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Background: Despite the approval of immune checkpoint inhibitors (ICIs), prognosis of small cell lung cancer (SCLC) remains poor. DLL3 is upregulated in SCLC whereas expression in normal tissues is minimal, representing the favorable profile as a therapeutic target. T cell engager (TCE) is a potent immunotherapy that redirects T cells to tumors expressing a specific antigen. Unlike ICIs, TCEs do not require the recognition of tumor antigens by T cells and thus could be an alternative approach to target tumors where the benefit of ICIs is limited such as SCLC. CD137 (4-1BB) is a costimulatory molecule that promotes T cell activation, proliferation, and survival. Since CD137 agonists synergize with CD3-mediated T cell activation, inducing concurrent CD137 costimulation is a promising strategy to unleash the potential of TCEs. We therefore developed a DLL3/CD3/CD137 trispecific T cell engager composed of two CD3/CD137 dual specific Fabs and one extra DLL3 Fab (DLL3 trispecific, RG6524). Results: We initially investigated the CD3 and CD137 signal transduction in Jurkat cells harboring NFAT or NF-κB reporter cocultured with DLL3 positive cells. DLL3 trispecific showed dose-dependent increase in NFAT and NF-κB activity, indicating that target engagement successfully activated CD3 and CD137 signaling. In the in vitro assay using human PBMC, DLL3 trispecific induced cytotoxicity against SCLC cell lines with EC50s in the two-digit pM range. In vivo efficacy was evaluated in SCLC xenograft models established by engrafting SCLC cell lines into immune humanized NOG mice. DLL3 trispecific showed greater tumor growth inhibition compared to a traditional bispecific T cell engager. Furthermore, flow cytometry-based immunophenotyping revealed that DLL3 trispecific increased T cell number in tumors and improved IFN-γ production from CD8 T cells. We also explored the combination with platinum-based drugs, which are widely used for SCLC, and showed that DLL3 trispecific enhanced the efficacy of platinum-based drugs. These data demonstrated that DLL3 trispecific has the potent in vivo efficacy and the potential for clinical use. We next evaluated whether cytokine release syndrome (CRS) mitigating approaches affect antitumor efficacy. Although prophylactic use of a steroid significantly reduced cytokine production, tumor growth inhibition by DLL3 trispecific remained unchanged. Likewise, tocilizumab treatment did not reduce the efficacy, suggesting that CRS mitigation did not abrogate the therapeutic benefit. We finally assessed the tolerability in non-human primates. DLL3 trispecific did not reach the maximum tolerated dose and was well tolerated up to 16 mg/kg Q2D, the highest dose tested. Conclusion: Our data showed that DLL3 trispecific has potent activity and is well suited for clinical application in SCLC. These findings provide a rationale for the clinical testing of DLL3 trispecific. Citation Format: Hirofumi Mikami, Shu Feng, Sotaro Naoi, Yumiko Azuma, Yoko Kayukawa, Toshiaki Tsunenari, Kentaro Asanuma, Ryutaro Iwabuchi, Hiroaki Nagano, Junko Shinozuka, Masaki Yamazaki, Haruka Kuroi, Siok Wan Gan, Priyanka Chichili, Shun Shimizu, Yutaka Matsuda, Shinya Ishii, Shogo Kamikawaji, Yasuko Kinoshita, Yuichiro Shimizu, Akihisa Sakamoto, Masaru Muraoka, Noriyuki Takahashi, Tatsuya Kawa, Hirotake Shiraiwa, Kenji Kashima, Futa Mimoto, Mika Kamata-Sakurai, Takehisa Kitazawa, Tomoyuki Igawa. A DLL3/CD3/CD137 trispecific T cell engager shows potent antitumor activity in small cell lung cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1872.

Agonistic antibodies targeting CD137 have been clinically unsuccessful due to systemic toxicity. Because conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigens, we exploited extracellular adenosine triphosphate (exATP), which is a hallmark of the tumor microenvironment and highly elevated in solid tumors, as a broadly tumor-selective switch. We generated a novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the presence of exATP. STA551 demonstrated potent and broad antitumor efficacy against all mouse and human tumors tested and a wide therapeutic window without systemic immune activation in mice. STA551 was well tolerated even at 150 mg/kg/week in cynomolgus monkeys. These results provide a strong rationale for the clinical testing of STA551 against a broad variety of cancers regardless of antigen expression, and for the further application of this novel platform to other targets in cancer therapy.Significance:Reported CD137 agonists suffer from either systemic toxicity or limited efficacy against antigen-specific cancers. STA551, an antibody designed to agonize CD137 only in the presence of extracellular ATP, inhibited tumor growth in a broad variety of cancer models without any systemic toxicity or dependence on antigen expression.See related commentary by Keenan and Fong, p. 20.This article is highlighted in the In This Issue feature, p. 1

STA551, a novel anti-CD137 switch antibody, binds to CD137 in an extracellular ATP (exATP) concentration dependent manner. Although STA551 was assumed to show higher target binding in tumor than normal tissues, quantitative detection of the target binding of switch antibody in vivo is technically challenging. In this study, we investigated the target binding of STA551 in vivo using intravital imaging with two-photon microscopy. Tumor-bearing human CD137 knock-in mice were intravenously administered 1 mg/kg of fluorescent-labeled antibodies at day 0 and 3. Flow cytometry analysis of antibody-binding cells and intravital imaging using two-photon microscopy was conducted at day4. Higher CD137 expression in tumor than spleen was detected by flow cytometry analysis, and T cells and NK cells were major CD137 expressing cells. In the intravital imaging experiment, conventional and switch anti-CD137 antibody showed binding in tumor. However, in spleen, the fluorescence of switch antibody was much weaker than conventional anti-CD137 antibody and comparable with isotype control. In conclusion, we could assess switch antibody biodistribution in vivo through intravital imaging with two-photon microscopy. These results suggested that the tumor selective binding of STA551 leads to a wide therapeutic window and potent antitumor efficacy without systemic immune activation.

STA551, a novel anti-CD137 switch antibody, binds to CD137 in an extracellular ATP concentration-dependent manner. Although STA551 is assumed to show higher target binding in tumor tissues than in normal tissues, quantitative detection of the target binding of the switch antibody in vivo is technically challenging. In this study, we investigated the target binding of STA551 in vivo using intravital imaging with two-photon microscopy. Tumor-bearing human CD137 knock-in mice were intravenously administered fluorescently labeled antibodies. Flow cytometry analysis of antibody-binding cells and intravital imaging using two-photon microscopy were conducted. Higher CD137 expression in tumor than in spleen tissues was detected by flow cytometry analysis, and T cells and NK cells were the major CD137-expressing cells. In the intravital imaging experiment, conventional and switch anti-CD137 antibodies showed binding in tumors. However, in the spleen, the fluorescence of the switch antibody was much weaker than that of the conventional anti-CD137 antibody and comparable with that of the isotype control. In conclusion, we were able to assess switch antibody biodistribution in vivo through intravital imaging with two-photon microscopy. These results suggest that the tumor-selective binding of STA551 leads to a wide therapeutic window and potent antitumor efficacy without systemic immune activation.

CD137 is an attractive target for cancer immunotherapy, but its expression in normal tissues induces some adverse effects in patients receiving CD137-targeted therapy. To overcome this issue, we developed a switch antibody, STA551, that binds to CD137 only under high ATP concentrations around cells. This study quantified biodistribution of murine switch antibodies in human CD137 knock-in mice to show the viability of the switch antibody concept in vivo. We utilized four antibodies: Sta-MB, Ure-MB, Sta-mIgG, and KLH-MB. Sta-MB is a switch antibody having the variable region of STA551. The MB is a murine Fc highly binding to murine Fcγ receptor II. Ure-MB has a variable region mimicking the clinically available anti-CD137 agonist antibody urelumab, binding to CD137 regardless of ATP concentration. Sta-mIgG has the same variable region as Sta-MB but has the standard murine constant region. KLH-MB binds to keyhole limpet hemocyanin. The four antibodies were radiolabeled with In-111, SPECT/CT imaging was conducted in human CD137 knock-in mice, and the uptake in regions of interest was quantified. In-labeled Sta-MB and Sta-mIgG showed high uptake in tumors but low uptake in the lymph nodes and spleen in human CD137 knock-in mice. On the other hand, Ure-MB highly accumulated not only in tumors but also in the lymph nodes and spleen. KLH-MB showed low uptake in the tumors, lymph nodes, and spleen. The present study provides evidence that the switch antibody concept works in vivo. Our findings encourage further clinical imaging studies to evaluate the biodistribution of STA551 in patients.

Background: CD137 (4-1BB) is a co-stimulatory molecule which exerts activation signals in multiple immune cell subsets. However, agonist antibodies targeting CD137 have not been clinically successful due to severe systemic toxicity. Therefore, we generated STA551, a tumor selective CD137 agonist antibody. STA551 binds to CD137 only in the presence of ATP, which is known to be present at high levels in tumor tissue, and activates immune cells such as T cells. Previous studies have shown that STA551 exhibits antitumor efficacy without inducing a systemic immune response in a murine model. Combination studies with anti-PD-L1 antibody have also shown synergistic efficacy without inducing a systemic immune response. However, it was unclear why they exhibit strong combination effects. In this study, we elucidated the mechanism of STA551 and anti-PD-L1 antibody efficacy through comprehensive analysis of gene expression using single cell RNA sequencing. Method & Result: In a colon 38-bearing human CD137 knock-in mouse model, combination therapy with STA551 and anti-PD-L1 antibody showed potent anti-tumor efficacy. From immunohistochemistry of tumor tissue, increased numbers of CD8+ T cells and elevated expression of PD-L1 molecules were observed. To perform a detailed mechanistic analysis, tumor tissue was collected after antibody administration and CD3 positive and CD3 negative fractions were separated and gene expression was comprehensively analyzed using single cell RNA-seq. Next, the fraction expressing Cd8a gene was extracted from CD3 positive fraction and clustered. One cluster was found to be markedly increased in STA551 and anti-PD-L1 antibody combination compared to vehicle or monotherapy treatment. Differential gene expression analysis revealed that the cluster was enriched for genes related to cytokines and chemokines. This result revealed that the combination of STA551 and anti-PD-L1 antibody induced highly activated CD8+T cells. Conclusion: The combination of STA551 and PD-L1 showed synergistic anti-tumor efficacy in a colon38-bearing human CD137 knock-in mouse model. Detailed mechanistic analysis showed that the combination of STA551 and PD-L1 induced highly activated CD8+T cells expressing chemokines and cytokines compared to monotherapy. This cluster also express chemokines, suggesting that other immune cells have been recruited. These results suggest that even the activation of only tumor tissue, which is a characteristic of STA551, can induce a strong immune response. Citation Format: Ryo Uchikawa, Kazuya Takakuwa, Hirofumi Mikami, Shoichi Metsugi, Kenji Taniguchi, Yoshinori Narita, Mika Kamata-Sakurai. Single cell RNA sequencing revealed that the mechanism of the synergistic efficacy of STA551 and immune checkpoint inhibitor results from the induction of highly activated T cells in tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5518.

Background Agonistic antibodies targeting CD137 in clinic have failed due to severe hepatotoxicity, leading to the development of bispecific approaches that must rely on high tumor-associated antigen expression to crosslink CD137. Here we report on STA551, a novel anti-CD137 agonist antibody which binds to CD137 only in the presence of ATP. Extracellular ATP concentration is well-known to be elevated in tumor tissue while remaining tightly regulated in non-tumor tissue, suggesting that STA551 can activate immune cells only in tumor tissue and not elsewhere. Thus, STA551 has great potential to overcome the limitations of conventional CD137-targeted antibodies. Methods We evaluated in vitro STA551’s effect on IFN-γ production from human CD8+ T cells. We also evaluated in vivo STA551’s effect on tumor growth, RNAseq-based immune-related gene expression, immunohistochemistry, and T cell activation in tumor and non-tumor tissues in human CD137 knock-in mice treated with mouse surrogate STA551 (Sta-MB) or urelumab (Ure-MB) in combination with anti-PD-L1 antibody. Results In a human T cell assay, STA551 induced IFN-γ only in the presence of ATP. In contrast, urelumab induced IFN-γ regardless of ATP concentration. In mice with Colon 38 tumors, Sta-MB inhibited tumor growth at least as strongly as Ure-MB, but whereas Ure-MB elicited systemic immune responses in draining lymph node, spleen, and liver, Sta-MB appeared to evade such responses. To confirm immune responses in tumors, we evaluated immune-related gene expression and found changes after treatment with Sta-MB or Ure-MB. These results suggest that STA551 works only in tumor tissue. Furthermore, Sta-MB with anti-PD-L1 antibody synergistically inhibited tumor growth and dramatically changed immune-related gene expression, CD8+ T cell infiltration, and PD-L1 expression without systemic immune responses. Also, it was well-tolerated in cynomolgus monkey in a repeated-dose toxicity study*. Conclusions STA551 is a novel anti-CD137 agonist antibody that exerts agonistic activity selectively in tumors without on-target toxicity in non-tumor tissues, regardless of tumor-associated antigen expression. These results strongly support the clinical testing of STA551 for the treatment of solid tumors. STA551 is currently being tested in a phase 1 clinical study. Ethics Approval All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC).

Background: CD137 (4-1BB) is a co-stimulatory molecule which exerts activation signals in multiple immune cell subsets. However, agonist antibodies targeting CD137 have not been clinically successful due to severe systemic toxicity. Since conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigen, we instead exploited extracellular adenosine triphosphate (exATP), which is known to be elevated in tumor microenvironment (TME) while remaining tightly regulated in normal tissues, as a broadly tumor selective switch. Here we report a novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the presence of exATP. Methods & Results: STA551 bound to human CD137 in an ATP dependent manner. Consistent with the binding profile, STA551 induced IFN-γ only in the presence of ATP, whereas urelumab induced IFN-γ regardless of ATP concentration in a human T cell assay. We examined the anti-tumor efficacy of Sta-MB, which has the same variable region as STA551 using a surrogate mouse Fc, in human CD137 knock-in (hCD137-KI) mice. STA551 demonstrated potent and broad anti-tumor efficacy against all the tumors we tested both as a monotherapy and in combination with anti-PD-L1 antibody. To confirm the immune responses in tumors, we evaluated gene expression and analyzed tumor infiltrating lymphocytes by flow cytometry. Immune-related gene expression changed after treatment with Sta-MB. We also confirmed that Sta-MB treatment increased the number of activated CD8+ T cells in tumors. For all tumor responses, Sta-MB tended to be more potent than Ure-MB. Whereas other anti-CD137 conventional antibodies showed significant CD137 mediated clearance and systemic T cell activation in draining lymph node (DLN), spleen, and liver, STA551 demonstrated significant intratumor T cell activation, but not in normal tissues and without CD137 mediated systemic clearance in mice. Furthermore, STA551 was well-tolerated in cynomolgus monkeys in a repeated-dose toxicity study*. Conclusion: These results suggest that STA551, due to its tumor selective agonistic activity, has potent anti-tumor efficacy and a wide therapeutic window, providing a strong rationale for its clinical testing against a broad variety of cancers regardless of antigen expression, and for the further application of this novel platform to other targets in cancer therapy. STA551 is currently being tested in a phase 1 clinical study. We will here disclose the clinical study design for the first time. Citation Format: Mika Kamata-Sakurai, Yoshinori Narita, Ryo Uchikawa, Kenji Taniguchi, Sayuri Horikawa, Koki Hamada, Yuji Hori, Takayuki Nemoto, Shoichi Metsugi, Michiyasu Inatani, Tomoyuki Igawa. Responses of a novel tumor selective anti-CD137 agonist antibody activated by elevated extracellular ATP in tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1851.

Immune checkpoint inhibitors such as anti-PD1 antibodies have shown promising clinical responses in several solid tumors, however there remain patients who do not show an adequate response. Recent biomarker studies have revealed that the presence of neoantigens in the tumor can determine the level of response, and thus the next challenge will be how to target tumors with a neoantigen level that is too low to be recognized by endogenous cytotoxic T cells. Hope in this area is offered by a T cell-redirecting antibody (TRAB), which bispecifically engages CD3 and a tumor antigen, even at very low expression levels, to activate the inherent cytolytic potential of T cells against target tumor cells. A TRAB is highly potent because T cells are activated only in the presence of the targeted antigens and are not restricted by the specificity of the T cell receptor. Given this very potent cytotoxicity, the key to successfully achieving strong antitumor efficacy while avoiding on-target off-tumor toxicity is to select a highly tumor-selective antigen. Our fully humanized IgG TRAB recognizes CD3 and a highly tumor-selective antigen, glypican-3 (GPC3), which is a fetal protein expressed in a wide variety of tissues during development but suppressed in most adult tissues. On the other hand, an inct101e in GPC3 expression has been reported in hepatocellular carcinoma, gastric cancer, lung squamous cell carcinoma, and other cancers. In nonclinical in vitro pharmacology studies, the anti-GPC3 TRAB elicited activation and proliferation of T cells and T cell-dependent cellular cytotoxicity against a wide variety of GPC3-expressing tumor cells, and showed long-lasting in vivo efficacy against tumor expressing very low levels of GPC3 at a few thousand molecules per cell. Furthermore, in an immunocompetent mouse model using human CD3 transgenic mice, anti-GPC3 TRAB showed strong antitumor efficacy against poorly immunogenic tumors, whereas both the immune checkpoint inhibitors and a conventional ADCC-inducing antibody recognizing GPC3 did not show significant efficacy. Pharmacokinetics and toxicology studies in nonhuman primates showed a plasma half-life comparable to a standard IgG drug, allowing a QW or Q2W regimen in humans, with toxicity which was manageable and reversible; the main observations of transient cytokine elevation and associated clinical symptoms were markedly reduced by steroid premedication. Our anti-GPC3 TRAB, which is supported by proprietary antibody engineering technology (ART-Ig) that enables large-scale GMP manufacturing, has promise as a new approach in cancer immunotherapy. Citation Format: Takahiro Ishiguro, Yasuko Kinoshita, Yuji Sano, Yumiko Azuma, Toshiaki Tsunenari, Natsuki Ono, Yoko Kayukawa, Mika Kamata-Sakurai, Hirotake Shiraiwa, Akihisa Kaneko, Werner Frings, Shunichiro Komatsu, Junichi Nezu, Mika Endo. First-in-class T cell-redirecting bispecific antibody targeting glypican-3: a highly tumor-selective antigen. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr DDT01-05.

The extracellular adenosine triphosphate (ATP) concentration is highly elevated in the tumor microenvironment (TME) and remains tightly regulated in normal tissues. Using phage display technology, we establish a method to identify an antibody that can bind to an antigen only in the presence of ATP. Crystallography analysis reveals that ATP bound in between the antibody-antigen interface serves as a switch for antigen binding. In a transgenic mouse model overexpressing the antigen systemically, the ATP switch antibody binds to the antigen in tumors with minimal binding in normal tissues and plasma and inhibits tumor growth. Thus, we demonstrate that elevated extracellular ATP concentration can be exploited to specifically target the TME, giving therapeutic antibodies the ability to overcome on-target off-tumor toxicity.

The antibody drug market is rapidly expanding, and various antibody engineering technologies are being developed to create antibodies that can provide better benefit to patients. Although bispecific antibody drugs have been researched for more than 30 years, currently only a limited number of bispecific antibodies have achieved regulatory approval. Of the few successful examples of industrially manufacturing a bispecific antibody, the “common light chain format” is an elegant technology that simplifies the purification of a whole IgG–type bispecific antibody. Using this IgG format, the bispecific function can be introduced while maintaining the natural molecular shape of the antibody. In this article, we will first introduce the outline, prospects, and limitations of the common light chain format. Then, we will describe the identification and optimization process for ERY974, an anti-glypican-3 × anti-CD3e T cell–redirecting bispecific antibody with a common light chain. This format includes one of Chugai’s proprietary technologies, termed ART-Ig technology, which consists of a method to identify a common light chain, isoelectric point (pI) engineering to purify the desired bispecific IgG antibody from byproducts, and Fc heterodimerization by an electrostatic steering effect. Furthermore, we describe some tips for de-risking the antibody when engineering a T cell redirecting antibody.

Cancer care is being revolutionized by immunotherapies such as immune checkpoint inhibitors, engineered T cell transfer, and cell vaccines. The bispecific T cell-redirecting antibody (TRAB) is one such promising immunotherapy, which can redirect T cells to tumor cells by engaging CD3 on a T cell and an antigen on a tumor cell. Because T cells can be redirected to tumor cells regardless of the specificity of T cell receptors, TRAB is considered efficacious for less immunogenic tumors lacking enough neoantigens. Its clinical efficacy has been exemplified by blinatumomab, a bispecific T cell engager targeting CD19 and CD3, which has shown marked clinical responses against hematological malignancies. However, the success of TRAB in solid tumors has been hampered by the lack of a target molecule with sufficient tumor selectivity to avoid "on-target off-tumor" toxicity. Glypican 3 (GPC3) is a highly tumor-specific antigen that is expressed during fetal development but is strictly suppressed in normal adult tissues. We developed ERY974, a whole humanized immunoglobulin G-structured TRAB harboring a common light chain, which bispecifically binds to GPC3 and CD3. Using a mouse model with reconstituted human immune cells, we revealed that ERY974 is highly effective in killing various types of tumors that have GPC3 expression comparable to that in clinical tumors. ERY974 also induced a robust antitumor efficacy even against tumors with nonimmunogenic features, which are difficult to treat by inhibiting immune checkpoints such as PD-1 (programmed cell death protein-1) and CTLA-4 (cytotoxic T lymphocyte-associated protein-4). Immune monitoring revealed that ERY974 converted the poorly inflamed tumor microenvironment to a highly inflamed microenvironment. Toxicology studies in cynomolgus monkeys showed transient cytokine elevation, but this was manageable and reversible. No organ toxicity was evident. These data provide a rationale for clinical testing of ERY974 for the treatment of patients with GPC3-positive solid tumors.

Monoclonal antibodies (mAbs) against mucin 21 (MUC21), a human counterpart of mouse epiglycanin/Muc21, were prepared using human embryonic kidney 293 cells transfected with MUC21 as the immunogen. The specificity of these mAbs was examined by flow cytometry, immunoprecipitation and western blotting focusing on the differential glycosylation of MUC21 expressed in variant Chinese hamster ovary (CHO) cells (ldlD cells and Lec2 cells) and CHO-K1 cells. One of these mAbs, heM21D, bound to both the unmodified core polypeptide of MUC21 and MUC21 attached with N-acetylgalactosamine (Tn-MUC21). Six antibodies, including mAb heM21C, bound to MUC21 with Tn, T or sialyl-T epitopes but not the unmodified core polypeptide of MUC21. Esophageal squamous carcinomas and adjacent squamous epithelia were immunohistochemically examined for the binding of these mAbs. MUC21 was expressed in esophageal squamous epithelial cells, and its O-glycan extended forms were observed in the luminal portions of squamous epithelia. As revealed by the binding of mAb heM21D and the absence of reactivity with mAb heM21C, esophageal squamous carcinoma cells produce MUC21 without the attachment of O-glycans. This is the first report to show that there is a change in the glycoform of MUC21 that can be used to differentiate between squamous epithelia and squamous carcinoma of the esophagus. Thus, these antibodies represent a useful tool to characterize squamous epithelial differentiation and carcinogenesis.

The molecular structure of mouse Mucin 21 (Muc21)/epiglycanin is proposed to have 98 tandem repeats of 15 amino acids and three exceptional repeats with 12 or 13 amino acids each, followed by a stem domain, a transmembrane domain, and a cytoplasmic tail. A cDNA of Muc21 having 84 tandem repeats of 15 amino acids was constructed and transfected using a Venus vector into HEK 293T cells. The fluorescent cells, which were considered to express Muc21, were nonadherent. This antiadhesion effect was lessened when constructs with smaller numbers of tandem repeats were used, suggesting that the tandem repeat domain plays a crucial role. Cells expressing Muc21 were significantly less adherent to each other and to extracellular matrix components than control cells. Antibody binding to the cell surface integrin subunits alpha5, alpha6, and beta1 was reduced in Muc21 transfectants in a tandem repeat-dependent manner, whereas equal amounts of proteins were detected by Western blot analysis. Muc21 was expressed as a large glycoprotein that was highly glycosylated with O-glycans at the cell surface, as detected by flow cytometry, Western blotting, and lectin blotting. Although at least a portion of Muc21 was glycosylated with sialylated glycans, removal of sialic acid did not influence the prevention of adhesion.

We present efficacy data for the T cell-redirecting antibody (TRAB) with highly potent anti-tumor efficacy. Anti-Glypican-3 (GPC3) TRAB is a humanized IgG4 bispecific antibody that simultaneously binds to GPC3 on the cancer cell surface and to CD3 on the T cell surface. Anti-GPC3 TRAB utilizes T cells as effectors to induce strong TRAB dependent cellular cytotoxicity (TDCC) in the presence of GPC3-expressing cells. Treatment with anti-GPC3 TRAB first activates T cells by increasing the expression of CD25 and CD69 and also upregulating cytokines IL-2, IL-4, IL-6, IL 10, IFNγ, and TNF, and then it enhances the proliferation of T cells. Anti-GPC3 TRAB showed antitumor activity against xenograft tumors derived from various cancer types — MKN-74 (human gastric adenocarcinoma), PC-10 (human lung squamous cell carcinoma), TOV-21G (human ovarian clear cell carcinoma), and KYSE70 (human esophageal squamous cell carcinoma) — in a NOD-SCID mouse model injected with human T cells. Although recent immunotherapy, as represented by immune check point inhibitors PD-1, PD-L1, and CTLA-4 antibodies, showed promising efficacy in human, not every patient can benefit from this immunotherapy, because the significant efficacy shown in patients by a blockade of immune checkpoints is closely related to the tumor microenvironment. The immune check point inhibitors show high efficacy against inflamed tumors, because these have been sufficiently infiltrated by cytotoxic T cells that recognize cancer-specific antigens. However, they do not have efficacy against non inflamed tumors. In an immunocompetent mouse model using human CD3 transgenic mice, neither the inhibitors that block immune checkpoints (such as PD-1, PD-L1 and CTLA-4) nor a conventional ADCC antibody recognizing GPC3 could show significant efficacy against a poorly immunogenic LLC1/hGPC3 tumor. However, anti-GPC3 TRAB showed efficacy against this poorly immunogenic tumor by utilizing any kind of T cell as effectors irrespective of TCR specificity, including not only CD8-positive but also CD4-positive T cells. The studies we present show that anti-GPC3 TRAB is a promising drug with high efficacy utilizing all kinds of T cells as effectors. The compound is expected to have efficacy even in patients with poorly immunogenic tumors, in which an immune checkpoint blockade fails to show efficacy. Citation Format: Yasuko Kinoshita, Takahiro Ishiguro, Yuji Sano, Yumiko Azuma, Toshiaki Tsunenari, Natsuki Ono, Yoko Kayukawa, Otoya Ueda, Naoko A. Wada, Hiroshi Hino, Koichi Jishage, Hirotake Shiraiwa, Mika Kamata-Sakurai, Junichi Nezu, Mika Endo. Anti-GPC3 TRAB, a first-in-class T cell-redirecting bispecific antibody targeting glypican-3 with potent in vitro and in vivo antitumor efficacy against solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1482.

TPS2592Background: Despite recent advance of immunotherapy, not all patient benefit. Reasons for the limited therapeutic effect include a low level of target protein expression or tumor immunogenic...

Hemophilia A is a bleeding disorder resulting from coagulation factor VIII (FVIII) deficiency. Exogenously provided FVIII effectively reduces bleeding complications in patients with severe hemophilia A. In approximately 30% of such patients, however, the 'foreignness' of the FVIII molecule causes them to develop inhibitory antibodies against FVIII (inhibitors), precluding FVIII treatment in this set of patients. Moreover, the poor pharmacokinetics of FVIII, attributed to low subcutaneous bioavailability and a short half-life of 0.5 d, necessitates frequent intravenous injections. To overcome these drawbacks, we generated a humanized bispecific antibody to factor IXa (FIXa) and factor X (FX), termed hBS23, that places these two factors into spatially appropriate positions and mimics the cofactor function of FVIII. hBS23 exerted coagulation activity in FVIII-deficient plasma, even in the presence of inhibitors, and showed in vivo hemostatic activity in a nonhuman primate model of acquired hemophilia A. Notably, hBS23 had high subcutaneous bioavailability and a 2-week half-life and would not be expected to elicit the development of FVIII-specific inhibitory antibodies, as its molecular structure, and hence antigenicity, differs from that of FVIII. A long-acting, subcutaneously injectable agent that is unaffected by the presence of inhibitors could markedly reduce the burden of care for the treatment of hemophilia A.

The effects of MUC1 DNA vaccination on the orthotopic growth and liver metastasis of colon carcinoma cells were investigated in mice. Vaccination with MUC1 DNA resulted in immune responses that were effective in suppressing mouse colon carcinoma cells transfected with MUC1 cDNA. CD4+ T cells but not CD8+ T cells mediated this antitumor response as shown by the in vivo depletion of lymphocyte subpopulations with the use of anti-CD4 or anti-CD8 antibody. The effects of neutralizing antibodies in vivo revealed that the predominant effector molecule in preventing orthotopic tumor growth was FasL, whereas the effector molecule effective in preventing liver metastasis was tumor necrosis factor-α. Colon carcinoma cells isolated from tumors growing in the ceca, spleens, and livers were shown to be equally sensitive to FasL and tumor necrosis factor-α. The results strongly suggest that elimination of tumor cells initiated by DNA vaccination in the present protocol is mediated by antigen-specific CD4+ T cells and the effector mechanisms in the cecum and in the liver are distinct due to a unique organ microenvironment. (Cancer Sci 2008; 99: 2477–2484)

The gene for the human orthologue of mouse epiglycanin, a mucin expressed on mammary carcinoma TA3-Ha cells but not TA3-St cells, was identified by homology search to a mouse epiglycanin cDNA fragment identified by representational difference analysis between TA3-Ha and TA3-St cells. The open reading frame of this gene was cloned from human cervical carcinoma ME-180 cells. It consists of a mucin domain with 28 nonidentical tandem repeats of 45 nucleotides each corresponding to a threonine/serine-rich peptide, a stem domain, a transmembrane domain, and a cytoplasmic tail. The cloned cDNA with a FLAG sequence was expressed in K562 cells. A combination of immunoprecipitation with a polyclonal antibody specific for the cytoplasmic tail and Western blotting analysis with an anti-FLAG antibody and lectins revealed a mucin-like component as the gene product. Analysis by the use of tissue cDNA libraries indicated that the gene is expressed in lung, large intestine, thymus, and testis among 16 normal tissues tested. The polyclonal antibody specific for a synthetic peptide from the cytoplasmic tail, when tested for its reactivity with normal lung tissues, reacted with epithelia of bronchi and bronchioli but not with alveoli. All of 24 lung adenocarcinomas specimens tested were reactive with the antibody, whereas reactivity was observed with only 2 out of 24 squamous and none out of 24 small cell lung carcinomas. This is a novel transmembrane mucin and designated as MUC21.