CA2157929A1 - Determination of concentration by affinity titration and competitive displacement in drug delivery - Google Patents

Abstract

A method for determining the amount of analyte in a sample utilizes a series of test regions with systematically varied, preferably monotonically increasing, affinity for a specific binding partner for an analyte or for the analyte itself. By determining the portion of test regions which are capable of binding specific binding partner in competition with the analyte in the sample, or which bind the analyte, the amount of analyte may be estimated. Thus, titration of affinity replaces titration of concentration, allowing assays to be performed without the need for serial dilution steps. Alternatively, advantage is taken of competition of various ligands for components of complexes to regulate the release of desired components from the complexes. In one embodiment, clearance of small moieties can be delayed by combining them with size-enhancing agents which can be released or dissociated on cue by action of a releasing agent. In other embodiments, a target itself is a successful competitor in releasing the active form of the moiety from the complex.

Description

W094/23298 21 S ~ 9 2 ~ PCT~S94/03490 DETERMINATION OF CONCENTRATION BY AFFINITY TITRATION
AND
COMPETITIVE DISPLACEMENT IN DRUG DELIVERY

Field of the Invention 5The 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 vitro environments.

Backqround Art AssaYs Assays to determine the concentration of analyte in clinical, environmental, or other settings generally involve the use of serial dilution. The purpose of such dilution is twofold: it may be necessary to bring the concentration of the sample within the range of the assay; if the sample is too concentrated, a meaningful reading may not result.
Additionally, 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. In other cases, the level of dilution itself may be used as an assessment of concentration. In this instance, 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 W094/23298 ~ PCT~S94/03490 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. By taking account of 5 the level of dilution at which a response is no longer visible, and comparing the results to those obtained with standards, the concentration of analyte in the original sample can be back-calculated.
Methods that employ serial dilution are useful, but 10 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 cont~m;n~nts in soil at the location where field testing is appropriate should be accomplished by 15 methods that require only the application of a single sample volume, rather than the more complex and error-prone process of obtaining multiple dilutions. Similarly, in clinical settings, shortages of trained and reliable personnel manually to conduct serial dilutions.for assessment is a recognized problem in 20 supplying health care; instrumentation to make such dilutions mechanically is expensive and of limited reliability.
Furthermore, it would be desirable to conduct clinical assays on extremely small samples so as to minimize the invasive nature of sample taking. Conduct of serial dilution on samples in the 25 microliter range, for example, is inherently inaccurate.
One approach to this problem has been set forth in U.S. patents 4,654,310 and 4,923,800 to Ly. In the methods described, systematically varying amounts of test reagents in multiple test portions are used to obtain semiquantitative 30 results for the same solution of analyte without necessity for serial dilutions of the sample. One easily understood disclosed approach takes advantage of two competing catalytically controlled reactions using varying relative amounts of the two catalysts. In its simplest form, two enzymes which utilize the J
35 analyte as the substrate compete for conversion of the substrate to product. One of the products gives an all-or-none detectable result; the other does not give a detectable response. If there is a high concentration of analyte, even large amounts of W094/23298 ~ 1~ 7 ~ PCT~S94/03490 competing enzyme which take away from conversion to thedetectable product don't matter; however, at low concentrations of analyte, not enough will be left to see the result.
Therefore, high concentrations of analyte will be capable of giving a detectable result in the presence even of high concentrations of the competing enzyme; low concentrations of analyte will only give a detectable result at low concentrations of competing enzyme. In a somewhat different, but related, approach, described in U.S. patent 4,042,329 to Hochstrasser, 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. In contrast to the above-mentioned techniques, 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.

Drug Delivery Discovery of biologically active compounds or of compounds which are capable of labeling tissues for diagnosis is only one part of a complex problem of using these materials to their desired ends. In order for drugs, for example, to be effective they must remain in the environment of their intended targets for a sufficient time to exert their effects. They also benefit from selective delivery to these targets in many cases.
The same is true of the use of labels to localize specific tissues. A mechanism must be found to permit such localization to occur and then to clear the remaining background label from the system so that the localized label can be detected. A
number of approaches are known for providing these effects. For example, it has long been suggested that toxins which are designed to destroy, for example, tumor cells be complexed with specific affinity reagents such as immunoglobulins which are W094/23298 ~ PCT~S94/03490 capable of binding the tumors. Presumably by collecting the combination of specific binding agent and toxin at the surface of the tumor, the tumor is destroyed at the expense of tissues to which the specific affinity reagent is not attracted.
Similarly, techniques are available for specific imaging of particular tissues by combining scintigraphic labels, for example, with agents having specific affinity for their targets.
More generally, a number of drugs have been modified to enhance their overall half-life by coupling them to polymers such as polyethylene glycol. However, it has not been possible carefully to calibrate the pharmacokinetics so that the biologically active material to be delivered is brought exclusively to the desired target and cleared from the remaining tissues when homing has been accomplished. Nor has it been possible, in many cases, even to accumulate sufficient drug at the desired location.
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 vitro environments.

Disclosure of the Invention First, 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. In the preferred competitive mode, since 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.
Thus, in one preferred aspect, 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. The test regions contain W094/23298 21~ 7 9 2 9 PCT~S94/03490 ligands which have varying affinities for a specific bindingpartner that is capable of binding the desired analyte. The test regions are arranged sequentially--i.e., in such a manner that a monotonic increase of affinity of the contained ligands for the specific binding partner can be discerned. Of course, 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.
At the time the sample is contacted with the multiplicity of test regions, a quantity of specific binding partner is also supplied to each, and, indeed, can be contained in each test region ab initio. After contact of the test regions with the sample in the presence of specific binding partner, 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 re~;n1ng 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.
Unlike the semiquantitative data available from conducting the methods of Ly and Hochstrasser described above, 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.
In the alternative, 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. At low concentrations of analyte, only those test portions which contain ligands with high affinities for the analyte will bind sufficient amounts of analyte to be detectable. 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 æs W094l23298 PCT~S94/03490 different from that of the competitive format and can be conducted using routine experimentation.
In other aspects, 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.
Thus, 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.
In one embodiment, the complex contains as the control agent a size-enhancing agent which provides complexes that have low clearance rates in in ~ivo 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 W094/23298 21~ ~ 9 ~ ~ PCT~S94/03490 not released or dissociated until sufficient desired component has accumulated at the target.
In another embodiment, 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. Alternatively, an external releasing agent can be used.
In a third illustrative embodiment, 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.

Brief Description of Drawings 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.
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.

W094/~298 ~ PCT~S94/03490 Modes of Carryinq Out the Invention In the invention method, titration of affinity replaces titration of concentration for generating a readable signal. The general principle can be described as follows:
Figure 6 illustrates three specific binding agents or ligands (A, B and C) for which the response to di~ferent doses of analyte are offset due to differèntial affinity of the binding agent for the analyte or for a competitor species. As shown, 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.
While the diverse panels of ligands described in the above-referenced patent are a preferred source of the ligands with monotonically varying binding capability, other sources of such ligands could also be used. A series of monoclonal antibodies of varying affinities for their specific binding partner, for example, could be used. Similarly, peptides having random or systematically varied sequences can be generated using techniques by now well known in the art.
While it is relatively inconvenient to do so, it is not impossible to obtain a range of ligands of varying affinities essentially by trial and error among suitable candidates based on the nature of the target to which the ligand W094/23298 21~ 7 ~ ~ ~; PCT~S94/03490 _ g _ is to be bound. For example, if the 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. If 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.
Thus, a multiplicity of approaches for obtaining the desired collection of ligands with systematically varying affinities for analyte or specific binding partner for analyte is available in the art.
The preferred competitive form of the method uses a "specific binding partner" for analyte. As used herein, "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. In addition, of course, if the analyte is itself an antibody, 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.
However, it would also be possible to format the assay for homogeneous solutions, e.g. 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 as, for example, microtiter plates, can conveniently be used. Alternatively, flat, hydrophilic surfaces that have been divided into test regions by application of hydrophobic boundaries can be used. For example, a cellulose backing with wax cross-hatchings so as to define a multiplicity of rectangular or square regions arranged linearly could be used.

~s~
W094/23298 ~ PCT~S94/03490 The design of the array of test regions is a matter of convenience and simplicity of interpreting the results.
Preferably, 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.
Alternatively, 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 nature of the coupling of the individual ligands to the test regions, if such coupling is desired, also varies widely, depending on the nature of the solid substrate and the nature of the ligand used. 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.
In setting up the test device, 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.
For conduct in the competition format, 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. Alternatively, the specific binding partner may be supplied as a separate solution at the time sample is applied. In the conduct of the test, the sample is applied to W094t23298 ~ g ~ PCT~S94/03490 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. Depending on the method of detection of binding of specific binding partners to the ligand, it may or may not be necessary or desirable, after the incubation period, to wash the test portions so that only specific binding partner bound to ligand in the test region remains.
In any event, 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. For example, in a format involving coupling of ligand to solid support, 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.
Alternatively, 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. For example, if 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 W094/23298 ~5 PCT~S94/03490 labeling are well known in the art, including radiolabeling, enzymatic labeling, fluorescent labeling, and combinations of these.
When detection has been effected, the pattern of binding is then observed. In a single series of test regions, 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. Thus, 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.
Alternatively, in the direct format, 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.
In this format, 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 W094/23298 2 1 ~ 7 ~ 2 ~ PCT~S94/03490 conventional approaches involving removing sample containing unbound analyte and then using a secondary binding agent containing label are preferred. Washing, however, 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.
The results are then read by comparing the number of test regions binding analyte in standard concentration controls as compared to the number of regions binding analyte in the sample to be tested. In this format, higher concentrations of analyte will show detectable binding in a greater number of test regions.
Whether conducted in a direct or competitive format, the assay is conducted using a multiplicity of test regions.
Preferably, the test regions are arranged in such a way that the result in each can be measured and associated with a particular ligand. For direct reading devices, 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. Alternatively, other orderly arrangements such as spirals or even two-dimensional arrays could be used, as long as the results are intelligible. If the 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 re~uired number of test regions comprises a basic hydrophilic matrix wherein regions of the matrix are separated from each other by hydrophobic barriers. Thus, a cellulose mat might be subdivided into squares or other suitably shaped regions by lines of wax or other hydrophobic barrier.

W094/~298 ~ PCT~S94/03490 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. 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. By adjusting the affinity of the control agent for the desired substance or the strength of its aggregation with respect to the affinity of the releasing agent for the substance of interest or the control agent, 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. For example, if the releasing agent is designed to dissociate the complex by virtue of outcompeting the active component for binding to a control agent, the affinity of the releasing agent W094/23298 21~ 7 ~ ~ 9 PCT~S94/03490 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. In order to provide releasing agents of suitable affinities, 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.
Such panels with systematically varying properties are described, for example, in U.S. patent 5,133,866 as set forth above. This patent describes panels of ligands with maximally varying characteristics so as to minimize the number of candidates in the panel required to span the range of desired affinities. The use of such panels as sources for materials for construction of the complexes as well as releasing agents is preferred but not required. Any method for obtaining a range of affinities so that a material of the required affinity for the complex component (or the releasing agent) can be used.
The general principle is diagrammed in Figures la-lc.
In its simplest form, 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. In Figure 2a, 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 W094/~298 2~ PCT~S94/03490 interest, the active component (AC). In the absence or low concentration of releasing agent, as shown in Figure 2a, the SEA
is aggregated to form the complex. However, in the presence of suitable concentrations of releasing agent, as shown in Figure 2b, the size-enhancing agent is disassociated so that the active component is present as an effectively lower aggregate weight moiety.
The following examples are intended to illustrate but not to limit the invention.

Example 1 AssaYs The assays can be illustrated using materials described by Scott, J. K. et al. Proc Natl Acad Sci USA (1992) 89:5398-5402. Briefly, the lectin Concanavalin A (ConA) specifically binds the sugar methyl-alpha-Man (MeMan).
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.
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.
Conversely, Con A could be viewed as the analyte and dextran B-1355-5 as the specific binding agent, with MeMan and the peptides as competing agents that allow a range of ConA
concentrations to be determined.

The drug delivery aspect of the invention as it relates to dru~ delivery is illustrated in the following embodiments. It will be apparent from review of these W094/23298 2 ~ ~ ~ 9 2 9 PCT~Sg4/03490 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.

5Example 2 Delivery of Monoclonal Antibodies Cou~led with Toxins or Radioactive Metals for Use in Treatment or Diaqnosis Many attempts have been made to utilize monoclonal antibodies as specific affinity reagents to target malignant cells and mark them either for detection or destruction. Thus, a vast literature describing immunoconjugates for delivery of label, toxins or other drugs, or other desired substances to tumor targets is now available. In this embodiment of the present invention, the immunoconjugate is, therefore, the substance of interest. The tumor cells constitute the target.
One problem that has been encountered with the delivery of immunoconjugates is that although a low molecular weight, preferably less than 70 kd, and more preferably less than 20 kd, is desired to permit penetration of the tumor mass, immunoconjugates of such low molecular weights clear rapidly through the kidneys. It is estimated that localization to the tumor requires 24-36 hours; by this time, such low molecular weight substances have mostly cleared. On the other hand, once the appropriate amount of material has accumulated at the tumor by virtue of the ability of the monoclonal antibody to bind, rapid clearance becomes desirable. If the purpose of the immunoconjugate is labeling, such clearance reduces the background and enhances the accuracy of detection. If the immunoconjugate is intended as a therapeutic, such clearance prevents adverse effects to healthy tissue.
Complexes of >70 kd, however, are not cleared rapidly.
If the immunoconjugate could be made part of a complex that exceeds this molecular weight for a sufficient time to permit homing to the tumor tissue, and could then be released to allow penetration and clearance, an ideal pharmacodynamic situation would be achieved.

2~
W094/~298 PCT~S94/03490 This ideal situation can be achieved by supplying an appropriate size-enhancing agent which can be maintained in a complex with the immunoconjugate until a releasing agent is supplied. A variety of designs of the size-enhancing agent and the complex might be envisioned.
For example, the monoclonal antibody or preferably an immunoreactive portion thereof -- i.e. the Fv 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. Although a fusion protein of the Fv region is illustrated, the control agent (in this case a size-enhancing agent represented by the peptide extension) could be, instead, a covalently bound moiety, including a peptide, but also including other appropriate chemistries such as oligonucleotides.
As shown in Figure 3a, the antibody or Fv 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 Fv 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.
In general, 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.
A somewhat more sophisticated version of this design is shown in Figure 4. In this illustration, the size-enhancing agent extension has at least two binding arms formed by branching of the fused peptide. The binding arms might then be W094/23298 PCT~S94/03490 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.
Alternatively, the binding arms may comprise an epitope/paratope pair to effect binding. The peptide with the branched arms fused to the Fv 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. If the binding arms are polyionic, 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.
The foregoing embodiment is illustrated schematically in Figure 4. Figure 4a shows the intact aggregate assembled by virtue of the oppositely charged polyionic arms of the branched extension. At high concentration of 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. In this instance, the releasing agent comprises the ambient conditions W094/23298 ~ ~S~ ~ PCT~S94/03490 that control diffusion. Suitable polymers include porous polymers such as polyacrylamide, collagen, and the like.
Alternatively, a radioisotope label or toxin or drug may be coupled to the Fv 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. Some of the 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.
This is illustrated in Figure 5. 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. For example, 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 W094/23298 2 ~ 5 7 ~ 2 9 PCT~S94/03490 1) immunospecific regions designed to bind the targetspecifically; 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.

Example 3 Maskinq and Unmaskina of Active Sites 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. This has been done, in one protocol, by administering 25 times the usual levels of insulin along with sufficient glucose to offset the hypoglycemic effect of these high insulin levels. This approach is, on its face, cumbersome and less than satisfactory.
In the invention method, the active site of insulin is masked by a ligand that has a higher affinity for hepatic-specific cell surface antigens than for insulin. In another W094/~298 ~5~ 22 - PCT~S94/03490 alternative, 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.

Example 4 Substrate-Affinity or Pseudosubstrate Affinity Com~etition In a third embodiment, 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. For example, 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. In this illustration, glucose might be considered a "pseudosubstrate" for insulin since although insulin does not interact with glucose directly, it is responsible for its metabolism.
It is, of course, desirable to enhance the levels of available insulin when high concentrations of glucose are present. By coupling the insulin to an antibody or lectin which has an affinity for dextran, a complex can be formed which adversely affects the capacity of insulin to effect glucose metabolism. High concentrations of glucose will release the antibody- or lectin-bound insulin from the immobilizing dextran and permit the insulin, though still coupled to lectin or antibody, to perform its metabolic function. In this illustration, 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.

W094/23298 21 S ~ 9 2 ~ PCT~S94/03490 Example 5 Use of Bivalent Mimotopes In still another embodiment, 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. At low concentrations of the tumor antigen, such as those resulting in blood from antigen-shedding, the bivalent mimotope is complexed through both valences to its complementary bivalent antibody and the complex is effectively inert.
However, at high concentration of tumor antigen as would be found at the tumor site, at least the portion of the bivalent mimotope which mimics the tumor antigen is displaced from its binding site on the bivalent antibody effectively resulting in binding of the bivalent antibody to the tumor. Due to the now decreased affinity of the mimotope for antibody, wherein only one of the antibody valences interacts with one of the mimotope valences, the bivalent mimotope is eventually shed. Thus, 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.

Summary of Examples 2-5 In examples 2-5, 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 W094/23298 ~ PCT~S94/03490 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.

Claims (23)

Claims

1. A method to determine the concentration of an analyte in a sample, which method comprises:
(a) applying said sample to a multiplicity of test regions, said test regions being arranged to permit orderly retrieval of the results in each and which contain a series of ligands of systematically varying affinity for said analyte or for a specific binding partner capable of binding said analyte;
wherein when said systematically varying affinity is for a specific binding partner capable of binding said analyte said applying is conducted in the presence of a constant amount of said specific binding partner, under conditions wherein said ligand and said analyte compete for the specific binding partner;
(b) detecting said analyte or specific binding partner bound to the ligand in each test region; and (c) determining the concentration of analyte by assessing the portion of test regions to which analyte or specific binding partner is bound.

2. The method of claim 1 wherein said ligands are coupled to a solid support.

3. The method of claim 1 or 2 which further includes, after step (a), the step of removing analyte or specific binding partner that is not bound to the ligand contained in each test region.

4. The method of any of claims 1-3 wherein said ligands are polymers having maximally diverse characteristics with respect to at least two parameters selected from the group consisting of hydrophobicity, charge distribution, and corrugation factor.

5. The method of any of claims 1-4 wherein said bound analyte or specific binding partner is detected by an enzyme or radioactive label or wherein said bound analyte or specific binding partner is detected by assessing a difference in a characteristic of the test region.

6. The method of any of claims 1-5 wherein said systematically varying affinity is for analyte and said test regions further contain said specific binding partner.

7. The method of claim 2 wherein said test regions are arranged linearly on a planar surface as a series of hydrophilic matrices separated by hydrophobic barriers.

8. The method of any of claims 1-7 wherein said test regions are arranged sequentially with ligands of monotonically increasing affinity for said analyte or specific binding partner.

9. A series of test regions suitable for the conduct of the method of any of claims 1-8.

10. A test device which device comprises a series of test regions arranged to permit orderly retrieval of the result in each and which contain a series of ligands of systematically varying affinity for analyte or for a specific binding partner capable of binding a desired analyte.

11. The device of claim 10 wherein said series of test regions is arranged sequentially with respect to said monotonic increase of affinity, and/or wherein said ligand is coupled to a solid support.

12. The device of claim 11 wherein said test regions are comprised of a hydrophilic matrices separated by hydrophobic barriers.

13. The device of any of claims 10-12 wherein said systematically varying affinity is for analyte and test regions further contain said specific binding partner.

14. 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 contained in a complex that includes a control agent to the environment; and effecting the release of the desired substance from the complex by providing a sufficient concentration of releasing agent, wherein the releasing agent competes with the control agent for the desired substance or with the desired substance for the control agent, or wherein said releasing agent disaggregates said control agent.

15. The method of claim 14 wherein said releasing agent is endogenous to the environment or wherein said releasing agent is associated with the target, or wherein the releasing agent is administered to the environment at such time that release is to be effected.

16. The method of claim 14 or 15 wherein the control agent inactivates a biological activity characteristic of the desired substance when in the complex, or wherein the control agent is a size-enhancing agent.

17. The method of claim 16 wherein the size-enhancing agent comprises a bifunctional ligand capable of aggregation, or wherein the size-enhancing agent comprises a polymer coupled to at least one affinity ligand directed to the desired substance or control agent.

18. A method to deliver a moiety having specific affinity for a target located in an animal subject to said target wherein said moiety has an aggregate weight of less than 70 kD, which method comprises administering to said subject a complex that includes said moiety and at least one size-enhancing agent, said complex having an aggregate weight of more than 70 Kd;

permitting the complex to home to the target whereby at least some of the moiety is bound to the target;
administering to said subject a releasing agent that either selectively binds the size-enhancing agent at the expense of said moiety in an amount effective to displace said moiety from the complex or disaggregates the bulking agent; and permitting any moiety not bound to the target to clear from the environment.

19. A complex of an aggregate weight greater than 70 kD which consists essentially of an immunoconjugate of less than 70 kD and at least one size-enhancing agent, which size-enhancing agent is capable of self-aggregation.

20. A method to deliver a biologically active substance to a target which method comprises administering to an environment containing said target a complex consisting essentially of said active substance and a control agent, wherein the control agent inactivates the substance when in the complex, and wherein said control agent is removed from the complex by interaction with a releasing agent, and providing said releasing agent to dissociate the complex.

21. The method of claim 20 wherein the releasing agent is endogenous to the target, and said providing comprises permitting the complex to home to the target so as to remove the control agent and activate the active substance.

22. The method of claim 21 wherein the releasing agent is glucose, the substance comprises insulin, and the control agent is a dextran.

23. A method to regulate the half-life of a biologically active substance in a subject, which method comprises administering to a subject a complex containing said biologically active substance and a size-enhancing agent, which complex has an aggregate weight of more than 70 kD;
permitting the complex to remain in the subject for the desired time; and administering a releasing agent that dissociates the complex into components of <70 kd, thus permitting the components to clear the subject.