PL2563813T3

Anti-c5a antibodies and methods for using the antibodies

Abstract

This record has no abstract on file.

Term

4.6 yearsto projected expiry

Projected expiry 29 April 2031, counted from filing; an application has no term until it is granted.

1 claim: 1 independent, 0 dependent

1
Zastrzeżenia patentowe 1. Izolowane przeciwciało lub jego fragment wiążący antygen, które wiążą się do wolnego polipeptydu C5a, przy czym wolny C5a oznacza ludzki polipeptyd (hC5a) o sekwencji aminokwasowej przedstawionej w SEQ ID NO:1, i przy czym przeciwciało lub jego fragment wiążący antygen wiążą się do wolnego polipeptydu hC5a in vitro z KD, która jest niższa niż 1,25 x 10 -9 M jak zmierzono za pomocą rezonansu plazmonów powierzchniowych (SPR) w obecności molarnego nadmiaru nierozszczepionego, natywnego ludzkiego C5, i przy czym przeciwciało lub jego fragment wiążący antygen zawieraj ą: I. (a) polipeptyd łańcucha lekkiego zawieraj ący: (i) CDR1 łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:20;CDR2 łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:21;i CDR3 łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:22;lub (ii) CDR1 łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:20;CDR2 łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:38;i CDR3 łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:22;oraz (b) polipeptyd łańcucha ciężkiego zawieraj ący: (i) CDR1 łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:28;CDR2 łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:29;i CDR3 łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:30;lub (ii) CDR1 łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:28;CDR2 łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:46;i CDR3 łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:47;II. (a) polipeptyd łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:37 i polipeptyd łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:27;150 (b) polipeptyd łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:36 i polipeptyd łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:33;(c) polipeptyd łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:19 i polipeptyd łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:27;(d) polipeptyd łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:17 i polipeptyd łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:25;(e) polipeptyd łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:42 i polipeptyd łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:21;(f) polipeptyd łańcucha lekkiego zawieraj ący sekwencj ę aminokwasową przedstawioną na SEQ ID NO:40 i polipeptyd łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:33;(g) polipeptyd łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:17 i polipeptyd łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:33;(h) polipeptyd łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:19 i polipeptyd łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:45;(i) polipeptyd łańcucha lekkiego zawierający sekwencj ę aminokwasową przedstawioną na SEQ ID NO:17 i polipeptyd łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:44;(j) polipeptyd łańcucha lekkiego zawierający sekwencj ę aminokwasową przedstawioną na SEQ ID NO:17 i polipeptyd łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:49;(k) polipeptyd łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:37 i polipeptyd łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:45;lub (1) polipeptyd łańcucha lekkiego zawieraj ący sekwencję aminokwasową przedstawioną na SEQ ID NO:36 i polipeptyd łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:49;lub III. (a) polipeptyd łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:42 i polipeptyd łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:45;151 (b) polipeptyd łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:40 i polipeptyd łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:49;lub 2. Izolowane przeciwciało lub jego fragment wiążący antygen według zastrzeżenia 1, przy czym przeciwciało lub jego fragment wiążący antygen zawierają: (a) polipeptyd łańcucha lekkiego zawierający: (i) CDR1 łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:20;CDR2 łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:21;i CDR3 łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:22;i polipeptyd łańcucha ciężkiego zawierający: (i) CDR1 łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:28;CDR2 łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:29;i CDR3 łańcucha ciężkiego zawieraj ący sekwencję aminokwasową przedstawioną na SEQ ID NO:30. 3. Izolowane przeciwciało lub jego fragment wiążący antygen według zastrzeżenia 1, przy czym przeciwciało lub jego fragment wiążący antygen zawierają: (a) polipeptyd łańcucha lekkiego zawierający: (i) CDR1 łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:20;CDR2 łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:21;i CDR3 łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:22;i polipeptyd łańcucha ciężkiego zawierający: CDR1 łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:28;CDR2 łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:46;i CDR3 łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:47. 4. Izolowane przeciwciało lub jego fragment wiążący antygen według zastrzeżenia 1, przy czym przeciwciało lub jego fragment wiążący antygen zawierają: (a) polipeptyd łańcucha lekkiego zawierający: (i) CDR1 łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:20;CDR2 łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:38;i CDR3 łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:22;i polipeptyd łańcucha ciężkiego zawierający: (i) CDR1 łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:28;CDR2 łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:29;i CDR3 łańcucha ciężkiego zawieraj ący sekwencję aminokwasową przedstawioną na SEQ ID NO:30. 5. Izolowane przeciwciało lub jego fragment wiążący antygen według zastrzeżenia 1, przy czym przeciwciało lub jego fragment wiążący antygen zawierają: (a) polipeptyd łańcucha lekkiego zawierający: (i) CDR1 łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:20;CDR2 łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:38;i CDR3 152 łańcucha lekkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:22;i polipeptyd łańcucha ciężkiego zawierający: CDR1 łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:28;CDR2 łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:46;i CDR3 łańcucha ciężkiego zawierający sekwencję aminokwasową przedstawioną na SEQ ID NO:47. Piotr Godlewski Rzecznik patentowy 153 154 155 Dni po wystąpieniu choroby 156 Dni po wystąpieniu choroby 157 HC FR1 HC FR2 Fl0?5 158 159 Omiń CZAS . 5 min 160 161 [Antygen, nM] 1GG000 162 163 120-, StężeniefiiMj (ia%)dOO osoumAiw 164 BNJ383 8.00 10.00. 12.00 14.00 16.00 18.00 20.00 Minuty 165 Anty-C5 mAb Minuty nv 166 Minuty #S.I ην 167 168 169 OO Ο BNJ383 (pg/ml) 4500-, τ-1-1-1-1-r (jui/Bd) ego 170 171 120-ι 172

1,344 paragraphs in 131 sections, as filed

[0001] The patent application uses priority and uses US applications (provisional applications) No. 61 / 330.260, filed April 30, 2010 and 61 / 471,465, filed April 4, 2011.

Field of the invention [0002] The field of the invention is medicine, immunology, molecular biology and protein chemistry.

Background [0003] The complement system works in conjunction with other body immune systems to defend against the entry of cellular and viral pathogens. There are at least 25 complement proteins that are found as a complex collection of plasma proteins and membrane cofactors. Plasma proteins make up about 10% of globulin in vertebrate serum. The components of the complement system fulfill their immune defense functions by interacting in a series of complicated but precise events of enzymatic cleavage and membrane binding. The resulting cascade of the complement system leads to the production of products with opsonizing, immunoregulatory and lytic functions. A concise summary of the biological activities associated with the activation of the complement system is provided, for example, in the Merck Manual, issue 16.

[0004] The complement cascade progresses through the classical pathway, alternative pathway or lectin pathway. These pathways have many common elements, and while they differ in their initial stages, they converge and have the same "complement terminal" components (C5 to C9) responsible for the activation and destruction of target cells.

[0005] The classical pathway (CP) is typically initiated by recognition by the antibody and binding to an antigenic site on the target cell. The alternative pathway (AP) may be independent of the antibody, and may be initiated by certain molecules on the surface of pathogens. In addition, the lectin pathway is typically initiated by binding mannose-binding lectin (MBL) to mannose-rich media. These pathways converge at the point where the complement component C3 is cleaved by the active protease, resulting in C3a and C3b. Other pathways that activate complement may act later in the sequence of events, leading to various aspects of complement function.

[0006] C3a is anaphylatoxin. C3b binds to bacterial and other cells; as well as to some viruses and immune complexes, and means they are removed from the circulation. (C3b in this role is known as opsonin). The opsonizing function of C3b is generally considered to be the most important anti-infective function of the complement system. Patients with genetic damage that block C3b function are susceptible to infection by a wide range of pathogenic organisms, while patients with damage at a later stage of the complement cascade sequence, i.e. patients with damage that block C5 function appear to be more susceptible to Neisseria infection, and only to some extent.

[0007] C3b also forms a complex with other components, unique to each pathway, to form a classic or alternative C5 convertase that intersects C5 to C5a and C5b. C3 is therefore considered the central protein in the complement sequence, as it is relevant to both the alternative and classical pathways. This property of C3b is regulated by serum protease factor I, which acts on C3b to produce iC3b. While it is still functional as opsonin, iC3b cannot form active C5 convertase.

[0008] C5 is 190 kDa beta globulin found in normal serum at a concentration of approximately 75 μg / ml (0.4 μΜ). C5 is glycosylated, with about 1.5 to 3 percent of its weight attributed to carbohydrate. Mature C5 is a 999 amino acid heterodimer, 115 kDa alpha chain, which is a disulfide linked to 655 amino acid 75 kDa beta chain. C5 is synthesized as a single-chain protein product precursor, a single copy of the gene (Haviland et al. (1991) J Immunol 146: 362-368). The cDNA sequence of the transcript of this gene predicts a secreted pro-C5 precursor of 1658 amino acids, along with an 18 amino acid leader sequence (see, e.g., US Patent No. 6,355,245).

[0009] The precursor pro-C5 is cleaved after amino acids 655 and 659, giving the beta chain as a fragment at the amino terminus (amino acid residues +1 to 655 of the above sequence) and the alpha chain as a fragment at the carboxyl terminus (amino acid residue 660 to 1658 of the above sequence) , with four amino acids (amino acid residues 656-659 of the above sequence) deleted between the two [fragments].

[0010] C5a is cleavable from the C5 alpha chain by alternative or classical C5 convertase as the amino terminal fragment containing the first 74 amino acids of the alpha chain (i.e. amino acid residues 660-733 of the above sequence). Approximately 20 percent of the 11 kDa mass of C5a is attributed to carbohydrate. The cleavage site for convertase occurs at, or next to, amino acid residue 733 of the above sequence. A compound that will bind to or adjacent to this cleavage site will have the potential to block access of C5 converting enzymes to the cleavage site and thereby act as a complement inhibitor.

[0011] C5 can also be activated by other means than C5 convertase activity. Limited digestion with trypsin (see e.g. Minta and Man (1997) J Immunol 119: 1597-1602 and Wetsel and Kolb (1982) J Immunol 128: 2209-2216), thrombin, and acid treatment (Yamamoto and Gewurz (1978) J Immunol 120: 2008 and Damerau et al. (1989) Molec Immunol 26: 1133-1142) can also cut C5 and produce active C5b.

[0012] C5 cleavage releases C5a, a strong anaphylatoxin and chemotactic agent, and C5b, which through a series of protein interactions leads to the formation of the lytic complement terminal complex, C5b-9. C5a and C5b-9 also have pleiotropic cell activating properties by amplifying the release of downstream inflammatory factors such as hydrolytic enzymes, reactive oxygen species, arachidonic acid metabolites and various cytokines.

[0013] C5b binds to C6, C7 and C8 to form a C5b-8 complex on the surface of the target cell. After binding of several C9 molecules, the membrane attack complex (MAC, C5b-9, terminal complement complex - TCC) is formed. When enough MACs enter the target cell, the holes in the membranes they produce (MAC pores) mediate the rapid osmotic lysis of the target cells. Lower, non-lytic MACs concentrations may produce other effects. In particular, penetration into the membrane of a small number of C5b-9 complexes into endothelial cells and platelets can cause deleterious cell activation. In some cases, activation may precede cell lysis.

[0014] As mentioned above, C3a and C5a are anaphylatoxins. These activated complement components can trigger mast cell degranulation that release histamine from basophils and mast cells, and other inflammatory mediators, resulting in smooth muscle spasm, increased vascular permeability, leukocyte activation, and other inflammatory phenomena, including cell proliferation, resulting in nadkomórkowością. C5a also functions as a chemotactic peptide that serves to attract pro-inflammatory granulocytes to the site of complement activation.

[0015] C5a receptors are found on the surfaces of bronchial epithelial and pulmonary alveolar cells and smooth bronchial muscle cells. C5a receptors have also been found on eosinophils, mast cells, monocytes, neutrophils and activated lymphocytes.

[0016] EP 0245 993 A2 discloses monoclonal antibodies that bind to C5a. Larric: et al (Infection and Immunity, American Society for Microbiology, USA, vol. 55, No. 8, 1987, pages 1867-1872) discloses the characteristics of murine monoclonal antibodies that recognize neutralizing epitopes on human C5a.

Summary of the invention [0017] The disclosure relates, inter alia, to the production by the inventors of a series of humanized monoclonal antibodies that specifically bind to free C5a protein (i.e., C5a that has been proteolytically cleaved from the C5 protein), but not paralog fragments of the free C4a protein or free C3a [antibodies are often referred to herein as anti-C5a antibodies or anti-C5a neoepitope antibodies]. As described herein and detailed in the working examples, generated anti-C5a antibodies exhibit high affinity for free C5a. For example, all of the humanized anti-C5a antibodies described herein bind to free C5a with KD, which is lower than 1.25 nanomolar. Many antibodies bind to free C5a (e.g. free human C5a) with KD that is lower than 300 picomolar; several antibodies bind to free C5a with KD, which is lower than 100 picomolar. In addition, humanized antiC5a antibodies described herein also inhibit C5a signaling. Further structural and functional properties of the antibodies described herein are discussed in detail below and detailed in working examples.

[0018] The inventors have also demonstrated, using an animal model of rheumatoid arthritis (RA) and a replacement anti-mouse C5a antibody with properties similar to those of a humanized antibody, the effectiveness of anti-C5a antibodies in the treatment of RA. Experiments demonstrating the positive therapeutic effects of a humanized anti-C5a antibody in a human animal model of C5a induced neutropenia have also been shown in working examples.

[0019] Accordingly, the inventors believe that the anti-C5a antibodies, or antigen-binding fragments thereof, described herein are useful in many diagnostic and therapeutic methods associated with disorders in which signaling in which C5a is involved contributes to pathogenesis. For example, the inventors claim that the humanized anti-C5a antibodies described herein are useful for treating or preventing RA and other complement-related disorders, including but not limited to atypical hemolytic uremic syndrome (aHUS), age-related macular degeneration (AMD), sepsis, burns (e.g. severe burns), antiphospholipid syndrome (APS), acute respiratory distress syndrome (ARDS), inflammatory pain, asthma, lupus nephropathy, intrauterine growth retardation (IUGR), HELLP syndrome (hemolytic anemia, elevated liver enzymes and low platelet counts) blood), Goodpasture syndrome and chronic obstructive pulmonary disease (COPD). Additional disorders that are particularly amenable to treatment with a humanized anti-C5a antibody, or antigen-binding fragment thereof, are known in the art and cited herein.

[0020] The humanized anti-C5a antibodies described herein have many advantages, e.g., against agents that bind to and inhibit cleavage of full-length, mature C5. Like such agents, the anti-C5a antibodies (and antigen-binding fragments thereof) described herein are capable of inhibiting the anaphylatoxic effects of C5 downstream activation mediated by the C5-C5a fragment. That is, the anti-C5a antibodies described herein can inhibit the C5a-mediated inflammatory response that is known to play an integral role in the pathogenesis of complement-related disorders such as, but not limited to sepsis, RA, and asthma. However, since the C5 concentration in human serum is approximately 0.37 μM (Rawal and Pangburn (2001) J Immunol 166 (4): 2635-2642), the use of high concentrations and / or frequent administration of anti-C5 antibodies is often necessary for effectively inhibiting C5, and thereby inhibiting a C5a-mediated inflammatory response in a human. In contrast to C5, C5a is present in blood at much lower concentrations and is often limited to specific areas of local complement activation such as e.g. lungs in patients with asthma, RA patients' joints, or drusen in the eyes of patients with AMD. Thus, the anti-C5a antibodies described herein can be administered (e.g., topically administered to complement activation sites) to a human at a much lower dose and / or less frequently than, e.g. anti-C5 antibody, and effectively provide the same or higher C5a inhibition in human. The ability to administer a lower dose of anti-C5a antibody, compared to the required dose of anti-C5 antibody, also allows the use of additional delivery routes such as, e.g., subcutaneous administration, intramuscular administration, pulmonary delivery, and administration using biologically degradable microspheres . The lower concentration of the C5a antigen relative to C5 also favors the longer half-life of the anti-C5a antibody, compared to, e.g., the therapeutic half-life of the antibody that targets the terminal portion of the complement, due to the reduced contribution of the antigen-regulated antibody clearance.

[0021] In addition, the anti-C5a antibodies described herein can also be distinguished from therapeutics that inhibit the terminal portion of complement (such as C5 inhibitors) by their safety profile. A noteworthy consequence of inhibiting the terminal components of the complement system such as C5, C5b, C6, C7, C8, or C9 is a reduced protection by the host's immune system against enveloped bacteria that are usually lysed by the terminal part of the complement - for example, Neisseria meningitides and Neisseria gonorrhoeae. See. e.g. Haeney et al (1980) Clin Exp Immunol 40: 16-24 and Brodsky (2009) Blood 113 (26): 6522-6527. Since anti-C5a antibodies inhibit the C5a-mediated inflammatory response, but do not prevent the formation of a terminal complement complex that lyses these enveloped bacteria, patients receiving the anti-C5a therapeutic antibody described herein will not require protective vaccination, e.g., Neisseria vaccination meningitides and Neisseria gonorrhoeae.

[0022] Accordingly, an isolated antibody or antigen-binding fragment thereof that binds to a free C5a polypeptide is provided, wherein free C5a is a human polypeptide (hC5a) having the amino acid sequence set forth in SEQ ID NO: 1, and wherein the antibody or an antigen-binding fragment thereof binds to a free hC5a polypeptide in vitro with KD that is less than 1.25 x 10-9 M as measured by surface plasmon resonance (SPR) in the presence of a molar excess of un cleaved native human C5, and wherein the antibody or antigen binding fragment thereof comprises:

AND.

(a) a light chain polypeptide comprising:

(i) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 20; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 21; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 22; or (ii) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 20; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID

NO: 38; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 22; and (b) a heavy chain polypeptide comprising:

(i) a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 28; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 29; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 30; or (ii) a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 28; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 46; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 47;

II.

(a) a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 37 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 27;

(b) a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 36 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 33;

(c) a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 19 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 27;

(d) a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 17 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 25;

(e) a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 42 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 27;

(f) a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 40 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 33;

(g) a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 17 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 33;

(h) a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 19 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 45;

(i) a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 17 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 44;

(j) a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 17 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 49;

(k) a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 37 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 45; or (l) a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 36 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 49; or

III.

(a) a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 42 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 45;

(b) a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 40 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 49; or (c) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 20; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 21; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 22; Heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 28; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 46; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 47.

[0023] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises: (a) a light chain polypeptide comprising: (i) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 20; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 21; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 22; and a heavy chain polypeptide comprising: (i) a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 28; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 29; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 30.

[0024] In another embodiment, the isolated antibody or antigen-binding fragment thereof comprises: (a) a light chain polypeptide comprising: (i) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 20; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 21; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 22; and a heavy chain polypeptide comprising: a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 28; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 46; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 47.

[0025] In another embodiment, the isolated antibody or antigen-binding fragment thereof comprises: (a) a light chain polypeptide comprising: (i) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 20; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 38; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 22; and a heavy chain polypeptide comprising: (i) a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 28; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 29; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 30.

[0026] In another embodiment, the isolated antibody or antigen-binding fragment thereof comprises: (a) a light chain polypeptide comprising: (i) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 20; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 38; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 22; and a heavy chain polypeptide comprising: a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 28; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 46; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 47.

[0027] Hence, an isolated antibody or antigen-binding fragments thereof that binds to free C5a are described. The antibody or antigen-binding fragment thereof may bind to free human C5a (hC5a; e.g., human C5a protein comprising or consisting of the amino acid sequence depicted in SEQ ID NO: 1). In some aspects of the disclosure, the antibodies can bind to the arginine-free form of free C5a, e.g. the arginine-free form of human C5a containing, or consisting of, the amino acid sequence depicted in SEQ ID NO: 2. The antibody may bind to a free C5a neoepitope, which epitope is not present on the unseparated C5 or is present only on the minor fraction of the total un cleaved C5a.

[0028] While the disclosure is in no way limited to any particular theory or mechanism of action, in some aspects of the disclosure, the anti-C5a antibody or antigen-binding fragment thereof binds to free C5a (e.g., free hC5a) and can also bind to an unvaccinated, C5 treated (e.g. C5 plasma) subpopulation of less than 10 (e.g. less than 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, 0.4, 0.3, 0.2, or less than 0.1)% of the total full-length C5 population in the sample (e.g. blood or plasma sample or sample containing recombinant full length

C5), which subpopulation is, in whole or in part, denatured such that the otherwise covered C5a neoepitope is exposed to which the anti-C5a antibody or fragment thereof binds. Hence, the anti-C5a antibody or antigen-binding fragment thereof described herein may, in some aspects of the disclosure, bind to free C5a, but not to un cleaved C5 protein with 90% or more of the un cleaved native C5 population. In some aspects of the disclosure, the partially or fully denatured C5 subpopulation described above is inactive or has reduced activity (e.g., less than 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5% activity of fully functional, full-length C5 protein) in any number of appropriate assays useful for testing C5 activity, e.g. hemolytic or CH50eq assay (see below). Suitable methods for testing the activity of a minor subpopulation to which the anti-C5a antibody described herein can bind are known in the art and described herein.

[0029] In some aspects of the disclosure, any of the anti-C5a antibodies or antigen-binding fragments thereof described herein do not inhibit C5 activity in an in vitro hemolytic assay or in vitro CH50eq assay in the presence of at least, equal to or higher than 5- (eg. 5,6, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200)-fold excess of anti-C5a antibody or antigen-binding fragment thereof relative to non-cleaved C5 (e.g., non-cleaved, native C5). In some aspects of the disclosure, any of the anti-C5a antibodies or antigen-binding fragments thereof described herein do not inhibit C5 activity in an in vitro hemolytic assay or in vitro CH50eq assay even in the presence of between about 5 to 200 times (e.g. between about 5-fold and 100-fold, between about 10-fold and 100-fold, between about 20-fold and 100-fold, or between about 10-fold and 150-fold excess of anti-C5a antibody or binding fragment thereof antigen relative to non-cleaved native C5. Inhibition, e.g. as it relates to C5 activity, includes at least 5 (e.g. at least 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 )% decrease in activity of un cleaved native C5 in, e.g. hemolytic assay or CH50eq assay compared to effects on a control antibody (or antigen-binding fragment thereof) under similar conditions and at equimolar concentration. Significant inhibition, as used herein, refers to inhibition of a given activity (e.g. C5 activity) by at least 40 (e.g. at least 45, 50, 55.60, 65, 70, 75, 80, 85, 90 or 95 or more)% . In some embodiments, C5 is obtained from plasma (e.g., purified from or present in plasma, e.g., human plasma).

[0030] In some aspects of the disclosure, the antibody or antigen binding fragment thereof binds to a C5a protein (e.g., a human C5a protein) with a KD that is less than 2 nM. In some aspects of the antibody, o or antigen binding fragment thereof binds to a C5a protein with KD that is less than 1 nM [also referred to herein as "subnanomolar affinity"].

[0031] In some aspects of the disclosure, an anti-C5a antibody or antigen-binding fragment thereof binds to free C5a with subnanomolar affinity [e.g. KD less than or equal to 9.9 x 10-10 (e.g., less than or equal to 9 x 10-10, 8 x 10-10, 7 x 10-10, 6 x 10-10, 5 x 10-10, 4 x 10-10, 3 x 10-10, 2.5 x 10-10, 2 x 10-10, 1 x 10-10, 8.0 x 10-11, 7.0 x 10-11, 6.0 x 10-11, 5.0 x 10-11, 4.0 x 10-11, or 3.0 x 10-11) M] in the presence of a molar excess of un cleaved native C5 (e.g. purified and / or recombinant C5). In some aspects of the disclosure, any of the anti-C5a antibodies or antigen-binding fragments thereof described herein have at least 100- (e.g., at least 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000) fold affinity (e.g. represented by KD) for free C5a than for non-cleaved native C5 protein.

[0032] Hence, in another aspect, the disclosure provides an antibody or antigen-binding fragment thereof that (a) binds to free C5a (e.g., hC5a) with subnanomolar affinity, and (b) binds to free C5a with an affinity that is at least 100 (e.g. at least 110, 120, 130, 140, 150, 160,170,180, 190, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7,000, 8,000, 9,000, or 10,000) fold higher than its corresponding affinity for un cleaved native C5 protein. For example, the anti-C5a antibody or antigen-binding fragment thereof described herein, may, in some embodiments, bind to free hC5a with a KD of 100 nM and to at least a subpopulation of un-cleaved human C5 protein with KD that is at least 100-fold higher (e.g. at least 10 nM).

[0033] In another aspect, the disclosure provides an isolated antibody or antigen binding fragment thereof that binds to a free human C5a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, wherein the antibody or antigen binding fragment thereof binds to human C5a polypeptide with KD, which is less than 1.25 x 10-9 M in the presence of a molar excess (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400 or 500-fold molar excess) of un-cleaved native human C5 versus human C5a (hC5a). In some aspects of the disclosure, the antibody or antigen-binding fragment thereof binds to free hC5a polypeptide with subnanomolar affinity (e.g. any of the subnanomolar KDs cited in the description) in the presence of at least, or more than, a 2-fold molar excess, but not more or not less than 500 (e.g. 500, 450, 400, 350, 300, 250, 200, 150, 100 , 90, 80, 70, 60, 50, 40, 30, 25, 20 or 15) times a molar excess of un cleaved, native C5 relative to free hC5a. In some aspects of the disclosure, the antibody or antigen-binding fragment thereof binds to free hC5a polypeptide with subnanomolar affinity (e.g. any of the subnanomolar KDs cited in the description) in the presence of at least or more than a 2-fold molar excess, but no more or less than 500 (e.g. 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20 or 15) -molar excess of un cleaved native N5 relative to free hC5a. In some aspects of the disclosure, the antibody or antigen-binding fragment thereof binds to free hC5a polypeptide with subnanomolar affinity (e.g. any of the subnanomolar KDs cited herein) in the presence of a 2-fold to 20-fold molar excess of un-cleaved native C5 relative to free hC5a. In some aspects of the disclosure, the antibody or antigen-binding fragment thereof binds to free hC5a polypeptide with subnanomolar affinity (e.g. from subnanomolar KD cited herein) in the presence of a 10-fold to 20-fold molar excess of unseparated native C5 relative to free hC5a. In some aspects of the disclosure, the antibody or antigen-binding fragment thereof binds to free hC5a polypeptide with subnanomolar affinity (e.g., any of the subnanomolar KDs cited herein) in the presence of a 5-fold to 15-fold molar excess of un-cleaved, native C5 relative to free hC5a. In some aspects of the disclosure, the antibody or antigen-binding fragment thereof binds to free hC5a polypeptide with subnanomolar affinity (e.g. any of the subnanomolar KDs cited herein) in the presence of a 2-fold, but not more than 20-fold molar excess of un-cleaved native C5 relative to free hC5a. Such measurements can mean measurements made in vitro using, e.g., standard affinity determination techniques, many of which are cited and / or described herein.

[0034] In another aspect, the disclosure provides an isolated antibody or antigen binding fragment thereof that binds to a human C5a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, wherein the antibody or antigen binding fragment thereof binds to the human C5a polypeptide from KD, which is less than 1.25 x 10-9 M and wherein the antibody or antigen-binding fragment thereof does not significantly inhibit, compared to an equimolar amount of the control antibody or antigen-binding fragment thereof, C5 activity, even in the presence of less than or equal to 10 fold molar excess of anti-C5a antibody or fragment antigen-binding to un cleaved native C5.

[0035] In some aspects of the disclosure of any anti-C5a antibodies or antigen-binding fragments thereof described herein, the antibody or antigen-binding fragment thereof binds to free human C5a and is cross-reactive with free C5a from at least one non-human mammal species . For example, in some aspects of the disclosure, an anti-C5a antibody (or antigen-binding fragment thereof) binds to free C5a from a human (e.g. with subnanomolar affinity) and also bind to free C5a from non-human primates (e.g. cynomolgus macaque, rhesus macaque, monkey, baboon, chimpanzee, orangutan or gorilla), rodent (e.g. mouse, rat, hamster, guinea pig or rabbit) ), cow, goat, donkey, pig, dog, cat or horse. In some aspects of the disclosure, the anti-C5a antibody or antigen-binding fragment thereof described herein binds to free hC5a with a KD less than or equal to 9.9 x 10-10 (e.g., less than or equal to x 10-10, 8 x 10-10, 7 x 10-10, 6 x 10-10, 5 x 10-10, 4 x 10-10, 3 x 10-10, 2.5 x 10-10, 2 x 10-10, 1 x 10-10, 8.0 x 10-11, 7.0 x 10-11 6.0 x 10-11, 5.0 x 10-11, 4.0 x 10-11, or 3.0 x 10-11) M and also bind to free C5a from Cynomolgus macaque (or other non-human primate), with affinity (e.g. represented by KD) for human C5a not greater than 500 (e.g. not greater than 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 350, 400, 450, or 475) higher than the affinity for C5a cynomolgus (or other species) non-human chief). For example, in some aspects of the disclosure, the anti-C5a antibody binds to free hC5a with an affinity that is not greater than 50-fold higher than the corresponding affinity of the non-human primate C5a antibody (e.g., KD for 100 nM and KD free hC5a for a non-human primate C5a not greater than 5 nM). In some aspects of the disclosure, the anti-C5a antibody or antigen-binding fragment thereof described herein binds to free hC5a with a KD of less than or equal to 9.9 x 10-10 (e.g., less than or equal to 9 x 10-10, 8 x 10-10, 7 x 10-10, 6 x 10-10, 5 x 10-10, 4 x 10-10, 3 x 10-10, 2.5 x 10-10, 2 x 10-10, 1 x 10-10, 8.0 x 10-11, 7.0 x 10-11, 6.0 x 10-11, 5.0 x 10-11, 4.0 x 10-11, or 3.0 x 10-11) M and also bind to rodent C5a (e.g. mouse, rat or rabbit), with affinity (e.g. represented by their KD) for human C5a not greater than 1000 (e.g. not greater than 5, 10, 20 , 30, 40, 50, 60, 70, 80, 90, 100,125, 150, 200, 250, 300, 350, 400, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900 , 950, or 975) higher than the affinity for rodent C5a. In some aspects of the disclosure, any anti-C5a antibodies or antigen-binding fragments thereof described herein bind to subnanomolar affinity for both human C5a and C5a from a non-human mammal (e.g. rodent or non-human primate, such as cynomolgus macaque) ). The antibody or antigen binding fragment thereof may, in some aspects of the disclosure, bind to human C5a and C5a from non-human primates with equal affinity (e.g. equivalent to KD).

[0036] For example, the disclosure provides an antibody or antigen-binding fragment thereof that binds to free human C5a with subnanomolar affinity [e.g. KD less than or equal to 9.9 x 10-10 (e.g. less than or equal to 9 x 10-10, 8 x 10-10, 7 x 10-10, 6 x 10-10, 5 x 10-10, 4 x 10-10, 3 x 10-10, 2.5 x 10-10, 2 x 10-10, 1 x 10-10, 8.0 x 10-11, 7.0 x 10-11, 6.0 x 10-11, 5.0 x 10-11, 4.0 x 10-11, or 3.0 x 10-11) M] and is cross-reactive with free C5a from cynomolgus (or other non-human primate), the antibody or antigen-binding fragment thereof binds to C5a from cynomolgus (or other non-human primate) from KD less than 10 x 10-9, 9 x 10-9, 8 x 10-9, 7 x 10-9, 6 x 10-9, 5 x 10-9, 4 x 10-9, 3 x 10-9, 2x10-9, 1 x 10-9, 9.9 x 10-10 (e.g., less than 9 x 10-10, 8 x 1010, 7 x 10-10, 6 x 10-10, 5 x 10-10, 4 x 10-10, 3 x 10-10, 2.5 x 10-10, 2 x 10-10, 1 x 10-10, or 8.0 x 10-11) M], where the affinity for human C5a is not more than 500 (e.g. not more than 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250 , 300, 350, 400, 450, or 475) higher than the affinity for C5a from cynomolgus (or non-human primate) (e.g. KD for human C5a of order 100 nM and KD for non-human primate C5a not greater than 50 nM). Suitable methods for determining the affinity of an antibody or antigen-binding fragment thereof for a given antigen are known in the art and described and detailed in the specification.

[0037] In some aspects of the disclosure, the cross-reactive anti-C5a antibody or antigen-binding fragment thereof functionally inhibits both free hC5a and the non-human mammalian C5a to which they bind. For example, the antibody inhibits at least (e.g., at least 75, 80, 85, 90, or 95 or more)% human C5a-dependent neutrophil activation in humans at a molar ratio of 1: 1 (antigen binding site: C5a) and inhibits by at least 70 (e.g. at least 75, 80, 85, 90, or 95 or more)% non-human, C5-dependent mammalian activation of neutrophils (neutrophils from the same species as the non-human C5a mammal to which the antibody binds) at a 1: 1 molar ratio (antigen binding site: C5a).

[0038] In another aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to a free hC5a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, wherein the antibody or antigen-binding fragment thereof binds to a human C5a polypeptide from KD, which is less than 1.25 x 10-9 M and wherein the antibody or antigen-binding fragment thereof binds to both hC5a and C5a from a non-human mammal species. A non-human mammal species can mean, for example, a non-human primate, such as Crab-eating macaque, Rhesus macaque or baboon. In some aspects of the disclosure of a non-human mammalian species is a rodent such as mouse, rat, rabbit, guinea pig, gerbil or hamster. In some aspects of the disclosure, the o-antibody or antigen-binding fragment thereof binds to hC5a with an affinity not higher than 100-fold higher than the corresponding affinity for C5a from a non-human mammal species. In some aspects of the disclosure, the antibody or antigen-binding fragment inhibits at least 50% human C5a-dependent neutrophil activation in humans at a molar ratio of 1: 1 (antigen binding site: C5a).

[0039] In some aspects of the disclosure, the antibody or antigen-binding fragment thereof binds to free C5a from a non-human primate (e.g., cynomolgus or rhesus macaque), a free C5a protein having an amino acid sequence comprising, or consisting of, the amino acid sequence depicted in SEQ ID NO: 179 or SEQ ID NO: 180.

[0040] As described in the working examples, the inventors also found the existence of a bivalent anti-C5a antibody, BNJ383, which binds to free C5a (in this case human C5a) with high affinity and, with much lower affinity, to un cleaved human C5 (hC5 ), in which the composition (e.g. aqueous solution) under equilibrium physiological conditions, and in the presence of a molar excess of un cleaved human C5 compared to the molar amount of the antibody antigen binding site, at least 95% of many antibodies, each binding not more than one hC5 molecule. A second antigen binding site for at least 95% of many antibodies remains available (e.g., extensively available) for binding to free C5a. While the disclosure is not limited in any way to any particular theory or mechanism of action, the inventors believe that the bivalent anti-C5a antibody binds to un-cleaved C5 in this manner (e.g. with such an epitope) that spatial hindrance excludes, or at least significantly inhibits, the binding of the second antigen-binding site of the anti-C5a antibody to the second, un cleaved C5 protein, although the antibody can easily contain binding to two hC5a molecules. Hence, the antibody, even in molar excess of un cleaved C5, retains the ability to bind to free C5a with high affinity and thus retains, even in molar excess, the ability to inhibit C5a pro-inflammatory activity.

[0041] One skilled in the art will readily and readily appreciate the myriad therapeutic benefits of such an anti-C5a antibody. For example, as noted above, the concentration of circulating C5 in human serum is very high. Thus, when introduced into a mammal, an anti-C5a antibody that is able to bind to two un-cleaved C5 molecules simultaneously will be rapidly inactivated in molar excess of C5 and will no longer be able to bind to free C5a when complement is activated. And, as with anti-C5 antibodies, it will be necessary to use high concentrations and / or frequent administration of this type of anti-C5a antibody to effectively inhibit C5a, if it were produced. In contrast, the anti-C5a antibody described herein, which retains the ability to bind to free C5a, even in a molar excess of un cleaved C5, can therefore be administered to a human at a much lower dose and / or less frequently than, e.g. anti-C5 antibody and effectively provides the same or higher inhibition of C5a in human.

[0042] Accordingly, an isolated antibody comprising two antigen binding sites is also described herein, wherein each antigen binding site can independently bind to free human C5a (hC5a) or un cleaved human C5 (hC5), in aqueous solution containing: (i) many of these antibodies; and (ii) a molar excess of hC5 compared to the molar number of antigen binding sites, at equilibrium and in physiological conditions, at least 95 (e.g., at least 95.5, 96, 96.5, 97, 97 , 5, or 97.7)% of said number of antibodies binds to no more than one hC5 molecule, i.e. no more than 5% of the antibodies bind two hC5 molecules at equilibrium.

[0043] In another aspect, the disclosure provides an isolated antibody comprising two antigen binding sites, wherein each antigen binding site can independently bind to free human C5a (hC5a) or un-cleaved human C5 (hC5), at equilibrium and under conditions physiological, in an aqueous solution containing: (i) a plurality of said antibodies; and (ii) a molar excess of hC5 compared to the molar number of antigen binding sites (or antibodies), at least 95% of said number of antibodies retains at least one antigen binding site available for binding to free hC5a.

[0044] In another aspect, the disclosure provides an isolated antibody comprising two antigen binding sites, wherein each antigen binding site can independently bind to free human C5a (hC5a) or un cleaved human C5 (hC5), at equilibrium and under conditions physiological, in an aqueous solution containing: (i) a number of said antibodies and (ii) a molar excess of hC5 compared to the molar number of antigen binding sites (or antibodies), each antigen binding site for not more than 5% of said number of antibodies is bound to the hC5 molecule.

[0045] In some aspects of the disclosure of any of the isolated antibodies described herein, molar excess is at least two-fold (e.g., at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or even 10-fold) molar excess.

[0046] In some aspects of the disclosure of any of the isolated antibodies described herein, physiological conditions are 3.9 mM NaH2PO4, 6.1 mM

Na2HPO4 and 150 mM NaCl, at pH 7.0.

[0047] In some aspects of the disclosure of any of the isolated antibodies described herein, each antigen binding site can independently bind to free hC5a with KD that is less than 1.25 x 10-9 M. In some embodiments, each antigen binding site can independently bind to free hC5a with subnanomolar affinity (see above).

[0048] In some embodiments of any of the isolated antibodies described herein, the isolated antibody comprises a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 42 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 45.

[0049] In some embodiments of any of the isolated antibodies described herein, the isolated antibody comprises: (i) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 20; (ii) a light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 21; (iii) a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 22; (iv) a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 28; (v) a heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 46; and (vi) a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 47.

[0050] In some aspects of the disclosure, the isolated antibody may comprise any of the sets of light chain CDRs described herein, any of the light chain variable regions described herein (e.g., any of the humanized light chain variable regions), any of the sets of CDRs heavy chain depicted herein, any of the heavy chain variable regions depicted herein (e.g. any of the humanized heavy chain variable regions), or any combination thereof; see. e.g. Tables 1 or 2.

[0051] In another aspect, the disclosure provides a method of treating a human affected by a complement related disorder (e.g., C5a related complement disorder), the method comprising administering to the human any of the isolated antibodies described herein in an amount sufficient to treat the disorder associated with complement system.

[0052] In another aspect, the disclosure provides a method for treating a human affected by a C5a related disorder of the complement system, the method comprising administering to a human at least 0.6 (e.g., at least 0.7, 0.8, 0.9 or 1 ) mg of any of the isolated antibodies described herein per kg of human body weight to thereby partially or completely bind and demarcate nanogram free C5a levels for longer than, evenly, or at least 12 (e.g. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) days.

[0053] In another aspect, the disclosure provides a method for treating a human affected by a C5a related disorder of the complement system, the method comprising administering to the human being at least 10 mg of any of the isolated antibodies described herein per kg of human body weight, thereby partially or completely bind and demarcate nanogram free C5a levels for at least 24 days.

[0054] In another aspect, the disclosure provides a method for treating a human suffering from a C5a-related complement disorder, wherein the method comprises administering to a human any of the isolated antibodies described herein (or, for example, a pharmaceutical composition comprising any of the isolated antibodies described herein) in an amount sufficient to (a) achieve molar Cmax values equal to or less than the physiological molar concentration of unseparated hC5 and ( b) partial or complete binding and demarcation of pathophysiological levels of free C5a.

[0055] In some aspects of any of the methods described herein, the antibody is administered to the subject (e.g., human) in an amount sufficient to achieve a molar Cmax value that is significantly lower than the physiological molar concentration of un cleaved C5 (e.g., hC5).

[0056] In some aspects of any of the methods described herein, the Cmax value is, e.g., not more than 80 nM (or approximately 0.6 mg / kg). In some aspects, Cmax levels are not higher than 70 (e.g., 60, 50, 40, 30 or 20) nM. In some aspects, the Cmax value is not higher than approximately 100 nM. In some aspects, the Cmax value is not higher than 200 nM. In some aspects of any of the methods described herein, the Cmax is not, e.g., higher than 400 nM (or approximately 3 mg / kg). In some aspects, the Cmax value is not higher than 400 (e.g. 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10) nM.

[0058] In another aspect, the disclosure provides a method for treating a human affected by a C5a related disorder of the complement system, the method comprising administering to a human any of the isolated antibodies described herein (or, for example, a pharmaceutical composition comprising any of the isolated antibodies described herein) in sufficient to (a) achieve molar Cmax values equal to, less than, or significantly lower than the molar physiological concentration of un cleaved hC5 and (b) partial or complete binding and demarcation of nanogram free C5a levels for at least 12 days (e.g., at least 24 days). The corresponding Cmax values are described above.

[0059] In some aspects of any of the methods described herein, C5a-related complement disorder may be, e.g. selected from the group consisting of sepsis, acute respiratory distress syndrome (ARDS), septic shock, antiphospholipid syndrome, catastrophic antiphospholipid syndrome, disseminated intravascular coagulation, lupus nephropathy, Goodpasture syndrome, burns or severe burns, asthma, HELLP anemia , elevated liver enzymes and low platelet count), inflammation-induced pain, C5a-regulated neutropenia, age-related macular degeneration (AMD), chronic obstructive pulmonary disease and rheumatoid arthritis.

[0060] In yet another aspect, the disclosure provides a composition comprising a plurality of isolated antibodies, each of a plurality of antibodies comprising two antigen binding sites, wherein each antigen binding site can independently bind to free human C5a (hC5a) or un cleaved human C5 (hC5) , and wherein, in the presence of human C5 (hC5) and in physiological conditions, no more than 5% of many of the equilibrium antibodies contain two antigen binding sites simultaneously bound to un-cleaved hC5.

[0061] In some aspects, any of the compositions described herein, the percentage of many in any particular binding configuration can be assessed using high performance liquid chromatography (HPLC). In some aspects of the disclosure, the physiological conditions under which antibodies are evaluated include the following conditions: hC5 incubation (e.g., molar excess (e.g. 2 fold molar excess (hC5) with multiple antibodies, at 4 ° C for 84 hours in an aqueous solution containing 3.9 mM NaH2PO4, 6.1 mM Na2HPO4, and 150 mM NaCl, at pH 7.0. For the purposes of disclosure, a solution obtained after 84 hours at 4 ° C is considered to be in equilibrium.

[0062] In some aspects of any of the compositions described herein, no more than 5% of the antibodies of many contain two antigen binding sites simultaneously bound to unseparated hC5 under physiological conditions and in the presence of at least a 2-fold molar excess of hC5 relative to the antibody.

[0063] In some aspects of any of the compositions described herein, no more than 5% of the multiple antibodies contain two antigen binding sites simultaneously bound to non-cleaved hC5 molecules, as assessed by HPLC, followed by incubation with multiple antibodies with hC5 at 4 ° C for 84 hours in an aqueous solution containing 3.9 mM NaH2PO4, 6.1 mM Na2BPO4, and 150 mM NaCl, at pH 7.0.

[0064] In another aspect, the disclosure provides a composition comprising a plurality of isolated antibodies, each of the multiple antibodies comprising two antigen binding sites, wherein each antigen binding site can independently bind to free human C5a (hC5 a) or un cleaved human C5 (hC5) , and wherein no more than 5% of the antibodies of many contain two antigen binding sites simultaneously bound to un cleaved hC5, as assessed (e.g. using HPLC) after incubation with multiple antibodies with hC5 at 4 ° C for 84 hours in an aqueous solution containing 3.9 mM NaH2PO4, 6.1 mM Na2HPO4, and 150 mM NaCl, at pH 7.0.

[0065] In yet another aspect, the disclosure provides a composition comprising a plurality of isolated antibodies, each of a plurality of antibodies comprising a first antigen binding site and a second antigen binding site, wherein each antigen binding site can independently bind to free human C5a (hC5a) or un cleaved human C5 (hC5), wherein each antigen binding site can independently bind to free KD hC5a polypeptide, which is less than 1.25 x 10-9 M, and wherein, in the presence of human C5 (hC5) and as assessed (e.g. using high performance liquid chromatography (HPLC)) under physiological conditions, two antigen binding sites for at least 95% of many antibodies are occupied by unseparated hC5 in the following configurations: (i) the first antigen binding site binds the un cleaved hC5 and the second antigen binding site is unbound; or (ii) the first antigen binding site is unbound and the second antigen binding site binds un cleaved hC5.

[0066] In yet another aspect, the disclosure provides a composition comprising a plurality of isolated antibodies, each of a plurality of antibodies, comprising two antigen binding sites, wherein each antigen binding site can independently bind to free human C5a (hC5a) or un cleaved human C5 (hC5), and wherein, in the presence of human C5 (hC5) and as evaluated (e.g. using high performance liquid chromatography (HPLC)) under physiological conditions, at least 95% of the antibodies of many contain at least one antigen binding site capable of binding to free hC5a.

[0067] In some aspects of any of the compositions described herein, many antibodies are evaluated in the presence of at least a 2-fold molar excess of hC5: antibody. In some aspects of any of the compositions described herein, many antibodies are evaluated in the presence of at least a 2-fold molar excess of hC5: antigen binding sites.

[0068] In yet another aspect, the disclosure provides an isolated antibody comprising two antigen binding sites, wherein the antibody binds to free C5a or un cleaved C5, and wherein one of the antigen binding sites of the antibody remains available for binding to free C5a in the presence of molar excess (e.g., at least or more than 2-fold, 5-fold, 10-fold, 15-fold, or even 20-fold molar excess) of un cleaved C5.

[0069] In some aspects of the disclosure, the antigen binding sites have the same specificity (eg, the CDRs of each of the two antigen binding sites have identical amino acid sequences). In some embodiments, free C5a is human C5a. In some aspects of the disclosure, the antibody is cross-reactive between human C5a and C5a from a non-human mammalian species. The antibody may, in some aspects, bind to free C5a with subnanomolar affinity. In some aspects of the disclosure, the antibody has an affinity for C5a that is at least 100 times higher than its corresponding affinity for un cleaved C5.

[0070] In another aspect, the disclosure provides an isolated antibody comprising two antigen binding sites, wherein each antigen binding site independently binds to free human C5a (hC5a) or un-cleaved human C5 (hC5), and wherein at any un-cleaved concentration hC5 (e.g., in molar excess of un-cleaved hC5 relative to hC5a), at least one antigen binding site of the antibody remains available for binding to free hC5a (e.g. under physiological conditions for humans, e.g. in human blood or serum).

[0071] In another aspect, the disclosure provides a composition comprising a plurality of isolated antibodies, wherein each of the multiple antibodies has two antigen binding sites, each of which antigen binding sites can independently bind to free human C5a (hC5a) or un cleaved human C5 ( hC5), and wherein, at a 1: 1 molar ratio (antibody: hC5), no more than or less than 5 (e.g. not more than or less than 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9 , 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2 , 6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4 , 1,3, 1,2, 1,1 or 1)% of the antibodies of many contain two antigen binding sites simultaneously bound to un cleaved hC5. In some aspects of the disclosure, each antigen binding site can independently bind to free hC5a with KD that is less than 1.25 x 10-9 M (or, for example, with subnanomolar affinity).

[0072] In another aspect, the disclosure provides a composition comprising a plurality of isolated antibodies, wherein each of the multiple antibodies comprises two antigen binding sites, wherein each of the antigen binding sites can independently bind to free human C5a (hC5a) or un cleaved human C5 ( hC5), and wherein, in the presence of physiological levels of un cleaved hC5, antibodies from many partially or completely bind and demarcate nanogram levels of free C5a for longer than, evenly, or at least 12 (e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) days when administered to humans at doses of 1 mg / kg or higher.

[0073] In another aspect, the disclosure provides a composition comprising a plurality of isolated antibodies, wherein each of the multiple antibodies comprises two antigen binding sites, wherein each of the antigen binding sites can independently bind to free human C5a (hC5a) or un cleaved human C5 ( hC5), and wherein, in the presence of physiological levels of un cleaved hC5, antibodies from many partially or completely bind and separate nanogram levels of free C5a for longer than, evenly, or at least 12 (e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) days when administered to humans at doses of 10 mg / kg or higher.

[0074] In another aspect, the disclosure provides a composition comprising a plurality of isolated antibodies, wherein each of the multiple antibodies comprises two antigen binding sites, wherein each of the antigen binding sites can independently bind to free human C5a (hC5a) or un cleaved human C5 ( hC5), and wherein, in the presence of physiological levels of un cleaved hC5, antibodies from many partially or completely bind and separate nanogram levels of free C5a for longer than, evenly, or at least 12 (e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) days when administered at doses reaching molar Cmax values significantly lower than the molar physiological concentration of unsealed hC5.

[0075] In another aspect, the disclosure provides a composition comprising a plurality of isolated antibodies, wherein each of the multiple antibodies comprises two antigen binding sites, wherein each of the antigen binding sites can independently bind to free human C5a (hC5a) or un cleaved human C5 ( hC5), and wherein, in the presence of physiological levels of un cleaved hC5, many antibodies partially or completely bind and separate nanogram levels of free C5a when administered at doses reaching molar Cmax values significantly lower than the molar physiological concentration of un cleaved hC5.

[0076] In another aspect, the disclosure provides isolated antibodies having a first antigen binding site and a second antigen binding site, wherein each antigen binding site can independently bind to free human C5a (hC5a) or un cleaved human C5 (hC5), and wherein when both antigen binding sites are fully occupied (and, e.g., in human physiological conditions, e.g. in human blood or serum), the following binding configurations are possible: (i) the first antigen binding site binds free hC5a and the second antigen binding site binds un cleaved hC5; (ii) the first antigen binding site binds free hC5a and the second antigen binding site binds free hC5a; or (iii) the first antigen binding site binds unseparated hC5 and the second antigen binding site binds free hC5a.

[0077] In yet another aspect, the disclosure provides isolated antibodies having a first antigen binding site and a second antigen binding site, wherein each antigen binding site can independently bind to free human C5a (hC5a) or un cleaved human C5 (hC5), each antigen binding site may independently bind to free hC5a with KD, which is less than 1.25 x 10-9 M (or, for example, with subnanomolar affinity), and wherein, in a physiological solution containing multiple antibodies, at least 95% of the antibodies have the following configurations: (i) the first antigen binding site binds free hC5a and the second antigen binding site unseparated hC5; (ii) the first antigen binding site binds free hC5a and the second antigen binding site binds free hC5a;

(iii) the first antigen binding site binds un cleaved hC5 and the second antigen binding site binds free hC5a; (iv) the first antigen binding site binds unsealed hC5 and the second antigen binding site is unbound; (v) the first antigen binding site binds hC5a and the second antigen binding site is unbound;

(vi) the first antigen binding site is unbound and the second antigen binding site binds un cleaved hC5; (vii) the first antigen binding site is unbound and the second antigen binding site binds hC5a; and (viii) the first antigen binding site is unbound and the second antigen binding site is unbound.

[0078] In another aspect, the disclosure provides an isolated antibody comprising two antigen binding sites, wherein each antigen binding site can independently bind to free human C5a (hC5a) or un-cleaved human C5 (hC5), and wherein, in molar excess of un cleaved hC5 versus hC5a, the antibody inhibits at least 50% of hC5a-dependent human neutrophil activation in a 1: 1 molar ratio (antigen binding site: hC5a).

[0079] In some aspects of any of the antibodies described herein, configurations are possible under human physiological conditions with fully folded native human C5a and C5 proteins.

[0080] In some aspects of any of the antibodies described herein, the antibody binds to free hC5a with KD, which is less than 1.25 x 10-9 M (or, for example, with subnanomolar affinity).

In yet another aspect, the disclosure provides an antibody that (a) binds to free C5a (e.g. hC5a) with subnanomolar affinity, and (b) binds to free C5a with an affinity that is at least 100 (e.g. at least 110, 120, 130,140,150,

160, 170, 180, 190, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000) - times higher than its corresponding affinity for unseparated C5 protein. In a physiological composition containing multiple antibodies, for at least 95% of antibodies, only one antibody antigen binding site binds to v C5 protein, while the other antigen binding site remains available for binding to free C5a. (HC5a may have the amino acid sequence depicted in SEQ ID NO: 1.) [0082] In another aspect, the disclosure provides a method of treating a human being suffering from a C5a-related complement disorder, a method comprising administering to the human a composition comprising a plurality of isolated antibodies, each antibody having many contain two antigen binding sites, wherein each of the antigen binding sites can independently bind to free human C5a (hC5a) or un cleaved human C5 (hC5), wherein at least 1 mg of antibodies per kg of human body weight is administered to a human, and wherein the administration of the antibodies is effective for targets for partial or complete binding and separates nanogram free C5a levels for at least 12 days.

[0083] In another aspect, the disclosure provides a method of treating a human being affected by a complement related disorder (e.g. C5a related disorder of the complement system), a method comprising administering to a human composition comprising multiple isolated antibodies, wherein each of the multiple antibodies has two antigen binding sites, each of which antigen binding sites can independently bind to free human C5a (hC5a) or unsealed human C5 (hC5), with at least 10 mg of antibodies per kg human body weight administered to a human being, and wherein the administration of the antibodies is effective for the purpose of partial or complete binding and separates nanogram free C5a levels for at least 24 days.

[0084] In another aspect, the disclosure provides a method of treating a human being with it and a complement related disorder (e.g. C5a related disorder of the complement system), a method comprising administering to a human composition comprising multiple isolated antibodies, wherein each of the multiple antibodies has two antigen binding sites, each of which antigen binding sites can independently bind to free human C5a (hC5a) or unsealed human C5 (hC5), wherein the antibodies are administered at a dose sufficient to: (a) achieving molar Cmax values significantly lower than the molar physiological concentration of unsealed hC5 and (b) partial or complete binding and demarcation of nanogram free C5a levels for at least 12 days.

[0085] In another aspect, the disclosure presents a method of treating a human suffering from a complement related disorder (e.g. C5a related disorder of the complement system), a method comprising administering to a human composition comprising multiple isolated antibodies, wherein each of the multiple antibodies has two antigen binding sites, each of which antigen binding sites can independently bind to free human C5a (hC5a) or unsealed human C5 (hC5), wherein the antibodies are administered at a dose sufficient to: (a) achieving molar Cmax values significantly lower than the molar physiological concentration of unsealed hC5 and (b) partial or complete binding and demarcation of nanogram free C5a levels for at least 24 days.

[0086] In another aspect, the disclosure presents a method of treating a human being affected by a complement related disorder (e.g. C5a related disorder of the complement system), the method comprising administering to a human composition comprising multiple isolated antibodies, wherein each of the multiple antibodies has two antigen binding sites, each of which antigen binding sites can independently bind to free human C5a (hC5a) or un-cleaved human C5 (hC5), wherein the antibodies are administered at a dose sufficient to: (a) achieving molar Cmax values significantly lower than the molar physiological concentration of unsealed hC5 and (b) partial or complete binding and demarcation of pathophysiological levels of free C5a.

[0087] It is understood that any of the compositions (e.g., having multiple antibodies) or isolated antibodies (e.g., which retain, in the presence of C5 or molar excess of C5, a free Fab frame capable of binding to free C5a) as described herein may be : (a) formulated as pharmaceutical compositions as disclosed, (b) included in therapeutic kits (described herein), or (c) included in the pre-filled syringes described herein.

[0088] As described in the working examples provided herein, the inventors have also found the existence of an antibody, BNJ383 (see below), which not only binds with high affinity (subnanomolar affinity) to free hC5a, but also at excess concentrations of un cleaved C5 inhibits formation of the terminal complement complex (TCC) in a dose-dependent manner. Even at concentrations of anti-C5a antibody in excess of 6.5 fold excess of C5, however, inhibition of TCC is not complete. While the disclosure is not limited in any way by any particular theory or mechanism of action, the antibody may inhibit the formation of TCC by binding to at least the non-cleaved C5 fraction and preventing its cleavage and / or preventing otherwise successfully binding to C5 with additional TCC ingredients. The inventors have recognized that such an antibody is useful in the treatment of complement-related disorders, e.g., in which C5a plays an important role and C5b-containing TCC may play a less significant role. Such disorders may include, e.g. sepsis, acute respiratory distress syndrome (ARDS), septic shock, antiphospholipid syndrome, catastrophic antiphospholipid syndrome, disseminated intravascular coagulation, lupus nephropathy, Goodpasture syndrome, burns or severe burns, asthma, HELLP syndrome (hemolytic anemia and hemolytic anemia) low platelet count), inflammation-induced pain, C5a-regulated neutropenia, age-related macular degeneration (AMD), chronic obstructive pulmonary disease and rheumatoid arthritis.

[0089] The inventors have also recognized that the use of such an anti-C5a antibody to treat these conditions, among others, can provide an even more favorable safety profile compared to the use of terminal complement inhibitor drugs. As noted above, one notable consequence of inhibiting the terminal components of the complement system such as C5, C5b, C6, C7, C8 or C9 is a reduced protection by the host's immune system against enveloped bacteria that the terminal part of the complement system usually lyses - for example, Neisseria meningitides and Neisseria gonorrhoeae. Because the anti-C5a antibodies described in this section inhibit the C5a-mediated inflammatory response, but do not completely inhibit the formation of the terminal complement complex that lys these enveloped bacteria, patients receiving the therapeutic anti-C5a antibody described herein may not require protective vaccinations, e.g. vaccination against Neisseria meningitides and Neisseria gonorrhoeae. Partial TCC inhibition, while not completely abrogating the antimicrobial response of the terminal complement, can actually reduce TCC-induced inflammation in the form of tissue injury. Partial TCC inhibition, in combination with C5a inhibition, is thought to make the anti-C5a antibody an even more potent anti-inflammatory compound.

[0090] Accordingly, in another aspect, the disclosure provides an antibody or antigen-binding fragment thereof that binds to free C5a, wherein free C5a is human C5a having the amino acid sequence set forth in SEQ ID NO: 1, wherein the antibody inhibits binding C5a to the C5a receptor, and wherein the antibody partially inhibits the formation of the terminal complement complex (TCC). Partial inhibition by an anti-C5a antibody or antigen-binding fragment thereof described herein can mean, e.g., complement activity that is, in the presence of the antibody, up to, or no more than, 80 (e.g. 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or 25)% complement activity in the absence of the antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof binds to free C5a (e.g. free hC5a) with subnanomolar affinity. In some aspects of the disclosure, the antibody or antigen-binding fragment thereof has an affinity for free C5a that is at least 100-fold higher than the corresponding affinity of the antibody or antigen-binding fragment for un cleaved C5. In some embodiments, the antibody or antigen-binding fragment thereof inhibits by at least 50% the formation of TCC at concentrations exceeding 200 (e.g. 210, 220, 230, 240, 250, 260,

270, 280, 290, 300, 320, 340, 360, 380, or 400 or more) μg / ml as measured using the CH50eq assay. In some aspects of the disclosure, the antibody or antigen-binding fragment thereof inhibits by at least 50% the classical complement activation pathway at concentrations exceeding 200 (e.g. 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 320, 340, 360, 380, or 400 or more) μg / ml as measured using the Wieslab® kit for the classic complement pathway, as described in the working examples.

[0091] In another aspect, the disclosure provides a method of treating a human affected by a complement related disorder (e.g., a C5a related complement disorder or a complement related inflammatory disorder). The method comprises administering to a human an effective amount of an antibody or antigen-binding fragment thereof that inhibits C5a binding to the C5a receptor, and wherein the antibody partially inhibits the formation of the terminal complement complex (TCC). See. above. The disorder may be one of those known in the art or described herein.

[0092] In another aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to a human C5a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, but does not bind to the alpha chain of an un cleaved native C5, wherein the antibody or an antigen-binding fragment thereof binds to a human C5a polypeptide with KD that is less than 1.25 x 10-9 M.

[0093] In another aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to a human C5a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, wherein the antibody inhibits at least 50% of human C5a dependent on neutrophil activation in human in a 1: 1 molar ratio (antibody binding site: C5a). In some aspects of the disclosure, the antibody inhibits at least 50% of human C5a-dependent neutrophil migration in a human assay in which 0.4 nM antibody was used to inhibit neutrophil activation - 2 nM activity of human C5a as described in Example 5. In some aspects disclosing the antibody does not comprise the exemplary CDR pair 3 shown in Table 1. In some aspects of the disclosure, the antibody is not BNJ371.

[0094] In some aspects of the disclosure, an isolated antibody or antigen binding fragment thereof described herein binds to a human C5a polypeptide having the amino acid sequence depicted in SEQ ID NO: 2.

[0095] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a light chain polypeptide comprising: a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 20; A light chain CDR2 containing the amino acid sequence depicted in

SEQ ID NO: 21; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 22.

[0096] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a light chain polypeptide comprising: a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 20; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 38; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 22.

[0097] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a heavy chain polypeptide comprising: a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 28; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 29; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 30.

[0098] In some aspects of the disclosure, the isolated antibody or antigen binding fragment thereof described herein comprises a heavy chain polypeptide comprising: a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 28; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 67; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 30.

[0099] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a heavy chain polypeptide comprising: a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 28; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 46; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 47.

[0100] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 37 or SEQ ID NO: 36:

[0101] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 27 or SEQ ID NO: 33.

[0102] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 37 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 27.

[0103] In some embodiments, the isolated antibody or antigen binding fragment thereof described herein comprises a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 36 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 33.

[0104] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 19 or SEQ ID NO: 17.

[0105] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 27 or SEQ ID NO: 25.

[0106] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 19 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 27.

[0107] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 17 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 25.

[0108] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 42 or SEQ ID NO: 40:

[0109] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 27 or SEQ ID NO: 33.

[0110] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 42 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 27.

[0111] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 40 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 33.

[0112] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 17 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 33.

[0113] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a heavy chain polypeptide comprising the amino acid sequence depicted in any of SEQ ID NO: 45, SEQ ID NO: 44 or SEQ ID NO: 49.

[0114] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 19 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 45.

[0115] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 17 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 44.

[0116] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 17 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 49.

[0117] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 37 or SEQ ID NO: 36:

[0118] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 45 or SEQ ID NO: 49.

[0119] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 37 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 45.

[0120] In some embodiments, the isolated antibody or antigen binding fragment thereof described herein comprises a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 36 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 49.

[0121] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 42 or SEQ ID NO: 40:

[0122] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 45 or SEQ ID NO: 49.

[0123] In some embodiments, the isolated antibody or antigen binding fragment thereof described herein comprises a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 42 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 45.

[0124] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein comprises a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 40 and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 49.

[0125] In some aspects of the disclosure, an isolated antibody or antigen-binding fragment thereof described herein binds to hC5a with KD that is less than 7 x 10-10 M.

[0126] In some aspects of the disclosure, the isolated antibody or antigen-binding fragment thereof described herein binds to hC5a with KD that is less than 5 x 10-10 M.

[0127] In some aspects of the disclosure, the isolated antibody or antigen-binding fragment thereof described herein binds to hC5a with KD that is less than 3 x 10-10 M.

[0128] In some aspects of the disclosure, the isolated antibody or antigen-binding fragment thereof described herein binds to hC5a with KD that is less than 2.5 x 10-10 M.

[0129] In some aspects of the disclosure, the isolated antibody or antigen-binding fragment thereof described herein binds to hC5a with KD, which is less than 1.5 x 10-10 M.

[0130] In some aspects of the disclosure, an isolated antibody or antigen-binding fragment thereof described herein binds to hC5a with a KD that is less than 1.0 x 10-10 M.

[0125] In some aspects of the disclosure, an isolated antibody or antigen-binding fragment thereof described herein binds to hC5a with KD, which is less than 8.0 x 10-11 M.

[0132] In some aspects of the disclosure, the antibody inhibits at least 70 (e.g., at least 75, 80, 85, 90, or 95) or more human C5a-dependent neutrophil activation in humans at a molar ratio of 1; 1 (antigen binding site : C5a). In some aspects, the antibody does not comprise the exemplary CDR pair 3 shown in Table 1. In some aspects, the antibody is not BNJ371.

[0133] In yet another aspect, the disclosure provides an isolated antibody or antigen binding fragment thereof that comprises a set of light chain CDRs as given in Table 3 or Table 7. For example, an isolated antibody or antigen binding fragment thereof of the disclosure may comprise a light chain polypeptide comprising: (i) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 140; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 96; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 142; (ii) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 156; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 157; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 158; (iii) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 164; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 165; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 166; (iv) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 172; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 173; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 174; (v) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 84; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 85; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 86; (vi) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 92; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 89; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 93; (vii) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 88; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 89; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 90; (viii) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 95; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 96; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 97; (ix) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 99; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 100; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 101; (x) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 84; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 85; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 103; (xi) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 105; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 106; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 107; (xii) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 92; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 89; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 108; (xiii) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 20; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 110; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 111; or (xiv) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 20; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 21; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 113. In some aspects of the disclosure, the antibody or antigen-binding fragment thereof comprising a set of light chain CDRs also comprises a heavy chain polypeptide comprising any of the sets of heavy chain CDRs as given in Table 8.

[0134] In another aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof that comprises a set of heavy chain CDRs as given in Table 3 or Table 8. For example, in some aspects, the disclosure is an isolated antibody or antigen-binding fragment thereof described herein contain a heavy chain polypeptide comprising:

(i) a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 115; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 144; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 117; (ii) a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 28; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 67; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 30; (iii) a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 160; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 161; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 162; (iv) a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 168; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 169; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 170; (v) a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 176; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 177; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 178; (vi) a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 115; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 116; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 117; (vii) a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 119; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 120; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 121; (viii) a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 115; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 123; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 117; (ix) a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 115; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 124; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 117; (x) a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 119; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 126; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 127; (xi) a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 115; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 129; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 117; (xii) a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 131; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 132; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 133; (xiii) a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 28; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 46; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 47; or (xiv) a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 136; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 137; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 138. In some aspects of the disclosure, the antibody or antigen-binding fragment thereof comprising a set of heavy chain CDRs also includes a light chain polypeptide comprising any of the light chain CDR sets as are given in Table 7.

[0135] In another aspect of the disclosure, an isolated antibody or antigen-binding fragment thereof comprising a set of light chain CDRs from Table 7 and its related heavy chain CDR set as set out in Table 8. In another aspect, the disclosure presents isolated antibodies having or an antigen binding fragment thereof comprising a paired set of light chain and heavy chain CDRs as given in Table 3 or Table 9. For example, in some aspects of the disclosure, an isolated antibody, or antigen-binding fragment thereof, described herein comprises: (i) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 140; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 96; A light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 142; Heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 115; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 144; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 117; (ii) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 20; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 21; and a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 22; Heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 28; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 67; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 30; (iii) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 156; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 157; A light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 158; Heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 160; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 161; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 162; (iv) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 164; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 165; A light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 166; Heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 168; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 169; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 170; (v) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 172; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 173; A light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 174; Heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 176; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 177; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 178; (vi) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 88; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 89; A light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 90; Heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 119; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 120; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 121; (vii) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 105; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 106; A light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 107; Heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 115; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 124; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 117; (viii) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 84; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 85; A light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 86; Heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 115; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 116; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 117; (ix) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 20; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 110; A light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 111; Heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 136; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 137; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 138; (x) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 20; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 21; A light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 113; Heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 28; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 46;

and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 47; (xi) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 99; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 100; A light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 101; Heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 119; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 126; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 127; (xii) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 95; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 96; A light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 97; Heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 115; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 144; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 117;

(xiii) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 140; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 96; A light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 142; Heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 115; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 123; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 117; (xiv) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 105; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 106; A light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 107; Heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 115; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 123; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 117; (xv) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 92; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 89; A light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 108; Heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 115; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 144; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 117; (xvi) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 92; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 89; A light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 93; Heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 115; Heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 123; and a heavy chain CDR3 comprising the amino acid sequence depicted in

SEQ ID NO: 117; (xvii) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 92; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 89; A light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 93; Heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 115; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 144; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 117; (xviii) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 84; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 85; A light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 103; Heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 115; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 129; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 117; (xix) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 95; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 96; A light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 97; Heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 115; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 123; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 117; (xx) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 84; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 85; A light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 103; Heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 115; Heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 144; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 117; or (xxi) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 20; A light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 21; A light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 113; Heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 131; A heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 132; and a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 133.

[0136] In some aspects of the disclosure, the antibody or antigen-binding fragment thereof comprises a paired set of light chain CDRs and a set of heavy chain CDRs as given in Table 3. In some aspects of the disclosure, the antibody or antigen-binding fragment thereof comprises a paired set of CDRs light chain and heavy chain CDR set as given in Table 2. For example, the disclosure provides an antibody comprising: (i) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 20; (ii) a light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 21; and (iii) a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 22; (iv) a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 28; (v) a heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 46; and (vi) a heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 47.

[0137] In some aspects of the disclosure, the antibody or antigen-binding fragment thereof comprises a light chain variable region given in Table 2, wherein the light chain is paired with any of the heavy chain variable regions given in Table 2. For example, the disclosure provides an antibody (or antigen binding fragment thereof) comprising: (a) a light chain variable region having the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 42 and (b) a heavy chain variable region having the amino acid sequence comprising the amino acid sequence shown on SEQ ID NO: 45.

[0138] In some aspects of the disclosure, the antibody or antigen binding fragment thereof described herein comprises: (i) a heavy chain variable region framework 1 comprising the amino acid sequence depicted in SEQ ID NO: 68 or SEQ ID NO: 69; (ii) a heavy chain variable region framework 2 containing the amino acid sequence depicted in SEQ ID NO: 70 or SEQ ID NO: 71; and the heavy chain variable region framework 3 containing the amino acid sequence depicted in any of SEQ ID NOs: 72 to 74. In some aspects of the disclosure, the antibody or antigen binding fragment thereof comprises the heavy chain variable region framework 4 containing the amino acid sequence depicted in SEQ ID NO: 75. In some aspects of the disclosure, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence depicted in any one of SEQ ID NOs: 76 to 80. The antibody heavy chain may comprise any of the heavy chain CDR sets described herein. The heavy chain variable region may, in some embodiments, be paired with a polypeptide variable region comprising the amino acid sequence depicted in SEQ ID NO: 16.

[0139] In some aspects of the disclosure, the antibody or antigen-binding fragment thereof binds to a non-human C5a protein. For example, the antibody or antigen binding fragment thereof may bind to murine C5a and / or arginine-deficient murine C5a protein. In some aspects of the disclosure, an isolated antibody or antigen-binding fragment thereof may bind to murine C5a (and / or arginine-deficient murine C5a) and comprise: (i) a light chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 54; (ii) a light chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 55; (iii) a light chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 56: (iv) a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 62; (v) a heavy chain CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 63; and (vi)

A heavy chain CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 64. In some aspects of the disclosure, the anti-mouse C5a antibody may comprise a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 59; and a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 66.

[0140] In some aspects of the disclosure, an isolated antibody or antigen-binding fragment thereof described herein inhibits the interaction between C5a and a C5a receptor. The C5a receptor may be, e.g., C5aR1 or C5L2. [0141] In some aspects of the disclosure, the isolated antibody or antigen-binding fragment thereof described herein does not significantly inhibit complement regulated red blood cell hemolysis in vitro and / or in vivo.

[0142] In some aspects of the disclosure, the isolated antibody (and any antigen-binding fragment thereof accordingly) is a monoclonal antibody, a humanized antibody, or a fully human antibody.

[0143] In some aspects of the disclosure, the isolated antibody or antigen-binding fragment thereof described herein is selected from the group consisting of a recombinant antibody, single chain antibody, diabody, intrabody, chimerized or chimeric antibody, deimmunized human antibody, Fv fragment, Fd fragment, fragment Fab, Fab 'fragments and F (ab') fragment 2.

[0144] In some aspects of the disclosure, the isolated antibody or antigen-binding fragment thereof described herein is multispecific (e.g., bispecific) in the sense that the antibody or fragment binds to at least two different epitopes. Two different epitopes may be, e.g., two different epitopes from the same protein (e.g., C5a), or the antibody may bind to the first epitope of the first protein (e.g., C5a) and the second epitope of the second protein.

[0145] In some aspects of the disclosure, the isolated antibody or antigen-binding fragment thereof described herein comprises a heterologous moiety. A heterologous moiety may be, e.g., sugar. For example, the antibody or antigen binding fragment thereof may be glycosylated. A heterologous moiety can be, e.g. a detectable label such as, but not limited to, a fluorescent label, luminescent label, heavy metal label, radioactive label or enzyme label.

[0146] In some aspects of the disclosure, an isolated anti-C5a antibody or antigen-binding fragment thereof described herein is modified with a moiety that improves the stabilization and / or retention of the antibody in circulation. For example, the modification may be PEGylation or hesylation. In another embodiment, the anti-C5a antibody may comprise an altered constant region that has reduced (or not) effector function, compared to the effector function of its corresponding unchanged constant region. In some embodiments, the anti-C5a antibody comprises an altered constant region that has between about 0 to about 20% of the effector function of the unchanged constant region. Exemplary embodiments of such antibodies with reduced effector function are described herein.

[0147] In another aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof that cross-blocks the binding of any of the above antibodies.

[0148] In yet another aspect, the disclosure provides a pharmaceutical composition comprising one or more of any of the isolated antibodies or antigen-binding fragments thereof described herein and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0149] In another aspect, the disclosure provides: (i) a nucleic acid encoding one or more of any of the antibodies or antigen-binding fragments thereof described herein; (ii) a nucleic acid containing vector; (iii) an expression vector containing nucleic acid; and / or (iv) a cell containing the expression vector or vector. In another aspect, the disclosure provides a method of producing a polypeptide (such as any of the antibodies or antigen-binding fragments thereof described herein). The method comprises culturing the above-mentioned cell (containing the expression vector) under conditions and for a period of time sufficient to allow the cell to express the antibody or antigen-binding fragment encoded by the nucleic acid in the vector. The method may also include an isolated antibody or antigen-binding fragment from the cell or medium in which the cell is cultured.

[0150] In another aspect, the disclosure provides an isolated nucleic acid encoding any of the amino acid sequences described herein, or a polypeptide having an amino acid sequence comprising, or consisting of, any of the amino acid sequences described herein. The nucleic acid may be included in a vector, e.g., an expression vector, and / or may be present in the cell.

[0151] In yet another aspect, the disclosure provides a therapeutic kit comprising: (i) one or more isolated antibodies or antigen binding fragments described herein (e.g., one or more of any of the humanized antibodies or antigen binding fragments thereof described herein) ; and (ii) means for delivering the antibody or antigen-binding fragment to a subject. Measures may be suitable for, e.g. subcutaneous delivery, ocular delivery, or delivery delivery of the antibody or antigen-binding fragment thereof to a subject. The agents may mean, e.g., a syringe, a two-cylinder syringe, or two separate syringes implemented for use in the administration of a therapeutic antibody or antigen-binding fragment thereof, while withdrawing fluid from the knee (e.g. for analysis) in a push-pull manner [pressure and withdrawal] . In some aspects of the disclosure, the agents are for ocular delivery and include an intradural insert or contact lens, each containing an antibody binding fragment. In some aspects of the disclosure, the agents are suitable for pulmonary delivery. For example, the agent may be an inhaler or nebulizer. In some aspects of the disclosure, the agent is a pre-filled syringe, such as a pen device. The pre-filled syringe may contain, e.g. at least one pharmaceutical unit dosage form of one or more antibodies or antigen-binding fragments thereof as provided herein.

[0152] In some aspects of the disclosure, the therapeutic kits described herein may contain at least one additional active agent for use in treating a complement related disorder in a subject. The additional active agent may be, e.g., any of the additional agents described herein.

[0153] In yet another aspect, the disclosure provides a method for treating or preventing a disorder associated with the complement system. The method includes administering to a human in need thereof a therapeutic antibody or antigen-binding fragment thereof described herein in an amount sufficient to treat a disorder associated with the complement system of an afflicted human being. The method may also include identifying the individual as suffering from a complement related disorder. The complement-related disorder may be, e.g., complement-related inflammatory disorder, atypical haemolytic uraemic syndrome, age-related macular degeneration, rheumatoid arthritis, sepsis or antiphospholipid syndrome. In some embodiments, the complement related disorder is complement related pulmonary disorder. For example, complement-related pulmonary disorder can be, e.g., asthma or chronic obstructive pulmonary disease. Other disorders associated with the complement system, amenable to treatment or prevention as indicated in the method are described herein. The mode of administration, which may vary depending on the type of disorder associated with the complement system to be treated, can be, e.g., intravenous administration, intrapulmonary administration, ocular administration, subcutaneous administration; or delivery by delivery.

[0154] In some aspects of the disclosure, the antibody or antigen-binding fragment thereof is administered to a human in an amount and with a frequency sufficient to maintain a reduced level of systemic C5a activity throughout the duration of treatment. In some aspects of the disclosure, the methods may include, after administration, monitoring a human for an improvement in one or more symptoms of a complement related disorder.

[0155] In some aspects of the disclosure, the methods may include administering to a human one or more additional therapeutics.

[0156] In yet another aspect, the disclosure provides an industrial article that comprises: (i) a container with a label, and (ii) a composition comprising the antibody or antigen binding fragment thereof described herein. The marker may indicate that the composition is to be administered to a person suspected of having or at risk of having a disorder associated with the complement system. An industrial article may also include one or more additional active ingredients.

[0157] Throughout the disclosure, the term "antibody" refers to the entire molecule or intact antibody (e.g., IgM, IgG, IgA, IgD, or IgE) that is generated by any of a number of methods that are known in the art and described herein . The term "antibody" includes a polyclonal antibody, monoclonal antibody, chimerized or chimeric antibody, humanized antibody, de-immunized human antibody and fully human antibody. The antibody can be obtained from or derived from any of a number of species, e.g. mammals such as humans, non-human primates (e.g. monkeys, baboons or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats and mice. The antibody may be a purified or recombinant antibody.

[0158] As used herein, the term "antibody fragment," "antigen-binding fragment," or similar terms refer to an antibody fragment that retains the ability to bind to an antigen (e.g., an epitope present in C5a, but not in the alpha chain of an un cleaved native C5 protein) , e.g. a single chain antibody (scFv), Fd fragment, Fab fragment, Fab 'fragment or F (ab') 2 fragment. scFv means a single polypeptide chain that includes both the heavy and light chain variable regions of the antibody from which scFv is derived. In addition, diabodies (Poljak (1994) Structure 2 (12): 1121-1123; Hudson et al. (1999) J Immunol Methods 23 (1-2): 177-189), minicrafts, tricycles (Schoonooghe et al. (2009 ) BMC Biotechnol 9:70), domain antibodies (also known as "heavy chain immunoglobulins" or camelids; Holt et al. (2003) Trends Biotechnol 21 (11): 484-490); and intrabodies (Huston et aL (2001) Hum Antibodies 10 (3-4): 127142; Wheeler et al. (2003) Mol Ther 8 (3): 355-366; Stocks (2004) Drug Discov Today 9 (22): 960-966) are included in the definition of antibody fragments and can be incorporated into the compositions, and used in the methods described herein.

[0159] Unless otherwise defined, all technical and scientific terms used in the description have the same meaning as commonly understood by those of ordinary skill in the art to which disclosure belongs. In the event of non-compliance, disclosure, including definitions, is paramount. Although any methods and materials similar to or equivalent to those described herein can be used in the testing or practice of the present invention, preferred methods and materials are described below. All of the publications, patent applications, patent specifications and other references mentioned in the description are incorporated in their entirety.

[0160] Other features and advantages of the disclosure, eg, methods of treating a complement related disorder in a subject, will be apparent from the description, examples, and claims.

Short description of the figures [0161]

Fig. 1 is a Venn diagram showing the extent of epitope overlap in human C5a bound by a selected set of murine anti-human C5a antibodies:

5an101ME, 5an180ME, 5an048ME, 5an179ME and 5an178ME.

Fig. 2 is a line graph showing antagonism of C5a-mediated signal transduction in vitro using a neutrophil activation assay. The Y axis represents the optical density (OD) measurement of a chromogenic substrate as a function of myeloperoxidase release by freshly isolated human neutrophils. The X axis represents the concentration of antibodies incubated with the cells.

Tested humanized antibodies - BNJ367, BNJ369, BNJ371, BNJ378, BNJ381, BNJ383, and humanized anti-C5 antibody - are identified in the legend.

Fig. 3 is a line graph showing the effect of several therapeutic antibodies on arthritis in a mouse model of rheumatoid arthritis. The Y axis represents the thickness of the initial inflamed knee in millimeters. The X axis represents the days after the onset of disease symptoms. Tested therapeutic antibodies - 5an195ME (mouse anti-mouse C5a antibody) and a control antibody with the same Fc region as the anti-C5a antibody - are identified in the legend.

Fig. 4 is a line graph showing the effect of several therapeutic antibodies on overall disease severity in a mouse model of rheumatoid arthritis. The Y axis represents the arthritis index. The X axis represents the days after the onset of disease symptoms. Tested therapeutic antibodies - 5an195ME (mouse anti-mouse C5a antibody) and control antibody with the same Fc region as the anti-C5a antibody - are identified in the legend.

Figure 5 sets out a series of humanized heavy chain variable region sequences. In order from highest to lowest: heavy chain variable region of the humanized anti-C5a BNJ3 antibody (SEQ ID NO: 76); heavy chain variable region of the humanized anti-C5a BNJ346 antibody (SEQ ID NO: 77); heavy chain variable region of the humanized anti-C5a BNJ347 antibody (SEQ ID NO: 78); heavy chain variable region of the humanized anti-C5a BNJ354 antibody (SEQ ID NO: 79); and the heavy chain variable region of the humanized anti-C5a BNJ350 antibody (SEQ ID NO: 80). One skilled in the art will recognize the distinction between heavy chain framework regions 1, 2, 3, and 4, and heavy chain 1, 2, and 3 CDRs. Such demarcations as defined in Kabat et al. (infra) is shown in the figure. "HC FR1" refers to the heavy chain variable region 1, "HC FR2" refers to the heavy chain variable region 2, "HC FR3" refers to the heavy chain variable region 3, and "HC FR4" refers to the heavy chain variable region heavy 4. "HC CDR1" refers to the heavy chain complementarity determining region (CDR) 1, "HC CDR2" refers to the heavy chain variable region CDR2, and "HC CDR3" refers to the heavy chain variable region CDR3.

Fig. 6 is a line graph showing the percentage of circulating neutrophils in the blood of mice after administration of hC5a to mice. On the Y axis, the number of neutrophils is expressed as a percentage "for baseline," which is the number of neutrophils at time 0 (or 100% neutrophils). The X axis represents the time in minutes. Mouse cohorts were administered intravenously with a control antibody [protective antigen anthrax, IgG2 / G4 isotype] ("control"; five mice) or anti-human C5a BNJ383 antibody at one of the following doses: 24 mg / kg (five mice); 12 mg / kg (five mice); 6 mg / kg (five mice); and 3 mg / kg (five mice) followed by hC5a. See. Example 13. Six mice, "sham", were not administered human C5a.

Fig. 7 is a bar graph showing myeloperoxidase (MPO) levels in mouse plasma before and after administration of human C5a to mice. The Y-axis represents the concentration (ng / ml) of MPO in mouse plasma. The X axis represents the time in minutes. Mouse cohorts were intravenously administered a control antibody [protective anthrax 63 against anthrax, IgG2 / G4 isotype] ("control"; eight mice) or anti-human C5a BNJ383 antibody at one of the following doses: 24 mg / kg (five mice); 12 mg / kg (five mice); 6 mg / kg (five mice); and 3 mg / kg (five mice) followed by hC5a. Four mice, "sham", were not administered human C5a.

Fig. 8 is a line graph showing the change in the level of human C5a in mouse plasma (administered human C5a) in the presence or absence of different concentrations of anti-hC5a antibody (BNJ383). The Y axis represents the concentration (ng / ml) of hC5a in mouse plasma. The x-axis represents the time in minutes. Mouse cohorts were dosed intravenously with a control antibody [protective anthrax 63 against anthrax, IgG2 / G4 isotype] ("control"; six mice) or anti-human C5a BNJ383 antibody at one of the following doses: 24 mg / kg (three mice); 12 mg / kg (three mice); 6 mg / kg (three mice); and 3 mg / kg (three mice) followed by hC5a. Four mice, "sham", were not administered human C5a.

Fig. 9 is a line graph showing binding competition to human C5a in vitro. 250 pM ruthenium-labeled anti-C5a antibody (BNJ383) were incubated with 1 nM biotinylated hC5a, along with various concentrations (e.g. 400, 133, 44.4, 14.8, 4.9, 1.6, and 0.5 nM ) one of the following: (a) human arginine-depleted C5a protein in phosphate buffered saline, (b) human plasma, (c) cynomolgus plasma, (d) Balb / C plasma (mouse) or (e) DBA / 2J plasma (mouse). For plasma components (b), (c), (d) and (e), the concentration refers to the approximate final concentration of C5 antigen in the incubated mixture. The Y axis represents arbitrary fluorescence units as a function of the detected amounts of ruthenium-labeled anti-C5a antibody. The X axis represents the concentration (nM) of the antigenic competitor.

Fig. 10 is a line graph showing the effect of several systemic protein inhibitors on the complement pathway (AP). The Y-axis represents the percentage of AP complement activity compared to baseline (BL; activity level in the absence of complement inhibitor). The X axis represents the concentration of a given complement inhibitor (μΜ). The effect of anti-hC5a antibody, BNJ383, together with anti-C5 antibody on AP activity was evaluated.

Fig. 11 is a line graph showing the effect of several complement protein inhibitors on the classical complement pathway (CP). The Y axis represents the percentage of CP complement activity compared to baseline (BL; activity level in the absence of complement inhibitor). The X axis represents the concentration of a given complement inhibitor (μΜ). The effect of anti-hC5a antibody, BNJ383, together with anti-C5 antibody on CP activity was evaluated.

Figures 12A, 12B, 12C and 12D are a series of chromatographs showing the retention times of anti-C5a antibody (BNJ383) and anti-hC5 antibody in the presence or absence of hC5 protein. For all panels, the X axis represents retention time in minutes, and the Y axis represents absorbance units at 214 nm. The insert subpanel on each panel represents the magnification of the peaks shown.

Fig. 12A shows the retention time for BNJ383 in the absence of hC5 protein.

Fig. 12B shows the retention time for anti-C5 antibody in the absence of hC5 protein.

Fig. 12C shows the retention time for BNJ383 in the presence of hC5 (2.1 fold molar excess of hC5 relative to BNJ383). From right to left, these peaks mean: (a) uncomplexed BNJ383 or hC5; (b) BNJ383 with one antigen-binding site bound to undissolved hC5; and (c) minimizes the fraction coherent with the BNJ383 double facing with open cut hC5.

Fig. 12D shows the retention time for an anti-C5 antibody in the presence of an equimolar amount of hC5. From right to left, said peaks mean: (a) uncomplexed anti-C5 or hC5 antibody; (b) anti-C5 antibody with one antigen-binding site bound to undissolved hC5; and (c) an anti-C5 antibody bound to two uncleaved C5 molecules.

Fig. 13 is a line graph showing binding of anti-C5a BNJ383 antibody to hC5a deficient in arginine in the presence of hC5 using ELISA. The X axis represents the concentration (ng / ml) of the antibody. The Y axis represents optical density at 450 nm.

Fig. 14 is a scatter plot showing the concentration of free C5a / C5a lacking arginine present in cynomolgus plasma as a function of the plasma concentration of anti-C5a antibody (BNJ383). The Y-axis represents the concentration (pg / ml) of C5a / C5a lacking arginine detected in crayfish plasma samples at time points in the range of 1 day to 30 days after a single intravenous dose of BNJ383 at 1 mg / kg, 10 mg / kg, 100 mg / kg, 250 mg / kg or

400 mg / kg body weight of the animal. The X axis represents the BNJ383 concentration (pg / ml) in each sample.

Fig. 15A is a scatter plot showing the percentage of hemolytic activity in serum samples relative to baseline (Y-axis) values initiated via the classical pathway as a function of circulating anti-C5a (BNJ383) antibody concentration (x-axis).

Fig. 15B is a scatter plot showing the percentage of terminal complement formation that is initiated via the classical pathway in serum samples measured in the CH50eq assay relative to baseline (Y axis) as a function of anti-C5a antibody concentration (BNJ383) in the circulation (X axis) .

Fig. 15C is a scatter plot showing the percentage of terminal complement formation that is initiated via the classical pathway in serum samples measured in the CCP assay relative to baseline (Y axis) as a function of anti-C5a antibody concentration (BNJ383) in the circulation (X axis) .

Detailed Description of the Invention [0162] The disclosure provides antibodies and antigen-binding fragments thereof that bind to free C5a (e.g., free human C5a), compositions comprising antibodies or fragments thereof, and methods of using any of the following to treat or prevent system-related disorders complement, such as, but not limited to, aHUS, macular degeneration (e.g. AMD), RA, sepsis, antiphospholipid syndrome, burns (e.g. severe burns), Goodpasture syndrome, lupus nephropathy, or complement-related pulmonary disorder such as asthma or chronic obstructive pulmonary disease (COPD). The disclosure also provides anti-C5a antibodies (and fragments thereof) that are cross-reactive between free C5a from humans and free C5a from a non-human mammal species, such as non-human primates (e.g., cynomolgus or rhesus macaque). In the complete absence of limiting nature, exemplary antibodies (and antigen-binding fragments), compositions (e.g., pharmaceutical compositions and formulations), and methods of using the compositions are discussed in detail below and detailed in the working examples.

Anti-C5a antibodies and antigen-binding fragments thereof [0163] The disclosure provides antibodies that bind to a component of the C5a complement. As discussed above, the pro-form C5, having 1676 amino acid residues, a protein precursor, is processed in a series of proteolytic cleavage events. The first 18 peptides (numbers -18 to -1) are a signal peptide that is cleaved from the protein precursor. The remaining 1658 amino acid portion of the protein is cleaved in two places to form alpha and beta chains. The first cleavage event occurs between amino acid residues 655 and 656. The second cleavage occurs between amino acid residues 659 and 660. Two cleavage events result in the formation of three separate polypeptide fragments: (i) a fragment containing amino acids 1 to 655, which is referred to as the beta chain; (ii) a fragment containing amino acids 660 to 1658, which is referred to as the alpha chain; and (iii) a tetrapeptide fragment consisting of amino acids 656 to 659. The polypeptide fragments of the alpha chain and beta chain are joined together by a disulfide bond and constitute a mature C5 protein. CP or AP C5 convertase activates mature C5 by cleaving the alpha chain between residues 733 and 734, resulting in the release of the C5a fragment (amino acids 660 to 733). The remainder of mature C5 is a C5b fragment that contains residues 734 to 1658 of the alpha chain disulfide bound to the beta chain.

[0164] In vivo, C5a is rapidly metabolized by the serum enzyme carboxypeptidase B to an amino acid form, called "arginine-free C5a," which has lost the carboxyterminal residue of arginine. Accordingly, in some aspects of the disclosure, the antibody that binds to free C5a also binds to arginine-depleted C5a. In some aspects of the disclosure, the antibody that binds to C5a does not bind to arginine-depleted C5a.

[0165] In some aspects of the disclosure, the anti-C5a antibody binds to a neoepitope present in C5a, i.e. an epitope that becomes exposed when C5a is released from the mature C5 alpha chain fragment. That is, in some aspects, the anti-C5a antibody described herein binds to C5a and / or C5a lacking arginine, but not to un cleaved, native (fully folded) C5.

[0166] As described above, in some aspects of the disclosure, an anti-C5a antibody or antigen-binding fragment thereof may bind to an unsprayed, C5 treated (e.g., C5 plasma) subpopulation of less than 10 (e.g., less than 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, 0.4, 0.3, 0.2, or less than 0.1)% of the total full-length C5 population in the sample (e.g. blood or plasma sample or sample containing recombinant full length C5), which subpopulation is wholly or partly denatured such that otherwise covered C5a neoepitope is exposed. Thus, the anti-C5a antibody or antigen-binding fragment thereof described herein may, in some embodiments, bind to free C5a, but not to C5 with 90% or a higher degree of an un cleaved native C5 population. In some embodiments, the partially or fully denatured subpopulation is inactive or has reduced activity (e.g., less than 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20 , 15, 10, 5% activity of fully functional, full-length C5 protein) in any number of suitable assays useful for testing C5 activity, e.g. hemolytic assay or CH50eq assay. Suitable methods for testing the activity of a minor subpopulation to which it may bind, in some embodiments, the anti-C5a antibody described herein is known in the art and described herein.

[0167] In some aspects of the disclosure, an anti-C5a antibody binds to a mammalian (e.g., human) C5a protein. For example, an anti-C5a antibody may bind to human C5a protein having the following amino acid sequence:

TLQKKIEEIAAKYKHSVVKKCCYDGACVNNDETCEQRAARISLGPRCIKAFTE CCVVASQLRANISHKDMQLGR (SEQ ID NO: 1). In some aspects of the disclosure, the anti-C5a antibody can bind to an arginine-deficient human C5a protein having the following amino acid sequence:

TLQKKIEEIAAKYKHSVVKKCCYDGACVNNDETCEQRAARISLGPRCIKAFTE CCVVASQLRANISHKDMQLG (SEQ ID NO: 2). The anti-C5a antibody described herein can bind to both full-length human C5a and non-arginine-terminated human C5a.

[0168] In some aspects of the disclosure, the antibody may bind to human C5a at an epitope within or overlapping a structural fragment of a protein having the amino acid sequence: TLQKKIEEIAAKYK (SEQ ID NO: 3); HSVVKKCCYDGAC (SEQ ID NO: 4); VNNDE (SEQ ID NO: 5); TCEQRAAR (SEQ ID NO: 6); ISLG (SEQ ID NO: 7); PRCIKAFTECCVVASQLRANIS (SEQ ID NO: 8); HKDMQLG (SEQ ID NO: 9); or HKDMQLGR (SEQ ID NO: 10), see e.g. Cook et al. (2010) Acta Cryst D66: 190-197. In some aspects of the disclosure, the antibody may bind to C5a at an epitope within or overlapping the amino acid sequence of a C5a peptide fragment containing at least two paired cysteine residues. For example, an anti-C5a antibody may bind to a fragment containing, or consisting of, the amino acid sequence: CCYDGACVNNDETC (SEQ ID NO: 11); CYDGACVNNDETCEQRAARISLGPRCIKAFTEC (SEQ ID NO: 12; and CEQRAARISLGPRCIKAFTECC (SEQ ID NO: 13), wherein, in each of the peptide fragments, the first and last cysteine residues are paired using a disulfide bond. In some aspects of the disclosure, the anti-C5a antibody described herein may bind to human C5a protein at an epitope within or overlapping the amino acid sequence: YDGACVNNDETCEQRAAR (SEQ ID NO: 14) or CYDGACVNNDETCEQRAA (SEQ ID No: 15). In some aspects of the disclosure, the antibody may bind to human C5a protein or a fragment thereof comprising an amino acid sequence that comprises or consists of at least four (e.g. at least four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, or 17 or more) of the consecutive amino acids depicted on any of SEQ ID NOs: 1 to 15. In some aspects disclosure, the anti-C5a antibody described herein binds to a ternary epitope containing two or more (e.g., at least two, three, or four) discontinuous peptide regions of a C5a protein, e.g. two or more discontinuous C5a peptide regions joined together by a disulfide bond.

[0169] Methods for identifying an epitope to which a particular antibody binds (e.g., anti-C5a antibody) are also known in the art. For example, binding of an epitope within C5a (or lacking arginine C5a) to which an anti-C5a antibody binds can be identified by measuring the binding of the antibody to several (e.g. three, four, five, six, seven, eight, nine, 10, 15, 20, or 30 or more) overlapping peptide fragments of the C5a protein, a component of the complement system (e.g., several overlapping fragments of the human C5a protein with the amino acid sequence depicted in SEQ ID NO: 1 or SEQ ID NO: 2). Each of the different overlapping peptides is then bound to a unique address on a solid support, e.g., separate wells of a multi-well assay plate. Then, the anti-C5a antibody is analyzed by contacting it with each of the peptides in the assay plate, for a period of time and under conditions that allow the antibody to bind to its epitope. Unbound anti-C5a antibody is removed by washing each of the wells. Then, the detectably labeled secondary antibody that binds to the anti-C5a antibody, if present in the plate well, is contacted with each well and unbound secondary antibody is removed in the washing steps. The presence or amount of detectable signal produced by the detectably labeled secondary antibody in the well is an indication that the anti-C5a antibody binds to a particular peptide fragment associated with the well. See. for example. Harlow and Lane (supra), Benny KC Lo (supra), and U.S. Patent Application Publication No. 20060153836.

[0170] The specific epitope to which the antibody binds can also be identified using BIAcore chromatography techniques (see, e.g., Pharmacia BIAtechnology Handbook, "Epitop Mapping," Section 6.3.2 (May 1994); and Johne et al. (1993) J Immunol Methods 160: 20191-8).

[0171] In some aspects of the disclosure, the anti-C5a antibody described herein comprises a specific set of light chain complementarity determining regions (CDRs) and / or a specific set of heavy chain CDRs. For example, in some aspects of the disclosure, the anti-C5a antibody or antigen-binding fragment thereof described herein may comprise a set of light chain CDRs obtained from a light chain polypeptide comprising the amino acid sequence depicted in any of SEQ ID NOs: 19, 37 or 42 . In some embodiments, the anti-C5a antibody or antigen-binding fragment thereof described herein can comprise a heavy chain CDR set obtained from a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 27 or 45. Exemplary light chain CDR sets and heavy chain obtained from the above-mentioned light chain variable regions and heavy chain variable regions are described in more detail below (see Table 1).

[0172] The exact boundaries of the CDRs and framework regions were defined differently according to different methods. In some aspects of the disclosure, the positions of CDRs or framework regions within a light or heavy chain variable domain may be as defined by Kabat et al [(1991) "Sequences of Proteins of Immunological Interest". NIH Publication No. 91-3242, US Department of Health and Human Services, Bethesda, MD]. In such cases, the CDRs may be referred to as "Kabat CDRs" (e.g. "Kabat LCDR2" or "Kabat HCDR1"). In some aspects of the disclosure, the positions of the light or heavy chain variable region CDRs may be as defined by Chothia et al. (1989) Nature 342: 877-883. Accordingly, these regions may be referred to as "Chothia CDRs" (e.g., "Chothia LCDR2" or "Chothia HCDR3"). In some aspects of the disclosure, the CDR positions of the light chain and heavy chain variable regions can be as defined in the Kabat-Chothia combined definition. In such aspects of the disclosure, these regions may be referred to as "Kabat-Chothia-linked CDRs". In some aspects of the disclosure, the positions of CDRs and / or framework regions within a light or heavy chain variable domain may be as defined by Honnegger and Piucktliun (2001) J Mol Biol 309: 657-670. Identification of CDRs within a light chain or heavy chain variable region using the above-mentioned definitions is well known in the art of antibody modification. For example, Thomas et al. [(1996) Mol Immunol 33 (17/18): 1389-1401] discusses the identification of light chain and heavy chain CDR boundaries as defined by Kabat and Chothia.

[0173] Accordingly, in some aspects of the disclosure, the anti-C5a antibody or antigen-binding fragment thereof described herein may comprise Kabat-defined, Chothia-defined, or Kabat / Chothia-jointly defined set of light chain CDRs derived from a chain polypeptide light containing the amino acid sequence depicted in any of SEQ ID NOs: 19, 37 or 42. In some aspects of the disclosure, the anti-C5a antibody or antigen-binding fragment thereof described herein may comprise Kabat-defined, Chothia-defined, or Kabat / Chothia-collectively defined heavy chain CDRs derived from a heavy chain polypeptide having the amino acid sequence depicted in SEQ ID NO: 27 or 45.

[0174] In some embodiments, the anti-C5a antibody described herein comprises a light chain variable region comprising one or more light chain CDR1 comprising or consisting of the following amino acid sequence: RASESVDSYGNSFMH (SEQ ID NO: 20); A light chain CDR2 comprising or consisting of the following amino acid sequence: RASNLES (SEQ ID NO: 21); and a light chain CDR3 comprising or consisting of the following amino acid sequence: QQSNEDPYT (SEQ ID NO: 22). In some embodiments, the antiC5a antibody described herein comprises a light chain variable region comprising each of the light chain CDR1 comprising or consisting of the following amino acid sequence: RASESVDSYGNSFMH (SEQ ID NO: 20); A light chain CDR2 comprising or consisting of the following amino acid sequence: RASNLES (SEQ ID NO: 21); and a light chain CDR3 comprising or consisting of the following amino acid sequence: QQSNEDPYT (SEQ ID NO: 22). Exemplary anti-C5a antibodies containing such a light chain variable domain include, e.g., anti-C5a antibodies BNJ364, BNJ367, BNJ378, BNJ366, BNJ369 and BNJ383 described herein (vide infra; Table 2).

[0175] In some embodiments, the anti-C5a antibody described herein comprises a light chain variable region comprising one or more of: a light chain CDR1 comprising or consisting of the following amino acid sequence: RASESVDSYGNSFMH (SEQ ID NO: 20); A light chain CDR2 comprising or consisting of the following amino acid sequence: WASTRES (SEQ ID NO: 38); and a light chain CDR3 comprising or consisting of the following amino acid sequence:

QQSNEDPYT (SEQ ID NO: 22). In some embodiments, the antiC5a antibody described herein comprises a light chain variable region comprising each of the light chain CDR1 comprising or consisting of the following amino acid sequence: RASESVDSYGNSFMH (SEQ ID NO: 20); A light chain CDR2 comprising or consisting of the following amino acid sequence: WASTRES (SEQ ID NO: 38); and a light chain CDR3 comprising or consisting of the following amino acid sequence: QQSNEDPYT (SEQ ID NO: 22). Exemplary anti-C5a antibodies containing such a light chain variable domain include, e.g., anti-C5a BNJ371 and BNJ381 antibodies described herein (vide infra; Table 2).

[0176] In some embodiments, the anti-C5a antibody described herein comprises a heavy chain variable region comprising one or more of: a heavy chain CDR1 comprising or consisting of the following amino acid sequence: DYSMD (SEQ ID NO: 28); A heavy chain CDR2 comprising or consisting of the following amino acid sequence: AINPNSGGTNYNQKFKD (SEQ ID NO: 29); and a heavy chain CDR3 comprising or consisting of the following amino acid sequence: SGSYDGYYAMDY (SEQ ID NO: 30). In some embodiments, the anti-C5a antibody described herein comprises a heavy chain variable region comprising each of the heavy chain CDR1 comprising or consisting of the following amino acid sequence: DYSMD (SEQ ID NO: 28); A heavy chain CDR2 comprising or consisting of the following amino acid sequence: AINPNSGGTNYNQKFKD (SEQ ID NO: 29); and a heavy chain CDR3 comprising or consisting of the following amino acid sequence: SGSYDGYYAMDY (SEQ ID NO: 30). Exemplary anti-C5a antibodies containing such a heavy chain variable domain include, e.g., anti-C5a BNJ364, BNJ367, BNJ371 and BNJ378 antibodies described herein (vide infra; Table 2).

[0177] In some embodiments, the anti-C5a antibody described herein comprises a heavy chain variable region comprising one or more of: a heavy chain CDR1 comprising or consisting of the following amino acid sequence: DYSMD (SEQ ID NO: 28); A heavy chain CDR2 comprising or consisting of the following amino acid sequence: AIHLNTGYTNYNQKFKG (SEQ ID NO: 46); and a heavy chain CDR3 comprising or consisting of the following amino acid sequence: GFYDGYSPMDY (SEQ ID NO: 47). In some embodiments, the anti-C5a antibody described herein comprises a heavy chain variable region comprising each of the heavy chain CDR1 comprising or consisting of the following amino acid sequence: DYSMD (SEQ ID NO: 28); A heavy chain CDR2 comprising or consisting of the following amino acid sequence: AIHLNTGYTNYNQKFKG (SEQ ID NO: 46); and a heavy chain CDR3 comprising or consisting of the following amino acid sequence: GFYDGYSPMDY (SEQ ID NO: 47). Exemplary anti-C5a antibodies containing such a heavy chain variable domain include, e.g., anti-C5a BNJ366, BNJ369, BNJ381, and BNJ383 antibodies described herein (vide infra; Table 2).

[0178] In some aspects of the disclosure, the anti-C5a antibody or antigen-binding fragment thereof described herein may comprise a heavy chain CDR2 region comprising the amino acid sequence: AINPNSGGTNYSQKFKD (SEQ ID NO: 67). For example, the anti-C5a antibody described herein may comprise a heavy chain variable region containing one or more heavy chain CDR1 comprising or consisting of the following amino acid sequence: DYSMD (SEQ ID NO: 28); A heavy chain CDR2 comprising or consisting of the following amino acid sequence: AINPNSGGTNYSQKFKD (SEQ ID NO: 67); and a heavy chain CDR3 comprising or consisting of the following amino acid sequence: SGSYDGYYAMDY (SEQ ID NO: 30). In some aspects of the disclosure, the anti-C5a antibody described herein comprises a heavy chain variable region comprising each of the heavy chain CDR1 comprising or consisting of the following amino acid sequence: DYSMD (SEQ ID NO: 28); A heavy chain CDR2 comprising or consisting of the following amino acid sequence: AINPNSGGTNYSQKFKD (SEQ ID NO: 67); and a heavy chain CDR3 comprising or consisting of the following amino acid sequence: SGSYDGYYAMDY (SEQ ID NO: 30). An example of an anti-C5a antibody described herein that contains such a heavy chain polypeptide and binds to human C5a with a KD that is less than 1 nM is the 5an101ME antibody described below.

[0179] In some embodiments, the anti-C5a antibody or antigen-binding fragment thereof described herein comprises one of exemplary light chain CDR sets and 1 to 4 paired heavy chain CDR sets shown in Table 1.

_Table 1. Examples of heavy and light chain CDR pairs_

<td rowspan="2">Examples of CDR pairs</td><td colspan="3">Light chain</td><td colspan="3">Heavy chain</td><td rowspan="2">Exemplary antibodies with such pairs</td>

<td colspan="8">"SIN" means "SEQ ID NO". The amino acid sequences represented by SEQ ID NO in Table 1 are given in Table 2.</td>

[0180] In some embodiments, the anti-C5a antibody does not include an exemplary pair of CDRs 3. In some embodiments, the anti-C5a antibody is not BNJ371.

[0181] In some embodiments, the anti-C5a antibody light chain polypeptide described herein may be a λ light chain polypeptide (e.g., fully human or humanized λ light chain polypeptide). In some embodiments, the anti-C5a antibody light chain polypeptide described herein is a κ light chain polypeptide (e.g., fully human or humanized κ light chain polypeptide). The amino acid sequences of numerous light chain polypeptides (e.g., numerous human light chain polypeptides) are well known in the art and are provided in, e.g., Kabat et al. (1991), supra. Exemplary κ amino acid light chain polypeptide sequences are given in Table 2.

[0182] In some embodiments, the anti-C5a antibodies described herein may contain a light chain constant region. For example, the light chain constant region may be a light chain constant region polypeptide or κ light chain constant region polypeptide. The amino acid sequence of many human light chain constant regions λ, and κ is known in the art and described in, e.g., Kabat et al. (1991), supra. Exemplary κ light chain polypeptide amino acid sequences are provided in the Table

[0183] The heavy chain polypeptide may comprise a constant region (e.g., heavy chain constant region 1 (CH1), heavy chain constant region 2 (CH2), heavy chain constant region 3 (CH3), heavy chain constant region 4 (CH4), or a combination of any of the above). The heavy chain polypeptide may comprise an Fc portion of an immunoglobulin molecule. The Fc region can be, e.g. an Fc region from an IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD immunoglobulin molecule or a combination of parts of each. More specifically, the anti-C5a antibodies described herein may be an isotype, e.g., IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD. The amino acid sequences of many human heavy chain constant regions are known in the art and described in, e.g., Kabat et al. (1991), supra.

[0184] In some embodiments, the heavy chain polypeptide may comprise a hybrid constant region, or a portion thereof, such as the G2 / G4 hybrid constant region (see, e.g., Burton et al. (1992) Adv Immun 51: 1-18; Canfield et al. (1991) JExp Med 173: 1483-1491; I Mueller et al. (1997) Mol Immunol 34 (6): 441-452). For example (and according to Kabat numbering), the IgG1 and IgG4 constant regions contain residues G249G250, while the IgG2 constant region does not contain residue 249 but contains G250. In the G2 / G4 hybrid constant region, where the 249-250 region is derived from the G2 sequence, the constant region may be further modified to introduce a glycine residue at position 249 to form a G2 / G4 fusion having G249 / G250. Other constant domain hybrids that contain G249 / G250 may also form part of the modified antibodies as disclosed. Exemplary amino acid sequences for the heavy chain polypeptide are given in Table 2.

[0185] The anti-C5a antibody may be, e.g., one of the specific antibodies specified in the working examples: BNJ364, BNJ367, BNJ378, BNJ366, BNJ369, BNJ383, BNJ371 or BNJ381. The amino acid sequences for these specified anti-C5a antibodies that can be used in conjunction with any of the methods described herein are given in Table 2.

<td>h humanized antv-C5a antibodies</td><td>Amino acid sequence</td><td>MVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERAT] NCRASESVDSYGNSFMHWYQQKPGQPP KLLIYRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGGGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC</td><td>DIVMTQSPDSLAVSLGERATINGRASESVDSYGNSFMHWYQQKPGQPPKLLrYRASNLESGVPDRFSGS GSGTDFTLnSSLQAEDVAVYYCQQSNEDPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPYTKSFNRGEC '</td><td>MVLQTQVFISLLLWISGAYG</td><td>What ABOUT WHAT and g > about What ω at FROM· What at and CU ABOUT ABOUT and θ '<4 C / M > ω · b 1§ Sh every 5 * him 2 a> ś Irt S o iS ' Zi What & QP S5id C / Fe HQ 2H > O C. What OO</td><td>RASESVDSYGNSFMH</td><td>RASNLES</td><td>QQSNEDPYT</td>

<td rowspan="3">Table 2. Amino acid sequences selected]</td><td>5Λ '5 about</td><td>Full light chain sequence with peptide signal</td><td>Full light chain sequence without peptide signal</td><td>Light chain variable region signal peptide sequence</td><td>Light chain variable region sequence</td><td>Kabat light chain variable region sequence LCDR1</td><td>Kabat light chain variable region sequence LCDR2</td><td>Kabat LCDR3 light chain variable region sequence</td>

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HCDR2

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<td>Kabat heavy chain variable region sequence HCDR3</td><td>Heavy chain constant region sequence</td><td>Full light chain sequence with peptide signal</td><td>Full light chain sequence without peptide signal</td><td>Variable region signal peptide sequence light chain</td><td>Light chain variable region sequence</td><td>Kabat light chain variable region sequence LCDR1</td><td>Kabat light chain variable region sequence LCDR2</td>

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<td>signal</td><td>Variable region signal peptide sequence light chain</td><td>Light chain variable region sequence</td><td>Kabat light chain variable region sequence LCDR1</td><td>Kabat light chain variable region sequence LCDR2</td><td>Kabat light chain variable region sequence LCDR3</td><td>Light chain constant region sequence</td><td>Full heavy chain sequence with peptide signal</td>

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<td>MDWTWRVFCLLAVAPGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSR4DWVRQAPGQGLE WMGAINPNSGGTNYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGSYDGYYAMDYW GQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSWTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKD TLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLN GKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK</td><td>F * ¢ 0 Ρ 5 £ g & S o Z ił · & from 1/5 C ij h h e. O> O 2> ϋ § <Ł ZC Gi> £ A 5 g K fc 5> in Η ϋ z 3 Ł J Bi sd g OP 52 £ 2 £ E • 5 O £ 5 W Cl, 2 orf 3 3 sts > about SH oo 3 £ WA 3 S go> S 5 2 3 § 5 may ggsgggg £> mg; £ w rf ϊλ g U ω W θ 'g m H H Φ z <SUŁŁo o A 's? «6 in 3 MSSEP Ot Q EOF > 1 S> OO ii b oo 2> So Ο> υϋω <ξ> > From Cu H cl, CJ · _) σα «3> q% £</td><td>MDWTWRVFCLLAVAPGAHS</td><td>QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLEWMGAINPNSGGTNYNQKFK DRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGSYDGYYAMDYWGQGTTVfVSS</td><td>DYSMD</td><td>Q σ from H ABOUT about HIM FROM plh FROM M <</td><td>SGSYDGYYAMDY</td>

<td></td><td>Full heavy chain sequence without peptide signal</td><td>Variable region signal peptide sequence heavy chain</td><td>Heavy chain variable region sequence</td><td>Kabat heavy chain variable region sequence HCDR1</td><td>Kabat heavy chain variable region sequence HCDR2</td><td>Kabat heavy chain variable region sequence HCDR3</td>

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<td>Heavy chain constant region sequence</td><td>Full light chain sequence with peptide signal</td><td>Full light chain sequence without peptide signal</td><td>Variable region signal peptide sequence light chain</td><td>Light chain variable region sequence</td><td>Kabat light chain variable region sequence LCDR1</td><td>Kabat light chain variable region sequence LCDR2</td><td>Kabat light chain variable region sequence LCDR3</td><td>Light chain constant region sequence</td>

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<td></td><td>Full heavy chain sequence with peptide signal</td><td>Full heavy chain sequence without peptide signal</td><td>Variable region signal peptide sequence heavy chain</td><td>Heavy chain variable region sequence</td><td>Kabat heavy chain variable region sequence HCDR1</td><td>Kabat heavy chain variable region sequence HCDR2</td>

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<td>LCDR1</td><td>Kabat light chain variable region sequence LCDR2</td><td>Kabat light chain variable region sequence LCDR3</td><td>Light chain constant region sequence</td><td>Full heavy chain sequence with peptide signal</td><td>Full heavy chain sequence without peptide signal</td><td>Variable region signal peptide sequence heavy chain</td><td>Heavy chain variable region sequence</td>

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working examples.

[0186] In some aspects of the disclosure, the anti-C5a antibody described herein comprises a Chothia-defined light chain CDR set or a combined Kabat / Chothia-defined light chain CDR set obtained from any of the light chain variable regions described in Tables 2 or 3. In some aspects of the disclosure, the anti-C5a antibody, or antigen-binding fragment thereof described herein comprises a Chothia-defined heavy chain CDR set or a Kabat / Chothia-linked heavy chain CDR set obtained from any of the heavy chain variable regions described in Tables 2 or

[0187] In preferred aspects of the disclosure, the described anti-C5a antibody binds to C5a, but not to native full length C5. In some aspects of the disclosure, the anti-C5a antibody binds to C5a, but does not bind to the alpha chain of un cleaved native C5a. As used herein, "un cleaved C5" refers to a C5 protein that has not been cleaved into C5a and C5b fragments by AP or CP C5 convertase. An exemplary amino acid sequence for the human C5 alpha chain is given in Haviland et al. (1991), supra.

[0188] In some aspects of the disclosure, the anti-C5a antibody described herein does not bind to human C5 paralogs, such as human C3a or human C4a.

[0189] The disclosure also provides antibodies that cross-block the binding of an anti-C5a antibody described herein (eg, cross-block any of BNJ364, BNJ367, BNJ378, BNJ366, BNJ369, BNJ371, BNJ381 or BNJ383). As used herein, the term "cross-blocking antibody" refers to an antibody that reduces the amount of binding (or prevents binding) of an anti-C5a antibody to an epitope on a complement component of a C5a protein, relative to the amount of binding of an anti-C5a antibody to an epitope in the absence of a cross-blocking antibody . Suitable methods for determining whether the first antibody cross-blocks the binding of the second antibody to the epitope are known in the art. For example, cross-blocking antibodies can be identified by comparing the monoclonal binding of anti-C5a BNJ364 antibody in the presence and absence of the test antibody. The reduced binding of the BNJ364 antibody in the presence of the test antibody compared to the binding of the BNJ364 antibody in the absence of the test antibody indicates that the test antibody is a cross-blocking antibody.

[0190] In some aspects of the disclosure, antibody binding to C5a may inhibit C5a biological activity. Methods for measuring C5a activity include, e.g., chemotaxis assays, radioimmunoassays (RIAs), or immunoassay enzyme (ELISA) assays (see, e.g., Ward and Zvaifler (1971) J Clin Invest 50 (3): 606-16 and Wurzner et al (1991) Complement Inflamm 8: 328-340). In some aspects of the disclosure, the binding of an antibody or antigen binding fragment thereof to C5a may inhibit the activation of neutrophils in vitro. Suitable methods for determining whether an anti-C5a antibody inhibits C5a-mediated activation of neutrophils in vitro, or the extent to which the antibody inhibits activation, are known in the art and detailed in the working examples below. For example, human neutrophils obtained from healthy donors can be isolated and contacted with isolated human C5a in the presence or absence of the tested anti-C5a antibody. C5a-dependent activation of human neutrophils can be measured as a function of myeloperoxidase (MPO) release from cells in the presence of C5a. Inhibition of the amount of MP released from cells in the presence of C5a and test antibody, compared to the amount of MPO released from cells in the presence of C5a and control antibody, indicates that the test antibody inhibits C5a-mediated neutrophil activation.

[0191] In some aspects of the disclosure, the anti-C5a antibody or antigen-binding fragment thereof does not inhibit (or significantly inhibit) the activity of the complement component, C5, compared to the level of inhibition (if present) observed for the corresponding control antibody or its antigen binding fragment (i.e., an antibody that does not bind to free C5a or CS). C5 activity can be measured as a function of its ability to lyse cells in a subject's body fluids. Cell lysis capacity, or reduction thereof, for C5 can be measured by methods well known in the art such as, for example, a conventional hemolytic assay, such as the hemolytic assay described by Kabat and Mayer (eds), "Experimental Immunochemistry, 2nd Edition ., "135-240, Springfield, IL, CC Thomas (1961), pages 135-139, or a conventional variation of this assay, such as a method for hemolysis of chicken erythrocytes as described in, e.g., Hillmen et al. (2004) NEngl J Med 350 (6): 552.

[0192] In some aspects of the disclosure, C5 activity, or its inhibition, is quantified using the CH50eq assay. The CH50eq assay is a way to measure the total activity of classical complement in serum. This test is a lytic assay that uses antibody sensitized erythrocytes as an activator of the classical complement pathway, and various dilutions of test serum to determine the amount required to obtain 50% lysis (CH50). The percentage of hemolysis can be determined, for example, using a spectrophotometer. The CH50eq assay provides an indirect measure of terminal complement formation (TCC), as TCC itself is directly responsible for the hemolysis that is measured.

[0193] The assay is well known and widely practiced by one of ordinary skill in the art. Briefly, to activate the classical complement pathway, undiluted serum samples (e.g., human serum samples) are added to microtiter wells containing antibody sensitized erythrocytes to thereby produce TCC. Then, activated sera are diluted in microtiter wells that are coated with a capture reagent (e.g. an antibody that binds to one or more TCC components). The TCC present in activated samples bind to monoclonal antibodies covering the surface of the microtiter wells: The wells are washed and a detection reagent is added to each well that is detectably labeled and recognizes bound TCC. A detectable tag can be, e.g., a fluorescent tag or an enzyme tag. The results of the determination are expressed in equivalents of CH50 units per milliliter (CH50 U Eq / ml).

[0194] Additional methods for detecting and / or measuring C5 activity in vitro are given and detailed in the working examples.

[0195] In some aspects of the disclosure, antibody binding to C5a may inhibit the interaction between C5a and C5aR1. Suitable methods for detecting and / or measuring the interaction between C5a and C5aR1 (in the presence and absence of an antibody) are known in the art and described in, e.g. Mary and Boulay (1993) Eur J Haematol 51 (5): 282-287; Kaneko et al. (1995) Immunology 86 (1): 149-154; Giannini et al. (1995) J Biol Chem 270 (32): 19166-19172; and U.S. Patent Application Publication No. 20060160726. For example, the binding of detectably labeled (e.g., radioactively labeled) C5a to C5aR1 expressing mononuclear peripheral blood cells can be evaluated in the presence and absence of antibody. The decrease in the amount of detectably labeled C5a that binds to C5aR1 in the presence of the antibody, compared to the amount of binding in the absence of the antibody, is an indication that the antibody inhibits the interaction between C5a and C5aR1.

[0196] In some aspects of the disclosure, antibody binding to C5a may inhibit the interaction between C5a and C5L2. Methods for detecting and / or measuring the interaction between C5a and C5L2 are known in the art and described in, e.g., Ward (2009) J Mol Med 87 (4): 375-378 and Chen et al. (2007) Nature 446 (7132): 203-207. Additional methods for assessing the biological effect of the anti-C5a antibody described herein are detailed in the working examples below.

[0197] In some aspects of the disclosure of anti-C5a antibodies, it specifically binds to the human complement component, the C5a protein (e.g., the human C5a protein having the amino acid sequence depicted in SEQ ID NO: 1 or SEQ ID NO: 2). The terms 'specific binding' and 'specific binding' and similar grammatical concepts, as used herein, refer to two molecules forming a complex (e.g. complex between the antibody and the complement component - the C5a protein) which are relatively stable under physiological conditions. Typically, the binding is considered specific when the association constant (ka) is higher than 106 M1s-1. Hence, the antibody can specifically bind to a C5a protein of at least (or more than) 106 (e.g., at least or more than 107, 108, 109, 1010, 1011, 1012, 1013, 1014, or 1015 or more) M-1s-1. In some embodiments, the anti-C5a antibody described herein has a dissociation constant (kd) of less than or equal to 10-3 (e.g., 8 x 10-4, 5 x 10-4, 2 x 10-4, 10-4; or 10-5) p-1.

[0198] In some aspects of the disclosure, the anti-C5a antibody described herein has KD less than 10-8, 10-9, 10-10, 10-11, or 10-12 M. Equilibrium constant KD is the ratio of kinetic constants - kd / ka. In some aspects of the disclosure, the anti-C5a antibody described herein has a KD of less than 1.25 x 10-9 M. Examples of anti-C5a antibodies that bind to C5a with a KD that is less than 1.25 x 10-9 M include, e.g., anti-C5a BNJ364, BNJ367, BNJ371, BNJ378, BNJ366, BNJ369, BNJ381 and BNJ383 antibodies.

[0199] In some aspects of the disclosure, the anti-C5a antibody described herein has a KD of less than 1x10-9 M. Examples of anti-C5a antibodies that bind to C5a with a KD that is less than 10-9 M include, e.g., anti-C5a antibodies BNJ364, BNJ367, BNJ378, BNJ366, BNJ369, BNJ381 and BNJ383.

[0200] In some aspects of the disclosure, the anti-C5a antibody described herein has a KD of less than 5 x 10-10 M. Examples of anti-C5a antibodies that bind to C5a with a KD that is less than 5 x 10-10 M include, e.g., anti-C5a antibodies BNJ367, BNJ378, BNJ366, BNJ369, BNJ381 and BNJ383.

[0201] In some aspects of the disclosure, the anti-C5a antibody described herein has a KD of less than 2 x 10-10 M. Examples of anti-C5a antibodies that bind to C5a with a KD that is less than 2 x 10-10 M include, e.g., anti-C5a antibodies BNJ367, BNJ366, BNJ369, BNJ381 and BNJ383.

[0202] In some aspects of the disclosure, the anti-C5a antibody described herein has a KD of less than 1x10-10 M. Examples of anti-C5a antibodies that bind to C5a with a KD that is less than 1x10-10 M include, e.g., anti-C5a antibodies BNJ369, BNJ381 and BNJ383.

[0203] In some aspects of the disclosure, the anti-C5a antibody described herein has a KD of less than 7.5 x 10-11 M. Examples of anti-C5a antibodies that bind to C5a with a KD that is less than 7.5 x 10-11 M include, e.g., anti-C5a BNJ369 and BNJ383 antibodies.

[0204] Methods for determining the affinity of an antibody for a protein antigen are known in the art. For example, the affinity of an antibody for a protein antigen can be quantified using a number of techniques such as, but not limited to, Western hybridization, point hybridization, biological layer interferometry, surface plasmon resonance (SPR) methods (e.g. BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ), or immuno specific enzyme assays (ELISA). See. e.g. Harlow and Lane (1988) "Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Benny KC Lo (2004) "Antibody Engineering: Methds and Protocols," Humana Press (ISBN: 1588290921); Borrebaek (1992) "Antibody Engineering, A Practical Guide," WH: Wolneman, i Co., NY; Borrebaek (1995) "Antibody Engineering," 2nd Edition, Oxford University Press, NY, Oxford; Johne et al. (1993) Immunol Meth 160: 191-198; Jonsson et al. (1993) Ann Biol Clin 51: 19-26; and Jonsson et al. (1991) Biotechniques 11: 620-627.

[0205] Any of the light chain CDR sets or light chain variable regions described herein may be paired with any of the heavy chain CDR sets or heavy chain variable regions described herein. It is within the knowledge of the skilled artisan to, e.g., confirm (test) that an anti-C5a antibody produced by such pairing had the desired affinity or activity. Suitable methods for confirming the activity and / or affinity of the anti-C5a antibody are described herein.

[0206] In some aspects of the disclosure, the anti-C5a antibodies described herein bind to both human C5a (hC5a) and C5a from a non-human mammal, such as a non-human primate (e.g., cynomolgus macaque). In some aspects of the disclosure, the anti-C5a antibody or antigen-binding fragment thereof described herein does not bind to human C5a paralogs, such as C3a or C4a, from the same non-human mammal species.

[0207] In some aspects of the disclosure, the anti-C5a antibody or antigen-binding fragment thereof described herein binds to free hC5a and Cynomolgus C5a protein comprising, or consisting of, the following amino acid sequence:

MLQEKIEEJAAKYKHLVVKK CCYDGVRINH DETCEQRAAR ISVGPRCVKA FTECCVVASQLRANNSHKDLQLGR (SEQ ID NO: 179). In some aspects of the disclosure, the anti-C5a antibody or antigen-binding fragment thereof described herein binds to free hC5a and rhesus macaque C5a protein comprising, or consisting of the amino acid sequence depicted in SEQ ID NO: 179.

[0208] In some aspects of the disclosure, the antibody, or antigen-binding fragment thereof, can bind to the non-terminal arginine form of the C5a protein from a non-human mammalian species (e.g., a non-human primate). For example, the antibody or antigen-binding fragment thereof may bind to free Arginine-depleted C5a protein from cynomolgus or rhesus macaque, a protein containing, or consisting of the following amino acid sequence: MLQEKIEEIAAKYKHLVVKK CCYDGVRINH DETCEQRAAR ISVGPRCVKA FTECLQVASQ.

[0209] In some aspects of the disclosure, the anti-C5a antibodies described herein bind to mouse C5a (i.e., free C5a from mice). In some aspects of the disclosure, the anti-C5a antibodies described herein bind to mouse C5a, but not to human C5a. In some aspects of the disclosure, the anti-C5a antibody described herein does not bind to un-cleaved, native (fully-folded) mouse C5. In some aspects of the disclosure, the anti-C5a antibody described does not bind to mouse C5a paralogs such as mouse C3a or mouse C4a.

[0210] Anti-mouse C5a antibody, or antigen-binding fragment thereof, can bind to mouse C5a protein comprising, or consisting of, the following amino acid sequence:

LRQKIEEQAAKYKHSVPKKCCYDGARVNFYETCEERVARVTIGPLCIRAFNECCT IANKIRKESPHKPVQLGR (SEQ ID NO: 51). See also, e.g., Wetsel et al. (1987) Biochem 26: 737-743. In some aspects of the disclosure, the anti-mouse C5a antibody, or antigen-binding fragment thereof, can bind to the arginine-free form of the murine C5a protein comprising, or consisting of, the following amino acid sequence:

LRQKIEEQAAKYKHSVPKKCCYDGARVNFYETCEERVARVTIGPLCIRAFNECCT IANKIRKESPHKPVQLG (SEQ ID NO: 52). In some aspects of the disclosure, the anti-mouse C5a antibody binds to both the full-length mouse C5a protein and the arginine-free form of the mouse C5a protein.

[0211] The anti-mouse C5a antibody described herein may, e.g., comprise a set of light chain CDRs obtained from a light chain variable region polypeptide having the following amino acid sequence:

EIVLTQSPAIMSASLGEKVTMSCRASSSVNYTYWYQQKSDASPKLWIYYTSNLAP GVPARFSGSGSGNSYSLTISSMEGEDAATYYCQQFTSSPLTFGVGTKLELKR (SEQ ID

NO: 53). For example, the anti-mouse C5a antibody may comprise: (i) Kabat-defined light chain CDR1 comprising, or consisting of, the following amino acid sequence: RASSSVNYIY (SEQ ID NO: 54); (ii) Kabat-defined light chain CDR2 comprising or consisting of the following amino acid sequence: YTSNLAP (SEQ ID NO: 55); and / or (iii) Kabat-defined light chain CDR3 comprising or consisting of the following amino acid sequence: QQFTSSPLT (SEQ ID NO: 56).

[0212] The anti-mouse C5a antibody may comprise a light chain constant region, e.g., a mouse light chain kappa constant region comprising, or consisting of, the following amino acid sequence:

ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSW TDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNKNEC (SEQ ID NO: 57).

[0213] In some aspects of the disclosure, the anti-mouse C5a antibody described herein may comprise an amino-terminal signal peptide, e.g., a signal peptide comprising, or consisting of, the following amino acid sequence:

MGWSCIILFLVATATGVHS (SEQ ID NO: 58).

[0214] In some aspects of the disclosure, the anti-mouse C5a antibody described herein may comprise a light chain polypeptide comprising, or consisting of the following amino acid sequence:

REIVLTQSPAIMSASLGEKVTMSCRASSSVNYIYWYQQKSDASPKLWIYYTSNLA PGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQQFTSSPLTFGVGTKLELKRAD AAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTD QDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 59) or

MGWSCIILFLVATATGVHSREIVLTQSPAIMSASLGEKVTMSCRASSSVNYIYWY QQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQQF TSSPLTFGVGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVK WKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTS TSPIVKSFNRNEC (SEQ ID NO: 60). In some aspects of the disclosure, the anti-mouse C5a antibody described herein comprises a light chain polypeptide having the amino acids 2 to 214 in SEQ ID NO: 59. In some aspects of the disclosure, the anti-mouse C5a antibody described herein comprises a light chain polypeptide having the amino acids 1 to 19 and 21 to 233 in SEQ ID NO: 60.

[0215] The anti-mouse C5a antibody described herein may, e.g., comprise a heavy chain CDR set obtained from a heavy chain variable region polypeptide having the following amino acid sequence:

LEVQLQQSGPELVKPGASVKISCKASGYTFTDYYYINWVKQSHGKSLEWIGYIYP NDGDTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARPYYSDYGM DYWGQGTSVTVSS 61) SEQ ID NO. For example, the anti-mouse C5a antibody may comprise: (i) Kabat-defined heavy chain CDR1 comprising, or consisting of, the following amino acid sequence: DYYYIN (SEQ ID NO: 62); (ii) Kabat-defined heavy chain CDR2 comprising, or consisting of, the following amino acid sequence: YIYPNDGDTNYNQKFKG (SEQ ID NO: 63); and / or (iii) a heavy chain CDR3 defined by Kabat comprising or consisting of the following amino acid sequence: PYYSDYGMDY (SEQ ID NO: 64).

[0216] Anti-mouse C5a antibody may contain a heavy chain constant region. In some aspects of the disclosure, the anti-mouse C5a antibody described herein may comprise an amino terminal signal peptide, e.g., a signal peptide containing, or consisting of, the following amino acid sequence: MGWSCIILFLVATATGVHS (SEQ

ID NO: 65).

[0217] In some aspects of the disclosure, the anti-mouse C5a antibody described herein may comprise a heavy chain polypeptide comprising, or consisting of, the following amino acid sequence:

LEVQLQQSGPELVKPGASVKISCKASGYTFTDYYYINWVKQSHGKSLEWIGYIYP NDGDTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARPYYSDYGM DYWGQGTSVTVSS (SEQ ID NO.

MGWSCIILFLVATATGVHSLEVQLQQSGPELVKPGASVKISCKASGYTFTDYYYI NWVKQSHGKSLEWIGYIYPNDGDTNYNQKFKGKATLTVDKSSSTAYMELRSLT SEDSAVYYCARPYYSQYGM. NO. In some aspects of the disclosure, the anti-mouse C5a antibody described herein comprises a heavy chain polypeptide having the amino acids 2 to 121 of SEQ ID NO: 66. In some aspects of the disclosure, the anti-mouse C5a antibody described herein comprises a heavy chain polypeptide having the amino acids 1 to 19 and 21 to 140 of SEQ ID NO: 67. In some aspects of the disclosure, the anti-mouse C5a antibody described herein comprises a heavy chain constant region polypeptide having one or more amino acid substitutions from the sequence described above.

[0218] In some aspects of the disclosure, the anti-mouse C5a antibody described herein comprises a light chain polypeptide comprising: (i) a light chain CDR1 comprising, or consisting of, the amino acid sequence depicted in SEQ ID NO: 54; (ii) a light chain CDR2 comprising, or consisting of, the amino acid sequence depicted in SEQ ID NO: 55; and (iii) a light chain CDR3 comprising, or consisting of, the amino acid sequence depicted in SEQ ID NO: 56; (iv) a heavy chain CDR1 comprising, or consisting of, the amino acid sequence depicted in SEQ ID NO: 62; (v) a heavy chain CDR2 comprising, or consisting of, the amino acid sequence depicted in SEQ ID

NO: 63; and / or (vi) a heavy chain CDR3 comprising, or consisting of, the amino acid sequence depicted in SEQ ID NO: 64.

[0219] In some aspects of the disclosure, the anti-mouse C5a antibody described herein comprises a light chain polypeptide comprising, or consisting of, the amino acid sequence depicted in SEQ ID NO: 59 and a heavy chain polypeptide comprising, or consisting of, the amino acid sequence depicted in SEQ ID

NO: 66.

[0220] In some aspects of the disclosure, the anti-C5a antibody described herein can bind to human C5a and to murine C5a.

Methods for making anti-C5a antibodies and antigen-binding fragments thereof [0221] The disclosure also provides methods for producing any anti-C5a antibodies or antigen-binding fragments thereof described herein. In some aspects of the disclosure, methods of producing antibodies described herein may include immunizing an individual (e.g., a non-human mammal) with a suitable immunogen. Suitable immunogens for generating any of the antibodies described herein are provided herein. For example, to generate an antibody that binds to C5a, one of skill in the art may immunize for a suitable individual (e.g. a non-human mammal such as a rat, mouse, gerbil, hamster, dog, cat, pig, goat, horse or non-human primate) full-length C5a polypeptide such as full-length C5a polypeptide containing the amino acid sequence depicted in SEQ ID NO: 1 or an arginine-depleted form of C5a (e.g., arg-depleted human C5a containing the amino acid sequence depicted in SEQ ID NO: 2). In some aspects of the disclosure, the non-human mammal has a C5 deficiency, e.g. C5 deficient mouse, described in, e.g., Levy and Ladda (1971) Nat New Biol 229 (2): 51-52; Crocker et al. (1974) J Clin Pathol 27 (2): 122-124; Wetsel et al. (1990) J Biol Chem 265: 2435-2440; and Jungi and Pepys (1981) Immunology 43 (2): 271-279. Human C5a can be purified from human serum as described in, e.g., McCarthy and Henson (1979) J Immunol 123 (6): 2511-2517 and Manderino et al. (1982) J Immunol Methods 53 (1): 41-50. See. also working examples. Human C5a can also be produced in vitro as described in, e.g., Vallota and Miiller-Hberhard (1973) J Exp Med 137: 1109. Purified human C5a is also commercially available from, e.g., Complement Technology, Inc. (catalog number A144; Tyler, Texas). Recombinant C5a can also be generated by one of skill in the art as described in, e.g., Toet al. (1994) Prot Sci 3: 1159-1168.

[0222] A suitable individual (e.g., a non-human mammal) may be immunized with the appropriate antigen along with subsequent booster immunizations, a sufficient number of times to induce the production of antibody by the mammal. The immunogen may be administered to an individual (e.g., a non-human mammal) with an adjuvant. Adjuvants useful in the production of antibodies in a subject include, but are not limited to, an adjuvant protein; bacterial adjuvants, e.g. complete bacteria (BCG, Corynebacterium parvum or Salmonella minnesota) and bacterial components including cell wall skeleton, trehalose dimicolinate, monophosphoryl lipid A, methanol extracted residue (MER) tuberculosis tuberculosis, complete or incomplete Freund's adjuvant; viral adjuvants; chemical adjuvants, e.g., aluminum hydroxide, and iodine acetate and cholesteryl hemisuccinate. Other adjuvants that can be used in methods of inducing an immune response include, e.g. cholera toxin and parapoxvirus proteins. See. also Bieg et al. (1999) Autoimmunity 31 (1): 15-24. See. also, e.g. Lodmell et al. (2000) Vaccine 18: 1059-1066; Johnson et al. (1999) J Med Chem 42: 4640-4649; Baldridge et al. (1999) Methods 19: 103-107; and Gupta et al (1995) Vaccine 13 (14): 1263-1276.

[0223] In some of the aspects of the disclosure, the methods include obtaining a hybridoma cell line that secretes a monoclonal antibody that binds to the immunogen. For example, a suitable mammal such as a laboratory mouse is immunized with a C5a polypeptide as described above. Antibody-producing cells (e.g. spleen B cells) of the immunized mammal can be isolated two to four days after at least one immunogen boost, and then cultured briefly, before fusing with the cells of the corresponding myeloma cell line. Cells may be fused in the presence of a fusion promoter such as e.g. vaccinia virus or polyethylene glycol. Fused hybrid cells are cloned, and cell clones secreting the desired antibodies are selected. For example, the spleen cells of Balb / c mice immunized with the appropriate immunogen may be fused with myeloma cell line PAI or myeloma cell line Sp2 / 0-Ag 14. After the fusion, the cells are propagated in the appropriate culture medium, which is supplemented with selective medium. for example, HAT medium at regular intervals to prevent normal myeloma cells from overgrowing the desired hybridoma cells. The resulting hybrid cells are then screened for the secretion of desired antibodies, e.g., an antibody that binds to C5a and inhibits the interaction between C5a and the C5a receptor (e.g. C5aR1).

[0224] In some aspects of the disclosure, one skilled in the art can identify anti-C5a antibody from a library without non-immunological bias as described in, e.g., US Patent No. 6,300,064 (for Knappik et al; Morphosys AG) and Schoonbroodt et al (2005) Nucleic Acids Res 33 (9): e81.

[0225] In some aspects of the disclosure, the methods described herein may relate to, or be used in conjunction with, e.g. phage display technologies, bacterial display, yeast surface display, eukaryotic virus display, mammalian cell display, and cell free ( e.g., ribosome display) techniques for antibody screening (see, e.g., Etz et al. (2001) J Bacteriol 183: 6924-6935; Comelis (2000) Curr Opin Biotechnol 11: 450-454; Klemm et al (2000) Microbiology 146: 3025-3032; Kieke et al (1997) Protein Eng 10: 1303-1310; Yeung et al. (2002) Biotechnol Prog 18: 212-220; Boder et al. (2000) Methods Enzymology 328: 430-444; Grabherr et al. (2001) Comb Chem High Throughput Screen 4: 185-192; Michael et al. (1995) Gene Ther 2: 660-668; Pereboev et al. (2001) J Virol 75: 7107-7113; Schaffitzel et al. (1999) J Immunol Methods 231: 119-135; and Hanes et al. (2000) Nat Biotechnol 18: 1287-1292).

[0226] Methods for identifying antibodies using various phage display methods are known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. Such phages can be used to display antigen-binding domains of antibodies, such as Fab, Fv, or disulfide-stabilized Fv fragments of antibodies expressed from the repertoire or combinatorial library of antibodies (e.g., human or mouse). The phage used in these methods are typically filamentous phages such as fd and M13. Antigen binding domains are expressed as a recombinantly coupled protein with any of the phage coat proteins, pIII, pVIII, or pIX. See. e.g. Shi et al. (2010) JMB 397: 385-396. Examples of phage display methods that can be used to produce immunoglobulins, or fragments thereof, described herein include those disclosed in Brinkman et al. (1995) J Immunol Methods 182: 41-50; Ames et al. (1995) J Immunol Methods 184: 177-186; Kettleborough et al. (1994) Eur J Immunol 24: 952-958; Persic et al. (1997) Gene 187: 9-18; Burton et al. (1994) Advances in Immunology 57: 191-280; and PCT Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, and WO 95/20401. Suitable methods are also described, e.g., in US Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

[0227] In some aspects of the disclosure, antibody phage display libraries can be produced using mRNA harvested from B cells from immunized mammals. For example, a spleen cell sample containing B cells may be isolated from mice immunized with the C5a polypeptide as described above. mRNA can be isolated from cells and converted to cDNA using standard molecular biology techniques. See. e.g., Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual, 2nd Edition," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane (1988), supra; Benny KC Lo (2004), supra; and Borrebaek (1995), supra. CDNA encoding variable regions of the heavy chain and immunoglobulin light chain polypeptides are used to construct a phage display library. Methods for making such a library are described in, e.g., Merz et al. (1995) J Neurosci Methods 62 (1-2): 213-9; Di Niro et al (2005) Biochem J 388 (Pt 3): 889-894; and Engberget al. (1995) Methods Mol Biol 51: 355-376.

[0228] In some aspects of the disclosure, a combination of selection and screening can be used to identify the subject antibody from, e.g., hybridoma antibody populations or the antibody phage display library. Suitable methods are known in the art and are described in, e.g., Hoogenboom (1997)

Trends in Biotechnology 15: 62-70; Brinkman et al. (1995), supra; Ames et al (1995), supra; Kettleborough et al. (1994), supra; Persic et al. (1997), supra; and Burton et al. (1994), supra.

For example, many phagemid vectors are generated, each encoding a bacteriophage coat protein fusion protein (e.g., pIII, pVIII, or pIX M13) and various regions of the combined antigens using standard molecular biology techniques and then introduced into bacterial populations (e.g., E. coli ). Bacteriophage expression in bacteria may, in some embodiments, require helper phage. In some embodiments, no helper phage is required (see e.g. Chasteen et al. (2006) Nucleic Acids Res 34 (21): e145). The phages produced in bacteria are recovered and then contacted with, e.g., a solid (immobilized) target antigen. Phage can also be contacted with the antigen in solution, and the complex is subsequently bound to a solid support.

[0229] In some aspects of the disclosure, immobilized phage is the subject phage. Accordingly, unbound phages are removed by washing the medium. After the washing step, the bound phage is then eluted from the solid support, e.g. using a low pH buffer or free antigen target competitor, and recovered by infecting bacteria. In some aspects of disclosing a phage that is not immobilized is object phage. In such aspects of the disclosure, the phage population may be contacted with the antigen two or more times to remove any of the phages that bind to the medium from the population. Unbound phages are then collected and used for further screening steps.

[0230] To enrich the phage population with phage particles that contain antibodies having a higher affinity for the target antigen (while reducing the percentage of phages that can bind to the antigen non-specifically), the eluted phage (described above) can be used to re-infect bacterial populations host cells. The expressed phage is then isolated from bacteria and re-contacted with the target antigen. Antigen concentration, pH, temperature and the inclusion of detergents and adjuvants during contact can be modulated to enrich for higher affinity antibody fragments. Unbound phages are removed by washing the solid support. The number of cycles, duration, pH, temperature and the inclusion of detergents and adjuvants during contact can also be modulated to enrich for antibody fragments with higher affinity. After the washing step, the bound phage is then eluted from the solid support. Any one to six repetitive cycles of selective uptake can be used to enrich for phage containing antibodies having higher affinity for the target antigen. In some embodiments, a deselecting step may also be performed in conjunction with any of the selective capture approaches described herein.

[0231] Individual phages from the population can be isolated by infecting bacteria and then plating, with a density that allows monoclonal antibodies to be obtained.

[0232] For example, the following selective capture approach can be used to identify an antibody using phage display techniques, which antibody binds to C5a, but not to C5. The population may first be contacted with a surface containing bound native, full-length human C5. The method can be repeated two or more times, with the collection of unbound phage each time. The population may also be contacted with a solid support containing C4 and / or C3 surface bound proteins. The unbound phage from the above steps is then contacted with a surface containing C5a bound or C5a-depleted arginine. The phages that bind to C5a are eluted from the surface and recovered by infecting bacteria. You can perform repeated rounds of phage selection. After one to six rounds of selection, individual recovered phagemids can be screened for expression of antibody fragments with the desired specificity and affinity.

[0233] A subpopulation of antibodies screened using the above methods can be characterized for their specificity and binding affinity for a particular immunogen (e.g., C5a) using any immunology or biochemistry-based method known in the art. For example, specific binding of an antibody to C5a, as compared to native full-length C5, can be determined, for example, using immunology or biochemistry-based methods, such as, but not limited to, ELISA, SPR, immuno-precipitation, affinity chromatography, and dialysis. equilibrium as described above. Immunoassays that can be used to analyze the immuno-specific binding and cross-reactivity of antibodies include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western hybridization, RIA, ELISA (enzyme-linked immunosorbent assay), sandwich immunoassays, immunoassay assays, assays immunodiffusion, agglutination assays, complement binding assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such assays are routine and well known in the art.

[0234] Antibodies can also be determined using any SPR-based assay known in the art to characterize the kinetic parameters of the interaction of the antibody with C5a. Any SPR instrument commercially available including, but not limited to, BIAcore Instruments (Biacore AB; Uppsala, Sweden) can be used; lAsys instruments (Affinity Sensors; Franklin, Massachussetts); IBIS system (Windsor Scientific Limited; Berks, UK), SPR-CELLIA systems (Nippon Laser and Electronics Lab; Hokkaido, Japan), and SPR Detector Spreeta (Texas Instruments; Dallas, Texas) in the methods described herein. See. e.g., Mullett et al. (2000) Methods 22: 77-91; Dong et al. (2002) Reviews in Mol Biotech 82: 303-323; Fivash et al. (1998) Curr Opin Biotechnol 9: 97101; and Rich et al (2000) Curr Opin Biotechnol 11: 54-61.

[0235] It is understood that the above methods can also be used to determine whether, e.g., anti-C5a antibody does not bind to full-length native C5, C3, and / or C4 proteins. The above methods can also be used to determine whether an antibody that binds to C5a also inhibits the interaction between C5a and the C5a receptor. The above methods can also be used to determine whether an antibody that binds to C5a also inhibits C5a activity.

[0236] As described in the above references, after phage selection, the antibody coding regions can be isolated and used to generate whole antibodies, including human antibodies or any other desired fragment, and expressed in the desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, e.g. as described in detail below. For example, techniques for recombinantly producing Fab, Fab 'and F (ab') 2 fragments can also be used using methods known in the art, such as those disclosed in PCT Publication No. WO 92/22324; Mullinax et al. (1992) BioTechniques 12 (6): 864-869; and Sawai et al. (1995) Am J Repr Immunol 34: 26-34; and Better et al. (1988) Science 240: 1041-1043. Examples of techniques that can be used to produce single chain Fvs and antibodies include those described in US Patent Nos. 4,946,778 and 5,258,498; Huston et al. (1991) Methods in Enzymology 203: 46-88; Shu et al. (1993) Proc Nat Acad Sci USA 90: 7995-7999; and Skerra et al. (1988) Science 240: 1038-1040.

[0237] Phage display technology can also be used to, e.g., increase the affinity of antibodies for their related antigen. The technology, referred to as affinity maturation, can utilize mutagenesis or CDR displacement and re-selection to identify antibodies that bind to higher affinity for the antigen compared to the parent or parent antibody. See. e.g. Glaser et al. (1992) J Immunol 149: 3903-3913. Libraries can be constructed consisting of a pool of variants of clones, each differing by one or more amino acid substitutions. Mutants with increased binding affinity for the antigen can be selected by contacting the immobilized mutants with the labeled antigen or any combination of the methods described above. Any prior art screening method known to identify mutated antibodies with increased antigen affinity (e.g. SPR or ELISA techniques).

[0238] In some aspects of the disclosure, epitope mapping can be used to identify, e.g., a C5a region that interacts with an antibody, e.g., a C5a region that binds to C5aR1. Methods for identifying the epitope to which a particular antibody binds are also known in the art and are described above.

[0239] Antibodies and fragments thereof identified herein may or may be made "chimeric". Chimeric antibodies and antigen-binding fragments thereof contain parts from two or more different species (e.g., mouse and human). Chimeric antibodies can be produced with murine variable regions of the desired specificity conjugated to human constant domains (for example, US Patent No. 4,816,567). In this way, non-human antibodies can be modified to make them more suitable for human clinical applications (e.g., methods for treating or preventing complement regulated disorder in a subject).

[0240] Monoclonal antibodies of the disclosure include "humanized" forms of non-human (e.g., murine) antibodies. Humanized mAbs or CDR-transplanted mAbs are particularly useful as therapeutics for humans because they are not cleared from the circulation as quickly as murine antibodies and do not typically cause an unwanted immune response. In general, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "imported" residues that are typically taken from the "imported" variable domain. Methods for producing humanized antibodies are well known in the art. For example, humanization can generally be carried out according to the method of Winter et al. (See, e.g., Jones et al. (1986) Nature 321: 522-525; Riechmann et al. (1988) Nature 332: 323-327; and Verhoeyen et al. (1988) Science 239: 1534-1536), by substituting framework sequences or rodent CDR sequences for the corresponding sequences of a human antibody. Also see e.g. Staelens et al. (2006) Mol Immunol 43: 1243-1257. In some embodiments, humanized non-human forms (e.g. mouse) antibodies are human antibodies (recipient antibody) in which the amino acid residues of the non-human CDR antibody region (e.g., mouse, rat, rabbit or non-human primate antibody) having the desired specificity, affinity and binding ability are transplanted onto a human framework scaffold antibodies. Additional humanization methods are described below in working examples.

[0241] Methods for grafting CDR sequences from a donor antibody (e.g., a non-human antibody) into the acceptor antibody framework regions (e.g., a human antibody) are well known in the art and are described in, e.g. Jones et al. (1986) Nature 321: 522-525; Verhoeyen et al. (1988) Science 239 (4847): 1534-1536; Riechmann et al. (1988) Nature 332: 323-327; Queen et al. (1989) Proc Natl Acad Sci USA 86: 10029-10033; PCT Publication No. WO 93/011237; Kettleborough et al. (1991) Protein Engineering, Design and Selection 4: 773-783; Benny KC Lo (2004) "Antibody Engineering: Methds and Protocols," Humana Press (ISBN: 1588290921); Borrebaek (1992) "Antibody Engineering, A Practical Guide," WH Wolneman i Co., NY; and Borrebaek (1995) "Antibody Engineering" 2nd Edition, Oxford University Press, NY, Oxford. For example, CDRs from the donor antibody may be grafted into the framework regions of the acceptor antibody using polymerase chain reaction overlapping fragmentation (PCR) techniques as described in, e.g., Daugherty et al. (1991) Nucleic Acids Res 19 (9): 2471-2476; Roguska et al. (1996) Protein Engineering 9 (10): 895-904; and Yazaki et al. (2004) Protein Engineering "Design & Selection 17 (5): 481-489.

[0242] In aspects of the disclosure in which selected CDR amino acid sequences are short sequences (e.g., less than 10-15 amino acids in length), nucleic acids encoding CDRs can be chemically synthesized as described in, e.g., Shiraishi et al . (2007) Nucleic Acids Symposium Series 51 (1): 129-130 and U.S. Patent No. 6,995,259. For a given nucleic acid sequence encoding an acceptor antibody, the region of the nucleic acid sequence encoding CDRs can be replaced with chemically synthesized nucleic acids using standard molecular biology techniques. The 5 'and 3' ends of chemically synthesized nucleic acids can be synthesized to contain sticky ends with respect to cleavage sites by restriction enzymes for use in cloning nucleic acids into the nucleic acid encoding the variable region of the donor antibody.

[0243] In some cases, one or more amino acid residues of the human immunoglobulin framework are also replaced with the corresponding amino acid residues of a non-human antibody (so-called "restoration mutations"). In addition, phage display libraries can be used to differentiate amino acids at selected positions within the antibody sequence. The selection of the human framework also affects the properties of the humanized antibody. In addition, humanized and chimerized antibodies can be modified to contain y residues that are not found in the recipient antibody or in the donor antibody to further improve the properties of the antibody, such as, for example, affinity or effector function.

[0244] Fully human antibodies are also included in the disclosure. The term "human antibody" includes antibodies having variable and constant regions (if present) derived from human immunoglobulin sequences, preferably human germline sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g. mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from another mammalian species, such as a mouse, have been transplanted into human framework sequences (i.e., humanized antibodies). Fully human or human antibodies may be from transgenic mice carrying human antibody genes (carrying variable (V), differentiated (D), connecting (J), and constant (C) exons or from human cells. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, after immunization, of producing a full repertoire of human antibodies in the absence of production of endogenous immunoglobulins. See. e.g. Jakobovits et al. (1993) Proc Natl Acad Sci USA 90: 2551; Jakobovits et al. (1993) Nature 362: 255-258; Bruggemann et al. (1993) Year in Immunol. 7:33; and Duchosal et al. (1992) Nature 355: 258. Transgenic mouse strains can be modified to contain gene sequences from unrearranged human immunoglobulin genes. One example of such a mouse is the huMAb® mouse (Medarex, Inc.), which contains human immunoglobulin transgene minilots that encode unrearranged human μ heavy chain and κ immunoglobulin light chain sequences, along with targeted mutations that inactivate endogenous μ chain loci and κ. See. e.g. Lonberg, et al. (1994) Nature 368 (6474): 856-859. Preparation and use of HuMab mice and genomic modifications found in such mice are further described in Taylor et al. (1992) Nucleic Acids Res 20: 6287-6295; Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc Natl Acad Sci USA 90: 3720-3724; Choi et al. (1993) Nature Genetics 4: 1 17123; Tuaillon et al. (1994) J Immunol 152: 2912-2920; Taylor et al. (1994) International Immunology 6: 579-591; and Fishwild et al. (1996) Nature Biotechnol 14: 845-851. An alternative transgenic mouse system for expressing human immunoglobulin genes is referred to as Xenomouse (Abgenix, Inc.) and is described in, e.g., US Patent Nos. 6,075,181; 6,114,598; 6,150,584; and 6,162,963. Like the huMAb® mouse system, the Xenomouse mouse system involves the disruption of endogenous mouse heavy chain and light chain genes, and the introduction into the mouse genome of transgenes bearing unarranged human heavy chain and light immunoglobulin loci that contain human variable and constant region sequences. Other prior art systems for expressing human immunoglobulin genes include the KM mouse system

Mouse®, described in detail in PCT Publication No. WO 02/43478 and the TC mouse system described in Tomizuka et al. (2000) Proc Natl Acad Sci USA 97: 722-727.

[0245] Human sequences can encode both heavy and light chains of human antibodies and will function properly in mice undergoing rearrangement to provide a broad antibody repertoire similar to that of humans. Transgenic mice can be immunized with the target protein immunogen to form a diverse range of specific antibodies and RNA encoding them. Nucleic acids encoding components of the antibody chains for such antibodies can then be cloned from the animal into a presentation vector. Typically, separate populations of nucleic acids encoding heavy and light chain sequences are cloned, and the separate populations are then combined upon insertion into the vector, so that any copy of the vector receives a random combination of heavy and light chains. The vector is designed to express antibody chains so that they can be assembled and displayed on the outer surface of the presentation package containing the vector. For example, antibody chains can be expressed as fusion proteins, with a phage coat protein on the outer surface of the phage. Subsequently, presentation packages can be selected and screened for the presentation of antibodies binding to the target.

[0246] Additionally, phage display libraries screened above may include human antibodies (Hoogenboom et al. (1992) J Mol Biol 227: 381; Marks et al. (1991) J Mol Biol 222: 581-597; and Vaughan et al . (1996) Nature Biotech 14: 309). Synthetic phage libraries can be created that use randomized combinations of synthetic human antibody V regions. By antigen selection, fully human antibodies in which the V regions are very human can be obtained. See. e.g. US patents no. 6,794,132; 6,680,209; 4,634,666; and Ostberg et al. (1983) Hybridoma 2: 361-367.

[0247] For the generation of human antibodies, see also Mendez et al. (1998) Nature Genetics 15: 146-156 and Green and Jakobovits (1998) J Exp Med 188: 483-495.

[0248] Human antibodies are further discussed and outlined in US Patent Nos. 5,939,598; 6,673,986; 6,114,598; 6,075,181; 6,162,963; 6,150,584; 6,713,610; and 6,657,103 as well as in US Patent Publication Nos. 20030229905 A1, 20040010810 A1, 20040093622 A1, 20060040363 A1, 20050054055 A1, 20050076395 A1, and 20050287630 A1. See. also international publications nos. WO 94/02602, WO 96/34096, and WO 98/24893, and European Patent No. EP 0 463 151 B1.

[0249] In an alternative approach, others, including GenPharm International, Inc., used the "minilocus" approach. The minilocus approach mimics the exogenous Ig locus by incorporating fragments (individual genes) from the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, mu constant region and second constant region (preferably gamma constant region) are formed into a construct for introduction into an animal. This approach is described in, e.g. U.S. Patent Nos. 5,545,807; 5,545,806; 5,625,825; 5,625,126; 5,633,425; 5,661,016; 5,770,429; 5,789,650; and 5,814,318; 5,591,669;

5,612,205; 5,721,367; 5,789,215; 5,643,763; 5,569,825; 5,877,397; 6,300,129; 5,874,299; 6,255,458; and 7,041,871.

[0250] See also European Patent No. 0 546 073 B1, International Patent Publication Numbers WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852 and WO 98/24884.

[0251] See referred to as Taylor et al. (1992) Nucleic Acids Res 20: 6287; Chen et al. (1993) Int Immunol 5: 647; Tuaillon et al. (1993) Proc Natl Acad Sci USA 90: 3720-4; Choi et al. (1993) Nature Genetics 4: 117; Lonberg et al. (1994) Nature 368: 856-859; Taylor et al. (1994) International Immunology 6: 579-591; Tuaillon et al. (1995) J. Immunol 154: 6453-65; Fishwild et al. (1996) Nature. Biotechnology 14: 845; and Tuaillon et al. (2000) EurJImmunol. 10: 2998-3005.

[0252] In some aspects of the disclosure, de-immunized antibody forms, or antigen-binding fragments as described herein are provided. De-immunized antibodies or antigen-binding fragments thereof are antibodies that have been modified to make the antibody or antigen-binding fragment thereof non-immunogenic or less immunogenic for a given species. De-immunization can be achieved by modifying the antibody or antigen-binding fragment thereof, using any of a number of techniques known to one of skill in the art (see, e.g., PCT Publication Nos. WO 04/108158 and WO 00/34317). For example, an antibody or antigen binding fragment thereof can be de-immunized by identifying potential T cell epitopes and / or B cell epitopes within the amino acid sequence of the antibody or antigen binding fragment thereof and removing one or more potential T cell epitopes and / or cell epitopes B from an antibody or antigen binding fragment thereof, for example, using recombinant techniques. The modified antibody or antigen binding fragment thereof can then optionally be prepared and tested to identify antibodies or antigen binding fragments thereof that have retained one or more desired biological activities, such as, for example, binding affinity but have reduced immunogenicity. Methods for identifying potential T cell epitopes and / or B cell epitopes can be carried out using techniques known in the art, such as, for example, computational methods (see e.g. PCT Publication No. WO 02/069232), in vitro or in silico techniques, and biological assays or physical methods (such as, for example, determining the binding of peptides to MHC molecules, determining the binding of peptide: MHC complexes to T cell receptors having a species take the antibody or antigen-binding fragment thereof, testing the protein or peptide part thereof with transgenic animals with MHC molecules of the species to receive the antibody or antigen-binding fragment thereof, or testing using transgenic animals with reconstituted cells of the immune system for the species having the antibody or antigen-binding fragment thereof, etc.). In various aspects of the disclosure, the de-immunized antibodies described herein include de-immunized antigen binding fragments, Fab, Fv, scFv, Fab 'and F (ab') 2, monoclonal antibodies, mouse antibodies, fully human antibodies, modified antibodies (such as, for example, chimeric, single chain, CDR-grafted, humanized, and artificially selected antibodies), synthetic antibodies, and semisynthetic antibodies.

[0253] Bispecific antibodies are contemplated in the therapeutic aspects of the disclosure. Bispecific antibodies are monoclonal antibodies, preferably human or humanized, that have binding specificities for at least two different antigens. In the present case, one binding specificity is for C5a and the other is for any other antigen.

[0254] Methods for making bispecific antibodies are well known to those skilled in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, with the two heavy chains having different specificities (Milstein and Cuello (1983) Nature 305: 537-539). Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be conjugated to immunoglobulin constant domain sequences. The heavy chain variable region fusion preferably occurs with an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge regions, CH2 and CH3. DNAs encoding immunoglobulin heavy chain fusions and, if desired, immunoglobulin light chain fusion are introduced into separate expression vectors, and are co-transfected into the appropriate host organism. Further details regarding illustrations of currently known methods for generating bispecific antibodies are provided in, e.g., Suresh et al. (1986) Methods in Enzymology 121: 210; PCT Publication No. WO 96/27011; Brennan et al. (1985) Science 229: 81; Shalaby et al., J Exp Med (1992) 175: 217-225; Kostelny et al. (1992) J Immunol 148 (5): 1547-1553; Hollinger et al. (1993) Proc Natl Acad Sci USA 90: 6444-6448; Gruber et al. (1994) J Immunol 152: 5368; and Tutt et al. (1991) J Immunol 147: 60. Bispecific antibodies also include cross-linked or heteroconjugated antibodies. The antibody heteroconjugate can be obtained using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in US Patent No. 4,676,980, along with a number of cross-linking techniques.

[0255] Various techniques have also been described for the production and isolation of bispecific antibody fragments directly from recombinant cell culture. For example, bispecific antibodies have been produced using leucine zippers. See. e.g. Kostelny et al. (1992) J Immunol 148 (5): 1547-1553. Leucine zipper peptides from the Fos and Jun proteins were combined with the Fab 'portions of two different antibodies by gene fusion. Antibody homodimers were reduced in the hinge region to form monomers, followed by re-oxidation to form antibody heterodimers. This method can also be used to produce antibody homodimers. The "diabody" technology described in Hollinger et al. (1993) Proc Natl Acad Sci USA 90: 6444-6448 provided an alternative mechanism for producing bispecific antibody fragments. The fragments contain a heavy chain variable domain (VH) connected to a light chain variable domain (VL) via a linker that is too short to allow pairing between two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, while simultaneously creating two antigen binding sites. Another strategy for producing bispecific antibody fragments by the use of single chain Fv dimers (sFv) has also been described. See. e.g. Gruber et al. (1994) J Immunol 152: 5368. Alternatively, the antibodies may be "linear antibodies" as described in, e.g., Zapata et al. (1995) Protein Eng. 8 (10): 1057-1062. Briefly, these antibodies contain a pair of tandem Fd segments (VHCH1-VH-CH1) that form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

[0256] Antibodies with more than two valences (e.g., trispecific antibodies) are contemplated and described in, e.g., Tutt et al. (1991) J Immunol 147: 60.

[0257] The disclosure also includes variant forms of multispecific antibodies, such as the dual variable domain of immunoglobulin molecules (DVD-Ig) described in Wu et al. (2007) Nat Biotechnol 25 (11): 1290-1297. DVD-Ig molecules are designed such that the variable domains of two different light chains (VL) from two different parent antibodies are tandemly linked directly or by a short linker, by recombinant DNA techniques, with a subsequent light chain constant domain. Similarly, the heavy chain contains two different heavy chain (VH) variable domains tandemly connected, with a subsequent constant CH1 domain and an Fc region. Methods for producing DVD-Ig molecules from two parent antibodies are further described in, e.g., international PCT publications nos. WO 08/0241188 and WO 07/024715.

[0258] The disclosure also provides camelid or bactrian camel antibodies (e.g., antibodies derived from Camelus bactrianus, Calelus dromaderius, or Lama paccos). Such antibodies, as opposed to typical two-chain (fragment) or four-chain (whole antibody) antibodies, as in most mammals, generally lack light chains. See. U.S. Patent No. 5,759,808; Stijlemans et al. (2004) J Biol Chem 279: 1256-1261; Dumoulin et al. (2003) Nature 424: 783788; and Pleschberger et al. (2003) Bioconjugate Chem 14: 440-448. Modified libraries of camelid antibodies and fragments of camelid antibodies are commercially available, for example, from Ablynx (Ghent, Belgium). As with other antibodies of non-human origin, the amino acid sequence of the camelid antibody can be changed by recombination to obtain a sequence that more closely resembles the human sequence, i.e. the nanobody can be "humanized" to thereby further reduce the potential immunogenicity of the antibody.

[0259] In some aspects of the disclosure, anti-C5a antibodies herein comprise an altered heavy chain constant region with reduced (or absent) effector function relative to the corresponding unchanged constant region. Effector functions associated with the anti-C5a constant region can be modulated by altering the properties of the constant region or Fc region. Altered effector functions include, for example, the modulation of one or more of the following activities: antibody dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), apoptosis, binding to one or more Fc receptors and pro-inflammatory responses. Modulation refers to the increase, decrease or elimination of effector function activity exhibited by the subject antibody comprising the altered constant region as compared to the activity of the unchanged form of the constant region. In particular aspects of the disclosure, modulation includes situations where activity is abolished or completely absent.

[0260] An altered constant region with altered FcR binding affinity and / or ADCC activity and / or altered CDC activity means a polypeptide that has increased or decreased FcR binding activity and / or ADCC activity and / or CDC activity as compared to the unchanged form of the constant region . An altered constant region that exhibits increased binding to FcR binds at least one FcR with higher affinity than the unchanged polypeptide. An altered constant region that exhibits reduced binding to FcR binds at least one FcR with lower affinity than the unchanged form of the constant region. Such variants that show reduced binding to FcR may have little or no noticeable binding to FcR, e.g. 0 to 50% (e.g. less than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) binding to FcR compared to the level of binding of the native immunoglobulin constant region or Fc region sequence to FcR. Similarly, an altered constant region that exhibits modulated ADCC and / or CDC activity may exhibit increased or decreased ADCC and / or CDC activity as compared to the unchanged constant region. For example, in some aspects of the disclosure, an anti-C5a antibody containing the altered constant region may exhibit approximately 0 to 50% (e.g. less than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of ADCC activity and / or CDC of the unchanged form of the constant region. An anti-C5a antibody described herein containing an altered constant region exhibiting reduced ADCC and / or CDC may exhibit reduced - or absent - ADCC and / or CDC activity as specified herein.

[0261] In some aspects of the disclosure, the altered constant region has at least one amino acid substitution, insertion and / or deletion, compared to the native constant region sequence or to an unchanged constant region, e.g. from about one to about one hundred substitutions, insertions and / or deletion of amino acids in the native constant region sequences or in the constant region of the parent polypeptide. In some aspects of the disclosure, the altered constant region herein has at least about 70% homology (similarity) or identity with the unchanged constant region, and in some cases at least about 75% and in other cases at least about 80% homology or identity to them, and in others embodiments of at least about 85%, 90% or 95% homology or identity thereto. The altered constant region may also contain one or more amino acid deletions or insertions. In addition, the altered constant region may contain one or more amino acid substitutions, deletions or insertions, resulting in altered post-translational modifications, including, for example, altered glycosylation pattern (e.g. addition of one or more sugar components, loss of one or more sugar components, or change in the composition of one or more sugar components relative to the unchanged constant region).

[0262] Antibodies with altered - or absent - effector function can be produced by modifying or producing antibodies with differentiated constant regions, Fc, or heavy chains; recombinant DNA technology and / or cell cultures and expression conditions can be used to produce antibodies with altered function and / or activity. For example, recombinant DNA technology can be used to modify one or more amino acid substitutions, deletions, or insertions into regions (such as, for example, Fc or constant regions) that affect antibody function, including effector functions. Alternatively, changes can be made to post-translational modifications, such as, e.g., glycosylation patterns, by manipulating cell culture and expression conditions by which the antibody is produced. Suitable methods for introducing one or more substitutions, additions or deletions into the Fc region of an antibody are well known in the art and include, e.g., standard DNA mutagenesis techniques as described in, e.g., Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual, 2nd Edition," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane (1988), supra; Borrebaek (1992), supra; Johne et al. (1993), supra; PCT Publication No. WO 06/53301; and U.S. Patent No. 7,704,497.

[0263] In some aspects of the disclosure, the anti-C5a antibody described herein exhibits reduced - or absent - effector function. In some aspects of the disclosure, the anti-C5a antibody comprises a hybrid constant region, or a portion thereof, such as the G2 / G4 hybrid constant region (see, e.g., Burton et al. (1992) Adv Immun 51: 1-18; Canfield et al. ( 1991) J Exp Med 173: 1483-1491; and Mueller et al. (1997) Mol Immunol 34 (6): 441-452). See. above.

[0264] In addition to using the G2 / G4 construct as described above, the anti-C5a antibody described herein having reduced effector function can be produced by introducing other types of changes in the amino acid sequence of specific regions of the antibody. Such amino acid sequence changes include, but are not limited to, Ala-Ala mutations described in, e.g., PCT International Publication Nos. WO 94/28027 and WO 98/47531; and Xu et al. (2000) Cell Immunol 200: 16-26. Hence, in some aspects of the disclosure, an anti-C5a antibody with one or more constant region mutations, including the Ala-Ala mutation, has reduced or absent effector function. In accordance with these aspects, an antibody constant region may comprise a substitution to alanine at position 234 or a mutation to alanine at position 235. In addition, the altered constant region may contain a double mutation: a mutation to alanine at position 234 and a second mutation to alanine at position 235. In one aspect of the disclosure, the anti-C5a antibody comprises an IgG4 framework, wherein the Ala-Ala mutation would describe a mutation (mutations) from phenylalanine to alanine at position 234 and / or a mutation from leucine to alanine at position 235.

In another aspect of the disclosure, anti-C5a antibodies comprise the IgG1 b-strand, where the Ala-Ala mutation would describe the mutation (s) from leucine to alanine at position 234 and / or the mutation from leucine to alanine at position 235. Anti antibody -C5a may alternatively or additionally carry other mutations, including the K322A point mutation in the CH2 domain (Hezareh metal. (2001) J Virol 75: 12161-12168). The antibody with said mutation (s) in the constant region may further be a blocking or non-blocking antibody.

[0265] Additional substitutions that, when introduced into the heavy chain constant region, result in reduced effector function are given in, e.g., Shields et al. (2001) J.

Biol Chem 276 (9): 6591-6604. See. in particular Table 1 ("Binding of human IgG1 variants to human FcRn and FcyR) in Shields et al.: which disclosure is incorporated herein by reference in its entirety. By screening anti-IgE antibody libraries, each of the antibodies in the library differing by one or more substitutions in the heavy chain constant region for binding to a panel of Fc receptors (including FcRn, FcyRI, FcyRIIA, FcyRIIB, and FcyRIIIA), the authors have identified many substitutions that modulate specific Fc-Fc receptor interactions. For example, the IgG2a variant of the heavy chain constant region in which the CH2 domain contains D265A substitution (heavy chain amino acid numbering according to Kabat et al. (Supra)) results in complete loss of interaction between the constant region variant and the IgG Fc Fc? RIIB, Fc? RIII, Fc? RI and Fc? RIV receptors . Shields et al. (2001) on page 6595, Table 1. See also Baudino et al. (2008) J Immunol 181: 6664-6669 (supra).

[0266] Changes within the hinge region also affect effector functions. For example, deletion of the hinge region may reduce the affinity for Fc receptors and may reduce complement activation (Klein et al. (1981) Proc-Natl Acad Sci USA 78: 524-528). The disclosure also applies to antibodies with changes in the hinge region.

[0267] In some aspects of the disclosure, the anti-C5a antibody may comprise an altered constant region showing increased or reduced complement dependent cytotoxicity (CDC). Modulation of CDC activity can be achieved by introducing one or more amino acid substitutions, insertions or deletions in the antibody Fc region. See. e.g. US Patent No. 6,194,551. Alternatively or additionally, a cysteine residue (s) may be introduced into the Fc region, thereby allowing interchain formation of disulfide bonds in this region. The homodimeric antibody thus produced may have improved or reduced internalization capability and / or increased or reduced complement-regulated cell killing. See. e.g. Caron et al. (1992) J Exp Med 176: 1191-1195 and Shopes (1992) Immunol 148: 2918-2922; PCT publications Nos. WO 99/51642 and WO 94/29351; Duncan and Winter (1988) Nature 322: 738-40; and US patents no. 5,648,260 and 5,624,821.

[0268] Further potential agents for modulating the effector function of antibodies include glycosylation changes that are listed in, e.g., Raju (2003) BioProcess International 1 (4): 4453. According to Wright and Morrison, the microheterogenicity of human IgG oligosaccharides may affect biological functions such as CDC and ADCC, binding to various Fc receptors and binding to Clq protein. (1997) TIBTECH 15: 26-32. Antibody glycosylation patterns may vary depending on the producing cell and cell culture conditions (Raju, supra). Such differences can lead to changes in both effector function and pharmacokinetics. See. e.g. Israel et al. (1996) Immunology 89 (4): 573-578; Newkirk et al. (1996) Clin Exp Immunol 106 (2): 259-264. Differences in effector function may be related to the ability of IgG to bind to Fcy receptors (Fc? Rs) on effector cells. Shields et al. have shown that IgG, with changes in the amino acid sequence that improved binding to Fc? R, can show up to 100% increased ADCC using human effector cells. (2001) J Biol Chem 276 (9): 6591-6604. While these changes include changes in amino acids not found in the interfaces of the binding, both the nature of the sugar component as well as its structural formula may also contribute to the observed differences. In addition, the presence or absence of fucose in the IgG oligosaccharide component may improve binding and ADCC. See. e.g. Shields et al. (2002) J Biol Chem 277 (30): 26733-26740. IgG lacking fucosylated carbohydrate associated with Asn297 showed normal receptor binding to the FcγRI receptor. In contrast, binding to the Fc? RIIIA receptor was 50-fold improved, and was associated with an increase in ADCC, especially at lower antibody concentrations.

[0269] Shinkawa et al. showed that the anti-human IL-5 receptor antibody produced in a rat hybridoma showed more than 50% higher ADCC compared to that produced in Chinese hamster ovary (CHO) cells (Shinkawa et al. (2003) JBiol Chem 278 (5): 3466- 73). Monosaccharide composition and oligosaccharide profiling showed that IgG produced in the rat hybridoma had a lower fucose content than the protein produced by CHO. The authors conclude that the lack of IgG1 fucosylation is critical in increasing ADCC activity.

[0270] Another approach was used by Umana et al., Changing the glycosylation pattern of chCE7, an anti-neuroblastoma chimeric IgG1 antibody. (1999) Nat Biotechnol 17 (2): 176180). Using tetracycline, they regulated the activity of the glycosyltransferase enzyme (GnTIII), which cleaves oligosaccharides associated with ADCC activity. The parent antibody ADCC activity was slightly above background level. Measurement of ADCC activity in chCE7 produced at different levels of tetracycline showed an optimal GnTIII expression range for maximal in vitro ADCE activity of chCE7. This activity correlated with the level of the constant region-bound oligosaccharide complex cleaved in half. The newly optimized variants showed significant ADCC activity. Similarly, Wright and Morrison produced antibodies in a CHO cell line deficient in glycosylation and showed that the antibodies produced in this cell line were unable to complement mediated cytolysis. (1994) J Exp Med 180: 1087-1096. Hence, as known changes that affect effector function include modifications to the glycosylation pattern or change in the number of glycosylated residues, the disclosure relates to an anti-C5a antibody wherein the glycosylation is altered to increase or decrease effector functions, including ADCC and CDC. Altered glycosylation includes a decrease or increase in the number of glycosylated residues as well as a change in the pattern or location of glycosylated residues.

[0271] There are other approaches to altering the effector function of antibodies. For example, the antibody producing cells may be hypermutagenic, thereby generating antibodies with randomly changed residues in the polypeptide for the entire antibody molecule. See. e.g. PCT Publication No. WO 05/011735. Hypermutagenic host cells include cells deficient in DNA mismatch repair. Antibodies produced in this way may be less antigenic and / or have beneficial pharmacokinetic properties. In addition, such antibodies can be selected for properties such as increased or decreased effector functions. Additional details of the molecular biology techniques useful for obtaining the antibody or antigen-binding fragment thereof described herein are given below.

Expression and purification of the recombinant antibody [0272] Antibodies or antigen-binding fragments thereof described herein can be produced using a variety of techniques known in the art, molecular biology and protein chemistry. For example, a nucleic acid encoding one or both heavy and light chains of an antibody polypeptide may be inserted into an expression vector that contains transcriptional and translational regulatory sequences that include, e.g., promoter sequences, ribosome binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcription termination signals, polyadenylation signals and enhancer or activator sequences. Regulatory sequences include the promoter and transcriptional start and stop sequences. In addition, the expression vector may include more than one replication system, such that it can be maintained in two different organisms, for example in mammalian or insect cells, for expression, and in a prokaryotic host, for cloning and amplification.

[0273] Several potential vector systems are available for expressing cloned heavy chain and light chain polypeptides from nucleic acids in mammalian cells. One class of vectors is based on the integration of desired gene sequences into the host cell genome. Cells that have stably integrated into DNA can be selected by simultaneously introducing drug resistance genes such as E. coli gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA 78: 2072) or Tn5 neo (Southern and Berg (1982) Mol. Appl Genet 1: 327). The selectable marker gene can be combined with DNA sequences for expression or introduced into the same cell by co-transfection (Wigler et al. (1979) Cell 16:77). The second class of vectors uses DNA elements that confer the ability of autonomous replication of an extrachromosomal plasmid. These vectors may be derived from animal viruses such as bovine papilloma virus (Sarver et al. (1982) Proc. Natl Acad Sci USA, 79: 7147), cytomegalovirus, polyoma virus (Deans et al. (1984) Proc Natl Acad Sci USA 81: 1292), or SV40 virus (Lusky and Botchan (1982) Nature 293: 79).

[0274] The expression vector may be introduced into cells in a manner suitable for subsequent expression of the nucleic acid. The method of introduction depends largely on the type of target cell, as discussed below. Exemplary methods include precipitation

CaPO4, liposome fusion, cationic liposomes, electroporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.

[0275] Suitable host cells for expressing antibodies or antigen-binding fragments thereof include yeast, bacterial, insect, plant and mammalian cells. Bacteria such as E. coli, fungi such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as SF9, mammalian cell lines (e.g. human cell lines) as well as major cell lines are of particular interest.

[0276] In some aspects of the disclosure, the antibody or fragment thereof can be expressed in, and purified from, transgenic animals (e.g., transgenic mammals). For example, the antibody may be produced in a non-human transgenic mammal (e.g. rodent) and isolated from milk as described in, e.g., Houdebine (2002) Curr Opin Biotechnol 13 (6): 625-629; van Kuik-Romeijn et al. (2000) Transgenic Res 9 (2): 155-159; And Pollock et al. (1999) J Immunol Methods 231 (1-2): 147-157.

[0277] Antibodies and fragments thereof can be produced in cells, by culturing a host cell transformed with an expression vector containing a nucleic acid encoding the antibodies or fragments, under conditions and for a period of time sufficient to allow expression of the protein. Such conditions for protein expression will vary, depending on the choice of expression vector and host cell, and will be easily determined by one of ordinary skill in the art by routine experimentation. For example, antibodies expressed in E. coli may be refolded from inclusion bodies (see, e.g., Hou et al. (1998) Cytokine 10: 319-30). Bacterial expression systems and methods for their use are well known in the art (see Current Protocols in Molecular Biology, Wiley & Sons, and Molecular Cloning A Laboratory Manual - 3rd Edition, Cold Spring Harbor Laboratory Press, New York (2001)). The choice of codons, appropriate expression vectors and appropriate host cells will vary depending on the number of factors, and can be easily optimized as needed. The antibody (or fragment thereof) described herein may be expressed in mammalian cells or other expression systems, including but not limited to yeast, baculovirus and in vitro expression systems (see, e.g., Kaszubska et al. (2000) Protein Expression and Purification 18 : 213-220).

[0278] After expression, the antibodies and fragments thereof can be isolated. The term "purified" or "isolated" in relation to any of the proteins (antibodies or fragments) described herein refers to a polypeptide that has been separated or purified from components (e.g., proteins or other naturally occurring biological or organic molecules) that accompany in natural conditions, e.g. other proteins, lipids and nucleic acid in a prokaryotic protein-expressing organism. Typically, the polypeptide is purified when it is at least 60 (e.g., at least 65, 70, 75, 80, 85, 90, 92, 95, 97, or 99)%, by weight, of total protein in the sample.

[0279] The antibody or fragment thereof can be isolated or purified in a number of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobicity, affinity, and reverse phase HPLC. For example, the antibody may be purified using a standard anti-antibody column (e.g. protein-A or protein-G column). Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. See. e.g. Scopes (1994) "Protein Purification, 3rd Edition," Springer-Verlag, New York City, New York. The degree of purification necessary will vary depending on the desired application. In some cases, no purification of the expressed antibody or fragments thereof will be necessary.

[0280] Methods to determine the yield or purity of the purified antibody or fragment thereof are known in the art and include, e.g., Bradford assay, UV spectroscopy, biuret protein assay, Lowry assay protein, amido black protein assay, high pressure liquid chromatography (HPLC) ), mass spectrometry (MS), and electrophoretic separation methods in gels (e.g. using a protein stain (such as Coomassie Blue or colloidal silver for staining).

[0281] In some aspects of the disclosure, endotoxins can be removed from antibodies or fragments. Methods for removing endotoxin from a protein sample are known in the art and detailed in working examples. For example, endotoxin can be removed from a protein sample using a number of commercially available reagents, including, but not limited to ProteoSpin ™ endotoxin removal kits (Norgen Biotek Corporation), Detoxi-Gel endotoxin removal gel (Thermo Scientific; Pierce Protein Research Products) , MiraCLEAN® endotoxin removal kit (Mirus), or Acrodisc ™ - Mustang® E membrane (Pall Corporation).

[0282] Methods for detecting and / or measuring the amount of endotoxin present in a sample (both before and after purification) are known in the art and commercial kits are available. For example, endotoxin concentration in a protein sample can be determined using the QCL1000 Chromogenic kit (BioWhittaker), sets based on Limulus amebocyte lysate (LAL) such as Pyrotell®, Pyrotell®-T, Pyrochrome®, Chromo-LAL and CSE kits available from the Cape Cod Incorporated association.

[0283] In the absence of complete limitation, exemplary methods for making the antibodies described herein are provided in the working examples. Modification of Antibodies or Antigen Binding Fragments [0284] Antibodies or antigen binding fragments thereof can be modified after expression and purification. Modifications can be covalent or non-covalent modifications. Such modifications can be made to antibodies or fragments by reacting target amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Suitable modification sites can be selected using any of a number of criteria, including, e.g., structural analysis or amino acid sequence analysis of antibodies or fragments.

[0285] In some aspects of the disclosure, the antibodies or antigen-binding fragments thereof can be conjugated to a heterologous moiety. A heterologous moiety can be, e.g., a heterologous polypeptide, a therapeutic (e.g. toxin or drug), or a detectable tag, such as, but not limited to, a radioactive tag, an enzyme tag, a fluorescent tag, a heavy metal tag, a luminescent tag or an affinity tag, such like biotin or streptavidin. Suitable heterologous polypeptides include, e.g., an antigenic tag (e.g., FLAG (DYKDDDK (SEQ ID NO: 50)), polyhistidine (6-His; HHHHHH (SEQ ID NO: 81), hemagglutinin (HA; YPYDVPDYA (SEQ ID-NO: 82), glutathione-S-transferase (GST), or maltose binding protein (MBP) for use in purifying antibodies or fragments. Heterologous polypeptides also include polypeptides (e.g. enzymes) that are useful as diagnostic or detectable markers, for example, luciferase, fluorescent protein (e.g., green fluorescence protein (GFP)), or chloramphenicol acetyl transferase (CAT). Suitable radiolabels include, for example32P 33P 14C 125AND, 131AND, 131AND, 35S and 3H. Suitable fluorescent labels include, but are not limited to, fluorescein, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), DyLight ™ 488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor® 700, Cy5 , allophycocyanin and Cy7. Luminescent labels include, e.g., any of a series of luminescent lanthanide chelates (e.g., europium or terbium). For example, suitable europium chelates include europium chelate of diethylenetriaminepentaacetic acid (DTPA) or tetraazacyclododecane-1,4,7,10 tetraacetic acid (DOTA). Enzyme labels include, e.g., alkaline phosphatase, CAT, luciferase, and horseradish peroxidase.

[0286] Two proteins (e.g., antibody and heterologous moiety) can be cross-linked using any of a number of known chemical cross-linking agents. Examples of such crosslinkers are those that link two amino acid residues with a bond that includes "disruptive" disulfide bonding. In these bonds, the disulfide bond within the crosslinking unit is protected (by interfering groups on each side of the disulfide bond) from reduction due to, for example, reduced glutathione or the disulfide reductase enzyme. One suitable reagent, 4-succinimidyloxycarbonyl-α-methyl-α (2-pyridyldithio) toluene (SMPT), creates such a bond between two proteins using terminal lysine from one protein and terminal cysteine from the other. It is also possible to use heterobifunctional reagents that cross-link via different cross-linking moieties on each protein. Other useful cross-linking agents include, but are not limited to, reagents that connect two amino groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g. 1,4-bismaleimidobutane), an amino group and a sulfhydryl group (e.g. m-maleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and a carboxyl group (e.g. 4- [pazosalosalicylamido] butylamine), and an amino group and a guanidinium group that is present in the arginine side chain (e.g., p-azidophenylglyoxal monohydrate).

[0287] In some aspects of the disclosure, the radioactive label can be directly conjugated to the antibody amino acid backbone. Alternatively, the radioactive tracer may be captured as part of a larger molecule (e.g.125And in Meta: [125I] iodophenyl-N-hydroxysuccinimide ([125I] mIPNHS) which binds to free amino groups to form meta-iodophenyl (mIP) derivatives of the proteins concerned (see e.g. Rogers et al. (1997) J Nucl Med 38: 1221-1229) or chelate (e.g. DOTA or DTPA) which is in turn bound to the protein backbone. Methods for conjugating radioactive labels or larger molecules / chelates containing them to the antibodies or antigen-binding fragments described herein are known in the art. Such methods include incubating the protein with the radioactive label under conditions (e.g., pH, salt concentration and / or temperature) that facilitate the binding of the radioactive label or chelate to the protein (see, e.g., US Patent No. 6,001,329) [0288] Fluorescent label conjugation methods ( sometimes referred to as a "fluorophore") to a protein (eg, an antibody) are known in the art in protein chemistry. For example, fluorophores can be coupled with free amino groups (e.g. lysines) or sulfhydryl groups (e.g., cysteines) of a protein using succinimidyl ester (NHS) or tetrafluorophenyl ester (TFP) attached to fluorophores. In some aspects of the disclosure, the fluorophores can be coupled to a heterobifunctional cross-linking moiety such as sulfo-SMCC. Suitable conjugation methods include incubating the antibody protein, or fragment thereof, with a fluorophore under conditions that facilitate the binding of the fluorophore to the protein. See. for example. Welch I Redvanly (2003) "Handbook of Radiopharmaceuticals: Radiochemistry and Applications," John Wiley and Sons (ISBN 0471495603).

[0289] In some aspects of the disclosure, the antibodies or fragments can be modified, e.g., by a moiety that improves the stabilization and / or retention of the antibodies in the circulation, e.g., in blood, serum, or other tissues. For example, the antibody or fragment may be pegylated as described in, e.g., Lee et al. (1999) Bioconjug Chem 10 (6): 973-8; Kinstler et al. (2002) Advanced Drug Deliveries Reviews 54: 477-485; and Roberts et al. (2002) Advanced Drug Delivery Reviews 54: 459-476 or HESylated (Fresenius Kabi, Germany; see e.g. Pavisic 'et al. (2010) Int J Pharm 387 (1-2): 110119). A stabilizing moiety can improve the stability, or retention, of an antibody (or fragment) by at least 1.5 (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) fold.

[0290] In some aspects of the disclosure, the antibodies or antigen-binding fragments thereof described herein may be glycosylated. In some aspects of the disclosure, the antibody or antigen binding fragment thereof described herein may be subjected to enzymatic or chemical treatment, or produced in cells, such that the antibody or fragment has reduced or absent glycosylation. Methods for producing antibodies with reduced glycosylation are known in the art and described in, e.g., US Patent No. 6,933,368; Wright et al. (1991) EMBO J 10 (10): 2717-2723; I Co et al. (1993) Mol Immunol 30: 1361.

Pharmaceutical compositions [0291] Compositions comprising the antibody or antigen-binding fragment thereof described herein can be formulated as a pharmaceutical composition, e.g. for administration to a subject for treating or preventing a complement related disorder. Pharmaceutical compositions will generally contain a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to and includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The compositions may contain a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see, e.g., Berge et al. (1977) J Pharm Sci 66: 1-19).

[0292] The compositions may be formulated according to standard methods. The pharmaceutical formulation is well established in the art, and is further described in, e.g., Gennam (2000) "Remington: Science and Practice of Pharmacy, Twentieth Edition, Lippincott, Williams & Wilkins (ISBN: 0683306472); Ansel et al. (1999) "Pharmaceutical Dosage Forms and Drug Delivery Systems," 7th Edition, Lippincott Williams & Wilkins Publishers (ISBN: 0683305727); and Kibbe (2000) "Handbook of Pharmaceutical Excipients American Pharmaceutical Association" 3rd edition (ISBN: 091733096X). In some aspects of the disclosure, the composition may be formulated, for example, as a buffered solution at a suitable concentration and suitable for storage at 2-8 ° C (e.g., 4 ° C). In some aspects of the disclosure, the composition may be formulated for storage at a temperature below 0 ° C (e.g., -20 ° C or -80 ° C). In some aspects of the disclosure, the composition may be formulated for storage, up to 2 years (e.g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 1 year, 1 and 1/2 years, or 2 years) at 2-8 ° C (e.g. 4 ° C). Hence, in some aspects of the disclosure, the compositions described herein are stable when stored for at least 1 year at 2-8 ° C (e.g., 4 ° C).

[0293] The pharmaceutical compositions may have a variety of forms. These forms include, e.g., liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., for injection and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends, in part, on the intended route of administration and therapeutic application. For example, compositions containing an antibody or fragment intended for systemic or local delivery may be in the form of injectable or infusion solutions. Accordingly, the compositions may be formulated by parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal or intramuscular injection). "Parenteral administration", "parenteral administration" and other grammatically equivalent phrases as used herein refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, injection and infusion: intravenous, intranasal, intraocular, intramuscular, intraarterial, intrathecal, intracapsular, intraocular, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intracranial, intracranial, intracranial .

[0294] The compositions may be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure, suitable for stable storage at high drug concentration. Sterile, injectable solutions may be prepared by incorporating the antibody (or antibody fragment) in the required amount, in a suitable solvent, with one or a combination of the ingredients listed above, as appropriate, followed by filtration sterilization. Generally, dispersions are obtained by incorporating the antibody or fragment described herein into a sterile transducer that contains a basic dispersion medium and other required components from those specified above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and lyophilization, which result in the antibody powder, or antigen-binding fragment thereof described herein plus any additional desired components (see below) from the previously sterilized method filtration of its solution. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, the maintenance of the correct particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition a reagent that delays absorption, such as monostearate salts and gelatin.

[0295] Anti-C5a antibodies, or antigen-binding fragments thereof, described herein can also be formulated in immunoliposomal compositions. Liposomes containing the antibody can be obtained by methods known in the art such as, e.g., the methods described in Epstein et al. (1985) Proc Natl Acad Sci USA 82: 3688; Hwang et al. (1980) Proc Natl Acad Sci USA 77: 4030; and US patents no. 4,485,045 and 4,544,545. Liposomes with extended circulation time are disclosed in, e.g. U.S. Patent No. 5,013,556.

[0296] In some aspects of the disclosure, the antibody or antigen-binding fragment thereof can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as poly (ethylene-co-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid). Many methods for preparing such formulations are known in the art. See. e.g., JR Robinson (1978) "Sustained and Controlled Release Drug Delivery Systems," Marcel Dekker, Inc., New York.

[0297] In some aspects of the disclosure, the antibody or antigen-binding fragment described herein can be formulated in a composition suitable for intrapulmonary administration (e.g., for administration by a nebulizer; see below) to a mammal such as a human. Methods for making such compositions are known in the art and described in, e.g., US Patent Application Publication. 20080202513; U.S. Patent No. 7,112,341 and 6,019,968; and publication of PCT Patent Application Nos. WO 00/061178 and WO 06/122257, the disclosures of which are incorporated herein by reference in their entirety. Formulations for dry powder inhalers and suitable systems for administering formulations are described in, e.g., US Patent Application Publication No. 20070235029, PCT Publication No. WO 00/69887; and U.S. Patent No. 5,997,848.

[0298] In some aspects of the disclosure, the anti-C5a antibody or antigen-binding fragment thereof described herein can be formulated in a composition suitable for ocular delivery. In some aspects of the disclosure, one or more anti-C5a antibodies (or antigen-binding fragments thereof) described herein can be administered topically, for example, by topical application or intravitreal injection. For example, in some embodiments, anti-C5a antibodies can be formulated for administration with eye drops.

[0299] A therapeutic preparation for treating an eye may contain one or more anti-C5a antibodies at a concentration of from about 0.01 to about 1% by weight, preferably from about 0.05 to about 0.5% in a pharmaceutically acceptable solution, suspension or ointment. The preparation will preferably be in the form of a sterile aqueous solution containing, e.g. additional ingredients such as, but not limited to, preservatives, buffers, tonics, antioxidants and stabilizers, nonionic wetting or clarity enhancing agents, and viscosity enhancing agents.

[0300] Suitable preservatives for use in such a solution include benzalkonium chloride, benzethonium chloride, chlorobutanol, thimerosal and the like. Suitable buffers include, for example, boric acid, sodium and potassium bicarbonate, sodium and potassium borates, sodium and potassium carbonate, sodium acetate, and sodium diphosphate, in an amount sufficient to maintain the pH between about pH 6 and pH 8, and preferably, between about pH 7 and pH 7.5. Suitable tonicity agents are dextran 40, dextran 70, dextrose, glycerin, potassium chlorides, propylene glycol and sodium chloride.

[0301] Suitable antioxidants and stabilizers include sodium bisulfite, sodium metabisulfite, sodium thiosulfite and thiourea. Suitable wetting and clarity enhancing agents include polysorbate 80, polysorbate 20, poloxamer 282 and tyloxapol. Suitable viscosity enhancing agents include dextran 40, dextran 70, gelatin, glycerin, hydroxyethyl cellulose, hydroxymethylpropyl cellulose, lanolin, methyl cellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone and carboxymethyl cellulose. The preparation may be administered topically to the eye of a subject in need of treatment (e.g., subject affected by AMD) by conventional means, e.g. in the form of drops, or by rinsing the eye in a therapeutic solution containing one or more anti-C5a antibodies.

[0302] In addition, a number of devices have been developed for introducing drugs into the vitreous cavity of the eye. For example, US Patent Application Publication No. 20020026176 describes a cork containing a pharmaceutical that can be inserted through the sclera so that it protrudes deep into the vitreous for delivery of the pharmaceutical into the vitreous. In a further example, US Patent No. 5,443,505 describes an implantable device for insertion into the supravasal space or into the non-vascularized area for the purposes of sustained release of the drug into the eye. US Patent Nos. 5,773,019 and 6,001,386 disclose, each, an implantable drug delivery device attached to the surface of the sclera of the eye. The device has an inner core containing an effective amount of low solubility agent coated with a non-erosionable polymer that is permeable to the low solubility agent. During operation, the low solubility agent penetrates through the bioerodible polymer sheath for sustained release from the device. Additional methods and devices (e.g. a patch extending through the sclera and delivery through contact lenses) for delivery of the therapeutic to the eye are described in, e.g., Ambati and Adamis (2002) Prog Retin Eye Res 21 (2): 145151; Ranta and Urtti (2006) Adv Drug Delivery Rev 58 (11): 1164-1181; Barocas and Balachandran (2008) Expert Opin Drug Delivery 5 (1): 1-10 (10); Gulsen and Chauhan (2004) Invest Opthalmol Vis Sci 45: 2342-2347; Kim et al. (2007) Ophthalmic Res 39: 244-254; and PCT Publication No. WO 04/073551.

[0303] Nucleic acids encoding the antibody (or antigen-binding fragment thereof) can be incorporated into a gene construct for use as part of a gene therapy protocol to provide nucleic acids that can be used to express and produce agents within a cell (see below) . Expression constructs of such components can be administered in any therapeutically effective carrier, e.g. any formulation or composition capable of efficiently delivering a gene component to cells in vivo. Approaches include the introduction of the gene in question in viral vectors, including recombinant retroviruses, adenovirus, adeno-associated virus, lentivirus, and herpes simplex virus-1 (HSV-1), or recombinant bacterial or eukaryotic plasmids. Viral vectors can transfect cells directly; plasmid DNA can be delivered by, for example, cationic liposomes (lipofectins) or derivatized (e.g. antibody conjugates), polylysine conjugates, gramicidin S, artificial viral envelopes or other such intracellular carriers as well as direct injection of the gene construct or CaPO4 precipitation ( see e.g. WO04 / 060407) carried out in vivo. (See. also, "Ex vivo Approaches," below.) Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are known to those skilled in the art (see, e.g., Eglitis et al. (1985) Science 230: 1395-1398; Danos and Mulligan (1988) Proc Natl Acad Sci USA 85: 6460-6464; Wilson et al. (1988) Proc Natl Acad Sci USA 85: 3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87: 6141-6145; Huber et al. (1991) Proc Natl Acad Sci USA 88: 8039-8043; Ferry et al. (1991) Proc Natl Acad Sci USA 88: 8377-8381; Chowdhury et al. (1991) Science 254: 1802-1805; van Beusechem et al. (1992) Proc Natl Acad Sci USA 89: 7640-7644; Kay et al. (1992) Human Gene Therapy 3: 641-647; Dai et al. (1992) Proc Natl Acad Sci USA 89: 10892-10895; Hwu et al. (1993) J Immunol. 150: 4104-4115; US patents no. 4,868,116 and 4,980,286; PCT publications No. WO89 / 07136, WO89 / 02468, WO89 / 05345 and WO92 / 07573).

Another viral gene delivery system uses adenovirus-based vectors (see, e.g., Berkner et al. (1988) BioTechniques 6: 616; Rosenfeld et al. (1991) Science 252: 431-434; and Rosenfeld et al. (1992) Cell 68: 143-155). Suitable adenoviral vectors derived from the adenovirus strain Ad type dl324 or other adenovirus strains (e.g. Ad2, Ad3, Ad7, etc.) are known to those skilled in the art. Still another viral vector system useful for delivering a gene to a subject is a virus

100 associated with adenoviruses (AAV). See. e.g. Flotte et al. (1992) Am J Respir Cell Mol Biol 7: 349-356; Samulski et al. (1989) J Virol 63: 3822-3828; and McLaughlin et al. (1989) J Virol 62: 1963-1973.

[0304] In some aspects of the disclosure, the antibody, or antigen-binding fragment thereof described herein can be formulated with one or more additional active ingredients useful for treating or preventing a complement related disorder in an individual. Additional agents for treating a complement-related disorder in a subject will vary depending on the particular disorder being treated, but may include, but are not limited to, an antihypertensive drug (e.g., an angiotensin converting enzyme inhibitor), an anticoagulant, a corticosteroid (e.g., prednisone ), or an immunosuppressant (e.g. vincristine or cyclosporin A). Examples of anticoagulants include, e.g. warfarin (Coumadin), heparin, fenindione, fondaparinux, idraparinux, and thrombin inhibitors (e.g. argatroban, lepirudin, bivalirudin, or dabigatran). The antibody or fragment thereof described herein may also be formulated with a fibrinolytic agent (e.g. ancrod, ε-aminocaproic acid, anti-plasmin-a1, prostacyclin and defibrotide) for complement mediated treatment of the disorder. In some aspects of the disclosure, the antibody may be formulated with a lipid lowering agent, such as a hydroxymethylglutaryl-CoA reductase inhibitor. In some aspects of the disclosure, the antibody may be formulated with, or for use with, an anti-CD20 agent such as rituximab (Rituxan ™; Biogen Idec, Cambridge, MA). In some aspects of the disclosure, e.g. for the treatment of RA, the antibody or antigen-binding fragment thereof can be formulated with one or both of infliximab (Remicade®; Ceptocor, Inc.) and methotrexate (Rheumatrex®, Trexall®). In some aspects of the disclosure, the antibody or antigen-binding fragment thereof described herein can be formulated with a nonsteroidal anti-inflammatory drug (NSAID). Many different NSAIDs are available, some over the counter, including ibuprofen (Advil®, Motrin®, Nuprin®) and naproxen (Alleve®), and many more are available on prescription, including meloxicam (Mobic®), etodolac (Lodine® ), nabumetone (Relafen®), sulindac (Clinoril®), tolementin (Tolectin®), choline magnesium salicylate (Trilasate®), diclofenac (Cataflam®, Voltaren®, Arthrotec®), diflusinal (Dolobid®), indomethacin (Indocin ®), ketoprofen (Orudis®, Oruvail®), oxaprozin (Daypro®), and piroxic (Feldene®). In some aspects of the disclosure, the antibody or fragment thereof may be formulated for use with an antihypertensive agent, anticonvulsant (e.g., magnesium sulfate), or anticoagulant. Antihypertensives include, e.g., labetalol, hydralazine, nifedipine, calcium channel blockers, nitroglycerin, or sodium nitroprusside. See. e.g. Mihu et al. (2007) J Gasrointestin LiverDis 16 (4): 419-424. Anticoagulants include, e.g. heparin, antithrombin, prostacyclin, or a low dose of aspirin.

[0305] In some aspects of the disclosure, the antibody or antigen-binding fragment thereof can be formulated for administration to a subject along with intravenous gamma globulin (IVIG) therapy, plasmapheresis, or plasma exchange. In some aspects of the disclosure

101 the anti-C5a antibody or antigen-binding fragment thereof can be formulated for use before, during or after kidney transplantation.

[0306] When the antibody or antigen-binding fragment thereof is to be used in combination with a second active agent, the agents may be formulated separately or in combination. For example, the relevant pharmaceutical compositions may be mixed, e.g., just before administration, and administered together, or may be administered separately, e.g., at the same time or at different times (see below).

[0307] As described above, the composition may be formulated to contain a therapeutically effective amount of an anti-C5a antibody or antigen binding fragment thereof described herein. In some aspects of the disclosure, the composition may be formulated to contain a sub-therapeutic amount of the antibody (or fragment) and a sub-therapeutic amount of one or more additional active ingredients, such that the total ingredients are therapeutically effective to treat or prevent a disorder associated with complement system. Methods for determining a therapeutically effective dose of an agent such as a therapeutic antibody are known in the art and described herein.

Uses [0308] Antibodies, antigen-binding fragments thereof, conjugates and compositions of any of the above can be used in a variety of diagnostic and therapeutic applications. For example, detectably labeled anti-C5a antibodies (e.g., anti-human C5a antibodies or anti-mouse C5a antibodies) can be used in assays to detect the presence or amount of C5a present in a biological sample. Determination of the amount of C5a in the sample, e.g. the patient's blood sample, it may be useful to evaluate the level of complement activation in the sample. Suitable methods for using antibodies in diagnostic assays are known in the art and include, but are not limited to, ELISA techniques, fluorescence resonance energy transfer applications, Western hybridization, and point hybridization. See. e.g. Sambrook et al., supra and Ausubel et al., supra.

[0309] In some aspects of the disclosure, the antibodies and antigen-binding fragments described herein can be used as positive controls in assays designed to identify additional new compounds for the treatment of complement regulated disorders. For example, an anti-C5a antibody that inhibits C5a activity can be used as a positive control in the assay to identify additional compounds (e.g. small molecules, aptamers or antibodies) that inhibit C5a or C5a-dependent signaling via the C5a receptor.

[0310] In some aspects of the disclosure, cross-reactive anti-C5a antibodies or antigen-binding fragments thereof (e.g., cross-reactive with human C5a and, e.g., C5a cynomolgus macaque) described herein can be used for preclinical testing in non-human mammals, e.g. pharmacokinetic or pharmacodynamic studies in non-human primates. Accordingly, a researcher wishing to evaluate the efficacy of an anti-C5a antibody in the treatment of a related related disorder

102 with a complement system (e.g. RA or sepsis) can use the cross-reactive anti-C5a antibody described herein in the respective non-human primate model of the disease. If the researcher, for example, establishes the effectiveness of the antibody in a non-human primate model, these results may provide sufficient proof-of-concept evidence to apply for authorization to use the antibody in human therapy. Alternatively, or in addition, the investigator may administer a cross-reactive antibody to a non-human primate for analysis, e.g., antibody clearance and / or pharmacodynamic properties. Based on such studies, using a cross-reactive antibody, the researcher can more accurately determine the dose required to treat the disease in humans.

[0311] In some aspects of the disclosure, anti-mouse C5a antibodies or antigen-binding fragments thereof described herein, as well as antibodies that cross-react with human and mouse C5a, can be used as antibody substitutes in mouse models of human disease. This may be particularly useful when humanized anti-human C5a antibody does not cross-react with mouse C5a and / or when there is a likelihood of eliciting a response to an anti-human antibody in a mouse that is administered the humanized antibody. Accordingly, a researcher wishing to analyze the effect of an anti-C5a antibody on the treatment of a disease (e.g., ischemia-reperfusion injury) may use the anti-mouse C5a antibody described herein in the respective mouse disease model. If the researcher is able to establish efficacy in a mouse disease model using anti-mouse C5a antibody, the results can be used to develop the so-called proof-of-concept for the use of anti-human C5a antibody in the treatment of human disease. Working examples disclose an exemplary study using an anti-mouse C5a antibody replacement in a mouse RA model, establishing a proof-of-concept for the use of anti-human C5a antibody for the treatment of RA in humans.

[0312] The anti-C5a antibodies described herein can also be used in methods for purifying C5a from a sample (e.g., biological sample). In some aspects of the disclosure, the anti-C5a antibody can be immobilized on a solid support using methods well known in the art. The sample containing the purification antigen, in this case C5a, is contacted with the antibody on a solid support under conditions and for a period of time sufficient to allow the antigen to bind to the antibody. The solid support is then washed one or more times with an appropriate buffer to remove unbound material. The solid support can then be contacted with a second buffer, resulting in the release of the antigen from the antibody. The released antigen is then collected and characterized (e.g. in terms of purity and activity) using well known methods in the art.

[0313] Anti-C5a antibodies and antigen-binding fragments thereof described herein can also be used in therapeutic methods as detailed below.

Methods of treatment [0314] The compositions described above are useful in, inter alia, methods of treating or preventing multiple disorders associated with the complement system in a subject.

103

The compositions can be administered to an individual, e.g. a human, using a variety of methods that depend in part on the route of administration. The route may mean, e.g. intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal injection (IP) or intramuscular injection (IM).

[0315] Administration can be accomplished by, e.g., local infusion, injection, or by implant. The implant may be porous, non-porous or a gelatinous material, including membranes, such as sialastic membranes, or fibers. The implant may be configured to extend or periodically release the composition to the subject. See. e.g. US Patent Application Publication No. 20080241223; U.S. Patent Nos. 5,501,856; 4,863,457; and 3.710,795; EP488401; and EP 430539. The composition can be delivered to an individual by means of an implantable device based on, e.g., diffusion, erodable or convective systems, e.g. osmotic pumps, biodegradable implants, electrode diffusion systems, electroosmosis systems, steam pressure pumps, electrolytic pumps, turbulent pumps, piezoelectric pumps , erosion-based systems or electromechanical systems.

[0316] In some aspects of the disclosure, an anti-C5a antibody or antigen-binding fragment thereof is therapeutically delivered to a subject by topical administration. As used herein, "topical administration" or "topical delivery" refers to delivery that is not based on transporting the composition or agent to the intended target tissue or site via the vascular system. For example, the composition may be delivered by injection or implantation of the composition or agent, or by injection or implantation of a device containing the composition or agent. After topical administration, adjacent to the target tissue or site, the composition or agent, or one or more components thereof, can diffuse into the intended target tissue or site.

[0317] In some aspects of the disclosure, an anti-C5a antibody or antigen-binding fragment thereof may be locally administered to the joint (e.g., articulation). For example, in embodiments where the complement related disorder is arthritis, the complement inhibitor may be administered directly into the joint (e.g., into the joint space) or adjacent to the joint. Examples of joints with intra-articular local delivery of an anti-C5a antibody or antigen-binding fragment thereof are, e.g., hip, knee, elbow, wrist, sternoclavicular joint, temporomandibular joint, carpal joint, interstitial joint, ankle joint, and any other subject matter joints under arthritis. An anti-C5a antibody or antigen-binding fragment thereof may also be administered intrathecally, such as, e.g. shoulder, double-headed radial, elbow-radial, deltoid, patellar, sciatic, and all other wallets known in the art in medicine.

[0318] In some aspects of the disclosure, an anti-C5a antibody or antigen-binding fragment thereof can be topically administered to the eye. As used herein, the term "eye" refers to all and all anatomical tissues and structures associated with the eye. The eye has a wall consisting of three separate layers: the outer sclera, the middle layer of the choroid,

104 and internal volleyball. The chamber behind the lens is filled with a jelly-like fluid called the vitreous. At the back of the eye is the retina, which detects light. The cornea is an optically transparent tissue that transmits images to the back of the eye. The cornea includes one pathway for the penetration of drugs into the eye. Other anatomical tissue structures associated with the eye include a tear removal system that includes a secretory system, a distribution system, and an excretory system. The secretory system contains secretory elements that are stimulated by blinking and temperature, due to the evaporation of tears, and reflex secretory elements that have access to parasympathetic efferent nerves and release tears in response to physical or emotional stimulation. The distribution system covers the eyelids and meniscus tears around the edge of the eyelid of the open eye, which spreads the tears over the surface of the eyeball by blinking, hence the appearance of dry areas is reduced.

[0319] In some aspects of the disclosure, an anti-C5a antibody or antigen binding fragment thereof is administered to the posterior chamber of the eye. In some aspects of the disclosure, an anti-C5a antibody or antigen binding fragment thereof is administered to the vitreous. In some aspects of the disclosure, the anti-C5a antibody or antigen-binding fragment thereof is administered transdural.

[0320] In some aspects of the disclosure, eg, in aspects for treating or preventing complement-related pulmonary disorders, such as COPD or asthma, the anti-C5a antibody or antigen binding fragment thereof described herein can also be administered to a subject to the lung. Pulmonary drug delivery can be achieved by inhalation, and administration by inhalation as described herein can be oral and / or nasal. Examples of pharmaceutical devices for pulmonary delivery include pressurized metered dose inhalers, dry powder inhalers (DPIs) and nebulizers. For example, the anti-C5a antibody or antigen-binding fragment thereof can be administered to the lung of a subject using a dry powder inhaler. These inhalers are propellant-free devices that deliver dispersible and stable dry powder formulations to the lungs. Dry powder inhalers are well known in the art of medicine and include, but are not limited to: TurboHaler® (AstraZeneca; London, England) AIR® inhaler (Alkermes®; Cambridge, Massachusetts); Rotahaler® (GlaxoSmithKline; London, England); and Eclipse ™ (Sanofi-Aventis; Paris, France). See. also, e.g. PCT publications WO 04/026380, WO 04/024156, and WO 01/78693. DPI devices were used to deliver polypeptides such as insulin and growth hormone to the lungs. In some aspects of the disclosure, the anti-C5a antibody or antigen-binding fragment thereof can be administered intrapulmonary by means of a pressurized metered dose inhaler. These inhalers are based on propellant gas to deliver a small dose of the compound to the lungs. Examples of compounds administered with pressurized metered dose inhalers include, e.g., Astovent® (Boehringer-Ingelheim; Ridgefield, Connecticut) and Flovent® (GlaxoSmithKline). See. also, e.g. U.S. Patent Nos. 6,170,717; 5,447,150; and 6,095,141.

[0321] In some aspects of the disclosure, an anti-C5a antibody or antigen binding fragment thereof can be administered to the lungs of a subject by means of a nebulizer. Nebulizers use

105 compressed air to deliver the compound in the form of a liquefied aerosol or mist. A nebulizer may be, e.g., a jet nebulizer (e.g., air or liquid stream nebulizers) or an ultrasonic nebulizer. Additional devices and methods of intrapulmonary administration are given in, e.g., US Patent Application Publication Nos. 20050271660 and 20090110679.

[0322] In some aspects of the disclosure, the antibodies or antigen-binding fragments thereof provided herein are present in unit dosage forms, which may in particular be suitable for self administration. The formulated product of the disclosure can be enclosed within a container, typically, for example, a vial, cartridge, pre-filled syringe or disposable pen. A dispenser, such as the dispensing device described in US Patent No. 6,302,855, may also be used, for example, with the injection system of the disclosure.

[0323] The injection system of the disclosure may use a delivery pen as described in US Patent No. 5,308,341. Pen devices, most often used for self-delivery of insulin by diabetic patients, are well known in the art. Such devices may contain at least one injection needle (e.g. a 31 gauge needle (about 5 to 8 mm long), are typically pre-filled with one or more therapeutic unit doses of the therapeutic solution, and are useful for quickly delivering a solution to a subject, as painlessly as possible.

[0324] One pen for drug delivery includes a vial holder in which an insulin or other drug vial can be placed. The vial holder is an elongated, generally tubular structure with proximal and distal ends. The distal end of the vial holder includes fastening means for accommodating the double-ended needle cannula. The proximal end also includes fastening means for accommodating the pen body, which includes a controller and a dose determining device. Containing a one-off drug (e.g. solution with a high concentration of anti-C5a antibody or antigen-binding fragment thereof) a vial for use with a prior art vial holder has a distal end having a pierceable elastomeric septum which can be pierced with one end of a double-ended needle cannula. The proximal end of this vial includes a stopper, slidably positioned in fluid, in close contact with the cylindrical wall of the vial. This drug delivery pen is used by inserting the medicine vial into the vial holder. The pen body then contacts the proximal end of the vial holder. The pen body includes a dose setting device for designing a drug dose to be delivered through a pen and a control device for displacing the stopper in the distal direction along the vial to a distance corresponding to the selected dose. The pen user attaches the double-ended needle cannula to the distal end of the vial holder such that the proximal point of the needle cannula pierces the septum of the vial. The patient then selects a dose, and manipulates the pen to move the cork distally to deliver the selected dose. The dose selector returns to the zero point after the selected dose has been injected. The patient then removes and discards the needle cannula, and stores the drug delivery pen in a convenient location for the next,

106 required drug administration. The medicine in the vial will be depleted after several such administrations. The patient then separates the vial holder from the pen body. The empty vial can then be removed and discarded. A new vial can be inserted into the vial holder, and the vial holder and pen body can be assembled and used as explained above. Accordingly, the drug delivery pen generally has a control mechanism for accurate dosing and ease of use.

[0325] A dosing mechanism, such as a rotary knob, allows the user to accurately adjust the amount of drug to be injected through the pen from a pre-prepared drug vial. To inject a dose, the user inserts the needle under the skin and presses the dial once as much as possible. The pen may be a complete mechanical device or it may be connected to an electrical circuit to accurately configure and / or indicate the dosage of the drug being injected into the user. See. U.S. Patent No. 6,192,891.

[0326] In some aspects of the disclosure, the pen device needle is disposable, and the kits include one or more disposable replacement needles. Pen devices suitable for delivering any of said antibodies or antigen-binding fragments thereof are also described in, e.g., US Patent Nos. 6,277,099;

6,200,296; and 6,146,361.

[0327] A micro needle based pen device is described in, e.g., US Patent No. 7,556,615.

[0328] See also the Precision Pen Injector (PPI), Molly ™, manufactured by Scandinavian Health Ltd.

[0329] The disclosure also presents controlled release or sustained release formulations suitable for chronic and / or self administration of a drug, such as an anti-C5a antibody or antigen binding fragment thereof described herein. Different formulations can be administered to a patient in need of treatment, with a bolus drug or by continuous infusion over a period of time.

[0330] In some aspects of the disclosure, the highly concentrated anti-C5a antibody (or antigen-binding fragment thereof) described herein is formulated for administration in a delayed release, sustained release, timed release, controlled release, or continuous release form. In some aspects of the disclosure, tissue embedded depot formulations are used to administer the antibody to a subject in need thereof. In this way, the antibody is formulated with one or more carriers providing a gradual release of the active agent over a period of many hours or days. Such formulations are often based on a degradable matrix that gradually disperses in the body, releasing the active agent.

[0331] In some aspects of the disclosure, the C5a binding fragment (e.g., single chain antibody, diabody or Fab 'fragment) of the anti-C5a antibody described herein is administered by intrapulmonary administration to a subject in need thereof. For example, an antibody (e.g. human) form may be provided to the subject

107 single chain for any of the anti-C5a antibodies described herein, by means of a nebulizer or inhaler, affected by a complement-related pulmonary disorder such as asthma or COPD.

[0332] The appropriate dose of the antibody or fragment thereof described herein, which dose is capable of treating or preventing a complement related disorder in a subject, may depend on a number of factors, including, e.g., the age, sex and weight of the subject to be subjected. treatment, and the specific inhibitor compounds used. For example, different doses of complete anti-C5a antibody may be required to treat a subject with RA, compared to the dose of C5a binding Fab 'antibody fragment required to treat the same subject. Other factors affecting the dose administered to the subject include, e.g., the type or severity of complement-mediated disorder. For example, a subject having RA may need to be administered a different dose of anti-C5a antibody than the subject with AMD. Other factors may include, e.g. other health disorders, currently or previously affecting the subject, the subject's overall health, propensity, diet, time of administration, secretion rate, drug combination, and any other additional therapies that are administered to the subject. It should also be understood that the specific dosage and treatment regimen for any particular subject will also depend on the assessment of the treatment by the healthcare professional (e.g., physician or nurse).

[0333] The antibody described herein may be administered, at a fixed dose, or at a dose in milligrams per kilogram (mg / kg). In some aspects of the disclosure, the dose may also be selected to reduce or eliminate the production of antibodies or other host immune responses against one or more active antibodies in the composition. In the complete absence of limiting nature, exemplary dosages of an antibody such as anti-C5a antibody include, e.g., 11,000 μβ / kg, 1-100 μβ / kg, 0.5-50 μg / kg, 0.1-100 μg / kg, 0.5-25 μg / kg, 1-20 μg / kg, and ΙΙΟ μg / kg, 1-100 mg / kg, 0.5-50 mg / kg, 0.1-100 mg / kg, 0.5 -25 mg / kg, 1-20 mg / kg, 0.100 mg / kg to 1 mg / kg, and 1-10 mg / kg. Exemplary dosages of the antibody or antigen binding fragment thereof described herein include, but are not limited to, 0.1 μg / kg, 0.5 μg / kg, 1.0 μg / kg, 2.0 μg / kg, 4 μg / kg and μg / kg, 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 4 mg / kg, 8 mg / kg and 20 mg / kg.

[0334] The pharmaceutical composition may comprise a therapeutically effective amount of an anti-C5a antibody or antigen-binding fragment thereof described herein. Such effective amounts can be readily determined by one of skill in the art based, in part, on the effect of the administered antibody, or on the combinatorial effect of the antibody and one or more additional active ingredients when more than one agent was used. The therapeutically effective amount of an antibody or fragment thereof described herein may also vary, depending on factors such as the condition, age, sex, and body weight of the individual, and the ability of the antibody (and one or more additional active ingredients) to induce the desired a person’s response, e.g., improvement of at least one fitness parameter, e.g. improvement of at least one

108 complement regulated disorder. For example, a therapeutically effective amount of an anti-C5a antibody may inhibit (reduce the severity or eliminate the occurrence) and / or prevent a specific disorder, and / or any of the symptoms of a particular disorder, known in the art or described herein. A therapeutically effective amount is also the one for which any toxic or harmful effects of the composition are outweighed by the therapeutically beneficial effects.

[0335] Appropriate dosages for humans with any of the antibodies or fragments thereof described herein can be further evaluated, e.g., in Phase I studies: dose escalation. See. e.g. van Gurp et al. (2008) Am J Transplantation 8 (8): 1711-1718; Hanouska et al. (2007) Clin Cancer Res 13 (2, part 1): 523-531; and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50 (10): 3499-3500.

[0336] The terms "therapeutically effective amount" or "therapeutically effective dose" or similar terms used herein are intended to mean the amount of the agent (e.g., anti-C5a antibody or antigen-binding fragment thereof) that will elicit the desired biological or medical response (e.g. (improvement in one or more symptoms of a complement related disorder). In some aspects of the disclosure, the composition described herein comprises a therapeutically effective amount of an antibody, or antigen-binding fragment thereof, that specifically binds to a neo-epitope present in C5a. In aspects of the disclosure, the composition comprises any antibodies and or antigen-binding fragments thereof described herein and one or more (e.g. two, three, four, five, six, seven, eight, nine, 10, or 11 or more) additional therapeutics such that the composition as a whole is therapeutically effective. For example, the composition may comprise the anti-C5a antibody described herein and an immunosuppressant agent, wherein the antibody and agent are each at such a concentration that when combined they are therapeutically effective for treating or preventing complement related disorders (e.g. the complement related inflammatory disorder (COPD, asthma, sepsis or RA) in the subject.

[0337] The toxicity and therapeutic efficacy of such compositions can be determined by known pharmaceutical procedures, in cell cultures or in experimental animals (e.g., animal models of any of the complement-regulated disorders described herein). The use of anti-C5a antibody in the RA animal model is detailed in working examples. These procedures can be used, e.g. to determine LD50 (lethal dose for 50% of the population) and ED50 (therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. An antibody or antigen-binding fragment thereof that exhibits a high therapeutic index is preferred. While compositions that exhibit toxic side effects can be used, care should be taken to design a delivery system that directs such compounds to localize the affected tissue to minimize potential damage to normal cells and thereby reduce side effects.

109 [0338] Data obtained from cell culture assays or animal studies can be used in formulating a dosage range for human applications. The dosage of such antibodies or antigen-binding fragments thereof is generally within a range of concentrations of circulating antibodies or fragments that include ED50 with little or no toxicity. Dosage may vary within this range, the range depending on the dosage form used and the route of administration used. For the anti-C5a antibodies described herein, the therapeutically effective dose can be estimated initially from cell culture assays. The dose may be formulated in animal models to achieve a range of circulating plasma concentrations that include IC50 (i.e. antibody concentration for which half maximal inhibition of symptoms is achieved) as found in cell culture assays. This information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography. In some aspects of the disclosure, e.g. when topical administration is desired (e.g. eye or joint), cell culture or animal models can be used to determine the dose required to achieve therapeutically effective concentrations within the local site.

[0339] In some aspects of the disclosure, the methods can be carried out in conjunction with other therapies for complement-related disorders. For example, the composition may be administered to a subject at the same time, before or after, plasmapheresis, IVIG therapy or plasma exchange. See. e.g. Appel et al. (2005) J Am Soc Nephrol 16: 1392-1404. In some aspects of the disclosure, the composition may be administered to the subject at the same time, before or after kidney transplantation.

[0340] A "subject," as used herein, may mean a mammal. For example, the subject may be a human, non-human primate (e.g., monkey, baboon or chimpanzee), horse, cow, pig, sheep, goat, dog, cat, rabbit, guinea pig, gerbil, hamster, rat or mouse. In some embodiments, the subject is an infant (e.g., a human infant).

[0341] As used herein, an individual "in need of prevention," "in need of treatment," or "in need of it" refers to one that, as judged by the appropriate health care professional (e.g., physician, nurse, or a trainee nurse for humans; a veterinarian for mammals) non-human), it will reasonably benefit from the treatment provided (such as treatment with a composition comprising an anti-C5a antibody).

[0342] The term "prevention" is recognized in the art, and when used to refer to a disease, is well understood in the art, and includes administering a composition that reduces the incidence, or delays the onset of symptoms of a disease in an individual relative to an individual, who did not receive the composition. Thus, the prevention of a complement related disorder such as asthma includes, for example, a reduction in the extent or incidence of coughing, wheezing or chest pain in a population of patients receiving prophylactic treatment for an untreated control population, and / or delaying the onset of cough or

110 wheezing in the treated population relative to the untreated control population, e.g., statistically and / or clinically significant amount.

[0343] As described above, the antibodies and biologically active fragments described herein can be used to treat a number of complement-related disorders, such as, but not limited to: rheumatoid arthritis (RA); lupus nephropathy; ischemia-reperfusion injury; atypical haemolytic uraemic syndrome (aHUS); typical or infectious haemolytic uraemic syndrome (tHUS); thick deposit disease (DDD); paroxysmal nocturnal hemoglobinuria (PNH); multiple sclerosis (MS); macular degeneration (e.g. age-related macular degeneration (AMD)); hemolysis, syndrome with elevated levels of liver enzymes and low levels of platelets (HELLP); sepsis; dermatomyositis; diabetic retinopathy; thrombotic thrombocytopenic purpura (TTP); spontaneous miscarriages; silent immunodeficiency (pauci-immuno); bullous epidermal separation; recurrent miscarriages; multiple sclerosis (MS); and traumatic brain injury. See. for example. Holers (2008) Immunological Reviews 223: 300-316 and Holers and Thurman (2004) Molecular Immunology 41: 147-152. In some aspects of the disclosure, complement-mediated disorder is complement-mediated vascular disorder, such as, but not limited to, cardiovascular disorder, myocarditis, cerebrovascular disorder, peripheral (e.g. musculoskeletal) vascular disorder, renal vascular disorder, mesenteric / intestinal vascular disorder, revascularization for transplantation and / or reimplanted organs, vasculitis, IgA-associated vasculitis, vasculitis associated with systemic lupus erythematosus, vasculitis associated with rheumatoid arthritis , immune complex vasculitis, Takayasu's disease, capillary leak syndrome, dilated cardiomyopathy, diabetic angiopathy, chest / abdominal aortic aneurysm, Kawasaki disease (arteritis), gas venous embolism (VGE), and stent placement restoration, rotational aterectomy and percutaneous coronary angioplasty (PTCA). (See e.g. U.S. Patent Application Publication No. 20070172483.) In some embodiments, the complement-related disorder is myasthenia gravis, cold agglutinin disease (CAD), paroxysmal cold hemoglobinuria (PCH), dermatomyositis, scleroderma, autoimmune hemolytic anemia with antibodies present increased temperature, Graves disease, Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA), idiopathic thrombocytopenic purpura (ITP), Goodpasture syndrome, antiphospholipid syndrome (APS), Degos disease and catastrophic APS (CAPS).

[0344] In some aspects of the disclosure, the anti-C5a antibody or antigen-binding fragment thereof described herein, alone or in combination with a second anti-inflammatory agent, may be used to treat an inflammatory disorder such as, but not limited to, RA (above), inflammatory bowel disease, sepsis (above), septic shock, acute lung injury, disseminated intravascular coagulation (DIC), or Crohn's disease. In some aspects of the disclosure, the second anti-inflammatory agent may be

111 selected from the group consisting of NSAIDs, corticosteroids, methotrexate, hydroxychloroquine, anti-TNF agents such as etanercept and infliximab, a B cell depressant such as rituximab, an interleukin-1 antagonist or a costimulatory T cell blocking agent such as abatacept .

[0345] In some aspects of the disclosure, a complement related disorder is a complement related neurological disorder such as, but not limited to, amyotrophic lateral sclerosis (ALS), brain injury, Alzheimer's disease, and chronic inflammatory demyelinating neuropathy.

[0346] Complement related disorder also includes complement-related pulmonary disorders such as, but not limited to, asthma, bronchitis, chronic obstructive pulmonary disease (COPD), interstitial lung disease, α-1 anti-trypsin deficiency, emphysema, obstructive inflammation. bronchioles, alveolitis, sarcoidosis, pulmonary fibrosis and collagen vascular disorders.

[0347] For complement-related hemolytic disorders, such as PNH, CAD, and PCH, a healthcare professional will recognize that the C5 C5b fragment (as a terminal complement complex) contributes significantly to the pathogenesis of these disorders. See. e.g. Kaplan (2002) Curr Opin Investig Drugs 3 (7): 1017-23; Hill (2005) Clin Adv Hematol Oncol 3 (11): 849-50; and Rother et al. (2007) Nature Biotechnology 25 (11): 1256-1488. Accordingly, a healthcare professional may choose to administer one or more of the anti-C5a antibodies described herein in conjunction with one or more additional treatments for a haemolytic disorder, such as a complement inhibitor that prevents the formation of the C5b-9 terminal complement complex. . In some aspects of the methods described herein, the complement related disorder is not complement related hemolytic disorder. In some aspects of the disclosure, an anti-C5a antibody or antigen-binding fragment thereof is administered to a subject to treat, prevent or ameliorate at least one symptom associated with an complement inflammatory response (e.g. an aspect of the complement-associated inflammatory response of a complement-related disorder) in a subject. For example, the anti-C5a antibody described herein can be used to treat, prevent and / or ameliorate one or more symptoms associated with complement-associated inflammatory response, such as transplant rejection / graft versus host disease (GVHD), reperfusion injury (e.g. . after extracorporeal circulation or tissue transplantation), and tissue damage after other forms of traumatic injury, such as a burn (e.g. severe burn), blunt trauma, back injury or frostbite. See. e.g. Park et al. (1999) Anesth Analg 99 (1): 42-48; Tofukuji et al. (1998) J Thorac Cardiovasc Surg 116 (6): 1060-1068; Schmid et al. (1997) Shock 8 (2): 119124; and Bless et al. (1999) Am J Physiol 276 (1): L57-L63.

[0348] In some aspects of the disclosure, the anti-C5a antibody or antigen-binding fragment thereof described herein can be administered to a subject as monotherapy. Alternatively, as described above, the antibody or fragment thereof may be administered to the subject as a combination therapy with another treatment, e.g., another treatment for the disorder

112 complement-related or complement-related inflammatory response. For example, combination therapy may include administering to the subject (e.g., a human patient) one or more additional agents (e.g., anticoagulants, antihypertensives, or anti-inflammatory drugs (e.g., steroids)) that provide therapeutic benefit to the subject who suffers from, or is at risk of sepsis. In a further example, the combination therapy may comprise administering to the subject one or more additional agents (e.g., anti-IgE antibody, anti-IL-4 antibody, anti-IL-5 antibody or anti-histamine) that provide therapeutic benefit to the subject suffering from is at risk of, or suspected to have, a complement related pulmonary disorder such as COPD or asthma. In some aspects of the disclosure, an anti-C5a antibody and one or more additional active ingredients are administered at the same time. In other aspects of the disclosure, an anti-C5a antibody is administered temporarily first and one or more additional active ingredients are administered second. In some aspects of the disclosure, one or more additional active ingredients are administered first and anti-C5a antibody is administered second.

[0349] The anti-C5a antibody or antigen-binding fragment thereof described herein can replace or enhance prior or current therapy. For example, after treatment with an anti-C5a antibody or antigen-binding fragment thereof, administration of one or more additional active ingredients may be discontinued, e.g., administration at a lower level. In some aspects of the disclosure, administration of prior therapy may be maintained. In some aspects of the disclosure, prior therapy will be maintained until the level of anti-C5a antibody reaches a level sufficient to provide a therapeutic effect. Two therapies can be given in combination.

[0350] Monitoring a subject (e.g. a human patient) for improvement of a complement related disorder (e.g. sepsis, severe burns, RA, lupus nephropathy, Goodpasture syndrome or asthma) as defined herein means performing condition assessments an individual for a change in disease parameter, e.g., improvement in one or more symptoms of a given disorder. Symptoms of the complement-related disorder are well known in the art in medicine. In some aspects, disclosure is made at least at least one (1) hour, e.g. at least every 2, 4, 6, 8, 12, 24, or 48 hours, or at least every 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or more, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more after administration. The subject may be evaluated for one or more of the following periods: before treatment begins; during treatment; or after administration of one or more components of treatment. The assessment may include assessing the need for further treatment, e.g., whether the dosage, frequency of administration, or duration of treatment should be changed. It may also include an assessment of the need to add or omit a selected therapeutic modality,

113 e.g., adding or skipping any of the complement-related disorder therapies described herein.

Therapeutic and diagnostic kits [0351] The disclosure also provides therapeutic and diagnostic kits, including one or more anti-C5a antibodies, and / or antigen-binding fragments thereof, described herein. Therapeutic kits may contain, e.g., appropriate means for delivering the antibody or antigen-binding fragment to a subject. In some aspects of the disclosure, the agent is suitable for subcutaneously delivering an antibody or antigen-binding fragment thereof to a subject. The agent may mean, e.g., a syringe or osmotic pump. That is, the therapeutic kit described herein may include a syringe pre-filled with anti-C5a antibody or antigen-binding fragment thereof (e.g., a pen device containing the antibody or fragment) described herein, or the kit may include a pump (e.g. osmotic pump) and one or more disposable cassettes configured for use with the pump, cassettes pre-filled with anti-C5a antibody or antigen-binding fragment described herein (e.g., pre-filled with an aqueous solution containing anti-C5a antibody or antigen-binding fragment). In a further example, the kit may include a transdural or implantable delivery device (e.g. cork) which is pre-filled (or otherwise contains) a solution containing the anti-C5a antibody or antigen binding fragment thereof described herein.

[0352] In some aspects of the disclosure, the means for delivering an anti-C5a antibody or antigen-binding fragment thereof is a pen delivery device.

[0353] In some aspects of the disclosure, the agent is suitable for intrapulmonary delivery of an antibody or antigen-binding fragment thereof to a subject, e.g., for use in the treatment or prevention of complement-associated pulmonary disorder, such as, but not limited to COPD or asthma. Accordingly, the agent may be, e.g., an oral or nasal inhaler (see above). An inhaler can mean, e.g. pressure metered dose inhaler (MDI), dry powder inhaler (DPI), or nebulizer. Such a kit may also, optionally, contain instructions for administration (e.g., for self administration) of an anti-C5a antibody or antigen binding fragment thereof to a subject.

[0354] Therapeutic kits may contain, e.g., one or more additional active ingredients to treat or prevent complement related disorders and / or to alleviate their symptoms. For example, therapeutic kits designed for use in the treatment or prevention of complement-associated pulmonary disorder may include one or more additional active ingredients including, but not limited to, another therapeutic antibody (e.g. anti-IgE antibody, anti-IL-4 antibody or anti-IL-5 antibody), small molecule anti-IgE inhibitor (e.g. montelukast sodium), sympathomimetic (e.g. albuterol), antibiotic (e.g. tobramycin), deoxyribonuclease (e.g. pulmozym), agent anticholinergic (e.g. ipratropium bromide), corticosteroid (e.g. dexamethasone), β-adrenoreceptor agonist, leukotriene inhibitor (e.g. zileuton), 5-lipoxygenase inhibitor, phosphodiesterase inhibitor

114 (PDE), CD23 antagonist, IL-13 antagonist, cytokine release inhibitor, HI histamine receptor antagonist, anti-histamine, anti-inflammatory agent (e.g., sodium cromoglycate or any other anti-inflammatory agent known in the art or described), or a release inhibitor histamine.

[0355] In some aspects of the disclosure, the agents may be suitable for ocular administration of an anti-C5a antibody, or antigen-binding fragment thereof, described herein, to a subject in need thereof, e.g., a subject afflicted with AMD or any other complement-related disorder, in relation to the eyeball. The agent may mean, e.g., a syringe, an intradural insert, or even contact lenses containing an antibody or fragment. The agent may, in some aspects of the disclosure, be an eye drop dispenser, wherein the anti-C5a antibody or antigen-binding fragment thereof is formulated for administration. Such therapeutic kits may also include, e.g., one or more additional therapeutics for use in treating an eye disorder associated with the complement system. Therapies can mean, e.g. bevacizumab or Fab fragment of bevacizumab, ranibizumab, both sold by Roche Pharmaceuticals, Inc., or pegaptanib sodium (Mucogen®; Pfizer, Inc.). Such a kit may also, optionally, comprise instructions for administering an anti-C5a antibody or antigen-binding fragment thereof to a subject.

[0356] In some aspects of the disclosure, the agents may be suitable for intra-articular administration of an anti-C5a antibody, or antigen-binding fragment thereof described herein, to a subject in need thereof, e.g., a subject affected by RA. Means may mean, e.g., a syringe or a two-cylinder syringe. See. e.g. US patents no. 6,065,645 and 6,698,622. The two-cylinder syringe is useful for administering two different compositions to the joint in just one injection. Two separate syringes can be implemented for use in therapeutic administration, with fluid draining from the knee for analysis (puncture of the cavity) in a push-pull manner. Additional therapeutics that may be administered with anti-C5a antibodies or fragments in association with a two-cylinder syringe, or which may otherwise be included in the therapeutic kits described herein, include, e.g. NSAIDs, corticosteroids, methotrexate, hydroxychloroquine, anti-TNF agents such as etanercept and infliximab, a B cell depressant such as rituximab, an interleukin-1 antagonist, or a co-stimulant T cell blocking agent such as abatacept. Such a kit may also, optionally, comprise instructions for administering an anti-C5a antibody or antigen-binding fragment thereof to a subject. It will be appreciated that the disclosure includes kits comprising one or more anti-C5a antibodies described herein and one or more anti-inflammatory agents selected from the group consisting of NSAIDs, corticosteroids, methotrexate, hydroxychloroquine, anti-TNF agents such as etanercept and infliximab , a B cell decreasing agent such as rituximab, an interleukin-1 antagonist or a T cell blocking agent, such as abatacept. Antibodies and agents can, e.g., be formulated separately or in combination. The kits can be used to treat an inflammatory condition such as RA, Crohn's disease, inflammatory bowel disease, or

115 any other inflammatory disorder known in the art or cited herein.

[0357] Diagnostic kits comprising anti-C5a antibodies or antigen-binding fragments thereof described herein are also provided. For example, the kits may contain a detectably labeled form of anti-C5a antibody (e.g., anti-C5a antibody or anti-mouse C5a antibody) described herein for use in detecting or determining the amount of C5a in a biological sample. In some aspects of the disclosure, the kits may contain an isolated C5a protein (e.g. one or both human and mouse C5a protein) and / or a control sample containing one or both human and mouse C5a protein. In some aspects of the disclosure, the kit comprises a multi-well plate coated with a first anti-C5a antibody having a first specificity. The kit also includes a second anti-C5a antibody (e.g., a detectably labeled second anti-C5 antibody) having a second specificity. Such a kit is intended for use in capturing, with the first antibody bound to the plate, the C5a protein (e.g., human C5a protein) in a sample (e.g. biological sample) contacting the plate and then detecting the captured C5a protein at using a second antibody. In some aspects of the disclosure, diagnostic kits include both anti-mouse C5a antibody and anti-human C5a antibodies described herein. In some aspects of the disclosure, diagnostic kits include an anti-C5a antibody that binds to both mouse C5a and human C5a.

[0358] The following examples are intended to illustrate but not limit the invention.

Examples

Example 1. Immunization methods [0359] The anti-C5a antibodies currently described are humanized forms of mouse antibodies generated in accordance with the following immunization protocol. Immunizations to obtain antibodies against human C5a-deficient Arginine were carried out on four mice, including two mice from the DBA / 2J strain and two mice from the A / J strain. These strains were chosen because they carry the Hc allele0which causes them to be defective for endogenous C5. All immunizations were repeated 14 days apart, performing a total of three immunizations. All animals received subcutaneous booster immunization with approximately 50 μg purified C5a in 200 μl adjuvant emulsion, approximately 14 days after the last immunization and 5 to 7 days before harvesting. Serum titration from immunized mice, using the ELISA assay, showed that the mice showed a strong response to the anti-human arginine-deficient C5a immunogen.

Example 2. Determination of the specificity of murine antibodies to human C5a [0360] A subset of five murine anti-human Fabs C5a that were representative of neoepitope selective Fabs were converted to full length murine IgG2a antibodies designated as 5an048ME, 5an101ME, 5an178ME, 5an179ME and 5an179ME. These antibodies were evaluated for specificity using layer interferometry

116 biological on an Octet device (ForteBio Inc.). (The amino acid sequences of the light chain and heavy chain CDR sets of each antibody as defined in Kabat are given in Table 3.) Briefly, human C5a, human C5a des Arg, human full length C5, or human C3a C5a paralogs and human C4a biotin conjugated at stoichiometry <1 (biotin): 1 (antibody) via amino groups and immobilized at the tip with streptavidin. The loaded tips were then exposed to a solution containing 20 nM anti-C5a IgG antibody. Each of the antibodies bound to C5a and lacking arginine C5a. None of the anti-C5a IgG antibodies bound to C3a or C4a. However, 5an178ME and 5an179ME each bound to full-length human C5. A small amount of binding was observed between 5an048ME and full length human C5. However, the binding of 5an048ME to C5 was much weaker than the binding observed for C5a.

[0361] These results confirmed that the murine anti-human C5a antibodies - 5an048ME, 5an101ME and 5an180ME - bound to a C5a neoepitope that was blocked with native full-length C5 or was generated after C5 cleavage into C5a and C5b fragments. The results also indicated that three antibodies were selective for human C5a compared to C3a or C4a paralogs.

Table 3. Amino acid sequences for five mouse anti-human C5a antibodies

5an048 ME

<td>SIN:</td><td>Description</td><td>Amino acid sequence</td>

<td> 151</td><td>VL amino acid sequence</td><td>EIVLTQSPAIMSASPGEKVTMTCRASSSVSSS YLHWYQQKSGASPKLWIYSTSNLASGVPAR FSGSGSGTSYSLTISSVEAEDAATYYCQQYS GYPLTFGGGTKLEIKR</td>

<td> 140</td><td>Light chain CDR1</td><td>RASSSVSSSYLH</td>

<td> 96</td><td>Light chain CDR2</td><td>STSNLAS</td>

<td> 142</td><td>Light chain CDR3</td><td>QQYSGYPLT</td>

<td> 152</td><td>VH amino acid sequence</td><td>EVRLQQSGPELVKPGASVRJSCKASGYTFN DYYYMNWVKQSHGKSLEWIGYIFPKTGGT HYNQRFKGKATLTVDKSSSTAYMELRSLTS EDSAVYYCASGPFAYWGQGTLVTVSA</td>

<td> 115</td><td>Heavy chain CDR1</td><td>DYYYMN</td>

<td> 144</td><td>Heavy chain CDR2</td><td>YIFPKTGGTHYNQRFKG</td>

<td> 117</td><td>Heavy chain CDR3</td><td>GPFAY</td>

117

<td rowspan="8">5an101 ME</td><td> 153</td><td>VL amino acid sequence</td><td>DIVMTQSPASLAVSLGQRATISCRASESVDS YGNSFMHWYQQKPGQPPKLLIYRASNLESG IPARFSGSGSRTDFTLTINPVEADDVATYYC OOSNEDPYTFGGGTKLEIKR</td>

<td> 20</td><td>Light chain CDR1</td><td>RASESVDSYGNSFMH</td>

<td> 21</td><td>Light chain CDR2</td><td>RASNLES</td>

<td> 22</td><td>Light chain CDR3</td><td>OOSNEDPYT</td>

<td> 154</td><td>VH amino acid sequence</td><td>EVQLQQSGPELVKPGSSVKISCKASGYTFTD YSMDWVKQSHGKSLEWIGAJNPNSGGTNY SQKFKDKATLTVDKSSSTAYMELRSLTSED SAVYYCASSGSYDGYYAMDYWGQGTSVT vss</td>

<td> 28</td><td>Heavy chain CDR1</td><td>DYSMD</td>

<td> 67</td><td>Heavy chain CDR2</td><td>AINPNSGGTNYSQKFKD</td>

<td> 30</td><td>Heavy chain CDR3</td><td>SGSYDGYYAMDY</td>

<td rowspan="7">5an180 ME</td><td> 155</td><td>VL amino acid sequence</td><td>DIQMTQSPASLSASVGETVTITCRASENIYSY LAWYQQKQGKSPQLLVYNAKTLAEGVPSR FSGSGSGTQFSLKJNSLQPEDFGSYYCQHHY gtpytfgggtkleikr</td>

<td> 156</td><td>Light chain CDR1</td><td>RASENIYSYLA</td>

<td> 157</td><td>Light chain CDR2</td><td>NAKTLAE</td>

<td> 158</td><td>Light chain CDR3</td><td>QHHYGTPYT</td>

<td> 159</td><td>VH amino acid sequence</td><td>EVQLQQPGAEIVRPGASVKLSCRASGYTFT DYWMN WVKQRPGQGLEWIGTIDPSDSYTI YNQKFKGKATLTVDTSSTTAYIQLSSLTSED SAVYFCARGEDYDVSSYTMDYWGQGTSVT VSS</td>

<td> 160</td><td>Heavy chain CDR1</td><td>DYWMN</td>

<td> 161</td><td>Heavy chain CDR2</td><td>TIDPSDSYTIYNQKFKG</td>

118

<td></td><td> 162</td><td>Heavy chain CDR3</td><td>GEDYDVSSYTMDY</td>

<td rowspan="9">5an178 ME</td><td> 163</td><td>VL amino acid sequence</td><td>EIVLTQSPASLAVSLGQRATISCSASESVEYF GTSLMQWYQQKPGQPPKLLIYAASNVESG VPARFSGSGSGTDFSLNIHPVEEDDIAMYFC</td>

<td></td><td></td><td>qqsrkvpwtfgggtkleikr</td>

<td> 164</td><td>Light chain CDR1</td><td>sasesveyfgtslmq</td>

<td> 165</td><td>Light chain CDR2</td><td>aasnves</td>

<td> 166</td><td>Light chain CDR3</td><td>QQSRKVPWT</td>

<td> 167</td><td>VH amino acid sequence</td><td>evklvesggglvqpggsrklscaasgftfs DYGMVWVRQAPGKGLEWVAFISSGSSNIY YADTVKGRFTISRDNPKNTLFLQMNSLRSE DTAIYYCGRAFSFYYGYDYWGQGTTLTVSS</td>

<td> 168</td><td>Heavy chain CDR1</td><td>DYGMV</td>

<td> 169</td><td>Heavy chain CDR2</td><td>fissgssniyyadtvkg</td>

<td> 170</td><td>Heavy chain CDR3</td><td>afsfyygydy</td>

<td rowspan="6">5an179 ME</td><td> 171</td><td>VL amino acid sequence</td><td>DWMTQTPLSLPVSLGDQASISCRSSQSLVH SNGNTYLHWYLQKPGQSPKLLIYKVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYF CSQSTHVPLTFGAGTKJLELKR</td>

<td> 172</td><td>Light chain CDR1</td><td>RSSQSLVHSNGNTYLH</td>

<td> 173</td><td>Light chain CDR2</td><td>KVSNRFS</td>

<td> 174</td><td>Light chain CDR3</td><td>SQSTHVPLT</td>

<td> 175</td><td>VH amino acid sequence</td><td>EVQLQQSGPELVKPGASVRMSCKASGYTFT SYLIHWVKQKPGQGLEWIGYIYPFNDGTKN NENFKGKATLTSDKSSSTVYMEVSSLTSEDS AVYYCARSHGPHYYGGSYGYHFDYWGQG TTLTVSS</td>

<td> 176</td><td>Heavy chain CDR1</td><td>sylih</td>

119

<td rowspan="2"></td><td> 177</td><td>Heavy chain CDR2</td><td>YIYPFNDGTKNNENFKG</td>

<td> 178</td><td>Heavy chain CDR3</td><td>SHGPHYYGGSYGYHFDY</td>

<td colspan="4">"SIN" in the table refers to "SEQ ID NO." * CDR amino acid sequence defined according to Kabat et al. (Supra)</td>

[0362] A series of sandwich assays using Octet was performed on a selected subset of murine anti-human IgG2a C5a antibodies to determine the degree of overlap of C5a epitopes for each of five representative IgG2a antibodies. Briefly, the first antibody was biotinylated and immobilized on a streptavidin coated tip on the Octet platform. Then, human C5a was captured from a 20 nM solution onto the immobilized antibody. The tip carrying the antibody-C5a complex was then exposed to a solution containing 20 nM unlabeled second anti-C5a IgG antibody. The stimulation of an additional association profile in the sensogram would indicate that two antibodies bound C5a simultaneously to a triple complex, and that epitope binding of the two antibodies was non-overlapping. Failure to obtain a second association profile after the addition of a second antibody would indicate that the two antibodies competitively bound C5a, i.e. the C5a epitope to which the second antibody bound was covered by binding by the first antibody. In contrast to non-competitive binding, competitive binding need not necessarily indicate that the first and second antibodies recognize the same, or even overlapping, epitopes on human C5a. Using this approach, the binding sites of five representative anti-C5a antibodies were assigned to 4 separate epitopes on human C5a (Figure 1). Antibodies 5an048ME, 5an180ME and 5an101ME competed with each other. While 5an048 and 5an180 also competed with the non-selective 5an179ME neoepitope, 5an101ME did not compete, indicating that 5an048ME and 5an180ME recognize a neoepitope that is different than the epanope recognized by 5an101ME. In addition, while the non-selective 5an178ME antibody competed with the non-selective 5an179ME neoepitope, only the latter competed with 5an180ME and 5an048ME, which shows that 5an179ME and 5an178ME bind different epitopes that are available in both C5 and 5 The results also indicate that some combinations of antibodies can be used in sandwich assays to detect and / or determine the amount of C5a in a sample.

Example 3. Humanization of selected mouse anti-human C5a antibodies [0363] Variable regions of two related mouse anti-human C5a antibodies 5an101ME and 5an185ME - selected for humanization as full length IgG antibody. Humanization of the light chain and heavy chain variable regions was based on identifying individual framework regions from a human antibody (with preference for germline V genes) with a high degree of sequence identity to the original murine parent antibody. Methods for identifying suitable framework candidates are described in US Patent No. 7,393,648 to US Pat

120

Rother and Wu. Definition of the framework (FW) and complementarity determining regions (CDRs) was carried out according to the methods described in Kabat, Chothia and IMGT® (International ImmunoGenetics Information System; France). Briefly, database queries were conducted independently for both light chain and heavy chain variable regions for a number of antibody fragments including: intact murine variable regions from FW1 to FW4, intact murine variable regions excluding CDRs, and all potential fragments of murine variable regions, including one, two or three frameworks with or without their flanking CDRs. Human frameworks were selected from this pool of candidates based on their overall sequence identity to original mouse antibodies and fragments thereof. Routine molecular biology methods were used to assemble small combinatorial libraries of less than 103 members in which each set of mouse CDRs was surrounded by all potential combinations of selected human frameworks. These humanized antibodies were expressed as soluble Fabs and evaluated for binding to arginine-depleted C5a by ELISA. Fabs that bound to C5a were then subjected to DNA sequence analysis.

[0364] Of these binding members, a subset of six humanized Fabs was reformatted as full-length IgGs (human IgG2 or human IgG2 / G4). Additional humanization was performed for two antibodies (BNJ371 and BNJ381) by replacing the mouse residues in the light chain CDR2 with their corresponding human germline amino acids. The amino acid sequences of humanized anti-C5a antibodies - BNJ364, BNJ367, BNJ371, BNJ378, BNJ366, BNJ369, BNJ381, and BNJ383 - are given in Table 2 above.

Example 4. Affinity determination of humanized C5a anti-human antibodies for

C5a [0365] Humanized antibodies were subjected to BIAcore analysis to quantify their individual affinities for human C5a. See. e.g. Karlsson and Larsson (2004) Methods Mol Biol 248: 389-415. Briefly, each of the humanized antibodies were screened using 3-4 concentrations of human C5a (antigen) using capture techniques. Antibodies were captured with Anti-Fc (human) CM5 sensor directly immobilized on the chip at various concentrations in the range of 0.6 nM to 5.9 nM human C5a moved over the surface of the sensor chip. The surface was regenerated with 20 mM HCl, 0.02% P20 after each cycle to remove bound antibody and antigen. Data were evaluated using the Biacore BIAevaluation software using a 1: 1 Langmuir fit model (Rmax: Global Fit; RI: Local Fit). Kinetic information such as (ka: social rate constant), (kd: dissociation rate constant) and KD (equilibrium dissociation constant) was obtained from matching. The results of the analyzes are given in Table 4. These experiments were conducted as screening tests, with the minimum number of analyte concentrations (3 to 4) in 1 replicate. Therefore, approximate kinetic values are given in Table 4.

Table 4. Affinity measurements for selected humanized anti-C5a antibodies

121

<td>Antibody determination</td><td>ka (1 / Ms) (x106)</td><td> -4 kd (1 / s) (x10 )</td><td>Kd (M) (x10-12)</td><td>X2</td>

[0366] All of the humanized antibodies specifically bind to human C5a with a kD of less than 1.20 nanomolar. All antibodies except BNJ371 bound to human C5a with a KD of less than 1-nanomolar. Three antibodies, BNJ369, BNJ381, and BNJ383 bind to human C5a with a KD of less than 100 picomolar.

Example 5. Anti-C5a antibodies inhibit C5a mediated signal transduction in vitro [0367] An in vitro neutrophil activation assay was used to assess the activity of humanized antibodies. The designation is generally described in, e.g., Paczkowski et al. (1999) Br J Pharmacol 128 (7): 1461-1466, and is used to determine the amount of myeloperoxidase (MPO) produced by neutrophils as a measure of neutrophil activation. Briefly, multinucleated cells, most of which are neutrophils, were isolated using density gradient centrifugation (catalog number for separating mononuclear leukocytes from multinucleated mono-poly resolved medium: 91698049; MP Biochemicals; Solon, Ohio) from whole blood from a healthy donor . Cells were washed once with phosphate buffered saline (PBS) and red blood cells (RBC) were removed from the cell population by lysis in hypotonic solution (ACK lysis buffer, catalog number 10-548E; Lonza). After a further two washes with PBS, RBC-free cells were resuspended at a concentration of 4 x 106 cells / ml in Hank's balanced salt solution (HBSS; Mediatech, catalog number: 21-023-CV), to which calcium and magnesium were added, and additionally 0.1% gelatin (Sigma Aldrich; St. Louis, Missouri) [in the rest of the buffer for determination].

[0368] Cytochalazine B (Sigma Aldrich) was added to the cell suspension in an amount sufficient to reach a concentration of 10 μg / ml. The suspension was then incubated for 10 minutes at 37 ° C. 100 μl of cells were added to the wells of 96-well plates with a U-shaped bottom. The wells of the plates were grouped into several different sets. Each of several different sets of wells contained anti-C5a antibody: each well of set 1 contained a humanized antibody that binds to un-cleaved native C5 but not to free C5a; each well of set 2 contained BNJ367 122 humanized anti-C5a antibody; each well of set 3 contained BNJ369 humanized anti-C5a antibody; each well of set 4 contained BNJ371 humanized anti-C5a antibody; each well of set 5 contained BNJ378 humanized anti-C5a antibody; each well of set 6 contained BNJ381 humanized anti-C5a antibody; and each well of set 7 contained BNJ383 humanized anti-C5a antibody. Each well of the eighth set of wells did not contain any antibody. A series of antibody concentrations were evaluated in each of the well sets, ranging from 0.08 nM, 0.4 nM, 2 nM and 10 nM antibody.

[0369] C5a (obtained from Complement Technologies, Inc.) was evaluated at a concentration of 2 nM. A 10X working concentration of 20 nM was obtained in the aforementioned assay buffer and 20 μl was added to each well. After adding C5a to the wells, the plate was incubated for 10 minutes at 37 ° C. After incubation, 60 Pl PBS was added to each well of the plate. Plates were centrifuged at 1200 rpm (approximately 335 xg) for 10 minutes at room temperature. 100 μl of the supernatant from each well was transferred to the corresponding well of the second plate. 25 ml substrate (Sigma Aldrich catalog number T0440) was added to each well of the second plate and allowed to react with peroxidase, which lasted approximately two to five minutes. The reaction was quenched by the addition of 25 μl 1N HCl. OD at 450 nm was recorded.

[0370] As shown in Figure 2, all humanized anti-C5a antibodies inhibited neutrophil activation in vitro. These results indicate that the humanized anti-C5a antibodies described herein are potent inhibitors of C5a mediated signal transduction in vitro and support the inventors' conclusion that the antibodies are useful in the treatment of a number of complement-related disorders (e.g., complement-related inflammatory disorders) in people.

Example 6. Characterization of replacement mouse anti-mouse C5a antibodies [0371] A series of sandwich assays were performed for the selected mouse anti-mouse C5a IgG antibody - 5an195ME - to determine the specificity of the C5a antibody. Briefly, the wells of the assay plate were coated with 5an195ME antibody. The plate was washed thoroughly to remove unbound antibody. Then, wells containing 5an195ME were contacted with mouse C5a for a period of time and under sufficient conditions to allow antigen binding to the antibody. Unbound protein was removed by washing. After the washing step, the wells were additionally coated with a solution containing the second biotinylated anti-C5a antibody. The wells were washed again to remove any unbound second antibody. The amount of binding of the second antibody per well was quantified using streptavidin-conjugated horseradish peroxidase (HRP). The amount of binding of the second antibody was the function of binding C5a to 5an195ME.

[0372] In a parallel experiment, a set of 5an195ME coated wells were incubated with full-length mouse C5 protein, rather than C5a. After the washing step, the wells were contacted with the solution containing the second antibody: biotinylated anti-mouse C5 antibody. The amount of binding of the second antibody as a function of the amount of C5 bound

123 by 5an195ME, quantified using a streptavidin-coupled HRP construct. No binding of the second antibody indicates that 5an195ME does not bind to full-length mouse C5.

[0373] While 5an195ME bound to C5a in a dose-dependent manner, no binding between antibody and full-length mouse C5 was detected using this assay. These results indicate that 5an195ME binds to the neo-epitope present in C5a.

[0374] The relative binding affinity of 5an195ME for mouse C5a was further quantified using BIAcore. The kinetics of 5an195ME were measured using a capture technique. Antibodies were captured using Anti-Fc (mouse) CM5 sensor directly immobilized on the chip at various concentrations, ranging from 0.4 nM to 25 nM mouse C5a moved over the surface of the sensor chip. Replicates for each concentration were also analyzed. The chip surface was regenerated with 10 mM glycine, HCl pH 1.7 after each cycle to remove bound antibody and antigen. Data were evaluated using the Biacore BIAevaluation software using a 1: 1 Langmuir fit model (Rmax: Global Fit; RI: Local Fit). Kinetic information, such as ka (constant sociation rate), kd (constant dissociation rate) and KD (equilibrium dissociation constant) was obtained from matching. The results of kinetic analyzes are shown in Table 5.

<td colspan="3">Table 5. 5an195M kinetics measurements</td><td colspan="2">E for mouse C5a</td>

<td>Parameter:</td><td>ka (1 / Ms)</td><td>kd (1 / s)</td><td>KD (M)</td><td>X2</td>

[0375] These results indicate that mouse anti-mouse C5a antibody is not only specific for C5a, compared to full length mouse C5, but also that the antibody binds with high affinity to mouse C5a.

Example 7. Use of the 5an195ME replacement anti-mouse C5a antibody in the RA animal model. [0376] 5an195ME anti-mouse C5a antibody was evaluated in a collagen-induced arthritis mouse model. Male DBA / 1LacJ mice (9 to 12 weeks old) were immunized by intradermal injection into the base of the tail with 300 μg of bovine type II collagen, emulsified with equal volumes of Freund's complete adjuvant. The procedure was repeated two weeks after the first immunization. Mice were monitored daily to identify the onset of inflammation in the knee joint. After identifying the onset of inflammation, mice were administered intraperitoneally three times weekly anti-mouse C5a 5an195ME antibody (40mg / kg) or control antibody (40 mg / kg). The thickness of the initial inflamed joint (in mm) was measured daily until day 12.

[0377] As shown in Fig. 3, 5an195ME reduced knee thickness compared to the control antibody. 5an195ME seemed to provide an advantage by keeping the knee thickness below 4.5 mm.

124 [0378] In addition to assessing the ability of the 5an195ME antibody to reduce initial swelling of the inflamed knee, the ability of the 5an195ME anti-C5a antibody to prevent inflammation migration into new joints was also assessed. The number of newly recruited joints was measured daily, from day 1 to day 12. The results of the experiment are given in Table 6.

Table 6. 5an195ME efficacy in the RA model

<td>Treatment</td><td>number mice</td><td>Number of joints inflamed on day 1</td><td>Number of newly inflamed joints on day 12</td><td>Average number of newly inflamed joints per mouse</td>

<td>inspection ab</td><td> 6</td><td> 7</td><td> 12</td><td> 2</td>

[0379] As shown in Table 6, 5an195ME-treated mice had clearly fewer newly inflamed joints compared to animals treated with control Ab by day 12. 5an195ME-treated mice also had on average significantly less newly inflamed joints.

[0380] Arthritis in mice was also monitored and defined using a clinical arthritis point indicator. Each limb was assessed daily according to an established scoring system (0, normal joint; 1, mild / moderate visible erythema and edema; 2, severe erythema and edema affecting the entire paw or joint; 3, deformed paw or stiffened joint.), With a maximum score of twenty-four per animal. See. e.g. Wang et al. (2000) J Immunol 164: 4340-4347. As shown in Figure 4, mice treated with anti-mouse C5a antibody 5an195ME showed a marked reduction in clinical score (mean score less than 1), compared to control antibody treated mice (mean score over 6), throughout the study.

[0381] In summary, these results indicate that interchangeable anti-mouse C5a is effective in the treatment of RA - both in the initial joint and under conditions of migration of inflammation to the secondary joints, in the mouse disease model. The results also strongly suggest that therapeutic anti-human C5a antibody, such as any of the humanized anti-C5a antibodies described herein, is useful for treating people with RA.

Example 8. Use of anti-C5a antibody for the treatment of rheumatoid arthritis [0382] A human patient is identified by a healthcare professional as having rheumatoid arthritis in a single articulated joint. Soon thereafter, the patient was administered intra-arterially or intraperitoneally a composition containing the humanized anti-C5a antibody described herein in an amount sufficient to reduce C5a-mediated C5aR1 signaling locally within space

125 pond. The patient and healthcare professional observe significant improvement for at least two known symptoms of rheumatoid arthritis after treatment. The patient receives intravenously administered "maintenance doses" of the antibody every month, which prevents the reappearance of symptoms, to prevent the progression of RA in a single joint, or to prevent the migration of RA symptoms to another joint.

Example 9. Use of anti-C5a antibody for the treatment of sepsis [0383] A human patient is identified by a healthcare professional as having sepsis. The patient was shortly thereafter administered a composition containing the humanized anti-C5a antibody described herein at a dose of approximately 600 to 900 mg by intravenous infusion. The patient and healthcare professional observe significant improvement for at least two known symptoms of sepsis during treatment. The patient receives intravenous boosters every two weeks until the patient leaves the hospital.

Example 10. Use of anti-C5a antibody for the treatment of complement-related pulmonary inflammatory disorders [0384] A human patient is identified by a healthcare professional as having severe COPD. The patient was administered once every two weeks a composition containing humanized anti-C5a antibody at a dose of approximately 600 to 900 mg by intravenous infusion. The patient and healthcare professional observe significant improvement in at least two known COPD symptoms during initial treatment. For example, a patient receiving anti-C5a antibody has a reduced incidence and / or severity of COPD-related exacerbations. The patient continues to receive intravenous "maintenance doses" of the antibody every two weeks to maintain a reduced incidence and / or severity of COPD-related exacerbations.

[0385] A human patient is identified by a healthcare professional as having severe asthma. The patient is prescribed therapeutic, humanized anti-C5a antibody for administration via an inhaler device. During the next attack of bronchospasm, the patient self-administers anti-C5a in an amount sufficient to reduce the C5a-mediated inflammatory response in the patient's lungs. The patient continues to use the inhaler, where necessary, to prevent or reduce the severity of asthma attacks.

Example 11. Additional anti-C5a antibodies identified in immunized mice [0386] Several additional antibodies were obtained from immunized mice (see Example 1) and were further identified by ELISA as capable of binding to human C5a. Additional antibodies include 15 unique light chain CDR sets (listed in Table 7) and 14 unique heavy chain CDR sets (as listed in Table 8).

Table 7. Amino acid sequences of several unique V1 and light CDR chain sequences defined according to Kabat from additional murine anti-human C5a antibodies

126

<td>ab</td><td>SIN:</td><td>Description</td><td>Amino Acid Sequence *</td>

<td rowspan="4">5an110</td><td> 83</td><td>Amino acid sequence wa VL</td><td>DIVMTQSQKFMSTSVGDRVSVTCKASQNVGT NVAWYQQKPGQSPKALIYSASYRYSGVPDRF TGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYP FTFGSGTKLEIKR</td>

<td> 84</td><td>CDR1 chain light</td><td>KASQNVGTNVA</td>

<td> 85</td><td>CDR2 chain light</td><td>SASYRYS</td>

<td> 86</td><td>CDR3 chain light</td><td>QQYNSYPFT</td>

<td rowspan="4">5an177</td><td> 87</td><td>Amino acid sequence wa VL</td><td>EIVLTQSPAIMSASPGEKVTMTCSASSSVSYMH WYQQKSGTSPKRWIYDTSKLASGVPARFSGS GSGTSYSLTISSMEAEDAATYYCQQWSSNPLT FGAGTKLELKR</td>

<td> 88</td><td>CDR1 chain light</td><td>SASSSVSYMH</td>

<td> 89</td><td>CDR2 chain light</td><td>DTSKLAS</td>

<td> 90</td><td>CDR3 chain light</td><td>QQWSSNPLT</td>

<td rowspan="4">5anG12</td><td> 91</td><td>Amino acid sequence wa VL</td><td>QIVLTQSPAJMSASPGEKVTMTCSASSSISYMH WYQQKPGTSPKRWIYDTSKLASGVPARFSGS GSGTSYSLTISSMEAEDAATYYCHQRSSYPWT FGGGTKLEIKR</td>

<td> 92</td><td>CDR1 chain light</td><td>SASSSISYMH</td>

<td> 89</td><td>CDR2 chain light</td><td>DTSKLAS</td>

<td> 93</td><td>CDR3 chain light</td><td>HQRSSYPWT</td>

127

<td rowspan="4">5an052</td><td> 94</td><td>Amino acid sequence VL</td><td>QIVLTQSPAIMSASPGEKVTLTCSASSSVSSSYL YWYQQKPGSSPKLWIYSTSNLASGVPARFSGS GSGTSYSLTISTVEAEDAASYFCHQWSSYPPTF GGGTKLEIKR</td>

<td> 95</td><td>CDR1 chain light</td><td>SASSSVSSSYLY</td>

<td> 96</td><td>CDR2 chain light</td><td>STSNLAS</td>

<td> 97</td><td>CDR3 chain light</td><td>HQWSSYPPT</td>

<td rowspan="4">5an107</td><td> 98</td><td>Amino acid sequence VL</td><td>DIQMTQSPAPMLVSVGETVTITCRGSENIYSNL AWYQQKQGKSPQLLVYAATNLADGVPSRFSG SGSGTQYSLKINSLQSEDFGSYYCQHFWGTPR TFGGGTKLEIKR</td>

<td> 99</td><td>CDR1 chain light</td><td>RGSENIYSNLA</td>

<td> 100</td><td>CDR2 chain light</td><td>AATNLAD</td>

<td> 101</td><td>CDR3 chain light</td><td>QHFWGTPRT</td>

<td rowspan="4">5anE11</td><td> 102</td><td>Amino acid sequence wa VL</td><td>DIVMTQSQKFMSTSVGDRVSVTCKASQNVGT NVAWYQQKPGQSPKALIYSASYRYSGVPDRF TGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYP WTFGGGTKLEIKR</td>

<td> 84</td><td>CDR1 chain light</td><td>KASQNVGTNVA</td>

<td> 85</td><td>CDR2 chain light</td><td>SASYRYS</td>

<td> 103</td><td>CDR3 chain</td><td>QQYNSYPWT</td>

128

<td></td><td></td><td>light</td><td></td>

<td rowspan="4">5an054</td><td> 104</td><td>Amino acid sequence wa VL</td><td>QIVLTQSPVIMSASPGEKVTMTCSASSSVSYM YWYQQKPGSSPRLLIYDTSNLASGVPVRFSGS GSGTSYSLTISRMEAEDAATYYCQQWSSYPPT FGAGTKLELKR</td>

<td> 105</td><td>CDR1 chain light</td><td>SASSSVSYMY</td>

<td> 106</td><td>CDR2 chain light</td><td>DTSNLAS</td>

<td> 107</td><td>CDR3 chain light</td><td>QQWSSYPPT</td>

<td>5anG10</td><td> 141</td><td>Amino acid sequence wa VL</td><td>QIVLTQSPAJMSASPGEKVTMTCSASSSISYMH WYQQKPGTSPKRWIYDTSKLASGVPARFSGS GSGTSYSLTISSMEAEDAATYYCHQRRSYPWT FGGGTKLEIKR</td>

<td> 92</td><td>CDR1 chain light</td><td>SASSSISYMH</td>

<td> 89</td><td>CDR2 chain light</td><td>DTSKLAS</td>

<td> 108</td><td>CDR3 chain light</td><td>HQRRSYPWT</td>

<td rowspan="3">5an188</td><td> 109</td><td>Amino acid sequence wa VL</td><td>DIVLTQSPASLAVSLGQRATISCRASESVDSYG NSFMHWYQQKPGQPPKLLIYLASNLESGVPAR FSGSGSRTDFTLTIDPVEADDAATYYCQQNNE DPLTFGAGTKLELKR</td>

<td> 20</td><td>CDR1 chain light</td><td>RASESVDSYGNSFMH</td>

<td> 110</td><td>CDR2 chain light</td><td>LASNLES</td>

129

<td></td><td> 111</td><td>CDR3 chain light</td><td>QQNNEDPLT</td>

<td rowspan="4">5an185</td><td> 112</td><td>Amino acid sequence VL</td><td>DIVLTQSPASLAVSLGQRATISCRASESVDSYG NSFMHWYQQKPGQPPKLLIYRASNLESGIPAR FSGSGSRTDFTLTINPVEADDVATYYCQQSNE DPLTFGAGTKLELKR</td>

<td> 20</td><td>CDR1 chain light</td><td>RASESVDSYGNSFMH</td>

<td> 21</td><td>CDR2 chain light</td><td>RASNLES</td>

<td> 113</td><td>CDR3 chain light</td><td>QQSNEDPLT</td>

"SIN" in table refers to "SEQ ID NO." * CDR amino acid sequences are defined according to Kabat et al. (Supra)

Table 8. Amino acid sequences of several unique VH and heavy chain CDR sequences defined according to Kabat from an additional murine anti-human antibody

C5a

<td>ab</td><td>SIN:</td><td>Description</td><td>Amino Acid Sequence *</td>

<td rowspan="4">5an110</td><td> 114</td><td>Amino acid sequence VH</td><td>EVQLQQSGPELVKPGASVKISCKASGYTFSDY YYMNWVKKSHGKSLEWIGYIFPKTGGTNYS QRFKGKATLTVDKSSSTAYMELRSLTSEDSA VYYCASGPFAYWGQGTLVTVSA</td>

<td> 115</td><td>CDR1 chain heavy</td><td>DYYYMN</td>

<td> 116</td><td>CDR2 chain heavy</td><td>YIFPKTGGTNYSQRFKG</td>

<td> 117</td><td>CDR3 chain heavy</td><td>GPFAY</td>

130

<td rowspan="4">5an177</td><td> 118</td><td>Amino acid sequence VH</td><td>EVKLVESGGGLVKPGGSLKLSCAASGITFSSY YMAWVRQTPDKRLEWVATISSGGSYTYYPD NVKGRFTISRDNAKNTLYLQMSSLKSEDTAM YYCTRYYEDDAMDYWGQGTSVTVSS</td>

<td> 119</td><td>CDR1 chain heavy</td><td>SYYMA</td>

<td> 120</td><td>CDR2 chain heavy</td><td>TISSGGSYTYYPDNVKG</td>

<td> 121</td><td>CDR3 chain heavy</td><td>YYEDDAMDY</td>

<td rowspan="4">5an055</td><td> 122</td><td>Sequence amino acid waVH</td><td>EVQLQQSGPELVKPGASVKISCKASGYTFSDY YYMNWVKKSHGKSLEWIGYIFPKTGGTNYN QRFKGKATLTVDKSSSTAYMELRSLTSEDSA VYYCASGPFAYWGQGTLVTVSA</td>

<td> 115</td><td>CDR1 chain heavy</td><td>DYYYMN</td>

<td> 123</td><td>CDR2 chain heavy</td><td>YIFPKTGGTNYNQRFKG</td>

<td> 117</td><td>CDR3 chain heavy</td><td>GPFAY</td>

<td rowspan="4">5an054</td><td> 145</td><td>Amino acid sequence VH</td><td>EVQLQQSGPELVKPGASVKISCKASGYTFTDY YYMNWVKQSHGKSLEWIGYIFPNTGGTTYN QRFKGKATLTVDKSSSTAYMELRSLTSEDSA VYYCASGPFAYWGQGTLVTVSA</td>

<td> 115</td><td>CDR1 chain heavy</td><td>DYYYMN</td>

<td> 124</td><td>CDR2 chain heavy</td><td>YIFPNTGGTTYNQRFKG</td>

<td> 117</td><td>CDR3</td><td>GPFAY</td>

131

<td></td><td></td><td>chain heavy</td><td></td>

<td rowspan="4">5an107</td><td> 125</td><td>Amino acid sequence wa VH</td><td>EVKLVESGGGLVKPGGSLKLSCAASGYTFSS YYM AWVRQTPDKRLEWVATIS SGGS YTYYR DNVKGRFTISRDNAKNTLYLQMSSLKSEDTA MYYCTRYFEDYPMDYWGQGTSVTVSS</td>

<td> 119</td><td>CDR1 chain heavy</td><td>SYYMA</td>

<td> 126</td><td>CDR2 chain heavy</td><td>TISSGGSYTYYRDNVKG</td>

<td> 127</td><td>CDR3 chain heavy</td><td>YFEDYPMDY</td>

<td rowspan="4">5an111</td><td> 128</td><td>Amino acid sequence wa VH</td><td>EVQLQQSGPELGKPGASGKISCKASGYTFTDY YYMNWVKQSHGKSLEWIGYIFPNTGGTSYN QRFKDKATLTVDKSS STAYMELRSLTSEDSA VYYCASGPFAYWGQGTLVTVSA</td>

<td> 115</td><td>CDR1 chain heavy</td><td>DYYYMN</td>

<td> 129</td><td>CDR2 chain heavy</td><td>YIFPNTGGTSYNQRFKD</td>

<td> 117</td><td>CDR3 chain heavy</td><td>GPFAY</td>

<td rowspan="4">5an183</td><td> 130</td><td>Amino acid sequence wa VH</td><td>EVQLQQPGSVLVRPGATVKLSCKASGFTFTSS WMHWAKQRPGQGLEWIGEIHTSGHTNYNEK FKGKATLTLDTSSSTAYVDISSLTSEDSAVYY CARGGLRRGYAMDYWGQGTSVTVSS</td>

<td> 131</td><td>CDR1 chain heavy</td><td>SSWMH</td>

<td> 132</td><td>CDR2 chain heavy</td><td>EIHTSGHTNYNEKFKG</td>

<td> 133</td><td>CDR3</td><td>GGLRRGYAMDY</td>

132

<td></td><td></td><td>chain heavy</td><td></td>

<td>5an185</td><td> 134</td><td>Amino acid sequence wa VH</td><td>EVQPQQSGPELVKPGSSVKISCKASGYTFTDY SMDWVKQSHGKSLEWIGAJHLNTGYTNYNQ KFKGKATLTVDKSSSTAYMELRSLTSEDSAV YYCARGFYDGYSPMDYWGQGTSVTVSS</td>

<td> 28</td><td>CDR1 chain heavy</td><td>DYSMD</td>

<td> 46</td><td>CDR2 chain heavy</td><td>AiHLNTGYTNYNQKFKG</td>

<td> 47</td><td>CDR3 chain heavy</td><td>GFYDGYSPMDY</td>

<td rowspan="4">5an188</td><td> 135</td><td>Amino acid sequence VH</td><td>EVQLQQSGAELVKPGTSVKLSCKASGYTFTS YWMHWVKQRPGQGLEYIGEIHPSSGHTNYH EKFKSKATLTVDKSSSTAYMQLSSLTSEDSAV YYCARAŚLLRAYAMDYWGQGTSVTVSS</td>

<td> 136</td><td>CDR1 chain heavy</td><td>SYWMH</td>

<td> 137</td><td>CDR2 chain heavy</td><td>EiHPSSGHTNYHEKFKS</td>

<td> 138</td><td>CDR3 chain heavy</td><td>ASLLRAYAMDY</td>

"SiN" in the table refers to "SEQ iD NO".

* CDR amino acid sequences are defined according to Kabat et al. (Supra).

[0387] VL and VH pairs leading to 5an177ME, 5an054ME, 5an110ME, 5an188ME, 5an185ME and 5an107ME antibodies are evident from Tables 7 and 8. Additional exemplary pairs of heavy chain and light chain variable regions and / or sets of CDRs are given in Table 9 below.

133

Table 9. Amino acid sequences for additional mouse anti-human C5a antibody CDR sets

<td>ab</td><td>SIN:</td><td>Description</td><td>Amino Acid Sequence *</td>

<td rowspan="8">5an047</td><td> 139</td><td>Amino acid sequence VL</td><td>EIVLTQSPAIMSASPGEKVTMTCRASSSVSSSY LHWYQQKSGASPKLWIYSTSNLASGVPARFS GSGSGTSYSLTISSVEAEDAATYYCQQYSGYP LTFGAGTKLELKR</td>

<td> 140</td><td>Light chain CDR1</td><td>RASSSVSSSYLH</td>

<td> 96</td><td>Light chain CDR2</td><td>STSNLAS</td>

<td> 142</td><td>Light chain CDR3</td><td>QQYSGYELT</td>

<td> 143</td><td>Amino acid sequence VH</td><td>EVQLQQSGPELVKPGASVRISCKASGYTFSDY YYMNWVKKSHGKSLEWIGYIFPKTGGTHYN QRFKGKATLTVDKSSSTAYMELRSLTSEDSA VYYCASGPFAYWGOGTLVTVSA</td>

<td> 115</td><td>Heavy chain CDR1</td><td>DYYYMN</td>

<td> 144</td><td>Heavy chain CDR2</td><td>YIFPKTGGTHYNQRFKG</td>

<td> 117</td><td>Heavy chain CDR3</td><td>GPFAY</td>

<td>5an181</td><td> 139</td><td>Amino acid sequence VL</td><td>EIVLTQSPAIMSASPGEKVTMTCRASSSVSSSY LHWYQQKSGASPKLWIYSTSNLASGVPARFS</td>

<td rowspan="6"></td><td></td><td></td><td>GSGSGTSYSLTISSVEAEDAATYYCQQYSGYP LTFGAGTKLELKR</td>

<td> 140</td><td>Light chain CDR1</td><td>RASSSVSSSYLH</td>

<td> 96</td><td>Light chain CDR2</td><td>STSNLAS</td>

<td> 142</td><td>Light-Chain CDR3</td><td>QQYSGYPLT</td>

<td> 122</td><td>Amino acid sequence VH</td><td>EVQLQQSGPELVKPGASVKISCKASGYTFSDY YYMNWVKKSHGKSLEWIGY1FPKTGGTNYN QRFKGKATLTVDKSSSTAYMELRSLTSEDSA VYYCASGPFAYWGQGTLVTVSA</td>

<td> 115</td><td>Heavy chain CDR1</td><td>DYYYMN</td>

134

<td rowspan="2"></td><td> 123</td><td>Heavy chain CDR2</td><td>YIFPKTGGTNYNQRFKG</td>

<td> 117</td><td>Heavy chain CDR3</td><td>GPFAY</td>

<td rowspan="8">5an109</td><td> 146</td><td>Amino acid sequence VL</td><td>EIVLTQSPAIMSASPGEKVTMTCSASSSVSYM YWYQQKPGSSPRLLIYDTSNLASGVPVRFSGS GSGTSYSLTISRMEAEDAATYYCQQWSSYPP TFGGGTKLEIKR</td>

<td> 105</td><td>Light chain CDR1</td><td>SASSSVSYMY</td>

<td> 106</td><td>Light chain CDR2</td><td>DTSNLAS</td>

<td> 107</td><td>Light chain CDR3</td><td>QQWSSYPPT</td>

<td> 122</td><td>Amino acid sequence VH</td><td>EVQLQQSGPELVKPGASVKISCKASGYTFSDY YYMNWVKKSHGKSLEWIGYIFPKTGGTNYN QRFKGKATLTVDKSSSTAYMELRSLTSEDSA VYYCASGPFAYWGQGTLVTVSA</td>

<td> 115</td><td>Heavy chain CDR1</td><td>DYYYMN</td>

<td> 123</td><td>Heavy chain CDR2</td><td>YIFPKTGGTNYNQRFKG</td>

<td> 117</td><td>Heavy chain CDR3</td><td>GPFAY</td>

<td rowspan="6">5anG10</td><td> 141</td><td>Amino acid sequence VL</td><td>QIVLTQSPAIMSASPGEKVTMTCSASSSISYM HWYQQKPGTSPKRW1YDTSKLASGVPARFSG SGSGTSYSLTISSMEAEDAATYYCHQRRSYP WTFGGGTKLEIKR</td>

<td> 92</td><td>Light chain CDR1</td><td>SASSSISYMH</td>

<td> 89</td><td>Light chain CDR2</td><td>DTSKLAS</td>

<td> 108</td><td>Light chain CDR3</td><td>HQRRSYPWT</td>

<td> 147</td><td>Amino acid sequence VH</td><td>EVQLQQSGPELVKPGASVRISCKASGYTFND YYYMNWVKQSHGKSLEWIGYIFPKTGGTHY NQRFKGKATLTVDKSSSTAYMELRSLTSEDS AVYYCASGPFAYWGQGTLVTVSA</td>

<td> 115</td><td>Chain CDR1</td><td>DYYYMN</td>

135

<td rowspan="3"></td><td></td><td>heavy</td><td></td>

<td> 144</td><td>Heavy chain CDR2</td><td>yifpktggthynqrfkg</td>

<td> 117</td><td>Heavy chain CDR3</td><td>GPFAY</td>

<td rowspan="8">5an053</td><td> 148</td><td>Amino acid sequence VL</td><td>EIVLTQSPVIMSASPGEKVTMICSASSSISYMH WYQQKPGTSPKRWIYDTSKLASGVPARFSGS GSGTSYSLTISIMEAEDAATYYCHQRSSYPWT FGGGTKLEIKR</td>

<td> 92</td><td>Light chain CDR1</td><td>SASSSISYMH</td>

<td> 89</td><td>Light chain CDR2</td><td>DTSKLAS</td>

<td> 93</td><td>Light chain CDR3</td><td>hqrssypwt</td>

<td> 149</td><td>Amino acid sequence VH</td><td>EVQMQQSGPELVKPGASVKISCKASGYTFSD YYYMNWVKKSHGKSLEWIGYIFPKTGGTNY NQRFKGKATLTVDKSSSTAYMELRSLTSEDS AVYYCASGPFAYWGQGTLVTVSA</td>

<td> 115</td><td>Heavy chain CDR1</td><td>DYYYMN</td>

<td> 123</td><td>Heavy chain CDR2</td><td>yifpktggtnynqrfkg</td>

<td> 117</td><td>Heavy chain CDR3</td><td>GPFAY</td>

<td rowspan="5">5anG12</td><td> 91</td><td>Amino acid sequence VL</td><td>QIVLTQSPAIMSASPGEKVTMTCSASSSISYM HWYQQKPGTSPKRWIYDTSKLASGVPARFSG · SGSGTSYSLTISSMEAEDAATYYCHQRSSYPW TFGGGTKLEIKR ·.</td>

<td> 92</td><td>Light chain CDR1</td><td>SASSSISYMH</td>

<td> 89</td><td>Light chain CDR2</td><td>DTSKLAS</td>

<td> 93</td><td>Light chain CDR3</td><td>hqrssypwt</td>

<td> 147</td><td>Amino acid sequence VH</td><td>EVQLQQSGPELVKPGASVRISCKASGYTFND YYYMNWVKQSHGKSLEWIGYIFPKTGGTHY NQRFKGKATLTVDKSSSTAYMELRSLTSEDS AVYYCASGPFAYWGQGTLVTVSA</td>

136

<td rowspan="3"></td><td> 115</td><td>Heavy chain CDR1</td><td>DYYYMN</td>

<td> 144</td><td>Heavy chain CDR2</td><td>YIFPKTGGTHYNQRFKG</td>

<td> 117</td><td>Heavy chain CDR3</td><td>GPFAY</td>

<td rowspan="6">5an052</td><td> 94</td><td>Amino acid sequence VL</td><td>QIVLTQSPAJMSASPGEKVTLTCSASSSVSSSY LYWYQQKPGSSPKLWIYSTSNLASGVPARFS GSGSGTSYSLTISTVEAEDAASYFCHQWSSYP PTFGGGTKLEIKR</td>

<td> 95</td><td>Light chain CDR1</td><td>SASSSVSSSYLY</td>

<td> 96</td><td>Light chain CDR2</td><td>STSNLAS</td>

<td> 97</td><td>Light chain CDR3</td><td>HOWSSYPPT</td>

<td> 147</td><td>Amino acid sequence VH</td><td>EVQLQQSGPELVKPGASVRISCKASGYTFND YYYMNWVKQSHGKSLEWIGYIFPKTGGTHY NQRFKGKATLTVDKSSSTAYMELRSLTSEDS AVYYCASGPFAYWGQGTLVTVSA</td>

<td> 115</td><td>Heavy chain CDR1</td><td>DYYYMN</td>

<td rowspan="2"></td><td> 144</td><td>Heavy chain CDR2</td><td>YIFPKTGGTHYNQRFKG</td>

<td> 117</td><td>Heavy chain CDR3</td><td>GPFAY</td>

<td rowspan="4">5an111</td><td> 102</td><td>Amino acid sequence VL</td><td>DIVMTQSQKFMSTSVGDRVSVTCKASQNVGT NVAWYQQKPGQSPKALIYSASYRYSGVPDRF TGSGSGTDFTLTISNVQSEDLAEYFCQQYNSY PWTFGGGTKLEIKR '</td>

<td> 84</td><td>Light chain CDR1</td><td>KASQNVGTNVA</td>

<td> 85</td><td>Light chain CDR2</td><td>SASYRYS</td>

<td> 103</td><td>Light chain CDR3</td><td>QQYNSYPWT</td>

137

<td rowspan="4"></td><td> 128</td><td>Amino acid sequence VH</td><td>EVQLQQSGPELGKPGASGKJSCKASGYTFTDY yymnwvkqshgkslewigyifpntggtsyn QRFKDKATLTVDKSSSTAYMELRSLTSEDSA vyycasgpfaywgqgtlvtvsa</td>

<td> 115</td><td>Heavy chain CDR1</td><td>DYYYMN</td>

<td> 129</td><td>Heavy chain CDR2</td><td>yifpntggtsynqrfkd</td>

<td> 117</td><td>Heavy chain CDR3</td><td>gpfay</td>

<td rowspan="8">5an055</td><td> 150</td><td>Amino acid sequence VL</td><td>eivltqspaimsaspgekvtltcsasssvsssy lywyqqkpgsspklwiystsnlasgvparfs gsgsgtsysltissmeaedaasyfchqwssyp PTFGGGTKLEIKR</td>

<td> 95</td><td>Light chain CDR1</td><td>sasssvsssyly</td>

<td> 96</td><td>Light chain CDR2</td><td>stsnlas</td>

<td> 97</td><td>Light chain CDR3</td><td>HQWSSYPPT</td>

<td> 122</td><td>Amino acid sequence VH</td><td>EVQLQQSGPELVKPGASVKJSCKASGYTFSDY YYMNWVKKSHGKSLEWIGYIFPKTGGTNYN QRFKGKATLTVDKSSSTAYMELRSLTSEDSA VYYCASGPFAYWGQGTLVTVSA</td>

<td> 115</td><td>Heavy chain CDR1</td><td>DYYYMN</td>

<td> 123</td><td>Heavy chain CDR2</td><td>yifpktggtnynqrfkg</td>

<td> 117</td><td>Heavy chain CDR3</td><td>gpfay</td>

<td rowspan="3">5anE11</td><td> 102</td><td>Amino acid sequence VL</td><td>DIVMTQSQKFMSTSVGDRVSVTCKASQNVGT NVAWYQQKPGQSPKALIYSASYRYSGVPDRF TGSGSGTDFTLTISNVQSEDLAEYFCQQYNSY PWTFGGGTKLEIKR</td>

<td> 84</td><td>Light chain CDR1</td><td>kasqnvgtnva</td>

<td> 85</td><td>Light chain CDR2</td><td>sasyrys</td>

138

<td rowspan="5"></td><td> 103</td><td>Light chain CDR3</td><td>QQYNSYPWT</td>

<td> 143</td><td>Amino acid sequence VH</td><td>evqlqqsgpelvkpgasvrisckasgytfsdy YYMNWVKKSHGKSLEWIGYIFPKTGGTHYN QRFKGKATLTVDKSSSTAYMELRSLTSEDSA VYYCASGPFAYWGQGTLVTVSA</td>

<td> 115</td><td>Heavy chain CDR1</td><td>DYYYMN</td>

<td> 144</td><td>Heavy chain CDR2</td><td>YIFPKTGGTHYNQRFKG</td>

<td> 117</td><td>Heavy chain CDR3</td><td>GPFAY</td>

<td rowspan="8">5an183</td><td> 112</td><td>Amino acid sequence VL</td><td>DIVLTQSPASLAVSLGQRATISCRASESVDSY GNSFMHWYQQKPGQPPKJLLIYRASNLESGIP ARFSGSGSRTDFTLTINPVEADDVATYYCQQS NEDPLTFGAGTKLELKR</td>

<td> 20</td><td>Light chain CDR1</td><td>rasesvdsygnsfmh</td>

<td> 21</td><td>Light chain CDR2</td><td>RASNLES</td>

<td> 113</td><td>Light chain CDR3</td><td>QQSNEDPLT</td>

<td> 130</td><td>Amino acid sequence VH</td><td>EVQLQQPGSVLVRPGATVKLSCKASGFTFTSS WMHWAKQRPGQGLEWIGEIHTSGHTNYNEK FKGKATLTLDTSSSTAYVDISSLTSEDSAVYY CARGGLRRGYAMDYWGQGTSVTVSS</td>

<td> 131</td><td>Heavy chain CDR1</td><td>SSWMH</td>

<td> 132</td><td>Heavy chain CDR2</td><td>EIHTSGHTNYNEKFKG</td>

<td> 133</td><td>Heavy chain CDR3</td><td>GGLRRGYAMDY</td>

"SIN" in the table refers to "SEQ ID NO".

* CDR amino acid sequences are defined according to Kabat et al. (Supra).

Example 12. Additional humanized anti-human C5a antibodies [0388] Several additional heavy chain variable regions of humanized anti-C5a antibodies were generated, all of which, when pairing with a common light chain variable region (light chain with amino acid sequence

139 shown in SEQ ID NO: 16) bound to human C5a with a KD of less than 1 nM as determined by Biacore analysis (see above for methodology). All of these additional humanized antibodies bound specifically to human C5a, but did not bind to native, fully-folded human C5, C4a, or C3a as determined by Octet analysis (see above for methodology). Additional humanized antibodies contained: (i) framework heavy chain variable region 1 containing one of the following amino acid sequences:

QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 68) or QVQLVQSGSELKKPGASVKVSCKASGYTFT (SEQ ID NO: 69); (ii) framework heavy chain variable region 2 comprising one of the following amino acid sequences: WVRQAPGQGLEWMG (SEQ ID NO: 70) or WVRQASGKGLEWVG (SEQ ID NO: 71); (iii) framework heavy chain variable region 3 containing one of the following amino acid sequences:

RVTITRDTSASTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 72);

RVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 73); or RVTITRDRSMSTAYMELSSLRSEDTAMYYCAR (SEQ ID NO: 74); and the heavy chain variable region framework 4 containing the following amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 75). Exemplary additional humanized heavy chain variable regions containing one or more additional humanized framework kits described in this section are given in Figure 5.

Example 13. Use of anti-human C5a antibody in a mouse neutropenia model [0389] A mouse C5a-neutropenia model was used to evaluate the efficacy of anti-human free C5a antibody in vivo. To induce neutropenia, purified, native human C5a (hC5a) was administered by intravenous tail vein injection of Balb / c mice. The number of circulating neutrophils was assessed up to five minutes after administration of hC5a.

[0390] Administration of 300 μβ / kg hC5a was found to consistently induce neutrophil activation as measured by myeloperoxidase release (MPO) assay (see Example 5 for use with serum compared to cell culture supernatant, supra) and neutropenia (reduction of circulating neutrophil count) ). In addition, plasma levels of hC5a and human anti-C5a BNJ383 antibody (when administered to mice, infra) were also measured to determine the pharmacodynamic response (see below).

[0391] The number of neutrophils in peripheral blood was measured before hC5a challenge or vehicle control. Compared to sham-treated control mice (1.37 ± 0.09 x106/ ml), neutrophil count in mice treated with anti-human free C5a antibody (1.32 ± 0.13 x106 per ml 24 mg / kg; p> 0.05) or mAb isotype control (1.31 ± 0.10 x106 per ml, 24 mg / kg each; p> 0.05) remained the same. These results indicate that this antibody alone did not induce changes in the circulating neutrophil count.

[0392] To assess the effectiveness of the anti-C5a antibody in inhibiting hC5a-induced neutropenia in mice, different dosages (24 mg / kg, 12 mg / kg, 6 mg / kg and 3 mg / kg) of the anti-human C5a BNJ383 antibody were administered to mice Balb / c 24 hours before hC5a injection. Anti-C5a antibody administration 24 hours before hC5a enabled analysis

140 pharmacodynamic properties of the antibody during the β phase of antibody clearance in mice.

[0393] As shown in Fig. 6, the number of neutrophils after treatment is expressed as a percentage "for baseline" (where the number for time 0 is 100%). In sham-treated control mice, the neutrophil count was 79.02 ± 5.71%, 67.42 ± 3.23%, and 59.54 ± 2.11% for baseline at 1, 3 and 5 minutes, respectively hC5a. Mice treated with a control isotype antibody showed a significant reduction (p <0.01) in neutrophil counts to 6.76 ± 0.81% after 1 minute, 6.68 ± 0.81% after 3 minutes, and 8.29 ± 0, 79% 5 minutes after the intravenous injection of hC5a. Anti-C5a antibody showed a dose dependent effect on hC5a-induced neutropenia. At the highest dose, 24 mg / kg, anti-C5a antibody completely blocked neutropenia. The number of neutrophils was 70.35 ± 8.64% after 1 minute, 63.35 ± 6.08% after 3 minutes, and 59.65 ± 6.51% after 5 minutes, which was comparable to the level of neutrophils in sham patients control animals at the same time points. Lower doses of 12 mg / kg or 6 mg / kg anti-C5a antibody also significantly inhibited the reduction in neutrophil count (12 mg / kg: 42.61 5.12% after 1 minute, 45.33 ± 8.29% after 3 minutes, and 41.02 ± 7.08% after 5 minutes, p <0.01; 6 mg / kg: 18.00 ± 3.8 after 1 minute, 26.20 ± 4.44% after 3 minutes, and 28, 03 ± 4.51% after 5 minutes, p <0.05) after hC5a administration, compared to the control isotype antibody group (6.76 6 0.81% after 1 minute, 6.68 ± 0.81% after 3 minutes, and 8.29 0.79% after 5 minutes). The control isotype antibody is an antibody that binds protective antigen anthrax 63 and contains the human IgG2 / 4 isotype Fc region. The lowest dose of anti-C5a antibody (3 mg / kg) did not significantly reduce neutropenia (6.28 ± 0.88% after 1 minute, 6.71 ± 2.14% after 3 minutes, and 8.75 ± 2.98 % after 5 minutes, p> 0.05). See. fig. 6.

Anti-human C5a antibody inhibits hC5a-induced MPO release in vivo [0394] As discussed above, human C5a activates neutrophils by cross-reactive binding to the mouse C5a receptor. Release of myeloperoxidase (MPO) is a consequence of neutrophil activation by binding of C5a to C5aR. See. Darren et al. (2004) Mol Pharm 65 (4): 868-879. Intravenous injection of recombinant human C5a mice can induce neutropenia and activate neutrophils in the circulation. The ability of anti-human free C5a antibody to inhibit MPO release due to in vivo activation of neutrophils was evaluated using an anti-C5a antibody to bind free hC5a and prevent binding to mouse C5aR.

[0395] An in vivo experiment (in which human C5a was administered to mice) was performed as described above. The MPO plasma level at time 0 was 79.25 ± 22.88 ng / ml in the sham-treated control group. Five minutes after intravenous injection of buffer vehicle, MPO levels were not significantly changed (77.46 ± 21.21 ng / ml, p> 0.05). Prior to intravenous injection of hC5a, MPO levels in animals treated with a control isotype antibody (75.17 ± 14.66 ng / ml) or treated with an anti-C5a antibody (87.57 ± 14.86 ng / ml) were comparable to those observed in treated with sham control animals. After intravenous injection of hC5a, MPO levels for

141 all doses of C5a increased and remained at significantly higher levels after 5 minutes (Figure 7).

[0396] Compared with animals treated with the control isotype antibody (221.00 ± 51.02 ng / ml), animals treated with anti-hC5a antibody showed a dose-dependent reduction in MPO levels (114.83 ± 23.26 ng / ml, p <0.05, in the cohort 24 mg / kg; 104.80 ± 29.83 ng / ml, p <0.05, in the cohort 12 mg / kg; and 126.90 ± 36.40 ng / ml, p = 0.08, in the cohort 6 mg / kg). MPO levels levels in animals treated with low dose (3 mg / kg) anti-C5a antibody (176.55 ± 23.05 ng / ml) were not significantly different from those treated with animals with control isotype (p> 0.05) .

Anti-C5a antibody reduces circulating levels of hC5a in mice [0397] As noted above, C5a is a potent inflammatory peptide with several biological functions. These above studies have shown that human C5a cross-reacts with mouse C5aR on neutrophils, as intravenous injection of recombinant human C5a may induce neutropenia. While the disclosure is in no way limited by any particular theory or mechanism of action, the anti-C5a antibody can inhibit human C5a-induced neutropenia by forming complexes with hC5a and preventing binding of hC5a to mouse C5aR expressed on the cell surface. HC5a levels were measured in mouse plasma before and 1.3, and 5 minutes after intravenous administration of hC5a to confirm that the anti-hC5a antibody interactions in vivo were associated with its binding dependent inhibition of hC5a.

[0398] The experiment was performed as described above using the mouse-hC5a induced neutropenia model. hC5a was not detected in plasma in any group of mice prior to hC5a administration at time 0 using an enzyme immunosorbent assay (ELISA). The plasma hC5a level, however, increased to peak after 1 minute (7783.50 ± 327.73 ng / ml), then decreased to 4788.38 ± 260.51 ng / ml after 3 minutes, and then to 3855, 75 ± 298.99 ng / ml 5 minutes after intravenous injection of hC5a (in mice treated with mAb isotype control). Compared to control-treated mice, hC5a levels in mice treated with 24 mg / kg anti-C5a antibody showed 43, 30, and 23-fold decrease after 1 minute (178.4 6 14.14 ng / mL), respectively, 3 minutes (158) , 4 6 10.43ng / ml) and 5 minutes (167.2 6 15.61 ng / ml). Plasma hC5a levels in the 12 mg / kg and 6 mg / kg cohorts were 235.00 ± 22.33 and 609.20 ± 78.75 ng / ml, respectively, after 1 minute, 210.80 ± 19.59 and 527.60 ± 52.25 ng / ml after 3 minutes, 192.20 ± 7.40 and 505.00 ± 45.96 ng / ml after 5 minutes. Anti-C5a-treated mice showed significantly reduced levels of hC5a in a dose-dependent manner during neutropenia after intravenous injection of hC5a (p <0.001). Although mice receiving the lowest dose of anti-C5a antibody (3mg / kg) were not free of hC5a-induced neutropenia, mice nevertheless had a significant reduction in plasma hC5a (3130.40 ± 433.58 ng / ml after 1 minute; 1932.00 ± 268.92 ng / ml after 3 minutes; 1593.00 ± 169.68 ng / ml after 5 minutes) compared to plasma hC5a levels found in mice treated with the control isotype antibody (p <0.05). See. Figure 8. These dates indicated that administration of the anti-C5a antibody to mice significantly

142 reduced free hC5a plasma levels, resulting in significant improvements in hC5a-induced neutropenia.

[0399] Overall, the results presented in this section indicated that the anti-human C5a antibody described herein can inhibit the biological effect of human C5a in the disease system in vivo and provided strong evidence that the antibodies (and antigen-binding fragments thereof) are useful. in. in the treatment or prevention of disorders associated with the complement system, such as any of those cited herein.

Example 14. Anti-human C5a antibody cross-reacts with C5a from non-human primates [0400] Several humanized anti-hC5a antibodies were tested for their ability to cross-react with C5a from one or more non-human mammal species. As noted above, the benefits of such an anti-C5a antibody are numerous, e.g. enabling the researcher or healthcare professional to model the efficacy of the therapeutic anti-C5a antibody in a non-human disease model prior to administration of the antibody to humans. Tests conducted in non-human mammals may also allow determination or approximation of the appropriate dosage of anti-C5a antibody required to achieve efficacy in humans.

[0401] Briefly, BNJ369, BNJ366, BNJ364, and BNJ383 (described above) were evaluated to determine whether they could cause co-immunoprecipitation of the C5a protein in activated serum from several non-human primates, including baboon, rhesus macaque and cynomolgus. The serum was activated by adding zymosan. After the overnight incubation of each antibody with activated serum was completed, the antibodies were separated from the solution phase using protein A-coupled agarose beads. The beads were washed thoroughly and then boiled in β-mercaptoethanol buffer for applying samples on a polyacrylamide gel, for electrophoresis with addition of sodium dodecyl sulfate (SDS-PAGE). The boiled samples were then subjected to SDS-PAGE electrophoresis. Non-human primate C5a was detected by Western blot using a commercially available anti-C5a antibody for neoepitope # 2942 (Abeam, Cambridge, MA). Each of the tested antibodies was able to immunoprecipitate C5a (or C5a free of arginine) from baboon, rhesus macaque, and cynomolgus macaque, which indicates that the antibody cross-reacts with C5a from these species as well as with human C5a.

[0402] Determination of cynomolgus C5a binding affinity parameters was determined as described above. Briefly, the BNJ383 antibody was screened against 3-4 concentrations of C5a recombinant cynomolgus (antigen) using the capture technique as described above. Antibodies were captured using Anti-Fc (human) CM5 sensor directly immobilized on the chip at various concentrations in the range of 0.6 nM to 5.9 nM C5a Crab macaque moved over the surface of the sensor chip. The surface was regenerated with 20 mM HCl, with 0.02% P20 surfactant after each cycle to remove bound antibody and antigen. Data were evaluated using the Biacore BIAevaluation software using

143 Langmuir 1: 1 fit model (Rmax: Global Fit; RI: Local Fit). These experiments were conducted as screening tests, with a minimum number of analyte concentrations (3 to 4) in 1 replicate. The approximate KD of cynomolgus C5a antibody is 3.3 nM. See. Table 10

Table 10. Determination of affinity for non-human C5a protein

<td>Crab-eating macaque</td><td> 1,28</td><td> 42,3</td><td> 3300</td><td> 1,45</td>

<td>Mouse</td><td> 2,8</td><td> 10,6</td><td> 379*</td><td> 2,38</td>

<td colspan="5">* This is only an approximation KD based on the quality of the curve fit.</td>

[0403] Antibody BNJ383 was also screened against 3-4 concentrations of recombinant mouse C5a (antigen) using the capture technique as described above to determine its affinity for mouse protein. Antibodies were captured as described above using anti-Fc (human) CM5 sensor directly immobilized on the chip at various concentrations in the range of 0.6 nM to 5.9 nM mouse C5a moved over the surface of the sensor chip. The surface was regenerated with 20 mM HCl, 0.02% P20 after each cycle to remove bound antibody and antigen. Data were evaluated using the Biacore BIAevaluation software using a 1: 1 Langmuir fit model (Rmax: Global Fit; RI: Local Fit).

[0404] The above results indicate that several of the humanized anti-hC5a antibodies described herein cross-react with C5a from several non-human primate species, including Crab macaque, Rhesus macaque and baboon. The BNJ383 antibody, e.g., also cross-reacts with mouse C5a. In addition, the results described in this section indicate that the anti-human C5a antibody, such as BNJ383, is useful not only in clinical applications for the treatment of complement-related disorders, but also in a number of preclinical applications in non-human mammals that are necessary for , or supportive for obtaining authorization for human clinical use.

Example 15. Competition on binding to C5a [0405] An experiment was performed to assess the binding of the anti-C5a antibody described herein, BNJ383, in the presence of potentially competitive antigens. Briefly, ruthenium-labeled BNJ383 (250 pM) was incubated for two hours at room temperature with 1 nM biotinylated C5a, along with various concentrations (e.g. 400, 133, 44.4, 14.8, 4.9, 1.6 and 0.5 nM) of one of the following: (a) human C5a protein lacking arginine in phosphate buffered saline, (b) human plasma, ( c) cynomolgus plasma, (d) Balb / C plasma (mouse) or (e) DBA / 2J plasma (mouse). For plasma components (b), (c), (d) and (e), the concentration refers to the approximate final concentration of C5 antigen in the incubated mixture.

144 [0406] After the incubation period, the samples were contacted with the respective individual wells of the streptavidin coated assay plate under conditions that allowed binding of biotinylated C5a to streptavidin in the wells of the plate. The wells were washed thoroughly to remove unbound material. The amount of BNJ383 binding to C5a in the presence of competitor was determined by detecting the amount of signal produced from the detectable ruthenium tag. The results are shown in Figure 9.

[0407] While human C5a deficient in arginine was an effective competitor, essentially no competition was observed in the presence of mouse serum (17% reduction in detectable signal observed for a ratio of approximately 400: 1 C5 derived from mouse Balb / C plasma relative to biotinylated human C5a and % reduction in detectable signal observed for a ratio of approximately 400: 1 C5 derived from plasma DBA2 / J relative to biotinylated human C5a). No change in the level of BNJ383 binding to biotinylated human C5a was observed up to approximately 15: 1 C5 derived from human plasma or cynomolgus versus biotinylated human C5a.

[0408] As noted above, while the disclosure is not limited in any way to any particular theory or mechanism of action, the inventors hypothesize that the anti-C5a antibody may bind to a subpopulation of non-cleaved, C5-treated (e.g., C5 plasma) representing less than 10% of the total full-length C5 population in the sample (e.g. plasma sample), which subpopulation is fully or partly denatured such that otherwise the covered C5a neoepitope to which the anti-C5a antibody or binding fragment thereof binds becomes exposed. Hence, it is believed that the antibody does not bind to the fully functional and / or fully functional type of C5 and hence does not actually bind to non-cleaved native C5. Human plasma is at least approximately 30 to 100 times weaker competitor in the fight for binding to biotinylated C5a than to human C5a lacking arg.

[0409] Notwithstanding these considerations, the results further indicate that the anti-human C5a antibodies described herein, such as BNJ383, preferentially bind to free human C5a even in the presence of up to an approximately 20-fold excess of unresected, but not necessarily completely native , human plasma derived C5 protein.

Example 16. Effect of anti-C5a antibody on AP and CP activity in vitro [0410] An experiment was performed to check the effect of the anti-C5a antibody described herein (BNJ383) on the complement pathway (AP) activity of the complement system in vitro using pooling normal human serum (PNHS). The experiment used the Wieslab® kit for the alternative complement pathway (Wieslab® COMPL AP330, Euro-Diagnostica, Sweden) and the attached protocol was followed, with only routine optimization within the discretion of the skilled person. Briefly, PNHS aliquots were incubated in wells of the lipopolysaccharide coated plate for one hour (at 37 ° C) with various concentrations (0.778, 0.389, 0.194, 0.097, 0.049, 0.024, 0.012, 0.006, 0.003 and 0.002 μΜ) of anti-hC5 antibody or

145 anti-hC5a antibodies (BNJ383). Anti-C5 antibody inhibits the cleavage of human C5 into fragments C5a and C5b; as a negative control, several wells were incubated with PNHs under the same conditions, but in the absence of anti-hC5 antibody or anti-hC5a antibody.

[0411] After incubation, the wells were thoroughly washed with the 1X wash buffer provided in the kit. The level of activation of the alternative complement pathway was measured by absorbance at 405 nm, after contact of each well with the enzyme conjugate (anti-C5b-9 antibody conjugated with alkaline phosphatase conjugated) and fluorogenic substrate (on which the enzyme acts) and the ongoing min incubation at room temperature. The results are shown in Figure 10.

[0412] While the anti-C5 antibody completely inhibited the activity of the alternative complement pathway at concentrations higher than 0.1 μΜ, the anti-hC5a antibody did not significantly inhibit complement activity even at the highest concentrations tested.

[0413] An experiment was performed to evaluate the effect of the anti-C5a antibody described herein (BNJ383) on the classical pathway (CP) activity of the complement system in vitro using PNHs. The experiment used the Wieslab® kit for the classic complement pathway (Wieslab® COMPL CP310, Euro-Diagnostica, Sweden) and the attached protocol was followed, only with routine optimization within the discretion of the skilled person. Briefly, aliquots of PNHs were incubated in the wells of a plate coated with human IgM for one hour (at 37 ° C) together with various concentrations (7.2, 3.6, 1.8, 0.9, 0.45, 0.2, 0.1, 0.05, 0.02 or 0.01 μΜ) anti-hC5 antibodies or anti-hC5a antibodies (BNJ383). The anti-C5 antibody inhibits the cleavage of human C5 into C5a and C5b fragments. As a control, several wells were incubated with PNHs under the same conditions, but in the absence of anti-hC5 antibody or anti-hC5a antibody.

[0414] After incubation, the wells were thoroughly washed with the 1X wash buffer provided in the kit. The level of activation of the alternative complement pathway was measured by absorbance at 405 nm, after contact of each well with the enzyme conjugate (anti-C5b-9 antibody conjugated with alkaline phosphatase conjugated) and fluorogenic substrate (on which the enzyme acts) and the ongoing min incubation at room temperature. The results are shown in Figure 11.

[0415] While the anti-C5 antibody completely inhibited the activity of the classical complement pathway at concentrations higher than 0.1 μΜ, the anti-hC5a antibody did not significantly inhibit complement activity even at the highest concentrations tested.

Taken together, these results indicate that in vitro, the anti-hC5a antibody, BNJ383, did not significantly affect C5b-9 formation (activation of the complement terminal system) in the course of a classic or alternative complement pathway, hence this confirms that the anti-hC5a antibodies described herein specifically target the free C5a of the anaphylatoxin arm in complement activation.

146

Example 17. Anti-C5a antibody maintains a free antigen binding site available for binding to C5a even in the presence of molar excess of hC5 [0417] Anti-C5a antibody BNJ383 (listed above) was incubated at 4 ° C for 84 hours in the presence of 2.1- fold molar excess of human C5 (hC5) to allow complete formation of the C5 antibody complex. A parallel experiment was performed using an antibody that binds to human C5 at 2: 1 stoichiometry (hereinafter anti-C5 antibody). Antibody: C5 complexes were separated on a TSK ™ G4000 SW size exclusion column (Tosoh, Tokyo) using a Waters ™ 2690/5 HPLC system with a Waters ™ W2487 dual wavelength detector to determine binding site binding. Peaks were monitored at 214 nm. The mobile phase in the HPLC analysis contained a buffer with the following composition: 3.9 mM NaH2PO4, 6.1 mM Na2HPO4, 150 mM NaCl, at pH7.0. The flow rate was 1.0 ml per minute and the analysis time was 20 minutes. Data was collected and analyzed using Waters Empower ™ 2 chromatography software.

[0418] As shown in Fig. 12, BNJ383 alone (Fig. 12A) and hC5 alone (Fig. 12B) each separated into a single peak approximately 10.2 minutes and> 95% anti-C5a antibody complexes: hC5 separated as a single peak approximately for 9.2 minutes (Figure 12C). In contrast, hC5 and anti-C5 antibody complexes separated into two peaks at 8.6 min (39%) and 9.2 min (61%) (Figure 12D). Thus, even in a molar excess of hC5, 95.2% BNJ383 had a free Fab arm that may be able to bind to human C5a.

[0419] These samples were further analyzed to determine if BNJ383 antibody retained the ability to bind C5a in the presence of saturating C5 concentrations. Free antibody or antibody complexes: C5 was titrated from 500 to 0.5 ng / ml on a streptavidin coated plate on which biotin-conjugated hC5a was immobilized. Captured antibody was detected with horseradish peroxidase conjugated anti-human Fc antibody. The results presented in Fig. 13 demonstrate that even BNJ383 in complex with hC5 is capable of binding C5a and that the concentration of antibody available for C5a binding is not detectably reduced in the presence of saturating C5. Hence, the results presented herein indicate that the antibody, even in the presence of a molar excess of un cleaved C5, retains the ability to bind to free C5a with high affinity and thus retains, even in molar excess, the ability to inhibit C5a pro-inflammatory activity.

Examples 18. BNJ383 is a potent C5a antagonist but is an incomplete / partial antagonist of terminal complement formation in vivo [0420] Cynomolgus macaques were administered intravenously with a single dose of BNJ383 anti-C5a antibody at 1 mg / kg, 10 mg / kg, 100 mg / kg , 250 mg / kg or 400 mg / kg. Plasma samples were collected from macaques at time points ranging from 1 day to 30 days after antibody administration. Plasma levels of arginine-deficient C5a / C5a were determined by an electrochemiluminescent assay (ECL) in which free argent-free C5a / C5a was captured on a microtiter plate coated with an antibody specific for

147 neoepitope on C5a / C5a devoid of arg and was detected by means of a non-competitive C5a antibody conjugated with a ruthenium containing an ECL moiety and read on a SECTOR 2400 ™ plate reader (MesoScale Discovery). Circulating antibody concentrations were determined using an enzyme immunosorbent assay (ELISA) in which free antibody (BNJ383) was captured on an arga-free human C5a coated microtiter plate detected with mouse anti-human horseradish peroxidase conjugated antibody (HRP).

[0421] As shown in Figure 14, similar to the results described in Example 13, circulating concentrations of BNJ383 as low as 10 Lig / ml reduce plasma levels of C5a / C5a depleted of arg below the detection limits of cynomolgus macaques. Also these results indicate that the antibody, even in the presence of a molar excess of un cleaved C5, retains the ability to bind to free C5a with high affinity and thus retains, even in molar excess, the ability to inhibit C5a pro-inflammatory activity.

[0422] To determine if BNJ383 had an effect on the hemolytic activity of macaque serum, the antibody was evaluated in a hemolytic red cell assay in vitro.

[0423] The hemolytic assay for red blood cells is generally described in detail in, e.g., Rinder et al. (1995) J Clin Invest 96: 1564-1572. Briefly, serum samples obtained from macaques treated with BNJ383 (as described above) were added to many wells of a 96-well assay plate such that the serum concentration in each well was approximately 10%. Serum samples, grouped for the time points at which they were obtained, contained different concentrations of BNJ383 antibody. Hemolytic activity of serum from macaques that did not receive BNJ383 served as negative control and as hemolytic activity at baseline.

[0424] Chicken erythrocytes (Lampire Biological Laboratories, Pipervilie, PA) were washed and resuspended in buffer at a final concentration of 5 x 107 cells / ml. Erythrocytes were sensitized to lysis by incubating cells with a polyclonal antibody composition against chicken erythrocytes. Sensitized erythrocytes were added to the wells of a 96 well plate, and the plate was incubated at 37 ° C for 30 minutes. Hemoglobin release was measured by apparent absorbance at 415 nm using a microplate reader.

[0425] As shown in Figure 15A, even high concentrations of BNJ383 did not significantly inhibit erythrocyte hemolysis under these experimental conditions of an ex vivo hemolytic assay.

[0426] Antibody BNJ383 was also evaluated to determine whether it had an effect on complement activation in macaque serum using an ex vivo CH50eq assay. The CH50eq assay is a way of measuring the overall activity of classical complement in serum. This test is an enzyme-linked immunosorbent assay that uses human gammaglobulin and mouse monoclonal antibodies as activators of the classic complement pathway, and captures terminal complement complexes

148 (TCC) generated in the well of a microtiter plate coated with a TCC neoepitope specific antibody. Captured TCC was detected with a goat anti-TCC antibody conjugated to horseradish peroxidase. The CH50eq assay provides a direct measure of the formation of the terminal complement complex (TCC).

[0427] As shown in Figure 15B, high concentrations of BNJ383 present in the serum of macaques were able to significantly inhibit TCC formation under these ex vivo experimental conditions. These results indicate that the BNJ383 antibody is not only able to bind to and separate free C5a, but is also able, as a function of concentration, to partially or significantly inhibit TCC formation.

[0428] An ex vivo experiment was also performed to assess the effect of BNJ383 on the complement classical pathway (CP) activity using macaque serum samples described above. The experiment used the Wieslab® kit for the classical complement pathway (Wieslab® COMPL CP310, Euro-Diagnostica, Sweden) and the attached protocol was followed, with only routine optimization within the discretion of the skilled person. Briefly, aliquots of macaque serum samples were incubated in the wells of a plate coated with human IgM for one hour. As a control, several wells were incubated under the same conditions with serum from macaques not treated with BNJ383 antibody.

[0429] After incubation, the wells were thoroughly washed with the 1X wash buffer provided in the kit. The level of activation of the alternative complement pathway was measured by absorbance at 405 nm, after contact of each well with the enzyme conjugate (anti-C5b-9 antibody conjugated with alkaline phosphatase conjugated) and fluorogenic substrate (on which the enzyme acts) and the ongoing min incubation at room temperature. The results are shown in Fig. 15C.

[0430] Anti-hC5a antibody did indeed, although not completely, inhibit complement activity in a dose-dependent manner. Taken together, the results presented herein indicate that BNJ383 is not only a potent C5a antagonist, but is also an incomplete / partial antagonist of the formation of the terminal complement complex in vivo. hence, the antibody and the antibodies sharing its properties are useful for treating a number of complement-related disorders in which C5a-mediated inflammation is the primary contributor to harmful pathological effects and TCC may play a less significant or even beneficial role in the pathological state .

[0431] While the disclosure has been described with reference to specific embodiments, those skilled in the art should be aware that various changes can be made, and equivalents can be made. In addition, many modifications can be made to suit a particular situation, material, matter composition, method, step or method steps.

Piotr Godlewski Patent Attorney

149

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