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WO1994023298A1 - Determination de la concentration d'un constituant par titrage par affinite et par deplacement par concurrence, utilisee dans l'apport de medicaments - Google Patents

Determination de la concentration d'un constituant par titrage par affinite et par deplacement par concurrence, utilisee dans l'apport de medicaments Download PDF

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Publication number
WO1994023298A1
WO1994023298A1 PCT/US1994/003490 US9403490W WO9423298A1 WO 1994023298 A1 WO1994023298 A1 WO 1994023298A1 US 9403490 W US9403490 W US 9403490W WO 9423298 A1 WO9423298 A1 WO 9423298A1
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WO
WIPO (PCT)
Prior art keywords
analyte
complex
agent
specific binding
binding partner
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/US1994/003490
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English (en)
Inventor
Lawrence M. Kauvar
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Telik Inc
Terrapin Technologies Inc
Original Assignee
Telik Inc
Terrapin Technologies Inc
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from US08/039,786 external-priority patent/US5356784A/en
Application filed by Telik Inc, Terrapin Technologies Inc filed Critical Telik Inc
Priority to EP94913987A priority Critical patent/EP0692096A1/fr
Priority to JP6522341A priority patent/JPH08508568A/ja
Priority to AU66229/94A priority patent/AU693442B2/en
Publication of WO1994023298A1 publication Critical patent/WO1994023298A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • 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

Definitions

  • the invention in one aspect is related to determination of concentration of an analyte in a simplified, field-usable form suitable for use with small sample volumes. Another application of the principles involved in such determination relates to delivery systems wherein competition between a portion of a complex and a releasing agent determines the fate of a complex that includes the substance of interest.
  • the assays of the invention involve a systematically variable competition immunoassay to determine concentration without the necessity for serial dilution, either to place the concentration of analyte within the -1.5 log-unit range of a typical immunoassay or to obtain multiple readings in the dynamic range.
  • The- drug delivery aspect invention concerns use of affinity competition to regulate the availability and/or activity of certain binding moieties in in vivo or in in vi tro environments.
  • serial dilution may accommodate and span a dynamic range wherein variable readings over a series of concentrations is obtained, thus enhancing the precision of the result.
  • the level of dilution itself may be used as an assessment of concentration.
  • immunoassays or other specific binding assays can be used to assess the quantity of an analyte in a sample by using a multiplicity of test regions or portions in combination with serial dilutions of the sample.
  • a variety of test formats is used wherein the same test format is used in these multiple test regions, but the sample containing analyte is used in lower and lower concentrations until a discernible response disappears.
  • concentration of analyte in the original sample can be back- calculated.
  • serial dilution Methods that employ serial dilution are useful, but quite labor- or machine-intensive, and are not suited for semiquantitative determinations as might be needed in testing for analytes outside of a laboratory context. For example, ascertaining the levels of contaminants in soil at the location where field testing is appropriate should be accomplished by methods that require only the application of a single sample volume, rather than the more complex and error-prone process of obtaining multiple dilutions.
  • shortages of trained and reliable personnel manually to conduct serial dilutions .for assessment is a recognized problem in supplying health care; instrumentation to make such dilutions mechanically is expensive and of limited reliability.
  • variable stoichiometric reagent concentrations are used to achieve semiquantitative results in a series of test regions.
  • the assay aspect of the present invention similarly provides a method that permits quantitative analyte concentration determination using a series of test regions without the need for serial dilution.
  • the invention method takes advantage of the variable binding affinity of a multiplicity of ligands either with the analyte itself or with a specific binding partner of the analyte. In either case, the invention takes advantage of a multiplicity of ligands which react with varying degrees of efficacy for a single substance.
  • the tumor is destroyed at the expense of tissues to which the specific affinity reagent is not attracted.
  • techniques are available for specific imaging of particular tissues by combining scintigraphic labels, for example, with agents having specific affinity for their targets.
  • a number of drugs have been modified to enhance their overall half-life by coupling them to polymers such as polyethylene glycol .
  • polymers such as polyethylene glycol .
  • the drug delivery aspect of the present invention provides strategies which improve the precision with which these general approaches can achieve their effects, and is applicable to delivery of substances of interest to targets in both in vivo and in vi tro environments.
  • the invention provides a method which is straightforward and quantitative for simple determination of the concentration of an analyte of interest under field or clinical conditions, and takes advantage of varying affinities of ligands either for analyte or for a specific binding moiety, wherein the specific binding moiety is reactive with the analyte.
  • the reagents offer varying degrees of competition for the specific binding partner, the competitive success of the test sample with regard to an orderly array of competitors can be used as an index of its amount following calibration runs with known concentrations of analyte.
  • the invention is directed to a method to determine the quantity of an analyte in the sample, wherein the method comprises applying the sample to a multiplicity of test regions.
  • test regions contain ligands which have varying affinities for a specific binding partner that is capable of binding the desired analyte.
  • the test regions are arranged sequentially--!.e. , in such a manner that a monotonic increase of affinity of the contained ligands for the specific binding partner can be discerned.
  • this is preferably done in the simplest possible way--e.g., in a linear arrangement wherein ligands of increasing affinity are arranged, for example, from left to right.
  • a quantity of specific binding partner is also supplied to each, and, indeed, can be contained in each test region ab ini tio .
  • the specific binding partner that is not bound to the ligand coupled to the test region is washed away, if necessary. As is known in the art, for some methods of detection, washing is not necessary. The remaining bound specific binding partner is then detected. The portion of test regions containing bound specific binding partner is then used as a measure of the analyte. Intermediate values between all-or-none in each zone provide further precision in quantitation.
  • the results obtained using the method of the present invention are susceptible to fully quantitative analysis if desired or can be interpreted more qualitatively.
  • the precision of the answer obtained can be increased by augmenting the number of and appropriate selection of reagents for the test portions.
  • the multiplicity of test portions is used in a direct, noncompetitive format wherein the series of specific binding ligands are of varying affinities for the analyte itself.
  • the series of specific binding ligands are of varying affinities for the analyte itself.
  • This form of the method of the invention can be conducted either in a kinetically controlled or equilibrium- controlled format. The optimization of the range of binding affinities required to provide quantitative results will be different from that of the competitive format and can be conducted using routine experimentation.
  • the invention is directed to test devices for use in the method of the invention.
  • These test devices contain a multiplicity of test regions containing ligands of varying affinity for the specific binding partner (or, with respect to the second format, for the analyte) ; the test regions are arranged in such a manner that regions containing ligands of increasing affinity for the specific binding partner (or analyte) can readily be discerned.
  • the drug delivery aspect of the invention provides a means to control the absolute and relative amounts of materials of interest in desired locations by effecting competition between a releasing agent and a modulating material contained in complexes with the substance of interest .
  • the releasing agent may be supplied at the appropriate time, or may be endogenous to the targeted location.
  • the complex is maintained intact until the releasing agent is supplied or encountered; the component of interest is then able to exert its effects.
  • the invention is directed to a method to modulate the activity of a desired substance with respect to a target in an environment, which method comprises supplying said desired substance as a complex that includes a control agent; and effecting the release of the desired substance from the complex by interaction of the complex with a releasing agent.
  • the releasing agent competes with the control agent for the desired substance or with the desired substance for the control agent, or dissociates an aggregate effected by a control agent.
  • the complex contains as the control agent a size-enhancing agent which provides complexes that have low clearance rates in in vivo systems. Therefore the component of interest can be maintained in the subject as a complex of high aggregate weight. Clearance can be effected by providing a releasing reagent that destroys the complex and releases the desired component. This method can be refined so that the desired component also includes a specific affinity component which selectively binds a target. The size-enhancing agent is not released or dissociated until sufficient desired component has accumulated at the target.
  • an active component is complexed with a partner which inactivates it while it remains in the complex. If this inactivating material is also capable of specific binding to the target for which the biologically active material is intended, and if binding to the target destroys the complex, the active substance will be active only in the presence of the target.
  • the target or materials in its proximity thereby function as releasing agents.
  • an external releasing agent can be used.
  • an active substance is administered complexed to a competitor for its intended substrate or pseudosubstrate. High concentrations of its intended substrate or pseudosubstrate then release the active material.
  • Figures la-lc show diagrammatically the effect of increasing concentrations of releasing agent (RA) on a complex between active component (AC) and a control agent (CA) .
  • Figures 2a-2b show diagrammatically the effect of increasing concentrations of releasing agent on an aggregated complex that includes a self-aggregating size-enhancing agent (SEA) as the control agent.
  • Figures 3a-3b show diagrammatically the effect of increasing concentrations of releasing agent on an aggregated complex that includes size-enhancing agent and an immunoconjugate.
  • SEA self-aggregating size-enhancing agent
  • Figures 4a and 4b show a more elaborate version of the complexes of Figures 3a-3b.
  • Figures 5a and 5b show the effect of competition of an epitope target at high concentration on the binding of an immunoconjugate to a polymer containing an epitope analog.
  • Figure 6 shows illustrative affinity titration curves for various reagents reactive with an analyte.
  • titration of affinity replaces titration of concentration for generating a readable signal.
  • Figure 6 illustrates three specific binding agents or ligands (A, B and C) for which the response to different doses of analyte are offset due to differential affinity of the binding agent for the analyte or for a competitor species.
  • the displaced dose-response curves generate a distinguishable response pattern for different concentration zones (numbered 1- 5) of analyte.
  • the concentration zone of an unknown specimen can thus be determined by comparing its response pattern with respect to these three ligands and comparing this response to that of standards. Quantitation within a zone can be accomplished by standard methods, if desired, or by increasing the number of different binding agents within this range.
  • U.S. Patent 5,113,866 which is incorporated herein by reference, describes the production of diverse panels of ligands with maximally varying characteristics. These panels are particularly useful in supplying ligands of varying affinity for a single moiety, including a specific binding partner for analyte or for the analyte itself. While maximal diversity is not required for the ligands used in the present invention, such diversity is advantageous since it permits systematic control of the binding of the ligands for the analyte or specific binding partner.
  • analyte or the specific binding partner for analyte is an enzyme
  • variations on the substrate molecule or inhibitor molecules for the enzyme may be used.
  • the analyte or the specific binding partner for analyte is a receptor
  • variations on the ligand known to bind to the receptor might be used; conversely, if the target compound is a moiety which is known to bind to a receptor, variations in the binding site of the receptor protein can be employed.
  • the preferred competitive form of the method uses a "specific binding partner" for analyte.
  • “specific binding partner” refers to a substance which is known to bind with considerable affinity for the desired analyte; typical such specific binding partners would be antibodies or immunologically reactive fragments thereof, such as Fab, Fab' or Fab' 2 fragments or, for example, when the analyte is a ligand which matches a receptor, the receptor for the ligand, etc.
  • the specific binding partner can be the antigen to which the antibody is responsive.
  • the assay of the invention is preferably conducted on a solid support matrix to which the ligands of varying affinity for the specific binding partner or analyte are coupled.
  • a solid support matrix to which the ligands of varying affinity for the specific binding partner or analyte are coupled.
  • a fluorescent energy transfer as a detection method is available in a homogeneous solution phase; no wash or attachment to solid support is required.
  • the solid matrix may be of any design, so long as discrete test regions can be defined at its surface.
  • Conventional substrates of this type, such ⁇ for example, microtiter plates, can conveniently be used.
  • flat, hydrophilic surfaces that have been divided into test regions by application of hydrophobic boundaries can be used.
  • a cellulose backing with wax cross-hatchings so as to define a multiplicity of rectangular or square regions arranged linearly could be used.
  • the design of the array of test regions is a matter of convenience and simplicity of interpreting the results.
  • the regions are arranged in such a manner that a linear array of ligands of increasing affinity for the specific binding partner or analyte can be coupled to the backing.
  • the ligands can be arranged as a circle or a spiral or any other convenient, orderly design pattern.
  • a multiplicity of a series of such ligands of monotonically increasing affinity for specific binding partners of the same analyte or different analytes can be arranged on the surface of the same substrate or solid support.
  • the binding may be covalent or by adsorption. If peptides are used as a source of ligands having multiple affinities, linker moieties capable of forming ester, amide, or disulfide bonds with the peptide and suitable covalent bonds with the substrate may be employed. However, additional types of ligands, including nucleic acids, carbohydrates, and other polymers could also be used. Some of these are described in the above-referenced patent. Coupling is through conventional procedures; for example, binding to cellulose substrates may be effected by cyanogen bromide, alkyl chloroformates or carbonyl diimidazole to form cyclic carbonate or carbonate active esters.
  • each test region is separately coupled to the ligand of specified affinity in a known pattern.
  • the coupling thus results in a series of test regions of varying affinity for the specific binding partner or analyte.
  • the support containing the test regions may optionally be supplied with a known, preferably constant, amount of specific binding partner contained in, but not coupled to, each test region in advance of the test itself.
  • the specific binding partner may be supplied as a separate solution at the time sample is applied.
  • the sample is applied to - li ⁇ the entire series of test regions along with a constant amount of specific binding partner.
  • the sample and specific binding partner are allowed to remain in contact with the series of test regions for sufficient time to permit competition for the specific binding partner between the ligand and the analyte contained in the sample.
  • the presence or absence of ligand-bound specific binding partner in a specific test region is detected after incubation in competition with the sample containing analyte.
  • This detection can be conducted in a variety of ways. In some methods, it is not necessary to wash away solution containing unbound specific binding partner.
  • the solid support may be provided with a fluorescence-emitting compound wherein the fluorescence can be quenched by a moiety attached to the specific binding partner. Only bound specific binding partner is capable of quenching the fluorescence, and the presence of unbound partner in the solution does not interfere with the reading.
  • This method can also be used in homogeneous medium where the ligand is in solution in the test region.
  • optical devices which detect the presence or absence of a signal, such as a fluorescence signal, only at the surface and not elsewhere can also be employed.
  • More traditional methods such as, for example, adding a substrate solution to the series of test regions wherein the specific binding partner is coupled to an enzyme for the detection of bound enzyme may require prewashing of the test regions. If the test regions are, indeed, washed free of ' unbound specific binding partner, the presence or absence of the specific binding partner is then detected for each test region using such conventional methods.
  • the specific binding partner is an antibody
  • this antibody may itself contain a label or may be labeled using a second antibody specifically immunoreactive with it.
  • Various conventional methods of labeling are well known in the art, including radiolabeling, enzymatic labeling, fluorescent labeling, and combinations of these.
  • test regions When detection has been effected, the pattern of binding is then observed.
  • samples with large concentrations of analyte will result in failure to bind specific binding partner in the majority of test regions .
  • Samples containing only low amounts of analyte which are poorly capable of competing with coupled ligand will result in a series wherein most of the test regions show the presence of label.
  • the proportion of test regions showing binding is roughly inversely proportional to the level of analyte in the sample.
  • the test is made semiquantitative by suitable standardization with known amounts of analyte.
  • the level of precision may be varied according to the desired need for same by adjusting the relative affinities of the test portions for the specific binding partner. A large number of such test portions having ligands with only moderate increments of affinity can be used to enhance the precision of the assay.
  • the multiplicity of test portions is contacted with the sample putatively containing analyte.
  • the ability of the analyte to bind to ligand in a particular test portion will depend on the affinity of the ligand residing in that test portion for the analyte itself. Binding can be judged on a kinetic or equilibrium basis; if judged on a kinetic basis, short-term incubations which terminate prior to establishment of equilibrium are used.
  • analyte will bind to ligand in those test portions containing ligand for which it has the highest affinity preferentially; at low concentrations, only test portions having ligands with very high affinity for the analyte will succeed in binding detectable amounts of analyte. At higher concentrations of analyte even test portions with ligand having relatively small affinities will be able to bind analyte.
  • the detection of ligand-bound analyte in this format may also be conducted by measuring changes in characteristics of the surface of the solid support due to binding; however, more conventional approaches involving removing sample containing unbound analyte and then using a secondary binding agent containing label are preferred.
  • Washing is generally not indicated since this may alter the binding characteristic of the sample.
  • One example of this approach would employ, for example, an antibody or fragment thereof capable of specifically binding analyte wherein the antibody or fragment itself contains a radioactive, fluorescent, enzyme, or other label.
  • test regions binding analyte in standard concentration controls as compared to the number of regions binding analyte in the sample to be tested.
  • higher concentrations of analyte will show detectable binding in a greater number of test regions.
  • the assay is conducted using a multiplicity of test regions.
  • the test regions are arranged in such a way that the result in each can be measured and associated with a particular ligand.
  • some orderly arrangement will be necessary, such as a linear arrangement of ligands with increasing affinity for analyte or increasing affinity for the specific binding reagent.
  • test regions are simply wells of a microtiter plate or test tubes in a rack, the arrangement is flexible and at the option of the practitioner. Random physical arrangements may also be used using computer processing to sort out the position of the ligands of various affinities. In principle, however, in a single formatted test, simply the number of test portions which provide positive results will be determinative.
  • One particularly convenient method to construct a device with the required number of test regions comprises a basic hydrophilic matrix wherein regions of the matrix are separated from each other by hydrophobic barriers.
  • a cellulose mat might be subdivided into squares or other suitably shaped regions by lines of wax or other hydrophobic barrier.
  • the invention also provides methods to control the behavior of materials in specified environments, such as metabolic systems, by controlling the interaction of substances of interest with other materials through a process of "affinity competition" .
  • the material or substance of interest is included in a complex with a "control" agent, which controls the behavior of the substance of interest, either by inactivating it, changing its size, or otherwise affecting its properties.
  • the complex is dissociated by a "releasing agent" which competes either with the substance of interest for the control agent or with the control agent for the substance of interest, or which causes dissociation of a self-aggregate formed by the control agent.
  • a "releasing agent” which competes either with the substance of interest for the control agent or with the control agent for the substance of interest, or which causes dissociation of a self-aggregate formed by the control agent.
  • the presence of the releasing agent in the proximity of the complex between the "control agent” and the substance of interest is achieved either arbitrarily by administering the releasing agent to the appropriate environment or by the location of high concentrations of the releasing agent at a target position.
  • the release of the desired substance from the complex is essentially the result of an affinity titration with respect to the concentration of the releasing agent. As the concentration of releasing agent becomes higher and higher, its capacity to release the desired substance becomes substantially greater.
  • the appropriate concentration of releasing agent can be determined. If the releasing agent is endogenous, the affinities of the components of the system must be adjusted to accommodate the concentration of releasing agent found in the environment.
  • Successful application of the present method of the invention depends upon correct selection of the affinities of the releasing agent for a component of the complex and of this component for the remaining components of the complex.
  • the affinity of the releasing agent for the control agent as compared to the affinity of the active component to the control agent must be such that the achievable concentration of the releasing agent is sufficient to tip the balance of affinities in favor of the releasing agent/control agent interaction.
  • recourse can be had to panels of substances having systematically varied properties. This will provide a range of affinities for the control agent so that appropriate selection of a releasing agent can be made. Conversely, such panels can provide candidates for a control agent suitable to respond to a predetermined releasing agent concentration.
  • the active component (AC in the figure) is administered in the form of a complex with a control agent (CA) into an environment that generally has low concentrations of a releasing agent as shown in Figure la. At these concentration levels, the complex remains intact. However, when the releasing agent concentration is increased, the complex is disassociated due to the affinity of the releasing agent (RA) either for the CA as shown in Figure lb or for the active component as shown in Figure lc. If the releasing agent is also the target for the active component or is associated with it in some way, its effective concentration will be highest at the target location.
  • An alternative form of this concept is diagrammed schematically in Figures 2a and 2b.
  • a high aggregate weight complex is formed by virtue of the self- association of the control agent, in this case, a size-enhancing agent (SEA) .
  • the SEA may be covalently bound to a substance of interest, the active component (AC) .
  • the SEA is aggregated to form the complex.
  • the size-enhancing agent is disassociated so that the active component is present as an effectively lower aggregate weight moiety.
  • ConA Concanavalin A
  • Multivalent ConA can also bind various bacterial-derived dextrans such as that from strain B-1355-5, to form an insoluble complex.
  • Precipitation of ConA-dextran can be inhibited in a dose-dependent manner by MeMan.
  • MeMan By screening a phage epitope display library, several peptides (e.g. MYWYPY and VGRAFS) were identified which also inhibit Con A-dextran precipitation, with distinctive 50% inhibition values.
  • Measurement of MeMan as an analyte can thus in principle be accomplished using peptides as competing ligands or for blocking the precipitation of ConA-dextran, effectively expanding the range of MeMan concentrations that can be measured.
  • Dextran and peptides could also in principle be immobilized and used to trap ConA competitively with MeMan.
  • Con A could be viewed as the analyte and dextran B-
  • the drug delivery aspect of the invention as it relates to drug delivery is illustrated in the following embodiments. It will be apparent from review of these embodiments that they share the general characteristics of providing a complex between a control moiety and a substance of interest which is dismantled in the presence of a releasing agent.
  • the monoclonal antibody or preferably an immunoreactive portion thereof -- i.e. the F v region -- might be constructed as a fusion protein with a peptide which can be modified to contain both a chelating moiety for a radioisotopic label and an extension which serves as a size-enhancing agent as shown in Figure 3.
  • the control agent in this case a size-enhancing agent represented by the peptide extension
  • the antibody or F v region thereof coupled to the radiolabel or drug shown as an asterisk in the figure) is extended by a size-enhancing agent (SEA) .
  • the label can be placed either on the SEA or on F v and can be added before or after coupling.
  • the SEA is designed so as to encourage dimer or multimer formation. This can be effected by using two mutually attractive extensions, SEA and SEA' , which are oppositely charged; alternatively, extensions can be used which have a natural affinity such as hydrophobic extensions.
  • size-enhancing agents which simply self-aggregate are preferred since only one set of reagents needs be prepared in this case.
  • the affinity of the extensions for each other can be modified by design of epitope/paratope pairs using panels of candidates of maximal diversity as described above or by using the screening techniques described in U.S. patent 5,217,869, incorporated herein by reference.
  • the releasing agent, RA has sufficient affinity for at least one of the SEA extensions so that at appropriate concentrations the dimer is dissociated as shown in Figure 3b.
  • Figure 4 A somewhat more sophisticated version of this design is shown in Figure 4.
  • the size-enhancing agent extension has at least two binding arms formed by branching of the fused peptide.
  • the binding arms might then be comprised of polyionic regions, such as polylysine (poly + ) or polyglutamic or polyaspartic (poly " ) which can be configured to permit aggregation using the electrostatic attraction between the positive and negative arms to hold the complex together.
  • the binding arms may comprise an epitope/paratope pair to effect binding.
  • the peptide with the branched arms fused to the F v or Mab is thus a size-enhancing agent which is capable of effecting aggregation of the individual fusion peptides.
  • the aggregate is then administered to the subject animal body and the complex is allowed to accumulate at target cells or tissues. When sufficient time has elapsed to allow the accumulation to have occurred, a releasing agent is added.
  • the releasing agent is in the form of, for example, a positively or negatively charged peptide (polylysine or polyglutamic or polyaspartic) . If the binding arms are an epitope/paratope pair, the releasing agent will be the paratope or epitope or mimic thereof. Interaction of the releasing agent with aggregated complexes dissociates them and reduces the aggregate weight so that the clearance and penetration powers of the fusion protein are realized.
  • Figure 4a shows the intact aggregate assembled by virtue of the oppositely charged polyionic arms of the branched extension.
  • a releasing agent having a positive charge for example, as shown in Figure 4b, the complex is dissociated.
  • the active moiety may also be contained in a controlled release setting, such as polymeric beads with interstices bearing moieties which interact with a controlling region of the active component.
  • the active component along with its controlling region, is released from the interstices by diffusion.
  • the rate of release can be controlled by the specific affinity between a portion of the active component and a "control agent" contained in the polymer, where the effective local concentration of the polymer-contained moiety can be adjusted to effect the appropriate rate of diffusion.
  • the releasing agent comprises the ambient conditions that control diffusion.
  • Suitable polymers include porous polymers such as polyacrylamide, collagen, and the like.
  • a radioisotope label or toxin or drug may be coupled to the F v unit either covalently, as in the case of drugs or toxins, or by chelation as in the case of radioisotopes, and the resulting immunoconjugate adsorbed to a polymer having at least one affinity ligand (such as the relevant antigen, or an epitope thereof) coupled to the polymer.
  • Suitable polymers include, for example, inert biocompatible materials such as polyethylene glycol, polyacrylamide, polymethacrylate, and the like.
  • the affinity polymer size- enhancing agent then maintains the immunoconjugate in the environment in which the target is included until sufficient immunoconjugate accumulates at the target.
  • affinity polymer size-enhancing agent may be released when the complex reaches the target due to competition for the immunoconjugate from the endogenous antigen (as the releasing agent) ; for circulating complex, however, this can be released at an appropriate time by administration of, for example, a peptide representing the target epitope.
  • the smaller epitope will then replace the affinity polymer effectively lowering the aggregate weight of the substitute complex so that it can be cleared effectively.
  • Figure 5 shows the immunoconjugate complexed by affinity to an epitope or epitope analog region contained on a polymer. At low concentrations of the epitope as shown in Figure 5a, the complex remains intact. When the local concentration of the epitope is increased, as shown in Figure 5b, the complex is dissociated.
  • Another strategy provides, in the immunoconjugate, a separate region of affinity for the size-enhancing agent. In this strategy, the releasing agent is not provided by the target.
  • the size-enhancing agent may also be polyvalent.
  • the immunoreactive regions of the desired antibody may be coupled covalently to a second immunoreactive region unrelated to that directed to the target; the immunoconjugate will also contain the desired moiety such as toxin, drug or radioisotope.
  • This immunoconjugate therefore, contains 1) immunospecific regions designed to bind the target specifically; 2) an immunospecific region designed to bind the size-enhancing agent; and 3) a label or drug.
  • the size- enhancing agent may comprise one or more ligands specifically immunoreactive with region of the immunoconjugate designed as its specific binding partner covalently bound to polymer.
  • the complex then comprises the size-enhancing agent (such as the derivatized polymer) complexed with the immunoconjugate through this second immunoreactive region.
  • the accumulation of the complex at the target in this case does not result in any appreciable dissociation of the complex.
  • This is effected by administering, as a releasing agent, the affinity ligand used to couple the complex to the size-enhancing agent or a mimic thereof .
  • the invention method may also be used to unmask the active site of a biologically active molecule at its site of action.
  • This embodiment depends on either a differential affinity of the masking agent for a releasing agent present specifically at the site at which biological activity is to be produced or on differential affinity of the biologically active agent with respect to its target as compared to the masking (control) agent .
  • An application of this embodiment relates to the administration of insulin. Insulin is now administered by injection; however, normal hepatic insulin levels are 10-50 times blood levels. In order to obtain the desired hepatic insulin levels, it would be necessary to increase dramatically the dose of insulin administered by injection.
  • the active site of insulin is masked by a ligand that has a higher affinity for hepatic- specific cell surface antigens than for insulin.
  • insulin could be supplied as an immunoconjugate specific for hepatic cell antigens and released from a masking agent-containing complex in the manner described in Example 1 above. The insulin is thus protected from activity in the blood until it reaches the hepatic sites wherein the cell surface antigens displace the masking agent and result in active insulin.
  • a biologically active moiety which operates on an endogenous substrate directly or indirectly can be titrated out depending on the concentration of the substrate by supplying the moiety coupled to a substitute substrate with which the biologically active substance binds, but does not recognize as a substrate or pseudosubstrate.
  • insulin can be supplied as a complex with antibody or lectin which binds to dextran; the dextran is then displaced by high endogenous glucose levels.
  • glucose might be considered a "pseudosubstrate" for insulin since although insulin does not interact with glucose directly, it is responsible for its metabolism.
  • the dextran plays the role of a control agent, glucose is a releasing agent, and the desired substance is insulin; the active component of the complex is the coupled lectin-insulin or antibody-insulin.
  • Example 5 Use of Bivalent Mimotopes
  • advantage is taken of the use of the combination of a bivalent mimotope, the valences of which are complementary, with suitable affinity constants, to the valences of a bivalent antibody or immunoreactive portion thereof .
  • At least one of the valences of the bivalent mimotope has a controlled affinity for its counterpart region on the antibody as compared to a target antigen -- e.g. a tumor antigen. In a sense, then, this valency mimics the tumor antigen.
  • the bivalent mimotope is complexed through both valences to its complementary bivalent antibody and the complex is effectively inert .
  • the administered complex is designed to permit the integrity of the complex to be qualitatively affected by the difference of endogenous concentrations of the tumor antigen -- i.e. the releasing agent.
  • An alternative arrangement utilizes a chimeric protein that contains an extension to the antibody where the extension contains an epitope analog.
  • the epitope analog binds to the immunoreactive portion of another molecule of the chimera, thus forming a dimer.
  • the dimer is dissociated when the epitope is displaced by the releasing agent, e.g. a tumor antigen.
  • a complex is formed that includes a desired substance for e.g., therapy or diagnosis and a control agent which imparts a desired characteristic to the complex -- e.g. size, inactivity of the desired substance, etc.
  • the complex remains intact until dissociated when the releasing agent is either supplied endogenously or administered separately at an appropriate time and at an appropriate concentration.

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Abstract

L'invention se rapporte à un procédé pour déterminer la quantité d'un analyte présent dans un échantillon, ce procédé utilisant une série de régions de test ayant une affinité pour un partenaire de liaison spécifique d'un analyte ou pour l'analyte lui-même, cette affinité étant amenée à varier systématiquement et à croître de préférence de façon monotonique. En déterminant les parties des régions de test qui sont capables de se lier au partenaire de liaison spécifique en concurrence avec l'analyte présent dans l'échantillon ou qui se lient à l'analyte, on peut obtenir une estimation de la quantité d'analyte présent. Ainsi, le titrage par affinité remplace le titrage par concentration, ce qui permet d'effectuer des dosages sans qu'il soit nécessaire de recourir à des étapes de dilution en série. Dans une variante, on profite de la concurrence de divers ligands pour des constituants de complexes, afin de réguler la libération de constituants désirés à partir de ces complexes. Dans un premier mode de réalisation, on peut retarder l'opération d'épuration permettant de séparer de petites fractions du complexe, en combinant ces petites fractions avec des agents d'agrandissement de la taille, lesquels peuvent être libérés ou dissociés par action d'un agent de libération au moment où on en donne le signal. Dans d'autres modes de réalisation, une cible elle-même peut être un concurrent capable de l'emporter sur les autres concurrents et d'être ainsi celle qui libérera la forme active d'une telle fraction à partir d'un tel complexe.
PCT/US1994/003490 1993-03-30 1994-03-30 Determination de la concentration d'un constituant par titrage par affinite et par deplacement par concurrence, utilisee dans l'apport de medicaments Ceased WO1994023298A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP94913987A EP0692096A1 (fr) 1993-03-30 1994-03-30 Determination de la concentration d'un constituant par titrage par affinite et par deplacement par concurrence, utilisee dans l'apport de medicaments
JP6522341A JPH08508568A (ja) 1993-03-30 1994-03-30 親和性滴定による濃度の決定とドラッグデリバリーにおける競合的置換
AU66229/94A AU693442B2 (en) 1993-03-30 1994-03-30 Determination of concentration by affinity titration and competitive displacement in drug delivery

Applications Claiming Priority (4)

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US08/039,786 1993-03-30
US08/039,786 US5356784A (en) 1993-03-30 1993-03-30 Determination of concentration by affinity titration
US16365993A 1993-12-06 1993-12-06
US08/163,659 1993-12-06

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Cited By (3)

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WO1996023879A1 (fr) * 1995-01-30 1996-08-08 Terrapin Technologies, Inc. Corps agglutinants - multiplicite de proteines capables de lier diverses petites molecules
GB2324866A (en) * 1997-04-21 1998-11-04 Randox Lab Ltd Device for multianalyte assays.
US7396689B2 (en) 2005-02-04 2008-07-08 Decision Biomarkers Incorporated Method of adjusting the working range of a multi-analyte assay

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JP3479100B2 (ja) * 1993-06-02 2003-12-15 帝国臓器製薬株式会社 免疫化学的簡易半定量方法および装置

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WO1991006356A1 (fr) * 1989-10-31 1991-05-16 Terrapin Technologies, Inc. Procede d'identification de ligands de liaison d'analytes
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WO1993003367A1 (fr) * 1991-07-29 1993-02-18 Serex, Inc. Affinites de liaison differentielles et methodes de detection de dissociation basees sur ces affinites
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US5217869A (en) * 1987-10-13 1993-06-08 Terrapin Technologies, Inc. Method to produce immunodiagnostic reagents
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WO1991006356A1 (fr) * 1989-10-31 1991-05-16 Terrapin Technologies, Inc. Procede d'identification de ligands de liaison d'analytes
US5116944A (en) * 1989-12-29 1992-05-26 Neorx Corporation Conjugates having improved characteristics for in vivo administration
WO1992004052A1 (fr) * 1990-09-07 1992-03-19 Techniclone, Inc. Anticorps modifies a temps de clairance regule
WO1992017784A1 (fr) * 1991-04-02 1992-10-15 Terrapin Technologies, Inc. Procede permettant de classer les analytes a l'aide de profils de concentrations d'analytes contenues dans des echantillons
WO1993003367A1 (fr) * 1991-07-29 1993-02-18 Serex, Inc. Affinites de liaison differentielles et methodes de detection de dissociation basees sur ces affinites

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996023879A1 (fr) * 1995-01-30 1996-08-08 Terrapin Technologies, Inc. Corps agglutinants - multiplicite de proteines capables de lier diverses petites molecules
GB2324866A (en) * 1997-04-21 1998-11-04 Randox Lab Ltd Device for multianalyte assays.
GB2324866B (en) * 1997-04-21 2001-11-14 Randox Lab Ltd Device and apparatus for the simultaneous detection of multiple analytes
US6498010B1 (en) 1997-04-21 2002-12-24 Randox Laboratories, Ltd Method for making a device for the simultaneous detection of multiple analytes
US7396689B2 (en) 2005-02-04 2008-07-08 Decision Biomarkers Incorporated Method of adjusting the working range of a multi-analyte assay

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CA2157929A1 (fr) 1994-10-13
AU6622994A (en) 1994-10-24
AU693442B2 (en) 1998-07-02
EP0692096A1 (fr) 1996-01-17

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