Obinutuzumab as bridging therapy for successful manufacturing of axicabtagene ciloleucel for transformed follicular lymphoma with circulating cells

To the Editor:

Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is approved for treatment of relapsed/refractory diffuse large B-cell lymphoma (DLBCL) and variants. In DLBCL, axicabtagene ciloleucel (axi-cel) and tisagenlecleucel (tisa-cel) were approved in the United States based on the results of the ZUMA-1 and JULIET trials, respectively.^1,2 Recent reports, including patients treated with axi-cel as a standard of care outside clinical trials, confirm its efficacy and durability of response in this poor risk population, including transformed follicular lymphomas (TFL).³ Some patients with transformed lymphomas may have circulating lymphoma cells. A high burden of circulating lymphoma cells is a concern for CAR T-cell manufacturing due to a potential risk of CAR transgene insertion into malignant B-cells and, consequently, disease progression, as recently reported in a case of acute leukemia.⁴ This potential scenario could adversely impact the manufacturing process in aggressive B-cell lymphoma with circulating malignant cells. The CD3+ T-cell selection could overcome the problem, but it is not an FDA approved process for axi-cel manufacturing (in contrast to tisa-cel). Successful treatment strategies are needed to clear circulating malignant cells in patients planned for CAR T-cell therapy. Here, we describe two cases of transformed follicular lymphoma with circulating malignant cells who received the anti-CD20 antibody obinutuzumab to facilitate successful axi-cel manufacturing.

Case 1:

A 52 y. old woman diagnosed eight years earlier with stage II follicular lymphoma was treated with several lines of therapy, prior to transformation into a high-grade refractory B-cell lymphoma. Prior therapies included: single agent rituximab, bendamustine-rituximab, R-CHOP, ofatumumab-CHOP and lenalidomide-ofatumumab. The patient was deemed eligible for axi-cel therapy and underwent leukapheresis; however, the first attempt for axi-cel manufacturing was unsuccessful, possibly due to a high content of circulating CD10 and CD20 positive, lambda light chain restricted follicular lymphoma cells in the leukapheresis product (Table 1). Flow cytometry of the peripheral blood showed 21.9% of circulating follicular lymphoma cells out of the total lymphoid events (12.2% of the total analyzed events). She was given two doses of obinutuzumab 1000 mg in addition to a chemotherapy combination of gemcitabine plus oxaliplatin (GEMOX) resulting in significant decrease of the circulating follicular lymphoma cells, down to 2.7% of the total lymphoid events (0.8% of the total analyzed events). She received two additional doses of obinutuzumab, and the repeated flow cytometriy analysis showed 8% follicular lymphoma cells of total lymphoid events (3% of total analyzed events). A pre-CAR T-cell infusion bone marrow (BM) aspirate and biopsy still showed diffuse involvement by follicular lymphoma, expressing CD20, PAX-5, BCL-6, and BCL-2. This occupyied approximately 80% of marrow space; and a staging PET-CT showed extensive adenopathy in the head/neck, mediastinum, retroperitoneal, mesenteric, and inguinal adenopathy. The second leukapheresis led to a successful manufacturing of axi-cel. A dose of 2 ×10⁶ CAR T-cells was eventually infused after a standard lymphodepleting regimen of fludarabine and cyclophosphamide (FluCy). The post-CAR-T infusion course was complicated by cytokine release syndrome (grade 2 per Lee Criteria) and grade 4 neurotoxicity by the CARTOX 10 assessment score. This manifests as severe encephalopathy and non-convulsive status epilepticus. That required mechanical ventilation for airway protection, and several antiepileptic medications apart from dexamethasone 10 mg/day (later switched to solumedrol 1000 mg/day) and one dose of intravenous immunoglobulins 0.4 g/kg. A BM aspirate and biopsy on day +30 showed no morphologic evidence of follicular lymphoma. The patient was discharged from the hospital on day +42 post axi-cel, and a PET-CT on day +48 showed CR. Repeat PET-CT on days +90 and +207 post axi-cel shows sustained CR. Her BM remains free of lymphoma. At the time of her last follow up, corresponding to day +319 post axi-cel, she was clinically asymptomatic.

TABLE 1.

Initial WBC, ALC and % circulating lymphoma cells pre-and post-obinutuzumab administration

	Patient 1	Patient 2
WBC pre-obinutuzumab	39.28 × 109/L	8.4 × 109/L
ALC pre obinutuzumab	33.85 × 109/L	2.1 × 109/L
% Circulating lymphoma cells pre-obinutuzumab	21.9%	20.8%
Number of obinutuzumab doses	4	1
WBC post obinutuzumab	4.7 × 109/L	1.88 × 109/L
ALC post obinutuzumab	1.06 × 109/L	0.19 × 109/L
% Circulating lymphoma cells post-obinutuzumab	8%	0

Abbreviations: ALC, absolute lymphocyte count; WBC, white blood cell counts.

Case 2:

A 66-year-old man presented ficw years earlier with stage IVB diffuse large B-cell lymphoma, germinal center subtype. He achieved complete remission with six cycles of R-CHOP. Four years later he developed recurrent lymphadenopathy with restaging imaging showing diffuse lymphadenopathy including a bulky abdominal mass. A cervical node biopsy revealed low-grade follicular lymphoma, which was also found in the bone marrow. The biopsy of the abdominal mass was consistent with DLBCL. His disease failed to show response after 2 cycles of GDP-R. He was planned for CAR T-cell therapy with axi-cel; but prior to leukapheresis circulating B-cell atypical lymphocytes at a level of 2.1 × 10⁹/L were observed on work up (Table 1). Flow cytometry confirmed a population of circulating CD10 positive cells representing 20.8% of the total population. He received a single dose of obinutuzumab 1000 mg IV which rapidly cleared atypical lymphocytes and facilitated leukapheresis. In fact, flow cytometry of the peripheral blood on the morning of apheresis confirmed absence of the CD10 positive population. Between the time of apheresis and CAR T infusion he received bridging radiation therapy to the large abdominal mass as well as dexamethasone 40 mg orally × 4 days. His axi-cel product was deemed to be within manufacturing specifications, and therefore he proceeded with lymphodepleting chemotherapy with fludarabine and cyclophosphamide and eventually axi-cel infusion. He tolerated the procedure well without significant toxicity. He did not show clinical signs of CRS but developed grade 1 neurologic toxicity per CTCAE 5.0 criteria. At day +30 post-axi-cel infusion his PET/CT showed a complete metabolic remission, and the circulating lymphoma cells remain undetectable since his initial obinutuzumab treatment.

Data show that the final leukapheresis MNC product could affect the CAR T-cell manufacturing process, especially in the presence of excessive monocytes, non-T cell lymphocytes and circulating malignant cells.⁵ While there are several methods for the separation of non-T cell lymphocytes and monocytes, this could be more challenging in the presence of circulating malignant B-cells for the manufacturing of axi-cel for aggressive B-cell lymphomas. Thus, effective alternatives that eradicate circulating lymphoma cells before leukapheresis are certainly needed. Obinutuzumab is a type II glycol-engineered anti-CD20 monoclonal antibody (MAb) that has higher antibody-dependent cellular cytotoxicity and phagocytosis, and less complement activation compared to rituximab.⁶ Obinutuzumab also has a more pronounced direct cell death effect when compared to other type 1 MAbs such as rituximab and ofatumumab.⁶ These properties can lead to a more rapid reduction of circulating malignant B-cells. Obinutuzumab is currently approved by the US FDA for chronic lymphocytic leukemia and follicular lymphoma.

These two cases demonstrate the efficacy of obinutuzumab in reducing circulating follicular lymphoma cell burden, and ultimately enabling successful manufacturing of axi-cel. It is not considered a required practice to perform flow cytometry for detection of monoclonal B-cells in patients being considered for CAR T-cell therapy. However, our cases illustrate that in patients with suspected circulating lymphoma cells standard flow cytometry will be required. That facilitates the successful axi-cel manufacturing, and reduces the risk of unintended transfer of the CAR gene into a malignant B-cell.