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United States Patent Application 20170152323
Kind Code A1
Chang; Tse-Wen ;   et al. June 1, 2017

Anti-inflammatory molecules with tissue-targeting functions

Abstract

The present disclosure provides various molecular constructs having a targeting element and an effector element. Methods for treating various diseases using such molecular constructs are also disclosed.


Inventors: Chang; Tse-Wen; (Taipei, TW) ; Chen; Jou-Han; (Taipei, TW) ; Chu; Hsing-Mao; (Taipei, TW)
Applicant:
Name City State Country Type

Immunwork Inc.

Taipei

TW
Assignee: Immunwork Inc.
Taipei
TW

Family ID: 1000002448112
Appl. No.: 14/997849
Filed: January 18, 2016


Related U.S. Patent Documents

Application NumberFiling DatePatent Number
62104405Jan 16, 2015
62114427Feb 10, 2015
62137737Mar 24, 2015

Current U.S. Class: 1/1
Current CPC Class: C07K 16/468 20130101; C07K 2319/33 20130101; C07K 16/241 20130101; C07K 16/244 20130101; C07K 14/70578 20130101; C07K 16/2875 20130101; C07K 2317/524 20130101; C07K 2317/526 20130101; C07K 2317/622 20130101; C07K 2317/31 20130101; C07K 2317/55 20130101; C07K 2317/21 20130101; C07K 2319/32 20130101; C07K 2317/94 20130101; A61K 2039/505 20130101; C07K 2317/76 20130101; C07K 16/18 20130101
International Class: C07K 16/46 20060101 C07K016/46; C07K 16/28 20060101 C07K016/28; C07K 14/705 20060101 C07K014/705; C07K 16/18 20060101 C07K016/18; C07K 16/24 20060101 C07K016/24

Claims



1. A molecular construct comprising, a pair of CH2-CH3 segments of an IgG.Fc; a first pair of effector elements, wherein the effector element is an antibody fragment specific for tumor necrosis factor-.alpha. (TNF-.alpha.), interleukin-17 (IL-17), IL-17 receptor (IL-17R), IL-1, IL-6, IL-6R, IL-12, IL-23, B cell activating factor (BAFF), or receptor activator of nuclear factor kappa-B ligand (RANKL); or a soluble receptor of TNF-.alpha. or IL-1; and a first pair of targeting elements, wherein the targeting element is an antibody fragment specific for .alpha.-aggrecan, collagen I, collagen II, collagen III, collagen V, collagen VII, collagen IX, collagen XI, or osteonectin, wherein, when the first pair of effector elements is linked to the N-termini of the pair of CH2-CH3 segments, then the first pair of targeting elements is linked to the C-termini of the pair of CH2-CH3 segments, and vice versa, or when the first pair of effectors elements and the first pair of targeting elements are both in the form of single-chain variable fragments (scFvs), then the first pair of targeting elements is linked to the N-termini of the first pair of effector elements in a tandem or diabody configuration, thereby forming a pair of bispecific scFvs that are linked to the N-termini of the pair of CH2-CH3 segments.

2. The molecular construct of claim 1, wherein the pair of CH2-CH3 segments is derived from human .gamma.4 or .gamma.1 immunoglobulin.

3. The molecular construct of claim 1, wherein when the first pair of effector elements is in the form of an antigen-binding fragment (Fab), and the first pair of targeting elements is in the form of scFvs, and vice versa; then the Fab and scFvs are respectively linked to the N-termini and C-termini of the CH2-CH3 segments, so that molecular construct adopts an extended IgG configuration.

4. The molecular construct of claim 1, further comprising a second pair of effector elements or a second pair of targeting elements, wherein the second pair of effector or targeting elements is linked to the free C-termini of the CH2-CH3 segments.

5. The molecular construct of claim 1, wherein, the effector element is an scFv specific for TNF-.alpha.; and the targeting element is an scFv specific for collagen II, collagen IX, or .alpha.-aggrecan.

6. The molecular construct of claim 1, wherein, the two effector elements are in the form of a Fab antibody specific for TNF-.alpha.; and the targeting element is an scFv specific for collagen II or collagen IX.

7. The molecular construct of claim 1, wherein, the effector element is an scFv specific for IL-17; and the targeting element is an scFv specific for collagen I or collagen VII.

8. The molecular construct of claim 1, wherein, the two effector elements are in the form of a Fab antibody specific for IL-17; and the targeting element is an scFv specific for collagen I or collagen VII.

9. The molecular construct of claim 1, wherein, the effector element is an scFv specific for BAFF; and the targeting element is an scFv specific for collagen I or collagen VII.

10. The molecular construct of claim 1, wherein, the two effector elements are in the form of a Fab antibody specific for BAFF; and the targeting element is an scFv specific for collagen I or collagen VII.

11. The molecular construct of claim 1, wherein, the effector element is an scFv specific for TNF-.alpha.; and the targeting element is an scFv specific for collagen III or collagen V.

12. The molecular construct of claim 1, wherein, the two effector elements are in the form of a Fab antibody specific for TNF-.alpha.; and the targeting element is an scFv specific for collagen III or collagen V.

13. The molecular construct of claim 1, wherein, the effector element is an scFv specific for RANKL; and the targeting element is an scFv specific for collagen I or osteonectin.

14. The molecular construct of claim 1, wherein, the two effector elements are in the form of a Fab specific for RANKL; and the targeting element is an scFv specific for collagen I or osteonectin.

15. A method for treating an immune disorder, comprising the step of administering to a subject in need thereof an effective amount of the molecular construct according to claim 1.

16. The method of claim 15, wherein the immune disorder is an autoimmune disease.

17. The method of claim 16, wherein, the autoimmune disease is rheumatoid arthritis, psoriatic arthritis, or ankylosing spondylitis; the effector element is an antibody fragment specific for TNF-.alpha., IL-12/IL-23, IL-1, IL-17, or IL-6; and the targeting element is an antibody fragment specific for collagen II, collagen IX, collagen XI, or .alpha.-aggrecan.

18. The method of claim 16, wherein, the autoimmune disease is psoriasis; the effector element is an antibody fragment specific for TNF-.alpha., IL-12/IL-23, or IL-17; and the targeting element is an antibody fragment specific for collagen I or collagen VII.

19. The method of claim 16, wherein, the autoimmune disease is systemic lupus erythematosus, cutaneous lupus, or Sjogren's Syndrome; the effector element is an antibody fragment specific for BAFF; and the targeting element is an antibody fragment specific for collagen I or collagen VII.

20. The method of claim 16, wherein, the autoimmune disease is an inflammatory bowel disease; the effector element is an antibody fragment specific for TNF-.alpha.; and the targeting element is an antibody fragment specific for collagen III or collagen V.

21. The method of claim 20, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

22. A method for treating osteoporosis, comprising the step of administering to a subject in need thereof an effective amount of the molecular construct according to claim 1.

23. The method of claim 22, wherein, of the effector element is an antibody fragment specific for RANKL; and the targeting element is an antibody fragment specific for collagen I or osteonectin.
Description



CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application relates to and claims the benefit of U.S. Provisional Application No. 62/104,405, filed Jan. 16, 2015, U.S. Provisional Application No. 62/114,427, filed Feb. 10, 2015, and U.S. Provisional Application No. 62/137,737, filed Mar. 24, 2015; the contents of the applications are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present disclosure relates to the field of pharmaceuticals; more particularly, to multi-functional molecular constructs, e.g., those having targeting and effector elements for delivering the effector (e.g., therapeutic drug) to targeted sites.

[0004] 2. Description of the Related Art

[0005] The continual advancement of a broad array of methodologies for screening and selecting monoclonal antibodies (mAbs) for targeted antigens has helped the development of a good number of therapeutic antibodies for many diseases that were regarded as untreatable just a few years ago. According to Therapeutic Antibody Database, approximately 2,800 antibodies have been studied or are being planned for studies in human clinical trials, and approximately 80 antibodies have been approved by governmental drug regulatory agencies for clinical uses. The large amount of data on the therapeutic effects of antibodies has provided information concerning the pharmacological mechanisms how antibodies act as therapeutics.

[0006] One major pharmacologic mechanism for antibodies acting as therapeutics is that, antibodies can neutralize or trap disease-causing mediators, which may be cytokines or immune components present in the blood circulation, interstitial space, or in the lymph nodes. The neutralizing activity inhibits the interaction of the disease-causing mediators with their receptors. It should be noted that fusion proteins of the soluble receptors or the extracellular portions of receptors of cytokines and the Fc portion of IgG, which act by neutralizing the cytokines or immune factors in a similar fashion as neutralizing antibodies, have also been developed as therapeutic agents.

[0007] Several therapeutic antibodies that have been approved for clinical applications or subjected to clinical developments mediate their pharmacologic effects by binding to receptors, thereby blocking the interaction of the receptors with their ligands. For those antibody drugs, Fc-mediated mechanisms, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytolysis (CMC), are not the intended mechanisms for the antibodies.

[0008] Some therapeutic antibodies bind to certain surface antigens on target cells and render Fc-mediated functions and other mechanisms on the target cells. The most important Fc-mediated mechanisms are antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytolysis (CMC), which both will cause the lysis of the antibody-bound target cells. Some antibodies binding to certain cell surface antigens can induce apoptosis of the bound target cells.

[0009] Antibodies can also serve as carriers of cytotoxic molecules or other therapeutic agents without the antibodies' serving obvious therapeutic effector functions. In general, those antibodies bind to "tumor-associated" antigens on target cells, but cannot cause cell lysis by themselves. Antibodies specific for CD19 and CD22 on B lymphomas are well known. For many years, those antibodies have been explored as carriers for cytotoxic agents, including radioactive nuclides with very short half-lives, such as .sup.90Y, .sup.131I, and .sup.177Lu. Some antibodies have also been studied as targeting agents for liposomes loaded with cytotoxic drugs, such as doxorubicin, paclitaxel, and amphotericin B. The field of antibody drug conjugates (ADC) has experienced an explosive phase of research and development in recent years, mainly attributing to the development of extremely cytotoxic drugs, such as auristatin, maytansine, calicheamicin, and camptothecin, and of methodologies for conjugating the cytotoxic molecules onto antibody molecules. Those ADCs have been designed to target diffusive (or liquid) tumors of the blood, lymphoid system, and bone marrow, including various types of lymphomas and leukemia, expressing one or more unique CD markers. Some ADCs are also being developed for solid tumors. A few of this new generation of antibody drug conjugates have been approved for clinical uses and many are in clinical trials.

[0010] However, in the first generation of ADCs, the cytotoxic drug molecules are linked non-selectively to cysteine or lysine residues in the antibody, thereby resulting in a heterogeneous mixture of ADCs with different numbers of drug molecules per ADC. This approach leads to some safety and efficacy issues. For example, the first FDA-approved ADC, gemtuzumab ozogamicin, for treating acute myelogenous leukemia, is now withdrawn from the market due to unacceptable toxicity.

[0011] The concept and methodology for preparing antibodies with dual specificities germinated more than three decades ago. In recent year, the advancement in recombinant antibody engineering methodologies and the drive to develop improved medicine has stimulated the development bi-specific antibodies adopting a large variety of structural configurations.

[0012] For example, the bi-valent or multivalent antibodies may contain two or more antigen-binding sites. A number of methods have been reported for preparing multivalent antibodies by covalently linking three or four Fab fragments via a connecting structure. For example, antibodies have been engineered to express tandem three or four Fab repeats.

[0013] Several methods for producing multivalent antibodies by employing synthetic crosslinkers to associate, chemically, different antibodies or binding fragments have been disclosed. One approach involves chemically cross-linking three, four, and more separately Fab fragments using different linkers. Another method to produce a construct with multiple Fabs that are assembled to one-dimensional DNA scaffold was provided.

[0014] Those various multivalent Ab constructs designed for binding to target molecules differ among one another in size, half-lives, flexibility in conformation, and ability to modulate the immune system. In view of the foregoing, several reports have been made for preparing molecular constructs with a fixed number of effector elements or with two or more different kinds of functional elements (e.g., at least one targeting element and at least one effector element). However, it is often difficult to build a molecular construct with a particular combination of the targeting and effector elements either using chemical synthesis or recombinant technology. Accordingly, there exists a need in the related art to provide novel molecular platforms to build a more versatile molecule suitable for covering applications in a wide range of diseases.

SUMMARY

[0015] The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the present invention or delineate the scope of the present invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

[0016] In a first aspect, the present disclosure is directed to a fragment crystallizable (Fc)-based molecular construct that has at least one targeting element and at least one effector element linked, directly or indirectly, to a CH2-CH3 domain of an immunoglobulin. The design of the present Fc-based molecular construct allows for numerous combinations of a wide range of targeting and effector elements. Hence, the present Fc-based molecular construct may serve as a platform for constructing multi-valent molecules.

[0017] According to certain embodiments of the present disclosure, the Fc-based molecular construct comprises a pair of CH2-CH3 segments of an IgG.Fc, a first pair of effector elements, and a first pair of targeting elements.

[0018] In some embodiments, the present Fc-based molecular constructs are intended to be used in the treatment of immune diseases (in particular, autoimmune diseases) or osteoporosis. In this case, the first pair of effector elements consists of two effector elements, in which each of the two effector elements is an antibody fragment specific for tumor necrosis factor-.alpha. (TNF-.alpha.), interleukin-17 (IL-17), IL-17 receptor (IL-17R), IL-1, IL-6, IL-6R, IL-12/IL-23, B cell activating factor (BAFF), or a receptor activator of nuclear factor kappa-B ligand (RANKL); or a soluble receptor of TNF-.alpha. or IL-1. Further, the first pair of targeting elements consists of two targeting elements, in which each of the two targeting elements is an antibody fragment specific for .alpha.-aggrecan, collagen I, collagen II, collagen III, collagen V, collagen VII, collagen IX, collagen XI, or osteonectin. In the case where the first pair of effector elements is linked to the N-termini of the pair of CH2-CH3 segments, the first pair of targeting elements is linked to the C-termini of the pair of CH2-CH3 segments, and vice versa. Alternatively, when the first pair of effectors elements and the first pair of targeting elements is both in the form of single-chain variable fragments (scFvs), then the first pair of targeting elements is linked to the N-termini of the first pair of effector elements in a tandem or diabody configuration, thereby forming a pair of bispecific scFvs that are linked to the N-termini of the pair of CH2-CH3 segments.

[0019] In certain embodiments, the pair of CH2-CH3 segments is derived from human IgG heavy chain .gamma.4 or human IgG heavy chain .gamma.1.

[0020] In some examples, the first pair of effector elements or the first pair of the targeting elements takes a Fab configuration (i.e., consisting of the V.sub.H-CH1 domain and the V.sub.L-C.kappa. domain); this Fab fragment is linked to the N-termini of the first and second heavy chains, so that the Fc-based molecular construct adopts an IgG configuration. In these cases, the pair of elements that is not in the Fab configuration is linked to the C-termini of the pair of CH2-CH3 segments.

[0021] According to other embodiments, the Fc-based molecular construct further comprises a second pair of effector elements, which consists of two additional effector elements that are both selected from the group described above for the effector elements. According to various embodiments, the elements of the second pair of effector elements are different from those of the first pair of effector elements. In these embodiments, the second pair of effector elements is linked to the free C-termini of the CH2-CH3 segments.

[0022] Alternatively, the present Fc-based molecular construct further comprises a second pair of targeting elements, in which the two targeting elements are both selected from the group described above regarding the targeting elements. According to various embodiments, the elements of the second pair of targeting elements are different from those of the first pair of targeting elements. In these embodiments, the second pair of targeting elements is linked to the free C-termini of the CH2-CH3 segments.

[0023] According to various optional embodiments, the targeting elements and effector elements described above can be combined as desired, so as to attain the intended therapeutic effect. Some exemplary combination of the effector element(s) and targeting element(s) for treating immune diseases are provided in the appended claims and discussed in the description section bellow.

[0024] In a second aspect, the present disclosure is directed to methods for treating various diseases. Generally, the methods involve the step of administrating an effective amount of the Fc-based molecular constructs according to the first aspect and any of the associated embodiments, to a subject in need of such treatment.

[0025] In certain embodiments, the present method is directed to the treatment of an immune disease; in particular, an autoimmune disease.

[0026] According to some embodiments of the present disclosure, the autoimmune disease is rheumatoid arthritis, psoriatic arthritis, or ankylosing spondylitis. In this case, the effector element is an antibody fragment specific for TNF-.alpha., IL-12/IL-23, IL-1, IL-17, or IL-6, while the targeting element may be an antibody fragment specific for collagen II, collagen IX, collagen XI, or .alpha.-aggrecan.

[0027] According to various embodiments, the autoimmune disease is psoriasis. In this case, the effector element is an antibody fragment specific for TNF-.alpha., IL-12/IL-23, or IL-17, while the targeting element is an antibody fragment specific for collagen I or collagen VII.

[0028] According to some other embodiments, the autoimmune disease is systemic lupus erythematosus, cutaneous lupus, or Sjogren's Syndrome. In this case, the effector element is an antibody fragment specific for BAFF, and the targeting element is an antibody fragment specific for collagen I, or collagen VII.

[0029] According to some embodiments, the autoimmune disease is an inflammatory bowel disease, such as Crohn's disease or ulcerative colitis. In this case, the effector element is an antibody fragment specific for TNF-.alpha., and the targeting element is an antibody fragment specific for collagen III or collagen V.

[0030] Another disease treatable by the method proposed herein is osteoporosis. According to embodiments of the present disclosure, the effector element for treating osteoporosis comprises an antibody fragment specific for RANKL, while the targeting element comprises an antibody fragment specific for collagen I or osteonectin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present description will be better understood from the following detailed description read in light of the accompanying drawings briefly discussed below.

[0032] FIGS. 1A to 1F are schematic diagrams illustrating Fc-based molecular constructs according to various embodiments of the present disclosure.

[0033] FIGS. 2A and 2B are schematic diagrams illustrating Fc-based molecular constructs according to various embodiments of the present disclosure.

[0034] FIG. 3 shows the SDS-PAGE analysis of (scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4Fc.

[0035] FIGS. 4A and 4B show the ELISA results analyzing the binding of (scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4Fc to collagen II and TNF-.alpha..

[0036] FIGS. 5A and 5B respectively show the SDS-PAGE and ELISA analysis of the 2-chain (scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc-(scFv .alpha. IL-17).

[0037] FIGS. 6A and 6B respectively show the SDS-PAGE and ELISA analysis of 2-chain (soluble TNF-.alpha. receptor)-IgG1.CH2-CH3-scFv .alpha. collagen II.

[0038] FIG. 7 shows the SDS-PAGE analysis of the 2-chain fusion protein containing intact antibody for human TNF-.alpha. and scFv specific for collagen II.

[0039] FIGS. 8A and 8B respectively show the SDS-PAGE and ELISA analyses of the 2-chain fusion protein containing intact antibody for human IL-17 and scFv specific for collagen VII.

[0040] FIGS. 9A and 9B respectively show the SDS-PAGE and ELISA analysis of scFv .alpha. collagen VII-IgG4.CH2-CH3-scFv .alpha. BAFF.

[0041] FIGS. 10A and 10B respectively show the SDS-PAGE and ELISA analyses of the 2-chain fusion protein containing intact antibody for human BAFF and scFv specific for collagen VII.

[0042] FIGS. 11A and 11B respectively show the SDS-PAGE and ELISA analyses of the 2-chain (scFv .alpha. SPARC)-(scFv .alpha. RANKL)-hIgG4.Fc molecular construct.

[0043] FIGS. 12A and 12B respectively show the SDS-PAGE and ELISA analyses of the 2-chain fusion protein containing intact antibody for human RANKL and scFv specific for human osteonectin.

[0044] FIG. 13 shows the immunostaining of mouse epiphyseal bone with (scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4Fc and 2-chain (scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc-(scFv .alpha. IL-17).

[0045] FIG. 14 shows the immunostaining of mouse epiphyseal bone with 2-chain (soluble TNF-.alpha. receptor)-IgG1.CH2-CH3-scFv .alpha. collagen II.

[0046] FIG. 15 shows bio-distribution of fluorescence-labeled (scFv .alpha. SPARC)-(scFv .alpha. RANKL)-hIgG4Fc in vivo in BALB/c mice.

[0047] In accordance with common practice, the various described features/elements are not drawn to scale but instead are drawn to best illustrate specific features/elements relevant to the present invention. Also, like reference numerals and designations in the various drawings are used to indicate like elements/parts, where possible.

DESCRIPTION

[0048] The detailed description provided below in connection with the appended drawings is intended as a description of the present examples and is not intended to represent the only forms in which the present example may be constructed or utilized. The description sets forth the functions of the example and the sequence of steps for constructing and operating the example. However, the same or equivalent functions and sequences may be accomplished by different examples.

[0049] For convenience, certain terms employed in the specification, examples and appended claims are collected here. Unless otherwise defined herein, scientific and technical terminologies employed in the present disclosure shall have the meanings that are commonly understood and used by one of ordinary skill in the art.

[0050] Unless otherwise required by context, it will be understood that singular terms shall include plural forms of the same and plural terms shall include the singular. Specifically, as used herein and in the claims, the singular forms "a" and "an" include the plural reference unless the context clearly indicated otherwise. Also, as used herein and in the claims, the terms "at least one" and "one or more" have the same meaning and include one, two, three, or more. Furthermore, the phrases "at least one of A, B, and C", "at least one of A, B, or C" and "at least one of A, B and/or C," as use throughout this specification and the appended claims, are intended to cover A alone, B alone, C alone, A and B together, B and C together, A and C together, as well as A, B, and C together.

[0051] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in the respective testing measurements. Also, as used herein, the term "about" generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term "about" means within an acceptable standard error of the mean when considered by one of ordinary skill in the art. Other than in the operating/working examples, or unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages such as those for quantities of materials, durations of times, temperatures, operating conditions, ratios of amounts, and the likes thereof disclosed herein should be understood as modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and attached claims are approximations that can vary as desired. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Ranges can be expressed herein as from one endpoint to another endpoint or between two endpoints. All ranges disclosed herein are inclusive of the endpoints, unless specified otherwise.

[0052] This present disclosure pertains generally to molecular constructs, in which each molecular construct comprises a targeting element (T) and an effector element (E), and these molecular constructs are sometimes referred to as "T-E molecules", "T-E pharmaceuticals" or "T-E drugs" in this document.

[0053] As used herein, the term "targeting element" refers to the portion of a molecular construct that directly or indirectly binds to a target of interest (e.g., a receptor on a cell surface or a protein in a tissue) thereby facilitates the transportation of the present molecular construct into the interested target. In some example, the targeting element may direct the molecular construct to the proximity of the target cell. In other cases, the targeting element specifically binds to a molecule present on the target cell surface or to a second molecule that specifically binds a molecule present on the cell surface. In some cases, the targeting element may be internalized along with the present molecular construct once it is bound to the interested target, hence is relocated into the cytosol of the target cell. A targeting element may be an antibody or a ligand for a cell surface receptor, or it may be a molecule that binds such antibody or ligand, thereby indirectly targeting the present molecular construct to the target site (e.g., the surface of the cell of choice). The localization of the effector (therapeutic agent) in the diseased site will be enhanced or favored with the present molecular constructs as compared to the therapeutic without a targeting function. The localization is a matter of degree or relative proportion; it is not meant for absolute or total localization of the effector to the diseased site.

[0054] According to the present invention, the term "effector element" refers to the portion of a molecular construct that elicits a biological activity (e.g., inducing immune responses, exerting cytotoxic effects and the like) or other functional activity (e.g., recruiting other hapten tagged therapeutic molecules), once the molecular construct is directed to its target site. The "effect" can be therapeutic or diagnostic. The effector elements encompass those that bind to cells and/or extracellular immunoregulatory factors. The effector element comprises agents such as proteins, nucleic acids, lipids, carbohydrates, glycopeptides, drug moieties (both small molecule drug and biologics), compounds, elements, and isotopes, and fragments thereof.

[0055] Although the terms, first, second, third, etc., may be used herein to describe various elements, components, regions, and/or sections, these elements (as well as components, regions, and/or sections) are not to be limited by these terms. Also, the use of such ordinal numbers does not imply a sequence or order unless clearly indicated by the context. Rather, these terms are simply used to distinguish one element from another. Thus, a first element, discussed below, could be termed a second element without departing from the teachings of the exemplary embodiments.

[0056] Here, the terms "link," "couple," and "conjugates" are used interchangeably to refer to any means of connecting two components either via direct linkage or via indirect linkage between two components.

[0057] The term "polypeptide" as used herein refers to a polymer having at least two amino acid residues. Typically, the polypeptide comprises amino acid residues ranging in length from 2 to about 200 residues; preferably, 2 to 50 residues. Where an amino acid sequence is provided herein, L-, D-, or beta amino acid versions of the sequence are also contemplated. Polypeptides also include amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. In addition, the term applies to amino acids joined by a peptide linkage or by other, "modified linkages" (e.g., where the peptide bond is replaced by an .alpha.-ester, a .beta.-ester, a thioamide, phosphonamide, carbomate, hydroxylate, and the like.

[0058] In certain embodiments, conservative substitutions of the amino acids comprising any of the sequences described herein are contemplated. In various embodiments, one, two, three, four, or five different residues are substituted. The term "conservative substitution" is used to reflect amino acid substitutions that do not substantially alter the activity (e.g., biological or functional activity and/or specificity) of the molecule. Typically, conservative amino acid substitutions involve substitution one amino acid for another amino acid with similar chemical properties (e.g., charge or hydrophobicity). Certain conservative substitutions include "analog substitutions" where a standard amino acid is replaced by a non-standard (e.g., rare, synthetic, etc.) amino acid differing minimally from the parental residue. Amino acid analogs are considered to be derived synthetically from the standard amino acids without sufficient change to the structure of the parent, are isomers, or are metabolite precursors.

[0059] In certain embodiments, polypeptides comprising at least 80%, preferably at least 85% or 90%, and more preferably at least 95% or 98% sequence identity with any of the sequences described herein are also contemplated.

[0060] "Percentage (%) amino acid sequence identity" with respect to the polypeptide sequences identified herein is defined as the percentage of polypeptide residues in a candidate sequence that are identical with the amino acid residues in the specific polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percentage sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, sequence comparison between two polypeptide sequences was carried out by computer program Blastp (protein-protein BLAST) provided online by Nation Center for Biotechnology Information (NCBI). The percentage amino acid sequence identity of a given polypeptide sequence A to a given polypeptide sequence B (which can alternatively be phrased as a given polypeptide sequence A that has a certain % amino acid sequence identity to a given polypeptide sequence B) is calculated by the formula as follows:

X Y .times. 100 % ##EQU00001##

[0061] where X is the number of amino acid residues scored as identical matches by the sequence alignment program BLAST in that program's alignment of A and B, and where Y is the total number of amino acid residues in A or B, whichever is shorter.

[0062] The term "PEGylated amino acid" as used herein refers to a polyethylene glycol (PEG) chain with one amino group and one carboxyl group. Generally, the PEGylated amino acid has the formula of NH.sub.2--(CH.sub.2CH.sub.2O).sub.n--COOH. In the present disclosure, the value of n ranges from 1 to 20; preferably, ranging from 2 to 12.

[0063] As used herein, the term "terminus" with respect to a polypeptide refers to an amino acid residue at the N- or C-end of the polypeptide. With regard to a polymer, the term "terminus" refers to a constitutional unit of the polymer (e.g., the polyethylene glycol of the present disclosure) that is positioned at the end of the polymeric backbone. In the present specification and claims, the term "free terminus" is used to mean the terminal amino acid residue or constitutional unit is not chemically bound to any other molecular.

[0064] The term "antigen" or "Ag" as used herein is defined as a molecule that elicits an immune response. This immune response may involve a secretory, humoral and/or cellular antigen-specific response. In the present disclosure, the term "antigen" can be a protein, a polypeptide (including mutants or biologically active fragments thereof), a polysaccharide, a glycoprotein, a glycolipid, a nucleic acid, or a combination thereof.

[0065] In the present specification and claims, the term "antibody" is used in the broadest sense and covers fully assembled antibodies, antibody fragments that bind with antigens, such as antigen-binding fragment (Fab/Fab'), F(ab').sub.2 fragment (having two antigen-binding Fab portions linked together by disulfide bonds), variable fragment (Fv), single chain variable fragment (scFv), bi-specific single-chain variable fragment (bi-scFv), nanobodies, unibodies and diabodies. "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen-binding region or variable region of the intact antibody. Typically, an "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The well-known immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, with each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V.sub.L) and variable heavy chain (V.sub.H) refer to these light and heavy chains, respectively. According to embodiments of the present disclosure, the antibody fragment can be produced by modifying the nature antibody or by de novo synthesis using recombinant DNA methodologies. In certain embodiments of the present disclosure, the antibody and/or antibody fragment can be bispecific, and can be in various configurations. For example, bispecific antibodies may comprise two different antigen binding sites (variable regions). In various embodiments, bispecific antibodies can be produced by hybridoma technique or recombinant DNA technique. In certain embodiments, bispecific antibodies have binding specificities for at least two different epitopes.

[0066] The term "specifically binds" as used herein, refers to the ability of an antibody or an antigen-binding fragment thereof, to bind to an antigen with a dissociation constant (Kd) of no more than about 1.times.10.sup.-6 M, 1.times.10.sup.-7 M, 1.times.10.sup.-8 M, 1.times.10.sup.-9 M, 1.times.10.sup.-10 M, 1.times.10.sup.-11 M, 1.times.10.sup.-12 M, and/or to bind to an antigen with an affinity that is at least two-folds greater than its affinity to a nonspecific antigen.

[0067] The term "immune disorder" as used herein refers to a disorder involving deficiency of humoral immunity, deficiency of cell-mediated immunity, combined immunity deficiency, unspecified immunity deficiency, and autoimmune disease.

[0068] The term "treatment" as used herein includes preventative (e.g., prophylactic), curative or palliative treatment; and "treating" as used herein also includes preventative (e.g., prophylactic), curative or palliative treatment. In particular, the term "treating" as used herein refers to the application or administration of the present molecular construct or a pharmaceutical composition comprising the same to a subject, who has a medical condition a symptom associated with the medical condition, a disease or disorder secondary to the medical condition, or a predisposition toward the medical condition, with the purpose to partially or completely alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms or features of said particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition, and/or to a subject who exhibits only early signs of a disease, disorder and/or condition, for the purpose of decreasing the risk of developing pathology associated with the disease, disorder and/or condition.

[0069] The term "effective amount" as used herein refers to the quantity of the present recombinant protein that is sufficient to yield a desired therapeutic response. An effective amount of an agent is not required to cure a disease or condition but will provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered or prevented, or the disease or condition symptoms are ameliorated. The effective amount may be divided into one, two, or more doses in a suitable form to be administered at one, two or more times throughout a designated time period. The specific effective or sufficient amount will vary with such factors as particular condition being treated, the physical condition of the patient (e.g., the patient's body mass, age, or gender), the type of subject being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives. Effective amount may be expressed, for example, as the total mass of active component (e.g., in grams, milligrams or micrograms) or a ratio of mass of active component to body mass, e.g., as milligrams per kilogram (mg/kg).

[0070] The terms "application" and "administration" are used interchangeably herein to mean the application of a molecular construct or a pharmaceutical composition of the present invention to a subject in need of a treatment thereof.

[0071] The terms "subject" and "patient" are used interchangeably herein and are intended to mean an animal including the human species that is treatable by the molecular construct, pharmaceutical composition, and/or method of the present invention. The term "subject" or "patient" intended to refer to both the male and female gender unless one gender is specifically indicated. Accordingly, the term "subject" or "patient" comprises any mammal, which may benefit from the treatment method of the present disclosure. Examples of a "subject" or "patient" include, but are not limited to, a human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird and fowl. In an exemplary embodiment, the patient is a human. The term "mammal" refers to all members of the class Mammalia, including humans, primates, domestic and farm animals, such as rabbit, pig, sheep, and cattle; as well as zoo, sports or pet animals; and rodents, such as mouse and rat. The term "non-human mammal" refers to all members of the class Mammalis except human.

[0072] The present disclosure is based, at least on the construction of the T-E pharmaceuticals that can be delivered to target cells, target tissues or organs at increased proportions relative to the blood circulation, lymphoid system, and other cells, tissues or organs. When this is achieved, the therapeutic effect of the pharmaceuticals is increased, while the scope and severity of the side effects and toxicity is decreased. It is also possible that a therapeutic effector is administered at a lower dosage in the form of a T-E molecule, than in a form without a targeting component. Therefore, the therapeutic effector can be administered at lower dosages without losing potency, while lowering side effects and toxicity.

[0073] Diseases that can Benefit from Better Drug Targeting

[0074] Drugs used for many diseases can be improved for better efficacy and safety, if they can be targeted to the disease sites, i.e., if they can be localized or partitioned to the disease sites more favorably than the normal tissues or organs. Following are primary examples of diseases, in which drugs can be improved if they can be preferentially distributed to the disease sites or cells.

[0075] I Immune Disorder

[0076] According to the design of molecular constructs of the present disclosure, the diseases, conditions, and/or disorders treatable with the present method is an immune disorder; for example, an autoimmune disorder that includes, but is not limited to, psoriasis, systemic lupus erythematosus (SLE), cutaneous lupus, Sjogren's syndrome, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and inflammatory bowel disease.

[0077] Most of the autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, psoriasis, Crohn's disease, inflammatory bowel diseases, and others affect connective tissues. Regardless of the etiological nature, whether it is environmental, genetic, epigenetic, or their combinations, the affected tissues are damaged by prolong inflammatory processes. It is rationalized in this invention that in bringing anti-inflammatory therapeutic agents, such as anti-TNF-.alpha., anti-IL-17, anti-BAFF, anti-IL-6, anti-IL-12/IL-23, to the diseased connective tissues, the components of the extracellular matrix may be employed as target antigens. The target antigens that may be considered include the various types of collagens, laminins, elastins, fibrillins, fibronectins, and tenascins. Connective tissues fill in nearly all parts of the human body. However, due to the structural and functional requirements of the connective tissues in different locations, the types of those extracellular matrix components are different, providing excellent choices for target tissue specificity.

[0078] The advantages of choosing extracellular components over cell surface antigens for targeting the anti-inflammatory therapeutic agents are that the choices of selectivity among the various types of matrix proteins and the abundant amounts of the extracellular matrix proteins. Furthermore, because cells are not used as antigenic targets, the potential harmful effects of direct binding to cells by anti-inflammatory agents can be avoided.

[0079] I-(i) Rheumatoid Arthritis, Psoriatic Arthritis, or Ankylosing Spondylitis

[0080] Several antibodies against TNF-.alpha., e.g., infliximab and adalimumab, and fusion proteins of TNF-.alpha. receptor and IgG.Fc (e.g. etanercept) are approved or in human clinical trials for use to treat rheumatoid arthritis, ankylosing spondylitis, and other autoimmune diseases. The extracellular portion the receptor for interleukin-1 (IL-1), anakinra, is approved for treating rheumatoid arthritis. Antibodies against the shared p40 protein of IL-12 and IL-23, e.g., ustekinumab and briakinumab, are approved for psoriatic arthritis or in trials for rheumatoid arthritis. An antibody against IL-6 receptor (tocilizumab) is approved for rheumatoid arthritis and systemic juvenile idiopathic arthritis, and several antibodies against IL-6, e.g., sarilumab and olokizumab, are in clinical trials for treating rheumatoid arthritis. An antibody specific for IL-17 (secukinumab) is approved for psoriasis and in clinical trials for rheumatoid arthritis and ankylosing spondylitis.

[0081] While those therapeutic agents can alleviate severe symptoms better than previously available medications, they cause a range of serious side effects in some treated patients. For example, infliximab can cause serious blood disorders, like leukopenia and thrombocytopenia, serious infections, lymphoma and other solid tumors, reactivation of hepatitis B and tuberculosis, and other serious problems. Anakinra causes frequent infections, and severe side effects on the gastrointestinal and the respiratory tracts and the blood forming organs. It is important that the serious side effects of these widely used therapeutic agents be minimized, while retaining or even enhancing their therapeutic effects.

[0082] In rheumatoid arthritis, joints of the knees, fingers, toes, and other joints are affected, and in ankylosing spondylitis, joints of the spine and the sacroiliac joint of the pelvis are affected. In the diseased joints, the surface of the bones and the articular cartilage lining the bone surfaces are attacked by the inflammatory immune components in the joints. The articular cartilage in the joints is a smooth cartilage that contains an extracellular matrix. The cartilage is avascular and approximately 60% of the weight is water and the remaining content is composed of collagens and .alpha.-aggrecan, a proteoglycan, and other matrix molecules. Collagen II forms the major fibril in the cartilage. Aggrecan is the second most abundant component in the cartilage. Collagen XI is bound to the surface of the collagen II fibril helping to form fibril networks and collagen IX is associated with collagen II and collagen XI. The cartilage has a large surface and the .alpha.-aggrecan has a structure and shape like a feather. In addition to the cartilage formation, the joints have also ligaments, which connect adjacent bones, such as the cruciate ligaments, and tendons, which connect muscles to the bones. The ligaments and tendons are formed by fibrous network of collagen types I, II, and III, and elastin and fibrillins 1 and 2.

[0083] The present invention rationalizes that the antagonist for TNF-.alpha., IL-1, and IL-12/IL-23 can be carried to the diseased joints by using antibody fragments, such as scFv, specific for collagen II, .alpha.-aggrecan, collagen XI or collagen IX, or alternatively, collagen I, elastin or fibrillin 1 as the targeting agent. A preferred anti-collagen II antibody is one that binds to native collagen II in the joints and does not bind to N-terminal and C-terminal propeptides, which are cleaved off during fibril assembly. A preferred anti-aggrecan antibody is one that binds to whole native .alpha.-aggrecan molecules and does not bind to fragments that are cleaved off and released into the blood circulation. By adopting the present molecular construct with scFv of anti-collagen II as targeting agent, in comparison with regular IgG against TNF-.alpha., IL-1, and IL12/IL-23, larger proportions of the present therapeutic agents can be carried to the diseased sites and less amounts of the therapeutic agents will be present in other irrelevant, normal tissues, especially, lymphoid organs, and hence fewer side effects will occur.

[0084] I-(ii) Psoriasis

[0085] Most patients with psoriasis or plaque psoriasis present inflammatory symptoms primarily in the skin and not in other tissues and organs. Psoriasis involves mainly keratinocytes in part of skin in the affected patients. A systematic administration of monoclonal antibodies anti-TNF-.alpha., anti-IL-12/IL-23, and anti-IL-17 or anti-IL-17 receptor (anti-IL-17R) or other anti-inflammatory agents, such as anti-IL6, causes unwanted side effects, as discussed in the preceding section. The serious adverse side effects of all these immune modulating antibodies have been well documented.

[0086] A number of membrane or extracellular proteins, such as filaggrin, collagen I, which are expressed at much higher levels in the skin tissues than most of other tissues, probably can be considered as the target proteins to shuffle therapeutic agents to the skin. Filaggrin is present in the tight junction between cells and is probably accessible by antibodies in the diseased tissue sites. While collagen I is also present in the bone matrix and many parts of the body, it is present in the dermis layer of the skin in abundant proportions.

[0087] For damping the inflammatory activity caused by the diseased keratinocytes, which manifests psoriatic symptoms, it is not necessary to deliver the anti-inflammatory antibody drugs to be in contact with the keratinocytes. The keratinocytes are in the outmost, epidermis layer of the skin; blood vessels, sweat glands, and collagen fibers are in the middle dermis layer of the skin. The inner layer is hypodermis, where adipose tissues are. The three layers of human skin together are 2-3 mm thick. If the anti-inflammatory antibodies are delivered to the dermis layer by scFv specific for collagen I, they can diffuse into the other layers. Or, the antibodies can trap inflammatory cytokines in the three layers of the skin.

[0088] Several proteins present at the dermo-epidermal junction may also be employed as targets for carrying therapeutic agents to the skin. These include type VII collagen, type XVII collagen, and laminins type 5, 6, or 10. The dermo-epidermal junction is the area of tissue that joins the epidermal and dermal layers of the skin. The basal cells in the stratum basale of epidermis connect to the basement membrane by the anchoring filament of hemidesmosomes. The cells of the papillary layer of the dermis are attached to the basement membrane by anchoring fibrils, which consist of type VII collagen. Type XVII collagen, a transmembrane protein (also referred to as BP180) expressed on keratinocytes, is a structural component of hemidesmosomes, multiprotein complexes at the dermal-epidermal basement membrane zone that mediate adhesion of keratinocytes to the underlying membrane. Laminins are structural non-collagenous glycoproteins present in basement membranes. Among the many types of laminins, types 5, 6, and 10 are specific of the basal lamina present under stratified epithelia.

[0089] I-(iii) Systemic Lupus Erythematosus (SLE), Cutaneous Lupus, or Sjogren's Syndrome

[0090] Systemic lupus erythematosus (SLE) is an autoimmune disease involving multiple autoantigens, such as nucleic acids, histones, and other nuclear proteins. Sjogren's syndrome is an autoimmune disease, in which the immune system attacks the exocrine glands, specifically the salivary and lacrimal glands, which produce saliva and tears, respectively, resulting the symptoms of dry eyes and dry mouth, leading to infections and various other problems. Both of these diseases occur 9 times more frequently in women than in men, especially in women of child-bearing ages 15 to 35. SLE is a systemic autoimmune connective tissue disease and affects many organs and tissues. In general, those tissues and organs, such as the heart, lungs, bladder, and kidneys, which exhibit elasticity and can expand and contract, contain collagen network. In several types of SLE, cutaneous manifestation of inflammatory symptoms is prominent.

[0091] For more than 50 years, not a single new therapeutic agent had been developed for SLE, until belimumab, a human monoclonal antibody specific for BAFF was developed and approved. However, the therapeutic effect of belimumab for SLE has been considered to be marginal. Belimumab causes a host of side effects, including more incidences of serious infections and deaths in the treatment group than the placebo group. Interestingly, in a phase II trial on Sjogren's syndrome, belimumab showed more successful results than in SLE.

[0092] In addition to BAFF, researchers have been searching other therapeutic targets for SLE. While not a single inflammatory cytokine has been identified as mainly responsible for the pathological process in SLE, the expression of a group of genes known as downstream events of type 1 interferon stimulation, which is termed "type 1 interferon signature", has been documented in many studies. The pathogenesis of SLE has been found to be associated with the activation of toll-like receptors 7 and 9 (TLR 7 and TLR9), which induce the expression of a group of genes similar to that resulting from the activation by IFN-.alpha..

[0093] Several monoclonal antibodies specific for IFN-.alpha., including rontalizumab, sifalimumab, and anifrolumab have been studied in clinical trials for the treatment of SLE. Since IFN-.alpha. is involved in many functions, a systemic administration of an antibody against IFN-.alpha. without localized targeting to disease sites may render serious side effects.

[0094] I-(iv) Inflammatory Bowel Disease

[0095] Anti-TNF-.alpha. (such as adalimumab) has also been approved for treating Crohn's disease and ulcerative colitis (a form of inflammatory bowel disease). However, as described in an earlier section, the administration of anti-TNF-.alpha. is associated with a range of series side effects, including severe infectious diseases and B cell lymphoma. Therefore, in treating patients with Crohn's disease or ulcerative colitis with anti-TNF-.alpha., it will be desirable to distribute the administered anti-TNF-.alpha. in favor of the intestine and colon. It has been found collagen III and type V are relatively abundant in the connective tissues in the intestine and bowel.

[0096] II Osteoporosis Disease

[0097] An antibody specific for RANKL (CD254), the ligand of RANK (RANK, receptor activator of nuclear factor .kappa.B), denosumab, is approved for the treatment of osteoporosis. The development of denosumab represents a major advancement in the care for osteoporosis. However, the administration of denosumab causes common side effects, such as infections of the urinary and respiratory tracts, cataracts, constipation, rashes, and joint pain. It is hence desirable that the therapeutic agent is carried preferentially to the bone.

[0098] RANKL is a membrane protein, belonging to the tumor necrosis factor ligand family. RANKL is detected at high levels in the lung, thymus, and lymph nodes. It is also detected at low levels in the bone marrow, stomach, peripheral blood, spleen, placenta, leukocytes, heart, thyroid and skeletal muscle. Since IgG anti-RANKL, such as denosumab, can serve a therapeutic agent for osteoporosis, the molecular constructs of this invention should provide as better therapeutic agents than IgG anti-RANKL.

[0099] Another target for antibodies for the treatment of osteoporosis is sclerostin, encoded by SOST gene. The glycoprotein is produced and secreted by osteocytes and negatively regulates osteoblastic bone formation. The loss or defective mutation of SOST gene causes progressive bone thickening. A defective mutation in the SOST gene increases bone formation. Antibodies against sclerostin cause increased bone formation, bone mineral density, and stronger bones. The phase I and II clinical trials of two humanized monoclonal antibodies against sclerostin, blosozumab and romosozumab, indicated that the antibody treatment is associated with increased bone mineral density and bone formation and decreased bone resorption.

[0100] In light of the foregoing discussion, molecular platforms for constructing the T-E molecules of this invention are provided in the present disclosure. Detailed discussions relating to the structure of the molecular construct having the "Fc" configuration are provided below, as well as the practical applications of each molecular construct.

[0101] PART I Anti-Inflammatory Molecules with Tissue-Targeting Functions

[0102] In the broad sense of the Fc-based configuration, immunoglobulin antibody can serve as the base of a targeting or effector element, and its corresponding effector or targeting element can be incorporated at the C-terminal of its two heavy .gamma. chains in the form of scFv domains. For a typical "Fc-based" configuration, two-chain IgG.Fc is used as the base of the molecular platform. Each of the polypeptide chain is fused with one or two targeting and one or two effector elements, for a total of two to three elements on each chain. The T-E molecule with an Fc-based configuration will have a total of four to six elements (e.g., scFv, growth factor, or cytokines). Optionally, the Fc portion of the molecular constructs also carries Fc-mediated effector functions, ADCC, and/or complement-mediated activation. While in certain other applications, such Fc-mediated effector functions are avoided.

[0103] In designing the Fc-based molecular constructs, targeting elements are positioned at the N- or C-terminus. If the effector elements function by binding to a cell surface component, such as CD3, CD16a, PD-1, PD-L1, or CTLA-4, they should also be positioned at the terminus. If the effector elements function by binding to and neutralizing soluble factors, such as VEGF, TNF-.alpha., IL-17, or BAFF, they can be positioned between a terminal targeting or effector element and CH2-CH3.

[0104] In some embodiments of the present disclosure, both the effector element and the targeting element carried by the CH2-CH3 segment (or CH2-CH3 chain) are mostly comprised of amino acid residues, and for the sake of discussion, these molecular constructs are referred to anti-inflammatory molecules with tissue-targeting functions or anti-inflammatory Fc-based molecular construct. For example, the effector element may be an antibody fragment or a soluble receptor, while the targeting element is also an antibody fragment. Some illustrative structures of this Fc-based molecular construct are discussed in this section.

[0105] Referring to FIG. 1A, which is a schematic diagram illustrating an Fc-based molecular construct 800A according to certain embodiments of the present disclosure. As illustrated, the Fc-based molecular construct 800A comprises two identical CH2-CH3 chains 810, a first pair of effector elements E1 linked to the N-termini of the CH2-CH3 chains 810, and a first pair of targeting elements T1 linked to the C-termini of the CH2-CH3 chains 810. In this illustrative configuration, both the targeting element T1 and effector element E1 are antibody fragments.

[0106] In some embodiments, the CH2-CH3 chains are adopted from human immunoglobulins .gamma.1 or .gamma.4. In general, .gamma.1 is chosen, when Fc-mediated functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated activity (inflammatory activation or target cell lysis), are desired. In the case where Fc-mediated functions are avoided, .gamma.4 is chosen for constructing the present Fc-based molecular constructs.

[0107] The Fc-based molecular construct 800B illustrated in FIG. 1B is quite similar to the Fc-based molecular construct 800A of FIG. 1A in structure, except that the two effector elements E1 are respectively linked to the C-termini of the CH2-CH3 chains 810, while the two targeting effectors are respectively linked to the C-termini of the CH2-CH3 chains 810.

[0108] According to certain embodiments, both the effector elements and targeting elements are linked to the N-termini of the CH2-CH3 chains. For example, when both the effector element and the targeting element are in the form of single-chain variable fragments (scFvs), the effector element and the targeting element may be linked in a tandem or diabody configuration, thereby forming a bispecific scFv that is linked to the N-terminus of the CH2-CH3 chain.

[0109] The Fc-based molecular construct 800C (FIG. 1C) comprises an Fc portion, and accordingly, each CH2-CH3 chain 810 has a T1-E1 bispecific scFv linked to the N-terminus thereof.

[0110] As discussed above, the anti-inflammatory Fc-based molecular constructs can also use a soluble receptor (e.g., the soluble receptor of TNF-.alpha. or IL-1) as the effector element, according to certain embodiments. In these cases, the Fc-based molecular construct 800D (FIG. 1D) may have two effector elements E1 respectively linked to the N-termini of the CH2-CH3 chains 810, and two targeting elements T1 respectively linked to the C-termini of the CH2-CH3 chains 810. It is also possible that the effector elements and the targeting elements are respectively arranged at the C- and N-termini of the CH2-CH3 chains; see, for example, the Fc-based molecular construct 800E of FIG. 1E.

[0111] In some examples, the first pair of effector elements or the first pair of the targeting elements takes a Fab configuration (i.e., consisting of the V.sub.H-CH1 domain and the V.sub.L-C.kappa. domain); this Fab fragment is linked to the N-termini of the CH2-CH3 chains, so that the Fc-based molecular construct adopts an IgG configuration. In these cases, the pair of elements that is not in the Fab configuration may be linked to the C-termini of the pair of CH2-CH3 segments.

[0112] For example, in the Fc-based molecular construct 800F of FIG. 1F, each of the two targeting elements T1 comprises the V.sub.H-CH1 domain 820 and the V.sub.L-C.kappa. domain 825, thereby forming a Fab configuration 830 that is linked to the N-termini of the CH2-CH3 chains 810, so that the Fc-based molecular construct 800F adopts the IgG configuration. In this case, the pair of effector elements E1 is linked to the C-termini of the pair of CH2-CH3 chains 810.

[0113] As described above, the present Fc-based molecular construct may carry a total of six elements at most. The additional elements may be a second pair of effector elements or a second pair of targeting elements.

[0114] According to other embodiment, the Fc-based molecular construct 900A (FIG. 2A) comprises a second pair of targeting elements T2. In these cases, the targeting elements T1 and T2 are linked in a tandem or diabody configuration to form a bispecific scFv that is linked to the N-terminus of the CH2-CH3 chain 910, and the effector element E1 is linked to the C-terminus of the CH2-CH3 chain 910.

[0115] According to embodiments exemplified in FIG. 2B, the Fc-based molecular construct 900B comprises a second pair of effector elements E2. In these cases, the effector element E1 and E2 are linked in a tandem or diabody configuration to form a bispecific scFv that is linked to the N-terminus of the CH2-CH3 chain 910, and the targeting element T1 is linked to the C-terminus of the CH2-CH3 chain 910.

[0116] Now that the basic structural arrangements of the anti-inflammatory Fc-based molecular constructs have been discussed above, certain combinations of particular effector element(s) and targeting element(s) are provided below for the illustration purpose.

[0117] According to some embodiments, the effector element is an scFv specific for TNF-.alpha., and the targeting element is an scFv specific for collagen II or collagen IX, or .alpha.-aggrecan. According to some embodiments, each of the two effector elements is an scFv specific for IL-17, while the targeting element is an scFv specific for collagen I or collagen VII. Still alternatively, each of the two effector elements is an scFv specific for BAFF, and the targeting element is an scFv specific for collagen I or collagen VII. In some embodiments, each of the two effector elements is an scFv specific for TNF-.alpha., and the targeting element is an scFv specific for collagen III or collagen V. In some other embodiments, the two effector elements are in the form of a Fab specific for RANKL, and the targeting element is an scFv specific for collagen I or osteonectin. For example, such molecular construct may take the configuration of any of those depicted in FIGS. 1A-1C and 1F.

[0118] In some embodiments, the first pair of effector elements includes an scFv specific for TNF-.alpha. and an scFv specific for IL-17, while the first pair of targeting elements includes an scFv specific for collagen II and an scFv specific for collagen IX. In some alternative embodiments, the first pair of effector elements includes an scFv specific for TNF-.alpha. and an scFv specific for IL-17, while the first pair of targeting elements includes an scFv specific for collagen I and an scFv specific for collagen VII. Alternatively, each of the two effector elements is an scFv specific for BAFF, and the targeting element is an scFv specific for collagen I or collagen VII. Still alternatively, each of the two effector elements is an scFv specific for RANKL, and the targeting element is an scFv specific for collagen I or osteonectin. These molecular constructs may take the configuration of any of those depicted in FIGS. 1B, 1D, and 1F.

[0119] In certain embodiments, the two effector elements are in the form of a Fab antibody specific for TNF-.alpha., while the targeting element is an scFv specific for collagen II or collagen IX. In some embodiments, the two effector elements are in the form of a Fab antibody specific for IL-17, and the targeting element is an scFv specific for collagen I or collagen VII. Alternatively, the two effector elements are in the form of a Fab antibody specific for BAFF, and the targeting element is an scFv specific for collagen I or collagen VII. Still alternatively, the two effector elements are in the form of a Fab antibody specific for TNF-.alpha., and the targeting element is an scFv specific for collagen III or collagen V.

[0120] The essence of this invention is the rationalization and conception of the specific combination or pairing of the targeting and effector elements. The adoption of Fc-fusion configuration in the molecular constructs is a preferred embodiment. It is conceivable for those skilled in the arts to link the pairs of targeting and effector elements of this invention employing other molecular platforms, such as peptides, proteins (e.g., albumin), polysaccharides, polyethylene glycol, and other types of polymers, which serve as a structural base for attaching multiple molecular elements.

[0121] PART II Uses of Anti-Inflammatory Molecules with Tissue-Targeting Functions

[0122] Another aspect of the present disclosure is directed to the use of the anti-inflammatory Fc-based molecular constructs discussed above in PART I.

[0123] As could be appreciated, the description in Part IV-(i) regarding the rationales underlying the selection of suitable targeting and effector elements is also applicable in this section. For example, anti-inflammatory Fc-based molecular constructs used for treating various immune disorders contain an antibody fragment (e.g., scFv, Fab, and the like) specific for collagen II, collagen XI, or .alpha.-aggrecan used as targeting elements and an antibody fragment (e.g., scFv, Fab, and the like) specific for TNF-.alpha. and IL-17 as effector elements.

[0124] According to various embodiments of the present disclosure, the present treatment method involves the administration of a suitable anti-inflammatory Fc-based molecular construct to a subject in need of such treatment. Specific examples of anti-inflammatory Fc-based molecular constructs for treating various immune disorders, in particular, autoimmune diseases, are discussed below.

[0125] According to certain embodiments, the present method is used to treat rheumatoid arthritis, psoriatic arthritis, or ankylosing spondylitis. In these cases, each effector element of the anti-inflammatory Fc-based molecular construct is an antibody fragment specific for TNF-.alpha., IL-12/IL-23, IL-1, IL-17, or IL-6, while each targeting element is an antibody fragment specific for collagen II, collagen IX, collagen XI, or .alpha.-aggrecan. For example, each effector element of the first pair of effector elements is an scFv specific for TNF-.alpha., while each targeting element of the first pair of targeting elements is an antibody fragment specific for collagen II. In other embodiments, the effector element is an scFv specific for TNF-.alpha., while the targeting element is an antibody fragment specific for collagen IX. Alternatively, the effector element is an scFv specific for TNF-.alpha., while the targeting element is an antibody fragment specific for .alpha.-aggrecan. According to various embodiments, the above-mentioned anti-inflammatory Fc-based molecular constructs may have the configuration of 800A, 800B, or 800C discussed above.

[0126] Another anti-inflammatory Fc-based molecular construct for treating rheumatoid arthritis, psoriatic arthritis, or ankylosing spondylitis comprises two effector elements that are in the form of a Fab antibody specific for TNF-.alpha.. In these cases, both targeting elements of the first pair of targeting elements is scFvs specific for collagen II or scFvs specific for collagen IX. Configurations of these Fc-based molecular constructs are illustrated in FIG. 1F, for example.

[0127] The present methods are also applicable in the treatment of psoriasis. For example, the anti-inflammatory Fc-based molecular construct may comprise effector elements of an antibody fragment specific for TNF-.alpha., IL-12/IL-23, or IL-17, and targeting elements of an antibody fragment specific for collagen I or collagen VII. According to some embodiments, the effector elements are scFvs specific for IL-17, while the targeting elements are scFvs specific for collagen I. Alternatively, the effector elements are scFvs specific for IL-17, while the targeting elements are scFvs specific for collagen VII. These anti-inflammatory Fc-based molecular constructs have the configuration of 800A, 800B, or 800C discussed above.

[0128] Another anti-inflammatory Fc-based molecular construct for treating psoriasis comprises two effector elements that are in the form of a Fab antibody specific for IL-17. In these cases, both targeting elements of the first pair of targeting elements is scFvs specific for collagen I or scFvs specific for collagen VII. Configurations of these Fc-based molecular constructs are illustrated in FIG. 1F, for example.

[0129] Another set of diseases treatable by the present method using the anti-inflammatory Fc-based molecular constructs are systemic lupus erythematosus, cutaneous lupus, or Sjogren's Syndrome. In these embodiments, each effector is an antibody fragment specific for BAFF, and each targeting element is an antibody fragment specific for collagen I or collagen VII. These anti-inflammatory Fc-based molecular constructs may have the configuration illustrated in FIGS. 1A to 1C. As could be appreciated, the pair of effector elements may also take the form of a Fab antibody specific for BAFF, and the pair of targeting elements may be scFvs specific for collagen I or collagen VII, which takes the configuration of the molecular construct 800F illustrated in FIG. 1F.

[0130] In other embodiments, the present method is used to treat inflammatory bowel disease, such as Crohn's disease or ulcerative colitis. In these cases, each effector is an antibody fragment specific for TNF-.alpha., and each targeting element is an antibody fragment specific for collagen III or collagen V. Configurations of these Fc-based molecular constructs are illustrated in FIGS. 1A to 1C, for example. Of course, the pair of effector elements may also take the form of a Fab antibody specific for TNF-.alpha., while the pair of targeting elements may be scFvs specific for collagen III or collagen V, thereby giving the configuration illustrated in FIG. 1F.

[0131] The present anti-inflammatory Fc-based molecular constructs are also applicable in the treatment of osteoporosis. For example, the effector elements are antibody fragments specific for RANKL, while the targeting elements are antibody fragments specific for collagen I or osteonectin. As could be appreciated, the antibody fragments specific for RANKL may be scFvs so that the Fc-based molecular construct has the configuration illustrated in FIG. 1A to 1C, or they may take the form of a Fab so that the Fc-based molecular construct has the configuration illustrated in FIG. 1F.

[0132] It should be noted that above-examples are given for the purpose of illustration, and treatments using anti-inflammatory Fc-based molecular constructs with other T-E combinations are within the scope of the present disclosure.

EXPERIMENTAL EXAMPLES

Example 1: Construction of Gene Segments Encoding 2-Chain IgG4.Fc Fusion Protein Containing scFv Specific for Human Collagen II and scFv Specific for TNF-.alpha.

[0133] Mouse B cell hybridoma II-116B3 producing anti-collagen II antibody was purchased from Developmental Studies Hybridoma Bank at the University of Iowa. Poly(A)+ RNA was reverse-transcribed with a SuperScript III RT-PCR system (Invitrogen, Waltham, USA), and first strand cDNA was synthesized. The V.sub.H and V.sub.L nucleotide and amino acid sequences of II-116B3 had not been published. To determine the sequences of variable regions of II-116B3, cDNA of V.sub.H and V.sub.L were amplified by PCR using a set of DNA primers provided by Ig-primer Sets (Novagen, Madison, USA) per the manufacturer's instructions. The amino acid sequence of V.sub.H and V.sub.L of II6B3 monoclonal antibody specific for collagen type II (CII, or COL2) are described in SEQ ID NOs: 3 and 4. The sequences of V.sub.L and V.sub.H of scFv specific for TNF-.alpha. were those of V.sub.L and V.sub.H of adalizumab.

[0134] Illustrated below is the configuration of 2-chain IgG4.Fc fusion protein molecular construct. The scFv1-scFv2-CH2-CH3 (human .gamma.4) recombinant chain was configured by fusing two scFvs, one specific for human collagen II and the other specific for human TNF-.alpha., to the N-terminal of CH2 domain of IgG4.Fc through a flexible hinge region. The first scFv (specific for collagen II) had an orientation of V.sub.L-linker-V.sub.H and the second scFv (specific for TNF-.alpha.) was in V.sub.H-linker-V.sub.L. The V.sub.L and V.sub.H in each of the two scFv were connected by a hydrophilic linker, GSTSGSGKPGSGEGSTKG. The two scFvs were connected via a flexible linker, (GGGGS).sub.3. The sequence of the recombinant chain in the IgG4.Fc fusion protein molecular construct illustrated below is described in SEQ ID NO: 5.

[0135] Illustrated below is the configuration of the present 2-chain (scFv .alpha. collagen II)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc molecular construct.

Example 2: Expression and Purification of Recombinant 2-Chain (scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc Fusion Protein

[0136] In this Example, the gene-encoding sequence was placed in pcDNA3 expression cassette. Expi293F cells were seeded at a density of 2.0.times.10.sup.6 viable cells/ml in Expi293F expression medium and maintained for 18 to 24 hours prior to transfection to ensure that the cells were actively dividing at the time of transfection. On the day of transfection, 7.5.times.10.sup.8 cells in 255-ml medium in a 2-liter Erlenmeyer shaker flask were transfected by ExpiFectamine.TM. 293 transfection reagent. The transfected cells were incubated at 37.degree. C. for 16 to 18 hours post-transfection in an orbital shaker (125 rpm) and the cells were added ExpiFectamine.TM. 293 transfection enhancer 1 and enhancer 2 to the shaker flask, and incubated for another 7 days. Culture supernatants were harvested and recombinant 2-chain (scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc fusion proteins in the media were purified using Protein A chromatography. Following buffer exchange to PBS, the concentration of (scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4Fc proteins was determined and analyzed by SDS-PAGE; see, FIG. 3; II-116B3 (lane 1) and (scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4Fc (lane 2) were analyzed in 10% SDS-PAGE. The Fc-fusion molecular construct was revealed as the major band at about 80 kDa, consistent with the expected size.

Example 3: ELISA Analysis of the Binding of Recombinant 2-Chain (scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc Fusion Protein

[0137] To examine the binding ability of recombinant 2-chain (scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc fusion protein to type II collagen, ELISA assay was performed, using adalimumab and mouse parental monoclonal antibody II-116B3 for comparison. ELISA plates were coated with 5 .mu.g/mL of human type II collagen (human COL2), mouse type II collagen (mouse COL2), and chicken type II collagen (chick COL2). 1D11 was a human IgG1 antibody against mite allergen as an isotype control. Recombinant 2-chain (scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc fusion protein, purified anti-collagen II antibody (II-116B3) and adalimumab were detected by HRP-conjugated goat anti-human IgG4.Fc, goat anti-mouse IgG.Fc, and goat anti-human IgG1.Fc, respectively. The ELISA results were summarized in FIG. 4A.

[0138] To examine the binding ability of 2-chain (scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc fusion protein to human TNF-.alpha., ELISA assay was performed, along with adalimumab and mouse parental monoclonal antibody II-116B3. ELISA plates were coated with 1 .mu.g/mL of human TNF-.alpha. and 1 .mu.g/mL of human serum albumin as a control. Recombinant 2-chain (scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc fusion protein, purified anti-collagen II antibody (II-116B3) and Adalimumab were detected by HRP-conjugated goat anti-human IgG4.Fc, goat anti-mouse IgG.Fc, and goat anti-human IgG1.Fc, respectively. The results, as summarized in FIG. 4B, showed that recombinant 2-chain (scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc fusion protein displayed significant binding activity toward human TNF-.alpha.; HSA (human serum albumin) was used as control.

Example 4: Preparation of 2-Chain IgG4.Fc Fusion Protein Containing scFv Specific for Human Collagen II, scFv Specific for TNF-.alpha. and scFv Specific for Human IL-17

[0139] The scFv1-scFv2-CH2-CH3-scFv3 (human .gamma.4) recombinant chain was configured by fusing three scFvs, in which the first one specific for human collagen II and the second one specific for TNF-.alpha. were fused to the N-terminal of CH2 domain of IgG4.Fc through a flexible hinge region, while the third one specific for IL-17 was fused to the C-terminal of CH3 domain.

[0140] The V.sub.H and V.sub.L of the scFv specific for collagen II were from monoclonal antibody II-116B3; the V.sub.H and V.sub.L of the scFv specific for TNF-.alpha. were from monoclonal antibody adalimumab; V.sub.H and V.sub.L of the scFv specific for IL-17 were from secukinumab. The first scFv (specific for collagen II) had an orientation of V.sub.L-linker-V.sub.H, the second scFv (specific for TNF-.alpha.) was in the orientation of V.sub.H-linker-V.sub.L, and the third scFv (specific for IL-17) was in the orientation of V.sub.L-linker-V.sub.H. The V.sub.L and V.sub.H in each of the three scFv were connected by a hydrophilic linker, GSTSGSGKPGSGEGSTKG. The three scFv were fused via a flexible linker, (GGGGS).sub.3.

[0141] The sequence of the recombinant chain in the IgG4.Fc fusion protein molecular construct of the construct illustrated below is shown in SEQ ID NO: 6. The expression of the constructed genes in Expi293F cells and the purification of the expressed fusion protein were performed as in preceding Examples. Characterization of the new construct was performed with SDS-PAGE and ELISA. The antibody Secukinumab (Cosentyx) was purchased from Chang Gung Hospital (Taipei, Taiwan); human IL-17 was from Peprotech (NJ, USA). FIG. 5A shows the SDA-PAGE results, indicating that the recombinant chain of the new construct had a size of about 110 kDa, consistent with the expected size. FIG. 5B shows the ELISA results, indicating that the present recombinant Fc-fusion protein had the binding activity to human collagen II, human TNF-.alpha., and human IL-17.

[0142] Illustrated below is the configuration of the present 2-chain (scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc-(scFv .alpha. IL-17) molecular construct.

Example 5: Preparation of 2-Chain IgG1.Fc Fusion Protein Containing TNF-.alpha. Soluble Receptor and scFv Specific for Collagen II

[0143] The (TNF-.alpha. receptor)-CH2-CH3-scFv .alpha. collagen II (human .gamma.1) recombinant chain was configured by fusing human TNF-.alpha. receptor, IgG1.Fc, and scFv specific for collagen II. The sequences of TNF-.alpha. receptor and IgG1.Fc were those of etanercept. Etanercept and scFv were fused via a flexible linker, (GGGGS).sub.3. The sequence of the recombinant chain in the IgG4.Fc fusion protein molecular construct is shown in SEQ ID NO: 7.

[0144] The expression of the constructed genes in Expi293F cells and the purification of the expressed fusion protein were performed as in the preceding Examples. Characterization of the new construct was performed with SDS-PAGE and ELISA. Etanercept (Enbrel) was purchased from Chang Gung Hospital (Taipei, Taiwan). FIG. 6A shows the SDS-PAGE results, indicating that the recombinant chain in the new molecular construct had a size of about 100 kDa, consistent with the expected size (an Etanercept molecule had a molecular weight of 150 kDa and one chain of it was about 75 kDa in size; an scFv was about 27 kDa in size). FIG. 6B shows the ELISA results, indicating the present molecular construct bound to human TNF-.alpha., human collagen II, and mouse collagen II.

[0145] Illustrated below is the configuration of the present 2-chain (soluble TNF-.alpha. receptor)-IgG1.CH2-CH3-scFv .alpha. collagen II molecular construct.

Example 6: Preparation of 2-Chain Fusion Protein Containing Intact Antibody for Human TNF-.alpha. and scFv Specific for Collagen II

[0146] The IgG-scFv (human .gamma.1) recombinant chain was configured by fusing the intact antibody specific for human TNF-.alpha. and scFv specific for collagen II (illustrated below). The sequences of intact antibody were those of adalimumab. Adalimumab and scFv were fused via a flexible linker, (GGGGS).sub.3.

[0147] The construction of the heavy and light chains of recombinant genes was built by inserting the two genes into a pG1K expression cassette with the multiple cloning site. To prepare the 2-chain fusion protein containing intact antibody for human TNF-.alpha. and scFv specific for collagen type II, transfection of the expression vectors into Expi293F cells was performed as in preceding Examples. The amino acid sequence of the heavy chain of 2-chain fusion protein containing intact antibody for human TNF-.alpha. and scFv specific for collagen type II is indicated in SEQ ID NO: 8, and the amino acid sequence of the light chain of the 2-chain fusion protein is indicated in SEQ ID NO: 9.

[0148] FIG. 7 shows the SDS-PAGE result, indicating that the heavy recombinant chain in the present extended IgG molecular construct had a size of about 75 kDa, consistent with the expected size.

[0149] Illustrated below is the configuration of the present anti-human TNF-.alpha. in extended IgG configuration with scFv specific for human collagen II at the C-terminal.

Example 7: Preparation of 2-Chain Fusion Protein Containing Intact Antibody for Human IL-17 and scFv Specific for Collagen VII

[0150] The IgG-scFv (human .gamma.1) recombinant chain was configured by fusing the intact antibody specific for human IL-17 and scFv specific for collagen type VII (SEQ ID NO: 20). The sequences of the intact antibody were those of secukinumab. Secukinumab and the scFv were fused via a flexible linker, (GGGGS).sub.3.

[0151] The construction of the heavy and light chains of recombinant genes was built by inserting the two genes into a pG1K expression cassette with the multiple cloning site. To prepare the 2-chain fusion protein containing intact antibody for human IL-17 and scFv specific for collagen type VII, transfection of the expression vectors into Expi293F cells was performed as in the preceding Example. The amino acid sequence of the heavy chain of 2-chain fusion protein containing intact antibody for human IL-17 and scFv specific for collagen type VII is indicated in SEQ ID NO: 10, and the amino acid sequence of the light chain of the 2-chain fusion protein is indicated in SEQ ID NO: 11.

[0152] FIG. 8A shows the SDS-PAGE results indicating that the recombinant heavy chain of the new molecular construct had a size of 75 kDa, consistent with the expected size. FIG. 8B shows the ELISA results, indicating that the present molecular construct bound to human IL17 and human collagen VII. The relatively low binding to collagen VII was due to a low concentration the antigen used for coating the ELISA plates

[0153] Illustrated below is the present anti-human IL-17 in extended IgG configuration with scFv specific for human collagen VII at the C-terminal.

Example 8: Preparation of 2-Chain IgG4.Fc Fusion Protein Containing scFv Specific for Human Collagen VII and scFv Specific for BAFF

[0154] cDNA of Mouse B cell hybridoma LH7.2 mAb producing anti-collagen VII antibody was a gift from Dr. Purdie, cancer research UK skin tumor laboratory at Queen Mary University of London. The V.sub.H and V.sub.L sequences of LH7.2 monoclonal antibody (SEQ ID NOs: 1 and 2) were determined in an earlier Example. The sequences of V.sub.L and V.sub.H of scFv specific for BAFF were those of V.sub.L and V.sub.H of belimumab.

[0155] The scFv1-CH2-CH3-scFv2 (human .gamma.4) recombinant chain (SEQ ID NO: 12) was configured by fusing two scFv, spaced with IgG4.Fc, one specific for collagen VII and the other specific for BAFF, to the C-terminal of CH3 domain of IgG4.Fc through a flexible linker, (GGGGS).sub.3 (illustrated below). The first scFv (specific for collagen VII) had an orientation of V.sub.L-linker-V.sub.H and the second scFv (specific for BAFF) was in V.sub.H-linker-V.sub.L. The V.sub.L and V.sub.H in each of the two scFv were connected by a hydrophilic linker, GSTSGSGKPGSGEGSTKG.

[0156] The expression of the constructed genes in Expi293F cells and the purification of the expressed fusion protein were performed as in an earlier Example. Characterization of the new construct was performed with SDS-PAGE and ELISA. The antibody Belimumab (Belynsta) was purchased from Chang Gung Hospital (Taipei); human BAFF was from GenScript (NJ, USA). FIG. 9A shows the SDA-PAGE results, indicating that the recombinant chain in the new molecular construct had a size of about 80-90 kDa, consistent with or somewhat larger than the expected size. FIG. 9B shows the ELISA results, indicating that the new construct bound specifically to human BAFF and human collagen VII.

[0157] Illustrated below is the configuration of the present 2-chain Fc-fusion molecular construct with scFv .alpha. collagen VI I-IgG4.CH2-CH3-scFv .alpha. BAFF.

Example 9: Preparation of 2-Chain Fusion Protein Containing Intact Antibody for Human BAFF and scFv Specific for Collagen VII

[0158] The IgG-scFv (human .gamma.1) recombinant chain was configured by fusing the intact antibody specific for human BAFF and scFv specific for collagen type VII. The sequences of intact antibody were those of belimumab. Belimumab and the scFv were fused via a flexible linker, (GGGGS).sub.3.

[0159] The construction of the heavy and light chains of recombinant genes was built by inserting the two genes into a pG1K expression cassette with the multiple cloning site. To prepare the 2-chain fusion protein containing intact antibody for human BAFF and scFv specific for collagen type VII, transfection of the expression vectors into Expi293F cells was performed as in preceding Examples.

[0160] The amino acid sequence of the heavy chain of 2-chain fusion protein containing intact antibody for human BAFF and scFv specific for collagen type VII is indicated in SEQ ID NO: 13, and the amino acid sequence of the light chain of the 2-chain fusion protein is indicated in SEQ ID NO: 14. FIG. 10A shows the SDS-PAGE results, indicating that the recombinant heavy chain of the new molecular construct had a size of about 80 kDa, consistent with the expected size. FIG. 10B shows the ELISA results, indicating that the new extended IgG construct bound specifically to human BAFF and human collagen VII.

[0161] Illustrated below is the configuration of the present anti-human BAFF extended IgG configuration with scFv specific for human collagen VII at the C-terminal.

Example 10: Preparation of 2-Chain IgG4.Fc Fusion Protein Containing scFv Specific for Human Osteonectin and scFv Specific for RANKL

[0162] Mouse B cell hybridoma AON-1 producing anti-osteonectin (SPARC) antibody was purchased from Developmental Studies Hybridoma Bank at the University of Iowa. Poly(A)+ RNA was reverse-transcribed with a SuperScript III RT-PCR system (Invitrogen), and the first strand cDNA was synthesized. The V.sub.H and V.sub.L nucleotide and amino acid sequences of AON-1 had not been published. To determine the sequences of variable regions of AON-1, cDNA of V.sub.H and V.sub.L were amplified by PCR using a set of DNA primers provided by Ig-primer Sets (Novagen) according to the manufacturer's instructions. The V.sub.H and V.sub.L sequences of AON-1 monoclonal antibody specific for osteonectin are shown in SEQ ID NOs: 15 and 16. The sequences of V.sub.L and V.sub.H of scFv specific for RANKL were those of V.sub.L and V.sub.H of denosumab.

[0163] The scFv1-scFv2-CH2-CH3 (human .gamma.4) recombinant chain (SEQ ID NO: 17) was configured by fusing two scFv, one specific for human osteonectin and the other specific for RANKL, to the N-terminal of CH2 domain of IgG4.Fc through a flexible hinge region (illustrated below). The first scFv (specific for osteonectin) had an orientation of V.sub.L-linker-V.sub.H and the second scFv (specific for RANKL) was in V.sub.H-linker-V.sub.L. The V.sub.L and V.sub.H in each of the two scFv were connected by a hydrophilic linker, GSTSGSGKPGSGEGSTKG. The two scFv were fused via a flexible linker, (GGGGS).sub.3.

[0164] The expression of the constructed genes in Expi293F cells and the purification of the expressed fusion protein were performed as in an earlier Example. The characterization of the binding of the fusion protein to human osteonectin and RANKL by ELISA was performed as in a preceding Example. Characterization of the new construct was performed with SDS-PAGE and ELISA. The antibody Denosumab (Prolia) was purchased from Chang Gung Hospital; human RANKL and human osteonectin (SPARC) were from GenScript. FIG. 11A shows the SDS-PAGE results, indicating that the recombinant chain in the new molecular construct had a size of about 80 kDa. FIG. 11B shows the ELISA results, indicating that the new Fc-fusion construct bound specifically to human SPARC and human RANKL.

[0165] Illustrated below is the configuration of the present 2-chain (scFv .alpha. SPARC)-(scFv .alpha. RANKL)-hIgG4.Fc molecular construct.

Example 11: Preparation of 2-Chain Fusion Protein Containing Intact Antibody for Human RANKL and scFv Specific for Human Osteonectin

[0166] The IgG-scFv (human .gamma.1) recombinant chain was configured by fusing the intact antibody specific for human RANKL and scFv specific for human osteonectin. The sequences of intact antibody were those of denosumab. Denosumab and the scFv were fused via a flexible linker, (GGGGS).sub.3.

[0167] The construction of the heavy and light chains of recombinant genes was built by inserting the two genes into a pG1K expression cassette with the multiple cloning site. To prepare the 2-chain fusion protein containing the intact antibody for human RANKL and scFv specific for human osteonectin, transfection of the expression vectors into Expi293F cells was performed as in preceding Examples.

[0168] The amino acid sequence of the heavy chain of 2-chain fusion protein containing intact antibody for human RANKL and the scFv specific for human osteonectin is indicated in SEQ ID NO: 18 and the amino acid sequence of the light chain of the 2-chain fusion protein is indicated in SEQ ID NO: 19. FIG. 12A shows the SAS-PAGE results, indicating that the recombinant heavy chain in the extended IgG molecular construct had a size of about 80 kDa. FIG. 12B shows the ELISA results, indicating that the new Fc-fusion construct bound specifically to human SPARC and human RANKL.

[0169] Illustrated below is the configuration of the present anti-human RANKL in extended IgG configuration with scFv specific for human osteonectin at the C-terminal.

Example 12: Immunohistologic Chemical Analysis of 2-Chain IgG4.Fc Fusion Protein Containing scFv Specific for Human Collagen II and scFv Specific for TNF-.alpha. in Binding to Joint Cartilage

[0170] Immunohistologic analysis was performed in the histology core facility of Genomics Research Center, Academia Sinica to examine whether the molecular construct, 2-chain (scFv .alpha. collagen II)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc (the configuration illustrated in an earlier Example), had affinity for binding to cartilage. Mouse bone and cartilage samples were obtained from FVB/N mice sacrificed by using CO.sub.2. Femur connected with tibia and knee femoral ends were harvested and fixed with 10% neutral buffered formaldehyde at room temperature for 48 hours. Samples were then decalcified in 10% EDTA, pH 7.4, for 7 days, with daily renewal of the solution. After decalcification, samples were post-fixed in 10% neutral buffered formaldehyde at room temperature for 24 hours and stored in 70% ethanol at 4.degree. C. until dehydration by ASP6025 Tissue Processor (Leica) and paraffin embedding.

[0171] Safranin 0 staining was performed according to the protocol described in Schmitz et al., 2010. For immunostaining, 3-.mu.m-thick sections were deparaffinized and rehydrated using Leica AutoStainer XL, followed by staining procedures described in the Tyramide Signal Amplification Biotin kit (PerkinElmer). In brief, sections were quenched for endogenous peroxidase activity in 3% H.sub.2O.sub.2 for 15 minutes, followed by antigen retrieval with 1 mg/mL hyaluronidase (Sigma Aldrich) in 37.degree. C. for 20 minutes and 20 .mu.g/mL proteinase k (TOOLS) at room temperature for 10 minutes. Sections were next blocked in TNB buffer of the TSA kit. For staining with mouse II-116B3 antibody, additional mouse IgG blocking reagent (Vector Laboratories) was added preceding TNB blocking. Both primary antibodies II-116B3 and (scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4Fc were used at 50 .mu.g/mL. Goat anti-mouse IgG Fc and goat anti-human IgG Fc (Jackson ImmunoResearch) were use at 1.6 .mu.g/mL for incorporating HRP, which reacted with the subsequently added biotin-tyramide. The biotin labels were then probed with streptavidin-HRP and chromogenically visualized with diaminobenzidine substrate (BioGenex). Sections were counterstained using hematoxylin and mounted with Leica CV5030 Coverslipper.

[0172] FIGS. 13 panel A to 13 panel C showed the immunostaining of mouse epiphyseal bone with monoclonal antibodies II-116B3 (FIG. 13 panel A), adalimumab (FIG. 13 panel B), and (scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4Fc, i.e., the configuration illustrated in an earlier Example (FIG. 13 panel C), followed by HRP-labeled goat anti-mouse or goat anti-human secondary antibodies and tyramide amplification. Type II collagen was revealed at epiphyseal articular cartilage (AC) and growth plate (GP) in FIG. 13 panel A and FIG. 13 panel C. The results showed that the anti-collagen II monoclonal antibody II-116B3 stained the AC and GP parts prominently (FIG. 13 panel A), while adalimumab had no significant staining (FIG. 13 panel B). The present construct also stained positively (FIG. 13 panel C). The positive staining originally revealed in brown color was converted to black/gray. The scale bar represents 250 .mu.m.

Example 13: Immunohistologic Chemical Analysis of 2-Chain IgG1.Fc Fusion Protein Containing scFv Specific for Collagen II and scFv Specific for TNF-.alpha. and scFv Specific for IL-17 in Binding to Joint Cartilage

[0173] The preparation of tissue thin sections, staining with the molecular construct, 2-chain (scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc-(scFv .alpha. IL-17) (the configuration illustrated in an earlier Example) and controls were performed as in the preceding Examples.

[0174] FIGS. 13 panel D to 13 panel E showed the immunostaining of collagen II in mouse epiphyseal bone. 3-.mu.m thick sections of the femoral end of mouse knee were stained with monoclonal antibodies, an anti-IL17a mouse antibody (purchased from PeproTech, NJ, USA) (FIG. 13 panel D), and the present construct 2-chain (scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc-(scFv .alpha. IL-17) (FIG. 13 panel E), followed by HRP-labeled goat anti-mouse or goat anti-human secondary antibodies and tyramide amplification. The results showed that anti-IL17 monoclonal antibody had no significant staining, while the present construct had moderately positive staining.

Example 14: Immunohistologic Chemical Analysis of 2-Chain IgG1.Fc Fusion Protein Containing TNF-.alpha. Soluble Receptor and scFv Specific for Collagen II in Binding to Joint Cartilage

[0175] The preparation of tissue thin sections, staining with the molecular construct, 2-chain (soluble TNF-.alpha. receptor)-IgG1.CH2-CH3-scFv .alpha. collagen II (the configuration illustrated in an earlier Example) and controls were performed as in the preceding Example.

[0176] The staining procedure was the same as in preceding examples. The mouse epiphyseal bone tissue section samples were from the same batch. The results showed that the positive control II-116B3 stained strongly (FIG. 14 panel A), etanercept had no significant staining (FIG. 14 panel B), and the present construct stained collagen II-containing components AC and GP positively (FIG. 14 panel C).

Example 15: Bio-Distribution of Recombinant 2-Chain IgG4.Fc Fusion Protein Containing scFv Specific for Human Osteonectin (SPARC) and scFv Specific for RANKL Using In Vivo Imaging System

[0177] A Dylight 680 Antibody Labeling Kit (Thermo Scientific) was used to conjugate denosumab and (scFv .alpha. SPARC)-(scFv .alpha. RANKL)-hIgG4Fc (scheme 67 of Example 60), according to the manufacturer's instructions. 8 to 10-week-old BALB/c mice were injected intravenously with PBS or 40 .mu.g labeled antibodies. At various time points, mice were anaesthetized with isoflurane in O.sub.2 and placed in the IVIS Spectrum In Vivo Imaging System (PerkinElmer) with a supine position. Fluorescent images were captured with ex/em=675/720, using the Living Image Software V3.2.

[0178] To investigate the targeting effect of (scFv .alpha. SPARC)-(scFv .alpha. RANKL)-hIgG4Fc, tissue distribution of antibodies in mice was compared, through observing fluorescent signals from the abdominal aspect. Fluorescent images from BALB/c mice were acquired and analyzed at 30 minutes, 3 hours, and 28 hours after the administration of DyLight 680-conjugated antibodies. 30 minutes after the injection, the penetration of anti-SPARC, (scFv .alpha. SPARC)-(scFv .alpha. RANKL)-hIgG4Fc, and BoneTag into limbs was greater than that observed with denosumab. In addition to bladder accumulation, the distribution of denosumab, anti-SPARC, and (scFv .alpha. SPARC)-(scFv .alpha. RANKL)-hIgG4Fc was more dispersed after 3 hours, while BoneTag was restricted to the head and the limbs. Bone structures were clearly resolved with remaining anti-SPARC 28 hours after the antibody administration.

[0179] FIG. 15 showed bio-distribution of fluorescence-labeled antibodies in vivo. BALB/c mice were intravenously injected with PBS (1), denosumab (2), anti-SPARC mAb (3), (scFv .alpha. SPARC)-(scFv .alpha. RANKL)-hIgG4Fc (4), and BoneTag (5). Images were captured at indicated time points using IVIS Spectrum imager and analyzed with Living Image software. Spectral unmixing was performed to distinguish tissue autofluorescence from DyLight 680 signals. The results showed that in comparison to denosumab, the present construct was distributed more similarly to anti-SPARC monoclonal antibody at 30 minutes after treatment. At longer time points, the distribution was influenced by half-lives of the reagents. Anti-SPARC and denosumab were both antibodies and had similar serum half-lives.

[0180] It will be understood that the above description of embodiments is given by way of example only and that various modifications may be made by those with ordinary skill in the art. The above specification, examples and data provide a complete description of the structure and use of exemplary embodiments of the invention. Although various embodiments of the invention have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those with ordinary skill in the art could make numerous alterations to the disclosed embodiments without departing from the spirit or scope of this invention.

Sequence CWU 1

1

201112PRTArtificial Sequence1F10-VH 1Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ser Met His Trp Val Lys Gln Ala Pro Gly Glu Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Tyr Tyr Gly Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 110 2111PRTArtificial Sequence1F10-VL 2Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Gly Tyr Met Asp Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Phe Ala Ala Ser Asn Leu Lys Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Thr Asn 85 90 95 Glu Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 3134PRTArtificial SequenceII6B3-VH 3Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Lys Leu Ser Cys Val Ala Ser Gly Ile Thr Phe Ser Asp Tyr 20 25 30 Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Glu Ile Arg Leu Lys Ser Ser Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr 85 90 95 Tyr Cys Thr Pro Leu Thr Gly Gly Phe Val Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro 115 120 125 Leu Val Pro Gly Ser Leu 130 4129PRTArtificial SequenceII6B3-VL 4Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Phe Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Lys 115 120 125 Leu 5741PRTArtificial Sequence(scFv anti-collagen II)-(scFv anti-TNF-alpha)-hIgG4.Fc 5Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Phe Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 115 120 125 Thr Lys Gly Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln 130 135 140 Pro Gly Gly Ser Met Lys Leu Ser Cys Val Ala Ser Gly Ile Thr Phe 145 150 155 160 Ser Asp Tyr Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu 165 170 175 Glu Trp Val Ala Glu Ile Arg Leu Lys Ser Ser Asn Tyr Ala Thr His 180 185 190 Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser 195 200 205 Lys Ser Ser Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr 210 215 220 Gly Ile Tyr Tyr Cys Thr Pro Leu Thr Gly Gly Phe Val Tyr Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Gly Gly Gly 245 250 255 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu 260 265 270 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Arg Leu 275 280 285 Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Ala Met His Trp 290 295 300 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Thr 305 310 315 320 Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val Glu Gly Arg Phe 325 330 335 Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn 340 345 350 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Ser 355 360 365 Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly Gln Gly Thr Leu 370 375 380 Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser 385 390 395 400 Gly Glu Gly Ser Thr Lys Gly Asp Ile Gln Met Thr Gln Ser Pro Ser 405 410 415 Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala 420 425 430 Ser Gln Gly Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly 435 440 445 Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly 450 455 460 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 465 470 475 480 Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln 485 490 495 Arg Tyr Asn Arg Ala Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu 500 505 510 Ile Lys Arg Ala Ser Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 515 520 525 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 530 535 540 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 545 550 555 560 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 565 570 575 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 580 585 590 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 595 600 605 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 610 615 620 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 625 630 635 640 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 645 650 655 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 660 665 670 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 675 680 685 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 690 695 700 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 705 710 715 720 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 725 730 735 Leu Ser Leu Gly Lys 740 61015PRTArtificial Sequencefusion protein containing scFv specific for human collagen type II, scFv specific for TNF-alpha and scFv specific for human IL-17 6Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Phe Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 115 120 125 Thr Lys Gly Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln 130 135 140 Pro Gly Gly Ser Met Lys Leu Ser Cys Val Ala Ser Gly Ile Thr Phe 145 150 155 160 Ser Asp Tyr Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu 165 170 175 Glu Trp Val Ala Glu Ile Arg Leu Lys Ser Ser Asn Tyr Ala Thr His 180 185 190 Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser 195 200 205 Lys Ser Ser Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr 210 215 220 Gly Ile Tyr Tyr Cys Thr Pro Leu Thr Gly Gly Phe Val Tyr Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Gly Gly Gly 245 250 255 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu 260 265 270 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Arg Leu 275 280 285 Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Ala Met His Trp 290 295 300 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Thr 305 310 315 320 Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val Glu Gly Arg Phe 325 330 335 Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn 340 345 350 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Ser 355 360 365 Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly Gln Gly Thr Leu 370 375 380 Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser 385 390 395 400 Gly Glu Gly Ser Thr Lys Gly Asp Ile Gln Met Thr Gln Ser Pro Ser 405 410 415 Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala 420 425 430 Ser Gln Gly Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly 435 440 445 Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly 450 455 460 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 465 470 475 480 Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln 485 490 495 Arg Tyr Asn Arg Ala Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu 500 505 510 Ile Lys Arg Ala Ser Gly Gly Ser Pro Pro Cys Pro Ser Cys Pro Ala 515 520 525 Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 530 535 540 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 545 550 555 560 Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 565 570 575 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 580 585 590 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 595 600 605 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 610 615 620 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 625 630 635 640 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 645 650 655 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 660 665 670 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 675 680 685 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 690 695 700 Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe 705 710 715 720 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 725 730 735 Ser Leu Ser Leu Ser Leu Gly Lys Gly Thr Gly Gly Gly Gly Ser Gly 740 745 750 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser 755 760 765 Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys 770 775 780 Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln 785 790 795 800 Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg 805 810 815 Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 820 825 830 Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr 835 840 845 Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Cys Thr Phe Gly Gln Gly Thr 850 855 860 Arg Leu Glu Ile Lys Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly 865 870 875 880 Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly 885 890 895 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 900 905 910 Ser Gly Phe Thr Phe Ser Asn Tyr Trp Met Asn Trp Val Arg Gln Ala 915 920 925 Pro Gly Lys Gly Leu Glu Trp Val Ala Ala Ile Asn Gln Asp Gly Ser 930 935 940 Glu Lys Tyr Tyr Val Gly Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 945 950 955 960 Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Val 965 970 975 Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg Asp Tyr Tyr Asp Ile Leu 980 985 990 Thr Asp Tyr Tyr Ile His Tyr Trp Tyr Phe Asp Leu Trp Gly Arg Gly 995 1000 1005 Thr Leu Val Thr Val Ser Ser 1010 10157735PRTArtificial Sequencefusion protein

containing TNF-alpha soluble receptor and scFv specific for collagen type II 7Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser 1 5 10 15 Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys 20 25 30 Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr 35 40 45 Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu 50 55 60 Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser 65 70 75 80 Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys 85 90 95 Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys 100 105 110 Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala 115 120 125 Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro 130 135 140 Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 145 150 155 160 Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala 165 170 175 Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val 180 185 190 His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr 195 200 205 Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly 210 215 220 Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys Gly Gly Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly 465 470 475 480 Gly Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser 485 490 495 Leu Gly Asp Gln Ala Ser Ile Phe Cys Arg Ser Ser Gln Ser Ile Val 500 505 510 His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly 515 520 525 Gln Ser Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly 530 535 540 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 545 550 555 560 Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe 565 570 575 Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu 580 585 590 Ile Lys Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu 595 600 605 Gly Ser Thr Lys Gly Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu 610 615 620 Val Gln Pro Gly Gly Ser Met Lys Leu Ser Cys Val Ala Ser Gly Ile 625 630 635 640 Thr Phe Ser Asp Tyr Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys 645 650 655 Gly Leu Glu Trp Val Ala Glu Ile Arg Leu Lys Ser Ser Asn Tyr Ala 660 665 670 Thr His Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 675 680 685 Asp Ser Lys Ser Ser Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu 690 695 700 Asp Thr Gly Ile Tyr Tyr Cys Thr Pro Leu Thr Gly Gly Phe Val Tyr 705 710 715 720 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr 725 730 735 8719PRTArtificial Sequenceheavy chain of fusion protein containing intact antibody for human TNF-alpha and scFv specific for collagen type II 8Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Asp Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Arg Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys Gly Gly Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly 450 455 460 Gly Ser Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro 465 470 475 480 Gly Gly Ser Met Lys Leu Ser Cys Val Ala Ser Gly Ile Thr Phe Ser 485 490 495 Asp Tyr Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu 500 505 510 Trp Val Ala Glu Ile Arg Leu Lys Ser Ser Asn Tyr Ala Thr His Tyr 515 520 525 Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys 530 535 540 Ser Ser Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly 545 550 555 560 Ile Tyr Tyr Cys Thr Pro Leu Thr Gly Gly Phe Val Tyr Trp Gly Gln 565 570 575 Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Gly Ser Thr Ser 580 585 590 Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Val 595 600 605 Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln 610 615 620 Ala Ser Ile Phe Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly 625 630 635 640 Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys 645 650 655 Val Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg 660 665 670 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg 675 680 685 Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly Ser His 690 695 700 Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 705 710 715 9214PRTArtificial Sequencelight chain of fusion protein containing intact antibody for human TNF-alpha and scFv specific for collagen type II 9Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 10721PRTArtificial Sequenceheavy chain of fusion protein containing intact antibody for human IL-17 and scFv specific for collagen type VII 10Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ala Ile Asn Gln Asp Gly Ser Glu Lys Tyr Tyr Val Gly Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Asp Tyr Tyr Asp Ile Leu Thr Asp Tyr Tyr Ile His Tyr Trp 100 105 110 Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala 115 120 125 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 130 135 140 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 145 150 155 160 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 165 170 175 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 180 185 190 Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 195 200 205 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg 210 215 220 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 225 230 235 240 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 245 250 255 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 260 265 270 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 275 280 285 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 290 295 300 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 305 310 315 320 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 325 330 335 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 340 345 350 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 355 360 365 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 370 375 380 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 385 390 395 400 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 405 410 415 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 420 425 430 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 435 440 445 Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Ala Ser Gly Gly 450 455 460 Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Met Leu Val Glu Ser Gly 465 470 475 480 Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala 485 490 495 Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Thr 500 505 510 Pro Glu Lys Arg Leu Glu Trp Val Ala Thr Ile Thr Ser Gly Gly Asp 515 520 525 Tyr Thr Phe Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 530 535 540 Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Arg Arg Ser 545 550 555 560 Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg His Gly Glu Ile Gly Tyr

565 570 575 Gly Ser Ser Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 580 585 590 Val Ser Ala Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu 595 600 605 Gly Ser Thr Lys Gly Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu 610 615 620 Ala Ala Ser Pro Gly Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys 625 630 635 640 Ser Ile Ser Lys Tyr Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr 645 650 655 Asn Lys Leu Leu Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro 660 665 670 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 675 680 685 Ser Ser Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His 690 695 700 Asn Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 705 710 715 720 Arg 11215PRTArtificial Sequencelight chain of fusion protein containing intact antibody for human IL-17 and scFv specific for collagen type VII 11Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Cys Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 12742PRTArtificial Sequencefusion protein containing scFv specific for human collagen type VII and scFv specific for BAFF 12Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly 1 5 10 15 Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30 Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile 35 40 45 Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Ser Thr Ser 100 105 110 Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val 115 120 125 Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu 130 135 140 Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met 145 150 155 160 Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Thr 165 170 175 Ile Thr Ser Gly Gly Asp Tyr Thr Phe Tyr Pro Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln 195 200 205 Met Ser Ser Arg Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg 210 215 220 His Gly Glu Ile Gly Tyr Gly Ser Ser Ala Trp Phe Ala Tyr Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Gly Gly Ser Pro Pro 245 250 255 Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 260 265 270 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 275 280 285 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 290 295 300 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 305 310 315 320 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 325 330 335 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 340 345 350 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 355 360 365 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 370 375 380 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 385 390 395 400 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 405 410 415 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 420 425 430 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 435 440 445 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 450 455 460 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly 465 470 475 480 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln 485 490 495 Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Arg 500 505 510 Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asn Asn Asn Ala Ile Asn 515 520 525 Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Gly Ile 530 535 540 Ile Pro Met Phe Gly Thr Ala Lys Tyr Ser Gln Asn Phe Gln Gly Arg 545 550 555 560 Val Ala Ile Thr Ala Asp Glu Ser Thr Gly Thr Ala Ser Met Glu Leu 565 570 575 Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser 580 585 590 Arg Asp Leu Leu Leu Phe Pro His His Ala Leu Ser Pro Trp Gly Arg 595 600 605 Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 610 615 620 Gly Ser Gly Gly Gly Gly Ser Ala Phe Ser Ser Glu Leu Thr Gln Asp 625 630 635 640 Pro Ala Val Ser Val Ala Leu Gly Gln Thr Val Arg Val Thr Cys Gln 645 650 655 Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro 660 665 670 Gly Gln Ala Pro Val Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser 675 680 685 Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser 690 695 700 Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 705 710 715 720 Ser Ser Arg Asp Ser Ser Gly Asn His Trp Val Phe Gly Gly Gly Thr 725 730 735 Glu Leu Thr Val Leu Gly 740 13717PRTArtificial Sequenceheavy chain of fusion protein containing intact antibody for human BAFF and scFv specific for collagen type VII 13Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asn Asn Asn 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Met Phe Gly Thr Ala Lys Tyr Ser Gln Asn Phe 50 55 60 Gln Gly Arg Val Ala Ile Thr Ala Asp Glu Ser Thr Gly Thr Ala Ser 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Arg Asp Leu Leu Leu Phe Pro His His Ala Leu Ser Pro 100 105 110 Trp Gly Arg Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys Gly Gly Gly Ala Ser Gly Gly Gly Gly Ser Gly 450 455 460 Gly Gly Gly Ser Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val 465 470 475 480 Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr 485 490 495 Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg 500 505 510 Leu Glu Trp Val Ala Thr Ile Thr Ser Gly Gly Asp Tyr Thr Phe Tyr 515 520 525 Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 530 535 540 Asn Thr Leu Tyr Leu Gln Met Ser Ser Arg Arg Ser Glu Asp Thr Ala 545 550 555 560 Met Tyr Tyr Cys Ala Arg His Gly Glu Ile Gly Tyr Gly Ser Ser Ala 565 570 575 Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Gly 580 585 590 Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys 595 600 605 Gly Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro 610 615 620 Gly Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys 625 630 635 640 Tyr Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu 645 650 655 Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser 660 665 670 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 675 680 685 Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro 690 695 700 Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 705 710 715 14214PRTArtificial Sequencelight chain of fusion protein containing intact antibody for human BAFF and scFv specific for collagen type VII 14Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Val Arg Val Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Ser Ser Gly Asn His 85 90 95 Trp Val Phe Gly Gly Gly Thr Glu Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210 15140PRTArtificial SequenceVH of SPARC 15Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp His 20 25 30 Thr Ile His Trp Met Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Thr Tyr 65 70 75 80 Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Leu Ala Phe Gly Ile Ile Thr Thr Val Val Arg Tyr Phe Asp 100 105 110 Val Trp Gly Thr Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr Thr 115 120 125 Ala Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Leu 130 135 140 16125PRTArtificial SequenceVL of SPARC 16Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45 Tyr Asn Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Lys Leu Gly 115 120 125 17751PRTArtificial SequenceFc fusion protein containing scFv specific for human osteonectin and scFv specific for RANKL 17Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Gly Ser Thr Ser 100 105 110 Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val 115 120 125 Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys Pro Gly Ala Ser Val 130 135 140 Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp His Thr Ile 145 150 155 160 His Trp Met Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Tyr 165 170 175 Ile Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe Lys Gly 180 185 190 Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Thr Tyr Met Gln 195 200 205 Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg 210 215 220 Leu Ala Phe Gly Ile Ile Thr Thr Val Val Arg Tyr Phe Asp Val Trp 225 230 235 240 Gly Thr Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr Thr Gly Gly 245 250 255 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln 260 265 270 Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 275 280 285 Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser 290 295 300 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile 305 310 315 320 Thr Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg 325 330 335 Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met 340 345 350 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Asp 355 360 365 Pro Gly Thr Thr Val Ile Met Ser Trp Phe Asp Pro Trp Gly Gln Gly 370 375 380 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Ser Thr Ser Gly 385 390 395 400 Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Ile Val 405 410 415 Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala 420 425 430 Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Gly Arg Tyr Leu Ala 435 440 445 Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly 450 455 460 Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly 465 470 475 480 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp 485 490 495 Phe Ala Val Phe Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Arg Thr Phe 500 505 510 Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Ala Ser Gly Gly Ser Pro 515 520 525 Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 530 535 540 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 545 550 555 560 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 565 570 575 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 580 585 590 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 595 600 605 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 610 615 620 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 625 630 635 640 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 645 650 655 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 660 665 670 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 675 680 685 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 690 695 700 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 705 710 715 720 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 725 730 735 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 740 745 750 18721PRTArtificial Sequenceheavy chain of fusion protein containing intact antibody for human RANKL and scFv specific for human osteonectin 18Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Thr Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Pro Gly Thr Thr Val Ile Met Ser Trp Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys Gly Gly Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly 450 455 460 Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys 465 470 475 480 Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe 485 490 495 Thr Asp His Thr Ile His Trp Met Lys Gln Arg Pro Glu Gln Gly Leu 500 505 510 Glu Trp Ile Gly Tyr Ile Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn 515 520 525 Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 530 535 540 Thr Thr Tyr Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val 545 550 555 560 Tyr Phe Cys Ala Arg Leu Ala Phe Gly Ile Ile Thr Thr Val Val Arg 565 570 575 Tyr Phe Asp Val Trp Gly Thr Gly Thr Thr Val Thr Val Ser Ser Ala 580 585 590 Lys Thr Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu 595 600 605 Gly Ser Thr Lys Gly Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu 610 615 620 Ser Ala Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly 625 630 635 640 Asn Ile His Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser 645 650 655 Pro Gln Leu Leu Val Tyr Asn Ala Lys Thr Leu Ala Asp Gly Val Pro 660 665 670 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile 675 680 685 Asn Ser Leu Gln Pro Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe 690 695 700 Trp Ser Thr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 705 710 715 720 Arg 19215PRTArtificial Sequencelight chain of fusion protein containing intact antibody for human RANKL and scFv specific for human osteonectin 19Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Gly Arg 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Phe Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 20260PRTArtificial SequenceCVII scFv 20Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly 1 5 10 15 Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30 Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile 35 40 45 Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Ser Thr Ser 100 105 110 Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val 115 120 125 Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu 130 135 140 Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met 145 150 155 160 Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Thr 165 170 175 Ile Thr Ser Gly Gly Asp Tyr Thr Phe Tyr Pro Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln 195 200 205 Met Ser Ser Arg Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg 210 215 220 His Gly Glu Ile Gly Tyr Gly Ser Ser Ala Trp Phe Ala Tyr Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly 245 250 255 Gly Gly Ser Cys 260

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