Hermanson G. Bioconjugate Techniques, Second Edition

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850 21. Immunotoxin Conjugation Techniques

SMCC

Succinimidyl-4-( N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) is a crosslinker with

signiﬁ cant utility in protein conjugation (Chapter 5, Section 1.3). It is a popular choice among

heterobifunctional reagents, especially for the preparation of antibody–enzyme and hapten–

carrier conjugates (Hashida and Ishikawa, 1985; Dewey et al., 1987). The NHS ester end of

the reagent can react with primary amine groups on proteins to form stable amide bonds. The

maleimide end of SMCC is speciﬁ c for coupling to sulfhydryls when the reaction pH is in the

range of 6.5–7.5 (Smyth et al., 1964). The nature of the reactive groups of SMCC allow for

highly controlled crosslinking procedures to be performed wherein the resulting products can

be closely limited to a 1:1 ratio in the ﬁ nal complex. Thus, low-molecular-weight conjugates

can be made which make ideal reagents for in vivo purposes.

However, since SMCC forms nonreversible thioether linkages with sulfhydryl groups, it

only can be used in the preparation of immunotoxins if intact A–B toxins are employed in the

conjugate. In such conjugates, the A chain still have the potential for reductive release from

the B-chain subunit after cellular docking and internalization. Immunotoxins prepared with

A-chain or single-subunit toxins will not display cytotoxicity if crosslinked with SMCC, since

the crosslinker does not create cleavable disulﬁ de bonds upon conjugation.

SMCC has been used to prepare immunotoxins with CVF (Vogel, 1987) and was compared

to other crosslinkers in the preparation of gelonin and PAP conjugates (Lambert et al ., 1985).

The following protocol is a suggested method for the conjugation of SMCC-activated anti-

bodies with 2-iminothiolane-modiﬁ ed, intact toxin molecules ( Figure 21.13 ). It utilizes the

water-soluble analog of SMCC, sulfo-SMCC, which contains a negatively charged sulfonate

group on its NHS ring.

Protocol

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

handling.

Note: This protocol requires mixing activated antibody with thiolated toxin at a ratio of

2.25 mg of antibody per mg of toxin. This ratio should be taken into account before starting

the reactions.

Activation of Antibody with Sulfo-SMCC

1. Dissolve 10 mg of speciﬁ c antibody in 1 ml of 0.1 M sodium phosphate, 0.15 M NaCl,

pH 7.2.

2. Weigh out 2 mg of sulfo-SMCC (Thermo Fisher) and add it to the above solution. Mix

gently to dissolve. To aid in measuring the exact quantity of crosslinker, a concentrated

stock solution may be made in water and an aliquot equal to 2 mg transferred to the reac-

tion solution. If a stock solution is made, it should be dissolved rapidly and used immedi-

ately to prevent extensive hydrolysis of the active ester. Alternatively, a stock solution of

sulfo-SMCC may be prepared in DMSO and an aliquot added to the aqueous reaction.

3. React for 1 hour at room temperature.

4. Immediately purify the maleimide-activated protein by applying the reaction mixture to a

desalting column. Do not dialyze the solution, since the maleimide activity will be lost over

the time course required to complete the operation. To obtain good separation between

the protein peak (eluting ﬁ rst) and the peak representing excess reagent and reaction by-

products (eluting second), the applied sample size should be no more than 5–8 percent of

the column bed volume.

5. Collect the peak containing the activated antibody (eluting ﬁ rst) and concentrate to 10 mg/ml

using centrifugal concentrators. Use immediately for conjugating to a thiolated toxin.

Thiolation of Intact A–B toxin

1. Dissolve the toxin (e.g., intact ricin) at a concentration of 10 mg/ml in 50 mM trieth-

anolamine hydrochloride, pH 8.0, containing 10 mM EDTA. The buffer should be

Figure 21.13 Sulfo-SMCC may be used to activate antibody molecules for coupling to thiolated toxin compo-

nents. An intact A–B toxin molecule can be modiﬁ ed to contain sulfhydryls by treatment with 2-iminothiolane.

Thiolation with this reagent retains the cytotoxic properties of the toxin while generating a sulfhydryl for conju-

gation. Reaction of the thiolated toxin with the maleimide-activated antibody creates the immunotoxin through

thioether bond formation.

2. Preparation of Immunotoxin Conjugates 851

852 21. Immunotoxin Conjugation Techniques

degassed under vacuum and bubbled with nitrogen to remove oxygen that may cause

sulfhydryl oxidation after thiolation.

2. Dissolve 2-iminothiolane in degassed, nitrogen-bubbled deionized water at a concentra-

tion of 20 mg/ml (makes a 0.14 M stock solution). The solution should be used imme-

diately. Add 70 l of the 2-iminothiolane solution to each ml of the toxin solution (ﬁ nal

concentration is about 10 mM).

3. React for 1 hour at 0°C (on ice) under a nitrogen blanket.

4. Purify the thiolated toxin from unreacted Traut ’s reagent by gel ﬁ ltration using 0.1 M

sodium phosphate, 0.15 M NaCl, pH 7.5, containing 10 mM EDTA. The presence of

EDTA in this buffer helps to prevent oxidation of the sulfhydryl groups with resultant

disulﬁ de formation. The degree of  SH modiﬁ cation in the puriﬁ ed protein may be

determined using the Ellman ’s assay (Chapter 1, Section 4.1).

5. Concentrate the thiolated toxin to 10 mg/ml using centrifugal concentrators.

Conjugation of SMCC-Activated Antibody with Thiolated Toxin

1. Mix the thiolated toxin with SMCC-activated antibody at a ratio of 2.25 mg of antibody

per mg of toxin. Protect the solution from light.

2. React for 18 hours at room temperature.

3. To block unreacted sulfhydryl groups, add iodoacetamide to the solution to a ﬁ nal con-

centration of 2 mM. React for an additional 1 hour at room temperature.

4. Isolation of the ideal 1:1 antibody–toxin conjugate can be done by gel ﬁ ltration separa-

tion using a column of Sephacryl S-300.

5. Removal of nonspeciﬁ c binding potential in the B chain must be done before using an

A–B intact toxin conjugate in vivo. A large proportion of the binding sites on the B

chains usually are blocked during the above conjugation process, and the galactose bind-

ing potential is signiﬁ cantly impaired. Further puriﬁ cation to remove conjugates that

have galactose binding potential can be done on an acid-treated agarose chromatogra-

phy column (which contains galactose residues) or on a column of asialofetuin bound to

agarose (Cumber et al., 1985). Conjugate fractions that do not bind to both afﬁ nity gels

contain no nonspeciﬁ c binding potential toward non-targeted cells.

MBS

m -Maleimidobenzoyl- N-hydroxysuccinimide ester (MBS) is a heterobifunctional crosslinking

agent containing an NHS ester and a maleimide group. The NHS ester can react with primary

amines in proteins and other molecules to form stable amide bonds, while the maleimide end

reacts with sulfhydryl groups to create stable thioether linkages (Chapter 5, Section 1.4). The

reagent can be used in many different conjugation protocols to crosslink amine-containing pro-

teins with sulfhydryl-containing proteins. Since the thioether bond formed at the maleimide end

is nonreversible, MBS can be used for immunotoxin preparation only if the conjugate involves

crosslinking intact A–B toxins with antibody molecules. Using intact toxins (as opposed to

single-chain or A-chain isolates), the A chain still is able to release from the complex after cel-

lular docking and inactivate ribosomal activity (Youle and Nevelle, 1980; Dell ’Arciprete et al .,

1988; Myers et al ., 1989).

MBS contains a benzoic acid derivative as its cross-bridge. In many applications involving

NHS–maleimide crosslinkers, non-aromatic cross-bridges are considered superior to aromatic

ones. This is reﬂ ected in the stability of the maleimide group to hydrolysis prior to conjugating

with a sulfhydryl group. For immunotoxin preparation, however, aromatic maleimides resulted

in better conjugate yield and more potent cytotoxic effects when compared to aliphatic ones

(Myers et al., 1989). MBS, therefore, may be a crosslinker of choice when making conjugates

with intact toxin molecules.

The following protocol is adapted from Myers et al. (1989). It involves activation of ricin

with MBS and conjugation with a partially reduced antibody ( Figure 21.14 ).

Protocol

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

handling.

Figure 21.14 Activation of an intact A–B toxin molecule with MBS with subsequent conjugation with a

reduced antibody fragment to produce an immunotoxin.

2. Preparation of Immunotoxin Conjugates 853

854 21. Immunotoxin Conjugation Techniques

This method uses a molar ratio of 15:1 for ricin:antibody. This requires 6.24 mg of ricin per

mg of antibody. This ratio should be considered when determining how much starting materi-

als to use for each step.

Activation of Ricin with MBS

1. Dissolve ricin at a concentration of 10 mg/ml in 0.1 M sodium phosphate, 0.15 M NaCl,

pH 7.5.

2. Dissolve MBS (Thermo Fisher) in DMF at a concentration of 2 mg/ml.

3. Add 76 l of the MBS solution to each ml of the ricin solution. This represents a 3:1

molar ratio of crosslinker to protein.

4. React for 30 minutes at room temperature.

5. Immediately purify the MBS-activated toxin by gel ﬁ ltration using a column of desalting

resin. Apply no more sample than represents 5–8 percent of the gel volume. Isolate the

protein peak by its absorbance at 280 nm and concentrate to 10 mg/ml using centrifugal

concentrators with a MW cutoff of 10,000.

Partial Reduction of Antibody with DTT

1. Dissolve the antibody in 0.1 M sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.5,

at a concentration of 10 mg/ml.

2. Add DTT to a ﬁ nal concentration of 50 mM.

3. Reduce for 30 minutes at room temperature.

4. Purify the reduced antibody using gel ﬁ ltration on a column of Sephadex G-25.

Concentrate the protein to 10 mg/ml using centrifugal concentrators.

Conjugation of MBS-Activated Ricin with Partially Reduced Antibody

1. Mix the MBS-activated ricin with the partially reduced antibody in a molar ratio of

15:1 (or 6.24 mg activated ricin per mg of reduced antibody). This represents a volume

ratio (at 10 mg/ml for both proteins) of 1 ml ricin solution mixed with 160 l antibody

solution.

2. React for 18 hours at room temperature.

3. Puriﬁ cation of the immunotoxin conjugate from unconjugated ricin can be done using

a column of TSK3000 SW (Toya Soda, Japan) according to the method of Myers et al .

(1989).

4. Removal of nonspeciﬁ c binding potential in the B chain must be done before using an

A–B intact toxin conjugate in vivo. See step 5 of the MBS conjugation protocol discussed

previous to this section.

SMPB

Succinimidyl-4-( p-maleimidophenyl)butyrate (SMPB), is a heterobifunctional analog of MBS

containing an extended cross-bridge (Chapter 5, Section 1.6). The crosslinker has an amine-

reactive NHS ester on one end and a sulfhydryl-reactive maleimide group on the other.

Conjugates formed using SMPB thus are linked by stable amide and thioether bonds.

As in the case of MBS, discussed previously, SMPB was found to be more effective than

aliphatic crosslinkers in producing immunotoxin conjugates with ricin that have high yields of

cytotoxicity (Myers et al., 1989). This was attributed to the reagent ’s aromatic ring structure.

A comparison with SPDP produced immunotoxin conjugates concluded that SMPB formed more

stable complexes that survive in serum for longer periods (Martin and Papahadjopoulos, 1982).

The method for the preparation of immunotoxins with SMPB is identical to that used for

MBS (above). Since the thioether bonds formed with sulfhydryl-containing molecules are non-

cleavable, A-chain isolates or single-chain toxin molecules can not be conjugated with anti-

bodies with retention of cytotoxicity. Only intact A–B toxin molecules may be used with this

crosslinker, since the A chain still is capable of being reductively released from the complex.

2.3. Preparation of Immunotoxin Conjugates via Reductive Amination

Conjugations involving aldehyde groups and amine-containing molecules can be done through

Schiff base formation with subsequent reduction using sodium cyanoborohydride to form sta-

ble secondary amine linkages (Chapter 2, Section 5.3). Carbohydrates, glycoproteins, and other

polysaccharide-containing molecules can be oxidized to contain aldehyde residues by sodium peri-

odate or speciﬁ c oxidases (Chapter 1, Section 4.4). Some antibodies and toxin molecules are glyc-

oproteins and contain sufﬁ cient carbohydrate to be utilized for reductive amination crosslinking.

A second method of immunotoxin preparation by reductive amination involves the use a

polysaccharide spacer. Soluble dextran may be oxidized with periodate to form a multifunctional

crosslinking polymer. Reaction with antibodies and cytotoxic molecules in the presence of a reduc-

ing agent forms multivalent immunotoxin conjugates. The following sections discuss these options.

Periodate Oxidation of Glycoproteins Followed by Reductive Conjugation

Antibody molecules usually contain carbohydrate in their Fc regions. Similarly, many toxins,

such as ricin and abrin, are glycoproteins that contain abundant polysaccharide. These car-

bohydrate residues can be oxidized with 10 mM sodium periodate to form reactive aldehyde

groups capable of being conjugated with primary amines (Chapter 1, Section 4.4). Mixing an

aldehyde-containing glycoprotein with another amine-containing molecule in the presence of

sodium borohydride or sodium cyanoborohydride reduces the intermediate Schiff bases that

are formed to stable secondary amine bonds. Since functional groups on the antibody and the

toxin components are the only ones necessary for this type of conjugation strategy, it is often

referred to as a zero-length crosslinking procedure (Chapter 3). In other words, no additional

crosslinking reagents are introduced into the site of the crosslink. This method of conjugation

is used with great success in the formation of antibody–enzyme conjugates, especially using the

glycosylated enzyme, horseradish peroxidase (HRP) (Chapter 26, Section 1.1).

The disadvantage of this type of conjugation approach for producing immunotoxins is that

many of the monoclonal antibodies or antibody fragments used for immunotoxin conjugation

are devoid of carbohydrate. Especially when using small Fv fragments or single-chain anti-

bodies produced by recombinant techniques, there are typically no polysaccharide portions

attached to them. In this case, creation of aldehydes on the targeting component is not possi-

ble. In addition, not all toxin molecules contain carbohydrate. Ricin, abrin, and CVF are glyc-

oproteins and can be oxidized and coupled to antibodies without difﬁ culty (Olsnes and Pihl,

2. Preparation of Immunotoxin Conjugates 855

856 21. Immunotoxin Conjugation Techniques

Figure 21.15

A periodate-oxidized dextran polymer may be reacted with both an antibody and an intact toxin

component using reductive amination to form a multivalent immunotoxin complex.

1982a, b; Vogel and Muller-Eberhard, 1984). However, it is not well known if immunotoxin

conjugates formed by this procedure retain their ability to inhibit ribosomal activity.

Suggested procedures for using reductive amination techniques may be found in Chapter 1,

Section 4.4 and Chapter 3, Section 4.

Periodate-Oxidized Dextran as Crosslinking Agent

Dextran polymers consist of glucose residues bound together predominantly in -1,6 link-

ages. The main repeating unit is an isomaltose group. Most preparations of dextran contain

some branching, mainly incorporating 1,2, 1,3, and 1,4 linkages. The degree of branching is

characteristic of its source—the strain and species of yeast or bacteria from which the dextran

originated. The terminating monosaccharide in a dextran polymer is often a fructose group.

Dextrans of MW 10,000–40,000 provide long, hydrophilic arms that can accommodate multi-

ple attachment points for macromolecules along their length. Soluble dextrans can be oxidized

in aqueous solution to create numerous aldehyde residues suitable for use in reductive amina-

tion techniques (Hurwitz et al., 1978, 1985; Manabe et al., 1983; Sela and Hurwitz, 1987).

Periodate oxidation results in the cleavage of the carbon–carbon bonds between the No. 2 and

3 carbons within each monosaccharide unit of the chain, transforming the associated hydroxyl

groups into aldehydes (Chapter 1, Section 4.4).

Periodate-oxidized dextran can be used as a protein modiﬁ cation or crosslinking agent (Chapter

25, Section 2). Conjugation of antibody molecules to toxins can be done with dextran to produce

immunotoxins suitable for in vivo administration. Mixing of the antibody and toxin together with

the oxidized dextran under alkaline conditions results in the formation of Schiff base interactions

with the amines on both proteins. Reduction of these Schiff base linkages with sodium borohy-

dride or sodium cyanoborohydride results in stable amide bonds, covalently attaching multiple

antibody and toxin molecules along the length of the polysaccharide chain ( Figure 21.15 ).

Chemoimmunoconjugates consisting of drugs attached to antibody-targeting molecules also

can be formed using oxidized dextran carriers. Cancer therapeutic agents such as adriamy-

cin, bleomycin, and daunomycin can be coupled to the oxidized dextran through their amine

groups. After formation of Schiff base linkages between these drugs and the carrier, the anti-

body is added and a reducing agent used to create the ﬁ nal amide bond linkages (Sela and

Hurwitz, 1987). The dextran backbone provides many more drug molecules associated with

each antibody than could be accomplished by direct conjugation to the antibody itself.

Although dextran can be a versatile crosslinking agent for the preparation of many forms of

macromolecular conjugates, immunotoxin conjugation may be impeded by the nonreversibility

of the multiple amide bond linkages formed during reductive amination. Certainly, only intact

A–B toxins have a chance of succeeding with this method, since A-chain or single-subunit

toxins would not be capable of release from the complex after cellular docking. Even intact

two-subunit toxins, however, may not be capable of releasing an A-chain unit, due to the mul-

tivalent nature of the oxidized dextran linker. For this reason, activated dextran may be more

useful for constructing antibody conjugates consisting of some cytotoxic component other than

protein toxins—for example, drug, hormone, or radioactive complexes.

Methods for using oxidized dextran, including reductive amination techniques, can be

found in Chapter 1, Section 4.4, Chapter 3, Section 4, and especially Chapter 25, Section

2). Reference also should be made to the use of dendrimers as carriers for making cytotoxic-

targeting complexes (Chapter 7).

2. Preparation of Immunotoxin Conjugates 857

858

A fast growing ﬁ eld that heavily depends on bioconjugate technology involves the use of

liposomes. At one time, liposomes were studied only for their interesting structural character-

istics in solution. Their physicochemical properties were investigated extensively as models of

membrane morphology. Today, they are being put to use as macromolecular carriers for nearly

every application of bioconjugate chemistry imaginable. They are used as delivery devices to

encapsulate cosmetics, drugs, ﬂ uorescent detection reagents, and as vehicles to transport nucleic

acids, peptides, and proteins to cellular sites in vivo. Targeting components such as antibod-

ies can be attached to liposomal surfaces and used to create large antigen-speciﬁ c complexes.

In this sense, liposomal derivatives are being used to target cancer cells in vivo, to enhance

detectability in immunoassay systems, and as multivalent cross-bridges in avidin–biotin-based

assays. Covalent attachment of antigens to the surface of liposomes provides excellent immu-

nogen complexes for the generation of speciﬁ c antibodies or as vaccine carriers to elicit protect-

ive immunity.

The end-products of liposome technology are used in retail markets, for the diagnosis of

disease, as therapeutic agents, as vaccines, and as important components in assays designed to

either detect or quantify certain analytes.

The following sections discuss the properties and applications of liposome technology as

well as the most common methods of preparing conjugates of them with proteins and other

molecules.

1. Properties and Use of Liposomes

1.1. Liposome Morphology

Liposomes are artiﬁ cial structures primarily composed of phospholipid bilayers exhibiting

amphiphilic properties. Other molecules, such as cholesterol or fatty acids also may be included

in the bilayer construction. In complex liposome morphologies, concentric spheres or sheets of

lipid bilayers are usually separated by aqueous regions that are sequestered or compartmen-

talized from the surrounding solution. The phospholipid constituents of liposomes consist of

hydrophobic lipid “tails” connected to a “head” constructed of various glycerylphosphate

Preparation of Liposome Conjugates and Derivatives

derivatives. The hydrophobic interaction between the fatty acid tails is the primary driving

force for creating liposomal bilayers in aqueous solution.

However, the organization of liposomes in aqueous solution may be highly complicated. The

nature of the lipid constituents, the composition of the medium, and the temperature of the solu-

tion all affect the association and morphology of liposomal construction. Small “ monomers ” or

groupings of lipid molecules may assemble to create larger structures having several main forms

(Figure 22.1 ). Aggregation of these monomers may fuse them into spherical micelles, wherein

the polar head groups are all facing outward toward the surrounding aqueous medium and the

hydrophobic tails are all pointing inward, excluding water. In addition, aggregation may result

in bilayer construction. In this case, sheets of lipid molecules, all with their head groups fac-

ing one direction and their tails facing the other way, are fused with another lipid sheet having

their tails and heads facing the opposite direction. Thus, the inside of the bilayer contains only

hydrophobic tails from both sheets, while the outside contains the hydrophilic heads facing the

outer aqueous environment.

Figure 22.1 The amphiphilic nature of phospholipids in solution drives the formation of complex structures.

Spherical micelles may form in aqueous solution, wherein the hydrophilic head groups all point out toward the

surrounding water environment and the hydrophobic tails point inward to the exclusion of water. Larger lipid

bilayers may form by similar forces, creating sheets, spheres, and other highly complex morphologies. In non-

aqueous solution, inverted micelles may form, wherein the tails all point toward the outer hydrophobic region

and the heads point inward forming hexagonal shapes.

1. Properities and Use of Liposomes 859