(575c) Electrostatic-Driven Interactions Enhance Intratumoral Retention, Distribution, and Antitumor Efficacy of Immune Checkpoint Blockade Antibodies

To effectively treat cancers, it is critical for the current therapies to reach as many cancer cells as possible for improved efficacy. However, drug carriers achieve poor, heterogeneous distribution in tumors in part due to limited transport through the tumor extracellular matrix (ECM). Tumor ECM forms a net negative charged network that interacts with and hinders the transport of molecules partly based on electrostatic interactions. The focus on drug delivery in solid tumors has traditionally been on the development of neutral charge coatings to minimize interactions with the ECM for improved transport. Contrary to the literature, we recently found a positively charged peptide that enhanced the penetration, uptake, and retention of nanoparticles in tumor ECM and tumor tissue. Mechanistically, the peptide-coated nanoparticle is partitioned into the tumor tissue due to electrostatic interactions—further weak and reversible binding with the tumor bed allowed it to penetrate deep within the tumor tissue. Additionally, a high number of intra-tissue binding sites in the tumor environment enabled the peptide-coated nanoparticle to be retained in the tissue for a longer time. Based on this previous study, here we hypothesize that the electrostatically driven interactions of the positively charged peptide will improve the binding, retention, and intratumor distribution of immune checkpoint blockade antibodies, which would ultimately enhance their antitumor efficacy. The combination of immune checkpoint blockade antibodies, αCTLA4 and αPDL1, which have been FDA-approved as monotherapies for melanoma and other cancers, can have synergistic antitumor effects to achieve even greater efficacy.

Methods

We prepared peptide antibody conjugates (PACs) by conjugating the positively charged peptide to immune checkpoint blockade antibodies, αCTLA4 and αPDL1 Abs, using copper-free click chemistry. The click chemistry was also done in parallel with the antibodies without the peptide. We validated the peptide conjugation by running the PACs and unmodified Abs on standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and nonreducing conditions. Then we quantified the average number of peptides per Ab using liquid chromatography-mass spectrometry (LC-MS). We next measured the binding affinities of the PACs and the unmodified Abs to a tumor-like ECM model consisted of collagen and hyaluronic acid using enzyme-linked immunosorbent assay (ELISA). Using a murine melanoma cancer model, we investigated whether the peptide conjugation enhanced the tumor regression of the immune checkpoint blockade Abs by improving the tumor infiltration of activated T cells. For this, we inoculated B16F10 murine cancer cells intradermally into the left flank of C57BL/6 immunocompetent mice on day 0. We then injected treatments (PBS, 100 µg of unmodified Abs, or 100 µg of PACs) peritumorally on days 4 and 7. We measured the tumors with a digital caliper starting day 4 and calculated the volumes. On day 10, mice were sacrificed humanely, and we harvested spleens, tumor-draining lymph nodes, and tumors. Next, we prepared single-cell suspensions by gently disrupting the organs and then analyzed the number and frequency of T cells using flow cytometry.

Results

We confirmed an increase in the molecular weight of both heavy and light chains of the Abs after peptide conjugation, with ~average of 1-2 peptides per Ab. The PACs recognized their target antigens with similar affinities to the unmodified Abs (~300 pM for αCTLA4 and ~50 pM for αPDL1), suggesting that the peptide conjugation did not affect the antigen recognition by the Abs. Upon peptide conjugation, PACs demonstrated binding affinity (367 nM for αCTLA4-peptide and 9.5 nM for αPDL1-peptide) against the tumor-like ECM, whereas the unmodified Abs did not bind to the ECM. Together, this confirmed the positively charged peptide conjugation improved the binding of the Abs to the tumor ECM without affecting their antigen recognition capacities. We observed significant tumor regression in the murine melanoma model by both unmodified Abs and PACs compared to the PBS control. PACs improved the tumor regression by 1.7-fold compared to the unmodified Abs after 10 days. We quantified a significantly higher population of tumor-infiltrating CD8⁺ T cells upon PAC administration than the PBS control. Additionally, we observed significant depletion of regulatory T cells in the tumor and tumor-draining lymph nodes upon PAC treatment.

In conclusion, our findings suggest that the positively charged peptide conjugation improved the antitumor activity of immune checkpoint blockade Abs. The positively charged peptide electrostatically interacted with the net negatively charged tumor ECM and enhanced the tumor tissue binding of the Abs. Because of this binding, the Abs were retained longer in the tumor environment. The longer retention of the PACs recruits a higher number of activated tumor-infiltrating T-cells, resulting in delayed tumor growth.

Implications

This study indicates that net negative tumor ECM acts as a drug-delivery depot for positively charged solutes toward improved intratumoral drug penetration and retention. Leveraging favorable interactions between the tumor ECM with therapeutics results in improved drug accumulation and retention for improved antitumor efficacy. As a result, this work can upend the current dogma of designing inert drug delivery systems and exploit electrostatic interactions to significantly improve therapeutic index of drugs and in the long-term, improve therapeutic outcomes in solid cancers.