https://orcid.org/0000-0001-7704-9185
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ABSTRACT
The urokinase-type plasminogen activator receptor (uPAR) is an essential regulator for cell signaling in tumor cell proliferation, adhesion, and metastasis. The ubiquitous nature of uPAR in many aggressive cancer types makes uPAR an attractive target for immunotherapy. Here, we present a rapid and successful workflow for developing cross-reactive anti-uPAR recombinant antibodies (rAbs) using high-throughput optofluidic screening of single B-cells from human uPAR-immunized mice. A total of 80 human and cynomolgus uPAR cross-reactive plasma cells were identified, and selected mouse VH/VL domains were linked to the trastuzumab (Herceptin®) constant domains for the expression of mouse-human chimeric antibodies. The resulting rAbs were characterized by their tumor-cell recognition, binding activity, and cell adhesion inhibition on triple-negative breast cancer cells. In addition, the rAbs were shown to enact antibody-dependent cellular cytotoxicity (ADCC) in the presence of either human natural killer cells or peripheral blood mononuclear cells, and were evaluated for the potential use of uPAR-targeting antibody-drug conjugates (ADCs). Three lead antibodies (11857, 8163, and 3159) were evaluated for their therapeutic efficacy in vivo and were shown to suppress tumor growth. Finally, the binding epitopes of the lead antibodies were characterized, providing information on their unique binding modes to uPAR. Altogether, the strategy identified unique cross-reactive antibodies with ADCC, ADC, and functional inhibitory effects by targeting cell-surface uPAR, that can be tested in safety studies and serve as potential immunotherapeutics.
Abbreviations
| ADCC | = | antibody-dependent cellular cytotoxicity |
| ACSs | = | antibody-secreting cells |
| ADCs | = | antibody-drug conjugates |
| BLI | = | biolayer interferometry |
| CDR | = | complementarity-determining region |
| Cyno | = | cynomolgus monkeys |
| FACS | = | fluorescence-activated cell sorting |
| FcγR | = | Fc gamma receptor |
| GPI | = | glycosylphosphatidylinositol |
| HER2 | = | human epidermal growth factor receptor-2 |
| NGS | = | next-generation sequencing |
| NK cells | = | natural killer cells |
| OEP | = | opto-electropositioning |
| PBMCs | = | peripheral blood mononuclear cells |
| rAbs | = | recombinant antibodies |
| suPAR | = | soluble urokinase-type plasminogen activator receptor |
| Tras | = | trastuzumab |
| TNBC | = | triple-negative breast cancer |
| uPAR | = | urokinase-type plasminogen activator receptor |
| uPA | = | urokinase-type plasminogen activator |
| VN | = | vitronectin |
Acknowledgments
The authors thank Dr. Cheng-I Wang for helpful comments and discussion during the preparation of this article. We also acknowledge the Nikon Imaging Center at UCSF for providing access to the spinning disk confocal microscope.
Disclosure statement
Charles S. Craik is a co-founder of Dandelion Biosciences. Robert Drakas is the president of ShangPharma Innovation.
Data availability
The data that support the findings of this study are available from the corresponding author, CSC, upon reasonable request.
Supplementary data
Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2023.2184197