preparation and purification of antibody-enzyme conjugates for therapeutic applications

Preparation

advanced

drug delivery reviews

Advanced Drug Delivery Reviews 22 ( 1996) 2XY-30 I

and purification of antibody-enzyme conjugates for therapeutic applications

Roger G. Melton”

Cetltw ,fiw Applied Microbiology ard Resecrr-ch, Portm Lhwr, Sulisbury, Wilts, SP4 OJG, UK

Accepted I6 July I996

Abstract

The successful use of antibody-enzyme conjugates for therapeutic applications requires conjugates of defined composition and low molecular weight, dictating the use of heterobifunctional coupling agents under carefully controlled and optimised conditions. The coupling chemistry chosen for the production of antibody-enzyme conjugates has been based almost exclusively on the use of thioether linkages, because of their greater stability in vivo compared with the disulphide linkages commonly used for the production of antibody-toxin conjugates. Using this type of conjugation chemistry, the modification of the proteins takes place in a semi-random fashion, any exposed modifiable amino acids being potential coupling sites, and there is no homogeneity of the product in the sense that location of modified residues is not controllable. Whilst the yield of the conjugation step is typically about 35-5076, purification is complicated by the changes in the charge properties of the proteins, which occur as a result of heterobifunctional and thiolation agents modifying positively charged lysine residues. This has meant that although affinity and ion exchange chromatography techniques have been used to some extent, the most common method of purifying conjugates is size exclusion chromatography. Incomplete separation means that the overall yield\ of purified product are typically only IO-IS’% at best. New technologies for the construction of antibody-enzyme conjugates are gradually emerging, which may eventually supplant the current chemical conjugation techniques, although it seems likely that chemical conjugation will continue to be of value for preliminary studies. Revcrsc protcolytic methods seek to extend the usefulness of chemical conjugation by achieving conjugation at defined sites on the proteins. The development of antibody-enzyme fusion proteins is becoming common, and bifunctional antibodies, based on an antigen binding arm together with an enzyme capture or catalytic antibody arm have also been described. These offer the prospect of homogeneity of product, simple purification using immunoglobulin binding proteins, and straightforward routes to the development of non-immunogenic proteins using conventional humanisation techniques.

Kcy~~~rzl.s: ADEPT: Conjugate; Coupling; Hcterobifunctional reagents: Antibody-enzyme conjugates: Puritication

Contents

I. Introduction ............................................................................................................................................................................

2. Production of antibody fragments ............................................................................................................................................. 3. Chemical coupling of antibodies to enzymes .............................................................................................................................

3. I. Choice of linkage.. ...........................................................................................................................................................

3.2. Insertion of maleimide groups into proteins.. ......................................................................................................................

1.3. Thiolation of proteins .......................................................................................................................................................

3.4. Coupling conditions .........................................................................................................................................................

290

290

291

292

292

294

295

“Corresponding author: Tel: + 44 I980 6 12430; fdx: + 44 1980 6 IO3 I I ; e-mail: I003 [email protected]

0169-409X/96/$32.00 Copyt-i&t 0 1996 Elsevier Science B.V. All rights reserved 1’11 so 169.409X( 96)00447-4

290 R.G. Melton I Ad,wrced Drug Delivery Reviews 22 (1996) 289-301

3.5. Site-specific coupling of proteins ....................................................................................................................................... 295 4. Alternatives to chemical coupling.. ........................................................................................................................................... 296

4.1, Biapecific antibodies ........................................................................................................................................................ 296 4.2. Fusion proteins ................................................................................................................................................................ 291

5. Purification of conjugates.. ....................................................................................................................................................... 297 6. Future developments __..._.......,.,._.......,,..,,,,.,..........,.,,,,.,.........

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . .

References _. ._. __. ._. . . __. _.

299 . 299

299

1. Introduction

The generation of chemically-linked antibody-en-

zyme conjugates is a long-established practice, particularly the manufacture of such conjugates for use

in enzyme linked immunosorbent assays (ELISAs),

but the methodology used in the production of conjugates for therapeutic applications differs from

that normally employed to make conjugates for

ELISA purposes. For the latter application, the primary concern is to achieve high yields with good

retention of bioactivity. For therapeutic use, although

the criteria of yield and activity retention are still

crucial, it is also important to have a product which

is as closely defined as possible. Thus, the therapeu-

tic antibody-enzyme product is required to be primarily 1:l antibody/enzyme conjugate, to minimise

the conjugate size in order to achieve maximum extravasation and penetration of the target tumour,

and to be free of large aggregates which, even if soluble, tend to be rapidly taken up by cells of the

reticuloendothelial system. Classical methods of antibody-enzyme coupling, such as periodate oxida-

tion or glutaraldehyde activation [l] have therefore

been largely supplanted by techniques based on

heterobifunctional reagents, using methodology which was initially developed for the production of

antibody-toxin conjugates 121, themselves highly specialised enzymes in the case of the ribosome inactivating proteins such as ricin. Using such re-

agents, the stoichiometry of the reaction can be carefully controlled to optimise the formation of the desired 1: 1 conjugate. The methods used for the production of antibody-glucose oxidase conjugates

for possible use as anticancer agents, which can perhaps be regarded the precursors of true antibody

directed enzyme prodrug therapy (ADEPT) systems, represent an intermediate phase in the development of conjugation methodology. Some workers in this field used periodate oxidation [3,4] or glutaraldehyde

I51 whilst others described the use of the

homobifunctional reagent diethylmalonimidate [6]. There appears to have been little effort made to

generate conjugates of defined composition and the

conjugates which were produced were typically not

purified beyond centrifugation to remove insoluble aggregates [3,4]. The conjugates thus produced were used only in vitro, where the presence of high

molecular weight aggregates would be of relatively

low importance to their efficacy.

2. Production of antibody fragments

The manufacture of the basic enzyme and anti-

body components will not be discussed in great detail here; however, in many cases it is desirable to use F(ab’), or Fab fragments of monoclonal anti-

bodies, rather than intact antibodies. Fab/c fragments

have been described [7] but have not found an application in any ADEPT system to date. The lower

molecular weights of antibody fragments potentially leads to more rapid clearance of conjugate, better

tissue penetration and improved specificity of locali-

sation as a result of diminished interaction with

non-specific Fc receptors. As yet, there is no standardised method of producing Fv fragments of antibodies by chemical digestion, however methods

for the production of F(ab’), and Fab are well established and commercial kits are readily available for this purpose, although such kits tend to be based

only on the long established enzymes pepsin and papain. Fv fragments are normally produced by recombinant technology in the single chain Fv form, and provide the basis of fusion protein constructs [8]

although Fab fragments have also been used for the production of fusion proteins [9]. One group has

described the enzymatic digestion (using clostripain) of a mutated IgG2a, in which the entire CHl domain

is deleted, to yield stable Fv fragments [lo], however

KG. Melton I Advanced Dr~r,q De/harry R~tinvs 22 (1996? 289%301 291

this is not a widely applicable route to the production fragmentation. The use of a rat IgG2a, ICR12, has

of Fv fragments on a routine basis. Other enzymes also been reported [ 151, but in this case whole

such as ficin and elastase have also been studied as antibody was used, partly due to the known difficul-

possible candidates for the generation of F(ab’)z and ties of fragmenting antibodies of this subclass. Other

may offer some advantages, particularly for anti- proteolytic enzymes such as lysyl endopeptidase

bodies which are difficult to fragment successfully in have different specificities and can be successfully

high yield [ 111, but for most antibodies of the IgGl used to digest immunoglobulins of subclasses such

subclass, bromelain or papain will probably work as IgG2a or IgG2b which can be resistant to the

satisfactorily. classical enzymes such as pepsin and papain [ 16).

The early method of pepsin digestion for the

production of F(ab’), fragments is still used. but has

been largely superseded to a large extent by the use

of the thiol-activated enzymes papain or bromelain in

the absence of reducing agent. These have the advantage of being more efficient than pepsin, permitting the use of less protease and therefore

diminishing the problem of removal of the protease from the final product. Some commercially available

kits use pepsin or papain immobilised on an insolu-

ble support, permitting their removal by centrifugation. The yields obtained using thiol activated papain

or bromelain also tend to be higher than those

achievable with pepsin [ 12,131. Typically it is possible to use about l-2% bromelain or papain by

weight, compared with 3% or more of pepsin.

Depending on the conditions used, bromelain and papain can be used to generate either F(ab’), or Fab,

the product depending on whether incubation takes place in the presence or absence of thiol groups. In either case, the proteolytic enzyme must be activated

by pre-incubation with thiol. Fab can also be gener-

ated from F(ab’)l by reduction of F(ab’), with about 20 mM of a thiol reducing agent such as dithio-

threitol (DTT). However this process must be care-

fully controlled, as over-reduction leads to the cleavage of interchain disulphide bridge and dissociation

of heavy and light chains. The pH at which reduction

is carried out is also important, low pH facilitating the reduction of interchain disulphide bonds. If dissociation of heavy and light chains does occur, it

is possible to reoxidise the heavy and light chains to reform Fab, using dehydroascorbic acid. Prolonged incubation with this agent can also lead to the

reformation of F(ab’),. However this latter process occurs very slowly, and in practice, the reaction can be easily contained at the Fab stage 1141.

3. Chemical coupling of antibodies to enzymes

The differing modes of action of toxin- and

enzyme-antibody conjugates have lead to precise

details of the respective coupling protocols being adapted to suit the differing conjugate stability

requirements of the two approaches, but the basic underlying chemistry is essentially very similar. In

all the commonly used coupling protocols, a heterobifunctional reagent is used to couple via

modified lysine residues on one protein to sulphydryl

groups on the second protein. The modification of

lysine residues involves use of a heterobifunctional reagent comprising an N-hydroxysuccinimide func-

tional group, together with a maleimide or protected sulphydryl group. The linkage takes the form of one of two basic types. a disulphide bridge or a thioether

bond (Fig. 1) depending on whether the introduced group was a sulphydryl or maleimide. respectively.

The thiol group on the second protein may be an

0

? Enzyme-NH-C-X-N

3 0

S-Antibody

a. Thioether linkage (X = spacer group)

‘il Toxin-NH-C-CH2--CHz--S -S-Antibody

b. Disulphide linkage

The majority of monoclonal antibodies used for ADEPT applications to date have been of the IgGl subclass and generally present few problems in

Fig. I. The two main types of protein-protein linkages typically

used for the production of antibody-enzyme or antibody-toxin

conjugates. The relative positions of the proteins with respect to

the linkage may be reversed.

292 R.G. Melton I Advanced Drug Delivery Reviews 22 (1996) 289-301

endogenous free sulphydryl if one is available, or it

may be chemically introduced, again by modification of lysine residues.

3.1. Choice of linkage

The choice of disulphide or thioether linkage is

largely determined by the required stability of the

linkage. Disulphide linkages appear to be inherently unstable in the plasma [ 17,181, but a certain degree

of instability appears to be required for antibody- toxin conjugates to allow the free toxin to be

released intracellularly, and non-reducible conjugates

have reduced potency [ 181. For ADEPT applications, such instability is definitely undesirable in vivo

because a period of some days may be required to elapse between administration of antibody-enzyme

conjugate and prodrug, in order to allow unbound

conjugate to clear from normal tissues [19]. In the

case of a conjugate of CPG, with ICR12, an intact

IgG2a directed against c-erbB2, a period of 14 days

was necessary between administration of conjugate and prodrug when used in a mouse xenograft model, in order to allow sufficient clearance of non-localised

conjugate from the blood, yet the antibody-enzyme conjugate remained active at the end of this period and an impressive anti-tumour effect was attained

[ 151. Although accelerated clearance systems have

been developed [20,21], a minimum of 24 h is

usually allowed between administration of conjugate

and clearing agent in order to achieve maximal

tumour localisation of conjugate. The problem of the

inherent instability of disulphide-linked conjugates has been addressed to some extent by the development of sterically hindered disulphide crosslinking agents such as N-succinimidyloxycarbonyl-a-(2-

pyridyldithio)-toluene SMPT [22] or N-succinimidyl- 3-( 2_pyridyldithio)-butyrate (SPDB) [ 181; however these reagents still do not confer stability properties

comparable with those of thioether-linked conjugates. With very few exceptions, therefore, the

universally preferred linkage for conjugates intended to be used in ADEPT systems has been the thioether bond, with the use of disulphide-linked conjugates so far confined to in vitro studies [23]; there have been

no reports of conjugates intended for use in ADEPT being produced using hindered disulphide linkers.

3.2. Insertion of maleimide groups into proteins

The maleimide-introducing component for the

generation of thioether linkages is essentially common to all the systems described, with N-suc-

cinimidyl-4-(N-maleimidomethyl)-cyclohexane- l-

carboxylate (SMCC) or its more soluble sulphated

form (Sulfo-SMCC) being the most commonly used

reagent [23-291, although N-succinimidyl-4-(p-

maleimidophenyl)-butyric acid (SMPB) [30] and N- maleimidobenzoyl-N-hydroxysuccinimide (MBS) [31] have also been used. These compounds differ

only in the carbon spacer length between the N-

hydroxysuccinimide (NHS) and maleimide functional groups, with SMPB having the greatest spacer

length and MBS the shortest (Fig. 2). In all these compounds, the NHS ester moiety reacts with the primary amines of lysine residues at slightly alkaline

pH (ca. 7.5) to form an amide bond, linking the

maleimide group to the protein and releasing N-

hydroxysuccinimide (Fig. 3a), which can be re-

moved, together with unreacted reagent, by dialysis

or gel filtration. Proprietary prepacked columns, such as the PDlO columns containing Sephadex G25 marketed by Pharmacia are ideal for this purpose. After removal of excess reagent, the maleimide-

activated protein is mixed with the second, thiolated, protein. The maleimide group then reacts with the

artificially inserted or endogenous thiol groups in the

second protein under slightly acidic to neutral (pH

0

MBS - 9.9A

0

T 0

a N-0-C-CH2-CH,

+0

a> N SMPB - 1457

$-0-!+H$ SMCC - 11.6A

0 0

Fig. 2. Structures of commonly used heterobifunctional coupling

agents showing spacer lengths.

R.G. Melton I Advuanced Drug Delivery Reviews 22 (1996) 289-301 293

Protein1 -NH-C-X-N ’ 3 + d-_-OH

0 0

W 0 0 0

Proteinl--NH4$-X-N 3 + HS-Protein2 -

0 -Protein2

Fig. 3. The generalised reaction scheme for the coupling of proteins using N-hydroxysuccinimide ester/maleimide heterobifunctional agents,

where X represents spacer groups of differing chain lengths.

6.5-7.5) conditions to form a thioether bond (Fig. 3b). Both reactions are rapid and are essentially

complete within 30 min at room temperature under

the conditions outlined. It is clearly necessary that

the protein to have the maleimide group attached

should be free of sulphydryl groups in the form of cysteine residues, otherwise homopolymerisation

may occur. If both proteins contain such groups they

may be capped on one prior to reaction with the

heterobifunctional reagent, by the addition of N-

ethylmaleimide which can also be used to cap

unreacted thiol residues once conjugation is com-

plete, to prevent subsequent aggregation when fur-

ther manipulation of the conjugate takes place e.g.

concentration for loading onto columns. Treatment of a slight molar excess of N-ethylmaleimide with

0

+ Protein pH>7.0

0 S-S-CHz --CHz-NH-Protein

DTT $I * HS-CHs-CH,-C-NH-Protein t

a. SPDP (N-succinimidyl3-[2-pyridyldithiolpropionate)

+ Protein 51 c1

w H&--C--S-CHZ-C-NH-Protein

0% Y s H

0

. HO-N

3 0

9 ? H&-C-S--CHz-C-NH-Protein

NH>OH.HCI R * HS-CH+Z-NH-Protein + CHsCOOH

b. SATA (N-succinimdyl 2-mercapto-[S-acetyilacetic acid)

Fig. 4. Reaction schemes for two N-hydroxysuccinimide-based reagents commonly used for insertion of thiol residues into proteins

294 R.G. Melton I Advanced Drug Delivery Reviews 22 (1996) 289-301

respect to protein, followed by a slightly larger

excess of mercaptoethanol will cap all unreacted

thiol and maleimide groups.

3.3. Thiolation of proteins

The method of inserting a thiol group into the

second component protein is less standardised. His- torically, in common with antibody-toxin conjuga-

tion methodology, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) has often been used, and

the reaction proceeds rapidly at neutral or slightly

alkaline pH, being essentially complete within 15

min. SPDP inserts a 2-pyridyldisulphide-protected

thiol which must be reduced using a suitable reduc-

ing agent such as dithiothreitol to generate the free

thiol required (Fig. 4a), thus the process is somewhat

complicated by the need for an additional step which is necessary to remove excess reducing agent prior to

coupling, since this would otherwise provide a

competing source of thiol groups. The release of pyridine-Zthione residues in this process can be used

as a measurement of the number of thiol groups inserted, providing a useful and rapid in-process test

[32]. The reduction is normally carried out at pH 4.5

to prevent the reduction of internal disulphide

bridges within the protein, but this can prove proble-

matical as not all proteins tolerate such low pHs

well. In the absence of internal disulphide bridges, however, the reduction step can be carried out at

neutral PH. N-Succinimidyl-2-mercapto-[S-

acetyllacetic acid (SATA) [33] also inserts a protected thiol and is a closely related reagent to SPDP in terms of its reaction conditions and prop-

erties, but in its case the protecting S-acetyl group may be cleaved off by hydroxylammonium hydro-

chloride at neutral pH (Fig. 4b) and excess cleaving

reagent does not have to be removed prior to the coupling reaction. In common with SPDP, the

protected thiol generated by the use of SATA has the

advantage that the thiolated protein can be stored in the protected form prior to use. A disadvantage of

SATA, however, is that the deprotection reaction cannot be used to measure the degree of modification

achieved in an analogous way to SPDP and separate

assays to determine thiol incorporation are therefore

necessary.

The common feature of SPDP and SATA is the

N-hydroxysuccinimide function via which the

protected thiol group is attached to lysine residues, but in the case of some enzymes modification of

lysine residues by NHS reagents results in extensive loss of enzyme activity [28], and reversal of the

system, i.e. introducing the maleimide group on to

the enzyme, does not help because the maleimide-

insertion reaction also uses NHS esters. In such instances 2-iminothiolane hydrochloride (Traut’s Re-

agent, 2-IT) provides a useful alternative to both SPDP and SATA, and can thiolate without loss of

enzyme activity [28], (R.G. Melton, unpublished

results). It has the further advantage that, as shown in Fig. 5, it does not alter the charge properties of the

modified protein, since it converts the normally positively charged lysine residue to another positive-

ly charged group. In contrast, modification with NHS-based reagents converts positively charged

lysines to neutral groups, which may be the cause of

loss of bioactivity if the charged residue so modified is involved in the catalytic or binding site, or is

involved with the stabilisation of protein folding.

Unlike the NHS-based reagents, however, 2-IT reacts

relatively slowly and progressively with time, therefore in optimising coupling conditions it is necessary

to determine the rate of thiolation over a period of time and maintain and control this incubation period

(G.M. Anlezark, unpublished results). Occasionally, there are endogenous free thiol

residues present on one of the proteins, which may be used to eliminate the need for a thiolation step,

one such example being the thiol groups exposed by

cleavage of the hinge region of F(ab’), fragment of

immunoglobulin to yield Fab fragments [26,34]. Conversely, there may be free thiol residues present which, while they are not available for coupling

n + l-&--!Jrotein

pH 7-10 YH2+

* HS-CH2CH2CH2-C-NH+rtein

Fig. 5. Thiolation of proteins using 2-iminothiolane,

purposes due to steric hindrance, may nevertheless

form internal disulphide bridges if the protein is

thiolated leading to loss of functionality [28].

-3.4. Coupling conditions

Optimisation of the coupling chemistry has been carried out for the conjugation of carboxypeptidase

G2 to F(ab’), fragment of A5B7, an anti-carcinoembryonic antigen monoclonal antibody [30], where it was found that insertion of l-3 active groups per

protein molecule gave the best yields of 1: 1 conju-

gate with minimal formation of high molecular weight aggregates. It should be noted, however, that

although these conditions provide a general approxi-

mate starting point, detailed study of the precise

requirements for any given system is necessary. In general, however, it is advisable to use the minimum

level of substitution compatible with obtaining an acceptable yield of product. High substitution levels

not only tend to cause degradation of enzymic activity and antigen binding properties, but also give an increased risk of formation of high molecular

weight aggregates. An important third, interrelated

variable is the concentration of the components on

mixing, and if high substitution levels are needed to obtain a good yield of conjugate it is usually possible

to control unwanted aggregation by reducing the

concentration of the components at the mixing step.

Conversely, it may be possible to use low substitution levels and high protein concentrations in order

to preserve bioactivity. For large scale production of conjugate, this latter approach is preferable, since it minimises the volumes of materials to be handled.

The availability on the native protein of lysine or thiol residues for use in coupling may be readily

determined by conventional analytical biochemistry [35,36], as may be the substituent levels [30]. Determining optimal coupling conditions in this

empirical manner is relatively costly in terms of

materials but is essential if reproducibility of batches

of conjugate for in vitro and, more particularly, in vivo studies is to be attained. The overall yields of purified conjugate are not high, typical figures of about 30% yield in the crude incubation mix (as

estimated by integration of A280 traces from analytical gel filtration columns) but 15% or less as

purified conjugate being the norm.

3.5. Site-spec$ic coupling qf proteins

The coupling methods described above have some disadvantages. There is little or no control over the

location of the substitution site, so that linkage may occur via any of the available lysine residues.

Specificity of location can be ensured by the use of endogenous sulphydryl residues because these are

relatively rare and their position is fixed in the amino acid sequence of the protein. As described above,

modification of amino acids in critical areas of the protein may result in deleterious effects on the

protein’s function, e.g. loss of enzymic activity or

antigen binding function. There has been very little effort devoted to devising methods of protecting the

active sites of enzymes or antibodies until recently.

Where intact antibodies are employed a study of

more traditional coupling strategies based on per-

iodate oxidation of glycosyl residues might be worthy of consideration, since the majority of

glycosylation of immunoglobulins is associated with the Fc domain. This could therefore be used to

achieve domain-specific coupling away from the variable regions of the antibody which constitute its

binding site [37] and indeed such an approach has

recently been described for the construction of

antibody hapten conjugates, with the added sophisti-

cation of using an engineered unique glycosylation

site [38].

An alternative approach is to bind the protein to be modified to an affinity matrix or to carry out the

reaction in the presence of substrate or a reversible inhibitor in order to protect the active site residues

whilst they are undergoing modification. Such an approach has been described for the modification of

yeast invertase 1391: the enzyme was bound to a column of concanavalin A-Sepharose and activated

with glutaraldehyde, uricase was allowed to react with the invertase and the conjugate eluted from the

column. This method gave conjugates of fairly well

defined molecular weight and presumably provided protection for the active site of invertase, if not

uricase. Similar such systems may have potential for

the conjugation of enzymes and antibodies which are very sensitive to modification, although affinity systems based on proteinaceous ligands would present alternative reactive sites for coupling reagents

and the use of dye-based pseudo-affinity ligands might be preferable where these are suitable. A

296 R.G. Melton I Advanced Drug Delivep Reviews 22 (1996) 289-301

dye-affinity system using Procion red H8BN was

used for the purification of carboxypeptidase G?, apparently binding via the zinc atoms which are

implicated in the active site 1401; however no further

work has been done to explore whether such a system could confer protection on the active site

during coupling reactions. Recent publications by Fisch et al. [41] and Rose

et al. [42] have described a novel approach to protein

conjugation which does produce conjugates with

well-defined linkage sites. This approach, illustrated in Fig. 6, works under mild conditions and is based

on reverse proteolysis. A carbohydrazide residue is

specifically attached to the carboxyl terminus of one protein using a proteolytic enzyme under conditions

which force it to act as a ligase. This carbohydrazide

group is then linked to an oxidised aldehyde or ketone group on the second protein. If the second

protein has a suitable amino acid (serine or threonine) as its N-terminus this can be specifically

oxidised to provide a second defined linkage site.

The technique has been successfully used to conju-

gate both carboxypeptidase G, [43] and p-lactamase [44] to suitable antibody fragments and in the

absence of the desired serine or threonine N-terminus

an N-terminal threonine was generated on carboxypeptidase G, by site-directed mutagenesis. This

conjugation method appears to offer significant

benefits by comparison with less specific chemical conjugation techniques and could become the approach of first choice in the future, although it seems

likely to be largely superseded by the development

/NH-NH2

of antibody-enzyme fusion proteins and may well

represent the end of the line for chemical conjugation techniques. The yields obtainable are at least com-

parable with those achieved by more conventional means [43].

4. Alternatives to chemical coupling

The avoidance of chemical conjugation methods

for the production of antibody-enzyme conjugates is an obvious goal, given the relatively low yields of

material attained and the problems of conjugate

heterogeneity and loss of bioactivity. These problems are gradually being addressed. Although covered

elsewhere in this volume, a brief outline of the

available systems is appropriate here.

4.1. Bispecij’ic antibodies

Sahin et al. [45] have described the generation of a

bispecific antibody, one arm of which binds to the

CD30 antigen and the other to alkaline phosphatase, providing a capture mechanism for this enzyme. In

this case the two fragments were coexpressed in a

mammalian cell line, thus random recombination of the fragments results in some 50% of the total

antibody produced being present as the bispecific

form, which can be isolated by two affinity purification steps, one for each binding function - only the bifunctional antibody is able to bind to both columns. De Sutter and Fiers [46] have taken this

OX

Proteinl-COOH ‘NH-NH2

Protease * Proteinl- CO-NH-NH2

R

0 ~H-OH OH

Protein2-NHd!-dH-NH;I + 104 B Protein2--NH~--d=O + NH3 + RCHO

(R = H: serine; R = Cb: threonine)

F/t;’ 0 ProteinlXO-NH-NH, + Protein2+JH<-C=O - Protein1 --J!--NH-N=CH-CO-NH-Protein2 + Hz0

Fig. 6. Coupling of proteins by site-specific reverse proteolytic attachment of carbohydrazide to C-terminal carboxylic acid residues and

super-mild oxidation of N-terminal serine or threonine.

process a step further and produced a mouse-human

chimeric antibody in which one antigen binding arm has been replaced by bacterial B-lactamase.

4.2. Fusion proteins

A number of workers have begun to produce

fusion proteins for use in ADEPT applications, the first to be described being that of Bosslet et al., who

fused placental B-giucuronidase to the Fab fragment

of a CDR-grafted humanised antibody to carcinoem-

bryonic antigen [9]. Similarly, Goshorn et al. have

produced a fusion of B-lactamase from Bacillus cereus with a single chain Fv form of the L6

antibody, which binds to a tumour-associated

glycoprotein on a range of tumour cell types [8]. Both of these enzymes are monomeric proteins,

circumventing the potential problems of subunit assembly which may occur with dimeric proteins

such as carboxypeptidase Gz. A major problem identified in pilot clinical trials

with chemically linked A5B7 F(ab’),-carboxypep-

tidase G2 conjugate is that of the immunogenicity of

the conjugate. Human antibody responses to both the antibody and enzyme components of the adminis-

tered antibody-enzyme conjugate developed within

10 days of conjugate administration [47,48] necessi- tating the use of immunosuppressive drugs if repeat cycles of therapy are to be possible. Fusion proteins

could possibly circumvent this problem if con-

structed using a human enzyme and human, or humanised, antibody components. The construct of

Bosslet et al. ]9] comes closest to this ideal in its use

of a humanised murine antibody fragment and

human placental glucuronidase, but there have been

no data reported regarding the immunogenicity of this construct as yet. The ultimate constructs may be

derived from human bispecific catalytic antibodies. Although catalytic antibodies have been reported to

be capable of catalysing a wide range of reactions with specificity equal to or greater than the corresponding enzyme, in general the rates of reaction are not comparable with those of enzymes. The technology of catalytic antibodies is in its infancy at present, but developments in the field are rapid and it can be

confidently predicted that catalytic antibodies will eventually play an important role in ADEPT systems of the future.

5. Purification of conjugates

Once coupled, the desired conjugate must be separated from the uncoupled components because

free antibody will compete with the conjugate for antigen binding sites, whilst free enzyme may persist in the circulation and cause non-specific activation of

prodrug in plasma. In practice, the rate of clearance

of free enzyme tends to be much greater than that of

conjugate or free antibody and it is therefore proba-

bly more important to aim for complete removal of free antibody.

The most common method of purification used is

size exclusion chromatography on Sephadex-type

matrices, although its success is governed by the

difference in molecular weight between the conju-

gate and its components and the fractionation range of the gel. In general, conjugates which have only a small difference in molecular weight between the

conjugate and one of the components will not be well separated from the uncoupled components.

Thus, a I: I conjugate constructed of F(ab’)? with a

low molecular weight enzyme will be poorly separated from the antibody component, although well

separated from the free enzyme. The disadvantages

of this type of chromatography are the relatively low

sample capacity and flow rates of gel filtration columns based on the older Sephadex gels. Modern

gel tiltration matrices such as Sephacryl and Super- dex have alleviated the problem of how rates con-

siderably and their use is practically universal,

although sample capacities remain limited by volume and viscosity considerations. A purification protocol

based on two gel filtration steps, using Sephacryl

S300 and Superdex G200, has been successfully

used to produce A5B7 F(ab’)2-carboxypeptidase G2

conjugates on milligram and multi-gram scales for

pilot clinical trials (R.G. Melton, unpublished data), and the conditions successfully adapted for the manufacture and purification of a murine anti-Ly

2.1-carboxypeptidase Gz conjugate, in this case using intact antibody [49].

Ion exchange chromatography would normally be

the method of choice for the purification of conjugates. The capacities of ion exchange matrices are

high compared with those of size exclusion gels. whilst elution conditions are milder than those

commonly used for immunoaftinity columns. The

technique has, however, found relatively little favour

298 R.G. Melton I Advanced Drug Delivery RevieM;J 22 (1996) 289-301

for the purification of antibody-enzyme conjugates, although a number of workers have described the use of anion [24,28,50,51] or cation exchange [52]

matrices for the separation of uncoupled antibody

from conjugate. The reason for this probably lies in the fact that, as discussed earlier, NHS-based re-

agents modify charge properties, converting the

initially homogeneous charge of a protein to become

heterogeneous on modification, resulting in elution

profiles which are ill defined under salt or pH

gradient elution conditions by comparison with that of the native protein. Under these circumstances the

use of 2-iminothiolane, which does not modify the

charge of the group it modifies, may be helpful in assisting the development of ion exchange purifica-

tion techniques. Protein A-Sepharose immunoaffinity chromatog-

raphy has been used for the purification of an

antibody-p-glucuronidase conjugate [23] and can be

used to remove free enzyme, but does not separate

free antibody from conjugate. Its use is largely

restricted to conjugates produced using intact anti-

body, since binding of antibody to protein A occurs primarily via the Fc domain. A more recently

developed immunoglobulin-binding protein, protein

L from Peptostreptococcus magnus, binds via the Fv domain and may offer significant advantages for the

purification of conjugates constructed using antibody

fragments [53]. It should be of particular use for the purification of antibody-enzyme fusion proteins,

where there is no background of unreacted com-

ponent antibody. A possible disadvantage of im-

munoaffinity chromatography systems is that rela-

tively harsh conditions, e.g. low pH, may be required to elute bound material, and although antibodies may tolerate such conditions, enzymes may not. An

example of this is carboxypeptidase Gz, which is irreversibly inactivated at low pH, but each case must be judged on its individual merits. The purification of an anti_CD30/alkaline phosphatase bispecihc

antibody has been achieved using immobilised calf alkaline phosphatase, with elution by a pH gradient. In this instance the single binding moiety - i.e. the bispecific antibody - could be recovered by elution

at pH 5.5, whereas the bifunctional form required a pH of 3.5 for elution [45]. Similarly, an immuno-

affinity column using rabbit polyclonal anti-p-lactamase has been used to purify a murine::human

chimeric antibody in which one antigen binding arm has been replaced by p-lactamase [46]. Elution in this case required the use of 0.1 M glycine at pH 3.0.

It is possible that affinity chromatography systems based on enzyme/substrate interactions may have

utility for the purification of antibody-enzyme conju-

gates, for example carboxypeptidase GZ has been

successfully purified by dye affinity chromatography [40] and although some problems were encountered

with dye leaching from the column and coeluting

with the enzyme, dye affinity matrices with improved stability are now commercially available [54]

and may solve this problem. Such a system may

therefore represent a feasible approach to the removal of free antibody from conjugate preparations; the feasibility of such an approach has been demon-

strated by its use in the purification of ricin immunotoxins using a cibachron blue F3GA-Sepharose

matrix [55].

More conventionally, P-lactamase fusion proteins

have been purified on phenylboronic acid affinity

columns with elution at pH 7.0, by means of a salt

gradient 1561, and chemically linked p-lactamase

conjugates have been similarly purified on boronic

acid affinity columns [57]. Although affinity purification techniques are

powerful, it is probable that their use will be reiatively restricted. The cost of immunoaffinity

matrices based on antibodies is high and their capacity limited by comparison with ion exchange

matrices, for example. The large scale purification of

conjugates for clinical trial purposes does not appear

to be a feasible proposition based on such technolo-

gy, and is complicated by the need to quantify the rate of leaching of the immobilised ligand from the

column for quality assurance purposes. The use of the immunoglobulin binding proteins would appear to be more appropriate for large-scale production

purposes, if an affinity-based system is required, since such matrices are commonly used for the

production of monoclonal antibodies, and the rate of loss of immobilised protein is well studied for such

materials [SS]. The specificity of enzyme-substrate interactions

gives rise to the possibility of developing methods of

conjugate purification based on such interactions, using non-substrate analogues: such compounds may well be generated in the course of prodrug optimi-

R.G. Meltm I Advanced Drug Delivery Revierss 22 (1996) 289-301 299

sation screens, and if they act as reversible inhibitors

may be able to form the basis of an affinity matrix from which bound conjugate could be eluted by use of a competitive substrate. The latter material, being of low molecular weight could be removed by conventional desalting or buffer exchange tech-

niques.

6. Future developments

It seems likely that the current widespread use of

chemically linked antibody-enzyme conjugates in ADEPT will eventually fall into relative disuse as

more sophisticated approaches, such as fusion proteins, become more practical, but the chemically linked conjugate is likely to play an important role in

early preclinical studies, given the relative ease of

their production. As antibody-enzyme fusion proteins become more

widely used, it is likely that purification protocols based on the use of histidine tags, permitting the use

of immobilised metal affinity chromatography [59],

or alternative systems using cleavable affinity tags which can be removed after purification of the

protein, will become important routes for conjugate purification. Such systems based on glutathione-S- transferase [60] or maltose binding protein [61], use

a site-specific protease such as thrombin or Factor Xa to cleave off the tag, and the plasmid vector

includes a suitable recognition site sequence immedi- ately upstream of the fusion protein cloning site.

There are some potential problems with these sys-

tems, notably ensuring complete consistency of removal of tags. For this reason, histidine tags,

which are not normally removed and appear to have

no adverse effects in vivo, at least in the mouse, [62] may be the preferred option. However, there is relatively limited information on the in vivo interactions of the histidine tag as yet.

Acknowledgments

The author wishes to thank Drs G.M. Anlezark, G.

Jack, M.A. Sims and Mr R.J. Ling for helpful discussions and the Cancer Research Campaign for their support for much of the work described here.

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preparation and purification of antibody-enzyme conjugates for therapeutic applications

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