Anti-C5a binding moieties with high blocking activity

Abstract

The present invention relates to binding moieties that specifically bind to a conformational epitope of C5a, in particular human C5a. Preferred binding moieties are anti-C5a antibodies that bind to this conformational epitope. The binding moieties described herein are useful as active agents in pharmaceutical compositions for the treatment and prevention of various acute and chronic diseases, in particular acute inflammatory diseases, such as the systemic inflammatory response syndrome (SIRS), and different degrees of sepsis including sepsis, severe sepsis, and septic shock.

Claims

1. An antibody or an antigen-binding fragment thereof comprising a VL and a VH domain, wherein the VL domain comprises a light chain CDR3 sequence having SEQ ID NO: 8, two tyrosine residues preceeding SEQ ID NO: 8 and a light chain FR4 comprising the sequence of SEQ ID NO: 58 or conservative amino acid substitution variants of SEQ ID NO: 58, wherein said antibody or said antigen-binding fragment thereof specifically binds to human C5a; and wherein said antibody or antigen-binding fragment thereof has a binding constant to human C5a with a K.sub.d value of 100 nM or less.

2. The antibody or the antigen-binding fragment thereof according to claim 1, wherein said antibody or said antigen-binding fragment thereof exhibits one or more of the following properties: said antibody or said antigen-binding fragment thereof has a binding constant to C5a with a K.sub.d value of 10 nM or less; said antibody or said antigen-binding fragment thereof exhibits at least 80% blocking activity for biological effects induced by one molecule C5a, wherein said biological effects are mediated by C5a-05aR interaction; said antibody or said antigen-binding fragment thereof does not inhibit CH50 activity in human plasma; and said antibody or said antigen-binding fragment thereof is capable of reducing E. coli induced IL-8 production in human whole blood.

3. The antibody or the antigen-binding fragment thereof according to claim 1, wherein said antibody or said antigen-binding fragment is an antibody selected from the group consisting of polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and human antibodies.

4. The antibody or the antigen-binding fragment thereof according to claim 1, wherein said antibody or said antigen-binding fragment is an antigen-binding fragment of an antibody selected from the group consisting of Fab fragments, Fab fragments, F(ab).sub.2 fragments, Fv fragments, disulfide-linked Fvs (dsFv), and single chain Fv (scFv) antibodies.

5. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to claim 1 and further comprising one or more pharmaceutically acceptable carriers, diluents, excipients, fillers, binders, lubricants, glidants, disintegrants, adsorbents, and/or preservatives.

6. A method of blocking C5a-induced biological effects in a patient with acute or chronic inflammation, wherein said method comprises administering to a patient in need of such blocking of C5a-induced biological effects, the pharmaceutical composition of claim 5.

7. A method for the treatment of a disease involving acute or chronic inflammation, wherein said method comprises administering, to a patient in need of such treatment, the pharmaceutical composition of claim 5.

8. The method of claim 7, wherein the disease involving acute or chronic inflammation is selected from the group consisting of systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock, ischemia/reperfusion related injuries, acute lung injury, pneumonia, acute and chronic graft rejection in transplant patients, graft versus host reactions, renal glomerular diseases, glomerulonephritis, entities of renal failure, rheumatoid arthritis, auto-immune diseases, Bechterew's disease, lupus-type diseases, inflammatory bowel disease, Crohn's disease, tumor growth, and solid organ cancer.

9. The antibody or the antigen-binding fragment thereof according to claim 1, wherein the conservative amino acid substitution variants of SEQ ID NO: 58 are hydrophobic amino acid substitutions.

10. The antibody or the antigen-binding fragment thereof according to claim 9, wherein at least one hydrophobic amino acid substitution is a leucine to valine substitution.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 Shows the Effect of C5a Mutants on Enzyme Releases from Blood Cells

(2) Potential amino acids in C5a molecules for constitution of antibody epitopes were mutated into alanine, and these C5a mutants were tested for their bioactivity to induce lysozyme releases from human whole blood cells. C5a site mutation resulting in more than 50% bioactivity loss in comparison to human C5a was considered as a critical site for C5a biological function. These sites are 24, 29, 31, 37, 68, and 69.

(3) FIG. 2 Shows the Binding Capability of INab308 to C5a Mutants

(4) C5a and C5a mutants were coated on a 96-well plate. The binding capabilities of INab308 to these proteins were assessed by ELISA approach. The loss of binding capability greater than 50% is considered significant. The data indicate that INab308 binds to two regions, 31-37 and 68-69.

(5) FIG. 3 Shows the Binding Capability of INab708 to C5a Mutants

(6) C5a and C5a mutants were coated on a 96-well plate. The binding capabilities of INab708 to these proteins were assessed by ELISA approach. The loss of binding capability greater than 50% is considered significant. The data indicate that INab708 binds to two regions, 31-37 and 68-70.

(7) FIG. 4: INab308 and INab708 do not Affect Human Plasma CH50 Activity

(8) Human plasma hemolytic activity was determined by classical CH50 assay. Mabs to human C5a including INab708, INab308, and F20, were pre-incubated with human plasma, and then CH50 assay was performed subsequently. Among these antibodies, F20 strongly inhibits CH50 activity, while INab708 and INab308 have no influence when used at a concentration of approx. 5 M, which is significantly higher than the C5 concentration occurring in human whole blood (approx. 0.4 M).

(9) FIG. 5A-C Shows a Comparison of the Blocking Effects of INab308, INab708, and L2B23 on C5a Bioactivity

(10) Blocking activity of INab308, INab708 and L2B23 was assessed by C5a-induced lysozyme release assay. The molar ratio of antibody to C5a was set to 1:2 to evaluate the blocking activity of one antibody to two C5a molecules-elicited biological effect. The data show that INab308 and INab708 possess very high blocking activity (>90%) to C5a bioactivity, while L2B23 only shows a minimal effect.

(11) FIG. 6 Shows the Inhibitory Effect of INab308 and INab708 on E. coli-Induced IL-8 Production in Human Whole Blood

(12) E. coli was incubated with whole blood for 4 hours, and IL-8 levels were assessed by ELISA. In the presence of INab308 and INab708 during the incubation, IL-8 levels were significantly attenuated (P<0.01), while there was no significant reduction in the presence of L2B23.

EXAMPLES

1. Methods

(13) 1.1 Recombinant C5a and C5a Mutant Preparation:

(14) A DNA sequence encoding human C5a was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) using RNA isolated from peripheral blood leukocytes. C5a mutants were generated using PCR methods by introducing the GCT (alanine) into the mutation site. The C5a DNA was then ligated with pET-32a (NOVAGEN|m, Gibbstown, N.J.), and the ligation mixture was used to transform JM109-competent cells. The expression plasmids were transformed into BL21 using a standard calcium chloride method. A single colony from a Luria-Bertani broth (LB) plate was picked up, inoculated into LB medium with ampicillin, and incubated at 37 C. overnight. The culture was transferred to 2 L of LB medium and incubated at 37 C. until the mid-exponential phase (A6000.6), and then isopropyl--D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM. The cells were allowed to continue to grow at 30 C. overnight and harvested by spinning the culture at 7,000 rpm, 4 C., for 15 min. After washing with phosphate-buffered saline (PBS; 10 mM PB, 150 mM NaCl [pH 7.4]) once, the bacterial pellet was resuspended in PBS and sonicated on ice. After centrifugation at 12,000 rpm, 4 C., for 15 min, the soluble fraction was separated from insoluble pellet. To purify human recombinant C5a, the supernatant of cell lysate was loaded on a nickel-chelated affinity column preequilibrated with PBS. Then the column was washed with 50 mM imidazole and 200 mM imidazole in PBS, respectively. Finally, the bound proteins were eluted by 500 mM imidazole, dialyzed against PBS overnight, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

(15) 1.2 Immunization and Hybridoma Screening by ELISA:

(16) Monoclonal antibodies were made using hybridoma methods. Immunization and production of MAbs were carried out using standard protocols. Five 4-week-old female BALB/c mice were subcutaneously immunized with 100 g of purified recombinant C5a in complete Freund's adjuvant per animal. The animals were boosted twice at 4-week intervals using 100 g of antigen in incomplete Freund's adjuvant. Three days after the final booster, mice were sacrificed, and its splenocytes were fused with NS-1 at a 5:1 ratio, and 200 L of cells was plated in each well on five 96-well plates. Hybrids were selected in a Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 20% fetal calf serum and 510.sup.3 M hypoxanthine, 210.sup.5 M aminopterin, and 810.sup.4 M thymidine (HAT).

(17) After 8 days, cell clones secreting antibodies against human C5a were screened by enzyme-linked immunosorbent assay (ELISA). Briefly, a 96-well plate was coated with 2 g/mL recombinant human C5a at 4 C. overnight. After being blocked with 5% nonfat milk in PBS at 37 C. for 1 h, 50 L of culture medium of the growing clones were added to each well and incubated at 37 C. for 1 h, followed by 100 L of goat anti-mouse antibody labeled with horseradish peroxidase (HRP) for 1 h. The peroxidase reaction was developed with color development solution containing 5.5 mM o-phenylene-diamine hydrochloride (OPD) and 8.5 mM H.sub.2O.sub.2. The light absorbance was measured at 492 nm with an ELISA reader (ANTHOS, Wals/Salzburg, Austria).

(18) 1.3 Production and Purification of Monoclonal Antibodies:

(19) To produce the Mab in large quantity, 510.sup.6 hybridoma cells were injected into the peritoneal cavity of mice. After 14 days, ascites were withdrawn and centrifuged at 1500 rpm, 4 C., for 5 min. The supernatant was collected and applied to a column of protein A-Sepharose 4B, which had been pre-equilibrated in PBS. The bound Mab was eluted with citric acid (pH 4.0) and dialyzed against PBS overnight. The purified proteins were analyzed by SDS-PAGE.

(20) 1.4 Enzyme Release Assay for C5a Bioactivity

(21) Induction of enzyme releases by degranulation is an important biological feature of C5a. In our study, fresh human whole blood from healthy volunteers was used to assess the effect of C5a on lysozyme releases. The lysozyme levels released from whole blood cells were analyzed by EnzChek Lysozyme Assay Kit (INVITROGEN, CA, USA). To study the blocking activity of anti-05a antibodies, rhC5a (100 nM) was mixed with different concentration of antibody. Thereafter, whole blood cells were immediately added to avoid pre-incubation of antibodies with C5a. After incubation, 50 I of the sample supernatants were added to 50 l of diluted substrate solution. Plate was incubated at 37 C. for 30 min in the dark and read out thereafter with PERKINELMER 1420 multilabel Counter (Massachusetts, USA). Fluorescence intensity was measured with an excitation at 490 nm and an emission at 525 nm and zero standard value (blank) was subtracted from all samples. Blocking activity of the antibody was calculated after the subtraction of fluorescence intensity of rhC5a-independent lysozyme release (buffer control) from fluorescence intensity of rhC5a-induced lysozyme release. Blocking activity was calculated with the following formula, blocking activity=C5a.sub.Fluorescence(C5a+Ab).sub.Fluorescence/C5a.sub.FluorescenceBuffer Control.sub.Fluorescence.

(22) 1.5 ELISA Analysis of INab308 and INab708 Binding Capability to Human C5a or C5a Mutants

(23) A binding Elisa was carried out to determine the binding activities of INab308 and INab708 to human C5a and C5a mutants. C5a mutants are produced by replacing the correspondent amino acid with alanine by introduction of GCT into the mutation site from the cDNA level. These C5a mutants include site mutation on 24, 29, 30, 31, 32, 35, 36, 37, 30/37 double mutation, 40, 53, 64, 65, 66, 68, 64/68, 66/68, 69, 70, and C-del (12 amino acids were deleted from C5a C terminus).

(24) Human C5a (Sigma C5788), recombinant C5a, and C5a mutants (2 g/mL) were coated on 96-well EIA plate (COSTAR 9018) at 4 C. overnight. After being blocked with 5% nonfat milk in PBS at 37 C. for 1 h, 0.08, 0.4, 2 g/ml of anti-05a antibodies (INab308 and INab708) prepared with dilution buffer were added to each well and incubated at 37 C. for 1 h, followed by 100 L of goat anti-mouse antibody labeled with horseradish peroxidase (HRP) for 1 h. The peroxidase reaction was developed with color development solution containing 5.5 mM o-phenylene-diamine hydrochloride (OPD) and 8.5 mM H.sub.2O.sub.2. The light absorbance was measured at 492 nm with an ELISA reader (ANTHOS, Wals/Salzburg, Austria). The OD value for recombinant C5a was set as 100% binding activity. The binding capability of C5a mutants is calculated by OD.sub.C5a mutant/OD.sub.C5a.

(25) 1.6 Plasma Hemolytic Activity (CH50):

(26) In brief, sheep red blood cells (sRBC) were prepared from fresh sheep whole blood by centrifugation, and then were sensitized with anti-sRBC. Plasma samples from healthy volunteers were serially diluted and incubated with sensitized sRBC. Half an hour after incubation, the unlysed cells were spun down, and the supernatants were read at 542 nm on a plate reader. To determine the effect of anti-C5a antibodies on C5 activation, equal volumes of anti-C5a antibody and plasma were pre-incubated for 1 hour prior to the addition of sensitized sRBC.

(27) 1.7 IL-8 Production in the Whole Blood Model of E. coli Infection:

(28) To assess the efficacy of anti-C5a antibodies in the setting close to clinical sepsis, 250 l of whole blood from healthy volunteers were spiked with anti-C5a antibodies, and 250 l of E. coli diluted in saline buffer with a concentration of 110.sup.7/ml were then added. After 4-hour incubation at 37 C., the supernatants were spun down and collected for ELISA analysis for IL-8. IL-8 levels in the supernatants were analyzed by IL-8 ELISA kit (BIOLEGEND, USA).

(29) 1.8 Assay for Screening Antibodies Binding to the New Conformational Epitope:

(30) In 3-D structure of C5a obtained from computer modeling method, the spatial epitopes containing peptide C5a 28-40 (VNNDETCEQRAAR, SEQ ID NO: 67) and C5a peptide 65-70 (ISHKDM, SEQ ID NO: 68) can be viewed as random coils. When the two peptides are linked by a flexible peptide linker, GGGGS (SEQ ID NO: 69), the spatial epitopes is reconstructed resembling the parent antigen conformation, as the weak hydrophobic interaction from the two peptides ensures a pocket-shape conformation. Computer modeling analysis of the peptide NH.sub.2-28-40-Linker(GGGGS)-65-70-COOH maintains the same conformation as the parent antigen. This new 24-AA peptide can be synthesized and conjugated with keyhole limpet hemocyanin (KLH) to form an immunogen to immunize mice, and the traditional hybridoma technology can be subsequently applied to obtain INab308 and INab708 using the new 24-AA peptide based ELISA as a screening tool.

(31) A 96-well ELISA plate is coated with 1-2 g/mL synthetic peptides with the conformational epitope at 4 C. overnight. After being blocked with 5% nonfat milk in PBS at 37 C. for 1 h, 50 L of culture media of the hybridoma growing clones are added to each well and incubated at 37 C. for 1 h, followed by 100 L of goat anti-mouse antibody labeled with horseradish peroxidase (HRP) for 1 h. The peroxidase reaction is developed with color development solution containing 5.5 mM o-phenylene-diamine hydrochloride (OPD) and 8.5 mM H.sub.2O.sub.2. The light absorbance is measured at 492 nm with an ELISA reader.

2. Results

(32) 2.1 Identification of Amino Acids Relevant for C5a Activity

(33) Several amino acids within the C5a molecule that constitute possible antibody epitopes were mutated into alanine. In particular, C5a mutants include site mutations on 24, 29, 30, 31, 32, 35, 36, 37, 30/37 double mutation, 40, 53, 64, 65, 66, 68, 64/68, 66/68, 69, 70, and C-del (12 amino acids were deleted from the C-terminus of C5a). The C5a mutants were tested for their bioactivity to induce lysozyme release from human whole blood cells (FIG. 1).

(34) C5a site mutation resulting in more than 50% bioactivity loss in comparison to human C5a was considered as a critical site for C5a biological function. Thus, amino acid residues 24, 29, 31, 37, 68, and 69 were identified as sites critical for function (FIG. 1).

(35) 2.2 Characterization of the Epitopes on C5a Bound by Antibodies INab308 and INab708

(36) 2000 cell clones secreting antibodies against human C5a were screened with the functional assay (enzyme release). Only two antibodies exhibited superior blocking activities.

(37) These two antibodies, INab308 and INab708, were further characterized with respect to the particular amino acids on C5a recognized by the two antibodies.

(38) In particular, several mutants of C5a were generated in which one or more amino acids were replaced by alanine. Antibodies INab308 and INab708 were contacted with these mutants and the extent of binding was determined by ELISA (see section 1.5 above). A loss of binding capability greater than 50% (as compared to wild-type C5a) was considered significant.

(39) The data indicate that INab308 binds to two regions, 31-37 and 68-69 (FIG. 2). Likewise, INab708 binds to two regions, 31-37 and 68-70 (FIG. 3).

(40) Notably, the regions identified for both antibodies cover four amino acid residues (31, 37, 68, and 69) that were identified as sites critical for C5a function in section 2.1 above.

(41) 2.3 Effect of Antibodies INab308, INab708, and F20 on Human Plasma Hemolytic Activity

(42) The total hemolytic complement titer (CH50) is a conventional method for determination of the activation of classical complement pathway (see section 1.6).

(43) Monoclonal antibodies to C5a (INab708, INab308, and F20) were pre-incubated with human plasma at a concentration of approx. 5 M, and then the CH50 assay was performed. Among these antibodies, F20 strongly inhibits CH50 activity, while INab708 and INab308 have no influence (FIG. 4).

(44) These results demonstrate that INab708 and INab308 do not interfere with the complement activation mediated by C5b.

(45) 2.4 Effect of Antibodies INab308, INab708, and L2B23 on C5a Bioactivity

(46) Blocking activity of antibodies INab308, INab708, and L2B23 on C5a was assessed by C5a-induced lysozyme release assay (see section 1.4). The molar ratio of antibody to C5a was set to 1:2 to evaluate the blocking activity of one antibody to two C5a molecules-elicited biological effect. These antibodies are bivalent antibodies. Accordingly, by choosing the above molar ratio of 1:2, the paratopes of the antibodies on the one hand and C5a on the other hand are present in equimolar concentrations.

(47) The data in FIG. 5 show that INab308 and INab708 possess very high blocking activity to C5a bioactivity (94% and 100%, respectively), while L2B23 only shows a minimal effect (about 12%).

(48) 2.5 Effect of Antibodies INab308 and INab708 on E. coli-Induced IL-8 Production in Human Whole Blood

(49) To assess the efficacy of anti-C5a antibodies in a setting close to clinical sepsis, E. coli-induced IL-8 production in whole blood was determined. This assay can be regarded as a model of E. coli infection (see also section 1.7).

(50) In the presence of INab308 and INab708 during the incubation, IL-8 levels were significantly attenuated (P<0.01), while there was no significant reduction in the presence of L2B23 (FIG. 6).

3. Summary

(51) Important properties of preferred antibodies of the invention INab308 and INab708 are summarized in Table 3 below. Further included are comparative antibodies 8g8, MAb 137-26, Ab11876, G57, F20, L2B23, and G13 which do not bind simultaneously to both amino sequences of the conformational epitope identified in the present invention, i.e. amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3. Comparative antibodies 8g8, MAb 137-26, F20, G57, L2B23, and G13 bind to only one of these two amino acid sequences (8g8, MAb 137-26, F20, G57, and G13) or to a different amino acid sequence (L2B23). The target epitope of Ab 11876 is not known.

(52) Monoclonal antibodies to human C5a were generated using classical hybridoma technology. Mabs' binding sites to human C5a were determined by alanine screening method. Mabs' blocking activities were quantitated by inhibition of C5a-induced lysozyme releases from human whole blood cells.

(53) TABLE-US-00004 TABLE 3 Neutralizing antibodies to C5a and the epitopes Affinity Ratio of Ab to Blocking Antibody Binding sites (Kd: nM) C5a activity INab308 31-37, 68-69 0.50 1/2:1 90% INab708 31-37, 68-70 0.51 1/2:1 90% 8g8 69-74 0.27 1/2:1 60% MAb 137-26* 35-46 0.06 1/2:1 55% Ab11876** ND ND 4:1 40% G57 31-37 ND 4:1 40% F20 68-69 7.06 10:1 70% L2B23 64-66 ND 10:1 50% G13 53, 66-68 ND 10:1 60% ND: Not determined; *ATCC clone no. PTA-3650 relating to an antibody disclosed in European patent application 1 878 441 A2; **Ab11876 is a monoclonal mouse anti C5/C5a antibody obtainable from ABCAM (Cambridge, UK).

(54) As shown in Table 3, some comparative antibodies (8g8, MAb 137-26) exhibit higher binding affinities to C5a than the preferred antibodies of the invention, INab308 and INab708. Nevertheless, INab308 and INab708 exhibit higher blocking activities than any comparative antibody studied. More specifically, each one of INab308 and INab708 exhibits a very high blocking activity (>90%), even when used in stoichiometric amounts, i.e. at a ratio of 1 paratope per 1 epitope; i.e. 0.5 antibody molecules per 1 target molecule. Antibodies of the prior art (MAb 137-26, Ab11876) achieved reasonable blocking activities only when used in superstoichiometric amounts. These findings demonstrate that high binding affinity cannot always be equated with high blocking activity.

(55) Summarizing, the present invention provides for the first time antibodies that exhibit an extremely high blocking activity, even when used just in stoichiometric amounts.

(56) The amino acid sequences of the complementarity determining regions of the heavy and light chains of antibodies INab308 and INab708 are listed below in Table 4.

(57) TABLE-US-00005 TABLE4 CDRandFRsequencesofantibodiesINab308andINab708 (Chothiaclassificationmode) INab308: INab708: HeavyChain: HeavyChain: FR1: FR1: QVQLQQSGPQLVRPGTSVKIS VQLLESGAELMKPGASVKIS (= SEQIDNO:51) (SEQIDNO:59) CDR1: CDR1: CKASGYSFTTFWMD CKATGNTFSGYWIE (= SEQIDNO:14) (= SEQIDNO:15) FR2: FR2: WVKQRPGQGLEWIGR WVKQRPGHGLEWIGE (SEQIDNO:52) (SEQIDNO:60) CDR2: CDR2: IDPSDSESRLDQ ILPGSGSTNYNE (= SEQIDNO:10) (= SEQIDNO:11) FR3: FR3: RFKDRATLTVDKSSSTVYMQLSSPTSEDSAVYY KFKGKATLTADTSSNTAYMQLSSLTSEDSAVYY (SEQIDNO:53) (SEQIDNO:61) CDR3: CDR3: CARGNDGYYGFAY CTRRGLYDGSSYFAY (= SEQIDNO:6) (= SEQIDNO:7) FR4: FR4: WGQGTLVTVSSA WGQGTLVTVSA (SEQIDNO:54) (SEQIDNO:62) Lightchain: LightChain: FR1: FR1: DIVLTQSPASLAVSLGQRATIS DIVLTQSPASLAVSLGQRATIS (SEQIDNO:55) (SEQIDNO:63) CDR1: CDR1: CKASQSVDYDGDSYMK CKASQSVDYDGDSYMN (= SEQIDNO:16) (= SEQIDNO:17) FR2: FR2: WYQQKPGQPPKLL WYQQKPGQPPKLL (SEQIDNO:56) (SEQIDNO:64) CDR2: CDR2: IYAASNL IYAASNL (= SEQIDNO:12) (= SEQIDNO:13) FR3: FR3: QSGIPARFSGSGSGTDFTLNIHPVEEEDAATYY GSGIPARFSGSGSGTDFTLNIHPVEEEVAATYY (SEQIDNO:57) (SEQIDNO:65) CDR3: CDR3: CQQSNEDPYT CQQNNEDPLT (= SEQIDNO:8) (= SEQIDNO:9) FR4: FR4: FGGGTKLEIK FGAGTLLELK (SEQIDNO:58) (SEQIDNO:66)

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SEQUENCE LISTING FREE TEXT INFORMATION

(59) SEQ ID NO: 6 INab308 CDR3 heavy chain

(60) SEQ ID NO: 7 INab708 CDR3 heavy chain

(61) SEQ ID NO: 8 INab308 CDR3 light chain

(62) SEQ ID NO: 9 INab708 CDR3 light chain

(63) SEQ ID NO: 10 INab308 CDR2 heavy chain

(64) SEQ ID NO: 11 INab708 CDR2 heavy chain

(65) SEQ ID NO: 12 INab308 CDR2 light chain

(66) SEQ ID NO: 13 INab708 CDR2 light chain

(67) SEQ ID NO: 14 INab308 CDR1 heavy chain

(68) SEQ ID NO: 15 INab708 CDR1 heavy chain

(69) SEQ ID NO: 16 INab308 CDR1 light chain

(70) SEQ ID NO: 17 INab708 CDR1 light chain

(71) SEQ ID NO: 18 Consensus sequence of C5a in the region of amino acids 30-38

(72) SEQ ID NO: 19 Consensus sequence of C5a in the region of amino acids 66-72

(73) SEQ ID NO: 20 Consensus sequence of C5a in the region of amino acids 31-37

(74) SEQ ID NO: 21 Consensus sequence of C5 in the region of amino acids 67-71

(75) SEQ ID NO: 51 INab308 FR1 heavy chain

(76) SEQ ID NO: 52 INab308 FR2 heavy chain

(77) SEQ ID NO: 53 INab308 FR3 heavy chain

(78) SEQ ID NO: 54 INab308 FR4 heavy chain

(79) SEQ ID NO: 55 INab308 FR1 light chain

(80) SEQ ID NO: 56 INab308 FR2 light chain

(81) SEQ ID NO: 57 INab308 FR3 light chain

(82) SEQ ID NO: 58 INab308 FR4 light chain

(83) SEQ ID NO: 59 INab708 FR1 heavy chain

(84) SEQ ID NO: 60 INab708 FR2 heavy chain

(85) SEQ ID NO: 61 INab708 FR3 heavy chain

(86) SEQ ID NO: 62 INab708 FR4 heavy chain

(87) SEQ ID NO: 63 INab708 FR1 light chain

(88) SEQ ID NO: 64 INab708 FR2 light chain

(89) SEQ ID NO: 65 INab708 FR3 light chain

(90) SEQ ID NO: 66 INab708 FR4 light chain

(91) SEQ ID NO: 69 Peptide linker