A bispecific antibody NXT007 exerts a hemostatic activity in hemophilia A monkeys enough to keep a nonhemophilic state.

BACKGROUND: Emicizumab, a factor (F) VIIIa-function mimetic bispecific antibody (BsAb) to FIXa and FX, has become an indispensable treatment option for people with hemophilia A (PwHA). However, a small proportion of PwHA still experience bleeds even under emicizumab prophylaxis, as observed in the long-term outcomes of clinical studies. A more potent BsAb may be desirable for such patients. OBJECTIVES: To identify a potent BsAb to FIXa and FX, NXT007, surpassing emicizumab by in vitro and in vivo evaluation. METHODS: New pairs of light chains for emicizumab's heavy chains were screened from phage libraries, and subsequent antibody optimization was performed. For in vitro evaluation, thrombin generation assays were performed with hemophilia A plasma. In vivo hemostatic activity was evaluated in a nonhuman primate model of acquired hemophilia A. RESULTS: NXT007 exhibited an in vitro thrombin generation activity comparable to the international standard activity of FVIII (100 IU/dL), much higher than emicizumab, when triggered by tissue factor. NXT007 also demonstrated a potent in vivo hemostatic activity at approximately 30-fold lower plasma concentrations than emicizumab's historical data. In terms of dose shift between NXT007 and emicizumab, the in vitro and in vivo results were concordant. Regarding pharmacokinetics, NXT007 showed lower in vivo clearance than those shown by typical monoclonal antibodies, suggesting that the Fc engineering to enhance FcRn binding worked well. CONCLUSION: NXT007, a potent BsAb, was successfully created. Nonclinical results suggest that NXT007 would have a potential to keep a nonhemophilic range of coagulation potential in PwHA or to realize more convenient dosing regimens than emicizumab.

Engineering a bispecific antibody with a common light chain: Identification and optimization of an anti-CD3 epsilon and anti-GPC3 bispecific antibody, ERY974.

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-CD3ε 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.

An anti-glypican 3/CD3 bispecific T cell-redirecting antibody for treatment of solid tumors.

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.

Generation of a humanized anti-glypican 3 antibody by CDR grafting and stability optimization.

Glypican 3 (GPC3), a glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan, is expressed in a majority of hepatocellular carcinoma tissues. The murine monoclonal antibody GC33 that specifically binds to the COOH-terminal part of GPC3 causes strong antibody-dependent cellular cytotoxicity against hepatocellular carcinoma cells and exhibits strong antitumor activity in the xenograft models. To apply GC33 for clinical use, we generated a humanized GC33 from complementarity-determining region grafting with the aid of both the hybrid variable region and two-step design methods. The humanized antibody bound to GPC3 specifically and induced antibody-dependent cellular cytotoxicity as effectively as a chimeric GC33 antibody. To improve stability of the humanized GC33, we further optimized humanized GC33 by replacing the amino acid residues that may affect the structure of the variable region of a heavy chain. Substitution of Glu6 with Gln in the heavy chain significantly improved the stability under high temperatures. GC33 also has the risk of deamidation of the -Asn-Gly- sequence in the complementarity-determining region 1 of the light chain. As substitution of Asn diminished the antigen binding, we changed the neighboring Gly to Arg to avoid deamidation. The resulting humanized anti-GPC3 antibody was as efficacious as chimeric GC33 against the HepG2 xenograft and is now being evaluated in clinical trials.