A novel anti-CTLA-4 nanobody-IL12 fusion protein in combination with a dendritic cell/tumour fusion cell vaccine enhances the antitumour activity of CD8+ T cells in solid tumours

Abstract

Background: We previously developed a nanobody targeting CTLA-4 and demonstrated that it can boost antitumour T-cell responses in vitro; however, the resulting responses after the injection of T cells into cancer models are usually weak and transient. Here, we explored whether fusing our nanobody to IL-12 would enable it to induce stronger, longer-lasting T-cell immune responses after exposure to immature dendritic cell and tumour cell fusions.

Results: The fusion protein enhanced the response of CD8⁺ T cells to tumour antigens in vitro and led to stronger, more persistent immune responses after the T cells were injected into mice bearing different types of xenografts.

Conclusion: Our in vitro and in vivo results suggest the anticancer potential of our nanobody-interleukin fusion system and support the clinical application of this fusion approach for various nanobodies.

Keywords: Adoptive therapy; CD8+ T cell; CTLA-4; IL-12; Nanobody.

Fig. 1

Schematic of the experimental procedures. Upper panel: Gene sequences encoding the nanobody Nb16 against CTLA-4 and human IL-12 were spliced together with a flexible polypeptide linker, and hexahistidine and FLAG epitope tags were added to the ends. The fusion protein was overexpressed in bacteria and then purified using affinity chromatography against the hexahistidine tag. Lower panel: Each of the three types of tumour cells (see Methods) was fused with immature dendritic cells (DCs), which were mixed with CD8⁺ T cells in the presence of the Nb16-IL12 fusion. The resulting activated T cells were injected into mice bearing xenografts of the same tumour type

Fig. 2

Expression of Nb16-IL12 and confirmation of its specific binding to CTLA-4. (a) SDS‒PAGE followed by Coomassie staining to track the purification of the 100 ~ kDa Nb16-IL12 (arrow) from E. coli using affinity chromatography against the hexahistidine tag. Lanes: M- molecular weight marker, 1- Nb16-IL12 fusion protein. (b) Enzyme-linked immunosorbent assay to evaluate the binding of Nb16-IL12 to human CTLA-4 or human IL-12 receptor. The blank samples contained no Nb16-IL12. (c) Flow cytometry was used to evaluate the binding of Nb16-IL12 to activated T cells (left) or activated Jurkat cells (right). Images are representative of three independent experiments. lrr, Irrelevant; mAb, monoclonal antibody

Fig. 3

Ability of Nb16-IL12 to activate CD8⁺T cells and stimulate their proliferation in vitro CD8⁺ T cells were prelabelled with carboxyfluorescein succinimidyl ester, cocultured with fusions of dendritic tumour cells (FCs) in the presence or absence of Nb16-IL12, isolated and stained with a PE-conjugated monoclonal antibody against CD25 or CD69. The tumour cell types were hepatocellular carcinoma (HepG2, left column), breast cancer (MCF-7, middle column) or melanoma (C8161, right column). (A) Flow cytometry based on immunolabelling against CD25 to assess T-cell activation. (B) Flow cytometry based on immunolabelling against CD69 to assess T-cell activation. (C) Flow cytometry based on carboxyfluorescein fluorescence was used to assess T-cell proliferation. The data in the plots are presented as the mean ± SD of three independent experiments. **p < 0.01, ***p < 0.001, between the indicated groups

Fig. 4

Antitumour activity of CD8⁺ T cells in vitro after stimulation with Nb16-IL12 and dendritic-tumour cell fusions CD8⁺ T cells were stimulated in vitro in the indicated ways and then cocultured with each of the three types of tumour cells. (a) Enzyme-linked immunosorbent assays of interleukin (IL)-2, interferon (IFN)-γ and tumour necrosis factor (TNF)-α in the medium of cocultures. The data are presented as the mean ± SD from three independent experiments. *p < 0.05, ***p < 0.01, ***p < 0.001. (b) CD8⁺ T cells from the different groups were coincubated with CFSE-prestained HepG2, CFSE-MCF7, or CFSE-C8161 tumour cells at E/T ratios of 1:1, 10:1, or 20:1, respectively, with or without Nb16-IL12 for 24 h. The DNA dye propidium iodide (PI) was used for lysis and cell staining. The ratios of the CFSE⁺ PI⁺ cell population were measured using flow cytometry. Line graphs show the mean percentages of specifically lysed cells (CFSE⁺ PI⁺) from three experiments. The results demonstrated that Nb16-IL12 enhanced the cytotoxicity of CD8⁺ T cells induced by DC/tumour-FC to the corresponding tumour cells in vitro. We considered that cells labelled with carboxyfluorescein and propidium iodide had been lysed by cytotoxic T cells. The data are presented as the mean ± SD from three independent experiments. **p < 0.01, ***p < 0.001, between the indicated groups

Fig. 5

Antitumour activity of CD8⁺ T cells in vivo after stimulation with Nb16-IL12 and dendritic-tumour cell fusions. BALB/c nude mice bearing xenografts of each of the three types of tumours were injected with human CD8⁺ T cells that had been stimulated in the indicated ways. As a negative control, T cells were incubated only in PBS. The mice were injected with cells every 7 days for a total of four injections. On Day 33, tumours were collected from some animals in each group, while other animals were monitored for tumour diameter and survival. (a) Schematic of the experimental procedure. s.c., subcutaneous; i.v., intravenous. (b-e) Analysis of animals bearing HepG2 xenografts: (b) representative photographs and (c) weights of HepG2 tumours from animals on Day 33; (d) tumour volume and (e) animal survival as a function of time after treatment. (f-i) Analysis of animals bearing MCF-7 xenografts. (j-m) Analysis of animals bearing C8161 xenografts. The data in the plots are the mean ± SD of four animals per group. *p < 0.05, **p < 0.01, ***p < 0.001, between the indicated groups