Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/531—Production of immunochemical test materials
  • G01N33/532—Production of labelled immunochemicals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
  • A61K51/1069—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from blood cells, e.g. the cancer being a myeloma
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
• • 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
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/3061—Blood cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2121/00—Preparations for use in therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2123/00—Preparations for testing in vivo
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention is directed to recombinant antigen binding proteins having multiple disulfide bonds useful for the preparation of diagnostic and therapeutic conjugates.
• this invention is directed to recombinant antibodies comprising an IgG 3 hinge region and lacking a CH2 constant domain.
• This invention also is directed to immunoconjugates comprising such a recombinant antibody which is covalently bound to a diagnostic or therapeutic agent through one or more reduced disulfide bonds in the antibody, hinge region.
• This invention is further directed to methods for preparing such immunoconjugates.
• This invention also is directed to methods of diagnosis and therapy using such immunoconjugates.
• Monoclonal antibodies can be conjugated to a variety of haptens to form immunoconjugates for use in diagnosis and therapy.
• agents include chelates, which allow the immunoconjugate to form a stable bond with radioisotopes, and cytotoxic agents such as toxins and chemotherapy drugs.
• cytotoxic agents that normally would be too toxic to patients when administered in a systemic fashion can be coupled to anti-cancer antibodies in such a manner that their toxic effects become directed only to the tumor cells bearing the target antigens.
• the diagnostic or therapeutic efficacy of immunoconjugates depends upon several factors. Among these factors, the molar ratio of the diagnostic or therapeutic agent to antibody and the antibody binding activity of the immunoconjugate are of major concern.
• haptens particularly in a multiple manner, at nonspecific sites on monoclonal antibodies (Mabs) can lead to a reduction, or a complete loss, of the ability of the Mab to bind antigen.
• a lysine residue is used for nonspecific substitutions due to the ease of performing chemical substitutions at the free amino group.
• One approach is to attach haptens via Mab carbohydrate groups, which normally are located in the constant (CH2 domain) and distant from antigen-binding domains. According to this technique, vicinal diols of carbohydrate are oxidized to aldehydes, and the amino group of a hapten is coupled to the aldehydes to form a
• the Schiff base can be reduced to an amino bond.
• the immunoconjugate contained approximately 30-50 molecules of drug per molecule of immunoglobulin.
• indirect conjugation of a diagnostic or therapeutic agent to a carbohydrate moiety in the Fc region of an antibody provides a means to obtain immunoconjugates with functional antigen binding activity and a high substitution level.
• a disadvantage of using the carbohydrate moiety in the Fc region as an attachment site is that the entire antibody is required for the immunoconjugate.
• the use of antibody fragments, particularly Fab, Fab' and F(ab') 2 provide an advantage over the use of an entire antibody because such fragments are better able to diffuse out of capillaries and into target tissues.
• Brown "Clinical Use of Monoclonal Antibodies," in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al . , eds. Chapman & Hall, pp.227-249 (1993) .
• haptens are attached to Mabs via thiol groups that have been generated by reducing Mab disulfide bonds. Although a significant proportion of inter-chain disulfide bonds may be reduced, the Mab heavy chains and light chains are held together via electrostatic and hydrophobic interactions.
• disulfide bonds are not located near antigen-binding domains, the reduction of the disulfide bonds will not interfere with antigen binding by the hapten-antibody conjugate.
• disulfide (or thiol) groups are universally present in antibodies including humanized Mab constructs prepared by bacterial fermentation.
• sulfhydryl-containing proteins prepared from intrinsic Mab disulfide bonds do not produce aggregated Mab side-products.
• F(ab) 2 /F (ab' ) 2 fragments of Mabs are taken up by the liver at a much lower rate than intact Mabs due to the lack of Fc domains.
• blood retention times are shorter with F(ab) 2 /F(ab' ) 2 fragments, compared with intact Mabs.
• Haptens also can be bound to Fab/Fab' fragments which have even shorter half-lives in circulation than F(ab) 2 /F(ab' ) 2 fragments. These smaller molecules, however, are characterized by a lower rate of uptake in target tissue and by shorter retention time in target tissue due to the monovalent character of the fragments. Unlike the smaller monovalent fragments, F(ab) 2 /F(ab' ) 2 fragments retain the divalent character of intact Mabs that provides the high level of antigen binding capacity.
• Fab/Fab' fragments are less suitable as therapeutic agents, compared with F(ab) 2 /F(ab' ) 2 fragments, because the uptake of a therapeutic agent in a target is a critical determinant of therapeutic efficacy.
• F(ab) 2 /F(ab' ) 2 fragments are less amenable to successful non-site-specific hapten attachment compared with intact Mab. This is so because the likelihood that a hapten will bind with a lysine residue in a antigen- binding site is increased in the smaller molecules. Moreover, the fragments typically lack carbohydrate residues because the CH2 constant region is destroyed during enzymatic cleavage of the intact Mab. Finally, reduction of F(ab) 2 /F(ab' ) 2 fragments for thiol attachment can cleave the divalent fragments to produce their monovalent counterparts. The Fab/Fab' fragments lack constant domains to promote reassociation to the divalent form.
• an antigen-binding molecule that combines the antigen-binding capacity and hapten- binding capacity of intact antibodies with the rapid clearance and high tissue penetrability of antibody fragments.
• a mutated antibody comprising a heavy chain that contains a hinge region with multiple disulfide bonds, but lacks a CH2 domain.
• Another object of this invention is to provide methods for selectively targeting diagnostic and therapeutic agents to tumor cells or to infectious agents, while avoiding major toxic side effects to normal organs .
• a mutated antibody comprising a heavy chain having (a) a variable region, (b) a CHI domain, (c) a hinge region having three or more disulfide bonds, and
• the present invention also is directed to such mutated antibodies wherein the hinge region is a human IgG 3 hinge region.
• the present invention is further directed to mutated antibodies having a variable region that binds with an antigen associated with a tumor or an infectious agent.
• the present invention also contemplates mutated antibodies that further comprise a light chain comprising a carbohydrate moiety attached at about amino acid 18 in the variable region of the light chain.
• the present invention also is directed to immunoconjugates comprising a mutated antibody and at least one diagnostic or therapeutic agent.
• diagnostic agents include a radioactive label, photoactive agent or dye, florescent label, enzyme label, bioluminescent label, chemiluminescent label, colloidal gold and paramagnetic ion.
• Suitable therapeutic agents are selected from the group consisting of radioisotope, boron addend, immunomodulator, toxin, photoactive agent or dye, cancer chemotherapeutic drug, antiviral drug, antifungal drug, antibacterial drug and antiprotozoal drug.
• Figure 1 shows a representation of the model mutated antibody described herein.
• Figure 2 shows high-pressure liquid chromatography traces of an antibody-streptavidin conjugate prepared by two different methods.
• Figure 3 shows a map of the plasmid, LL2-14pGlg.
• Figure 4 shows maps of the vectors, CHpBSK and ⁇ C2h3pBSK.
• Figure 5 shows a map of the plasmid, hMN-14 ⁇ C2h3pGlg.
• Figure 6 shows a map of the plasmid, hMN-14pdHL2.
• Figure 7 shows a map of the plasmid, hMN- 14 ⁇ C2h3pdHL2.
• Figure 8 presents the structure of calicheamicin, where R' is rhamnose, R' ' is an aminosugar, and R' ' ' is a CH 3 -S group that forms a trisulfide bond with sulfur.
• the figure also illustrates how a calicheamicin immunoconjugate would cleave DNA in vivo.
• hIMMU-14 humanized antibody
• the humanized antibody, hMN-14 (“hIMMU-14")
• the hMN-14 antibody is an anti-CEA antibody having the human IgG,/ ⁇ isotype. Shevitz et al . , J. Nucl . Med. 35:112 (1994) .
• hIMMU-14- ⁇ CH2-IgG 3 the IgG, hinge region is replaced with an IgG 3 hinge region.
• the IgG, CH2 domain is deleted.
• the resultant structure of the hIMMU-14- ⁇ CH2- IgG 3 heavy chain is: [hMN14 VH] - [CHI (IgG,)]- [hinge (IgG 3 ) ] - [CH3 (IgG,) ] .
• the VK domain of hMN-14 may be engineered with an N- linked glycosylation site.
• This construct retains the advantageous properties of divalent Mabs and fragments, but lacks the disadvantages of each form. Specifically, the lack of CH2 domain leads to reduced hepatic uptake and liver toxicity, compared with intact Mabs . The greater size of the new construct results in reduced renal uptake and kidney toxicity, compared with F (ab) 2 /F(ab' ) 2 fragments. This is so because the new construct has a molecular weight above the level where kidney sequestration is observed. However, the extended disulfide hinge region together with the CHI region inhibits cleavage of the mutated antibody to monovalent fragments in blood.
• the extended hinge region with 11 sulfhydryl groups provides for the multiple substitution of haptens via thiol groups, unlike F (ab) 2 /F (ab' ) 2 fragments.
• Multi-drug loading with minimal impingement on antibody function is an important feature of the new construct.
• Figure 1 illustrates the model mutated antibody described herein with the extended hinge region.
• the new construct associates to a dimeric fragment from monomeric units in an efficient manner due to the presence of multiple disulfide bonds and the retention of a significant portion of the constant region. This feature is not only important for the efficient production of the constructs in vi tro, but also is significant because mild reduction for hapten attachment will not cause cleavage to monomeric subunits. Moreover, the new construct has greater penetrative capability than the larger intact Mabs due to the smaller size of the new construct.
• VK-appended carbohydrate moiety at an engineered glycosylation site can serve as an additional conjugation site to increase the loading capacity or to conjugate haptens that are different from those conjugated at the hinge region.
• a mutated antibody comprises a heavy chain that contains hinge region having three or more disulfide bonds, but lacks a CH2 constant domain.
• a diagnostic or therapeutic agent is a molecule or atom which is conjugated to an antibody moiety to produce an immunoconjugate that is useful for diagnosis and for therapy.
• diagnostic or therapeutic agents include drugs, toxins, immunomodulators, chelators, boron compounds, photoactive agents or dyes, radioisotopes, fluorescent agents, paramagnetic ions or molecules and marker moieties.
• an immunoconjugate is a molecule comprising a mutated antibody and a diagnostic or therapeutic agent.
• An immunoconjugate retains the immunoreactivity of the mutated antibody, i . e . , the antibody moiety has roughly the same, or only slightly reduced, ability to bind the antigen after conjugation as before conjugation.
• mRNA messenger RNA
• Promoter A DNA sequence which directs the transcription of a structural gene to produce mRNA.
• a promoter is located in the 5' region of a gene, proximal to the start codon of a structural gene. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent . In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter.
• Enhancer A promoter element.
• An enhancer can increase the efficiency with which a particular gene is transcribed into mRNA irrespective of the distance or orientation of the enhancer relative to the start site of transcription.
• Complementary DNA is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Typically, a primer complementary to portions of mRNA is employed for the initiation of reverse transcription.
• cDNA refers to a double-stranded DNA molecule consisting of such a single-stranded DNA molecule and its complement.
• Expression is the process by which a polypeptide is produced from a structural gene. The process involves transcription of the gene into mRNA and the translation of such mRNA into polypeptide (s) .
• Cloning vector A DNA molecule, such as a plasmid, cosmid, phagemid, or bacteriophage, which has the capability of replicating autonomously in a host cell and which is used to transform cells for gene manipulation.
• Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences may be inserted in a determinable fashion without loss of an essential biological function of the vector, as well as a marker gene which is suitable for use in the identification and selection of cells transformed with the cloning vector.
• Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance.
• Expression vector A DNA molecule comprising a cloned structural gene encoding a foreign protein which provides the expression of the foreign protein in a recombinant host.
• the expression of the cloned gene is placed under the control of (i.e., operably linked to) certain regulatory sequences such as promoter and enhancer sequences. Promoter sequences may be either constitutive or inducible.
• a recombinant host may be any prokaryotic or eukaryotic cell which contains either a cloning vector or expression vector. This term is also meant to include those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell.
• suitable hosts see Sambrook et al . , MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989) .
• a tumor associated antigen is a protein normally not expressed, or expressed at very low levels, by a normal counterpart.
• tumor associated antigens include -fetoprotein and carcinoembryonic antigen (CEA) .
• CEA carcinoembryonic antigen
• Many other illustrations of tumor associated antigens are known to those of skill in the art. See, for example, Urban et al . , Ann . Rev. Immunol . 10 : 617 (1992) .
• an infectious agent denotes both microbes and parasites.
• a "microbe” includes viruses, bacteria, rickettsia, mycoplasma, protozoa, fungi and like microorganisms.
• a "parasite” denotes infectious, generally microscopic or very small multicellular invertebrates, or ova or juvenile forms thereof, which are susceptible to antibody-induced clearance or lytic or phagocytic destruction, such as malarial parasites, spirochetes, and the like.
• Humanized antibodies are recombinant proteins in which murine complementary determining regions of monoclonal antibodies have been transferred from heavy and light variable chains of the murine immunoglobulin into a human variable domain.
• antibody component includes both an entire antibody and an antibody fragment. 3. Methods for Preparing a Mutated Antibody That Contains Multiple Disulfide Bonds in the Hinge Region
• a mutated antibody of the present invention may be derived from a rodent monoclonal antibody.
• Rodent mono ⁇ clonal antibodies to specific antigens may be obtained by methods known to those skilled in the art. See, for example, Kohler and Milstein, Nature 256 : 495 (1975) , and
• monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybrido as, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
• Mabs can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines et al . , "Purification of Immunoglobulin G (IgG) ,” in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992) .
• IgG Immunoglobulin G
• rodent monoclonal antibodies that bind with adenocarcinoma-associated antigen (Cat. No. 121730), human chorionic gonadotropin
• human alpha-fetoprotein Cat. No.
• rodent monoclonal antibodies that bind with antigenic determinants of infectious agents such as Escherichia coli (HB 8178), Legionella pneumophila (CRL 1770) , Schistosoma mansoni (HB 8088) , Streptococcus , Group A (HB 9696) , Treponema pallidum (HB 8134), hepatitis B (CRL 8017) , herpes simplex (HB 8181) , human immunodeficiency virus (HB 9101) , among others, can be obtained from American Type Culture Collection (Rockville, MD) .
• murine monoclonal antibodies against merozoites and sporozoites of Plasmodium fal ciparu can be prepared as described by Goldenberg, U.S. patent No. 5,332,567 (1994) , which is incorporated by reference.
• a mutated antibody of the present invention may also be derived from a subhuman primate antibody.
• General techniques for raising therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al . , international patent publication No. WO 91/11465 (1991), and in Losman e ⁇ al . , Int . J. Cancer 46 : 310 (1990), which is incorporated by reference.
• a mutated antibody may be derived from a "humanized" monoclonal antibody.
• Humanized monoclonal antibodies are produced by transferring mouse complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human -In ⁇
• variable domain variable domain
• the use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions.
• General techniques for cloning murine immunoglobulin variable domains are described, for example, by the publication of Orlandi et al . , Proc . Nat ' l Acad. Sci . USA 86 : 3833 (1989) , which is incorporated by reference in its entirety.
• Techniques for producing humanized Mabs are described, for example, by Jones et al . , Na ture 321 : 522 (1986) , Riechmann et al .
• a mutated antibody of the present invention may be derived from human antibody fragments isolated from a combinatorial immunoglobulin library. See, for example, Barbas et al . , METHODS: A Companion to Methods in Enzymology 2 : 119 (1991), and Winter e ⁇ al . , Ann . Rev. Immunol . 12 : 433 (1994) , which are incorporated by reference.
• Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, CA) .
• a mutated antibody of the present invention may be derived from a human monoclonal antibody.
• Such antibodies are obtained from transgenic mice that have been "engineered” to produce specific human antibodies in response to antigenic challenge.
• elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci.
• the transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas.
• a mutated antibody of the present invention comprises a heavy chain that contains a variable domain, a CHI constant domain, a hinge region with multiple sulfhydryl groups and a CH3 constant domain.
• the lack of CH2 domain results in reduced hepatic uptake and liver toxicity, as discussed above.
• the presence of CHI and CH3 constant domains and the hinge region promotes the efficient formation of mutated antibodies in vivo and/or in vi tro, and provides for the stability of the divalent structure of mutated antibody in the blood.
• a suitable hinge region contains three or more sulfhydryl groups for the formation of interchain disulfide bonds.
• the hinge region of human IgG 2 contains four interchain disulfide bonds.
• a preferred hinge region is obtained from human IgG 3 , which contains 11 or more interchain disulfide bonds.
• the extended IgG 3 hinge region, described herein, contains 11 sulfhydryl groups.
• DNA molecules encoding suitable variable regions, constant domains, and hinge regions can be synthesized using the polymerase chain reaction with RNA from antibody-producing hybridomas that produce such antibodies.
• General techniques for the synthesis of antibody components are described, for example, by
• the mutated antibody of the present invention may lack such an attachment site.
• a glycosylation site can be introduced into the light chain variable domain, as described by Hansen et al . , U.S. patent No. 5,443,953 (1995) .
• the engineered carbohydrate moiety can serve as an additional conjugation site to increase the loading capacity or as a site for conjugation of haptens that are different from those conjugated at the hinge region.
• the DNA sequence must be operably linked to regulatory sequences controlling transcriptional expression in an expression vector and then, introduced into either a prokaryotic or eukaryotic host cell.
• regulatory sequences controlling transcriptional expression in an expression vector and then, introduced into either a prokaryotic or eukaryotic host cell.
• expression vectors include translational regulatory sequences and a marker gene which is suitable for selection of cells that carry the expression vector.
• Suitable promoters for expression in a prokaryotic host can be repressible, constitutive, or inducible.
• Suitable promoters are well-known to those of skill in the art and include promoters capable of recognizing the T4, T3 , Sp6 and T7 polymerases, the P R and P L promoters of bacteriophage lambda, the trp, recA, heat shock, and lacZ promoters of E. coli , the ⁇ -amylase and the ⁇ 28 - specific promoters of B .
• subtilis subtilis, the promoters of the bacteriophages of Bacillus, Streptomyces promoters, the int promoter of bacteriophage lambda, the J la promoter of the / 3-lactamase gene of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene.
• Prokaryotic promoters are reviewed by Glick, J. Ind. Microbiol . 1:277-282 (1987) ; Watson et al., MOLECULAR BIOLOGY OF THE GENE, 4th Ed., Benjamin Cummins (1987) ; Ausubel et al . , supra , and Sambrook et al . , supra .
• a preferred prokaryotic host is E. coli .
• Preferred strains of E. coli include Y1088, Y1089, CSH18, ER1451, and ER1647 (see, for example, Brown (Ed.) , MOLECULAR BIOLOGY LABFAX, Academic Press (1991) ) .
• An alternative preferred host is Bacillus subtilus , including such strains as BR151, YB886, MI119, MI120, and B170 (see, for example, Hardy, "Bacillus Cloning Methods, " in DNA CLONING: A PRACTICAL APPROACH, Glover (Ed.) , IRL Press (1985) ) .
• the mutated antibody is preferably expressed in eukaryotic cells, and especially mammalian, insect, and yeast cells.
• mammalian cells provide post-translational modifications to the cloned polypeptide including proper folding and glycosylation.
• such mammalian host cells include COS-7 cells (ATCC CRL 1651) , non-secreting myeloma cells (SP2/0-AG14; ATCC CRL 1581), Chinese hamster ovary cells (CH0-K1; ATCC CCL 61) , rat pituitary cells (GH,; ATCC CCL 82) , HeLa S3 cells (ATCC CCL 2.2) , and rat hepatoma cells (H-4-II-E; ATCC CRL 1548) .
• COS-7 cells ATCC CRL 1651
• SP2/0-AG14 ATCC CRL 1581
• CH0-K1 Chinese hamster ovary cells
• GH rat pituitary cells
• HeLa S3 cells ATCC CCL 2.2
• H-4-II-E ATCC CRL 1548
• the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, and simian virus.
• promoters from mammalian expression products such as actin, collagen, or myosin, can be employed.
• a prokaryotic promoter such as the bacteriophage T3 RNA polymerase promoter
• the prokaryotic promoter is regulated by a eukaryotic promoter (for example, see Zhou et al . , Mol . Cell . Biol . 10:4529-4537 (1990) ; Kaufman et al . , Nucl . Acids Res . 15:4485-4490 (1991) ) .
• Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the genes can be modulated.
• eukaryotic regulatory regions will include a promoter region sufficient to direct the initiation of RNA synthesis.
• eukaryotic promoters include the promoter of the mouse metallothionein I gene
• Strong regulatory sequences are the most preferred regulatory sequences of the present invention.
• preferred regulatory sequences include the SV40 promoter-enhancer (Gorman, "High Efficiency Gene Transfer into Mammalian cells," in DNA CLONING: A PRACTICAL APPROACH, Volume II, Glover (Ed.) , IRL Press pp. 143-190 (1985)), the hCMV-MIE promoter-enhancer (Bebbington et al . , Bio/Technology 10:169-175 (1992)) , and antibody heavy chain promoter (Orlandi et al . , Proc . Natl . Acad. Sci . USA 86: 3833 - 3831 (1989)) .
• kappa chain enhancer for the expression of the light chain and the IgH enhancer (Gillies, "Design of Expression Vectors and Mammalian Cell Systems Suitable for Engineered Antibodies," in ANTIBODY ENGINEERING: A PRACTICAL GUIDE, C. Borrebaeck (Ed.), W.H. Freeman and Company, pp. 139-157 (1992) ; Orlandi et al . , supra) .
• the mutated antibody-encoding sequence and an operably linked promoter may be introduced into eukaryotic cells as a non-replicating DNA molecule, which may be either a linear molecule or, more preferably, a closed covalent circular molecule.
• the expression of the protein may occur through the transient expression of the introduced sequence.
• permanent expression occurs through the integration of the intro ⁇ quizd sequence into the host chromosome.
• the introduced sequence will be incorporated into a plasmid or viral vector that is capable of autonomous replication in the recipient host.
• vector systems are available for this purpose.
• One class of vectors utilize DNA elements which provide autonomously replicating extra-chromosomal plasmids, derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, or SV40 virus.
• a second class of vectors relies upon the integration of the desired genomic or cDNA sequences into the host chromosome.
• Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.
• the cDNA expression vectors incorporating such elements include those described by Okayama, Mol . Cell . Biol . 3 : 280 (1983) , Sambrook e ⁇ al . , supra, Ausubel et al . , supra , Bebbington et al . , supra, Orlandi et al. , supra, and Fouser et al . , Bio/Technology 10:1121-1127 (1992) ; Gillies, supra. Genomic DNA expression vectors which include intron sequences are described by Orlandi et al .
• an expression vector comprising an antibody light chain can be co-transfected into mammalian cells with a mutated antibody heavy chain expression vector.
• mammalian cells containing-a mutated heavy chain expression vector can be transfected with an expression vector comprising an antibody light chain, and mammalian cells containing an expression vector comprising a light chain can be transfected with a mutated heavy chain expression vector.
• mammalian cells can be transfected with a single expression vector comprising DNA fragments that encode an antibody light chain, as well as DNA fragments that encode mutated antibody heavy chain. See, for example, Gillies, supra; Bebbington et al . , supra . Any of these approaches will produce transfected cells that express whole antibody molecules that have the mutated heavy chain. Similar approaches can be used to produce recombinant antibodies comprising a mutated heavy chain and a light chain with an engineered glycosylation site.
• Standard transfection techniques are well known in the art. See, for example, Sambrook et al . , supra ; Ausubel et al . , supra .
• Transfected cells that carry the expression vector are selected using the appropriate drug.
• G418 can be used to select transfected cells carrying an expression vector having the aminoglycoside phosphotransferse gene. Southern et al . , J. Mol . Appl . Gen . 1:327-341 (1982) .
• hygromycin-B can be used to select transfected cells carrying an expression vector having the hygromycin-B- phosphotransferase gene. Palmer et al . , Proc . Natl . Acad. Sci . USA 54:1055-1059 (1987) .
• aminopterin and mycophenolic acid can be used to select transfected cells carrying an expression vector having the xanthine-guanine phosphoribosyltransferase gene. Mulligan et al . , Proc . Na tl . Acad. Sci . USA 78 : 2072 -2016 (1981) .
• Transfected cells that produce a mutated antibody can be identified using a variety of methods. For example, any immunodetection assay can be used to identify such "transfectomas. "
• Drug molecules can be attached to free thiol groups which have been generated by reduction of antibodies using standard methods for site-specific reaction at sulfur.
• the nucleophilic nature of the thiol group at moderate pH (pH 5 - 7) allows for protein alkylation with organic halides, epoxides, aziridines, tosylates, mesylates, maleimides, and the like, in rapid reactions that have minimal effects on other functional groups of protein.
• Many such alkylating agents are well-known anti-cancer drugs. Examples of alkylation drugs can be found in Holland et al . , CANCER MEDICINE (Lea & Fibiger 1982) .
• Common drugs such as doxorubicin and methotrexate have available functional groups which can be modified to carry a thiol-specific reacting group for reaction with protein.
• modifications include the use of cross-linking agents such as amine/active ester or oxidation/reduction chemistries ( e . g. , hydrazones) .
• cross-linking agents such as amine/active ester or oxidation/reduction chemistries (e . g. , hydrazones) .
• a nitrogen mustard also can be used as a hapten of an immunoconjugate.
• Compounds such as mechlorethamine, L-phenylalanine mustard (Melphalan) , cyclophosphamide and chlorambucil can be attached to reduced mutated antibody thiol groups using an excess of drug, leaving one chloroethyl group free to react upon delivery to the target cell .
• Trifunctional alkylating agents such as Trenimon are particularly useful because two alkylating functions are available after the compounds are bound to the mutated antibody. Accordingly, the ability to cross ⁇ link will remain after attachment.
• Bizelesin a potent bisalklyating agent, also can be utilized by reaction with a thiol-reduced mutated antibody construct in the presence of excess drug. Bizelesin and related compounds are described by Aristoff et al . , DN&P 6 : 229 (1993) .
• Antitumor antibiotics such as the calicheamicins and esperamicins, also are useful for the preparation of immunoconjugates.
• the top portion of Figure 8 illustrates the calicheamicin structure, where R' is rhamnose, R' ' is an aminosugar, and R' ' ' is a CH 3 -S group that forms a trisulfide bond with sulfur. This methyl trisulfide bond plays a crucial role in the mechanism of action of the compound.
• the enediyene system of calicheamicin can undergo a Bergmann cyclization, forming a benzene ring with the generation of two highly reactive single electrons on the benzene ring.
• the triggering event for this transformation is a nucleophilic attack on the trisulfide by a thiol such as glutathione, which is present in high concentrations within cells.
• the free radical form of the drug can induce double-stranded DNA cleavage.
• Calicheamicins and esperamicins can be bound to mutated antibodies that contain an engineered carbohydrate group.
• a suitable conjugation method is described by Hinman et al . , Cancer Res . 53 : 3336 (1993) .
• such compounds can be conjugated to a reduced disulfide bond of a mutated antibody.
• the trithiol of the drug is replaced by a mixed disulfide bond formed between the reduced disulfide bond of the antibody moiety and the drug.
• the sulfur atom that is proximal to the R' ' ' group would belong to a cysteine unit of the antibody moiety.
• the lower portion of Figure 8 illustrates how such an immunoconjugate would cleave DNA in vivo .
• bacterial toxins e . g. , Pseudomonas exotoxin
• polymers that alter the biodistribution properties of antibody conjugates e. g. , polylysine, polyglutamic acid, copolymers of amino acids, (polyethylene)glycol and (polyethylene) imine
• substituted polymers capable of carrying increased loads of haptens such as polyamino acids ( e . g.
• Mutated antibodies of the present invention are particularly suitable for preparation of immunoconjugates that deliver antigenic peptides to cells for antigen presentation. See, for example, Wyss-Coray et al . , Cell . Immunol . 139 : 268 (1992), which describes the use of an antibody-peptide construct to deliver antigenic peptides to T cells.
• antigenic peptides are constructed with a single cysteine residue that is used to attach the peptide to the hinge region of a reduced mutated antibody via disulfide bond formation.
• antigenic peptides examples include the tetanus toxoid peptide p2 with an N-terminal cysteine, CQYIKANSKFIGITEL (C + tt830-844; C-ttp2; SEQ ID NO:4) , and tetanus toxoid peptide p30 with a C-terminal cysteine, FNNFTVSFWLRVPKVSASHLEC (tt947-967 + C; SEQ ID NO:5) .
• General techniques for such conjugation are well-known in the art. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991) .
• hapten may not be available for coupling to an oxidized carbohydrate group for costly amino group-bearing polymers such as streptavidin, Pseudomonas exotoxin and ribonuclease.
• the hinge region thiol moieties of the mutated antibodies described herein provide the site-specificity required for polymer conjugation.
• intrinsic thiol groups i.e., those derived from reduced Mab disulfide bonds
• Figure 2 shows two, superimposed preparative size-exclusion high- pressure liquid chromatography (HPLC) traces.
• HPLC preparative size-exclusion high- pressure liquid chromatography
• the background trace shows hIMMU-14-IgG- streptavidin prepared by reaction of thiolated antibody and maleimido-streptavidin wherein the sulfhydryl groups are appended to the Mab by 2-iminothiolane (Traut's reagent) .
• the smaller foreground trace shows hIMMU-14- IgG-streptavidin prepared by reaction of reduced antibody and maleimido-streptavidin wherein the sulfhydryl groups are derived by reduction of Mab disulfide bonds.
• Lower weight materials are on the left-hand side, with the peak at the farthest left being the unreacted hIMMU-14 Mab. The peak to the immediate right of the Mab peak is the desired conjugate, hIMMU-14-streptavidin.
• the background trace shows a clear discrimination between the Mab and the hIMMU-14-streptavidin peak, whereas the foreground trace shows the hIMMU-14- streptavidin peak appearing as a shoulder on the Mab peak.
• This observation indicates that the hIMMU-14- streptavidin prepared via reduction . of intrinsic disulfide bonds forms a species of lower apparent molecular weight than the same reagent prepared using a thiolation reagent, suggesting that, in the former case, the two proteins must come closer together so that reaction with the buried thiol groups can occur.
• the result is a tighter fit protein-protein conjugate, as opposed to two distinct proteins held apart by a relatively long-chain organic linker.
• the tighter-fit protein-protein conjugates are expected to be less recognizable by the reticuloendothelial cell system, behaving in vivo more as single protemaceous entities. Consequently, these conjugates are expected to have longer circulation times and superior biodistribution properties.
• Metals useful in diagnosis and therapy can be attached to a reduced hinge region of a mutated antibody.
• Chelators for radiometals or magnetic resonance enhancers are well-known in the art. Typical are derivatives of ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) . These chelators typically have groups on the side chain by which the chelator can be attached to a carrier. Such groups include, e . g. , benzylisothiocyanate, by which the DTPA or EDTA can be coupled to the amine group of a carrier. Alternatively, carboxyl groups or amine groups on a chelator can be coupled to a carrier by activation or prior derivatization and then coupling, all by well- known means.
• Labels such as enzymes, fluorescent compounds, electron transfer agents, and the like can be linked to a carrier by conventional methods well known to the art. These labeled carriers and the immunoconjugates prepared from them can be used for immunochemical detection, as described below.
• Boron addends such as carboranes, can be attached to antibody components by conventional methods.
• carboranes can be prepared with carboxyl functions on pendant side chains, as is well known in the art. Attachment of such carboranes to a carrier, e . g. , aminodextran, can be achieved by activation of the carboxyl groups of the carboranes and condensation with amines on the carrier to produce an intermediate conjugate. Such intermediate conjugates are then attached to antibody components to produce therapeutically useful immunoconjugates, as described below.
• carboranes can be attached to mutated antibodies via pendent thiol groups.
• borocaptate salts can be coupled via mixed disulfide bond formation with reduced mutated antibody, or by conversion to a maleimide derivative by reaction with excess bi s ( ma1 e imide ) cros s - 1 inkers , such as bis (maleimide)hexane.
• the second maleimido group is then used for reaction with reduced mutated antibody.
• the first method produces a cleavably linked carborane, while the second approach provides a carborane that is not cleavable from the antibody moiety.
• the carbohydrate group can be used to increase the loading of the same hapten that is bound to a thiol group, or the carbohydrate moiety can be used to bind a different hapten.
• the carbohydrate moiety can be used to attach polyethyleneglycol in order to extend the half-life of a mutated antibody in blood, lymph, or other extracellular fluids.
• the general method involves reacting an antibody component having an oxidized carbohydrate portion with a carrier polymer that has at least one free amine function and that is loaded with a plurality of drug, toxin, chelator, boron addends, or other diagnostic or therapeutic agent. This reaction results in an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.
• the present invention contemplates the use of immunoconjugates to screen biological samples in vi tro for the expression of tumor associated antigens or for the presence of antigens associated with infectious agents.
• the immunoconjugates of the present invention can be used to detect the presence of CEA in tissue sections prepared from a biopsy specimen.
• General immunochemistry techniques are well-known to those of ordinary skill. See, for example, Ponder, "Cell Marking Techniques and Their Application, " in MAMMALIAN DEVELOPMENT: A PRACTICAL APPROACH, Monk (ed.), pages 115- 38 (IRL Press 1987) , Volm e ⁇ al . , Eur. J. Cancer Clin . Oncol .
• an antibody moiety that binds the c-erb B2 proto-oncogene product may be more suitable for a particular breast cancer than an antibody moiety that binds carcinoembryonic antigen.
• Immunochemical detection can be performed by contacting a biological sample with a mutated antibody and then contacting the biological sample with a detectably labeled molecule which binds to the mutated antibody.
• the detectably labeled molecule can comprise an antibody moiety that binds the mutated antibody.
• the mutated antibody can be conjugated with avidin/streptavidin (or biotin) and the detectably labeled molecule can comprise biotin (or avidin/streptavidin) . Numerous variations of this basic technique are well-known to those of skill in the art.
• a mutated antibody can be conjugated with a diagnostic agent to form an immunoconjugate .
• Antibodies can be detectably labeled with any appropriate marker moiety, for example, a radioisotope, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label or colloidal gold.
• a marker moiety for example, a radioisotope, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label or colloidal gold.
• the marker moiety can be a radioisotope that is detected by autoradiography.
• Isotopes that are particularly useful for the purpose of the present invention are 3 H, 125 I, 13I I , 35 S and 14 C.
• Immunoconjugates also can be labeled with a fluores ⁇ cent compound. The presence of a fluorescently-labeled antibody component is determined by exposing the immunoconjugate to light of the proper wavelength and detecting the resultant fluorescence.
• Fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
• immunoconjugates can be detectably labeled by coupling to a chemiluminescent compound.
• the presence of the chemiluminescent-tagged immunoconjugate is determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
• chemiluminescent labeling compounds include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester.
• Bioluminescent compound can be used to label immunoconjugates of the present invention.
• Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
• Bioluminescent compounds that are useful for labeling include luciferin, luciferase and aequorin.
• immunoconjugates can be detectably labeled by linking a mutated antibody to an enzyme.
• the enzyme moiety reacts with the substrate to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means.
• enzymes that can be used to detectably label immunoconjugates include /3-galactosidase, glucose oxidase, peroxidase and alkaline phosphatase.
• the above-described immunochemical detection methods can be used to assist in the diagnosis or staging of a pathological condition. These techniques also can be used to identify the most suitable composition of mutated antibody immunoconjugate for subsequent in vivo diagnosis and therapy.
• immunoconjugates for in vivo diagnosis.
• immunoconjugates can be used to diagnose cardiovascular disease.
• immunoconjugates comprising anti-mysosin fragments can be used for imaging myocardial necrosis associated with acute myocardial infarction.
• Immunoconjugates comprising an antibody component that binds platelets and fibrin can be used for imaging deep-vein thrombosis.
• immunoconjugates that bind to activated platelets can be used for imaging atherosclerotic plaque.
• immunoconjugates of the present invention can be used to localize particular tumors and infectious agents.
• radioisotopes may be bound to a mutated antibody either directly, or indirectly by using an intermediary functional group.
• Bifunctional chelates can be attached to hinge region thiols using the alkylation reactions described above.
• examples of such agents include bromoacetyl- or maleimide derivatives such a s 2 - ( p - b r o m o a c e t y l a m i d o ) b e n z y l - diethylenetriaminepentaacetic acid (DTPA) , and 2-(p- bromoacetylamido)benzyl-1,4,7, 10-tetraazadodecane-N, N' , N" , N' " -tetraacetic acid (DOTA) .
• DTPA 2-(p- bromoacetylamido)benzyl-1,4,7, 10-tetraazadodecane-N
• Certain metals can be bound directly to the free thiol groups in a stable manner to give useful metalloproteins, including chemically reduced technetium- 99m, technetium-94m and rhenium isotopes, as well as "soft" metals such as silver-Ill and the copper isotopes.
• Site-specific attachment of these metals to mutated antibody via bifunctional chelating agents or directly onto reduced hinge region disulfide bonds allows for labeling of the mutated antibody with these elements in procedures which are amenable to an easy-to-use kit formulation. For example, see Shih et al . , supra , and U.S. patent No. 5,057,313. Also, see Griffiths, U.S. patent No. 5,128,119 (1992).
• the radiation dose delivered to the patient is maintained at as low a level as possible through the choice of isotope for the best combination of minimum half-life, minimum retention in the body, and minimum quantity of isotope which will permit detection and accurate measurement.
• radioisotopes that can be bound to antibodies and are appropriate for diagnostic imaging include ⁇ -emitters and positron-emitters such as
• Preferred ⁇ -emitters have a gamma radiation emission peak in the range of 50-500 Kev, primarily because the state of the art for radiation detectors currently favors such labels.
• Examples of such 7-emitters include "Tc, 67 Ga, 13 I, 125 I and I31 I.
• the mutated antibodies of the present invention overcome practical problems associated with the use of rhenium-186 and silver-Ill.
• Rhenium-186 is a carrier-added nuclide and the ability to load multiple radiometal units at an antigen-distant site is a significant advantage over other methodologies used for rhenium labeling of Mabs.
• Multiple loading of silver-Ill (and numerous other similarly produced nuclides) onto a mutated antibody also confers a significant additional practical advantage to this type of conjugate because the major factor limiting use of this radiometal is the cost of production from isotopically-enriched and expensive palladium-110. Multiple loading of radiometal allows for the use of silver-Ill and similar nuclides at lower specific activity.
• Antibodies also can be labeled with paramagnetic ions for purposes of in vivo diagnosis. Elements that are particularly useful for magnetic resonance imaging include Gd, Mn, Dy and Fe ions.
• Gd, Mn, Dy and Fe ions are particularly useful for magnetic resonance imaging.
• a high background level of non- argeted antibody provides a major impediment to in vivo diagnosis methodology.
• the ratio of target to nontarget radiolabeled antibody can be enhanced through the use of a nonlabeled second antibody which scavenges and promotes the clearance of the nontargeted circulating radiolabeled antibody.
• the second antibody may be whole IgG or IgM, or a fragment of IgG or IgM, so long as it is capable of binding the radiolabeled antibody to form a complex which is cleared from the circulation and nontarget spaces more rapidly than the radiolabeled antibody alone.
• suitable second antibodies may bind with either the Fc portion (i.e., CH3 domain) or variable region of a radiolabeled immunoconjugate. See, for example, Goldenberg, U.S. patent No. 4,624,846, Goldenberg, international publication No. WO 92/19273, and Sharkey et al . , Int . J. Cancer 51 : 266 (1992), which are incorporated by reference.
• the location of CEA-bearing tumor cells can be determined by parenterally injecting a mammal with an immunoconjugate comprising a variable domain that binds with CEA and a diagnostic agent. Subsequently, the mammal is injected with an antibody or antibody fragment that binds with the immunoconjugate in an amount that is sufficient to decrease the level of circulating immunoconjugate by about 10-85% within 2 to 72 hours. The mammal is then scanned with a detector to locate the site or sites of uptake of the immunoconjugate . See Goldenberg, U.S. patent No. 4,624,846.
• detection methods are improved by taking advantage of the binding between avidin/streptavidin and biotin.
• Avidin found in egg whites, has a very high binding affinity for biotin, which is a B-complex vitamin.
• Streptavidin isolated from Streptomyces avidinii , is similar to avidin, but has lower non-specific tissue binding and therefore, streptavidin often is used in place of avidin.
• a basic diagnostic method comprises administering a mutated antibody conjugated with avidin/streptavidin (or biotin) , injecting a clearing composition comprising biotin (or avidin/streptavidin) , and administering a conjugate of a diagnostic agent and biotin (or avidin/streptavidin) .
• the biotin (or avidin/streptavidin) component of the clearing composition is coupled with a carbohydrate moiety (such as dextran) or a polyol group ( e . g. , polyethylene glycol) to decrease immunogenicity and permit repeated applications.
• a modification of the basic method is performed by parenterally injecting a mammal with a mutated antibody that has been conjugated with avidin/streptavidin (or biotin) , injecting a clearing composition comprising biotin (or avidin/streptavidin) , and parenterally injecting an immunoconjugate according to the present invention, which further comprises avidin/streptavidin (or biotin) .
• a mammal with a mutated antibody that has been conjugated with avidin/streptavidin (or biotin)
• injecting a clearing composition comprising biotin (or avidin/streptavidin)
• an immunoconjugate according to the present invention, which further comprises avidin/streptavidin (or biotin) .
• improved detection can be achieved by conjugating multiple avidin/streptavidin or biotin moieties to a polymer which, in turn, is conjugated to an antibody component.
• immunoconjugates can be produced which contain multiple avidin/streptavidin or biotin moieties.
• improved detection is achieved by injecting a targeting mutated antibody conjugated to biotin (or avidin/streptavidin) , injecting at least one dose of an avidin/streptavidin (or biotin) clearing agent, and injecting a diagnostic composition comprising a conjugate of biotin (or avidin/streptavidin) and a naturally occurring metal atom chelating protein which is chelated with a metal detection agent.
• Suitable targeting proteins according to the present invention would be ferritin, metallothioneins, ferredoxins, and the like. This approach is disclosed by Goldenberg e ⁇ al . , international application No. PCT/US94/05149, which is incorporated by reference.
• hIMMU-14 antibody a high-affinity Mab raised against carcinoembryonic antigen.
• Mutated antibody constructs of the hIMMU-14 Mab are most usefully coupled to a protein such as streptavidin, and then used for localizing biotin- isotope/drug conjugates to the target, since such as two- step delivery system requires an initial targeting agent which remains on the outside of the targeted cell .
• Immunoconjugates comprising a radiolabel also can be used to detect tumor cells or infectious agents in the course of intraoperative and endosc ⁇ pic examination using a small radiation detection probe.
• a photoactive agent or dye such as dihematoporphyrin ether (Photofrin II)
• Photofrin II dihematoporphyrin ether
• a subject is injected parenterally with an immunoconjugate comprising a variable domain that binds with an antigen that is associated with a tumor or infectious agent and a photoactive agent or dye.
• Sites of accretion are detected using a light source provided by an endoscope or during a surgical procedure.
• the detection of immunoconj gates during intraoperative or endoscopic examination can be enhanced through the use of second antibody or avidin/streptavidin/biotin clearing agents, as discussed above.
• the detection means can be inserted into a body cavity through an orifice, such as, the mouth, nose, ear, anus, vagina or incision.
• an orifice such as, the mouth, nose, ear, anus, vagina or incision.
• the term "endoscope” is used generically to refer to any scope introduced into a body cavity, e . g. , an anally introduced endoscope, an orally introduced bronchoscope, a urethrally introduced cystoscope, an abdominally introduced laparoscope or the like. Certain of these may benefit greatly from further progress in miniaturization of components and their utility to practice the method of the present invention will be enhanced as a function of the development of suitably microminiaturized components for this type of instrumentation.
• Highly miniaturized probes which could be introduced intravascularly, e. g. , via catheters or the like, are also suitable for use in the embodiments of the invention for localizing tumor cells or infectious agents .
• the present invention also contemplates the use of immunoconjugates for immunotherapy.
• An objective of immunotherapy is to deliver cytotoxic doses of radioactivity, toxin, or drug to target cells, while minimizing exposure to non-target tissues.
• the immunoconjugates of the present invention are expected to combine the high antigen-binding capacity and high hapten-binding capacity of intact antibodies with the rapid clearance and high tissue penetrability of antibody fragments.
• a therapeutic immunoconjugate may comprise an ⁇ -emitting radioisotope, a /J-emitting radioisotope, a ⁇ -emitting radioisotope, an Auger electron emitter, a neutron capturing agent that emits a- particles or a radioisotope that decays by electron capture.
• Suitable radioisotopes include 198 Au, 32 P, 125 I, l31 I, 90 Y, 186 Re, ,88 Re, 67 Cu, 211 At, and the like.
• a radioisotope can be attached to an antibody directly or indirectly, via a chelating agent.
• 67 Cu considered one of the more promising radioisotopes for radioimmunotherapy due to its 61.5 hour half-life and abundant supply of beta particles and gamma rays, can be conjugated to an antibody using the chelating agent, p-bromoacetamido-benzyl- tetraethylaminetetraacetic acid (TETA) . Chase, supra .
• TETA p-bromoacetamido-benzyl- tetraethylaminetetraacetic acid
• ⁇ which emits an energetic beta particle, can be coupled to an antibody using diethylenetriaminepentaacetic acid (DTPA) .
• DTPA diethylenetriaminepentaacetic acid
• boron addends such as carboranes can be attached to mutated antibodies.
• Carboranes can be prepared with carboxyl functions on pendant side chains, as is well-known in the art. Attachment of carboranes to a carrier, such as aminodextran, can be achieved by activation of the carboxyl groups of the carboranes and condensation with amines on the carrier. The intermediate conjugate is then conjugated to the antibody. After administration of the immunoconj gate, a boron addend is activated by thermal neutron irradiation and converted to radioactive atoms which decay by ⁇ -emission to produce highly toxic, short-range effects.
• therapeutically useful immunoconjugates can be prepared in which a mutated antibody is conjugated to a toxin or a chemotherapeutic drug.
• toxins which are suitably employed in the preparation of such conjugates are ricin, abrin, human ribonuclease, pokeweed antiviral protein, gelonin, diphtherin toxin, and Pseudomonas endotoxin. See, for example, Pastan et al . , Cell 47 : 641 (1986), and Goldenberg, CA - A Cancer Journal for Clinicians 44 : 43 (1994) .
• Other suitable toxins are known to those of skill in the art.
• Useful cancer chemotherapeutic drugs for the preparation of immunoconjugates include nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purine analogs, antibiotics, epipodophyllotoxins, platinum coordination complexes, hormones, and the like.
• Chemotherapeutic drugs that are useful for treatment of infectious agents include antiviral drugs (such as AZT, 2',3'- dideoxyinosine and 2' ,3' -dideoxycytidine) , antimalarial drugs (such as chloroquine and its congeners, diaminopyrimidines, mefloquine) , antibacterial agents, antifungal agents, antiprotozoal agents, and the like.
• Suitable chemotherapeutic agents are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed. (Mack Publishing Co. 1990), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co.
• Immunoconjugates of the present invention also can be used for prodrug therapy. In this approach, a mutated antibody-enzyme conjugate is used to localize the enzyme to target tissue. Subsequent administration of prodrug results in the site-specific activation of the prodrug at target cells.
• therapeutically useful immunoconjugates can be obtained by conjugating photoactive agents or dyes to a mutated antibody.
• Fluorescent and other chromogens, or dyes, such as porphyrins sensitive to visible light have been used to detect and to treat lesions by directing the suitable light to the lesion (cited above) .
• this has been termed photoradiation, phototherapy, or photodynamic therapy (Jori et al . (eds. ) , PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES (Libreria Progetto 1985) ,- van den Bergh, Chem . Bri tain 22 : 430 (1986) ) .
• the present invention contemplates the therapeutic use of immunoconjugates comprising photoactive agents or dyes.
• the general methodology is described above in relation to the use of such immunoconjugates for diagnosis.
• the present invention also contemplates two-, three- or four-step targeting strategies to enhance antibody therapy.
• General techniques include the use of mutated antibodies conjugated with avidin, streptavidin or biotin, and the use of second antibodies that bind with the primary immunoconjugate, as discussed above. See, for example, Goodwin et al . , Eur. J. Nucl . Med. 9 : 209 (1984), Goldenberg e ⁇ al . , J. Nucl . Med. 25:1604 (1987), Hnatowich et al . , J. Nucl . Med. 28 : 1294 (1987) , Paganelli et al . , Cancer Res .
• a mammal having a disease caused by an infectious agent may be treated by parenterally injecting the mammal with an immunoconjugate comprising a variable domain that binds with an antigen that is associated with the infectious agent and a therapeutic agent. Subsequently, the mammal is injected with an antibody or antibody fragment that binds with the immunoconjugate in an amount that is sufficient to decrease the level of circulating immunoconjugate by about 10-85% within 2 to 72 hours.
• An alternative approach to enhancing the therapeutic index comprises administering a mutated antibody conjugated with avidin/streptavidin (or biotin) , injecting a clearing composition comprising biotin (or avidin/streptavidin) , and administering a conjugate of a therapeutic agent and avidin/streptavidin (or biotin) , as discussed above.
• LL2 is a murine Mab that has been shown to be effective for the diagnosis and treatment of non-Hodgkins B-cell lymphoma. Goldenberg et al . , J. Clin . Oncol .
• this Mab is internalized very rapidly, making a mutated antibody construct of this Mab very useful with protein agents such as ribonuclease and deoxyribonuclease, which can cleave RNA and DNA, respectively, in the target cell.
• protein agents such as ribonuclease and deoxyribonuclease, which can cleave RNA and DNA, respectively, in the target cell.
• an internalizing construct would be expected to have superior therapeutic properties.
• Immunoconjugates may be administered alone, or in conjugation with the conventional chemotherapeutic agents described above .
• Modes of chemotherapeutic administration and suitable dosages are well known to those of skill in the art. See, for example, REMINGTON'S
• the dosage of administered immunoconjugates will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history. Typically, it is desirable to provide the recipient with a dosage of immunoconjugate which is in the range of from about 1 pg/kg to 10 mg/kg (amount of agent/body weight of patient) , although a lower or higher dosage also may be administered as circumstances dictate.
• Immunoconjugates to a patient can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, by perfusion through a regional catheter, or by direct intralesional injection.
• the administration may be by continuous infusion or by single or multiple boluses.
• Immunoconjugates having a boron addend-loaded carrier for thermal neutron activation therapy will normally be effected in similar ways. However, it will be advantageous to wait until non-targeted immunoconjugate clears before neutron irradiation is performed. Clearance can be accelerated using an antibody that binds to the immunoconjugate. See U.S. patent No.
• the immunoconjugates of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby immunoconjugates are combined in a mixture with a pharmaceutically acceptable carrier.
• a composition is said to be a "pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient patient.
• Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier.
• Other suitable carriers are well-known to those in the art. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed. (1990) .
• an immunoconjugate and a pharmaceutically acceptable carrier are administered to a patient in a therapeutically effective amount.
• a combination of an immunoconjugate and a pharmaceutically acceptable carrier is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant.
• An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.
• an agent is physiologically significant if its presence results in the inhibition of the growth of target cells.
• Additional pharmaceutical methods may be employed to control the duration of action of an immunoconjugate in a therapeutic application. Control release preparations can be prepared through the use of polymers to complex or adsorb the immunoconjugate.
• biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebacic acid. Sherwood et al . , Bio/Technology 10: 1446 (1992) . The rate of release of an immunoconjugate from such a matrix depends upon the molecular weight of the immunoconjugate, the amount of immunoconjugate within the matrix, and the size of dispersed particles. Saltzman et al . , Biophys . J. 55: 163 (1989) ; Sherwood et al . , supra . Other solid dosage forms are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th ed. (1990) .
• a staging vector designated as CHpBSK, is shown in Figure 4.
• CHpBSK A staging vector, designated as CHpBSK, is shown in Figure 4.
• the Pstl site is located in the intron sequence immediately adjacent and upstream of the exon sequence encoding the IgG, hinge region, while the Sfil site is located in the intron sequence about 35 base pairs downstream of the exon sequence encoding the CH2 domain.
• the fragment encoding the human IgG, hinge region and CH2 domain is excised from CHpBSK by digestion with Pstl and Sfil.
• a DNA fragment containing the genomic sequence for human IgG 3 hinge region is subsequently inserted into this site, replacing the IgG, hinge region and the CH2 domain, as follows.
• the human IgG 3 hinge region amino acid sequence is : ELKTPLGDTT HTCPRCPEPK SCDTPPPCPR CPEPKSCDTP PPCPRCPEPK SCDTPPPCPR CP [SEQ ID NO:l] . Kabat et al . , SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, VOLUME II (1991) .
• a DNA fragment encoding the human IgG 3 hinge region is obtained by polymerase chain reaction (PCR) as follows.
• the h3BACK(+) primer is a 39-mer with the nucleotide sequence of 5' -ctt etc tct gca gAG CTC AAA ACC CCA CTT GGT GAC ACA-3' [SEQ ID NO:2] , where lower case letters represent the intron sequence, uppercase letters represent the sequence encoding the first nine amino acids of the human IgG 3 hinge region, and the underlined portion indicates the Pstl site at the intron/exon junction.
• the antisense primer h3F0R(-) is a 63-mer with the nucleotide sequence of 5'-ggt ggg ccg age egg cct ggc cct cgc ace cca egg gtc cca cCT TTG GCT TTG GAG ATG GTT-3' [SEQ ID NO: 3] , where lower case letters represent the intron sequence, uppercase letters represent the sequence encoding the last six amino acids of the human IgG 3 hinge region, and the underlined portion indicates the Sfil site.
• the PCR reactions are performed in a volume of 100 ⁇ l by mixing 3 ⁇ g of pdHL2-Imm ⁇ 3Fab with 5 ⁇ l of 5 ⁇ M of the flanking primer pairs in the presence of 10 ⁇ l of lOx PCR buffer (500 mM KC1, 100 mM Tris-HCI (pH 8.3), 15 mM MgCl 2 ) and 5 units of AmpliTaq DNA polymerase (Perkin Elmer Cetus; Norwalk, CT) .
• the reaction mixture is subjected to 30 cycles of PCR reaction consisting of denaturation at 94°C for one minute, annealing at 55°C for 1.5 minutes, and polymerization at 72°C for 1.5 minutes.
• Double-stranded PCR-amplified products (about 650 base pairs in length) are gel-purified, digested with Pstl and Sfil, and subcloned into the complementary sites of CHpBSK.
• the resultant construct designated as ⁇ C2h3pBSK ( Figure 4) , contains sequences encoding a human constant region with the structure of [CHI (IgG,) ] - [IgG 3 hinge] - [CH3 (IgG,)] . Sanger's dideoxy sequencing reactions are carried out to confirm the sequence.
• the human ⁇ C2h3IgG, constant region fragment is excised from ⁇ C2h3pBSK by digestion with BamHI and EagI and subcloned into the corresponding sites of the vector hMN14pGlg, replacing the human IgG, constant region.
• the resultant expression vector, hMN14- ⁇ C2h3pGlg ( Figure 5)
• hMN14pKh when co-transfected with the light-chain expression vector, hMN14pKh, into SP2/0 cells will express a divalent hMN14 antibody having a long hinge region and lacking a CH2 domain.
• Co- transfection conditions are described by Leung et al . , Hybridoma 13 : 469 (1994) .
• the fragment encoding the ⁇ CH2h3IgG, constant region can be subcloned into the high-level expression vector, hMN14pdHL2 (Immunomedics, Inc.; Morris Plains, NJ) . See Figure 6.
• This vector contains both heavy and light chain sequences.
• the vector also includes a dihydrofolate reductase gene to facilitate subsequent amplification with increasing concentrations of methotrexate.
• the IgG, constant region fragment containing CHI, IgG, hinge, CH2 and CH3 is removed from the vector by Hindlll/Xmnl digestion.
• Hindlll cleaves at the intron sequence joining the constant region to the heavy chain variable domain, while Xmnl cleaves at the coding sequences of the CH3 domain.
• the Hindlll /Xmnl DNA fragment from ⁇ C2h3pBSK is inserted into the corresponding site of the hMN14pdHL2 construc .
• the resultant construct, hMN-14 ⁇ C2h3pdHL2 ( Figure 7) , when transfected into SP2/0 cells, or other mammalian cell lines, will express a hMN14 antibody with the CH2 domain deleted and carrying an IgG 3 hinge region instead of an IgG, hinge region.
• the mutation is introduced by PCR using a long oligonucleotide (about a 65-mer) that has an identical sequence to the 5' region of the light chain sequence of interest, except at amino acid position 18 - 20 (according to Rabat's numbering system) where the sequence for carbohydrate addition is engineered, and a short 3 '-end oligonucleotide with a sequence identical to the C-terminus of the antibody of interest.
• a vector containing DNA that encodes a mutant light chain when co-transfected with the heavy chain vector hMN14 ⁇ C2h3pGlg into SP2/0 cells, will express a CEA-specific antibody carrying an IgG 3 hinge region with the CH2 domain deleted, and a light chain variable region glycosylated at amino acid position 18 - 20.
• reaction conditions can be varied within certain parameters to manipulate the nature of the final product.
• a mutated antibody is first reduced with 1 - 500 mM solutions of thiols to reduce the desired number of hinge-region disulfide bonds.
• Thiols such as cysteine, cysteamine, glutathione, mercaptoethanol, mercaptoethylamine, dithiothreitol and dithioerythritol may be used in the practice of the invention.
• a thiol-containing mutated antibody is advantageously further substituted within 24 hours of preparation, although storage at low temperature (4°C or frozen) under an argon atmosphere may be employed to stabilize the reduced construct prior to further reactions .
• the thiol-containing construct can be compounded for technetium-99m radiolabeling with or without stannous ion. If stannous ion is compounded with the reduced construct, the construct can be labeled with pertechnetate taken directly from a Tc-99m/Mo-99 generator. Alternatively, the mutated antibody can be compounded without the stannous ion and radiolabeled with reduced Tc-99m-tartrate and the like. Technetium-99m labeling of mutated antibodies can be carried out at specific activities up to 150 mCi per milligram of protein. Most preferably, specific activities of 20 - 40 mCi/mg protein are employed. Tc-99m labeling is carried out most usefully in volumes of about 0.5 - 2 milliliters.
• anthracyclines Two classes of antitumor drugs that have been conjugated to Mabs most frequently are anthracyclines and methotrexate.
• Doxorubicin or cyanomorpholino anthracycline is reacted with bromoacetic acid hydrazide to yield the bromoacetyl hydrazone of the drug. That is, doxorubicin is activated for thiol reaction while an acid-labile hydrazone group has been placed within the linkage.
• the second generation, more toxic, antitumor agent, cyanomorpholinyl anthracycline is attached to the construct in the same manner.
• This aspect of the invention describes the attachment of hydrazone-drug conjugates to free thiol groups of a mutated antibody in a site-specific manner.
• the purity of the immunoconjugates are evaluated initially by size-exclusion HPLC using a Zorbax-250 or Bio-Sil sizing column and appropriate molecular weight standards. Dual wavelength detection at 280 nm for antibody detection and at 495 nm for drug detection are used. Samples are often re-analyzed using buffers with increasing percentages (up to 20%) of organic buffers to discern if any non-covalently bound drug is associated with the Mab. Ion-exchange columns also are used as a secondary system for the same purpose.
• ILC Instant thin-layer chromatography
• PAGE Polyacrylamide gel electrophoresis
• Immunoprecipitation is used to determine immunoreactivity retention of the drug conjugates.
• radiolabeled immunoconjugates are incubated with rabbit anti-mouse IgG for 1 hour at 4°C.
• Immunoprecipi in (Gibco BRL; Gaithersburg, MD) is added and, after an additional incubation, the mixture is centrifuged and the pellet separated. The supernatant is treated with 3 N HCl to hydrolyze any drug-Mab conjugate and the aqueous solution neutralized and extracted with an organic solvent.
• the amount of doxorubicin is quantified by UV spectroseopy at 495 nm, and compared with the amount of radioactivity located in the pellet or in the supernatant to gauge the percentage of non- immunoreactive conjugate.
• the reduced antibody (10 ⁇ mol) in phosphate-buffered 0.9% sodium chloride (pH 7.5) is treated with cyanomorpholino anthracycline-2-bromoacetic acid hydrazone (400 ⁇ mol) at 25°C. Residual free thiol groups are tested during the course of the reaction by Ellman testing of reaction aliquots at hourly time intervals. The conjugation reactions proceeds for about six hours before the Ellman reaction is negative.
• the cyanomorpholinylanthracycline-hydrazonyl-2-acetamido-S- hIMMU-14- ⁇ CH2-IgG 3 conjugate is purified by size- exclusion chromatography and the substitution ratio of the drug onto the protein is determined spectrophotometrically, as described above. Approximately 5 - 15 drug molecules are bound to the mutated antibody framework.
• the hIMMU-14- ⁇ CH2-IgG 3 construct (10 ⁇ mol) is reduced with 2-mercaptoethanol (150 mM) at pH 8.7 for 10 minutes at 4°C.
• 2-mercaptoethanol 150 mM
• the reduction mixture is applied to a spin-column of Sephadex G-50-80 in 0.1 M sodium phosphate (pH 7.5) .
• the number of free sulfhydryl groups in the effluent is determined by the Ellman reaction, and protein concentration is determined by absorbance at 280 nm.
• the reduced antibody (10 ⁇ mol) in phosphate-buffered 0.9% sodium chloride (pH 7.5) is treated with cyanomorpholino anthracycline, 2- (bromoacetyl) -6- aminohexanoic acid hydrazone (400 ⁇ mol) at 25°C. Residual free thiol groups are tested during the course of the reaction by Ellman testing of reaction aliquots at hourly time intervals. The conjugation reactions proceeds for about six hours before the Ellman reaction is negative.
• the cyanomorpholinyl anthracycline- hydrazonyl-hexanoyl-6-acetamido-S-hlMMU-14- ⁇ CH2-IgG 3 conjugate is purified by size-exclusion chromatography and the substitution ratio of the drug onto the protein is determined spectrophotometrically, as described above. Approximately 5 - 15 drug molecules are bound to the mutated antibody framework. 3. Preparation of alkylating drug conjugates of the hIMMU-14- ⁇ CH2-IgG 3 construct
• the hIMMU-14- ⁇ CH2-IgG 3 construct is solubilized in
• 0.2 M Tris buffer pH 8.7 at a concentration of 5 - 20 mg/ml, and treated with 2-mercaptoethanol in the same buffer to a final concentration of 1 - 100 mM of the free thiol.
• the reduced construct is purified by Sephadex G-50-80 size-exclusion chromatography developed in 0.2 M PBS (pH 7.4 - 8.5) that contains 10 mM EDTA to preserve thiol groups in the reduced state.
• the reduced mutated construct is then treated with a 10 - 1000 fold molar excess of a triaziridine chemotherapeutic alkylating agent, Trenimon, for 0.15 - 3 hours with stirring.
• Purified drug-hlMMU- 14- ⁇ CH2-IgG 3 conjugate is obtained by size-exclusion chromatography and stored at pH 4 - 5 to maintain the stability of the appended chloroethyl groups.
• the number of reactable chloroethyl groups per mutated antibody is determined by duplicate/triplicate reaction with known amounts of low molecular weight thiol, followed by estimation of residual unreacted thiol using a procedure such as the Ellman reaction. Remaining thiol is determined by measuring absorbance at 410 nm, and the level of chlorambucil substitution is determined by subtracting this number from the total amount of free thiol added.
• Thiol-reduced hIMMU-14- ⁇ CH2-IgG 3 construct is solubilized in 0.1 M phosphate-buffered saline (pH 7.4 - 8.5) at a temperature of 4 - 25°C.
• the construct is incubated for 1 - 5 hours with stirring in 10 - 15% dimethylsulfoxide (DMSO) with 10 - 50-fold molar excess
• DMSO dimethylsulfoxide
• Streptavidin is activated for specific thiol coupling to mutated antibody by reaction with the amino-thio cross - l inker sul f osucc inimidyl - 4 - [N - maleimidomethyl] cyclohexane-1-carboxylate (sulfo-SMCC; Pierce Chemical Co., Rockford, IL) .
• a streptavidin solution (2.9 ml) containing 29 mg of streptavidin dissolved in 0.1 M phosphate buffered saline (PBS) , pH 7.3, is treated with 2.42 ml of a freshly prepared solution of 10 mM sulfo-SMCC in water.
• the protein is purified from low molecular weight materials on a Sephadex G-50-80 size-exclusion column run in 0.1 M PBS (pH 7.0) .
• the maleimido-streptavidin is reconcentrated to about 5 to 6 ml for reaction with reduced mutated antibody.
• a solution of 12 mg of 2-mercaptoethylamine in 53 ⁇ l of 0.1 M ethylenediaminetetraacetic acid (EDTA) and one milliliter of 0.1 M PBS (pH 7.3) is freshly prepared. Six hundred microliters of this solution is added to 2.5 ml of an 18 mg/ml solution of the mutated antibody in 0.1 M PBS (pH 7.3) . After a 45 minute incubation at room temperature, the reduced construct is purified by size exclusion chromatography on a Sephadex G-50-80 column run in 0.1 M PBS (pH 7) . The product is concentrated to 3 -
• Each of the maleimido-streptavidin and the reduced mutated antibody is added in six aliquots to 8 ml of 0.1 M PBS (pH 7) with rapid stirring over a period of 5 to 6 minutes. Stirring is continued for one hour at room temperature. Then, 25 mg of sodium tetrathionate are added to the mixture to block unreacted maleimides and stirring is continued for another five minutes.
• the product, 1:1 mutated antibody-streptavidin is purified from aggregated and unreacted proteins and low molecular weight materials by preparative size-exclusion chromatography on a 600 x 21.5 mm TSK-SW-3000 column run at 1.5 ml/min in 0.2 M PBS (pH 6.8) . 2. Substitution of cysteinyl-peptide haptens onto an hIMMU-14- ⁇ CH2-IgG 3 construct
• the hIMMU-14- ⁇ CH2-IgG 3 construct is solubilized in
• the modified construct is purified by size-exclusion chromatography in 0.1 M borate buffer
• Mutated antibody is reduced with 2-mercaptoethanol (25 mM) at pH 8.7 for 10 minutes at 4°C.
• the reduction mixture is applied to a spin column of Sephadex G-50-80 in 0.1 M sodium phosphate (pH 8.1) .
• the number of sulfhydryl groups in the effluent is determined by the Ellman reaction.
• a mixture of reduced mutated antibody (7.3 mg) and 8 ⁇ l of 2- (p-bromoacetamido) benzyl-DTPA (Br-Bz-DTPA) (0.23 ⁇ mole) is incubated at 37°C for 30 minutes at pH 8.1.
• the conjugate is purified on a one milliliter spin column of Sephadex G-50-80 in 50/150 mM acetate/sodium chloride (pH 5.3) .
• the conjugate concentration of the effluent is determined by absorbance at 280 nm to be 13.3 mg/ml.
• the chelate/antibody ratio is determined by metal binding assay.
• ⁇ l InCl 3 is purchased from a supplier such as New England Nuclear. It is diluted with 0.1 M ammonium citrate (pH 5.58) to give a solution of 15 ⁇ Ci/ ⁇ l . This mixture is incubated for one hour at room temperature for 30 minutes prior to use.
• mutated antibody- Bz-DTPA (104 ⁇ l, 1.5 g) is treated with 280 ⁇ l of m In (5.4 mCi) in 0.1 M ammonium citrate.
• the radiolabeling mixture is incubated at room temperature for one hour.
• Nonspecifically bound indium is removed by treating the reaction mixture with EDTA (final concentration: lOmM) for ten minutes at room temperature.
• ITLC in 10 mM EDTA shows 84% of the radioactivity bound to antibody.
• the mutated antibody-Bz-DTPA- m In is purified on a one milliliter spin column of Sephadex G-50-80, 50/150 mM acetate/sodium chloride (pH 5.3) .
• HPLC of the effluent shows the product eluting at 8.6 minutes as a single peak.
• ITLC shows 99% of the radioactivity bound to protein, while immunoreactivity measurement on a CEA-affigel column is measured at 90%. Mutated antibody can be labeled with yttrium-90 as follows.
• a solution of mutated antibody-S-Bz-DTPA (60 ⁇ l, 1 mg) in 0.1 M sodium acetate buffer (pH 6.5) is added to a solution of yttrium-90 (0.5 ml, 5mCi) in 0.5 M sodium acetate buffer (pH 6) .
• the solution is mixed and incubated at room temperature for one hour.
• the Y-90-mutated antibody is diluted to 5 ml using 2% human serum albumin in 0.1 M PBS (pH 7) , containing 1 mM EDTA.
• ITLC an aliquot of the diluted labeling mixture, run in 0.01 M EDTA shows 96% of the radioactivity associated with the protein construct.
• a bifunctional chelate can be labeled with a radioactive isotope, and the radiolabeled bifunctional chelate is then conjugated with a mutated antibody.
• the activated bifunctional che l at e , 2 - p - ( bromoacet amido ) benzyl - diethylenetriaminepentaacetic acid (Br-Bz-DTPA) (5 ⁇ l,
• the hIMMU-14- ⁇ CH2-IgG 3 (10 ⁇ mol) constructed as described above, is reduced with 2-mercaptoethanol (150 mM) at pH 8.7 for 10 minutes at 4°C.
• the reduction mixture is applied to a spin column of Sephadex G-50-80 in 0.1 M sodium acetate (pH 5.5) that contains 0.9% sodium chloride (ABS buffer) .
• the number of free sulfhydryl groups in the effluent is determined to be 20 per mole of protein by the Ellman reaction. Protein concentration is determined by measuring absorbance at 280 nm.
• the reduced mutated antibody is aliquoted into 100 ⁇ g - 2 mg fractions and stored under argon frozen (or at 4°C) prior to radiolabeling.
• the reduced mutated antibody is compounded with stannous ion and lyophilized for radiolabeling in the f ture.
• each 200 ⁇ g of the hIMMU-14- ⁇ CH2-IgG 3 -SH construct (5-10 SH/mole protein) is mixed with a solution of 25 ⁇ g of stannous chloride in 150 ⁇ l of 50 mM ABS buffer containing sodium potassium tartrate (9.2 mM) and lyophilized in a two milliliter glass vial.
• the lyophilizate is stored under partial vacuum/argon or under full vacuum in a septum-sealed vial.
• Tc-99m radiolabeling is performed by adding 5 mCi (1 ml) of Tc-99m pertechnetate generator-eluate to achieve a specific activity of 25 mCi/mg.
• the labeling may be performed on the freshly formulated material without the lyophilization step.
• Radio-HPLC analysis at 30 minutes post-labeling shows 95-100% incorporation of radioactivity into the antibody construct.
• Re-188-hIMMU-14- ⁇ CH2-IgG 3 A solution of stannous chloride dihydrate in 6N HCl at a stannous ion concentration of 100 mg/ml together with a solution of 100 mM sodium potassium tartrate (a stannous ion complexor) in 50 mM sodium acetate buffer
• Rhenium-188 is conveniently obtained from a tungsten- 188/rhenium-188 generator system such as the one developed at Oak Ridge National Laboratory. Re-188- perrhenate is eluted with sterile physiological saline to provide the isotope (up to a Curie) in a 5 to 20 milliliter volume of solution. The vial containing the hIMMU-14- ⁇ CH2-IgG 3 -SH is reconstituted with Re-188 perrhenate, mixed to ensure dissolution and allowed to stand for two hours at room temperature .
• the radiolabeled product is analyzed by HPLC on a Bio-Sil 250 size-exclusion column (Bio-Rad; Hercules, CA) fitted with an in-line radioactivity detector, and run at 1 ml/minute in 0.2 M phosphate buffer (pH 6.8) .
• ITLC on a silica gel impregnated glass fiber strips (Gellman Sciences; Ann Arbor, MI) activated according to the manufacturer's instructions also are used for analysis. These analyses show greater than 90% incorporation of the Re-188 isotope into the hIMMU-14- ⁇ CH2-IgG 3 -SH construct. 3.
• Silver-Ill nitrate is diluted with water to 5 mCi/ml. A labeling volume of 1 ml is used to reconstitute (if necessary) a sample of lyophilized hIMMU-14- ⁇ CH2-IgG 3 -SH (at pH 5) in 50 - 100 mM ABS buffer (1 mg) . After thorough mixing, the solution is incubated at room temperature for 30 minutes, and the Ag-lll labeled conjugate is then tested.

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