Systemic Antitumor Effects of Intratumoral Administration of the Novel Immunotherapeutic Combination Anti-CTLA4, Anti-CD137, and Anti-OX40 in Mouse Models of Lymphoma and Solid Tumor
Authors: Jonathan Hebb , Kohrt Holbrook E 
Published: October 2019

Abstract
Immunotherapy with agents such as immune checkpoint inhibitors and tumor necrosis factor (TNF) receptor superfamily agonists is a groundbreaking development with proven benefits and great potential in the treatment of solid tumors and hematologic malignancies. Combination immunotherapy creates even more potential for efficacy by targeting synergistic immune pathways. However, systemically administered immunomodulators can activate T-cells non-specifically resulting in off-target toxicity, which is dose-limiting and potentially lethal in combination. Intratumoral administration of immunotherapeutic agents has several advantages including 1) higher concentrations of agents in close proximity to target antigens and tumor-infiltrating lymphocytes 2) lower doses overall with less systemic exposure and toxicity 3) potentially a novel mechanism of action such as depletion of intratumoral Tregs 4) potential for an abscopal effect, essentially acting as an in-situ cancer vaccine.
Here we report a novel combination of immunomodulators, anti-CTLA4, CD137 and OX40 administered by intratumoral route in a mouse lymphoma model (A20) as well as a colon cancer model (MC38). Additionally, anti-PD1 and CpG were used as adjunctive therapies in the lymphoma model. CTLA4 and PD1 are immune checkpoint inhibitors, CD137 and OX40 are members of the TNF receptor superfamily, and CpG is a TLR9 agonist. For the A20 model, 2 tumors were created by injecting 1 x 10^7 A20 cells on each flank of BalbC mice. Treatment was started on day 8 when there were visible tumors and intratumoral injection was to the left tumor only. Groups included 1) PBS 2) triple combination of immunomodulators (anti-CTLA4, anti-CD137 and anti-OX40) at 30 mg each in 50 ml volume x 4 biweekly doses 3) triple combination x 2 biweekly doses followed by 2 doses of anti-PD1 at 150 mg in 100ul given intraperitoneally 4) anti-PD1 x 2 biweekly doses followed by 2 doses of triple combination and 5) CpG 50 mg concurrent with triple combination. For the MC38 model, 5 x 10^5 cells were injected bilaterally on the flanks of C57BL/6 mice. Mice were treated with either PBS or triple combination 3x/week for 6 doses and intratumoral injection was to the left tumor only.
For the A20 model, all treatment groups had a significant anti-tumor response compared to PBS with systemic regressions including both the treated and untreated tumor. Complete response was observed in 100% of the triple combination group, 100% of the CpG and triple combination group, and in 86% of the triple combination followed by PD1 group. Group 3, PD1 followed by triple combination, tumors initially progressed with PD1 alone, albeit slower than untreated, then regressed with triple combination treatment with 57% ultimately tumor-free. For the MC38 model, the triple combination resulted in a systemically effective anti-tumor response with near complete regression of treated and untreated sites and a significant survival advantage.
Thus we demonstrate this novel combination of immunomodulators delivered intratumorally induces a dramatic anti-tumor effect. Anti-PD1 given prior to the combination appears to abrogate the response. Overall these preliminary results of intratumoral administration of combination anti-CTLA4, CD137 and OX40, all agents in clinical trials, are promising for future clinical development via this safe and highly efficacious route of administration.

Disclosures
No relevant conflicts of interest to declare.