These results suggest that NOG-dKO mice might serve as a good humanized immunotherapy model to evaluate the efficacy of anti-PD-1 antibody prior to the clinical treatment. Clin Cancer Res; 23(1); 149-58. ©2016 AACR.

Purpose: The B7 homolog 4 (B7-H4, VTCN1) is an immune checkpoint molecule that negatively regulates immune responses and is known to be overexpressed in many human cancers. Previously, we generated a mouse anti-human B7-H4 mAb that did not have a significant antitumor effect in vivo probably because of molecule instability. In this study, we designed a B7-H4/CD3-bispecific antibody (BsAb) and investigated its antitumor activity in vitro and in vivo using a humanized mouse model. Experimental Design: cDNAs of the antibody-binding fragment (Fab)-single-chain variable fragment (scFv) and scFv-scFv of the anti-B7-H4/CD3 BsAb were synthesized, and the BsAb antibodies were produced in HEK293 cells. The antitumor activity against human breast cancer cells by human peripheral blood mononuclear cells (hPBMC) with BsAb was measured by lactate dehydrogenase release in vitro, and in vivo using hPBMC-transplanted MHC class I-and class II-deficient NOG mice. Results: hPBMCs with anti-B7-H4/CD3 BsAbs successfully lysed the human breast cancer cell line MDA-MB-468 (EC 50 : 0.2 ng/mL) and other B7-H4 þ cell lines in vitro. When BsAb was injected in a humanized mouse model, there was an immediate and strong antitumor activity against MDA-MB-468, HCC-1954, and HCC-1569 tumors and CD8 þ and granzyme B þ CTL infiltration into the tumor, and there were no adverse effects after long-term observation. CD8 þ T-cell depletion by an anti-CD8 antibody mostly reduced the antitumor effect of BsAb in vivo. Conclusions: An anti-B7-H4/CD3 BsAb may be a good therapeutic tool for patients with B7-H4 þ breast cancers.

Abstract. Background: Temozolomide-resistant (TMZ-R) glioblastoma is very difficult to treat, and a novel approach toGlioblastoma (GB) is one of the most malignant and aggressive tumors and has a very poor prognosis, with a mean survival time of less than 2 years even with the recent development of temozolomide (TMZ)-based intensive treatment (1, 2). Once recurrence develops, there are few therapeutic approaches to control the growth of glioblastoma. Therefore, TMZ-resistant GB is very difficult to treat, and a novel approach to overcome resistance is needed.With regard to the mechanism of TMZ resistance, O 6 -methylguanine-DNA -methyltransferase (MGMT) removes methylation from the O 6 position of guanine and contributes to TMZ resistance induction (3). It is generally accepted that high MGMT expression through the methylation of the MGMT promoter is one of the mechanisms responsible for TMZ resistance. Alternatively, several novel biomarkers linked to MGMT expression and the methylation status such as the HOX signature and epidermal growth factor receptor (EGFR) expression (4), somatic mutation of mismatch repair gene mutS homolog (MSH)6 (5), prolyl 4-hydroxylase, beta polypeptide (P4HB), EGFR mutation (EGFRvIII) (6), CD74 and signal transducer and activator of transcription (STAT)3 signaling have been reported. Kohsaka et al. reported the association of STAT3 expression with MGMT expression level using small interfering (si)RNA-mediated STAT3 gene inhibition (7).Additionally, mammalian target of rapamycin (mTOR) signaling is activated in TMZ-resistant glioma cells as a result of EGFR, phosphoinositide 3 kinase (PI3K) and Akt signaling activation. mTOR is a Ser/Thr kinase that belongs to the phosphoinositide kinase-related family of protein kinases (PIKKs). mTOR acts as an essential integrator of growth-factor-activated and nutrient-sensing pathways to control and coordinate various cellular functions, including survival, proliferation, differentiation, autophagy and metabolism (8)(9)(10)(11) This article is freely accessible online. *These authors contributed equally to this study.Abbreviations: GB: Glioblastoma, TMZ: temozolomide, MGMT: O 6 -methylguanine-O 6 -methylguanine-DNAmethyltransferase, STAT: signal transducer and activator of transcription, mTOR: mammalian target of rapamycin, shRNA: small hairpin RNA.

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+ , exhibited an upregulation of cytotoxic T lymphocyte (CTL) killing-associated genes, T-cell activation genes, antigen-presentation and dendritic cell (DC) maturation genes, and T-cell-attracting chemokine genes, which promoted Th1 antitumor responses. By contrast, type C, with PD-L1and CD8B-, exhibited a low expression of T-cell-activating genes and an upregulation of cancer driver gene signaling, which suggested an immune-suppressive status. With regard to hypermutator tumors, PD-L1 + hypermutator cases exhibited a specific upregulation of the IL6 gene compared with the PD-L1cases. On the whole, our data indicate that the classification of the TME immune types may prove to be a useful tool for evaluating the immunological status and predicting antitumor responses and prognosis.