Engineered antibody for inhibition of fibrosis

Abstract

A chimeric, humanized or single-chain antibody contains a light chain variable region containing the complementarity determining regions of SEQ ID NO: 1, SEQ ID NO:2 and SEQ ID NO:3, and a heavy chain variable region containing the complementarity determining regions SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. The antibody or antibody fragment thereof is capable of binding the C-terminal telopeptide of the α2(I) chain of human collagen I, and is useful in the treatment of diseases or disorders associated with excessive collagen fibril.

Description

REFERENCE TO GOVERNMENT GRANT

The benefit of the filing date of U.S. Provisional Patent Application No. 61/636,073, filed Apr. 20, 2012, is hereby claimed. The entire disclosures of the aforesaid application are incorporated herein by reference.

REFERENCE TO GOVERNMENT GRANT

The invention was made with government support under grant 5RO1 AR048544-05 and IR21AR061118-01 awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on The complementarity determining regions are double-underlined.heavy light chain variable region comprising the three complementarity determining regions having the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and a light heavy chain variable region comprising the three complementarity determining regions having the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. The engineered antibodies are capable of binding the C-terminal telopeptide of the α2(I) chain of human collagen I.

In one embodiment, the engineered antibody is a chimeric antibody comprising the mouse light chain variable region (mV_L) having the amino acid sequence of SEQ ID NO:10, and the mouse heavy chain variable region (mV_H) having the amino acid sequence of SEQ ID NO:11. The mouse light chain variable region and mouse heavy chain variable regions, with predicted signal peptides, are shown in FIGS. 1 and 2, respectively. The mV_L and mV_H complementarity determining regions are shown by double underlining in FIG. 1 and FIG. 2. The first fifteen amino acids in FIG. 1 (indicated by single underlining) comprise the signal sequence for the mV_L. The first nineteen amino acids in FIG. 2 (indicated by single underlining) comprise the signal sequence for the mV_H.

Using the sequences encoding the above light and heavy chain variable regions, chimeric antibodies may be produced by any of the well-known techniques for production of chimeric antibodies (Morrison et al., 1984, Proc. Natl. Acad. Sci., 81:6851-5; Neuberger et al., 1984, Nature, 312:604-8; Takeda et al., 1985, Nature, 314:452-4). Accordingly, a DNA molecule encoding the light chain variable region SEQ ID NO:10, e.g., the DNA molecule having the nucleotide sequence of SEQ ID NO:22, is prepared. The DNA molecule may further encode a signal sequence for the light chain variable region, such as the fifteen amino acid signal sequence shown in FIG. 1 by single underlining. SEQ ID NO:7 is the amino acid sequence of the polypeptide comprising the signal sequence and light chain variable region shown in FIG. 1. SEQ ID NO:7 may be encoded by, for example, the nucleotide sequence of SEQ ID NO:24.

A DNA molecule encoding the heavy chain variable region SEQ ID NO:11, e.g., the DNA molecule having the nucleotide sequence of SEQ ID NO:23, is prepared. The DNA molecule may further encode a signal sequence for the heavy chain variable region, such as the nineteen amino acid signal sequence shown in FIG. 2 by single underlining. SEQ ID NO:8 is the amino acid sequence of the polypeptide comprising the signal sequence and heavy chain variable region shown in FIG. 2. SEQ ID NO:8 may be encoded by, for example, the nucleotide sequence of SEQ ID NO:25.

The DNA molecules encoding the light and heavy chain variable regions are then ligated into vector DNA and expressed using, for example, any of the available ligation kits to construct a recombinant vector. See e.g., J. Sambrook et, Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989.

In one embodiment, a chimeric antibody may be prepared by ligating a mouse leader sequence and a variable region sequence present in a cloned mouse cDNA to a sequence coding for a human antibody constant region already present in an expression vector of a mammalian cell. Alternatively, a mouse leader sequence and a variable region sequence present in a cloned cDNA are ligated to a sequence coding for a human antibody constant region followed by ligation to a mammalian cell expression vector. The mouse leader sequence may comprise, for example, the signal sequences shown in FIG. 1 or FIG. 2.

The polypeptide comprising human antibody constant region can comprise any of the heavy or light chain constant regions of human antibodies, including, for example, γ1, γ2, γ3 or γ4 for heavy chains, and κ for light chains.

To prepare a chimeric antibody, two expression vectors are constructed. A first expression vector contains DNAs coding for the light chain variable region and human light chain constant region under the control of an expression control element such as an enhancer/promoter system. A second expression vector contains DNAs coding for the heavy chain variable region and human heavy chain constant region under the control of an expression control element such as an enhancer/promoter system. Host cells such as mammalian cells (for example, COS cell) are cotransformed with these expression vectors. The transformed cells are cultivated in vitro or in vivo to produce a chimeric antibody, See, e.g. WO91/16928.

As an alternative, mouse leader sequence present in cloned cDNA and DNAs coding for mouse light chain variable region and human light chain constant regions, as well as a mouse leader sequence and DNAs coding for mouse heavy chain variable region and human heavy chain constant region, are introduced into a single expression vector (see, for example, WO94/11523). The single vector is used to transform a host cell. The transformed host is cultured in vivo or in vitro to produce a desired chimeric antibody.

The vector for the expression of the heavy chain of the chimeric antibody can be obtained by introducing cDNA comprising a nucleotide sequence coding for the mouse heavy chain variable region into a suitable expression vector containing genomic DNA comprising a nucleotide sequence coding for the heavy chain constant region of human antibody, or cDNA coding for the heavy chain constant region. As indicated above, the heavy chain constant region may comprise, for example, γ1, γ2, γ3 or γ4.

Expression vectors comprising genomic DNA coding for a heavy chain constant region include, for example, HEF-PMh-g gamma 1 (WO92/19759) and DHER-INCREMENT E-RVh-PM1-f (WO92/19759). Alternatively, a human constant region library can be prepared using cDNA from human peripheral blood mononuclear cells, as described by Liu. et al., Proc. Natl. Acad. Sci. USA, 84:3439-43 (1987) or Reff et al., Blood 83(2): 435-45 (1994), for example.

The cDNA coding for the mouse heavy chain variable region of SEQ ID NO:11 (e.g., the nucleotide sequence SEQ ID NO:23, which does not contain a signal sequence segment; or the nucleotide sequence of SEQ ID NO:25, which contains a signal sequence) is treated with suitable restriction enzyme(s) and inserted into genomic DNA coding for a heavy chain constant region, to construct a chimeric heavy chain expression vector containing the genome DNA coding for the heavy chain constant region. Insertion is by ligation of the cDNA encoding the heavy chain constant region, and insertion into an expression vector such as pQCXIH (Clontech), to construct an expression vector containing the complete cDNA encoding the complete chimeric heavy chain. Alternatively, the cDNA encoding the mouse heavy chain variable region may be ligated into an appropriate commercially available cloning plasmid that expresses the constant region of a human heavy chain, e.g., pFUSE-CHIg-hG1 (InvivoGen, San Diego, Calif.).

In a similar fashion, a vector for the expression of the light chain of the chimeric antibody can be constructed by ligating a cDNA coding for the mouse light chain variable region (e.g., the nucleotide sequence SEQ ID NO:22, which does not contain a signal sequence; or the nucleotide sequence of SEQ ID NO:24, which contains a signal sequence) and a genomic DNA or cDNA coding for the light chain constant region of a human antibody, and introduction into a suitable expression vector. The light chain constant region includes, for example, κ or λ, chains. Any of the four known λ chain constant region isotypes may be utilized. Alternatively, the cDNA encoding the mouse light chain variable region may be ligated into an appropriate commercially available cloning plasmid that expresses the constant region of a human κ light chain, e.g., pFUSE2-CLIg-hκ (InvivoGen, San Diego, Calif.).

FIG. 11 shows the DNA (SEQ ID NO:20) and amino acid (SEQ ID NO:8) sequences of a construct encoding a mouse heavy chain variable region (mV_H) containing a leader signal peptide, for the preparation of a further construct for expressing a chimeric antibody heavy chain consisting of the mV_H and the constant region of a human γ chain (mV_H-hγ). FIG. 12 is a schematic of a plasmid for expression of the mV_H-hγ, utilizing the mV_H construct of FIG. 11. The mV_H construct was cloned into the pFUSE-CHIg-hG1 plasmid as shown in FIG. 12. Restriction sites utilized in the cloning strategy are indicated.

FIG. 13 shows the DNA (SEQ ID NO:21) and amino acid (SEQ ID NO:7) sequences of a construct encoding a mouse light chain variable region (mV_L) containing a leader signal peptide, for the preparation of a plasmid for expressing a chimeric antibody heavy chain consisting of the mV_L and the constant region of a human κ chain (mV_L-hκ). FIG. 14 is a schematic of a plasmid for expression of the chimeric mV_L-hκ, utilizing the mV_L construct of FIG. 13. The mV_L construct was cloned into the pFUSE2-CLIg-hκ plasmid as shown in FIG. 14. Restriction sites utilized in the cloning strategy are indicated.

In another embodiment, the engineered antibody is a humanized antibody. The humanized antibody comprises a human antibody framework region and advantageously further comprises a human antibody constant region. The six complementarity determining regions (CDRs) SEQ ID NOS:1-6 are grafted to a human antibody. The general genetic recombination procedure for producing humanized antibodies are described, for example, in EP 125023 and WO 96/02576. Accordingly, a DNA sequence is designed in which DNA encoding the aforementioned CDRs are ligated through framework regions. The DNA sequence is synthesized by a polymerase chain reaction method using oligonucleotide primers which are designed to have regions overlapping the terminal regions of the CDRs and the framework regions. The resultant DNA is ligated to DNA encoding the human antibody constant region, and the ligation product is integrated into an expression vector. The resultant recombinant expression vector is introduced into a host, thereby producing the humanized antibody. See, e.g., WO 96/02576.

The framework regions ligated through the CDRs are selected so that the CDRs can form a functional antigen binding site. If necessary, an amino acid(s) in the framework regions of the antibody variable region may be replaced so that the CDRs of the resulting humanized antibody can form an appropriate antigen binding site. See Sato et al., Cancer Res. 53:851-6 (1993).

The amino acid sequences of the framework regions are preferably selected to reflect a high homology to the framework sequences of a human antibody. In this regard, a comparison may be undertaken between the variable regions SEQ ID NO:10 and SEQ ID NO:11 and the variable regions of structurally elucidated human antibodies using, e.g., the Protein Data Bank. Other research tools that may consulted include the Kabat Database of Sequences of Proteins of Immunological Interest, www<<dot>>kabatdatabase<<dot>>com and the search tools included as part of that database; and Kabat, E. A. et al., (1991) Sequences of Proteins of Immunological Interest, 5^th edition. U.S. Department of Health and Human Services.

A humanized antibody variable region is selected as a basis for the humanized antibody variable region. For example, the framework region of a human antibody variable region having a high homology, e.g., greater than about 80%, or greater than about 90%, or greater than about 95%, with the framework regions of SEQ ID NOS:10 and 11 (the non-CDR regions of SEQ ID NOS:10 and 11) is selected. A polypeptide comprising the framework regions of the humanized antibody and the CDRs of SEQ ID NOs:1-6 can be produced by “CDR-grafting”, a PCR-based method utilizing a DNA fragment of a human antibody as a template. For an example of CDR grafting, see Kettleborough et al., Protein Eng. 4(7):773-783 (1991); “Antibody Humanization by CDR Grafting”, Methods of Molecular Biology, 248(11):135-159 (2004).

Humanized antibodies may be prepared, as exemplified in Jones et al., 1986 Nature 321:522-525, which describes replacing the complementarity-determining regions in a human antibody with those from a mouse. Also see Riechmann, 1988, Nature 332:323-327; Queen et al., 1989, Proc. Nat. Acad. Sci. USA 86:10029 (preparation of humanized antibody binding the interleukin 2 receptor); and Orlandi et al., 1989, Proc. Natl. Acad. Sci. USA 86:3833 (describing the cloning of immunoglobulin variable domains for expression by the polymerase chain reaction).

Any suitable expression system may be used to produce the chimeric or humanized antibody. For example, eukaryotic cells include animal cells such as established mammalian cell lines, fungal cells, and yeast cells; prokaryotic cells include bacterial cells such as Escherichia coli. Mammalian host cells are preferred. The expression system may incorporate conventional promoters useful for the expression in mammalian cells, e.g., the human cytomegalovirus (HCMV) immediate early promoter. Promoters for expression in mammalian cells may include virus promoters, such as those of retrovirus, polyoma virus, adenovirus and simian virus (SV) 40, and mammalian cell derived promoters, such as those of human polypeptide chain elongation factor-1 alpha (HEF-1 alpha).

The expression system may include a replication origin such as those derived from SV40, polyoma virus, adenovirus or bovine papilloma virus (BPV). The expression vector may comprise a gene for phosphotransferase APH(3′) II or I (neo), thymidine kinase (TK), E. coli xanthine-guanine phosphoribosyltransferase (Ecogpt) or dihydrofolate reductase (DHFR) as a selective marker for increasing the gene copy number in a host cell system.

The expressed chimeric or humanized antibody is produced by culturing the thus-transformed host cells, and isolating and purifying the antibody from the cells according to well-known techniques. The concentration of the resulting purified antibody can be determined by, for example, enzyme-linked immunosorbent assay (ELISA). Antigen-binding activity can be confirmed by known methods antibody, techniques such as ELISA, enzyme immunoassay, radioimmunoassay or fluorescent assay.

Fragments of the chimeric or humanized antibody retaining antigen-binding activity may be prepared, e.g., Fab, F(ab′)₂, and Fv fragments. Antibody fragments can be produced by cleaving the antibody with an enzyme (e.g., papain, pepsin) into antibody fragments, or by constructing a gene encoding the antibody fragment and inserting the gene into an expression vector and introducing the resultant recombinant expression vector into a suitable host cell, thereby expressing the antibody fragment (see, for example, Co et al., J. Immunol. 152:2968-76 (1994)).

In another embodiment, the engineered antibody is a single-chain antibody (SCA), also referred to herein as a single-chain variable fragment (scFv), comprising a light chain variable region comprising the complementarity determining regions (CDRs) having the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and a heavy chain variable region comprising the complementarity determining regions having the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. The scFv can be produced by ligating a heavy chain variable region comprising the CDRs of SEQ ID NOS:4, 5 and 6 to a light chain variable region comprising the CDRs of SEQ ID NOS:1, 2 and 3 through a linker. The linker is preferably a peptide linker, i.e., the linker is composed of amino acid residues. The residues for the linker may be selected from naturally occurring amino acids, non-naturally occurring amino acids, and modified amino acids. The linker will typically connect the carboxy terminus of the heavy chain variable region to the amino terminus of said light chain variable region. The reverse is also possible, i.e., using the linker to connect the carboxy terminus of the light chain variable region to the amino terminus of the heavy chain variable region. The linker may comprise any number of amino acids. The linker may thus comprise, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or more amino acids. In some embodiments, the linker may be composed of from 3 to 60 amino acid residues, from 3 to 40 amino acids, from 3 to 30 amino acids, from 3 to 24 amino acids, from 3 to 18 amino acids, or from 3 to 15 amino acids. The linker may comprise, for example, a repeating sub-sequence of 2, 3, 4, 5 or more amino acid residues, comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more repeats of the sub-sequence.

In one embodiment, the linker comprises the amino acid sequence Gly-Ser, or repeats thereof. See, e.g., Huston, et al., Methods in Enzymology, 203:46-88 (1991). In another embodiment, the linker comprises the amino acid sequence Glu-Lys, or repeats thereof. See, e.g., Whitlow et al., Protein Eng., 6:989 (1993)). In another embodiment, the linker comprises the amino acid sequence Gly-Gly-Ser, or repeats thereof. In another embodiment, the linker comprises the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:29), or repeats thereof. In certain specific embodiments, the linker contains from 2 to 12 repeats of Gly-Gly-Ser or Gly-Gly-Gly-Gly-Ser (SEQ ID NO:29).

An scFv of the invention having the amino acid sequence SEQ ID NO:14 is illustrated in FIG. 3. The scFv comprises from amino terminus to carboxy terminus, (i) an initial methionine residue, (ii) the mV_L sequence SEQ ID NO:9, which comprises the parental mV_L of SEQ ID NO:10 without the terminal Arg residue of SEQ ID NO:10, (iii) Val-Asp, (iv) a linker, (v) Leu-Glu, (vi) the mV_H sequence SEQ ID NO.11, and (vii) a FLAG-Tag for affinity chromatography purification.

The antibody or antibody fragments of the invention may be utilized to bind to the C-terminal telopeptides of the α2-chain of collagen to inhibit the formation of collagen fibrils, and thereby prevent excessive deposition of collagen fibrils that is characteristic of fibrotic processes. The antibody or antibody fragments of the invention may thus be utilized to reduce localized and systemic fibrotic lesions, including but not limited to fibroses occurring in internal organs, the dermus or the eye.

Types of fibrosis that may be treated or prevented include, for example, pulmonary fibrosis, idiopathic pulmonary fibrosis, cirrhosis, endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, Crohn's Disease, keloid formation, myocardial infarction, scleroderma/systemic sclerosis, arthrofibrosis, and adhesive capsulitis. The fibrotic lesion treated may be triggered by trauma, accidental injury or surgical procedures, among other causes. The antibody or antibody fragments of the invention may be administered, for example, after surgery in the abdomen to avoid the formation of excessive scar tissue around abdominal organs; after plastic surgery to the face to reduce scar formation; to the eye following glaucoma surgery performed to maintain a lamellar channel from the subconjunctival space to the anterior chamber, to prevent excessive scar formation that may function to closes the pressure-reducing channel and cause intraocular pressure to rise; and following implantation of medical devices and materials implanted in the human body, which would otherwise trigger a fibrotic response. In one embodiment, the antibody or antibody fragments are administered to teat or prevent the formation of keloids,

Pharmaceutical compositions for use in accordance with the present invention can be formulated in conventional manner using one or more pharmaceutically acceptable carriers or excipients. The antibodies or fragments thereof can be formulated for administration in accordance with the route of administration. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the antibody can be in lyophilized powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

In some embodiments, the subject of treatment is human. In other embodiments, the subject is a veterinary subject.

Treatment may involve administration of one or more antibodies or antibody fragments of the invention, alone or with a pharmaceutically acceptable carrier. The active agent may be administered in combination with one or more other therapeutic, diagnostic or prophylactic agents. The administered composition may thus further comprise an additional agent selected from the group consisting of corticosteroids, antiinflammatories, immunosuppressants, antimetabolites, and immunomodulators, for example.

The pharmaceutically acceptable carrier may comprise any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption enhancing or delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, acetate buffer with sodium chloride, dextrose, glycerol, polyethylene glycol, ethanol and the like, as well as combinations thereof. In some cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are surfactants, wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody.

The compositions used in the practice of the invention may be in a variety of forms, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions, dispersions or suspensions, tablets, pills, lyophilized cake, dry powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. In some embodiments, the antibody or antibody fragment may be administered by using a pump, enema, suppository, or indwelling reservoir or such like.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, lyophilized cake, dry powder, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile solutions can be prepared by incorporating the antibody or fragment in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization. In the case of sterile powders for the preparation of sterile solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile solution thereof. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. The desired characteristics of a solution can be maintained, for example, by the use of surfactants and the required particle size in the case of dispersion by the use of surfactants, phospholipids and polymers. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts, polymeric materials, oils and gelatin.

In certain embodiments, the antibody or antibody fragment compositions of the invention may be prepared with a carrier that will protect the antibody against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems (J. R. Robinson, ed., Marcel Dekker, Inc., New York (1978)).

The compositions of the invention may be administered parenterally. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

The compositions of the invention may be given orally, in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch.

The compositions of the invention may be administered topically. For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Topically-transdermal patches may also be used. For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Topical or other localized administration is advantageously utilized at the site of a localized fibrosis, e.g., by localized injection. The location of the fibrosis may comprise, for example, a wound, particularly a wound edge.

The dosage of active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. Detection and measurement of indicators of efficacy may be measured by a number of available diagnostic tools, including, for example, by physical examination including blood tests, pulmonary function tests, and chest X-rays; CT scan; bronchoscopy; bronchoalveolar lavage; lung biopsy and CT scan. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount.

Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a pre-determined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

As a non-limiting example, an effective amount of an antibody or antibody fragment active agent is from about 0.025 to about 50 mg/kg, or from about 0.1 to about 50 mg/kg, or from about 0.1-25 mg/kg, or from about 0.1 to about 10 mg/kg, or from about 0.1 to about 3 mg/kg. Dosage may vary with the type and severity of the condition to be alleviated. For any particular treatment subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

The practice of the invention is illustrated by the following non-limiting example. The invention should not be construed to be limited solely to the compositions and methods described herein, but should be construed to include other compositions and methods as well. One of skill in the art will know that other compositions and methods are available to perform the procedures described herein.

EXAMPLES

Example 1: Single-Chain Antibody by Bacterial Expression

A construct for preparing a single chain antibody, also referred to herein as a single-chain variable fragment (scFv), was prepared containing DNA sequences encoding the light chain variable region of SEQ ID NO:7 and the heavy chain variable region of SEQ ID NO:8. The DNA sequences were obtained by PCR from a hybridoma expressing an IgA class antibody to collagen α2-chain (Chung et al., J Biol. Chem. 283(38):25879-25886 (2008)). Total RNA was prepared from hybridoma cells with the use of an RNA-isolation kit according to the manufacturer's protocol (QIAGEN). PCR products spanning the V_H of the α and the V_L of the κ chains were cloned into the pETBlue-1 Blunt vector and sequenced.

A. Bacterial Expression System

For the bacterial expression of the scFv, a construct was created in which the V_L region was connected with the V_H region via a 15 amino acid linker comprising three repeats of the Gly-Gly-Gly-Gly-Ser (SEQ ID NO:29) motif. A DNA sequence encoding a FLAG tag was fused to the 3′ end of the construct to facilitate downstream purification of the recombinant scFv (FIG. 3). Minor additions to the original amino acid sequences of the V_L (SEQ ID NO:10) and V_H (SEQ ID NO:11) regions were created as a consequence of the cloning process (FIG. 3). Moreover, the native signal peptides shown in FIGS. 1 and 2 (single underlining) were omitted in the scFv construct. The constructs were thus arranged to provide a scFv comprising, from N-terminus to C-terminus, light chain variable region-linker-heavy chain variable region, where the linker connected the N-terminus of the heavy chain to the C-terminus of the light chain. More specifically, the scFv comprised, from amino terminus to carboxy terminus, (i) an initial methionine residue, (ii) the mV_L sequence SEQ ID NO:9, which comprises the parental mV_L of SEQ ID NO:10 without the terminal Arg residue of SEQ ID NO:10, (iii) Val-Asp, (iv) a linker, (v) Leu-Glu, (vi) the mV_H sequence SEQ ID NO.11, and (vii) a FLAG-Tag. The fidelity of the construct was confirmed by DNA sequencing.

The scFv DNA construct was initially cloned into the NcoI/SacI site of the pET-28 (not shown), a vector that does not include any signaling sequences for the secretion of exogenous proteins into the periplasm (EMD Biosciences). Because the expression of exogenous proteins in bacteria is frequently associated with the formation of insoluble aggregates packed into the inclusion bodies, the pET-22 vector was also employed (FIG. 4) (EMD Biosciences). In contrast to the pET-28 vector, the pET-22 vector carries an N-terminal pelB signal sequence for potential periplasmic localization of the expressed protein, a situation in which the produced proteins are potentially more soluble. The two above vectors were tested for their ability to facilitate the production of the scFv variant.

Bacteria expressing the scFv cloned into the pET-22 and pET-28 vectors were cultured in the presence of ampicillin or kanamycin, respectively. After reaching the optical density (OD₆₀₀) of 0.5 units, bacterial cultures were treated with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) to induce the expression of the scFv. Subsequently, the bacterial cultures were incubated for an additional 3 h at 37° C.

Bacteria were collected by centrifugation and then lysed with the B-PER II Protein Extraction Reagent (Thermo Scientific) in the presence of lysozyme, and then the scFv-rich insoluble inclusion bodies were collected by centrifugation. Subsequently, the inclusion bodies were solubilized in 6M guanidinium hydrochloride (GdnHCl) in the presence of the reducing agent dithiothreitol (DTT). Refolding conditions were tested by running pilot-scale refolding assays with the use of the Protein Refolding Kit (Thermo Scientific). As a result of these assays, the denatured scFv molecules present in the GdnHCl-solubilized material were refolded by diluting them into 100 volumes of a refolding buffer consisting of 55 mM Tris, 21 mM NaCl, 0.88 mM KCL, pH 8.2. Subsequently, the sample was concentrated 100-fold by ultrafiltration.

B. scFv Purification and Analyses

Pilot purification of the scFv was carried out with the agarose-conjugated anti-FLAG antibody according to the manufacturer's protocol (Sigma-Aldrich). The purity of purified soluble scFv was analyzed by polyacrylamide gel electrophoresis and by Western blot assay with the anti-FLAG antibody (Sigma-Aldrich) run in denaturing and reducing conditions. The result is shown in FIG. 5A. The electrophoretic migration of the purified scFv indicated the predicted mass.

In addition, due to the relatively high cost of the anti-FLAG antibody, an ion exchange chromatography on the MonoQ Sepharose resin (GE Healthcare Life Sciences) was employed to purify the semi-preparative amounts of the scFv variant. In brief, a sample containing the refolded scFv was loaded onto a MonoQ Sepharose column, and then bound proteins were eluted with a 0-1 M NaCl gradient. The collected peaks were then checked for the presence of the scFv variant by gel electrophoresis assays and Western blot assays with the use of the anti-FLAG antibody (Sigma-Aldrich) (FIG. 5B) in denaturing and reducing conditions. The fraction including the scFv was eluted with 200 mM NaCl, while contaminating proteins were eluted with 500 mM NaCl (FIG. 5B).

The molecular mass of the purified native scFv was analyzed by high pressure liquid size exclusion chromatography (SEC HPLC) run in non-denaturing conditions. The chromatography profile is shown in FIG. 6. The three main peaks in FIG. 6 indicate the single chain antibody monomers and oligomers.

The binding specificity of the scFv for its intended epitope, the α2-chain of collagen, was confirmed by a Western blot-based assay as follows. Purified human procollagen I was electrophoresed in a polyacrylamide gel. Subsequently, procollagen chains were transferred to a nitrocellulose membrane. Next, the membranes were incubated with FLAG-tagged single chain antibody. The bound single chain antibody was detected with chemiluminescence by an anti-FLAG antibody conjugated to horseradish peroxidase. The results are shown in FIG. 7. The lines in FIG. 7 depict different repeats of the assay, and were identified as procollagen I α2 chains, thereby indicating specific binding of the scFv to collagen α2-chain.

Example 2: Single Chain Antibody Inhibition of Collagen Fibril Deposition

Assays of the inhibition of collagen fibril deposition by the scFv of Example 1 were done in cultures of keloid-derived fibroblasts. In brief, the fibroblasts were seeded into the wells of a 96-well plate at the density of 4×10³ cells/well. Six hours after seeding, the attached cell layers were washed and fresh media supplemented with 40 μg/ml of L-ascorbic acid phosphate magnesium salt n-hydrate (Wako Pure Chemical Co.) was added to the cells. Three experimental groups of cells were analysed: (I) non treated, (II) treated with tissue growth factor β (TGF-β) and the scFv, and (III) treated only with TGF-β. TGF-β was added at a concentration of 1 ng/ml, while scFV was added at 1, 5 or 10 μg/ml. The purpose of adding TGF-β, an activator of collagen biosynthesis, was to stimulate cells to produce and secrete high amounts of collagen. After the 48-h culture in the presence or the absence of TGF-β and scFv, layers consisting of collagen-rich extracellular matrix and cells were solubilized by adding a lysis buffer and analyzed by Western blot assays for the presence of collagen I, a main constituent of collagen fibrils. The results shown in FIG. 8 indicate a marked decrease of collagen deposits in a group treated with the scFv. An equal number of cells in the analyzed groups is indicated by comparable amounts of GAPDH. The analyzed groups and the collagen I marker (Cm) are indicated.

Example 3: Constructs for Single-Chain Antibody Production by Yeast Expression

Constructs for the expression of the scFv of Example 1 in yeasts were also engineered. The rationale for employing yeasts to produce the scFv variants was dictated by the fact that those organisms are eukaryotes, a characteristic that potentially may improve the folding and solubility of the recombinant scFv variants.

Accordingly, the following scFv variants were designed: (i) A_L_K, a construct in which a cassette for the V_H of the mouse heavy α chain was linked with a cassette for the V_L of the mouse light κ chain; and (ii) K_L_A, a construct in which a cassette for the V_L of the light κ chain was linked with a cassette for the V_H of the heavy α chain. In comparison to the 15-amino acid linker designed for the bacterial scFv construct (FIG. 3), the scFv constructs for yeast expression consisted of a 30-amino acid linker. See FIGS. 9A-9C. By increasing the length of the linker, the predicted solubility of the A_L_K and K_L_A scFv variants will be higher than that with a shorter linker. Moreover, the increased length of the linker may decrease the potential of scFv variants to form oligomers. If necessary, that the length of the linker may be readily changed by DNA engineering technology.

The nucleotide (SEQ ID NOS:19 and 30) and amino acid (SEQ ID NO:18) sequences of the A_L_K construct are shown in FIGS. 9A-9C. A schematic of the same construct cloned into the pPIC-9K plasmid is shown in FIG. 10. In both figures, restrictions sites utilized in the cloning strategy are indicated—SnaBI and NotI restriction sites were incorporated at the 5′ and 3′ ends of the construct, respectively, to facilitate its downstream cloning into the pPIC-9K yeast-expression vector (Invitrogen). A construct for the K_L_A scFv was prepared in a similar way (not shown). The entire DNA constructs for the A_L_K and K_L_A variants were synthesized commercially (Blue Heron Biotechnology, Bothell, Wash. 98021 USA). In both constructs, a His-tag-coding sequence was incorporated to facilitate the downstream purification process (FIGS. 9A-9C). The fidelity of constructs was confirmed by sequencing. The nucleotide and amino acid sequences of the V_H-linker-V_L portion of the A_L_K construct (without added restriction sites and His-Tag), comprise SEQ ID NO:17 (nucleotide) and SEQ ID NO:16 (amino acid).

Example 4: Preparation of Chimeric Antibody by Mammalian Cell Expression

Constructs for the mammalian-cell expression of full-length mouse/human chimeric IgG were created. The constructs consist of sequences encoding a mouse variable region fused to sequences encoding constant regions of human to γ and κ chains. Two such DNA constructs were prepared as follows for the expression of a chimeric mouse/human antibody. A first construct encoded a chimera of the mouse-derived heavy chain variable region and a human-derived heavy γ chain (mV_H-hγ), while the second construct encoded a chimera of the mouse-derived light chain variable region and a human-derived light κ chain (mV_L-hκ).

A. Preparation of DNA Construct for Chimeric Mouse V_H-Human Heavy γ Chain (mV_H-hγ)

The DNA sequence encoding the V_H region of the heavy mouse α chain of the original anti-α2Ct IgA was cloned into the pETBlue-1 Blunt vector (EMD Biosciences/Novagen). To facilitate downstream cloning into the pFUSE-CHIg-hG1 vector that includes the sequence encoding the constant region (CH) of the heavy γ chain of the human IgG1 (InvivoGen), the EcoRI and NheI restriction sites were introduced via PCR to the DNA sequence encoding the V_H region. See FIG. 11. (The nucleotide and amino acid sequences shown in FIG. 11 are SEQ ID NO:20 and SEQ ID NO:8, respectively.) Subsequently, an insert encoding the V_H region of the heavy mouse α chain was cloned into corresponding restriction sites of the pFUSE-CHIg-hG1 vector (FIG. 12). The fidelity of the mV_H-hγ construct was confirmed by DNA sequencing.

B. Preparation of DNA Construct for Chimeric Mouse V_L-Human Light κ Chain (mV_L-hκ)

DNA sequence encoding the V_L region of the light mouse κ chain of the original anti-α2Ct IgA was cloned into the pETBlue-1 Blunt vector (EMD Biosciences/Novagen). To facilitate downstream cloning into the pFUSE2-CLIg-hκ vector that includes the sequence encoding the constant region (CH) of the human light κ chain (InvivoGen), the AgeI and BsiWI restriction sites were introduced via PCR to the DNA sequence encoding the V_L region of the light mouse κ chain. See FIG. 13. (The nucleotide and amino acid sequences shown in FIG. 13 are SEQ ID NO:21 and SEQ ID NU:7, respectively.) Subsequently, the insert encoding the V_L region of the light mouse κ chain was cloned into the corresponding restriction sites of the pFUSE2-CLIg-hk vector (FIG. 14). The fidelity of the mV_L-hκ construct was confirmed by DNA sequencing.

C. Selection of Clones Expressing Chimeric mV_H-hγ and mV_L-hκ Variants in Mammalian Cells

Chimeric variants were expressed in Chinese hamster ovary cells (CHO). In brief, CHO cells were transfected with a DNA construct encoding the mV_H-hγ variant. Zeocin-resistant clones were selected and screened for the presence of the mV_H-hγ. Specifically, proteins secreted to the media by selected clones were analyzed by Western blot for the presence of the mV_H-hγ variant with the use of the goat anti-human γ chain polyclonal antibody conjugated with HRP (Sigma-Aldrich). Next, the selected γ chain-positive CHO clone was expanded in cell culture and then transfected with a DNA construct encoding the mV_L-hκ variant. Subsequently, cell culture media from the double-transfected clones resistant to Zeocin and Blasticidin were analyzed by Western blot for the production of the mV_L-hκ chain with the use of the polyclonal goat anti-human k chain antibodies conjugated with HRP. Selected clones stably co-expressing mV_H-hγ and mV_L-hκ chains were expanded in cell culture and then cryopreserved in liquid nitrogen.

D. Purification of the Chimeric Antibody Consisting of the mV_H-hγ and mV_L-hκ Chains

Cell culture media from selected CHO cells producing the chimeric antibody were collected. Subsequently, proteins secreted to the media were precipitated with ammonium sulfate added to a 50-% saturation. Precipitated proteins were collected by centrifugation and then the protein pellet was solubilized in phosphate buffered saline (PBS). Insoluble material was removed by centrifugation while the supernatant was collected for further processing. The chimeric IgG was purified by employing the Protein-L agarose (Thermo Scientific), a resin that specifically binds to the κ chain of human but not bovine origin.

Purified chimeric antibodies were analyzed in reducing conditions for the presence of the γ and κ chains. Specific antibodies against particular chains confirm the production of the γ and κ chains by CHO cells (FIG. 15A). To determine if the two chains of the chimeric IgG antibody, i.e., mV_H-hγ and mV_L-hκ chains, co-assemble into native-like molecules consisting of both types of chains linked via disulfide bonds, the purified proteins secreted from CHO cells were separated in a polyacrylamide gel in non-reducing conditions. Subsequent Western blot assays with the anti-human γ chain and the anti human κ chain antibodies indicate that mV_H-hγ and mV_L-hκ chains were covalently linked via disulfide bonds, thereby indicating the formation of chimeric antibody molecules consisting of heavy and light chains of expected molecular mass (FIG. 15B.). The presence of a high molecular band indicates production of chimeric IgG molecules by CHO cells. Human IgG with the chain was used as a positive marker (h-IgG(κ).

E. Binding of the Chimeric IgG to its Designated Target

Western blot assays were employed to test the binding of purified chimeric IgG to its designated target, i.e., the C terminal telopeptide of the α2 chain of human procollagen I (α2Ct). In brief, the al and α2 chains of procollagen I purified from the cultured of human dermal fibroblasts were separated in a polyacrylamide gel followed by their transfer into a nitrocellulose membrane. Subsequently, the membrane was incubated with chimeric IgG solubilized in a blocking buffer. The binding of the chimeric IgG to the α2Ct was detected by chemiluminescence with the use of the anti-human γ antibodies conjugated with horseradish peroxidase (HRP), as shown in FIG. 16. Positive bands were identified as those corresponding to intact procollagen I α2 chain (upper band) and its partially degraded form. Native human IgG with the κ chain was used as a positive marker (Bethyl Laboratories Inc.).

Example 5: Binding Kinetics of Anti-α2Ct Variants to Human Procollagen

The binding of the anti-α2Ct variants (scFv and chimeric-Ig) to human procollagen I was analyzed with the use of the SensiQ Pioneer biosensor (ICx Nomadics). In brief, purified human procollagen I, a protein that includes a native α2 C telopeptide, was covalently immobilized on a sensor chip (COOH2, ICx Nomadics). Subsequently, the kinetics of the binding of the original anti-α2Ct mouse IgA antibody, the binding of the bacterial-derived scFv variant of Example 1, and the binding of the mouse-human chimeric IgG of Example 4, were analyzed. Human IgG with the light κ chain was used as a control (Bethyl Laboratories, Inc.). The dissociation equilibrium constant (K_D) values for each interaction were calculated with the use of the QDat software (ICx Nomadics). The results are show in FIG. 17. In each panel, the curves represent association and dissociation events during the interaction between immobilized procollagen I and free antibody variants present at concentrations ranging from 4×10⁻⁷ M to 1.25×10⁻⁸ M. For each assay, the association rate constants (k_on) and the dissociation rate constants (k_off) were obtained, and the equilibrium dissociation constants (K_D) values were calculated from a ratio of k_off/k_on. Although specific K_D values differ, all variants are characterized by a strong binding affinity to procollagen I. The lack of binding of control human IgG to procollagen I indicates the high specificity of the presented binding assays.

Example 6: Expression, Purification and Testing of scFv Variants Produced in Yeast

A. Expression of scFv in Yeasts

Yeast clones expressing the A_L_K or K_L_A scFv variants cloned into the pPIC-9K yeast-expression vector according to Example 3 were selected by culturing them in the absence of histidine (His(−) conditions) according to manufacturer's suggestions (Invitrogen). Subsequently, the selected clones were tested for production of the scFv variants. In brief, the selected clones were cultured in the presence of methanol as a sole source of carbon. After six days cell culture media were tested for the presence of secreted scFv variants. Specifically, His-tagged scFv variants were detected by Western blot assays in which the anti-His tag antibody was employed. In addition to Western blot assays, PCR was employed to confirm the presence of DNA encoding the specific scFv variants in the yeasts' genome.

The results are shown in FIGS. 18A (Western blot assay) and 18B (PCR assay). Because of noticeably smaller yield of the K_L_A variant (not shown), only the A_L_K scFv construct was chosen for further analyses. As shown in FIG. 18A, Western blot assays of the A_ L_K scFv variant secreted by various yeast clones cultured in His(−) conditions stained positive for His-tagged scFv variant. Cell culture media from non-transformed yeast cells (Ctrl) are negative for the scFv variant. As shown in FIG. 148, PCR assay confirmed the presence of DNA constructs encoding the A_L_K and K_L_A variants in the genome of analyzed clones.

B. Purification of Yeast scFv Variants

Purification of the scFv variants was carried out with a nickel column according to the manufacturer's protocol (Invitrogen). The His-tagged A_L_K was purified on the nickel column. The purified scFv variants were analyzed by gel electrophoresis and by Western blot assays run in denaturing and reducing conditions. Furthermore, the molecular mass of the purified native scFv was analyzed by high pressure liquid size exclusion chromatography (SEC HPLC) run in non-denaturing conditions.

As indicated in FIG. 19, the A_L_K scFv variant eluted from the nickel column contributed the main band seen in the electrophoretic gel. In the denaturing/reducing conditions of the assay, the A_L_K scFv variant purified from yeast cultures migrated in the electrophoretic field according to its predicted mass of 28 kDa. SEC HPLC assays (not shown), however, demonstrate that in the applied native conditions, most of the concentrated scFv molecules (concentration of 0.6 mg/ml) aggregate to form high-molecular mass assemblies. Such aggregate formation is a common characteristic of recombinant proteins, and may be remedied by optimization of experimental conditions in which aggregate formation is minimized.

C. Western Blot Assays of scFv Binding to its Designated Target

Western blot assays were employed to test the binding of purified scFv to its designated target i.e. the C terminal telopeptide of the α2 chain of human procollagen I (α2Ct). In brief, the Pro-al and Pro-α2 chains of procollagen I purified from the culture of human dermal fibroblasts were separated in a polyacrylamide gel followed by their transfer into a nitrocellulose membrane. Subsequently, the nitrocellulose membrane was incubated with scFv. The binding of the His-tagged scFv to the α2Ct was detected by chemiluminescence with the use of the anti-His antibodies conjugated with horseradish peroxidase (HRP). The results, shown in FIG. 20. Lane (A) represents the electrophoresis of the purified human procollagen I in a polyacrylamide gel (Coomassie blue-stained chains are indicated). Lane B represents the nitrocellulose membrane incubated with His-tagged yeast-derived scFv. The presented protein band (Lane B) was identified as procollagen I α2 chain, thereby indicating specific scFv-α2Ct binding.

The disclosures of each and every patent, patent application, publication and GenBank record cited herein are hereby incorporated herein by reference in their entirety.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. While the invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope used in the practice of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. A chimeric, humanized, or single-chain antibody which comprises a light chain variable region comprising complementarity determining regions comprising the amino acid sequences SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and a heavy chain variable region comprising complementarity determining regions comprising the amino acid sequences SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or a fragment of said chimeric or humanized antibody, which antibody or antibody fragment binds the C-terminal telopeptide of the α2(I) chain of human collagen I.

2. The antibody or antibody fragment according to claim 1, which is a chimeric antibody or fragment of a chimeric antibody, which fragment binds the C-terminal telopeptide of the α2(I) chain of human collagen.

3. The A chimeric, humanized, or single-chain antibody according to claim 2, which comprises comprising an antibody light chain comprising three complementarity determining regions of a light variable region having the amino acid sequence shown in SEQ ID NO:10, and an antibody heavy chain comprising three complementarity determining regions of a heavy chain variable region having the amino acid sequence SEQ ID NO:11, or a fragment of said chimeric antibody which, wherein the antibody or the fragment each binds the C-terminal telopeptide of the α2(I) chain of human collagen I.

4. The chimeric, humanized, or single-chain antibody or the fragment according to claim 3, wherein said antibody light chain comprises a human antibody light chain constant region, and said antibody heavy chain comprises a human antibody heavy chain constant region, or a fragment of said chimeric antibody which binds the C-terminal telopeptide of the α2(I) chain of human collagen.

5. The chimeric, humanized, or single-chain antibody according to claim 4, wherein the human constant regions comprise IgG constant regions.

6. The antibody or antibody fragment according to claim 1, which is a humanized antibody or fragment of a humanized antibody, which fragment binds the C-terminal telopeptide of the α2(I) chain of human collagen.

7. The humanized antibody according to claim 6, which comprises a human antibody framework region and/or a human antibody constant region, or a fragment of said humanized antibody which binds the C-terminal telopeptide of the α2(I) chain of human collagen.

8. The humanized antibody according to claim 6, which comprises a human antibody constant region.

9. The humanized antibody according to claim 8, wherein the constant region comprises an IgG constant region.

10. The antibody according to claim 1 3, which is a single chain antibody.

11. The single chain antibody according to claim 10, wherein the single chain antibody comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO:10, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:11.

12. The single chain antibody according to claim 11 10, wherein the antibody further comprises a linker comprising a sequence of one or more amino acids linking said light chain variable region and said heavy chain variable region.

13. The single chain antibody according to claim 12, wherein the linker comprises the amino acid sequence Gly-Gly-Ser or the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:29).

14. The single chain antibody according to claim 12, wherein the linker connects the carboxy terminus of said light chain variable region to the amino terminus of said heavy chain variable region.

15. The single chain antibody according to claim 13, wherein the linker comprises from two to twelve repeats of the amino acid sequence Gly-Gly-Ser, or from two to twelve repeats of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:29).

16. The single chain antibody according to claim 15, comprising the amino acid sequence shown in SEQ ID NO:12.

17. The single chain antibody according to claim 12, wherein the linker connects the carboxy terminus of said heavy chain variable region to the amino terminus of said light chain variable region.

18. The single chain antibody according to claim 17, wherein the linker comprises from two to twelve repeats of the amino acid sequence Gly-Gly-Ser, or from two to twelve repeats of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:29).

19. The single chain antibody according to claim 18, comprising the amino acid sequence shown in SEQ ID NO:16.

20. A pharmaceutical composition comprising the antibody or antibody the fragment according to claim 1 3and a pharmaceutically acceptable carrier.

21. The chimeric, humanized, or single-chain antibody or the fragment according to claim 3, wherein the antibody or fragment each comprises the light variable region having the amino acid sequence shown in SEQ ID NO:10 and the heavy chain variable region having the amino acid sequence SEQ ID NO:11.