HK1081205A - Anti-c5ar antibodies and uses thereof - Google Patents

Description

anti-C5 aR antibodies and uses thereof

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Technical Field

The present invention relates to antibodies that bind C5aR and that are useful in diagnostic and therapeutic methods.

Background

Proteolytic cleavage of each complement protein C3-C5 produces a cationic amino-terminal fragment (6-9) with a signal molecule called anaphylatoxins (anaphylatoxins). The most effective of these fragments, C5a, elicited the broadest effect. C5a is a "complete" proinflammatory mediator, believed to be a component of the inflammatory response of leukocyte migration and infiltration, granule-bound proteolytic enzymes (granules-bound proteolytic enzymes), production of reactive oxygen and nitrogen source free radicals, altered blood flow and capillary permeability, and having the ability to contract smooth muscle. C5a elicits chemochemotaxis of all myeloid lineage cells (neutrophils, eosinophils and basophils, macrophages and monocytes) at sub-nanomolar to nanomolar levels and causes increased vascular permeability, which is significantly enhanced by prostaglandins and circulating leukocytes. Higher nanomolar concentrations of C5a triggered degranulation and activation of NADPH oxidase. The breadth of this biological activity is in sharp contrast to other inflammatory mediators. C5a [1, 2] has been implicated in the pathogenesis of rheumatoid arthritis, psoriasis, sepsis, reperfusion injury, and adult respiratory distress syndrome.

C5aR includes an extended N-terminal extracellular domain. The large N-terminal domain is typically a G-protein binding receptor that binds to peptides including the IL-8 and fMet-Leu-Phe (FMLP) receptor families, and the structure of C5aR corresponds to seven transmembrane receptor families, followed immediately by the extracellular N-terminus by seven transmembrane helices joined by an interhelical domain, which serve as the intracellular and extracellular loops, ending with the intracellular C-terminal domain.

Inhibition of the C5a effect with C5aR antagonists may reduce the acute inflammatory response mediated by C5a without affecting other complement components. To date, peptide antagonists of C5aR and anti-C5 a receptor antibodies have been previously described. For example, WO95/00164 describes antibodies directed against the N-terminal peptide (residues 9-29) of the C5a receptor. However, there is still a need for alternative and/or improved C5aR antagonists.

Disclosure of Invention

The present inventors have now developed a novel monoclonal antibody reactive with a region other than the N-terminal domain of C5aR, which is highly effective in inhibiting the binding of C5a to C5 aR. These monoclonal antibodies have been designated 7F3, 6C12 and 12D 4.

Thus, in one aspect, the invention provides an antibody reactive with an extracellular loop other than the N-terminal domain of C5aR, wherein the antibody reduces or inhibits the binding of C5a to C5 aR.

By "extracellular loop" we mean the first extracellular loop (residues 95 to 110), or the second extracellular loop (residues 175 to 206) or the third extracellular loop (residues 265-283) of C5 aR.

In a preferred embodiment, the antibody is reactive with an epitope comprising the second extracellular loop (residues 175 to 206) of C5 aR.

In another aspect, the invention provides an antibody reactive with the same epitope of C5aR as MAb7F3, wherein the antibody reduces or inhibits the binding of C5a to C5 aR.

In another aspect, the invention provides an antibody reactive with the same epitope of C5aR as MAb6C12, wherein the antibody reduces or inhibits the binding of C5a to C5 aR.

In another aspect, the invention provides an antibody reactive with the same epitope of C5aR as MAb12D4, wherein the antibody reduces or inhibits the binding of C5a to C5 aR.

In another aspect, the invention provides an antibody that binds to C5aR, wherein the antibody competitively inhibits the binding of MAb7F3 to C5 aR.

In another aspect, the invention provides an antibody that binds to C5aR, wherein the antibody competitively inhibits the binding of MAb6C12 to C5 aR.

In another aspect, the invention provides an antibody that binds to C5aR, wherein the antibody competitively inhibits the binding of MAb12D4 to C5 aR.

In a preferred embodiment of these aspects of the invention, the relative binding specificity is determined by antibody-antibody competition assay in the presence of C5aR or a polypeptide comprising the extracellular loop of C5 aR.

In yet another aspect, the present invention provides a polypeptide comprising a sequence defined by SEQ ID NOs: 19 and SEQ ID NO: 21, wherein the antibody reduces or inhibits binding of C5a to C5 aR.

In yet another aspect, the present invention provides a polypeptide comprising at least one amino acid sequence represented by seq id NO: 26. SEQ ID NO: 27 or SEQ ID NO: 28, a CDR loop sequence substantially identical to a variable heavy chain CDR1, CDR2, or CDR3 loop sequence, wherein the antibody reduces or inhibits the binding of C5a to C5 aR.

In a preferred embodiment, the antibody comprises at least two, more preferably at least three, cdrs of at least one polypeptide consisting of SEQ ID NOs: 26. SEQ ID NO: 27 and SEQ ID NO: 28, a CDR loop sequence substantially identical to the variable heavy chain CDR1, CDR2, or CDR3 loop sequences.

In a further preferred embodiment, the antibody comprises at least one heavy chain variable region consisting essentially of seq id NO: 19, or a CDR loop sequence defined by amino acid residues 24 to 39, 55 to 61, or 94 to 102 of the variable light chain sequence set forth in seq id No. 19. Preferably, the antibody comprises at least two, more preferably at least three heavy chain variable domains consisting essentially of SEQ ID NO: 19, and CDR loop sequences determined at amino acid residues 24 to 39, 55 to 61, and 94 to 102 of the variable light chain sequence set forth in seq id No. 19.

In yet another aspect, the present invention provides a polypeptide comprising a sequence identical to SEQ ID NO: 15 and SEQ ID NO: 17, wherein the antibody reduces or inhibits binding of C5a to C5 aR.

In yet another aspect, the present invention provides a polypeptide comprising at least one amino acid sequence represented by seq id NO: 29. SEQ ID NO: 30 or SEQ ID NO: 31, a CDR loop sequence substantially identical to a variable heavy chain CDR1, CDR2, or CDR3 loop sequence, wherein the antibody reduces or inhibits the binding of C5a to C5 aR.

In a preferred embodiment, the antibody comprises at least two, more preferably at least three, cdrs of at least one polypeptide consisting of SEQ ID NOs: 29. SEQ ID NO: 30 and SEQ ID NO: 31, a CDR loop sequence substantially identical to the variable heavy chain CDR1, CDR2, or CDR3 loop sequences.

In a further preferred embodiment, the antibody comprises at least one heavy chain variable region consisting essentially of seq id NO: 15, or a CDR loop sequence defined by amino acid residues 24 to 39, 55 to 61, or 94 to 102 of a variable light chain sequence set forth in seq id No. 15. Preferably, the antibody comprises at least two, more preferably at least three heavy chain variable domains consisting essentially of SEQ ID NO: 15, or a CDR loop sequence defined by amino acid residues 24 to 39, 55 to 61, and 94 to 102 of the variable light chain sequence set forth in seq id No. 15.

In yet another aspect, the present invention provides a polypeptide comprising a sequence identical to SEQ ID NO: 23 and SEQ ID NO: 25, wherein the antibody reduces or inhibits binding of C5a to C5 aR.

In yet another aspect, the present invention provides a polypeptide comprising at least one amino acid sequence represented by seq id NO: 32. SEQ ID NO: 33 or SEQ ID NO: 34, a variable heavy chain CDR1, CDR2, or CDR3 having substantially the same loop sequence of the CDR loop sequences, wherein the antibody reduces or inhibits the binding of C5a to C5 aR.

In a preferred embodiment, the antibody comprises at least two, more preferably at least three, cdrs of at least one polypeptide consisting of SEQ ID NOs: 32. SEQ ID NO: 33 and SEQ ID NO: 34, and a CDR loop sequence substantially identical to the variable heavy chain CDR1, CDR2, or CDR3 loop sequences.

In a further preferred embodiment, the antibody comprises at least one heavy chain variable region consisting essentially of seq id NO: 23, or a CDR loop sequence defined by amino acid residues 24 to 39, 55 to 61, or 94 to 102 of the variable light chain sequence set forth in seq id No. 23. Preferably, the antibody comprises at least two, more preferably at least three heavy chain variable domains consisting essentially of SEQ ID NO: 23, and CDR loop sequences determined at amino acid residues 24 to 39, 55 to 61, and 94 to 102 of the variable light chain sequence set forth in seq id No. 23.

In a preferred embodiment of the invention, C5aR is human C5 aR.

In one embodiment of the invention, the antibody also inhibits activation of neutrophils via other neutrophil chemoattractants (chemoattractants), in particular CXCR1 and CXCR2 ligands such as IL-8.

In a preferred embodiment of the invention, the antibody is a monoclonal or recombinant antibody. Preferably, the monoclonal or recombinant antibody is a chimeric antibody or a humanized antibody.

The antibody may be of any isotype (isotype). In a further preferred embodiment of the invention, however, the antibody is an antibody of the IgG2a type or IgG3 type.

In a preferred embodiment of the invention, the antibody is a monoclonal antibody selected from the group consisting of MAb7F3, MAb6C12, and MAb12D 4.

In another aspect, the invention provides a hybridoma deposited with ECACC under accession number 00110609.

In another aspect, the invention provides a hybridoma deposited with ECACC under accession number 02090226.

In another aspect, the invention provides a hybridoma deposited with ECACC under accession number 02090227.

It will be appreciated that various chemical derivatives of the antibodies of the invention may also be produced. For example, immunoconjugates comprising an antibody of the invention conjugated to a label, such as a radioisotope or other tracer molecule, can be prepared by techniques known in the art. Likewise, an antibody may be conjugated to a molecule for therapeutic use, which may be directed to a desired site of action due to the specificity of antibody binding.

Accordingly, in another aspect of the invention, the invention provides a conjugate comprising an antibody of the invention and a therapeutic agent.

It is understood that a range of therapeutic agents may be used in the present invention. Preferred drugs include drugs that mediate cell death or protein inactivation. The therapeutic agent can be any of a number of toxins known in the art. The toxin may be pseudomonas exotoxin or a derivative thereof. In a preferred embodiment, the toxin is PE 40.

In yet another aspect of the invention, the invention provides a conjugate comprising an antibody of the invention and a detectable label.

The detectable label may be any suitable label known in the art. For example, the label may be a radioactive label, a fluorescent label, an enzymatic label, or a contrast agent.

In yet another aspect of the invention, the invention provides an isolated nucleic acid molecule comprising a sequence encoding an antibody of the invention.

In yet another aspect of the invention, the invention provides a composition comprising an antibody of the invention and a pharmaceutically acceptable carrier.

In yet another aspect of the invention, the invention provides a method of inhibiting the interaction of cells bearing C5aR with their ligands, the method comprising exposing the cells to an antibody of the invention.

In yet another aspect of the invention, the invention provides a method of inhibiting C5aR activity in a cell, the method comprising exposing the cell to an antibody of the invention.

In yet another aspect of the present invention, the present invention provides a method of treating a disease involving neutrophil migration (migration) in a target subject, the method comprising administering to the target subject an antibody of the present invention.

It will be appreciated by those skilled in the art that the antibodies of the invention may also be used to determine, quantify and/or locate cells expressing C5 aR.

Thus, in another aspect of the invention there is provided a method of diagnosing a disease involving neutrophil migration in a subject of interest, the method comprising contacting a sample from the subject of interest with a conjugate of the invention and determining immunospecific binding between the conjugate and the sample.

Various immunoassays may be used in the diagnostic methods. Such immunoassays include the use of competitive and non-competitive detection systems such as radioimmunoassays, ELISAs, "sandwich" immunoassays, precipitation reactions, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, and the like. Both in vitro and in vivo assays can be used.

The sample from the target subject may comprise any bodily fluid, such as peripheral blood, plasma, lymph, peritoneal fluid, cerebrospinal fluid, or pleural fluid, or any body tissue. In vitro binding can be performed using histological samples or tissue fragments or body fluids. In vivo binding can be accomplished by injection of the conjugate using any method known in the art (e.g., intravenous, intraperitoneal, intraarterial, etc.), and immunospecific binding can be determined accordingly.

In addition, imaging techniques may be used in which the primary antibody is bound to a suitable imaging label. The labeled antibody may be administered in vivo to determine the localization of C5aR in the target subject.

Accordingly, in another aspect of the invention there is provided a method of diagnosing a disease involving neutrophil migration in a subject, the method comprising administering an antibody of the invention labelled with an imaging agent to the subject under conditions such that a complex between the antibody and a cell presenting C5aR is formed, and imaging the complex.

In a preferred embodiment of the invention, the disease involving neutrophil migration is a C5aR mediated disease. Preferably, the disease is an immunological disease.

In a further aspect, the invention provides a method of delivering a therapeutic agent to a site of inflammation in a target subject, the method comprising administering to the target subject a conjugate of the invention.

In a further aspect of the invention there is provided a method of introducing genetic material into a cell presenting C5aR, the method comprising contacting the cell with an antibody of the invention, wherein the antibody is linked or associated with the genetic material.

In a preferred embodiment, the cells presenting C5aR are selected from the group consisting of granulocytes, leukocytes such as monocytes, macrophages, basophils and eosinophils, mast cells and lymphocytes including T cells, dendritic cells, and non-myeloid cells such as endothelial cells and smooth muscle cells.

Also encompassed by the present invention are methods of determining other ligands or other agents that bind to C5aR, including inhibitors and/or enhancers of mammalian C5aR function. For example, a reagent having the same binding specificity as the antibody of the present invention or a functional fragment thereof can be determined by a competitive assay using the above-mentioned antibody or fragment. Thus, the invention also includes identifying ligands or other agents that bind to C5aR, including inhibitors (e.g., antagonists) or enhancers (agonists) of receptor function. In one embodiment, cells that naturally express C5aR or host cells that have been processed to express C5aR or a variant encoded by a nucleic acid introduced into the host cell are used in assays to determine and evaluate the effectiveness of inhibitors or enhancers of ligand, receptor function. These cells are also used to determine the function of the expressed receptor protein or polypeptide.

Drawings

FIG. 1 shows the results of flow cytometric detection of monoclonal antibody 7F 3. These results indicate that 7F3 reacts specifically with L1.2 cells transfected with C5 aR.

FIG. 2 shows a monoclonal antibody comprising a panel of monoclonal antibodies including 7F3¹²⁵I C5a ligand binding assay.

FIG. 3 shows the monoclonal antibody 7F3 pair¹²⁵I C5a ligand binding dose-responsive inhibition.

FIG. 4 shows the results of chemochemotaxis assays performed with L1.2 cells transfected with C5aR and a panel of monoclonal antibodies including 7F3, 6C12 and 12D 4.

FIG. 5 shows the complete inhibition of chemochemotaxis of C5 aR-transfected L1.2 cells by monoclonal antibody 7F 3.

FIG. 6 shows the complete inhibition of C5 a-mediated chemotaxis of neutrophils by monoclonal antibody 7F 3.

FIG. 7 shows the inhibition of C5 a-mediated neutrophil chemotaxis by monoclonal antibodies 7F3, 6C12 and 12D 4.

FIG. 8 shows the inhibition of IL-8 mediated neutrophil chemotaxis by monoclonal antibodies 7F3, 6C12 and 12D 4.

FIG. 9 shows the results of an assay to determine the competitive inhibition of the binding of the C5aR N-terminal peptide PEPI to anti-C5 aR MAb to L1.2 cells transfected with human C5 aR.

FIG. 10 shows the results of an assay to determine FACS staining of purified neutrophils by MAb7F3 in the presence or absence of the C5aR N-terminal peptide PEPI.

FIG. 11 shows an alignment of the variable light chain DNA sequences of MAb7F3, 6C12 and 12D 4.

Fig. 12 shows an alignment of the variable heavy chain DNA sequences of MAb7F3, 6C12, and 12D 4.

Fig. 13 shows an alignment of the variable light chain protein sequences of MAb7F3, 6C12, and 12D 4.

Fig. 14 shows an alignment of the variable heavy chain protein sequences of MAb7F3, 6C12, and 12D 4.

Description of sequence listing

SEQ ID NO: 1 human C5aR protein sequence

SEQ ID NO: 26C 12 variable light chain PCR primers

SEQ ID NO: 36C 12 variable light chain PCR primers

SEQ ID NO: 46C 12 variable heavy chain PCR primer

SEQ ID NO: 56C 12 variable heavy chain PCR primer

SEQ ID NO: 67F 3 variable light chain PCR primers

SEQ ID NO: 77F 3 variable light chain PCR primers

SEQ ID NO: 87F 3 variable heavy chain PCR primer

SEQ ID NO: 97F 3 variable heavy chain PCR primer

SEQ ID NO: 1012D 4 variable light chain PCR primers

SEQ ID NO: 1112D 4 variable light chain PCR primers

SEQ ID NO: 1212D 4 variable heavy chain PCR primer

SEQ ID NO: 1312D 4 variable heavy chain PCR primer

SEQ ID NO: 146C 12 variable light chain (DNA) sequence

SEQ ID NO: 156C 12 variable light chain (protein) sequence

SEQ ID NO: 166C 12 variable heavy chain (DNA) sequence

SEQ ID NO: 176C 12 variable heavy chain (protein) sequence

SEQ ID NO: 187F 3 variable light chain (DNA) sequence

SEQ ID NO: 197F 3 variable light chain (protein) sequence

SEQ ID NO: 207F 3 variable heavy chain (DNA) sequence

SEQ ID NO: 217F 3 variable heavy chain (protein) sequence

SEQ ID NO: 2212D 4 variable light chain (DNA) sequence

SEQ ID NO: 2312D 4 variable light chain (protein) sequence

SEQ ID NO: 2412D 4 variable heavy chain (DNA) sequence

SEQ ID NO: 2512D 4 variable heavy chain (protein) sequence

SEQ ID NO: 267F 3 variable heavy chain CDR1 Loop

SEQ ID NO: 277F 3 variable heavy chain CDR2 Loop

SEQ ID NO: 287F 3 variable heavy chain CDR3 Loop

SEQ ID NO: 296C 12 variable heavy chain CDR1 Loop

SEQ ID NO: 306C 12 variable heavy chain CDR2 Loop

SEQ ID NO: 316C 12 variable heavy chain CDR3 Loop

SEQ ID NO: 3212D 4 variable heavy chain CDR1 Loop

SEQ ID NO: 3312D 4 variable heavy chain CDR2 Loop

SEQ ID NO: 3412D 4 variable heavy chain CDR3 Loop

Detailed Description

C5aR structure

SEQ ID NO: 1 provides the amino acid sequence of human C5 aR.

The various domains of human C5aR are shown below:

amino acids 1-37 extracellular domain-N-terminal

Amino acid 38-61 transmembrane domain

Amino acid 62-71 intracellular domain

Amino acid 72-94 transmembrane domain

Amino acids 95-110 extracellular domain-extracellular loop 1

Amino acid 111-132 transmembrane domain

Amino acid 133.-.149 intracellular domain

Amino acid 150.-.174 transmembrane domain

Amino acid 175-. 206 extracellular domain-extracellular loop 2

Amino acid 207-. 227 transmembrane domain

Amino acid 228.-.242 intracellular domain

Amino acid 243-. 264 transmembrane domain

Amino acids 265' -.283 extracellular domain-extracellular loop 3

Amino acid 284.-.307 transmembrane domain

Amino acid 308-. 350 intracellular domain-C-terminus

Details of microorganism preservation

A hybridoma producing a monoclonal antibody designated 7F3 was deposited with ECACC at 11/6 of 2000 with accession number 00110609.

A hybridoma producing a monoclonal antibody designated 6C12(6C 12M 12) was deposited with ECACC at 9/2 of 2002 with accession number 02090226.

A hybridoma producing a monoclonal antibody designated 12D4(12D4-P9) was deposited with the ECACC at 9.2.2002 under accession number 02090227.

These deposits were made under the budapest treaty. This ensures that viable cultures can be preserved for 30 years from the date of preservation. These organisms are available from ECACC under the terms of the budapest treaty, which ensures that progeny of the culture are available to the public for long periods of time and without restriction after authorization.

The assignee of the present application has agreed that if a deposited culture is inactivated or lost or destroyed under appropriate culture conditions, it will be replaced with a viable specimen of the same culture immediately upon notification. The availability of the deposited culture is not meant to be an admission that the invention may be practiced under the patent rights granted under the authority of any government in the light of its patent laws.

Monoclonal and recombinant antibodies

The inventors have generated murine monoclonal antibodies specific for C5aR and designated 7F3, 6C12 and 12D4 as described herein. Surprisingly, these monoclonal antibodies (mabs) substantially or completely block the binding of C5a to C5 aR. In particular, MAb7F3 is a fully neutralizing antibody.

Unlike other known anti-C5 aR antibodies, 7F3, 6C12, and 12D4 react with regions other than the N-terminal domain of C5 aR. It is believed that 7F3, 6C12, and 12D4 reacted primarily with the second extracellular loop (residues 175 to 206) of C5 aR. For example, the reaction of MAb12D4 with C5aR was almost completely stopped by mutating residues 181 and 192 of the second extracellular loop from tyrosine to phenylalanine. This inhibition was observed in binding studies involving the C5aR mutation L2-FF (Farzan et al, J.Exp.Med., 193: 1059-1065, 2001).

Because the extracellular loops and the N-terminal domain are similarly conformationally and closely related, these MAbs can also bind to other extracellular loops or to a region of the N-terminal domain.

Surprisingly, it has been shown that MAb7F3, 6C12, and 12D4 also inhibit the activation of neutrophils by other chemoattractant ligands (chemoattractrant ligands). Examples of these other chemoattractant ligands include CXCR1 and CXCR2 ligands IL-8, ENA-78 and GPC-2. This ability to inhibit the function of different chemoattractant receptors (chemoattractrant receptors) provides a rare and unexpected benefit not seen with other known anti-C5 aR molecules. In particular, anti-C5 aR molecules that inhibit the function of multiple neutrophil chemoattractant receptors are extremely effective therapeutic agents for the treatment of immune diseases.

In one aspect, the invention provides antibodies that bind to the extracellular loop of C5aR alone, preferably the second extracellular loop, or in combination with other loops or domains. In a preferred aspect, the invention provides antibodies that bind to C5aR and have an antigenic determinant specificity identical or similar to that of any of MAb7F3, 6C12 and 12D 4.

"antibodies" as used herein include intact molecules as well as fragments thereof which bind to an antigenic determinant, e.g., Fab, F (ab') 2, and Fv. These antibody fragments retain some ability to selectively bind to their antigen or receptor and are defined as follows:

fab, a fragment containing a monovalent antigen-binding fragment of an antibody molecule, which can be generated by degrading the entire antibody with the enzyme papain to generate an intact light chain and a portion of a heavy chain.

Fab', fragments of an antibody molecule obtainable by treatment of the whole antibody with pepsin and subsequent reduction to yield one complete light and part of the heavy chain; two Fab' fragments were obtained per antibody molecule.

(Fab') 2, fragments of an antibody obtainable by treating the whole antibody with pepsin without subsequent reduction; (Fab ') 2 is a dimer of two Fab' fragments linked by two disulfide bonds;

fv, defined as a genetically engineered fragment containing the variable regions of the light chain and the variable regions of the heavy chain, represented as two chains; and

5. single chain antibodies ("SCA"), are defined as genetically engineered molecules comprising a light chain variable region and a heavy chain variable region linked by an appropriate polypeptide linker and as genetically fused single chain molecules.

Methods for making such fragments are known in the art. (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), incorporated herein by reference).

The term "epitope" as used herein refers to any antigenic determinant on an antigen that binds to the antigen-binding portion of an antibody. Antigenic determinants generally include chemically active surface groups of the molecule, such as amino acids or sugar side chains and generally have specific three-dimensional structural characteristics as well as specific charge characteristics.

Antibodies of the invention can be prepared using cells expressing C5aR, intact C5aR, or fragments containing one or more extracellular loops as the vaccinating antigen. If desired, the peptide used to inoculate the animal can be derived from translated cDNA or chemically synthesized, purified and ligated to a carrier protein. Such commonly used carriers for chemical conjugation to peptides include Keyhole Limpet Hemocyanin (KLH), thyroglobulin, Bovine Serum Albumin (BSA), tetanus toxoid. The binding peptide can then be used to inoculate an animal (e.g., a mouse or rabbit).

If desired, the polyclonal antibody may be further purified, for example, by binding the antibody to a peptide bound to a substrate and eluting it. Those skilled in the art are aware of the techniques commonly used in various immunological techniques for purifying and/or concentrating polyclonal and monoclonal antibodies, see, e.g., Coligan, et al, Unit 9, Current Protocols in Immunology, Wiley Interscience, 1991, incorporated by reference).

Monoclonal antibodies can be prepared using any technique for the production of antibody molecules by continuous culture cell lines, such as hybridoma technology, human B-cell hybridoma technology, and EBV hybridoma technology (Kohler et al Nature 256, 495-.

Antibodies that bind to the extracellular loop of C5aR can be identified and isolated from antibody expression libraries by methods known in the art. For example, one method for identifying and isolating antibody binding domains that bind to the extracellular loop of C5aR is a phage vector system. This vector system has been used to express a combinatorial library of murine antibodies from E.coli (Huse, et al., Science, 246: 1275-shocking 1281, 1989) and Fab fragments from the human antibody library (Mullinax, et al., Proc. Nat. Acad. Sci., 87: 8095-shocking 8099, 1990). The method may also be applied to hybridoma cell lines expressing monoclonal antibodies capable of binding to preselected ligands. Hybridomas secreting the desired monoclonal antibodies can be generated in a variety of ways, using techniques well known to those of ordinary skill in the art, and need not be repeated here. Details of these techniques are described in the literature references such as monoconal Antibodies hybrids: ANew Dimension in Biological Analysis, Edited by Roger H.Kennett, et al, Plenum Press, 1980; and U.S.4,172,124, incorporated by reference.

Additionally, methods of producing chimeric antibody molecules with different combinations of "humanized" antibodies are known in the art and include combinations of murine variable regions and human constant regions (Cabily, et al. Proc. Natl. Acad. Sci. USA, 81: 3273, 1984), or by grafting the human-antibody variant derivatives (CDRs) onto the human framework (Riechmann, et al., Nature 332: 323, 1988).

The invention further provides chimeric antibodies to the anti-C5 aR antibodies of the invention or biologically active fragments thereof. The term "chimeric antibody" as used herein refers to an antibody in which the variable region of an antibody from one species is combined with the constant region of an antibody from a different species or equally to a CDR-grafted antibody. The construction of chimeric antibodies using recombinant DNA techniques has been described, for example, in Shaw, et al, j.immun, 138: 4534(1987), Sun, LK., et al, proc.natl.acad.sci.usa, 84: 214-218(1987).

Any of the above antibodies or biologically active antibody fragments can be used to produce CDR grafted and chimeric antibodies. "CDRs" or "complementarity determining regions" or "hypervariable regions" are defined as the amino acid sequences of the light and heavy chains of an antibody that form a three-dimensional loop structure that contributes to the formation of an antigen-binding site.

The term "CDR-grafted" antibody as used herein refers to an antibody having at least a portion of one or more CDR sequences of the light chain and/or variable domain replaced with an amino acid sequence derived from a homologous portion of a CDR sequence of an antibody having a different binding specificity for a given antigen or receptor.

The homologous CDR sequences are said to be "grafted" onto the substrate or recipient antibody. A "donor" antibody is an antibody that provides CDR sequences, and an antibody that accepts substituted sequences is a "substrate" antibody. These CDR-grafted antibodies can be readily produced by those skilled in the art using the teachings provided herein in combination with methods well known in the art (see, Borrebiack, C.A., Antibody Engineering: A Practical Guide, W.H.Freeman and Company, New York, 1992, incorporated by reference).

The invention also provides cell lines producing the monoclonal antibodies of the invention. Isolation of cell lines producing monoclonal antibodies of the invention can be accomplished using conventional screening techniques that recognize the basic reaction pattern of the relevant monoclonal antibody. Thus, if the monoclonal antibody tested binds to C5aR and blocks C5 a-mediated biological activity, the monoclonal antibody tested is equivalent to the monoclonal antibody produced by the cell line of the invention.

Antibodies with the same or similar epitope specificity as MAb7F3, 6C12 or 12D4 can be recognized using the ability to compete with specific mabs that bind to C5aR (e.g., cells with C5aR, e.g., transfectants with C5aR, monocytes, dendritic cells, macrophages and basophils). The binding site for any of the antibodies of MAb7F3, 6C12, or 12D4 can be mapped using receptor chimerization (Rucker et al, Cell 87: 437-446(1996)) or other techniques known in the art.

It is also possible to determine, without undue experimentation, whether a monoclonal antibody has the same specificity as a monoclonal antibody of the invention, as determined by whether the former inhibits the binding of the latter to a peptide comprising the extracellular loop of C5 aR. If the monoclonal antibody being tested competes with the monoclonal antibody of the invention, as indicated by a decrease in binding of the monoclonal antibody of the invention, then the two monoclonal antibodies bind to the same or closely related epitopes.

Another method of determining whether a monoclonal antibody has specificity to a monoclonal antibody of the invention is to pre-incubate the monoclonal antibody being tested with a peptide with which the antibody is predicted to react, and then add the monoclonal antibody of the invention to determine whether the monoclonal antibody of the invention is inhibited from binding to the peptide. If the monoclonal antibodies of the invention are inhibited, all of the monoclonal antibodies that may be tested have the same or functionally equivalent specificity for the antigenic determinant of the monoclonal antibodies of the invention. Screening of the monoclonal antibodies of the invention and determining whether the monoclonal antibodies block the binding of C5a to C5aR can also be performed using appropriate peptides.

By using the monoclonal antibody of the invention, anti-idiotypic antibodies can be generated which can be used to screen monoclonal antibodies to identify whether the antibody has the same binding specificity as the monoclonal antibody of the invention. These antibodies can also be used for vaccination purposes (Herlyn, et al., Science, 232: 100, 1986). These anti-idiotype antibodies can be produced using well-known hybridoma techniques (Kohler and Milstein, Nature, 256: 495, 1975). An anti-idiotype antibody is an antibody that recognizes unique determinants present on monoclonal antibodies produced by related cell lines. These determinants are located in the hypervariable regions of the antibody. This is the region that binds to a given epitope (antigen binding portion) and thus it determines the specificity of the antibody. Anti-idiotype antibodies can be prepared by inoculating an animal with a relevant monoclonal antibody. The vaccinated animals will recognize and respond to the idiotypic determinants of the vaccinating antibody and generate antibodies against these idiotypic determinants. By using anti-idiotype antibodies specific for the vaccinated animal of the monoclonal antibody of the invention produced by the cell line used to vaccinate the second animal, other clones having the same antigenic determinants as the hybridoma antibody used for vaccination can be identified. The idiotypic identity of the monoclonal antibodies of both cell lines demonstrates that both monoclonal antibodies recognize the same epitope identically. Thus, by using anti-idiotype antibodies, other hybridomas expressing monoclonal antibodies with the same epitope specificity can be identified.

Monoclonal antibodies that mimic antigenic determinants can also be generated using anti-idiotypic techniques. For example, an anti-idiotype monoclonal antibody made against a first monoclonal antibody will have a binding domain in the hypervariable region which is the "image" of the epitope to which the first monoclonal antibody binds. Thus, since the binding domain of an anti-idiotype monoclonal antibody can effectively act as an antigen, the anti-idiotype monoclonal antibody can be used for immunization.

Antibody fragments comprising an epitope binding site of any of the mabs of the invention can be generated by known techniques. For example, suitable antibody fragments can be obtained by first obtaining MAb7F3 from a stored hybridoma and then processing the antibody (e.g., by proteolytic digestion) to obtain its hypervariable regions.

Similarly, DNA encoding the hypervariable regions may be cloned in a suitable host using standard recombinant DNA procedures, for example as described herein.

Preferred antibodies of the invention comprise a variable region or one or more CDR loops substantially identical to the variable region or one or more CDR loops of MAb7F3, 6C12, or 12D 4. It is understood that the variable regions or CDR loops shown in the sequence listing can be modified for use in the present invention. In general, modifications are made to maintain the binding specificity of the sequence. Conservative substitutions may be made, for example, without affecting the binding specificity of the antibody. Thus, in one embodiment, amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 to provide modified sequences which retain substantially the same binding specificity. However, in an alternative embodiment, modifications of the amino acid sequence of the antibodies of the invention may be intentionally made to reduce the biological activity of the antibody. For example, a modified antibody that retains the ability to bind to C5aR but lacks a functional effector domain may be used as an inhibitor of the biological activity of C5 aR.

Amino acid substitutions may also include the use of non-naturally occurring analogs, e.g., to increase the plasma half-life of an administered therapeutic antibody.

Typically, less than 20%, 10% or 5% of the amino acids of a preferred variant or derivative are altered compared to the corresponding variable region or CDR loop described in the sequence listing.

Throughout the present invention, a sequence "substantially identical" to one of the regions of a variable region as indicated in the sequence listing may include an amino acid sequence that is at least 80%, 85% or 90% identical, preferably at least 95 or 98% identical at the amino acid level to at least 20, preferably at least 50 amino acids of the variable region. Homology should be considered for those regions of the sequence known to be critical for binding specificity, rather than those adjacent sequences that are not critical.

Homology comparisons can be made visually or, more commonly, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percent homology between two or more sequences.

The percent homology of adjacent sequences can be calculated by aligning one sequence with the other and comparing the amino acids of one sequence directly with the corresponding amino acids of the other sequence, residue by residue. This is called an "ungapped" alignment. Typically, these unnotched alignments can only be performed over a relatively small number of residues (e.g., less than 50 contiguous amino acids).

Although this is a very simple and consistent approach, it fails to take into account, for example, that when making an overall comparison, an insertion or deletion of one residue in an otherwise perfectly identical pair will cause the amino acid residues following it to be out of alignment, thus significantly causing a% reduction in homology. Thus, most comparison methods are designed to generate optimal alignments that take into account possible insertions and deletions without greatly reducing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment so that local homology is maximised as far as possible.

Most alignment programs can modify gap compensation. However, when using sequence comparison software, it is preferred to use default values. For example, when using the GCG Wisconsin Bestfit package (see below), the default complement of amino acid sequences is gap-12 and each extension-4.

Therefore, the calculation of the maximum value of the percentage of homology requires first generating the optimal alignment taking into account gap compensation. A suitable computer program for performing these alignments is the GCG Wisconsin Bestfit program package (University of Wisconsin, U.S. A.; Devereux et al, 1984, Nucleic acids research 12: 387). Other examples of software that can be used for sequence comparison include, but are not limited to, the BLAST package (see Ausubel et al, 1999 ibid-Chapter 18), FASTA (Atschul et al, 1990, J.mol.biol., 403- "410), and GENEWORKS comparison kit. Both BLAST and FASTA can be used for offline or online searches (see Ausubel et al, 1999ibid, pages 7-58 to 7-60)). Preferably, however, the GCG Bestfit software package is used.

Although the final% homology can be determined in terms of homology, the alignment program itself is generally not based on a non-perfect, i.e., perfect, pair-wise comparison. Instead, a similarly scaled integration model is often used that assigns an integration to each pair comparison based on chemical similarity or evolutionary distance. An example of such a model that is commonly used is the BLOSUM62 model-the default model of the BLAST suite of programs. GCG Wisconsin programs often use public default values or custom symbol comparison tables if provided (see user manual for further details). Preferably using the common default values of the GCG package or a default model in other software, such as BLOSUM 62.

Once the software has generated the optimal alignment, percent homology, preferably percent sequence identity, can be calculated. Software typically compares this as part of the sequence comparison and generates a numerical result.

Humanization of antibodies

It is preferred that the antibodies of the invention be humanized, i.e., antibodies generated using molecular modeling techniques in which the humanized portion of the antibody is maximized with little or no loss of binding affinity formed by the variable regions of the murine antibody. Thus, in one embodiment, the invention provides a chimeric antibody comprising the amino acid sequence of a human framework region for humanizing or rendering a non-immunogenic hypervariable region from a murine monoclonal antibody, such as 7F3, C612 or 12D4, and the amino acid sequence of a constant region from a human antibody.

The methods described below are applied to humanize a wide variety of animal antibodies. A two-step method can be used that includes (a) selecting human antibody sequences for use as human frameworks for humanization, and (b) determining which variable region residues of an animal monoclonal antibody should be selected for insertion into the selected human framework.

The first step involves the selection of the best available human framework sequence for the available sequence information. This selection method is based on the following selection criteria.

(1) Percentage of homology

The sequences of the heavy and light chain variable regions of the animal monoclonal antibody to be humanized are optimally aligned and preferably compared to the heavy and light chain variable region sequences of all known human antibodies.

Once the sequences are so compared, the homology of the residues is recorded and the percent homology is determined. All other factors being equal, it is desirable to select a human antibody that has the highest percentage homology to the animal antibody.

(2) Uncertainty of sequence

The known human antibody chain sequences are then evaluated for the presence of unrecognized residues and/or ambiguous sequences, which is the uncertainty of the sequence. The most common such uncertainty is the incorrect identification of one amino acid as another during sequence analysis due to the loss of ammonia, e.g., the inaccurate identification of a glutamic acid residue, whereas the residue actually present in the protein is a glutamine residue. All other factors being equal, it is desirable to select human antibody chains with as little uncertainty as possible.

(3) Needle zone Spacing (Pin-region Spacing)

The variable region of the antibody chain contains intra-domain disulfide bonds. The distance between cysteine residues (number of residues) that make up these disulfide bonds is called the pin spacing [ Chothia et al, j.mol.biol.196: 901(1987)]. All other factors are the same, and it is most desirable that the pin spacing of the selected human antibody be similar or identical to the pin spacing of the animal antibody. It is also required that the spacing of the pin regions of the human sequence be similar to that of the 3-dimensional structure of known antibodies to facilitate computer model design.

Based on the aforementioned criteria, a human antibody (or antibodies) with the best overall combination of desired properties is selected as the framework for humanization of the animal antibody. The heavy and light chains selected may be from the same or different human antibodies.

The second step of the method of the invention involves determining which animal antibody variable region sequences should be selected for transplantation into a human framework. The selection method is based on the following selection criteria:

(1) residue selection

Two types of potential variable region residues are evaluated in animal antibody sequences, the first being referred to as "minimal residues". These minimized residues comprise the CDR structural loops plus any other residues required to support and/or position the CDR structural loops as shown in the computer model design.

Other types of potential variable region residues are referred to as "maximizing residues". They comprise the minimized residues plus any other residues that fall within the CDR structural loop residues of about 10 * and have a surface accessible to aqueous solutions as shown by computer model design [ Lee et al, j.biol.chem.55: 379(1971)]

(2) Computer model design

In order to identify potential variable region residues, in (a) the variable region sequences of an animal antibody to be humanized; (b) selected human antibody framework sequences, and (c) all possible combinatorial antibodies including human antibody framework sequences grafted with various minimum and maximum animal antibody residues.

Computer model design was performed using software suitable for protein model design, and structural information was obtained from (a) an antibody having a variable region amino acid sequence that almost completely coincides with the variable region amino acid sequence of an animal antibody and (b) an antibody having a known 3-dimensional structure. An example of software that may be used is SYBYL Biopolymer Module software (Tripos Associates). The antibody from which structural information can be obtained can be, but need not be, a human antibody.

Based on the results obtained in the foregoing analysis, a recombinant chain containing the animal variable region closest to the variable region of the animal antibody generated by computer model design was selected for humanization.

Antibody isotype (isotypes)

Under certain conditions, monoclonal antibodies of one isotype may be preferred over monoclonal antibodies of another isotype for their diagnostic or therapeutic effect. For example, it is known from studies of antibody-mediated cell lysis that unmodified murine monoclonal antibodies of the gamma-2 a and gamma-3 isotype are generally more effective at lysing target cells than antibodies of the gamma-1 isotype. This differential effect is believed to be due to the ability of the gamma-2 a and gamma-3 isoforms to more efficiently participate in the lytic destruction of target cells. Specific isotypes of monoclonal antibodies are subsequently produced from maternal hybridomas secreting monoclonal antibodies of different isotypes by separately converting the variants using a daughter selection technique (Steplewski, et al, Proc. Natl. Acad. Sci.U.S.A., 82: 8653, 1985; Spira, et al, J.Immunol. methods, 74: 307, 1984). Thus, the monoclonal antibodies of the invention will include type-converting variants specific for any one of the antibodies MAb7F3, 6C12, and 12D 4.

In vitro assay

The monoclonal antibodies of the invention are suitable for in vitro applications, for example they may be applied to a liquid phase or bound to a solid phase support in an immunoassay. These antibodies can be used to monitor C5aR levels in a sample. Likewise, anti-idiotype antibodies are used to determine the level of C5a in a sample. In addition, monoclonal antibodies in these immunoassays can be detectably labeled in various ways. Examples of the types of immunoassays that can utilize the monoclonal antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the Radioimmunoassay (RIA) and sandwich (immunoassay) assays. Immunoassays in a forward, reverse, or simultaneous manner, including immunohistochemical detection of physiological samples, can be used to detect antigens using the monoclonal antibodies of the invention. Other immunoassay formats that do not require undue experimentation will be known or readily apparent to those skilled in the art.

The antibodies of the invention can be bound to a variety of different carriers and used to determine the presence of C5 aR. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, natural and modified celluloses, polyacrylamides, agarose, and magnetite. The properties of these antibodies may be soluble or insoluble for the purposes of the present invention. Those skilled in the art will know of other suitable vectors for binding monoclonal antibodies or will be able to define such vectors using routine experimentation.

In one embodiment, cells that naturally express C5aR or cells that contain a recombinant nucleic acid sequence encoding C5aR or a variant thereof are used in the binding assays of the invention. The cells are maintained under conditions suitable for expression of the receptor. These cells are contacted with the antibody or fragment under conditions suitable for binding (e.g., in a suitable binding buffer) and binding is determined using standard techniques. To determine binding, the degree of binding is determined relative to an appropriate control (e.g., compared to background determined in the absence of antibody, compared to binding to a second antibody (i.e., a standard antibody), compared to binding of antibody to non-transfected cells). Cell fragments, such as membrane fragments, containing the receptor or liposomes containing the receptor can be used in place of whole cells.

Binding inhibition assays may also be used to identify antibodies or fragments thereof that bind to C5aR and inhibit the binding of C5a and C5aR or functional variants. For example, a binding assay may be performed in which a decrease in C5a binding (with antibody present) is detected or determined as compared to C5a binding in the absence of antibody. Compositions comprising individual and/or recombinant mammalian C5aR or functional variants thereof can be contacted with C5a and the antibody simultaneously, or sequentially one after the other. The reduction in the extent of ligand binding in the presence of the antibody is an indication of inhibition of binding by the antibody. For example, binding of the ligand may be reduced or eliminated.

Other methods of determining the presence of antibodies that bind to C5aR are available, such as other suitable binding assays, or methods of monitoring events triggered by receptor binding, including signaling functions and/or stimulation of cellular responses (e.g., leukocyte aggregation). Antibodies recognized in this manner can be further evaluated to determine whether they can act to inhibit other functions of C5aR upon binding or to evaluate their therapeutic use.

Signal analysis

Binding of a ligand or enhancer such as an agonist to C5aR can form a signal through the G protein binding receptor and stimulate the activity of G proteins and other intracellular signaling molecules. The induction of signal function by a compound (e.g., an antibody or fragment thereof) can be monitored using any suitable method. Such a test may be used to identify an antibody agonist to C5 aR. The inhibitory activity of the antibodies or their functional fragments can be determined in an assay using a ligand or enhancer and the ability of the antibodies to inhibit the activity induced by the ligand or enhancer is assessed.

Methods known in the art or other suitable methods (see, e.g., Neote, K.et al., Cell, 72: 415-425, 1993; Van Riper et al., J.Exp.Med., 177: 851-856, 1993; Dahinden, C.A.et al., J.Exp.Med., 179: 751-756, 1994) can detect G protein activity, e.g., hydrolysis of GTP to GDP, or receptor binding triggered subsequent signaling events, e.g., the induction of a rapid and transient increase in intracellular (cytosolic) free calcium concentration.

For example, functional assays using Sledziewski et al, where a hybrid G protein binds to a receptor, can be used to test the ability of ligands or enhancers to bind to the receptor and activate the G protein (Sledziewski et al, U.S. Pat. No.5,284,746).

These assays can be performed in the presence of the antibody or fragment thereof being assayed, as well as the ability of the antibody or fragment to elicit activity by a ligand or enhancer using known methods and/or methods described herein.

Detection of chemochemotaxis and cellular stimulation

Chemochemotaxis assays can also be used to assess the ability of antibodies or their functional fragments to block ligand binding to C5aR and/or inhibit functions associated with ligand and receptor binding. These assays are based on the functional migration of cells induced extracellularly or intracellularly by the compounds. Chemochemotaxis can be assessed in any suitable manner, for example, by detection using a 96-well chemochemotactic plate, or by other methods of technical validation. For example, Springer et al, (Springer et al, WO 94/20142, published Sep.15, 1994; see alsoBerman et al, Immunol. invest.17: 625-. Transendothelial migration into collagen has also been described (Kavanaugh et al, J.Immunol., 146: 4149-. Stable transfectants of murine L1-2 pre-B cells or other suitable host cells capable of chemochemotaxis can be used for chemochemotaxis assays.

Generally, chemochemotaxis assays monitor the movement or migration of appropriate cells, such as leukocytes (e.g., lymphocytes, eosinophils, basophils), directly to or through a barrier (e.g., endothelium, or filter), toward increasing levels of compounds, from a first surface of the barrier to an opposite second surface. The membrane or filter provides a convenient barrier so that the movement or migration of suitable cells directly to or through the filter towards increasing levels of the compound can be monitored from a first surface of the filter to an opposite second surface. In some assays, the membrane is coated with a substance such as ICAM-1, fibronectin, or collagen to promote adhesion. These assays provide an in vitro approximation of leukocyte "homing".

For example, inhibition of cell migration in a suitable container (a containment device) from a first compartment into or through a microporous membrane into a second compartment containing a test antibody, separated from the first compartment by a membrane, can be detected or measured. Membranes with appropriate pore size, e.g. nitrocellulose, polycarbonate, are selected for monitoring the specific migration of the reactive compound. For example, pore sizes of about 3-8 microns, and preferably about 5-8 microns, may be used. The pore size on the filter membrane may be uniform or within a suitable range of pore sizes.

To assess migration and inhibition of migration, the distance of migration into the filter, the number of cells that remain adhered to the second surface of the filter across the filter, and/or the number of cells that have accumulated in the second chamber can be determined using standard techniques (e.g., microscopy). In one embodiment, the cells are labeled with a detectable label (e.g., a radioisotope, fluorescent label, antigen or epitope label) and migration in the presence or absence of the antibody or fragment can be determined by measuring the label attached to the membrane and/or present in the second chamber by a suitable method (e.g., by measuring radioactivity, fluorescence, immunodetection). The extent of migration induced by the antibody agonist relative to a suitable control (e.g., compared to background migration measured in the absence of antibody, compared to the extent of migration induced by a second compound (i.e., a standard compound), compared to migration induced by antibody of untransfected cells) can be determined. In one embodiment, transendothelial migration may be monitored, particularly for T cells, monocytes, or cells expressing C5 aR. In this embodiment, migration through the endothelial cell layer is measured. To prepare the cell layer, endothelial cells may be cultured on the microfiltration membrane or membrane, optionally encapsulated with a substance such as collagen, fibronectin, or other extracellular matrix protein to facilitate attachment of the endothelial cells. Preferably, the endothelial cells are cultured until a confluent monolayer of cells is formed. Various mammalian endothelial cells can be used to form a monolayer of cells, including, for example, venous, arterial, or microvascular endothelial cells, such as human umbilical vein endothelial cells (Clonetics Corp, San Diego, Calif.). Endothelial cells of the same species are preferred for the evaluation of chemochemotaxis in response to specific mammalian receptors. Furthermore, endothelial cells from heterologous mammalian species or genera may also be used.

Typically, the detection is carried out by detecting the directional migration of cells into or through a membrane or filter, the direction of migration being towards increasing levels of the compound, from a first surface of the filter to an opposite second surface of the filter, wherein the filter contains a layer of endothelial cells at the first surface. Directional migration occurs from the region adjacent the first surface, into or through the membrane, towards the opposite side of the filter where the compound is located. The concentration of the compound in the region adjacent to the second surface is higher than the concentration of the compound in the region adjacent to the first surface.

In one embodiment for determining antibody inhibitors, a composition comprising cells capable of migrating and expressing C5aR is placed in a first chamber. A composition comprising one or more ligands or enhancers capable of inducing chemochemotaxis of cells in the first chamber (having chemochemotactic function) is placed in the second chamber. Preferably, the composition comprising the test antibody is placed in, preferably the first chamber shortly before, or in synchrony with, the cells.

Antibodies or functional fragments thereof that bind to the receptor and inhibit chemochemotaxis of C5 aR-expressing cells induced by the ligand or enhancer are inhibitors of receptor function (e.g., inhibitors of stimulatory function) in the present assay. The reduction in the extent of ligand or enhancer induced migration in the presence of the antibody is indicative of inhibitory activity. Separate binding studies can be performed to determine whether inhibition is the result of antibody binding to the receptor or occurs by a different mechanism.

In vitro monitoring of leukocyte infiltration in tissues in response to in vivo injection of compounds (e.g., chemical factors or antibodies) is described below (see inflammation model). These in vivo homing models measure the ability of cells to respond to ligands or enhancers and to chemotact to sites of inflammation by migration and chemistry, and measure the ability of antibodies or their fragments to block such migration.

In addition to the methods described, the effect of an antibody or fragment on the stimulatory function of C5aR is determined by monitoring the cellular response induced by an active receptor using a suitable host cell containing the receptor.

Identification of additional ligands, inhibitors or enhancers of C5aR

The above assays, which can be used to determine the binding and function of the antibodies and fragments of the invention, can be used to identify other ligands and other substances that bind to C5aR or functional variants thereof, as well as inhibitors and/or enhancers of C5aR function. For example, competitive assays using the antibodies or portions thereof can identify substances with the same or similar binding specificity as the antibodies of the invention or functional portions thereof. Thus, the present invention also includes methods of identifying ligands or other agents for the receptor that bind to C5aR, as well as inhibitors (antagonists) or enhancers (agonists) of receptor function. In one embodiment, cells having C5aR protein or functional variants thereof (e.g., leukocytes, cell lines, or suitable host cells that have been processed to express mammalian C5aR protein or functional variants encoded by nucleic acids introduced into the cells) are used in methods of identifying and evaluating the effect of ligands or other agents that bind to receptors, including inhibitors or enhancers of receptor function. These cells are also used to evaluate the function of the expressed receptor protein or polypeptide.

According to the invention, ligands and other substances which bind to the receptor, inhibitors and enhancers of receptor function can be identified in a suitable assay and the therapeutic effect can be further evaluated. Antagonists of receptor function can be used to inhibit (reduce or prevent) receptor activity, and ligands and/or agonists can be used to induce (trigger or enhance) normal receptor function as indicated therein. Accordingly, the present invention provides a method of treating inflammatory diseases, including autoimmune diseases and transplant rejection, comprising administering an antagonist of receptor function to an individual (e.g., a mammal). The invention further provides a method of stimulating receptor function by administering to an individual a novel ligand or agonist of receptor function, providing a novel means of selectively stimulating leukocyte function, e.g., which is useful in the treatment of infectious diseases and cancer.

As used herein, a "ligand" of a C5aR protein refers to a specific type of substance that binds to a mammalian C5aR protein, including natural ligands and synthetic and/or recombinant forms of natural ligands. In a preferred embodiment, the ligand that binds to the C5aR protein has a high affinity.

An "antagonist" as used herein is a substance that inhibits (reduces or prevents) at least one functional characteristic of C5aR protein, such as binding activity (e.g., ligand binding, enhancer binding, antibody binding), signaling activity (e.g., activation of mammalian G protein, induction of rapid and transient increases in plasma free calcium concentration), and/or cellular response function (e.g., stimulation of chemochemotaxis, extracellular secretion of leukocytes, or release of inflammatory mediators). The term antagonist includes substances that bind to the receptor (e.g., antibodies, mutants of natural ligands, small molecular weight organic molecules, other competitive inhibitors of ligand binding) and substances that inhibit the function of the receptor without binding thereto (e.g., anti-idiotypic antibodies).

An "agonist" as used herein is a substance that enhances (induces, causes, enhances or increases) at least one functional characteristic of C5aR protein, such as binding activity (e.g., ligand, inhibitor and/or enhancer binding), signaling activity (e.g., activation of mammalian G protein, induction of rapid and transient increases in plasma free calcium concentration), and/or cellular response function (e.g., stimulation of chemochemotaxis, extracellular secretion of leukocytes, or release of inflammatory mediators). The term agonist includes substances that bind to the receptor (e.g., antibodies, homologues of natural ligands from other species), as well as substances that promote the function of the receptor without binding thereto (e.g., by activating the associated protein). In a preferred embodiment, the agonist is a homologue of the natural ligand.

Thus, the invention also relates to a method for detecting or identifying substances that bind to C5aR or variants of their binding partners, including ligands, antagonists, agonists and other substances that bind to C5aR or functional variants. According to the present methods, the tested substances, antibodies or antigen-binding fragments of the invention (e.g., antibodies having the same or similar antigenic determinant specificity as 7F3, and antigen-binding fragments thereof), and compositions comprising C5aR or ligand binding variants thereof, can be combined. The above components are mixed under conditions suitable for binding of the antibody or antigen-binding fragment to C5aR, and binding of the antibody or fragment to C5aR is detected or determined, directly or indirectly, according to the methods described herein or other suitable methods. A decrease in the amount of complex formed relative to an appropriate control (e.g., in the absence of the substance being tested) indicates that the substance binds to the receptor or variant described above. Compositions comprising C5aR may be membrane fragments of cells bearing recombinant C5aR or their ligand binding variants. The antibodies or fragments thereof may be labeled with a label such as a radioisotope, a rotational label, an antigen or epitope label, an enzymatic label, a fluorescent group, and a chemiluminescent group.

Inflammation model

In vivo models of inflammation are available which can be used to evaluate the effect of the antibodies and fragments of the invention as therapeutic agents in vivo. For example, chemokines and antibodies or fragments thereof reactive with C5aR are injected subcutaneously into suitable animals, such as rabbits, mice, guinea pigs, or macaques, and leukocyte infiltration is monitored (see, e.g., Van Damme, J.et al, J.exp.Med., 176: 59-65 (1992); Zachariae, C.O.C.et al, J.exp.Med.171: 2177-. In one embodiment, skin biopsy histology evaluates infiltration of leukocytes (e.g., eosinophils, granulocytes). In another embodiment, labeled cells capable of chemochemotaxis and extravasation (e.g., stably transfected cells expressing C5aR) are administered to an animal. The test antibody or fragment is administered to the test animal before, simultaneously with, or after the labeled cells are administered to the test animal. The reduction in the degree of infiltration in the presence of antibody compared to the degree of infiltration in the absence of inhibitor is indicative of inhibition.

Diagnostic and therapeutic applications

The antibodies and fragments of the invention are useful in a variety of applications, including research, diagnostic and therapeutic applications. In one embodiment, these antibodies are labeled with a suitable label (e.g., a fluorescent label, a chemiluminescent label, an isotopic label, an antigen or epitope label, or an enzymatic label). For example, they can be used to isolate and/or purify receptors and their portions, and to study the structure (e.g., conformation) and function of receptors.

In addition, various antibodies of the invention can be used to detect C5aR or to assay, e.g., T cells (e.g., CD 8)⁺Cell, CD45RO⁺Cells), monocytes, and/or cells transfected with a receptor gene. They can therefore also be used for applications for diagnostic or research purposes, such as cell sorting (e.g. flow cytometry, fluorescence activated cell sorting).

The anti-C5 aR antibodies of the invention are useful in diagnostic applications. Typically, diagnostic assays are used to determine the formation of a complex formed by the binding of antibodies or fragments thereof to C5 aR. For diagnostic purposes, the antibody or antigen binding fragment is labeled or unlabeled. The antibody or fragment may be directly labeled. A variety of labels may be used, including, but not limited to, radionuclides, fluorescers, enzyme substrates, enzyme cofactors, enzyme inhibitors, and ligands (e.g., biotin, haptens). A variety of suitable immunoassays are known to those of skill in the art (see, e.g., U.S. Pat. No. 3,817, 827; 3,850,752; 3,901,654 and 4,098,876). Immunohistochemistry of tissue samples may also be used in the diagnostic methods of the invention. When the antibody or fragment is unlabeled, the antibody or fragment can be assayed using a suitable method, such as agglutination detection. The unlabeled antibody or fragment can also be used in combination with another (i.e., one or more) suitable reagent that can be used to detect, for example, an antibody that is a labeled antibody (e.g., a second antibody) that reacts with a first antibody (e.g., an anti-idiotypic antibody or other antibody specific for unlabeled immunoglobulin), or other suitable reagent (e.g., labeled protein A).

Kits for detecting the presence of C5aR protein in a biological sample may also be prepared. These kits will include an antibody or fragment thereof that binds to C5aR, and one or more auxiliary reagents suitable for detecting the presence of a complex between the antibody or fragment and C5 aR. The antibody compositions of the invention may be provided in lyophilized form, which may be used alone or in combination with other antibodies specific for other antigenic determinants. The labeled or unlabeled antibody may be included in the kit with auxiliary components (e.g., buffers such as Tris, phosphate and carbonate, stabilizers, excipients, biocides, and/or inert proteins such as bovine serum albumin). For example, a lyophilized mixture of the antibody and the adjunct ingredient can be provided, or the adjunct ingredients can be provided separately to the user for combined use. These auxiliary materials are generally less than 5% by weight, based on the amount of active antibody, and the total amount is at least 0.001% by weight, based on the antibody concentration. The kit may provide the secondary antibodies using a container, such as a separate vial or canister, to which the antibodies can bind. If a second antibody is present, it is usually labeled and prepared in the same manner as described above for antibody preparation.

Likewise, the invention also relates to a method for detecting and/or quantifying C5aR expressed by a cell, wherein a composition comprising the cell or a fragment thereof (e.g., a membrane fragment) is contacted with an antibody or functional fragment thereof that binds to C5aR under conditions suitable for binding of the antibody or fragment, and binding is monitored. Detection of the antibody and the suggestion that a complex formed between the antibody and C5aR indicates the presence of the receptor. For example, the binding of an antibody to a cell can be determined as described under the heading "binding assay". The method may be used to detect expression of C5aR on cells from an individual (e.g., a sample, such as a bodily fluid, such as blood, saliva, or other suitable sample). The expression level of C5aR on the surface of T cells or monocytes can also be determined, for example, by flow cytometry, and the level of expression (e.g., staining intensity) can be correlated with disease susceptibility, progression, or risk.

The receptor functions to chemochemotactic leukocytes for systemic migration, particularly to sites of inflammation. The migration of inflammatory cells out of blood vessels is regulated by a three-step process involving the interaction of leukocytes and endothelial Cell adhesion proteins and Cell-specific chemoattractants and activators (Springer, T.A., Cell, 76: 301-314 (1994); Butcher, E.C., Cell, 67: 1033-1036 (1991); Butcher, E.C., and Picker, L.J., Science (Wash.D.C.), 272: 60-66 (1996)). They are: (a) low affinity interactions between leukoselectin and endothelial cell sugars; (b) high affinity interactions between leukocyte chemoattractant receptors and chemoattractant/activators; and (c) tight binding between leukocyte integrins and endothelial cell adhesion proteins of the immunoglobulin superfamily. Different subpopulations of leukocytes express different selectins, chemoattractants and integrin populations. In addition, inflammation alters the expression of endothelial adhesion proteins as well as the expression of chemoattractants and leukocyte activation factors. Thus, there is a large difference in the selectivity of regulating leukocyte circulation to sites outside the blood vessel. The second step is critical, where activation of the leukocyte chemoattractant receptor is thought to cause a transition from selectin-mediated cell turnover to integrin-mediated tight binding. This prepares the leukocytes for migration to the perivascular site. Chemoattractant/chemoattractant receptor interactions are also critical for transendothelial migration and localization within tissues (Campbell, J.J., et al, J.cell biol., 134: 255-266 (1996); Carr, M.W., et al, Immunity, 4: 179187 (1996)). This migration is directed by the concentration gradient of the chemoattractant and towards the inflammatory foci.

C5aR has an important effect on leukocyte aggregation. C5aR may be a key chemoattractant receptor acting on neutrophils, eosinophils, T cells or T cell subsets or monocytes migrating to specific sites of inflammation, so anti-C5 aR mabs may be used to inhibit (reduce or prevent) leukocyte migration, particularly migration associated with neutrophil arrest injury, such as reperfusion injury and stroke, T cell dysfunction, such as autoimmune disease, or allergic or monocyte mediated diseases, such as atherosclerosis.

Thus, the antibodies and their fragments of the invention may also be used to modulate receptor function in research and therapeutic applications. For example, the antibodies and functional fragments described herein can act as inhibitors that inhibit (reduce or prevent) (a) binding to a receptor (e.g., a ligand, inhibitor or enhancer), (b) receptor signaling function, and/or (c) stimulating function. Antibodies that are inhibitors of receptor function may block binding of ligands or enhancers directly or indirectly (e.g., by causing a conformational change). For example, an antibody may inhibit receptor function by inhibiting binding of a ligand or by desensitization (with or without inhibition of ligand binding). Antibodies that bind to a receptor may also act as agonists of receptor function, triggering or stimulating receptor function, e.g., binding to a receptor-initiated signal or stimulating function of the receptor (e.g., leukocyte aggregation).

Accordingly, the present invention provides a method of inhibiting leukocyte aggregation in a mammal (e.g., a human patient) comprising administering to the mammal an effective amount of an antibody or functional fragment of the invention. The invention also provides a method of inhibiting other effects associated with C5aR activity, such as histamine release by basophils and granules release by eosinophils, basophils and neutrophils. Administration of the antibodies or fragments of the invention may result in amelioration or elimination of the disease state.

Monoclonal antibodies may also be used in the immunotherapy of immune related diseases. The terms "immunotherapeutically" or "immunotherapy" as used herein in connection with the monoclonal antibodies of the invention refer to both prophylactic and therapeutic administration. Thus, monoclonal antibodies can be administered to high risk patients to reduce the likelihood and/or severity of immune disease, as well as to patients who have demonstrated active disease, such as sepsis due to gram-negative bacterial infection.

The antibodies or functional fragments thereof may be used for the treatment of allergy, atheromatous plaque formation, allergic reactions, malignancies, chronic and acute inflammation, histamine and IgE mediated allergic reactions, shock, and rheumatoid arthritis, atherosclerosis, multiple sclerosis, allograft rejection, fibrotic diseases, asthma, inflammatory glomerular diseases or any immune complex related disease.

Diseases or conditions of humans or other species that may be treated with inhibitors of C5aR receptor function (including antibodies or suitable fragments thereof) include, but are not limited to:

(a) inflammatory or allergic diseases and pathologies, including respiratory allergic diseases, such as asthma, allergic rhinitis, hypersensitivity lung disease, hypersensitivity pneumonitis, Interstitial Lung Disease (ILD) (e.g., idiopathic pulmonary fibrosis, or ILD associated with rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, systemic sclerosis, sjogren's syndrome, polymyositis, or dermatomyositis); allergic or hypersensitivity reactions, drug allergies (e.g., to penicillins, cephalosporins), insect sting allergies; inflammatory bowel diseases, such as crohn's disease and ulcerative colitis; spondyloarthropathy; scleroderma; psoriasis and inflammatory skin lesions such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria; vasculitis (e.g., necrotizing, cutaneous, and hypersensitive vasculitis);

(b) autoimmune diseases, such as arthritis (e.g., rheumatoid arthritis, psoriatic arthritis), multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, juvenile onset diabetes, renal diseases such as glomerulonephritis, autoimmune thyroiditis, behcet's disease;

(c) graft rejection (e.g., in transplantation), including allograft rejection or graft versus host disease;

(d) atherosclerosis;

(e) tumors with leukocyte skin or organ infiltration;

(f) other diseases or conditions that may be treated (including C5 aR-mediated diseases or conditions) in which an undesirable inflammatory response is inhibited include, but are not limited to, reperfusion injury, stroke, adult respiratory distress syndrome, certain hematologic tumors, cytokine-related toxicities (e.g., septic shock, endotoxic shock), polymyositis, dermatomyositis, pemphigoid, Alzheimer's disease, and granulomatous diseases including sarcoidosis.

The anti-C5 aR antibodies of the invention can block the binding of one or more ligands, thus blocking the chain reaction leading to one or more events downstream of the disease.

In a preferred embodiment, the antibodies of the invention are used to treat sepsis, stroke, or adult respiratory distress syndrome.

Diseases or conditions of humans or other species that can be treated with enhancers of C5aR function include, but are not limited to, immunosuppression, such as those individuals with immunodeficiency syndrome, such as AIDS, individuals undergoing radiation or chemotherapy, treatment of autoimmune diseases, or other drug treatment (e.g., glucocorticoid treatment), which all cause immunosuppression; and immunosuppression due to congenital defects in receptor function or other causes.

Mode of administration

One method of immunotherapy according to the invention entails administering the therapeutic agents of the invention by injection or infusion either before (prevention) or after (treatment) the onset of the immune disease.

One or more antibodies or fragments of the invention may be administered to an individual by a suitable route, either alone or in combination (before, simultaneously or after) with other drugs or agents. For example, the antibodies of the invention may be used prophylactically or therapeutically in combination with other monoclonal or polyclonal antibodies (e.g., in combination with antibodies that bind to a chemokine receptor, including but not limited to CCR2 and CCR3) or with anti-TNF or other anti-inflammatory drugs or with plasma preparations such as commercially available gamma globulin and immunoglobulin preparations. The antibodies or fragments of the invention can be used as compositions for separate administration and administered in combination with antibiotics and/or antimicrobial drugs.

An effective amount of the antibody or fragment (i.e., one or more antibodies or fragments) is administered. The preferred dose is an amount effective to achieve the desired therapeutic (including prophylactic) effect under the conditions of administration, e.g., an amount effective to inhibit C5aR function and thus the inflammatory response.

Various routes of administration are possible, including but not necessarily limited to: oral, dietary, topical, parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous injection), inhalation (e.g., tracheal, ocular, nasal or oral inhalation, nasal drip), depending on the disease or condition being treated. Other suitable methods of administration may also include rechargeable or biodegradable devices and sustained release polymer devices. The pharmaceutical compositions of the present invention may also be administered as part of a combination therapy with other drugs.

The form of the antibody or fragment administered will vary depending on the route of administration and the form chosen (e.g., solution, emulsion, capsule). A suitable pharmaceutical composition comprising the antibody or functional fragment thereof may be prepared in a physiologically acceptable vehicle or carrier. Mixtures of antibodies and/or fragments may also be used. For solutions or emulsions, suitable carriers include, for example, aqueous or ethanol/water solutions; for emulsions or suspensions, suitable carriers include saline and buffered media. Parenteral vehicles may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Various suitable aqueous carriers well known to those skilled in the art include water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution, and sugar gums. Intravenous vehicles may include various additives, preservatives, or liquid, nutrient or electrolyte supplements (commonly found in Remington's pharmaceutical Science, 16th Edition, Mack, ed. 1980). The compositions may optionally contain pharmaceutically acceptable auxiliary substances to bring them into close proximity with the physiological state, such as pH adjusting and buffering agents, toxicity adjusting agents, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate. The antibodies and fragments of the invention can be lyophilized for storage and reconstituted in a suitable carrier according to lyophilization and reconstitution techniques of known art. The optimum concentration of the active ingredient in the chosen vehicle can be determined empirically according to methods well known in the art and will depend on the desired final pharmaceutical form. For inhalation, the antibody or fragment can be dissolved and loaded into a suitable dispenser for administration (e.g., a nebulizer, atomizer, or pressurized aerosol dispenser).

The dosage range at which the monoclonal antibody of the present invention is administered is a dosage large enough to produce the desired effect, wherein the symptoms of immune disease are ameliorated or the likelihood of infection or overstimulation of the immune system is reduced. The dosage should not be so large as to cause adverse side effects such as high viscosity syndrome, pulmonary edema, congestive heart failure, etc. Generally, the dosage will vary with the age, general condition, sex and extent of the disease of the patient and will be determined by one skilled in the art. The individual family physician can adjust the dosage for any complication. The dosage may vary from about 0.1mg/kd to about 300mg/kg, preferably from about 0.2mg/kg to about 200mg/kg, most preferably from about 0.5mg/kg to about 20mg/kg, administered in one or more doses per day for one or more days.

It will be appreciated by those skilled in the art that the antibodies of the invention may be introduced into a subject of interest by administering a composition comprising a nucleic acid molecule encoding the antibody. The nucleic acid molecule may be in the form of DNA or RNA or a chimeric molecule containing DNA or RNA. The nucleotide sequence encoding the antibody may be cloned into an expression vector, wherein the drug-encoding sequence and the expression regulatory elements are operably linked. Expression regulatory elements are well known in the art and include, for example, promoters, enhancers and appropriate start and stop codons.

Various methods can be used to introduce nucleic acid molecules encoding antibodies into target cells in vivo. For example, naked nucleic acid can be injected at the target site, can be encapsulated into liposomes, or can be introduced via a viral vector.

Direct injection of nucleic acid molecules alone or encapsulated in, for example, cationic liposomes, can be used to transfer a nucleic acid-stable gene encoding TSP-1 into non-dividing or dividing cells in vivo (Ulmer et al, Science 259: 1745. multidot. 1748 (1993)). In addition, nucleic acids can be transferred into various tissues using particle bombardment methods (Williams et al, Proc. Natl. Acad. Sci. USA 88: 2726-.

Viral vectors can be used to genetically transfer nucleic acid molecules encoding antibodies into specific cell types in vivo. Viruses are specific infectious agents that infect and propagate within specific cell types. This specificity for infecting a particular cell type is particularly suited for targeting antibodies to selected cells in vivo. The choice of viral vector will depend in part on the cell type targeted.

Specific viral vectors capable of targeting to specific cell types are well known in the art. Such vectors include, for example, recombinant adeno-associated viral vectors with universal or tissue-specific promoters (Lebkowski et al U.S. Pat.No.5,354,678). An additional advantage of recombinant adeno-associated viral vectors is that recombinant viruses can stably integrate into the chromosome of even quiescent non-proliferating cells (Lebkowski et al, mol. cell. biol. 8: 3988-.

Viral vectors can be constructed by incorporating tissue-specific promoters or enhancers into the vector, allowing further control over the cell type expressing the encoded antibody (Dai et al, Proc. Natl. Acad. Sci. USA 89: 10892-10895 (1992)).

Retroviral vectors are also suitable for carrying out in vivo methods of delivering nucleic acid molecules encoding antibodies. These vectors can be constructed to act as infectious particles or as non-infectious particles that undergo only a single first round of infection.

Receptor-mediated DNA transfer methods can also be used to transfer nucleic acid molecules encoding antibodies into cells in a tissue-specific manner using tissue-specific ligands or antibodies that are non-covalently linked to the nucleic acid molecules by bridge molecules (Curiel et al, hum. Gene Ther.3: 147-154 (1992); Wu and Wu, J.biol. chem.262: 4429-4432 (1987)).

For example, gene transfer to obtain antibody expression in a target subject may also be performed by transfection of homologous cells in vitro. Suitable cells for such in vitro transfection include blood cells, as these cells are readily manipulated and reintroduced into the target subject using methods well known in the art.

Gene transfer by in vitro transfection of cells can be performed by a variety of methods, for example, calcium phosphate precipitation, diethylaminoethyl dextran, electroporation, lipofection, or viral infection. Such methods are well known in the art (see, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Springs harbor Laboratory Press (1989)). Once these cells are transfected, they are then transplanted or migrated back into the target subject being treated. Once these cells introduced into the body are able to produce antibodies, these antibodies can enter the circulation and inhibit platelet aggregation at the site of disease or pathology.

Throughout this application the word "comprise", or "comprises", or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, materials, schemes, articles or the like which has been included in this application is for the purpose of serving solely as a background to the application and is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention in australia as at the priority date of each claim of this application.

The following examples will now illustrate the invention, which is not intended to be limiting in any way. The contents of all references cited herein are incorporated by reference.

Examples

Materials and methods

1. Monoclonal antibody generation and flow cytometry

By 10⁷L1.2C 5aR transfected cells [8]Five to six intraperitoneal C57BL/6 mice were inoculated every 2 weeks to generate monoclonal antibodies (MAbs) reactive with C5 aR. The last inoculum was injected intravenously. Four days later, spleens were removed and cells were fused as described with the SP2/0 cell line [9 ]]. L1.2 cells transfected with C5aR, untransfected L1.2 cells, or L1.2 cells transfected with an unrelated receptor such as CXCR2 or CX3CR1(V28), staining with fluorescence and transfection with FACScan * (Becton Dickinson)&Co., Mountain View, CA) to identify mabs that reacted with C5 aR. As previously described [10 ]]MAb staining of cells was performed using standard procedures.

2. Ligand binding assays

Recombinant human C5a was from Sigma chemical company (st.¹²⁵I-Bolton-Hunter-labeled complement C5a was purchased from NEN-Dupont (Boston, MA) and has a specific activity of 2200 Ci/mM. As previously described [9, 11]Binding of C5a to L1.2C 5aR transfectants was performed. Briefly, cells were washed once in PBS and 10 times⁷The suspension was resuspended in binding buffer (50mM Hepes, pH7.5, 1mM CaCl, 5mM MgCl)₂0.5% BSA and 0.05% azide). 50ml (5X 10)⁵Cells) in water was dispersed into a microcentrifuge tube, followed by addition of cold competitor and 1nM radiolabeled C5 a. The final reaction volume was 200. mu.l. After incubation at room temperature for 60 minutes, the cells were washed three times with 1ml binding buffer containing 0.5M NaCl. The cells were then counted. Background binding was obtained by incubation of cells with radiolabeled C5a and was at least 400-fold higher than unbound C5 a. Duplicate wells were used throughout the experiment and the standard deviation was always < 10% of the mean.

3. Chemochemotaxis assay for transfectants

C5aR transfected L1.2 cells were scraped off and washed in migration medium and resuspended at 107 cells/ml. Tissue culture inserts (Becton Dickinson) were placed into each well of a 24-well tissue culture plate&Co., Mountain View, CA), which forms an upper and lower chamber separated by a polyethylene terephthalate membrane (polyethylene terephthalate membrane) with a 3mm diameter hole. Chemotactic C5a (diluted in assay medium) was added to 600. mu.l of assay medium in 24-well tissue culture plates to give a final concentration of 1 nM. 100 ten thousand cells in 100T1 and the supernatant from the antibody-containing hybridoma were cultured in advance for 30 minutes. Add cell-supernatant mixture or purified mAb to the upper chamber of the well and allow cells to stand at 5% CO₂The cells were transferred to the lower chamber at 37 ℃ for 18 hours. After migration the inserts were removed and the cells were counted using FACScan *. Relative cell counts were obtained by taking 30 second events. This method was found to be extremely reproducible and allows gating of leukocytes and removal of debris.

4. Neutrophil chemotaxis assay

Cell preparation: the leukocyte fraction was first obtained by a dextran precipitation step at room temperature for 40 minutes, and then the cell fraction was added to Ficoll-Paque (Amersham biosciences) and subjected to density gradient centrifugation at 2500rpm at room temperature, thereby separating neutrophils from peripheral blood. After hypotonic lysis of the remaining erythrocytes, the neutrophils were resuspended in the same volumes of RPMI 1640(Invitrogen Inc.), M199(Invitrogen Inc.) and 2% fcs (hyclone).

And (3) chemical chemotaxis detection: anti-C5 aR MAb6C12, 7F3 and 12D4 at concentrations ranging from 0.5 to 10 μ g/ml were added to neutrophils (1X 10)⁷Per ml). The cells were then placed in the upper chamber of a 24-well insert (corning inc., NY) with a 3.0 μm pore size polycarbonate membrane and incubated for 10 minutes at room temperature. The insert is then placed into the lower chamber containing a human neutrophil chemoattractant, e.g., C5a (0.1 to 100nM) and IL-8(1.12ng/ml to 11.2 ng/ml). The neutrophils were then incubated at 37 ℃ for 30 min. Flow cytometry quantified the number of neutrophils migrating through the membrane into the lower chamber.

5. Competitive inhibition assay

anti-C5 aR MAb at 50. mu.g/ml was added to the N-terminal, synthetically produced peptide of C5aR (Biosource; Eldridge) called "PEPI" at concentrations ranging from 1 to 100. mu.M. Then C5a receptor transfected and resuspended in 1% bovine serum albumin (BSA; GibcoBRL) (1X 10)⁷/ml) of mouse L1.2 cells to make a total volume of 100. mu.l. Cells were incubated at 4 ℃ for 30 minutes and washed once with 0.1% BSA. Fluorescein-bound sheep anti-mouse IgG, F (ab') 2(Jackson Immunoresearch Laboratories Inc.) was used as the secondary antibody (1: 200) and incubated for 15 minutes at 4 ℃ followed by an additional wash step with 0.1% BSA. Cells were resuspended in 0.1% BSA and analyzed by flow cytometry.

ELISA detection

The ELISA was performed as described in the immunological general protocol (Unit 2.1) (Edited by J.F.Coligan, A.M.Kruisbeam, D.B.Margulies, E.M.Shevach and W.Strober), John Wiley and Sons, New York. Briefly, 96-well flat-bottomed ELISA plates (Maxisorp; Nunc) were coated with 1Tg/ml protein PBS solution at 37 ℃ for 1 hour, and then blocked with BSA at 4 ℃ overnight. The plates were then washed, incubated with antibody, washed and incubated with peroxidase-conjugated sheep anti-mouse IgG antibody. The substrate used was TMB substrate reagent (PharMingen).

Example 1: MAb production and flow cytometry

Transfectants [8] expressing high levels of C5aR were used to inoculate mice, and ten mabs specifically reactive with C5aR transfected L1.2 cells, but not CX3CR1(V28) or CXCR2 transfected L1.2 cells, were identified by flow cytometry. These ten mabs are called 12D4, 10G1, 5H11, 6C12, 10D4, 5F3, 7F3, 8D6, 11B9, and 1D 12.

FIG. 1 is a set of histograms showing that MAb7F3 reacted with C5aR transfectants (L1.2C 5aR) and human neutrophils, but not with CX3CR1 transfected cells (L1.2V 28) or CXCR2 transfected cells (L1.2 CXCR 2). These MAb7F3 results are representative of the ten antibodies identified.

Example 2: inhibition of binding of C5a to C5aR transfected cells

MAb inhibition was tested¹²⁵Ability of I-labeled C5a to bind to C5aR transfectants. FIG. 2 shows that MAb7F3 completely inhibits¹²⁵I-labeled C5a bound to the transfectants and this inhibition was stronger than that obtained with 400nM C5 a. This indicates that MAb7F3 completely blocked the binding of C5a to C5 aR. MAb6C12 and 12D4 also show pairs¹²⁵Fundamental inhibition of binding of I-labeled C5a to C5aR transfectants. Fig. 3 shows the dose-dependent inhibition of binding of C5a to C5aR by MAb7F 3.

Example 3: inhibition of human C5 a-mediated C5aR transfectant migration by MAb7F3

L1.2 cells transfected with C5aR chemochemotaxis assays were performed as described above. FIG. 4 shows that 7F3, 6C12, and 12D4 completely or substantially inhibited chemotaxis of C5a for C5aR-L1.2 cells. FIG. 5 shows dose-dependent inhibition of the chemochemotaxis of C5a on C5aR-L1.2 cells by mAb7F 3.

Example 4: inhibition of human C5 a-mediated neutrophil migration by MAb7F3

anti-C5 aR MAb was dialyzed in 1xPBS (GibcoBRL) and 5. mu.g/ml of both dialyzed and non-dialyzed 7F3 MAb were added to neutrophils (1X 107/ml). Negative controls (no Ab addition, and 1XPBS addition) were included. The cells were then placed into the upper chamber of a 24-well insert (Corning inc., NY) with a 3.0 μm pore size polycarbonate membrane and incubated for 10 minutes at room temperature. The insert was then placed into the lower chamber containing the human neutrophil chemoattractant, C5a (0.1 to 100 nM). Neutrophils were then incubated at 37 ℃ for 30 minutes. Flow cytometry (FACSCalibur; BD Biosciences) quantified the number of neutrophils migrating through the membrane into the lower chamber.

Figure 6 shows that the addition of MAb7F3 (either dialyzed or not) caused inhibition of neutrophil migration compared to the two negative controls.

Example 5: inhibition of human C5 a-mediated neutrophil migration by MAb7F3, 6C12, and 12D4

Three anti-C5 aR MAb7F3, 6C12 and 12D4 at 5. mu.g/ml were added to neutrophils (1X 107/ml). Negative controls (no Ab addition, and 1XPBS addition) were included. The cells were then placed into the upper chamber of a 24-well insert (cominginc., NY) with a 3.0 μm pore size polycarbonate membrane and incubated for 10 minutes at room temperature. The insert was then placed into the lower chamber containing the human neutrophil chemoattractant, C5a (1.12 to 1120 ng/ml). Neutrophils were then incubated at 37 ℃ for 30 minutes. Flow cytometry (FACSCalibur; BD Biosciences) quantified the number of neutrophils migrating through the membrane into the lower chamber.

The results in fig. 7 show that all three mabs showed inhibition of neutrophil migration towards C5a, compared to the two negative controls. In particular, the 7F3 MAb showed the most effective inhibition, resulting in a 140-fold reduction in the number of neutrophil migrations over background levels.

Example 6: inhibition of human IL-8 mediated neutrophil migration by MAb7F3, 6C12 and 12D4

Three anti-C5 aR MAb7F3, 6C12 and 12D4 at 5. mu.g/ml; and 7F3 were added to neutrophils (1X 107/ml) and placed in the upper chamber of a 24-well insert. Negative controls (no Ab addition, and 1XPBS addition) were also included. After incubation at room temperature for 10 minutes. The insert was then placed into the lower chamber containing IL-8(1.12 to 1120ng/ml), a human neutrophil chemoattractant that binds to CXCR1 and CXCR2 receptors expressed on the surface of neutrophils. Neutrophils were then incubated at 37 ℃ for 30 minutes. Flow cytometry (FACSCalibur; BD Biosciences) quantified the number of neutrophils migrating through the membrane into the lower chamber.

FIG. 8(a) shows that all three MAbs exhibit inhibition of neutrophil migration towards IL-8. Both the 7F3 MAb (dialyzed and dialyzed) were the most potent inhibitor, resulting in a 5-fold decrease in the number of neutrophil migration.

The ability of MAB7F3 to inhibit other neutrophil chemoattractants, particularly CXCR1 and CXCR2 ligands, was also determined. Table 1 shows the true inhibition of neutrophil migration to a number of neutrophil chemoattractants, particularly CXCR1 and CXCR2 ligands, in the neutrophil chemochemotaxis assay.

TABLE 1

Chemoattractant (112 ng/ml)% inhibition

C5a 98

IL-8 81

GCP-2 91

ENA-78 83

Example 7: competitive inhibition of MAb7F3, 6C12, and 12D4 binding to C5aR transfectants by C5aRN terminal peptides (9-29).

The binding of MAb7F3, 6C12, and 12D4 to C5aR transfected cells was determined by staining with Fluorescein (FITC) -bound sheep anti-mouse IgG. The ability of the C5aR N-terminal peptide (9-29) to inhibit this binding was then determined according to the method described above. The C5aR N-terminal peptide (9-29) has the sequence PDYGHYDDKDTLDLNTPVDKT and is referred to herein as "PEPI".

Fig. 9(a) shows that increasing concentrations of PEPI did not inhibit fluorescent staining of the three anti-C5 aR mabs. Even at 100. mu.M concentration of PEPI, the fluorescent staining was stable.

FIG. 9(b) shows that PEPI (50. TM. concentration) does not inhibit FACS staining of purified neutrophils by MAb7F 3.

Example 8: reactivity of MAb7F3, 6C12, and 12D4 with C5aRN terminal peptides (9-29) ("PEPI") and OPG

The reactivity of MAb7F3, 6C12, 12D4 and PEPI and OPG was determined by ELISA assay as described above. OPG is a member of the TNF receptor superfamily, which specifically binds to its ligand TNFSF 11/OPGL. More specifically, OPG is a decoy receptor secreted by osteoblasts and acts as a negative regulator of bone resorption

MAb7F3, 6C12, and 12D4 at a concentration of 1. mu.g/ml were used in ELISA as purified proteins. MAb 9C1 (specific for OPG) and MAb 11B9 (recognizing PEPI) were used as positive controls. These control mabs were used as undiluted tissue culture supernatants.

FIG. 10 shows that MAb7F3, 6C12 and 12D4 did not react with PEPI, and MAb7F3 and OPG had weak cross-reactivity.

Example 9: sequencing of anti-C5 aR MAb7F3, 6C12 and 12D4

The nucleotide sequences of the anti-C5 aR antibodies 7F3, 6C12 and 12D4 were determined from RNA extracted from antibody-expressing hybridoma cells. To determine the primers for amplifying the variable regions of the heavy and light chains, the protein sequences of the variable regions of the three antibodies were determined by Biogen inc, and isotypes of the antibodies were determined using a mouse monoclonal antibody isotyping kit-isosttip (Roche cat. No. 1493027). Thus, the 5 'framework 1 primer is from Biogen inc. protein sequence, while the 3' primer is based on the isotype of the antibody.

The isotype of each anti-C5 aR antibody was as follows:

6C 12: light chain Kappa

6C 12: heavy chain IgG3

7F 3: light chain Kappa

7F 3: heavy chain IgG2a

12D 4: light chain Kappa

12D 4: heavy chain IgG3

Total RNA was isolated from hybridoma cells using Trizol reagent (Invitrogen, Cat. No. 15596-018). RNA was isolated as described by the manufacturer. Briefly, approximately 5 × 10⁶Cells were lysed in 1ml Trizol reagent. Cell debris was removed with 200T1 chloroform and centrifuged. The aqueous layer containing the RNA was removed and the RNA was precipitated with 250T1 isopropanol.

cDNA was prepared from total RNA (2Tg) using AMV reverse transcriptase (Promega Cat. No. M5101). The sequence 6C12 light chain variable region primer encoding the variable region was then amplified using cDNA as a template using the following primers: mIgkapFR 15': GATGTTTTGATGACCCAAACTCC (SEQ ID NO: 2) mIgkapcon 3': ACACTCATTCCTGTTGAAGCTCTTG (SEQ ID NO: 3)6C12 heavy chain variable region primer: mIgVh 25 'SAGGTCCAGCTGCARCAGTC (SEQ ID NO: 4) FR1 VhIIA family mIgG3con 3' TGGGCATGAAGAACCTGG (SEQ ID NO: 5) hinge region 7F3 light chain variable region primer: mIgkapFR 15': GATGTTTTGATGACCCAAACTCC (SEQ ID NO: 6) mIgkapcon 3': ACACTCATTCCTGTTGAAGCTCTTG (SEQ ID NO: 7)7F3 heavy chain variable region primer: mIgVh 25': SAGGTCCAGCTGCARCAGTC (SEQ ID NO: 8) FR1 VhIIA family mIgG2acon 3': TTTGCATGGAGGACAGGG (SEQ ID NO: 9)12D4 light chain variable region primer: mIgkapFR 15': GATGTTTTGATGACCCAAACTCC (SEQ ID NO: 10) mIgkapcon 3': ACACTCATTCCTGTTGAAGCTCTTG (SEQ ID NO11)12D4 heavy chain variable region primer: mIgVh 15': CAGGTGCAGCTGAAGSAGTC (SEQ ID NO: 12) FR1 VhIB family mIgG3con 3': TGGGCATGAAGAACCTGG (SEQ ID NO: 13) hinge region

Polymerase Chain Reaction (PCR) was performed with high fidelity Pfu polymerase (Promega Cat. No. M7741), annealing at 60 ℃ and primer extension at 72 ℃ for 3 min. The resulting PCR fragment of approximately 700bp was cloned into pGEM-Teasy (Promega Cat. No. A1360). Individual clones were isolated and sequenced using commercially available sequencing tools.

The sequence of results provided herein is as follows:

6C12 light chain variable region (DNA) sequence: SEQ ID NO: 14

6C12 light chain variable region (protein) sequence: SEQ ID NO: 15

6C12 heavy chain variable region (DNA) sequence: SEQ ID NO: 16

6C12 heavy chain variable region (protein) sequence: SEQ ID NO: 17

7F3 light chain variable region (DNA) sequence: SEQ ID NO: 18

7F3 light chain variable region (protein) sequence: SEQ ID NO: 19

7F3 heavy chain variable region (DNA) sequence: SEQ ID NO: 20

7F3 heavy chain variable region (protein) sequence: SEQ ID NO: 21

12D4 light chain variable region (DNA) sequence: SEQ ID NO: 22

12D4 light chain variable region (protein) sequence: SEQ ID NO: 23

12D4 heavy chain variable region (DNA) sequence: SEQ ID NO: 24

12D4 heavy chain variable region (protein) sequence: SEQ ID NO: 25

Example 10: analysis of the identity and similarity of the DNA and protein sequences of MAb7F3, 6C12, and 12D 4.

The DNA and protein sequences of three anti-C5 aR antibodies (7F3, 6C12, and 12D4) were compared using MacVector 6.5.3. The ClustalW (1.4) multiple alignment program was used for this analysis.

(i) Analysis of DNA sequence of light chain variable region

FIG. 11 shows an alignment of the light chain variable region DNA sequences of 7F3, 6C12, and 12D 4.

ClustalW (1.4) multiple sequence alignment analysis gave the following results:

3 Sequences Aligned. Alignment Score＝6612

Gaps Inserted＝0 Conserved Identities＝315

Pairwise Alignment Mode：Slow

Pairwise Alignment Parameters：

Open Gap Penalty＝10.0 Extend Gap Penalty＝5.0

Multiple Alignment Parameters：

Open Gap Penalty＝10.0 Extend Gap Penalty＝5.0

Delay Divergent＝40％ Transitions：Weighted

Processing time：0.4 seconds

1.7F3 Vk vs.6c12 Vk

Aligned Length＝336 Gaps＝0

Identities＝320(95％)

2.7F3 Vk vs.12d4 Vk

Aligned Length＝336 Gaps＝0

Identities＝320(95％)

3.6c12 Vk vs.12d4 Vk

Aligned Length＝336 Gaps＝0

Identities＝326(97％)

(ii) analysis of DNA sequence of heavy chain variable region

Fig. 12 shows an alignment of the heavy chain variable region DNA sequences of 7F3, 6C12, and 12D 4.

ClustalW (1.4) multiple sequence alignment analysis gave the following results:

3 Sequences Aligned. Alignment Score＝5346

Gaps Inserted＝3 Conserved Identities＝200

Pairwise Alignment Mode：Slow

Pairwise Alignment Parameters：

Open Gap Penalty＝10.0 Extend Gap Penalty＝5.0

Multiple Alignment Parameters：

Open Gap Penalty＝10.0 Extend Gap Penalty＝5.0

Delay Divergent＝40％ Transitions：Weighted

Processing time：0.5 seconds

1.7F3 Vh vs.6c12 Vh

Aligned Length＝363 Gaps＝0

Identities＝333(91％)

2.7F3 Vh vs.12d4 Vh

Aligned Length＝363 Gaps＝3

Identities＝210(57％)

3.6c12 Vh vs.12d4 Vh

Aligned Length＝363 Gaps＝3

Identities＝210(57％)

(iii) analysis of light chain variable region protein sequence

Fig. 13 shows an alignment of the light chain variable region protein sequences of 7F3, 6C12, and 12D 4.

ClustalW (1.4) multiple sequence alignment analysis gave the following results:

3 Sequences Aligned. Alignment Score＝1902

Gaps Inserted＝0 Conserved Identities＝99

Pairwise Alignment Mode：Slow

Pairwise Alignment Parameters：

Open Gap Penalty＝10.0 Extend Gap Penalty＝0.1

Similarity Matrix：blosum

Multiple Alignment Parameters：

Open Gap Penalty＝10.0 Extend Gap Penalty＝0.1

Delay Divergent＝40％ Gap Distance＝8

Similarity Matrix：blosum

Processing time：0.1 seconds

1.7F3 Vk vs.6c12 Vk

Aligned Length＝112 Gaps＝0

Identities＝102(91％) Similarities＝5(4％)

2.7F3 Vk vs.12d4 Vk

Aligned Length＝112 Gaps＝0

Identities＝103(91％) Similarities＝4(3％)

3.6c12 Vk vs.12d4 Vk

Aligned Length＝112 Gaps＝0

Identities＝104(92％) Similarities＝4(3％)

(iv) analysis of heavy chain variable region protein sequences

Fig. 14 shows an alignment of the heavy chain variable region protein sequences of 7F3, 6C12, and 12D 4.

ClustalW (1.4) multiple sequence alignment analysis gave the following results:

3 Sequences Aligned. Alignment Score＝1432

Gaps Inserted＝2 Conserved Identities＝51

Pairwise Alignment Mode：Slow

Pairwise Alignment Parameters：

Open Gap Penalty＝10.0 Extend Gap Penalty＝0.1

Similarity Matrix：blosum

Multiple Alignment Parameters：

Open Gap Penalty＝10.0 Extend Gap Penalty＝0.1

Delay Divergent＝40％ Gap Distance＝8

Similarity Matrix：blosum

Processing time：0.1 seconds

1.7F3 Vh vs.6c12 Vh

Aligned Length＝121 Gaps＝0

Identities＝107(88％) Similarities＝6(4％)

2.7F3 Vh vs.12d4 Vh

Aligned Length＝121 Gaps＝2

Identities＝52(42％) Similarities＝25(20％)

3.6c12 Vh vs.12d4 Vh

Aligned Length＝121 Gaps＝2

Identities＝54(44％) Similarities＝25(20％)

it will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. Accordingly, all portions of the present embodiments are to be considered in all respects as illustrative and not restrictive.

Reference documents:

1.Gerard，C.and N.P.Gerard，C5A anaphylatoxin and its seventransmembrane-segment receptor.Annual Review of Immunology，1994.12：p.775-808.

2.Murdoch，C.and A.Finn，Chemokine receptors and their role ininflammation and infectious diseases.Blood，2000.95(10)：p.3032-43.

3.Watanabe，H.，et al.，Analysis of C5a receptorby monoclonal antibody.Journal of Immunological Methods，1995.185(1)：p.19-29.

4.Pellas，T.C.，et al.，Novel C5a receptor antagonists regulate neutrophilfunctions in vitro and in vivo.Journal of Immunology，1998.160(11)：p.5616-21.

5.Konteatis，Z.D.，et al.，Development of C5a receptor antagonists.Differential loss of functional responses.Journal of Immunology，1994.153(9)：p.4200-5.

6.Kaneko，Y.，et al.，Antagonistic peptides against human anaphylatoxinC5a.Immunology，1995.86(1)：p.149-54.

7.Morgan，E.L.，et al.，Anti-C5a receptor antibodies.Characterization ofneutralizing antibodies specific for a peptide，C5aR-(9-29)，derived from thepredicted amino-terminal sequence of the human C5a receptor.Journal ofImmunology，1993.151(1)：p.377-88.

8.Campbell，J.J.，et al.，Biology of chemokine and classicalchemoattractant receptors：differential requirements for adhesion-triggeringversus chemotactic responses in lymphoid cells.J Cell Biol，1996.134(1)：p.255-66.

9.Heath，H.，et al.，Chemokine receptor usage by human eosinophils.Theimportance of CCR3 demonstrated using an antagonistic monoclonalantibody.J Clin Invest，1997.99(2)：p.178-84.

10.Ponath，P.D.，et al.，Molecular cloning and characterization of a humaneotaxin receptor expressed selectively on eosinophils[see comments].J ExpMed，1996.183(6)：p.2437-48.

11.Ponath，P.D.，et al.，Cloning of the human eosinophil chemoattractant，eotaxin.Expression，receptor binding，and functional properties suggest amechanism for the selective recruitment of eosinophils.J Clin Invest，1996.97(3)：p.604-12.

sequence listing

<110> G2 therapy Co., Ltd

<120> anti-C5 aR antibody and use thereof

<130>501129

<150>USSN 60/350,961

<151>2002-01-25

<160>34

<170>PatentIn version 3.1

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Met Asn Ser Phe Asn Tyr Thr Thr Pro Asp Tyr Gly His Tyr Asp Asp

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Lys Asp Thr Leu Asp Leu Asn Thr Pro Val Asp Lys Thr Ser Asn Thr

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Leu Arg Val Pro Asp Ile Leu Ala Leu Val Ile Phe Ala Val Val Phe

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Leu Val Gly Val Leu Gly Asn Ala Leu Val Val Trp Val Thr Ala Phe

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Glu Ala Lys Arg Thr Ile Asn Ala Ile Trp Phe Leu Asn Leu Ala Val

65 70 75 80

Ala Asp Phe Leu Ser Cys Leu Ala Leu Pro Ile Leu Phe Thr Ser Ile

85 90 95

Val Gln His His His Trp Pro Phe Gly Gly Ala Ala Cys Ser Ile Leu

100 105 110

Pro Ser Leu Ile Leu Leu Asn Met Tyr Ala Ser Ile Leu Leu Leu Ala

115 120 125

Thr Ile Ser Ala Asp Arg Phe Leu Leu Val Phe Lys Pro Ile Trp Cys

130 135 140

Gln Asn Phe Arg Gly Ala Gly Leu Ala Trp Ile Ala Cys Ala Val Ala

145 150 155 160

Trp Gly Leu Ala Leu Leu Leu Thr Ile Pro Ser Phe Leu Tyr Arg Val

165 170 175

Val Arg Glu Glu Tyr Phe Pro Pro Lys Val Leu Cys Gly Val Asp Tyr

180 185 190

Ser His Asp Lys Arg Arg Glu Arg Ala Val Ala Ile Val Arg Leu Val

195 200 205

Leu Gly Phe Leu Trp Pro Leu Leu Thr Leu Thr Ile Cys Tyr Thr Phe

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gatgttttga tgacccaaac tcc 23

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tgggcatgaa gaacctgg 18

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gatgttttga tgacccaaac tcc 23

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acactcattc ctgttgaagc tcttg 25

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tttgcatgga ggacaggg 18

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gatgttttga tgacccaaac tcc 23

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<220>

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acactcattc ctgttgaagc tcttg 25

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<213>Artificial Sequence

<220>

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caggtgcagc tgaagsagtc 20

<210>13

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<213>Artificial Sequence

<220>

<223>PCR primer

<400>13

tgggcatgaa gaacctgg 18

<210>14

<211>336

<212>DNA

<213>Mus musculus

<400>14

gatgttgtga tgacccaaat tccactctcc ctgcctgtca gtcttggaga tcaaacctcc 60

atctcttgca gatctagtca gagccttata cacagtaatg gaaacaccta tttacattgg 120

tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240

agcagagtgg aggctgagga tatgggagtt tatttctgct ctcaaagtac acatgttcct 300

ccgacgttcg gtggaggcac caagctggaa atcaaa 336

<210>15

<211>112

<212>PRT

<213>Mus musculus

<400>15

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

1 5 10 15

Asp Gln Thr Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Ile His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Met Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210>16

<211>363

<212>DNA

<213>Mus musculus

<400>16

caggttcagc tgcagcagtc tggacctgag gtggtgaagc ctggggcctc agtgaagatt 60

tcctgcaagg cttctggcta cgcattcagt aggtcctgga tgaactgggt gaagcagagg 120

cctggaaagg gtcttgagtg gattggacgg attgatgctg gagatggaga tactaaatac 180

aatgggaagt tcaagggcaa ggccacactg actgcagaca aatcctccag cacagcctac 240

atgcaactca gcagcctgac atctgaggac tctgcggtct acttctgtgc aagccttctc 300

attactacgg tagtgggagc tatggactac tggggtcaag gaacctcagt caccgtctcc 360

tca 363

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<212>PRT

<213>Mus musculus

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Arg Ser

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Ala Gly Asp Gly Asp Thr Lys Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Ser Leu Leu Ile Thr Thr Val Val Gly Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210>18

<211>336

<212>DNA

<213>Mus musculus

<400>18

gatgttgtga tgacccaatc tccactctcc ctgcctgtca gtcttggaaa tcaagcctcc 60

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tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttctc actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acttgttccg 300

ctcacgttcg gtgctgggac caagctggaa ctgaaa 336

<210>19

<211>112

<212>PRT

<213>Mus musculus

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Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asn Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr Leu Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210>20

<211>363

<212>DNA

<213>Mus musculus

<400>20

caggttcagc tgcagcagtc tggacctgag ctggtgaagc ctggggcctc agtgaagatt 60

tcctgcaagg cttctggcta cgcattcagt aactcctgga tgaactgggt gaagcagagg 120

cctggaaagg gtcttgagtg gattggacgg atttatcctg gagatggaga tactaagtac 180

aatgggaagt tcaagggcaa ggccacactg actgcagaca aatcctccag cacagcctac 240

atgcaactca gcagcctgac atctgaggac tctgcggtct atttctgtgc aagattccta 300

cttattagta cggtaacagc cgttgactac tggggccaag gcaccactct cacagtctcc 360

tca 363

<210>21

<211>121

<212>PRT

<213>Mus musculus

<400>21

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Ser

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Lys Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Phe Leu Leu Ile Ser Thr Val Thr Ala Val Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210>22

<211>336

<212>DNA

<213>Mus musculus

<400>22

gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgta gatctagtca gagccttgta cacagtagtg gaaacaccta tttacattgg 120

tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtctc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cacatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggaatt tatttctgct ctcaaagtac acttgttcct 300

ccgacgttcg gtggaggcac caagctggaa atcaaa 336

<210>23

<211>112

<212>PRT

<213>Mus musculus

<400>23

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Ser Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr His Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Phe Cys Ser Gln Ser

85 90 95

Thr Leu Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210>24

<211>357

<212>DNA

<213>Mus musculus

<400>24

caggtgcagc tgaaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccatc 60

acatgcactg tctctgggtt ctcattaacc agctatggtg tagactgggt tcgccagtct 120

ccaggaaagg gtctggagtg gctgggagta atatggggtg ttggaagcac aaattataat 180

tcagctctca aatccagact gagcatcagc aaggacaact ccaagagcca agttttctta 240

aaaatgaaca gtctgcaaac tgatgacgca gccatgtact actgtgccag ccactatggt 300

tacgacggtc tggggtttgc ttactggggc caagggactc tggtcactgt ctctgta 357

<210>25

<211>119

<212>PRT

<213>Mus musculus

<400>25

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

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val Asp Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Gly Val Gly Ser Thr Asn Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Ala Ala Met Tyr Tyr Cys Ala

85 90 95

Ser His Tyr Gly Tyr Asp Gly Leu Gly Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Val

115

<210>26

<211>5

<212>PRT

<213>Mus musculus

<400>26

Asn Ser Trp Asn Asn

1 5

<210>27

<211>17

<212>PRT

<213>Mus musculus

<400>27

Arg Ile Tyr Pro Gly Asp Gly Asp Thr Lys Tyr Asn Gly Lys Phe Lys

1 5 10 15

Gly

<210>28

<211>12

<212>PRT

<213>Mus musculus

<400>28

Phe Leu Leu Ile Ser Thr Val Thr Ala Val Asp Tyr

1 5 10

<210>29

<211>5

<212>PRT

<213>Mus musculus

<400>29

Arg Ser Trp Met Asn

1 5

<210>30

<211>17

<212>PRT

<213>Mus musculus

<400>30

Arg Ile Asp Ala Gly Asp Gly Asp Thr Lys Tyr Asn Gly Lys Phe Lys

1 5 10 15

Gly

<210>31

<211>12

<212>PRT

<213>Mus musculus

<400>31

Leu Leu Ile Thr Thr Val Val Gly Ala Met Asp Tyr

1 5 10

<210>32

<211>5

<212>PRT

<213>Mus musculus

<400>32

Ser Tyr Gly Val Asp

1 5

<210>33

<211>16

<212>PRT

<213>Mus musculus

<400>33

Val Ile Trp Gly Val Gly Ser Thr Asn Tyr Asn Ser Ala Leu Lys Ser

1 5 10 15

<210>34

<211>11

<212>PRT

<213>Mus musculus

<400>34

His Tyr Gly Tyr Asp Gly Leu Gly Phe Ala Tyr

1 5 10

Claims (51)

1. An antibody reactive with an extracellular loop other than the N-terminal domain of C5aR, wherein the antibody reduces or inhibits binding of C5a to C5 aR.

2. The antibody of claim 1, wherein the antibody is reactive with an epitope comprising the second extracellular loop (residues 175 to 206) of C5 aR.

3. An antibody reactive with the same epitope of MAb7F3 and C5aR, wherein the antibody reduces or inhibits the binding of C5a to C5 aR.

4. An antibody reactive with the same epitope of MAb6C12 and C5aR, wherein the antibody reduces or inhibits the binding of C5a to C5 aR.

5. An antibody reactive with the same epitope of MAb12D4 and C5aR, wherein the antibody reduces or inhibits the binding of C5a to C5 aR.

6. An antibody that binds to C5aR, wherein the antibody competitively inhibits the binding of MAb7F3 to C5 aR.

7. An antibody that binds to C5aR, wherein the antibody competitively inhibits the binding of MAb6C12 to C5 aR.

8. An antibody that binds to C5aR, wherein the antibody competitively inhibits the binding of MAb12D4 to C5 aR.

9. The antibody of any one of claims 6 to 8, wherein the relative binding specificity is determined by antibody competitive detection in the presence of C5aR or a polypeptide comprising the extracellular loop of C5 aR.

10. A polypeptide comprising a sequence consisting of SEQ ID NO: 19 and SEQ ID NO: 21, wherein the antibody reduces or inhibits the binding of C5a to C5 aR.

11. An antibody comprising at least one CDR loop sequence that hybridizes to a sequence consisting of SEQ ID NO: 26. SEQ ID NO: 27 or SEQ ID NO: 28, and a variable heavy chain CDR1, CDR2, or CDR3 loop sequence that is substantially the same, wherein the antibody reduces or inhibits the binding of C5a to C5 aR.

12. The antibody of claim 11, wherein the antibody comprises at least two cdrs that are substantially identical to sequences respectively represented by SEQ ID NOs: 26. SEQ ID NO: 27 and SEQ ID NO: 28, a CDR loop sequence substantially identical to the variable heavy chain CDR1, CDR2, or CDR3 loop sequences.

13. The antibody of claim 11 or 12, wherein the antibody further comprises at least one light chain variable region consisting essentially of SEQ ID NO: 19, or a CDR loop sequence defined by amino acid residues 24 to 39, 55 to 61, or 94 to 102 of the variable light chain sequence set forth in seq id No. 19.

14. The antibody of claim 13, wherein the antibody comprises at least two cdrs consisting essentially of SEQ ID NOs: 19, and CDR loop sequences determined at amino acid residues 24 to 39, 55 to 61, and 94 to 102 of the variable light chain sequence set forth in seq id No. 19.

15. A polypeptide comprising a sequence consisting of SEQ ID NO: 15 and SEQ ID NO: 17, wherein the antibody reduces or inhibits binding of C5a to C5 aR.

16. An antibody comprising at least one CDR loop sequence that hybridizes to a sequence consisting of SEQ ID NO: 29. SEQ ID NO: 30 or SEQ ID NO: 31, and wherein the antibody reduces or inhibits binding of C5a to C5aR, wherein the variable heavy chain CDR1, CDR2, or CDR3 loop sequences are substantially identical.

17. The antibody of claim 16, wherein the antibody comprises at least two cdrs that are substantially identical to sequences respectively represented by SEQ ID NOs: 29. SEQ ID NO: 30 and SEQ ID NO: 31, a CDR loop sequence substantially identical to the variable heavy chain CDR1, CDR2, or CDR3 loop sequences.

18. The antibody of claim 16 or 17, wherein the antibody further comprises at least one light chain variable region consisting essentially of SEQ ID NO: 15, or a CDR loop sequence defined by amino acid residues 24 to 39, 55 to 61, or 94 to 102 of a variable light chain sequence set forth in seq id No. 15.

19. The antibody of claim 18, wherein the antibody comprises at least two cdrs consisting essentially of SEQ ID NOs: 15, or a CDR loop sequence defined by amino acid residues 24 to 39, 55 to 61, and 94 to 102 of the variable light chain sequence set forth in seq id No. 15.

20. A polypeptide comprising a sequence consisting of SEQ ID NO: 23 and SEQ ID NO: 25, wherein the antibody reduces or inhibits binding of C5a to C5 aR.

21. An antibody comprising at least one CDR loop sequence that hybridizes to a sequence consisting of SEQ ID NO: 32. SEQ ID NO: 33 or SEQ ID NO: 34, and a variable heavy chain CDR1, CDR2, or CDR3 loop sequence that is substantially the same, wherein the antibody reduces or inhibits the binding of C5a to C5 aR.

22. The antibody of claim 21, wherein the antibody comprises at least two cdrs that are substantially identical to sequences respectively represented by SEQ ID NOs: 32. SEQ ID NO: 33 and SEQ ID NO: 34, and a CDR loop sequence substantially identical to the variable heavy chain CDR1, CDR2, or CDR3 loop sequences.

23. The antibody of claim 21 or 22, wherein the antibody further comprises at least one light chain variable region consisting essentially of SEQ ID NO: 23, or a CDR loop sequence defined by amino acid residues 24 to 39, 55 to 61, or 94 to 102 of the variable light chain sequence set forth in seq id No. 23.

24. The antibody of claim 23, wherein the antibody comprises at least two cdrs consisting essentially of SEQ ID NOs: 23, and CDR loop sequences determined at amino acid residues 24 to 39, 55 to 61, and 94 to 102 of the variable light chain sequence set forth in seq id No. 23.

25. The antibody of any one of claims 1 to 24, wherein the antibody also inhibits neutrophil activation by a chemoattractant ligand other than C5 a.

26. The antibody of any one of claims 1 to 25, wherein the antibody is a monoclonal antibody or a recombinant antibody.

27. The antibody of any one of claims 1 to 25, wherein the antibody is a chimeric antibody or a humanized antibody.

28. The antibody of any one of claims 1 to 27, wherein the antibody is an IgG2 a-type or IgG 3-type antibody.

29. A monoclonal antibody selected from the group consisting of MAb7F3, MAb6C12, and MAb12D 4.

30. A hybridoma as deposited with ECACC under accession number 00110609.

31. A hybridoma as deposited with ECACC under accession number 02090226.

32. A hybridoma as deposited with ECACC under accession number 02090227.

33. A conjugate comprising the antibody of any one of claims 1 to 29 and a therapeutic agent.

34. The conjugate of claim 33, wherein the therapeutic agent is a toxin.

35. The conjugate of claim 33, wherein the toxin is pseudomonas exotoxin or a derivative thereof.

36. A conjugate comprising the antibody of any one of claims 1 to 29 and a detectable label.

37. The conjugate of claim 36, wherein said label is selected from the group consisting of a radioactive label, a fluorescent label, an enzymatic label, and a contrast agent label.

38. An isolated nucleic acid molecule comprising a sequence encoding the antibody of any one of claims 1 to 29.

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

40. A method of inhibiting the interaction of a C5 aR-bearing cell with its ligand, the method comprising exposing the cell to the antibody of any one of claims 1 to 29.

41. A method of inhibiting C5aR activity in a cell, the method comprising exposing the cell to the antibody of any one of claims 1 to 29.

42. A method of treating a disease involving neutrophil migration in a target subject, the method comprising administering to the target subject an antibody according to any one of claims 1 to 29.

43. A method of diagnosing a disease involving neutrophil migration in a target subject, the method comprising contacting a sample obtained from the target subject with the conjugate of claim 36 or 37, and detecting immunospecific binding between the conjugate and the sample.

44. The method of claim 43, wherein said method is performed in vitro using a histological sample or tissue fragment or body fluid obtained from said target subject.

45. The method of claim 43, wherein the method is performed in vivo.

46. A method of diagnosing a disease involving neutrophil migration in a target subject, the method comprising administering an antibody according to any one of claims 1 to 19 with an imaging substance to the target subject under conditions which enable formation of a complex between the antibody and a cell presenting C5aR within the target subject, and imaging the complex.

47. The method of any one of claims 42 to 46, wherein the disease is an immunological disease.

48. A method of delivering a therapeutic agent to a site of inflammation in a target subject, the method comprising administering to the target subject a conjugate according to any one of claims 33 to 35.

49. A method of introducing genetic material into a cell presenting C5aR, the method comprising contacting the cell with the antibody of any one of claims 1 to 29, wherein the antibody has attached or bound to genetic material.

50. The method of claim 49, wherein said C5 aR-presenting cell is selected from the group consisting of granulocytes, leukocytes such as monocytes, macrophages, basophils and eosinophils, mast cells, and lymphocytes including T cells, dendritic cells, and non-myeloid cells such as endothelial cells and smooth muscle cells.

51. A method of treating a disease involving neutrophil migration in a subject of interest, the method comprising introducing a polynucleotide encoding an antibody of any one of claims 1 to 29 into cells of the subject of interest such that the antibody is expressed in vivo.