Rituximab improves Clinical Outcomes of CAR-T therapy for r/r B-ALL via sensitizing Leukemia Cells to CAR-T-mediated cytotoxicity and reducing CAR-T exhaustion

doi:10.21203/rs.3.rs-3621440/v1

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Rituximab improves Clinical Outcomes of CAR-T therapy for r/r B-ALL via sensitizing Leukemia Cells to CAR-T-mediated cytotoxicity and reducing CAR-T exhaustion

https://doi.org/10.21203/rs.3.rs-3621440/v1

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Despite chimeric antigen receptor (CAR) T-cell therapy has achieved great advances in recent year, approximately 50% of relapsed/refractory B cell acute lymphoblastic leukemia (r/r B-ALL) patients treated with CAR-T experience relapse 6 months post CAR-T treatment. CD20 express on 30 to 50% of B-ALL, which makes CD20 Monoclonal Antibody as one of the potential therapy strategies to decrease the tumor burden and improve the efficacy of CAR-T therapy. Adding Rituximab to chemotherapy protocol had been demonstrated to improve the outcome for CD20-positive ALL. However, rare study explored the influence of Rituximab combined with CAR-T therapy. We retrospectively analyzed 20 r/r B-ALL patients who received CAR-T therapy, all of whom had failed multiple lines of therapy. Before CAR-T infusion, we administered Rituximab to 10 patient with high CD20 expression at a dose of 375mg/m2 for 1 day. Meanwhile, we selected 10 patients with the comparable features who underwent CAR-T treatment without Rituximab in the same period as the control group. In vitro, the surface molecule expression and killing of CAR-T post Rituximab-treated B-ALL cells co-incubated with CAR-T cells were detected by flow cytometry. Rituximab combined with CAR-T therapy was effective for improving the long-term outcome of B-ALL patients who have failed multiple lines of therapy. In vitro, we observed that rituximab potentially improved CAR-T efficacy by sensitizing Leukemia Cells to CAR T-mediated cytotoxicity and reducing CAR-T exhaustion.

Chimeric antigen receptor T cell

Rituximab

r/r B-ALL

CD20 express on 30 to 50% of B-ALL[6-9], which makes CD20 Monoclonal Antibody as one of the potential therapy strategies to decrease the tumor burden and improve the efficacy of CAR-T therapy.

Intravenously administered rituximab was the first therapeutic mAb（Monoclonal Antibody）to be used in the field of oncology, establishing a new class of anticancer drugs. Adding rituximab to chemotherapy protocols has been shown to improve outcomes in CD20-positive ALL. In preclinical studies, rituximab augmented CD19 chimeric antigen receptor (CAR) T-cell function and increased tumor reduction and survival in murine models via synergistic targeting with CAR T-cells (引用：Mihara K, et al. Br J Haematol. 2010). However, rare study explored how rituximab improves CAR-T efficacy. Therefore, we evaluated the efficacy and safety of rituximab administration before CAR T therapy in r/r B-ALL and investigated the influence of rituximab for CAR-T as well as B-ALL cells in vitro.

1.Study Design and Patient Population

We retrospectively analyzed 10 r/r B-ALL patients who received CAR-T therapy at the First Affiliated Hospital of Soochow University between May 2017 and September 2022，all of whom had failed multiple lines of therapy and with high CD20 expression. Before CAR-T infusion, we administered Rituximab to all patient at a dose of 375mg/m2 for 1 day. Meanwhile, we selected the patients with the comparable features who did not receive Rituximab treatment in the same period as the control group. We matched control group with Rituximab group at the ratio of 1:1 based on: (1) age; (2) sex; (3) molecular mutations;(4) cytogenetic abnormalities; (5) CAR-T type; (6) conditioning regimen; (7) donor type. The study regimen was approved by the Ethics Committee of the First Affiliated Hospital of Soochow University, and all patients provided written informed consent.

2.Manufacture of tandem CD19/CD22 CAR-T cells

CAR-T cells were provided by Shanghai Unicar-Therapy Bio-medicine Technology Co. Ltd. Manufacture of CD19/CD22 CAR-T cells mainly consisted of retroviral vector (mCD19-mCD22 scFv.CD28/4-1BB/z) production, T cell transduction and CAR-T cell expansion(Figure 4A). CAR constructs (mCD19-mCD22 ) were synthesized and cloned into a third-generation lentiviral plasmid backbone. CAR constructs contained a CD8 transmembrane domain in tandem with CD28, OX40 and CD3ζ transmembrane domain. CAR-T cells were successfully manufactured and qualified tests were done before infusion.

3.Lymphodepletion chemotherapy

Patients received lymphodepletion regimens: FC (Fludarabine 30mg/m2/day and Cyclophosphamide 300 mg/m2/day) on days -5 to -3，with or without Decitabine (DAC) (total dose 100mg/m2 in 3 days from day -6 to -4). The median dose of CAR-T cells was 1× 10⁷ cells/kg（1-4×10⁷ cells/kg）at dose escalation.

4.Clinical response assessment

All disease assessments were conducted per protocol or institutional practice. Response assessment was performed 28 days after CAR-T cell infusion. Complete remission(CR) was defined as less than 5% bone marrow (BM) blasts, the absence of circulating blasts, and no extrame-dullary sites of disease, regardless of cell count recovery. Complete response with incomplete count recovery was a complete response with cytopenia. MRD negativity was defined as less than 0.01% bone marrow blasts by flow cytometry. Relapsed disease was defined as the reappearance of blasts in the blood or bone marrow or in an extramedullary site after a complete remission.

5.Toxicity assessment

Adverse events that occurred within one month after CAR-T therapy were recorded. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) was graded according to ASTCT Consensus Grading[11]. The diagnostic criteria for macrophage activation syndrome (MAS) were the diagnosis from MD Anderson for patients post CAR-T cells therapy[12]. Other toxicities were assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) version 5.0.

6.Cell Lines

The B lymphocyte leukemia cell line Nalm-6 was purchased from ATCC (Manassas, VA). Primary human ALL specimens were acquired from National Clinical Research Center for Hematologic Diseases. All cell lines were cultured in RPMI-1640 medium (Pricella, China)) supplemented with 10% foetal bovine serum (Sigma-Aldrich, USA), 2 mM L-glutamine (Pricella) and 100 U/mL peni-cillin/streptomycin (Pricella, China). This study design was approved by the First Affiliated Hospital of Soochow University Ethics Board. Written informed consent for publication of their clinical details was obtained from the patient/relative of the patient.

7.In vitro functional assays

The proliferation of T cells was determined by CSFE staining in vitro. Individual groups of CAR T cells (1 × 10⁶ cells/tube) were labeled with CSFE (Beyotime, China）at 37 °C for 30 min. After being washed, individual groups of cells were stimulated with the same number of target cells that had previously been treated by mitomycin. CAR-T cells were collected after 5 days, and fluorescence was detected by flow cytometry.

To detect the optimized E:T ratio, CAR T cells were co-cultured with target cells at different E:T ratios（0.5:1，1:1，5:1） in 96-well plate. CCK8 were added to each well, and the optical density(OD) were measured after 4H by Epoch (BioTek). The killing rate was calculated by the following equation: 1-([OD of sample]-[OD of negative control])/([OD of positive control]-[OD of negative control]).

For Apoptosis assay, CAR-T cells were incubated with target cells at E/T ratios（1:1）for 24h, and the target cells stained by CSFE and PI were tested with Flow cytometry to evaluate the killing rate of effect cells. Effector cells and target cells were cultured at an E:T ratio of 1:1 in RPMI 1640 media for 24 h. Supernatant of culture was analyzed by Cytometric bead array according to the manufacturer’s instructions (BD Biosciences ).

TUNEL assay using the BrightRed Apoptosis Detection Kit (Vazyme, China), according to the manufacturer’s instruction. Images were obtained under a microscope (Nikon, Japan)。

8.Activation Marker and Exhaustion Marker Staining

CAR-T cells were co-cultured with Nalm-6 cells in the medium at the ratio of 1:1 E:T for 24 h, 48 h, and 72 h. The following anti-human antibodies were also used in this study: CD3 (Percp-cy5.5, 1:100 dilution), CD4 (FITC, 1:100 dilution), CD8 (APC, 1:100 dilution), CD25 (BB700, 1:100 dilution) , CD69(PE, 1:100 dilution), CD62L (PE, 1:100 dilution), PD-1 (PE, 1:100 dilution), TIM-3 (BB515, 1:100 dilution), LAG-3 (AF674, 1:100 dilution) were purchased from BD Biosciences. The resulting datas were analyzed by FlowJo software.

9.Statistical Analysis

OS was defined as the time from CAR-T cell infusion to death or the end of follow-up. LFS was defined as the time from CAR-T cell infusion to first relapse or death, the end of follow-up. OS and LFS were censored at the last follow-up. The probabilities of OS and LFS were estimated by the Kaplan-Meier (KM) method and assessed with the use of the log-rank test. Median follow-up time was estimated by using the reverse KM method. Categorical variables were performed with Fisher’s exact tests. Comparisons of two groups were determined by two-tailed parametric t tests for unpaired or paired data. All the reported P values were two-sided and statistically significant was defined as P < 0.05. All the analyses were performed in the GraphPad Prism 8. The statistical test used for each figure is described in the corresponding figure legend.

1. Patient characteristics

We retrospectively analyzed 20 r/r B-ALL patients who received CAR-T treatment between June 2017 and September 2021 at the First Affiliated Hospital of Soochow University. Ten patients received treatment with Rituxima before CAR-T cells infusion (Rituximab group), and ten patients did not receive treatment with rituximab (Control group). Demographic and clinical characteristics of patients with r/r B-ALL were shown in Table 1. Baseline characteristics of patients in both groups had no significant differences. In addition, both groups were treated during the s ame period so that there would be no significant differences in supportive care (Table 1).

2. Clinical Response

28 days after CAR-T cells infusion, 8 of the 10 patients (80%) achieved minimal residual disease negative complete remission (MRD-negative CR) in the Rituximab group, who remained CR for the median time of 11.15 months(0.46-46.93) post-CAR-T therapy. 6 of the 10 patients (60%) achieved MRD-negative CR in the Control group, who remained CR for the median time of 5.03 months(0.13-28) post-CAR-T therapy (p = 0.158)(Table 1).At the time of the analysis，8 of the 10 patient (80%) remained alive in the Rituximab group. 3 of the 10 patients (30%) remained alive in the Control group (Figure 1A). In the Rituximab group, 6 patients (60%) proceeded to allo-HSCT, and 5 (5/6, 83.3%) patients were alived with CR. However, 8 patients (80%) in the Control group underwent allo-HSCT, and just 3 (3/8, 37.5%) patients were alive with CR( Figure 1B-C). Immune-modulating cytokines, including TNF-α, IFN-γ, IL-2, IL-6, IL-4 and IL-10, were induced in patients following 19-22 CAR-T and rituximab infusion and were more prominently elevated in Rituximab Group vs Control Group( Figure 1 D).

3. Long-term Survival

The median follow-up of Rituximab and Control group were 29.27 and 9.83 months. We found that adding Rituximab may confer a favorable prognosis compared with Control group. The 2-year overall survival (OS) and leukemia-free survival (LFS) rates both were longer in the Rituximab group (90% vs 26.7%, p=0.0342; 41.7% vs 25%, p=0.308)(Figure 1E-F). Particularly, subgroup analysis showed that Rituximab could markedly improve the OS and LFS for relapsed patients (88.9% vs 25%, p=0.0203; 87.5% vs 15%, p=0.0112). (Figure 2 A-B).In addition, CAR-T combined with Rituximab improved long-term prognosis in patients who had failed multiple lines of therapy (>5) (OS 100% vs 28.5%, p=0.0205; LFS 50% vs 0%, p=0.0119) (Figure 2 C-D). After CAR-T treatment, we analyzed subgroups of patients who were treated with or without bridging allo-HSCT. We found that the long-term prognosis of the Rituximab group was superior to the Control group. For patients who received allo-HSCT after CAR-T therapy，OS in the Rituximab group was significantly longer than the Control group (83.3%vs33.3%, p=0.0469)(Figure 2 E-F). For patients who did not receive allo-HSCT after CAR-T therapy，LFS in the Rituximab group was significantly superior to that in the Control group (33.3%vs 0%, p=0.0389)(Figure 2 G-H）.

4. Safety

There were no significant differences between the two groups in terms of hematological and non-hematological toxicities. All patients experienced grade 1-3 cytokine release syndrome (CRS) symptoms, such as fever and hypotension, which were effectively managed through symptomatic treatment. Two patients in the Rituximab group developed headache, while patients in the Control group had no neurotoxicity syndromes. Moreover, all patients suffered from hematological toxicities in our study. Median time to platelet and neutrophil counts recovery were similar in both groups. In addition, 50% (5/10) of patients in the Rituximab group experienced infection, and 30% (3/10) in the control group. All adverse events were reversible and manageable (Table 1).

5. Expansion and Killing of CD19/CD22 CAR-T Cells was improved by Pre-treatment of Tumor Cells With Rituximab

Our clinical data indicated that Rituximab combined with CAR-T treatment was effectively improved the long-term prognosis of r/r B-ALL patients. Subsequently, we evaluated the killing ability of Rituximab combined with CAR-T on tumor cells and the proliferation of CAR-T in vitro.

Firstly, we evaluated the tumor killing effect by selecting CD19, CD20 and CD22 simultaneously antigen-expressing B-ALL cells (NALM-6) as well as primary cells (Figure 3B). Subsequently, we identified the concentration of Rituximab in vitro that were effective for killing tumors cell (NALM-6,Primary cell) without impacting CAR-T activity (Figure 3C，Supplymentary 1A). We used a concentration of 350 nM Rituximab to treat tumor cells followed by flow cytometry to detect the expression of CD19 and CD22, and the results showed that the expression of CD19 and CD22 had not obviously altered (Supplymentary 1C). We also selected an efficacy target ratio based on the killing of NALM-6 by Rituximab combined with CAR-T cells , and eventually we selected 1:1 efficacy target ratio for the subsequence experiments (Figure 3D).

In proliferation experiments，we found CD19-CD22 CAR-T cells rapidly proliferated upon Nalm-6 challenge, with a peak level of expansion occurring at day 5 (Figure 3E). Rituximab presensitized tumor cells facilitated the CAR-T cell proliferation.

We divided mitomycin-treated NALM-6, and primary cells into two groups according to the pretreatment with or without Rituximab, following which they were incubated with CD19/CD22 CAR-T cells. FCM analysis showed that Rituximab group significantly promoted tumor cell apoptosis compared to the Control group（p<0.001），Terminal deoxynucleotidyl transferase dUTP nickend labeling (TUNEL) assays displayed that the numbers of apoptotic cells in Rituximab groups were significantly greater than that of Control groups(Figure3F, Supplymentary 1D).

To examine the effector function of rituximab combined with CAR-T , a panel of cytokines were measured during the in vitro cytotoxicity assay. Compared with control group, a broad range of cytokines awere produced by rituximab combined with CD19-CD22 CAR-T when co-cultured with NALM-6. Increased secretion of effector cytokines and chemokines such as interferon-γ(IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2) was observed (Figure 4A).

6. Pre-treatment of tumor cells with Rituximab affects the surface molecule expression of CD19-CD22 CAR-T

To investigate the effects of Rituximab on the expression of surface marker molecules on CD19/CD22 CAR-T cells, we co-cultured Rituximab-treated tumor cells with CAR-T cells for 24 hours.Subsequently, we assessed the expression of activation molecules and memory molecules on CAR-T cells using flow cytometry. The analysis revealed that CD25+, CD8+CD69+, and CD4+CD62L+ molecules on the surface of CD19/CD22 CAR-T cells were significantly upregulated in the Rituximab pre-treatment group compared to the group without Rituximab treatment (p<0.001) (Figure 4A-C).

To investigate the influence of Rituximab on the persistence of CD19/CD22 CAR-T cells, the expression of inhibitory molecules PD-1, LAG-3 and Tim-3 on CD19/CD22 CAR-T cells were monitored. FCM analysis at 24h showed that the expression of CD19/CD22 CAR-T cell surface exhaustion molecules (LAG-3, PD-1, TIM-3) were significantly lower in the Rituximab group than in the Control group (p<0.001) (Figure5 A-C). Interestingly, compared to the control group, FCM analysis at 96h showed that pretreatment of tumor cells with rituximab did not significantly upregulate CD19/CD22 CAR-T surface exhaustion molecules(PD-1,TIM-3) (Figure5 B-C).

CAR-T cells have emerged as a potent therapeutic weapon against B cell hematologic malignancies, leading to an exponential increase of clinical trials [13]. Even though ALL has the highest success rate, it still has an 40% recurrence rate, strong activation in clinical therapy drives CAR-T cells exhaustion and even apoptosis, which limits their antitumor efficacy[14-15]. A Long-Term Follow-up study of CD19 CAR-T Therapy revealed that patients with a low disease burden had a significantly longer EFS and OS than did those with a high disease burden [4]. Hence, reducing tumor burden prior to CAR-T therapy would be a strategy with beneficial effects on the long-term survival of r/r B-ALL.

Rituximab has revolutionized the treatment of B-cell malignancies, being part of the standard of care for FL [16-17], DLBCL [17-18], and CLL[19-20]. Recent clinical studies have showed that Rituximab also exhibits good antitumor effects in other B lineage hematologic tumors. There had been clinical trials (or trial) demonstrated that Rituximab combined with chemotherapy can increase EFS compared to only chemotherapy in Philadelphia chromosome negative acute lymphoblastic leukemia[21]. Thus, we successfully treated r/r B-ALL patients who failed multiple lines of therapy by Rituximab combined with CAR-T. Our clinical data showed that Rituximab combined with CAR-T improved the long-term prognosis of r/r B-ALL patients. Whether or not they were bridged to transplantation after CAR-T treatment, Rituximab combined with CAR-T still improved the long-term prognosis of patients. Besides, for the patients infused with CD19/CD22 CAR-T cells , rituximab combined with CAR-T effectively improved the LFS of r/r B-ALL patients. We observed that pretreatment of tumor cells with Rituximab enhanced the expansion of CD19/CD22 CAR-T in vitro. Concurrently, FCM analysis showed that CD25+, CD8+CD69, CD4+CD62L+ on the surface of CD19/CD22 CAR-T cells were also significantly up-regulated (p<0.001). Subsequently, in Cytotoxicity Assays, we demonstrated that Rituximab pretreatment of tumor cells increased the killing activity of CAR-T cells. Our data in vitro demonstrated that Rituximab was efficient in reducing tumor burden, moreover, the CAR-T combined with Rituximab enhanced the killing efficiency of CAR-T in vitro. Accordingly, we conclude that rituximab might be enhancing the efficacy of CAR-T cells by reducing tumor burden.

In a subgroup analysis, we found that rituximab combined with CAR-T was effective in improving the long-term prognosis of relapsed patients. Besides, rituximab combined with CAR-T significantly improved LFS in patients who failed multiple lines of therapy, so we suggested that rituximab may be effective in relapsed patients, particularly for those who failed multiple lines of therapy. CAR-T cells are depleted due to repetitive antigen stimulation in relapsed patients, as evidenced by upregulation of CAR-T exhaustion molecules and dysregulation of activation molecules [22-23]. Nevertheless, there was limited evidence on whether rituximab was an effective approach to reduce CAR-T exhaustion. To investigate whether rituximab has an effect on CAR-T persistence, we stimulated CAR-T cells repeatedly in vitro with rituximab-treated nalm-6 to evaluate the changes in CAR-T surface exhaustion molecules at different times. We found that the expression of CAR-T cell surface exhaustion molecules (LAG-3, PD-1, TIM-3) were significantly lower in the Rituximab group than in the Control group. Besides, the upregulation of CAR-T cell surface exhaustion molecules was also significantly slower in the rituximab group versus the control group. We only observed alterations in exhaustion molecules without further exploring the mechanism, so the evidence was limited. We speculated that rituximab combined with CAR-T treatment could potentially enhance CAR-T efficacy via reducing its exhaustion.

To the best of our knowledge, this is the first report to assess the efficacy and safety of rituximab combined with CAR-T therapy in patients with r/r B-ALL. This study represented the successful application of rituximab combined with CAR-T therapy in patients with r/r B-ALL who have failed multiple lines of therapy. In our study, rituximab combined with CAR-T therapy showed an excellent safety record with manageable adverse effects in patients. Furthermore, we performed in vitro assays to investigate how rituximab enhanced CAR-T capabilities. Despite the limitations of the data, we made a pioneer to observe the clinical benefits of rituximab combined with CAR-T therapy and to further investigate the effect of rituximab on CAR-T cells in vitro, which partly illustrated the effectiveness of rituximab combined with CAR-T therapy.

Acknowledgment

The authors would like to thank all members of the study team, the patient and their family, and Shanghai Unicar-Therapy Bio-medicine Technology Co., Ltd.

Funding

This work was supported by research grants from National Natural Science Foundation of China (82070162), Frontier Clinical Technical Project of Suzhou Science and Technology plan (SKY2022001), Bethune Charitable Foundation (BCF-IBW-XY-20220930-13), Suzhou diagnosis and treatment project of Clinical Key Diseases (LCZX202201), China International Medical Foundation (Z-2018-31-2102-4), Boxi clinical research project of The First Affiliated Hospital of Soochow University (BXLC005), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Contributions

YL collected, analyzed research data, designed the research, performed statistical analyses, wrote and edited the manuscript; SL, LL and JG collected research data; XT, QC, ML, ZL, XZ, and WC treated the patients; LK and LY designed the clinical CAR vector, supervised the production of CAR T-cell Product; DW and XT conceived of the study and revised the paper. All authors have read and approved the final manuscript.

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Table 1 is available in the Supplementary Files section.

No competing interests reported.

S1.png
Supplymentary 1 A. The inhibition of primary cells by rituximab at different drug concentrations as measured by CCK8. Curves were plotted to calculate IC50（323.7nM) B. The impact of rituximab on CAR-T cell activity at different drug concentrations was measured by CCK8. (n =3 samples examined over three independent experiments). C. Formalin-fixed cells were stained for TUNEL assays. Quantitative analysis of apoptotic cells. Data are representative images (magnification×20) or expressed as the mean±SD from three randomly selected fields of cells. The quantitative analysis was used by one-way ANOVA with multiple comparisons test. D. Expression of CD19 and CD22 antigens on the surface of target cells (NALM-6,primary cells) after rituximab treatment by flow cytometry
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Rituximab improves Clinical Outcomes of CAR-T therapy for r/r B-ALL via sensitizing Leukemia Cells to CAR-T-mediated cytotoxicity and reducing CAR-T exhaustion

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