Secretory effector cell

Abstract

Provided is an engineered cell, wherein a gene of a fusion molecule formed by means of linking a T cell receptor (TCR) to an immune effector molecule in a specific manner is transduced. The fusion molecule is expressed by the cell, is secreted extracellularly, and is capable of binding to both an effector cell and a tumor cell. Further provided is the use of the cell as a drug.

Description

A secretory effector cell Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to an engineered cell capable of secreting and expressing a specific fusion molecule. The present invention also relates to the use of the cell. Background Art

[0002] Cellular immunotherapy is a promising and emerging cancer treatment modality. T cell therapy, a highly anticipated and effective treatment approach, primarily includes CAR-T cell (Chimeric Antigen Receptor Therapy) and TCR-T cell (T Cell Receptor Therapy). TCRs are proteins on the surface of T cells, which are part of the human immune system and play a crucial role in defending against infection and disease, including cancer. Antigens are cleaved and degraded within cells by proteases before being presented on the cell membrane by the major histocompatibility complex (MHC), where they are recognized by the TCR on T cells. The TCR on the surface of cytotoxic CD8 T cells recognizes specific class I MHCs and the peptide-MHC complexes they present (pMHC). HLA (human leukocyte antigen) is a specific MHC for humans. Specific binding activates T cells, which then release cytotoxic substances or death receptors to specifically kill tumor cells.

[0003] However, TCR-T cell therapy also has limitations and challenges. For example, the preparation process is relatively complex and time-consuming, and can be affected by various factors, leading to low production efficiency or reduced cell activity. Furthermore, the high production costs associated with infusing hundreds of millions of T cells into TCR-T cell therapy limit its application. Furthermore, long-term use can lead to immune tolerance or cell exhaustion, compromising therapeutic efficacy.

[0004] T cell engager TCE (T Cell Engager), a fusion molecule of T cell receptor and immune effector molecule, can specifically guide effector cells to tumor cells, thereby achieving a strong anti-tumor effect. It is a new immunotherapy strategy. However, although TCE has significant potential therapeutic effects, it also has some shortcomings and limitations. First, the pharmacokinetic properties of TCE may not be ideal. In the body, the metabolism and clearance rate of TCE may be affected by multiple factors, which may lead to drug accumulation in the body and unstable drug concentrations, further affecting the therapeutic effect. Secondly, due to the short effective period of TCE, multiple doses are often required, affecting patient compliance.

[0005] The present invention has developed a novel cell that secretes a fusion protein expressing a T cell receptor (TCR) and an immune effector molecule. Through the T cell receptor binding to antigenic peptide-MHC epitopes on the tumor cell surface and the immune effector molecule binding to the effector molecule receptor on the effector cell surface, effector T cells are recruited to the vicinity of tumor cells to perform their killing function. TCE-secreting cells can achieve a low efficiency-target ratio while being effective, thus preventing immune escape. Furthermore, even exhausted T cells can still secrete TCE to recruit bystander T cells to continue killing tumors. Summary of the Invention

[0006] The object of the present invention is to provide an engineered cell capable of secreting and expressing a fusion molecule.

[0007] In the first aspect of the present invention, an engineered cell is provided, characterized in that it is transduced with a gene of a fusion molecule composed of a T cell receptor (TCR) and an immune effector molecule connected in a specific manner, and the fusion molecule is expressed by the cell and secreted outside the cell.

[0008] In a preferred embodiment, the engineered cells are cells that can stably secrete and express exogenous genes. Preferably, the cells can be T cells, NK cells, NK-T cells, 293T cells or LCL cells.

[0009] In a preferred embodiment, the immune effector molecule in the fusion molecule is an anti-CD antibody.

[0010] In a preferred embodiment, the anti-CD antibody is an anti-CD3 antibody.

[0011] In a preferred example, the fusion molecule is a single-chain structure formed by connecting the TCR variable regions Vα and Vβ via a flexible linker, and the light chain (VL) and heavy chain (VH) of an anti-CD3 antibody via a flexible linker.

[0012] In a preferred embodiment, the fusion molecule transduced into the cell is composed of Vα and Vβ of the TCR molecule, and VH and VL of the anti-CD3 antibody molecule.

[0013] In a preferred embodiment, the TCR and the anti-CD3 antibody molecule are connected by a short linker sequence between the Vα of the TCR and the VL of the anti-CD3 antibody, and a short linker sequence between the Vβ of the TCR and the VH of the anti-CD3 antibody.

[0014] In a preferred example, the short linker sequence is SEQ ID NO: 18.

[0015] In a preferred embodiment, the VH and VL of the anti-CD3 antibody are connected via a long linker sequence.

[0016] In a preferred example, the long linker sequence is SEQ ID NO: 19.

[0017] In a preferred embodiment, the VH and VL of the anti-CD3 antibody are linked by a disulfide bond.

[0018] In a preferred embodiment, the Vβ of the TCR and the VL of the anti-CD3 antibody are linked via a disulfide bond.

[0019] In a preferred embodiment, the disulfide bond is formed by introducing SEQ ID NO: 20 into the C-terminus of the anti-CD3 antibody VL and SEQ ID NO: 21 into the C-terminus of the TCRVβ.

[0020] In a preferred embodiment, the disulfide bond is formed by introducing SEQ ID NO: 22 into both the C-terminus of TCRVβ and the C-terminus of the anti-CD3 antibody VL.

[0021] In a preferred example, the fusion molecule secreted and expressed by the cells can bind to effector cells and tumor cells at the same time.

[0022] In a preferred embodiment, the effector cells are T cells.

[0023] In a preferred embodiment, the effector cells are NK cells.

[0024] In a preferred embodiment, the effector cells are NK-T cells.

[0025] In a preferred embodiment, the T cell receptor is a T cell receptor with high affinity for the pMHC (peptide-major histocompatibility complex) to which it is targeted. Preferably, the affinity is at least 100 times, preferably at least 500 times, and more preferably at least 1000 times, the affinity of a wild-type TCR for the corresponding antigen.

[0026] Specifically, the dissociation equilibrium constant of the TCR to the pMHC complex to which it is directed is KD≤1 μM; preferably, KD≤100 nM; more preferably, KD≤1 nM.

[0027] In another preferred embodiment, the immune effector molecule in the fusion molecule is a cytokine.

[0028] In a preferred embodiment, the cytokine is interleukin (IL).

[0029] In a preferred embodiment, the cytokine is TGF-β.

[0030] In a preferred embodiment, the cytokine is IFN-γ.

[0031] In a preferred embodiment, the cytokine is TNFα.

[0032] In a preferred embodiment, the gene of the fusion molecule is transduced into the cell via lentivirus.

[0033] In another preferred embodiment, the gene of the fusion molecule is transduced into the cell by electroporation.

[0034] In the second aspect of the present invention, a nucleic acid molecule is provided, which comprises a nucleic acid sequence encoding the fusion molecule in the cell of the first aspect of the present invention or a complementary sequence thereof.

[0035] In the third aspect of the present invention, a vector is provided, wherein the vector contains the nucleic acid molecule described in the second aspect of the present invention.

[0036] In a fourth aspect of the present invention, a pharmaceutical composition is provided, comprising the cells described in the first aspect of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a schematic diagram of the structure of scDART in the form of a fusion molecule;

[0038] FIG2 is a schematic diagram of the structure of the fusion molecule dcDART;

[0039] Figure 3 is a schematic diagram of the structure of the fusion molecule dcDART-QG;

[0040] Figure 4 is a schematic diagram of the structure of the fusion molecule dcDART-F;

[0041] Figure 5 is a schematic diagram of the structure of the fusion molecule dcDART-L;

[0042] Figure 6 is a schematic diagram of the structure of the fusion molecule DART-Fc;

[0043] FIG7 is a schematic diagram of the structure of the fusion molecule scCIS;

[0044] Figures 8 to 14 are affinity binding curves of the TCR end of fusion protein molecules scDART, scDART-QG, dcDART, dcDART-QG, dcDART-F, dcDART-L and scCIS and their corresponding antigen peptide complexes pMHC;

[0045] Figures 15 to 21 are affinity binding curves of the anti-CD3 antibody end of the fusion protein molecules scDART, scDART-QG, dcDART, dcDART-QG, dcDART-F, dcDART-L and scCIS with the CD3 molecule;

[0046] FIG22 is a diagram showing the staining results of TCE on T cells in Example 2;

[0047] FIG23 is a diagram showing the results of the ELISpot assay to verify the TCE-T cell activation function in Example 3;

[0048] FIG24 is a graph showing the results of the LDH test to verify the TCE-T cell killing function in Example 4;

[0049] FIG25 is a diagram showing the results of ELISpot assays to verify TCE-T cell activation with different fusion molecule formats in Example 5;

[0050] FIG26 is a graph showing the results of the LDH test in Example 6 verifying the TCE-T cell killing effect of different fusion molecule forms;

[0051] FIG27 is a graph showing the results of the ELISpot assay in Example 7 to verify the TCE-T cell activation function of different TCR molecules;

[0052] FIG28 is a graph showing the results of the LDH test in Example 8 to verify the TCE-T cell killing function of different TCR molecules;

[0053] Figures 29a, 29b, and 29c are the results of ELISpot experiments to verify the function of TCE-transfected 293T cells;

[0054] FIG30 is the functional results of LDH experiments verifying TCE-transfected 293T cells;

[0055] FIG31 shows the results of LDH experiment to verify the function of TCE-transfected LCL cells. DETAILED DESCRIPTION

[0056] Through extensive and in-depth research, the present invention has developed an engineered cell capable of secreting and expressing a fusion protein. This cell can secrete and express a fusion protein of a T cell receptor and an immune effector molecule. Through the T cell receptor binding to the antigen peptide-MHC epitope on the surface of tumor cells and the immune effector molecule binding to the effector molecule receptor on the surface of the effector cell, effector T cells are recruited to the vicinity of tumor cells to perform a killing function.

[0057] Specifically, the cells of the present invention are transduced with genes of fusion molecules (TCEs) composed of T cell receptors (TCRs) and immune effector molecules connected in a specific manner. The fusion molecules are expressed by the cells and secreted outside the cells, and the fusion molecules can simultaneously bind to effector cells and tumor cells.

[0058] The immune effector molecule in the fusion molecule is a cytokine; preferably, the cytokine is IL interleukin, TGF-β, IFN-γ or TNFα.

[0059] The cells are T cells, NK cells or NK-T cells; preferably, the cells are T cells.

[0060] The immune effector molecule in the fusion molecule is usually an anti-CD antibody, preferably an anti-CD3 antibody.

[0061] The fusion molecule is a single-chain structure formed by connecting the TCR variable regions Vα and Vβ via a flexible linker, and the light chain (VL) and heavy chain (VH) of an anti-CD3 antibody via a flexible linker.

[0062] The fusion molecule may be composed of Vα, Vβ of a T cell receptor molecule (TCR) and VH and VL of an anti-CD3 antibody molecule, wherein Vα and Vβ of the TCR are cross-linked to VH and VL of the anti-CD3 antibody molecule, respectively. The cross-linking means that when Vα of the TCR is linked to VL of the anti-CD3 antibody molecule, Vβ of the TCR is linked to VH of the anti-CD3 antibody molecule; and when Vα of the TCR is linked to VH of the anti-CD3 antibody molecule, Vβ of the TCR is linked to VL of the anti-CD3 antibody molecule.

[0063] The variable regions Vα and Vβ of the two TCR chains are cross-linked with the variable regions of the CD3 antibody light chain VL and heavy chain VH. They are connected by a short flexible linker sequence so that they cannot be paired in space. The linker sequence is generally 5-9 amino acids long. When the two chains formed by the cross-link are put together, they will fold back in the middle, allowing the heavy and light chains of the antibody and the Vα and Vβ of the TCR to pair in space to form a dimer structure. The two chains can be connected by a longer linker sequence to form a single-chain construct (scDART). The linker sequence is usually flexible and consists of amino acids without bulky side chains. It is usually 15-24 amino acids in length. The two chains can also be connected to form a dimer structure by introducing a disulfide bond at the C-terminus or between anti-CD antibody molecules (dcDART-F, dcDART-L, dcDART-QG).

[0064] There are 8 connection methods for scDART, in which different domains (specifically Vα, VL, VH, and Vβ) are connected by linker sequences, SL represents a short linker sequence, and LL represents a long linker sequence: Vα-SL-VL-LL-VH-SL-Vβ, Vα-SL-VH-LL-VL-SL-Vβ, Vβ-SL-VL-LL-VH-SL-Vα, Vβ-SL-VH-LL-VL-SL-Vα, VL-SL-Vα-LL-Vβ-SL-VH, VL-SL-Vβ-LL-Vα-SL-VH, VH-SL-Vα-LL-Vβ-SL-VL, VH-SL-Vβ-LL-Vα-SL-VL. dcDARTs are linked by four different linker combinations, where SL represents a short linker sequence: Vα-SL-VL and VH-SL-Vβ, VL-SL-Vα and Vβ-SL-VH, Vα-SL-VH and VL-SL-Vβ, and VH-SL-Vα and Vβ-SL-VL. dcDARTs are linked by disulfide bonds, which can be formed by mutations within the anti-CD27 antibody molecule or by introducing FNRGEC and VEPKSC pairings or LGGC and LGGC pairings at the C-termini of the two chains.

[0065] Binding affinity (inversely proportional to the dissociation equilibrium constant KD) and binding half-life (expressed as T1 / 2) can be measured by any suitable method. It should be understood that doubling the affinity of a TCR will result in KD being halved. T1 / 2 is calculated as In2 ​​divided by the dissociation rate (Koff). Therefore, doubling T1 / 2 will result in Koff being halved. Preferably, the same experimental protocol is used to detect the binding affinity or binding half-life of a given TCR several times, for example 3 times or more, and the average value of the results is taken. In a preferred embodiment, these detections are performed using the surface plasmon resonance (BIAcore) method in the examples herein.

[0066] In some embodiments, the T cell receptor is a T cell receptor with high affinity for the pMHC (peptide-major histocompatibility complex) to which it is directed. Preferably, the affinity is at least 100 times, preferably at least 500 times, and more preferably at least 1000 times, the affinity of a wild-type TCR for the corresponding antigen.

[0067] Specifically, the dissociation equilibrium constant of the TCR to the pMHC complex to which it is directed is KD≤1 μM; preferably, KD≤100 nM; more preferably, KD≤1 nM.

[0068] In some embodiments of the present invention, the TCR may be a TCR comprising only two domains, Vα and Vβ, and the anti-CD3 antibody may also comprise only two domains, VH and VL.

[0069] In some embodiments of the present invention, a TCR with high affinity for the AFP antigen was selected for fusion protein construction. Specifically, the TCR exhibits high affinity for the complex of the AFP-derived antigenic peptide FMNKFIYEI and HLA A0201, with a KD value of 7.26E-11M, approximately one million times the affinity of a wild-type TCR for the corresponding complex. This TCR molecule is named AFP1, and its Vα and Vβ sequences are SEQ ID NO:1 and SEQ ID NO:2, respectively. The anti-CD3 antibody used is UCHT1, and its VH and VL sequences are SEQ ID NO:3 and SEQ ID NO:4, respectively.

[0070] The scDART fusion protein of the present invention is constructed by connecting Vα to VL and Vβ to VH. In one embodiment of the present invention, the structural schematic diagram of the construct is shown in Figure 1, and the connection method is Vα-SL-VL-LL-VH-SL-Vβ. The short linker sequence between Vα and VL is GGGSGGGGG (SEQ ID NO: 18), the short linker sequence between VH and Vβ is GGGSGGGGG, and the long linker sequence between VL and VH is GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 19). In one embodiment of the present invention, its sequence is SEQ ID NO: 5. In addition, a disulfide bond can be introduced between VH and VL, and the fusion protein construct is named scDART-QG. In one embodiment of the present invention, its sequence is: SEQ ID NO: 25. The site for introducing the disulfide bond is the classic Kabat disulfide bond in antibodies: H44-L100, that is, a disulfide bond is introduced between the VH and VL of the anti-CD3 antibody UCHT1. The positions where cysteine ​​residues were introduced in the sequence of SEQ ID NO: 25 are indicated by bold letters.

[0071] In one embodiment of the present invention, the dcDART fusion protein is constructed by connecting Vα to VL and Vβ to VH, and its structural diagram is shown in Figure 2. The short linker sequence between Vα and VL is SEQ ID NO: 18, and the short linker sequence between VH and Vβ is SEQ ID NO: 18. The sequences of Vα, Vβ, VH, and VL are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively. SEQ ID NO: 6 and SEQ ID NO: 7 are the sequences of the two cross-peptide chains (Vα-SL-VL, VH-SL-Vβ), respectively.

[0072] In one embodiment of the present invention, a disulfide bond VL:Q101C-VH:G45C is introduced between the VH and VL of the above-mentioned dcDART fusion protein construct to form a new construct dcDART-QG, the schematic diagram of which is shown in Figure 3. The sequences of the two chains (Vα-SL-VL, VH-SL-Vβ) are SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

[0073] In one embodiment of the present invention, the sequences FNRGEC (SEQ ID NO: 20) and VEPKSC (SEQ ID NO: 21) were introduced into the C-termini of the VL and Vβ of the above-mentioned dcDART fusion protein construct to form a disulfide bond, thereby forming a new construct dcDART-F, the structure of which is shown in Figure 4. The sequences of the two chains (Vα-SL-VL, VH-SL-Vβ) are SEQ ID NO: 10 and SEQ ID NO: 11, respectively.

[0074] In one embodiment of the present invention, the sequence LGGC (SEQ ID NO: 22) is introduced into the C-termini of both the VL and Vβ of the dcDART fusion protein construct to form a disulfide bond, forming a new construct dcDART-L, a schematic diagram of which is shown in Figure 5. The sequences of the two chains (Vα-SL-VL, VH-SL-Vβ) are SEQ ID NO: 12 and SEQ ID NO: 13, respectively.

[0075] In one embodiment of the present invention, the sequence GGCGGGKVAALKEKVAALKEKVAALKEKVAALKE (SEQ ID NO: 23) is introduced into the VL C-terminus of the above-mentioned dcDART fusion protein construct, and the Vβ C-terminus is connected to Fc by introducing the sequence GGCGGGEVAALEKEVAALEKEVAALEKEVAALEKGGG (SEQ ID NO: 24), which is shown in FIG6 . It is expected that a pair of disulfide bonds and a coil-coil structure will be formed between the linker at the C-terminus of VL and the linker at the C-terminus of Vβ, and two pairs of disulfide bonds will be formed between the N-terminus of Fc1 and the N-terminus of Fc2. The sequences of Fc1 and Fc2 are SEQ ID NO: 14 and SEQ ID NO: 15, respectively. The sequences of Vα-SL-VL and VH-SL-Vβ-Fc1 in the construct are SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0076] The scCIS structure of the present invention is a single-chain structure composed of the variable regions Vα and Vβ of a TCR connected by a flexible linker, and the light chain (VL) and heavy chain (VH) of an anti-CD3 antibody connected by a flexible linker. A schematic diagram of its structure is shown in Figure 7. In one embodiment of the present invention, the AFP1 molecule was selected to prepare the construct, and its sequence is SEQ ID NO: 26.

[0077] In another embodiment of the present invention, the TCR used to construct the scCIS structure is a TCR molecule with high affinity for a complex composed of the GP100-derived antigen peptide YLEPGPVTA and HLA A0201. This TCR is designated GP100. The sequence of the scCIS fusion molecule composed of the light chain (VL) and heavy chain (VH) of an anti-CD3 antibody is shown in SEQ ID NO: 27.

[0078] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, and the scope of the present invention will be limited only by the appended claims.

[0079] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0080] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[0081] the term

[0082] The terms used in the present invention have the following meanings:

[0083] TCR: T cell receptor

[0084] TCR-T: T cells transduced with exogenous TCR

[0085] MHC: Major histocompatibility complex

[0086] pMHC: peptide-major histocompatibility complex

[0087] HLA: Human leukocyte antigen, the major histocompatibility complex in humans

[0088] Vα: T cell receptor α chain variable region

[0089] Vβ: T cell receptor β chain variable region

[0090] VH: antibody heavy chain variable region

[0091] VL: antibody light chain variable region

[0092] TCE: A fusion molecule consisting of a T cell receptor (TCR) and an immune effector molecule connected in a specific way

[0093] TCE-T: T cells transduced with TCE

[0094] ELIspot: enzyme-linked immunospot assay

[0095] LDH: lactate dehydrogenase

[0096] Detailed Description of the Invention

[0097] The following specific examples further illustrate the present invention. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, were generally performed under conventional conditions, such as those described in (Sambrook and Russell et al., Molecular Cloning: A Laboratory Manual (3rd Edition) (2001) CSHL Press), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0098] Unless otherwise specified, the materials used in the embodiments of the present invention are all commercially available products.

[0099] Example 1 Expression and purification of fusion protein molecules.

[0100] The expression and purification process of the constructs scDART, scDART-QG, dcDART, dcDART-QG, dcDART-F, dcDART-L and scCIS fusion proteins of the present invention are as follows:

[0101] The fusion protein gene was connected to the pCDNA3.4 eukaryotic expression vector and used to transfect Gibco's Expi293F cells.

[0102] Expression: According to the instructions of Gibco Expi293F mammalian cell transient expression system.

[0103] Purification: In this example, the first step was purified using GE HisTrap HP, and the second step was purified using GE HiPrep 16 / 60S-100 molecular sieve.

[0104] The first step is HisTrap HP purification:

[0105] The HisTrap HP was equilibrated with 20mM PB, 300mM NaCl, pH 7.8 buffer (hereinafter referred to as Buffer A). The filtered eukaryotic expression sample was injected into the HisTrap HP at a rate of 4mL / min. After loading, the column was rinsed with Buffer A for 3-5 column volumes, and then eluted with 20mM PB, 300mM NaCl, 500mM imidazole, pH 7.8 (hereinafter referred to as Buffer B) at a rate of 2mL / min. The eluted peak was collected and analyzed on a non-reducing SDS-PAGE gel. Fractions containing the fusion protein were pooled and concentrated using a 50K concentrator for further purification.

[0106] The second step of molecular sieve purification:

[0107] A GE HiPrep 16 / 60 Sephacryl S-100 molecular sieve chromatography column was equilibrated with PBS. The concentrated HisTrap HP purified fractions were injected, not exceeding 2% of the column volume. The eluted peaks were collected and analyzed on reducing and non-reducing SDS-PAGE gels. Fractions containing the fusion protein were pooled, concentrated using a 50K concentrator, quantified using a BCA kit, and stored.

[0108] The affinity was detected using the BIAcore T200 real-time analysis system. Figures 8 to 14 are the affinity binding curves of the TCR end of scDART, scDART-QG, dcDART, dcDART-QG, dcDART-F, dcDART-L and scCIS with pMHC, and their KD values ​​were 9.150E-11M; 1.11E-10M; 1.03E-10M; 8.07E-11M; 1.05E-10M; 9.89E-11M, and 1.57E-10M, respectively. Figures 15 to 21 show the affinity binding curves of the CD3 antibody end of scDART, scDART-QG, dcDART, dcDART-QG, dcDART-F, dcDART-LUCHT1, and scCIS to CD3, with KD values ​​of 1.277E-08M, 1.31E-08M, 1.03E-08M, 1.06E-08M, 1.08E-08M, 1.22E-08M, and 4.49E-09M, respectively. After purification, the above-mentioned fusion molecules all maintain their good spatial folding and affinity.

[0109] Example 2 TCE transfection of T cells

[0110] A gene encoding the scCIS molecule AFP1 was transduced into T cells to generate TCE-expressing cells. Flow cytometry was used to assess the transfection efficiency and potential target cell binding of the TCE. The transfected TCE-T cells were stained with a tetramer (pep-HLA tetramer) containing pHLA, a TCR-specific target on the TCE, and then analyzed. The results are shown in Figure 22. The results showed that the GFP-positive rate of transfected T cells exceeded 80%, indicating successful transfection. Furthermore, nearly all GFP-positive and non-positive cells were stained with the pep-HLA tetramer, indicating that the secreted TCE bound to uninfected T cells and that the specific TCR was recognized and bound by the pHLA tetramer.

[0111] Example 3 ELISpot experiment to verify the function of TCE-T cells (the molecular form of TCE is scCIS, and the molecule used is AFP1)

[0112] IFN-γ release was measured using the BD ELISPOT (enzyme-linked immunospot) assay. This yield was used as a readout for cytotoxic T lymphocyte activation and to evaluate the efficacy of the effector peptide in the ability of TCE-T cells to mediate T cell activation and recruit bystander T cells to target tumor cells. In this experiment, T cells not transfected with TCE were designated as NC-T cells. The TCE molecular form used in this experiment was scCIS, and the molecule used was AFP1, whose sequence is SEQ ID NO: 26.

[0113] Procedure: First, prepare the ELISPOT plate. Activate the ELISPOT plate with ethanol and coat it at 4°C overnight. On the first day of the experiment, remove the coating solution, wash and block the plate, incubate at room temperature for two hours, remove the blocking solution, and add the test components to the ELISPOT plate: target cells are 2×10 4 cells / well, cells (CD8+ T cells) are 5-10×10 3 Each well was plated with two replicates. Effector T cells included TCE-T cells transfected with SEQ ID NO: 26 and untransfected NC-T cells from the same source. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, the plates were washed and subjected to secondary detection and color development. The plates were dried, and the spots formed on the membranes were counted using an ELISPOT READER system (AID20).

[0114] The test results are shown in Figure 23. The results show that the two different cell dosages of untransfected NC-T cells have no function. TCE-T cells can be activated by positive target cells HepG2 and JHH7 (A0201) in the presence of target cells, and their function becomes stronger as the number of T cells increases. In addition, the results when TCE-T and NC-T co-exist indicate that NC-T can be recruited in the presence of TCE-T, can be activated by positive target cells and function. At the same time, no non-specific reaction of T cells to negative target cells was observed during the entire reaction process.

[0115] Example 4. LDH Experiment Verification of TCE-T Cell Function (TCE Molecular Form is scCIS, Molecule Used is AFP1)

[0116] This example uses a non-radioactive cytotoxicity test to measure the release of LDH, thereby verifying the killing function of cells transduced with the TCR of the present invention. This test is a colorimetric alternative to the 51Cr release cytotoxicity test, which quantitatively measures lactate dehydrogenase (LDH) released after cell lysis. A 30-minute coupled enzyme reaction is used to detect LDH released in the culture medium. In the enzyme reaction, LDH can convert a tetrazolium salt (INT) into red formazan. The amount of red product generated is proportional to the number of cells lysed. A standard 96-well plate reader can be used to collect 490nm visible light absorbance data. In this experiment, T cells not transfected with TCE are recorded as NC-T. In this experiment, the molecular form of TCE is scCIS, and the molecule used is AFP1, whose sequence is SEQ ID NO: 26.

[0117] First, prepare the LDH plate and add the test components to the plate in the following order: 3 × 10 target cells 4 Each group of effector T cells was loaded in triplicate, with a different number of cells / well. Effector cells included TCE-T cells transfected with SEQ ID NO: 26 and untransfected NC-T cells from the same source. Incubate overnight (37°C, 5% CO2). On day 2 of the experiment, color development was detected, and after terminating the reaction, absorbance was recorded at 490 nm using a microplate reader (Bioteck).

[0118] The test results are shown in Figure 24. The results show that the two different cell dosages of untransfected NC-T cells were not functional. TCE-T cells were able to kill positive target cells HepG2 in the presence of target cells, and the intensity increased with the increase of T cells. In addition, the results when TCE-T and NC-T co-existed showed that NC-T could be recruited to kill target cells in the presence of TCE-T. At the same time, no non-specific reaction of T cells to negative target cells was observed during the entire reaction process.

[0119] Example 5 ELISpot assay to verify TCE-T cell function (TCE molecular forms are scDART and dcDART-F, and the molecule used is AFP1)

[0120] The BD ELISPOT (enzyme-linked immunospot) assay was used to measure the release of IFN-γ and measure the production as a readout value after activation of cytotoxic T lymphocytes to evaluate the efficacy of the effector molecule polypeptide in the ability of TCE fusion proteins secreted by TCE-T to mediate T cells and recruit bystander T cells to target tumor cells. In this experiment, T cells not transfected with TCE were recorded as NC-T. The molecular forms of TCE in this experiment were scDART and dcDART-F, and the molecule used was AFP1. The sequence of scDART is SEQ ID NO: 5, and the sequences of the two chains of dcDART-F (Vα-SL-VL, VH-SL-Vβ) are SEQ ID NO: 10 and SEQ ID NO: 11, respectively. The positive tumor cell line used in the example is HepG2, and the negative tumor cell line used is NCI-H1650.

[0121] Procedure: First, prepare the ELISPOT plate. Activate the ELISPOT plate with ethanol and coat it at 4°C overnight. On the first day of the experiment, remove the coating solution, wash and block the plate, incubate at room temperature for two hours, remove the blocking solution, and add the test components to the ELISPOT plate: target cells are 2×10 4 cells / well, effector cells (CD8+ T cells) were 5 or 10×10 3 Each well was plated with two replicates. The effector T cells included TCE-T cells transfected with the aforementioned AFP1 in the molecular form of scDART and dcDART-F, and NC-T cells transfected with scAFP-TCE from the same source. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, the plates were washed and subjected to secondary detection and color development. The plates were dried, and the spots formed on the membrane were counted using an ELISPOT READER system (AID20).

[0122] The test results are shown in Figure 25. The results show that the two different cell dosages of untransfected NC-T cells have basically no function. TCE-T cells can be activated by positive target cells HepG2 in the presence of target cells, and their function becomes stronger as the number of T cells increases. In addition, the results when TCE-T and NC-T co-exist indicate that NC-T can be recruited in the presence of TCE-T, can be activated by positive target cells and function. At the same time, no non-specific reaction of T cells to negative target cells was observed during the entire reaction process.

[0123] Example 6. LDH test to verify TCE-T cell function (TCE molecular forms are scDART and dcDART-F, and the molecule used is AFP1)

[0124] This example uses a non-radioactive cytotoxicity experiment to measure the release of LDH, thereby verifying the killing function of cells transduced with the TCR of the present invention. In this experiment, T cells not transfected with TCE are recorded as NC-T. In this experiment, the molecular forms of TCE are scDART and dcDART-F, and the molecule used is AFP1. The sequence of scDART is SEQ ID NO: 5, and the sequences of the two chains of dcDART-F (Vα-SL-VL, VH-SL-Vβ) are SEQ ID NO: 10 and SEQ ID NO: 11, respectively. The positive tumor cell line used in the embodiment is, and the negative tumor cell line used is.

[0125] Procedure: First, prepare the LDH plate and add the test components to the plate in the following order: 3×10 target cells 4 cells / well, and the amount of effector T cells added to each group was different (3 or 6×10 4 Each well was set up in triplicate. Effector T cells included TCE-T cells transfected with the aforementioned AFP1 in the form of scDART and dcDART-F, and untransfected NC-T cells of the same source. The cells were incubated overnight (37°C, 5% CO2). On the second day of the experiment, color development was detected, and after terminating the reaction, absorbance was recorded at 490 nm using a microplate reader (Bioteck).

[0126] The test results are shown in Figure 26. The results show that the two different cell dosages of untransfected NC-T cells were ineffective. TCE-T cells were able to kill positive target cells HepG2 in the presence of target cells, and the intensity increased with the increase of T cells. In addition, the results when TCE-T and NC-T co-existed showed that NC-T could be recruited to kill target cells in the presence of TCE-T. At the same time, no non-specific reaction of T cells to negative target cells was observed during the entire reaction process.

[0127] Example 7 ELISpot experiment to verify the function of TCE-T cells (the molecular form of TCE is scCIS, and the molecule used is GP100)

[0128] In this experiment, T cells not transfected with TCE were designated as NC-T. The TCE molecular form used in this experiment was scCIS, and the molecule used was GP100. The sequence of the fusion molecule is SEQ ID NO: 27. The positive tumor cell line used in the examples was Mel526, and the negative tumor cell line used was NCI-H1650.

[0129] Procedure: First, prepare the ELISPOT plate. Activate the ELISPOT plate with ethanol and coat it at 4°C overnight. On the first day of the experiment, remove the coating solution, wash and block the plate, incubate at room temperature for two hours, remove the blocking solution, and add the test components to the ELISPOT plate: target cells are 2×10 4 cells / well, effector cells (CD8+ T cells) were 1 or 2×10 4 Each well was plated with duplicate wells. Effector T cells were transfected with TCE-T cells containing the aforementioned GP100 in the form of scCIS, and untransfected NC-T cells containing the same T cell source, GP100-TCE. The cells were incubated overnight (37°C, 5% CO2). On the second day of the experiment, the plates were washed and subjected to secondary detection and color development. The plates were dried, and the spots formed on the membranes were counted using an ELISPOT READER system (AID20).

[0130] The test results are shown in Figure 27. The results show that the two different cell dosages of untransfected NC-T cells have basically no function. TCE-T cells can be activated by positive target cells Mel526 in the presence of target cells, and their function becomes stronger as the number of T cells increases. In addition, the results when TCE-T and NC-T co-exist indicate that NC-T can be recruited in the presence of TCE-T, can be activated by positive target cells and function. At the same time, no non-specific reaction of T cells to negative target cells was observed during the entire reaction process.

[0131] Example 8. LDH Experiment Verification of TCE-T Cell Function (TCE Molecular Form is scCIS, Molecule Used is GP100)

[0132] This example demonstrates the cytotoxicity of cells transduced with the TCR of the present invention by measuring LDH release through a non-radioactive cytotoxicity assay. In this experiment, T cells not transfected with TCE are designated as NC-T cells. The TCE in this experiment is in the form of scCIS, and the molecule used is GP100. The sequence of the fusion molecule used is SEQ ID NO: 27. The positive tumor cell line used in this example is Mel526, and the negative tumor cell line used is NCI-H1650.

[0133] First, prepare the LDH plate and add the test components to the plate in the following order: 3 × 10 target cells 4 cells / well, and the amount of effector T cells added to each group was different (0.6 or 1.2×10 5Each well was set up in triplicate. Effector T cells included TCE-T cells transfected with the aforementioned GP100 in the form of scCIS and untransfected NC-T cells from the same source. The cells were incubated overnight (37°C, 5% CO2). On the second day of the experiment, color development was detected, and after terminating the reaction, absorbance was recorded at 490 nm using a microplate reader (Bioteck).

[0134] The test results are shown in Figure 28. The results show that the two different cell dosages of untransfected NC-T cells were not functional. TCE-T cells were able to kill the positive target cells MEWO in the presence of target cells, and the intensity increased with the increase of T cells. In addition, the results when TCE-T and NC-T co-existed showed that NC-T could be recruited to kill target cells in the presence of TCE-T. At the same time, no non-specific reaction of T cells to negative target cells was observed during the entire reaction process.

[0135] Example 9 ELISpot experiment to verify the function of TCE-transfected 293T cells

[0136] In this experiment, TCE-transfected 293T cells were used as secretory cells, and T cells not transfected with TCE were recorded as NC-T. The molecular forms of TCE in this experiment were scCIS, scDART, and dcDART, and the molecule used was AFP1. The positive tumor cell line used in the examples was NCI-H1650 loaded with the short antigen peptide PX551 (FMNKFIYEI) corresponding to the AFP1 molecule, and the negative tumor cell line used was NCI-H1650, purchased from ATCC.

[0137] Procedure: First, prepare the ELISPOT plate. Activate the ELISPOT plate with ethanol and coat it at 4°C overnight. On the first day of the experiment, remove the coating solution, wash and block the plate, incubate at room temperature for two hours, remove the blocking solution, and add the test components to the ELISPOT plate: target cells are 1×10 4 500 TCE-transfected 293T cells were plated per well, with duplicate wells set up for each group. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, wash the plates and perform secondary detection and color development. Dry the plates, and count the spots formed on the membrane using an ELISPOT READER system (AID20).

[0138] The experimental results are shown in Figures 29a, 29b, and 29c. In the absence of 293T cells, NC-T cells were nonfunctional. In the presence of 293T cells, NC-T cells were recruited and activated, and their activity gradually increased with increasing cell number, with no response to negative cells.

[0139] Example 10 LDH experiment verifies the function of TCE-transfected 293T cells

[0140] In this experiment, 293T cells transfected with TCE served as secretory cells, while T cells not transfected with TCE were designated as NC-T cells. TCEs were administered in the form of scCIS, scDART, and dcDART, and the molecule used was AFP1. The positive target cell line used in this experiment was HepG2, and the negative target cell line used was NCI-H1650.

[0141] First, prepare the LDH plate and add the test components to the plate in the following order: 3 × 10 target cells 4 cells / well, 3 or 6×10 cells / well for TCE-transfected 293T cells 4 Each well was incubated overnight (37°C, 5% CO2). On the second day of the experiment, the color development was detected and the absorbance was recorded at 490 nm using a microplate reader (Bioteck) after the reaction was terminated.

[0142] The experimental results are shown in Figure 30. In the absence of 293T cells, NC-T cells were inactive. However, in the presence of 293T cells transfected with different molecular forms, NC-T cells were recruited through the fusion molecules secreted by the 293T cells and exhibited strong cytotoxicity against target cells.

[0143] Example 11 LDH experiment verifies the function of TCE-transfected LCL cells

[0144] In this experiment, LCL cells transfected with TCE served as secretory cells, while T cells not transfected with TCE were designated as NC-T cells. TCEs were administered in the form of scCIS, scDART, and dcDART, and the molecule used was AFP1. The positive target cell line used in this experiment was HepG2, and the negative target cell line used was NCI-H1650.

[0145] First, prepare the LDH plate and add the test components to the plate in the following order: 3 × 10 target cells 4 cells / well, and the number of LCL cells transfected with TCE was 1.5 or 3×10 4 / (calculated based on the positive rate), with three replicates per group. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, detect color development, terminate the reaction, and record the absorbance at 490 nm using a microplate reader (Bioteck).

[0146] The experimental results are shown in Figure 31. In the absence of LCL cells, NC-T cells were inactive. However, in the presence of LCL cells transfected with different molecular forms, NC-T cells were recruited through the fusion molecules secreted by the LCL cells and exhibited strong cytotoxicity against target cells.

[0147] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An engineered cell, characterized in that The gene of a fusion molecule composed of a T cell receptor (TCR) and an immune effector molecule connected in a specific manner is transduced, and the fusion molecule is secreted and expressed outside the cell.

2. The cell according to claim 1, wherein The immune effector molecule in the fusion molecule is an anti-CD antibody.

3. The cell according to claim 2, wherein The anti-CD antibody is an anti-CD3 antibody.

4. The cell according to claim 1, wherein The cells are T cells, NK cells or NK-T cells; preferably, the cells are T cells.

5. The cell according to claim 1, wherein The immune effector molecule in the fusion molecule is a cytokine; preferably, the cytokine is IL interleukin, TGF-β, IFN-γ or TNFα.

6. The cell according to claim 3, wherein The fusion molecule is a single-chain structure formed by connecting the TCR variable regions Vα and Vβ via a flexible linker, and the light chain (VL) and heavy chain (VH) of an anti-CD3 antibody via a flexible linker.

7. The cell according to claim 1, wherein The fusion molecule transduced into the cell is composed of Vα and Vβ of the TCR molecule, and VH and VL of the anti-CD3 antibody molecule.

8. The cell according to claim 7, wherein The TCR and the anti-CD3 antibody molecule are connected in such a way that the Vα of the TCR and the VL of the anti-CD3 antibody are connected via a short linker sequence, and the Vβ of the TCR and the VH of the anti-CD3 antibody are connected via a short linker sequence.

9. The cell according to claim 8, wherein The short linker sequence is SEQ ID NO:

18.

10. The cell according to claim 8, wherein The VH and VL of the anti-CD3 antibody are connected by a long linker sequence.

11. The cell according to claim 10, wherein The long linker sequence is SEQ ID NO:

19.

12. The cell according to claim 8, wherein The VH and VL of the anti-CD3 antibody are linked by a disulfide bond.

13. The cell according to claim 8, wherein The Vβ of TCR and the VL of anti-CD3 antibody are linked by a disulfide bond.

14. The cell according to claim 13, wherein The disulfide bond was formed by introducing SEQ ID NO: 20 into the C-terminus of the anti-CD3 antibody VL and SEQ ID NO: 21 into the C-terminus of the TCRVβ.

15. The cell according to claim 13, wherein The disulfide bond was formed by introducing SEQ ID NO: 22 into both the C-terminus of TCRVβ and the C-terminus of the anti-CD3 antibody VL.

16. The cell according to claim 1, wherein The fusion molecule secreted and expressed by the cells can simultaneously bind to effector cells and tumor cells; preferably, the effector cells are T cells.

17. The cell according to claim 1, wherein The gene of the fusion molecule is transduced into the cells via lentivirus or electroporation.

18. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleic acid sequence encoding the fusion molecule of claim 1 or a complementary sequence thereof.

19. A carrier, characterized in that The vector contains the nucleic acid molecule according to claim 8.

20. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the cells according to claim 1.

21. A method for treating a disease, characterized in that: The method comprises administering the cell of claim 1 to a subject in need of treatment.

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