See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/312890378 Bispecific Antibodies (bsAbs): Promising Immunotherapeutic Agents for Cancer Therapy Article  in  Protein and Peptide Letters · January 2017 DOI: 10.2174/0929866524666170120095128 CITATIONS 2 READS 878 7 authors, including: Some of the authors of this publication are also working on these related projects: Histoprotective potential of cocoa View project Currently, we are evaluating antimicrobial potency of essential oil on common sexually transmitted infections (STIs) and pathogenic members of the Enterobacteriaceae. View project Desmond Omane Acheampong University of Cape Coast 59 PUBLICATIONS   155 CITATIONS    SEE PROFILE Christian K. 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Acheampong1*, Christian K. Adokoh2,*, Paulina Ampomah1, Daniel S. Agyirifo3, Isaac Dadzie4, Francis A. Ackah1 and Ernest A. Asiamah2 1Department of Biomedical Sciences, School of Allied Health Sciences, University of Cape Coast, Cape Coast, Ghana; 2Department of Forensic Sciences, School of Biological Sciences, University of Cape Coast, Cape Coast, Ghana; 3Department of Molecular Biology and Biotechnology, School of Biological Sciences, University of Cape Coast, Cape Coast, Ghana; 4Department of Medical Laboratory Technology, School of Allied Health Sciences, University of Cape Coast, Cape Coast, Ghana A R T I C L E H I S T O R Y Received: October 1, 2016 Revised: January 10, 2017 Accepted: January 11, 2017 DOI: 10.2174/09298665246661701200 95128 Abstract: Antibodies have become the preferred therapeutic treatment option for cancers. Antibody therapy is associated with low toxic profile and specific in its activity, unlike chemotherapy and ra- diotherapy. Types of tumor are known to express multiple receptors that cross-talk to activate per- petual growth, proliferation and metastasis, and inhibit apoptosis in such tumors. Bispecific antibod- ies (BsAbs) are therefore the preferred agent for the treatment of such cancers due to its unique char- acteristics. This review discusses up to date therapeutic potentials of BsAbs. Keywords: Antibody, bispecific antibody (BsAb), immunotherapeutic agent, cancer therapy, diabody, antitumor. 1. INTRODUCTION Monoclonal antibody (mAb) therapy has become argua- bly the mainstay of modern medicines with proven potency and better safety profile, which has considerably improved outcomes of disease management and benefited millions of patients with serious conditions such as cancer [1].This has generated a lot of interest in its construction, development and use in recent times. The advancement in antibody engi- neering has resulted in the generation of different types of antibodies. These include fusion antibodies with bispecific properties. Bispecific antibodies are recombinant molecules, capable of binding two different targets simultaneously [2, 3]. They can therefore be applied to redirect effector cells such as Natural Killers (NK) cells and T-cells to cancers [2, 3]. Developing recombinant bispecific antibodies has be- come imperative, especially, in cancer therapy, because looking at it from the perspective of cancer biology, many cancers rely on multiple pathways for survival through cros- stalk between signaling cascades. There is therefore the need to simultaneously target the different signal pathways to fully inhibit cancer proliferation and growth [4, 5]. Single targeting does not appear to be the most efficient strategy to *Address correspondence to these authors at the Department of Biomedical Sciences, School of Allied Health Sciences, University of Cape Coast, Cape Coast, Ghana; Tel: +233 543710234; E-mails: dacheampong@ucc.edu.gh; do.acheampong@gmail.com; do.acheampong@yahoo.com; 2Department of Forensic Sciences, School of Biological Sciences, Univer- sity of Cape Coast, Cape Coast, Ghana; E-mail: cadokoh@ucc.edu.ghmailto: christattom@yahoo.com destroy such cancer cells. Thus, treatment of cancer with mAbs has not been as successful as expected due to some of these functional limitations. Therefore, producing bispecific recombinant antibody could be the way forward in improv- ing cancer therapy. The quest to improve the treatment out- comes has generated an interest in the development and us- age of bsAbs [6, 7]. The review therefore seeks to discuss the possible techniques that can be employed to design and con- struct bsAbs to improve their antitumor efficacy. 2. STRUCTURE AND FUNCTION OF THERAPEUTIC ANTIBODIES Antibodies, produced by B cells, are involved in the hu- moral branch of the adaptive immunity. They recognize pathogens or foreign molecules through specific binding to antigens expressed on them [8, 9]. Specific interaction can neutralize the threat pose by these pathogens or foreign molecules or trigger effector functions of the immune system to eliminate them. Examples of the effector functions are opsonization, activation of complement and antibody- dependent cell-mediated cytotoxicity (ADCC) [9, 10]. Anti- body is made up of two identical light (L) chains and two identical heavy (H) chains (Figure 1), with the molecular weight of the light and heavy chain being about 25 and 50 kDa respectively [11, 12]. Each light chain (L) is joined to a heavy chain via a disulfide bond and a combination of non- covalent interactions such as hydrogen bonds, salt bridges and hydrophobic interaction [13]. The parts of an antibody have specific functions. The arms of the Y shaped antibody 2 Protein & Peptide Letters, 2017, Vol. 24, No. 3 Acheampong et al. consist of the sites that can bind two antigens and, therefore, recognize specific foreign molecules [14]. This region of the antibody is referred to as the fragment antigen binding (Fab) region. It is made up of one constant and one variable do- main from each heavy and light chain of the antibody, with VH, VL, CH and CL representing variable heavy chain, variable light chain, constant heavy chain and constant light chain respectively [14]. The amino-terminal regions of the VH and VL vary greatly among antibodies with different specificities [15]. Within the variable regions, sequence vari- ability is concentrated in several hyper-variable regions. These hyper-variable regions constitute the antigen-binding site of an antibody, and are called complementarity- determining regions (CDRs) [16]. Remaining domains of VL and VH, which exhibit far less variation, are called the framework regions (FW). The Y shaped antibody base per- forms an important role in controlling immune cell activity and the region is known as the Fragment crystallizable (Fc) region. This region is made up of two heavy chains that con- tribute two or three constant domains depending on the class of the antibody [16]. For a given antigen to bind to a specific protein, Fc fragment guarantees that the antibody generates appropriate immune response [17]. Additionally, the Fc fragment can bind to different kinds of cell receptors, for example, Fc receptors, and other immune related molecules, such as complement proteins to mediate different physio- logical effects such as opsonization, cell lysis, and degranu- lation of mast cells, eosinophil’s and basophils [18]. Fc and Fab fragments can be generated from an antibody in the laboratory. Thus, the enzyme papain can be employed to cleave an immunoglobulin into two Fab fragments and an Fc fragment. Additionally, the enzyme pepsin can cleave below the hinge region to produce a F(ab’)2 fragment and a pFc’ fragment. Employing mild reduction, the F(ab’)2 fragment can be split into two Fab’ fragments. (Figure 1) [19]. This implies that the structure of a therapeutic antibody can be re- engineered using modern technology to improve its efficacy, and has actually informed the trend of research in this field of study in recent times. 3. THERAPEUTIC ANTIBODY AND CANCER MANAGEMENT Therapeutic Antibody and Cancer Management Antibod- ies have been used in several roles in cancer management. These include diagnosis, monitoring, and treatment of the disease [20, 21]. In diagnosis, flow cytometry has been used in the identification of different subsets of non-Hodgkin's lymphoma. Antibodies (Abs) have also been employed to monitor cancer progression, via the carcinoembryonic anti- gen measurement in colon cancer [22-24]. Essentially, Abs have been applied directly as therapeutic agent in cancer treatment [25, 26]. In relationship to treatment, Abs can bind to particular antigens on tumor cells to enhance the patient's immune surveillance. More interestingly, Abs can be pro- grammed to inhibit growth factors which promote cancer proliferation and growth, thereby blocking cancer cell growth [27]. Additionally, Abs can carry chemotherapy drug or radioactive particle. These categories of Abs are called conjugated antibodies. These Abs are used as a homing de- vice to transport one of these substances directly to the can- cers. The Abs moves through the body until it finds and hooks onto the target. It then delivers the drug or radioactive substance to where it is mostly needed. This obviously re- duces the toxicity to normal cells in other parts of the body because of the specific nature of the conjugate monoclonal antibodies [28]. Conjugated Abs are also sometimes referred to as tagged, labeled, or loaded antibodies because of their well-defined activity. Ab could also be used to select normal stem cells from bone marrow or blood in preparation for a hematopoietic stem cell transplant in cancer patients [29]. There are a number of things to consider when using Ab for any of the above stated cancer management applications. First, a target antigen on the cancer must be selected. It is imperative that this antigen is expressed uniquely on the tu- Figure 1. A schematic diagram of an IgG1 showing the various components of the heavy (H) and light (L) chains which constitute the IgG1. As indicated on diagram, the whole structure is divided into two main parts; fragment antigen binding (Fab) and crystallizable fragment (Fc). Fab is made up of variable light chain (VL), variable heavy chain (VH), constant light chain (CL) and constant heavy chain (CH) coupled with two binding antigen sites. The Fc is made up of four constant heavy chains (CH) linked with the Fab with disulfide bonds. Bispecific Antibodies (bsAbs): Promising Immunotherapeutic Protein & Peptide Letters, 2017, Vol. 24, No. 3 3 mor cells and not on normal cells. Thus, the selected antigen must be over-expressed on the cancer but not/under- expressed by normal cells. The immunogenicity of the Ab should also be considered. These therapeutic antibodies are often derived from non-human sources, therefore, they are capable of eliciting an immune response themselves. An- other important factor to consider is the Half-life. The Half- life should be long enough to produce the expected result [30]. Finally, logistical challenges such as cost and availabil- ity of the therapeutic antibody, and also the decision as to whether or not the mAb should be used alone or in combina- tion with other agent(s) should be considered in order to de- rive the desired effect. 4. SUITABLE CANCER ANTIGENS FOR ANTIBODY TARGETED THERAPY One way the immune system attacks foreign substances in the body is by the production of large numbers of antibod- ies. These antibodies circulate through the body until they find and attach to a target. Once attached, they are capable of recruiting other relevant parts of the immune system to at- tack the cancer cells [31]. Researchers are now able to design and produce antibodies that specifically target specific anti- gens, such as those found on cancers. Many copies of these antibodies are now produced in laboratories for such pur- poses. The key factor that is considered when engineering or producing Ab is to identify the antigen the antibody will at- tack. Using Ab for cancer treatment is not always easy, be- cause they have proven to be more useful against some can- cers than others [32]. The efficacy and safety profile of Ab in oncology vary depending on the nature of the target. More importantly, the target should be abundantly and homogene- ously expressed. Additionally, the target antigen should be accessible, consistently and exclusively expressed on the target cancer cells [33]. Secretion of the target antigen must be minimal as possible to derive the desire effect. This is because secreted antigens could possibly bind to the anti- body and inhibit sufficient antibody from binding to the tar- get antigen on the tumor surface [34]. Therapeutic Abs tar- geted antigens on tumor can be categorized into different groups. Abs target antigens associated with hematopoietic differentiation are glycoproteins usually associated with cluster of differentiation (CD) groupings which include CD20, CD30, CD33 and CD52 [35]. Cell surface differentia- tion antigens which are a diverse group of glycoproteins and carbohydrates found on the surface of both normal and tumor cells can also be targeted with antibodies in cancer therapy [36]. Additionally, growth factors and their receptors in- volved in growth and differentiation signaling could be tar- geted by Abs as suitable antigens for cancer therapy [36]. These growth factors includes: epidermal growth factor re- ceptor (EGFR) [36], ERBB2 [37], ERBB3 [38], MET [39], insulin- like growth factor 1 receptor (IGF1R) [40], ephrin receptor A3 (EPHA3) [41], tumor necrosis factor (TNF)- related apoptosis-inducing ligand receptor 1 (TRAILR1), TRAILR2 and the receptor activator of nuclear factor- κBligand (RANKL) [42, 43]. Angiogenesis related antigens are mostly growth factors that support the formation of new microvasculature and therefore are also suitable candidates for Abs targeted therapy. These include vascular endothelial growth factor (VEGF) and the receptor VEGFR, integrin αVβ3 and integrin α5β1 [44]. Finally, antigens of the stromal and extracellular matrix could serve as therapeutic targets for cancer treatment, and these include fibroblast activation pro- tein (FAP) and tenascin [45]. 5. ANTIBODY ENGINEERING AND DEVELOPMENT Advent of new techniques in molecular biology in the early 1990s made it possible to clone genes of IgG molecules and their derivatives in eukaryotic expression vectors [46]. This has paved way to develop and produce recombinant types of Ab from different cell lines in a reproducible fash- ion, and has undoubtedly solved some of the production challenges that come about as a result of the instability asso- ciated with most hybridoma lines [47]. Cloning of genes of antibody allowed for antibodies modification through sub cloning, random or directed mutagenesis and molecular evo- lution procedures. This has made it probable to augment the production of recombinant antibodies and kicked started antibody engineering [48]. Antibody engineering application has resulted in generation of chimeric antibody, which is made up of fully human Fc portion and therefore about 70% human (Figure 2). Chimeric antibody is created by fusing murine variable domains, responsible for binding antigens, with human constant domains responsible for binding effec- tor cells [49] resulting in the development of a new genera- tion of therapeutic candidates [50]. In humans, humanized Fc portion renders chimeric antibodies substantially less immu- nogenic. With significant progress in antibody engineering methods, it has become probable to reduce further the mur- ine part of mAbs by replacing the hyper-variable loops of the murine antibody by hyper-variable loops of a fully human antibody, employing a technique known as complementarity- determining region grafting [51]. These antibodies are there- fore 85–90% human and have a reduced amount of immuno- genic tendencies than chimeric antibodies (Fig. 2). Notwith- standing, the complementarity-determining region grafting require special expertise to successfully carry out, compared to the mere fusion. For instance, directed mutagenesis ap- proach is often required to restore the affinity in the murine parental antibody. An antibody generated with this technol- ogy is called humanized antibodies, and most of the clini- cally approved Abs currently in use are either chimeric or humanized. The in vitro selection techniques development, espe- cially, the phage display is a major improvement in the de- velopment of therapeutic antibodies. The synthesis of reper- toires of antibody fragment genes are made possible via the improved antibody engineering from immunized or non- immunized animals, including humans. This therefore im- plied that a powerful selection technique was required to isolate antibodies from this large number of potential ligands, capable of binding specific antigen. This selection method like most in vitro methods relies prominently on the ability to establish a physical connection among a particular protein and the gene encoding it. Thus, establishing a link between a protein fused to a filamentous phage capsid pro- tein (p3 or p8) displayed on the phage M13 surface and the matching gene confined in the encapsidated DNA [52]. Im- munopurifying the said molecule via conjugating to antigen of interest makes the gene of interest readily accessible and therefore allows for sequencing and further multiplication of 4 Protein & Peptide Letters, 2017, Vol. 24, No. 3 Acheampong et al. the particular clone. One advantage of in vitro selection techniques is that, it has made it probable to efficiently select fully functional human antibody fragments against virtually any antigen, using large non-immunized libraries [53]. Addi- tionally, in vitro selection techniques can be used to improve the affinity of a therapeutic antibody through the creation of a secondary library comprising of first candidate of mutants and carrying out vigorous in vitro specificity against the an- tigen of interest [54]. More importantly, Phage display and equally efficient ribosome display have successfully been used to produce ligands with higher affinities for the antigen of interest compared to most of the conventional Abs [55]. The quest to generate fully human antibodies in the last dec- ade has led to the development of a complementary approach in the production of therapeutic antibodies. The idea was that creating 8 humanized mice could provide the vehicle for the production of fully human antibodies. Transgenic ‘human- ized’ mice were therefore generated by substituting the whole mouse IgG repertoire with a human repertoire [56]. These humanized mice produce human IgGs upon immuni- zation (Figure 2). The already popular hybridoma technique can then be used to clone the human antibodies with the mandatory properties [57]. Notable advantages of this ap- proach are that, it yields already matured in vivo antibodies, and therefore require no additional affinity maturation. More importantly, it directly yields full-length IgG, the preferred format for therapy. Nevertheless, one major limitation to this technique is that the humanized mice cannot be used effi- ciently when the immunogen has high toxicity or if the anti- gen of interest demonstrates high degree of homology shar- ing with its murine ortholog [57]. 6. BISPECIFIC ANTIBODY: THE PREFERRED AGENT FOR CANCER THERAPY Bispecific antibody derivatives (bsAbs) have come to be accepted over time as the next generation of targeted drugs for cancer therapy. The general concept underlying the de- velopment of bsAbs is about the physical connection of the recombinant antibody-derived moieties with at least two binding specificities [1]. Thus, bsAbs bind to two antigens or epitopes simultaneously, which is not the case in the conven- tional antibodies, it is therefore an improvement over the conventional antibodies. The bsAbs combine specificities of two different antibodies or proteins and concurrently address different antigens or epitopes (Figures 3 and 4). Aside its ability to interact with multiple cell surface receptors and ligands, bsAbs can also place target and effector cells in close proximity to trigger cytotoxicity in the target cell [58].These therapeutic bsAbs are mostly generated as re- combinant proteins which are either large IgG-like proteins with Fc moieties or as smaller moieties with bispecific anti- gen binding fragments but without Fc moieties. The first bsAb was approved in 2009 after many years of research, and as recent as December 2014, another bsAb was intro- duced into the market coupled with the fact that many of these bsAbs are at different stages of clinical trials [1, 59]. Basically, bsAbs can be grouped into two main classes based on the absence or presence of Fc moiety. The bsAbs without Fc moiety are smaller than those with Fc moiety (Figure 4). BsAbs with Fc fragment present with certain advantages which include the Fc region’s ability to facilitate its purifica- tion, employing the already established protocol for the puri- fication of IgG molecule [60]. Additionally, bsAbs with Fc moiety are relatively large and as a result has lengthy half- life which could enhance its potency as drug (Figure 3). Fur- thermore, Fc moiety mediated effector functions such as antibody-dependent mediated cytotoxicity (ADCC) and complement fixation (CDC) are associated with bsAbs carry- ing Fc moiety [1]. This bispecific IgG molecule is commonly developed via two diverse light and heavy domains ex- pressed in the same cell. However, due to the random as- sembly of the different chains that constitute the molecule, it leads to the formation of substantial amount of IgG mole- cules which are nonfunctional [61]. This bottleneck could be resolved by fusing another bind- ing moiety such as single chain fragment Fv to the N or C terminal of the light or heavy chains, resulting in the forma- tion of tetravalent molecules with two different reacting sites for particular antigen. Also a second VH and VL can be fused to the VH and VL respectively, forming double vari- Figure 2. Schematic diagram of the construction of monoclonal antibody (mAb) from the light and heavy chain gene, and the possibility of generating chemeric, humanized and fully human mAbs. Bispecific Antibodies (bsAbs): Promising Immunotherapeutic Protein & Peptide Letters, 2017, Vol. 24, No. 3 5 able domain (DVD) IgG [62]. Additionally, the knob into holes technology has been used in the generation of bivalent bispecific IgG. Thus, heavy-chain heterodimerization was generated by the introduction of different mutations into the two CH3 moieties, leading to the formation of asymmetric antibodies [63]. There could be variation in this approach which includes the usage of alternative mutations, hybrid CH3 moiety obtained from IgG and IgA antibodies or elec- trostatic steering effect [64]. Nevertheless, these technolo- gies or approaches are faced with random pairing challenge commonly referred to as light chain problem, which is ran- dom combination of the two chains of the heavy chain het- erodimer with the two different light chains [65]. Reportedly, this problem can be prevented by using common light chains instead of the different light chains, which allows binding to both antigens [66]. Although this approach has proved effi- cient in resolving the light chain problem, it has not been successful for many antibodies. In situation like this, bacte- rial expression containing light and heavy chain combination can be employed [1]. In contrast, the therapeutic potentials of bsAbs without Fc moiety depend solely on their antigen-binding capacity. However, due to their relatively smaller molecular size they are able to diffuse faster and therefore reach the target cells easily [1]. Production of this antibody is mostly done in ei- ther yeast or bacterial expression factory, which are known to be relatively cheaper but robust. In view of this, purifica- tion cannot be done employing the already established tech- nology for the purifying full IgG. As result, tagged amino acid such as His-tag which facilitates protein purification is added to the original bsAb during its construction for purifi- cation purposes. Small bsAbs without Fc moiety are either made up of variable VH and VL domains of two different antibodies or Fab moieties. Utilizing for instance the Bis- pecific T cell Engager (BiTE) technology, two scFv moieties are fused genetically, resulting in the formation of tandem scFv molecule. Two scFv fragments form independent fold- ing molecules and are linked with flexible peptide linker [67]. Alternatively, the diabody technology can be em- ployed. Thus the variable domains VH and VL from two antibodies (A and B) are expressed as two polypeptide chains VLB -VHA and VHB-VLA (Fig. 4). These domains are linked via short flexible peptide linkers, resulting in het- erodimerization of the two chains forming the diabody [68]. The diabody can further be modified to improve its therapeu- tic activities by transformation into a solitary chain to form scDb and dimeric tetravalent byproducts with two reacting sites for each antigen [1], and also the dual- affinity retarget- Figure 3. A schematic diagram of conventional IgG and bispecific IgG. (A) Conventional IgG with the same antigen binding sites; (B) Bis- pecificIgG with different antigen binding sites. Figure 4. Schematic diagram of bispecific antibody fragments without Fc moiety. (A) Single chain variable fragment (scFv); (B) Two single chain variable fragments fused with a linker employing BiTE technology, which is bispecific in function; (C) Bispecificdiabody (Db). 6 Protein & Peptide Letters, 2017, Vol. 24, No. 3 Acheampong et al. ing molecules DART which are disulfide-stabilized variants [1]. Construction and utilization of bsAbs became imperative when it became evident through research that growth, prolif- eration, metastasis of cancer cells can be attributed to more than one tumoral receptor (commonly two), which simulta- neously contribute to the survival of the cancer cells through cross-talk [69]. This situation is a common place in most cancer conditions. This has rendered most conventional anti- bodies ineffective in the treatment of cancers with these characteristics. In such cases, the best therapeutic option is to co-target the two receptors to derive the optimum benefit of the treatment. The suitable antitumor agent for this purpose therefore is undoubtedly bsAb and its derivatives. A case in point is the VEGFR2-EGFR (Vascular endothelial growth factor receptor2-Endothelial growth factor receptor) pathway cross-talk which plays critical role in the angiogenesis of the cancer cells. The extensive degree of EGFR-VEGFR path- way cross-talk has provided a rationale for joint targeting of these two pathways [70]. Reports from other studies else- where have also demonstrated that, combining EGFR and VEGFR2 antagonists increased the antitumor effect, and combined EGFR and VEGFR2 targeted therapy demon- strated significant therapeutic potential [71, 72]. My research team has successfully constructed and produced both recom- binant human IgG-like bispecific antibody (Bi-Ab) and bis- pecificdiabody (Db), targeting both VEGFR2 and EGFR [72, 73]. Our Bi-Ab exhibited improved antitumor activity and demonstrated slightly superior activity over their combina- tion (cetuximab and mAb-04) treatment. It has shown prom- ise as a therapeutic agent, especially, to treat cancers co- expressing EGFR2 and EGFR. Also, the Db demonstrated similar antitumor trend. Thus, the Db exhibited enhanced antitumor activity compared to their combination (anti- VEGFR2 scFv and anti-EGFR scFv) treatment and the treatment with individual components (anti-VEGFR2 scFv or anti-EGFR scFv) of the Db. Reports from these two stud- ies and others elsewhere give credence to the fact that, bsAbs are the preferred candidates for cancer therapy, especially tumor cells that express more than one tumoral receptor [72,73]. 7. MODE OF ACTION OF bsABs The mode of action of bsAbs on cancers can be catego- rized into three, based on the probable operational therapeu- tic paths. These are: recruitment of the appropriate immune cells, blocking receptor signaling and inactivating of ligand signaling, and targeted or pretargeted payload delivery bsAbs as summarized in Figure 5 [1]. As demonstrated in Table. 1, recruitment of immune cells such as an NK and T cell to the site of the cancer is an important step towards the elimination of the cancer cells. Although the ability of bsAbs to effectively recruit appropriate immune cells to eliminate or reduce the cancer cell burden in patients, only a few are already in the clinic with majority at various stages of pre- clinical trials. Anti-CD3 bsAb called catumaxomab was the first to received approval for clinical use [74]. It was devel- oped by a German Pharmaceutical Company called Trion Pharma as a trifunctional bsAb, a hybrid of murine IgG2a and rat IgG2b. The bsAb functionally targets a tumor anti- gen, and recruits T effector cells through CD3 binding and possibly activates NK cells, monocytes, macrophages and dendritic cells via its Fc moiety [75]. Catumaxomab has proven potent for the treatment of malignant ascites, and Figure 5. Mode of action of bispecific antibodies: (A) recruitment of the appropriate immune cells, (B) Blocking receptor ligand signaling pathway, and (C) Forced protein association (application of bsAbs in antibody mediated forced assembly of the coagulation Xasc complex). Bispecific Antibodies (bsAbs): Promising Immunotherapeutic Protein & Peptide Letters, 2017, Vol. 24, No. 3 7 therefore, it is being tried on ovarian, epithelial and gastric cancers. In addition to catumaxomab, there are other bsAbs with Fc moiety which have similar mode of action as catu- maxomab which were successful at the preclinical trials. Out of these, ertumaxomab which targets the cancer cells via HER2 and recruits T cells via its anti-CD3 arm, and lym- phomun that targets CD20 antigen on B cell lymphoma and recruits T cells through its anti-CD3 arm are currently being tried clinically [75, 76]. Additionally, there are bsAbs with- out Fc moieties such as blinatumomab (BiTE) which targets CD19 on acute lymphoblastic leukemia has demonstrated potency in the treatment of this cancer [76]. Another com- mon bsAb without Fc moiety which recruits immune cells is MGD006. MGD006 is DART that targets CD123 on hema- tological cancers and recruits T cells via CD3 [77]. Unlike the other bsAbs without Fc, AFM13 which is a TandAb re- cruits NK cells or macrophages via CD16A on these effector cells [78, 79]. The second mode of action of bsAbs is by blocking re- ceptor and inactivation of ligand signaling that promotes tumor growth. Receptor tyrosine kinases (RTKs) such as HER family which is made up of four members: HER1/EGFR, HER2, HER3 and HER4 which promote can- cer growth when activated [80]. They are therefore target for cancer therapy. Thus, mAbs such as panitumumab and cetuximab which target HER1/EGFR as well as pertuzumab and trastuzumab that target HER2 are known cancer thera- peutic agents [80]. Notwithstanding, most cancers are able to express more than one tumoral receptor which crosstalk to promote cancer growth, therefore using monospecific RTKs targeting antibody may not be the appropriate strategy to completely inhibit cancer growth. The bsAbs which are ca- pable of binding these receptors simultaneously are the best agents to completely inhibit the growth of such cancers. For instance, using pertuzumab and trastuzumab do not com- pletely inhibit ligand induce HER3 activation, however tar- geting both HER3 and HER2 simultaneously with bsAb completely inhibits cancer growth [80, 81]. Notable exam- ples of these kinds of bsAbs are MM111 (phase I trial) that targets HER2 and HER3, MM141 (phase I trial) that targets HER3 and insulin-link growth factor (IGF-IR) which is not a member of the HER family, and RG7597 (phase II trial) that binds HER1 and HER3 [1, 79]. Targeted or pretargeted payload delivery bsAbs is em- ployed to deliver payloads such as drugs, radiolabels and nanoparticles to the tumor site. The attached drugs or nanoparticles are usually too powerful to be used directly, because of their high toxic level. Additionally, bsAbs can be used to enrich payloads in tumor sites which prolong the serum retention time and improves the tumor/blood ratio [79, 82]. The use of radiolabel bsAbs is an efficient strategy em- ployed in diagnosing tumor or locating tumor site [83]. Thus, used for tumor imaging and radioimmunotherapy. The bsAb TF2 is a notable example of targeted delivery bsAbs, de- signed for such purpose. It works by binding to CEA and 99mT-labeled hapten histamine-succinyl-glycine (HSG) which is found on the surface of most solid tumors [84]. TF2 is currently undergoing phase I trial in patients with colorec- tal cancer [84]. Other possible applications TF2 include binding to Lutetium-177-labeled HSG or Indium-111 HSG and CEA for radioimmunotherapy in patients with advanced colorectal tumor, and it is in the phase I of the clinical trial [79, 84]. CONCLUSION This review has focused mainly on the therapeutic poten- tial of antibody for cancer treatment. The advent of new powerful antibody engineering has resulted in a generation of bispecific antibodies capable of simultaneously binding Table 1. Some common bsAbs in clinical development. bsAb Format Biological Function Target Status and Cancer type Catumaxomab TrioMab T cell recruitment, Fc- mediated effector functions EPCAM x CD3 Market: Malignant ascitis; phase II: ovarian, gastrointesti- nal, breast cancer; phase I/II: peritoneal carcinomatosis Blinatumomab BiTE T cell recruitment CD19 x CD3 Phase II: non Hodgkin lymphoma, acute lymphomatic leukemia Ertumaxomab   TrioMab T cell recruitment, Fc- mediated effector functions HER2 x CD3 Phase I: metastatic breast cancer Lymphomun   TrioMab T cell recruitment, Fc- mediated effector functions   CD20 x CD3 Phase I-II: BCL MGD006   DART T cell recruitment   CD123 x CD3 Phase I: AML AFM13   TandAb   NK cell recruitment CD30 x CD16 Phase I: Hodgkin Disease   MM111   scFv2-HSA Two-RTK inactivation HER2 x HER3 Phase I: breast, stomach and esophageal cancer MM141   IgG-scFv Two-RTK inactivation IGF1R x HER3 Phase I: solid cancers RG7597   DAF-IgG Two-RTK inactivation HER1 x HER3 Phase II: head and neck cancer, metastatic colorectal cancer TF2   D&L-Fab3 Payload delivery CEA x Hapten Phase I: colorectal cancer 8 Protein & Peptide Letters, 2017, Vol. 24, No. 3 Acheampong et al. multiple targets on cancer cell to inhibit its progression. This unique characteristic has been found to be very crucial in controlling cancers possessing multiple pathways for sur- vival and proliferation. Apart from its acceptable toxic pro- file, the agent has been specific in its activity, which ac- counts for its potent anti-tumor activity. Due to the proven therapeutic potential of bsAbs compared to the conventional Abs, a number of bsAbs are in clinical trials, given credence to the fact that bsAbs are gradually becoming the preferred therapeutic agents for the treatment of cancers. Nevertheless, identifying optimal application modes and potentially modu- lating the PK properties of bsAbs, especially small sized bsAbs molecules are some of the important aspects that can be addressed to further improve the efficacy of these cancer therapeutic agents. CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. 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