EP0105360A1 - Antikörper mit zweifacher spezifizität, deren herstellung und verwendungen - Google Patents

Antikörper mit zweifacher spezifizität, deren herstellung und verwendungen

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Publication number
EP0105360A1
EP0105360A1 EP83901672A EP83901672A EP0105360A1 EP 0105360 A1 EP0105360 A1 EP 0105360A1 EP 83901672 A EP83901672 A EP 83901672A EP 83901672 A EP83901672 A EP 83901672A EP 0105360 A1 EP0105360 A1 EP 0105360A1
Authority
EP
European Patent Office
Prior art keywords
antibody
process according
hybrid
antibodies
hapten
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP83901672A
Other languages
English (en)
French (fr)
Other versions
EP0105360A4 (de
Inventor
Joanne Martinis
Richard M. Bartholomew
Gary S. David
Thomas H. Adams
James M. Frincke
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hybritech Inc
Original Assignee
Hybritech Inc
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Filing date
Publication date
Application filed by Hybritech Inc filed Critical Hybritech Inc
Publication of EP0105360A1 publication Critical patent/EP0105360A1/de
Publication of EP0105360A4 publication Critical patent/EP0105360A4/de
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • 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
    • A61K51/1084—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 the antibody being a hybrid immunoglobulin
    • A61K51/109—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 the antibody being a hybrid immunoglobulin immunoglobulins having two or more different antigen-binding sites or multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2123/00—Preparations for testing in vivo

Definitions

  • This invention relates to antibodies having du specificities. In another aspect it relates to immun diagnostic and immunotherapeutic processes. In y another aspect it relates to hybridomas and relat monoclonal antibody technology.
  • the antigen-antibody reaction is already routine exploited in a variety of practical applications and being widely investigated to establish its value in othe as yet unproven, utilities.
  • serum antibodi produced by a host animal's immune response to an i mun gen can be used in affinity purification procedures isolate the immunogen from solutions in which it i present in only minute quantities.
  • the immunogen is a diseas associated antigen
  • its presence in a patient's serum o other body fluid can be detected using immunoassay o immunometric techniques.
  • detection of HBs using a radioimmunometric technique is the current metho of choice.
  • serum antibodies to ferri tin obtained from New Zealand white rabbits and labele with 131l, have been reported as showing promise for th treatment of liver tumors. (See Order et al, Internation al Journal of Radiation Oncology, Biology and Physics, j 703 (1980)).
  • Serum antibodies for example, those obtained fro rabbits, urine species or other mammals are "polyclonal in nature since the immune system of the host is stimula ted to produce a mixture of specific antibodies directe
  • each antibody specie is distinct molecule and the differences in peptide sequen between different ' species affect their general specifici ties as well as the particular epitopes they recognize an their affinities for the antigen.
  • a monoclonal antibod will have a constant specificity for a single site on th antigen molecules and a well defined affinity.
  • T immortality of the hybridoma guarantees an almost u limited supply of the antibody it secretes and alleviat problems associated with variance in antibody titer a overall affinity from animal to animal used to produ serum antibodies.
  • Monoclonal antibodies obtained fr hybridomas have, for example, been put to practic application in diagnostic kits. A selection of such ki is available from Hybritech, Inc., assignee of th application.
  • An antibody molecule can generally be considered express a single specificity which is exhibited towar the immunogen to which the host's immune system respond by production of the antibody.
  • the antibody is compos of two identical halves, each of which is comprised of heavy and light chain pair and each of which recognize the same antigenic determinant as the other.
  • the fol lowing is a representation of the arrangement of heavy (H and light (L) chains in an antibody molecule:
  • the -S-S- disulfide bridges which link the two (H chains together at the location of cysteine moieties ca usually be cleaved selectively j ⁇ n vitro by a mild reduc tion, and the half molecules disassociated by subsequen acidification. The half molecules can then be recombine
  • OM (renatured), again in vitro, at neutral pH, the reassocia- tion taking place through non-covalent interaction. If antibodies of different specificities are sub ⁇ jected to a selective cleavage of the disulfide bridges between the heavy chains and conditions conducive to renaturation subsequently established, reassociation between half molecules may occur randomly to produce a population of antibodies, at least some of which are hybrids in that one half of an antibody molecule of one specificity combines with one half of an antibody molecule of a different specificity.
  • hybrid antibodies can be obtained in the manner described above, the yields are often very low, the reactions used to make them difficult to reproduce and the hybrid antibodies usually suffer significant, irreversible denaturation. Such denaturation can reduce immunore- activity and would be expected to result in different metabolic characteristics in vivo. As a result, the hybrid antibody today remains largely a laboratory curi ⁇ osity which is difficult to obtain.
  • Antibodies having dual specificities may also be prepared by conjugating pairs of intact antibodies, monoclonal or otherwise, using a variety of coupling or crosslinking agents such as protein A (from Staphylococcus aureus), carbodiimide and bifunctional compounds such as N-succinimidyl-3-(2-pyridyldithio) propionate to obt dimeric and higher antibody multimers to which each mem of the antibody pair contributes its specificity.
  • protein A from Staphylococcus aureus
  • carbodiimide and bifunctional compounds such as N-succinimidyl-3-(2-pyridyldithio) propionate to obt dimeric and higher antibody multimers to which each mem of the antibody pair contributes its specificity.
  • F example, Mandoche et al have reported the formation multivalent antibodies having dual specificities, by sequential reaction of antibodies with protein A, wh have been shown to be capable of detecting cell surf antigens jLn vitro. See
  • the present invention provides, among other things, novel, completely biological method for reliably obtaini hybrid monoclonal antibodies in good yields witho denaturation.
  • hybrid antibody will be used to designate a sing antibody molecule having two different specificities. T individual specificities may be to antigenic determinan on two different antigens or to different antigen determinants (epitopes) on the same antigen. Furthermor unless otherwise indicated, the term "antigen" al embraces haptens.
  • hybrid antibodies having a dual specificity are obtain by fusion of a hybridoma, preferably a selectively d structible hybridoma, which secretes an antibody against preselected antigenic determinant with a fusible B-lymph cyte or a second hybridoma, the B-lymphocyte or secon hybridoma secreting a second antibody against a differe antigenic determinant, to form a second generation hybri doma (hereinafter "polydoma").
  • a hybridoma preferably a selectively d structible hybridoma, which secretes an antibody against preselected antigenic determinant with a fusible B-lymph cyte or a second hybridoma
  • the B-lymphocyte or secon hybridoma secreting a second antibody against a differe antigenic determinant
  • the ter “selectively destructible hybridoma” means a hybridom which lacks, or at least substantially lacks, the capabi ity of surviving in the medium in which the polydoma i cultured.
  • the polydoma i addition secretes a high percentage of a monoclonal hybri antibody having a dual specificity, i.e., a capability t bind with either of the antigenic determinants recognize by the individual antibodies produced by the parent cell or with both determinants at the same time.
  • the hybri monoclonal antibody obtained in this way has not suffere the undesireable denaturation which characterizes hybrid obtained from the process of chemical recombination o antibody half molecules. Furthermore, the process of th invention permits the hybrid to be obtained reliably a in large amounts.
  • these processes employ a monoclonal antibody or polyclona antibodies having a first specificity against a targe antigen and a second specificity against a substance, fo example, another antigen or hapten, which permits diagnosis to be made of the target antigen or whic permits delivery of, or is itself, an agent which i lethal to the target antigen or the tissue with which i is associated.
  • polydoma can be obtained according to the present inven tion which will secrete an antibody having one specificit for a target antigen and a second specificity for a moiet useful in diagnosis or therapy.
  • antibod half molecules can be recombined using _in vitro chemica means or individual intact mono-specific antibodies can b coupled or crosslinked by chemical means to obtain anti body multi ers (which may be a dimer, trimer or highe multimer) having a dual specificity and having the same o a similar utility as a hybrid monoclonal antibody havin the same dual specificity made according to the presen invention.
  • antibody includes anti ⁇ body fragments having immunochemical properties such as Fab or F(ab) 2 fragments.
  • the process for obtaining a hybrid monoclonal antibody according to the present invention requires, as one parent, a hybridoma, and preferably a selectively destructible hybridoma, which secretes a monoclonal antibody against a preselected antigenic determinant or epitope.
  • a selective destructible hybridoma as a parent has the advantage that it prevents the cells obtained by fusion of the selectively destructible hybridoma with a B-lymphocyte or a second hybridoma, i.e., the polydoma, from being over ⁇ grown by a population of the parent hybridoma when the cells obtained in the fusion process are cultured and to provide a means by which the polydoma cells can be isola ⁇ ted from parental hybridoma cells.
  • hybri ⁇ domas useful in our invention can be obtained from hybri ⁇ domas secreting an antibody having one of the desired specificities made by the classic Kohler-Milstein process, i.e., hybridomas obtained by fusion of a myeloma cell and a B-lymphocyte such as that found in the spleen cells of a mouse.
  • hybridoma is subjected to a back selection process to obtain the hybridoma which is selectively destructible.
  • selective destructibility can be obtained by back selection to a hybridoma which lacks a genetic component which is necessary to its survival in a medium of choice in which the polydoma produced by the fusion can be cultured because of a genetic contribution from the fusion partner of the selectively destructible hybridoma, i.e., the B-lymphocyte or second hybridoma.
  • the presently preferred back selection process involves culturing a hybridoma which secretes an antibody having one of the desired specificities to be incorporated into the hybrid antibody in a growth medium containing 8-azaguanine.
  • a growth medium containing 8-azaguanine any cell which incorpora ⁇ tes 8-azaguanine and can, therefore, grow in the medium are ones which lack the enzyme hypoxanthine-guanine phosphoribosyl transferase (HPRT).
  • HPRT hypoxanthine-guanine phosphoribosyl transferase
  • treatment of the selected hybridoma with a analog of glutamine such as azaserine or 5-diazo-5-oxa-L norleucine (DON) irreversibly inhibits the enzyme formyl- glycinamide ribonucleotide amidotransferase by formatio of a covalent bond with a cysteine residue at the enzyme's active site. This inhibition will ultimately result in cell death.
  • the hybridoma can be rescued by fusion with the second parent of the polydoma which supplies the necessary enzyme.
  • the selectively destruc- tible hybridoma is fused with complementary B-lymphocytes, typically obtained as spleen cells taken from a host which has previously been immunized with an antigen, which may be a hapten bound to a carrier protein, selected to cause the host to generate an immune response which produces antibodies having the second specificity desired in the hybrid antibody.
  • an antigen which may be a hapten bound to a carrier protein
  • the host is usually a mouse but species of rabbits, humans and other animals may also be used although interspecies fusion may exhibit a low order of stability.
  • the process for immunizing such a host is, of course, well known and details need not be given here.
  • Fusion of the selectively destructible hybridoma wit the B-lymphocytes to obtain the polydoma can be accom plished by combining the two groups of cells in a maxim containing an agent known to promote cell fusion such a polyethylene glycol or Sendi virus according to know methods.
  • the polydoma is obtained b fusing the selectively destructible hybridoma using suitable fusion agent with a second hybridoma which i also selectively destructible.
  • the second parent hybri doma is obtained in the same manner as the first, i.e., b a process of back selection, irreversible enzyme inhibi tion or by any other suitable technique.
  • the second hybridoma must be able to complement th first.
  • the first selectively destructibl hybridoma lacks the enzyme HPRT
  • the second must b capable of contributing to the polydoma a gene which wil enable the polydoma to express HPRT.
  • th second selectively destructible hybridoma lacks the enzym
  • selection for ouabain resistance can be done first either on the parental myeloma line or the hybridoma derived therefrom, followed by back selection or other technique to confer selective destructibility.
  • Cells obtained by fusion of the two hybridomas in polyethylene glycol or other fusion agent are transferred to HAT medium containing ouabain in a concentration lethal to the second hybridoma parent.
  • the selectively destruc ⁇ tible hybridoma parent cannot survive in the HAT medium either, lacking, for example, the HPRT or other enzyme, even though ouabain resistant.
  • the polydoma cells can grow in the medium since they will possess the enzymes and ouabain resistance necessary for survival.
  • Yet another technique for obtaining a polydoma which employs a universal parent, i.e., one which has both a positive and a negative marker, which can be fused with any "off the shelf" hybridoma involves the use of recom- binant DNA vectors carrying various drug resistance markers.
  • SV40 carrying a gene for neomycin resistance can be used.
  • Example 1 A hybrid monoclonal antibody having a dual spec ficity for hepatitis B surface antigen (HBsAg) and pr static acid phosphatase (PAP) was- made in accordance wi the present invention in the following manner:
  • a hybridoma secreting a monoclonal antibody to P was grown in HAT medium for one week and then transferr to and grown in a non-selective medium. After variou lengths of time of growth under non-selective conditions 2 ml aliquots of cells were placed in medium containin 10-4 M 8-azaguanine which prevented cells from growin by incorporating 8-azaguaine in their DNA instead o guanine. Cells lacking the HPRT enzyme survived and gre in this medium and these cells necessarily did not surviv in HAT.
  • Clones that grew in the medium ' containing 8-aza guanine were tested for sensitivity to HAT and anti-PA production.
  • One clone which still produced anti-PAP an exhibited HAT sensitivity with a reversion frequency o less than 4 x 10 ⁇ "8 was subcloned. All of the subclone behaved like the parental clone.
  • Cells from one of the HAT sensitive subclones were fused in polyethylene glycol with spleen cells obtaine
  • the ascites from the polydoma clone bound both labeled HBsAg and labeled PAP, the former attributable to the presence of some non-hy- brid, mono-specific antibody to HbsAg in the ascites and the latter attributable to a hybrid that can bind and bridge the HBsAg on the ball and the trace labeled PAP in solution.
  • the experiment using a mixture of labeled PAP and anti-PAP from the parental hybridoma confirms that the anti-PAP specificity of the hybrid is for the same epitope as the antibody secreted by the parent since only background radiation is observed due to inhibition by the parental antibody of binding of labeled PAP to the hybrid antibody.
  • HBsAg Material representing each of the DEAE peaks was tested for antigen binding using radiolabeled HBsAg and PAP.
  • the first peak bound HBsAg but not PAP.
  • the middle peak is hybrid antibody having a dual specificity to HBsAg and PAP comprised of at least two subspecies.
  • the hybrid antibody obtained as the middle peak of the DEAE chromatography was radiolabeled with 125 ⁇ . After labeling, 85% of the labeled antibody would bind to PAP and 88% would bind to HBsAg.
  • the affinity of the hybrid for PAP was found to be slightly lower than that of the monoclonal antibody to PAP produced by the parental line. This difference in affinity was about the same as that observed by us between a monoclonal antibody and its Fab fragment.
  • the widel available HAT-sensitive mouse myeloma P3.653 was selecte for a second genetic marker, ouabain resistance, b introducing 1 mM ouabain into the growth medium. Whil most cells died, approximately 1/100,000 cells had b random mutation acquired resistance to the drug an so survived and multiplied to form the new myeloma popula tion which was HAT-sensitive and ouabain resistant.
  • This HAT-sensitive, ouabain-resistant myeloma was then fused with spleen cells obtained from Balb/c mice hyperimmunized with IgD using the previously cited techni ⁇ que of Gerhard.
  • Hybrids were selected in HAT medium (without ouabain) and clones were screened for production of monoclonal antibody directed against IgD. From among the positive clones, one which produced an IgG against IgD was selected for further study. This clone was tested for retention of the trait of ouabain resistance by adding 1 mM ouabain to the growth medium. Approximately one- third of the cells retained this genetic marker. When the culture was growing exponentially in ouabain, the cells
  • OM were subcloned. Ouabain-resistant subclones were tested for continued production of the monoclonal anti-IgD antibody. One of the subclones was further backselected by the procedure of Example 1 to obtain a population of cells sensitive to HAT. This subclone was grown for two weeks under non-selective conditions and then placed into medium containing 6-thioguanine. As noted above, the mechanism of action of 6-thioguanine is similar to that for 8-azaguanine. Cells which incorporate 6-thioguanine into their DNA instead of guanine will not grow. Cells lacking HPRT enzyme will not utilize 6-thioguanine from the medium and therefore can grow but are consequent ⁇ ly sensitive to HAT.
  • mice hybridoma which secretes a monoclonal antibody directed against prolactin.
  • the antiprolactin monoclonal antibody is of the same subclass (IgG i) as the anti-IgD ex ⁇ pressed by the parent line and it is easily separated from that antibody on Ornstein-Davis gels. Such a separation is indicative of greatly different charge on the anti ⁇ bodies and so should allow easy isolation of a hybrid antibody by DEAE-Sephadex chromatography.
  • a pol styrene bead coated with another anti-prolactin monoclon antibody was incubated 5 hours with 200 U.1 of a 100 ng/ prolaction solution.
  • the antibody used binds prolactin a distinct site from that of the antibody produced by t fused hybridoma cell line.
  • the bead was washed, th incubated overnight with the clone supernatants. The ne day, following several washes, 125 ⁇ labeled IgD w added.
  • Hybrid antibody bound to the bead by one functiono al arm could bind the radiolabeled IgD with the fr anti-IgD functionality whereas neither parental ty antibody IgD-Ig.D or Prolactin-Prolactin could form th bridge between the Prolactin bead and 125 ⁇ -igD trace
  • Results of a typical assay for clones producing hybri bifunctional antibody are presented in Table 2 belo
  • Anti-Prolactin 8721 21 of 36 clones exhibited significant bifunction activity by this assay. Ascites generated from 2 clon available to date have been shown to react in the bi functional assay. These ascites contain antibodies whi separate into three distinct bands on Ornstein-Davi gels: two bands coincide exactly with antibodies produce by the parent hybridomas (anti-IgD and anti-prolactin) The third band migrates midway between the parenta monoclonal antibody bands as expected of the hybri antibody.
  • the hybr antibody can be used as a component of an assay for pr lactin. Tailor-making other hybrid antibodies offe similar opportunities for other assays.
  • a CEA solution 600 ng or 25 ng was allowed to adsorb overnight to each well of plastic 96-well microtiter plate. The next day, unad sorbed material was washed out of the wells with PBS-Twee 20. Clone supernatants were added and incubated 2-1/ hours at 35 ⁇ C and then washed off the plate.
  • CEA-CEA an CEA-arsenate antibody would remain attached to the plat via the adsorbed antigen.
  • the second antigen, arsenyli acid coupled to the enzyme alkaline phosphatase was adde to the wells for 3 hours at 35°C.
  • an antibody useful in immunohistology would have a first specificity- for a suspect antigen, for example, a tumor associated antigen such as CEA, PAP or ferritin, and a second specificity against a hapten or antigen which will participate in a color reaction such as an enzym which causes a color reaction in the presence of suitable substrate.
  • a tumor associated antigen such as CEA, PAP or ferritin
  • a second specificity against a hapten or antigen which will participate in a color reaction such as an enzym which causes a color reaction in the presence of suitable substrate.
  • suitable enzymes to which th second specificity of the antibody may be directed i prostatic acid phosphatase (PAP) , horse radish peroxidase glucose oxidase, and alkaline phosphatase.
  • a tissu section is first treated with the antibody of dual speci ficity. Prior to doing so, the hybrid can have alread been allowed to bind the enzyme which catalyzes th staining reaction. If not, the section is then treate with a second solution containing the enzyme and rinse after an appropriate incubation and then treated with th substrate which undergoes a color change in the presenc of the enzyme. The formation of the color produce by the enzyme and substrate in the tissue sample is positive indication of the presence in the tissue of th target antigen.
  • the hybrid antibody against HBsAg and PA whose preparation is described herein, has been found t bind to HBsAg on a test substrate (polystyrene balls) and to PAP in a simulated staining experiment using p-nitro- phenyl phosphate as the enzyme substrate. After incuba ⁇ tion of the hybrid antibody with PAP and the HBsAg, the addition of the p-nitrophenyl phosphate resulted in the balls undergoing the characteristic yellow to brown color change.
  • an antibody of dual speci ⁇ ficity can also be used in immunoassays and immunometric assays.
  • an immunometric assay for HBsAg can be performed using an immobilized monoclonal antibody to HBsAg as a solid phase to extract HBsAg from a serum or other liquid sample suspected of containing the antigen.
  • the sample is incubated with a ball, beads, test tube or other substrate which has the anti-HBsAg bound or coated on its surface.
  • the incubation with the serum sample can be followed by, done simulta eously with or preceded by an incubation with a solutio of the hybrid.
  • the result will be the formation of a sandwich o the immobilized antibody, HBsAg if present in the sample and the hybrid antibody.
  • PAP i permitted to bind with the hybrid antibody. This can b done during or after formation of the sandwich, or in t alternative, the antibody-PAP complex can be preformed After formation of the sandwich, the solid phase is washe to remove sample residue and unbound hybrid antibody a then contacted with a solution containing a substrate suc as p-nitrophenyl phosphate or c ⁇ -napthol phosphate whi undergoes a color change in the presence of PAP. Occu rence of the color change confirms the presence of targe antigen in the sample.
  • Detection means other than enzymatically catalyzed reactions are also possible.
  • the second specificity of the hybrid or other antibody having a dual specificity can be directed against a hapten or antigen which is radiolabeled or which is fluorescent or which is detectable in the sandwich by any other- suitable means.
  • a preferred process which utilizes a hybrid antibody or other antibody having a dual specificity in an immuno ⁇ assay exploits the phenomenon of fluorescence quenching.
  • one specificity of the antibody is directed against a target antigen and the other against, for example, a hapten bearing a fluorescing chromophore.
  • the chromophore is either bound to the hapten or, in appropriate cases, may be the hapten itself.
  • the two chromophores are selected so that the first of them fluoresces at a wavelength which can be absorbed (quenched) by the other if they are positioned closely enough together so that the photon emitted by the fluores- cer can be captured by the quencher.
  • the two chromophores should be within about 100 angstroms and, preferably, within about 50 angstroms of each other. This positioning will occur when the fluorescing chromophore is bound at one antibody binding site and the quenching chromophore is bound to added antigen at the other.
  • a suitable pair of chromophores includes fluorescein as the fluorescing chromophore and rhodamine as the quenching chromophore.
  • the measured fluorescence will vary inversely with the amount of native antigen in the sample since, in the absence of native antigen, all of the antigen bound to the antibody will be labeled with the quenching chromo ⁇ phore and be positioned to absorb fluorescence by the chromophore carried by the hapten. Comparison of the measured fluorescence with that of a control sample containing a known amount of antigen permits a qualitative and quantitative determination of the presence of antigen in the sample.
  • This kind of immunoassay can, for example, be used to determine the levels in serum of drugs such as dilantin which must be closely monitored.
  • the target antigen would, of course, be dilantin.
  • this process can be used to detect other antigens as well including, in particular, tumor associated antigens.
  • Another preferred process which utilizes a hybri antibody or other antibody having a dual specificity in a immunoassay relies upon an enzymatic reaction.
  • one of the antibody speci ficities is directed, of course, to the target antigen an the other to an enzyme or a hapten to which is bound a enzyme.
  • the assay is conducted by incubating the antibod with a sample suspected of containing the target antige to which has been added a predetermined quantity of th target antigen that has been modified by binding to it substance that interacts with the enzyme to produce eithe a detectable substance or in some other way to permi detection of formation of the antigen-antibody complex Detection may be, for example, by fluorimetry, lumines cence, spectrophotometry or the like.
  • the added target antige may have the enzyme bound to it in which case the antibod has one of its specificities directed against the sub stance which interacts with the enzyme or against a ' hapte to which the substance is bound.
  • the substance which interacts with the enzyme can itself be another enzyme.
  • one of the enzymes catalyzes production of a product required by the other.
  • the antibody binds both the added target antigen, to which is bound one of the enzymes, and the other enzyme, the product of the first enzymatic reaction is formed in proximity to the second enzyme and can undergo a reaction catalyzed by the latter enzyme before significant diffusion of the product into the surrounding medium can occur.
  • An example of such a process utilizes the two enzymes hexokinase (HK) and glucose-6-phosphate dehydrogenase (G-6-PDH) in the following reaction scheme.
  • HK hexokinase
  • G-6-PDH glucose-6-phosphate dehydrogenase
  • ATP adenosine diphosphate + glucose-6-phosphate
  • the added target antigen will have either HK or G-6-PDH bound to it and the hybrid antibody will have one of its specificities di- rected against the other (or a hapten bearing it).
  • the sample has added to it, in addition to the hybrid antibody and the predetermined amount of enzyme labeled antigen, glucose, ATP and the coenzyme NAD + .
  • the hybrid antibody preferably has it the other enzyme already bound to it. Alternatively, this enzyme can be added to the sample with the other reagents.
  • the amount of NADH formed varies inversely with the amount of native antigen in the sample, i.e., its maximum produc ⁇ tion occurs when there is no target antigen in the sample being assayed. Comparison of the amount of the NADH formed with a control sample permits a qualitative and quantitative determination of the presence of antig in the sample.
  • This kind of. assay can be used to monitor the lev of dilantin or other drugs in serum.
  • t drug is the target antigen.
  • an assay c also be used to detect other serum antigens such as tho associated with tumors or other diseases.
  • In vivo immunodiagnosis can also be performed using hybrid or other dual specificity antibody.
  • the antibod having one specificity against a target antigen such as tumor associated antigen and the second ' against a hapt to which is bound a suitable radionuclide, preferably o which emits o-radiation, is first administered to the hos After a sufficient time has passed during which t antibody has localized at the target site and unbou antibody has been permitted to clear from healthy tiss in the host, the hapten bearing the radionuclide i administered and binds to the localized antibody. After a suitable interval to permit unbound hapten t clear the host, scanning of the host with a suitabl camera is conducted to determine whether there are area in which radiation has been concentrated.
  • This process has several advantages over that .
  • the radionuclide In suc cases, the radionuclide must have a long enough half lif that a sufficient quantity remains after the time neces sary for substantial localization of the antibody at th target site has elapsed.
  • the antibody may be retained for a period of time in th liver or other non-target tissues which are then subjecte to the radiation carried by the antibody.
  • the presen invention permits the use of radio nuclides having shorter half-lives than those used wit monospecific antibodies.
  • the radionuclide bearing hapten Being a relatively smal particle, the radionuclide bearing hapten has a hi mobility ⁇ n vivo and will travel rapidly through the ho and either bind to the antibody which has localized at t target site or clear the body without spending appreciab time in non-target tissue. For this reason, isotopes short half-life can be administered in quantities whi pose the minimum risk to healthy tissue even thou administered in substantial excess.
  • the hapten is an agent to which t radionuclide is directly bound or which will complex wi the radionuclide.
  • a chelating agent for the radionucli bound to a hapten may be used for the latter purpos Those skilled in the art will appreciate that a wi variety of chelating agents and radionuclides are suit for this purpose.
  • Phenylarsenate to which ethylen diaminetetraacetic acid (EDTA) is bound as a chelati agent is a suitable hapten.
  • a radionuclide suit for use with this hapten is U ln.
  • the toxin or drug can itself function as a hapten, it ne not be bound to any other moiety.
  • the hapten can have t radionuclide bound directly to it or the hapten can be, have bound to it, an agent such as a chelating agent whi will form a complex with the radionuclide.
  • a hapten to . which a radionuclide directly bound is 6- 211 At-astato-2-methyl-l,4-napth quinol bis(disodium phosphate) which is described "International Journal of Applied Radiation and Isotopes" 33, 75 (1982).
  • the 211&t is an emitter of ⁇ -radiatio
  • there a numerous suitable radionuclides which can be bound di rectly to haptens or complexed with a hapten by means any of a wide variety of chelating agents.
EP19830901672 1982-04-12 1983-04-12 Antikörper mit zweifacher spezifizität, deren herstellung und verwendungen. Ceased EP0105360A4 (de)

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CH (1) CH672796A5 (de)
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FI (1) FI834529A0 (de)
GB (4) GB2128631B (de)
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WO2004010957A2 (en) 2002-07-31 2004-02-05 Seattle Genetics, Inc. Drug conjugates and their use for treating cancer, an autoimmune disease or an infectious disease
WO2005089269A2 (en) 2004-03-16 2005-09-29 Temple University - Of The Commonwealth System Of Higher Education Substituted phenoxy- and phenylthio- derivatives for treating proliferative disorders
WO2007011968A2 (en) 2005-07-18 2007-01-25 Seattle Genetics, Inc. Beta-glucuronide-linker drug conjugates
EP2260858A2 (de) 2003-11-06 2010-12-15 Seattle Genetics, Inc. Monomethylvalinverbindungen mit Ligandenkonjugationsfähigkeit
EP2377527A1 (de) 2007-01-22 2011-10-19 Genentech, Inc. Abscheidung von Polyelektrolyten und Reinigung von Antikörpern
WO2011133658A1 (en) 2010-04-22 2011-10-27 Boston Medical Center Corporation Compositions and methods for targeting and delivering therapeutics into cells
EP2511299A1 (de) 2005-04-19 2012-10-17 Seattle Genetics, Inc. Humanisierte, anti-CD70-bindende Wirkstoffe und ihre Verwendung
EP2609932A2 (de) 2006-12-01 2013-07-03 Seattle Genetics, Inc. Veränderliche Target-Bindemittel und Verwendungen davon
WO2014144871A1 (en) 2013-03-15 2014-09-18 The Centre For Drug Research And Development Cytotoxic and anti-mitotic compounds, and methods of using the same
WO2015095953A1 (en) 2013-12-27 2015-07-02 The Centre For Drug Research And Development Sulfonamide-containing linkage systems for drug conjugates
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US11730822B2 (en) 2017-03-24 2023-08-22 Seagen Inc. Process for the preparation of glucuronide drug-linkers and intermediates thereof
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EP0710723A1 (de) 1994-05-17 1996-05-08 Bristol-Myers Squibb Company Klonierung und Exprimierung von einem Gen der für Bryodin 1 kodiert von Bryonia dioica
EP2357006A2 (de) 2002-07-31 2011-08-17 Seattle Genetics, Inc. Arzneimittelkonjugate und deren Verwendung zur Behandlung von Krebs, einer Autoimmunerkrankung oder einer Infektionskrankheit
EP2353611A2 (de) 2002-07-31 2011-08-10 Seattle Genetics, Inc. Arzneimittelkonjugate und deren Verwendung zur Behandlung von Krebs, einer Autoimmunerkrankung oder einer Infektionskrankheit
WO2004010957A2 (en) 2002-07-31 2004-02-05 Seattle Genetics, Inc. Drug conjugates and their use for treating cancer, an autoimmune disease or an infectious disease
EP3120861A1 (de) 2003-11-06 2017-01-25 Seattle Genetics, Inc. Zwischenverbindungen zur herstellung von konjugaten von auristatinderivaten mit linkern
EP2260858A2 (de) 2003-11-06 2010-12-15 Seattle Genetics, Inc. Monomethylvalinverbindungen mit Ligandenkonjugationsfähigkeit
EP2478912A1 (de) 2003-11-06 2012-07-25 Seattle Genetics, Inc. Konjugate von Auristatin mit gegen HER2- oder CD22-gerichtete Antikörper und deren therapeutische Verwendung
EP3434275A1 (de) 2003-11-06 2019-01-30 Seattle Genetics, Inc. Auf die verwendung von auristatin-konjugaten mit antikörpern basierter nachweis für krebszellen
EP3858387A1 (de) 2003-11-06 2021-08-04 Seagen Inc. Zur konjugierung an liganden fähige monomethylvalinverbindungen
EP3138832A1 (de) 2004-03-16 2017-03-08 Temple University - Of The Commonwealth System of Higher Education Substituierte phenoxy- und phenylthioderivate zur behandlung proliferativer erkrankungen
WO2005089269A2 (en) 2004-03-16 2005-09-29 Temple University - Of The Commonwealth System Of Higher Education Substituted phenoxy- and phenylthio- derivatives for treating proliferative disorders
EP3505191A1 (de) 2004-11-12 2019-07-03 Seattle Genetics, Inc. Auristatine mit einer aminobenzoesäureeinheit am n-ende
EP2511299A1 (de) 2005-04-19 2012-10-17 Seattle Genetics, Inc. Humanisierte, anti-CD70-bindende Wirkstoffe und ihre Verwendung
WO2007011968A2 (en) 2005-07-18 2007-01-25 Seattle Genetics, Inc. Beta-glucuronide-linker drug conjugates
EP3248613A1 (de) 2005-07-18 2017-11-29 Seattle Genetics, Inc. Beta-glukuronid-linker-wirkstoffkonjugate
EP4026840A1 (de) 2005-07-18 2022-07-13 Seagen Inc. Beta-glukuronid-linker-wirkstoffkonjugate
EP2609932A2 (de) 2006-12-01 2013-07-03 Seattle Genetics, Inc. Veränderliche Target-Bindemittel und Verwendungen davon
EP2377527A1 (de) 2007-01-22 2011-10-19 Genentech, Inc. Abscheidung von Polyelektrolyten und Reinigung von Antikörpern
US11319526B2 (en) 2008-05-02 2022-05-03 Seagen Inc. Methods and compositions for making antibodies and antibody derivatives with reduced core fucosylation
WO2011133658A1 (en) 2010-04-22 2011-10-27 Boston Medical Center Corporation Compositions and methods for targeting and delivering therapeutics into cells
US10918735B2 (en) 2012-12-04 2021-02-16 Massachusetts Institute Of Technology Substituted pyrazino[1′,2′:1,5]pyrrolo[2,3-b]indole-1,4-diones for cancer treatment
WO2014144871A1 (en) 2013-03-15 2014-09-18 The Centre For Drug Research And Development Cytotoxic and anti-mitotic compounds, and methods of using the same
EP3590922A1 (de) 2013-03-15 2020-01-08 Zymeworks Inc. Zytotoxische und antimitotische verbindungen und verfahren zur verwendung davon
WO2015095953A1 (en) 2013-12-27 2015-07-02 The Centre For Drug Research And Development Sulfonamide-containing linkage systems for drug conjugates
WO2016041082A1 (en) 2014-09-17 2016-03-24 CDRD Ventures, Inc. Cytotoxic and anti-mitotic compounds, and methods of using the same
EP4029873A1 (de) 2014-09-17 2022-07-20 Zymeworks Inc. Cytotoxische und antimitotische verbindungen und verfahren zur verwendung davon
EP3165532A2 (de) 2015-11-03 2017-05-10 Industrial Technology Research Institute Auristatin derivate, linker-arzneimittel und ligandenarzneimittelkonjugate
EP3165237A1 (de) 2015-11-03 2017-05-10 Industrial Technology Research Institute Antikörperwirkstoffkonjugat und herstellungsverfahren dafür
US11844839B2 (en) 2016-03-25 2023-12-19 Seagen Inc. Process for the preparation of pegylated drug-linkers and intermediates thereof
US10918627B2 (en) 2016-05-11 2021-02-16 Massachusetts Institute Of Technology Convergent and enantioselective total synthesis of Communesin analogs
US11730822B2 (en) 2017-03-24 2023-08-22 Seagen Inc. Process for the preparation of glucuronide drug-linkers and intermediates thereof
US11932650B2 (en) 2017-05-11 2024-03-19 Massachusetts Institute Of Technology Potent agelastatin derivatives as modulators for cancer invasion and metastasis
US10640508B2 (en) 2017-10-13 2020-05-05 Massachusetts Institute Of Technology Diazene directed modular synthesis of compounds with quaternary carbon centers
WO2020229982A1 (en) 2019-05-10 2020-11-19 Takeda Pharmaceutical Company Limited Antibody drug conjugates
WO2021138264A1 (en) 2019-12-30 2021-07-08 Seagen Inc. Methods of treating cancer with nonfucosylated anti-cd70 antibodies
US11820827B2 (en) 2019-12-30 2023-11-21 Seagen Inc. Methods of treating myelodysplastic syndrome and acute myeloid leukemia with nonfucosylated anti-CD70 antibodies
WO2022097117A1 (en) 2020-11-09 2022-05-12 Takeda Pharmaceutical Company Ltd. Antibody drug conjugates
WO2023278377A1 (en) 2021-06-29 2023-01-05 Seagen Inc. Methods of treating cancer with a combination of a nonfucosylated anti-cd70 antibody and a cd47 antagonist

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JPH0753600A (ja) 1995-02-28
ES521370A0 (es) 1985-04-16
ES537257A0 (es) 1986-04-01
GB2167086A (en) 1986-05-21
AU550486B2 (en) 1986-03-20
ES8607386A1 (es) 1986-05-16
AU1555983A (en) 1983-11-04
FI834529A (fi) 1983-12-09
CH672796A5 (de) 1989-12-29
WO1983003679A1 (en) 1983-10-27
GB2169921B (en) 1987-03-04
ES8606655A1 (es) 1986-04-01
GB2128631A (en) 1984-05-02
JP2562002B2 (ja) 1996-12-11
ES533930A0 (es) 1985-12-01
GB2168998B (en) 1987-03-04
ES8506091A1 (es) 1985-06-16
GB2169921A (en) 1986-07-23
FI834529A0 (fi) 1983-12-09
EP0105360A4 (de) 1986-07-08
IT8320548A0 (it) 1983-04-12
IT1219778B (it) 1990-05-24
GB8332646D0 (en) 1984-01-11
ES8503441A1 (es) 1985-02-16
CA1213229A (en) 1986-10-28
JPH0753119B2 (ja) 1995-06-07
GB2167086B (en) 1987-03-04
ES8504461A1 (es) 1985-04-16
ES527963A0 (es) 1985-02-16
GB2168998A (en) 1986-07-02
GB8530308D0 (en) 1986-01-22
GB8530310D0 (en) 1986-01-22
ES533931A0 (es) 1985-06-16
ES8604424A1 (es) 1986-02-01
ATA901883A (de) 1991-10-15
GB2128631B (en) 1987-02-25
GB8530309D0 (en) 1986-01-22
AT394577B (de) 1992-05-11
ES8603080A1 (es) 1985-12-01
JPS6312276A (ja) 1988-01-19
ES545247A0 (es) 1986-05-16
ES538727A0 (es) 1986-02-01

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