Multispecific Targeting with Synthetic Ankyrin Repeat Motif Chimeric Antigen Receptors

Abstract

Purpose: The outgrowth of antigen-negative variants is a significant challenge for adoptive therapy with T cells that target a single specificity. Chimeric antigen receptors (CAR) are typically designed with one or two scFvs that impart antigen specificity fused to activation and costimulation domains of T-cell signaling molecules. We designed and evaluated the function of CARs with up to three specificities for overcoming tumor escape using Designed Ankyrin Repeat Proteins (DARPins) rather than scFvs for tumor recognition.

Experimental design: A monospecific CAR was designed with a DARPin binder (E01) specific for EGFR and compared with a CAR designed using an anti-EGFR scFv. CAR constructs in which DARPins specific for EGFR, EpCAM, and HER2 were linked together in a single CAR were then designed and optimized to achieve multispecific tumor recognition. The efficacy of CAR-T cells bearing a multispecific DARPin CAR for treating tumors with heterogeneous antigen expression was evaluated in vivo.

Results: The monospecific anti-EGFR E01 DARPin conferred potent tumor regression against EGFR⁺ targets that was comparable with an anti-EGFR scFv CAR. Linking three separate DARPins in tandem was feasible and in an optimized format generated a single tumor recognition domain that targeted a mixture of heterogeneous tumor cells, each expressing a single antigen, and displayed synergistic activity when tumor cells expressed more than one target antigen.

Conclusions: DARPins can serve as high-affinity recognition motifs for CAR design, and their robust architecture enables linking of multiple binders against different antigens to achieve functional synergy and reduce antigen escape.

FIGURE 1:

EGFR recognition by E01 DARPin CARs. (A) Schematic of the structure of EGFR-specific CARs consisting of the EF-1 promoter (EF-1p), GM-CSF receptor leader, E01 DARPin or Cet scFv, spacer sequences, CD28 transmembrane, 4-1BB costimulation and CD3ζ activation domain with a truncated CD19 transduction marker after the T2A element. (B) Cytotoxicity of E01 DARPin and cetuximab scFv specific cells against K562 cells and K562 cells transduced to express full-length EGFR (n=3). (C) Cytotoxicity of E01 DARPin and cetuximab scFv specific cells against tumor lines expressing EGFR (n=3). (D) Immunoblot with anti CD3ζ measuring CAR and endogenous CD3ζ on sort-purified specific cells (E) Cytotoxicity of CD8⁺ E01 DARPin specific cells against MDA-MB-231 tumor cells with short and long spacer (+/−(G₄S)₂ linker) (n=3). (F,G) Cytokine secretion and proliferation of CD8⁺ E01 DARPin specific cells with short and long spacer (+/−(G₄S)₂ ) against MDA-MB-231 target cells (n=3). (H) Bioluminescent imaging of MDA-MB-231 breast cancer cells transduced with firefly luciferase in NSG mice treated with 5 million CAR-T cells (CD8:CD4:1:1). (I,J) Average tumor radiance and survival of E01 DARPin CAR with short and long spacer (+/− (G₄S)₂ linker) compared to the cetuximab scFv CAR (n=8 mice from 2 donors). (**-p<0.05, ns-not significant)

FIGURE 2:

Design of multispecific DARPin CARs. (A) Bispecific DARPin CARs constructed with either the Ec1 DARPin or E01 DARPin as the membrane-proximal DARPin separated by (G₄S)₂ or (G₄S)₄ linkers. (B,C) Cytotoxicity and cytokine production of each bispecific DARPin CAR against single positive or double positive target cells (n=3). (D) Trispecific CAR designs and recognition of single positive tumor targets measured by IFN-γ production by specific cells (n=4) (E) Cytotoxicity of trispecific specific cells against Raji/HER2. (**-p<0.05, ns-not significant)

FIGURE 3:

Trispecific CAR function against target cells expressing single antigens. (A) Surface expression of monospecific and trispecific DARPins CARs measured by staining of N-terminal MYC tag. (B,C) Cytotoxicity and cytokine production of monospecific versus trispecific DARPin specific cells against Raji cells expressing EGFR or EpCAM or HER2 (n=3). (D) Proliferation of the trispecific G3-Ec1-E01 specific cells against Raji cells expressing single targets. (**-p<0.05, ns-not significant).

FIGURE 4:

In vivo anti-tumor function of trispecific specific cells against heterogeneous tumor cells. (A) NSG mice were inoculated with a 1:1:1 mix (0.5 million) of Raji/EGFR, Raji/EpCAM, and Raji/HER2 intravenously (IV) followed by IV injection of CAR-T cells (15 million CD8:CD4 1:1) 1 week after tumor cell injection. (B) Bioluminescence imaging of mice treated with trispecific CAR-T cells, monospecific CAR-T cells, or a mix of monospecific CARs at the same total T cell dose. (C,D) Average tumor radiance and survival of mice in each treatment cohort. (n=7-12 from 2 donors). (E) CAR-T cell frequencies in blood for monospecific versus trispecific CAR (n=7). (F) Antigen expression on tumor cells harvested on euthanasia in the heterogeneous tumor model in each treatment cohort (**- p<0.05, ns-non significant)

FIGURE 5:

Trispecific CAR G3-Ec1-E01 T cells demonstrate synergy against tumor cells expressing multiple antigens. (A) Bioluminescence imaging of tumors expressing single (EGFR, EpCAM or HER2) antigens or both HER2 and EpCAM in mice treated with 9 million trispecific CAR-T cells. (B) Survival curves of cohorts of mice after treatment with 9 million trispecific CAR-T cells. (C) Bioluminescence imaging of tumors expressing single, double or all 3 antigens (EGFR, HER2 and EpCAM) in mice treated with 5 million trispecific CAR-T cells. (D) Survival curves of cohorts of mice after treatment with 5 million trispecific CAR-T cells. (**-p<0.05, ns-not significant)