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- A new generation of virus-free CAR-T cell therapy
CAR-T cell therapy for pediatric acute lymphoblastic leukemia-past, present and future
CAR-T cell therapy for pediatric acute lymphoblastic leukemia. In this commentary dedicated to commemorating the 60th anniversary of the founding of the British Society of Hematology, we focus on our community’s contribution to the success of CD19-oriented CAR-T cell therapy in children. We will place the current practice of CAR-T cell therapy in the context of future challenges to be solved in order to realize its “rule-changing” therapeutic potential.
The modern concept of T cell immunotherapy began with the first bone marrow transplantation and the understanding of the anti-leukemia effect of the transplant, which was established on the basis of groundbreaking observations made between 1979 and 1990. TCR genetic engineering methods are limited to patients with a specific human leukocyte antigen (HLA) background. Therefore, in order to be relevant to a wider range of patients, there is increasing interest in generating synthetic receptors that recognize antigens in an unlimited manner.
Construction of Chimeric Antigen Receptor
Chimeric antigen receptors combine antigen recognition domains usually derived from antibodies (such as single-chain variable fragments or scFv) with hinge/stem regions, and transmembrane domains that connect the receptor to the cell membrane and connect within the cell To provide this attraction to the CD3ζ inner domain in a single linear molecule (Figure 1).
Figure 1 The structure of different generations of chimeric antigen receptors. scFV, single-chain variable fragment.
CD19 CAR-T cell therapy
CAR-T cells have shown promising results for CD19-positive primary B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) cells in vitro [33-35]. Then there are promising CD19 CAR-T cell therapy in vivo models [34,36-38]. However, the immunodeficiency mouse model is not enough to study the persistence of CAR-T cells. The first generation of CD19 CAR-T cells is used for difficult Clinical studies of therapeutic follicular lymphoma have shown a lack of persistence . Subsequent studies using second-generation CAR-T cells have shown that this design leads to longer-lasting expansion/persistence of CAR-T cells, which is indisputably proved in the study of Savoldo et al. Jointly manage the first and second generation CAR-T cells, and notice that the latter has obvious advantages in expansion and proliferation [37, 40].
For the success of CAR-T cell therapy, the doubling time of aggressive ALL is considered too fast, but the first two pediatric patients with refractory ALL treated with second-generation CD19 CAR-T cells on the basis of sympathy achieved Complete remission (CR), CD19-negative (CD19-) disease recurred after 2 months . Subsequent studies have consolidated this information, showing that 70–96% of patients with advanced disease obtain CR, and 60-93% of patients achieve measurable residual disease (MRD) negative status after receiving CAR-T cell therapy[42-45 ].
After a successful single-center study at the University of Pennsylvania, the multi-center ELIANA study proved the efficacy of tisagenlecleucel (Kymriah, Novartis) , a CD19 CAR-T cell product containing the FMC63 CD19 binding agent in the second generation form , Has the costimulatory domain of 4-1BB pediatric relapsed or refractory ALL . The overall survival rate and event-free survival rate at 12 months were 81% and 50%, respectively. This pivotal study was approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) to treat children and young people with relapsed and refractory ALL.
The main adverse events reported included cytokine release syndrome (CRS), neurotoxicity, cytopenias, and B cell dysplasia leading to hypogammaglobulinemia. Overall, CRS was observed in 77% of patients, and severe (grade 3/4) CRS was observed in 46% of patients. Any level of neurotoxicity was observed in 40% of patients. The median time to onset of CRS is 3 days (range 1-51), and the median duration of CRS is 8 days (range 1-36). The median onset time of neurotoxicity is 6 days (range 1-359), and the median duration is 6 days .
This promising data provided hope for a cure for patients who relapsed after stem cell transplantation, and attracted the attention of Sir Simon Stevens. After the approval of the NHS Rapid Approval (NHSE) in the United Kingdom, it supported the delivery framework of tisagenlecleucel in the United Kingdom (Figure 2). ).
Figure 2 timeline shows the major milestones in the development and implementation of CAR-T cell therapy in clinical practice.
Referral to NHSE-funded CAR-T cell therapy
The National Health Service of England (NHSE) has established a National CAR-T ALL Clinical Team (NCCP ALL) to confirm eligibility for tisagenlecleucel and provide timely access. The eligibility criteria are roughly similar to the inclusion criteria of the ELIANA study.
Based on geographic distance, patient preferences and local capacity limitations, eligible patients are assigned to CAR-T cell therapy centers accredited by the Joint Certification Committee-International Cell Therapy and EBMT Association (JACIE). Currently, there are 10 centers in the UK that provide tisagenlecleucel for children and young people. Funding is provided by the Cancer Drug Fund, and the cost of service delivery is paid by national tariffs.
Made by Tisagenlecleucel
After evaluating the patient at the CAR-T cell center, once an appropriate interval from the last treatment is observed, peripheral blood mononuclear cells (PBMC) are collected by leukopenia and then transferred for manufacturing. PBMC are cultured with T cell mitogenic stimulators and transduced with lentiviral vectors to express CAR (Figure 3). This process usually takes 4-6 weeks.
Figure 3 Tisagenlecleucel CAR-T cell manufacturing process.
According to the relapsed/refractory nature of the cohort, 87% of patients in the ELIANA study received bridging chemotherapy after leukocyte removal . The choice of bridging chemotherapy depends on the assessment of previous toxicity and disease response, as well as the expected interval between admissions for CAR-T cell infusion and pre-existing disease burden. Patients with low-level diseases can maintain the maintenance program; for patients with a heavier disease burden, consider gradually increasing vincristine and methotrexate (such as Capizzi mid-term maintenance but without asparaginase), drug induction [3-4 ], cyclophosphamide/cytarabine or inotuzumab.
The principle of bridging chemotherapy is to provide disease control and limit disease burden at CAR-T cell treatment points, while minimizing toxicity. This represents a shift in the treatment goals of relapsed/refractory diseases compared with pre-hematopoietic stem cell transplantation and first-line treatment, in which reducing MRD to a predetermined level is the goal. The advantage of reducing disease burden is to reduce the risk of severe CRS ; however, this needs to be balanced with the provision of CD19 (on disease or normal B cells) to promote the expansion and persistence of CAR-T. Limited data in the adult population suggests that higher-intensity bridging therapy is associated with a higher risk of infection complications, but is not beneficial to CAR-T results . In addition, in patients with a CD19+ bone content of less than 15%, it was noted that the duration of CAR-T cells was shorter than the bone marrow cells before infusion .
There is evidence that blinatumumab [a bispecific T cell junction agent (BiTE) targeting CD19] should be avoided as a bridging therapy as much as possible because of the reduced expression of CD19, which is related to the reduced response rate in a single-center retrospective study. .
Lymphatic removal reduces competition for steady-state cytokines (such as IL-7 and IL-15), deletes regulatory T cells and other potential regulatory immune subgroups, and eliminates the anti-CAR immune response in some patients. Compared with the combination of cyclophosphamide and fludarabine, the CAR-T cell duration was shorter when using single-agent cyclophosphamide . This highlights the importance of the intensity of lymphatic clearance, although in some studies, lymphatic clearance is optional for leukopenia . Common lymphatic removal protocols for pediatric ALL indications include fludarabine and cyclophosphamide.
Complications of CAR-T cell therapy
Cytokine release syndrome
Cytokine release syndrome is a recognized complication. In the mildest case, fever may occur, but in severe cases, hypotension, hypoxia, respiratory failure, renal failure, capillary leakage, and coagulopathy may occur  . The CRS definition has multiple iterations, including different grading systems. The American Society for Transplantation and Cell Therapy provided consensus guidelines in 2018 to define the severity of CRS based on clinical parameters (ie, fever, hypoxia, and hypotension). This consensus is increasingly accepted in the UK and worldwide . The severity of CRS is related to higher tumor burden , but the clear association with anti-tumor effects has not been confirmed [42, 43, 54-56].
The treatment of CRS depends on its severity and is based on supportive care and judicious use of the IL-6 blocking antibody tocilizumab. In the ELIANA study, 30% of patients reported grade 1 and 2 CRS, and 46% of patients reported grade 3 and 4 CRS.
In rare cases, patients with severe CRS do not respond to tocilizumab and require corticosteroids [48, 53, 57]. However, the use of corticosteroids may inhibit T cell function and proliferation [56, 58].
The pathophysiology of neurotoxicity is not fully understood, but it may involve damage to the blood-brain barrier after activation of the endothelial cell and monocyte/macrophage system [59, 60]. The neurotoxicity of CAR-T cell therapy, called immune effector cell-related neurotoxicity syndrome (ICANS) can be manifested as delirium, encephalopathy, aphasia, lethargy, inattention, agitation, tremor, seizures, and rare cerebral edema .
The neurotoxicity grading system was revised in 2018, when four levels were defined based on signs of encephalopathy, low level of consciousness, seizures, motor weakness, and increased intracranial pressure . However, diagnosing ICANS is still a challenge for young children or those who lack sufficient cognitive abilities to be assessed using existing encephalopathy assessment tools [53, 61].
The severity of neurotoxicity depends on the CAR-T cell product used. CARs containing CD28 costimulatory domain have a higher incidence of neurotoxicity in children and adults [62, 63]. In the ELIANA study, 40% of patients had neurotoxicity, and 13% of patients had grade 3 neurotoxicity (no neurotoxicity 4).
The treatment of neurotoxicity depends on the severity of the symptoms. In most cases, with only supportive care, the symptoms will disappear within a few weeks. Level 2 neurotoxicity in the case of CRS should be treated with tocilizumab blocking the IL-6 axis. However, grade 3/4 neurotoxicity that does not respond to tocilizumab or does not have CRS should be treated with corticosteroids [45, 59, 64, 65].
B cell hypoplasia
Targeting CD19 with CAR-T cells will cause normal B cell depletion outside the target tumor, so B cell hypoplasia is a useful marker of CAR-T cell persistence. The resulting hypogammaglobulinemia is more likely to occur in children than adults, and this may be due to the lack of mature (CD19-) plasma cell pools . However, this can easily be controlled by regularly replacing immunoglobulins [45, 64]. Current pediatric practice uses a threshold of 5 grams per liter. However, there is a lack of prospective data on immunoglobulin replacement after CAR-T cell therapy [64, 70].
Cytopenia is a recognized adverse reaction that may last for several weeks after CAR-T cell therapy. Although the exact pathophysiology of cytopenia is not fully understood, it seems that the important contributing factors are cytokines released during CRS, lymphocyte clearance protocol, previous chemotherapy or SCT, and macrophage activation [57, 67, 68].
In the ELIANA study, 53% of patients with grade 3 or 4 neutropenia did not resolve by day 28. Similarly, after the 28th day, 41% of patients had grade 3 or 4 thrombocytopenia. Bone marrow growth factor may support prolonged neutropenia, but it is not recommended within the first 3 weeks after tisagenlecleucel or until CRS subsides .
Cytopenia and subsequent infections are related to the severity of CRS [69, 70]. Approximately 20-40% of patients will develop an infection within the first month after receiving CAR-T cell therapy . The incidence of infection in children and young people seems to have declined after the first 4 months . The severity and incidence of late-stage infections after CAR-T cell therapy have not been well described, but most seem to be mild, mainly viral respiratory diseases [69, 72].
The future of CAR-T cell therapy and the contribution of British hematologists
CD19 CAR-T cell therapy in the treatment of B-cell malignancies still has major challenges to overcome, although this therapy has now been established as a game changer for patients with relapsed/refractory ALL. The initial scientific progress was mainly promoted by the cooperation of US research groups with pharmaceutical companies, such as Novartis and Kite, which were approved by the FDA.
However, the qualifications of the UK CAR-T cell research community discussed below support the rapid implementation of extensive clinical research. The portfolio of University College London (UCL) is one of the most advanced and diversified institutions in Europe, such as those outside the United States. One of the highest number of patent registrations measured .
It is committed to providing next-generation CAR-T products that serve unmet clinical needs.
This expertise was developed through transatlantic collaboration and the guidance of British hematologists (including Dr. Martin Pule and Professor Persis Amrolia), and was developed by Professor Malcolm Brenner (formerly Royal Free Hospital and Great Ormond Street Hospital), but then Established a T cell engineering laboratory in the UK. Baylor College of Medicine in Houston, Texas. Since returning to the UK, Dr. Pule and Professor Amrolia have implemented one of the first studies of CAR-T cell therapy (CD19 TPALL study) in Europe, and a group has been actively researching a series of indications for CAR-T cell therapy, such as development Next-generation CD19 CAR-T cell therapy. Dr. Pule founded Autolus Ltd, which has an extensive CAR-T cell research portfolio, including ALL, B-cell non-Hodgkin’s lymphoma (NHL), myeloma and T-cell malignancies.
Other CAR-T researchers at UCL include Professor Karl Peggs, Dr. Claire Roddie, Professor Waseem Qasim (immunologist), Professor John Anderson, and Dr. Karin Straathof (both oncologists), who have translated the generic CD19 targeting T Cell and GD2CAR-T cell therapy are in neuroblastoma respectively. Memorial Sloan Kettering Cancer Center Sadelain Laboratory graduates, such as Professor John Maher (oncologist) and Dr. Reuben Benjamin, established in the pre-clinical and clinical research of CAR-T cells for the treatment of solid organs and hematological malignancies, respectively A good record. Professor Katy Rezvani was initially trained at Hammersmith Hospital and studied under the guidance of Professor John Barrett of the National Institutes of Health. Later, she established a laboratory at the MD Anderson Cancer Center in Texas, where she developed a A pioneering alternative source of CAR effector cells. CD19 CAR natural killer (NK) cells are used to treat CLL and NHL patients from umbilical cord blood donors, and because they lack endogenous TCR, they do not mediate GVHD and therefore undergo The test can be universally applied .
Broadly speaking, the biggest challenge is to expand the accessibility of this approach to other cancer environments, including the treatment of solid organ malignancies. But even in the treatment of B-ALL, CAR-T cell therapy needs to be improved. The persistent recurrence and loss of CAR-T cells is still the biggest obstacle, so at least 50% of patients need to continue to receive further treatment , usually involving stem cell therapy. The latter represents a significant toxicity burden for patients who usually undergo a large amount of pretreatment, as well as a resource burden for healthcare services. However, in some studies, especially in the case of non-persistent CAR-T products, the results have improved compared with patients receiving CAR-T cell therapy but not undergoing adjuvant transplantation [44, 75, 76].
Disease recurrence is caused by antigen escape (usually in the case of persistent CAR-T cells) or persistent loss of CAR-T cells. In this case, the recurrence usually continues to express CD19. The relative proportion of CD19+ and CD19- recurrences varies depending on the CAR-T cell product studied, and patients who receive longer-lasting CD19-CAR treatment have a higher recurrence rate.
CD19 disease will recur in 7% to 25% of all patients receiving targeted CD19 CAR-T cell therapy . Therefore, antigen escape is an important challenge for the further development of CAR-T cell therapy, especially because this type of relapse is not suitable for treatment with blinatumomab, which is a bispecific antibody that can effectively connect relapsed patients with stem cell transplantation [78 ].
Theoretically, CAR-T cells targeting multiple antigens rather than a single antigen may overcome the random mechanism of antigen negative escape, because it is unlikely that two such events will occur at the same time.
The dual targeting method for B cell malignancies has different strategies. Schultz et al. A bivalent CAR method for CD19/CD22 was used in a joint pediatric and adult study. In their interim data analysis, 92% of these patients achieved CR on day 28, and only one patient developed severe CRS and ICANS. Due to the short duration of the product, three patients experienced recurrence of CD19-positive disease .
Co-administration of separate CD19 and CD22 targeting CAR-T cells is another strategy. In a study in China, 96% of patients achieved CR. However, the persistence of CAR-T cells is also limited, and nearly 50% of patients will relapse CD19+/CD22+ disease . Gardner et al. studied the co-administration of single CD19 and CD22 CAR-T cells and bispecific CD19/CD22 CAR-T cells in three different populations. Patients achieved an MRD-negative CR rate of 87%, but the short duration of CD22CARs was observed, so most recurrences are related to diseases that express CD22 .
In the preliminary report on the UK AMELIA study, this is one of the first clinical studies using the bicistronic CD19/CD22 CAR vector to ensure that all CAR-T cells express two CARs. Seven out of seven patients (100% ) Receive intermediate or higher therapeutic cell doses (>3 × 106/kg) to achieve molecular remission. Three recurrences were reported, one of which was CD19-negative/CD22 low expression 1 year after treatment. No severe (Grade 3 or 4) CRS has been observed . This method proved to have good toxicity characteristics, the same response rate, but the duration of CAR-T was shorter than the ELIANA study (median 180 days), and the CD19-recurrence rate was lower despite the dual targeting strategy, but it was still visible.
Compared with tisagenlecleucel, the CAR-T persistence noted in all these studies is shorter, which may be related to the steric hindrance of CD22: CD22 CAR interaction and poor T cell activation, which will need to be used in future CAR design solve.
Improve the persistence of CAR-T cells
Persistent loss of CAR-T cells can also lead to relapse. Preclinical studies have shown that generating CAR-T cells with a lower degree of differentiation can improve results [82, 83]. Globally, different groups have studied different strategies to achieve this goal, for example, pre-selecting pivotal memory T cells (TCM) or stem cell-like memory T cells (TSCM) populations [51, 75, 84, 85] For CAR transduction, or the production of CAR-T cells in the presence of mediators that maintain an early differentiation state . CAR-T cells can be used with additional stimulatory ligands, such as 4-1BBL [31, 32], that are commonly used to improve the efficacy, expansion and persistence of CAR-T cells.
Paradoxically, strategies to enhance CAR-T cell signaling/potency and overcome co-suppressive signaling may impair the adaptability of CAR-T cells by enhancing T cell depletion. CAR usually uses antibody-derived binding domains to bind antigens with an affinity range much higher than that of natural TCR. In addition, the selection of specific transmembrane domains and costimulatory domains can lead to T cell depletion [87, 88]. Recently, engineering strategies to reduce CAR signal intensity have shown promise, for example, reducing the number of immunoreceptor tyrosine-based activation motifs (ITAM) in the CAR signal domain or changing the promoter used to reduce CAR expression. We have translated a new type of CD19 binder that has a lower affinity for CD19.
Compared with Kymriah, the CAR has been shown to have comparable anti-tumor efficacy, while showing improved expansion (the maximum CAR-T cell concentration achieved is three times that seen with tisagenlecleucel) and durability, despite the fact that the treated patient’s bone marrow The disease burden is very low. Among the 14 evaluable patients, the median duration of CAR-T duration was 215 days (range 14-728 days). The one-year overall survival rate and event-free survival rate are comparable to those in the ELIANA study, being 63% and 46%, respectively . The toxicity profile is very favorable. Even if patients with significant recurrence are treated, severe CRS does not occur. This is confirmed by the use of the same CAR structure in the adult patient population with advanced ALL , which eventually led to the launch of a licensing study for this product .
Before obtaining regulatory approvals from the FDA, EMA, and the National Institute for Health and Clinical Optimization (NICE), patients could only receive CAR-T cell therapy in clinical studies. In the UK, thanks to the pioneering work of hematologists such as Professor Persis Amrolia, Dr. Martin Pule, Professor Paul Veys and other UCL researchers, children with relapsed/refractory B-ALL have been able to obtain a series of open clinics since 2014 Research is conducted at the Institute of Child Health, University College London and Great Ormond Street Hospital. The United Kingdom has always supported the method of centrally funding licensed CAR-T cell products through the NHS, and has ensured that everyone who meets the conditions can use CAR-T cell therapy . However, even for patients with permitted indications, CAR-T cell therapy still has many obstacles.
A major factor leading to the unfairness of global CAR-T cell therapy is its price, which of course precludes its widespread use in developing countries. The huge cost comes from clinical-grade products produced on a per-patient basis in centralized production facilities and meeting strict quality standards . There is also the high cost of key reagents (such as lentivirus transfer vectors). Progress has been made in the CAR-T cell manufacturing process, including non-viral transduction and the use of semi-automated platforms, such as those developed by Dr. Claire Roddie and Professor Mark Lowdell of University College London to provide decentralized/point-of-care production [95, 96]. However, these advancements have not yet developed into licensed products.
In addition to limited medical resources, the availability of CAR-T cell therapy may be hindered by factors such as autologous harvest, manufacturing failure, rapid disease progression, and patient comorbidities due to the reduction of lymphocytes in patients. Therefore, a large number of patients eligible for screening failed to participate in ELIANA (32 of the 107 patients screened) and CARPALL (3 of the 17 patients screened) [45, 90]. These patients will benefit from access to generic CAR products on closed shelves without the 5-8 week delay inherent in self-manufacturing and related bridging treatments. Global research work has provided many ways to provide universally applicable CAR products, including providing CAR-transduced effector immune cells without the need for immune response to allogeneic receptors (such as NK cells)  without the need for genetic Engineering to achieve universal application. In UCL, Pule et al. In cooperation with the biopharmaceutical company Cellectis, the concept of T cells destroyed by the TCR gene of the transcriptional activator-like effector nuclease (TALEN) was developed, which also developed resistance to alemtuzumab through accompanying CD52 destruction. Therefore, these CAR-T cells lacking endogenous TCR will not be able to mediate the anti-host response and will not be deleted from the host by using alemtuzumab as a modulator. After promising preclinical work and good manufacturing practices, a successful pilot study of two infants with refractory ALL at Great Ormond Street Hospital led to a multicenter global clinical study . Transcription activator-like effector nucleases have potential for gene damage mediated by genotoxicity (TALEN), which has led people to consider safer next-generation gene editing platforms, such as base editing  and innovative protein engineering methods. 
New goals, new structures, etc.
Ongoing clinical trials are evaluating the impact of CD19 CAR-T cell therapy on pediatric patients with relapsed/refractory B-cell NHL [100, 101]. However, there is a greater unmet clinical need to find treatment options for non-B-cell hematological malignancies. Relapsed/refractory T-cell ALL and lymphoblastic lymphoma represent one such challenge. Finding suitable target antigens Is controversial . Finding new targets is a challenge, especially if the lack of tumor-specific antigens leads to possible shutdown of tumors, targeted toxicity, if a single antigen is targeted or CAR-T cell failure, if the targeted antigen is widely expressed. Even when the best target is set, the immunosuppressive tumor microenvironment will disable efficient CAR-T cells. In these cases, CAR-T cell therapy may need to be provided with multiple immunomodulatory therapies, or Be “armed” to resist adverse immune regulation.
Finding the best CAR-T cell construct for acute myeloid leukemia (AML) faces similar obstacles. AML is a disease with diverse phenotypes, and it represents a greater challenge because AML blasts share a common antigen with normal bone marrow cells and hematopoietic stem cells (HSC). Strategies to reduce the effect of extra-tumor targeting include multi-targeting CAR, using complex gating strategies to selectively identify AML primitive cells or editing HSC to prevent the expression of CAR targeted antigens [103, 104]. Finding the best CAR-T therapy for the treatment of AML and improving the outcome of this disease in children and adults with this disease is one of our main goals. Together with our mentors and professors including Dr. Martin Pule and Professor Persis Amrolia Colleagues and other British researchers work together. For example, Professor Waseem Qasim is about to start a clinical study (CARAML study) on a series of ready-made generic CAR-T cells for a series of AML antigens.
Standing on the shoulders of the giants in the field of adoptive therapy and stem cell transplantation in the United Kingdom, we hope that in the next 60 years of British hematology, we will be closer to the dream of every stem cell transplant doctor: to provide high-precision adoptive cancer therapy with minimal side effects.
(source:internet, reference only)