Hermanson G. Bioconjugate Techniques, Second Edition

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conjugate into the cell. Preparation of the conjugate should maintain the antigen binding char-

acter of the attached antibody and at the same time not block the ribosome-inactivating activ-

ity of the toxin component.

Studies have been done to investigate the importance of using a cleavable linker between the

antibody and the toxin. This conﬁ guration in the immunoconjugate would mimic the natural

state of two-subunit toxins like ricin that are held together by disulﬁ de bonds. There is evi-

dence that disulﬁ de reduction and cleavage of the A chain from the B chain is necessary for

cytotoxicity in native toxins (Olsnes, 1978). There is similar evidence that the creation of cyto-

toxic immunotoxins using only A-chain subunits requires that the conjugation be done with a

monoclonal using a crosslinker that possesses a disulﬁ de bond in its cross-bridge or creates a

disulﬁ de linkage upon coupling (Masuho et al., 1982). Using disulﬁ de-cleavable crosslinkers

in the preparation of immunotoxins results in the antibody taking on the role of the B chain

in recognizing and binding to antigenic determinants on the surface of cells. After binding,

some mechanism internalizes the conjugate where the two components then are separated by

disulﬁ de reduction. The A-chain subunit is then freed to enter the cytoplasmic space where

enzymatic degradation of the ribosomal proteins occurs.

Other investigators, however, have demonstrated that conjugations of antibody with intact,

two-subunit toxins can be done using non-cleavable crosslinkers such as NHS ester–maleimide

heterobifunctionals (Chapter 5, Section 1) (Myers et al., 1989). Presumably, the toxin is still able

to release the A chain after the antibody has bound to the cell, since the conjugation process

does not permanently attach the two toxin subunits together—only the toxin to the antibody.

Thus, two main strategies can be used in making immunotoxin conjugates ( Figure 21.3 ).

In the most often used method, the isolated A chain of two-subunit toxins (or the intact polypep-

tide of single-subunit toxins like gelonin) is conjugated to a monoclonal using a crosslinker

that can introduce a disulﬁ de bond. When using only puriﬁ ed A chain, it is common (but not

absolutely required) to couple through the sulfhydryl that is freed during A–B chain cleavage by

disulﬁ de reduction. The single-chain toxins like gelonin, however, have no free sulfhydryls, so

a thiolating agent such as 2-iminothiolane (Chapter 1, Section 4.1) may be used to create them

(Lambert et al., 1985).

When using ricin A chains, it has been found that chemical deglycosylation of the subunit

prevents its nonspeciﬁ c binding to receptors for mannose on certain cells of the reticuloendothe-

lial system (Vitetta and Thorpe, 1985; Ghetie et al., 1988, 1991; O ’Hare et al., 1988). Thus,

immunotoxin conjugates consisting of deglycosylated ricin A chain (dgA) have been shown to

survive longer in vivo and are more efﬁ cient at reaching their intended target cells. In addition,

if the antibody component does not contain Fc region, but consists of only F(ab )

, Fab , Fab,

or smaller Fv fragments, then nonspeciﬁ c binding of the immunotoxin in vivo will be reduced to

a minimum. One study found that constructing immunotoxin conjugates with molar ratios of

two dgA per antibody molecule resulted in a 7-fold increase in cytotoxicity over a 1:1 conjugate

ratio (Ghetie et al., 1993).

A-chain immunotoxins, however, may not be quite as cytotoxic as conjugates formed from

intact toxin molecules (Manske et al., 1989). In an alternative approach to A chain use, the

intact toxin of two-subunit proteins is directly conjugated to a monoclonal without isolation

of the A chain. Conjugation of an antibody with intact A–B chain toxins can be done with-

out a cleavable linker, as long as the A chain can still separate from the B chain once it is

internalized. Therefore, it is important to avoid intramolecular crosslinking during the conju-

gation process which can prevent release of the A–B complex. In addition, since the B chain

830 21. Immunotoxin Conjugation Techniques

possesses a recognition site for most cell surfaces, it still has the ability to nonselectively bind

and kill non-tumor cells. To maintain antibody speciﬁ city in intact toxin conjugates toward

only one cell type (and thus prevent nonspeciﬁ c cell death), all cell binding capability within

the toxin itself must be removed. Fortunately, a large proportion of the binding sites on the B

chains usually are blocked during the conjugation process, and the galactose binding potential

is signiﬁ cantly impaired. Further puriﬁ cation to remove conjugates that have remaining galac-

tose binding potential can be done on an acid-treated agarose chromatography column (which

contains galactose residues) or on a column of asialofetuin bound to agarose (Cumber et al .,

1985). Conjugate fractions which do not bind to both afﬁ nity gels contain no nonspeciﬁ c bind-

ing potential toward non-targeted cells.

More elaborate methods of blocking or eliminating the B-chain galactose binding site also

can be done to prevent nonspeciﬁ c cytotoxicity. For instance, the crosslinking agent may have a

Figure 21.3 The strategies involved in creating an immunotoxin conjugate are numerous. Intact antibody

molecules or enzymatic fragments may be used as the targeting component. Even small recombinant Fv frag-

ments that are genetically engineered to limit host immune response can be employed. Conjugates can include

two-subunit toxins or puriﬁ ed A-chain components. If intact toxins are used, the B-chain binding site must be

blocked to prevent nonspeciﬁ c cell death. If A-chain subunits are used, to induce cytotoxic effects in the conju-

gate the crosslinking agent must generate a disulﬁ de bond that can allow toxin release.

2. Preparation of Immunotoxin Conjugates 831

lactose molecule designed into it that can block B-chain activity. The lactose portion possesses

natural afﬁ nity for the B-chain binding site, and thus it occupies that area while a nearby reac-

tive group covalently attaches the crosslinker to neighboring functional groups on the protein.

Moroney et al. (1987) used this approach in creating a ricin conjugate. Lactose was modi-

ﬁ ed at its reducing end with cystamine via reductive amination (Chapter 2, Section 5.3). The

cystamine was reduced with dithiothreitol (DTT) and immobilized on an afﬁ nity gel which was

activated with a pyridyl dithiol group (Chapter 2, Section 2.6). The coupled lactose then was

modiﬁ ed with a chlorotriazine derivative through the secondary amine created by the reductive

amination process. Next, the ricin molecule was immobilized by the additional reactive group on

the chlorotriazine ring. Since this reactive group was immediately adjacent to the lactose residue,

the ricin bound the sugar at its B-chain binding site and it was covalently coupled through a

nearby amine. This process effectively blocked and eliminated all nonspeciﬁ c binding potential in

the ricin dimer. After removal of the blocked ricin from the support by DTT, the free sulfhydryl

group on the protein was conjugated to an SMCC-modiﬁ ed antibody, forming the ﬁ nal conjugate

(Figure 21.4 ). Although this process worked in preparing an effective immunotoxin conjugate,

most conjugation schemes are less elaborate.

Regardless of their method of preparation, the required and ideal characteristics of immuno-

toxin conjugates can be summarized in the following points:

1. The conjugation process must leave the antigen binding sites on the antibody component

free to interact with its intended target. Crosslinker modiﬁ cation or blockage of these

binding sites by the attached toxin must be kept to a minimum.

2. The toxin component of the conjugate must be able to elicit cytotoxicity by ribosomal

damage as it could in its native state. This means that the cell penetration and enzymatic

properties of the toxin remain unaltered, although an antibody molecule is conjugated

to it.

3. The activity of toxin binding to cells through the B chain must be eliminated in the con-

jugate to prevent nonspeciﬁ c binding and nonselective cell death. This may be accom-

plished by using only the A-chain subunit or by blocking the B-chain binding site in the

intact toxin conjugate.

4. Avoid covalently linking the A and B chains together during the crosslinking process.

This can be done by using heterobifunctional crosslinkers that are more controllable in

their reactivity than homobifunctional reagents.

5. The crosslinking process must minimize polymerization of either the antibody or the

toxin. Low molecular weight, 1:1 or 1:2 conjugates of antibody-to-toxin are best.

6. The crosslinker used to form the bond between the antibody and toxin must be able to

survive in vivo and not be cleaved by enzymatic or reductive means before reaching the

targeted cells.

7. The conjugate must reach its intended target without being intercepted, bound and

destroyed by the host immune system.

8. Administration of the immunotoxin should result in cell death and complete elimination

of all target cells.

The last two points are the most difﬁ cult to realize. The following methods describing the

conjugation strategies used to prepare immunotoxins work well in creating complexes contain-

ing active antibody and toxin. The majority of research today is not so much concerned with

further optimization of the crosslinking process, but primarily is directed at overcoming the

832 21. Immunotoxin Conjugation Techniques

host immune system and making the conjugates more effective in accomplishing complete tar-

geted tumor destruction.

2.1. Preparation of Immunotoxin Conjugates via Disulﬁ de Exchange Reactions

Since the cytotoxic potential of most common toxins relies on their subunit disulﬁ de cleavabil-

ity with subsequent release of associated A chains, most successful conjugation techniques for

preparing immunotoxins involve the use of disulﬁ de exchange reactions. Heterobifunctional

crosslinking agents containing an amine-reactive group at one end and a disulﬁ de bond with a

Figure 21.4 In an elaborate strategy to block the B-chain binding site in the construction of immunotox-

ins using intact A–B subunit toxins, cystamine was ﬁ rst coupled to the reducing end of lactose by reductive

amination. DTT was then used to reduce the cystamine disulﬁ de group, revealing the free thiol. A pyridyl

disulﬁ de-activated agarose gel then was used to couple the lactose derivative through its sulfhydryl. Next,

trichloro-s-triazine was reacted with the support to modify the secondary amine on the cystamine component,

forming a reactive gel. Finally, addition of an intact toxin to the afﬁ nity support caused binding with the lactose

group at the B-chain binding site. Since the B chain was now in proximity to the chlorotriazine ring, covalent

coupling occurred with available amines on the protein toxin, thus permanently blocking the binding site. After

removal of the modiﬁ ed toxin from the gel using a disulﬁ de reducing agent, the free thiol of the cystamine group

was used to conjugate with an SMCC-activated immunoglobulin.

2. Preparation of Immunotoxin Conjugates 833

good leaving group on the other end are common choices for making these conjugates (Chapter 5,

Section 1). The leaving group on the disulﬁ de portion of the crosslinker permits efﬁ cient disulﬁ de

interchange with a free sulfhydryl on the antibody or toxin. The resultant covalent bond thus is

a cleavable disulﬁ de which mimics the native cleavability inherent in the toxin dimer.

Pyridyl Disulﬁ de Reagents

The most common reactive group for initiating disulﬁ de interchange reactions is a pyridyl

disulﬁ de. Attack of a nucleophilic thiolate anion dissociates the pyridine-2-thione leaving

group from this group, forming a new disulﬁ de bond with the incoming sulfhydryl compound.

Several crosslinking reagents containing these groups are popular choices for producing anti-

body–toxin conjugates.

SPDP

SPDP, N-succinimidyl 3-(2-pyridyldithio)propionate, is by far the most popular heterobifunc-

tional crosslinking agent used for immunotoxin conjugation (Chapter 5, Section 1.1). The acti-

vated NHS ester end of SPDP reacts with amine groups in one of the two proteins to form an

amide linkage. The 2-pyridyldithiol group at the other end reacts with sulfhydryl groups in

the other protein to form a disulﬁ de linkage (Carlsson et al., 1978). The result is a crosslinked

antibody–toxin conjugate containing cleavable disulﬁ de bonds that can emulate the activity of

native two-subunit toxin molecules.

LC-SPDP (Chapter 5, Section 1.1) is an analog of SPDP containing a hexanoate spacer arm

within its internal cross-bridge. The increased length of the extended crosslinker is important

in some conjugations to avoid steric problems associated with closely linked macromolecules.

However, for the preparation of immunotoxins, no advantages were observed for LC-SPDP

over SPDP (Singh et al ., 1993).

SPDP is also useful in creating sulfhydryls in one of the two proteins being conjugated

(Chapter 1, Section 4.1). Once modiﬁ ed with SPDP, the protein can be treated with DTT (or

another disulﬁ de reducing agent) to release the pyridine-2-thione leaving group and form the

free sulfhydryl. The terminal  SH group then can be used to conjugate with any crosslinking

agents containing sulfhydryl-reactive groups, such as maleimide or iodoacetyl (for covalent con-

jugation) or 2-pyridyldithiol groups (for reversible conjugation).

In the preparation of immunotoxins, some procedures call for the modiﬁ cation of the anti-

body with SPDP to introduce reactive thiols (Cumber et al., 1985). The NHS ester end of the

crosslinker is reacted at slightly alkaline pH with the primary amines on the antibody. After

removal of excess reagent by gel ﬁ ltration, the pyridyl disulﬁ de groups are reduced by DTT.

The reductant causes the removal of pyridine-2-thione groups and the creation of sulfhydryl

groups on the immunoglobulin. The reason the antibody is thiolated in this manner and not

the toxin is to avoid exposing the intact toxin to reducing conditions that could disassociate

the A and B subunits. To activate the toxin, SPDP again can be used to modify the intact A–B

component. After puriﬁ cation of the modiﬁ ed toxin from excess crosslinker, the SPDP–toxin is

mixed with the thiolated antibody to effect the ﬁ nal conjugate ( Figure 21.5 ).

This multi-step crosslinking method employing SPDP on both molecules has been used to

prepare a number of immunotoxin conjugates (Edwards et al., 1982; Thorpe et al., 1982;

Colombatti et al., 1983; Wiels et al., 1984; Vogel, 1987; Reiter and Fishelson, 1989). While

834 21. Immunotoxin Conjugation Techniques

this method has worked well for many different toxins, its main potential disadvantage is expo-

sure of the antibody to reducing conditions that potentially could cleave the disulﬁ de bonds

holding its heavy and light chains together. Alternative methods using SPDP in a nonreducing

environment may result in better conjugates.

For instance, if toxin A chain–antibody conjugates are to be prepared, the antibody can be

similarly activated with SPDP, but in this case not treated with reductant. After removal of

Figure 21.5 SPDP can be used to modify both an antibody and a toxin molecule for conjugation purposes. In

this case, the antibody is thiolated to contain a sulfhydryl group by modiﬁ cation with SPDP followed by reduc-

tion with DTT. A toxin molecule is then activated with SPDP and reacted with the thiolated antibody to effect

the ﬁ nal conjugate through a disulﬁ de bond.

2. Preparation of Immunotoxin Conjugates 835

excess crosslinker, the activated antibody can be directly mixed with isolated A chain to create

the conjugate ( Figure 21.6 ). This procedure makes use of the indigenous sulfhydryl residues

produced during reductive separation of the A and B chains and therefore does not require

crosslinker thiolation of one of the proteins.

Another way of utilizing SPDP is to again activate the antibody to create the pyridyl disulﬁ de

derivative, but this time thiolate the toxin component using 2-iminothiolane (Chapter 1,

Figure 21.6 SPDP can be used to activate an antibody molecule through its available amine groups to form

a sulfhydryl-reactive derivative. Toxin molecules containing disulﬁ de-linked A–B chains may be reduced with

DTT to isolate the A-chain component containing a free thiol. The SPDP-activated antibody is then mixed with

the reduced A chain to effect the ﬁ nal conjugate by disulﬁ de bond formation.

836 21. Immunotoxin Conjugation Techniques

Section 4.1). 2-Iminothiolane reacts with primary amines in a ring-opening reaction that cre-

ates a terminal sulfhydryl group without reduction. Intact A–B toxins and toxins containing

only one subunit, like gelonin, PAPs, and Pseudomonas toxin A, can be coupled to antibodies

using this procedure (Lambert et al., 1985; Bjorn et al., 1986; Scott et al., 1987; Lambert et al .,

1988; Ozawa et al., 1989). Mixing the SPDP-activated antibody with the thiolated toxin effects

the ﬁ nal conjugation ( Figure 21.7 ).

SPDP also can be used to conjugate other targeting molecules to toxins, such as transferrin,

epidermal growth factor, 

-macroglobulin, and human chorionic gonadotropin (Fizgerald et al.,

1980; Helenius et al., 1980; Keen et al., 1982; Oeltmann, 1985). To create these conjugates, one

of the two components must be activated with SPDP to generate the sulfhydryl-reactive pyridyl

disulﬁ de groups, while the other component must be modiﬁ ed to contain the  SH functionality.

Mixing these modiﬁ ed molecules together forms the toxin conjugate.

Figure 21.7 An intact A–B subunit toxin molecule may be activated with 2-iminothiolane with good retention

of cytotoxic activity. The thiolated toxin then may be conjugated with SPDP-activated antibody to generate the

immunotoxin conjugate through a disulﬁ de bond.

2. Preparation of Immunotoxin Conjugates 837

The following methods are generalized to provide an overview of how SPDP can be used in

these conjugation techniques. The appropriate optimization for a particular toxin conjugate

should be done.

Caution! Toxins are highly toxic even in very low amounts. Handle all toxin molecules and

their isolated subunits with extreme care.

Protocol for Thiolation of Antibody with SPDP and Conjugation to an SPDP-Activated Toxin

Caution: toxin molecules are dangerously toxic even in small amounts. Use extreme care in

handling.

Note: In this protocol, for every mg of toxin employed, 2.5 mg of antibody is required to

obtain the correct molar ratios in the ﬁ nal conjugate.

Activation of Toxin with SPDP

1. Dissolve the toxin to be conjugated in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.5,

at a concentration of 10 mg/ml. Some protocols use as an SPDP reaction buffer, 50 mM

sodium borate, 0.3 M NaCl, 0.5 percent n-butanol, pH 9.0. Both buffer systems work

well for the NHS ester modiﬁ cation reaction, although the pH 9 buffer is at the higher

end of effective derivatization with active esters, since the hydrolysis rate is dramatically

increased at this level of alkalinity.

2. Dissolve SPDP (Thermo Fisher) in DMF at a concentration of 3 mg/ml. Add 20 l of this

solution to each ml of the toxin solution. Gently mix to effect dissolution. Retain the

SPDP stock solution for use in the antibody modiﬁ cation step, below.

3. React for 30 minutes at room temperature.

4. Purify the SPDP-activated toxin from excess reagents and reaction by-products by gel

ﬁ ltration using a desalting resin. For the chromatography use 0.1 M sodium phosphate,

0.15 M NaCl, pH 7.5, containing 10 mM EDTA.

5. Concentrate the toxin to 10 mg/ml using a centrifugal concentrator with a MW cutoff of

10,000. Retain this solution for the conjugation reaction.

Thiolation of Antibody with SPDP

1. Dissolve the antibody to be conjugated in 0.1 M sodium phosphate, 0.15 M NaCl, pH

7.5, at a concentration 10 mg/ml. Note: some protocols use the borate buffer system

described in step A. Use 2.5 mg of antibody per mg of toxin to be conjugated.

2. Dissolve SPDP in DMF at a concentration of 3 mg/ml. Add 24 l of this solution to each

ml of the antibody solution with gentle mixing to effect complete dissolution.

3. React for 30 minutes at room temperature.

4. Remove excess crosslinker by gel ﬁ ltration using a desalting resin. Perform the chro-

matography using 0.1 M sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.5. The

buffer should be degassed under vacuum and nitrogen bubbled through it to remove oxy-

gen. The presence of EDTA stabilizes the free sulfhydryls formed in the following steps

against metal-catalyzed oxidation.

5. Concentrate the fractions containing protein from the gel ﬁ ltration step to 10 mg/ml

using a centrifugal concentrator (MW cutoff of 10,000).

838 21. Immunotoxin Conjugation Techniques

6. To reduce the pyridyl dithiol groups and create reactive sulfhydryls, dissolve DTT in water

at a concentration of 17.2 mg/ml and immediately add 500 l of this solution to each ml

of concentrated antibody solution. Mix to dissolve and react for 30 minutes at room

temperature.

7. Remove excess DTT by gel ﬁ ltration using the same buffer as in step 4. Pool the fractions

containing protein and concentrate to 10 mg/ml.

Conjugation of SPDP-Activated Toxin with Thiolated Antibody

1. Immediately mix the concentrated, thiolated antibody solution from part B with the

SPDP-activated toxin from part A.

2. React for 18 hours at room temperature to form the ﬁ nal conjugate. Isolation of the ideal

1:1 or 1:2 antibody–toxin conjugate can be done through gel ﬁ ltration separation using a

column of Sephacryl S-300 or the equivalent.

Protocol for Activation of Antibody with SPDP and Conjugation to a Toxin A Chain

Caution: toxin molecules are dangerously toxic even in small amounts. Use extreme care in

handling.

Since the A chain of toxin molecules contains a free sulfhydryl group, there is no need in this

conjugation strategy to thiolate one of the molecules. The following protocol calls for 1.73 mg

of antibody per mg of toxin A chain to produce a conjugate possessing the correct molar ratio

of components. Best results for creating a highly cytotoxic immunotoxin will be obtained if

deglycosylated ricin A chain is used.

1. Dissolve the antibody to be conjugated in 0.1 M sodium phosphate, 0.15 M NaCl, pH

7.5, at a concentration 10 mg/ml.

2. Dissolve SPDP (Thermo Fisher) in DMF at a concentration of 3.0 mg/ml. Add 30 l of

this solution to each ml of the antibody solution with gentle mixing to effect complete

dissolution.

3. React for 30 minutes at room temperature.

4. Remove excess crosslinker by gel ﬁ ltration using a desalting resin. Perform the chroma-

tography using 0.1 M sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.5.

5. Concentrate the fractions containing SPDP-activated antibody from the gel ﬁ ltration step

to 10 mg/ml using a centrifugal concentrator (MW cutoff of 10,000).

6. Mix the activated antibody solution with a solution of deglycosylated toxin A chain

(dgA) dissolved in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.5, containing 10 mM

EDTA. The ratio of mixing should equal 1.73 mg of antibody per mg of A chain or 580  l

of A-chain solution at 10 mg/ml per ml of activated antibody solution at 10 mg/ml. The

A-chain solution must not contain any reducing agents left over from the disassociation

of the toxin subunits during the A subunit isolation. Reductants will compete for the

SPDP activation sites on the antibody molecule.

7. React for 18 hours at room temperature. Isolation of the 1:1 or 1:2 antibody–toxin con-

jugate can be done through a gel ﬁ ltration separation using a column of Sephacryl S-200.

2. Preparation of Immunotoxin Conjugates 839