibco Invitrogen cat # 1 1360070), 5ml 0.1 % penicillin-streptomycin (Gibco Invitrogen cat # 15140148). The medium was filtered before use. Expansion medium was made by combining the following : 1000ml serum free medium (Gibco Hybridoma SFM # 12045) , 1 00ml 10% HyClone SuperLow IgG Defined FBS #

SH30898.03 and 10ml penicillin/streptomycin. Freezing medium was 45ml heat inactivated FBS (HyClone SH30070.03) and 5ml DMSO, filter sterilized. Other materials included the following : HAT (50x) was obtained from Sigma-Aldrich (# H0262) ; Hybridoma Fusion and Cloning Supplement (50X) (Roche Diagnostics 1 1 363 735 001 ) ; Trypan Blue Stain 0.4% (Invitrogen cat # 15250-061 or T10282) ; PEG 1500 in 75 mM Hepes 50% w/v (Roche cat # 783641 (10783641001 ) . All the reagents except HAT and Hybridoma Fusion and cloning supplement were used at 37°C.

Table 2. Pe tide Reagents Used in Immunization and Screenin

Briefly, three or four days before the fusion, the mouse was boosted with an antigen of interest either intraperitonealy or intravenously. On the day of the fusion, the mouse was sacrificed in C02 chamber, blood was collected by cardiac puncture and the spleen was taken out and placed into 10 ml of serum free IMDM in a Petri dish. Fusion partner cells myeloma: FO (ATCC ref CRL-1646)/ X63 Ag8.653 (ATCC ref CRL1580) were grown at a log phase, then split one day before the fusion (1 :2 and 1 :5), and collected into 20 ml centrifuge tubes, spun and resuspended the pellet in 1 0ml IMDM. The pellet was washed two times with serum free IMDM medium. All the centrifugations are performed at 1570 rpm for 5 min. Final resuspension was in 10ml serum free IMDM. The connective tissue was dissected away from the spleen. The spleen was injected with 1 ml of serum free IMDM preheated to 37° C by 1 ml syringe and 25-gauge needle. Splenocytes are squeezed out of the fibroelastic coat by forceps and washed two times in 10 ml of serum free IMDM (including initial spin) and were resuspended in 10ml serum free IMDM. Cells were counted in Countess Automated Cell Counter.

Fusion partner cells and splenocytes were combined in one 50ml tube at ratio of 1 :2 to 1 : 0 (by cell number) and spun down at 970 rpm for 10 min (slow spin) to form a loose pellet. After the "slow" spin, supernatant was taken out with the precaution not to disturb the pellet, but minimize the amount of liquid over the cells in order not to dilute PEG 1 500. The last remaining medium was reserved and added back after the PEG is added (below). Preheated PEG 1 500 (37°C, total 1 ml) was added drop by drop to the cell pellet over 1 minute period of time and cells were mixed after every drop of PEG was added. Pellet was incubated with PEG for another 1 minute followed by addition of 10 ml of serum-free IMDM medium over 1 minute, so that the first 1 ml out of 10 is added over 30 sec. Cells underwent slow spin at 970 rpm for 10 min and supernatant decanted. Into (2) 100ml troughs, the following was added: 70ml IMDM with 10% FBS, 2ml HATand 2ml Hybridoma and Fusion Cloning Supplement. Cells were resuspended in 10ml IMDM with 10% FBS and split into (2) 50ml tubes (5ml cells/tube) and 25ml IMDM with 1 0% FBS was added. The resulting 30ml was transferred to the troughs containing 70ml HBSS/HAT/cloning supplement and 200ul cells/well were pipetted into (10) 96-well plates. Fusion was ready for screening by ELISA (50ul) about 10 to 14 days later, or when medium in the wells turns yellow. After the primary screening, positive clones are selected, numbered and moved to a 24-well plate in 500 ul per well of IMDM with 10% FBSH I. Hybridoma supernatants were screened by ELISA on streptavidin plated coated with N- and C-term biotinylated peptides (see below).

Example 2: Characterization and Selection of Hybridomas Expressing Antibodies Against Human BKR1 Ligands

Hybridoma supernatants were screened by ELISA on streptavidin plated coated with N- and C-term biotinylated peptides (see e.g., those set forth in Table 2) and then antibody binding kinetics were determined for confirmed positive hybridoma clones.

The ability of the antibodies in hybridoma supernatants to bind to BKR1 ligand peptide was evaluated with an ELISA assay. DAKD-biotin or KD-biotin peptides was coated on a 96-well SA

plate in phosphate buffered saline (PBS) buffer for an hour at room temperature at 5 ug/ml, and the nonspecific binding sites were blocked with 1 % bovine serum albumin (BSA) in PBS buffer. This plate was used to perform primary and secondary screening of the crude hybridoma supernatants. Hybridoma supernatants were added to the plates for binding to the coated KD or DAKD peptides. After 1 hour incubation, the plate was washed and bound antibodies were detected using horseradish peroxidase (HRP) conjugated secondary antibody (HRP-goat anti-mouse IgG (H+L) : Jackson ImmunoResearch Labs # 1 15-035-166) and developed using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) substrate (Roche diagnostics # 1 1 204 521 001 ). Data was analyzed using Excel . The antibodies showing positive signals (2 fold higher than 1 :10000 serum dilution ELISA signal) were selected and re-screened in duplicates for confirmation. Confirmed positive hybridoma clones were selected and subjected to binding dissociation rate ranking by Biacore.

For antibody binding kinetics, the instruments used were the B IACORE 2000 or BIACORE 3000 (GE Healthcare), designed for biomolecular interaction analysis (BIA) in real time. The sensor chip used was SA chip (GE Healthcare) with streptavidin covalently immobilized on a carboxym ethyl ated dextran matrix. Each sensor chip has four parallel flow cells (Fc). Every biotinylated BKR1 or BKR2 ligand peptides were immobilized to one of the flow cells 2 to 4 (Fc2 to Fc4) in the SA chip for binding dissociation rate screening and selectivity screening. Flow cell 1 (Fc1 ) was reserved and immobilized with a random peptide (biotinylated at one terminus) with equal or close peptide length in comparison to the testing ligand peptides as the negative control. In screening assays, cell culture supernatants of the hybridoma clones selected through primary screening of of transiently expressed humanized variants were injected over immobilized peptides. Hybridoma cell culture media was also injected over the chip surface as blank to establish a baseline. After subtracting signals of Fc1 and blank buffer runs, the dissociation rate of the antibodies from the supernatants to each peptide was analyzed and ranked using

BIAevaluation software. Only the antibody clones that demonstrated superior (kd < 10 -4 1 /s) binding dissociation rate were selected for subcloning and further characterization. In kinetics analysis, the corresponding biotin-peptides identified in screening for the testing antibody were immobilized in Fc2 to Fc4 while Fc1 with a random peptide used as reference cell. Each purified antibody selected from screenings were made into a series of two fold dilutions in running buffer (1 x HBS-EP buffer, GE Healthcare) between 0.1 to 10 nM. Binding association rate, dissociation rate and the overall affinity were calculated in B IAevaluation. Antibody binding kinetics for each antibody was always confirmed in triplicate assays using Biacore.

A total of 8 mice were immunized with mixed KLH-KD/KD-KLH and KLH-DAKD/DAKD-KLH and the spleens were fused using the above protocols. After primary screening of about 7680 hybridoma clones in ELISA with DAKD-biotin and KD-biotin, only 76 clones were confirmed

positive and selected for binding dissociation rate ranking in Biacore 3000/2000 over the immobilized DAKD-biotin and KD-biotin on Streptavidin (SA) chips. Among those, 8 hybridoma clones with binding dissociation rate <=of 10"4 were subcloned, sequenced, purified and further characterized (see Table 3).

Table 3 : Immunization Results with KLH-KD/KD-KLH and KLH-DAKD/DAKD-KLH

-* = residual binding (low RU in Biacore) Based on results seen in Table 3, five clones with unique sequences were selected for kinetic studies. These antibodies were highly selective for DAKD-biotin, KD-biotin, DAKLP-biotin and KLP-biotin binding (see Table 4) . They do not bind to other kinin peptides or to peptides biotinylated at the N-terminus.

Table 4. Summary of Kinetics of Selected anti-DAKD/KD Antibody Candidates

Additional immunization were performed with an array of immunogens (see list of peptides, Table 2) for generating antibodies blocking the rodent BKR1 ligands, DABK and DAKD as well as antibodies with other binding specificities against different member of kinin family of peptides. Table 5 lists the heavy and light sequences of the antibodies generated.

EXAMPLE 3: Generation of Surrogate Antibody for Murine Animal Studies

A surrogate antibody to be used in murine animal studies needed to be able to bind and neutralize rodent BKR1 ligands, DABK and DAKLP (mouse equivalent of DAKD) . In order to generate the required surrogate antibody, mice were first immunized with DABK and/or DAKD with KLH directly conjugated to the N-terminals of the peptides. Biotin-DABK/biotin-DAKD (biotinylation directly on N-terminus of the peptide) positive hybridoma clones from ELISA screening were selected for scaling up and purification. The antibodies listed in Family 7 (see Table 12) that demonstrated high binding affinities to biotin-DABK, biotin-DAKLP and biotin- DAKD were selected based on Biacore direct binding assay (Table 10). However, these Family 7 antibodies showed no binding to the native, unmodified DABK and DAKD peptides in competitive ELISA, and lacked neutralizing functionality in a calcium influx assay with Functional Drug Screening System (FDSS) (Hamamatsu Photonics K.K., Japan). Moreover, the biotin-DABK and biotin-DAKD completely lost bioactivity in the FDSS assay in comparison to the native, unmodified DABK and DAKD peptides (data not shown).

It was hypothesized that the direct N-terminus conjugation of KLH and biotin prevented the native confirmation of DABK and DAKD to form . With the aim to restore the native conformation in KLH- and biotin- conjugated peptides, linkers were designed and added to the N-terminus of DABK and/or DAKD with the intention to "cushion" the KLH/biotin conjugation effects on peptide conformation. Poly-glycine linkers were first attempted and tested because of their simple, non-polar and neutral properties based on modeling results. The FDSS assay results indicated that the gly-gly-gly (3G) linker was the best according to its ability to restore the bioactivities of KLH and biotin conjugated DABK and DAKD peptides (data not shown). Therefore, KLH-3G-DABK was chosen to immunize mice. And biotin-3G-DABK and biotin-3G-KD were used in binding based screening assays (ELISA and Biacore). Several DABK/DAKD specific antibodies (Family 3, see Table 13) were identified in this new round of surrogate antibody hybridoma selection. EE1 was selected as the lead surrogate antibody based on its superior binding affinity and neutralization activity against native DABK/DAKD and lack of cross- reactivity to other peptides ( see Tables 6 - 12)

Antibodies with different specificities were generated when using the different immunogens listed in Table 13. Family 4 antibodies were specific to the BKR2 receptor ligands, BK and KD. Family 5 antibodies specifically bind to the C terminus of BK and DABK. Family 6 antibodies bind BK, DABK and DAKD but do not bind to KD.

Additional linkers were evaluated for binding to the surrogate EE1 antibody for their ability to fit into the DABK/DAKD binding pocket in EE1 , including longer poly-glycine linkers, poly-alanine linkers and preexisting linkers such as polyethylene glycol (PEG2) linker and aminohexanoic acid (Ahx) linker (a 6-carbon inert linker). All linker peptides were custom synthesized by Abgent (Can Diego, CA). All tested biotinylated peptides with linkers (biotin-linker-DABK/DAKD) bound well to EE1 , indicating that any inert N-terminus linkers helped DABK and DAKD peptides to retain their native bioactive conformation when conjugated with biotin and other molecules. In contrast, no binding or poor binding to EE1 was observed with biotin-DABK and biotin-DAKD, peptides that have direct N-terminal biotin conjugation (see Figure 1 ).

The binding kinetics of generated antibodies are summarized in Tables 5-1 1 . Then, all antibodies generated were sorted into families and their binding specificities are summarized below in Table 12. Table 13 provides the heavy and light chain sequences of antibodies that were placed into family 1 and family 2 based on their binding specificity (see Table 12).

Table 6. Summary of Antibody Kinetics to b-3G-DABK and b-3G-DAKD Peptides

Table 7. Summary of Antibody Kinetics to b-3G-DAKLP and b-3G-BK Peptides

Table 8. Summary of Antibody Kinetics to b-3G-KLP and b-3G-KD Peptides

Table 9. Summary of Antibody Kinetics to DABK-b and DAKLP-b Peptides

Table 10. Summary of Antibody Kinetics to BK-b and b-BK Peptides

Table 11. Summary of Antibody Kinetics to b-DABK and b-DAKD Peptides

Table 12. Summary of Antibody Kinetics to b-DAKLP and b-KD Peptides

Table 13. Summary of Anti-kinin peptide antibody generation

Example 4: Characterization of des-arg-Kinin Ligand Depletion using Calcium Mobilization A functional assay was used to further characterize the seven families of generated antibodies. The Bradykinin B1 Receptor signaling is Gq coupled, therefore receptor activation can be monitored using Gq activation of IP3 and downstream calcium mobilization. HEK m BKR1 (recombinant mouse bradykinin B1 receptor) cells or MRC5 (endogenous expression of bradykinin B2 receptors; (ATCC CCL-171 )) were used to measure calcium mobilization.

Briefly, the mouse Bdkrbl gene (sequence provided below) was amplified from mouse lung cDNA (Biochain, Cat# C1334152) using PCR primers 804_cGWY_F:

5'-AAAAGCAGGCTTAGGAGCGGCCGCCATGGCGTCCCAGGCCTCGCTG-3' (SEQ ID NO: 107) and 804_cGWY_R:

5'-CAAGAAAGCTGGGTCGGATCCTTATAAAGTTCCCAGAACCCTGGTC-3' (SEQ ID NO: 108) and Pfu Polymerase (Agilent Technologies, Cat# 600264) and cloned into pDON R201 using BP clonase enzyme mix (Invitrogen, Cat# 1 1789-020). In parallel, the pEAK8 expression vector (EDGE Biosystems) was modified by inserting a N-terminal HA tag

(GCATACCCATACGACGTCCCAGACTACGCT, GenBank SEQ ID NO:109 CY100443) into pEAK8 linearized with EcoRI and Hindl ll (vector pEAK8-nHA) and subsequent insertion of the Gateway cassette B (Invitrogen, Cat# 1 1 828-029) into pEAK8_nHA digested with EcoRI and NotI and blunt-ended with Klenow polymerase (N EB, cat# M0210S) resulting in vector

pEAK8_nHA_DEST. Next mouse Bdkrbl was subcloned into pEAK8_nHA_DEST using LR

clonase (Invitrogen, Cat# 1 1791 -100). 293-PSC cells were then transfected with pEAK8-Bdkrb1 plasmid using Fugene 6 transfection reagent. The cells were put under antibiotic (puromycin) selection 24 hours after transfection, and selection was maintained to generate a stable cell line. Presence of the Bdkrbl gene in the resultant stable cell lines was confirmed using real time RT-PCR, and by agarose gel electrophoresis. Cell surface expression of the Bradykinin B1 receptor was performed by using an antibody against the N-terminal-HA tag (Covance, Cat # MMS-101 P) on the Bradykinin B1 R on a FACS instrument. Functional activity of the Bradykinin B1 receptor was demonstrated in calcium mobilization assay with selective agonists.

Bdkrbl gene subcloned into cells:

ATGGCGTCCCAGGCCTCGCTGAAGCTACAGCCTTCTAACCAAAGCCAGCAGGCCCCTCCCAACATCACCTC CTGCGAGGGCGCCCCGGAAGCCTGGGATCTGCTGTGTCGGGTGCTGCCAGGGTTTGTCATCACTGTCTGTT TCTTTGGCCTCCTGGGGAACCTTTTAGTCCTGTCCTTCTTCCTTTTGCCTTGGCGACGATGGTGGCAGCAG CGGCGGCAGCGCCTAACCATAGCAGAAATCTACCTGGCTAACTTGGCAGCTTCTGATCTGGTGTTTGTGCT GGGCCTGCCCTTCTGGGCAGAGAACGTTGGGAACCGTTTCAACTGGCCCTTTGGAAGTGACCTCTGCCGGG TGGTCAGCGGGGTCATCAAGGCCAACCTGTTCATCAGCATCTTCCTGGTGGTGGCCATCAGTCAGGACCGC TACAGGTTGCTGGTATACCCCATGACCAGCTGGGGGAACCGGCGGCGACGGCAAGCCCAAGTGACCTGCCT GCTCATCTGGGTAGCTGGGGGCCTCTTGAGCACCCCCACGTTCCTTCTGCGTTCCGTCAAAGTCGTCCCTG ATCTGAACATCTCTGCCTGCATCCTGCTTTTCCCCCACGAAGCTTGGCACTTTGTAAGGATGGTGGAGTTG AACGTTTTGGGTTTCCTCCTCCCATTGGCTGCCATCCTCTACTTCAACTTTCACATCCTGGCCTCCCTGAG AGGACAGAAGGAGGCCAGCAGAACCCGGTGTGGGGGACCCAAGGACAGCAAGACAATGGGGCTGATCCTCA CACTGGTAGCCTCCTTCCTGGTCTGCTGGGCCCCTTACCACTTCTTTGCCTTCCTGGATTTCCTGGTCCAG GTGAGAGTGATCCAGGACTGCTTCTGGAAGGAGCTCACAGACCTGGGCCTGCAGCTGGCCAACTTCTTTGC TTTTGTCAACAGCTGCCTGAACCCACTGATTTATGTCTTTGCAGGCCGGCTCTTTAAGACCAGGGTTCTGG GAACTTTATAA (GenBank NM_007539; SEQ ID NO: 110)

HEK 1T1 BKRI or MRC5 cells were plated into 384 well clear bottom plates in growth medium , and allowed to attach overnight. Then growth media was removed, cells were washed in assay buffer (HBSS, 20mM HEPES, 2.5 mM probenecid), then dye-loaded with 0.5 uM Fluo-4AM, a cell permeable calcium sensing dye, with 0.04% Pluronic Acid for 1 hr at 37C. The AM ester is cleaved, and the calcium dye is retained in the cytoplasm . After 1 hr, the cells were washed to remove excess dye, and 20ul of residual buffer remained on the cells. Treatments were added as 2x solutions on the Functional Drug Screening System from Hamamatsu (FDSS), and the calcium mobilization was monitored kinetically for at least 4 minutes. B1 R or B2R receptor activation results in Galpha q mediated activation of phospholipase C and IP3

mediated calcium mobilization. The Fluo-4 dye chelates the released calcium , and a

robust change in fluorescence is observed. The results were exported as max-min relative fluorescence units to normalize for differences between cell density or dye loading across the plate.

Ligand potency was determined each day by running concentration response curves of ligand, and an approximate EC70-80 concentration of ligand was selected for incubation with antibodies. An EC80 concentration was selected because it is on the linear range of the detection curve and there was ample window to see a decrease with antagonists or ligand depleting antibodies. Dose response curve of antibodies were allowed to bind a EC80 concentration ligand, and the extent of ligand depletion was monitored using change in fluorescence. Results were normalized to buffer and EC80 ligand response, and an EC50 for ligand depletion was calculated. The results were then reported as molar ratio which corresponds to the Antibody concentration that reduces depletes 50% of the ligand response (i.e., EC50 of Ab) divided by the ligand concentration used. The theoretical max should be 0.5 because one unit of antibody should be able to deplete 2 units of ligand, but we have seen lower values in practice but that may be a reflection of the insensitivity of the detection method for low ligand concentrations, rather than a stochiometric constraint for the antibody. The results of these experiments are set forth in Tables 14-16.

All family 1 and family 2 antibodies (see Table 13) demonstrated superior binding kinetics by Biacore (Table 3) and neutralization activity as measured by calcium mobilization against DAKD and KD peptides (Tables 14 and 15). The antibodies were further analyzed for their thermal stability and sequence suitability for humanization. F151 was advanced for humanization because it was thermally stable, there were no problematic residues in the CDR regions and it was cross-reactive to the mouse ligand KLP and DAKLP.

Table 14: Characterization of des-arg-Kinin Ligand Depletion using Calcium Mobilization in HEK mBKR1 cells

4 NR15 ND ND ND

4 NR1 ND ND ND

5 UR29 0.60 0.12 5 IA200 3 IA300 4

6 UR1 1 6.99 1 .61 3 19.65 14.95 3 1 1 .09 3.13 2

7 LR4 IA100 1 IA400 1 IA400 1

7 LR6 IA100 1 IA100 1 ND

7 LR12 IA100 1 IA100 1 ND

7 LR16 IA100 1 IA100 1 ND

Antibodies were pre-incubated with a set concentration of ligand, usually an EC70-80 for activating calcium mobilization at the Bradykinin B1 Receptor. The antibody-ligand mixture was added to HEK mBKR1 cells pre-loaded with a calcium sensing dye (Fluo-4AM or Fluo-8AM) on the Hamamatsu FDSS6000 instrument, and calcium mobilization was monitored. Data was exported as a max-min relative fluorescence of the biological response, and IC50 for ligand depletion was calculated using sigmoidal curve fit in Graph Pad Prism V4.03. Data reported as molar ratio for ligand depletion by the antibody to standardize the different concentration of ligand that was used for the various experiments.

Molar Ratio for ligand depletion = [IC50 of Antibody]/ [Ligand]

SD = Standard Deviation; ND = not determined; IA100 = Inactive at 100nM; IA200 = Inactive at 200nM; IA300 = Inactive at 300nM; IA400 = Inactive at 400nM

Table 15: Characterization of Kinin Ligand Depletion using Calcium Mobilization in MRC5 Fetal Lung Fibroblasts cells

4 MBK3 22.1 1 14.10 9 3.46 2.64 6 9.45

1

4 NR15 15.26 1 1 .51 5 4.34 2.55 5 1 1 .18

1

4 NR1 39.31 1 42.15 1 32.58

1

5 UR29 1.15 0.86 5 0.30 0.08 2 0.41

1

6 UR1 1 5.41 0.80 2 25.21 4.54 2 1 .53

1

7 LR4 IA100 1 IA100 1 ND

7 LR6 IA100 1 IA100 1 ND

7 LR12 IA100 1 IA100 1 ND

7 LR16 IA100 1 IA100 1 ND

Antibodies were pre-incubated with a set concentration of ligand, usually an EC70-80 for activating calcium mobilization at the Bradykinin B2 Receptor. The antibody-ligand mixture was added to MRC5 Fetal Lung Fibroblasts (ATCC CCL-171 ) pre-loaded with a calcium sensing dye (Fluo-4AM or Fluo-8AM) on the Hamamatsu FDSS6000 instrument, and calcium mobilization was monitored. Data was exported as a max-min relative fluorescence of the biological response, and IC50 for ligand depletion was calculated using sigmoidal curve fit in Graph Pad Prism V4.03. Data reported as molar ratio for ligand depletion by the antibody to standardize the different concentration of ligand that was used for the various experiments.

Molar Ratio for ligand depletion = [IC50 of Antibody]/ [Ligand]

SD = Standard Deviation; ND = not determined; IA100 = Inactive at 100nM; IA150 = Inactive at 150nM; IA300 = Inactive at 300nM; IA400 = Inactive at 400nM; IA600 = Inactive at 600nM

Example 5: Engineering of F151 : Humanization, Stabilization and Mutation of Unwanted Sequence Motifs

1. HUMANIZATION

The humanization protocol used has been described in PCT/US08/74381 (US201 10027266), herein incorporated by reference in its entirety. The variable light (VL) and variable heavy (VH) sequences of murine F151 were used to build a homology model of anti-DAKD/KD F151 LC and HC in Molecular Operating Environment (MOE; v. 2009.10; Chemical Computing Group). The following templates were used: light chain framework - 1 SBS (93% identity in the framework regions), heavy chain framework - 2VXT (84% identity in the framework regions), L1 - 1 LVE (93% identity) , L2 - 1 EEU (100% identity), L3 - 2R56 (93% identity), H1 - 1 NJ9 (95% identity) , H2 - 2VXU (76% identity) and H3 - 1 H IL (49% identity). Templates were available at the RCSB Protein Data Bank found on the world wide web at rcsb.org, a website managed by Rutgers and the University of California San Diego (Berman, H.M; Westbrook J. ; Feng. Z.; Gilliland, G.; Bhat, T.N.; Weissig, H.; Shindyalov, I.N. ; Bourne, P.E. The Protein Data Bank, Nucleic Acids

Research, 2000, 28, 235-242.). The homology model was subsequently energy minimized using

the standard procedures implemented in MOE. A molecular dynamics (MD) simulation of the minimized 3D homology model of the murine F151 was subsequently performed, with constraints on the protein backbone at 500 K temperature for 1 .1 nanoseconds (ns) in Generalized Born implicit solvent. Ten diverse conformations were extracted from this first MD run every 100 picoseconds (ps) for the last 1 ns. These diverse conformations were then each submitted to a MD simulation, with no constraints on the protein backbone and at 300 K temperature, for 2.3 ns. For each of the 10 MD runs, the last 2,000 snapshots, one every ps, from the MD trajectory were then used to calculate, for each murine F151 amino acid, its root mean square deviations (rmsd) compared to a reference medoid position. By comparing the average rmsd on the 10 separate MD runs of a given amino-acid to the overall average rmsd of all F151 murine amino-acids, one decides if the amino-acid is flexible enough, as seen during the MD to be considered as likely to interact with T-cell receptors and responsible for activation of the immune response. 62 amino-acids were identified as flexible in the murine F151 antibody, excluding the CDR and its immediate 5 A vicinity.

The motion of the 28 most flexible murine F151 amino acids, during the 20 ns (10 x 2 ns), were then compared to the motion of the corresponding flexible amino-acids of 49 human germline homology models, for each of which were run the 10 x 2 ns MD simulations. The 49 human germline models were built by systematically combining the 7 most common human germline light chains (vk1 , vk2, vk3, vk4, vlambdal , vlambda2, vlambda3) and 7 most common human germline heavy chains (vh1 a, vh1 b, vh2, vh3, vh4, vh5, vh6). The vk1 -vh1 b human germline antibody showed 0.80 4D similarity of its flexible amino-acids compared to the flexible amino-acids of the murine F151 antibody; the vk1 -vh1 b germline antibody was therefore used to humanize F151 antibody focusing on the flexible amino-acids. For the pair wise amino-acid association between murine F151 vk1 -vh1 b amino-acids, the 2 sequences were aligned based on the optimal 3D superposition of the alpha carbons of the 2 corresponding homology models (see Figure 15 for an alignment of F151 LC and F151 HC with vk1 and vhl b, respectively).

2. STABILIZATION

Two approaches were used to improve the stability of the antibody.

a) KNOWLEDGE-BASED APPROACH

The amino acids of the light and heavy chains with low frequency of occurrence vs. their respective canonical sequences, excluding the CDRs, were proposed to be mutated into the most frequently found amino acids (AAGth > 0.5 kcal/mol ; E. Monsellier, H. Bedouelle. J. Mol. Biol . 362, 2006, p. 580-593). This first list of consensus mutations for the light chain (LC) and heavy chain (HC) was restricted to the amino acids found in the closest human germline (vk1 -vh1 b). Suggested changes in the immediate vicinity of the CDRs (5 Angstroms "Vernier" zone, J. Mol . Biol. 224, 1992, p. 487-499) were removed from consideration. This resulted in 5 stabilizing mutations in the LC (see Table 19) and 4 stabilizing mutations in the HC (see Table 20). Other criteria were taken into account to consider these mutations for potentially stabilizing the anti-DAKD/KD F151 antibody. These criteria were a favorable change of hydropathy at the surface or a molecular mechanics based predicted stabilization of the mutant. Also, additional stabilizing mutations reported to be successful in the literature (E. Monsellier & H. Bedouelle, J. Mol. Biol., 362, 2006, p. 580-593; B.J. Steipe et al. J. Mol. Biol, 1994, 240, 188-192) were considered (see Tables 16-22). One of these changes was incorporated as a stabilizing mutation (D89E) in sequences HC2a, HC2b and HC2c below. Another suggested change (Q62E) was incorporated in variant HC2b.

b) 3D AND MD-BASED APPROACHES

3D and MD- based approaches have been previously reported (Seco J, Luque FJ, Barril X., J Med Chem . 2009 Apr 23;52(8) :2363-71 ; Malin Jonsson et al., J. Phys. Chem . B 2003, 107, 551 1 -5518). Hydrophobic regions of the antibody were explicitly identified by analyzing the molecular dynamics simulation of the Fab in a binary solvent (20% isopropanol in water, 20 ns production simulation). Lysine mutations were then introduced in the vicinity of these regions as an attempt to prevent the aggregation. Additional analysis using a hydrophobic surface map within

Schrodinger's maestro software (v. 8.5.207) was completed. Using a combination of these two techniques, 2 Lys mutations, 1 in the heavy chain and 1 in the light chain, are suggested.

3. HUMAN IZATION BY GRAFTING

Humanization using grafting grafting techniques has previously been reported (Peter T. Jones, Paul H. Dear, Jefferson Foote, Michael S. Neuberger & Greg Winter

Nature, 1986, 321 , 522-525). The humanization process which was used started by identifying the closest human germlines to anti-DAKD/KD light and heavy chains. This is done by performing a BLAST search vs. all the human germlines which were systematically enumerated (all possible combinations of the V & J domains for the kappa and lambda chains; V, D and J domains for the heavy chains).

The following closest human germlines were identified with 83% and 62% sequence identity to anti-DAKD/KD F151 light chains (LC) and heavy chains (HC), respectively (see Figure 16). Using the internal VBASE germline, the light chain is found to be close to V IV-B3 (-83% identity) locus and the heavy chain close to 1 -08 & 1 -18 (-62% identity) locus of the VH1 sub- family. CDR regions (as defined by MOE), and Vernier regions (as defined in Foote & Winter, J. Mol . Biol., 1992, 224, 487-499) are indicated in boldface The humanizing mutations in underlining were obtained by performing a pairwise comparison of the 2 aligned sequences, excluding the CDR & Vernier zone residues as defined above. In another variant of the humanization, only the CDRs were excluded in the comparison.

4. MUTATION OF UNWANTED SEQUENCE MOTIFS

The following motifs of sequences were considered: Asp-Pro (acid labile bond), Asn-X-Ser/Thr (glycosylation, X=any amino-acid but Pro) , Asp-Gly/Ser/Thr (succinimide/iso-asp formation in flexible regions), Asn-Gly/His/Ser/Ala/Cys (exposed deamidation sites), and Met (oxidation in exposed areas). Among other criteria, the VL & VH domains of murine F151 was selected from other murine antibodies because murine F151 did not have exposed unwanted sequence motifs, but they are introduced in some humanized variants.

LC3a, LC3b, HC3a and HC3b each have potentially problematic succinimide sites that were identified. These sites were not modified in the proposed sequences as the residues involved are potentially involved in H-bond network (visual inspection of the homology model). These positions are also found in a number of other antibody structures. Additionally, in both HC3a and HC3b, a strict humanization by grafting would include a substitution of Ser1 15 to Met. This Methionine is exposed. A substitution to Leucine at this position is suggested as a humanizing mutation as it is a common residue among many close human germline sequences.

The resulting humanized sequences were blasted for sequence similarity against the

International Epitope Database (IEDB) database (found on the world wide web at

immuneepitope.com ; version June 2009; Vita R, Zarebski L, Greenbaum JA, Emami H, Hoof I, Salimi N, Damle R, Sette A, Peters B. The immune epitope database 2.0. Nucleic Acids Res. 2010 Jan;38(Database issue) :D854-62. Epub 2009 Nov 1 1 ) to ensure that none of the sequences contain any known human B- or T-cell epitopes (sequence identity of 70% used as cut-off for the results obtained through BLAST search and considering only the results from human species) .

5. ORIGINAL SEQUENCES OF MURINE F151 VARIABLE DOMAINS

CDRs are highlighted in bold and Vernier regions (as defined in Foote & Winter, J. Mol . Biol., 1992, 224, 487-499) are underlined.

Light Chain (SEQ ID NO:26)

D^VMSQSPS S LAVSVGEKVTMSCKSSQSLLYSSNQKNYLA

WYQQKPGQSP KPLIYWASTRESGVPDRFTGSGSGTDFTLT

ISSVKAEDLA IYYCQQYYSYPWTFGGGTKLEIK

Germinality index = 83% with Z4661 5_1 _V_X67858_1_J [V IV-B3]

Heavy Chain (SEQ ID N0:19) :

EIQLQQSGPELVKPGTSVKVSCKASGYSFTDYNIYWVKQS HGKSLEWIGY FDPYNGNTGYNQKFRGKATLTVDKS SSTAF

MHLSSLTSDDSAVYYCANYYRYDDHAMDYWGQGTSVTVSS

Germinality index = 62% with Z1231 6_1 _VX97051_4_D_X97051 _5_J [VH1 1 -18]

6. ENGINEERED SEQUENCES

a) Background

5 versions for the light chain (LC1 , LC2a, LC2b, LC3a, and LC3b) and 5 versions of the heavy chain (HC1 , HC2a, HC2b, HC3a, and HC3b) were proposed.

LC1 contains 5 humanizing mutations identified using the 4D humanization protocol. LC2a introduced an additional 5 stabilizing mutations. LC2b added 1 Lysine mutations to help prevent aggregation. LC3a contains 15 mutations derived from grafting to the closest human germline sequence and retaining the murine CDR and Vernier zone residues. LC3b contained 16 mutations derived from CDR-grafting with one additional humanizing mutation.

HC1 has 6 humanizing mutations identified by the in-house protocol. HC2a introduced 5 additional stabilizing mutations while HC2b contains 6 additional stabilizing mutations as compared to HC1 . HC2c contains 1 Lys mutation, in addition to the stabilizing mutations of HC2a, to help prevent aggregation. HC3a contains 19 mutations derived from grafting to the closest human germline sequence and retaining the murine CDR and Vernier zone residues. HC3b contains 25 mutations derived from CDR grafting.

6 combinations in total were proposed (summarized in Table 16) :

• LC1 x HC1 (mutations addressing humanization only)

• LC2a x HC2a (mutations addressing humanization and stabilization)

· LC2a x HC2b (mutations addressing humanization and stabilization)

• LC2b x HC2c (mutations addressing humanization, stabilization and "anti-aggregation")

• LC3a x HC3a (mutations addressing mostly humanization by grafting + Vernier)

• LC3b x HC3b (mutations addressing humanization by grafting)

Table 16: Summary of the 6 LCxHC combinations proposed

Table 17: Mutations of the 5 LC variants of the anti-DAKD/KD F151 antibody

Val15 Val15 Leu Leu

Glu17 Glu17 Asp Asp Asp

Lys18 Lys18 Arg Arg Arg Arg Arg

Val19 Val19 Ala Ala

Met21 Met21 lie He He He

Ser22 Ser22 Asn Asn

Gln48 Gln42 Lys Lys Lys

Ser49 Ser43 Pro Pro

Pro52 Pro46 Leu

Thr69 Thr63 Ser Ser Ser Ser

Val84 Val78 Leu Leu

Lys85 Lys79 Gin Gin Gin Gin Gin

Leu89 Leu83 Lys Val Val

He91 Ile85 Thr Thr Val Val

Gly106 Gly100 Gin Gin

Leu1 10 Leu 104 Val Val

Mutations: 5 10 1 1 15 16

Table 18: Mutations of the 6 HC variants of the anti-DAKD/KD F151 antibody

Phe80 Phe79 Tyr Tyr Tyr Tyr Tyr

His82 His81 Glu Glu

Ser84 Ser82A Arg Arg

Leu 86 Leu82C Lys

Thr87 Thr83 Arg Arg

Asp89 Asp85 Glu Glu Glu Glu

Asp90 Asp86 Glu Glu Glu

Ser91 Ser87 Thr Thr

Ser1 15 Ser108 Leu Leu

Mutations: 6 1 1 12 12 19 25

a) Engineered light chain sequences:

No potentially problematic known T-cell or B-cell epitopes were found in all the variants proposed.

LC1 (SEQ ID NO:27) , humanizing mutations are underlined, CDRs and vernier zones are in bold:

DIVMSQSPS SLAASVGDRVTMSCKSSQSLLYSSNQKNYLA WYQQKPGKSP KPLIYWASTRESGVPDRFTGSGSGTDFTLT ISSVQAEDLAIYYCQQYYSYPWTFGGGTKLEIK

LC2a (SEQ ID NO:28), humanizing mutations are underlined, CDRs and vernier zones are in bold, stabilization mutations are in italics (T at position 5, S at position 12, I at position 21 , S at position 69, T at position 91 shown below) :

DIVMTQSPSSLSASVGDRVTJSCKSSQSLLYSSNQKNYIiA

WYQQKPGKSPKPLIYWASTRESGVPDRFSGSGSGTDFTLT ISSVQAEDLA TYYCQQYYSYPWTFGGGTKLEIK

LC2b (SEQ ID NO:29) humanizing mutations are underlined, CDRs and vernier zones are in bold, stabilization mutations are in italics (T at position 5, S at position 12, I at position 21 , S at position 69, T at position 91 shown below) and an anti-aggregation mutation is K at position 89:

DIVMTQSPSSLSASVGDRVTJSCKSSQSLLYSSNQKNYIiA

WYQQKPGKSPKPLIYWASTRESGVPDRFSGSGSGTDFTLT ISSVQAEDKA TYYCQQYYSYPWTFGGGTKLEIK

LC3a (SEQ ID NO:30), grafted mutations shown in underline and CDRs and vernier zones shown in bold:

DIVMTQSPDSLAVSLGERATI CKSSQSLLYSSNQKNYLA WYQQKPGQPPKPLIYWASTRESGVPDRFSGSGSGTDFTLT ISSLQAEDVAVYYCQQYYSYPWTFGQGTKVEIK

LC3b (SEQ ID NO:31 ), grafted mutations shown in underline and CDRs and vernier zones shown in bold:

DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLA WYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLT ISSLQAEDVAVYYCQQYYSYPWTFGQGTKVEIK

Note that L at position 52 is a vernier residue that is mutated to human,

c) Engineered Heavy chain sequences

HC1 (SEQ ID NO:20), humanizing mutations are underlined, CDRs and vernier zones are in bold:

EIQLVQSGPEVKKPGASVKVSCKASGYSFTDYNIYWVKQS PGKSLEWIGYFDPYNGNTGYNQKFRGKATLTVDKSSSTAF MHLSSLTSEDSAVYYCANYYRYDDHAMDYWGQGTSVTVSS

HC2a (SEQ ID NO:21 ), humanizing mutations are underlined, CDRs and vernier zones are in bold, stabilization mutations are in italics (Q at position 1 , A at position 9, G at position 44, Y at position 80 and E at position 90 shown below) :

QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYNIYWVKQS PGKGLEWIGYFDPYNGNTGYNQKFRGKATLTVDKSSSTAY MHLSSLTSE£SAVYYCANYYRYDDHAMDYWGQGTSVTVSS

HC2b (SEQ ID NO:22), humanizing mutations are underlined, CDRs and vernier zones are in bold, stabilization mutations are in italics (Q at position 1 , A at position 9, G at position 44, E at position 62, Y at position 80 and E at position 90 shown below) :

QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYNIYWVKQS PGKGLEWIGYFDPYNGNTGYNKKFRGKATLTVDKSSSTAY MHLSSLTSE£SAVYYCANYYRYDDHAMDYWGQGTSVTVSS

No human epitopes were identified for sequence HC2b in IEDB database.

HC2c (SEQ ID NO:23), humanizing mutations are underlined, CDRs and vernier zones are in bold, stabilization mutations are in italics (Q at position 1 , A at position 9, G at position 44, Y at position 80 and E at position 90 shown below) and an anti-aggregation mutation at K at position 86:

QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYNIYWVKQS

PGKGLEWIGYFDPYNGNTGYNQKFRGKATLTVDKSSSTAY MHLSSKTSE£SAVYYCANYYRYDDHAMDYWGQGTSVTVSS

HC3a (SEQ ID NO:24), grafted mutations shown in underline and CDRs and vernier zones shown in bold:

QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYNIYWVRQA PGQGLEWIGYFDPYNGNTGYNQKFRGRATLTVDKSTSTAY MELRSLRSDDTAVYYCA YYRYDDHAMDYWGQGTLVTVSS

LC3b (SEQ ID NO:25), grafted mutations shown in underline and CDRs and vernier zones shown in bold:

QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYNIYWVRQA

PGQGLEWMGYFDPYNGNTGYNQKFRGRVTMTTDTSTSTAY MELRSLRSDDTAVYYCANYYRYDDHAMDYW

GQGTLVTVS S

Note that the following Vernier Residue are mutated to human: V at position 2, M at position 48, V at position 68, M at position 70 and T at position 74.

No human epitopes were identified for sequence HC3b in IEDB database.

HC3b germ inal ity i ndex = 83% with Z12316_1 _V_J00235_1 _D_U42590_1 _J [1 -18/D P-14] .

Table 19: Stabilizing Changes Proposed in Light Chain

Table 20: Stabilizing Changes Proposed in Heavy Chain

sn- rg . o - ern er reg on

Table 21 : Combinations of stabilizing mutations evaluated

*Note: Sequential numbering used to refer to residues

Example 6: Characterization of Humanization Variants

Based on the in silico modeling presented in Table 16, the variable region of the light chain (VL) and heavy chain (VH) DNA of humanized F151 were codon optimized for HEK293 expression and gene synthesized by GeneArt (subsidiary of Life Technologies). The synthesized DNA fragments were cloned into the constant region of the light chain (CL) encoding vectors, pFF0362 (A. Human Kappa LC vector) at ApaLI/BsiWI sites and the constant regions of the heavy chain (CH1 , CH2 and CH3) encoding vectors, pFF0363 (B. Human lgG1 HC vector) at ApaLI/Apal sites respectively. The resulted plasmids pFF0460 containing the full sequence of LC and pFF0466 containing the full length of HC of humanized F151 variants were co-transfected and

transiently expressed in Freestyle™ 293 Expression System (Invitrogen/Life Technologies, catalog no. K9000-01 ).

The six humanized variants shown in Table 16 were characterized by various parameters such as binding kinetics (discussed above) as well as chemical and physical properties such as thermostability that are routinely used in the art.

The characterization was done in two tiers. Tier I included differential scanning calorimetry (DSC) shown in Table 24 and Figure 2. Briefly, for the DCS experiments, the antibodies were dialyzed against phosphate-buffered saline solution. Antibody concentrations were measured by UV absorbance. The antibodies were diluted to 1 mg/mL using PBS. Scans were performed using a Calorimetry Sciences Corporation N-DSC II instrument using a 0.3268 m l_ capillary cell with PBS in the reference cell. The scan rate was 2O/min and the samples were scanned from 20Ό to 100Ό.

All variants, except for HC3b/LC3b showed comparable binding affinities to the parental antibody. Variant HC3a/LC3a was selected over the other variants based on other

physiochemical properties such as SEC data, stability and lack of aggregation (see Tables 23- 25).

Table 23 . Comparison of Kinetics of the Humanized F151 Variants

DAKLP-b 4.46E+05 1 .30E-05 2.97E-1 1 9.03E+05 1 .10E-05 1.21 E-1 1

KLP-b 4.01 E+05 2.20E-06 5.76E-12 5.16E+05 1 .02E-05 1.98E-1 1

HC3a LC3a HC3b/LC3b

Ka(1/Ms) Kd(1/s) KD(M) Ka(1/Ms) Kd(1/s) KD(M)

DAKD-b 5.06E+05 1 .28E-05 2.53E-1 1 3.85E+05 5.15E-05 1.35E-10

KD-b 4.27E+05 2.95E-06 6.78E-12 2.51 E+05 3.02E-06 1.44E-1 1

DAKLP-b 4.65E+05 1 .42E-05 3.05E-1 1 7.04E+04 2.76E-03 4.05E-08

KLP-b 5.02E+05 5.43E-07 1.06E-12 5.39E+05 2.72E-04 5.26E-10

For comparison: Ka(1/Ms) of mF151 was 7.84E+05 for DAKD-b, 8.30E+05 for KD-b, 1.81 E+06 for DAKLP-b, and 1.12E+06 for KLP-b

Table 25. Tier 2 comparison of humanization variants

Table 26. Comparison of Parental F151 and humanized variant F151 (HC3a/LC3a)

For alignment of light and heavy chains of parental F151 to humanized F151 variant (HC3a/LC3a) , see Figure 3.

Example 7: Crystal Structure of Humanized Antibody F151 against BRK1 Ligand Kallidan and des-arg10-Kallidin

The crystal structures of humanized F151 (HC3a/LC3a) Fab bound to kallidan or des-arg10- kallidin was determined and the molecular interactions analyzed.

Kail idin powder was purchased from Phoenix Pharmaceuticals (Cat. No. 009-37) . For Fab protein generation, the DNA of heavy chain (HC) VH region from humanized F151 HC3a was cloned into 6XHis tagged CH1 vector pFF0366. The light chain (LC) plasmid used here was the same as of the original F151 LC3a used in F151 humanization (see Example 5). The two plasmids were co-transfected into free style HEK293 cells for Fab expression. The Fab protein was purified using cobalt-resin, buffer exchanged to 50 mM MES pH6.0, 50mM NaCI before being concentrated to about 9 mg/m L. Purified F151 Fab protein was mixed with kallidin in a molar ratio of 1 :2 and set up for crystallization screening. Crystallization screening was done with a wide range of conditions. The best crystal was observed under condition B10, B12 and G10 of Hampton Research screening kit PEG/ION HT. The crystals were cryo-protected with 20% glycerol in well buffer and frozen for diffraction data collection. The X-ray diffraction data for both complexes were collected at Canadian Light Source, beamline CMCF-08ID. The Rmerge for the F151 -KD complex is 8.9% and l/s(l) = 20.2 , while those for the F151 -DAKD are 7.7% and 18.5, respectively. The F151 -KD structure was solved by molecular replacement in Phaser using Fab coordinates from PDB entry 3QOS, treating the VL-VH and CL-CH1 domains as independent units. The structure was refined in autoBuster at 2.07 A resolution in space group P2i2i2i to an Rfactor of 0.205 and an Rfree of 0.228. The F151 -DAKD structure was solved using the F151 - KD coordinates. The structure was refined in autoBuster at 1 .86 A resolution in space group P212121 to an Rfactor of 0.232 and an Rfree of 0.238.

The electron density maps shown in Figures 4 and 5 depict the binding of kallidin (KD) and Des- Arg10-kallidin (DAKD) to the F151 Fab and unambiguously determine the positions of each amino acid. For kallidin, the electron density for the extreme C-terminal residue Arg10 is not present. This is in agreement with the observation that DAKD, which is missing the C-terminal residue arginine (shown in Table 27 below), binds equally well to F151 as KD. The IC50 values of F151 in the neutralization FDSS cellular assay towards KD and DAKD are 0.12 nM and 0.09 nM, respectively. In both cases the electron density is weaker towards the C-termini of the peptides. Since Phe9 in KD has slightly better electron density than that in DAKD, it is possible that the presence of the additional arginine at the C-terminus of KD stabilizes the C-terminus of this peptide when binding to F1 51 although this arginine itself is not stable enough to be observed by X-ray. Since the two structures are essentially identical (rms between KD and DAKD is 0.139 for C atoms and 0.328 for all atoms), all the following discussions are based on the F151 -KD structure.

Table 27. A selected list of kinin peptides

KD is bound with its N-terminus buried in the interface between Fv subunits of the light and heavy chains, as shown in Figure 6. The interface between light and heavy chains are packed with aromatic amino acids, including Tyr-L42, Tyr-L93, Tyr-L100, Trp_L102, Phe-L104 and Tyr- H35, Trp-H47, Tyr-H50, Tyr-H99, Trp-H1 10, stabilizing each other through stacking and hydrophobic interactions. Residues from each of the CDR's of light and heavy chains contribute to the binding. The residues along the light and heavy chains that are involved in interactions with KD as mapped on the CDRs are shown in Figures 7 and 8. CDR H3 of the heavy chain is the longest loop and the one most frequently used in the interactions with KD, forming a side cover for KD. The loop was stabilized mostly through interactions with the other two CDRs, H1 and H2 of the heavy chain, namely, Salt bridge between Asp-H101 and Arg-H52 (stabilizing H1 and H3), arene-H interaction between Tyr-H102 and Tyr-H54 (stabilizing H2 and H3), H-bond between Asp-H108 and Tyr-H35 and H-bond between His-H105 and Tyr-L55 (stabilizing H3 and L2).

Comparing the KD interacting residues among the antibodies generated, it can be seen that there is similarity among the antibodies, and some were more related in use of particular amino acids for KD-interaction than others. For example, in the light chain F1 51 , C63 and I22 use more similar amino acids in their CDRs to bind KD, while B21 and I54 were more similar. In the heavy chain, F151 and C63 were surprisingly unique from each other and from B21 , I22 and I54. The latter three appear to form a group in similarity. C63 is particularly interesting in its heavy chain, that the loop length in H2 and H3 are more different from all others. Considering the Fab as a whole, B21 and I54 were most closely related.

In the crystal structure, we found that KD is involved in systematic hydrogen bond and hydrophobic interactions with the Fab. The N-terminus of KD is buried in the Fab and harbors more intensive interactions, while the C-terminus is essentially solvent exposed. Except for the first 4 residues (Lys-Arg-Pro-Pro), the other residues of KD gradually extend into the bulk solvent. The amidinium group of Lys1 sidechain is anchored by Glu-L61 (L: light chain) through salt bridges, while the amino terminal amino group Lys 1 forms a salt bridge with Asp-H108 (H : heavy chain). The amidinium group of Lys1 sidechain also hangs over the aromatic ring of Tyr-L55, involved in cation interactions. Such intensive interactions involved with Lys1 tightly anchor the amino terminus of KD in the Fab. This also accounts for the importance of Lys1 in the binding of KD to F151 . Without it (i.e. bradykinin), no detectable binding to hF151 or F151 can be measured. Like Lys1 , Arg2 interacts with the Fab through a salt bridge. The guanidium group of Arg2 interacts with the sidechain of Asp-H104. The sidechain of Arg2 is also H-bonded with the mainchain carbonyl oxygen of Arg-H101 . Also, the mainchain oxygen of Pro8 is H-bonded to the sidechain of Arg-H101 . Tyr-H102 is half-way intercalated into Phe8 and Pro9, involving in hydrophobic interactions with KD. In addition to direct interaction, numerous water-mediated H-bonds between KD and the Fab are also seen. It is also interesting to notice that tyrosine residues are most frequently used in the interaction compared to other amino acids; 9 out of the 16 residues marked with asterisks in Figures 7 and 8 are tyrosines. All the residues in F151 surrounding KD appear to play a role in ligand binding, except for Asn-H33, which is close to

Phe6 sidechain but incompatible in polarity and lack of other important interactions. Substitution with aromatic/hydrophobic residues, such Trp or Tyr to interaction with Phe8 appears to be a quick pick if affinity maturation is considered. These two aromatic amino acids are in fact seen in other antibodies (Trp in C63, and Tyr in B21 , I22, I54). Table 28 below provides a detailed analysis of 16 KD-interacting amino acid residues marked in Figures 7 and 8 and sets forth functional substitutions that can be made in the CDR regions that should not disrupt antigen binding.

Table 28. A list of amino acid residues found around the KD binding pocket, and their roles in KD binding and potential functional substitutions (light chain residues in grey-colored cells and heavy chain residues in unshaded cells)

• Part of aromatic interface between H and L chains • Mutate to small aromatic a. a. except W,

Tyr-H99

• H-bond with Asn-H33 such as Phe and His

• Tight space

Arg- • H-bond with amide of Pro8 • Mutate as a pair with Asp-H52 to H101 • supported by Asp-H52 (salt bridge) reversely charged a. a., such Arg- H52 Asp-H101 , or a pair of hydrophobic a. a. to form a cluster with Phe6 of KD

Tyr-H102 • Half-way intercalating into Phe9 and Pro8, hydrophobic • Phe can be better, Trp or His may be OK interactions with KD too

Asp- • Key residue to salt-bridge with Arg2 • Glu to maintain salt bridge with Arg2 Hi 04 • Bigger a.a to fill the gap from Pro3, such as Tyr as seen in B21 , I22 and I54

Asp- • Key residue to salt bridge with N-term -NH3+ of KD • Glu

Hi 08 • H-bond with Tyr-H33, stabilizing H3 loop

• Conserved residue! Not in CDR

Analysis of the conformational epitope of kallidin (KD) or desArgl O-Kallidin (DAKD) revealed that it adopts a "Pro4 kink" conformation. As depicted in Figure 1 7, a hallmark of the "Pro 4 kink" conformation is a type I I tight turn in the main chain polypeptide backbone of KD or DAKD at Proline 4 (see Richardson JS. "The anatomy and taxonomy of protein structure." Adv Protein Chem . 1981 ;34:1 67-339, which is incorporated by reference herein). The "Pro 4 kink" conformation may further defined by all or substantially all of the remaining amino acids of KD (1 -2 and 6-9) or DAKD adopting repeats of a sigmoid shape which align the hydrophobic side chains in a spatially stacking mode.

Example 8: In vivo Pharmacology of anti-BKR1 -Ligand Antibodies in Pain Models

The examples of the present invention illustrate the in vivo efficacy of anti-BKR1 receptor-ligand antibodies in different preclinical models of acute and chronic pain according to modified procedures described in (a) Saddi GM and Abbott FV., Pain (2000), 89:53-63; (b) Chen et al., Molecular Pain (2010), 2:6-13 and (c) Bennett GJ and Xie YK., Pain (1988), 33:87-107.

Animals

Experiments were carried out using adult male OF1 mice (20-30 gr) for formalin studies and adult male C57BI/6J mice (25-30 gr) for both CFA and CCI studies. The mice were kept in a controlled temperature room under a 12-h light-dark cycle. Food and water were provided ad libitum. For all of the experiments, mice were acclimatized to the laboratory room for at least 2 hours before testing. No randomization was performed for the studies. Experimenters performing the behavioral tests were not blind to treatment; however they were not aware of the study hypothesis. All procedures have been approved by the "Comite d'Experimentation pour la Protection de Γ Animal de Laboratoire" (Animal Care and Use Committee) of sanofi-aventis recherche & developpement and were carried out in accordance with French legislation (Decree n ¾7-848 - 19 October 1987 - and decision -19 April 1988) implementing European directive 86/609/EEC.

A. Formalin-induced acute inflammatory pain

The formalin test was used to measure nociceptive and inflammatory pain. Indeed, intraplantar injection of formalin induces an initial acute nociceptive behavioral response (0-12 minutes), followed by a second inflammatory-mediated response (1 5-45 minutes) , which is attributed to spinal cord excitability.

Formaldehyde (37%, Sigma) was diluted in saline (v/v) to obtain a 2.5% formaldehyde concentration (i.e.≡ 6.25% formalin concentration). Mice were gently restrained and 20 μΙ_ of this solution was injected subcutaneously into the dorsal part of one hind paw. Behavioral responses were scored immediately after formalin injection, then at 3 minutes intervals over 45 minutes as follows: (0) : normal weight bearing of the injected paw; (1 ) : injected paw resting lightly on floor; (2) : lifting-elevation of the injected paw; (3) : licking or biting the injected paw. Group sizes were 1 1 -12 male OF1 mice.

Scores were plotted versus time and areas under the curves (AUC) were calculated from the mean scores (+SEM) for both the early (0-12 min) and the late (15-45 min) phases. Reversal of pain-like behaviors was expressed as change in AUC in %.

EE1 antibody inhibited the pain-like behavior in the late phase of the formalin test in male OF1 mice. EE1 antibody, when administered intravenously 48 hours before intraplantar injection of formalin, showed a dose dependent reversal of the pain-like behavior only in the late phase with a Minimal Effective Dose (MED)=2.5 mg/kg, as depicted in Figure 9. Indeed, when administered at 2.5, 10 and 30 mg/kg, EE1 reversed the late phase by 35+5%, 33+5% and 45+7%, respectively, as depicted in Table 29.

In contrast, F151 weakly inhibits the pain-like behavior in the late phase of the formalin test when administered 48 hours before intraplantar injection of formalin. Indeed, when administered at 2.5 and 10 mg/kg, F151 reversed the late phase by 1 5+7% and 21+5%, respectively, as depicted in Table 29.

Table 29. Effect of EE1 and F151 antibodies on formalin-induced pain-like behavior in male OF1 mice

B. CFA (Complete Freund's adjuvant)-induced chronic inflammatory pain

Chronic inflammation was induced under brief anesthesia (Isoflurane, 3%) by an intraplantar administration of 25 μΙ_ of Complete Freund's Adjuvant (CFA) containing i Mg/ML heat-killed Mycobacterium tuberculosis in mineral oil and mannide monooleate (Sigma). Group sizes were 8 male C57BI/6 mice.

EE1 antibody was administered intravenously 22 hours after intraplantar CFA injection at 2.5 and 30 mg/kg and mechanical and thermal hypersensitivities were assessed at Day 1 (D1 ), Day 4 (D4) and Day 7 (D7) post-CFA intraplantar administration.

B1. Mechanical Hypersensitivity

Mechanical hypersensitivity was assessed by measuring the Frequency of withdrawal Response (FR, in %) following 10 applications of a 0.6 g Von Frey filament (Bioseb, France) onto the plantar surface of the injected paw.

To investigate the efficacy of EE1 antibody on pain-like behavior, we calculated the reversal of mechanical hypersensitivity (in %) as follows:

Percent reversals were calculated as (Mean FR-isotype-controlpostdose - FR-lpsipostdose) / (Mean FR-isotype-controlpostdose - Mean FR-shamp0stdose) for each mouse.

At D1 , D4 and D7 after intraplantar injection of CFA, a significant increase of FR to the Von Frey filaments was observed in the isotype-control 1 B7.1 1 -treated group in comparison with the naive group, demonstrating the development of mechanical hypersensitivity. EE1 antibody, when administered intravenously 22 hours after intraplantar CFA, was able to significantly decrease this FR at the different times studied compared with that obtained in the isotype-control 1 B7.1 1 -treated group. (Figure 10).

Reversal of mechanical hypersensitivity was 41+8% and 22+8% at D1 , 36+9% and 32+9% at D4 and 27+10% and 50+9% at D7 for a 2.5 mg/kg and 30 mg/kg intravenous administration of EE1 antibody, respectively (Table 30).

Table 30. Effect of EE1 antibody on CFA-induced mechanical hypersensitivity in male

C57BI/6 mice

B2. Thermal Hypersensitivity

For thermal hypersensitivity, measures of Paw Withdrawal Latencies (PWL, in seconds) in response to a radiant heat using a plantar apparatus (IITC, Woodland Hills, USA) were assessed.

To investigate the efficacy of EE1 antibody on pain-like behavior, we calculated the reversal of thermal hypersensitivity (in %) as follows:

Percent reversals were calculated as (PWLpostdose - Mean isotype-controlpostdose) / (Mean isotype- controlpredose - Mean isotype-controlpostdose) for each mouse.

Thermal hypersensitivities were not different between all groups at baseline, before intraplantar injection of CFA (data not shown).

At D1 , D4 and D7 after intraplantar injection of CFA, a significant decrease in paw withdrawal latency of the injected paw was observed in isotype-control 1 B7.1 1 -treated group of mice, demonstrating that CFA induced a thermal hypersensitivity (data not shown).

EE1 antibody, administered intravenously 22 hours after intraplantar CFA injection (i.e. on Day 1 post-intraplantar CFA injection), and was not able to increase the Paw Withdrawal Latency at D1 , whatever the dose tested (Figure 1 1 ). However, EE1 significantly increased the Paw Withdrawal Latency at D4 and this effect was also present at D7 (Figure 1 1 ).

Reversal of thermal hypersensitivity was 41 +15% and 58+21 % at D4 and 46+10% and 52+17% at D7 for a 2.5 mg/kg and 30 mg/kg intravenous administration of EE1 , respectively (Table 31 ).

Table 31. Effect of EE1 antibody on CFA-induced thermal hypersensitivity in male C57BI/6 mice

C. CCI (Chronic Constriction lnjury)-induced neuropathic-like pain (Bennett's model)

CCI model was used as a model of peripheral nerve injury. Briefly, mice were anesthetized with Isoflurane (3%), and the right sciatic nerve was exposed at mid thigh level through a small incision. Three loose ligatures of 6.0 chromic gut (Ethicon) at 1 mm space were placed around the sciatic nerve. The surgical procedure was completed by closing the muscles and skin. The day of CCI surgery was considered as Day 0. Group sizes were 6-10 male C57BI/6 mice.

EE1 antibody was administered intravenously at 2.5 and 30 mg/kg on Day 1 1 post surgery and mechanical and thermal hypersensitivities were assessed on Day 12 (D12), Day 14 (D14) and Day 18 (D18) post-surgery which corresponded to Day 1 (D1 ) , Day 3 (D3) and Day 7 (D7) post-treatment.

C1. Mechanical Hypersensitivity

Mechanical hypersensitivity was assessed by measuring hind paw withdrawal thresholds (on both injured [i.e. Ipsi] and non-injured [i.e. Contra] paws) to an increasing pressure (in g) stimulus using a Dynamic Plantar Aesthesiometer (Ugo-Basile, Italy) ; a steel rod was applied to the hind paws of the mice with an increasing force (5 grams in 10 seconds).

To investigate the efficacy of EE1 antibody on pain-like behavior, we determined the reversal of mechanical hypersensitivity as follows: percent reversals were calculated as (lpsipostdose -Ipsipredose) / (Contrapredose - lpsipredose) for each mouse.

Following surgery, operated mice developed a robust sensitization to mechanical stimulus on the injured paw, whereas the non-injured paw was not affected. At Day 1 1 , the mechanical sensitization on the injured paw reached a plateau (data not shown).

EE1 antibody, administered intravenously on Day 1 1 demonstrated a slight tendency to reverse CCI-induced mechanical hypersensitivity on D12, D14 and D18 with 15.2+4.9% and 1 5.2+5.7% on D12, 26.8+5.7% and 25.7+4.5% on D14 and 30.3+7.1 % and 20.8+5.9% on D18, at 2.5 and 30 mg/kg respectively (Figure 12 and Table 32).

Table 32. Effect of EE1 antibody on CCI-induced mechanical hypersensitivity in male C57BI/6 mice

C2. Thermal Hypersensitivity

For thermal hypersensitivity, measures of Paw Withdrawal Latencies (in seconds) in response to a radiant heat using a plantar apparatus (IITC, Woodland Hills, USA) were assessed on the injected hind paw.

To investigate the efficacy of EE1 antibody on pain-like behavior, we calculated the reversal of thermal hypersensitivity (in %) as follows:

Percent reversals were calculated as ( lpsipostdose - Mean isotype-controlpostdose) / (Mean naivePostdose - Mean isotype-controlp0stdose) for each mouse.

Following surgery, operated mice developed a robust sensitization to thermal stimulus on the injured paw, whereas the non-injured paw was not affected. At Day 1 1 , the thermal sensitization on the injured paw reached a plateau (data not shown) .

EE1 antibody, administered intravenously on Day 1 1 did not significantly increase Paw

Withdrawal Latency of the injured paw on D12, even if a trend was observed. However, from D14, EE1 antibody significantly increased the Paw Withdrawal (Figure 13).

Reversal of thermal hypersensitivity was 41 +16% and 56+24% at D12 and 51+16% and 98+48% at D14 and 78+19% and 84+22% at D18, for a 2.5 mg/kg and 30 mg/kg intravenous administration of EE1 antibody, respectively (Table 33).

Table 33. Effect of EE1 antibody on CCI-induced thermal hypersensitivity in male C57BI/6 mice

Kinetic evaluation
We Claim :

1 . An isolated monoclonal antibody or antigen binding fragment thereof that:

a) specifically binds to Kallidin or des-Arg10-Kallidin but not to Bradykinin or des-Arg9-Bradykinin;

b) specifically binds to Kallidin or des-Arg10-Kallidin with a KD of less than 1 x1 0"10 M; c) specifically binds to Kallidin or des-Arg10-Kallidin with a Koff of less than 1 x1 04 s"1 ; and/or

d) specifically binds to Kallidin or des-Arg10-Kallidin and inhibits binding to the bradykinin B1 receptor.

2. The antibody or antigen binding fragment thereof of any of the preceding claims that binds to the N-terminal Lysine residue of Kallidin or des-Arg10-Kallidin.

3. The antibody or antigen binding fragment thereof of any of the preceding claims that inhibits the binding of Kallidin or des-Arg10-Kallidin to a bradykinin-1 receptor.

4. The antibody or antigen binding fragment thereof of any of the preceding claims that binds specifically to mouse Kail idin-like peptide (KLP).

5. The antibody or antigen binding fragment thereof of any of the preceding claims comprising a heavy chain variable domain comprising an HCDR3 amino acid sequence selected from the group consisting of:

a) SEQ ID NO: 7 [Χ,Υ Χζ X3D X4HAM X5Y], wherein

X! is Y, F or H,

X2 is R, D, A, V, L, I, M, F, Y or W,

X3 is Y, F, W or H,

X4 is D, E or Y, and,

X5 is D or E;

b) SEQ ID NO: 63 [X1 EYDGX2YX3X4LDX5], wherein

X! is W or F,

X2 is N or no amino acid;

X3 is Y or S,

X4 is D or P, and

X5 is F or Y;

c) SEQ ID NO: 13;

d) SEQ ID NO: 32

e) SEQ ID NO: 40

f) SEQ ID NO: 47; and

g) SEQ ID NO: 55.

6. The antibody or antigen binding fragment thereof of any of the preceding claims further comprising an HCDR2 amino acid sequence selected from the group consisting of:

a) SEQ ID NO: 8 [YFXT PXZNGNTGYNQKFRG], wherein

X! is D, R, A, V, L, I, M, F, Y or W, and

X2 is Y, D, E, N , or Q;

b) SEQ ID NO: 64 [WX! DPENGDXzXgYAPKFQG], wherein

X2 is T, or S, and

X3 is G, or D;

c) SEQ ID NO: 1 4

d) SEQ ID NO: 33;

e) SEQ ID NO: 41 ;

f) SEQ ID NO: 48; and

g) SEQ ID NO: 56.

7. The antibody or antigen binding fragment thereof of any of the preceding claims further comprising an HCDR1 amino acid sequence selected from the group consisting of:

a) SEQ ID NO: 9 [GYSFTDYXT IY], wherein X, is N, W or Y;

b) SEQ ID NO: 65 [GFN IKDYYXi H], wherein X, is L, or M;

c) SEQ ID NO: 1 5;

d) SEQ ID NO: 34;

e) SEQ ID NO: 42;

f) SEQ ID NO: 49; and

g) SEQ ID NO: 57.

8. The antibody or antigen binding fragment thereof of any of the preceding claims further comprising a light chain variable domain comprising an LCDR3 amino acid sequence selected from the group consisting of:

a) SEQ ID NO: 1 0 [QQ X, X2S X3P X4T], wherein

X2 is Y, F, H or W,

X4 is W. Y, F, H or L:

b) SEQ ID NO: 66 [QX1X2X3SX4PX5T], wherein

X! is Q or N,

X2 is Y, F, D or H,

X3 is Y, F, H or W,

X4 is Y, F, T or H, and

X5 is W. Y, F, H or L;

c) SEQ ID NO: 69 [XiQGTHFPYT], wherein is L or M;

d) SEQ ID NO: 16;

e) SEQ ID NO: 35;

f) SEQ ID NO: 43;

g) SEQ ID NO: 50; and

h) SEQ ID NO: 58.

9. The antibody or antigen binding fragment thereof of any of the preceding claims further comprising an LCDR2 amino acid sequence selected from the group consisting of:

a) SEQ ID NO: 1 1 [WASTRX^, wherein is E, D, Q or N ;

b) SEQ ID NO: 67 [X2ASTRX2], wherein

X2 is E, D, Q or N ;

c) SEQ ID NO: 1 7;

d) SEQ ID NO: 36;

e) SEQ ID NO: 51 ; and

f) SEQ ID NO: 59.

10. The antibody or antigen binding fragment thereof of any of the preceding claims further comprising an LCDR1 amino acid sequence selected from the group consisting of:

a) SEQ ID NO: 12 [KSSQSLL X^SNQKN X2LA], wherein

X! is W, H, Y or F, and

X2 is H or Y;

b) SEQ ID NO: 68 [KSSQSLLX1X2SX3QX4NX5LA], wherein

X2 is S or G,

X3 is N or D,

X4 is K or R,

X5 is H or Y.

c) SEQ ID NO: 70 [KSSQSLLYSNGXiTYLN], wherein is K or E;

b) SEQ ID NO: 18;

c) SEQ ID NO: 37;

d) SEQ ID NO: 44;

e) SEQ ID NO: 52; and

f) SEQ ID NO: 60.

1 1 . The antibody or antigen binding fragment thereof of any one of claims 1 -4 comprising a light chain variable domain comprising an LCDR3 amino acid sequence selected from the group consisting of:

a) SEQ ID NO: 10 [QQ X, X2S X3P X4T], wherein

X! is Y, F or H,

X2 is Y, F, H or W,

X3 is Y, F, T or H, and,

X4 is W, Y, F, H or L:

b) SEQ ID NO: 66 [QX1X2X3SX4PX5T], wherein

X2 is Y, F, D or H,

X3 is Y, F, H or W,

X4 is Y, F, T or H, and

X5 is W. Y, F, H or L;

c) SEQ ID NO: 69 [X^GTHFPYT], wherein is L or M;

d) SEQ ID NO: 16;

e) SEQ ID NO: 35;

f) SEQ ID NO: 43;

g) SEQ ID NO: 50; and

h) SEQ ID NO: 58.

12. The antibody or antigen binding fragment thereof of claim 1 1 further comprising an LCDR2 amino acid sequence selected from the group consisting of:

a) SEQ ID NO: 1 1 [WASTRX^, wherein is E, D, Q or N ;

b) SEQ ID NO: 67 [X2ASTRX2], wherein

X2 is E, D, Q or N ;

c) SEQ ID NO: 1 7;

d) SEQ ID NO: 36;

e) SEQ ID NO: 51 ; and

f) SEQ ID NO: 59.

13. The antibody or antigen binding fragment thereof of claim 12 further comprising an LCDR1 amino acid sequence selected from the group consisting of:

a) SEQ ID NO: 12 [KSSQSLL Xi SSNQKN X2LA], wherein

X! is W, H, Y or F, and

X2 is H or Y;

b) SEQ ID NO: 68 [KSSQSLLX1X2SX3QX4NX5LA], wherein

X2 is S or G,

X3 is N or D,

X4 is K or R,

X5 is H or Y.

c) SEQ ID NO: 70 [KSSQSLLYSNGXiTYLN], wherein X, is K or E;

b) SEQ ID NO: 18;

c) SEQ ID NO: 37;

d) SEQ ID NO: 44;

e) SEQ ID NO: 52; and

f) SEQ ID NO: 60.

14. The antibody or antigen binding fragment thereof of any one of claims 1 -4 comprising a heavy chain variable region comprising the HCDR3, HCDR2 and HCDR1 region amino sequences set forth in SEQ ID NOs 13, 14, and 15, respectively, and one or more amino acid substitution at positions selected from the group consisting of H 1 , H5, H9, H1 1 , H12, H 16, H38, H40, H41 , H43, H44, H66, H75, H79, H81 , H82A, H83, H87, and H108 according to Kabat.

15. The antibody or antigen binding fragment thereof of claim 14 further comprising a light chain variable region comprising the LCDR3, LCDR2 and LCDR1 region amino sequences set forth in SEQ ID NOs 1 6, 1 7, and 18, respectively, and one or more amino acid substitution at positions selected from the group consisting of L5, L9, L15, L1 8, L19, L21 , L22, L43, L63, L78, L79, L83, L85, L100 and L104, according to Kabat.

16. The antibody or antigen binding fragment thereof of any one of claims 1 -4 comprising a light chain variable region comprising the LCDR3, LCDR2 and LCDR1 region amino sequences set forth in SEQ ID NOs 16, 1 7, and 18, respectively, and one or more amino acid substitution at positions selected from the group consisting of L5, L9, L1 5, L18, L19, L21 , L22, L43, L63, L78, L79, L83, L85, L100 and L104 according to Kabat.

17. The antibody or antigen binding fragment thereof of any one of claims 1 -4 comprising a heavy chain variable region amino acid sequence with at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, 21 , 22, 24, 25, 38, 45, 53, and 61 .

18. The antibody or antigen binding fragment thereof of claim 17 further comprising a light chain variable domain amino acid sequence with at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29, 29, 30, 31 , 39, 46, 54, and 62.

19. The antibody or antigen binding fragment thereof of any one of claims 1 -4 comprising a light chain variable region amino acid sequence with at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29, 29, 30, 31 , 39, 46, 54, and 62.

20. The antibody or antigen binding fragment thereof of any one of claims 1 -4 comprising a heavy chain variable domain comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 19, 20, 21 , 22, 24, 25, 38, 45, 53, and 61 .

21 . The antibody, or antigen binding fragment thereof, of claim 20, further comprising a light chain variable domain amino acid sequence selected from the group consisting of: SEQ ID NO: 26, 27, 28, 29, 29, 30, 31 , 39, 46, 54, and 62.

22. The antibody or antigen binding fragment thereof of any one of claims 1 -4 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 26, 27, 28, 29, 29, 30, 31 , 39, 46, 54, and 62.

23. The antibody or antigen binding fragment thereof of any one of claims 1 -4 comprising the heavy chain and light chain variable region amino acid sequences set forth in SEQ ID NO: 19 and 26, SEQ ID NO: 20 and 27, SEQ ID NO: 21 and 28; SEQ ID NO: 22 and 28; SEQ ID NO: 23 and 29; SEQ ID NO: 24 and 30; SEQ ID NO: 25 and 31 ; SEQ ID NO: 38 and 39, SEQ ID NO: 45 and 46, SEQ ID NO: 53 and 54, or SEQ ID NO: 61 and 62, respectively.

24. An antibody, or antigen binding fragment thereof, that specifically binds to Kallidin and des-Arg10-Kallidin, wherein the antibody, or antigen binding fragment thereof, competes for binding to Kallidin and des-Arg10-Kallidin with an antibody comprising the heavy chain and light chain variable region amino acid sequences set forth in SEQ ID NO: 1 9 and 26, SEQ ID NO: 38 and 39, SEQ ID NO: 45 and 46, SEQ ID NO: 53 and 54, or SEQ ID NO: 61 and 62, respectively.

25. An isolated monoclonal antibody or antigen binding fragment thereof that competes for binding to Kallidin or des-Arg10-Kallidin with the antibody of any one of the preceding claims, and does not bind to Bradykinin or desArg9-Bradykinin.

26. An isolated monoclonal antibody or antigen binding fragment thereof that specifically binds to a conformational epitope of kail idin (KD) or desArgl O-Kallidin (DAKD) which adopts a Pro4 kink conformation comprising a type II tight turn at Proline 4 of the KD or DAKD).

27. The antibody or antigen binding fragment of claim 26, wherein the Pro 4 kink conformation of KD or DAKD further comprises amino acid repeats of a sigmoid shape which align the hydrophobic side chains of the amino acids in a spatially stacking mode.

28. The antibody or antigen binding fragment of claim 26 or 27, which

(a) specifically binds Kallidin or des-Arg10-Kallidin but not to Bradykinin or des-Arg9- Bradykinin;

b) specifically binds to Kallidin or des-Arg10-Kallidin with a KD of less than 1 x1 0"10 M; c) specifically binds to Kallidin or des-Arg10-Kallidin with a Koff of less than 1 x1 04 s"1 ; and/or

d) specifically binds to Kallidin or des-Arg10-Kallidin and inhibits binding to the bradykinin

B1 receptor.

29. An antibody of any one of the preceding claims conjugated to a diagnostic or therapeutic agent.

30. An isolated nucleic acid encoding the amino acid sequence of the antibody, or antigen binding fragment thereof, of any one of the preceding claims.

31 . A recombinant expression vector comprising the nucleic acid of claim 30.

32. A host cell comprising the recombinant expression vector of claim 31 .

33. A method of producing an antibody that binds specifically to Kallidin and des-Arg10-Kallidin, comprising culturing the host cell of claim 32 under conditions such that an antibody that binds specifically to Kallidin and des-Arg10-Kallidin is produced by the host cell.

34. A pharmaceutical composition comprising the antibody, or antigen binding fragment thereof, of any one of claims 1 -28 and one or more pharmaceutically acceptable carrier.

35. A method for treating a Kallidin or des-Arg10-Kallidin-associated disease or disorder, the method comprising administering to a subject in need of thereof the pharmaceutical composition of claim 34.

36. The method of claim 35, wherein the disease or disorder is chronic pain.

37. A method of generating an antibody that specifically binds to des-Arg9- Bradykinin and des-Arg10-Kallidin-like peptide comprising: immunizing an animal with an immunogen comprising a peptide, wherein the peptide consists of the amino acid sequence set forth in SEQ ID No.1 1 , and wherein the amino terminal arginine of the peptide is indirectly coupled to a carrier moiety through a linker moiety, such that an antibody that specifically binds to des-Arg9- Bradykinin, des-Arg10-Kallidin and des-Arg10-Kallidin-like peptide is produced by the immune system of the animal.

38. The method claim 37, further comprising isolating from the animal , the antibody, a nucleic isolating encoding the antibody, or an immune cell expressing the antibody.

39. The method claim 37, wherein the carrier moiety is a protein.

40. The method claim 39, wherein the protein is Keyhole limpet hemocyanin (KLH).

41 . The method claim 40, wherein the linker moiety comprises [Gly-Gly-Gly]n, wherein n is at least 1 .