Abstract 2728: Discovery of a multispecific CD73/PD-1 targeting drug Fc-conjugate (DFC), which improves tumor reduction compared to PD-1 monotherapy in a humanized mouse model
Authors: Levin James , Döhrmann Simon , Dedeic Nicholas , Almaguer Amanda , Zuill Doug , Abelovski Elizabeth , Cole Jason N. , Locke Jeffrey B. , Fortier Joanne , Zhao Qiping , Hernandez Maria , Amundson Karin , Moniz Madison , Chen Hongyuan , Panickar Dhanya , Lam Thanh , Brady Tom , Borchardt Allen , Hough Grayson , Tari Leslie W. 
Published: March 2024

Abstract
Background: The approval of immune checkpoint therapies has revolutionized cancer treatment and established immuno-therapy as an additional treatment option. However, while anti-PD-(L)1 therapies have demonstrated durable responses, only a small subset of patients respond. One mechanism of tumor escape is via the release of immunosuppressive metabolites such as adenosine. CD73, an extracellular enzyme expressed on immune cells and some tumors, catalyzes the conversion of AMP to adenosine, the rate limiting step in adenosine production. As a strategy to improve response rates we developed a first in class CD73/PD-1 targeting DFC, comprised of a multivalent conjugate of a small molecule CD73 inhibitor stably linked to a proprietary human IgG1 Fc-fusion with a PD-1 inhibitor peptide. This multispecific DFC has the potential for differentiation and increased therapeutic potency compared to approved PD-(L)1 inhibitors.
Methods: Binding of CD73/PD-1-001 to biotinylated hPD-1 was determined using ELISA and to human CD8+ T cells by flow cytometry. CD73 inhibition was measured in cell-free and cell-based assays. Efficacy was determined in a humanized mouse model optimized for PD-1 activity against a transgenic MC-38 colon cell line expressing hPD-L1 (Genoway, France). Tumor volumes were recorded and statistical analysis was conducted by t-test (Mann-Whitney) or two-way ANOVA.
Results: In vitro, CD73/PD-1-001 demonstrated potent activity against both checkpoint targets. CD73/PD-1-001 bound to hPD-1 with an IC50 of &lt; 1 nM and demonstrated functional inhibition of CD73 with an EC50 of &lt;10 nM, which is on par with respective single agent molecules. Importantly, addition of the CD73 targeting moiety resulted in no significant loss in PD-1 binding relative to PD-1-001, a DFC containing only the PD-l inhibitor peptide. Efficacy was determined in a humanized mouse model against a transgenic MC-38 cell line. Both CD73/PD-1-001 and PD-1-001 DFCs were highly potent in this model with tumor growth inhibition (TGI) of &gt;70% at dose concentrations of 3 mg/kg or greater (p≤0.01). However, at the lowest test concentration of 1 mg/kg, only CD73/PD-1-001 retained significant potency with a TGI of 56.0 % (p ≤ 0.05). Tumor reduction in the 1 mg/kg PD-1-001 group did not reach statistical significance (36.5%, p &gt; 0.05).
Conclusions: This work demonstrates that a multispecific CD73/PD-1 targeting DFC, is a potent inhibitor of tumor growth in a humanized mouse model. Furthermore, CD73/PD-1-001 was more potent than PD-1 monotherapy, demonstrating the therapeutic benefit of targeting two important checkpoints with a single agent molecule. Collectively, preclinical testing of a CD73/PD-1 DFC has validated the benefits of targeting the PD-(L)1 axis in combination with adenosine suppression, resulting in improved anti-tumor effects.
Citation Format: James Levin, Simon Döhrmann, Nicholas Dedeic, Amanda Almaguer, Doug Zuill, Elizabeth Abelovski, Jason N. Cole, Jeffrey B. Locke, Joanne Fortier, Qiping Zhao, Maria Hernandez, Karin Amundson, Madison Moniz, Hongyuan Chen, Dhanya Panickar, Thanh Lam, Tom Brady, Allen Borchardt, Grayson Hough, Leslie W. Tari. Discovery of a multispecific CD73/PD-1 targeting drug Fc-conjugate (DFC), which improves tumor reduction compared to PD-1 monotherapy in a humanized mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2728.