HK1081448B - Bispecific anti-erb-b antibodies and their use in tumor therapy - Google Patents

Description

Bispecific anti-ERB-B antibodies and their use in tumor therapy

Technical Field

The present invention relates to novel bispecific antibodies and their use in the treatment of tumors. The novel antibodies are capable of binding to ErbB receptors, particularly ErbB1 receptors, that are overexpressed on a variety of cancer tissues. Because the different specificities of the antigen binding sites are directed to different epitopes within the binding domains of the same or different ErbB receptors, these antibodies are more effective at inhibiting and down-regulating ErbB receptors and the corresponding signaling cascade. The invention also relates to pharmaceutical compositions comprising the bispecific antibody or fragment thereof and other pharmaceutically effective agents such as monospecific antibodies, immunoconjugates and/or cytotoxic agents.

Background

Over a period of more than 20 years, biomolecules, such as monoclonal antibodies (mabs) or other proteins/polypeptides, as well as small chemical compounds directed against a variety of receptors and other antigens on the surface of tumor cells are known to be useful in tumor therapy. With respect to antibody approaches, most of these mabs are chimerized or humanized to improve tolerance of the human immune system. Mabs or the above-mentioned chemical entities bind specifically to their target structure on tumor cells and in most cases also on normal tissues and can lead to different effects depending on their epitope specificity and/or the functional properties of the specific antigen. Mabs directed against orphan receptors (orphan receptors) or other non-functional cell surface molecules, as well as mabs directed against structures outside the ligand-binding site of functionally active receptors (e.g. growth factor receptors with kinase activity), are expected to induce immune effector functions (antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC)) primarily directed against target cells. Furthermore, depending on the nature of the antigen and Mab, binding of the antibody may result in cross-linking of the receptor. The resulting internalization of the receptor-antibody complex can result in a long-term down-regulation of receptor density on the cell surface.

Mabs that bind to an epitope within or in direct proximity to the ligand-binding site compete with the natural ligand for binding to its receptor, thereby reducing or completely inhibiting ligand binding and displacing ligand that has bound to its receptor. Blockade of this receptor inhibits ligand-dependent receptor activation and downstream signaling. For example, blockade of ErbB receptors, such as Epidermal Growth Factor Receptor (EGFR), by monoclonal antibodies results in a variety of cellular effects, including inhibition of DNA synthesis and proliferation, induction of cell cycle arrest and apoptosis, and anti-metastatic and anti-angiogenic effects.

In the eighties of the twentieth century, ErbB receptors were the typical receptor tyrosine kinases involved in cancer. Tyrosine kinases are a class of enzymes that catalyze the transfer of the terminal phosphate of adenosine triphosphate to tyrosine residues in protein substrates. Tyrosine kinases are believed to play a key role in the signaling of many cellular functions through substrate phosphorylation. Although the exact mechanism of signal transduction is still unclear, tyrosine kinases have been shown to be important contributing factors in cell proliferation, carcinogenesis and cell differentiation.

Tyrosine kinases can be classified into receptor type and non-receptor type. Both receptor-type and non-receptor-type tyrosine kinases are involved in cellular signaling pathways that can lead to a number of pathological conditions including cancer, psoriasis, and hyperimmune responses. Many tyrosine kinases are involved in cell growth and angiogenesis.

Non-receptor tyrosine kinases also consist of a number of subfamilies including Src, Frk, Btk, Csk, Abl, Zap70, Fes/Fps, Fak, Jak, Ack and LIMK. Each of these subfamilies may also be subdivided into distinct receptors. For example, the Src subfamily is one of the largest subfamilies, which includes Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr, and Yrk. Enzymes in the Src subfamily are involved in tumor formation. For a more detailed discussion of non-receptor type tyrosine kinases, see bolen oncogene, 8: 2025-2031(1993).

Receptor-type tyrosine kinases have an extracellular, transmembrane and intracellular portion, whereas non-receptor-type tyrosine kinases are entirely intracellular. Receptor-linked tyrosine kinases are transmembrane proteins that comprise an extracellular ligand binding domain, a transmembrane sequence, and a cytoplasmic tyrosine kinase domain. Receptor-type tyrosine kinases consist of a large number of transmembrane receptors with diverse biological activities.

Receptor-type tyrosine kinases of different subfamilies have been identified. The tyrosine kinases involved include Fibroblast Growth Factor (FGF) receptors, Epidermal Growth Factor (EGF) receptors and platelet-derived growth factor (PDGF) receptors in the ErbB major family class. Also contemplated are Nerve Growth Factor (NGF) receptors, brain-derived neurotrophic factor (BDNF) receptors, and neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) receptors.

A receptor type tyrosine kinase subfamily, designated HER or ErbB subfamily, consisting of EGFR (ErbB1), HER2(ErbB2 or p185neu), HER3(ErbB3) and HER4(ErbB4 or tyr 02). Ligands for this subfamily of receptors include Epidermal Growth Factor (EGF), TGF-a, amphiregulin (ampheregulin), HB-EGF, beta-cell regulator (betacellulin), heregulin and neuregulin. The PDGF subfamily includes the FLK family consisting of kinase insertion domain receptors (KDRs).

EGFR, encoded by the ErbB1 gene, is causally related to human malignancies. In particular, enhanced expression of EGFR has been observed in breast, bladder, lung, head, neck and stomach cancers as well as glioblastomas. The enhancement of EGFR receptor expression is often accompanied by an increase in the production of the EGFR ligand, transforming growth factor alpha (TGF-. alpha.), in the same tumor cells, leading to receptor activation via the autocrine stimulatory pathway (Baselga and Mendelsohn, Pharmac. Ther.64: 127-.

The EGF receptor is a transmembrane glycoprotein with a molecular weight of 170,000 and is found on many epithelial cell types. It can be activated by at least three ligands, EGF, TGF-alpha (transforming growth factor alpha) and amphiregulin. Both Epidermal Growth Factor (EGF) and transforming growth factor alpha (TGF-. alpha.) have been shown to bind to EGF receptors and cause cell proliferation and tumor growth. These growth factors do not bind to HER2 (Ulrich and SCH1 singer, 1990, Cell 61, 203). Unlike several families of growth factors that induce receptor dimerization by virtue of their self-dimerization properties (e.g., PDGF), monomeric growth factors such as EGF have 2 receptor binding sites and therefore can cross-link two adjacent EGF receptors (Lemmon et al, 1997, EMBO j.16, 281). Receptor dimerization is essential for activating the intrinsic catalytic activity of growth factor receptors and for autophosphorylation of growth factor receptors at tyrosine residues. The latter can serve as docking sites for a variety of adapter proteins or enzymes that can initiate many signaling cascades simultaneously. In higher eukaryotic cells, a simple linear pathway evolves into a fully interactive multilayer network, where the combined expression and activation of components allows for the development of context-specific biological responses throughout development. The ErbB network integrates not only its own input signals, but also heterologous signals, including hormones, lymphokines, neurotransmitters and stress inducers.

It should be noted that receptor Protein Tyrosine Kinases (PTKs) are capable of both homodimerization and heterodimerization, where the mitogenic and transforming effects (no or weak initiation of signal transduction) of homodimeric receptor combinations are lower than those of the corresponding heterodimeric combinations. Heterodimers comprising ErbB2 are the most potent complexes (see reviews: Yarden and Sliwkowski, 2001, Naturereviews, Molecular cell Biology, volume 2, 127-.

Antibodies against the EGF receptor have been shown to inhibit tumor cell proliferation when blocking EGF and TGF- α binding to the receptor. In view of these findings, a number of mouse and rat monoclonal anti-EGF receptor antibodies have been developed and tested for their ability to inhibit tumor cell growth in vivo and in vitro (Modjtahedi and Dean, 1994, J. Oncology 4, 277). Both humanized 425(hMAb425, U.S. Pat. No. 5,558,864; EP 0531472) and chimeric 225(cMAb 225, U.S. Pat. No. 3, 4,943,533 and EP 0359282) monoclonal antibodies were directed against the EGF receptor and were shown to be effective in clinical trials. The C225 antibody (cathximab) has been shown to inhibit tumor cell growth mediated by EGF in vitro, as well as human tumor formation in nude mice. Moreover, and most importantly, the antibody has also been shown to eradicate human tumors in vivo in a xenograft mouse model in synergy with certain chemotherapeutic agents, namely doxorubicin (doxorubicin), paclitaxel, and cisplatin. Ye et al (1999, Oncogene 18, 731) reported that human ovarian cancer cells could be successfully treated by the combined use of chimeric MAb225 and humanized MAb4D5 directed against the HER2 receptor.

The second member of the ErbB family, HER2(ErbB2 or p185neu), was originally identified as the transforming gene product of neuroblastoma from chemically treated rats. The activated form of the neu proto-oncogene is caused by a point mutation (valine to glutamic acid) in the transmembrane region of the encoded protein. Amplification of the neu human homolog can be observed in breast and ovarian cancers, which is associated with poor prognosis (Slamon et al, Science, 235: 177-182 (1987); Slamon et al, Science, 244: 707-712 (1989); U.S. Pat. No. 4,968,603). ErbB2(HER2) has a molecular weight of approximately 185,000, although to date the specific ligand for HER2 has not been clearly established, ErbB2(HER2) has a rather high homology with the EGF receptor (HER 1).

It was further found that antibody 4D5 directed against the HER2 receptor sensitizes breast tumor cell lines overexpressing ErbB2 to the cytotoxic effects of TNF α (US 5,677,171). Humanized recombinants of murine anti-ErbB 2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2 or HERCEPTIN)US 5,821,337) has clinical activity in patients with ErbB 2-overexpressing metastatic breast cancer who have been previously treated for substantial anticancer therapy (Baselga et al, j.clin.oncol.14: 737-744(1996)). HERCEPTINWas officially approved in 1988 for marketing to treat patients with metastatic breast cancer whose tumors overexpress the ErbB2 protein.

In addition to anti-ErbB antibodies, a variety of small chemical molecules are known to be potent inhibitors of ErbB receptor molecules that block the binding site for natural ligands (see detailed description), or block tyrosine residues of the binding site for receptor kinases, thereby preventing phosphorylation and further cascade signaling.

One representative of the high efficacy shown in clinical trials is Iressa^TM(ZD-1839), which can be used for NSCLC indications (non-small cell lung cancer).

Although there are some drugs and methods in development and marketing that hold promise for the treatment of tumors, there is still a need for additional agents and pharmaceutical compositions and combinations with improved properties and increased efficacy.

Summary of The Invention

The present invention is based on the following findings of the present inventors: certain receptor tyrosine kinases, such as ErbB receptor molecules, that are overexpressed on the surface of diseased cells, e.g., tumor cells, have specific epitope sites within the natural ligand binding domain to which different antibodies or, in general, different specificities can bind simultaneously with no or only negligible mutual hindrance. It is clear that the epitopes to which these antibodies or specificities bind are relatively small in three-dimensional configuration compared to the full size of the binding domain of the receptor molecule. Which may induce an increased down-regulation of the activity of the signaling pathway, preferably an increased blocking of the ErbB receptor, and thus an increased blocking of the entire signaling cascade.

The present invention describes for the first time the novel concept of tumor therapy, i.e. the administration of a bispecific antibody or a functionally effective fragment thereof to an individual, blocking or inhibiting an ErbB receptor, preferably the EGF receptor (EGFR), by binding of a first specific antigen-binding site of said bispecific antibody to a first epitope and binding of a second specific antigen-binding site to a second, different epitope of the same or a different receptor.

It was found that the bispecific antibody can bind to different epitopes in the natural ligand binding domain of the same receptor molecule (e.g. EGFR or Her-2) or different receptor molecules (e.g. EGFR and Her-2) simultaneously via its two different antigen binding sites without significant mutual hindrance between the different antigen binding sites of the antibody, thereby allowing a higher antibody density at the receptor and leading (by reducing the binding capacity to natural (agonistic) ligands such as EGF or TGF a) to a much stronger inhibition of the signaling cascade of the corresponding receptor molecule in monomeric or dimeric units. This should lead to a stronger inhibition of tumor growth and/or to an increased apoptosis of solid tumors or tumor metastases. Preferred antibodies are especially the anti-EGFR and anti-Her 2 antibodies described above and below, and fragments thereof, preferably bispecific F (ab') 2 fragments, because of their small size. In a preferred embodiment of the present invention, a bispecific antibody (fragment) (BAb) is described consisting of a first antigen-binding site derived from a humanized, chimeric or murine MAb425 and a second antigen-binding site derived from a humanized, chimeric or murine MAb225<425，225>，F(ab′<425>，ab′<225>)). In other embodiments of the invention, bispecific antibodies (fragments) (babs) are described that consist of a first antigen-binding site derived from a humanized, chimeric or murine MAb425 and a second antigen-binding site derived from a humanized, chimeric or murine MAb4D5<425，4D5>，F(ab′<425>，ab′<4D5>)). In other embodiments of the invention, bispecific antibodies (fragments) (babs) are described that consist of a first antigen-binding site derived from a humanized, chimeric or murine MAb225 and a second antigen-binding site derived from a humanized, chimeric or murine MAb4D5<225，4D5>，F(ab′<425>，ab′<4D5>)). In the above embodiments, humanized MAb425, chimeric MAb225 (CETUXIMAB)) And humanized 4D5(HERCEPT 1N)) Preferably as the source antibody. In principle the invention also encompasses hybrid antibodies (heteroantibodies) or fragments thereof. Synthetically prepared hybrid antibodies may even consist of three different antigen binding site portions derived from three different anti-EGFR antibodies or fragments thereof (e.g.<425，225，4D5>)。

It has been found that bispecific antibodies according to the invention are capable of enhancing the cross-linking/dimerization of different or identical ErbB receptors, enhancing the blocking/inhibition of ErbB receptors, enhancing the induction of modulation of ErbB receptor-specific signaling pathways, as compared to corresponding monospecific antibodies. Interestingly, this cross-linking can be further enhanced by a mixture comprising said bispecific antibody (fragment) and a monospecific anti-ErbB antibody (fragment), wherein said monospecific antibody (fragment) preferably has the same antigen binding site as said first or second antigen binding site of the bispecific antibody (fragment). In other words: for example, (i) MAb425 or MAb225 or MAb4D5 and BAb <425, 225 >; or (ii) MAb425 or MAb225 or MAb4D5 with BAb <425, 4D5 >; or (iii) a mixture of MAb425 or MAb225 or MAb4D5 with BAb <4D5, 225> causes increased inhibition and down-regulation of ErbB receptors as compared to administration of the MAb or BAb as a single agent at the same concentration.

Although the above findings have been made only for the ErbB receptors as target receptor molecules, it should be noted that the scientific principles disclosed by the present inventors and described above and below are also applicable to biological receptors other than ErbB.

Optionally, the compositions of the present invention further comprise therapeutically active compounds that can support and enhance the efficacy of the above molecules.

The agent may be a cytotoxic agent, preferably an antagonistic molecule such as a tyrosine kinase antagonist, other ErbB antagonist, hormone receptor antagonist, protein kinase antagonist or an anti-angiogenic agent. The molecules useful in the present invention will be described in more detail below.

The therapeutically active agents of the present invention may also be provided in the form of a pharmaceutical kit comprising a package containing one or more of the antagonists in separate containers or in a single container. Methods of treatment using this combination may optionally include radiation therapy. In general, such drug administration may be concomitant with radiation therapy, wherein radiation therapy may be performed substantially simultaneously with, or before, or after, drug administration. The administration of the different agents in the combination therapy according to the invention may also be carried out substantially simultaneously or sequentially. Tumors that have receptors on their cell surface that are involved in tumor angiogenesis can be successfully treated with the combination therapies of the present invention.

There are several alternative routes known for tumor development and growth. If one route is blocked, they can often switch to another by expressing and using other receptor and signaling pathways. Thus, the combination of the drugs of the invention has several advantages since it can block several of these possible tumor development strategies. The combinations according to the invention are useful for the treatment and prevention of tumors, tumor-like and neoplastic diseases and tumor metastases that develop and grow by activating the relevant hormone receptors present on the surface of tumor cells.

The different agents of the combination of the invention are preferably used in combination at low doses, i.e. at doses lower than those used in conventional clinical conditions. The advantages of reduced dosages when administering the compounds, compositions, medicaments and treatments of the present invention to an individual include a reduced incidence of high dose-related side effects. For example, by reducing the dosage of the agents as described above and below, the frequency and severity of nausea and vomiting are reduced compared to the effects observed at higher doses. It is expected that reducing the incidence of side effects will improve the quality of life of cancer patients. The benefits of reducing the incidence of side effects also include improved patient compliance, reduced number of hospitalizations for side effects, and reduced use of analgesics in treating pain associated with side effects. In addition, the methods and combinations of the present invention also maximize therapeutic efficacy at high doses.

The above combinations show surprising synergistic effects. The actual shrinkage and disintegration of the tumors using this drug combination was observed in clinical studies, while no significant drug side effects were detected.

The present invention relates generally to:

a bispecific antibody, or a functionally effective fragment thereof, comprising a first antigen binding site that binds to a first epitope of a first ErbB receptor and a second, different antigen binding site that binds to a second epitope of a second ErbB receptor.

A corresponding bispecific antibody or fragment thereof, wherein said first and/or said second epitope is located within the binding domain of a natural ligand of said receptor.

A corresponding bispecific antibody or fragment thereof which enhances the blocking and/or inhibition of an ErbB receptor and enhances the induction of a down-regulation of an ErbB receptor-specific signaling pathway compared to a corresponding monospecific antibody.

A corresponding bispecific antibody or fragment thereof which enhances the induction of cross-linking and/or dimerization of receptor molecules with the same or different specificities.

A corresponding bispecific antibody or fragment thereof, wherein a first epitope of said first ErbB receptor is different from a second epitope of a second ErbB receptor.

An accordingly specified antibody or fragment thereof according to claim 5 wherein said first ErbB receptor is different from said second ErbB receptor.

A corresponding bispecific antibody or fragment thereof, wherein said first and second ErbB receptors are identical.

A corresponding bispecific antibody or fragment thereof, wherein the first ErbB receptor is an EGF receptor (EGFR).

A corresponding bispecific antibody or fragment thereof, wherein said second ErbB receptor is ErbB-2 (Her-2).

A corresponding bispecific antibody or fragment thereof, wherein the first and second ErbB receptors are EGF receptors (EGFR).

A corresponding bispecific antibody or fragment thereof, wherein the first antigen-binding site is derived from humanized, chimeric or murine MAb 425.

A corresponding bispecific antibody or fragment thereof, wherein the first antigen-binding site is derived from a humanized, chimeric or murine MAb 225.

A corresponding bispecific antibody or fragment thereof, wherein the first antigen-binding site is derived from humanized, chimeric or murine MAb425 and the second antigen-binding site is derived from humanized, chimeric or murine MAb225, each antigen-binding site binding to a different epitope in the natural ligand-binding domain of the same EGF receptor molecule.

A corresponding bispecific antibody or fragment thereof wherein the first ErbB receptor is EGF receptor (EGFR) and the second ErbB receptor is ErbB-2 (Her-2).

A corresponding bispecific antibody or fragment thereof, wherein the first antigen-binding site is derived from humanized, chimeric or murine MAb425 or 225 and the second antigen-binding site is derived from MAb4D5 (Herceptin))。

A corresponding bispecific antibody or fragment thereof, wherein the fragment is F (ab') 2.

A pharmaceutical composition comprising a bispecific antibody or functionally effective fragment thereof according to any one of the preceding claims and optionally a pharmaceutically acceptable carrier, diluent or excipient.

A corresponding pharmaceutical composition further comprising a monospecific anti-ErbB antibody or a functionally effective fragment thereof.

A corresponding pharmaceutical composition, wherein said monospecific anti-ErbB antibody or functionally effective fragment thereof is selected from MAb425, MAb225, or MAb4D5 (Herceptin))。

A corresponding pharmaceutical composition, which also comprises a cytotoxic agent.

A corresponding pharmaceutical composition, wherein the cytotoxic agent is a chemotherapeutic agent.

A corresponding pharmaceutical composition, wherein the chemotherapeutic agent is selected from: cisplatin, adriamycin, gemcitabine, docetaxel, paclitaxel and bleomycin.

A corresponding pharmaceutical composition, wherein the cytotoxic agent is an ErbB receptor inhibitor, a tyrosine kinase inhibitor, a protein kinase a inhibitor, or an anti-angiogenic agent.

A pharmaceutical kit comprising

(i) A first package comprising at least the bispecific antibody or functionally effective fragment thereof described above, and

(ii) a second package comprising at least a monospecific anti-ErbB antibody or a functionally effective fragment thereof.

A corresponding pharmaceutical kit comprising a first package comprising BAb < h425, c225> or a F (ab') 2 fragment thereof and a second package comprising humanized MAb425(h 425), chimeric MAb225(c 225) or humanized MAb4D5 or a functionally effective fragment thereof.

A corresponding pharmaceutical kit, further comprising a third package comprising a further medicament.

A corresponding pharmaceutical kit, wherein the other agent is a cytotoxic agent.

A corresponding pharmaceutical kit, wherein the cytotoxic drug is selected from: cisplatin, doxorubicin, gemcitabine, docetaxel, paclitaxel, bleomycin, ErbB receptor inhibitors, tyrosine kinase inhibitors, protein kinase a inhibitors, and anti-angiogenic agents.

Use of the bispecific antibody or pharmaceutical composition/kit described above in the manufacture of a medicament for the treatment of tumors overexpressing the ErbB receptor and tumor metastases.

A method of treating tumors overexpressing ErbB receptors and tumor metastases in an individual comprising administering to said individual a therapeutically effective amount of the bispecific antibody or functionally effective fragment thereof or pharmaceutical composition/kit as described above and in the claims.

A method of enhancing the down-regulation of ErbB receptor-specific signaling pathways in tumors that overexpress ErbB receptors by administering to an individual a therapeutically effective amount of the bispecific antibody or functionally effective fragment thereof or pharmaceutical composition/kit described above.

A corresponding method, further comprising administering to the patient an effective amount of a cytotoxic agent.

A corresponding method, wherein the cytotoxic agent is a chemotherapeutic agent and is selected from: cisplatin, adriamycin, gemcitabine, docetaxel, paclitaxel and bleomycin.

A corresponding method, wherein the cytotoxic agent is an ErbB receptor inhibitor, a tyrosine kinase inhibitor, a protein kinase a inhibitor, or an anti-angiogenic agent.

In a preferred embodiment of the invention, the first ErbB receptor type bound by one antigen binding site of the bispecific antibody of the invention is ErbB1 receptor (EGFR).

Thus, the invention more specifically relates to:

a bispecific antibody or fragment thereof capable of binding to a different epitope on the same or a different ErbB receptor molecule type, said antibody comprising a first antigen binding site that binds to an epitope of a first receptor type ErbB1 and a second, different antigen binding site that binds to a different epitope of a second ErbB receptor molecule type.

A bispecific antibody wherein the second ErbB receptor molecule type is ErbB1 (EGFR).

A bispecific antibody wherein the second ErbB receptor molecule type is ErbB2 (Her-2).

A bispecific antibody wherein at least one of said epitopes is located within the receptor binding domain.

A bispecific antibody wherein the receptor binding domain is a native ligand binding domain of the receptor.

A bispecific antibody wherein the first or second antigen binding site binds to an epitope within the natural ligand binding domain of said ErbB receptor molecule type.

A bispecific antibody wherein the first and second antigen binding sites bind to an epitope within the natural ligand binding domain of said ErbB receptor molecule type.

Bispecific antibodies wherein the antigen binding site binds to a different epitope located on the same ErbB receptor molecule type.

Bispecific antibodies wherein the antigen binding site binds to a different epitope located on a different ErbB receptor molecule type.

A bispecific antibody wherein the first and second antigen-binding sites each bind to a different epitope within the natural ligand-binding domain of said ErbB receptor, thereby blocking and/or inhibiting the receptor, thereby enhancing the blocking and/or inhibition of the ErbB receptor and enhancing the induction of down-regulation of an ErbB receptor-specific signaling pathway as compared to a corresponding monospecific antibody.

Bispecific antibodies wherein the induction of cross-linking and/or dimerization of different ErbB receptor molecules with the same or different specificities is enhanced as compared to the binding of the bispecific antibody to an epitope on the same ErbB receptor molecule type.

A bispecific antibody, wherein the first antigen-binding site is derived from humanized, chimeric or murine MAb 425.

An accordingly bispecific antibody according to any one of claims 1-11, wherein the first antigen-binding site is derived from a humanized, chimeric or murine MAb 225.

A bispecific antibody designated "BAb < h425, c225 >", wherein the first antigen-binding site is derived from a humanized, chimeric or murine MAb425 and the second antigen-binding site is derived from a humanized, chimeric or murine MAb225, each antigen-binding site binding to a different epitope on an ErbB1 receptor (EGFR) molecule.

Bispecific antibodies wherein the different epitopes are located within the binding domain of a natural ligand.

A bispecific antibody wherein the second antigen-binding site binds to an ErbB2 receptor molecule (Her-2) or a VEGF receptor molecule.

The bispecific antibody of claim 16, wherein the second antigen-binding site is derived from MAb4D5 (Herceptin))。

A bispecific antibody fragment derived from a bispecific antibody according to any one of the preceding and claims, wherein the fragment is F (ab') 2.

A pharmaceutical composition comprising one or more bispecific antibodies or fragments thereof as described above and in the claims, and optionally a pharmaceutically acceptable carrier, diluent or excipient.

A pharmaceutical composition further comprising a monospecific anti-ErbB antibody or a functionally effective fragment thereof.

Pharmaceutical composition, wherein the monospecific antibody isThe ErbB antibody or functionally effective fragment thereof is selected from the group consisting of MAb425, MAb225, and MAb4D5 (Herceptin))。

A pharmaceutical composition further comprising a cytotoxic agent.

A pharmaceutical composition, wherein the cytotoxic agent is a chemotherapeutic agent.

A pharmaceutical composition, wherein the chemotherapeutic agent is selected from: cisplatin, adriamycin, gemcitabine, docetaxel, paclitaxel and bleomycin.

A pharmaceutical composition, wherein the cytotoxic agent is an ErbB receptor inhibitor, a VEGF receptor inhibitor, a tyrosine kinase inhibitor, a protein kinase a inhibitor, an anti-angiogenic agent, an anti-hormonal agent, or a cytokine.

An immunoconjugate comprising the bispecific antibody described above fused at the C-terminus to a biologically effective peptide, polypeptide or protein, either directly or via a linker molecule, wherein preferably the protein or polypeptide is a cytokine.

Pharmaceutical kit comprising

(i) A first package comprising at least a bispecific antibody or immunoconjugate as described above and in claim, and

(ii) a second package comprising at least a monospecific anti-ErbB antibody or a functionally effective fragment thereof.

A pharmaceutical kit comprising a first package comprising the bispecific antibody "BAb < h425, c225 >" or F (ab') 2 fragment thereof or an immunoconjugate thereof and a second package comprising the humanized MAb425(h 425), chimeric MAb225(c 225) or humanized MAb4D5 or a functionally effective antibody fragment thereof or an immunoconjugate thereof.

A pharmaceutical kit further comprising a third package comprising a cytotoxic drug.

A pharmaceutical kit, wherein the cytotoxic drug is selected from: cisplatin, doxorubicin, gemcitabine, docetaxel, paclitaxel, bleomycin, ErbB receptor inhibitors, VEGF receptor inhibitors, tyrosine kinase inhibitors, protein kinase a inhibitors, anti-hormonal agents, and anti-angiogenic agents.

Use of a bispecific antibody or pharmaceutical composition/kit as described above for the preparation of a medicament for the treatment of tumors overexpressing the ErbB receptor and tumor metastases and related diseases.

Detailed Description

The present invention is based on the observation that two or more mabs with specificity for different immunogenic structures can bind their epitopes simultaneously and unimpeded or with only insignificant hindrance, which epitopes may be located within the same receptor, even within the same receptor domain, e.g. the ligand binding domain. Thus, a bispecific antibody with specificity for different epitopes of the same receptor can bind to its two specific epitopes thereby forming a bivalent receptor-antibody complex, which is mostly not visible on a single receptor when using monospecific mabs.

Alternatively, the antigen binding site of the bispecific antibody may interact with other similar receptors in the vicinity, thereby creating a complex between these receptors. In addition, bispecific antibodies directed against antigenic structures on different receptors of the same or different receptor families can also be used to construct complexes between these receptors.

The use of one or more bispecific antibodies or a combination of monospecific and bispecific antibodies directed against the same or different receptors can greatly improve the therapeutic effect compared to the therapeutic effect with only one monospecific antibody:

each antigen binding site of a bispecific antibody binds independently to its specific epitope on a target receptor (e.g., EGFR).

The two antigen binding sites of a bispecific antibody can interact with their specific epitopes, which epitopes can be located on the same receptor and possibly within the same receptor domain or on different receptors.

Bispecific antibodies are capable of binding independently to two different epitopes on the same receptor. The avidity of a bispecific antibody compared to a monospecific antibody is in most cases limited by monovalent binding to the receptor, which increases the overall avidity of the bispecific antibody for a single receptor.

Due to the higher affinity for a single receptor, a lower concentration of bispecific antibody would be required to effectively block the receptor compared to a monospecific MAb.

Because bispecific antibodies are more effective in receptor blockade than monospecific mabs, they can induce more significant inhibition of receptor activation and downstream signaling.

Similarly, one or more bispecific antibodies or a mixture of monospecific and bispecific antibodies with specificity for different epitopes within or adjacent to the ligand binding domain can increase the efficacy of receptor blockade.

Because receptor blockade by the combination of two or more monospecific and/or bispecific antibodies directed against the same receptor domain is more effective than receptor blockade by only a single monospecific or bispecific antibody, a more effective inhibition of ligand-binding can be obtained, resulting in more effective receptor inactivation.

This more efficient receptor inactivation results in a more efficient inhibition of downstream receptor signaling and thus in an enhanced effect on ligand-dependent cellular function.

Due to this more efficient receptor blocking, the dose (or concentration) of each monospecific and/or bispecific antibody used can be reduced without loss of efficacy. This may be of great interest when using therapeutic antibodies that still show dose-limiting toxicity or side effects below the optimal therapeutic dose.

Bispecific antibodies that bind to different receptors on the same cell, as well as monospecific antibodies, will first form dimeric receptor-antibody complexes. However, due to their different antigen specificities, bispecific antibodies can form receptor-antibody complexes that are not limited to dimers of the same receptor. Thus, receptor aggregates formed by bispecific antibodies may contain a large, theoretically unlimited number of receptors.

These large receptor-antibody complexes improve receptor internalization and thus may be more effective for removing receptors from the cell surface and subsequently down-regulating receptor-dependent cellular function.

The formation of strong receptor-antibody complexes can be further enhanced by combining two or more monospecific and/or bispecific antibodies, whereby receptor internalization and down-regulation of receptor-dependent cellular function can be further enhanced.

Bispecific antibodies directed against the same or different receptors and combinations of two or more monospecific and/or bispecific antibodies may be used to treat tumors with the appropriate receptor. EGFR positive tumors are a typical example, but the application of the therapeutic principles described in this invention is not limited to this indication. Thus, a wide variety of tumors bearing other receptors, receptor families, or other antigenic structures can be treated using the same principles.

Therapy with bispecific antibodies or combination therapy with two or more monospecific and/or bispecific antibodies directed against different antigens on the same or different receptors, it being possible for the combination therapy to be carried out in combination with chemotherapeutic agents and/or radiation.

Therapy with bispecific antibodies or combination therapy with two or more monospecific and/or bispecific antibodies directed against different antigens on the same or different receptors, can also be used in combination with other therapeutic principles including, but not limited to, hormone antagonists or agonists, angiogenesis inhibitor therapy and other therapies.

The therapeutic principles of using bispecific antibodies or a combination of monospecific and bispecific antibodies with specificity for different antigenic structures on the same or different receptors are described herein as examples of treatment of EGFR-positive tumors. However, the principle is not limited to EGFR, which may be adapted for use with any other target antigen.

Unless otherwise defined, the terms and words used herein have the meanings and definitions set forth below. Moreover, these definitions and meanings describe the invention in more detail, including preferred embodiments.

"receptor" or "receptor molecule" refers to a soluble or membrane-bound/associated protein or glycoprotein that contains one or more domains that can bind to a ligand to form a receptor-ligand complex. By binding to a ligand, which may be an agonist or antagonist, the receptor may be activated or inactivated, thereby initiating or blocking a signaling pathway.

The term "receptor molecule type" or "ErbB (ErbB1) receptor molecule type" refers to a specific receptor type such as ErbB1, ErbB2, etc., rather than a specific single molecule of that receptor type. In other words: a bispecific antibody of the invention can bind a first epitope of an individual ErbB1 receptor molecule via its first antigen binding site, while a second antigen binding site of the antibody binds a second, different epitope of the same individual ErbB1 receptor molecule. It is also possible that a second antigen binding site of the antibody binds to a second, different epitope of another individual receptor molecule of the same type (ErbB 1). In addition, it is possible that a second antigen binding site of the antibody binds to a second, different epitope on an individual receptor molecule of a different ErbB receptor molecule type (e.g., ErbB 2).

"ligand" or "receptor ligand" refers to a natural or synthetic compound that can bind to a receptor molecule to form a receptor-ligand complex. The term ligand includes agonists, antagonists and compounds having partial agonist/antagonist activity.

"agonist" or "receptor agonist" refers to a natural or synthetic compound that binds to a receptor to form a receptor-agonist complex, which is activated to initiate signaling pathways and further biological processes.

"antagonist" or "receptor antagonist" refers to a natural or synthetic compound that has a biological effect opposite to that of an agonist. Antagonists bind to the receptor and block the action of receptor agonists by competing with the agonist for the receptor. An antagonist is defined by its ability to block the action of an agonist. The receptor antagonist may also be an antibody or an immunotherapeutically active fragment thereof. Preferred antagonists of the present invention are listed and discussed below.

By "ErbB receptor" is meant a receptor protein tyrosine kinase belonging to the ErbB receptor family (as described above), including the EGFR (ErbB1), ErbB2, ErbB3 and ErbB4 receptors, as well as other members of this family to be identified in the future. ErbB receptors generally contain an extracellular domain that binds to an ErbB ligand; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxy-terminal signal domain containing several tyrosine residues that can be phosphorylated. The ErbB receptor may be a "native sequence" ErbB receptor or an "amino acid sequence variant" thereof. Preferred ErbB receptors are the native sequence human ErbB receptors. ErbB1 refers to a gene that encodes the EGFR protein product. Most preferred is the EGF receptor (EGFR, HER 1). The expression of "ErbB 1" and "HER 1" and "EGFR" are used interchangeably herein and both refer to the human HER1 protein. "ErbB 2" and "HER 2" are used interchangeably herein and both refer to human HER2 protein. The ErbB1 receptor (EGFR) is preferred in the present invention.

An "ErbB ligand" is a polypeptide that binds to and/or activates an ErbB receptor. ErbB ligands that bind to EGFR include, for example, EGF, TGF- α, amphiregulin, betacellulin (betacellulin), HB-EGF and epiregulin (epiregulin), preferably EGF and TGF- α.

In the context of the present invention, an "ErbB receptor binding domain" is a local region (binding sequence/loop/pocket) of an ErbB receptor that binds to a natural ligand or an antagonistic or agonistic drug. The region may comprise not only one specific binding site or epitope, but also two or more epitopes or binding sites. One specific binding epitope within this domain binds to a class of antagonistic or agonistic drugs or ligands. However, it is believed that binding of different agents to different epitopes within or adjacent to a binding domain of the same receptor type will generally result in a unique and unique signaling pathway, through inhibition or activation, that is specific to the antibody. Furthermore, it is to be noted that the phrase "within the binding domain" as used in the present specification and claims also includes the position of the actual binding domain next to the corresponding natural ligand.

An "ErbB binding epitope or binding site" refers to the conformation and/or configuration of amino acids within or immediately adjacent to the binding domain of an ErbB receptor that binds to a ligand or receptor antagonist/agonist.

The "same ErbB/ErbB1 receptor molecule" does not necessarily mean the same receptor molecule, but also includes other receptor molecules of the same type. Preferably, the same acceptor molecule is meant.

The term "ErbB receptor antagonist/inhibitor" refers to a biologically effective molecule that binds to and blocks or inhibits an ErbB receptor. Thus, by blocking the receptor, the antagonist prevents ErbB ligand (agonist) binding and activation of the agonist/ligand receptor complex. ErbB antagonists may be directed against HER 1(ErbB1, EGFR), HER2(ErbB2) and ErbB3 and ErbB 4.

Preferred antagonists of the invention are directed against the EGF receptor (EGFR, HER 1). The ErbB receptor antagonist may be an antibody or an immunotherapeutically active fragment of an antibody or a non-immunobiological molecule such as a peptide, polypeptide protein. Chemical molecules are also included, but preferred antagonists of the invention are anti-EGFR antibodies and anti-HER 2 antibodies.

Preferred antibodies of the invention are anti-HER 1 and anti-HER 2 antibodies, more preferably anti-HER 1 antibody. Preferred anti-HER 1 antibodies are MAb425, preferably humanized MAb425(hMAb425, U.S. Pat. No. 5,558,864; EP 0531472) and chimeric MAb225 (CETUXIMAB)). Most preferred is monoclonal antibody h425, which has shown high efficacy and reduced adverse and side effects in monotherapy. The most preferred anti-Her 2 antibody is HERCEPTIN marketed by Genentech/Roche

The potent EGF receptor antagonists of the present invention may also be natural or synthetic chemical compounds. Some examples of such preferred molecules include organic compounds, organometallic compounds, salts of organic compounds and organometallic compounds. Examples of chemical HER2 receptor antagonists are: styryl-substituted heteroaryl compounds (US 5,656,655); bi-monocyclic and/or bicyclic aryl heteroaryl, carbocyclic and heterocarbocyclic compounds (US 5,646,153); tricyclic pyrimidine compounds (US 5,679,683); quinazoline derivatives having receptor tyrosine kinase inhibitory activity (US 5,616,582); heteroarylalkenediyl or heteroarylalkenediylaryl compounds (US 5,196,446); a compound named 6- (2, 6-dichlorophenyl) -2- (4- (2-diethyl-aminoethoxy) phenylamino) -8-methyl-8H-pyrido (2, 3) -5-pyrimidin-7-one (Panek, et al, 1997, j.pharmacol. exp. therapeutic.283, 1433) which can inhibit the EGFR, PDGFR and FGFR receptor families.

According to the present invention, the term "tyrosine kinase antagonist/inhibitor" refers to natural or synthetic agents capable of inhibiting or blocking tyrosine kinases, including receptor tyrosine kinases. Thus, the term substantially includes ErbB receptor antagonists/inhibitors as described above. In addition to the anti-ErbB receptor antibodies mentioned above and below, the more preferred tyrosine kinase antagonists under this definition are chemical compounds that have been shown to be effective in single drug therapy for breast and prostate cancer. Suitable indolocarbazole-type tyrosine kinase inhibitors may be found in, for example, U.S. patent 5,516,771; 5,654,427, respectively; 5,461,146, respectively; 5,650,407, etc. Us patent 5,475,110; 5,591,855, respectively; 5,594,009 and WO96/11933 disclose pyrrolocarbazole type tyrosine kinase inhibitors and prostate cancer. One of the most promising anticancer agents in this context is gefitinib (IRESSA)Astra Zeneca), which has been reported to have significant therapeutic effects and good tolerance in patients with non-small cell lung cancer (NSCLC) as well as advanced head and neck cancer.

The preferred dosage of the chemical tyrosine kinase inhibitor as defined above is from 1pg/kg body weight to 1g/kg body weight per day. More preferably, the dosage of the tyrosine kinase inhibitor is from 0.01mg/kg body weight to 100mg/kg body weight per day.

The biomolecule according to the invention is preferably an antibody or a fragment thereof or any variant of an antibody such as an immunoconjugate.

In this context, the term "antibody" or "immunoglobulin" is used in its broadest sense and specifically includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) comprised of at least 2 intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity. The term generally includes hybrid antibodies consisting of 2 or more antibodies or antibody fragments with different binding specificities linked together.

Complete antibodies can be divided into different "antibody (immunoglobulin) types" according to the amino acid sequence of the antibody constant region. There are 5 major classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA 2. The heavy chain constant regions corresponding to different antibody types are referred to as α, δ, ε, γ, and μ, respectively. The preferred main classes of antibodies of the present invention are IgG, more specifically IgG1 and IgG 2.

Antibodies are often glycoproteins of molecular weight about 150,000, consisting of 2 identical light (L) chains and 2 identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between heavy chains varies for different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end. The constant region of the light chain is juxtaposed with the first constant region of the heavy chain, and the variable region of the light chain is juxtaposed with the variable region of the heavy chain. It is believed that the particular amino acid residues form the interface between the light and heavy chain variable regions. The "light chains" of antibodies of vertebrate species can be divided into 2 clearly distinct classes, kappa (κ) and lambda (λ), based on the amino acid sequences of their constant regions.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., each antibody within the population is identical except for possible naturally occurring mutations, which may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Moreover, unlike polyclonal antibody preparations, polyclonal antibodies include different antibodies directed against different determinants (epitopes), whereas each monoclonal antibody is directed against only one determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they can be synthesized without contamination by other antibodies. Monoclonal antibodies can be prepared by methods including those described in Kohler and Milstein (1975, Nature 256, 495) and "monoclonal antibody technology, preparation and characterization of rodent and human hybridomas" (1985, Burdon et al, Eds, Laboratory technique Biochemistry and Molecular Biology, Volume 13, Elsevier science publishers, Amsterdam), or by well-known recombinant DNA methods (see, for example, U.S. Pat. No. 4,816,567). For example, Clackson et al, Nature, 352: 624-: 58, 1-597(1991) from phage antibody libraries.

The term "chimeric antibody" refers to an antibody having a portion of its heavy and/or light chain identical to or homologous to corresponding sequences in an antibody from a particular species or an antibody belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in an antibody from another species or an antibody belonging to another antibody class or subclass, and to fragments of such an antibody, so long as it possesses the desired biological activity (e.g., U.S. Pat. No. 4,816,567; Morrison et al, Proc. Nat. Acad. Sci. USA, 81: 6851-6855 (1984)). Methods for producing chimeric and humanized antibodies are well known to those skilled in the art. For example, methods for making chimeric antibodies include those described in Boss (Celltech) and Cabilly (genentech) (U.S. Pat. No. 4,816,397; U.S. Pat. No. 4,816,567).

A "humanized antibody" is an antibody in the form of a non-human (e.g., rodent) chimeric antibody that contains minimal amounts of sequences derived from non-human immunoglobulins. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity and performance. Sometimes, human immunoglobulin Framework Region (FR) residues are replaced by corresponding non-human residues. In addition, humanized antibodies may contain residues that are not found on both the recipient and donor antibodies. These modifications can further refine antibody performance. In general, a humanized antibody will comprise substantially all of the variable domains (at least one, and typically two variable domains), in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs have human immunoglobulin sequences. The humanized antibody may also optionally contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for the preparation of humanized antibodies are described, for example, in Winter, U.S. Pat. No. 5,225,539 and Boss (Celltech, U.S. Pat. No. 4,816,397).

An "antibody fragment" includes a portion of an intact antibody, preferably comprising the antigen binding or variable region of the antibody. Examples of antibody fragments include Fab, Fab ', F (ab') 2, Fv and Fc fragments, diabodies (diabodies), linear antibodies, single-chain antibody molecules; and multispecific antibodies composed of antibody fragments. By "intact" antibody is meant an antibody comprising a variable region that binds antigen, as well as a light chain constant region (CL) and a heavy chain constant region (CH1, CH2, and CH 3).

Preferably, the intact antibody has one or more effector functions. Papain digestion of antibodies produces 2 identical antigen-binding fragments (referred to as "Fab" fragments, each containing an antigen-binding site and a CL region and a CH1 region) and a residual "Fc" fragment (the name reflecting its ability to crystallize readily).

The "Fc" region of an antibody typically contains the hinge region of the main class of CH2, CH3, and IgG1 or IgG2 antibodies. The hinge region is a group having about 15 amino acid residues, which combines the CH1 region and the CH2-CH3 region.

Pepsin treatment produces an "F (ab') 2" fragment that contains 2 antigen binding sites and still has the ability to cross-link antigens.

"Fv" is the smallest antibody fragment that contains one complete antigen recognition and antigen binding site. This region consists of a dimer of one heavy chain variable region and one light chain variable region joined together by a tight, non-covalent linkage. In this configuration the 3 hypervariable regions (CDRs) of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Overall, the 6 hypervariable regions confer antigen-binding specificity to the antibody. However, even just one variable domain (or half of an Fv comprising only 3 antigen-specific hypervariable regions) has the ability to recognize and bind antigen, albeit with less avidity than the entire binding site.

The "Fab" fragment also includes the constant region of the light chain and the first constant region of the heavy chain (CH1) and has only one antigen binding site. The "Fab'" fragment differs from the Fab fragment by the addition of several residues at the carboxy terminus of the heavy chain CH1 region, including one or more cysteines from the antibody hinge region.

F (ab ') 2 antibody fragments were originally produced as pairs of Fab ' fragments with a cysteine hinge between the two Fab ' fragments. Other chemical couplings of antibody fragments are also known (see, e.g., Hermanson, Bioconjugate Techniques, Academic Press, 1996; U.S. Pat. No. 4,342,566).

"Single chain Fv'" or "scFv" antibody fragments comprise the V, and V, regions of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding. Single chain FV Antibodies can also be produced from, for example, Pl ü ckthun (The Pharmacology Of monoclonal Antibodies, Vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315(1994)), WO 93/16185; US 5,571,894; US 5,587,458; huston et al (1988, Proc. Natl. Acad. Sci.85, 5879) or Skerra and Plueckthun (1988, Science 240, 1038).

The term "variable" or "FR" refers to the fact that there are quite different sequences between antibodies in certain parts of the variable region that are used for the binding and specificity of each particular antibody for its particular antigen. However, this variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments called hypervariable regions of the light and heavy chain variable regions. The higher conserved portions of the variable regions are called Framework Regions (FR). The variable regions of natural heavy and light chains each comprise four FR's (FR1-FR4) that mainly adopt a β -sheet configuration, linked by three hypervariable regions that form loops connecting, and sometimes forming part of, the β -sheet structure. The hypervariable regions Of each chain are held together in close proximity by the FRs and, together with the hypervariable regions Of the other chain, contribute to the formation Of the antigen-binding site Of an antibody (see Kabat et al, Sequences Of Proteins Of immunological interest, 5th Ed. public Health Service, National Institutes Of Health, Bethesda, Md. (1991). although the constant regions are not directly involved in the binding Of an antibody to an antigen, they exhibit a variety Of effector functions, such as participation in antibody-dependent cellular cytotoxicity (ADCC).

The term "hypervariable region" or "CDR" as used herein refers to the amino acid residues of an antibody which are responsible for binding to an antigen. Hypervariable regions typically comprise the amino acid residues of a "complementarity determining region" or "CDR" (e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) of the light chain variable region, 31-35 (H1), 50-65 (H2) and 95-102 (H3) of the heavy chain variable region); and/or amino acid residues of "hypervariable loops" (e.g., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) of the light chain variable region, residues 26-32 (H1), 53-55 (H2) and 96-101 (H3) of the heavy chain variable region; Chothia and Lesk J.mol.biol.196: 901-917 (1987)). "framework region" or "FR" residues refer to those variable region residues other than the hypervariable region residues defined herein.

The term "monospecific" refers to an antibody according to the invention wherein both binding sites of the antibody have the same specificity and are thus capable of binding to the same epitope on the receptor. Preferably, according to the present invention, the pharmaceutical composition comprises a monospecific antibody.

A "bispecific antibody (BAb)" is a single, bivalent antibody (or immunotherapeutically active fragment thereof) containing 2 antigen binding sites of different specificities. According to the present invention, the BAbs are characterized by the BAb < MAb 1, MAb 2>, wherein < MAb 1> and < MAb 2> are named antigen binding sites derived from MAb 1 and MAb 2. For example, the first antigen binding site is directed to an angiogenic receptor (e.g., integrin or VEGF receptor) and the second antigen binding site is directed to an ErbB receptor (e.g., EGFR or HER 2). Bispecific antibodies can be prepared by chemical methods (see, e.g., Kranz et al (1981) proc.natl.acad.sci.usa 78,5807) or by "polydoma" techniques (see US4,474,893) or recombinant DNA techniques, all of which are essentially known techniques. Other methods are described in WO 91/00360, WO 92/05793 and WO 96/04305. Bispecific antibodies can also be prepared using single chain antibodies (see, e.g., Huston et al (1988) Proc. Natl. Acad. Sci.85, 5879; Skerra and Plueckthun (1988) Science 240, 1038). These are antibody variable region analogs produced as a single polypeptide chain. To construct a bispecific binding agent, single chain antibodies can be coupled together by chemical or genetic engineering methods well known to those skilled in the art. Bispecific antibodies of the invention can also be prepared using leucine zipper sequences. The sequences used may be derived from the leucine zipper region of the transcription factors Fos and Jun (Landshulz et al, 1988, Science 240, 1759; reviewed in Maniatis and Abel, 1989, Nature 341, 24). Leucine zippers are special amino acid sequences that are about 20-40 residues long, typically one leucine for every 7 residues. This zipper sequence forms an amphipathic α -helix with leucine residues aligned on the hydrophobic side to form a dimer. Peptides corresponding to the leucine zipper of Fos and Jun proteins preferentially form heterodimers (O' Shea et al, 1989, Science 245, 646). Zipper-containing bispecific antibodies and methods for their preparation are also disclosed in WO92/10209 and WO 93/11162.

The term "fusion protein" refers to a natural or synthetic molecule consisting of one or more of the above biomolecules, wherein two or more peptide or protein (including glycoprotein) based molecules with different specificities are fused together, optionally through chemical or amino acid based linker molecules. This linkage can be achieved by C-N fusion or N-C fusion (in the 5 '→ 3' direction), preferably C-N fusion.

However, preferred fusion proteins according to the invention are immunoconjugates as described below.

The term "immunoconjugate" refers to a fusion protein, in the sense of an antibody or immunoglobulin or immunologically active fragment thereof fused by covalent bond to a non-immunological effector molecule. The fusion partner (partner) is preferably a peptide or protein that can be glycosylated. The non-antibody molecule may be linked to the C-terminus of the antibody heavy chain constant region or to the N-terminus of the light and/or heavy chain variable regions. The fusion partners may be linked by linker molecules, typically peptides containing 3-15 amino acid residues. The immunoconjugates of the invention are fusion proteins composed of an immunoglobulin against an ErbB receptor or an immunotherapeutically effective fragment thereof and preferably a cytokine such as TNF α, IFN γ or IL-2, or other toxic agents. Preferably, these peptide-or protein-based molecules are linked via their N-terminus to the C-terminus of the immunoglobulin (i.e., the Fc portion thereof).

A "hybrid antibody" is a fusion protein consisting essentially of two or more antibodies or antibody-binding fragments fused together conventionally by a chemical cross-linking agent, wherein the antibodies each have a different binding specificity. Hybrid antibodies can be prepared by conjugating two or more antibodies or antibody fragments together.

Preferred hybrid antibodies consist of cross-linked Fab/Fab' fragments. A wide variety of coupling or crosslinking reagents are available for conjugation of antibodies. Such as protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA) and N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) (see, for example, Karpovsky et al (1984) J.EXP.Med.160, 1686; Liu et al (1985) Proc.Natl.Acad.Sci.USA 82,8648). Other methods are also available, Paulus, Behring Inst.Mitt., No.78, 118 (1985); brennan et al (1985) Science 30, 81 or Glennie et al (1987) J.Immunol.139, 2367. Another method uses ortho-phenylenedimaleimide (oPDM) to couple together three Fab' fragments (WO 91/03493).

Multispecific antibodies of the invention are also suitable and may be prepared, for example, according to the teachings of WO94/13804 and WO 98/50431. Preferred hybrid antibodies of the invention are fusion proteins (e.g., MAB 425-MAB 225) comprising two anti-EGFR antibodies (each directed against a different epitope of the same receptor) linked together as described.

The term "cytokine" is a generic term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; (ii) prorelaxin; glycoprotein hormones such as Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH) and Luteinizing Hormone (LH); a liver growth factor; fibroblast growth factor; prolactin; placental lactogen; mouse gonadotropin-related peptides; a statin; an activin; vascular Endothelial Growth Factor (VEGF); an integrin; thrombopoietin (TPO); nerve growth factors such as NGF β; platelet growth factor; transforming Growth Factors (TGF) such as TGF α and TGF β; erythropoietin (EPO); interferons such as IFN α, IFN β and IFN γ; colony stimulating factors such as M-CSF, GM-CSF and G-CSF; interleukins such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; and TNF α or TNF β. Preferred cytokines of the present invention are interferon and TNF α and IL-2.

Antibody "effector functions" refer to those biological activities caused by the Fc region of an antibody (either the native sequence Fc region or the amino acid sequence variant Fc region). Examples of antibody effector functions include complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors), and the like.

The term "ADCC" (antibody-dependent cell-mediated cytotoxicity) refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (fcrs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. The primary cells mediating ADCC, NK cells, express Fc γ R III only, whereas monocytes may express Fc γ R I, Fc γ R II and Fc γ R III. To estimate the ADCC activity of a molecule of interest, it can be performed using an in vitro ADCC assay, for example as described in the prior art (U.S. Pat. No. 5,500,362; U.S. Pat. No. 5,821,337). Useful effector cells in this experiment include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells.

The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one that binds an IgG antibody (gamma receptor) and includes receptors of the Fc γ R I, Fc γ R II and Fc γ R III subclasses, including allelic variants and alternatively spliced forms of these receptors. For reviews of FcR see, e.g., ravatch and Kinet, annu. 457-92(1991).

As a specific embodiment, the therapeutic methods of the invention include the administration of other therapeutically effective agents that can aid in the desired effect, such as tumor toxicity or cytostatic efficacy, or reduce or prevent unwanted side effects. Thus, the invention includes the combination of the agent, which may be typically other ErbB receptor antagonists, VEGF receptor antagonists, cytokines, cytokine immunoconjugates, anti-angiogenic agents, anti-hormonal agents, or cytotoxic agents, with the pharmaceutical compositions described above and below and defined herein. It is also an object of the present invention to combine the compositions described herein with radiation therapy according to known methods.

The term "cytotoxic agent" as used herein has a very broad definition and refers to a substance that inhibits or prevents cellular function and/or causes cell destruction (cell death) and/or produces an anti-neoplastic/anti-proliferative effect, e.g., directly or indirectly prevents the progression, maturation or spread of neoplastic tumor cells. The term also expressly includes agents which cause only cytostatic effects and no purely cytotoxic effects. The term includes chemotherapeutic agents, as well as other ErbB antagonists (such as anti-ErbB antibodies), anti-angiogenic agents, tyrosine kinase inhibitors, protein kinase a inhibitors, cytokine family members, radioisotopes, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, as described in detail below.

The term "chemotherapeutic agent" is a subset of the term "cytotoxic agent" which specifically refers to a chemical agent that has an anti-tumor effect, preferably acts directly on tumor cells, and less indirectly through mechanisms such as biological response modification. Suitable chemotherapeutic agents of the invention are preferably natural or synthetic chemical compounds. A large number of existing anti-neoplastic chemotherapeutic agents in commercial use, clinical evaluation, and preclinical development stages are encompassed by the present invention for the treatment of tumors/neoplasias by combination with the receptor antagonists described herein. It should be noted that chemotherapeutic agents may optionally be administered with the ErbB receptor antagonist of the present invention, preferably the anti-EGFR antibody.

Examples of chemotherapeutic agents include alkylating agents such as nitrogen mustards, aziridine compounds, alkyl sulfonates and other compounds having an alkylating effect such as nitrosoureas, cisplatin and dacarbazine; antimetabolites such as folic acid, purine or pyrimidine antagonists; mitotic inhibitors such as vinca alkaloids and podophyllotoxin derivatives; cytotoxic antibiotics and camptothecin derivatives.

Preferred chemotherapeutic agents are amifostine (ethiol), cisplatin, Dacarbazine (DTIC), dactinomycin, mechlorethamine, streptozotocin, cyclophosphamide, carrnsustine (BCNU), lomustine (CCNU), doxorubicin liposomes (doxil), gemcitabine (gemzar), daunorubicin liposomes (daunoxame), procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil (5-FU), vinblastine, vincristine, bleomycin, taxol (taxol), taxotere (taxotere), aldesleukin (aldeslin), asparaginase, busulfan, carboplatin, camptothecin, CPT-11, 10-hydroxy-7-ethyl-camptothecin (SN38), gefitinib, dacarbazine, fludarabine, idarubicin, mesna, interferon alpha, interferon beta, irinotecan (irinotecan), mitoxantrone, topotecan, leuprolide, megestrol, antineoplastic, mercaptopurine, plicamycin (plicamycin), chlorhexadiene, asparaginase (pegaspragase), pentostatin (pentostatin), pipobroman, plicamycin, streptozotocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil, and combinations thereof.

The most preferred chemotherapeutic agents of the present invention are cisplatin, gemcitabine, doxorubicin, paclitaxel (taxol) and bleomycin.

An anti-angiogenic agent "refers to a natural or synthetic compound that blocks or interferes to some extent with angiogenesis. The anti-angiogenic molecule can be, for example, a biomolecule that binds to and blocks angiogenic growth factors or growth factor receptors. Preferred anti-angiogenic molecules herein may bind to a receptor, preferably to an integrin receptor or to a VEGF receptor. This term also includes prodrugs of the anti-angiogenic agents in the context of the present invention. The term also includes agents that have the described effects and can also be classified as cytotoxic agents, preferably chemotherapeutic agents.

There are many molecules of different structures and origins that can cause anti-angiogenic properties. Most relevant classes of suitable angiogenesis inhibitors or blockers of the present invention are, for example:

(i) antimitotic agents, such as fluorouracil, mitomycin C, paclitaxel;

(ii) estrogen metabolites such as 2-methoxyestradiol;

(iii) matrix Metalloproteinase (MMP) inhibitors that inhibit zinc metalloproteases (e.g., betamastat, BB16, TIMPs, minocycline, GM6001, or those (substances) mentioned in "inhibition of matrix metalloproteases: therapeutic applications" (Golub, Annals of the New Yorkacademy of Science, Vol.878a; Greenwald, Zucker (Eds.), 1999);

(iv) anti-angiogenic multifunctional agents and factors, such as IFN alpha (US 4,530,901; US4,503,035; 5,231,176); angiostatin and plasminogen fragments (e.g., kringle1-4, kringle5, kringle 1-3(O 'Reilly, M.S. et al, Cell (Cambridge, Mass.)79 (2): 315-328, 1994; Cao et al, J.biol.Chem.271: 29461-29467, 1996; Cao et al, J.biol chem 272: 22924-22928, 1997); endostatin (endostatin) (O' Reilly, M.S. et al, Cell 88(2), 277,1997 and WO 97/15666), thrombospondin (TSP-1; Frazier, 1991, Curr Opin Biol 3 (5: 792); platelet factor 4(PF 4);

(v) plasminogen activator/urokinase inhibitor;

(vi) a urokinase receptor antagonist;

(vii) a heparinase;

(viii) fumagillin analogs such as TNP-470;

(ix) tyrosine kinase inhibitors such as SU 10 (many ErbB receptor antagonists mentioned above and below (EGFR/Her 2 antagonists) are also tyrosine kinase inhibitors and thus may show anti-EGF receptor blocking activity leading to inhibition of tumor growth and anti-angiogenic activity leading to inhibition of vascular development and endothelial cell development, respectively);

(x) Suramin and suramin analogs;

(xi) Angiostatic (angiostatic) steroids;

(xii) VEGF and bFGF antagonists;

(xiii) VEGF receptor antagonists such as anti-VEGF receptor antibodies (DC-101);

(xiv) flk-1 and flt-1 antagonists;

(xv) cyclooxygenase-II inhibitors such as COX-II;

(xvi) Integrin antagonists and integrin receptor antagonists such as α v antagonists and α v receptor antagonists, for example, anti- α v receptor antibodies and RGD peptides. Integrin (receptor) antagonists are preferred in the present invention.

The term "integrin antagonist/inhibitor" or "integrin receptor antagonist/inhibitor" refers to a natural or synthetic molecule that blocks and inhibits integrin receptors. Sometimes, this term includes ligands directed against the integrin receptor (e.g., for α)_vβ₃: vitronectin, fibrin, fibrinogen, von willebrand factor, thrombospondin, lamininZonulin; for alpha_vβ₅: vitronectin; for alpha_vβ₁: fibronectin and vitronectin; for alpha_vβ₆: fibronectin).

The present invention is preferably directed to antagonists of integrin receptors. Integrin (receptor) antagonists can be natural or synthetic peptides, non-peptides, peptidomimetics (peptidomiactics), immunoglobulins such as antibodies or functional fragments of antibodies, or immunoconjugates (fusion proteins).

Preferred integrin inhibitors of the invention are directed against alpha_vIntegrin receptors (e.g.. alpha.)_vβ₃，α_vβ₅，α_vβ₆And subclass) of inhibitors. Preferred integrin inhibitors are α_vAntagonists, especially alpha_vβ₃An antagonist. Preferred alpha of the invention_vThe antagonist is an RGD peptide, a peptidomimetic (non-peptide) antagonist and an anti-integrin receptor antibody such as blocking alpha_vAn antibody to the receptor. Exemplary non-immunological alpha_vβ₃Antagonists are taught in US 5,753,230 and US 5,766,591. Preferred antagonists are RGD-containing linear and cyclic peptides. Cyclic peptides are generally more stable and have a longer half-life in serum. However, the most preferred integrin antagonist of the present invention is cyclo (Arg-Gly-Asp-DPhe-NMeVal) (EMD121974, CilengitideMerck KgaA, germany; EP 0770622) which effectively block integrin receptor alpha_vβ₃、α_vβ₁、α_vβ₆、α_vβ₈、α_nbβ₃。

Alpha is described in both scientific and patent literature_vβ₃/α_vβ₅/α_vβ₆Suitable peptidic and peptidic mimetic (non-peptidic) antagonists of integrin receptors. See, for example, Hoekstra and Poulter, 1998, curr.med.chem.5, 195;WO 95/32710; WO 95/37655; WO 97/01540; WO 97/37655; WO 97/45137; WO 97/41844; WO 98/08840; WO 98/18460; WO 98/18461; WO 98/25892; WO 98/31359; WO 98/30542; WO 99/15506; WO 99/15507; WO 99/31061; WO 00/06169; EP 0853084; EP 0854140; EP 0854145; US 5,780,426; and US 6,048,861. Benzazoles and related benzodiazepines and benzocycloheptenes alpha also suitable for use in the present invention are disclosed_vβ₃Patents for integrin receptor antagonists include WO 96/00574, WO 96/00730, WO 96/06087, WO96/26190, WO 97/24119, WO 97/24122, WO 97/24124, WO 98/15278, WO 99/05107, WO 99/06049, WO 99/15170, WO 99/15178, WO 97/34865, WO97/01540, WO 98/30542, WO 99/11626 and WO 99/15508. In WO 98/08840; WO 99/30709; WO 99/30713; WO 99/31099; WO 00/09503; US 5,919,792; US 5,925,655; US 5,981,546; and other integrin receptor antagonists characterized by a backbone conformational loop constraint are described in US 6,017,926. A series of nonanoic acid derivatives are disclosed in US 6,048,861 and WO00/72801, which are effective alpha_vβ₃Integrin receptor antagonists. Other chemical small molecule integrin antagonists (mostly vitronectin antagonists) are disclosed in WO 00/38665. Other alpha_vβ₃Receptor antagonists have been shown to be effective in inhibiting angiogenesis. For example, synthetic receptor antagonists such as (S) -10, 11-dihydro-3- [3- (pyridin-2-ylamino) -1-propoxy]-5H-dibenzo [ a, d1 cycloheptene-10-acetic acid (named SB-265123) has been tested in a number of mammalian model systems. (Keenan et al, 1998, Bioorg. Med. chem. Lett.8(22), 3171; Ward et al, 1999, Drug Metab. Dispos.27(11), 1232). Screening assays for integrin antagonists suitable for use as antagonists are described, for example, in Smith et al, 1990, J.Bio1.chem.265, 12267 and in the referenced patent literature.

Antibodies against integrin receptors are also well known. Can be used for treating suitable anti-integrin (such as alpha)_vβ₃，α_vβ₅，α_vβ₆) Is modified to include the antigen thereofBinding fragments (including F (ab)₂Fab and engineered Fv or single chain antibody). Against integrin receptors alpha_vβ₃A suitable and preferably used monoclonal antibody of (I) is identified as LM609(Brooks et al, 1994, Cell 79, 1157; ATCC HB 9537). A strongly specific anti-alpha is disclosed in WO 97/45447_vβ₅The antibody, P1F6, is also preferred for use in the present invention. Another suitable alpha_vβ₆The selective antibody is alpha selective for integrin receptors_vMab 14d9.f8(WO 99/37683, DSMACC2331, Merck KGaA, germany) and Mab 17.E6(EP 0719859, DSMACC2160, Merck KGaA). Another suitable anti-integrin antibody is marketed Vitraxin

As used herein, the term "anti-hormonal agent" includes natural or synthetic organic or peptide compounds that act to modulate or inhibit the action of hormones on tumors. More specifically, "anti-hormonal agents" (1) inhibit the production of serum androgens, (2) block the binding of serum androgens to androgen receptors, or (3) inhibit the conversion of testosterone to DHT, or a combination of two or more such compounds. The anti-hormonal agents of the present invention generally include steroid receptor antagonists, more specifically anti-estrogenic agents such as those including tamoxifen, raloxifene, aromatase inhibiting 4(5) -imidazole, 4-hydroxyttamoxifen, hydronaproxen, keoxifene, LY117018, onasterone and toremifene (terresifene) (Fareston); and antiandrogens such as flutamide (flutamide), nilutamide (nilutamide), bicalutamide, leuprolide (1euprolide) and goserelin (goserelin); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. The term also includes agonists and/or antagonists of glycoprotein hormones, such as Follicle Stimulating Hormone (FSH), Thyrotropin (TSH) and Luteinizing Hormone (LH) and LHRH (luteinizing hormone releasing hormone). The LHRH agonist useful in the present invention is capromorelin acetate, which is known under the tradename ZOLADEX(Zeneca). Another example of a suitable LHRH antagonist is cyproterone acetate (CPA) and megestrol acetate, which are commercially available as MEGACE(Bristol-Myers, Oncology). Steroidal antiandrogens block the prostatic androgen receptor. It also inhibits the release of LH. Preferably, CPA doses in the range of 100 mg/day to 250 mg/day are administered to human patients. Nonsteroidal antiandrogens block the androgen receptor. They may also cause an increase in serum LH levels and serum testosterone levels. A preferred non-steroidal antiandrogen is flutamide (2-methyl-N- [4-20 nitro-3- (trifluoromethyl) phenylpropionamide]Under the trade name EULEXIN(Schering Corp.). Flutamide exerts an anti-androgen effect that inhibits androgen uptake, inhibits nuclear binding of androgen in the target tissue, or both. Another non-steroidal antiandrogen agent is nilutamide, which is known by the chemical name 5, 5-dimethyl-3- [ 4-nitro-3- (trifluoromethyl-4' -nitrophenyl) -4, 4-dimethyl-imidazolidine-dione. In some embodiments of the invention, the anti-hormonal agent is a combination of an LHRH agonist such as leuprolide acetate and an anti-androgenic agent such as flutamide or nilutamide. For example, leuprolide acetate can be administered by subcutaneous, intramuscular, or intravenous injection, and flutamide can be administered orally at the same time. The anti-hormonal agents of the present invention include, as described above, antagonists of steroid/thyroid hormone receptors, including antagonists of other non-permissive receptors, such as RAR, TR, VDR, and the like. One skilled in the art will readily appreciate that a variety of synthetic and natural Retinoic Acid Receptor (RAR) antagonists may be used in accordance with the present invention.

The bispecific antibodies according to the invention may be combined with other drugs. These drugs are preferably selected from:

tyrosine kinase inhibitors, such as Iressa

Anti-angiogenic agents, preferably integrin inhibitors, more preferably RGD peptides, including cyclic peptides such as cyclo- (Arg-Gly-Asp-DPhe-NMeVal) (CilengitideMerck KGaA)；

An anti-VEGF receptor antibody, such as DC-101, or a VEGF antagonist;

COX-II inhibitors;

cytokines such as TNF- α, IFN- α, IFN- β, IFN- γ, IL-2;

type I Protein Kinase A (PKAI) inhibitors, such as mixed backbone antisense oligonucleotides, such as HYB 165 (see, e.g., Totorta et al, 1999, Clin. cancer Res., 875-881);

anti-hormonal agents such as goserelin, boserelin, leuprolide, tamoxifen.

The terms "cancer" and "tumor" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Tumors, such as breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head and neck, ovary, prostate, brain, pancreas, skin, bone marrow, blood, thymus, uterus, testis, cervix and liver tumors can be treated by administering the pharmaceutical composition of the present invention. More specifically, the tumor is selected from: adenomas, angiosarcoma, astrocytomas, epithelial cancers, germ cell tumors, glioblastoma, glioma, hamartoma, angioendothelioma, angiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma and teratoma. In detail, the tumor is selected from: acropigmented melanoma, actinic keratosis, adenocarcinoma, cystadenocarcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, astrocytoma, bartholinia carcinoma, basal cell carcinoma, bronchogenic adenocarcinoma, capillary hemangioma, carcinoma, carcinosarcoma, cavernous cholangiocarcinoma, chondrosarcoma, choriocapillaris papilloma/carcinoma, clear cell carcinoma, cystadenoma, endoblastoma, endometrial hyperplasia, endometrial interstitial sarcoma, endometrial adenocarcinoma, ependymal sarcoma, epithelioid sarcoma, ewing's sarcoma, fibrolamellar carcinoma, focal nodular hyperplasia, gastrinoma, germ cell tumor, glioblastoma, glucagon tumor, hemangioblastoma, hemangioma, hepatic adenoma, hepatoadenomatosis, hepatocellular carcinoma, insulinoma, intraepithelial neoplasia, inter-epithelial squamous cell carcinoma, invasive squamous cell carcinoma, large cell carcinoma, leiomyosarcoma, malignant pigmented plaque-type melanoma, malignant mesothelioma, medulloblastoma, medullo-epithelial carcinoma, melanoma, meningeal tumors, mesothelial tumors, metastatic tumors, mucoepithelial carcinoma, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, oat cell carcinoma, oligodendroglioma, osteosarcoma, pancreatic polypeptide, papillary serous adenocarcinoma, pineal cell tumor, pituitary tumor, plasmacytoma, pseudosarcoma, pneumocblastoma, renal cell tumor, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin-secreting cell tumor, squamous cell carcinoma, sub-mesothelial, superficial spreading melanoma, undifferentiated carcinoma, uveal melanoma, warty carcinoma, vasoactive intestinal polypidoma, well-differentiated carcinoma, and nephroblastoma.

Preferred tumors that can be treated with the antibody molecules according to the invention are solid tumors or tumor metastases that abundantly express the ErbB receptor, in particular the ErbB1 receptor, such as breast cancer, prostate cancer, head and neck cancer, SCLC, pancreatic cancer.

The term "biologically/functionally effective" or "therapeutically effective amount" refers to a drug/molecule that causes a biological function or a change in a biological function in vivo or in vitro, and that is effective in treating a disease or condition in a mammal, preferably a human, in a specified amount. For cancer, a therapeutically effective amount of the drug can reduce the number of cancer cells; reducing the size of the tumor; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) metastasis of the tumor; inhibit tumor growth to some extent; and/or to alleviate one or more symptoms associated with cancer to some extent. A drug may be cytostatic and/or cytotoxic if it can prevent the growth of and/or kill existing cancer cells. For the treatment of cancer, efficacy can be determined, for example, by estimating time to disease progression (TTP) and/or determining Response Rate (RR).

The term "immunotherapeutically effective" refers to a biomolecule that elicits an immune response in a mammal. More specifically, the term refers to molecules that can recognize and bind antigens. Typically, antibodies, antibody fragments containing their antigen binding sites (complementarity determining regions, CDRs), and antibody fusion proteins are immunotherapeutically effective.

"radiotherapy": tumors may also be treated with radiation or radiopharmaceuticals according to the present invention. The radiation source may be located either outside or inside the patient to be treated. If the radiation source is located outside the patient's body, the treatment method is called External Beam Radiotherapy (EBRT). If the radiation source can be located in the patient, the treatment is called Brachytherapy (BT). Some typical radioactive atoms that have been used include radium, cesium-137, and iridium-192, americium-241 and gold-198, cobalt-57; copper-67; technetium-99; iodine-123; iodine-131; and indium-111. The agents of the invention may also be labelled with radioisotopes. Current radiation therapy is the standard treatment for controlling unresectable or inoperable tumors and/or tumor metastases. It has been found that the combination of radiotherapy and chemotherapy can improve the efficacy. Radiotherapy is based on the principle that high doses of radiation projected onto a target area will cause the death of proliferating cells in tumor and normal tissues. The radiation dose plan is generally determined in terms of radiation absorbed dose (rad), time and fractions, and must be carefully determined by an oncologist. The amount of radiation a patient receives will depend on various factors, but the two most important factors are the location of the tumor relative to other important structures or organs of the body, and the extent to which the tumor has spread. A preferred treatment regimen for radiotherapy administration to a patient is a course of treatment lasting 5 to 6 weeks with a total dose of 50-60Gy administered to the patient in 1.8-2.0Gy doses administered in portions 1 time per day for 5 days per week. Gy is an abbreviation for gray and refers to a 100rad dose. If tumors are treated with the anti-ErbB antibodies of the present invention in the context of radiotherapy, a positive or even synergistic effect is often observed. In other words, the inhibition of tumor growth will be enhanced if the compound is combined with radiation and/or a chemotherapeutic agent. Radiation therapy may optionally be used in accordance with the present invention. Which is recommended and preferred in case an effective amount of the medicament according to the invention cannot be administered to a patient.

"drug therapy": the method of the invention comprises various embodiments in terms of steps. For example, the agents of the invention may be used simultaneously, sequentially or independently. Additionally, the agents may be administered separately and within about 3 weeks of each other, i.e., the second agent begins administration substantially immediately after the first agent is administered to begin administration no more than about 3 weeks after the first agent is administered. The method may be performed after surgery. Alternatively, the surgery may be performed during the interval between administration of the first active agent and administration of the second active agent. An example of such a method is the use of the method of the invention in combination with surgical tumor removal surgery. Treatment according to the methods of the invention typically comprises administration of the present therapeutic compositions in one or more cycles of administration. For example, when administered simultaneously, a therapeutic composition containing 2 agents is administered for a period of about 2 days to about 3 weeks. Thereafter, the treatment cycle may be repeated as necessary at the discretion of the practitioner. Similarly, if sequential administration is performed, the time of administration of each therapeutic agent can be adjusted to typically cover the same time. The interval between cycles can vary from about 0 to 2 months.

The agents of the invention may be administered parenterally by injection or by gradual infusion over time. The tissue to be treated in vivo is generally treated by systemic administration, and thus the most commonly used method is intravenous administration of the therapeutic composition, although other tissues and methods of administration are contemplated as the target tissue may contain the target molecule. Thus, the agents of the invention may be administered intraocularly, intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, by orthotopic injection and infusion, and may also be administered by peristaltic pump means. For example, therapeutic compositions of the invention, including, for example, integrin antagonists, are typically administered intravenously, e.g., by injection in unit doses.

The therapeutic compositions of the present invention comprise a physiologically tolerable carrier and, dissolved or dispersed therein, as active ingredient, the relevant agents described herein.

As used herein, the term "pharmaceutically acceptable" refers to compositions, carriers, diluents, and agents that are capable of being administered to a mammal without producing undesirable physiological effects such as nausea, dizziness, nausea and the like. The preparation of pharmaceutical compositions in which the active ingredient is dissolved or dispersed is well known to those skilled in the art and need not be defined on a formulation basis. Typically, such compositions are formulated as injectables, e.g., as liquid solutions or suspensions, but may also be formulated in solid form suitable for solution or suspension in liquid prior to use. The formulation may also be emulsified. The active ingredient may be mixed with excipients in amounts suitable for pharmaceutical use in the methods of treatment described herein and compatible with the active ingredient.

Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like and combinations of these. In addition, if desired, the compositions may also include minor amounts of auxiliary substances which enhance the effectiveness of the active ingredient, such as wetting or emulsifying agents, pH buffering agents and the like. Pharmaceutically acceptable salts of the ingredients may be included in the therapeutic compositions of the present invention. Pharmaceutically acceptable salts include the acid addition salts (which form salts with the free amino groups of the polypeptide) which are inorganic acids, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, tartaric, mandelic, and the like. Salts with free carboxyl groups can also be obtained from inorganic bases, for example, sodium, potassium, ammonium, calcium or iron hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like. The use of HCl salt is particularly preferred in formulations of cyclic peptide α v antagonists. Physiologically tolerable carriers are well known to those skilled in the art. Examples of liquid carriers are sterile aqueous solutions which may contain the active ingredient and water alone or may also contain buffering agents such as sodium phosphate at physiological pH, physiological saline or both, e.g., phosphate buffered saline.

In addition, the aqueous carrier may contain one or more buffer salts as well as salts such as sodium chloride and potassium chloride, glucose, polyethylene glycol and other solutes. The liquid composition may also contain a liquid phase with or without water. Examples of such other liquid phases are glycerol, vegetable oils such as cottonseed oil and water-oil emulsions.

Typically, for example, for immunotherapeutic agents in the form of ErbB (ErbB1) receptor blocking bispecific antibodies, integrin receptor blocking antibodies or antibody fragments or antibody conjugates, or anti-VEGF receptor blocking antibodies, fragments or conjugates, a therapeutically effective amount is an amount sufficient to achieve a plasma concentration of from about 0.01 micrograms (μ g) per milliliter (ml) to about 100 μ g/ml, preferably from about 1 μ g/ml to about 5 μ g/ml, usually about 5 μ g/ml, when administered in a physiologically tolerable composition. In other words, the dosage may vary from about 0.1mg/kg to about 300mg/kg, preferably from about 0.2mg/kg to about 200mg/kg, most preferably from about 0.5mg/kg to about 20mg/kg, in one or more daily administrations for one or more days. When the immunotherapeutic agent is in the form of a fragment or conjugate of a monoclonal antibody, its amount can be readily adjusted according to the ratio of the mass of the fragment/conjugate relative to the mass of the whole antibody. Preferred plasma molarity is about 2 micromolar (. mu.M) to about 5 millimolar (mM), preferably about 100. mu.M to 1mM, of antibody antagonist.

For agents of the invention that are non-immunotherapeutic peptide or protein polypeptides or other biomolecules of similar size, a therapeutically effective amount is typically an amount of the polypeptide sufficient to achieve a plasma concentration of about 0.1 micrograms (μ g) per milliliter (ml) to about 200 μ g/ml, preferably about 1 μ g/ml to about 150 μ g/ml, when administered in a physiologically tolerable composition. Preferred plasma molarity is about 2 micromolar (. mu.M) to about 5 millimolar (mM), preferably about 100. mu.M to 1mM, of polypeptide antagonist, calculated on the basis of about 500 grams mass of polypeptide per mole.

Typical dosages for the active agent, which is preferably a chemocytotoxic agent or chemotherapeutic agent (neither an immunotherapeutic agent nor a non-immunotherapeutic peptide/protein) of the invention, are from 10mg to 1000mg, preferably from about 20 to 200mg, more preferably from 50 to 100mg per kg body weight per day.

The "pharmaceutical composition" of the present invention may comprise an agent that reduces or avoids the side effects associated with the combination therapy of the present invention ("adjuvant therapy"), including, but not limited to, agents that reduce the toxic effects of anti-cancer drugs, such as bone resorption inhibitors, cardioprotective agents. The adjuvant can prevent or reduce the incidence of nausea and vomiting caused by chemotherapy, radiotherapy or surgery, or reduce the incidence of infection caused by the administration of myelosuppressive anticancer drugs. Auxiliary agents are well known to those skilled in the art. In addition, the immunotherapeutic agent of the present invention can also be used together with adjuvants such as BCG and immune system stimulants. Furthermore, the compositions may include immunotherapeutic or chemotherapeutic agents containing radiolabeled isotopes having cytotoxic effects or other cytotoxic agents such as cytotoxic peptides (e.g., cytokines) or cytotoxic drugs, and the like.

The term "pharmaceutical kit" for the treatment of tumors or tumor metastases refers to a package and, generally, instructions for the use of the agent in a method for the treatment of tumors and tumor metastases. The agents of the kits of the invention are generally formulated as therapeutic compositions as described herein and may therefore take any form suitable for placement within the kit. These forms may include liquid, powder, tablet, suspension, etc. formulations to provide therapeutic molecules of the invention, preferably anti-ErbB 1 antibodies. These agents may be provided in separate containers suitable for separate administration according to the methods of the present invention, or may be provided in combination in the composition within a single container in such a package. The package may contain an amount of the agent sufficient to be administered one or more times in accordance with the treatment methods described herein. The kits of the invention also include "instructions for use" of the materials contained in the package.

Examples

Example 1:

preparation of F (AB') 2-fragments of MAB425 and MAB225

The anti-EGFR antibody humanized MAb425 and chimeric MAb225 were converted to F (ab') 2 fragments using restricted proteolysis. The production of F (ab') 2 antibodies must be optimized for each antibody. The general scheme for this preparation is as follows.

Pepsin cleavage was optimal for both antibodies, however papain cleavage was also suitable. Residual intact antibody and Fc fragment were removed from the protein a sepharose column. The yield of F (ab') 2 fragments was close to 100%. F (ab') 2 fragments can be stored at-20 ℃ without losing any activity for a prolonged period of time.

General scheme:

growth fermentation → centrifugation → ultrafiltration → protein A chromatography → dialysis/ultrafiltration → lysis → protein A chromatography → dialysis/ultrafiltration → F (ab') 2 product

The detailed contents are as follows:

PBS，pH：7.4

protein content: 5.06mg/ml (Pierce).

Reagent:trisodium citrate dihydrate, citric acid, tris (hydroxymethyl) aminomethane, pepsin, glycine, sodium chloride

Buffer solution:

0.1M sodium citrate buffer pH 3.5,

10mg/mL pepsin in pH 3.5 sodium citrate buffer,

1M Tris pH 11

1.5M Glycine +3M NaCI pH 8.9

0.1M citric acid pH 2.5

And (3) a pepsin digestion process:

the composition was prepared by mixing the ingredients in 0.1M sodium citrate, pH: MAb was dialyzed overnight at 3.5 to adjust pH and buffer conditions. Pepsin was added to the dialyzed immunoglobulins in a ratio of 1: 33 w/w.

The mixture was stirred continuously in a water bath at 37 ℃. After 75 minutes, digestion was stopped by adding 7ml of 1M Tris solution. In this step, the pH of the reaction should be set to about 8.5. The mixture was then transferred to a protein a column to remove residual IgG and/or Fc fragments.

Protein-a-agarose:

the pepsin digest was added to the equilibrated protein-a-sepharose column and washed with equilibration buffer until the chromatogram returned to baseline. The direct flow fractions were collected, reduced in volume in Amicon chambers (Membrane YM 30), and then diluted with PBS pH: 7.4 dialysis was performed. pH with 0.1M citric acid: 2.5 elution of potential contaminants such as Fc fragments or unmodified antibodies from protein-A-Sepharose columns.

Chromatography conditions are as follows:

the size of the column bed is 5cm × 2.5 cm. 1ml of protein A agarose was expected to bind 10mg IgG, using 1.5M glycine +3M NaCl, pH: 8.9 equilibrium column, flow rate: 60ml/h, detection: OD 280nm, 0.2/2.0Abs-Range, Uvicord SI1, recording paper speed: 0.1mm/min, 5ml of each fraction was collected.

Yield of F (ab') 2 preparation (Pierce)

Considering that the Fc-portion is approximately one-third of the molecule, the yield of F (ab') 2 preparations of both antibodies approaches 100%. The concentration of the sample should be 6-7 mg/ml. The purity of the F (ab') 2 preparations was monitored by SDS-PAGE.

Example 2: preparation of bispecific antibody BAB <425, 225>

Bispecific antibodies were generated by chemical recombination of IgG fragments using the method described by Brennan et al (Science, 1985, 229, 81-83). The steps of the modification method of the invention are as follows:

f (ab ') 2 product → reduction to Fab' → gel chromatography → derivatization → gel chromatography → conjugation → gel chromatography → ultrafiltration → sterile filtration → BAbs

Both specific F (ab ') 2 fragments were converted to Fab'. The success of the conjugation step depends on the selection of the appropriate Fab' fragment for elmann modification. For babs derived from MAb 225/MAb 425, the <225> component is modified. After introduction of these modifications, individual BAb yields were 20-30%. The anti-225 Fab 'was modified with Epermann reagent and conjugated to a 425 specific Fab'. Bispecific antibody was recovered by gel filtration.

The detailed contents are as follows:

in this step, both antibodies are reduced with DTT to produce Fab' fragments.

Fab 'derived from MAb225 was modified with elmann' prior to conjugation. However, Fab 'derived from MAb425 may also be modified with Ellman's reagent.

Fragment (b):

(i)F(ab′<425>)2 7.4mg/ml

(ii)F(ab′<225>)2 6.9mg/ml

solution/buffer:

PBS pH7.4, PBS +0.65M NaCl +2.5mM EDTA, pH7.4, 51mM dithiothreitol in PBS, 0.1M sodium phosphate buffer, pH 8.0, 35mM Epermann's reagent in 0.1M sodium phosphate buffer, pH 8.0, 250mM EDTA, pH7.4

Reagent:

1, 4-dithiothreitol, Elman's reagent, sodium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, EDTA, Titriplex III, Superdex 200(26/60) Pharmacia,

the first step of the synthesis:

MAb <225> Fab' -TNB was prepared.

6550μl F(ab′)2MAb 22540mg+65.4μl DTT 51mM+65.4μl EDTA250mM.

The final concentration of DTT was 0.5mM, while that of EDTA was 2.5 mM.

The reaction was covered with argon and incubated in a water bath at 30 ℃ for 40 minutes with constant stirring. After incubation, 1120. mu.l of Ellman's reagent was added to the reaction mixture, which reversibly blocked free SH groups in the resulting Fab'.

The final concentration of the Elmann's reagent in the reaction was 5.0 mM. The reaction mixture was stirred at room temperature for 30 minutes in order to block all SH groups. The color of the reaction mixture changed from clear to yellow. The reaction mixture was purified by Superdex 200(26/60) column with PBS +0.65M NaCl +2.5mM EDTA buffer to separate the reduced Ellman modified Fab ' molecules from potential contaminants such as unreduced F (ab ') 2, Fab ' and excess reagents. The Fab-TNB fractions were pooled, covered with argon and stored on ice prior to the coupling reaction.

The second step of the synthesis:

MAb <425> Fab' was prepared.

6135μl F(ab′)2MAb 42540mg+80μl EDTA 250mM+80μl DTT 51mM。

The reaction should not be initiated before the Fab-TNB preparation is almost complete. The final concentration of the reaction was 0.5mM DTT and 2.5mM EDTA. The reaction mixture was covered with argon and incubated at 30 ℃ for 40 minutes. Immediately after incubation, the reaction mixture was transferred to an equilibrated Superdex 200(26/60) column and Fab 'was separated from the non-reduced F (ab') 2 and DTT using PBS +0.65M NaCl +2.5mM EDTA pH7.4 buffer. The buffer and manifold have been separately saturated with argon to prevent oxidation of free SH groups. The fraction containing Fab' was collected directly in a tube saturated with argon.

The third step of the synthesis:

conjugation of Fab '< 425> and Fab' <225> -TNB

Coupling reaction: 32.5ml MAb225 Fab '-TNB, 0.9mg/ml, 31.6mg +23.5ml MAb425 Fab' 1.5mg/ml, 34.8 mg. The Fab 'and Fab' -TNB antibodies were combined and the volume was reduced to approximately 5ml (using argon) in an Amicon chamber containing YM 10 membrane. The reaction mixture was blanketed with argon and stirring was continued at 4 ℃ overnight. The conjugate was purified by passing through a Superdex 200(26/60) column, both the buffer and the column being saturated with helium. Bispecific F (ab') 2 was recovered, (peak 1). Peak 1: purified bispecific antibody (166-: the Fab' remained.

To verify sample identity and purity, samples were added to a non-reducing 10% SDS-Page gel. The yield of purified BAb <425, 225> F (ab') 2 in this representative example was 11mg (16.7%).

Claims (10)

1. A bispecific antibody or fragment thereof capable of binding to an EGF receptor, said antibody or fragment thereof comprising a first antigen-binding site that binds to a first epitope of said EGF receptor and a second, different antigen-binding site that binds to a second epitope of said EGF receptor, wherein said first antigen-binding site is derived from a humanized, chimeric or murine MAb425, said second antigen-binding site is derived from a humanized, chimeric or murine MAb225, and said first and second antigen-binding sites both bind to different epitopes on the same EGF receptor molecule.

2. The bispecific antibody according to claim 1, wherein the different epitopes are located within the binding domain of a natural ligand of the EGF receptor.

3. The bispecific antibody according to claim 1, wherein at least one of said epitopes is located within the binding domain of a natural ligand of the EGF receptor.

4. The bispecific antibody according to claim 1, wherein the first or second antigen binding site binds to an epitope within the binding domain of a natural ligand of the EGF receptor molecule.

5. The bispecific antibody according to claim 1, wherein the first and second antigen binding sites bind to an epitope within the binding domain of a natural ligand of the EGF receptor molecule.

6. A bispecific antibody fragment derived from the bispecific antibody of claim 1, wherein the fragment is F (ab') 2.

7. An immunoconjugate comprising a bispecific antibody or fragment thereof according to claim 1 fused at the C-terminus to a cytokine, either directly or via a linker molecule.

8. A pharmaceutical composition for the treatment of cancer comprising the bispecific antibody of claim 1, and optionally a pharmaceutically acceptable carrier, diluent or excipient.

9. The pharmaceutical composition of claim 8, further comprising a chemotherapeutic agent or cytokine.

10. Use of the bispecific antibody of claim 1 or the pharmaceutical composition of claim 8 for the manufacture of a medicament for the treatment of EGF receptor-associated tumors and tumor metastases.