Kinase inhibitors such as ibrutinib have advanced treatment of chronic lymphocytic leukemia (CLL). However, there remains a need for adjunct treatments capable of deepening response or overcoming resistance to kinase inhibitors. CD19/CD3 bispecific antibodies (bsAbs) recruit endogenous T cells to form cytolytic synapses with CD19+ tumor cells. Blinatumomab, a CD19/CD3 bsAb designed in the 54 kDa BiTE format, is FDA approved for the treatment of some forms of acute lymphoblastic leukemia, and has potential for use in other B-cell malignancies. However, due to its short half-life of 2.1 hours, blinatumomab requires continuous intravenous dosing for efficacy. We have developed a novel CD19/CD3 bsAb in the 100 kDa single chain-Fv Fc format (19/3-scFv-Fc). With a half-life of approximately 7 days, 19/3-scFv-Fc may be suitable for weekly dosing, providing a significant logistical advantage. Here we investigated the potential use of 19/3-scFv-Fc for treatment of CLL.
We first cultured peripheral blood mononuclear cells (PBMCs) from treatment-naïve CLL patients with bsAbs and measured CLL cell death by flow cytometry. After 12 days of exposure, median specific-killing compared to the non-targeting control HER2/CD3-scFv-Fc was 91.2% for blinatumomab (P= 0.0009) and 92.1% for 19/3-scFv-Fc (P= 0.003)(n = 12). Both CD19/CD3 bsAbs induced over 25-fold expansion of autologous CD8 and CD4 T cells, as demonstrated by increase in absolute cell counts and by CFSE proliferation assays. Activation markers CD69 and CD25, as well as granzyme B, were also increased in both CD8 and CD4 T cell subsets cultured with blinatumomab or 19/3-scFv-Fc.
As blinatumomab and 19/3-scFv-Fc demonstrated comparable activity against CLL ex vivo, we next evaluated efficacy of bsAbs in vivo using the NOD/SCID/IL2Rγnull (NSG) patient-derived xenograft model. Fifty million CLL PBMCs were injected per mouse, with 2-5 mice tested per treatment group and patient combination. bsAbs were then given once-weekly, with blinatumomab dosed at 0.25 mg/kg and 19/3-scFv-Fc at 0.5 mg/kg to achieve equal molar concentrations. Treatment with 19/3-scFv-Fc resulted in elimination of over 98% of CLL cells in the blood (P < 0.0001) and spleen (P= 0.0026) compared to treatment with HER2/CD3-scFv-Fc (n = 4). Blinatumomab failed to induce any response, either with once-weekly dosing or daily dosing for 8 days.
Ibrutinib has been shown to improve T cell dysfunction characteristic of CLL, suggesting immunotherapy may work well with concurrent ibrutinib treatment. Culture of CLL PBMCs from ibrutinib-treated patients (n = 10, 12 ± 1 months on ibrutinib) with 19/3-scFv-Fc induced T cell activation and increased granzyme B expression comparable to that seen in cells obtained from treatment-naïve patients. However, treatment with 19/3-scFv-Fc drove significantly more killing of ibrutinib-treated CLL cells compared to treatment-naïve CLL cells after 3 days of exposure (43.7% versus 2.23% median specific-killing, P= 0.004). By day 7, 95.3% of ibrutinib-treated and 45.3% of treatment-naïve CLL cells were eliminated (P= 0.02).
Resistance to ibrutinib has been commonly linked to mutations in BTK and/or PLCG2 and is associated with early mortality. We assessed activity of 19/3-scFv-Fc in 3 patients with acquired ibrutinib resistance, manifest 34 to 44 months from the start of ibrutinib. Multiple BTK and/or PLCG2 mutations were present in all 3 patients, with cancer cell fractions of the most abundant mutation in each patient ranging from 8-32%. 19/3-scFv-Fc treatment in culture resulted in >90% specific-killing of CLL cells from all 3 ibrutinib-resistant patients (range 91.50 - 97.83%, P= 0.0066). After engraftment of ibrutinib-resistant cells in NSG mice, 1 dose of 19/3-scFv-Fc eliminated >90% of circulating CLL cells, while HER2/CD3-scFv-Fc had no anti-leukemic activity (P < 0.0001), indicating that 19/3-scFv-Fc can effectively target mutant CLL clones resistant to ibrutinib. Taken together, these data support the investigation of 19/3-scFv-Fc as a promising immunotherapy for CLL, either in combination with ibrutinib or as rescue therapy in ibrutinib-resistant disease.
Wiestner: Pharmacyclics: Research Funding; Acerta Pharma: Research Funding.
Asterisk with author names denotes non-ASH members.
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