MXPA99000376A - Adhesives heteromultimeras quimeri - Google Patents

Abstract

Chimeric heteromultimeric adhesives that bind to the ligand of natural heteromultimeric receptors and uses thereof are presented. The chimeric molecules of the heteromultimeric adhesins comprise an extracellular region of a heteromultimeric receptor monomer and a multimerization region for the stable interaction of the chimeric molecules in the adhesin. Specifically heteromultimeric adhesins comprising the extracellular regions of ErbB2 and ErbB3óErbB2 and ErbB4 were presented. The chimeric ErbB heteromultimer adhesins of the present invention are used as competitive antagonist antagonists of a neurogulin for the treatment of diseases such as various cancer

Description

ADHESINAS HETEROMULTIMERAS QUIMÉRICAS This application is generally concerned with chimeric multicomferential adhesins comprising extracellular binding regions of heteromultimeric receptors, the heteromultimer adhesins that bind to the ligand of the natural receptor. The invention additionally concerns antibodies of heteroadhesins, methods of making adhesins and methods of using heteroadhesins and antibodies.

Background of the Invention The transduction of indicators that regulate cell growth and differentiation is regulated in part by phosphorylation of several proteins. Proteins tyrosine kinases are enzymes that catalyze this process. The protein tyrosine kinase receptor is reliable for directing cell growth via ligand-stimulated tyrosine phosphorylation of intracellular substrates.

The single-space ErbB family, the tyrosine kinase receptor consists of four members: epidermal growth factor receptor (EGFR), ErbB2 (HER2 / neu), ErbB (HER3) and ErbB4 (HER4). A number of ligands, all REF.29127 which are a product of a different gene, have been identified to bind and activate EGFR. (reviewed in Groenen et al., 1994). In contrast, a single neurogulin gene codes for a large number of isoform proteins that result from the alternative binding of mRNA transcripts (examined in (Lemc, G. (1996) Mol Cell.

Neurosci. 2; 247-262). ErB3 (Carra ay, K. L. et al. (1994) J. Biól. Chem. 269: 14303-14306) or ErbB4 (Plowman, G.

D. et al. (1993) Nature 366: 473-475) can serve as receptors for neurogulins. These receptors and ligands play key roles in normal cell growth and differentiation.

The protein tyrosine kinase receptor growth factor of the subfamily of class 1 includes the epidermal growth factor receptor 170 kDa (EGFR) encoded by the 'er gene > Bl. The er_Bl has been causally implicated in human malignancy. In particular, increased expression of this gene has been observed in more aggressive carcinomas of the breast, bladder, lung and stomach (Modjtahedi, H. and Dean, C. C. (1994) Int. J. Oncol 4: 277-296).

The second member of the class I subfamily, pl85ne_, was originally identified as the product of the transforming gene of neuroblastomas from chemically treated rats. The neu gene (also called er¿> B2 and HER2) which codes for the 185 kDa protein tyrosine kinase receptor. The amplification and / or overexpression of the human HER2 gene correlates with a poor prognosis in breast and ovarian cancers (Slamon, DJ et al, Science 235: 177-182 (1987); and Slamon et al., Science 244: 707 -712 (1989)). Over-expression of HER2 has been correlated with other carcinomas that include carcinomas of the stomach, endometrium, salivary glands, lung, kidney, colon and bladder. Accordingly, Slamon et al. In US Patent No. 4,968,603 discloses and claims several diagnostic assays for determining amplification or expression of the HER2 gene in tumor cells. Slamon et al. Have found that the presence of copies of the multiple oncogene HER2 gene in tumor cells indicates that the disease is more similarly extended to sites farther from the primary tumor, and that the disease may therefore require more aggressive treatment than could be expected. another way be indicated by other diagnostic factors. Slamon and colleagues concluded that the HER2 gene amplification test, together with the determination of the condition of the lymph node, provides mostly improved utility prognoses.

A related additional gene, called er £ > B3 or HER3 has also been described. See US Patent No. 5,183,884; Kraus et al., Proc. Nati Acad. Sci. USA 86: 9193-9197 (1989); Patent Application EP No. 444,961A1; and Kraus et al., Proc. Nati Acad. Sci. USA 90_: 2900-2904 (1993). Kraus et al. (1989) has found that markedly elevated levels of er £ > B3 nRNA was present in certain mammary tumor cell lines indicating that er £ > B3, similar to erBl and erbB2, may play a role in human malignancy. Also, Kraus and collaborators (1993) have shown that EGF-dependent activation of the ErbB3 catalytic region of a chimeric EGFR / ErbB3 receptor resulted in a proliferative response in cells NIH-3T3 transfected. In addition, these researchers have shown that some human mammary tumor cell lines display a significant elevation in the stable status of tyrosine phosphorylation in ErbB3, in addition to indicating that this receptor may play a role in human malignancy. The role of ér £ > B3 in cancer has been explored by others. It has been found to be over-expressed in breast (Lemoine et al., Br. J. Cancer 66: 1116-1121 (1992)), gastrintestinal (Poller et al., J. Pathol. 168: 275-280 (1992), Rajkumer. and collaborators, J. Pathol, 170: 271-278 (1993), and Sanidas et al., Int. J. Cancer 54: 935-940 (1993)), and pancreatic cancer (Lemoine et al., J. Pathol. 269-273 (1992), and Fries et al., Clinical Cancer Research 1: 1413-1420 (1995)).

ErbB3 is the only one among the receptors of the ErbB family that has little or no intrinsic tyrosine kinase activity (Guy et al, Proc Nati Acad Sci USA 93: 8132-8136 (1994) and Kim et al J. Biol. Chem. 269: 24747-55 (1994)). When ErbB3 is co-expressed with ErbB2, an active indicator complex is formed and antibodies directed against ErbB2 are capable of disrupting this complex ((Sliwkowsky et al., J. Biol. Chem. 269 (20): 14661-14665 (1994 ).) Additionally, the affinity of ErbB3 for her'egulin (HRG) is increased to a higher affinity condition when co-expressed with ErbB2 See also, Levi et al., Journal of Neuroscience 15: 1329-1340 ( 1995), Morrisey et al., Proc. Nati, Acad. Sci. USA 92: 1431-1435 (1995), Lewis, GD et al., Cancer Res., 5_6: 1457-1465 (1996); Pinkas-Kra arski, R and collaborators (1996) EMBO J. 15: 2452-2467; Beerli, R. et al. (1995) Mol. Cell. Biol. 15: 6496-6505; and Karunagaran, D. et al. (1996) EMBO J. 35 : 254-264 with respect to the ErbB2-ErbB3 protein complex in vivo.

The class I subfamily protein tyrosine kinase growth factor receptor has been further extended to include the HER4 / Erb4 receptor.

See EP Patent Application No. 599,274; Plow an et al., Proc. Nati Acad. Sci. USA 90: 1746-1750 (1993); and Plowman et al., Nature 366: 473-475 (1993).

Plowman and colleagues found that increased expression of HER4 correlates closely with certain carcinomas of epithelial origin, including breast adenocarcinomas. Diagnostic methods for detecting human neoplastic conditions (especially breast cancers) that evaluate the expression of HER4 are described in EP Patent Application No. 599,274.

The search for the HER2 oncogene activator is important in the discovery of a family of heregulin polypeptides. These proteins appear to result from the alternating binding of a single gene that was projected for the short arm of human chromosome 8 by Lee, J. and Wood, W. I. (1993) Genomics 16: 790-791).

Holmes et al. Isolated and cloned a family of activating polypeptides for the HER2 receptor that they called heregulin-a (HRG-a), heregulin-βl (HRG-ßl), heregulin-β2 (HRG-ß2), heregulin ß2 bis (HRG- ß2-similar) (HRG-ß2-similar), and heregulin-ß3 (HRG-ß3). See Holmes, W. E. et al., Science 256: 1205-1210 (1992); WO 92/20798; and US Patent 5,367,060. The 45 kDa polypeptide, HRG-a, was purified from the conditioned medium of the human breast cancer MDA-MB-231 cell line. These investigators have demonstrated the ability of the purified heregulin polypeptides to activate the tyrosine phosphorylation of the HER2 receptor in MCF breast tumor cells. further, the mitogenic activity of the heregulin polypeptides on SK-BR-3 cells (which express high levels of the HER2 receptor) was illustrated. Other similar growth factors belonging to the EGF family, soluble HRG polypeptides, appear to be derived from a membrane-bond precursor (called pro-HRG) that is proteolytically processed to release the 45 kDa soluble form. These pro-HRGs lack an N-final indicator peptide.

While the heregulins are substantially identical in the first 213 amino acid residues, they are classified into two major types, a and ß, based on two variants of the EGF-like regions that differ in their C-end portions. However, these EGF-like regions are identical in the range of six cysteine residues contained inside, based on a comparison of amino acid sequences, Holmes et al. Found that between the first and the sixth cysteines in the EGF-si region , HRGs were 45% similar for heparin-like growth factor EGF-like (HB-EGF), 35% identical to anfiregulin (AR), 32% identical to TGF-a, and 27% identical to EGF.

The differentiation factor 44 Da neur which is the equivalent rat of human HRG, was first described by Peles et al., Cell, _69: 205-216 (1992); and Wen et al., Cell, 69: 559-572 (1992). Like the HRG polypeptides, NDF has an Immunoglobulin (Ig) with region of homology followed by an EGF-like region and lack an N-final reporter peptide. Subsequently, Wen et al., Mol. Cell. Biol., 14 (3): 1909-1919 carried out "exhaustive cloning" to extend the family of NDFs. This work revealed six different fibroplastic pro-NDFs. Adopting the nomenclature of Holmes et al., NDFs are classified as either a or b polypeptides based on the sequences of the EGF-like regions. Isoforms 1 to 4 are characterized on the basis of the elastic variable membrane (between the EGF-like region and the transmembrane region). Also, isoforms a, b and c are described as having long, variable cytoplasmic regions. These researchers concluded that the different NDF isoforms are generated by alternative bonds and different tissue-specific performances. See also EP 505 148; WO 93/22424; and WO 94/28133 concerning NDF.

While the heregulin polypeptides were first identified on the basis of their ability to activate the HER2 receptor (see Holmes et al., Supra), it was discovered that certain neutropen transplants expressing neurofibroblasts and ovary cells did not bind or link to the NDF, nor responded to NDF by being subjected to tyrosine phosphorylation (Peles et al., EMBO J. 12: 961-971 (1993)). This indicated that other cellular components were necessary to confer total sensitivity to heregulin. Carraway et al. Subsequently demonstrated that? 2sI-rHRGli77-24 binds to the NIH-373 fibroblast stably transfected with bovine erbB3 but not to non-transfected parental cells. Consequently, they concluded that ErbB3 is a receptor for HGR and mediator in the phosphorylation of intrinsic tyrosine residues as well as the phosphorylation of the ErbB2 receptor in cells expressing both receptors. Carraway et al., J. Biól. Chem. 269 (19): 14303-14306 (1994). sliwkovski et al., J. Biol. chem. 269 (20): 14661-14665 (1994) found that cells transfected with HER3 only show low affinities for heregulin, while cells transfected with both HER2 and HER3 show higher affinities.

This observation correlates with the "receptor-interferent" previously described by Kokai et al., Cell 58: 287-292 (1989); Stern et al., EMBO J. 7: 995-1001 (1988). These investigators found that the EGF binding to EGFR resulted from the activation of the EGFR kinase region and cross-phosphorylation of P185HER2. This is believed to be a result of receptor heterodimerization induced by the ligand and concomitant cross-phosphorylation of the receptors without the heterodimer (Wada et al., Cell 61: 1339-1347 (1990)).

Plowman and his colleagues have similarly studied the activation of P180HER / P185HER2. They expressed P185HER2 alone, P180HEP4 alone, or the two receptors together on human T lymphocytes and demonstrated that heregulin is capable of stimulating tyrosine phosphorylation of P180HER4, but could only stimulate the phosphorylation of P185HER2 in cells expressing both receptors. Plowman et al., Nature 336: 473-475 (1993). Thus heregulin is an example of a member of the EGF family of growth factors that can interact with several receptors (Carraway and Cantley, Cell 7_8: 5-8 (1994).) Additionally, the β-cellulin ligand has been demonstrated which binds to EGF and HER4 receptors, but does not link to HER3 (Riese II, DJ et al. (1996) Oncogene 12: 345-353).

The biological role of heregulin has been investigated by several groups. For example, Falls et al. (Cell 72: 801-815 (1993)) found that ARIA plays a role in myotube differentiation, nominally affecting the synthesis and concentration of neurotransmitter receptors in post-synaptic muscle cells of motor neurons. Corfas and Fischbach showed that ARIA also increases the number of sodium channels in chicken muscle. Corfas and Fischback, J. Neuroscience, 13 (5): 2118-2125 (1993). It has also been shown that GGFII is itogenic for immobile subconfluent human myoblasts and that differentiation of human cloned myoblasts in the continuous presence of GGFII resulted in increased amounts of myotubes after six days of differentiation- (Sklar et al., J. Cell. Biochem., Abst. W462, 18D, 540 (1994)). See also WO 94/26298 published November 24, 1994.

Holmes et al., Supra, found that HRG exerted a mitogenic effect on mammary cell lines (such as SK-BR-3 and MCF-7). The mitogenic activity of GGFs on Schwann cells has also been reported. See, for example, Brockes et al., J. Biol. Chem. 255 (18): 8374-8377 (1980); Lemke and Brockes, J. Neurosci 4: 75-83 (1984); Brockes et al J. Neuroscience _4 (l): 75-83 (1984); Brockes et al., Ann. Neurol. 20 (3): 317-322 (1986); Brockes, J., Methods in Enzim. , 147: 217-225 (1987) and Marchioni et al., Supra. Schwann cells provide myelin sheathing around axons of neurons, thereby forming individual nerve fibers. In this way it is obvious that Schwann cells play an important role in the development, function and regeneration of peripheral nerves- The implications of this from a therapeutic point of view have been directed by Levi et al., J. Neuroscience 14 (3) : 1309-1319 (1994). Levi and colleagues discuss the potential for building a cell prosthesis that includes human Scwann cells that could be transplanted into damaged areas of the spinal cord. Methods for culturing ex vivo Scwann cells have been described. See WO 94/00140 and Li et al., J. Neuroscience 16 (6): 2012-2019 (1996).

Pinkas-Kramarski and colleagues found that NDF appear expressed in adult rat brain and embryonic cells and neurons and primary cultures of rat brain cells, and suggest that it may act as a maturation and survival factor for astrocytes (Pinkas- Kamrski et al., PNAS, USA 91: 9387-9391 (1994)). Meyer and Birchmeier, PNAS, USA 91: 1064-1068 (1994) analyzed the expression of heregulin during embryogenesis of mouse and perinatal animal using in situ hybridization and RNase protection experiments. These authors concluded that, based on the expression of this molecule, heregulin plays an in vivo role as an esenchymal and neuronal factor. In this way, its finding implies that heregulin functions in the development of the epithelium. Similarly, Danilenko et al., Abstract 3101, FASEB 8 (4-5): A535 (1994), found that the interaction of NDF and the HER2 receptor is important in the direction of migration and epidermal differentiation during wound healing.

Interactions of members of the ErbB family have been investigated in vitro and in vivo. The transactivation of ErbB2 as a result of the interaction of the ligand with other members of the ErbB family is a common and physiologically important event (Dougall, WC et al., (1993) J. Cell. Biochem. 53: 61-73; Earp, HS et al., (1995) Breast Cancer Res. Treatment 35: 115-132). The co-expression of ErbB2 with ErB3 leads to the formation of a high affinity binding site in heregulin (HRG) (Sliwkowski, M. X. et al., (1994) J. Biol. Chem. 269: 14661-14665). ErbB2 modulates the affinity of ErbB3 for HRG and appears to provide tyrosine kinase activity to the ErbB3-HRG complex, since ErbB3 is a dysfunctional indicator receptor that lacks intrinsic tyrosine kinase activity (Guy, P. M. et al., (1994) PNAS USA 93.: 8132-8136). Physico-chemical studies have not shown an association of ErbB2 and ErbB3 ECDs in vitro (Horan et al., J. Biol. Chem. 270: 24604-24608 (1995)). In addition, the binding of neu differentiation factor (NDF) to soluble HER3 was not enhanced by the presence of soluble HER2.

Brief description of the invention The invention concerns the unexpected discovery of soluble chimeric heteromultimers comprising extracellular regions of the monomers of a heteromultimeric receptor that bind to the ligand. receiver . The invention also concerns methods for making chimeric heteromultimers, methods for using them as receptor ligand antagonists, chimeric heteromultimeric antibodies that function as receptor ligand antagonists or agonists, and methods of treating disease conditions related to the ligand-receptor interaction. .

In one aspect the invention includes a chimeric heteromultimer comprising a first amino acid sequence, the sequence of which forms a chimeric monomer and comprises an extracellular region (ECD) or ligand binding fragment thereof, or a first monomer of a natural heteromultheric receptor and a multimerization region, characterized in that the ECD is fused to the multimerization region. The chimeric heteroadhesin of the invention further comprises an additional amino acid sequence forming an additional chimeric monomer comprising an extracellular region, an additional monomer of the natural heteromultimeric receptor and a multimerization region. According to this aspect of the invention, the extracellular regions of the first and the additional monomers of the natural heteromultimeric receptor are associated in a cell to form a natural heteromultimeric receptor which is activated by the binding of a ligand, and is characterized in that the adhesin Soluble chimeric heteromultimer has 1/10 x up to 1/106 times affinity for the ligand in relation to a monomer of the natural receptor or a homomultimer of the natural receptor. In a preferred embodiment of the invention, the chimeric heteromultimer adhesin is a water-soluble adhesin.

In one embodiment of the invention, the chimeric heteromultimer adhesin is a ligand antagonist that binds to the extracellular region of the natural heteromultimeric receptor.

In another embodiment of the invention, the multimerization region of the first amino acid sequence is capable of interacting with the multimerization region of each of the additional amino acid sequences to form a heteromultimer.

In yet another embodiment of the invention, the chimeric heteromultimer adhesin comprises a multimerization region that includes an immunoglobulin region, preferably a constant region immunoglobulin, such as those of IgG1, IgG2, IgG3, IgG4, IgM, and IgF.

In yet another embodiment of the invention, the chimeric heteroadhesin includes a multimerization region capable of forming a stable protein-protein interaction. Such protein-protein interaction regions (or multimerization regions) include a closing leucine, an amino acid sequence comprising a complementary protuberance in an amino acid sequence comprising a space, a hydrophobic region, a hydrophilic region, and a sequence of amino acid comprising a free thiol portion capable of reacting to form an intermolecular disulfide bond with a multimerization region of an additional amino acid sequence.

A further embodiment of the invention is a chimeric heteromultimer adhesin in which the first amino acid sequence comprises an extracellular region of the ErbB2 receptor monomer, an additional amino acid sequence comprising an extracellular region of the ErbB3 monomer receptor, and a multimerization region of the first and additional amino acids each comprising a constant region immunoglobulin. The multimerization region is provided for the formation of a stable protein-protein interaction between the first and the additional amino acid sequences. A preferred ligand for this chimeric heteroadhesin is the ligand, heregulin.

A further embodiment of the invention is a chimeric heteromultimer adhesin in which the first amino acid sequence comprises an extracellular region of the receptor monomer ErbB2, an additional amino acid sequence comprising an extracellular region of the receptor monomer ErbB4, and a multimerization region of the former and additional amino acids each comprising a constant region immunoglobulin. The multimerization region is provided for the formation of a stable protein-protein interaction between the first and the additional amino acid sequences. A preferred ligand for this chimeric heteroadhesin is the ligand, heregulin.

In another aspect, the invention includes an isolated nucleic acid sequence encoding the chimeric heteromultimer adhesin of the invention.

In another embodiment, the invention provides an isolated nucleic acid molecule encoding the chimeric amino acid sequence of a heteromultimetric adhesin monomer such as, for example, ErbB2-IgG, ErbB3-IgG, or ErbB4-IgG. For example, the nucleic acid molecule can be selected from a group consisting of: (a) a nucleic acid comprising the nucleotide sequence of the extracellular region (eg ligand binding region or ligand binding fragment thereof) of a monomer of a natural heteromultimeric receptor covalently linked in phase and in the direction of transcription of a nucleic acid sequence encoding a multimerization region, such as a constant region immunoglobulin; and (b) a nucleic acid corresponding to the sequence of (a) in the scope of the degeneracy of the genetic code. The optionally isolated nucleic acid molecule and additionally comprises a promoter operably linked thereto.

The isolated nucleic acid can also be used for gene therapy in vivo or ex vivo. This embodiment of the invention encompasses the administration of the nucleic acid of the invention, a vector comprising the nucleic acid, or a cell comprising the nucleic acid of a mammal such that the chimeric chimeric adhesin is expressed in the mammal and acts as an antagonist of your ligand. For example, ErbB2 / 3-IgG expressed in a mammal is used to reduce the local concentration of heregulin near an ErbB2 / 3 receptor and inhibit the growth of a cell that has the receptor on its surface. Preferably the expressed ErbB2 / 3-IgG is used to treat a proliferative cell disease, such as a cancer, in which the binding antagonist heregulin of its receptor inhibits cell growth.

In one embodiment of the invention, the nucleic acid sequence isolated from the chimeric amino acid encoding an extracellular region or linker thereof fragment of the ErbB2 receptor, and in the multimerization region comprising a constant region immunoglobulin.

In yet another embodiment of the invention, the nucleic acid sequence isolated from the chimeric amino acid sequence encodes an extracellular region or linker fragment of the ECB receptor of ErbB3, and in the multimerization region comprises a constant region immunoglobulin.

In another embodiment of the invention, the nucleic acid sequence isolated from the chimeric amino acid sequence encodes an extracellular region or links a fragment thereof to the ECD of the ErbB4 receptor, and characterizes the multimerization region because it comprises a constant region immunoglobulin.

Another embodiment of the invention includes a promoter operably linked to the nucleic acid molecule.

In yet another embodiment, the invention includes a vector comprising the isolated nucleic acid of the invention. For example, the invention provides a vector comprising the nucleic acid molecule (eg, a vector expression comprising the nucleic acid molecule operably linked to the control sequences recognized by a host cell transformed with the vector); a host cell comprising the nucleic acid molecule; and a method of using a nucleic acid molecule encoding a chimeric heteromultimer adhesin, such as an ErbB-IgG, for adhesin production purposes comprising the step of culturing the host cell and recovering the adhesin from the cell culture. In a related embodiment the method of using the nucleic acid for the production effect of the adhesin includes multiple introduction of nucleic acid sequences encoding different chimeric adhesins and expressing a mixture of chimeric adhesins. For example, a nucleic acid encoding ErbB2-IgG and a nucleic acid encoding ErbB3-IgG are introduced into a host cell, expressed, and a mixture of the homodimers and heterodimers is isolated from the cell or culture medium.

One embodiment of the invention further includes a host cell comprising the nucleic acid of the invention. Preferably the host cell is capable of expressing the nucleic acid, which expression includes the translation and production of the chimeric heteroadhesin of the invention. The embodiment of the invention encompasses a host cell comprising and expressing a chimeric monomer of heteroadhesin, while in another host cell of the invention an additional chimeric monomer of heteroadhesin is expressed. Alternatively, the modality encompasses the expression of more than one chimeric monomer in a single host cell.

In a preferred embodiment of the invention, the host cell comprises a first isolated nucleic acid sequence, coding for the first amino acid sequence of the soluble chimeric heteromultimer of the invention, characterized in that the extracellular region is of the ErbB2 receptor and in that region of multimerization comprises a constant region immunoglobulin; and a second isolated nucleic acid sequence encoding an additional amino acid sequence of the soluble chimeric heteromultimer of the invention, characterized in that the extracellular region is of the ErbB3 receptor and that the multimerization region comprises a constant region immunoglobulin.

In another preferred embodiment of the invention, the host cell comprises a first isolated nucleic acid sequence encoding the first amino acid sequence of the soluble chimeric heteromultimer of the invention, which is characterized in that the extracellular region is of the ErbB2 receptor and in that region of multimerization comprises a constant region immunoglobulin; and a second isolated nucleic acid sequence coding for an extracellular region is of the ErbB4 receptor and is characterized in that the multimerization region comprises a constant region immunoglobulin.

Another aspect of the invention includes a chimeric heteromultimeric adhesin antagonist antagonist antibody of the invention, which is characterized in that the antibody binds to the natural heteromultimeric receptor and inhibits its activation Another aspect of the invention includes a chimeric heteromultimeric adhesin agonist antibody of the invention, in which the antibody binds the natural heteromultimer receptor and activates it. In preferred embodiments of the invention, the agonist antibody is capable of activating the natural heteromultimeric receptor from 1/10 x up to 106 times the activity of the natural ligand.

The chimeric specific-arm antibodies can be used, inter alia, in a method for detecting heteromultimeric receptors, comprising the step of contacting a sample suspected of containing the heteromultimer receptor with the antibody (which is optionally labeled) and detecting whether it takes place the link. The antibody can also be used in a method for purifying the heteromultimer receptor, comprising the step of passing a mixture containing the heteromultimeric receptor onto a solid phase in which the antibody is bound and the fraction containing the heteromultimeric receptor recovered. Preferably, in one embodiment of the invention, the heteromultimeric receptor is ErbBB2 / ErbB4 and the chimeric heteromultimeric adhesin is ErbB2-IgG / ErbB4-IgG. In another preferred embodiment, the heteromultimeric receptor is ErbB2 / ErbB3 and the chimeric heteromultimer adhesin is ErbB2-IgG / ErbB3-IgG.

In yet another aspect, the invention includes a method of forming a chimeric heteromultimeric ligand-adhesin complex in a sample comprising the ligand. The method of the invention includes contacting the chimeric heteromultimer adhesin of the invention with the sample under conditions such that the ligand binds the heteromultimer to form a chimeric heterodimeric ligand-adhesin complex.

In one embodiment of the invention, the chimeric heteromultimer ligand-adhesin complex inhibits ligand binding to the natural heteromultimer receptor. Preferably the sample is a mammalian tissue or a mammalian fluid, such as a body fluid that includes, but is not limited to blood, serum, plasma, lymph, and urine. Preferably the mammal is a human.

In another aspect, the invention involves a method of inhibiting the activation of the natural heteromultimer receptor. The method includes the steps of 1) contacting chimeric heteromultimeric adhesins of the invention with a sample containing a ligand for the natural heteromultimeric receptor and the receptor, and 2) incubating the chimeric heteromultimeric adhesin with the ligand to form a complex such that it is inhibited activation of the natural heteromultimeric receptor by the ligand.

In one embodiment of the method of inhibiting the binding of the ligand to a natural heteromultimer receptor, the natural heteromultimeric receptor is ErbB and the soluble chimeric heteromultimer comprises the extracellular regions of ErbB2 and ErbB3.

In another embodiment of the method of inhibiting the binding of the ligand to the natural heteromultimer receptor, the natural heteromultimeric receptor is ErbB and the soluble chimeric heteromultimer comprises the extracellular regions of ErbB2 and ErbB4.

Another embodiment of the invention is a method of inhibiting the binding of the ligand to a natural heteromultimer receptor, in which the activation of the receptor is inhibited. The method comprises contacting the antagonist antibody of the invention with the natural heteromultimeric receptor to form an antibody-heteromultimer antagonist receptor complex, in which receptor activation is inhibited.

In another aspect, the invention involves a method of activating the natural heteromultimeric receptor which comprises contacting the agonist antibody of the invention with the natural heteromultimeric receptor to form the antibody-heteromultimeric agonist receptor complex, in which the receptor is activated.

In yet another aspect, the invention involves a method for the treatment of a condition of a disease comprising administering to a mammal in need thereof a therapeutically effective dose of a chimeric heteromultimeric adhesin of the invention. Modalities of the invention encompass conditions of diseases in which the disease is treatable by inhibition by contact between the ligand and the natural heteromultimeric receptor such as by selective binding of the heteroadhesin to the ligand.

In one embodiment of the invention, the chimeric heteromultimer adhesin is an ErbB2 / ErbB4-Ig heteroadhesin. In another embodiment, the chimeric heteromultimer is an ErbB2 / ErbB4-Ig heteroadhesin.

The invention encompasses a composition comprising the chimeric heteromultimer adhesin. The composition comprising the adhesin is preferably sterile. When the composition is an aqueous solution, preferably the adhesin is soluble. When the composition is a lyophilized powder, preferably the powder is reconstituted in an appropriate solvent.

In another embodiment of the invention, the method of treatment comprises the administration of chimeric heteromultimer adhesins comprising chimeric monomers, each prepared using an extracellular region of the heteromultimeric receptor monomers of interest. The extracellular regions are preferably from receptors selected from the following heteromultimeric receptors: Axl, Rse, epidermal growth factor receptor (EGF), hepatocyte growth factor receptor (HGF), IL-2, c-mer, Al-1, EPH, TrkA, TrkB, TrkC, TNF, IL-10, CRF-4, RXR, RON, ACHRa / d, TRa / RXRa, Tra / DR4, Tra / MHC-TRE, Tra / ME, Tra / F2, KDR / FLT-1, FLT / VEGF, VEGF121 / 165, Arnt / Ahr, CGA / CGB, EGFR / pl85-neu, prolactin receptor (PRL), T cell receptor (TCR), fibroblast growth factor (FGF), and Cak receptor ((Kishimoto, T , et al. (1994) Cell 76: 253-262; Kendall, RL et al. (1996) Biochem. Biophys., Res. Comm. 226: 324-328; Chang, WP and Clavenger, CV (1996) PNAS USA 93: 5947 -5952; Lala, DS et al. (1996) Nature 383: 450-453;; Collesi, C. et al. (1996) Mol Cell. Biol. 3A: 5518-5526; Tzahar, E et al. (1996) Mol. Cell Biol. 16: 5276-5287; Shtrom, S. S. and Hall, Z. W. (1996) J. Biol. Chem. 271: 25506-25514; Nagaya, T. and collaborators (1996) Biochem. Biophys. Res. Comm. 226: 426-430; Dendall, R. L. et al. (1996) Biochem Biophys. Res. Comm. 226: 324-328; Kainu, T et al (1995) Neuroreport 6: 2557-2560; Yoo, S. H. and Lewis, M.S. (1996) J. Biol. Chem. 271: 17041-17046; Murali, R. and collaborators (1996) PNAS USA 93: 6252-6257; Dietrich J. et al. (1996) Oncogene 1_2: 1469-1477). The extracellular regions are more preferably of receptors selected from the following: IL-6 / gpl30, IL-ll / gpl30 leukemia inhibitory factor (LIF) / gpl30, cardiotrophin-l / gpl30 (CT-1), IL-ll / gpl30, ciliary neurotropic factor (CNTF / gpl30, oncostatin M (SM) / gpl30, inferred ?, and inferred, ß (Kishimoto, T. and collaborators (1994), supra, Taga, T. (1996) J. Neurochem. ^ 57: 1-10; Pennica, D. et al. (1995) J. Biól. Chem. 270: 10915-10922; Marsters, SA (1995) PNAS USA 92: 5401-5405; and Wollert, KC et al. (1996) J. Biol. Chem. 271: 9535-9545). More preferably, the extracellular regions are selected from the receptors of the ErbB family.

Modalities of the treatment method encompass a condition or conditions of diseases such as immunological disorders, cancer, and neurological disorders.

In embodiments wherein the heteroadhesin is an ErbB2 / ErbB3-Ig heteroadhesin or an ErbB4-Ig, the treatment method encompasses a disease condition selected from the group consisting of inflammatory diseases, cancer, neurological disorders such as neurofibromatosis and peripheral neuropathy, and disorders. cardiac conditions such as cardiac hypertrophy.

The invention further provides a method for treating a mammal, which comprises administering a therapeutically effective amount of a chimeric heteromultimeric adhesin, such as ErbB2 / 3-IgG or ErbB2 / 4-IgG to the mammal. For example, the mammal may be suffering from a neurological disorder or proliferative cell disease. The mammal is one that could benefit from a reduction in the levels of HGR / biological activity (for example cancer).

In another aspect, the invention includes pharmaceutical compositions. In another embodiment of the invention, the pharmaceutical compositions comprise a chimeric heteromultimer adhesin of the invention, which heteroadhesins 1) comprises an ECD or binding fragment thereof of a natural heteromultimer receptor, and 2) is a ligand antagonist that binds the receptor ECD heteromultimer natural.

In another embodiment of the invention, the pharmaceutical composition comprises an antibody of a chimeric heteromultimeric adhesin of the invention, which anti-heteroadhesin antibody 1) comprises an ECD or binding fragment thereof of a natural heteromultimer receptor, and 2) binds to the receptor ECD natural heteromultimeric and is a ligand antagonist that binds the ECD of the natural heteromultimeric receptor.

In yet another embodiment of the invention the pharmaceutical composition comprises an antibody of a chimeric heteromultimeric adhesin of the invention, which anti-heteroadhesin antibody 1) comprises ECD and / or binding fragment thereof of a natural heteromultimer receptor, and 2) binds to the ECD of the natural heteromultimeric receptor and is a ligand agonist that binds the ECD of the natural heteromultimeric receptor.

In yet another aspect, the invention includes articles of manufacture comprising a container, a mark on the container, and a composition contained in the container. In one embodiment of the invention, the composition comprises the chimeric heteromultimeric adhesin composition of the invention, which heteroadhesin is a ligand antagonist. The composition is effective to antagonize ligand bonds of its natural heteromultimer receptor, and the label on the container indicates that the composition can be used to stabilize ligand bonds at the natural heteromultimer receptor. In a preferred embodiment the chimeric heteromultimer adhesin is selected from the group consisting of ErbB2 / ErbB3-Ig or ErbB2 / ErbB4-Ig.

In another embodiment of the article of manufacture, the composition comprises a heteromultimer anti-chimeric adhesin antibody, which antibody is a ligand antagonist. The composition is effective to antagonize ligand linkages at its natural heteromultimeric receptor, and the label on the container indicates that the composition can be used to antagonize ligand bonds at the natural heteromultimeric receptor. In a preferred embodiment the heteromultimer anti-chimeric adhesin antibody is an antibody produced from a chimeric heteroadhesin selected from the group consisting of ErbB2 / ErbB3-Ig or ErbB2 / ErbB4-Ig.

In yet another embodiment of the article of manufacture, the composition comprises a heteromultimer anti-chimeric adhesin antibody, which antibody is an agonist of. a ligand The composition is effective to activate the natural heteromultimeric receptor of the ligand, and the label in the container indicates that the composition can be used to activate the natural heteromultimeric receptor. In a preferred embodiment the anti-chimeric heteromultimer adhesin antibody is an antibody produced from a chimeric heteroadhesin selected from the group consisting of ErbB2 / ErbB3-Ig or ErbB2 / ErbB4-Ig.

These and other aspects of the invention will be apparent to those skilled in the art in consideration of the following detailed description.

Brief description of the Drawings.

Fig. 1 is a diagram of the ErbB family of chimeric homomers and heterodimers. The extracellular regions (ECD) and immunoglobulin region (Fe) of the chimeras are indicated. The extracellular regions are derived from the natural heteromultimeric receptor and are fused by recombinant means to a multimerization region, the immunoglobulin region.

Fig. 2 is a graphical diagram showing the binding analysis of the chimeric immunoadhesin. The ErbB3-IgG and ErbB4-IgG homodimeric were able to specifically bind 125I-HRG, while non-discernible bonds were detected with the ErbB2-IgG construct.

Fig. 3A-3D are graphical results of 25l-heregulin binding studies for each of the chimeric immunoadhesins ErbB2 / 3-IgG, ErbB2 / 4-IgG and ErbB3 / 4-IgG. As shown in Figure 3A, a high affinity to the binding site HRG could be detected with the heterodimer containing ErbB2 but not with the ErbB3 / 4-IgG.

Fig. 4A and 4B are graphical results of binding studies of monoclonal anti-ErbB2 antibodies (2C4), in which the binding activity of chimeric ErbB homodimers is compared to that of chimeric ErbB heterodimers in the presence of 2C4.

Fig. 5 is a bar graph indicating the ability of ErbB-IgG proteins to inhibit HRG-dependent thymidine incorporation in the breast carcinoma cell line, MCF7. By varying the concentrations of the different ErbB-IgG proteins they were incubated with 1 nM rHRG and added in cultures of MCF7 cells in a serum-deprived layer. Cells were labeled with 3H-thymidine to measure DNA synthesis. Receptor fusions capable of HRG bonds inhibited the mitogenic HRG-mediator response in one dose in the manner discussed. The heterodimeric IgGs, ErbB3 / 2-IgG and ErbB4 / 2-IgG, were more potent than their corresponding homodimeric protein fusions.

Fig. 6 is a graphic description of possible models for the interaction of ErbB2 with ErbB3 or ErbB4, a "contact" model (left) and a "confirmation" model (right).

Detailed Description of the Invention Prior to the present chimeric heteromultimetric adhesins, methods of making them, and uses of these are described, it is clear that this invention is not limited to the particular adhesins or processes described as well as compounds and methods can, of course, vary. It is also clear that the terminology used is characterized by the purpose of describing particular modalities only, and is not an attempt to limit since the scope of the invention will be limited only by the claims annexes. • I. Of iniciones In general, the following words or phrases will have the indicated definition when they are used in the description, examples and claims.

Unless otherwise indicated, the term "ErbB" when used herein refers to any one or more than 20 mammalian ErbB receptors (eg, ErbBl or epidermal growth factor receptor (EGF); HER2, ErbB3 or HER3 receptors, ErbB4 or HER4 receptors, and any other member (s) of this tyrosine kinase class I family to be identified in the future) and "erbB" refers to the genes erbB of mammals encoding these receptors.

"HRG" ("or heregulin") is defined herein as any polypeptide sequence that possesses at least one biological property (as defined below) of the native HRG sequence ((US Application a.Serial No.: 60 / 021,640) , PR1043, supra.) This definition encompasses not only the polypeptide isolated from a source such as human MDA-MB-175 cells or from another source, such as other animal species, but also the polypeptide prepared by synthetic or recombinant methods. variant forms including functional derivatives, allelomorph variants, naturally occurring isoforms and analogs thereof Sometimes HRG is "native HRG" which refers to endogenous HRG polypeptides that have been isolated from a mammal. "Native HRG sequence" encompassing the polypeptide produced by synthetic or recombinant means "Mature HRG" is soluble or secreted HRG released from the cell (eg sequences lacking amino-end). "HRG isoforms" are naturally occurring polypeptides that comprise at least part of the N-final region of HRG.

The term "immunoadhesin" as used herein refers to antibody-typical molecules that combine the binding region of a protein such as an extracellular region (the adhesin portion) of a receptor cell surface with effector functions of a constant region immunoglobulin. Immunoadhesins may possess many of the valuable chemical and biological properties of human antibodies. Since immunoadhesins can be constructed from a human protein sequence with a desired binding specificity for an appropriate human essential immunoglobulin and constant region (Fe) sequence, the binding specificity of interest can be achieved using fully human components. Such immunoadhesins are minimally immunogenic in the patient, and are safe for repeated or chronic use.

The immunoadhesins reported in the literature include fusions of the T cell receptors (Gascoigne et al., Proc. Nati, Acad. Sci. USA 84: 2936-2940 (1987)); CD4 (Capón et al., Nature 337: 525-531 (1989); Traunecker et al., Nature 339: 68-70 (1989); Zettmeissl et al., DNA Cell Biol. USA 9: 347-353 (1990); and Byrn et al., Nature 344: 667-670 (1990)); L-selectin or guiding receptor (Watson et al., J. Cell.

Biol. 110: 2221-2229 (1990); and Watson et al., Nature 349: 164-167 (1997)); CD44 (Aruffo et al., Cell 61: 1303-1313 (1990)); CD28 and B7 (Linsley et al., J.

Exp. Med. 173: 721-730 (1991)); CTLA-4 (Lisley et al., J. Exp. Med. 174: 561-569 (1991)); CD22 (Stamenkovic et al., Cell 66: 1133-1144 (1991)); TNF receptors (Ashkenazi et al., Proc. Nati, Acad. Sci. USA 88: 10535-10539 (1991); Lesslauer et al.

Eur. J. Immunol. 27: 2883-2886 (1991); and Peppel et al., J. Exp. Med. 174: 1483-1489 (1991)); receivers NP (Bennett et al., J. Biol. Chem. 266: 23060-23067 (1991)); inferred? receptor (Kurschner et al., J. Biol. Chem. 267: 9354-9360 (1992)); 4-1BB (Chalupny et al., PNAS USA 89: 10360-10364 (1992)) and IgE α receptors (Ridgway and Gorman, J. Cell. Biol. 115, abstract No. 1448 (1991)).

Examples of homomultimeric immunoadhesins that have been described for therapeutic use include the CD4-IgG immunoadhesin to block HIV binding on the CD4 cell surface. Data obtained from the phase Y of clinical trials in which CD4-IgG was administered in pregnant women just before giving birth suggest that this immunoadhesin may be useful in the prevention of maternal-fetal transfer of HIV. Ashkenazi and collaborators, Intern. Rev. Inmunol. 10: 219-227 (1993). An immunoadhesin that binds tumor necrosis factor (TNF) has also been developed. "TNF is a proinflammatory cytokine that has been shown to be an important mediator in septic shock, based on a model of septic shock in mice., a TNF receptor immunoadhesin has shown promise as a candidate for clinical use in treatment of septic shock (Ashkenazi, A et al. (1991) PNAS USA 88: 10535-10539). Immunoadhesins also have non-therapeutic uses. For example, the L-selectin receptor immunoadhesin was used as a reagent for histochemical staining of elevated endothelial venules of peripheral lymph nodes. This reagent was also used to isolate and characterize the ligand L-selectin (Ashkenazi et al., Supra).

If the two arms of the immunoadhesin structure have different specificities, the immunoadhesin is termed "bispecific immunoadhesin" by analogy to bispecific antibodies • Dietsch et al., J. Immunol. Methods 162: 123 (1993) - that is to say, a bispecific immunoadhesin that combines the extracellular regions of the adhesion molecules, E-selectin and P-selectin, each of the selectins is expressed in a different type of cells in nature . Linkage studies indicated that the bispecific protein-immunoglobulin fusion thus formed had an improved ability to bind to a myeloid cell line compared to the monospecific immunoadhesin from which it was derived.

The term "heteroadhesin" is used interchangeably with the term "chimeric heteromultimeric adhesin" and refers to the complex of chimeric molecules (amino acid sequences) in which chimeric molecules combine a biologically active portion, such as the extracellular region of each of the heteromultimeric receptor monomers, with a multimerization region. The "multimerization region" promotes the stable interaction of the chimeric molecules in the heteromultimer complex. The multimerization regions may interact via an immunoglobulin sequence, closing leucine, a hydrophobic region, or a free thiol that forms an intermolecular disulfide bond between the chimeric molecules of the chimeric heteromultimer. The multimerization region may comprise a constant region immunoglobulin. A possible multimerization region used in the present invention is found in U.S. to. Do not. 07/440, 625, P565P1 (incorporated herein by reference) in which hybrid immunoglobulins are described. In addition, a multimerization region can be designed so that spherical interactions not only promote stable interaction, but additionally promote the formation of heterodimers on homodimers of a monomer mixture. See, for example, U.S. to. No. 08/399, 106, P0927 (incorporated herein by reference in its entirety)) in which a "protuberance-intra-cavity" strategy for an interface between a first and a second polypeptide is exposed by hetero-oligomerization. "Protuberances" are constructed by replacing small amino acid side chains of the first polypeptide interface with longer side chains (eg, tyrosine or tryptophan). "Compensatory" cavities of similar or identical size of the protuberances are optionally created on the interface of the second polypeptide by replacement of large amino acid side chains with smaller ones (for example alanine or tronin). The immunoglobulin sequence preferably, but not necessarily, is a constant region immunoglobulin. The immunoglobulin portion in the chimeras of the present invention can be obtained from subtypes IgGi, IgG2, IgG3 or IgG4, IgA, IgE, IgD or IgM, but preferably IgGi or IgG3.

The term "tainted epitope" as used herein, refers to a chimeric ppolypeptide comprising the chimeric heteroadhesin, or a fragment thereof, fused to a "tag polypeptide". the tag polypeptide has enough residues to provide an epitope against which an antibody can be made, it is still insufficient to not interfere with the activity of the chimeric heteroadhesin, the tag polypeptide is preferably clearly unique so that the antibody against that does not substantially cross-react with other appropriate tag epitopes. Polypeptides generally have at least 6 amino acid residues and usually between about 8-50 amino acid residues (preferably between about 9-30 residues). One embodiment of the invention encompasses a chimeric heteroadhesin attached to an epitope tag, whose tag is used to detect the adhesin in a sample or recover the adhesin from a sample.

"Isolated chimeric heteromultimeric adhesin", "highly purified chimeric heteromultimer adhesin" and "substantially homogeneous chimeric heteromultimeric adhesin" are used interchangeably "and means adhesin that has been purified from a source or has been prepared by synthetic or recombinant methods and is sufficiently free of other peptides or homogeneity proteins by chromatographic techniques or other purification techniques, such as SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining. Homogeneity here means less than 5% contamination with other proteins. As discussed here (below), the chimeric heterobrassins ErbB2 / 3-IgG or ErbB2 / 4-IgG of the invention bind with sufficiently greater affinity in relation to the homodimers than the use of a mixture of homodimers and heterodimers, is thus considered a useful embodiment of the invention. The terms "chimeric heteromultimeric adhesin", "chimeric heteroadhesin" and "CHA" are used interchangeably herein.

"Biological property", when used in conjunction with "chimeric heteromultimeric adhesin" means an ability to bind a ligand and function as a ligand antagonist to bind to the natural receptor. "Biological property" when used in conjunction with "an antibody of a chimeric heteromultimer adhesin" means an ability to bind the extracellular regions encoded in the adhesin or the extracellular regions of the natural heteromultimeric receptor such that the antibody acts as an antagonist or a ligand agonist.

"Biological activity" used in conjunction with a chimeric heteroadhesin such as the ErbB heteroadhesins includes functioning as an antagonist of the activation of the heregulin receptor (eg antagonist of ErbB2 receptor activation, ErbB3 and / or ErbB4) by binding of the associated cell membrane to the heregulina or associated with the hereguilina; inhibition of growth of cellular expressions of ErbB receptors on its surface; inhibition of the differentiation and / or proliferation of cellular expressions of these receptors (for example SK-BR-3 cells, Schwann cells, hepatocytes, glioblastoma cells, epithelial cells (such as breast, ovarian, prostate, lung , of pancreas, colon and rectum), muscle cells, astrocytes and / or oligodendrocytes); inhibition of binding receptors (for example of the ErbB2 / 3 receptor, ErbB2 / 4, ErbB3 and / or ErbB4); inhibition of mitogenic activity; inhibition of acetylcholine receptor synthesis in the neuromuscular capsule; and inhibition of the formation of a synaptic capsule between a neuron and a muscle, nerve or glandular cell.

"Biological activity" 'used in conjunction with a chimeric agonist heteroadhesin antibody such as an anti-ErbB agonist heteroadhesin antibody includes functioning as an agonist of the heregulin receptor activation (eg activation of the ErbB2, ErbB3 and / or ErbB4 receptor); link and activate the receiver (for example the ErbB2 / 3 receptor, ErbB2 / 4, ErbB3 and / or ErbB4); promote the growth of cellular expressions of ErbBB receptors on its surface; promote the differentiation and / or proliferation of cellular expressions of these receptors (eg SK-BR-3 cells, Schwann cells, hepatocytes, glioblastoma cells, epithelial cells (such as breast, ovarian, prostate, lung, pancreas, colon) and rectum), muscle cells, astrocytes and / or oligodendrocytes); promote mitogenic activity; promote the synthesis of the acetylcholine receptor in the neuromuscular junction; and promote the formation of a synaptic junction between a neuron and a muscle, nerve or glandular cell.

"Percent identity of the amino acid sequence", with respect to the chimeric heteromultimer adhesin is defined herein as the percentage of amino acid residues in the extracellular region of the candidate sequence that are identical with the extracellular region residues of the a monomer of the natural heteromultimeric receptor, after aligning the sequences and introducing voids, if necessary, to achieve the maximum percentage of sequence identity, and not considering any conservative substitution as part of the sequence identity. None of the N-end, C-end, or internal extensions, deletions or insertions in the adhesin sequence will be constructed in order to affect the sequence identity or homology.

The term "disease stage" refers to a physiological stage of a cell or of a complete mammal in which an interruption, cessation, or disorder in the functions in the cellular or bodily systems, or of the organs, takes place.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by irregular cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small cell lung cancer, non-minute cell lung cancer, gastric cancer, pancreatic cancer, cell tumors such as glioblastoma and neurofibromatosis, cervical cancer, cancer of the ovary, cancer in liver, bladder cancer, hepatoma, breast cancer, colon cancer, colon cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma and various types of cancer in the neck and head Where the chimeric heteroadhesin of the invention in an ErbB-Ig heteroadhesin, the cancer treated is preferably cancerous development of cells expressing ErbB receptors, such as cancerous growth in breast, ovarian, prostate, lung, pancreatic and colorectal cells.

The term "inflammatory disorder" refers to a fundamental pathological process consisting of a dynamic complex of cytological and histological reactions that occur in affected blood vessels and adjacent tissues in response to damage or abnormal stimulation caused by a physical, chemical agent or biological, which includes: 1) local reactions and resulting morphological changes, 2) destruction or removal of damaged material, 3) responses that lead to repair and healing. Inflammatory disorders treatable by the invention are those in which inflammation is associated with cytokine-induced disorders, such as those associated with interleukin and with cytokine inhibitory factors of leukemia. Such disorders include abnormalities in thrombopoiesis, development and differentiation of macrophages, proliferation of hematopoietic progenitors, and the like.

The term "neurological disorder" refers to or describes the physiological condition in mammals that is typically characterized by development, differentiation of nerve cells, or indicator cells. Examples of neurological disorders include, but are not limited to, neurofibromatosis and peripheral neuropathy.

The term "cardiac disorder" refers to or describes the physiological condition in mammals that is typically characterized by the development and differentiation of cardiac cells. An example of cardiac disorders includes, but is not limited to, cardiac hypertrophy and heart failure, including congestive heart failure, myocardial infarction, and tachyarrhythmia. "Heart failure" refers to an abnormality of cardiac function in which the heart does not pump blood in the proportion needed for tissue metabolism requirements.

"Condition" determinant of the disease ", refers to the act of determining the probability of survival of the patient and relapse time for neoplastic diseases, particularly breast, ovary, stomach, endometrium, salivary glands, lung, kidney, colon, and carcinomas of bladder, in particular, an antibody of the invention (produced in the chimeric heteroadhesin of the invention and capable of interacting with the extracellular regions) can be used to quantitate the overexpression of the heteromultimeric receptor (e.g., ErbB2, ErbB3 or ErbB4, but usually ErbB2) in cancerous tissue taken from a patient suffering from carcinoma.This can also be referred to as "determining the appropriate course of treatment for patients suffering from carcinoma." For example, those patients characterized by overexpressions of ErbB2 or who have increased amounts of surface receptors of ErbB2 / 3 or ErbB2 / 4 cells may require more aggressive treatments (eg high doses of chemo or radiotherapy treatment) that may otherwise be indicated by other diagnostic factors. This phrase includes patients diagnosed with a high degree of ductal carcinoma in situ, which includes extensive intraductal carcinoma. See, for example, Disis et al., Cancer Research, 54: 16-20 (1994).The word "sample" refers to tissue, body fluid or a cell of a patient. Normally, the tissue or cell will be removed from the patient, but in vivo diagnosis is also contemplated. In the case of solid tumor, a tissue sample can be taken from a tumor surgically removed and prepared for testing by conventional techniques. In the case of lymphomas and leukemias, lymphocytes, leukemic cells, or lymphatic tissues will be obtained and prepared appropriately. Other patient samples, including urine, tears, serum, cerebrospinal fluid, stool, sputum, cell extracts etc. They will also be used for particular tumors.

The term "labeled" as used herein, refers to a molecule (e.g., chimeric heteroadhesin such as ErbB2 / 3-IgG) that has been conjugated, directly or indirectly, with a detectable compound or composition. The label may be detectable by itself (eg, radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze a chemical alteration of a compound or substrate composition that is detectable.

By "solid phase", it is meant a non-aqueous matrix in which a reagent of interest (for example ErbB2 / 3-IgG, ErbB2 / 4-IgG or an antibody thereof) can adhere. Examples of solid phases encompassed herein include those formed partially or wholly of glass (for example controlled porous glass), polysaccharides (for example, agarose). polyacrylamides, polystyrene, polyvinyl alcohol and silicones. in certain embodiments, depending on the context, the solid phase may comprise the cavity of a test plate; in others it is a purification column (for example an affinity chromatographic column). This term also includes a discontinuous solid phase of discrete particles such as those described in U.S.A. do not. 4,275,149.

The phrase "Activating an ErbB receptor" refers to the act of causing the intracellular kinase region of an ErbB receptor to phosphorylate tyrosine residues. Generally, this will involve the binding of an increased antibody in ErbB2 / 3-Ig, for example, whose antibody is tested for its ability to act as a heregulin agonist by binding a receptor to the complex of two or more ErbB receptors (e.g. , an ErbB2 / ErbB3 or ErbB2 / ErbB4 complex) that activates a kinase region of one or more of those receptors and consequently results from the phosphorylation of tyrosine residues on additional polypeptide substrates (s). The phosphorylation of the ErbB receptor can be quantified using the tyrosine phosphorylation assays described abjao. The phrase "inhibiting an ErbB receptor" refers to the antagonistic property of a chimeric ErbB heteroadhesin or an antagonist antibody set against which, when linked to an ErbB receptor, prevents activation of the receptor (eg, inhibits kinase function).

The term "cell survival decrease" refers to the act of decreasing the period of a cell's existence, relative to an untreated cell that has not been exposed to chimeric ErbB-IgG (or an antagonistic antibody raised from it). ) either in vivo or in vitro. The term "cell proliferation decrease" refers to a decrease in the number of cells in a population exposed to chimeric ErbB-IgG (or an increased antagonistic antibody thereof) either in vitro or in vivo, relative to a cell not treated.

The term "increase the survival of a cell" or "increase in cell proliferation" refers to an increase in the existence or increase in the number of cells in a population exposed to an agonist antibody added to a Chimeric ErbB-IgG of the invention, either in vitro or in vivo, in relation to an untreated cell. An increase or decrease in cell proliferation in cell cultures can be detected by the number of cells before and after exposure to the anti-ErbB-IgG agonist antibody.

The degree of proliferation can be quantified by microscopic examination of the degree of confluence. Cell proliferation can also be quantified by measuring the uptake of 3H-thymidine by the cells.

By "improvement of the differentiation of a cell" is meant the act of increasing the degree of acquisition or possession of one or more characteristics or functions that differ from these of the original cells (for example cellular specialization). This can be detected by examining a change in the phenotype of the cell (for example identifying morphological changes in the cell). Improving the differentiation of a cell also refers here to cell maturation in which, for example, unique proteins associated with mature cells are synthesized.

A "glial cell" is derived from the central and peripheral nervous system and can be selected from oligodendroglial, astrocyte, ependymal, or microglial cells as well as satellite cells from neurolema cells around peripheral nerve fibers.

"Muscle cells" includes skeletal, cardiac or smooth muscle tissue cells. These terms encompass those cells - which differentiate more specialized muscle cells (for example myoblasts).

"Isolated nucleic acid" is RNA or DNA free of at least one nucleic acid contaminant source with which it is normally associated in the natural source and preferably substantially free of RNA or DNA of any other mammal. The phrase "free of at least one nucleic acid contaminating source with which it is normally associated" includes the case in which the nucleic acid is present in the original or natural cell but which is in a different chromosomal location or is otherwise flanked by nucleic acid sequences that are not normally found in the cells of origin.

The isolated nucleic acid is RNA or DNA encoding a biologically active chimeric heteromultimeric adhesin in which each extracellular region contributes at least 75%, more preferably at least 80%, even more preferably at least 85%, at the most preferably 90% level , and more preferably 95% to the identity of the sequence with the extracellular region of the natural receptor monomer from which it was derived.

"Severe conditions" are those that (a) employ low ionic strength and high temperature to rinse, for example, 0.015 M NaCl / 0.0015 M sodium citrate / 0.1% NaDodS04 (SDS) at 50 ° C, or (b) used during the hybridization a denaturing agent such as formamide, for example, formamide 50% by volume with 0.1% bovine albumin serum / 0.1% FicoII / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 with 750 mM of NaCl, 75 mM sodium citrate at 42 ° C. Another example is the use of 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium phosphate, 5 x Denhardt's solution, Sperm salmon DNA sonicated (50 μg / mL), 0.1% SDS, and 10% Dextran Sulfate at 42 ° C, with rinses at 42 ° C in 0.2 x SCC and 0.1% SDS.

"Moderately severe conditions" are described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), and include the use of rinsing solutions and hybridization conditions (e.g., temperature, strength ion, and% SDS) less severity than described above, an example of moderately severe conditions is a condition such as incubating the night before at 37 ° C in a solution comprising: 20% formamide, 5 x SSC (150 mM of NaCl, 15 mM trisodium citrate '), 50 mM sodium phosphate (pH 7.6), 5 X Denhardt's solution, 10% dextran sulfate, and 20 mg / mL denatured reduced sperm DNA, followed by rinsing the filter in 1 x SSC at approximately 37-50 ° C. The person skilled in the art will recognize how to adjust the temperature, ionic strength, etc. as necessary to adapt factors such as length tests and the like.

The term "control sequences" refers to DNA sequences necessary for the expression of a coding sequence that operably links in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally a sequencer operator, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation indicators, and enhancers.

The nucleic acid is "operably linked" when it is located in a functional correspondence with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences that are linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, breeders do not have to be contiguous. The union is accompanied by the binding at the convenient restriction site. If such sites do not exist, the synthetic oligonucleotide or binding binders are used according to conventional practice.

An "antagonist" of HRG, is a molecule that prevents, or interferes with, an effector function of HRG (eg, a molecule that prevents or interferes with the binding and / or activation of an ErbB receptor of HRG). Such molecules can be examined for their ability to competitively inhibit the activation of the ErbB receptor of HRG in the tyrosine phosphorylation tests set forth herein, for example. Preferred antagonists are those that do not substantially interfere with the interaction of other heregulin polypeptides with ErbB2 / 4 receptor (s). Examples of HRG antagonists include neutralizing antibodies against the chimeric heteroadhesins ErbB2 / 3-Ig or ErbB2 / 4 of the invention.

The term "antibody" is used in the broadest sense and specifically includes only anti-chimeric heteroadhesins (such as anti-ErbB2 / 3-IgG or anti-ErbB2 / 4-IgG) antichimeric heteroadhesin antibody compositions and monoclonal antibodies with polyepitopic specificity (which includes neutralizing and non-neutralizing antibodies). The antibody of particular interest here is one that does not significantly cross-react with other heteromultimeric receptors, such as those described in the preceding background section and is therefore one that "binds specifically" to a heteromultimeric receptor, such as ErbB2 / 3 or ErbB2 / 4. In such modality, the binding of the antibody to the receptors does not. ErbB will be less than 10% as determined by radioimmunoprecipitation (RIA), for example.

The term "monoclonal antibody" as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, for example, individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minors amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Additionally, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant in the antigen.

The monoclonal antibodies herein include hybrid and recombinant antibodies produced by binding a variable region of an anti-chimeric heteroadhesin antibody to a constant region (eg "humanized" antibodies), or a light chain with a heavy chain, or a chain of species with a chain of other species, or fusions with heterologous proteins, without considering species of origin or designation of classes or subcals of immunoglobulins. so that fragments of antibodies (e.g., Fab, F (ab) 2, and Fv), as long as they exhibit a desired biological activity, (see for example, US Patent No. 4,816,567 and Mage &; La Oyi, in Monoclonal Antibody Production Techniques and Applications, pp.79-97 (Marcel Dekker, Inc.), New York (1987)).

Thus, the "monoclonal" modifier indicates the character of the antibody as it is obtained from a substantially homogeneous population of antibodies. For example, the monoclonal antibodies used in accordance with the present invention can be made by the hybridoma method first described by Kohler & Milstein, Nature 256: 495 (1975), or can be made by recombinant DNA methods (US Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage stocks generated using the techniques described in McCafferty et al., Nature 348: 552-554 (1990), for example.

"Humanized" or non-human forms of antibodies are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fav, FabA F (ab) 2 or other subsequences of antigen-binding antibodies) that contain minimal sequence derived from immunoglobulin non-human For the most part, humanized antibodies are human immunoglobulins (receptor antibody) in which residues of the complementary determining regions (CDRs) of the receiving antibody) are replaced by residues of the CDRs of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity, in some cases, residues of the structural region (FR) of human immunoglobulin are replaced by the corresponding non-human FR residues. In addition, the humanized antibody can comprise residues of which none are found in the receptor antibody or in the imported Fr or CDR sequences. These modifications are made to add refinement and optimize the performance of the antibodies, in general, the humanized antibody will comprise substantially all or at least one, and typically two, variable regions, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR residues are those of a sequence consensually related to a human immunoglobulin. The humanized antibody optimally will also comprise at least a portion of an immunoglobulin constant region (Fe), typically that of a human immunoglobulin.

By "neutralizing antibody" is meant an antibody molecule as defined herein that is capable of blocking or significantly reducing an egector function of the native HRG sequence. For example, a neutralizing antibody can inhibit or reduce the ability of HRG to activate an ErbB receptor in the tyrosine phosphorylation assay described herein. The neutralizing antibody can also neutralize the mitogenic activity of HRG in the cell proliferation test described herein.

"Treatment" refers to both therapeutic and prophylactic / preventive measures. Those that need treatment even those already with disorders as well as those prone to have the disorder or those in which the disorder is being prevented.

"Mammal" for treatment purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, or pet animals, such as sheep, dogs, horses, cats, cows, ... etc . Preferably the mammal here is the human.

"Pharmaceutically acceptable" vehicles, excipients, or stabilizers are ones that are not toxic to the cell or to mammals exposed to them, in the doses and concentrations employed. Often physiologically acceptable vehicles is an aqueous solution at regulated pH. Examples of physiologically acceptable carriers include regulators, such as phosphate, citrate, and other organic acids; antioxidants that include ascorbic acid; low molecular weight polypeptides (less than about 10 residues); proteins, such as albumin serum, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, spargin, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salts-formations of anions such as sodium; and / or non-ionic surfactants such as Tween ™, polyethylene glycol (PEG), and pluronics ™.

II. Methods for implementing the invention 1. Production of a chimeric heteromultimer adhesin A chimeric heteroadhesin of the invention is preferably produced by expression in and isolated from a host cell. A host cell is generally transformed with the nucleic acid of the invention. Preferably the nucleic acid is incorporated into a vector expression. Suitable host cells for cloning or expressing the vectors here are prokaryotic host cells (such as E. coli, Bacillus strains, Pseudomonas and other bacteria), yeasts and other eukaryotic microbes, and larger eukaryotic cells (such as hamster ovary cells). Chinese (CHO) and other mammalian cells). The cells may be present in live animals (for example, in cows, goats or sheep). Insect cells can be used. Cloning and expression methods are well known in the art.

To obtain the expression of a chimeric heteromultimer such as ErbB2-IgG, ErbB3-IgG, and / or ErbB4-IgG, a vector expression is introduced into host cells by transformation or transfection and the resulting recombinant host cells are cultured in nutrient media. conventional, properly modified by induction of promoters, selection of recombinant cells, or amplification of ErbB-IgG DNA. In general, principles, protocols, and technical practices to maximize the productivity of mammalian cell cultures in vitro can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, de. (IRL Press, 1991).

The terms "Transformation" and "transfection" are used interchangeably herein and refer to the process of introducing DNA into the cell. Following transfection or transformation, the nucleic acid of the invention can be integrated into the genome of the host cell, or it can exist as an extrachromosomal element. If prokaryotic cells or cells containing substantially constructed cell walls are used as hosts, the preferred transfection methods of the cells with DNA is the calcium treatment method described by Cohen, S. N. et al., Proc. Nati Acad. Sci. U.S. to. 69: 2110-2114 (1972) or the polyethylene glycol method of Chung et al., Nuc. Acids Res. 16: 3580 (1988). If yeasts are used as hosts, the transfection is generally accompanied by the use of polyethylene glycol, as disclosed by Hinnen, proc. Nati Acad. Sci. U.s. a., 75: 1929-1933 (1978). If mammalian cells are used as host cells transfection is generally carried out by the calcium phosphate precipitation method, Graham et al., Virology 52: 546 (1978), Gorman et al., DNA and Protein Eng. Tech. 2 : 3-10 (1990). However, other known methods for introducing DNA into prokaryotic and eukaryotic zelules, such as nuclear injection, electroporation, protoplast ophusion, are also suitable for use in this invention.

Particularly useful in this invention are expressions of vectors that provide for the transient expression in mammalian cells of DNA encoding a chimeric heteroadhesin such as ErbB2 / 3-Ig or ErbB2 / 4-Ig. In general, transient expression involves the use of a vector expression that is capable of efficiently replicating in a host cell, such that the host class accumulates many copies of the expression of the vector, in turn, synthesizes high levels of a desired polypeptide encoded by the expression of the vector. Transient expression systems, comprise a suitable vector expression and a host cell, which are taken into account for the convenient positive identification of polypeptides encoded by cloned DNAs, as well as for the rapid screening of such polypeptides for the desired biological and physiological properties .

A chimeric heteroadhesin is preferably recovered from the culture medium as a secreted polypeptide, although it can also be recovered from the used host cell. As a first step, the particulate debris, either from host cells or lysate fragments, is removed, for example, by centrifugation or ultrafiltration; optionally, the protein can be concentrated with a commercially available protein concentration filter, followed by separating the chimeric heteroadhesin from other impurities by one or more purification procedures selected from: fractionation in a column by in aafinity: fractionation in an exchange column ionic; precipitation with ethanol or ammonium sulfate; HPLC in inverted phase; chromatography on silica; chromatography on heparin Sepharose; chromatography on a cation exchange resin; chromatography by focusing; SDS-PAGE; and filtration on gel.

The preparation of the taguised epitope of the chimeric heomeromultimer, such as ErbB-IgG, facilitates purification using an immunoaffinity column containing the epitope antibody to adsorb the fusion polypeptide. Immunoaffinity columns such as a rabbit polyclonal anti-ErbB column can be used to absorb ErbB-IgG by binding to an immune epitope of ErbB.

Variants of the amino acid sequence of the extracellular region of the native sequence included in the chimeric heteroadhesin are prepared by introducing appropriate changes in the nucleotide into the DNA of the extracellular region of the native sequence, or by in vitro synthesis of the monomer polypeptide of the native sequence. the desired chimeric heteroadhesin. Such a variant includes, for example, deletions of, or insertions or substitutions of, residues in the amino acid sequence of the chimeric heteroadhesin.

Variations in the native sequence as previously cescribed can be made using any of the techniques and guides for conservative and non-conservative mutations set forth in US Patent No. 5,364,934. See especially Table i here and the discussion around this table for orientation in the selection of amino acids for change, addition or elimination.

Nucleic acid molecules encoding amino acid sequence variants of extracellular regions of the native sequence (such as ErbB) are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from an original source (in the case of variants of naturally occurring amino acid sequences) or preparations by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cartridge mutagenesis of a previously prepared variant or a non-variant version of native sequences of ErbB2, -3, and / or -4.

A preferred type of chimeric amino acid sequence is a fusion protein comprising an extracellular region, from an ErbB monomer, linked to a heterologous polypeptide, as well as a multimerization region (Immunoglobulin constant region and the like). In the same way a sequence can be constructed using recombinant DNA phenols. alternatively, heterologous polypeptides can be covalently linked to the extracellular region of the polypeptide by techniques well known in the art such as the use of heterobifunctional crosslinking reagents. Exemplary coupling agents include N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), inminothiolate (IT), bifunctional derivatives of imidoesters (such as dimethyl adipidimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazonium benzoyl) -ethylenediamine), diisocyanates (such as tolylene 2, 6 -diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

In one embodiment, a chimeric heteroadhesin polypeptide comprises a fusion of a chimeric heteroadhesin monomer with a tag polypeptide that delivers an epitope to which an anti-tag anti-body can selectively bind. similar forms of the taguised epitope of the chimeric heteroadhesin that are used, as well as the presence of these can be detected using an antibody labeled against the tag polypeptide. Also, providing the epitope tag facilitates the chimeric heteroadhesin to be easily purified by purification affinity using the anti-tag antibody. Tag polypeptides and their respective antibodies are well known in the art. Examples include the influenza HA tag polypeptide and its 12CA5 antibodies, (Field et al., Mol.Cell. Biol. 8: 2159-2165 (1988), the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10, antibodies to these (Evan et al., Molecular and Cellular Biology 5 (12): 3610-3616 (1985); and Herpes Simplex glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein Engineering 3 ( 6): 547-553 (1990)).

When preparing the chimeric heteroadhesins of the present invention, the nucleic acid encoding an extracellular region of a natural heteromultimer recpetor is C-fused to the nucleic acid encoding the N-terminus of a constant region immunoglobulin sequence, notwithstanding the fusions N-endings are still possible. Typically, in such fusions the encoded chimeric polypeptide will retain at least functionally an active essential point, CH2 and CH3 regions of the constant region of a heavy chain immunoglobulin. The fusions are also made in the C-terminus of the Fe portion of a constant region, or immediately N-terminus in the CH1 of the heavy chain or in the corresponding region of the light chain. The construction of the resulting DNA fusion is expressed in suitable host cells.

Another type of covalent modification of a chimeric heteromultimer comprises linking a monomer polypeptide of the heteromultimer to one of a variety of monoproteinaceous polymers, for example, polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. A chimeric heteromultimer may also be entrapped in microcapsules prepared, for example, by coacervation or interfacial polymerization techniques (for example, hydroxymethylcellulose or gelatin microcapsules and polymethylmetacylate microcapsules), respectively, in colloidal drug release systems (for example, example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are set forth in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A. De. (1980).

Generally, the ErbB chimeric heteromultimers of the invention will have any one or more of the following properties: (a) the ability to compete with a natural heteromultimer receptor to bind to a neurogulin, such as heregulin; (b) the ability to form complexes ErbB2-IgG / ErbB3-IgG and / or ErbB2-IgG / ErbB4-IgG; and (c) the ability to inhibit the activation of a natural heteromultimer receptor by inheritance of the heregulin from the natural receptor habitat, thereby inhibiting the proliferation of cells expressing the ErbB2 receptor and ErbB3 and / or the ErbB2 and ErbB4 recpetor.

In property tests (a) the ability of the heteromultimeric chimeric ErbB adhesin to bind to? -uregulin can be easily determined in vitro. For example, immunoadhesin forms of these receptors can be generated (see below) and immunoadhesins ErbB2 / 3-Ig or ErbB2 / 4-Ig can be immobilized on a solid phase. { for example in coatings tests of plates with anti-human-goat antibody). The ability of HRG to bind immobilized immunoadhesin can then be determined, for example, determination of competitive displacement by other heregulin molecules. For more details, see the linkage test 25l-HRG described in the following example.

As in property (c), the tyrosine phosphorylation test using MCF7 cells described in the example provides a means for examination of activation of ErbB receptors. In an alternative embodiment of the invention, the KIRA-ELISA described in WO 95/14930 can be used to qualitatively and quantitatively measure the ability of a chimeric ErbB heteroadhesin to inhibit the activation of an ErbB receptor.

The ability of a chimeric heteroadhesin such as ErbB2 / 3-Ig or ErbB2 / 4 to stimulate the proliferation of a cell expressing the ErbB2 and ErbB3 receptor and / or the ErbB2 and ErbB4 recpetor can easily be determined in cell culture. Cells useful for these experiments include MCF7 and SK-BR-3 cells obtainable from the ATCC and Schwann cells (see, for example, Li et al., J.

Neuroscience 16 (6): 2012-2019 (1996)). These tumor cell lines can be plated on cell culture plates and kept adhered to them. Ligand HRG is added in the presence or absence of a chimeric ErbB heteroadhesin. The monolayers can be stained / fixed with crystal violet. Inhibition or delcular growth can therefore be quantified as described.

Other heteromultimeric receptors in which the present invention can be applied for the preparation of useful chimeric heteroadhesins include the following: Axl, Rse, epidermal growth factor receptor (EFG), hepatocyte growth factor receptor (HGF), IL-2, Al-1, EPH, TrkA, TrkB, TrkC, TNF, IL-100, CRF2-4, RXR, RON, AChRa / d, TRa / RXP.a, Tra / DR4, Tra / MHC-TRE, Tra / ME, Tra / F2, KDR / FLT-1, FLT / VEGF, VEGF121 / 165, Armt / Ahr, CGA / CGB, EGFR / pl85-neu, prolactin receptor (PRL), T cell receptor (TCR), fibroblast growth factor (FGF), Cak receptor, IL-6 / gpl30, leukemia inhibitory factor IL-ll / gpl30 ( LIF) / gpl30, cardiotropin-l / gpl30 (CT-1), IL-11 / gpl30, ciliary neurotropic factor (CNTF) / gpl30, oncostatin M (OSM) / gpl30, inferred?, And interferon a, ß.

A chimeric heteroadhesin of the invention comprises the extracellular regions of a naturally occurring heteromultimeric receptor, wherein an ECD (or linker ligand thereof fragment) of a receptor monomer is fused to a multimerization region as described above. The chimeric monomers of the heteroadhesin are stably associated via the multimerization region to form the chimeric heteroadhesin. The heteroadhesins of the invention bind the ligand of the natural receptor from which the ECDs are obtained and are used as ligand antagonists. Such antagonists are used in the treatment of disease stages resulting from the binding of the ligand and the activation of the natural heteromultimeric receptor. 2. Compositions and Therapeutic Methods The use of the chimeric heteroadhesins of the invention as therapeutic compositions is a modality of the invention. The uses generally set forth herein are provided as a guide for the use of chimeric heteroadhesins in general. The ErbB chimeric heteroadhesins are set forth as examples for further guidance.

HRG promotes the development, maintenance, and / or regeneration of neurons in vivo, including central neurons (brain and spinal cord), peripheral neurons (sympathetic, parasympathetic, sensory and enteric neurons), and motor neurons. Accordingly, an agonist HRG such as an agonist anti-ErbB-Ig antibody can be used in methods for the diagnosis and / or treatment of a variety of "neurological diseases or disorders" that affect the nervous system of a mammal such as a human . According to this embodiment of the invention, the agonist antibody cultured in the chimeric heteroadhesin ErbB cross-reacts with and activates the ErbB receptor.

Such diseases or disorders may arise in patients in whom the nervous system has been damaged by trauma, surgery, apolplegia, ischemia, infection, metabolic disease, nutritional deficiency, malignant or toxic agents. The agent is desognado to promote the survival, proliferation or differentiation of neurons. For example, anti-ErbB chimeric heteroadhesin agonist antibodies can be used to promote the survival or proliferation of motor neurons that are damaged by trauma or surgery. They can also be used to treat motoneuron disorders, such as amyotrophic lateral sclerosis (Lou Gehring's Disease), Bell's Palsy, and various conditions involving spinal muscular atrophy, or paralysis. The agonist antibody can be used to treat human "neurovegetative disorders", such as Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, Huntington's disease, Down syndrome, nervous deafness, and Meniere's disease.

In addition, an anti-ErbB chimeric heteroadhesin agonist antibody can be used to treat neuropathy and especially peripheral neuropathy. "Peripheral neuropathy" refers to a disorder that affects the peripheral nervous system, more often manifested as one or more combinations of motor dysfunctions, sensory, sensorimotor, or autonomic neural. The wide variety of morphologies exhibited by peripheral neuropathies can each be attributed only to an equally large number of causes. For example, peripheral neuropathies can be genetically acquired, can result from a systemic disease, or can be induced by a toxic agent. Examples include but are not limited to distal sensomotor neuropathy, or autonomic neuropathy such as reduced mobility of the gastrointestinal tract or atony of the urinary bladder. Examples of neuropathies associated with systemic disease include post-polio syndrome; examples of hereditary neuropathies include Charcot-Marie-Tooth disease, Refsu disease, Abetalipoproteinemia, early Tangier age, Krabbe disease, metachromatic leucodysterias, Fabry disease, and Dejerine-Sottas syndrome; and examples of neuropathies caused by a toxic agent include aquila caused by treatment with a chemotherapeutic agent.An anti-ErbB chimeric heteroadhesin agonist antibody of the invention can also be used to treat muscle cells and medical conditions that affect them. For example, HRG can be used to treat a pathophysiological condition of the musculature in a mammal, such as a skeletal muscle disease (eg, myopathy or dystrophy), a disturbance of the heart muscle. (such as atrial cardiac arrhythmias), cardiomyopathy, ischemic damage, congenital disease, or cardiac trauma), or a smooth muscle disorder (e.g., arterial sclerosis, vascular injury, or congenital vascular disease); to treat muscle damage; to decrease atrophy of muscle cells; to increase the survival, proliferation and / or regeneration of muscle cells in a mammal; to treat hypertension; and / or to treat a muscle cell that had functional insufficiency of acetylcholine receptors (as in a patient with myasthenia gravis or tachycardia).

An anti-ErbB chimeric heteroadhesin agonist antibody can be used to induce the formation of ion channels in a supeficie membrane of a cell and / or to improve the formation of synaptic junctions in an individual. HRG can also be used as a memory enhancer and can eliminate "anxiety" for nicotine.

The anti-ErbB chimeric heteroadhesin agonist antibody can be used to improve, restore and / or regenerate tissues that produce ErbB receptor (s), especially the ErbB2 receptor. For example, the anti-ErbB chimeric heteroadhesin agonist antibody can be used to treat skin lesions; gastrointestinal disease; Barrett's esophagus; cystic or non-cystic enferemdad in the final kidney stage; and inflammatory bowel disease. Similarly, these molecules can be used to promote reepithelialization in the gastrointestinal, respiratory, reproductive or urinary tracts in humans.

It may be desirable to treat the mammal with an HRG antagonist, such as a chimeric ErbB-Ig heteroadhesin, particularly where excessive levels of HRG and / or excessive activation of ErbB receptors are present by the HRG found in the mammal. Exemplary conditions or disorders are treated with an HRG antagonist including malignant or benign tumors (eg, renal, kidney, bladder, breast, gastric, ovarian, colon and neck tumors); leukaemias and malignant lymphoid; other disorders such as neuronal, glial, astrocital, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic disorders; inflammatory, angiogenic and immunological disorders; formation of psoriasis and scars on the skin. HGR antagonists can be used to reverse the resistance of tumor cells to the immune response, to inhibit pathological angiogenesis and to stimulate the immune system.

In still other embodiments of the invention, an anti-ErbB chimeric heteroadhesin as well as an antagonist HRG can be administered to patients suffering from neurological diseases, or disorders characterized by excessive production of HRG and / or excessive activation of the ErbB receptor by HRG. An anti-ErbB chimeric heteroadhesin antagonist antibody can be used in the prevention of aberrant regeneration of sensory neurons as it can happen postoperatively, or in selective ablation of sensory neurons, for example, in the treatment of chronic pain syndromes.

There are two major approaches for introducing the nucleic acid (optionally contained in a vector) into cells of patients; in vivo and ex vivo. For in vivo releases the nucleic acid is injected directly into the patient, usually at the site where the chimeric heteroadhesin is required. For ex vivo treatment, the patient's classrooms are removed, the nucleic acid is introduced into these isolated cells and the modified cells are administered to the patient either directly or for example, encapsulated in porous membranes that are implanted in the patient (see , for example, US Pat. Nos. 4,892,538 and 5,283,187).

There are a variety of techniques available to introduce nucleic acids into viable cells. The techniques vary depending on whether the nucleic acid is transferred in cell cultures in vitro, or in vivo in the cells of the intended host. Techniques available for the transfer of nucleic acid in mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the method of calcium phosphate precipitation, etc. A vector commonly used for ex vivo gene releases is a retrovirus.

Currently preferred in vivo nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus, Herpes Simplex I virus), or lipid-associated viruses) and lipid-based systems (lipids useful for lipid-media transfer of the DOTMA, DOPE and DC-Chol genes, for example). In some situations it is desirable to provide the original nucleic acid with an agent that hits the target cells, such as an antibody specific for a membrane surface of the target cell or protein cell, a ligand for a receptor on the target cell, etc. Where liposomes are employed, proteins that bind to the surface cell membrane of protein associated with endocytocytes can be used to objectify and / or facilitate uptake, eg, capsid proteins or fragments of these, topical for a particular type of protein. cell, antibodies for proteins that undergo internalization in cyclase, and proteins whose intracellular objective is the localization and improvement of the intracellular half-life. The technique of endocytosis by means of a receptor is described, for example, by Wu et al., J. Biol. Chem. 262: 4429-4432 (1987); and Wagner et al., Proc. Nati Acad. Sci. USA 87: 3410-3414 (1990). For review of gene therapy and gene tagging protocols commonly known see Anderson et al., Science 256: 808-813 (1992). See also WO 93/25673 and the references cited herein.

Therapeutic formulations of a chimeric heteroadhesin or an antibody against it are prepared to be admixed by mixing the heteroadhesin or antibody having the desired purity grade with physiologically acceptable vehicles, excipients or stabilizers (Remington's Pharmaceutical Sciences, 16th Edition, Osol. , A. De., (1980)), in the form of lyophilized cake or aqueous solutions. Pharmaceutically acceptable carriers, excipients, or stabilizers that are in non-toxic containers at the doses and concentrations employed, and include regulators such as phosphate, citrate, and other organic acids; antioxidants that include ascorbic acid; low molecular weight polypeptides (less than about 10 residues); proteins, such as albumin serum, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, aspargin, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; anion-forming salts such as sodium; and / or non-ionic surfactants such as Tween ™, Pluronics ™, or polyethylene glycol (PEG).

A chimeric heteroadhesin or anti-chimeric heteroadhesin antibody, is used for in vivo administration must be sterile. This is easily achieved by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. The formulation will ordinarily be stored in lyophilized form or in solution.

Therapeutic chemical heteroadhesins or. Anti-chimeric heteroadhesin antibody compositions are generally placed in a container having a sterile access port, for example, an intravenous solution bag or a bottle having a prepourable cap for a hypodermic needle for injection.

The route of administration of the chimeric antibody or heteroadhesin conforms, with known methods, for example, intravenous, intraperitoneal, intracerebral, intramuscular, infraocular, intraarterial, or intralesional injection or infusion, or by sustained release systems as will be noted below. . The heteroadhesin or antibody is administered continuously by infusion or by bolus injection.

Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the protein, which matrices are in the form of molded articles, eg, films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels, (eg, poly (2-hydroxyethyl-methacrylate) as described by Langer et al, J. Bio ed. Mater. Res., 15: 167-277 (1981) and Langer, Chem. Tech., 12: 98-105 (1982) or poly (vinylalcohol)), polylactides (US Patent No. 3,773,919, EP 58,881), copolymers of L-glutamic acid and ethyl-L-glutamate range (Sidman et al. Biopolymers, 22: 547-556 (1983), non-degradable ethylene vinyl acetate (Langer et al., Supra), lactic acid-degradable lactic acid copolymers such as Lupron Depot ™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D - (-) - 3-hydroxybutyric acid (EP 133,988).

Sustained-release compositions of chimeric heteroadhesins or antagonists of anti-heteroadhesin antagonists also include liposomally trapped drugs. Liposomes containing HRG are prepared by known methods "per se": 3,218,121; Epstein et al., Proc. Nati Acad. Sci. USA 82: 3688-3692 (1985); Hwang et al., Proc. Nati Acad. Sci. USA 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Patent Application 83-118008; US Patent Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the small unilamellar type in which the lipid content is greater than about 30 mol% of cholesterol, the selected proportion being adjusted for optimal therapy. Particularly useful liposomes that can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and phosphatidylamine-PEG derivatives (PEG-PE). The liposomes are extruded through pore size filters to produce liposomes with the desired diameter. A chemotherapeutic agent (such as Doxorubicin) is optionally contained in the liposome. "See Gabizon et al., J. National Cancer Inst. 81 (19): 1484 (1989).

The chimeric ErbB-Ig heteroadhesin of the invention can be used to bind and sequester the HRG ligand thereby inhibiting the activation of ErbB in the cell and inhibiting a disturbance in cell proliferation in a cancer patient.

A cancer patient who is treated with a heregulin antagonist ErbB2 / 3-Ig or ErbB2 / 4-Ig or an anti-ErbB-Ig antibody as well as an antagonist discussed here, may also receive radiation therapy, alternatively, or In addition, a chemotherapeutic agent can be administered to the patient. Indications for the preparation and dosage for such chemotherapeutic agents can be used according to the manufacturer's instructions or as determined empirically by those skilled in the art. Indications for the preparation and dosage for such chemotherapy are also described in Chemotherapy Service De., M. C. Perry, Williams & Wilkins, Baltimore, MD (1992). The chemotherapeutic agent may precede or follow the administration of the antagonist or may be given simultaneously with it. Indications for cancer, it may also be desirable to administer antibodies against antigens associated with tumors or against antiogenic factors, such as antibodies that bind to EGFR, ErbB2, ErbB3, ErbB4, or vascular endothelial factor (VEGF). alternatively, or in addition one or more cytokines can be co-administered to the patient.

An effective amount of antagonist that is therapeutically used will depend, for example, on the therapeutic objectives, the route of administration, and the conditions of the patient. Therefore, it will be necessary for the therapist, titrate the dose and modify the route of administration as required to obtain the maximum therapeutic effect. A typical dose will be in the range of approximately 1 μg / Kg. up to 100 mg./Kg. of the patient's body weight, preferably about 10 μg / Kg. up to 10 mg./Kg. Typically, clinicians will administer an antagonist to an enriched dose until the desired effect is achieved by the treatment of the abovementioned disorders. 3. Non-therapeutic methods An anti-ErbB2 / 3-Ig antibody or anti-ErbB2 / 4 HRG agonist antibody can be used for cellular developments (such as muscle or muscle cells) ex vivo. It is desirable to have such cell populations in cell cultures for isolation of specific cellular factors for example P75NGFR which is a specific marker of Schwann Class. Such factors are used as a diagnostic instrument or, in the case of P75NGFR, antigens can be used to generate antibodies for diagnostic use. It is also beneficial to have populations of mammalian cells (e.g., Schwann cells) to be used as prostheses for implantation in patients (e.g., in areas of damaged spinal cord in an effort for interruned regeneration of central axons, to aid in nerve recovery. damaged peripherals and as an alternative for multiple autografts.

In accordance with the in vitro methods of the invention, cells comprising an ErbB receptor are provided and located in a cell culture medium. Suitable culture media for knitting are well known to those skilled in the art, and include, but are not limited to, Minimum Essential Medium (MEM), RPMI-1640, and Dulbecco's Modified Eagle Medium (DMEM). These tissue culture media are commercially available from Sigma Chemical Company (St. Louis, MO) and GIBCO (Grand Island, NY). The cells are then cultured in the cell culture medium under conditions sufficient for the cells to remain viable and grow in the presence of an effective amount of agonistic antibody. Classrooms can be grown in a variety of ways, including culturing in a clot, agar, or culture liquid.

Anti-ErbB-Ig antibodies can be used in the diagnosis of cancers characterized by overexpression and / or erbB amplification (e.g. erbB2), in which anti-chimeric heteroadhesin antibodies are used that cross-react with the ErbB receptor. Such diagnostic tests may be used in combination with other prognostic / diagnostic evaluations such as the determination of the "status" of the lymph node, size of primary tumor, histological grade, estrogen and progesterone status, DNA content in tumor (ploid) ), or cell proliferation (fraction S-phrase). See Muss et al., New Eng. J. Med., 330 (18).-1260-1266 (1994).

The sample as defined here, is obtained from the primary lesion of a patient, fixed to formalin, paraffin-embedded blocks were prepared. See Muss et al., Supra and Press et al., Cancer Research 54: 2771-2777 (1994). Tissue sections (eg, 4μM) were prepared according to known techniques. The degree of binding of the anti-ErbB2 / 3-Ig antibody or anti-erbB2 / 4-Ig in the tissue sections is then quantified.

Generally, the chimeric heteroadhesin or anti-chimeric heteroadhesin antibody will be labeled either directly or indirectly with a detectable label.

Numerous brands that are available can usually be grouped into the following categories: (a) Radioisotopes, such as 35S,? .C, 125I, 3H, and 131Y. The? -HRG or the antibody can be labeled with the radioisotope using the techniques described in Current Protocols in Immunology, De. Coligen et al., Wiley Publishers, Vols 1 & 2, for example, and radioactivity can be measured using count per scintillator. (b) Fluorescent markers such as rare earth chelates (europiom chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lisamine, Phycoerythrin and Texas Red are available. The fluorescent labels can be conjugated with the chimeric heteroadhesin or the antibody using the techniques set forth in Current Protocols in Immunology, supra, for example. Fluorescence can be quantified using a fluorimeter (Dynatech). (c) Various enzyme substrate markers are available and US Pat. No. 4,275,149 provides a review of some of these. The enzyme generally catalyzes a chemical alteration of the chromogenic substrate that can be measured using various techniques. For example, the enzyme can catalyze a color change in a substrate, which can be measured spectrophotometrically. alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a fluorescence change were previously described. The chemiluminescent substrate begins to be electronically excited by a chemical reaction and can then emit light rays that can be measured (using a Dynatech ML300 chemiluminometer, for example) or donate energy to a fluorescent acceptor. Examples of enzymatic labels include luciferases (eg, firefly luciferase and bacterium luciferase; US Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazineadicketones, dihydrohydrogenase malate, urease, peroxidase such as horseradish peroxidase (HRPO), phosphatase alkaline, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (for example glucose oxidase, galactose oxidase, and glucose-6-phosphate dihydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, icroperoxidase, and the like. Techniques for conjugation of protein enzymes are described in O'Sullivan et al.

Méthodes for the Preparation of Enzyme-Antibody Conjugates for use in Enzime Immunoassay, in Methods in Enzim. (by J. Langone &H. Van Vunakis), Academic Press, New York, 73: 147-166 (1981).

Examples of enzyme-substrate combinations, for example: (a) Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, in which hydrogen peroxidase oxidizes a dye precursor (eg, orthophenylene diamine (OPD)) or 3, 3 ', 5, 5'- tetramethyl benzidine hydrochloride (TMB)); (b) alkaline phosphatase (AP) with para-nitrophenyl phosphate as the chromogenic substrate (for example p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate 4-methylumbelliferyl-β-D-galactosidase.

Numerous other combinations of enzyme-substrates are available to those skilled in the art. For a general review of these, see US Patents Nos .: 4,275,149 and 4,318,980.

Optionally, the label is indirectly conjugated to the chimeric heteroadhesin or anti-CHA antibody. The knowledgeable expert will be aware of various techniques to achieve this. For example, the CHA or anti-CHA antibody may be conjugated to biotin and any of the three broad categories of markers mentioned above may be conjugated to avidin, or vice versa. Biotin binds selectively to avidin and thus, the label can be conjugated to the CHA or anti-CHA antibody in this indirect manner. See, Current Protocols in Immunology, supra, for a review of techniques involving biotin-avidin conjugations. Alternatively, to achieve indirect conjugation of the marker with the CHA or anti-CHA antibody, the CHA or anti-CHA antibody is conjugated to a small hapten) for example digoxin) and one of the different types of markers mentioned above is conjugated with an anti-CHA. anti-hapten body (for example anti-dioxin antibody). Therefore indirect conjugation of the label with CHA or anti-CHA antibody can be achieved.

In another embodiment of the invention, the CHA or anti-CHA antibody does not need to be labeled, and the presence of these can be detected using a labeled anti-CHA antibody or antibody (for example conjugated with HRPO).

In the preferred embodiment, the HRG or antibody is labeled with an enzymatic label that catalyzes a color change of a substrate (such as tetramethyl benzimidine (TMB), or ortaphenylene diamine (OPD)). Thus, the use of radioactive materials is avoided. A color change of the reagent can be determined spectrophotometrically at a suitable wavelength (for example 450 nm for TMB and 490 nm for OPD, with a reference wavelength of 650 nm).

Cellular conceptions capable of expressing a ligand such as HRG are exposed to the labeled ErbB CHA and the intensity of the spot of the determined cell culture medium. While in vitro analysis is normally contemplated, in vivo diagnosis using labeled conjugated ErbB CHA in a detectable (eg unimaginable) portion may result as well. See for example, US Patent No. 4,938, 948.

CHAs or anti-CHA antibodies are also used in a radioimmunoassay, enzyme-linked immunoassay, or radio-receptor tests), in affinity purification techniques (for example for HRG, or for an ErbB receptor such as ErbB3 or ErbB4), and in tests of binding type competitive receptors when marked with readiode, enzymes, fluorophores, labeled spins, and the like. Thus, CHAs are used as immunogens to generate anti-CHA antibodies for diagnostic use. 4. Anti-chimeric Hetroadhesin Antibodies and Uses of these.

Techniques for generating antibodies, such as monoclonal and polyclonal antibodies are well known in the medium. Polyclonal antibodies are generally cultured by immunization of animals with CHA or fragments thereof (optionally conjugated to a protein heterologous which is immunogenic in the species to be immunized). Monoclonal antibodies directed towards a CHA can be produced using any method provided for the production of antibody molecules by continuous cultures of cell lines. Examples of suitable methods for preparing monoclonal antibodies include the original hybridoma method of Kohler et al., Nature 256: 495-497 (1975), and the human B-cell hybridoma method, Kozbor, J. Immunol. 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc. New York, 1987); and Boemer et al., J. Immunol. 147: 86-95 (1991). • DNA encoding the monoclonal antibodies of the invention is easily isolated and sequenced using conventional procedures (for example, for use in oligonucleotide assays that are capable of binding specifically to genes encoding the heavy and light chains of antibodies). The hybridoma cells of the invention • serve as a preferred source of such DNA. Once In isolation, DNA can be located in vector expressions, which are then transfected into host cells such as E. Coli cells, Cos-simian cells, Chinese hamster ovary (CHO) cells, or myeloma cells that are not produced from another so that by immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in ce Recombinant host classes.

The DNA can also be modified, for example, by substitution of the coding sequence for constant regions of light and heavy human chain instead of homologous murine sequences (Morrison et al., Proc., Nati. Acad. Sci. USA 81: 6851 (1984)), or by covalently linking to the immunoglobulin coding sequence all or in part with the coding sequence for a non-immunoglobulin polypeptide. Thus, "chimeric" or "hybrid" antibodies are prepared having the binding specificity of an anti-CHA monoclonal antibody here.

Methods for humanizing non-human antibodies are well known in the medium. Generally, a humanized antibody has one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable region. Humanization can be essentially effected following the method of Winter et al. (Jones et al., Nature 321: 522-525 (1986); Riechman et al., Nature 332: 323-327 (1988); and Verhoeyen et al., Science 239: 1534-1536 (1988)), - by substitution of rodent CDR or CDR sequences by the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (US Patent No. 4,816,567), wherein substantially less than an intact human variable region has been replaced by the corresponding sequence of a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues from analogous sites in anti-rodent bodies.

The selection of the human variable region, both light and heavy, to be used in the preparation of humanized antibodies is very important to reduce antigenicity. Accordingly to the so-called "best-selected" method, the sequence of the variable region of a rodent antibody is examined in contrast to the complete stock of seceunces of known human variable regions. The human sequence that is closest to that of the rodent is then accepted as the human structure (FR) for the humanized antibody (Sims et al., J.

Immunol. 151: 2296 (1993); and Chothia and Lesk, J. Mol. Biol. 196: 901 (1987). Another method that uses a particular structure derived from the consensus sequences of all human antibodies of a particular subgroup of light and heavy chains. The same structure can be used for several different humanized antibodies (Cárter et al, Proc Nati Acad Sci USA 89: 4285 (1992) and Presta et al J. Immunol. 151: 2623 (1993)).

It is also important that the antibodies are humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this objective, according to the preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and several conceptual humanized products using three-dimensional models of the humanized and parenteral sequences. Three-dimensional models of immunoglobulins are commonly available and are familiar from those known to those skilled in the art. Available computer programs illustrate and exhibit probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. The inspection of these exposures allows the analysis of similar roles of the residues in the functioning of the candidate immunoglobulin sequence, for example, the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this manner FR residues can be selected and combined from consensus and import sequences as well as the desired characteristic antibody, such as increased affinity for the target antigen, is achieved. In general, CDR residues are directly and substantially involved in influencing the binding antigen.

According to an alternative method for producing human antibodies, transgenic animals (e.g., mice) that are available are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of production of endogenous immunoglobulins. For example, it has been described that the homozygous deletion of the heavy chain antibody that binds the gene (JH) region with original mutant and chimeric lines of mice results from the complete inhibition of the production of endogenous antibodies. The transfer of the genetic array to the original human immunoglobulin line in such an original mouse mutant line will result from the production of human antibodies by the antigen tested. See, for example, Jakobovits et al., Proc. Nati Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); and Bruggermann et al., Year Immuno. 7:33 (1993).

Alternatively, the phage display technology (McCafferty et al., Nature 348: 552-553 (1990)) can be used to produce human antibodies and fragments of antibodies in vitro, from the genetic repertoires of variable region immunoglobulins (V) from non-immunized donors. , according to this technique, variable region genes of antibodies are cloned in structure in either a major or minor protein gene layer of a filamentous bacteriophage, such as M13 or fd, and exposed as functional antibody fragments on the surface of The phage particle Because the filamentous particles contain a DNA copy of a single strand of the phage genome, selections based on the functional properties of the antibody also result from the selection of the gene encoding the antibody that exhibits these properties. phage simulates some of the properties of the B cell. Phage display can be carried out in a variety For example, for review see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion Structural Biology 3: 564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al., Nature, 352: 624-628 (1991) isolated a different array of anti-oxazolone antibodies from a small combinatorial pool of orange blossom V genes derived from the kidney of immunized mice. A repertoire of V genes from immunized human donors can be constructed and antibodies in a different array of antigens (including antigens themselves) can be isolated essentially following the techniques described by Marks et al., J. Mol. Biol. 222: 581-597 (1991), or Griffith et al., EMBO J. 12: 725-734 (1993).

Bi-specific antibodies are antibodies that have binding specificity for at least two different antigens. In the present case, one of the binding specificities is towards one CHA (preferably the CHD ECDs) the other is towards any other antigen. Bispecific antibodies can be prepared as full-length antibodies or fragments of antibodies (for example, bispecific antibodies) F (ab ') 2. Procedures described below are used for the preparation of bispecific antibodies as well as the preparation of multimerization regions of CHAs of the invention.

Methods for the preparation of bispecific antibodies are known in the medium. The traditional production of full-length bispecific antibodies is based on the coexpression of two heavy chain-light chain immunoglobulins, in which the two chains have different specificities (Millstein et al., Nature 305: 537-539 (1983)). Because of the orange blossom classification of heavy and light chain immunoglobulins, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule, which is usually given by chromatographic affinity steps, is rather difficult to handle, and the yields of the product are low. Similar procedures are set forth in WO 93/08829, and in Trauckecker et al., EMBO j. 10: 3655-3659 (1991).

According to different approaches, antibodies of variable regions with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin sequences of constant regio. The fusion preferably is with heavy chain constant region immunoglobulins, comprising at least part of the axis, CH2, and CH3 regions. It is preferred to have the first heavy chain constant region (CH1) containing the necessary site to bind light chain present in at least one of the fusions. DNAs encoding the heavy chain immunoglobulin fusion, if desired, the light chain immunoglobulin, are inserted into separate vector expressions, and are co-transfected into a suitable host organism. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments in which unequal proportions of the three polypeptide chains used in the construction provide optimum performance. It is, however, possible to insert the coding sequences for two or more polypeptide chains into a vector expression when the expression of at least two polypeptide chains in equal proportions results in high yields or when the results are not of particular significance.

In a preferred embodiment of this approach, bispecific antibodies are composed of a heavy chain immunoglobulin hybrid with a first binding specificity in one arm, and a heavy chain-chain immunoglobulin hybrid pairs (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitated the separation of the desired bispecific compounds from immunoglobulins of unwanted chain combinations, as the presence of a light chain immunoglobulin in only one half of the bispecific molecule provides an easy way of separation. This approach is set forth in WO 94/04690. For further details of bispecific antibody generation see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

According to another approach, the interface between a pair of antibody molecules can be designed to maximize the percentage of heterodimers that are recovered from recombinant cell culture. The preferred interface comprises at least a portion of the CH3 region of a constant region antibody. in this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with longer side chains (eg, tyrosine or tryptophan). Compensatory spaces of identical or similar size in the long side chains are created on the interface of the second antibody molecule by replacement of the long side chains of the amino acid with small ones (for example alanine or trionine). This provides a mechanism to increase the performance of the heterodimer over other undesirable end products such as homodimers.

Bispecific antibodies include "cross-linked" or "heteroconjugate" antibodies. "For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin, such antibodies have, for example, been proposed for target cells of the immune system of undesirable cells (US Patent No. 4,676,980), and - for the treatment of HIV infections (WO 91/00360), heteroconjugate antibodies can be made using any of the convenient cross-linking methods. known in the medium, and are disclosed in US Patent No. 4,676,980, together with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragments can be prepared using chemical linkage. Brennan et al., Science 229: 81 (1985) describes a procedure in which intact antibodies are proteolytically divided to generate F (ab ') 2 fragments. These fragments were reduced in the presence of the dithiol of the complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formations. The generated Fab 'fragments are then converted into thionitrobenzoate derivatives (TNB). One of the Fab'-TNB derivatives is then reconverted to Fab '-thiol by reduction with mercaptoethylane and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Recent advances have facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describes the production of a completely humanised bispecific antibody (ab ') 2 molecule. Each Fab 'fragment was separately secreted in E. coli and subjected to direct chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was capable of binding to overexpressed erbB cells and normal human T cells, as well as initiator of the lytic activity of human cytotoxic lymphocytes in contrast to target human breast tumors.

Several techniques for making and isolating bispecific antibody fragments directly from recombinant cell cultures have also been described. For example, bispecific antibodies have been produced using terminators-leucine. Kostelny et al., J. Immunol. 148 (5): 1547-1553 (1992). The leucine-ter peptides of the Fos and Jun proteins were linked to the Fab 'portions of two different antibodies by gene fusion. The homodimer antibodies were reduced in the central region to form monomers and then reoxidized to form the heterodimer antibodies. This method can also be used for the production of homodimer antibodies. The "diantibody" technology described by Hollinger et al. Proc. Nati Acad. Sci. USA, 90: 6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy chain (VH) region connected to a light chain variable region (VL) by a linker that is also short to maintain pairing between the two regions of the same chain. Accordingly, the VH and VL regions of one fragment are forced to pair with the complementary regions VL and VH of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv dimer (sFv) have also been reported. See Gruber et al. J. Immunol. 152: 5368 (1994).

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tut et al. J. Immunol. 147: 60 (1991).

To manufacture a neutralizing antibody, antibodies are made using techniques for regeneration of these molecules elaborated above. The preferred neutralizing antibody is specific for the extracellular region of the CHA and cross-reacts with the extracellular reegion of the natural heteromultimeric receptor, but does not cross-react with other receptors. Following the production of a series of antibodies, the antibodies are subject to screening process. effect of identifying those molecules that meet the desired criteria (for example, those that are capable of neutralizing a biological activity of the natural heteromultimeric receptor either in vitro or in vivo). For example, the ability of erbB-Ig CHA to block ErbB activity in any one or more of the tests described above can be evaluated. Those CHAs or anti-CHA antibodies that block the ability of HRG to bind and / or activate an ErbB receptor and / or the mitogenic activity of HRG on cells can be selected as neutralizing antibodies CHAs or CHA antibodies.

The antibodies can be coupled to a cytotoxic agent or enzyme (for example, above activating-prodrug = in a manner similar to that described above for a CHA.In addition, the antibodies can be labeled as described above, especially especially where the Antibodies have been used in diagnostic tests.

. Diagnostic Equipment and Articles of Manufacture.

Since the invention provides at least two types of diagnostic tests (for example detecting cancer using anti-ErbB-Ig antibody, for example, and to detect the presence of Hrg in a sample using ErbB-Ig, for example) as a matter of of convenience, reagents for these tests can be provided in a piece of equipment, for example, a combination of packaged reagents, to be combined with the sample to be tested. The components of the equipment will normally be provided in predetermined proportions. Accordingly, a kit can comprise the CHA or anti-CHA antibody labeled directly or indirectly with a suitable label. Where the detectable label is an enzyme, the equipment will include substrates and cofactors required by the enzyme (for example, precursor substrate that provides the detectable chromophore or fluorofor). furtherOther additives may be included such as stabilizers, regulators and the like. The relative amounts of the various reagents can vary widely to provide solution concentrations of reagents that substantially optimize the sensitivity of the test. Particularly, the reagents can be provided as dry powders, usually lyophilized, including excipients which in solution will provide a reactive solution having the appropriate concentration. The equipment also suitably includes instructions for carrying out the bio-assay.

In another embodiment of the invention, there is provided an article of manufacture containing materials useful for the treatment of the disorders described above. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, bottles, syringes, and test tubes. The containers may be formed of a variety of materials such as glass or plastic. The container contains a composition that is effective to treat the condition and can have a sterile access port (for example the container can be a bag for intravenous solution or bottles having a plug pierceable by a hypodermic injection needle). The active agent in the composition is CHA or an anti-CHA antagonist HRG antagonist thereof. The label on, or associated with, the container indicates that the composition is used to treat the selected condition. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable regulator, such as phosphate saline regulator, Ringer's solution, and dextrose solution. It may also include other desirable materials from the commercial and user's point of view, which include other regulators, diluents, filters, needles, syringes, and packaging with inserted instructions for use.

EXAMPLES The following examples are offered by way of illustration and not in a limiting manner. The examples are provided so as to provide those skilled in the art with a complete disclosure and description of how to make and use the compounds, compositions, and methods of the invention and do not attempt to limit the scope that the inventors considered their invention. Efforts were made to ensure accuracy with respect to the numbers used (eg, quantities, temperature, etc.) but some experimental errors and deviations could be explained.Unless otherwise indicated, parts are parts by weight, temperature is in degrees Celsius and pressure is at or almost atmospheric.Exposure of all citations in the specification are expressly incorporated herein as a reference.

Example 1: Materials and Methods These examples describe the construction, isolation and biochemical characterization of the chimeric amino acid sequences ErbB2-IgG, ErbB3-IgG, and ErbB4-IgG and the resultant chimeric heteromultimers of the present invention.

Reagents: The EGF-typical region of HRGßl (i77-244) was expressed in E. Coli, purified and radioiodinated as previously described (Sliwkowski, M. et al. J. Biol. Chem. 269: 14661-14665 (1994)) . Full length rHRGβ, which was expressed in Chinese hamster ovary cells, was used in Western blot analysis. Anti-ErbB2, 2C4 and 4D5 monoclonal antibodies have been described elsewhere (Fendly et al. Cancer Research 50: 1550-1558 (1990)).

Immunoadhesins ErbB2-, ErbB3-, and ErbB4-: A single site Mlu I was designed on a human heavy chain IgG d-expressor plasmid (pDR, a gift from J. Ridgeway and P. Carter, Genentec, Inc.) in the coding region of the immunoglobulin central region. Mlu I sites were also designed in an expression set of ErbB plasmids in the coding region of the ECD / TM junctions of these receptors. All mutagenesis was done using the Kunkel method (Kunkel, T. Proc. Nati, Acad. Sci. USA 82: 488 (1985)). The Mlu I sites were used to elaborate the proper construction of the ErbB-IgG fusion. The fusion of junctions of the various ErbB-IgG chimeras were: for ErbB2, E raised to the power 646 ErbB2 index raised to the power - (TR) -DKTH raised to the power 224 VH index; for ErbB3, L raised to power 636 ErbB3 index raised to power - (TR) -DKTH raised to power 224 VH index; for ErbB4, G raised to power 640 ErbB4 index raised to potency - (TR) -DKTH raised to potency 224 VH index, where the amino acid numbering ErbB polypeptides is described in Plowman et al. (Plowman, GD and collaborators, (1993a) PNAS USA 90: 1746-1750). The conserved TR sequence is derived from the Mlu I site. The sequence of the Fe region used in the preparation of the fusion construct is found in Ellison, J. W. et al. (Ellison, J. W. et al. (1982) NAR 10: 4071-4079). The construction of the final expression was in a pRK-type main plasmid which is characterized in that the eukaryotic expression is driven by a CMV promoter (Gorman et al., Prot. Eng. Eng. Tech. 2: 3-10 (1990)).

To obtain proteins for in vitro experiments, HEK-293 adherent cells (ATCC No. CRL-1573) were transfected with the expression of appropriate plasmids using standard methods of calcium phosphate (Gorman et al., Supra and Huang et al., Nucleic Acids. Res. 18: 937-947 (1990)). Serum-containing medium was replaced by serum-free medium 15 hours post-transfection and transfected cells incubated for 5-7 days. The resulting conditioned medium was collected and passed through Protein A columns (1 mL Pharmacia HiTrap ™). Purified IgG fusions were eluted with 0.1 M citric acid (pH 4.2) in tubes containing 1 M Tris pH 9. The eluted proteins were subsequently dialyzed against PBS and concentrated using Centri-prep 30 (amicon) filters. Glycerol was added to a final concentration of 25% and the material was stored at -20 ° C. The concentrations of the material were determined via an Fc-ELISA. 125I-HRG binding tests: Link tests were performed on the Nunc plate separation immuno-module. The dishes were well covered at 4 ° C overnight with 100 μg / ml. of anti-human-goat antibody (Boehringer Mannheim) in 50 mM carbonate buffer (pH 9.6). Plates were rinsed twice with 200 μl of wash buffer (PBS / 0.05% Tween ™) followed by a brief incubation with 100 μl of 1% BSA / PBS for 30 min. at room temperature. The regulator was removed and each well was incubated with 100 μl of IgG fusion protein in 1% BSA / PBS under vigorous shaking from side to side for one hour. The plates were rinsed three times with regulator of lavdo and the competitiov link was carried out - by adding several amounts of cold competitor? -HRG and 25l-HRGpl and incubated at room temperature for 2-3 hours with vigorous rotation from side to side. The receptacles were quickly rinsed three times with draining buffer, drained and the individual receptacles were counted using a "100 Series Iso Data? -counter". Scatchard analysis was performed using a modified Ligand program (Munson, P. and Robard, D. (1980) Analytical Biochemistry 107: 220-239). 3H-Thymidine incorporation test: Tritiated thymidine incorporation tests were performed in a format-96 receptacle. MCF7-7 cells were placed in dishes of 10,000 cells / covers with 50:50 F12 / DMEM (high glucose) 0.1% calf fetal serum (100 mL). The cells were maintained to pellet for 3 hours, after which the ErbB-IgG and / or heregulin fusion proteins were added into the containers (the final volume of 200 mL) and the plates incubated for 15 hours in a culture incubator. epithelial at 37 ° C. Tritiated thymidine was added to the containers (20 mL of tritiated thymidine diluted 1/20 standard: Amersham TRA 120 B363, 1 mCi / mL) and the dishes were incubated for 3 additional hours. The tritiated material was collected on GF / C unifilters (96 format containers) using a "Packard Filtermate 196" collector. The filters were counted using a "Packard Topcount" device Example 2: ErbB3-IgG and ErbB4-IgG proteins that bind HRG.

As described above, a series of plasmid constructs that were prepared allowed the eukaryotic expression of the extracellular regions (ECDs) of ErbB receptors fused to the constant regions of human IgG. As described in Fig. 1, these receptor-IgG constructs exist in solution as dimers bound to disulfides. IgG homodimer receptors for ErbB2, ErbB3 and ErbB4 were individually expressed in HEK-293 cells and the receptor secreted by the resulting fusion proteins, was purified by affinity chromatography on protein A. Chen et al. (Chen X. et al., (1996) J. Biol. Chem. 271: 7620-7629) reported a similar construction of the immunoadhesins ErbB4- and ho odi érica ErbB3-, which were used as immunogens for the generation of the receptor-specific monoclonal antibody. Binding analysis of the chimeric immunoadhesin proteins was carried out using a microtiter plate format (see example 1). As shown in Fig. 2, the homodimeric ErbB3-IgG and ErbB4-IgG were able to specifically bind 125I-HRG, considering the link that was detected with the ErbB2-IgG construct not discernible. Scatchard analysis of HRG to ErbB3-IgG links exposes a single binding affinity site with a Kd of 9.3 ± 2.9 nM. Conjugate constants for detergents -ErbB3 solubilized expressed in insect cells (Carraway et al., 1994), ErbB3 expressed in C0S7 cells (sliwkowski et al., (1994) supra) and ErbB3 expressed in K563 cells arranged between 0.8 to 1.9 nM. ErbB3-homodimeric IgG has a higher constant affinity for HRG than the 26 nM value recently reported by Horan et al. (Horan et al., (1995) J. Biol. Chem. 270: 24604-24608) in an analysis that used a Soluble monovalent ECD of ErbB3. These data suggest that the optimal conformation of the binding site HRG on ErbB3 can be stabilized by a lipid bilayer. A greater loss of binding affinity relative to the intact receptor has also been reported for soluble versions of the EGF receptor (Brown, P. M. et al., (1994) Eur. J. Biochem. 225: 223 ^ 23; and Zhou, M. et al. (1993) Biochemistry 32: 8193-8198). The affinity constant measured for ErbB4 = Ig was 5.0 ± 0.8 nM. This value is in close agreement with that reported by Tzahar and collaborators of 1.5 nM for full-length ErbB4 expressed in C0S7 cells (Tzahar, E. et al., (1994) J. Biol. Chem. 269: 25226-25233).

However, neurogulins are a family of proteins originating in the alternative RNA binding, the receptor binding is through the EGF-typical quality present in every active isoform. Chimeric homodimeric immunoadhesins containing the multiple bonding forms of ErbB3 or ErbB4 ECDs of the neurogulin family provided that the EGF-typical regions of these proteins are present. The variants of heregulins that bind these homodimers included rHRGßl? -2 4, rHRGβ? -244, thioredoxin-HRGß? .4-24, and thioredoxin-? HRG, rHRGa? -23 ..

Example 3: Heterodimeric ErbB-IgG fusion proteins that form a high-affinity HRG binding site when ErbB2 is present with ErbB3 or ErbB4 Heterodimeric versions of the constructed IgG receptor were generated by coXransfection of two expressions of plasmids encoding different receptors in the same cell (see Example 1). The secreted forms of the resulting IgGs receptor are mixtures of two types of homodimers and the expected heterodimer. Three different co = transfections were carried out to generate the following ErbB mixtures: ErbB2 / 3-IgG, ErbB2 / 4-IgG and ErbB3 / 4-IgG. Binding affinities for each of the mixtures were then determined. As shown in Fig. 3A, a high affinity HRG binding site could be detected with heterodimers containing ErbB2 but not ErbB3 / 4-IgG. Scatchard plots of these data were curvilinear for mixtures of heterodimers containing ErbB2 (Fig. 3B and 3C) suggesting the presence of two distinct types of binding sites (Munson, P. and Robard, D. (1980) Analytical Biochem. : 220-239). AKd of 0.013 nM was measured for the high affinity binding site, considering that the low affinity binding site had a Kd of 12 nM. The high affinity binding constant is in agreement with the measured values when ErbB3 is expressed in cells containing high levels of ErbB2 (Carraway et al., (1994) supra) or when high-affinity HRG binding sites are determined from a 2-site suitable to link data in high ErbB3 basic. (Sliwkowski et al. (1994) supra). ErbB2 / 4-IgG (Fig. 3CJ also exhibited a similar ainity that changes when compared to the ErbB4-IgG homodimer.) The measured ainity constant for ErbB2 / 4-IgG was 0.017 nM, again using a suitable 2-site to a binding site of low affinity, a Kd of 5 nM was measured.This value is closely in agreement with the Kd measured for the ErbB4-IgG homodimer, In contrast, the ErbB3 / ErbB4-IgG protein (Fig. 3D) did not present a high affinity site, but in exchange a Kd of 6 nM was measured, which was comparable to that found for the ErbB3-IgG and ErbB4-IgG homodimers. The formation of a binding high affinity binding site correlated with the expression of the ErbB2 ECD with an ECD from another member of the ErbB family suggests that ErbB2 was required for the formation of a high affinity site. A summary of binding constants for the ErbB-IgG fusion proteins is shown in Table I. The high-affinity binding site that was formed for the odimeric protein ErbB2 / 3-IgG or ErbB2 / 4-IgG was 300-700 times greater than that corresponding to homodimeric species.

Table I. Linking Constants for ErbB-IgG Homodimers and Heterodimer Immunoadhesins.

Constructions ErbB-IgG Kd (nM) ErbB2 NB ErbB3 9.24 + 2.94 ErbB4 4.9810.80 ErbB2 / 3 0.013 + 0.004 ErbB2 / 4 0.01710.009 ErbB3 / 4 5.9810.70 N.B. indicates non-measurable links.

In addition to testing the hypothesis that ErbB2 contributed to the formation of the high affinity binding site, the effect of an anti-ErbB2 ECD antibody to inhibit high affinity binding in the ErbB immunoadhesins was examined. Binding reactions were conducted in the presence of a 2C4 antibody, which is specific for ErbB2 ECD (Lewis, G. D. et al. (1996) Cancer Res. 56: 1457-1465; Sliwkowski et al., (1994) supra). As shown in Fig. 4A, adding monoclonal antibody 2C4 had a marked inhibitory effect on the HRG bond by the ErbB2 / ErbB3-IgG heterodimer but not by the corresponding ErbB3-IgG homodimer. Similarly the anti-ErbB2 monoclonal antibody also performed HRG binding in the ErbB2 / ErbB4-IgG heterodimer (Fig. 4B) but not in the corresponding ErbB4-IgG homodimer. These data indicate that the physical interaction of the ErbB2 ECD with the ECD of either ErbB3 or ErbB4 results from the formation of a high affinity growth factor binding site in this soluble receptor system.

Example 4: ErbB-IgG Iusion Proteins That Inhibit the Biological Effects of HRG With HRG treatment, a number of different cell types have been known to undergo proliferative responses. The ability of ErbB-IgG proteins to inhibit HRG-thymidine-dependent incorporation was tested in the breast carcinoma cell line, MCF7 (Lewis et al., (1996) supra). Variable concentrations of the different EebB-IgG proteins were incubated with 1 nM of HRG and then added to frozen monolayer cultures of MCF7 cells (see example 1). Following 24 hours of incubation, the cells were then labeled with 3H-thymidine to measure DNA synthesis. As shown in Fig. 5, all fusions of receptors capable of HRG binding inhibited the HRG-by mitogenic response in a dose in a relative manner. The heterodimeric IgGs, ErbB3 / 2-IgG and ErbB4 / 2 were more potent than their corresponding homodimeric fusion proteins.

DISCUSSION The extracellular region of ErbB2 modulates the binding of HRG to ErbB3 and ErbB4.

Immunoadhesins offer numerous advantages for in vitro analysis (see Chamow, S.M. and Ashkenazi, A. (1996) Trends in Biotechnology 14: 52-60, for review). It is the dimerization capacity of the IgG fusions that seem to mimic the putative heterodimerization in vivo of the ErbB family of receptors resulting from the generation of the binding site of high affinity heregulin. Analysis of Hrg linkages showed that heterodimeric mixtures including ErbB2, for example, ErbB2 / ErbB3-IgG and ErbB3-IgG or ErbB4-IgG, produced a binding site of heregulin with more than 300 times higher affinity than that found in the ErbB3- homodimers IgG or ErbB4-IgG or in the ErbB3 / ErbB4-IgG heterodimer. The low affinity HRG binding site present in the ErbB3 / ErbB4-IgG heterodimer suggests that the creation of a binding site in high affinity heregulin can not be made by the combination of any two different ErbB-IgGs, but is preferably specific to mixtures containing ErbB2-IgG. Further evidence for the requirements of ErbB2 to generate this high affinity binding site was determined with monoclonal antibodies directed against ErbB2 (Lewis et al., (1996) supra; Sliwkowski et al., (1994) supra). When linkage studies were conducted with heterodimers containing ErbB2 in the presence of these antibodies, a significant decrease in the affinity of enl'ce HRG was observed.

The formation of the HRG-ErbB3-ErbB2 complex occurs sequentially in cell lines that express normal levels of these receptors. Specifically, HRG links for ErbB3 and ErbB3es are then supplied for this busy HRG receiver. Complex formation results in a decrease in the ligand dissociation ratio, which generates a high affinity binding site (Karunagaran, D. et al. (1996) EMBO J. 15: 254-264). It is now reported that the formation of the high affinity complex also took place in a soluble receptor system in the absence of intracellular and transmembrane regions, provided that a dimerization ability was present. In contrast, Horan et al. (Horan, T. et al. (1995) J. Biól. Chem. 270: 24604-24608) reported no apparent increments of > HRG binder of ErbB3-ECD when adding ErbB2-ECD. In agreement with these findings, a similar result was obtained if homErbB-homodimeric IgGs were produced from individually transfected cells that were mixed and tested to bind heregulin. The resulting mixtures of ErbB2-IgG homodimers mixed with ErbB3-IgG or ErbB4-IgG homodimers did not exhibit any higher ligand affinity than ErbB3-IgG or ErbB4-IgG alone. The dimerization ability replaced by the Fe component is therefore an important feature in the formation of binding sites of high affinity ligands. On the other hand, the flexibility of the main region can also help by facilitating these ligand-receptor interactions. Without being limited to any of the theories, with intact receptors fixed in a cell membrane, other abilities, such as the transmembrane regions or the intracellular region, may also contribute to the stabilization of ErbB2 containing hetero-oligometric complexes.

The role of ErbB2 in an oligomeric complex heregulin-indicator receptor.

Oligomerization of an induced ligand-receptor is a common paradigm for the passage of individual transmembranes to receptors (Ulrich, A. and Schlessinger, J. (1990) Cell 61: 203-212; and Wells, JA (1994) Curr Opin Cell Biol. 6: 163-173). Based on the discussion here of a soluble chimeric heterodimer composed of either ErbB3 or ErbB4 with ErbB2, it is concluded that such a chimeric heterodimer is sufficient for the formation of a high affinity binding condition. Two possible models are proposed that are consistent with these data (Fig. 6). The "contact" model is analogous to that developed for hormonal development and its receptor (Wells, J. A. (1996) PNAS USA 93: 1-6), except that the lreside site in ErbB3 or ErbB4 and site 2 is contributed by ErbB2. This model predicts that the affinity for binding HRG at site 1 would be similar to that measured for the ErbB3 or ErbB4 homodimers. ErbB2 is then included in the ErbB3-HRG or ErbB4-HRG complex, and contacts the HRG-ErbB3 (or ErbB4) link. The formation of the ErbB3-HRG-ErbB2 complex decreases the dissociation of HRG and generates the high affinity binding condition. Alternatively, the "conformation" of the model postulates that ErbB2 modulates the interaction of Hrg with ErbB3 or ErbB4, but contact between ErbB2 and HRG does not take place. In this model, the interaction of ErbB2 with ErbB3 or ErbB4 alters the conformation of these receptors and creates a high affinity binding condition.

Using crosslinked Hrg-crosslinked chemical techniques on cells expressing ErbB3 and ErbB2 (Holmes, EE et al. (1992) Science 256: 1205-1210; Sliwkowski et al., (1994) supra), cross-linked complexes corresponding to proteins with Molecular sizes of approximately 190 kDa and greater than 500 kDa were observed. These results suggest that the oligomeric structure of the receptor complex may include multiple copies of ErbB3 and ErbB2. In addition, since ErbB3 is devoid of intrinsic tyrosine kinase activity (Guy or collaborators (1994) supra), this hypothesis offers a clarification for the increase of the ligand-dependent tyrosine phosphorylation that is observed for both ErbB2 and ErbB3. For example, a complex containing two copies of ErbB3 and two copies of ErbB2 could consider the phosphorylation of ErbB3, and likewise the transphosphorylation of secondary ErbB2 receptors. Immunoadhesins homodimers of the TNF receptor (Ashkenazi, A. et al., (1991) PNAS USA 88: 10535-10539) appear to simulate the TNF receptor system in which the cell surface of the TNF receptor is a trimer (Banner, DW et al. (1993). ) Cell 73-431-445).

Biological implications of the modulation of ErbB2 in ErbB3 and ErbB4.

Since ErbB2 was discovered, it has been assumed that there should be a ligand that only acts on and activates ErbB2. However, numerous candidate proteins have been successively placed as putative ligands for ErbB2 (reviewed in Hynes, NE and Stern, DF (1994) Biochem, Biophys, Acta 1198: 165-184), the proteins have not been characterized at the molecular levels that Fill these criteria. Other studies have suggested that ErbB2 seems to play a multifaceted role in both EGF and heregulin receptor complexes (Earp et al., (1995) supra: Karunagaran et al. (1996) supra.) The functions of ErbB2 in these complexes include altering affinity. of the binding region of the ligand, contributing to contribute very potent tyrosine kinase components and provide tyrosine residues that with phosphorylation provide activation and amplification in several ways of transduction indicators.The activation of ErgB2 heregulin is physiologically relevant in neural junctions. nuscular (Altiok, N. et al., (1995) EMBO j. 14: 4258-4266; Chu, GC et al., (1995) Neuron 14: 329-339; and Jo, SA et al (1995) Nature 373: 158-161) and in schwanneural cell junctions (Dong, Z. et al. (1995) Neuron 15: 585-596; Marchioni, MA et al. (1993) Nature 362: 312-318; and Morrissey, TK et al. (1995) PNAS USA 92: 1431-1435). In experimental cell cultures, human tumor cell lines were used, several reports show that eliminating ErbB2 interactions with either ErbB3 or ErbB4 decreases downstream indicators as well as subsequent biological responses such as growth.

(Karunagaran et al. (1996) supra; Lewis et al. (1996) supra; Pinkas-Kramarski, R. et al. (1996) EMBO J. 15: 2452-2467). The concept of ErbB2 as a permanent "orphan" receptor (Lonardo et al., 1990) is also supported by recent reports on the definitive ErbB2 and neuregulin phenotypes. In both cases, mice that are homozygous for the two mutations were lethal in embryo near E10.5 (Lee, KF., Et al. (1995) Nature 378: 394-398; and Meyer, D. and Birchmeier, C. (1995 ) Nature 378: 386-390) In each case, the dead embryos of a similar cardiac phenotype, the lack of ventricular trabeculation. Both embryos also had rigorously similar malformations of the metencephalon. These observations also suggest that ErbB2 is critical to translate HRG indicator. Under normal biological circumstances, the sole function of ErbB2 ° appears to mediate HRG responses and EGF ligand as a common member of these complex receptors.

Although the invention has been described with reference to the presently preferred embodiment, it could be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Having been described as above, it is claimed as property in the following,

Claims (24)

1. An isolated recombinant chimeric heteromultimer adhesive which is characterized in that it comprises: a first amino acid sequence characterized in that it comprises an extracellular region of a monomeric ErbB2 receptor or fragments thereof and a first heterologous multimerization region; an additional amino acid sequence which is characterized in that it comprises an extracellular region of a monomeric ErbB receptor or fragments thereof, and a second heterologous multimerization region, which is characterized in that the multimerization region of the first amino acid sequence and each of the additional amino acid sequences comprise a constant region immunoglobulin or fragments thereof; which is characterized in that the extracellular region of the first amino acid sequence and the extracellular region of the additional amino acid sequence are brought together via interaction of the multimerization region of the first amino acid sequence and the multimerization region of the additional amino acid sequence to form a binding region of a chimeric heteromultimeric adhesin having 1/101 up to 6 fold, affinity for the ligand relative to the monomer of the receptor or to a receptor heteromultimer and is characterized in that the chimeric heteromultimeric adhesin is water-soluble.

2. The isolated recombinant chimeric heteromultimeric adhesin of claim 1 is characterized in that the extracellular region of the additional amino acid sequence is any one of: (i) a monomeric ErbB3 receptor, and which is characterized in that the chimeric heteromultimeric adhesin is an ErbB2-IgG / ErbB3-IgG adhesin, or (ii) a monomeric ErbB4 receptor and which is characterized in that the chimeric heteromultimeric adhesin is an ErbB2-IgG / ErbB4-IgG adhesin.

3. The chimeric heteromultimeric adhesin of claim 1 or claim 2, which is characterized in that the ligand is a neuregulin.

4. The chimeric heteromultimeric adhesin of any one of the preceding claims which is characterized in that the chimeric heteromultimer is a ligand antagonist.

5. An isolated nucleic acid sequence encoding an amino acid sequence of the chimeric heteromultimeric adhesin of any one of the preceding claims.

6. The isolated nucleic acid of claim 5 which is characterized in that it additionally comprises a promoter operably linked to the nucleic acid molecule.

7. A vector that is characterized in that it comprises the isolated nucleic acid of claim 5 or claim 6.

8. A host cell which is characterized in that it comprises the nucleic acid of claim 5 or claim 6, or the vector of claim 7.

9. An antibody of the chimeric heteromultimeric adhesin of any of claims 1 to 4, which is characterized in that the antibody which is a ligand antagonist, binds to the heteromultimer receptor as it naturally occurs and activates it.

10. An antibody of the chimeric heteromultimeric adhesin of any one of claims 1 to 4 which is characterized in that the antibody which is a ligand agonist, binds to the heteromultimer receptor as it naturally occurs and activates it.

11. A method of forming a chimeric heteromultimeric ligand-adhesin complex in a sample comprising the ligand, said method is characterized or comprises: contacting the chimeric heteromultimer of any one of claims 1 to 4 with the sample under conditions such that the ligand binds to the chimeric heteromultimeric adhesin to form a heterodimeric ligand-adhesin complex.

12. The method of claim 11 which is characterized in that the complex inhibits the binding of the ligand to the heteromultimer as it is naturally found, and that it is characterized in that the sample is from a mammal and is selected from the group consisting of a tissue, a fluid, and a body fluid.

13. A method of inhibiting the activation of a heteromultimer receptor as found naturally, which is characterized in that the method comprising; contacting the chimeric heteromultimeric adhesin of claim 1 with a sample comprising a ligand of the heteromultimeric receptor as found naturally and the receptor, and incubating the chimeric heteromultimeric adhesin with the ligand to form a complex such that activation of the heteromultimeric receptor as is found naturally by the ligand, is inhibited.

14. The method of claim 13 which is characterized in that the additional amino acid sequence comprises any one of: (i) the extracellular region of ErbB3 or fragments thereof, or (ii) the extracellular region of ErbB4 or fragments thereof.

15. A method of inhibiting the activation of the heteromultimeric ErbB receptor as found naturally, the method is characterized in that it comprises contacting the antagonist antibody of claim 9 with the heteromultimeric receptor as it is naturally found to form a heteromultimer receptor-antagonist antibody complex, which is characterized in that the activation of the receptor is inhibited.

16. A method of activating a natural heteromultimeric receptor that is characterized in that the method comprises contacting the agonist antibody of claim 10 with the heteromultimeric receptor as it is naturally found to form a heteromultimeric receptor-agonist antibody complex, which is characterized in that the recpetor is activated .

17. A pharmaceutical composition which is characterized in that it comprises the chimeric heteromultimeric adhesin of any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

18. A pharmaceutical composition which is characterized in that it comprises the antagonist antibody of claim 9 and a pharmaceutically acceptable carrier.

19. A pharmaceutical composition which is characterized in that it comprises the agonist antibody of claim 10 and a pharmaceutically acceptable carrier.

20. An article of manufacture, which is characterized in that it comprises: a container; a label on said container; and a composition contained in said container; the composition is characterized in that it comprises the chimeric heteromultimeric adhesin of any one of claims 1 to 4, and the composition is characterized in that it is effective to antagonize ligand bonds to its heteromultimeric receptor as it is antially, and the label on the container indicates that the composition can be used to antagonize ligand bonds to the heteromultimeric receptor as it is naturally found.

21. A manufacturing article that is characterized in that it comprises: a container; a label on said container; and a composition contained in said container; the composition is characterized in that it comprises the anti-chimeric heteromultimeric adhesin antibody of claim 9, and the composition is characterized in that it is effective to antagonize ligand bonds to its heteromultimeric recpeptor as naturally found, and the label on the container indicates that the composition can be used to antagonize ligand bonds to its heteromultimeric receptor as it is naturally found.

22. A manufacturing article that is characterized in that it comprises. a container; a label on said container; and a composition contained in said container; the composition is characterized in that it comprises the antibody of the heteromultimeric anti-chimeric adhesin of claim 10, and the composition is characterized in that it is effective to agonize the activation of the heteromultimeric receptor as it is naturally found, and the label on the container indicates that the composition it can be used to agonize the activation of the heteromultimeric receptor as it is naturally found.

23. A method of producing the antibody of claim 9 or of claim 10, said method is characterized in that it comprises the step of immunizing an animal with the chimeric heteromultimeric adhesin of any of claims 1 to 4.

24. The chimeric heteromultimeric adhesin of any of claims 1 to 4; the antibody of claim 9 or claim 10: or the pharmaceutical composition of any of claims 17 to 20 for the treatment of any one or more of the following disease conditions: inflammatory disorders, cancer, neurofibromatosis; peripheral neuropathologies; cardiac hypertrophy.