WO1996010176A1

WO1996010176A1 - Means for reversibly inactivating biological molecules

Info

Publication number: WO1996010176A1
Application number: PCT/US1995/012310
Authority: WO
Inventors: Thomas Reichert
Original assignee: Becton Dickinson and Co
Current assignee: Becton Dickinson and Co
Priority date: 1994-09-28
Filing date: 1995-09-26
Publication date: 1996-04-04
Anticipated expiration: 1997-03-28

Abstract

This invention discloses high affinity complex or compound reagents formed by a plurality of low to moderate affinity species for a particular target, connected by linkers which can be easily disrupted or lysed. Once lysed, the individual species exhibit reduced affinity for the target, thereby becoming reversibly bound.

Description

MEANS FOR REVERSIBLY INACTIVATING BIOLOGICAL MOLECULES

Background of Invention

The use of binding molecules having specific affinity foi a particular moleculai species is a well-known technique in both the diagnostic and therapeutic fields The binding molecule, generally an antigen or antibody specific foi its target is ordinarily selected for its ability to bind tightly to its target

In certain applications, the binding molecule will exert an inhibitory effect on the activity of its target, permitting that taiget to be effectively removed fiom the sv stem Reversal of the binding to achieve ie-activation of the taiget is generally not practical due to the severe nature of the conditions or agents needed to achieve such reversal such such conditions oi agents will often denatuie oi otherwise altei the target, rendenng U permanently inactive

The use of brute force methods of reveisal of binding in theiapeutic applications compounds this difficulty, as many such conditions oi agents aie unsuitable foi in vivo use

Furthermore, the use of less tightly bound, binding molecules is not a viable option in most situations, since it is desirable, and peihaps even necessary, to keep the binding molecule taiget complexes intact until leveisal is desπed Lowei affinity binding molecules will become oie easily dissociated fiom the taiget, thereby pioducing a i educed effect with attendant loss of sensitivity and usually also, specificity

Thus, theie exists a leal need for binding molecules which will tightly bind to a taiget which binding can be ieadily ieversed on demand b\ tieatments which will not affect the target. This need exists with separate requirements for in vivo and m vitro applications

Summary of Invention

The instant invention presents a means whereby the activity of a biomolecular substance may be temporarily suspended and subsequently reactivated when desired

In vitro, this invention will permit the effectively complete, yet reversible inhibition of biomolecular substances. Such a capability will permit stable sample collection, storage, and transport, with a controllable reinstitution of the activity of the biomolecular-molecular substance very close to the conditions actually present m vivo at the time of sample collection. In vivo, this invention presents two separate effects In the first, similar to the w vitro application, an inhibitor can be introduced m vivo such that the target biomoleculai substance can be tightly bound to the inhibitor, but with the activity later remstituted by disrupting the binding. Alternatively, the lnhibitoi-biomolecule complex can be formed w vitro and subsequently introduced in vwojwit . subsequent re-activation in settings when the desired conditions, location, target, etc., are realized This invention also permits the generation of very high affinity and specificity reagents from relatively low affinity and low specificity components.

Briefly, the invention involves the creating of a complex oi compound reagent, which is formed by linking together a plurality of molecular binding segments which, each individually, express a low or moderate affinity for a particular taiget species Such compound reagents possess a much greater affinity for the taiget than the meie sum of the low affinities for the target of each of the molecular binding segments and, indeed, if the compound reagents could be constructed such that each molecular binding segment would act independently of the others, the affinity of the compound reagent would be the product of the individual affinities. Further, because the binding occurs at multiple sites, the "off-time" will be quite long. By proper choice of molecular binding segments, die affinity and/or off- time of any compound reagent can be optimized for a given application

These molecular binding segments are connected or linked together by molecular S95/12310

linkers which are selected for their ability to be disrupted or lysed by treatments which do not deleteriously affect the target system and, in a preferred embodiment, even the molecular binding segments. By use of appropriate molecular linkers, a compound reagent, comprised of alternating molecular binding segments and molecular linkers can be formed having virtually any desired binding affinity for a given target. However, once the molecular linkers are lysed, since the molecular binding segments are, generally, low affinity reagents, they will rapidly dissociate from the target, leaving it unfettered. Thus, the binding affinity can be conveniently manipulated by use of appropriate linkers and binding segments.

A wide array of applications can benefit from the use of this invention, expressed as a target molecule, which can be reversibly bound to a compound reagent. If the reagent exerts an inhibitory effect on the activity of the target, the target can be inhibited until desired, and then "switched on" by lysing the linkers. Alternatively, if the reagent exerts a catalytic or positive effect on the target molecule, the lysis can be utilized to "switch off the activity. This permits significant latitude in the ultimate end use.

Detailed Description of the Invention Several important features of this invention are summarized as follows:

1. Low (relative to the generally observed levels for useful single-binding- site reagents) affinity reagents which bind to different portions of a target, and which have low "Off times for such reagents (< 1 sec.) are utilized for the binding segments. While they exhibit low affinity, generally the specificity must be relatively high, at least for one. and preferably, most reagents comprising the compound reagent; in some reagents, on the other hand (especially those for which binding sites are rare) some degree of lack of specificity is tolerable. Furthermore, preferably at least one of the affinity reagents should be selected for very high "on" rates. These affinity reagents could be any binding element, including small molecular species, peptides, nucleic acids, antibodies, lipids and polysaccharides.

2. A compound reagent is formed by connecting two or more of these relatively low affinity reagents by linkers These linkers could conceivably be specifically crafted to precisely separate the affinity reagents, and position then in the desned conformation Other embodiments could utilize flexible hydrophihc hnkeis open chain in structure, but at least of very low affinity for the target and having no effect, othei than connection to the low affinity reagents These Imkers could be comprised of amino acids. nucleic acids, or even simpler chemical species such as polyethylene glycol, or othei ohgomers

3 The linkers must be chosen to have a specific sensitivity to degradation which is not necessarily (though possibly) shared by the low affinity reagents In general the affinity leagent segments, when bound in the compound reagent target complex will be somewhat shielded fiom degradation

4 The compound reagent will have an affinity greatei than any of the low affinity reagent substituents, in general, much greatei Because of the multipartite binding the "off-time" of such a leagent should inciease even moie than the proportionate inciease m affinity The compound reagent will usually be "constructed" so that it will have a compound affinity at least as gieat as a very high affinity antibody, with an "off time of hours to days The specificity will, by virtue of the requirement for binding of the multiple low affinity reagents, simultaneously, be very great, thus, one oi more of the low affinitv reagents may have a level of specificity for the target less than would be toleiable in a single reagent

5 At least one of the low-affinity reagents should be chosen to bind eithei at oi so as to inhibit access to, the active site of the taiget, to permit effective inhibition of the target

Candidates for Binding Segments

The low affinity reagent segments can be any linkable elements, which exhibit both binding affinity for the target combined with the desired degree of specificity Such elements include antibodies, haptens. nucleic acids, certain carbohydrates, and certain protems

One preferred embodiment, because of the ease with which affinity can be controlled. and because of die simplicity of the linkage chemistiy, is the use of nucleic acid constructs produced by the process descπbed m U S Patent No 5,270, 163 to Gold, et al, incorpoiated herein by reference These can be discovered by truncating the SELEX process lelatively early, when affinities are still in the high nanomolai oi low miciomolai iange Blocking d e site of binding of such a leagent, for the puipose of using SELEX oi any othei affinity ieagent generating piocess to geneiate affinity ieagents to other sites, can be accomplished using a highei affinity reagent such as those geneiated latei in the SELEX piocess specific for the same site

Candidates for Linkers

There aie a large number of chemical entities which could be suitable foi use as lysable linkeis Several prefeπed candidates aie summaiized below, but the list is not mtended to be exclusive

The use of 2' amino- or 2' fluoro- nucleosides in ohgonucleotide linkage segments is useful, since the pynmidine-specific endonucleases piesent in human serum will be ineffective in lysing such linkers Such linkage segments would, howevei, be quite susceptible to puπne-specific endonucleases from bacterial species Divalent zinc is known to slowly degrade RNA at physiological pH, and thus might be a candidate for effectuating time release, m vivo.

Rn ozymes could be constructed with a high degiee of specificity for specific RN Λ sequences used as linkers Such agents would selectively lyse linkage segments both in vitro

Restriction endonucleases nick nucleotide stiands at a point determined by particulai local sequences Such sequences could be embedded in eithei DNA oi RNA linkers

Since extracellular RNA and DNA has no known physiological function, agents lytic for polynucleotide strands need not be particulai ly specific to be effective in lysing nucleotide linkage segments in the situation in which the application is either extracellular o cell surface. For in vivo applications of this sort, of course, considerations of potentia immune reactions may dominate, so that either a very small or non-immunologically reactiv cleavage agent, or an array of such cleavage agents, might be necessary. An alternative to the design and generation of a synthetic linkage segment is th

"walking" extension of the SELEX method of Turek and Gold described in U.S. Patent No 5,270, 163. previously incorporated by reference.

The phenomenon of enantiomer preference is known to exist for proteases, e.g. certain plant proteases are specific for d-amino acids, while many animal proteases ar specific for 1-amino acids. Therefore, linkage segments constructed of appropriate optica isomeric amino acids, augmented by certain known sequence preferences, could be used t provide both specifically lysable linkages and a degree of time release.

Similarly, many inexpensive, readily available synthetic materials could be used t generate hydrophilic and flexible linkage segments. One example of such is organic oligomers. The -O-CH2 repeat unit, for example, is both flexible and interacts with aqueous solvent by hydrogen bonding. Specific cleavability could be achieved by condensing two heterobifunctional oligoethyleneglycols with a homobifunctional unit containing a specifically cleavable unit. For example,

THPO O OH HO O OTHP

+

In the case in which X is a diol, cleavage conditions in vitro could be very mild, utilizing periodate (0.015M).

In the case in which X is a disulfide, very mild cleavage conditions are available both in vitro and in vivo. The disulfide unit is cleavable with sulfhydryl reagents, such as glutathione, and the rate of cleavage can be engineered by adjusting differential charge in the environment.

In the case in which X is a peptide, the amino sequence chosen can be specific for a peptidase.

In the case in which X is comprised of ester units, these may be chosen to be those recognized by an esterase. As a diagnostic touch, if nitrophenyl esters are employed, a strong UV signal (@ 400nm) appears upon cleavage.

In the case in which X is an acetal linkage, cleavage may be effected by mild acidic conditions, with slow cleavage at pH 5-6. and rapid cleavage pH < 4.

X can also be a linkage disruptable by an input of radiation. A particularly useful type of this phenomenon would be a link particularly susceptible to energy deliverable by a focused energy source such as a laser or maser. Depending on the wavelength of energy required for bond disruption, this approach could be useful both in vitro and in vivo. There is nothing unique about polyethyleneglycol as the linkage backbone. The general principle is merely that a flexible, hydrophilic structure be generated into which a specifically lysable link is inserted, so that the linkages could be lysed, either in vitro as part of the analytical process or in vivo.

EXAMPLES

The following examples are illustrative of certain preferred embodiments of the invention, but are not intended to be illustrative of all embodiments.

Example 1 The small protein, hirudin, originally isolated from the salivary gland of the leech, is an extraordinarily specific and avid binder of thrombin. The three dimensional structure of this protein is essentially a perfect fit for each of the three more or less adjacent binding sites of the thrombin molecule. Theoretically, a NeXamer™ could be constructed which mimics the entn e structui e of hirudin

Alternatively, by the blocking strategy discussed above, and pieviously descnbed by NeXagen, thiee sepaiate ieagent segments could be cieated

If the thiee affinity ieagent segments aie joined by two linkage segments, a compound NeXamei™ is formed The linkage segment monomei elements, oi at least a particulai link in the linkage segment, could be chosen to have a susceptibility to specific degradation Alternatively , these linkage segments can be fashioned eithei wholly 01 in part from several different monomei elements having a diffeiential susceptibility to degradation 01 the linkage segments can be made of a controllably variable mixtuie of the diffeient monomei elements. whichever pioduces the desiied diffeiential lysabiliry. thus ci eating a time-ielease effect

Even if each element in the compound NeXamei had a low affinity (on the order of miciomolai) foi the taiget, if each linked element weie geneialK independent of the otheis, the compound NeXamei could have an affinity appioaching 10^{~ 1 X} M This essentially irreversible binding could then be reversed by lysing the molecular hnkeis This mechanism may also create an "Embedded Antidote" effect, which is most clearly achieved by the use of an agent (preferably a small non-immunogenic moiety) which, when administeied, induces disruption of the lysable link undei m vivo conditions This disruption could occui either by the direct action of the lysing agent itself oi by the action of active moieties geneiated by the introduced agent, if the lysing moieties slowly degiade the complex, a time lelease effect could be geneiated The time-ielease effect pei se is likely to be useful onK theiapeutically, but may also be a ke\ to diffeiential lysabiliry in certain w vitro systems b\ . foi example, facilitating the action of a degiadative enzyme not usually found in the taiget system Once the lysis occuis, by whatevei means, the taiget would be fiee to act on the s\ stem It

be necessary, in certain particulai taiget applications, foi one of the binding reagent elements to be lelatively high in affinity, in oidei to piovide sufficiently long "on" time foi the othei low ei affinity elements to find then positions (Such a binding ieagent might be called an "anchoi " ieagent) As long as that elatively high affinity element does not attach in or near the active site of the target, or in any other way affect, by its binding the function of the target system, there is no other effect, and the principle is unchanged. Example 2

A specific application is anti-coagulation of blood by the specific inhibition of thrombin, with the selective option of disrupting other enzyme systems, and with the additional option of reversing any inhibition emplaced, without disrupting chemical environments necessary for physiological function, e.g., divalent ion dependent chemistiy

A more general application is the performance of tests of the coagulation system by reversing the inhibition of thrombin immediately prior to the initiation of measurements of any of the components of the coagulation cascade (prothrombin time, activated thromboplastin time, clotting time, etc.)

A still more general application is the inhibition of any particular enzyme at the time of sample collection, transport/storage of the inhibited sample; and reversal of the inhibition as the first step in the process of analyzing the inhibited enzyme system. The inhibition and reversal of the target enzyme or other biomolecular system may be separated or combined with the inhibition of other biomolecular systems. For example, the target may be a biomolecular species best measured in whole blood. Thrombin can then be inhibited by a process not reversible by the process which reverses the inhibitor of the target. Alternatively, the taiget may be a molecule or molecular system best stored in whole blood, but best analyzed in serum. In this instance, the mechanism of reversal of the inhibitor of the taiget system might be the same as that used to inhibit thrombin. Example 3

Many processes for generating New Chemical Entities for therapeutic applications commonly produce reagents which have some activity, but insufficient specificity and/or affinity (iNCEs). If two or more of such iNCE's bind to different sites on the target biomolecular system, at least one of which inhibits the function of that system, then, a compound specie, comprised of the iNCEs which bind to different sites linked together, will, in general, have an affinity higher than any of its components. The specificity of the compound iNCE will be much greater than that of any component iNCE.

Very high affinity therapeutic agents, (for example, hirudin) have the disadvantage that their effects are difficult to reverse. A compound iNCE with linker segments comprised of elements which are controllably biodegradable in situ might be said to contain an Embedded Antidote.

If the links for a compound iNCE can be constructed of at least two types of linker units, one resistant to ambient degradative effects (e.g. endonucleases), and one sensitive thereto, then the compound iNCE could be synthesized to be completely resistant to degradation, completely sensitive to degradation, and

(if either other linker types are intermediately sensitive to degradation or if mixing linker types in the same linker segment produces intermediate sensitivity) variably sensitive to degradation.

A mixture of all three types of a particular compound iNCEs would produce a time- release foπnulation in which the action of the compound agent would decrease (possibly controllably so) as the links were digested.

It is apparent that many modifications and variations of this invention as herein set forth may be made without departing from the spirit and scope hereof. The specific embodiments described are given by way of example only and the invention is limited only by the terms of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A reagent capable of reversibly binding to a target comprising a plurality of molecular binding segments linked together by molecular linkers characterized in that each of said molecular binding segments possesses a low to moderate affinity for at least a portion of said target, and some or all of said molecular linkers can be disrupted or lysed by treatments which will not affect the target.

2. The reagent of Claim 1 wherein each of the molecular binding segments is selected from the group consisting of peptides, antibodies, haptens. nucleic acids, and polymers thereof.

3. The reagent of Claim 1 wherein each of the molecular linkers is selected from the group consisting of 2'-aminonucleosides, 2'-fluoronucleosides, RNA, DNA, d-amino acids, and ethylene glycol oligomers linked by a bifunctional unit.

4. The reagent of Claim 3 wherein the bifunctional unit is a diol.

5. The reagent of Claim 3 wherein the bifunctional unit is a disulfide.

6. The reagent of Claim 1 which further comprises at least one molecular binding segment having a high affinity for the target.

7. A method for reversibly binding a target compound comprising:

(i) mixing said target with an effective amount of a reagent which comprises a plurality of molecular binding segments linked together by molecular linkers characterized in that each of said molecular binding segments possesses a low to moderate affinity for at least a portion of said target, and each of said molecular linkers can be disrupted or lysed by treatments which will not affect the target, such that said target becomes rightly, but reversibly bound to said reagent in a complex; and

(ii) subsequently exposing said complex to an effective amount of a material or mixture of materials capable under appropriate conditions of disrupting or lysing said molecular linkers such that some or all of the low to moderate affinity molecular binding segments become unlinked, thereby reducing the affinity of the reagent to that of the binding segments.

8 The method of Claim 7 wherein each of the moleculai binding segments is selected from the group consisting of peptides. antibodies, haptens. nucleic acids, and polymers thereof.

9. The method of Claim 7 wherein each of the moleculai linkeis is selected fiom the group consisting of 2'-aminonucleosιdes. 2'-fluoronucleosιdes. RNA, DNA. d-amino acids, and ethylene glycol oligomers linked by a bifunctional unit

10 The method of Claim 9 wherein the bifunctional unit is a diol

1 1. The method of Claim 9 wherein the bifunctional unit is a disulfide

12 The method of Claim 7 which furthei compiises at least one moleculai binding segment having a high affinity for the target.

13 The method of Claim 7 wherein the target and reagent are mixed m vitro and the moleculai linkers are disrupted or lysed in vivo.

14. A method for reversibly inhibiting an active target compound:

(i) mixing said target with an effective amount of a reagent which comprises a plurality of molecular binding segments linked together by molecular linkers characteπzed in that each of said molecular binding segments possesses a low to moderate affinity foi at least a portion of said target, and each of said molecular hnkeis can be disrupted or lysed by treatments which will not affect the target, such that said target becomes tightly but leversibly bound to said reagent m a complex, wheiein at least one of said molecular binding segments binds to the target in such a manner as to inhibit its activity. and

(π) subsequently exposing said complex to an effective amount of a mateπal or mixtuie of mateπals capable, undei appiopnate conditions, of disrupting oi lysing said moleculai linkers such that some oi all of the low to modeiate affinitv moleculai binding segments become unlinked, theieby reducing the affinity of the reagent to that of the binding segments, and the activity ceases to be inhibited

15 The method of Claim 14 wherein each of the moleculai binding segments is selected from the group consisting of peptides, antibodies haptens nucleic acids and polymeis theieof

16 The method of Claim 14 wherein each of the molecular linkers is selected from the group consisting of 2'-amιnonucleosιdes, 2'-fluoronucleosιdes, RNA. DNA d-amino acids, and ethvlene glycol oligomers linked by a bifunctional unit

17 The method of Claim 16 wherein the bifunctional unit is a diol

18 The method of Claim 16 wherein the bifunctional unit is a disulfide

19 The method of Claim 14 which furthei comprises at least one moleculai binding segment having a high affinity for the target

20 The method of Claim 14 wherein the target and reagent are mixed m vitro and the molecular linkers aie disrupted or lysed in vivo