HK1157663B - Compositions and methods for antibodies targeting complement protein c5 - Google Patents

Description

Compositions and methods of antibodies targeting complement protein C5

Introduction to the invention

The present invention relates to antibodies and compositions targeting complement protein C5 and methods of use thereof.

Background

The normal role of complement is in host defense, and complement is part of the innate immune system. Complement protects against bacterial infection, links adaptive and innate immunity, and handles immune complexes and products of inflammatory injury.

Defense functions are accomplished by biologically active products produced during complement activation that opsonize pathogens, promote inflammation, or lyse susceptibility targets (Marzari et al, Eur JImmunol 32: 2773-. The complement system consists of approximately 25-30 plasma proteins that play a role in the immune system. The complement cascade is activated by at least three major pathways. The classical pathway is usually activated by immune complexes, the alternative pathway can be activated by unprotected cell surfaces, and the mannose-binding lectin (MBL) pathway is initiated by MBL binding to cell-surface carbohydrates (Trendelenburg, Swiss Med Wkly 137: 413-417 (2007)).

All three pathways lead to cleavage of C5 by the C5 convertase. The result of this cleavage is the release of a potent inflammatory molecule, fragment C5a, and C5b that initiates the Membrane Attack Complex (MAC). Complement products, once released, cannot differentiate between exogenous and self targets and, if not tightly regulated, often cause extensive damage to innocent cells and tissues in clinical settings associated with unrestricted complement activation (Marzari et al, 2002).

C5 is expressed intracellularly as a single pre-C5 peptide of 1676 amino acids consisting of an 18 residue signal sequence located between the mature N-terminal β chain and the C-terminal α chain and an Arg-rich linker sequence (RPRR). Mature C5 has a molecular weight of approximately 190kDa and consists of two polypeptide chains linked by disulfide bonds (α, 115 kDa; β,75 kDa). The C5 convertase cleaves C5 between residues 74 and 75 of the alpha chain, releasing a 74 amino acid C5a peptide and a C5b fragment that is subsequently incorporated into the Membrane Attack Complex (MAC).

Macular degeneration is a medical condition that is found primarily in the elderly, where the center of the lining of the eye, known as the macula area of the retina, suffers thinning, atrophy, and in some cases, bleeding. This can result in loss of central vision, which makes it impossible to see fine details, read or identify faces. The pathogenesis of new choroidal vascularisation is poorly understood, but factors such as inflammation, ischemia, and local production of angiogenic factors are thought to be important.

Although therapeutic options currently exist for treating diseases and conditions associated with the classical or alternative complement pathways, particularly AMD, there remains a need to find specific targets that produce effective and well-tolerated treatments.

Summary of The Invention

The present invention provides isolated complement C5-binding molecules (e.g., C5-binding antibodies or antigen-binding fragments thereof), pharmaceutical compositions comprising such molecules, methods of making such molecules and compositions, and methods of use thereof.

In some embodiments, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds C5 protein, wherein the antibody has at least 1x10⁷M^-1、10⁸M^-1、10⁹M^-1、10¹⁰M^-1Or 10¹¹M^-1Affinity constant (K) of_A)。

In some embodiments, the invention provides an isolated antibody, or antigen-binding fragment thereof, that specifically binds C5 protein and has an IC of about 20pM to about 200pM as determined by an in vitro hemolytic assay₅₀The domain inhibits the alternative complement pathway.

In some embodiments, the invention provides an isolated antibody, or antigen-binding fragment thereof, that specifically binds to C5 protein and cross-competes with an antibody described in table 1 below. In some embodiments, the invention provides an isolated antibody or antigen-binding fragment thereof that binds to the same epitope of C5 protein as an antibody described in table 1 below.

In some embodiments, the antibodies of the invention are isolated monoclonal antibodies that specifically bind to C5 protein. In some embodiments, the antibodies of the invention are isolated human or humanized monoclonal antibodies that specifically bind to C5 protein. In some embodiments, the antibodies of the invention are isolated chimeric antibodies that specifically bind to C5 protein. In some embodiments, the antibodies of the invention comprise a human heavy chain constant region and a human light chain constant region.

In some embodiments, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds C5 protein, wherein the antibody is a single chain antibody. In some embodiments, the antibodies of the invention are Fab fragments. In some embodiments, the antibody of the invention is an scFv.

In some embodiments, the invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds human C5 and cynomolgus C5. In some embodiments, the antibodies of the invention are of the IgG isotype.

In some embodiments, the invention provides an isolated antibody or antigen-binding fragment thereof comprising a framework in which amino acids have been replaced with antibody frameworks from the respective human VH or VL germline sequences.

In some embodiments, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds C5 protein, wherein the antibody comprises an amino acid sequence that specifically binds to SEQ ID NO: 1. 2, 3,4, 5,6, 17, 18, 19, 20, 21, 22, 33, 34, 35, 36, 37, 38, 49, 50, 61, 62, 63, 64, 65, 66, 77, 78, 89, 95, 101, 107, 113, 119, 120, 131, 132, 133, 134, 135, 136, 145, 146, 147, 148, 149, 150, 159, 160, 161, 162, 163, 164, 173, 174, 175, 176, 177, 178, 195, 196, 197, 198, 199, 200, 209, 226, 235, 236, 237, 238, 239, or 240 has at least 90%, 95%, 97%, 98%, or at least 99% sequence identity.

In some embodiments, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds C5 protein, wherein the antibody comprises an amino acid sequence that specifically binds to SEQ ID NO: 1. 2, 3, 17, 18, 19, 33, 34, 35, 49, 61, 62, 63, 77, 95, 107, 113, 119, 132, 131, 133, 145, 146, 147, 159, 160, 161, 173, 174, 175, 195, 196, 197, 226, 235, 236, or 237.

In some embodiments, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds C5 protein, wherein the antibody comprises an amino acid sequence that specifically binds to SEQ ID NO: 4.5, 6, 20, 21, 22, 36, 37, 38, 50, 64, 65, 66, 78, 89, 101, 120, 134, 135, 136, 148, 149, 150, 162, 163, 164, 176, 177, 178, 198, 199, 200, 209, 238, 239, or 240.

In some embodiments, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds C5 protein, wherein the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1. heavy chain CDR1 of 17, 33, 61, 131, 145, 159, 173, 195, and 235; selected from the group consisting of SEQ ID NOs: 2. 18, 34, 49, 62, 77, 95, 107, 113, 119, 132, 146, 160, 174, 196, 226, and 236; and selected from the group consisting of SEQ ID NOs: 3. 19, 35, 63, 133, 147, 161, 175, 197 and 237. In some embodiments, such antibodies or antigen-binding fragments thereof further comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 4. 20, 36, 64, 134, 148, 162, 176, 198, and 238 light chain CDR 1; selected from the group consisting of SEQ ID NOs: 5. 21, 37, 65, 135, 149, 163, 177, 199, and 239, light chain CDR 2; and selected from the group consisting of SEQ ID NOs: 6. 22, 38, 50, 66, 78, 89, 101, 120, 136, 150, 164, 178, 200, 209, and 240.

In some embodiments, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds C5 protein, wherein the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4. 20, 36, 64, 134, 148, 162, 176, 198, and 238 light chain CDR 1; selected from the group consisting of SEQ ID NOs: 5. 21, 37, 65, 135, 149, 163, 177, 199, and 239, light chain CDR 2; and selected from the group consisting of SEQ ID NOs: 6. 22, 38, 50, 66, 78, 89, 101, 120, 136, 150, 164, 178, 200, 209, and 240.

In some embodiments, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds C5 protein, wherein the antibody comprises an amino acid sequence that specifically binds to SEQ ID NO: 7. 23, 39, 51, 67, 79, 96, 108, 114, 121, 137, 151, 165, 179, 187, 201, 210, 218, 227, 241, 253, 257, 273, 277 or 281 a heavy chain variable region having at least 90%, 95%, 97%, 98% or at least 99% sequence identity. In some embodiments, such antibodies or antigen-binding fragments thereof further comprise a heavy chain variable region that differs from SEQ ID NO: 8. 24, 40, 52, 68, 80, 90, 102, 122, 138, 152, 166, 180, 188, 202, 211, 219, 228, 242, 261, 265, 269, 285, and 289 have at least 90%, 95%, 97%, 98%, or at least 99% sequence identity.

In some embodiments, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds C5 protein, wherein the antibody comprises an amino acid sequence that specifically binds to SEQ ID NO: 8. 24, 40, 52, 68, 80, 90, 102, 122, 138, 152, 166, 180, 188, 202, 211, 219, 228, 242, 261, 265, 269, 285, and 289 have at least 90%, 95%, 97%, 98%, or at least 99% sequence identity.

In some embodiments, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds C5 protein, wherein the antibody comprises an amino acid sequence that specifically binds to SEQ ID NO: 9. 25, 41, 53, 69, 81, 97, 109, 115, 123, 139, 153, 167, 181, 189, 203, 212, 220, 229, 243, 249, 254, 258, 274, 278 or 282 heavy chains having at least 90%, 95%, 97%, 98% or at least 99% sequence identity. In some embodiments, such antibodies further comprise a heavy chain variable region identical to SEQ ID NO: 10. 26, 42, 54, 70, 82,91, 103, 124, 140, 154, 168, 182, 190, 204, 213, 221, 230, 244, 251, 262, 266, 270, 286, or 290, a light chain having at least 90%, 95%, 97%, 98%, or at least 99% sequence identity.

In some embodiments, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds C5 protein, wherein the antibody comprises an amino acid sequence that specifically binds to SEQ ID NO: 10. 26, 42, 54, 70, 82,91, 103, 124, 140, 154, 168, 182, 190, 204, 213, 221, 230, 244, 251, 262, 266, 270, 286, or 290, a light chain having at least 90%, 95%, 97%, 98%, or at least 99% sequence identity.

The present invention also encompasses pharmaceutical compositions comprising one or more C5-binding molecules of the present invention (e.g., a C5 binding antibody or antigen-binding fragment thereof) and a pharmaceutically acceptable carrier.

In some embodiments, the present invention provides a nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising a nucleotide sequence identical to SEQ ID NO: 7. 23, 39, 51, 67, 79, 96, 108, 114, 121, 137, 151, 165, 179, 187, 201, 210, 218, 227, 241, 253, 257, 273, 277 or 281 a heavy chain variable region having at least 90%, 95%, 97%, 98% or at least 99% sequence identity.

In some embodiments, the present invention provides a nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising a nucleotide sequence identical to SEQ ID NO: 8. 24, 40, 52, 68, 80, 90, 102, 122, 138, 152, 166, 180, 188, 202, 211, 219, 228, 242, 261, 265, 269, 285, and 289 have at least 90%, 95%, 97%, 98%, or at least 99% sequence identity.

The invention also provides vectors and host cells comprising such nucleic acids. In one embodiment, the present invention provides an isolated host cell comprising (1) a recombinant DNA segment encoding a heavy chain of an antibody of the present invention, and (2) a second recombinant DNA segment encoding a light chain of an antibody of the present invention; wherein said DNA segments are each operably linked to a first and a second promoter and are capable of expression in said host cell. In another embodiment, the invention provides an isolated host cell comprising recombinant DNA segments encoding the heavy and light chains, respectively, of an antibody of the invention, wherein said DNA segments are operably linked to a promoter and are capable of expression in said host cell. In some embodiments, the host cell is a non-human mammalian cell line. In some embodiments, the antibody or antigen-binding fragment thereof is a human monoclonal antibody, or antigen-binding fragment thereof.

The invention also provides therapeutic or diagnostic methods of using the C5 binding molecules of the invention (e.g., C5 binding antibodies or antigen binding fragments thereof). In one embodiment, the invention provides a method of treating age-related macular degeneration comprising administering to a subject in need thereof an effective amount of a composition comprising an antibody or antigen-binding fragment thereof of the invention.

In another embodiment, the invention provides a method of treating a disease comprising administering to a subject in need thereof an effective amount of a composition comprising an antibody or antigen-binding fragment thereof of the invention, wherein the disease is asthma, arthritis, autoimmune heart disease, multiple sclerosis, inflammatory bowel disease, ischemia-reperfusion injury, barth's syndrome, hemodialysis, systemic lupus erythematosus, psoriasis, multiple sclerosis, transplantation, alzheimer's disease, glomerulonephritis, or MPGNII.

The invention also provides a method for treating Paroxysmal Nocturnal Hemoglobinuria (PNH), comprising administering to a subject in need thereof an effective amount of a composition comprising an antibody or antigen-binding fragment thereof of the invention.

The invention also provides a method of ameliorating a symptom associated with extracorporeal circulation comprising administering to a subject in need of treatment an effective amount of a composition comprising an antibody or antigen-binding fragment thereof of the invention.

3.1 definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "antibody" as used herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. A naturally occurring "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH 3. Each light chain comprises a light chain variable region (abbreviated VL) and a light chain constant region. The light chain constant region comprises a domain CL. The VH and VL regions can be further subdivided into hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with more conserved regions termed Framework Regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

As used herein, the term "antigen-binding portion" of an antibody refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., C5). The antigen binding function of an antibody can be performed by a fragment of the intact antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include Fab fragments, which are monovalent fragments consisting of the VL, VH, CL and CH1 domains; f (ab)₂A fragment which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; an Fd fragment consisting of the VH and CH1 domains; (ii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; single domain antibody (dAb) fragments (Ward et al, 1989Nature 341: 544-546) consisting of a VH domain; and an isolated Complementarity Determining Region (CDR).

Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by an artificial peptide linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al, 1988 Science 242: 423-. Such single chain antibodies include one or more "antigen-binding portions" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and screened for use in the same manner as intact antibodies.

Antigen binding moieties may also be incorporated into single domain antibodies, macroantibodies (maxibodes), minibodies (minibodies), intrabodies, diabodies, triabodies, tetrabodies, v-NARs and bis-scFvs (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). The antigen-binding portion of the antibody can be grafted into a scaffold based on a polypeptide such as fibronectin type III (Fn3) (see U.S. patent No. 6,703,199, which describes fibronectin polypeptide monoclonal antibodies).

The antigen-binding portion can be incorporated into a single chain molecule comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) that together with a complementary light chain polypeptide form a pair of antigen-binding regions (Zapata et al, 1995 Protein Eng.8 (10): 1057-1062; and U.S. Pat. No. 5,641,870).

As used herein, the term "affinity" refers to the strength of an antibody's interaction with an antigen at a single antigenic site. In each antigenic site, the variable region of the antibody "arm" interacts with the antigen at many sites through weak non-covalent forces; the more interactions, the stronger the affinity.

As used herein, the term "avidity" refers to an informative measure of the overall stability or strength of an antibody-antigen complex. It is controlled by three main factors: antibody epitope affinity; titers of both antigen and antibody; and structural arrangements of interacting moieties. Ultimately these factors determine the specificity of the antibody, i.e., the likelihood that a particular antigen will bind to a precise epitope.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a similar manner to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified by, for example, hydroxyproline, γ -carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon, a carboxyl group, an amino group, and an R group that is bound to a hydrogen, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

As used herein, the term "binding specificity" refers to the ability of each antibody binding site to interact with only one antigenic determinant. The binding site of the antibody is located in the Fab portion of the molecule and is constructed from hypervariable regions of the heavy and light chains. The binding affinity of an antibody is the strength of the reaction between a single epitope and a single binding site on the antibody. Which is the sum of the attractive and repulsive forces acting between the antigenic determinant and the antibody binding site.

Specific binding between two entities represents the equilibrium constant (K)_A) Is at least 1x10⁷M^-1、10⁸M^-1、10⁹M^-1、10¹⁰M^-1Or 10¹¹M^-1In combination with (1). The phrase "specifically (or selectively) binds" to an antibody refers to a binding reaction that determines the presence of a close relative antigen (e.g., human C5 or cynomolgus C5) in a heterogeneous population of proteins and other biological products. In addition to the equilibrium constants (KA) noted above, the C5-binding antibodies of the invention typically have an equilibrium constant of about 1x10^-2s^-1、1x10^-3s^-1、1x10^-4s^-1、1x10^-5s^-1Or a lower dissociation rate constant (Kd), and binds C5 with an affinity that is at least two times higher than its affinity for binding non-specific antigens (e.g., C3, C4, BSA). The phrases "antibody recognizing an antigen" and "antibody specific for an antigen" are used interchangeably herein with "antibody that specifically binds to an antigen".

The term "chimeric antibody" is an antibody molecule in which (a) the constant regions or portions thereof are altered, replaced, or exchanged such that the antigen binding site (variable region) is linked to a different or altered class of constant regions, effector function, and/or species, or an entirely different molecule that confers novel properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region or a portion thereof is altered, replaced or exchanged by a variable region having a different or altered antigenic specificity. For example, a mouse antibody can be modified by replacing the mouse constant region with a constant region from a human immunoglobulin. Chimeric antibodies can retain their specificity in recognizing antigens due to replacement with human constant regions, while having reduced antigenicity in humans compared to the original mouse antibody.

The terms "complement C5 protein" or "C5" are used interchangeably and refer to C5 protein in different species. For example, human C5 has the amino acid sequence as shown in SEQ ID NO: 296, macaque C5 has the sequence set forth in SEQ ID NO: 297 (Macaca fascicularis) (see table 1). Human C5 was available from Quidel (cat. number a 403). Macaque C5 can be produced as illustrated in the examples section below.

The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each position where the codon determines alanine, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variations. Each nucleic acid sequence herein that encodes a polypeptide also describes each possible silent variation of the nucleic acid. One skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, each silent variation of a nucleic acid encoding a polypeptide is implied in each such sequence.

With respect to polypeptide sequences, "conservatively modified variants" includes each substitution, deletion or addition to a polypeptide sequence which results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. The following 8 groups contain amino acids that are conservative substitutions for each other: 1) alanine (a), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M) (see, e.g., Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modification" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence.

The terms "cross-block", "cross-blocked", and the like are used interchangeably herein to refer to the ability of an antibody or other binding agent to interfere with the binding of the other antibody or binding agent to C5 in a standard competitive binding assay.

Standard competitive binding assays can be used to determine the ability or extent to which an antibody or other binding agent is able to interfere with the binding of another antibody or binding molecule to C5, and thus whether it can be referred to as a cross-blocking of the invention. One suitable assay includes the use of Biacore technology (e.g. by using a Biacore 3000 instrument (Biacore, Uppsala, Sweden)), which can measure the extent of interaction using surface plasmon resonance technology. Another assay for measuring cross-blocking uses an ELISA-based method.

The term "epitope" refers to a protein determinant capable of specifically binding to an antibody. Epitopes generally consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and generally have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes can be distinguished in that binding to the former is lost in the presence of denaturing solvents, while not the latter.

As used hereinThe term "high affinity" for an IgG antibody means having 10 to the target antigen^-8M or less, 10^-9M or less, 10^-10M, or 10^-11M or lower KD. However, "high affinity" binding may vary for other antibody isotypes. For example, "high affinity" binding for IgM isotype means having 10^-7M or less, or 10^-8M or lower KD.

As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human-derived sequences. Furthermore, if the antibody contains constant regions, the constant regions are also derived from such human sequences, e.g., human germline sequences, or mutated forms of human germline sequences. The human antibodies of the invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

The term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity having variable regions in which both the framework and CDR regions are derived from human sequences. In one embodiment, the human monoclonal antibodies are produced by a hybridoma that includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

A "humanized" antibody is one that retains the reactivity of a non-human antibody but is less immunogenic in humans. This can be achieved, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody (i.e., the constant regions and the framework portions of the variable regions) with their human counterparts. See, e.g., Morrison et al, proc.natl.acad.sci.usa, 81: 6851 6855, 1984; morrison and Oi, adv.immunol., 44: 65-92, 1988; verhoeyen et al, Science, 239: 1534 1536, 1988; padlan, molec. immun., 28: 489-498, 1991; and Padlan, molec. immun., 31: 169-217, 1994. Other examples of ergonomic techniques include, but are not limited to, Xoma technology disclosed in US 5,766,886.

The term "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if they have a specified percentage (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over the full-length sequence) of identical amino acid residues or nucleotides, as determined using the following sequence comparison algorithm or by manual alignment and visual inspection, compared and aligned for maximum correspondence over a comparison window or within a specified region. Optionally, identity exists over a region of at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region of at least 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

As used herein, a "comparison window" includes reference to a segment of any number of contiguous positions selected from 20 to 600, typically about 50 to about 200, and more typically about 100 to about 150, wherein a sequence can be compared to a reference sequence of the same number of contiguous positions after optimal alignment of the two sequences. Methods of sequence alignment for comparison are well known in the art. For example by Smith and Waterman (1970) adv.appl.math.2: 482c by Needleman and Wunsch, j.mol.biol.48: 443, 1970 by search of Pearson and Lipman, proc.nat' l.acad.sci.usa 85: 2444, 1988, by computerized implementation of these algorithms (Wisconsin Genetics Software Package, Genetics computer group, 575 Science Dr., Madison, GAP, BESTFIT, FASTA and TFASTA in Wis.), or by manual alignment and visual inspection (see, e.g., Brent et al, Current protocols in Molecular Biology, John Wiley & Sons, Inc. (ringer Bou editor, 2003)).

Two examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al, nuc. acids res.25: 3389 3402, 1977; and Altschul et al, j.mol.biol.215: 403-. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology information. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. Word hits are extended in both directions of each sequence as long as the cumulative alignment score can be increased. Cumulative scores were calculated for nucleotide sequences using the parameters M (reward score for a pair of matching residues; total > 0) and N (penalty score for mismatching residues; total < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. When: (ii) the cumulative alignment score falls off its highest achieved value by an amount of X; (ii) the cumulative alignment score reaches zero or less due to one or more negative scoring residue alignments accumulation; or the end of any sequence is reached, stopping the extension of word hits in each direction. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses default word length (W)11, expectation (E)10, M-5, N-4 and a comparison of the two strands. For amino acid sequences, the BLASTP program uses a default word length of 3, and an expectation (E)10 and BLOSUM62 scoring matrix (see Henikoff and Henikoff, proc. natl. acad. sci. usa 89: 10915, 1989) alignment of (B)50, expectation (E)10, M-5, N-4 and two-strand comparisons.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

The percentage identity between two amino acid sequences can also be determined using the algorithm of e.meyers and w.miller (comput.appl.biosci., 4: 11-17, 1988) which has been integrated into the ALIGN algorithm (version 2.0), using a PAM120 weighted residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, software packages that have been integrated into the GCG (as may be found inwww.gcg.comObtained above) of the GAP program (j.mol, biol.48: 444-453, 1970) algorithm using either the Blossom 62 matrix or the PAM250 matrix, together with the gap weights 16, 14, 12, 10, 8, 6, or 4 and the length weights 1,2, 3,4, 5, or 6 to determine percent identity between two amino acid sequences.

In addition to the percentage of sequence identity indicated above, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with an antibody raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, the polypeptide is typically substantially identical to a second polypeptide, e.g., where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.

The term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds C5 is substantially free of antibodies that specifically bind antigens other than C5). However, an isolated antibody that specifically binds C5 may be cross-reactive to other antigens. Furthermore, the isolated antibody may be substantially free of other cellular material and/or chemicals.

The term "isotype" refers to the class of antibodies (e.g., IgM, IgE, IgG such as IgG1 or IgG4) provided by the heavy chain constant region genes. Isoforms also include modified forms of one of these classes in which modifications have been made to alter Fc function, for example to enhance or diminish effector function or binding to Fc receptors.

As used herein, the term "Kassoc" or "Ka" is intended to refer to the on-rate of a particular antibody-antigen interaction, while as used herein, the term "Kdis" or "Kd" is intended to refer to the off-rate of a particular antibody-antigen interaction. As used herein, the term "K_D"is intended to mean the dissociation constant, which is obtained as the ratio of Kd to Ka (i.e., Kd/Ka) and expressed as molar concentration (M). The K of an antibody can be determined using methods well known in the art_DThe value is obtained. For determination of antibody K_DBy using surface plasmon resonance, or by using biosensor systems such asProvided is a system.

As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The term "nucleic acid" is used herein interchangeably with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. The term includes nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties to the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral-methylphosphonate, 2-O-methyl ribonucleotide, Peptide Nucleic Acid (PNA).

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. In particular, degenerate codon substitutions may be accomplished by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed base and/or deoxyinosine residues, as described in detail below (Batzer et al, Nucleic Acid Res.19: 5081, 1991; Ohtsuka et al, J.biol.chem.260: 2605-.

The term "operably linked" refers to the functional relationship of two or more polynucleotide (e.g., DNA) segments. Generally, it refers to the functional relationship between transcriptional regulatory sequences and transcribed sequences. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences operably linked to a transcribed sequence are in physical spatial proximity to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically adjacent or in close proximity to the coding sequence (an enhancer enhances its transcription).

As used herein, the term "optimized" means that the nucleotide sequence has been altered to encode an amino acid sequence using codons preferred in a production cell or organism, typically a eukaryotic cell, such as a pichia cell, a chinese hamster ovary Cell (CHO), or a human cell. The optimized nucleotide sequence is engineered to retain, completely or to the greatest extent possible, the amino acid sequence originally encoded by the starting nucleotide sequence (which is also referred to as the "parent" sequence). The optimized sequences herein have been engineered to have codons that are preferred in mammalian cells. However, optimized expression of these sequences in other eukaryotic or prokaryotic cells is also contemplated herein. The amino acid sequence encoded by the optimized nucleotide sequence is also referred to as optimized.

The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, conservatively modified variants thereof are also encompassed by a particular polypeptide sequence.

As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies isolated from animals transgenic or transchromosomes for human immunoglobulin genes (e.g., mice) or hybridomas prepared therefrom, antibodies isolated from host cells transformed to express human antibodies, such as antibodies isolated from transfectomas, antibodies isolated from recombinant, combinatorial human antibody libraries, and antibodies prepared, expressed, produced or isolated by any other means, including splicing all or portions of human immunoglobulin genes, sequences into other DNA sequences. Such recombinant human antibodies have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and, thus, the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, when derived from and related to human germline VH and VL sequences, may not naturally occur in the human antibody germline repertoire in vivo.

The term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. It is understood that such terms are not intended to refer to a particular subject cell, but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

The term "subject" includes both human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. The terms "patient" or "subject" are used interchangeably herein, except where indicated.

The term "treating" includes administering the composition or antibody to prevent or delay the onset of symptoms, complications, or biochemical indicators of a disease (e.g., AMD), to alleviate symptoms, or to arrest or inhibit further development of the disease, condition, or sign. Treatment may be prophylactic (to prevent or delay the onset of disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic inhibition or alleviation of symptoms following disease manifestation.

The term "vector" is intended to mean a polynucleotide molecule capable of transporting a polynucleotide which has been linked to another polynucleotide. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop to which additional DNA segments have been ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which the vector is introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" are used interchangeably, as plasmids are the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

Brief Description of Drawings

Figure 1 shows the variable region alignment of the selected antibody with its closest human germline sequence.

Figure 2 shows a hemolytic assay in which human C5 was titrated into human C5 depleted serum to determine C5 activity.

Figure 3 shows cynomolgus monkey serum titrated into human C5 depleted serum to determine optimal cynomolgus monkey C5 concentration for alternative pathway hemolysis assay.

Figure 4 shows an example of a classical pathway hemolysis assay using 20% human serum.

Figure 5 shows an example of an alternative pathway hemolytic assay, using 100pM purified human C5 added to human C5 depleted serum.

Figure 6 shows an example of an alternative pathway hemolysis assay using 0.025% cynomolgus monkey serum added to human C5 depleted serum.

Fig. 7 shows an example of a classical pathway hemolysis assay (20% human serum) using mature Fabs in comparison to their respective parents.

Fig. 8 shows an example of a classical pathway hemolysis assay (5% cynomolgus monkey serum) using mature Fabs.

Figure 9 shows the characterization of affinity matured Fab in an alternative pathway hemolytic assay using 100pM human C5 added to 20% human C5 depleted serum.

Figure 10 shows the characterization of affinity matured Fab in an alternative pathway hemolysis assay using 20% human serum.

Figure 11 shows the characterization of affinity matured fabs in an alternative pathway hemolytic assay added to 20% human C5 depleted serum using 100pM cynomolgus C5.

Figure 12 shows the characterization of germline IgGs in a classical pathway hemolysis assay using 20% human serum.

Figure 13 shows the characterization of germline IgGs in a classical pathway hemolytic assay using 5% cynomolgus monkey serum.

FIG. 14 shows the characterization of germline IgGs in an alternative pathway hemolytic assay using 100pM human C5.

Figure 15 shows the characterization of final germline IgGs in an alternative pathway hemolysis assay using 20% human serum and a C5a production ELISA.

Figure 16 shows the characterization of affinity matured fabs in C5a ELISA using supernatants from 20% human serum hemolysis assay.

Figure 17 shows a specific solution ELISA on human C3, C4, C5, and cynomolgus C5 test antibody 7091 and derivatives thereof.

Figure 18 shows serum stability assays using Fabs (binding to human C5 in the presence of 50% serum).

Fig. 19 shows epitope binding of some of the affinity-enhanced Fabs.

Figure 20 shows ELISA of antibody binding to mouse-human chimeric C5 or human C5 to determine alpha chain versus beta chain binders. Competition with 5G1.1 was determined by presentation of C5 by 5G 1.1.

Figure 21 shows ELISA for testing alpha chain versus beta chain binders with 5G1.1 capture.

FIG. 22 shows the results of a hemolytic assay for testing alpha chain versus beta chain binders.

FIG. 23 shows thermolysin proteolysis of parent Fabs at 37 deg.C (0, 30, 60, and 90 minutes).

FIG. 24 shows thermolysin proteolysis of parent Fabs at 55 deg.C (0, 30, 60, and 90 minutes).

FIG. 25 shows the thermolysin sensitivity of mature Fabs at 37 ℃.

FIG. 26 shows the thermolysin sensitivity of mature Fabs at 55 ℃.

Figure 27 shows an example of Fab inhibition of the variable pathway in a MAC deposition assay.

Detailed Description

The invention provides antibodies that specifically bind complement C5 protein (e.g., human C5, cynomolgus C5), pharmaceutical compositions, methods of production, and methods of using such antibodies and compositions.

5.1C5 antibody

The present invention provides antibodies that specifically bind to C5 (e.g., human C5, cynomolgus C5). In some embodiments, the invention provides antibodies that specifically bind to human and cynomolgus C5. Antibodies of the invention include, but are not limited to, human monoclonal antibodies isolated as described in the examples (see section 6 below).

The present invention provides antibodies that specifically bind to C5 protein (e.g., human and/or cynomolgus C5) comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7. 23, 39, 51, 67, 79, 96, 108, 114, 121, 137, 151, 165, 179, 187, 201, 210, 218, 227, 241, 253, 257, 273, 277 or 281. The invention also provides antibodies that specifically bind to C5 protein (e.g., human and/or cynomolgus C5), said antibodies comprising VH CDRs having the amino acid sequence of any one of the VH CDRs listed in table 1 below. In particular, the invention provides antibodies that specifically bind to a C5 protein (e.g., human and/or cynomolgus C5), said antibodies comprising (or consisting of) one, two, three, four, five or more VH CDRs having the amino acid sequence of any one of the VH CDRs listed in table 1 below.

The present invention provides antibodies that specifically bind to C5 protein (e.g., human and/or cynomolgus C5) comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 8. 24, 40, 52, 68, 80, 90, 102, 122, 138, 152, 166, 180, 188, 202, 211, 219, 228, 242, 261, 265, 269, 285 or 289. The invention also provides antibodies that specifically bind to a C5 protein (e.g., human and/or cynomolgus C5), said antibodies comprising VL CDRs having the amino acid sequence of any one of the VL CDRs listed in table 1 below. In particular, the invention provides antibodies that specifically bind to a C5 protein (e.g., human and/or cynomolgus C5) comprising (or consisting of) one, two, three or more VL CDRs having the amino acid sequence of any one of the VL CDRs listed in table 1 below.

Other antibodies of the invention include amino acids that have been mutated to still have at least 60, 70, 80, 90, or 95 percent identity in the CDR regions to the CDR regions described in the sequences set forth in table 1. In some embodiments, it includes mutant amino acid sequences, wherein no more than 1,2, 3,4, or 5 amino acids in a CDR region have been mutated when compared to a CDR region described in a sequence set forth in table 1.

The invention also provides nucleic acid sequences encoding the VH, VL, full length heavy chain and full length light chain of an antibody that specifically binds to C5 protein (e.g., human and/or cynomolgus C5). Such nucleic acid sequences can be optimized for expression in mammalian cells (e.g., table 1 shows optimized nucleic acid sequences for the heavy and light chains of antibodies 8109, 8110, 8111, 8113, 8114, 8112, 8125, 8126, 8127, 8128, 8129, 8130, 8131, 8132, and 8091).

TABLE 1 examples of the C5 antibody and C5 protein of the present invention

Other antibodies of the invention include those in which the amino acids or nucleic acids encoding the amino acids have been mutated, but still have at least 60, 70, 80, 90 or 95 percent identity to the sequences described in table 1. In some embodiments, it includes mutant amino acid sequences in which no more than 1,2, 3,4, or 5 amino acids have been mutated within the variable region when compared to the variable region in the sequences depicted in table 1, but retain substantially the same therapeutic activity.

Because each of these antibodies can bind C5, the VH, VL, full length light chain and full length heavy chain sequences (amino acid sequences and nucleotide sequences encoding the amino acid sequences) can be "mixed and matched" to produce other C5 binding antibodies of the invention. Such "mixed and matched" C5 binding antibodies can be tested using binding assays known in the art (e.g., ELISA, and other assays described in the examples section). When these chains are mixed and matched, the VH sequences from a particular VH/VL pairing should be replaced with structurally similar VH sequences. Likewise, the full-length heavy chain sequence from a particular full-length heavy chain/full-length light chain pairing should be replaced with a structurally similar full-length heavy chain sequence. Likewise, VL sequences from a particular VH/VL pairing should be replaced with structurally similar VL sequences. Likewise, full-length light chain sequences from a particular full-length heavy chain/full-length light chain pairing should be replaced with structurally similar full-length light chain sequences. Accordingly, in one aspect, the present invention provides an isolated monoclonal antibody, or antigen binding region thereof, having: comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7. 23, 39, 51, 67, 79, 96, 108, 114, 121, 137, 151, 165, 179, 187, 201, 210, 218, 227, 241, 253, 257, 273, 277, and 281; and comprises a sequence selected from the group consisting of SEQ ID NOs: 8. 24, 40, 52, 68, 80, 90, 102, 122, 138, 152, 166, 180, 188, 202, 211, 219, 228, 242, 261, 265, 269, 285, and 289; wherein the antibody specifically binds to C5 (e.g., human and/or cynomolgus C5).

In another aspect, the invention provides (i) an isolated monoclonal antibody having: comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 9. 25, 41, 53, 69, 81, 97, 109, 115, 123, 139, 153, 167, 181, 189, 203, 212, 220, 229, 243, 249, 254, 258, 274, 278, and 282, which has been optimized for expression in a mammalian cell; and comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 10. 26, 42, 54, 70, 82,91, 103, 124, 140, 154, 168, 182, 190, 204, 213, 221, 230, 244, 251, 262, 266, 270, 286, and 290, which has been optimized for expression in a mammalian cell; or (ii) a functional protein comprising an antigen-binding portion thereof.

In another aspect, the invention provides a C5 binding antibody comprising heavy and light chain CDR1s, CDR2s and CDR3s as described in table 1, or a combination thereof. The amino acid sequence of VH CDR1s of the antibody is shown in SEQ ID NOs: 1. 17, 33, 61, 131, 145, 159, 173, 195 and 235. The amino acid sequence of VH CDR2s of the antibody is shown in SEQ ID NOs: 2. 18, 34, 49, 62, 77, 95, 107, 113, 119, 132, 146, 160, 174, 196, 226 and 236. The amino acid sequence of VH CDR3s of the antibody is shown in SEQ ID NOs: 3. 19, 35, 63, 133, 147, 161, 175, 197 and 237. The amino acid sequence of VL CDR1s of the antibody is shown in SEQ ID NOs: 4. 20, 36, 64, 134, 148, 162, 176, 198 and 238. The amino acid sequence of VL CDR2s of the antibody is shown in SEQ ID NOs: 5. 21, 37, 65, 135, 149, 163, 177, 199, and 239. The amino acid sequence of VL CDR3s of the antibody is shown in SEQ ID NOs: 6. 22, 38, 50, 66, 78, 89, 101, 120, 136, 150, 164, 178, 200, 209, and 240. The CDR regions were delineated using the Kabat system (Kabat, E.A., et al, 1991 Sequences of Proteins of Immunological Interest, fifth edition, U.S. department of Health and Human Services, NIH publication No. 91-3242).

If each of these antibodies can bind C5 and provide antigen binding specificity primarily through the CDR1, 2, and 3 regions, the VH CDR1, 2, and 3 sequences and the VL CDR1, 2, and 3 sequences can be "mixed and matched" (i.e., the CDRs from different antibodies can be mixed and matched, although each antibody must contain VH CDRs 1,2, and 3 and VL CDRs 1,2, and 3 to produce the other C5 binding molecules of the invention). Such "mixed and matched" C5-binding antibodies can be tested using binding assays known in the art and those described in the examples (e.g., ELISA). When VH CDR sequences are mixed and matched, the CDR1, CDR2, and/or CDR3 from a particular VH sequence should be replaced with structurally similar CDR sequences. Similarly, when VL CDR sequences are mixed and matched, CDR1, CDR2, and/or CDR3 sequences from a particular VL sequence should be replaced with structurally similar CDR sequences. It will be apparent to those of ordinary skill in the art that one or more of the VH and/or VL CDR regions may be replaced with structurally similar sequences from the CDR sequences shown herein for the monoclonal antibodies of the invention to generate novel VH and VL sequences.

Accordingly, the present invention provides an isolated monoclonal antibody, or antigen binding region thereof, comprising: comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1. 17, 33, 61, 131, 145, 159, 173, 195 and 235, or a light chain variable region CDR1 of an amino acid sequence of seq id No. 17, 33, 61, 131, 145, 159, 173, 195 and 235; comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2. 18, 34, 49, 62, 77, 95, 107, 113, 119, 132, 146, 160, 174, 196, 226, and 236; comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3. 19, 35, 63, 133, 147, 161, 175, 197 and 237, or a light chain variable region CDR 3; comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4. 20, 36, 64, 134, 148, 162, 176, 198, and 238, or a light chain variable region CDR 1; comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5. 21, 37, 65, 135, 149, 163, 177, 199, and 239, or a light chain variable region CDR 2; comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6. 22, 38, 50, 66, 78, 89, 101, 120, 136, 150, 164, 178, 200, 209, and 240; wherein the antibody specifically binds to C5.

In certain embodiments, the antibody that specifically binds C5 comprises SEQ ID NO:1 heavy chain variable region CDR 1; SEQ ID NO: 2, CDR 2; SEQ ID NO:3, CDR 3; SEQ ID NO:4 CDR 1; SEQ ID NO:5 light chain variable region CDR 2; and SEQ ID NO: 6 light chain variable region CDR 3. In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 17, a heavy chain variable region CDR 1; SEQ ID NO: 18, heavy chain variable region CDR 2; SEQ ID NO: 19 heavy chain variable region CDR 3; SEQ ID NO: 20 light chain variable region CDR 1; SEQ ID NO: 21 light chain variable region CDR 2; and SEQ ID NO: 22 CDR 3.

In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 33, a heavy chain variable region CDR 1; SEQ ID NO: 34, a heavy chain variable region CDR 2; SEQ ID NO: 35, a heavy chain variable region CDR 3; SEQ ID NO: 36, light chain variable region CDR 1; SEQ ID NO: 37, a light chain variable region CDR 2; and SEQ ID NO: 38, CDR 3. In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 17, a heavy chain variable region CDR 1; SEQ ID NO: 49, CDR 2; SEQ ID NO: 19 heavy chain variable region CDR 3; SEQ ID NO: 20 light chain variable region CDR 1; SEQ ID NO: 21 light chain variable region CDR 2; and SEQ ID NO: 50 CDR3 of the light chain variable region.

In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 61, CDR 1; SEQ ID NO: 62, a heavy chain variable region CDR 2; SEQ ID NO: 63, heavy chain variable region CDR 3; SEQ ID NO: 64, light chain variable region CDR 1; SEQ ID NO: 65 light chain variable region CDR 2; and SEQ ID NO: 66, CDR 3. In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 61, CDR 1; SEQ ID NO: 77 CDR 2; SEQ ID NO: 63, heavy chain variable region CDR 3; SEQ ID NO: 64, light chain variable region CDR 1; SEQ ID NO: 65 light chain variable region CDR 2; and SEQ ID NO: 78 CDR 3in the light chain variable region.

In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 61, CDR 1; SEQ ID NO: 77 CDR 2; SEQ ID NO: 63, heavy chain variable region CDR 3; SEQ ID NO: 64, light chain variable region CDR 1; SEQ ID NO: 65 light chain variable region CDR 2; and SEQ ID NO: 89 light chain variable region CDR 3. In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 61, CDR 1; SEQ ID NO: 62, a heavy chain variable region CDR 2; SEQ ID NO: 63, heavy chain variable region CDR 3; SEQ ID NO: 64, light chain variable region CDR 1; SEQ ID NO: 65 light chain variable region CDR 2; and SEQ ID NO: 89 light chain variable region CDR 3.

In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 61, CDR 1; SEQ ID NO: 95 heavy chain variable region CDR 2; SEQ ID NO: 63, heavy chain variable region CDR 3; SEQ ID NO: 64, light chain variable region CDR 1; SEQ ID NO: 65 light chain variable region CDR 2; and SEQ ID NO: 89 light chain variable region CDR 3. In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 17, a heavy chain variable region CDR 1; SEQ ID NO: 49, CDR 2; SEQ ID NO: 19 heavy chain variable region CDR 3; SEQ ID NO: 20 light chain variable region CDR 1; SEQ ID NO: 21 light chain variable region CDR 2; and SEQ ID NO: 101, CDR 3.

In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 17, a heavy chain variable region CDR 1; SEQ ID NO: 107, CDR 2; SEQ ID NO: 19 heavy chain variable region CDR 3; SEQ ID NO: 20 light chain variable region CDR 1; SEQ ID NO: 21 light chain variable region CDR 2; and SEQ ID NO: 22 CDR 3. In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 17, a heavy chain variable region CDR 1; SEQ ID NO: 107, CDR 2; SEQ ID NO: 19 heavy chain variable region CDR 3; SEQ ID NO: 20 light chain variable region CDR 1; SEQ ID NO: 21 light chain variable region CDR 2; and SEQ ID NO: 101, CDR 3.

In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 17, a heavy chain variable region CDR 1; SEQ ID NO: 113, heavy chain variable region CDR 2; SEQ ID NO: 19 heavy chain variable region CDR 3; SEQ ID NO: 20 light chain variable region CDR 1; SEQ ID NO: 21 light chain variable region CDR 2; and SEQ ID NO: 22 CDR 3. In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 17, a heavy chain variable region CDR 1; SEQ ID NO: 113, heavy chain variable region CDR 2; SEQ ID NO: 19 heavy chain variable region CDR 3; SEQ ID NO: 20 light chain variable region CDR 1; SEQ ID NO: 21 light chain variable region CDR 2; and SEQ ID NO: 101, CDR 3.

In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO:1 heavy chain variable region CDR 1; SEQ ID NO: 119, a heavy chain variable region CDR 2; SEQ ID NO:3, CDR 3; SEQ ID NO:4 CDR 1; SEQ ID NO:5 light chain variable region CDR 2; and SEQ ID NO: 120, CDR 3. In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 131 CDR 1; SEQ ID NO: 132, CDR 2; SEQ ID NO: 133, a heavy chain variable region CDR 3; SEQ ID NO: 134 light chain variable region CDR 1; SEQ ID NO: 135 CDR 2; and SEQ ID NO: 136, CDR 3.

In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 145, CDR 1; SEQ ID NO: 146, heavy chain variable region CDR 2; SEQ ID NO: 147 of CDR 3; SEQ ID NO: 148, light chain variable region CDR 1; SEQ ID NO: 149 CDR 2; and SEQ ID NO: 150, CDR 3. In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 159, CDR 1; SEQ ID NO: 160, heavy chain variable region CDR 2; SEQ ID NO: 161 heavy chain variable region CDR 3; SEQ ID NO: 162, CDR 1; SEQ ID NO: 163 light chain variable region CDR 2; and SEQ ID NO: 164, CDR 3.

In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 173 heavy chain variable region CDR 1; SEQ ID NO: 174, CDR 2; SEQ ID NO: 175, CDR 3; SEQ ID NO: 176, light chain variable region CDR 1; SEQ ID NO: a light chain variable region CDR2 of 177; and SEQ ID NO: 178, CDR 3. In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 195, CDR 1; SEQ ID NO: 196, CDR 2; SEQ ID NO: 197 the heavy chain variable region CDR 3; SEQ ID NO: 198, CDR 1; SEQ ID NO: 199, light chain variable region CDR 2; and SEQ ID NO: 200, CDR3 of the light chain variable region.

In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 61, CDR 1; SEQ ID NO: 77 CDR 2; SEQ ID NO: 63, heavy chain variable region CDR 3; SEQ ID NO: 64, light chain variable region CDR 1; SEQ ID NO: 65 light chain variable region CDR 2; and SEQ ID NO: 209, CDR 3. In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 17, a heavy chain variable region CDR 1; SEQ ID NO: 49, CDR 2; SEQ ID NO: 19 heavy chain variable region CDR 3; SEQ ID NO: 20 light chain variable region CDR 1; SEQ ID NO: 21 light chain variable region CDR 2; and SEQ ID NO: 22 CDR 3.

In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 33, a heavy chain variable region CDR 1; SEQ ID NO: 226, CDR 2; SEQ ID NO: 35, a heavy chain variable region CDR 3; SEQ ID NO: 36, light chain variable region CDR 1; SEQ ID NO: 37, a light chain variable region CDR 2; and SEQ ID NO: 38, CDR 3. In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 235, CDR 1; SEQ ID NO: 236, CDR 2; SEQ ID NO: 237 heavy chain variable region CDR 3; SEQ ID NO: 238 light chain variable region CDR 1; SEQ ID NO: 239 light chain variable region CDR 2; and SEQ ID NO: 240, CDR 3.

In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO:1 heavy chain variable region CDR 1; SEQ ID NO: 119, a heavy chain variable region CDR 2; SEQ ID NO:3, CDR 3; SEQ ID NO:4 CDR 1; SEQ ID NO:5 light chain variable region CDR 2; and SEQ ID NO: 6 light chain variable region CDR 3. In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO:1 heavy chain variable region CDR 1; SEQ ID NO: 2, CDR 2; SEQ ID NO:3, CDR 3; SEQ ID NO:4 CDR 1; SEQ ID NO:5 light chain variable region CDR 2; and SEQ ID NO: 120, CDR 3.

In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 61, CDR 1; SEQ ID NO: 62, a heavy chain variable region CDR 2; SEQ ID NO: 63, heavy chain variable region CDR 3; SEQ ID NO: 64, light chain variable region CDR 1; SEQ ID NO: 65 light chain variable region CDR 2; and SEQ ID NO: 209, CDR 3. In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 61, CDR 1; SEQ ID NO: 95 heavy chain variable region CDR 2; SEQ ID NO: 63, heavy chain variable region CDR 3; SEQ ID NO: 64, light chain variable region CDR 1; SEQ ID NO: 65 light chain variable region CDR 2; and SEQ ID NO: 209, CDR 3.

In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 61, CDR 1; SEQ ID NO: 77 CDR 2; SEQ ID NO: 63, heavy chain variable region CDR 3; SEQ ID NO: 64, light chain variable region CDR 1; SEQ ID NO: 65 light chain variable region CDR 2; and SEQ ID NO: 66, CDR 3. In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 61, CDR 1; SEQ ID NO: 77 CDR 2; SEQ ID NO: 63, heavy chain variable region CDR 3; SEQ ID NO: 64, light chain variable region CDR 1; SEQ ID NO: 65 light chain variable region CDR 2; and SEQ ID NO: 78 CDR 3in the light chain variable region.

In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 61, CDR 1; SEQ ID NO: 77 CDR 2; SEQ ID NO: 63, heavy chain variable region CDR 3; SEQ ID NO: 64, light chain variable region CDR 1; SEQ ID NO: 65 light chain variable region CDR 2; and SEQ ID NO: 89 light chain variable region CDR 3. In another specific embodiment, the antibody that specifically binds C5 comprises the amino acid sequence of SEQ ID NO: 17, a heavy chain variable region CDR 1; SEQ ID NO: 107, CDR 2; SEQ ID NO: 19 heavy chain variable region CDR 3; SEQ ID NO: 20 light chain variable region CDR 1; SEQ ID NO: 21 light chain variable region CDR 2; and SEQ ID NO: 22 CDR 3.

In certain embodiments, the antibody that specifically binds C5 is an antibody described in table 1.

As used herein, a human antibody comprises a heavy or light chain variable region or a full-length heavy or light chain that is "produced from" or "derived from" a particular germline sequence if the variable region or full-length chain of the antibody is obtained from a system using human germline immunoglobulin genes. Such systems include immunization of transgenic mice carrying human immunoglobulin genes with the antigen of interest or screening of phage-displayed human immunoglobulin gene libraries with the antigen of interest. A human antibody "produced from" or "derived from" a human germline immunoglobulin sequence can be identified, for example, by comparing the amino acid sequence of the human antibody to the amino acid sequence of a human germline immunoglobulin and selecting the human germline immunoglobulin sequence that is the closest in sequence (i.e., the highest% identity) to the human antibody. A human antibody "produced from" or "derived from" a particular human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence, for example due to naturally occurring somatic mutations or deliberate introduction of site-directed mutations. However, in the VH or VL framework regions, the selected human antibody is typically at least 90% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that, when compared to germline immunoglobulin amino acid sequences from other species (e.g., murine germline sequences), identify the human antibody as human. In certain instances, a human antibody has at least 60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or 99% identity in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, recombinant human antibodies differ from human germline immunoglobulin genes by no more than 10 amino acids in the VH or VL framework regions of the encoded amino acid sequences. In some cases, the human antibody differs from the amino acid sequence encoded by the germline immunoglobulin gene by no more than 5, or even no more than 4,3, 2, or 1 amino acid.

Homologous antibodies

In another embodiment, the invention provides antibodies or antigen-binding fragments thereof comprising amino acid sequences homologous to the sequences set forth in table 1, and which bind to C5 protein (e.g., human and/or cynomolgus C5) and retain the desired functional properties of those antibodies set forth in table 1.

For example, the present invention provides an isolated monoclonal antibody (or functional antigen-binding fragment thereof) comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain variable region that differs from a light chain variable region selected from the group consisting of SEQ ID NOs: 7. 23, 39, 51, 67, 79, 96, 108, 114, 121, 137, 151, 165, 179, 187, 201, 210, 218, 227, 241, 253, 257, 273, 277 or 281, an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical; the light chain variable region comprises a light chain variable region substantially identical to a light chain variable region selected from the group consisting of SEQ ID NOs: 8. 24, 40, 52, 68, 80, 90, 102, 122, 138, 152, 166, 180, 188, 202, 211, 219, 228, 242, 261, 265, 269, 285, or 289, or an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical; the antibodies specifically bind to C5 (e.g., human and/or cynomolgus C5), and the antibodies can inhibit erythrolysis in a hemolysis assay. In particular examples, such antibodies have in a hemolytic assay when using C5 depleted serum reconstituted with 100pM human C5IC of 20-200pM₅₀The value is obtained.

In other embodiments, the VH and/or VL amino acid sequences may be 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequences set forth in table 1. In other embodiments, the VH and/or VL amino acid sequences may be identical, with amino acid substitutions at no more than 1,2, 3,4, or 5 amino acid positions. The nucleic acid sequences of SEQ ID NOs: 7. 23, 39, 51, 67, 79, 96, 108, 114, 121, 137, 151, 165, 179, 187, 201, 210, 218, 227, 241, 253, 257, 273, 277 or 281; and 8, 24, 40, 52, 68, 80, 90, 102, 122, 138, 152, 166, 180, 188, 202, 211, 219, 228, 242, 261, 265, 269, 285, or 289, to obtain antibodies having VH and VL regions with high (i.e., 80% or greater) identity to those described in table 1, and then testing the encoded altered antibodies for retained function using the functional assays described herein.

In other embodiments, the full length heavy chain and/or full length light chain amino acid sequence is 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence set forth in table 1. Antibodies having an amino acid sequence substantially identical to a sequence of a nucleic acid molecule encoding such a polypeptide, respectively, can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of the nucleic acid molecule encoding such a polypeptide, respectively, having an amino acid sequence identical to a sequence of SEQ ID NOs: 9. 25, 41, 53, 69, 81, 97, 109, 115, 123, 139, 153, 167, 181, 189, 203, 212, 220, 229, 243, 249, 254, 258, 274, 278 and any of the full-length heavy chains of SEQ ID Nos 10, 26, 42, 54, 70, 82,91, 103, 124, 140, 154, 168, 182, 190, 204, 213, 221, 230, 244, 251, 262, 266, 270, 286, and 290 have high (i.e., 80% or more) identity with the full-length light chain, and the encoded altered antibody is then tested for retained function using the functional assays described herein.

In other embodiments, the full length heavy chain and/or full length light chain nucleotide sequences are 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequences set forth above.

In other embodiments, the variable region of the heavy and/or light chain nucleotide sequence is 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth above.

As used herein, the percent identity between two sequences is a function of the number of identical positions common to the sequences (i.e.,% identity equals the number of identical positions/total number of positions x100), wherein the gaps need to be introduced for optimal alignment of the two sequences, taking into account the number of gaps and the length of each gap. As described in the non-limiting examples below, comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

Additionally or alternatively, the protein sequences of the invention may further be used as "query sequences" to search against public databases, for example to identify related sequences. For example, Altschul et al, 1990 J.mol.biol.215: BLAST programs from 403-10 (version 2.0) perform such searches.

Antibodies with conservative modifications

In certain embodiments, the antibodies of the invention have a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein one or more of these CDR sequences has a particular amino acid sequence based on the antibodies described herein or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the C5 binding antibodies of the invention. Accordingly, the present invention provides an isolated monoclonal antibody, or a functional antigen-binding fragment thereof, consisting of a heavy chain variable region comprising the sequences of CDR1, CDR2 and CDR3 and a light chain variable region comprising the sequences of CDR1, CDR2 and CDR3, wherein: the heavy chain variable region CDR1 amino acid sequence is selected from SEQ ID NOs: 1. 17, 33, 61, 131, 145, 159, 173, 195, and 235, and conservative modifications thereof; the heavy chain variable region CDR2 amino acid sequence is selected from SEQ ID NOs: 2. 18, 34, 49, 62, 77, 95, 107, 113, 119, 132, 146, 160, 174, 196, 226, and 236, and conservative modifications thereof; the heavy chain variable region CDR3 amino acid sequence is selected from SEQ ID NOs: 3. 19, 35, 63, 133, 147, 161, 175, 197, and 237, and conservative modifications thereof; the light chain variable region CDR1 amino acid sequence is selected from SEQ ID NOs: 4. 20, 36, 64, 134, 148, 162, 176, 198, and 238, and conservative modifications thereof; the light chain variable region CDR2 amino acid sequence is selected from SEQ ID NOs: 5. 21, 37, 65, 135, 149, 163, 177, 199, and 239, and conservative modifications thereof; the light chain variable region CDR3 amino acid sequence is selected from SEQ ID NOs: 6. 22, 38, 50, 66, 78, 89, 101, 120, 136, 150, 164, 178, 200, 209, and 240, and conservative modifications thereof; the antibody or antigen-binding fragment thereof specifically binds to C5 and inhibits erythrocyte lysis in a hemolysis assay as described herein.

In other embodiments, an antibody of the invention optimized for expression in a mammalian cell has a full-length heavy chain sequence and a full-length light chain sequence, wherein one or more of these sequences has a particular amino acid sequence based on the antibody described herein or conservative modifications thereof, and wherein the antibody retains the desired functional properties of a C5-binding antibody of the invention. Accordingly, the present invention provides an isolated monoclonal antibody optimized for expression in a mammalian cell, consisting of a full length heavy chain and a full length light chain, wherein: the full-length heavy chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 9. 25, 41, 53, 69, 81, 97, 109, 115, 123, 139, 153, 167, 181, 189, 203, 212, 220, 229, 243, 249, 254, 258, 274, 278, and 282, and conservative modifications thereof; and the full length light chain has an amino acid sequence selected from SEQ ID NOs: 10. 26, 42, 54, 70, 82,91, 103, 124, 140, 154, 168, 182, 190, 204, 213, 221, 230, 244, 251, 262, 266, 270, 286, and 290, and conservative modifications thereof; the antibody specifically binds C5 (e.g., human and/or cynomolgus C5); and the antibody inhibits erythrocyte lysis in a hemolysis assay as described herein. In particular embodiments, such antibodies are in solution when human C5 depleted serum reconstituted with 100pM human C5 is usedHas an IC of 20-200pM in a blood assay₅₀The value is obtained.

Antibodies binding to the same epitope

The present invention provides antibodies that bind to the same epitope as the C5 binding antibodies described in table 1. Additional antibodies can therefore be identified based on their ability to cross-compete with other antibodies of the invention (e.g., competitively inhibit binding in a statistically significant manner) in a C5 binding assay. The ability of a test antibody to inhibit binding of the antibody of the invention to a C5 protein (e.g., human and/or cynomolgus C5) indicates that the test antibody can compete with the antibody for binding to C5; according to non-limiting theory, the antibody and the antibody with which it competes bind to the same or related (e.g., structurally similar or spatially close) epitope on C5. In one embodiment, the antibody that binds to the same epitope on C5 as the antibody of the invention is a human monoclonal antibody. Such human monoclonal antibodies can be prepared and isolated as described herein.

Engineered and modified antibodies

Antibodies of the invention may also be prepared using antibodies having one or more of the VH and/or VL sequences set forth herein as starting materials to engineer modified antibodies that may have altered properties from the starting antibody. Antibodies can be engineered by modifying one or more residues within one or both variable regions (i.e., VH and/or VL), e.g., one or more CDR regions and/or one or more framework regions. In addition or alternatively, antibodies may be engineered by modifying residues within the constant region, for example to alter the effector function of the antibody.

One type of variable region modification that can be performed is CDR grafting. Antibodies interact with a target antigen primarily through amino acid residues located in the six heavy and light chain Complementarity Determining Regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse between antibodies than sequences outside the CDRs. Because the CDR sequences are responsible for most of the antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular naturally occurring antibody by constructing an expression vector that includes CDR sequences from the particular naturally occurring antibody grafted onto framework sequences of different antibodies with different properties (see, e.g., Riechmann, L. et al, 1998Nature 332: 323-.

Accordingly, another embodiment of the present invention is directed to an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising heavy chain variable regions comprising, respectively, a heavy chain variable region having a heavy chain variable region selected from the group consisting of SEQ ID NOs: 1. a CDR1 sequence of the amino acid sequences of 17, 33, 61, 131, 145, 159, 173, 195 and 235; has a sequence selected from the group consisting of SEQ ID NOs: 2. 18, 34, 49, 62, 77, 95, 107, 113, 119, 132, 146, 160, 174, 196, 226, and 236; has a sequence selected from the group consisting of SEQ ID NOs: 3. 19, 35, 63, 133, 147, 161, 175, 197 and 237; and light chain variable regions comprising light chain variable regions having amino acid sequences selected from SEQ ID NOs: 4. 20, 36, 64, 134, 148, 162, 176, 198, and 238; has a sequence selected from the group consisting of SEQ ID NOs: 5. 21, 37, 65, 135, 149, 163, 177, 199, and 239; has a sequence selected from SEQ ID NOs: 6. 22, 38, 50, 66, 78, 89, 101, 120, 136, 150, 164, 178, 200, 209, and 240, and a CDR3 sequence. Thus, such antibodies contain the VH and VL CDRs of the monoclonal antibodies, but still contain different framework sequences from these antibodies.

Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. For example, germline DNA Sequences for Human heavy and light chain variable region genes can be found in the "VBase" Human germline sequence database (available on the Internet www.mrc-cpe. cam. ac. uk/VBase), as well as Kabat, E.A., et al, 1991 Sequences of proteins of Immunological Interest, fifth edition, U.S. department of health and Human Services, NIH publication Nos. 91-3242; tomlinson, i.m., et al, 1992 j.fol.biol.227: 776-798; and Cox, j.p.l. et al, 1994eur.j immunol.24: 827-836; the contents of each are expressly incorporated herein by reference.

Examples of framework sequences for use in antibodies of the invention are those that are structurally similar to the framework sequences used in selected antibodies of the invention, e.g., the consensus sequences and/or framework sequences used in monoclonal antibodies of the invention. The VH CDR1, 2 and 3 sequences, and VL CDR1, 2 and 3 sequences may be grafted onto framework regions having the same sequence as the sequences found in the germline immunoglobulin gene from which the framework sequences are derived, or the CDR sequences may be grafted onto framework regions containing one or more mutations compared to the germline sequences. For example, it has been found advantageous in some cases to mutate residues within the framework regions to maintain or enhance the antigen binding capacity of the antibody (see, e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al).

Another type of variable region modification is mutation of amino acid residues within the VH and/or VL CDR1, CDR2, and/or CDR3 regions, thereby improving one or more binding properties (e.g., affinity) of the antibody of interest, referred to as "affinity maturation. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutations and to assess the effect on antibody binding or other functional properties of interest in vitro or in vivo assays as described herein and provided in the examples. Conservative modifications (as discussed above) may be introduced. The mutation may be an amino acid substitution, addition or deletion. In addition, typically no more than 1,2, 3,4 or 5 residues within a CDR region are altered.

Thus, in another embodiment, the present invention provides an isolated C5 binding monoclonal antibody, or antigen binding fragment thereof, consisting of a heavy chain variable region having: consisting of a polynucleotide selected from the group consisting of SEQ ID NOs: 1. 17, 33, 61, 131, 145, 159, 173, 195 and 235, or a sequence identical to SEQ ID NOs: 1. 17, 33, 61, 131, 145, 159, 173, 195 and 235 has a VH CDR1 region consisting of an amino acid sequence of 1,2, 3,4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence; has a sequence selected from the group consisting of SEQ ID NOs: 2. 18, 34, 49, 62, 77, 95, 107, 113, 119, 132, 146, 160, 174, 196, 226, and 236, or a sequence identical to SEQ ID NOs: 2. 18, 34, 49, 62, 77, 95, 107, 113, 119, 132, 146, 160, 174, 196, 226 and 236 having an amino acid sequence of 1,2, 3,4 or 5 amino acid substitutions, deletions or additions compared to the VH CDR2 region; has a sequence selected from the group consisting of SEQ ID NOs: 3. 19, 35, 63, 133, 147, 161, 175, 197 and 237, or a sequence identical to SEQ ID NOs: 3. 19, 35, 63, 133, 147, 161, 175, 197 and 237 VH CDR3 region having an amino acid sequence with 1,2, 3,4 or 5 amino acid substitutions, deletions or additions compared to; has a sequence selected from SEQ ID NO: 4. 20, 36, 64, 134, 148, 162, 176, 198 and 238, or a sequence identical to SEQ ID NOs: 4. 20, 36, 64, 134, 148, 162, 176, 198 and 238 having an amino acid sequence of 1,2, 3,4 or 5 amino acid substitutions, deletions or additions to the VL CDR1 region; has a sequence selected from the group consisting of SEQ ID NOs: 5. 21, 37, 65, 135, 149, 163, 177, 199 and 239, or a sequence identical to SEQ ID NOs: 5. 21, 37, 65, 135, 149, 163, 177, 199, and 239, a VL CDR2 region having an amino acid sequence of 1,2, 3,4, or 5 amino acid substitutions, deletions, or additions; and has a sequence selected from SEQ ID NOs: 6. 22, 38, 50, 66, 78, 89, 101, 120, 136, 150, 164, 178, 200, 209, and 240, or a sequence identical to SEQ ID NOs: 6. 22, 38, 50, 66, 78, 89, 101, 120, 136, 150, 164, 178, 200, 209, and 240 has a VL CDR3 region having an amino acid sequence of 1,2, 3,4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence.

Grafting of antigen binding domains into alternative frameworks or scaffolds

A variety of antibody/immunoglobulin frameworks or scaffolds may be used, so long as the resulting polypeptide includes at least one antigen-binding region that specifically binds C5. Such frameworks or scaffolds include the 5 major idiotypes of human immunoglobulins or fragments thereof, and include immunoglobulins of other animal species, preferably animal species with humanization. Single heavy chain antibodies, such as those identified in camels, are important in this regard. New frameworks, scaffolds and fragments are continually discovered and developed by those skilled in the art.

In one aspect, the invention relates to the production of non-immunoglobulin-based antibodies using non-immunoglobulin scaffolds onto which the CDRs of the invention can be grafted. Known or unknown non-immunoglobulin frameworks and scaffolds may be used, provided they comprise a binding region specific for the target C5 protein (e.g., human and/or cynomolgus C5). Known non-immunoglobulin frameworks or scaffolds include, but are not limited to, fibronectin (Compound Therapeutics, inc., Waltham, MA), ankyrin (molecular partners AG, Zurich, Switzerland), domain antibodies (domanis, ltd., Cambridge, MA, and Ablynx nv, Zwijnaarde, Belgium), lipocalin (pierisprotolab AG, freesing, Germany), small modular immunopharmaceuticals (truonpharmaceuticals inc., Seattle, WA), maxbodiies (Avidia, inc., mountain view, CA), a protein (Affibody AG, Sweden), and affilin (gamma-crystal protein or ubiquitin) (Scil Proteins GmbH, halrule, Germany).

Fibronectin scaffolds are based on fibronectin type III domains (e.g., the tenth module of fibronectin type III (10Fn3 domain)). Fibronectin type III domains have 7 or 8 beta strands distributed between two beta sheets that pack themselves into each other forming the core of the protein and further contain loops (similar to the CDRs) that link the beta strands to each other and are exposed to solvents. At least three such loops are present at each edge of the beta sheet sandwich, where the edge is the protein boundary perpendicular to the direction of the beta strand (see US 6,818,418). These fibronectin based scaffolds are not immunoglobulins, although the overall folding is very similar to the smallest functional antibody fragment, the heavy chain variable region comprising the complete antigen recognition unit in camel and vicuna IgG. Because of this structure, non-immunoglobulin antibodies mimic antigen-binding properties similar to those in nature and affinities similar to those of antibodies. These scaffolds can be used in vitro loop randomization and shuffling strategies, which are similar to the affinity maturation process of antibodies in vivo. These fibronectin based molecules can be used as scaffolds in which the loop regions of the molecule are replaced with the CDRs of the invention using standard cloning techniques.

Ankyrin technology is based on the use of proteins with ankyrin-derived repeating modules as scaffolds carrying variable regions that can be used to bind different targets. The ankyrin repeat module is a 33 amino acid polypeptide consisting of two antiparallel alpha helices and beta turns. Binding of the variable regions was maximally optimized by using ribosome display.

Avimers are derived from a native A domain containing, for example, LRP-1 protein. These domains are naturally used for protein-protein interactions, and in humans more than 250 proteins are structurally based on the a domain. Avimers consist of a large number of different "A-domain" monomers (2-10) connected by amino acid linkers. For example, U.S. patent application publication No. 20040175756; 20050053973, respectively; 20050048512 and 20060008844, produce Avimers that can bind to the target antigen.

Affibody affinity ligands are small, simple proteins consisting of a triple helix bundle based on a scaffold for one IgG binding domain of protein a. Protein A is a surface protein from the bacterium Staphylococcus aureus (Staphylococcus aureus). The scaffold domain consists of 58 amino acids, 13 of which were randomized to generate affibody libraries with a large number of ligand variants (see e.g., US 5,831,012). The Affinibody molecules mimic antibodies, which have a molecular weight of 6kDa compared to the molecular weight of 150kDa antibodies. Despite its small size, the binding site of affibody molecules is similar to that of antibodies.

Anticalins are products developed by Pieris ProteoLab AG. They are derived from lipocalins, a large class of small and robust proteins that are generally involved in physiological transport or in the storage of chemically sensitive or insoluble compounds. Several native lipocalins are produced in human tissues or body fluids. The protein structure is shown as an immunoglobulin with hypervariable loops on top of a rigid framework. However, in contrast to antibodies or their recombinant fragments, lipocalins consist of a single polypeptide chain with 160 to 180 amino acid residues, only slightly larger than a single immunoglobulin domain. The tetracyclic group constituting the binding pocket shows significant structural plasticity and tolerates a variety of side chains. The binding sites can thus be remodeled in a proprietary way to recognize defined target molecules of different shapes with high affinity and specificity. One protein of the lipocalin family, the posterior bile pigment binding protein (BBP) of Pieris Brassicae, has been used to develop anticalins by mutagenizing tetracyclic groups. One example of a patent application describing anticalins is in PCT publication No. WO 199916873.

Affilin molecules are small, non-immunoglobulin proteins designed to have specific affinity for proteins and small molecules. New affilin molecules can be selected very rapidly from two libraries, each based on scaffold proteins from different humans. The Affilin molecules do not show any structural homology to immunoglobulin proteins. Currently, two affilin scaffolds are used, one of which is the gamma crystallin, the human structural lens protein, and the other is the "ubiquitin" superfamily protein. Both human scaffolds were very small, showed high temperature stability, and were almost resistant to pH changes and denaturants. This high stability is mainly due to the extended beta sheet structure of the protein. Examples of gamma crystallin derived proteins are described in WO200104144, and examples of "ubiquitin-like" proteins are described in WO 2004106368.

Protein Epitope Mimic (PEM) is a medium-sized, circular, peptide-like molecule (MW 1-2kDa) that mimics the protein β hairpin secondary structure, the main secondary structure involved in protein-protein interactions.

In some embodiments, Fabs are converted to the silent IgG1 format by altering the Fc region. For example, CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 293) can be used to replace the "X" in the heavy chain amino acid sequence and to use: CS (if the light chain is. lamda.), or C (if the light chain is. kappa.) in place of the "X" in the light chain amino acid sequence, to convert the antibodies 6525-7910 of Table 1 to the silent IgG1 form. As used herein, a "silent IgG 1" is an IgG1Fc sequence in which the amino acid sequence has been altered to reduce Fc-mediated effector functions (e.g., ADCC and/or CDC). Such antibodies typically have reduced binding to Fc receptors and/or C1 q.

In some other embodiments, the Fabs are converted to the IgG2 form. For example, with the constant sequence of the IgG2 heavy chain: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 295) was substituted for the constant sequence ASTKGPSVFPLAPSSKSTSGGTTAALGCLVKDYFLVTVSWNSGALTSVHTFPWLSKLQSSGLYSLSSSVVPSSSSSQTYLCTYPNHNHKPSNTKVDKKVEPKSX (SEQ ID NO: 294) and CS (if the light chain was. lamda.), or C (if the light chain was. kappa.), was substituted for the "X" in the light chain amino acid sequence to convert antibody 6525-7910 in Table 1 to the IgG2 form.

Human or humanized antibodies

The present invention provides fully human antibodies that specifically bind to C5 protein (e.g., human and/or cynomolgus C5). The human C5-binding antibodies of the invention have further reduced antigenicity when administered to a human subject as compared to the chimeric or humanized antibodies.

Human C5 binding antibodies can be generated using methods known in the art. For example, human engineering techniques are used to transform non-human antibodies into engineered human antibodies. U.S. patent publication No. 20050008625 describes an in vivo method of replacing the non-human antibody variable regions with human variable regions in an antibody while maintaining the same or providing better binding properties than those of non-human antibodies. The method relies on epitope-directed replacement of the variable region of a non-human reference antibody by a fully human antibody. The resulting human antibody is generally structurally unrelated to the reference non-human antibody, but binds to the same epitope on the same antigen as the reference antibody. Briefly, a continuous epitope-directed complementation approach is made possible by establishing a competition between "competitors" in cells and a library of multiple hybrids of a reference antibody ("test antibody") for binding a limited amount of antigen in the presence of a reporter system that responds to binding of the test antibody to the antigen. The competitor may be a reference antibody or derivative thereof, such as a single chain Fv fragment. The competitor may also be a natural or artificial antigen ligand which binds to the same epitope as the reference antibody. The only requirement of the competitor is that it can bind the same epitope as the reference antibody and that it competes for binding to the antigen with the reference antibody. The test antibody has one common antigen-binding V-region from a non-human reference antibody, and other V-regions randomly selected from a repertoire of components from different sources, such as human antibodies. The common V-region from the reference antibody serves as a leader, placing the test antibody on the same epitope on the antigen, and in the same orientation, so that the choice is biased toward having the highest antigen binding fidelity to the reference antibody.

Many types of reporter systems are available for detecting the desired interaction between the test antibody and the antigen. For example, complementary reporter fragments can be linked to an antigen and a test antibody, respectively, such that reporter activation of fragment complementarity occurs only when the test antibody binds to the antigen. When the test antibody and antigen reporter fragment fusion are co-expressed with a competitor, reporter activation becomes dependent on the ability of the test antibody to compete with the competitor, which is proportional to the affinity of the test antibody for the antigen. Other reporter systems that may be used include the reactivator of the self-inhibiting reporter reactivation system (RAIR) as disclosed in U.S. patent application Ser. No. 10/208,730 (publication No. 20030198971), or the competitive activation system disclosed in U.S. patent application Ser. No. 10/076,845 (publication No. 20030157579).

Selection was performed to identify cells expressing a single test antibody with competitor, antigen and reporter components using a sequential epitope-directed complementation replacement system. In these cells, each test antibody competes one-to-one with the competitor for binding to a limited amount of antigen. The activity of the reporter is proportional to the amount of antigen bound to the test antibody, which in turn is proportional to the affinity of the test antibody for the antigen and the stability of the test antibody. When expressed as a test antibody, the test antibody is initially selected on the basis of its activity relative to a reference antibody. The result of the first round of selection is a set of "hybrid" antibodies, wherein each antibody comprises the same non-human V-region from the reference antibody and a human V-region from the library, and each antibody binds to the same epitope on the antigen as the reference antibody. One or more hybrid antibodies selected in the first round have an affinity for the antigen comparable to or higher than the reference antibody.

In a second V region replacement step, the human V regions selected in the first step are used as a guide to select a diverse library of cognate human V regions to replace the remaining non-human reference antibody V regions. The hybrid antibodies selected in the first round can also be used as competitors for the second round of selection. The result of the second round of selection is a panel of fully human antibodies that differ in structure from the reference antibody, but compete with the reference antibody for binding to the same antigen. Some of the selected human antibodies bind to the same epitope on the same antigen as the reference antibody. Of these selected human antibodies, one or more antibodies bind the same epitope with an affinity comparable to or higher than that of the reference antibody.

Using one of the mouse or chimeric C5 binding antibodies described above as a reference antibody, this method can be readily used to generate human antibodies that bind human C5 with the same binding specificity and the same or higher binding affinity. In addition, such human C5-binding antibodies are also commercially available from companies that typically produce human antibodies, such as kalobis, Inc.

Camel (camelid) antibodies

Antibody proteins obtained from members of the camel and dromedarius families, including new world members such as vicuna species (Lama paccos, Lama glama and Lama vicugna), have been characterized in terms of size, structural complexity and antigenicity in human subjects. Certain IgG antibodies from this mammalian family found in nature lack a light chain and are therefore structurally distinct from the typical four-chain quaternary structure of the two heavy and two light chains of antibodies from other animals. See PCT/EP93/02214 (WO 94/04678 published 3.3.1994).

Regions of camelid antibodies identified as small single variable domains of VHH can be obtained by genetic engineering to produce small proteins with high affinity for the target, resulting in low molecular weight antibody-derived proteins, known as "camelid nanobodies". See U.S. patent No. 5,759,808 filed on 2.6.1998; see also Stijlemans, b. et al, 2004J Biol Chem 279: 1256-1261; dumoulin, m. et al, 2003Nature 424: 783-788; pleschberger, M. et al 2003Bioconjugate Chem 14: 440-448; cortex-Retamozo, V. et al 2002 Int Jcancer 89: 456 to 62; and Lauwereys, M. et al 1998 EMBO J17: 3512-3520. Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium. Like other antibodies of non-human origin, the amino acid sequence of camelid antibodies can be recombinantly altered to obtain sequences more similar to human sequences, i.e., nanobodies can be "humanized". Thus, the natural low antigenicity of camelid antibodies to humans can be further reduced.

Camelid nanobodies have a molecular weight of roughly one tenth that of human IgG molecules, and the proteins are only a few nanometers in physical diameter. One consequence of the small size is that camelid nanobodies are able to bind to functionally invisible antigenic sites of larger antibody proteins, i.e. camelid nanobodies may be used as reagents for antigens that are not detectable using classical immunological techniques and may be used as therapeutic agents. Thus, another consequence of the small size is that camelid nanobodies may therefore inhibit specific site binding in the groove or slit of the target protein and may thus have the ability to function more like typical low molecular weight drugs than typical antibodies.

The low molecular weight and compact size also result in camelid nanobodies that are extremely thermostable, stable to extreme pH and proteolytic digestion, and weak in antigenicity. Another result is that camelid nanobodies are easily transferred from the circulation to tissues, even across the blood-brain barrier, and can treat conditions affecting neural tissues. The nanobody may further facilitate drug transport across the blood-brain barrier. See U.S. patent application 20040161738, published on 8/19/2004. These features, combined with low antigenicity in humans, indicate great therapeutic potential. In addition, these molecules can be fully expressed in prokaryotic cells, such as E.coli (E.coli), and expressed as fusion proteins with phage, and are functional.

Thus, the present invention features camelid or nanobodies with high affinity for C5. In certain embodiments herein, the camelid antibodies or nanobodies are naturally produced in camelids, i.e., by immunizing a camelid with C5 or a peptide fragment thereof using the techniques described herein for other antibodies. Alternatively, the panning procedure using C5 as a target as described in the examples herein was engineered to produce C5-binding camelid nanobodies by, for example, selection from a phage library displaying appropriately induced camelid nanobody proteins. Engineered nanobodies may be further customized by genetic engineering to have a half-life in the recipient subject of from 45 minutes to two weeks. In a particular embodiment, a camelid or nanobody is obtained by grafting the CDRs sequences of the heavy or light chain of a human antibody of the invention into nanobody or single domain antibody framework sequences, as described in PCT/EP 93/02214.

Bispecific molecules and multivalent antibodies

In another aspect, the invention features bispecific or multispecific molecules comprising a C5-binding antibody or fragment thereof of the invention. The antibody or antigen-binding fragment thereof of the invention can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or ligand of a receptor), to produce a bispecific molecule that binds to at least two different binding sites or target molecules. The antibodies of the invention may in fact be derivatized or linked to more than one other functional molecule to produce multispecific molecules that bind to more than two different binding sites and/or target molecules; the term "bispecific molecule" as used herein is also intended to include such multispecific molecules. To produce a bispecific molecule of the invention, an antibody of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent binding, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, in order to produce a bispecific molecule.

Accordingly, the present invention includes bispecific molecules comprising at least a first binding specificity for C5, and a second binding specificity for a second target epitope. For example, the second target epitope is another epitope of C5 that is different from the first target epitope.

Furthermore, for the invention wherein the bispecific molecule is multispecific, the molecule may comprise a third binding specificity in addition to the first and second target epitopes.

In one embodiment, bispecific molecules of the invention comprise as binding specificity at least one antibody, or antibody fragment thereof, including, for example, Fab ', F (ab') 2, Fv or single chain Fv. The antibody may also be a light or heavy chain dimer, or any minimal fragment thereof, such as an Fv or single chain construct as described in U.S. Pat. No. 4,946,778 to Ladner et al.

Diabodies are bivalent, bispecific molecules in which the VH and VL domains are expressed on a single polypeptide chain, connected by a linker that is too short to pair between the two domains on the same chain. The VH and VL domains pair with complementary domains of the other chain, thereby generating two antigen binding sites (see, e.g., Holliger et al, 1993Proc. Natl. Acad. Sci. USA 90: 6444-. Diabodies can be produced by expressing two polypeptide chains with the structures VHA-VLB and VHB-VLA (VH-VL configuration), or VLA-VHB and VLB-VHA (VL-VH configuration) in the same cell. Most diabodies are expressed in bacteria in soluble form. Single chain diabodies (scDb) are generated by joining two diabodies to form a polypeptide chain with a linker of about 15 amino acid residues (see Holliger and Winter, 1997Cancer immunol. immunother., 45 (3-4): 128-30; Wu et al, 1996Immunotechnology, 2 (1): 21-36). scDb is expressed in bacteria as soluble, active monomers (see Holliger and Winter, 1997Cancer immunol., 45 (34): 128-30; Wu et al, 1996 immunology, 2 (1): 21-36; Pluckthun and Pack, 1997 immunology, 3 (2): 83-105; Ridgway et al, 1996 Protein eng., 9 (7): 617-21). Diabodies can be fused to Fc to produce "di-diabodies" (see Lu et al, 2004j. biol. chem., 279 (4): 2856-65).

Other antibodies that may be used in bispecific molecules of the invention are murine, chimeric and humanized monoclonal antibodies.

Bispecific molecules of the invention can be prepared by conjugation component binding specificity using methods known in the art. For example, each binding specificity of a bispecific molecule can be generated separately and then conjugated to each other. When the binding specificity is a protein or peptide, a variety of coupling or crosslinking agents may be used for covalent conjugation. Examples of crosslinking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5' -dithiobis (2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), and sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-l-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al, 1984J. exp. Med. 160: 1686; Liu, MA et al, 1985Proc. Natl. Acad. Sci. USA 82: 8648). Other methods include those described in Paulus, 1985Behring Ins.Mitt.No.78, 118-; brennan et al, 1985Science 229: 81-83 and Glennie et al, 1987J. Immunol.139: 2367 and 2375. The conjugating agents were SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

When the binding specificities are antibodies, they may be conjugated by thiol bonding of the C-terminal hinge region of the two heavy chains. In particular embodiments, the hinge region is modified to contain an odd number of thiol residues, for example 1, prior to conjugation.

Alternatively, both binding specificities can be encoded in the same vector, and expressed and assembled in the same host cell. This method is particularly useful where the bispecific molecule is a mAb x mAb, mAb x Fab, Fab x F (ab') 2, or ligand x Fab fusion protein. The bispecific molecules of the invention may be single chain molecules comprising one single chain antibody and a binding determinant, or single chain bispecific molecules comprising two binding determinants. A bispecific molecule can comprise at least two single chain molecules. For example, in U.S. Pat. nos. 5,260,203; U.S. patent nos. 5,455,030; U.S. patent nos. 4,881,175; U.S. Pat. nos. 5,132,405; U.S. Pat. nos. 5,091,513; U.S. patent nos. 5,476,786; U.S. patent nos. 5,013,653; U.S. Pat. nos. 5,258,498; and U.S. patent No. 5,482,858 describes methods for making bispecific molecules.

For example, binding of a bispecific molecule to its specific target is verified by enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (REA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays generally detects the presence of particularly important protein-antibody complexes by using a labeled reagent (e.g., an antibody) specific for the complex of interest.

In another aspect, the invention provides multivalent compounds comprising at least two identical or different antigen binding portions of an antibody of the invention that binds C5. The antigen binding portions may be linked together by protein fusion or covalent or non-covalent bonds. Alternatively, ligation methods have been described for bispecific molecules. The tetravalent compound is obtained, for example, by crosslinking the antibody of the present invention with a constant region, such as an Fc or hinge region, which binds the antibody of the present invention.

Trimerization domains are described, for example, in the Borean patent EP 1012280B 1. Pentameric modules are described, for example, in PCT/EP 97/05897.

Antibodies with extended half-life

The present invention provides antibodies with extended half-lives in vivo that specifically bind to the C5 protein.

Many factors can influence the half-life of a protein in vivo. For example, kidney filtration, metabolism in the liver, degradation by proteolytic enzymes (proteases) and immunogenic responses (e.g., protein neutralization of antibodies and uptake by macrophages and dendritic cells). Various strategies can be used to extend the half-life of the antibodies of the invention. For example, by chemically linking polyethylene glycol (PEG), reCODE PEG, antibody scaffolds, polysialic acid (PSA), hydroxyethyl starch (HES), albumin binding ligands, and carbohydrate protective layers; proteins that bind to serum proteins by genetic fusion, such as albumin, IgG, FcRn, and transfer; by coupling (genetically or chemically) to other binding moieties that bind serum proteins, such as nanobodies, Fabs, DARPins, avimers, affibodies and anticalins; by genetically fusing rPEG, albumin, domains of albumin, albumin binding protein, and Fc; or by incorporation into nanceriers, slow release formulations and medical devices.

To prolong serum circulation of the antibody in vivo, inert polymeric molecules such as high molecular weight PEG can be attached to the antibody or fragment thereof by site-specific conjugation of the PEG to the N-or C-terminus of the antibody or by the epsilon amino groups present on lysine residues, with or without the use of multifunctional linkers. To pegylate an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), such as an active ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or fragment thereof. Pegylation can be performed by acylation or alkylation using a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to include any PEG form that has been used to derivatize other proteins, such as mono (C1-C10) alkoxy or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Linear or branched polymer derivatization with minimal loss of bioactivity was used. The extent of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure correct conjugation of the PEG molecule to the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate by size exclusion or by ion exchange chromatography. The PEG-derivatized antibodies can be tested for binding activity and in vivo efficacy using methods well known to those skilled in the art, for example, by the immunoassay methods described herein. Methods of pegylating proteins are known in the art and can be applied to the antibodies of the invention. See, for example, EP 0154316 to Nishimura et al and EP 0401384 to Ishikawa et al.

Other modified pegylation techniques include the reconstruction of chemoorthogonal directed engineering techniques (ReCODE PEG) that incorporate chemically specific side chains into biosynthetic proteins via a reconstruction system that includes tRNA synthetases and trnas. This technique enables the incorporation of more than 30 new amino acids into biosynthetic proteins in E.coli, yeast and mammalian cells. The tRNA incorporates an unnatural amino acid anywhere where the amber codon is located, converting the amber codon from a stop codon to a codon that signals the incorporation of a chemically specific amino acid.

Recombinant pegylation technology (rPEG) can also be used for serum half-life extension. The technique involves the fusion of a 300-600 amino acid unorganized protein tail to an existing drug protein. Because the apparent molecular weight of the unorganized protein chain is about 15 times greater than its actual molecular weight, the serum half-life of the protein is greatly extended. In contrast to conventional pegylation, which requires chemical conjugation and re-purification, the preparation process is extremely simplified and the product is homogeneous.

Polysialylation is another technique that uses the natural polymer polysialic acid (PSA) to prolong active life and improve the stability of therapeutic peptides and proteins. PSA is a polymer of sialic acid (sugar). When used for protein and therapeutic peptide drug delivery, polysialic acid provides a protective microenvironment on conjugation. This increases the active life of the therapeutic protein in the circulation and prevents it from being recognized by the immune system. PSA polymers are naturally found in the human body. It is adopted by certain bacteria, which have evolved over millions of years, coating their walls with the PSA polymer. These natural polysialylated bacteria are then able to defeat the body's defence system by molecular mimicry. PSA is a natural ultimate theft technique that can be easily produced in large quantities from such bacteria and has predetermined physical characteristics. Even when coupled to proteins, bacterial PSA is completely non-immunogenic because it is chemically identical to PSA in humans.

Another technique involves the use of hydroxyethyl starch ("HES") derivatives linked to antibodies. HES is a modified natural polymer from waxy corn starch and is metabolized by body enzymes. HES solutions are typically administered to replace insufficient blood volume and improve the rheological properties of the blood. Hydroxyethylation of antibodies enables extended circulation half-lives by increasing the stability of the molecule, as well as decreasing renal clearance, resulting in increased biological activity. By varying different parameters, such as the molecular weight of HES, a wide range of HES antibody conjugates can be tailored.

Antibodies with increased half-life in vivo may also be generated by introducing one or more amino acid modifications (i.e., substitutions, insertions, or deletions) into the IgG constant domain or FcRn binding fragment thereof, preferably an Fc or hinge Fc domain fragment. See, for example, international publication nos. WO 98/23289; international publication nos. WO 97/34631; and U.S. Pat. No. 6,277,375.

In addition, the antibody may be conjugated to albumin to make the antibody or antibody fragment more stable in vivo or have a longer half-life in vivo. The art is well known in the art, see, e.g., international publication nos. WO 93/15199, WO 93/15200, and WO 01/77137; and european patent No. EP 413,622.

The half-life enhancing strategy is particularly useful for nanobodies, fibronectin-based binders, and other antibodies or proteins for which enhanced half-life in vivo is desired.

Antibody conjugates

The present invention provides antibodies or fragments thereof that specifically bind to C5 protein recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to a heterologous protein or polypeptide (or fragment thereof, preferably a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids) to produce a fusion protein. In particular, the invention provides fusion proteins comprising an antigen-binding fragment (e.g., a Fab fragment, Fd fragment, Fv fragment, f (ab)2 fragment, VH domain, VH CDR, VL domain, or VL CDR) of an antibody described herein and a heterologous protein, polypeptide, or peptide. Methods for fusing or conjugating proteins, polypeptides or peptides to antibodies or antibody fragments are known in the art. See, e.g., U.S. Pat. nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; european patent nos. EP 307,434 and EP 367,166; international publication Nos. WO 96/04388 and WO 91/06570; ashkenazi et al, 1991, proc.natl.acad.sci.usa 88: 10535-10539; zheng et al, 1995, j.immunol.154: 5590-; and Vil et al, 1992, proc.natl.acad.sci.usa 89: 11337-11341.

Additional fusion proteins can be produced by techniques of gene shuffling, motif shuffling, exon shuffling, and/or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to alter the activity of an antibody or fragment thereof of the invention (e.g., an antibody or fragment thereof with higher affinity and lower off-rate). See generally U.S. Pat. nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; patten et al, 1997, curr. opinion biotechnol.8: 724-33 parts of; harayama, 1998, Trends Biotechnol.16 (2): 76-82; hansson et al, 1999, j.mol.biol.287: 265 to 76; and Lorenzo and blasto, 1998, Biotechniques 24 (2): 308-313 (each of these patents and publications is incorporated by reference in its entirety). The antibody or fragment thereof, or the encoded antibody or fragment thereof, can be altered prior to recombination by random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods. Polynucleotides encoding antibodies or fragments thereof that specifically bind to C5 protein may be recombined with one or more components, motifs, fragments, parts, domains, fragments, etc. of one or more heterologous molecules.

In addition, the antibody or fragment thereof may be fused to a tag sequence, such as a peptide, to facilitate purification. In a preferred embodiment, the tag amino acid sequence is a hexa-histidine peptide, such as the tag provided in the pQE vector (QIAGEN, inc., 9259Eton Avenue, Chatsworth, CA, 91311), many of which are commercially available. Such as Gentz et al, 1989, Proc.Natl.Acad.Sci.USA86: 821-824 provides for convenient purification of the fusion protein. Other peptide tags that may be used for purification include, but are not limited to, the hemagglutinin ("HA") tag, which corresponds to an epitope from the influenza hemagglutinin protein (Wilson et al, 1984, Cell 37: 767) and the "flag" tag.

In other embodiments, the antibodies or fragments thereof of the invention are conjugated to diagnostic or detection reagents. Such antibodies can be used to monitor or predict the onset, development, progression and/or severity of a disease or disorder as part of a clinical testing procedure, such as determining the efficacy of a particular treatment. Such diagnosis and detection can be accomplished by coupling the antibody to a detectable substance, including but not limited to a variety of enzymes, such as but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups such as, but not limited to, streptavidin biotin and avidin/biotin; fluorescent substances such as, but not limited to, umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials such as, but not limited to, luminol; bioluminescent materials such as, but not limited to, luciferase, luciferin, and aequorin; radioactive substances such as, but not limited to, iodine (131I, 125I, 123I, and 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115In, 113In, 112In, and 111In), technetium (99Tc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, and 117 Tin; and positron emitting metals using various positron emission tomography, and nonradioactive paramagnetic metal ions.

The invention also includes the use of an antibody or fragment thereof conjugated to a therapeutic moiety. The antibody or fragment thereof may be conjugated to a therapeutic moiety, such as a cytotoxin, e.g. a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g. an alpha particle emitter. A cytotoxin or cytotoxic agent includes any agent that is harmful to a cell.

In addition, the antibody or fragment thereof may be conjugated to a therapeutic or drug moiety that modifies a given biological response. The therapeutic moiety or drug moiety is not to be construed as limited to a typical chemotherapeutic agent. For example, the drug moiety may be a protein, peptide or polypeptide having a desired biological activity. Such proteins may include, for example, toxins such as abrin, ricin a, pseudomonas exotoxin, cholera toxin, or diphtheria toxin; proteins such as tumor necrosis factor, interferon-alpha, interferon-beta, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, apoptosis agents, anti-angiogenesis agents; or biological response modifiers, such as lymphokines.

In addition, the antibody may be conjugated to a therapeutic moiety, such as a radioactive metal ion, such as an alpha particle emitter, such as 213Bi or macrocyclic chelator for conjugating radioactive metal ions (including but not limited to 131In, 131LU, 131Y, 131Ho, 131Sm) to the polypeptide. In certain embodiments, the macrocyclic chelator is 1,4, 7, 10-tetraazacyclododecane-N, N ', N ", N'" -tetraacetic acid (DOTA), which can be attached to an antibody through a linker molecule. Such linker molecules are well known in the art and are described in Denadro et al, 1998, Clin Cancer Res.4 (10): 2483-90; peterson et al, 1999, bioconjugate. chem.10 (4): 553-7; and Zimmerman et al, 1999, nuclear.med.biol.26 (8): 943-50, each of which is incorporated by reference in its entirety.

Techniques For conjugating therapeutic moieties to Antibodies are well known, see, e.g., Arnon et al, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", Monoclonal Antibodies And Cancer Therapy, Reisfeld et al (ed.), pages 243-56 (orange r. loss, inc. 1985); hellstrom et al, "Antibodies For Drug Delivery," Controlled Drug Delivery (Robinson et al, ed. 2, pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibodies Carriers Of cytotoxic reagents In Cancer Therapy: A Review", Monoclonal Antibodies 84: Biological Antibody Applications, Pincher et al, pp. 475-.

The antibodies can also be attached to a solid support, which is particularly useful for immunoassay or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

5.2 methods of producing antibodies of the invention

5.2.1 nucleic acids encoding antibodies

The present invention provides a substantially purified nucleic acid molecule encoding a polypeptide comprising a segment or domain of a C5 binding antibody chain as described above. Some nucleic acids of the invention comprise a nucleic acid sequence encoding a polypeptide shown in SEQ ID NO: 7. 23, 39, 51, 67, 79, 96, 108, 114, 121, 137, 151, 165, 179, 187, 201, 210, 218, 227, 241, 253, 257, 273, 277 or 281 and/or a nucleotide sequence encoding the heavy chain variable region shown in SEQ ID NO: 8. 24, 40, 52, 68, 80, 90, 102, 122, 138, 152, 166, 180, 188, 202, 211, 219, 228, 242, 261, 265, 269, 285 or 289. In particular embodiments, the nucleic acid molecules are those identified in table 1. Some other nucleic acid molecules of the invention comprise nucleotide sequences that are substantially identical (e.g., at least 65, 80%, 95%, or 99%) to the nucleotide sequences of those nucleic acid molecules identified in table 1. When expressed from an appropriate expression vector, the polypeptides encoded by these polynucleotides are capable of exhibiting C5 antigen binding capability.

Also provided by the present invention are polynucleotides encoding at least one CDR region and generally all three CDR regions from the heavy and light chains of the C5 binding antibodies set forth above. Some other polynucleotides encode all or substantially all of the variable region sequences of the heavy and/or light chains of the C5 binding antibodies set forth above. Because of the degeneracy of the codons, a variety of nucleic acid sequences encode each immunoglobulin amino acid sequence.

The nucleic acid molecules of the invention may encode the variable and constant regions of an antibody. Some of the nucleic acid sequences of the present invention comprise nucleotides encoding a mature heavy chain variable region sequence that hybridizes to the mature heavy chain variable region sequence set forth in SEQ ID NO: 7. 23, 39, 51, 67, 79, 96, 108, 114, 121, 137, 151, 165, 179, 187, 201, 210, 218, 227, 241, 253, 257, 273, 277, or 281 is substantially identical (e.g., at least 80%, 90%, or 99%). Some other nucleic acid sequences comprise nucleotides encoding a mature light chain variable region sequence that hybridizes to the mature light chain variable region sequence set forth in SEQ ID NO: 8. the mature light chain variable region sequences set forth in 24, 40, 52, 68, 80, 90, 102, 122, 138, 152, 166, 180, 188, 202, 211, 219, 228, 242, 261, 265, 269, 285, or 289 are substantially identical (e.g., at least 80%, 90%, or 99%).

Polynucleotide sequences can be generated by de novo solid phase DNA synthesis or by PCR mutagenesis of existing sequences (e.g., the sequences described in the examples below) encoding C5-binding antibodies or binding fragments thereof. Can be prepared by methods known in the art, such as Narang et al, 1979, meth.enzymol.68: 90 phosphotriester process; brown et al, meth.enzymol.68: 109, 1979; beaucage et al, tetra. 1859, 1981, the diethylphosphoramidite method; and the solid support method of U.S. Pat. No. 4,458,066. Such as PCR Technology: principles and Applications for DNA Amplification, h.a. erlich (editors), Freeman Press, NY, 1992; PCR Protocols: a guides to Methods and applications, Innis et al (eds.), Academic Press, San Diego, CA, 1990; mattila et al, Nucleic Acids Res.19: 967, 1991; and Eckert et al, PCR methods and Applications 1: 17, 1991 by PCR to introduce mutations into polynucleotide sequences.

The present invention also provides expression vectors and host cells for the production of the C5 binding antibodies described above. A variety of expression vectors can be used to express a polynucleotide encoding a C5-binding antibody chain or binding fragment. Both viral-based and non-viral expression vectors can be used to produce antibodies in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors, typically with expression cassettes for expression of proteins or RNA, and artificial chromosomes (see, e.g., Harrington et al, Nat Genet 15: 345, 1997). For example, non-viral vectors useful for expressing C5-binding polynucleotides and polypeptides in mammalian (e.g., human) cells include pThioHis a, B & C, pcdna3.1/His, pEBVHis a, B & C (Invitrogen, San Diego, CA), MPSV vectors, and many other vectors known in the art for expressing other proteins. Useful viral vectors include retroviral, adenoviral, adeno-associated viral, herpes virus based vectors, SV40, papilloma virus, HBP EB virus, vaccinia virus vectors and Semliki Forest Virus (SFV) based vectors. See, Brent et al, supra; smith, annu, rev, microbiol.49: 807, 1995; and Rosenfeld et al, Cell 68: 143, 1992.

The choice of expression vector will depend on the intended host cell in which the vector is to be expressed. Typically, the expression vector contains a promoter and other regulatory sequences (e.g., enhancers) operably linked to a polynucleotide encoding a C5 binding antibody chain or fragment. In some embodiments, an inducible promoter is used to prevent expression of the inserted sequence (except under inducing conditions). Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters or heat shock promoters. The culture of the transformed organism can be expanded under non-inducing conditions without biasing the population of coding sequences, the host cell being better able to tolerate the expression products of said coding sequences. In addition to the promoter, other regulatory elements are also needed or desired for efficient expression of C5-binding antibody chains or fragments. These elements typically include the ATG initiation codon and adjacent ribosome binding sites or other sequences. Furthermore, the efficiency of expression can be enhanced by including enhancers suitable for the cell system in use (see, e.g., Scharf et al, ResultsProbl. cell Differ.20: 125, 1994; and Bittner et al, meth.Enzymol., 153: 516, 1987). For example, the SV40 enhancer or the CMV enhancer may be used to increase expression in a mammalian host cell.

The expression vector may also provide a secretion signal sequence position to form a fusion protein with the inserted polypeptide encoded by the C5 binding antibody sequence. More often, the inserted C5 binding antibody sequence is linked to a signal sequence prior to inclusion in the vector. The vector to be used to receive the sequences encoding the antibody light and heavy chain variable domains to which C5 binds sometimes also encodes a constant region or portion thereof. Such vectors allow the expression of the variable regions as fusion proteins with the constant regions, thereby resulting in the production of whole antibodies or fragments thereof. Typically, such constant regions are human constant regions.

The host cell used to carry and express the C5-binding antibody chain may be a prokaryotic cell or a eukaryotic cell. Coli is a prokaryotic host used for cloning and expressing the polynucleotides of the present invention. Other microbial hosts suitable for use include Bacillus, such as Bacillus subtilis, and other Enterobacteriaceae (Enterobacteriaceae), such as Salmonella (Salmonella), Serratia (Serratia), and various Pseudomonas species. In these prokaryotic hosts, expression vectors can also be prepared, which typically contain expression control sequences (e.g., origins of replication) that are compatible with the host cell in question. In addition, there are any number of a variety of well-known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or a promoter system from bacteriophage lambda. The promoter is typically expressed, optionally with an operator sequence, and has a ribosome binding site sequence or the like for initiating and completing transcription and translation. Other microorganisms, such as yeast, may also be used to express the C5-binding polypeptides of the invention. Insect cells may also be used in combination with baculovirus vectors.

In some preferred embodiments, mammalian host cells are used to express and produce the C5-binding polypeptides of the invention. For example, they may be hybridoma cell lines expressing endogenous immunoglobulin genes (e.g., the 1d6.c9 myeloma hybridoma clones described in the examples) or mammalian cell lines carrying exogenous expression vectors (e.g., the SP2/0 myeloma cells exemplified below). These include any normally lethal or normal or abnormal immortalized animal or human cell. For example, a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cell cultures TO express polypeptides is generally discussed in, for example, Winnacker, FROM GENES TO CLONES, VCH Publishers, n.y., 1987. Expression vectors for use in mammalian host cells can include expression control sequences such as origins of replication, promoters and enhancers (see, e.g., Queen et al, Immunol. Rev.89: 49-68, 1986), and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors typically contain promoters from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type specific, stage specific, and/or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (e.g., the human immediate early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

The method used to introduce the expression vector containing the polynucleotide sequence of interest varies depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation may be used for other cellular hosts. (see generally Sambrook, et al, supra). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, biolistic methods, virosomes, immunoliposomes, polycations: nucleic acid conjugates, naked DNA, artificial virions, fusions to the herpes virus structural protein VP22 (Elliot and O' Hare, Cell 88: 223, 1997), agent-enhanced uptake and ex vivo transduction of DNA. For long-term, high-yield production of recombinant proteins, stable expression is often desired. For example, cell lines stably expressing C5-binding antibody chains or binding fragments can be prepared using expression vectors of the invention that contain viral origins of replication or endogenous expression elements and a selectable marker gene. After introduction of the vector, cells may be allowed to grow in the enrichment medium for 1-2 days before switching to the selective medium. The purpose of the selectable marker is to confer resistance to selection and its presence allows cells that successfully express the introduced sequence to grow in selective media. Stably transfected cells that are resistant to proliferation are propagated using tissue culture techniques appropriate to the cell type.

5.2.2 Generation of monoclonal antibodies of the invention

The antibody can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as Kohler and Milstein, 1975 Nature 256: 495 to produce monoclonal antibodies (mAbs). Many techniques for producing monoclonal antibodies can be used, for example, viral transformation or oncogenic transformation of B lymphocytes.

The animal system used to prepare the hybridomas is the murine system. Hybridoma production in mice is a well established procedure. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the invention may be prepared based on the sequence of murine monoclonal antibodies prepared as described above. DNA encoding the heavy and light chain immunoglobulins can be obtained from murine hybridomas of interest using standard molecular biology techniques and engineered to contain non-murine (e.g., human) immunoglobulin sequences. For example, to generate chimeric antibodies, murine variable regions can be joined to human constant regions using methods known in the art (see, e.g., U.S. Pat. No. 4,816,567 to Cabilly et al). To generate humanized antibodies, murine CDR regions can be inserted into human frameworks using methods known in the art. See, for example, U.S. patent No. 5225539 to Winter, and U.S. patent No. 5530101 to Queen et al; 5585089, respectively; 5693762, and 6180370.

In one embodiment, the antibodies of the invention are human monoclonal antibodies. Such human monoclonal antibodies to C5 can be produced using transgenic or transchromosomal mice carrying portions of the human immune system rather than the mouse system. These transgenic and transchromosomal mice include what are referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "human Ig mice".

Small HuMAb(Metarex, Inc.) contains a human immunoglobulin gene minilocus (minioci) encoding unrearranged human heavy (mu and gamma) and K light immunoglobulin sequences, as well as targeted mutations that inactivate endogenous mu and K chain loci (see, e.g., Lonberg, et al, 1994 Nature368 (6474): 856-. Thus, mice were shown to reduce mouse IgM or K expression and in response to immunization, the introduced human heavy and light chain transgenes underwent class switching and somatic mutation, generating high affinity human IgGK monoclonal antibodies (Lonberg, N. et al, 1994 supra; reviewed in Lonberg, N., 1994 Handbook of Experimental Pharmacology 113: 49-101; Lonberg, N. and Huszar, D., 1995 Intern.Rev.Immunol.13: 65-93, and Harding, F. and Lonberg, N., 1995 Ann.Y.Acad.Sci.764: 536-. The preparation and use of HuMAb mice and the genomic modifications carried by such mice are further described in Taylor, l. et al, 1992 Nucleic Acids Research 20: 6287-6295; chen, J. et al, 1993 International Immunology 5: 647-656; tuaillon et al, 1993Proc.Natl.Acad.Sci.USA 94: 3720-3724; choi et al, 1993 Nature Genetics 4: 117-; chen, J. et al, 1993EMBO J.12: 821-830; tuaillon et al, 1994J. Immunol.152: 2912-2920; taylor, L. et al, 1994 International Immunology 579-; and fisherworld, d. et al, 1996Nature Biotechnology 14: 845-851, the contents of all references are expressly incorporated by reference in their entirety. See further U.S. Pat. nos. 5,545,806, both Lonberg and Kay; 5,569,825; 5,625,126, respectively; 5,633,425, respectively; 5,789,650, respectively; 5,877,397, respectively; 5,661,016, respectively; 5,814, 318; 5,874,299 and 5,770,429 Surani et al, U.S. Pat. No. 5,545,807; PCT publication Nos. WO 92103918, WO93/12227, WO 94/25585, WO 97113852, WO 98/24884 and WO 99/45962, all to Lonberg and Kay; and PCT publication No. WO 01/14424 to Korman et al.

In another embodiment, the human antibodies of the invention can be produced using mice carrying human immunoglobulin sequences on transgenes and transchromosomes, such as mice carrying human heavy chain transgenes and human light chain transchromosomes. Such mice, referred to herein as "KM mice", are described in detail in PCT publication WO 02/43478 to Ishida et al.

Further, alternative transgenic animal systems for expressing human immunoglobulin genes are available in the art and can be used to produce the C5-binding antibodies of the invention. For example, an alternative transgene system known as xenolouse (Abgenix, Inc.). In the fields of, for example, U.S. Pat. Nos. 5,939,598 to Kucherlapati et al; 6,075,181; 6,114,598, respectively; such mice are described in 6,150,584 and 6,162,963.

In addition, alternative transchromosomal animal systems for expressing human immunoglobulin genes are available in the art and can be used to produce the C5-binding antibodies of the invention. For example, a mouse carrying a human heavy chain transchromosome and a human light chain transchromosome, referred to as a "TC mouse," can be used; in Tomizuka et al, 2000proc.natl.acad.sci.usa 97: such mice are described in 722-727. Additionally, cows carrying human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al, 2002 Nature Biotechnology 20: 889-894) and can be used to produce the C5-binding antibodies of the invention.

The human monoclonal antibodies of the invention can also be prepared using phage display methods for screening human immunoglobulin gene libraries. Such phage display methods for isolating human antibodies are established in the art or described in the examples below. See, for example: U.S. Pat. Nos. 5,223,409 to Ladner et al; 5,403,484; and 5,571,698; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et al; U.S. patent nos. 5,969,108 and 6,172,197 to McCafferty et al; and U.S. patent No. 5,885,793 to Griffiths et al; 6,521,404; 6,544,731, respectively; 6,555,313, respectively; 6,582,915, and 6,593,081.

The human monoclonal antibodies of the invention can also be prepared using SCID mice in which human immune cells have been reconstituted such that a human antibody response results after immunization. Such mice are described, for example, in Wilson et al, U.S. Pat. Nos. 5,476,996 and 5,698,767.

5.2.3 framework or Fc engineering

Engineered antibodies of the invention include those in which framework residues within the VH and/or VL have been modified, for example, to improve antibody properties. These framework modifications are typically made to reduce the immunogenicity of the antibody. For example, one approach is to "back mutate" one or more framework residues into the corresponding germline sequence. More particularly, antibodies that have undergone somatic mutation contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. In order to restore the framework sequences to their germline configuration, somatic mutations can be "back-mutated" into the germline sequences, for example by site-directed mutagenesis. Such "back-mutated" antibodies are also intended to be encompassed by the present invention.

Another type of framework modification involves mutating one or more residues within the framework region, or even one or more CDR regions, to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This method is also known as "deimmunization" and is described in further detail in U.S. patent publication No. 20030153043 to Carr et al.

In addition to or as an alternative to modifications within the framework or CDR regions, the antibodies of the invention can be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. In addition, the antibodies of the invention can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter their glycosylation, again for altering one or more functional properties of the antibody. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This process is further described in U.S. Pat. No. 5,677,425 to Bodmer et al. The number of cysteine residues in the hinge region of CH1 is altered, for example to facilitate light and heavy chain assembly, or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of an antibody is mutated to reduce the biological half-life of the antibody. More particularly, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc hinge fragment such that the antibody has impaired staphylococcal protein a (SpA) binding relative to native Fc hinge domain SpA binding. This method is further described in U.S. Pat. No. 6,165,745 to Ward et al.

In another embodiment, the antibody is modified to increase its biological half-life. A variety of approaches are possible. For example, one or more of the following mutations may be introduced: T252L, T254S, T256F, as described in U.S. patent No. 6,277,375 to Ward. Alternatively, to increase biological half-life, antibodies can be altered within the CH1 or CL regions to contain a salvage receptor binding epitope taken from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. patent nos. 5,869,046 and 6,121,022 to Presta et al.

In other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids may be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand, but retains the antigen binding ability of the parent antibody. The effector ligand for which the affinity is altered may be, for example, an Fc receptor or the C1 component of complement. This process is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260 to Winter et al.

In another embodiment, one or more amino acids selected from the group consisting of amino acid residues may be replaced with a different amino acid residue such that the antibody has altered C1q binding and/or reduced or eliminated Complement Dependent Cytotoxicity (CDC). This method is described in further detail in U.S. Pat. No. 6,194,551 to Idusogene et al.

In another embodiment, one or more amino acid residues are altered, thereby altering the ability of the antibody to fix complement. This process is described in further detail in PCT publication WO 94/29351 to Bodmer et al.

In yet another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for the fey receptor by modifying one or more amino acids. This process is described in further detail in PCT publication WO 00/42072 to Presta. Furthermore, the binding sites for Fc γ Rl, Fc γ RII, Fc γ RIII and FcRn have been mapped on human IgG1 and variants with improved binding have been described (see Shields, R.L. et al, 2001 J.biol.Chen.276: 6591-6604).

In yet another embodiment, the glycosylation of the antibody is modified. For example, aglycosylated antibodies (i.e., the antibodies lack glycosylation) can be made. Glycosylation can be altered, for example, to increase the affinity of an antibody for an "antigen". Such carbohydrate modifications can be accomplished, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made, thereby removing glycosylation at that site, which results in the removal of one or more variable region framework glycosylation sites. This aglycosylation may increase the affinity of the antibody for the antigen. This process is described in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 to Co et al.

Additionally or alternatively, antibodies with altered types of glycosylation can be made, such as low fucosylated antibodies with reduced amounts of fucose residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to enhance the ADCC ability of the antibody. Such carbohydrate modifications are accomplished, for example, by expressing the antibody in a host cell with an altered glycosylation machinery. Cells having altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention, thereby producing antibodies having altered glycosylation. For example, EP 1,176,195 to Hang et al describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase such that antibodies expressed in such cell line exhibit low fucosylation. PCT publication WO 03/035835 to Presta describes a variant CHO cell line Lecl3 cell that has a reduced ability to attach fucose to Asn (297) -linked carbohydrates, also resulting in low fucosylation of antibodies expressed in the host cell (see also Shields, R.L. et al, 2002 J.biol.chem.277: 26733-26740). PCT publication WO 99/54342 to Umana et al describes an engineered expression of a glycoprotein-modified glycosyltransferase (e.g.,. beta. (1, 4) -N acetylglucosaminyltransferase III (GnTIII)) such that the antibody expressed in the engineered cell line exhibits an increased bisecting GlcNac structure that results in increased ADCC activity of the antibody (see also Umana et al, 1999 Nat. Biotech.17: 176-180).

5.2.4 methods of engineering altered antibodies

As discussed above, the C5-binding antibodies shown herein having VH and VL sequences or full-length heavy and light chain sequences can be used to generate novel C5-binding antibodies by modifying the full-length heavy and/or light chain sequences, VH and/or VL sequences, or constant regions attached thereto. Thus, in another aspect of the invention, the structural features of the C5-binding antibodies of the invention are used to generate structurally related C5-binding antibodies that retain at least one functional property of the antibodies of the invention, such as binding to human C5, and that inhibit one or more functional properties of C5 (e.g., inhibit erythrolysis in a hemolytic assay).

For example, one or more CDR regions of an antibody of the invention or mutations thereof can be recombined with known framework regions and/or other CDRs to produce additional recombinantly engineered C5-binding antibodies of the invention, as discussed above. Other types of modifications include those described in the previous section. Starting materials for the engineering methods are one or more VH and/or VL sequences provided herein, or one or more CDR regions thereof. To produce an engineered antibody, it is not necessary to actually prepare (i.e., express as a protein) an antibody having one or more of the VH and/or VL sequences provided herein, or one or more CDR regions thereof. Rather, the information contained in the sequence is used as starting material to generate a "second generation" sequence from the original sequence, which is then prepared and expressed as a protein.

Thus, in another embodiment, the invention provides methods for making a C5-binding antibody; altering at least one amino acid residue within the heavy chain variable region antibody sequence and/or the light chain variable region antibody sequence to produce at least one altered antibody sequence; and expressing the altered antibody sequence as a protein, the C5 binding antibody consisting of: has a sequence selected from the group consisting of SEQ ID NOs: 1. 17, 33, 61, 131, 145, 159, 173, 195 and 235, a CDR1 sequence selected from SEQ ID NOs: 2. 18, 34, 49, 62, 77, 95, 107, 113, 119, 132, 146, 160, 174, 196, 226, and 236, and/or a CDR2 sequence selected from SEQ ID NOs: 3. 19, 35, 63, 133, 147, 161, 175, 197 and 237 of CDR3 sequences; and has a sequence selected from SEQ ID NOs: 4. 20, 36, 64, 134, 148, 162, 176, 198 and 238, a CDR1 sequence selected from SEQ ID NOs: 5. 21, 37, 65, 135, 149, 163, 177, 199 and 239, and/or a CDR2 sequence selected from SEQ ID NOs: 6. 22, 38, 50, 66, 78, 89, 101, 120, 136, 150, 164, 178, 200, 209, and 240, and a light chain variable region antibody sequence.

Thus, in another embodiment, the invention provides methods for making C5-binding antibodies optimized for expression in mammalian cells; altering at least one amino acid residue within a full-length heavy chain antibody sequence and/or a full-length light chain antibody sequence to produce at least one altered antibody sequence; and expressing the altered antibody sequence as a protein, the C5 binding antibody consisting of: has a sequence selected from the group consisting of SEQ ID NOs: 9. 25, 41, 53, 69, 81, 97, 109, 115, 123, 139, 153, 167, 181, 189, 203, 212, 220, 229, 243, 249, 254, 258, 274, 278, and 282; and a full length light chain antibody sequence having a sequence selected from the group consisting of 10, 26, 42, 54, 70, 82,91, 103, 124, 140, 154, 168, 182, 190, 204, 213, 221, 230, 244, 251, 262, 266, 270, 286, and 290.

Altered antibody sequences can also be prepared by screening antibody libraries with fixed CDR3 sequences or minimal essential binding determinants as well as diversity in CDR1 and CDR2 sequences as described in US 20050255552. Screening can be performed according to any screening technique suitable for screening antibodies from antibody libraries, such as phage display technology.

Standard molecular biology techniques can be used to prepare and express altered antibody sequences. The antibodies encoded by the altered antibody sequences are antibodies that retain one, some, or all of the functional properties of the C5 binding antibodies described herein, including, but not limited to, specific binding to human and/or cynomolgus C5; and the antibody inhibits erythrocyte lysis in a hemolysis assay.

The functional properties of the altered antibodies can be assessed using standard assays available in the art, such as those illustrated in the examples (e.g., ELISA).

In certain embodiments of the methods of the invention for engineering antibodies, mutations may be introduced randomly or selectively along all or part of the C5 binding antibody coding sequence, and the resulting modified C5 binding antibodies may be screened for binding activity and/or other functional properties as described herein. Methods of mutagenesis have been described in the art. For example, PCT publication WO 02/092780 to Short describes methods for generating and screening antibody mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof. Alternatively, PCT publication WO 03/074679 to Lazar et al describes methods for optimizing antibody physicochemical properties using computational screening methods.

5.3 characterization of the antibodies of the invention

Antibodies of the invention can be characterized by a variety of functional assays. For example, they can be characterized by their ability to inhibit red blood cell lysis in a hemolytic assay, affinity for C5 protein (e.g., human and/or cynomolgus C5), epitope packaging (binding), their resistance to proteolysis, and the ability to block the complement cascade, e.g., their ability to inhibit MAC formation.

A variety of methods are available for determining the presence of complement pathway molecules and activation of the complement system (see, e.g., U.S. Pat. No. 6,087,120; and Newell et al, J Lab Clin Med, 100: 437-44, 1982). For example, inhibition of complement-mediated lysis (hemolysis) of red blood cells can be determined by (i); (ii) determining the ability to inhibit C3 or C5 cleavage; and (iii) inhibition of alternative pathway mediated hemolysis.

The two most commonly used techniques are hemolytic assays (see, e.g., Baatrup et al, Ann Rheum Dis, 51: 892-7, 1992) and immunological assays (see, e.g., Auda et al, Rheumatotol Int, 10: 185-9, 1990). The hemolysis technique measures the functional ability of the complete sequence in the classical or alternative pathway. Immunological techniques measure the protein concentration of a particular complement component or lysate. Other assays that may be used to detect complement activation or to measure the activity of complement components in the Methods of the invention include, for example, T cell proliferation assays (Chain et al, J immunological Methods, 99: 221-8, 1987) and Delayed Type Hypersensitivity (DTH) assays (Forstrom et al, 1983, Nature 303: 627-629; Halliday et al, 1982, Assessment of Immune Status by the Leucocyte Adherence initiation Test, Academic, New York, pages 1-26; Koppi et al, 1982, cell. immunological.66: 394-406; and U.S. Pat. No. 5,843,449).

In the hemolysis technique, all complement components must be present and functional. Thus, hemolysis techniques can screen for functional integrity and deficiencies of the complement system (see, e.g., Dijk et al, J Immunol methods 36: 29-39, 1980; Minh et al, Clin Lab Haematol.5: 23-341983; and Tanaka et al, J Immunol 86: 161-170, 1986). To determine the functional capacity of the classical pathway, sheep red blood cells coated with hemolysin (rabbit IgG directed against sheep red blood cells) or chicken red blood cells sensitized with rabbit anti-chicken antibody were used as target cells (sensitized cells). These Ag-Ab complexes activate the classical pathway and lead to lysis of the target cells when the components are functional and present in sufficient concentration. To determine the functional capacity of the alternative pathway, rabbit erythrocytes were used as target cells (see, e.g., U.S. patent No. 6,087,120).

To test the ability of the antibodies to inhibit MAC (membrane attack complex) formation, a MAC deposition assay can be performed. Briefly, zymosan can be used to activate the alternative pathway and IgM can be used to activate the classical pathway. Fabs were preincubated with human serum and added to plates coated with zymosan or IgM. Percent inhibition of MAC deposition for each sample relative to baseline (EDTA-treated human serum) and positive control (human serum) can be calculated.

The ability of the antibody to bind to C5 can be detected by directly labeling the antibody of interest, or indirectly detecting binding without labeling the antibody and using a variety of sandwich assays known in the art.

In some embodiments, the C5-binding antibody of the invention blocks or competes for binding of the reference C5-binding antibody to the C5 polypeptide. These may be fully human C5 binding antibodies as described above. They may also be other mouse, chimeric or humanized C5 binding antibodies that bind the same epitope as the reference antibody. The ability to block or compete for reference antibody binding indicates that C5 in the test binds to the same or similar epitope defined by the reference antibody, or an epitope sufficiently close to the epitope bound by the reference C5 binding antibody. Such antibodies may have, inter alia, advantageous properties identified for reference antibodies. The ability to block or compete with a reference antibody can be determined by, for example, a competitive binding assay. The antibodies in the test are tested for their ability to inhibit specific binding of a reference antibody to a common antigen, such as a C5 polypeptide, using a competitive binding assay. If an excess of test antibody substantially inhibits binding of the reference antibody, the test antibody competes with the reference antibody for specific binding to the antigen. By substantially inhibited is meant that the test antibody reduces specific binding of the reference antibody by typically at least 10%, 25%, 50%, 75% or 90%.

There are many known competitive binding assays that can be used to assess that the C5-binding antibody competes with the reference C5-binding antibody for binding to the C5 protein. Such assays include, for example, solid phase direct or indirect Radioimmunoassays (RIA), solid phase direct or indirect Enzyme Immunoassays (EIA), sandwich competition assays (see Stahli et al, Methods in Enzymology 9: 242-253, 1983); solid phase direct biotin-avidin EIA (see Kirkland et al, J.Immunol.137: 3614-3619, 1986); solid phase direct labeling assay, solid phase direct labeling sandwich assay (see Harlow & Lane, supra); direct labeling of RIA using an I-125 labeled solid phase (see Morel et al, mol. Immunol.25: 7-15, 1988); solid phase direct Biotin-avidin EIA (Cheung et al, Virology 176: 546-552, 1990); and directly labeled RIA (Moldenhauer et al, Scand. J. Immunol.32: 77-82, 1990). Typically, such assays involve the use of purified antigen bound to a solid surface or cells carrying either an unlabeled test C5 binding antibody or a labeled reference antibody. Competitive inhibition is determined by measuring the amount of label bound to a solid surface or cells in the presence of the test antibody. The test antibody is typically present in excess. Antibodies identified by competition assays (competitor antibodies) include antibodies that bind the same epitope as the reference antibody and antibodies that bind a nearby epitope that is close enough to the epitope bound by the reference antibody to be sterically hindered.

To determine whether the selected C5-binding monoclonal antibody binds a unique epitope, each antibody can be biotinylated using commercially available reagents (e.g., reagents from Pierce, Rockford, IL). Competition studies using unlabeled and biotinylated monoclonal antibodies can be performed using ELISA plates coated with C5 polypeptide. Biotinylated MAb binding can be detected using a streptavidin-avidin-alkaline phosphatase probe. To determine the isotype of the purified C5-binding antibody, an isotype ELISA can be performed. For example, the wells of a microtiter plate may be coated with 1. mu.g/ml of anti-human IgG overnight at 4 ℃. After blocking with 1% BSA, the plates were reacted with 1. mu.g/ml or less of monoclonal C5-binding antibody or purified isotype control for 1 to 2 hours at room temperature. The wells are then reacted with human IgGl or human IgM specific alkaline phosphatase conjugated probes. The plates were then developed and analyzed to determine the isotype of the purified antibody.

To demonstrate binding of the monoclonal C5 binding antibody to hepatocytes expressing C5 polypeptide, flow cytometry can be used. Briefly, a cell line expressing C5 (grown under standard growth conditions) was mixed with various concentrations of C5 binding antibody in PBS containing 0.1% BSA and 10% fetal bovine serum and incubated at 37 ℃ for 1 hour. After washing, the cells were reacted with fluorescein-labeled anti-human IgG antibody under the same conditions as the primary antibody staining. Samples can be analyzed by FACScan instruments using light and side scatter properties to gate individual cells. Alternative assays using fluorescence microscopy may be used (in addition to or instead of) flow cytometry assays. Cells can be stained as described above and detected by fluorescence microscopy. This method allows the display of single cells but with reduced sensitivity depending on the density of the antigen.

The reactivity of the C5-binding antibodies of the invention with C5 polypeptides or antigen fragments can be further tested by western blotting. Briefly, purified C5 polypeptide or fusion protein, or cell extracts from cells expressing C5, can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens were transferred to nitrocellulose membranes, blocked with 10% fetal bovine serum, and probed with the monoclonal antibodies to be tested. Human IgG binding can be detected using anti-human IgG alkaline phosphatase and developed with BCIP/NBT substrate tablets (Sigma chem.co., st.louis, MO).

Examples of functional assays are also described in the examples section below.

5.4 prophylactic and therapeutic uses

The invention provides methods of treating a disease or condition associated with increased complement activity by administering to a subject in need thereof an effective amount of an antibody of the invention. In particular embodiments, the invention provides methods of treating age-related macular degeneration (AMD) by administering to a subject in need thereof an effective amount of an antibody of the invention.

Antibodies of the invention may be used, inter alia, to prevent the progression of dry AMD to wet AMD, to slow and/or prevent the progression of geographic atrophy (geographic atrophy), and to ameliorate vision loss resulting from the progression of dry AMD. It may also be used in combination with anti-VEGF therapy to treat wet AMD patients.

In some embodiments, the invention provides methods of treating a complement-associated disease or disorder by administering to a subject in need of treatment an effective amount of an antibody of the invention. Examples of known complement-associated diseases or disorders include: neurological disorders, multiple sclerosis, stroke, Guillain-Barre syndrome, traumatic brain injury, Parkinson's disease, diseases in which complement activation is inappropriate or undesirable, hemodialysis complications, hyperacute allograft rejection, xenograft rejection, interleukin 2-induced toxicity during IL-2 therapy, inflammatory diseases, autoimmune disease inflammation, Crohn's disease, adult respiratory distress syndrome, thermal injury including burns or cold injury, post-ischemic reperfusion conditions, myocardial infarction, balloon angioplasty, post-pump syndrome in cardiopulmonary bypass or renal bypass, hemodialysis, renal ischemia, peri-reconstruction mesenteric artery reperfusion, infectious disease or sepsis, immune complex disorders and autoimmune diseases, rheumatoid arthritis, Systemic Lupus Erythematosus (SLE), SLE nephritis, proliferative disorders, autoimmune diseases, Hemolytic anemia, and myasthenia gravis. In addition, other known complement-associated diseases are pulmonary diseases and disorders such as dyspnea, hemoptysis, ARDS, asthma, Chronic Obstructive Pulmonary Disease (COPD), emphysema, pulmonary embolism and infarction, pneumonia, fibrotic dust diseases, inert dusts and minerals (e.g., silicon, coal powder, beryllium and asbestos), pulmonary fibrosis, organic dust diseases, chemical injury (due to irritating gases and chemicals such as chlorine, phosgene, sulfur dioxide, hydrogen sulfide, nitrogen dioxide, ammonium, and hydrochloric acid), smoke injury, thermal injury (e.g., burns, frostbite), asthma, allergies, bronchoconstriction, hypersensitivity, parasitic diseases, goodpasture's syndrome, pulmonary vasculitis, and immune complex-related inflammation.

In particular embodiments, the invention provides methods of treating a complement-associated disease or disorder by administering to a subject in need of treatment an effective amount of an antibody of the invention, wherein the disease or disorder is asthma, arthritis (e.g., rheumatoid arthritis), autoimmune heart disease, multiple sclerosis, inflammatory bowel disease, ischemia-reperfusion injury, barth's syndrome, hemodialysis, systemic lupus erythematosus, psoriasis, multiple sclerosis, transplantation, central nervous system diseases such as alzheimer's disease and other neurodegenerative conditions, aHUS, glomerulonephritis, bullous pemphigoid, or MPGNII.

In certain embodiments, the invention provides methods of treating glomerulonephritis by administering to a subject in need thereof an effective amount of a composition comprising an antibody of the invention. Symptoms of glomerulonephritis include, but are not limited to, proteinuria; reduced Glomerular Filtration Rate (GFR); serum electrolyte changes including azotemia (uremia, excessive blood urea nitrogen-BUN) and salt retention, resulting in water retention, leading to hypertension and edema; hematuria and abnormal urine deposits, including red blood cell casts; hypoalbuminemia; hyperlipidemia; and fatty urine. In particular embodiments, the invention provides methods of treating Paroxysmal Nocturnal Hemoglobinuria (PNH) by administering to a subject in need thereof an effective amount of a composition comprising an antibody of the invention.

In particular embodiments, the invention provides methods of reducing immune and hemostatic system dysfunction associated with extracorporeal circulation by administering to a subject in need of treatment an effective amount of a composition comprising an antibody of the invention. The antibodies of the invention may be used in any method that involves circulating blood in a patient's blood vessel through a catheter and back into the patient's blood vessel, the catheter having a mesh surface comprising a material capable of causing at least one of complement activation, platelet activation, leukocyte activation, or platelet-leukocyte adhesion. Such methods include, but are not limited to all forms of ECC, as well as methods involving the introduction of artificial or foreign organs, tissues or vessels into the blood circulation of a patient.

Other therapeutic agents can also be administered to a subject to be treated with a therapeutic agent of the present invention using known methods of treating conditions associated with macular degeneration, such as antibiotic treatment as described in U.S. Pat. No. 6,218,368. In other therapies, immunosuppressive agents such as cyclosporine are therapeutic agents capable of suppressing the immune response. These therapeutic agents include cytotoxic drugs, corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDs), specific T lymphocyte immunosuppressive agents, and antibodies or fragments thereof (see Physicians' Desk Reference, 53 th edition, Medical Economics Company inc., Montvale, n.j. (1999)). Immunosuppressive therapy is typically continued at weekly, monthly, trimester, sixty-monthly or yearly intervals. In some patients, the treatment is administered for the remainder of the patient's life.

When a therapeutic agent of the present invention is administered with another therapeutic agent, the two therapeutic agents may be administered sequentially in any order or simultaneously. In some aspects, an antibody of the invention is administered to a subject who is also receiving treatment with a second therapeutic agent (e.g., verteporfin). In another aspect, the binding molecule is administered in conjunction with a surgical treatment.

Suitable therapeutic agents for combination therapy with C5-binding antibodies include those known in the art that are capable of modulating the activity of complement components (see, e.g., U.S. patent No. 5,808,109). Other therapeutic agents have been reported to reduce complement-mediated activity. Such therapeutic agents include: amino acids (Takada, Y., et al Immunology 1978, 34, 509); phosphonates (Becker, l.biochem.biophy.acta1967, 147, 289), polyanionic materials (Conrow, r.b. et al j.med.chem.1980, 23, 242); sulfonyl fluorides (Hansch, c.; Yoshimoto, m.j.med.chem.1974, 17, 1160, and references cited therein); polynucleotides (DeClercq, p.f. et al biochem. biophysis. res. commun.1975, 67, 255); pimaric acid (Glovsky, m.m. et al j.immunol.1969, 102, 1); porphine (Lapidus, m. and Tomasco, j. immunopharmacol.1981, 3, 137); some anti-inflammatory agents (burger, j.j. et al j.immunol.1978, 120, 1625); phenols (Muller-Eberhard, H.J.1978, Molecular Basis of Biological DegradativeProcesses, Berlin, R.D., et al, ed., Academic Press, New York, page 65); and benzamidines (Vogt, W. et al Immunology 1979, 36, 138). Some of these agents act by generally inhibiting proteases and esterases. Other therapeutic agents are not specific for any particular intermediate step in the complement pathway, but inhibit more than one step of complement activation. Examples of the latter compounds include benzamidines, which block the utilization of C1, C4 and C5 (see e.g. Vogt et al immunol.1979, 36, 138).

Additional therapeutic agents known in the art that inhibit the activity of complement components include the fungal metabolite K-76 from Stachybotrys (Stachybotrys) (Corey et al, J.Amer.chem.Soc.104: 5551, 1982). K-76 and K-76COOH have been shown to inhibit complement primarily at step C5 (Hong et al, J.Immunol.122: 2418, 1979; Miyazaki et al, Microbiol.Immunol.24: 1091, 1980) and to prevent production of chemokines from normal human complement (Bumpers et al, Lab.Clinc. Med.102: 421, 1983). In high concentrations of K-76 or K-76COOH, some inhibition of the reactions of C2, C3, C6, C7, and C9 with their respective previous vehicles was shown. K-76 or K-76COOH has also been reported to inhibit the C3b activation system of complement (Hong et al, J.Immunol.127: 104-108, 1981). Other suitable therapeutic agents for practicing the methods of the invention include griseofulvin (Weinberg, Principles of Medicinal Chemistry, 2 nd edition, Foye, W.O., ed., Lea & Febiger, Philadelphia, Pa., p.813, 1981), isopannarin (Djura et al, Aust.J. Chem.36: 1057, 1983) and the metabolites of Siphonodictyronecalli-phagum (Sullivan et al, Tetrahedron 37: 979, 1981).

The combination treatment regimen may be additive, or it may produce a synergistic result (e.g., a decrease in complement pathway activity beyond that expected from the combined use of the two therapeutic agents). In some embodiments, the invention provides combination therapy for the prevention and/or treatment of AMD, or another complement-associated disease as described above, using a C5 binding antibody of the invention and an anti-angiogenic agent, such as an anti-VEGF therapeutic.

5.5 diagnostic uses

In one aspect, the invention includes diagnostic assays for determining C5 protein and/or nucleic acid expression and C5 protein function in the context of a biological sample (e.g., blood, serum, cells, tissue) or in an individual suffering from a disease or disorder, or at risk of developing a disorder associated with AMD.

Diagnostic assays, such as competitive assays, rely on the ability of a labeled analog ("tracer") to compete with the test sample analyte for a limited number of binding sites on a common binding partner. The binding partner is typically not solubilized prior to or after competition, and the tracer and analyte bound to the binding partner are then separated from the unbound tracer and analyte. The separation is accomplished by decantation (where the binding partner is not dissolved beforehand) or by centrifugation (where the binding partner is precipitated after the competition reaction). The amount of test sample analyte is inversely proportional to the amount of bound tracer, as determined by the amount of marker substance. A dose-response curve for a known amount of analyte is prepared and compared to the test results to quantitatively determine the amount of analyte present in the test sample. When enzymes are used as detection markers, these assays are referred to as ELISA systems. In this format of the assay, competitive binding between the antibody and the C5 binding antibody results in the bound C5 protein, preferably the C5 epitope of the invention, being a measure of, most particularly neutralizing, the antibody in the serum sample.

A significant advantage of the assay is that the neutralizing antibodies (i.e., those that interfere with binding to the C5 protein, particularly the epitope) are assayed directly. This assay, and in particular the ELISA test format, has considerable application in clinical settings and routine blood screening.

Immunological Techniques use polyclonal or monoclonal antibodies directed against different epitopes of various complement components (e.g., C3, C4, C5) to detect, for example, the cleavage products of the complement components (see, e.g., Hugli et al, Immunoassays Clinical Laboratory Techniques 443-460, 1980, Gorski et al, J Immunol Meth 47: 61-73, 1981; Linder et al, J Immunol Meth 47: 49-59, 1981; and Burger et al, J Immunol 141: 553-. Competitive binding of the antibody to the lysate and a known concentration of labeled lysate can then be determined. A variety of assays are available for detecting complement lysis products, such as radioimmunoassays, ELISA's and radiodiffusion assays.

Immunological techniques provide a high sensitivity to detect complement activation because they allow the determination of the formation of lysate in blood from test and control subjects with or without a macular degeneration-related disorder. Thus, in some embodiments of the invention, abnormal complement activation is determined by quantifying the soluble cleavage products of complement components in the plasma of a test subject to obtain a diagnosis of a disorder associated with an ocular disorder. As in Chenoweth et al, N Engl J Med 304: 497-502, 1981; and Bhakdi et al, Biochim Biophys Acta 737: 343-372, 1983. Preferably, only complement activation formed in vivo is measured. This can be accomplished by collecting a biological sample (e.g., serum) from the subject in a medium containing an inhibitor of the complement system, and then determining complement activation (e.g., quantification of the cleavage product) in the sample.

In the clinical diagnosis or monitoring of patients suffering from ocular diseases or condition-related disorders, comparison of the detection of complement proteins with levels in corresponding biological samples from normal subjects indicates that the patients suffer from a condition associated with macular degeneration.

In vivo diagnosis or imaging is described in US 2006/0067935. Briefly, these methods generally comprise administering or introducing to a patient a diagnostically effective amount of a C5 binding molecule operatively attached to a label or tag detectable by non-invasive methods. Allowing sufficient time for the antibody-label conjugate to localize and bind to complement proteins in the eye. The patient is then exposed to a detection device to identify the detectable label, thereby forming a positional image of the C5 binding molecule in the patient's eye. The presence of C5-binding antibody or antigen-binding fragment thereof is detected by detecting whether the antibody-label binds to a component of the eye. Detection of increased levels of a selected complement protein or combination of proteins as compared to a normal individual not having AMD disease indicates a predisposition to and/or onset of a condition associated with macular degeneration. These aspects of the invention are also preferred for use in methods of imaging the eye and combined methods of diagnosis and treatment of angiogenesis.

The invention also relates to the field of predictive medicine, where diagnostic assays, prognostic assays, pharmacogenomics and monitoring clinical trials are used for prognostic (predictive) purposes, thereby prophylactically treating an individual.

The invention also provides prognostic (or predictive) assays for determining whether an individual is at risk for developing a condition associated with dysregulation of complement pathway activity. For example, a mutation in the C5 gene in a biological sample can be determined. Such assays may be used for prognostic or predictive purposes, thereby prophylactically treating an individual prior to the onset of a condition characterized by or associated with C5 protein, nucleic acid expression or activity.

Another aspect of the invention provides methods for determining C5 nucleic acid expression or C5 protein activity in an individual, thereby selecting an appropriate therapeutic or prophylactic agent for the individual (referred to herein as "pharmacogenomics"). Pharmacogenomics allows for the selection of therapeutic agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., examining the genotype of an individual to determine the ability of the individual to respond to a particular therapeutic agent).

Another aspect of the invention relates to monitoring the effect of a therapeutic agent (e.g., a drug) on the expression or activity of C5 protein in a clinical trial.

5.6 pharmaceutical compositions

The present invention provides pharmaceutical compositions comprising a C5 binding antibody (intact or binding fragment) formulated with a pharmaceutically acceptable carrier. The compositions may additionally contain one or more other therapeutic agents suitable for treating or preventing complement-associated diseases (e.g., AMD). The pharmaceutical carrier enhances or stabilizes the composition or facilitates its preparation. Pharmaceutically acceptable carriers include, physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.

The pharmaceutical compositions of the present invention can be administered by a variety of methods known in the art. The route and/or mode of administration will vary depending upon the desired result. Intravenous, intramuscular, intraperitoneal, or subcutaneous administration, or administration near the target site is preferred. In particular embodiments, the antibodies of the invention are formulated such that they can be administered intravitreally to the eye. The pharmaceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compounds, i.e. antibodies, bi-and multispecific molecules, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

The composition should be sterile and fluid. Coatings such as lecithin may be used to maintain the desired particle size in the case of dispersion and surfactants may be used to maintain proper fluidity. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion in the composition of a substance delaying absorption, for example, aluminum monostearate or gelatin.

The pharmaceutical compositions of the present invention may be prepared according to methods well known in the art and conventionally practiced. See, for example, Remington: the Science and Practice of Pharmacy, Mack publishing Co., 20 th edition, 2000; and Sustained and Controlled Release drug delivery Systems, J.R. Robinson, eds., Marcel Dekker, Inc., New York, 1978. The pharmaceutical composition is preferably prepared under GMP conditions. Typically, a therapeutically effective dose or effective dose of a C5 binding antibody is used in the pharmaceutical compositions of the invention. The C5-binding antibody is formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art. The dosage regimen is adjusted to provide the most desirable response (e.g., therapeutic response). For example, rapid perfusion may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. Formulating parenteral compositions in dosage unit form is particularly advantageous for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention can be varied to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular composition of the invention or ester, salt or amide thereof employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds and/or substances used in combination with the particular composition employed, the age, sex, body weight, condition, general health, and prior medical history of the patient being treated.

The physician or veterinarian can begin administering the dose of the antibody of the invention used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. In general, the effective dosage of the compositions of the present invention for treating the allergic inflammatory conditions described herein will vary depending upon a number of different factors, including the means of administration, the target site, the physiological state of the patient, whether the patient is a human or an animal, the other agent administered, and whether the treatment is prophylactic or therapeutic. Titration of the therapeutic dose is required to optimize safety and efficacy. For systemic administration of the antibody, the dosage will range from about 0.0001 to 100mg/kg of host body weight, and more typically between 0.01 and 15mg/kg of host body weight. Exemplary treatment regimens allow systemic administration once every two weeks or once a month or once every 3 to 6 months. For intravitreal administration of the antibody, the dose ranges between about 0.0001 to about 10 mg. Exemplary treatment regimens allow systemic administration once every two weeks or once a month or once every 3 to 6 months.

Antibodies are generally administered in a variety of situations. The interval between single doses may be weekly, monthly or yearly. The intervals may also be irregular, as indicated by determining the blood level of C5-bound antibody in the patient. In some methods of systemic administration, the dose is adjusted to achieve a plasma antibody concentration of 1-1000 μ g/ml, and in some methods 25-500 μ g/ml. Alternatively, the antibody may be administered as a sustained release formulation, in which case less frequent administration is required. The dose and frequency will vary depending on the half-life of the antibody in the patient. Generally, humanized antibodies exhibit a longer half-life than chimeric and non-human antibodies. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the remainder of the life. In therapeutic applications, it is sometimes desirable to have a relatively high dose within a relatively short interval until the progression of the disease is slowed or stopped, and preferably until the patient exhibits a partial or complete improvement in disease symptoms. Thereafter, a prophylactic regimen may be administered to the patient.

6. Examples of the embodiments

The following examples are provided to further illustrate the invention, but not to limit its scope. Other variations of the invention will be apparent to those of ordinary skill in the art and are encompassed by the appended claims.

Example 1: generation of macaque C5 and human C5

1. Generation of macaque C5

Cynomolgus C5 was successfully purified from cynomolgus serum by affinity chromatography using MOR07086 hu IgG 1. Macaque C5 was detected qualitatively by SDS-PAGE, Western blot, mass spectrometry and hemolytic assay. The quality of the purified cynomolgus C5 was shown to be high by SDS-PAGE and western blot. No C3 contamination was confirmed by SDS and western blot. In addition, the identity of the cynomolgus C5 sequence was determined by mass spectrometry and the activity of purified cynomolgus C5 was tested in a haemolysis assay. In the hemolytic assay, the new preparation was equivalent to human C5 (e.g., sample 6 used in the affinity maturation panning restored complement activity of 20% human C5 depleted serum, with activity similar to purified human C5).

2. Quality control of biotinylated and non-biotinylated C5 proteins from human and cynomolgus monkey

The biological activity of purified human C5 was characterized and verified by alternative pathway hemolytic activity. C5 was injected into C5 depleted human serum at various concentrations to obtain EC 50. EC50 values in the range of 0.02-0.1nM are considered acceptable.

Before use, the biological activity of each purified batch of human C5 protein was tested in a hemolytic assay. Identical quality control was performed on cynomolgus monkey C5 after purification from cynomolgus monkey serum. After biotinylation of human and cynomolgus C5 was performed, the biological activity of the substances was also tested in a hemolysis assay to analyze whether biotinylation resulted in loss of activity.

Example 2: slave HuCALGeneration of C-specific antibodies in libraries

Use of a commercial phage display library MorphoSys HuCALThe library serves as a source of antibody variant protein, and the C5 antibody is generated by selecting clones with high binding affinity.

HuCALThe library is a Fab library (Knappik et al, 2000) in which all six CDRs are diversified by appropriate mutations, and which uses the CysDisplayTM technique to link Fab to phage surface (WO01/05950,etc., 2001).

1. Selection by panning C5-specific antibodies from the library

For the selection of antibodies against C5, two different panning strategies were applied. Each panning bank was performed in three separate rounds for six different banks: (a) solid phase panning, in which the antigen (human and cynomolgus C5) was coated directly onto a Maxisorp 96-well microtiter plate (Nunc, wissbaden, Germany); or (b) panning using a solution of biotinylated human and cynomolgus C5 in which phage-antigen complexes were captured by streptavidin magnetic beads (Dynabeads M-280; Dynal).

HuCAL was amplified in 2XYT medium (2XYT-CG) containing 34. mu.g/ml chloramphenicol and 1% glucoseA library. After infection with VCSM13 helper phage with an OD600nm of 0.5 (30 min without shaking at 37 ℃ C.; 30 min with shaking at 250rpm at 37 ℃ C.), the cells were centrifuged (4120 g; 5 min; 4 ℃ C.), resuspended in 2 XYT/34. mu.g/ml chloramphenicol/50. mu.g/ml kanamycin/0.25 mM IPTG and incubated overnight at 22 ℃. From the supernatant PEG precipitation of phage, it in PBS/20% glycerol resuspension and stored at-80 ℃. Phage amplification between two panning cycles was performed as follows: infection of mid-log E.coli TG1 cells with eluted phageAnd plated on LB agar (LB-CG plates) supplemented with 1% glucose and 34. mu.g/ml chloramphenicol. After overnight incubation at 30 ℃, the TG1 colonies were scraped from the agar plates and used to inoculate 2xYT-CG until an OD600nm of 0.5 was reached. VCSM13 helper phage was added as described above for infection.

A total of 354 clones from all panning strategies were sequenced, yielding 64 unique clones with the desired properties: binds to human and cynomolgus C5, but not to the anti-targets C3 and C4.

45 clones from solid phase panning and 19 clones from solution panning were selected for protein expression and purification. 4 Fabs from solid phase panning (MOR06525, MOR06756, MOR06757 and MOR06763) and 6 Fabs from solution panning (MOR07086, MOR07087, MOR07091, MOR07092, MOR07093 and MOR07094) entered affinity maturation.

Solid phase panning against C5 on directly coated proteins

The first panning variant was solid phase panning alternating between human C5 (first and third round of selection) and cynomolgus C5 (second round of selection).

Three wells of a Maxisorp plate (F96 Nunc-immunoplate) were coated with 200. mu.l of 50nM C5 overnight at 4 ℃. The coated wells were washed 2 times with 400 μ l PBS and blocked with 350 μ l 5% MPBS on a microtiter plate shaker at room temperature for 2 hours. For each panning, blocking with an equal volume of PBST/5% milk powder at room temperature for about 10¹³HuCALPhage antibody 2 hours. After completion of the blocking procedure, the coated wells were washed 2 times with 400 μ l PBS. Add 200. mu.l of pre-blocked HuCAL to each coated wellPhage antibodies and incubate for 2 hours at room temperature on a shaker. Washing was performed by adding five times 350. mu.l PBS/0.05% Tween, followed by five additional washes with PBS. For theSome panning conditions, more stringent washing procedures were applied.

Phage were eluted from the plate for 10 min using 200. mu.l of 20mM DTT in 10mM Tris/HCl pH8 per well. To 15ml of E.coli TG1 was added DTT phage eluate, which was grown to an OD600 of 0.6-0.8 at 37 ℃ in 2YT medium and incubated for 45 minutes at 37 ℃ in 50ml plastic tubes without shaking for phage infection. After centrifugation at 4120Xg for 5 minutes, the bacterial pellets were resuspended in 600. mu.l of 2XYT medium each, inoculated onto 3XYT-CG agar plates, and incubated overnight at 37 ℃. Colonies were scraped from the plates, phage recovered and amplified as described above.

The second and third rounds of solid phase panning were performed according to the protocol of the first round. For some panning conditions in the second round of selection, the output of the first round was used for selection on cynomolgus C5 to enrich for cynomolgus cross-reactive antibodies.

For some panning conditions, wash stringency was increased and antigen concentration was decreased in three rounds of selection to generate high affinity antibodies.

HuCALThe phagemid library was used to select specific Fab antibody fragments directed against human C5. The first strategy was solid phase panning (panning method as described above) on directly coated human C5 protein.

After the third panning round, the enriched phage pool was removed from23 library vectors (allowing efficient antibody display on phage surface) subcloning into vectors mediating periplasmic expression of soluble Fabsx9_ Fab _ MH expression vector. Single clones were picked and soluble Fabs were expressed from these single clones.

In total 6624 clones were analyzed in the primary screen by binding Fabs directly from bacterial lysates to human C5 immobilized on Maxisorp microtiter plates. 1660 hits with a signal 5 times greater than background were obtained from the primary screen of human C5. 384 hits were further analyzed in a secondary screen to confirm binding at human C5 and to screen for binding to the anti-targets human C3 and C4.

Many primary hits could be confirmed on human C5 and were not cross-reactive with human C3 and C4, but only 6 Fabs had weak cross-reactivity with cynomolgus C5.

As a first result, new solid phase elutriations were performed alternately on human and cynomolgus C5. In parallel, quality control of the purified kiwifruit C5 lot showed high amounts of kiwifruit C3 within the kiwifruit C5 lot. In view of this result, a novel method was applied to screen cynomolgus monkey cross-reactive antibodies. Cynomolgus C5 was captured from cynomolgus sera using C5 binding polyclonal antibodies (see example 3, part 3). Using this method, the initial primary hits were screened again on cynomolgus C5 and 56 clones were confirmed to bind cynomolgus C5.

For alternate solid phase panning, the first round output of the most successful 12 human solid phase panning was used for selection on macaque C5 (protein batches contaminated with macaque C3; unknown in panning). In the second screen 376 clones were confirmed to bind to human C5 and 361 clones bound to cynomolgus C5 captured from cynomolgus serum.

Solution panning on biotinylated C5 protein

The second panning variant was solution panning against biologically active (in a haemolysis assay) biotinylated human C5 and biotinylated cynomolgus C5.

For this panning, 200. mu.l of streptavidin magnetic beads (Dynabeads M-280; Dynal) were washed once with PBS and blocked with Chemibocker for 2 hours at room temperature. Phage eluted with 300. mu.l PBS were also blocked on a rotator for 1-2 hours at room temperature using Chemibocker. Blocked phage were pre-adsorbed twice for 30 min against 50. mu.l of blocked streptavidin magnetic beads. The phage supernatant was transferred to a new closed 2ml reaction tube, human biotinylated C5 was added, and incubated on a rotator at room temperature for 1 hour. To each panning pool 100. mu.l of blocked streptavidin magnetic beads were added and incubated for 10 minutes on a rotator. The beads were collected with a particle separator (Dynal MPC-E) for approximately 2.5 minutes and the solution was carefully removed.

The beads were then washed 7 times in PBS/0.05% Tween using a rotator, followed by 3 more washes with PBS. Phage were eluted from Dynabeads by adding 200. mu.l of 20mM DTT in 10mM Tris/HCl pH8 to each tube and incubated for 10 min. Dynabeads were removed by a magnetic particle separator and the supernatant was added to 15ml of E.coli TG-1 culture grown to an OD600nm of 0.6-0.8. The beads were then washed once with 200. mu.l PBS and the PBS was added to 15ml of E.coli TG-1 culture with additional phage removed. For phage infection, the cultures were incubated for 45 minutes at 37 ℃ in 50ml plastic tubes without shaking. After centrifugation at 4120Xg for 5 minutes, the bacterial pellets were resuspended in 600. mu.l of 2XYT medium each, inoculated onto 3XYT-CG agar plates, and incubated overnight at 37 ℃. Colonies were scraped from the plates, phage were recovered and amplified as described above. The second and third round selections are made in the same manner as the first round selections.

Other panning strategies were to use human C5 and alternate solutions of human and cynomolgus C5 for panning (cynomolgus C3 contaminated protein batches, unknown during panning). The protein is thus biotinylated and the biological functionality remaining after the biotinylation process is confirmed in a haemolytic bioassay.

The phage-antigen complex is captured on streptavidin magnetic beads by the biotin portion of the antigen. After washing, only specifically bound phage were eluted (panning method as described above).

The first screen was done on directly coated proteins (see example 3, part 1) and only 80 clones could be confirmed on human C5. Because of this fact, antigens remain in solution during panning, new screening methods were developed. In a solution ELISA, Fabs were incubated with biotinylated antigen on NeutrAvidin plates. Using this solution screening method, a significantly greater number of human and cynomolgus C5-specific clones can be selected. These results demonstrate that many Fabs from solution panning only recognize C5 in solution or when captured (e.g., by polyclonal C5 binding antibodies).

2. Subcloning and expression of selected Fab fragments

To facilitate rapid expression of soluble Fabs, HuCAL was selectedFab-encoding inserts of phage were subcloned into E.coli expression vectors by XbaI and EcoRIx9_ MH. The Fab fragment carries a C-terminal Myc tag, and a 6 XHis-tag (Chen et al, Gene 139: 73-75(1994)) as a second C-terminal tag. After transformation of the expression plasmids into E.coli TG 1F-cells, chloramphenicol resistant single clones were picked up in the wells of sterile 384-well microtiter plates pre-filled with 60. mu.l of 2XYT-CG medium and incubated overnight at 30 ℃. Mu.l of each E.coli TG-1 overnight culture were transferred to fresh sterile 96-well microtiter plates pre-filled with 40. mu.l of 2XYT medium (supplemented with 34. mu.g/ml chloramphenicol) per well. The microtiter plates were incubated at 30 ℃ on a microplate shaker at 400rpm with shaking until the cultures were slightly cloudy and had an OD600nm of 0.5 (. about.2-4 hours). To these expression plates were added 10. mu.l of 2XYT medium supplemented with 34. mu.g/ml chloramphenicol and 3mM IPTG (isopropyl-. beta. -D-thiogalactopyranoside) (final concentration of 0.5mM IPTG) per well. The microtiter plates were sealed with gas permeable tape and incubated overnight at 30 ℃ with shaking at 400 rpm. To each well of the expression plate, 15. mu.l BEL buffer containing 2.5mg/ml lysozyme, 4mM EDTA and 10U/. mu.l Benzonase was added, and the mixture was shaken in a microtiter plate (400 r)pm) at 22 ℃ for 1 hour, followed by an optional freezing step at-80 ℃ for at least 2 hours. BEL extracts were used for binding analysis by ELISA or Fab SET screening after affinity maturation.

TG-1 cells in shake flask cultures using 750ml 2XYT medium supplemented with 34. mu.g/ml chloramphenicolExpression of x9_ Fab _ MH encoded Fab fragments. The culture was shaken at 30 ℃ until OD600nm reached 0.5. Expression was induced by addition of 0.75mM IPTG20 hours at 30 ℃. The cells were disrupted using lysozyme and the Fab fragments were isolated by Ni-NTA chromatography (Qiagen, Hilden, Germany). Buffer exchange was performed to 1 xDulbecco's PBS (pH 7.2) using a PD10 column. The sample was sterile filtered (0.2 μm, Millipore). The purity of the samples in the denatured, reduced state was determined by SDS-PAGE (15% Criterion Gels, BioRad) and in the native state by size exclusion chromatography (HP-SEC). Protein concentrations were determined by UV-spectrophotometry (Krebs et al, J.Immunol. methods 254: 67-84 (2001)).

At the Fab level, the total expression rate and percentage of monomeric fraction in SEC (size exclusion chromatography) was between the acceptable range to the good range for most of the identified antibody fragments. 64 parental Fabs were expressed and 61 Fabs could be purified. 60 affinity matured Fabs were purified at mg level. Most Fabs are good expressors and have no aggregation propensity.

Example 3: slave HuCALIdentification of C5-specific antibodies in libraries

In the following, four different enzyme-linked immunosorbent assay (ELISA) methods describe the screening for specificity and anti-C5-binding antibodies on the antigen (as bacterial BEL lysates or purified Fabs).

1. Screening on directly coated proteins

Maxisorp (Nunc, Rochester, NY, USA)384 well plates were coated with 20. mu.l of PBS per well, 2.5. mu.g/ml antigen (human C5 and anti-proteins human C3 and C4) at pH 7.4 overnight at 4 ℃. Meanwhile, each well was coated with 20. mu.l of PBS, 5. mu.g/ml Fd fragment-specific sheep anti-human IgG (The Binding Site, Birmingham, UK) diluted at pH 7.4 to examine The expression level of Fab.

The plates were blocked with 5% milk powder in PBS/0.05% Tween20 (PBST) for 1-2 hours at room temperature. After washing the wells with PBST, BEL-extract diluted in PBS, purified HuCAL was addedFabs or control Fabs and incubated for 1 hour at room temperature. To detect Fab binding, anti-HIS 6 antibody (Roche) conjugated to peroxidase was used.

For detection of POD conjugates, the fluorogenic substrate quantablu (pierce) was used according to the manufacturer's instructions. Between all incubation steps, wells of the microtiter plates were washed three times with PBST, and five times with PBST after the final incubation with secondary antibody. Fluorescence was measured on a Tecan GENios Pro plate reader.

2. Solution screening using biotinylated proteins

Following solution panning using biotinylated complement proteins, HuCAL was screened using the ELISA method described belowFabs。

NeutrAvidin plates were blocked overnight at 4 ℃ with 1X Chemibocker (Chemicon) diluted in PBS. These plates were used to screen for binding to human C5 and to the anti-targets C3 and C4. In parallel, Maxisorp 384 was coated with 20. mu.l of PBS per well, 5. mu.g/ml Fd fragment-specific sheep anti-human IgG (The Binding Site, Birmingham, UK) diluted at pH 7.4Well plates (Nunc, Rochester, NY, USA). These plates were used to check Fab expression levels and non-specific biotin binding. The following day, the coated Maxisorp plates were washed 2 times with PBST and blocked with 3% BSA in TBS for 1-2 hours at room temperature. Add Fabs or purified HuCAL to blocked NeutrAvidin and Maxisorp platesPeriplasmic BEL extracts of Fabs.

Subsequently, 20 μ l biotinylated human C5 (to detect specific binding) was added to each well of the NeutrAvidin plate, and biotinylated human C3 and C4 (to detect unwanted binding) were added in parallel. The biotinylated antigen is conjugated to HuCALThe Fabs were incubated at room temperature for 1-2 hours. Biotinylated unrelated antigen transferrin was then added to the Maxisorp plate to detect biotin-bound Fabs (in this case, previously captured by anti-Fd antibody-a Fab fragment).

The following secondary antibodies were used for detection: adding goat anti-human Alkaline Phosphatase (AP) -conjugated streptavidin-AP affinity fragment F (ab') 2 to the Maxisorp expression plates; anti-HIS 6 peroxidase-conjugated mouse antibody (Roche) was added to NeutrAvidin plates and streptavidin-alkaline phosphatase ZYMED and biotinylated transferrin were added to Maxisorp plates.

For the detection of the AP conjugate, the fluorogenic substrate Attophos (Roche Diagnostics, Mannheim, Germany) was used according to the manufacturer's instructions, and for the detection of the POD conjugate, the fluorogenic substrate QuantaBlu (Pierce) was used. Fluorescence was measured in a Tecan GENios Pro plate reader.

Using this method, antibodies can be screened that recognize anti-human C5Fabs of human C5 in solution and exclude the biotin moiety that binds to the target antigen.

3. Determination of Cross-reactivity with Kiwi C5

Polyclonal C5 binding antibody (US Biological Cat # C7850-24) was used to capture cynomolgus C5 from cynomolgus sera.

384-well Maxisorp plates were coated with 20. mu.l/well 5g/ml polyclonal C5 binding antibody in PBS and incubated overnight at 4 ℃. The following day, the plates were washed 3 times with PBST and blocked with 100. mu.l/well of diluent (4% BSA/0.1% Tween 20/0.1% Triton-X100/PBS) for 2 hours at room temperature. Macaque serum (at an approximate concentration of 4g/ml for macaque C5) was diluted 1: 20 in diluent (4% BSA/0.1% Tween 20/0.1% Triton-X100/PBS) and 20. mu.l was added to each well of the 2 XPBST washed Maxisorp plate. After 1 hour incubation at room temperature, the plates were washed with 3x PBST, the BEL lysate containing Fab fragments or purified Fabs was added, and incubated for 1 hour at room temperature. The plates were washed again and the detection antibody anti-HIS 6-POD (Roche #1965085) was added. Addition of the POD substrate: soluble BM Blue (Roche Applied Science) was used in combination with 1M H₂SO₄The reaction was terminated. Absorbance was read at 450nm using a BMG Reader instrument.

Example 4: affinity maturation

1. Construction of affinity maturation libraries of selected C5-binding Fabs

To increase the affinity and biological activity of the selected antibody fragments, the L-CDR3 and H-CDR2 regions were optimized in parallel by cassette mutagenesis using trinucleotide-directed mutagenesis (see, e.g., Virnekas et al, Nucleic Acids Res.22: 5600-. All parental Fab fragments are recovered from the corresponding expression vectors before cloning for affinity maturation: (x9_ MH) was transferred to CysDisplayTM vector via XbaI/EcoRI25 (c). HuCAL by removing one BssHII site interfering with library cloningDisplay carrier23 generation25, for H-CDR2 optimization. To optimize the L-CDR3 of the parent Fabs, the L-CDR3 of the light chain of the binder (405bp), framework 4 and constant region were removed by BpiI/SphI and replaced with the diversified L-CDR3 and framework 4 and all components of the constant region.

The 10 parental C5 binding Fabs were divided into 7 pools according to different selection criteria and Fabs with the same framework only were put together: (1) MOR 07086; (2) MOR06525+6756 (same framework); (3) MOR 06757; (4) MOR 06763; (5) MOR 07087; (6) MOR07091+7092 (same framework); (7) MOR07093+7094 (same framework).

Approximately 1.5. mu.g of individual Fab vector fragments and Fab libraries were ligated with a3 to 5 fold molar excess of the insert carrying the diversified L-CDR3 s. In the second library set, H-CDR2(xhoI/BssHII) was diversified, while the ligation framework regions remained unchanged. To monitor cloning efficiency, the parent H-CDR2 was replaced with a mimetic prior to cloning the diversified H-CDR2 cassette.

Ligation mixtures of different libraries were electroporated into E.coli TOP 10F' cells (Invitrogen) to yield 2x10⁷To 2x10⁸Individual colonies. Amplifying the library. For quality control, several single clones of each library were then picked and sequenced using primers CFR84(VL) and OCAL _ Seq _ hp (vh).

As described above, 7 mature sub-pools were generated and kept separate during the subsequent selection process.

By passingStandard cloning methods generated 14 different affinity maturation libraries (one LCDR3 and one HCDR3 library for each guide or pool) and transformed the diversified clones into electrocompetent e.coli TOP 10F' cells (Invitrogen). Library size was appropriate at 2X10⁷-5x10⁸Within the range. Sequencing of subsequently picked clones revealed 100% diversity. No parental binders were found, but derivatives of all the respective parental import binders were found. Finally, phage were prepared for all 14 libraries individually.

TABLE 2 summary of the maturation libraries

MOR0	Maturation of the plant	VH/VL type	Library size
				6757	HCDR2	VH3	3.70x10E7
6763	HCDR2	VH3	4.95x10E7
				7086	HCDR2	VH1A	1.58x10E8
7087	HCDR2	VH1A	7.85x10E7
				6525+6756	HCDR2	VH5	5.22x10E7
7091+7092	HCDR2	VH5	3.51x10E7
				7093+7094	HCDR2	VH2	2.01x10E7
6757	LCDR3	Vkappa1	1.89x10E7
				6763	LCDR3	Vlambda2	7.35x10E7
7086	LCDR3	Vlambda3	7.54x10E7
				7087	LCDR3	Vkappa1	5.46x10E7
6525+6756	LCDR3	Vlambda2	8.50x10E7
				7091+7092	LCDR3	Vlambda3	4.93x10E8
7093+7094	LCDR3	Vlambda2	1.33x10E8

2. Preparation of antibody-phage for affinity maturation

Amplification in 2XYT Medium containing 34. mu.g/ml chloramphenicol and 1% glucose (2XYT-CG)And (4) maturing the library. After infection with VCSM13 helper phage with an OD600nm of 0.5 (30 min without shaking at 37 ℃ C.; 30 min with shaking at 37 ℃ C., 250 rpm), the cells were centrifuged (4120 Xg; 5 min; 4 ℃ C.), resuspended in 2 XYT/34. mu.g/ml chloramphenicol/50. mu.g/ml kanamycin/0.25 mM IPTG and incubated overnight at 22 ℃. The phages were PEG precipitated twice from the supernatant, resuspended in PBS and used for maturation as described belowAnd (4) elutriation.

3. Standard solution maturation panning on biotinylated C5 protein

As described above, under very stringent conditions, from the affinity maturation library generated in about 10 recovery¹²Individual phages were subjected to panning to select affinity-enhanced C5-specific Fabs.

Solution panning using the respective phage pools was performed using biotinylated C5 or alternatively biotinylated human and cynomolgus C5 proteins. To increase panning stringency and select for increased off-rates, antigen concentrations were reduced and extended wash times were applied (wash conditions are listed in table 3).

TABLE 3 improved wash conditions during selection cycles of solution maturation panning

Previously blocked phages (1: 2 mixture with 2X Chemibocker incubated for 1h at room temperature) were incubated with a low concentration of biotinylated C5 protein for 1-2 hours at room temperature. The panning strategy was similar to the standard solution panning described above. The phage antigen complex was captured by the biotin moiety of C5 onto pre-blocked streptavidin magnetic beads for 30 minutes at room temperature. The beads were then washed more rigorously than normal panning. Elution and amplification of phage was performed as described above.

The second and third rounds of selection were performed in the same manner as the first round, but with more stringent wash conditions and lower antigen concentrations. For each antibody guide or pool, several different panning were performed. For each panning strategy, different stringency conditions are applied. The panning strategy is summarized in table 4.

Table 4 summary of solution maturation panning on biotinylated human C5 and biotinylated cynomolgus monkey C5 1783 and 1784

After maturation panning, the enriched phagemid library was subcloned intox9_ MH expression vector.

4. Cross-combination of optimized VL (L-CDR3) with optimized VH (H-CDR2)

For further improvement of affinity and potency, independently optimized heavy and light chain combinations of mature antibodies from the same parental clones were used (see, e.g., Rauchenberger et al, J.biol. chem.278: 38194-38205 (2003); Chen et al, J.mol. biol.293: 865-881 (1999); and Schier et al, J.mol. biol.263: 551-567 (1996)). This method, called cross-cloning, is applied to binders from the same parental clone.

5. Affinity screening and maturation panning results

A total of 2640 clones from all panning were screened as bacterial lysates for improved affinity on human C5. Initial affinity was assessed by Solution Equilibrium Titration (SET). Based on their estimated affinities, clones from each parental Fab or Fab library were sequenced. Table 5 shows the number of sequenced clones and the number of unique sequences obtained for each panning condition.

TABLE 5 summary of affinity-enhancing clones selected for sequence analysis

Parental/maturation	Antigens	Sequencing of	Unique	Unique parent
					MOR06525+6756 HCDR2	hu/hu/hu	10	9	6525
MOR07086 HCDR2	hu/hu/hu	10	4	7086
					MOR06763 HCDR2	hu/hu/hu	22	10	6763(8x)，7086(2x)
MOR07087 HCDR2	hu/hu/hu	10	4	7087
					MOR06757 HCDR2	hu/hu/hu	10	0
MOR07091+7092 HCDR2	hu/hu/hu	24	7	7092
					MOR07093+7094 HCDR2	hu/hu/hu	10	10	7093
MOR06525+6756 LCDR3	hu/hu/hu	20	5	6756
					MOR07086 LCDR3	hu/hu/hu	10	5	7086
MOR06763 LCDR3	hu/hu/hu	10	8	7086
					MOR07087 LCDR3	hu/hu/hu	6	1	7086
MOR06757 LCDR3	hu/hu/hu	16	0
					MOR07091+7092 LCDR3	hu/hu/hu	6	6	7091(1x)，7092(5x)
MOR07093+7094 LCDR3	hu/hu/hu	10	9	7094
					MOR06525+6756 HCDR2	hu/cyno/hu	10	8	6525
MOR07086 HCDR2	hu/cyno/hu	10	6	7086
					MOR06763 HCDR2	hu/cyno/hu	22	5	6763
MOR06757 HCDR2	hu/cyno/hu	15	2	6757
					MOR07091+7092 HCDR2	hu/cyno/hu	15	6	7091(3x)，7092(3x)
MOR07093+7094 HCDR2	hu/cyno/hu	10	10	7093
					MOR07087 HCDR2	hu/cyno/hu	10	6	7087(5x)，7086(1x)
MOR06525+6756 LCDR3	hu/cyno/hu	12	0
					MOR07086 LCDR3	hu/cyno/hu	10	1	7086
MOR06763 LCDR3	hu/cyno/hu	10	0
					MOR06757 LCDR3	hu/cyno/hu	9	1	7094
MOR07091+7092 LCDR3	hu/cyno/hu	11	9	7091(6x)，7092(3x)
					MOR07093+7094 LCDR3	hu/cyno/hu	10	7	7094
MOR07087 LCDR3	hu/cyno/hu	10	0
					Sum of		338	139

6. Sequence analysis and selection of affinity optimized Fabs for protein production

Very good diversity was maintained by recovering derivatives of all 10 parent Fabs. The nucleotide sequences of the heavy chain (VH) of 188 HCDR2 improved clones and the light chain (VL) variable region of 150 improved LCDR3 clones were determined. 87 unique HCDR2 and 52 unique LCDR3 sequences were selected for detailed analysis of sequence diversity within the mature CDRs. Fabs with potential glycosylation sites in the CDRs were omitted from further characterization.

VH and VL sequence analysis and affinity data show that all 10 parental Fabs produce inheritors with improved affinity. The parents Fabs MOR06525, MOR06757, MOR06763, MOR07087 and MOR07094 produced clones improved only in HCDR2, and the parents MOR06756 and MOR07093 produced clones improved only in LCDR 3. MOR07086, MOR07091 and MOR07092 have mature clones for VH and VL. This latter allows cross-cloning of the VH and VL mature chains. From all data, 60 clones with the greatest affinity and highest diversity among the mature CDRs were selected for Fab expression. Selected VH and VL amino acids and nucleotide sequences are listed in table 1.

Example 5: IgG conversion

1. Conversion to the human IgG2 form

For expression of full-length immunoglobulins (Ig), variable domain fragments of the heavy (VH) and light (VL) chains are isolated fromx9_ MH Fab expression vector subcloned into2_ h _ Ig vector series for human IgG 2. Restriction enzymes MfeI and BlpI for subcloning VH Domain fragments into2_ h _ IgG 2. VL domain fragmentation by EcoRV and BsiWI sitesSubcloning of 2_ h _ Ig kappa, whereas the cloning was accomplished using EcoRV and HpaISubcloning of 2_ h _ Ig lamda 2.

All 10 parental Fabs (MOR06525, 6756, 6757, 6763, 7086, 7087, MOR07091, 7092, 7093 and 7094) were converted to adult IgG 2. IgGs are also expressed.

2. Conversion to the human IgG1AA form

To express full-length immunoglobulins, variable domain fragments of the Fab heavy (VH) and light (VL) chains were subcloned from the Fab expression vector into the IgG1 expression vector. Restriction enzymes MfeI and BlpI for subcloning VH Domain fragments into2_ h _ IgG1AA, in which the leucines at positions 234 and 235 were mutated to alanines to eliminate FcR γ binding and attenuate effector function. Restriction enzymes EcoRV and HpaI for subcloning VL domain fragments into2_ h _ Ig λ 2.

The following mature Fabs with the desired profile were subcloned into the human IgG1AA form: MOR07832, 7834, 7872, 7876, 7829, 7871, 7865, 7873, 7830, 7878, 7910. Cross-cloning at the IgG level was accomplished by transfecting cells with a combination of light and heavy chain constructs. For example, MOR08114 is the product of a germlined heavy chain from MOR07829 and a germlined light chain from MOR 07871. Table 6 summarizes the germlined IgGs of the most relevant cross-clones.

TABLE 6 summary of germlined IgGs for the most relevant cross-clones

3. Transient expression and purification of human IgG

Eukaryotic HKB11 and HEK293 cells were transfected with equimolar proportions of IgG heavy and light chain expression vector DNA. Cell culture supernatants were collected 3 or 7 days post-transfection and subjected to standard protein a affinity chromatography (rProteinA FF or MabSelect SURE, GE Healthcare). Unless otherwise stated, buffer exchanged to 1 × Dulbcecco's PBS (pH 7.2, Invitrogen) and sterile filtered samples (0.2 μm). The purity of IgG in the denatured, reduced and non-reduced conditions was analyzed in SDS-PAGE or by using an Agilent BioAnalyzer, and the purity of IgG in the native state was analyzed by HP-SEC.

Example 6 germlining

Use ofDirected mutagenesis kit (Stratagene) the IgG constructs were germlined by site-directed mutagenesis. The N-terminal DI of MOR 08111V λ 2 was changed to ES to match the human germline sequence and avoid the terminal Q (the N-terminal Q may form pyroglutamate). The N-terminal DI of MOR 08110V λ 3, MOR08113V λ 3 and MOR 08114V λ 3 were germlined into SY, which is the most common sequence in the human λ 3 gene. The N-terminus QVQ of MOR08111 VH2 was germlined into an EVT to match the λ 2 gene and avoid terminal Q. The N-terminal Q of MOR08109 VH5, MOR08110 VH5, MOR08113VH5 and MOR08114 VH5 was also mutated to E.

The framework sequence of MOR 08109V λ 3 was synthesized to match the human λ 3j gene and cloned into the expression vector using NheI and HpaI restriction sites. The sequence alignment of the antibody variable domains with their respective closest human germline sequences is shown in figure 1.

Example 7: affinity assay

1. Kon/Koff and K for anti-human C5 antibodies using surface plasmon resonance (Biacore)_DMeasurement of (2)

It was determined that the anti-Fab antibodies used to immobilize the Fabs on the Biacore chip affect the binding affinity of each Fab to human C5 to varying degrees, thus making comparison between Fabs difficult. Biacore analysis was performed on IgG antibodies.

CM4 chips were coated with 50. mu.g/ml goat anti-human Fc antibody (500-2000RU) in 10mM acetate buffer, pH 4.5, using standard EDC-NHS amine coupling chemistry. Each anti-human C5IgG in HBS-EP buffer was captured on the chip at a constant flow rate of 10. mu.l/min for a constant time, resulting in a ligand density of approximately 20 RU. After capture of anti-human C5IgG, different concentrations of human or cynomolgus C5 in the range of 0.156nM to 2.5nM were injected. Each cycle was completed with two regeneration steps with phosphoric acid. All run conditions were performed in 1x HBS-EP buffer at 25 ℃. The resulting signal is adjusted by subtracting the refractive index value from the binding step in the reference flow cell and in the absence of analyte by means of a double reference. Data were collected at 10Hz and analyzed using Biacore T100 Evaluation Software Version 1.1 (GE). The program used a global fit analysis method for determining the rate and affinity constant of each interaction.

The specificity of the antibody was determined. Preferably, the Kon and Koff values for the combination of human and cynomolgus C5 are as follows: kon > 1x10⁵，Koff＜1x10^-4. These assays for germlined IgGs were performed in Biacore and the resulting data are listed in table 7.

TABLE 7K of germlined IgGs as determined in Biacore_DKon and Koff values

2. Pemolar affinity (Meso Scale Discovery (MSD)) of purified Fabs or Fabs bacterial lysates was determined using Solution Equilibrium Titration (SET)

For K by Solution Equilibrium Titration (SET)_DThe monomeric fraction of the Fab protein (at least 90% of the monomer content by analytical SEC; Superdex75, Amersham Pharmacia) was used for the determination of (1). Affinity assays in solution were performed essentially as described in the reference (Friguet al, J.Immunol Methods 77: 305-319 (1985)). To improve the sensitivity and accuracy of the SET method, a switch was made from the classical ELISA to ECL-based techniques (Haenel et al, Anal biochem 339: 182- & 184 (2005)).

1mg/ml goat anti-human (Fab) was labeled with ECL Sulfo-TAGTM NHS-Ester (Meso Scale discovery, Gaithersburg, Md., USA) according to the manufacturer's instructions₂Fragment-specific antibodies (Dianova). Experiments were performed in polypropylene microtiter plates and PBS pH 7.4 containing 0.5% BSA and 0.02% Tween20 was used as assay buffer. In the ratio of K_DAt least 10 times higher concentration, starting with 2ⁿUnlabeled antigen was serially diluted. Wells without antigenFor determining the Bmax value; wells with neither antigen nor Fab were used as assay background. After addition of e.g. 10pM Fab (final concentration in a final volume of 60 μ l), the mixture is incubated overnight at room temperature. Fab concentrations used with expected K_DSimilar or lower than expected K_D。

Streptavidin MSD plates were coated with 0.2. mu.g/ml biotinylated human C5 (30. mu.l/well) and blocked with 5% BSA in PBS. Subsequently, equilibrated samples were transferred to those plates (30 μ l per well) and incubated for 20 minutes. After washing, a final dilution of 1: 1500 of sulfo-tagged detection antibody (goat anti-human (Fab)2) was added to the MSD plate and incubated for 30 minutes on an Eppendorf shaker (700 rpm).

After washing and addition of 30. mu.l/well of MSD Read buffer T containing surfactant, the electrochemiluminescence signal was detected using a Sector imager 6000(Meso Scale Discovery, Gaithersburg, Md., USA).

Data were evaluated using xlfit (idbs) software applying a custom fit model. For data evaluation, i.e. K of Fab molecules_DThe following fitted model (model of Abraham et al, modified according to 200515) was used for the assay: y ═ Bmax- (Bmax/(2. cFab) ((x + cFab + KD) -4. x. cFab))); cFab: the Fab concentration used; x: total soluble antigen concentration (binding site) applied; sqrt: square root. The (monomer) affinity of the affinity optimized C5 binding Fabs was determined in solution using the assay conditions described above.

Parent Fabs

To further characterize the C5-binding antibodies, the affinity of the parent Fabs to human C5 was determined. Because the focus was characterized for efficacy in hemolytic assays, affinity assays were performed only for the most relevant Fabs. For a reliable determination of monovalent affinity only Fab batches were used for the determination, which showed a monomer fraction of > 90% in qualitative size exclusion chromatography.

The affinities of the 10 parental Fabs entering affinity maturation are summarized in table 8. The affinity varied between 72pM and 3.7 nM.

TABLE 8 affinity of 10 parent Fabs determined in SET

Mature Fabs

The monovalent affinity of purified Fabs to human C5 was determined in SET. The affinities were in the low pM range and the greatest affinities were obtained for derivatives of MOR07086, 7091, 7092 and 7093. Subsequent affinity assays of these derivatives with cynomolgus C5 showed affinities ranging from medium to low pM.

The affinity maturation process is very successful in producing all components with significantly improved affinity binders. Table 9 summarizes the affinity of cynomolgus C5 and human best improved binders. Certain Fabs have a K of ≦ 30pM for human C5_DFor macaque C5, K is less than or equal to 150pM_D。

TABLE 9 summary of the affinities of best affinity-improved Fabs to human and cynomolgus C5

3. Determination of K of IgG molecules using Solution Equilibrium Titration (SET)_D

The affinity of germlined IgGs (human IgG1AA form) for human and cynomolgus C5 was determined in SET as described below. A similar data set of two independent assays showed that the lead IgGs had a higher affinity for human C5 than the reference IgG 5G1.1 (see us patent No. 6,355,245). The final IgGs had an affinity in the range of 1 to 14pM for human C5 and 3 to 29pM for cynomolgus C5.

TABLE 10 Final in SETK for guiding IgGs (human IgG1AA format)_DValue determination

For K by Solution Equilibrium Titration (SET)_DThe monomeric fraction of IgG protein (at least 90% monomer content, as analyzed by SEC MALS; Tosoh TSKgelG3000SWXL, Wyatt Treos miniDAWN) was used for the assay of (1). Affinity assays in solution were performed essentially as described in the reference (Friguet al, J.Immunol Methods 77: 305-319 (1985)). To improve the sensitivity and accuracy of the SET method, a switch was made from the classical ELISA to ECL-based techniques (Haenel et al, Anal Biochem 339: 182- & 184 (2005)).

1mg/ml goat anti-human (Fab) was labeled with ECL Sulfo-TAGTM NHS-Ester (Meso Scale discovery, Gaithersburg, Md., USA) according to the manufacturer's instructions₂Fragment-specific antibodies (Dianova). Experiments were performed in polypropylene microtiter plates and PBS pH 7.4 containing 0.5% BSA and 0.02% Tween20 was used as assay buffer. In the ratio of K_DStarting at a concentration at least 10-fold higher, unlabeled antigen was serially diluted in 2n or 1.75n, respectively. Wells without antigen were used to determine Bmax values; wells containing neither antigen nor IgG were used as assay background. After addition of e.g. 10pMIgG (final concentration in a final volume of 60. mu.l), the mixture is incubated overnight at room temperature. IgG concentration used and expected K_DSimilar or lower than expected K_D。

Streptavidin MSD plates were coated with 0.2. mu.g/ml biotinylated human C5 (30. mu.l/well) and blocked with 5% BSA in PBS. The equilibrated samples were then transferred to those plates (30. mu.l per well) and incubated for 20 minutes. After washing, ECL sulfo-labeled detection antibody (goat anti-human (Fab)2) was added to the MSD plate at a final dilution of 1: 1500 and incubated on an Eppendorf shaker (700rpm) for 30 minutes.

After washing and addition of 30. mu.l/well of MSD Read buffer T containing surfactant, the electrochemiluminescence signal was detected using a Sector imager 6000(Meso Scale Discovery, Gaithersburg, Md., USA).

Data were evaluated using xlfit (idbs) software using a custom fit model. For data evaluation, i.e. K of IgG molecules_DThe following fitting model was used for IgG (modified according to Piehler et al 199717): y ═ Bmax/(cIgG/2) ((cIgG/2- ((x + cIgG + KD)/2- ((x + cIgG + KD) ^2/4-x ^ cIgG 0.5) ^ 2/(2X IgG)), (cIgG ^ the IgG concentration applied, the complete molecule (non-binding site), x ═ the total soluble antigen concentration applied (binding site), and sqrt: square root.

Example 8: characterization by hemolytic assay

Hemolytic assays are basic functional assays that detect complement activation and have been used to evaluate the ability of anti-human C5mAbs and Fab molecules to block lysis of Red Blood Cells (RBCs) through the complement pathway (see, e.g., Evans et al, Mol. Immunol 32: 1183-1195 (1995); Thomas et al, Mol Immunol 33: 1389-1401 (1996); Rinder et al, J Clin Invest 96: 1564-1572 (1995)). Briefly, for the classical pathway assay, sensitized erythrocytes are used as targets for lysis by complement proteins present in serum. This assay is important for the characterization and screening of high affinity anti-human C5 mAbs.

1. Classical pathway

The desired number of chicken erythrocytes was washed four times with cold gelatin phoronella buffer (GVB + +) and resuspended to 5X10⁷Cells/ml. To sensitize the cells, rabbit anti-chrbcigg was added to the RBC cell suspension to a final concentration of 1 μ g/ml IgG. After 15 minutes of incubation on ice, sensitized chrrbcs were centrifuged, washed twice with GVB + +, and diluted to 8.33x10⁷Individual cells/ml.

Round bottom 96-well plates were used for hemolysis assay. Antibodies were diluted in GVB + + buffer and added to wells (samples were considered to be diluted twice when serum was added when calculating the required concentration of C5 binding to Abs). To 50 μ l of the antibody dilution, 50 μ l of 40% human serum (diluted in GVB +) was added, resulting in a final serum assay concentration of 20%.

Control and blank wells were prepared as described herein: control wells: i) 0% lysis control → 100 μ l GVB + +, ii) 100% lysis control → 100 μ l 0.1% NP-40, iii) 20% serum control → 100 μ l 20% serum (0% Ab control). Blank wells: i) 20% serum blank → 100. mu.l 20% serum, ii) GVB + + blank → 100. mu.l GVB + +, iii) NP-40 blank → 100. mu.l 0.1% NP-40.

Add 2.5x10 to all sample and control wells⁶(30l) sensitized chRBCs/well. PBS was added to the blank wells instead of the cells. Assay plates were incubated at 37 ℃ for 30 minutes, centrifuged (2.000rpm, 5 minutes), and 85. mu.l of supernatant was transferred to a new flat-bottom 96-well plate. The new plate was centrifuged (2.000rpm, 3 minutes) to remove any blisters. Hemoglobin release was determined by reading the absorbance at 415 nm. Percent hemolysis relative to control and blank wells was calculated using the following calculation algorithm:

wherein

OD sample ═ average OD_{Sample (I)}]- [ average OD_{20% serum blank}]

OD negative control [. mean OD ]_{0% dissolution}]- [ average OD_{GVB + blank}]

OD Positive control [. mean OD ]_{100% dissolution}]- [ average OD_{NP-40 blank}]

Using this method, anti-human C5 antibodies that inhibit erythrocyte lysis can be identified. To screen for cross-reactivity with cynomolgus C5, the classical pathway was performed using 5% cynomolgus serum.

2. By-pass pathway

Hemolysis assay via the alternative pathway is performed in a similar manner to classical pathway hemolysis assay. In the alternative pathway, RBCs cells from rabbits are used and sensitization of the cells is not required. Rabbit RBCs differ from chicken RBCs in that they are sensitive to lysis by the alternative complement pathway.

The working buffer was GVB + +, supplemented with 10mM EGTA and 5mM Mg + +, since the C5 convertase of the alternative pathway was Mg + +, whereas the C5 convertase of the classical pathway was Ca + +.

Hemolytic assays of the alternative pathway were performed with i) 20% human serum, ii)100 pM human C5 added to 20% human C5-depleted serum, iii) 0.025% cynomolgus monkey serum added to 20% human C5-depleted serum, iv) 100pM cynomolgus monkey C5 added to 20% human C5-depleted serum, v) 10% cynomolgus monkey serum. These settings were used to screen for antibodies with high affinity to human and cynomolgus C5 protein, which C5 protein is capable of very effectively inhibiting alternative complement pathway induced erythrolysis.

3. Hemolytic assay Using parental Fabs

Hemolytic assays were used as a basic biofunctional assay to assess the ability of anti-human C5mAbs to block complement-mediated lysis of erythrocytes. The C5 convertase cleaves C5 into the C5a peptide and C5b fragment, which are subsequently incorporated into the Membrane Attack Complex (MAC), which leads to cell lysis. The C5 convertase of the classical pathway formed by the C3bC4bC2a complex has a different structure from the C5 convertase of the alternative pathway formed by the C3bC3bBb complex. HuCALAntibodies produced should be inhibitory in both the classical and alternative pathways, but focus on the alternative pathway, mainly due to the alternative pathway (factor H, factor B and factor H-related genes) involved in AMD.

Classical and alternative pathway assays were performed with 20% human serum (-80 nM C5). To improve the sensitivity of the alternative pathway assay, new assay formats have been developed. To human C5 depleted serum (but containing all other serum and complement components) was added 10-100pM purified human C5 or 0.025% cynomolgus monkey serum (. about.100 pM cynomolgus monkey C5).

FIG. 2 shows that a large amount of hemolysis was observed between the addition of 10 to 100pM purified human C5 to human C5 depleted serum. Cynomolgus monkey serum was added to human C5 depleted serum to test for cross-reactivity. FIG. 3 shows that addition of 0.025% cynomolgus monkey serum (-100 pMC5) to human C5 depleted serum restores hemolytic activity.

Classical pathway

The first Fab selection was performed in the classical pathway (20% human serum). About half of the 61 purified parental Fabs are weak to strong inhibitors of the classical pathway. The IC50 values for the best inhibitory Fabs were between 35 and 900 nM.

The measurements were performed and showed consistent results (as shown in figure 4). The% hemolysis relative to control and blank wells was calculated. Fab inhibition by cytolysis was compared to the highest lysis caused by 20% human serum (═ 100%). Irrelevant human Fab (chicken lysozyme binder MOR03207) was used as a negative control and anti-human C5IgG monoclonal antibody (Quidel) was used as a positive control. Fig. 4 shows an example with optimal inhibitory Fabs.

By-pass pathway

Fabs showing inhibitory activity in the classical pathway were further evaluated in the alternative pathway. Hemolysis assay was performed by adding 100pM purified human C5 or 0.025% cynomolgus monkey serum to human C5 depleted serum. IC of human alternative assay₅₀Values were between 0.1 and 90nM (an example of an assay with the most relevant Fabs is shown in fig. 5).

The positive control of the classical pathway (anti-human C5 antibody, quedel) was not inhibitory in the alternative pathway. Anti-complement factor P antibody (Quidel) was therefore used as a positive control. As shown in fig. 5, MOR07086 had the highest inhibitory activity, and NVS data showed higher potency than the reference antibody 5G 1.1.

To test the crossreactivity of cynomolgus monkeys, an alternative route hemolytic assay was performed with the addition of 0.025% cynomolgus monkey serum in human C5 depleted serum. Since 5G1.1 does not recognize macaque C5, a comparison with 5G1.1 is not possible. Anti-factor P antibody was used as a positive control. The assay results showed IC for the most inhibitory Fabs₅₀Values were between 0.1 and 400 nM. Again, MOR07086 showed the highest efficacy (shown in figure 6).

Constant inhibitory activity of Fabs was noted in both the classical and alternative pathways. Table 11 below summarizes the results of the hemolysis assay of the most relevant 22 Fabs. To have a reliable comparison between different experiments, the solubilization by 20% human serum was normalized to 100%.

TABLE 11 hemolytic assay overview of the most relevant Fabs

Impure onium as MH

＊＊pMx9_FS

4. Hemolytic assay using mature Fabs

Classical pathway

(1) Classical pathway using 20% human serum

Mature Fabs were tested in the classical pathway using 20% human serum. Derivatives of MOR07086, 7091, 7092 and 7093 showed the greatest efficacy (IC 50 values in the low nM range). Progeny of MOR07091, 7092 and 7093 showed significantly improved efficacy. Fig. 7 shows an example of a hemolytic assay using derivatives of MOR07086, 7091, 7092 and 7093.

(2) Classical pathway using 5% cynomolgus monkey serum

Assays for the complement pathway were also performed in the presence of 5% cynomolgus monkey serum to test for cross-reactivity. Derivatives of MOR07086, 7091, 7092 and 7093 were very effective in inhibiting erythrolysis. The negative control MOR03207 (anti-lysozyme Fab) had no effect on the complement pathway. The results of these assays are shown in fig. 8.

By-pass pathway

(1) Alternative pathway Using 100pM human C5

Mature Fabs were tested in an alternative pathway hemolytic assay using 100pM human C5. Some derivatives of MOR06525, 6757, 6763, and 7087 exhibit increased potency compared to their parent. MOR07086-, 7091-, 7092-, 7093-and 7094-derived Fabs showed the highest efficacy (IC 50 values in the low nM range). The progeny of MOR07091, 7092, 7093 and 7094 showed highly improved potency, many of which were more potent than the reference antibody 5G 1.1. FIG. 9 shows an example of the results of a hemolysis assay of affinity matured Fabs and 5G 1.1.

(2) Alternative pathway using 20% human serum

Mature Fabs were tested in an alternative pathway hemolysis assay using 20% human serum. Mor07086-, 7091-, 7092-and 7093-derived Fabs showed the highest inhibitory activity. Many of these Fabs have higher inhibitory activity than 5G 1.1. FIG. 10 shows an example of the results of a hemolysis assay of affinity matured Fabs and reference antibody 5G 1.1.

(3) Alternative pathway Using 100pM Kiwi C5

Mature Fabs were tested in an alternative pathway hemolytic assay using 100pM cynomolgus C5 supplemented with 20% human C5 depleted serum. MOR07091-, 7092-and 7093-derived Fabs showed the highest inhibitory activity; 5G1.1 did not cross-react with macaque C5. FIG. 11 shows an example of the results of a hemolysis assay of affinity matured Fabs.

5. Hemolytic assay using germlined IgGs (human IgG1AA format)

Classical pathway

(1) Classical pathway using 20% human serum

The classical pathway using 20% human serum was performed at MOR. The IC50 values for the final germlined hu IgGAA-MOR08109, 8110, 8113, 8114 were better than or similar to those of the reference IgG 5G1.1 (see FIG. 12).

(2) Classical pathway using 5% cynomolgus monkey serum

The comparison with 5G1.1 in the classical pathway using 5% cynomolgus monkey serum is not applicable because the reference antibody does not recognize cynomolgus monkey C5. The final germlined IgGs were able to completely inhibit red blood cell lysis induced by cynomolgus monkey sera except MOR 08111. The data are shown in figure 13.

By-pass pathway

(1) Alternative pathway Using 100pM human C5

Germlined IgGs were detected in an alternative pathway hemolytic assay using 100pM human C5. All antibodies showed potent inhibitory activity with an IC50 value between 28 and 128pM (except MOR08111, see fig. 14), all equal or similar to 5G 1.1. FIG. 14 shows an example of the results of the hemolytic assay for IgGs.

(2) Generation of ELISA using the alternative pathway of 20% human serum and C5a

Germlined IgGs were also tested in an alternative pathway hemolytic assay using 20% human serum. Most of the tested antibodies achieved complete inhibition with IC50 values below 80 nM. The reference antibody 5G1.1 did not completely inhibit hemolysis in this assay. FIG. 15 shows an example of the results of the hemolytic assay for IgGs. The final IgGs inhibited C5a production similarly to 5G1.1 (IC 50 values in the low nM range).

(3) Alternative pathway Using 100pM Kiwi C5

Reconstitution of 20% human C5 depleted serum with 100pM macaque C5 for hemolytic assay of the alternative pathway. The efficacy of the germlined final candidate against cynomolgus C5 was within 5-fold of human C5 efficacy (IC 50 values in the low pM range).

(4) Alternative pathway using 10% cynomolgus monkey serum

In a haemolysis assay using the alternative pathway of 10% cynomolgus monkey serum ([ C5] -40 nM), the potency of the germlined candidate was similar to that in human serum (success criteria were to have a potency no more than 5-fold lower than that of a functional assay using human C5).

Example 9: c5a production ELISA

The C5a-des-Arg ELISA was developed to measure the production of C5a during hemolysis to confirm that inhibitory antibodies in hemolysis assays also inhibited cleavage of C5 into C5a and C5 b.

Maxisorp plates were coated with 100. mu.l/well mouse anti-human C5a-des-Arg (US biologics) at 1g/ml in coating buffer (bicarbonate pH 9.5-9.8) and incubated overnight at 4 ℃. After 3 washes with PBST, the plates were blocked with 300 l/well diluent (Synblock, AbD Serotec) for 2 hours at room temperature. After withdrawal of the blocking solution, 100. mu.l of the sample or standard diluted with diluent was incubated at room temperature for 1 hour. The standards were prepared as follows: starting with 20ng/ml of standard (rC5a-des-Arg), 1: 4 serial dilutions were made for the 7-point curve. The hemolyzed samples were diluted 1: 5in diluent (the hemolyzed supernatant should be stored at-80 ℃ until used in C5 aELISA). Plates were washed 3 times with PBST in the middle. 0.4g/ml detection antibody (biotin-goat anti-human c5a, R) diluted 100 l/well in diluent was added&D Systems) and after 1 hour incubation at room temperature, 100 l/well Strep-HRP (poly-HRP streptavidin) diluted 1: 5000 in HRP diluent (poly-HRP diluent) was added for 30 minutes. After 4 washes with PBST, 100 l/well of TMB substrate (Ultra TMB substrate solution) was added for 5-10 min. Stop solution with 50 l/well (2N H)₂SO₄) The reaction was terminated. The absorbance (A450-A570) was read and the data was analyzed using SoftMax Pro.

Production of C5a during hemolysis was examined for mature Fabs to confirm that the inhibitory activity was due to blocking cleavage of C5 to C5a and C5 b. The supernatant from the hemolytic assay in 20% human serum was used to quantify the C5a formation.

All Fabs tested dropped the level of C5a down to baseline. Fig. 16 shows an example of the results of the C5a ELISA.

Example 10: specific ELISA on human C3, C4, C5 and macaque C5

All purified Fabs were analyzed in solution ELISA (method described above) for binding to human C3, C4, and C5. Fabs were incubated with biotinylated antigen on Neutravidin plates and detected by histidine tag.

Improved binding was seen for nearly all mature Fabs compared to their respective parents. No binding to the anti-targets human C4 and C3 was detected at up to 100nM Fab. These results hit the success criteria for specificity: binds to human and cynomolgus C5, but not to human complement proteins C3 and C4. An example of a derivative of the parent Fab MOR07091 is shown in figure 17.

Example 11: serum stability assay

Retained binding activity of C5-binding antibody to human C5 in a binding assay of 50% human serum was determined as described below.

Antibodies (Fab format) were incubated with 100% human C5 depleted serum or PBST/0.5% BSA (positive control) at 37 ℃ for up to 8 hours. The wells of the blocked polypropylene plate were used for incubation to ensure that the antibodies did not bind to the surface for a long incubation time. Samples were collected at various time points and stored at-20 ℃.

Samples were tested in solution ELISA on NeutrAvidin plates to check for binding to human C5. To a 1x ChemiBlocker-PBST closed overnight NeutrAvidin plate was added 20. mu.l serial dilutions of different collected samples. The first dilution of the sample was 1: 2 (final serum concentration 50%), followed by a 1: 3 dilution step. After 1 hour incubation, the plates were washed 3 times with PBST and 20. mu.l biotinylated human C5 to a concentration of 2.5. mu.g/ml was applied. After 1 hour, the plates were washed again 5 times with PBST (0.05% Tween) and anti-HIS 6-POD detection antibody was added.

The fluorescence of the substrate (Quanta Blue or AttoPhos) was measured after 5-10 minutes and the retained binding activity compared to each of the strongest signals (antibody incubated with PBST/0.5% BSA) was calculated.

One "must" criterion for C5 binding to antibodies is that 75-80% of the binding activity is retained in human serum in i) a 10% serum functional assay and ii) a 50% serum binding assay. Because the hemolytic assay was performed in the presence of 20% serum, the retained binding must only be shown in the binding assay in 50% serum.

Thus, mature final Fabs were incubated with 100% human C5 depleted serum at 37 ℃ for 8 hours. Samples were collected at various time points and tested for binding to human C5 in a solution ELISA. Fab + serum samples for ELISA were diluted to a concentration of 50% serum +10nM Fab.

Figure 18 illustrates the results of Fab form final C5 binding to the final antibody. Compared to the incubation in PBS, 70-93% of the binding activity was retained after an incubation time of 8 hours at 37 ℃ in 50% serum.

Example 12: characterization by epitope binning

This method was used to sort anti-human C5Fabs into different epitope boxes (bins) that bind the same or overlapping epitopes of C5 protein.

Competition of each biotinylated anti-human C5 antibody with a 100-fold excess of each unlabeled anti-human C5 antibody was detected in an ELISA (capture mode). It was compared to the strongest signal of each antibody (non-competing biotinylated Fab).

Human C5 was captured by polyclonal anti-human C5IgG (us biological) previously coated on 384-well blank Maxisorp plates at 4 ℃. The next day, the plates were washed 2 times with PBST and blocked with 3% BSA-PBST for 2 hours. After 3 washes with PBST, 20 μ l of human C5 was added and incubated for 2 hours at room temperature. The plates were washed 3 times with PBST before adding the Fabs.

To the Maxisorp plate 20. mu.l unlabeled Fab (200. mu.g/ml or 400. mu.g/ml) (100 fold excess) was added followed by 20ng/ml or 40ng/ml biotinylated Fab. The biotinylated and unlabeled Fabs were incubated at room temperature for 1 hour. The plates were washed 3 times with PBST and Strep-AP Zymax streptavidin-alkaline phosphatase, ZYMED, code: 43-8322, batch number: 50799648 was used to detect binding of biotinylated Fab to the plate via C5. Attophos substrate (Roche) was added to the plate and fluorescence was read after 5-10 minutes.

Parent Fabs

C5 was captured (by polyclonal antibody) and excess unlabeled FabY was applied to biotinylated FabX. Binding of biotinylated FabX to human C5 was detected. Six groups of Fabs may be defined: group 1: MOR06952, 6961; group 2: MOR06525, 6756, 6757, 6763; group 3: MOR 07087; group 4: MOR06764, 6776, 7081; group 5: MOR 07089; group 6: MOR07082, 7083, 7084, 7086, 7088, 7090, 7091, 7092, 7093, 7095.

Different approaches were also used to classify Fabs into different epitope-binding groups: FabX was immobilized and FabY pre-incubated with biotinylated C5 was then added. The following groups of Fabs may be defined: group 1: MOR06952, 6961; group 2: MOR06525, 6757, 7083; group 3: MOR 07087; group 4: MOR 06763; group 5: MOR 07081; group 6: MOR07082, 7083, 7084, 7086, 7088, 7091, 7092, 7093(7089 competes with 7084). Similar results were obtained using two different approaches to conclude.

Mature Fabs

To complete Fab characterization, the competition of biotinylated Fab with unlabeled Fab (100-fold excess application) was determined in a solution ELISA. The results were compared to the strongest signal (non-competing biotinylated Fab).

As shown in fig. 19, biotinylated Fabs competed with the same unlabeled Fabs, and all Fabs competed for binding to the same or overlapping epitopes. These results correlate with epitope binning data for the parental Fabs.

Example 13: screening for C5 alpha vs. beta chain binders and Competition assays

Two ELISA experiments and hemolytic assays were performed to detect whether Fab was an alpha or beta chain binder as described below.

In the first experiment, Fab was coated on plates and purified C5 or supernatant from chimeric C5 preparation (human α, mouse β chain) was added. In the second step, 5G1.1 was applied and detection was performed by anti-human IgG.

In a second experiment, 5G1.1 was coated on plates, purified C5 or supernatant from chimeric C5 preparation (human α, mouse β chain) was added, followed by Fab detection with anti-Myc antibody.

The reference IgG 5G1.1 recognized the alpha chain and was used to determine whether the Fabs produced by MorhpSys competed for binding with 5G 1.1. In a hemolytic assay, supernatants from chimeric C5 preparations were added to human C5 depleted sera and tested for hemolytic inhibition of Fabs.

Parent Fabs

FIG. 20 shows the results of an ELISA experiment in which Fabs were coated on plates, supernatants of C5 or chimeric C5 preparations (human α chain and mouse β chain) were added, followed by 5G 1.1. Fig. 21 shows the results of an ELISA experiment in which purified C5 and supernatant from chimeric C5 were captured by 5G 1.1.

MOR06525, 6756, 6763 is a beta strand binder (binds C5 but not chimeric C5). Most MOR070XX Fabs (from solution panning) are alpha chain binders (binding C5 and chimeric C5). MOR06952 and 6961 compete with 5G1.1, so they are negative for C5 and chimeric C5 and are therefore most likely alpha chain binders as with 5G1. MOR06757 behaves similarly to MOR06952 and 6961, i.e., it is likely to be an alpha chain binder. However, MOR06757 did not inhibit hemolysis of chimeric C5 supernatant added to C5 depleted serum, while all other alpha chain binders could be inhibited (see figure 22).

In a hemolytic assay, supernatants from chimeric C5 preparations were added to human C5 depleted sera and tested for hemolytic inhibition of Fabs. MOR06525, 6756, 6757 and 6763 and chimeric C5 did not inhibit hemolysis and therefore could be a β -strand binder. MOR06952, 6961, 7081, 7082, 7083, 7084, 7086, 7087, 7088, 7089, 7090, 7091, 7092, 7093, 7094, 7095 inhibit hemolysis and thus can be an alpha chain binder.

Example 14: resistance to proteolysis

To investigate the structural rigidity of Fabs, resistance of Fabs to proteolysis by thermolysin was performed (thermolysin bacterial protease, Calbiochem). Fab was incubated with thermolysin (Fab: thermolysin. RTM. 3: 1(w/w), 8. mu.L reaction volume) at either 37 ℃ or 55 ℃ (thermolysin activity was optimal at 55 ℃). The reaction was stopped by adding 4. mu.L of 0.5M EDTA and 4. mu.L of 4 XSDS sample buffer (Invitrogen), and the stopped samples were run on 4-12% SDS-PAGE under non-reducing conditions. The proteolysis of Fabs was analyzed by monitoring the disappearance of the Fab bands, which were revealed by coomassie staining.

Parent Fabs

The resistance of the parent Fabs to proteolysis by thermolysin was tested at 37 ℃ and 55 ℃. Fab from humanized IL-1. beta. antibody was used as a control. Most of the Fabs tested were resistant to degradation by thermal decomposition at 37 ℃ for up to 90 minutes. To further differentiate the structural rigidity of Fabs, proteolysis was performed at higher temperatures of 55 ℃. Many of the Fabs tested degraded rapidly at 55 ℃ (> 90% Fab degraded within 30 minutes), while some Fabs remained resistant to proteolysis after 90 minutes (e.g. 7094). Suggesting that the resistant Fabs have a more rigid structure, so that they show better pharmacokinetic properties in vivo. The results of these experiments are shown in fig. 23 and 24.

Mature Fabs

The Fabs with the highest potency in the hemolytic assay were tested for sensitivity to thermolysin at 37 ℃ and 55 ℃. In fig. 25 and 26, experiments using derivatives of MOR07086, 7091, 7092 and 7093 are shown.

The results of these tests revealed that derivatives of the parental MOR07091, 7092 and 7093 were less susceptible to proteolysis, whereas MOR07086 derivatives were more susceptible to proteolysis.

Example 15: MAC deposition assay

Because the terminal complement cascade terminates with the formation of MAC, inhibition of MAC formation is a further cue for the ability of antibodies to block the complement cascade. It is reasonable to have an additional setup that is independent of the cells and the cell behavior.

Zymosan (Sigma), an insoluble carbohydrate from yeast cell walls, was coated to activate the alternative pathway and igm (Sigma) to activate the classical pathway for the determination of MAC (membrane attack complex) deposition, especially in immunoassays for the alternative pathway. Fabs were preincubated with human serum (6% for AP, 2% for CP) and added to the plates. Percent (%) inhibition of MAC deposition was calculated for each sample relative to baseline (EDTA-treated human serum) and positive control (human serum) and used to generate IC using XLFit₅₀Curve line.

Parent Fabs

Parental Fabs were used at different concentrations and the maximum inhibition (and IC50 values, if applicable) was determined (example shown in figure 27). Most Fabs completely inhibited MAC deposition, indicating blockade of C5 cleavage. The efficacy and grade of Fabs were similar to the data from the hemolytic assay.

Claims (16)

1. An isolated monoclonal antibody, or antigen binding fragment thereof, comprising

(i) Heavy chain CDR1, which is the sequence of SEQ ID NO. 1; heavy chain CDR2, which is a sequence selected from SEQ ID NOs:2 and 119; and a heavy chain CDR3, which is the sequence of SEQ ID NO. 3; and

(ii) a light chain CDR1, which is the sequence of SEQ ID NO. 4; a light chain CDR2, which is the sequence of SEQ ID NO. 5; and a light chain CDR3, which is a sequence selected from SEQ ID NOs:6 and 120; wherein the antibody binds to human C5 protein.

2. The isolated antibody or antigen-binding fragment of claim 1, comprising a heavy chain variable region having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID No.7, 121, or 187, and a light chain variable region having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID No. 8, 122, or 188.

3. The isolated antibody or antigen-binding fragment of claim 2, comprising a heavy chain variable region of an amino acid sequence selected from SEQ ID NO 7, 121, or 187, and a light chain variable region of an amino acid sequence selected from SEQ ID NO 8, 122, or 188.

4. The isolated antibody or antigen-binding fragment of claim 1, comprising a heavy chain having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 9, 123, 189, or 249 and a light chain having at least 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO 10, 124, 190, or 251.

5. The isolated antibody or antigen-binding fragment of claim 4, comprising a heavy chain of an amino acid sequence selected from SEQ ID NO 9, 123, 189 or 249 and a light chain of an amino acid sequence selected from SEQ ID NO 10, 124, 190 or 251.

6. The antibody of any one of claims 1 to 5, which antibody has an IC of 20-200pM when determined by an in vitro hemolysis assay using 100pM human C5 reconstituted human C5 depleted serum₅₀Inhibiting the alternative complement pathway.

7. The antibody or antigen-binding fragment of any one of claims 1 to 6, wherein the antibody is a human, humanized or chimeric antibody.

8. The antibody or antigen binding fragment of any one of claims 1 to 7, wherein the antibody binds to human C5 and cynomolgus C5 with at least 1x10⁷M^-1、10⁸M^-1、10⁹M^-1、10¹⁰M^-1Or 10¹¹M^-1Affinity constant (K) of_A)。

9. The antibody of any one of claims 1 to 8, wherein the antibody is of the IgG isotype.

10. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of the preceding claims and a pharmaceutically acceptable carrier.

11. An isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising the heavy chain CDR1, CDR2, CDR3 and light chain CDR1, CDR2, CDR3 of claim 1 and comprising a heavy chain variable region having at least 95% sequence identity to SEQ ID No.7, 121 or 187 or a light chain variable region having at least 95% sequence identity to SEQ ID No. 8, 122 or 188.

12. The nucleic acid of claim 11, wherein the nucleotide sequence encoding the polypeptide comprising the heavy chain variable region is selected from SEQ ID NOs 11, 125 or 191 and the nucleotide sequence encoding the polypeptide comprising the light chain variable region is selected from SEQ ID NOs 12, 126 or 192.

13. A vector comprising the nucleic acid of claim 11 or 12.

14. An isolated host cell comprising (1) a recombinant DNA segment encoding the heavy chain of the antibody of claim 4 or 5, and (2) a second recombinant DNA segment encoding the light chain of the antibody of claim 4 or 5; wherein said DNA segment is operably linked to a promoter and is capable of expression in said host cell.

15. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 9 or a pharmaceutical composition according to claim 10 in the manufacture of a medicament.

16. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 or a pharmaceutical composition according to claim 10 in the manufacture of a medicament for the treatment of age-related macular degeneration, asthma, arthritis, autoimmune heart disease, multiple sclerosis, inflammatory bowel disease, ischemia-reperfusion injury, Barre syndrome, hemodialysis, systemic lupus erythematosus, psoriasis, multiple sclerosis, transplantation, Alzheimer's disease, glomerulonephritis, Paroxysmal Nocturnal Hemoglobinuria (PNH) or MPGNII.