We generated two humanized interleukin-13 receptor α2 (IL-13Rα2) chimeric antigen receptors (CARs), Hu07BBz and Hu08BBz, that recognized human IL-13Rα2, but not IL-13Rα1. Hu08BBz also recognized canine IL-13Rα2. Both of these CAR T cell constructs demonstrated superior tumor inhibitory effects in a subcutaneous xenograft model of human glioma compared with a humanized EGFRvIII CAR T construct used in a recent phase 1 clinical trial (ClinicalTrials.gov: NCT02209376). The Hu08BBz demonstrated a 75% reduction in orthotopic tumor growth using low-dose CAR T cell infusion. Using combination therapy with immune checkpoint blockade, humanized IL-13Rα2 CAR T cells performed significantly better when combined with CTLA-4 blockade, and humanized EGFRvIII CAR T cells’ efficacy was improved by PD-1 and TIM-3 blockade in the same mouse model, which was correlated with the levels of checkpoint molecule expression in co-cultures with the same tumor in vitro. Humanized IL-13Rα2 CAR T cells also demonstrated benefit from a self-secreted anti-CTLA-4 minibody in the same mouse model. In addition to a canine glioma cell line (J3T), canine osteosarcoma lung cancer and leukemia cell lines also express IL-13Rα2 and were recognized by Hu08BBz. Canine IL-13Rα2 CAR T cell was also generated and tested in vitro by co-culture with canine tumor cells and in vivo in an orthotopic model of canine glioma. Based on these results, we are designing a pre-clinical trial to evaluate the safety of canine IL-13Rα2 CAR T cells in dog with spontaneous IL-13Rα2-positive glioma, which will help to inform a human clinical trial design for glioblastoma using humanized scFv-based IL-13Rα2 targeting CAR T cells.

No abstract

We set out to clone Bax-specific CD8+ T cells from peripheral blood samples of patients with primary chronic lymphocytic leukaemia. A number of clones were generated using a Bax peptide pool and their T-cell epitope was mapped to two peptides sharing a common 9-amino-acid sequence (LLSYFGTPT), restricted by HLA-A*0201. However, when these T-cell clones were tested against highly purified syntheses (> 95%) of the same peptide sequence, there was no functional response. Subsequent mass spectrometric analysis and HPLC fractionation suggested that the active component in the original crude peptide preparations (77% pure) was a peptide with a tert-butyl (tBu) modification of the tyrosine residue. This was confirmed by modification of the inactive wild-type sequence to generate functionally active peptides. Computer modelling of peptide:HLA-A*0201 structures predicted that the tBu modification would not affect interactions between peptide residues and the HLA binding site. However, these models did predict that the tBu modification of tyrosine would result in an extension of the side chain out of the peptide-binding groove up towards the T-cell receptor. This modified product formed < 1% of the original P603 crude peptide preparation and < 0·05% of the original 23-peptide mixture used for T-cell stimulation. The data presented here, illustrate the potential for chemical modifications to change the immunogenicity of synthetic peptides, and highlight the exquisite capacity of T-cell receptors to discriminate between structurally similar peptide sequences. Furthermore, this study highlights potential pitfalls associated with the use of synthetic peptides for the monitoring and modulating of human immune responses.

under the Erasmus Scheme. Beamtime was supported by proposals mx10462 and mx14843.

Long-term remission for acute myeloid leukemia (AML) is still not achieved for the majority of patients and consequently there is a need for new treatments to consolidate current therapy. A promising approach is to augment the anti-tumor immune response in these patients; however most cancers do not activate immune effector cells because they express immunosuppressive ligands. Previously we showed that CD200 overexpression on AML blasts suppresses memory CD4+ and CD8+ T cell effector function through engagement with CD200 receptor (CD200R) on these cells. Blocking CD200:CD200R, however, only partially restored T cell activity, suggesting that alternative immunosuppressive mechanisms were involved. Recently, promising clinical outcomes have been reported for melanoma and non-small cell lung cancer using humanized antibodies targeting another immunosuppressive receptor, PD-1, and we therefore investigated whether this could be contributing to the immunosuppression of T cell effector responses in CD200hi AML patients. Initially, we investigated whether CD200 and the immunosuppressive ligand for PD-1, PD1-L1, were co-expressed in AML blasts at diagnosis. Affymetrix gene expression data from 158 AML blasts showed that AML patients in the upper quartile for CD200 expression (CD200hi) had 10-fold higher levels of PD1-L1 expression compared to CD200lo (lower quartile) patients. Analysis of CD200 and PD1-L1 protein expression on AML blast cells confirmed this association at the protein level (r2 = 0.49; p<0.01). The co-expression of CD200 and PD1-L1 on patient AML blast cells, suggested that they cooperated in immunosuppression. In support of this, we found that the CD200 and PD1-L1 cognate co-receptors (CD200R and PD-1 respectively) were present on CD4+ and CD8+ T cells from AML patients. Further characterization of PD-1+ T cells showed that the mean frequency of PD-1+ early differentiated T cells (CD57- CD28+) was increased for CD200hi AML patients CD4+ (19% ± 3 vs 13% ± 3; p<0.05) and CD8+ T cells (21% ± 3 vs 11% ± 2; p<0.05). We also found that the mean frequency of late differentiated CD8+ T cells that have poor anti-tumor function (CD57+ CD28- PD-1+) was almost twice that for CD200hi patients compared with CD200lo (38% ± 6 vs 21% ± 9 respectively; p<0.05). Expansion of these cells was also associated with a decreased CD4:CD8 ratio in these patients (2.1 ± 0.5 vs 3.7 ± 1 for CD200hi and CD200lo respectively; p<0.01). . These findings show for the first time a link between CD200 expression level on AML blast cells and the frequency of PD-1+ late differentiated CD8+ T cells. To directly test whether engagement of CD200 with CD200R was capable of mediating PD-1 up-regulation on CD8+ T cells, we co-cultured a CD8+ CD200R+ T cell clone (7E7) either with K562 cells stably overexpressing CD200 or K562 empty vector controls (negative for CD200). Co-culture with CD200+ cells, significantly increased the frequency of PD-1+ T cells (26% ± 3 vs 17% ± 4; p<0.05) and this was antagonized by CD200 blocking antibody (26% ± 3 vs 21% ± 3; p<0.01). These data show that CD200:CD200R interaction has the capacity to increase the frequency of PD-1+ CD8+ T cells. To model the functional implications of this, we created a series of K562 lines expressing CD200, PD1-L1 or both molecules in combination and analyzed the effect of T cell activation (via TNFα production). We found that both CD200 and PD1-L1 induced a similar (>50%) reduction in the frequency of activated 7E7 T cells; however, when both CD200 and PD1-L1 were co-expressed, T cell activation was almost ablated (~90% reduction; p<0.01). Moreover, the strength of the TNFα response was also reduced in co-culture assays where either CD200 or PD1-L1 were present, indicating a direct effect at the level of CD8+ T cell function (2.8 ± 0.5 vs 1.7 ± 0.5; p<0.05). These data demonstrate that CD200:CD200R and PD1-L1:PD-1 engagement on T cells can act in tandem to augment immunosuppression of CD8+ T cells. In summary, we show for the first time that the immunosuppressive molecules, CD200 and PD1-L1 appear to be co-regulated on AML blasts and that these can act in combination to profoundly suppress T cell activation. Further, we show that CD200:CD200R engagement induces PD-1+ CD8+ T cells. Taken together we propose a novel CD200/PD1-L1 immunotherapeutic synapse in AML which should be targeted by combining CD200:CD200R and PD1-L1:PD-1 blockade in immunotherapy of AML. Disclosures No relevant conflicts of interest to declare.