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WO1993012220A1 - ANTICORPS DE RECOMBINAISON ET CHIMERIQUES CONTRE c-erbB-2 - Google Patents

ANTICORPS DE RECOMBINAISON ET CHIMERIQUES CONTRE c-erbB-2 Download PDF

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Publication number
WO1993012220A1
WO1993012220A1 PCT/US1992/010437 US9210437W WO9312220A1 WO 1993012220 A1 WO1993012220 A1 WO 1993012220A1 US 9210437 W US9210437 W US 9210437W WO 9312220 A1 WO9312220 A1 WO 9312220A1
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sequence
erbb
antibody
recombinant dna
protein
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PCT/US1992/010437
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English (en)
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Laura K. Shawver
Hsiao-Lai C. Liu
Deborah L. Parkes
Michael P. Mcbrogan
John W. Brandis
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Berlex Laboratories, Inc.
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Publication of WO1993012220A1 publication Critical patent/WO1993012220A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • 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.
  • 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) .
  • Kd 185 kilodalton glycoprotein having tyrosine kinase activity
  • EGFR epidermal growth factor receptor
  • 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) .
  • the mouse 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.
  • 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.
  • mouse variable region exons specific for tumor antigens have been fused with human K or ⁇ 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.
  • 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.
  • 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 p ⁇ C19V H ;
  • Figure 2 illustrates the cloning of a PCR-amplified mouse V ⁇ (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 ⁇ fragment was cloned into the Hind III site to make the plasmid pIBI21V ⁇ ;
  • FIG 3 illustrates the cloning of a PCR-amplified mouse K enhancer.
  • Plasmid p ⁇ C19 was cut with Eco RI and treated with calf intestine alkaline phosphatase. The enhancer was ligated into the Eco RI site to make pl ⁇ enhancer;
  • Figure 4 illustrates the cloning of a PCR-amplified human ⁇ i 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 C ⁇ l fragment was then ligated into the Bam HI and Sal I sites to make plasmid p322 ⁇ i;
  • Figure 5 illustrates the cloning of a PCR-amplified human
  • Plasmid pUC19 was cut with Bam HI and Eco RI and then treated with calf intestine alkaline phosphatase. The Cj ⁇ 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 C ⁇ l fragment into plasmid pSV2neo.
  • the mouse V H and the human C ⁇ l fragments were excised from p322V H and p322 ⁇ i, 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 C ⁇ l fragments were ligated into the pSV2neo vector at the Bam HI site to make pSVNH-L;
  • FIG 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 ⁇ fragment was excised from pIBI21V ⁇ by cutting with Hind III. The V ⁇ fragment was ligated into pBR322 at the Hind III site to make p322V ⁇ ;
  • Figure 9 illustrates construction of p322 ⁇ RH. 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 p322 ⁇ RH;
  • Figure 10 illustrates the construction of a plasmid containing a mouse V ⁇ and human C ⁇ .
  • the mouse V ⁇ and the human C ⁇ region were excised from p322V ⁇ and pl9 ⁇ , respectively, by cutting with both Bam HI and Eco RI.
  • Plasmid p322 ⁇ RH was cut with Bam HI and treated with calf intestine alkaline phosphatase.
  • the V ⁇ and the C ⁇ fragments were ligated into the Bam HI site ' to make plasmid p ⁇ RH-light;
  • Figure 11 illustrates the construction of an intermediate plasmid containing a mouse V ⁇ and human C ⁇ .
  • Plasmid p ⁇ RH-light was cut with Eco RI and Xba I.
  • An Eco Rl-Xba I adapter wa ligated into the site to make p ⁇ -light;
  • Figure 12 illustrates the construction of a plasmi containing DNA encoding a mouse V ⁇ , a mouse K enhancer, an human C ⁇ region.
  • Plasmid p ⁇ -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 p ⁇ -light to make the plasmid p ⁇ L-enhancer containing chimeric K chain;
  • Figure 13 illustrates the final step in constructing 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;
  • 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.
  • 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.
  • 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- ⁇ i) when placed in contact with cells expressing the c-erbB-2 protein.
  • PLC- ⁇ i phospholipase C
  • 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
  • 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
  • Recombinant vectors, host cells, and chimeric antibody peptides are also encom ⁇ passed in the invention.
  • 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.
  • the present invention encompasses recombinant DNA sequences, recombinant vectors, host cells, and immunoglobulin peptides (including CDRs) encoded by recombinant sequences.
  • 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) .
  • EGFR epidermal growth factor receptor
  • the c-erbB-2 protein has an extracellular domain tha includes two cysteine-rich repeat clusters, a transmembran domain and an intracellular kinase domain.
  • 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.
  • 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.
  • 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.
  • proteins developed from othe host systems, e.g., proteins that are immunologically relate to the human c-erbB-2 protein.
  • a related ra gene designated neu has been reported in Schecter et al. Science, 229:976 (1985) .
  • Immunoglobu lins or antibodies are typically composed of four covalentl bound peptide chains.
  • 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 (NH 2 ) 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, V ⁇ )
  • the light chain variable region is referred to as V L (including V ⁇ 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 ⁇ (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) .
  • 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
  • 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.
  • 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 ⁇ , 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 ⁇ or C ⁇ in particular.
  • C H 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 ( ⁇ ) , delta ( ⁇ ) gamma ( ⁇ f ) , alpha ( ⁇ ) , and epsilon (e) segments of the heav chain constant region, respectively. In addition, there ar a number of ⁇ 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.
  • 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.
  • 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.
  • Fab 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 limitation 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.
  • 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".
  • “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.
  • 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.
  • 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 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.
  • chimeric anti bodies can include antibodies where the framework an complementarity- determining regions are from differen sources.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • the specificity of the peptides to c-erbB-2 can be determined by their affinity for the antigen.
  • 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.
  • R c-erbB-2
  • ligand or peptide L and receptor-ligand complex (R-L)
  • R-L receptor-ligand complex
  • 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.
  • 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.
  • 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.
  • Assays may b conducted where cells bearing the c-erbB-2 protein are place into contact with radioactive labelled 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.
  • cells or cell extracts may be incubated in the presence o absence of the peptide specific for c-erbB-2.
  • the immun complex may be incubated with ⁇ r 32 P-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.
  • 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
  • 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) .
  • 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.
  • 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.
  • 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.
  • a control sequence include a naturally-associated or heterologous promoter region.
  • the expression control sequence will be a eukaryotic promoter system in vector capable of transforming or transfecting a eukaryoti host cell.
  • 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.
  • 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.
  • 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.
  • yeast yeast
  • amphibian oocytes insect cells
  • mammalian host cell lines such as myelom cells, Cos, CHO or L cells.
  • 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) .
  • 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 interests 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.
  • 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 C ⁇ gene is described in Ellison et al., Nucl . Acid. Res.
  • 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) .
  • ATCC American Type Cultur Collection
  • 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)
  • polypeptide fragments comprising only a portio of the primary antibody structure may be produced, which frag ments possess binding and/or effector activities.
  • the immunoglobulin-related gene may 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.
  • promoters and enhancers specific for other host cells are known in the art. See, Kameyoma et al., supra.
  • 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.
  • 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.
  • antibody peptides are com ⁇ prised of the V H , amino acid Sequence ID No. 3, and the V ⁇ , 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 ⁇ ) .
  • 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 ⁇ i constant region.
  • Th TAb 250 V ⁇ 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.
  • 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 ⁇ C 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.
  • 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.
  • the linkage may be by way of heterobifunctional cross-linkers, e.g., SPDP, carbodiimide, glutaraldehyde, or the like.
  • 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.
  • 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.
  • 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.
  • the antibodies in the immunotoxins will be of the human IgM or IgG isotype, but other mammalian constant regions may be utilized as desired.
  • 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.
  • labels may be employed, such as radionuclides, fluors, enzymes, enzyme substrates, enzyme co-factors, enzyme inhibitors, ligands (particularly haptens) , etc.
  • radionuclides such as 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 .
  • EBV Epstein-Barr virus
  • 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.
  • idiotyp antibody Abl
  • Ab2 anti-idiotype
  • 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.
  • 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.
  • 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.,, IgG 2 , IgG 3 , or IgG 4 .
  • 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.
  • antibodies to framework-associated idiotopes, o regulatory idiotopes may select or amplify T and/or B cel clones with specificity for tumor antigens.
  • 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.
  • 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.
  • cDNA refers to DNA that is derived from a messenger RNA sequence (mRNA) , for example, using reverse transcriptase.
  • mRNA messenger RNA sequence
  • 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.
  • 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.
  • the expressio vectors have a selectable marker, for example, neomyci resistance, which permits the selection of cells containin the marker.
  • 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.
  • 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.
  • 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.
  • 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.
  • the pharmaceutical composition or formulation ma also include other carriers, adjuvants; or nontoxic, nonthera Treatmentic, nonimmunogenic stabilizers and the like.
  • 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.
  • 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.
  • the compositions may be admini ⁇ stered with an anti-neoplastic agent.
  • 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.
  • Target tumor cells are any tumor cells z ⁇ 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 ( 111 In 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.
  • 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.
  • 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.
  • 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 JH 3 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.
  • c-erbB-2 Monoclonal Antibodies
  • 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.
  • Fuse cells were gently washed and plated in 96-well plates a 1-4X10 6 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.
  • V H TAb 250 heavy chain variable region
  • V ⁇ TAb 250 light chain variable region
  • 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 MgCl 2 concentration was 1.5 mM.
  • both V H and V ⁇ cloning th PCR primers were designed to amplify the entire genes, including promoter elements, leaders and variable regions.
  • the amplified V render gene fragment also includes its own enhance sequence.
  • 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 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.
  • All primer sequences were run on a computer program t test for secondary structure.
  • the 5' primer were compared to the 3' primer by a computer program t eliminate the possibility of primer di ers.
  • All primers were 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 ⁇ l of reaction mixture containing 0.1 ⁇ g of genomic DNA; 10 mM Tris-HCl (pH 8.4); 2.5 mM MgCl 2 ; 50 mM KC1; 100 ⁇ g/ml gelatin; 200 ⁇ M of each dATP, dCTP, dTTP and dGTP; 0.25 ⁇ M of each PCR primer and 2.5 units of TAQ polymerase.
  • the primer concentration, magnesium concentration, primer annealing temperature and reaction cycles were varied in the reactions.
  • V H amplification 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 .
  • V ⁇ 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 ⁇ 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 ⁇ 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 ⁇ 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 ⁇ i constant region described below, except that ImM MgCl 2 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).
  • 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.
  • 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 C ⁇ l an C/c coding region. Each amplified C ⁇ l and CK gene fragmen contained several hundred base pair flanking sequences at bot the 5' and 3' end. A.
  • the ⁇ l 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 MgCl 2 , 10 ⁇ g/ml gelatin, 0.25 ⁇ M each PCR primer, 0.2 mM dNTPs, 2 ng/ ⁇ l genomic DNA, 0.025 u/ ⁇ l TAQ polymerase in 100 ⁇ l 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 ⁇ l constant region by Southern blot (see Sequence ID No.
  • 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 ⁇ l PCR DNA was then cloned into pBR322 to create plasmid 322 ⁇ i ( Figure 4) .
  • 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 MgCl 2 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.
  • 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 H mouse heavy chain variable region
  • pBR322 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) .
  • V/c The mouse light chain variable region (V/c) wa subcloned into pBR322: pIBI21V /c was digested with Hind III the V ⁇ 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 ⁇ (Figur 8).
  • the mouse K enhancer (cloned using primers of Sequence ID Nos. 9 and 10) was added to p ⁇ -light: pl9enhancer was digested with Eco RI and the enhancer fragment was gel isolated. p ⁇ -light was digested with Eco RI and treated with calf intestine alkaline phosphatase. The enhancer fragment was ligated into p ⁇ -light to create p ⁇ L-enhancer ( Figure 12) .
  • 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
  • PBS phosphate buffered saline
  • G418 antibiotic selection at concentration of 300 ⁇ g/ml.
  • 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 ⁇ g/ml.
  • Ninety six well plates were coated with 100 ⁇ l of anti-human IgG monoclonal antibody (Sigma) diluted 1:400 (v/v) in PBS or 10 ⁇ l of 2 ⁇ g/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 ⁇ l of 2% BSA in PBS for 1 hour. After washing with PBS, 100 ⁇ l 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 ⁇ l of goat anti-human IgG ⁇ 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.
  • HRP horseradish peroxidase
  • 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 ⁇ l of TMB/peroxidase substrate at 1:1 (v/v) (Kirkegaard an Perry Lab Inc., Gaithersburg, Maryland). The reaction wa terminated by adding 50 ⁇ l 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.) .
  • 1x10 cells were washe with selection medium (RPMI supplemented with 10% fetal cal serum; 2 mM Glutamine; 1 mM sodium pyruvate and 300 ⁇ g/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.
  • selection medium RPMI supplemented with 10% fetal cal serum; 2 mM Glutamine; 1 mM sodium pyruvate and 300 ⁇ g/m G4108
  • 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.
  • TAb 250 chimeric antibody designated BACh 250
  • EIA was performed in 96-well microtiter plates coated with glutaraldehyde fixed c-erbB-2 transfected NIH 3T3 cells at lxlO 4 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.
  • 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 125 I-TAb 250.
  • TAb 250 was radiolabeled usin Iodobeads (Pierce Chemical Company, Rockford, Illinois according to the manufacturer's specifications. Carrier-fre Na 125 I (400 ⁇ Ci of IMS.30, Amersham Corporation, Arlingto Heights, Illinois) was reacted with 25 ⁇ g TAb 250 in 100 m Na-phosphate buffer (200 ⁇ l, 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 ⁇ l 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.
  • 2 ⁇ l 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 ⁇ Ci/ ⁇ g.
  • 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 ⁇ l of 125 I-labeled TAb 250 at specific activity of 8 x 10 5 cpm/ ⁇ g, and 1x10 cells of prepared SKBR3 cells in a final volume of 100 ⁇ l, on ice in a shaker at 80 rpm for 4 hours.
  • binding buffer MEM medium supplemented with 0.1% BSA, 50 mM HEPES pH 7.0
  • BACh 250 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 ⁇ g/ml. At various times after the addition of antibodies, the cells were removed with trypsin and quantified using a Coulter Counter.
  • 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%) .
  • TAb 250 or BACh 250 (3.1-25 ⁇ g/ml) was added to 51 Cr- 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.
  • 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.
  • E:T effector to target ratios
  • mice 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 ⁇ g/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.
  • 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.
  • 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.
  • 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.
  • MOLECULE TYPE DNA (genomic)
  • AATATTTTTA AATGAATTGA GCAATGTTGA GTTGGAGTCA AGATGGCCGA TCAGAACCAG 1560
  • CCAGCCCCAC CAAACCGAAA GTCCAGGCTG AGCAAAACAC CACCTGGGTA ATTTGCATTT 1680
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

Cette invention concerne des procédés de production de peptides d'anticorps de recombinaison et chimériques qui reconnaissent l'oncogène c-erbB-2, ainsi que des vecteurs, des cellules hôtes et des séquences d'ADN utiles pour produire ces peptides d'anticorps.
PCT/US1992/010437 1991-12-12 1992-12-04 ANTICORPS DE RECOMBINAISON ET CHIMERIQUES CONTRE c-erbB-2 WO1993012220A1 (fr)

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US5648237A (en) * 1991-09-19 1997-07-15 Genentech, Inc. Expression of functional antibody fragments
US5840301A (en) * 1994-02-10 1998-11-24 Imclone Systems Incorporated Methods of use of chimerized, humanized, and single chain antibodies specific to VEGF receptors
US5861499A (en) * 1994-02-10 1999-01-19 Imclone Systems Incorporated Nucleic acid molecules encoding the variable or hypervariable region of a monoclonal antibody that binds to an extracellular domain
US5955311A (en) * 1994-02-10 1999-09-21 Imclone Systems Incorporated Monoclonal antibodies specific to VEGF receptors and uses thereof
US6627196B1 (en) 1999-08-27 2003-09-30 Genentech, Inc. Dosages for treatment with anti-ErbB2 antibodies
US6811779B2 (en) 1994-02-10 2004-11-02 Imclone Systems Incorporated Methods for reducing tumor growth with VEGF receptor antibody combined with radiation and chemotherapy
US6949245B1 (en) 1999-06-25 2005-09-27 Genentech, Inc. Humanized anti-ErbB2 antibodies and treatment with anti-ErbB2 antibodies
US7041292B1 (en) 1999-06-25 2006-05-09 Genentech, Inc. Treating prostate cancer with anti-ErbB2 antibodies
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WO2010108127A1 (fr) 2009-03-20 2010-09-23 Genentech, Inc. Anticorps anti-her di-spécifiques
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