Treatment of tumor: CAR-T Cell Therapy and its clinical application

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Chimeric antigen receptor T cell (chimeric antigen receptor T cell, CAR-T) immunotherapy is to genetically engineer T cells isolated from patients or allogeneic donors to express chimeric antigen receptor (chimeric antigen receptor). receptor, CAR), an adoptive cell therapy that specifically recognizes and kills tumor cells. CAR-T is one of the major breakthroughs in the field of cancer immunotherapy in recent years. It has great advantages in the treatment of hematological malignancies and has broad development prospects. This article reviews the basic structure and function of CAR-T cells, their applications in hematological malignancies, and the challenges they face.

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Structure and Function of CAR-T Cells

CAR-T uses gene modification technology to transduce genetic material with specific antigen recognition domains and T cell activation signals into T cells, so that T cells can target and recognize relevant antigens on the surface of tumor cells, thereby activating effector T cells. cells, thereby exerting an anti-tumor effect. This target recognition process does not depend on the interaction between the traditional T cell receptor (TCR) and the major histocompatibility complex (MHC), and avoids tumor cells by downregulating MHC expression. Immune escape. CAR-T can be divided into the extracellular domain, a transmembrane domain and intracellular domain according to its structure. The extracellular region is usually a single-chain variable fragment (scFv) that targets and recognizes the antigen. The transmembrane region is usually derived from CD28 or CD8 and can affect the interaction between CARs. The intracellular domain is the CD3ζ signaling domain, which contains three immunoreceptor tyrosine-based activation motifs (ITAMs), which can initiate intracellular signal transduction through phosphorylation activation, activate T cells, promote proliferation, Cytokine secretion and cytotoxicity.

The first generation of CARs, composed of scFv and CD3ζ signaling domains, possessed T cell killing toxicity in vitro, but showed no antitumor effect in clinical trials. The second generation of CARs added a costimulatory domain derived from CD28 or 4-1BB on the basis of the first generation, which extended the survival time of CAR-T cells in vivo, and enhanced the proliferation ability and killing toxicity. The third-generation CARs added two costimulatory molecules on the basis of the first-generation CARs, and their anti-tumor effects were further improved. The fourth generation of CARs, also known as "armored" T cells (T cells redirected for universal cytokine killing, TRUCKs), enhances the efficacy of CAR-T by genetic modification to secrete specific cytokines or express additional costimulatory ligands, while introducing The suicide gene system controls CAR-T activity and is activated when necessary to reduce cytotoxicity.

At present, clinical trials carried out around the world mainly focus on the second-generation CAR-T composed of CD28 or 4-1BB co-stimulatory domains. The CD28 or 4-1BB co-stimulatory domain is of great significance in promoting the proliferation and survival of CAR-T cells in vivo, and can significantly improve the complete remission rate of patients receiving CAR-T therapy. Studies have shown that the CD28 co-stimulatory domain can induce a more rapid anti-tumor response, but less durable; while 4-1BB has a slower tumor clearance rate, but can induce a sustained high level of response, but clinical studies have shown that expression of CD28 co-stimulatory response. The clinical efficacy of CAR-T cells in the stimulatory domain is similar to that of CAR-T cells expressing 4-1BB in the treatment of hematological malignancies. In addition to CD28 and 4-1BB, research on CAR-T cells derived from other costimulatory domains is also being actively carried out, such as OX40 (CD137), ICOS, etc.

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Clinical application of CAR-T cells

CAR-T immunotherapy has made a breakthrough in the treatment of hematological malignancies. At present, the U.S. Food and Drug Administration (FDA) has approved four CAR-T cell products targeting CD19, namely Tisagenlecleucel (tisa-cel, Kymriah), Axicabtagene Ciloleucel (axio-cel, Yescarta), Brexucabtagene Autoleucel (brexucel, Tecartus) and Lisocabtagene Maraleucel (liso-cel, Breyanzi), these products are mainly used for the treatment of children and young adults with relapsed/refractory acute B-cell lymphoblastic leukemia (B-cell acute lymphoblastic leukemia, B- ALL) and certain types of B cell non-Hodgkin lymphoma (B-NHL) in adults, including relapsed/refractory diffuse large B cell lymphoma (DLBCL), relapsed/refractory mantle cell lymphoma (mantle cell lymphoma, MCL) and relapsed/refractory follicular lymphoma (follicular lymphoma, FL), and the curative effect is significant. With the development of a large number of CAR-T clinical studies, CAR-T cells targeting different targets are also expected to be used in B-NHL, multiple myeloma (MM), chronic lymphocytic leukemia (chronic lymphocytic leukemia, CLL), acute myeloid leukemia (AML), T-cell lymphoma and solid tumors.

2.1 B-cell malignancies

CD19 is a B-cell-specific target that is widely expressed in almost all B-cell malignancies. CD19-targeted CAR-T therapy demonstrated rapid and durable efficacy in clinical trials of B-cell malignancies such as children and adults with relapsed/refractory B-ALL, relapsed/refractory DLBCL, relapsed/refractory FL, and CLL Antitumor effect. CD20 is expressed in B cells of all differentiation stages except plasma cells. CD20-targeted CAR-T cells are currently in the early clinical research stage and have a high response rate in patients with DLBCL and indolent non-Hodgkin lymphoma. In addition, CD19, CD20 bispecific CAR-T cells have shown low toxicity and high efficiency in clinical trials, and may become an effective means to overcome the recurrence of drug resistance caused by antigen downregulation. CD22, a member of the sialoadhesin family, is unique to B cells and is expressed in the vast majority of B cell malignancies. Since about 50% of patients receiving CD19-targeted CAR-T therapy experience drug resistance relapse, and the main mechanism is immune escape caused by antigen loss, the application of CD22-targeted CAR-T cells may be beneficial to CD19-targeted CAR-T cells. Patients who relapsed after T therapy were effective. In a clinical trial of B-ALL patients treated with CD22-specific CAR-T cells, the complete response rate was >70%, including a large number of patients who relapsed after CD19-targeted CAR-T therapy.

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2.2 MM

B cell maturation protein (BCMA) is a member of the TNF superfamily, expressed in some mature B cells, plasma cells and almost all MM cells. BCMA-targeted CAR-T cells showed satisfactory complete response rates in MM treatment, but patients were prone to relapse within 12 months after treatment. Ciltacabtagene Autoleucel (cilta-cel) is a CAR-T cell expressing dual BCMA epitopes, containing a co-stimulatory domain derived from 4-1BB and a CD3ζ intracellular signaling domain. Studies have shown that a single infusion of cilta-cel can produce deep and durable responses in patients with relapsed/refractory MM, with a complete response rate of 67% within 1 month of infusion and 62% within 3 months. In addition, other potential targets for the treatment of MM include SLAM7, CD38, CD138, immunoglobulin kappa light chain and GPRC5D, etc. These targets have also shown initial efficacy in clinical trials.

2.3 Hodgkin lymphoma

CD30 is a member of the TNF receptor superfamily, ubiquitously expressed in Reed-Sternberg cells, and is a safe and effective target for the treatment of relapsed/refractory Hodgkin lymphoma. In a phase I clinical trial, CD30-targeting CAR-T cells demonstrated significant antitumor activity and low toxicity in patients with relapsed/refractory Hodgkin lymphoma pretreated with high-intensity lymphodepletion.

2.4 T/NK cell malignancies

The CAR-T research on T/NK cell lymphoma is still in the early stage, and the development is relatively slow. The main limitation is that it is difficult to find suitable targets to avoid the "cannibalism" of CAR-T cells. The main targets currently in clinical research are CD5 and CD7. Among them, CD5-targeted CAR-T cells avoid "cannibalism" by downregulating the expression of endogenous CD5. In clinical trials, peripheral T cell lymphoma (PTCL) and acute T lymphocytic leukemia (T Cell acute lymphoblastic leukemia, T-ALL)) patients are effective. CD7-targeted CAR-T cells cannot significantly downregulate their own CD7 expression, but CD7-targeted gene disruption can make CAR-T cells resistant to "cannibalism" without affecting antigen recognition, thus confirming CD7-targeted therapy feasibility. In a clinical trial of donor-derived CD7-targeting CAR-T cells, CAR-T cells had higher remission rates and less toxicity in patients with relapsed/refractory T-ALL, although CD7-positive T-cell Cells and NK cells were rapidly cleared, but CD7-negative T cells and NK cells expanded dramatically and were effector to fungal and viral stimuli, suggesting that these cells were immunoprotective.

2.5 AML

AML cell surface antigens are shared with normal myeloid cell surface antigens, so direct targeting of AML antigens would have severe toxic side effects on normal myeloid cells, thereby limiting the therapeutic potential of CAR-T cells. A preclinical trial used gene-editing technology to transfer autologous CD33-knockout hematopoietic stem and progenitor cells into animal models established a hematopoietic system resistant to CD33-targeted therapy and enabled CD33-targeted CAR-T cells to Locate to AML. Although it is currently difficult to advance research into the clinic, the results of this trial provide a new way to solve the problem. Relevant research to find AML targets that are tolerated by normal tissues is also actively carried out.

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