CD45-Targeted Cytokine Delivery Drives Epitope Spreading for Enhanced Lung Cancer Immunotherapy

Introduction: : Cytokines are potent immunomodulatory signals that have been approved for use as cancer immunotherapy agents. Interleukin-12 (IL-12) is a potent immunostimulatory cytokine with therapeutic potential in cancer immunotherapy. As a canonical T cell priming signal produced by dendritic cells (DCs), IL-12 can potentiate cytotoxic CD8 T cell responses to tumor antigens. Clinical translation of IL-12, however, is limited by its short half-life and high toxicity. Previous attempts to use single-agent IL-12 at tolerable doses to treat orthotopic models of lung cancer have been unsuccessful. Engineering cytokine-antibody conjugates (termed immunocytokines) that bind to CD45 (Figure 1A) have been shown to improve the tolerability and efficacy of cytokines when delivered intratumorally in flank tumor models. By binding CD45—a surface marker on all immune cells with a long membrane residence time—immunocytokines improve cytokine targeting to immune cells and extend cytokine half-life by preventing receptor-mediated internalization. Lung tumors, however, are not conducive to intratumoral administration and are often refractory to immunotherapies due to tissue-specific immune suppression. We therefore sought to investigate the efficacy of single-agent CD45-targeted IL-12 (αCD45-IL12) in both transplantable and genetically engineered mouse models (GEMM) of lung cancer. We found that intraperitoneal (i.p.) administration of low-dose single-agent αCD45-IL12 potentiated a robust multiepitope anti-tumor immune response. These results elucidate the mechanism of action of αCD45-IL12 and demonstrate that CD45-targeted immunocytokines can be effective in cancers where intratumoral administration is not feasible.

Materials and

Methods: : Protein constructs were recombinantly produced in HEK cells and purified using a protein A column and size exclusion chromatography. Syngeneic transplantable KP.SIY tumors, with mutant KrasG12D and p53-/- (KP) driver mutations and expressing the model CD8 T cell antigen SIYRYYGL, were established by tail vein injection. Mice were treated i.p. on days 7 and 11 post-tumor inoculation with 5 μg of αCD45-IL12 or an untargeted control construct (IgG-IL12). Immune cell phenotyping was performed by flow cytometry on a FACSymphony A3 cytometer (BD Biosciences). ELISpot analysis was conducted on day 14 after a single dose of αCD45-IL12 on day 7 using an IFN-γ ELISpot kit (BD Biosciences). For the ELISpot, splenocytes from treated and control mice were cocultured with irradiated KP cells without the SIY antigen to quantify the number of T cells that specifically recognize tumor antigens. Irradiated B16 cancer cells were used as a control for non-specific activation. The lung cancer GEMM was established by intratracheally inoculating KrasG12D/+ p53fl/fl (KP) mice with lentivirus engineered to express the model CD8 T cell antigen Rpl18 and Cre recombinase, which triggers Kras mutation and P53 deletion in infected lung cells. Mice were given six weekly doses of 5 μg of αCD45-IL12 starting at week 6 post lentiviral inoculation. Tumor burden was analyzed at week 12 by quantifying the area of the lung that was tumor by histology.

Results, Conclusions, and Discussions:: KP.SIY lung tumors progress rapidly when left untreated and are refractory to immune checkpoint blockade. By day 21, approximately 40% of the lungs of untreated mice are covered by tumors (Figure 1B). Treating mice with αCD45-IL12 results in a 4-fold reduction in tumor burden with minimal toxicity. Treatment with an untargeted IL-12 immunocytokine (IgG-IL12) failed to reduce tumor burden compared to the control, suggesting that CD45 targeting is necessary for treatment efficacy. The reduction in tumor burden after αCD45-IL12 therapy was accompanied by a significant survival benefit over untreated mice (Figure 1C). Treating an autochthonous KP model of lung cancer with αCD45-IL12 similarly led to a 2-fold reduction in tumor burden, demonstrating that the engineered cytokine remains efficacious even in a challenging-to-treat lung cancer GEMM (Figure 1D). To understand the mechanism driving αCD45-IL12 treatment efficacy, we characterized the CD8 T cell compartment by flow cytometry in the tumor-draining lymph node. Treatment enhanced the cytotoxicity and activation of tumor-reactive CD8 T cells that recognize the model SIY antigen, identified by tetramer staining (Figure 1E). Surprisingly, this increase in effector molecule production was also observed in antigen-experienced CD44+ CD8 T cells that did not recognize the SIY antigen (Figure 1F). We hypothesized that these SIY non-reactive CD8 T cells are being primed by activated cross-presenting dendritic cells (cDC1) to recognize weaker endogenous cancer epitopes. Flow cytometry on myeloid cells revealed that αCD45-IL12 treatment increases the activation of migratory cDC1 and their expression of T cell co-stimulation markers like CD86 (Figure 1G). We then quantified epitope spreading using an IFN-γ ELISpot. Splenocytes from treated mice increased production of IFN-γ after being co-cultured with irradiated KP parental cells that lack the SIY antigen (Figure 1H). The antigen-specificity of this stimulation was confirmed by observing that the splenocytes did not respond to an irrelevant cancer cell line (B16-F10 melanoma cells). These results suggest that αCD45-IL12 improves lung cancer survival by increasing the stimulatory capacity of cross-presenting dendritic cells, potentiating a multi-epitope CD8 T cell immune response, and enhancing the effector function of these cytotoxic CD8 T cells.

Acknowledgements and/or References (Optional):: First and second author contributed equally.