The principle of CAR-T therapy is to use genetic engineering technology to extract a patient's own T cells and modify them to express a protein called chimeric antigen receptor (CAR). This CAR protein recognizes and binds to antigens on the surface of tumor cells, activating T cells to attack tumor cells. Specifically, CAR-T therapy involves the following steps: Collection of T cells: First, the doctor will collect a certain amount of T cells from the patient. Genetic modification: In the lab, doctors introduce a gene called a chimeric antigen receptor (CAR) into T cells to make them express the CAR protein. This process is usually implemented using viral vectors. Culture CAR-T cells: After genetic modification, doctors will grow T cells for several days to increase their numbers. Injection of CAR-T cells: Finally, the doctor will re-inject the cultured CAR-T cells into the patient. These CAR-T cells enter the patient's circulatory system and begin attacking tumor cells. Because CAR-T cells only recognize and attack tumor cells without causing harm to normal cells, the therapy has a higher therapeutic effect and fewer side effects.
Research Progress
CAR-T therapy was first proposed by American scientists in the 1980s. At that time, researchers discovered that introducing a protein called a chimeric antigen receptor (CAR) into T cells through genetic engineering could enable T cells to attack cancer cells without harming normal cells. After making some progress in the laboratory, CAR-T therapy entered the clinical trial phase. Early clinical trials were mainly conducted on patients with hematologic tumors, and certain curative effects were achieved. In 2017, the US FDA approved Kymriah, the first CAR-T therapy drug, for the treatment of acute lymphoblastic leukemia in children and young adults. Since then, a number of CAR-T therapy drugs have been approved by the FDA. With the continuous advancement of technology, the scope of application of CAR-T therapy is also expanding. At present, the therapy has been used in the treatment of many types of cancer, such as lymphoma, myeloma, lung cancer and so on.
Advantages of CAR-T Therapy
Highly targeted: CAR-T therapy is an individualized treatment method that uses the patient's own T cells to treat, avoiding the problems of rejection and immunosuppression that exist in allotransplantation. At the same time, CAR-T cells only attack tumor cells without causing harm to normal cells, so they have a higher therapeutic effect and fewer side effects. Long-lasting therapeutic effect: CAR-T cells can exist in the patient's body for a long time, so their therapeutic effect is relatively long-lasting. Some FDA-approved CAR-T therapy drugs, such as Kymriah and Yescarta, have therapeutic effects that can last for months or even years. Wide range of applications: The application of CAR-T therapy is constantly expanding. At present, it has been used in the treatment of many types of cancer, including blood tumors and solid tumors. With the continuous advancement of technology, CAR-T therapy is expected to become an important cancer treatment. New treatment means: CAR-T therapy is a new cancer treatment method, and its appearance has brought new hope for cancer treatment. Compared with traditional radiotherapy, chemotherapy and other treatment methods, CAR-T therapy has higher therapeutic effect and fewer side effects, and is expected to become one of the main means of cancer treatment in the future.
Evaluation Means
Creative BioMart offers a variety of pre-fluorescent-conjugated (R-PE Labeled Proteins and APC-labeled Proteins) and pre-biotinylated recombinant proteins, which are convenient for cell separation.
Fluorescently linked recombinant proteins can be used to track the distribution and behavior of CAR-T cells in the body. By binding fluorescent proteins to specific proteins or receptors on the surface of CAR T cells, visualization of CAR T cells can be achieved. This helps doctors and researchers monitor the status of CAR T cells in the body in real time, including their number, activity and distribution. In addition, the use of fluorescence-linked recombinant proteins can also evaluate the therapeutic effect of CAR T cells, for example by detecting the degree of infiltration of CAR T cells in tumor tissue.
Prebiotinized recombinant proteins can be used to optimize the therapeutic efficacy of CAR-T cells. Prebiotin is a protein that can bind to biotin, and it can be used to bind CAR T cells to biotin-labeled molecules or antibodies. In this way, positive or negative regulation of CAR T cells can be achieved to optimize their therapeutic effect. For example, CAR-T cells can be bound to specific antigens on the surface of tumor cells through biotin-avidin interaction, thereby enhancing the ability of CAR-T cells to attack tumor cells; Alternatively, CAR T cells can be bound to inhibitory molecules through biotin-avidin interactions, thereby inhibiting their ability to attack tumor cells.
Applications
Specific applications of fluorescentially linked and prebiotinized recombinant proteins in CAR-T therapy include:
Monitoring the status and behavior of CAR-T cells: Visualization of CAR-T cells can be achieved by binding fluorescent proteins to specific proteins or receptors on the surface of CAR-T cells. This helps doctors and researchers monitor the status of CAR T cells in the body in real time, including their number, activity and distribution. For example, fluorescently labeled anti-CD3 antibodies can be used to monitor the activity and function of CAR T cells.
Evaluating the efficacy of CAR-T cells: The efficacy of CAR-T cells can be evaluated by binding fluorescent proteins to specific antigens on the surface of tumor cells. For example, fluorescently labeled anti-PD-L1 antibodies can be used to evaluate the killing effect of CAR T cells on tumor cells.
Optimization of therapeutic strategies for CAR-T cells: By binding pre-biotinized molecules or antibodies to CAR-T cells, positive or negative regulation of CAR-T cells can be achieved to optimize their therapeutic efficacy. For example, biotin-avidin interactions can be used to bind CAR-T cells to specific antigens on the surface of tumor cells, thereby enhancing the ability of CAR-T cells to attack tumor cells; Or biotin-avidin interactions could be used to bind CAR-T cells to inhibitory molecules, thereby inhibiting their ability to attack tumor cells.
Study the biological characteristics of CAR-T cells: By combining fluorescent proteins or prebiotinized molecules with CAR-T cells, the biological characteristics of CAR-T cells can be studied, such as their activation, proliferation, apoptosis and other processes. This contributes to a deeper understanding of the mechanism of action of CAR T cells and provides a theoretical basis for optimizing CAR T cell therapy.
Case Study
Case Study 1: Active Recombinant Human CD19
T cells expressing anti-CD19 chimeric antigen receptors (CARs) have activity against chronic lymphocytic leukemia (CLL), but complete response rates range from 18% to 29%, so improvement is needed. Peripheral blood mononuclear cells (PBMCs) of CLL patients often contain high levels of CLL cells that can interfere with CAR T cell production, and T cells from CLL patients are prone to exhaustion and other functional defects. The researchers previously developed an anti-CD19 CAR designated Hu19-CD828Z. Hu19-CD828Z has a binding domain derived from a fully human antibody and a CD28 costimulatory domain. They aimed to develop an optimized process for producing Hu19-CD828Z-expressing T cells (Hu19-CAR T) from PBMC of CLL patients. They determined that supplementing Hu19-CAR-T cultures with interleukin (IL)-7 + IL-15 had advantages over using IL-2, including greater accumulation of Hu19-CAR T cells during in vitro proliferation assays. They determined that positive selection with anti-CD4 and anti-CD8 magnetic beads was the optimal method of T cell purification because this method resulted in high T cell purity.
(Christina Amatya, 2024) Fig1. On day 7 of clinical manufacturing, Hu19-CAR T derived from CLL PBMC were cultured alone or co-cultured with CD19-positive (NALM6, Toledo, CD19-K562) or CD19-negative (NGFR-K562) target cells.
Case Study 2: Active Recombinant Human EGFR Protein
Epidermal growth factor receptor (EGFR) is overexpressed in various cancers, including non-small cell lung cancer (NSCLC), and in some somatic cells at a limited level, rendering it an attractive antitumor target. In this study, the researchers engineered chimeric antigen receptor (CAR)-T cells using the piggyBac transposon system, autologous artificial antigen-presenting cells, and natural ligands of EGFR. The researchers showed that this approach yielded CAR-T cells with favorable phenotypes and CAR positivity. They exhibited potent antitumor activity against NSCLC both in vitro and in vivo. When administered to tumor-bearing mice and non-tumor-bearing cynomolgus macaques, they did not elicit toxicity despite their cross-reactivity to both murine and simian EGFRs. In total they tested three ligands and found that the CAR candidate with the highest affinity consistently displayed greater potency without adverse events.
(Thanyavi Chinsuwan, 2023) Fig2. Anti-EGFR CAR administration was safe in lymphodepleted cynomolgus macaques at clinically relevant doses. The amount of C-reactive protein (CRP), ferritin, and selected pro-inflammatory cytokines.
Case Study 3: Active Recombinant Human CD33
Acute myeloid leukemia (AML) is a malignant disorder derived from neoplastic myeloid progenitor cells characterized by abnormal proliferation and differentiation. Although novel therapeutics have recently been introduced, AML remains a therapeutic challenge with insufficient cure rates. In the last years, immune-directed therapies such as chimeric antigen receptor (CAR)-T cells were introduced, which showed outstanding clinical activity against B-cell malignancies including acute lymphoblastic leukemia (ALL). However, the application of CAR-T cells appears to be challenging due to the enormous molecular heterogeneity of the disease and potential long-term suppression of hematopoiesis. Here the researchers report on the generation of CD33-targeted CAR-modified natural killer (NK) cells by transduction of blood-derived primary NK cells using baboon envelope pseudotyped lentiviral vectors (BaEV-LVs). Transduced cells displayed stable CAR-expression, unimpeded proliferation, and increased cytotoxic activity against CD33-positive OCI-AML2 and primary AML cells in vitro. Furthermore, CD33-CAR-NK cells strongly reduced leukemic burden and prevented bone marrow engraftment of leukemic cells in OCI-AML2 xenograft mouse models without observable side effects.
(Nawid Albinger, 2022) Fig3. NK cells equipped with a CD33-CAR become highly cytotoxic against CD33-positive primary AML cells.
Case Study 4: Recombinant Human GPC3 protein
Available evidence regarding the most suitable treatment strategies for hepatocellular carcinoma (HCC) with inferior vena cava tumor thrombus (IVCTT) is extremely limited, and the median overall survival time for these patients after liver resection is only 17.76 months. Other local or systemic treatments for HCC with IVCTT result in a median overall survival time ranging from 5.88 to 15.36 months. Thus, new therapeutic strategies are urgently needed to improve the survival of HCC patients with IVCTT. Chimeric antigen receptor (CAR) T‐cell therapy has seen success in treating B‐cell neoplasms with impressive outcomes. However, this therapy alone has shown limited efficacy on solid tumors, such as HCC. In this study, the researchers put forward a proof‐of‐concept treatment strategy that local therapy plus CAR‐glypican‐3 (CAR‐GPC3) T‐cell therapy might be effective for advanced HCC patients and reported the application of this combination in two GPC3‐positive HCC patients with rapidly progressing IVCTT. In brief, both patients received local therapy to treat liver lesions and IVCTT, followed by sequential infusions of CAR‐GPC3 T‐cells, and achieved more than 5‐year disease‐free survival and more than 8‐year overall survival.
(Yaoping Shi, 2023) Fig4. Dynamic changes in the retroperitoneal lymphatic metastasis after CAR-GPC3 T-cell infusions.
Advantages
Wide range of products: We offer up to 200 different label proteins such as fluorescent labeling and biotinylation.
Customized services: Since each patient's cancer is unique and different research needs are unique, we are able to customize CAR-T-related proteins for each project.
Strict quality control: The whole process of the production line has a high standard of production delivery supervision standards to ensure high quality products.
Ongoing support: In addition to providing products, we also provide ongoing technical support and services to ensure your satisfaction.
FAQ
Q: What fluorescent-labeled proteins can you provide?
A: We mainly provide common CAR-T-associated protein labeling with R-PE or APC for the labeling of proteins inside and outside cells.
Q: What is the difference between R-PE or APC markings?
A: The excitation wavelength of R-PE is usually 561nm, and the fluorescence color is red. The excitation wavelength of APC is 488nm, and the fluorescence color is between orange and red.
References
Amatya C.; et al. Optimization of anti-CD19 CAR T cell production for treatment of patients with chronic lymphocytic leukemia. Mol Ther Methods Clin Dev. 2024;32(1):101212.
Albinger N.; et al. Primary CD33-targeting CAR-NK cells for the treatment of acute myeloid leukemia. Blood Cancer J. 2022;12(4):61.
Contact us or send an email at for project quotations and
more detailed information.
