Antibody targeting through a modular recognition domain

Abstract

The present invention provides antibodies containing one or more modular recognition domains (MRDs) for targeting the antibodies to specific sites. The use of the antibodies containing one or more modular recognition domains to treat disease, and methods of making antibodies containing one or more modular recognition domains are also provided in the invention.

Claims

1. An antibody fusion protein comprising a full length antibody and a modular recognition domain (MRD), wherein the MRD binds to integrin and consists of the amino acid sequence set forth in any one of SEQ ID NOs: 3-6, wherein the MRD is operably linked to the antibody, and wherein the antibody binds to ErbB2.

2. The antibody fusion protein of claim 1, wherein the antibody and the MRD are operably linked through a linker peptide.

3. The antibody fusion protein of claim 2, wherein the linker peptide comprises a sequence selected from the group consisting of: GGGS (SEQ ID NO: 1), SSGGGGSGGGGGGSS (SEQ ID NO: 2), and SSGGGGSGGGGGGSSRSS (SEQ ID NO: 19).

4. The antibody fusion protein of claim 1, wherein the MRD is operably linked to the C-terminal end of the heavy chain of the antibody, the N-terminal end of the heavy chain of the antibody, the C-terminal end of the light chain of the antibody, or the N-terminal end of the light chain of the antibody.

5. The antibody fusion protein of claim 1, wherein the antibody is a chimeric or humanized antibody.

6. The antibody fusion protein of claim 1, wherein the antibody is Trastuzumab.

7. The antibody fusion protein of claim 2, wherein the antibody is a chimeric or humanized antibody.

8. The antibody fusion protein of claim 2, wherein the antibody is Trastuzumab.

9. A method of treating a disease characterized by undesired angiogenesis, comprising administering to a subject in need thereof the antibody fusion protein of claim 1.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the schematic representation of different designs of tetravalent IgG-like BsAbs.

(2) FIG. 2 shows schematic representations of MRD-antibody fusions. (A) a typical peptibody as C-terminal fusion with Fc; (B) an antibody with a C-terminal MRD fusion with the light chain of the antibody; (C) an antibody with an N-terminal MRD fusion with the light chain of the antibody; and (D) an antibody with unique MRD peptides fused to each terminus of the antibody.

(3) FIG. 3 depicts the results of an ELISA in which integrin and Ang2 were bound by an anti-integrin antibody fused to an ang-2 targeting MRD.

(4) FIG. 4 depicts the results of an ELISA in which integrin and Ang2 were bound by an anti-integrin antibody fused to an ang-2 targeting MRD.

(5) FIG. 5 depicts the results of an ELISA in which an anti-ErbB2 antibody was fused to an MRD which targeted Ang2.

(6) FIG. 6 depicts the results of an ELISA in which an Ang2 targeting MRD was fused to a hepatocyte growth factor receptor binding antibody.

(7) FIG. 7 depicts the results of an ELISA in which an integrin targeting MRD was fused to an ErbB2 binding antibody.

(8) FIG. 8 depicts the results of an ELISA in which an integrin targeting MRD was fused to an hepatocyte growth factor receptor binding antibody.

(9) FIG. 9 depicts the results of an ELISA in which an insulin-like growth factor-I receptor targeting MRD was fused to an ErbB2 binding antibody.

(10) FIG. 10 depicts the results of an ELISA in which a VEGF-targeting MRD was fused to an ErbB2 binding antibody.

(11) FIG. 11 depicts the results of an ELISA in which an integrin targeting MRD was fused to a catalytic antibody.

(12) FIG. 12 depicts the results of an ELISA in which an Ang-2-targeting MRD was fused to a catalytic antibody.

(13) FIG. 13 depicts the results of an ELISA in which an integrin and Ang-2 targeting MRD was fused to an ErbB2 binding antibody.

(14) FIG. 14 depicts the results of an ELISA in which an integrin targeting MRD was fused to an ErbB2-binding antibody.

(15) FIG. 15 depicts the results of an ELISA in which an integrin, Ang-2, or insulin-like growth factor-I receptor-targeting MRD was fused to an ErbB2 or hepatocyte growth factor receptor-binding antibody with a short linker peptide.

(16) FIG. 16 depicts the results of an ELISA in which an integrin, Ang-2, or insulin-like growth factor-I receptor-targeting MRD was fused to an ErbB2 or hepatocyte growth factor receptor-binding antibody with a long linker peptide.

DETAILED DESCRIPTION OF THE INVENTION

(17) The term antibody used herein to refer to intact immunoglobulin molecules and includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies. An intact antibody comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH.sub.1, CH.sub.2 and CH.sub.3. Each light chain is comprised of a light chain variable region (abbreviated herein as V.sub.L) and a light chain constant region. The light chain constant region is comprised of one domain, C.sub.L. The V.sub.H and V.sub.L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V.sub.H and V.sub.L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

(18) A dual-specific antibody is used herein to refer to an immunoglobulin molecule which contain dual-variable-domain immunoglobins, where the dual-variable-domain can be engineered from any two monoclonal antibodies.

(19) An antibody combining site is that structural portion of an antibody molecule comprised of a heavy and light chain variable and hypervariable regions that specifically binds (immunoreacts with) an antigen. The term immunoreact in its various forms means specific binding between an antigenic determinant-containing molecule and a molecule containing an antibody combining site such as a whole antibody molecule or a portion thereof.

(20) The term peptibody refers to a peptide or polypeptide which comprises less than a complete, intact antibody.

(21) The term naturally occurring when used in connection with biological materials such as a nucleic acid molecules, polypeptides, host cells, and the like refers to those which are found in nature and not modified by a human being.

(22) Monoclonal antibody refers to a population of antibody molecules that contain only one species of antibody combining site capable of immunoreacting with a particular epitope. A monoclonal antibody thus typically displays a single binding affinity for any epitope with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different epitope, e.g., a bispecific monoclonal antibody.

(23) A modular recognition domain (MRD) or target binding peptide is a molecule, such as a protein, glycoprotein and the like, that can specifically (non-randomly) bind to a target molecule. The amino acid sequence of a MRD site can tolerate some degree of variability and still retain a degree of capacity to bind the target molecule. Furthermore, changes in the sequence can result in changes in the binding specificity and in the binding constant between a preselected target molecule and the binding site.

(24) Cell surface receptor refers to molecules and complexes of molecules capable of receiving a signal and the transmission of such a signal across the plasma membrane of a cell. An example of a cell surface receptor of the present invention is an activated integrin receptor, for example, an activated v3 integrin receptor on a metastatic cell.

(25) The target binding site or target site is any known, or yet to be described, amino acid sequence having the ability to selectively bind a preselected agent. Exemplary reference target sites are derived from the RGD-dependent integrin ligands, namely fibronectin, fibrinogen, vitronectin, von Willebrand factor and the like, from cellular receptors such as VEGF, ErbB2, vascular homing peptide or angiogenic cytokines, from protein hormones receptors such as insulin-like growth factor-I receptor, epidermal growth factor receptor and the like, and from tumor antigens.

(26) The term protein is defined as a biological polymer comprising units derived from amino acids linked via peptide bonds; a protein can be composed of two or more chains.

(27) A fusion polypeptide is a polypeptide comprised of at least two polypeptides and optionally a linking sequence to operatively link the two polypeptides into one continuous polypeptide. The two polypeptides linked in a fusion polypeptide are typically derived from two independent sources, and therefore a fusion polypeptide comprises two linked polypeptides not normally found linked in nature.

(28) The term linker refers to a peptide located between the antibody and the MRD. Linkers can have from about 2 to 20 amino acids, usually 4 to 15 amino acids.

(29) Target cell refers to any cell in a subject (e.g., a human or animal) that can be targeted by the antibody comprising an MRD of the invention. The target cell can be a cell expressing or overexpressing the target binding site, such as activated integrin receptor.

(30) Patient, subject, animal or mammal are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals. Animals include all vertebrates, e.g., mammals and non-mammals, such as sheep, dogs, cows, chickens, amphibians, and reptiles.

(31) Treating or treatment includes the administration of the antibody comprising an MRD of the present invention to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease, alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. Treatment can be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease. Treatment can be with the antibody-MRD composition alone, or it can be used in combination with an additional therapeutic agent.

(32) As used herein, the terms pharmaceutically acceptable, or physiologically tolerable and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.

(33) Modulate, means adjustment or regulation of amplitude, frequency, degree, or activity. In another related aspect, such modulation may be positively modulated (e.g., an increase in frequency, degree, or activity) or negatively modulated (e.g., a decrease in frequency, degree, or activity).

(34) Cancer, tumor, or malignancy are used as synonymous terms and refer to any of a number of diseases that are characterized by uncontrolled, abnormal proliferation of cells, the ability of affected cells to spread locally or through the bloodstream and lymphatic system to other parts of the body (metastasize) as well as any of a number of characteristic structural and/or molecular features. A cancerous, tumor, or malignant cell is understood as a cell having specific structural properties, lacking differentiation and being capable of invasion and metastasis. Examples of cancers are, breast, lung, brain, bone, liver, kidney, colon, head and neck, ovarian, hematopoietic (e.g., leukemia), and prostate cancer.

(35) Humanized antibody or chimeric antibody includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

(36) The present invention describes an approach based on the adaptation of target binding peptides or modular recognition domains (MRDs) as fusions to catalytic or non-catalytic antibodies that provide for effective targeting of tumor cells or soluble molecules while leaving the prodrug activation capability of the catalytic antibody intact. MRDs can also extend the binding capacity of non-catalytic antibodies providing for an effective approach to extend the binding functionality of antibodies, particularly for therapeutic purposes.

(37) One aspect of the present invention relates to development of a full-length antibody comprising a modular recognition domain (MRD). The interaction between a protein ligand and its target receptor site often takes place at a relatively large interface. However, only a few key residues at the interface contribute to most of the binding. Thus, molecules of peptide length (generally 2 to 60 amino acids) can bind to the receptor protein of a given large protein ligand. It is contemplated that MRDs of the present invention contain a peptide sequence that bind to target sites of interests and are about 2 to 60 amino acids.

(38) The role of integrins such as v3 and v5 as tumor-associated markers has been well documented. A recent study of 25 permanent human cell lines established from advanced ovarian cancer demonstrated that all lines were positive for v5 expression and many were positive for v3 expression. Studies have also shown that v3 and v5 is highly expressed on malignant human cervical tumor tissues. Integrins have also demonstrated therapeutic effects in animal models of Kaposi's sarcoma, melanoma, and breast cancer.

(39) A number of integrin v3 and v5 antagonists are in clinical development. These include cyclic RGD peptides and synthetic small molecule RGD mimetics. Two antibody-based integrin antagonists are currently in clinical trials for the treatment of cancer. The first is Vitaxin, the humanized form of the murine anti-human v3 antibody LM609. A dose-escalating phase I study in cancer patients demonstrated that it was safe for use in humans. Another antibody in clinical trials is CNT095, a fully human mAb that recognizes v integrins. A Phase I study of CNT095 in patients with a variety of solid tumors has shown that it is well tolerated. Cilengitide, a peptide antagonist of v3 and v5, has also proven safe in phase I trials. Furthermore, there has been numerous drug targeting and imaging studies based on the use of ligands for these receptors. These preclinical and clinical observations demonstrate the importance of targeting v3 and v5 and studies involving the use of antibodies in this strategy have consistently reported that targeting through these integrins is safe.

(40) An example of an integrin-binding MRD is an RGD tripeptide-containing binding site, and is exemplary of the general methods described herein. Ligands having the RGD motif as a minimum recognition domain are well known, a partial list of which includes, with the corresponding integrin target in parenthesis, fibronectin (31, 51, v1, IIb3, v3, and 31) fibrinogen (M2 and IIb1) von Willebrand factor (IIb3 and v3), and vitronectin (IIb3, v3 and v5).

(41) Examples of RGD containing targeting MRDs useful in the present invention have amino acid residue sequences shown below:

(42) TABLE-US-00003 (SEQID.NO.:3) YCRGDCT (SEQID.NO.:4) PCRGDCL (SEQID.NO.:5) TCRGDCY (SEQID.NO.:6) LCRGDCF

(43) A MRD that mimics a non-RGD-dependent binding site on an integrin receptor and having the target binding specificity of a high affinity ligand that recognizes the selected integrin is also contemplated in the present invention.

(44) Angiogenesis is essential to many physiological and pathological processes. Ang2 has been shown to act as a proangiogenic molecule. Administration of Ang2-selective inhibitors is sufficient to suppress both tumor angiogenesis and corneal angiogenesis. Therefore, Ang2 inhibition alone or in combination with inhibition of other angiogenic factors such as VEGF may represent an effective antiangiogenic strategy for treating patients with solid tumors.

(45) It is contemplated that MRDs useful in the present invention include those that bind to angiogenic receptors, angiogenic factors, and/or Ang-2. Examples of angiogenic cytokine targeting MRD sequences are listed below:

(46) MGAQTNFMPMDDLEQRLYEQFILQQGLE (SEQ ID. NO.: 7)

(47) MGAQTNFMPMDNDELLLYEQFILQQGLE (SEQ ID. NO.: 8)

(48) MGAQTNFMPMDATETRLYEQFILQQGLE (SEQ ID. NO.: 9)

(49) AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (SEQ ID. NO.: 10) (2Con4)

(50) MGAQTNFMPMDNDELLNYEQFILQQGLE (SEQ ID. NO.: 11)

(51) PXDNDXLLNY (SEQ ID. NO.: 12) where X is one of the 20 naturally-occurring amino acids

(52) MGAQTNFMPMDNDELLLYEQFILQQGLEGGSGSTASSGSGSSLGAQTNFMPMDNDE LLLY (SEQ ID NO: 20)

(53) AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (SEQ ID. NO.: 10)

(54) AQQEECEFAPWTCEHM ConFA (SEQ ID NO:21)

(55) core nEFAPWTn (SEQ ID NO: 22) where n is from about 0 to 50 amino acid residues

(56) AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFAPWTCEHMLE (SEQ ID NO: 23) 2ConFA

(57) AQQEECELAPWTCEHM (SEQ ID NO: 24) ConLA

(58) XnELAPWTXn where n is from about 0 to 50 amino acid residues and X is any amino acid (SEQ ID NO: 25)

(59) AQQEECELAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCEHMLE (SEQ ID NO: 26) 2ConLA

(60) AQQEECEFSPWTCEHM ConFS (SEQ ID NO: 27)

(61) XnEFSPWTXn where n is from about 0 to 50 amino acid residues and X is any amino acid (SEQ ID NO: 28)

(62) AQQEECEFSPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEHMLE 2ConFS (SEQ ID NO: 29)

(63) AQQEECELEPWTCEHM ConLE (SEQ ID NO: 30)

(64) XnELEPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO: 31) and wherein X is any amino acid

(65) AQQEECELEPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELEPWTCEHMLE 2ConLE (SEQ ID NO: 32)

(66) It should be understood that such peptides can be present in dimmers, trimers or other multimers either homologous or heterologous in nature. For example, one can dimerize identical Con-based sequences such as in 2ConFA to provide a homologous dimer, or the Con peptides can be mixed such that ConFA is combined with ConLA to create ConFA-LA heterodimer with the sequence:

(67) TABLE-US-00004 (SEQIDNO:33) AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPW TCEHMLE.

(68) Another heterodimer is ConFA combined with ConFS to create ConFA-FS with the sequence:

(69) TABLE-US-00005 (SEQIDNO:34) AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPW TCEHMLE.

(70) One of skill in the art, given the teachings herein, will appreciate that other such combinations will create functional Ang2 binding MRDs as described herein.

(71) In one aspect, the invention includes a peptide having the sequence:

(72) NFYQCIX.sub.1X.sub.2LX.sub.3X.sub.4X.sub.5PAEKSRGQWQECRTGG (SEQ ID NO:58), wherein X.sub.1 is E or D; X.sub.2 is any amino acid; X.sub.3 is any amino acid; X.sub.4 is any amino acid and X.sub.5 is any amino acid.

(73) The invention also includes peptides having a core sequence selected from:

(74) XnEFAPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO: 22);

(75) XnELAPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO: 25);

(76) XnEFSPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO: 28);

(77) XnELEPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO: 31); or

(78) Xn AQQEECEX.sub.1X.sub.2PWTCEHMXn where n is from about 0 to 50 amino acid residues and X, X.sub.1 and X.sub.2 are any amino acid (SEQ ID NO:57).

(79) Phage display selections and structural studies of VEGF neutralizing peptides in complex with VEGF have been reported. These studies have revealed that peptide v114 (VEPNCDIHVMWEWECFERL) (SEQ ID. NO.: 13) is VEGF specific, binds VEGF with 0.2 M affinity, and neutralizes VEGF-induced proliferation of Human Umbilical Vein Endothelial Cells (HUVEC). Since VEGF is a homodimer, the peptide occupies two identical sites at either end of the VEGF homodimer. An antibody containing an MRD that targets VEGF is contemplated in the present invention. Anti-VEGF antibodies can be found for example in Cancer Research 57, 4593-4599, October 1997; J Biol Chem 281:10 6625, 2006, herein incorporated by reference.

(80) Insulin-like growth factor-I receptor-specific MRDs can be used in the present invention. One example of an MRD sequence that targets the insulin-like growth factor-I receptor is SFYSCLESLVNGPAEKSRGQWDGCRKK (SEQ ID NO.: 14).

(81) Additional IGF-1R MRDs include the following:

(82) TABLE-US-00006 (SEQIDNO:35) NFYQCIEMLASHPAEKSRGQWQECRTGG (SEQIDNO:36) NFYQCIEQLALRPAEKSRGQWQECRTGG (SEQIDNO:37) NFYQCIDLLMAYPAEKSRGQWQECRTGG (SEQIDNO:38) NFYQCIERLVTGPAEKSRGQWQECRTGG (SEQIDNO:39) NFYQCIEYLAMKPAEKSRGQWQECRTGG (SEQIDNO:40) NFYQCIEALQSRPAEKSRGQWQECRTGG (SEQIDNO:41) NFYQCIEALSRSPAEKSRGQWQECRTGG (SEQIDNO:42) NFYQCIEHLSGSPAEKSRGQWQECRTG (SEQIDNO:43) NFYQCIESLAGGPAEKSRGQWQECRTG (SEQIDNO:44) NFYQCIEALVGVPAEKSRGQWQECRTG (SEQIDNO:45) NFYQCIEMLSLPPAEKSRGQWQECRTG (SEQIDNO:46) NFYQCIEVFWGRPAEKSRGQWQECRTG (SEQIDNO:47) NFYQCIEQLSSGPAEKSRGQWQECRTG (SEQIDNO:48) NFYQCIELLSARPAEKSRGQWAECRAG (SEQIDNO:49) NFYQCIEALARTPAEKSRGQWVECRAP

(83) A number of studies have characterized the efficacy of linking the vascular homing peptide to other proteins like IL-I2 or drugs to direct their delivery in live animals. As such, vascular homing MRDs are contemplated for use in the present invention. One example of an MRD sequence that is a vascular homing peptide is ACDCRGDCFCG (SEQ ID NO.: 15).

(84) Numerous other target binding sites are contemplated by the present invention, including epidermal growth factor receptor (EGFR), CD20, tumor antigens, ErbB2, ErbB3, ErbB4, insulin-like growth factor-I receptor, nerve growth factor (NGR), hepatocyte growth factor receptor, and tumor-associated surface antigen epithelial cell adhesion molecule (Ep-CAM). MRDs can be directed towards these target binding sites.

(85) Examples of MRD sequences that bind to EGFR are listed below:

(86) TABLE-US-00007 (SEQID.NO.:16) VDNKFNKELEKAYNEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAE AKKLNDAQAPK. (SEQID.NO.:17) VDNKFNKEMWIAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAE AKKLNDAQAPK.

(87) An example of an MRD sequence that bind to ErbB2 is listed below:

(88) TABLE-US-00008 (SEQID.NO.:18) VDNKENKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAE AKKLNDAQAPK.

(89) The sequence of the MRD can be determined several ways. MRD sequences can be derived from natural ligands or known sequences that bind to a specific target binding site can be used. Additionally, phage display technology has emerged as a powerful method in identifying peptides which bind to target receptors. In peptide phage display libraries, random peptide sequences can be displayed by fusion with coat proteins of filamentous phage. The methods for elucidating binding sites on polypeptides using phage display vectors has been previously described, in particular in WO 94/18221. The methods generally involve the use of a filamentous phage (phagemid) surface expression vector system for cloning and expressing polypeptides that bind to the pre-selected target site of interest.

(90) The methods of the present invention for preparing MRDs involve the use of phage display vectors for their particular advantage of providing a means to screen a very large population of expressed display proteins and thereby locate one or more specific clones that code for a desired target binding reactivity. Once the sequence of the MRD has been elucidated, the peptides may be prepared by any of the methods disclosed in the art.

(91) Variants and derivatives of the MRDs are included within the scope of the present invention. Included within variants are insertional, deletional, and substitutional variants as well as variants that include MRDs presented here with additional amino acids at the N- and/or C-terminus, including from about 0 to 50, 0 to 40, 0 to 30, 0 to 20 amino acids and the like. It is understood that a particular MRD of the present invention may contain one, two, or all three types of variants. Insertional and substitutional variants may contain natural amino acids, unconventional amino acids, or both.

(92) It is contemplated that catalytic and non-catalytic antibodies can be used in the present invention. Antibody 38C2 is an antibody-secreting hybridoma, and has been previously described in WO 97/21803. 38C2 contains an antibody combining site that catalyzes the aldol addition reaction between an aliphatic donor and an aldehyde acceptor. In a syngeneic mouse model of neuroblastoma, systemic administration of an etoposide prodrug and intra-tumor injection of Ab 38C2 inhibited tumor growth.

(93) Other antibodies of interest to this invention include A33 binding antibodies. Human A33 antigen is a transmembrane glycoprotein of the Ig superfamily. The function of the human A33 antigen in normal and malignant colon tissue is not yet known, however, several properties of the A33 antigen suggest that it is a promising target for immunotherapy of colon cancer. These properties include (i) the highly restricted expression pattern of the A33 antigen, (ii) the expression of large amounts of the A33 antigen on colon cancer cells, (iii) the absence of secreted or shed A33 antigen, and (iv) the fact that upon binding of antibody A33 to the A33 antigen, antibody A33 is internalized and sequestered in vesicles, and (v) the targeting of antibody A33 to A33 antigen expressing colon cancer in preliminary clinical studies. Fusion of a MRD directed toward A33 to a catalytic or non-catalytic antibody would increase the therapeutic efficacy of A33 targeting antibodies.

(94) The present invention also contemplates the preparation of mono-, bi-, tri-, tetra-, and penta-specific antibodies. It is contemplated that the antibodies used in the present invention may be prepared by any method known in the art.

(95) In the antibody-MRD fusion molecules prepared according to the present invention, the MRD may be attached to an antibody through the peptide's N-terminus or C-terminus. The MRD may be attached to the antibody at the C-terminal end of the heavy chain of the antibody, the N-terminal end of the heavy chain of the antibody, the C-terminal end of the light chain of the antibody, or the N-terminal end of the light chain of the antibody. The MRD may be attached to the antibody directly, or attached through an optional linker peptide, which can be between 2 to 20 peptides long. The linker peptide can contain a short linker peptide with the sequence GGGS (SEQ ID. NO.:1), a medium linker peptide with the sequence SSGGGGSGGGGGGSS (SEQ ID. NO.:2), or a long linker peptide with the sequence SSGGGGSGGGGGGSSRSS (SEQ ID NO.: 19). The present invention also provides for two or more MRDs which are linked to any terminal end of the antibody. It is also contemplated that two or more MRDs can be directly attached or attached through a linker peptide to two or more terminal ends of the antibody. The multiple MRDs can target the same target binding site, or two or more different target binding sites. Additional peptide sequences may be added to enhance the in vivo stability of the MRD.

(96) The antibody-MRD fusion molecules can be encoded by a polynucleotide comprising a nucleotide sequence. A vector can contain the polynucleotide sequence. The polynucleotide sequence can also be linked with a regulatory sequence that controls expression of the polynucleotide in a host cell. A host cell, or its progeny, can contain the polynucleotide encoding the antibody-MRD fusion molecule.

(97) The present invention contemplates therapeutic compositions useful for practicing the therapeutic methods described herein. Therapeutic compositions of the present invention contain a physiologically tolerable carrier together with at least one species of antibody comprising an MRD as described herein, dissolved or dispersed therein as an active ingredient. In a preferred embodiment, the therapeutic composition is not immunogenic when administered to a human patient for therapeutic purposes.

(98) The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art. Typically such compositions are prepared as sterile injectables either as liquid solutions or suspensions, aqueous or non-aqueous, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified. Thus, an antibody-MRD containing composition can take the form of solutions, suspensions, tablets, capsules, sustained release formulations or powders, or other compositional forms.

(99) The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient.

(100) The therapeutic composition of the present invention can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

(101) Physiologically tolerable carriers are well known in the art. Exemplary of liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, propylene glycol, polyethylene glycol, and other solutes.

(102) Liquid compositions can also contain liquid phases in addition to and to the exclusion of water.

(103) Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, organic esters such as ethyl oleate, and water-oil emulsions.

(104) A therapeutic composition contains an antibody comprising a MRD of the present invention, typically in an amount of at least 0.1 weight percent of antibody per weight of total therapeutic composition. A weight percent is a ratio by weight of antibody to total composition. Thus, for example, 0.1 weight percent is 0.1 grams of antibody-MRD per 100 grams of total composition.

(105) An antibody-containing therapeutic composition typically contains about 10 microgram (ug) per milliliter (ml) to about 100 milligrams (mg) per ml of antibody as active ingredient per volume of composition, and more preferably contains about 1 mg/ml to about 10 mg/ml (i.e., about 0.1 to 1 weight percent).

(106) A therapeutic composition in another embodiment contains a polypeptide of the present invention, typically in an amount of at least 0.1 weight percent of polypeptide per weight of total therapeutic composition. A weight percent is a ratio by weight of polypeptide to total composition. Thus, for example, 0.1 weight percent is 0.1 grams of polypeptide per 100 grams of total composition.

(107) Preferably, an polypeptide-containing therapeutic composition typically contains about 10 microgram (ug) per milliliter (ml) to about 100 milligrams (mg) per ml of polypeptide as active ingredient per volume of composition, and more preferably contains about 1 mg/ml to about 10 mg/ml (i.e., about 0.1 to 1 weight percent).

(108) In view of the benefit of using humanized or chimeric antibodies in vivo in human patients, the presently described antibody-MRD molecules are particularly well suited for in vivo use as a therapeutic reagent. The method comprises administering to the patient a therapeutically effective amount of a physiologically tolerable composition containing an antibody comprising a MRD of the invention.

(109) The dosage ranges for the administration of the antibody comprising a MRD of the invention are those large enough to produce the desired effect in which the disease symptoms mediated by the target molecule are ameliorated. The dosage should not be so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication.

(110) A therapeutically effective amount of an antibody comprising a MRD of this invention is typically an amount of antibody such that when administered in a physiologically tolerable composition is sufficient to achieve a plasma concentration of from about 0.1 microgram (ug) per milliliter (ml) to about 100 ug/ml, preferably from about 1 ug/ml to about 5 ug/ml, and usually about 5 ug/ml. Stated differently, the dosage can vary from about 0.1 mg/kg to about 300 mg/kg, preferably from about 0.2 mg/kg to about 200 mg/kg, most preferably from about 0.5 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or several days.

(111) The antibody comprising a MRD of the invention can be administered parenterally by injection or by gradual infusion over time. Although the target molecule can typically be accessed in the body by systemic administration and therefore most often treated by intravenous administration of therapeutic compositions, other tissues and delivery means are contemplated where there is a likelihood that the tissue targeted contains the target molecule. Thus, antibodies comprising a MRD of the invention can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, and can be delivered by peristaltic means.

(112) The therapeutic compositions containing a human monoclonal antibody or a polypeptide of this invention are conventionally administered intravenously, as by injection of a unit dose, for example. The term unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.

(113) The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for administration are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.

(114) The following examples are intended to illustrate but not limit the invention.

EXAMPLES

Example 1. Integrin Targeting Antibody-MRD Molecules

(115) Novel antibody-MRD fusion molecules were prepared by fusion of an integrin v3-targeting peptides to catalytic antibody 38C2. Fusions at the N-termini and C-termini of the light chain and the C-termini of the heavy chain were most effective. Using flow cytometry, the antibody conjugates were shown to bind efficiently to integrin v3-expressing human breast cancer cells. The antibody conjugates also retained the retro-aldol activity of their parental catalytic antibody 38C2, as measured by methodol and doxorubicin prodrug activation. This demonstrates that cell targeting and catalytic antibody capability can be efficiently combined for selective chemotherapy.

Example 2. Angiogenic Cytokine Targeting Antibody-MRD Molecules

(116) Angiogenic cytokine targeting antibody-MRD fusion molecules were constructed. The antibody used was 38C2, which was fused with a MRD containing the 2Con4 peptide (AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (SEQ ID. NO.: 10)). The MRD peptide was fused to either the N- or C-terminus of the light chain and the C-terminus of the heavy chain. Similar results were found with the other Ang-2 MRD peptides. Additional Ang-2 MRD peptides include:

(117) LM-2-32

(118) MGAQTNFMPMDNDELLLYEQFILQQGLEGGSGSTASSGSGSSLGAQTNFMPMDNDE LLLY (SEQ ID NO:20)

(119) AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (2Con4) (SEQ ID. NO.: 10)

(120) AQQEECEFAPWTCEHM ConFA (SEQ ID NO:21)

(121) core XnEFAPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:22)

(122) AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFAPWTCEHMLE 2ConFA (SEQ ID NO:23)

(123) AQQEECELAPWTCEHM ConLA (SEQ ID NO:24)

(124) XnELAPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:25)

(125) AQQEECELAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCEHMLE 2ConLA (SEQ ID NO:26)

(126) AQQEECEFSPWTCEHM ConFS (SEQ ID NO:27)

(127) XnEFSPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:28)

(128) AQQEECEFSPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEHMLE 2ConFS (SEQ ID NO:29)

(129) AQQEECELEPWTCEHM ConLE (SEQ ID NO:30)

(130) XnELEPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:31)

(131) AQQEECELEPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELEPWTCEHMLE 2ConLE (SEQ ID NO:32).

(132) It should be understood that such peptides can be present in dimmers, trimers or other multimers either homologous or heterologous in nature. For example, one can dimerize identical Con-based sequences such as in 2ConFA to provide a homologous dimer, or the Con peptides can be mixed such that ConFA is combined with ConLA to create ConFA-LA heterodimer with the sequence:

(133) TABLE-US-00009 (SEQIDNO:33) AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPW TCEHMLE.

(134) Another illustrative heterodimer is ConFA combined with ConFS to create ConFA-FS with the sequence:

(135) TABLE-US-00010 (SEQIDNO:34) AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPW TCEHMLE.

(136) One of skill in the art, given the teachings herein, will appreciate that other such combinations will create functional Ang2 binding MRDs as described herein.

Example 3. Antibody-MRD Fusions with Non-Catalytic Antibodies

(137) A humanized mouse monoclonal antibody, LM609, directed towards human integrin v3 has been previously described. (Rader, C. et. al., 1998. Rader C, Cheresh D A, Barbas C F 3rd. Proc Natl Acad Sci USA. 1998 Jul. 21; 95(15):8910-5).

(138) A human non-catalytic monoclonal Ab, JC7U was fused to an anti-Ang2 MRD containing 2Con4 (AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (SEQ ID. NO.: 10)) at either the N- or C-terminus of the light chain. 2Con4 (AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (SEQ ID. NO.: 10)) was studied as an N-terminal fusion to the Kappa chain of the antibody (2Con4-JC7U) and as a C-terminal fusion (JC7U-2Con4). Both fusions maintained integrin and Ang2 binding. As shown in the left panel of FIG. 3, both antibody constructs (2Con4-JC7U and JC7U-2Con4) specifically bound to recombinant Ang2 as demonstrated by ELISA studies. Binding to Ang2, however, is significantly higher with JC7U-2Con4, which has the 2Con4 (SEQ ID. NO.: 10) fusion at the C-terminus of the light chain of the antibody. The right panel of FIG. 3 depicts the binding of Ang2-JC7U and JC7U-Ang2 to integrin v3. The results show that fusion of 2Con4 (SEQ ID. NO.: 10) to either the N- or the C-light chain terminus does not affect mAb JC7U binding to integrin v3. FIG. 4 depicts another ELISA study using the same antibody-MRD fusion constructs.

Example 4. Herceptin-MRD Fusion Molecules

(139) Another example of MRD fusions to a non-catalytic antibody are Herceptin-MRD fusion constructs. The Herceptin-MRD fusions are multifunctional, both small-molecule v integrin antagonists and the chemically programmed integrin-targeting antibody show remarkable efficacy in preventing the breast cancer metastasis by interfering with v-mediated cell adhesion and proliferation. MRD fusions containing Herceptin-2Con4 (which targets ErbB2 and ang2) and Herceptin-V114 (which targets ErbB2 and VEGF targeting) and Herceptin-RGD-4C-2Con4 (which targets ErbB2, ang2, and integrin targeting) are effective.

Example 5. VEGF Targeting Antibody-MRD Molecules

(140) An antibody containing an MRD that targets VEGF was constructed. A MRD which targets v114 (SEQ ID. NO. 13) was fused at the N-terminus of the kappa chain of 38C2 and Herceptin using the long linker sequence (SEQ ID. NO. 2). Expression and testing of the resulting antibody-MRD fusion constructs demonstrated strong VEGF binding.

Example 6. IGF-1R Targeting Antibody-MRD Molecules

(141) Fusion of an MRD which targets the IGF-1R (SFYSCLESLVNGPAEKSRGQWDGCRKK (SEQ ID. NO.: 14)) to the N-terminus of the kappa chain of 38C2 and Herceptin using the long linker sequence as a connector was studied. Expression and testing of the resulting antibody-MRD fusion constructs demonstrated strong IGF-1R binding. Additional clones showing high binding to IGR-1R were also identified after several rounds of mutagenesis and screening. The preferred sequences listed below show no significant or no binding affinity to the insulin receptor. (see Table 2).

(142) TABLE-US-00011 TABLE1 Templateforfurthermutagenesis: Rm2-2-218 GTGGAGTGCAGGGCGCCG VECRAP SEQIDNO: 50,51 Rm2-2-316 GCTGAGTGCAGGGCTGGG AECRAG SEQIDNO: 52,53 Rm2-2-319 CAGGAGTGCAGGACGGGG QECRTG SEQIDNO: 54,55

(143) TABLE-US-00012 TABLE2 SEQ ID Mutant Aminoacidsequence Template NO: Rm4-31 NFYQCIEMLASHPAEKSRGQWQECRTGG Rm2-2-319 35 Rm4-33 NFYQCIEQLALRPAEKSRGQWQECRTGG Rm2-2-319 36 Rm4-39 NFYQCIDLLMAYPAEKSRGQWQECRTGG Rm2-2-319 37 Rm4-310 NFYQCIERLVTGPAEKSRGQWQECRTGG Rm2-2-319 38 Rm4-314 NFYQCIEYLAMKPAEKSRGQWQECRTGG Rm2-2-319 39 Rm4-316 NFYQCIEALQSRPAEKSRGQWQECRTGG Rm2-2-319 40 Rm4-319 NFYQCIEALSRSPAEKSRGQWQECRTGG Rm2-2-319 41 Rm4-44 NFYQCIEHLSGSPAEKSRGQWQECRTG Rm2-2-319 42 Rm4-45 NFYQCIESLAGGPAEKSRGQWQECRTG Rm2-2-319 43 Rm4-46 NFYQCIEALVGVPAEKSRGQWQECRIG Rm2-2-319 44 Rm4-49 NFYQCIEMLSLPPAEKSRGQWQECRTG Rm2-2-319 45 Rm4-410 NEYQCIEVFWGRPAEKSRGQWQECRTG Rm2-2-319 46 Rm4-411 NFYQCIEQLSSGPAEKSRGQWQECRTG Rm2-2-319 47 Rm4-415 NFYQCIELLSARPAEKSRGQWAECRAG Rm2-2-316 48 Rm4-417 NFYQCIEALARTPAEKSRGQWVECRAP Rm2-2-218 49

Example 7. ErbB2 Binding, Ang-2-Targeting Antibody-MRD Molecules

(144) An antibody was constructed which contains an MRD that targets Ang-2 (L17) fused to the light chain of an antibody which binds to ErbB2. Either the short linker sequence, the long linker sequence, or the 4th loop in the light chain constant region was used as a linker. FIG. 5 depicts the results of an ELISA using constructs containing an N-terminal fusion of Ang-2 targeting MRD with the ErbB2 antibody with the short linker peptide (GGGS (SEQ ID NO.: 1)) (L17-sL-Her), a C-terminal fusion of Ang-2 targeting MRD with the ErbB2 antibody with the short linker peptide (Her-sL-L17), a C-terminal fusion of Ang-2 targeting MRD with the ErbB2 antibody with the 4th loop in the light chain constant region (Her-lo-L17), or an N-terminal fusion of Ang-2 targeting MRD with the ErbB2 antibody with the long linker peptide (SSGGGGSGGGGGGSSRSS (SEQ ID NO.: 19)) (L17-lL-Her). ErbB2 was bound with varying degrees by all of the constructs. However, Ang-2 was bound only by Her-sL-L17 and L17-lL-Her.

Example 8. Hepatocyte Growth Factor Receptor Binding, Ang-2-Targeting Antibody-MRD Molecules

(145) Fusion of an MRD which targets Ang-2 (L17) was made to either the N-terminus or C-terminus of the light chain of the Met antibody, which binds to hepatocyte growth factor receptor. Either the short linker sequence or the long linker sequence were used as a connector. FIG. 6 depicts the results of an ELISA using constructs containing N-terminal fusion of Ang-2 targeting MRD with the Met antibody with the short linker peptide (GGGS (SEQ ID NO.: 1)) (L17-sL-Met), N-terminal fusion of Ang-2 targeting MRD with the Met antibody with the long linker peptide (SSGGGGSGGGGGGSSRSS (SEQ ID NO.: 19)) (L17-lL-Met), and C-terminal fusion of Ang-2 targeting MRD with the Met antibody with the long linker peptide (Met-iL-L17). Expression and testing of the resulting antibody-MRD fusion constructs demonstrated strong Ang-2 binding when the long linker peptide was used. Fusion of the Ang-2 targeting MRD to the C-light chain terminus of the antibody resulted in slightly higher binding to Ang-2 then fusion of the Ang-2 targeting to the N-light chain terminus of the antibody.

Example 9. ErbB2 Binding, Integrin-Targeting Antibody-MRD Molecules

(146) An antibody was constructed which contains an MRD that targets integrin v3 (RGD4C) fused to the light chain of an antibody Herceptin which binds to ErbB2 (Her). Either the short linker sequence, the long linker sequence, or the 4th loop in the light chain constant region was used as a linker. FIG. 7 depicts the results of an ELISA using constructs containing an N-terminal fusion of integrin v3 targeting MRD with the ErbB2 antibody with the short linker peptide (GGGS (SEQ ID NO.: 1)) (RGD4C-sL-Her), a C-terminal fusion of integrin v3 targeting MRD with the ErbB2 antibody with the short linker peptide (Her-sL-RGD4C), a C-terminal fusion of integrin v3 targeting MRD with the ErbB2 antibody with the 4th loop in the light chain constant region (Her-lo-RGD4C), or an N-terminal fusion of integrin v3 targeting MRD with the ErbB2 antibody with the long linker peptide (SSGGGGSGGGGGGSSRSS (SEQ ID NO.: 19)) (RGD4C-lL-Her). ErbB2 was bound with varying degrees by all of the constructs. However, integrin v3 was bound only by RGD4C-lL-Her.

Example 10. Hepatocyte Growth Factor Receptor Binding, Integrin-Targeting Antibody-MRD Molecules

(147) An antibody was constructed which contains an MRD that targets integrin v3 (RGD4C) fused to the light chain of an antibody which binds to the hepatocyte growth factor receptor (Met). Antibody-MRD constructs containing the long linker sequence were used. FIG. 8 depicts the results of an ELISA using constructs containing an N-terminal fusion of integrin v3 targeting MRD with the hepatocyte growth factor receptor antibody (RGD4C-lL-Met), or a C-terminal fusion of integrin v3 targeting MRD with the hepatocyte growth factor receptor antibody (Met-lL-RGD4C). The RGD4C-lL-Met demonstrated strong integrin v3 binding.

Example 11. ErbB2 Binding, Insulin-Like Growth Factor-I Receptor-Targeting Antibody-MRD Molecules

(148) Antibodies were constructed which contains an MRD that targets insulin-like growth factor-I receptor (RP) fused to the light chain of an antibody which binds to ErbB2 (Her). Either the short linker peptide, the long linker peptide, or the 4th loop in the light chain constant region was used as a linker. (Carter et al., Proc Natl Acad Sci USA. 1992 May 15; 89(10):4285-9.

(149) PMID: 1350088 [PubMedindexed for MEDLINE]; U.S. Pat. No. 5,677,171; ATCC Deposit 10463, all incorporated by reference herein). FIG. 9 depicts the results of an ELISA using constructs containing an N-terminal fusion of insulin-like growth factor-I receptor targeting MRD with the ErbB2 antibody with the short (RP-sL-Her), a C-terminal fusion of insulin-like growth factor-I receptor targeting MRD with the ErbB2 antibody and the short linker peptide (Her-sL-RP), a C-terminal fusion of insulin-like growth factor-I receptor targeting MRD with the ErbB2 antibody with the 4th loop in the light chain constant region (Her-lo-RP), an N-terminal fusion of insulin-like growth factor-I receptor targeting MRD with the ErbB2 antibody with the long linker peptide (RP-lL-Her), or a C-terminal fusion of insulin-like growth factor-I receptor targeting MRD with the ErbB2 antibody with the long linker peptide (Her-lL-RP). ErbB2 was bound with varying degrees by all of the constructs. Insulin-like growth factor-I receptor was bound by RP-lL-Her.

Example 12. ErbB2 Binding, VEGF-Targeting Antibody-MRD Molecules

(150) Fusion of an MRD which targets VEGF (V114) was made to the N-terminus of the the light chain of a ErbB2-binding antibody (Her). A medium linker peptide (SSGGGGSGGGGGGSS (SEQ ID NO.: 2)) was used as a connector. FIG. 10 depicts the results of an ELISA using a construct containing an N-terminal fusion of VEGF targeting MRD with the ErbB2-binding antibody with the medium linker peptide (V114-mL-Her). Expression and testing of the resulting antibody-MRD fusion construct demonstrated strong VEGF and ErbB2 binding.

Example 13. Integrin Targeting Antibody-MRD Molecules

(151) Fusion of an MRD which targets integrin v3 (RGD) to the N-terminus of the light chain of 38C2 using the medium linker peptide as a connector was studied. FIG. 11 demonstrates that expression and testing of the resulting antibody-MRD fusion construct had strong integrin v3 binding.

Example 14. Ang-2 Targeting Antibody-MRD Molecules

(152) Fusion of an MRD which targets Ang-2 (L17) to the C-terminus of the light chain of 38C2 using the short linker sequence as a connector was studied. FIG. 12 demonstrates that expression and testing of the resulting antibody-MRD fusion construct had strong Ang-2 binding.

Example 15. ErbB2 Binding, Integrin and Ang-2 Targeting Antibody-MRD Molecules

(153) An MRD which targets integrin v3 (RGD4C) was connected to the N-terminus of the light chain of an ErbB2 targeting antibody (Her) with a medium linker, and an Ang-2 (L17) targeting MRD was connected by a short linker to the C-terminus of the same ErbB2 targeting antibody (RGD4C-mL-Her-sL-L17). FIG. 13 demonstrates that the resulting antibody-MRD fusion construct bound to integrin, Ang-2, and ErbB2.

Example 16. ErbB2 Binding, Integrin-Targeting Antibody-MRD Molecules

(154) An antibody was constructed which contains an MRD that targets integrin v3 (RGD4C) fused to the N-terminus of the heavy chain of an antibody which binds to ErbB2 (Her) using the medium linker as a connector (RGD4C-mL-her-heavy). FIG. 14 depicts the results of an ELISA using the construct. Both integrin and ErbB2 were bound by the construct.

Example 17. ErbB2 or Hepatocyte Growth Factor Receptor Binding, and Integrin, Ang-2 or Insulin-Like Growth Factor-I Receptor-Targeting Antibody-MRD Molecules with the Short Linker Peptide

(155) Antibody-MRD molecules were constructed which contain ErbB2 or hepatocyte growth factor receptor binding antibodies, and integrin v3, Ang-2 or insulin-like growth factor-I receptor-targeting MRD regions were linked with the short linker peptide to the light chain of the antibody. FIG. 15 depicts the results of an ELISA using constructs containing an N-terminal fusion of Ang-2 targeting MRD fused to the ErbB2 antibody (L17-sL-Her), an N-terminal fusion of integrin-targeting MRD with the ErbB2 antibody (RGD4C-sL-Her), an N-terminal fusion of insulin-like growth factor-I receptor targeting MRD with the ErbB2 binding antibody (RP-sL-Her), a C-terminal fusion of Ang-2 targeting MRD with the hepatocyte growth factor receptor binding antibody (L17-sL-Met), a C-terminal fusion of Ang-2 targeting MRD with the ErbB2 binding antibody (Her-sL-L17), a C-terminal fusion of integrin targeting MRD with the ErbB2 binding antibody (Her-sL-RGD4C), or a C-terminal fusion of insulin-like growth factor-I receptor targeting MRD with the ErbB2 binding antibody (Her-sL-RP). ErbB2 was bound with varying degrees by the antibody-MRD constructs, with the exception of the construct containing the hepatocyte growth factor receptor-binding antibody. Antigen was bound only by the Her-sL-L17 construct.

Example 18. ErbB2 or Hepatocyte Growth Factor Receptor Binding, and Integrin, Ang-2 or Insulin-Like Growth Factor-I Receptor-Targeting Antibody-MRD Molecules with the Long Linker Peptide

(156) Antibody-MRD molecules were constructed which contain ErbB2 or hepatocyte growth factor receptor binding antibodies, and integrin v3, Ang-2 or insulin-like growth factor-I receptor-targeting MRD regions linked with the long linker peptide to the light chain of the antibody. FIG. 16 depicts the results of an ELISA using constructs containing an N-terminal fusion of Ang-2 targeting MRD fused to the ErbB2 antibody (L17-lL-Her), an N-terminal fusion of integrin-targeting MRD with the ErbB2 antibody (RGD4C-lL-Her), an N-terminal fusion of insulin-like growth factor-I receptor-targeting MRD with the ErbB2 binding antibody (RP-lL-Her), a C-terminal fusion of Ang-2 targeting MRD with the hepatocyte growth factor receptor binding antibody (L17-lL-Met), a C-terminal fusion of integrin targeting MRD with the hepatocyte growth factor receptor binding antibody (RGD4C-lL-Met), a C-terminal fusion of Ang-2 targeting MRD with the insulin-like growth factor-I receptor binding antibody (Her-lL-RP), a C-terminal fusion of Ang-2 targeting MRD with the the hepatocyte growth factor receptor binding antibody (Met-lL-L17), or a C-terminal fusion of integrin targeting MRD with the the hepatocyte growth factor receptor binding antibody (Met-lL-RGD4C). As shown in FIG. 16, antibody-MRD fusions are effective to bind antigen and ErbB2. Lu et al. J Biol Chem. 2005 May 20; 280(20):19665-72. Epub 2005 Mar. 9; Lu et al. J Biol Chem. 2004 Jan. 23; 279(4):2856-65. Epub 2003 Oct. 23,

(157) Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.