A second‐generation CD38‐CAR‐T cell for the treatment of multiple myeloma

Abstract Background Multiple myeloma (MM) is an aggressive plasma cell malignancy, causing a number of deaths worldwide every year. Chimeric antigen receptor (CAR) transduced T‐cell therapy has been a promising immunotherapy against hematological malignancies. Methods In this study, we developed a second‐generation CAR construct and generated CAR‐T cells targeting CD38 molecule. Then effects of CAR‐T cells against MM cell lines were evaluated. Results CD38‐CAR‐T cells showed higher cytotoxicity to MM cell lines and primary MM cells than that of control T cells in vitro. Over 50% MM1.s and RPMI8226 cells were killed by CAR‐T cells even at effector to target ratio of 1:100. CAR‐T cells also showed an enhanced cytotoxicity against primary MM cells. CAR‐T cells could be activated and produced a variety of cytokines in a target‐dependent manner. In vivo test indicated that CAR‐T cells also showed significant antitumor effect on xenograft mice models. Conclusion These results indicated a promising therapeutic strategy of CD38‐CAR‐T cells against MM.


| INTRODUCTION
Multiple myeloma (MM), an aggressive malignancy derived from plasma cells, remains the second most common hematological tumor in the world. Melphalan and prednisone used to be the first-line therapy for MM. However, the 5-year survival rate of MM patients was only 34.5%-49.6%. 1 In recent years, novel therapeutic methods, including proteasome inhibitors, immunomodulatory drugs, and targeted immunotherapy agents, have contributed to improved progression-free survival and median overall survival. 2 Nonetheless, drug resistance and poor prognosis of patients with relapse or refractory MM (RRMM) remain great challenges for hematologists and oncologists. 3 Thus, it is necessary to develop novel therapies to reduce the risk of recurrence and improve patient survival.
A new strategy, CAR-T-cell immunotherapy has shown tremendous potential in the treatment of hematological malignancies over the past decade. 1,2 CAR-T cells targeting different molecules such as CD19, CD20, CD22, and BCMA have been developed and investigated for the administration of leukemia, lymphoma, and MM. [3][4][5][6][7] CD38 plays an important role in pathogenesis and prognosis of human immunodeficiency virus infection and chronic lymphocytic leukemia. 8 Due to its high expression on the surface of MM cells, CD38 has been considered as a promising immunotherapy target for MM. [8][9][10][11][12][13] Anti-CD38 monoclonal antibodies such as daratumumab and isatuximab have been evaluated in the treatment of MM. However, the overall response rate (ORR) was only 36% in MM patients treated with daratumumab as a single agent. 12 While the ORR is 50%-60% in relapsed MM patients treated with isatuximab, lenalidomide, and dexamethasone. 17 There have been studies of CAR-T cells targeting CD38 molecule alone or in combination with B-cell maturation antigen (BCMA). [18][19][20][21] Clinical trials (NCT03464916 and NCT03767751) are being conducted to evaluate the efficacy of these CAR-T cells in MM patients.
In this study, we generated a second-generation CD38-CAR-T cells and assessed their antitumor effect in vitro and in vivo. We aimed to provide an immunotherapy for clinical practice and improve the treatment efficiency of myeloma.

| Immunohistochemistry (IHC) staining
Tissues were fixed in formalin, decalcified, and paraffinembedded. Following antigen retrieval, sections were blocked with 0.3% H 2 O 2 in methanol. Then they were boiled in citrate buffer and blocked with serum-free protein blocking solution. The sections were incubated with anti-CD38 (Servicebio, GB13014) antibody overnight at 4°C. Then they were counterstained with hematoxylin, dehydrated through a graded alcohol series, cleared in xylene, and covered with coverslips. The percentage of CD38-positive cells was scored as follows: 0 point represents negative expression (positive cells <5%), 1 point represents weakly positive expression (positive cells ranged from 5% to <25%), 2 points represent positive expression (positive cells ranged from 25% to <50%), 3 points represent strongly positive expression (positive cells ≥50%).

| CAR construct and generation of lentiviral particles
CAR sequence included a single chain variable fragment (scFv) format and a signaling sequence. The sequences of scFv domain of CD38 antibodies were synthetically produced by GENEWIZ (Suzhou). The scFv sequence was cloned into pEF-MCS-P2A-EGFP vector with a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ sequence.
The pRSV-Rev, pLP-VSVG, pCMV-Gag-Pol vectors, and CAR construct vectors were transfected using PEI transfection reagent (Sigma) into 293 T cells, cultured in DMEM medium supplemented with 10% FBS and antibiotics. Seventy-two hours after transfection, the supernatants containing lentiviral particles were collected and concentrated using an ultrafiltration device (Merck Millipore).

| Generation of CAR-T cells
Informed consent was obtained before the study started, and the study was approved by the Institutional Research Ethics Committee of the First Affiliated Hospital of Zhengzhou University (Lot No. 2022-KY-1062). T cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors using magnetic activated cell sorting (MACS) method with CD3 Dynabeads (Miltenyi Biotec). Then they were cultured and expanded in X VIVO-15 medium (LONZA) supplemented with 200 IU/ mL IL-2 for 3-5 days. T cells were transduced by incubation of CAR lentivirus (CD38-CAR-T) or vector lentivirus (Control T). Forty-eight hours later, transfection rate of T cells was detected by FACS with recombinant human CD38 protein (FITC-labeled, ACROBiosystems).

| Cell-mediated cytotoxicity assay
CD38-CAR-T or control T cells (effector) were cocultured with MM cell lines (target cells) at E:T (effector to target) ratio of 2:1, 1:1, 1:2, and 1:4 for 6 h. Then the cytotoxicity of CAR-T cells was determined by measuring lactate dehydrogenase (LDH) in the supernatant. LDH was detected by CytoTox 96® Non-Radioactive Cytotoxicity Assay kit (Promega).
To evaluate the cytotoxicity of CAR-T cells at lower E:T ratios, T cells were activated by CD3/28 dynabeads (Gibco) for 3 days and then transduced with CD38-CAR lentivirus. MM cell lines were labeled by CFSE and then cocultured with CD38-CAR-T or control T cells at a serial E:T ratios of 1:10, 1:20, 1:50, and 1:100 for 24 h. Then cells were stained with Annexin V and PI (Keygen Biotech) and analyzed by a FACS Calibur flow cytometer. Cytotoxicity of CD38-CAR-T cells against autologous PBMCs and primary tumor cells isolated from bone marrow in MM patients was also evaluated. PBMCs or primary tumor cells were co-incubated with CAR-T and control T cells at a E:T ratio of 1:1 or 1:10 for 24 h and analyzed by a FACS Calibur flow cytometer.

| Detection of T-cell activation and proliferation
To detect T-cell activation biomarkers, CD38-CAR-T cells or control T cells were incubated with MM cell lines (treatment group) at a ratio of 1:10 or medium (blank group) for 24 h. Then biomarkers in different subsets of T cells were detected by a FACS Calibur flow cytometer with anti-CD4, CD8, CD25, CD69, and HLA-DR fluorescein-conjugated antibodies (BD Biosciences).
To evaluate the proliferation of T cells, CD38-CAR-T and control T cells were labeled by CFSE and then incubated with MM cell lines (treatment group) at a ratio of 1:20 or medium (blank group) for 72 h. Then the FITC fluorescence intensity of T cells was detected and analyzed by a FACS Calibur flow cytometer (BD Biosciences).

| Cytokine measurements
Cytokine bead array (CBA) was used to detect the cytokines produced by CAR-T or control T cells. 2 × 10 4 CAR-T or control T cells were cocultured with 2 × 10 5 target MM cells (treatment group) or medium (blank group) in 200 μL volume for 24 h. Then 50 μL cell supernatant was incubated with specific capture beads (IL-2, IL-3, IL-8, IL-9, GM-CSF, granzyme B, TNF-α, and IFN-γ) and PE-labeled detection antibodies for 2 h. After washing, beads were analyzed by a standardized flow cytometry assay (BD Biosciences).

| In vivo experiment
NSG mice (3-4 weeks old, female, 15-20 g) used in this study were obtained from Shanghai Model Organisms Center. A total of 5 × 10 6 RPMI8226-Luciferase cells in 200 μL medium were mixed with isopycnic matrigel (Corning Incorporated) and implanted subcutaneously into the abdominal wall of mice. Ten days after tumor implantation, treatment groups of mice (n = 6 per group) received tail intravenous injection of CAR-T or control T cells at Days 12 and 17 with a total of 5 × 10 6 T cells per mice (about 2 × 10 6 transduced cells). Mice in blank group were untreated. Animal experiments were conducted in accordance with the Declaration of Helsinki and approved by the Institutional Research Ethics Committee of the First Affiliated Hospital of Zhengzhou University (Lot No. 2022-KY-1062). Mice were given D-Luciferin (150 mg/kg, i.p.) and anesthetized with isoflurane. After 5 min, luminescence was detected using the in vivo imaging system (IVIS) and the intensity was quantitated and normalized by the Living Image software (PerkinElmer).

| Statistical analysis
Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, Inc.). Results were reported as the mean ± standard deviation or median and interquartile range for repeated measurements. The t-test and Mann-Whitney test were applied to assess differences between normally distributed samples and non-normally distributed samples, respectively. p < 0.05 was considered statistically significant.

| Expression of CD38 in different tissues and MM cell lines
The expression of CD38 was detected in 178 tissues. Spleen, tonsil, fallopian tube, and breast tissue showed strongly positive expression of CD38. Prostate, lung, bladder, kidney, and rectum tissues showed varying degrees of CD38 expression (Table 1, Figure 1A).
MM cell lines, U266, MM1.s, and RPMI8226, were used as target cells in this study. FACS detection showed heterogeneous expression of CD38 on the three MM cell lines ( Figure 1B). The percentage of CD38 + subgroup was lowest in U266 cells (33.6%), while in MM1.s and 8226 cells it was over 90% (91.2% and 99.3%, respectively).

| Generation of CD38-CAR-T cells
The anti-CD38 scFv sequence is based on a CD38 antibody with binding affinity comparable to daratumumab. 14 It was cloned into pEF-MCS-P2A-EGFP vector ( Figure 1C). T cells were isolated and transduced with CD38-CAR lentivirus. The transduction efficiency was detected using FITC-labeled human CD38 protein ( Figure 1D).
CD38 is a T-cell activation marker and involved in chemotaxis, development of T cells, and humoral immune responses. 15,16 However, transduced CAR-T cells lost the expression of CD38 molecule ( Figure 1E). Therefore, we performed a variety of experiments to assess the function of CD38-CAR-T cells.

| Target-dependent activation and proliferation of CAR-T cells
Stimulation of T cells may lead to the alteration of cellular activation surface markers, including early (CD69) and late (CD25 and HLA-DR) markers. 17 To investigate the target-dependent activation of T cells, we assessed the expression of CD69, CD25, and HLA-DR on both CD4 + and CD8 + subsets of CD38-CAR-T and control T cells. Data showed that except CD69 expression in CD8 + subgroup of CAR-T and control T cells cocultured with U266 or 8266, expression of CD69, CD25, and HLA-DR in both CD4 + and CD8 + subgroups of CAR-T cells increased significantly compared with these markers in control T cells (Figure 2A,B).
There are differences in the CD4/CD8 ratio of CAR-T cells and control T cells stimulated by MM cells. After 24 h cocultivation with MM cell lines, CD4/CD8 ratio decreased in CAR-T cells and increased in control T cells ( Figure 2C).
To evaluate T-cell proliferation stimulated by MM cells, CFSE-labeled CAR-T and control T cells were cocultured with MM cell lines for 72 h. Then the CFSE fluorescence intensity of T cells was detected by FACS. T-cell proliferation was determined by the decreasing CFSE fluorescence intensity. Results indicated that both CD4 + and CD8 + subgroups of CAR-T cells proliferated significantly more than control T cells in response to MM cells ( Figure 2D,E).

MM cells
LDH released by lysed tumor cells was detected to evaluate the short-time cytotoxicity of CAR-T cells. After 6 h cocultivation, CAR-T cells lysed more than 70% MM1.s cells (average 72.95%) and 8226 cells (average 76.65%) at a E:T ratio of 2:1, which was significantly higher than control T cells (average 18.85% in MM1.s and 15.52% in 8226 cells). For U266 cells, CD38-CAR-T cells killed about 30% (average 30.87%) tumor cells, which was only 8.87% in control T cells ( Figure 3A).
For the determination of the antitumor effect at lower E:T ratios, T cells were activated by CD3/28 dynabeads before transduction. The activation markers T A B L E 1 CD38 expression in different tissues.  p < 0.05 was considered statistically significant. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

F I G U R E 3 Cytotoxicity and cytokine-releasing CAR-T cells. (A) CD38-CAR-T or control T cells were coincubated with MM cell lines for 6 h. The cytotoxic effect of CD38-CAR-T and control T cells was evaluated by detecting LDH released by tumor cells. (B) CD38-CAR-T or control T cells were co-incubated
with CFSE-labeled MM cell lines for 24 h. Cells were stained with Annexin V and PI to evaluate the cytotoxicity of T cells. (C) Expression of CD69, CD25, and HLA-DR in nonactivated T cells and CD3/28 dynabeads activated T cells.

(D) Differences between the cytotoxicity of CD38-CAR-T cells generated from nonactivated T cells and CD3/28 dynabeads activated T cells. (E) Cytokines released by CD38-CAR-T cells and control T cells were detected using the CBA kit.
Δpg/mL refers to the difference between cytokine concentration in the treatment group and the blank group. p < 0.05 was considered statistically significant. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.
were monitored. Data showed that T cells treated with CD3/28 dynabeads showed higher expression of CD69, CD25, and HLA-DR ( Figure 3C). CAR-T cells generated from activated T cells were cocultured with MM cell lines. Surprisingly, even at E:  Figure 3D).

| Cytokine release of CAR-T cells
The release of inflammatory cytokines was detected using a CBA kit. After 24 h cocultivation of CAR-T or control T cells and target MM cell lines, the cell supernatant was collected and incubated with specific capture beads and PE-labeled antibodies. Then the concentrations of IL-2, IL-3, IL-8, IL-9, GM-CSF, granzyme B, TNF-α, and IFN-γ were measured. Data showed that except the production of granzyme B by CAR-T cells cocultured with U266 similar to control T cells, CD38-CAR-T cells stimulated by MM cell lines generated significantly increased level of IL-2, IL-3, IL-8, IL-9, GM-CSF, granzyme B, IFN-γ, and TNF-α than control T cells ( Figure 3F).

| Effect of CD38-CAR-T cells on the treatment of MM in vivo
In vitro studies showed that CAR-T cells exhibited valid antitumor effect than control T cells. Hence, we further investigated the efficacy of CAR-T cells against MM cells in subcutaneously tumor xenograft NSG mice models. Mice were treated with CAR-T or control T cells. The tumor burden was monitored by luminescence intensity using the IVIS ( Figure 4A). Tumor growth showed aggressive progression of tumors in the untreated group. Compared to mice in the blank group, although treatment of control T cells showed a moderate therapeutic effect, the tumor progression was still rapid. While treatment with CAR-T cells had a significant antitumor effect. Tumor burdens showed significant differences between CAR-T-cell treatment group and blank group or control T-cell treatment group ( Figure 4B,C).

| Effect of CD38-CAR-T cells on autologous PBMCs and primary MM cells
Besides MM cells, CD38 also exists on the surface of normal hematopoietic and non-hematopoietic tissues. This may result in tissue damage that is not intended for treatment. Then we investigated the effect of CAR-T cells on autologous blood cells. CAR-T or control T cells were coincubated with both autologous PBMCs for 24 h. Then T, B, and natural killer cells were labeled and detected by FACS. Results showed that CAR-T cells showed limited cytotoxicity to T, B, and natural killer cells. (Figure 4D,E).
T cells and CD38 + primary tumor cells were isolated from the bone marrow of MM patients ( Table 2). T cells were transduced and cocultured with primary MM cells at a E:T ratio of 1:10 for 24 h. CAR-T cells showed an enhanced cytotoxicity against tumor cells ( Figure 4F).

| DISCUSSION
CD38 molecule is highly expressed on MM cells, but is low expressed on normal tissues. MM progenitor cells were suggested to be enriched in CD38 + CD138 − subgroups. 18 CD38 + MM cells in the minimal residual disease of bone marrow after autologous transplantation treatment may lead to reduced PFS in MM patients. 19,20 Hence, immunotherapy in MM targeting CD38 has drawn much attention. Daratumumab and isatuximab have shown efficacy in the treatment of MM. Daratumumab eliminates CD38 + MM cells by inducing Fc receptor-mediated apoptosis, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). 28 In addition, daratumumab can improve the antitumor immune response by eliminating CD38 + immunosuppressive cells. 29 While isatuximab acts on tumor cells through lysosomedependent pro-apoptotic activity and antibody-dependent cellular phagocytosis (ADCP) besides ADCC and CDC. 28 Isatuximab enhances the antitumor effect of T cells and NK cells by decreasing the number of regulatory T cells and production of IL-10. 29 In the past decade, a pioneering treatment, CAR-Tcell therapies have shown prominent antitumor effect in B-cell malignancies. 21,22 Our previous study showed that CAR-T cells targeting CD7 may be a promising strategy for T-lymphoblastic leukemia/lymphoma. 23 CAR-T-cell therapies in the administration of MM are in the early stage. BCMA is an ideal target due to its selective expression on mature B lymphocytes and plasma cells. 24 However, MM cells often lost expression of BCMA upon recurrence of disease after first infusion of BCMA-CAR-T cells. 25 CD138 is a specific biomarker for plasma cells. Reported data | 10813 demonstrated the safety and modest therapeutic response of CD138-CAR-T cells. 25,26 In this study, second-generation CD38-CAR-T cells were developed and effects of CAR-T cells on MM cells were evaluated. Cocultured with MM cell lines, CD38-CAR-T cells showed increased expression of T-cell activation markers in both CD4 + and CD8 + subgroups. Meanwhile, CD38 + MM cell lines significantly promoted the proliferation of CAR-T cells compared with control T cells. However, the stimulation of CAR-T cells and control T cells by MM cells seemed different. Cocultured with MM cell lines, CD8 + proportion of CAR-T cells increased markedly. While in control T cells, CD4 + proportion is elevated. This may be due to the use of 4-1BB as a costimulatory signal in the CAR construct. Studies have shown the principal role of 4-1BB in the activation of CD8 + T cells rather than CD4 + T cells. 27 Compared with control T cells, CAR-T cells could eliminate MM cells more rapidly and efficiently. Notably, CD38-CAR-T cells killed about 40% U266 cells, which was consent with its low expression of CD38 (33.6% positive rate). With more than 90% positive rate of CD38 expression, over 85% MM1.s and 8226 cells were lysed by CAR-T cells. These indicated that CAR-T cells can recognize and eliminate tumor cells in an HLA nonindependent manner. Similar to the results in MM cell lines, CAR-T cells also showed a significant cytotoxicity against CD38 + primary tumor cells isolated from MM patients. In vitro cytotoxicity of CAR-T cells was verified by the results of in vivo mouse models. Tumor progressed rapidly in mice without CAR-T-cell treatment. However, CD38-CAR-T cell therapy significantly inhibited tumor growth.
Activated by target antigens, CAR-T cells can release pro-inflammatory cytokines and lysis tumor cells. 28 In our study, CAR-T cells produced higher level of cytokines such as IL-2, IL-3, IL-8, IL-9, GM-CSF, granzyme B, IFN-γ, and TNF-α under the stimulation of MM cells. All these results indicated the robust anti-tumor activity of CD38-CAR-T cells against MM cells, even though CD38 was not expressed on transduced CAR-T cells.
Except plasma cells, CD38 was also expressed at lower levels on a fraction of normal cells and tissues. 14 This may arouse concern about organic damage risk of CD38-targeting immunotherapy. According to data of the Human Protein Profile Database (https://www.prote inatlas. org/), CD38 was only found strongly positive expression in prostate, lymph node, and tonsil. This is consistent with our IHC results. We further assessed the effect of CAR-T cells on autologous PBMCs. CAR-T cells showed no obvious cytotoxicity against T, B, and natural killer cells. Studies have also indicated the safety of immunotherapy targeting CD38 molecules. Drent et al. demonstrated that proliferation of blood progenitor cells was not inhibited by CD38-CAR-T cells. 14 An et al. reported a slight cytotoxicity of CD38-CAR-T cells against CD38 + blood cells. 29 Daratumumab treatment also showed little or no toxicity for prolonged administration periods. 8 However, cytotoxicity of CAR-T cells against CD38 + cells in non-hematopoietic tissues could not be evaluated. Optimization of the tumor cell affinity of CAR-T cells may improve the specificity for CD38 recognizing and reduce side effects.
Studies of CAR-T cells targeting CD38 have demonstrated their anti-tumor efficacy. The effective clearance  . (B, C) Tumor growth was monitored by IVIS living imaging. The mouse tumor burden was indicated by bioluminescence radiance. Mice treatment with CD38-CAR-T cells induced a significant antitumor effect compared to mice treated with control T cells or the blank group. While treatment of control T cells showed a certain therapeutic effect, tumor burden still progressed rapidly in the mice. (D, E) CD38-CAR-T cells showed limited cytotoxicity against CD3 + , CD19 + , and CD56 + subgroups of PBMCs. (F) CD38-CAR-T cells showed significant cytotoxicity against primary MM cells derived from patients. p < 0.05 was considered statistically significant. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001. of tumor cells by CD38-CAR-T cells requires a longer time or higher E:T ratios. [18][19][20] In our study, CAR-T cells generated from CD3/28 dynabeads activated T cells showed a significant cytotoxicity activity on MM1.s and RPMI8226 cells in a short period of 24 h and low E:T ratios. In this study, a CD38-CAR-T cell was constructed and exhibited robust cytolysis against MM cell lines. We first demonstrated the dynamic changes of activation markers and more cytokines generated by CAR-T cells with the stimulation of MM cells. It may provide a promising and effective solution for the treatment of MM and even CD38 + malignancies. Further clinical trials are required to evaluate its efficacy and safety.