Antigen-specific T cell Redirectors: a nanoparticle based approach for redirecting T cells

Redirection of T cells to target and destroy tumors has become an important clinical tool and major area of research in tumor immunology. Here we present a novel, nanoparticle-based approach to selectively bind antigen-specific cytotoxic T cells (CTL) and redirect them to kill tumors, termed ATR (Antigen-specific T cell Redirectors). ATR were generated by decorating nanoparticles with both an antigen-specific T cell binding moiety, either peptide loaded MHC-Ig dimer or clonotypic anti-TCR antibody, and a model tumor cell binding moiety, anti-CD19 antibody to engage CD19+ tumor cells. ATR stably bind tumor cells and CTL in a dose dependent fashion and stimulate antigen-specific conjugate formation between those cells. ATR induced redirected lysis of tumor cells in vitro, as demonstrated by 51Cr-release killing. In vivo ATR administration led to reduced tumor growth in a SCID/beige human lymphoma treatment model. In summary, ATR represent a novel, nanoparticle based approach for redirecting antigen-specific CTL to kill tumors.


Quality control of nanoparticle-based ATR
To evaluate the effective binding of the ATR to their targets, 2C T cells (Supplementary Figure S1 upper panel) or CD19 + tumor cells (Supplementary Figure S1 lower panel), were incubated with ATR and stained with an anti-mouse IgG1 PE mAb. The anti-mouse IgG1 PE mAb antibody is specific for the Fc portion of all molecules on the ATR and thus could be used to visualize ATR bound to cells. Anti-CD19 specific particles (1B2/CD19 and pep-MHC-Ig/CD19) bound to T2 cells (Supplementary Figure S1 lower panel). Particles containing TCR specific ligands (1B2, 1B2/CD19, pep-MHC-Ig and pep-MHC-Ig/ CD19 particles) bound to 2C T cells. This data indicates that particles coated with antibody (1B2 mAb) and pep-MHC-Ig ( SIY K b -Ig) can be generated and are capable of simultaneously binding 2C T cells and tumor cells.

Specificity, stability and ratio dependence of ATR to target cell binding
To set up an optimal protocol for subsequent killing assays, we initially investigated bead-to-cell binding conditions. First, we verified the specificity of a T cell ATR stain and determined the minimal staining time that resulted in a sufficient coating (Supplementary Figure S2A). 2C T cells were incubated with 50μl of either control or ATR (1B2/CD19). While no binding was detected at any time point when stained with control particles (Supplementary Figure S2A, left panel), good binding was detected on 2C T cells incubated with ATR (Supplementary Figure S2A, middle panel). Thereby 15 minutes displayed an optimal staining interval while MFI intensity increased only minimal at later time points. Furthermore, staining of gp-100 specific, transgenic Pmel T cells (Supplementary Figure  S2A, left panel) did not show any binding. Experiments utilizing SIY K b -Ig/CD19 ATR also demonstrated antigenspecific binding to 2C T cells (data not shown).
Subsequently, we investigated the stability of ATR to T cell interaction. ATR (1B2/CD19) labeled 2C T cells (Supplementary Figure S2B, black line) were transferred on 37°C after all ATR in excess were washed away. ATR coated 2C T cells were analyzed for decrease of MFI, similar to bead lose (Supplementary Figure S2B). Thus, the data indicate that ATR staining of 2C T cells was stable for ≤60 minutes; that represents a sufficient time interval to provide T to tumor cell interaction in a later killing assay.
We further examined how different bead to cell ratios interfere with an optimal staining outcome. In a first approach we incubated different amounts of 2C T cells with 1B2-(Supplementary Figure S2C

Oncotarget, Supplementary Materials 2016
Finally we investigated the potential of ATR to activated 2C T cells. Therefore cells were incubated with either control particles, OVA K b /CD19 (non-cognate) or SIY K b /CD19 (cognate) ATR and expression of CD107a has been analyzed by flow cytometry (Supplementary Figure S2E). Only 2C T cells co-cultured with cognate ATR demonstrated an activated CD107a + phenotype. Subsequently, activation of 2C T cells by cognate ATR resulted in a significant increase of proliferation. 2C T cell co-cultures with non-cognate ATR or un-stimulated cultures (control) demonstrated only background proliferation (Supplementary Figure S2F).

Supplementary Figure S1: Antigen-specific T cell Redirectors (ATR) bind to effector and target cells. Staining control of generated ATR. Effector cells (2C T cells) and target cells (T2 and
Raji) were stained with ATR at 4°C for 15 minutes washed and co-stained with anti-mouse-IgG1. pep-MHC represents staining of effector or target cells with SIY peptide loaded K b -Ig dimer generated ATR at 4°C for 45min.

Oncotarget, Supplementary Materials 2016
Supplementary Figure S2: Specificity, stability, activation and killing efficacy of ATR binding to target and effector cells. A. To test the specificity of ATR (1B2/CD19) binding 2×10 5 2C and Pmel T cells were incubated with 50 μl of ATR at 4°C for the duration indicated. B. To test the stability of ATR binding 2×10 5 2C T cell were incubate at 4°C for 15 minutes with 50 μl ATR (control), washed and subsequently transferred at 37°C. After indicated time points cells were analyzed for loose of ATR. ATR binding of 2C T cell and CD19 + tumor cells (T2) was investigated relating the impact of bead:cell ratio on staining intensity. Therefore, either different amounts of cells, as indicated C. were stained with 50 μl ATR or D. 2×10 5 2C T cells were stained with different amounts of ATR, as indicated. For detection of ATR cells were washed and secondary stained with anti-mouse IgG1. E. To determine the activation potential of ATR activated 2C T cells were incubated with 50 μl of either control particles, OVA peptide loaded K b /CD19 (non-cognate) or SIY peptide loaded K b / CD19 (cognate) ATR. After 4 hours expression of CD107a on cells was analyzed. F. Same ATR (50 μl) were bound to CFSE stained 2C T cells, washed and co-cultured for 3 days. Proliferation was determined by CFSE dilution compared to non-stimulated cells (control). G. To investigate ATR killing efficacy, specific lysis of CD19 + Raji cells was analyzed in a 51 Cr-release killing assay utilizing a 10:1 E:T ratio. 2×10 5 2C T cells were incubated with different amounts of ATR; 5×10 7 /ml (0.5μl), 5×10 8 /ml (5μl) and 5×10 9 /ml (50μl).