A ROR1 Bispecific T Cell Engager for the Treatment of Chronic Lymphocytic Leukaemia Demonstrates Enhanced Function Following Ibrutinib Treatment

ROR1 is expressed on a range of solid and haematological malignancies, including chronic lymphocytic leukaemia, mantle cell lymphoma and a subset of acute lymphoblastic lymphoma, making it an attractive therapeutic target. In keeping with this, ROR1 CAR T cells and a ROR1 antibody, are being investigated as novel treatments [NCT02222688, NCT02776917 & NCT02706392]. BiTEs are small, 55kDa antibody based therapeutics composed of two single chain variable fragments (scFv) linked in tandem by a flexible linker. One scFv binds to tumour antigens and the other to the CD3 complex, bringing target cells and T cells in close proximity, facilitating formation of cytotoxic synapses and resulting in target cell death. We have generated a fully humanised ROR1 BiTE which unlike conventional antibodies recruits and utilises the inherent cytotoxic potential of polyclonal resident T cells and does not require ex vivo cell manipulation. We therefore investigated the potential of a ROR1 BiTE for the treatment of CLL.

We first assessed the efficacy of our ROR1 BiTE against SKW6.4, Jeko1 and Kasumi-2 haematological cell lines which are ROR1 positive, confirming significant cytotoxicity even at low concentrations of 1ng/ml (Figure 1A). To demonstrate in vivo potential, mice were injected with SKW6.4 cells and disease established before treatment with T cells and 5 days of ROR1 BiTE or control PBS therapy. BiTE treated animals had improved survival compared to the control cohort (50 days vs 18 days), despite only a single infusion of T cells (Figure 1B).

We next assessed the potential of the BiTE to target primary CLL cells: allogeneic healthy donor T cells were co-cultured with CLL cells (3:1 effector:target ratio) in the presence of ROR1 BiTE with resultant decrease in CLL viability at 24 hours (Mean CLL survival 32%, Figure 1C). In contrast, we saw minimal cytotoxicity in co-cultures of autologous patient T cells and CLL cells with ROR1 BiTE under identical conditions (Mean CLL survival 92%, Figure 1D). We hypothesised this was secondary to the previously described multifactorial T cell dysfunction in CLL.

Ibrutinib, the first in class Bruton's tyrosine kinase inhibitor, demonstrates impressive efficacy in front line and relapsed/refractory CLL patients including those with high risk 17p deletion. In addition, it modulates the tumour micro-environment, skews T cells to a Th1 phenotype, reduces PD-1 expression and reverses pseudo-exhaustion. In view of this, we isolated T cells from patients receiving Ibrutinib and cultured them with autologous CLL cells and demonstrated significantly enhanced cytotoxicity compared to patients not exposed to Ibrutinib (Mean CLL survival 43% vs 85%, p=0.012) (Figure 1E). This cohort included patients who had failed multiple prior lines of therapy and/or 17p deleted cases. Ibrutinib treatment was also associated with markedly enhanced secretion of a range of pro-inflammatory mediators including IFNγ and TNFα (Figure 1F). To ensure this effect was not due to Ibrutinib increasing sensitivity of CLL cells to a ROR1 BiTE, we analysed matched samples from 2 patients pre and post treatment. Post Ibrutinib T cells demonstrated enhanced killing, irrespective of the CLL cells exposure to Ibrutinib, confirming enhanced T cell function as the driving force of improved BiTE mediated cytotoxicity (Figure 1G).

Taken together, we demonstrate the potential of a ROR1 BiTE to target haematological malignancies and highlight for CLL, the clinical context in which BiTEs are used is critical for effective responses. Baseline T cell dysfunction in untreated or relapsed CLL patients will likely limit efficacy, whilst Ibrutinib treatment allows for improved T cell effector function. In keeping with this CD19 CAR T cells demonstrate superior expansion, function and persistence when generated from CLL patients on Ibrutinib, confirming a global positive immunomodulatory role. Other small molecule inhibitors may improve T cell function by reducing tumour bulk but Ibrutinib inhibits other tyrosine kinases including ITK, which may play a role in T cell modulation. Despite Ibrutinib monotherapy being effective, patients often have a low level disease burden and minimal residual disease is rarely achieved. BiTE treatment for high risk patients in this situation represents a rational option to deepen responses. Overall, our data supports the assessment of our ROR1 BiTE in the post Ibrutinib setting for CLL.

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