In Situ Vaccination with IL-12Fc and TLR Agonist - a Crucial Role for B Cells in Generating Anti-Tumor T Cell Immunity

Interleukin 12 (IL-12) is a potent proinflammatory cytokine that plays a central role in regulating both innate and adaptive immune responses. This cytokine has been tested as a cancer immunotherapy agent based on its impressive anti-tumor effects in animal models. Clinically, systemic delivery of IL-12 incurred dose-limiting toxicities because of its pharmaco-kinetic extremes of peaks and troughs, a result of its short serum half-life. Here, we evaluated mDF6006, a mouse IL-12 Fc-fusion protein which was designed to have a prolonged half-life with equivalent potency to recombinant mouse IL-12.

We hypothesized that local intra-tumoral (IT) delivery of low doses IL-12Fc would lead to immune-therapeutic potential.

To screen candidates for in situ vaccination, we have employed a preclinical strategy whereby the same syngeneic tumor is implanted at two separate sites in the body. One tumor is then injected with the test agents and the resulting immune response is detected by the regression of the distant, untreated tumor.

Using this assay for abscopal therapeutic effects we have tested a variety of immune stimulatory ligands and antibodies against immune checkpoints and T cell co-stimulatory targets, alone and in multiple combinations.

Among these, the combination of unmethylated CG-enriched oligodeoxynucleotide (CpG), a TLR9 ligand, and IL-12Fc has provided the most impressive results. TLRs, expressed in antigen-presenting cells and are components of the innate immune system that recognize molecular patterns on bacterial, fungal, or viral pathogens.

In a mouse model of lymphoma, IT injection of CpG and IL-12Fc, combined to cause tumor eradication at both the injected site and the untreated tumor. All these results were dependent on T cells in the animal.

During this therapy we observed a significant increase in activated B cells within the treated tumor and especially in its draining lymph nodes (dLN). This B cell stimulation was contributed by both the CpG and the IL12-Fc. Surprisingly, depletion of the animals of B cells by an anti-CD20 antibody completely ablated the abscopal therapeutic effect.

B cells function as antigen-presenting cells, secrete cytokines and are precursors of antibody producing plasma cells. To evaluate which of these roles B cells play in the therapeutic efficacy of CpG and IL-12Fc we isolated B cells from the LN of the treated mice and co-cultured them with T cells. These B cells activated T cells in an antigen-specific manner that was MHC dependent. No such T cell activation occurred when MHC molecules were blocked with anti-MHC antibodies or when the cells were physically separated by a semi-permeable membrane. Thus, local CpG and IL12-Fc directly activated B cells to present tumor antigens to specific T cells in the tumor microenvironment, resulting ultimately in an abscopal tumor response.

IL-12Fc and CpG are both currently in phase-1/2 trials as single agents and in combinations with checkpoint antibodies. Our results provide strong rationale for using low doses of IL-12Fc with CpG as an in situ therapeutic vaccine for lymphoma.