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Emicizumab, a factor (F)VIIIa-function mimetic bispecific antibody (BsAb) to FIXa and FX, has become an indispensable treatment for people with hemophilia A (PwHA). Although emicizumab is very potent, long-term outcomes from the clinical studies suggest that a small proportion of PwHA still experiences bleeds. Additionally, non-clinical studies indicate that the maximum cofactor activity of emicizumab is lower than international standard activity (100 IU/dL of FVIII). An increased cofactor activity BsAb would benefit such patients. Here, we report NXT007, a BsAb binding FIXa and FX developed through further engineering of emicizumab. Emicizumab has a common light chain, but through advances in antibody engineering, we were able to create a more potent BsAb with two new non-common light chains. After extensive optimization of the heavy and light chains, the resulting BsAb, NXT007, exerted in vitro thrombin generation (TG) activity in hemophilia A plasma equivalent to 100 IU/dL of FVIII when triggered by tissue factor. NXT007 demonstrated potent hemostatic activity in an acquired hemophilia A model in non-human primates at a much lower dosage than emicizumab, consistent with an around 30-fold dose shift in the in vitro TG activity between NXT007 and emicizumab. Moreover, together with Fc engineering that enhanced FcRn binding and reduced in vivo clearance, we demonstrate that NXT007 could be effective at a much lower dosage with a longer dosing interval compared to emicizumab. These non-clinical results suggest that NXT007 could maintain a non-hemophilic range of coagulation potential in PwHA and provides a rationale for its clinical testing.

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.

An emerging structure for anti-tumor antibody drugs utilizes a bispecific antibody (BiAb) that recognizes a tumor surface antigen and CD3 on T cells. An impurity that commonly contaminates these BiAb products is an anti-CD3 monoclonal antibody (mAb). The most plausible cause of toxic activity by an anti-CD3 mAb is the induction of cytokines via T cell activation. In this in vitro study, we compared cytokine induction and T cell activation after treatment with an anti-glypican-3/CD3 BiAb (ERY974), anti-CD3 mAb impurity (aCD3), or ERY974 spiked with 5% aCD3. We found that contamination with up to 5% aCD3 did not affect cytokine release by ERY974. Cytokine levels induced by ERY974 in the presence of target cells were significantly higher than those induced by aCD3, but were very similar to those by the spiked treatment. The results supported the specification of a 5% limit for aCD3. OKT-3 had much higher activity to induce cytokines from peripheral blood mononuclear cells in an in vitro assay than aCD3. This suggests that specification limit should be decided for each type of anti-CD3 impurity that affects T cell-activating BiAb drug products. In vitro cytokine assays can provide useful information for determining these specification limits.

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.

Background Emicizumab (HEMLIBRA®) is a factor (F) VIII function-mimetic therapeutic bispecific antibody (BsAb) to FIXa and FX able to prevent bleeding in persons with hemophilia A (PwHA) when injected subcutaneously once every 1, 2 or 4 weeks. To develop a next generation version, we sought an agent able to keep hemostatic potential in non-hemophilic range with more convenient dosing regimen (dosing frequency/volume). We successfully created the emicizumab-based engineered four-chain BsAbs, NXT series. Among these, we selected NXT007 as a clinical candidate. Objectives The aim of this study is to clarify the in vitro and in vivo properties of NXT007 and predict its therapeutic potency non-clinically. Methods We evaluated the pharmacological activities of NXT007 in vitro using a thrombin generation assay (TGA) with FVIII-deficient patient plasma, and in vivo by inducing bleeding in FVIII-neutralizing antibody-treated acquired hemophilia A cynomolgus monkey (cyno) model. To clarify the FVIII-cofactor activity of NXT007, we performed an enzymatic kinetics analysis of FIXa-catalyzed FX activation with and without NXT007, as well as surface plasmon resonance analysis to determine the dissociation constant (KD) of NXT007 to FIX, FIXa, FX and FXa. We obtained its pharmacokinetic (PK) profile in non-human primates in a single dose SC/IV study. Results In vitro addition of NXT007 at 30 μg/mL increased the peak height of TGA in FVIII-deficient plasma to the same levels achieved by recombinant human FVIII at 40-100 IU/dL (FXIa-triggering) or 100-150 IU/dL (tissue factor-triggering). A single bolus intravenous injection of NXT007 (0.075 mg/kg) ameliorated bleeding symptoms in the cyno model to similar as a twice daily intravenous injection of recombinant porcine FVIII (20 U/kg). The in vitro and in vivo results were roughly concordant. NXT007 increased the turnover rate (kcat) of FIXa-catalyzed FX activation by approximately 4,000-folds compared to the condition without cofactor. The impact of NXT007 on the kcat was similar to that of emicizumab. As for binding affinities, the KD values of NXT007 to FIX, FIXa, FX and FXa were 1.08, 0.728, 0.0538 and 0.0231 μM, respectively in buffer solution. Compared to emicizumab, NXT007 bound more strongly to FX/FXa and with similar affinity to FIX/FIXa. This means that NXT007 would have an ability to form more FIX-BsAb-FX ternary complex than emicizumab. Calculated using the above KD values, at 30 μg/mL of BsAb the estimated concentration of FIX-NXT007-FX ternary complex in plasma is approximately 10-fold higher than that of the FIX-emicizumab-FX ternary complex which is roughly concordant with the difference in their FVIII equivalent thrombin generation activity. Prothrombin time (PT) was not clearly prolonged suggesting minimal impact on FX function by in vitro addition of NXT007 at up to 30 μg/mL, which was enough to induce sufficient thrombin burst in FVIII-deficient plasma as described above. A half-life of NXT007 was 19.6 to 24.4 days (0.02-2 mg/kg, SC) and SC bioavailability was 84.4% (2 mg/kg) in the in vivo cyno PK study, in which no obvious change in plasma FIX or FX levels was observed after 0.02-2 mg/kg single SC administration. Conclusions Based on the nonclinical results, NXT007, delivered in every-4-week SC injections, will keep a non-hemophilic range of equivalent FVIII thrombin generation in PwHA, Compared with emicizumab, NXT007's improved cofactor activity may be attributed to its more efficient ternary complex formation while keeping turnover rate with minimal impact on FX function suggested by PT value and antigen accumulation. A phase 1/2 clinical study of NXT007 is now on-going (NXTAGE; JapicCTI-194919). Disclosures Yamaguchi: Chugai Pharmaceutical Co., Ltd: Current Employment. Soeda:Chugai Pharmaceutical Co., Ltd.: Current Employment. Sato:Chugai Pharmaceutical Co., Ltd.: Current Employment. Shibahara:Chugai Pharmaceutical Co., Ltd.: Current Employment. Koga:Chugai Pharmaceutical Co., Ltd.: Current Employment. Ichiki:Chugai Pharmaceutical Co., Ltd.: Current Employment. Joyashiki:Chugai Pharmaceutical Co., Ltd.: Current Employment. Teranishi:Chugai Pharmaceutical Co., Ltd.: Current Employment. Nishimura:Chugai Pharmaceutical Co., Ltd.: Current Employment. Shiraiwa:Chugai Pharmaceutical Co., Ltd.: Current Employment. Kitamura:Chugai Pharmaceutical Co., Ltd.: Current Employment. Igawa:Chugai Pharmaceutical Co., Ltd.: Current Employment. Konishi:Chugai Pharmaceutical Co., Ltd.: Current Employment. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Current Employment.

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.

Osteopontin (OPN) has been shown to be expressed by cells in granulomas of various origins, but whether it plays a functional role in granuloma formation is not known. Here we used a cardiomyopathic hamster (TO2) model, to test the hypothesis that OPN contributes functionally to granuloma development. We immunized cardiomyopathic and normal hamsters by subcutaneous injection of bovine serum albumin in complete Freund's adjuvant, and assessed various tissues for both OPN RNA expression and granuloma formation. Cardiomyopathic hamsters expressed OPN, and formed granulomatous lesions, in heart tissue in both immunized and untreated animals. In addition, immunization induced expression of OPN in lung and lymph nodes of cardiomyopathic (but not normal) hamsters, and also induced granuloma formation in these organs. To test whether OPN expression could play a functional role in inducing granulomas, we produced an adenoviral vector containing the murine OPN gene, and introduced this vector intratracheally into the lungs of normal hamsters. The OPN-containing vector, but not the control vector, induced pulmonary granuloma formation. These studies provided direct in vivo evidence that OPN can contribute functionally to the formation of granulomatous lesions, and suggest that OPN expression may be a common factor involved in formation of granulomas of various origin.

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.

The humanized monoclonal antibody (mAb) against CD317 antigen (anti-HM1.24 antibody; AHM), which is highly expressed on multiple myeloma (MM), induces antibody-dependent cellular cytotoxicity (ADCC). However, the antitumor activity of AHM in the clinical setting has not been clearly demonstrated. In this study, we produced defucosylated AHM and evaluated its potency for clinical application by performing autologous ADCC assays against primary MM cells from patients. Defucosylated AHM that was produced in rat myeloma YB2/0 cells expressing a low level of fucosyltransferase (FUT8) showed significant ADCC activity against three out of six primary MM cells in the presence of autologous PBMC, whereas conventional AHM did not. The results indicate that the potency of AHM to induce ADCC against primary MM cells was insufficient, but was significantly augmented by defucosylation. To generate more homogenous defucosylated monoclonal antibodies (mAb) for fermentation, we disrupted the GFT gene that encodes a GDP-fucose transporter in a CHO/DXB11 cell line by sequential homologous recombination. Analysis of the N-linked oligosaccharide in the defucosylated AHM produced by the established GFT(-/-)CHO cell line showed that a majority (93.4%) of the oligosaccharide was fucose free. The GFT(-/-) cells stably produced defucosylated mAb over passages. These results demonstrate that GTF(-/-)CHO-produced defucosylated AHM (GFTKO-AHM) will be a promising new therapeutic antibody against MM in the clinical setting.