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4 Characterization of Conjugation Methods

4 Characterization of Conjugation Methods

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Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation

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fluorescence immunoassay for monitoring opiate drugs, Anal. Bioanal. Chem., 392, 215, 2008.

139. Tanaka, H., Yan, S., Miura, N., and Shoyama, Y., Preparation of anti-2,4-dichlorophenol and 2,4-dichlorophenoxyacetic acid monoclonal antibodies, Cytotechnology, 42, 101, 2003.

140. Shinkaruk, S., Lamothe, V., Schmitter, J. M., Fructus, A., Sauvant, P., Vergne, S., Degueil, M., Babin, P.,

Bennetau, B., and Bennetau-Pelissero, C., Synthesis of haptens and conjugates for ELISA of glycitein:

Development and validation of an immunological test, J. Agric. Food Chem., 56, 6809, 2008.

141. Shinkaruk, S., Bennetau, B., Babin, P., Schmitter, J. M., Lamothe, V., Bennetau-Pelissero, C., and Urdaci,

M. C., Original preparation of conjugates for antibody production against Amicoumacin-related antimicrobial agents, Bioorg. Med. Chem., 16, 9383, 2008.

142. Sathe, M., Derveni, M., Allen, M., and Cullen, D. C., Use of polystyrene-supported 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydroquinoline for the preparation of a hapten-protein conjugate for antibody development, Bioorg. Med. Chem. Lett., 20, 1792, 2010.

143. Hegde, V. L. and Venkatesh, Y. P., Generation of antibodies specific to D-mannitol, a unique haptenic

allergen, using reductively aminated D-mannose-bovine serum albumin conjugate as the immunogen,

Immunobiology, 212, 119, 2007.

144. Sreenath, K. and Venkatesh, Y. P., Reductively aminated D-xylose-albumin conjugate as the immunogen

for generation of IgG and IgE antibodies specific to D-xylitol, a haptenic allergen, Bioconjug. Chem., 18,

1995, 2007.

145. Bergeron, R. J., Bharti, N., Singh, S., McManis, J. S., Wiegand, J., and Green, L. G., Vibriobactin antibodies: A vaccine strategy, J. Med. Chem., 52, 3801, 2009.

146. Shim, J. Y., Kim, Y. A., Lee, E. H., Lee, Y. T., and Lee, H. S., Development of enzyme-linked immunosorbent assays for the organophosphorus insecticide EPN, J. Agric. Food Chem., 56, 11551, 2008.

147. Lee, J. K., Ahn, K. C., Stoutamire, D. W., Gee, S. J., and Hammock, B. D., Development of an enzymelinked immunosorbent assay for the detection of the organophosphorus insecticide acephate, J. Agric.

Food Chem., 51, 3695, 2003.

148. Krämer, P. M., Forster, S., and Kremmer, E., Enzyme-linked immunosorbent assays for the sensitive

analysis of 2,4-dinitroaniline and 2,6-dinitroaniline in water and soil, Anal. Bioanal. Chem., 391, 1821,


149. Lee, J. K., Park, S. H., Lee, E. Y., Kim. Y. J., and Kyung, K. S., Development of an enzyme-linked

immunosorbent assay for the detection of the fungicide fenarimol, J. Agric. Food Chem., 52, 7206,


150. Ramón-Azcón, J., Sánchez-Baeza, F., Sanvicens, N., and Marco, M.-P., Development of an enzymelinked immunosorbent assay for determination of the miticide bromopropylate, J. Agric. Food Chem., 57,

375, 2009.

151. Ahn, K. C., Watanabe, T., Gee, S. J., and Hammock, B. D., Hapten and antibody production for a sensitive immunoassay determining a human urinary metabolite of the pyrethroid insecticide permethrin,

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154. Montoya, A. and Castell, J. V., Long-term storage of peroxidase-labelled immunoglobulins for use in

enzyme immunoassay, J. Immunol. Methods, 99, 13, 1987.


Application of Chemical

Conjugation for the

Preparation of Immunotoxins

and Other Drug Conjugates

for Targeting Therapeutics


Drug conjugates developed as a result of systemic pharmacotherapy are target-specific cytotoxic

agents.1 The underlying concept involves coupling a therapeutic agent to a delivery molecule with

specificity for a defined target-cell population. Antibodies with high affinity for antigens are natural choices for targeting agents.2–5 With the availability of high-affinity monoclonal antibodies

(mAbs) and their fragments, the prospects of antibody-targeting therapeutics have become promising. Toxic substances that have been conjugated to mAbs include protein toxins, low-molecularweight drugs, biological response modifiers, and radionuclides.6 Antibody–toxin conjugates are

frequently termed immunotoxins, whereas immunoconjugates consisting of antibodies and lowmolecular-weight drugs such as methotrexate and adriamycin are called chemoimmunoconjugates.

Immunomodulators contain biological response modifiers that are known to have regulatory functions such as lymphokines, growth factors, and complement-activating cobra venom factor (CVF).

Radioimmunoconjugates consist of radioactive isotopes, which may be used as therapeutics to kill

cells with radiation or used for imaging.

In addition to antibodies, other molecules that have specific receptors or binding sites on target

cells have also been used as targeting agents. These include transferrin, α2-macroglobulin, epidermal growth factor (EGF), and hormones. When hormones are used as the targeting agents, the term

hormonotoxin is frequently used. Toxins have also been conjugated to antigens to selectively kill

antigen-responsive B cells.7

The coupling of targeting agents with toxic moieties is most commonly performed with heterobifunctional cross-linking reagents. Earlier attempts to conjugate toxins and antibodies by homobifunctional reagents generated nonspecific cross-linking products. More specific and efficient

cross-linking techniques have been developed.8 This chapter will review the procedures that have

been used to prepare immunotoxins and other cytotoxic drug conjugates. Theoretically, a crosslinking agent used to couple one toxin is applicable to other toxins. In fact, such is the case for most

of the toxins described.


13.2.1  Choice of Targeting Agents

In almost all cases, the cytotoxic chemotherapeutic agents, such as paclitaxel, cisplatin, and doxorubicin (DOX), cannot distinguish cancer cells from normal cells. Consequently, in the development or choice of tumor-specific delivery systems for anticancer agents, recognizing the intrinsic



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation

differences between normal and tumor cells is the most important success factor for efficacious

cancer chemotherapy. While new drug delivery systems continue to evolve, there are many choices

of targeting agents as listed below. Understanding the specificity of these agents facilitates the

development of tumor-directed drug conjugates.  Antibodies

As mentioned above, the toxin carriers in immunotoxins are antibodies.5,9 One of the most important aspects of targeting therapy is the specificity of the carrier agents toward the target.8 Among

all antibodies, mAbs raised against specific markers on the surface of tumor cells are the most

highly selective. Although these mAbs may have therapeutic value on their own, their cytotoxicities

are greatly enhanced by conjugating them to highly cytotoxic drugs, which may be too toxic to be

used alone. Thus, to achieve the full potential of immunotoxins, the antibody should be carefully

selected such that it binds selectively to the target tissue with high affinity and has little crossreactivity with healthy tissues. Ideally, the antibodies should be raised against those antigens that

are highly expressed on the cell surface for maximum therapeutic potential. To avoid immunogenicity of these antibodies, nonimmunogenic humanized forms of antibodies should be used. These

considerations are very important as demonstrated by the fact that a KS1/4 antibody–methotrexate

conjugate for nonsmall cell lung cancer10 and an antibody–DOX conjugate, BR96-Dox, for gastric

adenocarcinoma11 and metastatic breast cancer12 were all found to lack therapeutic benefit. On the

other hand, the antibody-maytansinoid conjugates seemed to provide superior antitumor activity.8

Also, chimeric antitransferrin receptor antibodies in which the constant region of the antibody is

substituted by a human-constant-region-inhibited proliferation and directly induced apoptosis in

hematopoietic-derived cell lines.13  Other Naturally Occurring Molecules

In addition to antibodies, many other naturally occurring biopolymers and substances have been

used as toxin carriers. These include vitamins, fatty acids, carbohydrates, transferrin, lectin, inulin,

and regulatory and signaling molecules. Chen et al.14 have developed a mechanism-based tumortargeting drug delivery system based on tumor-specific vitamin-receptor-mediated endocytosis.

In this case, biotin (a vitamin) is linked to cytotoxic agents such as taxoid through a disulfide

linkage attached to a phenyl group. Such a disulfide bond can be cleaved upon endocytosis by

endogenous thiols, for example, glutathione (GSH) and thioredoxin, to generate the desirable thiophenolate or sulfhydrylphenyl species and the free toxin. The synthesis involves pyridine disulfide

and N-hydroxysuccinimide. Other essential vitamins that have overexpressed receptors on cancer cell surface have also been used. These include folic acid,15 vitamin B12,16 and riboflavin.17

Unfortunately, in most cases, the synthesis of the linker between the vitamin derivatives and the

toxin is complicated involving many steps.

Another naturally occurring substance used as a drug carrier based on endocytosis is transferrin.13

Transferrins are iron-binding proteins. The human transferrin consists of two domains, homologous to each other with 679 amino acids. Iron-bound transferrin is internalized after binding to

transferrin-specific receptors by receptor-mediated endocytosis. Since the transferrin receptor

is expressed in inflammation and in proliferating malignant cells, transferrin may be used as a

drug carrier for therapy. Several drugs have been conjugated to transferrin and tested for efficacy against cancer cells. These drugs include adriamycin, cisplatin, chlorambucil, daunorubicin, protein synthesis inhibitors, plant-derived toxins, and many others.18 Adriamycin–transferrin

conjugate has been shown to be toxic to human leukemia, erythroleukemia, colorectal carcinoma, breast adenocarcinoma, mesothelioma, liver carcinoma, and cervical adenocarcinoma cell

lines.19 Cisplatin-transferrin inhibits the growth of the human epidermoid carcinoma cell line.20

The drugs chlorambucil and daunorubicin conjugated with transferrin show increased cytotoxicity in human breast cancer cell and small cell lung carcinoma compared with unconjugated

drug.21,22 The effect of other drug-transferrin conjugates has been reported.18 Many methods have

Preparation of Immunotoxins and Other Drug Conjugates


been used to prepare the transferring-drug conjugates such as Schiff base formation,18 acidsensitive maleimide derivatives, and glutaraldehyde.18–22

Polyunsaturated fatty acids (PUFAs) have also been used as tumor-specific molecules.

Representative naturally occurring PUFAs include linolenic acid (LNA), linoleic acid (LA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Kuznetsova

et al.23 prepared the conjugates of DHA, LNA, and LA with second-generation taxoids and studied their efficacy in vivo against ovarian and colon tumor xenografts. The coupling procedure for

esterification uses N,N′-dicyclohexylcarbodiimide (DCC) as a coupling agent, methylene chloride

as a solvent system, and dimethylaminopyridine (DMAP) as a catalyst. The authors demonstrated

that two of the PUFA-taxoid conjugates show total regression of drug-resistant and drug-sensitive

tumors in animal models.

Hyaluronic acid (HA, also called hyaluronan) is a polymer of disaccharides, composed of

d-glucuronic acid and d-N-acetylglucosamine, linked together via alternating β-1,4 and β-1,3 glycosidic bonds. HA can contain up to 25,000 disaccharide repeats in length with molecular weights

ranging from 5,000 to 20,000,000 Da. It is distributed widely throughout connective, epithelial,

and neural tissues. There are three main groups of cell receptors for HA: CD44, RHAMM (receptor for HA-mediated motility) and ICAM-1 (intracellular adhesion molecule-1). Because of these

receptors, HAs have been employed as tumor-specific modules to construct tumor-targeting drug

conjugates.24 Akima et al.25 coupled mitomycin C and epirubicin to HA by carbodiimide chemistry

and found that the former adduct was selectively taken up by, and was toxic to, a lung carcinoma

xenograft. Luo and Prestwich26 covalently attached taxol with N-hydroxysuccinimide to HA modified with adipic. The HA-taxol conjugates were shown to have selective toxicity toward the human

breast, colon, and ovarian cancer cell lines that overexpress HA receptors. Saravanakumar et al.27

modified HA with an amine-terminated hydrotropic N,N-diethylnicotinamide (DENA) oligomer to

synthesize hydrotropic HA (HydroHA) derivatives. Paclitaxel (PTX), a highly hydrophobic chemotherapeutic agent, was coupled to a HydroHA using carbodiimide chemistry. The HydroHA-PTX

conjugates were selectively taken up by the cancer cell line with overexpressing CD44. Luo et al.28

also prepared a modified HA with N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer and

conjugated to DOX. The HPMA-HA-DOX conjugate was found to have higher cytotoxicity against

human breast cancer, ovarian cancer, and colon cancer.

Lectins are sugar-binding proteins that are highly specific for the structures of sugar moieties. Lectin wheat germ agglutinin (WGA), for example, binds to N-acetyl-d-glucosamine and

sialic acid of carbohydrates. It has a high binding rate to intestinal cell lines of human origin,

human colonocytes, and prostate cancer cells. Moreover, it is also taken up into the cytoplasm

of enterocyte-like Caco-2 cells.29 Gabor et al.30 conjugated fluorescein-bovine serum albumin

(F-BSA) to lectin WGA using a homobifunctional cross-linker, divinyl sulfone, to react with the

amino groups of both proteins. The F-BSA-WGA conjugate were bound specifically to Caco-2

cells and exhibited uptake into the cells. The authors concluded that WGA-mediated drug delivery is a promising strategy. With another lectin, concanavalin A (Con-A), which binds specifically to mannose, Anande et al.31 prepared mucoadhesive microspheres of diloxanide furoate

(DF) for the effective treatment of amoebiasis. The carboxyl groups of Eudragit microspheres

of DF were linked to Con-A using ethyl-3,3-(dimethylaminopropyl) carbodiimide (EDC) and

N-hydroxysuccinimide. The conjugate increased the mucoadhesiveness and provided controlled

release of DF in simulated GI fluids.

Heparin is a highly sulfated glycosaminoglycan that has been conjugated to deoxycholic acid

(DOCA) for drug delivery.32 Park et al.32,33 prepared heparin-DOCA (HD) conjugates by covalently bonding N-(2-aminoethyl)deoxycholylamide to heparin via amide formation with EDC.

The HD conjugates were loaded with DOX through heparin nanoparticle formation. These

DOX-loaded heparin nanoparticles displayed a sustained drug release pattern and enhanced

therapeutic effect against squamous cell carcinoma and B16F10 melanoma. Wang et al.34 also

prepared various heparin conjugates by activating heparin to a mixed anhydride intermediate to


Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation

which paclitaxel and amino acid-paclitaxel derivatives have been linked. These conjugates were

shown to arrest MCF-7 cells in the G2/M phase of cell cycle.

Gellan gum, also known commercially as Phytagel or Gelrite, is a water-soluble polysaccharide

produced by bacterium, Sphingomonas elodea. The repeating unit of the polymer is a tetrasaccharide, which consists of two residues of d-glucose along with one residue of l-rhamnose and

one residue of d-glucuronic acid connected through an (α1 → 3) glycosidic bond. Krauland et al.35

conjugated l-cysteine to deacetylated gellan gum (DGG) by a carbodiimide. The DGG-cysteine

conjugate was capable of forming inter- and/or intramolecular disulfide bonds in aqueous solution.

The authors suggested that the conjugated polymer represents a promising novel excipient for various drug delivery systems.  Synthetic Peptides and Nucleotides

Many peptides, polypeptides, and proteins have been shown to posses the ability to traverse biological membranes. These matrices have been successfully used for the intracellular delivery of many

therapeutic agents including small molecules, proteins, peptides, oligonucleotides, plasmids, and

nanoparticles.36 Unbiased biopanning of phage-displayed peptide libraries has generated a suite of

cancer targeting peptidic ligands.37 Among these, cell-penetrating peptides, which are 9–35 mer

cationic and/or amphipathic peptides, can be linked to a variety of anticancer therapeutics, making

them an efficient, effective, and nontoxic mechanism for drug delivery.38

Specifically, receptors for certain peptide hormones are expressed in a relatively high concentration on a variety of cancer cells. These peptide hormones can serve as carriers for a local delivery of

cytotoxic agents or radiopharmaceuticals to the tumors. The most widely investigated of these peptide hormones is the hypothalamic hormone, somatostatin.39 The short plasma half-life of the native

forms of somatostatin prompted an avid search for more stable and more potent synthetic analogs

as demonstrated by the successful clinical use of radiolabeled somatostatin analog, Octreoscan, for

the detection and treatment of some somatostatin receptor-positive tumors. In recent years, a series

of other cytotoxic peptide hormone conjugates based on derivatives of hypothalamic hormones

such as luteinizing hormone-releasing hormone (LHRH) and the brain-gut hormone bombesin were

prepared. A derivative of DOX, 2-pyrrolino-DOX, which is 500–1000 times more active than its

parent compound was coupled to somatostatin octapeptide, to LHRH analogue, [D-Lys6]LHRH,

and to bombesin-like peptide. These conjugates were investigated for their effectiveness in various

cancers including ovarian and breast cancers,39 hepatocellular carcinoma,40 and prostate cancer.41

Oligonucleotides have also been used as targeting agent for drug delivery. Of particular importance are the aptamers, which are short single-stranded nucleic acids with a defined three-dimensional shape that allows them to interact with high affinity with a target molecule.42,43 They have

been used to target distinct cell subtypes and tissues. The most established and best characterized

aptamer in this regard is the prostate-specific membrane antigen (PSMA)-binding nucleic acid molecule A10. This aptamer has been conjugated with cytotoxic molecules, thereby allowing their selective delivery to cancer cells. For example, Farokzhad et al.44 have attached chemotherapeutics, such

as docetaxel, to the 5′-amino end of the aptamer by an NHS/EDC approach and showed the efficacy of such an aptamer-drug conjugate in an in vivo xenograft rat tumor model system. The same

group also directly complexed aptamer A10 with DOX and demonstrated that treatment of prostate

cancer cells with the complex resulted in a significant reduction of tumor-cell proliferation.45 Chu

et al.46 also conjugated the aptamer to the toxin gelonin, a ribosome-inactivating protein (RIP), with

N-succinimidyl-3-(2-pyridylodithio)propionate (SPDP). The aptamer conjugate not only promoted

uptake of gelonin into target cells but also decreased the toxicity of gelonin in nontarget cells.

In addition to chemotherapeutics, siRNA molecules have also been coupled to aptamer

A10  either directly by nucleotidic extensions or indirectly through the assembly of tetrameric

streptavidin–biotin complexes.47,48 Several other aptamers have also been used as aptamer-based

siRNA-delivering agents.49

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