Harnessing the immune response to target tumors

Development of “immune-based targeted therapy” in oncology has limited experience with signal pathway modulation. However, as we have become better versed in understanding immune function related to anticancer response, “hints” of specific targets associated with sensitivity and resistance have been identified with targeted immune therapy. This brief review summarizes the relationship of several targeted immune therapeutics and activity associated clinical responsiveness.

Introduction Although the immune system distinguishes self versus non-self antigens with the intent to eliminate cells expressing non-self antigens, cancer cells have developed mechanisms to escape or suppress the "non-self attack", thereby enabling tumor proliferation and progression. Increasing numbers of innovative immunotherapies are being developed that address immune modulation of non-self targets to reverse cancer defenses.

Checkpoint inhibitors
Several targets have been associated with evidence of clinical benefit, resulting in a broad spectrum of investigational and approved immunotherapies. These include cellular therapies (adoptive T-cell and dendritic cell therapy, cytokine-induced killer cells, tumor vaccines, and autologous tumor cell therapy) and checkpoint inhibitors (PD-1/L-1 and CTLA-4 inhibitors). Several checkpoint inhibitors have shown superior clinical benefit over standard of care 1-3 ; however, a high percentage of patients do not show durable response rates in monotherapies or combination therapies with checkpoint blockade. Factors like tumor-infiltrating lymphocyte (TIL) infiltration and PD-L1 expression levels are associated with response 4 . In addition, tumor mutation burden (TMB) appears to be a prognostic marker for immune response 5 . During cancer cell proliferation, somatic mutations increase the expression of a variety of tumor-associated antigens and neoantigens. Studies show that high TMB correlates with the amount of immunogenic neoantigens (P <0.0001), presented by major histocompatibility complex (MHC) molecules to immune effector cells, inducing higher durable immune responses (overall response rate of 63% versus 0%; P = 0.03) and progression-free survival prolongation (14.5 versus 3.7 months; P = 0.01) than in tumor types with lower mutation burden 6 . Regardless of histology type, tumors with a mean mutational load of more than 10 somatic mutations per megabase of coding DNA appear more likely to be immunogenic to effector T cells eliciting antitumor immunity 7,8 . Other cancer types, such as colorectal cancer, and interestingly subgroups with a high number of somatic mutations and potential mutation-associated neoepitopes appear to correlate with higher responses to checkpoint inhibitors in mismatch repair-deficient tumors 9 . Recent studies show that the overall response rate to PD-1/L-1 therapies in high TMB tumor types has been durable for years with delayed relapse or disease progression 10 . On the other hand, signal pathways, such as those associated with interferon receptor expression related to loss of JAK 1 or JAK 2 function, result in unresponsiveness to interferon gamma, a common antiproliferative cytokine associated with oncolytic activity. This effect has been well demonstrated in a subset of PD-1/L1-refractory patients. Zaretsky et al. 10 identified inactivating mutations in JAK 1 and 2 that silence the CD8 T cell-induced interferon gamma signaling cascade, an adaptive antitumor response. Another mechanism such as beta 2-microglobulin inactivation results in loss of MHC1 expression. Moreover, mutations of death receptors-like Fas or tumor necrosis factor-related apoptosis-inducing ligand-are associated with insensitivities against granzymes or perforin or both, which also play major roles in immune escape and resistance.
To address and overcome resistant mechanisms, ongoing studies are extensively investigating combination approaches (that is, with checkpoint inhibitors). For example, experiments of targeted inhibition of mitogen-activated protein kinase show synergy with PD-1/L1 pathway inhibition and increases in CD8 T-cell number within the tumor environment in association with increased tumor response 13 .

Adoptive T-cell therapies
Adoptive dendritic cell or T-cell therapies show clinically meaningful value in hematologic malignancies, and a small number of case reports support efficacy in solid tumors with demonstration of durable clinical responses [14][15][16] . For example, 20 to 25% of patients with metastatic melanoma showed durable responses to expanded TIL therapies 14,17 . This is most likely related to neoantigen signal identification. A remarkable case of response of adoptive T-cell therapy to a common neoantigen target was recently demonstrated to KRAS G12D mutation 16 and other, lesser-known mutations 14,15 . However, currently, the majority of cancer vaccines and adoptive T-cell approaches fall short of significant efficacy targeting pre-selected MHC-dependent (genetically modified T cells) or independent-chimeric antigen receptor-T (CAR-T)antigens showing limited activity in solid tumors, possibly related to the lack of knowledge of relevant neoantigens (Table 1). While CD19-targeting CAR-T cell therapies have demonstrated curative events in B-cell malignancies 18,19 , efficacy in solid tumors appears to be limited by heterogeneity, lack of relevant tumor-specific orassociated antigens and low immunogenicity 20 , in balance with other immunosuppressive pathways not addressed within the tumor microenvironment.
Adoptive T-cell therapy "exhaustion" may also be influenced by upregulation of pathways such as PD-L1 expression on tumor cells. Strategies to convert the negative signal of PD-L1 to co-stimulatory receptors by PD1:28 chimer alteration showed encouraging results in activation of CD8 effector T cells 21 . Tran et al. identified CD8 T-cell responses against mutant KRAS G12D and HLA-C*08:02 in a patient with colorectal cancer, receiving a single-dose infusion of 1.48 × 10 11 TILs (approximately 75% CD8 T cells) with durable regression of lung metastases with disease progression 9 months after treatment 16 .

Tumor signaling/microenvironment modulation
Overcoming tumor-induced immunosuppression can also involve tumor signal modulation and microenvironment influence. Altered expressions of survival genes (Bcl-xL), increasing the

CAR-T: selective antigen targets
Targeting driver mutations or their de novo neoepitopes are very attractive and appear to be very promising in effective anticancer therapies. There are several cancer-associated or -specific antigenloaded CAR-T cell therapies, selected by different algorithms, in clinical trials to investigate further efficacy in solid tumors ( Interestingly, one investigational personalized cellular immunotherapy product with a mechanism directly associated with autologous DNA engineered tumor cells called Vigil 32-35 shows evidence of enhanced tumor-specific antigen targeting via effector T-cell activation in correlation with clinical benefit in solid tumors. Autologous tumor cells include the full patient-and tumor-specific antigen repertoire. This is a unique aspect of the Vigil therapy.

Conclusions
The future is bright for combination immunotherapy, particularly as exact targets are identified with the tumor microenvironment, thereby enabling access to tumor "non-self" neoantigens.
Competing interests JN is a shareholder in Gradalis, Inc.

Grant information
The author(s) declared that no grants were involved in supporting this work.