*Brief Report* **Hyaluronidase Enhances Targeting of Hydrogel-Encapsulated Anti-CTLA-4 to Tumor Draining Lymph Nodes and Improves Anti-Tumor Efficacy**

**Airi Harui \* and Michael D. Roth**

Division of Pulmonary & Critical Care Medicine, Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1690, USA; mroth@mednet.ucla.edu

**\*** Correspondence: aharui@mednet.ucla.edu

**Abstract:** Immunotherapy targeting checkpoint inhibitors, such as CTLA-4 and/or PD-1, has emerged as a leading cancer therapy. While their combination produces superior efficacy compared to monotherapy, it also magnifies inflammatory and autoimmune toxicity that limits clinical utility. We previously reported that a peri-tumor injection of low-dose hydrogel-encapsulated anti-CTLA-4 produced anti-tumor responses that were equal to, or better than, systemic dosing despite a >80% reduction in total dose. Injection of hydrogel-encapsulated anti-CTLA-4 was associated with low serum exposure and limited autoimmune toxicity, but still synergized with anti-PD-1. In this report, we employ live and ex vivo imaging to examine whether peri-tumor administration specifically targets anti-CTLA-4 to tumor-draining lymph nodes (TDLN) and whether the incorporation of hyaluronidase enhances this effect. Tumor-free survival analysis was also used to measure the impact of hyaluronidase on tumor response. Compared to systemic dosing, peri-tumor injection of hydrogelencapsulated anti-CTLA-4/DyLight 800 resulted in preferential labeling of TDLN. Incorporating hyaluronidase within the hydrogel improved the rapidity, intensity, and duration of TDLN labeling and significantly improved tumor-free survival. We conclude that hydrogel-encapsulated anti-CTLA acts as a localized antibody reservoir and that inclusion of hyaluronidase optimizes the blockade of CTLA-4 in TDLN and thereby imparts superior anti-tumor immunity.

**Keywords:** checkpoint inhibitor blockade; immunotherapy; hydrogel; anti-CTLA-4; peri-tumor injection; hyaluronidase; antibody delivery; tumor draining lymph nodes

## **1. Introduction**

Monoclonal antibodies (mAbs) that block immune checkpoint inhibitors are rapidly expanding the treatment options for patients with soft tissue cancers [1,2]. However, individual mAbs are effective in only a minority of patients while combination therapy that blocks both CTLA-4 and PD-1 yields a significantly greater response and progression-free survival [3–7]. Unfortunately, systemic administration of combination therapy also increases acute inflammatory and autoimmune toxicity that can limit clinical utility [8,9]. Hydrogel-encapsulated checkpoint inhibitor therapy has recently been identified as an approach for improving this narrow benefit-to-risk ratio [10–13]. Using a mouse model, we previously reported that peri-tumor injection of a controlled-release hydrogel containing low-dose anti-CTLA-4 produces equal or greater tumor control than does high-dose systemic therapy [13]. This targeted low-dose approach still synergizes with systemic anti-PD-1 therapy and promotes lasting systemic protection against tumor re-challenge [13]. Local injection of anti-CTLA-4 alone (without a controlled-release hydrogel) produces only a limited response. Furthermore, using a NOD.H-2h4 model to assess anti-CTLA-4 associated autoimmune thyroiditis, systemic administration of anti-CTLA-4 induced high titers of anti-thyroglobulin antibodies while hydrogel-based administration did not. At the conclusion of that work, we hypothesized that controlled regional delivery of low-dose

**Citation:** Harui, A.; Roth, M.D. Hyaluronidase Enhances Targeting of Hydrogel-Encapsulated Anti-CTLA-4 to Tumor Draining Lymph Nodes and Improves Anti-Tumor Efficacy. *Gels* **2022**, *8*, 284. https:// doi.org/10.3390/gels8050284

Academic Editors: Wei Ji and Kunpeng Cui

Received: 14 March 2022 Accepted: 25 April 2022 Published: 3 May 2022

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anti-CTLA-4 results in a sustained perfusion of tumor draining lymph nodes (TDLN) that selectively expands and activates tumor-specific T cells while sparing autoimmune T cell activation at distant nodal sites. In related work, we demonstrated that the addition of hyaluronidase (HAse) to the hydrogel allowed us to fine tune the rate of mAb release and, by promoting autolysis of the hydrogel reservoir, facilitates repeated administration at the same location [14].

In the current brief report, we evaluate whether the incorporation of HAse into the hydrogel matrix has other beneficial effects on therapy. Injection of recombinant human HAse is approved by the FDA to permeabilize soft-tissue matrix in order to promote the uptake of fluids, drugs, and mAb therapies through existing lymphatic pathways [15]. Given our focus on regional perfusion of TDLN with anti-CTLA-4, we hypothesized that HAse might enhance lymphatic uptake and nodal targeting of administered anti-CTLA-4 and therefore promote even greater anti-tumor efficacy. In this study, live animal imaging is used to track anti-CTLA-4 distribution following either systemic injection, targeted peri-tumor injection of a standard hydrogel, or administration of a hydrogel that contains incorporated HAse. Lymph nodes were also excised and imaged ex vivo. Our established pre-clinical tumor immunotherapy model was then employed to evaluate the effects of HAse on anti-tumor activity and tumor-free survival [13].

#### **2. Results and Discussion**
