Novel two-chain structure utilizing KIRS2/DAP12 domain improves the safety and efficacy of CAR-T cells in adults with r/r B-ALL

Engineered T cells that express chimeric antigen receptors (CARs) have been a promising therapy for hematologic malignancies. The optimization of CAR structure using different signaling domains can alter a wide range of CAR-T cell properties, including anti-tumor activity, long-term persistence, and safety. In this study, we developed a novel CAR structure based on KIRS2/Dap12 for B cell acute lymphoblastic leukemia (B-ALL) antigen CD19 and compared the anti-tumor efficacy and safety of this construct in transduced T cells with standard second-generation CAR-T cells targeting CD19 for B-ALL in vitro and in vivo and in adult relapsed/refractory (r/r) B-ALL patients. We discovered that KIRS2/Dap12 receptor infused with 4-1BB co-stimulation domain could enhance anti-tumor efficacy by remarkably increasing the production of pro-inflammatory interleukin-2 (IL-2), especially when co-cultured with antigen-positive tumor cells. In addition, CD19-KIRS2/Dap12-BB CAR-T cells showed the inspiring outcome that complete responses were seen in 4 of 4 (100%) patients without neurotoxicity and a high rate of severe cytokine release syndrome (CRS) after CAR-T infusion in a phase I clinical trial. Given these encouraging findings, CD19-KIRS2/Dap12-BB CAR-T cells are safe and can lead to clinical responses in adult patients with r/r B-ALL, indicating that further assessment of this therapy is warranted. Engineered T cells that express chimeric antigen receptors (CARs) have been a promising therapy for hematologic malignancies. The optimization of CAR structure using different signaling domains can alter a wide range of CAR-T cell properties, including anti-tumor activity, long-term persistence, and safety. In this study, we developed a novel CAR structure based on KIRS2/Dap12 for B cell acute lymphoblastic leukemia (B-ALL) antigen CD19 and compared the anti-tumor efficacy and safety of this construct in transduced T cells with standard second-generation CAR-T cells targeting CD19 for B-ALL in vitro and in vivo and in adult relapsed/refractory (r/r) B-ALL patients. We discovered that KIRS2/Dap12 receptor infused with 4-1BB co-stimulation domain could enhance anti-tumor efficacy by remarkably increasing the production of pro-inflammatory interleukin-2 (IL-2), especially when co-cultured with antigen-positive tumor cells. In addition, CD19-KIRS2/Dap12-BB CAR-T cells showed the inspiring outcome that complete responses were seen in 4 of 4 (100%) patients without neurotoxicity and a high rate of severe cytokine release syndrome (CRS) after CAR-T infusion in a phase I clinical trial. Given these encouraging findings, CD19-KIRS2/Dap12-BB CAR-T cells are safe and can lead to clinical responses in adult patients with r/r B-ALL, indicating that further assessment of this therapy is warranted. IntroductionAdoptive T cell therapy (ACT) involves the manufacture of a patient's T cells followed by infusion of these engineered T cells into the patient with cancer.1Neelapu S.S. Locke F.L. Bartlett N.L. Lekakis L.J. Miklos D.B. Jacobson C.A. Braunschweig I. Oluwole O.O. Siddiqi T. Lin Y. et al.Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma.N. Engl. J. Med. 2017; 377: 2531-2544Google Scholar,2Leon E. Ranganathan R. Savoldo B. Adoptive T cell therapy: Boosting the immune system to fight cancer.Semin. Immunol. 2020; 49: 101437Google Scholar In recent years, chimeric antigen receptor (CAR) T cell immunotherapy, based on the infusion of engineered autologous T cells to recognize the tumor-associated antigens expressed on cancer cells, has changed the modality of treatment for hematological malignancies.3Abramson J.S. Palomba M.L. Gordon L.I. Lunning M.A. Wang M. Arnason J. Mehta A. Purev E. Maloney D.G. Andreadis C. et al.Lisocabtagene maraleucel for patients with relapsed or refractory large B-cell lymphomas (TRANSCEND NHL 001): a multicentre seamless design study.Lancet. 2020; 396: 839-852Google Scholar,4Schuster S.J. Svoboda J. Chong E.A. Nasta S.D. Mato A.R. Anak Ö. Brogdon J.L. Pruteanu-Malinici I. Bhoj V. Landsburg D. et al.Chimeric Antigen Receptor T Cells in Refractory B-Cell Lymphomas.N. Engl. J. Med. 2017; 377: 2545-2554Google Scholar Specifically, CAR-T cells targeting CD19 or B cell maturation antigen (BCMA) in treating B cell lymphoma, leukemia, and multiple myeloma have achieved unprecedented response rates.5Park J.H. Rivière I. Gonen M. Wang X. Sénéchal B. Curran K.J. Sauter C. Wang Y. Santomasso B. Mead E. et al.Long-Term Follow-up of CD19 CAR Therapy in Acute Lymphoblastic Leukemia.N. Engl. J. Med. 2018; 378: 449-459Google Scholar,6Raje N. Berdeja J. Lin Y. Siegel D. Jagannath S. Madduri D. Liedtke M. Rosenblatt J. Maus M.V. Turka A. et al.Anti-BCMA CAR T-Cell Therapy bb2121 in Relapsed or Refractory Multiple Myeloma.N. Engl. J. Med. 2019; 380: 1726-1737Google Scholar Synonymous with the outstanding clinical outcome of CAR-T cell therapy in hematological malignancies have been severe toxicities, including cytokine-release syndrome (CRS), neurotoxicity syndrome, CAR-T cell-related encephalopathy syndrome (CRES), and hemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS).7Anagnostou T. Riaz I.B. Hashmi S.K. Murad M.H. Kenderian S.S. Anti-CD19 chimeric antigen receptor T-cell therapy in acute lymphocytic leukaemia: a systematic review and meta-analysis.Lancet Haematol. 2020; 7: e816-e826Google Scholar Therefore, management of these toxicities has been a major concern for clinical implementation.A growing amount of evidence indicates that CAR toxicity may be linked to the synthetic nature of the receptor design.8Helsen C.W. Hammill J.A. Lau V.W.C. Mwawasi K.A. Afsahi A. Bezverbnaya K. Newhook L. Hayes D.L. Aarts C. Bojovic B. et al.The chimeric TAC receptor co-opts the T cell receptor yielding robust anti-tumor activity without toxicity.Nat. Commun. 2018; 9: 3049Google Scholar To improve the safety of CAR-transduced T cells, we previously designed a natural multi-chain immunoreceptor CAR based on the DNAX-activating protein of 12 kDa (Dap12) signaling domain for the first time, which triggers antigen-specific cytotoxicity, cytokine production, and proliferation that is comparable with CD3z-based CARs ex vivo/in vitro for hematological malignancies.9Chen B. Zhou M. Zhang H. Wang C. Hu X. Wang B. Wang E. TREM1/Dap12-based CAR-T cells show potent antitumor activity.Immunotherapy. 2019; 11: 1043-1055Google Scholar,10Wang E. Wang L.C. Tsai C.Y. Bhoj V. Gershenson Z. Moon E. Newick K. Sun J. Lo A. Baradet T. et al.Generation of Potent T-cell Immunotherapy for Cancer Using DAP12-Based, Multichain, Chimeric Immunoreceptors.Cancer Immunol. Res. 2015; 3: 815-826Google Scholar Dap12 is a transmembrane signaling adaptor protein containing a single immunoreceptor tyrosine-based activation motif (ITAM) that has low homology with ITAMs identified in the CD3z chain. The expression of Dap12 has been found in a variety of immune cells, such as natural killer (NK) cells, macrophages, and some T cells, indicating that Dap12 may have a general role in the immune response.11Parham P. Moffett A. Variable NK cell receptors and their MHC class I ligands in immunity, reproduction and human evolution.Nat. Rev. Immunol. 2013; 13: 133-144Google Scholar,12Angata T. Siglecs that Associate with DAP12.Adv. Exp. Med. Biol. 2020; 1204: 215-230Google Scholar Dap12 was originally found to activate NK cells when its ligand was ligated with an associated receptor, which then induced the SRC-family kinase activation and phosphorylation of tyrosine residues in the ITAM.13Boudreau J.E. Hsu K.C. Natural Killer Cell Education and the Response to Infection and Cancer Therapy: Stay Tuned.Trends Immunol. 2018; 39: 222-239Google Scholar,14Lanier L.L. DAP10- and DAP12-associated receptors in innate immunity.Immunol. Rev. 2009; 227: 150-160Google Scholar Currently, more than 20 Dap12-related receptors have been uncovered, including TREM1, TREM2, and KIRS et al.In contrast to the most common CARs utilizing a simplified format to recapitulate the signals necessary for T cell effector function and proliferation, we previously found that T cells expressing a KIRS2/Dap12 CAR showed anti-tumor activity even without the additional domains from costimulatory receptors.10Wang E. Wang L.C. Tsai C.Y. Bhoj V. Gershenson Z. Moon E. Newick K. Sun J. Lo A. Baradet T. et al.Generation of Potent T-cell Immunotherapy for Cancer Using DAP12-Based, Multichain, Chimeric Immunoreceptors.Cancer Immunol. Res. 2015; 3: 815-826Google Scholar Typically, the second- and third-generation CARs included one or two costimulatory domains derived from CD28 or 4-1BB that associate with the CD3ζ endo-domain.15van der Stegen S.J. Hamieh M. Sadelain M. The pharmacology of second-generation chimeric antigen receptors.Nat. Rev. Drug Discov. 2015; 14: 499-509Google Scholar,16Lim W.A. June C.H. The Principles of Engineering Immune Cells to Treat Cancer.Cell. 2017; 168: 724-740Google Scholar CD28 signaling is beneficial for T cell cytotoxicity, and 4-1BB/CD3ζ CARs could phosphorylate endogenous CD28 and activated the CD28 signaling pathway.17Klein Geltink R.I. O'Sullivan D. Corrado M. Bremser A. Buck M.D. Buescher J.M. Firat E. Zhu X. Niedermann G. Caputa G. et al.Mitochondrial Priming by CD28.Cell. 2017; 171: 385-397.e11Google Scholar,18Drent E. Poels R. Ruiter R. van de Donk N.W.C.J. Zweegman S. Yuan H. de Bruijn J. Sadelain M. Lokhorst H.M. Groen R.W.J. et al.Combined CD28 and 4-1BB Costimulation Potentiates Affinity-tuned Chimeric Antigen Receptor-engineered T Cells.Clin. Cancer Res. 2019; 25: 4014-4025Google Scholar However, encoding a fully functional CD28 signaling domain on a CAR polypeptide chain may yield excessive stimulation that increases the incidence of CRS, promotes T cell exhaustion, and reduces persistence. Considering the critical importance of co-stimulation for T cell activation and acquisition of effector function, we generated a panel of representative receptors through combining the KIRS2/Dap12 with costimulatory molecule 41-BB in this study. To determine whether they are able to induce a more controlled T cell response and improved T cell activity, we evaluated functional characterization of diverse KIRS2/Dap12 CAR structure and found that KIRS2/Dap12-BB CAR-conferred T cells enhanced antitumor cytotoxicity and increased cytokine production. Furthermore, we present experimental evidence for the efficacy and compatibility of the KIRS2/Dap12-BB platform with different targets in preclinical models of solid and hematological tumors. Finally, a phase I clinical trial using autologous T cells expressing CD19-specific CAR (CD19-KIRS2/Dap12-BB) in adult relapsed/refractory (r/r) B-ALL patients with lymphodepleting chemotherapy was conducted to evaluate the anti-tumor activity and safety of CD19-KIRS2/Dap12-BB CAR-T cells compared with second-generation CAR-T cells targeting CD19.Results4-1BB co-stimulation domain confers KIRS2/Dap12 CAR-T cells comparable cytotoxic activity and robust cytokine productionCo-stimulation domains in CARs can alter many effector functions of T cells. To directly compare the effector functions induced by 4-1BB co-stimulation, we introduce the signal transduction domains of 4-1BB in KIRS2/Dap12 receptor consisting of the same extracellular domain of anti-CD19 scFv (FMC63) with other CARs (Figure 1A). By using lentiviral vectors and transduction at a multiplicity of infection (2.5), the different CARs could be expressed ranging from 50% to 55% in primary human T cells (Figure 1B). Simultaneously, the CD4/CD8 ratios were also similar (∼20% CD4+ and ∼80% CD8+) (data not shown) among the 3 CAR-expressed T cells. Over 128-fold (population double > 9) expansion of CAR+ T cells could be achieved over the course of transduction and growth in 11 days. To evaluate the anti-tumor cytotoxicity of the 3 types of CAR-T cells, MCF-7 cells that express human CD19 antigen (MCF-7-CD19) were co-cultured with CAR-T cells in vitro at different effector:target (E:T) ratios ranging from 0:1 to 10:1 for 20 h. The results showed that these engineered T cells exhibit comparable lytic activity toward MCF-7-CD19 cells (Figure 1C). Furthermore, we observed that T cells expressing 4-1BB domain-infused KIRS2/Dap12 CAR produced greater quantities of interleukin-2 (IL-2) and lower interferon-γ (IFN-γ), and interleukin-6 (IL-6) when compared with cells expressing only KIRS2/Dap12 receptor or 4-1BB/CD3ζ (Figure 1D; Figure S1A). Meanwhile, the results of quantitative PCR (qPCR) analysis also showed that KIRS2/Dap12-BB CAR-T cells express higher IL-2, IFN-γ, granzyme B (GZMB), and IL12A than 4-1BB/CD3ζ CAR-T cells (Figure S1B).To further assess the primary human T cell differentiation over time, we used a multiparametric flow cytometric approach to distinguish naive-like (Tn, CD45RO−CCR7+), central memory (Tcm, CD45RO+CCR7+), effector memory (Tem, CD45RO+CCR7−), and effector (Teff, CD45RO−CCR7−) T cell subsets. During the expansion phase, the proportions of the Tem subset showed a robust increase from D9 to D11, and KIRS2/Dap12-BB CAR-T cells had higher ratio of Tcm subset than 4-1BB/CD3ζ CAR-T cells (Figure S1C). Moreover, the results of flow cytometric analysis showed that KIRS2/Dap12-BB CAR-T cells had lower PD-1 expression level than 4-1BB/CD3ζ CAR-T cells (Figure S1D).4-1BB infused with Dap12 confers CAR-T cells more IL-2 productionTo explore whether the infusion of 4-1BB with different chains of KIRS2/Dap12 influences the activity and cytokine production of T cells, we generated another CAR in which 4-1BB is infused with the KIRS2 chain and not the Dap12 chain (Figure 2A). The percentage of CAR-positive T cells expressing the two CARS on days 7 and 9 was comparable, with a range from 71% to 78% (Figure 2B). Further cytotoxicity assays showed that 4-1BB infused with different chains exhibits similar lytic activity toward target cells (Figure 2C). Interestingly, 4-1BB infused with the Dap12 chain confers T cells more robust IL-2 production than T cells expressing CAR in which 4-1BB is infused with the KIRS2 chain; however, the production of IFN-γ showed no difference between the two types of CAR-T cells (Figures 2D and 2E).Figure 2The effect of 4-1BB infusion with different chains on CAR-T cellsShow full caption(A) Schematic diagram of a CD19-specific KIRS2/Dap12 CAR infused with 4-1BB. (B) Flow cytometry analysis of the ratio of CAR+ T cells that express anti-mouse FMC63 scFv. (C) Cytotoxicity of CAR-T cells was evaluated by the RTCA system. (D and E) IFN-γ and IL-2 released in the culture supernatant by NTD and CAR-T cells were measured by ELISA (n = 3). ∗∗p < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT)KIRS2/Dap12-BB CAR-T cells show efficacy in hematological malignancies and solid tumor modelsWe next evaluated the anti-tumor cytotoxicity of CD19-KIRS2/Dap12-BB CAR-T cells compared with CAR-T cells engineered with a CD19-targeted 41BB-based second-generation CAR (termed CD19-BBζ CAR) in Nalm6 cell-derived xenograft tumors (Figure 3A). As shown in Figures 3B and 3C, both CD19-KIRS2/Dap12-BB and CD19 BBζ CAR-T cells displayed comparable anti-tumor activity in vivo. In striking contrast, untransduced T cells (NTD) showed no anti-tumor activity and tumor progression. Interestingly, analysis of the CD19 CAR-T cells in mouse tail vein blood indicated that KIRS2/Dap12-BB CAR-T cells exhibited stronger proliferative potential and longer persistence than CD19 BBζ CAR-T cells (Figure S1E). These data indicate that CD19-KIRS2/Dap12-BB-modified T cells exhibit control of leukemia as well as CD19-BBz-CAR-T cells, which have been shown to have potent lymphoma activity in humans.Figure 3The antitumor activity of CD19- and mesothelin-targeted KIRS2/Dap12 CAR-T cells in vivoShow full caption(A) Schema of the lymphoma xenograft model infused with CAR-T on day 15 after tumor inoculation. (B and C) Nalm6 tumors were subcutaneously (s.c.) established in the right flank of NCG mice and randomly grouped (n = 5 per group). The curve shows the change in tumor volume. (D) Schema of the pancreatic cancer xenograft model infused with CAR-T on day 18 after tumor inoculation. (E and F) AsPC-1 tumors were s.c. established in the right flank of NCG mice and randomly grouped (n = 5 per group). The curve showed the change in tumor volume following treatment with intravenous injections of 2 × 105 or 8 × 105 CAR-T cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further determine if the potent anti-tumor activity of T cells bearing a KIRS2/Dap12-CAR is dependent on the CD19 specificity, we generated an additional CAR targeting mesothelin (SS1 KIRS2/Dap12-BB). In an AsPC-1 cell-derived pancreatic adenocarcinoma xenograft model (Figure 3D), T cells expressing SS1-KIRS2/Dap12-BB achieved superior control of tumor growth when compared with untransduced T cells. In this experiment, we established two groups of increasing doses (2 × 105 and 8 × 105 SS1 KIRS2/Dap12-BB CAR-T cells) and found that although pancreatic adenocarcinoma relapsed by day 98 in the low-dose group, the tumor did not relapse for more than 100 days in the high-dose group (Figures 3E and 3F). These findings demonstrate that the activity of KIRS2/Dap12-BB CAR is not antigen dependent, and mesothelin-targeted SS1-KIRS2/Dap12-BB CAR-T cells may have high translation potential anti-tumor activity against mesothelin-positive solid tumors.Patient characteristicsGiven the encouraging data from the in vitro and in vivo experiments, we conducted a phase I, single-center, open-label clinical trial (ChiCTR1800016584) to evaluate the feasibility, safety, and clinical and biologic activity of manufacturing and administering CD19-targeted KIRS2/Dap12-BB and BBζ CAR-T cells to adult patients with r/r B-ALL. 11 r/r CD19-positive B-ALL patients, aged from 20 to 55 years, who had not previously received CAR-T therapy, signed a written informed consent between December 2018 and June 2020 and were enrolled into the present trial in the Nanjing Drum Tower Hospital. 3 patients failed to receive the CAR-T cell infusion due to disease progression. The remaining 8 patients (4 male and 4 female) received 3 to 20 intensive therapies, completed screening, and were infused with lentiviral-transduced CAR-T cells. Among them, 4 subjects were randomly enrolled in the BBζ CAR-T group, while 4 patients were randomly enrolled in the Dap12-BB group (Table 1).Table 1Patient characteristicsNo.CAR-T typeAge (years)GenderWeight (kg)Enrolled dateReceived treatmentPrevious treatmentDose (cells/kg)CAR-T positive (%)Relapse timeDeath timeCri (days)CR (days)CRSCRESCLHBBζCAR21female502018.12.2420VDCLP+MTX&L-Asp+CAM+MA+VDLP+COATD+CTX+PBSCT+MTX+6-MP+hormone+MAE+HyperCAVD&P+MTX&Ara-C+MAED+MTX6.0+VDCP+EA+FAMD+MOAP1.2 × 10644.1M5M8306040QMBBζCAR23female572019.05.213VDP+VDCP+HAAG1.2 × 10623––164520YZJBBζCAR27male422019.05.273VDP+VCP+dasatinib tablets1.2 × 10626.4M4M7306010XJJBBζCAR40male802019.04.016VDCP(3)+Hyper-CAVD(2)+MTX&HD-Ara-C1.5 × 1066.3M3–306030LHMDap-12 CAR24female422019.06.123VDP+VPAP+VDPAP1.2 × 10623.7M14–133010LMXDap-12 CAR48male622019.07.247CRRT+dasatinib tablets(2Y)+IA(2)+VP16A+PB&BMSCT+MA+MTX&Ara-C1.2 × 10622M9–143000SHWDap-12 CAR20male932020.05.268VDP(2)+VDCP+VDLP(2)+Ara-C(3)2 × 10648––142930CZLDap-12 CAR55female552020.06.166IVP+CAM+HD-MTX(2)+IVLP+Hyper-CVAD2 × 10643.3––73010Received treatment means the total number of different therapy or treatment that the patient has received.VDP, Vincristine + Daunorubicin + Prednison; VPAP, Vincristine + Prednison + Pegaspargase; VDPAP, Vincristine + Daunorubicin + Prednison + Pegaspargase; CRRT, continuous renal replacement therapy; VP-16, Etoposide Ara-C, vincristine + Daunorubicin + Prednison; PB&BMSCT, Peripheral blood & bone marrow stem cells transplantation; MA, Mitoxantrone + Cytarabine; MTX, Methotrexate; VDCP, Vincristine + Daunorubicin + Cyclophosphamide + Prednison; VDCLP, Cyclophosphamide + Vincristine + Daunorubicin + Lasparaginase + Prednisone; L-Asp, Lasparaginase; CAM, Lasparaginase + Cytarabine + 6-mercaptopurine; VDLP, Vincristine + Daunorubicin + Lasparaginase + Prednisone; COATD, Cyclophosphamide + Vindesine + Cytarabine + Tiniposide + dexamethasone; CTX, Cyclophosphamide; PBSCT, Peripheral blood stem cells transplantation; 6-MP, 6-mercaptopurine; MAE, Mitoxantrone + Cytarabine + Etoposide; HyperCAVD, Cyclophosphamide + Vincristine + Doxorubicin + dexamethasone; P, Prednisone; EA, Etoposide + Cytarabine; FAMD, 5-fluorouracil + adriamycin + mitomycin + cisplatin; MOAP, Mitoxantrone + Vincristine + Doxorubicin + Prednisone; HAAG, Recombinant human granulocyte colony stimulating factor + Aclamycin + cytarabine + Harringtonine. Open table in a new tab Safety of CD19-targeted CAR-T cell therapy in adult r/r B-ALL patientsAll enrolled patients underwent steady-state leukapheresis to collect T cells for CD19 CAR-T manufacturing. CAR-T cells for all the patients were successfully manufactured, achieving the dose of 1–2 × 106 CAR-T cells/kg. Patients received fludarabine and cyclophosphamide-based lymphodepletion chemotherapy consisting of fludarabine (30 mg/m2/day, days −5 to −2) and cyclophosphamide (500 mg/m2/day, days −5 to −4) before receiving T cell infusion. CD19 CAR-T cells were administered in an outpatient research unit over 2 days as split-dose intravenous infusions (30% [day 0] and 70% [day 1]) (Figures 4A and 4B ). The most common adverse event related to CD19 CAR-T cell treatment is the cytokine release syndrome (CRS), and the details are summarized in Table 2. In the BBζ CAR-T group, grade ≥ 3 CRS occurred in 2 patients, while 2 patients experienced grade 2 and 1 CRS. For the patients in the Dap12-BB group, only one patient experienced grade 3 CRS, and 2 patients experienced grade 1 CRS. The main symptoms of CRS are fever and hypotension, and tocilizumab and glucocorticoid were administered to control the CRS once the patient's temperature was over 38°C (Figure 4C; Table 2). The tocilizumab was administered with 5 mg/dose every 8 h until the patient's temperature was below 38°C. One patient in each group experienced hypotension, and they were treated with norepinephrine (0.2 μg/kg/min) to promote blood pressure to return to over 80 mm Hg. Moreover, lower IL-6 and IL-10 levels were observed in the patients' peripheral blood from the Dap12-BB group when compared with that from the BBζ CAR-T group (Figure 4D).Figure 4Phase I clinical trial design and the safety of CAR-T cell infusionShow full caption(A) Schema of the study design and patient enrollment. (B) Treatment schema. CTX, cyclophosphamide. ∗∗After the first 28 days, follow-up is every 4 weeks up to 6 months, then every 3 months up to 2 years. ∗∗∗Pre-tx, pretreatment, 2 to 5 days before CAR-T cell infusion. (C) The line graph shows the patient's body temperature change after receiving CAR-T cell infusion. (D) IL-6 and IL-10 levels in the subjects' peripheral blood were detected by ELISA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 2Summary of adverse events related to CD19-targeted CAR-T cellsAll subjects (n = 8)BBζ CAR-TKIRS2/Dap12-BB CAR-TGrade 1Grade 2Grade 3Grade 4Grade 1Grade 2Grade 3Grade 4CRS11112010Fatigue00000000Nausea10001000Vomiting60001000Confusion00000000Diarrhea00000000Dysgeusia00000000Fever1810504200Abdominal pain00001000Anorexia10001000Anxiety00000000Chills50001000Constipation00000000Dizziness10001000Myalgia00000000Paroxysmal atrial tachycardia10000000Pleural effusion10001000Sore throat00000000Abdominal distension32000000Acute kidney injury00000000Bacteremia00000000Hepatic failure00000000Hepatitis00000000Dyspnea00000000Hematologic eventsAnemia00000000DIC00000000Lymphocyte count decreased2Hypotension00100100Nonhematologic eventsAlkaline phosphatase increased00000000ALT increased00000000AST increased10000000Blood bilirubin increased00000000DIC, disseminated intravascular coagulation. Open table in a new tab Bioactivity and clinical response of CD19 CAR-T cellsThe anti-tumor efficacy and clinical response of CAR-T cells are dependent on the proliferative peak and persistence of CAR-T cells in vivo. The proliferation and survival of peripheral CD19 CAR-T cells were monitored by fluorescence-activated cell sorting (FACS) and qRT-PCR. In the KIRS2/Dap12-BB group, 4 patients achieved the CAR-T peak at ∼day 7, which is much faster than patients in the BBζ CAR-T group (∼day 14), followed by a decrease with a prolonged follow-up period (Figures 5A and 5B ). All 8 patients in two groups achieved complete response (CR) with minimal residual disease negative; even the patient with central nervous system leukemia (CNSL) relapse who received KIRS2/Dap12-BB CAR-T cells also achieved CR, as confirmed by cerebrospinal fluid. Interestingly, patients administered KIRS2/Dap12-BB CAR-T cells achieved CR at ∼day 30, which is much earlier than the patients infused with BBζ CAR-T cells, who ranged from 45 to 60 days (Figure 5C). Although a high CR rate was achieved, 3 of 4 patients infused with BBζ CAR-T cells relapsed in months 3–5, and 2 patients died 2 months later after relapse. Encouragingly, 2 of 4 patients administered KIRS2/Dap12-BB CAR-T cells relapsed in month 9–14, and all 4 patients were still alive, according to the recent follow-up data (Figure 5D). These findings demonstrated that adult r/r B-ALL patients can benefit from the KIRS2/Dap12-BB CAR-T cells.Figure 5The clinical response of CAR-T cellsShow full caption(A) CAR-T cell expansion and persistence in subjects' peripheral blood was examined by FACS. (B) CAR-T cell expansion and persistence in subjects' peripheral blood were examined by qRT-PCR. (C) Dot plot shows the time patients achieved CR. Each dot represents one patient. (D) Swimmer plot shows the time to disease relapse and patient death in months.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionCAR-T cell therapy is emerging as a promising therapeutic option for hematologic malignancies, with the potential for durable disease control following a single treatment.3Abramson J.S. Palomba M.L. Gordon L.I. Lunning M.A. Wang M. Arnason J. Mehta A. Purev E. Maloney D.G. Andreadis C. et al.Lisocabtagene maraleucel for patients with relapsed or refractory large B-cell lymphomas (TRANSCEND NHL 001): a multicentre seamless design study.Lancet. 2020; 396: 839-852Google Scholar,19Mikkilineni L. Kochenderfer J.N. CAR T cell therapies for patients with multiple myeloma.Nat. Rev. Clin. Oncol. 2021; 18: 71-84.Google Scholar We have shown that a KIRS2/DAP12 receptor with scFv triggered antigen-specific cytotoxicity, cytokine production, and proliferation that is comparable with second-generation CD3z-based CARs in a previous study. Incorporation of co-stimulatory domains in CARs has been one approach to enhance anti-tumor efficacy of CAR-T cells, and activation by co-stimulatory domain may confer differential T cell overall therapeutic efficacy. In recent years, co-stimulation receptors derived from 4-1BB or CD28, etc., were incorporated to create second-generation CARs, because the first-generation CAR-T cells engineered with a targeting domain and CD3ζ failed to elicit durable anti-tumor responses.20Weinkove R. George P. Dasyam N. McLellan A.D. Selecting costimulatory domains for chimeric antigen receptors: functional and clinical considerations.Clin. Transl. Immunology. 2019; 8: e1049Google Scholar, 21Savoldo B. Ramos C.A. Liu E. Mims M.P. Keating M.J. Carrum G. Kamble R.T. Bollard C.M. Gee A.P. Mei Z. et al.CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients.J. Clin. Invest. 2011; 121: 1822-1826Google Scholar, 22Guedan S. Posey Jr., A.D. Shaw C. Wing A. Da T. Patel P.R. McGettigan S.E. Casado-Medrano V. Kawalekar O.U. Uribe-Herranz M. et al.Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation.JCI Insight. 2018; 3: 96976Google Scholar It is now clear that incorporation of co-stimulatory signals in CARs is essential to maximize T cell expansion, persistence, and anti-tumor activity. For instance, clinical outcomes from anti-CD19 CAR-T cells demonstrate that inclusion of the CD28 or 4-1BB costimulatory domain enables CAR-T cell long persistence, enhanced cytotoxicity, and a safer profile.23Long A.H. Haso W.M. Shern J.F. Wanhainen K.M. Murgai M. Ingaramo M. Smith J.P. Walker A.J. Kohler M.E. Venkateshwara V.R. et al.4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors.Nat. Med. 2015; 21: 581-590Google Scholar, 24Salter A.I. Ivey R.G. Kennedy J.J. Voillet V. Rajan A. Alderman E.J. Voytovich U.J. Lin C. Sommermeyer D. Liu L. et al.Phosphoproteomic analysis of chimeric antigen receptor signaling reveals kinetic and quantitative differences that affect cell function.Sci. Signal. 2018; 11: eaat6753Google Scholar, 25Zhao Z. Condomines M. van der Stegen S.J.C. Perna F. Kloss C.C. Gunset G. Plotkin J. Sadelain M. Structural Design of Engineered Costimulation Determines Tumor Rejection Kinetics and Persistence of CAR T Cells.Cancer Cell. 2015; 28: 415-428Google