Correlation Between Clinical Responses and Immune Characteristics in Patients with Relapsed CLL Treated with Ofatumumab and Lenalidomide
Authors: Vitale Candida , Falchi Lorenzo , ten Hacken Elisa , Gao Hui , Shaim Hila , Van Roosbroeck Katrien , Calin George , O'Brien Susan , Faderl Stefan , Wang Xuemei , Wierda William , Rezvani Katayoun , Reuben James M , Burger Jan A. , Keating Michael , Ferrajoli Alessandra 
Published: October 2019

Abstract
Chronic lymphocytic leukemia (CLL) is characterized by phenotypical and functional alterations of different host immune components. Lenalidomide is an immunomodulatory agent which exerts both a direct antitumor effect and a pleiotropic activity on the immune system. Single-agent lenalidomide is active in patients with relapsed and refractory CLL, and the addition of an anti-CD20 monoclonal antibody to lenalidomide has shown synergism in vitro, as well as improved clinical responses in vivo.
We designed a phase II study with the aim of determining the efficacy and tolerability of combined ofatumumab and lenalidomide for the treatment of relapsed or refractory CLL. We also wanted to investigate whether immune system and tumor microenvironment characteristics, such as circulating T and NK cell number and function, and plasma levels of chemokines, cytokines, and angiogenic factors could be correlated with the response to treatment and could be modulated during the course of the therapy. Ofatumumab was administered at a dose of 300 mg on day 1, 1000 mg on days 8, 15, and 22 during course 1, 1000 mg on day 1 during courses 3-6, and once every other course on day 1 during courses 7-24 (28-day courses). Lenalidomide (10 mg daily) was started on day 9 and continued for as long as a clinical benefit was observed.
Thirty-four patients were enrolled. The overall response rate was 71%. Eight patients (24%) achieved a complete remission (CR) or CR with incomplete recovery of blood counts, including 9% with minimal residual disease-negative CR. The median progression-free survival was 16 months, and the estimated 5-year survival was 53%. The most common treatment-related toxicity was neutropenia (grade >2 in 18% of the 574 patient-courses). The most frequent infectious complications were pneumonia and neutropenic fever (24% and 9% of patients, respectively).
For the correlative studies, peripheral blood mononuclear cells and plasma samples were collected at baseline and after 3, 6, 9, and 12 months of therapy. Lymphocyte subsets were analyzed by flow cytometry. T and NK cell function was evaluated on 9 patients based on the expression of CD107a, IFNγ, TNFα, and IL-2 after the exposure to CD3/CD28 magnetic beads and K562 cell line, respectively. Chemokines, cytokines, and angiogenic factors were quantified through a multiplex bead-based immunoassay. Thirty-four age-matched healthy donors (HD) were used as controls. Treatment with ofatumumab and lenalidomide decreased the number of circulating T and NK cells. The greatest decrease was observed after 3 months of therapy as compared to baseline (p≤0.02 for all comparisons). We found that the baseline number of CD4+ T cells and CD57+ CD56+ NK cells was higher in CR patients as compared to non responder (NR) patients (median absolute number 1595/μl vs. 715/μl, p=0.04, and 358/μl vs. 70/μl, p=0.02). We also compared baseline T and NK cell populations of CR patients to HD, and found that the former had significantly higher numbers of CD16+ CD56+ and CD57+ CD56+ NK cells (median 356/μl vs. 80/μl, p=0.03, and 358/μl vs. 29/μl, p=0.0008). Interestingly, baseline numbers of CD16+ CD56+ and CD57+ CD56+ NK cells were comparable between NR patients and HD. We also observed that patients who achieved CR had significantly better T and NK cell function at baseline, whereas T and NK cell function was impaired in patients who only had partial or no response. Plasma levels of bFGF, VEGF and IFN-γ were significantly decreased at month 3 compared to baseline in all patients (p≤0.02 for all comparisons). Finally, significantly lower levels of circulating CCL2, CCL3, CCL4, IFNα, and IL-12p70 were detected during treatment in patients who achieved CR, compared to NR (p<0.05 for all comparisons).
In conclusion, the combination of ofatumumab and lenalidomide is a well tolerated regimen that induces durable responses in the majority of patients with relapsed CLL, and sustained MRD-negative CR in some. A higher number and preserved functional activity of T and NK cells were found in responding patients at baseline, as compared to NR, underscoring the importance of a competent immune system in supporting the effectiveness of this type of treatment, and suggesting a need to further investigate these immune features as markers for prediction of response to therapy.

Disclosures
Faderl: Celgene Corp.: Other: Advisory Board. Wierda:Celgene Corp.: Consultancy; Glaxo-Smith-Kline Inc.: Research Funding. Rezvani:Pharmacyclics: Research Funding. Burger:Pharmacyclics LLC, an AbbVie Company: Research Funding. Keating:Glaxo-Smith-Kline Inc.: Other: Advisory board; Celgene Corp.: Consultancy.