CA1322341C

CA1322341C - Method for the preparation of antibody-fragment conjugates

Info

Publication number: CA1322341C
Application number: CA000589927A
Authority: CA
Inventors: Michiel E. Ultee; Vernon L. Alvarez
Original assignee: Cytogen Corp
Current assignee: Cytogen Corp
Priority date: 1988-02-03
Filing date: 1989-02-02
Publication date: 1993-09-21
Anticipated expiration: 2010-09-21
Also published as: WO1989007269A1; EP0382796A4; EP0382796A1; US4937183A; JPH02503088A

Abstract

ABSTRACT OF THE DISCLOSURE A novel method for preparing soluble antibody fragment compositions having superior characteristics for targeted delivery when administered in vivo are disclosed. The antibody fragment compositions are characterized by substantially the same immunospecificity as the unconjugated antibody and aqueous solubility such that they are suitable for in vivo adminstration. Therapeutic and diagnostic methods utilizing the antibody fragment compositions are also disclosed.

Description

`~ ~322~ 2-1. FIELD OF THE INVENTION
The present invention is dir~cted generally to the area of antibody system~ for the dalivery of compounds to target sites in vivo. More particularly, the invention is .
directed to novel methods gor preparing conjugates comprising an antibody ~ragment 9it8 sp~cifically attached to a compound ~$a an oxidlz~d carbohydrate moiety of the antibody ~ragment. Th~ co~po~tions prepared according to 'he method of th~ lnvention ar~ advantageously employed to deliver co~pound~ to target 3it~ ~ollowing ln vivo administration.

2. ~ACXGROUND O~ THE INVENTION
United State3 P~tent No. 4,6~1,958 ~ssued to Rodwell et al. describes a method for SitQ ~p~Ci~iG covalent attach~ent oP a compound to an ant~body ~olecul~ by selectively oxidizlng a carbohydr~t~ moiety o~ th~ antibody, loca~ed outs~do tho antig~n binding region of th~ antibody, to ~orm an aldohyd~ group and th~n r~act~ng th~ re~ultant ald~hyd~ group with an a~ine group ~uch as a primary amine, secondary amine, hydrazin~, hydræzide, hydroxylamlne, phenylhydra~lnQ, or ~icar~az~do to ~or~ an antibody-compound conjugat~ which i~ charactorizlad by subst~ntially th~ sa~ c~p~ci~lcity al~ th~ unc:on~ugatl3d antibody.
European Patent Application No. 85401695 . 3 which was published as Publication No. EP 173629 A on March S, 1986 describes methods for prepa~ing and co~positions comprising aqueous soluble antibody-metal ion complexes in which a compatible chelator coordinately bound to a metal ion is coval~3ntly attached via an amine group of the chelator to an oxidized carbohydrate moiety of an antibody molecule, located outside the antigen binding region of the antibody.
The antibody-metal ion complexes are characterized ~y (1) substantially the same immunospeci:Eici~y as the unconjuyated antibody molecule; and .

1~1 , : , :, ' ' . ,~ :, . . .
.. .. .

, , , : ~ , ~ ' :: ,' ~3223~:~

(2) aqueous ~olubility such that they are ~uitable for in vivo administratiQn.
European Patent Application No. 85401776.1 published 5 March 26, 1986 describes methods for preparing and compositions comprising aqueous soluble antibody-therapautic agent conjugates in which a therapeutic agent is attached, either directly ox through a linker, via an amine group to an oxidized carbohydrate moiety of an antibody molecule, 10 located outside the antigen binding region of the antibody.
The antibody-therapeutic agent conjugates are characterized by (1) substantially the same immunospecificity as the unconjugated antibody molecule; and (2) aqueous solubility such that they are suitable for in vivo administration.
The methods described in the above references involve site specific attachment of a compound to either a whole antibody molecule or a preformed antibody fragment such as a half antibody molecule (i.e., a single heavy: light chain pair), a Fab, a (Fab' )2~ or a Fab~ fragment to form the 20 antibody-~ompound conjugates. Thus, according to conventional methods a preformed antibody fragment is coupled to a compound to form an antibody fragment conjugate.
In contrast, the present method involves cleavage of an antibody composition, comprising an antibody attached to a compound, a linker or a linker-compound intermediate, in which the attachment is site specific via an oxidized carbohydrate moiety of the whole antibody. Cleavage of the antibody composition according to the present invention 30 ~orms an antibody fragment composition. The present invention is based on the surprising discovery that antibody fragment conjugates prepared according to the methods disclosed herein behave significantly different from conventional conjugates when administered in ViVQ and 35 exhibit in vivo biodistribution which is advantageously used for targeted delivery to in vivo sites.

, ~ , . . .

.~ :

~3223~

3. SUMMARY OF THE INVFNTION
Th~ present invention provide~ a novel method for the preparation of agueous ~oluble antibody-~ragment compositions as well as a uniqus cla~s of antibody ~Fab')2 fragment co~positions prspared by ~uch method, Surprisingly antibody ~rag~ent compositions prepared according to the methods o~ th~ invenkion when administered _ vivo behave in a uperior mann2r for tarqeted delivery when compared to analogous antibody ~ragment compositions prepared by conventional ~ethod~. Thu~ the pre~ent compositions are adv~ntageou~ly used for th~ targeted delivery of a wide variety o~ compounds for th2rapeuti~ and diagnostic application~.
In it~ mo~t broad a~pect, the pre~ent invention provides a ~ethod for preparing an antibody compound conjugate (antibody linker inter~edi~t~) which comprises:
reac~ing a soluble antib~dy-compound conjugate (antibody-linker intarmediat~), which co~pri~es an antibody attached to a compound (link~r-intermQdiate) by a covalent bond to an oxidized carbohydrat~ of the antibody located outside the antiyen binding region o~ said antibody, with an activated thiol protease enzyme to form an antibody (Fab')2 co~pound conjugate (antlbody (Fab')2-linkex intsrmediate) , characteri~ed by (a~ sub~tantially the ~ame immunospecificity as the unconjugated antibody and (b) agueou~ ~olubility such that the antibody (~ab')~ compound con~ugate ~antibody (Fab')2-linker intermediate) is uitable ~or in vivo ad~ini~tration.
Th~ invention also provides nov~l an~ibody (Fab~)2 composition~ and m~thod3 utilizing such co~positions for th~rapeutic and diagno~tlc treatment o~ a variety of cellular disorder~.

~ .
~ ' ' .

~32~3~
~5-4. BRIEF DESCRIPTION OF THE FIGURES
The present invention may be ~ore fully understood by reference to the following detailed de~Gription of the invention, examples of specific a~bodiments and the appended ~igures in which:
FIG. l (A-B) is a graphic illu~trat:Lon of the in vitro bindin~ activity o~ B72.3 antibody compo~.itions. FIG. l~
shows binding activity o~ a B72.3 (Fab')2-linker intermediate prepared according to th~ reaction scheme described herein and o~ a conventionally prepared B72.3 (Fab')2 fragment. FIG. lB ~hows binding activity of a whole B~2.3 linker intermediate and of the native whole B72.3 antibody.
FIG. 2 is a representation of the carbohydrate moieties of B72.3 who}e antibody and (Fab'~2 fragments.

5. DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to a novel method for preparing aqueou~ ~oluble antibody compositions, comprising an (Fab')2 fragment o~ an antibody site specifically coupled to a co~pound either directly or via a linker. The antibody co~po~itions are advantageously u~ed for targeted delivery to an antigenic ~ite in vivo. The inven~ion i~ ba~ed on ~he ~urpri~ng ~iscovery tha~ antibody compoeition~ prepared according to the described method exhibit ~igni~icantly different advantageous behavior and biodistribution when admini~tered in vivo compared to analogous composition~ prepared by conventional methods.
As used throughout thi~ application, the term 30 nantibody ~Fab')2~ or ~antibody (Fab')2 fragment~ is meant to include ~ragments of an antibody which are ~ubstantially the same in an~igen binding activity and apparent ~olecular size or weight as those obtained using the pepsin digestion method described by Ni~onof et al., lg60r Arch. Biochem.
35 89:230~ The (Fab')2 fragments o~ the invention are not ~36223~ ~

obtained u~ing the pepsin digestion method of Nisonoff et al., supra. Rather they are prepared by digestion of an antibody-compound, an antibody-link~r inter~ediate or antibody-linker-compound conjugate using a pre-activated thiol protease enzyme, including but not limited to papain, chymopapain, bromelain, ficin, ~tc. in th~ reaction scheme de$cribed herein. The anti~ody ~Fab')2 fragm~nts do not exist as free (Fab')2 frag~ent~ according to the invention, but rather a~ part of antibody (Fab'~2-co~pound conjugates, antibody (Fab~)2-linker int~rmediates or antibody (Fab')2-linker-compound conjugates.

5. 1 7 ~2THOD OF PREPARATION
Antibody (Fab')2-compound conjugates are prepared by a method which results in a unique class of antibody fragment compositions diffsrent ~rom any other previously described antibody fragment compositions.
Thus, according to the pre~ent invention antibody (Fab')2 compound con~ugates ar~ prepared as follows:
An 3ntibody-compound conjugate, an antibodyolinker intermediake or an antibody-linker-compound conjugate is reacted with an ef~ective amount o~ an activated thiol protease to ~orm an antlbody ~Fab')2 compound conjugate/ an antibody tFab'32 linker int2rm~diate or an antibody (Fab')2-linker-co~pound conjugate. Suitable thiol protease enzy~es include, but are not limited to: papain, chymopapain, ~ro~elain, ficin, etc. The thiol protease enzyme~ are activated by reaction with cysteine or a low molecular weight ulfhydryl reducing agent such as 30 dithiothreitol, ~rcaptoethanol, ~ercaptoethylamine, etc.
In practice, an antibody-compound conjugate, an antibody-linker int~rmediate or an antibody-linker compound conjugate i~ react~d wl~h an amount o~ an activa~ed thiol proteas~ enzyme equivalent (on a weight basis) to about l-20% of the antibody composition. ~he reaction mixture may ~32234~

- be buffered using a buffer such as pho~phate, citrate or acetate buffer at a pH from about 5.0-~.0, preferably pH
5.0-6Ø The reaction mixture iB incubated at ~ temperature from about 4-37C, generally about roo~ te~perature ~about 25C). The raaction is prefer~bly perfon~ed in the presence of a chelating agent such as, Por example, ethylene-diaminetetraacetic acid (ED~) at about 1-3 ~M, which enhan es the stability of ~he thiol proteiase enzyme.
In practice, the thiol protease enzyme is pre~
activated by incubation with cysteine or ~ ~ulfhydryl r2ducing agent such as dithiothreitol, mercaptoethanol, mercaptoethylamine, 8tC. For exampl2, a thiol protease ~nzyme may be ac~ivated by di~solving th~ enzy~e at about 1 ~g/ml in 50 mM cy~teine-containing buffer and incubating the reaction m~xture Por about 1 hour at room temperature. The cysteine can ~e removed by gel filtration chromatography (~desalting~) and then the eluted activated enzyme can be used as described to digest an antibody co~position.
For convenience and ease o~ ~eparation o~ th~ antibody (Fab')2 composition~ formed, the en~ym~ can be im~obilized by attachment to a substrate ~uch a~ agarose, Sepharo~e~ or Sephadex~, polyac~yla~ide, or agaro~e-ac~ylamide beads, befor~ activation. ~or exa~ple, agarose beads activated by reaction with carbonyldiimidazole (co~mercially available as ~Reacti G~l Plerce~ Chemical Corporation) can be used to im~obilize the thiol protease enzyme employed in the present r~action sche~2.
When ~n antibody-linker ~ntermediate i~ u ed a~ the starting material, a compound i5 coupled to the antibody (Fab')2 link~r composition formed ~o form a usePul antibody (Fab)2-linker compound conjugate. ~hen an antibody-compound conjuga~e or an antibody-linker compound conjugate is used a~ the starting material, the antibody (Fab')2 compound conjug~te ~ormed is use~ul for a vareity of ln vivo applications.

. .. . .
~, .
.
.
- : ~

~3223~.

The antibody (Fab')2 compound conjugates prepared using the m~thod of the invention are characterized by (1) substantially the ~ame immunospecificity a~ the unconjugated antibody and (2) aqueous ~olubility such that they are suitable for in vivo administration.
_ The antibody-co~pound conjugates, antibody-linker inter~ediate and antibody-linker-co~poundl conjugates used in the pr~ent invention ~rQ prepared according to methods described in United States Pat. No. 4,671,958 issued to Rodwell et al., in European Patent Application No.
85401695.3 published o~ March 5, 1986 and European Patent ~ppln. No. ~5401776.1 published on ~arch 26, 1986.
Briefly, a carbohydrate ~ide chain of an antibody, located outside the antigen binding region of the antibody, is selectively oxidized or enzymatically modi~ied to form an aldehyde group. The resulting aldehyde i9 reacted with an amine group (2.g., ammonia derivatives such as primary amine, secondary a~ine, hydroxylamine, hydrazine, hydrazide, phenylhydrazine, emicarbazide or thiosemic~rbazide) to form a Schi~f's base or reduced Schiff's ba~e (e.g., imine, ena~ie, oxime, hydrazon~, phenylhydrazone, semicarbazone, thiosemicarbozone or reduc~d form~ thereo~).

5,1.1. HE~ICA~ METHODS OF OXIDATION
Oxidation of the carbohydrate portion or moiety of antibody molecules leads to format~on of aldehyde groups. A
variety o~ oxidizing ~gents can be used, such as periodic acid, paraperiodic acid, ~odium metaperiodate and potassium metaperiodate. Among thes~, oxygen acids and salts thereof 30 ar~ preferred ~ince ~econdary or undesira~le side reactions are less freguent. Por a general discussion, aee Jac~son, 1944, In Organic Reaction~ 2~ p.341: Bunton, 1965, Oxidation in Organic Chemistry, Vol. 1 ~Wibert, ed.), Academic Prees, New ~ork, p.367.

: ` :

~3223~

Oxidation of the antibodies with these oxidizing agents can be carried out by known methods. In the oxidation, the antibody i~ used generally in the form of an agueous solution, the conc~ntration being generally less than 100 mq/ml, preerably 1 to 20 mg~ml. ~hen an oxygen acid or a salt thereof i~ u~ed a~ the oxidizing agent, it is used generally in the ~or~ of ~n aqueou~ solution, and the concentration is generally 0.001 to 10 ~M and preferably 1.0 to 10 mM. The amount of ~he oxygen acid or sal~ thereo~
depen~s on the kind of antibody, but generally it is used in excess, for example, two to ten times a~ ~uch as the ~mount of the oxidizable carbohydrate. Ths opti~al a~ount, however, can be determined by routin~ experimentation.
In the process for oxidizing antibodie~ with oxygen acids or salt~ thereof, the optional r~nges include a pH of from about 4 to 8, a te~p~rature o~ fro~ 0~ to 37-C, and a reaction period of from about 15 ~inute~ to 12 hours.
During the oxidation of the antibody with an oxygen acid or a salt thereof, light ~ pr~ferably axcluded to prevent over oxidation of the glycoprotein.
.
5.1.2. NZY~ATIC ~THODS QF OXIDATION
Oxidation oP the c~rbohydrate portion of antibody molecule~ ~ay a~o b~ don~ wi~h ~he enzym~, galactose oxida~e (Cooper ~ al./ 1959, J. Biol. Che~. 234:445-448).
Tha ~ntibody i~ ussd in aqueous solution, the concentration b~ing gen~rally 0.5 to 20 ~g/~l. The enzy~e generally is su~d at ~bout 5 to 100 units per ml of solution, at a pH
ranging fro~ about 5.5 to about 8Ø The influence of pH, sub~trate concentration, buffers and buffer conc~ntrations on enzyme reaction aro reported in Cooper e~ al., supra.

5.1.3. TTACHM~NT AND STABILIZATION
Antibody coniugates (or antibody link2r-intermediates) are produced by r~actin~ the oxidized carbohydrate portion , . :

~ 3~341 --10~

of an antibody with a co~pound, a linker or a linker-compound having an availabl~ amine group selected from the group consistiny of primary amine, secondary a~ine, hydrazine, hydrazide, hydroxyla~ine, phenylhydrazin~, semicarbazide and thiosemicarbazide group.s. The immediately resulting products contain a carbon~nitrogen double bond resulting from elimination of a ~olecule of water from the initial addition products:

H
2 R ~~ ~~~> antibody-CH=N-R + H O
For a general discu~sion of the reaction of aldehydes with hydrazides, see ~arch, 1978, In Advanced Organic Chemistry:
Reactions ~echanisms and Structure, ~cGraw Hill Co., New York, pp.824-825.
A solution of the oxidized antibody at a concentration of from about 0.5 to 20 mg/ml is mixed with a compound a linker or a linker-compound (molar ratios of reactive amine group to antibody aldehyde ranging from about 1 to about 20 10,000) and the solution incubated for from about 1 to 10 hours. Suitable temperatures are fro~ 0 to 37C and pH may be from about 6 to 8.
After the antibody-conjugates (or antibody-linker intermediates) hav~ been form~d between the anti~ody and a 25 compound, a linker or a linker-compound they can optionally be sta~ilized with a ~uitable reducing agent, such as sodium cyanoborohydride or ~odium borohydride. Reducing agent is generally added to a ~olar excess of from about 10 to 100 fold molar exces~ over availalble aldehyde groups. ~or a 30 general di~cu3~ion, see Jentoft and Dearborn, 1979, J. Biol.
Chem. 254~4359 . :

:
,- .-~322~

5.2. COMPOUNDS ~ND LINKERS
According to the invention, a compound can be siteselectively attached to an oxidized carbohydrate moiety of an antibody directly through an a~ine group of the compound thus forming an antibody co~pound con~ugat~.
Alternatively, a co~pound can be si.te ~electively attached to an oxidiz2d carbohydr~te moiety of an antibody through an intermediate linker having at lea~t two reactive groups, one an amine group to react with the oxidized carbohydrate moiety of the antibody and one to react with the compound. The linker, which include~ any compatible compound, ~ust be chosen such that the reaction with antibody ~or cD~pound) does not adversely af~ect antibody reactivity and selectivity. Xoreover, i~ th~ co~pound attached is a therapeutic agent, ~uch attachment must not destroy the activity ~f ~he therapeutic agent. Suitable linkers for reaction with oxidized antibodies include those containing an amine elected fxom the group con~i~ting of primary amine, secondary amine, hydrazine, hydrazi~e, 20 hydroxylamine, phenylhydrazine, ~emicarbazide and ~hiosemicarbazide groups. Such reactive ~unctional groups may exist as p~rt of the ~tructure o~ the linker~ or may be introduced by ~uitable che~ical modi~icat~on o~ linkers not containing such groupsO
The compound can be attached to the linker before or after the link~r is attached to th2 antibody molecule~ In certain applications it may be desirabl~ to first produce an antibody linker intermediate in which the linker is free of an a~sociated compound. Depending upon the particular 30 application, a ~pecific compound can t~en be cQvalently or coordinately a~tached to ~he linker.
O~ additional interest are ~branched linkers~ which have multiple sites ~or attach~ent of co~pound~, For multiple site linkers, a ~ingle covalent attachment to an 35 antibody or an~ibody ~raq~ent would result in an antibody-~J
' .'` " ' "' !

~234~

linker interm~diate capable of bindinq a compound at anumber o~ sites.
In another embodi~ent, cleavable linkers can be used which are susceptible to cleavage by a variety of mechanisms. Peptide linker~ which are ~usceptible to cleavage by enzymes o~ the co~ple~en~ syste~, urokinase, ~issue plasminogen activator, tryp in, pl.asmin, or another enzyme having proteolytic activity may be! used. In one method o~ the present invsntion, a compoumd is attached via a linker susceptible to cleavage by co~ple~ent. An antibody selected from a class which can activate complement is then administered ~n co~bination with the antibody (Fab')2 linker compound of the invention. Th~ second antibody administered, thu., activate~ the oomplement cascade and 16 releases th~ compound Pro~ the antibody (Fab')2 linker compound conjugate at the target l~e. According to another embodiment of the pre~ent invention, a co~pound iæ attached via a linker usceptible to cleavage by enzyme~ having a proteolyti~ activity ~uch as urokinas~, a ti~sue plasminogen 20 activator, plas~in, or trypsin or the like.
In still another ~mbodiment, it may be necessary to construct the linker in such a way as to optim~ze t~e spacing between the compound and the antibody. This may be accomplished by use of a linker o~ the general structure ~5 W~(~H2~n~Q
wherein W i~ either -NH-CH2- or -C~2-;
Q i5 an amino acid, peptide; and n is an integer from 0 to 20.
In ~till other embodi~ents, the linker may comprise a 30 spacer element an~ a cleavable ele~en~. ThQ spacer element serves to position the cleavable element away from the core of the antibody ~olecule such that the cleavable element is more accessible to the enzyme respon~ible for cleavage.
Certain of the ~branched linkers~ described above may serve 35 as spacer elements.

, .

13223~1 When ~he compound to be attached is a metal ion, a linker which is a compatibla chelator is utilized to attach such compound to an antibody ~olecule. The term ~a compatible chelator~ i8 intended to ~ean any compound th~t ~l~ is able to donate electron~ and combi.ne by coordinate bonding with a metal lon to form ~tructures called chelates or chelation complexes and (2) i8 ~uitabl~ for covalent attachment to an antibody without 1085 of the ability to chelate metal ions or de~truction of the i~munospecificity tO of the antibody molecule. Co~patible chelators include but axe not limited to derivative of diethylenetraimine~
pentaacetic acid, ethylenediaminetetraacetic a~id (~DTA~, dimercaptosuccinic acid, 2,3-dimercaptopropanesul~onic acid, metallothio~in and cryptates, such as those described by Gansow et al., (1981, J. Heterocycl1c Chem. }8:297).
According to the present invention, suitable compatible chelators for reaction with an oxid~zed carbohydrate moiety of an antibody includ¢ those containiny an amine selected ~rom the group consisting of primary amine, secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide groups.
Such reactive ~un tional group~ may exist as part of the .
structure o the ch~lator, or may be introduced by suitable chemistry onto chelators not containing such groups.
For example, diethylenetriaminepentaacetic acid (DTPA) lacks an appropriat~ a~ine group ~or facile attachm~n~ ~o oxidized carbohydrat~. However, chemical modification can produce a variety of ~uitable deriv~tives, such a~ amine-containing derivatives o~ mixed anhydrides o~ DTPA
including, but not limited to p~a~inoaniline-DTPA, hydr~zide-DT~A~ phenylhydrazide-~TPA, hydroxylamine-DTPA, semicarbazide-DTPA, thioæemicarbazida-DTPA, polyethyl nei~ine-DTP~, p-phenylenediamine-DTPA, DTPA
mono~(4-aminophenyl)~ethyl] amide and amino acid containing derivativee o~ DTPA, includinq, but not limited to ~~N-.

~ 3~23~
`-14-DTPA-L-lysine, glycyl-tyrosyl-lysine-DTPA and L-lysine benzyl ester-DTPA.
The compound attached to the antibody compositions to prepare the antibody (Fab~)2 compound conjugates of the pre-sent invention is selected according to the purpose of theintended application ~e.g., killing, prevention of cell pro-liferation, hormone therapy, target imaging, or gene therapy, etc.). Such compounds include, for example, pharmaceutical agents, toxins, fragments of toxins, alkylating agents, en-zymes, antibiotlcs such as antibacterials, antifungals, anti-mycoplasmals, etc., antiviral agents, antimetabolites, anti-proliferative or antineoplastic agents, hormones, neurotrans-mitters, DNA, radioopaque dyes, radioactive isotopes includ-ing such as I-123, I-131 as well as radioactive metal ions, metal ions, fluorogenic compounds, marker compounds, lectins and compounds which alter ce]l membrane permeability. Table I of European Patent Application No. 85~01776.1 which was published as Publication No. EP 175617A on March 26, 1986, lists some therapeutic agents that can be used in the present invention. In no way is Table I, however, meant to be an e~-haustive list. Finally, a combination of compounds can be site specifically coupled to an antibody composition to pre-pare the antibody (Fab')2 compound conjugates of the inven-tion.
5.3. ANTIBODIES
Antibodies directed against any determinant of anyantigen or hapten may be used in the present invention.
While both conventional antibodies and monoclonal antibodies are suitable, monoclonal antibodies offer several advantages.
Each monoclonal antibody is specific for one antigenic deter-minant. Additionally, a large amount of substantially homo-genous monoclonal antibodies can be produced efficiently and economically using techniques known to those of skill in the art.

~ .

- "

~53223411 Classes of antibodies including IgG, IgM, and Ig~ can be used in the method of the present .invention.
As stated above, antibodies directe~d against any determinant of any antigen or hapten can bQ employed in the reaction scheme de~cribed herei~. Such deter~inants include, but are not limited to deter~ina:nts of~ tumor and malignant cells, bacterial, fungal, viral, parasitic, mycoplasmal, histoco~patab11ity, difer~ntiation and other cell membrane antigens, pathogen ~ur~ace antigens, tox.ins, enzymes, allergen~, drug~, any biologically active molecules, eto. In ~ome ~nstances, a combination of monoclonal antibodie~ reactive with different antigenic deter~inants can b~ used.

5.4. APP~I ATIONS
The aqueous soluble antibody (~ab')2 compoun~
conjugates o~ the present inv~ntion are advantageously u~eful for a variety of in vivo therapeutic and diagnostic applications~
In vivo admini~tration may involv~ u~e o~ a ~herapeutically e~fec~ive cG~position or a diagnostic composition compri~ing ~n aqueou~ sol~ble antibo~y (Fab')2 conjugate ln any 3uitable carrier, lncluding ~eru~ or phy~islogical ~alin~, with or without ~no~her protein, such 25 as human ~erum ~lbumin. Do~age~ o~ the antibody (Yab')2 con~ugate~ can be readily deter~in~d by one of ordinary skill and ~ay di~fer depending upon the nature of the co~pound atta~hed and the intended purpose of the conjugate.
The route of ad~inistration is generally parenteral, 30 with administration via an in~ravenou~ route generally preferred.
Therapeutic application~ g~nerally $nvolv~ treatment of a variety of cellular di~orders by admini~tering an eXfective a~ount of an antibody (Fab'~2 compound conjugate 35 in which the compound ~ ~ therapeutic agent. The '~
' .

~223~

properties of the antibody ~Fab'~2 in being immunospecific for and immunoreactiva with a particular antigen or antigen determinant associated with ~he cellular disorder render the compo~ition~ ideally suited for d~livery ~ the therapeutic agent to specific calls, tissues, or organs or any other 1n vivo æite having that antigenic determinant. The properties of th~ antibody (Fab')2 co~pound conjugatl 5 in having prolonged biological hal~-life compared to conventionally prepared antibody fra~ent conjugate render the pr~sent compositions especially suited for ~n ViYo delivery o~ a therapeutic agent when persistence o~ the agent at the target site is desired.
For therapeutic applications, the compound attached to the antibody (Fab')2 compositions of the invention is a thPrapeutic agent ~elected according to the intended purpose. Therapeutic agents ~uch as khos~ described above in Section 5.2 can be employed. In ~ome instances, the therapeutic agent iq a r~dioactive isotope ~uch a~ I-131, Yttrium-90, Copper-67, Rhenium-186, Rheniu~-188, Iron 59, Bismuth-212, Lead-212, ~tc.
As used in thi application the term ~cellular disorder~ is intended to includ~ all neoplasms, such as cancers, ~dQnomas, hyperplasias, etc~; certain immunological disorder~, includiny graft-ver~us-ho~t dis~ases (e.g., after bone marrow transplantation); immune suppressive disord~rs, (e.g., acquir~d i~muns dificiency ~yndrome ~AIDS), after kidney or bone marrow transplantation~; cardiovascular di~eases ~uch aY those associated with the ~ormation of atherloscelortic plaque~, etc,; in~ections induced by viral, bacterial, fungal, mycoplasmal, or parasitic agents, etc.
Diagnostic applications generally involve imaging of specific ti~sues or cellular disorders by administration of a ~u~ficient a~ount of an antibody ~Fab'32 compound conjugate to enable detection or localization o~ the 35 conjugate at the target tissue in an appropriate time frame.

, 1~2341 For diagnostic imaging purposes, the antibody (Fab')2 compound conjugates ~ay comprise a co~pound which i5 a non-metallic radioactive isotope such a~ I-123 coupled via a linker according to the method of the inv~ention.
Alternatively, the antibody (Fab')2 compound conjugates for diagnostic imaginy purpo es comprise a co~pound which is a metal ion attached to ~ linker which i~ a compatible chelator ac~ording to the ~ethod of thQ invention. Thus, the compositions for diagnostic appli~ation~ are pre~erably antibody ~Fab'32 chelator metal ion compllexes. Dosages and other aspects of admini~tration o~ the co~plexe ~re readily determin~d by one o~ ordinary ~kill in the art.
A wide variety of metal ions are suited for preparing the antibody (Fab')2 metal ion complexes including radioisotope~ such a~ Indium-lll, TQ~hnetium-99~, Copp~r-67, etc., positron ~mitting metal ion~ such as Scandium-43, Scandium-47, Iron-52, Cobalt-55, Gallium 68; and nonradioactive paramagn~tic mQtal ion~ ~uch as Iron-54;
Iron-56, Iron-57, Iron-58, Gadolium-57, Manganese-55, e~c which are detectible by nuclear magnetic resonance spectroscopy.
Ti~ues and c~llular di~orders which may b~ imaged include any solid n20plasms, eertain organs Such as lymph nodes, parathyroids, sple~n and kidney, ~ite~ o~ in~lamation or infection (e.g. ~acrophage~ at Ruch ~ite~ ycocardial infarction or thromboses, (naoantigenic deter~inants or fibrin, pla~lets), etc.

5.5. ADVANTAGE5 OF ~NTIBODY ~FAB'~2 CO~POUND CONJUGATES
The anti~ody (Fab')2 compound con~ugates of the present invention in which a compound is attached, either directly or via a linker, site specifically to a carbohydrate moiety of an antibody which i~ located outside the antigen binding region of the antibody, pos~ess 3 substantially the sam~ immunospecificity as the unconjugated ~3223~

whole antibody molecule. ~or~over, when administered in vivo the antibody ~Fab')~ compo~nd conjugates o~ the invention persist longer in the circulation and are not rapidly excreted, ~or example through the kidney, liver or spleen, compared to analogou~ (Fab')2 co~jugate~ pxepared by conventional methods using pre~ormed (Fab')2 ~ragments. The longer persistence or residenca time in the body and the specificity of the con~u~ate~ fox the intended target antigenic site provide ~ignificant advantages when the antibody (Fab')2 con~ugate~ are used for targeted ln vivo delivery systems for ther~peutic and/or diagnostic applications.
The following Examples are presented for purposes o~
illustration only and not by way of limitation on the scope of the present invention.

6. ENH~NCED BIODISTRIE3UTION ~lY 2.3 AN~IBODY (FAB'~2~GYX-DrPA- IN
The following experiments de~onstrate that a radiolabaled antibody (Fab')2 frag~ent-chelator metal ion complex prepared according to the ~ethod of the invention has significantly enhanced biodistribution compared to an analogou~ conventional co~plex when administere~ to experimental animal~.
The antlbody e~ployed in these experiments was a monoclonal antibody (Ig~l) reativ~ with human breast and colon cancer obtained ~rom hybridoma cell line ATCC No.
B72.3 (hereinafter ~72.3 antibody~) described in United States Pat. No. 4,522,918 icsued to Schlo~ et al. (See, Nuti et al., 1982, Int. J4 Canc~r 29: 539-45).
In one series o~ experiments, a ~72.3 antibody (Fab')2-chelator metal ion complex was prepaxed according to the method of the invention a~ follows:

. ~. .

~223~L~

~ETHOD A
tl) An antibody-chelator intermediate was formed using the method o~ Rodwell et al., 1986, Proc. Nat'l Acad.
Sci. US~ 83:263~-36.
Brie~ly, the carbohydrate ~oieties o~ the antibodies were oxidized by incubation in th~ dark with 10-30 mM NaI04 in phosphate bu~ered saline (0,15 M NaCl/O.Ol M sodium phosphate; PBS) at pH 6.0 in ice for 1 hour. The antibodies were purified using a Sephadex G~25 column equilibrated with PBS at pH 6Ø
The antibodie~ having oxidized carbohydrate ~oieties were then incubated with a derivative of the chelator diethylentriaminepentaacetic acid (DTPA), i.e., glycyltyrosylly~yl diethylentriaminepentaacetic acid (GYK-DTPA) at a one-thousand molar exces~ of GYK-DTPA for 1 hour at room temperatura. Sodium cyanoborohydride (Aldrich Chemical Co. Inc., Milwaukee, WI) wa~ added ~.o a final concentration of 10 m~. Th~ reaction mixture was lncubated for an additional 4 hours and then either dialyzed at 4C
against several change~ of PBS or pa~sed through ~ Sephadex~
G-50 gel column.
(2) The B72.3-GYR-DTPA antibody-chelator intermediate was then dige~ted with pre activated bromelain according to the ~ethod generally described by Parham (1983, J. Immunol. 131:2895-02) to ~orm a B72.3 (Fab')2-GYX-DTPA
intermediate. The bromelain (1 mg/ml) was activated by incubation ~or about 1 hour at room temperature with 50 mM
cys~eine in acetate buffer (100 ~M acetate, pH 5.5~
containing 3 m~ EDTA. Th~ cysteine wa5 removed from the 3~ activation mixture by desalting through Sephadex~ G~25.
B72 . 3 (Fab' ) 2-GYR-DTPA was then digested with a~tivated bromelain (5% bas~d on the ~eight o~ the antibody-linker intarmadiate) in acetate buffer. The dig~tion was monitored using size exclu~ion chro~atography (SEC) high 35 performance liquid chromatography (HPLC). The 872.3 (Fab')2 GYK-DTPA obtained wa puxi~içd using a Protein A column .
, ~ ~3223~

~f i-Gel Protein A, BioRad Laboratories, Richmond, CA), and then using a gel filtration column (AcA44, IBF Biotechnics, Savage, MD) in PBS, pH 6Ø Th~ product was analyzed using sodium dodecyl ~ulfate polyacrylamide gel. electrophoresis (SDS-PAGE) and H~LC-(SEC) to identify the! B72.3 (Fab')~
GYK-DTPA having a ~olecular weight o~ abr~ut lO0, oao daltons.
(3) A B72.3 ~Fab'j2 metal ion complex was then formed by incubatinq the ~72.3 ~Fab~)2-GYK-DTPA intermediate with lllInC13 (1 mCi) (NQW England Nucle~r, Boston, MA) in PBS pH 6.0 for abcut l hour at 37-C. The ~72.3 (Fab~)2-GYX-DTPA~ In formed wa~ separated ~ro~ ~ree lllIn by HPLC
using a TSK-G3000SW column.
In ~noth~r ~eries o~ experiments, a B72.3 antibody (Fab')2-chelator m~tal ion complex was prepared according to th~ inven~ion as follow~:
METHOD B
(1) An antibody-chelator intarmediata was formed as described above using the method o~ Rodwell et al., su~ra.
(2) An antlbody-chelator ~etal ion coMplex wa then prepared by incu~ating (l00 ~g) o~ the B72.3-GYX-DrPA
intermediat~ wi~h llllnC13 (l ~Ci) (Naw England Nuclear, Boston, M~) in PB5, pH 6.0 for 1 hour at 37-Co (3) Th~ B72.3-~YK-DTPA~ In complsx wa~ then digested with prQ-ac~iv~ted brom~lain as d~crib~d above excep~ that EDrA was omitted from th~ buf~r. The 1~72.3 (Fab')2 GYK-~rPA lllIn wa~ puri:tied by pa~sage through an TSKoÇ;3000 SW HPIC column.
An analogcus conventior~al B72 . 3 (Fab' ) 2 chelator metal 3~ ion compl~x wa~ ~lso prepar~d as ~ollow~:
72 . 3 an~ibody s~a~ d~ge ted with pr~
activat~d bro~elain accardir~g ~o ~h~ gen6~xal ~thod o~
Parham, supr~, to ~or~ B72.3 (~ab')2 ~ra~nent~. The B72.3 (Fab~ ) 2 ~ragment~ were ~eparated fro~ th~ Fc fragments and *Trade mark -. . :.. : .. . , .::

~322341 any residual whole antibody by chromatography using Protein A and a gel filtration column as described above.
(2) A B72.3 (Fab')2-~YK-DTPA intermediat~ was then for~ed by oxidizing the carbohydrate ~oi~ty o~ the B72.3 (Fab')2 fragment according to the method of ~odwell et al., supra, and reacting t~e aldehyde group ~o formed with a reactive amine of the chelator derivativ~ GYK-DTPA. The B72.3 ~Fab')2-GYK-DTPA intermediate was then stabilized by incubation with sodium cyanoborhydride as described above;
(3) The B72.3 (Fab')2 GYK-DTPA intermediate (100 ~g) was incu~ated with lllInC13 (1 mCi) (New England Nuclear, Boston, MA) in PBS pH 6.0 ~or 1 hour a~ 37~C. The B72.3 ~Fab')2-GYX-~TPA-1~1In oo~p}ex thus formed w~s purified by HP~C u~ing a TSK-G3 0008W column.
The B72.3-(Fab')2-GYR~ In oo~pl@xes prepared acoordinq to the present invention and according to conventional method~ were analyz~d using ~ize-exclusion chromatography (SEC) HPLC. S~C-HPLC o~ bnth the types of complexes showed that these complexes were of 9ub5tantially the same ~ize and homogenou~
A whole B72.3 antibody -GYR-~TpA~ CO~plex WaS
also prepared ~or co~pari~on, as desoribed above, without enzymatic digestion.
The biodl~tribution o~ ~72.3 (Fab')2 metal ion complexes prepar~d a~cording to the pre~ent invention and according to conventional ~e~hod~, a~ well a~ whole B72.3 metal ion co~plexe~, wa evaluated following in _ivo admini~tration to ~xp~rimental ani~al~.
In on~ ~r~Q~ oP experi~ent~, female nude (nu/nu) mice (Taconic Far~s, Ger~anto~n, NJ) bearing hu~an tumor xenoyraf~ were obtained by ~ubcutaneou~ in~ec~ion wi~h about lX106 LS174T (ATCC~ human colon ad~nocarcino~a cells (0.2 ~1) in the le~t rear flank. When the tu~ors were about 10-15 mm X 15-20 Dm, 200 ~1 of the ~72.3 (Fab')? complex (13 35 ~g~ of th~ pre~ent invention, and o~ the oonv~ntional B72.3 ~3223~1 (Fab')2-GYK-DTPA~ In complexes ~13 ~g) were each injected intravenously into 3 tumor-bearing and 3 non tumor bearing nude (nu/nu) tcontrol) animals. Whole B72.3 GYK-DTPAoll1In complexes (13 ~g) were si~ilarly administ: red into 2 tumor-bearing and 3 non-tumor bearing animals ~or comparison.
Animals were sacrificed and di~ected at 4 day~ post-injection to obtain quantitativ~ biodi~tribution data. At the time of dissection, tu~ors averaged 1.12 ~ O.59 g.
Re~ults obtain~d in tumor-bearin~ animal~; are illustrated in Table 1 and in non-t~mor bearing animals in Table 2.

:

. I

. . .-, ":

~223~

~IODISTRIBU~ION OF B72.3(FAB')~ GYX-DTPA
lllIN COMPLEXES IN TUMOR BEARING ANI~ALS
Tissue B72.3 (Fab')2Conventional Whole Organ:Bloo~ Complexes B72.3 (Fab')2 B72.3 b RatioOf the Inventiona Com~lexes Complexes Lung1.04 + 0.58 2.B1 + 0.22 0.77 + 0.19 Spleen1.26 ~ 0.~2 6.34 + 0067 0.86 + 0.21 Liver2.52 ~ 1.21 27.57 + 4.59 1.89 + 0.91 1~ Kidneys11.80 ~ 5.70168.96 ~ 41.181.73 t 0.73 Tumor4.93 + 3.24 6.18 + 1.99 7.0~ ~ 3.07 Muscle0.9B ~ 1.33 1.40 ~ 0.26 0.22 t 0.04 Tissue % Injacted Dose/qr~m Blood2.46 + 2.05 0.09 + 0.02 3.37 t 2.11 Lung1.81 + 0.30 0.24 + 0.05 2 ~1 ~ 0.98 Spleen2.24 + 0.53 0.54 ~ 0.09 ~ 7 + 1.09 Liver4.59 + 1.02 2.33 + 0.27 5.42 -t 0.91 Kidneys21.60 + 6.2314.37 t 3.984.88 ~ 0.52 Tumor8.39 + 2.03 0.52 + 0.1420.63 + 4.55 Muscle1.40 + 1~47 0.12 + 0.02 0.70 + 0.34 Isotope Half-LifeC3~3 + 0.1 0.9 + 0.1 6.1 + 1.2 .
b Values repre~ent x + SD, N 3.
Value~ reprQsent x ~ SD, N ~ 2.
c Half-life ~day~3 determinations were ~ade based on whole animal dose-calibrator mea6urements each day of the ~tudy, beqinning ~u~t after injection and ending jU5t before di~ect~on.
- ~ _ _ , ' ~ ~ - ` ~ .
.
'' ~

, ~ .
' .
~ , ~322~
: --24--BIODISTRIBUTION OF B72 . 3 (FAB' ) 2 GYX-DTPA
COMPLEXES IN NON-II~OR BEARING ANIMALSa Tissue B72 . 3 (Fab' ) Conventional Whole Organ:Blood CoTnplexes 2 B72.3 (Fab')2 B72.3 RatioOf the Invention C: omplexe~ Complexes Lung1.05 + 0.193.07 ~1.30 0.68 + 0.16 Spleen1.15 ~ 0.437.24 +2.~4 0.46 + O.lo Liver3 0 42 + 2 . 32 25. 29 ~ 4 . 36 0 . 81 ~ O . 65 Kidneys ll.91 ~ 2.76 195.31 + 107.35 0.65 + 0.18 Mu~cle2 . 01 + 1. 811. 70 +O. 65 0 .11 ~ o . 04 Tlssue % In; ected Dose/qram 1S Bloodl.99 + 0.350.08 +0.03 8.49 + 3.61 Lung ~ 0.550.24 +0.04 5.43 ~ 1.57 Spleen2.30 ~ 0.900.55 +0.08 3.63 + 0.82 Liver6.6~1 + 4.522.03 +0.26 7.0g -~ 3.86 Kidney~ 23.32 + 4.72 ~4.19 ~ 4.98 4.g8 -~ O.53 Muscle3 . 59 + 2 . 84 0 .13 + O . 03 0 . 85 ~ O . }1 Isotope b Half-Life3.1 + 0.2 O.B f0.2 5.8 -1- 0.9 25 a Value~ repre6ent X + SD, N - 3.
b Hal~-life (days) determinations were made based on whole animal do~e-calibrator measurem~nts each day of the ~tudy, beginning ~u~t after injection and ending ~ust before dissection~

:,; ' .' !,;,, .

::' ' ;' ' ' '' 13223~
-2~-As clearly demonstrated9 the mean residence time expressed as the Isotope Half~life of the B72.3 (Fab')2 metal ion complex of the invention was significantly longer than that of analogous conventionally prepared con~ugates.
Additionally the biodistribution pat_ern of the two differen~ly prepared antibody (Fab')2 conjugates diff~red significantly. In particular, the B72.3 (Fab')2 conjugate of the invention localized ~uch more ~pecifically to the target tumor site a~d less 50 to other non-target orgarls (see Tissue Organ: Blood Ratios).
In another series o~ experiments, female nude mice (nu/nu) bearing human LS174~ tumor x~nografts were obtained as described abov~. One or tws tu~or-bearing animals and two or three non-tumor bearing animals were injected intraperitoneally with 300 ~1 of B72.3 (Fab')2 GYK-DTPA-1 In o~ the present invention prepared by Method B above or300 ~1 (8 ~g) of a whol~ B72.3 GYX-DTPA~ In complex (3 g) in PB5. Three day~ post-injection, the animals were sacrificed and dis~ected. At the t~me of dissection, tumor weights averaged 1.13 + 0.54 g. Noroal Balb/c mice served as non-tumor bearing controls.
Results are illustrated in Tsble 3 for tumor-bearing and in Table 4 ~or non-tu~Qr bearing animals.

I
i .

.

-26- ~L 32234~

TA~LE 3 BIODISTRI~UTIOll OF B72 . 3 (FAB' 3 2 GYK-DTPA
lllIN COMPLEXES IN I~OR BEARIN~ ANIMALS
Tissu~3B72 . 3 (F~b' ~ 2W~ole Organ:Blood Complexes B72.3 RatioOf the Inv~ntionaomplexasb Lung 0.74 ~ 0.050.69 SpleenO . 44 ~ O . 020 ~ 95 Liver0.46 + 0.041.41 Kidneys11. 44 + 1. 202 . 03 Tumor1.40 + 0.025.11 MuscleO . 26 + O . 04O. 27 Tissu~
% Inj ected 1 5 Dose/gram Blood2.96 ~ 0.294.82 Lung 2.08 + 0.063.32 :~
Spleen1. 32 _ O . 06~ . 60 Liv~r1.36 + 0.026.78 Kidnays33 . 74 + 1. 01g . 75 Tumor4 .14 + O. 4824 . 64 Musc:leO, 78 + O. 051. 29 Isotope 25 Hal~ ~Life 2 . 6 + O days 9 . 5 days a Values r~present X + S . D., N = 2 .
b N 1.

,.,,.: , ~.
: :~ . :. :
.. .~. ~
. . .

-27- 132~34~

BIODISTRIBUTION OF B72 . 3 (FAEI' ) 2 GYX-DTPA
lllIN_ COMPLEXES IN NON-q~UMOR BEARIN~ ANIMALS
Tlssue E~72 . 3 (Fab' ~ 2 Whole Organ:Blood CoInplexe6 B72.3 RatioO~ the Inventiona ComplQx~ b Lung 0.79 + 0.03 0.06 + ID.02 Spleen0 . 53 + 0 . 07 0 . 48 + 0 . 01 Liver0.84 + 0.16 0.66 ~ ~D.06 Kidneys11. 08 + 0 . 87 0 . 92 + 0 . 08 2~uscle0.10 ~ 0. 02 U. 11 ~ O . 01 Tis~ue % In; ected 1 5 Dose/qram Blood5.36 t- 0.38 21.17 ~ o.a7 Lung4023 ~ 0.22 12.73 ~ 0.59 Spleen2 . 81 + O . 32 10 . ll3 ~ 0. 27 Liver3 . ~5 + 0 . 99 13 . 83 + 1. 56 Kidneys59 . 58 + 7 .16 19 . 48 + 1. 95 Muscle0. 94 + 0. 04 2 . 32 + 0.16 Isotope Half-Li~e3 . 3 + 0. 3 day~ 13 . 9 + 2 . 4 days 25 a~alu~s represent X + SD, N = 3.
bValu~ repre ~nt X + SD, N = 2.

~ , : ~ : ` ' .

:
.

-28- ~3~2~

The data in Tables 3 and 4 confi~m the results presented in Tables l and 2. The biodistribution of B72.3 (Fab')2-GYR~DTPA~ In co~plexes prepared according to the method of the invention was signi~icantly di~ferent from that of analogous complexes prepared according to tAe conentional method. At 4 days post-injection o~ complexes of the invention, about 8% of the injected complex was lo~alized at the tumor tar~et site, whereas at the same time only about 0.5% of the injected conventionally prepared complex was localized at the tumor site. ~oreover, the conventionally prepared (Fab')2 complexes were excreted very rapidly and showed high localization at the kidney and liver non-target sites. In contrast, the (Fab')2 comp~exes prepared according to the method o~ the invention persisted in the circulation about three times a~ long a~
conventionally prepared complexes and showed significantly less localization at the kidney and liver.

7. IN VITRO BINDING ACTIVITY OF B72.3 AND B72.3 (Fab')2 COMPOSITIONS
The ~ollowing experiment shows that an antibody (Fab')2-linker intemediate prepared according to the m~thod o~ the invention and an antibody (Fab'~2 fragment prepared using a conventional method had substantially the same bindinq activity when measur~d using an in Yitro binding assay. The ~periment rurther shows that the binding activity of the (Fab')2 co~positions was substantially th~
same a~ that o~ the unconjugat~d antibody.
A whole B72.3 linker intermediate, (i.e. B72.3-C~YK-DTPA) and a B72.3 (Fab'~2 linker inter~ediate (i.e. B72.3 30 (Fab' ) ~-&YK-DTPA w~re prepared as described in Seotion 6 above. A conventional B72.3 (Fab')2 fragment was also prepared as described above.
An enzyme-linked immunosorb~nt a~ay (ELISA) was used to assess the ~ vitro binding activity of the antibody and ~5 ~, .
, -29- ~ 322~41 (Fab')2 fragment compositions a~ follows: The ~ssays wer~
performed using microtiter plates on which were ~rown and then ~ixed human LS174T cells which contain the TA~-72 antigen for which B72.3 antibody ~5 specific. The antibody or [Fab')2 fra~ent compositions to be tested were serially diluted 1:2 on the plates from 10 ~g/ml to 0.04 ~g/ml ~corresponding to 67 nM/l to 0.26 nM/l ~or whole antibody and lOO nM/l to O.39 nM/l ~or (Fab')~ frag~ent, respe~tively). The bound antibody or ~Fab')2 fragment compositions were detected u~ing anti-~ouse IgG-Fab conjugated to peroxidase (Jackson Immunoresearch). The substrate used was tetramethylbenzidine. Result~ obtained are graphically illustrated in FIG. 1 (A and B).
As demonstrated in FIG. lA, both the B72.3 antibody (Fab')~ composition prepared according to ~he method of the invention and thQ conventional B72.3 ~Fab')2 had substantially the ~ame specific binding activity when measured using an in vitro ELISA binding assay. Moreover, as demonstrat~d in FIG. lB, the speci~ic binding activity of whole nativ~ B72.3 antibody a~d B72.3 antibody having a linker ~oiety ~ite speci~ically attached to an oxidized carbohydrate moiety also has sub~kantailly the same binding activity as the (Fab')2 fragments using the in vitro ELISA
assay. A control IgG1 antibody MOPC-21 had no activity in the ELISA assay (data not shown).

8. CARBOHYDRATE ANALYSIS OF
A B72.3 (FAB')2 FRAGMENT
~ he following experi~ent wa~ performed to evaluate the carbohydrate moieties of the whole B72.3 antibody molecule 30 and B72.3 (Fab')2 frag~ents analogou~ to those ~onmed in the reaction scheme of the present i~vention.
B72.3 (Fab')2 ~ragments were obtained by digesting an aliquot of B72.3 antibody with activated bromelain as described in S~ction 6 above. The carbohydrate moieties of ~ 30- ~32~341 both the whole B72.3 antibody molecule and the B72.3 (Fab~)2 fragments were analyz~d using three separate hydrolyses as follows.
~1~ amino sugars such as glucosamirle (Glc-Am) and galactosamine (Gal-Am) w~re analyzed a~ter hydrolyxis in 6 N
HCl a~ 100C for 3 hours:
(2) neutral sugar~ such as galactose, mannoæe and ~ucose were analyzed after hydrolyis in 4 M trifluoroacetic acid (TFA) at 100C for 2 hour~; and (3) sialic acid was analyzed a~ter hydrolysis in 20 mM H2SO4 ~t ~~ f~r 1 hour.
The neutral sugars were converted to amino-containing forms pri4r to analysi~. The hydrolyzed samples were analyz~d by cation-exchange HPLC, u~ing an ortho-phthaldehyde reactor and fluorescence detector. Results are graphically illustrated in FIG. 2.
As shown in FIG. 2, the carbohydra~e moieties of B72.3 ~Fab')2 frag~ents co~prise galactosamine, galactose, and sialic acid residues. Glucosamine, ~annose and ~ucose residues as well as an extra sialic acid residue pr~sent in the whole antibody ~olecule are not gound in t~e B72.3 (Fab')2 fragment. The composition o~ the carbohydrate moieties on the (Fab')2 ~ragment indicates that it is an 0- :
linked carbohydrate moie~y.
The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illu tr~tion~ o~ 6everal aspects of the invention. Any equivalent e~bodiments are intended to be within the scope 30 of this invent~on. Indeed, various ~odi~ications o~ the invention in additlon to those shown and de~cribed herein ~ill become apparent to those skilled in the art ~rom the ~oreg~ing description. such modifications are also intended to ~all within the scope o~ the appended clai~s.

Claims

WE CLAIM:

1. A method for preparing an antibody fragment compound conjugate, comprising: reacting a soluble antibody-compound conjugate, which comprises an antibody attached to an amine group of a compound selected from the group consisting of primary amine, secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide groups by a covalent bond to an oxidized carbohydrate of the antibody located outside the antigen binding region of said antibody, with an activated thiol protease enzyme to form an antibody (Fab')2 compound conjugate, characterized by (a) substantially the same immunospecificity as the unconjugated antibody and (b) aqueous solubility such that the antibody (Fab')2 compound conjugate is suitable for in vivo administration.

2. The method according to claim 1, in which the thiol protease is bromelain, papain, chymopapain or ficin.

3. The method according to claim l, in which the thiol protease is activated by reacting it with cysteine or a sulfhydryl reducing agent selected from the group consisting of dithiothrieitol, mercaptoethanol or mercaptoethylamine.

4. The method according to claim 1, in which the antibody compound conjugate is formed by a method, comprising:
(a) reacting an antibody with an oxidizing agent to form an aldehyde group in a carbohydrate 123876.1 moiety of the antibody located outside the antigen binding region of the antibody; and (b) reacting the aldehyde group of the resultant oxidized carbohydrate moiety of the antibody with an amine group of a compound selected from the group consisting of primary amine, secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide groups, to form a soluble antibody compound conjugate being characterized by (i) substantially the same immunospecificity as the unconjugated antibody and (ii) aqueous solubility such that the antibody compound conjugate is suitable for in vivo administration.

5. The method according to claim 4, in which the.
oxidizing agent is an enzyme.

6. The method according to claim 4, in which the oxidizing agent is an oxygen acid.

7. A method for preparing an antibody fragment compound conjugate, comprising:
(a) reacting an antibody-linker intermediate, which comprises a linker attached via a covalent bond between an amine group of the linker selected from the group consisting of primary amine, secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide groups, and an oxidized carbohydrate moiety of an antibody, in which the carbohydrate moiety is located outside the antigen binding region of the antibody, in which the antibody-linker intermediate is characterized by (i) substantially the same immunospecificity as the unconjugated antibody and 123876.1 (ii) aqueous solubility such that the antibody-linker intermediate is suitable for in vivo administration, with an activated thiol protease to form a soluble antibody (Fab')2-linker intermediate; and (b) attaching the linker portion of the antibody-(Fab')2 linker intermediate either by a covalent bond or a coordinate bond to a compound to form a soluble antibody (Fab')2 linker compound conjugate characterized by (i) substantially the same immunospecificity as the unconjugated antibody and (ii) aqueous solubility such that the antibody (Fab')2-linker compound conjugate is suitable for in vivo administration.

8. The method according to claim 7, in which the thiol protease is bromelain, papain, chymopapain or ficin.

9. The method according to claim 7, in which the antibody-linker intermediate is formed by a method, comprising:
(a) reacting an antibody with an oxidizing agent to form an aldehyde group in a carbohydrate moiety of the antibody located outside the antigen binding region of the antibody; and (b) reacting the aldehyde group of the resultant oxidized carbohydrate moiety of the antibody with an amine group of a linker selected from the group consisting of primary amine, secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine semicarbazide and thiosemicarbazide groups, to form a soluble antibody-linker intermediate being characterized by (i) substantially the same immunospecificity as the unconjugated antibody and 123876. 1 (ii) aqueous solubility such that the antibody-linker intermediate is suitable for in vivo administration.

10. The method according to claim 9, in which the oxidizing agent is an enzyme.

11. The method according to claim 9, in which the oxidizing agent is an oxygen acid.

12. A method for preparing an antibody fragment compound conjugate, comprising: reacting a soluble antibody-linker compound conjugate, which comprises a linker-compound moiety attached via a covalent bond between an amine group of the linker-compound selected from the group consisting of primary amine, secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide groups and an oxidized carbohydrate moiety of an antibody, in which the carbohydrate moiety is located outside the antigen binding region of the antibody, and in which the antibody-linker compound conjugate is characterized by (a) substantially the same immunospecificity as the unconjugated antibody and (b) aqueous solubility such that the antibody-linker compound conjugate is suitable for in vivo administration, with an activated thiol protease to form a soluble antibody (Fab')2-linker compound conjugate, characterized by (i) substantially the same immunospecificity as the unconjugated antibody and (ii) aqueous solubility such that the antibody (Fab')2-linker compound conjugate is suitable for in vivo administration.

123876.1

13. The method according to claim 12, in which the thiol protease is bromelain, papain, chymopapain or ficin.

14. The method according to claim 12, in which the antibody-linker compound conjugate is formed by a method, comprising: covalently or coordinately attaching a linker portion of an antibody-linker intermediate which comprises a linker covalently attached to an oxidized carbohydrate moiety of an antibody, by means of an amine group of the linker selected from the group consisting of primary amine, secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide groups, in which the carbohydrate moiety is located outside the antigen binding region of the antibody, to a compound to form a soluble antibody-linker compound conjugate being characterized by (a) substantially the same immunospecificity as the unconjugated antibody and (b) aqueous solubility such that the antibody-linker compound conjugate is suitable for in vivo administration.

15. The method according to claim 12, in which the antibody-linker compound conjugate is formed by a method, comprising:
(a) reacting an antibody with an oxidizing agent to form an aldehyde group in a carbohydrate moiety of the antibody, in which the carbohydrate moiety is located outside the antigen binding region of the antibody;
(b) reacting the aldehyde group of the resultant oxidized carbohydrate moiety of the antibody with an amine group of a linker containing an amine group selected from the group consisting of primary 123876.1 amine, secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide groups, to form an antibody-linker intermediate; and (c) covalently or coordinately attaching a compound to the linker portion of the antibody-linker intermediate to form an antibody-linker compound conjugate being characterized by (i) substantially the same immunospecificity as the unconjugated antibody and (ii) aqueous solubility such that the antibody-linker compound conjugate is suitable for in vivo administration.

16. The method according to claim 12, in which the antibody-linker compound conjugate is formed by a method, comprising: covalently attaching an amine group of a linker moiety of a linker-compound containing a reactive amine group selected from the group consisting of primary amine, secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide group to an oxidized carbohydrate moiety of an antibody to form an antibody-linker compound being characterized by (a) substantially the same immunospecificity as the unconjugated antibody and (b) aqueous solubility such that the antibody linker compound conjugate is suitable for in vivo administration.

17. The method according to claim 12, in which the antibody-linker compound conjugate is formed by a method, comprising:
(a) reacting an antibody with an oxidizing agent to form an aldehyde group in a carbohydrate moiety of the antibody, in which the carbohydrate 123876.1 moiety is located outside the antigen binding region of the antibody;
(b) reacting the aldehyde groups of the resultant oxidized carbohydrate moiety of the antibody with an amine group of a linker-compound containing an amine group selected from the group consisting of primary amine, secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide to form an antibody-linker compound conjugate, being characterized by (a) substantially the same immunospecificity as the unconjugated antibody and (b) aqueous solubility such that the antibody-linker compound conjugate is suitable for in vivo administration.

18. The method according to claim 1, 7 or 12, in which the antibody is a monoclonal antibody.

19. The method according to claim 7 or 12, in which the linker is a compatible chelator selected from the group consisting of amine-containing derivatives of diethylenetriaminepentaacetic acid, ethylenediaminetetraacetic acid, dimercaptosuccinic acid, 2,3-dimercaptopropanesulfonic acid, metallothioein and cryptates.

20. The method according to claim 7 or 12, in which the linker is selected from the group consisting of p-aminoanaline-diethylenetriaminepentaacetic acid, hydrazide-diethylenetriaminepentaacetic acid, phenylhydrazide-diethylenetriaminepentaacetic acid, hydroxylamine-diethylenetriaminepentaacetic acid, semicarbazide-diethylenetriaminepentaacetic acid, thiosemicarbazide-diethylenetriaminepentaacetic acid, polyethyleneimine-diethylenetriaminepentaacetic acid, 123876.1 polyethyleneimine - diethylenetriaminepentaacetic acid, p-phenylenediamine-diethylenetriaminepentaacetic acid, diethylenetriamine-pentaacetic acid mono[(4-aminophenyl)methyl]amide, .alpha.-N-diethylenetriaminepentaacetic acid-L lysine, glycyl-tyrosyl-lysine-diethylenetriaminepentaacetic acid and L-lysine benzyl ester -diethylenetriaminepentaacetic acid.

21. An antibody fragment compound conjugate, comprising: a soluble antibody (Fab')2 compound conjugate prepared by a method which comprises:
reacting a soluble antibody-compound conjugate, comprising an antibody attached to an amine group of a compound selected from the group consisting of primary amine, secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide groups, by a covalent bond to an oxidized carbohydrate of the antibody, located outside the antigen binding region of the antibody, with an activated thiol protease enzyme to form an antibody (Fab')2 compound conjugate, in which the antibody (Fab')2 compound conjugate is characterized (a) substantially the same immunospecificity as the unconjugated antibody and (b) aqueous solubility such that the antibody (Fab')2 compound conjugate is suitable for in vivo administration.

22. An antibody fragment compound conjugate, comprising: a soluble antibody (Fab')2 compound conjugate prepared by a method which comprises:
(a) reacting an antibody-linker intermediate which comprises a linker attached via a covalent bond between an amine group of the linker selected from the group consisting of primary amine, 123876.1 secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide groups, and an oxidized carbohydrate moiety of an antibody, in which the carbohydrate moiety is located outside the antigen binding region of the antibody and the antibody-linker intermediate is characterized by (i) substantially the same immunospecificity as the unconjugated antibody and (ii) aqueous solubility such that the antibody-linker intermediate is suitable for in vivo administration, with an activated thiol protease enzyme to form a soluble antibody (Fab')2 linker intermediate; and (b) attaching the linker portion of the antibody (Fab')2-linker intermediate either by a covalent or coordinate bond to a compound to form an antibody (Fab')2-linker compound conjugate being characterized by (i) substantially the same immunospecificity as the unconjugated antibody and (ii) aqueous solubility such that the antibody (Fab')2 compound conjugate is suitable for in vivo administration.

23. An antibody fragment compound conjugate, comprising: a soluble antibody (Fab')2 compound conjugate prepared by a method which comprises:
reacting a soluble antibody-linker compound conjugate, which comprises a linker compound moiety attached via a covalent bond between an amine group of the linker-compound selected from the group consisting of primary amines secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide groups, and an oxidized carbohydrate moiety of an antibody, in which the carbohydrate moiety is located outside the antigen binding region of the antibody and in which the antibody-linker 123876.1 compound conjugate is characterized by (a) substantially the same immunospecificity as the unconjugated antibody and (b) aqueous solubility such that the antibody-linker compound conjugate is suitable for in vivo administra-tion, with an activated thiol protease to form a soluble antibody (Fab')2 linker compound, being characterized by (a) substantially the same immunospecificity as the un-conjugated antibody and (b) aqueous solubility such that the antibody (Fab')2 compound conjugate is suitable for in vivo administration.

24. A method for in vivo imaging a specific tis-sue, comprising:
(a) administering to an animal or a human an effective amount of an aqueous soluble antibody (Fab')2 compound conjugate according to claim 21, 22 or 23 in which the antibody (Fab')2 compound conjugate is immuno-reactive with and immunospecific for an antigenic deter-minant of the specific tissue and non-immunospecific for non-specific tissue and in which the antigen determinant is not found in substantial amounts in non-specific tis-sue; and (b) detecting whether the antibody (Fab')2 compound conjugate localized at the specific tissue.

25. A pharmaceutical composition for use in the treatment of a cellular disorder in a patient compris-ing a therapeutically effective amount of an antibody (Fab')2 conjugate of claim 21, 22 or 23, together with a pharmaceutically acceptable carrier therefor, wherein said antibody (Fab')2 conjugate is immunoreactive with and immunospecific for a target site associated with said cellular disorder and substantially non-immunore-active with and non-immunospecific for tissue not as-sociated with said cellular disorder.