Radiotherapy can elicit systemic immune control of local tumors and distant nonirradiated tumor lesions, known as the abscopal effect. Although this effect is enhanced using checkpoint blockade or costimulatory antibodies, objective responses remain suboptimal. As radiotherapy can induce secretion of VEGF and other stress products in the tumor microenvironment, we hypothesized that targeting immunomodulatory drugs to such products will not only reduce toxicity but also broaden the scope of tumor-targeted immunotherapy. Using an oligonucleotide aptamer platform, we show that radiation-induced VEGF-targeted 4-1BB costimulation potentiated both local tumor control and abscopal responses with equal or greater efficiency than 4-1BB, CTLA-4, or PD1 antibodies alone. Although 4-1BB and CTLA-4 antibodies elicited organ-wide inflammatory responses and tissue damage, VEGF-targeted 4-1BB costimulation produced no observable toxicity. These findings suggest that radiation-induced tumor-targeted immunotherapy can improve the therapeutic index and extend the reach of immunomodulatory agents. .

Our laboratory has developed a process for generating mAbs with selectivity to unique peptides in the context of MHC molecules. Recently, we reported that RL4B, an mAb that we have called a TCR mimic (TCRm) because it recognizes peptide in the context of MHC, has cytotoxic activity in vitro and prevented growth of tumor cells in a prophylactic setting. When presented in the context of HLA-A2, RL4B TCRm recognizes the peptide GVLPALPQV derived from human chorionic gonadotropin (hCG)-β. In this study, we show that RL4B TCRm has strong binding affinity for the GVLPALPQV peptide/HLA-A2 epitope and fine binding specificity for cells that express endogenous hCGβ Ag and HLA-A2. In addition, suppression of tumor growth with RL4B TCRm was observed in orthotopic models for breast cancer. Using two aggressive human tumor cell lines, MDA-MB-231 and MCF-7, we provide evidence that RL4B TCRm significantly retards tumor growth, supporting a possible role for TCRm agents in therapeutic settings. Moreover, tumors in mice responded to RL4B TCRm therapy in a dose-dependent manner, eliminating tumors at the highest dose. RL4B TCRm strongly detects the hCGβ peptide/HLA-A2 epitope in human primary breast tumor tissue, but does not react or reacts weakly with normal breast tissue from the same patient. These results further illustrate the selective nature of TCRm Abs and the clinical relevance of the GVLPALPQV peptide/HLA-A2 epitope expression in tumor cells, because they provide the first evidence that Abs that mimic the TCR can be used to markedly reduce and suppress tumor growth.

mAbs that recognize peptides presented on the cell surface by MHC class I molecules are potential therapeutic agents for cancer therapy. We have previously demonstrated that these Abs, which we termed TCR mimic mAbs (TCRm), reduce tumor growth in models of breast carcinoma. However, mechanisms of TCRm-mediated tumor growth reduction remain largely unknown. In this study, we report that these Abs, in contrast to several mAbs used currently in the clinic, destroy tumor cells independently of immune effector mechanisms such as Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). We found that TCRm-mediated apoptosis of tumor cells was associated with selective and specific binding of these Abs to peptide/HLA class I complexes, which triggered the activation of JNK and intrinsic caspase pathways. This signaling was accompanied by the release of mitochondrial cytochrome c and apoptosis-inducing factor. TCRm-induced apoptosis in tumor cells was completely inhibited by soluble MHC tetramers loaded with relevant peptide as well as with inhibitors for JNK and caspases. Furthermore, mAbs targeting MHC class I, independent of the peptide bound by HLA, did not stimulate apoptosis, suggesting that the Ab-binding site on the MHC/peptide complex determines cytotoxicity. This study suggests the existence of mechanisms, in addition to ADCC and CDC, through which these therapeutic Abs destroy tumor cells. These mechanisms would appear to be of particular importance in severely immunocompromised patients with advanced neoplastic disease, since immune cell-mediated killing of tumor cells through ADCC and CDC is substantially limited in these individuals.