WO1993012220A1 - RECOMBINANT AND CHIMERIC ANTIBODIES TO c-erbB-2 - Google Patents

Abstract

This invention relates to methods for producing recombinant and chimeric antibody peptides that recognize the c-erbB-2 oncogene, and to vectors, host cells and DNA sequences for producing such antibody peptides.

Description

RECOMBINANT AND CHIME IC ANTIBODIES TO c-erbB-2

Field of the Invention This invention relates to methods for producing recom binant and chimeric antibody peptides that recognize th c-erbB-2 oncogene protein, and to vectors, host cells and DN seguences for producing such antibody peptides.

Background of the Invention

The mechanism for malignancy of mammalian cells has bee and continues to be the subject of intense investigation. number of oncogenes, some of which encode proteins that regu late cell growth and differentiation, have been shown to pla an important role in causing cancer. Many of the protein encoded by oncogenes function abnormally in malignant cells o are not regulated by normal control processes. This seems t play a part in the transformation of normal cells into cance cells.

The c-erbB-2 protein (also referred to as c-erbB-2 pro¬ tein receptor and sometimes designated HER-2 or neu) is a 185 kilodalton (Kd) glycoprotein having tyrosine kinase activity and is related to, but distinct from, the epidermal growth factor receptor (EGFR) . Over-expression of c-erbB-2 has been linked to poor clinical outcome and shortened disease-free survival of mammary and ovarian cancer patients. Slamon et al.. Science, 235:177 (1987); Berchuck, A., et al., Canc Res. , 50:4087 (1990).

Antibodies are molecules that recognize and bind to specific cognate antigen. Numerous applications of hybridoma produced monoclonal antibodies for use in clinical diagnoses treatment, and basic scientific research have been described Clinical treatments of cancer, viral and microbial infections B cell immunodeficiencies, and other diseases and disorders o the immune system using monoclonal antibodies appear promis ing. In particular, TAb 250, a mouse monoclonal antibod against the c-erbB-2 protein has been shown to be cytostati for tumor cell growth. See Hancock et al., Can. Res. , 51:457 (1991) .

In addition, promising monoclonal antibodies have bee shown to interact synergistically with anti-neoplastic drugs. For example, an antibody to c-erbB-2 protein has been shown t synergistically enhance the anti-tumor activity of the dru cisplatin. Hancock et al. , J. Cell. Biochem. Supp. , 148:342 (1990) and Hancock et al. , Can. Res. , supra. Mouse monoclonal antibody production is well known in th art, and mouse monoclonal antibodies can be generated against a wide array of antigens. Clinical results, however, of some immunotherapies have been disappointing. One possible source of the problems associated with cancer immunotherapy is the patient's own immune response to the therapeutic antibody. When used for clinical treatment in humans, the mouse antibody is recognized as antigen and the human immune system mounts a response. The human response to the mouse antibodies poses two potential problems. First, the anti-mouse immunoglobulin response can cause harmful symptoms. Second, the response can prematurely clear the therapeutic antibody from the patient's system, reducing its effectiveness and requiring higher doses. Although treatment with hximan monoclonal antibodies has been investigated, human hybridoma cell lines tend to be unstable and produce low amounts of immunoglobulin.

One approach to circumvent the immunogenicity of non- human antibodies in humans is to use recombinant DNA tech- niques to incorporate the segments of a non-human antibody that determine the antibody's specificity with other segments of a human antibody. Much of the immunogenicity resides in the constant domains of the non-human antibody. For example, mouse variable region exons specific for tumor antigens have been fused with human K or γ constant regions. See, for example, Sahagan et al., J. Immunol . , 1066 (1986); Liu et al., Proc. Natl . Acad. Sci. USA, 84:3439 (1987); and steplewski et al., Proc. Nat l . Acad. Sci. USA, 85:4852 (1988). A variation of this approach uses DNA sequences that code only complementarity- determining regions, also known as CDRs, from a mouse or other foreign antibody specific for the antigen of interest.

One advantage of such chimeric forms is that the variable regions can conveniently be derived from presently known sources using readily available hybridomas or B cells from non-human host organisms. These variable regions can be combined with constant regions derived from, for example, human cell preparations. Use of the human constant region is less likely to elicit an immune response when the antibodies are injected into a human subject. In addition, the human constant regions may interact more effectively with the human effector cells of the immune system. Thus, it would be beneficial to develop such chimeric antibodies in cancer therapy.

Brief Description of the Drawings Various other objects, features and attendant advantages of the present invention will be more fully appreciated as the same becomes better understood when considered in conjunction with the accompanying drawings, in which like reference characters designate the same or similar parts throughout the several views, and wherein:

Figure 1 illustrates the cloning of a PCR-amplified mouse V_H fragment from the DNA of hybridoma cells producing an antibody to c-erbB-2. Plasmid pUC19 was cut with Eco RI and then treated with calf intestine alkaline phosphatase. The purified V_H fragment was then ligated into the Eco RI site to make pϋC19V_H; Figure 2 illustrates the cloning of a PCR-amplified mouse V_κ (V_L) region from the DNA of the hybridoma producing the same antibody to c-erbB-2 as in Figure 1. Plasmid pIBI21 was cut with Hind III and treated with phosphatase. The purified V_κ fragment was cloned into the Hind III site to make the plasmid pIBI21V_κ;

Figure 3 illustrates the cloning of a PCR-amplified mouse K enhancer. Plasmid pϋC19 was cut with Eco RI and treated with calf intestine alkaline phosphatase. The enhancer was ligated into the Eco RI site to make plθenhancer; Figure 4 illustrates the cloning of a PCR-amplified human γi constant region from the DNA of ARH-77 cells. Plasmid pBR322 was cut with Bam HI and Sal I and then treated with calf intestine alkaline phosphatase. The Cγl fragment was then ligated into the Bam HI and Sal I sites to make plasmid p322γi;

Figure 5 illustrates the cloning of a PCR-amplified human

C_κ constant region from the DNA of ARH-77 cells. Plasmid pUC19 was cut with Bam HI and Eco RI and then treated with calf intestine alkaline phosphatase. The Cj_ς fragment was then ligated into the Bam HI/Eco RI site to make plasmid pl9/c;

Figure 6 illustrates the first step in construction of a chimeric heavy chain expression vector: cloning the mouse V_H region into pBR322. Plasmid pUC19V_H was digested with Eco RI and Sal I and purified V_H fragment was subcloned into pBR322 at the Bam HI and Sal I sites with the use of a Bam HI and Sal I oligonucleotide adapter, to create p322V_H;

Figure 7 illustrates the second step in construction of a chimeric heavy chain expression vector: subcloning the mouse V_H and human Cγl fragment into plasmid pSV2neo. The mouse V_H and the human Cγl fragments were excised from p322V_H and p322γi, respectively, by cutting with Bam HI and Sal I. Plasmid pSV2neo was cut with Bam HI and treated with calf intestine alkaline phosphatase. The V_H and Cγl fragments were ligated into the pSV2neo vector at the Bam HI site to make pSVNH-L;

Figure 8 illustrates the first step in construction of a chimeric light chain expression vector: subcloning mouse Vc fragment into pBR322. Plasmid pBR322 was cut with Hind III and treated with calf intestine alkaline phosphatase. The V_κ fragment was excised from pIBI21V_Λ by cutting with Hind III. The V_κ fragment was ligated into pBR322 at the Hind III site to make p322V_Λ; Figure 9 illustrates construction of p322ΔRH. Plasmid pBR322 was cut with Eco RI and Hind III and then treated with Klenow fragment of DNA polymerase. The plasmid ends were ligated to make p322ΔRH;

Figure 10 illustrates the construction of a plasmid containing a mouse V_κ and human C^. The mouse V_κ and the human C_κ region were excised from p322V_κ and pl9κ, respectively, by cutting with both Bam HI and Eco RI. Plasmid p322ΔRH was cut with Bam HI and treated with calf intestine alkaline phosphatase. The V_κ and the C_κ fragments were ligated into the Bam HI site^' to make plasmid pΔRH-light;

Figure 11 illustrates the construction of an intermediate plasmid containing a mouse V_κ and human C_κ. Plasmid pΔRH-light was cut with Eco RI and Xba I. An Eco Rl-Xba I adapter wa ligated into the site to make pΔ-light;

Figure 12 illustrates the construction of a plasmi containing DNA encoding a mouse V_κ, a mouse K enhancer, an human C_κ region. Plasmid pΔ-light was cut with Eco RI an treated with calf intestine alkaline phosphatase. The mous K enhancer was excised from pl9enhancer by cutting with Ec RI. The enhancer was ligated into the Eco RI site of the cu pΔ-light to make the plasmid pΔL-enhancer containing chimeric K chain;

Figure 13 illustrates the final step in constructing chimeric light chain expression vector. Plasmid pRSV-gpt wa cut with Bam HI and treated with calf intestine alkalin phosphatase. The chimeric K chain DNA was excised from pΔL enhancer by cutting with Bam HI. The Bam HI fragmen containing the chimeric light chain was ligated into the Ba HI site of pRSV-gpt to make pRGL-L, a chimeric light chain expression vector;

Figure 14 illustrates the ability of mouse/human chimeric antibodies (clones E8 and A7) to displace the binding of a murine monoclonal antibody, TAb 250, to SKBR-3 cells bearing the c-erbB-2 protein;

Figure 15 illustrates that BACh 250 mouse/human chimeric antibodies suppressed proliferation of SKOV3 cells bearing the c-erbB-2 protein in a manner similar to TAb 250, or the Fab or F(Ab')₂ fragments of TAb 250; and

Figures 16A and B indicate that BACh 250 fixes rabbit complement more effectively than TAb 250 by measuring chromium release from SKBR-3 cells in the presence of the antibodies and rabbi ^"complement. Summary of the Invention Chimeric or recombinant antibody peptides specific for c-rbB-2 can be used as important therapeutic or diagnostic agents for treating mammary and ovary cancers, or other tumors expressing significant levels of c-erbB-2. For example, these chimeric antibodies and peptides could be used to trigger the patient's endogenous immune effector response; be covalently linked to a toxin or radionuclide and used as an immunotoxin; or be administered in combination with a cytotoxic or cytostatic agent. A murine monoclonal antibody to c-erbB-2 protein, TAb 250, does not mediate human immune effector functions including complement dependent cellular cytotoxicity and antibody dependent cellular cytotoxicity. A mouse/human antibody improves such functions while maintaining the chemotherapeutic effects of the antibody.

The invention includes recombinant DNA sequences encoding at least one CDR region derived from an antibody specific for c-erbB-2 protein. The CDR region may be derived from a heavy chain or light chain variable region of the antibody. In a preferred embodiment the CDR regions will be derived from an antibody that activates the signalling pathway of the c-erbB-2 protein. Such activation is indicated, for example, when the antibody induces an increase in phosphorylation of the c-erbB- 2 protein, causes down modulation of the protein, or induces phosphorylation of substrates such as phospholipase C (PLC-γi) when placed in contact with cells expressing the c-erbB-2 protein.

Further included are recombinant DNA sequences that encode an antibody heavy chain variable region or light chain variable region specific for c-erbB-2 protein. These sequences may be those which encode variable regions that, when combined with a complementary chain (heavy/light) (including those set forth in Sequence ID Nos. 1 or 2) , specifically bind c-erbB-2.

The invention also includes recombinant DNA sequences that encode an antibody light chain variable region or heavy chain variable region which, when incorporated into immuno- globulin conformation, competes with an antibody produced by a hybridoma cell line bearing A.T.C.C. Accession No. HB10646

(TAb 250) for the binding to c-erbB-2. Preferred sequences include those that comprise DNA encoding an antibody heavy chain variable region or light chain variable region to c-rbB-2 protein having an amino acid sequence consisting essentially of that sequence set forth in Sequence

ID No. 3 or 4, respectively.

Recombinant vectors, host cells, and chimeric antibody peptides, e.g., humanized antibody peptides are also encom¬ passed in the invention. For example, included are recombi¬ nant DNA sequences that comprise DNA encoding a chimeric c-rbB-2 specific heavy chain peptide, the variable region derived from a first genetic source and a constant region derived from a second and different genetic source. Also included are sequences that comprise DNA encoding a chimeric c-erbB-2 specific light chain peptide, the variable region derived from a first genetic source and a constant region derived from a second and different genetic source.

Detailed Description

The present invention encompasses recombinant DNA sequences, recombinant vectors, host cells, and immunoglobulin peptides (including CDRs) encoded by recombinant sequences.

Particularly of interest are sequences encoding chimeric immunoglobulin peptides that specifically recognize the c-erbB-2 antigen. The c-erbB-2 protein (also referred to here simply c-rbB-2) is a 185 Kd (Kilodalton) membrane glycoprotein havi tyrosine kinase activity and is related to, but distinct fro the epidermal growth factor receptor (EGFR) . Like the EGF protein, the c-erbB-2 protein has an extracellular domain tha includes two cysteine-rich repeat clusters, a transmembran domain and an intracellular kinase domain. In addition, th amino acid sequence of the c-erbB-2 protein as well as th nucleotide sequence has been described by Coussens et al. Science, 230:1132 (1985), incorporated by reference herein The c-erbB-2 protein is encoded by the c-erbB-2 oncogen described in 1985 by three different research groups: Semb et al., Proc. Natl . Acad. Sci . USA , 82:6497 (designating the gen as c-erbB-2); Coussens et al., supra , (designating the gene a HER-2) ; and King et al. , Science, 229:1132 (designating th gene as v-erbB related) . Thus, the c-erbB-2 gene sequence an its corresponding protein sequence are well-known and de scribed in the art. Detection of the c-erbB-2 protein may b accomplished by well-known immunoassays employing antibodie specific to the c-erbB-2 protein, such as those describe here. Such antibodies are commercially available, for exam ple, from Chemicon International, Inc., Temecula, CA or may b prepared by standard immunological procedures. See, for exam ple, Harlow and Lane, Antibodies: A Laboratory Manual , Cold Sprin Harbor Publications, N.Y. (1988) , incorporated by referenc herein. It is intended herein that the c-erbB-2 protein defi nition will also include those proteins developed from othe host systems, e.g., proteins that are immunologically relate to the human c-erbB-2 protein. For example, a related ra gene (designated neu) has been reported in Schecter et al. Science, 229:976 (1985) .

One objective of this invention is to provide recombinan immunoglobulin peptides which bind to c-erbB-2. Immunoglobu lins or antibodies are typically composed of four covalentl bound peptide chains. For example, an IgG antibody has tw light chains and two heavy chains. Each light chain is co valently bound to a heavy chain. In turn each heavy chain i covalently linked to the' other to form a "Y" configuration, also known as an immunoglobulin conformation. Fragments o these molecules, or even heavy or light chains alone, may bin antigen. Antibodies, fragments of antibodies, and individual chains are also referred to herein as immunoglobulins. A normal antibody heavy or light chain has an N-terminal (NH₂) variable (V) region, and a C-terminal (-COOH) constant (C) region. The heavy chain variable region is referred to as V_H (including, for example, Vγ) , and the light chain variable region is referred to as V_L (including V_κ or V^) . The variable region is the part of the molecule that binds to the anti¬ body's cognate antigen, while the Fc region (the second and third domains of the C region) determines the antibody's effector function (e.g., complement fixation, opsonization) . Full-length immunoglobulin or antibody "light chains" (gene- rally about 25 Kd, about 214 amino acids) are encoded by a variable region gene at the N-terminus (generally about 110 amino acids) and a n (kappa) or λ (lambda) constant region gene at the COOH-terminus. Full-length immunoglobulin or antibody "heavy chains" (generally about 50 Kd, about 446 amino acids) , are similarly encoded by a variable region gene (generally encoding about 116 amino acids) and one of the constant region genes, e.g. gamma (encoding about 330 amino acids) . Typically, the "V_L" will include the portion of the light chain encoded by the V_L and/or J_L (J or joining region) gene segments, and the "V_H" will include the portion of the heavy chain encoded by the V_H, and/or D_H (D or diversity region) and J_H gene segments. See generally, Roitt et al., Immunology, Chapter 6, (2d ed. 1989) and Paul, Fundamental Immunology; Raven Press (2d ed. 1989) , both incorporated b reference herein.

An immunoglobulin light or heavy chain variable regio consists of a "framework" region interrupted by three hyper variable regions, also called complementarity-determinin regions or CDRs. The extent of the framework region and CDR have been defined (see. "Sequences of Proteins of Immunologi cal Interest," E. Kabat et al., U.S. Department of Health an Human Services, (1987); which is incorporated herein by refer ence) . The sequences of the framework regions of differen light or heavy chains are relatively conserved within species. The framework region of an antibody, that is th combined framework regions of the constituent light and heav chains, serves to position and align the CDRs in three di en sional space. The CDRs are primarily responsible for bindin to an epitope of an antigen. The CDRs are typically referre to as CDR1, CDR2, and CDR3, numbered sequentially startin from the N-terminus.

The two types of light chains, c and λ, are referred t as isotypes. Isotypic determinants typically reside in th constant region of the light chain, also referred to as the C in general, and C_κ or C^ in particular. Likewise, the constan region of the heavy chain molecule, also known as C_H, deter mines the isotype of the antibody. Antibodies are referred t as IgM, IgD, IgG, IgA, and IgE depending on the heavy chai isotype. The isotypes are encoded in the mu (μ) , delta (Δ) gamma (~f ) , alpha (α) , and epsilon (e) segments of the heav chain constant region, respectively. In addition, there ar a number of γ subtypes. The heavy chain isotypes determine different effecto functions of the antibody, such as opsonization or complemen fixation. In addition, the heavy chain isotype determines th secreted form of the antibody. Secreted IgG, IgD, and Ig isotypes are typically found in single unit or monomeric for Secreted IgM isotype is found in pentameric form; secreted I can be found in both monomeric and dimeric form.

Mouse monoclonal antibodies have been made against t extracellular portion of c-erbB-2. An example of such a antibody is TAb 250, which is deposited with the American Typ

Culture Collection, Rockville, Maryland (ATCC) bearin

Accession No. HB10646.

The DNA sequences of this invention comprise DN subsequences encoding amino acid sequences of the antibod heavy or light chains, or fragments thereof, which determin binding specificity for the oncogene c-erbB-2 protein such a those derived from TAb 250. These sequences may be ligated for example, into human constant region expression vectors and inserted into a host cell. The host cell can then expres a recombinant chimeric or hybrid antibody that is specific fo binding to a c-erbB-2 protein or polypeptide.

"Immunoglobulin" or "antibody peptide(s) " refers to a entire immunoglobulin or antibody or any functional fragmen of an immunoglobulin molecule. Examples of such peptide include complete antibody molecules, antibody fragments, suc as Fab, F(ab')₂, CDRs, V_L, V_H, and any other portion of a antibody. As described above, an IgG antibody molecule i composed of two light chains linked by disulfide bonds to tw heavy chains. The two heavy chains are, in turn, linked t one another by disulfide bonds in an area known as the hing region of the antibody. A single IgG molecule typically ha a molecular weight of approximately 150-160 kD and containin two antigen binding sites. An F(ab')₂ fragment lacks the C-terminal portion of the heavy chain constant region, and has a molecular weight of approximately 110 kD. It retains the two antigen binding sites and the interchain disulfide bonds in the hinge region, but it does not have the effector functions of an intact Ig molecule. An F(ab')₂ fragment may be obtained from an Ig molecule by proteolytic digestion with pepsin at pH 3.0-3. using standard methods such as those described in Harlow an Lane, supra.

An "Fab" fragment comprises a light chain and th N-terminus portion of the heavy chain to which it is linked b disulfide bonds. It has a molecular weight of approximatel 50 kD and contains a single antigen binding site. Fa fragments may be obtained from F(ab')₂ fragments by limite reduction, or from whole antibody by digestion with papain i the presence of reducing agents. (See, Harlow and Lane supra.) In certain cases, the concentration of reducing agen necessary to maintain the activity of papain in the presenc of atmospheric oxygen is sufficient to fully reduce th interchain disulfide bonds to the antibody. This can resul in loss of antigen recognition. To circumvent this problem papain may be activated and then exchanged into buffe containing a concentration of reducing agent compatible wit maintaining antigen binding activity. The antibody digestio is carried out under an inert atmosphere to preven deactivation of the papain.

The following protocol is an example of this process:

A) Activation of papain: Papain, supplied as 10 mg/m NHS0₄ suspension, is dissolved in 10 mM EDTA, 20 mM cysteine pH=8.0, to a final concentration of 2 mg/ml. The solution i degassed and allowed to incubate 2 hours at room temperatur under nitrogen.

B) The activated papain is exchanged into 20 mM NaP0₄ pH=7.0, 150 mM NaCl, 10 mM EDTA, 30 μM DTT.

C) Digestion of antibody: 1 mg of activated papain i added for every 100 mg of antibody, and the solution i dialyzed against a large excess of 20 mM NaP0 , pH=7.0, 150 m NaCl, 10 mM EDTA, 30 μM DTT, with continuous helium spargin Dialysis is necessary to maintain a molar excess of reducin agent during the course of the digestion.

D) After 2-4 hours at room temperature the digestion i terminated by addition of iodoacetamide.

E) Fab fragments are separated from undigested o partially digested antibody using standard chromatograph methods.

"Fab", or any other antibody fragment, has simila classifications according to the definition of the presen invention as does the general term "antibodies" or "immuno globulins". Thus, "mammalian" Fab protein, "chimeric Fab" and the like are defined analogously to the correspondin definitions set forth in the subsequent paragraphs. "Chimeric antibodies" or "chimeric peptides" refer t those antibodies or antibody peptides wherein one portion o the peptide has an amino acid sequence that is derived from or is homologous to a corresponding sequence in an antibody o peptide derived from a first gene source, while the remainin segment of the chain(s) is homologous to correspondin sequences of another gene source. For example, a chimeri antibody peptide may comprise an antibody heavy chain with murine variable region and a human constant region. The tw gene sources will typically involve two species, but will occasionally involve one species.

Chimeric antibodies or peptides are typically produced using recombinant molecular and/or cellular techniques. Typically, chimeric antibodies have variable regions of both light and heavy chains that mimic the variable regions of antibodies- derived from one mammalian species, while the constant portions are homologous to the sequences in anti¬ bodies derived from a second, different mammalian species. The definition of chimeric antibody, however, is n limited to this example. A chimeric antibody is any antibo in which either or both of the heavy or light chains a composed of combinations of sequences mimicking the sequenc in antibodies of different sources, whether these sources differing classes, differing antigen responses, or differi species of origin, and whether or not the fusion point is a the variable/constant boundary. For example, chimeric anti bodies can include antibodies where the framework an complementarity- determining regions are from differen sources. For example, non-human CDRs are integrated int human framework regions linked to a human constant region t make "humanized antibodies." See, for example, PCT Appli cation Publication No. WO 87/02671, U.S. Patent No. 4,816,567 EP Patent Application 0173494, Jones, et al.. Nature, 321:522 525 (1986) and Verhoeyen, et al.. Science, 239:1534-153 (1988) , all of which are incorporated by reference herein.

A "human-like framework region" is a framework region fo each antibody chain, and it usually comprises at least abou 70 or more amino acid residues, typically 75 to 85 or mor residues. The amino acid residues of the human-like framewor region are at least about 80%, preferably about 80-85%, an most preferably more than 85% homologous with those in a huma immunoglobulin. The term "humanized" or "human-like immunoglobulin refers to an immunoglobulin comprising a human-like framewor region and a constant region that is substantially homologou to a human immunoglobulin constant region, e.g., having a least about 80% or more, preferably about 85-90% or more an most preferably about 95% or more homology. Hence, most part of a human-like immunoglobulin, except possibly the CDRs, ar substantially homologous to corresponding parts of one or mor native human immunoglobulin sequences. "Hybrid antibody" refers to an antibody wherein eac chain is separately homologous with reference to a mammalia antibody chain, but the combination represents a novel assem bly so that two different antigens are recognized by th antibody. In hybrid antibodies, one heavy and light chai pair is homologous to that found in an antibody raised agains one epitope, while the other heavy and light chain pair i homologous to a pair found in an antibody raised agains another epitope. This results in the property of multi functional valency, i.e., ability to bind at least tw different epitopes simultaneously. Such hybrids may, o course, also be formed using chimeric chains.

The present invention, inter alia, encompasses a chimeri antibody, including a hybrid antibody or a humanized or human like antibody. It also encompasses a recombinant DNA sequenc encoding segments of said antibody or any peptide specific o c-erbB-2 protein. In a preferred embodiment, the variabl sequence originates from and is substantially identical to sequence of the murine TAb 250 antibody as described in Sequence ID Nos. 1 or 2, and is combined with human ~fl and K constant regions.

In the case of the sequences described herein, it should be understood that variants of these sequences are also in¬ cluded, such as substitution, addition, and/or deletion mutations, or any other sequence possessing substantially similar binding activity to the sequences from which they are derived or otherwise similar to.

For this invention, an antibody or other peptide is specific for a c-erbB-2 protein if the antibody or peptide binds or is capable of binding c-erbB-2 protein as measured or determined by standard antibody-antigen or ligand-receptor assays, for example, competitive assays, saturation assays, or standard immunoassays such as ELISA or RIA. This definition of specificity applies to single heavy and/or light chains CDRs, fusion proteins or fragments of heavy and/or ligh chains, that are also specific for c-erbB-2 protein if the bind c-erbB-2 protein alone or if, when properly incorporate into immunoglobulin conformation with complementary variabl regions and constant regions as appropriate, are then capabl of binding c-erbB-2 protein.

In competition assays the ability of an antibody or pep tide fragment to bind an antigen is determined by detectin the ability of the peptide to compete with the binding of compound known to bind the antigen. Numerous types of compe titive assays are known and are discussed herein. Alterna tively, assays that measure binding of a test compound in th absence of an inhibitor may also be used. For instance, th ability of a molecule or other compound to bind the c-erbB-2 protein can be detected by labelling the molecule of interes directly or it may be unlabelled and detected indirectly usin various sandwich assay formats. Numerous types of bindin assays such as competitive binding assays are known (see, e.g., U.S. Patent Nos. 3,376,110, 4,016,043, and Harlow an Lane, Antibodies: A Laboratory Manual , Cold Spring Harbo Publications, N.Y. (1988) , which are incorporated herein b reference) . Assays for measuring binding of a test compoun to one component alone rather than using a competition assa are also available. For instance, immunoglobulins can be use to identify the presence of the c-erbB-2 protein. Standar procedures for monoclonal antibody assays, such as ELISA, ma be used (see, Harlow and Lane, supra) . For a review of various signal producing systems which may be used, see, U.S. Patent No. 4,391,904, which is incorporated herein by reference.

Further, the specificity of the peptides to c-erbB-2 can be determined by their affinity for the antigen. Such speci¬ ficity exists if the dissociation constant (K_j, = 1/K, where K is the affinity constant) of the peptides is < lμM, preferab < 100 nM, and most preferably < 1 nM. Antibody molecules wi typically have a K,, in the lower ranges. K_j, = [R-L]/[R][ where [R], [L], and [R-L] are the concentrations at equili brium of the receptor or c-erbB-2 (R) , ligand or peptide (L and receptor-ligand complex (R-L) , respectively. Typically the binding interactions between ligand or peptide and recep tor or antigen include reversible noncovalent association such as electrostatic attraction, Van der Waals forces an hydrogen bonds.

Other assay formats may involve the detection of the pre sence or absence of various physiological or chemical change that result from the interaction, such as down modulation internalization or an increase in phosphorylation as describe in United States Patent Application No. 07/644,361 file January 18, 1991, incorporated by reference herein. See also Receptor-Effector Coupling - A Practical Approach, ed. Hulme, IR Press, Oxford (1990) .

A preferred peptide specific for c-erbB-2 protein induce an increase in the phosphorylation of the c-erbB-2 protei when placed in contact with tumor cells expressing th c-erbB-2 protein. A molecule that "induces an increase in th phosphorylation of c-erbB-2 protein" is one that causes detectable increase in the incorporation of phosphate into th protein over that which occurs in the absence of the molecule. Typically this detectable increase will be a two-fold o greater increase in phosphorylation, preferably greater tha a three-fold increase over controls. Phosphorylation may be measured by those methods known in the art for detectin phosphorylation of receptors. See, for example Cooper et al., Methods in Enzymology, 99:387-402 (1983); Antoniades and Pantazis, Methods in Enzymology, 147:36-40 (1987); and Lesniak et al., Methods in Enzymology, 150:717-723 (1987), which are al incorporated by reference herein.

Typically, phosphorylation can be measured by in viv phosphorylation of intact cells (Lesniak, supra) or by an i vitro autophosphorylation reaction (Antonaides, supra) . Fo measuring in vivo phosphorylation, for example, assays may b conducted where cells bearing the c-erbB-2 protein are place into contact with radioactive labelled phosphate. To detec phosphorylation of the c-erbB-2 protein receptor in the in viv assay, it is advantageous to incubate the test cells for abou 12 to about 18 hours, with the labeled phosphate. The cell are divided into two or more batches, where some are expose to the molecule expected to increase the phosphorylation o the receptor and some are separated out as controls. Th aliquots are subsequently immunoprecipitated, the receptor i recognized, for example, by SDS polyacrylamide gel or auto radiography methods, and an increase in phosphorylation i considered statistically significant when when there is a two fold or greater increase in the background of the aliquo exposed to the test molecule over the control aliquots.

To measure in vitro autophosphorylation, for example, cells or cell extracts may be incubated in the presence o absence of the peptide specific for c-erbB-2. Followin immunoprecipitation with an anti-c-erbB-2 antibody, the immun complex may be incubated with τr³²P-ATP and analyzed by SDS-PAG autoradiography.

Another preferred peptide specific for c-erbB-2 protei is one that causes down modulation of the c-erbB-2 protein. "Down modulation of the c-erbB-2 protein" is determined by detectable decrease in the presence on the tumor cells of th c-erbB-2 receptor. Such down modulation is detected by decrease in the ability of antibodies or other specifi binding moieties to bind to or recognize the c-erbB-2 recepto protein on the tumor cells. For example, down modulation c be determined by incubating tumor cells bearing the c-erbB protein receptor with the peptide of interest, washing t cells, then contacting the cells with labeled (preferabl radiolabelled) antibodies specific for the c-erbB-2 protei The extent of binding of the labelled anti-c-erbB-2 antibodie to the cells exposed to the peptide specific for c-erbB- protein is compared to the extent of binding of the antibodie to control cells (i.e., not exposed to the c-erbB-2 specifi peptide) . Preferably for these assays, the cells are directl subjected to the labeled anti-c-erbB-2 antibodies afte washing.

The down modulation observed is typically dose dependent i.e., the extent of down modulation increases with the amoun of peptide specific for c-erbB-2 protein exposed to th c-erbB-2 protein. Preferably, a peptide that causes decrease in 90% or greater of binding of the treated cell versus control cells to anti-c-erbB-2 antibodies is desirable Another preferred peptide specific for c-erbB-2 protei is one that binds tumor cells expressing c-erbB-2 protein an is internalized when placed in contact with such tumor cells. "Internalization" occurs when the receptor becomes sequestere in the cytoplasm of the cells. Once internalized, the recep tor may be degraded in the cell lysosomes or may be recycle to the cell surface. A method for determining internalizatio of a ligand-receptor complex is also described in Haigle et al., J. Biol. Chem. , 255:1239-1241 (1980), incorporated b reference herein.

This invention further includes recombinant DNA vectors comprising a gene expression control DNA sequence operably linked to the antibody peptide coding sequence. Examples of such a control sequence include a naturally-associated or heterologous promoter region. Preferably, the expression control sequence will be a eukaryotic promoter system in vector capable of transforming or transfecting a eukaryoti host cell. A control sequence for a prokaryotic host, how ever, can also be used. Once the vector has been incorporate into the appropriate host, the host is maintained under condi tions suitable for high level expression of the nucleotid sequence, and, as desired, the collection and purification o the light chain, heavy chain, light/heavy chain dimer o intact antibodies, binding fragments or other immunoglobuli form may follow. See generally for construction of expressio vectors, Kriegler, Gene Transfer and Expression, M.H. Freeman N.Y., N.Y. (1990), which is incorporated by reference.

The vectors of this invention also include recombinan DNA sequences encoding an antibody, or antibody peptide, alon with a relevant transcriptional element, such as an enhance and/or promoter. These transcriptional elements will be oper ably linked to the encoding gene to ensure their expression i the host cell system.

Other aspects of this invention include, for example, recombinant DNA sequences comprising one or more of the CDR of antibody peptides that compete with TAb 250 for binding t c-erbB-2. They may be interspersed among framework regions, including those derived from a different species.

This invention further includes suitable host cell lines. For example, the sequences encoding the anti-c-erbB-2 antibod peptides are placed into expression vectors for transfectio or transduction into bacteria, yeast, amphibian oocytes, insect cells or mammalian host cell lines, such as myelom cells, Cos, CHO or L cells. See generally, Kriegler, supra , Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed. 1989), European Patent Application Publication No. 0125023, published 11/14/84; Kameyama et al., FEBS. , 244:301 (1989); Better et al. , Science, 240:1041 (1988), all of which ar incorporated by reference.

Using standard methods that are well known in the art the variable regions and CDRs may be derived from a hybridom that produces a monoclonal antibody that is specific fo c-erbB-2. The nucleic acid sequences of the present inventio capable of ultimately expressing the desired chimeric anti bodies can be formed from a variety of different nucleotid sequences (genomic or cDNA, RNA, synthetic oligonucleotides etc.) and components (e.g., V, J, D, and C regions), as wel as by a variety of different techniques. Joining appropriat genomic sequences is presently a common method of production, but cDNA sequences may also be utilized (see, European Paten Publication No. 0239400 and Reichmann et al. , Nature, 332:323-327 (1988), both of which are incorporated herein b reference) .

In one preferred embodiment, the sequences encoding th V_L and V_H regions are cloned from a hybridoma's genomic DNA, or cDNA produced by reverse transcription of the hybridoma's RNA. See Sambrook et al., supra . Cloning can be accomplishe using traditional techniques, including the use of PCR primers that hybridize to the sequences lanking or overlapping with the variable regions or CDRs to amplify sequences of interes using cDNA or genomic DNA, as described below. See, Orlandi et al., Proc. Natl. Acad. Sci. USA, 86:3833 (1989), which is incorporated by reference. Exemplary primers for a variable heavy chain sequence are set out in Sequence ID Nos. 17 and 18. Exemplary primers for a variable light chain sequence are set out in Sequence ID Nos. 19 and 20. The amplified fragments can be subcloned into plasmids, such as pUC19. Ideally, the amplified DNA fragment should include a promoter. Promoter elements, leader, and other sequences such as enhancer elements upstream or downstream from the antibody peptides can be separately isolated an cloned into the plasmid containing the antibody encodin sequences.

A similar approach can be taken to isolate and subclon the sequences encoding the constant regions of the heavy an light chains that originate from another mammalian species The enhancers to the heavy and light chain can be included i the isolated heavy chain fragments, or can alternatively b isolated and subcloned. Human constant region DNA sequences are preferabl isolated from immortalized B-cells, see e.g., Heiter et al., Cel l , 22:197-207 (1980), incorporated by reference herein, bu can be isolated or synthesized from a variety of othe sources. The nucleotide sequence of a human immunoglobulin Cγ gene is described in Ellison et al., Nucl . Acid. Res. , 10:407 (1982); Beidler et al., J. Immunol . , 141:4053 (1988); Li et al., Proc. Natl . Acad. Sci . USA , 84:3439 (1987) (al incorporated by reference herein) .

The CDRs for producing the immunoglobulins of the presen invention preferably are derived from monoclonal antibodie capable of binding to the desired antigen, c-erbB-2 protein, and produced in any convenient mammalian source, including, mice, rats, rabbits, hamsters, or other vertebrate host cell capable of producing antibodies by well known methods. Suit able source cells for the DNA sequences and host cells fo immunoglobulin expression and secretion can be obtained fro a number of sources, such as the American Type Cultur Collection ("ATCC") ("Catalogue of Cell Lines and Hybrido as," Fifth edition (1985) Rockville, Maryland, U.S.A., which is incorporated herein by reference) .

In addition to the chimeric antibody peptides specifical¬ ly described herein, other "substantially homologous" modified i munoglobulins can be readily designed and manufactured uti lizing various recombinant DNA techniques known to thos skilled in the art. Modifications of the genes may be readil accomplished by a variety of well-known techniques, such a site-directed mutagenesis (see, Gillman and Smith, Gene 8:81-97 (1979) and Roberts, s., et al, Nature, 328:731-73 (1987) , both of which are incorporated herein by reference) Alternatively, polypeptide fragments comprising only a portio of the primary antibody structure may be produced, which frag ments possess binding and/or effector activities. Als because like many genes, the immunoglobulin-related gene contain separate functional regions, each having one or mor distinct biological activities, the genes may be fused t functional regions from other genes to produce fusion protein (e.g., immunotoxins) having novel properties or nove combinations of properties.

The cloned variable and constant regions can be isolate from plasmids and ligated together into a mammalian expression vector, such as pSV2-neo, and pRSV-gpt, to form a functional transcription unit. These expression vectors can then be transfected into host cells. Mouse myeloma cells, such as SP 2/0 or P3X cells, are a preferred host because they do not secrete endogenous immunoglobulin protein and contain all of the components used in immunoglobulin expression. Myeloma cells can be transfected using appropriate techniques as described above.

Other types of promoters and enhancers specific for other host cells are known in the art. See, Kameyoma et al., supra. For example, the DNA sequence encoding the chimeric antibody amino acid" sequence can be linked to yeast promoters and enhancers and transfected into yeast by methods well known in the art. See, Kriegler, supra . This same approach can be taken to isolate the c-erbB- specific CDRs from one source such as one mammalian specie and the framework regions of another source, such as different mammalian species. The CDRs can then be ligated t the framework regions and constant regions to form a chimeri antibody. See, PCT No. GB88/00731 (1989) , which i incorporated by reference. For example, the CDRs for th heavy chain of TAb 250, may be found within the variabl region at the following amino acid positions on Sequence ID. No. 3: 31-35 (CDR1) , 50-65(CDR2), and 98-105 (CDR3) . Th CDRs for the light chain may be found within the variable region at the following amino acid positions on Sequence ID. No. 4: 24-34 (CDR1) , 50-56 (CDR2) , and 89-97 (CDR3) . The CDRs could be cloned in an expression vector comprising, for example, human framework and constant regions.

Another example is a recombinant DNA sequence comprising the heavy and/or light chain CDR1, CDR2, and CDR3 of one species, such as mouse, and the framework regions of human heavy chain to encode an antibody specific for c-erbB-2. Other possibilities include using CDRs specific for c-erbB-2; using part of the variable region encompassing CDR1 and CDR2 from one mammalian species, and then ligating this sequence to another encoding the framework portions of a second mammalian species to the CDR3 of the first; or transfecting a host cell line with a recombinant DNA sequence encoding a c-erbB-2 specific heavy chain CDRs derived from a first mammalian species, interspersed within the framework of a second mammalian species with a light chain containing a variable region DNA sequence derived from the first species and the constant region derived from the second species.

In one preferred embodiment, antibody peptides are com¬ prised of the V_H, amino acid Sequence ID No. 3, and the V_κ, Sequence ID No. 4, which are derived from the murine TAb 250 antibody. The TAb 250 variable regions obtained from i rearranged configuration in the myeloma's genome have the D coding strand set forth in Sequence ID No. 1, base position 312-597 (V_H) and in Sequence ID No. 2 base positions 370-65 (V_κ) . The TAb 250 V_H D region is found at base position 600-609 in Sequence ID No. 1, the J4 region at base position 612-659 and the enhancer at base positions 1566-1813. The TA 250 V_H sequence is linked to a human γi constant region. Th TAb 250 V_κ sequence is linked to a human K constant region and murine K enhancer. The TAb 250 J2 region is found at bas positions 659-691 of Sequence ID No. 2. Recombinant DN expression vectors comprising these TAb 250 sequences may b transfected by electroporation into host cells. Standar selection procedures are used to isolate clones that produc the c-erbB-2 specific chimeric antibody.

Antibodies may be expressed in an appropriate folde form, including single chain antibodies, from bacteria such a E. coli . See, Pluckthun, Biotechnology, 9:545 (1991); Hus et al., Science, 246:1275 (1989) and Ward, et al., Nature, 341:544 (1989), all incorporated by reference herein.

The antibody peptide sequences may be amplified for clon ing by use of polymerase chain reaction, or PCR, a techniqu used to amplify a DNA sequence of interest using a thermo stable DNA polymerase, such as Taq polymerase, and polymeras and oligonucleotide primers, all as described in PCR Protocols, ed. Innis, et al., Academic Press, Inc. (1990), incorporate by reference herein. See also Orlandi, supra and Larric et al., Biotechnology, 7:934 (1989), incorporated by reference herein. A primer is an oligonucleotide which is capable of acting as a point of initiation of synthesis when placed under condi¬ tions typically which permit synthesis of a primer extension product which is complementary to a nucleic acid strand. These conditions typically include the presence of four diffe rent nucleoside triphosphates in an appropriate buffer o proper ionic strength, pH, cofactors, etc., and suitable tem perature. The oligonucleotide can be derived from a natura source, as a purified restriction fragment, or can be produce synthetically. PCR primers typically are preferably singl stranded oligodeoxyribonucleotides, about 15-30 residues i length. The primers are substantially complementary to th sequences to be amplified such that they can hybridize to th target sites.

Primers are chosen such that one primer hybridizes to th 5' end of sequence of interest, and a second primer hybridize to the 3' end of the sequence, but to the opposite strand.

In the first step of PCR, the reaction mixture is gene rally heated at about 90-100°C to denature the DNA. Th primers hybridize with the target sequence, typically, a about 40-60°C for about 10-60 seconds, followed by extensio of the primers, for example, at about 65-75^βC for approxi mately 1 minute for every kb of DNA to be amplified. Th products of synthesis become targets of the second primer, an a second cycle of hybridization and synthesis. The cycles ar repeated about 25-40 times, typically in an automated tempera ture cycling machine. A sequence can be amplified 10 time or more using PCR. Although polymerase chain reaction is a powerful clonin technique, problems can arise in choosing primers and esta blishing hybridization and extension conditions. Choosing a effective primer is often difficult. For example, a prime ideally has a high GC content, to optimize its ability t hybridize under high temperature conditions. Even mor problematic and less predictable is the secondary structure o the primer or the complementary DNA. If either is prone, fo example, to stem-loop formation, the primer might be unable t hybridize to the DNA. Small amounts of contaminating DNA ca result in the incorrect target being amplified.

The subject peptides may be used to make fusion protein such as immunotoxins. Immunotoxins are characterized by tw functional components and are particularly useful for killin selected cells in vitro or in vivo. One functional component is a cytotoxic agent which is usually fatal to a cell when attached or absorbed. The second functional component, known as the "delivery vehicle," provides a means for delivering the toxic agent to a particular cell type, such as cells compris¬ ing a carcinoma. The two components are commonly chemically bonded together by any of a variety of well-known chemical procedures. For example, when the cytotoxic agent is a protein and the second component is an intact immunoglobulin, the linkage may be by way of heterobifunctional cross-linkers, e.g., SPDP, carbodiimide, glutaraldehyde, or the like. Pro¬ duction of various immunotoxins is well-known within the art, and can be found, for example in "Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet," Thorpe et al., Monoclonal Antibodies in Clinical Medicine, Academic Press, pp. 168-190 (1982) and Waldmann, Science , 252:1657 (1991), both of which are incorporated herein by reference.

A variety of cytotoxic agents are suitable for use in immunotoxins. Cytotoxic agents can include radionuclides, such as Iodine-131, Yttrium-90, Rhenium-188, and Bismuth-212; a number of chemotherapeutic drugs, such as vindesine, metho- trexate, adriamycin, and cisplatin; and cytotoxic proteins such as ribosomal inhibiting proteins like pokeweed antiviral protein, Pseudomonas exotoxin A, ricin, diphtheria toxin, ricin A chain, ge^'lonin, etc., or an agent active at the cell sur¬ face, such as the phospholipase enzymes (e.g. , phospholipase C) . (See, generally, "Chimeric Toxins," Olsnes and Phil, Pharmac. Ther. , 25:355-381 (1982), and "Monoclonal Antibodies for Cancer Detection and Therapy," eds. Baldwin and Byers, pp. 159-179, 224-266, Academic Press (1985), which are bot incorporated herein by reference.)

The delivery component of the immunotoxin will include the peptides of the present invention. Intact immunoglobulins or their binding fragments, such as Fab, are preferably used. Typically, the antibodies in the immunotoxins will be of the human IgM or IgG isotype, but other mammalian constant regions may be utilized as desired. For diagnostic purposes, the antibody peptides may either be labeled or unlabeled. Unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies) that are reactive with the first antibody, such as antibodies specific for human immunoglobulin constant regions. Alterna- tively, the antibodies can be directly labeled. A wide variety of labels may be employed, such as radionuclides, fluors, enzymes, enzyme substrates, enzyme co-factors, enzyme inhibitors, ligands (particularly haptens) , etc. Numerous types of immunoassays are available and are well known to those skilled in the art.

Anti-idiotypic antibodies to c-erbB-2 protein may also be produced using the peptides of this invention by immunizing a host with the peptides, including one or more of the CDRs and then immortalizing cells which express nucleic acid sequences that encode antibodies or idiotopic regions thereof. The immortalization process may be carried out by hybridoma fusion techniques, by viral transformation of human antibody- producing lymphocytes, or by techniques that combine cell fusion and viral transformation methodologies. Monoclonal anti-idiotype antibodies may be prepared using a combination of Epstein-Barr virus (EBV) transformation and hybridoma fusion techniques such as those described by Kozbor et al., Proc. Natl . Acad. Sci . (1982) 79:6651, which is incorporated herein by reference. For instance, t hybridomas may be created by fusing stimulated B cell obtained from a human immunized with the primary (idiotyp antibody (Abl) to which the anti-idiotype (Ab2) is to be made with a mouse/human heterohybrid fusion partner. A variety o such fusion partners have been described. See, for example James and Bell, J. Immunol . Meths. (1987) 100:5-104 and U.S Patent No. 4,624,921, which are incorporated herein b reference. A mouse/human fusion partner may be constructed b fusing human lymphocytes stimulated or transformed by EBV wit readily available mouse myeloma lines such as NS-1 or P3NS-1 in the presence of polyethylene glycol, for instance. Th hybrid should be suitably drug-marked, which may b accomplished by cultivation of the hybrid in increasin concentrations of the desired drug, such as 6-thioguanine ouabain, or neomycin.

Anti-idiotype antibodies of interest may also be accomp lished using EBV transformation techniques. For example B-lymphocytes are derived from peripheral blood, bone marrow lymph nodes, tonsils, etc., of patients immunized with th idiotype antibody, and these lymphocytes are immortalize using EBV according to methods such as those described in U.S. Patent No. 4,464,465 and Chan et al. , J. Immunol . (1986) 136:106, which are incorporated herein by reference. The hybridomas or lymphoblastoid cells which secret anti-idiotypic antibody of interest may be identified b screening culture supernatants against antibody which i specific for c-erbB-2. Cells from wells possessing th desired activity are cloned and subcloned in accordance wit conventional techniques and monitored until stable immorta¬ lized lines producing the monoclonal antibody of interest are identified. The monoclonal antibodies thus produced may be of the IgG, IgM, IgA or IgD isotype, and may further be of any of the subclasses of IgG, such as IgG.,, IgG₂, IgG₃, or IgG₄. Onc an immortalized cell line is acquired, the antibodies can b used as a gene source for production of chimeric antibod peptides as described above. Anti-idiotypic antibodies which act as internal images o a tumor antigen may be used to prime a de novo response to th c-erbB-2 protein. By presenting these images of antigeni epitopes in a different molecular environment, responses ma be activated which would otherwise be silent. Nisonoff an Lamoyi, Cl in. Immunol . Immunopathol . (1981) 21:397. That is, whe the anti-idiotype represents a conformational image of th antigen, it may substitute for nominal antigen and elicit primary antibody-like response. Anti-idiotypic antibodie which do not bear the internal image of antigen may als induce antitumor responses by influencing the regulator idiotypic network. See, Bona, 1984, in Idiotypy in Biology an Medicine, Kohler et al., eds., Academic Press, pp. 29-42 Thus, antibodies to framework-associated idiotopes, o regulatory idiotopes, may select or amplify T and/or B cel clones with specificity for tumor antigens. Some evidence, however, suggests that this group of anti-idiotypic antibodie can prime a humoral response but are unable to cause matura tion of B cells without further challenge with the nomina antigen (Heyman et al., J. Exp. Med. (1982) 155:994), and thu combination with an internal image anti-idiotypic antibody ma be necessary to evoke a desired antitumor response.

Also contemplated here are those compounds that have de signed specificities based upon the CDRs specific to c-erbB- protein, such as those described here. Organic compounds ma be synthesized with similar biological activity by firs determining the relevant contact residues and conformatio involved in c-erbB-2 binding by an antibody peptide of thi invention. Computer programs to create models of protein such as antibodies are generally available and well known t those skilled in the art (see, Kabat et al. Sequences of Protein of Immunol ogical Interest, U.S. Department of Health and Huma Services, National Institutes of Health (1987); Loew et al. Int. J. Quant. Chem. , Quant. Biol . Sy p. , 15:55-66 (1988); Bruccoler et al.. Nature, 335:564-568 (1988); Chothia et al., Science, 233:755-758 (1986), all of which are incorporated herein b reference. Commercially available computer programs can b used to display these models on a computer monitor, t calculate the distance between atoms, and to estimate th likelihood of different amino acids interacting (see, Ferrin et al., J. Mol. Graphics, 6:13-27 (1988)). For example computer models can predict charged amino acid residues that were accessible and relevant in binding and then conformationally restricted organic molecules can be synthesized. See, for example, Saragovi et al. , Science, 253:792 (1991).

Other General Definitions.

"Recombinant" means that the subject product is the result of the manipulation of genes into new or non-native combinations.

"Restriction endonucleases" and "restriction enzymes" refer to enzymes which cut double stranded DNA at or near a specific nucleotide recognition sequence.

Complementary DNA, "cDNA" refers to DNA that is derived from a messenger RNA sequence (mRNA) , for example, using reverse transcriptase. Reverse transcriptase is an enzyme that polymerizes DNA using an RNA template.

"Transcriptional activating sequences" refer to DNA sequences, 'such as promoters and enhancers, that activate transcription of a gene. Such sequences, in a proper host, drive transcription of a correctly positioned DNA sequence encoding a peptide. For example, the K chain promoter and K chain enhancer will promote transcription of a correctl positioned DNA sequence in a myeloma or hybridoma host. Othe transcriptional regulator sequences will often be useful i analogous circumstances, for example, when deactivation may b desired.

"Coding strand sequence" is the region of a gene tha encodes the amino acid sequence of a protein.

A "promoter" is a DNA sequence 5' of the protein codin sequences which affects transcriptional activity. RN polymerase first binds to the promoter to initiat transcription of a gene.

An "enhancer" is a DNA sequence that can positivel affect transcriptional efficiency. A preferred enhancer fo the sequence encoding a heavy chain variable region of th antibodies described here is that found at base positions 156 to 1813 on Sequence Listing ID No. 1.

A "vector" is a sequence of DNA, typically in plasmid o viral form, which is capable of replicating in a host. vector.can be used to transport or manipulate DNA sequences. An "expression vector" includes vectors which are capable o expressing DNA sequences contained therein, typically pro ducing a protein product. The coding sequences are linked t other sequences capable of effecting their expression, such a promoters and enhancers. Expression vectors are capable o replicating in a host in episomal form; others can integrat into a host cell's chromosome. Ideally, the expressio vectors have a selectable marker, for example, neomyci resistance, which permits the selection of cells containin the marker. An "oligonucleotide" is a polymer molecule of two or mor nucleotides including either deoxyribonucleotides or ribo nucleotides. "Host cells" refer to cells which are capable or hav been transformed with a vector, typically an expressio vector. A host cell can be prokaryotic or eukaryotic including bacteria, insect, yeast and mammalian cells.

Pharmaceutical Applications.

The chimeric antibodies or antibody peptides of thi invention can be used in pharmaceutical compositions i dosages that are cytotoxic to tumor cells. Tumors or cancer to be treated with the compositions of this invention are an tumors which express, or are suspected of expressing, th c-erbB-2 oncogene protein or have amplification of th c-erbB-2 gene. These tumors include, for example: breast, ovarian, bladder, prostate, stomach, lung and thyroid cancers. The c-erbB-2 protein is reported to be expressed in: solid tumors by, for example, Gutman et al. , Int. J. Cancer, 44:802-805 (1989); in human adenocarcinomas (solid tumors) by Yokota et al. , The Lancet, April 5, 1986, p.765; in gastric and esophageal carcinomas by Houldsworth et al., Cancer Res. , 50:6417-22 (1990); in neoplastic cervix, vulva and vagina by Berchuck et al., Obstetrics. Gynecol . Surv. , 76:381 (1990); in renal cell carcinoma by Weidner et al., Cancer Res. , 50:4504 (1990); in lung adenocarcinomas by Kern et al., Cancer Res. , 50:5184-5191 (1990) and Schneider et al. , Cancer Res. , 49:4968-4971 (1989); in gastric cancer by Fukushige, et al., Mol. and Cell. Biol. , 6:955-958 (1986), Park et al. , Cancer Res. , 49:6605 (1989) ; in breast and ovarian cancer by Slamon et al., Science, 244:707 (1989); by Berchuck et al., Cancer Res. , 50:4087 (1990); by Van de Vijver et al., Mol . Cell Biol. , 7:2019-2023 (1987), by arley et al., Oncogene, 1:423-430 (1987), Bacus et al., Am. J. Path. , 137:103 (1990) all of which are incorporated by reference herein. The compositions can be used in either pre- or post operative treatment of cancer or both. The composition herein are preferably administered to human patients via oral intravenous or parenteral administrations and other systemi forms. The pharmaceutical formulations or compositions o this invention may be in the dosage form of solid, semi-solid or liquid such as, e.g., tablets, pills, powders, capsules, gels, ointments, liquids, suspensions, aerosols or the like. Preferably the compositions are administered in unit dosag forms suitable for single administration of precise dosag amounts. The compositions may also include, depending on th formulation desired, pharmaceutically-acceptable, non-toxi carriers or diluents, which are defined as vehicles commonl used to formulate pharmaceutical compositions for animal o human administration. The diluent is selected so as not t affect the biological activity of the combination. Example of such diluents are distilled water, physiological saline. Ringer's solution, dextrose solution, and Hank's solution. I addition, the pharmaceutical composition or formulation ma also include other carriers, adjuvants; or nontoxic, nonthera peutic, nonimmunogenic stabilizers and the like. Effectiv amounts of such diluent or carrier will be those amounts whic are effective to obtain a pharmaceutically acceptable formula tion in terms of solubility of components, or biologica activity, etc. Generally a dosage level of from 1-500 mg/m of body surface area may be used systemically for the antibod compositions, to be adjusted as needed depending upon othe agents used. For example, the compositions may be admini¬ stered with an anti-neoplastic agent. For pharmaceutical treatments and guidelines see generally, Goodman & Gi lman' s The Pharmacological Basis of Therapeutics, Seventh Ed., ed. Gilman et al., MacMillan Publishing Company (1985) and Remington's Pharmaceutical Science, Sixteenth Ed. , Mack Publishing Co. (1982) , all incorporated by reference herein.

The tumor cells that one wishes to kill or control the growth of are referred to as "target tumor cells." "Test tumor cells" are any tumor cells zπ vitro that express the c-erbB-2 protein.

Imaging agents comprising a radio- or other label attached to the peptides of this invention are also contem¬ plated for in vivo detection of cells expressing the c-erbB-2 protein. Radiolabels may be any labels appropriate for gamma camera imaging, such as radioiodines ( I or I) or radio- metals (¹¹¹In or """Tc) , for example. The peptide selectively binds to the cells expressing c-erbB-2 present in the patient, thereby concentrating the label in the area of the target cells. Alternatively, the peptides can be labeled with paramagnetic contrast agents, and detected by nuclear magnetic resonance methods. The labeled antibodies thus produce a detectable image of the tumor tissue.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. In the foregoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius and unless otherwise indicated, all parts and percentages are by weight.

The entire disclosures of all applications, patents and publications, cited above and below, are hereby incorporated by reference. E X A M P L E S

I. Cells, Vectors, Probes, and Primers

The hybridoma that produced monoclonal antibody TAb 25 was generated using c-erbB-2 transformed NIH 3T3 cells. Mous myelomas P3x63-Ag8.653 (P3X) and SP 2/0, and human cell lin ARH-77 (ATCC CRL 1621, human plasma cell leukemia periphera blood) were obtained from the American Type Culture Collectio (ATCC, Rockville, MD) . SKBR-3 cells and c-erb-2 transfecte NIH 3T3 cells were provided by Dr. S. Aaronson (NIH, Bethesda MD) . Genomic DNA was prepared either using A.S.A.P. kit fro Boehringer Mannheim according to the manufacturer's instruc tions or using protocol from Current Protocols in Molecular Biology, ed. Ausubel Green Publishing Associates and Wiley-Interscienc (1988) , incorporated by reference herein. Plasmid vector pSV2neo, pRSV5gpt (pRSVgpt) , pBR322 and pUC19 were obtaine from ATCC and pIBI 21 was obtained from Internationa Biochemical Inc. (New Haven, CT) . The mouse heavy chain J probe was a 1.0 Kb DNA fragment containing the JH₃ and JH region, which was isolated from plasmid pJ3J4. The mous light chain JK probe was a 1.8 kb DNA fragment containing th J region, which was isolated from plasmid pJKHB.l. Both pJ3J and pJKHB.l were from Dr. J. Donald Capra (Southern Medica Center, University of Texas) . The probes used for identifyin human constant heavy and light chain genes were oligonucleo tides, designed from coding sequences of both genes an synthesized on an Applied Biosystem 381A DNA synthesize (Applied Biosystems Inc., Foster City, California). All th primers used in polymerase chain reactions (PCR) and nucleo tide sequencings were synthesized on an Applied Biosystem 381 DNA synthesizer. II. Production of Monoclonal Antibody TAb 250 Example l

Preparation of c-erbB-2 Monoclonal Antibodies Production and characterization of TAb 250, a monoclona antibody to c-erbB-2 is described in Langton, et al. , Can Res. , 51:2593-2598 (1991), incorporated by reference herein Balb/c mice were immunized intraperitoneally and subcutaneous ly with 2x10 -1x10 NIH3T3 cells transfected with the huma c-erbB-2 oncogene, NIH3T3_t, (Science, 237:178-182) emulsifie 1:1 volume/volume in complete Freund's adjuvant. Sera wa collected every two weeks and tested for reactivity in a ELISA assay (described below) against formalin fixed NIH3T3 o fixed NIH3T3_t cells. Animals with positive titers wer boosted intraperitoneally or intravenously with cells in PBS, and animals were sacrificed 4 days later for fusion. Splee cells were fused with P3-X63Ag8.653 myeloma cells at a rati of 1:1 to 7.5:1 with PEG 4000, generally as described by th procedure of Kohler and Milstein (Nature, 256:495-497) . Fuse cells were gently washed and plated in 96-well plates a 1-4X10⁶ cells/ml in RPMI 1640 medium. Wells were fed with HA medium 24 hours after the fusion and then every 3 days for 2-3 weeks. When colony formation was visible, after 10-14 days, the supernatants were tested for reactivity in the ELISA assay. Prospective clones demonstrating good growth were expanded into 24-well plates and rescreened 7-10 days later. Positive wells were then assayed for external domain reac¬ tivity against live NIH3T3 and NIH3T3_t cells by flow sorting analysis. Clones which were positive both by ELISA assay and flow sorting analysis were recloned either by limiting dilu- tion or by single cell deposition using a flow cytometer. Cells were diluted and deposited into 96-well plates in the presence or absence of spleen feeder cells. Wells demon¬ strating growth were retested by ELISA and recloned an additional one to three times. Supernatants from hybridom clones were tested for isotype and subisotype, reactivity t surface expressed pl85 protein on NIH3T3_t cells by flo sorting analysis, and immunoprecipitation of a labeled pl85 protein from transfected cells. Positive hybridomas were grown in tissue and injected into pristane-primed Balb/c mice, Balb/c nude mice or IRCF, mice for ascites production. Monoclonal antibodies were purified from ascites fluid by HPLC using a Bakerbond ABx column. Purified TAb 250 antibodies were dialyzed against PBS and stored at -20°C. All purified antibodies were tested for isotype, subisotype, and contami¬ nating isotypes by radial immunodiffusion. Cell surface staining of pl85 expressing cell lines was detected and quantified by flow sorting analysis, ELISA assay against transfected and untransfected NIH3T3 cells, and radio- immunoprecipitation of pl85 from labeled c-erbB-2 expressing cell lines. The antibodies did not cross-react with the closely related EGF-receptor protein as shown by the failure to precipitate a radiolabeled 170 Kd protein from radiolabeled A-431 cells, and they were analyzed by SDS-PAGE and gel densitometry (all purified proteins are >90% immunoglobulin) .

III. Cloning Mouse V„ and V Genes from TAb 250

Two genomic DNA fragments, one containing TAb 250 heavy chain variable region (V_H) and the other containing TAb 250 light chain variable region (V_κ) , were both isolated from hybridoma genomic DNA by PCR cloning. The PCR for V_H cloning was performed with 100 μl of reaction mix containing 0.1 μg of TAb 250 genomic DNA; 10 mM Tris HC1 (pH 8.4); 2.5 mM MgCl₂; 50 mM KC1; 100 μg/ml gelatin; 200 μM of each dATP, dCTP, dTTP and dGTP; 0.25 μM of each PCR primer and 2.5 units of Taq polymerase. The reaction was carried out in a DNA thermal cycler (Perkin-Elmer-Cetus) as described above for 40 cycles with reaction cycle set up as follows: denaturation at 94° for 20 seconds; annealing at 50°C for 20 seconds, extension a 72°C for 3 minutes and autoextensions for 15 seconds. The PC for light chain cloning was performed using the sam conditions as for V_H amplification described above except th MgCl₂ concentration was 1.5 mM. In both V_H and V_κ cloning th PCR primers were designed to amplify the entire genes, including promoter elements, leaders and variable regions. The amplified V„ gene fragment also includes its own enhance sequence. By using GCG sequence analysis software packag version 7.0 (Devereux, et al., A Comprehensive Set of Sequenc Analysis Programs for VAX, Nuc. Acid Res. 12:387-95 (1984), three degenerative 5' primers were designed, of which one was effective (Sequence ID No. 18), and one specific 3' prime (Sequence ID No. 17) was designed for V_H cloning; and si degenerative 5* primers, of which one was effective (Sequence ID No. 20), and one specific 3' primer (Sequence ID No. 19), were designed for V_Λ cloning.

For the 3' primer of the heavy chain, mouse heavy chain immunoglobulin sequences from GCG were compared to determine a site downstream from the variable region that would be suitable for primer design and hybridization. Since it would be advantageous to include the region containing the enhancer element, a sequence just 3' to the putative enhancer was chosen. The enhancer has been shown by others to lie within a 312 bp (base pair) Pst I - Eco RI fragment.

Mouse K chain immunoglobulin sequences from GCG were compared to determine a site downstream from the variable region that would be suitable for primer design. Since the exact region of the K chain enhancer in relationship to the variable region has not been determined, a sequence 1280-1307 bp downstream of J5 was chosen. This region contained a Hind III site at the 3• end which could be used for cloning into a appropriate vector.

Mouse heavy and light chain immunoglobulin variabl region sequences from GCG were compared to determine a regio suitable for primer design that could be used in conjunctio with the 3• primer for amplification of variable regio sequences. Since the variable regions between immunoglobuli molecules are not identical, the sequences were analyzed fo regions of homology that could be used for designing degene rate oligonucleotide primers. Approximately 500 file containing heavy chain variable region sequences and 500 file containing light chain variable region sequences were analyze for homology. Three areas were examined: the 5' end of th sequences encoding the mature immunoglobulin molecule, the 5 end of leader sequences, and sequences surrounding putativ promoter elements upstream of the leader. Since it wa anticipated that expression of recombinant antibody would b attempted in murine myeloma cells, use of the endogenou murine immunoglobulin promoter was thought to be advantageous. For the heavy chain, there were 21 files that include promoter element sequences. From extensively examining thes sequences, it was found that they fell into one of thre roughly homologous groups. Therefore, it was decided t synthesize three degenerate oligonucleotides as possible 5' primers. For the light chain, there were also 21 files wit sequence information surrounding the promoter. Thes sequences fell into six groups with 2 sequences showing onl limited homology to the others. Six degenerate oligonucleo tides were synthesized for use as 5' primers to amplify ligh chain variable region sequences.

All primer sequences were run on a computer program t test for secondary structure. In addition, the 5' primer were compared to the 3' primer by a computer program t eliminate the possibility of primer di ers. All primers wer synthesized on an Applied Biosystems 381A DNA synthesizer an purified over an OPC column (Applied Biosystems) .

The standard reaction conditions for PCR amplification from genomic DNA is described as follows: 100 μl of reaction mixture containing 0.1 μg of genomic DNA; 10 mM Tris-HCl (pH 8.4); 2.5 mM MgCl₂; 50 mM KC1; 100 μg/ml gelatin; 200 μM of each dATP, dCTP, dTTP and dGTP; 0.25 μM of each PCR primer and 2.5 units of TAQ polymerase. To obtain optimal PCR protocol for V_H and V_κ cloning, the primer concentration, magnesium concentration, primer annealing temperature and reaction cycles were varied in the reactions. For V_H amplification, the following parameters were tested: primer concentration- 0.125 mM, 0.25 mM and 0.5 mM; magnesium concentration- 0.5 mM, 1.5 mM, 2.5 mM and 5.0 mM; TAQ polymerase- 1.25 units (u) , 2.5 units, 5.0 units and 7.5 units; primer annealing temperature- 32°C, 40°C and 50°C; reaction cycles- 30 and 40. Over 30 PCR amplification protocols at various conditions were performed in order to achieve the amplification of V_H. For V_κ amplifica- tion, the parameters tested were: magnesium concentration- 0.5 mM, 1.5 mM, 2.5 mM and 5.0 mM; primer annealing tempera¬ ture- 50°C and 55°C, and reaction cycles- 35 and 40. Over 40 PCR amplification protocols were performed to achieve the V_κ amplification. Southern hybridizations with JH and JL probes were performed to verify that the PCR amplified fragments were immunoglobulin heavy and light chain variable regions. The PCR cloned TAb 250 V,, and V_κ genes were subsequently subcloned into plasmid vector pUC19 at the Eco RI site (Figure 1) and pIBI21 at the Hind III site (Figure 2) respectively, and their nucleotide sequences were determined by plasmid DNA sequencing from both DNA strands. The mouse light chain enhancer, which was not included in the cloned V_κ gene fragment, was clone separately-from TAb 250 genomic DNA by PCR.

The mouse K enhancer was amplified from TAb 250 genomi DNA using PCR (Figure 3) . The primers used are found i Sequence ID No. 9 (5' primer) and Sequence ID No. 10 (3' primer) . Reaction conditions were the same as for the human γi constant region described below, except that ImM MgCl₂ was used. Amplification was performed in a Perkin-Elmer Cetus (Norwalk, CT) instrument with the following procedure: denature 95°C, 30 seconds, anneal 55°C, 30 seconds, extend 72°C, 90 seconds, for thirty cycles. The amplified DNA was extracted with chloroform, and primers were removed using Geneclean (BIO 101 Inc., La Jolla, California). The DNA was digested with Eco RI, run on an agarose gel, excised, and purified using Geneclean. Plasmid pUC19 was digested with Eco RI and treated with calf intestine alkaline phosphatase. The K enhancer DNA was then cloned into pUC19 to create plasmid p19enhancer. The c enhancer sequence was verified by restriction mapping. Attached as Appendix A is a sequence map of the heavy chain variable region of TAb250, with the location of such region indicated by the symbol VH. A promoter element, leader, D region, J4 region and enhancer region are also indicated. Attached as Appendix B is a sequence map of the light chain variable region of TAb250, with the location of such region indicated by the symbol vk. A promoter element, leader and J2 regions are also indicated.

IV. Cloning Human CΎI and C/c Genes

The human heavy chain 1 constant (C I) gene and the human light chain K (C_K) gene were cloned from human IgG producing cell line ARH-77 (ATCC CRL 1621) by using the similar PCR approach as described above and were verified by Southern hybridization using oligonucleotide probes to Cγl an C/c coding region. Each amplified Cγl and CK gene fragmen contained several hundred base pair flanking sequences at bot the 5' and 3' end. A. The γl constant region was amplified from ARH-7 genomic DNA using PCR. The primers used are found in Sequenc ID No. 5 (5' primer) and No. 6 (3' primer). Reactio conditions were 10 mM Tris, pH 8.4, 50 mM KCl, 1.5 mM MgCl₂, 10 μg/ml gelatin, 0.25 μM each PCR primer, 0.2 mM dNTPs, 2 ng/μl genomic DNA, 0.025 u/μl TAQ polymerase in 100 μl total volume. Amplification was performed in an ERICOMP, Inc. (San Diego, California) instrument with the following procedure: denature at 95°C, for 30 seconds, anneal at 55°C, for 30 seconds, and extend 72°C for 150 seconds, for thirty cycles. The ampli ied DNA was verified to be the γl constant region by Southern blot (see Sequence ID No. 11 for probe) and sequence analysis. The amplified DNA was extracted with chloroform and primers were removed using Geneclean (BIO 101) according to the manufacturer's directions. The DNA was digested with Bam HI and Sal I (New England Biolabs, Beverly, Massachusetts) according to the manufacturer's directions. The DNA was then run on an agarose gel, excised, and purified using Geneclean. Plasmid pBR322 was digested with Bam HI and Sal I and treated with calf intestine alkaline phosphatase (Boehringer Mannheim Biochemical, Indianapolis, Indiana) . The γl PCR DNA was then cloned into pBR322 to create plasmid 322γi (Figure 4) .

B. The K constant region was amplified from ARH-77 genomic DNA using PCR. The primers used are found in Sequence ID No. 7 (5' primer) and No. 8 (3' primer). Reaction conditions were the same as above, except that 2 mM MgCl₂ was used. Amplification was performed in ERICOMP with the following procedure: 10 cycles: denature at 94°C, for 30 seconds, anneal at 55°C, for 30 seconds, and extend at 72°C, for 150 seconds; followed by 10 cycles which were the same except that extension occurred for 210 seconds; followed by 10 cycles which were the same except that extension occurred for 270 seconds. The amplified DNA was verified to be the human K constant region by Southern blot. (See Sequence ID No. 12 for probe.) The amplified DNA was extracted with chloroform, and primers were removed using Geneclean. The DNA was digested with Bam HI and Eco RI, and purified using Geneclean. pUC19 was digested with Bam HI and Eco RI and treated with calf intestine alkaline phosphatase. The K PCR DNA was then cloned into pUC19 to create pl9_K (Figure 5) .

V. Construction of Expression Plasmids

A. Construction of plasmid SVNH-L (chimeric heavy chain expression plasmid) l) The mouse heavy chain variable region (V_H) was subcloned into pBR322: pUC 19V_H was digested with Eco RI and Sal I, the fragment was gel isolated and subcloned into pBR322 Bam Hi/Sal I with the use of an oligonucleotide adapter (see Sequence ID Nos. 13 and 14) to create plasmid 322V_H (Figure 6) .

2) The mouse V_H and the human γl constant region were subcloned into pSV2neo: p322V_H was digested with Bam HI and Sal I, and the V_H fragment was gel isolated. p322γl was digested with Bam HI and Sal I, and the Cγl fragment was gel isolated. Plasmid pSV2neo was digested with Bam HI and treated with calf intestine alkaline phosphatase. The V_H and Cγl fragments were ligated into pSV2neo to create plasmid SVNH-L (Figure 7) . B. Construction of plasmid RGL-L (chimeric light chai expression plasmid)

1) The mouse light chain variable region (V/c) wa subcloned into pBR322: pIBI21V_/c was digested with Hind III the V_κ fragment was gel isolated and subcloned into pBR32 which had been digested with Hind III and treated with cal intestine alkaline phosphatase to create plasmid 322V_Λ (Figur 8).

2) The mouse V_κ and the human K constant region (C_κ) were subcloned into p322ΔRH: p322V_κ was digested with Bam H and Eco RI and the V_κ fragment was gel isolated. Plasmid pl9c was digested with Bam HI and Eco RI and the C_κ fragment was gel isolated (Figure 9) . Plasmid p322ΔRH was digested with Bam HI and treated with calf intestine alkaline phosphatase. The v_κ and C^ fragments were ligated into p322ΔRH to create plasmid ΔRH-light (Figure 10) .

3) A portion of the intron 3* to the V_κ region was deleted from pΔRH-light: pΔRH-light was digested with Eco RI and Xba I and the vector fragment was gel isolated. The Eco Rl/Xba I oligonucleotide adapter (Sequence ID Nos. 15 and 16) was ligated into the vector to create plasmid Δ-light (Figure 11) .

4) The mouse K enhancer (cloned using primers of Sequence ID Nos. 9 and 10) was added to pΔ-light: pl9enhancer was digested with Eco RI and the enhancer fragment was gel isolated. pΔ-light was digested with Eco RI and treated with calf intestine alkaline phosphatase. The enhancer fragment was ligated into pΔ-light to create pΔL-enhancer (Figure 12) .

5) The light chain fragment containing V_Λ, K constant and enhancer regions from pΔL-enhancer was subcloned into pRSV-gpt: pΔL-erihancer was digested with Bam HI and the light chain fragment was gel isolated. pRSV-gpt was digested with Bam HI and treated with calf intestine alkaline phosphatase. The light chain fragment was ligated into pRSV-gpt to creat plasmid RGL-L (Figure 13) .

c. Gene Transfection

The chimeric heavy chain plasmid and the chimeric ligh chain plasmid were cut by restriction enzyme Pvu I and Bgl respectively . in a non-essential region of the plasmid

Samples of 5 μg to 50 μg of linear heavy and light chai plasmid DNA were cotransfected into 1x10 SP 2/0 cells in 1 m

PBS (phosphate buffered saline) by electroporation at 30 volts and 800 μF using cell-porator electroporation syste

(Bethesda Research Laboratories, Inc., Gaithersburg, MD

"BRL") . The electroporated cells were recovered in RPM medium (BRL) supplemented with 10% fetal calf serum under 5

C0₂ at 37°C for 24-48 hours. The cells were then dispense into 48-well tissue culture plates at lxlO⁴ cells/well unde

G418 antibiotic selection at concentration of 300 μg/ml.

IV. ELISA Analysis of Chimeric Antibody Expression

Culture supernatants harvested from G418 resistan transfectants were screened for chimeric heavy chain an chimeric light chain expression by a two antibody sandwic ELISA. Human IgG (Sigma Chemical Co., St. Louis, MO) was use as a standard for both heavy and light chain ELISA, prepare at concentrations ranging from 10 ng/ml to 1 μg/ml. Ninety six well plates were coated with 100 μl of anti-human IgG monoclonal antibody (Sigma) diluted 1:400 (v/v) in PBS or 10 μl of 2 μg/ml goat anti-human K light chain (Sigma) at 4°C fo 15-18 hours. Plates were washed once with 0.05% Tween 20 i PBS and blocked with 200 μl of 2% BSA in PBS for 1 hour. After washing with PBS, 100 μl of culture supernatants o human IgG standards were added to the wells and incubated fo 3 hours at room temperature. Plates were then washed si times with PBS and 100 μl of goat anti-human IgG γ chai specific HRP (horseradish peroxidase) conjugate (Zymed La Inc., South San Francisco, California) diluted at 1:2000 o goat anti-human IgG (H+L) - HRP conjugate (Zymed Lab Inc. diluted at 1:2000 was added to each well. Incubation wa carried on for 1 hour at room temperature. Plates were washe six times in 0.05% Tween 20 in PBS and developed by adding 10 μl of TMB/peroxidase substrate at 1:1 (v/v) (Kirkegaard an Perry Lab Inc., Gaithersburg, Maryland). The reaction wa terminated by adding 50 μl of 1 M phosphoric acid and th absorbance measured at a dual wave length of 450nm/595nm on microplate reader equipped with analysis software packag (Molecular Devices Corp.) . To measure the amount of antibod secreted by the transfected clone, 1x10 cells were washe with selection medium (RPMI supplemented with 10% fetal cal serum; 2 mM Glutamine; 1 mM sodium pyruvate and 300 μg/m G418) and resuspended in 1 ml of the same medium. After 48 hours at 37°C, the supernatants were recovered, a seria dilution of the culture supernatants was prepared and samples were assayed by ELISA as described above.

V. Characterization of Chimeric Antibody by Cell Labeling and Immunoprecipitation

An aliquot of 1x10 cells was pelleted and washed twice in methionine and cysteine free RPMI medium. The cell pellet was resuspended in 3 ml of the same methionine and cysteine free medium and labeled with 300 μCi of Trans ³⁵S-label S

( 35S-methionine, 35S-cysteine; ICN) for 6 hours at 37°C under 5% C0₂ with occasional swirling. Cell supernatants were collected for analysis of secreted antibody. Cell pellets were lysed in RIPA buffer (50 mM Tris-HCl pH 7.5; 150 mM NaCl; 1% sodium deoxycholate; 1% NP-40 and 0.1% SDS) and following centrifugation at 100,000 xg for 30 minutes, supernatants were collected for analyzing cytoplasmic antibody. Goat anti-human K light chain antibody (Sigma) was used in the immunopreci- pitation. After the culture supernatants or cell lysates were incubated with 10 μg of the antibody for 2 hours at 4°C, 50 μl of slurry of protein A sepharose CL-4B (Pharmacia LKB Biotech¬ nology, Inc. , New Jersey) was added and incubated for another 30 minutes. The antibody bound protein A beads were pelleted in a microfuge by a 30 second centrifugation, washed twice each in high salt (1 M NaCl) RIPA buffer and RIPA buffer, and resuspended in Laemmli sample buffer. The samples were analyzed directly under non-reducing conditions on a 4-20% SDS polyacrylamide gradient gel or under reducing conditions (by including 0.15 M mercaptoethanol in the sample buffer) on a 12% SDS polyacrylamide gel. The gels were fixed, treated with the autoradiography enhancer, Amplify (Amersham) , dried and exposed to Kodak XAR-5 film.

A. Binding Assays of Chimeric Antibody

The binding activity of TAb 250 chimeric antibody, designated BACh 250, was tested by EIA and competition binding assay. EIA was performed in 96-well microtiter plates coated with glutaraldehyde fixed c-erbB-2 transfected NIH 3T3 cells at lxlO⁴ cells per well. Culture supernatants at serial dilutions were added and incubated for 3 hours at room temperature. The unbound antibody was removed by two PBS washes. Goat anti-human IgG (H+L)-horseradish peroxidase conjugate (Zymed Lab Inc.) was added and incubated for two hours at room temperature. The plate was developed and analyzed as described for the ELISA.

B. I-TAb 250 Competition Assay TAb 250 and supernatants from BACh 250 secreting clones E8 and A7 were tested for their ability to influence the binding of ¹²⁵I-TAb 250. TAb 250 was radiolabeled usin Iodobeads (Pierce Chemical Company, Rockford, Illinois according to the manufacturer's specifications. Carrier-fre Na¹²⁵I (400 μCi of IMS.30, Amersham Corporation, Arlingto Heights, Illinois) was reacted with 25 μg TAb 250 in 100 m Na-phosphate buffer (200 μl, pH 7.4) in the presence of Iodobeads. This resulted in an approximate ratio of on iodine atom per IgG molecule. The incorporation was allowe to proceed at room temperature for 7.5 minutes with inter mi tent mixing. The reaction mixture was removed from th beads, and after 5 minutes, the volume was adjusted to 0.5 m with Na-phosphate buffer and 2 μl were taken to estimat specific activity (see below) . The remaining volume was de salted by gel filtration using a NAP-5 column (Pharmacia) equilibrated with PBS containing 0.1% BSA and 0.02% azide. The radiolabeled antibody was eluted in 1 ml column buffer an was stored at 4°C for up to 6 weeks with no apparent loss of binding activity. The de-salted material was essentially free of unincorporated iodine since >95% was TCA-precipitable. The specific activity of the radiolabeled antibody was estimated by TCA precipitation of the material before the de¬ salting step. Thus, 2 μl of the reaction mixture was diluted 500-fold in column buffer and duplicate aliquots mixed with an equal volume of ice-cold 20% TCA. After 15 minutes on ice the precipitated material was collected by centrifugation (10 min, 3000 xg) . Supernatants and pellets were counted separately, and the incorporation was expressed as the percent of TCA- precipitable counts. The incorporation obtained in separate iodinations ranged from 27% to 45%, yielding specific activity estimates from 3.9 to 7.2 μCi/μg. Before each binding experi- ment, an appropriate amount of 125I-TAb 250 was de-salted by gel filtration using a NAP-5 column equilibrated in binding buffer. This procedure removed the azide and yielded materia that was routinely >98% TCA-precipitable.

Single cell suspensions of SKBR3 cells and I-labele TAb 250 were prepared as previously described in commonl assigned U.S.S.N. 07/644,361. Sample dilutions were prepare in binding buffer (MEM medium supplemented with 0.1% BSA, 50 mM HEPES pH 7.0) . Samples of culture supernatants or cold TA 250 prepared at various concentrations were incubated wit 10 μl of ¹²⁵I-labeled TAb 250 at specific activity of 8 x 10⁵ cpm/μg, and 1x10 cells of prepared SKBR3 cells in a final volume of 100 μl, on ice in a shaker at 80 rpm for 4 hours. To terminate the reaction, 800 μl of ice-cold binding buffer was added to reaction mix and the supernatant was removed by centrifugation. The cell associated radioactivity was measured by counting the cell pellets in an Isodata γ counter. As shown in Figure 14, the BACh 250 antibodies displaced 125I-TAb 250 binding in a manner comparable to unlabeled TAb 250.

C. Effect of BACh 250 on Cell Proliferation The chimeric anti-c-erbB-2 antibody BACh 250 effect on cell proliferation was tested on the c-erbB-2 bearing tumor cell line SKOV3. SKOV3 cells were seeded in growth medium into 24-well dishes at 10,000 cells/well. After 24 hours at 37°C, monoclonal antibodies to c-erbB-2, TAb 250, BACh250, or purified Fab or F(ab')₂ fragments of TAb 250 were added to yield a final assay concentration of 10 μg/ml. At various times after the addition of antibodies, the cells were removed with trypsin and quantified using a Coulter Counter. In addi¬ tion, representative cell samples were stained with propidium iodide and analyzed using a FACS Scan (Becton-Dickinson, Mountain view, California) to determine the percentage of viable cells in each treatment group. Each point represents the mean of triplicates and is expressed as a percentage o the viable cell number compared to untreated control wells.

TAb 250 and BACh 250 had a similar effect on prolifera tion. The proliferation of SKOV3 cells was suppressed t approximately 60% of control cell number by TAb 250 after 10 days, while proliferation of SKOV3 cells was suppressed to approximately.58% of control cell number by BACh 250. Treat¬ ment with F(ab')₂ fragments of TAb 250 reduced cell growth to 76% of control levels. While control cells were assessed to be >98% viable, cells treated with these antibodies demon¬ strated a small but significant loss of viability (from 84 to 89%) .

D. Complement Fixation by BACh 250

TAb 250 or BACh 250 (3.1-25 μg/ml) was added to ⁵¹Cr- labeled SKBR-3 cells (which express high levels of c-erbB-2) , in the presence of rabbit complement. The cells were incu¬ bated at 37°C for 1 hour, and supernatants were harvested and counted in a gamma counter. The percent of specific Cr release was calculated as the difference between experimental and background release divided by the difference between total release and background release. Total release was calculated by lysing cells in 10% SDS, and the background release was determined in the absence of complement.

While the chromium release in the presence of TAb 250 and rabbit complement remained at essentially background levels, 6.25 μg/ml of BACh 250 in the presence of 1:10 dilution of rabbit complement caused approximately 67% of Cr release. See Figures 16A and B. These results indicate that BACh 250 can fix rabbit complement more effectively than TAb 250. E. Antibody dependent cellular toxicity mediated b human effector cells

Human effector cells were isolated by density gradien centrifugation. The effector cells were added to Cr-labele SKBR-3 cells at various effector to target ratios (E:T) in th presence of IgG, control antibody, TAb 250, or BACh 250. Plates were incubated for 24 hours at 37°C. Supernatants were harvested and counted in a gamma counter. The specific chro¬ mium release was more than twice that of the IgG, at optimal E:T ratios.

F. In vivo effect of BACh 250

Female Balb\c nu\nu mice were implanted with SKOV-3 cells. Treatments were started 7 days after tumor cell implant. Animals were treated with either an IgG, isotype control monoclonal antibody, TAb 250, or BACh 250 at 1000 μg/dose. Treatments were given interperitoneally on a schedule of one time a week for three weeks. After 36 days, the tumors in the BACh 250 animals were approximately one- third the volume of the tumors from the control animal tumors, while the tumors in the TAb 250 treated animals were approximately two-thirds the volume of the tumors from the control animals.

In the same experiment when TAb 250 or BACh 250 were combined with cisplatin, both were effective in reducing tumor size. After 36 days, tumors from the treated groups were approximately one third the volume of IgG or cisplatin treated control groups.

In a second experiment, the combination treatment of BACh 250 and cisplatin inhibited tumor growth an average of 85%. This was similar to the inhibitory effects observed with TAb 250 and cisplatin. However, in the BACh 250 and cisplatin groups, 3 of 8 mice showed no tumor growth or tumor regression as compared to 0 of 8 mice showing regressions in the control groups. One of eight mice showed no tumor growth in the TA 250 plus cisplatin groups. Thus, the chimeric antibod appears to be as effective as the parental antibody from whic it was derived, in inhibiting tumor growth when used above o in combination with chemotherapeutic drugs such as cisplatin.

The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Shawver, Laura

Liu, Hsaio-Lai C. Parkes, Deborah L. McGrogan, Michael P. Brandis, John W.

(ii) TITLE OF INVENTION: Recombinant and Chimeric Antibodies to c-erbB-2

(iii) NUMBER OF SEQUENCES: 20

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Edwin P. Ching

(B) STREET: 1501 Harbor Bay Parkway

(C) CITY: Alameda

(D) STATE: California

(E) COUNTRY: USA

(F) ZIP: 94501

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Ching, Edwin P.

(B) REGISTRATION NUMBER: 34,090

(C) REFERENCE/DOCKET NUMBER: A120

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 415-266-7476

(B) TELEFAX: 510-769-5308 (2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1824 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

( A ) NAME/KEY : promoter

( B ) LOCATION : 22. . 29 ( D ) OTHER INFORMATION : /label= promoter octamer

( ix ) FEATURE :

( A ) NAME /KEY : sig_peptide

( B ) LOCATION : 153..197

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 312..597

(D) OTHER INFORMATION: /label= VH

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 600..609

(D) OTHER INFORMATION: /label= D

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 612..659

(D) OTHER INFORMATION: /label= J4

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1566..1813

(D) OTHER INFORMATION: /note= "Enhancer"

(ix) FEATURE:

(A) NAME/KEY: region

(B) LOCATION: 402..416 (D) OTHER INFORMATION: /label= CDRl

(ix) FEATURE:

(A) NAME/KEY: region ^'

(B) LOCATION: 459..506 (D) OTHER INFORMATION: /label= CDR2 ( ix ) FEATURE:

(A) NAME /KEY: region

( B ) LOCATION : 603 . . 626

( D ) OTHER INFORMATION: /label= CDR3

( xi ) SEQUENCE DESCRIPTION: SEQ ID NO : l :

GAATTCATAT AGCAGGACCA TATGCAACTA AGCCTTCTCT CTGCCCATGA AAAACACCTC 60

GGCACTGACC CTGCAGCTCT GACAGAGGAG GCCAGTCCTG GATTCCCAGT TCCTCACATT 120

CAGTGATCAG CACTGAACAC GGACCCCTCA CCATGAACTT GGGGCTCAGC TTGATTTTCC 180

TTGTCCTTGT TTTAAAAGGT AATTTATTGA GAAGAGATGA CATCTGTTGT ATGCTCATGA 240

GACAGAAAAA TTGTTTGTTT TGTTAGTGAC AGTTTTCCAA CCAGCATTCT _< CTGTTTGCAG 300

GTGTCCAGTG TGAAGTGAAG CTGGTGGAGT CTGGGGGAGA CTTAGTGCAG CCTGGAGGGT 360

CCCTGAAACT CTCCTGTGCG ACCTCTGGAT TCTCTTTCAG TGACTTTTAC ATGTATTGGG 420

TTCGCCAGAC TCCAGAAAAG AGGCTGGAGT GGGTCGCATA TGTTAGTTCT GGAGGTGAGA 480

GCTATTATTC AGACACTATA AGGGGCCGAT TCACCTTCTC CAGAGACAGT GCCAAGAACA 540

CCCTGCACCT GCAAATGAGC CGTCTGAAGT CTGAGGACAC AGCCATGTAT TTCTGTGCAA 600

GATTTGGTGA CTCTGCTATG GACTACTGGG GTCAAGGAAC CTCAGTCACC GTCTCCTCAG 660

GTAAGAATGG CCTCTCCAGG TCTTTATCTT TACCCTTTGT TTTGGAGTTT TCTGAGCATT 720

GTAGACTATT CTTGGATATT TGTCCCTGAG GGAGCCGGCT GACAGAAGTT GGGAAATGAA 780

CTGTCTAGGG ATCTCAGAGC CTTTAGGGCA GATTATCTCC ACATCTTTGA AAAACTTAGA 840 .

ATCTGTGTGA TGGTGTTGGT GGAGTCCCTG GATGATGGGA TAGGGACTTT GGAGGCTCAT 900

TTGAGGGAGA TGCTAAAATA GTCCTATGGC TGGAGGGATA GTTGGGGCTG TAGTTGGAGA 960

TTTTCAGTTT TTAGAATAAA AGTATTAGCT GCGGAATACA CTTCAGACCA CCTCTGTGAC 1020

AGCATTTATA CAGTATGCAT AGGGACGTGG AGTGGGGCAC TTTCTTTAGA TTTGTGAGGA 1080

ATGTTCCACA CTAGATTGTT TAAAACTTCA TTTGTTGGAA GGAGAGCTGT CTTAGTGATT 1140 GAGTCAAGGG AGAAAGGCAT CTAGTCTCGG TCTCAAAAGG GTAGTTGCTG TCTAGAGAGG 1200

TCTGGTGGAG CCTGCAAAAG TCCAGCTTCA AAGGAACACA GAAGTATGTG TATGGAATAT 1260

TAGAAGATGT TGCTTTTACT CTTAAGTTGG TTCCTAGGAA AAATAGTTAA ATACTGTGAC 1320

TTTAAAATGT GAGAGGGTTT TCAAGTACTC ATTTTTTTAA ATGTCCAAAA TTTTTGTCAA 1380

TCAATTTGAG GTCTTGTTTG TGTAGAACTG ACATTACTTA AAGTTTAACC GAGGAATGGG 1440

AGTGAGGCTC TCTCATACCC TATTCAGAAC TGACTTTTAA CAATAATAAA TTAAGTTTAA 1500

AATATTTTTA AATGAATTGA GCAATGTTGA GTTGGAGTCA AGATGGCCGA TCAGAACCAG 1560

AACACCTGCA GCAGCTGGCA GGAAGCAGGT CATGTGGCAA GGCTATTTGG GGAAGGGAAA 1620

CCAGCCCCAC CAAACCGAAA GTCCAGGCTG AGCAAAACAC CACCTGGGTA ATTTGCATTT 1680

CTAAAATAAG TTGAGGATTC AGCCGAAACT GGAGAGGTCC TCTTTTAACT TATTGAGTTC 1740

AACCTTTTAA TTTTAGCTTG AGTAGTTCTA GTTTCCCCAA ACTTAAGTTT ATCGACTTCT 1800

AAAATGTATT TAGTCGACGA ATTC 1824

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1657 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: promoter

(B) LOCATION: 32..39

(D) OTHER INFORMATION: /label= promoter octamer

(ix) FEATURE:

(A) NAME/KEY: sig_peptide

(B) LOCATION: 120..179

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 370..659

(D) OTHER INFORMATION: /label= vk

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 659..691

(D) OTHER INFORMATION: /label= J2

(ix) FEATURE:

(A) NAME/KEY: region

(B) LOCATION: 440.. 72

(C) OTHER INFORMATION: /label= CDRl

(ix) FEATURE:

(A) NAME/KEY: region

(B) LOCATION: 518..538

(D) OTHER INFORMATION: /label= CDR2

(ix) FEATURE:

(A) NAME/KEY: region

(B) LOCATION: 635..661

(D) OTHER INFORMATION: /label= CDR3 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: AGCTTCATT TACTTCCTTA TTTGGTGACT ACTTTGCATA GCTGCCCATG ATTTATAAAC CATGTCTTTG CAGTGAGATC TGGGCATCAA GATGGAGTTT CAGACCCAGG TCTTTGTATT GTGAGACATT TAAAAGTATT ATAAAATCTT AAAAGTAATT ATAGGAAGCC AATATTAGGT CAGACAATAC CATTAAATAA TTGTATTATG AAGTCTTTGT ATATGTAAGT GTATAGTCAT TGTTGATGGA GACATTGTGA TGACCCAGTC TCAAAAATTC CAGGGTCAGC ATCACCTGCA AGGCCAGTCA GAATGTTCGC ACAGAGACCA GGGCAGTCTC CTAAAGCGCT GATTTACTTG AGTCCCTGAT CGCTTCACAG GCAGTGGATC TGGGACAGAT TGTGCAATCT GAAGACCTGG CAGATTATTT CTGTCTGCAA GTTCGGAGGG GGGACCAAGC TGGAAATAAA ACGTAAGTAG TAAGTCTAAC CTTGTTGAGT TGTTCTTTGC TGTGTGTTTT GTCAGCAAAT TCCATTCTCA GATCAGGTGT TAAGCAGGGA GGAACGATTT TCAGGCTAAA TTTCAGGCTT CTAAAGCAAA GGGATAAATG TCTTCCTTGG GAGGGTTTTG AGGTGGTAAA ATCACATTCA GTGATGGGAC CAGACTGGAA ATATAACCTA CTTGTGAAGT TTTGGTCCCA TTGTGTCCTT TGTATGAGTT GAACTATCCT TGTAACCCAA AACTTAAGTA GAAGAGAACC AGCTGAGCAA ACAGACTGAC CTCATGTCAG ATTTGTGGGA TTTCTCTGAA CTTAGCCTAT CTAACTGGAT CAGCCTCAGG CGCAGTGATA TGAATCACTG TGATTCACGT TCGGCTCGGG

GTAGGTTGAC TTTTGCTCAT TTACTTGTGA CGTTTTGCTT CTGTTTGGGT AACTTGTGTG 1380

ACTTTGTGAC ATTTTGGCTA AATGACCATT CCTGGCAACC TGTGCATCAT TAGAAGATCC 1440

CCCAGAAAAG AGTCAGTGTG AAAGCTGAGC GAAAAACTCG TCTTAGGCTT CTGAGACCAG 1500

TTTTGTAAGG GGAATGTAGA AGAGAGAGCT GGGCTTTTCC TCTGAATTTG GCCCATCTAG 1560

TTGGACTGGC TTCACAGGCA GGTTTTTTTA GAGAGGGACA TGTCATAGTC CTCACTGTGG 1620

CTCACGTTCG GTGCTGGGAC CAAGCTGGAG CTGAAAC 1657

2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 145 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 31..35

( D ) OTHER INFORMATION : / l abel = CDRl

( ix ) FEATURE :

(A) NAME/KEY: Region

(B) LOCATION: 50..65

( D ) OTHER INFORMATION : / l abel = CDR2

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 98..105

(D) OTHER INFORMATION: /label= CDR3

(ix) FEATURE:

(B) LOCATION: 51

(D) OTHER INFORMATION: /label= Residue may be Val or lie

(i ) FEATURE:

(B) LOCATION: 57

(D) OTHER INFORMATION: /label= Residue may be Asn or Ser

(ix) FEATURE:

(B) LOCATION: 69

(D) OTHER INFORMATION: /label= Residue may be lie or Phe

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Glu Val Lys Leu Val Glu Ser Gly Gly Asp Leu Val Gin Pro Gly Gly 1 5 10 15

Ser Leu Lys Leu Asp Cys Ala Thr Ser Gly Phe Ser Phe Ser Asp Phe 20 25 30 Tyr Met Tyr Trp Val Arg Gin Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45

Ala Tyr Xaa Ser Ser Gly Gly Glu Xaa Tyr Tyr Ser Asp Thr lie Arg 50 55 60

Gly Arg Phe Thr Xaa Ser Arg Asp Ser Ala Lys Asn Thr Leu His Leu 65 70 75 80

Gin Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met Tyr Phe Cys Ala

85 90 95

Arg Phe Gly Asp Ser Ala Met Asp Tyr Trp Gly Gin Gly Thr Ser Val 100 105 110

Thr Val Ser Ser Gly Lys Asn Gly Leu Ser Arg Ser Leu Ser Leu Pro 115 120 125

Phe Val Leu Glu Phe Ser Glu His Cys Arg Leu Phe Leu Asp lie Cys 130 135 140

Pro 145

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 109 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 24..34

(D) OTHER INFORMATION: /label= CDRl

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 50..56

(D) OTHER INFORMATION: /label= CDR2

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 89..97

(D) OTHER INFORMATION: /label= CDR3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Asp lie Val Met Thr Gin Ser^' Gin Lys Phe Thr Ser Thr Ser Val Gly 1 5 10 15

Asp Arg Val Ser lie Thr Cys Lys Ala Ser Gin Asn Val Arg Thr Ala 20 25 30

Val Ala Trp Phe Gin Gin Arg Pro Gly Gin Ser Pro Lys Ala Leu lie 35 40 45

Tyr Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Asn Val Gin Ser 65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gin His Trp Asn Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu lie Lys Arg Lys 100 105 (2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..33

(D) OTHER INFORMATION: /note= "gamma-1 constant region 5' primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CAAGTCGACA GCAGGTGCAC ACCCAATGCC CAT 33

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..31

(D) OTHER INFORMATION: /note= "gamma-1 constant region 3' primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CTAGGATCCA GAACCATCAC AGTCTCGCAG G 31

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..33

(D) OTHER INFORMATION: /note= "kappa constant region 5'primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GGTGAATTCC TGTCTGTCCC TAACATGCCC TGT 33

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..33

(D) OTHER INFORMATION: /note= "kappa constant region 3'primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GTTGGATCCT GACCGTAAGA CCTGTCACCC TTA 33

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

( A ) NAME/KEY: misc_feature

( B ) LOCATION: 1. .33

(D) OTHER INFORMATION: /note= "kappa enhancer 5'primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CTAGAATTCA GCTTTTGTGT TTGACCCTTC CCT 33

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..33

(D) OTHER INFORMATION: /note= "kappa enhancer 3 'primer'

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: CAGGAATTCA GCTAAACCTA CTGTATGGAC AGG 33

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 60 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..60

(D) OTHER INFORMATION: /note= "gamma oligo probe"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: ACGGTCACCA CGCTGCTGAG GGAGTAGAGT CCTGAGGACT GTAGGACAGC CGGGAAGGTG 60

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS: ' (A) LENGTH: 60 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..60

(D) OTHER INFORMATION: /note= "kappa oligo probe"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: TGCTGAGGCT GTAGGTGCTG TCCTTGCTGT CCTGCTCTGT GACACTCTCC TGGGAGTTAC 60

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..10

(D) OTHER INFORMATION: /note= "BamHI/EcoRI adapter oligo"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GATCCACTGG 10

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..10

(D) OTHER INFORMATION: /note= "BamHI/EcoRI adapter region"

( i) SEQUENCE DESCRIPTION: SEQ ID NO:14: AATTCCAGTG 10

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..10

(D) OTHER INFORMATION: /note= "Xbal/EcoRI adapter oligo"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: CTAGACAGTG 10

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(i ) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..10

(D) OTHER INFORMATION: /note= "Xbal/EcoRI adapter oligo"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: AATTCACTGT 10

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 37 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..37

(D) OTHER INFORMATION: /note= "3' Heavy Chain Variable Region PCR Primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GAGGAATTCG TCGACTAAAT ACATTTTAGA AGTCGAT 37

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..32

(D) OTHER INFORMATION: /note= "5' Heavy Chain Variable region PCR Primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GAGGAATTCM TATAGCAGRA MSAYATGCAA AT 32

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..28

(D) OTHER INFORMATION: /note= "3' Light Chain Variable region PCR Primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: CATTAAGCTT TTAATATAAC ACTGGATA 28

(2) INFORMATION FOR SEQ ID NO:20:

( i ) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..33

(D) OTHER INFORMATION: /note= "5' Light Chain Variable Chain Region PCR Primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: MAWTTACTTC CTTATTTGRT GACTRCTTTG CAT 33

Claims

WHAT IS CLAIMED IS;

1. A recombinant DNA sequence encoding at least one CD region derived from an antibody specific for c-erbB-2 protein.

2. The sequence of claim 1, wherein the CDR region i derived from a heavy chain variable region of the antibody.

3. The sequence of claim 2, wherein the CDR region is sequence selected from the group consisting of those that encode for the amino acid sequence at about position 31 to 35, 50 to 65, and 98 to 105 set forth on Sequence ID No. 3.

4. The sequence of claim 1, wherein the CDR region is derived from a light chain variable region of the antibody.

5. The sequence of claim 4, wherein the CDR region is a sequence selected from the group consisting essentially of those that encode for the amino acid sequence at about position 24 to 34, 50 to 56, and 89 to 97 set forth on Sequence ID No. 4.

6. The sequence of claim 1, wherein the antibody causes down modulation of c-erbB-2 protein or induces an increase in phosphorylation of c-erbB-2 protein when placed in contact with cells expressing the c-erbB-2 protein.

7. The sequence of claim 1, wherein the antibody is that produced by a hybridoma cell line bearing A.T.C.C. Accession No. HB10646.

8. The sequence of claim 1, wherein the sequence further encodes framework regions of an immunoglobulin.

9. The sequence of claim 8, wherein the framework regions are derived from a human immunoglobulin.

10. The sequence of claim 1, wherein the sequence further includes a control sequence for expression.

11. The sequence of claim 10, wherein the control sequence is a promoter.

12. The sequence of claim 10, wherein the control sequence is an enhancer.

13. A recombinant DNA sequence that encodes an antibody light chain variable region specific for c-erbB-2 protein.

14. The sequence of claim 13, wherein the light chain is specific for c-erbB-2 protein when combined with a heavy chain variable region, including that set forth in Sequence ID No. 3.

15. The recombinant DNA sequence of claim 13, that encodes an antibody light chain variable region which when incorporated into immunoglobulin conformation competes with an antibody produced by a hybridoma cell line bearing A.T.C.C. Accession No. HB10646 for the binding to c-erbB-2.

16. A recombinant DNA sequence that comprises DN encoding an antibody light chain variable region to c-erbB- protein having an amino acid sequence consisting essentiall of that sequence set forth in Sequence ID No. 4.

17. The recombinant DNA sequence of claim 16, wherein coding strand sequence is that set forth in Sequence ID No. at base positions 370 to 659.

18. The sequence of claim 13, wherein the sequenc comprises a CDR region selected from the group consisting of those that encode the amino acid sequence at about position 24 to 34, 50 to 56, and 89 to 97 set forth on Sequence ID No.. 4.

19. A recombinant DNA sequence that encodes an antibody heavy chain variable region specific for c-erbB-2 protein.

20. The sequence of claim 19, wherein the heavy chain is specific for c-erbB-2 protein when combined with a light chain variable region specific for c-erbB-2, set forth in Sequence ID No. 4.

21. The recombinant DNA sequence of claim 19, that encodes an antibody heavy chain variable region which when incorporated into immunoglobulin conformation competes with an antibody produced by a hybridoma cell line bearing A.T.C.C. Accession No. HB10646 for the binding to c-erbB-2.

22. A recombinant DNA sequence that comprises DNA encoding an antibody heavy chain variable region to c-erbB-2 having an amino acid sequence consisting essentially of that sequence set forth in Sequence ID No. 3.

23. The recombinant DNA sequence of claim 22 wherein a coding strand for the amino acid sequence is that set forth in Sequence ID No. 1 at about base positions 312 to 597.

24. The sequence of claim 19, wherein the sequence comprises a CDR region selected from the group consisting of those that encode the amino acid sequence at about position 31 to 35, 50 to 65, and 98 to 105 set forth on Sequence ID No. 3.

25. A recombinant DNA vector that comprises the DNA sequence of claim 1.

26. A recombinant DNA vector that comprises the DNA sequence of claim 13.

27. A recombinant DNA vector of claim 23 that further comprises a promoter or transcriptional activating sequence positioned to drive the expression of the DNA.

28. A recombinant DNA vector of claim 27 wherein one of the transcriptional activating sequences is an antibody enhancer.

29. A recombinant DNA vector that comprises the DNA sequence of claim 19.

30. A recombinant DNA vector that comprises the DNA sequence of claim 22.

31. A recombinant DNA vector of claim 29 that further comprises a promoter and transcriptional activating sequence positioned to drive the expression of said DNA.

32. A recombinant DNA vector of claim 31, wherein one o the transcriptional activating sequences is an antibod enhance .

33. A recombinant DNA sequence that comprises DN encoding a chimeric c-erbB-2 specific heavy chain peptide, th variable region derived from a first genetic source and constant region derived from a second and different geneti source.

34. The recombinant DNA sequence of claim 33, wherei the heavy chain variable region has an amino acid sequenc consisting essentially of Sequence ID No. 3.

35. The recombinant DNA sequence of claim 34, wherein a coding strand for the amino acid sequence is that of Sequence ID No. 1 at about base positions 312 to 597.

36. The recombinant DNA sequence of claim 33, wherein the heavy chain variable region has a CDR region selected from the group consisting of those that encode for the amino acid sequence at about position 31 to 35, 50 to 65, and 98 to 105 set forth on Sequence ID No. 3.

37. A recombinant DNA vector that comprises the DNA sequence of claim 33.

38. A recombinant DNA sequence that comprises DNA encoding a chimeric c-erbB-2 specific light chain peptide, a variable region derived from a first genetic source and a constant region derived from second and different genetic source.

39. The recombinant DNA sequence of claim 38, wherein the light chain variable region has an amino acid sequence consisting essentially of Sequence ID No. 4.

40. The recombinant DNA sequence of claim 39, wherein a coding strand for the amino acid sequence is that of Sequence ID No. 2 at about base positions 370 to 659.

41. The recombinant DNA sequence of claim 38, wherein the light chain variable region has a CDR region selected from the group consisting of those that encode for the amino acid sequence at about position 24 to 34, 50 to 56, and 89 to 97 set forth on Sequence ID No. 4.

42. A recombinant DNA vector that comprises the DNA sequence of claim 38.

43. A host cell that expresses chimeric antibodies chains specific for c-erbB-2.

44. The host cell line of claim 43, that expresses antibody which when in immunoglobulin conformation competes with an antibody produced by the hybridoma cell line bearing A.T.C.C. Accession No. HB10646 for the binding to c-erbB-2.

45. A host cell that expresses the protein encoded by the sequence of claim 1.