Bispecific effector-cell engagers, novel immunotherapeutics trained to fight cancer








Daniel BATY,PhD
Brigitte KERFELEC,PhD
Patrick CHAMES,PhD
Inserm, U1068, Centre de Recherche en Cancérologie de Marseille, Marseille, FRANCE
Aix-Marseille University
Marseille, FRANCE
CNRS, UMR7258, Centre de Recherche en Cancérologie de Marseille, Marseille, FRANCE
Institut Paoli-Calmettes
Marseille, FRANCE

Bispecific effectorcell engagers, novel immunotherapeutics trained to fight cancer


by D. Baty, B. Kerfelec, and P. Chames, France



Bispecific effector-cell engagers are bispecific antibodies that simultaneously target a tumor-associated antigen and an activating receptor at the surface of effector cells, such as natural killer or T cells. In recent years, the development of antibody engineering has fostered the emergence of several new bispecific formats, leading to remarkable preclinical results in vitro and in vivo. Many of these formats are currently under intense clinical investigation. Furthermore, the end of 2014 saw for the first time in history the approval of a recombinant bispecific T-cell engager by the US Food and Drug Administration, namely blinatumomab (BLINCYTOTM), for the treatment of relapsed or refractory B-cell precursor acute lymphoblastic leukemia. This review discusses the different formats designed to produce such immunotherapeutics with a special emphasis on molecules under clinical investigation. Several of these molecules are expected to dramatically improve treatment of a number of malignancies.

Medicographia. 2015;37:271-279 (see French abstract on page 279)



Antibody-based therapeutics are currently the fastest growing segment of the drug and biologics market. Since the launch of the first anti- CD3 antibody, muromonab-CD3 (OKT3), in 1986, close to 40 monoclonal antibodies (mAbs) and derivatives have been approved in the United States and Europe, and many of them are dedicated to cancer therapy. The first generation of approved antibodies were naked chimeric, humanized, or human antibodies endowed with several modes of action, mainly including the blockade of signaling pathways with induction of apoptosis, recruitment of the complement system (complement-dependent cytotoxicity [CDC]), or recruitment of effector cells such as natural killer (NK) cells (antibody-dependent cell-mediated cytotoxicity [ADCC]) or macrophages (antibody- dependent cell-mediated phagocytosis) through binding of the activating receptor FcϒRIIIA (or CD16A).1

Because ADCC is thought to be an important mode of action of several of these approved molecules, many efforts have been made to improve the interaction between the crystallizable fragment (Fc) of these molecules and FcϒRIIIA, through Fc or glycoengineering. This led to the approval of obinutuzumab, an anti-CD20 mAb that clearly outperforms rituximab, the first-generation anti-CD20 mAb. While many other ADCC-enhanced antibodies are in clinical trials, 2 new families of mAb-based therapeutics—antibody-drug conjugates (ADCs) and bispecific antibodies (bsAbs)— have emerged and led to exciting preclinical and clinical results. The high therapeutic potential of ADCs is out of the scope of this review and has been thoroughly described in recent publications.2 The second family corresponds to bsAbs. The recent blossoming of this field of research is in contrast with its long history, which began years ago, soon after scientists could produce mAbs in a reliable fashion. This is explained by immunogenicity issues faced by murine antibodies, by the very poor production yields afforded by initial approaches used to build these molecules, such as chemical cross-linking of 2 different antibodies or using quadroma technology, and by the difficulty of combining both favorable pharmacokinetic properties and easy large scale manufacturing into an unique new bispecific format.





The development of antibody engineering has brought about innovative solutions that have profoundly changed the situation.3 Many laboratories are currently exploring the numerous possibilities offered by bsAbs. A first therapeutic approach based on these molecules is the simultaneous blockage of 2 receptors or 2 ligands, allowing the simultaneous inhibition of redundant signaling pathways. The second approach, which constitutes the main focus of this review, is the recruitment and the activation of immune effector cells in the tumor microenvironment (Figure 1). The main actors of an immune response against tumor cells are NK cells, macrophages, and T cells. As a consequence, the large majority of bsAbs in this class target CD16 (FcϒRIII) expressed by NK and macrophages, or CD3, expressed by T cells. Here, we will review the main preclinical and clinical results obtained with the various proposed formats of anti-CD3 or anti-CD16 bsAbs (Table I).

bsAbs: a tale of formats

For 2 decades, low yield and heterogeneity of bsAb production relying on methods such as hybrid hybridomas and chemical linking have been significant obstacles to their development.3 A turning point was reached with the capability to produce recombinant fragments of antibodies that possess the full binding activity of the entire immunoglobulin G (IgG) molecule. The antigen-binding fragment (Fab) corresponds to the association of the entire light chain covalently linked via a disulfide bond to the variable (VH) and first constant domains (CH1) of the heavy chain. A smaller fragment could also be produced by linking the variable domains of the heavy and light chains (VH-VL) via a flexible peptide linker, leading to the so-called single-chain variable fragment (scFv). The possibility to produce these fragments in E coli and to combine them as building blocks to create multispecific molecules has led to a plethora of bispecific fragments that have been recently reviewed in detail.4 In this review, we will focus our attention on the formats used in clinical trials. Most bsAb formats can be categorized as either small bispecific formats or IgGlike molecules, the fundamental difference lying in the presence or absence of an intact Fc portion (Figure 2, page 274).

The main benefits of IgG-like formats include a long serum half-life, owing to neonatal Fc recptor (FcRn) binding via the Fc portion, and that their production and purification can be compatible with well-established processes developed for conventional antibodies. By contrast, small molecules devoid of Fc are characterized by a short, or even very short, serum half-life if their molecular weight is below the threshold of renal clearance (60-70 kDa). This drawback can be circumvented by continuous infusion protocols or partly solved by fusion with an albumin-binding domain.5,6 Moreover, the wide variety of size and valencies afforded by the modular nature of small formats and the frequent presence of linkers can also induce a lower global stability and, in some cases, aggregation issues, which in turn might increase their overall immunogenicity. On the other hand, the compact size might be an advantage when it comes to tumor penetration.7 It has also been suggested that small bsAb formats, by forcing close contacts between effector and target cells, trigger the formation of efficient immune synapses that could, in some cases, lead to the exclusion of some bulky receptors involved in negative costimulation.8 This fact might explain why formats such as Bispecific T-cell Engager (BiTE, see below) do not require costimulatory signals, such as CD28 engagement.


Figure 1
Figure 1. Recruitment of immune effector cells
by bispecific antibodies for cancer therapy.

Depending on the effector cells, the mode of action can
be either antibody-dependent cell-mediated cytotoxicity,
antibody-dependent cell-mediated phagocytosis, or cytotoxic
T-cell reaction.
Abbreviations: ADCC, antibody-dependent cell-mediated
cytotoxicity; ADCP, antibody-dependent cell-mediated
phagocytosis; bsFab, bispecific Fab fragments; NK, natural
killer; TAA, tumor-associated antigen; TCR, T-cell receptor.



Table I
Table I. Bispecific effector-cell engagers in clinical trials or approved.

Abbreviations: BiTE, Bispecific T-cell Engager; DART, Dual-Affinity Re-Targeting; EGFR, epidermal growth factor receptor; EMA, European Medicines Agency; EpCAM, epithelial cell adhesion molecule; FDA, US Food and Drug Administration; gpA33, glycoprotein A33 antigen; ERBB2, receptor tyrosine-protein kinase ErbB2; IgG, immunoglobulin G; PSMA, prostatespecific membrane antigen; scFv, single-chain Fv fragment; TaFv, tandem
scFv; TandAb, tandem diabody; TCR, T-cell receptor.



Figure 2
Figure 2. Schematic representation of various bispecific antibody
formats.

Immunoglobulin-like formats: bs-IgG obtained by knobs-intoholes engineering (pink dot in the CH3 domain represents the knobs-into-holes sites for heterodimerization; H, hinge region of IgG1); Triomab obtained by using an original subclass combination of mouse IgG2a and rat IgG2b mAb; bs-scFv-Fc obtained by fusion of different scFvs to the N-terminal and C-terminal parts of the Fc portion.
Fragment-based antibodies: DNL-Fab3 designed by the dock-and-lock approach (the peptides used for association are in brown and pink); Tribody designed by fusing a scFv at the C-terminus of the CH1 and Ck constant domains of a Fab fragment; bs-scFv-CH3: 2 scFv fused to CH3 domains (pink dot in the CH3 domain represents the knobs-into-holes sites for heterodimerization); TaFv, tandem single-chain Fv, also named BiTE; Db, diabody; scDb, single-chain diabody; DART, Dual-Affinity Re-Targeting; TandAb, tandem diabody; TasdAb, tandem single-domain (VHH) antibody from camelid; bsFab, a bispecific Fab (2 single-domain antibodies (VHH) fused to CH1 and Ck constant domains).
Short and long linkers are indicated by black and gray lines, respectively.
For a more complete list see reference 4.
Abbreviations: AD, anchoring domain; CH, constant domain of the heavy chain; CL, constant domain of the light chain; Fc, crystallizable fragment of IgG; IgG, immunoglobulin G; S-S, disulfide bond; SH, sulfhydryl group; VH, variable region of heavy chain; VL, variable region of light chain.



Small bispecific formats
♦ Tandem scFv

The simplest form of small bsAbs is perhaps the covalent association of 2 scFvs via a third flexible linker, leading to the socalled tandem scFv format (TaFv). At the end of the 1990s, this format was chosen by the company Micromet Inc (now part of Amgen) to generate their Bispecific T-cell Engagers (BiTE). BiTEs are generated by fusing an anti-CD3 scFv to an anti–tumor associated antigen scFv via a short 5-residue peptide linker (GGGGS). In 1995, Kufer and colleagues produced such a tandem scFv, targeting epithelial cell adhesion molecule (EpCAM) and human CD3 in Chinese hamster ovary (CHO) cells.9 This new kind of bsAb proved to be highly cytotoxic at nanomolar concentrations against various tumorcell lines, using unstimulated human peripheral blood mononuclear cells (PBMCs) and in the absence of cosignaling. Later, Löffler et al published similar data obtained with a fusion between a murine anti-CD19 scFv and a murine anti-CD3 scFv.10 This molecule demonstrated outstanding properties in vitro, including efficient cytotoxicity induced by subpicomolar concentrations of bsAb (10-50 pg.mL–1), at a low effector: target ratio (2:1), and in only 4 hours, without the need for prestimulation of T cells or, most surprisingly, the need for cosignaling (eg, through CD28). These results were in marked contrast with the majority of published studies based on anti- CD3 bispecific constructs. BiTEs have been demonstrated to induce immunological synapses identical to synapses induced by regular T-cell stimuli, even in the absence of major histocompatibility complex (MHC) class I molecules, as shown by the lysis of EpCAM-expressing K562 cells or -transfected rodent cells by human effector cells.11 The small size (60 kDa) of BiTEs, which ensures close proximity of T cells and target-cell membranes, might therefore be responsible for their high efficiency by leading to the active displacement of negative regulatory proteins from the forming synapse, as demonstrated in the case of CD45.8 In recent years, Micromet has developed a large BiTE platform, generating BiTEs against several tumor-associated targets such as EpCAM, receptor tyrosine-protein kinase ErbB2 (ERBB2, formerly HER2), carcinoembryonic antigen (CEA), Ephrin A2,CD33, and melanoma associated chondroitin sulfate proteoglycan (MCSP).12 The most advanced molecule, the anti-CD19xCD3 blinatumomab, rapidly demonstrated impressive success in early clinical trials13 and, as a consequence, Micromet was acquired in 2012 by Amgen for $1.2 billion. More recently, the US Food and Drug Administration (FDA) granted blinatumomab breakthrough therapy designation for patients with relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL). In a phase 2 multicenter single-arm open-label study for this indication, 42% of the 185 patients evaluated in the trial achieved complete remission or complete remission with partial hematologic recovery within 2 cycles of treatment. Among the responders, 75% even achieved a minimal residual disease response. Consequently, on December 2014, the FDA granted approval of blinatumomab, named BLINCYTO™, for the treatment of patients with Philadelphia chromosome–negative (Ph-) relapsed or refractory B-cell precursor ALL, making it the first bsAb approved by the FDA. Three other BiTEs, anti-EpCAMx- CD3 (MT110), anti-CEAxCD3 (MT111, MEDI-565) and anti– prostate-specific membrane antigen (PSMA)xCD3 (MT112, BAY 2010112) are currently under phase 1 clinical investigation for multiple solid cancers (NCT00635596), gastrointestinal adenocarcinomas (NCT01284231), and prostate cancer (NCT01723475), respectively.

Of note, the company Immunocore has produced a fusion between an affinity-matured soluble T-cell receptor (TCR) targeting the HLA-A2/gp100 peptide-MHC complex and the N-terminus of an anti-CD3 scFv. This molecule named IMCgp100, closely resembling a Fab-scFv bsAb, and active at picomolar-range concentrations in vitro,14,15 is under phase 2 clinical investigation for the treatment of late-stage melanoma (NCT01211262).

Diabody and Dual-Affinity Re-Targeting
Another effective format is the diabody (Db) format, originally developed by Holliger et al.16 Dbs are noncovalent dimers of scFv fragments from 2 different antibodies in which the reduced length of the peptide linker between VH-VL of the same scFv fragment impedes intramolecular pairing, thus promoting/ forcing the cross-pairing with the complementary domains of a second scFv fragment. These compact molecules are expressed at high yields in bacteria, and have been shown by structural experiments to adopt several conformations.17 This format has been further improved by the addition of an extra peptide linker between the 2 polypeptides in order to further decrease the amount of homodimers, yielding fragments called single-chain Dbs (scDb). Numerous studies have demonstrated the potency of these formats in preclinical studies.18 Examples of bsAb fragments with potential as therapeutic candidates include bispecific anti-CD19xCD3 and anti-CD19xCD16 Dbs which demonstrated a synergistic antitumor effect in a preclinical model of non-Hodgkin lymphoma (NHL),19 a promising anti–epidermal growth factor receptor (EGFR)xCD3 Db able to cure colon cancer–xenografted mice in combination with lymphokine-activated killer cells,20 and an effective anti- PSMAxCD3 Db for the treatment of prostate cancer–xenografted mice in the presence of peripheral blood lymphocytes.21 In 2010, Bonvini et al published a variation of the Db format targeting NK cells through CD16 and CD32B on B cells, by adding a free C-terminal cysteine on each chain of the Db, leading to an interchain disulfide bond upon chain association. This new format, named DART for Dual-Affinity Re-Targeting, showed extended storage and serum stability, combined with potent tumor cytolysis and autologous B-cell depletion in culture.22 In another study, they performed a side by side comparison of the DART and BiTE formats using the variable domains of blinatumomab. The CD19xCD3 DART molecules achieved an enhanced activity on all CD19-expressing target B cells evaluated using resting and prestimulated human PBMCs or purified effector–T-cell populations.23 Since then, the company MacroGenics has developed several DARTs. MGD006 (also encoded as S80880 by Servier, a partner of MacroGenics) targets the interleukin 3–receptor αchain (CD123), overexpressed on malignant cells in a wide range of hematological malignancies including acute myelocytic leukemia (AML) and myelodysplastic syndrome. MGD007 targets the glycoprotein A33 antigen (gpA33), a cell surface antigen expressed in more than 95% of primary and metastatic human colorectal cancers, including cancer stem cells. In preclinical studies, MGD007 mediated potent lysis of gpA33- positive colorectal cancer cell lines both in vivo and in vitro, and tumor growth inhibition was observed at very low doses. Recently, 2 phase 1 clinical trials were initiated with these 2 DARTs (NCT02152956 and NCT02248805 for MGD006 and MGD007, respectively).

Tandem Dbs
In a seminal work, Kiprianov et al proposed a new format based on the Db concept.24 They generated dimers of scDb (CD3xCD19) in an antiparallel orientation using middle linkers shorter than 12 amino acids. The new molecule called tandem Db (TandAb) was expressed in E coli as a highly stable bispecific and tetravalent dimer, which demonstrated increased valency and longer blood retention compared with scFv fragments and Dbs.24 With a size of approximately 110 kDa, TandAbs are smaller than an IgG molecule, which may enhance tumor penetration. However, their size is well above the renal threshold for first-pass clearance, offering a pharmacokinetic advantage over smaller bispecific formats. A TandAb platform has since been developed and is currently exploited by the company Affimed. One of their most advanced molecules, a CD30xCD16 TandAb called AFM13, is currently in a multicenter phase 2 clinical trial for the treatment of advanced relapsing/refractory Hodgkin lymphoma (NCT02321592). By specifically targeting CD16A, AFM13 is designed to recruit NK cells without interacting with neutrophils expressing CD16B, and is not affected by CD16A polymorphism that affects the Fc binding of conventional IgGs. Somehow surprisingly, the activation of NK cells was found strictly dependent on the presence of CD30+ target cells despite the TandAb bivalency for CD16A.25

The same strategy has been applied for the recruitment of T cells with the anti-CD19xCD3 TandAb, AFM11, made of fully human binding domains, which are isolated as scFvs from a phage-display library. This molecule is currently in a preclinical development stage for the treatment of NHL. As for most CD3-targeting bsAbs, AFM11 exhibits potent cytotoxic activity in vitro with half-maximal effective concentration (EC50) values in the low- to subpicomolar range, with complete lysis of CD19+ tumor cells typically observed within 2 hours. Unexpectedly, despite its bivalency and its very high affinity for CD3 (0.7 nM), the binding of AFM11 to CD3 in the absence of a tumor cell appears to be insufficient to activate T cells.26 A side by side in vitro comparison with blinatumomab on CD19+ cells (NALM-6, a human pre-B cell line) demonstrated a higher potency for the TandAb AFM11. In vivo studies on a Burkitt lymphoma xenograft model demonstrated high tumor-cell killing and advantageous pharmacokinetic properties, suggesting that AFM11 might not require administration by continuous infusion (Affimed’s presentation at the Essential Protein Engineering Summit [PEGS], October 2014). A phase 1 study was initiated in patients with relapsed and/or refractory NHL (NCT02106091). A third Tandab, AFM21, targeting CD3 and EGFR variant III (EGFRvIII) is also being developed for the treatment of solid tumors (Affimed website).

CH1/Ck domains as a heterodimerization motif: tribodies and bispecific Fab fragments
bsAbs containing constant IgG domains have also been developed. In 2010, Mertens et al published a way to obtain trivalent antibody fragments (tribodies) by fusing a scFv at the C-terminus of CH1 and Ck constant domains of a Fab fragment (Figure 2).27 This format was later used by Glorius et al to create a bispecific anti-CD20xCD16 tribody by fusing 2 anti-CD20 scFv at the C-termini of an anti-CD16 Fab.28 Interestingly, the potency and efficacy of lysis obtained with the tribody was significantly higher than that triggered by rituximab. Compared with rituximab, the tribody demonstrated depletion of autologous B cells in ex vivo whole blood assays at a 100-fold lower antibody concentration, as well as in mice with a reconstituted, humanized hematopoietic system. Tribodies display interesting pharmacokinetic properties such as biodistribution profiles similar to those of IgG and higher tumor-accumulation rates. The company Biotecnol is currently exploiting this format to develop Tb535, an anti-CD3 Tribody™, directed against the oncofetal antigen 5T4, found in various subtypes of malignant mesothelioma and absent from normal tissue. Tb535 demonstrated low picomolar EC50 values for cytotoxicity toward several human carcinoma cell lines (mesothelioma and others) in an in vitro assay using PBMCs from healthy human donors.

The natural in vivo heterodimerization of Fab fragments was also used to create a very compact format of bsAb, devoid of artificial linkers. The so-called bsFabs (for bispecific Fab fragments) relies on the useof single-domain antibodies (also called nanobodies) derived from heavy-chain antibodies, naturally occurring antibodies devoid of light chains found in camelids. In 2013, Rozan et al showed that an anti-CEAxCD16 bsFab29 could be efficiently produced by fusing anti-CD16 and anti- CEA nanobodies to the N-terminus of the human CH1 and Ck constant domains respectively, leading to a highly stable 50-kDa Fab-like bsAb able to elicit potent lysis of tumor cells by human NK cells at picomolar concentrations. This format was recently used to develop an anti-ERBB2xCD16 bsAb able to outperform trastuzumab, both in vitro and in vivo, on tumor cells expressing a low amount of ERBB2, thereby potentially enlarging the number of patients eligible for breast cancer immunotherapy.30

Fc fusions
In 2012, Kuo et al developed a straightforward bsAb format to target CD123+ leukemia cells by fusing an anti-CD123 scFv at the N-terminus of human IgG1 hinge-CH2-CH3 domains, followed by an anti-CD3 scFv at its C-terminus31 (Figure 2). Upon dimerization, this 160-kDa molecule, named BIf for bispecific immunofusion, is bispecific and tetravalent, and possesses an intact Fc portion allowing extended serum half-life and the ability to trigger ADCC.31 BIf shows cytolytic activities at low picomolar levels with effector:target ratios as low as 2. This molecule is bivalent for CD3, but the location of the anti-CD3 scFv at the C-termini of BIf reduces the affinity to CD3+ T cells by 2 orders of magnitude, which could help to prevent nonspecific T-cell activation. The company Trubion has developed a very similar bsAb format initially called SCORPION™. After their purchase by Emergent BioSolutions, that modular protein technology platform was renamed ADAPTIR™. In collaboration with MorphoSys, an anti-PSMAxCD3 bsAb named MOR209 or ES414 recently entered a phase 1 clinical trial evaluating the compound in patients with metastatic castration-resistant prostate cancer (NCT02262910).

bsAbs based on the dock-and-lock approach
The dock-and-lock (DNL) method was originally published in 200632 and represents a totally different way to create bsAbs. It relies on the spontaneous association of the 44–amino-acid peptide DDD2, derived from the regulatory subunit of human cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) with the 17-residue peptide AD2, derived from the anchoring domains (AD) of human A kinase anchor proteins (AKAPs). Upon association, 2 disulfide bonds are created, resulting in a covalent complex that is stable for more than a week at 37°C in human serum. This approach was recently developed to create T-cell retargeting bsAbs.33,34 (E1)- 3s is a T-cell–redirecting trivalent bsAb, comprising an anti- CD3 scFv covalently linked to a stabilized dimer of a humanized Trop-2–targeting Fab (from murine mAb RS7). (E1)-3s mediated a highly potent T-cell lysis of NCI-N87 target cells in vitro. In vivo, (E1)-3s effectively induced T-cell–mediated killing of Trop-2–expressing pancreatic and gastric cancers, which was enhanced with interferon α(INFα).33

IgG-like formats
Chemically cross-linked full-length antibodies
The simplest way to create anti-CD3 bsAbs is to chemically cross-link already approved therapeutic IgGs such as anti- CD3 mAb OKT3 and anti-ERBB2 trastuzumab, creating a heterogeneous mixture with high valency and bispecificity. As early as 2001, Lum et al used such a preparation to arm activated T cells (ATC, which are PBMC activated for 14 days with anti-CD3 mAb in the presence of interleukin 2), showing that the complex remains on ATC for 72 hours.35 Since then, they demonstrated that ATC armed with anti-CD3 x anti–tumor associated antigen bsAbs exhibit high levels of specific cytotoxicity against tumor cells expressing ERBB2,36 CD20,37 the ganglioside GD2,38 and EGFR39 via redirected non–MHC-restricted perforin/granzyme-dependent killing, both in vitro and in vivo. ATC armed with an anti-ERBB2xCD3 (HER2Bi) is currently in phase 2 clinical trials for the treatment of breast cancer (NCT01022138, NCT01147016). ATC/CD20Bi (prepared with rituximab) completed a phase 1 study for a combined treatment of patients with multiple myeloma with autologous hematopoietic stem cell transplantation (NCT00938626). ATC/GD2Bi (prepared with mAb 3F8) recently entered phase 1/2 clinical trials for the treatment of children with neuroblastoma and osteosarcoma (NCT02173093), while ATC/EGFRBi (prepared with cetuximab) is being tested in several phase 1 clinical trials for refractory, or metastatic non–small cell lung cancer and gastrointestinal cancer (NCT00569296 and NCT01420874, respectively).

Y-shaped bispecific IgG
♦ Triomabs

Because they rely on an adaptation of the conventional hybrid hydridoma technology, Triomabs represent the simplest format of IgG-like bsAbs. In 1995, Lindhofer et al published a paper describing a major improvement of the classical quadroma approach to produce bsAbs.40 By using an original subclass combination (mouse IgG2a and rat IgG2b), they demonstrated a preferential species-restricted heavy/lightchain pairing, in contrast to the random pairing in conventional mouse/mouse or rat/rat quadromas, as well as the use of sequential pH elution on protein A to easily separate the desired bsAb from the parental mAb. Surprisingly, the resulting hybrid rat/mouse Fc portion efficiently interacted with activating human Fc receptors (FcϒRI and FcϒRIII), but not with inhibitory ones (FcϒRIIB), thereby reaching the goal that other groups had hoped to achieve using human Fc engineering. 41,42 The investigators used this approach to create an anti-CD3xEpCAM bsAb, and demonstrated that this antibody was capable of binding to target cells and human T cells, but was also capable of activating dendritic cells (DCs), inducing NK-dependent ADCC and stimulating tumor-cell phagocytosis by macrophages.41,42 In short, this Fc adds 2 crucial functions to regular anti-CD3 x target bsAbs: additive tumor-killing capabilities through the efficient recruitment of macrophages and NK cells, and, most importantly, efficient costimulation of T cells through direct contact with accessory cells, such as macrophages and DCs or cytokine secretion. The most advanced Triomab, an anti EpCAMxCD3 bsAb called catumaxomab, has impressive preclinical data with total tumor eradication, but also with induction of immune protection.43 By early 2009, the results of a large international phase 2/3 pivotal study involving 258 patients demonstrated a statistically significant improvement of the primary end point, puncture-free survival, leading to the approval of the molecule by the European commission in April 2009 for the treatment of malignant ascites in patients with EpCAM-positive carcinomas in cases where standard therapy is not available or no longer feasible. Indeed, patients receiving catumaxomab had a 4-fold increase in puncture-free survival compared with those receiving paracentesis therapy only. Catumaxomab is thus the first approved bsAb in the history of immunotherapy. Ertumaxomab, a second Triomab targeting ERBB2, has also yielded impressive preclinical data44,45 and is currently in phase 1/2 trials for the treatment of patients with progressing ERBB2+ solid tumors (NCT01569412). A third Triomab, targeting CD20, called FBTA05 (or Lymphomun™ or Bi20), is under phase 1/2 clinical investigation for the treatment of chronic lymphocytic leukemia (CLL) and low- and high-grade NHL, in combination with donor lymphocyte infusions (NCT01138579).46

Intact IgG with engineered Fc
Several published approaches allow the generation of intact mAbs. The knobs-into-holes principle is a well-described Fc heterodimerization technology that consists of introducing complementary mutations (replacing a small amino acid with a larger one [“knob”] and vice versa [“hole”]) in each CH3 domain. The light-chain mispairing issue has also been solved via the use of common light chains, or more elegantly via the CrossMAb technology which avoids nonspecific light-chain mispairing by exchanging CH1 and CL constant domains in the Fab of one-half of the bsAbs (see reference 4 for detailed review). The use of such innovative formats for effector-cell retargeting is not yet well documented in the literature. The company Xencor has used structure- and sequence-based approaches to design Fc variants that preferentially heterodimerize to produce 3 anti-CD3 bsAbs targeting CD123, CD38, and CD20, all of them being in preclinical stage. Using an undisclosed technology, the company Regeneron Pharmaceuticals has generated an anti-CD20xCD3 bsAb, named REGN1979, which recently entered a multicenter phase 1 clinical trial for the treatment of patients with NHL and CLL (NCT02290951).

Conclusion

The journal Science has chosen cancer immunotherapy as “Breakthrough of the Year 2013,”47 mainly because of the clinical successes recorded with immunomodulatory mAbs such as anti–cytotoxic T-lymphocyte antigen 4 (CTLA-4), anti– programmed cell death protein 1 (PD-1), anti–programmed death-ligand 1 (PD-L1), and other promising approaches such as chimeric-antigen-receptor T cells and antibody drug conjugates. It might well be that, in the end, bi- and multispecific antibodies will have just as much impact as those approaches, if not more. The long-awaited results of ongoing clinical trials will surely tell.

References
1. Chames P, Van Regenmortel M, Weiss E, Baty D. Therapeutic antibodies: successes, limitations and hopes for the future. Br J Pharmacol. 2009;157: 220-233.
2. Beck A. Review of Antibody-Drug Conjugates, Methods in Molecular Biology series: a book edited by Laurent Ducry. . 2014;6:30-33.
3. Chames P, Baty D. Bispecific antibodies for cancer therapy: the light at the end of the tunnel? MAbs. 2009;1:539-547.
4. Kontermann RE. Dual targeting strategies with bispecific antibodies. Mabs. 2012;4:182-197.
5. Holt LJ, Basran A, Jones K, et al. Anti-serum albumin domain antibodies for extending the half-lives of short lived drugs. Protein Eng Des Sel. 2008;21: 283-288.
6. Roovers RC, Laeremans T, Huang L, et al. Efficient inhibition of EGFR signaling and of tumour growth by antagonistic anti-EFGR Nanobodies. Cancer Immunol Immunother. 2007;56:303-317.
7. Tijink BM, Laeremans T, Budde, M, et al. Improved tumor targeting of anti-epidermal growth factor receptor Nanobodies through albumin binding: taking advantage of modular Nanobody technology. Mol Cancer Ther. 2008;7:2288- 2297.
8. Wolf E, Hofmeister R, Kufer P, Schlereth B, Baeuerle PA. BiTEs: bispecific antibody constructs with unique anti-tumor activity. Drug Discov Today. 2005;10: 1237-1244.
9. Mack M, Riethmuller G, Kufer PA. Small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity. Proc Natl Acad Sci U S A. 1995;92:7021-7205.
10. Loffler A, Kufer P, Lutterbuse R, et al. A recombinant bispecific single-chain antibody, CD19 x CD3, induces rapid and high lymphoma-directed cytotoxicity by unstimulated T lymphocytes. Blood. 2000;95:2098-2103.
11. Offner S, Hofmeister R, Romaniuk A, Kufer P, Baeuerle PA. Induction of regular cytolytic T cell synapses by bispecific single-chain antibody constructs on MHC class I-negative tumor cells. Mol Immunol. 2006;43:763-771.
12. Huehls AM, Coupet TA, Sentman CL. Bispecific T-cell engagers for cancer immunotherapy. Immunol Cell Biol. 2015;93:290-296.
13. Bargou R, Leo E, Zugmaier G, et al. Tumor regression in cancer patients by very low doses of a T cell-engaging antibody. Science. 2008;321:974-977.
14. Liddy N, Bossi G, Adams KJ, et al. Monoclonal TCR-redirected tumor cell killing. Nat Med. 2012;18:980-987.
15. Bossi G, Buisson S, Oates J, Jakobsen BK, Hassan NJ. ImmTAC-redirected tumour cell killing induces and potentiates antigen cross-presentation by dendritic cells. . 2014;63:437-448.
16. Holliger P, Prospero T, Winter G. « Diabodies »: small bivalent and bispecific antibody fragments. Proc Natl Acad Sci U S A. 1993;90:6444-6448.
17. Lawrence LJ, Kortt AA, Iliades P, Tulloch PA, Hudson PJ. Orientation of antigen binding sites in dimeric and trimeric single chain Fv antibody fragments. FEBS Lett. 1998;425:479-484.
18. Muller D, Kontermann RE. Recombinant bispecific antibodies for cellular cancer immunotherapy. Curr Opin Mol Ther. 2007;9:319-326.
19. Kipriyanov SM, Cochlovius B, Schafer HJ, et al. Synergistic antitumor effect of bispecific CD19 x CD3 and CD19 x CD16 diabodies in a preclinical model of non-Hodgkin’s lymphoma. J Immunol. 2002;169:137-144.
20. Asano R, Sone Y, Makabe K, et al. Humanization of the bispecific epidermal growth factor receptor x CD3 diabody and its efficacy as a potential clinical reagent. Clin Cancer Res. 2006;12:4036-4042.
21. Buhler P, Wolf P, Gierschner D, et al. A bispecific diabody directed against prostate-specific membrane antigen and CD3 induces T-cell mediated lysis of prostate cancer cells. Cancer Immunol Immunother. 2008;57:43-52.
22. Johnson S, Burke S, Huang L, et al. Effector cell recruitment with novel Fvbased dual-affinity re-targeting protein leads to potent tumor cytolysis and in vivo B-cell depletion. J Mol Biol. 2010;399:436-449.
23. Moore PA, Zhang W, Rainey GJ, et al. Application of dual affinity retargeting molecules to achieve optimal redirected T-cell killing of B-cell lymphoma. Blood. 2011;117:4542-4551.
24. Kipriyanov SM, Moldenhauer G, Schuhmacher J, et al. Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics. J Mol Biol. 1999;293:41-56.
25. Reusch U, Burkhardt C, Fucek I, et al. A novel tetravalent bispecific TandAb (CD30/CD16A) efficiently recruits NK cells for the lysis of CD30+ tumor cells. MAbs. 2014;6:728-739.
26. McAleese F, Eser M. RECRUIT-TandAbs: harnessing the immune system to kill cancer cells. Future oncology. 2012;8:687-695.
27. Schoonooghe S, Kaigorodov V, Zawisza M, et al. Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris. BMC Biotechnol. 2009;9:70.
28. Glorius P, Baerenwaldt A, Kellner C, et al. The novel tribody [(CD20)2xCD16] efficiently triggers effector cell-mediated lysis of malignant B cells. Leukemia. 2013;27:190-201.
29. Rozan C, Cornillon A, Petiard C, et al. Single-domain antibody-based and linker-free bispecific antibodies targeting FcγRIII induce potent antitumor activity without recruiting regulatory T Cells. Mol Cancer Ther. 2013;12:1481-1491.
30. Turini M, Chames P, Bruhns P, Baty D, Kerfelec BA. FcγRIII-engaging bispecific antibody expands the range of HER2-expressing breast tumors eligible to antibody therapy. Oncotarget. 2014;5:5304-5319.
31. Kuo SR, Wong L, Liu JS. Engineering a CD123xCD3 bispecific scFv immunofusion for the treatment of leukemia and elimination of leukemia stem cells. Protein Eng Des Sel. 2012;25:561-569.
32. Rossi EA, Goldenberg DM, Cardillo TM, McBride WJ, Sharkey RM, Chang CH. Stably tethered multifunctional structures of defined composition made by the dock and lock method for use in cancer targeting. Proc Natl Acad Sci U S A. 2006;103:6841-6846.
33. Rossi EA, Rossi DL, Cardillo TM, Chang CH, Goldenberg DM. Redirected Tcell killing of solid cancers targeted with an anti-CD3/Trop-2-bispecific antibody is enhanced in combination with interferon-alpha. Mol Cancer Ther. 2014; 13:2341-2351.
34. Rossi DL, Rossi EA, Cardillo TM, Goldenberg DM, Chang CH. A new class of bispecific antibodies to redirect T cells for cancer immunotherapy. MAbs. 2014; 6:381-391.
35. Lum LG, Sen M. Activated T-cell and bispecific antibody immunotherapy for high-risk breast cancer. Bench to bedside. Acta Haematol. 2001;105:130-136.
36. Sen M, Wankowski DM, Garlie NK, et al. Use of anti-CD3 x anti-HER2/neu bispecific antibody for redirecting cytotoxicity of activated T cells toward HER2/ neu+ tumors. J Hematother Stem Cell Res. 2001;10:247-260.
37. Gall JM, Davol PA, Grabert RC, Deaver M, Lum LG. T cells armed with anti- CD3 x anti-CD20 bispecific antibody enhance killing of CD20+ malignant B cells and bypass complement-mediated rituximab resistance in vitro. Exp Hematol. 2005;33:452-459.
38. Yankelevich M, Kondadasula SV, Thakur A, Buck S, Cheung NK, Lum LG. Anti-CD3 x anti-GD2 bispecific antibody redirects T-cell cytolytic activity to neuroblastoma targets. Pediatr Blood Cancer. 2012;59:1198-1205.
39. Reusch U, Sundaram M, Davol PA, et al. Anti-CD3 x anti-epidermal growth factor receptor (EGFR) bispecific antibody redirects T-cell cytolytic activity to EGFR-positive cancers in vitro and in an animal model. Clin Cancer Res. 2006; 12:183-190.
40. Lindhofer H, Mocikat R, Steipe B, Thierfelder S. Preferential species-restricted heavy/light chain pairing in rat/mouse quadromas. Implications for a singlestep purification of bispecific antibodies. J Immunol. 1995;155:219-225.
41. Zeidler R, Mysliwietz J, Csánady M, et al. The Fc-region of a new class of intact bispecific antibody mediates activation of accessory cells and NK cells and induces direct phagocytosis of tumour cells. Br J Cancer. 2000;83:261-266.
42. Zeidler R, Reisbach G, Wollenberg B, et al. Simultaneous activation of T cells and accessory cells by a new class of intact bispecific antibody results in efficient tumor cell killing. J Immunol. 1999;163:1246-1252.
43. Ruf P, Lindhofer H. Induction of a long-lasting antitumor immunity by a trifunctional bispecific antibody. Blood. 2001;98:2526-2534.
44. Heiss MM, Strohlein MA, Jager M, et al. Immunotherapy of malignant ascites with trifunctional antibodies. Int J Cancer. 2005;117:435-443.
45. Jager M, Schoberth A, Ruf P, Hess J, Lindhofer H. The trifunctional antibody ertumaxomab destroys tumor cells that express low levels of human epidermal growth factor receptor 2. Cancer Res. 2009;69:4270-4276.
46. Buhmann R, Michael S, Juergen H, Horst L, Peschel C, Kolb HJ. Immunotherapy with FBTA05 (Bi20), a trifunctional bispecific anti-CD3 x anti-CD20 antibody and donor lymphocyte infusion (DLI) in relapsed or refractory B-cell lymphoma after allogeneic stem cell transplantation: study protocol of an investigator- driven, open-label, non-randomized, uncontrolled, dose-escalating Phase I/II-trial. J Transl Med. 2013;11:160.
47. Couzin-Frankel J. Breakthrough of the year 2013. Cancer immunotherapy. Science. 2013;342:1432-1433.



Keywords: bispecific antibodies; Bispecific T-cell Engager, cancer immunotherapy; CD3; CD16; Dual-Affinity Re-Targeting; FcɣRIIIA