Chimeric antigen receptor T-cell (CAR-T) immunotherapy is a treatment that uses genetic engineering to create CAR genes composed of a single-chain variable fragment (scFv) capable of antigen binding, a hinge-transmembrane domain, and intracellular signal transduction domains. These CAR genes are then introduced into T cells ex vivo using various vectors, transforming them into CAR-T cells, which are subsequently infused back into the patient.
CAR-T cell therapy has shown remarkable efficacy in various hematologic malignancies, particularly in relapsed or refractory conditions. It has significantly improved remission rates and extended relapse-free survival, representing a revolutionary milestone in 21st-century medicine. Since the FDA approved the first CD19-targeted CAR-T cell therapy for the treatment of relapsed or refractory acute B-cell lymphoblastic leukemia (ALL) in 2017, several commercial products have been approved for clinical use.
Development History
First-Generation CAR-T Cells
First-generation CAR-T cells consist of a CAR structure incorporating a classical antibody-derived scFv fragment, a hinge region, and a CD3ζ intracellular signaling domain. The scFv is fused into the T-cell receptor (TCR) constant region, endowing T cells with antibody-like recognition properties. These CAR-T cells combine direct antibody-like targeting with the cytotoxic features of T-cell immunity, enhancing their ability to specifically target and kill cells.
Second-Generation CAR-T Cells
Complete activation of T cells requires two signals: the primary signal transduced by extracellular TCR-antigen binding, and the secondary signal delivered by co-stimulatory molecules binding to their ligands. Second-generation CAR-T cells incorporate scFv, intracellular CD3ζ signaling domains, and co-stimulatory domains (e.g., CD28 or 4-1BB). These advancements enable improved T-cell activation and functionality.
Third-Generation CAR-T Cells
Third-generation CAR-T cells build upon the second-generation design with the addition of another co-stimulatory molecule domain. Common combinations include CD28 or 4-1BB paired with novel signaling molecules such as OX40, ICOS, CD27, or CD40-MyD88. While preclinical studies have shown greater potency and sustained activity compared to second-generation CAR-T cells, concerns have been raised about the lowered T-cell activation threshold, which may lead to excessive activation and complications such as cytokine overproduction.
Fourth-Generation CAR-T Cells
Fourth-generation CAR-T cells, also known as T-cells redirected for universal cytokine-mediated killing (TRUCK), incorporate a nuclear factor of activated T-cell (NFAT) transcriptional element within the CAR structure. This feature enables them to secrete specific transgene proteins, such as cytokines (e.g., IL-12, IL-18, chemokines), in the tumor microenvironment. These secretions modify the immunosuppressive tumor microenvironment, attract additional immune cells, and enhance immune responses against tumors.
Indications
CAR-T cells are categorized as "living cell" therapeutics. Several CAR-T cell products have received regulatory approval domestically and internationally following extensive clinical trials. These therapies are employed for second-line or later treatments of relapsed or refractory B-cell lymphoblastic leukemia, lymphoma, and multiple myeloma.
CAR-T products targeting CD19 include tisagenlecleucel, axicabtagene ciloleucel, lisocabtagene maraleucel, and relmacabtagene autoleucel, all of which are indicated for relapsed or refractory B-cell malignancies.
CAR-T products targeting B-cell maturation antigen (BCMA) include idecabtagene vicleucel, ciltacabtagene autoleucel, and others, which are used for relapsed or refractory multiple myeloma.
Additionally, patients with relapsed or refractory hematologic disorders may be eligible for enrollment in clinical trials for CAR-T therapies following comprehensive evaluation.
CAR-T Treatment Process
Patient Screening
Patients are selected based on the indications for various commercial CAR-T cell products or the inclusion and exclusion criteria of different clinical trials. Relevant information is collected, and comprehensive assessments are conducted, primarily including the following:
- Demographics and medical background: demographic characteristics, vital signs, general symptoms, medical history, and concomitant medications.
- Laboratory tests: complete blood count, urinalysis, stool examination (including occult blood), comprehensive biochemistry panel, immunoglobulin quantification, coagulation studies, cytokine measurements, lymphocyte subsets analysis, and virology tests.
- Bone marrow evaluation: bone marrow smear, biopsy, flow cytometry, chromosomal karyotyping, immunophenotyping, gene sequencing, and fluorescence in situ hybridization (FISH).
- Imaging and supplementary tests: electrocardiogram (ECG), echocardiography, ultrasound, CT, PET-CT.
- Lesion assessment: pathological biopsy of the lesion, immunohistochemistry, FISH, flow cytometry, as well as detection of lymphocyte antigen receptor gene rearrangement.
Cell Collection
Peripheral blood or mononuclear cells are collected from the patient for the isolation and purification of T cells, which are then used to prepare CAR-T cells. Researchers determine the collection method based on the patient’s hematologic profile, with apheresis being the preferred option. The collection volume depends on the required amount of CD3-positive cells and their concentration in the peripheral blood. Sterile and contamination-free procedures are followed, using closed-system collection methods to minimize the risk of microbial contamination. Successful T-cell collection is a critical prerequisite for the subsequent complex manufacturing of CAR-T cells.
Lymphodepleting Therapy
Lymphodepletion involves using chemotherapy to reduce the patient's lymphocyte count prior to CAR-T cell infusion. This process lowers tumor burden, depletes immune cells to reduce competition, and facilitates CAR-T expansion and persistence in the body. Low-dose chemotherapy is typically administered between Days -5 and -3 before CAR-T infusion. Common lymphodepleting regimens include cyclophosphamide, fludarabine, or bendamustine. The specific protocol is determined based on the patient’s condition. A typical regimen involves fludarabine administered intravenously at 25–30 mg/(m2·d) daily for three consecutive days (Days -5, -4, -3) and concurrent cyclophosphamide at 500 mg/(m²·d) intravenously on Day -3.
CAR-T Cell Manufacturing
CAR-T cell production is carried out in clean laboratory environments with ISO Class 7 (Grade C) certification, and T-cell culture takes place in ISO Class 5 (Grade A) laminar flow cabinets. T-cell separation and purification are initiated within 16 hours of obtaining the patient’s blood sample. The volume of blood processed is calculated based on the proportion of CD3-positive cells and leukocyte count in the sample. Lentiviral transduction or electroporation methods are used to manufacture CAR-T cells, which are then expanded in vitro. The effectiveness of CAR-T cells is verified through functional testing. Before infusion, cell supernatants are sent to accredited third-party laboratories for safety and quality control, including testing for bacteria, fungi, mycoplasma, chlamydia, and endotoxins.
CAR-T Cell Infusion
CAR-T cells that meet quality standards are delivered to healthcare providers for infusion. The cell dose is determined based on the product manual or the research protocol. Prior to infusion, the patient's vital signs (such as temperature, blood pressure, pulse, and respiration) are recorded. The infusion set is adjusted to the appropriate rate, flushed with saline to clear the tubing, and then connected to the CAR-T cell infusion bag containing the resuspended cells. The infusion procedure is initiated thereafter.
Monitoring and Management
Patients are generally monitored in the hospital for 14 days following CAR-T infusion to assess the risk of CAR-T-related adverse events and provide timely interventions as needed. Within the first month after infusion, patients undergo weekly follow-up assessments, with additional tests performed based on the clinical situation. For the first six months after infusion, monthly follow-ups are conducted to monitor treatment efficacy. After six months, follow-ups transition to every three months. Between one and two years after infusion, patients are assessed every six months. Beginning at three years after infusion, yearly follow-ups are conducted.
##Complications
CAR-T cell therapy has demonstrated high response rates and complete remission rates in B-cell hematologic malignancies. However, complications associated with CAR-T treatment remain a critical limitation to its broader application. These complications include cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), bone marrow suppression, CAR-T-associated coagulopathy (CARAC), infections, tumor lysis syndrome, and CAR-T-associated hemophagocytic lymphohistiocytosis (carHLH). CRS and ICANS are the primary complications that may significantly increase the risk during treatment and potentially lead to fatal outcomes. Enhancing management of these complications can improve patient outcomes and prognoses.
Cytokine Release Syndrome (CRS)
CRS is a clinical syndrome resulting from excessive cytokine release (e.g., IL-6) caused by lymphocyte activation. The incidence of CRS in CAR-T cell therapy for B-cell malignancies ranges from 50% to 90%, with severity influenced by factors such as the type of underlying disease, the CAR-T cell dose, and the type of co-stimulatory molecule used. The pathophysiology of CRS involves activation of the monocyte-macrophage system, inflammatory cytokine storms, and endothelial cell activation. Clinical manifestations are typically characterized by fever (≥38°C) and hemodynamic instability. Several grading systems are used in clinical trials to assess the severity of CRS, including the CTCAE scale, Lee scale, Penn grading scale, CARTOX criteria, and ASTCT consensus.
Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS)
ICANS refers to a spectrum of neurological symptoms that occur following immunotherapy, triggered by activation of endogenous or exogenous T cells and/or other immune effector cells. It is observed in 20% to 60% of patients undergoing CAR-T treatment. The pathophysiology underlying ICANS remains unclear but is thought to involve elevated levels of inflammatory cytokines in the serum and cerebrospinal fluid, endothelial dysfunction, central nervous system infiltration by inflammatory cells, and activation of astrocytes and microglia.
Symptoms of ICANS range from encephalopathy with altered consciousness and behavioral changes to expressive aphasia or other language impairments, difficulties with writing and articulation, fine motor dysfunction, tremors, myoclonus, and headaches. Severe cases may present with obtundation or seizures, requiring intubation to secure the airway. In rare cases, malignant cerebral edema may develop, leading to life-threatening complications. ICANS can occur concurrently with CRS or after CRS resolution, sometimes manifesting a month after infusion. Symptoms are typically self-limiting, lasting 5 to 17 days. The onset, duration, and severity of ICANS are associated with the CAR-T product used and the patient's disease status. The ICE (Immune Effector Cell-Associated Encephalopathy) score, with a maximum of 10 points, is utilized to evaluate the severity of neurological symptoms, including altered consciousness, motor weakness, seizures, increased intracranial pressure, and cerebral edema.
Treatment for ICANS primarily involves supportive care and corticosteroids. Tocilizumab is ineffective in ICANS and may even worsen it. Therefore, when ICANS and CRS co-occur, ICANS management takes priority over lower-grade CRS. While low-grade neurotoxicity may resolve spontaneously, ICANS of Grade 2 or higher typically requires corticosteroid intervention.
Bone Marrow Suppression
Bone marrow suppression is a common complication in patients with hematologic malignancies undergoing CAR-T therapy. Clinical manifestations include anemia, thrombocytopenia, and leukopenia. Potential causes encompass preconditioning chemotherapy, high levels of cytokine release during CRS, off-target effects, viral infections, and CRS-associated hemophagocytic syndrome.
CAR-T-Associated Coagulopathy (CARAC)
CARAC is an acute syndrome related to cytokine release, occurring shortly (typically within 28 days) after CAR-T cell infusion. It is characterized by bleeding and/or thrombosis accompanied by reduced platelet levels and coagulation abnormalities. The core mechanism involves endothelial activation or damage, presenting as prolonged PT, APTT, and TT, along with decreased fibrinogen levels. Severe cases may lead to disseminated intravascular coagulation (DIC) with associated symptoms such as bleeding, ecchymosis, hypotension, respiratory distress, and confusion. Treatment recommendations include supportive care and coagulation factor replacement therapy. Corticosteroids and IL-6 antagonists may be considered in cases involving concurrent CRS or severe bleeding complications.
Infections
Infections can include bacterial, viral, or fungal infections. Opportunistic infections are common during CAR-T therapy, with most occurring early after infusion but potentially arising weeks to months later. Infection prevention is crucial until immune reconstitution is achieved. Risk factors for infections include baseline patient characteristics prior to CAR-T therapy, CRS severity, and corticosteroid use. Symptoms of CAR-T-induced infections may include fever, nausea, increased fatigue, headache, myalgia, and general malaise. Late-onset infections are associated with prolonged B-cell depletion and corticosteroid treatment for CRS and/or ICANS. Therapy should be targeted, and prophylactic antibiotics are recommended for patients with prolonged cytopenias.
B-Cell Immunodeficiency
B-cell immunodeficiency arises from immune attacks by CAR-T cells targeting CD19, CD20, or CD22 on normal B cells or B cell precursors. It is characterized by sustained B-cell depletion and hypogammaglobulinemia. The duration of B-cell depletion serves as an indicator of the persistence of CAR-T cells in vivo. Hypogammaglobulinemia refers to serum levels of one or more immunoglobulins below the lower limit of normal, with IgG reduction being the most common feature.
CAR-T-Associated Hemophagocytic Lymphohistiocytosis (carHLH)
Hemophagocytic lymphohistiocytosis (HLH) is an inflammatory syndrome caused by pathological T-cell activation and associated with natural killer (NK) cell dysfunction. It is characterized by laboratory abnormalities such as hyperferritinemia, coagulation abnormalities, hypertriglyceridemia, and elevated liver transaminases. In some CAR-T therapy patients, HLH-like toxicity mimics secondary HLH/macrophage activation syndrome. Symptoms may include fever, splenomegaly, hepatomegaly, lymphadenopathy, rash, jaundice, and pulmonary manifestations such as cough and dyspnea.