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Antibody humanization methods – a review and update Yaghoub Safdari

a e

& Masoumeh Khalili

a

, Safar Farajnia , Mohammad Asgharzadeh

b

c d

a

Biotechnology Research Center, Tabriz University of Medical Sciences , Tabriz , Iran b

Paramedical Faculty, Tabriz University of Medical Sciences , Tabriz , Iran c

Drug Research Center, Mazandaran University of Medical Science , Sari , Iran d

Young Research Club, Azad University of Sari , Iran

e

Student Research Committee, Tabriz University of Medical Sciences , Tabriz , Iran Published online: 02 Aug 2013.

To cite this article: Yaghoub Safdari , Safar Farajnia , Mohammad Asgharzadeh & Masoumeh Khalili (2013) Antibody humanization methods – a review and update, Biotechnology and Genetic Engineering Reviews, 29:2, 175-186, DOI: 10.1080/02648725.2013.801235 To link to this article: http://dx.doi.org/10.1080/02648725.2013.801235

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Biotechnology and Genetic Engineering Reviews, 2013 Vol. 29, No. 2, 175–186, http://dx.doi.org/10.1080/02648725.2013.801235

Antibody humanization methods – a review and update

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Yaghoub Safdaria,e, Safar Farajniaa*, Mohammad Asgharzadehb and Masoumeh Khalilic,d a

Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; Paramedical Faculty, Tabriz University of Medical Sciences, Tabriz, Iran; cDrug Research Center, Mazandaran University of Medical Science, Sari, Iran; dYoung Research Club, Azad University of Sari, Iran; eStudent Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran b

(Received 4 October 2012; accepted 7 March 2013) This article reviews recent advances achieved during recent years on various aspects of antibody humanization theories and techniques. Common methods for producing humanized antibodies including framework-homology-based humanization, germline humanization, complementary determining regions (CDR)-homology-based humanization and specificity determining residues (SDR) grafting, as well as advantages and disadvantages of each of these methods and their applications are discussed. Keywords: humanized antibodies; germline humanization; framework homology; CDR homology; SDR grafting; affinity; immunogenicity

Introduction Antibodies have emerged as effective tools in the treatment and diagnosis of different human diseases. Non-human antibodies have been demonstrated to induce human immune responses, which result in neutralization of administered antibody and limits the application of such antibodies in treatment of human diseases. To overcome this problem the technology of antibody humanization has been developed. Antibody humanization is an efficient approach to eliminate or reduce the immunogenicity of these antibodies. So far, various methods have been innovated by researchers for humanization of non-human antibodies and to improve their affinity, specificity and other properties. Each of these methods has its advantages and disadvantages. During recent years, several studies have been done on humanization of antibodies, and a huge number of data have been obtained. In this article, we review some important studies in this area from the few last years, and we discuss resultant advances in antibody humanization methods. CDR grafting based on framework regions (FWR) homology, and the role of vernier zone residues A common method for humanization of non-human antibodies is complementary determining regions (CDR) grafting in which the CDRs of non-human antibodies are *Corresponding author. Email: [email protected] Ó 2013 Taylor & Francis

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grafted onto the human frameworks. Usually, human frameworks with highest homology to the framework regions of non-human antibody are chosen as an acceptor for CDR grafting (Pelat et al., 2008; Robert et al., 2010; Cheung, Guo, Hu, Tassev, & Cheung, 2012). The major problem associated with antibody humanization by this method is the loss of affinity to their specific targets (Pavlinkova et al., 2001). The straightforward grafting of CDR loops from murine antibodies onto human frameworks do not affect the antibody affinity in some cases, while in many more cases it reduces the affinity significantly. Some murine residues in framework regions, referred to as vernier zone residues, have been demonstrated to affect the conformation of CDR loops and affinity of antibody. These residues are located in the β-sheet framework regions closely underlying the CDRs (Foote & Winter, 1992; Makabe et al., 2008). Therefore, after the selection of desired human frameworks these residues are retained in humanized antibody. For example, in the case of murine D1.3 antibody the vernier zone comprises 30 amino acid residues in framework regions of variable domains: 16 in heavy-chain (residues at positions 2, 27, 28, 29, 30 in FWR-H1; residues at positions 47, 48, 49 in FWR-H2; residues at positions 67, 69, 71, 73, 78, 93, 94 in FWR-H3 and the residue at position 103 in FWR-H4) and 14 in light-chain variable regions (residues at positions 2 and 4 in FWR-L1; residues at positions 35, 36, 46, 47, 48, 49 in FWR-L2; residues at 64, 66, 68, 69, 71 in FWR-L3 and residue at 98 in FWR-L4 (Foote & Winter, 1992). CDR grafting with vernier zone retaining has been the most common method for production of humanized antibodies. For example, Herceptin (Trastuzumab), an FDA-approved anti-human epidermal growth factor receptor 2 (HER2) antibody, has been produced in 1992 by grafting of CDR loops of murine antibody 4-5D onto human immunoglobulin G (IgG) frameworks with retaining of vernier zone amino acids. Besides this antibody, numerous antibodies have been produced by these methods, which are shown in Table 1. Since some murine residues (CDRs and some vernier zone residues) are transferred to humanized antibodies in this technology, they may retain the immunogenicity of parental murine antibody. Considering the important role of vernier zone residues on CDR conformation, Makab and colleagues conducted an experiment to investigate their effects on affinity of humanized antibodies. They first produced a humanized version of 528, a murine antibody recognizing epidermal growth factor receptor (EGFR), by straightforward transplantation of CDRs and found a loss of affinity of 1/40. Then, to determine what vernier zone residue is predominantly responsible for reduction of antibody affinity, they produced several mutant versions of this humanized antibody in which some of the vernier zone residues were back-mutated into those in the parental murine antibody. By replacement of a murine residue in the vernier zone, both entropy and enthalpy increased. Unfavorable entropy change completely compensated the favorable enthalpy increase and therefore prevented the improvement of affinity. Consequently, these results emphasize the important role of vernier zone residues and indicate that regulation of conformational entropy change upon humanization of murine antibody must be carefully checked and optimized by methods like X-ray diffraction and isothermal titration calorimetry (Makabe et al., 2008). Germline humanization Human germline genes could be used as an alternative source of framework regions for humanization of murine antibodies. Compared with framework regions derived from IgG, the germline genes have less intraclonal somatic hypermutation (Pelat et al., 2008). Therefore, it is expected that humanized antibodies with germline frameworks show lower immunogenicity than humanized antibodies with IgG frameworks. So, these

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Table 1. The methods of humanization and application of some humanized antibodies. Antibody

Humanization method

Disease/application

Type

Reference

Anti-TAG-72

Human VL + humanized VH genes

scAb

Pavlinkova et al., 2001)

Hu 9.3(anti CD-28)

CDR homology + germline humanization

Fab

Tan et al., 2002

humanized H23 antiMUC1 AKA

CDR grafting

Human adenocarcinomas of the pancreas GVHD, autoimmune disease, transplantation Breast cancer

scAb

Mazor et al., 2005

h82D6A3

Variable domain resurfacing

Full length Full length

Yoon et al., 2006 Staelens et al., 2006

KD-247

Sequential immunization with V3 peptides Phage display technique

Full length scAb + full length Full length

Eda et al., 2006 Yang et al., 2007 Hwang et al., 2005

Anthrax

Fab

Multi inflammatory diseases Human B-cell malignancies

scFv

Pelat et al., 2008 Finlay et al., 2009 Kugler et al., 2009

Pancrease cancer

scAb

Lung carcinoma

Full length scAb

HzKR127

SDR grafting

Anti-lysozyme CDR homology-based antibody humanization D1.3. 35PA83 FR homology germline humanization Humanized FR homology germline anti-RAGE humanization Anti CD19 FR homology germline antibody humanization + point mutations AntiCDR grafting + conversion CEACAM6 of some residues in FWRs Humanized Mutational lineage guided EBV321 MLG hWO-2 Germline humanization with retaining the vernier zone h357 Resurfacing of both VH and VL domain Anti-HCV Phage display Anti-human integrin αvβ3 2C9 Anti-GD2 hDB32-6

Anti-tumor-associated glycoprotein-72 Inhibiting VWFinteraction to fibrillar collagen HIV-1 Hepatitis B –

Alzheimers TNF-α antibody Hepatitis C

scAb

Full length scAb

Phage antibody display

Anti-angiogenesis therapy

scFv

Fusion of m2c9 variable regions to human constant regions CDR grafting on human IgG1 and IgG4 frameworks CDR grafting on human IgG1 + mutation in all FRs

Yellow fever

Full length + chimeric Full length Full length

Neuroblastoma Dengue virus infection

Riley et al., 2009 Yu et al., 2010 Robert et al., 2010 Chiu et al., 2011 Lun et al., 2011 Liu et al., 2011 Thibodeaux et al., 2012 Cheung et al., 2012 Li et al., 2012

Notes: TAG-72, Tumor-associated_glycoprotein_72; MUC1, Mucin 1; CEACAM6, Carcinoembryonic antigenrelated cell adhesion molecule 6; RAGE, receptor for advanced glycation end products; HCV, hepatitis C virus.

features encouraged research on application of these sequences in antibody humanization. Robert et al. (2010) used this method to humanize murine antibody

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WO-2, a monoclonal antibody produced against Aβ peptide correlated with Alzheimer’s disease. With retaining murine vernier zone residues to preserve the affinity, they built three humanized formats of this antibody, including single-chain antibody, Fab fragments and chimeric Fab fragments containing human constant regions. They found that the single-chain and Fab fragment forms of this antibody bound to Aβ peptide with approximately two-fold lower affinity than recombinant chimeric Fab fragments and with five- to six-fold lower than papain-cleaved parental Fab fragments. Even though the humanization of murine WO-2 resulted in reduction of affinity, it was not undesirable because the humanized derivates still had high affinity (at nanomolar range) to Aβ (Robert et al., 2010). Tan and colleagues also reported a moderate reduction in avidity of an anti-CD-28 antibody 9.3 upon humanization through this method. They constructed three different Fab formats of this antibody, including humanized Fab fragment (by fusion of humanized variable (VH) and light-chain variable region (VL) genes to human immunoglobulin G1 heavy chain constant domain 1 (IgG1-CH1) and human kappa light chain constant domain (Ck) genes, respectively), chimeric Fab fragment (by fusion of murine VH and VL genes to the same human constant domain genes) and hybrid Fab fragment (by fusion of human VL gene to human Ck and murine VH gene to human IgG1 CH1 domain gene). Humanized Fab fragments showed a moderate reduction in avidity compared with chimeric Fab fragments. This humanized format had a biological activity comparable to those of chimeric and hybrid Fabs and was able to show immunosuppressive activity (Tan et al., 2002). Similarly, Pelat and colleagues observed a loss of affinity upon germline humanization of Fab fragments of non-human primate antibody 35PA83, but using some mutations in CDR and/or framework residues they could produce humanized Fab fragments with neutralizing properties similar to parental Fab fragment (Pelat et al., 2008). IgG- or germline-based humanization: Advantage and disadvantages Although germline sequences are potentially less immunogenic and therefore preferred in humanization of antibodies, IgG-derived sequences are sometimes more favorable. For example, Thibodeaux et al. (2012) showed that chimeric antibodies constructed by fusing the variable domain of murine antibody 2C9 to the constant region of IgG were effective in prophylaxis and therapy of yellow fever infection, but those with the constant region of IgM were not. They found the Fc region of IgM-humanized 2C9 antibody responsible for this inability. This conclusion derived from the fact that these two antibodies, 2C9-cIgM and 2C9-cIgG, had similar variable domains and differed only in Fc portions (Thibodeaux et al., 2012). It has previously been reported that loss of murine IgG Fc region reduces the protection capacity of antibody (Schlesinger & Chapman, 1995). Activity and properties of humanized antibodies also vary depending on the class of IgG (Thibodeaux et al., 2012). Cheung and colleagues found that humanized antibodies with the same variable domain, but with different Fc regions derived from IgG1 or IgG4, exhibited different properties. The humanized anti-GD2 antibody with a constant region of human IgG1, referred to as hu3F8-IgG1, had more potent peripheral blood mononuclear cell antibody-dependent cell-mediated cytotoxicity (PBMC-ADCC) and polymorphonuclear leukocytes (PMN)-ADCC than parental murine antibody (m3F8). The same antibody with an Fc region of IgG4, referred to as hu3F8IgG4, also had nearly no PBMC-ADCC and PMN-ADCC (Cheung et al., 2012).

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Affinity maturation of humanized antibody Several approaches have been used by researchers to increase the affinity of humanized antibodies, whereby given residues in framework or CDR regions of engineered antibodies are altered. Among all CDRs, alteration of the heavy-chain CDR3 (CDR-H3) has been used most frequently to increase the affinity or specificity of antibodies (Riley et al., 2009; McCarthy & Hill, 2001; Gupta et al., 2010). This CDR is the most variable CDR and its variation arises from somatic mutations and recombination of the variable (V), diversity (D) and joining (J) fragment-encoding sequences (Yang, Yoon, Jang, & Hong, 2007). By altering the amino acid residues in CDR-H3 of humanized HzKR127 antibody (hHzKR127), Yang and colleagues obtained 12 different clones of single-chain antibodies with enhanced affinity to Hepatitis B virus preS1 antigen. They also converted five affinity matured single-chain fragment variables (ScFvs) to whole antibodies by attaching them to human heavy- and kappa light-chain constant regions; two of them exhibited an approximately six-fold higher affinity, and three of them showed lower affinity than the parental humanized antibody hHzKR127. This revealed that antibody affinity can also be affected by constant regions (Yang et al., 2007). Humanized antibodies produced by CDR grafting are usually less immunogenic for human than murine or chimeric antibodies; however, they can be still immunogenic because the CDRs are not human. To overcome this problem, some researchers have proposed specificity determining residues (SDR) grafting for antibody humanization instead of CDR grafting. The immunogenicity of such antibodies is expected to be lower than those produced by CDR grafting methods because they contain a lower number of potentially immunogenic mouse residues. SDRs are residues within CDR regions that are involved in interaction with antigen but not in antigen binding (Padlan, 1991). Yoon and colleagues randomly mutated the SDRs in CDR-H3 of AKA, a humanized antibody-recognizing tumor-associated glycoprotein-72 antibody, and could improve its affinity up to 22-fold. This affinity matured antibody, called 3E8, also showed approximately two-fold higher tumor uptake than AKA (Yoon et al., 2006). Humanization via resurfacing Antibody resurfacing method, firstly described by Paldan in 1991, is another strategy for humanization of non-human antibodies. This method involves the replacement of potentially antigenic surface framework residues with the most common human residues at those positions (Padlan, 1991). The basis of this method is that human anti-mouse antibody (HAMA) response to the variable region is caused only by surface residues (Novotny et al., 1986; Staelens et al., 2006). Antibodies humanized by this method usually exhibit little change in stability and affinity. For example, the humanized antibody h82D6A3 has shown slight decrease in affinity compared with parental murine antibody 82D6A3. Variable domains of this humanized antibody contain necessary murine framework residues for CDR loop conformation while they differ from variable domains of parental murine antibody in 10 surface residues in its framework regions (Staelens et al., 2006). Murine anti-tumor necrosis factor-alpha(TNF-α) antibody m357 has also been humanized by this method. This antibody contains 17 non-conserved surface residues in its framework regions, of which six have been altered to those in PPS4, a human antibody with variable domains sequences homologous to m357. The other surface residues have been restored in humanized antibody, since they were thought to be necessary to support the conformation of CDR loops. The humanized

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antibody, called h357 IgG1, possesses human IgG1 heavy-chain constant region as well as human kappa light-chain constant region. This antibody has shown high antigen binding affinity and bioactivity on TNF-α binding and neutralizing assay. It is also able to trigger ADCC and to complement dependent cytotoxicity in cells bearing a transmembrane form of TNF-α (Chiu, Lai, & Chou, 2011).

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Humanization based on CDR homology For the first time, Hwang and colleagues devised a new method of antibody humanization based on homology of CDR regions. They hypothesized that frameworks of murine and human antibodies with similar CDRs can support CDR structure of each other with high affinity retention (Hwang, Almagro, Buss, Tan, & Foote, 2005) In this method, homology of framework regions is not considered to choose human frameworks, and the critical murine residues (vernier zone residues) are not restored in humanized antibody. Using this method, the formation of motifs that may be recognized as foreign is reduced. The antibodies produced by this method have been found to retain good degrees of affinity, relatively more than those produced by the framework-homologybased humanization method. The two antibodies humanized through this method, antilysozyme (D1.3) and anti-CD28 antibodies, have shown a small loss in avidity to their targets, only 6- and 30-fold, respectively (Tan et al., 2002; Hwang et al., 2005) Mutation in constant region of antibodies Mutation of amino acid residues outside of variable domains has been used to give new properties to humanized antibodies. By grafting CDR loops of murine antibody DB32-6 onto human IgG1 as well as conversion of two leucine residues to alanine in the CH2 domain, Li and colleagues obtained a humanized antibody with high affinity to Dengue virus while lacking antibody-dependent enhancement (ADE) phenomenon. ADE phenomenon is believed to enhance Dengue virus infection. The humanized antibody they produced was able to neutralize the virus infection without Fc receptor binding (Li et al., 2012). Modification of the Fc region for eliminating ADE phenomenon had been described before (Hessell et al., 2007; Beltramello et al., 2010; Goncalvez, Engle, St, Purcell, & Lai, 2007). Improvement the specificity and affinity of antibodies Several modifications, mainly in CDR regions of either heavy or light chains, have been examined by researchers in order to improve specificity and affinity of humanized antibodies. McCarthy and colleagues inserted an aspartic acid residue at position 101 in CDR-H3 of anti-Legionella antibody and obtained an antibody with enhanced specificity toward Legionella serotypes (McCarthy & Hill 2001). This improved specificity was attributed to the formation of a salt-bridge between this aspartic acid residue and an arginine residue at position 94 of the heavy chain. The role of this saltbridge in conformational structure of the CDR-H3 region of different antibodies had already been demonstrated (Chothia et al., 1989). Gupta and colleagues mutated two tyrosine residues (one in CDR-H2 and the other in CDR-H3) of murine antibody MR1-1, a single-chain antibody recognizing EGFRvIII, to phenylalanine residues in order to improve its specificity. MR1-1 had cross-reactivity with wild-type EGFR (170

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KDa) as well as with another receptor of this family referred to as mLEEK, a 45-kDa protein. After exerting these mutations they found that this double mutant antibody, RAbDMvIII, lost its cross-reactivity to mLEEK. Binding to wild-type EGFR was dependent on western blotting condition; under reducing condition it had reactivity to EGFR, but in non-reducing condition it recognized only EGFRvIII (Gupta et al., 2010). Krykbaev and colleagues constructed an antibody library containing randomized residues at positions 92–95 of CDR-L3 of 26–10, an antibody that equally binds to digoxin and digitoxin. These two antigens have similar structure and are distinguished from each other only by an additive hydroxyl group at carbon 12 of digoxin. They found that substitution of a tryptophan residue at position 94 of the light chain shifted the specificity toward digitoxin, up to 47-fold higher specificity to digitoxin than to digoxin (Krykbaev, Tsantili, Jeffrey, & Margolies, 2002). Based on previously determined immunochemical characteristics and molecular modeling, Lamminmaki and colleagues hypothesized that insertional mutation in CDR-H2 of anti-stradiol antibody can lead to increased affinity. They constructed a library containing two, three or four random amino acid insertions at the tip of the CDR-H2 loop, between threonine 52a and glutamine 53 and expressed them on M13 phage as Fab fragments. Compared with wild Fab fragment, most of the mutants, especially those with insertion of three or four amino acids, were found to exhibit higher affinity toward stradiol and significantly lower cross-reactivity with testosterone, a molecule with very similar structure (Lamminmaki et al., 1999). Chames and colleagues produced a library of single-chain antibodies derived from parsimonious mutagenesis in anti-cortisol single-chain antibody, 5A4. They randomly mutated amino acids in five positions, including histidine 95, threonine 100b and phenylalanine 100d in CDR-H3; and asparagine 94 and proline 96 in CDR-L3 of the light chain, so that each clone contained only one of these mutations. Among these clones two were found to display improved affinities to cortisol. Both of these clones contained either threonine or serine instead of original asparagine at position 94 in CDR-L3 (Chames, Coulon, & Baty, 1998). Yu and colleagues humanized the rabbit monoclonal antibody EBV321 – recognizing vascular endothelial growth factor (VEGF)-A – via a method known as mutational lineage guided (MLG) humanization. They substituted non-critical residues in CDR-L1 as well as in framework regions of both heavy and light chains with human residues and obtained a humanized antibody, hEBV321, with retained affinity and biological activity of the parental rabbit antibody (Yu et al., 2010). By mutation of amino acid residues at six positions, mostly in framework regions (1 and 53 of VL and 24, 37, 48 and 99 of VH), Riley and colleagues also obtained four single-chain humanized antibodies from murine monoclonal antibody 13.1. These humanized antibodies were able to bind with high affinity to CEACAM6, an important tumor-associated-antigen belonging to the carcinoembryonic antigen (CEA) family, and inhibit the growth of murine pancreatic ductal adenocarcinoma (PDA) xenograft (Riley et al., 2009).

Immunogenicity of humanized antibodies Humanization methods are aimed at eliminating or at least reducing the immunogenicity of non-human antibodies for humans; however, some humanized antibodies and even some fully human antibodies still exhibit some degrees of immunogenicity. Remained immunogenicity has been shown to vary from one humanized antibody to another, ranging from negligible to intolerable responses. The antibodies humanized through the frame-

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work-homology approach contain murine CDRs as well as some critical murine residues (vernier zone amino acids) in their framework regions and therefore carry a number of potentially immunogenic residues in both their framework and CDR regions. By contrast, antibodies humanized via the CDR-homology approach are expected to show lower immunogenicity, since their frameworks are fully human and murine vernier residues that are potentially immunogenic are not retained in humanized antibody (Hwang et al., 2005). To further illustrate the reasons for immunogenicity, Harding and colleagues studied a number of humanized antibodies with different VH and VL family members while carrying unique CDR loops and found that immunogenicity always would be present in some antibodies owing to the nature of antigen-specific combining sites. They suggested the modification of amino acids in CDR regions as an alternative to reduce or eliminate the immunogenicity of humanized antibody (Harding, Stickler, Razo, & Dubridge, 2010). The immunogenicity of engineered antibodies has been reviewed by Hwang and Foote in 2005. Based on immunogenicity in patients, they classified the immunogenicity of antibodies into three categories including negligible (< 2%), tolerable (2–15%) and

Table 2. The sequence and length of linker in some single-chain antibodies. Name of single-chain antibody MR1-1 (anti-EGFRvIII antibody) Anti-gp240 antibody

Length Amino acid sequence 15 18

SGGGSGGGGSGGGGS ⁄

GSTSGSGKPGSGEGSTKG

RASA (anti-human sperm surface antibody) 3C10, anti EGFRvIII antibody Wild-type anti-cortisol scFv4 Anti- stradiol 17β

15

GGGGSGGGGSGGGGS

15

GGGGSGGGGSGGGGS

2C11 (hamster antimurine CD3ɛ) 4F10

15

PV1 (hamster antimurine CD28) mAb 13-1: antiCEACAM6 Anti CD19 antibody

15

15

OVB3 antibody

14

45Î HscFv (derived from C2-45 HmAb) Anti-Legionella surface antigen Anti-RAGE antibody

15

18 15

26

16

18 16

Reference Lorimer et al., 1996 Rosenblum et al., 2003 Norton et al., 2001

Okamoto et al., 1996 ⁄ GSTSGSGKPGSGEGSTKG Chames et al., 1998 GGGGSGGGGSGGGGS Kobayashi et al., 2008 GGGGSGGGGSGGGGS Griffin et al., 2000 SSADDAKKDAAKKDDAKKDDAKKDAS Griffin et al., 2000 GGGGSGGGGSGGGGS Griffin et al., 2000 GGGGSGGGGS cys GGGGS Riley et al., 2009 GGGGSGGGGSGGGGS Kugler et al., 2009 EGKSSGSGSESKVD Chaudhary et al., 1990 GGGGSGGGGSGGGGS Shibaguchi et al., 2004 ⁄ GSTSGSGKPGSGEGSTKG McCarthy and Hill, 2001 DGGGSGGGGSGGGGSS Finlay et al., 2009

Notes: ⁄Whitlow linker (Whitlow et al., 1993). RASA, anti-human sperm surface antibody; RAGE, receptor for advanced glycation end products; CEACAM6, Carcinoembryonic antigen-related cell adhesion molecule 6.

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marked (> 15%). Unlike murine antibodies, they found most humanized antibodies to cause negligible or tolerable levels of immunogenicity. Hu23F2G, Hu2PLAP, Hu5c8, Hu5c8, Hu5c8, cantuzumab, mertansine, vitaxin and omalizumab are examples of humanized antibodies that have been reported to benon-immunogenic for patients (Hwang & Foote, 2005). It is noteworthy that even fully human antibodies, produced by phage display technique or transgenic mice, may show degrees of immunogenicity. For instance, adalimumab and golimumab, produced by phage display technique and genetically modified mice, respectively, are fully human antibodies that have been reported to induce marked immune responses (Mahler, Marquis, Brown, Roberts, & Hoogenboom, 1997; Inman et al., 2008). Linkers of single-chain antibodies Linkers are an important component of single-chain antibodies which influence the conformation of VH and VL domains. The characteristics of a linker are affected by its length and amino acid compositions. Various linkers with different lengths and amino acid compositions have been used to join VH and VL segments among them, the 15-amino-acid-length glycine-serine linker, (Gly4Ser)3, has been the most commonly used linker (Table 2). The amino acid composition of linkers has been reviewed in detail before (Ahmad et al., 2012); therefore we proceed to studies carried out on their other prospects. Freund and colleagues carried out an a nuclear magnetic resonance (NMR) spectroscopy analysis to assess the influence of the common linker peptide (Gly4-S)3 on conformation of antigen binding site in Fv fragments. They found that linkers do not disturb the conformation of VH and VL domains and the differences between the NMR spectra of scFv and Fv fragments were related to the signals of glysine and serine residues in the linker. The linker was also found to be more flexible than the rest of single-chain antibody (Freund, Ross, Guth, Pluckthun, & Holak, 1993). Wang and colleagues studied the effect of linker length on affinity of single-chain antibodies. They prepared various forms of a single-chain antibody, anti-deoxynivalenol (DON) antibody, which had the same VH and VL genes but contained linkers with different lengths (0, 3, 5, 8 12 and 15 amino acids). They found that the affinity constant of scFvs is increased when the lengths of the linker reduced. There were significant differences in affinity constant of scFvs with various linker lengths. They revealed that, when the length of a linker was more than 12 amino acids, the VH and VL fragments could form monomeric scFv. When linkers had 3–12 amino acids, the VH and VL were not able to assemble into a functional scFv but could bind to another scFv molecule to form dimers. Finally, they reported that, when the linkers were shorter than three residues, they would form trimers by interaction between VH and VL domains (Wang, Zheng, Liu, Zheng, & Wang, 2008). Conclusion Various methods have been developed for humanization of antibodies up to now; each has its advantages and disadvantages. Depending on the case, one of these methods may be preferred. All properties of antibodies including immunogenicity, affinity and specificity can be modified by alteration of some amino acid residues in either CDR or framework regions. Considering the broad scope and increasing application of humanized antibodies in treatment of human diseases, more development in this area is expected in the future.

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