Shohei Kishishita

Optimization of chemically defined feed media for monoclonal antibody production in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are the most commonly used mammalian host for large-scale commercial production of therapeutic monoclonal antibodies (mAbs). Chemically defined media are currently used for CHO cell-based mAb production. An adequate supply of nutrients, especially specific amino acids, is required for cell growth and mAb production, and chemically defined fed-batch processes that support rapid cell growth, high cell density, and high levels of mAb production is still challenging. Many studies have highlighted the benefits of various media designs, supplements, and feed addition strategies in cell cultures. In the present study, we used a strategy involving optimization of a chemically defined feed medium to improve mAb production. Amino acids that were consumed in substantial amounts during a control culture were added to the feed medium as supplements. Supplementation was controlled to minimize accumulation of waste products such as lactate and ammonia. In addition, we evaluated supplementation with tyrosine, which has poor solubility, in the form of a dipeptide or tripeptide to improve its solubility. Supplementation with serine, cysteine, and tyrosine enhanced mAb production, cell viability, and metabolic profiles. A cysteine-tyrosine-serine tripeptide showed high solubility and produced beneficial effects similar to those observed with the free amino acids and with a dipeptide in improving mAb titers and metabolic profiles.

Effect of temperature shift on levels of acidic charge variants in IgG monoclonal antibodies in Chinese hamster ovary cell culture

During the production of therapeutic monoclonal antibodies (mAbs), not only enhancement of mAb productivity but also control of quality attributes is critical. Charge variants, which are among the most important quality attributes, can substantially affect the in vitro and in vivo properties of mAbs. During process development for the production of mAbs in a Chinese hamster ovary cell line, we have observed that an improvement in mAb titer is accompanied by an increase in the content of acidic charge variants. Here, to help maintain comparability among mAbs, we aimed to identify the process parameters that controlled the content of acidic charge variants. First, we used a Plackett-Burman design to identify the effect of selected process parameters on the acidic charge variant content. Eight process parameters were selected by using a failure modes and effects analysis. Among these, temperature shift was identified from the Plackett-Burman design as the factor most influencing the acidic charge variant content. We then investigated in more detail the effects of shift temperature and temperature shift timing on this content. The content decreased with a shift to a lower temperature and with earlier timing of this temperature shift. Our observations suggest that Plackett-Burman designs are advantageous for preliminary screening of bioprocess parameters. We report here for the first time that temperature downshift is beneficial for effective control of the acidic peak variant content.

Abstract 3653: Combining ERY974, a novel T cell-redirecting bispecific antibody targeting glypican-3, with chemotherapy profoundly improved antitumor efficacy over its monotherapy in xenograft model

Background: ERY974 is a humanized IgG4 bispecific T cell-redirecting antibody (TRAB) currently in Phase 1 clinical trial (NCT02748837). ERY974 consists of a common light chain and two different heavy chains that respectively recognize glypican-3 (GPC3) and CD3. The Fc portion of ERY974 is modified to lose FcγR binding to prevent GPC3-independent Fc-mediated effector function. However, binding activity to FcRn, an important factor in the PK profile of IgG, is maintained. ERY974 simultaneously binds to GPC3 on the cancer cell surface and to CD3 on the T cell surface, and induces TRAB-dependent cellular cytotoxicity mediated by the potent effector function of T cells. ERY974 shows strong antitumor activity against gastric, lung, ovarian, head & neck, and esophageal cancer-derived xenograft tumors in a non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mouse model injected with human T cells. Cancer immunotherapy, as represented by immune checkpoint inhibitors such as PD-1, PD-L1, and CTLA-4 antibodies, has recently been demonstrating remarkable clinical benefit in various tumor types. However, the number of patients who have survival benefit is limited, and combining cancer immunotherapy with other agents is required to improve the efficacy. Although ERY974 monotherapy is expected to show clinical activity based on the preclinical data, we examined whether further improvement of ERY974-induced efficacy is attained by combination with chemotherapy. Method & Results: We evaluated the combination effect of ERY974 with chemotherapy against xenograft tumors of MKN45 (gastric cancer) or NCI-H446 (lung cancer) either in a NOD-SCID mouse model injected with human T cells or in a humanized non-obese diabetic/shi-scid/IL-2Rγnull model in which differentiated human T cells are constitutively supplied. Although ERY974 monotherapy shows only minor antitumor effect against MKN45 and NCI-H446, combination therapy remarkably enhanced efficacy. In particular, ERY974 in combination with paclitaxel or cisplatin in NCI-H446 tumors caused a tumor disappearance without regrowth for a long period. Conclusion: These preclinical data suggest the possibility that the strategy of combining ERY974 with chemotherapy may succeed in increasing the clinical benefit. Now the combination effect is being further investigated to clarify the mechanism. Citation Format: Yuji Sano, Yumiko Azuma, Toshiaki Tsunenari, Yasuko Kinoshita, Yoko Kayukawa, Hironori Mutoh, Yoko Miyazaki, Takahiro Ishiguro, Shohei Kishishita, Yoshiki Kawabe, Mika Endo. Combining ERY974, a novel T cell-redirecting bispecific antibody targeting glypican-3, with chemotherapy profoundly improved antitumor efficacy over its monotherapy in xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3653. doi:10.1158/1538-7445.AM2017-3653