dual targeting strategies with bispecific which antibodies双靶向策略具有双特异性抗体

dual targeting strategies with bispecific which antibodies双靶向策略具有双特异性抗体 作文 手机作文网 dual targeting strategies with bispecific which antibodies双靶向策 略具有双特异性抗体 REVIEWmAbs 4:2, 182-197; March/April 2012; ? 2012 Landes Bioscience182 mAbs Volume 4 Issue 2IntroductionWith more than 20 monoclonal antibodies (mAbs) approved for therapy, and many more in clinical development, this class of molecules has become an established treatment modality for a variety of diseases.1,2 Antibody engineering is routinely applied to adapt the composition and activity for therapeutic applications in humans, e.g., to reduce immunogenicity or to increase or abro-gate ADCC through the modification of Fc-mediated functions.3,4 Unmodified mAbs possess a defined specificity for a single epitope effector of an antigen, and thus can interact with only a singular target. However, complex diseases such as cancer or inflammatory disorders are usually multifactorial in nature, Correspondence to: Roland Kontermann; Email: roland.kontermann@izi.uni-stuttgart.deSubmitted: 11/08/11; Revised: 12/05/11; Accepted: 12/09/11, inflammatory and infectious diseases and other disorders. Most of the marketed antibodies are monospecific and therefore capable of interacting and interfering with a single target. However, complex diseases are often multifactorial in nature, and involve redundant or synergistic action of disease mediators or upregulation of different receptors, including crosstalk between their signaling networks. Consequently, blockade of multiple, different pathological factors and pathways may result in improved therapeutic efficacy. This result can be achieved by combining different drugs, or use of the dual targeting strategies applying bispecific antibodies that have emerged as an alternative to combination therapy. This review discusses the various dual targeting strategies for which bispecific antibodies have been developed and provides an overview of the established bispecific antibody formats.Dual targeting strategies with bispecific antibodiesRoland E. KontermannInstitut für Zellbiologie und Immunologie; Universit?t Stuttgart; Stuttgart, GermanyKey words: bispecific antibodies, dual targeting, dual retargeting, cancer therapy, inflammatory diseases, allergic diseasesAbbreviations: ADCC, antibody-dependent cell- mediated cytotoxicity; dAb, domain antibody; Db, diabody; DT, diphtheria toxin; DVD-Ig, dual-variable-domain immunoglobulin; EGF, epidermal growth factor; epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; ETA, EGFR, pseudomonas exotoxin A; GvHD, graft-versus-host disease; HER2, human epidermal growth factor receptor 2; HER3, human epidermal growth factor receptor 3; HSA, human serum albumin; IGF-1R, insulin-like growth factor 1 receptor; IL, interleukin; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; scDb, single-chain diabody; scFv, single-chain fragment variable; sctb, single-chain triple body; sVD, single variable domain; taFv, tandem scFv; TRAIL, TNF-related apoptosis-inducing ligand; TRAIL-R2, TRAIL receptor 2; uPA, urokinase-type plasminogen activator; uPAR, urokinase-type plasminogen activator receptor; VEGF, vascular endothelial growth factor; VEGFR2, VEGF receptor 2involving a redundancy of disease-mediating ligands and recep-tors, as well as crosstalk between signal cascades. For example, several proinflammatory cytokines such as TNF, IL-1 and IL-6 have 作文 手机作文网 作文 手机作文网 been identified as key players in inflammatory diseases.5 In cancer, tumor cells often upregulate different growth-promoting receptors that can act either independently or crosstalk intracel-lulary through signaling networks.6,7 Of note, an acquisition of resistance to therapy is often associated with upregulation of alternative receptors as well as pathway switching between two receptors.8,9 Consequently, therapy with mAbs that target only a singular antigen has limitations.Blockade of multiple targets or multiple sites on one tar-get should result in improved therapeutic efficacy. This can be achieved by combination therapy with mAbs10 but also other therapeutic compounds. Improved efficacy in cancer therapy has been demonstrated with combinations of mAbs targeting different receptor tyrosine kinases on cancer cells or growth factors involved in angiogenesis, or a combination of both. Furthermore, combinations of mAbs targeting two different epi-topes on a single target have shown promising results. In these studies, different mAbs, with a focus on approved antibodies such as cetuximab (Erbitux?), trastuzumab (Herceptin?) and bevacizumab (Avastin?), were combined to treat solid tumors, including metastatic pancreatic cancer and breast cancer known to be dependent on expression of tyrosine kinase receptors EGFR and HER2, as well as angiogenesis induced by VEGF. However, combination therapy requires the development and approval of the individual antibodies, which involves substantial investment of resources for manufacturing, clinical studies and regulatory review. New approaches for combination therapy, therefore, include use of oligoreactive (polyclonal) antibody mixtures for the treatment of complex diseases.11,12 For example, Sym004, a mixture of two anti-EGFR antibodies, has shown promising results in preclinical studies,13,14 and is currently undergoing evaluation in a Phase 2 study (NCT01417936) in patients with www.landesbioscience.com mAbs 183REVIEW REVIEWstructures, e.g., cell surface receptors or soluble factors (Table 1) and (ii) those that use dual targeting for delivery (retargeting) of a therapeutically active moiety, e.g., effector molecules and effec-tor cells (Table 2). Direct actions include binding and neutraliza-tion of two ligands or two receptors, neutralization of a receptor and a ligand, activation of two receptors, activation of one recep-tor and neutralization of another receptor or a soluble factor, but also neutralization by binding to different epitopes of one recep-tor or ligand (Fig. 1A–H). Indirect actions include ADCC and CDC mediated by an Fc region, retargeting of immune effector cells through a further binding site, targeting of an effector mol-ecule, e.g., a toxin, a cytokine or a prodrug-converting enzyme and targeting of drug-loaded nanoparticles (Fig. 1I–O). Direct and indirect actions can be combined within one molecule to further improve efficacy.Applications of dual targeting strategies are likewise mani-fold, with the main indications being cancer therapy and the squamous cell cancer of the head and neck who responded to pre-vious anti-EGFR mAb-based therapy and subsequently became resistant to that therapy.During the past decade, dual targeting with bispecific antibod-ies has emerged as an alternative to combination therapy or use of mixtures. The concept of dual targeting with bispecific antibodies is based on the targeting of multiple disease-modifying molecules with one drug. From a technological and regulatory perspective, this makes development less complex because manufacturing, preclinical and clinical testing is reduced to a single, bispecific molecule. Therapy with a single dual-targeting drug rather than combinations should also be less complicated for patients.Dual Targeting StrategiesDual targeting strategies using bispecific antibodies can be divided into two types: (i) those that directly act on target Table 1. Dual targeting approachesTargetingTarget 1Target 作文 手机作文网 作文 手机作文网 2FormatIndicationReference2 receptorsTRAIL-R2LTβRscFv-IgG, IgG-scFvCancer67EGFRIGFRDi-diabodyCancer51EGFRIGFRdiabodyCancer50EGFRIGFRscFv2-IgGCancer49EGFRIGFRPEGylated tandem Cancer52EGFRIGFRIgG-scFvCancer9VEGFR2VEGFR3diabodyCancer61VEGFR2VEGFR3Di-diabodAdnectin yCancer60VEGFR2PDGFRαdAb-IgGCancer57PDGFRαPDGFRβdAb-IgGCancer54HER2HER3scFv-HSA-scFv Cancer53EGFRHER2tandem AffibodyCancer151CD20CD22F(ab’)2Cancer65CD20CD22IgG-scFv2, DNL-Fab-IgGCancer62, 63FcεRICD32Bkih IgGallergic diseases74CD32BCD79BDART moleculeArthritis72MP65SAP-2tandem dAbinfectious diseases791 receptor or ligandIGFRIgG-scFv2Cancer80CCR5IgG-scFv2HIV infections24FcεRItandem DARPinallergic diseases81scorpion toxintandem Nanobodyenvenoming852 ligandsIL-17AIL-23taFv-Fc, scFv-Fc-scFv, IgG-scFvinflammatory diseases93IL-1αIL-1βDVD-Iginflammatory diseases25, 92IL-12IL-18DVD-Iginflammatory diseases25VEGFosteopontinDVD-IgCancer89VEGFAng-2CrossMabCancer30VEGFAng-2CovX-BodyCancer20LukS-PVLukF-PVHCAb-VHHinfectious diseases841 receptor and 1 ligandPDGFRβVEGFscFv-Fc-scFvCancer100HER2VEGFtwo-in-one IgGCancer26FcγRIIIgEF(ab’)2allergic diseases75 184 mAbs Volume 4 Issue 2(e.g., implementing humanized or human antibodies), the com-position (e.g., size, valency, isotype, presence or absence of an Fc region) and production (e.g., applying prokaryotic or eukaryotic expression systems).A large group of recombinant bispecific antibodies are IgG-like molecules. In most of these formats, binding sites of second specificity are fused to the N- or C-a terminus of the heavy or light chain, e.g., in the form of an scFv fragment or a variable single domain, resulting in bispecific, tetravalent molecules. Bispecific molecules generated through fusion of an scFv fragment to a mAb offer great flexibility (Fig. 2). ScFv molecules have been fused to the N-terminus but also the C-terminus of the of the heavy or light chain of a mAb,21-23 generally without compro-mising productivity or antigen-binding activity, although issues regarding stability have recently been addressed.24 This group of IgG-like bispecific molecules also includes DVD-Igs, where a second VH and VL domain is fused to the heavy and light chain, respectively, of a mAb,25 two-in-one antibodies, where a second specificity is introduced into the natural binding site of an IgG molecule,26 and mAb2 molecules, where a second specificity is build into the CH3 domain of the Fc region.27 A characteristic feature of all these molecules is a symmetry caused by dimeric assembly of two identical heavy chains, an intrinsic property of these chains. A different approach is the generation of asymmet-ric IgG molecules. This can be achieved with the knobs-into-holes strategy.28 Here, amino acids at the contact site between the CH3 domains are substituted by larger or smaller residues forcing a heterodimeric assembly of heavy chains. One drawback is, how-ever, that there is still random association with the light chains. This has been addressed by generating bispecific molecules with common light chains,29 or, more recently, by domain swapping between one heavy and light chain resulting in CrossMabs.30 Heavy chain heterodimerization was also achieved by engineer-ing a charged CH3 interface to introduce an electrostatic steering effect or using the strand-exchange engineered domain technol-ogy (SEEDbody) with CH3 sequences composed of alternating segments from human IgA and IgG.31,32 In contrast to the bispe-cific IgG-like molecules, these bispecific antibodies are bivalent treatment of inflammatory and infectious diseases (Tables 1 and 2). Here, the same mechanisms used for combination therapy of antibodies can be targeted with bispecific antibodies. Multiple diseases mediators and 作文 手机作文网 作文 手机作文网 signaling pathways thus can be addressed and simultaneously inhibited by the dual targeting antibody.15 This includes targets that act independently on different path-ways, but also that are capable of cross-talking. Of fur-ther interest are bispecific antibodies targets targeting different epitopes on a disease mediator, which can lead to increased binding and enhanced neutralization.Bispecific Antibody FormatsBispecific antibodies with defined dual specificity suitable for therapeutic use must be generated through biochemical or genetic means (Fig. 2).16,17 Bispecific IgG molecules can be produced by somatic hybridization of two antibody-secreting hybridoma cells. These hybrid hybridomas (quadromas) produce within a cell two different heavy and light chains. Random assembly results in a mixture of IgG molecules, some of which are bispecific. These bispecific IgGs must be purified by two-step -raphy. However, the isotypes of heavy and light chains depend on the affinity chromatog origin of the parental antibodies, which allows little flex-ibility. Interestingly, however, the first bispecific antibody (catu-maxomab) approved in the European Union for the treatment malignant ascites is produced from a mouse/rat quadroma cell line,18 demonstrating the of feasibility of this approach to generate therapeutic bispecific antibodies. Alternatively, bispecific anti-bodies can be generated from existing antibodies by chemical conjugation, e.g., of two IgG molecules or two Fab’ fragments, using homo- or hetero-bifunctional coupling reagents.19 A differ-ent chemical coupling approach is used to produce CovX-Bodies, which comprise a catalytic IgG molecule covalently coupled to reactive, bispecific peptides.20Bispecific antibodies can be also produced by genetic engineer-ing and more than 45 different formats have been established in the past two decades (Fig. 2).16 A clear advantage of this approach is the greater flexibility regarding the origin of the binding site Table 2. Dual retargeting approachesRetargetingTarget 1Target 2FormatIndicationReferencetoxinsCD4CD26IgG-ricinGvHD112CD4CD29IgG-ricinGvHD113CD19CD22taFv-ETACancer116CD19CD22DT-taFvCancer114, 115HER2EpCAMDT-taFvCancer116EGFRIL-13RDT-EGF-IL-13Cancer119–122uPARIL-13RDT- IL-13-uPACancer123–125EGFRuPAREGF-uPA-ETACancer128EGFRIL-4REGF-IL-4-ETACancer126, 127cytokinesCD20HLA-DRF(ab)2-IFNα2bCancer132effector cellsCD123CD33anti-CD16 sctbCancer105CD19CD33anti-CD16 sctbCancer106carrier systemsCD19CD20PEGylated immunoliposomesCancer145 www.landesbioscience.com mAbs 185(DNL). Here, antibody fragments are fused to a homodimerizing docking domain (DDD) from human cAMP-dependent protein kinase A (PKA) and the anchoring domain (AD) from A-kinase anchor protein (AKAP) leading to the formation of bispecific, trivalent molecules.42Many of the established bispecific antibody formats can also be combined with additional proteins and components, e.g., drugs, toxins, enzymes and cytokines, enabling dual targeting and delivery of a fusion partner. In addition, fusion to plasma pro-teins such as serum albumin or albumin-binding moieties can be applied to extend the plasma half-life of bispecific antibodies.43,44Dual Targeting of Two Receptors in Cancer TherapyTumor development and progression often depend on growth signals mediated by receptors, which are consequently upregu-lated or amplified in many tumor cells. Examples include mem-bers of the EGF receptor family, i.e., EGFR, HER2, HER3 and HER4,45 and the IGF-1 receptor (IGF-1R),46 which play essential roles in regulating cell proliferation, survival, differentiation and migration. These receptors, with some exceptions, e.g., HER3 which is by its own signaling incompetent, act through multiple downstream pathways including the Ras/Raf/ERK/MAPK and the PI3K/AKT signal pathways. An acquisition of resistance to antibody 作文 手机作文网 作文 手机作文网 therapy against a singular receptor is often associated with pathway switching between two receptors, i.e., through a compensatory upregulation and activation of the reciprocal receptor as shown for EGFR and IGF-1R, thus maintaining the malignant phenotype and leading to a relapse of the disease.7-9 Furthermore, co-expression and crosstalk of different growth-promoting receptors such as EGFR and IGF-1R have been found for many tumors.47,48 Consequently, it was postulated that with a size basically identical to that of IgG. Fc heterodimer-ization was recently applied to generate a trivalent, bispecific molecule fusing a VH and a VL domain to the C-termini of the engineered heavy chains (HA-TF Fc variant).33Bispecific antibodies with a molecular mass in the range of 50–100 kDa can be generated by combining the variable domains of two antibodies.16,34 For example, two scFv have been connected by a more or less flexible peptide linker in a tandem orientation (tandem scFv, taFv, tascFv), which can be extended further by additional scFv, e.g., generating bispecific or trispecific triple bodies (sctb).35 Diabodies are heterodimeric molecules composed of the variable domains of two antibodies arranged either in the order VHA-VLB and VHB-VLA (VH-VL orientation) or in the order VLA-VHB and VLB-VHA (VL-VH orientation). The linker connecting the two domains within one chain is approxi-mately 5 residues leading, after co-expression of the two chains within one cell, to a head-to-tail assembly and hence formation of a compact molecule with two functional binding sites.36 The diabody (Db) format was further stabilized by introducing inter-chain disulfide bonds (dsDb, DART molecules) or by generat-ing a single-chain derivative (scDb).37-39 ScDbs can be converted into tetravalent molecules by reducing the middle linker, result-ing in homodimerzation of two chains.40 Small bispecific mol-ecules have also been produced by fusing a scFv to the heavy or light chain of a Fab fragment.41 Furthermore, tandem scFv, diabodies and scDb have been fused to the Fc or a CH3 domain to generate tetravalent derivatives. Also, scFv can be combined with Fc or CH3 domains to generate tetravalent molecules, e.g., fusing scFvs to the N- and -terminus of an Fc fragment, or using the knobs-into-holes approach to generate bivalent C scFv-Fc or scFv-CH3 molecules. A different approach for the generation of bispecific antibodies is realized by the dock-and-lock method Figure 1. (A–H) Dual targeting strategies utilizing bispecific antibodies: (A) neutralization of two receptor-activating ligands, (B) neutralization of two receptors, (C) neutralization of a receptor and a ligand, (D) activation of two receptors, (E) activation of a receptor and inactivation of another receptor, (F) activation of a receptor and inactivation of a ligand, (G) blockage of two epitopes of one receptor, (H) blockage of two epitopes of one ligand. (I–O) Dual retargeting strategies utilizing bispecific antibodies: (I) binding to two receptors and Fc-mediated ADCC or CDC, (K) retargeting of cytotoxic ef-fector cells with a trispecific antibody, (L) targeting of a bispecific toxin (immunotoxin) or a bispecific antibody-drug conjugate (ADC) to two receptors, (M) targeting of a bispecific cytokine (immunocytokine) to two receptors, (N) targeting of an enzyme to two receptors, (O) targeting of a drug-loaded nanoparticle/liposome to two receptors. Strategies are exemplified with bispecific IgG and Fab molecules, respectively. 186 mAbs Volume 4 Issue 2Figure 2. Bispecific antibody formats. Variable heavy chain domains (VH) are shown in dark blue and dark red, variable light chain domains (VL) are shown in light blue and light red, red and blue indicating different specificities. Antibody constant domains are shown in white boxes and fusion proteins in white circles. 作文 手机作文网 作文 手机作文网 www.landesbioscience.com mAbs 187extent as the combination of the two monospecific antibodies. This was confirmed with a panel of tumor cell lines establish-ing expanded growth inhibition compared with the parental antibodies. Potent antitumor activities of EI-04 were demon-strated in tumor xenograft models. Here, the bispecific antibody administered at all dose levels was statistically more efficacious than the two parental antibodies alone using equimolar dosages. For one tumor cell line (BxPC3), EI-04 was also statistically more potent than the combination of the two parental antibod-ies, while with a second cell line (GEO) a similar efficacy was observed, further highlighting the complexity of target receptor biology.9Dual targeting of EGFR and IGF-1R was further investigated for bispecific Adnectins.52 AdnectinsTM represent an antibody-mimetic alternative scaffold derived from human fibronectin domain. The bispecific AdnectinsTM were generated by con-necting two a AdnectinsTM with a flexible linker composed of ten glycine-serine repeats. Affinities of these tandem molecules were similar to those of the monospecific AdnectinsTM. To increase plasma half-life, the tandem AdnectinsTM were coupled to a 40 kDa branched PEG chain, which resulted in a 10- to 20-fold reduction in binding, although inhibition of EGFR and IGF-1R phosphorylation in vitro was only slightly affected. Importantly, compared with the monospecific AdnectinsTM, the bispecific AdnectinsTM were more potent in inhibiting proliferation of lung cancer cell line H292 expressing high levels of EGFR and IGF-1R. A functional benefit of having both domains within one molecule was deduced from in vivo experiments with BxPC3 xenografts. Here, the tandem AdnectinTM showed significantly better tumor growth inhibition compared with the individual monospecific AdnectinsTM or a mixture of both molecules.Dual targeting of HER2- and HER3-expressing tumor cells was described for a bispecific molecule generated by fusing scFvs directed against HER2 and HER3 to the N- and C-terminus of human serum albumin (scFv-HSA-scFv).53 This molecule (MM-111) combines targeting of HER2-overexpressing tumor cells with potent inhibition of ligand-induced phosphoryla-tion of HER3 with IC50 values in the sub-nanomolar range. Computational physicochemical modeling was applied to opti-mize the monovalent binding affinities to increase potency and specificity for tumor cells. MM-111 is currently undergoing evaluation in three Phase 1 studies in patients with advanced HER2-amplified cancers (NCT00911898, NCT01097460 and NCT01304784).Simultaneous receptor blockage with bispecific antibodies was also applied for platelet-derived growth factor receptor α (PDGFRα) and β (PDGFRβ).54 These receptors are activated by members of the PDGF family and are capable of forming homo- as well as heterodimeric receptor complexes. PDGFRs have been identified on a number of tumor types and are involved in stimulation of tumor cells, but also angiogenesis.55,56 Bispecific IgG-like antibodies against the two PDGF receptors were gener-ated by fusion of an anti-mouse PDGFRα single variable domain (sVD) either to the N-terminus of the light chain (sVD-IgG) or the C-terminus of the heavy chain (IgG-sVD) of an anti-mouse PDGFRβ IgG (see also Fig. 2). Simultaneous binding of antigens targeting two different receptors on a tumor cell should increase the anti-proliferative effect and help to avoid the development of resistance.Several bispecific molecules targeting EGFR and IGF-1R have been developed, including bispecific diabodies, IgG-like tetravalent Di-diabodies, IgG-scFv fusion proteins and bispecific AdnectinsTM (Table 1).9,49-52 Bispecific diabodies targeting EGFR and IGF-1R were generated from the anti-EGFR antibody 11F8 and the anti-IGF-1R antibody A12 and retained binding activity for the respective receptors. However, it was found that the affin-ity was influenced by the domain orientation and arrangement, with the VL-VH orientation being superior over the VH-VL ori-entation.50 This diabody was further converted into an IgG-like 作文 手机作文网 作文 手机作文网 bispecific and tetravalent molecule by fusion of one of the chains to a human IgG Fc region, including the hinge region.51 This Di-diabody was produced in NS0 cells and the purified was shown to be capable of binding both antigens simultaneously. Inhibition of tumor protein cell proliferation was demonstrated in vitro, although the Di-diabody exhibited an approximately 25-fold lower inhibitory activity than the parental antibodies alone or in combination. Furthermore, the Di-diabody blocked signaling pathways stimulated by EGF and IGF-1, while the parental mAb showed inhibitory activity only for the respective pathway (Akt pathway for IGF-1R and MAPK p44/p42 for EGF). Importantly, the Di-diabody also mediated ADCC toward cells expressing either EGFR or IGF-1R, or both receptors, while the parental antibodies were only active toward tumor cells expressing their target antigen. Finally, antitumor activity was demonstrated in two xenograft mouse tumor models. In the HT29 tumor model, the bispecific tetravalent antibody was superior to treatment with the individual parental mAbs, with an activity similar to that of the combination of the two parental mAbs. The same group fur-ther developed tetravalent bispecific IgG-like molecules (scFv4-Ig) by fusing an anti-IGF-1R scFv to the constant VL domain and an anti-EGFR scFv to the first CH1 domain of the IgG heavy chain, or vice versa.49 With these constructs, similar results as for the Di-diabody were observed in vitro. The results also lead to the assumption that therapeutic response of targeting two differ-ent receptors may depend on the expression levels of the receptors and activation status of each receptor and its downstream signal-ing molecules.Another IgG-like anti-EGFR x anti-IGF-1R bispecific anti-body was generated by genetic fusion of a stability-engineered anti-IGF-1R scFv to the C-terminus of a chimeric IgG4.P/IgG1 antibody derived from an affinity-matured variant of an aglycosylated EGFRvIII-specific antibody isolated from a semi-synthetic phage library.9 This antibody (EI-04) demonstrated simultane-ous binding of both antigens with similar affinity as the paren-tal antibodies and concurrent blockade of ligand binding and receptor activation with IC50 values in the low nanomolar range. Interestingly, in a head and neck squamous cell carcinoma cell line, the bispecific antibody efficiently reduced EGFR phos-phorylation, while the parental anti-EGFR antibody showed little effect. This finding indicates that the bispecific antibody is capable of inhibiting receptor pathway crosstalk in this cell line. The bispecific antibody also inhibited proliferation to a similar 188 mAbs Volume 4 Issue 2increased anti-proliferative and cytotoxic effects without utilizing ADCC or CDC. Induction of cell death through apoptosis is also known for members of the death receptor family, e.g., TRAIL receptors, which has led to the development of agonistic antibod-ies for cancer therapy.66 The applicability of bispecific antibod-ies as agonists of death receptors was investigated with IgG-scFv fusion proteins targeting TRAIL receptor 2 (TRAIL-R2, DR5) and lymphotoxin-β receptor (LTβR), which are co-expressed on the surface of a variety of cell lines from tumors of epithe-lial origin.67 Bispecific IgG-scFvs were produced by fusing a stability-improved disulfide-linked anti-LTβR scFv either to the N- or C-terminus of the anti-TRAIL-R2 IgG heavy chain. An enhanced antitumor activity was observed for a subset of tumor cell lines in vitro relative to the combination of parental anti-bodies. In vivo, superior tumor growth inhibition was observed for MDA-MB-231 xenografts and a bispecific antibody with the scFv fused to the heavy chain C-terminus, while for another cell line (WiDr) effects were similar to that of the combination ther-apy, in accordance with the observed in vitro activity.Dual Targeting of Two Receptors for the Treatment of Inflammatory DiseasesSelf-perpetuating B lymphocytes have been 作文 手机作文网 作文 手机作文网 identified as con-tributors to the development and progression of inflammatory and autoimmune rheumatic diseases.68,69 Therefore, B cell deple-tion or inactivation represents viable therapeutic approach, as shown by the use of rituximab in the treatment of rheumatoid a arthritis.70 B cell activation is induced by antigen binding to the B cell receptor (BCR) and regulated by a negative feedback loop mediated by the inhibitory Fcγ receptor IIb (FcγRIIb; CD32B) induced by a close contact between the two receptors upon binding of antigen to the BCR and soluble immunoglobulins.71 To recapitulate this antigen-driven proximity of the activating and inhibitory receptors, a bispecific DART molecule directed against CD79b, which is a signaling-competent molecule of the BCR and CD32B was generated.72 Simultaneous binding to both receptors resulted in inhibition of B cell proliferation and secretion of immunoglobulins and reduced disease severity of collagen-induced arthritis in mice, indicating that bispecific antibodies as activation-dependent inhibitors are useful for the treatment of autoimmunity. A similar approach was applied for the treatment of allergic Crosslinking of the inhibitory CD32B receptor to the FcεRI delivers a diseases. dominant-negative sig-nal that efficiently suppresses all activating signals of the FcεRI receptor expressed by mast cells and basophils. This was shown previously with a dimeric dual targeting Fcγ-Fcε bifunctional fusion protein.73 A bispecific IgG recognizing FcεRI and CD32B was generated using a knobs-into-holes approach. This antibody inhibited the activation of mast cells in vitro and in a passive cutaneous anaphylaxis (PCA) model.74 Furthermore, coaggregation of FcεRI and CD32B and inhibition of hista-mine release was also achieved using a bispecific F(ab’)2 mol-ecule directed against IgE and FcγRII.75was demonstrated for the bispecific antibodies and both antibod-ies were capable of blocking binding of the ligands PDFG-AA and PDFG-BB to its receptors, which resulted in inhibition of ligand-mediated receptor phosphorylation. In these assays, the IgG-sVD fusion protein showed better effects than the sVD-IgG fusion protein indicating that the position of adding a second binding site has a direct influence on bioactivity.Another sVD-IgG construct was generated by fusing the anti-mouse PDGFRα sVD to the N-terminus of the light chain of an anti-mouse VEGFR2 IgG.57 Thus, this bispecific antibody targets another receptor involved in tumor angiogenesis.58 The bispecific antibody was able to recognize both receptors simulta-neously and to inhibit PDGF- and VEGF-induced stimulation of murine endothelial cells. Neutralization of stimulating recep-tors of tumor endothelial cells was also studied with a bispecific diabody directed against VEGFR2 (KDR) and VEGFR3 (Flt-4) or a Di-diabody (diabody-CH3 fusion protein) directed against VEGFR1 (Flt-1) and VEGFR2.59,60 For these constructs, inhi-bition of ligand binding and VEGF-induced cell migration was described.Based on results from a Phase 2 study of combination therapy with mAbs for the treatment of B cell lymphoma combining an anti-CD20 antibody (rituximab) with an anti-CD22 antibody (epratuzumab),61 it was postulated that bispecific antibodies should also be applicable for the treatment of hematologic malig-nancies. A tetravalent IgG-like bispecific antibody generated by fusing an anti-CD22 scFv from epratuzumab to the C-terminus of the heavy chain of an anti-CD20 IgG veltuzumab showed improved inhibition of cell proliferation as well as ADCC against Daudi cells in vitro.62 In vivo, beneficial therapeutic effects of the bispecific antibody were, however, seen only at the highest dose. Of interest was the finding that the bispecific antibody induces translocation and accumulation of B cell receptors (BCR) in lipid rafts, presumably due to a stronger interaction of CD22 with the BCR, which was thought to lead to increased growth inhibition and apoptosis. This approach was extended by the same group using a hexavalent IgG-Fab molecule produced by the 作文 手机作文网 作文 手机作文网 dock-and-lock method.63 Potent inhibition of cell proliferation was also observed for this bispecific antibody. Again, antitumor activity was similar to that observed for a combination of the parental antibody.63,64 In another study, a CD20 x CD22-specific bispe-cific F(ab’)2 molecule generated by chemical conjugation showed significantly improved antitumor effects compared with treat-ment of the parental antibodies rituximab (CD20) and HB22.7 (CD22) alone or in combination.65 All the studies established that the bispecific antibodies induce a stronger p38 phosphorylation, which might be responsible for the increased cytotoxic effects observed in vitro. Furthermore, it was discussed that the bispe-cific antibody prevents rapid internalization of CD22 through binding to non-internalizing CD20, thus affecting downstream signaling.The studies with anti-CD20 and anti-CD22 bispecific anti-bodies demonstrated that targeting of relevant cell surface recep-tors can lead to an efficient activation of signaling pathways and www.landesbioscience.com mAbs 189bispecific antibodies block two alternative docking sites of the CCR5 co-receptor of HIV. To improve the stability of the fusion proteins, the scFv fragment was further stabilized by introducing a disulfide-bond between the VH and VL domain or by increas-ing the linker length connecting the VH and VL domain from 15 residues to 30 residues, which drastically reduced the aggregation tendency. While binding to CCR5-expressing CHO cells was increased only 1.3- to 1.7-fold, an 18- to 57-fold increased antivi-ral activity was observed for the different variants compared with t... 作文 手机作文网