Poster CC13 - Engineered multiparatopic fusion antibodies for Intracellular Delivery of CRISPR-Cas9

Introduction: PD-L1 (programmed death-Ligand 1) blockade therapy has significant implications in the treatment landscape of malignancies, primarily due to its potential to re-activate the immune system for tumor control. Although PD-L1 blockade therapy has demonstrated clinical efficacy and durable responses across several types of cancers, more than 70% of the patients do not experience clinical benefit and have shown either intrinsic or acquired resistance, necessitating the development of alternative mechanistic approaches to enhance the efficacy of PD-L1-targeted therapy 1. To this end, our lab has developed a PD-L1-targeting multiparatopic antibody that can engage multiple distinct binding epitopes on PD-L1 to simultaneously block PD-L1 binding to its cognate receptor, (PD-1) and cross-link multiple PD-L1 to trigger its internalization and degradation. The capacity of this engineered multiparatopic antibody in mediating PD-L1 internalization offers an additional complementary mechanism to intracellularly deliver protein therapeutic cargoes, such as CRISPR-Cas9 complex, to further augment antitumor efficacy and prevent immune escape.

Materials and

Methods: To identify new PD-L1 binding domains that target different epitopes of PD-L1, we enriched and selected PD-L1 binding clones from a yeast-displayed synthetic antibody single-chain variable fragment (scFv) library and a single-domain antibody library derived from shark immunization. We then constructed a panel of bi- and tri-paratopic antibodies utilizing selected PD-L1 binding domains to identify multiparatopic constructs, and assessed their ability to induce PD-L1 downregulation in a panel of cancer cell lines. Both confocal and super-resolution microscopies were applied to understand the trafficking behavior of PD-L1 mediated by the multiparatopic antibodies. The immunostimulatory capacity and consequent immunotherapeutic potential of multiparatopic antibodies was assessed in both T cell-receptor activation reporter assays and patient-derived peripheral blood mononuclear cells. To examine the biodistribution and pharmacokinetic properties of the multiparatopic, we utilized positron emission tomography (PET) imaging to determine the PD-L1 availability in a mouse orthotopic xenograft model of triple negative breast cancer. To effectively re-route the multiparatopic antibody-mediated PD-L1 internalization away from the endolysosomal pathway and facilitate cytosolic delivery of CRISPR-Cas9 complex for manipulation of genes that are responsible for tumor development and progression, we further designed eight different multiparatopic antibodies that contain endosomal escape peptides (EEPs) to allow passage through the endosomal membrane. A pH-sensitive dye assay was applied to determine the escape efficacy mediated by panel of EEP-containing multiparatopic antibodies. These constructs were then evaluated in cellular gene editing assays.

Results, Conclusions, and Discussions: After four iterative rounds of magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS), three different anti-PD-L1 clones, D12 (scFv), A9 (single-domain antibody), and B8 (single-domain antibody), were selected from the two libraries. Cross-competition analysis of PD-L1 binding demonstrated the presence of three distinct binding epitopes amongst these newly discovered clones. We created panels of bi- and triparatopic antibodies using these clones and identified TS1521 is the most effective multiparatopic anti-PD-L1 antibody in inducing clustering and internalization of PD-L1 in the human breast cancer cell line MDA-MB-231, reducing the surface PD-L1 by 60-70% (Fig. 2)1. Confocal microscopy revealed that TS1521-mediated endocytosis led to lysosomal sorting and accumulation in H2444. We further observed that TS1521 exhibited robust tumor localization and durably attenuated PD-L1 availability in the mouse cancer model. Building on these results, we demonstrated that EEP-containing TS1521 antibodies exhibited a similar or better internalization capacity compared to the parental TS1521 (Fig. 2). Moreover, in terms of endosomal escape efficacy, we found that our engineered constructs showed an enhanced capacity to escape into the cytosol, thus harboring great potential as delivery vehicles.

PD-L1 pathway blockade therapy has emerged as a cornerstone in the cancer treatment landscape, providing hope for durable cures and prolonged survival in patients who previously had limited treatment options. To further enhance the clinical potential of anti-PD-L1 antibodies, we successfully developed multiparatopic antibodies that are capable of inducing rapid PD-L1 clustering and internalization, which can be leveraged to enable intracellular protein delivery and improve therapeutic outcomes.

We have successfully designed a multiparatopic antibody and demonstrated its utility as a protein delivery vehicle. Future in vivo studies involving the use of immunocompetent and translationally relevant models of cancer will establish in vivo gene editing of PD-L1-expressing tumor cells and illustrate the therapeutic activity of our engineered therapeutics.

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