Tony Ribas from University of California at Los Angeles elaborated on the mechanisms of acquired resistance to ICIs: (1) defects in antigen presentation, and (2) defects in the IFN-γ signaling pathway [
91,
92]. In situ vaccination has emerged as a candidate method for overcoming these defects, by facilitating TIME priming. Specifically, toll-like receptor 9 (TLR9) agonists [
93], oncolytic virus [
94], and IL-2 receptor agonists [
95], represent three major targets for in situ vaccination. Clinical trials using these approaches to prime the melanoma TIME and enhance the sensitivity of anti-PD-1 antibodies have been conducted. Preliminary results from these studies support using this novel strategy in overcoming the resistance to ICIs through activating T cells directly by activating type-I cytokine receptors or indirectly through activating innate immune responses.
Ronald Levy from Stanford University shared with the audience his recent research on the in situ vaccination strategy. Levy discussed intratumoral injection of the CpG oligodeoxynucleotide (CpG) as an in situ therapeutic vaccination to boost anti-cancer immunity. Unmethylated CpG commonly exists in microbial genomes, but rarely in vertebrates [
96]. Therefore, CpG is recognized via TLR-9 expressed by APCs, including dendritic cells and B cells [
97], which activates both innate and adaptive immune responses. In tumor-bearing mice, CpG induced an anti-tumor response only after direct intratumoral injection. When intratumoral vaccination with CpG was combined with low dose radiation during a multi-center Phase-I/II clinical trial in patients with low-grade B-cell lymphoma, durable responses were observed at distant tumor sites [
98]. With this success, several other combinations were tested both in the preclinical murine setting as well as in clinical trials. For example, Ibrutinib, a BTK and ITK inhibitor that can suppress myeloid-derived suppressor cells and regulatory T (T
reg) cells, produced synergistic anti-tumor activity when combined with CpG and low-dose radiation. In some cases, complete remission was observed in the treated lesions as well as distant lesions that were not treated with CpG. Flow cytometry and single-cell sequencing with paired biopsy specimens obtained pre- and post-treatment showed a decrease in tumor B cells post-treatment, while normal NK, B, and T cells increased. In addition, CpG vaccination not only stimulated an immune response but also induced the expression of OX40, also known as tumor necrosis factor receptor superfamily member 4, and a secondary co-stimulatory molecule expressed on T
regs and activated T cells. The combination of CpG vaccination and anti-OX40 therapy enhanced the anti-tumor immune response and eliminated established lymphoma as well as solid tumors in mice. In fact, this combination was more effective than the combination of CpG and anti-PD-L1 antibody [
99]. With these promising results in hand, two clinical trials are currently ongoing: (1) combination therapy with CpG, OX40 agonist, and low-dose radiation for non-Hodgkin’s lymphoma and (2) combination therapy with CpG and OX40 agonist in all cancer types.
Liang Deng from Memorial Sloan Kettering Cancer Center discussed a novel virotherapy based on vaccinia virus, another approach to the neoantigen-based in situ vaccination strategy. Similar to CpG, oncolytic virus is another kind of in situ therapy that can stimulate cancer immunotherapy as outlined in Fig.
1 (outermost circle). Oncolytic virus triggers an antitumor immune response through induction of immunogenic cell death, release of tumor-associated antigens (including damage-associated molecular patterns (DAMPs)), alteration of an immunosuppressive TIME, and promotion of dendritic cell maturation and antigen presentation. Hence, localized oncolytic virus can convert non-immunogenic “cold” tumors into immunogenic “hot” ones, inducing tumor infiltration by immune cells and overcoming resistance to ICIs [
100]. T-VEC is a replication-competent Herpes Simplex 1 (HSV-1) virus that expresses human GM-CSF (hGM-CSF). It was approved in the USA for treatment of advanced melanoma in 2015, making it the first oncolytic virus approved for this indication. However, compared to hGM-CSF control therapy, intratumoral injection of T-VEC only improved OS by 4.4 months [
101]. To further improve clinical efficacy, T-VEC is being tested in combination with immune checkpoint inhibitors, such as anti-CTLA-4 antibody [
102]. On the other hand, modified, attenuated vaccinia virus Ankara (MVA) is a new generation of smallpox vaccine that serves as a promising vaccine vector for infectious diseases and cancer. It has a deletion of 30 Kb from the parental vaccinia genome, which inhibits replication of the virus in mammals [
103]. Intratumoral injection of heat-inactivated MVA induces innate immunity via the STING pathway, which enhances tumor antigen presentation, promotes dendritic cell maturation, stimulates naïve T cell priming, increases tumor-specific T cell expansion and migration, and boosts cytotoxic T lymphocyte (CTL)-mediated killing of cancer cells [
104‐
106]. The anti-tumor effect of heat-inactivated MVA requires CD8
+ T cells, and the long-term anti-tumor memory response requires CD4
+ T cells. To generate the next generation of MVA with still greater efficacy, MVA with deletion of C7L (MVAΔC7L) was generated. MVAΔC7L can induce much higher levels of type I interferon, proinflammatory cytokines, and chemokines [
105,
107]. Additionally, MVA can be further engineered to express Flt3L, which is a growth factor for CD103
+ and plasmacytoid dendritic cells, and OX40L, which serves as co-stimulatory ligand for OX40 expressed by T cells. After intratumoral injection, Flt3L- and OX40L-expressing MVAΔC7L induced more CD8
+ and CD4
+ T cells responding in distant, non-injected tumors, and synergized with anti-PD-L1 antibody in multiple mouse tumor models as compared to heat-inactivated parental MVA. Currently, the Memorial Sloan Kettering Cancer Center has two vaccinia-based vectors: (1) recombinant MVA that expresses a non-replicative, safe immune activator, activates multiple innate immune pathways (including cGAS/STING), and can be used for intratumoral injection as a monotherapy or in combination with ICIs; (2) an oncolytic vaccinia platform that is replication-competent, has the capability to express large proteins (e.g., antibodies against immune checkpoint molecules), enhances anti-tumor activity and reduces immune-related toxicities.