Bewarder Moritz, Kiefer Maximilian, Moelle Clara, Goerens Lisa, Stilgenbauer Stephan, Christofyllakis Konstantinos, Kaddu-Mulindwa Dominic, Fadle Natalie, Regitz Evi, Neumann Frank, Hoth Markus, Preuss Klaus-Dieter, Pfreundschuh Michael, Thurner Lorenz
José Carreras Center for Immuno- and Gene Therapy, Saarland University Medical Center, Homburg, Germany.
Internal Medicine I, Saarland University Medical Center, Homburg, Germany.
Front Oncol. 2020 Nov 12;10:580364. doi: 10.3389/fonc.2020.580364. eCollection 2020.
Recently, neurabin-I and SAMD14 have been described as the autoantigenic target of approximately 66% of B-cell receptors (BCRs) of primary central nervous system lymphomas (PCNSL). Neurabin-I and SAMD14 share a highly homologous SAM domain that becomes immunogenic after atypical hyper-N-glycosylation (SAMD14 at ASN339 and neurabin-I at ASN1277). This post-translational modification of neurabin-I and SAMD14 seems to lead to a chronic immune reaction with B-cell receptor activation contributing to lymphoma genesis of PCNSLs. The selective tropism of PCNSL to the CNS corresponds well to the neurabin-I and SAMD14 protein expression pattern. When conjugated to Pseudomonas Exotoxin A (ETA´), the PCNSL reactive epitope exerts cytotoxic effects on lymphoma cells expressing a SAMD14/neurabin-I reactive BCR. Thus, the reactive epitopes of SAMD14/neurabin-I might be useful to establish additional therapeutic strategies against PCNSL. To test this possibility, we integrated the PCNSL-reactive epitope of SAMD14/neurabin-I into a heavy-chain-only Fab antibody format in substitution of the variable region. Specific binding of the prokaryotically produced SAMD14/neurabin-I Fab-antibody to lymphoma cells and their internalization were determined by flow cytometry. Since no established EBV-negative PCNSL cell line exists, we used the ABC-DLBCL cell lines OCI-Ly3 and U2932, which were transfected to express a SAMD14/neurabin-I reactive BCR. The SAMD14/neurabin-I Fab antibody bound specifically to DLBCL cells expressing a BCR with reactivity to SAMD14/neurabin-I and not to unmanipulated DLBCL cell lines. Eukaryotically produced full-length IgG antibodies are well established as immunotherapy format. Therefore, the PCNSL-reactive epitope of SAMD14/neurabin-I was cloned into a full-length IgG1 format replacing the variable domains of the light and heavy chains. The IgG1-format SAMD14/neurabin-I construct was found to specifically bind to target lymphoma cells expressing a SAMD14/neurabin-I reactive B cell receptor. In addition, it induced dose-dependent relative cytotoxicity against these lymphoma cells when incubated with PBMCs. Control DLBCL cells are not affected at any tested concentration. When integrated into the Fab-format and IgG1-format, the PCNSL-reactive epitope of SAMD14/neurabin-I functions as -cell receptor ntigen for everse targeting (BAR). In particular, the IgG1-format BAR-body approach represents a very attractive therapeutic format for the treatment of PCNSLs, considering its specificity against SAMD14/neurabin-I reactive BCRs and the well-known pharmacodynamic properties of IgG antibodies.
最近，神经素-I（neurabin-I）和含SAM结构域蛋白14（SAMD14）被描述为原发性中枢神经系统淋巴瘤（PCNSL）约66%的B细胞受体（BCR）的自身抗原靶点。神经素-I和SAMD14共享一个高度同源的SAM结构域，该结构域在非典型高N-糖基化后（SAMD14的天冬酰胺339位点和神经素-I的天冬酰胺1277位点）变得具有免疫原性。神经素-I和SAMD14的这种翻译后修饰似乎会引发慢性免疫反应，激活B细胞受体，从而促进PCNSL的淋巴瘤发生。PCNSL对中枢神经系统的选择性嗜性与神经素-I和SAMD14的蛋白表达模式高度吻合。当与铜绿假单胞菌外毒素A（ETA´）偶联时，PCNSL反应性表位对表达SAMD14/神经素-I反应性BCR的淋巴瘤细胞发挥细胞毒性作用。因此，SAMD14/神经素-I的反应性表位可能有助于建立针对PCNSL的额外治疗策略。为了验证这一可能性，我们将SAMD14/神经素-I的PCNSL反应性表位整合到仅重链的Fab抗体形式中，取代可变区。通过流式细胞术测定原核产生的SAMD14/神经素-I Fab抗体与淋巴瘤细胞的特异性结合及其内化情况。由于不存在已建立的EBV阴性PCNSL细胞系，我们使用了ABC-DLBCL细胞系OCI-Ly3和U2932，它们被转染以表达SAMD14/神经素-I反应性BCR。SAMD14/神经素-I Fab抗体特异性结合表达与SAMD14/神经素-I反应性BCR的DLBCL细胞，而不与未处理的DLBCL细胞系结合。真核产生的全长IgG抗体作为免疫治疗形式已得到充分确立。因此，将SAMD14/神经素-I的PCNSL反应性表位克隆到全长IgG1形式中，取代轻链和重链的可变结构域。发现IgG1形式的SAMD14/神经素-I构建体特异性结合表达SAMD14/神经素-I反应性B细胞受体的靶淋巴瘤细胞。此外，当与外周血单个核细胞（PBMC）一起孵育时，它对这些淋巴瘤细胞诱导剂量依赖性的相对细胞毒性。对照DLBCL细胞在任何测试浓度下均不受影响。当整合到Fab形式和IgG1形式中时，SAMD14/神经素-I的PCNSL反应性表位作为反向靶向的B细胞受体抗原（BAR）发挥作用。特别是，考虑到其对SAMD14/神经素-I反应性BCR的特异性以及IgG抗体众所周知的药效学特性，IgG1形式的BAR体方法代表了一种非常有吸引力的治疗PCNSL的形式。
