RU2773779C2 - ANTI-C5a ANTIBODIES AND THEIR APPLICATIONS - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular to an antibody that specifically binds to human C5a, as well as to a method for reducing the activity of the complement system of an individual using it. Also disclosed is a cell for producing an antibody, which contains a nucleic acid encoding the above antibody.

EFFECT: invention is effective for treating a complement-mediated disease or disorder in an individual.

19 cl, 7 dwg, 1 ex

Description

Cross-reference to related applications

This application claims priority in US Provisional Application No. 62/475,573, filed March 23, 2017, the contents of which are incorporated herein by reference in their entirety.

Statement of government financial support for research or development

The present invention was made with government support under NIH grant AI44970 from the National Institutes of Health (NIH). The government has certain rights to the invention.

State of the art

The complement system is part of innate immunity, which plays a key role in host defense. However, activated complement also has the potential to cause significant tissue damage and destruction, and dysregulation of complement activity has been found to be associated with a number of rare and common diseases such as paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome, rheumatoid arthritis, age-related macular degeneration, etc. Thus, anti-complement therapy is a promising way to treat these human disorders.

The C5 component of the complement system is a critical protein in the terminal complement pathway and is a precursor protein in the generation of the potent pro-inflammatory mediator C5a as well as the cytolytic membrane attack complex (MAC), C5b-9.

In some complement-mediated diseases, processes mediated by both C5a and MAC may contribute to pathogenesis, while in other diseases only C5a-mediated inflammation or MAC-mediated cell damage may be involved. Because complement mediators, including C5a and MAC, also play an important role in host defense against pathogenic infection, it is desirable that therapeutic agents develop selective anti-complement drugs, i.e., drugs that block only the negative effects of complement in damage. tissues, leaving its normal protective function for the host intact.

The cause of PNH hemolytic disease is the MAC. There are other anti-C5 mAbs available for the treatment of PNH. However, these antibodies excessively block C5a production, putting patients at greater risk of infection than a therapeutic agent that blocks only MAC. Similarly, there are complement-mediated diseases that can be mainly mediated by C5a-dependent inflammation (eg, sepsis), and for such pathological conditions, an anti-C5 mAb drug, although expected to be effective, would unnecessarily block MAC. as a side effect.

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

The essence of the invention

In one embodiment, the present invention relates to an antibody that specifically binds to C5a. In one embodiment, C5a is human C5a. In one embodiment, the antibody is a monoclonal antibody. In one embodiment, the antibody is a humanized antibody. In some embodiments, the antibody is a full length antibody. In some embodiments, the antibody is an antibody fragment that includes, but is not limited to, Fab, Fab', F(ab) ₂ , F(ab') ₂ , and scFv. In some embodiments, the antibody is part of a construct, such as a fusion construct, containing the antibody and a targeting molecule or effector molecule. In some embodiments, the antibody is part of a conjugate, such as an antibody-drug conjugate.

In one embodiment, the antibody is a chimeric antibody. In one embodiment, the antibody contains at least one of the CDRs selected from the group consisting of: VH-CDR1: SEQ ID NO: 3; VH-CDR2: SEQ ID NO: 4; VH-CDR3: SEQ ID NO: 5; VL-CDR1: SEQ ID NO: 8; VL-CDR2: SEQ ID NO: 9; and VL-CDR3: SEQ ID NO: 10, or a variant or variants thereof. In one embodiment, the antibody contains CDR: VH-CDR1: SEQ ID NO: 3; VH-CDR2: SEQ ID NO: 4; VH-CDR3: SEQ ID NO: 5; VL-CDR1: SEQ ID NO: 8; VL-CDR2: SEQ ID NO: 9; and VL-CDR3: SEQ ID NO: 10, or a variant or variants thereof. In one embodiment, the antibody contains a heavy chain containing the amino acid sequence of SEQ ID NO: 2 or a variant thereof. In one embodiment, the antibody contains a light chain containing the amino acid sequence of SEQ ID NO: 7 or a variant thereof. In one embodiment, the antibody contains a heavy chain containing the amino acid sequence of SEQ ID NO: 2 or a variant thereof, and a light chain containing the amino acid sequence of SEQ ID NO: 7 or a variant thereof. In one embodiment, the antibody is 7A12.

In one embodiment, the present invention relates to a method of treating a complement-mediated disease or disorder in an individual, comprising the step of administering to said individual an anti-C5a antibody described herein. In one embodiment, the disease or disorder is at least selected from the group consisting of: macular degeneration (MD), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, rheumatoid arthritis, lupus, ulcerative colitis, stroke, postoperative systemic inflammatory response syndrome, asthma, allergic asthma, chronic obstructive pulmonary disease (COPD), paroxysmal nocturnal hemoglobinuria (PNH), autoimmune hemolytic anemia (AIHA), Gaucher disease, myasthenia gravis, optic neuromyelitis, NMO, multiple sclerosis, delayed function transplant, 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 (including but not limited to: inflammation associated with cardiopulmonary shunts renal dialysis), C3 glomerulopathy, membranous nephropathy, IgA nephropathy, glomerulonephritis (including, but not limited to, antineutrophil cytoplasmic antibodies (ANCA)-associated glomerulonephritis, lupus nephritis, and combinations thereof), ANCA-associated vasculitis, shiga-induced HUS toxin, and antiphospholipid antibody-induced pregnancy loss, graft-versus-host disease (GVHD), or any combination thereof. In some embodiments, the AP-mediated disease is sepsis, septic shock, rheumatoid arthritis, autoimmune hemolytic anemia, GvHD, antiphospholipid syndrome, or Gaucher's disease. In one embodiment, administration of an anti-C5a antibody inhibits the activity of the C5a protein.

In one embodiment, the present invention relates to a method for reducing the activity of the complement system of an individual, where the method includes administering the antibody to the individual by administration selected from the group consisting of enteral administration, parenteral administration, and a combination thereof, and where the antibody contains six complementarity determining regions having the following amino acid sequences: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or a variant or variants thereof. In one embodiment, the antibody is an antibody fragment selected from the group consisting of Fab, Fab', F(ab) ₂ , F(ab') ₂ , scFv, and combinations thereof.

In one embodiment, the present invention describes an anti-human C5a antibody, wherein the antibody has a heavy chain variable region (VH) that has an amino acid sequence that is greater than about 90% (e.g., greater than 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 2 or a variant thereof. In one embodiment, the antibody is an antibody fragment selected from the group consisting of Fab, Fab', F(ab) ₂ , F(ab') ₂ , scFv, and combinations thereof.

In one embodiment, the present invention describes an anti-human C5a antibody, wherein the antibody has a light chain variable region (VL) that has an amino acid sequence that is greater than about 90% (e.g., greater than 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 7 or a variant thereof. In one embodiment, the antibody is an antibody fragment selected from the group consisting of Fab, Fab', F(ab) ₂ , F(ab') ₂ , scFv, and combinations thereof.

In one embodiment, the present invention is an anti-human C5a antibody, wherein the antibody has a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH region has an amino acid sequence that is greater than about 90% (e.g., greater than than 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 2 or a variant thereof, and wherein the VL region has the amino acid sequence, which is more than about 90% (e.g., more than 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 7, or her version. In one embodiment, the antibody is an antibody fragment selected from the group consisting of Fab, Fab', F(ab) ₂ , F(ab') ₂ , scFv, and combinations thereof.

In one embodiment, the present invention provides a cell containing at least one of the antibodies described elsewhere in this document. In one embodiment, the cell produces at least one of the antibodies described elsewhere in this document. In one embodiment, the cell is a hybridoma.

In one embodiment, the present invention provides a cell line containing at least one of the antibodies described herein. In one embodiment, the cell line produces at least one of the antibodies described herein. In one embodiment, the cell line is a hybridoma cell line.

Brief description of the figures

The foregoing summary of the invention, as well as the following detailed description of exemplary embodiments of the invention, will be better understood when read in conjunction with the accompanying figures. However, it should be understood that the invention is not limited to the preparation and instrumentation of the embodiments shown in the figures. On the figures:

In FIG. 1 including FIG. 1A and FIG. 1B shows results showing that mAb 7A12 binds to human C5. In FIG. 1A shows the reactivity of 7A12 and 2G1, a control anti-C5 mAb, with intact human C5, assessed using ELISA. The plate was coated with purified human C5. After incubation with serial dilutions of 7A12 or control anti-C5 mAb, bound mAbs were detected with HRP-conjugated rabbit anti-mouse IgG. 7A12 and control anti-C5a mAb showed high reactivity with human C5. In FIG. 1B shows using Western blotting that mAb 7A12 and control anti-C5a mAb recognized purified human C5 protein under non-reducing and reducing conditions, respectively. The observed 190 kDa 7A12 reactive band represents the complete C5 protein, while the 115 kDa band represents the C5 chain. NR: non-reducing conditions; R: reducing conditions.

In FIG. 2 shows the results showing that, unlike the control anti-C5 mAb 2G1, which inhibits erythrocyte lysis (RBC), 7A12 shows no activity in the hemolysis assay. Antibody-sensitized sheep erythrocytes were incubated with normal human serum (NHS) containing serial dilutions of 7A12 or control anti-C5a mAb at 37° C. for 1 hour. RBC lysis was determined by measuring absorbance at OD405 nm. As expected, the control anti-C5 mAb 2G1 inhibited 50% NHS-mediated lysis of sheep erythrocytes in a dose dependent manner. On the other hand, mAb 7A12 at doses of 0.975 to 120 μg/ml did not show 50% inhibition of NHS-mediated lysis of sheep erythrocytes.

In FIG. 3 including FIG. 3A and FIG. 3B, results are shown showing that, unlike the control anti-C5 mAb 2G1, which does not bind C5a, 7A12 binds human C5a in a dose dependent manner, but it does not bind murine C5a. The plate was coated with human C5 or mouse C5. After incubation with serial dilutions of 7A12 or control anti-C5 mAb, bound mAb was detected with HRP-conjugated rabbit anti-mouse IgG. In FIG. 3A shows that mAb 7A12 showed high reactivity to human C5a. Since mAb 7A12 reacts with complete C5 protein (as shown in Figure 1), it can be concluded that mAb 7A12 binds both the C5a fragment of native human C5 and free human C5a. On the other hand, binding of the control anti-C5a mAb to human C5a was not observed. In FIG. 3B shows that mAb 7A12 binding to C5a was specific to human C5a with little binding to mouse C5a.

In FIG. 4 including FIG. 4A and FIG. 4B shows the results of experiments evaluating the binding affinity of mAb 7A12 to human C5 and C5a. Purified human C5 or C5a was coupled on the CM4 chip using the amine coupling method. Biacore analysis was performed on a Biacore-2000 instrument. Between each binding, the chip was regenerated using 50 mM NaOH. mAb 7A12 binds to human C5 as shown in FIG. 4A and human C5a as shown in FIG. 4B, with the same affinity.

Figure 5 shows the results showing that mAb 7A12, but not the control anti-C5 mAb 2G1, inhibits C5a-mediated neutrophil migration. Human C5a at 10 nM was used to induce chemotaxis of a human monocytic cell line, U937, transfected with the human C5a receptor. Cells were placed in the upper chamber of the Transwell system in the presence of mAb 7A12 or control anti-C5 mAb 2G1, and cell migration was quantified by cell count in the lower chamber. mAb 7A12 showed complete inhibition of C5a-induced chemotaxis at a concentration of 10 μg/ml, while the control anti-C5 mAb 2G1 could not block C5a-induced chemotaxis.

In FIG. 6 including FIG. 6A-6D, results are shown showing that mAb 7A12, but not the control anti-C5 mAb 2G1, inhibits C5a-induced intracellular calcium mobilization in U937 cells. Calcium mobilization was absent in U937 cells expressing the human C5a receptor (U937-C5aR) in the absence of human C5a stimulation, as shown in FIG. 6A. Treatment with C5a (10 nM) resulted in a transient calcium influx in U937-C5aR cells as shown in FIG. 6B which could be inhibited by pre-incubation with mAb 7A12 (50 μg/ml) as shown in FIG. 6C but not with the control anti-C5 mAb 2G1 (50 μg/ml) as shown in FIG. 6d. Arrows indicate the time point at which C5a or a mixture of C5a and antibodies was added to the cell suspension.

In FIG. 7 including FIG. 7A and FIG. 7B shows the sequences of the heavy and light chain variable regions of mAb 7A12. In FIG. 7A shows the nucleic acid sequences and amino acid sequences of VH mAb 7A12. The signal peptide is underlined and the CDR1, CDR2 and CDR3 sequences are in bold and grayed out. In FIG. 7B shows the nucleic acid and amino acid sequences of VL mAb 7A12. The signal peptide is underlined and the CDR1, CDR2 and CDR3 sequences are in bold and grayed out.

Detailed description of the invention

The present invention relates to the inhibition of the complement signaling pathway using an anti-C5a antibody. In various embodiments, the invention relates to compositions and methods for treating a complement-mediated disease or complement-mediated disorder in an individual by contacting the individual with an anti-C5a antibody. Complement-mediated diseases and disorders that can be treated with the compositions and methods of the invention include, but are not limited to, macular degeneration (MD), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, rheumatoid arthritis, lupus, ulcerative colitis, stroke , postoperative systemic inflammatory response syndrome, asthma, allergic asthma, chronic obstructive pulmonary disease (COPD), paroxysmal nocturnal hemoglobinuria (PNH), autoimmune hemolytic anemia (AIHA), Gaucher disease, myasthenia gravis, neuromyelitis optic nerve, 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 (including but not limited to: inflammation associated with cardiopulmonary bypass and renal dialysis), C3 glomerulopathy, membranous nephropathy, IgA nephropathy, glomerulonephritis (including, but not limited to, antineutrophil cytoplasmic antibodies (ANCA)-associated glomerulonephritis, lupus nephritis, and combinations thereof), ANCA-associated vasculitis, Shiga-induced -HUS toxin, and antiphospholipid antibody-induced pregnancy loss, graft-versus-host disease (GVHD), or any combination thereof.

Definitions

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as generally understood by a person skilled in the art to which this invention pertains. While any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, illustrative methods and materials are described.

As used in this document, each of the following terms has the meaning associated with it in this section.

The terms "inhibit" and "inhibition" as used herein means the reduction, suppression, reduction or blocking of an activity or function by at least about 10% relative to a control value. In some embodiments, activity is inhibited or blocked by at least about 50% of a control value. In some embodiments, the activity is inhibited or blocked by at least about 75%. In some embodiments, the activity is inhibited or blocked by at least about 95%.

The terms "effective amount" and "pharmaceutically effective amount" refer to an amount of an agent sufficient to provide the desired biological result. Such a result may be a reduction and/or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired change in the biological system. A suitable effective amount in any particular case can be determined by one of ordinary skill in the art using routine experimentation.

The terms "patient", "individual", "individual" and the like are used interchangeably herein and refer to any animal, in some embodiments, a mammal, and in some embodiments, a human having a complement system, including a human in need of treatment or susceptible to the development of the condition or its complications. An individual may include, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats, monkeys, mice, and humans.

The term "abnormal", when used in the context of organisms, tissues, cells, or components thereof, refers to those organisms, tissues, cells, or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells, or components thereof that exhibit a "normal" (expected/homeostatic) corresponding characteristic. Characteristics that are normal or expected for one cell, tissue type, or individual may be abnormal for another cell or tissue type.

"Disease" is a state of health of an individual in which the individual cannot maintain homeostasis, and in which, if the disease is not relieved, the health of the individual continues to deteriorate.

In contrast, a "disorder" in an individual represents a state of health in which the individual can maintain homeostasis, but the individual's health is less favorable than it would be in the absence of the disorder. Without treatment, the disorder does not necessarily cause further deterioration in the health of the individual.

A disease or disorder is "ameliorated" if the severity of a sign or symptom of the disease or disorder, the frequency with which that sign or symptom, or both, experienced by the patient is reduced.

An "effective amount" or "therapeutically effective amount" of a compound is that amount of a compound that is sufficient to provide a beneficial effect to the individual to whom the compound is administered.

As used herein, the term "instructive material" includes a publication, record, diagram, or any other medium of expression that can be used to communicate the suitability of a compound, composition, vector, or delivery system of the invention, in a kit, to alleviate various diseases or disorders, specified in this document. Optionally or alternatively, the guidance material may describe one or more methods of alleviating diseases or disorders in a mammalian cell or tissue. The instructional material of a kit of the invention may, for example, be attached to a container that contains said compound, composition, vector, or delivery system of the invention, or shipped with a container that contains said compound, composition, vector, or delivery system. Alternatively, the instructional material may be sent separately from the container, with the intent that the instructional material and the connection be shared by the recipient.

As used herein, the term "operably linked" or "operably linked" may mean that the expression of a gene is under the control of the promoter to which it is spatially linked. A promoter can be located 5' (above) or 3' (below) from a gene that is under its control. The distance between a promoter and a gene can be about the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, changing this distance can be accommodated without loss of promoter function.

"Therapeutic treatment" is a treatment administered to an individual who exhibits signs of a disease or disorder with the aim of reducing or eliminating those signs.

As used herein, the term "treating a disease or disorder" means reducing the frequency and/or severity of a sign and/or symptom of a disease or disorder from which a patient is suffering.

As used herein, the expression "biological sample", "sample" or "sample" is intended to include any sample containing a cell, tissue or body fluid in which expression of a nucleic acid or polypeptide can be detected. The biological sample may include any biological material suitable for detecting the desired biomarkers and may contain cellular and/or non-cellular material obtained from the individual. Examples of such biological samples include, but are not limited to, blood, lymph, bone marrow, biopsy, and swabs. Samples that are liquid in nature are referred to herein as "biological fluids". Biological samples can be obtained from a patient in a variety of ways, including, for example, by scraping or taking a swab from the site, or using a needle to obtain body fluids. Methods for collecting various body samples are well known in the art.

As used herein, the term "antibody" refers to an immunoglobulin molecule that is capable of specifically binding to a particular antigen epitope. The antibodies may be intact immunoglobulins derived from natural sources or from recombinant sources and may be immunoreactive fragments of intact immunoglobulins. The antibodies of the present invention may be in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), Fv, Fab, Fab', F(ab) ₂ and F(ab') ₂ , and also single chain antibodies (scFv), heavy chain antibodies such as camel antibodies, and humanized antibodies (Harlow et al., 1999, 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).

As used herein, the term "synthetic antibody" means an antibody that is produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage. The term should also be construed as meaning an antibody that has been produced by the synthesis of a DNA molecule encoding an antibody, and such DNA molecule expresses an antibody protein or an amino acid sequence defining an antibody, where the DNA sequence or amino acid sequence has been obtained using synthetic DNA or amino acid sequencing technology which is available and well known in the art.

As used herein, the term "heavy chain antibody" or "heavy chain antibodies" includes immunoglobulin molecules derived from camelid species, either by immunization with a peptide and subsequent isolation of sera, or by cloning and expression of nucleic acid sequences encoding such antibodies. The term "heavy chain antibody" or "heavy chain antibodies" also encompasses immunoglobulin molecules isolated from an individual with heavy chain disease, or obtained by cloning and expression of VH (immunoglobulin heavy chain variable region) genes from an individual.

A "chimeric antibody" refers to a type of engineered antibody that contains a native variable region (light chain and heavy chain) derived from a donor antibody in association with light and heavy chain constant regions derived from an acceptor antibody.

"Humanized antibody" refers to a type of engineered antibody having CDRs derived from a non-human donor immunoglobulin and the remaining immunoglobulin-derived moieties are derived from one (or more) human immunoglobulin(s). In addition, framework residues can be altered to preserve binding affinity (see, e.g., 1989, Queen et al., Proc. Natl. Acad Sci USA, 86: 10029-10032; 1991, Hodgson et al., Bio/Technology , 9: 421). A suitable human acceptor antibody may be an antibody that is selected from a conventional database, such as the KABAT database, the Los Alamos database, and the Swiss Protein database, for homology with the nucleotide and amino acid sequences of the donor antibody. A human antibody having homology to the framework regions of the donor antibody (based on amino acids) may be suitable to provide a heavy chain constant region and/or a heavy chain variable region framework for insertion of the donor CDRs. A suitable acceptor antibody capable of donating light chain constant or variable framework regions can be selected in a similar manner. It should be noted that the heavy and light chains of an acceptor antibody are not necessarily derived from the same acceptor antibody. The prior art describes several methods for making such humanized antibodies (see, for example, EP-A-0239400 and EP-A-054951).

The term "donor antibody" refers to an antibody (monoclonal and/or recombinant) that donates the amino acid sequences of its variable regions, CDRs or other functional fragments, or analogues thereof, to a first immunoglobulin partner to provide an altered immunoglobulin coding region, and the resulting expressed altered antibody with antigenic specificity and neutralizing activity characteristic of the donor antibody.

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

"CDRs" are defined as the complementarity-determining regions of the amino acid sequences of an antibody, which are the hypervariable regions of the heavy and light chains of an immunoglobulin. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987). In the variable region of an immunoglobulin, there are three heavy chain CDRs and three light chain CDRs (or CDR regions). The structure and folding of an antibody protein may mean that other residues are considered part of the antigen-binding region and should be understood by one of skill in the art as such. See, for example, Chothia et al., (1989) Conformations of immunoglobulin hypervariable regions; Nature 342, p 877-883. One skilled in the art will appreciate that there are many methods and techniques used to predict CDR sequences. Thus, the CDR sequences of a particular antibody may vary slightly depending on which methods and techniques are used to predict the CDR sequences. Exemplary methods and techniques include, but are not limited to, those described by Lyskov et al., 2013, PLoS One, 8(5):e63906; Kunik, et al., 2012, Nucleic Acids Res. 40:W521-524; Marcatili et al., 2008, Bioinformatics, 24:1953; Chothia et al., 1989, Nature 342:887; Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD; Johnson et al., 2000, Nucleic Acids Res 28:214; Martin et al., 1989, P.N.A.S 86:9268; MacCallum et al., 1996, J. Mol. Biol. 5:732; and Dunbar et al., 20106, Nucleic Acids Res. 44:W474-478.

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

As used herein, the term "specifically binds" in relation to an antibody means an antibody that recognizes and binds to a particular target molecule, but does not substantially recognize or bind to other molecules in the sample. In some cases, the terms "specific binding" or "binding specifically" are used to mean that recognition and binding depend on the presence of a particular structure (eg, antigenic determinant or epitope) on the target molecule. If, for example, an antibody specifically binds to an "A" epitope, then the presence of an unlabeled molecule containing the A epitope (or a free, unlabeled A) in a reaction containing the labeled "A" and the antibody will result in a decrease in the amount of labeled A bound to the antibody. .

The "coding region" of a gene consists of nucleotide residues of the coding strand of the gene and nucleotides of the non-coding strand of the gene that are homologous or complementary, respectively, to the coding region of the mRNA molecule that is synthesized by transcription of the gene.

The "coding region" of an mRNA molecule also consists of the nucleotide residues of the mRNA molecule that correspond to the anticodon region of the transfer RNA molecule during translation of the mRNA molecule or that encode a stop codon. Thus, the coding region may include nucleotide residues containing codons for amino acid residues that are not present in the mature protein encoded by the mRNA molecule (eg, amino acid residues in the protein export signal sequence).

"Differentially down-expressed" or "down-regulated" refers to levels of a biomarker product that are at least 10% or more, such as 20%, 30%, 40% or 50%, 60%, 70%, 80 %, 90% lower or less, and/or 2.0 times, 1.8 times, 1.6 times, 1.4 times, 1.2 times, 1.1 times lower or less, and any and all total or partial spacings in between than controls.

"Differentially up-expressed" or "up-regulated" refers to levels of a biomarker product that are at least 10% or more, such as 20%, 30%, 40%, or 50%, 60%, 70%, 80 %, 90% higher or more and/or 1.1 times, 1.2 times, 1.4 times, 1.6 times, 1.8 times, 2.0 times or more, and any and all total or partial spacings in between than controls.

The term "complementary" as used with respect to a nucleic acid 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 the adenine residue of the first region of the nucleic acid is able to form specific hydrogen bonds ("base pairing") with the remainder of the second region of the nucleic acid, which is antiparallel to the first region, if the residue is a thymine or uracil. Similarly, it is also known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand that is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are anti-parallel, at least one nucleotide residue of the first region is capable of base-pairing with the remainder of the second region. In some embodiments, the first region comprises a first region and the second region comprises a second region, wherein when the first and second regions are anti-parallel, at least about 50%, or at least about 75%, or at least about 90% or at least about 95% of the nucleotide residues in the first region are capable of base pairing with nucleotide residues in the second region. In some embodiments, all nucleotide residues in the first region are capable of pairing with nucleotide residues in the second region.

As used herein, the term "DNA" is defined as deoxyribonucleic acid.

"Encoding" refers to the inherent property of specific nucleotide sequences in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as templates for the synthesis of other polymers and macromolecules in biological processes having either a specific nucleotide sequence (i.e., rRNA, tRNA, and mRNA ) or a particular amino acid sequence, and provide the resulting biological properties. Thus, a gene encodes a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both a coding strand whose nucleotide sequence is identical to an mRNA sequence and is commonly listed in sequence listings, and a non-coding strand used as a template for transcription of a gene or cDNA may be referred to as encoding a protein or other product of that gene or cDNA.

Unless otherwise indicated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate variants of each other and that encode the same amino acid sequence. The expression "a nucleotide sequence that codes for a protein or RNA" may also include introns, to the extent that a nucleotide sequence that codes for a protein may, in some embodiment, contain intron(s).

The term "hybridoma" as used herein refers to a cell resulting from the fusion of a B-lymphocyte and a fusion partner, such as a myeloma cell. The hybridoma can be cloned and maintained indefinitely in cell culture and is capable of producing monoclonal antibodies. A hybridoma can also be considered as a hybrid cell.

"Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide present in a normal context in a living individual in nature is not "isolated", but the same nucleic acid or peptide partially or completely separated from coexisting substances in its natural environment is "isolated". The isolated nucleic acid or protein may be in substantially purified form, or may be in a non-natural environment such as, for example, a host cell.

"Isolated nucleic acid" refers to a segment or fragment of a nucleic acid that has been separated from the sequences flanking it in its natural state, i.e., a DNA fragment that has been removed from sequences that are normally adjacent to the fragment, i.e. sequences adjacent to the fragment in the genome in which it occurs naturally. The term is also used in relation to nucleic acids that are essentially free of other components that accompany the nucleic acid in nature, ie, RNA or DNA, or proteins that accompany it in the cell in nature. Thus, the term includes, for example, recombinant DNA that has been incorporated into a vector, into an autonomously replicating plasmid or virus, or into prokaryotic or eukaryotic genomic DNA, or which is in the form of a single molecule (i.e., as cDNA or genomic , or a cDNA fragment obtained by PCR or restriction enzyme digestion), independent of other sequences. It also includes recombinant DNA, which is part of a hybrid gene encoding an additional polypeptide sequence.

In the context of the present invention, the following abbreviations are used for the most frequently occurring nucleic acid bases. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine and "U" refers to uridine.

As used herein, the term "polynucleotide" is defined as a chain of nucleotides. In addition, nucleic acids are polymers of nucleotides. Thus, the terms "nucleic acids" and "polynucleotides" as used herein are used interchangeably. The person skilled in the art will recognize that nucleic acids are polynucleotides that can be hydrolyzed to monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed to nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences that are obtained by any methods available in the art, including, without limitation, recombinant methods, i.e., cloning nucleic acid sequences from a recombinant library or cellular genome, using conventional cloning and PCR technologies and the like, and using synthetic methods.

As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limitation on the maximum number of amino acids that a protein or peptide sequence may include. Polypeptides include any peptide or protein containing two or more amino acids connected to each other by peptide bonds. As used herein, the term refers to both short chains, which are also commonly referred to in the art, such as peptides, oligopeptides, and oligomers, and longer chains, which are commonly referred to in the art as proteins, of which there are many. types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and others. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

As used herein, the term "progeny" refers to a progeny or progeny and includes the progeny of a mammal, and also includes a different or non-different progeny cell derived from a parent cell. In one usage, the term "offspring" refers to a progeny cell that is genetically identical to the parent. In another usage, the term "offspring" refers to a progeny cell that is genetically and phenotypically identical to the parent. In yet another usage, the term "offspring" refers to a descendant cell that is different from the parent cell.

As used herein, the term "RNA" is defined as ribonucleic acid.

As used herein, the term "recombinant DNA" is defined as DNA obtained by combining DNA fragments from different sources.

As used herein, the term "recombinant polypeptide" is defined as a polypeptide obtained using recombinant DNA techniques.

As used herein, the term "conjugated" refers to the covalent attachment of one molecule to a second molecule.

"Variant" as used herein is a nucleic acid sequence or peptide sequence that differs from the reference nucleic acid sequence or peptide sequence, respectively, but retains the basic biological properties of the reference molecule. Changes in the sequence of a variant nucleic acid may not result in a change in the amino acid sequence of the peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions, and truncations. Changes in the sequence of peptide variants are usually limited or conservative, so that the sequences of the reference peptide and the variant as a whole are very similar and in many areas identical. The variant and reference peptide may differ in amino acid sequence by one or more substitutions, additions, deletions, in any combination. The nucleic acid or peptide variant may be naturally occurring, such as an allelic variant, or may be a variant not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides can be obtained by mutagenesis or direct synthesis. In various embodiments, the variant sequence 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%, at least 85% identical to the reference sequence.

As used herein, the term "regulation" can mean any way to change the level or activity of the substrate. Non-limiting examples of protein regulation include effects on expression (including transcription and/or translation), effects on folding, effects on protein degradation or metabolism, and effects on protein localization. Non-limiting examples of enzyme regulation further include an effect on enzymatic activity. "Regulator" refers to a molecule whose activity includes affecting the level or activity of a substrate. The regulator can be direct or indirect. The regulator may function to activate or inhibit or otherwise modulate its substrate.

As used herein, the term "scan window" refers to a segment of a number of adjacent positions in which a sequence can be evaluated independently of any flanking sequence. The scan window is typically progressively shifted along the length of the sequence to be evaluated, with each new segment evaluated independently. The gradual shift may be 1 or more than one position.

As used herein, the term "vector" may mean a nucleic acid sequence containing an origin of replication. The vector may be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be a DNA or RNA based vector. The vector can either be a self-replicating extrachromosomal vector or a vector that integrates into the host's genome.

Ranges: Throughout this disclosure, various aspects of the invention may be presented in range format. It should be understood that the description in range format is for convenience and brevity only and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered as specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6 and so on, as well as individual numbers in this range, such as 1; 2; 2.7; 3; four; 5; 5,3 and 6. This applies regardless of the range width.

Description

The present invention relates to the inhibition of the signaling pathway and complement related diseases and disorders using an anti-C5a antibody. In one embodiment, the invention is directed to inhibition of the complement signaling cascade by specifically targeting the C5 component of complement and the C5a protein, its cleavage product, while the C5 C5b cleavage product remains functional. In one embodiment, the invention is directed to methods for treating and preventing inflammation and autoimmune diseases and disorders mediated by unwanted, uncontrolled, or excessive complement activation. In one embodiment, the invention is directed to treating a complement-mediated disease or complement-mediated disorder in an individual by contacting the individual with an anti-C5a antibody. In some embodiments, the invention relates to methods for treating C5a-mediated chemotaxis while leaving the C5b-mediated MAC assembly intact.

In one embodiment, the invention provides a method of treating a complement-mediated disease or disorder in an individual, comprising the step of administering to said individual an anti-C5a antibody, thereby selectively inhibiting the effects of the C5a protein. Examples of complement-mediated diseases and disorders that can be treated using the methods of the invention include, but are not limited to, macular degeneration (MD), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, rheumatoid arthritis, lupus, ulcerative colitis, stroke, postoperative systemic inflammatory response syndrome, asthma, allergic asthma, chronic obstructive pulmonary disease (COPD), paroxysmal nocturnal hemoglobinuria (PNH), autoimmune hemolytic anemia (AIHA), Gaucher disease, myasthenia gravis, neuromyelitis optic nerve, 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 (including, not limited to: cardio-related inflammation bypass and renal dialysis), C3 glomerulopathy, membranous nephropathy, IgA nephropathy, glomerulonephritis (including, but not limited to, antineutrophil cytoplasmic antibodies (ANCA)-associated glomerulonephritis, lupus nephritis, and combinations thereof), ANCA-associated vasculitis, Shiga-induced -HUS toxin, and antiphospholipid antibody-induced pregnancy loss, graft-versus-host disease (GVHD), or any combination thereof. In some embodiments, the AP-mediated disease is sepsis, septic shock, rheumatoid arthritis, autoimmune hemolytic anemia, GvHD, antiphospholipid syndrome, or Gaucher's disease.

The ability of the immune system to distinguish between self and non-self antigens is vital to the functioning of the immune system as a specific defense against invading microorganisms. "Non-self" antigens are antigens that are part of substances entering or present in the body that are markedly different or foreign compared to the individual's own components, while "self" antigens are those antigens that in a healthy individual are nothing are not markedly different or alien from their own components. In various embodiments of the methods, complement activation that is inhibited is activation that has been initiated by at least one of the group consisting of a microbial antigen, a non-biological foreign surface, an altered self tissue, or combinations thereof. One example of a non-biological foreign surface is tubing for blood lines, such as those used in cardiopulmonary bypass or kidney dialysis. Examples of altered self tissues include apoptotic, necrotic and ischemic tissues and cells, or combinations thereof.

In some embodiments, the anti-C5a antibodies of the invention inhibit the downstream effects of alternative complement pathway (AP), classical pathway (CP), or lectin pathway (LP) activation. As a rule, CP is initiated by antigen-antibody complexes, LP is activated by the binding of lectins to sugar molecules on the surfaces of microorganisms, while AP is constitutively active at a low level, but can rapidly increase on the surfaces of bacterial, viral, and parasitic cells due to the absence of regulatory proteins. Host cells are usually protected from AR complement activation by regulatory proteins. But in some situations, such as when regulatory proteins are defective or absent, AR can also become uncontrollably activated on host cells, leading to the development of a complement-mediated disease or disorder. CP consists of C1, C2, C4 components and converges with AP at the C3 activation stage. LP is composed of mannose-binding lectins (MBL) and MBL-associated serine proteases (MASP) and shares C4 and C2 components with CP. AP is composed of C3 components and several factors such as Factor B, Factor D, Roperdine, C5 and Factor H, a fluid phase regulator. Complement activation consists of three steps: (a) recognition, (b) enzyme activation, and (c) membrane attack leading to cell death. The first phase of CP complement activation begins at C1. C1 is composed of three separate proteins: the recognition subunit, C1q, and the serine protease subcomponents, C1r and C1s, which are linked together in a calcium-dependent tetramer complex, C1r2s2. The intact C1 complex is required for the physiological activation of C1. Activation occurs when an intact C1 complex binds to an immunoglobulin complexed with an antigen. This binding activates C1s, which then cleaves the C4 and C2 proteins to form C4a and C4b as well as C2a and C2b. Fragments C4b and C2a combine to form a C3 convertase, C4b2a, which in turn cleaves C3 to form C3a and C3b. LP activation is initiated by the binding of MBL to certain sugars on the target surface and this triggers the activation of MBL-associated serine proteases (MASPs), which then cleave C4 and C2 in a manner similar to the activity of C1s in the CP pathway, leading to the generation of a C3 convertase, C4b2a. Thus, CP and LP are activated by different mechanisms, but they share the same C4 and C2 components, and both pathways result in the same C3 convertase, C4b2a. Cleavage of C3 by C4b2a into C3b and C3a is the central event of the complement pathway for two reasons. It initiates the AP amplification loop, since surface-deposited C3b is the central AP intermediate. Both C3a and C3b are biologically important. C3a is pro-inflammatory and, together with C5a, is an anaphylatoxin. C3b and its further cleavage products also bind to complement receptors found on neutrophils, eosinophils, monocytes, and macrophages, thereby promoting phagocytosis and clearance of C3b-opsonized particles. Finally, C3b can bind to C4b2a to form the C5 convertase CP and LP to activate the terminal complement sequence, resulting in the formation of C5a, a potent pro-inflammatory mediator, and the assembly of the lytic membrane attack complex (MAC), C5-C9.

In one embodiment, the complement pathway activity that is inhibited using the method of the invention is complement pathway activation induced by at least one of the group selected from lipopolysaccharide (LPS), lipooligosaccharide (LOS), pathogen-associated molecular structures ( PAMP) and damage-associated molecular structures (DAMP). In another embodiment, the activity of the complement signaling pathway that is inhibited using the method of the invention is a C5a protein activity. In yet another embodiment, the complement pathway activity that is inhibited using the method of the invention is C5a dependent.

In one embodiment, the invention provides a method of inhibiting the initiation of an inflammatory cascade by activating the terminal complement component in an individual, comprising the step of administering to said individual an anti-C5a antibody, thereby inhibiting the initiation of C5a-dependent inflammation by activating the terminal complement component resulting from CP activation, LP or AP in an individual. Examples of these embodiments are patients with sepsis who suffer from complement-mediated systemic inflammation, and individuals suffering from conditions that can be caused by complement-mediated, organ-specific inflammation such as aHUS, AIHA, antiphospholipid syndrome, GVHD, asthma, ischemia/reperfusion injury, rheumatoid arthritis and ANCA-mediated kidney disease. In various embodiments, diseases and disorders that can be treated using the compositions and methods of the invention include, but are not limited to, complement-mediated hemolysis, complement-mediated aHUS, C3 glomerulopathy, optic neuromyelitis, myasthenia gravis, asthma, ischemia/reperfusion injury, sepsis, septic shock, rheumatoid arthritis, and ANCA-mediated diseases or disorders of the kidneys. In some embodiments, the AP-mediated disease is sepsis, septic shock, rheumatoid arthritis, autoimmune hemolytic anemia, GvHD, antiphospholipid syndrome, or Gaucher's disease.

In various other embodiments, provided herein are methods for identifying a potential anti-C5a antibody having an inhibitory effect on the complement signaling pathway. One such method includes the steps of: a) stable transfection of cells with the C5a receptor; b) seeding cells in chemotaxis assay buffer (RPMI medium with 0.5% BSA) in the upper chambers of Transwell inserts; c) adding 10 nM recombinant human complement C5a pretreated with antibody to chemotaxis assay buffer; c) incubation for 3 hours at 37°C; d) collecting migrated cells from the lower chambers and counting cells using a Coulter counter; f) comparing the number of seeded cells in relation to those migrated to the lower chamber of Transwell, which received recombinant human complement C5a, with the number of cells migrated to the lower chamber with a positive control in the Transwell system and a negative control in the Transwell system; where, when the number of migrated cells is reduced compared to the positive control, an anti-C5a antibody is identified.

In various other embodiments, provided herein are methods for identifying a potential anti-C5a antibody having an inhibitory effect on the complement signaling pathway. One such method includes the steps of: a) stable transfection of cells with the C5a receptor; b) washing the cells with HEPES buffer for calcium mobilization assay (25 mM HEPES, 119 mM NaCl, 5 mM KCl, 5.6 mM glucose, 0.4 mM MgCl ₂ and 1 mM CaCl ₂ ) containing 1 mg/ml bovine serum albumin (BSA); c) incubation of cells with 1 μM indo-1-acetoxymethyl ether at room temperature for 30 min; d) washing the cells twice and resuspending in 1.3 ml of the above buffer; e) mixing 10 nM human C5a protein and 50 μg/ml anti-C5a antibody to a final concentration of 10 nM protein and 50 μg/ml antibody at room temperature; f) intercellular Ca ²⁺ measurement at 360 nm excitation wavelength and 415 nm emission wavelength using the Infinite F200 Multi-Mode Microplate Reader; g) comparison of C5a-induced intercellular calcium mobilization in positive control and negative control treated cells; where, when calcium mobilization is reduced compared to the positive control, an anti-C5a antibody is identified.

Anti-C5a antibodies

In some embodiments, the invention includes compositions containing an antibody that specifically binds to C5 and C5a. In one embodiment, an anti-C5a antibody of the invention specifically binds to C5. In one embodiment, an anti-C5a antibody of the invention specifically binds to C5a. In one embodiment, an anti-C5a antibody of the invention binds specifically to both C5 and C5a. In one embodiment, an anti-C5a antibody of the invention specifically binds to both the C5a fragment of C5 and free C5a. In some embodiments, an anti-C5a antibody of the invention specifically binds to C5 but does not block the cleavage of C5 into C5a and C5b. In one embodiment, the anti-C5a antibody is a polyclonal antibody. In another embodiment, the anti-C5a antibody is a monoclonal antibody. In some embodiments, the anti-C5a antibody does not block the cleavage of C5 into C5a and C5b, but inhibits C5a-dependent biological activity. In some embodiments, the anti-C5a antibody is a chimeric antibody. In still other embodiments, the anti-C5a antibody is a humanized antibody. In some embodiments, the antibody is an antibody fragment. In some embodiments, C5a is human C5a.

In some embodiments, binding of an antibody or antibody fragment to human C5a is associated with a decrease in the expression level or half-life of C5a in an intact organism. In some embodiments, the invention provides a protein or polypeptide capable of binding to human C5a. In some embodiments, the antibody or antibody fragment, protein, or polypeptide binds to the corresponding region or fragment or epitope of human C5a, and binding of the antibody, or antibody fragment, or protein or polypeptide to the corresponding region of human C5a is associated with a decrease in the expression level or half-life of C5a in an intact organism.

In some embodiments, binding of an antibody or antibody fragment to human C5a is associated with a decrease in C5a activity in the complement pathway in an intact organism. In some embodiments, the invention provides a protein or polypeptide capable of binding to human C5a. In some embodiments, the antibody or antibody fragment, protein or polypeptide binds to the corresponding region or fragment or epitope of human C5a; and binding of the antibody or antibody fragment or protein or polypeptide to the corresponding region of human C5a is associated with a decrease in C5a activity in the intact organism.

In some embodiments, the human C5a-binding antibody or C5a-binding antibody fragment is further conjugated to a protein, peptide, or other compound. In some embodiments, the C5a-binding antibody or antibody fragment is conjugated to a protein, peptide, or other compound. In some embodiments, the protein, peptide, or other compound to which the C5-binding antibody or antibody fragment is conjugated is a targeting molecule (i.e., the targeting molecule specifically binds to a molecule other than C5a). In some embodiments, the protein, peptide, or other compound to which the C5a-binding antibody or antibody fragment is conjugated is an effector molecule (eg, a cytotoxic molecule).

In one embodiment, the anti-C5a antibody, or antigen-binding fragment thereof, comprises at least one of the CDRs selected from the group consisting of: VH-CDR1: SEQ ID NO: 3; VH-CDR2: SEQ ID NO: 4; VH-CDR3: SEQ ID NO: 5; VL-CDR1: SEQ ID NO: 8; VL-CDR2: SEQ ID NO: 9; and VL-CDR3: SEQ ID NO: 10, or a variant or variants thereof. In another embodiment, the anti-C5 antibody contains all of the CDRs from the group consisting of: VH-CDR1: SEQ ID NO: 3; VH-CDR2: SEQ ID NO: 4; VH-CDR3: SEQ ID NO: 5; VL-CDR1: SEQ ID NO: 8; VL-CDR2: SEQ ID NO: 9; and VL-CDR3: SEQ ID NO: 10, or a variant or variants thereof.

In some embodiments, the anti-C5a antibody, or antigen-binding fragment thereof, comprises: VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof containing up to about 3 (particularly about 1, 2, or 3) amino acid substitutions; and VL-CDR1: SEQ ID NO: 8 or a variant thereof containing up to about 3 (particularly about 1, 2 or 3) amino acid substitutions. In some embodiments, the anti-C5a antibody, or antigen-binding fragment thereof, comprises: VH-CDR1 containing the amino acid sequence of SEQ ID NO: 3; and VL-CDR1: SEQ ID NO: 8.

In some embodiments, the anti-C5a antibody, or antigen-binding fragment thereof, comprises: VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof containing up to about 3 (particularly about 1, 2, or 3) amino acid substitutions; and VL-CDR2: SEQ ID NO: 9 or a variant thereof containing up to about 3 (particularly about 1, 2 or 3) amino acid substitutions. In some embodiments, the anti-C5a antibody, or antigen-binding fragment thereof, comprises: a VH-CDR2 containing the amino acid sequence of SEQ ID NO: 4; and VL-CDR2: SEQ ID NO: 9.

In some embodiments, the anti-C5a antibody, or antigen-binding fragment thereof, comprises: VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof containing up to about 3 (particularly about 1, 2, or 3) amino acid substitutions; and VL-CDR3: SEQ ID NO: 10 or a variant thereof containing up to about 3 (particularly about 1, 2 or 3) amino acid substitutions. In some embodiments, the anti-C5a antibody, or antigen-binding fragment thereof, comprises: a VH-CDR3 containing the amino acid sequence of SEQ ID NO: 5; and VL-CDR3: SEQ ID NO: 10.

In some embodiments, the anti-C5a antibody, or antigen-binding fragment thereof, comprises: VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof containing up to about 3 (particularly about 1, 2, or 3) amino acid substitutions; VH-CDR2 containing the amino acid sequence of SEQ ID NO: 4, or a variant thereof, containing up to about 3 (in particular, about 1, 2 or 3) amino acid substitutions; VH-CDR3 containing the amino acid sequence of SEQ ID NO: 5, or a variant thereof containing up to about 3 (in particular, about 1, 2 or 3) amino acid substitutions; VL-CDR1 containing the amino acid sequence of SEQ ID NO: 8, or a variant thereof containing up to about 3 (in particular, about 1, 2 or 3) amino acid substitutions; VL-CDR2 containing the amino acid sequence of SEQ ID NO: 9, or a variant thereof, containing up to about 3 (in particular, about 1, 2 or 3) amino acid substitutions; and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 10, or a variant thereof, containing up to about 3 (in particular, about 1, 2 or 3) amino acid substitutions.

In some embodiments, the anti-C5a antibody, or antigen-binding fragment thereof, comprises: VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof containing up to about 3 (particularly any of 1, 2, or 3) amino acid substitutions ; VH-CDR2 containing the amino acid sequence of SEQ ID NO: 4, or a variant thereof, containing up to about 3 (in particular, about 1, 2 or 3) amino acid substitutions; VH-CDR3 containing the amino acid sequence of SEQ ID NO: 5; VL-CDR1 containing the amino acid sequence of SEQ ID NO: 8, or a variant thereof containing up to about 3 (in particular, about 1, 2 or 3) amino acid substitutions; VL-CDR2 containing the amino acid sequence of SEQ ID NO: 9, or a variant thereof, containing up to about 3 (in particular, about 1, 2 or 3) amino acid substitutions; and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the anti-C5a antibody, or antigen-binding fragment thereof, comprises: VH-CDR1 containing the amino acid sequence of SEQ ID NO: 3; VH-CDR2 containing the amino acid sequence of SEQ ID NO: 4; VH-CDR3 containing the amino acid sequence of SEQ ID NO: 5; VL-CDR1 containing the amino acid sequence of SEQ ID NO: 8; VL-CDR2 containing the amino acid sequence of SEQ ID NO: 9; and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the anti-C5a antibody, or antigen-binding fragment thereof, comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 2 or a variant thereof. In other embodiments, the anti-C5a antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof. In yet another embodiment, the anti-C5a antibody is a monoclonal antibody designated mAb 7A12. The anti-C5a monoclonal antibody, designated mAb 7A12, contains a heavy chain containing the amino acid sequence of SEQ ID NO: 2 or a variant thereof and a light chain containing the amino acid sequence of SEQ ID NO: 7 or a variant thereof. In some embodiments, the anti-C5a monoclonal antibody is a humanized antibody having one or more or all of the CDRs or variants thereof, mAb designated 7A12. In some embodiments, the anti-C5a monoclonal antibody is a chimeric antibody having one or more or all of the CDRs or variants thereof, mAb designated 7A12.

In some embodiments, the anti-C5a antibody, or antigen-binding fragment thereof, comprises at least one of a heavy chain CDR1, CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof. In other embodiments, the anti-C5a antibody, or antigen-binding fragment thereof, comprises at least one of a light chain CDR1, CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof.

In some embodiments, the antibodies are chimeric antibodies. In some embodiments, an anti-human C5a antibody may comprise human light chain and human heavy chain constant regions in combination with variable region CDR sequences, or a variant thereof, as described elsewhere in the specification. One skilled in the art can prepare and prepare a chimeric antibody using known techniques for substituting the appropriate domains for the specific antibodies of interest. Such an antibody is readily produced by grafting heterogeneous antibody domains, incorporating one or more of the CDR sequences described herein. Using known recombinant technology, it is possible to obtain and prepare a recombinant antibody containing heavy and light chain constant regions encoded by human heavy and light chain constant region nucleic acid sequences; and heavy and light chain variable regions containing CDRs encoded by nucleic acid sequences corresponding to the CDR sequences given in the disclosure. One of skill in the art can make an anti-human C5a antibody comprising one or more of the CDR sequences described in this disclosure, where portions of one light chain or portions of one heavy chain are replaced with regions from an antibody of a different species, such as a human. Human anti-human C5a antibody comprising variable regions having one or more CDR sequences selected from SEQ ID NOs: 3-5 and 8-10, or a variant or variants thereof, in combination with murine or non-mouse antibody structural elements outside the CDR region , can be obtained by conventional methods known in the art. In some embodiments, the antibodies or antibody fragments are further humanized using techniques known in the art.

In some embodiments, an anti-C5a antibody comprises an antibody having at least about 85% amino acid sequence identity with one or more of the CDR sequences described herein, shown in SEQ ID NOS: 3-5 and 8-10.

In one embodiment, the invention encompasses an anti-C5a antibody having CDR sequences at least about 85% identical to the CDR sequences described herein. The invention encompasses an anti-C5a antibody, or antigen-binding fragment thereof, having CDR sequences that are at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% 94%, 95%, 96%, 97%, 99%, or 100% identical to the CDR sequences described herein. In one embodiment, the anti-human C5a antibody has a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH region has an amino acid sequence that is at least about 85% identical to the sequence of SEQ ID NO: 2, and wherein the VL region has an amino acid sequence that is at least about 90% identical to the sequence of SEQ ID NO: 7. In some embodiments, the antibody or antibody fragment is modified. In some embodiments, the modifications include the fusion of an antibody or antigen-binding fragment thereof with regions of another protein or protein fragment. In some embodiments, an antibody or antibody fragment of the invention is modified to increase its in vivo circulating half-life. For example, a fragment antibody can be fused to an FcRn molecule, which is also known as the neonatal Fc receptor, to stabilize the antibody in vivo (Nature Reviews Immunology, 7: 715-725). One skilled in the art can prepare a human C5a binding single chain variable fragment (scFv) containing at least one CDR sequence selected from SEQ ID NOs: 3-5 and 8-10. The scFv may comprise at least one heavy chain variable region sequence shown in SEQ ID NOs: 3-5 and at least one light chain variable region shown in SEQ ID NOs: 8-10. CDR sequences included in scFvs having 80%, 81%, 82%, 83%, 84%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92% amino acid sequence identity , 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with the CDR sequences described in this disclosure are within the scope of this disclosure.

In some embodiments, the antibody or antibody fragment is modified. In some embodiments, the modifications include the fusion of an antibody or antigen-binding fragment thereof with regions of another protein or protein fragment. In some embodiments, an antibody or antibody fragment of the invention is modified to increase its in vivo circulating half-life. For example, a fragment antibody can be fused to an FcRn molecule, which is also known as the neonatal Fc receptor, to stabilize the antibody in vivo (Nature Reviews Immunology, 7: 715-725). In some embodiments, the antibody or antigen-binding fragment thereof is conjugated (eg, fused) to an effector molecule and/or another targeting molecule (such as an antibody or antibody fragment that recognizes another molecule, another antigen, or another epitope).

In various embodiments, any of the antibodies of the invention described herein having any of the variable regions described herein may comprise a human IgG4 heavy chain constant region. In some embodiments, an antibody of the invention comprises a human IgG4 heavy chain constant region having the S228P mutation.

One skilled in the art can prepare a C5a binding single chain variable fragment (scFv) containing at least one specific CDR sequence selected from SEQ ID NOs: 3-5, 8-10, or a variant or variants thereof. The scFv may comprise the heavy chain variable region sequences shown in SEQ ID NOs: 3-5, or a variant or variants thereof, and the light chain variable regions shown in SEQ ID NOs: 8-10, or a variant or variants thereof. CDR sequences included in scFvs having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 amino acid sequence identity %, 96%, 97%, 98%, 99%, or 100% with the CDR sequences described in this disclosure are within the scope of this disclosure.

Screening tests

The present invention finds use in various screening tests, including determining whether a candidate anti-C5a antibody can inhibit complement activity.

In some embodiments, the level of complement activity in the presence of a candidate anti-C5a antibody is compared to the complement activity established for a positive control. The positive control includes complement activation in the absence of added test compound or in the presence of another test compound that does not bind C5a. In some embodiments, a candidate anti-C5a antibody is identified as a complement inhibitor when complement activity in the presence of the candidate anti-C5a antibody is below about 70% of the complement activity found in the positive control; this corresponds to greater than about 30% inhibition of complement activity in the presence of the test compound. In still other embodiments, a candidate anti-C5a antibody is identified as a complement inhibitor when complement activity in the presence of the candidate anti-C5a antibody is below about 80% of the complement activity found in the positive control; this corresponds to greater than about 20% inhibition of complement activity in the presence of the test compound. In still other embodiments, a candidate anti-C5a antibody is identified as a complement inhibitor when complement activity in the presence of the candidate anti-C5a antibody is below about 90% of the complement activity found in the positive control; this corresponds to greater than about 10% inhibition of complement activity in the presence of the test compound. In some embodiments, the level of complement inhibition of a candidate anti-C5a antibody is compared to the level of inhibition found in a negative control.

A variety of immunoassay formats, including competitive and non-competitive immunoassay formats, antigen capture assays, sandwich-type assays with two antibodies, and sandwich-type assays with three antibodies, are suitable methods for the invention (Self et al., 1996, Curr. Opin. Biotechnol ., 7: 60-65). The invention should not be construed as being limited to any one type of known or hitherto unknown assay, provided that the assay is capable of detecting complement inhibition.

Enzyme-linked immunosorbent assay (ELISA) is suitable for use in the methods of the invention. An enzyme, such as, but not limited to, horseradish peroxidase (HRP), alkaline phosphatase, beta-galactosidase, or urease, may be associated, for example, with an antibody or secondary antibody for use in the method of the invention. A horseradish peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which in the presence of hydrogen peroxide gives a soluble product detectable at 450 nm. Other suitable enzyme coupled systems include, for example, an alkaline phosphatase detection system that can be used with the chromogenic substrate p-nitrophenyl phosphate to produce a soluble product readily detectable at 405 nm. Similarly, a beta-galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl-beta-D-galactopyranoside (ONPG) to produce a soluble product detectable at 410 nm. Alternatively, a urease detection system with a substrate such as urea-bromocresol purple (Sigma Immunochemicals, St. Louis, MO) can be used. Suitable enzyme-linked primary and secondary antibodies can be obtained from any number of commercial sources.

Chemiluminescent detection is also suitable for detecting complement inhibition. Chemiluminescent secondary antibodies can be obtained from any number of commercial sources.

Fluorescent detection is also useful for detecting complement inhibition. Suitable fluorochromes include, but are not limited to, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red, and antibodies labeled with lyssamine, fluorescein, or rhodamine.

Radioimmunoassay (RIA) is also useful in the methods of the invention. Such assays are well known in the art and are described, for example, in Brophy et al. (1990, Biochem. Biophys. Res. Comm. 167: 898-903) and Guechot et al. (1996, Clin. Chem. 42: 558-563). Radioimmunoassay is carried out, for example, using a primary or secondary antibody labeled with iodine-125 (Harlow et al., supra, 1999).

The signal emitted by the detectable antibody is analyzed, for example, using a spectrophotometer to detect the color of the chromogenic substrate; a radiation counter for detecting radiation, such as a gamma counter for detecting iodine-125; or a fluorometer for analyzing fluorescence in the presence of light of a specific wavelength. If an enzyme-linked assay is used, the quantitative analysis is carried out using a spectrophotometer. It is understood that the assays of the invention may be performed manually or, if desired, may be automated, and that the signal emitted from multiple samples may be detected simultaneously using many commercially available systems.

The methods of the invention also include the use of capillary electrophoresis immunoassays (CEIA), which can be automated if desired. Immunoassays can also be used in combination with laser-induced fluorescence, as described, for example, in Schmalzing et al. (1997, Electrophoresis, 18: 2184-2193) and Bao (1997, J. Chromatogr. B. Biomed. Sci., 699: 463-480). Liposomal immunoassays such as flow-injection liposomal immunoassays and liposomal immunosensors can also be used in accordance with the methods of the invention (Rongen et al., 1997, J. Immunol. Methods, 204: 105-133).

Quantitative Western blotting can also be used to determine the level of complement inhibition in the methods of the invention. Western blots are quantitatively analyzed using well known methods such as scanning densitometry (Parra et al., 1998, J. Vasc. Surg., 28: 669-675).

Methods of administration

The methods of the invention comprise administering a therapeutically effective amount of at least one anti-C5a antibody or antigen-binding fragment thereof (such as any of the antibodies or fragments thereof described elsewhere herein) to an individual identified as having, or suspected of having, mediated complement disease or disorder. In one embodiment, the individual is a mammal having a complement system. In one embodiment, the individual is a human. In various embodiments, at least one anti-C5a antibody, or antigen-binding fragment thereof, is administered topically, regionally, or systemically.

In various embodiments, the disease or disorder is at least selected from the group consisting of: macular degeneration (MD), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, rheumatoid arthritis, lupus, ulcerative colitis, stroke, postoperative systemic inflammatory response, asthma, allergic asthma, chronic obstructive pulmonary disease (COPD), paroxysmal nocturnal hemoglobinuria (PNH), myasthenia gravis, neuromyelitis optic nerve, 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 (including but not limited to: inflammation associated with cardiopulmonary bypass and kidney dialysis), C3 - glomerulopathy, membranosis nephropathy, IgA nephropathy, glomerulonephritis (including, but not limited to, antineutrophil cytoplasmic antibodies (ANCA)-associated glomerulonephritis, lupus nephritis, and combinations thereof), ANCA-associated vasculitis induced by Shiga toxin HUS, and antiphospholipid antibody-induced pregnancy loss, graft-versus-host disease (GVHD), or any combination thereof. In some embodiments, the AP-mediated disease is sepsis, septic shock, rheumatoid arthritis, autoimmune hemolytic anemia, GvHD, antiphospholipid syndrome, or Gaucher's disease. The methods of the invention may include administering at least one anti-C5a antibody or antigen-binding fragment thereof, but the present invention should in no way be construed as being limited to the anti-C5a antibodies described herein, but rather should be construed as encompassing any an anti-C5a antibody, both known and unknown, that reduces and decreases complement activation.

The method of the invention comprises administering a therapeutically effective amount of at least one anti-C5a antibody or antigen-binding fragment thereof to an individual, wherein the composition of the present invention comprises at least one anti-C5a antibody or antigen-binding fragment thereof, alone or in combinations with at least one other therapeutic agent. The invention can be used in combination with other treatments such as, for example, anti-inflammatory therapy and the like. Examples of anti-inflammatory therapy that can be used in combination with the methods of the invention include, for example, therapy that uses steroid drugs, as well as therapy that uses non-steroid drugs.

The method of the invention comprises administering to an individual a therapeutically effective amount of an anti-C5a antibody, or antigen-binding fragment thereof. In some embodiments, the invention provides a method of treating C5a related diseases involving dysregulation of the complement signaling pathway by administering a therapeutically effective amount of an antibody of the invention or a therapeutically effective amount of an antibody fragment such that a reduction in C5a activity occurs in an individual. In some embodiments, the invention provides a method of treating C5a related diseases involving dysregulation of the complement signaling pathway by administering a therapeutically effective amount of an antibody or antibody fragment. In some embodiments, the invention provides a method of treating C5a related diseases involving dysregulation of the complement signaling pathway by administering to an individual an effective amount of an antibody, antibody fragment, polypeptide, peptide, or conjugated peptide such that complement activity is reduced in the individual. In some embodiments, the method of treatment comprises systemically administering to an individual an effective dose of an antibody or antibody fragment, whereby the individual has a systemic decrease in C5a activity.

The introduction of an anti-C5a antibody or antigen-binding fragment thereof in the method of treatment according to the invention can be achieved in various ways using methods known in this field. Thus, the therapeutic and prophylactic methods of the invention encompass the use of pharmaceutical compositions containing an anti-C5a antibody.

Pharmaceutical compositions useful in the practice of the invention may be administered to deliver a dose of at least about 1 ng/kg, at least about 5 ng/kg, at least about 10 ng/kg, at least , about 25 ng/kg, at least about 50 ng/kg, at least about 100 ng/kg, at least about 500 ng/kg, at least about 1 μg/kg, at least , about 5 µg/kg, at least about 10 µg/kg, at least about 25 µg/kg, at least about 50 µg/kg, at least about 100 µg/kg, at least , about 500 µg/kg, at least about 1 mg/kg, at least about 5 mg/kg, at least about 10 mg/kg, at least about 25 mg/kg, at least , about 50 mg/kg, at least about 100 mg/kg, at least about 200 mg/kg, at least about 300 mg/kg, at least about 400 mg/kg, and at least least about 500 mg/kg body weight of the individual. In one embodiment, a dose is administered that provides a concentration of the anti-C5a antibody or antigen-binding fragment thereof of the present invention of at least about 1 pM, at least about 10 pM, at least about 100 pM, at least at least about 1 nM, at least about 10 nM, at least about 100 nM, at least about 1 μM, at least about 2 μM, at least about 3 μM, at least about 4 μM, at least about 5 μM, at least about 6 μM, at least about 7 μM, at least about 8 μM, at least about 9 μM, and at least about 10 μM , in an individual. In yet another embodiment, the invention provides for administering a dose that provides a concentration of an anti-C5a antibody or antigen-binding fragment of the present invention of at least about 1 pM, at least about 10 pM, at least about 100 pM, at at least about 1 nM, at least about 10 nM, at least about 100 nM, at least about 1 μM, at least about 2 μM, at least about 3 μM, at least at least about 4 μM, at least about 5 μM, at least about 6 μM, at least about 7 μM, at least about 8 μM, at least about 9 μM, and at least , about 10 μM, in the individual's plasma.

In some embodiments, pharmaceutical compositions useful in the practice of the invention may be administered to deliver a dose of no more than about 1 ng/kg, no more than about 5 ng/kg, no more than about 10 ng/kg, no more than about 25 ng/kg, not more than about 50 ng/kg, not more than about 100 ng/kg, not more than about 500 ng/kg, not more than about 1 μg/kg, not more than about 5 μg/kg , not more than about 10 µg/kg, not more than about 25 µg/kg, not more than about 50 µg/kg, not more than about 100 µg/kg, not more than about 500 µg/kg, not more than about 1 mg/kg, not more than about 5 mg/kg, not more than about 10 mg/kg, not more than about 25 mg/kg, not more than about 50 mg/kg, not more than about 100 mg/kg , not more than about 200 mg/kg, not more than about 300 mg/kg, not more than about 400 mg/kg, and not more than about 500 mg/kg body weight of the individual. In one embodiment, a dose is administered that provides a concentration of an anti-C5a antibody of the present invention of no more than about 1 pM, no more than about 10 pM, no more than about 100 pM, no more than about 1 nM, no more than about 10 nM, not more than about 100 nM, not more than about 1 µM, not more than about 2 µM, not more than about 3 µM, not more than about 4 µM, not more than about 5 µM, not more than about 6 µM, not more than about 7 µM, not more than about 8 µM, not more than about 9 µM, and not more than about 10 µM, in an individual. In yet another embodiment, the invention provides for administering a dose that provides a concentration of an anti-C5a antibody of the present invention of no more than about 1 pM, no more than about 10 pM, no more than about 100 pM, no more than about 1 nM, no more than more than about 10 nM, not more than about 100 nM, not more than about 1 µM, not more than about 2 µM, not more than about 3 µM, not more than about 4 µM, not more than about 5 µM, not more than than about 6 µM, not more than about 7 µM, not more than about 8 µM, not more than about 9 µM, and not more than about 10 µM, in an individual's plasma. Dose ranges between any of the doses disclosed herein are also contemplated.

Typically, doses that can be administered in the method of the invention to an individual, in some embodiments, a human, are in an amount of 0.5 μg to about 50 mg per kg of the individual's body weight. Although the exact dosage administered will vary depending on a number of factors, including, but not limited to, the type of individual and the type of disease state being treated, the age of the individual, and the route of administration. In some embodiments, the dosage of the compound will range from about 1 μg to about 10 mg per kg of the individual's body weight. In yet other embodiments, the implementation of the dose will vary from about 3 μg to about 1 mg per kg of body weight of the individual.

The antibody can be administered to the individual as often as several times a day, or it can be administered less frequently, such as once a day, twice a day, three times a day, once a week, twice a week, three times a week , once every two weeks, twice every two weeks, three times every two weeks, once a month, twice a month, three times a month, or even less frequently, such as once every few months, or even once or several times a a year or less. Dosing frequency will be apparent to those skilled in the art and will depend on a number of factors such as, but not limited to, the type and severity of the disease being treated, the type and age of the individual, and so on. The compositions of pharmaceutical compositions can be prepared by any method known or developed further in the field of pharmacology. Typically, such preparations include the step of bringing the active ingredient into association with the carrier or one or more other accessory ingredients, and then, if necessary or desired, shaping or packaging the product into the desired single or multi-dose unit.

Although the description of the pharmaceutical compositions provided herein is generally directed to pharmaceutical compositions that are suitable for ethical administration to humans, one of skill in the art will appreciate that such compositions are generally suitable for administration to individuals of all kinds. Modification of pharmaceutical compositions suitable for administration to humans to provide compositions suitable for administration to different individuals is well understood, and such modification can be designed and performed by a veterinary pharmacologist of ordinary skill with merely routine, if any, experimentation. Individuals to whom the pharmaceutical compositions of the invention are intended to be administered include, but are not limited to, humans and other primates, mammals, including commercial mammals such as non-human primates, cattle, pigs, horses, sheep, cats and dogs.

Pharmaceutical compositions that are useful in the methods of the invention may be formulated, packaged or marketed in formulations suitable for ophthalmic, oral, rectal, intravaginal, parenteral, topical, pulmonary, intranasal, buccal, intraocular, intravitreal, intramuscular, intradermal, and intravenous route of administration. Other contemplated formulations include engineered nanoparticles, liposomal formulations, active ingredient loaded and releasing red blood cells, and immunological formulations.

The pharmaceutical composition of the invention may be formulated, packaged or marketed as a single dose or multiple single doses. A unit dose is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of active ingredient is generally equal to the dose of the active ingredient that is administered to the individual, or a convenient fraction of such a dose, for example, half or a third of such a dose.

The relative amounts of active ingredient, pharmaceutically acceptable carrier and any additional ingredients in a pharmaceutical composition of the invention will vary depending on the identity, size and condition of the individual being treated, and further depending on the route by which the composition is to be administered. For example, the composition may contain from 0.1% to 100% (w/w) of the active ingredient. In various embodiments, the composition contains 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 , approx 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 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%, by m at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% (w/w) of the active ingredient.

In addition to the active ingredient, the pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents.

It is possible to prepare controlled or sustained release formulations of the pharmaceutical composition of the invention using conventional technology.

Parenteral administration of a pharmaceutical composition includes any route of administration characterized by a physical breach in the integrity of an individual's tissue and administration of the pharmaceutical composition through such a breach into the tissue. Parenteral administration may be local, regional or systemic. Thus, parenteral administration includes, but is not limited to, administering the pharmaceutical composition by injection of the composition, administering the composition through a surgical incision, administering the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is intended to include, but is not limited to, intravenous, intraocular, intravitreolar, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal injection, and intratumoral administration.

Formulations of a pharmaceutical composition suitable for parenteral administration include the active ingredient in combination with a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline. Such formulations may be formulated, packaged or marketed in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged or marketed in unit dosage form such as ampoules or multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained release or biodegradable formulations. Such formulations may further contain one or more additional ingredients, including, but not limited to, suspending, stabilizing or dispersing agents. In one embodiment of a parenteral formulation, the active ingredient is provided in dry (ie, powder or granular) form for reconstitution with a suitable vehicle (eg, sterile, pyrogen-free water) prior to parenteral administration of the reconstituted composition.

Pharmaceutical compositions can be formulated, packaged or marketed in the form of a sterile injectable aqueous or oily suspension or solution. Such a suspension or solution may be formulated in accordance with the prior art and may 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 parenteral acceptable diluent or solvent such as, for example, water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fatty oils such as synthetic mono- or diglycerides. Other formulations used for parenteral administration which are suitable include formulations that contain the active ingredient in microcrystalline form, in a liposomal formulation, or as a component of biodegradable polymer systems. Compositions for sustained release or implantation may contain pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a poorly soluble polymer, or a poorly soluble salt.

The pharmaceutical composition of the invention may be formulated, packaged or marketed in a formulation suitable for pulmonary administration through the oral cavity. Such a composition may contain dry particles that contain the active ingredient and that have a diameter in the range from about 0.5 to about 7 nm, and in some embodiments, from about 1 to about 6 nm. Such compositions are conveniently in the form of dry powders for administration using a device containing a dry powder reservoir into which a propellant stream can be directed to disperse the powder, or using a self-propelled solvent/powder distribution container, such as a device containing an active ingredient, dissolved or suspended in a low boiling propellant in a sealed container. In some embodiments, such powders contain particles wherein at least 98% of the particles by mass are greater than 0.5 nm in diameter and at least 95% of the particles by number are less than 7 nm in diameter. In some embodiments, at least 95% of the particles, by mass, have a diameter greater than 1 nm, and at least 90% of the particles, by number, have a diameter less than 6 nm. In some embodiments, dry powder compositions comprise a solid, finely divided powder diluent such as sugar and are conveniently administered in a unit dosage form.

Low boiling propellants typically include liquid propellants having an atmospheric boiling point below 65°F. Typically, the propellant may comprise from 50% to 99.9% (w/w) of the composition, and the active ingredient may comprise from 0.1% to 20% (w/w) of the composition. The propellant may also contain additional ingredients such as a liquid nonionic or solid anionic surfactant or a solid diluent (in some embodiments having a particle size of the same order of magnitude as the particles containing the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonary delivery may also provide the active ingredient in the form of a solution or suspension droplet. Such formulations may be formulated, packaged or marketed as aqueous or diluted alcoholic solutions or suspensions, optionally sterile, containing the active ingredient and may be conveniently administered using any spray or spray device. Such formulations may further contain one or more additional ingredients, including, but not limited to, a flavoring agent such as sodium saccharinate, a volatile oil, a buffering agent, a surfactant, or a preservative such as methyl hydroxybenzoate. In some embodiments, the droplets they provide by injection have an average diameter in the range of about 0.1 to about 200 nm.

The formulations are also suitable for intranasal delivery of the pharmaceutical composition of the invention.

Another formulation suitable for intranasal administration is a coarse powder containing the active ingredient and having an average particle size of from about 0.2 to 500 microns. Such a composition is administered in the same manner as snuffing tobacco, i.e. by rapid inhalation through the nasal passage from a container of powder held close to the nostrils.

Formulations suitable for intranasal administration may, for example, include from about 0.1% (w/w) to 100% (w/w) of the active ingredient and may also contain one or more additional ingredients.

The pharmaceutical composition of the invention may be formulated, packaged or marketed in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets or lozenges prepared using conventional methods and may, for example, contain from 0.1% to 20% (w/w) of the active ingredient, with the remainder comprising soluble or degradable in the oral cavity, the composition; and, optionally, one or more additional ingredients. Alternatively, formulations suitable for buccal administration may contain a powder or an aerosolized or sprayed solution or suspension containing the active ingredient. In some embodiments, such powder, aerosolized, or spray formulations, when dispersed, have an average particle or droplet size in the range of about 0.1 to about 200 nm, and may also contain one or more additional ingredients.

As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surfactants; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweeteners; flavors; dyes; preservatives; physiologically degradable compositions such as gelatin; aqueous carriers and solvents; oil carriers and solvents; suspending agents; dispersing or wetting agents; emulsifiers, softening agents; buffers; salt; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that may be included in pharmaceutical compositions of the 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 included in this document by reference.

Cells that produce antibodies and their antigen-binding fragments

In some embodiments, the invention is a cell or cell line (such as host cells) that produces at least one of the anti-C5a antibodies or antigen-binding fragments described herein. In one embodiment, the cell or cell line is a genetically modified cell that produces at least one of the anti-C5a antibodies or antigen-binding fragments described herein. In one embodiment, the cell or cell line is a hybridoma that produces at least one of the anti-C5a antibodies or antigen-binding fragments described herein.

Hybrid cells (hybridomas) are usually derived from bulk fusions of highly B-lymphocyte-rich mouse splenocytes and myeloma "fusion partner cells" (Alberts et al., Molecular Biology of the Cell (Garland Publishing, Inc. 1994); Harlow et al. , Garland Publishing, Inc. 1994); Harlow et al., Antibodies. A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988). The cells in confluence are then distributed into pools that can be assayed for the production of antibodies with the desired specificity. Positive pools can be further subdivided until single cell clones are identified that produce antibodies with the desired specificity. The antibodies produced by such clones are called monoclonal antibodies.

Also provided are nucleic acids encoding any of the antibodies or antibody fragments disclosed herein, as well as vectors containing the nucleic acids. Thus, the antibodies and fragments of the invention can be produced by expression of a nucleic acid in a cell or cell line, such as the cell lines commonly used to express recombinant or humanized immunoglobulins. Thus, the antibodies and fragments of the invention can also be obtained by cloning the nucleic acids into one or more expression vectors and transforming the vector into a cell line, such as the cell lines commonly used to express recombinant or humanized immunoglobulins.

Genes encoding immunoglobulin heavy and light chains, or fragments thereof, can be constructed according to methods including, but not limited to, polymerase chain reaction (PCR) known in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, N.Y., 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 heavy and light chains, or fragments thereof, can be cloned from the genomic DNA of an antibody-secreting cell, or cDNA is obtained by reverse transcription of cellular RNA. Cloning is carried out by conventional methods, including the use of PCR primers that hybridize to sequences flanking or overlapping the genes or gene segments to be cloned.

Nucleic acids encoding an antibody of the invention, or a heavy chain or a light chain, or fragments thereof, can be prepared and used in accordance with recombinant nucleic acid techniques to produce a particular immunoglobulin, immunoglobulin chain, or fragment or variant thereof, in various host cells or in the in vitro translation system. For example, nucleic acids encoding antibodies or fragments thereof can be placed in suitable prokaryotic or eukaryotic vectors, e.g., expression vectors, and introduced into a suitable host cell by an appropriate method, e.g., transformation, transfection, electroporation, infection, such that the acid becomes operably linked to one or more expression control elements, for example in a vector, or integrated into the genome of the host cell.

In some embodiments, heavy and light chains, or fragments thereof, can be placed in two different expression vectors that can be used to co-transfect a recipient cell. In some embodiments, each vector may contain two or more selectable genes, one for selection in a bacterial system and one for selection in a eukaryotic system. Such vectors enable the production and amplification of genes in the bacterial system, and also the subsequent co-transfection of eukaryotic cells and the selection of co-transfected cells. The selection procedure can be used to select the expression of antibody nucleic acids introduced in two different DNA vectors into a eukaryotic cell.

Alternatively, nucleic acids encoding heavy and light chains, or fragments thereof, can be expressed from a single vector. Although light and heavy chains are encoded by separate genes, they can be combined using recombinant methods. For example, two polypeptides can be joined by a synthetic linker that provides them as a single protein chain in which the V _L and V _H regions are paired to form monovalent molecules (known as single-chain Fvs (scFvs); see, for example, Bird et al. , 1988, Science 242: 423-426 and Huston et al., 1988, Proc Natl Acad Sci USA 85:5879-5883.

The invention provides an isolated nucleic acid molecule containing a nucleic acid sequence encoding a heavy chain and/or a light chain, as well as fragments thereof. A nucleic acid molecule containing both light and heavy chain coding sequences, or fragments thereof, can be designed to contain a synthetic signal sequence for secretion of the antibody or fragment when produced in a cell. In addition, the nucleic acid molecule may contain specific DNA linkages that allow the insertion of other antibody sequences and maintain the translational reading frame so as not to alter the amino acids normally found in antibody sequences.

In accordance with the present invention, nucleic acid sequences encoding an antibody can be inserted into a suitable expression vector. In various embodiments, the expression vector contains the necessary elements for transcription and translation of the inserted nucleic acid encoding an antibody so as to generate recombinant DNA molecules that direct the expression of antibody sequences to form an antibody or fragment thereof.

Nucleic acids encoding antibodies or fragments thereof can be subjected to various recombinant nucleic acid techniques known to those skilled in the art, such as site-directed mutagenesis.

A variety of methods can be used to express nucleic acids in a cell. Nucleic acids can be cloned into a number of different types of vectors. However, the present invention should not be construed as being limited to any particular vector. Instead, the present invention should be considered as covering a wide range of vectors that are readily available and/or known in the art. For example, the nucleic acid of the invention can be cloned into a vector, including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

In specific embodiments, the expression vector is selected from the group consisting of a viral vector, a bacterial vector, and a mammalian cell vector. Numerous vector expression systems exist that include at least some or all of the compositions discussed above. Systems based on prokaryotic and/or eukaryotic vector can be used for use in the present invention to obtain polynucleotides or their related polypeptides. Many such systems are commercially available and widely available.

Viral vector technology is well known in the art and is described, for example, in the monographs of Sambrook et al. (2012) and Ausubel et al. (1999), and other guides to virology and molecular biology. Viruses that are suitable as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes simplex 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 shown to efficiently express the desired nucleic acids in cells. However, the invention should not be limited to the use of the MSCV vector alone, rather, any method of retroviral expression is included in the invention. Other examples of viral vectors are Moloney mouse leukemia virus (MoMuLV) and HIV vectors. In some embodiments, a suitable vector contains an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction sites, and one or more selectable markers. (See, for example, WO 01/96584; WO 01/29058; and US Pat. No. 6,326,193).

You can use additional regulatory elements, such as enhancers that modulate the frequency of transcription initiation. A promoter may be an element that is naturally associated with a gene or nucleic acid sequence, which can be obtained by isolating 5' non-coding sequences located before the coding segment and/or exon. Such a promoter may refer to an "endogenous" promoter. Likewise, an enhancer may be an enhancer naturally associated with a nucleic acid sequence located upstream or downstream of that sequence. Alternatively, certain advantages are obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the nucleic acid sequence in its natural environment. Recombinant or heterologous enhancer also refers to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other prokaryotic, viral or eukaryotic cells, and promoters or enhancers not found in nature, for example, containing different elements of different transcriptional regulatory regions, and /or mutations that change expression. In addition to obtaining promoter and enhancer nucleic acid sequences synthetically, sequences can be obtained using recombinant cloning and/or nucleic acid amplification technology, including PCR, for the compositions disclosed herein (U.S. Patent No. 4,683,202, U.S. Patent No. 5,928,906). In addition, it is believed that control sequences can also be used for direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like.

Of course, it is important to use a promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. Molecular biologists generally know how to use promoters, enhancers, and combinations of cell types to express a protein, see, for example, Sambrook et al. (2012). The promoters used can be constitutive, tissue-specific, inducible and/or suitable under appropriate conditions for directing high-level expression of the introduced DNA segment, which is an advantage in large-scale production of recombinant proteins and their fragments.

An example of a promoter is the cytomegalovirus (CMV) immediate early promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of providing high-level expression of any polynucleotide sequence operably linked to it. However, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40 early promoter (SV40), the mouse mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the virus promoter Moloney, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and muscle creatine promoter. In addition, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter in the invention provides a molecular "switch" capable of "turning on" expression of the polynucleotide sequence to which it is operably linked when such expression is desired, or "turning off" expression when expression is undesired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter. In addition, the invention includes the use of a tissue-specific or cell-type-specific promoter, which is a promoter that is active only in the desired tissue or cell. Tissue-specific promoters are well known in the art and include, but are not limited to, the HER-2 promoter and PSA-related promoter sequences.

To evaluate the expression of nucleic acids, the expression vector to be introduced into a cell may also contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells that are transfected or infected with viral vectors. In other embodiments, the selectable marker may be carried in a single nucleic acid and used in a co-transfection procedure. Both selectable marker genes and reporter genes can be flanked by appropriate regulatory sequences to allow expression in host cells. Suitable selectable marker genes are known in the art and include, for example, antibiotic resistance genes such as neo and the like.

Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. Reporter genes that encode easily analyzed proteins are well known in the art. In general, a reporter gene is a gene that is absent or not expressed by the host organism or tissue and which encodes a protein whose expression exhibits some easily detectable property, such as enzymatic activity. The expression of the reporter gene is analyzed at the appropriate time after the introduction of the DNA into the recipient cells.

Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or a green fluorescent protein gene (see, for example, Ui-Tei et al., 2000 FEBS Lett., 479: 79-82). Suitable expression systems are well known and can be obtained using well known techniques or obtained from commercial sources. In general, a construct with a minimal 5' flanking region showing the highest level of reporter gene expression is identified as a promoter. Such promoter regions can be linked to a reporter gene and used to evaluate agents for their ability to modulate promoter-driven transcription.

Methods for introducing and expressing nucleic acids in a cell are known in the art. In the context of an expression vector, the vector can be easily introduced into a host cell, such as a mammalian, bacterial, yeast or insect cell, by any method known in the art. For example, the expression vector can be introduced into the host cell by physical, chemical, or biological means.

Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, laser poration, and the like. Methods for obtaining cells containing vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2012) and Ausubel et al. (1999).

Biological methods for introducing a nucleic acid of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, US Pat. Nos. 5,350,674 and 5,585,362.

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

Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose the cell to the nucleic acid of the present invention, various assays can be performed to confirm the presence of a recombinant DNA sequence in a host cell. Such assays include, for example, "molecular, biological" assays well known to those skilled in the art such as Southern blot and Northern blot, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, for example, by immunological methods (ELISA and Western blotting) or assays described herein, to identify agents falling within the scope of the invention.

Sets

The invention also includes a kit containing an anti-C5a antibody or combinations thereof according to the invention and instructional material that describes, for example, the administration of an anti-C5a antibody or combinations thereof to an individual as a therapeutic treatment or use in addition to treatment, as described elsewhere in this document. In one embodiment, the kit further comprises an (optionally sterile) pharmaceutically acceptable carrier suitable for dissolving or suspending a therapeutic composition containing an anti-C5a antibody or combinations thereof of the invention, for example, prior to administration of the antibody to an individual. Optionally, the kit includes an applicator for administering the antibody.

Experimental examples

The invention will now be described with reference to the following examples. These examples are provided for purposes of illustration only, and the invention should in no way be construed as being limited to the examples, but rather should be construed as covering any and all variations that become apparent from reading the inventive concept presented herein.

Without further description, it is believed that one skilled in the art, using the foregoing description and the following illustrative examples, can make and use the compounds of the present invention and practice the claimed methods. Therefore, the following working examples should not be construed as limiting the rest of the disclosure in any way.

Example 1

The complement system is part of innate immunity, which plays a key role in host defense. However, activated complement also has the potential to cause significant tissue damage and destruction, and dysregulation of complement activity has been found to be associated with a number of rare and common diseases such as paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome, rheumatoid arthritis, age-related macular degeneration, etc. Thus, anti-complement therapy is a promising way to treat these human disorders. The C5 component of the complement system is a critical protein in the terminal complement pathway and is a precursor protein in the generation of the potent pro-inflammatory mediator C5a as well as the cytolytic membrane attack complex (MAC), C5b-9.

A function-blocking, anti-human C5a monoclonal antibody (7A12) has been developed and described herein. This mAb blocks C5a-mediated activity but does not block MAC activity (as determined by hemolytic analysis).

The methods and materials used in this example will now be described.

Western blotting

Purified human C5 protein (1 μg) was boiled in sample buffer and loaded onto 6% SDS-PAGE gels under non-reducing or reducing conditions. Proteins were transferred to PVDF membranes which were probed with 10 μg/ml primary antibody (7A12 mAb or control anti-C5 mAb) for 1 h followed by detection using HRP-conjugated anti-mouse IgG (1:4000, Bio-Rad).

Human C5, C5a and mAb binding assay

Microtiter polystyrene plates were coated with purified human C5 or C5a (50 ng/well, Hycult) in PBS at 37°C for 1 h. After aspiration of the C5 or C5a solution, the wells were blocked with PBS containing 1% BSA in PBS at room temperature for 1 hour. Uncoated C5 or C5a wells served as background controls. Varying concentrations of 7A12 mAb or control anti-C5 mAb or chimeric 7A12 were added to wells at 50 μl/well in blocking solution. After incubation for 1 hour at room temperature, the wells were thoroughly washed with PBST. Human C5 or C5a bound mAbs were detected by adding HRP-conjugated anti-mouse IgG at a 1:4000 dilution in blocking solution, which was left to incubate for 1 hour at room temperature. After washing with PBST, the plate was developed with HRP substrate for 6-10 min. The reaction was stopped by adding 2 N H ₂ SO ₄ solution and the plate was analyzed at 450 nm on a microplate reader.

Mouse C5a and mAb binding assay

Microtiter polystyrene plates were coated with purified mouse C5a (50 ng/well, Hycult) in PBS at 37°C for 1 h. After aspiration of the C5a solution, the wells were blocked with PBS containing 1% BSA in PBS at room temperature for 1 h. uncoated C5a served as background control. Various concentrations of mAb 7A12 or control anti-C5a mAb or chimeric 7A12 were added to the wells, 50 μl/well in blocking solution. After incubation for 1 hour at room temperature, the wells were thoroughly washed with PBST. Mouse C5a bound mAbs were detected by adding HRP-conjugated anti-mouse IgG 1:4000 diluted 1:4000 in blocking solution, which was left to incubate for 1 hour at room temperature. After washing with PBST, the plate was developed with HRP substrate for 6-10 min. The reaction was stopped by adding 2 N H ₂ SO ₄ solution and the plate was analyzed at 450 nm on a microplate reader.

Production of antibodies against human C5a

Female B10.D2/oSnJ mice (Stock # 000461, Jackson Nursery) were immunized with 30 μg of purified human C5 (# A120, Complement Technology, Inc) emulsified with an adjuvant. On day 14, mice were immunized again with 30 μg of purified human C5 emulsified with adjuvant. Mice were injected with 33 μg of purified human C5 three times before confluence. Then the mice were sacrificed by dislocation of the cervical vertebrae and the spleen was isolated for preparation of a suspension of individual cells by mechanical destruction. The spleen cell suspension was washed once with serum-free hybridoma medium (HYB-SFM) (Invitrogen) + 10% FBS medium and cells were counted and mixed with X63-Ag8.653 myeloma cell line (ATCC) at a ratio of 2:1. The cell mixture was again washed with HYB-SFM medium and a cell pellet was obtained by centrifugation (1000 rpm, × 5 min). The cell pellet was gently disturbed and loosened, and then cell fusion was induced by slowly adding polyethylene glycol (PEG 1500) (1.5 ml PEG per 3×10 ⁸ cells). The cells were left for 1 min at 37°C and then 20 ml of HYB-SFM medium was added to the cells over 3 min (1 ml in the first minute, 3 ml in the second minute and 16 ml in the third minute). The mixture was centrifuged at 1000 rpm for 5 min and the cells were plated in 24-well plates in HAT medium (10 ml HAT [Sigma H0262], 5 ml Pen/Strep, 500 µl gentamicin and 10% FBS in 500 ml HYB- SFM). After 2 weeks, supernatants from wells with visible colonies were selected for reactivity screening with purified human C5 by ELISA. Positive clones were harvested and plated in 96-well plates by serial dilution to obtain single clones after a second round of ELISA screening. Positive clones were expanded in HT medium (10 ml HT, 5 ml Pen/Strep, 500 μl gentamicin and 10% FBS in 500 ml HYB-SFM medium). Before collecting antibodies, hybridoma cells were transferred to serum-free medium (SF-SFM) for 2-3 days. The cell culture medium was collected for mAb purification by protein G affinity chromatography.

mAb cloning

To clone the 7A12 cDNA, the total RNA fraction was isolated from hybridoma cells using the TRizol reagent (Sigma). First strand cDNA was synthesized by reverse transcription using primer Oligo(dT). The following primers were used to amplify the heavy chain cDNA (for IgG1, IgG2a/b) in PCR reactions: 5'-GAG GTG A AGCTG GTG G AG(T/A)C(T/A) GG-3' (SEQ ID NO: 11) and 5'-GGGGCCAGTGGATAGAC-3' (SEQ ID NO: 12). The following primers were used for light chain amplification: a mixture of 4 forward primers: 5'-CCAGTTCCGAGCTCCAGATGACCCAGACTCCA-3' (SEQ ID NO: 70); 5'-CCAGTTCCGAGCTCGTGCTCACCAGTCTCCA-3' (SEQ ID NO: 71); 5'-CCAGTTCCGAGCTCCAGATGACCCAGTCTCCA-3' (SEQ ID NO: 72); 5'-CC AGTTC CG A G CTC GTG ATG AC AC AGTCTCC A -3' (SEQ ID NO: 13); reverse primer: 5'-GTTGGTGCAGCATCAGC-3' (SEQ ID NO: 14). The PCR amplicons were cloned into the pCR TOPO TA 2.1 vector (Invitrogen) and sequenced. The signal peptide (leader) sequence of the mAb was obtained using the 5'-RACE method with a kit (GeneRacer) from Invitrogen. Whole cDNA of the variable region was amplified using specific primers determined using the 5'-RACE method and the original sequencing data.

Hemolysis analysis

Antibody-sensitized sheep erythrocytes (1×10 ⁷ cells, Complement Technology, Inc.) were incubated at 37° C. for 20 min with 50% NHS (Complement Technology, Inc.) in gelatin-veronal buffer (GVB2+, Sigma). Prior to addition to sheep erythrocytes, NHS was pre-incubated with 7A12 mAb or control anti-C5 mAb for 1 h at 4°C. The lysis reaction was stopped by adding an ice-cold 40 mM EDTA solution in PBS. The incubation mixtures were centrifuged for 5 min at 1500 rpm and the supernatant was collected and measured at OD405 nm. Samples without NHS addition or with EDTA were used as negative lysis controls, and a sample of sheep erythrocytes completely lysed with distilled water was used as a positive control (100% lysis) against which the % lysis in other samples was normalized.

Migration analysis in the Transwell system

U937 cells stably transfected with the C5a receptor (U937-C5aR cells) were plated (at 1 x 10 ⁶ cells per well in chemotaxis assay buffer) in the upper chambers of Transwell 24-well inserts with a 3.0 μm polycarbonate membrane filter (Corning). Lower Boyden chambers were loaded with 10 nM recombinant human complement C5a pretreated with antibody (1 μg/mL or 10 μg/mL) in chemotaxis assay buffer. After incubation for 3 hours at 37°C, migrated cells in the lower chambers were collected and counted using a Coulter counter (Beckman Coulter). (Chemotaxis assay buffer: RPMI 1640 medium with 0.5% BSA).

Calcium mobilization assay

1x10 ⁷ cells were washed twice with HEPES buffer for calcium mobilization assay (25 mM HEPES, 119 mM NaCl, 5 mM KCl, 5.6 mM glucose, 0.4 mM MgCl ₂ and 1 mM CaCl ₂ ) containing 1 mg/ml BSA, and then incubated with 1 μM indo-1-acetoxymethyl ether (Anaspec, Inc, Fremont, CA, USA) at room temperature for 30 minutes. The cells were then washed twice and resuspended in 1.3 ml of the same buffer. Human C5a and antibody were mixed to a final concentration of 10 nM and 50 μg/ml, respectively, for 10 minutes at room temperature. Finally, intercellular Ca ²⁺ measurement was performed using an Infinite F200 multi-mode microplate reader (Tecan Systems Inc, San Jose, CA, USA) with an excitation wavelength of 360 nm and an emission wavelength of 415 nm. The mixture was added 300 s after the start of the measurement.

The results of this example are now described.

As shown in FIG. 1, 7A12 has been shown to bind to human C5. The reactivity of 7A12 and 2G1, a control anti-C5 mAb, with intact human C5 was assessed by ELISA as shown in FIG. The ELISA plate was coated with purified human C5. After incubation with serial dilutions of 7A12 or control anti-C5 mAb, bound mAb was detected with HRP-conjugated rabbit anti-mouse IgG. 7A12 and control anti-C5a mAb showed high reactivity with human C5. Using Western blotting, the 7A12 mAb and control anti-C5a mAb were found to recognize purified human C5 protein under non-reducing and reducing conditions, respectively, as shown in FIG. 1b. The observed 190 kDa 7A12 reactive band represents the complete C5 protein, while the 115 kDa band represents the C5 α chain.

In contrast to the control anti-C5 mAb 2G1, which inhibited erythrocyte (RBC) lysis, 7A12 was shown to be inactive in the hemolysis assay as shown in FIG. 2. Antibody-sensitized sheep erythrocytes were incubated with normal human serum (NHS) containing serial dilutions of 7A12 or control anti-C5 mAb at 37° C. for 1 hour. RBC lysis was determined by measuring absorbance at OD405 nm. As expected, the control anti-C5 mAb inhibited 50% NHS-mediated lysis of sheep erythrocytes in a dose dependent manner. On the other hand, at doses of 0.975 to 120 μg/ml, mAb 7A12 did not show inhibition of 50% NHS-mediated lysis of sheep erythrocytes.

In contrast to the control anti-C5 mAb 2G1, which does not bind to C5a, 7A12 has been shown to bind to human C5a. The ELISA plate was coated with human C5a or mouse C5a. After incubation with serial dilutions of 7A12 or control anti-C5 mAb, bound mAb was detected with HRP-conjugated anti-mouse IgG. In FIG. 3A shows that mAb 7A12 showed high reactivity to human C5a. Since mAb 7A12 reacts with complete C5 protein (as shown in Figure 1), it can be concluded that mAb 7A12 binds to both the C5a fragment of native human C5 and free human C5a. On the other hand, no control anti-C5 mAb was found to bind to human C5a, as shown in Figure 3B. The binding of mAb 7A12 to C5a was found to be specific to human C5a with little binding to mouse C5a.

Binding affinity of mAb 7A12 for human C5 and C5a. Purified human C5 or C5a was bound to the CM4 chip using the amine coupling method. Biacore analysis was performed on a Biacore-2000 instrument. Between each binding, the chip was regenerated using 50 mM NaOH. mAb 7A12 binds to human C5 as shown in FIG. 4A and human C5a as shown in FIG. 4B with similar affinity.

The results shown in Figure 5 demonstrate that the 7A12 mAb, but not the control anti-C5 mAb 2G1, inhibits C5a-mediated neutrophil migration. Human C5a at 10 nM was used to induce chemotaxis of a human monocytic cell line, U937, transfected with the human C5a receptor. Cells were placed in the upper chamber of the Transwell system in the presence of mAb 7A12 or control anti-C5 mAb 2G1, and cell migration was quantified by cell count in the lower chamber. mAb 7A12 showed complete inhibition of C5a-induced chemotaxis at a concentration of 10 μg/ml, while the control anti-C5 mAb 2G1 could not block C5a-induced chemotaxis.

The results shown in FIG. 6 demonstrate that mAb 7A12, but not the control anti-C5 mAb 2G1, inhibits C5a-induced intracellular calcium mobilization in U937 cells. Calcium mobilization was absent in U937 cells expressing the human C5a receptor (U937-C5aR) in the absence of human C5a stimulation, as shown in FIG. 6A. C5a treatment resulted in a transient calcium influx in U937-C5aR cells as shown in FIG. 6B which could be inhibited by pre-incubation with mAb 7A12 (50 μg/ml) as shown in FIG. 6C but not with the control anti-C5 mAb 2G1 (50 μg/ml) as shown in FIG. 6d. The arrows refer to the time point when the mixture of C5a and antibodies was added to the cell suspension.

The disclosure of each patent, patent application and publication cited herein is incorporated by reference in its entirety.

Although the present invention has been disclosed with reference to specific embodiments, it is obvious that other embodiments and variations of the present invention may be developed by others skilled in the art without departing from the spirit and scope of the invention. The appended claims are intended to include all such embodiments and equivalents.

--->

SEQUENCE LIST

<110> JE TRUSTEE OF JE UNIVERSITY OF PENNSYLVANIA

SUN, Wenchao

SATO, Sayaka

MIWA, Takashi

GULLIPALLI, Damodar

<120> ANTI-C5a ANTIBODIES AND THEIR USES

<130> 046483-6157-00-WO.607245

<150> US 62/475,573

<151> 2017-03-23

<160> 17

<170>PatentIn version 3.5

<210> 1

<211> 499

<212> DNA

<213> Mus musculus

<400> 1

catggactga aggagtagaa atcttcagtc cctgaacaca ctgactgtaa caatggaatg 60

gagctgggtc tctctcttct tcctgtcagt aactacaggt gtccactccc aggttcagct 120

gcaacagtct gacgctgagt tggtgaaacc tggagcttca gtgaagattt cctgcaaggt 180

ttctggctac accttcactg accatattat tcactggatg aaccagaggc ctgaacaggg 240

cctggaatgg attggatata tttatcctag agatggtaat actaactaca atgaaaactt 300

caagggcaag gccacattga ctgcagacaa atcctccagc acagcctaca tgcagctcaa 360

cagcctgaca tctgaggact ctgcagtcta tttctgtgca agagaaagga acttggaata 420

ctttgactac tggggccaag gcaccactct cacagtctcc tcagccaaaa cgacaccccc 480

atctgtctat ccactggcc 499

<210> 2

<211> 149

<212> PROTEIN

<213> Mus musculus

<400> 2

Met Glu Trp Ser Trp Val Ser Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys

20 25 30

Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe

35 40 45

Thr Asp His Ile Ile His Trp Met Asn Gln Arg Pro Glu Gln Gly Leu

50 55 60

Glu Trp Ile Gly Tyr Ile Tyr Pro Arg Asp Gly Asn Thr Asn Tyr Asn

65 70 75 80

Glu Asn Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Phe Cys Ala Arg Glu Arg Asn Leu Glu Tyr Phe Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser

130 135 140

Val Tyr Pro Leu Ala

145

<210> 3

<211> 9

<212> PROTEIN

<213> Mus musculus

<400> 3

Tyr Thr Phe Thr Asp His Ile Ile His

fifteen

<210> 4

<211> 14

<212> PROTEIN

<213> Mus musculus

<400> 4

Trp Ile Gly Tyr Ile Tyr Pro Arg Asp Gly Asn Thr Asn Tyr

1 5 10

<210> 5

<211> 8

<212> PROTEIN

<213> Mus musculus

<400> 5

Arg Asn Leu Glu Tyr Phe Asp Tyr

fifteen

<210> 6

<211> 459

<212> DNA

<213> Mus musculus

<400> 6

catggactga aggagtagaa aaagcatcct ctcatctagt tctcagagat ggagacagac 60

acaatcctgc tatgggtgct gctgctctgg gttccaggct ccactggtga cattgtgctg 120

acccaatctc cagcttcttt ggctgtgtct ctagggcaga gggccaccat ctcctgcaag 180

gccagccaaa gtgttgatta tgatggtgat aattatatga actggtacca acagaaacca 240

ggacagccac ccaaactcct catctatgct gcatccaatc tagattctgg gatcccagcc 300

aggtttagtg gcagtggggtc tgggacagac ttcaccctca acatccatcc tgtggaggaa 360

gaagatgctg caacctatta ttgtcagcaa agtaatgagg atccgtacac gttcggaggg 420

gggaccaagc tggaaataaa acgggctgat gctgcacca 459

<210> 7

<211> 137

<212> PROTEIN

<213> Mus musculus

<400> 7

Met Glu Thr Asp Thr Ile Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala

20 25 30

Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser

35 40 45

Val Asp Tyr Asp Gly Asp Asn Tyr Met Asn Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Asp Ser

65 70 75 80

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys

100 105 110

Gln Gln Ser Asn Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125

Glu Ile Lys Arg Ala Asp Ala Ala Pro

130 135

<210> 8

<211> 12

<212> PROTEIN

<213> Mus musculus

<400> 8

Gln Ser Val Asp Tyr Asp Gly Asp Asn Tyr Met Asn

1 5 10

<210> 9

<211> 11

<212> PROTEIN

<213> Mus musculus

<400> 9

Leu Leu Ile Tyr Ala Ala Ser Asn Leu Asp Ser

1 5 10

<210> 10

<211> 8

<212> PROTEIN

<213> Mus musculus

<400> 10

Gln Gln Ser Asn Glu Asp Pro Tyr

fifteen

<210> 11

<211> 23

<212> DNA

<213> Mus musculus

<400> 11

gaggtgaagc tggtggagtc tgg 23

<210> 12

<211> 17

<212> DNA

<213> Mus musculus

<400> 12

ggggccagtggatagac 17

<210> 13

<211> 32

<212> DNA

<213> Mus musculus

<400> 13

ccagttccga gctccagatg acccagactc ca 32

<210> 14

<211> 32

<212> DNA

<213> Mus musculus

<400> 14

ccagttccga gctcgtgctc acccagtctc ca 32

<210> 15

<211> 32

<212> DNA

<213> Mus musculus

<400> 15

ccagttccga gctccagatg acccagtctc ca 32

<210> 16

<211> 32

<212> DNA

<213> Mus musculus

<400> 16

ccagttccga gctcgtgatg acacagtctc ca 32

<210> 17

<211> 17

<212> DNA

<213> Mus musculus

<400> 17

gttggtgcag catcagc

<---

Claims (37)

1. An antibody that specifically binds to human C5a, where the antibody contains a heavy chain variable region (VH) and a light chain variable region (VL), where VH contains i) VH-CDR1 containing the amino acid sequence of SEQ ID NO: 3, or a variant thereof, where the variant retains the basic biological properties and contains conservative substitutions containing up to 3, 2 or 1 amino acid substitutions; ii) VH-CDR2 containing the amino acid sequence of SEQ ID NO: 4, or a variant thereof, where the variant retains the basic biological properties and contains conservative substitutions containing up to 3, 2 or 1 amino acid substitutions; and iii) VH-CDR3 containing the amino acid sequence of SEQ ID NO: 5, or a variant thereof, where the variant retains the basic biological properties and contains conservative substitutions containing up to 3, 2 or 1 amino acid substitutions; and where VL contains i) VL-CDR1 containing the amino acid sequence of SEQ ID NO: 8, or a variant thereof, where the variant retains the basic biological properties and contains conservative substitutions containing up to 3, 2 or 1 amino acid substitutions; ii) VL-CDR2 containing the amino acid sequence of SEQ ID NO: 9, or a variant thereof, where the variant retains the basic biological properties and contains conservative substitutions containing up to 3, 2 or 1 amino acid substitutions; and iii) a VL-CDR3 containing the amino acid sequence of SEQ ID NO: 10, or a variant thereof, wherein the variant retains basic biological properties and contains conservative substitutions of up to 3, 2, or 1 amino acid substitutions. 2. The antibody of claim 1, wherein the antibody is a chimeric antibody. 3. The antibody according to claim 1, where the antibody contains a CDR: VH-CDR1: SEQ ID NO: 3; VH-CDR2: SEQ ID NO: 4; VH-CDR3: SEQ ID NO: 5; VL-CDR1: SEQ ID NO: 8; VL-CDR2: SEQ ID NO: 9; and VL-CDR3: SEQ ID NO: 10. 4. The antibody according to claim 1, where the antibody contains a heavy chain containing the amino acid sequence of SEQ ID NO: 2, or a variant thereof, where the variant retains the main biological properties and contains conservative substitutions. 5. The antibody according to claim 1, where the antibody contains a light chain containing the amino acid sequence of SEQ ID NO: 7, or a variant thereof, where the variant retains the main biological properties and contains conservative substitutions. 6. The antibody according to claim 1, where the antibody contains a heavy chain containing the amino acid sequence of SEQ ID NO: 2, or a variant thereof, where the variant retains basic biological properties and contains conservative substitutions, and a light chain containing the amino acid sequence of SEQ ID NO: 7 , or a variant thereof, where the variant retains basic biological properties and contains conservative substitutions. 7. A method of treating a complement-mediated disease or disorder in an individual, comprising the step of administering to said individual an anti-C5a antibody according to any one of claims 1-6. 8. The method of claim 7, wherein the disease or disorder is at least selected from the group consisting of macular degeneration (MD), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, rheumatoid arthritis, lupus, ulcerative colitis, stroke , postoperative systemic inflammatory response syndrome, asthma, allergic asthma, chronic obstructive pulmonary disease (COPD), paroxysmal nocturnal hemoglobinuria (PNH), autoimmune hemolytic anemia (AIHA), Gaucher disease, myasthenia gravis, neuromyelitis optic nerve, 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 (including but not limited to: inflammation associated with cardiopulmonary bypass and dialysis renal isom), C3-glomerulopathy, membranous nephropathy, IgA-nephropathy, glomerulonephritis (including, but not limited to, antineutrophil cytoplasmic antibodies (ANCA)-associated glomerulonephritis, lupus nephritis and combinations thereof), ANCA-associated vasculitis induced by Shiga-toxin HUS , and antiphospholipid antibody-induced pregnancy loss, graft-versus-host disease (GVHD), or any combination thereof. 9. A method of reducing the activity of an individual's complement system, wherein the method comprises administering the antibody to the individual by administration selected from the group consisting of enteral administration, parenteral administration, and combinations thereof, and wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region ( VL), where VH contains i) VH-CDR1 containing the amino acid sequence of SEQ ID NO: 3, or a variant thereof, where the variant retains the basic biological properties and contains conservative substitutions containing up to 3, 2 or 1 amino acid substitutions; ii) VH-CDR2 containing the amino acid sequence of SEQ ID NO: 4, or a variant thereof, where the variant retains the basic biological properties and contains conservative substitutions containing up to 3, 2 or 1 amino acid substitutions; and iii) VH-CDR3 containing the amino acid sequence of SEQ ID NO: 5, or a variant thereof, where the variant retains the basic biological properties and contains conservative substitutions containing up to 3, 2 or 1 amino acid substitutions; and where VL contains i) VL-CDR1 containing the amino acid sequence of SEQ ID NO: 8, or a variant thereof, where the variant retains the basic biological properties and contains conservative substitutions containing up to 3, 2 or 1 amino acid substitutions; ii) VL-CDR2 containing the amino acid sequence of SEQ ID NO: 9, or a variant thereof, where the variant retains the basic biological properties and contains conservative substitutions containing up to 3, 2 or 1 amino acid substitutions; and iii) a VL-CDR3 containing the amino acid sequence of SEQ ID NO: 10, or a variant thereof, wherein the variant retains basic biological properties and contains conservative substitutions of up to 3, 2, or 1 amino acid substitutions. 10. The method of claim 9 wherein the antibody is selected from the group consisting of Fab, Fab', F(ab) ₂ , F(ab') ₂ , scFv, and combinations thereof. 11. The antibody of claim 1, wherein the antibody has a heavy chain variable region (VH) with an amino acid sequence that is greater than 90% identical to the sequence of SEQ ID NO: 2. 12. The antibody of claim 11, wherein the antibody is selected from the group consisting of Fab, Fab', F(ab) ₂ , F(ab') ₂ , scFv, and combinations thereof. 13. An antibody according to claim 1, wherein the antibody has a light chain variable region (VL) with an amino acid sequence that is more than 90% identical to the sequence of SEQ ID NO: 7, where the VL region retains basic biological properties and contains conservative substitutions. 14. The antibody of claim 13, wherein the antibody is selected from the group consisting of Fab, Fab', F(ab) ₂ , F(ab') ₂ , scFv, and combinations thereof. 15. The antibody of claim 1, wherein the antibody has a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH region has an amino acid sequence that is greater than 90% identical to SEQ ID NO: 2, wherein the VH region retains basic biological properties and contains conservative substitutions, and where the VL region has an amino acid sequence that is more than 90% identical to the sequence of SEQ ID NO: 7, where the VL region retains basic biological properties and contains conservative substitutions. 16. The antibody of claim 15, wherein the antibody is selected from the group consisting of Fab, Fab', F(ab) ₂ , F(ab') ₂ , scFv, and combinations thereof. 17. A cell for producing an antibody, comprising an antibody according to at least one of claims 1-6 and 11-16, wherein the cell contains a nucleic acid encoding an antibody according to any one of claims 1-6 and 11-16. 18. A cell according to claim 17, wherein the cell produces an antibody according to at least one of claims 1-6 and 11-16. 19. The cell of claim 17, wherein the cell is a hybridoma.

Images