Immunotherapy of low grade lymphoid malignancies

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

We designed a clinical trial to investigate ofatumumab based therapy. In this phase 2 study investigated the efficacy of risk-adapted chemoimmunotherapy in treatment-nave CLL (NCT01145209). Patients with high-risk cytogenetics (deletion 17p or 11q) received induction with fludarabine, cyclophosphamide, and ofatumumab. Those without high-risk cytogenetics received fludarabine and ofatumumab. After induction, MRD positive (MRD+) patients received 4 doses of ofatumumab maintenance. MRD negative (MRD-) patients had no intervention. All evaluable patients responded to induction and 10 (36%) achieved MRD-. Two-year progression-free survival (PFS) was 71.4%. Ofatumumab maintenance did not convert MRD+ to MRD-. In the MRD+ group, CD20 antibody binding capacity markedly decreased during therapy, suggesting selective loss of CD20 antigens. In conclusion, ofatumumab maintenance did not improve the depth of response in MRD+ patients. The loss of targetable CD20 likely reduces the efficacy of maintenance therapy. Following administration of anti-CD20 mAbs CD20 expression is down-modulated by trogocytosis and internalization. While cells with low or absent CD20 expression can evade further anti-CD20 mAb binding, they are labelled by complement proteins. In particular, complement component C3d is covalently bound to the cell surface. We hypothesized that C3d constitutes a neoantigen that could be targeted by anti-C3d mAbs to enhance anti-CD20 therapy. We derived several hybridomas from mice immunized with human C3d and found to bind three distinct epitopes on C3d. One epitope was shared by all high affinity mAbs, while two other epitopes were bound by low affinity mAbs. From a high affinity murine mAb we derived a human IgG1 chimeric antibody that is highly selective for C3d, does not bind full length C3, and does not cross react with mouse C3d. The chimeric anti-C3d mAb bound immobilized C3d with a Kd of 17nM and cell-bound C3d with a kD of 3.0nM, similar to the kD of 2.4nM of ofatumumab binding to CD20. The anti-C3d antibody mediated complement-dependent cytotoxicity, NK cellular cytotoxicity, and phagocytosis of C3d opsonized cells. We tested activity of the anti-C3d mAb against primary tumor cells from patients with chronic lymphocytic leukemia (CLL) being treated with the anti-CD20 mAb ofatumumab. The first administration of ofatumumab decreased the tumor cell count in the blood of patients by 50% on average. As expected, CLL cells obtained 24 hours after administration of ofatumumab had lost CD20 antigen and carried abundant C3d on their cell surface. Importantly, the anti-C3d chimeric antibody specifically bound CLL cells but no other blood cells, indicating that targeting of C3d preserves the specificity of the initial complement fixing antibody. These studies provide proof of concept that targeting cell deposited C3d can enhance the potency of mAb therapy. Given that our anti-C3d mAb preserves the specificity of the initial mAb, it could augment the potency of many mAbs currently in clinical use by delivering a one-two punch. We conclude that targeting C3d deposited on cancer cells can eliminate antigen escape variants and potentiate existing therapeutic antibodies. We developed a novel CD19/CD3 bsAb in the single-chain Fv-Fc format (CD19/CD3-scFv-Fc) with a half-life of approximately 5 days. In in vitro experiments, both CD19/CD3-scFv-Fc and blinatumomab induced >90% killing of CLL cells from treatment-nave patients. Anti-leukemic activity was associated with increased autologous CD8 and CD4 T cell proliferation, activation, and granzyme B expression. In the NOD/SCID/IL2Rnull patient-derived xenograft mouse model, once-weekly treatment with CD19/CD3-scFv-Fc eliminated >98% of treatment-nave CLL cells in blood and spleen. We next explored the activity of CD19/CD3-scFv-Fc in the context of ibrutinib treatment and ibrutinib resistance. CD19/CD3-scFv-Fc induced more rapid killing of CLL cells from ibrutinib-treated patients than those from treatment-nave patients. CD19/CD3-scFv-Fc also demonstrated potent activity against CLL cells from patients with acquired ibrutinib-resistance harboring BTK and/or PLCG2 mutations in vitro and in vivo using patient-derived xenograft models. Compared to observations with samples from treatment-nave patients, T cells from patients being treated with ibrutinib expanded more rapidly and exerted superior cytotoxic activity in response to the bsAb. In addition to BTK, ibrutinib also inhibits IL2 inducible T cell Kinase (ITK). In contrast, acalabrutinib, does not inhibit ITK. Whether ITK inhibition contributes to the observed immune effects is unknown. To better understand how BTKis modulate T-cell function and cytotoxic activity, we cultured peripheral blood mononuclear cells (PBMCs) from BTKi-naive, and ibrutinib- or acalabrutinib-treated CLL patients with CD19/CD3 bsAb in vitro. T-cell expansion, activation, differentiation, and cytotoxicity were increased in PBMCs from patients on treatment with either BTKi compared to that observed for BKTi-nave patients. BTKi therapy transcriptionally downregulated immunosuppressive effectors expressed by CLL cells, including CTLA-4 and CD200. CTLA-4 blockade with ipilimumab in vitro increased the cytotoxic activity of the bsAb in BTKi-nave but not BTKi-treated PBMCS. Taken together, BTKis enhance bsAb induced cytotoxicity by relieving T cells of immunosuppressive restraints imposed by CLL cells. The benefit of combining bsAb immunotherapy with BTKis needs to be confirmed in clinical trials. To investigate the effect of ibrutinib on distinct T cell clonotypes, we performed deep sequencing of the TCR repertoire in 26 patients with CLL prior to treatment with ibrutinib, at the time of response, and either at disease progression or during sustained remission. The median productive clonality in CLL patients before treatment was more than double the clonality reported for healthy donors and was higher in patients with relapsed/refractory CLL compared to treatment-nave patients. Notably, CD8+ but not CD4+ T-cell counts were significantly correlated with clonality. Clonality increased at the time of response compared to baseline and remained high in patients with sustained anti-tumor response. In 12 patients who subsequently progressed, clonality decreased from the time of response to relapse. Expanding T cell clonotypes were patient-specific and correlated with CD8+ T cell numbers. Based on these preliminary data, we currently are pursuing the hypothesis that distinct CD8+ T-cell clonotypes recognize tumor antigens in a patient-specific manner and may contribute to anti-tumor activity during treatment with ibrutinib. In two open-label, single-arm clinical trials, we measured the effect of BTKis on de novo immune response against recombinant hepatitis B vaccine (HepB-CpG) and recall response against recombinant zoster vaccine (RZV) in CLL patients who were TN or on BTKi. The primary endpoint was serologic response to HepB-CpG (anti-HBs 10 mIU/mL) and RZV (4-fold increase in anti-glycoprotein E). The response rate to HepB-CpG was lower in patients on BTKi (3.8% 95% CI, 0.7%-18.9%) than TN patients (28.1% 95% CI, 15.6%-45.4%, P=.017). In contrast, the response rate to RZV did not differ significantly between the BTKi (41.5% 95% CI, 27.8%-56.6%) and TN cohorts (59.1% 95% CI, 38.7%-76.7%, P=.2). BTKis were associated with a decreased de novo immune response following HepB-CpG, whereas recall immune response following RZV was not significantly affected by BTKi therapy. Trials were registered at www.clinicaltrials.gov as NCT03685708 (Hep-CpG) and NCT03702231 (RZV). Ongoing analyses focus on the cellular response to the Shingrix vaccine.