CA2205373A1 - Phosphatase activated crosslinking, conjugating and reducing agents; methods of using such agents; and reagents comprising phosphatase activated crosslinking and conjugating agents - Google Patents

Abstract

The present invention provides crosslinking, conjugating and reducing agents which are functional with at least one phosphorothioate monoester group (-SPO3- 2). Crosslinking and conjugation methods as well as solid phase reagents and conjugates which are useful in immunoassays are also provided. Crosslinking and conjugating agents of the invention generally comprise a compound corresponding to the formula (I): Q-(S-PO3-2)n, wherein n is at least 1 and Q is a straight or branched monomer, polymer or oligomer having an average molecular weight between about 200 and about 1,000,000. Additionally, when n is 1, Q comprises at least 1 additional reactive functionality. The reducing agents that are provided conform to a compound of formula (Y) wherein (A) and (Z) can be independently selected from C1-C5 alkyl and CONH(CH2)p wherein p is an integer between 1 and 5.

Description

CA 0220~373 1997-0~-14 wo 96/17580 PCT/USg5/15586 PHOSPHATASE ACTIVATED CROSSLINKING, CONJUGATING AND REDUCING AGENTS; METHODS OF
USING SUCH AGENTS; AND REAGENTS COMPRISING
PHOSPHATASE ACTIVATED CROSSLINKING AND

Field of the Invention The present invention relates to cros~linking, conjugating and reducing agents and, in particular, relates to phosphorothioate monoester functional 1 0 cros~linking, conjugating and reducing agents.

Background of the Invention Tmmllnn~says have become a useful diagnostic tool for detecting the presence or amount of an analyte in a test sample. Various forms of 1 5 immunoassays, as well as the reagents and procedures necessary to pt;~ n such assays, are well known in the art.
One form of a conventional solid-phase immunoassay is a "sandwich assay" which involves cont~cting a test sample suspected of cont~ining an analyte with a subst~nti~lly solid inert plastic, latex or glass bead or 2 0 microparticle, or other support m~teri~l which has been coated with a protein or another substance capable of binding the analyte to the surface of the support.
The analyte and the protein or substance capable of binding the analyte are commonly referred to as a "binding pair" or individually known as "binding members", and a support m~tori~l coated with a binding member is variably 2 5 refe.r.red f~o as a "sol d phase rezger.t". Af~.er ~e ar.al~.e is bour.d to .he support m~t~ori~l the rr.. l -~ il lg test sample is removed from the support and the analyte bound support m~t~ori~l is treated with a second binding member. The second binding member can be conjugated to a signal generating group such as an enzyme, a fluorophore or a ch~omill~min~oscent label and collectively, the binding 3 0 member/signal generating group complex is variably referred to as a "conjugate"
or "inrlio~tor reagent". The conjugate becomes bound to the analyte which is bound on the support and the solid support, having the first binding member, theanalyte and conjugate bound thereon is separated from any unbound conjugate, typically with one or more wash steps. In the case of an enzyme immunoassay, 3 5 an indicator substance, for example, a chromogenic substrate, is added whichreacts with the enzyme to produce a color change. The color change can be CA 0220~373 1997-0~-14 observed visually, or more preferably by an in~ nt, to in~lic~te the presence or amount of an analyte in the test sample. For solid phase fluorescence or chPmilnminescence immlmo~es~ys~ fluorescent labeled binding members can be mnnitored using excit~tion at an ~p~lupliate wavelength, while 5 ch~-.milnmin~.sc~nt labeled binding members can be monitored after a reaction which chtq.mi~lly activates the cht~.mil--min~sc~.nt label and gen.or~tes light which can be detected by photometric means.
Tmmllno~c~y reagents such as a solid phase reagent or a conjugate are typicaUy m~nllf~ctured in buLlc and small amounts of the buL~ reagents are used 1 0 to perform individual assays. Rem~ining buLk reagents are then stored for subsequent assays. The stability of these reagents is paramount to providing analytical methods which exhibit precision and ullirOl~ y among individual assays. Instability of such reagents provides for unreproducible assay results as well as an increase in the costs of m~lic~l services because unstable bulk 1 5 reagents must be discarded.
Various methods have been used to increase the stability of immllno~c~y reagents by preserving the integrity and/or activity of the compounds that comprise the reagents. Some methods of preserving immunoassay reagents involve placing additives such as proteins or carbohydrates into solutions that 2 0 contain the reagents. Another method of preserving assay reagents includes adding redllcing agents (variously referred to as "anti-oxidants") to lyophili7ed assay reagents. Unfortunately, over time, recln~ing agents are themselves oxidized and consequently provide only short term reagent protection. Chemical cro~linking has also become accepted as a method for stabilizing 2 5 macromolecules and thereby preserving their integrity and activity.
Chemical cros~linking can effectively be accomplich~cl by int~rmnlecular cro.~linking or intramolecular crosslinking wherein molecules having a greater degree of cro~.~linking are generally more stable than molecules having a lesserdegree of cro~linking. Intramolecular cros~linking refers to covalent bonds or 3 0 cro~link~ that are formed within a single rmlltim~.ri~ or monomeric chemicalentity. Hence, disulphide bonds occurring within an antibody are exemplary of intramolecular cros~linking On the other hand, intermolecular cro.c~linking refers to covalent bonds or crosslinks that are formed between more than one distinct ch~-mi~l entity such as the bonds which are formed when one compound 3 5 is conjugated to another. Accordingly, an immunoassay's indicator reagent comprising, for example, an antibody attached or conjugated to an enzyme, is CA 0220~373 1997-0~-14 .~

exemrl~ry of int~-.rmoiecular cro.~.~linking. Additionally, an immunoassay's solid phase reagent or an affinity chromatography gel comprising an antibody bound to a cl~o",atographic gel are further ~x~mples of intermolecular cro.s.~linking.While intermolecular cros.slinkin.~, as exemrlifie~l above, is an effective means 5 of conjugating one ch~mi~l entity to another, generally, the degree of crosslinking is minim~l and the stability of compounds conjugated in this manneris seldom enhanced.
Cros~linking a ch~mi~l entity t~hrough multipoint int~rm~lecular cro~linking, however, can greatly enhance the compound's stability.
1 0 Multipoint int~-rmnlt-cular cros.~linking typically results in the formation of a plurality of bonds between a cro.~slinkin.~ agent and the compound which is cros~link~. Such cros~linking is most commonly associated with the bonds formed between a soluble entity such as, for example, a polymer and a protein such as, for example, an enzyme.
1 5 F.x~mples of intr~molecular and int.ormolecular cro.~slinking have previously been described. For example, Wong et al., Enzyme Microb.
Technol., vol 14, pg 866-874 (1992); generally outlines techniques and reagents for intramolecularly and intçrmolP.cularly crosslinking compounds.
Additionally, U.S. Patent No. 4,652,524 and U.S. Patent No. 4,657,853 2 0 disclose the cro~.clinking of mllltiple enzymes to a polymer, and further cro~slinking the polymeric enzyme to a binding member. European Patent Application No. 0 049 475 discloses a method for multipoint intermolecular crosslinking an enzyme with a soluble polymer. Unfortunately, however, the ~o~ ioned methods require harsh con~lition.~ to effect cros.slinking, lack 2 5 control over the cros.~linking process, and/or result in randomly polym--.ri7ed protein aggregates which are often non-soluble. Moreover the biological performance of the cros~linked entity is often negatively affected as manifestedby, for example, lower binding ~ffinities, ~limini.chçd enzymatic turnover, recognition impairment by specific lig~ntl.~, and the l~e.

CA 0220~373 1997-0~-14 WO 96/17~80 PCT/US95/15586 S--mm~ry of the Invention The present invention provides cros~linking, conjugating and reducing agents which are functional with at least one phosphorothioate monoester group (-SPo3-2). Agents of the inv~ntion can be activated by deprotecting or hydrolyzing the phosphate group(s) compri~ing the phosphorothioate monoester(s). Upon activation, the agents herein provided display an nucleophilic thiol group which can be used in cros~linking, reducing and/or conjugating c~pacitit-s The crosslinking, conjugating and redllcing agents herein provided can, for ex~mrle, be activated in a suitable pH environment but 1 0 the phosphate group(s) can also be hydrolyzed with a phosphate hydrolyzing enzyme. Advantageously, through enzymatic activation, innocuous phosphate ions and activated agent are the major activation reaction products.
Cros~linking and conjugating agents of the invention generally comprise a compound corresponding to the formula (I), shown below, wherein n is at 1 5 least 1 and Q is a straight or branched monomer, polymer or oligomer having an average molecular weight between about 200 and about 1,000,000.
Ad~litit n~lly, when n is 1, Q comprises at least 1 additional reactive functionality.
(I) 2 0 Q-(S-PO3-2)n A method for cro~linking and conjugating compounds which is provided herein comprises activating a compound corresponding to the formula a) to form an activated agent and contacting the activated agent with at least one compound which is functional with an electrophilic group. Preferably, the compound of the2 5 formula (I) is activated with a pH of between about 4.0 and about 5.5 or with a phosphatase enzyme.
Conjugates and solid phase reagents are also provided herein. A
conjugate as taught herein will generally comprise at least one binding member and at least one detect~hle moiety bound to the residue of a compound 3 0 corresponding to the formula (I). On the other hand, a solid phase reagent will generally comprise at least one binding member and a solid phase attached to theresidue of a compound having the formula (I).
Reducing agents are also provided which generally conform to a compound of the formula (Y), shown below, wherein (A) and (Z) can be 3 5 independently selected from Cl-Cs alkyl and CONH(CH2)p wherein p is an integer between 1 and 5.
-CA 0220~373 1997-0~-14 (Y) ~OH
,C C~,z spo3-2 SPO3 Brief Description of the Drawings Figure l(a)-(f) illustrate cros~linking and conjugation agents.
Figure 2(a)-(e) illustrate a method of st~bili7ing a compound.
Figure 3(a)-(f~ illustrate heterobifunctional conjugation agents.
Figure 4(a)-(d) illustrate a method of conjugating two distinct chlomiç~l entities.
1 0 Figure S(a)-(d) illustrate a method of conjugating a st~bili7t-d compound and a second compound.
Figure 6(a)-(d) illustrates site specific conjugation of a st~hili7ed compound with the Fc region of an antibody.
Figure 7(a)-(b) illustrate stable reducing agents.
1 5 Figure 8-13 graphically illustrate the various property improvements displayed by st~bili7e-1 compounds.
Figure 14(a)-(e) illustrate the conjugation of two ch~mic~l entities using a heterobifunctional conjugation agent.
Figure lS illustrates the effect stoichiometric manipulation has on the size 2 0 of the products produced in cros~linking reaction.

Detailed Description of the Invention I. Definitions The following clefiniti(lns are applicable to the invention:
2 5 The term "analyte", as used herein, refers to the compound or composition to be detected or measured and which has at least one epitope or binding site. The analyte can be any substance for which there exists a naturally occurring binding member or for which a binding mt-mh~r can be prepared.
Analytes include, but are not intended to be limited to, toxins, organic 3 0 compounds, proteins, peptides, microorganisms, amino acids, carbohydrates, nucleic acids, hormones, steroids, vitamins, drugs (inclu-ling those ~iminictered for therapeutic purposes as well as those ~minictered for illicit purposes), virus particles and metabolites of or antibodies to any of the above substances. For CA 0220~373 l997-0~-l4 r WO 96/17580 PCT/US95/15~86 example, such analytes include, but are not intended to be limited to, ferritin;cre~tinin~ kinase MB (CK-MB); digoxin; phenytoin; phenobarbitol;
carbanla;Gcyi,le; vancomy~l; gcll~lllycill; theophylline; valproic acid; qllini~ine;
lc~ g hormone (LH); follicle stimnl~ting hormone (FSH); estradiol, progesterone; IgE antibodies; vitamin B2 micro-globulin; glycated h~moglnbin (Gly. Hb); cortisol; digitoxin; N-acetylproc~in~mi(le (NAPA); proc~in~mitle;
antibodies to rubella, such as rube~la-IgG and rubella-IgM; antibodies to toxoplasmosis, such as toxoplasmosis IgG (Toxo-IgG) and toxoplasmosis IgM
(Toxo-IgM); testosterone; salicylates; ~cet~minophen; hepatitis B virus surface 1 0 antigen (HBsAg); antibodies to hepatitis B core antigen, such as anti hepatitis B
core antigen IgG and IgM (Anti-HBC); human immune deficiency virus 1 and 2 (HTLV); hepatitis B e antigen (HBeAg); antibodies to hepatitis B e antigen (Anti-HBe); thyroid stim~ ting hormone (TSH); thyroxine (T4); total triiodothyronine (Total T3); free triiodothyronine (Free T3); carcinoembryoic antigen (CEA); and 1 5 alpha fetal protein (AFP); and drugs of abuse and controlled substances, inclll-ling but not inten-ler7 to be limited to, amph~t~minto; methamph~l~"~h-e;b~bi~ulalcs such as amobarbital, secobarbital, pentobarbital, phenobarbital, andbarbital; benzodiazepines such as librium and valium; c~nn~hinoids such as hashish and nla ijuana; cocaine; fentanyl; LSD; opiates such as heroin, 2 0 morphine, codeine, hydromorphone, hydrocodone, methadone, oxycodone, oxymorphone and opium; phencyclidine; and propoxyphene as well as metabolites of the above drugs of abuse and controlled substances. The term "analyte" also in~ des any antigenic substances, haptens, antibodies, macromolecules and comhin~*nns thereof.
2 5 "Binding member", as used herein, means a member of a binding pair, i.e., two dirrclcn~ molecules where one of the molecules through chemical or physical means specifically binds to the other molecule. In addition to antigen and antibody specific binding pairs, other specific binding pairs include, but are not intt-n~iecl to be limited to, avidin and biotin, carbohydrates and lectins, 3 0 complementary nucleotide sequences, complemPnt~ry peptide sequences, effector and receptor molecules, an enzyme cofactor or substrate and an enzyme, an enzyme inhihitor and an enzyme, a peptide sequence and an antibody specific for the sequence or the entire protein, polymeric acids and bases, dyes and protein binders, peptides and specific protein binders (e. g., ribonuclease, S-3 5 peptide and ribonuclease S-protein),and the like. Furthermore, binding pairs can include members that are analogs of the original binding member, for example, CA 0220~373 l997-0~-l4 WO 96/17580 PCTtUS95/lS586 an analyte-analog or a binding member made by recombinant techniques or molecular ~nginçering If the binding member is an immunoreactant it can be, for ex~mplt-., a monoclonal or polyclonal antibody, a recomhin~nt protein or recombinant antibody, a chimtoric antibody, a ll~ e(S) or fragment(s) of the foregoing, as well as a preparation of such antibodies, peptides and nucleotidesfor which snit~hility for use as binding members is well known to those skilled in the art.
The term "detectable moiety", as used herein, refers to any compound or conventi~n~l detectable chemic~l group having a detectable physical or chemical 1 0 property and which can be used to label a binding member to form a conjugate therewith. Such detectable chçmic ~1 group can be, but is not int~ndetl to be limited to, ellzyma~ically active groups such as enzymes, enzyme substrates, prosthetic groups or coenzymes; spin labels; fluorescent molecules such as fluorophores and fluorogens; chromophores and chromogens; luminescent 1 5 molecules such as luminophores, cht~milllminophores and biolnminophores;
phosphorescent molecules; specifically binll~hl~ ligands such as biotin and avidin; electroactive species; radioisotopes; toxins; drugs; haptens; DNA; RNA;
polys~cch~rides; polypeptides; liposomes; colored particles and colored microparticles and the like.
2 0 A "solid phase", as used kerein, refers to any m~teri~l which is s~lkst,.nti~.lly insoluble. Tke solid phase can be chosen for its intrin~ic ability to attract and immobilize a binding member to form a capture reagent.
~lt~rn~tively, the solid phase can retain an additional receptor which has the ability to attract and immobilize a binding member to form a capture reagent.
2 5 The ~ ition~l receptor can include a charged subst~nce that is oppositely charged with respect to a binding member or to a charged sllbst~nce conjugated to a binding member. As yet another ,.ltern,.tive, the receptor molecule can be any specific binding member which is ~tt~chç~ to the solid phase and wkich has the ability to immobilize another binding mPmher through a specific binding 3 0 reactinn The receptor mol~cllle enables the indirect binding of a binding member to a solid phase m~t~ri:~l before the ~e~ lance of the assay or during the performance of the assay. The solid phase thus can be a latex, plastic, - derivatized plastic, m~gnçtic or non-magnetic metal, glass or silicon surface or surfaces of test tubes, microtiter wells, sheets, beads, microparticles, chips, and 3 5 other configurations known to those of ordinary skill in the art.

CA 0220~373 1997-0~-14 WO 96/17580 PCT/US9~i115586 It is contemplated and within the scope of the invention that the solid phase also can comprise any suitable porous m~tt-ri~l with sufficient porosity to allow access by in~ic~tor reagents. Microporous structures are generally p~rt;lled, but m~tt~ri~lc with gel structure in the hydrated state may be used as 5 well. Such useful solid supports incllllAe natural polymeric carbohydrates andtheir synthetically modi~led, cros~link~l or ~ub~tiluled derivatives, such as agar, agarose, cross-linked alginic acid, substituted and cross-lilLked guar gums, cellulose esters, especially with nitric acid and carboxylic acids, mixed cellulose esters, and cellulose ethers; natural polymers cont~inin~ nitrogen, such as 1 0 proteins and derivatives, including cross-linked or modified gelatins; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers which may be prepared with suitably porous structures, such as vinyl polymers, including polyethylene, polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and its partially hydrolyzed derivatives, polyacrylamides, polym~th~crylates, 1 5 copolymers and terpolymers of the above polyconllen~tes, such as polyesters,poly~mitles, and other polymers, such as polyurethanes or polyepoxides; porous inorganic m~teri~lc such as sulfates or carbonates of ~lk~lin~ earth metals and m~gne~illm, including barium sulfate, c~lcinm sulfate, calcium carbonate, ,tes of aL~ali and ~lk~lintq earth metals, ~l-.,..i-.-.l-- and m~gne~illm; and 2 0 ~ll.,~,i,,l,,,, or silicon oxides or hydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel, or glass (these m:3t~ri~1~ may be used as filters with the above polymeric m~t~ri~lc); and mixtures or copolymers or the above classes, such as graft copolymers obtained by initi~li7ing polym~-ri7~tinn of synthetic polymers on a pre-existing natural polymer. All of these m~t~ri~ may be used in suitable 2 5 shapes, such as films, sheets, or plates, or they may be coated onto or bonded or l~min~tecl to a~plopliate inert carriers, such as paper, glass, plastic ~llms, or fabrics.
The porous structure of nitrocellulose has excellent absorption and adsorption qualities for a wide variety of reagents in~lnAing monoclonal 3 0 antibodies. Nylon also possesses similar characteristics and also is sllit~hle The term "solid phase reagent", as used herein, means a solid phase to which a binding member has been immobilized. Those skilled in the art will recognize that a binding member can be immobilized to a solid phase through numerous known methods including, for ex~mI-le, any chemic~l means and/or 3 5 physical means that does not destroy the specific binding properties of the specific binding member.

CA 0220~373 1997-0~-14 WO 96tl7580 PCT/US95/15586 As used herein, the term "stable" as well as forms thereof, means that a ch~mi~l entity such as, for example, a binding member is efficacious in its environment of use and therefore has or retains at least the chemical and/or biological attributes or activity relevant for its intended use. Thus, for e~mple, 5 if a stabilized compound is a binding member used in an immunoassay, it will have the capacity to bind its cornplement~ry binding member to form a binding pair; if a st~hili7eA compound is an enzyme, it will have its ell~ylllatic activity; if the st~hili7eA compound is a Aetect~hlt- moiety, it will have its detect~hle property. It will be understood, of course, that it is not necessary that a 1 0 st~hili7e~1 compound have or retain every chemical attribute as long as the chemical attribute that is not retained is not relevant for its intended use.
Additionally, a st~hili7eA compound, as compared to an unst~hili7eA compound, generally resists the loss of its relevant chemic~l attributes when exposed to environment~l stresses such as, for example, temperature extremes, pH extremes 1 5 and organic solvents. Accordingly, a stabilized compound, as conl~al~d to anun~t~bili7eA compound, generally retains its relevant chemi~l attributes for longer periods of time.
The term "test sample", as used herein, refers to a m~t~.ri~l suspected of cont~ining the analyte. The test sample can be used directly as obtained from the 2 0 source or following a pre-tre~tm~-nt to modify the character of the sample. The test sample can be derived from any biological source, such as a physiological fluid, including, blood, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, miLk, ascites fluid, mucous, synovial fluid, peritoneal fluid, amniotic fluid and the like, and fermentation broths, cell cultures, and chemical reaction 2 5 mi~Lules and the lilce. The test sample can be p~ ealed prior to use, such as preparing plasma from blood, diluting viscous fluids, and the like. Methods of tre~tment can involve filtration, Ai~till~tion, extraction, concentration, inactivation of interfering components, and the addition of reagents. In addition to biological or physiological fluids, other liquid samples can be used such as 3 0 water, food products and the like for the perfnrm~nre of environm.ontAl or food production assays. In addition, a solid m~t~riAl suspected of cont~ining the analyte can be used as the test sample. In some instances, it may be ben~ficizll to modify a solid test sample to form a liquid meflillm or to release the analyte.

CA 0220~373 1997-0~-14 II. Pbosphatase Activatable Phosphorothioate Monoester A~ents The present invention provides novel compounds which display at least one phosphorothioate monoester group (-S-Po3-2). It has been discovered that these compounds have utility as (i) cros.clinking agents, (ii) conjugation agents, and (iii) redllçing agents. Prior to the present invention, compounds were typically crosslinked or conjugated under harsh chemic~l cr)ntlitionc.
Ul.ru~ulla~t;ly, such conditions can damage the chemical and/or biological l~ropti lies associated with the crocclinkPd or conjugated compounds. The agentsherein provided can be activated and thereafter employed to reduce, crosslilLk 1 0 and/or conjugate compounds under gentle conditions. Moreover, the by-products of such reactions are relatively innocuous. Accordingly, a crosclinkP~
compound, for example, does not require purification from the by-products of a croc.clinking reaction. Consequently, compounds that are crocclinkP~l, conjugated or reduced, as taught herein, do not run the risk of damage caused by1 5 harsh chemir.~l conditions.

A. Crosslinking and Conjugating Agents Crosclinking and conjugating agents of the present invention generally compri.ce a mnnnmPr, polymer or oligomer backbone that is functional with at 2 0 least two reactive moieties and at least one of the two reactive mniehes comprises a phosphorothioate monoester. The cros.clinking agents herein provided have the formula (I), shown below, wherein Q is a straight or branched monomer, polymer or oligomer and n is at least one.
(I) 2 5 Q-(S-P03~2)n As previously mentioned, cro.c.clinking and conjugating agents of the present invention will have at least two reactive moieties. Accordingly, when n is one, the monomer, polymer or oligomer backbone will comprice at least one other reactive moiety in addition to the phosphorothioate monoester. Such reactive 3 0 moieties can include electrophilic and nucleophilic groups such as, for example, haloalkyls, epoxides, hy~r~7.i~1es, hydrazines, thiolates, hydroxyls, and the like, preferably active esters, amines and carboxylic acids.
While the crosslinking and conjugating agents will comprise at least one phosphorothioate monoester group, it is plc~f~lled that such agents comprise 3 5 between about 2 and about SO phosphorothioate monoester groups, more preferably between about S and about 40 phosphorothioate monoester groups CA 0220~373 l997-0~-l4 and most preferably between about 10 and about 30 phosphorothioate monoester groups.
The cro.cslinking and conjugating agents herein provided are preferably hydrophilic and display a net negative charge which allows for adequate 5 solnbili7~tion of such agents. Accordingly, it is pler~ -~d that a crosslinking and conjugating agent's backbone is neutral or has a net negative charge.
~tlrlition~lly, it is preferable that the solubility of such agents is at least lx10-8 M
at 25C, more preferably at least lx10-7 M at 25C, and most preferably at leastlx10-6 M at 2~C.
1 0 The size of the backbone monomer, polymer or oligomer which compri~es a cro.~slinking and conjugating agent is largely a matter of choice based upon the compound or compounds which are to be cros.~link~-l or conjugated. Preferably the backbone will have an average molecular weight of between about 200 and about 1,000,000, more preferably between about 1,000 1 5 and about 850,000, and most preferably between about 2,000 and about 750,000. As it will be nn(l~-r.~tood, of course, the backbone will comprise at least one monomer that is suitable for deriv~ti7.~tir)n with at least one phosphorothioate monoester group. The backbone can be directly functional with the phosphorothioate monoester or the backbone may comprise a 2 0 phosphorothioate monoester which is pendent from a polymeric side chain or side chains. When present, side chains which may be pendant from the backbone polymer preferably comprise aliphatic chains from 1 to 40 carbon atoms which are optiGnally substituted with heteroatoms such as, for example, nitrogen (N), oxygen (O), and sulfur (S).
2 5 Several monomeric, polymeric or oligomeric backbones have been found to be especially suitable for forming the cro.c.~linking and conjugating agents herein provided. For example, suitable backbones include, but are not intenrle(lto be limited to straight or branched polypeptides comprising natural or synthetic amino acid residues such as, for example, polylysine, poly~mides, polygluL~Iluc 3 0 acid, and polyaspartic acid; oligonucleotides such as, for example, DNA andRNA; polycarbohydrates or polysacch~ri~les such as, for example, polyamylose, polyfuranosides, polypyranosides, carboxymethylamylose, and dextrans;
polystyrenes such as, for example, chloromethylated poly~Lylc;ne and bromomethylated polystyrene; polyacrylarnides such as, for example, 3 5 polyacrylarnide hydrazide; polyacids such as, for exarnple, polyacrylic acid;
polyols such as, for example, polyvinyl alcohol; polyvinyls such as, for W O96/17580 PCTrUS95/15~86 ~Y~mple, polyvinyI chl-)ri(le and polyvinyl bromide; polyesters; polyure.th~nç.~;
polyolefins; polyethers; C5-Cloo,ooo monomeric or polymeric straight or branched alkyl chains which may optionally contain, within such chains, he~loato,l,s which may comprise groups such as for example, amines, ~lisll~fi~les, thioethers, active esters, carbamates and the like; C10-C750,000 cycloalkyl chains; and the like as well as other monomeric, polymeric or oligomeric m~t~ri~ CO~ g reactive functional groups along the length of their chain which can be substitut~l with a phosphorothioate monoester group.
Synthesis of the cro.s~linking and conjugating agents can generally be 1 0 accomplished by filnchon~li7ing a monomer, polymer or oligomer with a phosphorothioate monoester functionality using methodologies which are well known to those skilled in the art. Backbones having, for example, carboxylate functionalities or hydroxyl functionalities such as, for example, polyglllt~miç
acid, polyacryIic acids, carboxymethyl amylose and the like, can be 1 5 functionalized with phosphorothioate monoester by (i) activating carboxylàte or hydroxyl functionalities with a suitable electrophilic activator such as, for e~mrl~ ethyl 3-(3-dimethylaminopropyl) carbodiimide (EDAC) or bromoacetic acid followed by EDAC and (ii) reacting the so-formed activated esters with cycte~rnin~o-s-phosphate~ Backbone polymers having haloaLkyl 2 0 styrene residues can be function~li7.efl with a phosphorothioate monoester by reacting a para or ortho phenyl alkyl halide with sodium thiophosphate (Na3SP03) as shown below in Scheme I. As it will be understood, of course, any halogenated monom~or, polymer or oligomer cont~ining~ or which has been mo-lifi~d to contain, a halide may be activated by reacting such polymer with 2 5 Na3SP03 in aqueous dimethyl r~, . . .~. . ,i.le according to Scheme I.

Scheme I
R--X f Na3SPO3 ~ R - SPO3 3 0 Scheme I generally depicts a method disclosed by Bieniarz C., Cornwell M.J., Tetrahedron Lett., 34, 939-942, (1993), for converting a primary or secondaly halide to a phosphorothioate monoester. According to Scheme I, the compound of the formula 1, which represents a primary or secondary halide wherein X is a halide, is converted to the corresponding phosphorothioate CA 0220~373 1997-0~-14 monoester of the formula 2 using sodium thiophosphate tribasic dodecahydrate or anhydrous sodium thiophosphate in a suitable solvent.

B. Chemical Crosslinking The cros~linkin~ and conjugating agents of the present invention (which will be referred to as cro~.~linking agents in this section) can be used to crosslink compounds by activating the cro~slinking agent and cont~eting the activated agent with at least one compound which displays electrophilic and/or nucleophilic groups. According to cros~linking emboAiment.c, multiple covalent 1 0 bonds are preferably formed between the cro.~.slinking agent and the compound which is cro.~slinkt-A As a result, a cros~linktoA compound is st~kili7.eA
A group of particularly plcfcllcd cro.~linking agents is shown in Figure 1. Figure l(a) represents poly(glutamic acid) poly(phosphorothioate) wherein m is an integer between about 1 and about 50 and n is an integer between about 1 1 5 and about 500; Figure l(b) represents carboxymethyl amylose poly(phosphorothioate) wherein m is integer between about 1 and about 500 and n is an integer between about 1 and about 500; Figure l(c) represents poly(acrylic acid) poly(hydræide) poly(phosphorothioate) wherein k is integer between about 1 and about 500, 1 is an integer between about O and about SOO, 2 0 m is integer between about O and about 500 and n is an integer between about 1 and about SOO; Figure l(d) represents bromomethylated poly(styrene) poly(phosphorothioate) wherein m is integer between about 1 and about 500 and n is an integer be~weell about 1 and about 100; Figure l(e) represents poly(acrylamide) poly(phosphorothioate) wherein m is integer between about 1 2 5 and about SOO and n is an integer between about 1 and about SOO; and Figure l(f) lc~,cs~;n~ dextran poly(phosphorothioate)wherein n is an integer be~wt;en about 1 and about SOO.
Cros.~linking agents of the present invention can be activated by deprotecting the thiol group comprising the phosphorothioate monoester.
3 0 D~utcc~ion generally involves hydrolysis of the phosphate group from the phosphorothioate monoester to expose the nucleophilic thiol group. For example, the thiol group of the phosphorothioate monoester can be dc~lo~ec~cd - under low pH conAition.~ Preferably, deprotection in this manner takes place at a pH in the range of between about ~.0 and about S.S, more preferably in the 3 5 range of between about 4.5 and about 5Ø

CA 0220~373 1997-0~-14 wo 96/17580 PCT/US9~/15586 In a particularly plt;rellc;d embodiment, a phosphate hydrolyzing enzyme (or phosph~t~e enzyme) is employed to hydrolyze the phosphate protecting group from the phosphorothioate monoester. Because enzymes have very specific catalytic activity, typically, a phosphatase enzyme will only hydrolyze5 phosphate groups and therefore only react with the cros~linking agent. Hence, compounds that are being cros~link~l are not exposed to d~ 1 chemical conditions. Enzym~*c~lly ac*vating a cros~linking agent is typically performed with a catalytic amount of phosph~t~e enzyme preferably in an amount between about 1x10-4 M and about 1x10-14 M, more preferably be~ween about 1x10-6 M
1 0 and about 1X10-12 M and most preferably between about 1x10-8 M and about lxlO-l M. Fx~mples of phosphatase enzymes include but are not inten~e l to be limited to na*ve and recombinant forms of ~lk~line phosphatase, acid phosphatase and the l~ke.
Upon activation of the cro~linking agent, the highly nucleophilic thiolate 1 5 groups can react with electrophilic groups displayed by compounds which willbe cro~link~A It has been discovered, that by controlling the stoichiometry of the cro.~linking agent and the compounds to be cros~link~d, efflcient cros~linking can be achieved. Surprisingly, reaction conditions can be adjusted so that monomeric, dimeric or trimeric crosslirLked compounds are generated and 2 0 uncontrolled polym~ri7~tion is substantially miti~tell The ratio of cros.~linking agent to compound to be cros~linkt~rl is preferably between about 2:1 and about 8:1, and more preferably between about 2:1 and about 4:1.
Generally, proteins (which will be used hereinafter as representative of compounds that can be cros~linkecl or conjugated) can be function~li7e~1 with 2 5 electrophilic groups through che~nic~l reaction with reactive groups naturally found on proteins such as, for example, -NH2, -SH and the like. Means, G.E.
and Feeny, R.E., Bioconju~ate Chemistry. 1: 2-12 (199Q) provides a ~UIlUll~y of methodologies for electrophilic addition. Electrophilic groups that can be used to function~li7e proteins include, but are not intencle~ to be limited to 3 0 hett;lobirulluLional linkers such as m-m~ imitlobGll~oyl-N-hydroxy~ucculu~ide ester (MBS), sulfosuccinimidyl 4-(p-maleimidophenyl) but-yrate (S-SMPB), m-m~ imitlobenzoylsulfosuccinimide ester (S-MBS) and N-~-maleimidobutyryloxysuccinimi(le ester (GMBS), succinimidyl 4-[N-maleimido-methyl] cyclohexane-1-carboxylate (SMCC), and 4-[(N-3 5 m~ imi(1omethyl)tncaproamido]-cyclohexane-1-carboxylate (STCM described in U.S. Patent No. 4,994,385); haloacetyl groups such as iodoacetyl, CA 0220=7373 1997-0=7-14 wo 96117580 . PCT/US95/15586 bromoacetyl, and chloroacetyl; acrylate groups such as methacrylates, quinone groups, and epoxide groups; thiopyridyl groups; as well as other protected disulfides such as, for example, cystamine; transition metal complexes or transition metals in various oxidation states or in colloidal forms which are 5 known to form stable coordinate bonds with thiols such as, for example iron, cobalt, nickel, copper, ruthenium, rhodium, p~ linm, silver, osmium, iriclillm, pl~tinllm, gold, c~lmillm and mercury; and the like. Preferably, m~leimide groups are used to electrophilically functionalize a protein and most preferablyCl-C3 aLkyl carboxylic acid active ester m~leimi~les and aryl carboxylic acid 1 0 active ester m~leimi~es having between about 6 and about 30 atoms between the two terminal functional groups. It will be understood, of course, that a proteinmay have functionalities that are suitable for reaction with non-phosphorothioate monoester functionalities that are present on the cros~linking agent.
Preferably, a cro.s.~linke-l protein is "wrapped" or "stitched" by the 1 5 cro.~linking agent as a result of multiple crosslinks which form between thecro.c~linking agent and the protein. Once wrapped by the polymer, a protein has less conro~ a~ional freedom and is therefore less l~kely to undergo structural distortion and in some cases denaturation. Consequently, a cro~.~linked protein is st~bili~e-l. Additionally, a reaction between a nucleophilic group on the 2 0 cro.~linking agent and an electrophilic group on a protein results in the form~hon of a "lilLking arm" which spans the distance between a cros~linking agent's backbone and the protein. Preferably, this distance is kept to a ,-,i~-ill,ll.-- to limit a cro.~.~linked compounds conroll,lational freedom.
It is worthy of note that enzymatic activation of a cros~linkin.~ agent can 2 5 be employed in a "self catalyzing reaction." Speci~lcally, the enzyme which catalyzes the activation of the cros~linking agent can be the compound which is to be cros~link-~l According to this mech~ni.~m, the enzyme can deprotect a crosslinking agent's thiol group or thiol groups which in turn react(s) with theenzyme which unm~ked the thiol group or thiol groups. Preferably, the amount 3 0 of enzyme employed in a self catalyzing reaction is between about 10-2 M and about 10-6M.
Figure 2 generally illustrates cro.c~linking a protein according to the instant invention. As e~cemrlified by Figure 2, the protein of Figure 2(a), which is functional with a plurality of amine groups, can be derivatized with 3 5 heterobifunctional linkers, such as SMCC, to yield the protein of Figure 2(b).
The cros.~linking agent of the instant invention, represented by Figure 2(c), can CA 0220~373 1997-0~-14 be activated with, for example, alkaline phosphatase enzyme to yield the activated cros~linking agent represented by Figure 2(d). Once activated, the cro~linkin~ agent readily reacts with the electrophilic groups on the protein ofFigure 2(b) to yield the stitched protein of Figure 2(e).
After a crosilinking reaction is s-lffici~ntly complete, the reaction may inherendy ttqrmin~t~o because there are no further groups capable of re~ction, or the reaction may be stopped. A cros~linking reaction can be stopped by capping the exposed thiol groups through the addition of any of the well known thiol capping groups such as, for e~r~mple, N-ethylmaleimide (NEM), iodo~cet~mifle, 1 0 iodoacetic acid and the like. ~ltern~tively, one of the reactants can be removed by, for ex~mrle, passing the reaction mixture over a sizing column. After a st~hili7erl protein is removed from a reaction mixture, unreacted thiol groups, if any, can be capped. As it will be understood, of course, a st~hili7ed protein asrepresented by Figure 2(e) can be conjugated to other proteins using unreacted 1 5 thiolates.
Proteins which are cro.~link~l as taught herein display an increased stability which can be manifested by, for example, a residual activity that lasts longer than the activity associated with an unst~bili7ed protein and/or a capacity to with~t~n-l environm~nt~l stress better than an unstabilized protein. For 2 0 example, a stabilized enzyme may m~int~in its activity when temperature stressed such as, for example, when the enzyme is stored for 7 days at 45C or stored at 25C for 30 days. A st~bili7~1 enzyme may retain its activity at pHs where the n~t~hili7ed enzyme does not have activity. Other potential effects of stabili7~tion may include stability of a protien in an organic solvent which would 2 5 ordinarily denature an unstabilized protein and enhancement of a binding member's ability to specifically bind and thereby form a binding pair.

C. Conjugating Compounds The crosslinking and conjugating agents of the present invention can also 3 0 be employed to conjugate compounds. In this section such agents will be referred to as conjugating agents. According to conjugation embo-1iment.~, at least two distinct ch~mic~l entities are bound or otherwise immobilized to a conjugating agent. For example, using a conjugating agent, detectable moieties can be immobilized to a binding member to form an indicator reagent, binding 3 5 members can be immobilized to a chromatographic gel to form affinity CA 0220~373 1997-0~-14 chromatographic gels, and binding members can be immobilized to a solid phase to thereby form a solid phase reagent.
While the compounds illustrated in Figure 1 can be employed as conjugating agents, formnl~e of heterobifunctional conjugating agents according 5 to the present invention include, but are not limited to, those found in Figure 3.
Figure 3(a) represents N-hycllo~y~uccinimidyl ~iy~le~ ophosphorothioate 4,5-tlithioheptyl l-carboxylate, Figure 3(b) represents N-hydroxysuccinimi-lyl ~;y~ lophosphorothioate 3-oxybutyl l-carboxylate, Figure 3(c) represents N-hydl:u~y~uccinimidyl cyste~mi-lophosphorothioate heptanoyl l-carboxylate, 1 0 Figure 3(d) represents cysteamidophosphorothioate heptanoyl l-hydrazide, Figure 3(e) represents cy~Lea~ dophosphorothioate heptanoyl 1-(aminoethyl)carboxamide, and Figure 3(f) represents p-nitrophenyl cysteamidophosphorothioate heptanoyl l-carboxylate.
Two or more proteins can be conjugated to each other with the 1 5 conjugation agent herein provided using the same reaction mech~ni~m previously outlined for cros~linking compounds. Specifically, a conjugating agent can be activated under suitable pH conditions or preferably with a phosphatase enzyme.
The activated conjugating agent can then be contacted with the proteins to be cros~link~A. The nucleophilic thiol groups of an activated conjugating agent 2 0 react with electrophilic groups present on the compounds to be conjugated tothereby conjugate the compounds. It will be understood, of course, that advantage may be taken of other non-phosphorothioate reactive functionalities displayed by a conjugating agent to conjugate proteins. It will also be understood that compounds may be modified with electrophilic groups, as 2 5 above, in order to allow them to react with the conjugating agent.
The ratio of re~ct~nt~ in a conjugation reaction are largely dependent upon the final product desired. Thus, for example, if an in~lic~tor reagent compri~ing multiple detectable moieties were desired, the amount of detectable moiety employed in a conjugation reaction would be greater than the amount of 3 0 either the binding member or conjugation agent employed. Typically, however,- the molar ratio of conjugation agent in a conjugation reaction designed to immobilize two compounds is 1:1:1.
- The conjugation agent can also be employed in a self catalyzing reaction.
For example, in cases where a phosphate hydrolyzing enzyme is being 3 5 conjugated, such an enzyme could serve as the activating agent for its own conjugation. Specifically, such an enzyme could hydrolyze the phosphate CA 0220~373 1997-0~-14 WO 96/17580 PCT/US95115~86 protecting group from the conjugation agent's phosphorothioate monoester and thereby allow a reaction between the conjugating agent and an electrophilically derivatized enzyme as well as any other electrophilic group functional compound being conjugated.
Figure 4 generally illu~L-~es the conjugation of mllltiplto compounds using the conjugation agent as a template. As shown by Figure 4, two proteins which have been functi-~n~li7~d with a maleimide linking group are represented by Figures 4(a) and 4(b). The conjugation agent is represented by Figure 4(c).
Upon activation with, for example, a phosphatase enzyme, the thiol groups of 1 0 the conjugation agent react with the maleimides to form a structure of Figure 4(d). Hence, the proteins represented by Figures 4(a) and 4(b) are conjugated.
As an ~ltt~rn~tive conjugation, a protein that has been crosslinked can be conjugated with other proteins using the cro~clinking agent as a conjugation agent as well. For example, Figure S shows the conjugation of a st~bili7ecl 1 5 protein to an antibody. As shown by Figure 5, an antibody depicted by Figure 5(a) can be modified to display a reactive region by, for example, (i) tre~tmt-.nt with periodate and (ii) tre~ nt with ~;y~L~u~ e and sodium cyanoborohydride to yield an antibody displaying ~ llphi~le bridges in the Fc region as depicted by Figure 5(b). The nucleophilic antibody can then be conjugated to a crosslinked 2 0 protein, such as that represented by Figure 5(c), to yield the antibody/stabilized protein conjugate depicted by Figure 5(d).
Similarly, site specific conjugation of an antibody and a st~bili7P~1 protein can be performed acccording to Figure 6 Wht:;lC;ill n is less than the number ofcarbohydrate functionalities present in the Fc region of an antibody. For 2 5 example, the Fc region of an antibody represented by Figure 6(a), can be oxidized by periodate and exposed to 4-~-m~leimi(lomethyl)cyclnh~x~ne-1-carboxyl hydrazide (M2C2H) to yield the the antibody represented by Figure 6(b). A stabilized protein represented by Figure 6(c) can then be reacted with the deri~ atized antibody to site speçific~lly add the stabilized protein to the Fc region 3 0 of the antibody and yield the conjugate of Figure 6(d).
Heterobifunctional conjugating agents such as those represented in Figure 3(a) through 3(f) can also be employed to conjugate compounds. For example, as shown in Figure 14, N-hydroxysuccinimidyl cysteamidophosphorothioate heptanoyl l-carboxylate ~ ;sell~d by Figure 3 5 14(a) is reacted under conditions well known to those skilled in the art with an amine functional protein depicted by Figure 14(b) to yield the protein of Figure CA 0220~373 1997-0~-14 14(c). A maleimide functional protein shown by Figure 14(d) is then reacted with the protein of Figure 14(c) in the presence of, for example, a catalytic amount of ~lk~line phosphatase. The ~lk~lin~ phosph~t~e hydrolyzes the phosphate groups from the heterobifunctional linker which allows a reaction between the nucleophilic thiol group and the electron rich region of the m~ imi~le to yield the conjugated proteins of Figure 14(e).

D. ReducingAgents The invention also provides a st~bili7e 1 retln~ing agent generally 1 0 represented by the compound of the formula (Y), shown below, wherein (A) and (Z) can be indepen(lt-ntly selected from Cl-Cs aL~yl and CONH(CH2)p wherein p is an integer between 1 and 5.

(~ ~OH
C C

1 5 Particularly plertllt;d stable re~ncing agents are shown in Figure 7 where the compound ~le~ignz~te-l 7(a) represents dithiothreitol disphosphate and the compound designated 7(b) represents 1,4-bisphosphorothioylethyl ~L~ ide.
The protected rerl~lcing agents herein provided can generally be synthesi7ed using methodologies previously described. For ~x~mple, according 2 0 to Scheme I, shown above, any primary or secondary halide such as, for example, 1,4-dibromo-2,3 ~t~nrliol can be converted to a st~bili7~d reclucing agent. As a further ~x~mple, carboxy functional compounds can be converted to stable reducing agents as previously taught. For in~t~n~e, tartaIic acid can be converted to a stable re~ cing agent by (i) activating the carboxylates with a 2 5 suitable electrophilic activator and (ii) reacting the so-formed activated esters with ~;y~le~lille-S-phosphate to yield the st~bili7~d re-ln~in~ agent.
Similarly to the conjugation and crosslinking agents, the thiol groups displayed by the reducing agents are protected and can be activated upon hydrolysis of the phosphate group. Hence, the reducing agents are useful in, for- 3 0 example, containers of liquid or lyophilized immunoassay reagents which require redllcing conditions at the time of use. When such a reducing environment is required, the reducing agents can be activated with, for example,a phosphate hydrolyzing enzyme or an appl opliate pH environment. After the phosphate groups are hydrolyzed from the phosphorothioate monoester function~lities, a reducing environrnent results because the thiol groups are nolonger protected.

5 m. Examples The following examples are provided to further ill~ tP. emborlim~-nt~ of the invention and should not be construed as a limitation on the scope of the invention. The m~tPri~l~ employed in the P"c~mples are co~ ially available or readily synthesi7~1 A general comr)il~tinn of m:~teri~l~ and their source can be1 0 found in Table 1 below.
Table 1 Material Obtained From Source Source LocationSource Amicon Beverly, MDC~ ),~)-30-Con~Pntr~tor~
Centricon-30-concenLla~ur Bio-Rad Hercules, CAEcono Column, Biû-Sil SEC-400 column, BIO-REX MSZ 501(D) resin Pharmacia LKB Piscataway, NJPharmacia Phastgel System Spectrum Houston, TXAlldialysistubing Hitachi Naperville~ ILHitachi F-4010 Fluorescence Spectropho~u",~;~l Abbott Laboratories Abbott Park, IL Abbott VP Biocllluma~ic Analyzer, horse radish peroxidase (HRPO), 3,5-dichloro-2-hydlu~y~ c..~P.sul~onic acid sodium salt (HDCBS), 4-amino~,li~,ylil,e (~AAP) Boehringer Tn~ n~polis, IN bovine alkaline phosphatase (ALP), ~l~nnhP.im glucose oxidase (GOD) Table 1 (cont.) SourceSource Location Material Obtained From Source Yamasa Shoyo Tokyo, Japan ~lllt~m~t~ oxidase (GlOX) Molecular Probes Eugene, OR R-phycoelyllllill (R-PE), amino dextran Pierce Rockford, IL 3,3'-dithiopropionic acid bis-active ester, SMCC linker, M2C2H linker, diglycolic acid bis-active ester, bis-active ester of suberic acid Sigma St. Louis, MO Sephadex G-25,N-ethylm~l~imi~le (NEM), succinimidyl bromoacetate, sodium thiophosphate, poly-L-glllt~mic acid, ~;y~ erS-phosphate, EDAC, carboxymethylamylose, sodium m-periodate, sodium cyanoborohydride, glucose, polyacrylamide hydrazide, p-nitrophenyl phosphate (PNPP), bovine serum albumin, ethyl~.ntoAi~minet~traacetic acid (EDTA) Seradyne Tn~ n~polis, IN ~min~3t~d microparticles Aldrich Milwaukee, WI hydrazine monohydrate, ethylen~i~minç, poly(acrylamide-co-acrylic acid), 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), glyceraldehyde, kathon, 1,4-dibromo-2,3-butanediol, silver nitrate, p~~ o~henol, dithiothreitol (DTT) CA 0220~373 l997-0~-l4 WO 96/17580 PCT/US95/lSS86 Example 1 Synthesis of Poly(phosphorothioate) Functionalized Polymers (a) Poly(glllt~mic acid) Poly(phosphorothioate) Poly-L-glllt~mi~ acid MW ~70,000 (1.0 g 14 ,~Lmol) and ~;y~Le~ S-phosphate (0.26 g, 1.4 mmol) were dissolved in 40 ml of deionized water.
EDAC (1.00 g, 5.2 mmol) was added in 100 mg lots every 30 minlltes for 5 hours. The polymer product was purified with a Centriprep-30-conce~ alu against d~-ioni7ed water and then lyophilized.

1 0 (b) PhosphorothioateAnalysis To 1.0 ml of a 5.0 ,uM solution of poly(glutamic acid) poly(phosphorothioate) in 0.1 M Tris buffer 1.0 mM MgC12, 0.1 mM ZnC12, pH 7.5 (Buffer A) was added 30 ~1 of DTNB (10 mM in buffer A). The solution was incubated for 5 minutes and the absorbance at 412 nm was 1 5 recorded. No free thiol was detected. ALP (10 ~1 of a 10 mg/ml solution) was added and the solution was incubated until no further increase in 412 nm absorbance was detected (~30 minutes). The concentration of free thiol (0.12 rnM) was calculated from the final 412 nm absorbance (1.54 AU) and the extinction coefficient of 2-nitro-5-thiobenzoic acid (13,000 M-lcm- 1 at pH 7.5).
2 0 There were found to be 24 moles of phosphorothioate per mole of polymer.

(c) Carboxymethylamylose Polyphosphorothioate Carboxymethylamylose MW ~60 000 (0.15 g, 2.5 llmol) and ~;y~ Le~ -S-phosphate were dissolved in 10 ml of deionized water. EDAC
2 5 (0.125 g, 0.65 mmol) was added in 25 mg lots every hour for 5 hours. The carbohydrate product was purified on a C~ ~-30-Conce"lla~ul against ~leit~ni7e~ water and then lyophilized. Phosphorothioate analysis (performed as described above) revealed 1 phosphorothioate per carboxymethylamylose polymer chain.
(d) Poly(acrylamide) Poly[acryloyl(2-(2-phosphorothioethyl)aminoethyl]
Hydrazide Polyacrylamide hydrazide MW ~180,000 (0.050 g, 0.28 ~mol) was dissolved in 20 ml of 0.05 M sodium acetate buffer pH 4.5 (buffer B).
3 5 Glyceraldehyde (0.080 g 0.89 rr rnol) and sodium cyanoborohydride (0.056 g,0.89 mmol) were added and the solution was stirred for 20 hours. The polydiol WO 96tl7580 PCT/US95/lS586 product was purified on a Centriprep-30-ConcenLI~lol against buffer B. Sodium periodate (0.095 g, 4.4 mmol) in 10 ml of buffer B was added to the polydiol and the ll~Lule was stirred for 1 hour in an ice-bath, allowed to warm to room temperature, and stirred again for another hour. The polyaldehyde product was 5 puri~led on a C~,Ir;l)~-30-Concentrator against 0.1 M sodium phosphate buffer pH 5.5.
To the polyaldehyde was added cyste~ine-S-phosphate (0.079 g, 0.44 mmol) and sodium cyanoborohydride (0.028 g, 0.44 mmol) and the ~ Lule was stirred overnight. The polyphosphorothioate product was purified on a 1 0 (~ç~ , ~-30-Concen~ldtor against deionized water and then lyophilized.
Phosphorothioate analysis (performed as described above) revealed 66 phosphorothioates per poly.ner.

(e) Poly(amino)dextran Poly(phosphorothioate) 1 5 Amino dextran (MW ~ 70,000, approximately 30 amines/polymer) is dissolved in deionized water. 20 equivalents of succinimidyl bromoacetate is dissolved in dim~;lhylr -~ I"~" ,ir~e (DMF) and a volume of this solution which is greater than 10% of the amino dextran solution is added to the amino dextran solution to form a reaction mixture. The reaction n~b~Lult; is stirred for 3 hours at 2 0 room temperature and the resulting bromoacetylated polymer is purified against deionized water with a C~"I, ip, ~-30-Concentrator. 50 equivalents of sodium thiophosphate in clei~ni7çd water is then added to the purified polymer and the resulting ll~ulC; is stirred for 2 hours at room temperature. The resulting phosphorothio~ted amino dextran is purified, as above, with a (~Ç~ c~-30-2 5 Conct;nLId~ol and lyophili7~A

(f) Poly(acrylamide-co-acrylic acid) Poly(phosphorothioate) Poly(acrylamide-co-acrylic acid) MW~200,000 (1.0 g, 5 ~mol) and cy~ e-S-phosphate (0.09g, O.5mmol) are dissolved in 40 ml of deionized 3 0 water. EDAC (0.5 g, 2.6 mmol) is added in 50 mg lots every 30 minutçs for 5hours. The polymer product is purified on a Cellllipl~-30-conce~ " against deionized water and then lyophilized.

Example 2 Polv(~lutamiç acid) Poly(phosphorothioate) Crosslinkin~ of Bovine ALkaline Phosphatase (ALP) (a) Crosslinking of AL~aline Phosphatase To 0.75 ml of 10 mg/ml (50 nmol) ALP was added 1.25 ml of 0.1 M
sodium phosphate, 0.1 M NaCl, 1.0 mM MgC12, 0.1 mM ZnCl2, pH 7.0 (buffer C). The enzyme was concentrated to approximately 0.2 ml using a Centricon-30-Concentrator. The concentrate was rediluted to 2.0 ml with buffer C, then reconcto~ ~l to 0.2 ml. This concentration/dilution procedure was 1 0 repeated three times. The volume of the enzyme solution was made up to 1.5 ml with buffer C and placed in a vial. To 75 ,ul of DMF was added 0.62 mg (1.87 ,umol) of SMCC. This solution was added to 1.46 ml of 4.8 mg/ml (46.7 nmol) washed aLkaline phosphatase and allowed to react for one hour at room temperature while rotating at 100 rpm on a rotary agitator. Coarse Sephadex G-1 5 25 thathad been previously rehydrated with 0.1 M sodium phosphate, 0.1 M
NaCl, 0.05% azide, pH 7.0 (buffer D) was poured to a bed height of 45 cm in a 1 x 50 cm Econo column. The column was equilibrated with three column volumes of buffer C. Following the incubation, the SMCC derivatized alk~lin~
phosphatase was applied to the G-25 column to remove unreacted SMCC. The 2 0 column was eluted with buffer C and 0.75 ml fractions were collected.
Fractions with A280 greater than 0.5 AU were pooled and the A280 of the pool was used to calculate the enzyme concentration of the SMCC derivatiæd ~lk~line phosphatase. To 300 ~11 of buffer C was added 2.43 mg (34.7 nmol) of 70,000 MW poly(~ t~mic acid) poly(phosphorothioate) (26 SPO3/PGA). This solution 2 5 was added to 1.86 ml of 1.40 mg/rnl (17.4 nmol) SMCC derivatized ~lk~line phosphatase and allowed to react overnight at 5C while rotating at 100 rpm on arotary agitator.

(b) Characterization of Cro.~link~cl ALP
3 0 Poly(glutamic acid) poly(phosphorothioate) cros~link~l ALP was evaluated by size exclusion chromatography using a Bio-Sil SEC-400 column.
Detection was at 280 nm. The mobile phase was 0.1 M sodium phosphate, 0.1 M NaCl, pH 7.0 (buffer E) running at a flow rate of 1.0 ml/minute. Results of this evaluation showed that the primary population generated had a ret~o.ntion time 3 5 corresponding to singlet cros~link~ enzyme with very little polymPri7~tion occurring. The cro~clinked ~lk~Tine phosphatase was also evaluated by SDS-.
WO 96/17580 . PCTIUS95/15586 PAGE using a Phastgel system. Gradient gels of 7%-10% polyacrylamide were run under non-reducing and reducing conditions. Results from the non-reducing conditions showed that the primary population generated was singlet cro.c.~link~en_yme with very little polymP,ri7~tif~n occurring. Results from the reducing 5 conditions showed that the cros~link~ ALP was not reduced under conditions that were snfficiçnt to fully reduce the native ALP into monom--.rs. The residual enzyme activity of the cro~.clink~,d ALP was evaluated and compared to the activity of the native ALP at the same concentration. To 1.0 ml of 7 mM PNPP
in 0.5 M diethanol~mine, 1.0 mM MgC12, 0.1 mM ZnC12, pH 10.2 buffer 1 0 (buffer F) was added 20 111 of 10 ~g/ml ALP. The rate (AU/second) of changein the 412 nm absorbance was calculated over a 14 second interval. The rate generated by the cro.~linkP,d ALP ~lc~ Lions was divided by the rate generated by the native ALP to calculate the percent residual enzyme activity for the cro.~linked preparations. The results of this evaluation showed that the 1 5 cro.~linked ALP had retained 80% of the initial enzyme activity.

(c) Thermal Stability Ev~ tion of Cr~ s.slink~ ALP
The thermal stability of poly(gl~ mic acid) poly(phosphorothioate) cro.~.~link~,d ALP was evaluated at 45C and compared to native ALP under the 2 0 same conditions. Both the native enzyme and the cro.s~link~l enzyme were diluted to 10 llg/ml with buffer A. These dilutions were stored in a 45C
incubator for the duration of the study. At day 0 and various time points along the course of the study the activity of the dilutions was ev~ln~terl To separate1.0 ml volumes of 7 mM PNPP in buffer F was added 20 ,ul of the 10 ~lg/ml 2 5 ALP dilutions. The rate of change in the 412 nm absorbance was calculated over a 14 second interval. The rate generated by the enzyme p~ Lions at the various time points was divided by the rate generated by the same enzyme pl~;~al~ion at day 0 to calculate the percent residual enzyme activity for the stressed plc;~ ions. The results of this evaluation are shown in Figure 8.
Example 3 Poly(,~lutamic acid) Poly(phosphorothioate) Cro~linkin~ of Glucose Oxidase (GOD) (Asper~eillus ni~er) (a) Cro.~linking GOD
3 5 To 200 mg (1.25 ,umol) of GOD was added 20 ml of buffer E. The enzyme was concentrated to a~lo~ lately 2 rnl using a Centriprep-30-wo 96/17580 PCT/US95/15586 Concen~tol with a MW cutoff of 30,000. The concentrate was re~ t~fl to 20 ml using buffer E then reconcentrated to 2 ml. This concentrationldilution procedure was repeated three times. The volume of the enzyme solution was made up to 6 ml with buffer E and placed in a vial. To 500 ,ul of DMF was added 9.39 mg (28.1 llmol) of SMCC. This solution was added to a 977 ul aliquot of 30.7 mg/ml (187 nmol) washed GOD and allowed to react for one hour at room temperature while rotating at 100 rpm on a rotary agitator. A
Sephadex G-25 was prepared as above with buffer E, and following the incubation, the SMCC derivatized GOD was applied to the G-25 column to 1 0 remove the unreacted SMCC. The column was eluted with buffer E and 0.75 ml fractions were collected. Practions with an A280 greater than 0.5 AU were pooled and the A280 of the pool used to calculate the enzyme concentration of the SMCC derivatized GOD. To 1.5 ml of buffer C was added 56.3 mg (938 nmol) of 60,000 MW poly(glllt~mic acid) poly(phosphorothioate) (19 SPO3/PGA).
1 5 To this solution was added 50 ,ul of 10 mg/ml (3.33 nmol) ALP to deprotect the phosphorothioate. This deprotection was allowed to proceed for three hours at room ~~ d~u~c; while rotating at 100 rpm on a rotary ~agitator. Following the incubation a 300 ~1 (188 nmol) aliquot of this solution was added to 1.26 ml of 7.95 mg/ml (62.6 nrnol) SMCC derivatized GOD and allowed to react overnight 2 0 at 5C while rotating at 100 rpm on a rotary agitator.

(b) ~h~ractt-ri7~tion of Cr cslink~ GOD
Poly(~ t~mic acid) poly(phosphorothioate) crosslinked GOD was evaluated by size exclusion chromatography using a Bio-Sil SE(:~-400 column.
2 5 Detection was at 280 nm. The mobile phase was buffer E running at a flow rate of 1.0 mVminute. Results of this evaluation showed that the primary population generated had a retention time corresponding to singlet cro.s~linkt d enzyme with very little polym~ri7ation occurring. The residual enzyme activity of the cros~linkt-A GOD was also evaluated and compared to the activity of the native 3 0 GOD at the same concentration. To 260 ~l of 2 mM 4-aminoantipyrine (~
AAP), 8 mM 2-hydroxy-3,5 dichlorobenzene acid (HDCBS), 1 U/ml horseradish peroxidase (HRPO) and 100 mM glucose, in buffer E was added 10 ~11 of 2,ug/ml GOD in buffer E. The rate (AU/minute) of change in the 550 nm absorbance was calculated over a 2 minute interval. The rate generated by the 3 5 crosslinked GOD preparations was divided by the rate generated by the native GOD to calculate the percent residual enzyme activity for the cros~linked ~l~ar~ions. The results of this evaluation showed that the cros~link~ GOD
had retained 85% of the initial enzyme activity.

(c) Thermal Stability Evaluation of Crosslink~d GOD at pH 7.4 The thermal stability of poly(glllt~mic acid) poly(phosphorothioate) croc~link~d GOD was evaluated at 37C and compared to native GOD under the same conditions. Both the native enzyme and the cros~link~c{ enzyme were diluted to 500 ,ug/ml with 0.1 M sodium phosphate, 1.0 mM EDTA, 0.1 M
NaCl, 0.05% Kathon, pH 7.4 buffer (buffer G). These dilutions were stored in 1 0 a 37C incubator for the duration of the study. At day 0 and various time points along the course of the study the activity of both plc~alaLions was ev~ te(l Prior to evaluation, the stressed enzyme preparations were diluted to 2 llg/ml using buffer E. To 260 111 of 2 mM 4-AAP, 8 mM HDCBS, 1 U/ml HRPO, 100 mM glucose in buffer E was added 10 ~1 of each of the 2 ~g/ml glucose 1 5 oxidase in buffer E dilutions. The rate (AU/minute) of change in the 550 nmabsorbance was calculated over a 2 minute interval. The rate generated by the enzyrne ~ Lions at the various tirne points was divided by the rate generated by the same enzyme ~r~ ;on at day 0 to calculate the percent residual ellzyl.-e activity for the stressed preparations. The results of this evaluation are shown in 2 0 Figure 9.

(d) Thermal Stability Evaluation of Cro~link~A GOD at pH 9.0 The thermal stability of poly(~lllt~mic acid) poly(phosphorothioate) crosslinked GOD was evaluated at 37C and compared to native GOD under the 2 5 same conditions. Both the native enzyme and the cros~link~d enzyme were diluted to 500 ~g/ml with 0.1 M sodium phosphate, 1.0 mM EDTA, 0.1 M
NaCl, 0.05% Kathon, pH 9.0 buffer (buffer H). These dilutions were stored in a 37C incubator for the duration of the study. At day 0 and various time pointsalong the course of the study the activity of both preparations was eval~ teA
3 0 Prior to ev~lu~tion the stressed enzyme ~,l~alations were diluted to 2 ,ug/rnl using buffer E. To 260 111 of 2 mM 4-AAP, 8 mM HDCBS, 1 U/ml HRPO, 100 rnM glucose in buffer E was added 10 ~1 of each of the 2 ~lg/ml GOD
dilutions. The rate (AU/minute) of change in the 550 nm absorbance was calculated over a 2 minute interval. The rate generated by the enzyme 3 5 preparations at the various time points was divided by the rate generated by the same enzyme preparation at day 0 to calculate the percent residual enzyme CA 02205373 1997-0~-14 W O96/17580 PCTrUS95/15586 activity for the stressed p~ ;ons. The results of this evaluation are shown in Figure 10.

Example 4 Poly(~lutamic acid) polv(phosphorothioate) Crosslinkin~ of Glutamate Oxidase (GlOX) (Streptomvces Sp. X119-6) (a) Crosslinking of GIOX
To 100 mg (714 nmol) of GlOX was added 20 ml of buffer E. This solution was conce~ tc;d to approximately 2 ml using a Ct;~ )r~-30-1 0 Concentrator with a MW cutoff of 30,000. The concentrate was re lilnte~l to 20 ml with buffer E then reconcentr~t~--cl to 2 ml. This concentration/dilution procedure was repeated three times. The volume of the enzyme solution was made up to 3 ml with buffer E and placed in a vial. To 800 ,ul of DMP was added 4.18 mg (12.5 ~mol) of SMCC. This solution was added to a 1.25 ml 1 5 aliquot of 28.0 mg/ml (250 nmol) washed GlOX and allowed to react for one hour at room temperature while rotating at 10n rpm on a rotary agitator. A
Sephadex G-25 column was prepared as above using buffer E, and following the incubation, the SMCC derivatized GlOX was applied to the G-25 column to remove the unreacted SMCC. The column was eluted with buffer E and 0.75 rnl 2 0 fractions were collected. Practions with an A410 greater than 1.0 AU were pooled and the A410 of the pool was from used to calculate the enzyme concentration of the SMCC derivatized GlOX. To 2.5 ml of buffer C was added 100 mg (1.67 ~mol) of 60,000 MW poly(glutamic acid) poly(phosphorothioate) (18 SPO3/PGA). To this solution was added 50 ~l of 10 mg/ml (3.33 nmol) 2 5 ALP to deprotect the phosphorothioate. The deprotection was allowed to proceed for three hours at room temperature while rotating at 100 rpm on a rotary agitator. Following the incubation, a 524 !11 (343 nrnol) aliquot of thissolution was added to 1.74 ml of 6.88 mg/ml (85.5 nmol) SMCC derivatized GlOX and allowed to react overnight at 5C while rotating at 100 rpm on a rotaly3 0 agitator.

(b) Characterization of Cro.c~link~ GlOX
Poly(glut~mir, acid) poly(phosphorothioate) cro~link~,cl GlOX was evaluated by size exclusion chromatography using a Bio-Sil SEC-400 column.
3 5 Detection was at 410 nm. The mobile phase was buffer E running at a flow rate of 1.0 ml/minute. Results of this evaluation showed that the primary population wo 96/17580 PCT/USg5/15586 generated had a retention time corresponding to doublet and triplet cros.slinkt~.d enzyme with very little poly" Ir.l ;~:11 ion occurring. The residual enzyme activity of the crosslinkeA GlOX was also evaluated and compared to the activity of the native GlOX at the same concentration. Activity measurements were performed using a VP bicl~ol~a~ic analyzer. The activity generated by the cros.slink~A
GlOX prepar~tiQns was divided by the activity of the native GlOX to calculate the percent residual enzyme activity for the cro.sslink~-l preparations. The results of this ev~ tinn showed that the crosslink~l GlOX had retained 81% of the initial enzyme activity.

(c) Thermal Stability Evaluation of Cro.s.slinkto.d GlOX
The thermal stability of poly(,~lutamic acid) poly(phosphorothioate) cros.slink~d GlOX was evaluated at 37C and compared to native GlOX under the same conrlititn.s. Both the native enzyme and the crosslink~d enzyme were 1 5 diluted to 500 ,ug/ml with buffer G. These dilutions were stored in a 37C
incubator for the clllratiQn of the study. At day 0 and various time points along the course of the study the activity of both yleyalaLions was ev~ln~teA Prior toevaluation, the stressed enzyme ylcy~dLions were diluted to 6 ,ug/ml using buffer E. Activity measurements were performed using the VP bichlul,-aLic 2 0 analyzer. The activity generated by the enzyme yl~;yal~Lions at the various time points was divided by the activity generated by the same enzyme yl~L)ald~ion at day 0 to calculate the percent residual enzyme activity for the stressed preparations. The results of this evaluation are shown in Figure 11.

2 5 Example 5 Poly(~lutamic acid) Polv(phosphorothioate) Cro.sslinkin,~ of R-phycoerythrin (R-PE) (Porphvra tenera) (a) Cro.sslinking of R-PE
To 2.5 ml of 10 mg/rnI R-PE from was added 2 ml of buffer E. This 3 0 solution was transferred to Spectrapore-2 dialysis tubing with a MVV cutoff of 12,000-14,000 and dialyzed for 24 hours each against three 4 liter changes of buffer E. To 200 111 of DMF was added 0.49 mg (1.47 ~Lmol) of SMCC. This solution was added to a 1.21 ml aliquot of 5.80 mg/ml (29.2 nmol) dialyzed R-PE and allowed to react for one hour at room temperature while rotating at 100 3 5 rpm on a rotary agitator. A Sephadex G-25 column was prepared as above withbuffer E, and following the incubation, the SMCC derivatized R-PE was applied CA 0220~373 1997-0~-14 to the G-25 column to remove unreacted SMCC. The column was eluted with buffer E and 0.75 ml fractions were collected. Fractions with an As66 greater than 1.0 AU were pooled and the As66 of the pool was used to calculate the enzyme concentr~ti-~n of the SMCC derivatized R-PE. To 1 ml of buffer C was added 10 mg (167 nmol) of 60,000 MW poly(glllt~mic acid) poly(phosphorothioate) (18 SPO3/PGA). To this solution was added 25 111 of 10 mg/ml (1.67 nmol) ALP to deprotect the phosphorothioate. This de~rot~.;Lion was allowed to proceed for three hours at room temperature while rotating at 100rpm on a rotary agitator. Following the incubation~ a 71 ~11 (11.5 nmol) aliquot1 0 of this solution was added to 1.91 ml of 1.44 mg/ml (11.5 nmol) SMCC
derivatized R-PE and allowed to react overnight at 5C while rotating at 100 rpmon a rotary agitator.

(b) Characterization of Crosslinked R-PE
1 5 Poly(gll-t~mic acid) poly(phosphorothioate) crocclink~ R-PE was evaluated by size exclusion chromatography using a Bio-Sil SEC-400 column.
Detection was at 280 nm. The mobile phase was buffer E running at a flow rate of 1.0 mVminute. Results of this evaluation showed that the primary population g~ner~tefl had a retention time corresponding to singlet and doublet croc.clink~2 0 protein with very lit~e polymPri7~tion occulTing. The residual fluorescent intensity of the crosslinked R-PEwas also evaluated and con~ya ed to the fluorescence of the native R-PE at the same concentration. ~luorescent intensitymeasurements were performed using an F-4010 fluorescence spectrophotometer.
The fluorescence generated by the crosclink~1 R-PE preparations was divided by 2 5 the fluorescence of the native R-PE to c~lc~ te the percent residual fluorescence for the crosclink~l ~r~ions. The results of this evaluation showed that the crosslinked R-PE had retained 92% of the initial fluorescent intensity.

(c) Thermal Stability Evaluation of Crosclink~ R-PE Fluorescence 3 0 The thermal stability of poly(~lut~mic acid) poly(phosphorothioate) crocclink~cl R-PE was evaluated at 45C and compared to native R-PE under the same conditions. Both the native protein and the crocclinked protein were diluted to 100 llg/ml with buffer E. These dilutions were stored in a 45C
incubator for the dllr~tion of the study. At day 0 and various time points along3 5 the course of the study the fluorescence intensity of both preparations wasevaluated. Prior to evaluation, the stressed protein preparations were diluted to CA 0220~373 1997-0~-14 1 ,ug/ml using buffer E. Fluorescence intensity measurements were performed using an F-4010 fluorescence spectrophotometer. The fluorescence by the R-PE
preparations at the various time points was divided by the fluorescence by the same preparation at day 0 to calculate the percent residual fluorescence intensity 5 for the stressed ~ tions. The results of this evaluation are shown in Figure 12.

(d) Thermal Stability Evaluation of Cro~slinkecl R-PE by Size The thermal stability of poly(glllt~mic acid) poly(phosphorothioate) 1 0 cros~link~l R-PE was evaluated at 45C and compared to native R-PE under the same conditions. A Bio-Sil SEC-400 column was used to follow the decomposition of the stressed R-PE into smaller subunit components. Detection was at 280 nm. The mobile phase was buffer E running at a flow rate of 1.0 ml~minute. Both the native protein and the cros~linkecl protein were diluted to 1 5 100 ~g/ml with buffer E. These dilutions were stored in a 45C incubator for the duration of the study. At day 0 and various time points along the course of the study the percentage of the total 280 nm absorbance which was due to the small components was ev~lu~tt-A The results of this evaluation are shown in Figure 13.
Example 6 Poly(glutamic acid) Poly(phosphorothioate) Crosslinked Bovine ALkaline Phosphatase (ALP)/Anti-TSH I~G Conju,eate (a) Deriv~ti7~ti~ n of Anti-TSH Antibody 2 5 To 1 ml of 6.6 mg/ml anti-TSH IgG was added 1 ml of buffer C. The antibody was conce~ teA to a~lo~ lately 0.2 ml using a Centricon-30-ConcenL~lol with a MW cutoff of 30,000. The concentrate was reAilllteA to 2 ml using buffer C, then reconcel~ d to 0.2 ml. This concentration/dilution procedure was repeated three times. The volume of the antibody solution was 3 0 made up to 1 ml with buffer C and placed in a vial. To 50 ~1 of DMF was added 0.56 mg (831 nmol) of succinimidyl (trica~luall~ido cyclohexylmethyl) N-m~leimi~le (STCM) linker. The linker was prepared as described in United States Patent No. 4,994,385 which is herein incorporated by reference. This solution was added to 0.47 ml of 5.30 mg/ml (16.6 nmol) washed antibody and 3 5 allowed to react for one hour at room lemL)~l~ture while rotating at 100 rpm on a rotary agitator. A Sephadex G-25 was prepared as above with buffer C and following the incllb~tion, the derivatized antibody was applied to the G-25 column to remove unreacted linker. The column was eluted with buffer C and 0.75 ml fractions were collected. Fractions with an A280 greater than 0.5 AU
were pooled and the A280 of the pool used to calculate the concentration of the 5 linker derivatized antibody. The antibody pool was stored on ice until conjugated.

(b) Conjugation of Linker Derivatiæd Anti-TSH IgG to Poly(gl-lt~mic acid) Poly(phosphorothioate) Crosslinked ALP
1 0 To a 0.72 ml aliquot of 0.83 mg/ml (4 nmol) linker derivatized anti-TSH
IgG was added 0.69 ml of 1.30 mg/ml (6 nmol) poly(glutarnic acid) poly(phosphorothioate) cros~linke,-l ALP (prepared in accordance with Exarnple 2). The resulting mixture was allowed to react overnight at 5C while rotating at 100 rpm on a rotar,y agitator.

Example 7 Poly(~lutamic acid) Poly(phosphorothioate) Crosslinked R-phvcot;lylh PE)/anti-CD8 I~G Conju~ate (a) Deriv:~ti7~tinn of Anti-CD8 Antibody.
2 0 To 1 ml of 4.1 mg/ml anti-CD8 IgG was added 1 ml of buffer E. The antibody was conct-ntTated to ~p,o~ lately 0.2 ml using a Centricon-30-Concent~dlul with a MW cutoff of 30,000. The concentrate was rediluted to 2 ml using buffer E then reconcentrated to 0.2 ml. This concentration/dilution procedure was repeated three times. The volume of the antibody solution was 2 5 made up to 1 ml with buffer E and placed in a vial. To 150 ~1 of DMF was added 0.15 mg (223 nmol) of STCM linker (as prepared in e~mrl~,6). This solution was added to 0.50 ml of 4.39 mg/ml (14.6 nmol) washed antibody and allowed to react for one hour at room temperature while rotating at 100 rpm on arotary agitator. A Sephadex G-25 column was prepared as above with buffer E
3 0 and following the incubation, the derivatized antibody was applied to the G-25 column to remove unreacted linker. The column was eluted with buffer E and 0.75 ml fractions were collected. Fractions with an A280 greater than 0.5 AU
were pooled and the A280 of the pool used to calculate the concentration of the linker derivatized antibody. The antibody pool was stored on ice until 3 5 conjugated.

CA 0220~373 1997-0~-14 (b) Conjugation of Linker Derivatized Anti-CD8 IgG to Poly(~lnt~mic acid) Poly(phosphorothioate) Crosslink~l R-PE
To a 0.53 ml aliquot of 1.71 mg/ml (6 nmol) linker derivatized anti-CD8 IgG was added 0.68 ml of 2.11 mg/ml (6 nmol~ poly(~lllt~mic acid) 5 poly(phosphorothioate) crosslinked R-PE (prepared in accordance with Example 5). The resulting ~ ulc was allowed to react overnight at 5C while rotating at 100 rpm on a rotary agitator.

Example 8 1 0 F~ ion of Goat Anti-alpha hCG Antibody/Bovine Alkaline Phosphatase (ALP) Conju~ate Usin~ Poly( lutamic acid) Polv(phosphorothioate) as a Template (a) Derivatization of Goat Anti-Alpha hCG Antibody A 1 ml aliquot of goat anti-alpha hCG antibody co~ i"g 2.8 mg (18.7 1 5 nmoV was diluted with 1 ml buffer E. The antibody was concenLl~lcd to apprnxim~tely 0.2 rnl by centrifugation at 5000 x g using a Centricon-30-ConcenL~Lur which contains a membrane sized to pass m~t-qri~l having a nulllbc average molecular weight of up to about 30,000. The conc~l~L dLt; was diluted to2 ml with buffer E and reconcentrated to approximately 0.2 ml. This 2 0 conce.ntr~ti- n and dilution procedure was repeated two more times. Next, the volume was made up to 1 ml with buffer E and the antibody solution was placed into a vial. To the antibody solution was added 0.19 mg (280 nmol) of STCM
linker (~ d as in Example 6) dissolved in 100 ,ul of DMF. The resulting reaction n~ulc was gently stirred on a rotary agitator for one hour at ambient 2 5 tel"~ d~Ulc. The derivatized antibody was purified by size exclusion chl~.",alography using a 1 x 45 cm column of Sephadex G25. The column was eqnilihr~teA and eluted with buffer E. Fractions of about 1 ml each were collected during elution and the absorbance at 280 nm was determined. The peak fractions were pooled and the concentration of the antibody in the pool was3 0 calculated from its absorbance at 280 nm using an extinction coefflcito.nt (Elcml%) of 13.9. The antibody pool was stored on ice until conjugation.

(b) Derivatization of ALP
A 0.7 ml aliquot cont~ining 7 mg (46.6 nmol) ALP was diluted to 2 ml 3 5 with buffer C, and concentrated to approximately 0.2 ml by centrifuging at 5000 x g using a Centricon-30-Concentrator. The concentrated enzyme was diluted CA 0220~373 1997-0~-14 wo 96/17580 Pcr/uss5lls586 again to 2 ml with buffer C and reconcentrated to about 0.2 ml. The volume was made up to 1 ml with buffer C and the enzyme solution was placed into a vial.
To the enzyme solution was added 0.63 mg (935 nmole) of STCM (prepared as in Example 6) dissolved in 200 ,ul of DMF. The resulting reaction ~ was gendy stirred on a rotary agitator for 30 minutes at ambient Lel.lp~ u.e and thederivatized enzyme was purified by siæ exclusion ch.olllatography using a 1 x 45 cm column of Sephadex G-25. The column was equilibrated and eluted with buffer C. Fractions of about 1 ml each were collected during elution and the absorbance at 280 nm was determined. The peak fractions were pooled and the 1 0 concentration of the enzyme in the pool was calculated from its absorbance at 280 nm using an extinction coefficient (Elcml%) of 10.

(c) Conjugation of Derivati~ed Enzyme and Antibody with Poly(C~ t~mic Acid) Poly(Phosphorothioate) as a Template 1 5 Three conjugates were prepared, with varying molar ratios of antibody:enzyme:poly(glutamic acid) poly(phosphorothioate), as follows:
Conjugate 1:
Antibody:Enzyme:Poly(Glutamic Acid) Poly(Phosphorothioate) (1:1:1) 1.0 ml aliquot (0.7 mg or 4.6 nmol) of the derivatized anti-alpha hCG
2 0 antibody was mixed with 0.45 ml (0.7 mg or 4.7 nmol) of the derivatized ALP, and 0.32 ml (0.32 mg or 4.6 nmol) of an aqueous solution of poly(~ t~mic acid) poly(phosphorothioate). The resnl~ing ~ u~t; was gently stirred on a rotary agitator overnight at 2-8C.
Conjugate 2:
2 5 Antibody:Enzyme:Poly(Glutamic Acid) Poly(Phosphorothioate) (1:3:1) 0.68 ml (0.5 mg or 3.3 nmol) of the solution of derivatized anti-alpha hCG antibody, was mixed with 0.96 ml (1.5 mg or 10 nmol) of the derivatized ALP, and 0.23 ml (0.23 mg or 3.3 nmol) of an aqueous solution of poly(glutamic acid) poly(phosphorothioate). The resulting mixture was gently 3 0 stirred on a rotary agitator overnight at 2-8C.
Conjugate 3:
Antibody:Enzyme:Poly(Glutamic Acid) Poly(Phosphorothioate) (1~
1.0 rr~ (0.7 mg or 4.6 nmol) of the solution of derivatized anti-alpha hCG antibody was mixed with 0.45 ml (0.7 mg or 4.6 nmol) of the derivatized 3 5 ALP and 0.17 ml (0.17 mg or 2.4 nmol) of an aqueous solution of poly(glllt~mic acid) poly(phosphorothioate). The resulting ~ Lule was gently stirred on a rotary agitator overnight at 2-8C.
AM three conjugates were evaluated by size exclusion HPLC. No residual antibody or enzyme were detected in conjugates 1 and 3. Conjugate 2, 5 however, contained about 20% residual starting m~tçri~l, pres~-m~bly enzyme.
The unreacted thiol groups on conjugated or any free poly(glut~mic acid) poly(phosphorothioate) were capped by treatment with N-ethylm~leimi(le, (NEM) for a period of 1 hour at ambient temperature. Aliquots of a 5 mM solution were added to the conjugate so that the final concentration of NEM in the conjugate 1 0 was about 0.3 mM.

Example 9 Preparation of Anti-pancreatic Thread Protein Antibody/Bovine Alkaline Phosphatase (ALP) Conju~ate Usin~ PolY(~elutamic acid) 1 5 Polv(phosphorothioate) as a Template (a) Derivatization of Anti-Pancreatic Thread Protein Antibody 4.0 ml of 1 mg/ml solution of anti-pancreatic thread protein antibody was concentr~tt-,-l to a~ ;" ,~te.ly 0.2 ml by centrifuging at 5000 x g using a Centricon-30-Conce~ ul and the concentrate was diluted to 2 ml with the 2 0 buffer E and reconcentrated to approximately 0.2 ml. The concentr~ti-m and dilution procedure was repeated two more times after which the volume was made up to 1 ml with buffer E. The antibody solution was placed into a vial and 0.27 mg of STCM linker (prepared as in Example 6) dissolved in 100 ~1 of DMF was added. The resulting reaction n~L~Lulc was gently stirred on a rotary 2 5 agitator for one hour at ~mhiçnt temperature. The derivatized antibody was purified by cl.lolllalography on a 1 x 45 cm column of Sephadex G-25. The column was eqnilihr~tecl and eluted with buffer E. Fractions of about 1 ml each were c-~llecteA during elution and the absorbance at 280 nm was det.o,rrnineA
The peak fractions were pooled and the concentration of the antibody in the pool3 0 was calculated from its absorbance at 280 nm using an extinction coe,fficient (Elcml%) of 13.9. The antibody pool was stored on ice until used in the conjugation reaction.

(b) Derivatization of ALP
3 5 A 0.7 ml aliquot of lO mg/ml (46.6 nmole) ALP was diluted to 2 ml with buffer C and concentrated to approximately 0.2 ml by centrifuging at 5000 x g CA 0220~373 1997-0~-14 using a Centricon-30-Concentrator. The concentr~tl cl enzyme was diluted again to 2 ml and reconcentrated to about 0.2 ml. The volume was made up to 1 ml with buffer C and the enzyme solution was placed into a vial. To the enzyme solution was added 0.63 mg (935 nmol) of STCM (prepared as in Example 6) dissolved in 200 ul of DMF. The resulting reaction ~ e was gendy stirred on a rotary agitator for 30 minutes at ~mhit~.nt ~~ c; and the derivatized enzyme was purified by chromatography on a 1 x 45 cm column of Sephadex G-25. The colurnn was equilibrated and eluted with buffer C. Fractions of about 1 ml each were collected during elution and the absorbance at 280 nm was 1 0 clet~rmin~l The peak fractions were pooled and the concentration of the enzyme in the pool was calculated from its absorbance at 280 nm using an extinction coefficient (Elcml%) of 10.

(c) Conjugation of Derivatized Enzyme and Antibody with Poly(Glutamic 1 5 Acid) Poly(Phosphorothioate) as a TP.mpl~te Two conjugates were prepared, with varying molar ratios of antibody:enzyme:poly(glutamic acid)poly(phosphorothioate), as follows:
Conjugate 1:
Ant~body:Enzyme:Poly(Glutamic Acid) Poly(Phosphorothioate) (1:1:1) 2 0 1.25 rnl (0.7 mg or 4.7 nmol) of the derivatized anti-pancreatic threadprotein antibody was rnixed with 0.45 ml (0.7 mg or 4.7) of the derivatized ALP, and 0.32 ml (0.32 mg or 4.6 nmol) of an aqueous solution of poly(~ t~mic acid) poly(phosphorothioate). The resulting ~ e was gently stirred on a rotary agitator overnight at 2-8C.
2 5 Conjugate 2:
Antibody:Enzyme:Poly(Glutamic Acid) Poly(Phosphorothioate) (1:3:1) 0.89 ml (0.5 mg or 3.3 nmol) of the solution of derivatized anti-pancreatic thread protein antibody was mixed with 0.96 ml (1.5 mg or 10 nmol) of the derivatized ALP and 0.23 ml (0.23 mg or ~.3 nmol) of an aqueous 3 0 solution of poly(glutamic acid) poly(phosphorothioate). The resulting mixture was gently stirred on a rotary agitator overnight at 2-8C.
The two conjugates were evaluated by size exclusion HPLC. No residual antibody or enzyme were detected in conjugates 1. Conjugate 2, however, contained about 20% residual starting m~tP.n~l, presumably enzyme.

The unreacted thiol groups on conjugated or any free poly(glutamic acid) poly(phosphorothioate) were capped by tre~tment with NEM for a period of 1 hour at ~mhit~,nt temperature as m~,ntioned in F.~mp1e 8c.

Example 10 Acid Deprotection of Cy~ int-,-S-phosphate Three 5 ,ul aliquots of 8.2 mM aqueous solution of cyste~minP-S-phosphate were placed into three separate vials. The three samples were diluted to 1 rnl with 0.1 M sodium acetate buffer, pH 4Ø The final pH of the samples 1 0 was found to be 4Ø The samples were left at ambient temperature and neutralized either at 1, 3, or 19 hours by addition of 50 lul of a~l,ro~illlately 5 M
sodium hydroxide and 2 ml of 0.1 M sodium phosphate, pH 8.5. The thiol groups generated were quantified colorimetrically after addition of 20 111 of lOmM solution of DTNB, The absorbance was read within S minutes of the 1 5 ~ lition of DTNB at 412 nm against a reagent blank with no cyste~mine-S-phosphate. The experim~,nt~lly determined molar extinction coefficient of 13,000 was used in thiol quantification. The results are sllmm~*7ed below in Table 2.
The expt~,rim-o,nt was repeated exactly as described above but with three 2 0 times higher concentration of ~;y~al~ e-S-phosphate. For controls, same amounts of cy~ e-S-phosphate were taken up in 0.1 M sodium phosphate buffer, pH 7.0, and subjected to thiol qll~ntification after appropriate intervals.
The results are !;ul "",~, ;7~d below in Table 3.

2 5 Table 2 Cy~le~llline-S-Phosphate pH 4.0 Exposure 1 hour 3 hours 19 hours Thiols Generated (nmol) 8.2 19.2 29.3 % Thiol D~lo~ec~ion20 46.8 71.5 Table 3 Cysteamine-S-Phosphate pH 4.0 Exposure - 1 hour 3 hours 19 hours Thiols Generated (nmol) 32.8 51.6 77.5 % Thiol Deprotection 26.7 42.0 63.0 The extent of acid catalyzed deprotection was found to be time dependent. A l~inPIeell hour incubation resulted in 63 to 71.5 % deprotection ofthe thiophosphate groups.

Example 11 Immobilization of Anti-TSH Antibody to Amino Microparticles (a) Function~1i7~tion of the Antibody An aliquot of anti-TSH antibody cont~ining 5 mg (33 nmol) is 1 0 extensively dialyæd against buffer E and placed in a vial. To the antibody solution is added 0.22 mg (660 nmol) of SMCC dissolved in 100 ul of DMF and the res-llting lli~Y,LLIle iS gently sti~red for 30 minutes at ~mhient temperature. The activated antibody is recovered by chromatography over a 1 x 45 cm colurnn of Sephadex G-25. The column is equilibrated and eluted with buffer E. Fractions 1 5 of about 1 rnl each are collected during elution and absorbance at 280 nm is ~letPrminerl The peak fractions are pooled and the concentration of the antibodyin the pool is calculated from its absorbance at 280 nm using an extinction coefficient (Elcml%) of 13.9.
To the activated antibody pool (4 mg or 26.7 nmol) is added 20 ~1 (0.2 2 0 mg or 1.3 nmol) of a solution of ALP and 8 mg (133 nmol) of poly(glutamic acid) poly(phosphorothioate) (MW = 60,000; 25 phosphorothioate groups/poly(~ t~mic acid) poly(phosphorothioate) dissolved in 0.8 rnl of buffer E. The resulting mixture is gently stirred overnight at 2-8C. The poly(glutarnic acid) poly(phosphorothioate) functionalized antibody is stored on ice until 2 5 coupling to rnicroparticles.

(b) Activation of Amino Microparticles One milliliter of arnino microparticles (diameter = 0.25 ,um, % solids =
10) is suspended in 3 rnl of distilled water followed by addition of 2 g of the 3 0 anion exchange resin, BIO-REX MSZ 501 (D). The resin/microparticle ~ Lult;
is rotated end over end for 1 hour at ~mhient temperature and then poured into acourse sintered glass funnel. The microparticles are pulled through the funnel under low vacuum and centrifuged at 18,000 rpm for 30 minlltes The sup~rn~t~nt is ~lec~nts~ carefully and the rnicroparticles are washed with 10 rnl of 3 5 water, and centrifuged. The supernatant is clecanted and the washed rnicroparticles are suspended in 4 ml of buffer E. 8 mg of STCM (prepared in accordance with Fx~mrle 6) is dissolved in 4 ml of DMF and added to the rnicroparticle suspension. The ~ is rotated end over end for one hour at ~mhi~-nt temperature and then poured into a centrifuge tube. Buffer E is added to final volume of 32 ml and the microparticles are centrifuged at 15,000 rpm for 30 minlltes. The sup~rn~t~nt is ~ec~nt~d and microparticles are resuspended in 30 rnl of buffer E. The washing with buffer E is repeated two more times and the washed microparticles are finally resuspended in 4 ml of buffer E.

(c) Coupling of Antibody to Microparticles 1 0 The poly(glnt~mi~ acid) poly(phosphorothioate) filnc~ion~li7eA antibody pool (3 ml colll~i,-;,,g 4 mg) is combined with the activated microparticles andthe resulting suspension is rotated end over end overnight at 2-8C. The rnicroparticles are poured into a centrifuge tube and buffer E cont~ining 1 mg/ml bovine serum albumin (BSA) is added to 35 ml. The microparticles are 1 5 centrifugçd and resuspended in 35 ml buffer E cont~inin~ BSA, after the supt~rn~t~nt has been dec~ntçd. The particles are washed two more times to ensure removal of any free antibody and then resuspended in an a~pl~lialt;
storage buffer and stored at 2-8C.

2 0 Example 12 Polv(~lutamic acid) poly(phosphorothioate) Crosslinked Bovine Alkaline Phosphatase (ALP) Site-Specificallv Conju~eated with Anti-Hepatitis B Surface Antigen I~G
(a) Derivatization of Anti-hep~titi~ B Surface Antigen (HBsAg) Antibody 2 5 To 10 ml of l. 1 mg/ml anti-Hepatitis B surface antigen IgG was added lOml of O.l M triethanol~mine (TEA), 0.16 M NaCl, pH 8.0 buffer (buffer I).
The antibody was concçntr~t~ to ~pro~ ately 1.5 ml using a Ct;l-L-ipl~-30-ConcenLlalo, with a MW cutoff of 30,000. The concentrate was redillltçd to 20 ml using buffer I then recon~tit~lted to 1.5 ml. This concentration/dilution 3 0 procedure was repeated three tirnes. The volume of the final conce .~ ed antibody solution was diluted to 2 ml with buffer I and placed in an amber vial.
To 1.71 rnl of the 4.68 mg/ml (53.3 nmol) washed IgG was added 160 ,ul of 200 mM (320 nmol) sodium m-periodate dissolved in buffer I. This ~
was allowed to react for one hour at room temperature while rotating on a rotary3 5 agitator. Following the incubation, the above reaction mixture, which contained oxidized antibody, was applied to the equilibrated G-25 colurnn which had been wo96/17580 Pcr/uS95/15586 prepared as above using buffer E. The column was eluted with buffer E and 0.75 ml fractions were collected. Frac~ons with an A280 greater than 0.3 AU
were pooled. The antibody pool was concentrated to 1 ml using a Centricon-30-Concç~ lol with a MW cutoff of 30,000. To the concentrated antibody pool was added 250 ~Ll of 0.75M (188 ~mol) cy~.la~ e dihydrochloride dissolved in buffer E. Following a 15 rninute incubation at room temperature with gentle stirring, 63 ,ul of 0.3M (18.9 ,umol) sodium cyanoborohydride dissolved in buffer E was added and the resl-lting mixture was allowed to react at room ture overnight. After the overnight incubation, the reaction mix lule was 1 0 applied to a Sephadex G-25 column that had been prepared as above using 0.1M sodium phosphate, 0.1 M NaCl, 2 mM EDTA, pH 7.0 buffer (buffer J).
The colurnn was eluted with buffer J and 0.75 rnl fractions were collected.
Fractions with an A280 greater than 0.3 AU were pooled. The antibody pool was concentrated to 1.5 rnl using a Centricon-30-Concentrator with a MW cutoff 1 5 of 30,000. To the concentrated antibody pool was added 75 ~1 of 40 mM (30 nmol) DTT dissolved in buffer J. This Il~ib~lule was allowed to react for 15 minntes at room le~ ,ture while rotating at 100 rpm on a rotary agitator. After the reaction, the ~ lu-t;; was applied to a Sephadex G-25 column that had been prepared as above using buffer J. The column was eluted with buffer J and 0.75 2 0 ml fractions were collected. Fractions with an A280 greater than 0.3 AU were pooled. The res-llting Fc-functionalized antibody with free thiols in the Fc region was stored on ice until conjugated.

(b) Derivatization of Poly(glut~mic acid) Poly(phosphorothioate) 2 5 Crosslin_ed ALP
To a 2.27 ml aliquot of 2.64 mg/ml (40 nmol) poly(glllt~mic acid) poly (phosphorothioate) cros~link--d ALP (prepared in accordance with Example 2) was added 100 ,ul of 0.16 M NEM (16,umol). The resulting llli~Lule was allowed to incubate for one hour while rotating at 100 rpm on a rotary agitator.3 To 50 ,ul of DMF was added 0.54 mg (831 nmol) of STCM linker which was prepared as described in Example 6. This linker solution was added to 1.18 ml of 2.53 mg/ml (20 nmol) NEM capped croc~link~l ALP and allowed to react for one hour at room temperature while rotating at 100 rpm on a rotary agitator.
After the reaction was complete, the mixture was applied to a Sephadex G-25 3 5 column which had been prepared as above except that the column was equilibrated with three column volumes buffer C. The column was eluted with CA 0220~373 1997-0~-14 buffer C and 0.75 ml fractions were collected. Fractions with an A280 greater than 0.3 AU were pooled. The linker functionalized crosslink~A ALP was stored on ice until conjugated.

5 (c) Conjugation of Fc Derivatized Anti-hepatitis B Surface Antigen IgG to Poly(ghlt~mic acid) Poly(phosphorothioate) Cros~linked ALP
To a 0.52 ml aliquot of 0.97 mg/ml (3.5 nmol) Fc derivatized anti-Hepatitis B surface antigen IgG was added 1.30 ml of 0.77 mg/ml (7 nmol) STCM lilLker derivatiæd NEM capped poly(glutamic acid) 1 0 poly(phosphorothioate) cros~linkP-l ALP. The resulting ~ e was allowed to react overnight at 5C while rotating at 100 rpm on a rotary agitator to yield the conjugated product.
Example 13 1 5 Poly(~lutamic acid) Poly(phosphorothioate) Crosslinked Bovine Alkaline Phosphatase (ALP)/Fc Site-Specifically Derivatized Anti-hCG IgG Conjugate (a) Derivatization of Anti-hCG Antibody.
To 8 ml of 1.1 mg/ml anti-hCG IgG was added 10 ml of buffer I and the antibody was conct;llLLa~ed to approxim~tely 1.5 ml using a Ce~ ~-30-2 0 Conce~ ol. The concentr~t~- was rediluted to 20 ml using buffer I then reconcentrated to 1.5 ml. This concentration/dilution procedure was repeated three times. The volume of the antibody solution was made up to 2 ml with buffer I and placed in an amber vial. To 2 ml of 4.0mg/ml (53.3 nmol) washed IgG was added 220 ml of 200 mM (440 nmol) sodium m-periodate dissolved in 2 5 buffer I. This l~ wc was allowed to react for one hour at room tenl~ldlule while rotating at 100 rpm on a rotary agitator. Following the incubation, the above reaction n~L~lult;, which contained the oxidized antibody, was applied to a G-25 column as prepared above using buffer E. The column was eluted with buffer E and 0.75 ml fractions were collected. Fractions with an A280 greater 3 0 than 0.3 AU were pooled. The antibody pool was concentrated to 1 ml using an Amicon Centricon-30-Concentrator. To the concentrated antibody pool was added 300 ml of 15 mM (4.81 mmol) 4-(N-maleimidomethyl) cyclohexane-I-carboxyl hydrazide (M2C2H) linker dissolved in buffer E. This mi~lult; was allowed to react for three hours at room ~ lp~ld~ while rotating at 100 rpm on 3 5 a rotary agitator. Following the incubation, the above reaction ~ lule was applied to a G-25 column as prepared above except 0.1 M sodium acetate, 0.1 M

CA 0220~373 1997-0~-14 NaCl, pH 6.0 (buffer K) was employed to remove unreacted linker. The column was eluted with buffer K and 0.75 ml fractions were collected.
Fractions with an A280 greater than 0.3 AU were pooled. The Fc-functionalized antibody with maleimides in the Fc region was stored on ice until conjugated.

(b) Conjugation of Fc Derivatized Anti-hCG IgG to Poly(glllt~nic acid) Poly(phosphorothioate) Crosslinked ALP
To a 1 ml aliquot of 2 mg/ml (13.3 nmol) Fc derivatized anti-hCG IgG is added 1 ml of 4 mg/ml (26.6 nmol) poly(gh~t~mic acid) poly(phosphorothioate) 1 0 cros~linkt-d ALP (prepared in accordance with the technique described in Example 2). The resulting ~ ure is allowed to react overnight at 5C while rotating at 100 rpm on a rotary agitator to yield the conjugated product.

Example 14 1 5 Synthesis of Dithiothreitol Diphosphate A solution of 1,4-dibromo-2,3-butanediol (1.0 g, 4.0 mmol) in 5 ml DMF is added to sodium thiophosphate dodecahydrate (3.8 gm, 10.1 mmol) in 20 ml H20. The ~ ; is stirred overnight at room temperature. A 5% silver nitrate solution is added to precipitate excess sodium thiophosphate. The 2 0 precipitate is filtered out and the filtrate dried under high vacuum. The solid residue is ~ d~d with methanol and filtered to yield 1.8 g (3.8 mmol) of threitol bis-phosphorothioate tetrahydrate.

Example 15 2 5 Dithiothreitol Diphosphate Deprotection When phosphorothioate deprotection of the bis-phosphorothioate threitol is desired, to activate its reducing ability, the cleavage of the phosphate bonds is accomplished by addition of 1 IlM solution of ALP. Due to the chelating nature of dithiothreitol, zinc and magnesium are added to the reaction meAillm to 3 0 ~the catalytic activity of the ALP.

wo 96/17580 PCT/USg5/15586 Example 16 Svnthesis of Phosphorothioate Heterobifunctional A~ents (a) N-hydroxy,~ucrinimi(lyl Cyste~mi~ophosphorothioate 4,5-Dithioheptyl l-Carboxylate 48.2 mg (269 llmol) of ~;y~le~ e-S-phosphate was dissolved in 2.5 ml of dto,ioni7e-1 water and the res-llting solution was added to a solution of 330 mg (816 llmol) of 3,3'-dithiopropionic acid bis-active ester in 2.5 ml of DMF. The two solutions were comhine(l while stirring atroom temperature and the stirring was continlleci for an additional 3 minutes after the solutions were combined.
1 0 After the 3 minute mixing period the solution was evaporated under reduced pressure for 18 minutes at room temperature. 20 ml of chloroform was added to the resulting white residue and the ~,~i,~Lur~ was stirred for 10 minutes. A white precipitate formed which was separated from the supern~t~nt liquid and dried under reduced pressure to yield the powder product N-hydroxysuccinimidyl 1 5 cysteamidophosphorothioate 4,5-dithioheptyl l-carboxylate.
FAB(-) mass ~e~iLl ulll data in(li~ated t,he presence of m~t~-,ri~l of m/e -1 =
445, the expected rn/e of the desired product is 446. FAB (+) spectrum data alsoinriirate~l the presence of the ~r~ iate ion (mJe+ Na+).

2 0 (b) N-hydroxysuccinimidyl Cysteami~lophosphorothioate 3-Oxybutyl 1-Carboxylate 50 mg (279 ~mol) of cysteamine-S-phosphate was dissolved in deionized water and the resulting solution was added to a solution of 370 mg (1.12 ~mol) of diglycolic acid bis-active ester dissolved in DMF. The solutions 2 5 were mixed at room temperature during the addition and the stirring was continued for an additional 3 minutes after the addtion was complete. After the stirring period was cnmp1ete, the reaction .,-,x lur~ was evaporated under reduced pressure at room temperature for 20 minutes. 35 ml of tetrahydrofuran (THF) was added to the resulting white residue and the resulting llli~Llllt; was stirred for 3 0 10 minutes. A white precipitate formed and was separated from the sup~,rn~t~nt - liquid and dried under reduced pressure to yield the product N-hydroxysuccinimidyl cysteamidophosphorothioate 3-oxybutyl l-carboxylate as a white powder.
FAB(-) mass spectrum data indicated the presence of m~teri~l of m-le -1 =
3 5 369, the expected mle of the desired product is 370. FAB (+) spectrum data also wo 96/l7sxo PCT/USg5/15586 inr~ ted the presence of the a~ iate ion (m/e+ Na+) however, the presence of large amounts of sodium ions produced a strong background.

(c) N-hy~u~y~uccinimidyl Cyste~miclophosphorothioate Heptanoyl 1-5 Carboxylate 50 mg (279 ~lmol) of cy~ llir~ S-phosphate sodium salt was dissolved in 3 ml of deionized water and added to a solution of 400 mg (1.086 ,umol) of the bis-active ester of suberic acid dissolved in 3 ml of DMF. The ~l(iition wasp~,rform~d over the course of 1 minute at 5C. The resulting reaction n i~ul~;
1 0 was stirred at room tempOEature for 1 hour and 45 minutes. The reaction ll~i~ G
was then evaporated to dryness under reduced pressure at room temperature for 18 minntes and the resulting solid residue was treated with 10 ml of THF. A
white preciI it~t~o formed and was collected and treated again with 10 ml of THF.
A white precipitate was again collected and dried under reduced pressure to yield 15 theproductN-hydlo~y~uccinimidylcy~ "~idnphosphorothioateheptanoyl 1-carboxylate.
FAB(-) mass spectrometry in~ tecl the presence of the molecular ion m/e-l = 409 which corresponds to the m/e of the desired m~tP,ri~l of m/e = 410.

2 0 (d) Cysteamidophosphorothioate Heptanoyl l-Hydrazide 0.10 gm (250 ,umol) of N-hydroxysuccinimidyl cysteamidophosphorothioate heptanoyl l-carboxylate is dissolved in 2 ml of a 1:1 solution of DMF in deionized water and the resulting solution is added to a solution of 1 mmol of hydrazine monohydrate dissolved in 2 ml of a 1:1 solution 2 5 of DMF in deionized water. The resulting reaction ll~Lx.Lul`e iS incubated while stirring for 10 minllt~s at 0C. After the incubation the reaction n~ U1G iS
evaporated until dry under reduced pressure. The solid residue is then washed three times with 10 ml of THF per wash. Precipitate formed after the last wash is then dried under reduced pressure.
(e) Cyste~mi(lophosphorothioate Heptanoyl l-(Aminoethyl)carbox~mi-le 0.1 gm (250 ,~Lmol) of N-hydroxysuccinimidyl cysteamidophosphorothioate heptanoyl l-carboxylate is dissolved in a 1.1 solution of DMF in deionized water and the res-llting solution is added to a 3 5 solution of 1 mrnol of ethylene~ mine dissolved in 2 ml of a 1:1 solution ofDMF in deionized water. The resulting reaction n~ixture is incubated while CA 0220~373 1997-0~-14 stirring for 10 minutes at 0C. After the incubation the reaction mi~lul~ is evaporated until dry under reduced pressure. The solid residue is then washed three times with 10 ml of THF per wash. Precipitate formed after the last wash is then dried under reduced ~lr,s~ulc;.

(f) p-NitrophenylCy~lr,~ idophosphorothioateHeptanoyl 1-Carboxylate 250 ~lmol of N-hyd~o~y~uccinimidyl cyste~mitlophosphorothioate heptanoyl 1-carboxylate [from Example l 6(c)] is dissolved in 3 ml of deionized water. l mmol of p-nitrophenol is dissolved in 3 ml of DMF and added to the 1 0 N-hydroxysuccinimidyl cyste~mi-lophosphorothioate heptanoyl 1-carboxylate solution. The resulting reaction nfi~ule is stirred at room temperature for 2 hours and then evaporated until dry under reduced pressure. The solid residue isthen washed three times with 10 ml of THF per wash. Precipitate formed after the last wash is then dried under reduced pressure.

Example 17 Stoichiometric Control of the Size of Poly(glutamic acid) Polv(phosphorothioate) Crosslinked Bovine Alkaline Phosphatase (ALP) , (a) ~l~a.dlion of Poly(glnt~mic acid) Poly(phosphorothioate) Cro~sslinkecl A number of poly(glut~mic acid) poly(phosphorothioate) crosslink~-l ALP
preparations were produced over a range of poly(glnt~mic acid) poly(phosphorothioate) to ALP ratios. Using the crosclinking techniques set forth in Example 2, poly(gl~lt~mic acid) poly(phosphorothioate)to ALP ratios of 1:1, 2:1, 3:1, 4:1, and 6:1 were employed to crosslink ALP. Theproducts of the crosslinking reactions were evaluated in order to detlo,rmine the effect of varying the molar ratio of reactants had on controlling the size of the crosslink~1 ALP.

3 0 b) Charact~ri7~tion of Crosslink~,d ALP
Poly(glutamic acid) poly(phosphorothioate) cro~slinkt-d ALP was evaluated by size exclusion chromatography using a Bio-Sil SEC-400 column.
Detection was at 280nm. The mobile phase was buffer E running at a flow rate of 1.0 ml/minute. From the HPLC chromatograms, the percentage of product 3 5 with a retention time corresponding to singlet crosslink~d ALP was calculated.
The result~s of this evaluation are shown in Figure 15. As shown by Figure 15, -W O 96/17580 PCT~US95/15586 the amount of monomeric crosslinked ALP produced per cros~linking reaction increased as a function of increasing the amount of poly(gl~lt~mic acid) poly(phosphorothioate). Additionally, the amount of mnltimt-rs produced decreased when the amount poly(glutamic acid) poly(phosphorothioate) was 5 increased. Hence, through stoichiometric manipulation, modulation of the size of the crosslinked ALP was possible.

While the invention has been described in detail and with reference to specific embodirnents thereof, it will be apparent to one skilled in the art that 1 0 various changes and modifications may be made therein without departing from the spirit and scope thereo Additionally, all references to patents or publications in this specification are incorporated herein by reference.

Claims (10)

Claims What is claimed is:

1. A crosslinking and conjugating agent comprising a compound corresponding to the formula (I):
(I) Q-(S-PO3-2)n wherein n is at least 1 and Q is a straight or branched monomer, polymer or oligomer having an average molecular weight between about 200 and about 1,000,000 and wherein when n is 1, Q comprises at least 1 reactive functionality.

2. The crosslinking and conjugating agent of claim 1 wherein said monomer, polymer or oligomer is selected from the group consisting of:
polystyrenes, polysaccharides, polyacrylamides, polypeptides, C5-C100,000 straight or branched alkyl chains, C5-C100,000 straight or branched alkyl chainshaving heteroatoms within said chains, and C10-C75,000 cycloalkyl.

3. The compound of claim 1 selected from the group consisting of:
carboxymethylamylose poly(phosphorothioate), poly(acrylamide) poly[acryloyl(2-(2-phosphorothioethyl)aminoethyl] hydrazide, poly(glutamic acid) poly(phosphorothioate), poly(styrene) poly(phosphorothioate), poly(acrylamide) poly(phosphorothioate), dextran poly(phosphorothioate), N-hydroxysuccinimidyl cysteamidophosphorothioate 4,5-dithioheptyl 1-carboxylate, N-hydroxysuccinimidyl cysteamidophosphorothioate 3-oxybutyl 1-carboxylate, N-hydroxysuccinimidyl cysteamidophosphorothioate heptanoyl 1-carboxylate, cysteamidophosphorothioate heptanoyl l-hydrazide, cysteamidophosphorothioate heptanoyl l-(aminoethyl)carboxamide, and p-nitrophenyl cysteamidophosphorothioate heptanoyl l-carboxylate.

4. A method of crosslinking and conjugating compounds comprising the steps of:
(a) activating a compound corresponding to the formula (I):
(I) Q-(S-PO3-2)n wherein n is at least 1 and Q is a straight or branched monomer, polymer or oligomer having an average molecular weight between about 200 and about 1,000,000 and wherein when n is 1, Q
comprises at least 1 reactive functionality, and (b) contacting said activated compound (I) with at least one compound that displays an electrophilic group.

5. The method of claim 4 wherein said compound (I) is selected from the group consisting of: carboxymethylamylose poly(phosphorothioate), poly(acrylamide) poly[acryloyl(2-(2-phosphorothioethyl)aminoethyl] hydrazide, poly(glutamic acid) poly(phosphorothioate), poly(styrene) poly(phosphorothioate), poly(acrylamide) poly(phosphorothioate), dextran poly(phosphorothioate), N-hydroxysuccinimidyl cysteamidophosphorothioate 4,5-dithioheptyl 1-carboxylate, N-hydroxysuccinimidyl cysteamidophosphorothioate 3-oxybutyl 1-carboxylate, N-hydroxysuccinimidyl cysteamidophosphorothioate heptanoyl 1-carboxylate, cysteamidophosphorothioate heptanoyl 1-hydrazide, cysteamidophosphorothioate heptanoyl 1-(aminoethyl)carboxamide, and p-nitrophenyl cysteamidophosphorothioate heptanoyl 1-carboxylate.

6. The method of claim 5 wherein activating said compound corresponding to the formula (I) comprises contacting said compound with a member of the group consisting of: a hydrogen ion concentration between about pH 4.0 and about 5.5, and a phosphatase enzyme.

7. A conjugate comprising: at least one binding member and at least one detectable moiety bound to the residue of a compound corresponding to the formula (I):
(I) Q-(S-PO3-2)n wherein n at least 1 and Q is a straight or branched monomer, polymer or oligomer having an average molecular weight between about 200 and about 1,000,000 and wherein when n is 1, Q comprises at least 1 reactive functionality.

8. The conjugate of claim 7 wherein said detectable moiety is stabilized by said compound corresponding to the formula (I).

9. A solid phase reagent comprising: a solid phase and at least one binding member bound to the residue of a compound corresponding to the formula (I):
(I) Q-(S-PO3-)n wherein n at least 1 and Q is a straight or branched monomer, polymer or oligomer having an average molecular weight between about 200 and about 1,000,000 and wherein when n is 1, Q comprises at least 1 reactive functionality.

10. A reducing agent comprising a compound of the formula (Y) (Y) wherein (A) and (Z) can be independently selected from C1-C5 alkyl and CONH(CH2)p wherein p is an integer between 1 and 5.