Bispecific antibodies have special mechanisms of action, including two most distinct and promising aspects: (1) as a bridge to link target cells with effector cells or drug moieties; (2) binding two epitopes on the same cell to simultaneously block two compensatory signaling pathways or to improve affinity and avoid side effects. The first aspect will be discussed in the following section. This section is to discuss the second aspect.
Bispecific antibodies that are capable of interacting with two different epitopes on the same antigen [
39] or different antigens on the same cell [
40] can induce receptor cross-linking and result in high affinity. The bispecific antibodies that target two different epitopes on HER2 cross-link HER2 and form a larger meshwork structure than a monoclonal antibody does [
23,
39]. Additionally, because of the surface diffusion, the distance of different antigens that previously exceeded the reach of the antigen-binding arms of the antibody can be pulled near each other by bispecific antibodies, which allows for the highly specific binding of bispecific antibodies to antigens on the same cell, despite these two antigens’ surface density being too low [
40]. These bispecific antibodies form a trimeric complex with the targeted antigens on the same cell [
20]. This demonstration can shed light on more novel drug discoveries. Anti-CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) and anti-PD-1 (programmed cell death protein 1) monoclonal antibodies were approved by the U.S. FDA for the treatment of melanoma and are undergoing clinical trials for the treatment of many other tumors. Though these checkpoint inhibitors have revolutionized the treatment of advanced cancers, they also results in a spectrum of adverse events [
41]. A bispecific antibody that consists of both of these two specificities may lead to a higher specificity and cause less adverse events.
3.1. Retargeting Cellular Immunity towards the Malignant Cells
These kinds of bispecific antibodies recruit effector cells from the immune system of the host, for the selective destruction of cells, especially tumor cells expressing the targeted antigens. One of their antibody moieties identifies the tumor cells via a tumor-specific antigen, while the second antibody binding site is used to recruit and activate a suitable leukocyte. T cells and NK cells are the main factors that help to kill tumor cells; therefore, T cells and NK cells are the most frequently recruited cells by bispecific antibodies [
42,
43]. Bispecific T cell engagers, termed BiTEs, with one scFv binding to a T cell-specific molecule, usually CD3, while the second scFv binds to a tumor-associated antigen, efficiently enhance the patient’s immune response to tumors. The T cells are activated and release cytokines to kill tumor cells when the synapse is formed between T cells and the tumor cells.
Blinatumomab is a murine-derived bispecific T cell engager (BiTE) antibody that directs CD3-positive T cells against CD19-positive B cells. It works by inducing the proliferation and the activation of T cells to exert cytotoxic activity on the malignant cells [
8]. Due to its excellent effect in clinical trials, blinatumomab received an accelerated approval from the FDA for the treatment of Philadelphia chromosome-negative relapsed or refractory acute lymphoblastic leukemia in December 2014 [
44]. It is currently investigated in pediatric ALL, because CD3 and CD19 are positive in pediatric, as well as adult patients [
45]. Blinatumomab might also benefit patients with non-Hodgkin’s lymphoma, especially for those with aggressive forms, like diffuse large B cell lymphoma [
46]. Another three BiTEs are also entering clinical trials: anti-EpCAM/CD3 MT-110 [
47], anti-CEA (carcino-embryonic antigen)/CD3 MT-111 [
48] and anti-PSMA (prostate specific membrane antigen)/CD3 AMG 212 [
50]. All of these BiTEs have already shown high antitumor activity in diverse xenograft models and were tested in the corresponding clinical trials. A study has also shown the capacity of MT-110 to eradicate cancer stem cells [
59].
DART proteins are the heterodimer of two peptides derived from two parent antibodies. Because DART protein is larger in size than BiTEs, it shows extended storage and serum stability. Functionally, the DART proteins have demonstrated extremely potent, dose-dependent cytotoxicity in redirecting human PBMC against targeted cell lines [
60]. MGD006 is a CD3 × CD123 DART molecule that induces T cell target-specific association, activation and proliferation. The high stability and excellent performance of DARTs enhanced the clinical application [
51]. As a result, it has entered the phase I clinical trial in relapsed/refractory acute myeloid leukemia (AML) or intermediate-2/high risk myelodysplastic syndromes (MDS) (ClinicalTrials.gov: NCT02152956).
Stadler et al. engineered a T cell-engaging bispecific single chain molecule (bi-(scFv)
2) with anti-CD3 and anti-CLDN6 specificities, with its format similar to BITEs. CLDN6 is the fetal tight junction molecule claudin, highly expressed in various solid tumors, whereas barely expressed in normal tissues. This kind of bispecific antibody also showed the efficacy of recruiting T cells and eradication of tumors in xenograft models [
61].
The bispecific NK cell engager (BiKE) is also constructed to recruit NK cells. Aifen Li et al. constructed a HER2-S-Fab with a binding site for CD16 to recruit NK cells to kill HER2-positive tumor cells [
34]. Schmohl et al. constructed another NK cell engager by incorporating IL-15 between the anti-EpCAM region and the anti-CD16 region. This new form showed the ability of both facilitating the ADCC effect and the induction of NK cell expansion [
62]. NKG2D is an activating receptor found on NK and CD8
+ T cells. Its ligand ULBP2 and MICA are attached through linkers to the scFvs, which target tumor antigens, like CD138, CD19, CD33 and CD24, to generate kinds of bispecific antibodies. A recombinant bispecific protein (ULBP2-BB4) binds the activating NK receptor NKG2D and the CD138, which is overexpressed on a variety of malignancies [
63]. A mono-targeting triple body ULBP2-aCD19-aCD19 and a dual-targeting triple body ULBP2-aCD19-aCD33 were constructed to recruit NK cells to kill CD19- or CD33-positive chronic lymphocytic leukemia cells [
64]. MICA was attached to rG7S, a single-chain antibody fragment (scFv) targeting the tumor-associated antigen CD24 to form a fusion protein rG7S-MICA to treat hepatocellular carcinoma (HCC) [
65]. All of these four recombinant proteins can strongly activate primary NK cells in vitro and in vivo and enhance the NK cell-mediated lysis of corresponding antigen-positive tumor cells [
63,
64,
65].
Because of offering a longer half-life compared to smaller antibody constructs, TandAbs are a hotspot to be developed. Unlike the BiTEs, TandAb has two binding sites for one antigen and another two binding sites for the second antigen. TandAb mediates the killing of tumor cells with little dependence on the potency or efficacy upon the effector:target ratio, and it can effectively kill tumor cells at lower effector: target ratios [
3,
14]. Thus, some promising TandAbs are coming to the fore, like CD33/CD3 TandAb [
13], CD19/CD3 AFM11[
14] and CD30/CD16A AFM13 [
52]. AFM11 induced dose-dependent growth inhibition and exhibited substantial cytotoxic activity both in NOD/SCID xenograft models and in vitro. Because of its excellent preclinical outcomes, AFM11 is entering clinical trials and currently recruiting patients with relapsed and/or refractory CD19-positive B cell non-Hodgkin lymphoma (NHL) (ClinicalTrials.gov: NCT02106091).
AFM13 is designed for the treatment of CD30-expressing malignant lymphomas by recruiting and activating natural killer (NK) cells via binding to CD16A. Preclinical data demonstrate a specific and efficient antitumor activity via the engagement of NK cells [
12]. In the phase I study, the treatment with AFM13 was well tolerated, and the treatment caused recruitment and activation of NK cells [
52]. It is now entering phase II clinical trials (ClinicalTrials.gov: NCT02321592).
Catumaxomab (anti-EpCAM/anti-CD3) is produced by a quadroma cell line. The quadroma cell line for the production of catumaxomab is obtained by the fusion of two parental hybridoma cell lines: a mouse hybridoma cell line expressing anti-human EpCAM antibody and a rat hybridoma cell line producing anti-human CD3 antibody. Catumaxomab has the ability to bind three different cell types: EpCAM positive tumor cells, T cells and accessory cells via its intact Fc region. It was approved in the European Union on 20 April 2009 for the treatment of malignant ascites (MA), a condition occurring in patients with EpCAM-positive metastasizing cancer where standard therapy is no longer feasible [
53]. It is safe and effective for catumaxomab to be given both to hospitalized patients and outpatients [
54]. Toxicity was generally manageable, with abdominal pain, nausea/vomiting, fatigue and fevers the predominant adverse effects; while its trifunction also causes some side effects, like liver injury. Catumaxomab binds to FcγR-positive Kupffer cells to stimulate the release of C-reactive protein, chemokines and cytokines, which induces liver injury [
66]. Besides catumaxomab, there are other similar bispecific antibodies composed of an anti-CD3 rat IgG2b half antibody for T cell recognition and the antigen binding site presented by the mouse IgG2a isotype: HER2/CD3 ertumaxomab for breast cancer treatment [
55] and CD20/CD3 FBTA05 for pediatric high-risk patients with recurrent CD20-positive B cell malignancies [
56]. The phase II clinical trial of ertumaxomab has terminated (clinicaltrials.gov: NCT00351858). FBTA05 is being tested in a phase I/II clinical trial evaluating the safety and efficacy of FBTA05 in combination with donor lymphocyte infusions for the treatment of relapsed or refractory disease in CD20-positive non-Hodgkin lymphoma after allogeneic transplantation (clinicaltrials.gov: NCT01138579).
CrossMab and knobs-into-holes technology are also widely used to overcome the obstacle of the light chain mispairing problem. CEA TCB is the IgG1-based bispecific heterodimeric antibody that binds with one arm to CD3 expressed on T cells and with two arms to CEA expressed on tumor cells. It adopted the crossmab and knobs-into-holes technology to make sure that the H and light chains pair correctly. The correct association of light chains of the antibody is enabled by introducing a CH1-CL crossover into the internal CD3-binding Fab. It also incorporated the engineered Fc region with completely abolished binding to Fcγ Rs and C1q [
17,
38]. CEA TCB is now entering clinical trials and getting promising results (ClinicalTrials.gov: NCT02324257).
However, more notably, the recruited T cells may be severely hampered by the dysfunction of infiltrated pre-existing T cells, as they may present with a high frequency of inhibitory checkpoint molecules like PD-1, thus resulting in significantly impaired tumor cell killing of recruited T cells. Jens Schreiner et al. characterized inhibitory receptors of pre-exiting T cells and found that highly expressed PD-1 infiltrated T cells defined a T cell subset with particularly high levels of multiple inhibitory receptors. Blocking PD-1 could restore the cytokine secretion of infiltrated T cells [
67]. Thomas Köhnke et al. also presented in a report a case of a 32-year-old male patient with refractory B-precursor ALL who was resistant to treatment with blinatumomab. Bone marrow immunohistochemistry showed the T cell infiltration and the increasing in the expression of PD-L1 as a potential immune escape mechanism [
68]. As the bispecific antibody can efficiently activate T cells, resulting in the production and release of a large number of inflammatory cytokines, bispecific antibody therapy may also trigger immune evasion of antibody-mediated tumor cell lysis of tumor cells. Christina Krupka et al. found that although the expression of PD-1 and PD-L1 was extremely low in patients with primary AML, their expression could be induced by AMG 330-mediated (a bispecific CD33/CD3 BiTE antibody construct) T cell activation. Additionally, blockade of the PD-1/PD-L1 interaction resensitizes target cells to AMG 330-mediated lysis [
69]. Regulatory T cells (Tregs) interfere with T cell function, leading to the failure to produce strong cellular and humoral immune responses that can affect the efficacy of recruited T cells. One clinical trial’s data suggest that cytotoxic T lymphocyte-associated antigen-4 (CTLA-4)-positive Tregs are associated with diminished T cell antitumor activity [
70]. Yano et al. used ipilimumab, an antagonistic monoclonal antibody against CTLA-4, to enhance bispecific antibody (BiAb)-redirected antitumor cytotoxicity of activated T cells by inhibiting the immunosuppressive activity of regulatory T cells (Tregs). They found that the presence of ipilimumab not only enhanced the amplification of activated T cells, but also enhanced the secretion of cytokines and the specific cytotoxicity of T cells [
71].
The method of adoptive T cell therapy combined with bispecific antibodies can be applied to address these limitations. Recent studies have shown that activated T cells armed with bispecific antibodies have promising efficacy in clinical trials and can be adapted to restore the sensitivity toward bispecific antibodies aiming to recruit immune cells [
72]. Briefly, activated T cells were produced by activating PBMC with soluble anti-CD3 monoclonal antibodies and expanded by adding IL-2 for 14 days. After culturing, activated T cells were harvested, washed, counted and resuspended, and then, they were armed with bispecific antibodies at different ratios for one hour at 4 °C. These T cells are derived from normal donors or cancer patients [
73]. Arming in vitro expanded T cells with bispecific antibodies may not only improve clinical responses by bypassing the depression of the tumor microenvironment, but also minimize toxicity by avoiding the cytokine storm that can occur by systemic infusion of bispecific antibodies alone [
74]. A phase I clinical trial was conducted, which included 23 women with metastatic breast cancer. Each one received the administration consisting of eight infusions of anti-CD3 × anti-HER2 bispecific antibody (HER2Bi) armed with anti-CD3 activated T cells (ATC) in combination with low dose interleukin 2 (IL-2) and granulocyte macrophage colony stimulating factor (ClinicalTrials.gov: NCT01147016). The results showed that infusions of activated T cells armed with bispecific antibodies induced anti-tumor responses, and the Th1 cytokines and IL-12 serum levels increased [
74].
Katarzyna Urbanska et al. also designed a combination platform to overcome these shortcomings. This platform includes unique bispecific antibodies and engineered T cells. Bispecific antibodies are bound with folate receptors and tumor-associated antigens. T cells were engineered to express a novel chimeric receptor comprised of an extracellular folate receptor fused to intracellular TCR and CD28 costimulatory signaling domains. In this way, bispecific antibodies can stimulate the engineered T cells to kill tumor cells and avoid the influence of the suppressive tumor environment [
75]. An article published recently pointed out another way to integrate bispecific antibody into T cells. T cells were engineered with synthetic Notch (synNotch) receptors, which can induce transcriptional activation, and with the genes that express bispecific antibodies, like BiTEs. As a result, when T cells were activated by one antigen expressed on tumor cells, the synNotch signaling will induce the expression and secretion of BiTEs; BiTEs will then activate T cells in turn [
76].
3.2. Delivering Cytotoxic Entities to the Malignant Cells
Bispecific antibodies that can simultaneously bind to cell surface antigens and payloads are a very ideal delivery system for therapeutic use. Digoxigenin (Dig), as a hapten, can be used as the payload scaffold to embark on some cytotoxic moieties, such as fluorophores, chelating agents, chemotherapeutics, nucleic acids, lipids, nanoparticles or peptides and proteins, and finally, form compounds, like Dig-Cy5, Dig-doxorubicin and Dig-GFP [
77]. Bispecific antibodies that target the cell membrane antigens and digoxin (DIG) were produced for targeted payload delivery. Targeting antibodies can bind the tumor antigens, such as HER2. A Dig-binding single-chain Fv was fused to the C terminus of the CH3 domains of targeting antibodies in a disulfide-stabilized form. Because Dig-bispecific antibodies can also effectively capture digoxigeninylated compounds under physiological conditions, separate administration of uncharged Dig bispecific antibodies followed by application of Dig payload is sufficient to achieve antibody-mediated targeting in vitro and in vivo [
78].
Delivering siRNA into tumor cells and knocking down some vital genes is also a promising method for cancer therapy. Hapten-based bispecific antibody is also an effective siRNA delivery system [
79]. siRNA was digoxigeninylated at its 3′-end and formulated into nanoparticles consisting of dynamic polyconjugates (DPCs) or into lipid-based nanoparticles (LNPs). This Dig-siRNA was bound in a 2:1 ratio to the bispecific antibodies, which bind tumor antigens, such as HER2, IGF1-R, CD22 and LeY. These bispecific antibody-siRNA complexes delivered siRNAs specifically to cells expressing the corresponding antigen, then the complexes were internalized into endosomes, and Dig-siRNAs were separated from bispecific antibodies. An in vitro study showed that the complexes induced the knockdown of targeting mRNA by specific siRNAs for a variety of bsAbs, siRNAs and target cells. An in vivo study in mice bearing tumor xenografts showed that CD31 mRNA was significantly knocked down in endothelial cells after systemic co-administration of bispecific antibodies and LNPs containing siRNAs that were targeted to the CD31 of tumor vasculature [
80].
Diphtheria toxin (DT) mediates potent cell-cycle-independent cell death and, therefore, can be particularly effective as an alternative therapy for chemotherapy refractory malignancies. Vallera et al. fused bispecific scFvs targeting human CD19 and CD22 cell surface receptors with DT as a new therapy against CD22
+CD19
+ systemic B cell malignancy. This new agent and its variant version DT2219ARL resulted in long-term tumor-free survivors measured in a bioluminescent xenograft imaging model [
57]. Because of the great anti-cancer activity, DT2219 entered phase 1 clinical trial and showed safety and effectivity [
58] (ClinicalTrials.gov:NCT02370160, NCT00889408).
As radio immunotherapy (RIT) enables the delivery of tumoricidal radiation doses to multiple systemically-dispersed sites simultaneously, it is feasible for diseases that have already metastasized to multiple organs. For this purpose, bispecific antibodies that target tumor antigens, as well as compounds containing radioactive materials were generated. A single-chain antibody fragment against DOTA complexed with β particle-emitting radio metals, such as 177Lu and 90Y, was attached to a full humanized monoclonal antibody targeting a tumor antigen GD2 [
81]. In the same way, another bispecific antibody with dual specificity for GPA33 tumor antigen and the DOTA-Bn (radio lanthanide metal) complex was tested [
82]. All of these radio immunotherapy agents can effectively bring radioactive materials into the tumor site and promisingly ablate corresponding tumors in mice, while sparing kidney and bone marrow accumulation.
Bacterial minicells are anucleate nanoparticles produced as a result of inactivating the genes that control normal bacterial cell division, thereby derepressing polar sites of cell fission. The minicells are purified through the use of a process to remove pollutants, such as parent bacterial cells, cell debris, cellular components, free nucleic acids and free endotoxin. Then, chemotherapeutic drugs were packaged into minicells. Finally, minicells loaded with chemotherapeutic drugs were targeted via bispecific antibodies to antigens on the membrane of cancer cells, which results in endocytosis, intracellular degradation and drug release. This new delivery system generates a significant tumor growth inhibition and regression in mouse xenografts [
83]. Because of the great outcomes of preclinical studies, a phase I clinical study of bispecific antibody-mediated EGFR-targeted, paclitaxel-loaded minicells was initiated. This minicell delivery system was well tolerated and safe in patients and has shown modest clinical efficacy [
84].
3.6. Bispecific Antibodies Used Besides for Tumors
Emicizumab, a humanized bispecific antibody binding to both the activated coagulation factor IX and factor X, was developed to decrease the bleeding rate in hemophilia A patients. It bridges the activated factor IX (factor IXa) and factor X, hence facilitating the intact cascade reaction. A phase I clinical study was conducted in healthy adults and in patients by single subcutaneous infusion of emicizumab [
97]. The median annual bleeding rates were significantly reduced. Additionally, it shows a high subcutaneous bioavailability with a prolonged plasma half-life [
98].
Wnt signaling is critical to promote osteoblast genesis and bone formation that occur during growth, bone homoeostasis or fracture repair. Sclerostin and Dickkopf-1 (DKK-1) are secreted factors that block Wnt signaling. A bispecific IgG against DKK-1 and sclerostin is constructed to treat and prevent bone disease, offering a promising therapeutic approach to the low bone mass disorders like osteoporosis. This bispecific IgG finally showed excellent bone repairing activity compared with parent mAb combinations [
99].
Pseudomonas aeruginosa is a common opportunistic pathogen with high drug resistance and poor clinical prognosis. Two antigenic proteins, PcrV and Psl, have been shown to play an important role in acute and chronic
Pseudomonas aeruginosa infection. The patients who were infected generally lack pre-existing immunity and cannot initiate an effective humoral response to these two antigens [
100]. Therapeutic strategies against PcrV and Psl are a promising method for the prevention of bloodstream infections of
Pseudomonas aeruginosa. A new bispecific antibody targeting PcrV and Psl (MEDI3902) has been reported [
101]. Compared with the parent monoclonal antibody, MEDI3902 was proven to have synergistic protective activity in the mouse model, which was considered to stem from enhanced targeting of the anti-PcrV arm via binding to the abundant surface Psl. Due to the outstanding effect of the bispecific antibody in preclinical experiments, it is the first bispecific antibody to enter clinical testing against a bacterial pathogen. MEDI3902 is currently under evaluation for safety and pharmacokinetics in healthy adults [
101].
The blood-brain barrier (BBB) is the main obstruction hampering the treatment of monoclonal antibodies that have therapeutic potential for treating diseases of the central nervous system. A bispecific antibody was designed to bind transferrin receptor (TfR) combined with a binding site directed against a target BACE1 in the brain. BACE1 is an aspartyl protease responsible for the accumulation of amyloid-β (Aβ) peptides, which are associated with Alzheimer’s disease. Inhibition of BACE1 also provides an ideal signal for the activity of antibodies that cross the BBB. These bispecific antibodies with low affinity of binding to the TfRs can successfully cross the BBB and reduce brain amyloid-b (Ab) in mice, as well as nonhuman primates [
102].
Broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) have exhibited extraordinary potency. Nonhuman primate studies have shown that HIV envelope antibodies can prevent viral infection and control viremia; while because of the high mutant rate, co-administration of different bNAbs is needed to target distinct epitopes. Recently, there have been some bispecific antibodies with exquisite potency against HIV-1, which show great ability for HIV prevention and treatment [
103,
104].