Optimizing TNFR2 antagonism for immunotherapy with tumor microenvironment specificity

Abstract

Most approved cancer immunotherapies lack T‐regulatory (Treg) or tumor specificity. TNF receptor 2 (TNFR2) antibody antagonism is emerging as an attractive immunotherapy due to its tumor microenvironment (TME) specificity. Here we show that the human TNFR2 receptor is overexpressed on both human tumor cells and on human tumor‐residing Tregs, but negligibly expressed on beneficial T effectors (Teffs). Further, we found widespread, if variable, TNFR2 expression on 788 human tumor cell lines from diverse cancer tissues. These findings provided strong rationale for developing a targeted immunotherapy using a TNFR2 antibody antagonist. We designed a novel, human‐directed TNFR2 antibody antagonist and tested it for function using three cell‐based TME assays. The antagonist showed TME specificity by killing of TNFR2‐expressing tumor cells and Tregs, but sparing Teffs, which proliferated. However, the antagonist shuffled between five isoforms, only one of which showed the desirable function. We designed and tested several new chimeric human versions of the antagonist, finding that the IgG2 isotype functioned better than the IgG1 isotype. To further improve function, we introduced targeted mutations to its amino acid sequence to stabilize the natural variability of the IgG2 isotype's hinge. Altogether, our findings suggest that optimal TNFR2 antagonists are of the human IgG2 isotype, have hinge stabilization, and have wide separation of antibody arms to bind to newly synthesized TNFR2 on rapidly growing tumor cells. Antagonistic antibodies with these characteristics, when bound to TNFR2, can form a nonsignaling cell surface dimer that functions with high TME specificity.

TNF receptor agonists induce distinct receptor clusters to mediate differential agonistic activity

“…molecular arrangement leading to super-agonism. It should also be noted that although in our experience the hIgG2 isotype has evoked agonism for CD40 and other TNFRs, there are examples where the heavy chain of an anti-TNFR2 mouse IgG2b has been replaced with that of hIgG2 to provide a more powerful antagonist 54 . It remains unclear if the Fc domain of these chimeric antibodies are required for their antagonistic function but indicates that the underpinning clustering geometry is important in each case.…”

TNFR2: Role in Cancer Immunology and Immunotherapy

“…25,80 Therefore, we (Xin Chen and Joost J. Oppenheim) and Dr. Faustman proposed that TNFR2 may be harnessed as a druggable target to enhance anti-tumor immune responses. 28,61,82,[84][85][86][87][88][89] This idea is supported by a number of studies, as summarized in Table 2.…”

“…82 A recent study of 788 commercially available human cancer cell lines from diverse cancer tissues indicates that various levels of TNFR2 are expressed, while hematopoietic and lymphoid cell lines express the highest levels of TNFR2. 86 An immunohistochemistry (IHC) study of 431 tissue specimens from esophageal squamous cell carcinoma (ESCC) patients found that TNFR2 expression is higher in • Concurrent or a prior dose of TY101 with anti-PD-1 showed maximum benefits.…”

“… 82 A recent study of 788 commercially available human cancer cell lines from diverse cancer tissues indicates that various levels of TNFR2 are expressed, while hematopoietic and lymphoid cell lines express the highest levels of TNFR2. 86 An immunohistochemistry (IHC) study of 431 tissue specimens from esophageal squamous cell carcinoma (ESCC) patients found that TNFR2 expression is higher in malignant tissue than non-tumor esophageal tissues, and the expression of TNFR2 is positively correlated with invasion depth, advanced clinical stage, low differentiation degree, and poor overall survival. 102 Moreover, TNFR2 expression is strongly correlated with the prognosis of middle thoracic ESCC patients than of lower thoracic ESCC patients.…”

“…The optimized version of anti-TNFR2 with IgG2 isoforms stabilize hinge region (disulfide double mutations at C232S and C233S) and the wide separation of antibody arms have demonstrated better TME specificity. 86 Besides, they also reported that the TNFR2 expression pattern provided the rationale for antagonistic treatment effectiveness. The TNFR2 antagonistic killing activity is more potent in the cancer cell line with high TNFR2 expression than the cancer cell line with low TNFR2 expression.…”