Hermanson G. Bioconjugate Techniques, Second Edition

Подождите немного. Документ загружается.

840 21. Immunotoxin Conjugation Techniques

Isolation of conjugates containing molar ratios of 1:2 antibody:dgA have resulted in

greater cytotoxicity behavior in vivo (Ghetie et al., 1993).

Protocol for the Conjugation of SPDP-activated Antibodies with

2-Iminothiolane-Modiﬁ ed Toxins

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

handling.

A third option for immunotoxin preparation is to again activate the antibody with SPDP,

while this time thiolating a single-chain toxin molecule to conjugate with it. This method works

especially well using 2-iminothiolane (Chapter 1, Section 4.1) to create sulfhydryls on gelonin

or PAPs. Gelonin is a single-polypeptide toxin containing no free sulfhydryls. A number of

options are available for thiolation, however the use of SPDP to add sulfhydryl groups inacti-

vates the toxin, while 2-iminothiolane preserves its activity, perhaps by maintaining the posi-

tive charge on the amines that are being modiﬁ ed (Lambert et al ., 1985).

Activation 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 of 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 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).

Thiolation of Gelonin (or Other Single-Polypeptide Toxins) with 2-Iminothiolane

(Traut ’s Reagent)

1. Dissolve gelonin at a concentration of 10 mg/ml in 50 mM triethanolamine hydrochloride,

pH 8.0, containing 10 mM EDTA. The buffer should be degassed under vacuum and bub-

bled with nitrogen to remove oxygen that may cause sulfhydryl oxidation after thiolation.

2. Dissolve 2-iminothiolane (Thermo Fisher) in degassed, nitrogen-bubbled deionized water

at a concentration of 20 mg/ml (makes a 0.14 M stock solution). The solution should be

used immediately. Add 70 l of the 2-iminothiolane solution to each ml of the gelonin

solution (ﬁ nal concentration is about 10 mM).

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

4. Purify the thiolated protein from unreacted Traut ’s reagent by gel ﬁ ltration on a desalting

resin 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

and resultant disulﬁ de formation. The degree of  SH modiﬁ cation in the puriﬁ ed pro-

tein 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. Immediately

use the modiﬁ ed protein in the conjugation reaction to prevent inactivation of

2-iminothiolane-modiﬁ ed molecules by recyclization (Chapter 1, Section 4.1).

Conjugation of SPDP-Activated Antibody with Thiolated Gelonin

1. Mix SPDP-activated antibody with thiolated gelonin in equal mass quantities (or equal

volumes if they are at the same concentration). This ratio results in about a 5-fold molar

excess of toxin over the amount of antibody.

2. React for 20 hours at 4°C under a nitrogen blanket.

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

centration of 2 mM.

4. React for an additional 1 hour at room temperature.

5. Remove unconjugated gelonin by passage of the conjugate solution over a column of

immobilized protein A (Thermo Fisher). Use 2 ml of the protein A column for each 10 mg

of conjugate to be puriﬁ ed. Equilibrate the column with 50 mM sodium phosphate, 0.15 M

NaCl, pH 7.5. Apply the conjugate sample and allow it to enter the gel. Continue to wash

the column with equilibration buffer while taking 2 ml fractions until baseline is reached

(monitored at an absorbance of 280 nm). Unconjugated gelonin will pass through the col-

umn unretarded. Elute bound conjugate with 0.1 M acetic acid, 0.15 M NaCl. Immediately

add 0.1 ml of 1 M potassium phosphate, pH 7.5 to each bound fraction for neutralization.

Alternatively, gel ﬁ ltration may be used to isolate the conjugate from lower-molecular-

weight antibody and gelonin. A column of Sephacryl S-200 works well for this purpose.

SMPT

Succinimidyloxycarbonyl--methyl--(2-pyridyldithio)toluene (SMPT) is a heterobifunctional

crosslinking agent similar to SPDP that contains an amine-reactive NHS ester on one end and a

sulfhydryl-reactive pyridyl disulﬁ de group on the other (Chapter 5, Section 1.2). Reaction with a

sulfhydryl-containing protein results in a cleavable disulﬁ de linkage, important for immunotoxin

activity. SMPT is an analog of SPDP that differs only in its cross-bridge, which contains an aro-

matic ring and a hindered disulﬁ de group (Thorpe et al., 1987; Ghetie et al., 1990). The spacer

arm of SMPT is slightly longer than SPDP, but the presence of the benzene ring and -methyl

group adjacent to the disulﬁ de sterically hinders the structure sufﬁ ciently to provide increased

half-life of immunotoxin conjugates in vivo .

SMPT often is used in place of SPDP for the preparation of immunotoxin conjugates. The hin-

dered disulﬁ de of SMPT has distinct advantages in this regard. Thorpe et al. (1987) showed that

SMPT conjugates had approximately twice the half-life in vivo as SPDP conjugates. Antibody–

toxin conjugates prepared with SMPT possess a half-life in vivo of up to 22 hours, presumably

due to the decreased susceptibility of the hindered disulﬁ de toward reductive cleavage.

Ghetie et al. (1991) developed a large-scale preparation procedure for antibody–deglycosylated

ricin A chain (dgA) conjugates utilizing this crosslinker. The following procedure describes a gen-

eralized method for using SMPT to prepare dgA–antibody conjugates. It is based on the Ghetie

protocol, but using smaller quantities of reagents. Figure 21.8 illustrates the reactions involved in

using SMPT.

Protocol

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

handling.

2. Preparation of Immunotoxin Conjugates 841

842 21. Immunotoxin Conjugation Techniques

The following method calls for mixing activated antibody with ricin A chain at a ratio of

2 mg antibody per mg of A chain. Adjustments to the amount of antibody and A chain initially

dissolved in the reaction buffers should be done to anticipate this ratio.

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

7.5, at a concentration of 10 mg/ml. If the antibody contains oligomers (as evidenced by

nondenaturing electrophoresis or HPLC gel ﬁ ltration analysis), then the monomeric IgG

Figure 21.8 SMPT may be used to form immunotoxin conjugates by activation of the antibody component to

form a thiol-reactive derivative. Reduction of an A–B toxin molecule with DTT can facilitate subsequent isola-

tion of the A chain containing a free thiol. Mixing the A-chain containing a sulfhydryl group with the SMPT-

activated antibody causes immunotoxin formation through disulﬁ de bond linkage. The hindered disulﬁ de of an

SMPT crosslink has been found to survive in vivo for longer periods than conjugates formed with SPDP.

form should be isolated by gel ﬁ ltration using a column of Sephacryl S-200HR. If no oli-

gomers are present, then omit the chromatographic puriﬁ cation.

2. Dissolve SMPT (Thermo Fisher) in DMF at a concentration of 4.8 mg/ml. Add 27 l of

this solution to each ml of the antibody solution. Mix gently. The ﬁ nal concentration of

SMPT in the reaction mixture is 0.13 mg/ml, which translates into about a 4.8-fold molar

excess of crosslinker over the amount of antibody present.

3. React for 1 hour at room temperature.

4. Remove unreacted SMPT and reaction by-products by gel ﬁ ltration on a desalting resin.

Pool fractions containing SMPT-activated antibody (the ﬁ rst peak eluting from the col-

umn) and concentrate them to 10 mg/ml using centrifugal concentrators with a MW cut-

off of 10,000.

5. Dissolve deglycosylated ricin A chain (dgA) in 0.1 M sodium phosphate, 0.15 M NaCl,

10 mM EDTA, pH 7.5, at a concentration of 10 mg/ml. The buffer should be degassed under

vacuum and nitrogen bubbled through it to remove oxygen. Prepare half the amount of

A-chain solution as the amount of antibody prepared in step 1. If the A-chain preparation is

done in bulk quantities or if the protein has been stored for lengthy periods, it may be neces-

sary to reduce the sulfhydryls with DTT prior to proceeding with the crosslinking reaction.

If A-chain sulfhydryl oxidation is suspected, add 2.5 mg of DTT per ml of A-chain solution.

React for 1 hour at room temperature. Purify the reduced ricin A chain by gel ﬁ ltration on

a desalting resin using the PBS-EDTA buffer. Apply no greater volume of sample to the gel

than is represented by 5 percent of the column volume to assure good removal of excess

DTT. Collect the protein and concentrate to 10 mg/ml using centrifugal concentrators.

6. Mix the reduced A-chain solution with activated antibody solution at a ratio of 2 mg of

antibody per mg of A chain. Sterile ﬁ lter the solution through a 0.22 m membrane, and

react at room temperature under nitrogen for 18 hours.

7. To block excess pyridyl disulﬁ de active sites on the antibody, add cysteine to a ﬁ nal con-

centration of 25  g/ml. React for an additional 6 hours at room temperature.

8. To isolate the conjugate, apply the immunotoxin solution to a column of Sephacryl

S-200HR. Collect the peaks with MW between 150,000 and 210,000. Further puriﬁ ca-

tion to remove excess unconjugated antibody can be done on a column of immobilized

Cibacron Blue (available commercially (Thermo Fisher) or for column preparation, see

Hermanson et al., 1992). Equilibration of the column with 50 mM sodium borate, 1 mM

EDTA, pH 9.0, will bind the conjugate, but not free antibody. Elution of puriﬁ ed immu-

notoxin conjugate can be done with 50 mM sodium borate, 1 mM EDTA, 0.5 M NaCl,

pH 9.0 (see Ghetie et al ., 1991).

3-(2-Pyridyldithio) Propionate

A lesser-used reagent to introduce sulfhydryl-reactive pyridyl disulﬁ de groups is 3-(2-

pyridyldithio)propionate (PDTP), the acid precursor of SPDP containing no NHS ester group on

the carboxylate. Sulfhydryl interchange reaction at the pyridyl dithiol end results in the forma-

tion of a disulﬁ de linkage with  SH-containing molecules. The carboxylate end is not further

derivatized to contain a reactive species, but may be coupled to amines by the carbodiimide

reaction (Chapter 3, Section 1). Reaction of PDTP with an antibody molecule in the presence of

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) results in the formation of amide link-

ages with the active pyridyl disulﬁ de groups still available for coupling to sulfhydryl-containing

2. Preparation of Immunotoxin Conjugates 843

844 21. Immunotoxin Conjugation Techniques

toxins ( Figure 21.9 ). Mixing the PDTP–antibody with puriﬁ ed ricin A chain results in disulﬁ de

crosslinks identical to those obtained using SPDP as the crosslinker (Jansen et al., 1980; Gros

et al., 1985). PDTP also has been used to activate transferrin to contain reactive pyridyl dithiol

groups for conjugation to ricin A-chain molecules (Raso and Basala, 1984, 1985).

Since activated molecules and crosslinks formed between two species are identical to those

formed using SPDP, it is of little advantage to use PDTP. Furthermore, an EDC-mediated reaction

of the carboxylate end of the crosslinker with amine groups on proteins can cause concomitant

zero-length crosslinking and polymerization of protein molecules. For these reasons, SPDP is the

better choice for preparing conjugates.

Use of Cystamine, Ellman ’ s Reagent, or S-Sulfonates

Other reagent systems can be used to form disulﬁ de linkages between antibody and toxin

molecules in immunotoxin conjugates. Cystamine can be incorporated into proteins by reac-

tion of its terminal amines with the carboxylates on the proteins via the carbodiimide reaction

(Chapter 3, Section 1). The resultant modiﬁ cations contain disulﬁ de linkages that can undergo

disulﬁ de interchange reactions with other sulfhydryl-containing molecules (Chapter 1, Section

4.1). For instance, a cystamine-modiﬁ ed-targeting component, such as an antibody, can be

mixed with the reduced A chain of a toxin molecule to cause conjugate formation through the

creation of a disulﬁ de bond ( Figure 21.10 ) (Oeltmann and Forbes, 1981). Epidermal growth

factor was modiﬁ ed with cystamine and coupled with reduced diphtheria toxin using this

approach (Shimisu et al ., 1980).

Similarly, Ellman ’s reagent [5,5 -dithiobis(2-nitrobenzoic acid)] can be used to activate one

thiol-containing molecule by disulﬁ de exchange and subsequently used to couple to a second

sulfhydryl-containing molecule by the same mechanism (Chapter 1, Section 5.2) ( Figure 21.11 ).

The disulﬁ de of Ellman ’s reagent readily undergoes disulﬁ de exchange with a free sulfhydryl to

form a mixed disulﬁ de with simultaneous release of one molecule of the highly chromogenic

5-sulﬁ do-2-nitrobenzoate, also called 5-thio-2-nitrobenzoic acid (TNB). The intense yellow color

produced by the TNB anion can be measured by its absorbance at 412 nm. Thus, the efﬁ ciency

of conjugation can be determined spectrophotometrically using this procedure (Pirker et al.,

Figure 21.9 PDTP may be used to modify antibody molecules using a carbodiimide reaction with EDC. The

derivative is the same as that obtained using SPDP activation and is highly reactive toward sulfhydryls.

1986; Fitzgerald et al., 1988). A method for the large-scale conjugation of Fab  fragments con-

taining an available sulfhydryl group and deglycosylated ricin A chain (also containing an  SH

group) were developed using Ellman ’s reagent as the crosslinker (Ghetie et al., 1988).

A ﬁ nal method of forming disulﬁ de crosslinks between toxins and targeting molecules is the

use of S-sulfonate formation using sodium sulﬁ te (Na

) in the presence of sodium tetrathion-

ate (Na

). Tetrathionate reacts with sulfhydryls to form sulfenylthiosulfate intermediates

(section 1.1.5.2). These derivatives are reactive toward other thiols to create disulﬁ de linkages

Figure 21.10 Cystamine may be used to make immunotoxin conjugates by a disulﬁ de interchange reaction.

Modiﬁ cation of antibody molecules using an EDC-mediated reaction creates a sulfhydryl-reactive derivative. A-

chain toxin subunits containing a free thiol can be coupled to the cystamine-modiﬁ ed antibody to form disulﬁ de

crosslinks.

2. Preparation of Immunotoxin Conjugates 845

846 21. Immunotoxin Conjugation Techniques

rapidly. Sulﬁ te ions react with disulﬁ des to form S-substituted thiosulfates, also known as

S-sulfonates, and a thiol. The combination of these reagents results in the transformation of

available thiols and disulﬁ des into reactive S-sulfonates that can be used to crosslink with sulf-

hydryl-containing molecules. S-sulfonate conjugation can be used to conjugate the A chain of

toxin molecules with sulfhydryl-containing Fab  fragments with good efﬁ ciency (Masuho et al.,

1979). Although the use of these alternative disulﬁ de generating agents has proven successful in

some applications, pyridyl disulﬁ de-containing crosslinkers, as discussed previously, are more

common for producing immunotoxin conjugates.

Figure 21.11 Fab  antibody fragments containing free thiols can be activated with Ellman ’s reagent to form a

sulfhydryl-reactive derivative. A-chain toxin subunits containing a free thiol group may be coupled to the acti-

vated Fab  molecule to produce an immunotoxin complex.

2.2. Preparation of Immunotoxin Conjugates via Amine- and Sulfhydryl-Reactive

Heterobifunctional Crosslinkers

Other forms of heterobifunctional crosslinkers that can be used for this purpose are the amine-

and sulfhydryl-reactive agents that produce a thioether bond with  SH-containing molecules

(Chapter 5, Section 1). The amine-reactive end of these crosslinkers is usually an NHS ester

group that can form a stable amide bond with amine-containing proteins. One of two main

reactive groups usually are used on the sulfhydryl-reactive end: an iodoacetyl group which cou-

ples to sulfhydryls with loss of HI or a maleimide group which undergoes a double bond addi-

tion reaction with  SH groups (see Chapter 2, Sections 2.1 and 2.2).

Since this type of crosslinker forms non-cleavable thioether bonds between toxin molecules

and the targeting component of the conjugates, they are not appropriate for use with A-chain

or single-chain toxins. This is because the crosslinker will not allow the conjugated A chain

to break free of the antibody by disulﬁ de reduction after docking at the cellular target. Since

release of the A chain is a prerequisite to ribosomal inactivation, such conjugates will prove to

be ineffective cytotoxic agents. One report found a 1,000-fold increase in cytotoxicity when

an immunotoxin-containing PAP or gelonin was prepared using a cleavable disulﬁ de linker as

opposed to a non-cleavable thioether linkage (Lambert et al ., 1985).

To make effective immunotoxin conjugates using the following crosslinkers, it is necessary

to crosslink intact A–B toxins to antibodies, not single chain or A-chain toxins. Using intact

two-subunit toxins allows the A chain to break free of the complex and perform its cytotoxic

duties upon entering the target cell. Two main criteria are especially important when using A–B

toxin conjugates: the B-chain binding site must be blocked or inoperative in the ﬁ nal immuno-

toxin complex to prevent nonspeciﬁ c cell death, and secondly, the two subunits of the toxin

must not be covalently crosslinked by the conjugation procedure, precluding them from being

separated in vivo .

Fortunately, satisfying these criteria is not difﬁ cult. The heterobifunctional crosslinkers

described in this section are sufﬁ ciently controllable as to prevent A–B chain crosslinking. In

addition, during the conjugation process, the B-chain binding site often becomes inactivated

or physically blocked by the attached antibody molecule. Subsequent cleanup of the conjugate

using afﬁ nity chromatography over a column containing an immobilized sugar can completely

eliminate any potential nonspeciﬁ city contributed by the B chain in the ﬁ nal preparation.

It should be noted that the use of the following crosslinkers to create other forms of toxic con-

jugates for cancer therapy is not restricted by the disulﬁ de bridge requirement (Trail et al., 1993;

Willner et al., 1993). Drug–toxin conjugates, hormonotoxins, lymphokine– or growth factor–

toxin conjugates all can be made using nonreversible thioether linkages without difﬁ culty.

SIAB

N-Succinimidyl(4-iodoacetyl)aminobenzoate (SIAB) is a heterobifunctional crosslinker contain-

ing amine-reactive and sulfhydryl-reactive ends (Chapter 5, Section 1.5). The NHS ester on one

end of the reagent can be used to couple with primary amine-containing molecules, forming

stable amide linkages (Chapter 2, Section 1.4). The other end contains an iodoacetyl group

that is speciﬁ c for coupling to sulfhydryl residues, potentially creating stable thioether bonds

(Chapter 2, Section 2.1).

2. Preparation of Immunotoxin Conjugates 847

848 21. Immunotoxin Conjugation Techniques

Conjugations using SIAB to create immunotoxins can be done by ﬁ rst reacting the NHS

ester end of the crosslinker with available amine groups on the antibody and then coupling

to a thiolated toxin dimer—or by ﬁ rst reacting it with the toxin and coupling to a thiolated

antibody ( Figure 21.12 ). Thiolation of the secondary component is usually done with SPDP

or 2-iminothiolane. Other crosslinkers containing an iodoacetyl group can be used in a similar

fashion.

Conjugations with iodoacetyl crosslinkers have been done using ricin and CVF (Thorpe et al.,

1984; Vogel, 1987; Myers et al., 1989). The following generalized protocol for using SIAB is

based on the method of Cumber et al. (1985).

Protocol

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

handling.

Figure 21.12 SIAB can be used to activate toxin molecules for coupling with sulfhydryl-containing antibod-

ies. In this case, the antibody molecule is thiolated using SATA and deprotected to reveal the free sulfhydryl.

Reaction with the SIAB-activated toxin forms the ﬁ nal conjugate by thioether bond formation.

To prepare an antibody–ricin conjugate using this protocol, 2.25 mg of antibody is needed

for every mg of toxin.

Activation of Toxin with SIAB

1. Dissolve intact ricin in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.5, at a concentra-

tion of 10 mg/ml.

2. Dissolve SIAB (Thermo Fisher) in DMSO at a concentration of 1.4 mg/ml. Prepare fresh

and protect from light to avoid breakdown of the active halogen group.

3. Add 160  l (225  g) of the SIAB solution to each ml of the ricin solution.

4. React for 30 minutes at room temperature in the dark.

5. Remove excess crosslinker from the activated ricin by gel ﬁ ltration using a desalting resin.

6. Concentrate the puriﬁ ed, SIAB-activated toxin to 10 mg/ml using centrifugal concentra-

tors with a MW cutoff of 10,000. Protect the activated toxin from light to prevent degra-

dation of the iodoacetyl-reactive group.

Thiolation of Speciﬁ c Antibody Molecule 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. Use 2.25 mg of antibody per mg of toxin to be conjugated.

2. Dissolve SPDP (Thermo Fisher) 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 column of desalting resin. Perform the

chromatography 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

oxygen. 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).

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

(Thermo Fisher) 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 SIAB-Activated Toxin with Thiolated Antibody

1. Mix activated toxin from part A with thiolated antibody from part B 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 in the dark.

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 or 1:2 antibody–toxin conjugate can be done by gel ﬁ ltration

separation using a column of Sephacryl S-300.

2. Preparation of Immunotoxin Conjugates 849