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Antibody Therapeutics, 2020, Vol. 3, No. 1 18–62 doi:10.1093/abt/tbaa003 Advance Access Publication on 17 February 2020 Review Article Biology drives the discovery of bispeciﬁc antibodies as innovative therapeutics 1, 1 2 2 Siwei Nie , Zhuozhi Wang , Maria Moscoso-Castro , Paul D’Souza , 2 1 1, Can Lei , Jianqing Xu and Jijie Gu 1 2 WuXi Biologics, 299 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, China and Clarivate Analytics, Friars House, 160 Blackfriars Road, London SE1 8EZ, UK Received: December 10, 2019; Revised: February 7, 2020; Accepted: Month 0, 2000 ABSTRACT A bispeciﬁc antibody (bsAb) is able to bind two different targets or two distinct epitopes on the same target. Broadly speaking, bsAbs can include any single molecule entity containing dual speciﬁcities with at least one being antigen-binding antibody domain. Besides additive effect or synergistic effect, the most fascinating applications of bsAbs are to enable novel and often therapeutically important concepts otherwise impossible by using monoclonal antibodies alone or their combination. This so-called obligate bsAbs could open up completely new avenue for developing novel therapeutics. With evolving understanding of structural architecture of various natural or engineered antigen-binding immunoglobulin domains and the connection of different domains of an immunoglobulin molecule, and with greatly improved understanding of molecular mechanisms of many biological processes, the landscape of therapeutic bsAbs has signiﬁcantly changed in recent years. As of September 2019, over 110 bsAbs are under active clinical development, and near 180 in preclinical development. In this review article, we introduce a system that classiﬁes bsAb formats into 30 categories based on their antigen-binding domains and the presence or absence of Fc domain. We further review the biology applications of approximately 290 bsAbs currently in preclinical and clinical development, with the attempt to illustrate the principle of selecting a bispeciﬁc format to meet biology needs and selecting a bispeciﬁc molecule as a clinical development candidate by 6 critical criteria. Given the novel mechanisms of many bsAbs, the potential unknown safety risk and risk/beneﬁt should be evaluated carefully during preclinical and clinical development stages. Nevertheless we are optimistic that next decade will witness clinical success of bsAbs or multispeciﬁc antibodies employing some novel mechanisms of action and deliver the promise as next wave of antibody-based therapeutics. Statement of Significance: This article comprehensively reviewed various bispeciﬁc antibody formats and the biology driving the design and selection of a right bispeciﬁc antibody to enable novel therapeutic concept and match intended therapeutic applications. The principles and the examples discussed could provide a general guidance for people interested in exploring bispeciﬁc antibody therapeutics. KEYWORDS: bispeciﬁc antibody; bsAb; multispeciﬁc antibody; msAb A BRIEF HISTORICAL VIEW OF BISPECIFIC sequences, with only one mAb (anti-CD3 muromonab) ANTIBODIES being approved. It took another decade for the field to solve the immunogenicity issues, and the lessons learned The invention of hybridoma technology in 1975 marked from the first wave of clinical trials of antibody therapeutics the arrival of new era of monoclonal antibody (mAb)- is the key driver leading to invention of innovative anti- based therapy . However, the first wave of clinical body humanization technologies represented by antibody attempts with mouse antihuman mAb therapeutics during chimerization, CDR graft, in vitro display of human 1975–86 largely failed, due to immunogenicity of mouse To whom correspondence should addressed. Jijie Gu or Siwei Nie. Email: email@example.com; firstname.lastname@example.org. © The Author(s) 2020. Published by Oxford University Press on behalf of Antibody Therapeutics. Antibody Therapeutics, 2020 19 antibody repertoire, and human immunoglobulin trans- commercial manufacturing, i.e., desired clinical efficacy, genic rodents. The approval of rituximab by the US appropriate safety profile, favorable pharmacokinetic/ FDA in 1997 marked the field entering into the booming pharmacodynamic (PK/PD) properties, appropriate physic- stage. About 100 antibody-based therapeutics have been ochemical properties, scalable manufacturability, and min- approved by the regulatory agencies worldwide, since then, imal or no immunogenicity risk—select a right molecule. antibody therapeutics have now become one of the main- Unfortunately, these six criteria, particularly those crit- stays for developing new medicines. The history of devel- ical for biological function (efficacy, safety, PK/PD, opment of bispecific antibodies (bsAbs) almost followed immunogenicity) and those critical for developability the footprint of the development of mAb therapeutics. (expression, homogeneity, solubility, stability, viscosity, As illustrated in a recent review article , starting in the formulation ability, etc.), often are not correlated with 1960s, scientists explored generation of antigen-binding each other, sometimes even counterbalance each other that fragments (Fabs) from two different polyclonal sera and requires balancing when selecting a therapeutic molecule. reassociated them into bispecific F(ab’)2 molecules. After Identification of a good therapeutic bispecific molecule hybridoma technology was established in 1975, chemical therefore usually requires starting with good therapeutic conjugation of two rodent mAbs or fusion of two antibody- molecular design defined by molecular product profile producing hybridomas (so-called quodroma) was explored (MPP) that is developed based on target product profile immediately to make bsAbs with defined specificities. (TPP), followed by rigorous molecular and functional screen, selection and characterization using pharmacologi- The first therapeutic bsAb catumaxomab (Removab ) cal assays, mechanistic and/or disease models, and other approved by the EMA in 2009 was made by this early preclinical translational systems relevant to the human technology. The bsAbs made by these two methods prior to disease one intends to treat. establishing antibody humanization technologies, however, suffered from the same issue of immunogenicity in addition to stability, solubility, and manufacturability challenges. The development of methods to produce recombinant THE MAKING OF RECOMBINANT BISPECIFIC antibodies in the 1980s enabled the rapid generation of ANTIBODIES various bsAbs with defined structure, composition, and In a recent review article, Brinkmann and Kontermann biochemical, functional, and pharmacological properties, thoroughly reviewed many experimentally verified formats but it still took scientists more than 2 decades to really that had been described in the literature as of September understand the unique structural features of various 2016 . We concur with their opinion that besides the antigen-binding building blocks such as Fab, Fv, scFv, freedom-to-operate (FTO) and the desire to generate pro- SDA, etc., to develop various innovative engineering prietary intellectual properties (IP) for competitive reason, solutions to generate homo- and heterodimerization one of the critical drivers for explosive diversity of so many building blocks necessary for making various bispecific bsAb formats is the plethora of desired functionalities and formats and most importantly understand the structural applications of bsAbs. Format variability is essential to biology of how to connect them together to enable serve diverse bsAb applications defined by different TPP. various biology concepts while maintaining favorable These formats may vary in size, domain composition and developability. In the later paragraphs, we will review the arrangement, binding kinetics and valencies, flexibility and evolution of some of those landmark solutions for bsAb geometry of their binding modules, as well as in their bio- construction. But before we get into detailed discussion of distribution and pharmacokinetic properties to fulfill a par- how to make various recombinant bsAbs, we will discuss the principles governing how to define and identify a good ticular clinical application. Small variations, such as minor bsAb therapeutics first. changes in linker length or composition of domains, can be crucial determinants for functionality. Some designed parameters may be deduced from structural modeling of drug-target interaction. In many cases, however, a suitable THE PRINCIPLES GOVERNING A GOOD molecule must be identified by generating and compar- THERAPEUTIC BISPECIFIC ANTIBODY ing the functionalities of different formats and different Though mAbs have demonstrated definitive therapeutic molecules in the systems relevant to clinical settings. benefits in multiple disease areas, it is believed that bsAbs Here we review various bsAb formats and classify them into 30 categories: (1) what are the building blocks of can further advance the success of therapeutic antibodies by enabling the molecules with new mechanisms of action antigen-binding and their combination, and (2) whether (MOAs) and by providing new functional advantages that they contain fragment of crystallizable region (Fc) domain. cannot be achieved by mAbs. We believe that identification From published reports and our practice, most bispecific of a good bsAb should be based on three principles (Fig. 1): formats contain the antigen-binding sites derived from (1) the molecule should be able to provide unique biological immunoglobulin domain of native antibodies. We identify function to achieve desired efficacy with appropriate safety single-domain antibody (SDA or VHH), variable fragment profile, driven by unique biology; (2) the format chosen (Fv), single-chain variable fragment (scFv), Fab, and single- should enable the molecule to fulfill its proposed function, chain antigen-binding fragment (scFab) as the five key match biology with an optimal format; and (3) the molecule building blocks of bispecific formats. As shown in Fig. 2, selected as a clinical development candidate should satisfy most of bsAb formats can be classified into 30 groups based the six criteria critical for clinical development and on the above classification. As there are more than 200 20 Antibody Therapeutics, 2020 Figure 1. The principles, criteria and screening funnel in discovering a good therapeutic bsAb. (A) Three principles of governing the discovery of a good bsAb, (B) Six criteria of defining a bsAb as a clinical development candidate. (C) Detailed function and developability screenings to identify a good therapeutic bsAb molecule. bispecific formats from published data and our practice, binding building block. It becomes obvious to employ these we do not intend to list all these formats in Fig. 2. Instead, fusion sites to make a bispecific format with desired binding we have just listed an example of each category to illustrate activity. the concept. Bispecific molecules containing non-antibody-binding In each category, the bispecific formats can be further domains such as peptides, ligands, receptors, or alternative classified by their geometry (such as homodimer vs. het- scaffolds may not fall into this classification system. erodimer) and valency (number of antigen-binding sites). A However, depending on how many polypeptide chains bsAb with one binding site to target A and one binding site of the antigen-binding sites are used, the non-antibody to target B is called 1 + 1 format. Similarly there are 1 + 2, bispecific molecules can be constructed using similar 1 + 3, and 2 + 2 formats. The formats with more than four approaches as the above bsAbs. antigen-binding sites are uncommon but growing, so they are just mentioned as examples in this review. Bispecific antibody fragments without Fc In addition to the building blocks, absence or presence of Fc, and different valency, multiple fusion sites of Fc- In this category, all antigen-binding sites are from the afore- containing formats increase the complexity of bispecific mentioned building blocks (SDA, Fv, scFv, Fab, and scFab) formats. As shown in Fig. 3A, an antigen-binding build- and the bsAbs do not contain Fc. Many different bispecific ing block can be fused to N-terminus or C-terminus of formats, including 1 + 1, 1 + 2, 1 + 3, and 2 + 2 formats, an Fc fragment or inserted between CH2 domain and and trispecific formats have been used for preclinical and CH3 domain. On a heterodimeric Fc-containing bispe- clinical development (Tables 1–6). BsAb fragments usually cific format, there are at least six fusion sites. If an Fc- are smaller than IgG and lack of Fc-related functions such containing format also comprises of CL, the fusion sites as Fcγ R-, FcRn-, and complement-binding and related increase to 12 (Fig. 3A). Moreover, theoretically all the activities. Due to large number of the bsAb fragment for- loops of each immunoglobulin domain (CL, CH1, CH2, mats, only some examples of bsAbs fragments are briefly and CH3) can be used as fusion sites to integrate an antigen- descripted below. Antibody Therapeutics, 2020 21 Table 1. Programs in clinical and preclinical stages to block the angiogenesis and/or tumorigenesis for cancer treatment Antibody name Organization Targets Highest phase Biological function Type of mechanism Format Clinical studies Dilpacimab, AbbVie VEGF × DLL4 Phase II Anti-angiogenesis Combinatorial effect Fab + Fv with Fc, NCT01946074, ABT-165 2 + 2 NCT01946074, NCT03368859, NCT03368859 MP0250 Molecular Partners AG VEGF × HGF Phase II Anti-angiogenesis Combinatorial effect Scaffold 1 + 1 + 1 NCT02194426, × albumin NCT03136653, NCT03418532 ABL-001, ABL Bio, TRIGR VEGF × DLL4 Phase I Anti-angiogenesis Combinatorial effect Fab + scFv with Fc, NCT03292783 NOV-1501, TR-009 Therapeutics 2 + 2 Vanucizumab, Roche, Harvard ANGPT2 × VEGF Phase I Anti-angiogenesis Combinatorial effect Fab + FabwithFc, NCT01688206, RG-7221 Medical School, 1 + 1 NCT02141295, National Cancer NCT02665416 Centre of Singapore BI-836880 Boehringer Ingelheim, ANGPT2 × VEGF, Phase I Anti-angiogenesis Combinatorial effect VH + VH, 1 + 1 + 1 NCT02674152, Sanofi albumin NCT02689505, NCT03468426, NCT03861234, NCT03972150 Navicixizumab, OncoMed VEGF × DLL4 Phase I Anti-angiogenesis Combinatorial effect Fab + FabwithFc, NCT02298387, OMP-305B83 Pharmaceuticals 1 + 1 NCT03030287, NCT03035253 KN-026 Jiangsu Alphamab HER2 × HER2 Phase II Anti-tumorigenesis Biparatopic Fab + FabwithFc, NCT03619681, Biopharmaceuticals 1 + 1 NCT03847168, NCT03925974, NCT04040699 ZW-25 Zymeworks, BeiGene HER2 × HER2 Phase II Anti-tumorigenesis Biparatopic Fab + scFv with Fc, NCT02892123, 1 + 1 NCT03929666 MCLA-128 Merus HER3 × HER2 Phase II Anti-tumorigenesis Combinatorial effect Fab + FabwithFc, NCT02912949, 1 + 1 NCT03321981 EMB-01, FIT-013a EpimAb EGFR × cMET Phase I/II Anti-tumorigenesis Combinatorial effect Fab + FabwithFc, NCT03797391 Biotherapeutics 2 + 2 JNJ-61186372, Janssen EGFR × cMET Phase I Anti-tumorigenesis Combinatorial effect Fab + FabwithFc, NCT02609776, JNJ-6372 1 + 1 NCT04077463 BCD-147 Biocad HER2 × HER2 Phase I Anti-tumorigenesis Biparatopic Fab + scFv with Fc, NCT03912441 1 + 2 MBS-301 Beijing Mabworks HER2 × HER2 Phase I Anti-tumorigenesis Biparatopic Fab + FabwithFc, NCT03842085 Biotech 1 + 1 Continued 22 Antibody Therapeutics, 2020 Table 1. Continued Antibody name Organization Targets Highest phase Biological function Type of mechanism Format Clinical studies BI-905677 Boehringer Ingelheim LRP5/6 Phase I Anti-tumorigenesis Biparatopic SDA + SDA, 1 + 1 NCT03604445 MP0274 Molecular Partners AG Her2 × Her2 Phase I Anti-tumorigenesis Biparatopic SCAFFOLD, 1 + 1 NCT03084926 VEGFR2/Ang2 Eli Lilly & Co VEGFR2 × ANGPT2 Preclinical Anti-angiogenesis Combinatorial effect Fab + scFv with Fc, NA 2 + 2 FS-101 F-star Therapeutics Ltd EGFR × HGF Preclinical Anti-angiogenesis Combinatorial effect Fab + SDA with Fc, NA 2 + 2 MP-E-8-3/1959 MediaPharma Endosialin × LGALS3BP Preclinical Anti-angiogenesis Combinatorial effect Not disclosed NA PMC-001 PharmAbcine Tie-2 × VEGFR2 Preclinical Anti-angiogenesis Combinatorial effect Fab + LIGAND NA with Fc, 2 + 2 PMC-201 PharmAbcine DLL4 × VEGFR2 Preclinical Anti-angiogenesis Combinatorial effect Not disclosed NA PMC-404 PharmAbcine ANGPT2 × VEGF-c Preclinical Anti-angiogenesis Combinatorial effect Not disclosed NA MCLA-129 Betta Pharmaceuticals; VEGF × cMET Preclinical Anti-angiogenesis, Combinatorial effect Fab + FabwithFc, NA Merus anti-tumorigenesis 1 + 1 MP-EV20/1959 MediaPharma HER3 × LGALS3BP Preclinical Anti-angiogenesis, Combinatorial effect Not disclosed NA anti-tumorigenesis CKD-702 Chong Kun Dang EGFR × cMET Preclinical Anti- tumorigenesis Combinatorial effect Fab + scFv with Fc, NA Pharmaceutical 2 + 2 CBA-0702 Sorrento Therapeutics Her3 × cMET Preclinical Anti-tumorigenesis Combinatorial effect scFv + scFv with Fc, NA 1 + 1 SRB-19 SunRock Biopharma EGFR × Her3 Preclinical Anti-tumorigenesis Combinatorial effect Not disclosed NA Anti-HER2 and Biocad Ltd Her2 × Her3 Preclinical Anti-tumorigenesis Combinatorial effect Not disclosed NA HER3 mAb BTA-106 Zenyaku Kogyo Co Ltd IgM × HLA-DR Preclinical Anti-tumorigenesis Combinatorial Fab + FabwithFc, NA effect? 1 + 1 TXB4-BC2 Ossianix Inc TfR × EGFRvIII Preclinical Anti-tumorigenesis Trojan horse Fab + SDA with Fc, NA 2 + 2 TXB4-BC1 Ossianix Inc TfR × CD20 Preclinical Anti-tumorigenesis Trojan horse Fab + SDA with Fc, NA 2 + 2 Antibody Therapeutics, 2020 23 Table 2. Programs in clinical and preclinical stages to enhance tumor immunity for cancer treatment Antibody name Organization Targets Highest phase Biological function Type of mechanism Format Clinical studies KN-046 Jiangsu Alphamab PD-L1 × CTLA-4 Phase II Enhance tumor Tumor or tissue SDA + SDA with NCT03529526, Biopharmaceuticals immunity localization Fc, 2 + 2 NCT03733951, NCT03838848, NCT03872791, NCT03925870, NCT03927495, NCT04040699 AK-104 Akeso Biopharma PD-1 × CTLA-4 Phase I/II Enhance tumor Combinatorial effect Fab + scFv with Fc, NCT03261011, immunity 2 + 2 NCT03852251 DuoBody-PD-L1x4- BioNTech, Genmab PD-L1 × 4-1BB Phase I/II Enhance tumor Tumor or tissue Fab + FabwithFc, NCT03917381 1BB, immunity localization 1 + 1 GEN-1046 REGN-5678 Regeneron PSMA × CD28 Phase I/II Enhance tumor Tumor or tissue Fab + FabwithFc, NCT03972657 immunity localization 1 + 1 FS118 mAb2, F-star PD-L1 × LAG-3 Phase I Enhance tumor Tumor or tissue Fab + SDA, 2 + 2 NCT03440437 FS-118, immunity localization LAG-3/PD-L1 mAb2 IBI-318 Innovent Biologics, Lilly PD-1 × PD-L1 Phase I Enhance tumor Promote Not disclosed NCT03875157 immunity downregulation LY-3434172 Eli Lilly PD-1 × PD-L1 Phase I Enhance tumor Promote Fab + FabwithFc, NCT03936959 immunity downregulation 1 + 1 MGD-013 MacroGenics, ZAI Lab PD-1 × LAG-3 Phase I Enhance tumor Combinatorial effect Fv + Fv with Fc, NCT03219268, immunity 2 + 2 NCT04082364 XmAb-23104 Xencor PD-1 × ICOS Phase I Enhance tumor Combinatorial effect Fab + scFv with Fc, NCT03752398 immunity 1 + 1 ABBV-428 AbbVie MSLN × CD40 Phase I Enhance tumor Tumor or tissue scFv + scFv with NCT02955251 immunity localization Fc, 2 + 2 ADC-1015, Alligator Bioscience OX40 × CTLA-4 Phase I Enhance tumor Combinatorial effect Fab + LIGAND NCT03782467 ATOR-1015 immunity with Fc, 2 + 2 INBRX-105-1, Inhibrx, Elpiscience PD-L1 × 4-1BB Phase I Enhance tumor Tumor or tissue SDA + SDA with NCT03809624 INBRX-105, ES-101 BioPharma immunity localization Fc, 2 + 2 MCLA-145 Merus, Incyte PD-L1 × 4-1BB Phase I Enhance tumor Tumor or tissue Fab + FabwithFc, NCT03922204 immunity localization 1 + 1 MEDI-5752 MedImmune PD-1 × CTLA-4 Phase I Enhance tumor Combinatorial effect Fab + FabwithFc, NCT03530397 immunity 1 + 1 MGD-019 MacroGenics PD-1 × CTLA-4 Phase I Enhance tumor Combinatorial effect Fv + Fv with Fc, NCT03761017 immunity 2 + 2 PRS-343 Pieris HER2 × 4-1BB Phase I Enhance tumor Tumor or tissue Fab + SCAFFOLD NCT03330561, immunity localization with Fc, 2 + 2 NCT03650348 RG-7769, Roche PD-1 × TIM-3 Phase I Enhance tumor Combinatorial effect Fab + FabwithFc, NCT03708328 RO-7121661 immunity 1 + 1 Continued 24 Antibody Therapeutics, 2020 Table 2. Continued Antibody name Organization Targets Highest phase Biological function Type of mechanism Format Clinical studies XmAb-20717 Xencor PD-1 × CTLA-4 Phase I Enhance tumor Combinatorial effect Fab + scFv with Fc, NCT03517488 immunity 1 + 1 XmAb-22841 Xencor CTLA-4 × LAG-3 Phase I Enhance tumor Combinatorial effect Fab + scFv with Fc, NCT03849469 immunity 1 + 1 RG-7827 Roche FAP × 4-1BB Phase I Enhance tumor Tumor or tissue Fab + LIGAND Company immunity localization with Fc, 1 + 3 development pipeline MP0310 Molecular Partners AG, FAP × CD40 Phase I Enhance tumor Tumor or tissue SCAFFOLD, 1 + 1 NCT04049903 Amgen immunity localization HX-009 HanX PD-1 × CD47 IND Filed Enhance tumor Combinatorial effect Fab + LIGAND NCT04097769 Biopharmaceuticals immunity with Fc, 2 + 2 AK-112 Akeso Biopharma VEGF × PD-1 IND Filed Enhanced tumor Combinatorial effect Fab + scFv with Fc, NCT04047290 immunity, 2 + 2 anti-angiogenesis INV-531 Invenra Inc OX40 biparatopic Preclinical Enhance tumor Biparatopic Fab + Fab with NA immunity Fc,1 + 2 ATOR-1144 Alligator Bioscience GITR × CTLA-4 Preclinical Enhance tumor Combinatorial effect Fab + LIGAND NA immunity with Fc, 2 + 2 BH-2996 h Beijing Hanmi PD-1 × PD-L1 Preclinical Enhance tumor Promote Fab + FabwithFc, NA Pharmaceutical immunity downregulation 1 + 1 GEN-1042 BioNTech; Genmab CD40 × 4-1BB Preclinical Enhance tumor Combinatorial effect Fab + FabwithFc, NA immunity 1 + 1 CB-213 Crescendo Biologics PD-1 × LAG- Preclinical Enhance tumor Combinatorial effect SDA + SDA + SDA, NA 3 × albumin immunity 1 + 1 + 2 FS-120 F-star Therapeutics OX40 × 4-1BB Preclinical Enhance tumor Combinatorial effect Fab + SDA, 2 +2NA immunity MEDI-3387 MedImmune LLC GITR × PD-1 Preclinical Enhance tumor Combinatorial effect Fab + LIGAND NA immunity with Fc, 2 + 2 MEDI-5771 MedImmune LLC GITR × PD-1 Preclinical Enhance tumor Combinatorial effect Fab + LIGAND NA immunity with Fc, 2 + 2 PT-302 Phanes Therapeutics LAG-3 × TIM-3 Preclinical Enhance tumor Combinatorial effect Not disclosed NA immunity TSR-075 TESARO Inc PD-1 × LAG-3 Preclinical Enhance tumor Combinatorial effect Not disclosed NA immunity PD-1/LAG-3 Xencor Inc PD-1 × LAG-3 Preclinical Enhance tumor Combinatorial effect Fab + scFv with Fc, NA bispecific mAbs immunity 1 + 1 AM-105 AbClon Inc EGFR × 4-1BB Preclinical Enhance tumor Tumor or tissue Not disclosed NA immunity localization Continued Antibody Therapeutics, 2020 25 Table 2. Continued Antibody name Organization Targets Highest phase Biological function Type of mechanism Format Clinical studies ALG-APV-527 Alligator; Aptevo 5T4 × 4-1BB Preclinical Enhance tumor Tumorortissue scFv + scFv with NA Therapeutics Inc immunity localization Fc, 2 + 2 BY-24.3 Beijing Beyond; VEGF × PD-1 Preclinical Enhance tumor Combinatorial effect Not disclosed NA Hangzhou Sumgen immunity BH-2922 Beijing Hanmi EGFR × PD-1 Preclinical Enhance tumor Combinatorial effect Fab + FabwithFc, NA immunity 1 + 1 BH-2950 Beijing Hanmi; Her2 × PD-1 Preclinical Enhance tumor Tumorortissue Fab + FabwithFc, NA Innovent immunity localization 1 + 1 DuoBody-PD-L1x4- BioNTech; Genmab PD-L1 × 4-1BB Preclinical Enhance tumor Tumorortissue Fab + FabwithFc, NA 1BB immunity localization 1 + 1 CDX-527 Celldex Therapeutics PD-L1 × CD27 Preclinical Enhance tumor Tumorortissue Fab + scFv with Fc, NA immunity localization 2 + 2 CB-307 Crescendo Biologics PSMA × 4- Preclinical Enhance tumor Tumorortissue SDA + SDA + SDA, NA 1BB × albumin immunity localization 1 + 1 + 1 ND-021 CStone; Numab PD-L1 × 4- Preclinical Enhance tumor Tumorortissue scFv + SDA + SDA, NA 1BB × albumin immunity localization 1 + 1 + 1 FS-222 F-star PD-L1 × 4-1BB Preclinical Enhance tumor Tumorortissue Fab + SDA, 2 +2NA immunity localization EGFR/CTLA-4 F-star EGFR × CTLA-4 Preclinical Enhance tumor Tumorortissue Fab + SDA, 2 +2NA bispecific mAb2 immunity localization IBI-323 Innovent Biologics PD-L1 × LAG-3 Preclinical Enhance tumor Tumorortissue Not disclosed NA immunity localization KY-1055 Kymab PD-L1 × ICOS Preclinical Enhance tumor Tumorortissue Fab + SDA with Fc, NA immunity localization 2 + 2 1D8N/CEGa1 LeadArtis EGFR × 4-1BB Preclinical Enhance tumor Tumorortissue scFv + SDA,3 +3NA immunity localization 4-1BBx5T4 MacroGenics 5T4 × 4-1BB Preclinical Enhance tumor Tumorortissue Fab + Fv with Fc, NA immunity localization 1 + 2 4-1BBxHER2 MacroGenics Her2 × 4-1BB Preclinical Enhance tumor Tumorortissue Fab + Fv with Fc, NA immunity localization 1 + 2 PD-L1x4-1BB MacroGenics Inc PD-L1 × 4-1BB Preclinical Enhance tumor Tumorortissue Fab + Fv with Fc, NA immunity localization 2 + 2 Continued 26 Antibody Therapeutics, 2020 Table 2. Continued Antibody name Organization Targets Highest phase Biological function Type of mechanism Format Clinical studies MEDI-1109 MedImmune PD-L1 × OX40 Preclinical Enhance tumor Tumor or tissue Fab + LIGAND NA immunity localization with Fc, 2 + 2 PRS-300 series A Pieris Her2 × CTLA-4 Preclinical Enhance tumor Tumor or tissue Not disclosed NA immunity localization PRS-342 Pieris GPC3 × 4-1BB Preclinical Enhance tumor Tumor or tissue SCAFFOLD + SCAF- NA immunity localization FOLD with Fc, 2 + 2 PRS-344 Pieris; Servier PD-L1 × 4-1BB Preclinical Enhance tumor Tumor or tissue Fab + SCAFFOLD NA immunity localization with Fc, 2 + 2 PD-1 × BTLA Xencor BTLA × PD-1 Preclinical Enhance tumor Combinatorial effect Fab + scFv with Fc, NA immunity 1 + 1 TXB4-BC3 Ossianix Inc TfR × PD-L1 Preclinical Enhance tumor Trojan horse Fab + SDA with Fc, NA immunity 2 + 2 CBA-0710 Sorrento cMET × PD-L1 Preclinical Enhance tumor Combinatorial effect Fab + FabwithFc, NA immunity, 1 + 1 anti-tumorigenesis Table 3. Programs in clinical and preclinical stages to modulate TME for cancer treatment Antibody name Organization Targets Highest phase Biological Type of mechanism Format Clinical studies function Bintrafusp alfa GlaxoSmithKline, PD-L1 × TGFbeta Phase III Modulate TME Tumor or tissue Fab + RECEPTOR with NCT04066491, Merck KGaA localization Fc, 2 + 2 NCT03840902, NCT03833661, NCT03631706, NCT03840915, NCT02699515, NCT02517398 AGEN-1423, Agenus, Gilead CD73 × TGFbeta Phase I Modulate TME Combinatorial effect Not disclosed NCT03954704 GS-1423 SHR-1701 Jiangsu Hengrui PD-L1 × TGFbeta Phase I Modulate TME Tumor or tissue Fab + RECEPTOR with NCT03710265, localization Fc, 2 + 2 NCT03774979 AK-123 Akeso Biopharma PD-1 × CD73 Preclinical Enhance tumor Tumor or tissue Not disclosed NA immunity, localization modulate TME UniTI-101 Elstar Therapeutics CCR2 × CSF1R Preclinical Modulate TME Combinatorial effect Fab + FabwithFc, 1 +1NA FmAb-2 Biocon; IATRICa EGFR × TGFbeta Preclinical Modulate TME Tumor or tissue Fab + RECEPTOR with NA localization Fc, 2 + 2 Antibody Therapeutics, 2020 27 Table 4. Programs in clinical and preclinical stages to promote target cell depletion for cancer treatment Antibody name Organization Targets Highest phase Biological Type of mechanism Format Clinical studies function Tebentafusp Immunocore gp100/HLA- Phase III Target cell Cytotoxic effector TCR + scFv, 1 + 1 NCT03070392, A 0201 × CD3 depletion engagement NCT02889861, NCT02570308, NCT02535078, NCT01211262, NCT01209676 OXS-1550, DT-2219 GT Biopharma CD19 × CD22 Phase II Target cell ADC scFv + scFv, 1 + 1 NCT00889408, depletion NCT02370160 AFM-13 Affimed CD16 × CD30 Phase II Target cell Cytotoxic effector Fv + Fv, 2 + 2 NCT01221571, depletion engagement NCT02321592, NCT02665650, NCT03192202, NCT04074746 Odronextamab, Regeneron CD3 × CD20 Phase II Target cell Cytotoxic effector Fab + FabwithFc, 1 + 1 NCT02651662, REGN-1979 depletion engagement NCT03888105 IMC-C103C Genentech; MAGE- Phase II Target cell Cytotoxic effector TCR + scFv, 1 + 1 NCT03973333 Immunocore A4/HLA A0201 × CD3 depletion engagement IMCnyeso GlaxoSmithKline; NY-ESO- Phase II Target cell Cytotoxic effector TCR + scFv, 1 + 1 NCT03515551 Immunocore 1/HLA A0201 × CD3 depletion engagement Mosunetuzumab, Genentech, Roche, CD3 × CD20 Phase I/II Target cell Cytotoxic effector Fab + FabwithFc, 1 + 1 NCT02500407, RG-7828 Chugai depletion engagement NCT03671018, NCT03677141, NCT03677154 OXS-3550, GT Biopharma, CD16 × CD33, IL-15 Phase I/II Target cell Cytotoxic effector scFv + scFv + LIGAND, NCT03214666 CD161533 TriKE Altor BioScience, U. depletion engagement 1 + 1 + 1 Minnesota GEN-3013 Genmab CD3 × CD20 Phase I/II Target cell Cytotoxic effector Fab + FabwithFc, 1 + 1 NCT03625037 depletion engagement MCLA-117 Merus CD3 × CLEC12 Phase I/II Target cell Cytotoxic effector Fab + FabwithFc, 1 + 1 NCT03038230 depletion engagement Flotetuzumab, MacroGenics, CD3 × CD123 Phase I/II Target cell Cytotoxic effector Fv + Fv, 1 + 1 NCT02152956, MGD-006 Servier depletion engagement NCT03739606 MGD-007 MacroGenics CD3 × GPA33 Phase I/II Target cell Cytotoxic effector Fv + Fv with Fc, 1 + 1 NCT02248805, depletion engagement NCT03531632 REGN-4018 Regeneron, Sanofi CD3 × MUC16 Phase I/II Target cell Cytotoxic effector Fab + FabwithFc, 1 + 1 NCT03564340 depletion engagement Cibisatamab, Genentech, Roche, CD3 × CEA Phase I/II Target cell Cytotoxic effector Fab + FabwithFc, 1 + 1 NCT02324257, RO-6958688, Chugai depletion engagement NCT02650713, RG-7802 NCT03337698, NCT03866239 Continued 28 Antibody Therapeutics, 2020 Table 4. Continued Antibody name Organization Targets Highest phase Biological Type of mechanism Format Clinical studies function huGD2-BsAb Y-mAbs CD3 × GD2 Phase I/II Target cell Cytotoxic effector Fab + scFv with Fc, 2 +2NA depletion engagement AMG-701 Amgen CD3 × BCMA Phase I Target cell Cytotoxic effector scFv + scFv, 1 + 1 NCT03287908 depletion engagement −337 A Generon (Shanghai) CD3 × EpCAM Phase I Target cell Cytotoxic effector Fab + scFv, 1 + 2 Company depletion engagement development pipeline AMG-160 Amgen CD3 × PSMA Phase I Target cell Cytotoxic effector scFv + scFv, 1 + 1 NCT03792841 depletion engagement AMG-330, Amgen CD3 × CD33 Phase I Target cell Cytotoxic effector scFv + scFv, 1 + 1 NCT02520427 MT-114 depletion engagement AMG-424 Amgen CD3 × CD38 Phase I Target cell Cytotoxic effector Fab + scFv with Fc, 1 + 1 NCT03445663 depletion engagement AMG-427 Amgen CD3 × FLT3 Phase I Target cell Cytotoxic effector scFv + scFv, 1 + 1 NCT03541369 depletion engagement AMG-562 Amgen CD3 × CD19 Phase I Target cell Cytotoxic effector scFv + scFv, 1 + 1 NCT03571828 depletion engagement AMG-596 Amgen CD3 × EGFRvIII Phase I Target cell Cytotoxic effector scFv + scFv, 1 + 1 NCT03296696 depletion engagement AMG-673 Amgen CD3 × CD33 Phase I Target cell Cytotoxic effector scFv + scFv, 1 + 1 NCT03224819 depletion engagement AMG-757 Amgen CD3 × DLL3 Phase I Target cell Cytotoxic effector scFv + scFv, 1 + 1 NCT03319940 depletion engagement AMV-564, Affimed, Fred CD3 × CD33 Phase I Target cell Cytotoxic effector Fv + Fv, 2 + 2 NCT03144245, TandAb T564 Hutch, Amphivena depletion engagement NCT03516591 APVO-436 Aptevo CD3 × CD123 Phase I Target cell Cytotoxic effector scFv + scFv with Fc, NCT03647800 depletion engagement 2 + 2 BI-836909, Amgen, Boehringer CD3 × BCMA Phase I Target cell Cytotoxic effector scFv + scFv, 1 + 1 NCT02514239, AMG-420 Ingelheim depletion engagement NCT03836053 RG-6026, Roche CD3 × CD20 Phase I Target cell Cytotoxic effector Fab + FabwithFc, 1 + 2 Company RO-7082859 depletion engagement development pipeline EM-901, Celgene CD3 × BCMA Phase I Target cell Cytotoxic effector Fab + FabwithFc, 1 + 2 NCT03486067 CC-93269 depletion engagement ERY-974 Chugai CD3 × GPC3 Phase I Target cell Cytotoxic effector Fab + FabwithFc, 1 + 1 NCT02748837 depletion engagement GBR-1302 Glenmark, Harbour CD3 × HER2 Phase I Target cell Cytotoxic effector Fab + scFv with Fc, 1 + 1 NCT02829372, BioMed depletion engagement NCT03983395 GBR-1342 Glenmark CD3 × CD38 Phase I Target cell Cytotoxic effector Fab + scFv with Fc, 1 + 1 NCT03309111 depletion engagement Continued Antibody Therapeutics, 2020 29 Table 4. Continued Antibody name Organization Targets Highest phase Biological Type of mechanism Format Clinical studies function GEM-333 GEMoaB, Celgene CD3 × CD33 Phase I Target cell Cytotoxic effector scFv + scFv, 1 + 1 NCT03516760 depletion engagement GEM-3PSCA, GEMoaB, Celgene CD3 × PSCA Phase I Target cell Cytotoxic effector scFv + scFv, 1 + 1 NCT03927573 GEM3PSCA depletion engagement IGM-2323 IGM Biosciences CD3 × CD20 Phase I Target cell Cytotoxic effector Fab + scFv with Fc, NCT04082936 depletion engagement 1 + 10 JNJ-67571244, Janssen Research & CD3 × CD33 Phase I Target cell Cytotoxic effector Fab + FabwithFc, 1 + 1 NCT03915379 JNJ-1244 Development depletion engagement JNJ-63709178, Janssen Research & CD3 × CD123 Phase I Target cell Cytotoxic effector Fab + FabwithFc, 1 + 1 NCT02715011 JNJ-9178 Development depletion engagement JNJ-64007957, Janssen Research & CD3 × BCMA Phase I Target cell Cytotoxic effector Fab + FabwithFc, 1 + 1 NCT03145181 JNJ-7957 Development depletion engagement JNJ-63898081, Janssen Research & CD3 × PSMA Phase I Target cell Cytotoxic effector Fab + FabwithFc, 1 + 1 NCT03926013 JNJ-8081 Development depletion engagement Orlotamab, MacroGenics CD3 × B7-H3 Phase I Target cell Cytotoxic effector Fv + Fv with Fc, 1 + 1 NCT02628535, MGD-009 depletion engagement NCT03406949 Pasotuxizumab, Amgen, Bayer CD3 × PSMA Phase I Target cell Cytotoxic effector scFv + scFv, 1 + 1 NCT01723475, AMG-212, depletion engagement NCT01723475 PF-06671008 Pfizer CD3 × CDH3 Phase I Target cell Cytotoxic effector Fv + Fv with Fc, 1 + 1 NCT02659631 depletion engagement PF-06863135, Pfizer CD3 × BCMA Phase I Target cell Cytotoxic effector Fab + FabwithFc, 1 + 1 NCT03269136 PF-3135 depletion engagement REGN-5458 Regeneron, Sanofi CD3 × BCMA Phase I Target cell Cytotoxic effector Fab + FabwithFc, 1 + 1 NCT03761108 depletion engagement RG-6194, Genentech CD3 × HER2 Phase I Target cell Cytotoxic effector Not disclosed NCT03448042 BTRC-4017A depletion engagement TNB-383B TeneoBio, AbbVie CD3 × BCMA Phase I Target cell Cytotoxic effector Fab + SDA with Fc, 1 + 2 NCT03933735 depletion engagement XmAb-13676, Xencor CD3 × CD20 Phase I Target cell Cytotoxic effector Fab + scFv with Fc, 1 + 1 NCT02924402 THG-338 depletion engagement XmAb-14045, Xencor, Novartis CD3 × CD123 Phase I Target cell Cytotoxic effector Fab + scFv with Fc, 1 + 1 NCT02730312 SQZ-622 depletion engagement XmAb-18087, Xencor CD3 × SSTR2 Phase I Target cell Cytotoxic effector Fab + scFv with Fc, 1 + 1 NCT03411915 XENP-18087 depletion engagement HPN-424 Harpoon CD3 × PSMA × albu- Phase I Target cell Cytotoxic effector SDA-SDA-scFv, NCT03577028 min depletion engagement 1 + 1 + 1 M-802 Wuhan YZY CD3 × HER2 Phase I Target cell Cytotoxic effector Fab + scFv with Fc, 1 +1NA Biopharma depletion engagement Continued 30 Antibody Therapeutics, 2020 Table 4. Continued Antibody name Organization Targets Highest phase Biological Type of mechanism Format Clinical studies function JNJ-64407564 Janssen CD3 × GPRC5D Phase I Target cell Cytotoxic effector Fab + FabwithFc, 1 + 1 NCT04108195, depletion engagement NCT03399799 RG-6160 Genentech CD3 × FcRH5 Phase I Target cell Cytotoxic effector Fab + FabwithFc, 1 + 1 NCT03275103 depletion engagement NI-1701, TG-1801 NovImmune, TG CD19 × CD47 Phase I Target cell Enhance Fab + FabwithFc, 1 + 1 NCT03804996 Therapeutics depletion phagocytosis MCLA-158 Merus EGFR × LGR5 Phase I Target cell Fc effector Fab + FabwithFc, 1 + 1 NCT03526835 depletion ZW-49 Zymeworks HER2 × HER2 Phase I Target cell ADC Fab + scFv with Fc, 1 + 1 NCT03821233 depletion A-319 Generon (Shanghai) CD3 × CD19 IND Filed Target cell Cytotoxic effector Fab + scFv, 1 + 2 NCT04056975 depletion engagement SAR-440234 Sanofi CD3 × CD123 Suspended (1/2) Target cell Cytotoxic effector Fab + Fv with Fc, 1 + 1 NCT03594955 depletion engagement AFM-11 Affimed CD3 × CD19 Suspended (1) Target cell Cytotoxic effector Fv + Fv, 2 + 2 NCT02106091, depletion engagement NCT02848911 cMet × EGFR Sorrento EGFR × cMET Preclinical Target cell ADC Not disclosed NA ADC depletion APLP2 × HER2 Regeneron APLP2 × HER2 Preclinical Target cell ADC Fab + FabwithFc, 1 +1NA ADC depletion ABP-150 Abpro Claudin 18.2 × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv with Fc, 2 +2NA depletion engagement ABP-110 Abpro GPC3 × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv with Fc, 2 +2NA depletion engagement ABP-140 Abpro; Luye CEA × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv with Fc, 2 +2NA depletion engagement ABP-130 Abpro; Luye CD38 × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv with Fc, 2 +2NA depletion engagement ABP-100 Abpro; MSK Her2 × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv with Fc, 2 +2NA Cancer Center depletion engagement CD16 × BCMA Affimed BCMA × CD16 Preclinical Target cell Cytotoxic effector Fv + Fv + Fv, 1 + 2 +1NA × CD200 × CD200 depletion engagement AFM-26 Affimed BCMA × CD16 Preclinical Target cell Cytotoxic effector Fv + Fv, 2 +2NA depletion engagement AFM-24 Affimed EGFR × CD16 Preclinical Target cell Cytotoxic effector Fv + Fv, 2 +2NA depletion engagement AFM-21 Affimed EGFRvIII × CD3 Preclinical Target cell Cytotoxic effector Fv + Fv, 2 +2NA depletion engagement B05/CD3 Affimed; Immatics MMP1-003/HLA- Preclinical Target cell Cytotoxic effector Fv + Fv, 2 +2NA A 02 × CD3 depletion engagement Continued Antibody Therapeutics, 2020 31 Table 4. Continued Antibody name Organization Targets Highest phase Biological Type of mechanism Format Clinical function studies CD3 × FLT3 Allogene; Maverick; FLT3 × CD3 Preclinical Target cell Cytotoxic effector Fab + FabwithFc, 1 +1NA Pfizer depletion engagement Fol-aCD3 Ambrx FolRa × CD3 Preclinical Target cell Cytotoxic effector Fab + LIGAND with Fc NA depletion engagement CD3 × MSLN Amgen MSLN × CD3 Preclinical Target cell Cytotoxic effector scFv + scFv, 1 +1NA depletion engagement CDH19 × CD3 Amgen Cadherin 19 × CD3 Preclinical Target cell Cytotoxic effector scFv + scFv with Fc, NA HLE BiTE depletion engagement 1 + 1 CD3 × EGFR Amgen; CytomX EGFR × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv with Fc, 2 +2NA Pb-TCB Therapeutics depletion engagement AMX-168 Amunix EpCAM × CD3 Preclinical Target cell Cytotoxic effector scFv + scFv, 1 +1NA depletion engagement APVO-425 Aptevo Therapeutics ROR1 × CD3 Preclinical Target cell Cytotoxic effector scFv + scFv with Fc, NA Inc depletion engagement 2 + 2 ARB-201 Arbele Cadherin-17 × CD3 Preclinical Target cell Cytotoxic effector scFv + scFv with Fc, NA depletion engagement 1 + 1 AVA-012 Avacta CD22 × CD3 Preclinical Target cell Cytotoxic effector Not disclosed NA depletion engagement CD3 × CD19 Avacta CD19 × CD3 Preclinical Target cell Cytotoxic effector Not disclosed NA depletion engagement CD3 × CD123 Baylor Scott & CD123 × CD3 Preclinical Target cell Cytotoxic effector scFv + scFv with Fc, NA White Research depletion engagement 2 + 2 Institute CD3 × HER2 Beijing Hanmi Her2 × CD3 Preclinical Target cell Cytotoxic effector Not disclosed NA depletion engagement CD3 × DLL3 Boehringer DLL3 × CD3 Preclinical Target cell Cytotoxic effector scFv + scFv with Fc, NA Ingelheim depletion engagement 1 + 1 ∗ ∗ CCW-702 CIBR ; Scripps PSMA × CD3 Preclinical Target cell Cytotoxic effector Fab + SMOL NA depletion engagement CBA-1535 Chiome Bioscience 5T4 × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv, 1 +2NA depletion engagement CTX-4419 Compass BCMA × NKp30 Preclinical Target cell Cytotoxic effector Fab + FabwithFc, 2 +2NA Therapeutics depletion engagement COVA-4231 Covagen; Fred CD33 × CD3 Preclinical Target cell Cytotoxic effector Fab + SCAFFOLD with NA Hutch depletion engagement Fc, 2 + 2 CD3 × EGFRvIII Duke University EGFRvIII × CD3 Preclinical Target cell Cytotoxic effector scFv + scFv, 1 +1NA depletion engagement Continued 32 Antibody Therapeutics, 2020 Table 4. Continued Antibody name Organization Targets Highest phase Biological Type of mechanism Format Clinical function studies ESK1 Eureka; MSK WT1p/HLA- Preclinical Target cell Cytotoxic effector scFv + scFv, 1 +1NA Cancer Center; A0201 × CD3 depletion engagement Novartis FPA-151 Five Prime BCMA × CD3 Preclinical Target cell Cytotoxic effector Not disclosed NA depletion engagement CD3 × CD79b Genentech Inc CD79b × CD3 Preclinical Target cell Cytotoxic effector Fab + FabwithFc, 1 +1NA depletion engagement CD3 × HER2 Genentech Her2 Preclinical Target cell Cytotoxic effector Fab + Fab + Fab with NA biparatopic biparatopic × CD3 depletion engagement Fc, 1 + 1 + 1 GBR-1372 Glenmark EGFR × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv with Fc, 1 +1NA depletion engagement PM-CD3-GEX Glycotope TA-MUC1 × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv with Fc, 2 +2NA depletion engagement HPN-217 Harpoon BCMA × CD3 × albu- Preclinical Target cell Cytotoxic effector SDA-SDA-scFv, NA min depletion engagement 1 + 1 + 1 HLX-31 Henlix; Henlix Her2 × CD3 Preclinical Target cell Cytotoxic effector Not disclosed NA depletion engagement p95HER2-TCB Hospital Vall Her2 × CD3 Preclinical Target cell Cytotoxic effector Fab + FabwithFc, 1 +2NA D’Hebron; MSK depletion engagement Cancer Center; Roche; U. Autonoma de Barcelona E1-3s IBC Trop2 × CD3 Preclinical Target cell Cytotoxic effector scFv + Fab, 1 +2NA Pharmaceuticals; depletion engagement Immunomedics CD123/CD3 bsAb IGM Biosciences CD123 × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv with Fc, NA depletion engagement 1 + 10 CD38/CD3 bsAb IGM Biosciences CD38 × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv with Fc, NA depletion engagement 1 + 10 IPH-61 Innate Pharma; TAA × NKp46 Preclinical Target cell Cytotoxic effector Fab + FabwithFc, 1 +1NA Sanofi depletion engagement CD123-CODV- INSERM; Sanofi CD123 × CD3 Preclinical Target cell Cytotoxic effector Fv + Fv with Fc, 2 +2NA TCE depletion engagement GNR-047 IBC Generium CD19 × CD3 Preclinical Target cell Cytotoxic effector Fv + Fv with Fc, 2 +2NA depletion engagement JNJ-0819 Janssen Heme × CD3 Preclinical Target cell Cytotoxic effector Fab + FabwithFc, 1 +1NA depletion engagement Continued Antibody Therapeutics, 2020 33 Table 4. Continued Antibody name Organization Targets Highest phase Biological Type of mechanism Format Clinical function studies Vγ 9/Vδ2 Lava EGFR × g9/d2 TCR Preclinical Target cell Cytotoxic effector SDA + SDA, 1 +1NA TCR × EGFR depletion engagement CD3 × 5T4 MacroGenics 5T4 × CD3 Preclinical Target cell Cytotoxic effector Fv + Fv with Fc, 1 +1NA depletion engagement Next-generation MacroGenics CD19 × CD3 Preclinical Target cell Cytotoxic effector Fv + Fv with Fc, 1 +1NA CD19 × CD3 depletion engagement DART CD123 × CD3 MacroGenics CD123 × CD3 Preclinical Target cell Cytotoxic effector Fv + Fv with Fc, 1 +1NA DART depletion engagement EphA2xCD3 MacroGenics Epha2 × CD3 Preclinical Target cell Cytotoxic effector Fv + Fv with Fc, 1 +1NA DART depletion engagement CD3 × IL13Ra2 MacroGenics IL-13Ra2 × CD3 Preclinical Target cell Cytotoxic effector Fv + Fv with Fc, 1 +1NA depletion engagement CD3 × ROR1 MacroGenics ROR1 × CD3 Preclinical Target cell Cytotoxic effector Fv + Fv with Fc, 1 +1NA depletion engagement CD3 × CD133 McMaster CD133 × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv, 1 +1NA University; depletion engagement University of Toronto h8F4-BiTE MD Anderson PR1/HLA-A2 × CD3 Preclinical Target cell Cytotoxic effector scFv + scFv, 1 +1NA Cancer Center depletion engagement ZW-38 Merck; Zymeworks CD19 × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv with Fc, 1 +1NA depletion engagement CD3 × HER2 Molecular Partners Her2 × CD3 Preclinical Target cell Cytotoxic effector SCALFFOLD, 1 +1NA depletion engagement CD3 × PSMA Regeneron PSMA × CD3 Preclinical Target cell Cytotoxic effector Fab + FabwithFc, 1 +1NA depletion engagement CD3 × CD20 Rinat-Pfizer CD20 × CD3 Preclinical Target cell Cytotoxic effector Fab + FabwithFc, 1 +1NA depletion engagement CD3 × ROR1 Scripps Research ROR1 × CD3 Preclinical Target cell Cytotoxic effector scFv + scFv with Fc, NA Institute depletion engagement 1 + 1 B-193 Shandong Danhong; CD19 × CD3 Preclinical Target cell Cytotoxic effector Not disclosed NA Shanghai Yanyi depletion engagement CD3 × Sialyl-Tn Siamab Sialyl-Tn × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv with Fc, 2 +2NA Therapeutics depletion engagement 19-3-19 SpectraMab CD19 × CD3 Preclinical Target cell Cytotoxic effector scFv + scFv, 1 +2NA depletion engagement SV-202 SYSVAX CD19 × CD3 Preclinical Target cell Cytotoxic effector SDA + SDA, 1 +1NA depletion engagement Continued 34 Antibody Therapeutics, 2020 Table 4. Continued Antibody name Organization Targets Highest phase Biological Type of mechanism Format Clinical function studies SV-201 SYSVAX Her2 × CD3 Preclinical Target cell Cytotoxic effector SDA + SDA, 1 +1NA depletion engagement TNB-585 TeneoBio PSMA × CD3 Preclinical Target cell Cytotoxic effector Fab + SDA with Fc, NA depletion engagement 1 + 1or1 + 1 + 1 TNB-486 TeneoBio CD19 × CD3 Preclinical Target cell Cytotoxic effector Fab + SDA with Fc, 1 +1NA depletion engagement TNB-381 M TeneoBio BCMA × CD3 Preclinical Target cell Cytotoxic effector Fab + SDA with Fc, 1 +1NA depletion engagement CD3 × CD19 Tianjin Chase Sun CD19 × CD3 Preclinical Target cell Cytotoxic effector Not disclosed NA Jinboda depletion engagement CD3 × MOSPD2 VBL Therapeutics MOSPD2 × CD3 Preclinical Target cell Cytotoxic effector scFv + scFv, 1 +1NA depletion engagement M-701 Wuhan YZY EpCAM × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv with Fc, 1 +1NA depletion engagement Y-150 Wuhan YZY CD38 × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv with Fc, 1 +1NA depletion engagement CD3 × EMP2 Wuhan YZY EMP2 × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv with Fc, 1 +1NA depletion engagement CD3 × EGFR Wuhan YZY EGFR × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv with Fc, 1 +1NA depletion engagement CD3 × CD19 Wuhan YZY CD19 × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv with Fc, 1 +1NA depletion engagement CD3 × CD20 Wuhan YZY CD20 × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv with Fc, 1 +1NA depletion engagement MS-133 Wuhan YZY CD133 × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv with Fc, 1 +1NA depletion engagement XmAb-14484 Xencor PSMA × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv with Fc, 1 +1NA depletion engagement YBL-013 Y-Biologics PD-L1 × CD3 Preclinical Target cell Cytotoxic effector Fab + Fv, 1 +2NA depletion engagement huCD33-BsAb Y-mAbs CD33 × CD3 Preclinical Target cell Cytotoxic effector Fab + scFv with Fc, 2 +2NA depletion engagement BI-905711 Boehringer Cadherin- Preclinical Target cell Enhance apoptosis Fab + scFv with Fc, 2 +2NA Ingelheim 17 × TRAIL-R2 depletion Novotarg Promethera CD20 × CD95 Preclinical Target cell Enhance apoptosis Fab + scFv, 1 +1NA depletion Continued Antibody Therapeutics, 2020 35 Table 4. Continued Antibody name Organization Targets Highest phase Biological Type of mechanism Format Clinical function studies ABP-160 Abpro PD-L1 × CD47 Preclinical Target cell Enhance Not disclosed NA depletion phagocytosis BH-29xx Beijing Hanmi PD-L1 × CD47 Preclinical Target cell Enhance Fab + FabwithFc, 1 +1NA depletion phagocytosis IMM-0306 Gateway Biologics; CD20 × CD47 Preclinical Target cell Enhance Fab + LIGAND with Fc, NA ImmuneOnco depletion phagocytosis 2 + 2 HMBD-004A Hummingbird CD33 × CD47 Preclinical Target cell Enhance Fab + scFv, 1 +1NA depletion phagocytosis HMBD-004B Hummingbird BCMA × CD47 Preclinical Target cell Enhance Fab + FabwithFc, 1 +1NA depletion phagocytosis IMM-2505 ImmuneOnco PD-L1 × CD47 Preclinical Target cell Enhance Fab + LIGAND with Fc, NA depletion phagocytosis 2 + 2 IMM-26011 ImmuneOnco FLT-3 × CD47 Preclinical Target cell Enhance Fab + LIGAND with Fc, NA depletion phagocytosis 2 + 2 IMM-0207 ImmuneOnco VEGF × CD47 Preclinical Target cell Enhance RECEPTOR + LIGAND NA depletion phagocytosis with Fc, 2 + 2 IMM-2902 ImmuneOnco Her2 × CD47 Preclinical Target cell Enhance Fab + LIGAND with Fc, NA depletion phagocytosis 2 + 2 IBI-322 Innovent PD-L1 × CD47 Preclinical Target cell Enhance Not disclosed NA depletion phagocytosis NI-1801 Novimmune MSLN × CD47 Preclinical Target cell Enhance Fab + FabwithFc, 1 +1NA depletion phagocytosis PT-886 Phanes Therapeutics Claudin 18.2 × CD47 Preclinical Target cell Enhance Not disclosed NA depletion phagocytosis PT-217 Phanes Therapeutics DLL3 × CD47 Preclinical Target cell Enhance Not disclosed NA depletion phagocytosis PMC-122 PharmAbcine PD-L1 × CD47 Preclinical Target cell Enhance Not disclosed NA depletion phagocytosis DuoHexaBody- Genmab CD37 biparatopic Preclinical Target cell Fc effector Fab + FabwithFc, 1 +1NA CD37 depletion PM-PDL-GEX Glycotope TA-MUC1 × PD-L1 Preclinical Target cell Fc effector Fab + scFv with Fc, 2 +2NA depletion CD38 × IGF-1R I’rom Group CD38 × IGF-1R Preclinical Target cell Fc effector scFv + scFv with Fc, NA depletion 1 + 1 CIBR, California Institute for Biomedical Research; SMOL, small molecule. 36 Antibody Therapeutics, 2020 Table 5. Programs in clinical and preclinical stages for inflammatory conditions Antibody name Organization Targets Highest Biological function Type of mechanism Format Clinical studies phase Ozoralizumab, Sanofi, Taisho, TNF × albumin Phase III Half-life extension Half-life extension SDA + SDA, 1 + 2 NCT00959036, TS-152, Eddingpharm NCT01007175, PF-5230896, NCT01063803, ATN-103 NCT04077567 Vobarilizumab AbbVie; Ablynx IL-6R × albumin Phase II Half-life extension Half-life extension SDA + SDA, 1 + 1 NCT02518620, NCT02437890, NCT02309359, NCT02287922 Romilkimab, Sanofi IL-4 × IL-13 Phase II Combinatorial effect Combinatorial effect Fab + Fv with Fc, NCT01529853, SAR-156597 2 + 2 NCT02345070, NCT02921971 M-1095, Avillion; Merck Serono IL-17A × albumin Phase II Combinatorial effect Combinatorial effect SDA + SDA, 1 + 1 + 1 NCT03384745, ALX-0761 × IL-17F NCT02156466 MGD-010, MacroGenics, Provention CD32B × CD79B Phase I/II Dominant negative Dominant negative Fv + Fv with Fc, 1 + 1 NCT02376036 PRV-3279 AMG-570, Amgen, Viela Bio, BAFF × ICOSL Phase I Combinatorial effect Combinatorial effect Fab + PEPTIDE with NCT02618967, MEDI-0700 AstraZeneca Fc, 2 + 2 NCT03156023, NCT04058028 Tibulizumab Eli Lilly BAFF × IL-17A Phase I Combinatorial effect Combinatorial effect Fab + scFv with Fc, Company 2 + 2 development pipeline JNJ-61178104 Janssen Research & TNF × IL-17A Phase I Combinatorial effect Combinatorial effect Fab + FabwithFc, NCT02758392 Development 1 + 1 ONO-4685 Ono CD3 × PD-1 Phase I Dominant negative Dominant negative Fab + FabwithFc, NCT04079062 1 + 1 ES-210, Aptevo Therapeutics CD86 - IL10 Phase I Tissue specificity Tumor or tissue scFv + scFv with Fc, NCT03768219 APVO-210 localization 2 + 2 CD19 × CD11c National Jewish Health CD19 × CD11c Preclinical Target cell depletion Fc effector Fab + FabwithFc, NA 1 + 1 AM-201 AbClon IL-6R × TNF Preclinical Anti-inflammation Combinatorial effect Fab + SCAFFOLD NA with Fc, 2 + 2 IL4Ralpha/IL-5 arGEN-X IL-4Ra × IL-5 Preclinical Anti-inflammation Combinatorial effect Fab + scFv with Fc, NA bsAb 2 + 2 Continued Antibody Therapeutics, 2020 37 SDA-based. Two different VHHs can be fused to form absAb. This format may be the smallest bsAb format with molecular weight approximately 25 KD. It has been reported that two different VHHs can be fused to coiled- coil peptide to form Combody. The peptide facilitates the oligomerization of the antibody and renders the antibody avidity effect . Two different VHH can also be engineered on the N-terminus of CH1 and CL to form a Fab-like 1 + 1 bsAb fragment . ScFv-based. Bispecific T cell engager (BiTE), one of the formats used to redirect T cells to tumor cells, com- prises two tandem linked scFvs: one scFv against a tumor- associated antigen and another binding to CD3 on T cells. The structure and mechanism of BiTE was well reviewed by Wolf . Two scFvs can also be indirectly linked, such as via a CL, to form a bsAb in scFv-CL-scFv format . Due to aggregative tendency of scFv, various techniques were employed to stabilize scFv. Brolucizumab (Beovu) was engi- neered using scFv-λcap platform . Similar technology was also used to build multispecific antibody (msAb)-based therapeutics by cognate heterodimerization (MATCH) , where up to four distinct binding sites can be integrated into a multispecific Fv- or scFv-based molecule. Fv-based. A diabody molecule is formed by two polypep- tides: one polypeptide contains VHa and VLb; another polypeptide contains VHb and VLa. Due to the short linker, VHa associates with VLa on another polypeptide and similarly VLb associates with VHb to form 1 + 1bsAb fragment. A diabody-based bispecific format is called dual- affinity retargeting antibody or DART [11–13]. DART molecules may contain Fc domain to extend in vivo half- life and grand effect functions. TandAb is another Fv-based bispecific fragment: two polypeptides are forced to fold in a head-to-tail fashion to form 2 + 2bsAbfragment. Combination. In a native antibody, VH and VL are on the N-terminus of Fab region and CH1 and CL on C- terminus. It was found that CH1 and CL can also facilitate the association of VH and VL on C-terminus of a Fab- Fv fusion protein. This Fab directed VH-VL association can be further improved by introducing a disulfide bond between the VH and VL on C-terminus . A VH on the C-terminus of a Fab-Fv may associate with a C-terminal VL on another Fab-Fv to form 2 + 2 tetramer Fab-Fv . Similarly, a scFv can be fused on the C-terminus of a Fab to form Fab-scFv fusion proteins. The so-called bibody has one Fab with one scFv, and “tribody” has one Fab with two scFvs . A tribody can be either bispecific or trispecific, depending on the specificity of the two attached scFvs. A VHH can be fused to a light chain C-terminus of aFab to form 1 + 1 bispecific antibody fragment . It was reported that three tandem linked VHHs can be fused with a scFv to form 1 + 3 bispecific fragment . A bsAb fragment containing a VHH or scFv specific to human serum albumin is a common strategy to extend serum half- life of such molecules. Table 5. Continued Antibody name Organization Targets Highest Biological function Type of mechanism Format Clinical studies phase BH-1657 Beijing Hanmi TNF × IL-17A Preclinical Anti-inflammation Combinatorial effect Fab + FabwithFc, NA 1 + 1 IL-4 × IL-13 Beijing VDJBio IL-4 × IL-13 Preclinical Anti-inflammation Combinatorial effect Not disclosed NA IL-1 × TNFα Beijing VDJBio IL-1 × TNF Preclinical Anti-inflammation Combinatorial effect Not disclosed NA CMX-02 Complix TNF × IL-23 Preclinical Anti-inflammation Combinatorial effect Fab + SCAFFOLD NA with Fc, 2 + 2 ND-016 Intarcia TNF × IL- Preclinical Anti-inflammation Combinatorial effect Fv + Fv + Fv, NA 17A × albumin 1 + 1 + 1 MT-6194 Mitsubishi Tanabe IL-6R × IL-17A Preclinical Anti-inflammation Combinatorial effect Fab + SCAFFOLD NA Pharma with Fc, 2 + 2 YBL-004 Y-Biologics TNF × IL-17A Preclinical Anti-inflammation Combinatorial effect Fab + scFv with Fc, NA 2 + 2 PT-001 Pandion MAdCAM × PD-1 Preclinical Anti-inflammation Tumor or tissue Fab + scFv with Fc, NA localization 2 + 2 ALXN-1720 Alexion C5 × albumin Preclinical Half-life extension Extended half-life scFv + scFv, 1 +1NA 38 Antibody Therapeutics, 2020 Table 6. Programs in clinical and preclinical stages for other conditions Antibody name Organization Targets Highest Biological function Type of mechanism Format Conditions Clinical studies phase Faricimab, Roche, Chugai VEGF × ANGPT2 Phase III Anti-angiogenesis Combinatorial effect Fab + Fab with Ocular, diabetic NCT01941082, RG-7716, Pharmaceutical Fc, 1 + 1 retinopathy NCT02484690, RO-6867461 NCT02699450, NCT03038880, NCT03622580, NCT03622593, NCT03823287, NCT03823300 IBI-302 Innovent VEGF × comple- Phase I Anti-angiogenesis; Combinatorial effect Not disclosed Ocular NCT03814291 ment anti-inflammation Gremubamab, MedImmune PcrV × PsI Phase II Combinatorial effect Combinatorial effect Fab + scFv Antibacterial NCT02255760, MEDI3902 with Fc, 2 + 2 NCT02696902 MEDI-7352 AstraZeneca NGF × TNF Phase II Combinatorial effect Combinatorial effect scFv + RECEP- Analgesic drugs NCT02508155, TOR with Fc, NCT03755934 2 + 2 10E8.4/iMab TaiMed, Aaron HIV-1 Env × CD4 Phase I Broaden protection Combinatorial effect Fab + Fab with HIV-1 NCT03875209 Diamond AIDS Fc, 1 + 1 Research Center SAR-441236 Sanofi, NIH HIV-1 Env Phase I Combinatorial effect Combinatorial effect Fab + Fv with HIV-1 NCT03705169 triparatopic Fc, 1 + 1 + 1 MGD-014 MacroGenics, CD3 × HIV-1 Env Phase I Target cell depletion Cytotoxic effector Fv + Fv with HIV-1 NCT03570918 NIAID protein engagement Fc, 1 + 1 BFKB-8488A, Genentech FGFR1 × beta- Phase I Tissue specificity Tumor or tissue scFv + scFv Diabetes NCT02593331, RG-7992 Klotho localization with Fc, 1 + 1 NCT03060538 ABP-201 AbMed VEGF × ANGPT2 Preclinical Anti-angiogenesis Combinatorial effect Fab + scFv Ocular NA with Fc, 2 + 2 SL-634 University of VEGF × ANGPT2 Preclinical Anti-angiogenesis Combinatorial effect Not disclosed Ocular NA Colorado System KSI-501 Kodiak Sciences VEGF × IL-6 Preclinical Anti-angiogenesis, Combinatorial effect Fab + RECEP- DME, Uveitis NA anti-inflammation TOR with Fc, 2 + 2 FIT-1 Humabs BioMed Zika E protein Preclinical Broaden protection Biparatopic Fab + Fab with Infection NA biparatopic Fc, 2 + 2 TMB-bispecific Aaron Diamond HIV gp120 × CD4 Preclinical Broaden protection Combinatorial effect Fab + Fab with Infection NA AIDS Research Fc, 1 + 1 Center; TaiMed VIS-RSV Visterra, Vir RSV F × RSV G Preclinical Broaden protection Combinatorial effect Fab + scFv Infection NA Biotechnology with Fc, 1 + 1 Continued Antibody Therapeutics, 2020 39 Fc-containing bispecific antibodies The Fc region, part of natural antibody, is homodimer of two polypeptide chains. Depending on the isotype of the antibody, each comprising two to three heavy chain constant domains (CH2, CH3, and CH4). The Fc region not only imparts an antibody effector functions due to Fcγ R binding and complement-binding but also extends its in vivo half-life via FcRn binding. When two different heavy chains and two different light chains of IgG antibodies are expressed in one cell, these different heavy chains and light chains may scramble randomly, possibly to form 10 different IgG antibody molecules. Among these, statistically only 12.5% would be desired bsAb. For symmetric Fc-containing bispecific formats, a challenge is to avoid heavy chain/light chain mispairing. For asymmetric Fc-containing bispecific formats, an additional challenge is to force heterodimeric heavy chain formation. Forming heterodimeric Fc can be achieved by engineer- ing the interface of two CH3 (for IgG) or CH4 (for IgM or IgE) domains, by changing size (knob-into-hole) [20– 22] and electrostatic steering . Computational methods have been used to identify the mutations that facilitate het- erodimeric association [24, 25]. Several groups also used the interface of different Ig proteins to design the heterodimeric Fc. Davis et al. developed derivatives of human IgG and IgA CH3 domains composed of alternating segments of human IgA and IgG CH3 sequences . Skegro et al. grafted some residues from T cell receptor (TCR) constant region to CH3 of IgG1 or CH4 of IgM . An alter- native strategy is to purify heterodimer from unwanted homodimers. With the modification on CH3 domain, the heterodimer and homodimer have different affinity binding on Protein A, and the bsAb with heterodimeric Fc can be isolated . In order to ensure cognate heavy chain and light chain pairing, several strategies have been reported. The first strategy is to use SDA, scFv, or scFab as antigen-binding building blocks. In addition, single-chain IgG has been reported , where two heavy chains, two light chains, and three linker sequences were expressed from one gene. Protease cleavage sites were integrated into these linkers, allowing protease digestion to cleave the linkers. The second strategy is using common light chain or common heavy chain. In the case of common light chain, an identical light chain is used as the partner for two different heavy chains [30–33]. Common heavy chain was also reported in a κλ- body case . The third strategy is to modify the interface of VH-CH1 and VL-CL, including developing orthogonal Fab interface [35, 36], altering the location of interchain disulfide bond [37–39], and addition of charged pairs and knob-into-hole [40, 41]. These strategies usually involve changes on both VH-VL interface and CH1/CL interface. Yet, Bonish et al. reported that the preferential association can be achieved by only engineering the CH1/CL interface . To associate cognate VH and VL, the CH2 domain from IgM or IgE have been engineered to form heterodimer to replace CH1/CL  [Dong, WO2017011342]. There are additional strategies to avoid heavy chain/light chain mispairing. Schaefer et al. described a CrossMab Table 6. Continued Antibody name Organization Targets Highest Biological function Type of mechanism Format Conditions Clinical phase studies Dual FZD and AntlerA FZD × LRP5/6 Preclinical Cofactor mimetic Cofactor mimetic Fv + Fv with Fc, Tissue repair NA LRP5/6 agonist Therapeutics biparatopic 2 + 1 + 1 ABL-301 ABL Bio ? × alpha-synuclein Preclinical Neutralizing Trojan horse Fab + scFv with Fc, Neurology/Psychiatric NA pathogenic target 2 + 2 ATV:BACE1/Tau Denali TfR × BACE1 or Preclinical Neutralizing Trojan horse Fab + Fab + SDA Neurology/Psychiatric NA TfR × Tau pathogenic target with Fc, 1 + 1 + 1 KY-1049 Kymab Ltd FIX × FX Preclinical Cofactor mimetic Cofactor mimetic Fab + FabwithFc, Hematologic NA 1 + 1 Bispecific fully Shire plc FIX × FX Preclinical Cofactor mimetic Cofactor mimetic Fab + FabwithFc, Hematologic NA human IgG 1 + 1 40 Antibody Therapeutics, 2020 Figure 2. The classification of bsAb formats based on assembly of antibody fragments as building blocks. The first row and column list the five basic building blocks (SDA, Fv, scFv, Fab, scFab). The different color and shape of VH and VL represent their origins from different parental antibodies. The assembly of different building blocks creates various bsAb formats classified into 30 groups. An exemplary format and its molecular weight of each group are listed. The diagonal line divides the formats into bispecific formats without Fc (top right with number 1-15) and bispecific formats with Fc (bottom left with number 16-30): 1, tandemly linked SDAs; 2, a SDA tandemly linked on the VH of a Fv; 3, a SDA tandemly linked on the VH of a scFv; 4, two SDAs are separately linked on the carboxyl-terminus of constant domain of a Fab; 5, a SDA tandemly linked on the VL of a scFab; 6, the VHs and VLs of two Fvs cross pair to each other to form diabody; 7, a scFv tandemly linked on the VH of a Fv; 8, the VH and VL of a Fv each linked on the carboxyl-terminus of CH1 and CL of a Fab; 9, the VH of a Fv linked to the CH1 of a scFab; 10, two tandemly linked scFvs; 11, a scFv linked on the VH of a Fab; 12, a scFab Antibody Therapeutics, 2020 41 approach: exchange of heavy chain and light chain domains aggregate. A scFv can be fused with heavy chain of IgG to within the Fab of one half of the bsAb . Moore et al. form symmetric 2 + 2bsAb, called Morrison format. described a Mab-Fv approach: the first pair of variable Morrison format is one of the earliest bispecific formats regions was present on the regular position of an IgG. The that have still been widely used. ScFv can also be fused second pair variable region VL and VH were fused to the with light chain . To form 1 + 1 format, several groups C-terminus of the heterodimeric heavy chain, respectively, used scFv to replace one of the Fabs on an IgG and used to form a 1 + 2bsAb. A similar 1 + 2 bsAb format engineered Fc heterodimer [55, 56]. Two different scFvs can with protease-inducible activity was also reported . be used to replace both Fabs on an IgG antibody to form Recently, a versatile bispecific platform named 1 + 1 bispecific format [57, 58]. ScFv can also be placed in the hinge region or CH3 domain to form 2 + 2 formats WuXiBody has been developed [Xu, WO2019057122A1]. [59, 60]. Kim et al. fused scFv with CH1-CH2-CH3 and co- The CH1/CL domains of one of the two parental antibodies expressed LC domain, potentially masking the hydropho- are replaced by stabilized TCR α and β constant regions bic part of scFv to make the molecule more stable . (Cα/Cβ) to ensure the cognate light chain-heavy chain pairing. This approach has been tested on more than 100 pairs of antibodies and compatible with most of the Fab/scFab-based. Several Fab- or scFab-based bispecific antibody pairs (data not shown). formats have been reported. Fab-based CrossMab, a 1 + 1 Below are some representative examples of Fc-containing bispecific IgG format , was mentioned above. Fabs-in- bispecific formats with SDA, Fv, scFv, Fab, and scFab. tandem immunoglobulin (FIT-Ig) is a format where a Fab is There are more examples that use the combination of these fused to the N-terminus of another IgG antibody: the light five building blocks. chain of the Fab is fused with the heavy chain of the IgG, and FD chain of the Fab and light chain of the IgG are SDA-based. As a small, flexible, and modular antigen- separate polypeptides. These three different polypeptides binding site, it is obvious that SDA or VHH can be fused can be co-expressed from single cells and be assembled to to N-terminus or C-terminus of heavy chain or light chain form the bsAb . In theory, the FD of antibody A and of an IgG antibody. As shown in Fig. 3, SDA can also the light chain of antibody B can associate to each other be inserted in to other fusion sites. Shen et al. reported to form a hybrid Fab, but this hybrid Fab can be removed that a SDA antibody can be fused with VL to form 2 + 2 in Protein A purification step. Bostrom et al. described a bispecific format [46, 47]. Shi et al. used two different SDAs two-in-one bsAb, a bsAb in regular IgG form, and each to replace VH and VL of an IgG, respectively, to form 2 + 2 arm can bind two distinct antigens [63–65]. Hu et al.even biparatopic antibody . In addition to variable domain, developed a four-in-one antibody . Strop et al. showed CH3 domain on Fc has been engineered as binding site that making mutations in hinge region and CH3 domain of . Broadly speaking, the engineered CH3 is a SDA that human IgG1 and IgG2 could facilitate heterodimerization can be integrated into a bispecific format, such as IgG-like of heavy chain. Two parental antibodies can be expressed BsAb . and purified separately and mixed together under appro- priate redox conditions, resulting in formation of a stable Fv-based. VH and VL domains are tandemly linked with bsAb . Labrijn et al. reported a similar platform, later VH and VL of another IgG antibody, to form a 2 + 2 called DuoBody . scFab can be used to construct 1 + 1 bispecific IgG format, named as “dual variable domain format , and it can also be used as one of the building immunoglobulin” or DVD-Ig . Fab + Fv-based 1 + 2 blocks to construct tetraspecific antibody . format called mAb-Fv  was mentioned above. Fv can also replace CH2 domain to form a 1 + 2 bispecific format Other binding modalities called TriFab . Seifert et al. employed diabody format combined with heterodimeric CH2 from IgM or IgE to As mentioned earlier, peptides, ligands, receptors, and construct 2 + 2 bispecific format . Aforementioned different protein scaffolds, either native form or engineered WuXiBody formats are also Fv-based formats, including form, can be used as antigen-binding sites. The non- 1 + 1, 1 + 2, and 2 + 2 formats. antibody scaffolds include Adnectin, DARPin, Affilins, alpha helix scaffold, avimer, Centyrin, Duocalin, Ecallan- ScFv-based. There are many Fc-containing bispecific tide, Fynomer, microprotein, peptide, Protein A domain, formats comprising of scFv, although scFv is prone to trimeric, TCR, etc. There are numerous possibilities to tandemly linked with a scFv; 13, two tandemly linked Fabs: the light chain of one Fab linked with the heavy chain of another Fab and vice versa; 14, a scFab linked on the VH of a Fab; 15, tandemly linked two scFab; 16, two tandemly linked SDA on Fc to form homodimer; 17, a SDA and the VH of a Fv linked to the FcA to pair with the VL of a Fv linked to another FcB to form heterodimer; 18, a diabody on FcA to pair with FcB to form heterodimer; 19, a SDA on FcA to pair with a scFv on FcB to form heterodimer; 20, a scFv and the VH of a Fv linked to FcA to pair with the VL of the Fv linked with FcB to form heterodimer; 21, two scFv tandemly linked to the amino- and carboxyl-terminus of a Fc to form homodimer; 22, a SDA tandemly link to the light chain of a IgG to form homodimer; 23, a TCR constant domain anchored Fv linked to FcA to pair with a half IgG with FcB to form heterodimer TM (WuXiBody ); 24, a scFv linked FcA to pair with a half IgG with FcB to form heterodimer; 25, two tandemly linked Fabs (the light chain of a Fab linked on the heavy chain of another Fab) on Fc to form homodimer (FIT-Ig); 26, a SDA on FcA to pair with a scFab on FcB to form heterodimer; 27, a scFab and the VH of a Fv linked on FcA to pair with the VL linked on FcB to form heterodimer; 28, a scFv linked on FcA to pair with a scFab linked on FcB to form heterodimer; 29, a half IgG with FcA paired with scFv linked on FcB to form heterodimer; 30, two scFab each linked on FcA and FcB to form heterodimer. Above mentioned FcA and FcB are engineered Fc pair to facilitate Fc heterodimerization. 42 Antibody Therapeutics, 2020 Figure 3. Fusion sites for antigen-binding building blocks. A) A heterodimeric Fc fragment has at least six fusion sites: amino-terminus (1 and 4), carboxyl- terminus (3 and 6) of Fc and between CH2 and CH3 (2 and 5). B) The fragment made of heterodimeric Fc and two differently heterodimerized CL-CH1 domains provides at least twelve fusion sites: amino-terminus of CL (1 and 7) and CH1 (3 and 9), carboxyl-terminus of CL (2 and 8) and CH3 (6 and 12), hinge region (4 and 10), between CH2 and CH3 (5 and 11). generate bispecific formats using these binding modalities. Accordingly, with the increase in bsAb development, the Recent examples include peptide , VEGF receptor , number of deals (excluding mergers and acquisitions) have TCR , and single-chain TRAIL . focused on clinical stage bsAbs resulting in an increase In the new paradigm of bsAbs, many novel formats of 140% in the last 3 years, while the total disclosed deal have been designed and tested. The general goal is to value decreased by 50%, from $3.2 billion to $1.6 billion; design a molecule to enable novel therapeutic mechanisms $6.8 billion is recorded across the whole period (Fig. 5A). and to make homogeneous product in large scale to meet From these deals, nine were worth more than $100 million, the need of clinical development and commercial manu- and most were structured with milestones, signifying the facturing. Additionally, more multispecific formats have balancing of risk and reward between the partners. Sanofi been reported in the recent years, including trispecific , and Regeneron’s 2015 co-development agreement focused tetraspecific , and pentaspecific . on various antibodies, including CD3 × MUC16 (REGN- 401) and CD3 × BCMA (REGN-5458) (Fig. 5B). From these deals, $3.8 billion were spent on oncology followed by $1 billion on infection diseases from a total of $6.8 THE RESURGENCE OF BISPECIFIC ANTIBODIES billion, probably due to the clinical and commercial poten- During the last few years, the number of clinical stud- tial of treating patients in these disease areas with bsAbs ies using bsAbs has increased exponentially (Fig. 4A). In (Fig. 5C). fact, this increase in 2014 matched with the launch of The global market of bsAb was worth $0.46 billion in Blincyto (Amgen), the first commercialized BiTE for the 2018, which was dominated almost equally by Blincyto treatment of acute lymphoblastic leukemia. However, it was ($230 million) and Hemlibra ($229 million). As predicted, not until 2017 that another bsAb, Hemlibra (Roche), was the market for Hemlibra will boom in the next few years launched for the treatment of hemophilia A. Currently, and grow up to $3.96 billion by 2024. Instead, Blincyto most bsAbs in clinical development are in early studies will only have a moderate increase. With the massive sales (67% in Phase I, 25% in Phase II) with only five products growth of Hemlibra and the potential approval of new in Phase III studies (Fig. 4B). The majority of clinical stage entrants, for instance, faricimab, gremubamab, MCLA- bsAbs (∼84%) are designed to treat cancer, especially solid 117, and XmAb-14045, the bsAb market will surpass $5.43 tumors, breast cancer, and acute myeloid leukemia. Nev- billion in 2024 (Fig. 5D). ertheless, there are some products designed to treat other conditions such as rheumatoid arthritis or autoimmune diseases (Fig. 4C). The company with more bsAbs under BIOLOGY DRIVES DEVELOPMENT OF VARIOUS active development is Amgen, followed by MacroGenics BISPECIFIC ANTIBODIES and then Lilly, Janssen, Roche, Sanofi, and Xencor. The strategy in nearly half of the developing bispecific products Most human diseases are complex, often driven by multiple is to deplete the malignant cells by engaging cytotoxic effec- redundant or distinct mechanisms; thereby single-target tor cells including T or natural killer (NK) cells using CD3 approach such as mAb may not be sufficient to achieve or FcGR3A (CD16) targeting arms. Another commonly optimal therapeutic efficacy. Especially, many therapeutic used strategy is to identify tumor or tissue-specific markers concepts need physical linkage of two or more targets. In to act only in the affected areas. For that, many companies this case, bsAbs or msAbs targeting two or more targets have designed their own technology to manufacture bsAbs, may offer novel therapeutic applications that are difficult including Amgen’s BiTE, MacroGenics’ DART, or Roche’s to achieve by mAbs. In a recent comprehensive review CrossMab platforms. article, Aran Labrijn, Maarten Janmaat, Janice Reichert, Antibody Therapeutics, 2020 43 Figure 4. Statistics showing the booming of bsAb programs. A). The number of clinical studies associated with bsAb in the past fourteen years (up to TM September 2019). The bsAb programs classified based on B) different clinical stages and C) different disease areas. Data source: Cortellis Competitive Intelligence (CCI) and CortellisTM Drug Discovery Intelligence (CDDI, formerly Integrity) as of Sept 23, 2019. Figure 5. Licenses and market analysis for bsAb programs. A). Licenses for clinical stage bsAbs. Line represents number of license signed each year. Blue and yellow bars represent the largest deal and total deal values for each year, respectively. B). The largest deals signed from 2014 to 2018. C). Deal values TM in disease area. D). BsAb market size in 2018 and forecast in 2024. Data source: Cortellis CCI as of Sept 23, 2019. and Paul Parren thoroughly reviewed global bispecific anti- tions (including IND filed—Phase III—and two programs body clinical pipeline using a mechanistic lens . Based on clinical hold). This highlights the increased interest of TM exploring bsAbs as a venue to develop novel antibody- on the analysis using Cortellis , a Clarivate Analytics based therapeutics. The most frequently studied target pairs solution, by the end of September 2019, there are 176 of those bispecific antibodies and the number of molecules bsAbs or bifunctional proteins with target disclosed under being explored are illustrated in a network graph (Fig. 6). active preclinical development, compared to 119 in clinical We took a step further and analyzed the disease areas cov- development for cancer, autoimmune, and other indica- 44 Antibody Therapeutics, 2020 Figure 6. A network graph characterizing the target pairs of most bispecific programs in both preclinical and clinical investigations. Each node in the network is one target, and each edge connecting two nodes represents one bispecific program. The circular edges are biparatopic programs. The node size shows the degree of a particular target being paired with other different targets. The colors of the edges are marked in black if only one program is available for that particular pair, otherwise in red if more than one are being explored. The popularity of that bispecific program is reflected from the thickness of TM the red edges. Source data are from Cortellis (Table 1-4). Tri-specific and albumin-relevant bispecific programs are not included. The albumin-relevant tri-specific are analyzed as bispecific projects. ered and mechanisms employed by these clinical molecules Anti-angiogenesis. As angiogenesis plays an essential and preclinical drug development candidates as well, with role in promoting tumor progression and metastasis, anti- the attempt to illustrate the principle of selecting a bispe- angiogenesis for cancer treatments have been extensively cific format to meet biology needs and selecting a bispecific explored. Though several therapies, such as bevacizumab molecule as a clinical development candidate by six critical (anti-VEGF) and ramucirumab (anti-VEGFR2), have criteria. been approved to treat several types of tumors, only moderate levels of antitumor activity were observed. Bispecific antibodies for cancer treatment Along with the booming of bispecific programs and better TM understanding of the angiogenesis process, new generation According to the Cortellis ’ analysis, the bsAb pipeline of anti-angiogenesis treatments are emerging. As shown in is composed predominantly by programs for cancer Supplementary Table S1 available online at ABT Online, treatment, with 99/119 programs in clinical stages and 12 programs are under active development. Majority of the 153/176 preclinical programs (Fig. 4C). As reviewed by programs are focusing on improving the anti-angiogenesis Hanahan D. and Weinberg RA., there are eight hallmarks effect by combinatory targeting two or even three molecules of cancer, and targeting these biological capabilities of that are involved in angiogenesis, such as VEGF, VEGFR2, cancer cells may lead to new therapeutic options for cancer DLL4, and ANGPT2. . Therefore, based on the biological functions, we Dilpacimab (AbbVie) targeting DLL4 and VEGF is categorize the bsAb programs into the following groups: one of the most advanced programs in this category. anti-angiogenesis, anti-tumorigenesis, enhancing tumor DLL4-Notch signaling plays a critical role in angiogenic immunity, modulating tumor microenvironment (TME), sprouting, and DLL4 blockade alone has shown inhibition and depletion of target cells. Antibody Therapeutics, 2020 45 in tumor growth [77, 78]. Dilpacimab was designed to members, Boehringer Ingelheim is developing a first- co-inhibit both DLL4 and VEGF signaling to achieve in-class biparatopic antibody to block the function of more prominent antitumor efficacy . Dilpacimab was LRP5/LRP6 and Wnt/β-catenin pathway. LRP5/LRP6 generated using DVD-Ig platform with the variable domain forms trimeric complex with the serpentine receptor (VD) of anti-DLL4 located at the outer position and anti- Frizzled and Wnt and mediates the stabilization of β- VEGF VD located at the inner position. Interestingly, in the catenin, the transcriptional activator of the Wnt targeting presence of VEGF, dilpacimab showed 20–50× enhanced genes. Aberrant Wnt/β-catenin pathway activation can capability of blocking DLL4 signaling, which may be due contribute to the carcinogenesis and has been observed to the VEGF homodimerization-mediated cross-linking of in many types of tumors. It has been suggested that dilpacimab, then enhanced its binding avidity to DLL4 LRP5/LRP6 can interact with different Wnt ligands at on the cell surface, and promoted the downregulation different domains, and mAbs blocking different domains of DLL4 . Considering higher levels of VEGF at showed different profile . Therefore, BI generated tumor sites than in peripheral circulation, this unique the LRP5/LRP6 biparatopic nanobody, BI 905677, with characteristic of dilpacimab may conditionally enhance high affinity and complete blockage of the binding of DLL4 neutralization activity only at tumor sites. Currently, Wnt ligands to LRP5/LRP6, thereby inhibiting the Wnt- the treatment of dilpacimab along or in combination mediated cancer cell proliferation and survival . This with chemotherapy in patients with advanced solid tumor molecule is currently at Phase I in patients with different have shown acceptable safety profile and demonstrated types of solid tumors. preliminary antitumor efficacy [80, 81]. To further expand the antitumor activity, strategies in combining the blockage of both angiogenic and tumorigenic pathways have been exploited, such as Anti-tumorigenesis. Anti-tumorigenesis by targeting targeting VEGF and cMET (Merus), as well as Her3 and oncogenic receptors is another well-validated anticancer LGALS3BP (MediaPharma). Both programs are still at treatment. Trastuzumab targeting Her2 was approved to preclinical stage (Table 1). treat Her2-overexpressing breast cancer in 1999. Later, pertuzumab recognizing a different epitope of Her2 was approved in 2012 for treatment of patients with Her2- Enhance tumor immunity. Though the idea of immunother- positive metastatic breast cancer in combination with apy dates back to the 1890s, it was not until the 2010s when trastuzumab and chemotherapy. To develop an ideal com- it had significant breakthrough with ipilimumab launched binatorial treatment with trastuzumab and pertuzumab, a in 2011 and Keytruda and Opdivo in 2014. The aim of handful of biparatopic Her2 bsAb programs are under early the immunotherapy is to boost the patients’ own immune clinical testing. ZW25, a biparatopic Her2 bsAb designed system to generate antitumor T cell responses. This can based on Azymetric platform, able to bind domains 2 and 4 be achieved by either blocking the inhibitory signals, such of extracellular region of HER2 simultaneously to promote as CTLA-4 and PD-1, or enhancing the co-stimulatory internalization of HER2 and to inhibit HER2/HER3 signals, such as 4-1BB and OX40. Till today, anti-CTLA-4 heterodimer formation, demonstrated promising clinical and anti-PD(L)1 treatments have shown promising efficacy efficacy in a Phase I study (ESMO-Asia 2019). Addi- and revolutionized cancer treatment. Nevertheless, only tionally, several other oncogenic targets are also under 10–30% of the patients benefit from the treatment [85, 86]. evaluation, such as EGFR, Her3, cMET, and lipoprotein The immune system is a fine-tuned system, with receptor-related proteins (LRP) 5/6. Leading players in this redundancy in most of the regulatory pathways to avoid area are Merus (Her3 × Her2), Jiangsu Alphamab (Her2 damage to the host while it remains effective to clear biparatopic), Zymeworks (Her2 biparatopic), Janssen infection and tumor cells. To further improve the antitumor (EGFR × cMET), and EpimAb (EGFR × cMET), efficacy of anti-CTLA-4 and anti-PD(L)1 therapies, several followed by Beijing Mabworks, Boehringer Ingelheim (BI), strategies are being evaluated, including combining the Molecular Partners, etc. (Table 1). anti-angiogenesis treatment with anti-PD-1 treatment In collaboration with Genmab, Janssen has developed (VEGF + PD-1) and combining the blockage of multiple JNJ-61186372 to concurrently block both EGFR and immune checkpoints (CTLA-4 + PD-1 , PD-1 + LAG- cMET pathways for treatment of patients who are resistant 3, etc.). Though the additive effect can be achieved by to EGFR tyrosine kinase inhibitors (TKIs). It has been combining two mAbs, bsAbs have the advantages in shown that JNJ-61186372 not only effectively blocks development as a single molecule entity, sometimes may the ligand binding-induced EGFR and cMET activation even have synergistic efficacy. As of September 2019, but also promotes the downregulation of both EGFR there are 11 bsAbs targeting multiple inhibitory immune and cMET. To further enhance its antitumor potency, modulatory pathways such as PD-1, CTLA-4, LAG-3, the antibody-dependent cellular cytotoxicity (ADCC) TIM-3, etc. under active development at clinical and 13 at function of JNJ-61186372 is augmented by production in preclinical stage (Table 2). For example, the co-expression a fucosylation defective CHO cell line . In the first-in- of PD-1 and LAG-3 on tumor-infiltrating lymphocytes human (FIH) Phase I study, JNJ-61186372 has been tested identifies the tumor-specific T cells , which are mostly on 142 patients who were progressed after EGFR TKI dysfunctional . The co-treatment of anti-PD-1 and therapies and has shown promising antitumor activity with anti-LAG-3 can effectively restore the T cell function  ∼30% partial response rate. and have showed antitumor activity in PD-1-resistant Though most of the companies are focusing on mod- patients . Based on this fact, several companies are ulating the signaling of the well-validated ErBB family evaluating PD-1 × LAG-3 bsAbs. Some of the molecules 46 Antibody Therapeutics, 2020 represent preferential binding on the double-positive T and chemotherapy. Bintrafusp alfa (aka M7824) has the cells and are more effective in upregulating the T effector TGFβRII extracellular domain fused to the C-terminal cell function, as compared to the combination of the of avelumab . In preclinical studies, M7824 exhibited two parental antibodies [WO2017019846; WO2018134279; strong antitumor activity and significantly decreased WO2018185043; WO2019158942]. the incidence of metastasis in mouse tumor models. In Despite the success achieved by the immune checkpoint clinical tests, M7824 displayed acceptable safety profile inhibitors, the development of co-stimulatory signal ago- and encouraging clinical efficacy in patients with heavily nists was hindered by the intriguing balance between safety pretreated advanced solid tumors . Other strategies tar- and efficacy. For instance, the co-stimulatory molecule geting TME, including CD73 × TGFβ, EGFR × TGFβ, 4-1BB is a promising target for cancer immunotherapy, and CCR2 × CSF1R, are also under development at early as it can activate T cells, NK cells, and other immune cells clinical stage or preclinical stage (Table 3). and has been clinically validated in CAR-T therapies to sustain T cell activation. However, the clinical development of anti-4-1BB monoclonal antibodies was stagnated by Target cell depletion. The last group represents the either liver toxicity or lack of efficacy . To minimize majority of the bsAb programs (clinical, 60/99; preclinical, the safety issue associated with the systemic activation 99/153) to promote the target cell depletion by different of 4-1BB, strategies have been employed to localize the mechanisms (Table 4). According to the MOAs, this group activation of 4-1BB at tumor site. Roche developed a can be further divided into subgroups, including cytotoxic 4-1BBL fusion protein targeting fibroblast activation effector engagement, Fc effector function (ADCC, ADCP, protein (FAP) with Fc region mutations to abrogate Fcγ R complement-dependent cytotoxicity [CDC]), enhanced binding but maintain favorable PK profile. The in vitro phagocytosis, enhanced apoptosis, and drug conjugation. functional tests suggested that, only in the co-presence Cytotoxic effector engagement is the largest subgroup of anti-CD3 signal and FAP-expressing cells, FAP-4- in this category. Two out of the three launched bsAbs, 1BBL can increase T cell activation and proliferation. catumaxomab, and blinatumomab are in this subgroup. Furthermore, preclinical studies showed that FAP-4-1BBL Catumaxomab contains the antigen-binding sites for CD3 cannot inhibit tumor growth by itself. The combined treat- on the T cells and EpCAM on the cancer cells . It was ment with relevant T cell-redirecting bispecific antibodies first authorized for market by the EMA in 2009 for the (TRBAs) or immune checkpoint inhibitor, such as anti-PD- treatment of malignant ascites , but was withdrawn in L1, can efficiently inhibit tumor growth without prominent 2017 due to commercial reasons. On the other hand, blina- liver toxicity . Recently, at the 34th SITC annual tumomab targeting CD3 and CD19 has shown impressive meeting, Pieris Pharmaceuticals reported the preliminary clinical results since launched in 2014 [98, 99]. results of the Phase I study of PRS-343 (Her2 × 4-1BB) in T cells identify the target cells by recognizing the peptides patients with Her2+ malignancies. Among the 18 patients presented by the major histocompatibility complex (MHC) who received active doses of PRS-343, 2 patients reached through TCR. Based on the dynamic segregation model, partial response, and 8 patients had stable diseases. This is the interaction of TCR with cognate peptide/MHC com- the first 4-1BB agonistic treatment reported with promising plex (pMHCs) brings the T cells and target cells in close efficacy as well as good safety profile. proximity (∼14 nm) and results in TCR clustering at the The tumor site localization strategy has also been center of immune synapse (IS) and exclusion of the large explored to selectively activate other co-stimulatory inhibitory tyrosine phosphatases from this region . receptors, such as OX40, CD27, CD28, CD40, and ICOS, Following TCR clustering, cytolytic granules move toward as well as to selectively inhibit co-inhibitory receptors, the center supramolecular activation cluster (cSMAC) and including CTLA-4 and PD-1. There are 10 bsAbs utilizing release perforin and granzymes into the target cells. Once this strategy under active development at clinical and 19 the target cells undergo cell death, the T cells quickly at preclinical stages, which reflects the growing interests in detach from the dying cells and move to the next target this area (Table 2). cell . TRBAs are a group of bsAbs that can simultaneously Modulate TME. To evade the immune surveillance, target a component of the TCR complex (most commonly tumor cells can commonly influence the microenviron- CD3ε) on a T cell and a target on the tumor cell surface ment around them by expressing immunosuppressive [101–103]. By this approach, TRBAs promote IS formation molecules, such as TGFβ and CD73, and by recruiting between T cells and cancer cells independent of ligation or promoting the differentiation of immunosuppressive of TCR with pMHCs, leading to T cell activation and cells, such as myeloid-derived suppressor cells (MDSCs), killing of the tumor cells [104–107]. Due to the clinical tumor-associated macrophages (TAMs), and regulatory T success of blinatumomab, the development of TRBAs cells (Tregs). A few bsAbs and bifunctional proteins are has gained substantial attention with 51 programs are at developed to overcome immunosuppressive TME, such clinical and 66 at preclinical stages. Molecules in different as bintrafusp alfa, an anti-PD-L1, and TGFβRII fusion formats are under evaluation to prove whether they can protein. TGFβ is a pleiotropic cytokine and plays dual deliver the proposed biological function or have therapeutic functions in cancer progression. Though TGFβ suppresses window. For examples, BiTE and half-life extended BiTE tumor progression at tumor initiation stage, at later stages, are used by Amgen; DART and DART-Fc format are TGFβ facilitates tumor progression and metastasis and evaluated by MacroGenics; common light chain format has been suggested to contribute to resistance to anti-PD-1 is under investigation by Regeneron; and DuoBody format Antibody Therapeutics, 2020 47 is under development for multiple projects by Genmab (E430G) in the IgG1 Fc region to enhance hexameriation and Janssen. Glenmark’s BEAT platform, Xencor’s XmAb upon antigen engagement and thereby enhance the CDC platform, Aptevo’s ADAPTIR platform, and Teneo- effect. By combining the HexaBody and DuoBody plat- Bio’s unique anti-CD3 platform are also under active forms, Genmab has developed a DuoHexaBody anti-CD37 exploration. Recently, IGM Biosciences announced the biparatopic antibody. In ex vivo CDC assays using samples initiation of FIH Phase I clinical trial of IGM-2323, an isolated from lymphoma patients, the DuoHexaBody anti- IgM-based CD20 × CD3 TRBA. Unlike other formats, CD37 biparatopic antibody showed more potent tumor cell containing only 1 or 2 binding units for the tumor- lysis, as compared to the control anti-CD20 antibodies, associated antigen (TAA), the IGM-2323 contains 10 rituximab, ofatumumab, and obinutuzumab . binding units for CD20. It is hypothesized that the higher CD47-SIRPα signaling plays an inhibitory effect on avidity to CD20 of IGM-2323 may provide an advantage to the phagocytosis by phagocytes, such as macrophages. low treat CD20 tumor cells over other formats. Moreover, the Tumor cells overexpress CD47 on the cell surface to IgM-based TRBAs can more efficiently mediate CDC than escape the elimination by phagocytes. Antibodies against IgG antibody. However, whether the IgM-based TRBAs CD47 and SIRPα have been developed to interrupt can deliver superior efficacy to other formats still needs to CD47-SIRPα signaling. The combined treatment of be confirmed in the clinical studies. a CD47 antagonist, Hu5F9-G4, with rituximab showed Approaches to engage effector cell populations other promising therapeutic efficacy in patients with non- than conventional αβ T cells, such as CD8 T cells, γδ T Hodgkin lymphoma (NHL) . By taking the advantage cells, NK cells, and iNKT cells, have also been explored of the CD47 antagonist, a series of bsAbs using anti-CD47 and were reviewed by Ellerman recently . The γδ T as one moiety to enhance the phagocytosis to the cancer cells represent 10% of the total T lymphocyte population cells have been developed. Though there is only 1 program in circulating blood. Unlike conventional T cells, γδ T at clinical Phase I (NI-1701, CD19 × CD47), 14 programs cells recognize stressed and malignant cells independent of are undergoing active development at preclinical phase with MHC molecules, and their activation does not require co- CD47 coupled with different tumor-associated antigens stimulatory signals . Besides strong cytotoxic activity, (Table 4), suggesting there is substantial growing interests one unique property of activated γδ T cells is that they in this area. Recently, the results published by Hatterer can cross-present tumor antigens to enhance CD8 T cell et al. have suggested that in addition to the enhancement response . A few strategies to improve the antitumor of phagocytosis, the co-engagement of CD47 and CD19 activity of the γδ T cells have been explored at preclini- by NI-1701 can prevent the colocalization of CD19 to cal and early clinical stages . A selective Vγ 9Vδ2T BCR cluster during B cell activation, therefore inhibiting cell engager (Her2 × Vγ 9) showed superior activity in activated B cell proliferation . inducing Vγ 9Vδ2 T cell-mediated tumor cell lysis than Unlike previously mentioned bispecific programs, which Her2 × CD3 TRBAs in vitro and exhibited antitumor activ- all rely on the cytotoxic function of the effector cells or the ity in combination with IL-2 and activated γδ T cells adop- complement system, two preclinical programs are focusing tive transfer treatments in the PancTu-1 xenograft mouse on actuating the apoptotic process of the cancer cells by model . activating the apoptotic receptors. BI and Promethera gen- It has been argued that NK cell engagement may have erated bsAbs targeting CDH17 × TRAILR2 (BI-905711) better safety profile over T cell engagement therapies, and CD20 × CD95 (Novotarg), respectively. According to while providing similar levels of clinical efficacy. CD16 the report published by BI on 2019 AACR Annual Meeting, is the most commonly used target for engaging NK BI-905711 induced TRAILR2 clustering on a CDH17- cells. Results published by Affimed have suggested that dependent manner and selectively triggered the apoptosis NK engagers may induce efficient target cell killing with of CDH17 expressing tumor cells. BI-905711 also demon- lower cytokine release risk, when compared to CD3 T strated significant antitumor activity in multiple colorectal cell engagers . Early clinical results reported at 60th cancer xenograft models . ASH meeting and 15th ICML meeting had shown that Lastly, there are a couple of bsAbs that are being used AFM-13 (CD16 × CD30) was well tolerated and effica- to deliver toxin into cells that are positive for either or both cious when administrated alone or in combination with targets based on the particular design of each molecule. For pembrolizumab [114, 115]. The definitive clinical benefit example, Regeneron is working on APLP2 × Her2 bispe- of NK cell engagement still remains to be demonstrated cific antibody-drug conjugate (ADC). Amyloid precursor- in ongoing clinical studies. Other NK cell-activating like protein 2 (APLP2) has been suggested to be involved receptors that are considered to have distinct advantages in increased tumor cell proliferation and migration, and to overcome certain deficiencies in TME, such as NKG2D, aberrant APLP2 expression was observed in multiple types NKp30, and NKp46, are under preclinical evaluation . of cancers, such as breast cancer . Though APLP2 is an Additionally, strategies that redirecting iNKT cells by using internalizing receptor, due to its ubiquitous expression and CD1d extracellular domain fusion protein is also at early the presence of secreted form, APLP2 is not an ideal target research stage . for ADC. Trastuzumab emtansine (T-DM1) has shown Along with the growing depth of knowledge in Fc effec- potent efficacy in cancer cells with high level of Her2 tor function, several approaches have been adopted for expression, but has little effect on cells with mid to low lev- bsAbs, including mutations in the Fc region to enhance els of Her2 expression. To improve the therapeutic efficacy the Fcγ R binding, and afucosylation. Genmab has estab- of Her2 ADC, Regeneron developed the Her2 × APLP2- lished a HexaBody platform, which contains mutations DM1. The bsAb binds to Her2-positive cells with the high- 48 Antibody Therapeutics, 2020 affinity Her2 binding arm and then bridges to the cell age-related macular degeneration (AMD). Anti-angiogenesis surface APLP2 with the low-affinity APLP2 binding arm, treatment, such as Lucentis, Eylea, and Beovu, has been which promotes rapid antibody internalization, lysosomal approved for treatment of this condition and has shown trafficking, and tumor cell killing . significant improvement in visual acuity and prevention of vision loss. Though over 90% of the patients can benefit from the treatment (i.e., maintain vision), eventually these Bispecific antibodies for inflammatory conditions patients become resistant to the treatment. New therapies Autoimmune disease is the second largest area for bsAbs’ are needed to further improve the therapeutic efficacy. As applications, with 10 clinical programs and 12 preclinical we mentioned above, several bsAbs have been developed programs ongoing. Most of these programs are aiming to to block the process of angiogenesis for cancer indications. block the function of multiple pro-inflammatory cytokines Similar strategy has also been exploited for the treatment by combining the neutralizing antibodies into one molecule of wet AMD and diabetic macular edema (DME). Roche’s entity, such as IL-1α × IL-1β,IL-17 × IL-13, IL-4 × IL-13, faricimab is an Ig-like bsAb targeting VEGF and ANGPT2 and BAFF × IL-17 (Table 5). using CrossMab technology. During clinical tests, faricimab In immune cells, when it is in-cis coupled with an activat- has shown superior efficacy and safety in patients with ing receptor, the inhibitory receptor can play a dominant DME, as compared to Lucentis . Phase III studies negative role by diminishing the transduction of the active to evaluate faricimab’s therapeutic efficacy are initiated in signal . It has been shown that MGD-010 targeting early 2019; and the filing for BLA is expected in 2021. CD79B, one component of the BCR complex and the inhibitory receptor CD32B, can decrease B cell response Neurology. Despite the tremendous efforts in developing without depleting the B cells in healthy donors . Ono biological therapeutics for neurodegeneration diseases, is developing ONO-4685 (CD3 × PD-1)toturndownthe effective treatment remains elusive. One of the obstacles T cell responses in autoimmune diseases. However, it is still in developing biological drugs for neurological disease is to not clear whether the effect of ONO-4685 is dependent on effectively deliver the large molecule into the central neuron the in-cis engagement of CD3 and PD-1 to block the T system. “Trojan horse” bsAb has one binding specificity cell activation or by in-trans interaction to deplete PD-1 responsible for the transportation of the antibody to expressing activated T cells. the location that otherwise cannot be reached naturally, whereas the other binding specificity fulfills its function. By Bispecific antibodies for other conditions using this approach, a group of bsAbs have been developed to cross the blood-brain barrier (BBB). These antibodies Hemophilia A. Hemophilia A is another successful usually have one binding arm recognizing the receptors example in the application of bsAbs, with the approval in the receptor-mediated transcytosis system, such as of emicizumab in 2017 as a landmark event. Emicizumab insulin receptor, transferrin receptor, and lipoprotein bridges factor IXa and X in spatially appropriate positions transport receptors , and the other arm targeting to facilitate the factor IXa-catalyzed factor X activation, the pathogenic molecules (Table 6). Bifunctional fusion which is usually mediated by factor VIII in healthy proteins or antibody-drug conjugates are also under active individuals, but is deficient in patients with hemophilia development as therapeutic drugs or diagnostic reagents A. Though the etiology of hemophilia A has been well for central nervous system diseases, but they are not under understood for a long period of time, the treatment options the scope of this review. are still limited. Recombinant factor VIII and human plasma-derived factor concentrates are the commonly used practices for hemophilia A. However, the short half-life Infectious diseases. Due to the high frequency of escape of factor VIII and development of anti-drug antibody mutations and development of drug resistance to single- (ADA) remains the major challenges for factor replacement agent treatment, combinatory treatment with mixture of therapy . Emicizumab was intentionally designed to mAbs or by bsAbs to broaden the protection spectrum and function as factor VIII with prolonged plasma half-life to decrease the chance to establish drug resistance is being [127, 128]. Clinical results in hemophilia A patients with developed to fight against infections. MEDI3902 was orig- factor VIII inhibitor showed that the weekly subcutaneous inally designed to achieve broader protection against Pseu- (SC) treatment of emicizumab significantly reduced the domonas aeruginosa by combining two clinically proven frequency of bleeding episodes with no detectable anti- anti-PcrV and anti-Psl antibodies into one molecule. Psl drug antibody . Based on its promising efficacy and PcrV are present in ∼90% of the P. aeruginosa clinical and superior regimen schedule, emicizumab was initially isolates, respectively. Theoretically, the bsAb targeting Psl launched in the USA for hemophilia A patients with factor and PcrV simultaneously can protect the host from the VIII inhibitor in 2017; and then its usage was quickly infection of 97–100% of the isolates, which express either expanded to patient without factor VIII inhibitor and was or both targets. Surprisingly, when compared to the mixture launched in EU and Japan in 2018. Similar programs are of the parental antibodies in preclinical studies, MEDI3902 under preclinical development by Kymab and Shire. showed enhanced efficacy. By further dissecting the MOA, it was found that the format of MEDI3902 rendered a high- Ocular. Excessive neovascularization, bleed, and fluid avidity low-affinity binding to Psl, which led to the accu- leakage from the abnormal blood vessels result in rapid mulation of MEDI3902 around the bacterium and more vision loss or even blindness in patients with wet form efficient blocking of PcrV-mediated cytotoxicity . Antibody Therapeutics, 2020 49 The “Trojan horse” strategy is also employed by some are required. For receptors depending on clustering to bsAbs with elegant design for infectious diseases treatment. activate, fast-on fast-off binding kinetics is preferred to During filoviruses (e.g., Ebola virus) infection, the mem- ensure efficient recruitment of receptors [136, 137]. On brane envelope glycoprotein (GP) is responsible for the cell the contrary, for receptors activated by ligand binding- attachment and membrane fusion. A unique feature about induced conformational change, the slow off binding the GP of filoviruses is that it first binds to the receptor kinetics would endorse more durable activating efficacy on the cell surface which induces the internalization of . Furthermore, there are evidences that the affinity the virus particle, and then in the late endosome, GP is to CD3 may significantly affect the function and safety cleaved to expose the highly conserved receptor-binding profile for TRBAs. It has been suggested that T cells require site (RBS) for Niemann-Pick C1 (NPC1), which mediates lower threshold for mediating cytotoxic killing than for the membrane fusion and cell entry . Therefore, to cytokine production perhaps due to different number of provide broad protection against filoviruses, bsAbs were ITAM motifs of TCR complex being phosphorylated, designed to block the intracellular GPCL-NPC1 interac- it may be possible to dissociate TRBAs’ potency from tion by coupling the GPCL-NPC1 blocking arm with a toxicity by modulating the CD3 affinity of the bsAbs. delivering arm targeting a broadly conserved epitope in As shown by Leong et al., by lowering the affinity to uncleaved GP. The delivering arm binds to the virus par- CD3, the CD3 × CLL1 bsAb with low affinity to CD3 ticles and goes into the endosome together with the virus, exhibited better safety profile and retained equivalent in where the blocking arm functions to abrogate the GPCL- vivo efficacy, as compared to the ones with high affinity to NPC1 interaction when it is exposed and prevents the CD3  when net impact on T cell activation, receptor viral entry . internalization, and PK all combined. Similar results were also shown by Zuch de Zafra et al. By comparing a series of CD38 × CD3 bsAbs with different affinities to CD3, they Diabetes. Fibroblast growth factor 21 (FGF21) plays found that lowering the affinity to CD3 can dramatically key roles in stimulating metabolism and has shown some decrease the cytokine release, but still maintain potency preliminary clinical benefits in obese patients with diabetes. in mediating cytotoxic killing . In November 2019, However, the poor PK profile and potential adverse effects AMG-424, the final lead from the aforementioned study, associated with long-term usage of recombinant FGF21 was granted with orphan drug designation for multiple limit its usage. RG7992 (FGFR1 × KLB) was therefore myeloma by the FDA. designed to mimic the function of FGF21 but selectively As for the affinities of TRBAs to TAAs, due to the differ- activate FGFR1/KLB complex in the liver, adipose, and ent expression profile of the TAAs in normal tissues versus pancreas tissues, where KLB is present, to avoid safety in tumors, and the tolerability and the ability of regener- concern associated with broad FGFRs activation, but still ation of TAA-positive cell populations in normal tissues, be able to provide clinical benefit in obesity and diabetes the TRBAs targeting different TAAs may require different . binding kinetics. For low-expression, tumor-specific anti- gens, a TRBA with high affinity to the antigen would be required to elicit efficient tumor cell killing. However, MATCH BIOLOGY WITH AN OPTIMAL BISPECIFIC for TAA with low expression on essential normal tissues/ FORMAT organs, to spare the normal cells and avoid on-target off- As discussed in the early section, format diversity is essen- tumor toxicity, low-affinity high-avidity TRBAs would be tial to serve the plethora of applications of bsAbs defined by preferred, which can be achieved by modulating the valency TPPs. Variances in affinity, valency, epitope, and geometry (see below). of their binding domains, linkers, as well as in size- and Fc- Moreover, for a bsAb, difference in affinities of two mediated distribution and pharmacokinetic properties to different antigen-binding specificities may determine which fulfill a particular clinical application define a bsAb format. arm drives tissue distribution, tissue penetration, and reten- In practice, many variances or attributes for selecting an tion of a therapeutic molecule at the site of MOA. For optimal format are intertwined and must be addressed for examples, high affinity to TAA and low affinity to CD3 selecting the right molecule. Therefore, we will discuss these may enable the preferential binding of TRBAs to the target attributes below. cells and implement serial killing of the target cells by a single T cell ; and as mentioned above, APLP2 × Her2 bispecific ADC with high affinity to Her2 and low affinity Antigen-binding affinity and valency to APLP2 preferentially binds to Her2-positive cells and Affinity. Even though one of the advantages of using then bridges APLP2 on the cell surface to mediate efficient antibody-based therapeutics is that they may interact endocytosis to avoid the toxicity associated with the pan with their antigens with substantially high affinities, expression of APLP2. higher affinity does not always translate into better For BBB crossing bsAbs, along with other consider- efficacy. Unlike antagonistic molecule, whose potency is ations, careful selection of the transport receptor and usually associated with its affinity, agonistic molecule is selection of a molecule with appropriate binding kinetics to more difficult to predict and to optimize its potency by the transport receptor is critical for success of this strategy. increasing the binding affinity. Based on different modes As reported by the scientists from Genentech, to ensure the receptor uses for activation, different binding kinetics the effectiveness of the transcytosis, the “Trojan horse” of the agonistic bsAb to reach optimal receptor activation antibody using the TfR pathway needs to have low affinity 50 Antibody Therapeutics, 2020 to TfR . Later, another study by the University of Epitope, geometry, and distance between different Wisconsin-Madison showed that TfR bsAb with high antigen-binding domains binding affinity to TfR at pH 7.4 but low affinity at Epitope. In respect of antagonistic bsAb, the binding pH 5.5 can effectually release the bsAbs from BBB into the epitope of the corresponding binding units are required brain and avoid the degradation of bsAb in the endosome to prevent the receptor/ligand engagement, or the receptor . signal complex formation, or any step that is crucial for the initiation or passage of signaling cascade into the cells to play its biological function. Valency. The valency for each target can dramatically In general, the receptor-binding epitope for agonistic affect the function of the bsAbs. For the TRBAs, mono- molecules is not as predictive as it is for antagonistic valency of anti-CD3 arms may help to avoid non-specific molecules. However, there is evidence to suggest that activation of the T cells without engagement of tumor cells, the binding epitopes do contribute significantly to the as shown by Bardwell et al.. Interestingly, Y-mAb bsAb efficacy. It was found that anti-CD3 binding arms and Abpro have CD3 scFv fused to the C-terminus of recognizing different epitopes on CD3-activated T cell the light chain. Even though the format ends up with two differently. TeneoBio, therefore, identified a dozen of binding sites for CD3, both companies claimed that this anti-CD3 antibodies with different binding epitopes and format was actually functional monovalent toward CD3. different binding kinetics to CD3 molecules to disassociate Additionally, Aptevo and Affimed also developed TRBAs the capabilities of TRBAs in inducing cytotoxic killing using bivalency to CD3. Preclinical evidence has suggested from promoting cytokine production post T cell activation. that the adoption of the ADAPTIR format can induce They identified a clone (F2B) that recognizes a unique potent T cell activation and target cell killing, but low levels epitope on CD3δε, but not CD3 γε, at a low affinity of cytokine release . AFM-11 (CD19 × CD3) also (34 nM). By comparing to another clone (F1F), which showed more potent T cell activation than BiTE control binds to both CD3δε and CD3 γε with high affinity and strict CD19-dependent T cell activation preclinically (<1 pM), they found that BCMA × CD3 bsAb using . However, due to one death and two life-threatening F2B arm (CD3_F2BxBCMA) can induce moderate levels events in clinical trial, AFM-11 was placed on clinical of cell killing but very weak cytokine production, as hold. compared to the one using F1F arm (CD3_F1FxBCMA) The valence for the TAA may vary based on the prop- in vitro. Moreover, the in vivo efficacy study showed erties of the TAA, such as tumor specificity, antigen size, that CD3_F2BxBCMA exhibited antitumor activity in a expression level on the tumor versus normal tissue, and the wide dose range (0.01–10 μg), while CD3_F1FxBCMA tolerance of complete elimination of TAA-positive cells. In completely lost its therapeutic efficacy at the high dose the case of some types of hematopoietic tumors, the deple- (10 μg) . tion of both normal and malignant cells expressing TAAs, As we mentioned above, to effectively redirect T cell such as CD19 and/or CD20, can be tolerated. However, for killing, the TRBAs must be able to induce the IS formation most of the other TAAs, the expression levels may be low between the T cells and target tumor cells. Besides the on normal tissues, but the killing of these low-expression format of the TRBAs, the tumor antigen selection, the size normal cells can lead to deleterious consequences. To dis- of the antigen, antigen surface density, as well as the dis- high low tinguish the target tumor cells from the target normal tance between the TRBAs binding epitope to target cell tissue, RG7802 (CEA × CD3) was optimized to have low- membrane, all can influence the formation of the IS. Com- affinity high-avidity 2 + 1 format in appropriate geometry paring to large antigens or antigens with protruding struc- high to facilitate the selection of CEA cells with a threshold of ture, the small antigens or antigens with structure close ∼10 000 CEA-binding sites/cell . to the cell membrane can more effectively promote the IS Based on the lessons learned from the initial mAb devel- formation . When the selected tumor antigen is large opment for cMET treatment, bivalency to cMET may in size, such as melanoma chondroitin sulfate proteoglycan elicit agonistic, instead of antagonistic, effect resulting from (MCSP) and FcRH5, the membrane-proximal the mAb-mediated dimerization of cMET. Though mono- epitope is desired. For cell surface targets that can be shed valent binding to cMET can function as an antagonist, into the bloodstream, to avoid antigen sink, the bsAbs it can only block the HGF-mediated cMET activation. should recognize the membrane-bound but not the soluble Later, Wang et al. demonstrated that ABT-700, a truly form of the antigen . antagonistic mAb against cMET, can bind to a unique epitope on cMET. The bivalency to cMET of ABT-700 and stringent hinge region was essential to inhibit both HGF- Geometry. Besides the distance between the epitope to dependent and HGF-independent activation of cMET and the target cell membrane, the distance between the two induce cMET downregulation . Interestingly, half of targets engaged by TRBAs also plays a crucial role in deter- the cMET bsAb programs are using monovalency against mining whether it can effectively promote IS formation and cMET to avoid agonistic effect, while the other half choose T cell activation. Considering the distances between the bivalency. EMB-01 (EGFR × cMET) has two binding sites TAA and CD3 epitopes to target cell and T cell, respec- for cMET, and has no obvious cMET activation in the tively, the format of the TRBAs needs to bring TAA and absence of ligands. Furthermore, it can effectively induce CD3 to a close proximity much less than 14 nm. Moreover, EGFR and cMET degradation, therefore preventing the the whole molecule has to be able to physically fit into the cMET activation . small junction between the two cells in a density to effec- Antibody Therapeutics, 2020 51 tively form a cluster with several engaged target pairs to region of the IgG subclass, the length, flexibility, and amino initiate TCR signaling. Despite its short serum half-life, the acid composition of the linkers used to connect the building small size of BiTE format with two binding units locating blocks (scFv, Fab, etc.) may determine the correct for- in opposite sides is extremely potent in redirecting T cell mation, functionality, and developability of the resulting cytotoxicity by inducing serial killing of tumor cells at an bispecific molecules, as shown by Le Gall et al.and effector-to-target ratio as low as 1:5 . In another case, DiGiammarino et al.. Aptevo fused the scFvs against the TAA and CD3 at the N- and C-terminus of Fc (scFv + scFv with Fc, 2 + 2), which Size ended up with longer distances between the two binding domains. The in vitro studies showed that this molecule had The bsAbs have made significant impact on hematologic more potent target cell killing, but less cytokine release, malignancy treatments. However, the therapeutic benefits as compared to the BiTE format . Unfortunately, the delivered by bsAb for solid tumor are still waiting to be clinical development for this molecule was discontinued unveiled. One of the concerns using bsAbs for solid tumor due to high frequency of anti-drug antibody development. treatment is how to increase the drug tumor penetration The same situation also applies to T cell co-stimulatory and accumulation. Though molecules with smaller size and co-inhibitory receptors, which co-cluster with TCR would have better chance entering the tumor site by during IS formation and regulate T cell activation. PD-1 increased tumor penetration, the molecules with size and PD-L1 interaction leads to the accumulation of PD-1 smaller than the threshold of renal clearance of proteins are microclusters at cSMAC and destabilizes the IS. When the rapidly cleared from the blood and therefore have decreased extracellular domain of PD-1 was elongated by inserting flux into the tumor . Using a compartmental model, extra Ig domains, the inhibitory role of PD-1 decreased Schmidt and Wittrup predicted that molecules with the along with the increase of the number of Ig domains size of 150 kDa would have the best tumor localization, inserted . Though current anti-PD-1 molecules all whereas molecules with the size of 25 kDa would have block the PD-1 signaling by inhibiting the PD-1/PD-L1 the worst tumor uptake . However, due to their large interaction, in theory, the designs that can prevent the PD- size, molecules at the size of ∼150 kDa have decreased 1 colocalization to cSMAC should also be able to diminish extravasation and normally take days to reach maximum the inhibitory role of PD-1 in T cell responses. On the tumor uptake. On the other hand, molecules of smaller size contrary, bsAbs to activate the co-stimulatory receptor reach the maximum tumor uptake within a short period of such as 4-1BB must exert its function at the site of IS time. The fast tumor penetration and systemic clearance ; therefore, a format that can meet these criteria is of small-sized molecule therefore lead to high tumor/blood necessary. As reported by Pieris, the geometry of the 4-1BB localization ratio, which is preferred for some applications, anticalin attachment significantly affected the function of such as imaging , as well as safety management to the Her2 × 4-1BB bispecific anticalins. PRS-343 with 4- decrease the systemic drug exposure-associated toxicity. 1BB anticalin attached at the C-terminus of the heavy chain To improve the serum half-life, while still retaining the showed the most effective T cell activation, as compared to fast extravasation property, Harpoon developed the Tri- other formats. One possible explanation is that the binding TAC platform, which targets TAA, CD3, as well as human sites for Her2 and 4-1BB are approximately 15 nm apart, albumin for extended half-life with a total size of ∼50 kDa. which is close to the distance of the IS. However, after It is believed that with its improved drug exposure and measuring the distances from the binding epitopes to the small size, TriTAC would enable faster and better tumor cell membrane, the distance between the target cell and the penetration, compared to large-sized bsAbs. effector cell might be much longer than 15 nm. On the other hand, ND-021 (PD-L1 × 4-1BB × HSA) is an Fc-lacking scFv-VHH-based molecule. With its small size and flexible Fc region structure, it may have better potential in colocalizing at The Fc region can substantially influence the bsAbs’ func- cSMAC and enhance TCR signaling. It will be interesting tion. It was found that the properties of IgG subclass hinge to see how it will perform in the clinical trials. region, such as length, sequences, flexibility, and disulfide Cases are also shown in bsAbs programs developed for bond structures, can influence the variable region presen- other conditions. For example, when the scientists at Med- tation and thereby affect the functionality of an antibody Immune tested their Psl × PcrV bsAbs, they examined sev- . While it is not always desired, the format with Fc can eral different formats with varying intramolecular distances prolong the bsAb serum half-life through FcRn-mediated between the two binding components. After comparison recycling and may provide Fc effector function through the of these formats in both in vitro and in vivo efficacy stud- interaction with Fcγ Rs. ies, BiS4aPa, with an intermediate distance, exhibited the most effective protection against P. aeruginosa infection and therefore was selected as the final therapeutic candidate IgG subclass. Recently, Kapelski et al. reported the format . influence of the IgG subclass on TRBAs. They found that due to its short and rigid hinge region, IgG2 cannot effec- tively promote the IS formation. However, by replacing the Linker design hinge region of the IgG2 with the hinge region of IgG4 or As reviewed by Brinkmann and Kontermann, various con- IgG1, the function of IgG2 chimeric bsAb can effectively necting linkers have been explored . Similar to the hinge induce IS formation and redirect T cell killing . 52 Antibody Therapeutics, 2020 Similarly, the Fc region also showed significant influence with good therapeutic and molecular design defined on the factor VIII-mimetic activity of emicizumab. After by MPP that is developed based on TPP, followed by comparison between different IgG subclasses, interchain vigorous in vitro and in vivo screening and characterizations. disulfide bonds, and mutations in hinge region and CH2 Below we will discuss these six criteria and how they can domain, IgG4 was selected as it presented with the most have significant impact on the outcome of the resulting potent factor VIII-mimetic activity . bsAbs: physiochemical properties, manufacturability, immunogenicity, PK/PD property, and, most importantly, efficacy and safety. Fc effector function. As mentioned above, several strategies have been developed to enhance the binding between Fc and Fcγ Rs to increase the Fc effector function. Physiochemical properties and manufacturability. As This could be important for bsAbs against TAAs for aforementioned, many strategies have been explored effective killing tumor cells or for bsAbs against infectious to solve the CMC quality issues, such as mispairing, agents for pathogen uptake and clearance. However, the Fc stability, aggregation, segmentation, solubility, viscosity, effector function and Fcγ R binding are usually abrogated purification, etc. A good bispecific clinical candidate for TRBAs and some other agonistic bsAbs to avoid should (1) be easily expressed with high percentage of the Fcγ R-mediated cross-linking, which may cause non- correctly assembled product in manufacturing scale; (2) specific activation of T cells and the targeted receptors, display no significant aggregation or low percentage of respectively. Advances in Fc engineering allow tailored aggregation that can be easily removed, as aggregation may modification of Fc effector functions for specific need. For affect the therapeutic efficacy and increase immunogenicity example, Xencor developed a series of TRBAs using the risk; (3) have good solubility, high stability, and low XmAb platforms, including AMG-424 (CD38 × CD3, viscosity to meet drug product formulation needs for Fab + scFv with Fc, 1 + 1), and used a combination intended clinical dosage and route of administration; and of mutations (E233P/L234V/L235A/G236del/S267K) to (4) have low manufacturing cost for economical reason. completely eliminate the binding of IgG1 Fc to Fcγ Rs . The stringency of those requirements may differ based on Because IgG4 only binds to Fcγ R1 with high affin- various clinical applications. For instance, reconstituted ity and mediates weaker effector function than IgG1, lyophilized formulation for intravenous infusion (IV) is it is commonly used for antagonistic antibodies tar- generally acceptable for oncology applications, while liquid geting immune cells, to avoid Fc effector function- formulation developed for SC administration may be mediated cell elimination. However, the research by Zhang preferred for most of autoimmune indications. For ocular et al. showed that the anti-PD-1/IgG4 antibody can disease, the high solubility, high stability, and low viscosity induce the phagocytosis of PD-1 T cells by activating are imperative for a competitive product. With the advance Fcγ RI macrophages. By introducing five additional of the bsAb technology, more and more reported bsAb mutations (E233P/F234VL235A/D265A/R409K), BGB- formats can be expressed and purified with reasonable A317 showed no binding to Fcγ R1, and more efficient yield and meet the reasonable physiochemical properties preclinical antitumor activity, as compared to the anti- for a given clinical application and are scalable for large- PD-1/IgG4 control . The recently reported results of scale manufacture, although some of the formats do require the pivotal study of BGB-A317 also exhibited its superior significant CMC optimization and longer development antitumor efficacy in patients with relapsed/refractory timeline than the others. classical Hodgkin lymphoma, with an overall response rate (ORR) of 87% and 63% complete response rate (CRR). Immunogenicity. Immunogenicity is one of the critical As we discussed above, the format contains many com- factors limiting clinical use of biological therapeutics, as the ponents that can be tweaked, their final impact on pharma- generation of ADA may lead to fast drug clearance, neutral- cological properties of a bsAb is intertwined, and here we ization of therapeutic effect, and even severe adverse events only mentioned some of them. The fine-tuned parts work in in clinic. The duration of the ADA response can be cate- concert with each other to determine the success of bsAbs. gorized into transient and persistent ADAs. The persistent To obtain the optimal therapeutic candidate, the selection ADA requires the T cell help and commonly leads to more of any component in the final format should be carefully deleterious consequences. The nature and the levels of the evaluated for specific target pairs; and the matched format ADA generated are influenced by both the patients’ physi- will not only facilitate the bsAbs to elicit biological function cal conditions (autoimmune-prone vs. immunosuppressive, but also may enable a molecule for further product devel- pre-existing ADAs, etc.) and the intrinsic properties of an opment, which otherwise may not be suitable for clinical antibody (i.e., sequences, impurities, format, MOAs, dos- application. ing regimens, etc.) . For example, the cancer patients are usually immunosuppressive, while the patients with autoimmune diseases are prone to develop auto-reactive SELECT A RIGHT MOLECULE TO MEET BOTH antibodies and ADAs. The antibodies that contain strong FUNCTION AND DEVELOPABILITY REQUIREMENT T cell epitopes have high risk to induce T cell-dependent As illustrated in the early section, the six criteria critical persistent ADA. Compared to bsAbs that deplete B cells, for clinical development and commercial manufacturing the bsAbs that enhance immune system response may have (Fig. 1) define a good bsAb. Identification of a good a higher chance to induce ADA. And the bsAbs dosed by therapeutic bispecific molecule usually requires starting SC and intramuscular (IM) administration may be easier Antibody Therapeutics, 2020 53 to be picked up by dendritic cells (DCs) and present bsAb- PD, described as what the drug does to the body, involves derived peptides to T cells, as compared to the ones given the target binding and the following effect. The PK/PD by IV infusion. profiles play an important role in effecting the drug efficacy As reviewed by Davda et al., using the approved and safety and therefore are critical for the development mAb clinical results, they found that although both ate- of bsAbs. Many factors of the bsAbs can influence the PK zolizumab and durvalumab were Fc-engineered anti-PD- profiles, including molecule format, size, physicochemical L1 mAbs, only atezolizumab showed higher rate of ADA, properties, Fcγ R binding, as well as target binding affin- as compared to the other anti-PD(L)1 mAbs. The combi- ity. For example, Harpoon is developing a novel protease- national treatment of the anti-PD(L)1 and anti-CTLA-4 activated T cell engager platform, ProTriTAC based on the could increase the rate of ADA: the ADA rates against aforementioned TriTAC platform. By modifying the non- nivolumab were increased from ∼12% (monotherapy) to CDR region, the anti-albumin SDA can bind and mask the 24–38% (combo therapy with ipilimumab). Furthermore, anti-CD3 arm while maintaining its binding to albumin. the antibodies mediating B cell depletion usually had Furthermore, a tumor-associated protease cleavage site is low ADA rates. As most of the reported bsAb formats introduced to the linker between the anti-CD3 binding are heavily engineered and with non-native Ig sequences domain and anti-albumin SDA. In the circulation, the introduced, it is very likely that the bsAbs have higher anti-albumin keeps anti-CD3 arm inactive and imparts the immunogenicity risk than regular mAbs. However, most molecule long serum half-life. Once it enters into the TME, of the bsAb programs are still at early clinical stages, and ProTriTACs are cleaved by tumor-associated proteases to only very limited information and results are available to lose the anti-albumin SDA and expose the anti-CD3 bind- evaluate the immunogenicity issue for bsAbs. As mentioned ing site to function. If the cleaved molecules enter into above, Aptevo developed APVO-414 (PSMA × CD3) the circulation again, they will be rapidly cleared from the using the ADAPTIR platform. In the initial Phase I system due to its small size. By using this strategy, they dose escalation study, 58% of the patients developed developed a ProTriTAC targeting EGFR, which was not ADA with the titers as high as 1:250 000, leading to fast easy to be targeted by TRBAs due to its wide expression in drug clearance from the blood. By modifying the dose the normal tissue. regimen from weekly IV to continuous IV infusion, the As long serum half-life may increase tissue penetration ADA titers were decreased dramatically to the range of and therapeutic efficacy, as well as require lower dosage 1:160–1:320. However, there were still 50% of the patients and less frequent drug administration, sometimes, a longer developed ADAs. Later, this program was discontinued as serum half-life is preferred. IgGs and albumin are both no therapeutic benefit was observed. abundant in plasma with long half-life due to the binding Due to its critical impact on the clinical outcomes, to FcRn, which rescues them from degradation in the methods to minimize the immunogenicity risk have been endo/lysosomal compartment. Therefore, enhancing the Fc exploited at early discovery phase. Firstly, more and more binding to FcRn, or by adding a HSA binding domain antibody therapeutics are utilizing humanized or even into the format (without Fc), is commonly used by bsAbs fully human antibodies or fragments to decrease the non- to improve the serum PK. Many mutations in the CH2- human sequences, thereby decreasing the immunogenicity CH3 region have been tested to increase the Fc binding to risk. Secondly, in silico approaches are being employed FcRn, yet only the YTE and LS mutation combinations to identify the immunogenic epitopes, especially T cell (YTE = M252Y/S254T/T256E; LS = M428L/N434S) have epitopes can be removed to prevent the generation of T been clinically validated . YTE mutations can increase cell-dependent persistent ADAs. Though these approaches the antibody serum half-life ∼4-fold in human, but also still need to be validated in clinical practice, various in silico decrease the ADCC activity of the antibody. VRC01LS algorithms have been developed to predict the presence containing the LS mutations also showed more than 4-fold of potential T cell epitopes. To compliment the in silico increase in serum half-life in human . Unlike YTE prediction, in vitro assays are utilized, which include HLA mutations, LS mutations have no impact on antibody’s binding assays, primary peripheral blood mononuclear cell ADCC activity. On the other hand, in some applications, (PBMC) assays, mixed lymphocyte reaction assays, and 3D when the prolonged half-life is undesired, mutations to models to mimic the conditions in specific tissues . decrease the Fc to FcRn binding can also be applied. The integrated results from in silico algorithm and in vitro Detailed methods in modulating FcRn binding to modify assays can provide some suggestive information and help PK were reviewed by Leipold . Though the effect of the bsAb development. For example, during rounds of FcRn on influencing serum half-life has been well studied, engineering and optimization, emicizumab adopted a de- it still remains controversial on how it affects the drug immunization strategy that the effects of each mutation on metabolism in other tissues, such as the brain and eyes. As immunogenicity were evaluated by using algorithm, and matter of fact, Lucentis (Fab) and Eylea (Fc fusion protein) any mutation that may increase immunogenicity risk was only showed slightly different ocular half-life in humans, avoided . The clinical data suggested that there were suggesting FcRn binding may not play a major role in low level or no ADA observed in treated patients. determining ocular half-life, while the molecular size may play some, but not determining roles on PK properties of molecules in retina. Pharmacokinetic and pharmacodynamic properties. PK, Due to the high binding affinity to the target, target- described as what the body does to a drug, refers to the mediated drug disposition (TMDD) is common for drug absorption, distribution, metabolism, and excretion. antibody-based therapeutics, especially for those targeting 54 Antibody Therapeutics, 2020 surface antigens. As mentioned in the previous section, influence each other. It is common that the drug showing decreasing the target binding affinity, in some cases, can high potency in discovery stage tends to be selected as the prolong the drug half-life and therefore improve the thera- therapeutic candidate. However, highly potent drug that peutic efficacy. As shown by Leong et al., the relationship induces toxicity at low dose leaves no or very limited ther- between CD3 affinity of the CD3 × CLL1 TRBA to its apeutic window, which may significantly hinder its clinical activity, PK and safety are quite complicated. During in application. On the other hand, the drug with a reasonable vitro characterization, they found that the one with low potency but better safety profile may have wide therapeutic affinity to CD3 (CLL1/CD3L) showed decreased potency, window, and the therapeutic efficacy may be improved by but had more favorable safety profiles, as compared to readily increasing the dose without inducing significant the one with high affinity to CD3 (CLL1/CD3H). More toxicity. Increasing drug exposure may be another way importantly, when they tested these molecules in vivo, they to enhance the efficacy and prolong the response dura- found that CLL1/CD3L had slower drug clearance (50%) tion, as we discussed above. However, increased systemic and increased drug exposure, which led to more durable exposure may also increase the chance and the severity antitumor responses, as compared to CLL1/CD3H . of adverse event. It is hard to predict which composition A similar case was also observed in an IL-15/Rα × PD- of the MPP can translate into an optimal TPP in clinical 1 bifunctional protein. The fusion protein was engineered application. to decrease the IL-15/Rα potency, thereby decrease the For example, despite its extreme potency in eliminating antigen sink, and increase half-life. Several variants with the tumor cells, the life-threatening adverse effect associ- decreased potency were generated and compared in vivo.As ated with the treatment of blinatumomab, as well as short they predicted, the low potency variants showed dramatic serum half-life, both significantly limit the application of half-life extension from 0.5 day (wild type) to 9 days blinatumomab . To improve the therapeutic efficacy [US20180118828]. and prolong the serum half-life, Affimed developed AFM- Besides affecting the serum PK, target binding affinity 11 (Fv + Fv, 2 + 2), a tetravalent CD19 × CD3 bsAb . may also influence the tumor/tissue distribution of the In vitro characterization studies showed that AFM-11 was bsAbs. For example, the affinity of the bsAb to the tumor more potent than BiTE molecule to elicit target cell killing. antigen can influence the tumor penetration. BsAbs with Though bivalent for CD3, AFM-11 showed stringent extremely high affinity to tumor antigen get stuck at the target-dependent activation of T cells. Using a NOD/SCID entrance and therefore have poor tumor penetration [168, xenograft model, AFM-11 showed favorable PK profiles 169]. While low-affinity bsAbs distribute further into the with preferential tumor localization over normal tissue and tumor, but bsAbs with small sizes may have decreased a half-life of ∼20 h. In Phase I dose escalation study, AFM- retention time in the tumor. The distribution of TRBAs 11 was dosed by continuous infusion (Week 1, 0.7 ng/kg/wk for solid tumors, as predicted by Friedrich et al., may be to 130 ng/kg/wk; Week 2+, 2 ng/kg/wk to 400 ng/kg/wk). significantly affected by the distribution of T cells, and During the study, among the 14 patients who completed modifying the affinity to CD3 or TAA may not be sufficient the dose limiting toxicity observation period, 3 patients to accumulate TRBAs and T cells into the tumor . showed complete response (CR), but 2 were transient and Other methods may be used to affect the bsAb distribution patients relapsed after cycle 2. Serum half-life was ranged inside the tumor tissue and include target selection, Fc, and from 7.14 to 10.6 h in four evaluable patients. Although no utilizing of transcytosis . cytokine release syndrome (CRS) was observed, two Grade The ultimate goal of all previously discussed strategies to 3 neurotoxicity and one fatal event were recorded in the two modulate PK was to enhance the overall clinical efficacy highest dose groups. AFM-11 was placed on clinical hold, and/or to minimize the toxicity of the therapeutic bsAbs. due to the severe adverse events. Similarly, improving the PD profiles can also be achieved by TRBAs in formats containing Fc may have improved modifying the antigen-binding activity and by modifying stability and manufacture profile, as well as prolonged the Fc-mediated effector function to further increase clini- serum half-life. The long-term drug exposure may provide cal potential of the bsAbs. Thus, the PK/PD profiles can be improved efficacy and more flexible dosing strategy, but modified by adjusting multiple factors, while most of these may be more difficult to handle if undesired effect is expe- factors are interdependent, which highlight the inherent rienced. Regeneron developed REGN-1979 (Fab + Fab challenges in therapeutic antibody design, and improving with Fc, 1 + 1), a CD20 × CD3 bsAb. In vitro assays one property can sometimes affect the others. Therefore, showed that REGN-1979 can effectively and specifically we should bear in mind that due to the complexity of the mediate the killing of CD20 cells. The preclinical phar- MOAs of bsAbs, the PK/PD profiles may not be the same as macology studies using cynomolgus monkeys showed that we expected (hoped). Robust technologies and tools (both REGN-1979 can cause durable and deep B cell depletion experimental and in silico) are critically needed to advance with a serum half-life of ∼14 days . In June 2019, the understanding of structural determinants of the bsAbs Regeneron reported the early-stage dose escalation trial that can impact the PK/PD properties and to guide the results of REGN-1979: 93% ORR and 71% CRR in 14 optimization of bsAbs. patients with follicular lymphoma treated with REGN- 1979 (5–320 mg); and 57% ORR in 7 patients with diffuse large B cell lymphoma (DLBCL) treated with REGN-1979 Efficacy and safety. A reasonable efficacy/safety win- (80–160 mg), which were all CR. Among the total of 81 dow is fundamental for a good clinical candidate; and evaluable patients, 7% experienced Grade 3 or higher CRS, PK/PD profiles, efficacy, and safety profiles commonly and at least 10% of patients experienced Grade 3 or higher Antibody Therapeutics, 2020 55 adverse event. The incidence and severity of CRS can be clinical candidate not only needs to show promising thera- mitigated by optimized premedication. Recently, in 2019 peutic potential but also needs to have good physiochemical ASH annual meeting, similar results were also reported properties and scalable manufacturability. Furthermore, for mosunetuzumab (CD20 × CD3, Roche) with ORR favorable PK properties and low immunogenicity are also and CRR of 62.7 and 43.3%, respectively, in patients with critical to assure the success of the candidate. Besides all slow-growing non-Hodgkin lymphoma. Both REGN-1979 the above mentioned factors, the efficacy/safety ratio is and mosunetuzumab showed benefit to patients who had one of the major determinants whether a bsAb moves into disease progressed post CAR-T therapies. development stages in the end. The comprehensive review regarding TRBAs published by Ellerman made a perfect case of how complex it can be to optimize a TRBA, and the change of a factor of the KEY CHALLENGES THE FIELD STILL FACING bsAb may influence multiple profiles of the molecule, and Though bsAbs development has made significant progress a molecule profile can be modulated by multiple factors. and several strategies have been exploited to solve some of For example, to uncouple the capabilities of TRBAs to the challenges, many still remain. We would like to review induce cytotoxic killing and cytokine production by the these challenges in two categories: technical challenges and T cells, the TRBAs can be modified by (1) decreasing the mechanistic or biology challenges. affinity to CD3, as T cell cytotoxic killing requires a lower activation threshold; (2) using a different CD3 binding epitope, as based on the “permissive geometry” model, Technical challenges different binding epitope may lead to different CD3 confor- Discovery. Compared to mAbs, bsAbs display signifi- mational change and T cell signaling; and (3) switching to cant complexity in the research and development stages. another format. In another case, to distinguish the antigen- Special testing systems are needed to characterize the overexpressing tumor cells and the low-expression normal potential therapeutic efficacy, toxicity, and PK/PD profiles cells, one can (1) decrease the binding affinity and use of the bsAb therapeutic candidates, and many of these multivalency to the antigen and (2) increase the distance of systems may be quite complicated, as compared to the the IS, by either choosing a membrane distal epitope on the systems used to evaluate mAbs. antigen or using a format with longer distance between the For example, artificial cell line used to evaluate bsAb two binding domain. From another aspect, decreasing the function needs to overexpress both targets and include both affinity of CD3 may diminish the target cell killing potency signaling pathways, and the generation of such cell line may in vitro; it may also increase the PK profile and tumor accu- have huge technical challenges. Also, the expression level mulation which ends up with comparable or even improved and temporal order of the two targets on the artificial cell in vivo efficacy and therapeutic window . line may not reflect the disease situation in human. For Another group of bsAbs that represents with challenges primary cell-based efficacy tests, a specific population of in leveraging the safety and efficacy is the agonistic bsAb cells may need to be isolated and cultured ex vivo to induce targeting co-stimulatory receptors, such as 4-1BB. Recently, the expression of both targets, which makes the assays the results reported for PRS-343 showed first sign of hope extremely time- and cost-consuming and low throughput. for development anti-4-1BB treatment (see above). Numab Furthermore, even though researchers try to mimic the developed ND-021, a monovalent trispecific antibody tar- real situation under which the bsAb plays its functional geting PD-L1, 4-1BB, and HSA. The in vitro efficacy tests −12 roles, the in vitro assay system cannot completely reflect the suggested that the ultrahigh affinity (2 × 10 M) to PD-L1 immune system, and therefore the effect of the bsAb cannot determined the potency of the molecule; binding to a distal be accurately evaluated in vitro. epitope on 4-1BB can promote the 4-1BB clustering more The selection of species and relevant disease model for effectively; and when the affinity to 4-1BB was way lower efficacy, pharmacology, and toxicology studies can be com- than the affinity to PD-L1, the effective dose range can be plicated, with considerations for the properties of both tar- significantly extended. As compared to the combinations gets, such as the cross-species specificity, the functionality of mAbs, ND-021 showed superior activity in enhancing of the bsAb, as well as the expression and function of the activated T cell responses. Due to the monovalency and lack targets. Although, transgenic animals and animals grafted of Fc region, ND-021 displayed strictly PD-L1-dependent with human immune systems are developed for bsAbs with- 4-1BB activation and spared antigen-presenting cells from out cross-species binding, it is still doubtful how closely depletion. In in vivo efficacy tests, ND-021 showed higher these models can reflect the actual clinical conditions and antitumor activity than combined treatment with mAbs in how accurately they predict the therapeutic efficacy, safety mice. Most importantly, ND-021 did not induce liver toxi- risk, and PK/PD profiles of a bsAb. city, and systemic T cell activation in cynomolgus monkey posts a single-dose IV injection  although it remains elusive how this may translate into safety in humans. Cur- CMC. With the advanced protein engineering technol- rently, this program is at IND-enabling study stage, and we ogy and elegantly designed bispecific formats, the physio- are looking forward to see its clinical results. chemical properties and manufacturability are no longer On the basis of the strong biological rationale, empow- significant hurdles in developing bispecific clinical candi- ered by the carefully harmonized format, and with the dates. However, different formats do vary in the degrees meticulously selected binding units, bispecific molecules of difficulty in Chemistry, Manufacturing and Controls just finish the first step to its final success. A good bispecific (CMC) development, and the ones that fulfill developabil- 56 Antibody Therapeutics, 2020 ity criteria no doubt would significantly lower development different domains of the bsAbs, as some of the bsAbs risk and shorten development timeline. are heavily engineered with potential immunogenic epitope introduced. Special attention needs to be taken on ADA against TRBAs and agonistic bsAbs using the tumor/tissue Preclinical pharmacology and toxicology. The preclinical localization strategy, as the presence of ADA may break the pharmacology and toxicology studies are very critical for TAA dependency of these bsAbs and lead to non-specific the development of bsAbs, as the results from these studies activation of immune cells and unpredictable severe adverse not only support the scientific rationale of the bsAbs but event. One should always follow FDA outlined and rec- also provide valuable information for selecting the FIH ommended adoption of a risk-based approach to evaluate dose. Though the scope of the bsAb preclinical studies may and mitigate immune responses or adverse immunologi- be similar to that for mAbs, the selection of the relevant cally related responses associated with therapeutic protein species may be more challenging for bsAbs due to the products that affect their safety and efficacy during clinical additional target. The relevant species should be selected development of a bsAb. based on the following: (1) both targets should have similar Furthermore, in some instances, combinational therapy expression profiles and biological functions as the targets provides the flexibility in adjusting the dosing regimen, in human, respectively, and (2) the bsAb should bind to which cannot be achieved by bsAbs. Although various both targets with similar properties as it binds to the human bispecific formats can provide some degree of flexibility in targets. In the case that Fc effector function is required, adjusting affinity and valency of a binding specificity to especially the ones with modified binding to Fcγ Rs, the suit different needs, once the format is determined, the ratio selected species should also be able to predict the Fc func- against two targets is fixed, and it cannot be adjusted based tion in human. If such a species is available, the FIH on the clinical results, which may pose clinical development dose may be selected based on the no-observed-adverse- challenge for a drug. Moreover, an optimal treatment may effect level (NOAEL). If a relevant species is not available, require sequential target intervention. For example, the in vitro pharmacology studies and in vivo pharmacology concurrent treatment of anti-PD-1 with anti-OX40 treat- studies using surrogate bsAb or transgenic animals may ment leads to substantial increase in serum cytokines and be required to provide supporting information. The FIH the expression of inhibitory receptors on T cells, as well as dose may be selected by using the minimum anticipated decreased T cell proliferation, thereby attenuating the anti- biological effect level (MABEL) approach if no relevant tumor efficacy of anti-OX40 treatment. However, delaying toxicity species are available, especially for molecules with the PD-1 treatment can increase the antitumor activity of agonistic activities. Several case studies of the bsAb preclin- anti-OX40 treatment . In another case, NK cells can ical studies were reviewed by Prell et al. and by Trivedi be activated and upregulate 4-1BB expression by exposing et al. to illustrate the complexity and challenge during to rituximab-coated CD20 tumor cells or trastuzumab- bsAb preclinical development. coated Her2-overexpressing breast cancer cells. The anti- 4-1BB treatment following the treatment of rituximab or trastuzumab can enhance the ADCC effect of NK cells Clinical development. Based on the draft guidance for to antibody-coated tumor cells [177, 178]. In such cases, bsAb development programs published by the FDA in the combinational therapy with mAbs offers the flexibility April 2019, several factors should be considered during which cannot be accomplished by current bsAb strategies. bsAb clinical development: (1) scientific rationale (e.g., MOA, therapeutic advantages over standard of care); (2) mode of action (e.g., bridge two target cells, simultaneous or sequential binding); (3) binding kinetics to each target; Mechanistic or biology challenges (4) special pharmacology studies (e.g., PK/PD assessment for active form of the bsAb, immunogenicity assessment for The most fascinating applications of bsAbs are to enable each domain of the bsAb); and (5) in certain cases, factorial novel biological function and therapeutic MOA otherwise design of clinical trials to inform risk/benefit ratio. impossible by using mAbs alone or in combination. How- TGN1412, an anti-CD28 agonistic antibody case, alerted ever, the novel MOA may also impose unknown safety risk us that cautions must be taken in regard to clinical develop- on bsAbs, which cannot be readily predicted or evaluated ment of bsAbs with novel MOAs, especially for agonistic in preclinical studies, and possibly result in severe or even molecules. Therefore, it is recommended that for bsAbs life-threatening adverse events during the clinical stage. playing agonistic function, especially for unprecedented Therefore, the uncertainty in function and safety of these target pairs, the selection of the initial dose of the FIH bsAbs represents a major challenge for development of trial should use MABEL approach. Additionally, agonis- bsAb therapeutics. tic bsAbs may have a bell-shaped dose-response that the When selecting the target pair, researchers should therapeutic efficacy peaks at a dose that receptor occu- consider the spatial and temporal presence of both targets. pancy is not saturated and then decreases along with the Whether both targets are expressed at the same location at increased drug dose . Therefore, an agonistic bsAb the same time? Whether their levels are within a reasonable with a narrowed bell-shaped dose-response curve may be range that can be effectively treated by a bsAb with fixed significantly difficult for researchers to select the optimal stoichiometry? Whether the two targets expressed on differ- doses for different patients. ent cells or on the same cells? Whether the bsAb will medi- Comprehensive examinations for anti-drug antibodies ate in-cis or in-trans engagement of the two targets? Will may be required to evaluate the immunogenicity risk of different engagement models result in different outcomes Antibody Therapeutics, 2020 57 in efficacy and safety? Those are all important questions for T cell-redirected cytotoxicity, a variety of formats, with one need to think through when embarking a bsAb project. difference in affinity, valency, domain geometry, Fc proper- Bispecific antibodies engaging CD32B and FcεRwere ties, and pharmacokinetic properties, have progressed into designed to employ the dominant negative role of CD32B clinical development. It will be interesting to see clinical and inhibit the activation of FcεR to alleviate IgE-mediated validation of various preclinical rationales behind the diseases. The bsAb 9202.1/5411 with IgG1 format was design of those molecules in the coming years. produced using Escherichia coli cell line and therefore had With the advent of gene therapy, RNA therapy, cell no Fc effector function due to lack of glycosylation. In vitro therapy, and various other new therapies, we should always analysis showed that this bsAb can inhibit IgE-mediated compare those different therapeutic options and pay close activation of mast cells and basophils. As mentioned by the attention to those new therapeutic modalities that may authors, several formats that were bivalent for FcεR might have disruptive potentials, for instance, both chimeric cross-link FcεR in the absence of CD32B, thereby activat- antigen receptors T cell (CAR-T) therapy and TRBAs have ing rather than inhibiting FcεR. One may speculate demonstrated dramatic effects in patients with hematologic in the worst scenario in vivo, sometime may be inevitable, tumors. One TRBA and two CAR-T cell products have if such a molecule formed aggregates, it may function to been approved by major regulatory agencies within the activate rather than inhibit FcεR as one initially designed. last 10 years for the treatment of hematological cancers, This becomes even more complicated for agonistic bsAbs and an additional approximately 60 TRBAs and 300 CAR to activate receptors. As a receptor is co-evolved with its cell constructs are in clinical trials today. CAR-Ts are cognate ligand, the signaling upon ligand-receptor engage- designed to activate T cells via intracellular T cell co- ment is evolved to be tightly controlled under physiological stimulatory signaling modules in tandem and to form a conditions. Due to the plasticity of receptors, polygamy cytolytic synapse with target cells that is very different widely exists for ligand-receptor interaction. When using from the classical immune synapse both physically and antibody-based therapeutics to mimic the function of a mechanistically, whereas the TRBA-induced synapse is ligand, the antibody may bind to the site on the receptor dif- similar to the classic immune synapse by bringing T cells ferent from its cognate ligand binding site, which may elicit close proximity to tumor cells via a bispecific molecule. different signals. The deviation from the cognate activation As published in 2018 ASH annual meeting, in patients may result in unexpected consequence, and their potential with relapsed and refractory multiple myeloma (r/r MM), safety risk is unknown. AMG-420 (BCMA × CD3, BiTE) showed 70% ORR For example, as reported by Gu et al., a panel of and 40% CRR. Similarly, bb-2121 (BCMA CAR-T) and biparatopic anti-Her2 antibodies in DVD-Ig format JCARH125 also demonstrated ∼80% ORR and ∼30% generated from the same parental mAbs only differed CRR. On the other hand, both TRBAs and CAR-T by VD orientations or linker length. Surprisingly, DVD- therapies showed similar adverse effect, which may be Ig molecules with one VD orientation showed agonistic due to their MOA in redirecting T cell cytotoxicity to effect and increased tumor cell proliferation, whereas tumor cells. Blincyto and CAR-T therapies, Kymriah and molecules with the opposite VD orientation remained Yescarta, are all targeting CD19 tumor cells and proved antagonistic. Further studies revealed that a particular for treatment of B cell lymphomas, and all of them have the VD orientation interrupted Her2/EGFR and Her2/Her3 block box warnings for CRS and neurological toxicities. interaction, resulting in increased Her2 homodimerization From the manufacturing aspect, due to the characteristics and activation . Similarly, a biparatopic anti-CTLA- of BiTE molecules, the manufacture of Blincyto still has 4 bsAb unexpectedly changed the signalosome assembly quite a few challenges, but this has been solved by the on the cytoplasmic domain of CTLA-4 and completely next generation of TRBAs in the clinical development. converted the inhibitory receptor into a stimulatory For autologous CAR-T therapies, a complicated and time- receptor . consuming (3–4 weeks) manufacturing process is required The preclinical and clinical development path have for each patient. Additionally, as the CAR-T therapies are largely paved for bsAbs with precedent mechanisms. live cells, the regulatory requirements for CAR-T therapies However, the development of bsAbs with novel biological are more complicated and stringent than regular biological mechanisms still faces a few challenges and pitfalls. It may therapeutics. Most CAR-T cells today are autologous, require more preclinical studies and early discussion with although significant strides are being made to develop regulatory agencies for clinical development plans. We off-the-shelf allogeneic CAR-based products. Therefore, believe that, in the future, biology will be the key driver for in general comparing these two therapeutic platforms, design and selection of a bsAb and the key consideration TRBAs are the off-the-shelf products and may be more for clinical development of bsAb drugs. convenient and affordable to patients in the near future when more TRBAs are available, while CAR-T therapy may be tedious but may have advantage to mobilize the entire T cell machinery in a very different mechanism to PERSPECTIVE fight cancer cells. Both platforms currently are facing the A growing number of recombinant bsAbs are now in clini- same moderate anticancer effects in solid tumor settings, cal development. These bsAbs represent quite different for- probably due to inaccessibility of immune effector cells to mats. The number of the formats may reflect the diversity solid tumors and complex immunosuppressive mechanisms in desired features of therapeutic applications and may also at TME. The knowledge learned from clinical trials for reflect the different understanding of biology. For instance, either one will definitely help to improve the design of both 58 Antibody Therapeutics, 2020 therapies with additional immunomodulatory features to by binding to porcine immunoglobulins. Vaccine 2005; 23: 4926–34. overcome the key challenges they are still facing. 5. Zhu, X, Wang, L, Liu, R et al. COMBODY: one-domain antibody Nevertheless, bsAbs and msAbs open up tremendous multimer with improved avidity. Immunol Cell Biol 2010; 88: opportunities to explore previously unexplored therapeutic 667–75. options. We believe that the next decade will witness 6. 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Antibody Therapeutics – Oxford University Press
Published: Feb 17, 2020
Keywords: bispecific antibody; bsAb; multispecific antibody; msAb
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