Antibody-based delivery of tumor necrosis factor (L19-TNFα) and interleukin-2 (L19-IL2) to tumor-associated blood vessels has potent immunological and anticancer activity in the syngeneic J558L BALB/c myeloma model
To analyze the impact of TNFα or IL2 on human lymphocytes in vitro and the anti-tumor and immune-modifying effects of L19-IL2 and L19-TNFα on subcutaneously growing J558L myeloma in immunocompetent mice.
PBMCs from three healthy volunteers were incubated with IL2, TNFα, or with IL2 plus addition of TNFα (final 20 h). BALB/c J558L mice with subcutaneous tumors were treated with intravenous L19-TNFα plus L19-IL2, or controls. Tumor growth and intra- and peri-tumoral tissues were analyzed for micro-vessel density, necrosis, immune cell composition, and PD1 or PD-L1 expressing cells.
Exposure of PBMC in vitro to IL2, TNFα, or to IL2 over 3 and 5 days plus TNFα for the final 20 h resulted in an approximately 50 and 75% reduction of the CD25low effector cell/CD25high Treg cell ratio, respectively, compared to medium control. IL2 or TNFα increased the proportion of CD4− CD25low effector lymphocytes while reducing the proportion of CD4+ CD25low Teff cells. In the J558L myeloma model, tumor eradication was observed in 58, 42, 25, and 0% of mice treated with L19-TNFα plus L19-IL2, L19-TNFα, L19-IL2, and PBS, respectively. L19-TNFα/L19-IL2 combination caused tumor necrosis, capillary density doubling, peri-tumoral T cell and PD1+ T cell reduction (− 50%), and an increase in PD-L1+ myeloma cells.
IL2, TNFα, or IL2 plus TNFα (final 20 h) increased the proportion of CD4− CD25low effector lymphocytes possibly indicating immune activation. L19-TNFα/L19-IL2 combination therapy eradicated tumors in J558L myeloma BALB/c mice likely via TNFα-induced tumor necrosis and L19-TNFα/L19-IL2-mediated local cellular immune reactions.
KeywordsJ558L myeloma Targeted immunocytokines IL-2 TNF-α, immune modulation of tumor microenvironment PD-L1 PD-1
We thank Paulina Kuczma, Ines Puschendorf, Katja Dörfel, and Edda von der Wall for thoroughly conducting the animal studies and for the immunohistological stainings.
HDM, DN, and HD designed the overall study concept and the animal studies. UH designed and performed the in vitro studies on human PBMCs. UE and BH performed the immunohistologic studies. HDM, UH, and HD wrote the manuscript with the help of UE and BH.
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
Statement of human rights
All procedures performed in studies involving human participants (blood samples obtained from human volunteers) were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Statement of animal welfare
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
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