CA1216231A - Bispecific antibody determinants - Google Patents
Bispecific antibody determinantsInfo
- Publication number
- CA1216231A CA1216231A CA000418116A CA418116A CA1216231A CA 1216231 A CA1216231 A CA 1216231A CA 000418116 A CA000418116 A CA 000418116A CA 418116 A CA418116 A CA 418116A CA 1216231 A CA1216231 A CA 1216231A
- Authority
- CA
- Canada
- Prior art keywords
- determinant
- molecule
- bispecific antibody
- antigenic
- composition
- 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.)
- Expired
Links
Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L35/00—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical, and containing at least one other carboxyl radical in the molecule, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Compositions of derivatives of such polymers
- C08L35/04—Homopolymers or copolymers of nitriles
-
- 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
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L55/00—Compositions of homopolymers or copolymers, obtained by polymerisation reactions only involving carbon-to-carbon unsaturated bonds, not provided for in groups C08L23/00 - C08L53/00
- C08L55/02—ABS [Acrylonitrile-Butadiene-Styrene] polymers
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L65/00—Compositions of macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain; Compositions of derivatives of such polymers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/002—Electrode membranes
- C12Q1/003—Functionalisation
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- 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
- G01N33/563—Immunoassay; Biospecific binding assay; Materials therefor involving antibody fragments
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/805—Test papers
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/81—Packaged device or kit
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/817—Enzyme or microbe electrode
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/962—Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/964—Chemistry: molecular biology and microbiology including enzyme-ligand conjugate production, e.g. reducing rate of nonproductive linkage
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/966—Chemistry: molecular biology and microbiology involving an enzyme system with high turnover rate or complement magnified assay, e.g. multi-enzyme systems
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/969—Multiple layering of reactants
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/807—Apparatus included in process claim, e.g. physical support structures
- Y10S436/808—Automated or kit
Abstract
Abstract of the Disclosure A homogenous sample of identical bispecific antibody determinants, each determinant being composed of two L-H
half-molecules linked by disulfide bonds, each L-H
half-molecule being specific for a different antigenic determinant and including at least the F(ab')2 portion of a monoclonal IgG antibody. The bispecific antibody determinants are useful, e.g., in the formation of multilamellar assemblies and enzyme electrodes.
half-molecules linked by disulfide bonds, each L-H
half-molecule being specific for a different antigenic determinant and including at least the F(ab')2 portion of a monoclonal IgG antibody. The bispecific antibody determinants are useful, e.g., in the formation of multilamellar assemblies and enzyme electrodes.
Description
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The IgG antibodies are known to consist of two half-molecules, each consisting of a light (L) chain and a heavy (H) chain. The H chains of the two halves are linked by disulfide bonds, which can be broken by selective redu~tion.
If this step is performed for two different IgG samples, the - half-molecules can be combined to form hybrid antibodies. This h-as been accomplished using intact rabbit globulins; Nisonoff et al. (1964) Science 134, 376-379.
Hybrids have also been formed using the F(ab')2 fragments of IgG antibodies, rather than intact antibodies;
i.e., the F(c) portions of the molecules, which do not provide immunospecificity, are, prior to hybridization, removed by digestion with an appropriate protease such as papain. This procedure has been described in Nisonoff et al. (1960) Arch.
Biochem. Biophys. 89, 230-244 and in Nisonoff and Rivers (1960) Arch. Biochem. Biophys. 93, 460-462. In a later discussion of the first paper Nisonoff wrote, in Current Contents (Nov. 2, 1981) 44, 25:
So far this procedure has had limited application, principally in the staining of cell surfaces with ferritin by using a hybrid of anti-ferritin antibody and antibody to a cell surface antigen. The use of hy~rid antibody has also been considered as a means of bringing a pharmacological agent specifically into contact with a desired tissue surface.
The use of such hybrids for the delivery of cytotoxic drugs has also been suggested in Raso and Griffin (1978) Fed.
Proc. 37, 1350.
(~ lZ't6Z3i Milstein ~1981) Proc. R. Soc~ Lond. B211, 393-412 sugg~sts the possibility of using "monoclonal antibodies as carriers of toxic substances for specific treatment of tumors,"
and states that "(i)t is possible that Fab fragments will be better targeting agents than intact antibody."
Hybrid antibodies have also been formed by fusing two cells, each -capable of producing different antibodies, to make a hybrid cell capable of producing hykrid antibodies. Such a method is described in Schwaber et al. (1974) P.N.A.S. USA 71, 2203-2207, Mouse myeloma cells were fused to human lymphocytes, and the resultant fused cells produced "hybrid antibody molecules containing components of mouse immunoglobulins assembled with human heavy and light chains~"
The human antibody component was not monoclonal, and was undefined.
Schwaber et al. also describes an in vitro experiment in which the mouse and human antibodies were reduced strongly enough to break bonds between L and H chains, and then "allowed to recombine randomly."
In Cotton et al. (1973) Nature 244, 42-43 there is described an experiment in which mouse myeloma cells were fused to rat tumor cells to produce fusions which produced "an extra component" which was "likely ... a hybrid mouse-rat light chain dimer" as well as "non-symmetrical molecules made up of one 25 light chain of each parental type."
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Another paper, Raso et al. (lg~l) Cancer Research 41, 2073-2078, describes the formation of an impure sample of rabbit antibody F(ab')2 fragments against human IgG F(ab')2 fragments; the rabbit antibody fragments were split by reduction and reassembled with antiricin A chain F(ab')2 fragments. The dual specificity dimers were used in targeted drug delivery experiments. The article states:
The 2 types of purified antibodies used for this work were isolated from conventional heteroantisera. Thus, a complicated array of affinity and specificity combination must arise upon annealing these 2 populations.
The advent of homogeneous hybridoma-derived antibodies will afford absolute control over the binding affinities of the constituent halves of a hybrid antibody, and this uniformity should greatly boost their ultimate effectiveness as delivery vehicles.
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The present invention provides a ~o~a~nou6-s-~mple-of identical bispecific antibody determinants, each bispecific determinant being composed of two L-H half molecules linked by disulfide bonds, each L-H half molecule being different from the other and being specific for a different antigenic determinant, and being composed of at least the F~ab')2 ~ortion of a monoclonal IgG antibody.
The bispecific antibody determinants of the invention are made according to the following procedure. Using conventional methods, two different monoclonal IgG antibody samples are produced, each antibody having one of two desired specificities. If desired, each sample is then exposed to an appropriate protease such as papain to cleave off the F(c) portion of the antibody molecules to produce F(ab')2 fragments. Each sample is then subjected to conditions sufficient to break at least some of the disulfide bonds linking the L-H half-molecules so that at least some of the antibodies are split into two half-molecules.
The two samples are then combined under conditions which permit at least some half-molecules of each determinant to chemically combine with at least some half-molecules of the other determinant to form the bispecific antibody determinants of the invention.
The bispecific antibody determinants molecules are then separated from the rest of the mixture. One separation method is contacting the mixture with an affinity matrix containing an antigen capable of specifically binding to either of the two halves of the bispecific antibody determinant, then eluting bound matrix-bound material, and contacting that material with an affinity matrix containing an antigen capable of specifically binding the other half-molecule. The material bound to this second matrix has the required dual specificity.
An alternative separation method can be used in a case where one of the halves of the bispecific antibody determinant has a specificity for an antigenic determinant which is a iZ16~3~L
macromolecule (a molecule having a molecular weight greater than about 1000 daltons). This method involves adding the macromolecular antigenic determinant to the sample containing the bispecific antibody determinant to be purified to form immune complexes which can be separated into subfractions having differen~ molecular weights by, e.g., gel filtration or electrophoresis. The subfraction having a molecular weight equivalent to the mole~ular weight of the complex of the desired bispecific antibody determinant with the macromolecular antigen is separated from the other subfractions, and, if desired, the macromolecular antigen is then removed using conventional methods.
In the drawings, Fig. l is a diagrammatic representation of two different antigenic determinants linked by a bispecific antibody determinant.
Figs. 2 and 3 are diagrammatic represenatations of electrodes employing bispecific antibody determinants.
Fig. 4 is a diagrammatic representation of a self-assembling network employing bispecific antibody determinants.
Fig. 5 is a diagrammatic representation of a multilamellar assembly useful for an assay method.
The bispecific antibody determinants of the invention 2S are useful for a wide range of applications. Referring to Fig.
1, these applications all flow from the ability of these ~L21~iZ3~
determinants to serve as highly specific linkers through specific sites A' and B', of any two antigenic determinants A
and B capable of stimulating antibody production in animals;
e.g., effective proteins, polypeptides, carbohydrates, nucleic acids, or haptens, either free or immobilized on surfaces or particles.
One application of the bispecific antibody determinants of the invention is their use as agents for bonding a desired antigenic entity to a desired surface which has a different antigenic determinant immobiliæed on it. For example, enzymes so immobilized on particles or membranes can be used as solid-state catalysts. Advantages of this type of immobilization over others are that antibodies can be selected which have no adverse effect on enzyme activity, and that pure enzymes can be immobilized from impure mixtures. Bispecific antibody determinants can also be used as highly specific bispecific reagents for immunoassay procedures which are used, e.g., in the diagnosis of medical disorders, or as molecular probes to study the relationships between antigenic determinants in biological systems.
An additional application of the bispecific antibody determinants is their use in electrodes. Currently-used enzyme electrodes frequently employ tissue slices as the enzyme source. For example, electrodes for measuring glutamine have been made using a conventional NH3 electrode in combination with kidney slices as the source of glutaminase, the enzyme ~Z1623~ ~
which breaks down glutamine to produce measurable NH3 ions;
Rechnitz (1981) Science 214, 2~7-2gl.
The present invention provides electrode apparatus for the measurement in a sample of an unknown amount of a substance which is acted on by one or more enzymes to evolve a measurable ion or compound, the ion or compound evolved being a measure of the unknown substance. The electrode apparatus includes means for measuring the measurable ion or compound, and, associated with that means, a membrane having associated therewith a plurality of molecules of each enzyme which acts on the substance to be measured and, bonded to the molecules of each enzyme, a plurality of identical, bispecific antibody determinants. Each determinant is composed of two different L-H half-molecules linked by disulfide bonds, and each half-molecule includes at least the F(ab')2 portion of a monoclonal IgG antibody. One said L-H half-molecule is specific for an antigenic site on the enzyme molecule to which it is bonded and the other half-molecule is specific for an antigenic determinant on the membrane to which the bispecific antibody determinant is bonded to become immobilizably associated with the membrane.
The electrode can be used to measure any substance which can be metabolized by an enzyme or combination of enzymes in a way which produces or consumes a measurable ion or compound such as NH3 , CO2, 2' or H , provided that -~` 1;21~23~L ~
each enzyme can bind specifically to a site on an immobilized bispecific antibody determinant.
The reaction can be one which requires more than one enzyme. It is required in such a case that all of the required enzymes be immobilized on bispecific antibody determinants which are immobilized in the electrode. Figs. 2 and 3 illustrate two modes of enzyme immobilization in a two-enzyme system in which the two enzymes catalyze consecutive reactions in the conversion of a substance to an ion or compound which can be measured by the appropriate ion or ccmpound-specific membrane electrode.
Referring to Fig. 3, membrane 2 of electrode 4 bears, on spacer arms 3 and 5, different haptens A and B, in the desired ratio, to which are immobilized different bispecific antibody determinants having, respectively, hapten-specific sites A' and B'. The second site on each bispecific antibody determinant is specific, respectively, for binding sites on enzymes C and D, which catalyze consecutive steps in the breakdown of the substance to be measured into a measurable compound or ion.
Referring to Fig. 4, membrane 6 of electrode 8 bears, on spacer 7, hapten A, to which is immobilized a bispecific antibody determinant having hapten A-specific site A' and a second site, B', which is specific for binding site B on one of the two enzymes necessary for the breakdown of the substances to be measured into a measurable compound or ion. The second ~2~23i bispecific antibody determinant has a site, C', specific for antigenic binding site C on the first enzyme, and a second site, D', specific for a different antigenic binding site D on the second enzyme required for the production of the measurable compound or ion. The advantage of the arrangement shown in Fig. 4 is that it assures that the two enzymes are closely linked so that the two reactions are efficiently coupled.
Enzyme electrodes made using bispecific antibody determinants possess several advantages over conventional enzyme electrodes. One advantage is their precise self-assembling property: the desired electrode assembly is generated simply by attaching the appropriate hapten or haptens to the membrane (either the electrode membrane or a separate membrane associated with the electrode) and then immersing the hapten-derived membrane into a solution containing the appropriate bispecific antibodies and enzymes. This ease of assembly also means that the electrode can be easily recharged after deterioration has occurred through prolonged use.
Another advantage of the electrodes is also a function of the specificity of the bispecific antibody determinants.
Any given enzyme will possess a number of antigenic sites capable of binding to a specific site of an antibody. However, coupling at many of these sites can cause inactivation of the enzyme. In the case of bispecific monoclonal antibody determinants, this problem is avoided because the determinants are selected so that they couple with the enzyme only at a site which does not cause deactivation of the enzyme.
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A further advantage is that assembly or recharging of the electrode can be done with impure enzyme mixtures beca~se the unique specificity of the bispecific antibody determinants assures the selection of the proper enzymes from the impure mixture.
In some instances the membrane containing the immobilized enzymes can be covered with a second semipermeable membrane to slow the deterioration of the electrode assembly, or the assembly can be stabilized by treatment with glutaraldehyde.
Yet another application for the bispecific antibody determinants is their use in the formation of self-assembling networks for use, e.g., as molecular microcircuits. Such a ne~work is illustrated diagrammatically in Fig. 4, wherein A, B, C, D, E, and F represent antigenic determinants and A', B', C', D', E', F', represent, respectively, corresponding antibody determinants. It can be seen that the number of linked specific determinants is virtually limitless and, further, that the network can be highly complex and in two or three ~0 dimensions. Most importantly, the network, no matter how complex, is entirely self-assembling in a uniquely defined way.
One example of such a self-assembling network is a multilamellar assembly for use, e.g., in chemical assays or in the production of specific chemicals in industrial processes.
Currently used assemblies for assays of substances in, e.g., serum, employ a series of layers of enzymes trapped between ~21623~
membranes of low porosity. The sample containing the substance to be measured is placed on the outer surface of the assembly and allowed to seep down through the layers, interacting successively with the trapped enzymes until, in the bottom layer, measurable result is produced, e.g. a flvorescence or a color change; this result is a measure of the substance being measured in the sample.
The multilamellar assembly of the invention employs bispecific antibody determinants to link two or more enzymes which can be sequentially acting, as illustrated in Fig. 4 (I-IV representing different enzymes). The low-porosity membranes of current assemblies are thus in many instances unnecessary, the spatial relationships among the enzymes already being fixed by théir attachment to bispecific antibody determinants. Furthermore, the use of bispecific antibody determinants to link enzymes enhances the efficiency of the reaction by reducing the diffusion time of intermediates.
- In the multilamellar assemblies of the invention, the antigenic determinants linked by the bispecific antibody determinants are, in some cases, not enzymes but other catalysts e.g., microbial cells. This will be the case in certain industrial processes, for example, in which the goal of the process is not the measurement of a compound but the production of a desired chemical via a series of chemical reactions.
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The following specific examples are intended to more particularly point out the invention, without acting as limitations upon its scope.
Example 1 The following procedure is used to prepare a homogeneous sample of identical bispecific antibody determinants in which each bispecific determinant has a site specific for a unique antigenic site on the enzyme glucose oxidase, and a site specific for a unique antigenic site on the enzyme ~ -galactosidase.
The first step is the preparation of monoclonal antibodies against the two enzymes glucose oxidase and ~-galactosidase. This is done by first immunizing one group of BALB/C mice against each enzyme using standard immunization procedures.
Following immunization, spleen cells of immunized animals are prepared and fused with a derivative of MOPC-21 myeloma cells (SP2/O-Agl4) using the procedure described in Galfre et al. (1981) Methods in Enzymology 73, 3-46. The hybrid cells are selected in hypoxanthine-aminopterin-thymidine medium, cloned, and screened for production of antibodies against the desired enzymes by the method described in Galfre et al. Id. The clones found to produce antibodies against the desired enzyme are then screened to select a clone which Z3~
produces an antibody of the IgG class which has a high affinity for the enzyme and which does not cause inactivation of the enzyme. The clones of interest are stored until use under liquid nitrogen. Antibody is prepared by propagating the cloned cells in spinner flasks in Bulbeccos's modified Eagles' medium containing 5~ fetal calf serum. Alternatively, a higher antibody yield is obtained by the standard technique of growing the cells as ascitic tumors in the peritoneal cavities of pristane-primed mice.
The desired IgG antibodies against glucose oxidase and ~-galactosidase are then purified from medium or ascites fluid by affinity chromatography on protein A-Sepharose, as described in Ey et al. (1978) Immunochemistry 15, 429-436.
Each of the two purified antibodies is then converted to F(ab')2 fragments by treatment with pepsin according to the procedure of Hackett et al. (1981) Immunology 4, 207-215, as follows. Four mg of purified immunoglobulins (IgG), dissolved in 0.1 M acetate buffer, pH 4.6, are incubated with 40 ~g of pepsin at 37C. After 20 hours, the mixture is adjusted to pH
8.1 with Tris buffer, passed through a column of protein A-Sepharose, and then purified by gel filtration on Sephadex* G-50.
The two types of F(ab')2 fragments are then combined to form bispecific determinants, as follows. First, one (either one) of the fragments is subjected to mild reduction with lQ mM mercaptoethylamine hydrochloride at 37C for 1 hour * Trade Mark `A~
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under a nitrogen atmosphere to separate the fragment into half-molecules without breaking the bonds between H and L chains.
The reducing agent is then removed by passing the mixture through a column of Dowex*-50 at pH 5. The effluent is then reacted immediately with 2 mM 5,5'-dithiobis (2-nitrobenzoic acid) in 0.02 M Na phosphate, pH 8.0, and 3 mM EDTA, as described in Raso and Griffin, J. Immunol. (1980) 125, 2610-2616. The Fabl-thionitrobenzoate derivative thus formed is then purified by gel filtration on Sephadex G0100 in 0.2 M
Na phosphate, pH 8Ø The other F(ab')2 fragment is likewise reduced and treated with Dowex-50, and the resulting Fab' derivative is mixed immediately with an equimolar amount of the Fab'-thionitrobenzoate derivative and incubated for 3h at 20C
to form a mixture containing a high yield of identiaal bispecific an~ibody determinants, each determinant being made up of two F(ab')2 L-H half molecules linked by disulfide bonds. To obtain a homogeneous sample of the identical b;specific antibody determinants, the mixture is passed through a column of Sepharose 4B equilibrated with 0.1 M Tris, pH 7.5, the Sepharose having covalently bonded to it ~-galactosidase.
The column is then washed with 0.1 M Tris, pH 7.5, and the anti-R-galactosidase determinants are then eluted with 0.1 M
glycine, pH 2.5, and then neutralized with Tris.
* Trade Mark ~`
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The eluate is then passed through a second column of Sepharose 4B which has glucose oxidase covalently bonded to it by CNBr activation. The col~mn is washed with 0.1 M Tris, pH
7.5, and the bispecific anti-glucose oxidase, anti-~-galactosidase determinants are then eluted with 0.1 M glycine pH 2.5, and then neutralized with Tris. The eluate constit~tes a homogenous sample of the desired identical bispecific antibody determinants.
Example 2 Using the same procedure employed in Example 1, a homogeneous sample of identical bispecific antibody determinants is prepared in which one antibody site is specific for a different antigenic site on the enzyme glucose oxidase from the site for which the bispecific antibody determinant of Example 1 is specific, and in which the second antibody site is specific for an antigenic site on Type I collagen.
Example 3 An enzyme electrode for the measurement of lactose is constructed according to the following procedure. First, a collagen membrane shaped to fit over a commercial 2 electrode is prepared by electrolysis of a collagen fibril suspension using platinum electrodes, as described in Karube et al. (1972) 47, 51-54.
A solution is prepared of the bispecific antibody determinants from Example 2 together with a 10-fold or higher molar excess of glucose oxidase, in 0.1 M phosphate buffer, pH
`23~
7.0; the glucose oxidase need not be pure. The collagen membrane is immersed in this solution and incubated for 1 h at 20C, after which time it is rinsed with buffer and then transferred to a solution containing the antibody from Example 1 together with a 10-fold or higher molar excess of ~-galactosidase in 0.1 M phosphate buffer, where it is incubated at 20C for 1 h. The membrane is then quickly rinsed in buffer and stabilized by immersion in 0.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.0, for 3 minutes.
The membrane is then placed over the oxygen-permeable teflon membrane of the commerical 2 electrode, rendering the electrode ready for use for the measurment of lactose, in a manner analogo~s to the method of measuring sucrose described in Satoh et al. (1976) Biotechnol. and Bioengineering 18, 269-272. A sample containing an unknown amo~nt of lactose is contacted with the membrane, and the immobilized ~-galactosidase catalyzes the breakdown of the lactose into glucose, which is then acted on by the immobilized glucose oxidase to release 2' which is measured as a measure of lactose in the sample.
In the preparation of the membrane described above, molar excesses of enzyme over antibody are employed because ~-galactosidase and glucose oxidase are each composed of several identical subunits. An excess of enzyme assures that, on average, only a single antigenic site on each enzyme molec~le is involved in complex formation. In the preparation f`. ~.`,'.
121623~ -of other electrode using monomeric enzymes, molar excesses of enzymes are not necessary. When equimolar amounts of enzymes and bispeci~ic antibody determinants are used, the reaction can be allowed to proceed in a single stage.
Example 4 The following is a description of an example of the type of assay assembly which employs the production of a colored or fluorescent substance, which can be measured colorimetrically, reflectometrically, or fluorometrically, as a measure of an unknown amount of a substance being assayed.
Fig. 5 is a diagrammatic representation of a colorimetric indicator for lactose. Biotin-substituted regenerated cellulose membrane 10 is used as the support for the immobilized enzymes which participate in the series of reactions by which lactose in a s-ample generates H2O2 to produce a colorimetrically measurable result, which is a measure of the amount of lactose in the sample.
The enzymes are immobilized, as shown in Fig. 5, by being bonded to three different bispecific antibody determinants, prepared according to the procedure described in Example 1. The first determinant has one site, A', specific for an antigenic site on the protein avidin, and the other site, B', specific for an antigenic site on the enzyme horseradish peroxidase. The second determinant has a site, C', specific for a different antigenic site on horseradish peroxidase, and the second site, D', specific for an antigenic .~ .
~6~3~ , site on glucose oxidose. The third determinant has an antibody site E', specific for a different antigenic site on glucose oxidase, and the second site, F', specific for an antigenic ~ site on ~-galactosidase.
Substituted cellulose membrane 10 is prepared by the cyanogen bromide procedure, e.g. Cuatrecasas et al. (1968) Proc. Nat'l. Acad. Sci. USA 61, 636-643, as follows.
Regenerated cellulose membranes are suspended in 0.1 M NaHCO3 at 4C and treated with an equal volume of 2.5% CNBr solution, the pH being continuously adjusted to 11 with 2N NaOH and the temperature kept at 4C. After 8 min, the cellulose membranes are washed with 0.1M NaHCO3 and then with water, 50~ acetone, and finally with 100% acetone. The cellulose membranes are then incubated at 4C for 20h in 0.2M NaHCO3, pH 9, containing 1 mg per ml of -N-biotinyl-L-lysine (Bayer et al.
(1974) Methods in Enzy_oloqy 34B, 265-267), followed by extensive washing with water.
The biotin-substituted cellulose membrane is then immersed in 0.1M phosphate buffer, pH 7.0, and incubated for lh at 20C with approximately equivalent molar amounts of avidin, horseradish peroxidase, and the bispecific antibody determinant having sites A' and B'. The membrane is then rinsed with buffer and transferred to a solution containing an approximately equivalent molar amount of the bispecific antibody determinant having sites C' and D', and a 10-fold molar excess of glucose oxidase. After 1 hour at 20C, the -- lg --23i (~
membrane is rinsed with buffer and transferred to a solution containing an approximately equivalent molar amount of the bispecific antibody determinant having sites E' and F', and a ~ 10-fold molar excess of ~- galactosidase, and incubated at 20C
for lh, followed by rinsing with buffer. If repeated use is anticipated, the membrane is stabilized by immersion in 0.5%
glutaraldehyde in O.lM phosphate buffer, pH 7, for 3 min.
The enzymes used in the above-described procedure need not be pure. In the example described, a molar excess of ~-galactosidase and glucose oxidase was necessary because these enzymes are composed of several identical subunits. In cases where only monomeric enzymes are used, molar excesses of enzymes are not necessary. When equimolar amounts of enzymes and bispecific antibody determinants are used, the reaction can be allowed to proceed in a single stage.
For the determination of lactose, membrane 10 is immersed in or wetted with a sample containing an unknown amount of lactose in O.lM phosphate buffer, p~ 7, and 0.01 o-dianisidine.
As shown in Fig. 5, lactose in the sample first acts on ~-galactosidose to form glucose, which in turn is acted on by glucose oxidase, in the presence of oxygen, to release H2O2, which, with peroxidase, oxidizes o-dianisidine to produce a yellow dye with absorbance at 460 mm. Various other chromogenic or fluorogenic substances can be substituted for o-dianisidine.
What is claimed is:
The IgG antibodies are known to consist of two half-molecules, each consisting of a light (L) chain and a heavy (H) chain. The H chains of the two halves are linked by disulfide bonds, which can be broken by selective redu~tion.
If this step is performed for two different IgG samples, the - half-molecules can be combined to form hybrid antibodies. This h-as been accomplished using intact rabbit globulins; Nisonoff et al. (1964) Science 134, 376-379.
Hybrids have also been formed using the F(ab')2 fragments of IgG antibodies, rather than intact antibodies;
i.e., the F(c) portions of the molecules, which do not provide immunospecificity, are, prior to hybridization, removed by digestion with an appropriate protease such as papain. This procedure has been described in Nisonoff et al. (1960) Arch.
Biochem. Biophys. 89, 230-244 and in Nisonoff and Rivers (1960) Arch. Biochem. Biophys. 93, 460-462. In a later discussion of the first paper Nisonoff wrote, in Current Contents (Nov. 2, 1981) 44, 25:
So far this procedure has had limited application, principally in the staining of cell surfaces with ferritin by using a hybrid of anti-ferritin antibody and antibody to a cell surface antigen. The use of hy~rid antibody has also been considered as a means of bringing a pharmacological agent specifically into contact with a desired tissue surface.
The use of such hybrids for the delivery of cytotoxic drugs has also been suggested in Raso and Griffin (1978) Fed.
Proc. 37, 1350.
(~ lZ't6Z3i Milstein ~1981) Proc. R. Soc~ Lond. B211, 393-412 sugg~sts the possibility of using "monoclonal antibodies as carriers of toxic substances for specific treatment of tumors,"
and states that "(i)t is possible that Fab fragments will be better targeting agents than intact antibody."
Hybrid antibodies have also been formed by fusing two cells, each -capable of producing different antibodies, to make a hybrid cell capable of producing hykrid antibodies. Such a method is described in Schwaber et al. (1974) P.N.A.S. USA 71, 2203-2207, Mouse myeloma cells were fused to human lymphocytes, and the resultant fused cells produced "hybrid antibody molecules containing components of mouse immunoglobulins assembled with human heavy and light chains~"
The human antibody component was not monoclonal, and was undefined.
Schwaber et al. also describes an in vitro experiment in which the mouse and human antibodies were reduced strongly enough to break bonds between L and H chains, and then "allowed to recombine randomly."
In Cotton et al. (1973) Nature 244, 42-43 there is described an experiment in which mouse myeloma cells were fused to rat tumor cells to produce fusions which produced "an extra component" which was "likely ... a hybrid mouse-rat light chain dimer" as well as "non-symmetrical molecules made up of one 25 light chain of each parental type."
~ lZ1~3~ ~
Another paper, Raso et al. (lg~l) Cancer Research 41, 2073-2078, describes the formation of an impure sample of rabbit antibody F(ab')2 fragments against human IgG F(ab')2 fragments; the rabbit antibody fragments were split by reduction and reassembled with antiricin A chain F(ab')2 fragments. The dual specificity dimers were used in targeted drug delivery experiments. The article states:
The 2 types of purified antibodies used for this work were isolated from conventional heteroantisera. Thus, a complicated array of affinity and specificity combination must arise upon annealing these 2 populations.
The advent of homogeneous hybridoma-derived antibodies will afford absolute control over the binding affinities of the constituent halves of a hybrid antibody, and this uniformity should greatly boost their ultimate effectiveness as delivery vehicles.
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The present invention provides a ~o~a~nou6-s-~mple-of identical bispecific antibody determinants, each bispecific determinant being composed of two L-H half molecules linked by disulfide bonds, each L-H half molecule being different from the other and being specific for a different antigenic determinant, and being composed of at least the F~ab')2 ~ortion of a monoclonal IgG antibody.
The bispecific antibody determinants of the invention are made according to the following procedure. Using conventional methods, two different monoclonal IgG antibody samples are produced, each antibody having one of two desired specificities. If desired, each sample is then exposed to an appropriate protease such as papain to cleave off the F(c) portion of the antibody molecules to produce F(ab')2 fragments. Each sample is then subjected to conditions sufficient to break at least some of the disulfide bonds linking the L-H half-molecules so that at least some of the antibodies are split into two half-molecules.
The two samples are then combined under conditions which permit at least some half-molecules of each determinant to chemically combine with at least some half-molecules of the other determinant to form the bispecific antibody determinants of the invention.
The bispecific antibody determinants molecules are then separated from the rest of the mixture. One separation method is contacting the mixture with an affinity matrix containing an antigen capable of specifically binding to either of the two halves of the bispecific antibody determinant, then eluting bound matrix-bound material, and contacting that material with an affinity matrix containing an antigen capable of specifically binding the other half-molecule. The material bound to this second matrix has the required dual specificity.
An alternative separation method can be used in a case where one of the halves of the bispecific antibody determinant has a specificity for an antigenic determinant which is a iZ16~3~L
macromolecule (a molecule having a molecular weight greater than about 1000 daltons). This method involves adding the macromolecular antigenic determinant to the sample containing the bispecific antibody determinant to be purified to form immune complexes which can be separated into subfractions having differen~ molecular weights by, e.g., gel filtration or electrophoresis. The subfraction having a molecular weight equivalent to the mole~ular weight of the complex of the desired bispecific antibody determinant with the macromolecular antigen is separated from the other subfractions, and, if desired, the macromolecular antigen is then removed using conventional methods.
In the drawings, Fig. l is a diagrammatic representation of two different antigenic determinants linked by a bispecific antibody determinant.
Figs. 2 and 3 are diagrammatic represenatations of electrodes employing bispecific antibody determinants.
Fig. 4 is a diagrammatic representation of a self-assembling network employing bispecific antibody determinants.
Fig. 5 is a diagrammatic representation of a multilamellar assembly useful for an assay method.
The bispecific antibody determinants of the invention 2S are useful for a wide range of applications. Referring to Fig.
1, these applications all flow from the ability of these ~L21~iZ3~
determinants to serve as highly specific linkers through specific sites A' and B', of any two antigenic determinants A
and B capable of stimulating antibody production in animals;
e.g., effective proteins, polypeptides, carbohydrates, nucleic acids, or haptens, either free or immobilized on surfaces or particles.
One application of the bispecific antibody determinants of the invention is their use as agents for bonding a desired antigenic entity to a desired surface which has a different antigenic determinant immobiliæed on it. For example, enzymes so immobilized on particles or membranes can be used as solid-state catalysts. Advantages of this type of immobilization over others are that antibodies can be selected which have no adverse effect on enzyme activity, and that pure enzymes can be immobilized from impure mixtures. Bispecific antibody determinants can also be used as highly specific bispecific reagents for immunoassay procedures which are used, e.g., in the diagnosis of medical disorders, or as molecular probes to study the relationships between antigenic determinants in biological systems.
An additional application of the bispecific antibody determinants is their use in electrodes. Currently-used enzyme electrodes frequently employ tissue slices as the enzyme source. For example, electrodes for measuring glutamine have been made using a conventional NH3 electrode in combination with kidney slices as the source of glutaminase, the enzyme ~Z1623~ ~
which breaks down glutamine to produce measurable NH3 ions;
Rechnitz (1981) Science 214, 2~7-2gl.
The present invention provides electrode apparatus for the measurement in a sample of an unknown amount of a substance which is acted on by one or more enzymes to evolve a measurable ion or compound, the ion or compound evolved being a measure of the unknown substance. The electrode apparatus includes means for measuring the measurable ion or compound, and, associated with that means, a membrane having associated therewith a plurality of molecules of each enzyme which acts on the substance to be measured and, bonded to the molecules of each enzyme, a plurality of identical, bispecific antibody determinants. Each determinant is composed of two different L-H half-molecules linked by disulfide bonds, and each half-molecule includes at least the F(ab')2 portion of a monoclonal IgG antibody. One said L-H half-molecule is specific for an antigenic site on the enzyme molecule to which it is bonded and the other half-molecule is specific for an antigenic determinant on the membrane to which the bispecific antibody determinant is bonded to become immobilizably associated with the membrane.
The electrode can be used to measure any substance which can be metabolized by an enzyme or combination of enzymes in a way which produces or consumes a measurable ion or compound such as NH3 , CO2, 2' or H , provided that -~` 1;21~23~L ~
each enzyme can bind specifically to a site on an immobilized bispecific antibody determinant.
The reaction can be one which requires more than one enzyme. It is required in such a case that all of the required enzymes be immobilized on bispecific antibody determinants which are immobilized in the electrode. Figs. 2 and 3 illustrate two modes of enzyme immobilization in a two-enzyme system in which the two enzymes catalyze consecutive reactions in the conversion of a substance to an ion or compound which can be measured by the appropriate ion or ccmpound-specific membrane electrode.
Referring to Fig. 3, membrane 2 of electrode 4 bears, on spacer arms 3 and 5, different haptens A and B, in the desired ratio, to which are immobilized different bispecific antibody determinants having, respectively, hapten-specific sites A' and B'. The second site on each bispecific antibody determinant is specific, respectively, for binding sites on enzymes C and D, which catalyze consecutive steps in the breakdown of the substance to be measured into a measurable compound or ion.
Referring to Fig. 4, membrane 6 of electrode 8 bears, on spacer 7, hapten A, to which is immobilized a bispecific antibody determinant having hapten A-specific site A' and a second site, B', which is specific for binding site B on one of the two enzymes necessary for the breakdown of the substances to be measured into a measurable compound or ion. The second ~2~23i bispecific antibody determinant has a site, C', specific for antigenic binding site C on the first enzyme, and a second site, D', specific for a different antigenic binding site D on the second enzyme required for the production of the measurable compound or ion. The advantage of the arrangement shown in Fig. 4 is that it assures that the two enzymes are closely linked so that the two reactions are efficiently coupled.
Enzyme electrodes made using bispecific antibody determinants possess several advantages over conventional enzyme electrodes. One advantage is their precise self-assembling property: the desired electrode assembly is generated simply by attaching the appropriate hapten or haptens to the membrane (either the electrode membrane or a separate membrane associated with the electrode) and then immersing the hapten-derived membrane into a solution containing the appropriate bispecific antibodies and enzymes. This ease of assembly also means that the electrode can be easily recharged after deterioration has occurred through prolonged use.
Another advantage of the electrodes is also a function of the specificity of the bispecific antibody determinants.
Any given enzyme will possess a number of antigenic sites capable of binding to a specific site of an antibody. However, coupling at many of these sites can cause inactivation of the enzyme. In the case of bispecific monoclonal antibody determinants, this problem is avoided because the determinants are selected so that they couple with the enzyme only at a site which does not cause deactivation of the enzyme.
6~
A further advantage is that assembly or recharging of the electrode can be done with impure enzyme mixtures beca~se the unique specificity of the bispecific antibody determinants assures the selection of the proper enzymes from the impure mixture.
In some instances the membrane containing the immobilized enzymes can be covered with a second semipermeable membrane to slow the deterioration of the electrode assembly, or the assembly can be stabilized by treatment with glutaraldehyde.
Yet another application for the bispecific antibody determinants is their use in the formation of self-assembling networks for use, e.g., as molecular microcircuits. Such a ne~work is illustrated diagrammatically in Fig. 4, wherein A, B, C, D, E, and F represent antigenic determinants and A', B', C', D', E', F', represent, respectively, corresponding antibody determinants. It can be seen that the number of linked specific determinants is virtually limitless and, further, that the network can be highly complex and in two or three ~0 dimensions. Most importantly, the network, no matter how complex, is entirely self-assembling in a uniquely defined way.
One example of such a self-assembling network is a multilamellar assembly for use, e.g., in chemical assays or in the production of specific chemicals in industrial processes.
Currently used assemblies for assays of substances in, e.g., serum, employ a series of layers of enzymes trapped between ~21623~
membranes of low porosity. The sample containing the substance to be measured is placed on the outer surface of the assembly and allowed to seep down through the layers, interacting successively with the trapped enzymes until, in the bottom layer, measurable result is produced, e.g. a flvorescence or a color change; this result is a measure of the substance being measured in the sample.
The multilamellar assembly of the invention employs bispecific antibody determinants to link two or more enzymes which can be sequentially acting, as illustrated in Fig. 4 (I-IV representing different enzymes). The low-porosity membranes of current assemblies are thus in many instances unnecessary, the spatial relationships among the enzymes already being fixed by théir attachment to bispecific antibody determinants. Furthermore, the use of bispecific antibody determinants to link enzymes enhances the efficiency of the reaction by reducing the diffusion time of intermediates.
- In the multilamellar assemblies of the invention, the antigenic determinants linked by the bispecific antibody determinants are, in some cases, not enzymes but other catalysts e.g., microbial cells. This will be the case in certain industrial processes, for example, in which the goal of the process is not the measurement of a compound but the production of a desired chemical via a series of chemical reactions.
~2~3~
The following specific examples are intended to more particularly point out the invention, without acting as limitations upon its scope.
Example 1 The following procedure is used to prepare a homogeneous sample of identical bispecific antibody determinants in which each bispecific determinant has a site specific for a unique antigenic site on the enzyme glucose oxidase, and a site specific for a unique antigenic site on the enzyme ~ -galactosidase.
The first step is the preparation of monoclonal antibodies against the two enzymes glucose oxidase and ~-galactosidase. This is done by first immunizing one group of BALB/C mice against each enzyme using standard immunization procedures.
Following immunization, spleen cells of immunized animals are prepared and fused with a derivative of MOPC-21 myeloma cells (SP2/O-Agl4) using the procedure described in Galfre et al. (1981) Methods in Enzymology 73, 3-46. The hybrid cells are selected in hypoxanthine-aminopterin-thymidine medium, cloned, and screened for production of antibodies against the desired enzymes by the method described in Galfre et al. Id. The clones found to produce antibodies against the desired enzyme are then screened to select a clone which Z3~
produces an antibody of the IgG class which has a high affinity for the enzyme and which does not cause inactivation of the enzyme. The clones of interest are stored until use under liquid nitrogen. Antibody is prepared by propagating the cloned cells in spinner flasks in Bulbeccos's modified Eagles' medium containing 5~ fetal calf serum. Alternatively, a higher antibody yield is obtained by the standard technique of growing the cells as ascitic tumors in the peritoneal cavities of pristane-primed mice.
The desired IgG antibodies against glucose oxidase and ~-galactosidase are then purified from medium or ascites fluid by affinity chromatography on protein A-Sepharose, as described in Ey et al. (1978) Immunochemistry 15, 429-436.
Each of the two purified antibodies is then converted to F(ab')2 fragments by treatment with pepsin according to the procedure of Hackett et al. (1981) Immunology 4, 207-215, as follows. Four mg of purified immunoglobulins (IgG), dissolved in 0.1 M acetate buffer, pH 4.6, are incubated with 40 ~g of pepsin at 37C. After 20 hours, the mixture is adjusted to pH
8.1 with Tris buffer, passed through a column of protein A-Sepharose, and then purified by gel filtration on Sephadex* G-50.
The two types of F(ab')2 fragments are then combined to form bispecific determinants, as follows. First, one (either one) of the fragments is subjected to mild reduction with lQ mM mercaptoethylamine hydrochloride at 37C for 1 hour * Trade Mark `A~
` ~21~23~
under a nitrogen atmosphere to separate the fragment into half-molecules without breaking the bonds between H and L chains.
The reducing agent is then removed by passing the mixture through a column of Dowex*-50 at pH 5. The effluent is then reacted immediately with 2 mM 5,5'-dithiobis (2-nitrobenzoic acid) in 0.02 M Na phosphate, pH 8.0, and 3 mM EDTA, as described in Raso and Griffin, J. Immunol. (1980) 125, 2610-2616. The Fabl-thionitrobenzoate derivative thus formed is then purified by gel filtration on Sephadex G0100 in 0.2 M
Na phosphate, pH 8Ø The other F(ab')2 fragment is likewise reduced and treated with Dowex-50, and the resulting Fab' derivative is mixed immediately with an equimolar amount of the Fab'-thionitrobenzoate derivative and incubated for 3h at 20C
to form a mixture containing a high yield of identiaal bispecific an~ibody determinants, each determinant being made up of two F(ab')2 L-H half molecules linked by disulfide bonds. To obtain a homogeneous sample of the identical b;specific antibody determinants, the mixture is passed through a column of Sepharose 4B equilibrated with 0.1 M Tris, pH 7.5, the Sepharose having covalently bonded to it ~-galactosidase.
The column is then washed with 0.1 M Tris, pH 7.5, and the anti-R-galactosidase determinants are then eluted with 0.1 M
glycine, pH 2.5, and then neutralized with Tris.
* Trade Mark ~`
3~
The eluate is then passed through a second column of Sepharose 4B which has glucose oxidase covalently bonded to it by CNBr activation. The col~mn is washed with 0.1 M Tris, pH
7.5, and the bispecific anti-glucose oxidase, anti-~-galactosidase determinants are then eluted with 0.1 M glycine pH 2.5, and then neutralized with Tris. The eluate constit~tes a homogenous sample of the desired identical bispecific antibody determinants.
Example 2 Using the same procedure employed in Example 1, a homogeneous sample of identical bispecific antibody determinants is prepared in which one antibody site is specific for a different antigenic site on the enzyme glucose oxidase from the site for which the bispecific antibody determinant of Example 1 is specific, and in which the second antibody site is specific for an antigenic site on Type I collagen.
Example 3 An enzyme electrode for the measurement of lactose is constructed according to the following procedure. First, a collagen membrane shaped to fit over a commercial 2 electrode is prepared by electrolysis of a collagen fibril suspension using platinum electrodes, as described in Karube et al. (1972) 47, 51-54.
A solution is prepared of the bispecific antibody determinants from Example 2 together with a 10-fold or higher molar excess of glucose oxidase, in 0.1 M phosphate buffer, pH
`23~
7.0; the glucose oxidase need not be pure. The collagen membrane is immersed in this solution and incubated for 1 h at 20C, after which time it is rinsed with buffer and then transferred to a solution containing the antibody from Example 1 together with a 10-fold or higher molar excess of ~-galactosidase in 0.1 M phosphate buffer, where it is incubated at 20C for 1 h. The membrane is then quickly rinsed in buffer and stabilized by immersion in 0.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.0, for 3 minutes.
The membrane is then placed over the oxygen-permeable teflon membrane of the commerical 2 electrode, rendering the electrode ready for use for the measurment of lactose, in a manner analogo~s to the method of measuring sucrose described in Satoh et al. (1976) Biotechnol. and Bioengineering 18, 269-272. A sample containing an unknown amo~nt of lactose is contacted with the membrane, and the immobilized ~-galactosidase catalyzes the breakdown of the lactose into glucose, which is then acted on by the immobilized glucose oxidase to release 2' which is measured as a measure of lactose in the sample.
In the preparation of the membrane described above, molar excesses of enzyme over antibody are employed because ~-galactosidase and glucose oxidase are each composed of several identical subunits. An excess of enzyme assures that, on average, only a single antigenic site on each enzyme molec~le is involved in complex formation. In the preparation f`. ~.`,'.
121623~ -of other electrode using monomeric enzymes, molar excesses of enzymes are not necessary. When equimolar amounts of enzymes and bispeci~ic antibody determinants are used, the reaction can be allowed to proceed in a single stage.
Example 4 The following is a description of an example of the type of assay assembly which employs the production of a colored or fluorescent substance, which can be measured colorimetrically, reflectometrically, or fluorometrically, as a measure of an unknown amount of a substance being assayed.
Fig. 5 is a diagrammatic representation of a colorimetric indicator for lactose. Biotin-substituted regenerated cellulose membrane 10 is used as the support for the immobilized enzymes which participate in the series of reactions by which lactose in a s-ample generates H2O2 to produce a colorimetrically measurable result, which is a measure of the amount of lactose in the sample.
The enzymes are immobilized, as shown in Fig. 5, by being bonded to three different bispecific antibody determinants, prepared according to the procedure described in Example 1. The first determinant has one site, A', specific for an antigenic site on the protein avidin, and the other site, B', specific for an antigenic site on the enzyme horseradish peroxidase. The second determinant has a site, C', specific for a different antigenic site on horseradish peroxidase, and the second site, D', specific for an antigenic .~ .
~6~3~ , site on glucose oxidose. The third determinant has an antibody site E', specific for a different antigenic site on glucose oxidase, and the second site, F', specific for an antigenic ~ site on ~-galactosidase.
Substituted cellulose membrane 10 is prepared by the cyanogen bromide procedure, e.g. Cuatrecasas et al. (1968) Proc. Nat'l. Acad. Sci. USA 61, 636-643, as follows.
Regenerated cellulose membranes are suspended in 0.1 M NaHCO3 at 4C and treated with an equal volume of 2.5% CNBr solution, the pH being continuously adjusted to 11 with 2N NaOH and the temperature kept at 4C. After 8 min, the cellulose membranes are washed with 0.1M NaHCO3 and then with water, 50~ acetone, and finally with 100% acetone. The cellulose membranes are then incubated at 4C for 20h in 0.2M NaHCO3, pH 9, containing 1 mg per ml of -N-biotinyl-L-lysine (Bayer et al.
(1974) Methods in Enzy_oloqy 34B, 265-267), followed by extensive washing with water.
The biotin-substituted cellulose membrane is then immersed in 0.1M phosphate buffer, pH 7.0, and incubated for lh at 20C with approximately equivalent molar amounts of avidin, horseradish peroxidase, and the bispecific antibody determinant having sites A' and B'. The membrane is then rinsed with buffer and transferred to a solution containing an approximately equivalent molar amount of the bispecific antibody determinant having sites C' and D', and a 10-fold molar excess of glucose oxidase. After 1 hour at 20C, the -- lg --23i (~
membrane is rinsed with buffer and transferred to a solution containing an approximately equivalent molar amount of the bispecific antibody determinant having sites E' and F', and a ~ 10-fold molar excess of ~- galactosidase, and incubated at 20C
for lh, followed by rinsing with buffer. If repeated use is anticipated, the membrane is stabilized by immersion in 0.5%
glutaraldehyde in O.lM phosphate buffer, pH 7, for 3 min.
The enzymes used in the above-described procedure need not be pure. In the example described, a molar excess of ~-galactosidase and glucose oxidase was necessary because these enzymes are composed of several identical subunits. In cases where only monomeric enzymes are used, molar excesses of enzymes are not necessary. When equimolar amounts of enzymes and bispecific antibody determinants are used, the reaction can be allowed to proceed in a single stage.
For the determination of lactose, membrane 10 is immersed in or wetted with a sample containing an unknown amount of lactose in O.lM phosphate buffer, p~ 7, and 0.01 o-dianisidine.
As shown in Fig. 5, lactose in the sample first acts on ~-galactosidose to form glucose, which in turn is acted on by glucose oxidase, in the presence of oxygen, to release H2O2, which, with peroxidase, oxidizes o-dianisidine to produce a yellow dye with absorbance at 460 mm. Various other chromogenic or fluorogenic substances can be substituted for o-dianisidine.
What is claimed is:
Claims (14)
PROPERTY OF PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A composition of identical bispecific antibody deter-minants, each said determinant comprising two L-H half-molecules linked by disulfide bonds, each said L-H half-molecule being specific for a different antigenic determinant, and comprising at least the F(ab')2 portion of a monoclonal IgG antibody.
2. A method of preparing a composition of identical bispecific antibody determinants, said method comprising the steps of providing samples of two different monoclonal IgG antibody determinants, each said determinant comprising two identical L-H
half-molecules linked by disulfide bonds, each said L-H molecule comprising at least the F(ab')2 portion of said monoclonal IgG
antibody, subjecting said antibody determinants in each sample to conditions sufficient to break at least some of said disulfide bonds linking said L-H half-molecules, whereby at least some of said determinants in each said sample are split into two half-molecules, combining said samples under conditions which permit at least some half-molecules of each determinant to chemically combine with at least some half-molecule of the other said determinant to form said identical bispecific antibody determinants, and separating said identical bispecific antibody determinants from said mixture.
half-molecules linked by disulfide bonds, each said L-H molecule comprising at least the F(ab')2 portion of said monoclonal IgG
antibody, subjecting said antibody determinants in each sample to conditions sufficient to break at least some of said disulfide bonds linking said L-H half-molecules, whereby at least some of said determinants in each said sample are split into two half-molecules, combining said samples under conditions which permit at least some half-molecules of each determinant to chemically combine with at least some half-molecule of the other said determinant to form said identical bispecific antibody determinants, and separating said identical bispecific antibody determinants from said mixture.
3. The composition of claim 1 wherein at least one of said antigenic determinants is a protein.
4. The composition of claim 3 wherein said protein is an enzyme.
5. The composition of claim 1 wherein one said antigenic determinant comprises an antigenic site on a solid matrix, whereby said bispecific antibody determinant is capable of being immobilized on said solid matrix by binding to said matrix at said antigenic site.
6. The composition of claim 5 wherein said other antigenic determinant is an antigenic site on an enzyme.
7. The composition of claim 5, wherein said antigenic site on said matrix is a site on a haptenic molecule attached to said matrix, said bispecific antibody determinant is bonded to said haptenic molecule, the other said antigenic determinant comprises a first anti-genic site on a first protein molecule, said bispecific antibody determinant being bonded to said protein molecule, and there is bonded to said first protein molecule, at a second antigenic site on said protein molecule, a second bispecific antibody determinant different from the determinant bonded to said haptenic molecule, each said second determinant comprising two L-H half-molecules linked by disulfide bonds, each said L-H
half molecule being specific for a different antigenic determinant, one said antigenic determinant being a second antigenic site on said first protein molecule, each said half-molecule comprising at least the F(ab')2 portion of a monoclonal IgG antibody.
half molecule being specific for a different antigenic determinant, one said antigenic determinant being a second antigenic site on said first protein molecule, each said half-molecule comprising at least the F(ab')2 portion of a monoclonal IgG antibody.
8. The composition of claim 7 wherein the other said antigenic determinant for which said second bispecific antibody determinant is specific is an antigenic site on a second protein molecule.
9. The composition of claim 8 wherein each said first and second protein is an enzyme.
10. The composition of claim 9 for the measurement of a substance, wherein said enzymes participate in a series of reactions which result in the production, from said substance, of a measurable effect which is a measure of said substance.
11. The composition of claim 1 wherein one half-molecule of each said bispecific antibody determinant is specific for an antigenic site on .beta.-galactosidase and the other half-molecule is specific for an antigenic site on glucose oxidase.
12. The composition of claim 1 wherein one half-molecule of said bispecific antibody determinant is specific for an antigenic site on glucose oxidase and the other half-molecule is specific for an antigenic site on Type I collagen.
13. The composition of claim 1 wherein one half-molecule of each said bispecific antibody determinant is specific for an antigenic site on peroxidase and the other half-molecule is specific for an antigenic site on glucose oxidase.
14. The composition of claim 1 wherein one half-molecule of said bispecific antibody determinant is specific for an antigenic site on peroxidase and the other half-molecule is specific for an antigenic site on avidin.
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US332,881 | 1981-12-21 | ||
US06/332,881 US4444878A (en) | 1981-12-21 | 1981-12-21 | Bispecific antibody determinants |
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---|---|---|---|
CA000418116A Expired CA1216231A (en) | 1981-12-21 | 1982-12-20 | Bispecific antibody determinants |
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---|---|
US (1) | US4444878A (en) |
EP (1) | EP0096076B1 (en) |
JP (2) | JPS58502182A (en) |
AT (1) | ATE21932T1 (en) |
AU (1) | AU549195B2 (en) |
CA (1) | CA1216231A (en) |
DE (2) | DE3249285T1 (en) |
DK (1) | DK379583A (en) |
FI (1) | FI68731C (en) |
GB (1) | GB2123030B (en) |
NO (1) | NO163255C (en) |
WO (1) | WO1983002285A1 (en) |
Families Citing this family (200)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4714681A (en) * | 1981-07-01 | 1987-12-22 | The Board Of Reagents, The University Of Texas System Cancer Center | Quadroma cells and trioma cells and methods for the production of same |
JPS58122459A (en) * | 1982-01-14 | 1983-07-21 | Yatoron:Kk | Measuring method utilizing association of enzyme |
US4659678A (en) * | 1982-09-29 | 1987-04-21 | Serono Diagnostics Limited | Immunoassay of antigens |
US4816567A (en) * | 1983-04-08 | 1989-03-28 | Genentech, Inc. | Recombinant immunoglobin preparations |
GB8314523D0 (en) * | 1983-05-25 | 1983-06-29 | Lowe C R | Diagnostic device |
GB8318575D0 (en) * | 1983-07-08 | 1983-08-10 | Cobbold S P | Antibody preparations |
US4783399A (en) * | 1984-05-04 | 1988-11-08 | Scripps Clinic And Research Foundation | Diagnostic system for the detection of cytomegalovirus |
US4818678A (en) * | 1984-05-04 | 1989-04-04 | Scripps Clinic And Research Foundation | Diagnostic system for the detection of cytomegalovirus |
DE3430905A1 (en) * | 1984-08-22 | 1986-02-27 | Boehringer Mannheim Gmbh, 6800 Mannheim | METHOD FOR DETERMINING AN IMMUNOLOGICALLY BINDABLE SUBSTANCE |
IL78034A (en) * | 1986-03-04 | 1991-08-16 | Univ Ramot | Biosensors comprising antibodies bonded to glassy carbon electrode for immunoassays |
JPH0721478B2 (en) * | 1986-03-31 | 1995-03-08 | 財団法人化学及血清療法研究所 | Working film for immunosensor |
US5260203A (en) * | 1986-09-02 | 1993-11-09 | Enzon, Inc. | Single polypeptide chain binding molecules |
US5869620A (en) * | 1986-09-02 | 1999-02-09 | Enzon, Inc. | Multivalent antigen-binding proteins |
US4946778A (en) * | 1987-09-21 | 1990-08-07 | Genex Corporation | Single polypeptide chain binding molecules |
FR2604092B1 (en) * | 1986-09-19 | 1990-04-13 | Immunotech Sa | IMMUNOREACTIVES FOR TARGETING ANIMAL CELLS FOR VISUALIZATION OR DESTRUCTION IN VIVO |
US4844893A (en) * | 1986-10-07 | 1989-07-04 | Scripps Clinic And Research Foundation | EX vivo effector cell activation for target cell killing |
JP2682859B2 (en) | 1987-07-27 | 1997-11-26 | コモンウェルス サイエンティフィック アンド インダストリアル リサーチ オーガニゼーション | Receptor membrane |
US5086002A (en) * | 1987-09-07 | 1992-02-04 | Agen Biomedical, Ltd. | Erythrocyte agglutination assay |
US5336603A (en) * | 1987-10-02 | 1994-08-09 | Genentech, Inc. | CD4 adheson variants |
US6710169B2 (en) * | 1987-10-02 | 2004-03-23 | Genentech, Inc. | Adheson variants |
US5389523A (en) * | 1988-05-31 | 1995-02-14 | The United States Of Americas, As Represented By The Secretary Of Commerce | Liposome immunoanalysis by flow injection assay |
US5601819A (en) * | 1988-08-11 | 1997-02-11 | The General Hospital Corporation | Bispecific antibodies for selective immune regulation and for selective immune cell binding |
SE8804074D0 (en) * | 1988-11-10 | 1988-11-10 | Pharmacia Ab | SENSOR UNIT AND ITS USE IN BIOSENSOR SYSTEM |
CA2006408A1 (en) * | 1988-12-27 | 1990-06-27 | Susumu Iwasa | Bispecific monoclonal antibody, its production and use |
US5116964A (en) | 1989-02-23 | 1992-05-26 | Genentech, Inc. | Hybrid immunoglobulins |
US6919211B1 (en) * | 1989-06-07 | 2005-07-19 | Affymetrix, Inc. | Polypeptide arrays |
US6406844B1 (en) | 1989-06-07 | 2002-06-18 | Affymetrix, Inc. | Very large scale immobilized polymer synthesis |
US5800992A (en) | 1989-06-07 | 1998-09-01 | Fodor; Stephen P.A. | Method of detecting nucleic acids |
US6955915B2 (en) * | 1989-06-07 | 2005-10-18 | Affymetrix, Inc. | Apparatus comprising polymers |
US6309822B1 (en) | 1989-06-07 | 2001-10-30 | Affymetrix, Inc. | Method for comparing copy number of nucleic acid sequences |
US5744101A (en) | 1989-06-07 | 1998-04-28 | Affymax Technologies N.V. | Photolabile nucleoside protecting groups |
US5424186A (en) | 1989-06-07 | 1995-06-13 | Affymax Technologies N.V. | Very large scale immobilized polymer synthesis |
US6416952B1 (en) | 1989-06-07 | 2002-07-09 | Affymetrix, Inc. | Photolithographic and other means for manufacturing arrays |
US6346413B1 (en) | 1989-06-07 | 2002-02-12 | Affymetrix, Inc. | Polymer arrays |
US6551784B2 (en) | 1989-06-07 | 2003-04-22 | Affymetrix Inc | Method of comparing nucleic acid sequences |
US5143854A (en) * | 1989-06-07 | 1992-09-01 | Affymax Technologies N.V. | Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof |
US5925525A (en) * | 1989-06-07 | 1999-07-20 | Affymetrix, Inc. | Method of identifying nucleotide differences |
US5547839A (en) * | 1989-06-07 | 1996-08-20 | Affymax Technologies N.V. | Sequencing of surface immobilized polymers utilizing microflourescence detection |
US5491097A (en) * | 1989-06-15 | 1996-02-13 | Biocircuits Corporation | Analyte detection with multilayered bioelectronic conductivity sensors |
US5156810A (en) * | 1989-06-15 | 1992-10-20 | Biocircuits Corporation | Biosensors employing electrical, optical and mechanical signals |
US5897861A (en) * | 1989-06-29 | 1999-04-27 | Medarex, Inc. | Bispecific reagents for AIDS therapy |
US5270194A (en) * | 1989-08-31 | 1993-12-14 | Instrumentation Laboratory Spa | Stabilized glucose oxidase from Aspergillus Niger |
US5583003A (en) * | 1989-09-25 | 1996-12-10 | Agen Limited | Agglutination assay |
EP0420151B1 (en) * | 1989-09-27 | 1996-04-10 | Hitachi, Ltd. | Anti-rhodopsin monoclonal antibody and use thereof |
US6506558B1 (en) | 1990-03-07 | 2003-01-14 | Affymetrix Inc. | Very large scale immobilized polymer synthesis |
EP0834576B1 (en) * | 1990-12-06 | 2002-01-16 | Affymetrix, Inc. (a Delaware Corporation) | Detection of nucleic acid sequences |
WO1992018866A1 (en) * | 1991-04-10 | 1992-10-29 | Biosite Diagnostics Incorporated | Novel conjugates and assays for simultaneous detection of multiple ligands |
AU656181B2 (en) * | 1991-05-03 | 1995-01-27 | Pasteur Sanofi Diagnostics | Heterobifunctional antibodies possessing dual catalytic and specific antigen binding properties and methods using them |
JP3951062B2 (en) | 1991-09-19 | 2007-08-01 | ジェネンテック・インコーポレーテッド | Expression of antibody fragments with cysteine present at least as a free thiol in E. coli for the production of bifunctional F (ab ') 2 antibodies |
US6468740B1 (en) | 1992-11-05 | 2002-10-22 | Affymetrix, Inc. | Cyclic and substituted immobilized molecular synthesis |
US6025165A (en) * | 1991-11-25 | 2000-02-15 | Enzon, Inc. | Methods for producing multivalent antigen-binding proteins |
US5635177A (en) | 1992-01-22 | 1997-06-03 | Genentech, Inc. | Protein tyrosine kinase agonist antibodies |
US7381803B1 (en) | 1992-03-27 | 2008-06-03 | Pdl Biopharma, Inc. | Humanized antibodies against CD3 |
US6129914A (en) * | 1992-03-27 | 2000-10-10 | Protein Design Labs, Inc. | Bispecific antibody effective to treat B-cell lymphoma and cell line |
GB9221657D0 (en) * | 1992-10-15 | 1992-11-25 | Scotgen Ltd | Recombinant bispecific antibodies |
GB2286189A (en) * | 1992-10-15 | 1995-08-09 | Scotgen Ltd | Recombinant specific binding protein |
WO1994012520A1 (en) * | 1992-11-20 | 1994-06-09 | Enzon, Inc. | Linker for linked fusion polypeptides |
ATE241642T1 (en) * | 1993-02-04 | 2003-06-15 | Denzyme Aps | IMPROVED METHOD FOR REFOLDING PROTEINS |
WO1995008637A1 (en) * | 1993-09-21 | 1995-03-30 | Washington State University Research Foundation | Immunoassay comprising ligand-conjugated, ion channel receptor immobilized in lipid film |
US5877016A (en) | 1994-03-18 | 1999-03-02 | Genentech, Inc. | Human trk receptors and neurotrophic factor inhibitors |
US6100071A (en) | 1996-05-07 | 2000-08-08 | Genentech, Inc. | Receptors as novel inhibitors of vascular endothelial growth factor activity and processes for their production |
US20020166764A1 (en) * | 1997-08-12 | 2002-11-14 | University Of Southern California | Electrochemical sensor devices and methods for fast, reliable, and sensitive detection and quantitation of analytes |
US6682648B1 (en) | 1997-08-12 | 2004-01-27 | University Of Southern California | Electrochemical reporter system for detecting analytical immunoassay and molecular biology procedures |
AU757800B2 (en) * | 1997-11-21 | 2003-03-06 | Inverness Medical Switzerland Gmbh | Improvements in or relating to electrochemical assays |
AU1539699A (en) | 1997-11-24 | 1999-06-15 | Johnson T. Wong | Methods for treatment of hiv or other infections using a t cell or viral activator and anti-retroviral combination therapy |
US6312689B1 (en) | 1998-07-23 | 2001-11-06 | Millennium Pharmaceuticals, Inc. | Anti-CCR2 antibodies and methods of use therefor |
US6545264B1 (en) | 1998-10-30 | 2003-04-08 | Affymetrix, Inc. | Systems and methods for high performance scanning |
JP5249482B2 (en) * | 1999-06-16 | 2013-07-31 | ボストン・バイオメデイカル・リサーチ・インステイテユート | Immunological control of β-amyloid levels in vivo |
AU2001283304B2 (en) * | 2000-08-11 | 2005-05-05 | Favrille, Inc. | Method and composition for altering a T cell mediated pathology |
US6911204B2 (en) | 2000-08-11 | 2005-06-28 | Favrille, Inc. | Method and composition for altering a B cell mediated pathology |
US7332585B2 (en) * | 2002-04-05 | 2008-02-19 | The Regents Of The California University | Bispecific single chain Fv antibody molecules and methods of use thereof |
US7332580B2 (en) * | 2002-04-05 | 2008-02-19 | The Regents Of The University Of California | Bispecific single chain Fv antibody molecules and methods of use thereof |
TWI353991B (en) | 2003-05-06 | 2011-12-11 | Syntonix Pharmaceuticals Inc | Immunoglobulin chimeric monomer-dimer hybrids |
WO2005035754A1 (en) * | 2003-10-14 | 2005-04-21 | Chugai Seiyaku Kabushiki Kaisha | Double specific antibodies substituting for functional protein |
WO2005035753A1 (en) * | 2003-10-10 | 2005-04-21 | Chugai Seiyaku Kabushiki Kaisha | Double specific antibodies substituting for functional protein |
JP2007521248A (en) | 2003-12-10 | 2007-08-02 | ミレニアム ファーマシューティカルズ, インコーポレイテッド | Humanized anti-CCR2 antibody and methods of using the antibody |
JP2007515493A (en) * | 2003-12-22 | 2007-06-14 | セントカー・インコーポレーテツド | Method for generating multimeric molecules |
DK1866339T3 (en) | 2005-03-25 | 2013-09-02 | Gitr Inc | GTR-binding molecules and their applications |
US10011858B2 (en) | 2005-03-31 | 2018-07-03 | Chugai Seiyaku Kabushiki Kaisha | Methods for producing polypeptides by regulating polypeptide association |
WO2006109592A1 (en) * | 2005-04-08 | 2006-10-19 | Chugai Seiyaku Kabushiki Kaisha | Antibody substituting for function of blood coagulation factor viii |
EP1899376A2 (en) | 2005-06-16 | 2008-03-19 | The Feinstein Institute for Medical Research | Antibodies against hmgb1 and fragments thereof |
EP1907001B1 (en) * | 2005-06-17 | 2015-07-15 | Merck Sharp & Dohme Corp. | Ilt3 binding molecules and uses therefor |
JP2009531324A (en) * | 2006-03-20 | 2009-09-03 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | Engineered anti-prostatic stem cell antigen (PSCA) antibody for cancer targeting |
US11046784B2 (en) | 2006-03-31 | 2021-06-29 | Chugai Seiyaku Kabushiki Kaisha | Methods for controlling blood pharmacokinetics of antibodies |
EP3345616A1 (en) | 2006-03-31 | 2018-07-11 | Chugai Seiyaku Kabushiki Kaisha | Antibody modification method for purifying bispecific antibody |
AU2007345745C1 (en) * | 2006-06-19 | 2013-05-23 | Merck Sharp & Dohme Corp. | ILT3 binding molecules and uses therefor |
US8580263B2 (en) * | 2006-11-21 | 2013-11-12 | The Regents Of The University Of California | Anti-EGFR family antibodies, bispecific anti-EGFR family antibodies and methods of use thereof |
EP2175884B8 (en) * | 2007-07-12 | 2017-02-22 | GITR, Inc. | Combination therapies employing gitr binding molecules |
WO2009032949A2 (en) | 2007-09-04 | 2009-03-12 | The Regents Of The University Of California | High affinity anti-prostate stem cell antigen (psca) antibodies for cancer targeting and detection |
US9096651B2 (en) | 2007-09-26 | 2015-08-04 | Chugai Seiyaku Kabushiki Kaisha | Method of modifying isoelectric point of antibody via amino acid substitution in CDR |
US8317737B2 (en) * | 2009-02-25 | 2012-11-27 | The Invention Science Fund I, Llc | Device for actively removing a target component from blood or lymph of a vertebrate subject |
US8246565B2 (en) * | 2009-02-25 | 2012-08-21 | The Invention Science Fund I, Llc | Device for passively removing a target component from blood or lymph of a vertebrate subject |
MX342623B (en) * | 2009-06-26 | 2016-10-06 | Regeneron Pharma | Readily isolated bispecific antibodies with native immunoglobulin format. |
EP3560962A1 (en) | 2010-07-09 | 2019-10-30 | Bioverativ Therapeutics Inc. | Processable single chain molecules and polypeptides made using same |
HUE038305T2 (en) | 2010-11-17 | 2018-10-29 | Chugai Pharmaceutical Co Ltd | Multi-specific antigen-binding molecule having alternative function to function of blood coagulation factor viii |
US20130245233A1 (en) | 2010-11-24 | 2013-09-19 | Ming Lei | Multispecific Molecules |
LT2717898T (en) | 2011-06-10 | 2019-03-25 | Bioverativ Therapeutics Inc. | Pro-coagulant compounds and methods of use thereof |
WO2013012733A1 (en) | 2011-07-15 | 2013-01-24 | Biogen Idec Ma Inc. | Heterodimeric fc regions, binding molecules comprising same, and methods relating thereto |
MX359384B (en) | 2011-10-11 | 2018-09-25 | Genentech Inc | Improved assembly of bispecific antibodies. |
EP2802606B1 (en) | 2012-01-10 | 2018-04-25 | Biogen MA Inc. | Enhancement of transport of therapeutic molecules across the blood brain barrier |
KR102494534B1 (en) | 2012-03-14 | 2023-02-06 | 리제너론 파마슈티칼스 인코포레이티드 | Multispecific antigen-binding molecules and uses thereof |
SG10201913874TA (en) | 2013-03-15 | 2020-03-30 | Biogen Ma Inc | Factor ix polypeptide formulations |
PT3050896T (en) | 2013-09-27 | 2021-08-24 | Chugai Pharmaceutical Co Ltd | Method for producing polypeptide heteromultimer |
WO2015070014A1 (en) | 2013-11-08 | 2015-05-14 | Biogen Idec Ma Inc. | Procoagulant fusion compound |
JOP20200094A1 (en) | 2014-01-24 | 2017-06-16 | Dana Farber Cancer Inst Inc | Antibody molecules to pd-1 and uses thereof |
JOP20200096A1 (en) | 2014-01-31 | 2017-06-16 | Children’S Medical Center Corp | Antibody molecules to tim-3 and uses thereof |
TWI777174B (en) | 2014-03-14 | 2022-09-11 | 瑞士商諾華公司 | Antibody molecules to lag-3 and uses thereof |
WO2015142675A2 (en) | 2014-03-15 | 2015-09-24 | Novartis Ag | Treatment of cancer using chimeric antigen receptor |
EP3172237A2 (en) | 2014-07-21 | 2017-05-31 | Novartis AG | Treatment of cancer using humanized anti-bcma chimeric antigen receptor |
WO2016014553A1 (en) | 2014-07-21 | 2016-01-28 | Novartis Ag | Sortase synthesized chimeric antigen receptors |
WO2016014530A1 (en) | 2014-07-21 | 2016-01-28 | Novartis Ag | Combinations of low, immune enhancing. doses of mtor inhibitors and cars |
US9777061B2 (en) | 2014-07-21 | 2017-10-03 | Novartis Ag | Treatment of cancer using a CD33 chimeric antigen receptor |
US20170209492A1 (en) | 2014-07-31 | 2017-07-27 | Novartis Ag | Subset-optimized chimeric antigen receptor-containing t-cells |
JP6919118B2 (en) | 2014-08-14 | 2021-08-18 | ノバルティス アーゲー | Treatment of cancer with GFRα-4 chimeric antigen receptor |
BR112017003104A2 (en) | 2014-08-19 | 2017-12-05 | Novartis Ag | cancer treatment using an anti-cd123 chimeric antigen receptor |
US10577417B2 (en) | 2014-09-17 | 2020-03-03 | Novartis Ag | Targeting cytotoxic cells with chimeric receptors for adoptive immunotherapy |
TWI701435B (en) | 2014-09-26 | 2020-08-11 | 日商中外製藥股份有限公司 | Method to determine the reactivity of FVIII |
MA40764A (en) | 2014-09-26 | 2017-08-01 | Chugai Pharmaceutical Co Ltd | THERAPEUTIC AGENT INDUCING CYTOTOXICITY |
TWI700300B (en) | 2014-09-26 | 2020-08-01 | 日商中外製藥股份有限公司 | Antibodies that neutralize substances with the function of FVIII coagulation factor (FVIII) |
TWI716362B (en) | 2014-10-14 | 2021-01-21 | 瑞士商諾華公司 | Antibody molecules to pd-l1 and uses thereof |
US20180334490A1 (en) | 2014-12-03 | 2018-11-22 | Qilong H. Wu | Methods for b cell preconditioning in car therapy |
EP3279216A4 (en) | 2015-04-01 | 2019-06-19 | Chugai Seiyaku Kabushiki Kaisha | Method for producing polypeptide hetero-oligomer |
IL254817B2 (en) | 2015-04-08 | 2023-12-01 | Novartis Ag | Cd20 therapies, cd22 therapies, and combination therapies with a cd19 chimeric antigen receptor (car) - expressing cell |
EP3286211A1 (en) | 2015-04-23 | 2018-02-28 | Novartis AG | Treatment of cancer using chimeric antigen receptor and protein kinase a blocker |
US11191844B2 (en) | 2015-07-06 | 2021-12-07 | Regeneran Pharmaceuticals, Inc. | Multispecific antigen-binding molecules and uses thereof |
WO2017019897A1 (en) | 2015-07-29 | 2017-02-02 | Novartis Ag | Combination therapies comprising antibody molecules to tim-3 |
SI3317301T1 (en) | 2015-07-29 | 2021-10-29 | Novartis Ag | Combination therapies comprising antibody molecules to lag-3 |
RU2018125603A (en) | 2015-12-17 | 2020-01-21 | Новартис Аг | COMBINATION OF C-MET INHIBITOR WITH ANTIBODY MOLECULE TO PD-1 AND ITS APPLICATIONS |
KR20180088907A (en) | 2015-12-17 | 2018-08-07 | 노파르티스 아게 | Antibody molecules to PD-1 and uses thereof |
EP3398965A4 (en) | 2015-12-28 | 2019-09-18 | Chugai Seiyaku Kabushiki Kaisha | Method for promoting efficiency of purification of fc region-containing polypeptide |
WO2017125897A1 (en) | 2016-01-21 | 2017-07-27 | Novartis Ag | Multispecific molecules targeting cll-1 |
KR20180118175A (en) | 2016-03-04 | 2018-10-30 | 노파르티스 아게 | Cells expressing multiple chimeric antigen receptor (CAR) molecules and their uses |
EP3432924A1 (en) | 2016-03-23 | 2019-01-30 | Novartis AG | Cell secreted minibodies and uses thereof |
HRP20230457T1 (en) | 2016-04-15 | 2023-07-21 | Novartis Ag | Compositions and methods for selective expression of chimeric antigen receptors |
EP3448891A1 (en) | 2016-04-28 | 2019-03-06 | Regeneron Pharmaceuticals, Inc. | Methods of making multispecific antigen-binding molecules |
EP3464375A2 (en) | 2016-06-02 | 2019-04-10 | Novartis AG | Therapeutic regimens for chimeric antigen receptor (car)- expressing cells |
CN110461315A (en) | 2016-07-15 | 2019-11-15 | 诺华股份有限公司 | Cytokines release syndrome is treated and prevented using with the Chimeric antigen receptor of kinase inhibitor combination |
BR112019001570A2 (en) | 2016-07-28 | 2019-07-09 | Novartis Ag | chimeric antigen receptor combination therapies and pd-1 inhibitors |
BR112019002035A2 (en) | 2016-08-01 | 2019-05-14 | Novartis Ag | cancer treatment using a chimeric antigen receptor in combination with an inhibitor of a m2 pro-macrophage molecule |
JP7125932B2 (en) | 2016-09-06 | 2022-08-25 | 中外製薬株式会社 | Methods of Using Bispecific Antibodies Recognizing Coagulation Factor IX and/or Activated Coagulation Factor IX and Coagulation Factor X and/or Activated Coagulation Factor X |
US10525083B2 (en) | 2016-10-07 | 2020-01-07 | Novartis Ag | Nucleic acid molecules encoding chimeric antigen receptors comprising a CD20 binding domain |
US11535662B2 (en) | 2017-01-26 | 2022-12-27 | Novartis Ag | CD28 compositions and methods for chimeric antigen receptor therapy |
US20200048359A1 (en) | 2017-02-28 | 2020-02-13 | Novartis Ag | Shp inhibitor compositions and uses for chimeric antigen receptor therapy |
EP3589650A1 (en) | 2017-03-02 | 2020-01-08 | Novartis AG | Engineered heterodimeric proteins |
WO2018201051A1 (en) | 2017-04-28 | 2018-11-01 | Novartis Ag | Bcma-targeting agent, and combination therapy with a gamma secretase inhibitor |
EP3615055A1 (en) | 2017-04-28 | 2020-03-04 | Novartis AG | Cells expressing a bcma-targeting chimeric antigen receptor, and combination therapy with a gamma secretase inhibitor |
JP7348844B2 (en) | 2017-06-07 | 2023-09-21 | リジェネロン・ファーマシューティカルズ・インコーポレイテッド | Compositions and methods for internalizing enzymes |
US11312783B2 (en) | 2017-06-22 | 2022-04-26 | Novartis Ag | Antibody molecules to CD73 and uses thereof |
WO2019006007A1 (en) | 2017-06-27 | 2019-01-03 | Novartis Ag | Dosage regimens for anti-tim-3 antibodies and uses thereof |
SG11201913137VA (en) | 2017-07-11 | 2020-01-30 | Compass Therapeutics Llc | Agonist antibodies that bind human cd137 and uses thereof |
CN111163798A (en) | 2017-07-20 | 2020-05-15 | 诺华股份有限公司 | Dosing regimens for anti-LAG-3 antibodies and uses thereof |
BR112020005834A2 (en) | 2017-09-29 | 2020-09-24 | Chugai Seiyaku Kabushiki Kaisha | multispecific antigen binding molecule having blood clotting factor viii cofactor function (fviii) replacement activity, and pharmaceutical formulation containing said molecule as an active ingredient |
WO2019089753A2 (en) | 2017-10-31 | 2019-05-09 | Compass Therapeutics Llc | Cd137 antibodies and pd-1 antagonists and uses thereof |
WO2019099838A1 (en) | 2017-11-16 | 2019-05-23 | Novartis Ag | Combination therapies |
EP3713961A2 (en) | 2017-11-20 | 2020-09-30 | Compass Therapeutics LLC | Cd137 antibodies and tumor antigen-targeting antibodies and uses thereof |
US20210038659A1 (en) | 2018-01-31 | 2021-02-11 | Novartis Ag | Combination therapy using a chimeric antigen receptor |
US20210147547A1 (en) | 2018-04-13 | 2021-05-20 | Novartis Ag | Dosage Regimens For Anti-Pd-L1 Antibodies And Uses Thereof |
WO2019210153A1 (en) | 2018-04-27 | 2019-10-31 | Novartis Ag | Car t cell therapies with enhanced efficacy |
WO2019226658A1 (en) | 2018-05-21 | 2019-11-28 | Compass Therapeutics Llc | Multispecific antigen-binding compositions and methods of use |
US20200109195A1 (en) | 2018-05-21 | 2020-04-09 | Compass Therapeutics Llc | Compositions and methods for enhancing the killing of target cells by nk cells |
WO2019227003A1 (en) | 2018-05-25 | 2019-11-28 | Novartis Ag | Combination therapy with chimeric antigen receptor (car) therapies |
US20210214459A1 (en) | 2018-05-31 | 2021-07-15 | Novartis Ag | Antibody molecules to cd73 and uses thereof |
SG11202011830SA (en) | 2018-06-13 | 2020-12-30 | Novartis Ag | Bcma chimeric antigen receptors and uses thereof |
US20210238268A1 (en) | 2018-06-19 | 2021-08-05 | Atarga, Llc | Antibody molecules to complement component 5 and uses thereof |
AR116109A1 (en) | 2018-07-10 | 2021-03-31 | Novartis Ag | DERIVATIVES OF 3- (5-AMINO-1-OXOISOINDOLIN-2-IL) PIPERIDINE-2,6-DIONA AND USES OF THE SAME |
WO2020021465A1 (en) | 2018-07-25 | 2020-01-30 | Advanced Accelerator Applications (Italy) S.R.L. | Method of treatment of neuroendocrine tumors |
BR112021008795A2 (en) | 2018-11-13 | 2021-08-31 | Compass Therapeutics Llc | MULTISPECIFIC BINDING CONSTRUCTS AGAINST CHECKPOINT MOLECULES AND THEIR USES |
CN113195539A (en) | 2018-12-20 | 2021-07-30 | 诺华股份有限公司 | Pharmaceutical combination |
EP3897637A1 (en) | 2018-12-20 | 2021-10-27 | Novartis AG | Dosing regimen and pharmaceutical combination comprising 3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives |
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Family Cites Families (14)
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JPS50155678A (en) * | 1974-06-03 | 1975-12-16 | ||
FR2334107A1 (en) * | 1975-12-05 | 1977-07-01 | Pasteur Institut | METHOD OF COUPLING BIOLOGICAL SUBSTANCES BY COVALENT BONDS |
JPS5344622A (en) * | 1976-09-30 | 1978-04-21 | Mochida Pharm Co Ltd | Immunologically measuring method |
US4208479A (en) * | 1977-07-14 | 1980-06-17 | Syva Company | Label modified immunoassays |
JPS5921500B2 (en) * | 1978-01-28 | 1984-05-21 | 東洋紡績株式会社 | Enzyme membrane for oxygen electrode |
JPS5510590A (en) * | 1978-05-04 | 1980-01-25 | Wellcome Found | Enzyme immunity quantity analysis |
US4235869A (en) * | 1978-05-16 | 1980-11-25 | Syva Company | Assay employing a labeled Fab-fragment ligand complex |
FR2437213A1 (en) * | 1978-09-28 | 1980-04-25 | Cm Ind | CYTOTOXIC PRODUCTS FORMED BY COVALENT BINDING OF THE CHAIN TO RICIN WITH AN ANTIBODY AND THEIR PREPARATION METHOD |
US4223005A (en) * | 1979-02-15 | 1980-09-16 | University Of Illinois Foundation | Antibody coated bacteria |
JPS5616418A (en) * | 1979-07-20 | 1981-02-17 | Teijin Ltd | Antitumor protein complex and its preparation |
US4278761A (en) * | 1979-12-26 | 1981-07-14 | President And Fellows Of Harvard College | Enzyme assay and kit therefor |
US4331647A (en) * | 1980-03-03 | 1982-05-25 | Goldenberg Milton David | Tumor localization and therapy with labeled antibody fragments specific to tumor-associated markers |
US4376110A (en) * | 1980-08-04 | 1983-03-08 | Hybritech, Incorporated | Immunometric assays using monoclonal antibodies |
US4474893A (en) * | 1981-07-01 | 1984-10-02 | The University of Texas System Cancer Center | Recombinant monoclonal antibodies |
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1981
- 1981-12-21 US US06/332,881 patent/US4444878A/en not_active Expired - Lifetime
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1982
- 1982-12-20 CA CA000418116A patent/CA1216231A/en not_active Expired
- 1982-12-20 DE DE19823249285 patent/DE3249285T1/en not_active Ceased
- 1982-12-20 AT AT83900528T patent/ATE21932T1/en not_active IP Right Cessation
- 1982-12-20 GB GB08321513A patent/GB2123030B/en not_active Expired
- 1982-12-20 AU AU11571/83A patent/AU549195B2/en not_active Expired
- 1982-12-20 WO PCT/US1982/001766 patent/WO1983002285A1/en active IP Right Grant
- 1982-12-20 DE DE8383900528T patent/DE3273080D1/en not_active Expired
- 1982-12-20 EP EP83900528A patent/EP0096076B1/en not_active Expired
- 1982-12-20 JP JP83500601A patent/JPS58502182A/en active Granted
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1983
- 1983-08-11 FI FI832897A patent/FI68731C/en not_active IP Right Cessation
- 1983-08-19 NO NO83832989A patent/NO163255C/en unknown
- 1983-08-19 DK DK379583A patent/DK379583A/en not_active Application Discontinuation
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1993
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GB8321513D0 (en) | 1983-09-14 |
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ATE21932T1 (en) | 1986-09-15 |
JPH0554066B2 (en) | 1993-08-11 |
DE3273080D1 (en) | 1986-10-09 |
US4444878A (en) | 1984-04-24 |
NO163255B (en) | 1990-01-15 |
GB2123030A (en) | 1984-01-25 |
EP0096076A4 (en) | 1984-05-03 |
GB2123030B (en) | 1985-03-13 |
FI68731C (en) | 1985-10-10 |
NO832989L (en) | 1983-08-19 |
JPH07108919B2 (en) | 1995-11-22 |
AU549195B2 (en) | 1986-01-16 |
EP0096076A1 (en) | 1983-12-21 |
NO163255C (en) | 1990-04-25 |
FI832897A0 (en) | 1983-08-11 |
DK379583D0 (en) | 1983-08-19 |
FI68731B (en) | 1985-06-28 |
FI832897A (en) | 1983-08-11 |
WO1983002285A1 (en) | 1983-07-07 |
JPH0690786A (en) | 1994-04-05 |
JPS58502182A (en) | 1983-12-22 |
DK379583A (en) | 1983-08-19 |
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