Role Of CLL1 CAR T-cells In Children With Refractory/Relapsed Acute Myeloid Leukemia

Part Ⅰ CLL1 CAR-T cells generation and confirmation of its targeting potential in vitro and in vivoBackground:Acute myeloid leukemia(AML)is a malignant disorder caused by hematopoietic stem cells(HSCs)mutations.The major reasons of recurrence in AML include high heterogeneity and the persistence of LSCs following treatment.The prognosis of refractory/relapsed AML(R/R-AML)remains dismal.Therefore,t there is an urgent need to find novel AML therapeutics.Chimeric antigen receptor(CAR)T cells are a novel immunotherapy that employs genetic engineering to precisely target tumor cells that express tumor-specific antigens.C-type lectin-like molecule 1(CLL1)is a type Ⅱtransmembrane protein expressed in AML cells,leukemia stem cells,and mature myeloid cells but not in normal hematopoietic stem cells and lymphocytes,indicating that CLL1 is a promising target.Objective:To create a CAR-T cell that targets CLL1 and test its capacity to eradicate AML cells in vitro and in vivo.Methods:1.CAR-T cell construction:Utilize hybridoma method to produce high-affinity CLL1 monoclonal antibodies,apply CAR lentivirus particles to transfect T cells.2.In vitro experiment:Co-culture CLL1 CAR-T cells with Raji cells,HL60 cells,and Raji-CLL1 cells for 24 hours,and then evaluate IFN-gamma production.3.Mice experiment:HL60luc cells were inoculated into immune-deficient B-NDG mice.The HL60 leukemia bearing mice were treated with vehicle,T-mock,low dosage CLL1 CAR-T cells,and high dose CLL1 CAR-T cells respectively.Following treatment,the mice were regularly monitored by an IVIS spectrum equipment and data was quantified with Living Image software.Meanwhile,the mice’s survival time was recorded.Results:1.CLL1 CAR-T cells were successfully generated.2.Coculturing CLL1 CAR-T cells with Raji-CLL1 cells or HL60 cells resulted in a substantial increase in IFN-gamma levels,but coculturing with Raji cells that did not express CLL1 resulted in no significant change in IFN-gamma production.3.On day 0,all mice showed weak fluorescence.The fluorescence intensity in the mice injected with the vehicle and T-mock steadily rose on Day 3.The fluorescence intensity in vivo reduced in both groups of mice injected with CLL1 CAR-T cells compared to day 0.From Day 7 to Day 14,the fluorescence intensity in animals injected with the vehicle and T-mock grew dramatically,but essentially no fluorescence was seen in mice injected with CLL1 CAR-T cells in both groups.4.The mice injected with T-mock and vehicle had the longest survival times of 29 and 31 days,respectively.The mice treated with low and high doses of CLL1 CAR-T cells survived for 39 and 37 days,respectively.Conclusions:1.CLL1 is a promising target for car-t cell therapy of AML.2.CLL1 CAR-T cells demonstrated potent tumor killing capactity in vitro and in vivo.Part Ⅱ Clinical Study of CLL1 CAR-T cells Therapy for Children with Refractory/Relapsed Acute Myeloid LeukemiaBackground:There have been significant advances in the treatment of children with refractory/relapsed acute myeloid leukemia with the updating of intensive chemotherapy regimens,the development of targeted agents,the optimization of hematopoietic stem cell transplantation technology,and the support of supportive care,but outcomes of children with AML continue to lag behind those of children with acute lymphoblastic leukemia.As a result,there is an urgent need for in-depth research into innovative treatment techniques.Many new drugs have been tested in the clinic,but no breakthrough in the treatment of AML has occurred.CAR T cells are genetically designed T cells that are chimeric with an antigen receptor,allowing the engineered T cell to interact and target proteins,resulting in T cell activation.The CLL1 is expressed in 77.5-92%of AML cells but not in CD34+progenitor cells or normal hematopoietic stem cells,it is possible to employ CLL1 as a target of CAR-T cells in an AML treatment regimen.Several in vitro experiments have shown that CLL1 CAR-T cells can effectively kill AML cells,and some clinical trials have also shown that CLL1 CAR-T cells achieve good outcomes in the treatment of R/R-AML patients,but data on the safety and efficacy of CLL1 CAR-T cells in AML treatment are still lacking.Objective:To investigate the safety and efficacy of CLL1 CAR-T cells therapy in children with R/R-AML.Methods:According to the inclusion and exclusion criteria,seven children with R/R-AML who underwent CLL1 CAR-T cell treatment at Guangzhou Women and Children’s Medical Center from August 2018 to February 2021 were recruited in the research.Before receiving CLL1 CAR-T cells,all patients had lymphodepleting chemotherapy,and patient medical records were collected to assess the effectiveness of CLL1 CAR-T cell therapy according to the 2021 NCCN AML recommendations version 3.0.The toxicity of CAR-T cell treatment was evaluated using the Common Terminology Criteria for Adverse Events(CTCAE)version 5.0 and the management methodology established by Antomasso et al.The persistence of CLL1 CAR-T cells was determined by quantitative real-time polymerase chain reaction(qPCR)measurement of car gene copy number in peripheral blood.Medical data were used to determine the patients’ medical bills and blood product infusions.Results:1.CLL1 CAR-T cell treatment was administered to 7 patients,4 males and 3 females,with a median age of 8.4 years(range 5.8 to 13.5 years).Before infusion,the median white blood cell count of patients was 0.7(range 0.1 to 1.5)109/L,and the median blast myeloid leukemia cell count was 9.5%(1.0%to 71.5%).The median number of CLL1 CAR-T cells administered to these individuals was 1.03(range 0.94 to 1.98)106/kg.2.Early early treatment response evaluation revealed that 5 patients obtained complete remission(CR),five patients achieved CR,three patients were negative for minimum residual disease(MRD),and two patients were positive for MRD.CLL1 CAR-T cell treatment did not work for two individuals.After 5 and 2 months of CLL1 CAR-T cell treatment,patients 2 and 6 died of disease progression,respectively.The remaining five patients’ follow-up periods ranged from 11 to 27 months.Patient 1 died on the 23rd month due to illness progression.Patient 3 had allogeneic hematopoietic stem cell transplantation(allo HSCT)two months after receiving CLL1 CAR-T cell treatment,but died 11 months later from gastrointestinal graft-versus-host disease(GVHD)and thrombotic microangiopathy.Patient 4 relapsed four months after receiving clll car-t cell treatment and was treated with a combination of hypomethylating agents-based chemotherapy and venetoclax.This patient relapsed again at the 12th month and had HSCT at the 15th month,but died of recurrence at the 23rd month.Patient 5 relapsed in the 13th month but was kept on venetoclax medication and is now in CR with MRD negative.After 18 months of CAR T-cell treatment,Patient 7 died of a recurrence.Patient 7 got HSCT three months after being treated with CLL1 CAR-T cells,but remained MRD positive and died of recurrence at the 18th month.3.In patients 2,4,6,and 7,CAR T-cells were not significantly increased.Patients 2 and 6 did not react to CAR T-cell treatment,whereas patients 4 and 7 achieved CR but had MRD.The CAR T-cells were effectively expanded in patients 1,3,and 5,and they all obtained CR with MRD negative over an extended period of time.4.All patients who received CLL1 CAR-T cell treatment exhibited grade 1-2 cytokine release syndrome(CRS).Patient 5 had immunological effector cell associated neurotoxicity syndrome of grade 2.(ICANS).Patients 1 and 5 had grade 3 liver function impairment,whereas patients 3 and 6 had grade 1 liver function impairment.These 7 individuals had a drop in WBC counts of 3-4,neutropenia,and different degrees of anemia.Patient 4 had grade 1 thrombocytopenia,whereas the remaining patients all had grade 4 thrombocytopenia.CAR-T cells had no effect on activated partial thromboplastin time(APTT),prothrombin time(PT),or international normalized ratio(INR).Toxic side effects such as macrophage activation syndrome(MAS),tumor lysis syndrome(TLS),hemophagocytic lymphohistiocytosis(HLH),allergies,and renal function impairment were not observed in any patient.5.The total medical costs for these seven individuals ranged from (?)29,165 to(?)549,750.Therapeutic medication expenses are the greatest,whereas transfusion and nursing costs are less than 10%.Antimicrobial expenditures accounted for more than half of all therapeutic medication costs.6.Patient 6 drank the most blood products,including 23.5 and 27 units of red blood cells and platelets,respectively.Patients 1,2,and 3 used 11,1,and 9 units of red blood cells,respectively,whereas patients 1,2,and 7 consumed 14,4,7,and 4 units of platelets,respectively.Conclusions:1.CLL1 CAR-T cells can be effective in treating children with R/R-AML.2.CLL1 CAR-T cell treatment has tolerable toxic side effects in children with R/R-AML.