Review
Antibody derivatization and conjugation strategies: Application in preparation of stealth immunoliposome to target chemotherapeutics to tumor

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Abstract

A great deal of effort has been made over the years to develop liposomes that have targeting vectors (oligosaccharides, peptides, proteins and vitamins) attached to the bilayer surface. Most studies have focused on antibody conjugates since procedures for producing highly specific monoclonal antibodies are well established. Antibody conjugated liposomes have recently attracted a great deal of interest, principally because of their potential use as targeted drug delivery systems and in diagnostic applications. A number of methods have been reported for coupling antibodies to the surface of stealth liposomes. The objective of this review is to enumerate various strategies which are employed in the modification and conjugation of antibodies to the surface of stealth liposomes. This review also describes various derivatization techniques of lipids prior and after their use in the preparation of liposomes. The use of single chain variable fragments and affibodies as targeting ligands in the preparation of immunoliposomes is also discussed.

Graphical Abstract

Preparation of PEGylated liposomes and conjugation of intact or derivatized monoclonal antibodies (PEGylated Immunoliposomes) through various functionalized PEG derivatives.

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Introduction

Cancer chemotherapy is generally accompanied by side effects. If an anticancer drug could be delivered only to the right site in the right concentration at the right time, cancer could be cured without any side effects. As delivery systems, liposomes are considered amongst the most useful of all targeted drug delivery system since liposomes are essentially non-toxic and biodegradable, their size, components, and modifications with various molecules are easily controlled, and they could deliver large amounts of either hydrophilic or hydrophobic agents [1]. Liposomes have been extensively evaluated as potential drug carrier systems for use in therapeutic applications because of their ability to alter pharmacokinetics and to reduce the toxicity of drugs associated with them [2]. After the administration of liposomes in vivo, opsonins are adsorbed onto their surface by a process called opsonisation, triggering recognition and liposome uptake by the MPS [3], [4] and, as a result, liposomes are rapidly eliminated from the blood circulation. The surface modification of liposomes with hydrophilic polymers such as PEG results in a decreased interaction with serum opsonins and subsequent recognition and phagocytosis by cells of the MPS, resulting in an increased circulation time [5]. The development of such PEGylated liposomes indicates that they have considerable potential for use in clinical applications [6]. Particularly, PEG is useful because of its ease of preparation, relatively low cost, controllability of molecular weight and linkability to lipids or protein including the antibody by a variety of methods [7].

Active targeting of liposomes to tumor cells is generally attempted by conjugating ligands to the liposomal surface which allow a specific interaction with the tumor cells. Several type of ligands have been used for this purpose, including antibodies or antibody fragments, vitamins, glycoproteins, peptides (RGD-sequences), and oligonucleotide aptamers. Among the different approaches of active targeting, immunoliposomes using antibody or antibody fragment as a targeting ligand and a lipid vesicle as a carrier for both hydrophilic and hydrophobic drugs, is a fascinating prospect in cancer therapy [8]. The use of an antibody molecule as a homing device has been especially facilitated by the development of the hybridoma technology, which makes it possible to produce a large quantity of a monoclonal antibody to a wide variety of cell determinants [9]. However, only a limited number of preclinical studies report successful targeting of immunoliposomes in vivo [10]. As systemic administration is the most practical route for the treatment, immunoliposomes must be developed so that physiological barriers can be overcome. Therefore, the development of liposomes with RES avoiding activity is a necessary first step before attempting the use of immunoliposomes. Given a suitable antibody with high specificity and affinity for the target antigen, the critical factor is the accessibility of target cells to immunoliposomes. Efficient target binding of the injected immunoliposomes occurs only when the target cell is in the intravascular compartment or can be accessible through leaky vascular structures. Thus, in terms of targeting drug delivery by immunoliposomes, two anatomical compartments can be considered as targeting sites. One is located at a readily accessible site in intravascular, such as the vascular endothelial surface, T cells, B cells or thrombus. Another is a much less accessible target site located in the extravascular compartment. This site involves a solid tumor, an infection site, or an inflammation site, which vascular structure is leaky [7]. The process of targeted drug delivery with immunoliposomes can be roughly divided into two phases: the transport phase, in which the immunoliposomes travel from the site of administration (often i.v. administration) to the target cells, and the effector phase that includes the specific binding of immunoliposomes to the target cells and the subsequent delivery of entrapped drugs [8]. Immunoliposomes for the treatment of tumor should satisfy a number of requirements aimed at maximum targeting effect of immunoliposome administered systemically in the bloodstream. The antigen binding site of the liposome-conjugated antibody must be accessible for unperturbed interaction with antigen on the surface of target cells. The blood clearance of immunoliposomes must be minimized in comparison with rate of extravasation into the tumor. Immunoliposome must allow efficient loading and retention of a selected anticancer drug. And finally, the drug and antibody incorporation must be stable enough to permit liposomal entry into the tumor tissue without the loss of either of these agents [7].

Previous research works on immunoliposomes are limited to use of only one or two functionalized PEG derivatives as linker and intact or monoclonal antibodies modified with a couple of specific techniques, but those papers do not give information on other chemical strategies which can also be employed in antibody and phospholipid modification and in immunoliposome preparation. In this review, we present some modification strategies for antibodies and phospholipids prior to and after their use in liposomes. We also mentioned some commonly used homo and hetero bifunctional PEG derivatives as linkers between liposomes and antibodies. This review article gives utmost knowledge, to the readers and researcher one who is working or wish to work on immunoliposomes, about chemical strategies involved in the preparation of immunoliposomes for selective targeting of chemotherapeutics to tumors. In this review, we described the chemical strategies of immunoliposome preparation based on (i). use of free amino groups, carboxyl groups and carbohydrate chains present in the antibody molecules, (ii). the modification of the existing functional groups (disulfide, amine, carboxyl, and carbohydrate groups) in the antibodies with suitable crosslinking reagents containing reactive functionalities, (iii). use of free functional groups present in phospholipids (for example hydroxyl and amine groups), (iv). modification of the existing functional groups of the phospholipids using suitable crosslinking reagents containing reactive functionalities, and (v). the use of various functionalised PEG derivatives, which act as a linker between antibodies and liposomes. Also, the applications of scFv and affibodies as targeting ligands in the preparation of immunoliposomes are discussed.

Section snippets

Modification of antibodies (Abs) for conjugation

The ability to conjugate an antibody to another protein or to drug delivery system is critically important for lots of applications in life science research, diagnostics, and therapeutics. Antibody conjugates have become one of the most important classes of biological agents associated with targeted therapy for cancer and other diseases. There are literally many markers that have been identified on tumor cells to which monoclonal antibodies have been developed for targeted therapy [11]. The

Thiolation of antibodies/proteins

The sulfhydryl group is a popular target in many modification strategies. The frequency of sulfhydryl occurrence in Abs/proteins or other molecules is usually low (or nonexistent) compared to other groups like amines or carboxylates. The use of sulfhydryl reactive chemistries thus can restrict modification to only a limited number of sites within a target molecule. Limiting modification greatly increases the chances of retaining activity after conjugation, especially in sensitive proteins like

Derivatization of liposomes/phospholipids

The lipid thioderivatives have been synthesized prior to their incorporation into the bilayer membrane during formation of liposomes, into the bilayer membrane. Under these circumstances, the thiol precursors are distributed on both sides of the membrane, and come into direct contact with solvents and entrapped solute. In cases where the entrapped material is sensitive to the action of these precursors, the presence of such thio-derivatives in the membrane during liposome manufacture is clearly

Functionalised PEG derivatives in antibody conjugation

In bioconjugation modification, PEG has been used repeatedly as a linker. As a polymer for in vivo use, it should exhibit certain minimum properties, such as biocompatibility, biodegradability, non-immunogenicity and non-toxicity. Besides the advantages, it can be obtained under GMP conditions and it is FDA-approved [50], [51]. The major role to play for PEG in bioconjugation for pharmaceutical and biotechnological use are giving stealth effect to biomolecule or carrier systems by shielding of

Chemical strategies of immunoliposome preparation

In this section we reviewed various chemical strategies of immunoliposome preparation. Table 1 includes composition of liposome along with functionalised PEG derivative used for antibody conjugation. It includes some techniques of derivatization of phospholipids, before and after their incorporation into liposomes, and monoclonal antibodies. In Table 1 we also included variety of monoclonal antibodies used in immunoliposome preparation, and cancer cell lines overexpressing specific tumor

Comparison of antibody-coupling methods

In general, coupling methods for the formation of ligand targeted liposomes (LTLs) should be simple, fast, efficient, and reproducible, yielding stable, non-toxic bonds. The biological properties of the ligand such as target recognition and binding efficiency should not be substantially altered. The LTLs should have stabilities and circulations half-lives long enough to allow them to reach and interact with the target cells. Further, the coupling reaction should not alter the drug loading

Employment of liposomal carboxyl groups

PE can be derivatized in such a way that the lipid moiety bears an active carboxyl group which, after incorporation into liposomes, is able to bind directly to amino groups on proteins and other molecules. This is achieved via the intermediary of a bifunctional straight chain α–ω dicarboxylic acid, which acts as a bridge between the PE and the protein. By choosing carboxylic acids containing different numbers of carbons, the length of this spacer group can be varied to suit one's purposes. A

Single chain antibodies (ScFv)

Recombinant antibody (rAb) fragments are becoming popular therapeutic alternatives to full length monoclonal Abs since they are smaller, possess different properties that are advantageous in certain medical applications, can be produced more economically and are easily amendable to genetic manipulation. Single-chain variable fragment (scFv) Abs are one of the most popular rAb format as they have been engineered into larger, multivalent, bi-specific and conjugated forms for many clinical

Actively tumor targeted liposomes under preclinical and clinical trials

The active targeting strategy consists of grafting a targeting ligand at the surface of nanocarriers (liposomes) to provide an enhanced selectivity and thus efficacy, as compared to the passive targeting. Although many authors report the evidence of this strategy in preclinical models, until now only two clinical trials have been conducted for ligand conjugated liposomes (Table 3), the GAH-targeted doxorubicin-containing immunoliposomes (MCC-465) [138] and the transferrin-targeted oxaliplatin

Conclusions

Modification and conjugation techniques are dependent on two interrelated chemistries: the reactive functionalities present on the various crosslinking or derivatizing reagents and the functional groups present on the target macromolecules to be modified. Without both types of functional groups being available and chemically compatible, the process of derivatization would be impossible. Reactive functionalities on crosslinking reagents, tags, and probes provide the means to specifically label

Acknowledgement

The authors would like to thank All India Council for Technical Education (AICTE), India, for grant support (F.N.:1-10/RID/NDF-PG(22)/2009–10). Also, authors thank Genzyme Pharmaceuticals LLC, Switzerland for providing phospholipids (DPPC, DSPE–mPEG2000 and DPPG), as gift samples, for the project on immunoliposomes.

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