Mesothelin (MSLN) is a 40-kDa glycosylphosphatidylinositol-anchored membrane glycoprotein that is normally expressed primarily on mesothelial cells lining the peritoneum, pericardium, and pleura [1
]. It is, however, significantly overexpressed in a number of malignancies, including mesothelioma, ovarian cancer, pancreatic cancer, head and neck cancer, cervical cancer, non-small cell lung cancer, and lung adenocarcinoma, in which it seems to be associated with aggressive phenotypes and a poor prognosis [2
]. MSLN overexpression in cancers enables tumor-specific targeting using monoclonal antibodies, as well as chimeric antigen receptor (CAR)-T cells containing single-chain variable domain fragments (scFvs) that bind to MSLN [3
]. Therefore, MSLN-targeted immunotherapies are being evaluated in phase I and/or phase II clinical trials [4
]. However, patients with malignant pleural mesothelioma or ovarian cancer require better systemic treatment, indicating a clear need for the development of novel modalities [5
Bispecific antibodies (bsAbs), which allow for dual targeting, have great potential as therapeutic strategies [7
]. Since the concept of bsAbs was originally described by Nisonoff and colleagues more than 50 years ago, technical innovations for generating bsAbs have progressed dramatically. To date, more than 85 bsAbs have been evaluated in clinical trials, and approximately half of all bsAb-related clinical studies have involved T-cell-engaging bsAbs [7
]. T-cell bsAbs recruit and engage T cells by binding to both CD3 of the T-cell receptor complex (TCR) and antigen on the target cell, resulting in target cell killing by T-cell proliferation and activation [8
]. In previous reports, T-cell bsAbs were constructed by combining several anti-CD3 antibodies that showed different affinities and epitopes to the T-cell receptor (TCR). A mucin core protein × CD3εΥ/δε (OKT3) bsAb was constructed for the treatment of bile duct carcinoma, and the antigen-specific cytotoxicity in vitro and inhibition of tumor growth in vivo were investigated [10
]. A HER2 × CD3ε (SP34) bsAb specifically killed HER2-expressing cancer cells by T-cell-killing activity and exhibited potent antitumor activity in animal models [11
]. In the BCMA × CD3δε (F2B) bsAb format, the anti-CD3δε arm showed low affinity and stimulated low levels of cytokine release, whereas the bsAb demonstrated robust antigen-specific tumor killing both in vitro and in vivo [12
]. However, bsAbs that target CD3 have potential safety concerns. Catumaxomab, the pioneering T-cell bsAb, provided important lessons regarding the clinical safety of CD3-targeting antibodies (Abs) [7
Blinatumomab is a CD19 × CD3ε T-cell bsAb that was approved for the treatment of relapsed/refractory B-cell acute lymphocytic leukemia in 2014. It contains two scFvs combined with a flexible linker [13
]. Although blinatumomab exhibits highly potent antitumor killing activity, its short serum half-life is a major drawback for clinical applications [8
], as it must be administered as a continuous intravenous infusion to achieve the desired trough concentrations. IgG-based T-cell bsAbs use a human Ig fragment-crystallized (Fc) region with diminished binding to Fc gamma receptors (FcγRs) to reduce immune effector functions, such as antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity. However, they maintain binding to neonatal Fc receptors (FcRns) to facilitate IgG recycling [14
Generation of bispecific heterodimeric/asymmetric IgG-based antibodies requires the use of several technologies to avoid the random association of heavy and light chains. Correct heavy chain heterodimerization is facilitated using the knob-into-hole (KiH) approach, and correct association of generic light chains is promoted using the common light chain approach or crossmab technology [16
]. These technologies allow for the construction of various bsAb IgG formats, including asymmetric heterodimeric bivalent 1 + 1 and trivalent 2 + 1 bispecific antibodies, as well as symmetric tetravalent 2 + 2 bispecific antibodies with different valencies [17
]. Trivalent 2 + 1 IgG antibodies can be generated by fusing a single antigen-binding fragment (Fab) or scFv to the N-terminus of the variable heavy chain (VH) or variable light chain (VL) domain, the C-terminus of the light chain, or the C-terminus of the Fc domain. Similarly, symmetric tetravalent bispecific 2 + 2 antibodies can be generated by fusing Fabs or scFvs via flexible linkers to the N-terminus of the VH or VL domain, the C-terminus of the VL domain, or the C-terminus of the Fc domain of an IgG molecule [17
]. In the case of T-cell-engaging bsAbs, a number of heterodimeric 1 + 1 or trivalent 2 + 1 IgG bsAbs have been created. For instance, REGN1979 and REGN4018 were generated by using a bivalent 1 + 1 IgG format, with each arm targeting CD20 × CD3 and Mucin 16 × CD3, respectively [20
]. RG7802, RG6026, EM801, and others were constructed as trivalent 2 + 1 IgG molecules [9
]. As these trivalent antibodies bind only monovalently to the CD3 of TCR chains, TCRs only become cross-linked and activated during concomitant binding of two tumor antigens, resulting in T-cell activation and tumor antigen-dependent T-cell killing of the target cell [9
Schanzer and colleagues constructed bsAbs using a single-chain Fab-fragment (scFab) to prevent mispairing of light chains with heavy chains [23
]. One binding arm was based on scFab, with the light chain attached to the N-terminus of the VH domain by a 32-amino acid (G4
GG linker to form the heavy chain. Dimerization of the two different heavy chains was facilitated by the KiH technology. Upon transient expression, purification yields of these bsAbs were comparable to those of conventional IgG, and the antibodies also exhibited a high level of purity. Thus, the use of two technologies to develop these bsAbs did not affect expression yields or purity.
In this article, we describe the use of scFab and KiH technology to develop two novel IgG-based bsAbs against MSLN and CD3ε for use as T-cell immunotherapy. These bsAbs had a 1 + 1 or 2 + 1 format. Their antitumor efficacy for MSLN-positive solid tumors was evaluated both in vitro and in vivo.
2. Materials and Methods
2.1. Construction of the Antibody Library and Selection of Antibodies Binding to rhMSLN
Mouse immunization was performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Green Cross Corporation (#GC-18-014A). Five Balb/C mice were immunized with an initial injection and three booster injections of recombinant human MSLN (rhMSLN; Acro Biosystems, Newark, DE, USA). After the final injection, the animals were euthanized, and total RNA was prepared from spleen tissues. Complementary DNA (cDNA) was synthesized using the ImProm-II reverse transcription system kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions. A phage-displayed mouse/human chimeric Fab Ab library was constructed using the pComb3XTT phagemid vector system, as previously described [24
]. Fab clones were selected from the library through four rounds of biopanning, as previously reported [24
]. For each round of biopanning, we used 96-well plates coated with 300 ng recombinant human (rh)MSLN protein. After the final round of biopanning, individual phage clones displaying Fab were generated from colonies grown on output plates and tested for reactivity to rhMSLN by using the phage enzyme immunoassay, as previously described [24
]. The selected mouse anti-human MSLN antibody was humanized by CDR-grafting methods, based on a previous study [25
]. After searching for VH and VL framework sequences in the ImMunoGeneTics database of human germline sequences, IGHV1-3*01 and IGKV1-12*01 were selected as framework templates. Production and purification of anti-MSLN (HMI323) IgG1
was performed as previously reported [26
2.2. Generation and Production of anti-CD3ε Antibody
The mouse anti-human CD3ε antibody SP34 (US2015/016661A1) was humanized by CDR-grafting methods, based on previous reports [27
]. We searched for homologous human Abs by using the BLAST sequence program to identify VH and VL framework sequences to use as templates; IGHV3-23*01 and IGLV7-46*01 were selected. The genes for the humanized anti-CD3 antibody were constructed with whole synthesized genes, which were then inserted into the pCI mammalian expression vector (Promega). Production and purification of anti-CD3ε (A15) IgG1
were performed as previously described [26
2.3. Enzyme-linked Immunosorbent Assay (ELISA)
A total of 250 ng rhMSLN or rhCD3ε (Acro Biosystems) was dissolved in 50 μL phosphate buffered saline (PBS) and added to the wells of a microtiter plate. After incubation overnight at 4 °C and washing three times with PBS containing 0.05% (v/v) Tween 20 (PBST), the microtiter plate was incubated for 1 h at 37 °C with 1% (w/v) bovine serum albumin (BSA) in PBS. After washing with PBST, the plate was incubated with 2-fold serially diluted HMI323 IgG1 or A15 IgG1 protein for 1 h at 37 °C, and then washed three times with PBST. The plate was incubated with anti-human Fab Ab, which was conjugated to horseradish peroxidase (HRP; Sigma, St. Louis, MO, USA) and diluted 5000-fold in PBS. After washing with PBST, 50 μL 3,3,5,5-tetramethylbenzidine substrate solution (Sera Care, Milford, MA, USA) and stop solution (Sera Care) were sequentially added to each well. Optical density was measured at 450 nm using a microtiter plate reader. All tests were conducted in duplicate.
2.4. Construction of the Fully Humanized Bispecific Expression Vector
A heterodimeric KiH Fc fragment vector of the human IgG1
isotype was constructed as previously described [28
]. The construct consisted of two scFabs reactive against MSLN and CD3ε. The heterodimeric 1 + 1 IgG design was monovalent for MSLN and CD3ε, whereas the trivalent 2 + 1 IgG was monovalent for CD3ε and bivalent for MSLN, with one scFab fragment fused to the N-terminus of the CD3ε-specific scFab via (G4
2.5. Production and Purification of Bispecific Antibodies
To prepare the 1 + 1 and 2 + 1 bsAb constructs, Expi293 cells were transfected with two expression vectors (knob and hole vectors) using an optimal expression vector ratio. The bsAbs were produced using the Expi293 Expression system (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) [29
]. Briefly, plasmid DNA and ExpiFectamin 293 reagent were mixed with Opti-MEM medium and incubated at room temperature for 30 min. The solution was then added to Expi293 cells cultured in Expi293 expression medium and incubated in a shaker incubator at 37 °C with a humidified atmosphere of 8% CO2
in air. After incubation for 18 h, ExpiFectamin 293 Transfection Enhancers 1 and 2 were added to each flask, and the transfected cells were incubated under the same conditions for 5 days.
Each bsAb was purified from the cell culture supernatant by affinity chromatography and size exclusion chromatography using PrismA resin and Superdex200 column (GE Healthcare, Chicago, IL, USA), respectively. SDS-PAGE was performed to confirm the size and purity of each bsAb.
2.6. Flow Cytometric Analysis
To perform flow cytometry, we used moderate MSLN-expressing human pancreas adenocarcinoma (AsPC-1), high MSLN-expressing human lung squamous carcinoma (H226), and human T lymphocyte (Jurkat) cell lines. A total of 5 × 105 cells were placed in suspension buffer (2% fetal bovine serum (FBS) containing PBS) and incubated with 5-fold serially diluted heterodimeric 1 + 1 MG1122-A bsAb or trivalent 2 + 1 MG1122-B bsAb for 1 h at 4 °C. After washing with suspension buffer, the cells were incubated for 30 min on ice with phycoerythrin-labeled antibodies against human IgG (Sigma). The cells were subsequently washed, resuspended in 200 μL suspension buffer, and subjected to flow cytometric analysis using the LSRFortesssa cell analyzer (BD Biosciences, San Jose, CA, USA).
2.7. Affinity Evaluation and Dual Binding Assay
Affinity and dual binding activity were evaluated using the Octet system (ForteBio, Fremont, CA, USA) in 96-well microplates at 25 °C, as previously described [30
]. Briefly, assays were performed by placing aminopropylsilane (APS) Biosensors (ForteBio) nickel-nitrilotriacetic acid (Ni-NTA) or anti-hIgG Fc capture (AHC) in the wells, followed by rinsing in PBS for 60 s, which served as the baseline. The sensors were then immobilized for 300 s with 200 μL rhMSLN or rhCD3ε (20 μg/mL) as antigens and subsequently washed in kinetic buffer (1 mM phosphate, 15 mM NaCl, 0.1 mg/mL BSA, 0.002% Tween-20) for 300 s. The antigen-captured sensors were then submerged in wells containing different concentrations of anti-MSLN (HMI323) IgG1
or anti-CD3ε (A15) IgG1
monoclonal antibodies as well as MG1122-A or MG1122-B bsAbs for 300 s, followed by 600 s of dissociation in kinetic buffer. Between assays, the sensors were regenerated with 10 mM glycine, pH 1.75. ForteBio Octet analysis software was used to generate the sensorgram and determine the association rate constant (ka
) and accuracy of the analysis. To analyze the simultaneous binding of the MG1122-A bsAb or MG1122-B bsAb to MSLN and to CD3ε, the antigen-captured sensors were submerged in wells containing 100 nM bsAbs for 300 s, followed by 300–500 s of dissociation in kinetic buffer. Then, the antigen-bsAb-captured sensors were submerged in wells containing 400 nM second antigens for 300 s. The sensors were regenerated as described above.
2.8. T-cell Activation Assay
T-cell activation assays were performed using a Promega kit, following the manufacturer’s protocol. Briefly, 3 × 104 AsPC-1 or H226 cancer cells were seeded into 96-well plates in Roswell Park Memorial Institute (RPMI)-1640 media with 10% FBS. After incubation overnight, the medium was aspirated, and bsAbs and 1 × 105 TCR/CD3 effector cells (NFAT) (Promega) were added. T-cell activation was assessed after 5 h of incubation at 37 °C in 5% CO2 atmosphere. Following mixing with Bio-Glo Reagent (Promega), luminescence was measured with a luminescence plate reader (Berthold Technologies, Bad Wildbad, Germany).
2.9. Preparation of Peripheral Blood Mononuclear Cells
Leukapheresis products were collected from healthy donors at Samsung Medical Center (SMC) after approval from the SMC institutional review board (No. 2018-01-089). Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation on a Ficoll density gradient (GE Healthcare) and stored in liquid nitrogen until use.
2.10. In Vitro bsAb/hPBMC Cytotoxicity Assay
AsPC-1 or H226 adherent target cells (3 × 104 cells) were transduced with red fluorescent protein using the Incucyte Red NucLight Lentivirus Reagent (Essen BioScience, Ann Arbor, MI, USA). After 24 h, the medium was replaced with fresh growth medium. Puromycin antibiotic was added to obtain a stable, homogenous cell population expressing nucleus-restricted red fluorescent protein. Selected AsPC-1 or H226 cells (1 × 104 cells) were seeded onto 96-well flat-bottom plates. After 24 h, various concentrations of bsAbs (MG1122-A or MG1122-B) or control molecules and human PBMC effector cells were added to the plates at an effector:target ratio of 10:1. All experiments were performed in triplicate. Target cell killing was assessed using the IncuCyte live-cell analysis system after 24 and 48 h of incubation at 37 °C in a 5% CO2 atmosphere. The percentage of specific cell lysis was calculated as follows: (effector cells red intensity [RCU × μm2/image] when co-cultured with target cells and agent/effector cells red intensity [RCU × μm2/image] when co-cultured with only target cells) × 100%. EC50 values were calculated using GraphPad Prism5 (GraphPad software, San Diego, CA, USA).
2.11. In Vivo Study Using a Tumor Xenograft Mouse Model
All NOG (NOD/Shi-scid
) mice studies were approved by the Institutional Animal Care and Use Committee of the GC Pharma (No. GC-17-008A). The xenograft mouse study was performed as previously reported [9
]. Female NOG mice (6-8 weeks old; CIEA Japan Inc., Kanagawa, Japan) were subcutaneously injected with AsPC-1 (5 × 106
cells) or H226 (1 × 107
cells) cells. Human T cells were selectively amplified by culturing human PBMCs using Dynabead Human T-Activator CD3/CD28 (Life Technologies, Carlsbad, CA, USA), after which they were injected intraperitoneally into mice as effector cells (effector:target ratio, 2:1) at 5 days after tumor cell implantation. MG1122-A (3 mg/kg, n
= 5), MG1122-B (3 mg/kg, n
= 5), or vehicle (PBS, n
= 5) was administered intraperitoneally 2 days after T-cell transfer and then daily over the next 3–4 days for a total of four injections. Twice per week, the length and width of each tumor were measured with calipers in two perpendicular dimensions, and the tumor volume was calculated using this formula: (width2
× length)/2. Clinical signs and body weight were also assessed twice per week.
2.12. Histologic Analysis
Tumor tissue samples were collected 1 week after treatment with bsAbs or PBS. Immunohistochemistry methods were based on previous reports [9
]. Samples were fixed with formalin and then embedded in paraffin blocks, which were cut into 4-μm sections. Following deparaffinization, the sections underwent heat antigen retrieval and were then stained with human CD3 (anti-CD3, Abcam, Cambridge, UK) or CD8 (anti-CD8, Abcam) using VECTASTAIN Elite ABC kits (VECTOR Lab, Burlingame, CA, USA). The tissues were subsequently counterstained with Mayer’s hematoxylin (Dako, Kyoto, Japan) and examined using an Olympus BX51 microscope (Olympus, Tokyo, Japan).
2.13. Bispecific Antibodies Pharmacokinetics in Nude Mice
All research procedures involving nude mice were approved by the Institutional Animal Care and Use Committee of GC Pharma (No. GC-17-008A). The pharmacokinetics study was performed as previously described [34
]. Briefly, 3 mg/kg MG1122-A or MG1122-B was injected through a tail vein of 6- to 8-week-old nude mice (Charles River Japan Lab, Kanagawa, Japan). Blood was then drawn from an intraorbital vein at set times ranging from 5 min to 672 h after injection of the bsAbs. Serum samples were stored at −80 °C. Serum MG1122-A concentrations were measured by sandwich ELISA using CD3ε and biotinylated MSLN (R&D systems, Minneapolis, MN, USA). Serum MG1122-B concentrations were detected using anti-human Fab antibody (Sigma) and HRP-conjugated anti-human Fc antibody (Sigma).
2.14. Statistical Analyses
Continuous variables were compared using two-way analysis of variance, with p < 0.01 representing a statistically significant difference between groups. GraphPad Prism (version 5.0) software was used for all statistical analyses.
The development of bsAbs has received considerable attention in the past 20 years [35
]. For treatment in oncology, one bsAb has received marketing approval, and more than 50 other bsAbs are undergoing clinical trials. These bsAbs have varying formats and mechanisms of action. Several previous studies presented evidence that the bsAb structure contributes significantly to the activity of these molecules. Most bsAbs recruit immune cells to kill tumor cells.
In the research described in this report, we generated two novel T-cell-engaging bsAbs for targeting MSLN-expressing solid tumors, using scFab and KiH technologies to prevent random association of heavy and light chains. MG1122-A is a heterodimeric 1 + 1 bispecific IgG molecule, with each antigen-binding fragment (scFab) targeting MSLN and CD3ε. MG1122-B is an asymmetric 2 + 1 bispecific antibody, which was constructed by head-to-tail fusion of the MSLN and CD3ε-binding Fab domains via a flexible linker. MG1122-B exhibits bivalent binding to MSLN and monovalent binding to CD3ε. The use of a bivalent tumor-targeting arm may enhance potency and tumor selectivity through enhanced binding avidity [37
]. Three 2 + 1 bsAbs—anti-CEA/CD3 (RG7802), anti-CD20/CD3 (RG6026), and anti-BCMA/CD3 (EM801)—were previously generated using crossmab and KiH technologies [9
]. They bind monovalently to CD3 with low affinity and bivalently to target cells with higher avidity to facilitate preferential target cell binding. Similarly, the bivalency of MG1122-B for tumor antigen promotes high binding avidity toward tumor cells, and its tumor targeting was superior to that of MG1122-A, which binds monovalently to MSLN. MG1122-B also exhibited better binding to tumor cells with high or moderate expression. The high-avidity binding to MSLN led to an enhanced selective killing of both moderate MSLN-expressing and high MSLN-expressing tumor cells both in vitro and in vivo. These findings suggest that MG1122-B could be useful for treating a wide variety of MSLN-expressing cancers. In contrast, MG1122-A activity was more dependent on MSLN expression, with higher in vivo potency observed in high MSLN-expressing tumors than in moderate MSLN-expressing tumors.
The administration of anti-CD3 antibody may produce acute systemic toxicity in both humans and mice; this has been attributed to cytokine release following antibody-induced T-cell activation [38
]. Several studies have demonstrated that the affinity of anti-CD3 antibodies dramatically affects the biodistribution of bsAbs [7
]. Achieving optimal T-cell cytotoxicity requires optimal kinetics for CD3 binding [39
], and high-affinity anti-CD3 molecules do not necessarily lead to enhanced T-cell activation. Lower-affinity anti-CD3 antibodies are preferable to facilitate good tumor distribution in vivo, without CD3-mediated plasma clearance or binding of the antibody within T-cell-containing tissues, such as the spleen and lymph nodes [40
]. In previous reports, anti-CD3 antibodies have exhibited affinities ranging from 1 to 200 nM, as demonstrated by surface plasmon resonance. For instance, the anti-CD3 affinities of blinatumomab and AFM11 were 100 and 70 nM, respectively [42
]. Head-to-tail geometry in a 2 + 1 format, in which the CD3ε binding region is introduced in an “inside” position, may increase the biodistribution of bsAbs by reducing CD3 affinity. For example, the anti-CD3 affinities of the head-to-tail 2 + 1 bsAbs RG7802 and EM801 were 100 nM and 70–100 nM, respectively [9
]. Both RG7802 and EM801 produce minimal nonspecific T-cell activation in the absence of target cells. In clinical trials, anti-CEA/CD3 (RG7802) and EM801 exhibited manageable toxicities [22
]. MG1122-A was generated by combining anti-MSLN scFab on the knob arm and anti-CD3 scFab on the hole arm. MG1122-B was constructed by fusing another anti-MSLN scFab to the N-terminus of anti-CD3 scFab via (G4
linkers. The CD3ε-binding region of MG1122-B was also introduced in an “inside” position, reducing the affinity to the CD3-binding arm compared with the affinity of MG1122-A. Therefore, MG1122-B may be less likely to bind to nonspecific T cells, leading to less systemic toxicity by avoiding the undesired activation of T cells in peripheral blood and less trapping of the antibody in the spleen or lymph nodes. However, when MG1122B reaches the target cells, its efficacy might be attributed to flexible tumor-targeting scFab arms via a flexible linker. This linker may allow for increased binding chances between the antibody and tumor and enhanced affinity to a potent CD3-targeting arm that can bind and activate T cells more easily at the tumor site, because of conformational changes in the tumor-targeting scFab upon binding.
MSLN is an attractive target for cancer therapy, and a growing number of clinical trials are currently exploring diverse MSLN-targeted strategies, including the use of immunotoxin, antibody drug conjugates, monoclonal antibodies, and CAR-T cells. The anti-MSLN monoclonal antibody amatuximab (also called MORAB-009) binds to MSLN and induces antibody-dependent, cell-mediated cytotoxicity. Phase II clinical trials have been conducted with amatuximab treatment alone or in combination with pemetrexed and cisplatin [44
]. Phase I studies with immunotoxin SS1P or the antibody drug-conjugate BAY 94-9343 have also been performed. Immunotoxin SS1P is limited by the development of neutralizing antibodies specific for the toxin portion and possibly also for the chimeric SS1 antibody [4
]. MG1122-A and MG1122-B are expected to have less immunogenicity, because humanized antibodies are associated with a lower risk of immune responses in humans than chimeric antibodies [45
]. Several phase I clinical trials have been initiated to determine the safety and maximally tolerated dose of MSLN CAR-T-cell therapy, but on-target/off-tumor toxicity is a concern. Moreover, the solid-tumor microenvironment provides several obstacles for MSLN CAR-T-cell therapy, and MSLN CAR-T cells must overcome immune barriers to infiltrate tumors. Our in vivo results demonstrating antitumor efficacy through T-cell infiltration of the solid-tumor microenvironment suggest that MG1122-A and MG112-B may be superior to CAR-T therapy for solid tumors. Other strategies have involved the production of bsAbs targeting MSLN and engaging other types of immune cells. For example, Bano and colleagues generated a Fab-like bsAb targeting MSLN and FCγRIII (CD16) that is expressed by natural killer cells [1
]. Similarly, Ye and colleagues constructed bsAbs containing single-chain Fv domains against MSLN and CD40 [46
]; to avoid systemic toxicity, their bsAbs had agonistic anti-CD40 activity. Notably, by limiting the antigen sink effect of CD3 in the periphery, MG1122-B was designed as an MSLN-targeting, T-cell-engaging bsAb with low systemic toxicity.
Recently, resistance to bsAb treatment has been observed in clinical trials [7
]. Downregulation of bsAb-specific, tumor-associated antigens on tumor cells is one potential mechanism of tumor escape, whereas other mechanisms may involve immune suppression by regulatory T cells or immune checkpoint molecules. For example, programmed cell death 1 (PD-1) and programmed cell death 1 ligand 1 expression, which may be induced by bsAbs, may limit the activity of T-cell bsAbs. Accordingly, combining a T-cell bsAb with a PD-1 antibody has been shown to enhance antitumor activity [7
]. Hence, combining MG1122-B with an immune checkpoint inhibitor may be a useful strategy for improving antitumor efficacy.