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Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody (mAb) by Neutralizing TNF Using an In Vitro Bioanalytical Method
Published on: September 16, 2017
Compromised anti-tumor responses in tumor necrosis factor-alpha knockout mice
C N Baxevanis1, I F Voutsas, O E Tsitsilonis
1Cancer Immunology and Immunotherapy Center, St. Savas Cancer Hospital, Athens, Greece.
Abstract:
The response of lymphokine-activated killer (LAK) and natural killer (NK) cells from mice lacking tumor necrosis factor-alpha (TNF-alpha-/- mice) was impaired in cytotoxicity assays against various tumor cell targets. Furthermore, allogeneic cytotoxic T lymphocyte (CTL) responses were also impaired as compared to TNF-alpha+/+ littermates (control mice). Cytotoxicity was restored both upon in vitro incubation of TNF-alpha-/- lymphocytes with recombinant TNF-alpha (rTNF-alpha) or upon in vivo treatment of TNF-alpha-/- mice with rTNF-alpha. Using combinations of monoclonal antibodies we were able to show that TNF-alpha-/- effector lymphocytes exhibit both perforin- and Fas ligand-based cytotoxicity. Furthermore, upon in vivo administration of rTNF-alpha these effectors, in addition to perforin and Fas ligand, are also armed with TNF-alpha cytotoxic molecules, thus resembling to the cytotoxic effectors from control mice. In a tumor model, immunized TNF-alpha-/- mice failed to reject the syngeneic fibrosarcoma MC57X, but did so when injected with rTNF-alpha. In vivo administration of anti-TNF-alpha mAb neutralized the effect of rTNF-alpha supporting the growth of MC57X cells. Our data provide novel evidence for TNF-alpha as an essential factor in (i) controlling cytotoxicity in vitro and in vivo and (ii) promoting tumor rejection in vivo.
Insights
Tumor necrosis factor-alpha (TNF-alpha) is crucial for immune cell cytotoxicity and tumor rejection. Restoring TNF-alpha in deficient mice enhances natural killer cell and cytotoxic T lymphocyte responses against tumors.
Area of Science:
- Immunology
- Cancer Biology
- Cellular Cytotoxicity
Background:
- Tumor necrosis factor-alpha (TNF-alpha) plays a role in immune responses.
- The specific contribution of TNF-alpha to the function of lymphokine-activated killer (LAK) and natural killer (NK) cells, as well as cytotoxic T lymphocyte (CTL) responses, requires further elucidation.
Purpose of the Study:
- To investigate the role of TNF-alpha in the cytotoxic activity of immune cells.
- To determine the impact of TNF-alpha on tumor rejection in vivo.
- To elucidate the mechanisms by which TNF-alpha influences cellular cytotoxicity.
Main Methods:
- Cytotoxicity assays using LAK, NK, and CTL cells from TNF-alpha deficient (TNF-alpha-/-) and wild-type (TNF-alpha+/+) mice.
- In vitro and in vivo administration of recombinant TNF-alpha (rTNF-alpha).
- Monoclonal antibody-mediated blockade of TNF-alpha.
- Tumor rejection models using syngeneic fibrosarcoma MC57X.
Main Results:
- TNF-alpha-/- lymphocytes exhibited impaired cytotoxicity against tumor targets compared to controls.
- Cytotoxicity was restored in TNF-alpha-/- cells upon rTNF-alpha treatment, both in vitro and in vivo.
- TNF-alpha-/- effector cells utilized perforin and Fas ligand pathways; rTNF-alpha treatment armed them with TNF-alpha cytotoxic molecules.
- Immunized TNF-alpha-/- mice failed to reject syngeneic tumors, but rejection was achieved with rTNF-alpha administration.
- Anti-TNF-alpha monoclonal antibody neutralized rTNF-alpha's tumor-promoting effects.
Conclusions:
- TNF-alpha is essential for optimal in vitro and in vivo immune cell cytotoxicity.
- TNF-alpha plays a critical role in promoting tumor rejection.
- Restoration of TNF-alpha activity can overcome deficits in immune cell-mediated tumor surveillance and rejection.
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