Fusion polypeptides capable of activating receptors

Abstract

A fusion polypeptide comprising (A).sub.x-M-(A′).sub.y, wherein A and A′ are each polypeptides capable of binding a target receptor. The fusion polypeptides of the invention form multimeric proteins which activate the target receptor. A and A′ may be each be an antibody or fragment derived from an antibody specific for a target receptor, such as the same or different scFv fragments, and/or a ligand or ligand fragment or derivative capable of binding the target protein, M is a multimerizing component, and X and Y are independently a number between 1-10.

Claims

1. A method of activating a Tie-1 and a Tie-2 receptor comprising exposing a cell that bears both Tie-1 and Tie-2 receptors to a homodimer formed from two fusion polypeptides of the form (A)-M-(A′), wherein component A comprises a single chain variable fragment (scFv) antibody capable of binding Tie-1, component M comprises a multimerizing component comprising an Fc domain of IgG, and component A′ comprises i) a fibrinogen domain of Ang1 or Ang2, or ii) a single chain variable fragment (scFv) antibody capable of binding Tie-2, wherein said homodimer binds to both Tie-1 and Tie-2 receptors and clusters four or more Tie receptors of the cell, resulting in phosphorylation of both Tie-1 and Tie-2 receptors.

2. The method of claim 1, wherein the cell is a mammalian cell.

3. The method of claim 1, wherein the cell is a human cell.

4. The method of claim 1, wherein A′ comprises a fibrinogen domain of Ang1.

5. The method of claim 1, wherein A′ comprises a fibrinogen domain of Ang2.

6. The method of claim 1, wherein M comprises an Fc domain of human IgG1.

7. The method of claim 1, wherein the fusion polypeptide comprises scFv.sub.1-1F11-Fc-FD1 (SEQ ID NO: 52).

8. The method of claim 1, wherein the fusion polypeptide comprises scFv.sub.1-1F11-Fc-FD2 (SEQ ID NO: 53).

9. The method of claim 1, wherein the scFv antibody capable of binding Tie-2 comprises a HCDR1 of amino acids 58-65 of SEQ ID NO:28, a HCDR2 of amino acids 83-90 of SEQ ID NO:28, a HCDR3 of amino acids 129-139 of SEQ ID NO:28, a LCDR1 of amino acids 192-201 of SEQ ID NO:28, a LCDR2 of amino acids 219-221 of SEQ ID NO:28, and a LCDR3 of amino acids 258-265 of SEQ ID NO:28.

10. The method of claim 1, wherein the scFv antibody capable of binding Tie-2 comprises a HCDR1 of amino acids 58-66 of SEQ ID NO:29, a HCDR2 of amino acids 84-90 of SEQ ID NO:29, a HCDR3 of amino acids 129-143 of SEQ ID NO:29, a LCDR1 of amino acids 196-201 of SEQ ID NO:29, a LCDR2 of amino acids 219-221 of SEQ ID NO:29, and a LCDR3 of amino acids 258-265 of SEQ ID NO:29.

11. The method of claim 1, wherein the scFv antibody capable of binding Tie-2 comprises a HCDR1 of amino acids 58-66 of SEQ ID NO:31, a HCDR2 of amino acids 84-90 of SEQ ID NO:31, s HCDR3 of amino acids 129-137 of SEQ ID NO:31, a LCDR1 of amino acids 196-201 of SEQ ID NO:31, a LCDR2 of amino acids 219-221 of SEQ ID NO:31, and a LCDR3 of amino acids 258-264 of SEQ ID NO:31.

12. The method of claim 1, wherein the fibrinogen domain is selected from the group consisting of amino acids 515-728 of SEQ ID NO:30 and amino acids 515-727 of SEQ ID NO:31.

13. The method of claim 1, wherein the scFv antibody capable of binding Tie-1 comprises a heavy chain variable region of amino acids 4-125 and a light chain variable region of amino acids 141-248 of SEQ ID NO:50.

14. The method of claim 1, wherein the scFv antibody capable of binding Tie-1 comprises a HCDR1 of amino acids 29-36 of SEQ ID NO:50, a HCDR2 of amino acids 54-61 of SEQ ID NO:50, a HCDR3 of amino acids 100-114 of SEQ ID NO:50, a LCDR1 of amino acids 167-172 of SEQ ID NO:50, a LCDR2 of amino acids 190-192 of SEQ ID NO:50, and a LCDR3 of amino acids 229-237 of SEQ ID NO:50.

15. The method of claim 1, wherein the scFv antibody capable of binding Tie-1 comprises a heavy chain variable region of of amino acids 4-122 and a light chain variable region of a sequence selected from the group consisting of amino acids 138-244 of SEQ ID NO:51 and amino acids 138-245 of SEQ ID NO:51.

16. The method of claim 1, wherein the scFv antibody capable of binding Tie-1 comprises a HCDR1 of amino acids 29-36 of SEQ ID NO:51, a HCDR2 of amino acids 54-61 of SEQ ID NO:51, a HCDR3 of amino acids 100-111 of SEQ ID NO:51, a LCDR1 of amino acids 164-169 of SEQ ID NO:51, a LCDR2 of amino acids 187-189 of SEQ ID NO:51, and a LCDR3 of amino acids 226-234 of SEQ ID NO:51.

17. A method of activating a Tie-1 and Tie-2 receptor comprising exposing a cell that bears both Tie-1 and Tie-2 receptors to a homodimer formed from two fusion polypeptides of the form (A)-M-(A′), wherein component A comprises i) a fibrinogen domain of Ang1 or Ang2, or ii) a single chain variable fragment (scFv) antibody capable of binding Tie-2, component M comprises a multimerizing component comprising an Fc domain of IgG, and component A′ comprises a single chain variable fragment (scFv) antibody capable of binding Tie-1, wherein said homodimer binds to both Tie-1 and Tie-2 receptors and clusters four or more Tie receptors of the cell, resulting in phosphorylation of both Tie-1 and Tie-2 receptors.

18. The method of claim 17, wherein the cell is a mammalian cell.

19. The method of claim 17, wherein the cell is a human cell.

20. The method of claim 17, wherein A comprises a fibrinogen domain of Ang1.

21. The method of claim 17, wherein A comprises a fibrinogen domain of Ang2.

22. The method of claim 17, wherein M comprises an Fc domain of human IgG1.

23. The method of claim 17, wherein the scFv antibody capable of binding Tie-2 comprises a HCDR1 of amino acids 58-65 of SEQ ID NO:28, a HCDR2 of amino acids 83-90 of SEQ ID NO:28, a HCDR3 of amino acids 129-139 of SEQ ID NO:28, a LCDR1 of amino acids 192-201 of SEQ ID NO:28, a LCDR2 of amino acids 219-221 of SEQ ID NO:28, and a LCDR3 of amino acids 258-265 of SEQ ID NO:28.

24. The method of claim 17, wherein the scFv antibody capable of binding Tie-2 comprises a HCDR1 of amino acids 58-66 of SEQ ID NO:29, a HCDR2 of amino acids 84-90 of SEQ ID NO:29, a HCDR3 of amino acids 129-143 of SEQ ID NO:29, a LCDR1 of amino acids 196-201 of SEQ ID NO:29, a LCDR2 of amino acids 219-221 of SEQ ID NO:29, and a LCDR3 of amino acids 258-265 of SEQ ID NO:29.

25. The method of claim 17, wherein the scFv antibody capable of binding Tie-2 comprises a HCDR1 of amino acids 58-66 of SEQ ID NO:31, a HCDR2 of amino acids 84-90 of SEQ ID NO:31, s HCDR3 of amino acids 129-137 of SEQ ID NO:31, a LCDR1 of amino acids 196-201 of SEQ ID NO:31, a LCDR2 of amino acids 219-221 of SEQ ID NO:31, and a LCDR3 of amino acids 258-264 of SEQ ID NO:31.

26. The method of claim 17, wherein the fibrinogen domain is selected from the group consisting of amino acids 515-728 of SEQ ID NO:30 and amino acids 515-727 of SEQ ID NO:31.

27. The method of claim 17, wherein the scFv antibody capable of binding Tie-1 comprises a heavy chain variable region of amino acids 4-125 and a light chain variable region of amino acids 141-248 of SEQ ID NO:50.

28. The method of claim 17, wherein the scFv antibody capable of binding Tie-1 comprises a HCDR1 of amino acids 29-36 of SEQ ID NO:50, a HCDR2 of amino acids 54-61 of SEQ ID NO:50, a HCDR3 of amino acids 100-114 of SEQ ID NO:50, a LCDR1 of amino acids 167-172 of SEQ ID NO:50, a LCDR2 of amino acids 190-192 of SEQ ID NO:50, and a LCDR3 of amino acids 229-237 of SEQ ID NO:50.

29. The method of claim 17, wherein the scFv antibody capable of binding Tie-1 comprises a heavy chain variable region of of amino acids 4-122 and a light chain variable region of a sequence selected from the group consisting of amino acids 138-244 of SEQ ID NO:51 and amino acids 138-245 of SEQ ID NO:51.

30. The method of claim 17, wherein the scFv antibody capable of binding Tie-1 comprises a HCDR1 of amino acids 29-36 of SEQ ID NO:51, a HCDR2 of amino acids 54-61 of SEQ ID NO:51, a HCDR3 of amino acids 100-111 of SEQ ID NO:51, a LCDR1 of amino acids 164-169 of SEQ ID NO:51, a LCDR2 of amino acids 187-189 of SEQ ID NO:51, and a LCDR3 of amino acids 226-234 of SEQ ID NO:51.

Description

DETAILED DESCRIPTION

(1) Before the present methods are described, it is to be understood that this invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

(2) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.

Definitions

(3) As used herein, the term “target receptor-related condition or disease” generally encompasses a condition of a mammalian host, particularly a human host, which is associated with a particular target receptor. Thus, treating a target receptor-related condition will encompass the treatment of a mammal, in particular, a human, who has symptoms reflective of decreased target receptor activation, or who is expected to have such decreased levels in response to a disease, condition or treatment regimen. Treating a target receptor-related condition or disease encompasses the treatment of a human subject wherein enhancing the activation of a target receptor with an activating dimer of the invention results in amelioration of an undesirable symptom resulting from the target receptor-related condition or disease. As used herein, an “target receptor-related condition” also includes a condition in which it is desirable to alter, either transiently, or long-term, activation of a particular target receptor.

(4) Target Receptors

(5) Examples of target receptors are members of the Eph family (e.g. EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB5, EphB6), Tie receptors (e.g. Tie-1 or Tie-2). Suitable ligands or fragments thereof include the soluble domain of an ephrin (e.g. ephrin-A1, ephrin-A2, ephrin-A3, ephrin-A4, ephrin-A5, ephrin-B1, ephrin-B2, ephrin-B3), and the fibrinogen domain of an angiopoietin (e.g. angiopoietin-1 (Ang1, Ang2, Ang3, Ang4).

(6) Suitable target receptors are receptors that are activated when multimerized. This class of receptors includes, but is not limited to, those that possess an integral kinase domain. Within this class of integral kinase receptors are those that form homodimers, or clusters of the same receptor, such as Tie-1, Tie-2, EGFR, FGFR, the Trk family and the Eph family of receptors, and those that form heterodimers, or clusters, such as the VEGF receptors VEGFR1, VEGFR2, the PDGF receptors PDGFRα and PDGFRβ, and the TGF-β family receptors. Suitable target receptors also include, but are not limited to, the class of receptors with associated kinases. These receptors include those that form homodimers, or clusters, such as the growth hormone receptor, EPOR and the G-CSF receptor CD114, and those that form heterodimers, or clusters, such as the GM-CSF receptors GMRα and GMRβ.

(7) Target Receptor-Specific Antibodies and Ligands

(8) In specific embodiments, the activating dimers of the invention comprise one or more immunoglobulin binding domains isolated from antibodies generated against a selected target receptor. The term “immunoglobulin” or “antibody” as used herein refers to a mammalian, including human, polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen, which, in the case of the present invention, is a target receptor or portion thereof. If the intended activating dimer will be used as a human therapeutic, immunoglobulin binding regions should be derived from the corresponding human immunoglobulins or be a humanized immunoglobulin. The human immunoglobulin genes or gene fragments include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant regions, as well as the 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. Within each IgG class, there are different isotypes (eg. IgG.sub.1, IgG.sub.2, etc.). Typically, the antigen-binding region of an antibody will be the most critical in determining specificity and affinity of binding.

(9) An exemplary immunoglobulin (antibody) structural unit of human IgG.sub.1 comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one light chain (about 25 kD) and one heavy chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 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.

(10) Antibodies exist as intact immunoglobulins, or as a number of well-characterized fragments produced by digestion with various peptidases, e.g., F(ab)′.sub.2, Fab′, etc. Thus, the terms immunoglobulin or antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) (scFv) or those identified using phase display libraries (see, for example, McCafferty et al. (1990) Nature 348:552-554). In addition, the target receptor-binding domain component of the fusion polypeptides of the invention include the variable regions of the heavy (V.sub.H) or the light (V.sub.L) chains of immunoglobulins, as well as target receptor-binding portions thereof. Methods for producing such variable regions are described in Reiter, et al. (1999) J. Mol. Biol. 290:685-698.

(11) Methods for preparing antibodies are known to the art. See, for example, Kohler & Milstein (1975) Nature 256:495-497; Harlow & Lane (1988) Antibodies: a Laboratory Manual, Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.). The genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody. Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity. Techniques for the production of single chain or recombinant antibodies (U.S. Pat. No. 4,946,778; U.S. Pat. No. 4,816,567) can be adapted to produce antibodies used in the fusion polypeptides, activating dimers and methods of the instant invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express human or humanized antibodies. Alternatively, phage display technology can be used to identify antibodies, antibody fragments, such as variable domains, and heteromeric Fab fragments that specifically bind to selected antigens. Phage display is of particular value to isolate weakly binding antibodies or fragments thereof from unimmunized animals which, when combined with other weak binders in accordance with the invention described herein, create strongly binding activating dimers.

(12) Screening and selection of preferred immunoglobulins (antibodies) can be conducted by a variety of methods known to the art. Initial screening for the presence of monoclonal antibodies specific to a target receptor may be conducted through the use of ELISA-based methods or phage display, for example. A secondary screen is preferably conducted to identify and select a desired monoclonal antibody for use in construction of the fusion polypeptides of the invention. Secondary screening may be conducted with any suitable method known to the art.

(13) Nucleic Acid Construction and Expression

(14) Individual components of the fusion polypeptides of the invention may be produced from nucleic acids molecules using molecular biological methods known to the art. Nucleic acid molecules are inserted into a vector that is able to express the fusion polypeptides when introduced into an appropriate host cell. Appropriate host cells include, but are not limited to, bacterial, yeast, insect, and mammalian cells. Any of the methods known to one skilled in the art for the insertion of DNA fragments into a vector may be used to construct expression vectors encoding the fusion polypeptides of the invention under control of transcriptional/translational control signals. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinations (See Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory; Current Protocols in Molecular Biology, Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, NY).

(15) Expression of the nucleic acid molecules of the invention may be regulated by a second nucleic acid sequence so that the molecule is expressed in a host transformed with the recombinant DNA molecule. For example, expression of the nucleic acid molecules of the invention may be controlled by any promoter/enhancer element known in the art.

(16) Immunoglobulin-Derived Components.

(17) The nucleic acid constructs include regions which encode binding domains derived from an anti-target receptor antibodies. In general, such binding domains will be derived from V.sub.H or V.sub.L chain variable regions. After identification and selection of antibodies exhibiting the desired binding characteristics, the variable regions of the heavy chains and/or light chains of each antibody is isolated, amplified, cloned and sequenced. Modifications may be made to the V.sub.H and V.sub.L nucleotide sequences, including additions of nucleotide sequences encoding amino acids and/or carrying restriction sites, deletions of nucleotide sequences encoding amino acids, or substitutions of nucleotide sequences encoding amino acids.

(18) The invention encompasses antibodies or antibody fragments which are humanized or chimeric. “Humanized” or chimeric forms of non-human (e.g., murine) antibodies are immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′) 2 or other antigen-binding subsequences of antibodies) that contain minimal sequences required for antigen binding derived from non-human immunoglobulin. They have the same or similar binding specificity and affinity as a mouse or other nonhuman antibody that provides the starting material for construction of a chimeric or humanized antibody. Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin gene segments belonging to different species. For example, the variable (V) segments of the genes from a mouse monoclonal antibody may be joined to human constant (C) segments, such as IgG1 and IgG.sub.4. Human isotype IgG1 is preferred. A typical chimeric antibody is thus a hybrid protein consisting of the V or antigen-binding domain from a mouse antibody and the C or effector domain from a human antibody. Humanized antibodies have variable region framework residues substantially from a human antibody (termed an acceptor antibody) and complementarity determining regions (CDR regions) substantially from a mouse antibody, (referred to as the donor immunoglobulin). See, Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989) and WO 90/07861, U.S. Pat. Nos. 5,693,762, 5,693,761, 5,585,089, 5,530,101 and 5,225,539. The constant region(s), if present, are also substantially or entirely from a human immunoglobulin. The human variable domains are usually chosen from human antibodies whose framework sequences exhibit a high degree of sequence identity with the murine variable region domains from which the CDRs were derived. The heavy and light chain variable region framework residues can be derived from the same or different human antibody sequences. The human antibody sequences can be the sequences of naturally occurring human antibodies or can be consensus sequences of several human antibodies. See WO 92/22653. Certain amino acids from the human variable region framework residues are selected for substitution based on their possible influence on CDR conformation and/or binding to antigen. Investigation of such possible influences is by modeling, examination of the characteristics of the amino acids at particular locations, or empirical observation of the effects of substitution or mutagenesis of particular amino acids. For example, when an amino acid differs between a murine variable region framework residue and a selected human variable region framework residue, the human framework amino acid should usually be substituted by the equivalent framework amino acid from the mouse antibody when it is reasonably expected that the amino acid: (1) noncovalently binds antigen directly; (2) is adjacent to a CDR region; (3) otherwise interacts with a CDR region (e.g. is within about 6 Å of a CDR region), or (4) participates in the V.sub.L-V.sub.H interface. Other candidates for substitution are acceptor human framework amino acids that are unusual for a human immunoglobulin at that position. These amino acids can be substituted with amino acids from the equivalent position of the mouse donor antibody or from the equivalent positions of more typical human immunoglobulins. Other candidates for substitution are acceptor human framework amino acids that are unusual for a human immunoglobulin at that position. The variable region frameworks of humanized immunoglobulins usually show at least 85% sequence identity to a human variable region framework sequence or consensus of such sequences.

(19) Fully human antibodies may be made by any method known to the art. For example, U.S. Pat. No. 6,596,541 describes a method of generating fully human antibodies. Briefly, initially a transgenic animal such as a mouse is generated that produces hybrid antibodies containing human variable regions (VDJ/VJ) and mouse constant regions. This is accomplished by a direct, in situ replacement of the mouse variable region (VDJ/VJ) genes with their human counterparts. The mouse is then exposed to human antigen, or an immunogenic fragment thereof. The resultant hybrid immunoglobulin loci will undergo the natural process of rearrangements during B-cell development to produce hybrid antibodies having the desired specificity. The antibody of the invention is selected as described above. Subsequently, fully-human antibodies are made by replacing the mouse constant regions with the desired human counterparts. Fully human antibodies can also be isolated from mice or other transgenic animals such as cows that express human transgenes or minichromosomes for the heavy and light chain loci. (Green (1999) J Immunol Methods. 231:11-23 and Ishida et al (2002) Cloning Stem Cells. 4:91-102). Fully human antibodies can also be isolated from humans to whom the protein has been administered. Fully human antibodies can also be isolated from immune compromised mice whose immune systems have been regenerated by engraftment with human stem cells, splenocytes, or peripheral blood cells (Chamat et al (1999) J Infect Dis. 180:268-77). To enhance the immune response to the protein of interest one can knockout the gene encoding the protein of interest in the human-antibody-transgenic animal.

(20) Receptor-Binding Domains.

(21) In accordance with the invention, the nucleic acid constructs include components which encode binding domains derived from target receptor ligands. After identification of a ligand's target receptor-binding domain exhibiting desired binding characteristics, the nucleic acid that encodes such domain is used in the nucleic acid constructs. Such nucleic acids may be modified, including additions of nucleotide sequences encoding amino acids and/or carrying restriction sites, deletions of nucleotide sequences encoding amino acids, or substitutions of nucleotide sequences encoding amino acids.

(22) The nucleic acid constructs of the invention are inserted into an expression vector or viral vector by methods known to the art, wherein the nucleic acid molecule is operatively linked to an expression control sequence. Also provided is a host-vector system for the production of the fusion polypeptides and activating dimers of the invention, which comprises the expression vector of the invention, which has been introduced into a suitable host cell. The suitable host cell may be a bacterial cell such as E. coli, a yeast cell, such as Pichia pastoris, an insect cell, such as Spodoptera frugiperda, or a mammalian cell, such as a COS, CHO, 293, BHK or NS0 cell.

(23) The invention further encompasses methods for producing the activating dimers of the invention by growing cells transformed with an expression vector under conditions permitting production of the fusion polypeptides and recovery of the activating dimers formed from the fusion polypeptides. Cells may also be transduced with a recombinant virus comprising the nucleic acid construct of the invention.

(24) The activating dimers may be purified by any technique which allows for the subsequent formation of a stable dimer. For example, and not by way of limitation, the activating dimers may be recovered from cells either as soluble polypeptides or as inclusion bodies, from which they may be extracted quantitatively by 8M guanidinium hydrochloride and dialysis. In order to further purify the activating dimers, conventional ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography or gel filtration may be used. The activating dimers may also be recovered from conditioned media following secretion from eukaryotic or prokaryotic cells.

(25) Screening and Detection Methods

(26) The activating dimers of the invention may also be used in in vitro or in vivo screening methods where it is desirable to detect and/or measure target receptor levels. Screening methods are well known to the art and include cell-free, cell-based, and animal assays. In vitro assays can be either solid state or soluble. Target receptor detection may be achieved in a number of ways known to the art, including the use of a label or detectable group capable of identifying an activating dimer which is bound to a target receptor. Detectable labels are well developed in the field of immunoassays and may generally be used in conjunction with assays using the activating dimer of the invention.

(27) Therapeutic Methods

(28) The ability of the activating dimers of the invention to exhibit high affinity binding for their receptors makes them therapeutically useful for efficiently activating their receptors. Thus, it certain instances it may be to increase the effect of endogenous ligands for target receptors, such as, for example, the ephrins. For example, in the area of nervous system trauma, certain conditions may benefit from an increase in ephrin responsiveness. It may therefore be beneficial to increase the binding affinity of an ephrin in patients suffering from such conditions through the use of the compositions described herein.

(29) The invention herein further provides for the development of an activating dimer described herein as a therapeutic for the treatment of patients suffering from disorders involving cells, tissues or organs which express the Tie-2 receptor. Such molecules may be used in a method of treatment of the human or animal body, or in a method of diagnosis.

(30) The target receptor known as Tie-2 receptor has been identified in association with endothelial cells and, as was previously demonstrated, blocking of agonists of the receptor such as Tie-2 ligand 1 (Ang1) has been shown to prevent vascularization. Accordingly, activating dimers of the invention wherein the target receptor is Tie-2 may be useful for the induction of vascularization in diseases or disorders where such vascularization is indicated. Such diseases or disorders would include wound healing, ischemia and diabetes. The ligands may be tested in animal models and used therapeutically as described for other agents, such as vascular endothelial growth factor (VEGF), another endothelial cell-specific factor that is angiogenic. Ferrara et al. U.S. Pat. No. 5,332,671 issued Jul. 26, 1994. Ferrara et al. describe in vitro and in vivo studies that may be used to demonstrate the effect of an angiogenic factor in enhancing blood flow to ischemic myocardium, enhancing wound healing, and in other therapeutic settings wherein neoangiogenesis is desired. According to the invention, such a Tie-2 specific activating dimer may be used alone or in combination with one or more additional pharmaceutically active compounds such as, for example, VEGF or basic fibroblast growth factor (bFGF).

(31) Methods of Administration

(32) Methods known in the art for the therapeutic delivery of agents such as proteins or nucleic acids can be used for the therapeutic delivery of an activating dimer or a nucleic acid encoding an activating dimer of the invention for activating target receptors in a subject, e.g., cellular transfection, gene therapy, direct administration with a delivery vehicle or pharmaceutically acceptable carrier, indirect delivery by providing recombinant cells comprising a nucleic acid encoding an activating dimer of the invention.

(33) Various delivery systems are known and can be used to administer the activating dimer of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction can be enteral or parenteral and include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, pulmonary, intranasal, intraocular, epidural, and oral routes. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

(34) Pharmaceutical Compositions

(35) The present invention also provides pharmaceutical compositions comprising an activating dimer of the invention and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. (See, for example, “Remington's Pharmaceutical Sciences” by E. W. Martin.

(36) In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

(37) The active agents of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

(38) Kits

(39) The invention also provides a pack or kit (e.g., a pharmaceutical pack or kit) comprising one or more containers filled with at least one activating dimer of the invention. The kits of the invention may be used in any applicable method, including, for example, diagnostically. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects (a) approval by the agency of manufacture, use or sale for human administration, (b) directions for use, or both.

(40) Transgenic Animals

(41) The invention includes transgenic non-human animals expressing a fusion polypeptide of the invention. A transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the transgene to particular cells. A transgenic non-human animal expressing an fusion polypeptide of the invention is useful in a variety of applications, including as a means of producing such fusion proteins Further, the transgene may be placed under the control of an inducible promoter such that expression of the fusion polypeptide may be controlled by, for example, administration of a small molecule.

EXAMPLES

(42) The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1. Production of Anti-Tie-2 Hybridomas

(43) Five 8-weeks old Balb/c mice were first immunized with purified human Tie-2-Fc (hTie-2-Fc); each mouse was injected subcutaneously with 200 μA emulsion containing 100 μg purified hTie-2-Fc protein and 100 μl Freund's complete adjuvant. Fifteen days after the primary injection, each mouse received subcutaneous injection of 200 μl emulsion containing 100 μg purified hTie-2-Fc in 100 μl PBS and 100 μl Freund's incomplete adjuvant. This injection was repeated for the five mice seven days later. One mouse was used for generation of hybridomas against hTie-2. Each of the four remaining mice were given subcutaneous injections of 200 μl emulsion each containing 100 μg purified rat Tie-2-Fc (rTie-2-Fc) in 100 μl PBS and 100 μl Freund's incomplete adjuvant six months after the primary injection of hTie-2-Fc. Eleven days later, the immune response of the mice to rTie-2-Fc was boosted by subcutaneous injection of 200 μl of emulsion containing 100 μg purified rTie-2-Fc in 100 μl PBS and 100 μl Freund's incomplete adjuvant for each mouse. Mouse sera were collected from tail veins three days after the injection, then the antibody titers against rTie-2-Fc were determined by ELISA. The two mice with the highest titers were given a final boost by tail vein injection of 100 μg purified rTie-2-Fc in 100 μl PBS. The mice were sacrificed three days later and their spleen cells were collected for fusion with Sp2/0-Ag14 cells.

(44) To generate hybridomas, mouse spleen cells were fused with Sp2/0-Ag14 myeloma cells using polyethylene glycol (PEG). Briefly, after the spleens were aseptically removed from the mice, one tip of each spleen was cut open and spleen cells collected. The spleen cells were washed twice with D-MEM and cell numbers were counted using a hemocytometer. 2×10.sup.8 spleen cells were combined with 3×10.sup.7 Sp2/0-Ag14 cells that were in log growth stage. The cell mix was washed with 30 mls D-MEM. 1 ml 50% PEG at 37° C. was slowly added to the cell pellet while stirring. D-MEM was added to the mix to bring the volume to 10 mls. The cells were spun down at 400×g for 10 minutes. After removal of supernatant, the cells were gently resuspended in 20 mls growth medium containing 60% D-MEM with 4.5 g/L glucose, 20% FCS, 10% NCTC109 medium, 10% hybridoma cloning factor, 1 mM oxaloacetate, 2 mM glutamine, 0.2 units/ml insulin, and 3 μM glycine. The cells were transferred to two T225 flasks, each containing 100 mls of the growth medium and were put into a tissue culture incubator. On the next day, 1×HAT was added to the medium to select against the myeloma cells that were not fused. Nine days after the fusion, the cultures were replenished with fresh medium. Human IgG was added to the cultures at 1 mg/ml. On the tenth day after the fusion, 2.6×10.sup.7 fused cells were stained sequentially with 1 μg/ml biotin-rTie-2-Fc for one hour and 2.5 μg/ml phycoerythrin (PE)-conjugated streptavidin for 45 minutes in growth medium at room temperature. As a control, 1×10.sup.6 fused cells were stained with 2.5 μg/ml PE-streptavidin for 45 minutes at room temperature. The cells were washed with 10 ml PBS after each stain. After staining, the cells were resuspended in PBS with 0.1% FCS and were analyzed by flow cytometry on a MoFlo (Cytomation). A population of cells (4% total cells) stained with both biotin rTie-2-Fc and PE-streptavidin exhibited fluorescence higher than the unstained cells and the cells stained with PE-streptavidin alone. The cells in this 4% gate were cloned by sorting single cells into two 96-well plates containing 200 μl growth medium per well. The cells were cultured for 10 days before splitting into two sets of 96-well plates. Cells in one set of plate were processed for RT-PCR of mouse IgG heavy chain variable region following by sequencing. The clones were grouped into 14 bins, with members of each bin having identical sequence in their heavy chain variable region. Conditioned medium of hybridoma cells in each bin was tested for its ability to stimulate phosphorylation of rTie-2 in cultured rat aortic endothelial cells (RAECs).

(45) Antibodies from two hybridomas, B2 and A12A, were chosen for further study because they were active in phosphorylation of Tie-2 in RAECs, and did not compete for binding to rTie-2 as determined by BIAcore analysis. In addition, these antibodies did not block binding of derivatives of angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2), the natural ligands of Tie-2.

Example 2. Construction of scFvs (B2 and A12A)

(46) Generally, antibody variable regions from hybridomas expressing antibodies specific for rTie-2 were cloned by first determining the DNA sequence of RT-PCR products using primers specific for mouse antibody variable regions, then using specific primers based on the determined sequence in order to amplify DNA fragments encoding scFvs. The scFv DNA fragments were cloned such that they could be cassette exchanged with multiple plasmids to yield all combinations of activating dimers. For example, one amplified scFv fragment could be fused to a signal sequence at the N-terminus and to a coding sequence for the IgG Fc domain at the C-terminus, or it could be fused to the C-terminus of an IgG Fc coding sequence such that the 3′ end of the scFv coding sequence contained a translation stop codon.

(47) The B2 hybridoma was found to express an antibody capable of inducing phosphorylation of the Tie-2 receptor in RAECs. Total RNA was isolated from this hybridoma using the promega SV96 Total RNA Isolation System (Promega) and variable heavy cDNA was synthesized using the Qiagen One-Step RT-PCR system (Qiagen) with heavy chain primers from the Ratner primer set (Wang et al. (2000) J. Immunol. Methods 233:167) that included equimolar amounts of the 5′ primers (SEQ ID NO:1-7) and the 3′ primer (SEQ ID NO:8). Similarly, the light chain variable regions were amplified from cDNA using equimolar amounts of the light chain-specific primers (SEQ ID NO:9 and 10). The amplified variable region fragments were cloned into the pCR2.1-TOPO vector (Invitrogen) and the DNA sequences were determined. Based on the determined variable region sequences for the B2 antibody, the variable heavy sequence was PCR amplified using the pCR2.1-TOPO cloned variable region as template and an equimolar mix of 5′ and 3′ primers (SEQ ID NO: 19 and SEQ ID NO: 20). The variable light sequence was PCR amplified using a similar strategy. The pCR2.1-TOPO cloned variable region was used as template and an equimolar mix of 5′ and 3′ primers (SEQ ID NO:21 and SEQ ID NO:22). The variable regions were joined by a (G4S)3 linker; scFv genes were assembled and PCR amplified using an equimolar mix of the above specific variable heavy and variable light PCR products and an equimolar mix of 5′ B2 heavy primer (SEQ ID NO:19) and the 3′ light primer (SEQ ID NO:22). PCR product was cloned into Invitrogen pCR2.1-TOPO (Invitrogen) to yield pRG1039. The sequence was confirmed before sub-cloning the 744 bp Ascl/Srfl to fuse the scFv gene to the N-terminus of a DNA encoding the human IgG1 Fc fragment (hFc), or the 753 bp Ascl/Notl restriction fragments to fuse the same scFv to the C-terminus of a DNA encoding hFc.

(48) The A12A hybridoma was also found to express an antibody capable of inducing phosphorylation of the Tie-2 receptor in RAECs. Total RNA was isolated from this hybridoma using the Quick Prep mRNA purification kit (Amersham Pharmacia Biotech) and variable heavy cDNA was synthesized using the Qiagen One-Step RT-PCR system, with equimolar amounts of primers from the from the Wright primer set (Morrison et al. (1995) Antibody Engineering, second edition, Borrebaeck, C. K. A. editor 267-293) that included the 5′ heavy chain primers (SEQ ID NO:11-13) and the 3′ primer (SEQ ID NO:8). Similarly, the light chain variable regions were amplified from cDNA with equimolar amounts of the 5′ heavy chain primers (SEQ ID NO:14-18) and the 3′ primer (SEQ ID NO:10). The amplified variable region fragments were cloned into the pCR2.1-TOPO vector (Invitrogen) and the DNA sequences were determined.

(49) Based on the determined variable region sequences for the A12A antibody, the variable heavy sequence was PCR amplified using the pCR2.1-TOPO cloned variable region as template and an equimolar mix of 5′ and 3′ primers (SEQ ID NO:23 and SEQ ID NO:24). The variable light sequence was PCR amplified using a similar strategy. The pCR2.1-TOPO cloned variable region was used as template and an equimolar mix of 5′ and 3′ primers (SEQ ID NO:25 and SEQ ID NO:26). The variable regions were joined by a (G.sub.4S).sub.3 linker; scFv genes were assembled and PCR amplified using an equimolar mix of the above specific variable heavy and variable light PCR products and an equimolar mix of 5′ A12A heavy primer (SEQ ID NO:23) and the 3′ light primer (SEQ ID NO:26). PCR product was cloned into Invitrogen pCR2.1-TOPO to yield pRG1090. The sequence was confirmed before sub-cloning the 747 bp Ascl/Srfl to fuse the scFv gene to the N-terminus of a DNA encoding the hFc fragment.

Example 3. Construction of Monospecific and Bispecific Activating Dimers

(50) The general scheme for constructing both monspecific and bispecific tetravalent activating dimers was based on the ability of either the B2 or A12A scFv genes to be inserted between the murine ROR1 signal sequence (SEQ ID NO:27) and the gene encoding hFc (nucleotides 85 to 765 of GenBank accession # X70421) when cut with one set of restriction enzymes, or after the hFc gene if cut with a different set of enzymes. This design of the scFv genes allowed the exchange of scFv cassettes among plasmids to obtain different combinations of scFv and hFc using standard known methods. All constructs have an optional three amino acid linker (spacer) between the cleavage site of the signal peptide and the start of the scFv gene, resulting from engineering a restriction site onto the 5′ end of the scFv genes. Similarly, fusion to the amino terminus of the hFc gene was facilitated by a three amino acid sequence (Gly-Pro-Gly), and fusion to the carboxy terminus of the hFc gene was facilitated by an eight amino acid sequence consisting of the residues Gly.sub.4-Ser-Gly-Ala-Pro (SEQ ID NO:32) As a consequence of the terminal restriction site linkers on the scFv genes, all constructs that have a carboxy terminal scFv end with the amino acids Gly-Pro-Gly.

(51) Two types of svFc-based chimeric molecules were constructed to assess the ability of scFv-based molecules to activate the rTie-2 receptor. One type of molecule used a single scFv fused to both the N-terminus and the C-terminus of hFc, the consequence of which was a monospecific tetravalent molecule capable of binding rTie-2. This molecule was expected to be capable of simultaneously binding four rTie-2 molecules. The plasmid pTE586 encodes the gene for scFv.sub.B2-Fc-scFv.sub.B2 (SEQ ID NO: 29) whose secretion is directed by the mROR1 signal peptide. The expression of scFv.sub.B2-Fc-scFv.sub.B2 in pTE586 was directed by the CMV-MIE promoter when transfected into CHO cells. This protein was easily purified by Protein A-Sepaharose affinity chromatography.

(52) Construction of an scFv-Fc-scFv molecule wherein the two scFv domains are derived from two different non-competing anti-rTie-2 antibodies would yield a molecule capable of clustering more than four receptors, in contrast to the scFv.sub.B2-Fc-scFv.sub.B2 described above, which can cluster only four receptors. It was determined by BIAcore analysis that the binding of the B2 antibody did not block binding of A12A to rTie-2, and A12A binding first did not block binding of B2. Consequently, scFv molecules made from these antibodies should be capable of clustering more than four receptors. To construct a bispecific tetravalent scFv-based molecule, the scFv.sub.A12A-gene was used in combination with the scFv.sub.B2 gene to yield scFv.sub.A12A-Fc-scFv.sub.B2 (SEQ ID NO: 28). The plasmid pTE585 encodes the gene for scFv.sub.A12A-Fc-scFv.sub.B2 and has the mROR1 signal peptide and CMV-MIE promoter when transfected into CHO cells. Both scFv.sub.B2-Fc-scFv.sub.B2 and scFv.sub.A12A-Fc-scFv.sub.B2 were expressed in CHO cells, and purified by Protein A-Sepharose affinity chromatography.

Example 4. Assays

(53) Antibodies to rTie-2, and chimeric molecules related to these antibodies, were evaluated for their ability to induce phosphorylation of Tie-2 in cultured rat aortic endothelial cells. Confluent RAECs, between passage 3 and 6 (Vec Technologies), were grown in MCDB-131 media (Vec Technologies) on 0.2% gelatin coated T-75 flasks. Cells were starved for 2 hrs. in serum-free DME-Hi glucose medium (Irvine Scientific) prior to incubation at 37° C. for 5 min. in 1.5 ml serum-free DME-Hi glucose medium with 0.1% BSA and the challenge molecule. The challenge medium was then removed and cells were lysed in 20 mM Tris, pH 7.6, 150 mMNaCl, 50 mM NaF, 1 mM Na orthovanadate, 5 mM benzamidine, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, with 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mM PMSF. Tie-2 was immunoprecipitated by incubating the lysates at 4° C. for 16 hrs. with 5 μg anti-Tie-2 mouse monoclonal antibody KP-m33, 10 μg biotinylated anti-mouse IgG (Jackson Laboratories), and 100 μl of neutravidin beads (Pierce). Beads were collected by centrifugation, washed 3 times with RIPA buffer, and bound proteins were eluted with 40 μl of 5× Laemmli buffer with 10% B-mercaptoethanol by heating at 100° C. for 5 min. After SDS-gel electrophoresis on a 4-12% Tris/glycine polyacrylamide gel (Novex), proteins were transferred to PVDF membranes and probed with mouse anti-phophotyrosine monoclonal antibody 4G10 (Upstate) then detected using goat anti-mouse IgG-HRP conjugate (Pierce) followed by ECL reagent (Amersham). The ability to induce Tie-2 phosphorylation in RAECs was determined for each activating dimer. Activity was evaluated by comparison to the level of stimulation obtained with FD1-Fc-FD1 (BA1)—a chimeric protein shown to be as active as Ang1 in binding and activation Tie-2 (Davis et al. (2003) Nature Struct. Biol. 10:38-44) (FD1 or FD2=human fibrinogen domain of Ang1 or Ang2, respectively). Maximum stimulation (ECmax) of Tie-2 in RAECs was observed when BA1 was used at about 0.5 to 1.0 μg/ml, and phosphorylation levels in mock treated cells were low. Similarly, the ECmax of scFv.sub.B2-Fc-scFv.sub.B2, scFv.sub.A12A-Fc-scFv.sub.B2, scFv.sub.B2-Fc-FD1, and scFv.sub.B2-Fc-FD2 were about 0.5 to 1.0 μg/ml. In all cases, the scFv-based molecules were capable of inducing a higher phosphorylation signal than observed for the related native antibodies isolated from hybridoma conditioned media.

(54) Purified scFv.sub.B2-Fc-scFv.sub.B2 and scFv.sub.A12A-Fc-scFv.sub.B2 were characterized for their ability to bind rTie-2 and induce phosphorylation. Binding to rTie-2 was determined by BIAcore analysis. Both the monospecific and the bispecific activating dimers were found to have significantly higher affinity for rTie-2 than FD1-Fc-FD1. In addition, both scFv.sub.B2-Fc-scFv.sub.B2 and scFv.sub.A12A-Fc-scFv.sub.B2 were able to stimulate phosphorylation of rTie-2 in RAECs comparable to FD1-Fc-FD1.

Example 5. Construction of scFv/Ligand Activating Dimers

(55) Bispecific tetravalent molecules were constructed to include both Tie-2 specific scFv and FD1 or FD2. The chimeric molecules were made by fusing the gene encoding scFv.sub.B2 to the N-terminus of hFc and the gene encoding Ang1 FD (Phe283 to Phe498 of Gen Bank accession #Q15389) or Ang2 FD (Phe281 to Phe496 of GenBank accession # O15123) to the C-terminus. Plasmid pTE514 encodes the gene for scFv.sub.B2-Fc-FD1 (SEQ ID NO: 30) and contained the mROR1 signal peptide and CMV-MIE promoter. Plasmid pTE614 encodes the gene for scFv.sub.B2-Fc-FD2 (SEQ ID NO: 31) and contained the mROR1 signal peptide and CMV-MIE promoter. Similar to scFv.sub.B2-Fc-scFv.sub.B2 and scFv.sub.A12A-Fc-scFv.sub.B2 the proteins expressed from pTE514 and pTE614 had a Gly-Ala-Pro linker between the mROR1 signal peptide and the scFv.sub.B2, a Gly-Pro-Gly linker between the N-terminal scFv.sub.B2 and hFc and a Gly.sub.4-Ser-Gly-Ala-Pro linker (SEQ ID NO:32) between the C-terminus of hFc and the N-terminus of the Ang FDs. Both scFv.sub.B2-Fc-FD1 and scFv.sub.B2-Fc-FD2 were expressed and purified as described above.

(56) Purified scFv.sub.B2-Fc-FD1 and scFv.sub.B2-Fc-FD2 were characterized for their ability to bind rTie-2 and induce phosphorylation as described in above. As determined by BIAcore analysis, the chimeric activating dimer scFv.sub.B2-Fc-FD1 was found to have significantly higher affinity for rTie-2 (0.04 nM) than FD1-Fc-FD1 (2 nM). Moreover, both scFv.sub.B2-Fc-FD1 and scFv.sub.B2-Fc-FD2 were able to stimulate phosphorylation of rTie-2 in RAECs comparable to FD1-Fc-FD1.

Example 6. Construction of Fully Human Activating Dimers

(57) Bispecific tetravalent molecules are formed from dimerized fusion constructs of the invention which include either two scFvs derived from human antibodies specific for hTie-2 or one scFv derived from a human antibody specific for hTie-2 and human FD1 or FD2. Human scFvs specific for hTie-2 are obtained by methods known to the art and as described above. In one embodiment, human scFvs are obtained recombinantly as described in Reiter et al. (1999) J. Mol. Biol. 290:685-698 and Gilliland et al. (1996) Tissue Antigens 47(1):1-20.

Example 7. Construction of scFvs (1-1F11 and 2-1G3)

(58) Anti-rTie-1 hybridomas were produced following the procedures described above for the production of anti-rTie-2 hybridomas. Briefly, mice were immunized three times with purified rat Tie-1-Fc protein and Freund's adjuvant. Spleen cells from the mouse with the highest anti-Tie-1 antibody titer were fused with Sp2/0-Ag14 myeloma cells using polyethylene glycol (PEG). After fusion, the cells were cultured in two T225 flasks. HAT was added to the cultures on the next day. Nine days after the fusion, the cultures were replenished with fresh medium. Human IgG was added to the cultures at 1 mg/ml. On the tenth day after the fusion, the HAT-resistant cells were stained sequentially with 1 μg/ml biotin-rat Tie-1-Fc for one hour and 2.5 μg/mIphycoerythrin (PE)-conjugated streptavidin for 45 minutes in growth medium at room temperature. After staining, the cells were analyzed by flow cytometery. Cells that bound rTie-1-Fc were cloned by sorting single cells into 96-well plates. The 96-well plate cultures were split into two sets ten days after sorting. RT-PCR of mouse IgG heavy chain variable region followed by sequencing were performed on one set of the 96-well plate cultures. Clones with unique IgG heavy chain variable region sequences were identified and expanded for the production of anti-rTie-1 antibodies. Antibodies were tested for binding rTie-1 protein and two clones, 1-1F11 and 1-2G3, were chosen for more detailed study.

(59) The 1-1F11 hybridoma was found to express an antibody capable of inducing phosphorylation of the Tie-1 receptor in RAECs. Messenger RNA was isolated and variable heavy cDNA synthesized as described above with heavy chain primers from the Wright primer set (Morrison et al. (1995) Antibody Engineering, second edition, Borrebaeck, C. K. A. editor 267-293) that included the 5′ heavy chain primers (SEQ ID NO:35-37) and the 3′ primer (SEQ ID NO:33). Similarly, the light chain variable regions were amplified from cDNA with equimolar amounts of the 5′ light chain primers (SEQ ID NO:38-41) and the 3′ primer (SEQ ID NO:34). The amplified variable region fragments were cloned into the pCR2.1-TOPO vector (Invitrogen) and DNA sequences determined. Based on the determined variable region sequences for the 1-1F11 antibody, the variable heavy sequence was PCR amplified using the pCR2.1-TOPO cloned variable region as template and an equimolar mix of 5′ and 3′ primers (SEQ ID NO:42 and SEQ ID NO:43). The variable light sequence was PCR amplified using a similar strategy. The pCR2.1-TOPO cloned variable region was used as template and an equimolar mix of 5′ and 3′ primers (SEQ ID NO:44 and SEQ ID NO:45). The variable regions were joined by a (G.sub.4S).sub.3 linker; scFv genes were assembled and PCR amplified using an equimolar mix of the above specific variable heavy and variable light PCR products and an equimolar mix of 5′ heavy primer (SEQ ID NO:42) and the 3′ light primer (SEQ ID NO:45). PCR product was cloned into Invitrogen pCR2.1-TOPO (Invitrogen) to yield pRG1192. The sequence was confirmed before sub-cloning the 747 bp Ascl/Srfl to fuse the scFv gene to the N-terminus of a DNA encoding the human IgG.sub.1 Fc fragment (hFc), or the 756 bp Ascl/Notl restriction fragments to fuse the same scFv to the C-terminus of a DNA encoding hFc.

(60) The 2-1G3 hybridoma was also found to express an antibody capable of inducing phosphorylation of the Tie-1 receptor in RAECs. Messenger RNA was isolated and variable heavy cDNA synthesized as described above with equimolar amounts of primers from the from the Wright primer set (Morrison et al. (1995) supra) that included the 5′ heavy chain primers (SEQ ID NO:35-37) and the 3′ primer (SEQ ID NO:33). Similarly, the light chain variable regions were amplified from cDNA with equimolar amounts of the 5′ heavy chain primers (SEQ ID NO:38-41) and the 3′ primer (SEQ ID NO:34). The amplified variable region fragments were cloned into the pCR2.1-TOPO vector (Invitrogen) and the DNA sequences were determined.

(61) Based on the determined variable region sequences for the 2-1G3 antibody, the variable heavy sequence was PCR amplified using the pCR2.1-TOPO cloned variable region as template and an equimolar mix of 5′ and 3′ primers (SEQ ID NO:46 and SEQ ID NO:47). The variable light sequence was PCR amplified using a similar strategy. The pCR2.1-TOPO cloned variable region was used as template and an equimolar mix of 5′ and 3′ primers (SEQ ID NO:48 and SEQ ID NO:49). The variable regions were joined by a (G.sub.4S).sub.3 linker; scFv genes were assembled and PCR amplified using an equimolar mix of the above specific variable heavy and variable light PCR products and an equimolar mix of 5′ 2-1G3 heavy primer (SEQ ID NO:46) and the 3′ light primer (SEQ ID NO:49). PCR product was cloned into Invitrogen pCR2.1-TOPO (Invitrogen) to yield pRG1198. The sequence was confirmed before sub-cloning the 738 bp Ascl/Srfl to fuse the scFv gene to the N-terminus of a DNA encoding the hFc fragment or the 747 bp Ascl/Notl restriction fragments to fuse the same scFv to the C-terminus of a DNA encoding h Fc.

Example 8. Construction of Monospecific and Bispecific Activating Dimers

(62) Two types of scFv-based chimeric molecules were constructed to assess the ability of scFv-based molecules to activate the rTie-1 receptor. One type of molecule used a single scFv fused to both the N-terminus and the C-terminus of hFc, the consequence of which was a monospecific tetravalent molecule capable of binding rTie-1. This molecule should be capable of simultaneously binding four rTie-1 molecules. The plasmid pTE778 encodes the gene for scFv.sub.1-1F11-FC-ScFv.sub.1-1F11 (SEQ ID NO:50) and contains the mROR1 signal peptide and CMV-MIE promoter. The protein was expressed and purified as described above.

(63) Construction of an scFv-Fc-scFv molecule where the two scFv domains are derived from two different non-competing anti-rTie-1 antibodies is expected to yield a molecule capable of clustering more than four receptors, in contrast to the scFv.sub.1-1F11-Fc-scFv.sub.1-1F11 described above, which can cluster only four receptors. It was determined by BIAcore analysis that the binding of the 1-1F11 antibody did not block binding of 2-1G3 to rTie-1, and 1-1F11 binding first did not block binding of 2-1G3. Consequently, scFv molecules made from these antibodies should be capable of clustering more than four receptors. To construct a bispecific tetravalent scFv-based molecule, the scFv.sub.2-1G3 gene was used in combination with the scFv.sub.1-1F11 gene to yield scFv.sub.2-1G3-Fc-scFv.sub.1-1F11 (SEQ ID NO:51). Both constructs were expressed and purified as described above.

(64) The following Tie-binding proteins were also made: a Tie-binding protein that is a dimer of a polypeptide that has an scFv that binds rat Tie-1 linked to a multimerizing component that is an Fc portion, linked to an FD1 (scFv.sub.1-1F11-Fc-FD1) (SEQ ID NO:52); and a Tie-binding protein that is a dimer of a polypeptide that has an scFv that binds rat Tie-1 linked to a multimerizing component that is an Fc portion, linked to an FD2 (scFv.sub.1-1F11-Fc-FD2) (SEQ ID NO:53).

Example 9. Activity of Activating Dimers

(65) SCR.sub.1-1F11-Fc-FD1 was tested for its ability to activate rTie-1 and rTie-2 in a RAEC assay as described herein. Exposure of scFv.sub.1-1F11-Fc-FD1 to RAECs bearing rTie-1 and rTie-2 resulted in a high degree of phosphorylation of both Tie-1 and Tie-2, as measured by phosphotyrosine blot of immunoprecipitates generated using anti-Tie-1 or anti-Tie-2. Phosphotyrosine blots showed a level of phosphorylation of both Tie-1 and Tie-2 that appeared to be at least double the phosphorylation exhibited by exposing the cells to scFv.sub.1-1F11-Fc-FD2 and at least 10-fold or more over a mock challenge (i.e., no antibody); in the same assay, a monospecific antibody that binds rTie-1 showed little to no phosphorylation of either Tie-1 or Tie-2.

(66) SCR.sub.11-1F11-Fc-FD1 was also compared in a RAEC assay with the following Tie-binding proteins: scFv.sub.B2-Fc-FD1, scFv.sub.B2-Fc-FD2, scFv.sub.1-1F11-Fc-ccFV.sub.B2, scFv.sub.1-1F11-Fc-FD2, and FD1-Fc-FD1. Blots of the Tie-1 phosphorylation assays revealed that scFv.sub.1-1F11-Fc-FD1 resulted in the highest phosphorylation of Tie-1 for Tie-binding proteins tested and among the highest activation of Tie-2 for the Tie-binding proteins tested.