Antibody-cytokine complexes may offer new tools to treat cancer. Here, we show how TNF-linked antibodies, which recognize tumor-selective splice isoforms of fibronectin (F8-TNF), can be exploited to eradicate sarcomas in immunocompetent mice. We treated mice bearing WEHI-164 fibrosarcoma with a combination of F8-TNF and doxorubicin, curing the majority of treated animals (29/37). Notably, cured mice were resistant to rechallenge not only by WEHI-164 cells but also heterologous C51 or CT26 colorectal tumor cells in a CD8þ T-cell-dependent process.Mechanistic analyses revealed that each tumor cell line presented AH1, a common endogenous retroviral peptide. Numbers of AH1-specific CD8 þ T cells exhibiting cytotoxic capacity were increased by F8-TNF plus doxorubicin treatment, arguing that cognate CD8 þ T cells contributed to tumor eradication. Sequence analysis of T-cell receptors of CD8 þ T cells revealed the presence of/AH1-specific T cells and an expansion of sequence diversity in treated mice. Overall, our findings provide evidence that retroviral genes contribute to tumoral immunosurveillance in a process that can be generally boosted by F8-TNF and doxorubicin treatment. Cancer Res; 77(13); 3644-54. Ó2017 AACR.

As an alternative to polyethylene glycol (PEG), electric field pulses offer, in theory, fusion conditions whose parameters are better controllable. In 1985 (1) we reported on the successful generation of hybridoma clones by means of electrofusion performed in a batch-type manner similar to that usually employed with PEG, and applicable to any type of antigens. Here we summarize the results of a series of fusions performed since then in which both electric field and PEG induced fusion were directly compared. Different types of antigens were used. Electrofusion resulted in a 3.8 to 33.0 times higher yield of hybridomas per unit number of spleen cells. Moreover, hybridomas grew more vigorously after fusion and, therefore, were earlier visible. Other parameters examined revealed no differences between hybridomas generated by either method.

A modification of the Hellström method for measuring cell‐mediated immunity to tumor‐associated antigens is described using mouse mammary tumors as a model. Target tumor cells are incubated with lymphocytes for 1 h and then cloned by the dilute agar method. The number of surviving cell colonies following incubation with immune lymphocytes is compared to the number of surviving tumor cell colonies incubated with control lymphocytes. Significant inhibition is obtained with as few as 10 immune lymphocytes for every target cell. Serum from immune animals can be shown capable of blocking lymphocyte‐mediated inhibition of tumor colony formation. The data obtained with this method are very similar to those obtained with the Hellström test. The advantages and disadvantages of the modification, as compared to the Hellström test, are discussed.

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<div>Abstract<p>Antibody–cytokine complexes may offer new tools to treat cancer. Here, we show how TNF-linked antibodies, which recognize tumor-selective splice isoforms of fibronectin (F8-TNF), can be exploited to eradicate sarcomas in immunocompetent mice. We treated mice bearing WEHI-164 fibrosarcoma with a combination of F8-TNF and doxorubicin, curing the majority of treated animals (29/37). Notably, cured mice were resistant to rechallenge not only by WEHI-164 cells but also heterologous C51 or CT26 colorectal tumor cells in a CD8⁺ T-cell–dependent process. Mechanistic analyses revealed that each tumor cell line presented AH1, a common endogenous retroviral peptide. Numbers of AH1-specific CD8⁺ T cells exhibiting cytotoxic capacity were increased by F8-TNF plus doxorubicin treatment, arguing that cognate CD8⁺ T cells contributed to tumor eradication. Sequence analysis of T-cell receptors of CD8⁺ T cells revealed the presence of H-2L^d/AH1-specific T cells and an expansion of sequence diversity in treated mice. Overall, our findings provide evidence that retroviral genes contribute to tumoral immunosurveillance in a process that can be generally boosted by F8-TNF and doxorubicin treatment. <i>Cancer Res; 77(13); 3644–54. ©2017 AACR</i>.</p></div>

<p>FACS analysis of AH1-specific CD8+ T cells</p>

<p>FACS analysis of AH1-specific CD8+ T cells</p>

<p>Immunofluorescence analysis with MHC I tetramers on spleen and tumor sections</p>