CLDN18.2 and 4-1BB bispecific antibody givastomig exerts antitumor activity through CLDN18.2-expressing tumor-directed T-cell activation.

BACKGROUND: Claudin18.2 (CLDN18.2) is a tight junction protein that has been identified as a clinically proven target in gastric cancer. Stimulation of 4-1BB with agonistic antibodies is also a promising strategy for immunotherapy and 4-1BB+ T cells were reported to be present within the tumor microenvironment of patients with gastric cancer. However, hepatotoxicity-mediated by 4-1BB activation was observed in clinical trials of agonistic anti-4-1BB monoclonal antibodies. METHODS: To specifically activate the 4-1BB+ T cells in tumor and avoid the on-target liver toxicity, we developed a novel CLDN18.2×4-1BB bispecific antibody (termed 'givastomig' or 'ABL111'; also known as TJ-CD4B or TJ033721) that was designed to activate 4-1BB signaling in a CLDN18.2 engagement-dependent manner. RESULTS: 4-1BB+ T cells were observed to be coexisted with CLDN18.2+ tumor cells in proximity by multiplex immunohistochemical staining of tumor tissues from patients with gastric cancer (n=60). Givastomig/ABL111 could bind to cell lines expressing various levels of CLDN18.2 with a high affinity and induce 4-1BB activation in vitro only in the context of CLDN18.2 binding. The magnitude of T-cell activation by givastomig/ABL111 treatment was closely correlated with the CLDN18.2 expression level of tumor cells from gastric cancer patient-derived xenograft model. Mechanistically, givastomig/ABL111 treatment could upregulate the expression of a panel of pro-inflammatory and interferon-γ-responsive genes in human peripheral blood mononuclear cells when co-cultured with CLDN18.2+ tumor cells. Furthermore, in humanized 4-1BB transgenic mice inoculated with human CLDN18.2-expressing tumor cells, givastomig/ABL111 induced a localized immune activation in tumor as evident by the increased ratio of CD8+/regulatory T cell, leading to the superior antitumor activity and long-lasting memory response against tumor rechallenge. Givastomig/ABL111 was well tolerated, with no systemic immune response and hepatotoxicity in monkeys. CONCLUSIONS: Givastomig/ABL111 is a novel CLDN18.2×4-1BB bispecific antibody which has the potential to treat patients with gastric cancer with a wide range of CLDN18.2 expression level through the restricted activation of 4-1BB+ T cells in tumor microenvironment to avoid the risk of liver toxicity and systemic immune response.

Grabody B, an IGF1 receptor-based shuttle, mediates efficient delivery of biologics across the blood-brain barrier.

Effective delivery of therapeutics to the brain is challenging. Molecular shuttles use receptors expressed on brain endothelial cells to deliver therapeutics. Antibodies targeting transferrin receptor (TfR) have been widely developed as molecular shuttles. However, the TfR-based approach raises concerns about safety and developmental burden. Here, we report insulin-like growth factor 1 receptor (IGF1R) as an ideal target for the molecular shuttle. We also describe Grabody B, an antibody against IGF1R, as a molecular shuttle. Grabody B has broad cross-species reactivity and does not interfere with IGF1R-mediated signaling. We demonstrate that administration of Grabody B-fused anti-alpha-synuclein (α-Syn) antibody induces better improvement in neuropathology and behavior in a Parkinson's disease animal model than the therapeutic antibody alone due to its superior serum pharmacokinetics and enhanced brain exposure. The results indicate that IGF1R is an ideal shuttle target and Grabody B is a safe and efficient molecular shuttle.

LAG-3xPD-L1 bispecific antibody potentiates antitumor responses of T cells through dendritic cell activation.

Several preclinical studies demonstrate that antitumor efficacy of programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade can be improved by combination with other checkpoint inhibitors. Lymphocyte-activation gene 3 (LAG-3) is an inhibitory checkpoint receptor involved in T cell exhaustion and tumor immune escape. Here, we describe ABL501, a bispecific antibody targeting LAG-3 and PD-L1 in modulating immune cell responses against tumors. ABL501 that efficiently inhibits both LAG-3 and PD-L1 pathways enhances the activation of effector CD4+ and CD8+ T cells with a higher degree than a combination of single anti-LAG-3 and anti-PD-L1. The augmented effector T cell responses by ABL501 resulted in mitigating regulatory-T-cell-mediated immunosuppression. Mechanistically, the simultaneous binding of ABL501 to LAG-3 and PD-L1 promotes dendritic cell (DC) activation and tumor cell conjugation with T cells that subsequently mounts effective CD8+ T cell responses. ABL501 demonstrates its potent in vivo antitumor efficacy in a humanized xenograft model and with knockin mice expressing human orthologs. The immune profiling analysis of peripheral blood reveals an increased abundance of LAG-3hiPD-1hi memory CD4+ T cell subset in relapsed cholangiocarcinoma patients after gemcitabine plus cisplatin therapy, which are more responsive to ABL501. This study supports the clinical evaluation of ABL501 as a novel cancer immunotherapeutic, and a first-in-human trial has started (NCT05101109).

Novel anti-4-1BB×PD-L1 bispecific antibody augments anti-tumor immunity through tumor-directed T-cell activation and checkpoint blockade.

BACKGROUND: Stimulation of 4-1BB with agonistic antibodies is a promising strategy for improving the therapeutic efficacy of immune checkpoint inhibitors (ICIs) or for overcoming resistance to ICIs. However, dose-dependent hepatotoxicity was observed in clinical trials with monoclonal anti-4-1BB agonistic antibodies due to the activation of 4-1BB signaling in liver resident Kupffer cells. METHODS: To avoid this on-target liver toxicity, we developed a novel bispecific antibody (4-1BB×PD-L1 bispecific antibody, termed "ABL503") uniquely designed to activate 4-1BB signaling only in the context of PD-L1, while also blocking PD-1/PD-L1 signaling. RESULTS: Functional evaluation using effector cells expressing both 4-1BB and PD-1 revealed superior biological activity of ABL503 compared with the combination of each monoclonal antibody. ABL503 also augmented T-cell activation in in vitro assays and further enhanced the anti-PD-L1-mediated reinvigoration of tumor-infiltrating CD8+ T cells from patients with cancer. Furthermore, in humanized PD-L1/4-1BB transgenic mice challenged with huPD-L1-expressing tumor cells, ABL503 induced superior anti-tumor activity and maintained an anti-tumor response against tumor rechallenge. ABL503 was well tolerated, with normal liver function in monkeys. CONCLUSION: The novel anti-4-1BB×PD-L1 bispecific antibody may exert a strong anti-tumor therapeutic efficacy with a low risk of liver toxicity through the restriction of 4-1BB stimulation in tumors.

Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis.

Several angiogenesis inhibitors targeting the vascular endothelial growth factor (VEGF) signaling pathway have been approved for cancer treatment. However, VEGF inhibitors alone were shown to promote tumor invasion and metastasis by increasing intratumoral hypoxia in some preclinical and clinical studies. Emerging reports suggest that Delta-like ligand 4 (Dll4) is a promising target of angiogenesis inhibition to augment the effects of VEGF inhibitors. To evaluate the effects of simultaneous blockade against VEGF and Dll4, we developed a bispecific antibody, HD105, targeting VEGF and Dll4. The HD105 bispecific antibody, which is composed of an anti-VEGF antibody (bevacizumab-similar) backbone C-terminally linked with a Dll4-targeting single-chain variable fragment, showed potent binding affinities against VEGF (KD: 1.3 nM) and Dll4 (KD: 30 nM). In addition, the HD105 bispecific antibody competitively inhibited the binding of ligands to their receptors, i.e., VEGF to VEGFR2 (EC50: 2.84 ± 0.41 nM) and Dll4 to Notch1 (EC50: 1.14 ± 0.06 nM). Using in vitro cell-based assays, we found that HD105 effectively blocked both the VEGF/VEGFR2 and Dll4/Notch1 signaling pathways in endothelial cells, resulting in a conspicuous inhibition of endothelial cell proliferation and sprouting. HD105 also suppressed Dll4-induced Notch1-dependent activation of the luciferase gene. In vivo xenograft studies demonstrated that HD105 more efficiently inhibited the tumor progression of human A549 lung and SCH gastric cancers than an anti-VEGF antibody or anti-Dll4 antibody alone. In conclusion, HD105 may be a novel therapeutic bispecific antibody for cancer treatment.

Development of antibody-based c-Met inhibitors for targeted cancer therapy.

Signaling pathways mediated by receptor tyrosine kinases (RTKs) and their ligands play important roles in the development and progression of human cancers, which makes RTK-mediated signaling pathways promising therapeutic targets in the treatment of cancer. Compared with small-molecule compounds, antibody-based therapeutics can more specifically recognize and bind to ligands and RTKs. Several antibody inhibitors of RTK-mediated signaling pathways, such as human epidermal growth factor receptor 2, vascular endothelial growth factor, epidermal growth factor receptor or vascular endothelial growth factor receptor 2, have been developed and are widely used to treat cancer patients. However, since the therapeutic options are still limited in terms of therapeutic efficacy and types of cancers that can be treated, efforts are being made to identify and evaluate novel RTK-mediated signaling pathways as targets for more efficacious cancer treatment. The hepatocyte growth factor/c-Met signaling pathway has come into the spotlight as a promising target for development of potent cancer therapeutic agents. Multiple antibody-based therapeutics targeting hepatocyte growth factor or c-Met are currently in preclinical or clinical development. This review focuses on the development of inhibitors of the hepatocyte growth factor/c-Met signaling pathway for cancer treatment, including critical issues in clinical development and future perspectives for antibody-based therapeutics.

Enhancement of the tumor penetration of monoclonal antibody by fusion of a neuropilin-targeting peptide improves the antitumor efficacy.

The limited localization and penetration of monoclonal antibodies (mAb) into solid tumors restricts their antitumor efficacy. Here, we describe a solid tumor-targeting antibody with enhanced tumor penetration activity. We designed a 22-residue peptide (A22p), which was extracted from the C-terminal basic region of semaphorin 3A (Sema3A) but modified to have higher affinity with neuropilin receptors (NRP), and genetically fused it to the C-terminus of Fc of human immunoglobulin G1 via a 15-residue (G4S)3 linker, generating Fc-A22p, for the bivalent binding to NRPs. In contrast to Fc or the monovalent A22p peptide alone, Fc-A22p homed to tumor vessels and induced vascular permeability through VE-cadherin downregulation and penetrated tumor tissues by interacting with NRPs in mice bearing human tumor xenografts. We extended the Fc-A22p platform by generating mAb-A22p antibodies of two clinically approved solid tumor-targeting mAbs, the anti-EGF receptor mAb cetuximab (erbitux), and the anti-Her2 mAb trastuzumab (herceptin). The mAb-A22p antibodies retained the intrinsic antigen binding, natural Fc-like biophysical properties, and productivity in mammalian cell cultures, comparable with those of the parent mAbs. In mouse xenograft tumor models, the mAb-A22p antibodies more efficiently homed to tumor vessels and spread into the extravascular tumor parenchyma, which significantly enhanced antitumor efficacy compared with the parent mAbs. Our results suggest that mAb-A22p is a superior format for solid tumor-targeting antibodies due to its enhanced tumor tissue penetration and greater antitumor efficacy compared with conventional mAbs.

Valproic acid sensitizes TRAIL-resistant anaplastic thyroid carcinoma cells to apoptotic cell death.

BACKGROUND: Anaplastic thyroid carcinoma (ATC) is an aggressive human tumor associated with a median survival of 2-6 months. TRAIL, as a ligand of death receptors, is known to induce apoptotic cell death in several cancer cells. However, TRAIL treatment alone is not effective against TRAIL-resistant cancer cells. This study was designed to investigate whether valproic acid (VPA) enhances apoptotic cell death of TRAIL-resistant ATC cells and to identify the mechanism of cell death of ATC cells by combination treatment with VPA and TRAIL. METHODS: To evaluate the cytotoxic effect of TRAIL and/or VPA on ATC cells, we used the MTT assay. The effects of VPA and TRAIL on apoptosis were assessed using FACS analysis (Annexin-V/PI stain) and Western blotting. RESULTS: The combination of VPA with TRAIL significantly induced apoptotic cell death compared with 8505C and ARO cells treated with TRAIL alone. The protein levels of cleaved caspase-8, -3, and PARP were increased in VPA and TRAIL co-treated ARO cells. The combination induced the activation of JNK and the phosphorylation of FADD and c-Jun but not p38. However, pretreatment with caspase inhibitors reduced the expression of cleaved caspase-8, -3, and PARP in co-treated ARO cells. SP600125 remarkably reduced the expression of cleaved caspase-8, -3, and PARP and the phosphorylation of FADD and c-Jun, as well as apoptotic cell death. CONCLUSIONS: VPA sensitized TRAIL-resistant ATC cells to apoptotic cell death through involvement of the JNK pathway. Thus, the combination of VPA and TRAIL may be a promising therapy for ATC.

The proteasome inhibitor MG132 potentiates TRAIL receptor agonist-induced apoptosis by stabilizing tBid and Bik in human head and neck squamous cell carcinoma cells.

Head and neck squamous cell carcinoma (HNSCC) is often resistant to conventional chemotherapy and thus requires novel treatment regimens. Here, we investigated the effects of the proteasome inhibitor MG132 in combination with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or agonistic TRAIL receptor 1 (DR4)-specific monoclonal antibody, AY4, on sensitization of TRAIL- and AY4-resistant human HNSCC cell lines. Combination treatment of HNSCC cells synergistically induced apoptotic cell death accompanied by caspase-8, caspase-9, and caspase-3 activation and Bid cleavage into truncated Bid (tBid). Generation and accumulation of tBid through the cooperative action of MG132 with TRAIL or AY4 and Bik accumulation through MG132-mediated proteasome inhibition are critical to the synergistic apoptosis. In HNSCC cells, Bak was constrained by Mcl-1 and Bcl-X(L), but not by Bcl-2. Conversely, Bax did not interact with Mcl-1, Bcl-X(L), or Bcl-2. Importantly, tBid plays a major role in Bax activation, and Bik indirectly activates Bak by displacing it from Mcl-1 and Bcl-X(L), pointing to the synergistic mechanism of the combination treatment. In addition, knockdown of both Mcl-1 and Bcl-X(L) significantly sensitized HNSCC cells to TRAIL and AY4 as a single agent, suggesting that Bak constraint by Mcl-1 and Bcl-X(L) is an important resistance mechanism of TRAIL receptor-mediated apoptotic cell death. Our results provide a novel molecular mechanism for the potent synergy between MG132 proteasome inhibitor and TRAIL receptor agonists in HNSCC cells, suggesting that the combination of these agents may offer a new therapeutic strategy for HNSCC treatment.

An agonistic antibody to human death receptor 4 induces apoptotic cell death in head and neck cancer cells through mitochondrial ROS generation.

The proapoptotic death receptor 4 (DR4), along with DR5, is currently regarded as a promising target for development of agonistic anti-cancer agents due to its tumor-selective apoptosis-inducing ability with no significant cytotoxicity to normal cells. In this study, we examine susceptibility of various head and neck cancer (HNC) cells and mechanism of cell death to an anti-DR4 agonistic monoclonal antibody (mAb), AY4. AY4 as a single agent induced caspase-dependent apoptotic cell death of KB and HN9, but not in SNU899 and FaDu cell lines. AY4 treatment resulted in accumulation of intracellular reactive oxygen species (ROS) generated from mitochondria in AY4-sensitive cells. Blockade of ROS production by N-acetyl-l-cysteine (NAC) resulted in protection of AY4-sensitive cells against AY4-induced apoptosis. ROS generation induced by AY4 treatment triggered down-regulation of anti-apoptotic molecules of Bcl-xL and X-linked inhibitor of apoptosis (XIAP) without affecting the expression levels of DR4, Mcl-1, and survivin. AY4 also inhibited growth of pre-established HN9 tumors in a nude mouse xenograft model and did not show noticeable cytotoxicity in a zebrafish model. Our results provide further insight into the mechanism of DR4-mediated cell death and potential use of AY4 mAb as an anti-cancer therapeutic agent in AY4-sensitive HNC types.

Histone deacetylase inhibitors synergistically potentiate death receptor 4-mediated apoptotic cell death of human T-cell acute lymphoblastic leukemia cells.

Cell-death signaling through the pro-apoptotic tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptors, death receptor 4 (DR4) and DR5, has shown tumor-selective apoptotic activity. Here, we examine susceptibility of various leukemia cell lines (HL-60, U937, K562, CCRF-CEM, CEM-CM3, and THP-1) to an anti-DR4 agonistic monoclonal antibody (mAb), AY4, in comparison with TRAIL. While most of the leukemia cell lines were intrinsically resistant to AY4 or TRAIL alone, the two T-cell acute lymphoblastic leukemia (T-ALL) lines, CEM-CM3 and CCRF-CEM cells, underwent synergistic caspase-dependent apoptotic cell death by combination of AY4 or TRAIL with a histone deacetylase inhibitor (HDACI), either suberoylanilide hydroxamic acid (SAHA) or valproic acid (VPA). All of the combined treatments synergistically downregulated several anti-apoptotic proteins (c-FLIP, Bcl-2, Bcl-X(L), XIAP, and survivin) without significant changing the expression levels of pro-apoptotic proteins (Bax and Bak) or the receptors (DR4 and DR5). Downregulation of c-FLIP to activate caspase-8 was a critical step for the synergistic apoptosis through both extrinsic and intrinsic apoptotic pathways. Our results demonstrate that the HDACIs have synergistic effects on DR4-specific mAb AY4-mediated cell death in the T-ALL cells with comparable competence to those exerted by TRAIL, providing a new strategy for the targeted treatment of human T-ALL cells.

Engineering of a human kringle domain into agonistic and antagonistic binding proteins functioning in vitro and in vivo.

Here, we report the development of target-specific binding proteins based on the kringle domain (KD) ( approximately 80 residues), a ubiquitous modular structural unit occurring across eukaryotic species. By exploiting the highly conserved backbone folding by core residues, but using extensive sequence variations in the seven loop regions of naturally occurring human KDs, we generated a synthetic KD library on the yeast cell surface by randomizing 45 residues in the loops of a human KD template. We isolated KD variants that specifically bind to anticancer target proteins, such as human death receptor 4 (DR4) and/or DR5, and that function as agonists to induce apoptotic cell death in several cancer cell lines in vitro and inhibit tumor progression in mouse models. Combined treatments with KD variants possessing different recognition sites on the same target protein exerted synergisitic tumoricidal activities, compared to treatment with individual variants. In addition to the agonists, we isolated an antagonistic KD variant that binds human tumor necrosis factor-alpha (TNFalpha) and efficiently neutralizes TNFalpha-induced cytotoxicity in vitro and in vivo. The KD scaffold with seven flexible loops protruding from the central core was strongly sequence-tolerant to mutations in the loop regions, offering a potential advantage of distinct binding sites for target recognition on the single domain. Our results suggest that the KD scaffold can be used to develop target-specific binding proteins that function as agonists or antagonists toward given target molecules, indicative of their potential use as biotherapeutics.

Humanization of an agonistic anti-death receptor 4 single chain variable fragment antibody and avidity-mediated enhancement of its cell death-inducing activity.

Development of agonistic monoclonal antibodies (mAbs) against the pro-apoptotic molecule death receptor 4 (DR4) [or tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) receptor 1] is an attractive anti-cancer strategy because of their potential for inducing tumor-specific cell death. In this study, we humanized an agonistic anti-DR4 AY4 scFv raised in mice (mAY4) by grafting the complementarity-determining regions (CDRs) onto a fixed human framework, while preserving the so-called Vernier zone residues, a group of framework (FR) residues directly underneath the CDRs, with the murine residues in the humanized antibody, hAY4. The humanized hAY4 scFv maintained the antigen binding affinity and epitope specificity of mAY4. To investigate how the valence of hAY4 scFv affects DR4-mediated cell death, bivalent and trivalent forms of hAY4 scFv were generated by linking a hinge region to the coiled-coil domain of a dimerizing leucine zipper and trimerizing isoleucine zipper, respectively. Compared to the monovalent and bivalent forms, the trivalent hAY4 scFv induced more potent caspase-dependent apoptotic cell death as evidenced by increased activation of caspase-8 and downstream pro-apoptotic molecules. Our results suggest that like other TNF family receptors, avidity-mediated oligomerization of DR4 augments the receptor-mediated apoptotic cell death by promoting intracellular cell death signaling.

A novel agonistic antibody to human death receptor 4 induces apoptotic cell death in various tumor cells without cytotoxicity in hepatocytes.

The proapoptotic tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptors death receptor (DR) 4 and DR5 are attractive targets to develop the receptor-specific agonistic monoclonal antibodies (mAb) as anticancer agents because of their tumor-selective cell death-inducing activity. Here, we report a novel agonistic mAb, AY4, raised against human DR4 in mice. ELISA analysis revealed that AY4 specifically bound to DR4 without competition with TRAIL for the binding. Despite distinct binding regions of AY4 on DR4 from those of TRAIL, AY4 as a single agent induced caspase-dependent apoptotic cell death of several tumor types through the extrinsic and/or intrinsic pathways without substantial cytotoxicity to normal human hepatocytes. Further, the AY4-sensitive cells followed the same cell death characteristics classified as type I and type II cells by the response to TRAIL, suggesting that the cell death profiles in responses to DR4 and/or DR5 stimulation are determined by the downstream signaling of the receptor rather than the kind of receptor. Noticeably, AY4 efficiently induced cell death of Jurkat cells, which have been reported to be resistant to other anti-DR4 agonistic mAbs, most likely due to the unique epitope property of AY4. In vivo administration of AY4 significantly inhibited tumor growth of human non-small cell lung carcinoma preestablished in athymic nude mice. Conclusively, our results provide further insight into the DR4-mediated cell death signaling and potential use of AY4 mAb as an anticancer therapeutic agent, particularly for DR4-responsive tumor types.