Processes and products with modified antibodies of human milk fat globule specificity

Abstract

An analogue peptide that comprises the variable regions of the light or heavy chains of an antibody of a first species selectively binding to a carcinoma antigen has 1 to 46 amino acids of the framework regions per chain substituted with amino acids such as those present in equivalent positions in antibodies of a species other than the first species, or fragments thereof comprising 1 to 3 variable region CDRs per chain and optionally flanking regions thereof of 1 to 10 or more amino acids, alone or with an N-terminal fragment of 1 to 10 or more amino acids, combinations or mixtures thereof. The polypeptide may also comprise an effector agent and/or be glycosylated, and is presented as a composition with a carrier. The analogue peptides are used in diagnostic kits for carcinomas and methods for in vivo imaging and treating a primary or metastasized carcinoma, and in vitro diagnosing a carcinoma, ex vivo purging neoplastic cells from a biological fluid. RNAs and DNAs encode the analogue peptide, and a hybrid vector carrying the nucleotides and transfected cells express the peptides and a method produces the analogue peptide. An anti-idiotype polypeptide comprises polyclonal antibodies raised against an anti-carcinoma antibody or the analogue peptide of this invention, monoclonal antibodies thereof, Fab, Fab′, (Fab′) 2 , CDR, variable region, or analogues or fragments thereof, combinations thereof with an oligopeptide comprising a TRP trimer, tandem repeats thereof, or combination or mixtures thereof. An anti-idiotype hybrid polypeptide with an effector agent and the anti-idiotype polypeptide, an anti-carcinoma vaccine, an anti-carcinoma vaccination kit, a method of vaccinating against carcinoma and a method of lowering the serum concentration of a circulating antibody or polypeptide are provided.

1 . A pure, isolated analogue peptide which selectively binds to an antigen that is present on the surface or in the cytoplasm of a carcinoma cell or that is released by the cell, the peptide being selected from the group consisting essentially of 
 at least one CDR of the light or heavy chain of an antibody of a first species having affinity and specificity for an antigen found on the surface or the cytoplasm of a carcinoma cell or released by the cell;    at least one variable region of the light or heavy chains of an antibody of the first species having affinity and specificity for an antigen found on the surface or the cytoplasm of a carcinoma cell or released by the cell, wherein about 1 to at least 46 amino acids in the FRs are substituted per chain with amino acids selected from the group consisting of amino acids present in equivalent positions in antibodies of a species other than the first species, or fragments thereof comprising 1 to 3 CDRs per chain and flanking regions thereof, each of about 1 to at least 10 amino acids, alone or with an N-terminal fragment of about 1 to at least 10 amino acids;    combinations thereof, wherein each analogue peptide is operatively linked to at least one other variable region peptide or analogue thereof; and    mixtures thereof.    
     
     
         2 . The anti-carcinoma analogue peptide of  claim 1 , wherein the antibody of the first species is selected from the group consisting of murine, rat, goat, rabbit, canine, primate, guinea pig, bovine, equine, feline and porcine antibodies.  
     
     
         3 . The anti-carcinoma analogue peptide of  claim 1 , being able to compete with the antibody secreted by the hybridoma cell ATCC No. HB 10028 for the human mammary fat globule antigen.  
     
     
         4 . The anti-carcinoma analogue peptide of  claim 1 , having an amino acid sequence selected from the group consisting of 
 amino acid Sequence ID Nos: 67 through 73;    amino acid Sequence ID Nos: 75 through 81;    fragments thereof comprising at least one CDR or one CDR and two flanking regions thereof per chain;    combinations thereof, wherein each analogue peptide is operatively linked to at least one other analogue peptide; and    mixtures thereof.    
     
     
         5 . A glycosylated analogue peptide which specifically binds to an antigen present on the surface or in the cytoplasm of a carcinoma cell or that is released by the cell, comprising 
 the analogue peptide of  claim 1;  and    at least one glycosyl residue operatively linked to the peptide.    
     
     
         6 . A composition of matter, comprising 
 the analogue peptide of  claim 1;  and    a carrier.    
     
     
         7 . An in vivo carcinoma therapy and diagnosis kit, comprising 
 the composition of  claim 6  in pharmaceutically-acceptable form; and    instructions for its use.    
     
     
         8 . The kit of  claim 7 , further comprising a molecule capable of selectively binding the analogue peptide and comprising an effector agent operatively linked thereto.  
     
     
         9 . The kit of  claim 7 , wherein the effector agent comprises a molecule selected from the group consisting of therapeutic, immunogenic and diagnostic agents, radioisotopes, DNA monomers, RNA monomers, chemical linkers, transmitter molecules, combinations thereof, and combinations thereof with peptide and non-peptide polymers or copolymers.  
     
     
         10 . An in vitro carcinoma diagnostic kit, comprising 
 the composition of  claim 6;     a solid support having operatively linked thereto an antigen which selectively binds to the analogue peptide; and    instructions for its use.    
     
     
         11 . The kit of  claim 10 , further comprising a molecule capable of selectively binding the analogue peptide and comprising an effector agent operatively linked thereto.  
     
     
         12 . The diagnostic kit of  claim 10 , further comprising anti-constant region immunoglobulins, protein G, protein A, other antibody binding molecules or binding fragments thereof.  
     
     
         13 . A hybrid analogue polymer, comprising 
 at least one analogue peptide of  claim 1  and at least one effector agent operatively linked to the peptide;    combinations thereof; and    mixtures thereof.    
     
     
         14 . The hybrid analogue polymer of  claim 13 , wherein the effector agent is selected from the group consisting of monomers, and non-peptide and peptide polymers other than the constant region of an antibody of the same species.  
     
     
         15 . The hybrid analogue polymer of  claim 13 , wherein the effector agent comprises a radioisotope, an enzyme, or a phosphorescent or a fluorescent label.  
     
     
         16 . The hybrid analogue polymer of  claim 14 , wherein the effector agent comprises a molecule selected from the group consisting of therapeutic, immunogenic and diagnostic agents, radioisotopes, phosphorescent and fluorescent agents, enzymes, DNA and RNA monomers, chemical linkers, transmitter molecules, combinations thereof, and combinations thereof with peptide and non-peptide polymers or copolymers.  
     
     
         17 . The hybrid analogue polymer of  claim 14 , wherein the effector agent comprises a non-peptide polymer selected from the group consisting of ester, ether, vinyl, amido, imido, alkylene, arylalkylene, cyanate, urethane, and isoprene polymers, halogenated polymers, DNA and RNA polymers, copolymers thereof, and copolymers thereof with peptide polymers or monomers.  
     
     
         18 . The hybrid analogue polymer of  claim 14 , wherein the effector agent comprises a peptide polymer selected from the group consisting of the constant region of antibodies or fragments thereof, the CDRs or variable regions of antibodies, whole antibodies, Fab and Fab′, (Fab′) 2 , or antibody fragments of a species other than the first species, analogues thereof, hormones, enzymes, peptide transmitters, combinations thereof, and combination thereof with non-peptide polymers, copolymers or monomers.  
     
     
         19 . The hybrid analogue polymer of  claim 18 , wherein 
 the analogue peptide comprises amino acid sequences of non-human origin; and    the peptide polymer comprises at least one constant region of the light or heavy chains of a human antibody or fragments thereof capable of binding to anti-constant region immunoglobulins, protein G or protein A or other antibody binding molecules.    
     
     
         20 . The hybrid analogue polymer of  claim 19 , being selected from the group consisting of 
 an analogue peptide comprising two heavy and two light chains, each light and heavy chain comprising at least one non-human CDR or analogue variable region peptide and at least one human constant region or fragment thereof;    at least one human-non-human analogue Fab, Fab′, or (Fab′) 2  fragment thereof;    fragments thereof;    combinations thereof; and    mixtures thereof.    
     
     
         21 . The hybrid analogue polymer of  claim 20 , wherein each pair of the analogue heavy and light chains has a different predetermined specificity.  
     
     
         22 . The hybrid analogue polymer of  claim 13 , wherein the analogue peptide and the effector agent are operatively linked by a polymer.  
     
     
         23 . The hybrid analogue polymer of  claim 13 , being selected from the group consisting of 
 at least one CDR or analogue variable region of the heavy chain of a first antibody of the first species or fragment thereof comprising at least one CDR and two flanking regions thereof and a first effector agent;    at least one CDR or analogue variable region of the light chain of a second antibody of the first species or fragments thereof comprising at least one CDR and two flanking regions thereof, and a second effector agent, wherein each chain has a predetermined specificity;    combinations thereof; and    mixtures thereof.    
     
     
         24 . The hybrid analogue polymer of  claim 13 , wherein 
 at least one first pair of light and heavy chains comprising at least one CDR or analogue variable region or fragments thereof is linked to at least one second pair of the analogue light and heavy chains comprising at least one CDR or one analogue variable region or fragments thereof.    
     
     
         25 . An anti-carcinoma composition, comprising 
 the hybrid analogue polymer of  claim 13;  and    a carrier.    
     
     
         26 . An in vivo carcinoma therapy and diagnosis kit, comprising 
 the anti-carcinoma composition of  claim 25  in pharmaceutically-acceptable form; and    instructions for its use.    
     
     
         27 . The kit of  claim 26 , further comprising a molecule capable of selectively binding the analogue peptide and comprising an effector agent operatively linked thereto.  
     
     
         28 . The kit of  claim 27 , wherein the effector agent comprises a molecule selected from the group consisting of therapeutic, immunogenic and diagnostic agents, radioisotopes, DNA monomers, RNA monomers, chemical linkers, transmitter molecules, combinations thereof, and combinations thereof with peptide and non-peptide polymers and copolymers.  
     
     
         29 . A diagnostic kit for carcinomas, comprising 
 the anti-carcinoma composition of  claim 25;     a solid support having operatively linked thereto an antigen which specifically binds to the hybrid analogue peptide; and    instructions for its use.    
     
     
         30 . The kit of  claim 29 , further comprising a molecule capable of selectively binding to the hybrid analogue polymer, the molecule having an effector agent operatively linked thereto.  
     
     
         31 . The diagnostic kit of  claim 29 , further comprising anti-constant region immunoglobulins, protein G, protein A, other antibody binding molecules or binding fragments thereof.  
     
     
         32 . A method of determining the presence of carcinoma cells in a tissue, comprising contacting a tissue suspected of comprising carcinoma cells with the anti-carcinoma analogue peptide of  claim 1  and allowing the analogue peptide to bind to any antigen associated with carcinoma cells present in the tissue to form an analogue peptide-cell antigen complex; and 
 detecting the presence of any complexes formed.    
     
     
         33 . An in vitro method of determining the presence of carcinoma cells in a tissue comprising the method of  claim 32 , wherein the tissue is excised from a subject prior to being contacted with the analogue peptide in vitro.  
     
     
         34 . An in vitro method of diagnosing a carcinoma comprising the method of  claim 33 , and comparing the results with a standard cut-off value determined by comparing results obtained for normal and carcinoma samples, wherein a result above the cut-off value is diagnostic of a carcinoma.  
     
     
         35 . The in vitro method of diagnosing a carcinoma of  claim 34 , conducted by 
 contacting a biological sample obtained from a subject suspected of having a carcinoma with a known amount of the anti-carcinoma analogue peptide of  claim 1  in the presence of a solid supported antigen molecule that is selectively bound by the analogue peptide and allowing the formation of solid supported analogue peptide-antigen molecule complexes and analogue peptide-sample antigen complexes with any carcinoma cell antigen present in the sample;    detecting any complex formed between the analogue peptide and the solid supported carcinoma antigen; and    comparing the result obtained with the standard cut-off value.    
     
     
         36 . An in vivo method of imaging a carcinoma present in a subject, comprising the method of  claim 32 , wherein 
 a pharmaceutically-acceptable composition comprising an effective amount of the analogue peptide in radiolabeled form to reach the carcinoma and bind thereto is administered to a subject suspected of having a primary or metastasized carcinoma; and    the detection is of any binding of the labeled analogue peptide to the antigen on the surface or in the cytoplasm of the carcinoma cells.    
     
     
         37 . An in vivo method of diagnosing the presence of a carcinoma in a subject comprising the method of  claim 36 , and comparing the results with a standard cut-off value determined by comparing results obtained for normal and carcinoma samples, wherein a result above the cut-off value is diagnostic of a carcinoma.  
     
     
         38 . A method of inhibiting the growth or reducing the size of a primary or metastasized carcinoma in a subject comprising administering to a subject in need of the treatment an effective amount of the anti-carcinoma composition of  claim 25  in pharmaceutically-acceptable form, wherein the effector agent comprises a therapeutic agent.  
     
     
         39 . The method of  claim 38 , further comprising administering to the subject a molecule capable of selectively binding to the analogue peptide and having an effector agent operatively linked thereto.  
     
     
         40 . A method of purging carcinoma cells from a biological fluid, comprising the method of  claim 32:  and 
 separating any analogue peptide-cell complexes present from the remainder of the fluid.    
     
     
         41 . An ex vivo method of purging carcinoma cells from a biological sample obtained from a subject, comprising 
 obtaining a biological sample from a subject suspected of having a carcinoma;    conducting the method of  claim 40  with the biological sample; and    replenishing the purged biological sample to the subject.    
     
     
         42 . An in vitro histochemical method of assessing the presence of carcinoma cells in a tissue, comprising 
 obtaining from a patient suspected of having a carcinoma a tissue sample and preparing therefrom a tissue substrate;    contacting the tissue substrate with the analogue peptide of  claim 1  and allowing the analogue peptide to bind to any carcinoma cells present therein; and    detecting the presence of any complexes formed.    
     
     
         43 . A hybridoma cell expressing the analogue peptide of  claim 1 .  
     
     
         44 . A composition, comprising the hybridoma cell of  claim 43 , and a diluent or a carrier.  
     
     
         45 . A substantially pure, isolated analogue polydeoxyribonucleotide, comprising an analogue oligodeoxyribonucleotide encoding the analogue peptide of  claim 1 .  
     
     
         46 . The analogue polydeoxyribonucleotide of  claim 45 , wherein the analogue oligodeoxyribonucleotide comprises a DNA sequence selected from the group consisting of 
 DNA Sequence ID No: 64;    DNA Sequence ID No: 65;    fragments thereof encoding at least one CDR or one CDR and two flanking regions per chain;    redundant DNA sequences thereof;    combinations thereof; and    mixtures thereof.    
     
     
         47 . A hybrid analogue vector, comprising a vector having the analogue polydeoxyribonucleotide of  claim 46  operatively linked thereto.  
     
     
         48 . The hybrid vector of  claim 47 , further comprising a polydeoxyribonucleotide comprising an oligodeoxyribonucleotide encoding an effector peptide, the effector peptide-encoding polydeoxyribonucleotide being operatively linked to the vector.  
     
     
         49 . A transfected host cell, carrying the hybrid analogue vector of  claim 47 .  
     
     
         50 . A pure, isolated analogue polyribonucleotide, comprising an analogue oligoribonucleotide encoding the analogue peptide of  claim 1 .  
     
     
         51 . A method of producing an analogue peptide which specifically binds to an antigen on the surface or in the cytoplasm of carcinoma cell, or is released by the cell, comprising 
 a) cloning the analogue polydeoxyribonucleotide of  claim 40  into a vector to form a hybrid vector;    b) transfecting a host cell with the hybrid vector and allowing the expression of the analogue peptide; and    c) isolating the analogue peptide or mixtures thereof.    
     
     
         52 . The method of  claim 51 , further comprising 
 site-specifically modifying the codon of at least one amino acid in the framework region of a non-human antibody prior to step a) to obtain the analogue polydeoxyribonucleotide.    
     
     
         53 . The method of  claim 51 , wherein 
 steps a) and b) are conducted by cloning analogue polydeoxyribonucleotides encoding analogue peptides selected from the group consisting of at least one CDR or analogue variable region of the first species or fragments thereof comprising at least one CDR and two flanking regions of the heavy or light chains of the antibody of the first species; and the method further comprises    d) allowing the expressed analogue peptides to interact with one another to form double chains.    
     
     
         54 . A method of producing a hybrid analogue peptide comprising an analogue peptide which specifically binds to an antigen on the surface or in the cytoplasm of a carcinoma cell or to an antigen released by the cell and an effector peptide, the method comprising 
 a) transfecting a host cell with the hybrid analogue vector of  claim 47  and allowing the expression of the anti-carcinoma hybrid analogue peptide; and    b) isolating the anti-carcinoma hybrid analogue peptide or mixtures thereof.    
     
     
         55 . An anti-idiotype polymer, comprising 
 polyclonal antibodies raised against anti-carcinoma antibodies or the analogue peptide of  claim 1;     monoclonal antibodies thereof capable of specifically binding the anti-carcinoma antibody;    fragments thereof selected from the group consisting of CDRs, Fab, Fab′, (Fab′) 2 , variable regions, and fragments thereof;    analogues thereof selected from the group consisting of antibodies, Fab, Fab′, (Fab′) 2 , and variable regions wherein about 1 to at least 46 amino acids in the FR are substituted per chain with amino acids selected from the group consisting of amino adds present in equivalent positions in antibodies of species other than the first species, or fragments thereof comprising 1 to 3 CDRs per chain and flanking regions thereof, each of about 1 to at least 10 amino adds, alone or with an N-terminal fragment of about 1 to at least 10 amino acids; combinations thereof, and combinations thereof with an oligopeptide comprising the amino acid sequence APDTRPAPG or fragments thereof comprising the TRP trimer hexamers thereof comprising the trimer, or tandem repeats thereof, wherein each analogue peptide is operatively linked to at least one other analogue peptide; and    mixtures thereof.    
     
     
         56 . A hybrid anti-idiotype polymer, comprising 
 the anti-idiotype polypeptide of  claim 55;  and    an effector agent operatively linked to the anti-analogue idiotype polypeptide.    
     
     
         57 . An anti-carcinoma vaccine, comprising 
 the anti-idiotype polypeptide of  claim 55;  and    a pharmaceutically-acceptable carrier.    
     
     
         58 . The anti-carcinoma vaccine of  claim 57 , in unit dose form.  
     
     
         59 . A anti-carcinoma vaccination kit, comprising 
 the vaccine of  claim 57  and a diluent in separate sterile containers; and    instructions for its use.    
     
     
         60 . A method of vaccinating against carcinoma comprising administering to a subject an effective amount of the composition of  claim 57 .  
     
     
         61 . The vaccination method of  claim 60 , wherein the anti-idiotype polypeptide is administered in an amount of about 0.1 to 5000.00 mg/kg body weight/dose.  
     
     
         62 . A method of lowering the serum concentration of a circulating antibody or polypeptide that binds to an antigen on the surface or in the cytoplasm of a carcinoma cell or that is released by the cell, comprising administering to a subject in need of the treatment a pharmaceutically-acceptable composition comprising an amount of the anti-idiotype polypeptide of  claim 55  effective to bind the circulating antibody or polypeptide and accelerate its clearance.  
     
     
         63 . The method of  claim 62 , wherein the anti-ideotype peptide is administered in an amount of about 0.01 to 5,000,00 mg/kg body weight/dose.  
     
     
         64 . A method of inhibiting the growth or reducing the size of a primary or metastasized carcinoma tumor in a subject, comprising administering to the subject an effective amount of a pharmaceutically-acceptable composition comprising an anti-carcinoma hybrid polymer comprising an effector agent selected from the group consist of radioisotopes and therapeutic drugs, and an anti-carcinoma polypeptide which specifically binds to an antigen on the surface or in the cytoplasm of a carcinoma cell or that is released by the cell; 
 allowing the hybrid polymer to reach the tumor and the anti-carcinoma polypeptide to bind thereto; and    administering to the subject a pharmaceutically-acceptable composition comprising an amount of the anti-idiotype polypeptide of  claim 55  effective to bind any residual or unbound circulating anti-carcinoma hybrid polypeptide and accelerate the clearance of the hybrid polymer.