A revolution in cancer immunotherapy has recently

A revolution in cancer immunotherapy has recently emerged with the development of novel immunomodulatory antibodies, classified as immunostimulatory agonist monoclonal chemical this article (mAbs) or checkpoint blocking mAbs. CD40/CD40L, as an important costimulatory pathway, mediates a broad spectrum of systemic immune processes, including T cell-dependent humoral responses (Kawashita et al., 2014). Malignant tumors were shown to inhibit the expression of CD40L on T lymphocytes, leading to arrest of antigen-presenting cells (APCs) and contributing to tumor immune escape (Yang et al., 2013). SGN-40 is an agonistic anti-CD40 mAb that has been used in the treatment of various hematological and lymphoid tumors (Chawla et al., 2013). However, mAbs have a high molecular weight and poor tissue penetration. To achieve a satisfactory therapeutic concentration, drugs must maintain a high plasma concentration, and the high metabolic burden of the liver and kidneys that accompanies treatment should not be ignored (Singer et al., 2008). Therefore, it is particularly important to rationally modify the antibody structure and develop multiple functional antibodies. Recently, recombinant antibody (RAb) technology has provided an alternative approach for engineering low-cost antibodies with desirable affinities and specificities (Yau et al., 2003; Kramer and Hock, 2003). Bispecific antibodies (BsAbs), which are capable of simultaneous binding to two different targets, could overcome many defects of mAb therapies (Schrama et al., 2006). Bispecificity can be used in cancer immunotherapy to crosslink tumor cells to immune cells, such as cytotoxic T cells. This crosslinking accelerates the disruption of tumor cells by immune cells, which could improve antitumor therapy efficiency and lower costs by decreasing the doses needed for therapy (Cao, 2003; Kufer et al., 2004). In our previous research, a high-affinity CD40 single-chain antibody clone was screened, and its function was verified. In this study, we used gene recombination technology to splice CD40 single-chain antibody clones and HER2 single-chain antibody clones to obtain an anti-CD40 × HER2 single chain diabody (ScDb). The HER2 targeting function and immune activation function of this molecule were verified both in vitro and in vivo.
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Results
Discussion The limited options in the treatment of malignancy have encouraged the search for new strategies. One promising approach is the recruitment of cytotoxic immune effector cells to tumor cells by BsAbs or diabodies. Studies have shown that activation of tumor-specific cytotoxic T cells (CTLs) can inhibit tumor proliferation in mouse tumor models (Yan et al., 2015). Therefore, the induction of specific T cell activation against tumors is currently one of the cores of tumor immunotherapy research (Lei et al., 2016). To date, several studies have compared various small BsAb formats, including multimeric formats, and their differences in function; however, no consensus has been established on the most appropriate format (Korn et al., 2004; Kipriyanov et al., 2003; Mølhøj et al., 2007). In the present study, we developed CD40 × HER2 ScDb, a diabody targeting HER2 and CD40, and examined the function of small BsAb formats using cytotoxicity assays, ELISA, western blot, and IHC. CD40/CD40 L is the main second signal system. Studies have found that tumor cells can inhibit the expression of the second signaling system by expressing several inhibitory immune factors, which contribute to blocking the maturation of DCs and inhibiting the production of effector T cells. Immune tolerance was ultimately induced (Yang et al., 2013). Kosaka (Kosaka et al., 2014) showed that agonistic antibodies against CD40 could bind to CD40 on DCs, promote their maturation and act as CD4+ helper T cells. In a study by Khong (Khong et al., 2013), FGK45 (CD40 mAb) was shown to promote DC cell maturation and induce effector T cell activation by binding to CD40, thereby inhibiting tumor proliferation.