Humanized anti-C5a antibody and its uses

Abstract

Inhibition of complement signaling using a humanized anti-C5a antibody is provided. A method of treating a complement-related disease or disorder in an individual by contacting the individual with the humanized anti-C5a antibody is provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of International Patent Application PCT / CN2021 / 121959, filed September 29, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 792252000941SEQLIST.TXT, Recorded: September 26, 2022, Size: 88,835 bytes).

[0003] The present invention relates to a humanized anti-C5a antibody and its use. [Background technology]

[0004] The complement system is part of the innate immunity that plays an important role in host defense. However, activated complement can also cause significant tissue damage and destruction, and dysregulated complement activity has been found to be associated with many rare and common diseases, including paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome, rheumatoid arthritis, and age-related macular degeneration. Therefore, anti-complement therapy is a promising approach to treat these disorders in humans.

[0005] Complement C5 is a crucial protein in the terminal pathway of complement activation and is the precursor protein that generates the potent proinflammatory mediator C5a as well as the cytolytic membrane attack complex (MAC), C5b-9.

[0006] In some complement-related diseases, both C5a- and MAC-mediated processes may contribute to pathogenesis, whereas in others, only C5a-related inflammation or MAC-related cellular injury may be involved. Because complement mediators, including C5a and MAC, also play important roles in host defense against pathogen infection, it is desirable to develop anticomplement drugs that are selective, i.e., that block only the deleterious effects of complement in tissue damage while leaving its normal host defense functions intact.

[0007] The hemolytic disease PNH is caused by MAC. Other anti-C5 mAbs exist to treat PNH. However, these antibodies unnecessarily block the production of C5a, putting patients at greater risk of infection than therapeutics that block MAC alone. Similarly, there are complement-related diseases that may be primarily mediated by C5a-dependent inflammation (e.g., sepsis), and for such conditions, anti-C5 mAb drugs, while expected to be effective, may unnecessarily block MAC as a side effect.

[0008] Thus, there is a need in the art for anti-human C5a mAbs that can inhibit C5a-mediated activities but do not block MAC activity.The present invention addresses these and other needs.

[0009] All references cited herein, including patent applications, patent application publications, and Genbank accession numbers, are incorporated by reference to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference in its entirety. Summary of the Invention

[0010] The present application provides humanized anti-C5a antibodies, including humanized anti-C5a antibodies that bind to C5a and C5 (the precursor of C5a, which the antibody also binds to, but does not block function) in a pH-dependent manner.

[0011] In some embodiments, an isolated humanized antibody that specifically binds to human C5a and C5 is provided, the antibody comprising a heavy chain variable domain (VH) comprising mutations I48M, D54E, and N56W, and a light chain variable domain (VL) comprising mutations D28E and D30F, wherein said VH mutations refer to SEQ ID NO: 1 under the Kabat numbering system, and said VL mutations refer to SEQ ID NO: 2 under the Kabat numbering system. In some embodiments, the antibody comprises i) a heavy chain CDR1 ("H-CDR1") comprising the amino acid sequence of SEQ ID NO:3, or a variant thereof comprising one, two, or three amino acid substitutions; ii) a heavy chain CDR2 ("H-CDR2") comprising the amino acid sequence of SEQ ID NO:4, or a variant thereof comprising one, two, or three amino acid substitutions; iii) a heavy chain CDR3 ("H-CDR3") comprising the amino acid sequence of SEQ ID NO:5, or a variant thereof comprising one, two, or three amino acid substitutions; iv) a light chain CDR1 ("L-CDR1") comprising the amino acid sequence of SEQ ID NO:6, or a variant thereof comprising one, two, or three amino acid substitutions; v) a light chain CDR2 ("L-CDR2") comprising the amino acid sequence of SEQ ID NO:7, or a variant thereof comprising one, two, or three amino acid substitutions; and vi) a light chain CDR3 ("L-CDR3") comprising the amino acid sequence of SEQ ID NO:8, or a variant thereof comprising one, two, or three amino acid substitutions.

[0012] In some embodiments, the antibody further comprises the mutation F29H in VH and the mutation Y96H in VL (wherein the VH mutation refers to SEQ ID NO: 1 under the Kabat numbering system and the VL mutation refers to SEQ ID NO: 2 under the Kabat numbering system).

[0013] In some embodiments, the antibody further comprises an additional substitution in the VH or VL, hi some embodiments, the mutation is selected from the group consisting of E54H in VH, N97H in VH, and N92H in VL (wherein the VH mutation refers to SEQ ID NO: 1 under the Kabat numbering system, and the VL mutation refers to SEQ ID NO: 2 under the Kabat numbering system).

[0014] In some embodiments, the antibody comprises i) a VH comprising the amino acid sequence SEQ ID NO:9, or a variant thereof that is at least about 85% identical (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, or 99%) to SEQ ID NO:9; and ii) a VL comprising the amino acid sequence of SEQ ID NO:10, or a variant thereof that is at least about 85% identical (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, or 99%) to SEQ ID NO:10.

[0015] In some embodiments, the antibody comprises i) an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 11; ii) an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 12; iii) an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 13; iv) an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 14; v) an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 15; and vi) an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 16.

[0016] In some embodiments, the antibody comprises i) a VH comprising the amino acid sequence of SEQ ID NO:17, and ii) a VL comprising the amino acid sequence of SEQ ID NO:18.

[0017] In some embodiments, the antibody comprises i) an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 19; ii) an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 20; iii) an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 21; iv) an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 22; v) an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 23; and vi) an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 24.

[0018] In some embodiments, the antibody comprises i) a VH comprising the amino acid sequence of SEQ ID NO:25, and ii) a VL comprising the amino acid sequence of SEQ ID NO:26.

[0019] In some embodiments, the antibody comprises i) an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 27; ii) an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 28; iii) an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 29; iv) an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 30; v) an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 31; and vi) an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 32.

[0020] In some embodiments, the antibody comprises i) a VH comprising the amino acid sequence of SEQ ID NO:33, and ii) a VL comprising the amino acid sequence of SEQ ID NO:34.

[0021] In some embodiments according to any one of the antibodies described herein, the antibody is selected from the group consisting of a full length antibody, a Fab, a Fab', a F(ab)2, a F(ab')2, and a scFv.

[0022] In some embodiments, the antibody further comprises an Fc region. In some embodiments, the Fc region comprises an IgG4 sequence. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 43, or a variant thereof. In some embodiments, the Fc region comprises one or more mutations selected from the group consisting of S228P, M428L, and N434A (wherein the mutations are compared to SEQ ID NO: 43 under the EU numbering system). In some embodiments, the Fc region comprises the mutations S228P, M428L, and N434A. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 44.

[0023] In some embodiments according to any one of the antibodies described herein, the neutral pH dissociation factor of the antibody dissociating from C5 is about 40% to about 70%. In some embodiments, the neutral pH dissociation factor of the antibody dissociating from C5 is about 0% to about 10%. In some embodiments, the ratio of neutral pH dissociation to neutral pH dissociation is 6 or greater.

[0024] In some embodiments, the antibody inhibits binding between human C5a and C5aR.

[0025] In some embodiments, the antibody has a serum half-life in humans of at least about 25 days.

[0026] In some embodiments, the antibody is produced in CHO cells.

[0027] In some embodiments, a nucleic acid encoding any one of the antibodies described herein is provided, comprising the sequence of any one of SEQ ID NOs: 46-53.

[0028] In some embodiments, a vector is provided that comprises a nucleic acid of any one of SEQ ID NOs: 46-53.

[0029] In some embodiments, methods are provided for producing any one of the antibodies described herein under conditions sufficient to allow expression of the antibody by a cell.

[0030] In some embodiments, a pharmaceutical composition is provided comprising any one of the antibodies described herein and a pharma- ceutical acceptable carrier.

[0031] In some embodiments, a method of treating an individual with a complement-related disease or condition is provided comprising administering to the individual an effective amount of a pharmaceutical composition described herein. In some embodiments, the selected disease or disorder is selected from macular degeneration (MD), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, rheumatoid arthritis, lupus, ulcerative colitis, stroke, postoperative systemic inflammatory syndrome, asthma, allergic asthma, chronic obstructive pulmonary disease (COPD), paroxysmal nocturnal hemoglobinuria (PNH) syndrome, autoimmune hemolytic anemia (AIHA), Gaucher disease, myasthenia gravis, neuromyelitis optica, (NMO), multiple sclerosis, delayed graft function, antibody-mediated rejection, atypical hemolytic uremic syndrome (aHUS), central retinal vein occlusion (CRVO), central retinal artery occlusion (CRAO), epidermolysis bullosa, sepsis, septic shock, organ transplantation, inflammation (including, but not limited to, and / or combinations thereof), C3 nephropathy, membranous nephropathy, IgA nephropathy, glomerulonephritis (non-limiting examples of which include inflammation associated with cardiopulmonary bypass surgery and kidney dialysis), ANCA-associated vasculitis, Shiga toxin-induced HUS, and antiphospholipid antibody-induced pregnancy loss, graft-versus-host disease (GVHD), bullous pemphigoid, hidradenitis suppurativa, dermatitis herpetiformis, Sweet's syndrome, pyoderma gangrenosum, palmoplantar pustulosis and pustular psoriasis, rheumatic neutrophilic dermatosis, subcorneal pustulosis, gut-associated dermatosis-arthritis syndrome, neutrophilic eccrine hidradenitis, linear IgA disease, or any combination thereof.

[0032] In some embodiments, a method is provided for reducing activity of the complement system in an individual, comprising administering to the individual an effective amount of a pharmaceutical composition described herein.

[0033] In some embodiments according to any one of the antibodies described herein, the antibody cross-reacts with cynomolgus monkey C5a or C5.

[0034] These and other aspects and advantages of the present invention will become apparent from the following detailed description and the appended claims. It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. [Brief description of the drawings]

[0035] [Figure 1A] FIG. 1A shows the results of an affinity ranking ELISA with combination mutant scFvs of anti-C5a constructs using cynomolgus monkey antigen. [Figure 1B] FIG. 1B shows the results of an affinity ranking ELISA with combination mutant scFvs of anti-C5a constructs using cynomolgus monkey antigen.

[0036] [Figure 2A] FIG. 2A shows the results of an affinity ranking ELISA with combination mutant scFvs of anti-C5a constructs using human antigen. [Figure 2B] FIG. 2B shows the results of an affinity ranking ELISA with combination mutant scFvs of anti-C5a constructs using human antigen.

[0037] [Diagram 3] Figure 3 shows the results of characterization of anti-C5a antibody binding to C5a. Four humanized anti-C5a constructs (16D10, E54, N92, and N97) were serially diluted and added to a plate pre-coated with C5a, and OD450 was measured at 450 nm by a microplate reader. Each data point is the average of two replicates.

[0038] [Figure 4] Figure 4 shows the results of in vitro activity of anti-C5a antibody in sheep RBC lysis assay. Antibody-sensitized sheep RBC was incubated with 5% normal human serum in the presence of different concentrations of anti-C5a antibody. OD405 was measured at 405 nm by a microplate reader.

[0039] [Diagram 5] Figure 5 shows the in vitro activity of anti-C5a antibodies in a ligand blocking assay. High control: fluorescent signal of binding of biotinylated C5a without anti-C5a antibodies; Low control: fluorescent signal of cells alone without biotinylated C5a.

[0040] [Figure 6] FIG. 6 shows the in vitro activity of anti-C5a antibodies in a human C5a-induced transmigration assay using U937 cells expressing C5aR.

[0041] [Figure 7] FIG. 7 shows the in vitro activity of anti-C5a antibodies in a C5a-dependent intracellular calcium mobilization assay on a Fluorescence Imaging Plate Reader (FLIPR).

[0042] [Figure 8] FIG. 8 shows the characterization of anti-C5a antibody binding to human C5.

[0043] [Figure 9] FIG. 9 shows Gator tracings of the association and dissociation of human C5 with recombinant E54, N97, N92, and WT(16D10) at pH 5.8 and pH 7.4.

[0044] [Figure 10] FIG. 10 shows the percentage of C5 dissociated from the peak values ​​for recombinant E54, N97, N92, and WT(16D10) at pH 5.8 and pH 7.4.

[0045] [Figure 11] FIG. 11 shows the pharmacokinetic profile of anti-C5a antibodies in C5 / FcRn-humanized SCID mice.

[0046] [Figure 12]FIG. 12 shows Western blots of human C5 protein in serum of C5 / FcRn-humanized SCID mice at various time points after a single intravenous dose of anti-C5a antibody.

[0047] [Figure 13] FIG. 13 shows the results of a sandwich ELISA to detect human C5 protein in the serum of C5 / FcRn-humanized SCID mice after a single intravenous injection of anti-C5a antibody at 25 mg / kg.

[0048] [Figure 14] FIG. 14 shows the results of C5 titration in a sheep RBC lysis assay using C5-deficient normal human serum.

[0049] [Figure 15] FIG. 15 shows the results of C5 titration in a rabbit RBC lysis assay using C5-deficient normal human serum.

[0050] [Figure 16] Figure 16 shows single doses of N92H administered intravenously to cynomolgus monkeys (one male and one female per dose group) at 5, 10, and 30 mg / kg. Concentrations were determined using an ELISA method.

[0051] [Figure 17] Figure 17 shows a single dose of N92H administered intravenously at 5 mg / kg to cynomolgus monkeys (one male and one female). CD11b expression was measured in vitro by stimulating whole blood samples with 10 nM and 30 nM C5a.

[0052] [Figure 18] Figure 18 shows that C5a (10 μg / kg) was administered intravenously at 6 hours (hr), 2, 7, and 14 days after infusion of N92H. Blood samples were collected 1 min before and 1 min after C5a injection and neutrophils were counted.

[0053] [Figure 19]Figure 19 shows the percent change in neutrophil counts in blood collected 1 min after C5a injection compared to samples collected 1 min before C5a injection. Each bar represents the mean ± SEM from two individual monkeys. Cynomolgus monkeys pre-treated with N92H showed complete inhibition of C5a-induced neutropenia, and this rescue effect could persist for at least 14 days. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] The present invention relates to the inhibition of complement signaling using humanized anti-C5a antibodies. In another embodiment, the present invention is directed to inhibiting the complement signaling cascade by specifically inhibiting the function of C5a protein while minimizing the disruption of the C5-b-mediated complement pathway. In one embodiment, the present invention is directed to a method for treating and preventing inflammatory and autoimmune diseases and disorders associated with unwanted, uncontrolled, or excessive complement activation. In one embodiment, the present invention is directed to treating a complement-associated disease or disorder in an individual by contacting the individual with a humanized anti-C5a antibody.

[0055] In various embodiments, the present invention is directed to compositions and methods for treating a complement-related disease or disorder in an individual by contacting the individual with an anti-C5a antibody. Non-limiting examples of complement-related diseases and disorders that can be treated with the compositions and methods of the present invention include macular degeneration (MD), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, rheumatoid arthritis, lupus, ulcerative colitis, stroke, postoperative systemic inflammatory syndrome, asthma, allergic asthma, chronic obstructive pulmonary disease (COPD), paroxysmal nocturnal hemoglobinuria (PNH) syndrome, autoimmune hemolytic anemia (AIHA), Gaucher disease, myasthenia gravis, neuromyelitis optica, (NMO), multiple sclerosis, delayed organ transplant function, antibody-mediated rejection, atypical hemolytic uremic syndrome (aHU), and other conditions. S), central retinal vein occlusion (CRVO), central retinal artery occlusion (CRAO), epidermolysis bullosa, sepsis, septic shock, organ transplant, inflammation (non-limiting examples of which include inflammation associated with cardiopulmonary bypass surgery and kidney dialysis), C3 nephropathy, membranous nephropathy, IgA nephropathy, glomerulonephritis (non-limiting examples of which include antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, lupus nephritis, and combinations thereof), ANCA-associated vasculitis, Shiga toxin-induced HUS, and antiphospholipid antibody-induced pregnancy loss, graft versus host disease (GVHD), or any combination thereof.

[0056] The present application provides novel humanized anti-human C5a antibodies that comprise a heavy chain variable domain (VH) and a light chain variable domain (VL) and that specifically bind to and inhibit the function of human C5a. In some aspects, the anti-C5a antibody contains mutations in its VH and / or VL domains. In one aspect, the mutations in the VH and VL render the binding of the antibody to the antigen pH sensitive.

[0057] In another aspect, methods are provided for inhibiting complement activation and / or treating diseases (such as complement-associated diseases) by administering any one or more of anti-C5a antibodies or constructs thereof.

[0058] In another aspect, exemplary nucleic acids encoding any one or more of the anti-C5a antibodies or constructs thereof are provided, as well as vectors or host cells containing such nucleic acids. Methods of making anti-C5a antibodies are also described.

[0059] I. Definition Unless otherwise defined, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, representative methods and materials are described.

[0060] As used herein, "treatment" or "treating" is an approach to obtain beneficial or desired results, including clinical results. For purposes of this application, non-limiting examples of beneficial or desired clinical results include one or more of the following: reducing one or more symptoms resulting from a disease, decreasing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the progression of the disease), preventing or slowing the onset or recurrence of the disease, slowing or slowing the progression of the disease, improving the disease state, providing remission (partial or complete) of the disease, reducing the dose of one or more other drugs required to treat the disease, slowing disease progression, improving quality of life, and / or prolonging survival. Also encompassed by "treatment" is the reduction of pathological consequences of the disease. The methods of this application contemplate any one or more of these aspects of treatment.

[0061] The terms "effective amount" and "therapeutically effective amount" are used herein to refer to an amount of an agent sufficient to provide a desired biological result, which can be either the reduction and / or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system.

[0062] As used herein, the terms "patient," "subject," "individual," and the like, are used interchangeably and refer to any animal with a complement system, and in some embodiments a mammal, and in some embodiments a human, including a human in need of treatment for or susceptible to a condition or its sequelae. Individuals can include, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats, monkeys, mice, and humans. In some embodiments, an individual is a human.

[0063] The term "antibody" is used herein in the broadest sense to encompass a variety of antibody structures, non-limiting examples of which include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired antigen-binding activity.

[0064] "Antibody" can refer to an immunoglobulin molecule or fragments thereof capable of specifically binding to a specific epitope of an antigen (comprising the basic four-chain antibody unit). Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources, and immunoreactive portions of intact immunoglobulins. Antibodies of the present invention can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), antigen-binding fragments (such as Fv, Fab, Fab', F(ab)2, and F(ab')2), as well as single chain antibodies (scFv), heavy chain antibodies (such as camelid antibodies), and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0065] The term "antigen-binding fragment" as used herein refers to antibody fragments, including, for example, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain Fvs (scFv), scFv dimers (bivalent diabodies), multispecific antibodies formed from a portion of an antibody containing one or more CDRs, camelized single domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure. An antigen-binding fragment can bind to the same antigen as the parent antibody or parent antibody fragment (e.g., parent scFv). In some embodiments, an antigen-binding fragment can include one or more CDRs from a particular human antibody grafted onto framework regions from one or more different human antibodies.

[0066] An "Fv" is the smallest antibody fragment and contains one complete antigen recognition and binding site. This fragment consists of a dimer of one heavy and one light chain variable region domain in tight non-covalent association. The folding of these two domains gives rise to six hypervariable loops (three loops each from the heavy and light chains) that contribute amino acid residues for antigen binding and thus confer antigen-binding specificity to the antibody. However, even a single variable domain (or even half of an Fv, containing only the three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, albeit with less affinity than the entire binding site.

[0067] A "single-chain Fv", also abbreviated as "sFv" or "scFv", consists of VH and VF linked together into a single polypeptide chain. L In some embodiments, the scFv polypeptide is an antibody fragment comprising a VH domain and a V LThe domains further comprise a polypeptide linker that enables the scFv to form the desired structure for binding to an antigen. For a review of scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0068] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five of these basic heterotetrameric units plus an additional polypeptide called the J chain, which contains 10 antigen-binding sites, whereas IgA antibodies contain two to five basic four-chain units, which can combine with the J chain to polymerize and form multivalent assemblies. In the case of IgG, the four-chain unit is generally about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, whereas the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the α and γ chains. H ) and four C for isotypes μ and ε H Each L chain has a variable domain (V L ) followed by a constant domain at the other end. L is aligned with VH, and C L is the first constant domain of the heavy chain (C H 1) and specific amino acid residues are thought to form an interface between the light and heavy chain variable domains. VH and V LWhen paired together they form a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, p. 71 and Chapter 6. Light chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Immunoglobulins are classified into two classes: H Depending on the amino acid sequence of the constant domain of the immunoglobulin, they can be assigned to different classes or isotypes. There are five classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ. Classes γ and α are H They are further divided into subclasses based on relatively slight differences in sequence and function; for example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0069] The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of the antibody are determined by sequences within the Fc region. This region is also recognized by Fc receptors (FcRs) found on the surface of certain types of cells.

[0070] An "isolated" antibody is an antibody that has been identified, separated, and / or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, an isolated polypeptide is free of any other components from its production environment. Contaminating components of its production environment (such as those resulting from recombinant transfected cells) are materials that would typically interfere with the antibody's use in research, diagnosis, or therapy, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide is purified (1) to greater than 95% by weight of the antibody, as determined, for example, by the Lowry method, and in some embodiments, greater than 99% by weight of the antibody; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions with Coomassie blue or silver staining, the latter being preferred. An isolated antibody includes the antibody in the recombinant cell itself, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated polypeptide or antibody will be prepared by at least one purification step.

[0071] "Variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody. The heavy and light chain variable domains can be referred to as "VH" and "VL", respectively. These domains are generally the most variable part of the antibody (relative to other antibodies of the same class) and contain the antigen binding site. Heavy-chain-only antibodies from camelid species have a single heavy chain variable region, which is referred to as a "VHH". A VHH is therefore a special type of VH.

[0072] The term "variable" refers to the fact that the sequences of certain sections within the variable domains vary widely among antibodies. The V domains mediate binding to antigens and define the specificity of a particular antibody for its corresponding antigen. However, the variability is not uniformly distributed throughout the span of the variable domains. Instead, it is concentrated in three sections, called hypervariable regions (HVRs), in both the light and heavy chain variable domains. The more conserved parts of the variable domains are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FR regions that mostly adopt a beta-sheet structure and are connected by three HVRs that form loops that connect, and in some cases form part of, the beta-sheet structure. The HVRs in each chain are held in close proximity to each other by the FR regions and, together with the HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0073] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies; i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by a hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies utilized in accordance with the present application may be produced by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2 nded. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display technology (see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004), and techniques for producing human or human-like antibodies in animals that have some or all of the human immunoglobulin loci, or genes encoding human immunoglobulin sequences (e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio / Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

[0074] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably and refer to an antibody in a substantially intact form, as opposed to an antibody fragment. In particular, full-length four-chain antibodies include antibodies having a heavy chain and a light chain, including an Fc region. The constant domains can be native sequence constant domains (e.g., native human sequence constant domains) or amino acid sequence variants thereof. In some cases, an intact antibody can have one or more effector functions.

[0075] The term "diabody" refers to a VH domain and a V L Bispecific diabodies refer to small antibody fragments prepared by constructing sFv fragments (see previous paragraph) with a short linker (approximately 5-10 residues) between the domains, resulting in interchain, rather than intrachain, pairing of the V domains, resulting in bivalent fragments (i.e. fragments with two antigen-binding sites). Bispecific diabodies are heterodimers of two "crossover" sFv fragments, in which the VH and VH domains of two antibodies are combined. L The domains are present on different polypeptide chains. Diabodies are described in more detail in, for example, EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

[0076] The monoclonal antibodies of the present invention specifically include "chimeric antibodies" in which a portion of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains are identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include the PRIMATTZFD® antibody, in which the antigen-binding region is derived from an antibody generated, for example, by immunizing macaque monkeys with an antigen of interest. As used herein, "humanized antibodies" is used as a subset of "chimeric antibodies."

[0077] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In some embodiments, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a HVR (defined below) of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) (such as mouse, rat, rabbit, or non-human primate) possessing the desired specificity, affinity, and / or capacity. In some cases, framework ("FR") residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications may be made to further refine antibody performance (such as binding affinity). Generally, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, which may contain substitutions of one or more individual FR residues that improve the performance of the antibody (binding affinity, isomerization, immunogenicity, etc.). The number of these amino acid substitutions in the FRs is typically no more than six in the H chain and no more than three in the L chain. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region. Suitable human acceptor antibodies can be selected from conventional databases (e.g., the KABAT database, the Los Alamos database, AbM, and the Swiss Protein database) by their homology with the nucleotide and amino acid sequences of the donor antibody. Human antibodies characterized by homology (on an amino acid basis) with the framework regions of the donor antibody may be suitable to provide heavy chain constant and / or heavy chain variable framework regions for insertion of the donor CDRs. A suitable acceptor antibody capable of donating a light chain constant or variable framework region can be selected in a similar manner. It should be noted that the heavy and light chains of the acceptor antibody do not have to be derived from the same acceptor antibody.The prior art describes several ways of producing such humanized antibodies (see, e.g., EP-A-0239400 and EP-A-054951). For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0078] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human, as disclosed herein, and / or produced using any of the techniques for producing human antibodies. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Human monoclonal antibodies can also be prepared using the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals (e.g., immunized xenomouse (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology) that have been engineered to produce such antibodies in response to antigen challenge, but in which the endogenous locus has been disabled. See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies produced by human B cell hybridoma technology.

[0079] The term "donor antibody" refers to an antibody (monoclonal and / or recombinant) that contributes the amino acid sequence of its variable regions, CDRs, or other functional fragments or analogs thereof to a first immunoglobulin partner, providing an altered immunoglobulin coding region and, as a result, an antibody that is expressed with an altered antigen specificity and neutralizing activity characteristic of the donor antibody.

[0080] The term "acceptor antibody" refers to an antibody (monoclonal and / or recombinant) that is heterologous to the donor antibody and contributes all (or any portion, but in some embodiments all) of the amino acid sequences encoding the heavy and / or light chain framework regions and / or the heavy and / or light chain constant regions to the first immunoglobulin partner. In certain embodiments, a human antibody is the acceptor antibody.

[0081] The terms "attach" or "attached" as used herein mean to connect or join by a bond, link, force, or ties to hold two or more components together, including either direct or indirect attachment, such as when a first polypeptide is directly attached to a second polypeptide or material, and also including when one or more intermediate compounds (e.g., amino acids, peptides, polypeptides, etc.) are disposed between the first polypeptide and the second polypeptide or material.

[0082] "CDR" is defined as the amino acid sequence of the complementarity determining region of an antibody, which is the hypervariable region of the heavy and light chains of the immunoglobulin. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987). There are three heavy chain CDRs (or CDR regions) and three light chain CDRs in the variable portion of an immunoglobulin. Thus, "CDR" herein refers to all three heavy chain CDRs, or all three light chain CDRs (or both all heavy chain CDRs and all light chain CDRs, as appropriate). The structure and protein folding of an antibody can mean that other residues are considered to be part of the antigen binding region and would be understood as such by one of skill in the art. See, e.g., Chothia et al., (1989) Conformations of immunoglobulin hypervariable regions; Nature 342, p 877-883.

[0083] As used herein, "immunoassay" refers to any binding assay that uses an antibody capable of specifically binding to a target molecule to detect and quantitate the target molecule.

[0084] The term "complementarity determining region" or "CDR" is used to refer to hypervariable regions defined by the Kabat system. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0085] As used herein, the phrases "specifically bind" or "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that specifically binds to a target (possibly an epitope) is an antibody that binds to this target with greater affinity, avidity, more readily, and / or for a longer period of time than it binds to other targets. In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding can include, but does not require, exclusive binding.

[0086] The term "specificity" means that an antigen-binding protein or antibody selectively recognizes a particular epitope of an antigen. For example, natural antibodies are monospecific. The term "multispecificity" is used herein to refer to an antigen-binding protein or antibody having two or more antigen-binding sites, at least two of which bind to different antigens or different epitopes of the same antigen. "Bispecificity" is used herein to refer to an antigen-binding protein or antibody having two different antigen-binding specificities. The term "monospecific" antibody is used herein to refer to an antibody having one or more binding sites, each of which binds to the same epitope of the same antigen.

[0087] An "effector cell" is a leukocyte that expresses one or more FcRs and performs an effector function. In one aspect, an effector cell expresses at least FcγRIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources (e.g., blood). Effector cells are generally lymphocytes associated with the effector phase and have the function of producing cytokines (helper T cells), killing pathogen-infected cells (cytotoxic T cells), or secreting antibodies (differentiated B cells).

[0088] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) bound to its cognate antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). Antibody variants with altered Fc region amino acid sequences and increased or decreased C1q binding ability are described in U.S. Patent No. 6,194,551B1 and WO 99 / 51642, the contents of which are specifically incorporated herein by reference. See also Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0089] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its corresponding partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high affinity antibodies generally bind antigens faster and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, any of which may be utilized for the purposes of this application. Exemplary and representative specific embodiments for measuring binding affinity are described below.

[0090] "On-rate", "rate of association", "association rate", or "k on " may also be determined using methods such as biolayer interferometry and surface plasmon resonance, as described herein above.

[0091] A "low pH dissociation factor" is defined herein as the percentage of an antibody that dissociates from an antigen at pH 5.8 at 25° C. (where the antibody is prebound to the antigen at pH 7.4). The low pH dissociation factor can be measured by associating an antibody with an antigen (e.g., a humanized anti-C5a antibody with human C5) for 600 seconds at pH 7.4, followed by a dissociation period of 600 seconds in a pH 5.8 buffer and calculating the percentage of the antibody that dissociates from the antigen at pH 5.8. A "neutral pH dissociation factor" is defined as the percentage of an antibody that dissociates from an antigen at pH 7.4 at 25° C. (where the antibody is prebound to the antigen at pH 7.4). The neutral pH dissociation factor can be measured by associating an antibody with an antigen (e.g., a humanized anti-C5a antibody with human C5) for 600 seconds at pH 7.4, followed by a dissociation period of 600 seconds in a pH 7.4 buffer and calculating the percentage of the antigen that dissociates from the antigen at pH 7.4. Antibody-antigen association and dissociation can be measured by a variety of methods within the art, such as biolayer interferometry.

[0092] "Percent (%) amino acid sequence identity" and "homology" with respect to peptide, polypeptide, or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that match those in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0093] "Isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in the normal context of a living subject is not "isolated," but the same nucleic acid or peptide that has been separated in part or in whole from the coexisting materials of its natural context is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment (such as, for example, a host cell).

[0094] The term "hybridoma" as used herein means a cell resulting from the fusion of a B lymphocyte with a fusion partner, such as a myeloma cell. Hybridomas can be cloned and maintained indefinitely in cell culture and are capable of producing monoclonal antibodies. Hybridomas can also be considered hybrid cells.

[0095] The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" can be used interchangeably and refer to a polymer of nucleotides. Such polymers of nucleotides can contain natural and / or non-natural nucleotides, non-limiting examples of which include DNA, RNA, and PNA. "Nucleic acid sequence" refers to a linear sequence of nucleotides that comprises a nucleic acid molecule or polynucleotide. "Isolated nucleic acid" refers to a section or fragment of a nucleic acid that is separated from sequences that are adjacent to it in nature, i.e., a DNA fragment that is removed from sequences that are normally adjacent to the fragment (i.e., sequences that are adjacent to the fragment in the genome in which the fragment naturally occurs). The term also applies to nucleic acids that have been substantially purified from other components that naturally accompany the nucleic acid (i.e., RNA or DNA) or protein that is naturally associated with it in a cell. Thus, the term includes recombinant DNA that is integrated into, for example, a vector, an autonomously replicating plasmid or virus, or genomic DNA of a prokaryote or eukaryote, or that exists as a separate molecule independent of other sequences (i.e., as a cDNA or genomic fragment or cDNA fragment generated by PCR or restriction enzyme digestion). The term also includes recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

[0096] "Complementary" as used herein to refer to nucleic acids refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue in a first nucleic acid region can form specific hydrogen bonds ("base pairing") with a residue in a second nucleic acid region antiparallel to the first region if this residue is thymine or uracil. Similarly, it is known that a cytosine residue in a first nucleic acid strand can form base pairs with a residue in a second nucleic acid strand antiparallel to the first strand if this residue is guanine. A first region of a nucleic acid is complementary to a second region of this nucleic acid or a different nucleic acid if at least one nucleotide residue in the first region can base pair with a residue in the second region when the two regions are positioned antiparallel. In some embodiments, the first region comprises a first portion and the second region comprises a second portion such that when the first and second portions are positioned antiparallel, at least about 50%, or at least about 75%, or at least about 90%, or at least about 95% of the nucleotide residues of the first portion can form base pairs with nucleotide residues in the second portion, in some embodiments, all nucleotide residues of the first portion can form base pairs with nucleotide residues of the second portion.

[0097] "Vector" as used herein can refer to a nucleic acid sequence that contains a replication origin.A vector can be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome.A vector can be a DNA vector or an RNA vector.A vector can be either a self-replicating extrachromosomal vector or a vector that is integrated into a host genome.

[0098] "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide (such as a gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids in biological processes, and the biological properties that result therefrom. Thus, a gene encodes a protein when that protein is produced in a cell or other biological system by transcription and translation of the mRNA that corresponds to that gene. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence, usually given in a sequence listing) and the non-coding strand (used as a template to transcribe the gene or cDNA) can be said to encode the protein or other product of that gene or cDNA.

[0099] The terms "polypeptide" and "peptide" are used interchangeably and refer to a polymer of amino acid residues, and are not limited to a minimum length. Such a polymer of amino acid residues can contain naturally occurring or non-naturally occurring amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The above terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, "polypeptide" includes modifications to the native sequence, such as deletions, additions, and substitutions (generally conservative in nature), so long as the polypeptide maintains the desired activity. These modifications can be artificial (e.g., through site-directed mutagenesis) or accidental (e.g., through mutations in the host producing the protein or errors resulting from PCR amplification).

[0100] As used herein, "complexed" means that one molecule is covalently bound to a second molecule.

[0101] The term "variant" as used herein refers to a nucleic acid or peptide sequence that differs in sequence from a reference nucleic acid or peptide sequence, respectively, but retains important biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not change the amino acid sequence of the peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, and truncations. Changes in the sequence of a peptide variant are typically limited or conservative, such that the sequences of the reference peptide and variant are generally similar and match in many regions. A variant and a reference peptide may differ in amino acid sequence by one or more substitutions, additions, or deletions, in any combination. A variant of a nucleic acid or peptide may be a naturally occurring (e.g., allelic) variant, or a variant that is not known to occur in nature. Non-natural variants of nucleic acids and peptides can be generated by mutagenesis techniques or direct synthesis. In various embodiments, the sequence of the variant is at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, or at least 85% identical to the reference sequence.

[0102] The term "modulate" as used herein can refer to any manner of altering the level or activity of a substrate. Non-limiting examples of modulation for proteins include affecting expression (including transcription and / or translation), affecting folding, affecting degradation or protein turnover, and affecting protein localization. Further non-limiting examples of modulation for enzymes include affecting enzyme activity. "Regulator" refers to a molecule whose activity includes affecting the level or activity of a substrate. Regulators can be direct or indirect. Regulators can function to activate, inhibit, or otherwise alter the corresponding substrate.

[0103] Ranges: Throughout this disclosure, various aspects of the invention may be expressed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, a description of a range should be considered to have all possible subranges specifically disclosed as well as individual numerical values ​​within that range. For example, a description of a range such as 1-6 should be considered to include the individual numbers within that range (e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6) as well as the specifically disclosed subranges (1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc.). This is true regardless of the breadth of the range. Reference to a value or parameter with "about" herein includes (and describes) a variation on that value or parameter itself. For example, a description that refers to "about X" includes the description of "X". Reference to "not" a value or parameter herein generally means and describes "other than" a value or parameter. For example, a method is not used to treat cancer of type X means that the method is used to treat cancer of a type other than X. The phrase "from about X to Y" has the same meaning as "from about X to about Y" herein.

[0104] The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0105] "Pharmaceutically acceptable carrier" means a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier common in the art for use with therapeutic agents that together comprise a "pharmaceutical composition" to be administered to a subject. A pharmaceutically acceptable carrier is non-toxic to a recipient at the dosage and concentration used and is compatible with the other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation in which it is used.

[0106] "Diluents" of interest herein are diluents that are pharma- ceutically acceptable (safe and non-toxic for administration to humans) and useful for preparing liquid formulations (e.g., formulations reconstituted after lyophilization). Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution. In an alternative embodiment, the diluent can include aqueous salt solutions and / or buffers.

[0107] A "preservative" is a compound that can be added to the formulation herein to reduce bacterial activity. Preservatives can be added, for example, to facilitate the manufacture of multi-use (multiple dose) formulations. Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols (such as phenol, butyl alcohol, benzyl alcohol), alkylparabens (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The most preferred preservative herein is benzyl alcohol.

[0108] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is to be administered. Such formulations may be sterile.

[0109] A "sterile" preparation is sterile or substantially free of live microorganisms and their spores.

[0110] A "stable" formulation is one in which the protein therein essentially retains its physical and chemical stability and integrity upon storage. A variety of analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature and for a selected period of time. For rapid screening, the formulation can be stored at 40°C for 2 weeks to 1 month, at which point stability is measured. If the formulation is stored at 2-8°C, it should generally be stable at 30°C or 40°C for at least 1 month, and / or at 2-8°C for at least 2 years. When a formulation is stored at 30° C., it should generally be stable at 30° C. for at least 2 years and / or at 40° C. for at least 6 months. For example, the degree of aggregation during storage can be used as an indicator of protein stability. Thus, a "stable" formulation can be one in which less than about 10%, preferably less than about 5%, of the protein is present as aggregates in the formulation. In other embodiments, any increase in aggregate formation during storage of the formulation can be accounted for.

[0111] A "reconstituted" formulation is one that has been prepared by dissolving a lyophilized protein or antibody formulation in a diluent to disperse the protein throughout. The reconstituted formulation is suitable for administration (e.g., subcutaneously) to a patient to be treated with the protein of interest, and in some embodiments may be suitable for parenteral or intravenous administration.

[0112] An "isotonic" formulation is one that has essentially the same osmolality as human blood. An isotonic formulation will generally have an osmolality of about 250-350 mOsm. The term "hypotonic" describes a formulation that has an osmolality lower than human blood. Correspondingly, the term "hypertonic" is used to describe a formulation that has an osmolality higher than human blood. Isotonicity can be measured, for example, using a vapor pressure or ice-type osmometer. The formulations of the present application can be hypertonic as a result of the addition of salts and / or buffers.

[0113] It should be understood that embodiments described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0114] As used in this specification and the appended claims, the singular forms "a," "an," "or," and "the" include the plural unless the context clearly dictates otherwise.

[0115] II. Anti-C5a antibody The present application provides novel humanized anti-human C5a antibodies and constructs. In some embodiments, the antibodies have mutations in their VH and / or VL domains. In some embodiments, the mutations render the antibodies pH sensitive in binding to antigens. In some embodiments, the anti-C5a antibodies have a mutation that reduces off-target binding to C5 by allowing the antibody to bind more strongly to C5 at neutral pH (e.g., pH about 7.4; e.g., pH found in blood) than at more acidic pH (e.g., pH about 5.8; e.g., pH found in endosomes). In some embodiments, the mutation is a histidine mutation (e.g., mutation F29H in the VH domain and mutation Y96H in the VL domain).

[0116] The humanized C5a antibody of the present application comprises at least one antigen-binding portion comprising a heavy chain variable domain (VH) and a light chain variable domain (VL). Non-limiting examples of representative antigen-binding fragments contemplated herein include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (such as scFv); and multispecific antibodies formed from antibody fragments. Such antigen-binding portions can be traditional full-length antibodies consisting of two heavy chains and two light chains, or antigen-binding fragments derived therefrom.

[0117] In some embodiments, the humanized C5a antibody comprises the mutations I48M, D54E, and N56W in the VH (wherein the mutations refer to SEQ ID NO:1 under the Kabat numbering system). In some embodiments, the humanized anti-C5a antibody comprises the mutations D28E and D30F in the VL (wherein the mutations refer to SEQ ID NO:2 under the Kabat numbering system). In some embodiments, the humanized anti-C5a antibody comprises a VH comprising the mutations I48M, D54E, and N56W under the Kabat numbering system with reference to SEQ ID NO:1, and a VL comprising the mutations D28E and D30F under SEQ ID NO:2. In some embodiments, the humanized anti-C5a antibody comprising the mutations I48M, D54E, and N56W in the VH has an improved affinity for human C5a that is greater than 10-fold greater than the anti-C5a antibody comprising SEQ ID NO:1. In some embodiments, a humanized anti-C5a antibody comprising the mutations I48M, D54E, and N56W in the VH has cross-species activity with cynomolgus C5a.

[0118] In some embodiments, the humanized C5a antibody further comprises one or more additional mutations in the variable domain of the light chain (VL), such as a histidine mutation. For example, in some embodiments, the humanized anti-C5a antibody comprises the mutation F29H in the VH and the mutation Y96H in the VL.

[0119] In some embodiments, the humanized C5a antibody further comprises mutations E54H or N97H in the VH (wherein the VH mutations refer to SEQ ID NO: 1 under the Kabat numbering system: QVQLQQSDAELVKPGASVKISCKVSGYTFTDHIIHWMNQRPEQGLEWIGYIYPRDGNTNYNENFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCARERNLEYFDYWGQGTTLTVSS (SEQ ID NO: 1)).

[0120] In some embodiments, the humanized C5a antibody further comprises one or more additional mutations in the variable domain (VL) of the light chain, such as a histidine mutation. For example, in some embodiments, the humanized anti-C5a antibody comprises the mutation N92H in the VL (wherein the VL mutation refers to SEQ ID NO: 2 under the Kabat numbering system: DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDNYMNWYQQKPGQPPKLLIYAASNLDSGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIK (SEQ ID NO: 2)).

[0121] In some embodiments, the humanized C5a antibody comprises a mutation selected from the group consisting of E54H in VH, N97H in VH, and N92H in VL (wherein said VH mutations refer to SEQ ID NO: 1 under the Kabat numbering system and said VL mutations refer to SEQ ID NO: 2 under the Kabat numbering system).

[0122] Thus, in some embodiments, the humanized C5a antibody comprises a VH comprising the mutations I48M, D54E, and N56W with reference to SEQ ID NO: 1, and a VL comprising the mutations D28E and D30F with reference to SEQ ID NO: 2. In some embodiments, the humanized anti-C5a antibody further comprises the mutation F29H in the VH with reference to SEQ ID NO: 1, and the mutation Y96H in the VL with reference to SEQ ID NO: 2. In some embodiments, the humanized anti-C5a antibody further comprises a mutation selected from the group consisting of E54H in VH, N97H in VH, and N92H in VL, where said VH mutations refer to SEQ ID NO: 1 under the Kabat numbering system, and said VL mutations refer to SEQ ID NO: 2 under the Kabat numbering system.

[0123] In some embodiments, the humanized C5a antibody comprises an Fc region (such as a human Fc region). In some embodiments, the Fc region is derived from an IgG molecule (such as any one of the subclasses IgG1, IgG2, IgG3, or IgG4). In some embodiments, the Fc region can mediate antibody effector functions (such as ADCC (antibody-dependent cellular cytotoxicity) and / or CDC (complement-dependent cytotoxicity)). For example, antibodies of subclasses IgG1, IgG2, and IgG3 with wild-type Fc sequences typically exhibit complement activation, including binding to CIq and C3, whereas IgG4 does not activate the complement system and does not bind CIq and / or C3. In some embodiments, the Fc region comprises a modification that reduces the binding affinity of the Fc region to an Fc receptor. In some embodiments, the Fc region is an IgG4 Fc. In some embodiments, the IgG4 Fc region comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the IgG4 Fc comprises a mutation. See, e.g., Armour KL et al., Eur J. Immunol. 1999; 29: 2613; and Shields RL et al., J. Biol. Chem. 2001; 276: 6591. In some embodiments, the Fc region comprises one or more mutations selected from the group consisting of S228P, M428L, and N434A (wherein the mutations are compared to SEQ ID NO: 43 under the EU numbering system). In some embodiments, the Fc region comprises the mutations S228P, M428L, and N434A. In some embodiments, the IgG4 Fc region comprises the amino acid sequence of SEQ ID NO: 44.

[0124] In some embodiments, the humanized C5a antibody (e.g., any one of the humanized C5a antibodies described herein comprising a VH comprising the mutations I48M, D54E, and N56W with reference to SEQ ID NO:1, and a VL comprising the mutations D28E and D30F with reference to SEQ ID NO:2) comprises a heavy chain CDR1 ("H-CDR1") comprising the amino acid sequence of SEQ ID NO:3, or a variant thereof comprising one, two, or three amino acid substitutions; a heavy chain CDR2 ("H-CDR2") comprising the amino acid sequence of SEQ ID NO:4, or a variant thereof comprising one, two, or three amino acid substitutions; a heavy chain CDR3 ("H-CDR3") comprising the amino acid sequence of SEQ ID NO:6, or a variant thereof comprising one, two or three amino acid substitutions; a light chain CDR1 ("L-CDR1") comprising the amino acid sequence of SEQ ID NO:7, or a variant thereof comprising one, two or three amino acid substitutions; a light chain CDR2 ("L-CDR2") comprising the amino acid sequence of SEQ ID NO:7, or a variant thereof comprising one, two or three amino acid substitutions; and a light chain CDR3 ("L-CDR3") comprising the amino acid sequence of SEQ ID NO:8, or a variant thereof comprising one, two or three amino acid substitutions. In some embodiments, the humanized anti-C5a antibody comprises a heavy chain CDR1 ("H-CDR1") comprising the amino acid sequence of SEQ ID NO:3; a heavy chain CDR2 ("H-CDR2") comprising the amino acid sequence of SEQ ID NO:4; a heavy chain CDR3 ("H-CDR3") comprising the amino acid sequence of SEQ ID NO:5; a light chain CDR1 ("L-CDR1") comprising the amino acid sequence of SEQ ID NO:6; a light chain CDR2 ("L-CDR2") comprising the amino acid sequence of SEQ ID NO:7; and a light chain CDR3 ("L-CDR3") comprising the amino acid sequence of SEQ ID NO:8.

[0125] In some embodiments (independently or in addition to the CDR sequences described herein), the humanized anti-C5a antibody comprises a VH comprising the amino acid sequence SEQ ID NO:9, or a variant thereof having at least about any one of 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% amino acid identity with SEQ ID NO:9; and a VL comprising the amino acid sequence of SEQ ID NO:10, or a variant thereof having at least about any one of 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% amino acid identity with SEQ ID NO:10. In some embodiments, the humanized anti-C5a antibody comprises a VH comprising the amino acid sequence SEQ ID NO:9, and a VL comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, the humanized anti-C5a antibody further comprises an Fc region (such as an IgG4 Fc region).

[0126] In some embodiments, the humanized C5a antibody (e.g., any one of the humanized C5a antibodies described herein comprising a VH comprising the mutations I48M, D54E, and N56W with reference to SEQ ID NO:1, and the mutations D28E and D30F with reference to SEQ ID NO:2) comprises a heavy chain CDR1 ("H-CDR1") comprising the amino acid sequence of SEQ ID NO:11, or a variant thereof comprising one, two, or three amino acid substitutions; a heavy chain CDR2 ("H-CDR2") comprising the amino acid sequence of SEQ ID NO:12, or a variant thereof comprising one, two, or three amino acid substitutions; a heavy chain CDR3 ("H-CDR3") comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising one, two, or three amino acid substitutions; a light chain CDR1 ("L-CDR1") comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising one, two, or three amino acid substitutions; a light chain CDR2 ("L-CDR2") comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising one, two, or three amino acid substitutions; and a light chain CDR3 ("L-CDR3") comprising the amino acid sequence of SEQ ID NO: 16, or a variant thereof comprising one, two, or three amino acid substitutions. In some embodiments, the humanized anti-C5a antibody comprises a heavy chain CDR1 ("H-CDR1") comprising the amino acid sequence of SEQ ID NO:11; a heavy chain CDR2 ("H-CDR2") comprising the amino acid sequence of SEQ ID NO:12; a heavy chain CDR3 ("H-CDR3") comprising the amino acid sequence of SEQ ID NO:13; a light chain CDR1 ("L-CDR1") comprising the amino acid sequence of SEQ ID NO:14; a light chain CDR2 ("L-CDR2") comprising the amino acid sequence of SEQ ID NO:15; and a light chain CDR3 ("L-CDR3") comprising the amino acid sequence of SEQ ID NO:16.

[0127] In some embodiments (independently or in addition to the CDR sequences described herein), the humanized anti-C5a antibody comprises a VH comprising the amino acid sequence SEQ ID NO: 17, or a variant thereof having at least about any one of 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% amino acid identity with SEQ ID NO: 17; and a VL comprising the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least about any one of 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% amino acid identity with SEQ ID NO: 18. In some embodiments, the humanized anti-C5a antibody comprises a VH comprising the amino acid sequence SEQ ID NO: 17, and a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the humanized anti-C5a antibody further comprises an Fc region (such as an IgG4 Fc region).

[0128] In some embodiments, the humanized C5a antibody (e.g., any one of the humanized anti-C5a antibodies described herein comprising a VH comprising the mutations I48M, D54E, and N56W with reference to SEQ ID NO:1, and a VL comprising the mutations D28E and D30F with reference to SEQ ID NO:2) comprises a heavy chain CDR1 ("H-CDR1") comprising the amino acid sequence of SEQ ID NO:19, or a variant thereof comprising one, two, or three amino acid substitutions; a heavy chain CDR2 ("H-CDR2") comprising the amino acid sequence of SEQ ID NO:20, or a variant thereof comprising one, two, or three amino acid substitutions; a heavy chain CDR3 ("H-CDR3") comprising the amino acid sequence of SEQ ID NO:22, or a variant thereof comprising one, two or three amino acid substitutions; a light chain CDR1 ("L-CDR1") comprising the amino acid sequence of SEQ ID NO:22, or a variant thereof comprising one, two or three amino acid substitutions; a light chain CDR2 ("L-CDR2") comprising the amino acid sequence of SEQ ID NO:23, or a variant thereof comprising one, two or three amino acid substitutions; and a light chain CDR3 ("L-CDR3") comprising the amino acid sequence of SEQ ID NO:24, or a variant thereof comprising one, two or three amino acid substitutions. In some embodiments, the humanized anti-C5a antibody comprises a heavy chain CDR1 ("H-CDR1") comprising the amino acid sequence of SEQ ID NO: 19; a heavy chain CDR2 ("H-CDR2") comprising the amino acid sequence of SEQ ID NO: 20; a heavy chain CDR3 ("H-CDR3") comprising the amino acid sequence of SEQ ID NO: 21; a light chain CDR1 ("L-CDR1") comprising the amino acid sequence of SEQ ID NO: 22; a light chain CDR2 ("L-CDR2") comprising the amino acid sequence of SEQ ID NO: 23; and a light chain CDR3 ("L-CDR3") comprising the amino acid sequence of SEQ ID NO: 24.

[0129] In some embodiments (independently or in addition to the CDR sequences described herein), the humanized anti-C5a antibody comprises a VH comprising the amino acid sequence SEQ ID NO:25, or a variant thereof having at least about any one of 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% amino acid identity with SEQ ID NO:25; and a VL comprising the amino acid sequence of SEQ ID NO:26, or a variant thereof having at least about any one of 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% amino acid identity with SEQ ID NO:26. In some embodiments, the humanized anti-C5a antibody comprises a VH comprising the amino acid sequence SEQ ID NO:25, and a VL comprising the amino acid sequence of SEQ ID NO:26. In some embodiments, the humanized anti-C5a antibody further comprises an Fc region (such as an IgG4 Fc region).

[0130] In some embodiments, the humanized C5a antibody (e.g., any one of the humanized C5a antibodies described herein comprising a VH comprising the mutations I48M, D54E, and N56W with reference to SEQ ID NO:1, and a VL comprising the mutations D28E and D30F with reference to SEQ ID NO:2) comprises a heavy chain CDR1 ("H-CDR1") comprising the amino acid sequence of SEQ ID NO:27, or a variant thereof comprising one, two, or three amino acid substitutions; a heavy chain CDR2 ("H-CDR2") comprising the amino acid sequence of SEQ ID NO:28, or a variant thereof comprising one, two, or three amino acid substitutions; a heavy chain CDR3 ("H-CDR3") comprising the amino acid sequence of SEQ ID NO:30, or a variant thereof comprising one, two or three amino acid substitutions; a light chain CDR1 ("L-CDR1") comprising the amino acid sequence of SEQ ID NO:30, or a variant thereof comprising one, two or three amino acid substitutions; a light chain CDR2 ("L-CDR2") comprising the amino acid sequence of SEQ ID NO:31, or a variant thereof comprising one, two or three amino acid substitutions; and a light chain CDR3 ("L-CDR3") comprising the amino acid sequence of SEQ ID NO:32, or a variant thereof comprising one, two or three amino acid substitutions. In some embodiments, the humanized anti-C5a antibody comprises a heavy chain CDR1 ("H-CDR1") comprising the amino acid sequence of SEQ ID NO:27; a heavy chain CDR2 ("H-CDR2") comprising the amino acid sequence of SEQ ID NO:28; a heavy chain CDR3 ("H-CDR3") comprising the amino acid sequence of SEQ ID NO:29; a light chain CDR1 ("L-CDR1") comprising the amino acid sequence of SEQ ID NO:30; a light chain CDR2 ("L-CDR2") comprising the amino acid sequence of SEQ ID NO:31; and a light chain CDR3 ("L-CDR3") comprising the amino acid sequence of SEQ ID NO:32.

[0131] In some embodiments (independently or in addition to the CDR sequences described herein), the humanized anti-C5a antibody comprises a VH comprising the amino acid sequence SEQ ID NO:33, or a variant thereof having at least about any one of 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% amino acid identity with SEQ ID NO:33; and a VL comprising the amino acid sequence of SEQ ID NO:34, or a variant thereof having at least about any one of 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% amino acid identity with SEQ ID NO:34. In some embodiments, the humanized anti-C5a antibody comprises a VH comprising the amino acid sequence SEQ ID NO:33, and a VL comprising the amino acid sequence of SEQ ID NO:34. In some embodiments, the humanized anti-C5a antibody further comprises an Fc region (such as an IgG4 Fc region).

[0132] In some embodiments, the humanized C5a antibody comprises the H-CDR1, H-CDR2, and H-CDR3 sequences of an antibody having a VH domain of SEQ ID NO: 54, and the L-CDR1, L-CDR2, and L-CDR3 sequences of an antibody having a VL domain of SEQ ID NO: 55.

[0133] In some embodiments, the humanized C5a antibody comprises the H-CDR1, H-CDR2, and H-CDR3 sequences of an antibody having a VH domain of SEQ ID NO: 56, and the L-CDR1, L-CDR2, and L-CDR3 sequences of an antibody having a VL domain of SEQ ID NO: 57.

[0134] In some embodiments, the humanized C5a antibody comprises the H-CDR1, H-CDR2, and H-CDR3 sequences of an antibody having a VH domain of SEQ ID NO: 58, and the L-CDR1, L-CDR2, and L-CDR3 sequences of an antibody having a VL domain of SEQ ID NO: 59.

[0135] In some embodiments, the humanized C5a antibody comprises the H-CDR1, H-CDR2, and H-CDR3 sequences of an antibody having a VH domain of SEQ ID NO: 60, and the L-CDR1, L-CDR2, and L-CDR3 sequences of an antibody having a VL domain of SEQ ID NO: 61.

[0136] In some embodiments, the humanized C5a antibody comprises the H-CDR1, H-CDR2, and H-CDR3 sequences of an antibody having a VH domain of SEQ ID NO: 62, and the L-CDR1, L-CDR2, and L-CDR3 sequences of an antibody having a VL domain of SEQ ID NO: 63.

[0137] In some embodiments, the humanized C5a antibody comprises the H-CDR1, H-CDR2, and H-CDR3 sequences of an antibody having a VH domain of SEQ ID NO: 64, and the L-CDR1, L-CDR2, and L-CDR3 sequences of an antibody having a VL domain of SEQ ID NO: 65.

[0138] In some embodiments, the humanized C5a antibody comprises the H-CDR1, H-CDR2, and H-CDR3 sequences of an antibody having a VH domain of SEQ ID NO: 66, and the L-CDR1, L-CDR2, and L-CDR3 sequences of an antibody having a VL domain of SEQ ID NO: 67.

[0139] In some embodiments, the humanized C5a antibody comprises the H-CDR1, H-CDR2, and H-CDR3 sequences of an antibody having a VH domain of SEQ ID NO: 68, and the L-CDR1, L-CDR2, and L-CDR3 sequences of an antibody having a VL domain of SEQ ID NO: 69.

[0140] In some embodiments, the humanized C5a antibody comprises the H-CDR1, H-CDR2, and H-CDR3 sequences of an antibody having a VH domain of SEQ ID NO: 70, and the L-CDR1, L-CDR2, and L-CDR3 sequences of an antibody having a VL domain of SEQ ID NO: 71.

[0141] In some embodiments, the humanized C5a antibody comprises the H-CDR1, H-CDR2, and H-CDR3 sequences of an antibody having a VH domain of SEQ ID NO: 72, and the L-CDR1, L-CDR2, and L-CDR3 sequences of an antibody having a VL domain of SEQ ID NO: 73.

[0142] In some embodiments, the humanized C5a antibody comprises the H-CDR1, H-CDR2, and H-CDR3 sequences of an antibody having a VH domain of SEQ ID NO: 74, and the L-CDR1, L-CDR2, and L-CDR3 sequences of an antibody having a VL domain of SEQ ID NO: 75.

[0143] In some embodiments, the humanized C5a antibody comprises the H-CDR1, H-CDR2, and H-CDR3 sequences of an antibody having a VH domain of SEQ ID NO: 76, and the L-CDR1, L-CDR2, and L-CDR3 sequences of an antibody having a VL domain of SEQ ID NO: 77.

[0144] In some embodiments, the humanized C5a antibody comprises the H-CDR1, H-CDR2, and H-CDR3 sequences of an antibody having a VH domain of SEQ ID NO: 78, and the L-CDR1, L-CDR2, and L-CDR3 sequences of an antibody having a VL domain of SEQ ID NO: 79.

[0145] In some embodiments, the humanized C5a antibody comprises the H-CDR1, H-CDR2, and H-CDR3 sequences of an antibody having a VH domain of SEQ ID NO: 80, and the L-CDR1, L-CDR2, and L-CDR3 sequences of an antibody having a VL domain of SEQ ID NO: 81.

[0146] In some embodiments, the humanized C5a antibody comprises the H-CDR1, H-CDR2, and H-CDR3 sequences of an antibody having a VH domain of SEQ ID NO: 82, and the L-CDR1, L-CDR2, and L-CDR3 sequences of an antibody having a VL domain of SEQ ID NO: 83.

[0147] The Kabat numbering system is used to refer to the mutations, however, it should be understood that the positions of the mutations can be represented under different numbering systems, as shown with representative sequences in Table 1 below. [Table 1]

[0148] C5a protein and C5a binding analysis The complement system plays a key role in the pathology of many autoimmune, inflammatory, and ischemic diseases. Inappropriate activation of complement and its deposition on host cells can lead to complement-mediated lysis and / or cell and target tissue damage, as well as tissue destruction due to the generation of potent mediators of inflammation. The complement system (also known as the complement cascade) is part of the immune system and enhances (complements) the ability of antibodies and phagocytes to eliminate microorganisms and damaged cells from the organism, promotes inflammation, and attacks the cell membranes of pathogens. The complement system is part of the innate immune system and is not adaptive, remaining unchanged throughout an individual's life. However, the complement system can be recruited and acted upon by antibodies generated by the adaptive immune system.

[0149] Without being bound by any theory or hypothesis, there are three known complement pathways: the alternative complement pathway (AP), the classical pathway (CP), and the lectin pathway (LP). In general, the CP is initiated by antigen-antibody complexes, and the LP is activated by lectins binding to sugar molecules on the microbial surface, whereas the AP is constitutively active at low levels but can rapidly amplify on the cell surface of bacteria, viruses, and parasites due to lack of regulatory proteins. Host cells are usually protected from AP complement activation by regulatory proteins. However, in some situations, such as when regulatory proteins are defective or missing, the AP can become activated and uncontrollable on the surface of host cells, leading to complement-related diseases or disorders. The CP consists of components C1, C2, and C4, which converge to the AP at the C3 activation step. The LP consists of mannose-binding lectin (MBL) and MBL-associated serine protease (MASP), and shares components C4 and C2 with the CP. The AP consists of component C3 and several factors, including factor B, factor D, properdin, and fluid-phase regulator H. Complement activation consists of three steps: (a) recognition, (b) enzymatic activation, and (c) membrane attack leading to cell death. The first step of CP complement activation begins with C1, which consists of three different proteins: the recognition subunit (C1q) and the serine protease subcomponents C1r and C1s. C1r and C1s bind to each other to form the calcium-dependent tetrameric complex C1r2 s2. An intact C1 complex is required for physiological activation of C1. Activation occurs when the intact C1 complex binds to immunoglobulins in complex with antigen. This binding activates C1s, which then cleaves both C4 and C2 proteins to generate C4a and C4b as well as C2a and C2b. The C4b and C2a fragments combine to form C3 convertase (C4b2a), which in turn cleaves C3 to form C3a and C3b.Activation of the LP is initiated by the binding of MBL to certain carbohydrates on the target surface, which initiates the activation of MBL-associated serine proteases (MAPS), which cleave C4 and C2 in a manner similar to the activity of C1s in the CP, generating the C3 convertase C4b2a. Thus, although CP and LP are activated by different mechanisms, they share the same components C4 and C2, and therefore generate the same C3 convertase, C4b2a, in both pathways. The cleavage of C3 by C4b2a to C3b and C3a is a central event in the complement pathway for two reasons. This cleavage initiates the AP amplification loop, because surface-deposited C3b is a central intermediate for the AP C3 convertase C3bBb. Both C3a and C3b are biologically important. C3a is proinflammatory and, together with C5a, is called an anaphylatoxin. C3b and its further cleavage products also bind to complement receptors present on the surface of neutrophils, eosinophils, monocytes, and macrophages, facilitating the phagocytosis and excretion of C3b-opsonized particles. Finally, C3b can associate with C4b2a or C3bBb to form C5 convertases in the CP and LP, and C5 convertase in the AP, respectively, activating terminal complement sequences, leading to the production of potent proinflammatory mediators, C5a, and the assembly of the lytic membrane attack complex (MAC), C5-C9.

[0150] Defective complement action contributes to several human glomerular diseases, including atypical hemolytic uremic syndrome (aHUS), antineutrophil cytoplasmic antibody-associated vasculitis (ANCA), C3 glomerulopathy, IgA nephropathy, immune complex membranoproliferative glomerulonephritis, renal ischemia-reperfusion injury, lupus nephritis, membranous nephropathy, and chronic transplant-associated glomerulopathy. Aberrant complement component activation has been proposed as a marker in various types of cancer and its clinical outcome. Lung cancer patients have been shown to have significantly higher plasma levels of complement proteins and activation fragments than control donors, and elevated complement levels correlate with lung tumor size. Complement-related proteins are also elevated in body fluids from patients with other types of tumors. See, for example, Pio et al. Semin Immunol. 2013 Feb; 25(1): 54-64. Inhibition of the complement cascade has been proposed for the treatment of glomerular diseases and cancer.

[0151] C5a is a 74 amino acid anaphylatoxin (SEQ ID NO: 45) that arises from the cleavage of complement component C5. Mature C5 is cleaved during activation of the complement pathway into C5a and C5b fragments. C5a is cleaved from the alpha chain of C5 by C5 convertase as an amino-terminal fragment containing the first 74 amino acids of the alpha chain. The remainder of mature C5 is fragment C5b, which contains the remainder of the alpha chain disulfide attached to the beta chain. Nearly 20% of the 11 kDa mass of C5a is attributed to carbohydrates. C5a acts as a highly inflammatory peptide, promoting the activation of complement, formation of MAC, recruitment of innate immune cells, and release of histamine, which are involved in allergic responses. C5a is an anaphylatoxin that causes increased expression of adhesion molecules on endothelin, contraction of smooth muscle, and increased vascular permeability. C5a des-Arg is a much weaker anaphylatoxin. Both C5a and C5a des-Arg can initiate mast cell degranulation, releasing the proinflammatory molecules histamine and TNF-α. C5a is also an effective chemoattractant, initiating the accumulation of complement and phagocytes at the site of infection or the recruitment of antigen-presenting cells to lymph nodes. C5a plays an important role in increasing the migration and adhesion of neutrophils and monocytes to the vessel walls. White blood cells are activated by upregulation of integrin avidity, lipoxygenase pathway, and arachidonic acid metabolism. C5a also alters the balance between activation and inhibition of IgG Fc receptors on white blood cells, thereby enhancing autoimmune responses. See, for example, Manthey HD, Woodruff TM, Taylor SM, Monk PN (November 2009). “Complement component 5a (C5a)”. The International Journal of Biochemistry & Cell Biology. 41 (11): 2114-7.

[0152] C5a binds to C5aR (also known as CD88) on the surface of target cells (such as macrophages, neutrophils, and endothelial cells) to initiate signaling and promote inflammatory responses. C5a / C5aR signals promote the proliferation of MDSCs, thus limiting T cell immunity. Inhibition of the C5a / C5aR pathway has been proposed to have potential in immuno-oncology to ameliorate inflammatory diseases.

[0153] The binding affinity and specificity of the humanized anti-C5a antibodies described herein can be experimentally determined by methods known in the art. For example, antibody binding to protein antigens can be detected and / or quantified using a variety of techniques, including, but not limited to, Western blot, dot blot, surface plasmon resonance (SPR) (e.g., the BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ), biolayer interference (BLI) (e.g., the Octet system, ForteBio), RIA, ECL, IRMA, EIA, peptide scan, and enzyme-linked immunosorbent assay (ELISA). See, e.g., Benny KC Lo (2004) "Antibody Engineering: Methods and Protocols." Humana Press (ISBN:1588290921);Borrebaek (1992) "Antibody Engineering, A Practical Guide." WH Freeman and Co., NY;Borrebaek (1995) "Antibody Engineering." 2nd Edition, Oxford University Press, NY, Oxford;Johne et al. (1993).J Immunol Meth 160:191-198;Jonsson et al. (1993) Ann Biol Clin 51:19-26;and Jonsson et al. (1991) Biotechniques 11:620-627. Additionally, methods for measuring affinity (e.g., dissociation and association constants by BLI) are illustrated.

[0154] Methods for determining whether a particular antibody described herein inhibits C5a are known in the art. As shown in Schraufstatter et al. J Immunol March 15, 2009, 182 (6) 3827-3836, inhibiting C5a can reduce chemotactic migration of cells. Thus, the function of C5a can be elucidated, for example, by cell migration assays. See, for example, Rousseau, Simon, et al. Cellular signalling 18.11 (2006): 1897-1905. C5a has been shown to induce calcium influx into cells (Moller et al., J Neurosci. 1997 Jan 15; 17(2): 615-624). Measurement of C5a activity using a calcium influx assay is possible using, for example, the calcium staining method described in Farkas et al., Neuroscience Volume 86, Issue 3, 1998, Pages 903-911, or radiometric techniques (Monk et al., Biochem J (1993) 295 (3): 679-684).

[0155] Methods for determining whether a particular antibody described herein inhibits C5 cleavage are known in the art. Inhibition of human complement component C5 can reduce the cytolytic ability of complement in a subject's body fluid. Such reduction in the cytolytic ability of complement present in a body fluid can be measured by methods well known in the art, such as a conventional hemolytic assay, such as the chicken erythrocyte hemolytic assay described in Hillmen et al. (2004) N Engl. J Med 350(6):552. Methods for determining whether a candidate compound inhibits the cleavage of human C5 into the forms C5a and C5b are known in the art, and are described, for example, in Thomas et al. (1996) Mol Immunol 33(17-18): 1389-401 and Evans et al. (1995) Mol Immunol 32(16): 1183-95. For example, the concentration and / or physiological activity of C5a and C5b in body fluids can be measured by methods well known in the art. Methods for measuring C5a concentration or activity include, for example, chemotaxis assays, RIA, or ELISA (see, e.g., Wurzner et al. (1991) Complement Inflamm 8:328-340). For C5b, lytic assays as discussed herein, or assays for soluble C5b-9, can be used. Other assays known in the art can also be used. These assays, or other suitable types of assays, can be used to screen candidate agents capable of inhibiting human complement component C5.

[0156] Hemolysis assays can be used to determine the inhibitory activity of anti-C5a antibodies against C5-mediated complement activation, and are therefore useful for determining potential off-target binding of anti-C5a antibodies. To determine the effect of humanized anti-C5a antibodies on classical complement pathway-mediated hemolysis in serum test solutions in vitro, sheep red blood cells coated with hemolysin or chicken red blood cells sensitized with anti-chicken red blood cell antibodies are used as target cells. The percentage of lysis is normalized by considering 100% lysis as equivalent to the lysis occurring in the absence of inhibitors. To determine the effect of humanized anti-C5a antibodies on alternative pathway-mediated hemolysis, unsensitized rabbit or guinea pig red blood cells are used as target cells. The percentage of lysis is normalized by considering 100% lysis as equivalent to the lysis occurring in the absence of inhibitors.

[0157] "TMDD" or "sink effect" and pH-dependent anti-C5a antibodies The majority of therapeutic monoclonal antibodies exhibit non-linear dose-dependent clearance due to target-mediated drug elimination or TMDD (also called the "sink effect").

[0158] For example, when an antibody is administered at low or sub-therapeutic doses, it is primarily bound to the target with very little free antibody. As the dose is increased, the proportion of free antibody increases over the antibody / antigen bound complex. When the dose is increased to very high levels, the antigen is fully saturated or bound and the majority of the total antibody is unbound or free. Free mAb has a longer half-life (due to the FcRn mechanism) than the mAb:Ag bound complex, which is cleared by the phagocytic process. Thus, at low doses, the half-life of total antibody (which is mostly bound) is shorter, and the half-life of total antibody increases as the dose is increased, reaching a plateau where the majority of the total antibody is unbound or free. Clearance of antibodies bound to membrane antigens follows the same paradigm; at low doses, clearance is more rapid. This is because unbound targets become "soaked up" antibodies and act as a sink (also called "antigen sink"; Keiser et al., Clin Pharmacokinet. 2010 Aug; 49(8):493-507; Eser et al., Curr Opin Gastroenterol. 2013 Jul; 29(4):391-6).

[0159] Since C5a is a part of C5, anti-C5a antibodies can bind well to C5. The large binding capacity of anti-C5a antibodies to C5 and the large excess of serum C5 concentration over C5a make C5 a binding sink for anti-C5a antibodies, leading to rapid clearance in vivo, i.e., the C5 "sink effect" for anti-C5a antibodies.

[0160] In some embodiments, the humanized anti-C5a antibodies described herein bind to C5 but do not inhibit cleavage of C5 by C5 convertase. In some embodiments, the humanized anti-C5a antibodies bind to C5 and reduce levels of C5 through an unknown mechanism of action. In some embodiments, the reduction in C5 levels is accompanied by a reduction in C5a levels and / or a desensitization of C5-mediated drug elimination. In some embodiments, the reduction in C5a levels and / or a desensitization of C5-mediated drug elimination leads to a sustained drug effect.

[0161] In some embodiments, the humanized anti-C5a antibodies described herein dissociate from C5 in a pH-dependent manner. Such pH-dependent binding provides greater persistence of the administered antibody or antibody fusion protein molecule, because immune complexes (i.e., humanized anti-C5a antibodies bound to C5) taken up by cells dissociate in the acidic environment of the endosome, allowing the free antibody or antibody fusion protein to be recycled out of the cell through the neonatal Fc receptor (FcRn), where it is available to bind new C5a molecules.

[0162] In some embodiments, the described humanized anti-C5a antibodies bind to C5 in a pH-dependent manner. As used herein, the term "pH-dependent binding" means that the antibody exhibits decreased binding to C5 at acidic pH (e.g., about pH 5.8; such as in an endosome) compared to neutral pH (e.g., about pH 7.4; such as in blood).

[0163] The pH dependence of the humanized anti-C5a antibodies described herein can be experimentally determined by methods known in the art (e.g., U.S. Pat. No. 9,079,949 and WO 2016 / 098356). The pH dependence may be reflected in differences in binding properties (e.g., binding affinity (e.g., dissociation constant), kinetic parameters (e.g., association and dissociation rates), and dissociation rates) at different pH levels. In some embodiments, the pH dependence of the humanized anti-C5a antibodies described herein can be expressed as a ratio of dissociation rates. In some embodiments, the dissociation rate can be expressed as a low pH dissociation factor and a neutral pH dissociation factor.

[0164] The pH dependency of humanized anti-C5a antibodies can be evaluated based on dissociation of C5-bound antibody at acidic pH (e.g., pH 5.8) or neutral pH (e.g., pH 7.4). A low pH dissociation factor, i.e., the percentage of antibody dissociated from antigen at pH 5.8 at 25° C. (wherein the antibody is pre-bound to antigen at pH 7.4), can be used to determine dissociation of C5-bound antibody at acidic pH. The low pH dissociation factor can be measured by associating antibody and antigen (e.g., humanized anti-C5a antibody and human C5) at pH 7.4 for 600 seconds, followed by a dissociation period of 600 seconds in a pH 5.8 buffer and calculating the percentage of antibody dissociated from antigen at pH 5.8. In some embodiments, the low pH dissociation factor for the humanized anti-C5a antibody of the present invention is about 5% to about 95%, about 10% to about 90%, about 15% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 25% to about 75%, about 25% to about 70%, about 25% to about 65%, In some embodiments, the neutral pH dissociation factor of the humanized anti-C5a antibody is within any one of the following ranges: about 25% to about 60%, about 30% to about 75%, about 30% to about 70%, about 30% to about 65%, about 30% to about 60%, about 35% to about 75%, about 35% to about 70%, about 35% to about 65%, about 35% to about 60%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, and about 40% to about 60%. In some embodiments, the neutral pH dissociation factor of the humanized anti-C5a antibody is any one of or more of about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%.

[0165] Neutral pH dissociation factor, i.e., the percentage of antibody that dissociates from antigen at pH 7.4 at 25° C. (wherein the antibody is pre-bound to antigen at pH 7.4 and can be used to determine dissociation of C5-bound antibody at neutral pH). The neutral pH dissociation factor can be measured by associating the antibody with antigen (e.g., humanized anti-C5a antibody with human C5) for 600 seconds at pH 7.4, followed by a dissociation period of 600 seconds in a pH 7.4 buffer and calculating the percentage of antibody that dissociates from antigen at pH 7.4. In some embodiments, the neutral pH dissociation factor of the humanized anti-C5a antibodies of the invention is less than or equal to about 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0166] In some embodiments, the ratio of low pH dissociation factor to neutral pH dissociation factor of a humanized anti-C5a antibody of the invention is any one of 1 or more, 1.5 or more, 2 or more, 2.5 or more, 3 or more, 3.5 or more, 4 or more, 4.5 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more. In some embodiments, the dissociation rate of the antibody for C5 at pH 5.8 relative to the dissociation rate of the antibody for C5 at pH 7.4 is at least 4, at least 5, or at least 6.

[0167] In some embodiments, the humanized anti-C5a antibody binds more strongly at neutral pH (e.g., pH 7.4) than at acidic pH (e.g., pH 5.8). In some embodiments, the low pH dissociation factor of the humanized anti-C5a antibody is greater than or equal to about any of 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%. In some embodiments, the neutral pH dissociation factor of the humanized anti-C5a antibody is less than or equal to about any of 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the ratio of low pH dissociation factor to neutral pH dissociation factor of the humanized anti-C5a antibody is about any of 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10. In some embodiments, the ratio of low pH dissociation factor to neutral pH dissociation factor of the humanized anti-C5a antibody is about any of 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 or more. In some embodiments, the ratio of low pH dissociation factor to neutral pH dissociation factor of the humanized anti-C5a antibody is 4 or more. In some embodiments, the ratio of low pH dissociation factor to neutral pH dissociation factor of the humanized anti-C5a antibody is 5 or more. In some embodiments, the ratio of low pH dissociation factor to neutral pH dissociation factor of the humanized anti-C5a antibody is 6 or greater.

[0168] Characteristics of pH-dependent anti-C5 antibodies The pH-dependent humanized anti-C5a antibodies described herein are suitable for development and use as pharmaceutical compositions.

[0169] The pH-dependent humanized anti-C5a antibodies described herein, in some embodiments, exhibit an extended serum half-life in vivo. In some embodiments, the humanized anti-C5a antibodies exhibit an extended serum half-life in mice, including transgenic mice. In some embodiments, the humanized anti-C5a antibodies exhibit an extended serum half-life in other test animals. Non-limiting examples of representative test animals include rats, chickens, rabbits, sheep, and cynomolgus monkeys. In some embodiments, the humanized anti-C5a antibodies exhibit an extended serum half-life in humans. In some embodiments, the humanized anti-C5a antibodies have a serum half-life in humans of at least one of about 2 hours, about 3 days, about 5 days, about 7 days, about 9 days, about 11 days, about 13 days, about 15 days, about 17 days, about 19 days, about 21 days, about 23 days, about 25 days. In some embodiments, the humanized anti-C5a antibodies have a serum half-life in humans of at least about 25 days.

[0170] In some embodiments, the pH-dependent humanized anti-C5a antibody has a binding affinity to human C5 comparable to a benchmark anti-C5a antibody.

[0171] III. Pharmaceutical Compositions Further provided by the present application is a pharmaceutical composition comprising any one of the humanized anti-C5a antibodies and a pharma- ceutical acceptable carrier. The pharmaceutical composition can be prepared in the form of a lyophilized formulation or an aqueous solution by mixing the humanized anti-C5a antibody of the desired degree of purification with optional pharma- ceutical acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

[0172] In some embodiments, the pharmaceutical composition further comprises additional components. Non-limiting examples of additional components include one or more of excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binders; lubricants; sweeteners; flavoring agents; coloring agents; preservatives; physiologically degradable compositions (such as gelatin); aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; weighting agents; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharma-ceutical acceptable polymeric or hydrophobic materials. Other "additional components" that can be included in the pharmaceutical composition of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0173] Additional excipients include agents that can function as one or more of the following: (1) bulking agents; (2) solubility enhancers; (3) stabilizers; and (4) agents that inhibit denaturation or adhesion to container walls.

[0174] Pharmaceutical compositions must be sterile for use in in vivo administration. Pharmaceutical compositions can be sterilized by filtration through sterile filtration membranes. The pharmaceutical compositions herein are generally placed into a container having a sterile access port, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0175] Sustained release preparations can be prepared.The composition for sustained release or implantation can comprise medicamentously acceptable polymeric materials or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers or sparingly soluble salts.Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing antagonist, which matrices are in the form of shaped articles, such as films or microcapsules.

[0176] The pharmaceutical compositions herein may also contain more than one active compound as necessary for the particular indication being treated, preferably active compounds with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may contain a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, or a growth inhibitory agent. Suitably, such molecules are present in combination in amounts effective for the intended purpose.

[0177] The active ingredient can also be encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules), prepared, for example, by coacervation techniques or interfacial polymerization, respectively, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 18th edition.

[0178] The formulations of pharmaceutical compositions can be prepared by any method known or hereafter developed in the art of pharmacology. Non-limiting examples of preparations include combining the active ingredient with a carrier or one or more other accessory ingredients, and then shaping or packaging the product into the desired single or multiple dose units, if necessary or desired. The pharmaceutical compositions can be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. The suspension or solution can be formulated according to known techniques and can contain, in addition to the active ingredient, additional ingredients, such as dispersing agents, wetting agents, or suspending agents. Such sterile injectable formulations can be prepared using a non-toxic, parenterally acceptable diluent or solvent, such as water or 1,3-butanediol. Non-limiting examples of other acceptable diluents and solvents include Ringer's solution, isotonic sodium chloride solution, and fixed oils, such as synthetic mono- or diglycerides.

[0179] The pharmaceutical compositions of the invention can be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such formulations can include dry particles containing the active ingredient and having diameters ranging from about 0.5 to about 7 nanometers (in some embodiments, from about 1 to about 6 nanometers). Such compositions are conveniently in the form of a dry powder for administration using a device with a dry powder reservoir and a propellant flowing toward the device to disperse the powder, or using a self-propelling solvent / powder delivery container (e.g., a device containing the active ingredient dissolved or suspended in a low boiling propellant in a sealed container).

[0180] The pharmaceutical composition of the present invention formulated for pulmonary delivery can also provide the active ingredient in the form of droplets of a solution or suspension. Such formulations can be prepared, packaged, or sold as aqueous or dilute alcoholic solutions or suspensions, optionally sterilized, containing the active ingredient, and can be conveniently administered using any spray or atomizing device. Such formulations can further include one or more additional components, non-limiting examples of which include flavoring agents (such as sodium saccharin), volatile oils, buffers, surfactants, or preservatives (such as methyl hydroxybenzoate).

[0181] The pharmaceutical compositions of the invention can be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations can be, for example, in the form of tablets or lozenges, using conventional techniques, and can contain, for example, 0.1-20% (w / w) of the active ingredient, with the remainder being a dissolvable or disintegrable oral composition and, optionally, one or more additional ingredients. Alternatively, a formulation suitable for buccal administration can include a powder containing the active ingredient, or can include an aerosolized or atomized solution or suspension containing the active ingredient. In some embodiments, such powdered, aerosolized, or aerosolized formulations have an average particle or droplet size when dispersed in the range of about 0.1 to about 200 nanometers, and can further include one or more additional ingredients.

[0182] IV. Method of Use Also provided are methods of inhibiting complement activation in an individual to treat a disease, such as a complement-related disease or disorder, by administering to the individual an effective amount of a humanized anti-C5a antibody. In some embodiments, the individual is a human.

[0183] Humanized anti-C5a antibodies can be used in combination with other therapeutic modalities, such as anti-inflammatory therapies. Examples of anti-inflammatory therapies that can be used in combination with the methods of the invention include, for example, therapies using steroid drugs, as well as therapies using non-steroid drugs.

[0184] In some embodiments, a method of inhibiting complement activation in an individual is provided, comprising administering to the individual (e.g., systemically, e.g., by subcutaneous or intravenous administration) an effective amount of a humanized anti-C5a antibody. In some embodiments, the humanized anti-C5a antibody comprises a VH comprising the mutations I48M, D54E, and N56W with reference to SEQ ID NO: 1, and a VL comprising the mutations D28E and D30F with reference to SEQ ID NO: 2. In some embodiments, the humanized anti-C5a antibody further comprises a mutation F29H in the VH with reference to SEQ ID NO: 1, and a mutation Y96H in the VL with reference to SEQ ID NO: 2. In some embodiments, the humanized anti-C5a antibody further comprises a mutation selected from the group consisting of E54H in the VH, N97H in the VH, and N92H in the VL, wherein said VH mutations refer to SEQ ID NO: 1 under the Kabat numbering system, and said VL mutations refer to SEQ ID NO: 2 under the Kabat numbering system. In some embodiments, the humanized anti-C5a antibody further comprises an IgG4 Fc region (e.g., the IgG4 Fc region comprises the PLA mutations: S228P, M428L, and N434A). In some embodiments, the humanized anti-C5a antibody binds more strongly at neutral pH (e.g., pH 7.4) than at acidic pH (e.g., pH 5.8). In some embodiments, the low pH dissociation factor of the humanized anti-C5a antibody is greater than or equal to about any of 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%. In some embodiments, the neutral pH dissociation factor of the humanized anti-C5a antibody is about 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less. In some embodiments, the ratio of low pH dissociation factor to neutral pH dissociation factor of the humanized anti-C5a antibody is about 1 or more, 1.5 or more, 2 or more, 2.5 or more, 3 or more, 3.5 or more, 4 or more, 4.5 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more. In some embodiments, the ratio of antibody dissociation at pH 5.8 for C5 to antibody dissociation at pH 7.4 for C5 is 6 or more. In some embodiments, the humanized anti-C5a antibody is administered by subcutaneous administration.

[0185] In some embodiments, the humanized anti-C5a antibody inhibits complement activation by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90%.

[0186] In some embodiments, the humanized anti-C5a antibody inhibits binding of C5a to C5aR and inhibits IC 50 The value is about 0.01 nM, about 0.1 nM, about 0.5 nM, about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 20 nM, about 50 nM, about 100 nM, or greater than 100 nM. In some embodiments, the inhibitor inhibits binding of C5a to C5aR and has an IC 50 The present invention provides a humanized anti-C5a antibody having a C5a concentration of about 1 nM to about 10 nM.

[0187] In some embodiments, the humanized anti-C5a antibodies inhibit C5a-induced calcium influx into cells and inhibit IC 50 The value is about 0.001 nM, about 0.005 nM, about 0.01 nM, about 0.05 nM, about 0.1 nM, about 0.15 nM, about 0.2 nM, about 0.25 nM, about 0.3 nM, about 0.4 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, about 1 nM, or greater than 1 nM. In some embodiments, the inhibitor inhibits C5a-induced calcium influx into cells and has an IC 50 Humanized anti-C5a antibodies having a C5A value of about 0.01 nM to about 0.3 nM are provided.

[0188] In some embodiments, the humanized anti-C5a antibody binds to human C5 with an affinity of about 0.001 nM, about 0.005 nM, about 0.01 nM, about 0.05 nM, about 0.1 nM, about 0.15 nM, about 0.2 nM, about 0.25 nM, about 0.3 nM, about 0.4 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, about 1 nM. In some embodiments, the humanized anti-C5a antibody binds to human C5 with low nanomolar affinity. In some embodiments, humanized anti-C5a antibodies are provided that bind to human C5 with an affinity of about 0.01 nM to about 1 nM.

[0189] In some embodiments, humanized anti-C5 antibodies are provided that do not affect C5-mediated complement activity.

[0190] In some embodiments, humanized anti-C5 antibodies are provided that do not inhibit C5 convertase-mediated activity.

[0191] In some embodiments, the humanized anti-C5a antibody reduces serum C5 levels in a subject up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after administration. In some embodiments, the humanized anti-C5a antibody reduces serum C5 levels in a subject by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% up to 15 days after administration. In some embodiments, a humanized anti-C5a antibody is provided that reduces serum C5 levels in a subject by at least 50% up to 15 days after administration.

[0192] In some embodiments, humanized anti-C5a antibodies that inhibit C5a-induced cell migration are provided. In some embodiments, the anti-C5a antibodies inhibit C5-induced cell migration and have a low nanomolar IC 50 In some embodiments, the IC of inhibition of C5a-induced cell migration by anti-C5a antibodies is 50 The value is about 0.01 nM, about 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, or greater than 10 nM. In some embodiments, the IC 50 Anti-C5a antibody inhibition is 0.01nM to 0.2nM.

[0193] In some embodiments, a method of inhibiting complement activation in an individual is provided, comprising administering to the individual (e.g., systemically, such as by subcutaneous or intravenous administration) an effective amount of a humanized anti-C5a antibody. In some embodiments, the humanized anti-C5a antibody comprises a VH comprising mutations I48M, D54E, and N56W with reference to SEQ ID NO: 1, and a VL comprising mutations D28E and D30F with reference to SEQ ID NO: 2. In some embodiments, the humanized anti-C5a antibody further comprises a mutation F29H in the VH with reference to SEQ ID NO: 1, and a mutation Y96H in the VL with reference to SEQ ID NO: 2. In some embodiments, the humanized anti-C5a antibody further comprises a mutation selected from the group consisting of E54H in VH, N97H in VH, and N92H in VL, where said VH mutations refer to SEQ ID NO: 1 under the Kabat numbering system, and said VL mutations refer to SEQ ID NO: 2 under the Kabat numbering system. In some embodiments, the humanized anti-C5a antibody further comprises an IgG4 Fc region (e.g., the IgG4 Fc region comprises the PLA mutations: S228P, M428L, and N434A). In some embodiments, the humanized anti-C5a antibody binds more strongly at neutral pH (e.g., pH 7.4) than at acidic pH (e.g., pH 5.8). In some embodiments, the low pH dissociation factor of the humanized anti-C5a antibody is greater than or equal to about any of 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%. In some embodiments, the neutral pH dissociation factor of the humanized anti-C5a antibody is less than or equal to about any of 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the ratio of low pH dissociation factor to neutral pH dissociation factor of the humanized anti-C5a antibody is less than or equal to about any of 1 or more, 1.5 or more, 2 or more, 2.5 or more, 3 or more, 3.5 or more, 4 or more, 4.5 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more.

[0194] In some embodiments, the selection of complement-related diseases or disorders, particularly diseases in which neutrophil activation plays a role in pathogenesis, includes macular degeneration (MD), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, rheumatoid arthritis, asthma, allergic asthma, lupus, ulcerative colitis, stroke, post-operative systemic inflammatory syndrome, asthma, allergic asthma, chronic obstructive pulmonary disease (COPD), paroxysmal nocturnal hemoglobinuria (PNH) syndrome, myasthenia gravis, neuromyelitis optica, (NMO), multiple sclerosis, delayed graft function, antibody-mediated rejection, atypical hemolytic uremic syndrome (aHUS), central retinal vein occlusion (CRVO), central retinal artery occlusion (CRAO), epidermolysis bullosa, sepsis, organ transplantation, inflammation, including but not limited to these. Examples include inflammation associated with cardiopulmonary bypass surgery and kidney dialysis), C3 nephropathy, membranous nephropathy, IgA nephropathy, glomerulonephritis (non-limiting examples of which include antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, lupus nephritis, and combinations thereof), ANCA-associated vasculitis, Shiga toxin-induced HUS, antiphospholipid antibody-induced pregnancy loss, COVID-19, graft-versus-host disease (GVHD), bullous pemphigoid, hidradenitis suppurativa, dermatitis herpetiformis, Sweet's syndrome, pyoderma gangrenosum, palmoplantar pustulosis and pustular psoriasis, rheumatic neutrophilic dermatosis, subcorneal pustulosis, gut-associated dermatosis-arthritis syndrome, neutrophilic eccrine hidradenitis, linear IgA disease, or any combination thereof.

[0195] Dose and Route of Administration The dosage and desired drug concentration of the pharmaceutical composition of the present application may vary depending on the specific application envisaged. The determination of the appropriate dosage or route of administration is well within the capabilities of one skilled in the art. Animal experiments provide reliable guidance for determining effective dosages for human treatment. Interspecies scaling of effective dosages can be performed according to the principles described in "The Use of Interspecies Scaling in Toxicokinetics," pp. 42-46 by Mordenti, J. and Chappell, W. in Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989.

[0196] Typically, the dose that can be administered to a subject (in some embodiments, a human) in the methods of the present invention ranges from an amount of 0.5 μg to about 50 mg per kilogram of the subject's body weight. Although the exact dose administered will vary depending on any number of factors, non-limiting examples of which include the type of subject and the type of disease state being treated, the age of the subject, and the route of administration. In some embodiments, the dose of the compound varies from about 1 μg to about 10 mg per kilogram of the subject's body weight. In other embodiments, the dose varies from about 3 μg to about 1 mg per kilogram of the subject's body weight.

[0197] In some embodiments, the humanized anti-C5a antibody is administered only once. In some embodiments, the humanized anti-C5a antibody is administered multiple times (e.g., any of 2, 3, 4, 5, 6, or more times). In some embodiments, the humanized anti-C5a antibody is administered once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once a month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, once every 7 months, once every 8 months, once every 9 months, or once a year. In some embodiments, the interval between administrations is any one of about 1 week to 2 weeks, 2 weeks to 1 month, 2 weeks to 2 months, 1 month to 2 months, 1 month to 3 months, 3 months to 6 months, or 6 months to 1 year. The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the art of medicine by monitoring the patient for symptoms of disease and adjusting the treatment accordingly. The frequency of administration will be apparent to one of skill in the art and will depend on any number of factors, including, but not limited to, the type and severity of the disease being treated, the type and age of the subject, etc.

[0198] The humanized anti-C5a antibody of the present application (non-limiting examples of which include reconstituted and liquid formulations) is administered to an individual (preferably a human) in need of treatment with the humanized anti-C5a antibody according to known methods (e.g., by intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, ocular, rectal, vaginal, parenteral, pulmonary, buccal, intraocular, or inhalation routes). Other formulations contemplated include planned nanoparticles, liposomal preparations, reclosed red blood cells containing active ingredients, and immunologically-based formulations. Parenteral administration of the humanized anti-C5a antibody includes any route of administration characterized by a physical break in the tissue of an individual and administration of the pharmaceutical composition through a break in the tissue. Parenteral administration can be local, regional, or systemic. Thus, non-limiting examples of parenteral administration include administration of the humanized anti-C5a antibody, for example, by injection of the composition, application of the composition through a surgical incision, application of the humanized anti-C5a antibody through a non-surgical wound that enters the tissue, etc. In particular, parenteral administration is contemplated, non-limiting examples of which include intravenous, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal injection, and intratumor.

[0199] The humanized anti-C5a antibody of the present invention can be prepared, packaged, or sold as a single unit dose or multiple single unit doses in bulk. A unit dose is a discrete amount of humanized anti-C5a antibody containing a predetermined amount of active ingredient. The amount of active ingredient is generally equal to the dose of active ingredient contemplated for administration to an individual, or a simple fraction of such a dose (e.g., 1 / 2 or 1 / 3 of such a dose).

[0200] The relative amounts of active ingredient, pharma- ceutical acceptable carrier, and optional additional ingredients in the pharmaceutical composition of the present invention will vary depending on the identity, size, and condition of the individual being treated, as well as the route by which the composition is administered. For example, the composition may contain between 0.1% and 100% (w / w) active ingredient. In various embodiments, the composition may contain at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%,At least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% (w / w) active ingredient.

[0201] V. Method of Preparation The present application also provides isolated nucleic acids encoding humanized anti-C5a antibodies, vectors and host cells containing such isolated nucleic acids, and recombinant methods for producing humanized anti-C5a antibodies.

[0202] Expression vectors and antibody-producing cells In some embodiments, the invention is a cell or cell line (such as a host cell) that produces at least one of the humanized anti-C5a antibodies described herein. In one embodiment, the cell or cell line is a genetically modified cell that produces at least one of the humanized anti-C5a antibodies described herein. In one embodiment, the cell or cell line is a hybridoma and produces at least one of the humanized anti-C5a antibodies described herein.

[0203] Hybrid cells (hybridomas) are typically generated from the fusion of a mass between a mouse spleen cell, which is highly enriched for B lymphocytes, and a myeloma "fusion partner cell" (Alberts et al., Molecular Biology of the Cell (Garland Publishing, Inc. 1994); Harlow et al., Antibodies. A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988). The cells in this fusion are then distributed into pools, which can be assayed for the production of antibodies with the desired specificity. Positive pools can be further divided until a single cell clone is identified that produces an antibody of the desired specificity. Antibodies produced by such clones are called monoclonal antibodies.

[0204] Nucleic acids encoding any of the humanized anti-C5a antibodies disclosed herein are also provided, as well as vectors containing the nucleic acids. Thus, the humanized anti-C5a antibodies of the invention can be produced by expressing the nucleic acid in a cell or cell line, such as a cell line typically used to express recombinant or humanized immunoglobulins. Thus, the antibodies and fragments of the invention can also be produced by cloning the nucleic acid into one or more expression vectors and transforming a cell line, such as a cell line typically used to express recombinant or humanized immunoglobulins, with the vector.

[0205] The genes encoding the heavy and light chains of the humanized anti-C5a antibody can be engineered according to methods known in the art, including, but not limited to, full-length gene chemical synthesis, polymerase chain reaction (PCR) (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, NY, 1989; Berger & Kimmel, Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques, Academic Press, Inc., San Diego, Calif., 1987; Co et al., 1992, J. Immunol. 148:1149). For example, genes encoding the heavy and light chains, or fragments thereof, can be cloned from genomic DNA of an antibody-secreting cell. Alternatively, cDNAs can be generated by reverse transcription of cellular RNA. Cloning is accomplished by conventional techniques, including the use of PCR primers that hybridize to sequences that flank or overlap the gene, or segment of the gene, to be cloned.

[0206] Nucleic acids encoding the humanized anti-C5a antibodies, or heavy or light chains, or fragments thereof, described herein, can be obtained and used in accordance with recombinant nucleic acid techniques to produce specific immunoglobulins, immunoglobulin chains, or fragments or variants thereof in a variety of host cells or in vitro translation systems. For example, a nucleic acid encoding an antibody, or a fragment thereof, can be introduced into a suitable host cell in a suitable prokaryotic or eukaryotic vector (e.g., an expression vector) by a suitable method (e.g., transformation, transfection, electroporation, infection) such that the nucleic acid is, for example, operably linked to one or more expression control elements in the vector or integrated into the genome of the host cell.

[0207] In some embodiments, the heavy and light chains, or fragments thereof, can be assembled in two different expression vectors and used to co-transfect recipient cells. In some embodiments, each vector can contain two or more selection genes, one for selection in bacterial systems and one for selection in eukaryotic systems. These vectors allow for the production and amplification of genes in bacterial systems, followed by co-transfection into eukaryotic cells and selection of the co-transfected cells. Selection procedures can be used to select for the expression of antibody nucleic acids introduced into eukaryotic cells on two different DNA vectors.

[0208] Alternatively, nucleic acids encoding the heavy and light chains, or fragments thereof, can be expressed from a single vector. Although the light and heavy chains are encoded by separate genes, they can be combined using recombinant techniques. For example, the two polypeptides can be joined by a synthetic linker that allows the two polypeptides to form a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single chain Fv (scFv); see, e.g., Bird et al., 1988, Science 242: 423-426 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883).

[0209] The present invention provides isolated nucleic acid molecules comprising nucleic acid sequences encoding the heavy and / or light chains, as well as fragments thereof. Nucleic acid molecules comprising sequences encoding both the light and heavy chains, or fragments thereof, can be engineered to contain synthetic signal sequences for secretion of the antibody or fragment when produced in a cell. Additionally, the nucleic acid molecules can contain special DNA links that allow for the insertion of other antibody sequences while maintaining the translational reading frame so as not to change amino acids normally found in antibody sequences. Representative nucleic acid sequences are shown in SEQ ID NOs: 33-62.

[0210] In accordance with the invention, a nucleic acid sequence encoding an antibody can be inserted into an appropriate expression vector. In various embodiments, the expression vector contains the necessary elements for the transcription and translation of the inserted nucleic acid encoding the antibody to generate a recombinant DNA molecule that directs the expression of the antibody sequence forming an antibody or fragment thereof.

[0211] Nucleic acids encoding antibodies, or fragments thereof, can be subjected to a variety of recombinant nucleic acid techniques known to those of skill in the art, such as site-directed mutagenesis.

[0212] A variety of methods can be used to express nucleic acids in cells. Nucleic acids can be cloned into many types of vectors. However, the invention should not be construed as limited to any particular vector. Instead, the invention should be construed to encompass a wide variety of vectors that are readily available and / or known in the art. For example, nucleic acids of the invention can be cloned into vectors, non-limiting examples of which include plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0213] In some embodiments, the expression vector is selected from the group consisting of viral vectors, bacterial vectors, and mammalian cell vectors. Numerous expression vector systems exist that contain at least some or all of the above compositions. Systems based on prokaryotic and / or eukaryotic vectors can be utilized in the present invention to produce polynucleotides, or their cognate polypeptides. Many such systems are commercially and widely available.

[0214] Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2012) and Ausubel et al. (1999), as well as other textbooks on virology and molecular biology. Non-limiting examples of viruses useful as vectors include retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In some embodiments, a mouse stem cell virus (MSCV) vector is used to express the desired nucleic acid. MSCV vectors have been demonstrated to efficiently express the desired nucleic acid in cells. However, the present invention should not be limited to the use of MSCV vectors alone, but rather any retroviral expression method is included in the present invention. Other examples of viral vectors are vectors based on Moloney murine leukemia virus (MoMuLV) and HIV. In some embodiments, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selection markers. (See, e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0215] Additional regulatory elements (e.g. enhancers) can be used to alter the frequency of transcription initiation. The promoter can be a promoter naturally associated with the gene or nucleic acid sequence, obtained by isolating the 5' non-coding sequence located upstream of the coding section and / or exon. Such a promoter can be called "endogenous". Similarly, the enhancer can be naturally associated with the nucleic acid sequence, located either downstream or upstream of the sequence. Alternatively, some advantages may be obtained by placing the coding nucleic acid section under the control of a recombinant or heterologous promoter, meaning a promoter that is not normally associated with the nucleic acid sequence in its natural environment. Recombinant or heterologous enhancer also means an enhancer that is not normally associated with the nucleic acid sequence in its natural environment. Such promoters or enhancers can include promoters or enhancers of other genes, promoters or enhancers isolated from any other prokaryotic, viral or eukaryotic cells, and promoters or enhancers that are not "natural", for example, containing different elements of different transcriptional regulatory regions and / or mutations that alter expression. In addition to producing promoter and enhancer nucleic acid sequences synthetically, sequences associated with the compositions disclosed herein can be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR (U.S. Patent Nos. 4,683,202 and 5,928,906).Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences in non-nuclear organelles (mitochondria, chloroplasts, etc.) can also be used.

[0216] Promoters and / or enhancers can be used that effectively direct the expression of the DNA segment in the cell type, organelle, and organism selected for expression. Those skilled in the art of molecular biology generally know how to use combinations of promoters, enhancers, and cell types to express proteins (see, for example, Sambrook et al. (2012)). The promoters used can be constitutive, tissue-specific, inducible, and / or useful for directing high levels of expression of the introduced DNA segment under appropriate conditions (such as being advantageous in large-scale production of recombinant proteins and their fragments).

[0217] An example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to it. However, other constitutive promoter sequences can also be used, non-limiting examples of which include the Simian Virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, Moloney virus promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, as well as human gene promoters (non-limiting examples of which include the actin promoter, myosin promoter, hemoglobin promoter, and muscle creatine promoter). Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter in the present invention provides a molecular switch that allows expression of a polynucleotide sequence operably linked to the inducible promoter to be turned on when desired and turned off when expression is not desired. Non-limiting examples of inducible promoters include metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters. Additionally, the present invention includes the use of tissue-specific or cell type-specific promoters (promoters that are active only in desired tissues or cells). Tissue-specific promoters are well known in the art, and non-limiting examples include the HER-2 promoter and PSA-related promoter sequences.

[0218] To assess expression of a nucleic acid, the expression vector introduced into a cell may also contain a selectable marker gene and / or a reporter gene, allowing expressing cells to be easily identified and selected from a population of cells to be transfected or infected with the viral vector. In other embodiments, the selectable marker may be carried on a separate nucleic acid and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to allow expression in the host cell. Useful selectable markers are known in the art and include, for example, antibiotic resistance genes (e.g., neo).

[0219] Reporter genes are used to identify potentially transfected cells and to assess the function of regulatory sequences. Reporter genes that encode proteins that are easily examined are well known in the art. In general, a reporter gene is a gene that is not present or expressed in the recipient organism or tissue, and that encodes a protein whose expression is manifested in some easily detectable characteristic (e.g., enzymatic activity). Expression of the report gene is examined at an appropriate time after the DNA has been introduced into the recipient cells.

[0220] Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (see, e.g., Ui-Tei et al., 2000 FEBS Lett. 479:79-82). Suitable expression systems are well known and can be prepared using well-known techniques or obtained commercially. Generally, the construct with the minimal 5' flanking region that exhibits the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions can be linked to the reporter gene and used to evaluate agents for their ability to alter promoter-driven transcription.

[0221] Methods for introducing and expressing nucleic acids into cells are known in the art. In the context of expression vectors, the vectors can be easily introduced into host cells (e.g., mammalian, bacterial, yeast, or insect cells) by any method in the art. For example, the expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0222] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, laser poration, etc. Methods for generating cells containing vectors and / or foreign nucleic acids are well known in the art. See, for example, Sambrook et al. (2012) and Ausubel et al. (1999).

[0223] Biological methods for introducing a nucleic acid of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells (e.g., human cells). Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type I, adenoviruses, and adeno-associated viruses, etc. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0224] Chemical means for introducing nucleic acids into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). The preparation and use of such systems is well known in the art.

[0225] Regardless of the method used to introduce exogenous nucleic acid into a host cell or the method of exposing the cell to a nucleic acid of the invention, a variety of assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include, for example, "molecular biological" assays well known to those of skill in the art (Southern blotting, Northern blotting, RT-PCR, PCR); "biochemical assays" (such as assays that detect the presence or absence of specific peptides, e.g., by immunological means (ELISA and Western blot) or the assays described herein, to identify agents within the scope of the invention).

[0226] 1. Vector Construction Polynucleotide sequences encoding the polypeptide components of the humanized anti-C5a antibody of the present application can be obtained using standard recombinant techniques. The desired polynucleotide sequence can be isolated from antibody producing cells (such as hybridoma cells) and sequenced. Alternatively, polynucleotides can be synthesized using a nucleotide synthesizer or PCR techniques. Once obtained, the polypeptide encoding sequence is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. Many vectors known and available in the art can be used for the purposes of the present application. Selection of an appropriate vector will depend primarily on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification and / or expression of heterologous polynucleotides) and compatibility with the specific host into which it is introduced. Non-limiting examples of vector components generally include an origin of replication, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid insert, and a transcription termination sequence.

[0227] Generally, plasmid vectors containing replicon and control sequences derived from a species compatible with the host cell are used in these hosts. The vector usually contains a replication site as well as marking sequences capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed with pBR322, a plasmid derived from an E. coli species. pBR322 contains genes encoding ampicillin (Amp) resistance and tetracycline (Tet) resistance, thus providing a simple means of identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophages can also contain, or be modified to contain, promoters that can be used by the microorganism to express endogenous proteins. Examples of pBR322 derivatives used to express specific antibodies are described in detail in Carter et al., U.S. Patent No. 5,648,237.

[0228] In addition, phage vectors containing replicon and control sequences compatible with the host microorganism can be used as transformation vectors for these hosts. For example, bacteriophages (such as GEM™-11) can be used to generate recombinant vectors that can be used to transform susceptible host cells (such as E. coli LE392).

[0229] The expression vectors described herein can contain two or more promoter-cistron pairs, each encoding a polypeptide component. A promoter is a non-translated regulatory sequence located upstream (5') of a cistron that modulates its expression. Prokaryotic promoters are typically classified into two classes: inducible and constitutive. Inducible promoters are promoters that initiate increased levels of transcription of the cistron under their control in response to a change in the culture conditions (e.g., the presence or absence of a nutrient, or a change in temperature).

[0230] A large number of promoters recognized by a variety of potential host cells are well known. A selected promoter can be operably linked to the cistron DNA encoding the light or heavy chain by removing it from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into a vector. Both the native promoter sequence and many heterologous promoters can be used to direct amplification and / or expression of the target gene. In some embodiments, heterologous promoters are used because they generally allow for higher transcription and higher yields of the expressed target gene when compared to the native target polypeptide promoter.

[0231] Suitable promoters for use in prokaryotic hosts include the PhoA promoter, the galactamase and lactose promoter systems, the tryptophan (trp) promoter system, and hybrid promoters (such as the tac or trc promoters). However, other promoters that function in bacteria are also suitable, such as other known bacterial promoters or phage promoters. These nucleotide sequences have been published and the skilled artisan can operably link them to the cistrons encoding the target light and heavy chains using linkers or adapters (Siebenlist et al. (1980) Cell 20: 269) and provide any necessary restriction sites.

[0232] In one aspect, each cistron in the recombinant vector contains a secretion signal sequence component that directs the translocation of the expressed polypeptide through the membrane. In general, the signal sequence can be a component of the vector or a portion of the target polypeptide DNA inserted into the vector. The signal sequence selected for the purposes of this application must be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In prokaryotic host cells that do not recognize and process the signal sequence native to the heterologous polypeptide, the signal sequence is replaced by a prokaryotic signal sequence selected from the group consisting of, for example, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leader, LamB, PhoE, PelB, OmpA, and MBP. In some embodiments, the signal sequence used in both cistrons of the expression system is the STII signal sequence or a variant thereof.

[0233] In some embodiments, production of the humanized anti-C5a antibody can occur in the cytoplasm of the host cell, so the presence of a secretion signal sequence in each cistron is not required. In some embodiments, the polypeptide components (a polypeptide encoding the VH domain of a first antigen-binding moiety, optionally fused to a second antigen-binding moiety, and a polypeptide encoding the VH domain of a first antigen-binding moiety, optionally fused to a second antigen-binding moiety) are L The polypeptide encoding the C5a domain is expressed, folded, and assembled to form a functional humanized anti-C5a antibody in the cytoplasm. - The β-actin (β-actin) gene of the present invention (Streptococcus cerevisiae) allows for proper folding and assembly of expressed protein subunits by providing cytoplasmic conditions that are favorable for the formation of disulfide bonds. Proba and Pluckthun Gene, 159:203 (1995).

[0234] 2. Protein production in prokaryotic host cells Suitable prokaryotic host cells for expression of the humanized anti-C5a antibodies of the present application include Archaea and Eubacteria (e.g., Gram-negative or Gram-positive organisms). Examples of useful bacteria include Escherichia (e.g., Escherichia coli), Bacillaria (e.g., Bacillus subtilis), Enterobacteriaceae, Pseudomonas species (e.g., Pseudomonas aeruginosa), Salmonella typhimurium, Serratia marcescens, Klebsiella, Proteus, Shigella, Rhizobium, Vitreosila, or Paracoccus. In some embodiments, Gram-negative cells are used. In some embodiments, Escherichia coli cells are used as hosts. Exemplary E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Accession No. 27,325) and its derivatives, including those of the genotype W3110 AfhuA(AtonA)ptr3 lac Iq lacL8 AompT A(nmpc-fepE) degP41 kan R A suitable strain is strain 33D3 (U.S. Pat. No. 5,639,635) carrying the replicon 1776 (ATCC 31,446), E. coli B, E. coli 1776 (ATCC 31,537), and E. coli RV308 (ATCC 31,608). Other strains and their derivatives are also suitable, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli 1776 (ATCC 31,537), and E. coli RV308 (ATCC 31,608). These examples are illustrative rather than limiting. Methods for constructing derivatives of any of the above bacteria with defined genotypes are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). It is generally necessary to select the appropriate bacterium by taking into account the replicability of the replicon in the bacterial cell. When using well-known plasmids (such as pBR322, pBR325, pACYC177, or pKN410) to provide the replicon, it may be preferable to use, for example, E. coli, Serratia, or Salmonella species as hosts.

[0235] Typically, the host cells should secrete minimal amounts of proteolytic enzymes and it may be desirable to incorporate additional protease inhibitors into the cell culture.

[0236] After transformation with the above expression vectors, host cells are cultured in conventional nutrient media modified as necessary to induce promoters, select transformants, or amplify genes encoding the desired sequences. Transformation means introducing DNA into a prokaryotic host so that the DNA can replicate, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is accomplished using standard techniques appropriate for such cells. A calcium treatment using calcium chloride is commonly used for bacterial cells that contain substantial cell wall barriers, and another method for transformation uses polyethylene glycol / DMSO. Yet another technique that may be utilized is electroporation.

[0237] Prokaryotic cells used to produce the humanized anti-C5a antibodies of the present application are grown in media known in the art and suitable for culturing the selected host cells. Examples of suitable media include Luria Broth (LB) plus necessary nutritional supplements. In some embodiments, the media also contains a selection agent selected based on the construction of the expression vector to selectively allow growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the media for the growth of cells expressing an ampicillin resistance gene.

[0238] Any necessary supplements other than sources of carbon, nitrogen, and inorganic phosphate may also be included at appropriate concentrations, introduced alone or in mixtures with other supplements or the medium (such as complex nitrogen sources).Optionally, the medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioerythritol, and dithiothreitol.

[0239] Prokaryotic host cells are cultured at an appropriate temperature. For example, for the growth of E. coli, the preferred temperature range is from about 20° C. to about 39° C., more preferably from about 25° C. to about 37° C., and even more preferably about 30° C. The pH of the medium depends primarily on the host organism, but can be any pH in the range of about 5 to about 9. For E. coli, the pH is preferably from about 6.8 to about 7.4, and more preferably about 7.0.

[0240] When an inducible promoter is used in the expression vector, expression of the protein is induced under conditions suitable for activating the promoter. In some embodiments, the PhoA promoter is used to control transcription of the peptide. The transformed host cell is then cultured in a phosphate-limiting medium for induction. The phosphate-limiting medium is preferably CRAP medium (see, e.g., Simmons et al., J. Immunol. Methods (2002), 263:133-147). As known in the art, a variety of other inducers can be used depending on the vector construct used.

[0241] The expressed humanized anti-C5a antibody of the present application is secreted into the periplasm of the host cell and recovered therefrom. Recovery of the protein typically involves disrupting the microorganism, generally by means such as osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris or whole cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transferred into the culture medium and isolated therein. The cells can be removed from the culture, and the culture supernatant can be filtered and concentrated to further purify the produced protein. The expressed polypeptide can be further isolated and identified using commonly known methods, such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays.

[0242] Alternatively, protein production can be achieved in large quantities by fermentation processes. A variety of large-scale fed-batch fermentation procedures are available for recombinant protein production. Large-scale fermentations have a volume of at least 1000 liters, and preferably from about 1,000 to 100,000 liters. These fermentors utilize impellers to distribute oxygen and nutrients, especially glucose (the preferred carbon / energy source). Small-scale fermentation generally refers to fermentation in fermentors with a volume of less than about 100 liters, with volumes ranging from about 1 liter to about 100 liters possible.

[0243] During the fermentation process, induction of protein expression is typically performed by culturing cells under appropriate conditions to a desired density (e.g., OD 550 Induction is initiated after proliferation to about 180-220 h, at which stage the cells are in early stationary phase. As known in the art and described above, a variety of inducers can be used depending on the vector construct used. The cells can be grown for a shorter period of time before induction. The cells are usually induced for about 12-50 hours, although longer or shorter induction times can be used.

[0244] Various fermentation conditions can be modified to improve the production yield and quality of the humanized anti-C5a antibody of the present application. For example, to improve proper assembly and folding of the secreted polypeptide, the host prokaryotic cells can be co-transformed with additional vectors overexpressing chaperone proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD, and / or DsbG) or FkpA (peptidyl prolyl cis-trans-isomerase with chaperone activity). Chaperone proteins have been demonstrated to facilitate proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Patent No. 6,083,715; Georgiou et al., U.S. Patent No. 6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem. 275: 17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol. Microbiol. 39:199-210.

[0245] Certain host strains deficient in proteolytic enzymes can be used in the present application to minimize proteolysis of expressed heterologous proteins, particularly those that are sensitive to proteolysis. For example, the host cell strain can be modified to introduce genetic mutations in genes encoding known bacterial proteases, such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof. Several E. coli protease-deficient strains are available and are described, for example, in Joly et al. (1998), supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S. Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

[0246] E. coli strains transformed with plasmids deficient in proteolytic enzymes and overexpressing one or more chaperone proteins can be used as host cells in expression systems encoding the humanized anti-C5a antibodies of the present application.

[0247] 3. Protein production in eukaryotic cells In some embodiments, the humanized anti-C5a antibodies described herein can be expressed in eukaryotic cells. Non-limiting examples of vector components for expression in eukaryotic cells generally include one or more of a signal sequence, an origin of replication, one or more marker genes and enhancer elements, a promoter, and a transcription termination sequence.

[0248] a) Selection and transformation of host cells Suitable host cells for cloning or expressing the DNA in the vectors herein include the higher eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become routine. Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line (293 cells, or 293 cells subcloned into suspension culture for propagation, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0249] After transformation of the host cells with the expression or cloning vectors described above to produce humanized anti-C5a antibody, the cells are cultured in conventional nutrient media modified as necessary to induce promoters, select transformants, or amplify genes encoding the desired sequences. In some embodiments, the humanized anti-C5a antibody is expressed in CHO cells. In some embodiments, the humanized anti-C5a antibody is expressed in Expi-CHO cells.

[0250] b) Cultivation of host cells The host cells used to produce the humanized anti-C5a antibodies of the present application can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium (MEM), (Sigma) RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Patent Reissue No. 30,985 can be used as media for the host cells. Any of these media may be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drugs), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other supplements required that would be known to one of skill in the art may also be included at appropriate concentrations. Culture conditions (temperature, pH, etc.) will be the same as those previously used with the host cell selected for expression and will be apparent to one of skill in the art.

[0251] c) Protein purification The humanized anti-C5a antibodies produced herein can be further purified to obtain substantially homogeneous preparations for further assays and uses. Standard protein purification methods known in the art can be used.

[0252] 4. Antibody Generation and Modification The components of the humanized anti-C5a antibody can be produced using any method known in the art, including those described below.

[0253] a) Monoclonal antibodies Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

[0254] For example, monoclonal antibodies can be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or can be made by recombinant DNA (U.S. Pat. No. 4,816,567).

[0255] In the hybridoma method, a mouse or other suitable host animal (such as a hamster) is immunized as described above to produce lymphocytes that produce, or are capable of producing, antibodies that will specifically bind to the immunizing protein. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent (such as polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0256] The immunizing agent will typically include an antigenic protein or a fusion variant thereof. Generally, peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using an appropriate fusing agent (such as polyethylene glycol) to form a hybridoma cell. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103.

[0257] Immortalized cell lines are usually myeloma cells originating from transformed mammalian cells (especially rodent, bovine and human). Usually, rat or mouse myeloma cell lines are used. The hybridoma cells thus prepared are seeded and grown in a suitable medium, preferably containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the medium for hybridomas will typically contain hypoxanthine, aminopterin and thymidine (HAT medium), which are substances that inhibit the growth of HGPRT-deficient cells.

[0258] Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium (such as HAT medium). Preferred among these are mouse myeloma lines (such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, CA, USA, and SP-2 cells (and derivatives thereof, e.g., X63-Ag8-653) available from the American Type Culture Collection, Manassas, VA, USA). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0259] The culture medium in which the hybridoma cells are growing is examined for the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0260] The culture medium in which the hybridoma cells are cultured can be examined for the presence of monoclonal antibodies directed against the desired antigen. Preferably, the binding affinity and specificity of the monoclonal antibody can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked assay (ELISA). Such techniques and assays are known in the art. For example, binding affinity can be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0261] After hybridoma cells producing antibodies with the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can be grown in vivo as tumors in a mammal.

[0262] The monoclonal antibodies secreted by the subclones are conveniently isolated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0263] Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567 and described above. DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures, e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of mouse antibodies. Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector which is transfected into host cells, and the monoclonal antibodies are synthesized in the recombinant host cells, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pliickthun, Immunol. Revs. 130:151-188 (1992).

[0264] In a further embodiment, antibodies can be isolated from antibody phage libraries generated using techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of mouse and human antibodies using phage libraries. Subsequent publications describe the generation of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)) as well as combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993)). These techniques are therefore viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0265] The DNA can also be modified, for example, by replacing the coding sequence for the human heavy and light chain constant domains with the homologous murine sequences (U.S. Patent No. 4,816,567; Morrison, et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Typically, such a non-immunoglobulin polypeptide is substituted for the constant domains of an antibody or for the variable domains of one antigen-binding site of an antibody, resulting in a bivalent chimeric antibody containing one antigen-binding site with specificity for one antigen and another antigen-binding site with specificity for a different antigen.

[0266] The monoclonal antibodies described herein can be monovalent, the preparation of which is well known in the art. For example, one method involves recombinant expression of an immunoglobulin light chain and a modified heavy chain. The heavy chain is typically truncated at a point within the Fc region to prevent cross-linking of the heavy chain. Alternatively, the relevant cysteine ​​residue can be replaced with another amino acid residue or removed to prevent cross-linking. In vitro methods are also suitable for the preparation of monovalent antibodies. Digestion of antibodies to generate fragments thereof, particularly Fab fragments, can be accomplished using routine techniques known in the art.

[0267] Chimeric or hybrid antibodies can also be prepared in vitro using known methods in synthetic protein chemistry, including methods involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide-exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0268] b) Humanized antibodies Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibodies) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) (such as mouse, rat, or rabbit) having the desired specificity, affinity, and capacity. In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin and all or substantially all of the FR regions are of a human immunoglobulin consensus sequence. The humanized antibody optimally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988), and Presta, Curr. Opin. Struct. Biol. 2: 593-596 (1992).

[0269] Methods for humanizing non-human antibodies are well known in the art. In general, a humanized antibody has one or more amino acid residues introduced from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues and are typically taken from an "import" variable domain. Humanization can be essentially performed according to the method of Winter and coworkers, Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988), or by replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody. Such "humanized" antibodies are thus chimeric antibodies (U.S. Pat. No. 4,816,567), in which substantially less than an intact human variable domain has been replaced by the corresponding sequences from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0270] The selection of human variable domains (both light and heavy chains) used to generate humanized antibodies is very important to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the rodent sequence is then accepted as the human framework (FR) for the humanized antibody. Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987). Another method uses a special framework derived from the consensus sequence of all human antibodies of a special subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies. Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993).

[0271] It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To this end, according to one preferred method, humanized antibodies are prepared by a method in which the parental sequences and various conceptual humanized products are analyzed using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commercially available and familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues which influence the ability of the candidate immunoglobulin to bind to its corresponding antigen. In this way, FR residues are selected and combined from the recipient and import sequences to achieve the desired antibody characteristic, such as increased affinity for the target antigen. In general, the CDR residues are directly and most substantially involved in influencing binding to the antigen.

[0272] Various forms of humanized antibodies are contemplated: for example, the humanized antibody can be an antibody fragment (such as a Fab), which is optionally conjugated to one or more cytotoxic agents to form an immunoconjugate, or the humanized antibody can be an intact antibody (such as an intact IgG4 antibody).

[0273] c) Human antibodies As an alternative to humanization, human antibodies can be produced. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, chimeric mice and mice with germline mutants of the antibody heavy chain joining region (J H It has been described that homozygous deletion of the .) gene completely inhibits endogenous antibody production. The transfer of the human germline immunoglobulin gene array in such germline mutant mice results in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993); U.S. Patent No. 5,591,669, and WO 97 / 17852. Transgenic mice or rats capable of producing fully human antibodies are known in the art. See, e.g., U.S. Patent Application Publication No. 20090307787A1, U.S. Patent No. 8,754,287, U.S. Patent No. 20150289489A1, U.S. Patent No. 20100122358A1, and WO 2004049794.

[0274] Alternatively, phage display technology can be used to generate human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. McCafferty et al., Nature 348:552-553 (1990);Hoogenboom and Winter, J. Mol. Biol. 227: 381 (1991). According to this technique, antibody V domain genes are cloned into frame with either the major or minor coat protein gene of a filamentous bacteriophage (such as M13 or fd) and functional antibody fragments are displayed on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody will result in selection of genes encoding antibodies exhibiting those properties. Thus, the phage mimics some of the properties of B cells. Phage display can be carried out in a versatile format, as reviewed, for example, in Johnson, Kevin S, and Chiswell, David J., Curr. Opin Struct. Biol. 3:564-571 (1993). Several sources of V gene compartments can be utilized for phage display. Clackson et al., Nature 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. Repertoires of V genes from unimmunized human donors can be constructed and antibodies against a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905.

[0275] Human monoclonal antibodies can also be prepared using the techniques of Cole et al. and Boerner et al. (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol. 147(1): 86-95 (1991). Similarly, human antibodies can be produced by introducing human immunoglobulin loci into transgenic animals (e.g., mice) in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in every respect, including gene rearrangement, assembly, and antibody repertoire. This approach has been described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following journal publications: Marks et al., Bio / Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-13 (1995); (1994), Fishwild et al., Nature Biotechnology 14: 845-51 (1996), Neuberger, Nature Biotechnology 14: 826 (1996), and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

[0276] Finally, human antibodies may be generated by in vitro activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275).

[0277] d) Antibody fragment In some situations, it is advantageous to use antibody fragments (such as antigen-binding fragments) rather than whole antibodies, as the smaller size of the fragments allows for rapid clearance and potentially improved access to solid tumors.

[0278] Various techniques have been developed to produce antibody fragments. Traditionally, these fragments are derived from proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., J Biochem Biophys. Method. 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli, allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from antibody phage libraries, as described above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Fab and F(ab')2 fragments with extended in vivo half-lives are described in U.S. Pat. No. 5,869,046. In another embodiment, the antibody of choice is a single chain Fv fragment (scFv). See WO 93 / 16185; U.S. Pat. No. 5,571,894, and U.S. Pat. No. 5,587,458. Antibody fragments can also be "linear antibodies," as described, for example, in U.S. Pat. No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

[0279] e) Effector function operation It may be desirable to modify the humanized anti-C5a antibodies of the present application with respect to Fc effector function, for example to alter (e.g., enhance or eliminate) the antigen-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. In a preferred embodiment, the Fc effector function of the humanized anti-C5a antibody is reduced or eliminated. This can be achieved by introducing one or more amino acid substitutions into the Fc region of the antibody. Alternatively, or in addition, cysteine ​​residues can be introduced into the Fc region to allow interchain disulfide bond formation in this region. The homodimeric humanized anti-C5a antibodies thus generated may have improved internalization capabilities and / or increased complement-associated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, BJ Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and has enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989).

[0280] To increase the serum half-life of the antibody, a salvage receptor binding epitope can be incorporated into the humanized anti-C5a antibody, for example, as described in U.S. Patent No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for the extended serum half-life of the IgG molecule in vivo.

[0281] f) Other amino acid sequence modifications Amino acid sequence modifications of the antibody (such as the single chain antibody or antibody components of the humanized anti-C5a antibody described herein) are contemplated. For example, this may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes (such as changing the number or position of glycosylation sites) can also alter post-translational processing of the antibody.

[0282] One useful method for identifying certain residues or regions of an antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells in Science, 244:1081-1085 (1989). Here, a residue or group of target residues is identified (e.g., the charged residues Arg, Asp, His, Lys, and Glu) and replaced with neutral or negatively charged amino acids (alanine or polyalanine are most preferred) to affect amino acid antigen interactions. Those amino acid positions that demonstrate function as sensitive to substitution are then further refined by introducing additional or other variants at or in place of the substitution sites. Thus, while the site for introducing the amino acid sequence change is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the performance of a mutation located at a given site, alanine scanning or random mutagenesis is performed at the target codon or region, and the expressed antibody variants are screened for the desired activity.

[0283] Amino acid sequence insertions include amino- and / or carboxy-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to a cytotoxic polypeptide. Other insertional variants of the antibody molecule include the fusion of the antibody to an enzyme or polypeptide which extends the serum half-life of the antibody (e.g., for ADEPT).

[0284] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced with a different residue. Sites of greatest interest for substitution mutagenesis include the hypervariable regions, although FR changes are also contemplated. Conservative substitutions are shown in the table below under the heading "preferred substitutions." If such substitutions alter biological activity, more substantial changes (shown in Table 2 as "representative substitutions" or further described below in relation to classes of amino acids) can be introduced and the products screened. [Table 2]

[0285] Substantial modification of the biological properties of the antibody is achieved by selecting substitutions that differ significantly in their effect on (a) maintaining the structure of the polypeptide backbone (e.g., sheet or helix) in the area of ​​the substitution, or (b) maintaining the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chain. Naturally occurring residues are divided into groups based on common side chain properties: (1) Hydrophobic: norleucine, met, ala, val, leu, ile; (2) Neutral hydrophilic: cys, ser, thr; (3) Acidic: asp, glu; (4) Basic: asn, gln, his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) Aromatic: trp, tyr, phe.

[0286] Non-conservative substitutions result in the exchange of a member of one of these classes for another class.

[0287] Any cysteine ​​residue not involved in maintaining the proper structure of the antibody also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking Conversely, cysteine ​​bond(s) can be added to the antibody to improve its stability, particularly where the antibody is an antibody fragment such as an Fv fragment.

[0288] A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variants selected for further development will have improved biological properties relative to the parent antibody from which they are generated.

[0289] Another type of amino acid variant of an antibody alters the native glycosylation pattern of the antibody, by which is meant removing one or more carbohydrate moieties found in the antibody, and / or adding one or more glycosylation sites that are not present in the antibody.

[0290] Glycosylation of antibodies is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine can also be used.

[0291] Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above tripeptide sequences (for N-linked glycosylation sites). The alteration may also be accomplished by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0292] Nucleic acid molecules encoding amino acid sequence variants to the humanized anti-C5a antibodies of the present application are prepared by a variety of methods known in the art, non-limiting examples of which include isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of previously prepared variant or non-variant versions.

[0293] g) Other modifications The humanized anti-C5a antibody of the present application can be further modified to contain additional non-proteinaceous moieties known in the art and readily available. Preferably, the moieties suitable for derivatization of the antibody are water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer can be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary and can be the same or different molecules when more than one polymer is attached. In general, the number and / or type of polymers used for derivatization can be determined based on a variety of considerations, including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc. Such techniques and other suitable formulations are disclosed in Remington: The Science and Practice of Pharmacy, 20th Ed., Alfonso Gennaro, Ed., Philadelphia College of Pharmacy and Science (2000).

[0294] kit The invention also includes a kit comprising a humanized anti-C5a antibody of the invention and instructional materials, e.g., describing administering the humanized anti-C5a antibody to an individual for therapeutic or non-therapeutic purposes, as described elsewhere herein. In one embodiment, the kit further comprises a pharma- ceutically acceptable (optionally sterile) carrier suitable for dissolving or suspending a therapeutic composition comprising a humanized anti-C5a antibody of the invention or a combination thereof, e.g., prior to administering the antibody to an individual. Optionally, the kit comprises an application device for administering the antibody. Unit dosage forms comprising the humanized anti-C5a antibody are also provided. EXAMPLES

[0295] The following examples are intended to be purely illustrative of the invention and should not be construed as limiting the invention in any manner. The following examples and detailed description are offered by way of illustration and not by way of limitation.

[0296] Example 1: Construction and selection of anti-C5a antibodies A murine anti-C5a IgG4 antibody / scFv comprising the VH amino acid sequence of SEQ ID NO: 1 and the VL amino acid sequence of SEQ ID NO: 2 is used to generate humanized anti-C5a antibodies and variants thereof. For further affinity optimization, a CDR grafting approach was used to generate a humanized anti-C5a construct in which the CDRs of the murine scFv were grafted onto the VH and VL frameworks according to SEQ ID NO: 9 and 10, respectively. The VH and VL of the CDR grafted anti-C5a construct are shown in SEQ ID NO: 84 and 85.

[0297] 1. Affinity Optimization Using site-directed mutagenesis, each amino acid in the six complementarity determining regions (CDRs) of the anti-C5a scFv clone containing the VH and VL sequences of SEQ ID NOs: 84 and 85 was individually mutated to a total of 20 amino acids. DNA primers containing NNS codons encoding the 20 amino acids were used to introduce mutations at each targeted CDR position. Degenerate primers were used in the site-directed mutagenesis reaction. Briefly, each degenerate primer was phosphorylated. PCR conditions were 94°C for 2 min, 16 cycles of (94°C for 30 s, 55°C for 30 s, 72°C for 5 min), and 72°C for 10 min. The PCR products were purified and then electroporated into BL21 for colony formation and scFv fragment generation. A construct named NM2 containing the VH and VL sequences of SEQ ID NOs: 82 and 83 was selected for screening the scFv mutant library.

[0298] 2. Primary scFv Mutant Library Screening The primary screen consisted of a single point scFv capture ELISA (SPE) performed as follows: 96-well Maxisorp Immunoplates were coated overnight at 4°C with anti-c-myc antibody (Bethyl-A190-204A) in coating buffer PBS pH 7.4. The next day, plates were blocked with casein in PBS pH 7.4 for 1 h at 25°C. ScFv containing PE was then added to the plates and incubated for 1 h at 25°C. After washing, biotinylated antigen was added to the wells and incubated for 1 h at 25°C. This was followed by incubation with SA-horseradish peroxidase (HRP) (Invitrogen SNN1004) conjugate for 1 h at 25°C. HRP activity was detected using tetra-methylbenzidine (TMB) (KPL-5120-0047) substrate and the reaction was quenched with 2M HCl. Plates were read at 450 nM. Clones showing optical density (OD) signals at 450 nm more than twice that of the parent clone were picked and sequenced.

[0299] Seven anti-C5a scFv constructs with sequence unique and improved binding were identified by SPR, and a summary of SPR binding is shown in Table 3. [Table 3]

[0300] 3. Combinatorial Screening of Anti-C5a scFv Library Point mutations in VH and VL that were determined to favor antigen binding were further combined to obtain additional binding synergy. Briefly, each degenerate primer was phosphorylated and then used with uridinylated ssDNA in a 10:1 ratio. The mixture was heated to 85°C for 5 min and then cooled to 55°C for 1 h. T4 ligase and T4 DNA polymerase were then added and the mixture was incubated at 37°C for 1.5 h. Typically, 200 ng of combinatorial library DNA was electroporated into BL21 for colony formation and scFv fragment generation.

[0301] Combinatorial mutants were expressed as scFvs and screened using capture ELISA. Clones showing optical density (OD) signals at 450 nanometers greater than twice that of the parental clones were sequenced and further confirmed by capture ELISA and SPR.

[0302] 4. Affinity Ranking ELISA Affinity ranking ELISA was performed as follows: scFv concentrations in PE were measured by quantitative ELISA using purified scFv samples as standards. Briefly, plates were coated with anti-His antibody (Genscript-A00186) in coating buffer PBS pH 7.4 overnight at 4°C. The next day, plates were blocked with casein in PBS pH 7.4 for 1 h at 25°C. ScFv containing PE was diluted in 3-fold serial dilutions and then added to the plate. Purified scFv reference samples of known concentration were used as standards. After incubation at 25°C for 1 h and washing 3 times with PBS-T, anti-c-myc-HRP (Bethyl-A190-104P) was added to the wells and incubated at 25°C for 1 h. HRP activity was detected with TMB substrate and the reaction was quenched with 2M HCl. Plates were read at 450 nM.

[0303] Plates were coated overnight at 4°C with anti-C5a cyno antigen and human antigen in PBS pH 7.4. The next day, plates were blocked with casein in PBS for 1 hour at 25°C. Normalized scFv containing PE was diluted using 3-fold serial dilutions starting at 100 nM, added to the plate and incubated at 25°C for 1 hour. After washing, anti-c-myc-HRP was added to the wells and incubated at 25°C for 1 hour. HRP activity was detected using TMB substrate and the reaction was quenched with 2M HCl. Plates were read at 450 nM. Data were fitted to a one-site binding equation using GraphPad Prism 5 software. Results for hits that bound to cyno antigen are shown in Figures 1A-1B. Results for hits that bound to human antigen are shown in Figures 2A-2B.

[0304] 5. SPR of Anti-C5a Hits immobilization An activator was prepared by mixing 400 mM EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and 100 mM NHS (N-hydroxysuccinimide) (GE) immediately prior to injection. The CMS sensor chip was activated with this mixture at a flow rate of 10 μL / min for 600 s. A mixture of anti-c-myc and anti-his antibodies in 10 mM NaAc (pH 4.5) was then injected over Fcl and Fc2 in channels 1 to 8 at a flow rate of 10 μL / min for 600 s. The chip was deactivated with 1 M ethanolamine-HCl (GE) at a flow rate of 10 μL / min for 420 s.

[0305] Ligand capture and running analytes Anti-C5a scFv was diluted to 0.5 μg / ml and other samples were diluted to 2 μg / ml in running buffer 1×HBS-EP+ (0.01 M HEPES, 0.15 M NaCl, 0.003 M EDTA, 0.05% surfactant P20). The same dilution scheme as for scFvs was used for NC (negative control) with TES (Tris-EDTA-sucrose) buffer. Diluted TES buffer was injected into channel Fc1 at a flow rate of 10 μL / min for 40 s and diluted scFv was injected into channel Fc2 at a flow rate of 10 μL / min for 40 s. Then, 60 nM cynomolgus antigen analyte or 5 nM human antigen analyte with running buffer was injected into Fc1-Fc2 at a flow rate of 100 μL / min for 60 s of association phase followed by dissociation for 1200 s. 10 mM glycine (pH 1.5) as regeneration buffer was injected after every dissociation phase. The chip was regenerated with 10 mM glycine (pH 1.5).

[0306] Data analysis Sensograms for the reference channel Fc1 and the buffer channel were subtracted from the test sensograms. The data was fitted by a 1:1 kinetic binding model. A molecular weight of 8.4 kDa was used to calculate the molar concentration of cyno antigen. A molecular weight of 8.2 kDa was used to calculate the molar concentration of human antigen. The results of binding to cyno antigen are shown in Table 4. The results of binding to cyno antigen are shown in Table 5. [Table 4] [Table 5]

[0307] To further optimize the pH-dependent antigen-binding properties, 16D10 was selected, which contains the VH amino acid sequence of SEQ ID NO: 9 and the VL amino acid sequence of SEQ ID NO: 10. Fc domain mutations were introduced into 16D10. The positions targeted by the designed mutations were introduced by QuikChange® (Agilent) or Q5® site-directed mutagenesis (NEB).

[0308] To account for the pH-dependent binding introduced, histidine substitutions were introduced at each of the six CDR regions of 16D10 by QuikChange® mutagenesis, a method known as histidine scanning. For each variant, expression plasmids carrying the mutated 16D10 heavy chain and wild-type light chain were transiently transfected into HEK293 cells and cultured in shake flasks with serum-free medium for 5-7 days. Light chain variants were similarly obtained by co-transfecting the mutated light chain and wild-type heavy chain. To characterize binding, expression supernatants were collected by centrifugation. Binding to C5 was measured at pH 7.4 and pH 5.8, respectively, using IgG from the expression supernatants, using a Gator™ (Probe Life, Inc.).

[0309] Mutant antibodies were selected that maintained the same binding dissociation constant at pH 7.4 but had a reduced binding dissociation constant at pH 5.8 compared to 16D10. pH-dependent binding variants with multiple histidine mutations were created by simultaneously transfecting single binding-favoring histidine mutations into the heavy or light chain. Variants with multiple binding-favoring histidine mutations within a chain were created by introducing those favorable mutations into the same chain by QuikChange® mutagenesis. The pH-dependent binding variants were further purified from expression supernatants by Protein-A affinity chromatography to characterize affinity. The purified proteins were dialyzed into 20 mM histidine buffer with 150 mM NaCl and quantified by Nanodrop (Thermo Fisher Scientific, Inc., USA) before being purified by Gator™. ( The affinity was measured using Probe Life, Inc.

[0310] Three humanized and affinity matured pH-dependent C5a binding construct variants were identified for further characterization: E54H, which contains the mutation F29H in VH CDR1, E54H in VH CDR2, and Y96H in VL CDR3 (VH: SEQ ID NO: 17; VL: SEQ ID NO: 18); N97H, which contains the mutation F29H in VH CDR1, N97H in VH CDR3, and Y96H in VL CDR3 (VH: SEQ ID NO: 25; VL: SEQ ID NO: 26); N92H, which contains the mutation F29H in VH CDR1, N92H in VL CDR2, and Y96H in VL CDR3 (VH: SEQ ID NO: 33; VL: SEQ ID NO: 34). pH-dependent data are described in Example 4.

[0311] The humanized anti-C5a antibodies were examined for affinity to human C5a by ELISA. Figure 3 shows that all humanized anti-C5a constructs (16D10, E54H, N97H, and N92H) bound to human C5a with comparable high affinity. The antibodies bound to C5 with higher affinity (Figure 8).

[0312] Because C5 provides a deep sink for drugs that target C5a, it is desirable for anti-C5a constructs to minimize disruption of the C5-mediated pathway. The C5 inhibitory efficacy of all anti-C5a antibody constructs was evaluated using a classical pathway-mediated sheep red blood cell lysis assay.

[0313] Antibody-sensitized sheep RBCs (2 × 10 7 Cells / assay, Solarbio) were incubated with 5% normal human serum (NHS, from Quidel) in gelatin veronal buffer (GVB2+, Complement Technology Inc; total assay volume: 50 μl) for 30 min at 37 °C. Anti-C5a constructs were incubated with NHS for 1 h at 4 °C and then added to sheep RBCs at various concentrations (Figure 4). The lysis reaction was stopped by adding ice-cold PBS containing 40 mM EDTA. The incubation mixtures were centrifuged at 1500 rpm for 5 min. The supernatants from each mixture were collected and OD was measured at 405 nm. EDTA, distilled water (denoted as DW), and the anti-C5 benchmark antibody Soliris were used as positive controls for RBC lysis. 5% serum was used as a negative control.

[0314] The results of the sheep RBC lysis assay are shown in Figure 4 and Table 6. The positive controls (EDTA, water, and Soliris) lysed sheep RBCs; Soliris effectively blocked sheep RBC lysis with an IC50 of 1.23 μg / ml. The humanized anti-C5a construct showed no inhibition in lysis, suggesting minimal disruption of the C5-mediated pathway. [Table 6]

[0315] Example 2: In vitro functional assays 1. Ligand Blocking Assay in U937 Cells U937 / C5aR cells were washed twice with PBS and diluted with 3 × 10 6 Cells were suspended at a density of 100 μl cells / ml. 100 μl of cells were added to a 96-well microplate, 50 μl of compound diluted in assay buffer and 50 μl of biotinylated ligand human C5a (final concentration 20 nM) were added sequentially to the corresponding wells, and the plate was incubated on ice for 120 min. Centrifuged at 1000 rpm for 3-5 min at 4°C, the supernatant was removed, and the cells were washed twice with pre-chilled PBS. 100 μl of FITC-labeled streptavidin (eBioscience) was added to the cells and incubated for another 30 min on ice. Centrifuged at 4°C, the supernatant was removed, and the cells were washed twice with pre-chilled PBS. 150 μl of 0.5% PFA was added to suspend the cells, and the signal was detected by FACS (Beckman, Cytoflex). IC 50 Values ​​were calculated by GraphPad Prism software. High control means that cells were incubated with biotinylated C5a without the addition of anti-C5a constructs; low control means that cells were incubated with assay buffer without the addition of anti-C5a constructs.

[0316] Four anti-C5a constructs (16D10, E54H, N97H, and N92H) were evaluated for their ability to block C5a receptor (C5aR) in U937 cells.

[0317] Anti-C5a constructs were titrated in U937 cells expressing C5aR at a series of concentrations as shown in Figure 5. Mean fluorescence intensity was measured at each antibody concentration. U937 cells without added antibody were used as low control. Cells incubated with biotinylated C5a without added anti-C5a constructs were used as high control.

[0318] The experiment was repeated and representative results are shown in Figure 5 and summarized in Table 7. All four anti-C5a constructs (16D10, E54H, N97H, and N92H) demonstrated potent ligand blocking at nanomolar concentrations. [Table 7]

[0319] 2. C5aR Antagonist Migration Assay Blockade of C5aR has been shown to inhibit lymphatic cell chemotaxis. U937 cells stably transfected with C5a receptor (U937-C5aR cells) were plated at 3 × 10 cells per well in the upper chamber of a 96-well Transwell insert in RPMI medium containing 0.5% BSA. 5 Cells were seeded at 100 μM. Transwell inserts contained 3.0-μm pore-sized polycarbonate membrane filters (Corning). The lower Boyden chamber contained 10 nM of recombinant human complement C5a pretreated with antibodies in RPMI medium containing 0.5% BSA. After 3 h of incubation at 37°C, cells that had migrated to the lower chamber were lysed by adding 50 μl of CellTiter-Glo (Promega), and luminescence intensity (LI) was read by a microplate reader (BioTek). Percentage of inhibition was calculated as follows: % inhibition = [1-(LI-LI [低対照] ) / (LI [高対照] -LI [低対照] )] × 100. I.C. 50 Values ​​were calculated by GraphPad Prism software. High control, C5a added only to the lower chamber; low control, assay buffer added only to the lower chamber.

[0320] Table 8 and Figure 6 show that in two independent experiments, all four anti-C5a constructs (16D10, E54H, N97H, and N92H) effectively inhibit migration of U937 cells at low nanomolar concentrations. [Table 8]

[0321] 3. C5aR Antagonist FLIPR Assay The FLIPR assay was used to examine the inhibitory activity of humanized anti-C5a constructs against C5a-induced calcium influx. Anti-C5a constructs were titrated in HEK293 cells prepared according to the instructions in the FLIPR assay kit. Briefly, cells were incubated at 1 × 10 6 The cells were suspended at a density of 100 cells / ml. 20 μl of the cell suspension was seeded into a 384-well plate and cultured overnight. 250 nl of the anti-C5a construct solution was transferred to the cell plate using an Echo and incubated for 60 minutes. The cells were loaded with Fluo-4 Direct TM dye and incubated at 37°C, 5% CO2 for 50 minutes and then at room temperature for 10 minutes. The cell plate was placed in a FLIPRTETRA (Molecular Devices). 10 μl of agonist human C5a was transferred to the cell plate. Fluorescence was measured at each antibody concentration point shown in Figure 7. IC50 was calculated by GraphPad Prism software. The results are shown in Figure 7 and Table 9. A sample with 250 nl of assay buffer instead of anti-C5a construct was used as a high control. [Table 9]

[0322] All four humanized anti-C5a constructs inhibited calcium influx into HEK293 cells at low nanomolar concentrations.

[0323] Example 3: C5 binding and the "sink effect" The affinity of anti-C5a constructs for human C5 was examined using ELISA. Off-target C5 binding reduces the availability of anti-C5a antibodies (C5-mediated "sink effect"). Four humanized anti-C5a constructs (16D10, E54, N92, and N97) were serially diluted and added to C5 pre-coated plates, and OD450 was measured at 450 nm by a microplate reader. Each data point was the average of two replicates. The results are shown in Figure 8 and Table 10.

[0324] All anti-C5a constructs (including the benchmark) also bound human C5 at neutral pH, although they showed minimal effects on sheep RBC lysis (Example 1). Mutants E54H, N97H, and N92H showed selective binding for C5a over C5 compared to 16D10 (increased C5a / C5 binding ratio compared to 16D10). [Table 10]

[0325] Example 4: pH-dependent C5 binding and reduced "sink effect" The pH dependence of the humanized anti-C5a constructs was investigated using a C5 binding assay. The pH-dependent dissociation of histidine mutants and benchmark antibodies was determined using a Biolayer Interferometer Gator™ (Probe life Inc., USA). Briefly, anti-human IgG Fc (FHC) probes were equilibrated in a kinetic (K) buffer of phosphate buffered saline (pH 7.4) containing 0.02% bovine serum albumin and 0.002% Tween® 20. Antibodies were captured on the surface of the sensors by immersing them in 200 μL of transfection supernatant at pH 7.4 for 600 seconds. The biosensors were then incubated for 600 seconds with human C5 (40 nM) prepared in K buffer (pH 7.4), followed by a dissociation period of 600 seconds in K buffer at pH 7.4 or pH 5.8. Data were processed and analyzed by Gator evaluation software.

[0326] The binding curves for 16D10, E54H, N97H, and N92H are shown in Figure 9. Percent dissociation values ​​at the end of 600 seconds are shown in Figure 10 and summarized in Table 11 below.

[0327] As shown in Figure 9, Figure 10, and Table 11, the histidine mutated constructs E54H, N97H, and N92H showed significant pH-dependent binding to human C5, with dissociation values ​​at pH 5.8 ranging from 49% to 61%. For E54H, N97H, and N92H, the ratios of dissociation rates at pH 5.8 and pH 7.4 were 11.3, 10.9, and 6.54, respectively. 16D10 and Benchmark did not show pH-dependent binding to human C5. These results demonstrated that the histidine mutated constructs could potentially avoid the "sink effect" and have greater in vivo persistence. This is because immune complexes taken up by the cell (i.e., the humanized anti-C5a antibody binds to C5, thereby undergoing a "sink effect") dissociate in the acidic environment of the endosome, allowing the released antibody or antibody fusion protein to be recycled out of the cell via the neonatal Fc receptor (FcRn), where it is available to bind to new C5a molecules. [Table 11]

[0328] Example 5: In vivo pharmacokinetic evaluation of anti-C5a constructs The in vivo pharmacokinetics of anti-C5a constructs were examined in C5-humanized mice on a SCID / human FcRn transgenic background (denoted HuC5 / Scid / FcRn, human FcRn transgenic, and mouse FcRn knockout). N97H, N92H, and 16D10 were examined.

[0329] Human IgG4 was detected in mice treated with anti-C5a antibody using a sandwich ELISA. 96-well plates were coated with anti-human kappa light chain antibody (antibody solution, AS75-P) at a final concentration of 2 μg / mL in bicarbonate buffer for 1 h at 37° C. After washing three times with PBS containing 0.05% Tween®-20, the plates were incubated with plasma samples diluted in blocking solution for 1 h at room temperature. After washing, the plates were incubated with anti-human IgG4 HRP (1:2000 dilution, Invitrogen, A10654) in blocking solution for 1 h at room temperature. After washing, the plates were developed with HRP substrate for 3 min. The reaction was stopped with 2N H2SO4 and the plates were read at 450 nm in a microplate reader. The pharmacokinetic profiles are shown in Figure 11 (Plasma concentration versus time profile of three anti-C5a humanized antibodies N92, N97, and 16D10 after a single intravenous injection at a dose of 25 mg / kg.

[0330] Figure 11 shows that both N97H and N92H had extended in vivo persistence up to 15 days after a single injection. 16D10 levels in human C5 / SCID / human FcRn mice declined by day 3 after a single injection.

[0331] Example 6: Effect of anti-C5a antibodies on C5 levels in vivo In FcRn / SCID mice expressing human C5, human C5 was detected using SDS-PAGE and sandwich ELISA after hydrodynamic injection of human C5 cDNA plasmid.

[0332] Figure 12 shows the levels of human C5 protein in serum at various time points after injection with anti-C5a antibodies N97H, N92H, and 16D10 (denoted as N97, N92, and WT in Figures 12 and 13, respectively). Both histidine mutant constructs reduced the levels of C5. 16D10 initially reduced the levels of C5 on days 1 and 3 after injection, but C5 levels recovered from day 5 onwards.

[0333] For sandwich ELISA, 96-well plates were coated with anti-human C5 antibody (Quidel, A217) at a final concentration of 2 μg / mL in bicarbonate buffer for 1 h at 37° C. After washing the plates with PBS containing 0.05% Tween®-20, they were incubated with the diluted plasma samples in blocking solution for 1 h at room temperature. The plates were then washed and incubated with biotinylated anti-human C5 mAb 9G6 in blocking solution for 1 h at room temperature, washed again, and incubated with avidin- or streptavidin-labeled horseradish peroxidase (BD pharmigen) in blocking solution for 1 h at room temperature. After the final wash, the plates were developed with HRP substrate for 3 min. The reaction was stopped with 2N H2SO4, and the plates were read at 450 nm in a microplate reader. The results are shown in FIG. 13.

[0334] Figure 13 shows that the levels of C5 after administration of N97H and N92H decreased to almost 25% of the pretreatment levels, and the levels of C5 after administration of 16D10 recovered from day 3 onwards, confirming the results from SDS-PAGE.

[0335] Example 7: Levels of C5 Required for Sufficient Complement Activation Sheep RBC lysis was performed to determine the level of C5 required for sufficient activation of the classical pathway. Samples incubated with normal human serum alone were used as a positive control for sufficient complement activity. Samples without added NHS or with added EDTA were used as negative lysis controls. As shown in Figure 14, approximately 25% of C5 was sufficient to induce sufficient complement activation through the classical pathway.

[0336] To assess the levels of C5 required for alternative pathway complement-associated hemolysis, a rabbit red blood cell lysis assay was performed. Rabbit RBCs (Rockland Immunochemicals Inc Cat. No. R403-0100) (1 × 10 per assay sample prepared in PBS) were used. 7 100 cells (Complement Technology Inc.) were incubated with gelatin veronal buffer (GVB2+EGTA, Sigma; total assay volume: 100 μL) containing 25% normal human serum (NHS, from Complement Technology Inc.) at 37° C. for 30 min. Samples incubated with NHS alone were used as positive controls for sufficient complement activity. Samples without NHS or with added EDTA were used as negative lysis controls. The lysis reaction was stopped by adding 40 mM EDTA in ice-cold PBS. The incubation mixture was centrifuged at 1500 rpm for 5 min. The supernatant was collected and OD405 nm was measured. FIG. 15 shows that approximately 25% of C5 is sufficient to induce sufficient complement activity through the alternative pathway. These results suggest that although anti-C5a antibodies reduced C5 levels in serum (Example 7), the remaining C5 was sufficient to induce sufficient complement activity.

[0337] Example 8: In vivo pharmacokinetic evaluation of N92H antibody in cynomolgus monkeys In vivo pharmacokinetic evaluation of N92H antibody was performed in cynomolgus monkeys by single intravenous administration of 5, 30, and 100 mg / kg N92H antibody. Blood samples were collected at the following time points: pre-dose (up to 2 days before dosing began), EOI (0-2 minutes before the end of infusion), and 1, 6, 24, 72, 168, 336, 504, and 672 hours after the end of infusion (EOI) on day 1. In addition, blood samples were collected from 5 mg / kg at 1728 (week 11), 2184 (week 14), and 2688 (week 17) hours after EOI on day 1.

[0338] The concentration of N92H antibodies was determined using an established ELISA method with a lower limit of quantification (LLOQ) of 37.3 ng / mL. This quantitative ELISA is based on a sandwich format and all steps were performed at room temperature, except for coating, which was applied at 4°C. Microwell plates were coated with a capture mouse anti-human IgG constant heavy chain 2 (CH2) antibody (Bio-Rad, MCA5748G) diluted in coating buffer. In the assay, standards, quality controls, and samples were dispensed into pre-coated microwells diluted at the minimum required dilution (MRD) in dilution buffer and the microwells were blocked. After incubation, the plates were washed to remove unbound material before proceeding to the well blocking step. Samples were then loaded into the wells and incubated at room temperature with agitation. After incubation, the wells were washed before the addition of a detection mouse anti-human IgG4 fragment crystallizable region (Fc)-horseradish peroxidase (HRP) conjugated antibody (Southern Biotech, 9200-05). The plates were washed and 3,3',5,5'-tetramethylbenzidine substrate solution (TMB) was added. After incubation, the reaction was stopped by adding sulfuric acid stop solution. Absorbance was read at 450 nm with a reference wavelength of 650 nm. Color development is proportional to the amount of N92H antibody present in the well.

[0339] Figure 16 shows that after a single intravenous infusion of N92H antibody over 30 minutes at 5, 30, and 100 mg / kg, plasma concentrations were quantifiable in all treatment groups throughout the 672-hour sampling period, including samples collected at weeks 11, 14, and 17 for 5 mg / kg. The time to maximum plasma concentration (tmax) of N92H antibody was observed at 0.5 hours (0–2 minutes before EOI) or 1.5 hours after SOI for 5 and 30 mg / kg, and at 0.5 hours after SOI for 100 mg / kg. With extended sampling, the terminal phase was well characterized at 5 mg / kg, with terminal half-life (T1 / 2) estimates of 576 and 595 hours, clearance (Cl) of 0.0922 and 0.109 mL / hr / kg, and volume of distribution (Vss) of 80.9 and 80.0 mL / kg for males and females, respectively.

[0340] Example 9: In vivo pharmacodynamic evaluation of N92H antibody in cynomolgus monkeys In vivo pharmacodynamic evaluation of the N92H antibody was performed in cynomolgus monkeys by administering a single intravenous dose of 5, 30, and 100 mg / kg N92H antibody. Blood samples were collected at the following time points: pre-dose (up to 2 days before dosing began), EOI (0-2 minutes before the end of infusion), and 1, 6, 24, 72, 168, 336, 504, and 672 hours after the end of infusion (EOI) on day 1. In addition, blood samples were collected from 5 mg / kg at 1728 (week 11), 2184 (week 14), and 2688 (week 17) hours after EOI on day 1.

[0341] A procedure was utilized to semi-quantify CD11b expression and reveal the pharmacodynamics of N92H antibody by flow cytometry after in vitro C5a stimulation in cynomolgus monkey whole blood. Blood samples were collected in K2-EDTA tubes and then transferred to FACS tubes within 1 h after the first collection time for intermediate processing. Recombinant cyno C5a (Sino Biological) was then added to the samples for stimulation and incubated with CD11b or isotype control antibodies (BD Biosciences, 557321 and 556650). Supernatants of red blood cells were obtained by mixing with lysis buffer (BD Biosciences, 555899). After fixing the cells with PFA (Alfa Aesar, J61899) / DPBS (Life Technologies, 14190136), FACS was performed on the samples to detect CD11b signals.

[0342] Figure 17 shows that C5a protein strongly increased the expression level of CD11b before administration, and this effect was inhibited throughout the study period in the study samples in the administered animals. In animals administered a low dose of N92H antibody (5 mg / kg), stimulation with C5a protein induced a strong increase in CD11b expression at both 10 and 30 nM before administration. Stimulation with C5a protein was inhibited and maintained up to 17 weeks. Inhibition of CD11b expression was relatively variable throughout all occasions, but remained below 20%.

[0343] Example 10: In vivo pharmacodynamic evaluation of N92H antibody in a model of C5a-induced neutropenia in cynomolgus monkeys C5a is one of the most potent proinflammatory mediators, inducing the expression of adhesion molecules and chemotactic migration of neutrophils. When C5a is produced locally in the bloodstream, nearby neutrophils bearing C5aR immediately upregulate adhesion molecules and adhere to the inner surface of blood vessels. When C5a is introduced systemically by intravenous injection, neutrophil adhesion occurs immediately throughout the vasculature, resulting in a transient and substantial reduction in the number of neutrophils still circulating in the bloodstream. This phenomenon is termed neutropenia. We evaluated the in vivo efficacy of the N92H antibody in a neutropenic model.

[0344] FIG. 18 illustrates the neutropenia model in monkeys. Briefly, human C5a (10 μg / kg) was administered intravenously 6 hours after administration of N92H antibody, and then on days 2, 7, and 14. Blood samples were collected immediately 1 minute (min) before and 1 min after each C5a injection, and neutrophil counts were analyzed within 2 hours by an automated hematology analyzer (Sysmex XT-2000iV). N92H antibody efficacy was calculated and expressed as the percentage change in neutrophils in blood samples 1 min before and 1 min after each C5a injection. The mean C5a-induced neutrophil reduction is approximately 90% for each of the C5a stimulations (data not shown). The data in FIG. 19 show that monkeys pretreated with an isotype control antibody had no effect on C5a-induced neutropenia. Conversely, monkeys pretreated with 5 mg / kg N92H antibody showed >90% rescue of C5-induced neutropenia, a rescue effect that could persist for at least 14 days. [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4]

Table 12-5

Table 12-6

Table 12-7

Table 12-8

Table 12-9

Claims

1. 1. An isolated humanized antibody that specifically binds to human C5a and C5, comprising a heavy chain variable domain (VH) comprising mutations I48M, D54E, and N56W, and a light chain variable domain (VL) comprising mutations D28E and D30F, wherein said VH mutations refer to SEQ ID NO: 1 under the Kabat numbering system, and said VL mutations refer to SEQ ID NO: 2 under the Kabat numbering system; and i) a heavy chain CDR1 (“H-CDR1”) comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising one, two, or three amino acid substitutions; ii) a heavy chain CDR2 (“H-CDR2”) comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising one, two, or three amino acid substitutions; iii) a heavy chain CDR3 (“H-CDR3”) comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising one, two, or three amino acid substitutions; iv) a light chain CDR1 (“L-CDR1”) comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising one, two, or three amino acid substitutions; v) a light chain CDR2 (“L-CDR2”) comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising one, two, or three amino acid substitutions; and vi) An antibody comprising a light chain CDR3 (“L-CDR3”) comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising one, two, or three amino acid substitutions.

2. 2. The antibody of claim 1, further comprising a mutation F29H in the VH and a mutation Y96H in the VL, wherein the VH mutation refers to SEQ ID NO: 1 under the Kabat numbering system and the VL mutation refers to SEQ ID NO: 2 under the Kabat numbering system.

3. The antibody of claim 2 further comprising a substitution in the VH or VL.

4. The antibody of claim 3, wherein the mutations are selected from the group consisting of E54H in VH, N97H in VH, and N92H in VL (wherein the VH mutations refer to SEQ ID NO: 1 under the Kabat numbering system and the VL mutations refer to SEQ ID NO: 2 under the Kabat numbering system).

5. i) a VH comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof that is at least about 85% identical to SEQ ID NO: 9; ii) The antibody of claim 1, comprising a VL comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof that is at least about 85% identical to SEQ ID NO:

10.

6. i) H-CDR1 comprising the amino acid sequence of SEQ ID NO: 11; ii) H-CDR2 comprising the amino acid sequence of SEQ ID NO: 12; iii) H-CDR3 comprising the amino acid sequence of SEQ ID NO: 13; iv) L-CDR1 comprising the amino acid sequence of SEQ ID NO: 14; v) L-CDR2 comprising the amino acid sequence of SEQ ID NO: 15; and vi) The antibody of claim 1, comprising an L-CDR3 comprising the amino acid sequence of SEQ ID NO:

16.

7. i) a VH comprising the amino acid sequence SEQ ID NO: 17; ii) The antibody of claim 6, comprising a VL comprising the amino acid sequence of SEQ ID NO:

18.

8. i) H-CDR1 comprising the amino acid sequence of SEQ ID NO: 19; ii) H-CDR2 comprising the amino acid sequence of SEQ ID NO: 20; iii) H-CDR3 comprising the amino acid sequence of SEQ ID NO: 21; iv) L-CDR1 comprising the amino acid sequence of SEQ ID NO: 22; v) L-CDR2 comprising the amino acid sequence of SEQ ID NO: 23; and vi) The antibody of claim 1, comprising an L-CDR3 comprising the amino acid sequence of SEQ ID NO:

24.

9. i) a VH comprising the amino acid sequence SEQ ID NO:25; ii) The antibody of claim 8, comprising a VL comprising the amino acid sequence of SEQ ID NO:

26.

10. i) H-CDR1 comprising the amino acid sequence of SEQ ID NO: 27; ii) H-CDR2 comprising the amino acid sequence of SEQ ID NO: 28; iii) H-CDR3 comprising the amino acid sequence of SEQ ID NO: 29; iv) L-CDR1 comprising the amino acid sequence of SEQ ID NO: 30; v) L-CDR2 comprising the amino acid sequence of SEQ ID NO: 31; and vi) The antibody of claim 1, comprising an L-CDR3 comprising the amino acid sequence of SEQ ID NO:

32.

11. i) a VH comprising the amino acid sequence SEQ ID NO: 33; ii) The antibody of claim 10, comprising a VL comprising the amino acid sequence of SEQ ID NO:

34.

12. 2. The antibody of claim 1, which is selected from the group consisting of a full-length antibody, a Fab, a Fab', a F(ab)2, a F(ab')2, and a scFv.

13. The antibody of claim 1 further comprising an Fc region.

14. The antibody of claim 13, wherein the Fc region comprises an IgG4 sequence.

15. The antibody of claim 14, wherein the Fc region comprises the amino acid sequence of SEQ ID NO: 43 or a variant thereof.

16. 16. The antibody of claim 15, wherein the Fc region comprises one or more mutations selected from the group consisting of S228P, M428L, and N434A (wherein said mutations are compared to SEQ ID NO: 43 under the EU numbering system).

17. 17. The antibody of claim 16, wherein the Fc region comprises the mutations S228P, M428L, and N434A.

18. The antibody of claim 17, wherein the Fc region comprises the amino acid sequence of SEQ ID NO:

44.

19. The antibody of claim 1, wherein the low pH dissociation factor of the antibody that dissociates from C5 is about 40% to about 70%.

20. The antibody of claim 1, wherein the neutral pH dissociation factor of the antibody that dissociates from C5 is about 0% to about 10%.

21. The antibody of claim 1, having a ratio of low pH dissociation to neutral pH dissociation of 6 or more.

22. The antibody of claim 1, which inhibits the binding between human C5a and C5aR.

23. The antibody of claim 1, having a serum half-life in humans of at least about 25 days.

24. The antibody of claim 1 , which is produced in CHO cells.

25. A nucleic acid encoding the antibody of any one of claims 1 to 24, comprising the sequence of any one of SEQ ID NOs: 46 to 53.

26. A vector comprising the nucleic acid of claim 25.

27. A host cell comprising the vector of claim 26.

28. A method for producing an antibody according to any one of claims 1 to 24 under conditions sufficient to allow expression of said antibody by a cell.

29. A pharmaceutical composition comprising the antibody of any one of claims 1 to 24 and a pharmaceutically acceptable carrier.

30. 30. A method of treating an individual having a complement-related disease or condition, comprising administering to said individual an effective amount of the pharmaceutical composition of claim 29.

31. The disease or disorder may be selected from macular degeneration (MD), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, rheumatoid arthritis, lupus, ulcerative colitis, stroke, postoperative systemic inflammatory syndrome, asthma, allergic asthma, chronic obstructive pulmonary disease (COPD), paroxysmal nocturnal hemoglobinuria (PNH) syndrome, autoimmune hemolytic anemia (AIHA), Gaucher disease, myasthenia gravis, neuromyelitis optica (NMO), multiple sclerosis, delayed graft function, antibody-mediated rejection, atypical hemolytic uremic syndrome (aHUS), central retinal vein occlusion (CRVO), central retinal artery occlusion (CRAO), epidermolysis bullosa, sepsis, septic shock, organ transplant, inflammation (non-limiting examples of which include cardiopulmonary bypass surgery and 31. The method of claim 30, wherein the inflammatory bowel disease is at least one of the group consisting of: inflammation associated with kidney dialysis, C3 nephropathy, membranous nephropathy, IgA nephropathy, glomerulonephritis (non-limiting examples of which include antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, lupus nephritis, and combinations thereof), ANCA-associated vasculitis, Shiga toxin-induced HUS, and antiphospholipid antibody-induced pregnancy loss, graft-versus-host disease (GVHD), bullous pemphigoid, hidradenitis suppurativa, dermatitis herpetiformis, Sweet's syndrome, pyoderma gangrenosum, palmoplantar pustulosis and pustular psoriasis, rheumatic neutrophilic dermatosis, subcorneal pustulosis, gut-associated dermatosis-arthritis syndrome, neutrophilic eccrine hidradenitis, linear IgA disease, or any combination thereof.

32. 30. A method of reducing the activity of the complement system in an individual, comprising administering to said individual an effective amount of the pharmaceutical composition of claim 29.

33. The antibody of any one of claims 1 to 24, which cross-reacts with C5a or C5 of cynomolgus monkeys.

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