Fusion protein, engineered immune cell, preparation method and application thereof

Abstract

The present application relates to the field of biomedical technology, and in particular to a fusion protein, an engineered immune cell, and a method for preparing and applying the same. From the extracellular domain to the intracellular domain, the fusion protein includes an IL-12p40 subunit and a membrane protein, and the membrane protein is used to anchor the IL-12p40 subunit to the cell membrane. The present application connects the p40 subunit of IL-12 to a membrane protein with an anchoring effect, so that the p40 subunit is anchored to the cell membrane surface of an immune cell (e.g., TIL) by a membrane protein, thereby achieving the anti-tumor killing effect of enhancing TIL, and at the same time, also improves the safety of IL-12 for anti-tumor treatment.

Description

Technical Field

[0001] The present application relates to the field of biomedical technology, and in particular to a fusion protein, an engineered immune cell, and a preparation method and application thereof. Background Art

[0002] Tumor infiltrating lymphocytes (TILs) are a heterogeneous cell population composed of CD8+T cells, CD4+T cells, B cells, NK cells, γδT cells, and innate lymphocytes (ILCs). After in vitro expansion and culture, CD3+T cells become the most important cell population (usually accounting for more than 95%). CD3+T cells express TCR receptors on their surface and play a key role in mediating the immune killing of cancerous cells. Different TCR receptors recognize different antigenic peptides presented by major histocompatibility complex (MHC) molecules, thereby giving T cells antigen specificity. Therefore, once T cells specific to an antigen are stimulated by a specific antigen, these cells can promote the programmed death of target cells by releasing cytotoxins and receptor-mediated mechanisms.

[0003] Unlike CAR-T (Chimeric Antigen Receptor T-Cell) / TCR-T cell therapy products, TILs usually play an anti-tumor role by recognizing tumor-associated antigens presented by tumor cells, such as neoantigens. Therefore, TIL therapy can achieve anti-tumor treatment by recognizing and targeting multiple tumor-associated antigens.

[0004] However, due to the regulation of various immunosuppression-related factors in the tumor microenvironment, TIL highly expresses immune checkpoint-related molecules, such as PD-1, CTLA4, TIM3, LAG3, TIGIT, etc. At the same time, due to the continuous stimulation of tumor-associated antigens, TIL presents a highly exhausted state, such as high expression of exhaustion-related transcription factors such as Tox and Id3. Even if TIL is separated and reactivated in vitro, the killing function of TIL is still limited. Therefore, how to enhance the continuous anti-tumor function of TIL is a technical problem that needs to be solved urgently. Summary of the invention

[0005] Based on this, one or more embodiments of the present application provide a fusion protein, an engineered immune cell, and a preparation method and application thereof.

[0006] One or more embodiments of the present application provide a fusion protein;

[0007] From one end of the extracellular domain to one end of the intracellular domain, the fusion protein includes an IL-12p40 subunit and a membrane protein, and the membrane protein is used to anchor the IL-12p40 subunit on the cell membrane.

[0008] In some embodiments of the present application, the membrane protein includes one or more of type I membrane protein, type II membrane protein, multi-spanning membrane protein and glycosylphosphatidylinositol (GPI) anchored protein.

[0009] In some embodiments of the present application, the membrane protein has a transmembrane domain or a partial region of the transmembrane domain of at least one of the following proteins: α chain of T cell receptor, β chain of T cell receptor, ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD48, CD55, CD64, CD80, CD86, CD90, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, ICOS, GITR, CD40, BAFFR, HVEM, SLAMF7, NKp80, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, TGF βRII, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, ITGB7, TNFR2, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, Ly9, PSGL1, CD100, SLAMF6, SLAM, BLAME, SELPLG, LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C.

[0010] In some embodiments of the present application, the IL-12p40 subunit and the membrane protein are directly or indirectly connected.

[0011] In some embodiments of the present application, the IL-12p40 subunit and the membrane protein are connected via one or more of a flexible linker and / or a hinge region.

[0012] In some embodiments of the present application, the hinge region is selected from the hinge region of at least one of the following proteins: IgG1, IgG4, IgG4m, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154.

[0013] In some embodiments of the present application, the fusion protein includes the IL-12p40 subunit, the flexible linker, the hinge region and the membrane protein in sequence from the extracellular domain to the intracellular domain.

[0014] In some embodiments of the present application, a signal peptide or an intrinsic signal anchor sequence fragment is connected to the N-terminus of the fusion protein.

[0015] One or more embodiments of the present application provide an engineered immune cell, which overexpresses the fusion protein.

[0016] In some embodiments of the present application, the engineered immune cells include one or more of CAR-T, TCR-T, TIL and CAR-NK.

[0017] One or more embodiments of the present application provide a method for preparing engineered immune cells, which comprises the following steps:

[0018] An immune cell to be transformed is provided, and the immune cell to be transformed is transformed to express the fusion protein, thereby constructing an engineered immune cell.

[0019] One or more embodiments of the present application provide a nucleic acid molecule encoding the above-mentioned fusion protein.

[0020] One or more embodiments of the present application provide a vector comprising the nucleic acid molecule.

[0021] In some embodiments of the present application, the vector comprises one or more of a lentivirus, a γ-retrovirus vector and a lipid nanoparticle.

[0022] One or more embodiments of the present application provide a use of the engineered immune cell, the nucleic acid molecule or the vector in the preparation of an anti-tumor drug.

[0023] In some embodiments of the present application, the anti-tumor drug prevents and / or treats the following tumors: solid tumors such as liver cancer, colon cancer, rectal cancer, ovarian cancer, cervical cancer, gastric cancer, pancreatic cancer, lung cancer, breast cancer, melanoma, etc.

[0024] One or more embodiments of the present application provide a drug comprising the engineered immune cells and a pharmaceutically acceptable carrier.

[0025] Compared with the traditional technology, the beneficial effects of this application include:

[0026] The present invention connects the p40 subunit of IL-12 to a membrane protein with an anchoring function, so that the p40 subunit is anchored on the cell membrane surface of immune cells (such as TIL) through the membrane protein, thereby unexpectedly enhancing the proliferation ability of immune cells and the anti-tumor killing effect. At the same time, it also improves the safety of IL-12 for anti-tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0028] Figure 1 It is a schematic diagram of the structure of membrane-bound IL-12;

[0029] Figure 2 Flow cytometry was used to detect the expression of membrane-bound IL-12 (p35 and p40);

[0030] Figure 3 Flow cytometry validation of membrane-bound IL-12 (p35 and p40);

[0031] Figure 4 The effect of overexpression of membrane-bound IL-12 (p35 and p40) on TIL proliferation and function;

[0032] Figure 5 To investigate the effect of overexpression of BN115 on TIL proliferation and function;

[0033] Figure 6 BN115 enhances the tumor cell killing effect of TIL (in vitro);

[0034] Figure 7 BN115 enhances the killing of CAR-T cells by tumor cells (in vitro);

[0035] Figure 8 BN115 enhances the tumor cell killing effect of TIL (in vivo). DETAILED DESCRIPTION

[0036] The present application will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and are not used to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms, is not limited to the embodiments and examples described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the present application, and the equivalent form obtained also falls within the protection scope of the present application. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present application, and it should be understood that the present application can be implemented without one or more of these details.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing implementation modes and embodiments and are not intended to limit this application.

[0038] the term

[0039] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0040] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0041] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0042] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0043] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0044] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of this application.

[0045] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0046] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0047] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0048] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0049] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0050] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0051] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0052] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0053] IL-12 is a pro-inflammatory cytokine secreted by antigen-presenting cells (such as dendritic cells, macrophages, and neutrophils) after antigen stimulation. IL-12 consists of two subunits, p35 (i.e., IL-12α) and p40 (i.e., IL-12β). Among them, p40 can form the cytokine IL-23 with p19, and p35 can form the cytokine IL-35 with Ebi3. These cytokines are collectively referred to as IL-12 family cytokines. During the immune response, IL-12 activates the downstream STAT4 signaling pathway by binding to its receptor (IL-12R, composed of IL-12Rb1 and IL-12Rb2) to promote the activation of NK cells and T cells. Among them, IL-12Rb2 is upregulated in activated T cells, which is crucial for IL-12 to enhance the immune response of T cells. IL-12 enhances the proliferation, survival, and killing of CD8+T cells by upregulating the expression of IFNg and TNFa in CD8+T cells. In NK cells, the signals activated by IL-12 and IL-2 interact with each other to upregulate IFNg expression in NK cells to enhance the immune response of NK cells. However, existing research results have shown that excessive or systemic IL-12 anti-tumor therapy has shown excessive toxic side effects. Therefore, through gene editing, in specific cells, while achieving overexpression of IL-12 (full-length IL-12 or IL-12a or IL-12b), the cytokine membrane is anchored on the cell membrane surface, which can ensure the safety of treatment while enhancing the anti-tumor therapeutic effect of T cells.

[0054] The document titled "Interleukin-23engineering improves CAR T cell function insolid tumors" enhances the sustained killing function of T cells by overexpressing the secretory form of p40 (a subunit of IL-12 and IL-23) in CAR-T cells. Since activated CAR-T does not express P35 (IL-12α, which forms the full-length IL-12 with p40), p19 is significantly upregulated, which combines with p40 to form IL-23 to promote the killing function of T cells. However, in the scheme, p40 combines with p35 and eventually exists as secretory IL-23, which is distributed to various tissues and organs through blood circulation, has a wide range of action, and still has certain toxic side effects.

[0055] The documents titled "Cell membrane-anchored and tumor-targeted IL-12 (attIL12)-Tcell therapy for eliminating large and heterogeneous solid tumors" and CN110267978A overexpress a membrane-bound p35 in T cells and combine it with secretory p40 to form membrane-bound IL-12, so as to limit the activation function of IL-12 to the local area on the surface of T cells. However, in this scheme, p40 is expressed in a secretory form and will be distributed to various tissues and organs through blood circulation. It has a wide range of action and still has certain toxic side effects.

[0056] The article titled "Reprogramming of IL-12 secretion in the PDCD1 locus improves the anti-tumor activity of NY-ESO-1TCR-T cells" knocked in the IL-12 gene downstream of the start code of the PD-1 gene in T cells, so that when TCR-T cells are activated, IL-12 is conditionally expressed through the activation of PD-1 expression, thereby enhancing the tumor killing effect of TCR-T cells. However, the IL-12 in this scheme is overexpressed in a secretory form and will be distributed to various tissues and organs through the blood circulation. It has a wide range of effects and still has toxic side effects.

[0057] The embodiment of the present application connects the p40 subunit of IL-12 to a membrane protein with an anchoring effect, so that the p40 subunit is anchored on the cell membrane surface of immune cells (such as TIL) through the membrane protein, thereby enhancing the anti-tumor killing effect of immune cells. At the same time, since the p40 subunit in this design cannot exist in a free form, its activation function is only limited to a local range, thereby overcoming the cytotoxic effect of IL-12 when activating T cell anti-tumor immunity. On the whole, the embodiment of the present application is more conducive to improving the therapeutic effect of solid tumors such as liver cancer, ovarian cancer, cervical cancer, and colorectal cancer by overexpressing the p40 subunit of membrane-bound IL-12. The following contents:

[0058] In a first aspect of the embodiments of the present application, a fusion protein is provided;

[0059] From the extracellular domain to the intracellular domain, the fusion protein includes an IL-12p40 subunit and a membrane protein, and the membrane protein is used to anchor the IL-12p40 subunit on the cell membrane.

[0060] “IL-12”, “IL-12 protein”, “IL-12 protein” may be used as concepts including “IL-12 protein and fragments thereof”. Unless otherwise specified, the terms “protein”, “polypeptide” and “peptide” may be used as interchangeable concepts.

[0061] "Subunit" refers to a part of the quaternary structure of a protein. In a protein composed of multiple amino acid chains, each amino acid chain is a subunit. IL-12 is composed of two subunits, p35 and p40.

[0062] The present application does not particularly limit the sequence of the IL-12p40 subunit, and the IL-12p40 may have a wild-type sequence (eg, SEQ ID NO. 2) or a conservative mutant thereof.

[0063] "Conservative mutants" refer to mutants modified on the basis of wild-type IL-12p40, and the activity or function (including affinity with receptors) of the resulting mutants are consistent with wild-type IL-12p40. Engineered immune cells with "conservative mutants" have consistent biological properties with engineered immune cells with wild-type IL-12p40.

[0064] For example, the conservative mutant of the p40 subunit has at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids replaced by amino acids with similar or similar properties compared to the p40 subunit of wild-type interleukin-12. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1 below.

[0065] Table 1

[0066]

[0067]

[0068] For example, a conservative mutant of the p40 subunit is prepared by a method of replacing, deleting or adding at least one amino acid in a wild-type p40 subunit without changing the function of IL-12, or a conservative mutant of the p40 subunit is prepared by a method of replacing, deleting or adding at least one amino acid in a wild-type p40 subunit without changing the function of the p40 subunit, and these conservative mutants have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with their corresponding wild types.

[0069] Traditionally, conservative mutants may have some conservative amino acid substitutions relative to wild-type subunits, and such conservative amino acid substitutions have no effect or weak effect on the overall protein charge, i.e., polarity or hydrophobicity. For conservative amino acid substitutions, reference may be made to Table 2 below.

[0070] Table 2

[0071]

[0072] For each amino acid, additional conservative substitutions include "homologues" of the amino acid. Specifically, "homologues" refer to amino acids obtained by inserting a methylene group (CH2) into the β-side chain of the amino acid side chain. Examples of "homologues" may include homophenylalanine, homoarginine, homoserine, etc., but are not limited thereto.

[0073] The present application does not specifically limit the species of the p40 subunit, which can be mammals (humans, mice, rats, guinea pigs, rabbits, macaques, goats, sheep, horses, cattle, pigs, dogs, etc.), or poultry such as chickens, geese, and ducks. In some embodiments, the p40 subunit is derived from mammals. Preferably, the p40 subunit is derived from humans, and IL-12β subunits from other species can replace human IL-12β subunits to achieve similar functions.

[0074] The present application does not specifically limit the type of membrane protein, which may be selected from but not limited to type I membrane proteins, type II membrane proteins, multi-transmembrane proteins and glycosylphosphatidylinositol-anchored proteins.

[0075] In some embodiments, the membrane protein has a transmembrane domain or a partial region of the transmembrane domain of at least one of the following proteins: α chain of T cell receptor, β chain of T cell receptor, ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD48, CD55, CD64, CD80, CD86, CD90, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, ICOS, GITR, CD40, BAFFR, HVEM, SLAMF7, NKp80, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, TGFβR II, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, ITGB7, TNFR2, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, Ly9, PSGL1, CD100, SLAMF6, SLAM, BLAME, SELPLG, LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C. It can be understood that the membrane protein in the embodiments of the present application can be the transmembrane domain of the above-mentioned protein, or a partial region of the transmembrane domain, or a conservative mutant that maintains the same function as the transmembrane domain. It is understood that, according to the needs, the transmembrane domain can also be modified to form a functional consistency modification. The source of the transmembrane domain is not particularly limited in the present embodiment, and can be derived from mammals or other species, preferably mammals, especially humans.

[0076] The present application does not specifically limit the connection mode of the IL-12p40 subunit and the membrane protein. The two can be directly connected through an amide bond, or indirectly connected through other auxiliary linkers. According to different membrane proteins, "direct connection", "indirect connection through a flexible linker", "indirect connection through a hinge region", "through a flexible linker + hinge region" and the like can all be the optimal solution. In some embodiments, the IL-12p40 subunit and the membrane protein are connected through one or more of the flexible linker and / or hinge region. In one embodiment, the IL-12p40 subunit and the membrane protein are connected through the hinge region. In one embodiment, the IL-12p40 subunit and the membrane protein are connected through a flexible linker and a hinge region. In one embodiment, other proteins (such as p35 subunits) are also connected between the IL-12p40 subunit and the membrane protein, and further, other proteins and the p40 subunit are connected through a flexible linker, and other proteins and the membrane protein are connected through a hinge region.

[0077] The hinge region is not particularly limited in the embodiments of the present application, and can be selected from but not limited to the hinge region of at least one of the following proteins: IgG1, IgG4, IgG4m, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154. It is understood that the hinge region in the embodiments of the present application can be the complete hinge region of the above-mentioned protein, or a partial region thereof, or a conservative mutant that maintains the same function as the hinge region. It is understood that, according to the needs, some modifications can also be made to the hinge region to form a functional consistency modification. The source of the hinge region is not particularly limited in the embodiments of the present application, and can be derived from mammals or other species, preferably from mammals, especially humans.

[0078] In some embodiments, the fusion protein includes the IL-12p40 subunit, the flexible linker, the hinge region, and the membrane protein in sequence.

[0079] In some embodiments, the N-terminus of the IL-12p40 subunit is linked to a signal peptide or an intrinsic signal anchor sequence fragment.

[0080] The second aspect of the embodiment of the present application provides an engineered immune cell, which overexpresses the fusion protein. The membrane protein anchors the IL-12p40 subunit on the cell membrane of the engineered immune cell.

[0081] In some embodiments, the engineered immune cells include one or more of CAR-T, TCR-T, TIL and CAR-NK. In a third aspect of the embodiment of the present application, a method for preparing the engineered immune cells is provided, which comprises the following steps:

[0082] An immune cell to be transformed is provided, and the immune cell to be transformed is transformed to express the fusion protein, thereby constructing an engineered immune cell.

[0083] A fourth aspect of the embodiments of the present application provides a nucleic acid molecule encoding the above-mentioned fusion protein.

[0084] As used herein, the term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence.

[0085] The nucleic acid molecules of the present application mainly refer to isolated nucleic acid molecules. "Isolated" refers to molecules that are substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates or other materials, such as cell debris and growth medium. Generally, the term "isolated" is not intended to refer to the complete absence of these materials or the absence of water, buffer or salts, unless they are present in an amount that significantly interferes with the experimental or therapeutic use of the compounds as described herein.

[0086] A fourth aspect of the embodiments of the present application provides a vector comprising the nucleic acid molecule.

[0087] The term "vector" is also "expression vector" and refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. In one embodiment, the vector is a viral vector, in which additional DNA segments can be connected to the viral genome. The vector disclosed herein can replicate autonomously in the host cell into which they have been introduced (e.g., bacterial vectors and additional mammalian vectors with bacterial replication origins) or can be integrated into the genome of the host cell after introduction into the host cell, thereby replicating with the host genome (e.g., non-additional mammalian vectors). Overall, the vector can be selected from, but not limited to, lentivirus, gamma retrovirus vectors, and lipid nanoparticles.

[0088] The fifth aspect of the embodiments of the present application provides the use of the engineered immune cells, the nucleic acid molecules or the vectors in the preparation of anti-tumor drugs.

[0089] "Anti" means "prevention", "treatment", "relief" and "inhibition".

[0090] In some embodiments, the anti-tumor drug prevents and / or treats the following but not limited to the following tumors: liver cancer, colon cancer, rectal cancer, ovarian cancer, cervical cancer, gastric cancer, pancreatic cancer, lung cancer, breast cancer, melanoma.

[0091] In a sixth aspect of the embodiments of the present application, a drug is provided, which includes the engineered immune cells described above, and a pharmaceutically acceptable carrier.

[0092] "Drug" refers to a mixture containing the engineered immune cells or other components. The purpose of a drug is to facilitate administration to an organism, facilitate the absorption of active ingredients, and thus exert biological activity.

[0093] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivering an active ingredient. The carrier can be an antiadhesive, adhesive, coating, disintegrant, filler or diluent, preservative (such as an antioxidant, antibacterial or antifungal agent), sweetener, absorption delay agent, wetting agent, emulsifier, buffer, etc. The example of a suitable pharmaceutically acceptable carrier includes water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) dextrose, vegetable oil (such as olive oil), saline, buffer, buffered saline and isotonic agents such as sugar, polyols, sorbitol and sodium chloride. It is to be understood that the present application embodiment is not particularly limited to the type and dosage of the carrier, and a suitable carrier can be selected according to clinical needs and pharmaceutical dosage forms.

[0094] A seventh aspect of the embodiments of the present application provides a method for treating tumors, comprising: administering an effective amount of the engineered immune cells or the drug to a subject.

[0095] "Administering," "giving," and "treating" when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administering," "giving," and "treating" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administering," "giving," and "treating" also mean in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnosis, a combination composition, or by another cell. "Treatment," when applied to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0096] "Treatment" means administering an internal or external therapeutic agent, such as any of the immune engineered cells and drugs disclosed herein, to a patient who has one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms in the treated patient or population to induce the regression of such symptoms or inhibit the development of such symptoms to any clinically measured extent. The amount of the therapeutic agent that effectively alleviates any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptoms.

[0097] "Effective amount" or "effective dose" refers to the amount of a drug, compound or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results. For preventive uses, beneficial or desired results include eliminating or reducing the risk, reducing the severity or delaying the onset of a condition, including the biochemical, histological and / or behavioral symptoms of the condition, its complications and the intermediate pathological phenotypes presented during the development of the condition. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of various related conditions mentioned in the application or improving one or more symptoms of the condition, reducing the dose of other agents required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of the patient's tumor of the present application embodiment.

[0098] The subject is an animal. An animal is a living multicellular vertebrate or invertebrate organism, and categories include, for example, mammals. The term mammal includes human mammals and non-human mammals. Similarly, the term "subject" includes humans and veterinary subjects, such as non-human primates. Therefore, administration to a subject may include administration to a human subject. Non-limiting examples of veterinary subjects include domestic animals (e.g., cats and dogs), livestock (e.g., cattle, horses, pigs, sheep, and goats), and experimental animals (e.g., mice, rabbits, rats, gerbils, guinea pigs, and non-human primates).

[0099] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0100] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0101] The structural design of each molecule is as follows Figure 1 As shown, Figure 1 It is a schematic diagram of the structure of membrane-bound IL-12.

[0102] Human IL-12α [abbreviated as p35], SEQ ID NO.1:

[0103] RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS.

[0104] Human IL-12β [abbreviated as p40], SEQ ID NO.2:

[0105] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATL SAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS.

[0106] Human CD8 Hinge domain [abbreviated as CD8 Hinge], SEQ ID NO.3:

[0107] FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD.

[0108] Human IgG1 Fc Hinge domain, SEQ ID NO.4:

[0109] ASESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0110] Human CD8 transmembrane domain, SEQ ID NO.5: IYIWAPLAGTCGVLLLSLVITLYCNHRN.

[0111] Connecting peptide G6S linker, SEQ ID NO.6: GGGGGGS.

[0112] Signal peptide 1, SEQ ID NO. 7: MCPARSLLLVATLVLLDHLSLA.

[0113] Signal peptide 2, SEQ ID NO. 8: MCHQQLVISWFSLVFLASPLVA.

[0114] The structure of the BN114 molecule from the amino terminus to the carboxyl terminus is as follows: human IL-12α [referred to as p35], human CD8 Hinge domain [referred to as CD8 Hinge], human CD8 transmembrane domain [referred to as CD8 TM]. The amino acid sequence of the BN114 molecule is shown in SEQ ID NO.9, and the BN114 with added signal peptide is shown in SEQ ID NO.10:

[0115] RNLPVATDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVID ELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN(SEQ ID NO.9).

[0116] MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQI FLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN(SEQ ID NO.10).

[0117] The structure of the BN115 molecule from the amino terminus to the carboxyl terminus is as follows: human IL-12β [abbreviated as p40], connecting peptide G6Slinker [abbreviated as G6S linker], human IgG1 Fc Hinge domain [abbreviated as IgG1 Fc Hinge], human CD8 transmembrane domain [abbreviated as CD8 TM], as shown in SEQ ID NO.11, and the BN115 molecule with added signal peptide is shown in SEQ ID NO.12.

[0118] (SEQ ID NO. 11).

[0119] (SEQ ID NO.12).

[0120] The structure of the BN116 molecule from the amino terminus to the carboxyl terminus is as follows: human IL-12β [abbreviated as p40], connecting peptide G6S linker [abbreviated as G6S linker], human IL-12α [abbreviated as p35], human CD8 Hinge domain [abbreviated as CD8 Hinge], human CD8 transmembrane domain [abbreviated as CD8 TM], as shown in SEQ ID NO.13. The BN116 molecule with added signal peptide is shown in SEQ ID NO.14.

[0121] (SEQ ID NO. 13).

[0122] (SEQ ID NO.14).

[0123] 1 Experimental Materials

[0124] Table 3

[0125]

[0126]

[0127] 2 Preparation and detection of TIL cells overexpressing membrane-bound IL-12

[0128] 2.1 Preparation of TIL cells overexpressing membrane-bound IL-12

[0129] Preparation step 1: Lentivirus preparation

[0130] Construct BN114, BN115, and BN116 lentiviral expression plasmids, respectively, and mix with lentiviral packaging plasmids pMDLg-pRRE, pRSV-Rev, and pMD2.G using polyethyleneimine transfection reagent, and co-transfect 293T cells. Replace with complete medium 6 hours after transfection. After 72 hours of culture, collect the viral supernatant, centrifuge at 3000 rpm for 10 min to 15 min at 4 ° C, and then filter through a 0.45 μm pore size filter membrane, and concentrate the virus. Store the collected lentiviral concentrate at -80 ° C.

[0131] Add 300 μL of complete medium (DMEM medium + 10% fetal bovine serum + 100 U / mL penicillin + 100 μg / mL streptomycin, % is volume percentage) and Jurkat cells to each well of a 24-well plate to a cell density of 1×10 5 cells / well.

[0132] The lentivirus concentrate was diluted 5-fold with Opti-MEM medium. The lentivirus of each dilution gradient was added to the above 24-well plate at a dosage of 200 μL / well, and the lentivirus was used to infect Jurkat cells (human PBMC cells in the negative control group were only added with Opti-MEM medium), and placed in a cell culture incubator for culture (culture temperature was 37°C, carbon dioxide concentration was 5%).

[0133] After 3 days of culture, the cells in each well were gently mixed and transferred to a 1.5 mL centrifuge tube. The virus titer was determined by qPCR. The titer determination results are shown in Table 4.

[0134] Table 4. Statistics of different batches of lentivirus titer detection results

[0135] Structure Name PCR titer (TU / mL) BN114 6.90E+08 BN115 2.17E+08 BN116 1.23E+07

[0136] Preparation step 2: Lentiviral transfection and detection of HCC-derived TILs

[0137] (1) TIL cell preparation

[0138] The TIL isolated and expanded from liver cancer tumor tissue was adjusted to 2×10 6 The cells were cultured at a cell density of 1:1 / mL in TIL expansion medium for 24 h (culture temperature: 37°C, carbon dioxide concentration: 5%), and TILs were collected.

[0139] (2) Lentiviral transduction of TILs

[0140] The obtained TILs were washed and the cell density was adjusted to 4 × 10 6 / mL. Add lentivirus at an MOI of 1TU / mL to 10TU / mL for transduction and culture in TIL expansion medium (the culture temperature of the cell culture incubator is 37°C and the carbon dioxide concentration is 5%). After 4 hours, adjust the cell density to 2×10 6 After 24 h, the cell density was maintained at 1×10 6 / mL~2×10 6 On the 6th day after transduction, the cells were collected and frozen in liquid nitrogen using a freezing medium (freezing medium containing 5 wt% human serum albumin: physiological saline = 1:1, v:v) for later use.

[0141] The obtained TILs were named after the corresponding molecular structures (i.e., BN114 cells, BN115 cells, and BN116 cells), and the TIL cells not transduced with lentivirus were named Mock TIL.

[0142] (3) Detection of TIL expressing membrane-bound p35

[0143] Wash the Mock TIL, BN114, BN115 and BN116 cells to be tested twice with PBS and resuspend them in FACS buffer. According to the antibody instructions, add IL-12p70 Capture Ab (Rat), BV421-labeled anti-human CD3 antibody and anti-Rat APC-labeled secondary antibody to the cell suspension to be tested and incubate at 4°C for 60 minutes. Using Mock TIL cells not transfected with lentivirus as negative control, flow cytometry was used to detect the expression rate of p35 on the cell membrane surface of BN114, BN115 and BN116 cells. FlowJo software was used for analysis.

[0144] (4) Detection of TIL expressing membrane-bound p40

[0145] Wash the Mock TIL, BN114, BN115 and BN116 cells to be tested twice with PBS and resuspend them in FACS buffer. According to the antibody instructions, add Biotin-labeled IL-12p70 Detection Ab, BV421-labeled anti-human CD3 antibody and APC-labeled Streptavidin to the cell suspension to be tested and incubate at 4°C for 60 minutes. Using Mock TIL cells not transfected with lentivirus as negative control, flow cytometry was used to detect the p40 expression rate on the cell membrane surface of BN114, BN115 and BN116 cells. FlowJo software was used for analysis.

[0146] Preparation step 3: Lentiviral transduction and detection of colorectal cancer-derived TILs

[0147] (1) TIL cell preparation

[0148] The TIL isolated and expanded from colorectal cancer tumor tissue was adjusted to 2×10 6 The cells were cultured at a cell density of 37° C. / mL in TILs expansion medium for 24 h (the culture temperature of the cell culture incubator was 37° C. and the carbon dioxide concentration was 5%), and TILs were collected.

[0149] (2) Lentiviral transduction of TILs

[0150] The obtained TILs were washed and the cell density was adjusted to 4 × 10 6 / mL. Add lentivirus at an MOI of 1TU / mL to 10TU / mL for transduction and culture in TIL expansion medium (the culture temperature of the cell culture incubator is 37°C and the carbon dioxide concentration is 5%). After 4 hours, adjust the cell density to 2×10 6 After 24 h, the cell density was maintained at 1×10 6 / mL~2×10 6 On the 6th day after transduction, the cells were collected and frozen in liquid nitrogen using a freezing medium (freezing medium containing 5 wt% human serum albumin: physiological saline = 1:1, v:v) for later use.

[0151] The obtained TILs were named after the corresponding molecular structures (i.e., BN114 cells, BN115 cells, and BN116 cells), and the TIL cells not transduced with lentivirus were named Mock TIL.

[0152] (3) Detection of TIL expressing membrane-bound p40

[0153] Wash the Mock TIL, BN114, BN115 and BN116 cells to be tested twice with PBS and resuspend them in FACS buffer. According to the antibody instructions, add Biotin-labeled IL-12p70 Detection Ab, BV421-labeled anti-human CD3 antibody and APC-labeled Streptavidin to the cell suspension to be tested and incubate at 4°C for 60 minutes. Using Mock TIL cells not transfected with lentivirus as negative control, flow cytometry was used to detect the p40 expression rate on the cell membrane surface of BN114, BN115 and BN116 cells. FlowJo software was used for analysis.

[0154] Preparation step 4: Lentiviral transduction and detection of CAR-T cells

[0155] (1) T cell preparation

[0156] The density of peripheral blood mononuclear cells from healthy donors was adjusted to 2×10 6 / mL, added 50ng / mL anti-CD3 antibody, 50ng / mL anti-CD28 antibody, and 300IU / mL recombinant IL-2, placed in a cell culture incubator for 24h (culture temperature: 37°C, carbon dioxide concentration: 5%), and collected T cells.

[0157] (2) Lentiviral transduction of T cells

[0158] Wash the obtained T cells and adjust the cell density to 4 × 10 6 / mL. CAR lentivirus and BN115 lentivirus were added at an MOI of 1TU / mL to 10TU / mL for transduction, and 50ng / mL anti-CD3 antibody, 50ng / mL anti-CD28 antibody, and 300IU / mL recombinant IL-2 were supplemented and cultured in a cell culture incubator (culture temperature was 37°C and carbon dioxide concentration was 5%). After 24h, the cell density was adjusted to 1.5×10 6 / mL~2×10 6 / mL, and supplemented with 300IU / mL of IL-2. On the 4th day after transduction, the cells were washed to remove the residual lentiviral particles in the supernatant, and then cultured in a cell culture incubator for 5 days (culture temperature was 37°C, carbon dioxide concentration was 5%), during which the cell density was maintained at 1×10 6 / mL~2×10 6 On the 10th day after transduction, the cells were collected and frozen in liquid nitrogen using a freezing medium (freezing medium containing 5% human serum albumin: physiological saline = 1:1) for later use.

[0159] The amino acid sequence of the CAR molecule of the CAR-T cell is shown in SEQ ID NO.15:

[0160] MALPVTALLLPLALLLHAARPSDVVVTQTPLSLPVSFGDQVSISCRSSQSLANSYGNTYLSWYLHKPGQS

[0161] PQLLIYGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPYTFGGGTKLEIKRGGGGSG

[0162] GGGSGGGGSQVQLQQSGAELVRPGASVKLSCKASGYTFSDFEMHWVKQTPVHGLEWIGDIDPGTGDTA

[0163] YNLKFKGKATLTTDKSSSTAYMELRSLTSEDSAVYYCTLGAFVYWGQGTLVTVSSESKYGPPCPPCPGQP

[0164] REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK

[0165] SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKCDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRK

[0166] KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD

[0167] VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0168] 2.2 Detection of TILs overexpressing membrane-bound IL-12

[0169] 2.2.1 Detection method

[0170] Experiment 1: In vitro functional assay

[0171] (1) Incucyte

[0172] The liver cancer cell line SK-Hep-1, which overexpresses liver cancer-related antigens and hepatitis B virus-related antigens, was selected as the target cell to test the killing effect of TIL expressing BN114, BN115, and BN116. The target cells were digested and counted, and 1×10 4 The target cells were seeded into a 96-well plate at a density of / well. 1×10 4 / hole or 2×10 4 The T cells were co-cultured at 100 μg / well. In the end, the long-term killing effect was detected using the Incucyte instrument (3-7 days).

[0173] The colon cancer cell line HCT116 was selected to test the killing effect of BN115 TIL. The target cells were digested and counted, with a value of 1×10 4 The target cells were seeded into a 96-well plate at a density of / well. 1×10 4 / hole or 2×104 The T cells were co-cultured at 100 μg / well. In the end, the long-term killing effect was detected using the Incucyte instrument (3-7 days).

[0174] The colon cancer cell line HCT116 was selected to test the killing effect of BN115 CAR-T. The target cells were digested and counted, with a value of 1×10 4 The target cells were seeded into a 96-well plate at a density of / well. 1×10 4 / hole or 2×10 4 The T cells were co-cultured at 100 μg / well. In the end, the long-term killing effect was detected using the Incucyte instrument (3-7 days).

[0175] The colorectal cancer cell line CaCo-2 was selected to test the killing effect of BN115 CAR-T. The target cells were digested and counted, with a value of 1×10 4 The target cells were seeded into a 96-well plate at a density of / well. 1×10 4 / hole or 2×10 4 The T cells were co-cultured at 100 μg / well. In the end, the long-term killing effect was detected using the Incucyte instrument (3-7 days).

[0176] (2) Flow cytometry to detect the intracellular expression level of IFNγ

[0177] Mock TIL and each group of TIL were co-cultured with the corresponding target cells in X-vivo medium without IL-2 (effect-target ratio was 4:1). After 12 hours, cytokine secretion blocker (BD GolgiPlug TM Protein Transport Inhibitor, BD biscience), cultured for 4 hours at 37°C and 5% carbon dioxide concentration. After the culture is completed, the Mock TIL to be tested and each group of TIL are washed twice with PBS and resuspended with FACS buffer. According to the antibody instructions, the anti-human CD8 antibody expressed by AF700, the anti-human CD4 antibody labeled with BV650, the anti-human CD3 antibody labeled with BV421, and the anti-human PD-1 antibody labeled with PE-Cy7 are added to the cell suspension to be tested and incubated at 4°C for 30 minutes. The Mock TIL to be tested and each group of TIL are washed twice with FACS buffer, and the cells are fixed and permeated using the BD Cytofix / Cytoperm kit. Then, FITC-labeled anti-human IFNγ antibody is added and incubated at 4°C for 30 minutes. IFNγ in each group of TIL is detected by flow cytometry. + CD8 + T cells or PD-1 +CD8 + The proportion of T cells was analyzed using FlowJo software.

[0178] Mock TIL cells not transfected with lentivirus were used as negative control, and the expression rate of p35 on the cell membrane surface of BN114 and BN116 cells was detected by flow cytometry. FlowJo software was used for analysis.

[0179] (3) ELISA to detect IFNγ secretion

[0180] Mock TIL and each group of TIL were co-cultured with the corresponding target cells in X-vivo medium without IL-2 (the effector-target ratio was 4:1). After 12 hours, the supernatant of the co-cultured cells was aspirated after centrifugation, and the sample was diluted with PBS containing 1wt% BSA. The IFNγ standard was dissolved with ddH2O, and placed at room temperature for 15-20 minutes to ensure full dissolution, and the standard was diluted according to the recommended gradient ratio. The standard and experimental samples were added to the corresponding reaction wells, 100μL per well. After incubation at room temperature for 1-3 hours, 1× cleaning solution was prepared, and each well was washed 3 times with 300μL cleaning solution, and the liquid in the well was patted dry. 100μL enzyme-labeled detection antibody was added to each well, and incubated at room temperature for 1-3 hours. Each well was washed 3 times with 300μL cleaning solution, and after the liquid in the well was patted dry, 100μL color substrate was added. After incubation at room temperature in the dark for 20 min, 50 μL of stop solution was added to each well, and the absorbance at 450 nm was measured using an enzyme-labeled instrument.

[0181] (4) ELISA to detect IL-12 secretion

[0182] TILs overexpressing BN115 were divided into two groups with equal cell numbers, 10 6Cells: One group was resuspended in 1mL X-vivo medium without IL-2, cultured for 12h, and centrifuged to obtain the supernatant of the co-cultured cells (referred to as the Supernatant group); the other group was lysed and resuspended in 1mL X-vivo medium without IL-2 (referred to as the Cell lysis group) for subsequent ELISA detection. The two groups of samples were diluted with PBS containing 1wt% BSA. The IL-12 standard was dissolved in ddH2O, placed at room temperature for 15-20min to ensure full dissolution, and the standard was diluted according to the recommended gradient ratio. The standard and experimental samples were added to the corresponding reaction wells, 100μL per well. After incubation at room temperature for 1h to 3h, 1× cleaning solution was prepared, and each well was washed 3 times with 300μL cleaning solution, and the liquid in the well was patted dry, and 100μL enzyme-labeled detection antibody was added to each well, and incubated at room temperature for 1h to 3h. Each well was washed three times with 300 μL of washing solution, and after the liquid in the well was patted dry, 100 μL of color substrate was added. After incubation at room temperature in the dark for 20 minutes, 50 μL of stop solution was added to each well, and the absorbance at 450 nm was measured with an ELISA reader.

[0183] (5) Cell proliferation assay

[0184] TILs were counted and then co-cultured with corresponding target cells in X-vivo medium without IL-2 (effector-target ratio was 4:1). After 96 h, the co-cultured cells were taken out and counted, and the total number of cells and expansion times were calculated.

[0185] Experiment 2: In vivo tumor inhibition experiment

[0186] The target cells in the logarithmic growth phase and in good growth condition were collected by trypsin digestion, washed once with physiological saline, and the cell density was adjusted to 2×10 7 / mL. 100 μl of cell suspension was injected subcutaneously on the right side of NOG mice near the armpit, that is, 2×10 6 The target cells were inoculated on day 0.

[0187] On the 7th day after inoculation of target cells (or when the average tumor volume is about 50 mm 3 ), BN115TIL (1×10 7 / pcs), Mock TIL(1×10 7 / mouse) and solvent (400μL / mouse), and the day of injection of the test substance was recorded as day 0 of treatment. Tumor size and mouse weight were measured 2 to 3 times a week. After 42 days of treatment, the mice were euthanized, and the tumor, heart, liver, spleen, lung, kidney, brain, ovary and other tissues were weighed. Tissues of 2 mice in each group were stored in an 80°C refrigerator for DNA extraction and detection of TIL infiltration in tumor tissues and their distribution in various organs; tissues of 2 mice in each group were fixed, and the morphology of tumor cells was detected by HE staining and the expression of antigens in tissues was detected by immunohistochemistry.

[0188] Expected tumor inhibition effect: BN115 TIL experimental group > Mock TIL control group > vehicle control group, indicating that overexpression of membrane-bound p40 can enhance the tumor cell-killing effect of TIL.

[0189] 2.2.2 Test results

[0190] (1) Validation of flow cytometry method for detecting membrane-bound IL-12

[0191] The present application example uses the antibody indirect labeling method of flow cytometry to detect liver cancer-derived TILs that overexpress full-length IL-12 (p70) and IL-12 subunits (p35 or p40). The information of flow cytometry anti-IL-12 antibodies is as follows: IL-12p70 Capture antibody (Cat. No.: 431704, Biolegend), IL-12p70 Detectionantibody (Cat. No.: 431704, Biolegend).

[0192] First, the examples of the present application verified the specificity of the above two antibodies, namely, Capture Ab (anti-p35 antibody) and Detection Ab (anti-p40 antibody), in detecting p35 and p40. Figure 2 The results showed that the Detecion Ab could specifically detect the expression of BN115 (i.e., membrane-bound p40), but could not detect the expression of BN114 (i.e., membrane-bound p35); accordingly, the Capture Ab could specifically detect the expression of BN114 (i.e., membrane-bound p35), but could not detect the expression of BN114 (i.e., membrane-bound p40). The above results suggest that the Capture Ab can specifically detect the expression of membrane-bound p35; the Detection Ab can specifically detect the expression of membrane-bound p40, and at the same time prove that both BN114 and BN115 structures can be effectively expressed in TIL cells.

[0193] In order to further verify the specificity of the above two antibodies for IL-12 subunit detection, the present invention embodiment was verified by simultaneously overexpressing BN114 and BN115, and by overexpressing BN116 (membrane-bound p70). Figure 3 The results showed that both Capture Ab and Detection Ab could bind to membrane-bound p35 and p40 co-overexpressed by BN114 and BN115, and Detection Ab could specifically detect the expression of membrane-bound p70; however, Capture Ab could not detect the expression of membrane-bound p70, possibly due to the influence of structural design. The above results show that Detection Ab can specifically detect the expression of membrane-bound p40 and p70, and Capture Ab can specifically detect the expression of membrane-bound p35.

[0194] (2) Effects of overexpression of membrane-bound IL-12 on TIL proliferation and function

[0195] Furthermore, the present application example compares the effects of overexpression of the following membrane-bound IL-12 on the proliferation and function of TILs: BN114, BN115, BN114+BN115, and BN116. After overexpression of the above membrane-bound IL-12, the number of proliferating cells of TILs was compared on day 7. The results are shown in Figure 4 .

[0196] according to Figure 4 It can be observed that the number of cells from most to least is BN116, BN114+BN115, BN115, BN114 and control TIL group ( Figure 4 Figure A, and Table 5). After co-culturing TILs with tumor cells, we unexpectedly found that BN115 (IL12 p40 subunit) had the highest increase in IFNγ and PD-1 expression levels in TIL cells compared with BN114 (IL-12p35 subunit) or BN116 (full-length IL-12), indicating that BN115 has the strongest activation effect on TIL cells ( Figure 4 Therefore, the subsequent research work of the present application embodiment will focus on BN115.

[0197] Figure 4 Middle: Figure A shows the TIL cell count results of the Mock group, BN114 group, BN115 group, BN114+BN115 group, and BN116 group on the seventh day after lentiviral transfection to overexpress membrane-bound IL-12. Figure B shows the expression of IFNγ and PD-1 in TIL after TIL overexpressing membrane-bound IL-12 was co-cultured with target cells for 12 hours.

[0198] Among them, HCC22004, HCC22009, and HCC22010 are TILs from different liver cancer patients.

[0199] Statistical methods: One-way ANOVA was used, and P < 0.05 was considered statistically significant.

[0200] Table 5. Statistics of cell counting results of TIL after transfection

[0201]

[0202] (3) Effects of overexpression of membrane-bound p40 on TIL proliferation and function

[0203] Based on the above results, the present application example selected mP40 (code: BN115) for subsequent development work, and the results are shown in Figure 5 First, in the present embodiment, BN115 was overexpressed by lentivirus in TIL derived from liver cancer. After the sixth day of transfection, the expression level of BN115 on TIL was detected by flow cytometry at 40% to 60% ( Figure 5 Moreover, compared with the control group (Mock group), the number of TIL cells overexpressing BN115 increased significantly ( Figure 5 Furthermore, after co-culturing BN115 TIL with liver cancer target cells, PD-1 of BN115 TIL could be detected. + CTLs ratio and IFNγ + The proportion of CTLs increased significantly ( Figure 5 In addition, during the co-culture process, the concentration of IFNγ secreted outside the cells also increased significantly. The above results suggest that BN115 can significantly upregulate the activation level of CTL and the secretion of tumor killing related factors.

[0204] Figure 5 Middle: Expression level of membrane-bound p40 (Figure A) and cell counting results (Figure B) on day 6 after lentiviral transfection to overexpress membrane-bound p40 (code name: BN115); PD-1 in TIL after co-culture with target cells + Cell ratio and IFNγ + The cell ratio (Figure C) and the level of IFNγ secreted by TIL to the extracellular space (Figure D) were analyzed by T test. P < 0.05 was considered statistically significant.

[0205] (4) Overexpression of membrane-bound p40 enhances the tumor cell killing effect of TIL (in vitro)

[0206] Furthermore, the present application example verifies the enhancement effect of BN115 on TIL killing tumor cells through in vitro experiments. First, liver cancer target cells are used to stimulate BN115 liver cancer TIL for 1 or 2 rounds. Then, the killing effect of BN115 TIL on liver cancer target cells after 1 or 2 rounds of stimulation is detected.

[0207] Depend on Figure 6 The results in Figure A show that, compared with the Mock group, the killing effect of BN115 TIL on target cells after one or more rounds of stimulation is stronger than that of the Mock group, indicating that BN115 can enhance the killing effect of TIL on tumor cells.

[0208] Furthermore, this implementation case used the IncuCyte live cell imaging analysis system to record in real time the killing effect of liver cancer or colorectal cancer TILs overexpressing BN115 on tumor cells, and then added target cells again at appropriate time points to repeatedly stimulate the TIL cells. Since the target cells carry fluorescent reporter genes, the IncuCyte software can be used to calculate the fluorescent signal of the target cells in each group. The lower the signal value, the fewer the number of target cells in the group, and the better the killing effect of TIL cells. The results showed that BN115 can significantly enhance the killing effect of TILs in the above two indications on liver cancer ( Figure 6 Middle B) or colorectal cancer ( Figure 6 (C) Sustained killing effect of tumor cell lines. Based on the above results, BN115 can significantly enhance the sustained killing effect of TIL on tumor cells in the in vitro tumor cell line killing model.

[0209] Figure 6 Figure A in the middle and Table 6 show the specific killing effect of liver cancer-derived BN115TIL on target cells after the first or second round of liver cancer target cell stimulation (at different effector-target ratios);

[0210] Figure 6 Figure B in the middle shows the sustained killing effect of liver cancer-derived BN115 TIL on target cells;

[0211] Figure 6 Figure C in the middle shows the sustained killing effect of colorectal cancer-derived BN115 TIL on target cells.

[0212] Table 6. Statistics of specific killing results of BN115 TIL on target cells at different effector-target ratios

[0213]

[0214]

[0215] (5) Overexpression of membrane-bound p40 enhances CAR-T cell killing (in vitro)

[0216] At the same time, the examples of this application verified the enhancement effect of BN115 on CAR-T killing tumor cells through in vitro experiments. Figure 7 The results showed that compared with the CAR-T group, the CAR+BN115 T cell group had a significantly enhanced killing effect on the pancreatic cancer cell line Caco-2; similarly, in the colorectal cancer HCT-116 cell line model, BN115 was also able to significantly enhance the function of CAR-T cells in killing tumor cells compared with the CAR-T group. Based on the above results, BN115 can significantly enhance the sustained killing effect of CAR-T on tumor cells in the in vitro tumor cell line killing model.

[0217] Figure 7 Figure A in the middle and Table 7 show the enhancement effect of BN115 on CAR-T killing of pancreatic cancer cell lines;

[0218] Figure 7 Figure B and Table 7 show the enhancing effect of BN115 on CAR-T killing of colorectal cancer cell lines.

[0219] Table 7. Cytotoxicity of BN115 CAR T cells against pancreatic cancer cell line (Caco-2) and intestinal cancer cell line (HCT116)

[0220]

[0221] (6) Overexpression of membrane-bound p40 enhances the tumor cell killing effect of TIL (in vivo)

[0222] In order to verify the results of the in vitro killing model, the present invention adopts liver cancer tumor cell line to carry out tumor in severe immunodeficient (NOG) mice. 3 Control or BN115 liver cancer TILs were infused back into the left and right sides.

[0223] Depend on Figure 8 As shown in Figure A, after the BN115 TIL was reinfused, the mice did not experience significant weight loss, which means that no toxic side effects of BN115 TIL were observed.

[0224] Depend on Figure 8 The results in Figure B show that BN115 can significantly enhance the anti-tumor effect of TIL in tumor-bearing mice without concomitant injection of IL-2.

[0225] In addition, the results of in vitro detection of BN115 TIL secretion of free IL-12 showed that ( Figure 8 In Figure C), only a small amount of free full-length IL-12 was detected, indicating that membrane-anchored p40 can significantly improve the safety of IL-12 in anti-tumor therapy.

[0226] Figure 8 The figure shows the anti-tumor effect of liver cancer-derived BN115TIL in severely immunodeficient (NOG) mice bearing liver cancer cell lines. Statistical methods: Two-way ANOVA was used, and P<0.05 was considered statistically significant; NS, no significant difference.

[0227] Overall, the present application overexpresses membrane-bound IL-12 in TIL to enhance the sustained anti-tumor killing function of TIL (independent of concomitant injection of IL-2). It was also unexpectedly found that compared with expressing membrane-bound IL-12p35 or full-length IL-12, expressing only the membrane-bound IL12 p40 subunit has the strongest activation effect on TIL cells. The present application has the following advantages:

[0228] (1) By linking the subunits of IL-12 to the transmembrane domain of CD8, these factors are anchored on the cell membrane surface and cannot exist in a free form, thus overcoming the cytotoxic effect of IL-12 when activating T cell anti-tumor immunity;

[0229] (2) By expressing IL-12p40, the safety of IL-12 used for immune activation can be further improved. Therefore, the present invention improves the proliferation ability and tumor killing ability of TIL by overexpressing membrane-bound IL-12, which is more conducive to improving the therapeutic effect of liver cancer and colorectal cancer.

[0230] (3) Only expressing IL-12p40 can achieve the optimal enhancement effect of TIL cells, which can reduce the requirements for the capacity of the expression vector, improve the expression level, and be more suitable for industrial applications.

[0231] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0232] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. An engineered immune cell, characterized in that: The engineered immune cells overexpress the fusion protein; From the extracellular domain to the intracellular domain, the fusion protein is composed of an IL-12 p40 subunit, a flexible linker, a hinge region and a membrane protein in sequence, and the membrane protein is used to anchor the IL-12 p40 subunit on the cell membrane.

2. The engineered immune cell according to claim 1, characterized in that The membrane proteins include type I membrane proteins, type II membrane proteins, multi-transmembrane proteins and glycosylphosphatidylinositol anchored proteins.

3. The engineered immune cell according to claim 1, characterized in that The membrane protein has the transmembrane domain of the following proteins or a partial region of the transmembrane domain: α chain of T cell receptor, β chain of T cell receptor, ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD48, CD55, CD64, CD80, CD86, CD90, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, ICOS, GITR, CD40, BAFFR, HVEM, SLAMF7, NKp80, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, TGFβRII, ITG A1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, ITGB 7. TNFR2, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, Ly9, PSGL1, CD100, SLAMF6, SLAM, BLAME, SELPLG, LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and NKG2C.

4. The engineered immune cell according to claim 1, characterized in that The hinge region is selected from the hinge regions of the following proteins: IgG1, IgG4, IgG4m, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154.

5. The engineered immune cell according to claim 1, characterized in that The N-terminus of the fusion protein is connected with a signal peptide or an internal signal anchor sequence fragment.

6. The engineered immune cell according to any one of claims 1 to 5, characterized in that The amino acid sequence of the fusion protein is shown in SEQ ID NO.11 or SEQ ID NO.

12.

7. The engineered immune cell according to any one of claims 1 to 5, wherein The engineered immune cells include one or more of CAR-T, TCR-T, TIL and CAR-NK.

8. The method for preparing the engineered immune cells according to any one of claims 1 to 7, comprising the following steps: An immune cell to be transformed is provided, and the immune cell to be transformed is transformed to express the fusion protein, thereby constructing an engineered immune cell.

9. Use of the engineered immune cell according to any one of claims 1 to 7 in the preparation of anti-tumor drugs.

10. The use according to claim 9, wherein: The anti-tumor drug prevents and / or treats the following tumors: liver cancer, colon cancer, rectal cancer, ovarian cancer, cervical cancer, gastric cancer, pancreatic cancer, lung cancer, breast cancer, and melanoma.

11. A medicine comprising the engineered immune cell according to any one of claims 1 to 7, and a pharmaceutically acceptable carrier.

12. A composition comprising the engineered immune cell according to any one of claims 1 to 7 and a CAR-T cell, wherein the amino acid sequence of the CAR molecule of the CAR-T cell is as shown in SEQ ID NO.15.

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