*2.3. Incorporation of HAse into the Hydrogel Matrix Enhances the Anti-Tumor Efficacy of Low Dose Anti-CTLA-4*

Targeting of TDLN occurs when a peri-tumor injection of hydrogel-incorporated anti-CTLA-4 is delivered, and this promotes effective tumor killing while limiting systemic exposure [13]. The addition of HAse improves anti-CTLA-4 release from the hydrogel, promotes mAb access to the lymphatic pathway, and results in higher and more prolonged targeting of CTLA-4 binding sites in TDLN. Anti-CTLA-4 results in an expansion of tumorreactive T cells, reduction in CTLA-4-expressing cells, and a relative expansion of cytotoxic CD8 cells [13]. The remaining question is whether greater TDLN perfusion by anti-CTLA-4 translates into better therapeutic outcomes. To test the hypothesis that HAse can improve immune activation resulting from low-dose anti-CTLA-4, tumor-bearing mice (N = 9 or 10) were treated with hydrogel-encapsulated anti-CTLA-4 (50 µg) that contained either 0, 50, or 250 U of HAse. Peri-tumor injections were delivered on days 6 and 11 after tumor implantation [13]. Tumor growth was monitored for 28 days (Figure 3). All tumors, regardless of assigned treatment group, grew in the first few days after starting therapy but thereafter the response was significantly different in the treatment group receiving 250 U HAse. At the end of 28 days, 5 mice (56%) in the control group (no HAse) were tumor free; 5 mice (50%) in the group receiving gels containing 50 U HAse were tumor free; but tumor-free survival was present in 8 mice (89%) that had received hydrogels containing 250 U HAse (*p* = 0.04; log-rank test for tumor-free survival; Figure S2). Additional controls demonstrated that these responses required the presence of anti-CTLA-4 as the difference in treatment response was highly significant when comparing animals treated with hydrogels containing only 250 U HAse and those receiving anti-CTLA-4 plus 250 U HAse (*p* < 0.001 by ANOVA; data not shown). The synergism that occurs between the delivery of anti-CTLA-4 and HAse, when delivered together in a peri-tumor injection of self-polymerizing hydrogel, therefore spans from enhanced effects on anti-CTLA-4 release, lymphatic uptake, and targeting of TDLN to a positive impact on anti-tumor efficacy.

**Figure 3.** Incorporation of HAse enhances the anti-tumor activity of hydrogel-encapsulated anti-CTLA-4. C57BL/6 mice injected with MC-38 tumor cells were treated with 50 µg of hydrogel-encapsulated anti-CTLA-4 by peri-tumor SQ injection at days 6 and 11 after tumor implantation. Groups of animals received either a standard hydrogel formulation (**a**), or a hydrogel formulated with either 50 U (**b**) or 250 U (**c**) of HAse. Tumor volume was measured every 3 or 4 days over a course of 28 days. Each line represents tumor growth in a single mouse. The number of tumor-free animals at day 28 are indicated (\* *p* < 0.05 compared to no HAse control by log-rank test). **Figure 3.** Incorporation of HAse enhances the anti-tumor activity of hydrogel-encapsulated anti-CTLA-4. C57BL/6 mice injected with MC-38 tumor cells were treated with 50 µg of hydrogelencapsulated anti-CTLA-4 by peri-tumor SQ injection at days 6 and 11 after tumor implantation. Groups of animals received either a standard hydrogel formulation (**a**), or a hydrogel formulated with either 50 U (**b**) or 250 U (**c**) of HAse. Tumor volume was measured every 3 or 4 days over a course of 28 days. Each line represents tumor growth in a single mouse. The number of tumor-free animals at day 28 are indicated (\* *p* < 0.05 compared to no HAse control by log-rank test).

#### **3. Conclusions 3. Conclusions**

In this study, a combination of whole-animal in vivo imaging and ex vivo imaging of recovered LNs provided direct evidence that a peri-tumor injection of hydrogel-encapsulated anti-CTLA-4 creates a local drug reservoir that targets mAb delivery to TDLN. While systemic anti-CTLA-4 therapy has the potential to disrupt tumor-associated immunosuppression through multiple pathways and at multiple sites, targeted delivery to TDLN has been shown in animal models to promote proportionally greater T-cell activation and systemic tumor control [11]. These outcomes occur even in the absence of direct perfusion of the tumor or depletion of intratumoral regulatory T cells [11]. This mechanism of action likely explains the capacity for hydrogel-delivered anti-CTLA-4 to induce effective antitumor immunity while sparing systemic exposure and drug-associated autoimmune toxicity [13]. Furthermore, we demonstrated that incorporating HAse into the hydrogel facilitates anti-CTLA-4 release, as well as the speed, intensity, and duration of its binding within TDLN. The end result is a significant improvement in systemic anti-tumor efficacy and tumor-free survival without an increase in delivered dose. According to convention, the dose of systemic anti-CTLA-4 therapy is directly linked to both its efficacy and toxicity [20,21]. The capacity to target TDLNs via a peri-tumor injection disrupts this relationship. As demonstrated here, incorporation of HAse into the hydrogel matrix further enhances the effectiveness of low-dose anti-CTLA-4 therapy and should further improve the benefit-to-risk ratio associated with its use. As such, these pre-clinical investigations define a clear pathway for employing hydrogel-encapsulated anti-CTLA-4 as a strategy for improving the tolerability and potency of cancer immunotherapy. As previously noted, the greatest promise from such an approach would be the ability to safely administer combination therapy that included hydrogel-encapsulated anti-CTLA-4, targeting TDLN, and a systemic PD-1 checkpoint inhibitor. In this study, a combination of whole-animal in vivo imaging and ex vivo imaging of recovered LNs provided direct evidence that a peri-tumor injection of hydrogel-encapsulated anti-CTLA-4 creates a local drug reservoir that targets mAb delivery to TDLN. While systemic anti-CTLA-4 therapy has the potential to disrupt tumor-associated immunosuppression through multiple pathways and at multiple sites, targeted delivery to TDLN has been shown in animal models to promote proportionally greater T-cell activation and systemic tumor control [11]. These outcomes occur even in the absence of direct perfusion of the tumor or depletion of intratumoral regulatory T cells [11]. This mechanism of action likely explains the capacity for hydrogel-delivered anti-CTLA-4 to induce effective anti-tumor immunity while sparing systemic exposure and drug-associated autoimmune toxicity [13]. Furthermore, we demonstrated that incorporating HAse into the hydrogel facilitates anti-CTLA-4 release, as well as the speed, intensity, and duration of its binding within TDLN. The end result is a significant improvement in systemic anti-tumor efficacy and tumor-free survival without an increase in delivered dose. According to convention, the dose of systemic anti-CTLA-4 therapy is directly linked to both its efficacy and toxicity [20,21]. The capacity to target TDLNs via a peri-tumor injection disrupts this relationship. As demonstrated here, incorporation of HAse into the hydrogel matrix further enhances the effectiveness of low-dose anti-CTLA-4 therapy and should further improve the benefit-to-risk ratio associated with its use. As such, these pre-clinical investigations define a clear pathway for employing hydrogel-encapsulated anti-CTLA-4 as a strategy for improving the tolerability and potency of cancer immunotherapy. As previously noted, the greatest promise from such an approach would be the ability to safely administer combination therapy that included hydrogel-encapsulated anti-CTLA-4, targeting TDLN, and a systemic PD-1 checkpoint inhibitor.

#### **4. Materials and Methods 4. Materials and Methods**

#### *4.1. Animals 4.1. Animals*

C57BL/6 mice were purchased from the Charles River Laboratory (Wilmington, MA, USA) and housed at the UCLA Division of Laboratory Animal Medicine facility. All protocols and procedures were approved by the UCLA Animal Research Committee. C57BL/6 mice were purchased from the Charles River Laboratory (Wilmington, MA, USA) and housed at the UCLA Division of Laboratory Animal Medicine facility. All protocols and procedures were approved by the UCLA Animal Research Committee.

#### *4.2. Reagents*

Mouse colorectal cancer cell line, MC-38, was obtained from the Division of Cancer Treatment and Diagnosis Tumor Repository, National Cancer Institute. Hydrogel

matrix components, CMHA-S and PEG-DA, were provided by Lineage Cell Therapeutics (Alameda, CA, USA). The anti-mouse CTLA-4 (clone # 9H10) was purchased from BioXcell (West Lebanon, NH, USA) and a matching fluorescent-conjugated anti-mouse CTLA-4/Dylight800 was prepared specifically for this study by Leinco Technology (St. Louis, MO). Purified bovine hyaluronidase (HAse) was from MP Biomedicals (Santa Ana, CA, USA).

## *4.3. Hydrogel Formulation*

Hydrogels were formulated by first mixing the anti-CTLA-4 9H10 clone (50 µg/gel) or the anti-CTLA-4/Dylight 800 fluorescent construct (25 or 50 µg/gel) with the solubilized PEG-DA and then combining with CMHA-S to initiate spontaneous cross-linking as described previously [13]. Briefly, CMHA-S and PEGDA were individually dissolved in degassed deionized water (pH 7.4) to prepare solutions of 1.25% (*w*/*v*) and 6% (*w*/*v*), respectively. Final component concentrations within 150 µL hydrogels were 0.8% *w*/*v* for CMHA-S and 1.2% *w*/*v* for PEG-DA as detailed in Table 1. Hydrogels that incorporated HAse (50 U–250 U) were formulated in the same manner except that the HAse component was pre-mixed with the PEG-DA solution prior to adding the other components.

**Table 1.** Hydrogel composition.


## *4.4. Tumor Model and Treatment with Anti-CTLA-4*

The immunotherapeutic activity of anti-CTLA-4 in tumor-bearing mice was assessed as prescribed previously [13]. In brief, C57BL/6 mice were implanted with MC-38 cells (3 <sup>×</sup> <sup>10</sup>5/mouse) by SQ injection into the right upper flank. For optical fluorescent imaging experiments, mice (2 mice/group) were treated with a single dose of anti-CTLA-4/Dylight 800 delivered by either IP injection (100 µg in PBS) or by peri-tumor SQ injection (25 or 50 µg) using a hydrogel formulation between 11 and 14 days after tumor implantation. For assessing the impact of hydrogel-encapsulated anti-CTLA-4 (50 µg/dose) on tumor growth, all animals were implanted with tumor on the same day and those with palpable tumors at Day 6 were randomly divided into treatment groups (N = 9–10/group). All three groups received a 150 µL injection of hydrogel-encapsulate anti-CTLA-4 delivered by peri-tumor SQ dosing on Days 6 and 11. One group received a standard formulation without HAse, one containing 50 U HAse, and one with 2500 U HAse. Tumor volumes were measured by calipers every 3 to 4 days up to day 28.

#### *4.5. Optical Fluorescence Imaging*

In vivo and ex vivo optical imaging were performed using an IVIS Lumina II system (Caliper Life Sciences, Inc.; Hopkinton, MA, USA) at the Crump Preclinical Imaging Technology Center at UCLA. In vivo whole-body imaging of mice was carried out under isoflurane anesthesia with serial assessments in the same animals. For ex vivo imaging of LN, LN were recovered by surgical resection in replicate cohorts of animals that had been euthanized at various time points (0–72 h) after administration of anti-CTLA-4/DyLight 800.

#### *4.6. Statistical Evaluations*

Biodistribution studies were performed in replicates, with all animals imaged under identical settings at the same session to facilitate comparison of fluorescent intensity, as indicated by a continuous red to yellow intensity spectrum. Where indicated, visual findings were quantitated by assessing the fluorescent emission (photons/second; 800 nm wavelength) within the region of interest. Mean values are represented. Tumor immunotherapy

responses are presented by individual spaghetti plots showing measured tumor volume over time for each animal in each treatment group. Comparison between groups was carried out by a tumor-free survival analysis employing a log-rank test. Impact on tumor growth over time was compared between groups using an ANOVA (two-factors with replication). *p* < 0.05 was considered as significant.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/gels8050284/s1, Figure S1: Effect of hyaluronidase (HAse) on the release of fluorescent-labeled anti-CTLA-4 from hydrogels; Figure S2: Inclusion of HAse within the hydrogel formulation enhances tumor-free survival; Table S1: Impact of hyaluronidase (HAse) on the delivery of hydrogel-encapsulated anti-CTLA-4 to TDLN.

**Author Contributions:** Conceptualization, methodology, formal analysis, resources, data curation, writing, reviewing, editing, project administration, and funding acquisition were all carried out by M.D.R. and A.H. Investigations were carried out by A.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded in part by the National Center for Advancing Translational Sciences, National Institutes of Health, which supported optical imaging experiments through UCLA CTSI grant no. UL1TR001881.

**Institutional Review Board Statement:** The study was conducted under approval from the UCLA Institutional Animal Care and Use Committee (protocol 2016-031; date of approval: 21 April 2016).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors thank Pat Leinert, Jr. and the production team at Leinco Technologies for the generation of anti-CTLA-4/DyLight 800; Thomas I. Zarembinski and BioTime Inc., for providing hydrogel reagents; Shili Xu and the Crump Preclinical Imaging Technology Center, Crump Institute for Molecular Imaging at UCLA (NIH/NCI 2P30CA016042-44).

**Conflicts of Interest:** The authors declare no financial conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

