JP2012050451A - TRANSGENIC NON-HUMAN MAMMAL COMPRISING POLYNUCLEOTIDE ENCODING HUMAN OR HUMANIZED C5aR - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a transgenic non-human mammal having somatic cells and germ-line cells including a stable built-in type polynucleotide encoding a human or humanized C5aR.SOLUTION: The invention relates to use of the transgenic non-human mammals for screening agonists, inverse agonists and antagonists of human C5aR, and for testing efficacy of C5aR agonists, inverse agonists and antagonists in various animal models of disease.

Description

The present invention relates to a transgenic non-human mammal comprising a polynucleotide encoding a human or humanized C5aR. The present invention also provides a transgenic non-human mammal for testing the effectiveness of C5aR agonists, inverse agonists, and antagonists in methods of screening for agonists, inverse agonists, and antagonists of human C5aR, and in various disease animal models. About the use of.

BACKGROUND OF THE INVENTION Complement proteins C3-C5, when proteolyzed, each yield an amino-terminal cationic fragment with a signaling molecule called anaphylatoxin. The most powerful of these, C5a, elicits the most extensive response. Considering factors of the inflammatory response, such as the marginal orientation and infiltration of leukocytes, the release of granule-bound proteolytic enzymes, the generation of reactive oxygen and nitrogen radicals, changes in blood flow and capillary leakage, and the ability to contract smooth muscle, The C5a molecule is a “complete” pro-inflammatory mediator. At subnanomolar to nanomolar levels, C5a molecules induce chemotaxis of all myeloid cell lineages (neutrophils, eosinophils and basophils, macrophages and monocytes) and are also induced by prostaglandins. Causes markedly enhanced vascular permeability and leukocyte circulation. Higher nanomolar concentrations induce degranulation and activation of NADPH oxidase. This breadth of biological activity is in contrast to other inflammatory mediators. C5a has been implicated in the development of rheumatoid arthritis, psoriasis, sepsis, reperfusion injury, and adult respiratory distress syndrome.

  C5a activity is mediated by C5a binding to its receptor (C5aR). C5aR belongs to a family of 7-transmembrane G protein-coupled receptors. C5aR is a high affinity receptor for C5a with a Kd of about 1 nM and is located on many different cell types including leukocytes. The number of receptors per cell is very large, up to 200,000 sites per leukocyte. Biological activation of the receptor occurs when the binding exceeds the saturation range.

  C5aR contains an extended N-terminal extracellular domain. This large N-terminal domain is typical of G protein-coupled receptors that bind to peptides, including the IL-8 and fMet-Leu-Phe (FMLP) receptor families. The C5aR structure follows the 7-transmembrane family of receptors, has an extracellular N-terminus, followed by a 7-transmembrane helix connected by inter-helical domains that alternate as intracellular and extracellular loops. Terminates with an inner C-terminal domain.

  C5aR agonists are useful for therapeutic purposes such as protection against bacterial infections, to promote the immunomodulatory effects of C5a, and to treat cancer, immunodeficiencies, and severe infections.

  C5aR antagonists are also useful therapeutic agents for treating, for example, inflammatory and autoimmune diseases. For example, C5aR antagonists include asthma, bronchial allergy, chronic inflammation, systemic lupus erythematosus, vasculitis, rheumatoid arthritis, osteoarthritis, gout, some autoallergic diseases, graft rejection, inflammatory bowel disease (eg, ulcerative) Colitis) Useful in the treatment of certain shock conditions, myocardial infarction, and post-viral encephalopathy. To this end, C5aR peptide antagonists and anti-C5a receptor antibodies have been described previously. For example, WO 95/00164 describes an antibody against the N-terminal peptide (residues 9-29) of the C5a receptor.

  Currently, other and / or improved C5aR antagonists and antagonists are desired, as are improved methods of screening for C5aR antagonists and antagonists.

  In vitro screening methods for detecting C5aR agonist / antagonists are well known in the art. For example, chemotaxis assays can be used to assess the ability of an antibody or functional fragment thereof to block ligand binding to C5aR and to inhibit functions associated with ligand binding to the receptor. These assays are based on the functional migration of cells in vitro induced by compounds. Chemotaxis is assessed by any suitable means, such as assays using 96-well chemotaxis plates or other methods for assessing chemotaxis recognized in the art. be able to. For example, the use of an in vitro transendothelial chemotaxis assay is described in Springer et al. (Springer et al., WO 94/20142 published September 15, 1994) (Berman et al., Immunol. Invest. 17: 625-677 (1998) (see also non-patent document 1)). Migration to collagen gel via the endothelium has also been described (Kavanaugh et al., J. Immunol., 146: 4149-4156 (1991) (Non-Patent Document 2)).

  An important requirement in the drug discovery process is to demonstrate in a relevant animal model that the new therapeutics identified by in vitro screening methods are safe and effective. In addition, it is often desirable to compare the in vivo efficacy of many selected drugs with respect to desired properties, including pharmacokinetic properties, efficacy on disease prognosis, and lack of side effects. One of the major obstacles in drug development is taking drug candidates from in vitro assays to demonstrate in vivo efficacy. In many cases, this is due to the fact that many drug candidates are species-specific. For example, antagonists developed against human chemical inducer receptors such as C5aR antagonize only human C5aR and cannot antagonize C5aR from mice, rabbits, or even higher primates. The inability of many drug compounds to “act” across species is a major reason for dropout in the preclinical phase.

  Accordingly, improved screening methods that can identify and / or test human C5aR agonists, inverse agonists, and antagonists in an in vivo environment are desirable.

International Publication No.94 / 20142

Berman et al., Immunol. Invest. 17: 625-677 (1998) Kavanaugh et al., J. Immunol., 146: 4149-4156 (1991)

  The inventors have found that many D5aR antagonists react with human C5aR but not with other species of C5aR. For example, monoclonal antibodies MAb 7F3, MAb 6C12, and MAb 12D4 (described in PCT / AU03 / 00084) bind to human C5aR but not mouse or baboon C5aR. This highlights the need for in vivo screening and verification systems that can detect and / or verify agonists / antagonists specific for human C5aR.

  The inventors have also found that the chemotaxis of mouse cells engineered to express human C5aR is suppressed by anti-human C5aR antibodies. This finding and the finding that mouse C5a binds to human C5aR with high affinity indicates that human C5aR is compatible with C5aR signaling mechanisms in other mammalian systems. Accordingly, the inventors have developed transgenic non-human mammals that express human C5aR and are useful for screening or testing for agonists / antagonists of C5aR, particularly agonists / antagonists specific for human C5aR.

  Accordingly, the present invention provides a transgenic non-human mammal comprising a polynucleotide encoding human C5aR or humanized C5aR.

  In one embodiment, the polynucleotide encodes human C5aR. Preferably, the polynucleotide encodes a polypeptide comprising the sequence shown in SEQ ID NO: 3 or an allelic variant thereof.

  In a further embodiment, the polynucleotide comprises the sequence shown in SEQ ID NO: 2 or an allelic variant thereof.

  In further embodiments, the polynucleotide encodes a humanized C5aR. By “humanized C5aR” is meant a non-human C5aR that has been modified to introduce or enhance at least one function that is unique to native human C5aR. Preferably, the non-human C5aR is C5aR endogenous to the transgenic mammal.

  For example, the modification can alter the binding specificity of a non-human C5aR such that the humanized C5aR binds to one or more ligands, agonists, inverse agonists, or antagonists of human C5aR. Alternatively, the modification can enhance the binding affinity of non-human 5CaR to one or more ligands, agonists, inverse agonists, or antagonists of human C5aR. In one preferred embodiment, the modification enhances the binding affinity of non-human 5CaR to one or more ligands, agonists, inverse agonists, or antagonists of human C5aR by at least 5-fold, more preferably at least 10-fold.

  The modification preferably includes substitution of at least one amino acid of the non-human 5CaR with the corresponding amino acid of human C5aR.

  Preferably, the modification comprises replacing at least one domain or a substantial part thereof with the corresponding domain of human C5aR or a substantial part thereof. For example, the modification may comprise replacing at least one extracellular domain of endogenous C5aR with the corresponding human C5aR extracellular domain. Accordingly, the humanized C5aR can comprise at least one extracellular domain of human C5aR. In one example, a humanized C5aR comprises an intracellular domain of endogenous C5aR and an extracellular domain of human C5aR.

  In a preferred embodiment, the transgenic mammal has somatic and germline cells comprising a polynucleotide encoding a human or humanized C5aR in a stable integrated form. In short, it is preferred that the transgenic mammal is a “knock-in” of human or humanized C5aR. It is understood that this can be achieved, for example, by introducing a polynucleotide construct encoding human C5aR or a polynucleotide construct encoding humanized C5aR into the mammalian genome by targeted integration into the mammalian genome. It is thought.

  Alternatively, a polynucleotide construct encoding a fragment of human C5aR can be incorporated within an endogenous C5aR sequence such that, after integration, the endogenous C5aR site comprises a polynucleotide encoding a humanized C5aR.

  In a further preferred embodiment, the transgenic mammal is homozygous for human or humanized C5aR.

  In further preferred embodiments, endogenous C5aR expression in the transgenic animal is undetectable or negligible. Reduction of endogenous C5aR expression can be achieved by any suitable means. For example, transgenic mammalian cells can be modified to express an antisense nucleic acid complementary to a nucleic acid encoding endogenous C5aR. Alternatively, the endogenous C5aR gene can be disrupted by homologous recombination. Preferably, the transgenic mammal is a homozygous “knockout” of endogenous C5aR.

  In a preferred embodiment of the invention, the “knockout” of endogenous C5aR coincides with the introduction of human or humanized C5aR. This is preferably accomplished by replacing the endogenous C5aR coding sequence or fragment thereof with the corresponding human C5aR coding sequence or fragment thereof by targeted homologous recombination. In one particular embodiment of the invention, one or more domains of endogenous C5aR are replaced with corresponding human domains.

  The transgenic animals of the invention may contain other genetic changes in addition to the presence of the human C5aR coding sequence. For example, the genome of a transgenic animal can be modified to include a marker gene or other genetic alteration compatible with the methods of the invention to affect the function of the endogenous gene.

  In a further preferred embodiment of the invention, the transgenic non-human mammal is from the group consisting of cattle, pigs, goats, sheep, camels, horses, cats, dogs, monkeys, baboons, rabbits, guinea pigs, rats, hamsters, and mice. Selected. Rodents such as rats, mice, and hamsters are preferred mammals. Preferably, the transgenic mammal is a mouse.

  The invention also provides an isolated tissue, isolated organ, isolated cell, primary cell culture, or established cell line obtained from the transgenic non-human mammal of the invention. Preferred organs or tissues include smooth muscle, endothelial tissue, contractile muscle, and heart.

  The present invention also provides a method for producing a transgenic non-human mammal, wherein a human C5aR, humanized C5aR, or human C5aR fragment is added to the genome of the non-human mammal to produce the transgenic non-human mammal. A method comprising the step of introducing a polynucleotide construct encoding

  In one embodiment, the polynucleotide construct encodes human C5aR. Preferably, the polynucleotide encodes a polypeptide comprising the sequence shown in SEQ ID NO: 3 or an allelic variant thereof.

  In a further embodiment, the polynucleotide construct comprises the sequence shown in SEQ ID NO: 2 or an allelic variant thereof.

  In a further embodiment, the polynucleotide construct encodes a humanized C5aR.

  In a further embodiment of the invention, the polynucleotide construct encodes a fragment of human C5aR. Preferably, the fragment includes at least one domain of human C5aR or a part thereof.

  In a preferred embodiment of the invention, the fragment includes at least one of the domains listed in Table 1. In one particular embodiment, the fragment includes at least one extracellular domain of human C5aR. In another embodiment, the fragment includes two or more of the domains listed in Table 1.

  In a further preferred embodiment, the method further comprises destroying endogenous C5aR in the non-human mammal. Preferably, the transgenic mammal is a homozygous “knockout” of endogenous C5aR.

  In a preferred embodiment, the method comprises the step of replacing the endogenous C5aR coding sequence or fragment thereof with the corresponding human C5aR coding sequence or fragment thereof by targeted homologous recombination. In one particular embodiment of the invention, the method comprises replacing one or more domains of endogenous C5aR with the corresponding human domain.

  In a further preferred embodiment, the polynucleotide construct comprises a selectable marker. Any suitable selectable marker can be used, but the preferred selectable marker is the PGK-neo gene. Preferably, the selectable marker is flanked by a loxP site so that the selectable marker can be removed after targeting by transient expression of Cre recombinase.

  In a further preferred embodiment, the polynucleotide construct comprises a region homologous to the 3 ′ and 5 ′ sequences adjacent to the endogenous C5aR coding sequence of the non-human mammal. Preferably, the polynucleotide construct comprises a homologous region at least 2 kb, more preferably about 3 kb upstream and downstream of exon 3 of the endogenous C5aR gene.

  It will be appreciated that the transgenic mammals of the invention are useful in identifying, evaluating, or validating novel agonists, inverse agonists, and antagonists of C5aR. For example, the transgenic mammals of the invention can be used to screen a number of candidate compounds to identify agonists, inverse agonists, or antagonists of human C5aR function. The transgenic animals of the invention can also be used to assess the therapeutic suitability of an agonist, inverse agonist, or antagonist of human C5aR function previously identified by the screening method.

  Accordingly, the present invention is also a method for assessing at least one pharmacokinetic and / or pharmacodynamic effect of a candidate compound, comprising the transgenic mammal of the present invention or an isolated tissue or There is provided a method comprising administering a candidate compound to a cell and testing at least one pharmacokinetic and / or pharmacodynamic effect of the candidate compound in a transgenic mammal.

  In preferred embodiments, the at least one pharmacokinetic effect that is tested is an absorption parameter, a distribution parameter, a metabolic parameter, or an excretion parameter.

  For example, at least one pharmacokinetic effect to be tested is: distribution volume, total clearance, protein binding, tissue binding, metabolic clearance, renal clearance, liver clearance, bile clearance, intestinal absorption, bioavailability, relative Bioavailability, intrinsic clearance, mean residence time, maximum metabolic rate, Michaelis-Menten constant, partition coefficient between tissue and blood or tissue and plasma, unchanged urinary excretion rate, rate of systemic conversion of drug to metabolite , Elimination rate constant, half-life, or secretory clearance.

  In order to clarify the whole aspect of the present invention and to help understanding thereof, “pharmacokinetic effect” refers to parameters such as absorption, distribution, metabolism, and excretion of candidate compounds and / or their metabolites in vivo. (ADME) refers to dynamics research on dynamic processes. The following pharmacokinetic terms used in the present invention are to be interpreted broadly and have their generally accepted meanings as described below.

  “Absorption” refers to the process of uptake of a candidate compound from the site of administration into the systemic circulation. Transfer of the candidate compound through the intestinal lumen is generally referred to as oral absorption, and transfer of the candidate compound through an external physiological barrier is referred to as general absorption. Absorption pharmacokinetic parameters can be estimated, for example, from biosensor data with a sensor chip having a plurality of suitable liposomes immobilized thereon.

  “Distribution” refers to the transfer of a candidate compound from the site of administration to the entire systemic circulation, then to extracellular and intracellular fluids and tissues. Distribution is usually a rapid and reversible process. The pharmacokinetic parameters of the distribution can be estimated, for example, from biosensor data with a sensor chip having a plurality of suitable plasma proteins, liposomes, and / or transport proteins immobilized thereon.

  “Metabolism” refers to the sum of all chemical reactions that occur in vivo with respect to the biotransformation of endogenous and exogenous substances. Metabolic pharmacokinetic parameters can be estimated, for example, from biosensor data with a sensor chip having a plurality of suitable metabolic enzymes immobilized thereon.

  “Excretion” refers to the final elimination or disappearance of the drug from the body. Exclusion includes both passive diffusion and relatively specific carrier-mediated exclusion. Candidate compounds can be excreted unchanged or as metabolites in the urine via the kidney or in the stool via the bile and / or intestine. Volatile compounds are often excreted into the breath by the lungs. Excretion pharmacokinetic parameters include, for example, antibodies that specifically detect candidate compounds, and sensor chips with immobilized other proteins / receptors with high affinity / specificity for drug candidates It can be estimated from biosensor data. Such antibodies and proteins / receptors can be used to quantify the concentration / amount of drug in various body fluids (eg, urine / feces) and tissues by direct binding assays.

  In another embodiment, the method comprises testing at least one pharmacodynamic effect of the candidate compound in the transgenic mammal. Preferably, at least one pharmacodynamic effect is modulation of C5aR activity. This can be assessed by monitoring at least one phenotype associated with C5aR signaling after administration of the candidate compound.

  Accordingly, in another embodiment, the present invention provides a method for identifying a compound that modulates C5aR activity, comprising: (i) under the condition that at least one phenotype associated with C5aR signaling is expressed. There is provided a method comprising administering a candidate compound to a transgenic mammal or isolated tissue or cells derived therefrom; and (ii) monitoring the development of at least one phenotype after administration of the compound.

  In a preferred embodiment, the method further comprises (iii) comparing the degree of phenotype in the transgenic mammal administered the compound or cells derived therefrom to the control mammal or cells derived therefrom, Any difference in phenotypic nature or extent when compared to indicates the potential of the compound to modulate C5aR activity.

  In one particular embodiment, the invention is a method of identifying a compound that inhibits or reduces C5aR activity, wherein (i) at least one phenotype associated with C5aR signaling is expressed. Administering a candidate compound to the transgenic mammal of the present invention or an isolated tissue or cell derived therefrom; and (ii) monitoring the development of at least one phenotype after administration of the compound. Methods are provided in which any reduction or inhibition of later phenotypic nature or extent indicates that the compound may inhibit or reduce C5aR activity. Preferably, the method further comprises the step of (iii) comparing the degree of phenotype in the transgenic mammal to which the compound has been administered to a control mammal, wherein the phenotypic nature or degree as compared to the control mammal. Any reduction or inhibition indicates that the compound may inhibit or reduce C5aR activity.

  In another embodiment, the invention provides a method of identifying a compound that promotes or enhances C5aR activity, wherein (i) at least one phenotype associated with C5aR signaling is expressed under conditions of the invention. Administering the candidate compound to the transgenic mammal or isolated tissue or cells derived therefrom; and (ii) monitoring the development of at least one phenotype after administration of the compound, the expression after administration Provided are methods where any enhancement of the type or nature of the type indicates that the compound is likely to promote or enhance C5aR activity. Preferably, the method further comprises the step of (iii) comparing the degree of phenotype in the transgenic mammal to which the compound has been administered to a control mammal, wherein the phenotypic nature or degree as compared to the control mammal. Any enhancement indicates that the compound may promote or enhance C5aR activity.

  As used herein, a “control” animal is any other mammal that expresses the same phenotypic index as that expressed in the mammal (ie, “test” mammal) from which the compound was tested. It's okay.

  In one embodiment, the control and test animals express comparable levels of functional human C5aR. For example, control animals and test equivalents are isogenic.

  In another embodiment, the control animal is a human or wild type animal that does not express humanized C5aR.

  The phrase “phenotype associated with C5aR signaling” encompasses any visible feature and / or behavior associated with biochemical processes involving C5aR signaling, including clinical symptoms of the disease Intended. The phenotype can be associated with normal or abnormal C5aR signaling. In one embodiment, the phenotype is a condition that is exacerbated by C5aR signaling, such as immune complex disorders, inflammatory or allergic diseases, graft rejection, or cancer. In another embodiment, the phenotype is a condition that is alleviated or ameliorated by increased C5aR signaling, such as a condition associated with immunosuppression.

  In one particular embodiment, the phenotype is inflammation. Inflammation can be induced, for example, by transferring the serum of an arthritic K / BxN mammal to a transgenic mammal of the invention.

  In another embodiment, the phenotype is an inflammatory tissue disorder such as ischemia-reperfusion injury.

  In another embodiment, the phenotype is leukocyte infiltration.

  In another embodiment, the phenotype is asthma.

  In another embodiment, the phenotype is sepsis, stroke, or respiratory distress syndrome.

  In another embodiment, the phenotype is a condition selected from the group consisting of: inflammatory or allergic diseases and conditions, such as respiratory allergic diseases such as asthma, allergic rhinitis, irritable lung disease, hypersensitivity Pneumonia, interstitial lung disease (ILD) (eg, ILD with idiopathic pulmonary fibrosis or rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, systemic sclerosis, Sjogren's syndrome, polymyositis, or skin Myositis); anaphylaxis or hypersensitivity response, drug allergy (eg to penicillin, cephalosporins), allergy from insect bites; inflammatory bowel diseases such as Crohn's disease and ulcerative colitis; spondyloarthropathy; scleroderma; Psoriasis and inflammatory skin diseases such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria; blood Inflammation (eg, necrotizing vasculitis, cutaneous vasculitis, and hypersensitivity vasculitis); autoimmune diseases such as arthritis (eg, rheumatoid arthritis, psoriatic arthritis), multiple sclerosis, systemic lupus erythematosus, myasthenia gravis , Juvenile diabetes, nephritis such as glomerulonephritis, autoimmune thyroiditis, Behcet's disease; graft rejection (eg at transplant), eg allograft rejection or graft-versus-host disease; atherosclerosis Cancer with leukocyte infiltration of skin or organs; reperfusion injury, stroke, adult respiratory distress syndrome, certain hematological malignancies, cytokine-induced toxicity (eg septic shock, endotoxic shock), polymyositis, Dermatomyositis, pemphigus, Alzheimer's disease, granulomatosis including sarcoidosis, immunodeficiencies such as AIDS, radiation therapy that brings about immunosuppression Therapy, therapy for autoimmune disease or other drug therapy (e.g., corticosteroid therapy); infections such and immunity due to birth defects suppression or severe acute respiratory syndrome (SARS).

  The range of candidate compounds contemplated herein includes C5aR inhibitory compounds, or inverse agonists or antagonists of the biological function of C5aR. In one embodiment, the compound is selected from the group consisting of: a peptide, eg, a C5aR or C5a-derived peptide or other non-C5aR peptide that can inhibit, reduce, or suppress C5aR function, C5aR dominant negative mutants; non-peptide inhibitors of C5aR; antibodies or antibody fragments that bind to C5aR and inhibit C5aR function; small organic molecules and nucleic acids such as peptides derived from C5aR or C5a or other non-C5aR peptides Nucleic acid encoding inhibitors, antisense nucleic acids against C5aR-encoding mRNA, or anti-C5aR ribozymes, or small interfering RNA (RNAi) targeting C5aR gene expression.

The present invention is also a method for identifying a compound that modulates C5aR activity comprising:
(a) administering a candidate compound to a transgenic mammal of the present invention or an isolated tissue or cell derived therefrom under conditions such that at least one phenotype associated with C5aR signaling is expressed; and Monitoring the development of at least one phenotype after administration of the compound
(b) optionally determining the structure of the candidate compound; and
(c) providing a method comprising providing a candidate compound or a name or structure of the candidate compound;

  Of course, for drugs that have not been previously tested for their function by the screening methods provided by the present invention, the determination of the structure of the compound is potentially included in step (b) . This is because a person skilled in the art knows the name and / or structure of the compound at the time of screening.

  As used herein, the term “providing a drug” is considered to include providing any chemical or recombinant synthetic means to produce a drug, or providing a drug previously synthesized by any person or means. Shall be.

  In an embodiment of the invention, the method further comprises formulating the identified compound for administration to a non-human animal or human. The formulation may be suitable for administration by subcutaneous, intravenous, intranasal, or intraperitoneal route. Alternatively, the formulation may be suitable for oral administration in the form of food additives, tablets, troches and the like.

  In another aspect, the present invention provides a compound identified by the present invention.

  Various features and aspects of the present invention belonged to the individual sections above and to other sections, mutatis mutandis as necessary. Therefore, features specified in one term can be combined with features specified in other terms as needed.

Sequence Listing Information SEQ ID NO: 1: Sequence of mouse C5aR (C5r1) gene locus and adjacent DNA.
SEQ ID NO: 2: Sequence of human C5aR cDNA.
SEQ ID NO: 3: amino acid sequence of human C5aR.
SEQ ID NO: 4: Primer F1C (specific for human C5aR exon 3).
SEQ ID NO: 5: Primer R2a (specific for human C5aR exon 3).
SEQ ID NO: 6: Primer F3C (specific for mouse C5aR gene).
SEQ ID NO: 7: Primer R4C (specific for mouse C5aR gene).

FIG. 3 shows the design of a targeting construct for generating human C5aR knock-in mice. FIG. 3 shows the design of a targeting construct for generating human C5aR knock-in mice. The figure which shows the transgenic mouse C5aR locus after deletion of the PGKneo gene by Cre recombinase. The figure which shows the selected restriction site and mouse C5aR (C5r1) gene exon. Location of oligonucleotide primers used in PCR genotyping assay. FACS analysis showing that human C5aR is expressed on neutrophils of mice carrying the hC5aR gene. Wild type (+ / +), heterozygous (H5Rf / +), and homozygous (H5Rf / H5Rf) mouse blood were incubated with human C5aR specific antibody 7F3 conjugated to FITC. The right part of the broken line in the neutrophil histogram shows positive staining of 7F3-FITC. Progression of RA-like inflammatory disease in homozygous human C5aR knock-in and wild-type mice. The left panel shows an increase in ankle thickness after i.p. injection of K / BxN serum on days 0 and 2. The right panel shows the clinical score. Mice # 10 and # 25 are homozygous for the human C5aR gene and mouse # 38 is a wild type litter. Both wild type and homozygous hC5aR gene mice developed ankle thickening and clinical disease in the typical manner described for this model (Lee et al (2002) Science, 297, 1689-1692) . Graph showing the progression and prevention of RA-like inflammatory disease in homozygous human C5aR knock-in mice. The left panel shows an increase in ankle thickness after i.p. injection of K / BxN serum on days 0 and 2. The right panel shows the clinical score. Mice # 7, # 10, # 24, and # 25 are homozygous for the human C5aR gene. Mice # 7 and # 24 were injected with human C5aR neutralizing antibody 7F3, and mice # 10 and # 25 were injected with isotype control antibody. Wild-type littermates (mouse # 38) were also treated with 7F3. The antibody was injected on days -1 and +3. Mice injected with control antibody developed inflammatory disease. In contrast, hC5aR knock-in mice injected with 7F3 were protected from inflammation. Wild type mice injected with 7F3 were not protected from inflammation.

Detailed Description of the Invention
General Techniques and Definitions Unless otherwise noted, the recombinant DNA techniques used in the present invention are standard techniques well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), TA Brown ( Editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DM Glover and BD Hames (editor), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996) , And sources such as FM Ausubel et al (editor), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all the latest editions up to the present invention). , Incorporated herein by reference. In particular, these references describe in detail how to transcribe or replicate nucleic acid molecules and the appropriate conditions necessary for that.

Definitions As used herein, the term “comprise”, or a conjugation such as “comprises” or “comprising”, is defined as an element, integer, or step, or element, integer, Alternatively, it is understood to include a group of stages but not to exclude other elements, integers or stages, or groups of elements, integers or stages.

  As used herein, a “ligand” of a C5aR protein refers to a specific class of substances that bind to a mammalian C5aR protein, including natural ligands, and synthetic and / or recombinant forms of natural ligands. . In a preferred embodiment, ligand binding of the C5aR protein occurs with high affinity.

  As used herein, an “antagonist” inhibits the binding of an agonist or inverse agonist to a C5aR protein and thus at least one functional property of the C5aR protein, eg, binding activity (eg, ligand binding, promoter binding, antibody Binding), signal transduction activity (eg, activation of mammalian G protein, rapid and transient induction of cytosolic free calcium concentration), and / or cell response function (eg, promotion of chemotaxis, exocytosis) Or the release of inflammatory mediators by leukocytes). The term antagonist includes substances that bind to the receptor (eg, antibodies, mutants of natural ligands, small molecular weight organic molecules, other competitive inhibitors of ligand binding), and receptors without binding to the receptor Contains substances that block function.

  As used herein, an “agonist” refers to at least one functional property of a C5aR protein, such as binding activity (eg, binding of a ligand, inhibitor, and / or promoter), signaling activity (eg, a mammal G protein activation, rapid and transient induction of cytosolic free calcium concentration), and / or cell response functions (eg, promoting chemotaxis, exocytosis, or release of inflammatory mediators by leukocytes) A substance that promotes (induces, induces, enhances, or increases). The term agonist includes substances that bind to the receptor (eg, antibodies, homologues of natural ligands from other species) and accept without binding to the receptor (eg, by activating related proteins). Contains substances that promote body function. In a preferred embodiment, the agonist is other than a homologue of the natural ligand.

  As used herein, an “inverse agonist” refers to a baseline intracellular response that binds to C5aR or otherwise interacts with C5aR and is initiated by active C5aR in the absence of an agonist. A molecule that suppresses below the normal baseline of observed activity.

  A “transgenic mammal” is a non-intrinsic entity that exists as an extrachromosomal element within a portion of a cell or is stably integrated within germline DNA (ie, in most or all genomic sequences of the cell). That is, it means a mammal (eg, mouse, rat, hamster, etc.) having a heterologous nucleic acid. Heterologous nucleic acid is introduced into the germline of such transgenic animals, for example, by genetic manipulation of embryos or embryonic stem cells of the host animal.

  The term “biologically active” or “functional” as used herein as a modifier of human C5aR functions as a functional property attributable to native human C5aR, eg, functions as a C5a receptor, or human A polypeptide that exhibits at least one such as the ability to bind one or more natural ligands of C5aR.

  By gene “knockout” is meant a change in gene sequence that preferably results in a decrease in the function of the target gene such that target gene expression is undetectable or negligible. Endogenous gene knockout means that the function of the gene is substantially diminished and expression is undetectable or present at only a minor level. A “knockout” transgenic may be a transgenic animal having a heterozygous knockout of a gene or a homozygous knockout of a gene. For example, when an animal that promotes recombination at the target gene site (for example, Cre in the Cre-lox system) is introduced into the “knock-out”, for example, when an animal is exposed to a substance that promotes a target gene change. Also included are conditional knockouts in which changes in the target gene occur in other ways to induce the target gene change after birth.

  A “knock-in” of a target gene is, for example, by introducing an additional copy of the target gene or by operably inserting a control sequence that provides enhanced expression of an endogenous copy of the target gene. Means changes in the host cell genome that result in altered expression (eg, increased (including ectopic)). A “knock-in” transgenic of interest of the invention is a transgenic animal having a human or humanized C5aR knock-in. Such a transgenic may be a heterozygous knock-in of a human or humanized C5aR gene or a homozygous knock-in of a human or humanized C5aR gene. “Knock-in” also encompasses conditional knock-in.

  “Construct” means a recombinant nucleic acid, generally recombinant DNA, made for the purpose of expressing a particular nucleotide sequence, or for use in the construction of other recombinant nucleotide sequences.

  “Functionally linked” means that the DNA sequence and the control sequence are combined in such a way that the gene can be expressed when an appropriate molecule (eg, a transcriptional activation protein) is bound to the control sequence. means.

  “Operatively inserted” means that the nucleotide sequence of interest directs the transcription and translation of the introduced nucleic acid sequence of interest (ie, production of a polypeptide encoded by, for example, a human C5aR sequence). Means flanking the nucleotide sequence.

  The term “corresponding” or “corresponding” means homologous to, or substantially equivalent to, or functionally equivalent to the designated sequence.

  The term “transgenic gene construct” refers to a nucleic acid molecule (eg, a vector) comprising a polynucleotide of interest (eg, a human C5aR polynucleotide or fragment thereof) operably linked in a manner that allows the polynucleotide to be expressed in a host cell. ). As used herein, the term “polynucleotide” includes deoxyribonucleotide nucleic acid (DNA) and, where applicable, ribonucleic acid (RNA). As used herein, this term also includes analogs of RNA or DNA made with nucleotide analogs, and single-stranded (sense or antisense) and double-stranded polys where applicable to the described embodiments. Includes nucleotides.

Transgenic animals Techniques for producing transgenic animals are well known in the art. A useful general textbook on this subject is Houdebine, Transgenic animals-Generation and Use (Harwood Academic, 1997)-an extensive review of techniques used to produce transgenic animals from fish to mice and cattle. Of particular interest in the present invention are transgenic non-human mammals such as cows, pigs, goats, horses, and the like, and particularly rodents (eg, rats, mice, hamsters, etc.). Preferably, the transgenic animal is a mouse.

  Advances in technology for embryo manipulation have now made it possible to introduce heterologous DNA into, for example, mammalian fertilized eggs. For example, totipotent or pluripotent stem cells are transformed by microinjection, calcium phosphate precipitation, liposome fusion, retroviral infection, or other means, and then the transformed cells are introduced into the embryo, and then the embryo Can be grown into transgenic animals. In a preferred method, a developing embryo is transfected with the desired DNA by electroporation, and a transgenic animal is produced from the infected embryo. However, in a further preferred method, appropriate DNA is co-injected into the pronucleus or cytoplasm of the embryo, preferably at the single cell stage, and the embryo is grown into a mature transgenic animal. These techniques are well known. Review of standard experimental procedures for microinjecting heterologous DNA into mammalian fertilized eggs, such as Hogan et al., Manipulating the Mouse Embryo, (Cold Spring Harbor Press 1986), Krimpenfort et al., Bio / Technology 9 : 844 (1991), Palmiter et al., Cell, 41: 343 (1985), Kraemer et al., Genetic manipulation of the Mammalian Embryo, (Cold Spring Harbor Laboratory Press 1985); Hammer et al., Nature, 315: See 680 (1985), Wagner et al., US Pat. No. 5,175,385; Krimpenfort et al., US Pat. No. 5,175,384. The contents of each of these are incorporated herein by reference.

  Another method used to generate transgenic animals involves microinjecting nucleic acids into pronuclear eggs by standard methods. The injected eggs are then cultured before being transplanted into the oviduct of a pseudopregnant recipient.

  Transgenic animals can also be produced by nuclear transfer techniques as described in Schnieke, AE et al., 1997, Science, 278: 2130, and Cibelli, JB et al., 1998, Science, 280: 1256. . In this way, donor animal fibroblasts are stably transfected with a plasmid incorporating the coding sequence of the binding domain or binding partner of interest under controlled regulation. Stable transfectants are then fused with enucleated oocytes, cultured, and transplanted into female recipients.

  Analysis of animals that may contain transgenic sequences is typically performed by PCR or Southern blot analysis according to standard methods.

  In a particular example of producing a transgenic mammal such as a bovine, a nucleotide construct comprising a sequence encoding a binding domain fused to GFP, for example using the techniques described in US Pat. No. 4,873,191, a mammal Microinjected into oocytes obtained from freshly removed ovaries. Oocytes are aspirated and fixed from the follicles, and then frozen sperm that have been qualified with heparin and pre-fractionated with a Percoll gradient to isolate the motile fraction are thawed and fertilized.

  Fertilized oocytes are centrifuged, for example, at 15,000 g for 8 minutes to visualize the pronuclei for injection, and then from the zygote tissue conditioned medium to the morula or blastocyst stage Incubate until. This medium is prepared using luminal tissue scraped from the oviduct and diluted in the medium. The zygote must be placed in the medium within 2 hours after microinjection.

  Coprostanol is then administered to synchronize the estrus of the intended recipient mammal, such as a cow. Estrus occurs within 2 days, and embryos are transferred to recipients 5-7 days after estrus. Successful transplantation can be assessed in offspring by Southern blot.

  Alternatively, the desired construct can be introduced into embryonic stem cells (ES cells), and the cells can be cultured to confirm the alteration by the transgene. The modified cells are then injected into the blastula stage embryo and the blastula transferred to a pseudopregnant host. The resulting progeny are chimeras of ES cells and host cells, and a non-chimeric strain containing only ES progeny can be obtained by conventional hybridization. This technique is described, for example, in W0 91/10741.

  Transgenic animals contain exogenous nucleic acid sequences that exist as extrachromosomal elements or are stably integrated into all or part of a cell, particularly a germ cell. Preferably the transgenic animal contains a stable change in germline sequence. Stable changes are generally achieved by introducing DNA into the cell's genome. Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACs, and the like. In the initial production of an animal, a “chimera” or “chimeric animal” is created in which only some cells have the modified genome. Chimeras are primarily used for mating purposes to produce the desired transgenic animals. Animals with heterozygous modifications are made by crossing chimeras. To make homozygous animals, male and female heterozygotes are typically mated.

  In a preferred embodiment, the transgenic non-human mammal of the invention is generated by introducing a human C5aR transgene into the germ line of a non-human animal. Embryonic stem cells (ES) are the primary target cell type for introducing human C5aR transgenes into non-human animals for homologous recombination. ES cells can be obtained from preimplantation embryos cultured in vitro and fused with embryos (Evans, MJ, et al. (1981) Nature 292, 154-156; Bradley, MO, et al. (1984) Nature 309, 255-258; Gossler, et al. (1986) Proc. Natl. Acad. Sci USA 83, 9065-9069; and Robertson, et al. (1986) Nature 322, 445-448). Transgenes can be efficiently introduced into ES cells by DNA transfection or by retrovirus-mediated transduction. Such transformed ES cells can then be combined with blastocysts derived from non-human animals. ES cells then settle in the embryo and contribute to the germline of the resulting chimeric animal. For a review, see Jaenisch, R. (1988) Science 240, 1468-1474. Transfected embryonic cells can be incubated for various times in vitro, or reimplanted into a surrogate host, or both.

Transgenic progeny of the surrogate host can be screened for the presence and / or expression of the transgene by any suitable method. Screening is often performed by Southern blot analysis or Northern blot analysis using a probe that is complementary to at least a portion of the transgene. As an alternative or further screening method for screening for the presence of the target gene product, Western blot analysis using antibodies against the protein encoded by the transgene can also be utilized. Typically, DNA is prepared from the tail tissue of a transgenic mouse and analyzed for the transgene by Southern analysis or PCR. Alternatively, tissues or cells that are thought to express the transgene at the highest level are tested for the presence and expression of the transgene by Southern analysis or PCR, although any tissue or cell type may be used for this analysis. The offspring of the transgenic animal can be obtained by mating the transgenic animal with an appropriate partner or by in vitro fertilization of eggs and / or sperm obtained from the transgenic animal.
The

  A method for producing transgenic mice by homologous recombination between an endogenous gene and a transgene construct is described by Hanks, M et al (Science 269: 679-682, 1995), which is incorporated herein by reference. Specifically incorporated into the description.

Human and humanized C5aR constructs The introduced polynucleotide construct may encode the wild type human C5aR sequence shown in SEQ ID NO: 3, or an allelic variant, biologically active derivative, or fragment thereof.

Non-limiting examples of allelic variants of SEQ ID NO: 3 include one or more of the following substitutions:
Amino acid number 2: Asp> Asn
Amino acid number 279: Asn> Lys.

  The polynucleotide construct comprises the sequence shown in SEQ ID NO: 2, or an allelic variant thereof.

Non-limiting examples of allelic variants of SEQ ID NO: 2 include one or more of the following base changes:
Base number 28: g> a
Base number 474: c> t
Base number 861: t> g
Insertion of base numbers 1313 to 1314: a Insertion of base numbers 1447 to 1448: a.

  The term “biologically active derivative” with respect to the amino acid sequence of human C5aR includes the resulting amino acid sequence having human C5aR activity, preferably at least 25-50% activity of the polypeptide shown in SEQ ID NO: 3, More preferably, any substitution, mutation, modification, exchange, deletion, or addition of one (or more) amino acids from or to the sequence shown in SEQ ID NO: 3 as long as it has at least substantially the same activity included. Preferably, human C5aR comprises a region sufficient to effect signal transduction by C5aR.

  Thus, the human C5aR sequence shown in SEQ ID NO: 3 can be modified for use in the present invention. Typically, modifications are made that maintain the original activity of the sequence. Thus, in one embodiment, amino acid substitutions, such as 1, 2, or 3, provided that the modified sequence maintains at least about 25-50% of C5aR signaling or substantially the same signaling as C5aR signaling. ˜10, 20, or 30 substitutions can be made. However, in another embodiment, modifications to the amino acid sequence of SEQ ID NO: 3 can be made with the intention of reducing the biological activity of the polypeptide.

  Generally, preferably less than 20%, 10%, or 5% of the amino acid residues of the biologically active derivative are modified compared to the corresponding region shown in SEQ ID NO: 3.

For example, conservative substitutions can be made according to the following table. Amino acids in the same block in the second column, preferably in the same row between the three columns, can be substituted for each other.

  In a preferred embodiment of the invention, the C5aR coding sequence is operably linked to a promoter, which may be endogenous or heterologous, constitutive or inducible, and other regulatory sequences required for expression in the host animal.

  Alternatively, the transfer construct can encode a humanized C5aR. Preferably, the humanized C5aR sequence is a modified version of a non-human C5aR, more preferably a modified version of the C5aR sequence within the transgenic mammal. In humanized C5aR, the modification introduces or enhances at least one functional property unique to native human C5aR compared to non-human C5aR or endogenous C5aR. For example, if a non-human C5aR does not bind to a specific ligand, agonist, inverse agonist, or antagonist of human C5aR, the modification allows the resulting humanized C5aR to bind to such a ligand, agonist, inverse agonist, or antagonist. In addition, the coupling characteristics can be changed. Or, if a non-human C5aR exhibits a low binding affinity for a particular ligand, agonist, inverse agonist, or antagonist of human C5aR, the modification may cause a binding affinity for such ligand, agonist, inverse agonist, or antagonist. However, enhanced humanized C5aR can be obtained over non-human C5aR. In one preferred embodiment, the modification causes the binding affinity of the humanized C5aR to one or more ligands, agonists, inverse agonists, or antagonists of human C5aR to be at least 5-fold, more preferably compared to non-human sequences. The enhancement is at least 10 times, more preferably at least 20 times, more preferably at least 50 times, more preferably at least 100 times.

  The modification preferably comprises substitution of at least one amino acid of non-human C5aR with the corresponding amino acid of human C5aR. More preferably, the modification comprises substitution of at least 2 amino acids of non-human C5aR, more preferably at least 5 amino acids, more preferably at least 10 amino acids, more preferably at least 20 amino acids with the corresponding amino acids of human C5aR.

  Preferably, the modification comprises replacing at least one domain of non-human C5aR or a substantial part thereof with the corresponding domain of human C5aR or a substantial part thereof. For example, the modification comprises replacing at least one extracellular domain of endogenous C5aR with the corresponding human C5aR extracellular domain. Accordingly, the humanized C5aR can comprise at least one extracellular domain of human C5aR. In one example, a humanized C5aR comprises an intracellular domain of endogenous C5aR and an extracellular domain of human C5aR.

  Various domains of human C5aR that can be introduced into humanized C5aR are listed in Table 1.

(Table 1)
Amino acids 1-37 Extracellular domain-N-terminal amino acids 38-60 Transmembrane domain amino acids 61-71 Intracellular domain amino acids 72-94 Transmembrane domain amino acids 95-110 Extracellular domain-extracellular loop 1
Amino acids 111-132 Transmembrane domain amino acids 133-153 Intracellular domain amino acids 154-174 Transmembrane domain amino acids 175-200 Extracellular domain-extracellular loop 2
Amino acids 201-226 transmembrane domain amino acids 227-242 intracellular domain amino acids 243-265 transmembrane domain amino acids 266-282 extracellular domain-extracellular loop 3
Amino acids 283 to 303 Transmembrane domain Amino acids 304 to 350 Intracellular domain-C-terminal

  Constructs for use in the present invention include any construct suitable for use in producing transgenic animals having a desired level of expression of a human or humanized C5aR coding sequence. These constructs can include cDNA, genomic sequences, or both. Methods for isolating and cloning the desired sequence and constructs suitable for expressing the selected sequence in the host animal are well known in the art. The construct may also contain sequences other than the C5aR coding sequence. For example, a marker gene such as lac Z can be included in the construct, where phenotypic changes are readily detected by up-regulating expression of the encoded sequence.

  The term “C5aR gene” is generally used to mean the human C5aR gene, and isoforms, altered forms, splice variants, mutant variants, etc. of this human gene. The term also includes an open reading frame that encodes a particular polypeptide, an intron, as well as an adjacent 5 'that is involved in the regulation of expression that spans up to about 1 kb beyond the coding region, but may also be in both directions. And is intended to mean 3 'non-coding nucleotide sequences. The DNA sequence encoding C5aR may be cDNA, genomic DNA, or a fragment thereof. The gene can be introduced into a suitable vector for extrachromosomal maintenance or integration into the host.

  Genomic sequences of particular interest include nucleic acids that exist between the start and stop codons, including all of the introns that are normally present in natural chromosomes. This may further include the 3 ′ and 5 ′ untranslated regions found in mature mRNA. This may further include specific transcriptional and translational control sequences, such as promoters, enhancers, etc., including flanking genomic DNA located at the 5 ′ or 3 ′ end of the transcribed region that is about 1 kb but may be longer. Genomic DNA can be isolated as a 100 kb or smaller fragment substantially free of flanking chromosomal sequences.

  The sequence of the 5 ′ region of the human C5aR gene, as well as additional 5 ′ upstream and 3 ′ downstream sequences, can be used as a promoter element that includes an enhancer binding site that provides expression in tissues where C5aR is normally expressed. Tissue specific expression serves to provide a promoter that mimics the natural expression pattern. Polymorphisms found naturally in the promoter region are useful for determining natural changes in expression, particularly changes that can be associated with a disease. Alternatively, mutations can be introduced into the promoter region to reveal the effects of altered expression in established experimental systems. Methods for identifying specific DNA motifs involved in transcription factor binding are well known in the art, such as sequence similarity to known binding motifs, gel retardation tests, and the like. For example, Blackwell et al. (1995) Mol Med 1: 194-205; Mortlock et al. (1996) Genome Res. 6: 327-33; and Joulin and Richard-Foy (1995) Eur J Biochem 232: 620-626. Please refer to.

  In one embodiment, a vector suitable for use in the present invention may comprise at least one expression control element operably linked to a nucleic acid sequence encoding human C5aR. Expression control elements can be inserted into the vector to regulate and control the expression of the nucleic acid sequence. Examples of expression control elements include, but are not limited to, the lac system, the phage lambda operator and promoter region, the yeast promoter, and promoters derived from polyoma, adenovirus, retrovirus, or SV40. . The vector may further include additional operational elements, which may be any necessary or preferred for proper transcription and / or translation of the leader sequence, stop codon, polyadenylation signal, and nucleic acid sequence encoding human C5aR. Other sequences are included, but are not limited to these.

  In a further preferred embodiment, the construct comprises regions homologous to the 3 ′ and 5 ′ sequences that flank the endogenous C5aR coding sequence of the non-human mammal. It will be appreciated that these regions of homology are useful for targeted integration of the construct into the endogenous C5aR locus of the transgenic mammal.

  Preferably, the polynucleotide construct comprises a homologous region at least 2 kb, more preferably about 3 kb upstream and downstream of exon 3 of the endogenous C5aR gene. For example, the upstream and downstream sequences can be derived from the sequence of nucleotides 7377 to 15045 of SEQ ID NO: 1.

  Such vectors are obtained by conventional methods (eg, Sambrook et al., (Eds.) (1989) “Molecular Cloning, A Laboratory Manual” Cold Spring Harbor Press, Plainview, NY; Ausubel et al., (Eds.) (1987) "Current Protocols in Molecular Biology", see John Wiley and Sons, New York, NY) .

  In some embodiments, it may be preferable to express human C5aR in tissues that mimic human natural expression patterns. A specific expression pattern is that the nucleic acid encoding human C5aR is placed under the control of an inducible or developmentally controlled promoter, or under the control of a tissue specific or cell type specific promoter. Can be achieved. As an example, a specific expression pattern can be achieved using the genomic sequence of human C5aR.

  In vitro mutagenesis techniques for cloned genes are well known. Examples of procedures for investigating mutations are Gustin et al., 1993 Biotechniques 14:22; Barany, 1985 Gene 37: 111-23; Colicelli et al., 1985 Mol Gen Genet 199: 537-9; and Prentki et al. al., 1984 Gene 29: 303-13. Site-directed mutagenesis methods are described in Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, CSH Press, pp. 15.3-15.108; Weiner et al., 1993 Gene 126: 35-41; Sayers et al., 1992 Biotechniques 13: 592-6; Jones and Winistorfer, 1992 Biotechniques 12: 528-30; Barton et al., 1990 Nucleic Acids Res 18: 7349-55; Marotti and Tomich, 1989 Gene Anal Tech 6: 67-70; Can be found in Zhu 1989 Anal Biochem 177: 120-4.

"Knockout" and "Knockin"
Although not necessary for the operability of the present invention, the transgenic animals described herein may also contain endogenous gene changes in addition to the genetic changes described above. For example, the host animal may be “knocked out” and / or “knocked in” with respect to the target gene, consistent with the objectives of the present invention (eg, the host animal's endogenous C5aR may be “knocked out”. And / or human C5aR may be “knocked in”). A knockout has a partial or complete loss of function in one or both alleles of the endogenous gene of interest (eg, C5aR). In the knock-in, a transgene having a gene sequence and / or function different from that of the endogenous gene is introduced. The two can be combined, for example, a naturally occurring gene loses function and a different form of gene is introduced. For example, it is preferable to knock out the endogenous C5aR gene of the host animal and introduce the human C5aR gene.

  In knockout, preferably the expression of the target gene is undetectable or negligible. For example, knockout of the C5aR gene means that the function of the C5aR gene is substantially reduced and expression is undetectable or present at a negligible level. This can be achieved by a variety of mechanisms, including destruction of the coding sequence, e.g. introduction of one or more stop codons, insertion of a DNA fragment, etc., deletion of the coding sequence, substitution of the coding sequence and stop codon, etc. . In some cases, the sequence of the exogenous transgene is ultimately deleted from the genome, leaving a net change to the native sequence. Another approach may be used to achieve “knockout”. Inducing chromosomal deletions in all or part of the native gene, including non-coding regions, especially promoter regions, 3 'regulatory sequences, enhancer deletions, or deletions of genes that activate C5aR expression Can do. Functional knockout can also be achieved by introduction of an antisense construct that blocks expression of the native gene (eg, Li and Cohen (1996) Cell 85: 319-329). For example, when an animal that promotes recombination at the target gene site (for example, Cre in the Cre-lox system) is introduced into the “knock-out”, for example, when an animal is exposed to a substance that promotes a target gene change. Also included are conditional knockouts in which changes in the target gene occur in other ways to induce the target gene change after birth.

  By “knock-in” of a target gene is meant a change in the host cell genome that results in a change in the expression or function of the natural target gene. Increased (including ectopic) or decreased expression can be achieved by introducing additional copies of the target gene or by operably inserting control sequences that provide enhanced expression of the endogenous copy of the target gene. Can be achieved. These changes may be constitutive changes or conditional changes, i.e. may depend on the presence of activators or inhibitors.

Identification and / or evaluation of C5aR ligands, agonists, inverse agonists, and / or antagonists Using this transgenic animal or cells derived therefrom, identify a ligand or substrate that modulates a phenomenon associated with C5aR signaling be able to. Depending on the particular assay, the entire transgenic animal of the invention can be used, or tissues, organs, or cells derived therefrom can be used. The cells may be freshly isolated from the transgenic animal or immortalized in culture. Cells of particular interest are smooth muscle, endothelium, contractile muscle, or heart.

  As used herein, the term “compound” refers to any molecule, such as a protein, small molecule, polynucleotide, or pharmaceutical agent, that has the ability to prevent or inhibit molecular and clinical phenomena associated with C5aR signaling. Refers to a molecule.

  Although candidate compounds include multiple chemical classifications, in preferred embodiments they are organic molecules, preferably small molecule organic compounds having a molecular weight greater than 50 and less than about 2,500 daltons. Candidate compounds preferably contain functional groups necessary for structural interactions with proteins, particularly hydrogen bonds, typically at least one amine group, carbonyl group, hydroxyl group, or carboxyl group, preferably these functional groups. Contains at least two of the chemical groups. Candidate agents often include cyclic carbon or heterocyclic structures and / or aromatic or polycyclic aromatic structures substituted with one or more of the above functional groups. Candidate compounds are also found from biomolecules including, but not limited to, sugars, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.

  Candidate compounds may be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or are readily generated. Furthermore, natural or synthetically generated libraries and compounds can be readily modified by conventional chemical, physical, and biochemical means and used to create combinatorial libraries. It is also possible to subject known pharmacological agents to directed or random chemical modifications such as acylation, alkylation, esterification, amidation to produce structural analogs.

  Screening can be performed on known pharmacologically active compounds and chemical analogs thereof.

  To obtain the desired result, the candidate agent is administered to the transgenic mammal of the present invention (or cells derived from the transgenic mammal) in any manner desirable and / or appropriate to deliver the agent. be able to. For example, the candidate agent is administered by injection (eg, intravenous injection, intramuscular injection, subcutaneous injection, or direct injection into the tissue where the desired effect is to be achieved), orally, or by any other desirable means. can do. Preferably, the in vivo screening is administered to a large number of animals with varying amounts and concentrations of the candidate compound (from those that do not contain the compound to the amount of the compound that reaches the upper limit that can be successfully delivered to the animal). And can include delivery of drugs in different dosage forms. The compounds may be administered alone or in combination of two or more, particularly when combined administration of drugs produces a synergistic effect. The effect of drug administration on transgenic rodents can be monitored by conventional methods.

  The range of candidate compounds contemplated herein includes C5aR inhibitory compounds, or antagonists of C5aR biological function. In one embodiment, the compound is selected from the group consisting of: a peptide, eg, a C5aR or C5a-derived peptide or other non-C5aR peptide that can inhibit, reduce, or suppress C5aR function, C5aR dominant negative mutants; non-peptide inhibitors of C5aR; antibodies or antibody fragments that bind to C5aR and inhibit C5aR function; small organic molecules and nucleic acids such as peptides derived from C5aR or C5a or other non-C5aR peptide inhibitors Nucleic acid encoding nucleic acids, antisense nucleic acids against C5aR-encoding mRNA, or anti-C5aR ribozyme, or small interfering RNA (RNAi) targeting C5aR gene expression. Many of these types of compounds are discussed below.

Small molecule candidate compounds include a number of chemical classes, but preferably they are organic molecules, preferably small molecule organic compounds having a molecular weight greater than 100 and less than about 2,500 daltons. Candidate compounds contain functional groups necessary for structural interactions with proteins, particularly hydrogen bonds, and typically include at least one amine group, carbonyl group, hydroxyl group, or carboxyl group, preferably these functional chemical groups. Including at least two. Candidate compounds often contain cyclic carbon or heterocyclic structures and / or aromatic or polycyclic aromatic structures substituted with one or more of the above functional groups. Candidate compounds are also found from biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, and derivatives, structural analogs, or combinations thereof. In fact, virtually any small organic molecule that could potentially bind to the biological target molecule of interest is sufficiently soluble and stable in aqueous solution to test for the ability to bind to the biological target molecule If so, it may be useful in the present invention.

  Candidate compounds are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, a number of means can be utilized for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or are readily generated. Furthermore, natural or synthetically produced libraries and compounds are readily modified by conventional chemical, physical, and biochemical means. Structural analogs can also be made by subjecting known pharmacological compounds to directed or random chemical modifications such as acylation, alkylation, esterification, amidation and the like.

Protein or peptide inhibitor In another embodiment, the candidate compound is a protein. As used herein, “protein” refers to at least two amino acids that are covalently linked, including proteins, polypeptides, oligopeptides, and peptides. Proteins may consist of natural amino acids and peptide bonds, or may consist of synthetic peptidomimetic structures. Thus, as used herein, the term “amino acid” or “peptide residue” refers to both natural and synthetic amino acids. For example, homophenylalanine, citrulline, and norleucine are considered amino acids in the present invention. “Amino acid” also includes imino acid residues such as proline and hydroxyproline. The side chain may be in the (R) configuration or the (S) configuration. In a preferred embodiment, the amino acid is in the (S) or (L) configuration. When using non-naturally occurring side chains, non-amino acid substituents can be used, for example, to prevent or retard in vivo degradation.

  In a further preferred embodiment, the candidate compound is a natural protein or a fragment of a natural protein. Thus, for example, cell extracts containing proteins, or random or directed digests of proteinaceous cell extracts can be used. In this way, libraries of prokaryotic and eukaryotic proteins can be generated. Particularly preferred in this embodiment are bacterial, fungal, viral, and mammalian protein libraries, with mammalian proteins being preferred and human proteins being particularly preferred.

  In a further preferred embodiment, the candidate compound is a peptide about 5 to about 30 amino acids long, preferably about 5 to about 20 amino acids, and particularly preferably about 7 to about 15 amino acids. The peptides may be digests of natural proteins, random peptides, or “biased” random peptides. As used herein, the term “randomized” or grammatically equivalent terms mean that each peptide consists essentially of random amino acids. In general, since these random peptides are chemically synthesized, the random peptides can contain any amino acid at any position. The synthetic process can be designed to generate random proteins so that all or most of the possible combinations can be formed over the length of the sequence, forming a library of randomized candidate bioactive proteinaceous compounds.

  Methods for preparing and screening combinatorial chemical libraries are well known to those skilled in the art. Such combinatorial chemical libraries include peptide libraries (eg, US Pat. No. 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37: 487-493, Houghton et al. (1991) Nature, 354: 84-88), but is not limited to this. Peptide synthesis is by no means the only approach envisioned and intended for use with the present invention. Other chemistries that create chemical diversity libraries can also be used. Such chemistry includes: peptoids (WO 91/19735, December 26, 1991), encoded peptides (WO 93/20242, October 14, 1993), random bio Diversomers such as oligomers (WO 92/00091, Jan. 9, 1992), benzodiazepines (US Pat. No. 5,288,514), hydantoins, benzodiazepines, and dipeptides (Hobbs et al., (1993) Proc. Nat. Acad. Sci. USA 90: 6909-6913), vinyl polypeptide (Hagihara et al. (1992) J. Amer. Chem. Soc. 114: 6568), non-peptide peptide with βD glucose skeleton Mimics (Hirschmann et al., (1992) J. Amer. Chem. Soc. 114: 92179218), similar organic synthesis of small compound libraries (Chen et al. (1994) J. Amer. Chem. Soc. 116: 2661), oligocarbamic acid (Cho et al., (1993) Science 261: 1303), and / or peptidylphosphones (Campbell et al, (1994) J. Org Chem 59:... 658) include, but are not limited to. Generally, Gordon et al., (1994) J. Med. Chem. 37: 1385, nucleic acid libraries, peptide nucleic acid libraries (see, eg, US Pat. No. 5,539,083), antibody libraries (eg, Vaughn et al. (1996) Nature Biotechnology, 14 (3): 309-314, and PCT / US96 / 10287), carbohydrate libraries (eg, Liang et al. (1996) Science, 274: 1520-1522, and US Pat. No. 5,593,853), and small organic molecule libraries (eg, benzodiazepines, Baum (1993) C & EN, Jan 18, page 33, isoprenoids, US Pat. No. 5,569,588, thiazolidinones and metathiazanones No. 5,549,974, pyrrolidine, US Pat. Nos. 5,525,735 and 5,519134, morpholino compounds, US Pat. No. 5,506,337, benzodiazepines, US Pat. No. 5,288,514, and the like).

  Equipment for preparing combinatorial libraries is commercially available (eg, 357 MPS, 390 MPS, Advanced Chem Tech, Kentucky, Louisville, Symphony, Rainin, Massachusetts, Woburn, 433A Applied Biosystems, Foster City, Calif. 9090 Plus, Millipore, Bedford, Mass.).

  In one embodiment, peptidyl C5aR inhibitors are synthesized chemically or recombinantly as oligopeptides (usually 10-25 amino acids in length) derived from C5aR or C5a sequences. Alternatively, a C5aR or C5a fragment is generated by digesting natural or recombinantly produced C5aR or C5a using a protease such as trypsin, thermolysin, chymotrypsin, or pepsin. Computer analysis (using commercially available software such as MacVector, Omega, PCGene, Molecular Simulation, Inc.) identifies protein cleavage sites. Proteolytic or synthetic fragments can contain as many amino acid residues as necessary to partially or fully inhibit C5aR function. Preferred fragments are at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more amino acids Including length.

  The protein or peptide inhibitor may also be a dominant negative variant of C5aR. The term “dominant negative variant” is a C5aR polypeptide that has been mutated from its native state and prevents C5aR from interacting with normally interacting proteins, thereby preventing endogenous native C5aR from forming an interaction. Point to.

Anti-C5aR Antibody The term “antibody” as used in the present invention includes complete molecules and fragments thereof, such as Fab, F (ab ′) ₂ , Fv, etc. that can bind to the epitope determinant of C5aR. These antibody fragments retain some ability to selectively bind with its antigen and are defined as follows:
(1) Fab, a fragment containing a monovalent antigen-binding fragment of an antibody molecule, is generated by digesting the entire antibody with the enzyme papain to produce one complete light chain and part of one heavy chain Can be;
(2) Fab ', this fragment of an antibody molecule can be obtained by treating the whole antibody with pepsin, followed by reduction, producing a complete light chain and part of a heavy chain; per antibody molecule Two Fab ′ fragments are obtained;
(3) F (ab ′) ₂ , this fragment of the antibody can be obtained by treating the whole antibody with the enzyme pepsin, followed by no reduction. F (ab ′) ₂ is a dimer of two Fab ′ fragments joined together by two disulfide bonds;
(4) Fv, defined as a recombinant fragment comprising the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and
(5) Single chain antibody ("SCA"), a genetically fused single chain molecule, a light chain variable region and a heavy chain variable region joined by an appropriate polypeptide linker Defined as a molecule.

  Methods for making these fragments are well known in the art. (See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), incorporated herein by reference).

  The antibody of the present invention can be prepared by using complete C5aR or a fragment thereof as an immunizing antigen. Peptides used to immunize animals may be derived from cDNA translation or chemical synthesis, and are purified and conjugated to a carrier protein as necessary. Commonly used carriers that are chemically coupled to peptides include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. This binding peptide can then be used to immunize an animal (eg, a mouse or rabbit).

  If necessary, the polyclonal antibody can be further purified by, for example, binding the peptide that produced the antibody to the bound substrate and eluting from it. Those skilled in the art will be familiar with various techniques common in the immunology field relating to the purification and / or enrichment of polyclonal and monoclonal antibodies (eg, Coligan, et al., Unit 9 incorporated by reference). , Current Protocols in Immunology, Wiley Interscience, 1991).

  Monoclonal antibodies can be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture, such as the hybridoma technique, the human B cell hybridoma technique, and the EBV hybridoma technique (Kohler et al. Nature 256, 495-497, 1975; Kozbor et al., J. Immunol. Methods 81, 31-42, 1985; Cote et al., Proc. Natl. Acad. Sci. USA 80, 2026-2030, 1983; Cole et al., Mol. Cell Biol. 62, 109-120, 1984).

  Antibodies that show binding to C5aR can be identified and isolated from antibody expression libraries by methods well known in the art. For example, a method for identifying and isolating antibody binding domains that exhibit binding to C5aR is a bacteriophage vector system. This vector system is derived from the mouse antibody repertoire in Escherichia coli (Huse, et al., Science, 246: 1275-1281, 1989) and from the human antibody repertoire (Mullinax, et al., Proc. Nat Acad. Sci., 87: 8095-8099, 1990) Used to express a combinatorial library of Fab fragments. This method can also be applied to hybridoma cell lines that express monoclonal antibodies that are binding to a preselected ligand. Hybridomas that secrete the desired monoclonal antibodies can be made in a variety of ways using techniques well understood by those skilled in the art, but will not be repeated herein. Details of these techniques are incorporated by reference, such as Monoclonal Antibodies-Hybridomas: A New Dimension in Biological Analysis, Edited by Roger H. Kennett, et al., Plenum Press, 1980; and US Pat. No. 4,172,124. It is described in.

  In addition, methods for making chimeric or “humanized” antibodies are well known in the art, which includes combining mouse variable regions with human constant regions (Cabily, et al. Proc. Natl. Acad. Sci). USA, 81: 3273, 1984), or by transplanting the mouse antibody complementarity determining region (CDR) into a human framework (Riechmann, et al., Nature 332: 323, 1988).

Antisense Compound The term “antisense compound” is complementary to at least a portion of a target mRNA molecule (Izant and Weintraub, 1984; Izant and Weintraub, 1985) and can interfere with post-transcriptional events such as mRNA translation or Includes RNA molecules. Antisense oligomers complementary to at least about 15 contiguous nucleotides of the target encoded mRNA are preferred because they are easily synthesized and are less likely to cause problems than larger molecules when introduced into target mRNA producing cells. The use of antisense methods is well known in the art (Marcus-Sakura, 1988).

Catalytic RNA molecules Catalytic RNA refers to RNA or RNA-containing molecules that specifically recognize different substrates and catalyze the chemical modification of this substrate (also known as “ribozymes”). Nucleobases in the catalytic nucleic acid can be bases A, C, G, T, and U, and derivatives thereof. Derivatives of these bases are well known in the art.

  Typically, a catalytic nucleic acid includes an antisense sequence for specifically recognizing a target nucleic acid, and a nucleic acid cleaving enzyme activity (also referred to herein as a “catalytic domain”).

  The types of ribozymes that are particularly useful in the present invention are hammerhead ribozymes (Haseloff and Gerlach 1988, Perriman et al, 1992) and hairpin ribozymes (Shippy et al, 1999).

  Ribozymes used in the present invention can be chemically synthesized using methods well known in the art. Ribozymes can also be prepared from DNA molecules (which produce RNA molecules upon transcription) operably linked to an RNA polymerase promoter, such as the promoter of T7 RNA polymerase or SP6 RNA polymerase. Similarly, ribozymes can be prepared in vitro by incubating with RNA polymerase and nucleotides when the vector includes an RNA polymerase promoter operably linked to a DNA molecule. In another embodiment, the DNA can be inserted into an expression cassette or transcription cassette.

dsRNA
dsRNA is particularly useful for specifically inhibiting the production of a particular protein. Without wishing to be bound by theory, Dougherty and Parks (1995) provide a model of the mechanism by which dsRNA can be used to reduce protein production. This model was improved and extended by Waterhouse et al (1998). This technique relies on the presence of dsRNA molecules that contain sequences that are essentially identical to the mRNA of the gene of interest. Conveniently, the dsRNA can be produced in a recombinant vector or in a single open reading frame within the host cell, in which the sense and antisense sequences are flanked by unrelated sequences, This allows the sense and antisense sequences to hybridize to form dsRNA molecules in which unrelated sequences form a loop structure. The design and generation of suitable dsRNA molecules that target the gene of interest are described in particular by Dougherty and Parks (1995), Waterhouse et al (1998), WO 99/32619, WO 99/53050, WO 99. / 49029 and WO 01/34815 are well within the ability of one skilled in the art.

  As used herein, the terms “small interfering RNA” and “RNAi” refer to homologous double-stranded RNA (dsRNA) that specifically targets a gene product, resulting in a null phenotype or a low trait phenotype. ). Specifically, the dsRNA contains two nucleotide sequences derived from the target RNA and having self-complementarity that can anneal to prevent expression of the target gene, possibly at the post-transcriptional level. RNAi molecules are described by Fire et al (1998) and reviewed by Sharp (1999).

Phenotypes Associated with C5aR Signaling In the methods of the present invention, a putative compound is administered to a transgenic animal and a transgenic response to the putative compound is measured. Preferably, the response of the transgenic mammal is compared to “normal” or wild type mice, or compared to a transgenic animal control (not receiving the compound).

  Accordingly, in one aspect of the present invention, there is provided a method for identifying a compound that modulates C5aR activity, comprising: (i) under the conditions where at least one phenotype associated with C5aR signaling is expressed. A method comprising: administering a candidate compound to a transgenic mammal; and (ii) monitoring the development of the phenotype after administration of the compound.

The phenotype to monitor can be any indicator of C5aR signaling, including:
(a) Inflammatory or allergic diseases and conditions such as respiratory allergic diseases such as asthma, allergic rhinitis, hypersensitivity lung disease, hypersensitivity pneumonia, interstitial lung disease (ILD) (eg idiopathic lung ILD with fibrosis or rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, systemic sclerosis, Sjogren's syndrome, polymyositis, or dermatomyositis); anaphylaxis or hypersensitivity responses, drug allergies (eg, penicillin, ceparo Allergic to insects bites; inflammatory bowel diseases such as Crohn's disease and ulcerative colitis; spondyloarthropathy; scleroderma; psoriasis and inflammatory skin diseases such as dermatitis, eczema, atopic skin Inflammation, allergic contact dermatitis, urticaria; vasculitis (eg, necrotizing vasculitis, cutaneous vasculitis, and hypersensitivity vasculitis);
(b) Autoimmune diseases such as arthritis (eg rheumatoid arthritis, psoriatic arthritis), multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, juvenile diabetes, glomerulonephritis nephritis, autoimmune thyroid Flame, Behcet's disease;
(c) graft rejection (eg at the time of transplantation), eg allograft rejection or graft-versus-host disease;
(d) atherosclerosis;
(e) cancer with leukocyte infiltration of the skin or organ;
(f) Diseases or conditions that may be treated with undesirable inflammatory responses to be suppressed, including but not limited to, C5aR-mediated diseases or conditions, such as reperfusion injury, stroke, adult respiratory distress Granulomatosis, including syndrome, certain hematological malignancies, cytokine-induced toxicity (eg, septic shock, endotoxic shock), polymyositis, dermatomyositis, pemphigoid, Alzheimer's disease, and sarcoidosis.

  Other phenotypes include immunosuppression, eg, individuals with immunodeficiencies such as AIDS, radiation therapy that results in immunosuppression, chemotherapy, autoimmune disease therapy, or other drug therapy (eg, corticosteroid therapy Immunosuppression as seen in individuals receiving); and immunosuppression due to congenital defects in receptor function or other causes.

  A number of in vivo models of inflammation are available and can be used to induce an appropriate C5aR related phenotype in the transgenic mammals of the invention. For example, collagen-induced arthritis (Trentham et al (1977) J Exp Med 146: 867-868), K / BxN serum-induced arthritis (Kouskoff et al (1996) Cell 87: 811-822), antigen-induced arthritis, or adjuvant induction Rheumatoid arthritis can be assessed using an animal (eg mouse) model of arthritis (Pearson CM (1956) Proc Soc. Expe Biblo Med 91: 95-101).

  Further examples of suitable animal models include: septic cecal ligation and puncture (CLP) model (Huber-Lang, MS, et al. (2002) Faseb J 16 (12): 1567-74); Rat model (Woodruff, TM, et al. (2002) Arthritis Rheum 46 (9): 2476-85); a sepsis pig model (Mohr, M., et al. (1998) Eur J Clin Invest 28 (3): 227-34); immune complex-induced lung disease; pancreatitis-related lung injury (Bhatia, M. et al. (2001) Am J Physiol Gastrointest Liver Physiol 280 (5): G974-8); acute lung injury; renal ischemia -Reperfusion injury; collagen-induced arthritis; and experimental airway disease (asthma-like model).

  In another example, leukocyte infiltration upon intradermal administration of a candidate compound can be monitored (eg, Van Damme, J. et al., J. Exp. Med., 176: 59-65 (1992); Zachariae , COC et al., J. Exp. Med. 171: 2177-2182 (1990); see Jose, PJ et al., J. Exp. Med. 179: 881-887 (1994)). In one embodiment, skin biopsy samples are evaluated histologically for infiltration of leukocytes (eg, eosinophils, granulocytes). Inhibition of C5aR signaling is indicated when the degree of invasion in the presence of the candidate compound is reduced compared to the degree of invasion in the absence of the candidate compound.

  Examples of preferred phenotypes are briefly discussed below.

Autoimmune diseases (including rheumatoid arthritis)
Autoimmune diseases affect 5-8% of the population. Immune complexes (IC) play an important role in the onset of several autoimmune diseases including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), glomerulonephritis, and immune vasculitis. While IC initiates an autoimmune response, other important factors that contribute to disease severity include the complement system, Fcg receptors, and neutrophils. Currently, it is recognized that complement's major contribution to IC inflammation is via C5aR signaling. IC activates both the classical and alternative complement pathways. C5a released by C5 cleavage during complement activation is a potent chemoattractant for neutrophils, monocytes, and macrophages. C5a binding to C5aR results in the release of additional chemotactic mediators (eg, CXC chemokines) and FcgR activation on macrophages resulting in further organization of the inflammatory response (up-regulation and inhibition of activated FcgRIII and FcgRI) A series of events are initiated, including down-regulation of FcgRIIB.

  The importance of C5a / C5aR has been demonstrated in various model systems of immune complex inflammation. For example, C5a has been shown to initiate an inflammatory cascade in a mouse peritonitis model of reverse passive Arthus reaction (Godau J, et al, J Immunol 173, 3437 (2004)). C5a has also been shown to regulate FcgR in IC-induced lung disease (Shushakova NJ, et al, J Clin Invest, 110, 1823-1830 (2002)). Furthermore, C5aR knockout mice can be treated with Rheumatoid arthritis Benoist-Mathis KRNxNOD serum transfer model (Ji H, et al, Immunity 16, 157 (2002)) or anti-collagen antibody induced arthritis model (Grant EP, et al, J Exp Med 196 , 1461 (2002)) does not develop inflammation.

Sepsis Sepsis is a serious disease caused by severe bloodstream infection by toxin-producing bacteria. It is recognized that innate sepsis can disrupt the innate immune system, as evidenced by extensive activation of inflammation, complement, and the coagulation system, and the appearance of cytokines and chemokines in plasma. This “cytokine storm”, as its name suggests, deregulates many inflammatory mediators that contribute to multiple organ dysfunction or multiple organ failure and death.

  A recent review of sepsis examines past attempts to find a cure for the disease and suggests future directions (Crowther and Marshall (2001) Jama 286 (15): 1894-6; Cohen, J (2002) Nature 420 (6917): 885-91; Cross and Opal (2003) Ann Intern Med 138 (6): 502-5).

  Hotchkiss and Karl (N Engl J Med 348 (2): 138-50, 2003) show that anticoagulation recombination activated protein C is effective in reducing septic mortality, in part, its anti-inflammatory effect. I guess that is due to. Some of the effects of activated protein C (direct and indirect) are blocking cytokine production, blocking cell adhesion, neutrophil recruitment, mast cell degranulation, and inhibiting platelet activation. C5a mediates leukocyte chemotaxis, release of histamine from mast cells, enhanced neutrophil-endothelial cell adhesion, and induction of cytokine production through binding to C5a receptors.

In human sepsis, disordered activation of the complement system results in excessive production of anaphylatoxins, particularly C3a and C5a, and subsequent neutrophil dysfunction. Later in sepsis, neutrophils suppress chemotactic response, decrease enzyme release, change intracellular pH, and respiratory burst failure (reduction of reactive oxygen species, especially H ₂ O ₂ production) ) Occurs, resulting in a bactericidal disorder. These defects have been shown to depend on C5a. In experimental sepsis, blood neutrophils have a greatly reduced ability to bind C5a, the chemotactic response to C5a is impaired, and H ₂ O ₂ production is lost. In CLP-induced sepsis, these defects can be prevented by treating animals with anti-C5a antibodies. This treatment alleviated bacteremia and significantly improved survival. This indicates that sepsis induces excessive production of C5a, which in turn leads to severe functional defects in neutrophils.

  Antibodies against C5aR can also protect animal models from death from sepsis. Both C5aR immunoreactivity and mRNA expression were shown to increase in lung, kidney, liver, heart, and thymus endothelial cells early in experimental sepsis. Mice that received antibodies to C5aR at the start of CLP showed a dramatic improvement in viability compared to animals that received nonspecific IgG. In CLP-treated mice, the serum levels of IL-6 and TNF-α and the number of bacteria in various organs were significantly reduced during CLP compared to control CLP animals. These studies demonstrated that C5aR blockade is highly protective against the lethal outcome of sepsis.

  It has been suggested that the position of C5a is important with regard to its potential to protect or harm. Local production of C5a in tissues is essential for early control of infection. A C5a gradient is established and leukocyte chemotaxis occurs. At high concentrations of C5a, chemotaxis is suppressed and cells produce toxic oxygen bursts. In sepsis, diffuse intravascular C5a is diffused by diffuse complement activation in the blood, which impairs neutrophil function and allows bacteria to grow indefinitely. At the same time, neutrophil sequestration during the microcirculation damages the lungs, kidneys, and liver. In this model, C5aR on leukocytes appears to be important rather than receptors on endothelial cells such as liver and lung. Intervention with C5aR antagonists may prove to be therapeutically beneficial.

Ischemia-reperfusion injury (IR)
The complement system is an important mediator of inflammatory tissue damage in a variety of diseases, including ischemia-reperfusion (IR) injury.

  The relative roles of C5a and the membrane attack complex C5b-9 in mediating IR injury may vary. In some models, C5a is an important mediator. Antagonists of C5aR protect mice and rats from IR injury in the small intestine and kidney (Heller et al. (1999) J Immunol 163 (2): 985-94; Arumugam et al. (2003) Kidney Int 63 (1) : 134-42; Arumugam et al. (2002) J Surg Res 103 (2): 260-7). In the porcine myocardial IR injury model, C5aR antagonist significantly reduced infarct size (Riley et al. (2000) J Thorac Cardiovasc Surg 120 (2): 350-8).

  Studies with antibodies to C5 also demonstrate a similar reduction in IR injury (Fitch et al. (1999) Circulation 100 (25): 2499-506; de Vries et al (2003) Transplantation 75 (3) : 375-82).

  Clinical and experimental studies have shown that IR of organs such as kidney, liver, lung, and heart induces rapid release of various cytokines. Many of these molecules decrease in levels after reperfusion, but IL-8 increases significantly. IL-8 is a powerful neutrophil chemoattractant. IL-8 levels correlate inversely with lung function, with high levels associated with mortality risk in lung transplantation. Intravenous administration of anti-IL-8 antibody at the beginning of reperfusion significantly reduces lung injury and neutrophil infiltration (de Perrot et al (2003) Am J Respir Crit Care Med 167 (4): 490-511 ).

  Evidence shows that IR injury occurs in a biphasic pattern. Macrophages activated during ischemia mediate early injury, whereas neutrophils and lymphocytes are primarily responsible for the second delayed phase. Neutrophil and lymphocyte recruitment occurs by the release of cytokines before and after reperfusion.

  C5a is a potent chemoattractant of various bone marrow cells, including macrophages and neutrophils, and blocking C5aR with specific antibodies to induce cytokine production, IL-8 release and neutrophils It is beneficial to prevent ball migration and is therefore expected to alleviate IR injury.

Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI)
ARDS and ALI are syndromes caused by pulmonary edema and inflammation. The development of ARDS / ALI is associated with a variety of clinical disorders including lung injury due to pneumonia infection, gastric contents aspiration, trauma, sepsis, acute pancreatitis, drug overdose, and cardiopulmonary bypass. Sepsis is the main cause of ARDS / ALI. Similar to IR, the inflammatory response in ALI is associated with the recruitment of numerous neutrophils and monocytes into the distal lung space. Pro-inflammatory molecules such as cytokines, active oxygen, and proteases play a role, and excessive inflammation can exacerbate ARDS / ALI (Ware and Matthay (2000) N Engl J Med 342 (18): 1334-49 Brower et al. (2001) Chest 120 (4): 1347-67).

  Anti-inflammatory strategies to reduce the number of neutrophils that migrate to the extravascular space of the lung, reduce neutrophil adhesion to the lung endothelium, and reduce chemotactic factor release may be beneficial It has been suggested (Brower et al. (2001) Chest 120 (4): 1347-67).

  Studies of ARDS patients reveal that high levels of IL-8 are present in BAL fluid or pulmonary edema fluid early in the disease. Furthermore, in the rabbit model, antibodies that neutralize IL-8 alleviated lung injury (Brower et al. (2001) Chest 120 (4): 1347-67).

  For ARDS and ALI, blocking C5aR is still considered a therapeutically viable approach. C5a is a potent chemotactic molecule and a promoter of cytokine release. The above studies using molecules that antagonize C5a or C5aR demonstrated the alleviation of injury in various inflammatory models.

Production of compounds identified by the screening methods of the invention In one embodiment of the invention, the method further comprises the step of producing or synthesizing a compound to be tested in the genetically modified animal.

  Peptidyl compounds are synthesized by Merrifield synthesis (Merrifield, J Am Chem Soc, 85,: 2149-2154, 1963) and the numerous improvements available for that technology (eg, Synthetic Peptides: A User's Guide, Grant, ed. 1992) WH Freeman & Co., New York, pp. 382; Jones (1994) The Chemical Synthesis of Peptides, Clarendon Press, Oxford, pp. 230); Barany, G. and Merrifield, RB (1979) , The Peptides (Gross, E. and Meienhofer, J. eds.), Vol. 2, pp. 1-284, Academic Press, New York; Wunsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls Methoden der Organischen Chemie (Muler, E., ed.), Vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart; Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky , M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474 Easy by method It is produced.

  Preferably, the peptide is further swollen with a lipophilic solvent such as dichloromethane or dimethylformamide (DMF), such as polystyrene gel beads comprising polystyrene cross-linked with divinylbenzene, preferably 1% (ww) divinylbenzene, etc. On a solid support. Polystyrene can be functionalized by adding chloromethane or aminomethyl groups.

  Alternatively, cross-linked and functionalized polydimethyl-acrylamide gels can be used by swelling and solvating with DMF or amphoteric aprotic solvents. Other solid phase supports well known to those skilled in the art can also be used for peptide synthesis, including polyethylene glycol derived beads made by grafting polyethylene glycol onto the surface of inert polystyrene beads. is there. Preferred commercially available solid supports include PAL-PEG-PS, PSC-PEG-PS, KA, KR, or TGR (Applied Biosystems, CA 94404, USA).

  In solid phase peptide synthesis, removal of the amino blocking group of the growing peptide chain and the protection of amino acid side chains during synthesis and masking of α-amino groups, carboxyl groups, or side chain functional groups and A blocking group is used that is stable to the iterations required for repeated amino acid coupling. Thus, a blocking group (also referred to as a protecting group or masking group) protects the amino group of an amino acid having an activated carboxyl group that participates in the coupling reaction or prevents the acylated amino group that participates in the coupling reaction. It protects the carboxyl group of the amino acid it has.

  In the synthesis process, coupling is performed after removal of the blocking group without destroying the peptide bond or the protecting group attached to another part of the peptide. In addition, the peptide-resin anchoring point protecting the C-terminus of the peptide is protected throughout the synthesis process until cleavage from the resin is required. Thus, judicious selection of ortho-protected α-amino acids adds amino acids to the desired position of the growing peptide while attached to the resin.

  Preferred amino blocking groups are easily removable, but are stable enough to surpass conditions such as coupling reactions and other manipulations such as side group modification. In one embodiment, the amino blocking group is selected from the group consisting of: (i) by catalytic hydrogenation at room temperature and atmospheric pressure, or by using sodium in liquid ammonia and hydrobromic acid in acetic acid. Easily removed benzyloxycarbonyl groups (Z or carbocenzoxy); (ii) urethane derivatives; (iii) introduced using t-butoxycarbonyl azide or di-tert-butyl dicarbonate, for example trifluoroacetic acid (T-butoxycarbonyl group (Boc) removed by weak acid such as HCl in 50% TFA in dichloromethane) or acetic acid / dioxane / ethyl acetate; (iv) eg primary or secondary amine (eg in dimethylformamide) 9-fluorenylmethyloxycyl which is cleaved under mildly basic, non-hydrolytic conditions such as (V) 2- (4-biphenylyl) propyl (2) oxycarbonyl group (Bpoc); (vi) 2-nitro-phenylsulfenyl group (Nps); and (vii) dithia-succinyl (Succionyl) group (Dts). In Fmoc chemistry, Boc is widely used to protect the N-terminus, and in Boc chemistry, Fmoc is widely used to protect the N-terminus.

  The side chain protecting group varies depending on the functional side chain of the amino acid forming the peptide to be synthesized. Side chain protecting groups are generally based on Bzl or tBu groups. Amino acids having an alcohol or carboxylic acid in the side chain are protected as Bzl ether, Bzl ester, cHex ester, tBu ether, or tBu ester. Side chain protection of Fmoc amino acids requires a blocking group that is ideally stable to bases and unstable to weak acids (TFA). Many different protecting groups for peptide synthesis have been described previously (see The Peptides, Gross et al. Eds., Vol. 3, Academic Press, New York, 1981). For example, 4-methoxy-2,3,6-trimethylphenylsulfonyl (Nd-Mtr) group is useful for arginine side chain protection, but deprotection of Arg (Mtr) requires long-term TFA treatment . To protect cysteine, many weak acids (TFA, thallium (III) trifluoroacetic acid / TFA) labile group, TFA stable but thallium (III) trifluoroacetic acid / TFA labile group, Alternatively, a stable group can be used for a soft acid.

  The two most widely used protection strategies are the Boc / Bzl strategy and the Fmoc / tBu strategy. In Boc / Bzl, Boc is used for amino protection, and the side chains of various amino acids are protected with protecting groups based on Bzl or cHex. Boc groups are stable under catalytic hydrogenation conditions and are used orthogonally with Z groups to protect many side chain groups. In Fmoc / tBu, Fmoc is used for amino protection and the side chain is protected with a tBu-based protecting group.

  Alternatively, peptidyl compounds are produced by recombinant expression of nucleic acids encoding the amino acid sequence of such peptides. Libraries that encode random peptides are particularly preferred for such purposes because they provide a wide variety of different compounds to test. Alternatively, natural nucleic acids can be screened. In this embodiment, the nucleic acid encoding the peptidyl compound is made by standard oligonucleotide synthesis, or is derived from a natural source, which is a promoter or other that can control expression in a cell-free or cell system. It is operably linked to control sequences and cloned into an appropriate expression vector.

  Oligonucleotides are preferably synthesized with adapter sequences at the 5 'and 3' ends so that they can be conveniently cloned into an appropriate vector system by standard techniques.

  Placing a nucleic acid molecule under the regulatory control of a promoter sequence, ie, “operably linked to” the promoter sequence, generally means that expression is achieved by placing the promoter 5 ′ (upstream) of the peptide coding sequence. It means to arrange the molecule so that it is controlled by the promoter sequence.

A prerequisite for producing a complete polypeptide in bacteria such as E. coli is the use of a strong promoter with an effective ribosome binding site. Typical promoters suitable for expression in bacterial cells such as E. coli include, but are not limited to, the lacz promoter, the temperature sensitive λ _L or λ _R promoter, the T7 promoter, or the IPTG inducible tac promoter. Many other vector systems for expressing nucleic acid molecules of the invention in E. coli are well known in the art, such as Ausubel et al (Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047150338, 1987) or Sambrook et al. al (Molecular cloning, A laboratory manual, second edition, Cold Spring Harbor Laboratory, Cold Spring Habor, NY, 1989). Many plasmids with a promoter sequence suitable for expression in bacteria and an effective ribosome binding site, such as pKC30 (λ _L : Shimatake and Rosenberg, Nature 292, 128, 1981); pKK173-3 (tac: Amann and Brosius, Gene 40, 183, 1985), pET-3 (T7: Studier and Moffat, J. Mol. Biol. 189, 113, 1986); pBAD / TOPO or pBAD / Thio-TOPO system containing arabinose-inducible promoter Vector (Invitrogen, Carlsbad, CA) (the latter is also designed to produce fusion proteins with thioredoxin to increase the solubility of the expressed protein); pFLEX-based expression vectors (Pfizer Inc. , USA, Connecticut); or pQE-based expression vectors (Qiagen, California).

  Typical promoters suitable for eukaryotic virus and eukaryotic expression include SV40 late promoter, SV40 early promoter, cytomegalovirus (CMV) promoter, CMV IE (cytomegalovirus immediate early) promoter, among others. Is included. Vectors suitable for expression in mammalian cells (eg, 293, COS, CHO, 10T cells, 293T cells) include, but are not limited to, the pcDNA vector system provided by Invitrogen, particularly the CMV promoter, and C PcDNA 3.1 myc-His-tag encoding the terminal 6xHis and MYC tags; and the retroviral vector pSRαtkneo (Muller et al., Mol. Cell. Biol., 11, 1785, 1991). The vector pcDNA 3.1 myc-His (Invitrogen) is particularly preferred for expressing secreted peptides in 293T cells, where the expressed peptide or protein is a standard using a nickel column that binds to the protein via a His tag. It can be purified without affinity proteins using affinity techniques.

  A wide range of additional host / vector systems suitable for peptide expression are publicly available, as described, for example, in Sambrook et al (Molecular cloning, A laboratory manual, second edition, Cold Spring Harbor Laboratory, Cold Spring Habor, NY, 1989) Has been.

  Means of introducing nucleic acids or gene constructs containing nucleic acids into cells for expression are well known to those of skill in the art. The technique used for a given organism depends on known successful techniques. Means for introducing recombinant DNA into animal cells include microinjection, DEAE-dextran mediated transfection, lipofectamine (Gibco, USA, Maryland) and / or cellfectin (Gibco, USA, Maryland). Liposomes mediated transfection as used, PEG mediated DNA uptake, electroporation, and using DNA coated tungsten or gold particles (Agracetus Inc., Wisconsin, USA) A fine particle gun.

  Techniques for synthesizing small organic compounds vary considerably from compound to compound, but such methods are believed to be well known to those skilled in the art. In one embodiment, combinatorial libraries are created using information science to select appropriate chemical building blocks from known compounds. For example, the QSAR (Quantitative Structure Activity Relationship) modeling approach uses linear regression or regression trees of compound structure to determine fit. Software from Chemical Computing Group, Inc. (Montreal, Canada) uses high-throughput screening experimental data on active and inactive compounds to create a stochastic QSAR model, which is then used to select lead compounds. Binary QSAR methods are based on three characteristic properties of a compound that form a “descriptor” that may or may not perform the required function of the particular compound: partial charge, molar refraction (bond interaction) , And logP (molecular lipophilicity). Each atom has a surface area in the molecule and has these three properties associated with it. All atoms of the compound with a specific range of partial charges are determined and the surface area (Van der Waals surface area descriptor) is summed. Next, an activity model or ADMET model is created using the Binary QSAR model, and a combinatorial library is constructed using these models. Thus, using information from known appetite suppressants and non-suppressants, including lead compounds identified in the initial screen, the list of compounds to be screened can be expanded, thereby identifying highly active compounds. it can.

  In one embodiment, the method further comprises formulating the identified compound for administration to a non-human animal or human. The formulation may be suitable for administration by subcutaneous, intravenous, intranasal, or intraperitoneal route. Alternatively, the formulation may be suitable for oral administration in the form of food additives, tablets, troches and the like.

  The compounds are conveniently formulated in suitable excipients or diluents, for example, non-toxic excipients such as aqueous solvents, non-aqueous solvents such as salts, preservatives, buffers. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters such as ethyl oleate. Aqueous solvents include parenteral excipients such as water, alcohol / water solutions, physiological saline, eg sodium chloride, Ringer's dextrose. Preservatives include antibacterial agents, antioxidants, chelating agents, and inert gases. The pH and exact concentration of the various components of the formulation suitable for administration to animals are adjusted according to routine techniques in the art. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents such as distilled water for injection, saline, fixed oils, polyethylene glycols, glycerin , Propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetic acid, citric acid, or phosphorus Acids and tonicity modifiers such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches and the like may contain any of the following ingredients or compounds of similar nature: binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose Disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring Agents such as peppermint, methyl salicylate, or orange flavor. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant (eg, a gas such as carbon dioxide) or a nebulizer. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally well known in the art and include, for example, surfactants for buccal administration, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated in ointments, salves, gels, or creams well known in the art.

  Optionally, the formulation also includes a carrier such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), ovalbumin, mouse serum albumin, rabbit serum albumin. Means of conjugating peptides to carrier proteins are also well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide, and bis-biazoated benzidine.

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

Example 1: Generation of a targeting construct for generating human C5aR knock-in mice To generate a mutant mouse strain by homologous recombination, two major elements are required. A targeting construct comprising an embryonic stem (ES) cell line that can contribute to the germline, and a target gene sequence with the desired mutation. Maintenance of ES cells in an undifferentiated state is achieved by culturing the cells on a layer of feeder cells. Cultured ES cells are then transfected with the targeting construct. ES cell lines are derived from the inner cell mass of blastocyst stage embryos. A small number of transfected cells undergo homologous recombination, resulting in the introduction of mutations present in the targeting construct within the target gene.

  Once a mutant ES cell clone has been identified, it can be microinjected into a normal blastocyst to create a chimeric mouse. Because many ES cell lines maintain the ability to differentiate into any cell type present in mice, chimeras have germline tissues derived from both normal blastocysts and mutant ES cells. obtain. By mating germline chimeras, animals that are heterozygous for the mutation introduced into ES cells are obtained, and these can be mated to produce homozygous mutant mice.

  As shown in Figure 1, the targeting construct used to replace the endogenous mouse C5aR sequence with the human C5aR sequence is derived from a positive selectable marker (PGK-Neo, a phosphoglycerate kinase I promoter driving the neomycin phosphotransferase gene. Two regions homologous to the target gene located on both sides of the hybrid gene). Homologous recombination proceeds by two crossover events and the target gene sequence is replaced by the replacement construct sequence. Since no sequence duplication occurs, normal genes cannot be regenerated.

  When the targeting construct is linearized, the neo gene is flanked by two regions complementary to the target gene. Selecting cells with an agent (eg, G418) removes the majority of cells that do not stably incorporate the construct.

  Many gene inactivation approaches involving homologous recombination still use constructs that leave a positive selection marker in the genomic DNA, but it has become increasingly clear that this can cause many unexpected effects . For example, the presence of a neo gene, often with its own promoter, can alter the expression of adjacent loci. This can be particularly problematic in gene clusters where adjacent genes belong to the same family. This is because the affected genes may have similar or identical functions. As a result, slight differences in the targeting construct have resulted in significant differences in phenotype.

  For this reason, the targeting constructs exemplified herein include a loxP site adjacent to the PKG-neo gene so that the selectable marker can be removed after targeting by transient expression of Cre recombinase. While this will leave a small loxP site in the genomic DNA, the construct can be engineered so that it is in a harmless position (Figure 2). Theoretically, even the loxP site may change the expression of adjacent genes, but no such case has been reported so far. The efficiency of Cre recombination by transient expression reported in the literature varies greatly from ˜2% to ˜15%. This ratio should be distinguished from the efficiency of Cre recombination in vivo, where Cre expression is derived from sequences integrated in the genome and thus shows long-lasting expression in almost all cases.

  SEQ ID NO: 1 shows the mouse genomic DNA sequence (˜22 kb) containing the C5aR (C5r1) gene. SEQ ID NO: 2 shows the human C5aR cDNA sequence and SEQ ID NO: 3 shows the human C5aR protein sequence.

The mouse genomic region (target locus) shown in SEQ ID NO: 1 is characterized as follows:
Exon 1: Nucleotides 757 to 784 (5 'untranslated region)
Exon 2: nucleotides 1048 to 1152 (5 'untranslated region and start codon)
Exon 3: nucleotides 10726-11778 (all coding sequences except ATG).

FIG. 3 shows a restriction enzyme map of the 22 kb sequence shown in SEQ ID NO: 1. Related restriction sites are as follows.

  The targeting vector used to generate the knock-in mouse contains a region homologous to about 3 kb of genomic DNA on both sides of exon 3 (ie, derived from about nucleotides 7377 to 15045 shown in SEQ ID NO: 1). In particular, the targeting vector contained a region of nucleotides 7377-15045 of SEQ ID NO: 1 except that nucleotides 10726-11778 were replaced with nucleotides 28-1077 of SEQ ID NO: 2. This means that, after integration, endogenous mouse exons 1 and 2 remain in the transgenic mouse, but exon 3 of the mouse locus is replaced with a sequence encoding human C5aR.

Example 2: Transfection and Identification of Mouse Embryonic Stem Cells Containing Human C5aR Gene The basic procedure for culturing embryonic stem (ES) cells and introducing targeting constructs into ES cells is provided below. This procedure also outlines how to identify clones in which the target gene has been modified by homologous recombination. The resulting homologous recombinant is heterozygous for human C5aR and can be used to generate transgenic mice that are homozygous for the human C5aR gene. In this example, ES cells were derived from C57BL / 6 mice.

Material-targeted construct embryonic stem (ES) cells
ES / LIF medium trypsin / EDTA: 0.25% (w / v) trypsin / 1 mM EDTA (20 mM HEPES, pH 7.3, optional)
ES medium electroporation buffer
G418
Freezing medium digestion buffer saturated NaCl
1% agarose

Construct transfection and ES cell selection
1. Incubate ES cells in ES / LIF medium. Cells are passaged every 2-3 days by seeding 1-2 × 10 ⁶ cells / plate in 100-mm gelatin-coated tissue culture plates. Leukemia inhibitory factor (LIF) prevents ES cells from differentiating. If planning blastocyst injection of cells, it may be preferable to pass the cells at high density (eg, 1.5 × 10 ⁶ cells per 25-cm ² flask).

2. Add trypsin / EDTA and incubate for ~ 5 minutes until cells are released from the plate surface and collect ~ 5 x 10 ⁶ to 1 x 10 ⁷ cells. Dissociate into single cells by pipetting 5-10 times. Add 5 ml of ES medium. Cells are pelleted and the cell pellet is resuspended in 1 ml electroporation buffer in the same tube. Typically, 10 ⁷ cells can be obtained from a nearly confluent 100-mm tissue culture plate.

3. Add 1 pmol of linearized sterile construct DNA.

4. Electroporate the mixture at 450 V and 250 uF in a 4-mm electroporation cuvette. Incubate for 10 minutes at room temperature. Many electroporation conditions can be used for ES cells.

5. Plate cells in ES medium at a rate of ˜2 × 10 ⁶ cells per 100-mm gelatin coated tissue culture plate. Incubate for 24 hours.

6. Replace ES medium with ES / LIF medium, add G418 to 0.2 mg / ml and GANC to 2 uM (final) to start selection.

7. Use ES / LIF medium and add G418 (final 0.2 mg / ml) until a single isolated colony is observed (typically 1 week after electroporation). Continue incubation with daily changes. Pick individual colonies from the plate using an autoclaved pipette tip and place in a trypsin / EDTA 35-ul drop for 5 minutes. Dissociate the cells by pipetting about 5 times. Transfer cells to a well of a gelatin-coated 24-well microtiter plate containing 1 ml of ES / LIF medium.

8. Incubate until cells do not differentiate and colonies are observed (typically 3-4 days). Half of the cells are passaged to a new gelatin-coated 24-well microtiter plate. Add remaining cells to 0.5 ml of freezing medium and place at -70 ° C. Freeze overnight and then transfer to liquid nitrogen. Undifferentiated cells grow as smooth round colonies. Differentiated cells are flat with clear intercellular boundaries. When half of the cells are in the freezer, proceed immediately to step 9.

Screening for homologous recombinants
9. Incubate ES cells in 24-well microtiter plate until almost confluent (usually 2-3 days) (step 14). At this stage, it is not important to prevent the differentiation of ES cells, so LIF can be removed from the medium; however, the presence of LIF may help maintain cell growth.

10. Add 300 ul of digestion buffer to each well. Transfer the contents of the wells to a 1.5-ml microcentrifuge tube and incubate overnight at 55 ° C.

11. Add 150 ul saturated NaCl and vortex vigorously (solution turns milky white). Add twice the volume of 95% ethanol (except for precipitated DNA, the solution turns clear). Some researchers precipitate DNA using 2 volumes of ethanol (or 1 volume of isopropanol) without adding salt. However, resuspension of the DNA pellet is easier when salt is added.

12. Resuspend the DNA pellet in 50 ul of water. Measure the absorbance at 260 nm to determine the DMA concentration.

13. Digest 10 ug of DNA (or 10 ul if DNA concentration has not been determined) with appropriate restriction enzymes.

14. Fractionate the digested DNA on a 1% agarose gel. The gene is transferred to a nylon membrane and hybridized with an appropriate target gene hybridization probe by Southern blotting to identify the target gene that has undergone homologous recombination with the unmodified target gene.

15. Shows two hybridizing fragments of approximately the same strength-one fragment that shows the expected size of the unmodified target gene and one fragment that shows the expected size of the target gene that has undergone homologous recombination Select ES cell colonies. If the two fragments are of unequal hybridization intensity, the cell population may not be a clone. Cells are frozen and stored in liquid nitrogen.

  A total of 672 ES cell colonies transfected with the hC5aR targeting construct were screened for homologous recombination between the targeting construct and the mouse genome.

DNA extracted from ES cell colonies was digested with EcoRV or XbaI and electrophoresed on an agarose gel. DNA was transferred to a filter (by Southern method) and hybridized with ³² P-labeled 5 ′ probe or 3 ′ probe DNA using standard conditions. After washing, the filter was exposed to X-ray film. From this screening, three colonies containing the human C5aR gene were obtained. Two of these colonies (5H1 and 6C8) were identified as correctly integrating the human C5aR gene into the mouse C5aR locus (data not shown).

Example 3: ES cell transplantation, chimera generation, and germline transmission of the human C5aR gene
The standard method for producing chimeras using ES cells uses 3.5 day old mouse embryos (blastocysts). The embryo at this stage has a large cavity filled with fluid that can introduce ES cells by injection. The blastocyst already exits the fallopian tube and is found in the uterine horn. When used for injection, blastocysts must be collected before hatching (disappearance of zona pellucida) and attachment to the uterine wall. Various mouse strains are used to generate blastocysts for injecting ES cells. In this example, the mouse strain Balb / c was used to generate blastocysts. This strain gives rise to a suitable number of embryos. This tends to yield excellent chimeras that can be easily distinguished by coat color when used with ES cells derived from various C57BL / 6 substrains.

  ES cells derived from colonies 5H1 and 6C8 were injected into ˜200 blastocysts. Blastocysts were transplanted into pseudopregnant female mice. About 17 hair color chimeras with 5-50% black hair were born. Thirteen male chimeras were bred with C57BL / 6 females to yield approximately 300 pups. Germline transmission of the human C5aR gene was observed in 19 offspring from 5 chimera x C57BL / 6 mating pairs.

  The presence of the human C5aR locus was confirmed by Southern blotting. Genomic DNA extracted from the mouse tail was digested with EcoRV, electrophoresed, and hybridized with the 3 ′ probe. The filter was then removed and reprobed with a Neo probe. Many mice have been shown to contain the C5aR gene. The genotype of these mice was named hC5aRfloxed (neoR) / wt (H5Rf / + for short).

Example 4: Mating and identification of mice homozygous for the human C5aR gene
Generation A H5Rf / + mice were bred with C57BL / 6 Cre-deficient mice to remove the neoR marker gene to produce H5R / wt / Cre mice. H5R / wt / Cre mice were crossed with C57BL / 6 mice to remove the Cre gene, and heterozygous H5R / wt mice (M5R / +) were prepared. H5R / H5R homozygous knock-in mice were obtained by crossing between H5R / + mice. Some H5Rf / + x H5Rf / + mating was also performed to produce H5Rf / H5Rf homozygotes.

  Confirmation that the mouse carries the human C5aR gene was performed by a genotyping assay based on PCR. The Southern blot genotype assay used above to identify mice carrying the human C5aR gene was based on hybridization with a mouse-specific DNA probe (not a human C5aR gene-specific probe). The PCR assay described below utilized primers that specifically amplify the human or mouse C5aR gene sequence.

Tail DNA preparation Place 3 mm tail tip in a 1.5 ml sterile Eppendorf tube and add 200 ul of DNA isolation buffer (67 mM Tris-Cl pH 8.0 (Calbiochem), 16.6 mM ammonium sulfate (Sigma), 6.5 mM magnesium chloride (Promega ), 1% β-mercaptoethanol (ICN Biomedical), and 0.05% Triton X100 (Sigma)) at 65 ° C. overnight. Transfer the supernatant to a new 1.5 ml tube containing 500 ul 100% ethanol (UNIVAR), mix and incubate for 2 hours at -20 ° C, then centrifuge for 15 minutes at 4 ° C and 13,000 rpm (Labofuge 400R, Heraeus Instruments) precipitated purified genomic DNA. The DNA pellet was washed with 70% ethanol, dried and then resuspended in 50 ul distilled water. In the following PCR assays, 1 ul aliquots were used as DNA templates.

いずれもヒトC5aRエキソン3に特異的；ならびに

いずれもマウスC5aR遺伝子に特異的。 PCR to amplify human and mouse C5aR genes
Each PCR reaction consisted of 5 ul of magnesium-free 10x PCR buffer, 5 ul of 25 mM magnesium chloride, 0.5 ul of Taq DNA polymerase (2.5 units / ul), 1 ul of 10 mM dNTP (Promega), each 10 mM oligonucleotide primer. (F1C, R2a, F3C, and R4C) 1 ul, template DNA 1 ul was included. The total volume was made up to 50 ul with distilled water. The oligonucleotide primers used were as follows:

Both specific for human C5aR exon 3; and

All are specific to the mouse C5aR gene.

  The target sequence was then amplified using a PCR System 2700 thermal cycler (Applied Biosystems). PCR started with a denaturation step at 95 ° C. for 5 minutes, followed by 25 cycles of 3 steps: 95 ° C. 30 seconds, 60 ° C. 30 seconds, and 72 ° C. 60 seconds. Final extension step: 72 ° C for 5 minutes, then stored at 4 ° C.

Gel electrophoresis The genotype of each sample was visualized by gel electrophoresis using 10 ul of PCR product and 6x loaded dye (Promega). The gel consisted of 80 ml of 1.5% DNA grade agarose (Progen) and 2 ul of ethidium bromide (MP Biomedicals) dissolved in 1 × TBE. Samples were run with a 1 kb DNA ladder (Promega) at 100 volts for 30 minutes. Homozygous H5Rf / H5Rf mouse DNA has a single 237 bp band, heterozygous H5Rf / + mice have 237 bp and 565 bp bands, and wild type mice have a single 565 bp band. That was expected. Analysis of the gel showed that mice # 10, 24, and 25 were homozygous (H5Rf / H5Rf) (data not shown). In some samples, an inappropriate transcript of 1139 bp was observed (amplification with primers F3C and R2a).

  The relative positions of the PCR primers F1C, R2a, F3C, and R4C used in the genotyping assay at the mouse / human C5aR locus are shown in FIG.

Example 5: Human C5aR is expressed in cells isolated from knock-in mice To confirm that the human C5a receptor is expressed in mice carrying the human C5aR gene, wild type (+ / +), heterozygous Neutrophils were isolated from body (H5Rf / +) and homozygous (H5Rf / H5Rf) blood.

Direct immunofluorescent staining of C5aR in mouse whole blood Approximately 30 ul of peripheral venous blood from homozygous (H5Rf / H5Rf), heterozygous (H5Rf / +), and wild type (+ / +) mice, EDTA (EDTA The final concentration was 5 mM). To detect the human C5aR receptor, 30 ul of blood was stained with 5 ul of FITC (fluorescein isothiocyanate, Sigma) labeled 7F3 (7F3 does not cross-react with mouse C5aR), an antibody specific for human C5aR. Blood and antibody were incubated for 15 minutes at room temperature in 12 x 75 mm tubes. To lyse the red blood cells and fix the cells, 500 ul of BD Lysing Solution (Becton Dickinson) was added to the blood / antibody solution. After 15 minutes, the cells were centrifuged at 1,200 rpm for 3 minutes at room temperature and the cell pellet was resuspended in 1 ml of wash buffer (1 × PBS, 0.1% BSA, 0.02% sodium azide). Cells were analyzed by FACS Calibur flow cytometry (Becton Dickinson) using Cell Quest software (Becton Dickinson). About 5,000 cells were counted for each sample.

  FIG. 5 shows the results of this analysis. Homozygous and heterozygous KI mouse neutrophils bound 7F3, an antibody specific for human C5aR. In contrast, wild type mouse neutrophils were not stained by 7F3. Lymphocytes from mice of either genotype were not stained with mAB 7F3. This is probably because C5aR is not expressed on lymphocytes. Homozygous and heterozygous knock-in mouse monocytes were partially stained with 7F3, indicating that a small percentage of cells expressed human C5aR. Wild type monocytes did not show positive for 7F3 staining.

  In summary, mice carrying the human C5aR gene express human C5aR on the surface of the expected cell type. This indicates that mouse cells can express and process human C5a receptor.

Example 6: Human C5aR knock-in mice can be screened for anti-inflammatory compounds using human C5aR knock-in mice To demonstrate the utility of human C5aR knock-in mice, homozygous (H5Rf / H5Rf) mice are modeled for inflammatory diseases, ie joints Rheumatic K / BxN model was used.

  K / BxN TCR transgenic (tg) mice are directed against self-peptides derived from the ubiquitously expressed glycolytic enzyme glucose-6-phosphate isomerase (GPI) presented by the MHC class II molecule Ag7 It expresses the transgene code (T cell receptor) TCR which shows reactivity. These animals spontaneously develop very severe arthritic conditions from 3-4 weeks of age. Like humans, the disease is chronic, progressive, and symmetric and exhibits most (but not all) the clinical, histological, and immunological features of human RA. Histological features include leukocyte infiltration, synovitis, pannus formation, cartilage and bone destruction. This mouse disease, which is highly dependent on both T and B cells, is joint-specific but develops by T cells that are autoreactive to the ubiquitously expressed antigen, GPI, and then persisted by B cells Turn into.

  Notably, transfer of arthritic K / BxN mouse serum (or affinity purified anti-GPI IgG) to healthy animals induces arthritis within a few days, even when the recipient lacks leukocytes.

Serum transfer procedure, antibody treatment, and arthritis scoring
Sera from 60-day-old arthritic K / BxN mice were pooled and transferred to homozygous (H5Rf / H5Rf) and wild-type (+ / +) mice 6-9 weeks old on days 0 and +2, respectively. Intraperitoneal (ip) injection (total volume 150 ul). On days -1 and +3 respectively, mice were injected ip with sterile antibody (200 ug mouse anti-human C5aR antibody 7F3-isotype IgG2a, or 200 ug isotype IgG2a control antibody).

Arthritis was scored daily for 10-14 days by clinical examination. The severity of arthritis in each affected foot was graded as follows:
0 points: no signs of inflammation
1 point: Slight inflammation (metatarsal phalangeal joints, individual phalanges, or local edema)
2 points: swelling is easily identified but limited to either the dorsal or ventral side of the foot
3 points: swelling of the entire paw The sum of all four paw scores for each mouse was defined as the clinical score (maximum score per animal = 12), which represents the overall disease severity and progression in the animal Is done.

  Ankle thickness was measured daily using a caliper. The thickness of each hind limb ankle was measured twice and the average of the four measurements was calculated. Ankle thickening was defined as the difference from the measured ankle thickness on day 0. Mice were weighed daily.

Histology Basic methods of joint section fixation, decalcification, paraffin section, and hematoxylin / eosin staining, as described (Kouskoff et al., 1996, supra). For immunohistology, cryostat sections that are not fixed and not decalcified are obtained. Briefly, an incised ankle joint without skin is embedded in OCT, frozen in dry ice isopentane, and placed on a -25 ° C. cryomicrotome support. After excising the tissue mass to a desired level, a transparent tape is applied on the section surface of the mass. Cut a sagittal section (6-8 mm thick) at the bottom of the tape, then transfer the tissue to an adhesive-coated slide. Slides are stored at −80 ° C. until use, then fixed with acetone for 30 seconds to 1 minute and air dried for 30 minutes. Counterstain nuclei with 50 ng DAPI (Molecular Probes).

Homozygous human C5aR knock-in mice develop inflammatory disease K / in homozygous human C5aR knock-in mice (C57BL / 6 background) and wild type / control (C57BL / 6) mice to induce inflammatory disease BxN serum was injected. Previous studies have determined the optimal dose of serum required and the expected time course of inflammation in the K / BxN model of rheumatoid arthritis. In this experiment, all mice were injected ip with 2 lots of 150 ul serum collected from arthritic K / BxN mice. Wild-type mice were expected to show signs of inflammation within 4 days of serum injection.

  We predicted that human C5aR knock-in mice will also show signs of inflammation if the hC5aR gene is expressed and the receptor protein is correctly processed and linked to the G protein signaling system. First, the affinity of human C5a for the C5a receptor in mouse and human cells is very similar (Woodruff et al, 2001, Inflammation, v25, p171-177), and the affinity of mouse C5aR for the human C5a receptor The sex is suggested to be similar to its affinity for mouse C5aR. Secondly, studies with C5aR-deficient mice show that the absence of C5aR prevents inflammation in the K / BxN model (Ji et al, 2002, Immunity, v16, p157-168). Thus, if the human C5aR gene is not expressed or the human C5aR receptor does not function correctly, homozygous hC5aR mice will not show signs of inflammation after K / BxN serum injection.

  In this model, the apparent clinical signs of inflammation are edema and redness of the feet, especially the hind limbs. Mice were weighed as described above, serum hind limb ankle thickness, and clinical score determined prior to and daily after serum injection. FIG. 6 shows that ankle thickness and clinical score increased in wild type and homozygous mice injected with K / BxN serum. Disease progression in homozygous hC5aR knock-in mice was similar to control mice, with inflammation evident from day 3 after serum injection. This demonstrates normal expression and processing of the human C5a receptor in knock-in mice.

  Histological examination of the ankle cross section taken from mice 13 days after serum injection in this model reveals leukocyte infiltration into bone erosions and synovial joints. The extent of tissue destruction and leukocyte accumulation was similar in homozygous hC5aR knock-in and control mice (data not shown).

Homozygous human C5aR knock-in mice can be used to test compounds for anti-inflammatory properties Human C5aR knock-in mice were developed as a useful tool for screening anti-human C5aR compounds for anti-inflammatory activity. To test the usefulness of this mouse, in the K / BxN model, both homozygous hC5aR mice and wild type (control) mice have antibodies specific to human C5aR (does not bind to mouse C5aR) or control antibodies. (Same isotype but unrelated specificity) was administered to determine the effect of the antibody on the progression of inflammatory disease. The antibody was injected ip twice (200 ug per dose) the day before and the next day after the first K / BxN serum injection. Mice were monitored as described above.

  FIG. 7 shows clinical disease progression and ankle thickness in mice throughout the course of the experiment. Homozygous hC5aR knock-in mice injected with the control antibody developed other signs of swelling and inflammation. In contrast, hC5aR knock-in mice injected with anti-human C5aR antibody 7F3 did not develop clinical signs of inflammation. Control (wild type) mice treated with 7F3 developed inflammation revealed by increased ankle thickness-7F3 specifically binds to human C5aR and not to mouse C5aR, as expected. It was.

  Furthermore, histological analysis shows that mice injected with anti-human C5aR antibody have no tissue damage when compared to the ankles of mice injected with control antibody-the structure of the synovial joint is a leukocyte with excessive bone erosion and excessive white blood cells It turned out to be normal with no signs of infiltration (data not shown).

  This data clearly demonstrates that human C5aR knock-in mice are useful for testing compounds for anti-inflammatory activity in inflammatory disease models.

  All publications referenced above are hereby incorporated by reference in their entirety.

  It will be appreciated by those skilled in the art that many changes and / or modifications can be made to the present invention as set forth in the specific embodiments without departing from the spirit or scope of the invention as broadly described. Accordingly, the aspects of the invention are to be considered in all respects only as illustrative and not restrictive.

Claims (41)

  A transgenic non-human mammal comprising a polynucleotide encoding human C5aR or humanized C5aR.   The transgenic non-human mammal according to claim 1, wherein the polynucleotide encodes human C5aR comprising the sequence shown in SEQ ID NO: 3 or an allelic variant thereof.   2. The transgenic non-human mammal according to claim 1, wherein the polynucleotide comprises the sequence shown in SEQ ID NO: 2 or an allelic variant thereof.   2. The transgenic non-human mammal of claim 1, wherein the polynucleotide encodes a humanized C5aR.   5. The transgenic non-human mammal of claim 4, wherein the humanized C5aR comprises a C5aR sequence endogenous to the non-human mammal wherein at least one extracellular or intracellular domain is replaced with a corresponding human C5aR domain.   5. The transgenic non-human mammal of claim 4, wherein the humanized C5aR comprises one or more intracellular domains of C5aR sequences endogenous to the non-human mammal and one or more extracellular domains of human C5aR.   The transgenic non-human mammal according to any one of claims 1 to 6, which has somatic cells and germline cells containing a polynucleotide encoding human or humanized C5aR in a stable integrated form.   The transgenic non-human mammal according to any one of claims 1 to 7, which is homozygous for human or humanized C5aR.   The transgenic non-human mammal according to any one of claims 1 to 8, wherein the expression of endogenous C5aR in the transgenic mammal is undetectable or negligible.   10. The transgenic non-human mammal according to claim 9, wherein the endogenous C5aR coding sequence or a fragment thereof is replaced with a corresponding human C5aR coding sequence or a fragment thereof by targeted homologous recombination.   8. The method of any one of claims 1-7, selected from the group consisting of cattle, pigs, goats, sheep, camels, horses, cats, dogs, monkeys, baboons, rabbits, guinea pigs, rats, hamsters, and mice. Transgenic non-human mammal.   The transgenic non-human mammal according to any one of claims 1 to 11, which is a rodent.   The transgenic non-human mammal according to any one of claims 1 to 12, which is a mouse.   14. An isolated cell, cell line, tissue, or organ obtained from a transgenic non-human mammal according to any one of claims 1 to 13, comprising a polynucleotide encoding human C5aR or humanized C5aR.   A method for producing a transgenic non-human mammal for testing for the effect of a compound on a phenotype associated with C5aR signaling, wherein the genome of the non-human mammal is used to produce a transgenic non-human mammal. Introducing a polynucleotide construct encoding a human C5aR, a humanized C5aR, or a fragment of human C5aR.   16. The method of claim 15, wherein the polynucleotide construct encodes human C5aR.   17. The method of claim 16, wherein the polynucleotide construct encodes a polypeptide comprising the sequence shown in SEQ ID NO: 3 or an allelic variant thereof.   17. The method of claim 16, wherein the polynucleotide construct comprises the sequence shown in SEQ ID NO: 2 or an allelic variant thereof.   16. The method of claim 15, wherein the polynucleotide construct encodes humanized C5aR.   16. The method of claim 15, wherein the polynucleotide construct encodes a fragment of human C5aR.   21. The method of claim 20, wherein the fragment comprises at least one domain of human C5aR or a portion thereof.   24. The method of claim 21, wherein the fragment comprises at least one extracellular domain of human C5aR.   23. The method of any one of claims 15-22, wherein the polynucleotide construct further comprises a selectable marker.   24. The method of claim 23, wherein the selectable marker is a PGK-neo gene.   25. The method of claim 23 or claim 24, wherein the selectable marker is flanked by loxP sites.   21. The method of any one of claims 15-20, further comprising destroying endogenous C5aR in the non-human mammal.   27. A method according to any one of claims 15 to 26, comprising replacing the endogenous C5aR coding sequence or fragment thereof with the corresponding human C5aR coding sequence or fragment thereof by targeted homologous recombination.   A method for assessing at least one pharmacokinetic and / or pharmacodynamic effect of a candidate compound, the transgenic mammal according to any one of claims 1 to 13, or an isolated obtained therefrom. Administering a candidate compound to a tissue or cell and testing at least one pharmacokinetic and / or pharmacodynamic effect of the candidate compound in a transgenic mammal.   30. The method of claim 28, wherein the at least one pharmacokinetic effect tested is an absorption parameter, a distribution parameter, a metabolic parameter, or an excretion parameter.   At least one pharmacokinetic effect tested is distribution volume, total clearance, protein binding, tissue binding, metabolic clearance, renal clearance, liver clearance, bile clearance, intestinal absorption, bioavailability, relative biology Availability, intrinsic clearance, mean residence time, maximum metabolic rate, Michaelis-Menten constant, tissue-to-blood or tissue-to-plasma partition coefficient, unchanged urinary excretion rate, systemic conversion rate of drug to metabolite, excretion 30. The method of claim 28 or claim 29, wherein the method is a rate constant, half-life, or secretory clearance.   29. The method of claim 28, wherein the at least one pharmacodynamic effect is a phenotypic modulation associated with C5aR signaling.   A transgenic mammal according to any one of claims 1 to 13 or an isolated tissue or cell derived therefrom under conditions in which (i) at least one phenotype associated with C5aR signaling is expressed 32. The method of claim 31, comprising: administering a candidate compound to; and (ii) monitoring the development of at least one phenotype after administration of the compound.   (iii) further comprising the step of comparing the degree of phenotype in the transgenic mammal administered the compound or cells derived therefrom with the control mammal or cells derived therefrom, wherein the phenotype of the phenotype as compared to the control mammal is 35. The method of claim 32, wherein any difference in nature or degree indicates that the compound modulates C5aR activity.   (i) administering a candidate compound to a transgenic mammal of the present invention or an isolated tissue or cell obtained therefrom under conditions such that at least one phenotype associated with C5aR signaling is expressed; ii) monitoring the development of at least one phenotype after administration of the compound; and optionally (iii) comparing the degree of phenotype in the transgenic mammal administered the compound with a control mammal, 32. The method of claim 31, wherein any reduction or inhibition of phenotypic nature or extent when compared to an animal indicates that the compound is likely to inhibit or reduce C5aR activity.   (i) administering a candidate compound to a transgenic mammal of the present invention or an isolated tissue or cell obtained therefrom under conditions such that at least one phenotype associated with C5aR signaling is expressed; ii) monitoring the development of the phenotype after administration of the compound; and optionally (iii) comparing the degree of phenotype in the transgenic mammal administered the compound with the control mammal and comparing with the control mammal 32. The method of claim 31, wherein any enhancement of phenotypic nature or extent when indicated indicates that the compound is likely to promote or enhance C5aR activity.   36. The method according to any one of claims 31 to 35, wherein the phenotype is a pathological condition worsened by C5aR signaling.   38. The method of claim 36, wherein the phenotype is immune complex disorder, inflammatory or allergic disease, graft rejection, or cancer.   36. The method of any one of claims 31 to 35, wherein the phenotype is a pathological condition that is alleviated or ameliorated by increased C5aR signaling.   40. The method of claim 38, wherein the phenotype is immunosuppression.   Peptides wherein the compound comprises: C5aR or a peptide derived from C5a or other non-C5aR peptide, C5aR dominant negative variant; which may inhibit, reduce or suppress C5aR function; non-peptide inhibitor of C5aR An antibody or antibody fragment that binds to C5aR and inhibits C5aR function; a small organic molecule, a nucleic acid encoding the peptide derived from C5aR or C5a or other non-C5aR peptide inhibitor, an antisense nucleic acid against C5aR-encoding mRNA, 40. The method according to any one of claims 28 to 39, selected from the group consisting of C5aR ribozyme and small interfering RNA (RNAi) targeting C5aR gene expression.   The nucleic acid encodes a peptide derived from C5aR or other non-C5aR peptide inhibitor, an antisense nucleic acid against C5aR-encoding mRNA, an anti-C5aR ribozyme, or a small interfering RNA (RNAi) that targets C5aR gene expression. 40. The nucleic acid according to 40.

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