Although cancer immunotherapy has yielded encouraging outcomes in hematologic malignancies, it has faced challenges in achieving the same level of effectiveness in numerous solid tumors, primarily because of the presence of immunosuppressive tumor microenvironments (TME). The immunosuppressive qualities of the TME have generated considerable interest, making it a focal point for treatments aimed at enhancing immune responses and inhibiting tumor progression. Fibroblast activation protein (FAP), an attractive candidate for targeted immunotherapy, is prominently expressed in the TME of various solid tumors. IL12, recognized as a key mediator of immune responses, has been explored as a potential candidate for cancer treatment. Nevertheless, initial efforts to administer IL12 systemically demonstrated limited efficacy and notable side effects, emphasizing the necessity for innovation. To address these concerns, our molecules incorporated specific IL12 mutations, called IL12mut, which reduced toxicity. This study explored the therapeutic potential of the FAP-IL12mut TMEkine—a novel immunotherapeutic agent selectively engineered to target FAP-expressing cells in preclinical cancer models. Our preclinical results, conducted across diverse murine cancer models, demonstrated that FAP-IL12mut significantly inhibits tumor growth, enhances immune cell infiltration, and promotes a shift toward a cytotoxic immune activation profile. These findings suggest that FAP-IL12mut could offer effective cancer treatment strategies.

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©2024 The Authors; Published by the American Association for Cancer Research
2024
American Association for Cancer Research
This open access article is distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license.
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License .
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Supplementary data

Figure S1

Supplementary Figure 1. In vitro characterization of hFAP-IL12mut. A. Surface plasmon resonance of hFAP-IL12mut on the CM5 chip immobilized with the anti-human IgG antibody. Each protein reacted on the chips and was exposed to a diluted solution of recombinant human FAP protein (hFAP). B. Binding specificity of hFAP-IL12 and hFAP-IL12mut to target cells was confirmed using flow cytometry with human FAP overexpressed HEK293T–hFAP cells and U-87 MG. HEK293T served as a negative control. The target cells were treated with the indicated antibodies (each 1 µg/mL) and then analyzed using flow cytometry with anti-human IgG antibodies to detect the antibodies.

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Figure S2

Supplementary Figure 2. In vitro characterization of mFAP-IL12mut. A. Binding specificity of mFAP-IL12 and mFAP-IL12mut to target the cells was confirmed using flow cytometry with mouse FAP overexpressed NIH3T3-mFAP, CT26-mFAP, and B16F10-mFAP cells. NIH3T3 cells served as a negative control. Target cells were treated with the indicated antibodies and then analyzed using flow cytometry. B. Dose-dependent induction of phosphorylated STAT4 of mFAP-IL12, mFAP-IL12mut, and recombinant mouse IL12 (rmIL12) was determined by phospho-STAT4 alphaLISA assay. C. IFNγ induction assay was assessed on activated mouse splenocytes. Data are presented as mean ± SEM. ****p < 0.0001, ns., not significant, two-tailed unpaired t-test.

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Figure S3

Supplementary Figure 3. FACS analysis of TILs. FACS analysis of tumor-infiltrating leukocyte populations from LLC1/NIH3T3-mFAP tumor-bearing mice on the indicated dates. LLC1/NIH3T3-mFAP tumor-bearing C57BL/6 mice were treated with 10 μg of mFAP-IL12mut once on day 0. For FACS analysis, tumors were harvested on the indicated dates after treatment initiation (n = 3 mice/group). The population of immune cell analysis of dissociated tumors was harvested on days 1, 3, 7, and 10 (n = 3/group). The population of NK cells (CD3−/CD49b+), macrophages (CD11b+/F4/80+), dendritic cells (CD11b+/CD11c+), CD4+ T cells (CD3+/CD4+), activated CD4+ T cells (CD44+/CD62L−/CD25+), effector memory CD4+ T cells (CD44+/CD62L−/CD25−), central memory CD4+ T cells (CD44+/CD62L+), CD8+ T cells (CD3+/CD8+), activated CD8+ T cells (CD44+/CD62L−/CD25+), effector memory CD8+ T cells (CD44+/CD62L−/CD25−), and central memory CD8+ T cells (CD44+/CD62L+) are shown. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ns., not significant, two-tailed unpaired t-test.

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Figure S4

Supplementary Figure 4. Systemic depletion of CD8+ T cells. The graph demonstrates the efficient depletion of CD8+ T cells (CD45+CD3+CD8+) from lymph nodes, spleen, and peripheral blood after six injections of CD8-depleting antibody. Data are presented as mean ± SEM: VC, vehicle control; LNs, lymph nodes.

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Figure S5

Supplementary Figure 5. Efficacy of anti-tumor effect of hFAP-IL12mut in a humanized NOG mouse model. A–B. On day 0, NOG mice were injected intravenously with 7 × 106 human PBMC cells. The humanized mice were subcutaneously inoculated with HT-29 on day 1 and treated with 100 μg or 20 μg of hFAP-IL12mut on day 2. The mean tumor growth over time (A) and individual tumor growth curve (B) (n = 8 mice/group). Data are presented as mean ± SEM. **p < 0.01 vs. control group. Significance was observed on day 14, with multiple unpaired t-tests. QW, once a week.

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