Immunomodulatory Effects of Novel Immune Costımulator Sa-4-1bbl In Nnk-Induced Lung Cancer Preclınıcal Model

Abstract

Immune checkpoint inhibitors hold promise in treating various malignancies, but their use is limited due to side effects and treatment resistance. Therefore, the immune costimulation pathway is an important target to enhance immune responses and overcome immune suppression in cancer immunotherapy. 4-1BB (CD137) is a potent molecule that initiates a costimulatory signal in the 4-1BB/4-1BBL pathway, making it a promising candidate for new therapeutic approaches that can be developed in the clinic. 4-1BB, also known as TNFR-9, is a costimulatory molecule belonging to the TNFR superfamily. The 4-1BB receptor is not continuously expressed on immune cells; its expression begins with immune activation of the cells. It is expressed in T cells, natural killer cells, and B cells, enhancing their survival and functionality. 4-1BBL is a protein that is functional only in its soluble form. Preclinical studies in mice after cloning the natural ligand for CD137 have shown that 4-1BBL provides therapeutic benefits in various solid and hematological malignancies. Agonistic 4-1BB antibodies have been used in both preclinical and clinical studies, but their application has been hindered due to reported hepatotoxicity. This limitation presents challenges for the broader clinical adoption of approaches involving agonistic 4-1BB monoclonal antibodies. Therefore, in this thesis, an alternative structure of the 4-1BBL protein has been designed. The recombinant form of the 4-1BBL protein was produced by fusing the extracellular segments of mouse 4-1BBL with streptavidin. In this thesis study, during the first 8 weeks, NNK, the most potent carcinogen in tobacco, was administered intraperitoneally to A/J mice weekly to induce lung cancer. To investigate the immunomodulatory effects of SA-4-1BBL, mice were injected subcutaneously with 100 μg SA-4-1BBL in the sixth and eighth weeks. The mice were observed for toxicity and side effects during an 18-week follow-up through their body weights and general examinations. Under the same conditions of NNK and SA-4-1BBL administration, some mice were separated, and in vivo depletion of different immune cell populations was performed. Depletion antibodies with in vivo activity and reactivity for CD4+, CD8+ T lymphocytes, and NK cells were administered intraperitoneally to the depletion group animals in the sixth and eighth weeks of NNK injections. The animals were monitored for clinical status and body weights to investigate possible side effects of in vivo depletion antibodies. In vivo cell depletion was observed to create no differences among experimental groups. The doses of in vivo depletion antibodies used were sufficient to cause depletion of CD4 and CD8 lymphocytes. At the end of the 18th week, the mice were sacrificed, and their lungs were evaluated for macroscopic and microscopic tumor formation. In this evaluation, it was observed that the number of macroscopic and microscopic tumors in the lung tissues of the mice treated with SA-4-1BBL was significantly reduced compared to the control group that did not receive the treatment. In vivo depletion of CD4+, CD8+ T lymphocytes, and NK cells abolished the protective effect of SA-4-1BBL monotherapy. A significant increase in macroscopic and microscopic tumor nodule formation was observed in the group with depleted CD4+ T cells. In vivo depletion of CD8+ T lymphocytes and NK cells resulted in a marked increase in microscopic tumor nodule formation, but no significant change was observed in macroscopic tumor nodule formation. These findings suggest that CD4+ T lymphocytes are essential for establishing an antitumor immune response, which aligns with a previous study using SA-4-1BBL. Given the nature of carcinogen-induced cancer models, it is possible to form metastases or tumor foci in different organs. Therefore, the mice were thoroughly evaluated on the termination day, and all organ systems were checked for metastases and tumor formation. Interestingly, one mouse in the in vivo CD4+ T lymphocyte depletion group showed a metastatic mass near the mandible. This lesion was also removed on the termination day and tissue was collected for histopathological analysis. The analyses showed that this lesion was metastatic and had the same histopathological phenotype as the nodules observed in the lungs. In flow cytometry analysis of leukocytes infiltrating the tumor, the mass showed a 14-fold increase in PD1+ CD4+ T lymphocytes and PD1+ CD8+ T lymphocytes (PD-1, programmed cell death protein 1) compared to the lungs with microscopic tumor nodules. Additionally, in vivo depletion of CD8+ T lymphocytes significantly reduced the protective effect of SA-4-1BBL monotherapy. The microscopic tumor nodules in mice treated with SA-4-1BBL but depleted of T lymphocytes or NK cells were significantly larger compared to the NNK control under comparable conditions. These data indicate that the immunotherapeutic efficacy of SA-4-1BBL relies on the presence of T lymphocytes and NK cells. Furthermore, lungs and lung-draining lymph nodes were collected and processed for detailed immunophenotyping to isolate immune cells infiltrating the tissue and compare differences in immune cell populations among different treatment groups. Compared to the control group (NNK + saline), it was shown that SA-4-1BBL significantly increased the absolute number of CD4+ central memory T cells (CD4+CD44highCD62Lhigh) in the lungs and lung-draining lymph nodes (p < 0.05). The number of naive CD4+ T lymphocytes (CD4+CD44lowCD62Lhigh) in the lung-draining lymph nodes of the SA-4-1BBL treatment group was significantly higher compared to the control group. Moreover, in the lung-draining lymph nodes of animals treated with SA-4-1BBL, a significant decrease was observed in the absolute number of PD-1+ CD8+ T lymphocytes due to the immunomodulatory effect of SA-4-1BBL. The programmed cell death protein 1 (PD-1) receptor is a critical immune checkpoint receptor primarily expressed on the cell membranes of activated T cells. PD-1/PD-L1 binding provides inhibitory signals that reduce immune activation. The decrease in PD-1+ CD8+ T lymphocytes in the SA-4-1BBL treatment group may serve as an additional mechanism to support its antitumoral immunomodulatory effect. Our study results observed that SA-4-1BBL treatment suppressed lung cancer development. T lymphocytes and NK (natural killer) cells played a significant role in this suppression. In vivo depletion of these immune cells was shown to be responsible for reducing the antitumoral protective effect of SA-4-1BBL. Our thesis study results suggest that SA-4-1BBL, an immune checkpoint stimulator, is a candidate that can create a protective immune response as a single agent in a chemically induced preclinical lung cancer model.