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

Journal of Controlled Release 150 (2011) 2–22

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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j c o n r e l

Review

Antibody derivatization and conjugation strategies: Application in preparation of stealth immunoliposome to target chemotherapeutics to tumor Arehalli S. Manjappa a, Kiran R. Chaudhari a, Makam P. Venkataraju a, Prudhviraju Dantuluri a, Biswarup Nanda a, Chennakesavulu Sidda a, Krutika K. Sawant a, Rayasa S. Ramachandra Murthy b,⁎ a b

TIFAC CORE in NDDS, Pharmacy Department, M.S. University of Baroda, Fatehgunj, Vadodara-390 002, Gujarat, India Department of Pharmaceutics, I.S.F. College of Pharmacy, Moga-142001, Punjab, India

a r t i c l e

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Article history: Received 28 July 2010 Accepted 19 October 2010 Available online 21 November 2010 Keywords: Monoclonal antibody Fab′ fragments scFv Affisomes Thiolation techniques Conjugation strategies

a b s t r a c t 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. © 2010 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modification of antibodies (Abs) for conjugation . . . . . . . . . . . . . . . . . . . Thiolation of antibodies/proteins . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. 2-Iminothiolane (Traut's reagent) modified antibodies . . . . . . . . . . . . . 3.2. N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) modified antibodies . . . 3.3. Succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT) modified 3.4. SATA modified antibodies . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: Ab, antibody; BSA, bovine serum albumin; Chol, cholesterol; CHEMS, cholesterylhemisuccinate; Cyanur–PEG2000–PE, cyanuric chloride–PEG2000–PE; DTT, dithiothreitol; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DSPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine; DDAB, dimethyl dioctadecyl ammonium bromide; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DPPE, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine; DPPEMBS, DPPE-m-maleimidobenzoyl-N-hydroxysuccinimide; DCP, dicetylphosphate; DiO, 3,3 -dioctadecyloxacarbocyanine perchlorate; DOTAP, 1,2-dioleoyl-3-trimethylammoniumpropane; DOGS-Ni-NTA, dioleoyl-glycero-succinyl-nitrilotriacetic acid; DiI, [1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine iodide]; EDTA, ethylene diamine tetraacetic acid; EPC, egg phosphatidyl choline; EGFR, epidermal growth factor receptor; EDCI, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride; FDA, food and drug administration; 5-FdU, 5-fluoro-deoxyuridine; GMP, good manufacturing practice; GD2, disialoganglioside antigen; GPLPLR peptide, Gly–Pro–Leu–Pro–Leu–Arg peptide; Hz, Hydrazide; HSPC, hydrogenated soya phosphatidyl choline; HER2, human epidermal growth factor receptor 2; HEPES, 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid; MPS, mononuclear phagocyte system; MEA, 2-mercaptoethylamine; Mal, maleimide; MPB–PE, maleimidophenylbutyrate-PE; MPB-mAbs, MPB-monoclonal antibodies; MCC–PE, maleimido-methyl-cyclohexanecarboxamide–PE; M-PE, maleimido phosphatidyl ethanolamine; MT1-MMP, membrane type-1 matrix metalloproteinase; MAN, paraaminophenyl-α-D-mannopyranoside; mPEG, methoxy PEG; NHS, N-hydroxy succinimide; NHSIA, N-hydroxysuccinimido-iodoacetate; NOAC-ETC, N4-octadecyl-1-β-D-arabinofuranosyl-cytosine-ethynylcytidine; Ni-NTA, Ninitrilotriacetic acid; NGR peptide, (asparagine–glycine–arginine) peptide; PI, phosphatidyl inositol; p-NP–PEG–PE, para nitrophenylcarbonyl-PEG-PE; PLA–PEG–Mal, poly-(D,L-lactic acid)-PEG-Mal; PEG, polyethylene glycol; PE, phosphatidyl ethanolamine; PDP–PE, pyridyldithiopropionate-PE; PDPH, 3-(2-pyridyldithio)propionic acid hydrazide; PC, phosphatidyl choline; POPC, 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine; RES, reticuloendothelial system; RGD peptide, Arg–Gly–Asp peptide; scFv, single chain variable fragment; SPDP, N-Succinimidyl 3-(2-pyridyldithio)propionate; SMPT, succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene; SATA, N-succinimidyl S-acetylthioacetate; SAMSA, S-Acetylmercaptosuccinic anhydride; SATP, succinimidyl acetylthiopropionate; SMPB, N-succinimidyl (-4-[p-maleimidophenyl]) butyrate; SPC, Soya phosphatidyl choline; Sulfo-MBS, m-maleimidobenzoyl-N-hydroxysulfosuccinimide; sulfo-SMCC, sulfosuccinimidyl-4-N-maleimidomethyl-cyclohexane-1-carboxylate; TCEP, tris (2-carboxyethyl)phosphine; TEM1, tumor endothelial marker 1; VCAM-1, vascular cell adhesion molecule-1. ⁎ Corresponding author. Tel.: + 91 9898368187(mobile); fax: + 91 0265 2418927/2423898. E-mail address: [email protected] (R.S. Ramachandra Murthy). 0168-3659/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2010.11.002

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3.5. S-Acetylmercaptosuccinic anhydride (SAMSA) modified antibodies . 3.6. Succinimidyl acetylthiopropionate (SATP) modified antibodies . . . 3.7. Comparison of antibody thiolation techniques . . . . . . . . . . . 4. Derivatization of liposomes/phospholipids . . . . . . . . . . . . . . . . 4.1. Comparison of liposome thiolation techniques . . . . . . . . . . 4.2. Formation of carbonyl groups . . . . . . . . . . . . . . . . . . 5. Functionalised PEG derivatives in antibody conjugation. . . . . . . . . . 6. Chemical strategies of immunoliposome preparation . . . . . . . . . . . 7. Comparison of antibody-coupling methods . . . . . . . . . . . . . . . 8. Alternative conjugation strategies . . . . . . . . . . . . . . . . . . . . 8.1. Employment of liposomal carboxyl groups . . . . . . . . . . . . 8.2. Employment of protein bound carboxyl groups . . . . . . . . . . 9. Recent activities of immunoliposome preparation . . . . . . . . . . . . 9.1. Single chain antibodies (ScFv) . . . . . . . . . . . . . . . . . . 9.2. Affibodies as targeting ligands (affisomes) . . . . . . . . . . . . 10. Actively tumor targeted liposomes under preclinical and clinical trials . . 11. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. 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.

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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

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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.

2. 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 preparation of antibody conjugates to find and destroy cancer cells in vivo has become one of the leading strategies of research into investigational new drugs [12]. The unique structural characteristics of antibody molecules supply a number of choices for modification and conjugation schemes [13,14]. The chemistry used to effect conjugate formation should be chosen to yield the best possible retention of antigen binding activity. A detailed illustration of antibody structure is shown in Fig. 1. The most fundamental immunoglobulin G molecule is composed of two light and two heavy chains, held together by noncovalent interactions as well as a number of disulfide bonds. The light chains are disulfide-bonded to the heavy chains in the CL and C1H regions, respectively. The heavy chains are in turn disulfide-bonded to each other in the hinge region. The heavy chains of each immunoglobulin molecule are identical. Depending on the class of immunoglobulin, the molecular weight of these subunits ranges from about 50,000 to around 75,000. Similarly, the two light chains of an antibody are identical and have a molecular weight of about 25,000. For IgG molecules, the intact molecular weight representing all four subunits is in the range of 150,000–160,000. There are five major classes of antibody molecules, each determined from their heavy chain type, and designated as IgG, IgM, IgA, IgE, or IgD. Three of these antibody classes, IgG, IgE, and IgD, consist of the basic Ig monomeric structure containing two light and two heavy chains. Both IgA and IgM contain an additional subunit, called the J chain—a very acidic polypeptide of molecular weight 15,000 that is very rich in carbohydrate. The heavy chains of immunoglobulin molecules also are glycosylated, typically in the C2H domain within the Fc fragment region, but also may contain carbohydrate near the antigen binding sites. There are two antigen binding sites on each of the basic Ig-type monomeric structures, formed

Fig. 1. Modified structure of an IgG antibody molecule.

by the heavy–light chain proximity in the N-terminal, hypervariable region at the tips of the “y” structure [15]. Useful enzymatic digests of antibody molecules may be prepared that still retain the antigen binding sites. Enzymatic digestion with papain produces two small fragments of the immunoglobulin molecule, each containing an antigen binding site (called Fab′ fragments), and one larger fragment containing only the lower portions of the two heavy chains (called Fc, for “fragment crystallizable”) (Fig. 2a) [16]. Alternatively, pepsin cleavage produces one large fragment containing two antigen binding sites [called F(ab′)2] and many smaller fragments formed from extensive degradation of the Fc region (Fig. 2b) [17]. The F(ab′)2 fragment is held together by retention of the disulfide bonds in the hinge region. Specific reduction of these disulfides using MEA or other reducing agents produces two Fab′ fragments each of which has one antigen binding site. Papain, pepsin, and similar enzymes including bromelain, ficin, and trypsin, cleave immunoglobulin molecules in the hinge region of the heavy chain pairs. Depending on the location of cleavage, the disulfide groups holding the heavy chains together may or may not remain attached to the antigen binding fragments that result. The immunogenic effect of the Fc portion and of the increased RES clearance through specific recognition by phagocytic cells carrying Fc receptor was eliminated by using antibody fragments instead of the whole antibody. Antibody fragments also allow better ways of conjugating moieties to the liposomes containing functionalised PEG derivatives through unique thiol groups in the hinge region [7]. Antibody molecules possess a number of functional groups suitable for modification or conjugation purposes. Crosslinking reagents may be used to target lysine ε-amine and N-terminal α-amine groups. Carboxylate groups also may be coupled to another molecule using the C-terminal end as well as aspartic acid and glutamic acid residues. Although, both amine and carboxylate groups are as abundant in antibodies as they are in most proteins, the distribution of them within the three-dimensional structure of an immunoglobulin is nearly uniform throughout the surface topology. For this reason, conjugation procedures that utilize these groups will crosslink somewhat randomly to nearly all parts of the antibody molecule. This in turn leads to a random orientation of the antibody within the conjugate structure, often blocking the antigen binding sites against the surface of another coupled protein or molecule. Obscuring the binding sites in this manner results in decreased antigen binding activity in the conjugate compared to that observed for the unconjugated antibody [18]. Conjugation chemistry finished with antibody molecules generally is more successful at preserving activity if the functional groups utilized are present in limiting quantities and only at discrete sites on the molecule. By proper selection of the conjugation chemistry and knowledge of antibody structure, the immunoglobulin molecule can be oriented so that its bivalent binding potential for antigen remains available. Two site-directed chemical reactions are especially useful in this regard. The disulfides in the hinge region that hold the heavy chains together can be selectively cleaved with a reducing agent [such as MEA, DTT, or TCEP] to create two half-antibody molecules, each containing an antigen binding site [19,20]. The second method of site-directed conjugation of antibody molecules takes advantage of the carbohydrate chains typically attached to the C2H domain within the Fc region. Mild oxidation of the polysaccharide sugar residues with sodium periodate will generate aldehyde groups [21]. A crosslinking or modification reagent containing a hydrazide functional group then can be used to target specifically these aldehydes for coupling to another molecule. Directed conjugation through antibody carbohydrate chains thus avoids the antigen binding regions while allowing for use of intact antibody molecules. This method often results in the highest retention of antigen binding activity within the ensuing conjugate. However, care should be taken in using this method, because some antibody molecules can be glycosylated near the antigen binding area, thus potentially interfering with activity upon conjugate formation [21,22].

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Another limitation to the use of this strategy is the necessity for the antibody molecule to be glycosylated. Antibodies of polyclonal origin (from antisera) are usually glycosylated and work well in this procedure, but other antibody preparations may not possess polysaccharide. In particular, some monoclonals may not be posttranslationally modified with carbohydrate after hybridoma synthesis. Recombinant antibodies grown in bacteria also may be devoid of carbohydrate. Before attempting to use a conjugation method that couples through polysaccharide regions, it is best to test the antibody to see whether it contains carbohydrate-especially if the immunoglobulin is of hybridoma or recombinant origin. 3. 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 some enzymes. Unfortunately, sulfhydryl groups often need to be generated (from reduction of indigenous disulfides) or created (from use of the appropriate thiolation reagent systems). The following sections describe the most popular techniques of creating these functionalities. Some of these reagent systems are specifically designed to form sulfhydryl groups, while others are crosslinkers that also can serve the dual purpose of sulfhydryl generating agents. Sulfhydryl groups are susceptible to oxidation and formation of disulfide crosslinks. To prevent disulfide bond formation it is necessary to remove oxygen from all buffers by degassing under vacuum and bubbling an inert gas (i.e., nitrogen) through the solution. In addition, EDTA (0.01–0.1 M) may be added to buffers to chelate metal ions, thus preventing metal ion catalyzed oxidation of sulfhydryls [23]. Some proteins of serum origin (particularly BSA) contain much contaminating metal ions (presumably iron from hemolyzed blood) so that 0.1 M EDTA is required to prevent this type of oxidation [23]. 3.1. 2-Iminothiolane (Traut's reagent) modified antibodies Perham and Thomas [24] originally prepared an imidoester compound containing a thiol group, methyl 3-mercaptopropionimidate hydrochloride. The imidoester group can react with amines to form a stable, charged linkage, while leaving a sulfhydryl group available for further coupling. Traut et al. [25] subsequently synthesized an analogous reagent containing one additional carbon, methyl 4-mercaptobutyrimidate. Later, this compound was found to cyclize as a result of the sulfhydryl group reacting with the intrachain imidoester, forming 2-iminothiolane. The cyclic imidothioester still can react with primary amines in a ring-opening reaction that regenerates the free sulfhydryl. Traut's reagent is fully water-soluble and reacts with primary amines in the range of pH 7–10. The cyclic imidothioester is stable to hydrolysis at acid pH values, but its half-life in solution decreases as the pH increases beyond neutrality [26]. At high pH (10.0), Traut's reagent is also reactive with aliphatic and aromatic hydroxyl groups, though the rate of reaction with these groups is only about 0.01% that of primary amines. In the absence of Fig. 2. Enzymatic digestion of IgG antibodies. (a). Papain digestion of IgG antibodies primarily results in cleavage in the hinge region above the interchain disulfides. This produces two heavy–light chain pairs, called Fab′ fragments, each containing one antigen binding site. The Fc region normally can be recovered intact. (b). Pepsin digestion of IgG class antibodies results in heavy chain cleavage below the disulfide groups in the hinge region. The bivalent fragments that are formed are called F(ab′)2. The remaining Fc region normally is severely degraded into smaller peptide fragments. F(ab′)2 fragments are then reduced with MEA·HCl which yield Fab′ fragments.

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Fig. 3. Thiolation of antibodies using Traut's reagent and conjugation of thiolated antibody to maleimide groups on the derivatized PEG.

amines, carbohydrates such as agarose or cellulose membranes can be modified to contain sulfhydryl residues [27]. Polysaccharides modified in this manner are effective in covalently crosslinking antibodies used in immunoassay procedures and in conjugation of antibodies to liposome membrane. Fig. 3 describes the thiolation of antibody using Traut's reagent in the preparation of immunoliposomes. Traut's reagent has been used successfully in the investigation of ribosomal proteins [26,28], RNA polymerase [29], progesterone receptor subunits [30], and in the synthesis of enzyme labeled DNA hybridization probes [31]. It is an excellent thiolation reagent for use in the preparation of immunotoxins. It has also been used to modify and introduce sulfhydryls into oligosaccharides from asparagine linked glycans [32]. Side reactions other than oxidation to disulfides also can take place using Traut's reagent. Once an amine on a protein is modified with 2-iminothiolane, the terminal thiol can recyclize by attacking the amidine carbon. This can then rearrange into an iminothiolane derivative, which effectively ties up the thiol [33]. Proteins and other molecules thiolated using Traut's reagent can lose substantial amounts of available thiol to recyclization in just hours. For this reason, the thiolated product of a Traut's reaction should be used immediately in a conjugation reaction to avoid significant loss of activity. 3.2. N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) modified antibodies SPDP is one of the most popular heterobifunctional crosslinking agents. The NHS ester end of SPDP reacts with amine groups to form an amide linkage, while the 2-pyridyldithiol group at the other end can react with sulfhydryl residues to form a disulfide linkage [34]. The reagent is also useful in creating sulfhydryls in proteins and other molecules. Once modified with SPDP, a protein can be treated with DTT (or other disulfide reducing agents, to release the pyridine-2-thione leaving group and form the free sulfhydryl (Fig. 4). The terminal –SH group then can be used to conjugate with any crosslinking agents containing sulfhydryl-reactive groups, such as maleimide [35] or iodoacetyl functionalities (for covalent conjugation) [36] or 2-pyridyldithiol groups (for reversible conjugation) [37]. The main disadvantage of using SPDP to create sulfhydryls is the necessity of using a reducing agent to remove the pyridine-2-thione group. Reducing agents also will affect indigenous disulfides within a protein molecule, cleaving and reducing them. This method therefore works well for proteins containing no sulfhydryls or no disulfides that are critical to function,

Fig. 4. Modification of antibodies with SPDP followed by reduction with DTT to yield free sulfhydryl groups for conjugation.

but it may cause loss of activity or subunit breakdown in proteins containing essential disulfides. 3.3. Succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT) modified antibodies SMPT contains an NHS ester end and a pyridyldisulfide end similar to SPDP but its hindered disulfide makes conjugates formed with this reagent more stable [38]. The reagent is especially useful in forming immunotoxin conjugates for in vivo administration. A water-soluble analog of this crosslinker containing an extended spacer arm is also commercially available as sulfo-LC-SMPT. SMPT or sulfo-LC-SMPT may be used as thiolation reagents by first reacting its NHS ester end with an amine-containing molecule and then releasing the pyridine2-thione leaving group with DTT to free the sulfhydryl (Fig. 5). The disadvantage of this approach is the necessity of using a reducing agent to create the –SH group modification. This method of thiolation only should be used if there are no disulfides in the target molecule that are critical to function. If a reductant cannot be used, choose a thiolation method that does not need DTT treatment, such as the use of Traut's reagent [33] or SATA [39]. Since SMPT is not soluble in aqueous solutions it must be first dissolved in organic solvent and an aliquot of this stock solution transferred to the reaction solution. The reagent is soluble in DMF and DMSO, but is much more stable in solutions of acetonitrile. 3.4. SATA modified antibodies A versatile reagent for introducing sulfhydryl groups into proteins is SATA [40]. The active NHS ester end of SATA reacts with amino groups in proteins and other molecules to form a stable amide linkage. The modified protein then contains a protected sulfhydryl that can be

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Fig. 6. Introduction of thioacetate groups into antibodies. (a). Reaction of SATA with antibodies results in thioacetate group. (b). Use of SAMSA results in the introduction of extra carboxy function, in addition to the thioacetate group. Antibodies derivatized with either agent can yield a free sulphydryl group upon incubation with hydroxylamine, as shown in figure.

3.5. S-Acetylmercaptosuccinic anhydride (SAMSA) modified antibodies

Fig. 5. SMPT modification of antibodies followed by reduction with DTT.

stored without degradation and subsequently deprotected as needed with an excess of hydroxylamine (Fig. 6a). Since the protecting group can be removed without adding disulfide reducing agents like DTT, disulfides indigenous to the native protein will not be affected. This is an important consideration if disulfides are vital to activity, such as in the case of antibodies and some protein toxins [41]. Most polyclonal antibody molecules may be modified to contain up to about 6 SATA molecules per immunoglobulin with minimal effect on antigen binding activity. Some sensitive monoclonal antibodies, however, may be susceptible to modification and should be tested on a case-bycase basis. Conjugates formed using SATA maintain a bivalent antibody structure, assuring a conjugate containing two antigen binding sites. This is an advantage over reduction schemes that break the antibody molecule into two heavy–light chain pairs to create sulfhydryls, since disulfide cleavage yields antibody fragments with only one antigen binding site. SATA has been used to form conjugates with avidin or steptavidin with excellent retention of activity. It also has been used in the formation of a therapeutically useful toxin conjugate with recombinant CD4 [42], to study syntaxin proteins [43], to prepare bispecific antibodies, and to make a unique polylysine conjugate as a vehicle for drug delivery [44]. SATA is freely soluble in many organic solvents. In use, it is typically dissolved as a stock solution in DMSO, DMF, or methylene chloride, and then an aliquot of this solution is added to an aqueous reaction mixture containing the protein to be modified.

SAMSA is an amine-reactive reagent containing a protected sulfhydryl much like SATA described previously. The anhydride portion opens in response to the attack of an amine nucleophile, yielding an amide linkage [45]. The ring-opening reaction, however, does produce a free carboxylate group that lends a negative charge to the modified molecule where once there may have been a positive charge. This charge reversal may affect the conformation and activity of some sensitive proteins. After the initial modification step, releasing the acetylated sulfhydryl protecting group with hydroxylamine forms the thiolated derivative (Fig. 6b). Antibodies or any other proteins can be modified using SATA and SAMSA as per protocol previously reported [41,46]. 3.6. Succinimidyl acetylthiopropionate (SATP) modified antibodies SATP is an analog of SATA containing one additional carbon atom in length [47]. The compound retains all the advantages of a protected sulfhydryl, including stability of the modified protein and selective release of the protecting group with hydroxylamine to free the sulfhydryl as needed (Fig. 7). SATP is soluble in DMF and methylene chloride. It is usually first solubilized in organic solvent and an aliquot added to an aqueous solution containing the macromolecules (proteins/antibodies) to be modified. It is particularly useful in adding an N-terminal sulfhydryl group at the completion of peptide synthesis. 3.7. Comparison of antibody thiolation techniques The above methods employed for thiolating antibodies/proteins depends to a large extent on the protein one wishes to use. If the protein has endogenous disulfide bridges which can be broken without destroying essential enzymatic, binding or immunogenic properties of the molecule, then this approach is to be preferred, since it appears that endogenous free thiols are often more reactive than those introduced as thiopropionyl derivatives, so that one may expect the rate of reaction, and the efficiency of conjugation to be increased. While the most widely used method for introduction of exogenous thiols employs SPDP, disadvantage of using this reagent is the large number of purifications which have to be carried out at each stage-

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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 undesirable [48]. Prior synthesis of lipid derivatives is also tedious and time-consuming, and their use during the preparation of liposomes is uneconomic since only a proportion of the functional groups, exposed on the outer surface of the liposome membrane, are available for coupling to ligand molecules. An alternative approach which overcomes these problems is to derivatize the PE or other lipids after the liposomes have been formed. In this way, only the residues on the outer membrane are transformed, and no reagents come into contact with entrapped materials. The method adopted for labelling is exactly the same as is employed for protein molecules, that is, the reagent [SPDP, SMPB, and NHSIA] is dissolved in concentrated form in an organic solvent such as dioxane, dimethyl formamide or ethanol, and is added with stirring to the liposome suspension. In the case of liposomes containing 50 mol% of cholesterol, the concentration of solvent added to the suspension can be tolerated up to a value of 5% without causing leakage of entrapped solutes. Good buffering capacity is required to ensure that the pH does not decrease upon hydrolysis of the reagents to the free acid. Aminolysis proceeds at a much faster than hydrolysis, but any unreacted reagent will have been hydrolysed after half an hour. It is possible to add the liposome suspension to the dry solid reagents (to give a final concentration of about 1 mg ml−1), with a molar excess of reagent to PE of between 10 and 20 fold. Separation can be carried out by dialysis or column chromatography at the same time as the protein is being prepared for conjugation [48].

Fig. 7. SATP reacts with amine group of antibody through its NHS ester end to create protected sulfhydryl derivatives in a manner similar to that of SATA. Deprotection can be done with hydroxylamine to free the thiol.

one to separate unreacted SPDP from protein, and one to remove DTT, with concomitant loss of protein on columns, etc. [41]. Hashimoto et al. [36] have pointed out that one separation step can be avoided by using the reagent SAMSA instead to introduce thiol groups (again employing an N-hydroxy succinimide ester). In this case, the sulphur atom is introduced not as a part of a disulfide bridge, but as a thioester, which is easily unblocked with hydroxylamine. Since the presence of hydroxylamine (unlike DTT) does not compete, or otherwise interfere, with the subsequent formation of either disulphide or thioether linkages, the two reactions (unblocking and conjugation) can be carried in the same without the need for removal of the hydroxylamine before addition of the antibody/protein to liposomes. One difficulty with SAMSA is that upon binding to the amine group of the protein, a free carboxyl group is revealed, which is retained as part of the linker molecule, thus changing the net charge of the protein radically after only a few substitutions. Such alterations, while sometimes being innocuous, may easily lead to increased aggregation, or nonspecific binding to cells. This problem is overcome with SATA which works in the same way as SAMSA, but releases the free carboxy moiety into the bulk medium (Fig. 6). 4. 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

Fig. 8. Modification of lipids containing amine groups with SPDP after their incorporation into bilayer membrane and conjugating antibody containing free thiol group.

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4.1. Comparison of liposome thiolation techniques The thiol reactive lipids are synthesized using the reagent SPDP (Fig. 8) and SMPB (Fig. 9). The former approach results in the reversible coupling of protein via a disulphide bond while the latter produces an irreversible thioether linkage. These reagents have been used by several laboratories for the conjugation of proteins to liposomes as both methods are similar or identical in a number of steps. PDP–PE binds rapidly and efficiently to thiolated proteins, and the reaction can tolerate a wider pH (6.0–8.0) range than can conjugation via MPB–PE (6.5–6.8). The disulphide linkage formed, however, is a reversible one, and can easily broken in the presence of thiols such as glutathione, which is present in high concentration in biological tissues. For in vivo use, therefore, SPDP derivatization may be inadvisable, except in cases where dissociation of the protein from liposomes is particularly desired. The MPB reaction, on the other hand, results in thioether linkage which is very stable in biological environments [21]. One objection to use of SMPB is the possibility that the large maleimido benzoyl residues may act as an immunogenic determinant in its own right (especially if conjugated with small proteins or haptens), and may interfere with the stimulation of immune responses by liposomes, or create difficulties in interpretation of the results of immunological experiments. It has been proposed that the introduction of iodoacetate residues onto the liposome surface (via NHSIA) may be a suitable alternative (Fig. 10). However, one group of workers found that the iodoacetate resulted in a very low efficiency of binding of protein while others found that use of a spacer group was necessary to overcome steric hindrance [36]. However, MPB is already large enough not require an extra spacer group.

Fig. 9. Schematic representation of conjugation of antibody containing sulphydryl groups to MPB–PE vesicle through a very stable thioether linkage.

Fig. 10. Conjugation of iodoacetate to liposome containing PE using NHSIA and incubation of thiolated antibodies with NHSIA modified liposomes to prepare immunoliposomes.

4.2. Formation of carbonyl groups The coupling of antibodies to liposomes can also be carried out utilizing the reaction of a Schiff base with primary or secondary amino groups. Since the endogenous amino groups of the protein can be used without modification and liposome bound glycolipids can be oxidized (Fig. 11) in situ without destroying the integrity of the liposomes, conjugation can be performed in a simple two step reaction. The protocol for periodate coupling of liposomes is well described by Heath et al. [49]. Although the optimal pH for periodate oxidation is 5.5, this reaction is carried out at higher pH values to avoid increase in the permeability of liposome membrane, and resultant loss of contents due to leakage at low pH. Also at low pH neutral liposomes are permeable to periodate, leading to oxidation of internal contents as well as surface glycolipids. Negatively charged liposomes are impermeable to periodate at low pH. If desired, periodate oxidation may be carried out at low pH provided the final periodate concentration is reduced to 10 mM or less. It is important to note that with glycolipids such as galactocerebroside, good coupling is not

Fig. 11. Linkage of liposome bound sugars to antibody via Schiff base. Glycolipids such as cerebrosides or gangliosides incorporated in to the liposome membrane can be used to link liposomes to antibody. Membrane bound sugars containing two adjacent (vicinal) hydroxyl groups can be oxidized by sodium periodate to aldehyde functional groups, which then react readily with the amino groups of antibody. Unreacted aldehyde functions and the newly formed imine linkage can be converted to unreactive species by reduction with sodium cyanoborohydride.

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observed at concentrations less than 20 mol%, presumably because only one sugar residue is available for oxidation per molecule. Linkage with PG (phosphatidyl glycerol) appears to be difficult, possibly because of steric considerations [41].

liposomal membrane. PEG remains as a spacer arm between liposome and conjugated antibody/protein. In this section we present some homo (Fig. 12) and heterobifunctional (Fig. 13) PEG derivatives, which can be employed successfully in the preparation of immunoliposomes using proper conjugation strategy.

5. Functionalised PEG derivatives in antibody conjugation 6. Chemical strategies of immunoliposome preparation 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, nonimmunogenicity 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 antigenic and immunogenic epitopes, shielding receptor-mediated uptake by the RES, and preventing recognition and degradation by proteolytic enzymes, increased body residence time, modification of organ disposition, drug penetration by endocytosis and new possibilities of drug targeting [52]. In addition to these properties, PEG facilitates conjugation by providing the functional groups required for conjugation. Now PEG derivatives are becoming available in a variety of activated and highly reactive end functional groups which need a minimum number of steps for conjugation. In a recent scenario more and more peptide and other macromolecules are delivered as a PEGylated form to overcome pharmacokinetic associated problems. Successful protein biopharmaceuticals include PEGylated interferons (PEGasys® and Intron®), PEGylated growth hormone receptor antagonist (Somavert®), PEGasparaginase (Oncospar®), adenosine deaminase (ADAGEN®), and granulocyte colony stimulating factor (Neulasta®). Under this section, we discuss some commonly used functionalised PEG derivatives in the conjugation of biomolecules such as antibody and protein over

Fig. 12. Chemical structures of some commonly used homobifunctional PEG derivatives.

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

Fig. 13. Chemical structures of some commonly used heterobifunctional PEG derivatives.

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Table 1 Chemical strategies of immunoliposome preparation (derivatization strategies of liposomes and antibodies before conjugation). Liposome composition with functional PEG derivative

Monoclonal antibody (mAb) and its modification (conjugation strategy)

(a): Conjugation through maleimide method (thioether linkage). PC:Chol(3:2, molar ratio):DSPE–PEG (≤12 mol%):M-PE Fab′ fragments of recombinant humanized monoclonal antibody rhuMAbHER2 with –SH group was coupled to liposome through thioether linkage [53]a. Refer Fig. 9. DDAB(0.6 μmol):POPC(18.6 μmol):DSPE–PEG2000 Anti-human insulin receptor monoclonal antibody 83-14 was activated with Traut's reagent (mAb-SH) and (0.6 μmol):DSPE–PEG2000–Mal conjugated to liposomes via thioether linkage [54]. Refer Fig. 3. HSPC:Chol:DSPE–PEG (50:45:5, molar ratio):DSPE– Mouse monoclonal anti-E-selectin antibody (Clone 2Q780), specific for MCa-4 mouse mammary tumors, was thiolated PEG–Mal (2 mol%) with Traut's reagent and conjugated to the distal end of functionalized PEG derivative [55]a. Refer Figs. 3 and 9. mAb, Herceptin, specific for HER2 expressing breast cancer cells (BT-474 and SK-BR-3 and MDA-MB-231) was S100PC:Chol:mPEG2000–DSPE (90:10:5 M% ratio):Mal– PEG2000–DSPE (0.2–2 mol%) thiolated using Traut's reagent and conjugated to liposomes by maleimide method [56a,57,58a]. Refer Figs. 3 and 9. HSPC:Chol(3:2, molar ratio):PEG2000–PE(0–6 mol%): Fab′ fragments (Fab9, scFv C6.5, scFv F5) of humanized monoclonal antibody, rhuMAb HER2 (trastuzumab, Mal-PE (2 mol%) and/or Mal–PEG–PE Herceptin), specific for HER2-overexpressing breast cancers (SK-BR-3 and MCF-7), containing a C-terminal cysteine were conjugated to liposomes by maleimide method [59a,60a,61,62a]. Refer Fig. 9. HSPC:Chol:PDP–PEG2000–DSPE (2:1:0.02, molar ratio): Intact mouse monoclonal anti-GD2 antibody, directed against disialoganglioside antigen (GD2) positive mPEG–DSPE (5 mol%). neuroblastoma (NB) cell lines (HTLA-230), was digested with lysyl endopeptidase to prepare F(ab′)2 fragments, were then reduced with β-mercaptoethylamine. The Fab′ fragments were coupled to the liposome through thioether linkage [63a,64,65]. Refer Figs. 2b and 9. EPC:Chol:DSPE–PEG2000:DSPE–PEG–Mal (3:2:0.17:0.09, The anti-CD33 (clone p67.6) and the isotype-matched control mouse IgG1 MOPC21 monoclonal antibodies, molar ratio) specific for CD33 (Gp67) surface antigen expressing cancer cells [CD33+ leukemic cell lines (HL60)], were oxidized first with cold sodium meta-periodate and then were modified with PDPH, then reduced with DTT. The thiolated antibodies were coupled liposomes through thioether linkage [22]. Refer Fig. 14. Intact OX26 monoclonal antibodies, directed against transferrin receptor expressing cells (OX26 and Y3.AG.1.2.3. Lipid nanocapsules (LNC) composed of Lipoid® S75-3 hybridoma cell lines), were first oxidized with sodium meta-periodate and then were reacted with PDPH [66]a, (soybean lecithin at 69% of PC and 10% PE), lipophilic Labrafac® WL 1349 (caprylic–capric acid triglycerides) followed by thiolation with DTT. F(ab′)2 fragments were obtained using the “ImmunoPure F(ab′)2 Preparation and non-ionic hydrophilic surfactant Solutol® HS15 Kit”. A solution of OX26 mAb in sodium acetate was mixed with immobilized pepsin. Then F(ab′)2 fragments in HEPES buffer. LNC were incubated with DSPE– were reduced using MEA·HCl. For conjugation LNC containing DSPE–PEG2000–Mal were incubated with thiolated PEG2000–Mal micelles for 2 h at 60 °C. intact OX26 MAb and Fab′ fragments [21]. Refer Figs. 2b and 14. EPC:Chol:DCP:mPEG–DSPE:MPB–mPEG–DSPE. Non-specific rat IgG antibody for lymph node targeting was modified using SPDP followed by reduction with DTT. (6.71:6.71:1:0.425:0.15, molar ratio) The thiolated antibody was coupled to liposomes through thioether linkage [67]. Refer Figs. 4 and 9. mAb My10 specific for CD34 antigen expressing cells (KG-1a cells; a human acute myelogenous leukemia cell PC:Chol (2:1, molar ratio):mPEG–PE (5 mol%):PDP– line) was modified with SMPB to obtain maleimide derivative (MPB–mAb) for conjugation to the distal end of PEG–PE (modified using SPDP). Liposomes containing PEG through sulphydryl group [68]. Refer Fig. 15. PDP–PEG–PE were reduced with DTT. Monoclonal anti-EGFR antibodies, specific for EGFR expressing cells (NSCLCs, A549 cells), were reacted with DOPE:CHEMS:PDP–PEG–DOPE (6:4:0.1, molar ratio). SMPB to obtain MPB–mAbs. The thiolated liposomes were incubated with MPB–mAbs to prepare Liposomes were reduced with DTT to obtain thiolated immunoliposomes [69]a. Refer Fig. 15. liposomes. Egg lecithin:Chol:DSPE–PEG2000: MPB–PE (23:16:1:0.4, Anti-myelin basic protein (MBP) mAb bind to schwann cells of the neural tissue was thiolated (SPDP modification molar ratio) followed by reduction with DTT) and conjugated to liposomes by maleimide method [35,70]. Refer Figs. 4 and 9. Murine monoclonal antibody D4, specific for human gliofibrillary acidic protein (GFAP), was thiolated with Traut's reagent and conjugated to liposomes by maleimide method [71]a. Refer Figs. 3 and 9. DPPC(18 mg):PI(2 mg):DPPE–MBS(0–4 mg) Monoclonal antibody (H17E2), specific to placental alkaline phosphatase (PLAP) (Human epidermoid carcinoma cell lines [72]), was derivatized using SATA method and conjugated to distal end of PEG through sulphydryl group [73]. Refer Figs. 6 and 16. PC:Chol:MPB–PE and/or MCC–PE (6:3:1, molar ratio) Single-chain Fv fragments (scFv A5), directed against human endoglin (CD105) expressing cells (HUVEC, HDMEC), containing C-terminus cysteine residue (scFv-His-Cys) were reduced with TCEP and incubated with preformed maleimide containing liposomes [74]a. Refer Fig. 17. (b) Conjugation through other methods (disulfide, hydrazone, amide, and other linkages). mAb My10 specific to CD34+ cells was used. 1. SPDP modified antibodies (mAb-PDP) were incubated with PC:Chol (2:1, molar ratio):mPEG–PE (5 mol%):PDP– PEG–PE. 1. Liposomes were thiolated with DTT. 2. thiolated liposomes. 2. mAb-PDP was thiolated with DTT and incubated with SPDP modified liposomes [37]. Refer Liposomes containing PDP–PEG–PE (SPDP modified) Fig. 18. BAFF (B cell activating factor) maintain normal B-cell development and homeostasis and its receptors are DSPC:Chol:PEG–DSPE:PDP–PEG–DSPE (2:1:0.08:0.02, significantly increased in B-cell malignancies (The human Burkitt's B lymphoma cell Raji). mBAFF (a soluble BAFF molar ratio). Liposomes were reduced with DTT to mutant with amino acid 217–224 being replaced by two glycine residues) may be used as a competitive inhibitor obtain thiolated liposomes. for BAFF to treat relevant malignant hematologic diseases. mBAFF was modified with SPDP and incubated with thiolated nontargeted liposomes [75]a. Refer Fig. 18. DPPC:Chol:PEG–DSPE:Stearylamine (SA):PDP–SA Anti-MY9, mouse IgG antibody specific for CD33 antigen (human leukemia HL-60 tumor cell line), was treated (10:5:1.4:1.4:1.5, molar ratio) with SPDP followed by reduction with DTT. The reduced mAb (mAb-SH) was attached to the liposome through disulfide linkage [76]a. Refer Figs. 4 and 19. mAb CC52 (mIgG1) recognizing a surface antigen on CC531 colon adenocarcinoma cells was modified with SATA 1. EPC:Chol:MPB–PE (23:16:1, molar ratio) 2. EPC:Chol: and attached to liposome via SH-Mal coupling method. For 3. Attached via a hydrazone-linkage between the MPB–PE (23:16:1, molar ratio):DSPE-PEG(4 mol%) hydrazide moiety and the oxidized carbohydrates of the antibody [39,77,78]. Refer Figs. 6 and 20. and 3. EPC:Chol:MPB–PE (23:16:1, molar ratio): DSPE–mPEG(1.5 mol%):Hz–PEG–DSPE (2.5 mol%). H S P C : C h o l : m P E G – D S P E : H z ( h y d r a z i d e ) – P E G – D S P E Human clinical grade C225 (monoclonal chimerized anti-EGFR) antibody specific for DU145 cells (EGFR-positive, (57:38:3.3:1.7, molar ratio) androgen-independent prostate carcinoma) was oxidized with sodium periodate and then conjugated to hydrazide containing liposomes [79]a. Refer Fig. 20. SPC:Chol (2:1, molar ratio):cyanuric chloride–PEG–PE(5 mol Murine anti (human) E-Selectin mAb (BBA 26) was conjugated to membrane anchors without previous %)/N-Glutaryl–PE(5 mol%). derivatizations. Upon activating the free carboxyl group of N-glutaryl–PE with carbodiimide (EDC), the antibodies were conjugated to liposomes through amide bond [80,81]. Refer Fig. 21. DOPE:PEG2000–DOPE (20:1, molar ratio):N-glutaryl HYB-241, a mouse anti-human p-glycoprotein monoclonal IgG1 antibody that targets p-glycoprotein rich bovine brain phosphatidylethanolamine (NGPE). micro vessel endothelial cells was conjugated to liposomes containing NGPE through amide bond [82,83]. Refer Fig. 21b. HSPC:Chol:DSPE–PEG2000:folate–PEG3350–Chol (60:35:5:0.5, mAb Cetuximab (Erbitux™ or C225) was modified with Sulfo-MBS (mAb–Mal). Folate binding protein (FBP) was mol/mol) modified with Traut's reagent (FAB-SH). FBP–mAb complex was prepared and attached to liposomes through folate–folate binding [84]. Refer Fig. 22. Nucleosome-specific monoclonal antibody 2C5, recognizing a broad variety of tumor cells (murine LLC, 4T1, C26 Commercial Doxil™, ALZA Corp., red liposomal dispersion and human BT-20, MCF-7, and PC3) via the tumor cell surface-bound nucleosomes, was modified with p-NP– composed of HSPC (9.58 mg/ml), Chol (3.19 mg/ml), PEG–PE and the modified antibodies (2C5–PEG–PE) were then incubated with commercial Doxil™ preparation and MPEG-DSPE (3.19 mg/ml) [85–87]. Refer Fig 23. (continued on next page)

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Table 1 (continued) Liposome composition with functional PEG derivative

Monoclonal antibody (mAb) and its modification (conjugation strategy)

HSPC:Chol:MPEG2000–DSPE (2:1:0.16, molar ratio). Liposomes were incubated with sterol–PEG1300–BSA.

Chemical activation of sterol-PEG1300 by tresylchloride (TRE). The resulting sterol–PEG1300–TRE is coupled to BSA (as an amine group containing model ligand) by acylation or alkylation to form sterol–PEG1300–BSA conjugates [88]. Refer Fig. 24.

a

Indicates references containing in vivo pharmacokinetic and/or tumor induced biodistribution study.

immunoliposome preparation, and cancer cell lines overexpressing specific tumor markers. We also summarized briefly the preparation of immunoliposomes with schematic representation. 7. 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 efficiency and drug release rates in a negative way [89]. Several end-functionalized derivatives of PEG (described in Section 5) have been synthesized for coupling antibodies to the PEGterminus of liposomes. Some commonly used PEG derivatives include pyridylditiopropionoylamino (PDP)-PEG-PE [68], hydrazide (Hz)-PEGPE [39,79] and maleimide (Mal)–PEG–PE [21]. The PDP–PEG and Mal–

Fig. 14. Treatment of the polysaccharide portion of antibody with sodium meta periodate to create aldehyde residues. The hydrazide end of PDPH reacts with carbonyl group of aldehyde results in hydrazone bond. The antibody was then thiolated with DTT which then incubated with liposomes containing DSPE–PEG–Mal to prepare immunoliposomes.

PEG coupling methods both rely on the formation of thioether bonds between proteins and liposomes, which results in efficient formation of a stable bond [21,59,90]. In the PDP–PEG method, a maleimide group is incorporated into the antibody molecules via reaction with SMPB and the antibodies are then incubated with thiolated PDP-PEG [68]. In the Mal–PEG method antibody molecules are thiolated (described under Section 3) and reacted with maleimide groups on the PEG-termini. The antibody coupling to liposomes using linkers SPDP and SMPB is described in detail under Sections 3.2, 3.7 and 4.1. Ansell et al. [66] have developed an alternative to SPDP for coupling monoclonals using PDPH as a crosslinker. They first formed a hydrazone derivative of the immunoglobulin through periodate oxidation followed by treatment with PDPH. The resulting product is then deprotected with DTT to a free thiol which is then conjugated to liposome containing maleimide group. The resulting liposomes-antibody conjugates were found to have in vitro properties similar to those of conjugates formed by the SPDP protocol but which were cleared less rapidly in circulation. These investigators conclude that PDPH is a viable alternative to SPDP, particularly for antibodies sensitive to amine modification [66]. In the Hz–PEG method, carbohydrates in the Fc-region of whole antibodies are oxidized to form reactive aldehydes, which form hydrazone linkages with the hydrazide groups on the PEG-terminus [39,79]. This coupling strategy avoids recognition of the immunoliposomes by the Fc-receptors of the macrophages and favours orientation of the antibody molecules so that their antigen-binding domains are exposed to the target epitopes. Hydrazone linkages formed in the process are stable at pH ≥ 7.5, but they hydrolyse very slowly in an acidic environment. Hydrazide groups have a pKa of approximately 3.0, which makes them uncharged at physiological pH. Their lack of charge makes PEG–Hz unlikely to interfere with in vivo pharmacokinetics, and it was found that liposomes containing 5 mol% DSPE– PEG2000–Hz have identical pharmacokinetics to liposomes containing

Fig. 15. Modification of liposomes with SPDP followed by thiolation with DTT which then incubated with SMPB modified antibodies to prepare immunoliposomes.

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Fig. 18. Preparation of immunoliposomes (a). Derivatization of liposomes with SPDP followed by thiolation with DTT which then incubated with SPDP modified antibody. (b). Modification of antibodies with SPDP followed by thiolation with DTT which then incubated with SPDP modified liposomes.

Fig. 16. Modification of phospholipid (DPPE) with m-Maleimidobenzoyl-N-hydroxysuccinimide (MBS) and incorporation of MBS modified DPPE to liposomes during preparation. Incubation of DPPE–MBS liposomes with SATA modified antibodies to prepare immunoliposomes.

Fig. 17. Reduction of disulfides of scFv A5 by TCEP and conjugation of reduced scFv A5 fragments to the liposome containing membrane anchor maleimido–methyl-cyclohexane-carboxamide (PE–MCC).

DSPE–PEG2000. No problems with liposome aggregation have been observed with this method [91]. A single hydrazone bond between a ligand and the particle surface may not provide enough stability to prevent leaching of ligand due to hydrolysis. There are two routes to overcome this instability: (1) reduce the hydrazone linkage using sodium cyanoborohydride or (2) create multiple hydrazone linkages between the ligand and the particle surface. Multi-site attachment provides sufficient ligand stability, because not all the hydrazones will hydrolyze simultaneously to release ligand, and when one hydrazone bond breaks, it will have enough time to reform before the other hydrazones hydrolyze. Thus, glycosylated proteins coupled after oxidation to hydrazide particles most likely will be stable due to the presence of more than one aldehyde group, but small ligands containing only a single carbonyl group probably should be treated with cyanoborohydride to stabilize the hydrazone bond [92]. Aldehyde forms temporary Schiff base interactions with amines on proteins and other biomolecules in aqueous solution and these are fully reversible and will rapidly exchange with a hydrazide or hydrazine, if present. The only potential problem of cross-reactivity for this chemoselective reaction pair with molecules of biological origin might occur from a hydrazine reacting with aldehyde or ketone containing metabolic intermediates, reducing sugars, or similar small organic molecules present within cells or cell lysates [93]. The hydrazine-aldehyde reaction has been used intracellularly to deliver

Fig. 19. Conjugation of SPDP derivatized antibodies to liposomes containing membrane anchor, SA-PDP through disulfide linkage.

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Fig. 20. The Oxidation of carbohydrate present in the Fc region of the antibody with sodium periodate to produce aldehyde group. Conjugation of oxidized antibody via a hydrazone linkage between the hydrazide moiety present at the distal end of the PEGchain and oxidized carbohydrate in the Fc-region of the antibody.

non-toxic drug components, which when linked to form a hydrazone bond in situ, become cytotoxic [94–96]. This same approach has been used to generate enzyme inhibitors in vivo, wherein the hydrazine and aldehyde precursors are not active, but when coupled together within cells to form a hydrazone linkage, become active site binders [97]. As the hydrazones can show cytotoxic and enzyme inhibition properties Fig. 22. Synthesis of FBP-mAb (C225) conjugate and anti-EGFR immunoliposomes (a) Schematic depiction of synthesis of FBP–C225 conjugate. (b) Construction of noncovalently coupled anti-EGFR C225-liposomes by incubating FBP–C225 with folate liposomes under a mild condition (neutral pH and room temperature).

Fig. 21. Conjugation of antibodies to liposomes containing membrane anchor (a) DPPE– PEG–Cyanuric chloride and (b) N-Glutaryl–PE without previous derivatizations.

one should keep these in mind as potential toxicities while preparing the immunoliposomes through hydrazone linkage. The methods described above, although effective, require a separate step of antibody modification prior to their attachment to liposomes. Torchilin et al. [87] have recently described a new method which allows a single step binding of ligands containing amino groups (for example antibodies) to the PEG-terminus of the liposomes using an amphiphilic PEG derivative, p-nitrophenylcarbonyl–PEG–PE (pNP– PEG–PE). pNP–PEG–PE incorporates into liposomes via its phospholipid residues, and binds amino groups via its water exposed pNP group, forming a stable, non-toxic urethane (carbamate) bond. The method permits the binding of several dozen protein molecules per 200 nm liposome, with retention of their specific activities. It was also shown that pNP–PEG–DOPE-liposomes with and without attached ligands demonstrate increased stability in mouse serum. Therefore, pNP–PEG–PE could serve as a very convenient tool for protein attachment to the distal ends of liposome grafted PEG chains [87]. Bendas et al. [81] have also introduced a new methodology for attaching antibodies, without their previous derivatization, on the PEG-terminus of liposomes. Anti E-selectin monoclonal antibodies were coupled, in mild basic conditions, to a new PEG-PE derivative that was end group functionalized with cyanuric chloride. Cyanuric chloride is a three-functional reagent with a strong reactive graduation and links antibodies via nucleophilic substitution at basic pH. The first two chloride substitutions can be achieved upon reaction with nucleophiles under slight basic conditions. At further states, reactivity of the third chloride is strongly depressed [81]. The nucleophilic substitution of chlorine atoms with primary or secondary

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Fig. 23. Conjugation of antibodies to membrane anchor PE–PEG–p-NP which then incubated with commercial Doxil™ to prepare immunoliposomes.

amines can be controlled with temperature. The first substitution on cyanuric chloride occurs in minutes at 0 °C, while the second substitution occurs in 12–24 h at ambient temperature, and finally

Fig. 25. The two alternative methods of conjugation using dicarboxylic acid as spacer group. The figure shows reaction schemes for the use of disuccinimidyl suberate (a) or the dicarboxylic acid anhydride (b) to introduce into liposome membranes as spacers of varying lengths terminated with a carboxyl functional group.

Fig. 24. Chemical activation of sterol-PEG1300 (a) by tresylchloride (b) and the resulting sterol–PEG1300–TRE (c) is coupled to BSA by acylation (d) or alkylation (e).

the third substitution typically occurs in 12–24 h and requires temperatures above 60 °C [98]. Using these characteristics of cyanuric chloride, Bendas et al. [81] prepared immunoliposomes by incubating monoclonal antibodies in borate buffer of pH 8.8 with liposome containing cyanuric chloride–PEG–PE at room temperature for about 16 h upon shaking. Therefore mAb does attach to the second chloride and the third chloride remains free. Also, the coupling reaction of antibodies to liposome surface at temperature above 60 °C is not preferable as most of the proteins are denaturate at temperature above 60 °C and might lose their biological activity. Although the free cyanuric chloride is regarded as a sensory respiratory irritant [99], comprehensive cell compatibility studies have proven that cyanuric chloride does not cause acute or chronic toxicity or genotoxic or mutagenic effects [100]. However, cyanuric chloride can be regarded as safe since its reactivity is pH-controlled. While substitutions occur in a basic medium around pH 8.5 (which is acceptable for proteins), cyanuric chloride is nearly non-reactive under neutral physiological pH [81]. Steenpaß et al. [88] conjugated BSA (as a model protein) to tresylated PEG (sterol–PEG1300–TRE) through secondary amide bond (alkylation) and/or sulfoacetamide linkage (acylation). While the secondary amide bond is stable in vitro and in vivo, sulfonyl linkages may be subject to slow hydrolytic cleavage. Furthermore, the formation of the stable secondary amine preserves the positive charge of the protein amino group and therefore may not decrease the biological function to the same extent as the alternative sulfoacetamide bond. As

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carbamate, p-nitrophenylcarbonyl or amide bonds, resulting from other amine-reactive coupling procedures also eliminate the positive charge, the coupling of proteins to liposomes may result in more effective ligand receptor interaction when tresyl-activated lipids are used for their attachment [88]. Also, they investigated that the covalent linkage between BSA and sterol–PEG1300 was stable (after incubating surface modified liposomes in human plasma (50% v/v) at 37 °C for 20 h) with regards to enzymatic stress, as well as a hydrophobic anchoring in the membrane which is resistant to interference by serum. However, further studies in animals can clarify if other destabilization or cleaving reactions occur under in vivo conditions. 8. Alternative conjugation strategies 8.1. Employment of liposomal carboxyl groups

Fig. 26. Employment of antibody carboxyl groups in immunoliposome preparation. In order to employ the carboxyl functions on proteins as linking groups for conjugation to liposomes, the endogenous amino groups must first be inactivated, to prevent antibody–antibody crosslinking from taking place after the carboxyl groups have been activated so that they may react with exogenous amino groups. The activating agents most commonly used are water soluble forms of carbodiimide. A convenient reversible blocking agent is citroconylate.

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 distance of 6–8 carbons in between the amide linkages has been found to be optimal in terms of the efficiency of linkage and minimization of non specific, non covalent binding of proteins. The lipid moiety containing the carboxylic acid spacer can be prepared in two ways. In the first the dicarboxylic acid is presented to the PE in the form of the NHS derivative [101], so that both the carboxy functions are activated: one of these reacts with the PE amino group to

Table 2 scFv′ immunoliposomes. Liposomal composition

Result

HSPC:Chol:DPPE:DiO (1:0.2:0.07:0.004, molar ratio)/sulfo-SMCC

ScFv-CM6 immunoliposomes loaded with NOAC-ETC showed increased binding affinity and up to 80% higher cytotoxic activity towards TEM1-expressing IMR-32 tumor cells (human neuroblastoma cell line IMR-32 and the human colon carcinoma cell line HT-29) compared to control liposomes [120]. Analysis of the blood circulation time of 3H-labeled Immunoliposomes (scFv A5, specific for endoglin CD 105) showed rapid clearance from the circulation with 50% values b 3 min. In contrast, the same liposomal formulation without coupling lipid had 50% values of approximately 1.5 h (t1/2α = 45 min, t1/2β = 9 h), indicating that Mal-PE is responsible for the drastically reduced circulation time [74]. Flow cytometry and fluorescence microscopy showed that EGFR-targeted immunoliposomes (Fab′ fragments derived from C225 or novel anti-EGFR scFv C10), but not nontargeted liposomes, were efficiently bound and internalized by EGFRoverexpressing cells, including glioma cells (U-87), carcinoma cells (A-431 and MDA-MB-468), and EGFRvIII stable transfectants (NR-6M) [121]. The scFv-cys (specific for transferrin receptor) targets the cationic liposome–DNA complex (lipoplex) to tumor cells and enhances the transfection efficiencies both in vitro and in vivo in a variety of human tumor models. This scFv-immunoliposome can deliver the complexed gene systemically to tumors in vivo, where it is efficiently expressed [122]. Radio labeled ScFv-liposomes (specific for fibronectin splice variant ED-B) accumulated in the tumors at 2–3 fold higher concentrations during the first 2 h after i.v. injection compared to unmodified liposomes. Animals treated i.v. with scFvliposomes containing 5-FdU-NOAC showed a reduction of tumor growth by 62–90% compared control liposomes [123]. Fluorescence microscopy studies confirmed binding of anti endoglin scFv A5 Ni-NTA-liposomes to HUVECs (endoglin positive human umbilical vein endothelial cells). In these experiments, no binding was observed with a control scFv or non-complexed Ni-NTA-liposomes [124]. Anti-FAP scFv 36 immunoliposomes specifically bound to Fibroblast activation protein (FAP)-expressing HT1080-FAPmo cells. No binding to these cells was observed for plain (untargeted) liposomes [125]. The fluorescence labeled anti-HER2 scFv proteins bind specifically to a human breast cancer cell line (SK-BR-3) that over express the HER2 receptor, indicating that the in vitro folded scFv fusion proteins are biologically active and the presence of conjugated multiple Alexa488 probes in their C-terminal end does not interfere with their binding to the antigen [126]. Binding of anti-CD22 scFv immunoliposomes (Mutated-HA22 scFv-liposomes) to BJAB cells was significantly greater than unconjugated liposomes. Mut-HA22-liposomes loaded with doxorubicin exhibited at least 2–3 fold more accumulation of doxorubicin in BJAB cells as compared to control liposomes [127]. Anti-PBV hdscFv25-immunoliposomes were found to be immunoreactive and they killed human hepatoma cells, SMMC-7721, in vitro with higher efficiency than nontargeted liposomes. They also showed high antitumor activity and resulted in a significant reduction in tumor size compared to nontargeted liposomes and PBV (Peptides in bee venom) [128]. Anti-endoglin scFv mE12 immunoliposomes but not unconjugated liposomes showed binding to mouse embryonic endothelioma cell line eEnd.2 cells. Cell binding was further increased by generating a bivalent scFv-Fc fusion protein composed of scFv mE12 and the human γ1 Fc part. Moreover, scFv mE12 was endowed with an additional cysteine residue in the linker region and applied for the generation of anti-endoglin scFv immunoliposomes [129].

PC:Chol:MPB–PE:MCC–PE (6:3:1, molar ratio)

DSPC:Chol (3:2, molar ratio):mPEG2000–PE (0.5–5 mol%):Mal–PEG2000–PE

DOTAP:DOPE (1:1, molar ratio) or DDAB:DOPE (1:1, molar ratio) with MPB-DOPE (5 mol%) SPC:Chol: NH2-PEG-PE (1:0.2:0.07, molar ratio)

EPC:Chol:DiI (7:3:0.03, mol ratio):Ni-NTADOGS EPC:Chol:mPEG–DSPE (6.5:3:0.5, molar ratio): Mal–mPEG–DSPE Alexa488 (fluorescence probe)-Anti-Her2ScFv (Glycosylated) conjugates DPPC (96 mol%):DSPE–PEG2000–Mal (4 mol%)

SPC:Cholesterol (5:4, m/m):Chol–PEG–COOH

EPC:Chol:Mal–PEG–DSPE (6.5:3:0.5, molar ratio)

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Table 3 Examples of active targeted liposomes under preclinical and clinical trials. Clinical data are obtained from http://www.clinicaltrials.gov. Targeting ligands/targets

Indications/tumor cells

Status

Reference

Anti-HER-2 MAb/HER2 receptors Anti-EGFR MAb/EGFR receptor Anti-MT1-MMP Fab′/MT1-MMP Anti-GAH Fab′/GAH Anti-VCAM-1 MAb/VCAM-1 NGR peptide/Aminopeptidase N Transferrin/Transferrin receptors Transferrin/Transferrin receptors Transferrin/Transferrin receptors Folate/Folate receptor Hyaluronan/CD44 receptor RGD peptide/Integrins (αvβ3) GPLPLR Peptide/MT1-MMP

BT-474/MCF-7 breast cancer MDA-MB-468, U87 glioma HT1080 cells Metastatic stomach cancer Human tumor cell line Colo677 Orthotopic neuroblastoma Ovarian cancer cell A2780 C6 glioma Metastatic solid tumor Human KB carcinoma B16F10 melanoma B16 melanoma Colon 26 NL-17 carcinoma

Preclinical Preclinical Preclinical Phase I Preclinical Preclinical In vitro Preclinical Phase I Preclinical In vitro Preclinical Preclinical

[140,141] [142] [143] [138] [144] [145] [146] [147] [139] [148] [149] [150] [151]

form a phospholipid containing a free carboxyl group also activated with NHS. This compound is incorporated into liposomes, which will then react spontaneously with antibodies/proteins via the amino groups (Fig. 25a). The only problem with this method is that the PE-spacer-CO. NHS derivative is moderately stable in organic solvents and breaks down rapidly in aqueous media, so that it can only be used in cases where liposomes are prepared by a fairly rapid procedure and little subsequent processing is required. In the second method [102], the dicarboxylic acid is converted to an internal acid anhydride by reaction with carbodiimide (CDI); the anhydride then reacts with PE to give a derivative containing a free carboxyl group at the end of a spacer chain (Fig. 25b). This compound is very stable in both organic and aqueous media. Upon incorporation in liposomes, the carboxyl function is activated with a carbodiimide at low pH. After excess free carbodiimide has been removed, protein is introduced into the liposome suspension which binds rapidly and efficiently as the pH is raised. Up to 60% binding efficiency has been reported using this method.

8.2. Employment of protein bound carboxyl groups The methods described previously have involved using the amino groups on the surface of proteins to conjugate them to linkage groups, and subsequently to liposomes. This is because the amino group (together with sulphydryl groups) is one of the few functional groups capable of forming covalent linkages under physiological conditions without the need for activation. Amide linkages can be formed by reaction with activated carboxyl groups (e.g. N-succinimido ester), where the later reagents have been synthesized prior to coupling, using much harsher chemical conditions. Circumstances will arise where linkage to proteins or polypeptides via amino groups is unsatisfactory because the biological activity of the molecule is reduced hence the methods need to be found for conjugating proteins using other functional groups. The approach using chemical modification of oligosaccharide moieties has already been mentioned at the beginning of this article. Another possibility is to make use of the free

Table 4 Conjugation strategies of active targeted liposomes under preclinical and clinical trials. Liposome composition HSPC:Chol (3:2, molar ratio):PEG–PE(0–6 mol%): M-PE(2 mol%) DSPC:Chol (3:2, molar ratio):mPEG–DSPE (0.5–5 mol%):Mal–PEG–DSPE

Conjugation strategy used

Anti-HER2 MAb fragments (Fab′ or single chain Fv with C-terminus cysteine residue), specific for HER2 expressing breast cancer cells (BT474), were conjugated to liposomes via thioether linkage [140,141]. Intact anti EGFR C225 mAb (cetuximab), specific for EGFR-overexpressing MDA-MB-468 tumor cells, was digested with pepsin and the resultant C225-F(ab′)2 was reduced with 2-mercaptoethylamine. Thiolated Fab′ fragments were covalently conjugated to maleimide groups of liposomes via thioether linkage [142]. HSPC:Chol (6:4, molar ratio):DSPE–PEG:DSPE– Intact anti-MT1-MMP mAb (222-1D8), specific for MT1-MMP overexpressing cells (HT1080), was digested first with pepsin PEG–Mal (9:1, molar ratio) and then 222-1D8-F(ab′)2 was reduced with cysteamine hydrochloride. Thiolated Fab′ fragment were then conjugated to liposomes via thioether linkage [143]. DPPC:Chol:Maleimidated DPPE (18:10:0.5, molar F(ab′)2 fragments of Intact anti-GAH monoclonal antibody, specific for metastatic tumor cancer, were prepared by pepsin ratio) digestion. The F(ab)2/Intact GAH was thiolated with 2-iminothiolane and conjugated to liposomes through thioether linkage [138]. SPC:Chol:cyanu-PEG2000–PE:DiO (64.5:30:5:0.5, Intact anti-VCAM-1 mAb, specific for human non-small cell lung tumor cell line Colo677 which forms solid tumors with mol%) VCAM-1 positive vessels, was conjugated to liposomes through chlorine group present in the PE-PEG2000-cyanuric chloride via nucleophilic substitution [144]. HSPC:CHOL:DSPE–PEG2000:DSPE–PEG2000–Mal NGR peptide which targets aminopeptidase N, a marker of angiogenic endothelial cells (human NB cell lines GI-ME-N, GI-LI(2:1:0.08:0.02, molar ratio) N, HTLA-230, IMR-32, and SH-SY5Y) was used. The additional residues were added to the peptide NH2 terminus to obtain peptides GNGRGGVRSSSRTPSDKYC with a NH2-terminal Cysteine, which were then conjugated to liposomes via thioether linkage [145]. EPC:Chol:DSPE–PEG2000:DSPE–PEG2000–COOH: First the liposomes were modified with MAN. In this reaction, the NH2 group of liposomes was coupled with MAN using DSPE–PEG2000–NH2 (52:43:4:0.5:0.5, glutaraldehyde as a coupling agent, as previously reported [152,153]. Then the COOH group of liposomes was activated with EDCI and NHS and transferin, specific for brain glioma cell C6, was conjugated to liposomes through amide linkage [147]. mmol/mmol) DSPC:Chol:DSPE–PEG (2K):DSPE–PEG(3K)–COOH The COOH group of liposomes was first activated with carbodiimide (EDC) and sulfo-NHS. Transferin, specific for (2:1:0.16:0.032, molar ratio). transferring receptor overexpressing solid Colon 26 tumor cells, was then conjugated to liposomes through amide linkage [139]. HSPC(55%):Chol(40%):mPEG2000–DSPE(4.7%): Folate ligand, specific for folate receptor-overexpressing tumors (mouse M109 and human KB carcinomas, and mouse J6456 Folate–PEG3350–DSPE lymphoma) was used [148]. SPC:Chol:DSPE–PEG:DSPE–PEG–RGD (20:10:1:1, RGD (Arg–Gly–Asp) peptide, specific for several different integrins (αvβ3 and α5β1) expressing murine B16 and human molar ratio) A375 melanoma cells, was conjugated to DSPE-PEG using DSPE–PEG–N-benzotriazole carbonate (BTC). The reaction takes place between α-amines of the RGD and BTC group of the lipid [150]. DSPC:Chol: stearoyl-GPLPLR (10:5:1, molar ratio) GPLPLR peptide, specific for MT1-MMP expressed specifically on the angiogenic endothelium as well as tumor cells (Colon 26 NL-17 carcinoma cells), was used as a ligand [151].

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carboxyl groups on proteins, and this can be accomplished by employing carbodiimide or analogues to catalyse the formation of an amide bond between the protein carboxyl group and the amino group of PE or of a linking reagent. Unfortunately, since proteins possess numerous carboxyl and amino groups on each molecule, extensive crosslinking will occur if carbodiimide is used on its own. Thus, before carbodiimide is used it is necessary to block the endogenous amino groups of the protein to prevent their participating in the coupling reaction (Fig. 26). One of the most satisfactory blocking agents which can be used for this purpose is probably citraconic acid, since this can be removed under conditions (dialysis against pH 4.4) which may not be too deleterious for the protein. This method has been used successfully for conjugating antibodies to PE [103], which is then inserted into membranes during formation of liposomes by detergent dialysis. If one wished to prepare liposomes by other methods, and thus attach the protein/peptide to preformed membranes, the same procedure could be employed that is, reaction of the blocked protein with liposomes in the presence of carbodiimide. One would then be faced with the task, however, of unblocking the protein at pH 4.4 in the presence of liposomes, which can cause some undesirable leakage or hydrolysis. This problem could be avoided by conjugating the protein first to a linkage group (e.g. an aminothiol, or aminothioester) which can then be unblocked separately, before conjugating it to liposomes, as described earlier, using the maleimido-derivative of PE [104].

9. Recent activities of immunoliposome preparation 9.1. 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 applications [105]. scFv Abs (26–28 kDa) are composed of VH and VL chains that are joined via a flexible peptide linker [106]. The first scFv molecules were developed independently by Huston et al. [107] and Bird et al. [108] and represent the smallest functional VH–VL domains of an Ab necessary for high-affinity binding of antigen. Originally derived from genes isolated from murine hybridoma cell lines, an scFv is capable of binding its target antigens with an affinity similar to that of the parent mAb [108]. Peptide linkers that join the VH and VL chains usually vary from 10 to 25 amino acids in length and typically include hydrophilic amino acids; the most common linker is the decapentapeptide (Gly4Ser)3. The easiest constructs to engineer are noncovalent diabody, triabody and tetrabody molecules that assemble according to changes in the linker length [109]. scFvs are predominantly monomeric when the VH and VL domains are joined by a linker of 12 or more amino acids. However, scFvs with a linker length of three to 12 residues cannot fold into a functional Fv domains and instead associate with a second scFv molecule to form a dimer (diabody, ~ 60 kDa) [110] due to pairing of the VH of one chain to the VL of another. Furthermore, reducing the linker length to 3 amino acids or less can force scFv association into trimers (triabodies ~90 kDa) [111] or tetramers (tetrabodies ~120 kDa) [112]. Another diabody format, a cysteine-modified diabody (Cys-diabody), has been engineered via the introduction of cysteine residues at specific locations for improved stability. Cys-diabodies have the same antigen binding as the noncovalent diabodies with the advantage of being able to be chemically modified (e.g. with a radiometal) following disulfide bond reduction [113].

In general, monovalent Ab fragments such as scFv, dsFv (VH and VL chains joined by disulfide bond) and Fv have a low functional affinity and a short in vivo half-life, due to their small size and valency, properties of which are detrimental to some therapeutic applications. However, because rAb fragments are easily and cost effectively expressed, and are easily subjected to genetic engineering, they remain attractive therapeutic candidates. As a result, Ab engineering endeavours have generated various multi-functional and multivalent scFv-based fragments that have proven to be superior therapeutic reagents (e.g. scFv-Fc, scFv-CH3: where, the scFvs are joined to the CH3 domain, and scFvSA, SA: streptavidin), compared to full-length mAbs, in various medical applications [105]. There are several potential advantages of using scFv fragments over whole antibodies or larger fragments for liposome targeting. These include: i) slower clearance than mAb-targeted liposomes, as Fc-mediated clearance is eliminated [114]; ii) theoretically lower production cost for scFv fragments generated from bacterial cultures relative to whole antibodies generated from animal ascites or cell culture [115]; iii) the ability to select scFv with the desired affinity and specificity using phage display [116]; iv) the option of engineering tags into scFv constructs, which can aid in their identification and purification [117]; and v) the ability to engineer fully human fragments or fragments with low levels of nonhuman content, which will reduce the risk of immunogenic reactions [118]. In order to achieve coupling of scFv to liposomes, one or more additional cysteine residues are attached to the C-terminus of scFv fragments [74]. This allows for site-directed conjugation, with the reactive sulfhydryl group(s) located opposite the antigen-binding site. Thus, similar to coupling of Fab′ fragments, conjugation of these scFv fragments does not interfere with target cell recognition. ScFv molecules are well established and have been used by several research groups for the generation of targeted liposomes (Table 2). Expression of scFv fragments in bacteria normally results in a mixture of monomeric and dimeric molecules, the latter being oxidation products of two scFv molecules. Thus, in order to achieve efficient coupling, scFv preparations have to be reduced under mild conditions prior to coupling [119]. 9.2. Affibodies as targeting ligands (affisomes) In recent times, a novel class of small molecules called “affibodies,” which can be considered antibody mimics, have been examined for liposome targeting. Affibody molecules are relatively small proteins (6–8 kDa) that offer the advantage of being extremely stable, highly soluble, and readily expressed in bacterial systems or produced by peptide synthesis. The binding affinities of affibody molecules are considerably higher compared with the corresponding antibodies [130]. The binding pocket of an affibody is composed of 13 amino acids, which can be randomized to bind a variety of targets. In contrast to monoclonal antibody, affibody has following advantages as a targeting ligand. First, the small size of affibody (MW: 6 kDa) guarantees its tissue/cell penetration ability. Second, its functional end groups for chemical conjugation are distanced from its binding site. Moreover, affibody has a robust structure, and can be easily synthesized in a large-scale manner [131]. All of these advantages make the affibody a valuable ligand for targeted drug delivery and tumor imaging. Wikman et al. [132], for the first time, identified an affibody (His6ZHER2/neu:4) which can specifically bind to the HER2 extracellular domain with a nanomolar affinity (∼ 50 nM). The His6-ZHER2/neu:4 affibody also showed selective binding to native HER2 on breast cancer cells [132]. Since then, anti-HER2 affibody has been widely used as an efficient tumor imaging tool after conjugating with radionuclide. Due to the short plasma circulation and fast blood clearance, affibodies are optimal for tumor imaging, but not for the affibody drug conjugates and radiotherapeutics [133]. Therefore,

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extension of the affibody survival time might be a prerequisite for affibody mediated targeted therapy. The albumin binding technology has been used to extend the plasma half life of affibody. Fusing affibody to the Albumin Binding Domain (ABD), a small protein domain (5 kDa), has been shown to elongate the half-life of affibody in mice [133,134]. Recently, anti-HER2 affibody was also employed as a targeting ligand for nano-scaled drug delivery systems [131,135]. Alexis et al. [131] conjugated the anti-HER2 affibody to poly-(D,L-lactic acid)– poly(ethylene glycol)–maleimide (PLA–PEG–Mal) copolymer, which was used to prepare paclitaxel encapsulated nanoparticles. This nanoparticle formulation was specifically internalized to HER2 positive tumor cells, and subsequently demonstrated cellular toxicity [135]. Furthermore, an adenovirus capsid was modified with antiHER2 affibody to change the natural tropism of the adenovirus vector. The adenovirus fiber was redesigned to include the anti-HER2 affibody without affecting the virion formation. The modified adenoviral vector selectively delivered a dual-function transgene into HER2 positive breast cancer cells [131,135]. Recently, Puri et al. [136] conjugated an 8.3-kDa HER2-specific affibody molecule (Z (HER2:342)-Cys) to the surface of thermosensitive liposomes (called “affisomes”) aimed at improving the targeting efficacy of these liposomes for breast cancer treatment. Another study by Beuttler et al. [137] used a bivalent, high-affinity epidermal growth factor receptor (EGFR)-specific affibody molecule (14-kDa) for targeting PEGylated liposomes to EGFR-expressing tumor cell lines. Enhanced cytotoxicity toward EGFR-expressing cells was detected with mitoxantrone loaded affibody targeted liposomes compared to untargeted liposomes in these studies [137]. Since the receptor-binding domains of affibodies may differ from that of antibodies, affisome uptake mechanisms may result in altered outcomes. Therefore, further studies in vitro and in animals are needed to establish the projected advantage of affibodies as targeting ligands for liposomes. 10. 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 GAHtargeted doxorubicin-containing immunoliposomes (MCC-465) [138] and the transferrin-targeted oxaliplatin containing liposomes [139]. On the other hand, a much larger number of preclinical studies are published, using various nanomedicines (liposomes) and targeting ligands (Table 3). In this section we discuss the type of conjugation strategies used in the preparation of these actively targeted liposomes along with their liposomal composition (Table 4). 11. 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 certain target groups on ligands, peptides, proteins, carbohydrates, lipids, synthetic polymers, nucleic acids, and oligonucleotides. Knowledge of the basic mechanisms by which the reactive groups couple to target functionalities provides the means to cleverly design a modification or conjugation strategy. Choosing the correct reagent systems that can react with the chemical groups available on

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target molecules forms the basis for successful chemical modification. A careful understanding of target molecule structure and reactivity provides the foundation for the successful use of all of the modification and conjugation techniques which are discussed in this review. 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. References [1] N. Oku, Anticancer therapy using glucuronate modified long-circulating liposomes, Adv. Drug Delivery Rev. 40 (1999) 63–73. [2] T.M. Allen, C.B. Hansen, Pharmacokinetics of stealth versus conventional liposomes: effect of dose, Biochim. Biophys. Acta 1068 (1991) 133–141. [3] J.H. Senior, Fate and behaviour of liposomes in vivo: a review of controlling factors, Crit. Rev. Ther. Drug Carrier Syst. 3 (1987) 123–193. [4] H.M. Patel, Serum opsonins and liposomes: their interaction and opsonophagocytosis, Crit. Rev. Ther. Drug Carrier Syst. 9 (1992) 39–90. [5] M. Woodle, D. Lasic, Sterically stabilized liposomes, Biochim. Biophys. Acta 1113 (1992) 171–199. [6] F.J. Martin, Clinical Pharmacology and Antitumor Efficacy of DOXIL (PEGylated Liposomal Doxorubicin). Medical Applications of Liposomes, Elsevier, Amsterdam, 1998, pp. 635–688. [7] K. Maruyama, O. Ishida, T. Takizawa, K. Moribe, Possibility of active targeting to tumor tissues with liposomes, Adv. Drug Delivery Rev. 40 (1999) 89–102. [8] E. Mastrobattista, G.A. Koning, G. Storm, Immunoliposomes for the targeted delivery of antitumor drugs, Adv. Drug Delivery Rev. 40 (1999) 103–127. [9] G. Kohler, C. Milstein, Continuous cultures of fused cells secreting antibody of predefined specificity, Nature 256 (1975) 495–497. [10] P.A.M. Peeters, G. Storm, D.J.A. Crommelin, Immunoliposomes in vivo: state of the art, Adv. Drug Deliv. Rev. 1 (1987) 249–266. [11] P. Carter, L. Smith, M. Ryan, Identification and validation of cell surface antigens for antibody targeting in oncology, Endocr.-Relat. Cancer 11 (2004) 11659–11687. [12] P.A. McCarron, S.A. Olwill, W.M.Y. Marouf, R.J. Buick, B. Walker, C.J. Scott, Antibody conjugates and therapeutic strategies, Mol. Interv. 5 (6) (2005) 368–380. [13] J.W. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, Orlando, 1986, pp. 6–58. [14] E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring, New York, 1988, pp. 23–135. [15] E.A. Padlan, Anatomy of the antibody molecule, Mol. Immunol. 31 (3) (1994) 169–217. [16] A. Coulter, R. Harris, Simplified preparation of rabbit Fab′ fragments, J. Immunol. Meth. 59 (1983) 199–203. [17] J. Rousseaux, R.R. Prevost, H. Bazin, Optimal conditions for the preparation of Fab′ and F(ab′)2 fragments from monoclonal IgG of different rat JgC subclasses, J. Immunol. Meth. 64 (1983) 141–146. [18] G.T. Hermanson, Bioconjugate Techniques, second ed, Academic press, USA, 2008, pp. 783–823. [19] J.L. Palmer, A. Nissonoff, Reduction and reoxidation of a critical disulfide bond in the rabbit antibody molecule, J. Biol. Chem. 238 (1963) 2393–2398. [20] M.M.C. Sun, K.S. Beam, C.G. Cerveny, K.J. Hamblett, R.S. Blackmore, M.Y. Torgov, F.G.M. Handley, P.D. Senter, S.C. Alley, Reduction-alkylation strategies for the modification of specific monoclonal antibody disulfides, Bioconjugate Chem. 16 (2005) 1282–1290. [21] A. Béduneau, P. Saulnier, F. Hindré, A. Clavreul, J.C. Leroux, J.P. Benoit, Design of targeted lipid nanocapsules by conjugation of whole antibodies and antibody Fab′ fragments, Biomater. 28 (33) (2007) 4978–4990. [22] P. Simard, J.-C. Leroux, pH-sensitive immunoliposomes specific to the CD33 cell surface antigen of leukemic cells, Int. J. Pharm. 381 (2) (2009) 86–96. [23] G.T. Hermanson, Bioconjugate Techniques, Second ed, Academic press, USA, 2008, p. 67. [24] R.N. Perham, J.O. Thomas, Reaction of tobacco mosaic virus with a thiolcontaining imidoester and a possible application to X-ray diffraction analysis, J. Mol. Biol. 62 (1971) 415–418. [25] R.R. Traut, A. Bollen, R.R. Sun, J.W.B. Hershey, J. Sundberg, L.R. Pierce, Methyl 4mercaptobutyrimidate as a cleavable cross-linking reagent and its application to the Escherichia coli 30s ribosome, Biochem. 12 (1973) 3266–3273. [26] R. Jue, J.M. Lambert, L.R. Pierce, R.R. Traut, Addition of sulfhydryl groups to Escherichia coli ribosomes by protein modification with 2-iminothiolane (methyl 4-mercap-tobutyrimidate), Biochem. 17 (1978) 5399–5405. [27] A.C. Alagon, T.P. King, Activation of polysaccharides with 2-iminothiolane and its uses, Biochem. 19 (1980) 4341–4345. [28] T.T. Sun, A. Bollen, L. Kahan, R.R. Traut, Topography of ribosomal proteins of the Escherichia coli 30S subunit as studied with the reversible cross-linking reagent methyl 4-mercaptobutyrimidate, Biochem. 13 (1974) 2334–2340.

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