International Journal of Health Sciences and Research www.ijhsr.org ISSN: 2249-9571

Review Article

Bispecific : Progress and Application

Ayesha Habeeb, SumaiyaAl Hajree

Pharm-D, Deccan School of Pharmacy, Hyderabad, India

Corresponding Author: SumaiyaAl Hajree

ABSTRACT

Bispecific antibodies also termed as “dual targeting” or “dual specificity” antibodies are one of the fastest growing classes of investigational drugs with around 100 different formats in the development. However, only two of these, and are approved till date. Most of the bispecific antibodies are being investigated for oncological indication with around 20 being investigated for non-oncological indications. BsAbs show therapeutic effect by various mechanisms of action such as blocking two different or mediators that play an important role in disease pathogenesis, inducing cell signalling pathways such as in proliferation or inflammation, retargeting to mediate dependent cell-mediated cytotoxicity, by delivering toxicities or by engaging T- cells. And some of the bispecific antibodies are designed in a way so as to improve the pharmacokinetic properties (PK-modulating BsAbs). In this review, we focus on the mechanism of action of bispecific antibodies that are approved or are in clinical trials and discuss the progress and latest advances in the development of these drugs.

Key words: Bispecific antibodies, BsAbs.

INTRODUCTION proliferation or inflammatory pathways. [2] Bispecific antibodies possess the BsAbs do not usually occurs in nature and capability of binding simultaneously two are required to be created through the use of different antigens or two distinct epitopes on recombinant technology or by somatically a same . In case of same antigen the fusing two hybridomas (hybrid hybridoma / effector site may bind to molecules such as quadroma) or through chemical means. [3] antigens, enzymes, drugs, , toxins, Based on the presence or absence of radio nucleotides, plasma proteins and cells Fc domain, bispecific antibodies are divided such as t cells, NK cells, and into two types, IgG-like and non IgG-like neutrophils. Whereas the targeting site may bispecific antibodies. IgG-like bsAbs have bind to molecules such as cytosine, growth conserved Ig constant domain, thus these factors, cellular targets like receptors and bear an Fc region which helps in adhesion molecules and infectious agents. [1] purification of the bsAb using protocols By simultaneously recognizing two different established for IgG molecules, and can targets, bsAbs can act as mediators to contribute to improved solubility and redirect immune effector cells, such as stability. Also these antibodies can exhibit Natural Killers cells and T-cells, to tumor Fc mediated effector functions, which might cells, enhancing their destruction. In be desirable add-ons for therapeutic addition, by targeting two different applications, such as antibody-dependent receptors in combination on the same cell, cellular cytotoxicity (ADCC), complement bsAbs can cause modifications of cell fixation (CDC), and enhanced long half-life signaling, including the inactivation of resulting from their larger size and FcRn- International Journal of Health Sciences & Research (www.ijhsr.org) 259 Vol.8; Issue: 1; January 2018 Ayesha Habeeb et al. Bispecific Antibodies: Progress and Application mediated recycling processes. Small heavy chain. Also the heterodimeric Fc part bispecific antibodies that lack constant can be stabilised by adding artificially domain on the other hand rely entirely on created disulfide bridges. [5,6] their antigen-binding capacity to exert their To ensure the light chain association therapeutic activities. These bsAbs have apart from ensuring heavy chain association been primarily designed as effector cells a modification of the above method was recruiters and T-cells engagers. [4] introduced called CrossmAb. In this BsAbs were first developed by technique the heavy and light chains of only Quadroma or Hybrid hydromas approach in one arm are modified by immunoglobulin 1980s. This technology consisted of domain crossover (Crossover of the producing bsAbs by somatic fusion of an antibody domain within one Fab arm of a antibody-drug producing lymphocyte and a bispecific IgG antibody whereas the heavy myeloma cell. Later a chimeric quadroma chain association can be corrected by was created from a murine and a rat Knobs-into-holes, electrostatic steering and hybridoma cell line with some other alternative approaches. This approach improvements. Recently a more advanced allows generation of various formats of method has been described using mild bispecific antibodies including bi (1+1), tri reducing agent 2-mercaptoethanesulfonic (2+1) and tetra (2+2) antibodies and also acid sodium salt to produce bsAbs from non Fc tandem Fab based antibodies. mAbs of a different or the same subclass Various alternative technologies have been and same species rapidly. In addition to developed further after the description of having two antigen-binding sites for CrossmAb technology allowing generation different antigens the intact Fc region can of IgG based bispecific antibodies from any bind to / macrophages, natural pair of antibodies. Some of these killer cells, dendritic cells or other Fc approaches include DVD-IgG and CODV- receptor expressing cells. Thus, because IgG, duo bodies, Dual action FA‟s (DABs) there are three regions binding, this type of or Dutadabs etc. Numerous CrossmAbs bsAbs is called trifunctional bispecific have been evaluated in preclinical studies, antibody or triomabs. [5,6] advanced four of which are currently in Another engineering is knobs-into- active phase 1/2 clinical trials. [6,7] holes which is based on transfection of Dual-variable-domain immunoglobulin modified human antibody coding genes in (DVD-Ig) format was first described in mammalian cells. Advantage over 2007. In this method the target binding Quadroma approach is that there is less variable domains of two preexisting immunogenicity as the formed bsAbs are of monoclonal antibodies are combined human nature instead of murine/ rat nature. through naturally occurring linkers to create The technology consists of developing a tetravalent antibody having dual heavy chain homodimers for specificities. The DVD-IG bsAbs preserve heterodimerization. It consists of creating the affinities of parent monoclonal „knob‟ variant by replacing small amino antibodies, hence both the antigen binding acid (T3664) on the CH3 domain of CD4- sites can function independent of one IgG immunoadhesin with large amino acid another as there is no stearic hindrance (Y407). „Hole‟ is produced by replacing posed. Monoclonal antibodies of different large amino acids with smaller amino acid nature (Human, murine or Chimeric) can be in the CH3 domain of humanised anti-CD3 combined/used in generating DVD-Ig antibody. And then, the knob is inserted into bispecific antibodies. [6,8] the hole.To optimise heavy chain Bispecific T-Cell engager antibodies association and stability of bsAbs some (BiTEs) consists of fusing only the variable mutations has been developed in the past, regions of monoclonal antibodies in the two in knob heavy chain and four in hole former of Single chain Fvs (ScFvs) by

International Journal of Health Sciences & Research (www.ijhsr.org) 260 Vol.8; Issue: 1; January 2018 Ayesha Habeeb et al. Bispecific Antibodies: Progress and Application linker peptides. DARTs are diabodies like granzymes and lysosomal enzymes which molecules. In these the VH of the first lead to apoptosis of the target cells. [9,10] variable region is linked to the VH of In addition to costimulatory receptor second binder and the VH of second T-cells also express inhibitory receptors variable regionis linked to VL of the first (CTLA4, PD1etc) that on binding to their binder. Stabilization is achieved by cognate ligands (B7-1,B7-2,PD-L1 etc)on introducing additional disulfide bridges. target cells can inhibit the activation of T- TandAbsarebi specific fusion proteins with cells. This can be prevented by non four binding sites. Two of the binding sites activating antibodies which can bind to are for binding to tumor cells surface either the co-inhibitory receptors (anti- antigens and the other two bind to immune CLA4, anti-PD1) or to their ligands (anti- cells. [6] PD-L1, Anti-B7-1, anti-B7-2 etc). Effector memory cells are the most commonly MECHANISM OF ACTION OF involved T-cells in the tumor killing. They BISPECIFIC ANTIBODIES: work by same mechanism except that 1. Activating and Redirecting Cellular they‟re already activated and just require Immunity towards the Tumor Cells: stimulation via CD3 signalling. [11] The Bispecific antibodies showing Blinatumomab is one such BiTE this function activates and recruits the that, for its excellent effects in clinical trials unstimulated effector cells of immune achieved an accelerated approval from the system for destruction of tumor cells FDA in December 2014 for the treatment of bearing the target antigens. One of the children and adults with Philadelphia antigen binding sites identifies and binds to chromosome-negative relapsed (that had the target antigen on the tumor cells and the returned after earlier treatment) or refractory other site binds to the suitable leukocyte. T- (those that failed to respond to standard Cells expressing CD3 and CD4 and NK therapies at all) B-cell precursor acute Cells expressing mostly CD16 are lymphoblastic leukemia. However on July frequently recruited leukocytes by bispecific 12, 2017under the change to full approval, antibodies. [9] FDA expanded the indication for Bispecific T-Cell engagers (BiTEs) blinatumomab to patients with Philadelphia are found to be very efficient in recruiting chromosome-positive ALL. One scFv binds the T-Cells and binding to tumor associated to CD19 positive lymphocytes, while the antigen enhancing the killing of the other binds to unstimulated primary human malignant cells. One scFv binds to the T- CD3. Aphase 3 randomised, open label Cell specific molecule, usually CD3 while multicenter clinical trial (TOWER, the other scFv binds to a tumor associated NCT02013167) was conducted in 405 antigen. Initial recognition events include patients with relapsed or refractory B-cell binding of T-cell to CD3 binding fragment ALL. Blinatumomab was administered at and tumor associated antigen. This activates 9mcg/day by continuous intravenous polyclonal T-cells in a non-MHC-restricted infusion for the first 7 dats of study fashion in the presence of appropriate followed by an escalated dose of 28mcg/day costimulatory receptors such as CD28,OX- starting from week 2 of treatment. 40,4-1BB etc to their cognate ligands viz. Statistically significant improvement was B7-1,B7-2,OX-40,4-1BBL etc. Other observed in patients treated with antibodies that can facilitate the activation blinatumomab compared to those treated of T-cells are anti-CD-28, anti-4-1BB etc by with Standard of care . The triggering the costimulatory receptors. Tcell long-term follow-up part of the study was activation by the signals is followed by their discontinued based on a recommendation degranulation releasing perforins, from an independent data monitoring committee (DMC) that the study be stopped

International Journal of Health Sciences & Research (www.ijhsr.org) 261 Vol.8; Issue: 1; January 2018 Ayesha Habeeb et al. Bispecific Antibodies: Progress and Application for benefit. It was also found to be effective molecules in the inhibition of metastases for patients who have recurred after formation. [14] chemotherapy and after allogeneic Dual affinity re-targeting (DART) hemopoietic stem cell transplantation. molecules are another set of bispecific Patients having non-Hodgkin‟s lymphoma antibodies which acts by recruiting immune also showed tumor regression with effector cells. The size of DART molecules blinatumomab. [12] is larger than that of BiTEs and this MT111/AMG 2111/ MEDI-656 contributes to serum stability and extended targets CEA, an immunoglobulin storage. MGD006 is a CD123×CD3 superfamily glycoprotein that is expressed bispecific antibody currently in phase 1 of on a variety of solid tumors and on GI the clinical trials for relapsed/refractory tumors.CEA (Carcinoembryonic antigen) is Acute Myeloid Leukemia (AML) or a glycosylated lumen oncofetal antigen intermediate-2/High risk myelodysplastic (antigen in normal cells is present in low syndromes. Other bsAbs of DART format concentrations and only in luminal portions which are currently in the phase 1 of clinical of cell) belonging to CEA related cell trials are MGD007 which is a GPA33×CD3 adhesion molecules(CEACAM) family of binding, PF-06671008 which is p- IgG gene superfamily. MT111 binds CEA cadherin×CD3 binding, MGD009 that is and CD3. In a multicenter phase 1, open B7H3×CD3 engaging, the former being label study, 39 patients with advanced GI studied against colorectal cancer and the adenocarcinoma were given MEDI 656 latter two against solid tumors. intravenously over 3 hours on days 1 Duvortuxizumab (MGD011, JNJ64052781) through 5 in 28 day cycles with is CD19×CD3 binding DART currently in dexamethasone premedication. At the end of the part-2 (dose expansion) phase-1 of the study 11 patients had stable disease. The clinical trials for relapsed/refractory B-cell median overall survival for 39 patients was malignancies. [15] 5.5 months and MTD of MEDI656 was TandAbsare tetravalent bispecific 5mg. Nausea, Vomiting, abdominal CD19/CD3 tandem diabodies comprising discomfort and fatigue were considered solely of Fvdomains, having two binding most common adverse effects. High level sites for each antigen. AFM13 is a NK-Cell antidrug antibodies were found in 19 recruiting TandAb, Fc portion of which patients. It showed rapid clearance and a binds to CD30 on the tumor cells and Fcγ short half life. [13] receptor IIIA (CD16A) and trigger antibody (MT110/AMG110), dependent cellular cytotoxicity (ADCC). BAY2010112(AMG212/MT112), CD16A is an antigen expressed on NK-Cell MOR209/ES414 are the other BiTEs which and macrophages and not on neutrophils. entered into clinical trials. Solitomab The crosslinking of CD30+ tumor cells and completed phase 1 while MT112 and NK-cells causes the activation of cytolytic MOR209 are in the phase 1 of clinical trials. activity of NK-cells. The cytolytic granules Solitomab can bindto polyclonal CD8+ and such as perforins and granzymes are CD4+ T-cells for highly potent redirected released. Perforins causes the formation of lysis of target cells. The mechanism pores in the target cells, cell lysing involves membrane damage, membrane components enter the cell and cause blessing, activation of procaspases 3 and 7, destruction. [16,17] AFM13 is now being fragmentation of nuclear DNA and cleavage studied in the phase 2 clinical trials as a of caspase substrate poly (ADP ribose) monotherapy against relapsed or refractory polymerase. Preclinical studies with MT110 Non-Hodgkin‟s lymphoma and in phase 1 in with a mouse surrogate molecule (muS110) combination with Merck‟s keytruda. have confirmed the efficacy of these AFM11 is a T-Cell binding TandAb with two binding sites for CD3 and two for

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CD19. By mechanism similar to AFM13 it REGN1979(anti-CD20×anti-CD3)for B-cell forms an immune synapse between CD3 and malignancies and ERY974 (anti- CD19 and causes destruction of tumor cells GPC3×anti-CD3) for advanced solid tumors expressing CD19. AFM11 entered phase 1 are in the clinical trials phase 1 currently of clinical trial for Relapsed or refractory recruiting participants. [15] (NCT03038230, CD19 positive B-cell Non-Hodgkin‟s NCT02290951, NCT02748837) lymphoma currently recruiting participants. scFv-Fc-Fab fused bispecific (Clinicaltrials.gov NCT02665650, antibodies such as Xmab 14045 (anti- NCT02321592NCT02106091). CD123×anti-CD3) for CD123 expressing Triomabs are another set of haematological malignancies and GBR1392 bispecific antibodies that enables immune (anti-Her2×anti-CD3) for HER2 positive redirection towards tumors by the formation carcinomas are in phase 1 of clinical trials of a tricellular complex comprising T-cells, currently recruiting participants. [15] tumor cells and expressing (NCT02730312, NCT02829372). accessory cells. Catumaxomabis a non BTCT44654 being studied for Non- humanised (rat/murine hybrid), Hodgkin‟s lymphoma(NHL) and chronic trifunctional, bispecific monoclonal lymphocytic lymphoma (CLL) as a single antibody combining two half antibodies of agent and in combination with both mouse and rat origin approved by (NCT02500407). JNJ European union in April, 2009 for the 63709178 is being studied in phase 1 intraperitoneal treatment of malignant Clinical trial currently recruiting ascites in patients with EpCAM positive participants. epithelial tumors for which a standard therapy is not available or is no longer 2. Delivering Cytotoxic Substances to the feasible. One Fab fragment (part of mouse Malignant Cells: hybridoma expressing anti-tumor EpCAM BsAbs that bind to the cell surface antibody, IgG2a) binds to EpCAM antigens and digoxigenin simultaneously are expressed on most epithelial tumors of non generated for targeted or pretargeted squamous differentiation and the other payload delivery. In this approach the fragment (part of rat hybridoma cell line targeting molecules are IgGs that bind to producing anti-tumor CD3 antibody, IgG2b) tumor antigens including Her2, IGFIR, binds to CD3 of T-cell receptor complex. CD22 or LeY. Payloads are The Fc region binds to accessory cells digoxigeninylated which comprises small expressing Fc gamma receptor I and III like compounds (Dig-Cy5, Dig-Doxorubicin) macrophages, dendritic cells and NK Cells. and proteins (Dig-GFP). Bispecific Formation of an immunological tricellular antibodies carrying unmodified hapten complex leads to release of cytokines binding module thats form non covalent derived from immune cells including IL-1 β, complexes between delivery vehicles and IL-2, IL-6, IL-12 and payload and can be separated on -1.The side effects were internalisation into the cell. This manageable, generally short lived and rarely intracellular payload release, facilitates the severeand most were related to cytokine uptake and improve the activity of rekease such as fever, chills, nausea, compounds whose molecular targets are vomiting, fatigue and abdominal pain. [18] located intracellularly. The additional Catumaxomab was found to be safe and stabilization of payloads by disulfide bonds appropriate in treating both hospitalised and renders stability in the circulation and out patients. [19] minimizes undesired premature payload cLc-Hetero-H chain IgG bispecific release. Once the complex reaches inside antibodies MCLA117 (anti-CLEC12A×anti- cells the payloads can be released by CD3) for acute myelogenous leukemia, reduction in disulfide bonds. Hapten based

International Journal of Health Sciences & Research (www.ijhsr.org) 263 Vol.8; Issue: 1; January 2018 Ayesha Habeeb et al. Bispecific Antibodies: Progress and Application bispecific antibodies deliver siRNA (Short- administered first which gets distributed and interfering RNA) effectively into the tumor bound to target sites and the non bound cells which knocks down some vital genes targeting vehicles are cleared from and can be considered a promising approach circulation. This is followed by for cancer therapy. SiRNA is haptenylated administration of haptenylated payloads by digoxigenin at its 3‟ end and formulated which becomes captured at the desired into nanoparticles consisting of dynamic target sites by hapten binding bsAbs. polyconjugates (DPCs) or into Lipid-based Initially pretargeting was done based on nanoparticles (LNPs). This is then conjugation of avidin/streptavidin modules completed with bsAbs in defined ratio of to bsAbs for accumulating biotinylated 2:1. The complex specifically targets cells (radioactive) payloads on target tissues. But expressing corresponding antigen and is this method induced immunogenicity due to then internalised into endosomes where the nonhuman haptens which was later Dig-siRNAs are separated from bsAbs. [20] improved by using standard haptensbinders. Tung et al. generated polyethylene glycol HSG(Histamine-Succinyl-Glysine) haptens (PEG) binding bsAbs. PEG is a polymer conjugated with chelating moieties such as that can be linked to various payloads such DTPA and DOTA were radiolabelled with as peptides, proteins, liposomes and In-111 and Ln-177/Y-90 respectively for nanoparticles in same manner as standard pretargeted radioimmunotherapy. A study haptens and hence can be used for targeted validated a SPECT/CT- based therapeutic nanoparticles delivery. In this the PRIT (pretargeting radioimmunotherapy) nanoparticles are coupled to other haptens. regimen -177Lu-DOTA-Bn that led to [21] 100% complete response and cure without Another method of payload delivery any treatment related toxicities with high through hapten binding is pretargeting in therapeutic indices for radiosensitive tissues which the targeting vehicles are such as blood and kidney. [20,22]

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Figure 1.Modes of Action of Bispecific Antibodies. [23]

A) Activate signalling by a ligand mimetic; toxin (DT390) fused with bispecific Single B) Inactivate signalling of two ligands or chain Fv of antibodies targeting human receptors by binding to them; C) Two- CD19 and CD22. It internalize into cell ligand inactivation: Two arms of the readily helping toxin entry into the cytosol bispecific antibody binds to different causing inhibition of protein synthesis and ligands on different cells belonging to the subsequent cell death through apoptosis. same population, such as DLL4 ×VEGF, Unlike cytostatic DT2219- TNFα × IL17A, IL14 × IL13 etc; D) Dimers mediated tumor toxicity is independent of Inhibition: BsAbs can bind to two cell cycle and p53 signalling. [24] receptor/ligands on the same cell Minicells are 400±20nm sized (HER2×HER3, HER2,HER4) eg. MM-111; bacterially derived anucleated nanoparticles E) Dual Inhibition: BsAbs can inhibit two created by inactivation of genes that different cytokines simultaneously, for controls normal cell division in bacteria. example, COVA322 that inhibits TNFα and These can be packaged with therapeutically IL17A; F) The antigen binding sites of significant concentrations of BsAb binds to Target cell receptor and T- chemotherapeutics, siRNA or shRNA after Cell receptor; Heavy chain site binds to NK complete and reproducible purification cells or dendritic cells or through procedures used to eliminate macrophages/phagosomes (e.g., contaminants such as parenteral bacterial Catumaxomab, , FBTA05); G) cells, debris etc, cross flow filtration and BiTEs bridges Target cells and T cells by centrifugation. These minicells loaded with binding to CD3/CD28 or chemotherapies is targeted to tumor cells CD19/CD20/CD22/CEA/EPCAM, through attachment of bsAbs to minicells respectively (e.g. Blinatumomab, MEDI- surface. One arm of bsAbs has specificity 565, MT110); H) towards liposaccharide content of minicells Transmembrane/Transcytosis: BsAbs cross and the inherent arm has specialty towards the membrane via receptor transport the tumor cells surface receptor. The (Transferrin receptor TfR) and bind to minicell bound to bsAbs is then internalised, enzyme receptor on the other side (BACE1). enfocytysed and degraded in the lysosomes. DT-2219 is a bispecific recombinant The minicell releases the cytotoxic drug immunotoxin containing the catalytic and allowing the tumor cell to commit suicide. translocation enhancing domain of diphteria Preclinical studies in xenograft models International Journal of Health Sciences & Research (www.ijhsr.org) 265 Vol.8; Issue: 1; January 2018 Ayesha Habeeb et al. Bispecific Antibodies: Progress and Application showed significant tumor killing by EGFR- advanced solid tumors was conducted in targeted-Paclitaxel-loaded minicells. Also which it was found to be safe and showed open label, phase 1 study in patients with clinical efficacy. [25,26]

Figure 2: Bispecific Antibody targeted, drug/SiRNA-packaged Minicells. [29]

Schematic showing the packaging of Bispecific antibody targeting HER2/HER3 anti-cancer drugs or SiRNAs into empty is useful in overcoming this resistance and minicells and targeting them to tumor cell- can restore the sensitivity of the drug GDC- surface receptor using bispecific antibodies 0941 towards prostate cancer cells. [28,29] where one arm of the antibody has minicell- surface O-polychharide specificity and the other arm has specificity for the tumor cell- surface receptor for example EGFR. Andreev J et al demonstrated that non covalently linking HER2 and PRLR at plasma membrane using anti-HER2×anti- PRLR bsAbs triggers HER2 degradation in the lysosomes and coupling an ADC to this rapidly internalising protein may be useful approach to enhance internalisation and cell killing activity. [27]

3. Overcoming Drug Resistance: The resistance developed against anticancer drugs is likely due to inhibitory checkpoint molecules as well as crosstalk Figure 3: ErbB3(Her3) interacts with ErbB2(Her2) to activate signaling pathways leading to multi-drug resistance in breast suggests between the signalling pathways. cancer. [30] Many therapeutic strategies are being developed to overcome this resistance out of In both luminal B and erbB2 which bispecific antibodies are best choice. subtypes of human breast cancer, Studies revealed that herugulin (a HER3 erbB2/erbB3 association may recruit IGF- ligand) induces resistance to GDC-0941, a 1R to form a trimeric complex activating PI- PI3K targeting drug against prostate cancer. 3K/AkT signalling and Src kinase and

International Journal of Health Sciences & Research (www.ijhsr.org) 266 Vol.8; Issue: 1; January 2018 Ayesha Habeeb et al. Bispecific Antibodies: Progress and Application resulting in transtuzumab resistance. Receptor tyrosine kinases (RTKs) (Huang: Cancer Res 2010); In erbB2+ are a superfamily of cell surface receptors breast cancer, association of erbB2 and that are involved in mediating intracellular erbB3 upregulates Survivin via a PI-3/ AkT- signalling by phosphorylating substrate dependent mechanism, and thereby confers proteins involved in cell proliferation, paclitaxel resistance. (Wang: Oncogene diffentiation, survival and migration. Thus, 2010); In luminal B breast cancer, the can stimulate and modulate the growth of erbB2/erbB3 heterodimers modulate ERα tumors. The Human Epidermal growth phosphorylation and activation through factor Receptors (HER) is a family in the MEK/MAPK and or PI-3 K/Akt signalling superfamily RTKs comprising of pathways and subsequently alter tamosifen EGFR (also known as ErbB1/HER1), sensitivity. (Liu: Int J Cancer 2007) HER2, HER3 and HER4. Although B7-H3 is a B7 family homologue 3, monospecific antibodies target EGFR, a transmembrane protein with IgG like HER2 or other receptors are already in structure belonging to B7 superfamily can therapeutic practice, Cancer cells are able to activate or inhibition T-Cell responses. It is escape the blocking of one signalling overexposed in malignant cells. Some pathway by taking up other signalling studies revealed that B7-H3 exhibited pathways. [28] inhibitory actions on host T-cells in cancer Bispecific antibodies mediating patients. BsAb targeting CD3 and B7-H3 simultaneous blockade of two pathways are was used to direct activated T-cells to kill now in Clinical development which are able tumor targets. In SCID-Beige mice model, to reduce the possibility of tumor escape ATC armed with B7-H3 bispecific antibody from such mechanisms. The showed increased cytotoxicity and cytokines phosphorylation of EGFR and HER3 production suppressing B7-H3 positive activates the downstream RAS/MAPK and cancer growth. [31] PI3K/AKT signalling pathways which Her2 is responsible for oncogenesis promotes cell growth and proliferation. and resistance to treatment of various solid Inhibition of either of the pathways alone tumors. Andrew et al developed a cannot completely inhibit the growth and HER2/CD3 targeting bsAbby replacing the proliferation of tumor cells. Sliwkowski et variable region of hu3F8-bsAb with al demonstrated the use of transtuzumab. In four humanized models of (MEHD7945A), a bispecific antibody in breast and ovarian cancer cells line vitro and in vivo, the results of which xenograft and Patient derived xenografts of revealed that MEHD7945A potently inhibits human breast and gastric cancer, it showed the phosphorylation of EGFR and HER3 as anti proliferative effects of transtuzumabas well as it enhances gemcitabine-mediated well as novel insensitivity to PD-1/PD-L1 cytotoxicity. [35] It is currently in phase 2 of immune checkpoint inhibition. [32] Clinical trials. Met oncogene have been reported to JNJ-61186372, a bispecific antibody be involved in acquired resistance of tumors targeting EGFR and cMET enhanced the towards EGFR inhibitors. [33] To solve this killing of EGFR mutant cells bispecific antibodies targeting MET/EGFR (NCT02609776).Bispecific antibody and MET/HER2 were developed that targeting EGFR and VEGFR2 showed successfully induced efficient internalisation potent antitumor activity by inhibiting of EGFR or HER2 and their degradation receptors and blocking PI3K/AKT and restoring the sensitivity of EGFR inhibitors. MAPK signalling pathways. [36] Negrinet al [34] investigated the ability of bispecific antibody targeting HER2 and Cancer 4. Inhibiting Multiple Signaling Antigen-125 (CA-125) with CIK cells Ligands/Pathways: against primary ovarian carcinomas. Results

International Journal of Health Sciences & Research (www.ijhsr.org) 267 Vol.8; Issue: 1; January 2018 Ayesha Habeeb et al. Bispecific Antibodies: Progress and Application showed that the cytotoxic activity of CIK (Clinicaltrials.gov Id: NCT02141295, cells with BsAbs was much higher than NCT03938880). It was also demonstrated CIKalone. [37] that simultaneous inhibition of VEGF/Ang- 2 normalises tumor vasculature and 5. Inhibition of tumor angiogenesis: prolongs survival in patients with Angiogenesis, the formation and glioblastoma by altering macrophages and development of new blood vessels is resident microglia. [38] stimulated and regulated by the release of DLL4, Delta like ligand-4 is a notch specific growth factors from the tumor cells, ligand which also plays an important role in endothelial cells or associated macrophages. vascular development. DLL4/Notch These growth factors include vascular signalling pathway regulates vasculature endothelial growth factor (VEGF), basic development as well as tumor angiogenesis. fibroblast growth factor (bFGF), Apart from VEGF gene, DLL4 is the only angiogenin, transforming growth factor gene whose haploinsufficiency leads to (TGF)-α, TGF-β, tumor necrosis factor embryonic lethalities and vascular defects. (TNF)-α, epidermal growth factor etc. Out However, there are many genes involved in of all these VEGF is considered most vascular development. Downstream of the powerful angiogenic agent in neoplastic VEGF/VEGF signalling pathway tissues, as well as in normal tissues, whose DLL4/Notch pathway plays a key negative expression is also associated with wide regulator. VEGF signalling pathway range of tumors in human. Production of activates the Notch pathway by up these growth factors is associated with regulating DLL4 expression. The Notch upregulation of physiological conditions activation causes down regulation of associated with tumors such as hypoxia VEGFR2 expression which in turn resulting from the increasing distance suppresses VEGF signalling pathway. This between the growing tumor cells and the results in inhibition of excessive vessel capillaries or from the inefficiency of new branching by prevention of endothelial tip vessels. Binding of vascular endothelial cell formation. Thus, this crosstalk suggests growth factor A (VEGF-A) to its endothelial that for the inhibition of tumor progression receptors leads to the progression of tumor, and angiogenesis, simultaneous inhibition of angiogenesis and vascular permeability. both signalling pathways. Angiopoietin 2 is primarily secreted by Navicixizumab/HD105, a bispecific endothelial cells. It is known to destabilise antibody blocks both VEGF/VEGFR and vessel assembly, increase vascular DLL4/Notch pathways in endothelial cells permeability, and cause endothelial cells resulting in inhibition of proliferation and sprouting and proliferation. Its upregulation signalling. [39] is evident in wide range of malignancies. In some preclinical and clinical studies VEGF 6. Mechanisms of Action of Bispecific inhibitors alone were shown to be antibodies in ailments other than cancer: promoting tumor invasion and metastasis by  Two factors dimerization: Emicizumab increasing the intratumoral hypoxia. binds to both the activated coagulation Simultaneously blocking two or more factor IX and to factor X, facilitating the angiogenetic factors can increase cascade reaction by mediating the therapeutic efficacy. activation of the factor X. This function /RG7221 and RG7716 is of coagulation factor VIII, which is are bispecific antibodies targeting Ang- deficient in haemophilia A patients. 2/VEGF currently being studied in phase 2 Phase 3 clinical trials HAVEN1, 2, 3 of clinical trials in patients with Colorectal and 4 are ongoing. [40] cancer and Wet Age-related macula  Targeted Apoptosis: RG7386 degeneration (AMD) respectively. simultaneously targets Fibroblast

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activation protein (FAP) on cancer TNF×IL17 respectively are currently in associated fibroblasts and Death phase 1 of clinical trials. [15] receptor-5 (DR-5) on cancer cells  Against Bacteria: Medi-3902 attacks leading to acidity driven hyper Pseudomonas aeroginosa bacterium and clustering of DR-5 and subsequently neutralized it‟s defences. One arm of it induces apoptosis in FAP positive tumor binds to Psl antigen and the other arm cells. Activation if DR-5 leads to binds to PcrV. PcrV and PRLR play an formation of death inducing signaling important role in acute and chronic complex followed by subsequent infection caused by Pseudomonas activation of caspase substrate 3,6 and 7 aeroginosa. Psl is an exopolysaccharide causing cell apoptosis. RG7386 showed that increases the availability of potent tumor cells apoptosis in vitro and neutrophils in recognising and in vivo in preclinical tumor models with phagocytosis the bacterium and PcrV is FAP positive stroma. [41] a serotype independent type III secretion  PK modulated receptor antagonists: system virulence factor, that prevents Vobarilizumab targets IL6 via its IL6 release of bacterium factors capable of receptor. It‟s one arm is anti-IL-6R neutralising phagocytosis process. nanobody linked to an anti-Human Patients infected generally were known Serum Albumin (HSA) nanobody. The to lack preexisting immunity and thus, HSA binding is meant to increase the in couldn‟t generate a humoral response vivo serum half life. IL6 is involved in against these antigens. MEDI-3902 stimulation of osteoclast differentiation showed superior synergistic protection and activation. Similar to against Pseudomonas aeruginosa- Vobarilizumab, Ozoralizumab binds its induced murine pneumonia compared to one arm with TNF, neutralising it and parent mAb combinations. [42] other arm with Albumin to increase its  Transmembrane transcytosis: The in vivo serum half life. [15] passage of large molecules across blood  Hormone mimetic action: RG-7992 is a brain barrier is restricted due to the bispecific antibody targeting Klotho beta presence of tight junctions between protein and Fibroblast growth factor endothelial cells in brain capillaries. receptor-1 (FGFR1), mimicking the Transport across bbb is allowed by metabolic hormone FHF1. Phase 1 of specific binding to receptors such as TfR clinical trial of RG-7992 has been that internalised and release the ligand completed. (NCT02593331) across the capillary endothelium. A  Two proteins/Two ligands inactivation: bispecific antibody binding to TfR with Many bispecific antibodies binding and one arm and BACE1 with other arm was inhibiting molecules are in clinical demonstrated. BACE1 (Beta amyloid development for various cancer as well precursor protein cleavage enzyme) is a as non-cancer diseases. XBI1034020 and enzyme that cleaves beta amyloid binding and inactivating beta amyloid precursor protein and release soluble 40 and 42 has been described for the Amyloid beta into brain interstitium. By treatment of Alzheimers and is currently binding it, the bsAb ensures it‟s being studied in phase 1 of clinical trials inhibition which leads to reduction in (NCT01958060).ALX0761 for soluble beta amyloid levels in the brain inflammatory diseases, RG7990 for preventing amyloid plaque formation. Asthma, MEDI 7352 for Osteoarthritis, [43] MEDI 0700 for Lupus arythromatosis,  Wnt Signalling: Wnt Signalling is an LY3114062 for rheumatoid arthritis important event in the osteogenesis and targeting IL17A×IL17F, IL13×IL17, bone formation that takes place during NGF×TNF, BAFF×b7RPI and growth, bone homeostasis or fracture

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repair. Sclerostin is a secreted factor 4. Kontermann, R.E. Strategies to extend produced by osteocytes that partly block plasma half-lives of recombinant antibodies. Wnt Signalling by binding to LRP5 and BioDrugs 23, 2009 : 93-109 LRP6 (Coreceptors expressed on surface 5. Suresh MR, Cuello AC, Milstein C: of bone cells). Dickkopf-1 is another Bispecific monoclonal antibodies from hybrid hybridomas. Meth. Enzymol. 1986; secreted factor that acts as Wnt 121:210–228. antagonist by blocking the binding of 6. SpasevskaI. An outlook on bispecific Wnt proteins to LRP5 and LRP6. antibodies: Methods of production and Monica et al demonstrated that dual therapeutic benefits. Biosciences Master inhibition of Sclerostin and DKK-1 by a Reviews. 2014;1-7 bispecific antibody leads to synergistic 7. Schaefer W, Regula JT, Bähner M, bone formation in rodents and Schanzer J, Croasdale R, Dürr H, Gassner nonhuman primates. [44] C, Georges G, Kettenberger H, Imhof-Jung  bNAbs: Broadly neutralizing antibodies S, et al.: Immunoglobulin domain (bNAbs) against the HIV-1 envelope crossover as a generic approach for the glycoprotein (Env) have been shown to production of bispecific IgG antibodies. PNAS 2011, 108:11187–11192. potently suppress viremia in animal 8. Wu, C. et al. Simultaneous targeting of models of HIV-1 and humans. To ensure multiple disease mediators by a dual- high potency without chance of mutant variable-domain immunoglobulin. Nat. escape, targeting distinct epitopes Biotechnol.2007; 25:1290-1297 required for the survival of virus is 9. Hoffmann, P.; Hofmeister, R.; Brischwein, needed. For this, Bispecific neutralizing K.; Brandl, C.; Crommer, S.; Bargou, R.; antibodies improving neutralisation Itin, C.; Prang, N.; Baeuerle, P.A. Serial activity are constructed. [45,46] killing of tumor cells by cytotoxic T cells redirected with a CD19-/CD3-bispecic CONCLUSION single-chain antibody construct. Int. J. Bispecific antibodies have shown Cancer 2005; 115, 98–104 10. Baeuerle, P.A. and Reinhardt, C. great therapeutic potential in oncological as Bispecific engaging antibodies well as non-oncological indications. The forcancer therapy. Cancer Res. 2009; growing field of immunotherapy needs 69:4941–4944 more understanding of mechanisms through 11. Schildberg FA, Klein SR et al. Co- which therapeutic efficacy can be obtained. inhibitory pathways in the B7-CD28 We have attempted to discuss the ligand receptor family. Immunity 2016; 44 mechanisms of working of bispecific (5):955-972 antibodies by focusing on the bsAbs under 12. Przepiorka, D.; Ko, C.W.; Deisseroth, A.; investigation. Yancey, C.L.; Candau-Chacon, R.; Chiu, H.J.; Gehrke, B.J.; Gomez-Broughton, C.; REFERENCES Kane, R.C.; Kirshner, S.; et al. FDA 1. Kontermann RE: Bispecific antibodies: approval: Blinatumomab. Clin. Cancer Developments and current perspectives Res. 2015, 21, 4035–4039. [Internet]. In Bispecific Antibodies. Edited 13. Peng L, Oberst MD, Huang J, Brohawn P, by Kontermann RE. Springer Berlin Morehouse C, Lekstrom K, et al. (2012) The Heidelberg; 2011:1–28. CEA/CD3-Bispecific Antibody MEDI-565 2. Kontermann RE, Brinkman U. Bispecific (MT111) Binds a Nonlinear Epitope in the antibodies. Drug Discovery Today 20, Full-Length but Not a Short Splice Variant 2015:838-847 of CEA. PLoS ONE7(5): e36412. 3. Mu¨ller D, Kontermann R. E. Recombinant 14. Ferrari Fc, Bellone S et al. Solitomab, an bispecific antibodies for cellular cancer EpCAM/CD3 bispecific antibody construct immunotherapy. Curr. Opin. Mol. Ther. (BiTE®), is highly active against primary 2007; 9:319–326 uterine and ovarian carcinosarcoma cell lines invitro. J ExpClin Cancer Res. 2015; 34:123

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15. Brinkmann U, Kontermann RE. The making 24. Bachanova, V.; Frankel, A.E.; Cao, Q.; of bispecific antibodies. MABS 2017, VOL. Lewis, D.; Grzywacz, B.; Verneris, M.R.; 9, NO. 2, 182–212 Ustun, C.; Lazaryan, A.; McClune, B.; 16. Wu J, Fu J, Zhang M, Liu D. AFM13: a Warlick, E.D.; et al. Phase I study of a first in class tetravalent bispecific anti- bispecic ligand-directed toxin targeting CD30/CD16A antibody for NK-cell CD22 and CD19 (DT2219) for refractory B mediated immunotherapy. Journal of cell malignancies. Clin. Cancer Res. 2015, Hematology& Oncology 2015;8:96 21, 1267–1272 17. Cochlovius, B. et al. Cure of Burkitt‟s 25. MacDiarmid, J.A.; Mugridge, N.B.; Weiss, lymphoma in severe combined J.C.; Phillips, L.; Burn, A.L.; Paulin, R.P.; immunodeficiency mice by T cells, Haasdyk, J.E.; Dickson, K.A.; Brahmbhatt, tetravalent CD3 CD19 tandem diabody, V.N.; Pattison, S.T.; et al. Bacterially andCD28 costimulation. Cancer Res. derived 400 nm particles for encapsulation 2000; 60, 4336:4341 and cancer cell targeting of 18. Seimetz D, Lindhofer H, Bokemeyer, C. chemotherapeutics. Cancer Cell 2007, 11, Development and approval of the 431–445 catumaxomab (anti- 26. MacDiamid, Brahmbhatt. Minicells as EPCAM × anti-CD3) as a targeted cancer versatile vectors for targeted delivery. immunotherapy. Cancer Treat. Rev. 2010 ; Current Opinion in biotechnology 2011, 36:458-467 22:909-916 19. Kurbacher, C.M.; Horn, O.; Kurbacher, 27. Andreev J, ThambiN, Bay EP, Frank JD, J.A.; Herz, S.; Kurbacher, A.T.; Martin JH, Kelly MP, Kirshner JR, Rafique Hildenbrand, R.; Bollmann, R. Outpatient A, Kunz A, Nittoli T et al. Bispecific intraperitoneal catumaxomab therapy for antibodies and Antibody Drug conjugates malignant ascites related to advanced Bridging Her2 and Prolactin receptor gynecologic . Oncologist 2015; Improve Efficacy Of Her2 ADCs. 20: 1333–1341. Molecular Cancer Therapeutics 2017. 20. Metz S,Haas AK, Daub K, Croasdale DOI: 10.1158/1535-7163.MCT-16-065827. R, Stracke J, Lau W, Georges G, Josel 28. Yang F, Wen W, Qin W. Bispecic HP, Dziadek S, Hopfner KP, Lammens A et Antibodies as a Development Platform for al. Bispecific Digoxigenin p-binding BP New Concepts and Treatment Strategies. antibodies for targeted payload delivery. International Journal Of Molecular Proc Natl AcadSci U S A.2011 May Sciences 2017, 18, 48; 17;108(20):8194-9 doi:10.3390/ijms18010048 21. Tung HY, Chen BM, Burnouf PA, Huang 29. Poovassery, J.S.; Kang, J.C.; Kim, D.; Ober, WC, Chuang KH, YanYT, ChengTL, R.J.; Ward, E.S. Antibody targeting of Roffler SR. Selective Delivery of PEGylated HER2/HER3 signaling overcomes Compounds to Tumor Cells by Anti-PEG heregulin-induced resistance to PI3K Hybrid Antibodies. Molecular Cancer inhibition in prostate cancer. Int. J. Cancer Therapeutics 2016Volume 14, Issue 6 ; 1317- 2015, 137, 267–277. 1326 30. Ma J, Lyu H, Jingcao H, Bolin L. Targeting 22. Cheal SM, Fung EK, Patel M, Xu H, Guo of erbB3 receptor to overcome resistance in HF, Zanzonico PB, Monette S, Wittrup cancer treatment. Molecular Cancer 2014; KD, Cheung NV, Larson SM. Curative 13:105 Multicycle Radioimmunotherapy Monitored 31. Ma J, Ma P, Zhao C, Xue X, Han H, Liu C, by Quantitative SPECT/CT Based Tao H, Xiu W, Cai J, Zhang M. B7-H3 as a Theranostics, Using Bispecific Antibody promising target for cytotoxicity T cell in Pretargeting Strategy in Colorectal Cancer. human cancer therapy. Oncotarget 2016. The Journal of Nuclear Medicine 2017; 32. Lopez-Albaitero A, Xu H, Guo H, et al. 58(11):1735-1742. Overcoming resistance to HER2-targeted 23. Trivedi A, Stienens S, Zhu M, Li H, therapy with a novel HER2/CD3 bispecific Yuraszeck T, Gibson J, Heather T et al. antibody. Oncoimmunology. 2017;6(3): Clinical Pharmacology and Translational e1267891.doi:10.1080/2162402X.2016.126 aspects of bispecific antibodies. 7891. ClinTranslSci2017; 10:147-162.

International Journal of Health Sciences & Research (www.ijhsr.org) 271 Vol.8; Issue: 1; January 2018 Ayesha Habeeb et al. Bispecific Antibodies: Progress and Application

33. Lin, L.; Bivona, T.G. Mechanisms of angiogenesis. MAbs 2016 Jul; 8(5): 892– resistance to epidermal growth factor 904. receptor inhibitors and novel therapeutic 40. Shima, M.; Hanabusa, H.; Taki, M.; strategies to overcome resistance in nsclc Matsushita, T.; Sato, T.; Fukutake, K.; patients. Chemother. Res. Pract. 2012, 2012, Fukazawa, N.; Yoneyama, K.; Yoshida, 1–9. H.; Nogami, K et al. Factor VIII-mimetic 34. Lee, J.M.; Lee, S.H.; Hwang, J.W.; Oh, S.J.; function of humanized bispecic antibody Kim, B.; Jung, S.; Shim, S.H.; Lin, P.W.; in hemophilia A. N. Engl. J. Med. 2016; Lee, S.B.; Cho, M.Y.; et al. Novel strategy 374:2044–2053. for a bispecic antibody: Induction of dual 41. Brunker P, Wartha K, FriessT, RichardsSG, target internalization and degradation. Waldhauer I, KollerCF, WeiserB, MajetyM, Oncogene 2016, 35, 4437–4446 Runza V, Niu H et al.RG7386, a Novel 35. Schaefer G1, Haber L, Crocker LM, Shia S, Tetravalent FAP-DR5 Antibody, Effectively Shao L, Dowbenko D, Totpal K, Wong A, Triggers FAP-Dependent, Avidity-Driven DR5 Lee CV, Stawicki S et al. A two-in-one Hyperclustering and Tumor Cell Apoptosis. antibody against HER3 and EGFR has MolCancer Ther. 2016 May;15(5):946-957 superior inhibitory activity compared with 42. DiGiandomenico A, Keller AE, Gao C, monospecific antibodies. Cancer Cell 2011; Rainey GJ, Warrener P, Camara MM, 20(4):472-86 Bonnellet al. A multifunctional bispecic 36. Chen Z, Xie W, Acheampong DO, Xu M, antibody protects against pseudomonas He H, Yang M, Li C, Luo C, Wang M, aeruginosa. Sci. Transl. Med. 2014,6, 262 Zhanga J. A Human IgG-like antibody co- ra155.doi:10.1126/scitranslmed.3009655. targeting epidermal growth factor receptor 43. Kanodia JS, Gadkar K, Bumbaca D, Zhang and the vascular endothelial growth factor Y, Tong RK, Luk W, Hoyte K, Lu Y, receptor 2 for enhanced antitumor activity. Wildsmith KR, Couch JA et al. Prospective Cancer BiolTher. 2016 Feb; 17(2): 139– design of anti-transferrin receptor bispecific 150. antibodies for optimal delivery into human 37. Chan JK, Hamilton CA, Cheung MK, brain. CPT Pharmacometrics Syst Karimi M, Baker J, Gall JM, et al. Enhanced Pharmacol2016; 5(5):283-91 killing of primary ovarian cancer by 44. Florio M, Gunasekaran K, Stolina M, Li retargeting autologous cytokine-induced X, Liu L, Tipton B, Salimi-Moosavi H, killer cells with bispecific antibodies: a Asuncion FJ, Li C, Sun B et al. A bispecic preclinical study. Clin Cancer Res. antibody targeting sclerostin and DKK-1 2006;12(6): 1859–1867. doi: 10.1158/1078- promotes bone mass accrual and fracture 0432.CCR-05-201 repair. Nat. Commun. 2016, 7, 11505. 38. Tampellini M, Sonetto C, Scagliotti GV. 45. Huang, Y.; Yu, J.; Lanzi, A.; Yao, X.; Novel anti-angiogenic therapeutic strategies Andrews, C.D.; Tsai, L.; Gajjar, M.R.; Sun, in colorectal cancer. Expert OpinInvestig M.; Seaman, M.S.; Padte, N.N.; et al. Drugs 2016;25(5):507-20. doi: Engineered bispecific antibodies with 10.1517/13543784.2016.1161754. exquisite HIV-1-neutralizing activity. Cell 39. Lee D, Kim D, ChoiYB, KangK, Sung ES, 2016;165:1621–1631. Ahn JH, Goo J, Yeom DH, Jang HS, Moon 46. Bournazos, S.; Gazumyan, A.; Seaman, KD. Simultaneous blockade of VEGF and M.S.; Nussenzweig, M.C.; Ravetch, J.V. DLL4 by HD105, a bispecific antibody Bispecific anti-HIV-1 antibodies with inhibits tumor progression and enhanced breadth and potency. Cell 2016; 165: 1609–1620.

How to cite this article: Habeeb A, Hajree S. Bispecific antibodies: progress and application. Int J Health Sci Res. 2018; 8(1):259-272.

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