Outsmart™ IL-2/15, a Novel Cytokine Designed to Enhance ROR1-Specific CAR T Anti-Tumor Activity While Minimizing Treg Activation and Systemic Cytokine Exposure
Authors: Moffett Howell , Weitzner Brian , Davenport Thaddeus , Tait Leah , Tan Tina , Baker Laura , Crowl John , Obenza Willimark , Hammerson Bradley , Kirkpatrick Robin , Sample Paul , Jones Lesley , Hermans Erik , Langan Robert , Haworth Kevin , Boyken Scott , Foster Aaron , Lajoie Marc 
Published: December 2023

Introduction: Effective treatment with chimeric antigen receptor T (CAR-T) cells against solid and some liquid tumors will require the safe production of immune-modulating cytokines to promote CAR-T cell expansion and to stimulate endogenous anti-tumor immune responses within the tumor microenvironment. OutSmart™ IL-2/15 is a computationally designed, CD8α-targeted, IL-2-based cytokine produced by engineered T cells via a T cell activation-dependent promoter. Here, we demonstrate that inducible production of CD8-targeted IL-2/15 drives proliferation and anti-tumor efficacy of ROR1-specific CAR-T cells, stimulates bystander CD8 + T and NK cells, and minimizes activation of immune suppressive T regulatory cells (Tregs).

Methods and Results: Wild-type (wt) IL-2 was modified using Rosetta protein design software package to eliminate IL-2Rα binding while retaining native IL-2Rβγ binding interfaces. Out of 10,355 designs, 38 were prioritized based on in silico analysis and ML-guided structure prediction, and evaluated for IL-2R binding as recombinant proteins. Consistent with the design criteria, none of the designs bound IL-2Rα, and 33 (87%) retained IL-2Rβγ binding and signaling activity. pSTAT5 analysis showed wt IL-2 highly activated CD4 +CD25 +FoxP3 + Treg cells, whereas the novel IL-2 designs showed a &gt;3-log reduction Treg stimulation. These designs were iteratively optimized to generate a version exhibiting improved thermostability and IL-2Rβγ binding affinity equivalent to wt IL-2. To increase the potency against immune effector cells, but not Tregs, an anti-CD8α VHH was tethered to the modified IL-2 molecules (CD8-IL-2/15) to mimic high-affinity IL-2Rα binding and selectively activate CD8 + T and NK cells. This prosthetic binding interaction dramatically increased CD8 + T cell stimulation and CD8α + NK cell activation (&gt;2-logs and 1-log by pSTAT5, respectively), while retaining its reduced capacity to stimulate Tregs. CD8-IL-2/15 gene expression was subsequently placed under the control of a T cell activation-dependent promoter and integrated into a lentiviral construct with a constitutively expressed ROR1-specific CAR. T cells transduced with a lentivirus expressing CD8-IL-2/15 and a ROR1-targeting CAR showed inducible cytokine production following exposure to ROR1 + tumor cells (H1975) leading to enhanced CAR-T cell proliferation and anti-tumor killing during repeat tumor challenge assays in vitro. Using immune-deficient mice (NSG) engrafted with H1975 tumors, inducible CD8-IL-2/15 enhanced ROR1-specific CAR-T cells showed durable tumor control at low doses (1e6 - 4e6 CAR T cells/animal), which was comparable to wt IL-2, whereas mock T cells and ROR1 CAR only T cells failed to control tumor growth ( Figure 1). In addition, CAR-T expansion with CD8-IL-2/15 increased &gt;4-fold over wt IL-2 (p&lt;0.0001), measured by cell counts in peripheral blood.

Conclusions: In summary, OutSmart™ IL-2/15 is a genetic module that produces CD8-IL-2/15 in response to T cell activation, promoting robust CAR-T expansion and enhanced anti-tumor efficacy. Furthermore, local production of a CD8-IL-2/15 enhances the effector function of bystander CD8 + T cells and NK cells, but only minimally activates Treg cells due to removing the IL-2Rα binding interface. Inducible production of CD8-IL-2/15 dramatically improves the potency of a ROR1 CAR for treating ROR1 + solid and liquid cancers. More generally, OutSmart™ protein design methods and control technologies can be applied to create other designed cytokines for oncology and beyond.