EA040917B1 - DELIVERY OF ANTIBODIES AGAINST EGFR VIA A MODULAR RECOGNITION DOMAIN - Google Patents

Description

The field to which the invention belongs

In general terms, the present invention relates to antibodies containing one or more of these modular recognition domains, and more specifically to the use of antibodies containing one or more of these modular recognition domains for the treatment of diseases and to methods for producing antibodies containing one or more of these modular recognition domains. .

Prior Art

Catalytically active monoclonal antibodies (Abs) can be used for selective prodrug activation and chemical transformations. Monoclonal antibodies Ab with aldolase activity act as highly efficient catalysts for a number of chemical transformations, in particular, in aldol and retroaldol reactions. The retro-aldolase activity of Ab antibodies such as 38C2 and 93F3 enables researchers to design, synthesize, and evaluate prodrugs of various chemotherapeutic agents that can be activated through retroaldol reactions. (The construction of 38C2 is described in WO 97/21803, which is incorporated herein by reference.) 38C2 contains an antigen binding site that catalyzes an aldol condensation reaction between an aliphatic donor and an aldehyde acceptor. In syngeneic mice with a neuroblastoma model, systemic administration of the etoposide prodrug and intratumoral injection of 38C2 resulted in inhibition of tumor growth.

One disadvantage of the use of catalytic Abs is the lack of a means to deliver the catalytic Ab to malignant cells. Preliminary studies have shown that in the antibody dependent enzyme mediated prodrug therapy (ADEPT) or antibody dependent abzyme mediated prodrug therapy (ADAPT) method, enzymes or catalytic antibodies can be delivered to tumor cells by chemical conjugation or recombinant attachment to the antibodies to be delivered. However, a more effective alternative may be to use a catalytic antibody attached to the peptide to be delivered and located outside the antigen binding site, which makes the active site available for prodrug activation. For example, attachment of Ab 38C2 to an αΎβ3 integrin binding peptide allows the antibody to selectively localize to the tumor and/or the tumor vasculature and trigger prodrug activation in that region. The possibility of implementing such a method of therapy was confirmed by the data of preclinical and clinical phase III studies and suggests that the peptides can be turned into full-fledged drugs by attaching them to the Fc regions of the antibody.

Production of bispecific or multispecific antibodies that can be delivered simultaneously to two or more cancer targets and/or activate prodrugs is a new and promising way to treat cancer and other diseases. Such antibodies are illustrated in FIG. 1 of this application. Bispecific antibody (BsAb) studies simultaneously targeting two tumor-associated antigens (eg, growth factor receptors) that have been performed to assess inhibition of multiple cell proliferation/survival pathways have confirmed the effectiveness of this method. Typically, bispecific antibodies are produced by chemically linking two different monoclonal antibodies or by fusing two hybridoma cell lines to produce a hybridoma hybridoma. Bispecific tetravalent IgG-like molecules or immunoglobulins consisting of two variable domains were constructed from two monoclonal antibodies. These immunoglobulins, consisting of two variable domains, have the ability to bind to both antigens in the presence of serum. However, such methods have certain disadvantages in terms of their industrial production, yield and purity.

A number of recombinant techniques have been developed to efficiently generate small BsAb fragments such as diantibody, minibody, and Fab-scFv fusion proteins. Such BsAb fragments, compared with full-length IgG-like molecules, may have certain advantages in terms of some clinical applications, such as tumor radioimaging and delivery to tumors, since these fragments have a higher level of tissue penetration and are more quickly cleared from the bloodstream. On the other hand, compared to smaller BsAb fragments, an IgG-like BsAb may be preferred for other in vivo applications, in particular in oncology, due to the presence of an Fc domain that confers a longer serum half-life and retains secondary immune function such as antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity. However, unlike fragment analogues, the construction and production of recombinant IgG-like BsAbs presents certain technical difficulties due to their large size (~150-200 kDa) and the complexity of their structure. As shown by the successful use of animal models, progress in this area has been very limited. A recent evaluation of a number of constructs indicates some progress in the efficient expression of BsAb molecules containing the Fc domain in mammalian cells.

Another method for delivering antibodies is the use of peptibodies. Peptide antibodies are essentially hybrids of peptides with Fc regions of antibodies. In view of the success of randomized peptide library studies to discover peptide ligands with high affinity for a wide range of targets, it has been concluded that the attachment of such peptides to the Fc regions of antibodies is a means that converts peptides into candidate therapeutic agents due to increased circulating half-life and increased potency as a result of increased valency.

Biochemical processes are based on the interaction of proteins with other molecules. Such protein interactions are receptor-ligand interactions, antibody-antigen interactions, cell contact, and interactions of pathogens with target tissues. Protein interactions may include contact with other proteins, carbohydrates, oligosaccharides, lipids, metal ions, and other substances. The main site of protein interaction is the region of the protein involved in contact and recognition, and this region is called the binding site or targeting site.

Peptides derived from a phage display library typically retain their binding properties when contacted with other molecules. Particular peptides of this type can be produced as specificity modular units or molecular recognition domains (MRDs) that can be combined to create a single protein having binding specificity for several defined targets.

An example of such a specific target binding site is an integrin. Integrins are a family of transmembrane cell-adhesion molecules that consist of α- and β-subunits and mediate the attachment of cells to proteins in the extracellular matrix (ECM). Currently, eighteen α-subunits and eight β-subunits are known, and these subunits form 24 different αβ-heterodimers with different specificities for different proteins that attach to ECM cells. The ligands for various integrins are fibronectin, collagen, laminin, von Willebrand factor, osteopontin, thrombospondin and vitronectin, all of which are components of the ECM. Some integrins can also bind to soluble ligands such as fibrinogen or other adhesion molecules on neighboring cells. Integrins are known to exist in different states of activation and have different affinities for the ligand. The recognition of soluble ligands by the action of integrins is strictly dependent on specific changes in the conformation of the receptor. This provides a switch in the type of molecules that regulate the ability of cells to aggregate by an integrin-dependent mechanism and to stop the cell cycle under conditions of dynamic circulation in the vascular system. Such a mechanism has been well studied for leukocytes and platelets circulating in the circulation in the resting cell phase, during which non-activated integrins are expressed. Following stimulation by proinflammatory and proplatelet agonists, this cell type rapidly responds to a range of molecular changes, including the transition of key integrins, β2 integrins for leukocytes and ave3 integrins for platelets, from a resting state to an activated state. This leads to a cessation of the cell cycle of this type of cells in the vascular system, to stimulation of cell adhesion and to the formation of blood clots.

It has been demonstrated that a metastatic subpopulation of human breast cancer cells expresses the ave3 integrin in a constitutively activated form. This disruption of ave3 expression plays a role in the metastasis of breast cancer cells, as well as prostate cancer, melanoma, and neuroblastoma. The activated receptor stimulates the migration of cancer cells to a high degree and leads to the arrest of the cell cycle in the bloodstream. Through this mechanism, ave3 activation imparts to metastatic cells key properties that are likely to play a critical role in their successful dissemination and colonization in target organs. Tumor cells that successfully invade the target organ can also use ave3 to grow in new environments, since ave3 interaction with the matrix can stimulate cell survival and proliferation. For example, binding of ave3 to osteopontin stimulates their malignancy, and an increase in osteopontin levels correlates with a poor prognosis of breast cancer.

For these reasons, and based on the already established role of the ave3 integrin in angiogenesis, this integrin is regarded as one of the best studied integrins. Antagonists of this molecule have significant potential for targeted drug delivery. One approach used to target the ave3 integrin is based on the high specificity of binding of peptides containing the Arg-Gly-Asp (RGD) sequence to ave3. This tripeptide, which is normally present in extracellular matrix proteins, is the primary binding site for the ave3 integrin. However, when using reporter probes based on RGD, there are certain problems associated with its rapid clearance from the bloodstream, high levels of absorption by the kidneys and liver, and rapid clearance from the tumor. It has been shown that chemical modification of cyclized RGD peptides leads to an increase in their stability and valency. These modified peptides are then coupled to radioactive isotopes and used either to image the tumor or to inhibit tumor growth.

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The ανβ3 integrin is one of the best characterized integrin heterodimers and one of several heterodimers involved in tumor-induced angiogenesis. Despite the low level of expression of αΎβ3 in mature blood vessels, this integrin is significantly activated during angiogenesis in vivo. Expression of ave3 correlates with the progression of breast and cervical cancer, as well as malignant melanoma. Recent studies suggest that αΎβ3 can be used as a diagnostic or prognostic indicator for some tumors. The αΎβ3 integrin is particularly attractive as a therapeutic target due to its relatively limited distribution in cells. It is not normally expressed on epithelial cells and is minimally expressed on other cell types. In addition, αΎβ3 antagonists, including RGD cyclic peptides and monoclonal antibodies, significantly inhibit cytokine-induced angiogenesis and the growth of solid tumors on the chicken chorioallantoic membrane.

Another integrin heterodimer, αΎβ5, exhibits broader expression on malignant tumor cells and appears to be involved in VEGF-mediated angiogenesis. It has been shown that ave3 and ave5 stimulate angiogenesis by different mechanisms: αΎβ3 stimulates angiogenesis through bFGF and TNF-α, and αΎβ5 through VEGF and TGF-α. It has also been shown that inhibition of Src kinase can block VEGF-induced but not FGF2-induced angiogenesis.

These results clearly indicate that FGF2 and VEGF activate different angiogenesis pathways that require the involvement of αΎβ3 and αΎβ5, respectively.

Integrins are also involved in the development of tumor metastases. Metastases are a major cause of cancer incidence and cancer mortality. The progression of malignant melanoma, glioma, ovarian cancer, and breast cancer is highly associated with the expression of the αΎβ3 integrin, and in some cases with the expression of αΎβ5. More recently, ave3 integrin activation has been shown to play a significant role in the development of human breast cancer metastases. A very strong correlation has been established between αΎβ3 expression and the development of breast cancer metastases, where normal breast epithelium does not contain αΎβ3, and approximately 50% of invasive lobular carcinomas and almost all bone metastases in breast cancer express αΎβ3. Studies conducted on breast cancer xenografts have shown that therapy using αΎβ3-binding cyclic peptide antagonists and radiation immunotherapy used together have a synergistic effect.

Angiogenesis, i.e. the formation of new blood vessels from existing vessels may play a major role in physiological and pathological processes. Normally, angiogenesis is tightly regulated by pro- and anti-angiogenic factors, however, in the case of diseases such as cancer, neovascular eye disease, arthritis, and psoriasis, this process can go down an abnormal path. The relationship of angiogenesis to disease progression makes the discovery of anti-angiogenic compounds particularly attractive. The most promising phage anti-angiogenic peptide, currently described and developed by Amgen, neutralizes the angiogenic cytokine Ang-2.

With regard to angiogenesis, the most widely distributed target molecules are VEGF and their receptors, and preclinical research programs targeting the recently discovered angiopoietin-Tie-2 pathway are currently being developed. Both families of these proteins are involved in ligand-receptor interactions and include members whose function is largely limited to postnatal endothelial cells and certain hematopoietic stem cell lineages. Tie-2 is a tyrosine kinase receptor with four known ligands, ie. angiopoietins 1-4 (Ang-1-Ang-4), of which only Ang-1 and Ang-2 are best studied. Ang-1 stimulates Tie-2 phosphorylation, and interaction of Ang-2 with Tie-2 has been shown to antagonize and agonize the Tie-2 receptor. An increase in the expression level of Ang-2 in the areas of normal and pathological postnatal angiogenesis indirectly indicates that Ang-2 may play a certain role in proangiogenesis. Induction of Ang-2, which is selective for certain vessels and associated with angiogenesis, has been observed in several diseases, including cancer. In patients with colon carcinoma, Ang-2 is ubiquitously expressed in the tumor epithelium, while Ang-1 expression in the tumor epithelium has been found to occur quite rarely. It has been suggested that a net increase in Ang-2 activity is a factor initiating tumor angiogenesis.

Other fusion proteins directed against cellular receptors are in clinical trials. Herceptin (trastuzumab), developed by Genentech, is a recombinant humanized monoclonal antibody directed against the extracellular domain of the human epidermal tyrosine kinase receptor 2 (HER2 or ErbB-2). The HER2 gene is overexpressed in 25% of invasive breast cancers and is associated with poor prognosis and altered chemotherapeutic responsiveness. Herceptin blocks the proliferation of ErbB-2-overexpressing breast cancer, and is currently for the treatment of ErbB2-overexpressing breast cancer metastases (MBC) by the Quality Control Authority.

- 3 040917 FDA (USA) approved therapy only with anti-ErbB-2 antibodies. In healthy adult cells, several ErbB-2 molecules are present on the cell surface, namely ~20,000 per cell, and therefore several heterodimers can be formed, hence growth signals are relatively weak and regulated. When ErbB-2 is overexpressed ~500,000 per cell, many ErbB-2 heterodimers are formed and cell signaling is enhanced, resulting in increased sensitivity to growth factors and an increase in the number of malignant cells. This explains why ErbB-2 overexpression is an indicator of poor breast tumor prognosis and can serve as a prognostic factor to predict response to treatment.

ErbB-2 is a promising and validated target in breast cancer and has been found at primary tumor sites and at metastatic sites. Herceptin induces rapid removal of ErbB-2 from the cell surface and reduces its ability to heterodimerize and stimulate tumor growth. The mechanisms of action of Herceptin observed in experimental models in vitro and in vivo include inhibition of proteolysis of the extracellular domain of ErbB-2, disruption of downstream signaling pathways such as phosphatidylinositol 3-kinase (P13K) and mitogen-activated protein kinase (MARK) cascades, arrest cell cycle in the G1 phase, inhibition of DNA repair, suppression of angiogenesis and induction of antibody-dependent cellular cytotoxicity (ADCC). However, the majority of patients with metastatic breast cancer who were initially susceptible to the action of Herceptin, one year after the start of treatment, progression of this disease was observed.

Another cellular target receptor is the insulin-like growth factor-1 type 1 receptor (IGF-1R), where IGF-1R is a tyrosine kinase receptor that plays a critical role in maintaining cell signaling and in cell proliferation. The regulation of the IGF system in cancer cells is often disrupted as a result of the formation of autocrine loops that play a role in the overexpression of IGF-I or -II and/or IGF-1R. In addition, epidemiological studies suggest an association between elevated IGF levels and the development of most human cancers such as breast cancer, colon cancer, lung cancer, and prostate cancer. The expression of IGF and their cognate receptors correlates with the stage of disease development, with a decrease in cell survival, with the development of metastases, and tumor dedifferentiation.

In addition to IGF-1R, the epidermal growth factor receptor (EGFR) is involved in the oncogenesis of various cancers. Efficient tumor inhibition has been achieved experimentally and clinically using a range of strategies that provide suppression of any receptor activity. Due to the large number of growth signaling pathways in tumor cells, inhibition of one receptor function (eg, EGFR) can be effectively offset by activation of pathways mediated by other growth factor receptors (eg, IGF-1R). For example, recent studies have shown that malignant glioma cell lines expressing equivalent EGFR have significantly different susceptibility to EGFR inhibition, depending on their ability to activate IGF-1R and downstream signaling pathways. Other studies have also demonstrated that overexpression and/or activation of IGF-1R in tumor cells may contribute to their resistance to chemotherapeutic agents, to radiation therapy or therapy using antibodies such as Herceptin. Therefore, inhibition of signal transduction under the action of IGF-1R leads to an increase in the susceptibility of tumor cells to Herceptin.

EGFR is a tyrosine kinase receptor that is expressed in many normal tissues as well as in tumor lesions of most organs. Overexpression of EGFR or expression of mutant forms of EGFR is observed in many tumors, and in particular in epithelial tumors, and is associated with a poor clinical prognosis. Inhibition of signal transmission through this receptor leads to the induction of an antitumor effect. Following FDA approval in February 2004 for the use of the Cetuximab antibody, also known as Erbitux (a mouse/human chimeric antibody), the main target for this antibodies approved for use as a drug for the treatment of metastases of rectal and colon cancer became EGFR. Erbitux was also approved by the FDA in March 2006 for the treatment of squamous cell carcinoma of the head and neck (SCCHN). More recently, a fully human anti-EGFR antibody has been approved for the treatment of metastatic colorectal cancer. None of these drugs is a stand-alone treatment for rectal and colon cancer, i.e. they have only been approved as adjuvant medicines to be used in conjunction with existing colorectal cancer regimens. For rectal and colon cancer, erbitux is used in combination with the drug irinotecan, and vectibix is administered after disease progression during or after chemotherapy using fluoropyrimidine, oxaliplatin, and irinotecan. Erbitux has been approved as the only treatment for recurrent or metastasized

- 4 040917 SCCHN, but only if previous platinum-based chemotherapy has failed. More and more clinical trials are currently underway in which these drugs are being used to target non-small cell lung carcinoma. The sequence of an Erbitux or an anti-EGFR antibody is well known to those skilled in the art (see, for example, Goldstein et al., Clin. Cancer Res., 1:1311, 1995; US Pat. No. 6,217,866, which are incorporated herein by reference).

A problem with the use of a catalytic antibody for the selective activation of a prodrug in cancer therapy is the systemic delivery of such an antibody to a tumor. The present invention describes a method that is based on the adaptation of target-binding peptides or modular recognition domains (MRDs) and their subsequent attachment to full-length antibodies effective for delivery to tumor cells or soluble molecules while maintaining the ability of the catalytic antibody to activate prodrugs. . Since the attachment of an MRD to an antibody is carried out in such a way that it does not significantly reduce the level of binding to the traditional antigen-binding site, the specificity of such an antibody after attachment of the MRD to it remains unchanged.

As shown in FIG. 2, the MRDs indicated by triangles, circles and squares can be attached to either end of the heavy or light chains of a typical antibody. The first scheme represents a simple peptibody with a peptide attached to the C-terminus of Fc. This method makes it possible to obtain bi-, tri-, tetra- and penta-specific antibodies. The presentation of a single MRD at each N- and C-terminus of IgG provides an octahedral display of the MRD. As an alternative to constructing bi- and multi-functional antibodies through a combination of antibody variable domains, the use of high-affinity peptides selected from phage display libraries or derived from natural ligands provides a highly versatile and modular method for constructing multi-functional antibodies that retains the advantages of traditional antibodies in terms of their binding and half-life. MRDs can also increase the binding ability of non-catalytic antibodies, which makes it possible to develop an effective method for increasing the functional binding of antibodies, used, in particular, for therapeutic purposes.

Description of the essence of the invention

The present invention relates to a full length antibody containing a modular recognition domain (MRD). The present invention also contemplates variants and derivatives of such MRD-containing antibodies.

In one aspect of the invention, the antibody and the MRD are operably linked to each other via a peptide linker. In one aspect of the invention, the peptide linker is 2-20 peptides long, or 4-10 or about 4-15 peptides long. In one aspect of the invention, the peptide linker contains a GGGS sequence (SEQ ID NO: 1), an SSGGGGSGGGGGGSS sequence (SEQ ID NO: 2), or an SSGGGGSGGGGGGSSRSS sequence (SEQ ID NO: 19). The present invention includes other linkers containing the GGGS core sequence shown in SEQ ID NO: 1, where the specified peptide linker has about 4-20 amino acids.

In accordance with another embodiment of the invention, the MRD is operably linked to the C-terminus of the heavy chain of an antibody. In another aspect of the invention, the MRD is operably linked to the N-terminus of an antibody heavy chain. In yet another aspect of the invention, the MRD is operably linked to the C-terminus of the antibody light chain. In another aspect of the invention, the MRD is operably linked to the N-terminus of an antibody light chain. In another aspect of the invention, two or more MRDs are operably linked to either end of an antibody. In another aspect of the invention, two or more MRDs are operably linked to two or more ends of an antibody.

The target for MRD is the epidermal growth factor receptor (EGFR).

In one embodiment, the MRD is a vascular homing peptide. In one aspect of the invention, the peptide sequence of the vascular homing peptide is ACDCRGDCFCG (SEQ ID NO: 15).

Said antibody or MRD binds to EGFR. In one aspect of the invention, the EGFR-targeting MRD peptide sequence is VDNKFNKELEKAYNEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 16). In one aspect of the invention, the peptide sequence of an EGFR-targeting MRD is VDNKFNKEMWIAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 17).

The present invention also relates to an isolated polynucleotide containing the nucleotide sequence of an antibody. In one aspect of the invention, the vector contains a polynucleotide. In yet another aspect of the invention, said polynucleotide is operably linked to a regulatory sequence that controls the expression of a sequence within the polynucleotide.

Brief description of graphic materials

In FIG. 1 is a schematic representation of various tetravalent IgG-like BsAb constructs.

In FIG. 2A shows an exemplary C-terminal fusion peptibody with Fc.

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In FIG. 2B shows an antibody having a C-terminal fusion of MRD with an antibody light chain.

In FIG. 2C shows an antibody having an N-terminal fusion of MRD with an antibody light chain.

In FIG. 2D shows an antibody having unique MRD peptides attached to each end of the antibody.

In FIG. 3 shows the results of an ELISA in which integrin and Ang-2 were linked via an anti-integrin antibody fused to an MRD targeting ang-2.

In FIG. 4 shows the results of an ELISA in which integrin and Ang-2 were linked via an anti-integrin antibody fused to an MRD targeting ang-2.

In FIG. 5 shows the results of an ELISA in which an anti-ErbB-2 antibody was attached to an MRD targeting Ang-2.

In FIG. 6 shows the results of an ELISA in which an MRD targeting Ang-2 was fused to an antibody that binds to the hepatocyte growth factor receptor.

In FIG. 7 shows the results of an ELISA in which an integrin-targeting MRD was fused to an ErbB-2 binding antibody.

In FIG. 8 shows the results of an ELISA in which an integrin-targeting MRD was fused to an antibody that binds to the hepatocyte growth factor receptor.

In FIG. 9 shows the results of an ELISA in which an MRD targeting the insulin-like growth factor-I receptor was fused to an ErbB-2 binding antibody.

In FIG. 10 shows the results of an ELISA in which an MRD targeting VEGF was fused to an ErbB-2 binding antibody.

In FIG. 11 shows the results of an ELISA in which an integrin-targeting MRD was attached to a catalytic antibody.

In FIG. 12 shows the results of an ELISA in which an MRD targeting Ang-2 was attached to a catalytic antibody.

In FIG. 13 shows the results of an ELISA in which an MRD targeting integrin and Ang-2 was fused to an ErbB-2 binding antibody.

In FIG. 14 shows the results of an ELISA in which an integrin-targeting MRD was fused to an ErbB-2 binding antibody.

In FIG. 15 shows the results of an ELISA in which an MRD targeting an integrin, Ang-2, or insulin-like growth factor-I receptor was attached to an antibody that binds to ErbB-2 or the hepatocyte growth factor receptor via a short linker peptide.

In FIG. 16 shows the results of an ELISA in which an MRD targeting an integrin, Ang-2, or insulin-like growth factor-I receptor was linked to an ErbB-2- or hepatocyte growth factor receptor-binding antibody via a long linker peptide.

Detailed description of the invention

As used herein, the term antibody refers to intact immunoglobulin molecules and includes polyclonal and monoclonal antibodies, chimeric antibodies, single chain antibodies, and humanized antibodies. An intact antibody includes at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (herein referred to as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH ₂ and CH ₃ . Each light chain is composed of a light chain variable region (hereinafter referred to as V _L ) and a light chain constant region. The light chain constant region consists of a single C _L domain. The V _H and V _L regions can also be subdivided into hypervariable regions called complementarity-determining regions (CDRs) and interspersed with more conserved regions called framework regions (FRs). Each of VH and VL consists of three CDRs and four FRs arranged in the amino to carboxy direction 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 the antigen.

As used herein, the term bispecific antibody means an immunoglobulin molecule containing immunoglobulins consisting of two variable domains, where said two variable domains can be constructed from any two monoclonal antibodies.

The antigen-binding site is a structural part of the antibody molecule, consisting of variable and hypervariable regions of the heavy and light chains, which specifically bind to the antigen (enter into an immune reaction with the antigen). The term immune response, as used in its various forms, means the specific binding of a molecule containing an antigenic determinant and a molecule containing an antigen-binding site, such as the whole or part of an antibody molecule.

The term peptibody means a peptide or polypeptide comprising a partial-length intact antibody.

The term natural, when used in conjunction with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to substances that occur naturally and have not been modified by humans.

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The term monoclonal antibody refers to a population of antibody molecules that contain an antigen-binding site of an antibody of only one species, capable of immunoreacting with a particular epitope. Thus, a monoclonal antibody generally has the same binding affinity for any epitope with which it interacts. Therefore, a monoclonal antibody may comprise an antibody molecule having a plurality of antigen-binding sites, each of which is immunospecific for a different epitope, and such an antibody is, for example, a bispecific monoclonal antibody.

A modular recognition domain (MRD) or target-binding peptide is a molecule, such as a protein, glycoprotein, or the like, that can specifically bind (ie, will not non-specifically bind) to a target molecule. The amino acid sequence of the MRD site may have some degree of variability and still retain sufficient ability to bind to the target molecule. In addition, changes in sequence may lead to changes in the binding specificity and binding constant of the preselected target molecule to the binding site.

The term cell surface receptor means molecules and their complexes capable of receiving a signal and transmitting such a signal across the plasma membrane of a cell. An example of a cell surface receptor according to the invention is an activated integrin receptor, for example an activated αΎβ3 integrin receptor on a metastatic cell.

A target binding site or targeting site is any known or not yet described amino acid sequence having the ability to selectively bind to a preselected agent. Representative standard targeting sites are derived from RGD-dependent integrin ligands, namely fibronectin, fibrinogen, vitronectin, von Willebrand factor, and the like, from cellular receptors such as VEGF, ErbB-2, vascular homing peptide, or angiogenic cytokines, from protein hormone receptors such as insulin-like growth factor-I receptor, epidermal growth factor receptor, and the like. and tumor antigens.

The term protein is defined as a biological polymer containing units derived from amino acids linked by peptide bonds, where said protein may consist of two or more chains.

A hybrid polypeptide is a polypeptide consisting of at least two polypeptides and optionally a linker sequence that operably links the two polypeptides to form one contiguous polypeptide. The two linked polypeptides that form the hybrid polypeptide usually come from two independent sources, and therefore the hybrid polypeptide contains two linked polypeptides that are not normally linked in nature.

The term linker means a peptide located between the antibody and the MRD. Linkers can be from about 2 to 20 amino acids, and typically they are from 4 to 15 amino acids.

The term target cell means any cell of an individual (eg, human or animal) that can be targeted by an MRD-containing antibody of the invention. The target cell can be a cell that expresses or overexpresses a target binding site, such as an activated integrin receptor.

As used herein, the terms patient, subject, animal, or mammal are used interchangeably and refer to mammals such as humans and non-human primates, as well as experimental animals such as rabbits, rats and mice, and other animals. Animals are all vertebrates such as mammals and non-mammals such as sheep, dogs, cows, chickens, amphibians and reptiles.

The terms treatment or therapy include administering an antibody comprising an MRD of the invention for the purpose of preventing or slowing the development of symptoms, complications, or biochemical signs of a disease, ameliorating symptoms, or stopping or inhibiting the further development of said disease, condition, or disorder. Treatment may be prophylactic (to prevent or slow the progression of a disease, or to prevent its clinical or subclinical symptoms), or it may be therapeutic and is used to suppress or relieve symptoms after the onset of symptoms of the disease. Treatment may be with the antibody-MRD composition alone, or with a combination with another therapeutic agent.

As used herein, the terms pharmaceutically acceptable or physiologically tolerant and their grammatical variants, if they refer to compositions, carriers, diluents and reagents, are synonymous and mean that these agents can be administered internally or externally to a person and do not produce undesirable physiological effects in him, such like nausea, dizziness, indigestion, etc.

The term to modulate means to adjust or adjust the range, frequency, degree or activity. In another related aspect of the invention, such modulation may be positive (for example, an increase in frequency, degree, or activity) or negative (for example, a decrease in frequency,

- 7 040917 degree or activity).

As used herein, the terms cancer, tumor, or malignant disease are synonymous and refer to any disease characterized by unregulated abnormal cell proliferation, the ability of affected cells to spread locally or travel through the circulatory and lymphatic systems to other parts of the body (i.e., the ability to metastasize) , and also mean any characteristic structural and/or molecular features. The terms cancer, tumor or malignant cells mean cells that have specific structural properties, are incapable of differentiation, and have the ability to invade and metastasize. Examples of cancers are cancers of the breast, lung, brain, bone, liver, kidney, colon, head and neck, ovary, hematopoietic system (eg, leukemia), and prostate.

The terms humanized antibody or chimeric antibody include antibodies in which CDR sequences derived from the germline of a mammal of another species, such as a mouse, have been fused to human framework sequences.

The present invention describes a method based on obtaining target-binding peptides or modular recognition domains (MRDs) as hybrids with catalytic or non-catalytic antibodies that provide their effective targeting to tumor cells or soluble molecules, but retain the ability of an intact catalytic antibody to prodrug activation. MRDs can also enhance the binding capacity of non-catalytic antibodies, which provides an effective tool for enhancing the functional binding of antibodies, in particular for therapeutic purposes.

In one of its aspects, the present invention relates to the production of a full length antibody containing a modular recognition domain (MRD). The interaction of a protein ligand with its receptor binding site often occurs over a relatively wide area of contact. However, binding occurs mainly in only a few key residues of this contact area. Thus, peptide molecules (typically 2-60 amino acids in length) can bind to the receptor protein of this large ligand protein. This provides that the MRD according to the invention contain a peptide sequence that binds to the target sites of interest and contains from about 2 to 60 amino acids).

The role of integrins such as αγβ3 and αγβ5 as tumor-associated markers has been well studied. A recent study of 25 stable human cell lines derived from advanced ovarian cancer cells demonstrated that all lines were positive for αγβ5 expression and many of them were positive for αγβ3 expression. Studies have also shown that αγβ3 and ανβ5 are highly expressed on malignant tumor tissues of the human cervix. Integrins have also been shown to have therapeutic effects in an animal model of Kaposi's sarcoma, melanoma, and breast cancer.

A number of ανβ3 and αγβ5 integrin antagonists are in clinical trials. Such antagonists are RGD cyclic peptides and synthetic low molecular weight RGD mimetics. Two antibody-based integrin antagonists are currently in clinical trials for use in cancer treatment. The first-line antagonist is Vitaxin, i.e. a humanized form of the mouse anti-human αγβ3 antibody LM609. A phase I dose escalation trial for cancer patients has shown that the agent is safe for human administration. Another antibody used in clinical trials is CNT095, ie. a fully human mAb that recognizes αν integrins. A phase I study conducted by administering CNT095 to patients with various solid tumors showed that the agent was well tolerated by patients. Phase I studies have also confirmed that cilengitide, a αγβ3 and αγβ5 peptide antagonist, is safe. In addition, there have been many studies on drug delivery and imaging, and these studies have been carried out using ligands for these receptors. These preclinical and clinical observations have demonstrated the important role of targeting αγβ3 and αγβ5, and antibody studies of this strategy have clearly shown that targeting these integrins is safe.

An example of an integrin-binding MRD is an RGD tripeptide-containing binding site, and representative general methods for preparing such an MRD are described herein. Ligands having an RGD motif as the minimum recognition domain are well known in the art and a non-exhaustive list includes fibronectin (α3β1, α5β1, αγβΐ, αΠββ3, αγβ3 and α3β 1), fibrinogen (αMβ2 and allbe 1), von Willebrand factor (αΙΦβ3 and αγβ3) and vitronectin (αΙΦβ3, αγβ3 and αγβ5), where the corresponding target integrin is indicated in brackets.

Representative RGD-containing targeting MRDs used in the present invention have the amino acid residue sequences shown below:

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YCRGDCT (SEQ ID NO: 3)

PCRGDCL (SEQ ID NO: 4)

TCRGDCY (SEQ ID NO: 5)

LCRGDCF (SEQ ID NO: 6)

The present invention also contemplates an MRD that mimics an RGD-independent binding site on an integrin receptor that has the target-binding specificity of a high affinity ligand and that recognizes a selected integrin.

Angiogenesis is a major factor in many physiological and pathological processes. Ang-2 has been shown to act as a pro-angiogenic molecule. Administration of Ang-2 selective inhibitors is sufficient to suppress tumor angiogenesis and corneal angiogenesis. Therefore, inhibition of Ang-2 alone or in combination with inhibition of other angiogenic factors such as VEGF may represent an effective anti-angiogenic strategy for the treatment of patients with solid tumors.

It is believed that the MRDs used in the present invention include MRDs that bind to angiogenic receptors, angiogenic factors and/or Ang-2. Examples of MRD sequences targeting angiogenic cytokines are listed below.

MGAQTNFMPMDDLEQRLYEQFILQQGLE (SEQ ID NO:7)

MGAQTNFMPMDNDELLLYEQFILQQGLE (SEQ ID NO:8)

MGAQTNFMPMDATETRLYEQFILQQGLE (SEQ ID NO:9)

AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (SEQ ID NO: 10) (2xCon4)

MGAQTNEMPMDNDELLNYEQFILQQGLE (SEQ ID NO: 11)

PXDNDXLLNY (SEQ ID NO: 12), where X is one of the 20 natural amino acids

MGAQTNFMPMDNDELLLYEQFILQQGLEGGSGSTASSGSGSSLGAQTNFMPMDNDELL

LY (SEQ ID NO: 20)

AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (SEQ ID NO: 10)

AQQEECEFAPWTCEHM ConFA (SEQ ID NO: 21) nEFAPWTn core sequence (SEQ ID NO: 22), where n is about 0 to about 50 amino acid residues

AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFAPWTCEHMLE (SEQ ID NO: 23) 2xConFA

AOQEECELAPWTCEHM (SEQ ID NO: 24) ConLA

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

AQQEECELAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCEHMLE (SEQ ID NO: 26) 2xConLA

AQQEECEFSPWTCEHM ConFS (SEQ ID NO: 27)

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

AOOEECEFSPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEHMLE

2xConFS (SEQ ID NO: 29)

AQQEECELEPWTCEHM CONLE (SEQ ID NO: 30)

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

AQQEECELEPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELEPWTCEHMLE 2xConLE (SEQ ID NO: 32)

Obviously, such peptides may be present in dimers, trimers, or other multimers that are homologous or heterologous in nature. So, for example, one peptide can dimerize like Con-containing sequences, such as 2xConFA, to form a homologous dimer, or Con-peptides can be mixed, for example, ConFA can be combined with ConLA to create a ConFA-LA heterodimer with the sequence

- 9 040917

AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCEHMLE (SEQ ID NO: 33)

Another heterodimer is ConFA, which when combined with ConFS forms ConFA-FS with the sequence

AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEHMLE (SEQ ID NO: 34)

One of skill in the art would appreciate that any other combinations described herein could be made to create the functional Ang-2 binding MRDs described herein.

In one of its aspects, the present invention includes a peptide having the sequence NFYQCIX ₁ X ₂ LX ₃ X ₄ X ₅ PAEKSRGQWQECRTGG (SEQ ID NO: 58), where X ₁ represents E or D;

X ₂ is any amino acid;

X ₃ is any amino acid;

X ₄ is any amino acid; And

X5 is any amino acid.

The present invention also includes peptides having a core sequence selected from

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

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

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

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

XnAQQEECEX1X ₂ PWTCEHMXn, where n is from about 0 to 50 amino acid residues, and X, X1 and X ₂ are any amino acid (SEQ ID NO: 57).

There are reports of phage display selection studies and structural studies of VEGF-neutralizing peptides in complex with VEGF. These studies showed that peptide v114 (VEPNCDIHVMWEWECFERL) (SEQ ID NO: 13) is VEGF-specific, binds to VEGF with an affinity of 0.2 μM, and neutralizes VEGF-induced proliferation of human umbilical vein endothelial cells (HUVEC). Because VEGF is a homodimer, the peptide occupies two identical sites at either end of the VEGF homodimer. The present invention contemplates an antibody containing an MRD targeting VEGF. A description of anti-VEGF antibodies can be found, for example, in Cancer Research 57, 4593-4599, Oct. 1997, J. Biol. Chem., 281:10 6625, 2006, which is incorporated herein by reference.

In the present invention, MRDs specific for the insulin-like growth factor-I receptor can be used. One example of an MRD sequence targeting the insulin-like growth factor-I receptor is

SFYSCLESLVNGPAEKSRGQWDGCRKK (SEQ ID NO: 14)

Additional MRDs targeting IGF-1R are the following sequences:

NFYQCIEMLASHPAEKSRGQWQECRTGG (SEQ ID NO:35)

NFYQCIEQLALRPAEKSRGQWQECRTGG (SEQ ID NO:36)

NFYQCIDLLMAYPAEKSRGQWQECRTGG (SEQ ID NO:37)

NFYQCIERLVTGPAEKSRGQWQECRTGG (SEQ ID NO:38)

NFYQCIEYLAMKPAEKSRGQWQECRTGG (SEQ ID NO:39)

NFYQCIEALQSRPAEKSRGQWQECRTGG (SEQ ID NO:40)

NFYQCIEALSRSPAEKSRGQWQECRTGG (SEQ ID NO:41)

NFYQCIEHLSGSPAEKSRGQWQECRTG (SEQ ID NO:42)

NFYQCIESLAGGPAEKSRGQWQECRTG (SEQ ID NO:43)

NFYQCIEALVGVPAEKSRGQWQECRTG (SEQ ID NO:44)

NFYQCIEMLSLPPAEKSRGQWQECRTG (SEQ ID NO:45)

NFYQCIEVFWGRPAEKSRGQWQECRTG (SEQ ID NO:46)

NFYQCIEQLSSGPAEKSRGQWQECRTG (SEQ ID NO:47)

NFYQCIELLSARPAEKSRGQWAECRAG (SEQ ID NO:48)

NFYQCIEALARTPAEKSRGQWVECRAP (SEQ ID NO:49)

In a number of studies, the efficacy of vascular homing peptide binding to other proteins such as IL-12 or drugs has been characterized in terms of the possibility

- 10 040917 their direct delivery to animals. The present invention contemplates the use of the vascular homing peptides themselves as an MRD. One example of an MRD sequence that is a vascular homing peptide is ACDCRGDCFCG (SEQ ID NO: 15).

The present invention contemplates a variety of other target binding sites including epidermal growth factor receptor (EGFR), CD20, tumor antigens, ErbB-2, ErbB-3, ErbB-4, insulin-like growth factor receptor-I, neural growth factor (NGR ), a hepatocyte growth factor receptor, and an epithelial cell-adhesion tumor-associated surface antigen molecule (Ep-CAM). MRDs can be directed to these target binding sites.

Examples of MRD sequences that bind to EGFR are listed below.

VDNKFNKELEKAYNEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 16)

VDNKFNKEMWIAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 17)

An example of an MRD sequence that binds to ErbB-2 is shown below.

VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 18)

The MRD sequence can be defined in several ways. MRD sequences can be derived from natural ligands or known sequences that bind to a specific target binding site that can be used. In addition, phage display technology has been developed as a highly efficient method for identifying peptides that bind to target receptors. In phage display peptide libraries, randomized peptide sequences can be displayed by attaching them to filamentous phage coat proteins. Methods for detecting binding sites on polypeptides using phage display vectors have already been described, in particular in WO 94/18221. Such methods essentially involve the use of a system of expression vectors on the surface of a filamentous phage (phagemid) to clone and express polypeptides that bind to a preselected target site of interest.

The methods of the invention for producing MRDs include the use of phage display vectors for their specific application to screen a very large population of expressed detectable proteins and to localize one or more specific clones encoding the desired reactive target binding site. Once the MRD sequence has been identified, said peptides can be obtained by any of the methods described in the literature.

Variants and derivatives of the MRD are within the scope of the present invention. Included within the scope of the present invention are variants with insertions, deletions, and substitutions, as well as variants comprising the MRDs provided herein with additional amino acids at the N- and/or C-terminus, including about 0-50, 0-40, 0-30, 0- 20 amino acids, etc. It should be noted that a particular MRD according to the invention may contain variants of one, two or all three types. Insertions and substitutions may contain naturally occurring amino acids, rare amino acids, or both.

The present invention contemplates the use of catalytic and non-catalytic antibodies. The 38C2 antibody is an antibody-secreting hybridoma, and such an antibody was previously described in WO 97/21803.38C2 and contains an antigen-binding site that catalyzes an aldol condensation reaction between an aliphatic donor and an aldehyde acceptor. In syngeneic mice with a neuroblastoma model, systemic administration of the etoposide prodrug and intratumoral injection of Ab 38C2 resulted in inhibition of tumor growth.

Other antibodies of interest according to the invention are A33 binding antibodies. The human A33 antigen is a transmembrane glycoprotein belonging to the Ig superfamily. The function of the human A33 antigen in healthy and malignant colon tissue is not yet clear, but some properties of the A33 antigen suggest that it may be a promising target for colon cancer immunotherapy. These properties are (i) a highly restricted expression profile of the A33 antigen;

(ii) expression of large amounts of the A33 antigen on colon cancer cells;

(iii) no secreted or shed A33 antigen;

(iv) after binding of the anti-A33 antibody to the A33 antigen, said anti-A33 antibody is internalized and sequestered in vesicles; and (v) as shown by preliminary clinical studies, the A33 antigen, expressed in colon cancer, is a target for anti-A33 antibodies.

Attaching an MRD directed against A33 to a catalytic or non-catalytic antibody would result in an increase in the therapeutic efficacy of the antibodies targeted to A33.

The present invention also contemplates the production of mono-, bi-, tri-, tetra- and penta-specific antibodies. It is also contemplated that the antibodies used in the present invention may be obtained by any method known to those skilled in the art.

In hybrid antibody-MRD molecules prepared in accordance with the present invention, the MRD may be attached to the antibody at the N-terminus or C-terminus via a peptide. The MRD can be attached to an antibody at the C-terminus of an antibody heavy chain, at the N-terminus of an antibody heavy chain, at the C-terminus of an antibody light chain, or at the N-terminus of an antibody light chain. The MRD may be attached to the antibody directly or via an optional peptide linker, which may be 2 to 20 peptides in length. The peptide linker may comprise a short linker peptide with the sequence GGGS (SEQ ID NO: 1), a medium length 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 relates to two or more MRD associated with any end of the antibody. It is also contemplated that two or more MRDs can be attached to two or more ends of an antibody, either directly or via a linker peptide. Multiple MRDs may target the same target binding site or two or more different target binding sites. Additional peptide sequences can be added to increase the in vivo stability of the MRD.

Hybrid antibody-MRD molecules can be encoded by a polynucleotide containing a nucleotide sequence. The vector may contain a polynucleotide sequence. The polynucleotide sequence may also be fused to a regulatory sequence that regulates the expression of the polynucleotide in the host cell. The host cell or progeny thereof may contain a polynucleotide encoding an antibody-MRD fusion molecule.

The present invention contemplates therapeutic compositions that can be used to implement the therapies described herein. The therapeutic compositions of the invention comprise a physiologically acceptable carrier together with at least one species of MRD-containing antibody as described herein, which is dissolved or dispersed in the carrier as the active ingredient. In a preferred embodiment of the invention, the therapeutic composition is not immunogenic when it is administered to a human for therapeutic purposes.

The preparation of a pharmacological composition containing active ingredients dissolved or dispersed therein is well known to those skilled in the art. Typically, such compositions are prepared as sterile injections, or as liquid aqueous or anhydrous solutions or suspensions, however, these compositions may also be prepared in solid forms suitable for solution or suspension in liquid prior to use. Such a preparation may also be emulsified. Thus, the composition containing the antibody-MRD can be prepared in the form of solutions, suspensions, tablets, capsules, sustained release preparations or powders, or in other compositional forms.

The active ingredient may be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and used in amounts suitable for their use in the therapies described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like. and their combinations. In addition, if necessary, said composition may contain small amounts of adjuvants, such as wetting or emulsifying agents, pH buffering agents, and the like, to increase the effectiveness of the active ingredient.

A therapeutic composition according to the invention may include pharmaceutically acceptable salts of the components of said composition. Pharmaceutically acceptable salts are acid addition salts (formed by the free amino groups of the polypeptide) which are formed from inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid, and the like. Salts formed by free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or iron(3) hydroxides, and from organic bases such as isopropylamine, trimethylamine, 2-ethylamine, ethanol, histidine, procaine, etc.

Physiologically acceptable carriers are well known to those skilled in the art. Representative liquid carriers are sterile aqueous solutions containing no substances other than the active ingredients and water, or containing a buffer such as sodium phosphate at physiological pH, saline, or both, such as phosphate buffered saline. In addition, aqueous vehicles may contain more than one buffer salt, as well as other salts such as sodium and potassium chlorides, dextrose, propylene glycol, polyethylene glycol, and other solutes.

Liquid compositions may also contain, in addition to or instead of water, liquid phases.

Examples of such additional liquid phases are glycerol, vegetable oils such as cottonseed oil, organic esters such as ethyl oleate, and water-in-water emulsions.

- 12 040917 oil.

The therapeutic composition contains an antibody comprising an MRD according to the invention, typically in an amount of at least 0.1% by weight of the antibody based on the weight of the entire therapeutic composition. The weight percent is the ratio of the weight of the antibody to the total weight of the composition. For example, 0.1 wt.% represents 0.1 grams of antibody-MRD per 100 grams of the total composition.

An antibody-containing therapeutic composition typically includes the antibody as an active ingredient in an amount of from about 10 micrograms (μg) per milliliter (mL) to about 100 milligrams (mg) per milliliter by volume of the entire composition, and more preferably from about 1 to 10 mg/mL ( i.e. about 0.1-1 wt.%.

In another embodiment of the invention, the therapeutic composition contains a polypeptide according to the invention, usually in an amount of at least 0.1 wt.% of the polypeptide by weight of the entire therapeutic composition. The weight percentage is the ratio of the weight of the polypeptide to the total weight of the composition. For example, 0.1 wt.% represents 0.1 g of the polypeptide per 100 g of the total composition.

Preferably, the polypeptide-containing therapeutic composition typically includes the polypeptide as an active ingredient in an amount of from about 10 μg/ml to about 100 mg/ml by volume of the entire composition, and more preferably from about 1 to 10 mg/ml (i.e., about 0 , 1-1 wt.%).

Due to the effective use of humanized or chimeric antibodies for administration to humans in vivo, the antibody-MRD molecules described herein are particularly suitable for their use in vivo as a therapeutic reagent. Said method comprises administering to a patient a therapeutically effective amount of a physiologically acceptable composition containing an antibody comprising an MRD of the invention.

Dosage intervals for administering an antibody containing an MRD of the invention are broad enough to achieve the desired effect, which results in the reduction of disease symptoms mediated by the target molecule. The dose should not be so high as to cause unwanted side effects such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like. Typically, such a dose may vary depending on the age, health, sex and severity of the disease of the patient and can be determined by the specialist. In the event of any complication, the dose administered to the patient may be adjusted by his attending physician.

A therapeutically effective amount of an MRD-containing antibody of the invention is typically that amount of antibody which, when administered as a physiologically acceptable composition, is sufficient to achieve a plasma concentration of about 0.1 to 100 μg/mL, preferably about 1 to 5 mcg/ml, and usually about 5 mcg/ml. In other words, the dose may vary from about 0.1 to 300 mg/kg, preferably from about 0.2 to 200 mg/kg, and most preferably from about 0.5 to 20 mg/kg, and one or more once a day for one day or several days.

The MRD-containing antibody of the invention may be administered parenterally by injection or by continuous infusion over a period of time. Although the target molecule present in the body is usually available by systemic administration, and therefore the most common method of treatment is intravenous administration of therapeutic compositions, however, administration to other tissues by other delivery methods can be carried out in the case where there is a possibility that the tissues being exposed treatment, contain the specified target molecule. Thus, MRD-containing antibodies according to the invention can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, into the cavity of the body, transdermally and can be delivered using peristaltic means.

Therapeutic compositions containing a human monoclonal antibody or polypeptide of the invention are typically administered intravenously, for example by unit dose injection. As used herein, the term unit dose, referring to a therapeutic composition according to the invention, means physically discrete units suitable for administration to an individual in the form of a unit dose, where each unit dose containing a predetermined amount of the active substance is calculated so that it gives the desired therapeutic effect in combinations with the required diluent, i.e. carrier or excipient.

The compositions are administered in a manner suitable for administering said drug, in a therapeutically effective amount. The amount of drug administered depends on the type of individual being treated, the ability of the individual's body to metabolize the active ingredient, and the level of therapeutic effect desired.

The exact amount of active ingredient required for administration depends on the prescription of the attending physician and is individual for each patient. However, suitable dosage ranges for systemic use as described herein depend on the route of administration. Suitable administration schedules may also vary, but typically the first dose and subsequent doses are administered one hour or several hours apart by injection or other route of administration. Alternatively, continuous intravenous infusion sufficient to maintain blood concentrations is considered.

- 13 040917 in time intervals typical for in vivo therapy.

The following examples are provided for illustrative purposes only and do not limit the scope of the invention.

Examples

Example 1 Antibody-MRD molecules targeting integrin.

New antibody-MRD hybrid molecules have been generated by attaching peptides targeting the αγβ3 integrin to the 38C2 catalytic antibody. The most effective hybrids are obtained at the N-terminus and C-terminus of the light chain and at the C-terminus of the heavy chain. Flow cytometry showed that the antibody conjugates effectively bind to human breast cancer cells expressing the αγβ3 integrin. The antibody conjugates also retained the retroaldol activity of the parent 38C2 catalytic antibody, as determined in the methodol and doxorubicin prodrug activation assay. This analysis showed that the method, which was based on the delivery of antibodies to cells and the catalytic function of these antibodies, can be effectively used in combination with selective chemotherapy.

Example 2 Antibody-MRD Molecules Targeting an Angiogenic Cytokine.

Were designed hybrid antibody-MRD molecules that target an angiogenic cytokine. The antibody used is a 38C2 antibody that has been fused to an MRD containing the 2xCon4 peptide (AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (SEQ ID NO: 10)). The MRD peptide was attached at the N- or C-terminus of the light chain and at the C-terminus of the heavy chain. Similar results were obtained for other MRD peptides targeting Ang-2. Additional MRD peptides that target Ang-2 are

LM-2X-32

MGAQTNFMPMDNDELLLYEQFILQQGLEGGSGSTASSGSGSSLGAQTNFMPMDNDELL

LY (SEQ ID NO: 20)

AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (2xCon4) (SEQ ID NO: 10)

AOQEECEFAPWTCEHM ConFA (SEQ ID NO: 21) XnEFAPWTXn core sequence, where n is about 0 to 50 amino acid residues (SEQ ID NO: 22)

AOQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSGSATHOEECEFAPWTCEHMLE

2xConFA (SEQ ID NO: 23)

AQQEECELAPWTCEHM ConLA (SEQ ID NO: 24)

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

AOQEECELAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCEHMLE

2xConLA (SEQ ID NO: 26)

AQQEECEFSPWTCEHM ConFS (SEQ ID NO: 27)

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

AQQEECEFSPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEHMLE

2xConFS (SEQ ID NO: 29)

AQQEECELEPWTCEHM CONLE (SEQ ID NO: 30)

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

AQQEECELEPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELEPWTCEHMLE

2xConLE (SEQ ID NO: 32)

Obviously, such peptides may be present in dimers, trimers, or other multimers that are homologous or heterologous in nature. So, for example, one of these peptides can dimerize like Con-containing sequences, such as 2xConFA, to form a homologous dimer, or Con-peptides can be mixed, for example, ConFA can be combined with ConLA to create a ConFA-LA heterodimer with the sequence

AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCEHMLE (SEQ ID NO: 33)

Another representative heterodimer is ConFA, which when combined with ConFS forms ConFA-FS with the sequence

- 14 040917

AQQEECEEAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEHMLE (SEQ ID NO: 34)

One of skill in the art would appreciate that any other combinations described herein could be made to create the functional Ang-2 binding MRDs described herein.

Example 3 Antibody-MRD hybrids with non-catalytic antibodies.

Humanized mouse monoclonal antibody, LM609, directed against human integrin α\'β3. has been described previously (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) .

A human non-catalytic Ab monoclonal antibody, JC7U, was fused to an anti-Ang2-MRD containing 2xCon4 (AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (SEQ ID NO: 10)), either at the N- or C-terminus of the light chain. 2xCon4 (AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (SEQ ID NO: 10)) was analyzed as an N-terminal hybrid with the antibody kappa chain (2xCon4-JC7U) and as a C-terminal hybrid (JC7U-2xCon4). Both hybrids retained the ability to bind to integrin and Ang-2. As shown in the left panel in FIG. 3, both antibody constructs (2xCon4-JC7U and JC7U-2xCon4) specifically bound to recombinant Ang-2 as indicated by ELISA assays. However, the level of binding to Ang-2 was significantly higher for JC7U-2xCon4, which has 2xCon4 (SEQ ID NO: 10) attached to the C-terminus of the light chain of the antibody. In the right panel of Fig. 3 illustrates the binding of Ang-2-JC7U and JC7UAng-2 to the ave3 integrin. The results showed that the addition of 2xCon4 (SEQ ID NO: 10) to the N- or C-terminus of the light chain did not adversely affect the binding of mAb JC7U to αγβ3 integrin. In FIG. 4 illustrates another ELISA assay performed using the same antibody-MRD fusion constructs.

Example 4 Herceptin-MRD hybrid molecules.

Another example of MRD hybrids with a non-catalytic antibody are Herceptin-MRD hybrid constructs. Herceptin-MRD hybrids are multifunctional, ie. they are small molecule av integrin antagonists, and such antibodies, chemically programmed to target integrin, are highly effective in preventing breast cancer metastasis due to their ability to suppress av-mediated cell adhesion and proliferation. MRD hybrids containing Herceptin-2xCon4 (which target ErbB-2 and Ang-2), Herceptin-V114 (which target ErbB-2 and VEGF), and Herceptin-RGD-4C-2xCon4 (which target ErbB-2, Ang-2 and integrin) are effective.

Example 5 Antibody-MRD Molecules Targeting VEGF.

Was designed MRD-containing antibody directed against VEGF. An MRD targeting v114 (SEQ ID NO: 13) was fused at the N-terminus of the kappa chain of the 38C2 antibody and Herceptin using a long linker sequence (SEQ ID NO: 2). Expression and analysis of the resulting antibody-MRD hybrid constructs indicated a high level of binding to VEGF.

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

Studies have been made to attach an MRD targeting IGF-1R (SFYSCLESLVNGPAEKSRGQWDGCRKK (SEQ ID NO: 14)) to the N-terminus of the kappa chain of the 38C2 antibody and Herceptin using a long linker sequence as a linker. Expression and analysis of the resulting antibody-MRD hybrid constructs indicated a high level of binding to IGF-1R. Additional clones showing a high level of binding to IGR-1R were also identified after several rounds of mutagenesis and screening. The preferred sequences listed below either did not have any significant binding affinity for the insulin receptor, or no such affinity at all (see Table 2).

Table 1

Rm2-2-218

Rm2-2-316

Rm2-2-319

Template for subsequent mutagenesis

GTGGAGTGCAGGGCGCCG VECRAP SEQ IDNO:

GCTGAGTGCAGGGCTGGG AECRAG SEQ IDNO:

CAGGAGTGCAGGACGGGG QECRTG SEQ IDNO:

50, 51

52, 53

54, 55

- 15 040917

table 2

SEQ Mutant Amino acid sequence Matrix ID 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 NFYQCIEHLLSGSPAEK.SRGQWQECRTG Rm2-2-319 42 Rm4-45 NFYQCIESLAGGPAEKSRGQWQECRTG Rm2-2-319 43 Rm4-46 NFYQCIEALVGVPAEKSRGQWQECRTG Rm2-2-319 44 Rm.4-49 NFYQCIEMLSLPPAEKSRGQWQECRTG Rm2-2-319 45 Rm4-410 NFYQCIEVFWGRPAEKSRGQWQECRTG 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 Antibody-MRD Molecules Binding to ErbB-2 and Targeting Anq-2.

An antibody was constructed containing an MRD targeting Ang-2 (L17) and attached to the light chain of an ErbB-2 binding antibody. The linker used was either a short linker sequence, a long linker sequence, or the 4th loop in the light chain constant region. In FIG. 5 shows the results of an ELISA assay performed using constructs containing

An N-terminal fusion of an Ang-2 targeting MRD with an anti-ErbB-2 antibody, wherein said fusion was generated using a short linker peptide (GGGS (SEQ ID NO: 1)) (L17-sL-Her);

C-terminal fusion of an Ang-2 targeting MRD with an anti-ErbB-2 antibody, wherein said fusion was generated using a short linker peptide (Her-sL-L17);

A C-terminal fusion of an Ang-2 targeting MRD with an anti-ErbB-2 antibody, wherein said fusion was generated using the 4th loop in the light chain constant region (Her-lo-L17); or

An N-terminal fusion of an Ang-2 targeting MRD with an anti-ErbB-2 antibody, wherein said fusion was generated using a long linker peptide (SSGGGGSGGGGGGSSRSS (SEQ ID NO: 19)) (L17-1L-Her).

For all constructs, the level of ErbB-2 binding varied. However, Ang-2 only binds via Her-sL-L17 and L17-1L-Her.

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

An Ang-2 (L17) targeting MRD hybrid was attached to the N-terminus or C-terminus of the light chain of an anti-Met antibody that binds to the hepatocyte growth factor receptor. A short linker sequence or a long linker sequence was used as a linker. In FIG. 6 shows the results of an ELISA assay performed using constructs containing

An N-terminal fusion of an Ang-2-targeted MRD with an anti-Met antibody, wherein said fusion was generated using a short linker peptide (GGGS (SEQ ID NO: 1)) (L17-sL-Met);

An N-terminal fusion of an Ang-2-targeting MRD with an anti-Met antibody, wherein said fusion was generated using a long linker peptide (SSGGGGSGGGGGGSSRSS (SEQ ID NO: 19)) (L17-1L-Met); And

A C-terminal fusion of an Ang-2 targeting MRD with an anti-Met antibody, wherein said fusion was generated using a long linker peptide (Met-IL-L17).

Expression and analysis of the resulting antibody-MRD hybrid constructs indicated a high level of binding to Ang-2 when using a long linker peptide. Attaching an Ang-2-targeted MRD to the C-terminus of the light chain of an antibody resulted in slightly higher binding to Ang-2 than attaching an Ang-2-targeted MRD to the N-terminus of the light chain of the antibody.

Example 9 ErbB-2 Binding Antibody-MRD Molecules Targeting Integrin.

An antibody was constructed containing an MRD targeting integrin ανβ3 (RGD4C) and attached to the light chain of a Herceptin antibody that binds to ErbB-2 (Her). The linker used was a short linker sequence, a long linker sequence, or the 4th loop in the light chain constant region. In FIG. 7 shows the results of an ELISA assay performed using constructs containing

An N-terminal fusion of an ανβ3 integrin-targeted MRD with an anti-ErbB-2 antibody, wherein said fusion was generated using a short linker peptide (GGGS (SEQ ID NO: 1)) (RGD4C-sL-Her);

- 16 040917

A C-terminal fusion of an ανβ3 integrin-targeted MRD with an anti-ErbB-2 antibody, wherein said fusion was generated using a short linker peptide (Her-sL-RGD4C);

A C-terminal fusion of an αΎβ3 integrin-targeted MRD with an anti-ErbB-2 antibody, wherein said fusion was generated using the 4th loop in the light chain constant region (Her-lo-RGD4C); or

An N-terminal fusion of an αΎβ3 integrin-targeted MRD with an anti-ErbB-2 antibody, wherein said fusion was generated using a long linker peptide (SSGGGGSGGGGGGSSRSS (SEQ ID NO: 19)) (RGD4C-1L-Her).

For all constructs, the level of ErbB-2 binding varied. However, the αΎβ3 integrin was bound only by RGD4C-1L-Her.

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

An antibody was constructed containing an MRD targeting integrin αΎβ3 (RGD4C) and attached to the light chain of an antibody that binds to the hepatocyte growth factor receptor (Met). Antibody-MRD constructs containing a long linker sequence were used. In FIG. 8 shows the results of an ELISA assay performed using constructs containing

N-terminal fusion of MRD targeting αΎβ3 integrin with an antibody against hepatocyte growth factor receptor (RGD4C-1L-Met); or

C-terminal fusion of MRD targeting integrin αΎβ3 with antibody against hepatocyte growth factor receptor (Met-1L-RGD4C).

RGD4C-1L-Met showed a high level of binding to the αΎβ3 integrin.

Example 11 ErbB-2 Binding Antibody-MRD Molecules Targeting the Insulin-Like Growth Factor-I Receptor.

Antibodies were constructed containing an MRD that targets the insulin-like growth factor-I (RP) receptor and is attached to the light chain of an ErbB-2-binding antibody (Her). Either a short linker peptide or a long linker peptide or the 4th loop of the light chain constant region was used as the linker. (Carter et al., Proc. Natl. Acad. Sci., USA, 1992 May 15, 89(10):4285-9).

PMID: 1350088 [PubMed is the catalog name for MEDLINE]; US patent No. 5677171; deposit ATCC 10463, all of these documents are introduced into the present description by reference. In FIG. 9 shows the results of an ELISA assay performed using constructs containing

An N-terminal fusion of an MRD targeting the insulin-like growth factor-I receptor with an anti-ErbB-2 antibody attached via a short linker (RP-sL-Her);

C-terminal fusion of an MRD targeting the insulin-like growth factor-I receptor with an anti-ErbB-2 antibody linked via a short linker peptide (Her-sL-RP);

C-terminal fusion of an MRD targeting the insulin-like growth factor-I receptor with an anti-ErbB2 antibody fused via the 4th loop in the light chain constant region (Her-lo-RP);

An N-terminal fusion of an MRD targeting the insulin-like growth factor-I receptor with an anti-ErbB-2 antibody linked via a long linker peptide (RP-1L-Her); or

C-terminal fusion of an MRD targeting the insulin-like growth factor-I receptor with an anti-ErbB-2 antibody linked via a long linker peptide (Her-1L-RP).

For all constructs, the level of ErbB-2 binding varied. The insulin-like growth factor-I receptor was bound by RP-1L-Her.

Example 12 ErbB-2 Binding Antibody-MRD Molecules Targeting VEGF.

A hybrid MRD targeting VEGF (V114) was fused to the N-terminus of the light chain of an ErbB-2 binding antibody (Her). A medium length linker peptide (SSGGGGSGGGGGGSS (SEQ ID NO: 2)) was used as a linker. In FIG. 10 shows the results of an ELISA assay using a construct containing an N-terminal fusion of an MRD targeting VEGF with an ErbB-2 binding antibody fused via a medium length linker peptide (V114-mL-Her). Expression and analysis of the resulting antibody-MRD fusion construct indicated a high level of binding to VEGF and ErbB-2.

Example 13 Integrin Targeting Antibody-MRD Molecules.

An MRD fusion targeting ave3 integrin (RGD) was studied with the N-terminus of the light chain of the 38C2 antibody fused using a medium length linker peptide as a linker. In FIG. 11 demonstrates that expression and analysis of the resulting antibody-MRD fusion construct indicated a high level of binding to the αΎβ3 integrin.

Example 14 Antibody-MRD Molecules Targeting Anq-2.

An MRD fusion targeting Ang-2 (L17) was studied with the light chain C-terminus of the 38C2 antibody attached using a short linker sequence as a linker. In FIG. 12 demonstrates that expression and analysis of the resulting antibody-MRD fusion construct indicated a high level of binding to Ang-2.

Example 15 ErbB-2 Binding Antibody-MRD Molecules Targeting Integrin and Anq-2.

The MRD targeting the αΎβ3 integrin (RGD4C) was attached to the N-terminus of the light chain of the anti-ErbB-2 antibody (Her) via a medium length linker, and the MRD targeted to Ang-2 (L17) was attached via a short linker to C -terminus of the same anti-ErbB-2 antibody (RGD4CmL-Her-sL-L17). In FIG. 13 shows that the resulting antibody-MRD fusion construct bound to integrin, Ang-2 and ErbB-2.

Example 16 ErbB-2 Binding Antibody-MRD Molecules Targeting Integrin.

An antibody containing an MRD targeting the αΎβ3 integrin (RGD4C) and attached to the N-terminus of the heavy chain of an ErbB-2 binding antibody (Her) was constructed using a medium length linker as a linker (RGD4C-mL-her-heavy chain). In FIG. 14 shows the results of an ELISA assay performed using this construct. In this construct, integrin and ErbB-2 were linked.

Example 17 Antibody-MRD molecules that bind to ErbB-2 or to the hepatocyte growth factor receptor and target an integrin, Anq-2 or insulin-like growth factor-I receptor, which were prepared using a short linker peptide.

Antibody-MRD molecules were constructed containing antibodies binding to ErbB-2 or the hepatocyte growth factor receptor and MRD regions targeting the αΎβ3 integrin, Ang-2 or insulin-like growth factor-I receptor, where these regions were attached via a short linker peptide to the antibody light chain. In FIG. 15 shows the results of an ELISA assay performed using constructs containing

N-terminal hybrid of MRD targeting Ang-2 with anti-ErbB-2 antibody (L17-sL-Her);

N-terminal fusion of integrin-targeted MRD with anti-ErbB-2 antibody (RGD4C-SL-Her);

N-terminal hybrid of MRD targeting insulin-like growth factor-I receptor with ErbB-2 binding antibody (RP-sL-Her);

C-terminal fusion of MRD targeting Ang-2 with an antibody that binds to the hepatocyte growth factor receptor (L17-sL-Met);

C-terminal hybrid of MRD targeting Ang-2 with ErbB-2 binding antibody (Her-sL-L17);

C-terminal fusion of an integrin-targeted MRD with an ErbB-2 binding antibody (Her-sL-RGD4C); or

C-terminal fusion of an MRD targeting the insulin-like growth factor-I receptor with an ErbB-2 binding antibody (Her-sL-RP).

In constructs, the ErbB-2 antibody-MRD had varying degrees of binding, with the exception of the construct containing the antibody that binds to the hepatocyte growth factor receptor. The antigen was bound only by the Her-sL-L17 construct.

Example 18 Antibody-MRD molecules that bind to ErbB-2 or the hepatocyte growth factor receptor and target an integrin, Anq-2 or insulin-like growth factor-I receptor, which were prepared using a long linker peptide.

Antibody-MRD molecules were constructed containing antibodies binding to ErbB-2 or the hepatocyte growth factor receptor and MRD regions targeting the αΎβ3 integrin, Ang-2 or insulin-like growth factor-I receptor, where these regions were attached via a long linker peptide to the antibody light chain. In FIG. 16 shows the results of an ELISA assay performed using constructs containing

N-terminal hybrid of MRD targeting Ang-2 with anti-ErbB-2 antibody (L17-1L-Her);

N-terminal fusion of an integrin-targeted MRD with an anti-ErbB-2 antibody (RGD4C-1LHer);

N-terminal hybrid of MRD targeting insulin-like growth factor-I receptor with ErbB-2 binding antibody (RP-1L-Her);

C-terminal fusion of MRD targeting Ang-2 with an antibody that binds to the hepatocyte growth factor receptor (L17-1L-Met);

C-terminal fusion of an integrin-targeted MRD with an antibody that binds to the hepatocyte growth factor receptor (RGD4C-1L-Met);

C-terminal fusion of MRD targeting Ang-2 with an antibody that binds to the insulin-like growth factor-I receptor (Her-1L-RP);

C-terminal fusion of MRD targeting Ang-2 with an antibody that binds to the hepatocyte growth factor receptor (Met-1L-L17); or

C-terminal fusion of an integrin-targeting MRD with an antibody that binds to the hepatocyte growth factor receptor (Met-1L-RGD4C).

As shown in FIG. 16, antibody-MRD hybrids efficiently bound antigen and ErbB-2. 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.

Although the present invention has been described with reference to the above examples, it should

- 18 040917 note that modifications and changes can be made to it that do not go beyond the essence and scope of the invention. Accordingly, the present invention is only limited by the appended claims.

Claims (13)

1. Fusion protein for targeted delivery of an antibody to a tumor, containing a full-length antibody and at least one modular recognition domain (MRD), where the specified MRD contains the sequence VDNKFNKELEKAYNEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 16) or the sequence VDNKFNKEMWIAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAQEAK and IDSEK (17) its target is the epidermal growth factor receptor (EGFR) and wherein said antibody binds to EGFR. 2. The fusion protein of claim 1, wherein said antibody and said MRD are operably linked via a linker peptide. 3. A fusion protein according to claim 2, wherein said linker peptide consists of 2-20 amino acids. 4. A fusion protein according to claim 2, wherein said linker peptide consists of 4-15 amino acids. 5. The fusion protein of claim 2, wherein said linker peptide contains the sequence GGGS (SEQ ID NO: 1). 6. The fusion protein of claim 2, wherein said linker peptide contains the sequence SSGGGGSGGGGGGSS (SEQ ID NO: 2). 7. The fusion protein of claim 2, wherein said linker peptide contains the sequence SSGGGGSGGGGGGSSRSS (SEQ ID NO: 19). 8. The fusion protein of claim 1, wherein two or more MRDs are operably linked to either end of the antibody. 9. The fusion protein of claim 1, wherein two or more MRDs are operably linked to two or more ends of the antibody. 10. The fusion protein of claim 1 wherein the antibody is Erbitux. 11. A polynucleotide containing a nucleotide sequence encoding a fusion protein according to claim 1. 12. An expression vector containing a polynucleotide according to claim 11. 13. An expression vector containing a polynucleotide according to claim 11, wherein said nucleotide sequence of the polynucleotide is operably linked to a regulatory sequence that regulates the expression of said polynucleotide in the host cell.