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CD19 CAR-NK cell therapy for the treatment of positive lymphoid tumors

CD19 CAR-NK cell therapy for the treatment of positive lymphoid tumors



Introduction to the clinical results of CD19 CAR-NK cell therapy for the treatment of positive lymphoid tumors.

Research background

Anti-CD19 chimeric antigen receptor (CAR) T cell therapy can effectively treat diffuse large B-cell lymphoma (DLBCL), a cancer with limited treatment options and poor outcomes, especially for relapsed or refractory cancers (r/r) Patients with disease.

However, CAR-T cells can produce a lot of toxic effects, and cell manufacturing is very complicated. Natural killer (NK) cells that have been modified to express anti-CD19 CAR may overcome these limitations.

Introduction to research methods

In this Phase 1 and Phase 2 trial, we used cord blood-derived patients with 11 patients with relapsed or refractory CD19-positive cancers (non-Hodgkin’s lymphoma or chronic lymphocytic leukemia [CLL]) HLA mismatches against CD19 CAR-NK cells.

NK cells are transduced with a retroviral vector that expresses genes encoding anti-CD19 CAR, interleukin 15 and inducible caspase 9 as safety switches. The cells are expanded in vitro and administered as a single infusion of one of three doses (1×105, 1×106 or 1×107 CAR-NK cells per kilogram of body weight) after lymphocyte depletion chemotherapy.

Introduction of Research Results

Patient characteristics

From June 2017 to February 2019, we continuously recruited 15 patients in accordance with the agreement. Of these patients, 4 withdrew before starting treatment due to disease progression, development of graft-versus-host disease, no detectable disease, and bacterial contamination of the product (1 patient per patient). Therefore, 11 patients received a single dose of CAR-NK cells (Table 1).

CD19 CAR-NK cell therapy for the treatment of positive lymphoid tumors

The median age of the patients was 60 years (range 47 to 70). These 11 patients have already received a median 4-line treatment (range, 3 to 11). Five patients had CLL (including two with Richter transforming or accelerated CLL) and had a history of disease progression when receiving ibrutinib plus at least three other treatments; all five patients had high-risk genetic characteristics. Six patients had lymphoma, of which 2 were diffuse large B-cell lymphoma, and 4 were follicular; 3 of them were transformed into high-grade lymphoma. Among the 6 patients with lymphoma, 4 had disease progression after autologous hematopoietic stem cell transplantation, and 2 had refractory disease.

Safety

After CAR-N cell infusion, all patients had no symptoms of cytokine release syndrome, neurotoxicity, or hemophagocytic lymphohistiocytosis. In addition, despite the HLA mismatch between patients and their CAR-NK products, we have not observed any cases of graft-versus-host disease. As expected, all patients had transient and reversible hematological toxicity events, mainly related to lymphocyte depletion chemotherapy. We cannot determine whether the injection of CAR-NK cells will cause hematological toxicity. There were no cases of tumor lysis syndrome or grade 3 or 4 non-hematological toxicity. The maximum tolerated dose of CAR-NK cells has not been reached. Table 2 lists all the adverse events observed in the study. No patients were admitted to the intensive care unit (ICU) due to the treatment of adverse events related to CAR-NK cells. However, patient 2 was admitted to the ICU for treatment of progressive lymphoma and subsequently died. In view of the absence of serious toxicity in the study, we did not activate the caspase 9 safety switch (using Rimidoxine) to eliminate CAR-NK cells.

CD19 CAR-NK cell therapy for the treatment of positive lymphoid tumors

Treatment response

At a median follow-up of 13.8 months (range 2.8 to 20.0), 8 patients (73%) achieved objective remission, of which 7 patients (3 CLL patients and 4 lymphoma patients) had a complete remission (Figure 1). Another patient with Richter transformation CLL (patient 5) completely relieved the high-grade lymphoma. According to positron emission tomography-computed tomography (PET-CT), no lesions of fluorodeoxyglucose uptake were found with CAR-NK infusion. Thirty days after the injection, there was still cytopenia, accompanied by bone marrow infiltration of CLL. Although this patient finally achieved complete remission when receiving post-remission treatment, we did not attribute this remission to CAR-NK treatment. In all 8 patients, the response to treatment occurred in the first month after the infusion. Of the 11 patients treated, 5 received products that did not match the KIR ligand. It is impossible to evaluate the effect of KIR ligand mispairing results in such a small series.

CD19 CAR-NK cell therapy for the treatment of positive lymphoid tumors

B cell aplasia

Since B cell hypoplasia has been used as a substitute for anti-CD19 CAR T cell activity, we measured the frequency of CD19-positive B cells in the peripheral blood of patients after CAR-NK cell infusion. With the exception of patients 1 and 5, all patients had B cell aplasia associated with previous B cell depletion therapy at the time of enrollment.

In patient 1, B cell aplasia occurred after CAR-NK treatment and lymphocyte depletion chemotherapy. Patient 5 had persistent CLL in the peripheral blood, despite a complete response to high-grade transformation, until he received venetoclax. Patient 3 has evidence of B cell recovery, which is consistent with a positive recurrence of minimal residual disease. During the follow-up period, none of the remaining patients returned to normal B cell counts.

CAR-NK expansion, migration and persistence

We use a quantitative real-time polymerase chain reaction assay to measure the in vivo expansion of CAR-NK cells based on the number of vector transgene copies per microgram of genomic DNA. Expansion was observed as early as 3 days after infusion, and CAR-NK cells persisted for at least 12 months (Figure 2A). The peak CAR-NK copy number is measured 3 to 14 days after infusion and depends on the dose.

After the 14th day, no dose-related differences were found in peripheral blood transcription levels or persistence of CAR-NK cells. As reported in patients receiving CAR-T cell therapy, the early expansion of CAR-NK cells in the 5, 6, and 23 patients who responded to the treatment in our study was significantly higher than that of patients who did not respond (Figure 2B ).

According to the degree of HLA mismatch with the recipient, we did not observe the persistence difference of CAR-NK cells (Table 1). These results were confirmed by flow cytometry.

In the two patients with available lymph node samples, more CAR-NK cells were found in the lymph nodes than bone marrow or peripheral blood. This finding supports the view of CAR-NK cells. Focus on the disease website. Similar levels of CAR-NK cells were detected in the bone marrow and peripheral blood of 10 patients with available samples.

The minimal number of contaminating CAR expressing T cells in the product will not cause detectable CAR T cell expansion after infusion, and CD3+ T cells will not cause the development of graft-versus-host disease (Figure 1). Although CD19 is expressed in tumor cells, low levels of CAR-NK cells can still be detected in patients who have not responded or relapsed, indicating that there are alternative immune escape mechanisms, such as inducing CAR-NK exhaustion. The function of CAR-NK cells remaining in relapsed patients has not been studied yet. At the time of relapse, persistent CAR-NK cells did not expand in the body.

Serum cytokine analysis

The inflammatory cytokines and interleukin-15 in the supernatant of a series of peripheral blood samples were measured, the latter being encoded by a retroviral vector used to produce CAR-NK cells. Compared with the baseline level, we did not observe an increase in the levels of inflammatory cytokines (for example, interleukin 6 and tumor necrosis factor alpha), nor did we find that the systemic level of interleukin 15 was higher than the value before treatment, which indicates that leukocytes The CAR-NK cells in the peripheral blood of interleukin 15 did not release to a significant systemic level after the infusion.

Induce an alloimmune antibody response against the donor

All patients received CAR NK products that did not match the HLA. Patients 1 to 9 received products that were partially matched to 4 of the 6 HLA molecules, while patients 10 and 11 were recipients of non-HLA matched CAR-NK cells. Therefore, we monitored the induction of donor-specific HLA antibodies. At all time points when the test was performed, no antibody induction against the mismatched HLA alleles of the infusion product was observed. The host cell response was not evaluated.

CD19 CAR-NK cell therapy for the treatment of positive lymphoid tumors


Conclusion analysis

Among the 11 patients with relapsed or refractory CD19-positive cancer, most of the patients responded to CAR-NK cell therapy, but no serious toxic effects occurred. (ClinicalTrials.gov number, NCT03056339.)

Engl J Med 2020;382:545-53. DOI:10.1056/NEJMoa1910607

Introduction to the clinical results of CD19 CAR-NK cell therapy for the treatment of positive lymphoid tumors.

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