Yasuko Kinoshita

Anti–Glypican 3 Antibody as a Potential Antitumor Agent for Human Liver Cancer

Human glypican 3 (GPC3) is preferentially expressed in the tumor tissues of liver cancer patients. In this study, we obtained a monoclonal antibody (mAb) against the COOH-terminal part of GPC3, which induced antibody-dependent cellular cytotoxicity (ADCC). The mAb, designated GC33, exhibited marked tumor growth inhibition of s.c. transplanted Hep G2 and HuH-7 xenografts that expressed GPC3 but did not inhibit growth of the SK-HEP-1 that was negative for GPC3. GC33 was efficacious even in an orthotopic model; it markedly reduced the blood alpha-fetoprotein levels of mice intrahepatically transplanted with Hep G2 cells. Humanized GC33 (hGC33) was as efficacious as GC33 against the Hep G2 xenograft, but hGC33 lacking carbohydrate moieties caused neither ADCC nor tumor growth inhibition. Depletion of CD56+ cells from human peripheral blood mononuclear cells markedly abrogated the ADCC caused by hGC33. The results show that the antitumor activity of hGC33 is mainly attributable to ADCC, and in human, natural killer cell-mediated ADCC is one possible mechanism of the antitumor effects by GC33. hGC33 will provide a novel treatment option for liver cancer patients with GPC3-positive tumors.

Identification of ROBO1 as a Novel Hepatocellular Carcinoma Antigen and a Potential Therapeutic and Diagnostic Target

Purpose: Hepatocellular carcinoma is the most common primary malignancy of the liver and accounts for as many as one million deaths annually worldwide. The present study was done to identify new transmembrane molecules for antibody therapy in hepatocellular carcinoma. Experimental Design: Gene expression profiles of pooled total RNA from three tissues each of moderately differentiated and poorly differentiated hepatocellular carcinoma were compared with those of normal liver, noncancerous liver tissue in hepatocellular carcinoma patients, 30 normal tissue samples, and five fetal tissue samples. Target genes up-regulated specifically in hepatocellular carcinoma were validated by immunohistochemical analysis and complement-dependent cytotoxicity assay using monoclonal antibodies generated against target molecules. Results: The human homologue of the Drosophila Roundabout gene, axon guidance receptor homologue 1, ROBO1/DUTT1, a member of the immunoglobulin superfamily, was highly expressed in hepatocellular carcinoma, whereas it showed only a limited distribution in normal tissues. On immunohistochemical analysis using a newly generated anti-ROBO1 monoclonal antibody, positive signals were observed in 83 of 98 cases of hepatocellular carcinoma (84.7%). The mAb B2318C induced complement-dependent cytotoxicity in ROBO1-expressing cell lines and in the liver cancer cell line PLC/PRF/5. Strikingly, the ectodomain of ROBO1 was detected not only in the culture medium of liver cancer cell lines (PLC/PRF/5, HepG2, etc.) but also in sera from hepatocellular carcinoma patients (6 of 11). Conclusions: This is the first report that ROBO1 is overexpressed in hepatocellular carcinoma and shed into serum in humans. These observations suggest that ROBO1 is a potential new serologic marker for hepatocellular carcinoma and may represent a new therapeutic target.

Anti-glypican 3 antibodies cause ADCC against human hepatocellular carcinoma cells

Glypican 3 (GPC3), a GPI-anchored heparan sulfate proteoglycan, is expressed in the majority of hepatocellular carcinoma (HCC) tissues. Using MRL/lpr mice, we successfully generated a series of anti-GPC3 monoclonal antibodies (mAbs). GPC3 was partially cleaved between Arg358 and Ser359, generating a C-terminal 30-kDa fragment and an N-terminal 40-kDa fragment. All mAbs that induced antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) against cells expressing GPC3 recognized the 30-kDa fragment, indicating that the C-terminal region of GPC3 serves as an epitope for mAb with ADCC and/or CDC inducing activities. Chimeric mAbs with Fc replaced by human IgG1 were created from GC33, one of the mAbs that reacted with the C-terminal 30-kDa fragment. Chimeric GC33 induced not only ADCC against GPC3-positive human HCC cells but also was efficacious against the Huh-7 human HCC xenograft. Thus, mAbs against the C-terminal 30-kDa fragment such as GC33 are useful in therapy targeting HCC.

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.

A Single-Chain Fv Diabody against Human Leukocyte Antigen-A Molecules Specifically Induces Myeloma Cell Death in the Bone Marrow Environment

Cross-linked human leukocyte antigen (HLA) class I molecules have been shown to mediate cell death in neoplastic lymphoid cells. However, clinical application of an anti-HLA class I antibody is limited by possible side effects due to widespread expression of HLA class I molecules in normal tissues. To reduce the unwanted Fc-mediated functions of the therapeutic antibody, we have developed a recombinant single-chain Fv diabody (2D7-DB) specific to the alpha2 domain of HLA-A. Here, we show that 2D7-DB specifically induces multiple myeloma cell death in the bone marrow environment. Both multiple myeloma cell lines and primary multiple myeloma cells expressed HLA-A at higher levels than normal myeloid cells, lymphocytes, or hematopoietic stem cells. 2D7-DB rapidly induced Rho activation and robust actin aggregation that led to caspase-independent death in multiple myeloma cells. This cell death was completely blocked by Rho GTPase inhibitors, suggesting that Rho-induced actin aggregation is crucial for mediating multiple myeloma cell death. Conversely, 2D7-DB neither triggered Rho-mediated actin aggregation nor induced cell death in normal bone marrow cells despite the expression of HLA-A. Treatment with IFNs, melphalan, or bortezomib enhanced multiple myeloma cell death induced by 2D7-DB. Furthermore, administration of 2D7-DB resulted in significant tumor regression in a xenograft model of human multiple myeloma. These results indicate that 2D7-DB acts on multiple myeloma cells differently from other bone marrow cells and thus provide the basis for a novel HLA class I-targeting therapy against multiple myeloma.

A new disulfide-linked dimer of a single-chain antibody fragment against human CD47 induces apoptosis in lymphoid malignant cells via the hypoxia inducible factor-1α pathway

CD47 belongs to the immunoglobulin superfamily and is associated with β‐integrins. Recently it was reported that CD47 ligation rapidly induces apoptosis in B‐chronic lymphocytic leukemia (CLL) cells. Chronic lymphocytic leukemia is still an incurable hematological malignancy even with the novel therapeutic agents; therefore, new and effective agents for the treatment of CLL in clinical settings are urgently needed. We generated a murine monoclonal antibody against an extracellular domain of human CD47 (designated MABL). Subsequently, we created a disulfide‐stabilized dimer of a single‐chain antibody fragment of MABL (S‐S diabody) to get rid of the adverse effect of MABL such as hemagglutination. In this study, we analyzed the effects of this new antibody on cellular proliferation, and the molecular mechanism of CD47‐mediated apoptosis in human lymphoid malignant cells. Treatment with S‐S diabody alone induced apoptosis of CD47‐positive primary B‐CLL and leukemic cells (MOLT‐4 and JOK‐1). In addition, administration of S‐S diabody significantly prolonged the survival of severe combined immunodeficiency (SCID) mice inoculated with JOK‐1 cells. In gene expression profiling of the S‐S diabody‐treated MOLT‐4 cells, hypoxia inducible factor (HIF)‐1α downstream genes (RTP801 and BNIP3) were upregulated, and the mRNA expression levels of HIF‐1α, RTP801 and BNIP3 were increased. Knockdown of HIF‐1α by siRNA repressed S‐S diabody‐induced apoptosis in MOLT4 cells. In conclusion, CD47 will be a molecular target for the treatment of lymphoid malignancies, and S‐S diabody might have potential as a novel therapeutic agent for B‐CLL. (Cancer Sci 2011; 102: 1208–1215)

Histopathological analyses of the antitumor activity of anti-glypican-3 antibody (GC33) in human liver cancer xenograft models: The essential role of macrophages

Previously, we demonstrated the antitumor efficacy of the anti-glypican-3 (GPC3) antibody GC33 in several human liver cancer xenograft models and the important role of antibody-dependent cellular cytotoxicity (ADCC) in the antitumor mechanism of GC33. Involvement of other mechanisms such as modulation of the functions of GPC3 in antitumor activity remains to be elucidated. In this study, we investigated histopathologically time-course changes in xenografts in mice following a single administration of GC33 to clarify the morphological changes contributing to the tumor growth inhibition of GC33, including the changes in GPC3-related factors/components (proliferation, extracelluar matrices (ECMs) and macrophage). Histopathological changes peaked 3–5 days after GC33 administration and included increased tumor cell death, tumor cells with round morphology, multinucleated tumor cells and small spindle/round-like cells (mostly F4/80-positive macrophages). No direct effects of GC33 on proliferation activity of tumor cells were observed. Meanwhile, alteration of ECM structures and a remarkable increase in macrophages was noted in the GC33-treated group. Increase in macrophages was observed mainly in the outer layer of tumor nodules; the area of the increase approximately included the area where the change in tumor cells and ECMs were observed. Interestingly, depletion of macrophages in the xenograft models resulted in a marked reduction of the antitumor activity of GC33. In the in vitro ADCC assay, ADCC was only slightly induced by mouse peritoneal macrophages. These data suggest that macrophages play an essential role in the antitumor activity of GC33 and the possible involvement of macrophage-mediated non-ADCC action.

Optimization of tissue processing for immunohistochemistry for the detection of human glypican-3

Glypican-3 (GPC3) is frequently upregulated in hepatocellular carcinoma (HCC) and data on the expression profile in HCC might be useful for therapeutic decision-making and prognostic prediction. This study was performed using HepG2 xenograft tissues to optimize the tissue processing method for GPC3 immunohistochemistry. The optimization was conducted in terms of using GPC3 immunohistochemistry for biological study of GPC3 (Experiment 1) and as a diagnostic tool (Experiment 2). In Experiment 1, GPC3 immunoreactivity (IR) and tissue architecture were compared among differently fixed and embedded specimens. In Experiment 2, using conventional formalin-fixed paraffin-embedded (FFPE) procedures, the effects of different fixation times and antigen retrieval treatments were assessed. In Experiment 1, the periodate-lysine-paraformaldehyde (PLP)-fixed and AMeX method-embedded (PLP-AMeX) specimen showed superior immunoreactivity and excellent tissue architecture preservation. In contrast, the other specimens, especially frozen specimens, resulted in poor IR. In Experiment 2, specimens fixed for 24h showed better IR than those fixed for 7 days and the most remarkable improvement in IR was achieved after protease treatment. These findings indicate that with GPC3 immunohistochemistry for biological studies, the PLP-AMeX specimen is preferable. For diagnostics using FFPE specimens, the fixation time should not be too long and protease should be used for the antigen retrieval treatment.

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

An anti–glypican 3/CD3 bispecific T cell–redirecting antibody (ERY974) is a promising therapeutic agent for solid tumors. Double trouble for solid tumors Because the endogenous immune response is not enough to clear a patient’s cancer, therapies are being designed to redirect T cells to tumor cells. This can be done by engineering the cells ex vivo, such as in CAR T cell therapy, or in vivo, such as with bispecific antibodies. Ishiguro et al. describe the development and preclinical testing of a bispecific antibody recognizing CD3 and glypican 3, a common antigen on solid tumors. This bispecific antibody was effective in a variety of mouse cancer models, even when treatment was initiated after the tumor was quite large. Treatment also appeared to be safe when administered to monkeys. These results suggest further development of this antibody for therapeutic use in multiple cancer types. 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.