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.

Corrigendum: Entire CD3ε, δ, and γ humanized mouse to evaluate human CD3-mediated therapeutics.

This corrects the article DOI: 10.1038/srep45839.

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.

Entire CD3ε, δ, and γ humanized mouse to evaluate human CD3-mediated therapeutics.

T cell-mediated immunotherapy is an attractive strategy for treatment in various disease areas. In this therapeutic approach, the CD3 complex is one of the key molecules to modulate T cell functions; however, in many cases, we cannot evaluate the drug candidates in animal experiments because the therapeutics, usually monoclonal antibodies specific to human CD3, cannot react to mouse endogenous Cd3. Although immunodeficient mice transfused with human hematopoietic stem or precursor cells, known as humanized mice, are available for these studies, mice humanized in this manner are not completely immune competent. In this study we have succeeded in establishing a novel mouse strain in which all the three components of the Cd3 complex - Cd3ε, Cd3δ, and Cd3γ - are replaced by their human counterparts, CD3E, CD3D, and CD3G. Basic immunological assessments have confirmed that this strain of human CD3 EDG-replaced mice are entirely immune competent, and we have also demonstrated that a bispecific antibody that simultaneously binds to human CD3 and a tumor-associated antigen (e.g. ERBB2 or GPC3) can be evaluated in human CD3 EDG-replaced mice engrafted with tumors. Our mouse model provides a novel means to evaluate the in vivo efficacy of human CD3-mediated therapy.

Differentiation and maintenance of mast cells from CD34(+) human cord blood cells.

OBJECTIVE: Establishment of a stable umbilical cord blood CD34(+) (UCB CD34(+)) cell culture system and identification of the cells in the cobblestone area differentiated from UCB CD34(+) long-term culture cells. MATERIALS AND METHODS: Human UCB CD34(+) cells were cultured on MS-5 mouse stroma cells in the presence of stem cell factor (SCF), flt-3 ligand (FL), and thrombopoietin (TPO) for 4 to 16 weeks. Cells in the culture medium and in the cobblestone area were collected and characterized by flow cytometry and microscopy. RESULTS: CD34(+) cells were stably expanded by culturing on MS-5 stroma cells in the presence of SCF, FL, and TPO for more than 4 months. Cells highly expressing CD117 (c-kit) appeared in the cobblestone area after 2 weeks and stably expanded. Isolation of cells highly expressing CD117 by fluorescence-activated cell sorter (FACS) revealed the cells were tryptase-positive and Fc epsilon receptor 1-negative mast cells. They showed typical mast cell morphology and released histamine upon stimulation by substance P or compound 48/80 in vitro. CONCLUSION: Human UCB CD34(+) cells were stably expanded on MS-5 stroma cells in the presence of SCF, FL, and TPO. Under this condition, multipotent CD34(+) cells and mast cells differentiated from UCB CD34(+) cells were expanded in the cobblestone area. The expanded mast cells showed histamine release after substance P or compound 48/80 stimulation. These human mast cells will be useful as a source of human cells for evaluating the allergic effects of drugs.