Abstract 1740: Engineered variant domain fusion proteins provide checkpoint inhibition and tumor antigen dependent CD28 costimulation resulting in potent anti-tumor immunity
Authors: Levin Steven D. , Maurer Mark F. , Gudgeon Chelsea , Chandrasekaran Siddarth , Ardourel Daniel , Demonte Daniel , Kuijper Joseph , Wolfson Martin , Garrett Logan , Kleist Kayla N. , Mudri Sherri , Nguyen Hieu , Seaberg Michelle , Wang Rachel , Yang Jing , Lewis Katherine E. , Dillon Stacey R. , Rixon Mark , Peng Stanford L. 
Published: July 2021

Abstract
Introduction: Checkpoint inhibition (CPI) has been shown to be an effective anti-tumor therapy, but CPI alone is frequently insufficient to control tumor growth, and costimulatory signals may also be required to produce clinically significant anti-tumor responses. PD1-PDL1 are established CPI targets and TMIGD2 is an inhibitory receptor expressed on T cells that is engaged by its cognate ligand HHLA2 on tumor cells, leading to inhibition of T cell responses. Novel biologics combining CD28 costimulation and CPI may yield promising tumor antigen-specific therapeutic candidates.
Methods: Variants of CD86 with increased CD28 affinity were engineered using our directed evolution platform. PD1 and TMIGD2 variants were also engineered for increased affinity to PDL1 and HHLA2, respectively. Fusion proteins were generated including either PD1 or TMIGD2 domains, an effectorless Fc domain, and an engineered CD86 domain to generate proteins to provide target-dependent costimulation (TDC) and evaluated in multiple in vitro T cell stimulation assays. Costimulatory effects were confirmed by evaluating T cell proliferation, cytokine release, and tumor cell killing. Fusion proteins were also tested in vivo in a mouse tumor model using MC38 cells expressing human PDL1 or HHLA2, and in a humanized tumor model where an HPV+ squamous cell carcinoma cell line expressing either PDL1 or HHLA2 was implanted into NSG mice. Tumor-bearing NSG mice were given primary human T cells expressing an HPV peptide-specific TCR, with or without the corresponding fusion protein, and tumor growth was measured over time.
Results: PD1-CD86 and TMIGD2-CD86 TDC proteins enhanced T cell costimulation in multiple in vitro T cell response assays, and costimulation was dependent on target cell lines expressing PDL1 or HHLA2, respectively. There was no effect on T cell responses if the target cells did not express the relevant target protein. PD1 and TMIGD2 domains alone had little effect on T cell responses, indicating these TDC proteins were driving enhanced responses. PD1-CD86 and TMIGD2-CD86 TDC proteins enhanced anti-tumor responses in vivo in a syngeneic MC38 implantation model only when MC38 cells expressed human PDL1 or HHLA2, respectively. Both proteins also enhanced antitumor responses in a humanized tumor implantation system using an HPV+ tumor cell line expressing the corresponding target antigen in the presence of human T cells expressing an HPV-specific TCR, but failed to control tumor growth when tumor cells lacked expression of the target protein.
Conclusions: Tumor antigen-specific antitumor therapy can be achieved with fusion proteins that combine engineered CPI and CD28 costimulatory domains. Such novel biologics may provide promising approaches to enhancing the efficacy of CPI monotherapies and to address checkpoint inhibitor-resistant tumors.
Citation Format: Steven D. Levin, Mark F. Maurer, Chelsea Gudgeon, Siddarth Chandrasekaran, Daniel Ardourel, Daniel Demonte, Joseph Kuijper, Martin Wolfson, Logan Garrett, Kayla N. Kleist, Sherri Mudri, Hieu Nguyen, Michelle Seaberg, Rachel Wang, Jing Yang, Katherine E. Lewis, Stacey R. Dillon, Mark Rixon, Stanford L. Peng. Engineered variant domain fusion proteins provide checkpoint inhibition and tumor antigen dependent CD28 costimulation resulting in potent anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1740.