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.

Recently, the first series of small molecule inhibitors of PD-1/PD-L1 were reported by Bristol-Myers Squibb (BMS), which were developed using a homogeneous time-resolved fluorescence (HTRF)-based screening investigation of the PD-1/PD-L1 interaction. Additional crystallographic and biophysical studies showed that these compounds inhibited the interaction of PD-1/PD-L1 by inducing the dimerization of PD-L1, in which each dimer binds one molecule of the stabilizer at its interface. However, the immunological mechanism of the antitumor effect of these compounds remains to be elucidated. In the present study, we focused on BMS-202 (a representative of the BMS compounds) and investigated its antitumor activity using in vitro and in vivo experiments. BMS-202 inhibited the proliferation of strongly PD-L1-positive SCC-3 cells (IC 50 15 μM) and anti-CD3 antibody-activated Jurkat cells (IC 50 10 μM) in vitro. Additionally, BMS-202 had no regulatory effect on the PD-1 or PD-L1 expression level on the cell surface of these cells. In an in vivo study using humanized MHC-double knockout (dKO) NOG mice, BMS-202 showed a clear antitumor effect compared with the controls; however, a direct cytotoxic effect was revealed to be involved in the antitumor mechanism, as there was no lymphocyte accumulation in the tumor site. These results suggest that the antitumor effect of BMS-202 might be partly mediated by a direct off-target cytotoxic effect in addition to the immune response-based mechanism. Also, the humanized dKO NOG mouse model used in this study was shown to be a useful tool for the screening of small molecule inhibitors of PD-1/PD-L1 binding that can inhibit tumor growth via an immune-response-mediated mechanism.

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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