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Engineered immune cell capable of inducing secretion of anti-CD47 antibody
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An immune cell and engineering technology, applied in the field of tumor immune cell therapy, can solve problems such as off-target toxicity and side effects
Inactive Publication Date: 2019-04-02
GRACELL BIOTECH SHANGHAI CO LTD
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However, since CD47 is generally expressed in normal tissues, systemic infusion of antibodies will bring many off-target side effects, such as anemia, neurotoxicity, etc.
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Embodiment 1
[0216] Example 1 CAR structure and iCD47scFv structure design and transduction
[0217] 1.1 CAR structure design targeting CD19 (CD19CAR for short)
[0218] CD19CAR structure: use the second-generation CD19 CARs, including the scFv from FMC63, the hinge and transmembrane region from CD28, the intracellular segment is CD28 and CD3ζ, the structural diagram is as follows figure 1 Shown, the amino acid sequence is shown in SEQ ID NO.:1.
[0231]Three days after activation, the isolated and purified primary T cells were infected with lentiviral expression vectors containing CD19-CAR and CD19-CAR-iCD47scFv, transferred to cell culture flasks, and placed at 37°C, 5% CO 2 cultured in a constant temperature incubator. On the 3rd and 7th day after infection, Protein L (Thermo Fisher Scientific) was used to detect the CAR positive rate of T cells, and half of the medium was replaced every 2-3 days. The CAR-T cells obtained after culture were CD19 CAR-T cells and iCD47scFv-CD19 CAR-T cells.
Embodiment 3
[0232] Example 3 CAR-T cytokine release detection
[0233] CD19 CAR-T cells and iCD47scFv-CD19 CAR-T cells obtained in Example 2 were combined with tumor cells (CD19 + K562) were mixed at a ratio of 1:1, and the control group was CD19 CAR-T cells and iCD47scFv-CD19 CAR-T cells were mixed with K562 cells at a ratio of 1:1. 6 / ml, 100ul each of CAR-T cells and tumor cells were placed in a 96-well plate, co-cultured overnight, the supernatant was collected, centrifuged and the supernatant was taken to detect the release levels of cytokines IFN-γ, IL-2 and CD47 antibodies, Elisa kit (Biolegend) was used for detection.
[0234] The results showed that the release of IFN-γ and IL-2 in CD19 CAR-T cells and iCD47scFv-CD19 CAR-T cell groups were significantly higher than those in the control group. The expression of CD47 scFv could only be detected in the experimental group activated by iCD47scFv-CD19 CAR-T cells.
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Abstract
The invention relates to engineered immune cell capable of inducing secretion of an anti-CD47 antibody. In particular, the present invention relates to an immune cell (such as a CAR-T cell) that induces secretion of the anti-CD47 antibody when CAR and / or exogenous TCR can be activated. The immune cell expresses the anti-CD47 antibody only when CAR and / or exogenous TCR can be activated, and is specifically secreted only in the tumor microenvironment, and the toxic and side effects are small. The immune cell of the invention can induce secretion of the anti-CD47 antibody, relieve the inhibitionof macrophages by CD47-positive tumor cells, reversely promote macrophages to attack tumor cells, and cooperate with CAR and / or exogenous TCR, the tumor cell killing effect is significantly enhanced,tumor cells and CD47-positive tumor cells expressing the CAR and / or exogenous TCR-targeted antigen can be simultaneously killed, tumor cells can be prevented from immune escape, and the immune cell isdifficult to target and relapse.
Description
technical field [0001] The invention belongs to the field of tumor immune cell therapy, and in particular relates to an engineered immune cell capable of inducing to secrete anti-CD47 antibody. Background technique [0002] Cellular immunotherapy is an emerging tumor treatment mode with significant curative effect, and it is a new type of autoimmune anti-cancer treatment. It is a method of using biotechnology and biological agents to culture and expand immune cells collected from patients in vitro and then infuse them back into the patient's body to stimulate and enhance the body's autoimmune function, so as to achieve the purpose of treating tumors. [0003] In recent years, chimeric antigen receptor gene-modified T (CAR-T) cells, as "living drugs", have achieved exciting results in the treatment of hematological tumors, and have become a new development direction for tumor treatment. The design of CAR has gone through the following process: the first-generation CAR has on...
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