CAR-T cell lmmunotherapy

Introduction of CAR-T technology

ChimericAntigen Receptor T-Cell immunotherapy (CAR-T) is one of the effective methods for the treatment of malignant tumors. The primary principle is to associate monoclonal antibody recognition site that specific against tumor antigens with the intracellular segments containing ITAM domain molecules. By replacing TCR with a sequence of antigen binding region of antibody, CAR-T cells can directly recognise natural proteins expressed on target cell surface, but don't need antigen processing, that breaks the restriction of the major histocompatibility complex (MHC) on T cell recognition antigen, finally leads to efficient killing effect of T cell on tumor cells. The advantage of CAR-T technology is the use of the patient's own immune cells to clear its own cancer cells, eventually, reach the therapeutic effect of cancer.

Structure of three generation of CAR molecules

The core part of the CAR-Tis CAR, which mainly includes: an extracellular antigen binding region, a transmem­ brane region and an intracellular signaling region.The CAR provides T cells with stronger ability to proliferate and resist apoptosis while acquiring target killing capability, and its tumor-killing effects are not limited by MHC.

immunotherapy based on CAR-T technology

Currently, the classic application field of CAR-T cell immunotherapy is in hematologic malignancies, for example, during the treatment of acute or chronic myeloid leukemia, the adoptive infusion of CAR-T cells is able to amplify in vivo (about  1000 times) and kilt the tumor cells effectively, eventually cure the tumor. In the meanwhile, the treatment has also made great progress in the study of solid tumors. For example, clinical trials for solid tumors such as melanoma have also achieved a significant effect.

Find ideal molecular targets for CAR-T cell immunotherapy

Although there have been pre-clinical trials of CAR-T cell immunotherapy for different targets in several cancers, but how to find the ideal tumor specific molecular target is significant for the development of CAR-T technology and its application in more tumors therapy. In this way, it can make CAR-T cell immunotherapy to be not only a cure of blood cancers, but also a treatmentof solid tumors. 

PD-1 fPD-L1
Programmed death 1 (PD-1) is an important immunosuppressive molecule,immune regulation with target of PD-1 plays an important role in anti-tumor, anti-infection, anti-autoimmune diseases and organ transplant surviva,l etc. Its ligand programmed death-ligand 1 (PD-L1) can also serve as a target.
 
lmmunotherapy of PD-1/PD·L1 has at!Jacted wide concern in various types of tumor therapy. In orde to support such a research, Cloud-Clone Corp. developed specific antibodies fol" PD-1 / PD-L 1. These antibodies were used for IHC test for the expression of P0-1and PD-L1 in human lymphoma and breast cancer tissues (Fig. 1 and 2). And they were further tested with knock-out cells model.

Fig.l lHC detection of P0-1 expression in lymphoma(lefl) andbreas t cancer( right)  Fig.2IHCdet&etlonofPO-L1 expressloninlymphoma(lefl} andbreast cancer(right} .

Combined with the lentiviral system and CRISPR/Cas9 technology, PD-1 and PD-L1 gene editing sites were located in Jurkat cells and MB231 cells, respectively. Then, the specific gene sites were knocked-out to obtain the knock-out cell lines (gene sequencing is shown in Fig. 3). After that, the specificity of anti PD-1 and PD-L1 antibodies were verified by western blot with normal and knock-out cells (Fig.4).

TIM -3
TIM-3 is a kind of immune checkpoint molecule, participate in regulating the immune function of the organism, and plays an important role in the anti-tumor immune response. lt"s mainly used in the treatment of carcinoma of the prostate and gynecologic neoplasm.
 SDF1
SOF1 and its receptor CXCR4 are a pair of important chemokine receptors, they can promote angiogenesis and maintain the growth of tumor cells, SOF1/CXCR4, as a target for treating tumors, can serve as a therapeutic strategy to block tumor formation
CD19
The specificity of C019 expression in B lymphocytes and its wide range expression in malignant tumors, make it become a potential molec­ular target for immunotherapy of B lymphocyte malignant tumors, Targeted immunotherapy with C019 has achieved encouraging therapeutic results in leukemia and lymphoma, effectively promotes the development of targeted immunotherapy.
CD13 7
Studies have shown that C0137 monoclonal antibody can realize the activation of antigen-specific memory T cells, organize tumor metas­ tasis and recurrence after operation. it plays an important role in the immune surveillance of tumor
CD138
CD138 can be used as a specific indicator of plasma cell dyscrasia, and accurately reflect the severity of malignant plasma cell dyscra-
CEA
CEA is a broad-spectrum tumor marker. it can reflect the existence of a variety of tumors, the curative effect judgment of colorectal cancer, breast cancer, and lung cancer, illness development, monitoring, and prognosis, it is a good tumor marker.
XP01
XP01 is a specific transporter of most tumor suppressor proteins, which can transport these proteins to the extracellular to promote the occurrence of tumors. The use of XP01 antagonists can keep the tumor suppressor proteins stay within the nucleus.