Chimeric signaling receptor targeting CD56 and cells for expressing the same

Abstract

The present invention belongs to the field of immune cell therapy technology, and specifically relates to a chimeric signal receptor for targeting leukocyte differentiation antigen 56 (CD56) and a cell for expressing the receptor. The structure of the chimeric signal receptor comprises a signal peptide, an extracellular antigen recognition region, an extracellular hinge region, a transmembrane region and an intracellular signal region; the chimeric signal receptor can specifically recognize the CD56 protein expressed by normal cells or tumor cells, and convert it into an activation signal inside the immune cells, participate in the proliferation and survival of immune cells, and enhance the activity of immune cells. The present invention also provides cells expressing the chimeric signal receptor. The cells of the present invention can be used as cell therapy drugs, and have good application prospects in the treatment of diseases such as tumors and autoimmune diseases.

Description

Technical Field

[0001] The invention belongs to the field of tumor immunotherapy, and specifically relates to a chimeric signal receptor targeting CD56, a cell expressing the signal receptor and an application thereof. Background Art

[0002] Cell therapy is considered one of the most promising methods of tumor immunotherapy. CAR-T cell therapy mainly transforms T cells through genetic engineering technology to express CAR and obtain specific and efficient killing activity. The full name of CAR is Chimeric Antigen Receptor. Its design for specific antigens on the surface of cancer cells is conducive to improving the specificity and effectiveness of anti-tumor immune response. It is widely used in immune cell therapy, especially the treatment strategy using T cells.

[0003] The structure of CAR can generally be divided into three parts: antigen recognition region, transmembrane region and intracellular signaling region. 1) Antigen recognition region: It is usually a single-chain antibody fragment (scFv), which is responsible for recognizing and binding tumor antigens. It has a high specificity for specific tumor antigens. 2) Transmembrane region: As a bridge between the antigen recognition domain and the signal transduction domain, it makes it possible to exchange information inside and outside the cell. 3) Intracellular signaling region: It usually includes CD3ζ chain and auxiliary stimulatory molecules (such as CD28, CD137, OX40, etc.). CD3ζ chain is involved in activating T-cell receptor (TCR) complex. Auxiliary stimulatory molecules help to fully activate T cells, improve their proliferation ability, prolong their survival, and enhance cytotoxic effects. According to the structure and complexity of the intracellular signaling region of CAR, CAR is divided into four generations. The intracellular signaling region of the first-generation CAR contains only CD3ζ chain, and one or more co-stimulatory signal molecules are added to the second and third generation CARs. Fourth-generation CARs (also known as TRUCKs) are also specifically designed to secrete immunomodulatory factors (such as IL-12) after antigen stimulation, with the main purpose of overcoming the suppressive immune microenvironment of the tumor.

[0004] The CD3ζ chain activates the downstream signaling pathways of T cells through the immunoglobulin ITAM (Immunoreceptor Tyrosine-based Activation Motif) in the cytoplasm. These signaling pathways include activation of protein tyrosine kinases, phosphorylation cascade reactions, etc., thereby triggering the activation and response of T cells. CAR-T design generally uses its intracellular segment, which contains three immunoreceptor tyrosine activation motifs (ITAM).

[0005] Immunoreceptor tyrosine activation motifs (ITAMs) play a key role in cell signal transduction. The ITAM domain is basically composed of: YXX (L / I). After the activated receptor binds to the corresponding ligand, its tyrosine is phosphorylated under the action of a class of protein tyrosine kinases PTK connected to the cell membrane, further recruiting other free protein kinases or adapter proteins in the cell to transmit activation signals into the cell, which is crucial to the signal transduction ability of TCR. For example, the intracellular tail of CD3ζ contains three ITAM motifs, and the intracellular tails of CD3γ, CD3ε and CD3δ molecules each contain an ITAM. CAR-T design generally uses the intracellular segment of CD3ζ.

[0006] CAR-T cell therapy is a revolutionary immunotherapy that has achieved remarkable success in the treatment of hematological malignancies such as acute lymphoblastic leukemia and non-Hodgkin's lymphoma. However, in the treatment of solid tumors, CAR-T cell therapy faces some challenges, including: 1. Local infiltration of tumor tissue: Cancer cells in solid tumors usually form a tight intratumoral environment, which limits the ability of CAR-T cells to infiltrate and contact cancer cells. CAR-T cells need to overcome this obstacle to effectively kill tumor cells. 2. Immunosuppression of the tumor microenvironment: There are many immunosuppressive factors in solid tumors, such as inhibitory cytokines and immune checkpoint molecules. These factors can inhibit the activity of CAR-T cells and limit their effects in solid tumors. 3. Lack of specific antigens: Compared with hematological tumors, solid tumors usually have fewer antigens, greater diversity, and differences between different patients. Therefore, it is a challenge to find suitable specific antigens for CAR-T cell therapy. 4. Side effects and toxicity: CAR-T cell therapy may cause serious side effects, such as cytokine release syndrome and neurotoxicity. In the treatment of solid tumors, because the intratumoral environment is more complex, the side effects and toxicity caused by treatment may be more prominent. 5. Durability of effect: The durability of CAR-T cell therapy in the treatment of solid tumors still needs to be improved. Due to the heterogeneity and complexity of solid tumors, CAR-T cells may not be able to completely eliminate all tumor cells, so there may be a risk of tumor recurrence and progression.

[0007] To overcome these challenges, scientists are actively conducting research and exploration. Some strategies include improving the design of CAR to improve its infiltration ability and persistence, developing CAR-T cells targeting multiple antigens, and combining CAR-T cell therapy with other treatments (such as immune checkpoint inhibitors) to enhance the therapeutic effect. In addition, understanding the intratumoral environment of solid tumors and its interaction with the immune system is also crucial to optimizing the application of CAR-T cell therapy in solid tumors.

[0008] CD56, also known as neuronal cell adhesion molecule (NCAM), is a glycoprotein that spans the cell membrane and plays an important role in cell-cell interactions and cell migration. CD56 is expressed on neurons, natural killer (NK) cells, some T cells, B cells and some tumor cells. In addition, CD56 is also often used as a cell surface marker and immune cell subtype identification. CD56 is encoded by five exons, and the polypeptide chains encoded by these exons are glycosylated and span the cell membrane. CD56 contains three complementary types (N-CAM, R-CAM, M-CAM); it has a wide N-terminal extracellular motif and a cell membrane anchoring region. The N-terminal extracellular motif contains glycosylated multivalent ligands, such as alpha-heparan sulfate, N-acetylceramide and heparin sulfate glucosamine ferric complex, which can participate in cell-to-cell adhesion through selective ligand binding. CD56 has diverse functions, mainly including: 1. Cell-to-cell adhesion: CD56 molecules contribute to the formation of neuronal networks through cell-to-cell adhesion. Neurons directly or indirectly participate in the formation of synaptic connections by expressing CD56, and promote sensory functions such as smell, vision, and hearing. In tumors, CD56 can promote cell-cell aggregation, migration, and invasion. 2. Immune surveillance: CD56 is also a marker for T cells and NK cells, and plays an important role in the immune surveillance process. NK cells express CD56 and kill activated tumor cells and virus-infected cells through directed cell killing. 3. Neural cell development and regeneration: CD56 also plays a key role in the development and regeneration of the nervous system. CD56 plays an important role in biological processes such as neuronal migration, differentiation and maturation of neural precursor cells, synapse formation, and neurotransmitter release. In short, CD56 is a transmembrane protein with a wide range of important functions. Its expression status, diversity, and changes in signal transduction pathways are closely related to the occurrence of many diseases.

[0009] Preclinical studies have shown that targeting CD56 can inhibit tumor growth 1 . However, there are many drawbacks to using CD56 as a target antigen for CAR-T cell therapy: 1. CAR-T cells targeting CD56 have a short survival time in vivo and require multiple infusions or the addition of IL-2 to fully exert their anti-tumor effects. 2. Non-tumor targeted toxicity. CD56 is expressed in neuronal cells, and direct targeting of CD56 can cause neuronal cell damage. In two studies, the drug-conjugated antibody IMGN901 reported some neuropathy and neurotoxicity 2,3 3. Antigen loss. Studies have found that CAR-T cells have a significant killing effect on tumor cells with high CD56 expression, but in low-expressing cells, some residual cell antigens will be lost after killing, leading to immune escape. Therefore, based on current research, CD56 is not an ideal target for CAR-T cell therapy. Summary of the invention

[0010] The technical problem to be solved by the present invention is: the persistent proliferation and survival of immune cells in peripheral circulation and tumor tissue.

[0011] The technical solution of the present invention to solve the above technical problems is to provide a chimeric signal receptor targeting leukocyte differentiation antigen 56 (CD56), characterized in that it comprises structural domains such as an extracellular antigen recognition region, an extracellular hinge region, a transmembrane region and an intracellular signal region; the extracellular antigen recognition region can recognize or bind to CD56; and further, the chimeric signal receptor does not contain an immunoreceptor tyrosine-based activation motif (ITAM); or, the intracellular signal region does not contain a CD3ζ chain.

[0012] Furthermore, the sequence of the immunoreceptor tyrosine-based activation motif (ITAM) in the chimeric signal receptor is YXX(L / I)X(6-8)YXX(L / I); wherein X is any amino acid. The extracellular antigen recognition region, the extracellular hinge region, the transmembrane region and the intracellular signal region are connected in the direction from the nitrogen end to the carbon end.

[0013] Furthermore, the nitrogen terminus of the chimeric signal receptor is also connected to a signal peptide.

[0014] Furthermore, the chimeric signal receptor satisfies at least one of the following 1) to 5):

[0015] 1) The signal peptide is at least one of the interleukin-2 signal peptide, leukocyte differentiation antigen-8 signal peptide, colony stimulating factor-2 receptor subunit α signal peptide, or a protein domain with a sequence homology of more than 90% with the aforementioned signal peptides;

[0016] 2) the extracellular antigen recognition region is at least one of a monoclonal antibody variable region or a heavy chain single domain antibody variable region that binds to CD56;

[0017] 3) The extracellular hinge region is a leukocyte differentiation antigen 8 extracellular hinge region, a leukocyte differentiation antigen 28 extracellular hinge region, a human immunoglobulin G1 (IgG1) hinge region, a human immunoglobulin G4 (IgG4) hinge region, a TRAIL extracellular hinge region, a leukocyte differentiation antigen 16 extracellular hinge region, a NKp30 extracellular hinge region, a NKG2C extracellular hinge region, a NKG2D extracellular hinge region, a 2B4 extracellular hinge region or a DNAM-1 extracellular hinge region; or at least one of the protein domains having a sequence homology of more than 90% with the foregoing extracellular hinge regions;

[0018] 4) the transmembrane region is at least one of the transmembrane region of leukocyte differentiation antigen 8, the transmembrane region of leukocyte differentiation antigen 28, the transmembrane region of interleukin 2 receptor β subunit, the transmembrane region of interleukin 7 receptor, the transmembrane region of TRAIL, the transmembrane region of leukocyte differentiation antigen 16, the transmembrane region of NKp30, the transmembrane region of NKG2C, the transmembrane region of NKG2D, the transmembrane region of 2B4 or the transmembrane region of DNAM-1, or a protein domain with a sequence homology of more than 90% with the aforementioned transmembrane region;

[0019] 5) The intracellular signaling region is co-stimulatory signaling region with leukocyte differentiation antigen 28, tumor necrosis factor receptor superfamily member 4, tumor necrosis factor receptor superfamily member 9, lymphocyte-specific protein tyrosine kinase, inducible T cell co-stimulatory molecule, leukocyte differentiation antigen 40 co-stimulatory signaling region, leukocyte differentiation antigen 27 co-stimulatory signaling region, DAP10 co-stimulatory signaling region, DAP12 co-stimulatory signaling region, TRAIL co-stimulatory signaling region, leukocyte differentiation antigen 16 co-stimulatory signaling region, NKp30 co-stimulatory signaling region, NKG2C co-stimulatory signaling region, NKG2D co-stimulatory signaling region, 2B4 co-stimulatory signaling region, DNAM-1 co-stimulatory signaling region, IL-2 receptor β subunit intracellular activation signaling region, IL-7 receptor intracellular signaling region; or at least one of the intracellular domains of protein domains having a sequence homology of more than 90% with the aforementioned intracellular signaling region.

[0020] The above-mentioned chimeric signal receptor further satisfies at least one of the following conditions:

[0021] 1) The signal peptide is a colony stimulating factor 2 receptor subunit α signal peptide; or a peptide having the same or similar function as the colony stimulating factor 2 receptor subunit α signal peptide obtained by substitution and / or deletion and / or addition of at least one amino acid;

[0022] Alternatively, the signal peptide is a leukocyte differentiation antigen 8 signal peptide, or a peptide having the same or similar function as the leukocyte differentiation antigen 8 signal peptide obtained by substitution and / or deletion and / or addition of at least one amino acid;

[0023] 2) The extracellular antigen recognition region is a monoclonal antibody variable region or a heavy chain single domain antibody variable region that recognizes CD56, or a peptide segment having the same or similar function as the monoclonal antibody variable region or the heavy chain single domain antibody variable region that recognizes CD56 obtained by substitution and / or deletion and / or addition of at least one amino acid;

[0024] 3) The extracellular hinge region is a human immunoglobulin G4 (IgG4) hinge region, or a peptide segment having the same or similar function as the human immunoglobulin G4 (IgG4) hinge region obtained by substitution and / or deletion and / or addition of at least one amino acid;

[0025] Or the extracellular hinge region is the leukocyte differentiation antigen 8 hinge region, or a peptide segment having the same or similar function as the leukocyte differentiation antigen 8 extracellular hinge region obtained by substitution and / or deletion and / or addition of at least one amino acid; 4) the transmembrane region is the leukocyte differentiation antigen 28 transmembrane region, or a peptide segment having the same or similar function as the leukocyte differentiation antigen 28 transmembrane region obtained by substitution and / or deletion and / or addition of at least one amino acid;

[0026] Alternatively, the transmembrane region is the leukocyte differentiation antigen 8 transmembrane region, or a peptide segment having the same or similar function as the leukocyte differentiation antigen 8 transmembrane region obtained by substitution and / or deletion and / or addition of at least one amino acid;

[0027] 5) The intracellular signal region is the intracellular domain of leukocyte differentiation antigen 28, or a peptide segment having the same or similar function as the intracellular domain of leukocyte differentiation antigen 28 obtained by substitution and / or deletion and / or addition of at least one amino acid;

[0028] Alternatively, the intracellular signal region is the intracellular co-stimulatory signal region of CD137 (also known as 4-1BB), or a peptide segment having the same or similar function as the intracellular co-stimulatory signal region of CD137 obtained by substitution and / or deletion and / or addition of at least one amino acid;

[0029] Alternatively, the intracellular signaling region is the intracellular activation domain of the interleukin-2 receptor β subunit, or a peptide segment having the same or similar function as the intracellular activation domain of the interleukin-2 receptor β subunit obtained by substitution and / or deletion and / or addition of at least one amino acid.

[0030] Furthermore, the amino acid sequence of the signal receptor of the present invention is shown in at least one of SEQ ID NO.11, SEQ ID NO.21 or SEQ ID NO.25.

[0031] The present invention also provides a nucleotide sequence encoding the above signal receptor. Further, the nucleotide sequence encoding the above signal receptor is shown in at least one of SEQ ID NO.12, SEQ ID NO.22 or SEQ ID NO.26.

[0032] The present invention also provides an expression vector capable of expressing the above-mentioned signal receptor.

[0033] Wherein, the above-mentioned expression vector is a recombinant plasmid vector, a recombinant virus vector or a combination thereof.

[0034] Furthermore, the recombinant virus is a lentiviral vector or a retroviral vector.

[0035] The present invention also provides a cell for treating tumors or autoimmune diseases. The cell for treating tumors or autoimmune diseases is a host cell containing the above expression vector.

[0036] Among them, the host cell described in the cells for treating tumors or autoimmune diseases is an immune cell. Further, the immune cell is at least one of a T cell, a tumor infiltrating lymphocyte or a natural killer cell. Further, the host cell is at least one of an αβT cell, a NKT cell, a γδT cell, a TCR-modified T cell or a chimeric antigen receptor T cell.

[0037] The present invention provides an expression vector capable of expressing the above-mentioned signal receptor, or use of a host cell containing the above-mentioned expression vector in preparing a drug for treating tumors or autoimmune diseases.

[0038] The present invention also provides a drug for treating tumors or autoimmune diseases, which is prepared by using the above-mentioned cells for targeted treatment of tumors or autoimmune diseases as active ingredients and pharmaceutically acceptable excipients or auxiliary ingredients.

[0039] Among them, the tumors described in the above-mentioned drugs for treating tumors or autoimmune diseases are small cell lung cancer, pancreatic endocrine tumors, medullary thyroid carcinoma, neuroendocrine tumors, malignant melanoma, leukemia, lymphoma, multiple myeloma, neuroblastoma, sarcoma, nephroblastoma, etc.; the autoimmune diseases described are rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), scleroderma, etc.

[0040] The beneficial effects of the CD56 chimeric signal receptor of the present invention are:

[0041] 1. Since some peripheral circulating immune cells express CD56, immune cells such as T cells modified with the chimeric signal receptor targeting CD56 of the present invention receive a large number of activation and proliferation signals in the peripheral blood after being infused back into the body, and can survive and proliferate in the body for a long time without quickly failing.

[0042] 2. Malignant tumors such as small cell lung cancer, lymphoma, neuroendocrine tumors, malignant melanoma, etc., whose tumor cells express CD56 antigen. After a small amount of immune cells such as T cells modified by the CD56 chimeric signal receptor of the present invention arrive at the tumor site, the CD56 on the surface of the tumor cells can cause specific T cells to proliferate locally, which can produce a significant anti-tumor effect.

[0043] 3. The CD56 chimeric signal receptor of the present invention will not directly kill CD56 positive cells, and has no cytotoxic effect on neurons, T and B cells expressing CD56, etc., and there is no "on target, off tumor" phenomenon. While exerting the function of anti-tumor and treatment of autoimmune diseases, it also effectively avoids damage to normal tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 , are schematic diagrams of the structures of three types of chimeric signal receptors prepared in the examples of the present invention.

[0045] Figure 2 , after T cells were infected with lentivirus containing the signal receptor A, signal receptor B and signal receptor C genes, the expression of receptors was detected by flow cytometry (the proteins of each signal receptor contain the G4S amino acid sequence). A, the expression of each signal receptor protein was detected by anti-G4S antibody; B, the expression of CD56 in lung cancer cell lines SHP77 and H446; C, the expression of DLL3 protein in lung cancer cell line SHP77.

[0046] Figure 3 , are the results of the T cell proliferation and survival test expressing the signal receptor. A, is the flow cytometry detection of the proliferation changes of CFSE-labeled T cells; B, is the T cell count analysis of the signal receptor gene modified.

[0047] Figure 4 , are the results of the in vitro killing ability test of T cells modified with signal receptors. A, are the in vitro killing effects of T cells modified with receptors on lung cancer cell line SHP77; B, are the levels of IFN-γ release.

[0048] Figure 5 , are the experimental results of the in vivo and in vitro killing ability of CAR-T cells after receptor modification. A, is the result of the determination of IFN-γ secretion; B, is the result of flow cytometry detection of tumor cell count; C, is the tumor volume after different modified CAR-T cells were used to treat the subcutaneous model of human SHP77 small cell lung cancer. DETAILED DESCRIPTION

[0049] As mentioned above, the current cell therapy still has many shortcomings, and CD56 is not an ideal CAR-T cell therapy target. In order to solve the above problems, the present invention creatively proposes a new chimeric signal receptor.

[0050] The present invention takes full account of the expression characteristics of CD56 (expressed on natural killer (NK) cells, certain T cells, B cells and certain tumor cells), and believes that it can be used as a stimulating signal for the proliferation and survival of immune cells in the body. Based on this, the present invention boldly designs a new type of chimeric signal receptor, using the CD56 antigen as an activation protein for the continuous proliferation and survival of immune cells in peripheral circulation and tumor tissues, thereby obtaining a stronger immune effect, without a domain that activates the killing effect of immune cells.

[0051] The chimeric signal receptor is a chimeric signal receptor targeting leukocyte differentiation antigen 56 (CD56), and its main structure includes an extracellular antigen recognition region, an extracellular hinge region, a transmembrane region and an intracellular signal region; the extracellular antigen recognition region can recognize or bind to CD56; and most importantly, the chimeric signal receptor does not contain an immunoreceptor tyrosine-based activation motif (ITAM). Or the intracellular signal region does not contain a CD3ζ chain.

[0052] Since the chimeric signal receptor of the present invention is a chimeric signal receptor targeting leukocyte differentiation antigen 56 (CD56), it can recognize or bind to CD56 through the extracellular antigen recognition region, thereby causing the activation and proliferation of immune cells such as T cells and NK cells, thereby achieving the expansion and long-term survival of immune cells.

[0053] The chimeric signal receptor does not contain the immune receptor tyrosine activation motif (ITAM), so it mainly triggers immune cell activation, proliferation, and survival effects. The traditional CAR mainly triggers immune cell activation, proliferation, killing and other effects. In other words, the chimeric signal receptor of the present invention does not have the function of triggering immune cell killing effects, and is essentially different from the traditional CAR in function. The advantage of this design is that it maintains the proliferation and survival of immune cells without direct damage to related tissue cells.

[0054] Therefore, the chimeric signal receptor of the present invention is a new technical solution that is different from traditional CAR and can overcome the shortcomings of traditional cell therapy.

[0055] The extracellular antigen recognition region in the chimeric signal receptor targeting leukocyte differentiation antigen 56 (CD56) of the present invention can recognize or bind to CD56, and can specifically be at least one of a monoclonal antibody variable region or a heavy chain single domain antibody (sdAb) variable region that can bind to CD56.

[0056] The extracellular hinge region, transmembrane region and intracellular signal region of the chimeric signal receptor targeting leukocyte differentiation antigen 56 (CD56) of the present invention can be selected from related components commonly used in the art. The directions of the above-mentioned domains such as the extracellular antigen recognition region, the extracellular hinge region, the transmembrane region and the intracellular signal region are connected in the direction from the nitrogen end to the carbon end. When a signal peptide is used, the signal peptide is connected to the nitrogen end of the chimeric signal receptor.

[0057] For example, the extracellular hinge region can be selected from the group consisting of the leukocyte differentiation antigen 8 extracellular hinge region, the leukocyte differentiation antigen 28 extracellular hinge region, the human immunoglobulin G1 (IgG1) hinge region, the human immunoglobulin G4 (IgG4) hinge region, the TRAIL extracellular hinge region, the leukocyte differentiation antigen 16 extracellular hinge region, the NKp30 extracellular hinge region, the NKG2C extracellular hinge region, the NKG2D extracellular hinge region, the 2B4 extracellular hinge region or the DNAM-1 extracellular hinge region, or at least one of the protein domains having a homology of more than 90% with these hinges;

[0058] For example, the transmembrane region can be selected from the group consisting of leukocyte differentiation antigen 8 transmembrane region, leukocyte differentiation antigen 28 transmembrane region, interleukin 2 receptor β subunit transmembrane region, interleukin 7 receptor transmembrane region, TRAIL transmembrane region, leukocyte differentiation antigen 16 transmembrane region, NKp30 transmembrane region, NKG2C transmembrane region, NKG2D transmembrane region, 2B4 transmembrane region or DNAM-1 transmembrane region, or at least one of the protein domains having a sequence homology of more than 90% with these transmembrane regions;

[0059] For example, the intracellular signaling region can be selected from leukocyte differentiation antigen 28, tumor necrosis factor receptor superfamily member 4, tumor necrosis factor receptor superfamily member 9, lymphocyte-specific protein tyrosine kinase, inducible T cell co-stimulatory molecule, leukocyte differentiation antigen 40 co-stimulatory signaling region, leukocyte differentiation antigen 27 co-stimulatory signaling region, DAP10 co-stimulatory signaling region, DAP12 co-stimulatory signaling region, TRAIL co-stimulatory signaling region, leukocyte differentiation antigen 16 co-stimulatory signaling region, NKp30 co-stimulatory signaling region, NKG2C co-stimulatory signaling region, NKG2D co-stimulatory signaling region, 2B4 co-stimulatory signaling region, DNAM-1 co-stimulatory signaling region, IL-2 receptor β subunit intracellular activation signaling region, IL-7 receptor intracellular signaling region, or at least one of the protein domains having a sequence homology of more than 90% with these intracellular signaling regions.

[0060] The intracellular signal region can be selected from one or more of the above-mentioned ones for use in combination. For example, the leukocyte differentiation antigen 28 co-stimulatory signal region and the CD137 intracellular co-stimulatory signal region can be used together. Furthermore, it can also be equipped with a special design that will secrete immune regulatory factors (such as IL-12) after being stimulated by antigens, which can overcome the inhibitory immune microenvironment of the tumor.

[0061] When constructing a chimeric signal receptor expressing the above-mentioned targeting CD56, it is generally necessary to design a coding sequence of a signal peptide at the 5' end of the coding sequence encoding the above-mentioned chimeric signal receptor in the vector, so that the chimeric signal receptor targeting CD56 of the present invention can reach the surface of the host cell after being expressed in the host cell and anchor to the cell membrane of the host cell through the transmembrane region.

[0062] The signal peptide is at least one of the interleukin 2 signal peptide, leukocyte differentiation antigen 8 signal peptide, colony stimulating factor 2 receptor subunit α signal peptide or a protein domain having a sequence homology of more than 90% with the above signal peptides.

[0063] Specifically, among the above available components:

[0064] The amino acid sequence of the colony stimulating factor 2 receptor subunit α signal peptide is shown in SEQ ID NO. 1. The encoding nucleotide sequence of the colony stimulating factor 2 receptor subunit α signal peptide is shown in SEQ ID NO. 2.

[0065] The amino acid sequence of the variable region of the monoclonal antibody that recognizes CD56 is shown in SEQ ID No. 3. The nucleotide sequence encoding the variable region of the monoclonal antibody that recognizes CD56 is shown in SEQ ID No. 4.

[0066] The amino acid sequence of the hinge region of human immunoglobulin G4 is shown in SEQ ID No. 5. The nucleotide sequence encoding the hinge region of human immunoglobulin G4 is shown in SEQ ID No. 6.

[0067] The amino acid sequence of the transmembrane region of leukocyte differentiation antigen 28 is shown in SEQ ID No. 7. The nucleotide sequence encoding the transmembrane region of leukocyte differentiation antigen 28 is shown in SEQ ID No. 8.

[0068] The amino acid sequence of the intracellular domain of the leukocyte differentiation antigen 28 is shown in SEQ ID No. 9. The nucleotide sequence encoding the intracellular domain of the leukocyte differentiation antigen 28 is shown in SEQ ID No. 10.

[0069] The amino acid sequence of the leukocyte differentiation antigen 8 signal peptide is shown in SEQ ID NO. 13. The encoding nucleotide sequence of the leukocyte differentiation antigen 8 signal peptide is shown in SEQ ID NO.14.

[0070] The amino acid sequence of the interleukin 2 signal peptide is shown in SEQ ID NO. 15. The encoding nucleotide sequence of the interleukin 2 signal peptide is shown in SEQ ID NO.16.

[0071] The amino acid sequence of the transmembrane region of leukocyte differentiation antigen 8 is shown in SEQ ID NO. 17. The encoding nucleotide sequence of the transmembrane region of leukocyte differentiation antigen 8 is shown in SEQ ID NO.18.

[0072] The amino acid sequence of the intracellular co-stimulatory signal region of CD137 is shown in SEQ ID NO. 19. The encoding nucleotide sequence of the intracellular co-stimulatory signal region of CD137 is shown in SEQ ID NO. 20.

[0073] The amino acid sequence of the leukocyte differentiation antigen 8 extracellular hinge region is shown in SEQ ID NO. 27. The nucleotide sequence encoding the leukocyte differentiation antigen 8 extracellular hinge region is shown in SEQ ID NO. 28.

[0074] The amino acid sequence of the intracellular activation region of the interleukin 2 receptor β subunit is shown in SEQ ID NO. 29. The encoding nucleotide sequence of the intracellular activation region of the interleukin 2 receptor β subunit is shown in SEQ ID NO. 30.

[0075] The amino acid sequence of the intracellular activation region of the interleukin-7 receptor is shown in SEQ ID NO. 23. The encoding nucleotide sequence of the intracellular activation region of the interleukin-7 receptor is shown in SEQ ID NO.24.

[0076] According to the above description, those skilled in the art can understand that the various components in the chimeric signal receptor targeting CD56 of the present invention can be selected from various alternative specific components for combination. That is, different types of specific signal peptides, extracellular antigen recognition regions (recognizing CD56), extracellular hinge regions, transmembrane regions, and intracellular signal regions can be combined arbitrarily to obtain the chimeric signal receptor of the present invention. Further, the signal peptide, extracellular antigen recognition region, extracellular hinge region, transmembrane region, and intracellular signal region and other domains are connected in the direction from the nitrogen end to the carbon end. Furthermore, the above-mentioned domains are directly connected.

[0077] On the basis of the above, the present invention further provides three specific configurations of chimeric signal receptors (see Figure 1 ).

[0078] Composition method 1:

[0079] The signal peptide is a colony stimulating factor 2 receptor α subunit signal peptide, or a peptide having the same or similar function as the colony stimulating factor 2 receptor α subunit signal peptide obtained by substitution and / or deletion and / or addition of at least one amino acid.

[0080] The extracellular antigen recognition region is a monoclonal antibody variable region or a heavy chain single domain antibody variable region that recognizes CD56, or a protein having the same or similar function as the monoclonal antibody variable region or the heavy chain single domain antibody variable region that recognizes CD56 obtained by substitution and / or deletion and / or addition of at least one amino acid.

[0081] The extracellular hinge region is a human immunoglobulin G4 (IgG4) hinge region, or a protein having the same or similar function as the human immunoglobulin G4 (IgG4) hinge region obtained by substitution and / or deletion and / or addition of at least one amino acid.

[0082] The transmembrane region is the leukocyte differentiation antigen 28 transmembrane region, or a protein having the same or similar function as the leukocyte differentiation antigen 28 transmembrane region obtained by substitution and / or deletion and / or addition of at least one amino acid.

[0083] The intracellular signal region is the intracellular domain of leukocyte differentiation antigen 28, or a protein having the same or similar function as the intracellular domain of leukocyte differentiation antigen 28 obtained by substitution and / or deletion and / or addition of at least one amino acid.

[0084] Composition method 2:

[0085] The signal peptide is the leukocyte differentiation antigen 8 signal peptide, or a peptide having the same or similar function as the leukocyte differentiation antigen 8 signal peptide obtained by substitution and / or deletion and / or addition of at least one amino acid.

[0086] The extracellular antigen recognition region is a monoclonal antibody variable region or a heavy chain single domain antibody variable region that recognizes CD56, or a protein having the same or similar function as a monoclonal antibody single chain variable region or a heavy chain single domain antibody variable region that recognizes CD56 obtained by substitution and / or deletion and / or addition of at least one amino acid.

[0087] The extracellular hinge region is the leukocyte differentiation antigen 8 hinge region, or a protein having the same or similar function as the leukocyte differentiation antigen 8 extracellular hinge region obtained by substitution and / or deletion and / or addition of at least one amino acid.

[0088] The transmembrane region is the leukocyte differentiation antigen 8 transmembrane region, or a protein having the same or similar function as the leukocyte differentiation antigen 8 transmembrane region obtained by substitution and / or deletion and / or addition of at least one amino acid.

[0089] The intracellular co-stimulatory signal region is the intracellular co-stimulatory signal region of CD137, or a protein having the same or similar function as the intracellular co-stimulatory signal region of CD137 obtained by substitution and / or deletion and / or addition of at least one amino acid.

[0090] Composition method three:

[0091] The signal peptide is the leukocyte differentiation antigen 8 signal peptide, or a peptide having the same or similar function as the leukocyte differentiation antigen 8 signal peptide obtained by substitution and / or deletion and / or addition of at least one amino acid.

[0092] The extracellular antigen recognition region is a monoclonal antibody single chain variable region or a heavy chain single domain antibody variable region that recognizes CD56, or a protein having the same or similar function as the monoclonal antibody variable region or the heavy chain single domain antibody variable region that recognizes CD56 obtained by substitution and / or deletion and / or addition of at least one amino acid;

[0093] The extracellular hinge region is a leukocyte differentiation antigen 8 hinge region, or is substituted and / or deleted and / or

[0094] or a protein having the same or similar function as the extracellular hinge region of leukocyte differentiation antigen 8 obtained by adding at least one amino acid;

[0095] The transmembrane region is the transmembrane region of leukocyte differentiation antigen 8, or a protein having the same or similar function as the transmembrane region of leukocyte differentiation antigen 8 obtained by substitution and / or deletion and / or addition of at least one amino acid;

[0096] The intracellular signal region is the intracellular activation domain of the interleukin 2 receptor β subunit, or a protein having the same or similar function as the intracellular activation domain of the interleukin 2 receptor β subunit obtained by substitution and / or deletion and / or addition of at least one amino acid.

[0097] The present invention is further described in detail below through the introduction of specific implementation methods.

[0098] Example 1 Structure and preparation of chimeric signal receptor A targeting CD56

[0099] The signal receptor provided in this embodiment is composed of a signal peptide, an extracellular antigen recognition region, an extracellular hinge region, a transmembrane region and an intracellular signal region.

[0100] The signal peptide is a leukocyte differentiation antigen 8 signal peptide, and its amino acid sequence is shown in SEQ ID NO. 13. Its nucleotide coding sequence is shown in SEQ ID NO. 14.

[0101] The extracellular molecule recognition region is a monoclonal antibody variable region that recognizes CD56, and its amino acid sequence is shown in SEQ ID NO.3. Its nucleotide coding sequence is shown in SEQ ID NO.4.

[0102] The extracellular hinge region is the leukocyte differentiation antigen 8 extracellular hinge region, and its amino acid sequence is shown in SEQ ID NO. 27. Its nucleotide coding sequence is shown in SEQ ID NO. 28.

[0103] The transmembrane region is the leukocyte differentiation antigen 8 transmembrane region, and its amino acid sequence is shown in SEQ ID NO. 17. Its nucleotide coding sequence is shown in SEQ ID NO. 18.

[0104] The intracellular co-stimulatory signal region is a CD137 intracellular co-stimulatory signal region, and its amino acid sequence is shown in SEQ ID NO. 19. Its nucleotide coding sequence is shown in SEQ ID NO. 20.

[0105] The amino acid sequence of the signal receptor A in this example is shown in SEQ ID NO. 21. The encoding nucleotide sequence is shown in SEQ ID NO. 22.

[0106] According to the coding nucleotide sequence of the signal receptor designed above, an expression vector based on a lentiviral backbone can be constructed, which can express the protein shown in SEQ ID NO.21.

[0107] Specifically, the gene fragment of signal receptor A was obtained by whole gene synthesis, and the following primers were designed to amplify the coding nucleotide fragment of receptor A:

[0108] 5' Primer (SEQ ID NO: 31):

[0109] aggtttaaactacggATGGCCCTCCCCGTGAC.

[0110] 3' Primer (SEQ ID NO: 32):

[0111] atgactagtcccgggTCACAGCTCGCAGCCGC.

[0112] The PCR reaction conditions were as follows: denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 68°C for 1 minute. The reaction was repeated for 25 cycles, followed by extension at 72°C for another 10 minutes.

[0113] The amplified gene fragment was directly inserted into the eukaryotic expression vector pWPXLd (purchased from Addgene, USA) by homologous recombination.

[0114] The specific construction process is as follows: the lentiviral vector pWPXLd is linearized using restriction endonucleases EcoR1 and BamH1, and the amplified gene fragment is inserted into the EcoR I / BamH I site of pWPXLd using the Tiangen EasyGeno Rapid Recombination Cloning Kit (Tiangen Biochemical Technology Co., Ltd., VI201). The recombinant product is transformed into Escherichia coli Stbl3, and 10 clones are randomly selected for sequencing identification. The sequencing results are consistent with the full-length sequence of each designed receptor. The plasmid is extracted and purified using Tiangen's large extraction plasmid purification kit. The purification steps refer to the kit instructions to obtain the expression vector of the receptor A gene. The expression vector of the prepared signal receptor A can be co-transfected with the auxiliary plasmid into the virus packaging cell 293T, and the concentrated lentivirus obtained can infect the target host cell. The host cells that can be transferred are αβT cells, NKT cells, γδT cells, tumor infiltrating lymphocytes, TCR-modified T cells, chimeric antigen receptor T cells or natural killer cells. Commonly used host cells are TCR-modified T cells and chimeric antigen receptor T cells.

[0115] Example 2 Structure and preparation of chimeric signal receptor B targeting CD56

[0116] The signal receptor provided in this embodiment is as follows Figure 1 As shown, it consists of a signal peptide, an extracellular antigen recognition region, an extracellular hinge region, a transmembrane region and an intracellular signal region.

[0117] The signal peptide is a colony stimulating factor 2 receptor subunit α signal peptide, and its amino acid sequence is shown in SEQ ID NO.1. Its nucleotide coding sequence is shown in SEQ ID NO.2.

[0118] The extracellular molecule recognition region is a monoclonal antibody variable region that recognizes CD56, and its amino acid sequence is shown in SEQ ID NO.3. Its nucleotide coding sequence is shown in SEQ ID NO.4.

[0119] The extracellular hinge region is a human immunoglobulin G4 (IgG4) hinge region, and its amino acid sequence is shown in SEQ ID No. 5. Its nucleotide coding sequence is shown in SEQ ID NO. 6.

[0120] The transmembrane region is the leukocyte differentiation antigen 28 transmembrane region, and its amino acid sequence is shown in SEQ ID NO.7. Its nucleotide coding sequence is shown in SEQ ID NO.8.

[0121] The intracellular costimulatory signal region is the intracellular domain of leukocyte differentiation antigen 28, and its amino acid sequence is shown in SEQ ID NO.9. Its nucleotide coding sequence is shown in SEQ ID NO.10.

[0122] The sequence of the signal receptor of this embodiment is shown in SEQ ID NO. 11. The nucleotide coding sequence thereof is shown in SEQ ID NO. 12.

[0123] According to the coding nucleotide sequence of the signal receptor designed above, an expression vector based on a lentiviral backbone was constructed, which can express the protein shown in SEQ ID NO.11.

[0124] Specifically, the gene fragment of receptor B was obtained by the method of total gene synthesis, and the following primers were designed to amplify the coding nucleotide fragment of signal receptor B:

[0125] 5' Primer (SEQ ID NO: 33):

[0126] aggtttaaactacggATGGTTTCTGCTGGTCAC.

[0127] 3' Primer (SEQ ID NO: 34):

[0128] atgactagtcccgggTCAGCTTCTGTAGGCGGC.

[0129] The reaction conditions are as follows:

[0130] PCR reaction: denaturation at 94°C for 30 seconds; annealing at 60°C for 30 seconds; extension at 68°C for 1 minute. Reaction for 25 cycles. Then extension at 72°C for another 10 minutes.

[0131] The amplified gene fragment was directly inserted into the eukaryotic expression vector pWPXLd (purchased from Addgene, USA) by homologous recombination.

[0132] The specific construction process is as follows: the lentiviral vector pWPXLd is linearized using restriction endonucleases EcoR1 and BamH1, the amplified gene fragment is inserted into the EcoR I / BamH I site of pWPXLd using the Tiangen EasyGeno Rapid Recombination Cloning Kit (Tiangen Biochemical Technology Co., Ltd., VI201), the recombinant product is transformed into Escherichia coli Stbl3, and 10 clones are randomly selected for sequencing identification. The sequencing results are consistent with the full-length sequence of each designed receptor. The plasmid is extracted and purified using Tiangen's large extraction plasmid purification kit. The purification steps refer to the kit instructions to obtain the expression vector of the receptor gene B.

[0133] The expression vector of the prepared signal receptor B can be co-transfected with the helper plasmid into the virus packaging cell 293T, and the concentrated lentivirus obtained can infect the target host cell. The host cells that can be transferred are αβT cells, NKT cells or γδT cells, tumor infiltrating lymphocytes, TCR-modified T cells, chimeric antigen receptor T cells or natural killer cells. Commonly used host cells are TCR-modified T cells and chimeric antigen receptor T cells.

[0134] Example 3 Structure and preparation of chimeric signal receptor C targeting CD56

[0135] The signal receptor provided in this embodiment is composed of a signal peptide, an extracellular molecule recognition region, an extracellular hinge region, a transmembrane region and an intracellular co-stimulatory signal region. The signal peptide is a leukocyte differentiation antigen 8 signal peptide, and its amino acid sequence is shown in SEQ ID NO.13. Its nucleotide coding sequence is shown in SEQ ID NO.14.

[0136] The extracellular molecule recognition region is a monoclonal antibody variable region that recognizes CD56, and its amino acid sequence is shown in SEQ ID NO.3. Its nucleotide coding sequence is shown in SEQ ID NO.4.

[0137] The extracellular hinge region is the leukocyte differentiation antigen 8 extracellular hinge region, and its amino acid sequence is shown in SEQ ID NO. 27. Its nucleotide coding sequence is shown in SEQ ID NO. 28.

[0138] The transmembrane region is the leukocyte differentiation antigen 8 transmembrane region, and its amino acid sequence is shown in SEQ ID NO. 17. Its nucleotide coding sequence is shown in SEQ ID NO. 18.

[0139] The intracellular costimulatory signal region is the intracellular activation domain of the interleukin 2 receptor β subunit, and its amino acid sequence is shown in SEQ ID NO. 29. Its nucleotide coding sequence is shown in SEQ ID NO. 30.

[0140] The sequence of the signal receptor of this embodiment is shown in SEQ ID NO. 25. The nucleotide coding sequence thereof is shown in SEQ ID NO. 26.

[0141] According to the coding nucleotide sequence of the signal receptor designed above, an expression vector based on a lentiviral backbone was constructed, which can express the protein shown in SEQ ID NO.25.

[0142] Specifically, the gene fragment of receptor C was obtained by whole gene synthesis, and the following primers were designed to amplify the receptor C fragment:

[0143] 5' Primer (SEQ ID NO: 35):

[0144] aggtttaaactacggATGGCCCTCCCCGTGAC.

[0145] 3' Primer (SEQ ID NO: 36):

[0146] atgactagtcccgggTCACACCAGATGTGTAGGATC.

[0147] The reaction conditions are as follows:

[0148] PCR reaction: denaturation at 94°C for 30 seconds; annealing at 60°C for 30 seconds; extension at 68°C for 2 minutes. Reaction for 25 cycles. Then extension at 72°C for another 10 minutes.

[0149] The amplified gene fragment was directly inserted into the eukaryotic expression vector pWPXLd (purchased from Addgene, USA) by homologous recombination.

[0150] The specific construction process is as follows: the lentiviral vector pWPXLd is linearized using restriction endonucleases EcoR1 and BamH1, and the amplified gene fragment is inserted into the EcoR I / BamH I site of pWPXLd using the Tiangen EasyGeno Rapid Recombination Cloning Kit (Tiangen Biochemical Technology Co., Ltd., VI201), and the recombinant product is transformed into Escherichia coli Stbl3, and 10 clones are randomly selected for sequencing identification. The sequencing results are consistent with the full-length sequence of each designed receptor. The plasmid is extracted and purified using Tiangen's large extraction plasmid purification kit, and the purification steps refer to the kit instructions to obtain the gene expression vector of signal receptor C.

[0151] The expression vector of the prepared signal receptor C can be co-transfected with the helper plasmid into the virus packaging cell 293T, and the concentrated lentivirus obtained can infect the target host cell. The host cells that can be transferred are αβT cells, NKT cells or γδT cells, tumor infiltrating lymphocytes, TCR-modified T cells, chimeric antigen receptor T cells or natural killer cells. Commonly used host cells are TCR-modified T cells and chimeric antigen receptor T cells.

[0152] Example 4 Lentivirus packaging and expression of T cell receptors

[0153] 1. Preparation and concentration of lentivirus:

[0154] One day before transfection, 293T cells cultured in DMEM medium containing 10% fetal bovine serum and 100U / ml penicillin / streptomycin were digested and plated, and cultured overnight at 5% CO2 and 37°C. Two hours before transfection, the medium was replaced with preheated 10% DMEM high-glucose medium without antibiotics. Transfection process: Tube A--Prepare the following DNA mixture in a 50ml BD tube (per 10cm plate / 500ul DMEM), psPAX2 7.5ug; pMD.2G 2.5ug; pWPXLd 10ug (pWPXLd is the lentiviral expression vector containing signal receptor A, signal receptor B or signal receptor C prepared in the above example). Tube B (per 10cm plate / 500ul DMEM)-EZ Trans transfection reagent 60ul (Li Ji Biology). Add the solution in tube B to tube A, mix gently, and let stand at room temperature for 15 minutes. Add 1 ml of transfection mixture to each dish and shake gently to mix, and continue culturing at 5% CO2 and 37. After 48 hours, collect the cell supernatant and filter with a 0.45μm filter. Add the virus concentrate (PEG6000) to the filtered virus solution at a volume of 1:3, mix evenly, incubate at 4°C overnight, and then centrifuge at 4°C, 1500g, 45min. After centrifugation, discard the supernatant culture medium, and invert the centrifuge tube on sterile filter paper to absorb the remaining culture medium. Use DMEM culture medium to resuspend the virus precipitate and divide it into EP tubes (100ul / tube), and store it in a -80°C ultra-low temperature refrigerator for later use. Prepared

[0155] 2. Preparation of receptor T cells:

[0156] 20 ml of peripheral blood from healthy people was collected and added to an equal volume of FICOLL lymphocyte separation medium. The cells were centrifuged at 32°C, 1000 g for 30 min. The lymphocytes in the middle buffy coat layer were aspirated and added to 20 mL of serum-free and antibiotic-free T cell culture medium and centrifuged at 500 g for 10 min. The supernatant was discarded and the lymphocyte pellet was resuspended in 10 mL of sterile lysate and incubated for 2-3 min. The cells were centrifuged at 500 g for 10 min. The supernatant was discarded and the cells were washed twice with 1640 culture medium, counted, and CD3 / CD28 Dynabeads (life technologies 11131D) (cells: beads = 1: 3) were added and resuspended in T cell culture medium supplemented with 50 U / ml IL-2, 10 ng / ml IL-7 and 10 ng / ml IL15. The cells were cultured at 5% CO2 and 37°C for 48 h.

[0157] 3. Recombinant virus infection of T cells

[0158] Before transfection, centrifuge T cells, resuspend T cells with medium containing virus supernatant (MOI=1:5), mix well, add to six-well plate, centrifuge at 32°C, 1000g, 2h. Take out the six-well plate and place it in a 37°C, 5% CO2 cell culture incubator for culture.

[0159] After the concentrated viruses expressing signal receptor A, signal receptor B and signal receptor C were infected with T cells for 48 hours, flow cytometry was used to detect the expression of each receptor protein using anti-G4S antibody (see Figure 2 A). The results showed that in cells transfected with receptor A, 43.02% of T cells were detected to express receptor A; in cells transfected with receptor B, 79.96% of T cells were detected to express receptor B; in cells transfected with receptor C, 64.65% of T cells were detected to express receptor C. The results showed that we successfully constructed T cells expressing different receptors. At the same time, the expression of CD56 in tumor cells was also detected ( Figure 2 B), the results showed that both SHP77 and H446 cells of small cell lung cancer expressed CD56 protein. Relatively speaking, the CD56 expression intensity of SHP77 cells was higher. In addition, we also detected the expression of DLL3 protein (another target antigen of small cell lung cancer, which will become the target antigen of CAR-T as a positive control) in SHP77 cells. The results showed that SHP77 cells highly expressed DLL3 antigen ( Figure 2 C).

[0160] Example 5 Determination of proliferation and survival ability of T cells expressing receptors.

[0161] CFSE dye was used to label T cells infected with the virus (Cell Trace TM CFSE Cell Proliferation Kit, Thermo Company). Effector T cells (such as control T cells, receptor AT cells, receptor BT cells and receptor CT cells) and target cells (SHP77 cells irradiated with 20 Gy X-ray) were added to a 12-well plate at an effector-target ratio of 1:1. The number of target cells in each well was 1*10 6 Set up control wells with only effector cells or target cells. Detect T cell CFSE changes by flow cytometry and count T cells.

[0162] The results are as follows Figure 3 As shown in A, the T cells modified by receptors A, B and C all proliferated significantly after being stimulated by antigens (target cells), but the cells in the control group did not change significantly. The T cells were further counted and analyzed (using Thermo's Count Bright TM absolute counting beads for counting), and the results are shown in Figure 3As shown in Figure B: T cells modified by receptors A, B and C all have a significant increase in cell number compared to the control group, among which the increase in T cells modified by receptor C is the most significant, which is consistent with the cell proliferation results indicated by CSFE detection. The experimental results show that T cells modified by signal receptors A, B and C prepared by the present invention all have significant proliferation and growth after being stimulated by CD56 antigen.

[0163] Example 6: Determination of the killing ability of receptor-modified T cells in vitro.

[0164] 1. Flow cytometry to detect tumor cell count and analyze CAR-T cell killing

[0165] SHP77 tumor cells were plated in 12-well plates, 105 / well; according to the experimental plan, different proportions of control T cells, receptor A-modified T cells, receptor B-modified T cells, receptor C-modified T cells and CAR-T cells targeting DLL3 (DLL3-CAR T as a positive control) were added, and the wells without T cells were used as blank groups; after 16 hours of co-culture, the cells were collected and stained with PE-labeled anti-human CD45 antibodies and Zombie NIR dyes; 300μl PBS was added to each well to resuspend the cells, and then 50ul counting magnetic beads (Count Bright TM from Thermo, 50,000 / tube) were added; flow cytometry detection: 10,000 magnetic beads were counted and collected, and the cells that were negative for both APC-CY7 and CD45 (i.e., live tumor cells) were calculated, and the number of live tumor cells was calculated based on the ratio of magnetic beads to tumor cells, and the killing efficiency was calculated by comparing with the untreated well plate group. The results are as follows Figure 4 As shown in A, DLL3 CAR-T cells can effectively kill SHP77 cells, but T cells modified with various receptor genes have no obvious direct killing effect on tumor cells.

[0166] 2. Determination of IFN-γ secretion.

[0167] Human IFNγ ELISA Kit (Thermo) was used to detect cytokine release. According to the experimental protocol, the supernatants of cells with different effector-target ratios were collected after 24 hours of co-culture. Sample concentrations were tested based on the operating procedures in the manual. The results are shown in Figure 2. Figure 4As shown in B: when the control T cells, T cells modified by receptor A, T cells modified by receptor B, and T cells modified by receptor C were co-incubated with CD56-positive SHP77 cells, they could not effectively activate T cell killing and release IFN-γ in large quantities. Their expression levels were all at the pg / ml level, which was an order of magnitude different from the cytokine release (ng / ml level) caused by CAR-T activation. This indicates that since the intracellular signal segment of the signal receptor of the present invention lacks the CD3ζ chain, its effect of activating T cell killing and releasing IFN-γ in large quantities is weaker than that of the CAR structure.

[0168] Example 7: Determination of the killing ability of CAR-T cells in vivo and in vitro after receptor modification.

[0169] 1. Determination of IFN-γ secretion.

[0170] Human IFNγ ELISA Kit (Thermo) was used to detect cytokine release. According to the experimental protocol, the supernatants of cells with different effector-target ratios were collected after 24 hours of co-culture. Sample concentration was detected based on the instructions. Control T cells, receptor A-modified T cells, receptor B-modified T cells, DLL3 CAR-T cells, receptor A-modified DLL3 CAR-T cells, and receptor B-modified DLL3 CAR-T cells were set up. Among them, receptor A-modified DLL3 CAR-T cells and receptor B-modified DLL3 CAR-T cells were obtained by co-transfecting T cells with the above-mentioned receptor A-containing virus, receptor B-containing virus, and DLL3 CAR virus.

[0171] The results are as follows Figure 5 As shown in A: when control T cells, T cells modified by receptor A, T cells modified by receptor B, DLL3CAR-T cells, DLL3 CAR-T cells modified by receptor A, and DLL3 CAR-T cells modified by receptor B were co-incubated with CD56-positive SHP77 cells, neither the T cells in the control group nor the T cells modified with the receptor alone could effectively activate the T cell killing effect and release a large amount of IFN-γ; compared with the DLL3 CAR-T cells alone, the IFN-γ released by the DLL3 CAR-T cells modified by receptor A and the DLL3 CAR-T cells modified by receptor B was significantly stronger, indicating that the signal receptor of the present invention can enhance the killing effect of CAR-T cells and release a large amount of IFN-γ.

[0172] 2. Flow cytometry to detect tumor cell count and analyze CAR-T cell killing

[0173] SHP77 tumor cells were plated in 12-well plates for 10 5 / well; according to the experimental scheme, different proportions of control T cells, receptor A modified T cells, receptor B modified T cells, DLL3 CAR-T cells, receptor A modified DLL3CAR-T cells, receptor B modified DLL3 CAR-T cells were added, and the wells without T cells were used as blank groups; after 16 hours of co-culture, the cells were collected and stained with PE-labeled anti-human CD45 antibody and Zombie NIR dye; 300μl PBS was added to each well to resuspend the cells, and then 50ul counting magnetic beads (Count Bright TM of Thermo Company, 50,000 / tube) were added; flow cytometry detection: 10,000 magnetic beads were counted and collected, and the cells that were negative for both APC-CY7 and CD45 (i.e., live tumor cells) were calculated, and the number of live tumor cells was calculated based on the ratio of magnetic beads to tumor cells, and the killing efficiency was calculated by comparing with the untreated well plate group. The results are as follows Figure 5 As shown in B, neither the control group T cells nor the T cells modified with only the receptor could effectively kill SHP77 cells; compared with DLL3 CAR-T cells alone, DLL3 CAR-T cells modified with receptor A and DLL3 CAR-T cells modified with receptor B had stronger in vitro killing effects.

[0174] 3. Differently modified CAR-T cells are used to treat the subcutaneous human SHP77 small cell lung cancer model.

[0175] The control T cells, DLL3 CAR-T cells, and receptor A-modified DLL3 CAR-T cells groups were set up. Because the above experiments have shown that receptor-modified T cells have no obvious killing effect on tumor cells in vitro, they were not set up separately in the in vivo experiment. Female NOD.Cg-PrkdcscidIl2rgtmWjl / SzJ (NSG) mice aged 8-12 weeks (7 mice per group) were selected and 1×10 6 SHP77 cells were subcutaneously inoculated to establish a human small cell lung cancer model. When the tumor grew to 50-100 mm3, 5×10 6 DLL3 CAR-T cells, receptor A-modified DLL3 CAR-T cells, or control T cells; 2 days later, the same dose was injected for the second time. Tumor size was measured every 2 days after the first CAR-T injection.

[0176] from Figure 5 It can be clearly seen from the results of C that the efficacy of DLL3 CAR-T cells modified with receptor A is significantly better than that of DLL3 CAR-T alone.

[0177] From the above experiments and their results, it can be seen that the chimeric signal receptors targeting CD56 involved in the present invention can promote the proliferation and survival of immune cells such as T cells by activating the intracellular signal domain after being stimulated by antigens; when co-modifying T cells with traditional CAR targeting DLL3, the anti-tumor effect of CAR-T can be enhanced. It can be seen that the chimeric signal receptors involved in the present invention can also enhance the killing effect of CAR-T cells or TCR-T cells of other targets, which helps to promote the clinical application of cell therapy in solid tumors. The signal receptors of the present invention and immune cells that can express the signal receptors can be used to prepare anti-tumor drugs and autoimmune disease drugs, and have good application prospects in the treatment of tumors and autoimmune diseases.

[0178] Amino acid and nucleotide sequences:

[0179]

[0180]

[0181]

[0182]

[0183]

[0184] References

[0185] 1. Crossland DL, Denning WL, Ang S, et al. Antitumor activity of CD56-chimeric antigen receptor T cells in neuroblastoma and SCLCmodels. Oncogene. 2018; 37(27): 3686-3697.

[0186] 2. Shah MH, Lorigan P, O'Brien MER, et al. Phase I study of IMGN901, aCD56-targeting antibody-drug conjugate, in patients with CD56-positive solidtumors. Investigational New Drugs. 2016; 34(3): 290-299.

[0187] 3.Socinski MA,Kaye FJ,Spigel DR,et al.Phase 1 / 2Study of the CD56-Targeting Antibody-Drug Conjugate Lorvotuzumab Mertansine(IMGN901)inCombination With Carboplatin / Etoposide in Small-Cell Lung Cancer PatientsWith Extensive-Stage Disease.Clinical Lung Cancer.2017;18(1):68-76.e62.

Claims

1. A chimeric signal receptor targeting leukocyte differentiation antigen 56 (CD56), characterized in that It comprises an extracellular antigen recognition region, an extracellular hinge region, a transmembrane region and an intracellular signal region; the extracellular antigen recognition region can recognize or bind to CD56; the intracellular signal region does not contain an immunoreceptor tyrosine-based activation motif (ITAM); or, the intracellular signal region does not contain a CD3ζ chain.

2. The chimeric signal receptor according to claim 1, characterized in that: The sequence of the immunoreceptor tyrosine-based activation motif (ITAM) is YXX(L / I)X(6-8)YXX(L / I).

3. The chimeric signal receptor according to claim 1 or 2, characterized in that: The chimeric signal receptor nitrogen end is also connected to a signal peptide.

4. The chimeric signal receptor according to claim 1, characterized in that Satisfy at least one of the following 1) to 5): 1) The signal peptide is at least one of the protein domains with a sequence homology of more than 90% with the interleukin-2 signal peptide, the leukocyte differentiation antigen-8 signal peptide, and the colony stimulating factor-2 receptor subunit α signal peptide; 2) the extracellular antigen recognition region is at least one of a monoclonal antibody variable region or a heavy chain single domain antibody variable region that binds to CD56; 3) the extracellular hinge region is at least one of the protein domains having a sequence homology of more than 90% with the extracellular hinge region of leukocyte differentiation antigen 8, the extracellular hinge region of leukocyte differentiation antigen 28, the hinge region of human immunoglobulin G1 (IgG1), the hinge region of human immunoglobulin G4 (IgG4), the extracellular hinge region of TRAIL, the extracellular hinge region of leukocyte differentiation antigen 16, the extracellular hinge region of NKp30, the extracellular hinge region of NKG2C, the extracellular hinge region of NKG2D, the extracellular hinge region of 2B4 or the extracellular hinge region of DNAM-1; 4) the transmembrane region is at least one of the protein domains having a sequence homology of more than 90% with the leukocyte differentiation antigen 8 transmembrane region, the leukocyte differentiation antigen 28 transmembrane region, the interleukin 2 receptor β subunit transmembrane region, the interleukin 7 receptor transmembrane region, the TRAIL transmembrane region, the leukocyte differentiation antigen 16 transmembrane region, the NKp30 transmembrane region, the NKG2C transmembrane region, the NKG2D transmembrane region, the 2B4 transmembrane region or the DNAM-1 transmembrane region; 5) The intracellular signaling region is at least one of the intracellular domains of a protein domain with a sequence homology of more than 90% with leukocyte differentiation antigen 28, CD137 intracellular co-stimulatory signaling region, tumor necrosis factor receptor superfamily member 4, tumor necrosis factor receptor superfamily member 9, lymphocyte-specific protein tyrosine kinase, inducible T cell co-stimulatory molecule, intracellular activation domain of interleukin 2 receptor β subunit, leukocyte differentiation antigen 40 co-stimulatory signaling region, leukocyte differentiation antigen 27 co-stimulatory signaling region, DAP10 co-stimulatory signaling region, DAP12 co-stimulatory signaling region, TRAIL co-stimulatory signaling region, leukocyte differentiation antigen 16 co-stimulatory signaling region, NKp30 co-stimulatory signaling region, NKG2C co-stimulatory signaling region, NKG2D co-stimulatory signaling region, 2B4 co-stimulatory signaling region, DNAM-1 co-stimulatory signaling region, and interleukin 7 receptor intracellular signaling region.

5. The chimeric signal receptor according to claim 1, characterized in that: At least one of the following is met: 1) The signal peptide is a colony stimulating factor 2 receptor subunit α signal peptide; or a peptide having the same or similar function as the colony stimulating factor 2 receptor subunit α signal peptide obtained by substitution and / or deletion and / or addition of at least one amino acid; Alternatively, the signal peptide is a leukocyte differentiation antigen 8 signal peptide, or a peptide having the same or similar function as the leukocyte differentiation antigen 8 signal peptide obtained by substitution and / or deletion and / or addition of at least one amino acid; 2) The extracellular antigen recognition region is a monoclonal antibody variable region or a heavy chain single domain antibody variable region that recognizes CD56, or a peptide segment having the same or similar function as the monoclonal antibody variable region or the heavy chain single domain antibody variable region that recognizes CD56 obtained by substitution and / or deletion and / or addition of at least one amino acid; 3) The extracellular hinge region is a human immunoglobulin G4 (IgG4) hinge region, or a peptide segment having the same or similar function as the human immunoglobulin G4 (IgG4) hinge region obtained by substitution and / or deletion and / or addition of at least one amino acid; Or the extracellular hinge region is the leukocyte differentiation antigen 8 hinge region, or a peptide segment having the same or similar function as the leukocyte differentiation antigen 8 extracellular hinge region obtained by substitution and / or deletion and / or addition of at least one amino acid; 4) the transmembrane region is the leukocyte differentiation antigen 28 transmembrane region, or a peptide segment having the same or similar function as the leukocyte differentiation antigen 28 transmembrane region obtained by substitution and / or deletion and / or addition of at least one amino acid; Alternatively, the transmembrane region is the leukocyte differentiation antigen 8 transmembrane region, or a peptide segment having the same or similar function as the leukocyte differentiation antigen 8 transmembrane region obtained by substitution and / or deletion and / or addition of at least one amino acid; 5) The intracellular signal region is the intracellular domain of leukocyte differentiation antigen 28, or a peptide segment having the same or similar function as the intracellular domain of leukocyte differentiation antigen 28 obtained by substitution and / or deletion and / or addition of at least one amino acid; Alternatively, the intracellular signal region is the CD137 intracellular co-stimulatory signal region, or a peptide segment having the same or similar function as the CD137 intracellular co-stimulatory signal region obtained by substitution and / or deletion and / or addition of at least one amino acid; Alternatively, the intracellular signaling region is the intracellular activation domain of the interleukin-2 receptor β subunit, or a peptide segment having the same or similar function as the intracellular activation domain of the interleukin-2 receptor β subunit obtained by substitution and / or deletion and / or addition of at least one amino acid.

6. The chimeric signal receptor according to any one of claims 1 to 5, characterized in that: The amino acid sequence of the colony stimulating factor 2 receptor subunit α signal peptide is shown in SEQ ID NO.1; further, the encoding nucleotide sequence of the colony stimulating factor 2 receptor subunit α signal peptide is shown in SEQ ID NO.

2.

7. The signal receptor according to any one of claims 1 to 6, characterized in that: The amino acid sequence of the variable region of the monoclonal antibody recognizing CD56 is shown as SEQ ID No.3; further, the encoding nucleotide sequence of the variable region of the monoclonal antibody recognizing CD56 is shown as SEQ ID No.

4.

8. The chimeric signal receptor according to any one of claims 1 to 7, characterized in that: The amino acid sequence of the hinge region of human immunoglobulin G4 is shown as SEQ ID No.5; further, the encoding nucleotide sequence of the hinge region of human immunoglobulin G4 is shown as SEQ ID No.

6.

9. The signal receptor according to any one of claims 1 to 8, characterized in that: The amino acid sequence of the transmembrane region of leukocyte differentiation antigen 28 is shown in SEQ ID No.7; further, the encoding nucleotide sequence of the transmembrane region of leukocyte differentiation antigen 28 is shown in SEQ ID No.

8.

10. The signal receptor according to any one of claims 1 to 9, characterized in that: The amino acid sequence of the intracellular domain of the leukocyte differentiation antigen 28 is shown in SEQ ID No.9; further, the encoding nucleotide sequence of the intracellular domain of the leukocyte differentiation antigen 28 is shown in SEQ ID No.

10.

11. The signal receptor according to any one of claims 1 to 10, characterized in that: The amino acid sequence of the leukocyte differentiation antigen 8 signal peptide is shown in SEQ ID NO.13; further, the encoding nucleotide sequence of the leukocyte differentiation antigen 8 signal peptide is shown in SEQ ID NO.

14.

12. The signal receptor according to any one of claims 1 to 11, characterized in that: The amino acid sequence of the interleukin-2 signal peptide is shown in SEQ ID NO.15; further, the encoding nucleotide sequence of the interleukin-2 signal peptide is shown in SEQ ID NO.

16.

13. The signal receptor according to any one of claims 1 to 12, characterized in that: The amino acid sequence of the leukocyte differentiation antigen 8 transmembrane region is shown in SEQ ID NO.17; further, the encoding nucleotide sequence of the leukocyte differentiation antigen 8 transmembrane region is shown in SEQ ID NO.

18.

14. The signal receptor according to any one of claims 1 to 13, characterized in that: The amino acid sequence of the CD137 intracellular co-stimulatory signal region is shown in SEQ ID NO.19; further, the encoding nucleotide sequence of the CD137 intracellular co-stimulatory signal region is shown in SEQ ID NO.

20.

15. The signal receptor according to any one of claims 1 to 14, characterized in that: The amino acid sequence of the extracellular hinge region of the leukocyte differentiation antigen 8 is shown in SEQ ID NO.27; further, the encoding nucleotide sequence of the extracellular hinge region of the leukocyte differentiation antigen 8 is shown in SEQ ID NO.

28.

16. The signal receptor according to any one of claims 1 to 15, characterized in that: The amino acid sequence of the intracellular activation region of the interleukin-2 receptor β subunit is shown in SEQ ID NO.29; further, the encoding nucleotide sequence of the intracellular activation region of the interleukin-2 receptor β subunit is shown in SEQ ID NO.

30.

17. The signal receptor according to any one of claims 1 to 16, characterized in that: The amino acid sequence of the intracellular activation region of the interleukin-7 receptor is shown in SEQ ID NO.23; further, the encoding nucleotide sequence of the intracellular activation region of the interleukin-7 receptor is shown in SEQ ID NO.

24.

18. The signal receptor according to any one of claims 1 to 17, characterized in that: The amino acid sequences of the signal receptors are shown in SEQ ID NO.11, SEQ ID NO.21 and SEQ ID NO.

25.

19. The signal receptor according to claim 18, characterized in that: The encoding nucleotide sequence of the signal receptor is shown as SEQ ID NO.12, SEQ ID NO.22 or SEQ ID NO.

26.

20. An expression vector, characterized in that: It can express the signal receptor described in any one of claims 1 to 19.

21. The expression vector according to claim 20, characterized in that: It is a recombinant plasmid vector, a recombinant virus or a combination thereof.

22. The expression vector according to claim 21, characterized in that: The recombinant virus is a lentiviral vector or a retroviral vector.

23. A cell for treating tumors or autoimmune diseases, characterized in that: A host cell containing the expression vector according to any one of claims 20 to 22.

24. The cell for treating tumors or autoimmune diseases according to claim 23, characterized in that: The host cell is an immune cell; further, the immune cell is at least one of a T cell, a tumor infiltrating lymphocyte or a natural killer cell; further, the host cell is selected from at least one of an αβT cell, a NKT cell, a γδT cell, a TCR-modified T cell, and a chimeric antibody receptor T cell.

25. A drug for treating tumors or autoimmune diseases, characterized in that: It is prepared by taking the cells for targeted treatment of tumors or autoimmune diseases as claimed in any one of claims 23 or 24 as active ingredients and adding pharmaceutically acceptable excipients or auxiliary ingredients.

26. The drug for treating tumors or autoimmune diseases according to claim 25, characterized in that: The tumor is at least one of small cell lung cancer, pancreatic endocrine tumor, medullary thyroid carcinoma, neuroendocrine tumor, malignant melanoma, leukemia, lymphoma, multiple myeloma, neuroblastoma, sarcoma or nephroblastoma.

27. The drug for treating tumors or autoimmune diseases according to claim 25, characterized in that: The autoimmune disease is at least one of rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) or scleroderma.

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