Protein combination, nucleic acid encoding same, recombinant vector comprising nucleic acid, recombinant lentivirus, engineered immune cell and use thereof

Abstract

The invention discloses a protein combination, nucleic acid for coding the protein combination, a recombinant vector containing the nucleic acid, a recombinant lentivirus, an engineered immune cell and application thereof. The protein combination comprises interleukin or a variant thereof, and PGC1 alpha or a variant thereof. Through overexpression of the protein combination in lymphocytes taking tumor infiltration lymphocytes as an example, the mitochondrial function of immune cells can be promoted to be reprogrammed, meanwhile, the in-vivo and in-vitro amplification capacity and the continuous tumor killing capacity of the immune cells are improved, and the treatment effect on cancers such as liver cancer and lung cancer can be better improved.

Description

Technical Field

[0001] This application relates to the field of cell therapy, specifically to a protein combination, a nucleic acid encoding the protein, a recombinant vector containing the nucleic acid, a recombinant lentivirus, engineered immune cells, and their uses. Background Technology

[0002] Cellular immunotherapy, hailed as a "powerful weapon against cancer," has demonstrated unprecedented therapeutic potential and hope in recent years, facing the severe challenge of approximately 20 million new cancer cases and nearly 10 million cancer deaths worldwide each year. Cellular immunotherapy, by activating or enhancing the patient's own immune system to fight cancer cells, has brought about a revolutionary breakthrough in cancer treatment. Compared with traditional anti-cancer methods, cellular immunotherapy has many advantages, such as precisely killing cancer cells and showing good therapeutic effects on refractory and recurrent hematological malignancies. However, cellular immunotherapy still has many areas that need improvement.

[0003] Taking tumor-infiltrating lymphocytes (TILs) as an example, traditional TIL end products have short survival times and weak proliferative capacity in vivo, and their killing effect on tumor cells depends on high-dose IL-2 concomitant injection. Furthermore, high-dose TIL reinfusion is required to observe an anti-tumor effect. For instance, some technologies have disclosed methods to enhance T cell killing with membrane-anchored IL-12, aiming to overcome the problem of excessive toxicity of secreted IL-12 in clinical use. However, during T cell activation / proliferation, when subjected to physical stimulation or damage, some mIL-12 inevitably detaches from the cell membrane and exists in a secreted form. Therefore, this combination still exhibits some mIL-12 toxicity. Another technology discloses a membrane-anchored IL-18 to enhance the killing effect of TILs on tumor cells. Its research approach is similar to the design of the membrane-anchored IL-12 mentioned above. Compared to expressing a single subunit of a cytokine, expressing the full-length cytokine is more toxic. Furthermore, IL-18 enhances T cell function by activating the NF-κB pathway, but its effect is weaker than that of IL-12. Other technologies report a method for constructing a membrane-bound IL-15 / IL-21 to enhance CAR-T / TCR-T cell killing, thus overcoming the toxicity problem of secreted IL-15 / IL-21 in clinical use. However, during T cell activation / proliferation, when subjected to physical stimulation or damage, some mIL-15 / IL-21 inevitably detaches from the cell membrane and exists in a secreted form, still exhibiting some cytokine toxicity.

[0004] Furthermore, unlike CAR-T (Chimeric Antigen Receptor T-Cell) / TCR-T cell therapy products, TILs typically exert their anti-tumor effects by recognizing tumor-associated antigens, such as neoantigens, presented by tumor cells. Therefore, TIL therapy can achieve anti-tumor therapeutic goals by recognizing and targeting multiple tumor-associated antigens. However, due to the regulatory effects of various immunosuppressive factors in the tumor microenvironment, TILs highly express immune checkpoint-related molecules, such as PD-1, CTLA4, TIM3, LAG3, and TIGIT. Simultaneously, due to continuous stimulation by tumor-associated antigens, TILs exhibit a highly depleted state, such as high expression of depletion-related transcription factors like Tox and Id3. Even after TIL isolation and reactivation in vitro, their cytotoxic function remains limited.

[0005] Therefore, how to enhance the sustained anti-tumor function of TIL has always been a technical problem that needs to be solved in this field. Summary of the Invention

[0006] Therefore, it is necessary to provide at least one protein combination, the nucleic acid encoding it, a recombinant vector containing the nucleic acid, a recombinant lentivirus, an engineered immune cell, and its uses.

[0007] In a first aspect of this application, a protein combination is provided comprising interleukin or a variant thereof, and PGC1α or a variant thereof.

[0008] In a second aspect of this application, a nucleic acid is provided that encodes a protein combination as described in the first aspect.

[0009] In a third aspect of this application, a recombinant vector is provided, comprising the nucleic acid as described in the second aspect.

[0010] In a fourth aspect of this application, a nucleic acid combination is provided, comprising nucleic acid 1 expressing the interleukin or a variant thereof, and nucleic acid 2 expressing the PGC1α or a variant thereof.

[0011] In a fifth aspect of this application, a recombinant vector is provided, comprising the nucleic acid combination as described in the fourth aspect.

[0012] In a sixth aspect of this application, a recombinant vector combination is provided, comprising a recombinant vector 1 containing nucleic acid 1 as defined in the fourth aspect, and a recombinant vector 2 containing nucleic acid 2 as defined in the fourth aspect.

[0013] In a seventh aspect of this application, an engineered immune cell is provided that expresses the protein combination described in the first aspect.

[0014] In an eighth aspect of this application, a cell-based drug composition is provided, comprising engineered immune cells as described in the seventh aspect, and a pharmaceutically acceptable carrier.

[0015] In a ninth aspect of this application, the use of recombinant vectors as described in the third or fifth aspect, combinations of recombinant vectors as described in the sixth aspect, or engineered immune cells as described in the seventh aspect is provided in the preparation of a medicament for treating cancer.

[0016] In a tenth aspect of this application, a method for treating cancer is provided, the method comprising administering to a subject engineered immune cells as described in the seventh aspect or a cell-pharmaceutical composition as described in the eighth aspect.

[0017] This application provides an exemplary protein combination and its application, which can promote the reprogramming of mitochondrial function of immune cells by overexpressing it in immune cells such as tumor-infiltrating lymphocytes, thereby enhancing the ability of immune cells to expand in vivo and in vitro and the ability to continuously kill tumors, which is more conducive to improving the treatment effect of cancers such as liver cancer and lung cancer. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without creative effort. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in illustrating this application.

[0019] Figure 1 This is a schematic diagram of the structure of the membrane-anchored IL-7 series PGC1α-NT in one embodiment of this application.

[0020] Figure 2A This shows a schematic diagram of the PGC1α molecular structure used for structure screening.

[0021] Figure 2B The positivity rate of TIL transduced with different structures of PGC1α molecules was shown by flow cytometry.

[0022] Figure 2C The mitochondrial mass of the PGC1α molecule TIL expressing the structure was detected by flow cytometry.

[0023] Figure 2D The ability to scavenge mitochondrial reactive oxygen species was demonstrated using flow cytometry.

[0024] Figure 2E The PGC1α-mediated TIL expression structure was used to demonstrate the killing effect of TILs on tumor target cells. Real-time fluorescence imaging was used to monitor the killing of target cells. Mock group: non-transduced control TIL group; BN118P4 group: BN118P4 TIL group transducing PGC1α; BN118-NTP4 group: BN118-NTP4 TIL group transducing PGC1α-NT; BN118-MutP4 group: BN118-MutP4 TIL group transducing PGC1α-Mut.

[0025] Figure 2F This study showed the expression levels of IFNγ and IL-2 after target cell killing by transduced PGC1αTIL. Statistical analysis: All results were obtained using one-way ANOVA with Dunnet correction. *P<0.05; **P<0.01; ***P<0.001.

[0026] Figure 2G The expression levels of GZMB and perforin were displayed. Statistical analysis: One-way ANOVA was used for all results, with Dunnet correction. *P<0.05; **P<0.01; ***P<0.001.

[0027] Figure 3A This shows a schematic diagram of the membrane-bound IL-7 molecule structure used for structure screening.

[0028] Figure 3B The expression rates of different membrane-bound IL-7 molecules transduced by TIL were shown using flow cytometry.

[0029] Figure 3C The study showed the killing effects of BWm7P4 and BWm7-GP4 TILs on the SK-HEP-1 hepatocellular carcinoma cell line. Real-time fluorescence imaging was used to monitor the killing of target cells. HCC22010 and HCC22050 refer to TILs from different patients; Mock, non-transduced control TIL group; BWm7P4, transduced BWm7P4 TIL group; and BWm7-GP4, transduced BWm7-GP4 TIL group.

[0030] Figure 4A This shows a schematic diagram of the molecular structure of the PGC1α-NT tandem membrane-bound IL-7 structure used for structure screening.

[0031] Figure 4B The expression rates of PGC1α and membrane-bound IL-7 after TIL transduction with different molecular structures were shown by flow cytometry.

[0032] Figure 4CThis study demonstrated the cytotoxic effect of PGC1α expressing different structures in combination with membrane-bound IL-7 TIL on the hepatocellular carcinoma cell line SK-HEP-1, and monitored the killing of target cells using a real-time fluorescence imaging system.

[0033] Figure 4D The changes in mitochondrial quality after killing the SK-HEP-1 liver cancer cell line with different structures of PGC1α combined with membrane-bound IL-7 TIL were shown using flow cytometry (MitoTracker Green).

[0034] Figure 4E The levels of mitochondrial reactive oxygen species (mtROS) were measured by flow cytometry after different structures of PGC1α combined with membrane-bound IL-7 TILs were shown, regardless of whether they killed the SK-HEP-1 liver cancer cell line. The TIL groups were: Mock (non-transduced control TIL group), BWm7-GP4 (transduced BWm7-GP4 TIL group), BN118-NTP4 (transduced BN118-NTP4 TIL group), BNC006P4 (transduced BNC006P4 TIL group), and BNC007P4 (transduced BNC007P4 TIL group).

[0035] Figure 5A The in vitro proliferation capacity of transduced BN118-NTP4, BWm7-GP4, and BNC007P4 TILs was compared. Statistical analysis: two-way ANOVA, Dunnet method correction; **P<0.01; ***P<0.001.

[0036] Figure 5B The proportion of central memory T cells transducing BN118-NTP4, BWm7-GP4, and BNC007P4 CD8+TILs was displayed; Statistical analysis: one-way ANOVA, Dunnet method correction; **P<0.01; ***P<0.001.

[0037] Figure 5C Displays the proportion of CD39-CD69-stem cell-like T cells; Statistical analysis: one-way ANOVA, Dunnet method correction; **P<0.01; ***P<0.001.

[0038] Figure 5D The killing effect of transduced BN118-NTP4, BWm7-GP4, and BNC007P4 CD8+TIL on SK-HEP-1 was demonstrated, and the killing of target cells was monitored using a real-time fluorescence imaging system.

[0039] Figure 5EThis study showed the expression levels of IFNγ and IL-2 during BNC007P4 cell killing. Statistical analysis: one-way ANOVA, corrected using the Dunnet method; **P<0.01; ***P<0.001.

[0040] Figure 5F The expression levels of GZMB and perforin were displayed using flow cytometry. Statistical analysis: one-way ANOVA, corrected for Dunnet's method; **P<0.01; ***P<0.001.

[0041] Figure 6 This application shows in vivo efficacy data of BNC007P4 according to one embodiment. Figure 6 The flowchart in section A shows the in vivo efficacy test of TILs transducing BN118-NTP4, BWm7-GP4, and BNC007P4. Figure 6 The B and C sequences show transductions BN118-NTP4, BWm7-GP4, and BNC007P4CD8. + The trend of body weight change in tumor-bearing mice after TIL reinfusion (B) and the tumor growth in tumor-bearing mice (C). Statistical analysis: (C) Two-way ANOVA, Dunnet method correction; **P<0.01; ***P<0.001. Detailed Implementation

[0042] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0043] Unless otherwise defined, 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 application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0044] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and other instances that otherwise indicate "one or more" shall be understood in the same way unless otherwise specified.

[0045] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0046] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method" etc., shall be defined as being able to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0047] In this application, terms such as "further," "even more," "particularly," "for example," "like," "example," and "exemplary" are used for descriptive purposes to indicate that different technical solutions preceding and following each other are related in terms of their coverage, but should not be construed as limiting the preceding technical solution or restricting the scope of protection of this application. In this application, unless otherwise specified, A (e.g., B) indicates that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0048] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it refers to either "with" or "without" a parallel solution. If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "option" is independent. Unless otherwise specified, the descriptions such as "optionally include" and "optionally contain" in this application, taking "optionally include" as an example, mean "may include or not include."

[0049] The terms “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0050] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0051] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0052] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only a non-exhaustive enumeration purpose and should be understood not to constitute a closed limitation on quantity.

[0053] In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0054] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0055] In a first aspect of this application, a protein combination is provided comprising interleukin or a variant thereof, and PGC1α or a variant thereof.

[0056] Unless otherwise specified, the term "combination" in this application may be understood as a composition or combination (such as a fusion protein molecule or recombinant protein molecule formed by two units).

[0057] Unless otherwise specified, the term "interleukin" refers to lymphokines that interact between leukocytes or immune cells, and is often abbreviated as "interleukin". At least 38 interleukins have been identified, with commonly used ones including IL-2, IL-4, IL-6, IL-7, IL-9, IL-12, IL-15, and IL-21. They possess a wide range of biological activities and play important roles in mediating cellular immunity, resisting microbial infections, and fighting tumors.

[0058] In some embodiments, the interleukin is interleukin-7.

[0059] Interleukin 7 (IL-7), primarily produced by stromal cells in lymphoid tissues, is a key cytokine essential for T cell development and peripheral persistence, and a member of the IL-2 superfamily. IL-7 possesses diverse biological activities, influencing various cell types through binding to IL-7R. The γ chain of IL-7R shares receptors with multiple interleukins, including IL-2, IL-4, IL-9, IL-15, and IL-21, and is expressed in various cell types. IL-7 functions by interacting with the receptor heterodimer complex (Il-7R) composed of IL-7γα / CD127 and the unprocessed γ chain, activating the intracellular JAK-STAT signaling pathway. The maintenance and survival of T cell subsets require the presence of IL-7. IL-7 influences T cell survival by regulating intrinsic apoptosis pathways. Tumor-infiltrating T cells are in a highly exhausted state; studies have shown that overexpression of IL-7 can enhance the in vitro and in vivo proliferation and survival of tumor-infiltrating T cells. However, excessive or systemic IL-7 anti-tumor therapy has significant toxic side effects. Therefore, by using gene editing techniques to overexpress IL-7 in specific cells while simultaneously anchoring this cytokine to the cell membrane surface, the safety of the treatment can be ensured while enhancing the anti-tumor therapeutic effect of T cells. In some embodiments, the amino acid sequence of the IL-7 includes the sequence shown in SEQ ID NO:5.

[0060] Unless otherwise specified, the term "PGC1α" refers to peroxisome proliferator-activated receptor-γ coactivator-1α, or PPARγ coactivator-1α. PGC-1α is a key regulator of mitochondrial biogenesis and respiration, acting as an inducible co-regulator in controlling energy homeostasis. PGC-1α is a co-regulator of GLUT4 and mitochondrial gene transcription, including components of the electron transport system. PGC-1α is highly expressed in tissues and cells with high energy demands, including brown adipose tissue, heart, skeletal muscle, kidney, and brain. Similarly, in immune cells, PGC-1α is upregulated in cells with high proliferative activity and rapid energy metabolism to maintain the metabolic adaptability required for immune function. Specifically, T cells, upon antigen activation, express large amounts of regulatory / killing-related cytokines and further proliferate and differentiate rapidly. This process generates a large amount of reactive oxygen species (ROS), which can disrupt mitochondrial metabolic function. Overexpression of PGC1α can significantly scavenge ROS, maintaining the adaptive function of T cell mitochondria. On the other hand, previous studies have shown that PGC1α expression is significantly downregulated in tumor-infiltrating T cells within the tumor microenvironment, leading to disordered mitochondrial metabolism. Therefore, overexpressing PGC1α in tumor-infiltrating T cells can reprogram their mitochondrial metabolic adaptations to enhance their anti-tumor function.

[0061] Unless otherwise specified, the term "variant" in this application refers to a protein obtained by mutating the wild-type protein through one or any combination of methods, including but not limited to amino acid substitution, deletion (truncation), insertion, and modification. For example, a variant of PGC1α contains some or all of the functions of wild-type PGC1α, or at least contains the functions of wild-type PGC1α.

[0062] In some embodiments, the variant of PGC1α is obtained by mutating (PGC1α mutant) or truncating (PGC1α truncated). The PGC1α mutant is, for example, obtained by replacing the 571st amino acid of wild-type PGC1α; exemplarily, the serine (S) at position 571 is mutated to alanine (A). The PGC1α truncated is, for example, a truncated wild-type PGC1α, retaining the first 270 amino acids from its N-terminus.

[0063] While not wanting to be limited by any theory, it has been found that truncated PGC1α variants, such as those that retain the first 270 amino acids from the N-terminus of wild-type PGC1α, when overexpressed in lymphocytes, such as TILs, can enhance the mitochondrial adaptability of TILs and strengthen their killing function in the tumor microenvironment.

[0064] In this application, the term "TIL" refers to tumor-infiltrating lymphocytes, a heterogeneous cell population composed of CD3+ αβ T cells, B cells, NK cells, γδ T cells, and innate lymphocytes (ILCs). After in vitro expansion and culture, CD3+ T cells become the dominant cell population (accounting for more than 90%). CD3+ T cells express TCR receptors on their surface and play a crucial role in mediating the immune killing of cancerous cells. Because different TCR receptors recognize different antigenic peptides presented by the major histocompatibility complex (MHC) molecule, T cells are endowed with antigen specificity. Therefore, once T cells specific to a particular antigen are stimulated by a specific antigen, these cells can promote programmed cell death of target cells through the release of cytotoxins and receptor-mediated mechanisms.

[0065] In some embodiments, the amino acid sequence of PGC1α comprises the amino acid sequence shown in SEQ ID NO:6.

[0066] In some embodiments, the amino acid sequence of the variant of PGC1α comprises the sequence shown in SEQ ID NO:7 or SEQ ID NO:8.

[0067] In some embodiments, the interleukin is a membrane-anchored form of interleukin.

[0068] Unless otherwise specified, the term "membrane" in this application refers to a biological membrane, such as a cell membrane. "Membrane anchoring form" refers to modifications of interleukin that enable it to anchor onto a biological membrane. Exemplarily, modifications used to anchor interleukin onto a biological membrane may include lipidation, transmembrane region modification, etc. Lipidation allows interleukin to interact with membrane lipids, thereby immobilizing interleukin onto the cell membrane. This modification involves certain amino acid residues of interleukin chemically reacting with fatty acids or other lipid molecules of membrane lipids to form covalent bonds, thereby enabling interleukin to be stably anchored onto the cell membrane.

[0069] Transmembrane region modification refers to the modification of interleukins using transmembrane regions, such as operatively linking them together. Through the embedding of the transmembrane region within the biological membrane, interleukins are anchored to the biological membrane. Common biological transmembrane regions include, but are not limited to, those selected from the group consisting of CD8, CD28, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, TCRα, TCRβ, TCRγ, TCRδ, TCRζ, OX40, ICOS, LAG-3, 2B4, BTLA, CTLA-4, and PD-1.

[0070] In some embodiments, the membrane-anchored form of interleukin comprises, from the N-terminus to the C-terminus, an interleukin, a hinge, and a transmembrane domain connected in sequence.

[0071] In some embodiments, the hinge is an IgG hinge. In some embodiments, the IgG hinge includes IgG4 Hinge and CH3 regions. In some embodiments, the amino acid sequence of the IgG hinge includes the sequence shown in SEQ ID NO:2. In some embodiments, the amino acid sequence of the IgG hinge is shown in SEQ ID NO:2.

[0072] In some embodiments, the hinge is a CD8 hinge. In some embodiments, the amino acid sequence of the CD8 hinge comprises the sequence shown in SEQ ID NO:3. In some embodiments, the amino acid sequence of the CD8 hinge is shown in SEQ ID NO:3.

[0073] While not wanting to be limited by any theory, it was found that when interleukin and the transmembrane domain were linked by an IgG hinge, the resulting membrane-anchored form of interleukin had a stronger killing effect on target cells (such as liver cancer cells, lung cancer cells, etc.).

[0074] In some embodiments, the membrane-anchored form of interleukin further comprises a signal peptide. In some embodiments, the signal peptide is located at the N-terminus of the interleukin.

[0075] In some embodiments, the membrane-anchored form of interleukin comprises, from the N-terminus to the C-terminus, a signal peptide, an interleukin, a hinge, and a transmembrane domain connected in sequence.

[0076] In some embodiments, the membrane-anchored form of interleukin includes, from the N-terminus to the C-terminus, a signal peptide, interleukin-7, an IgG hinge, and a CD8 transmembrane domain connected in sequence.

[0077] In some embodiments, the membrane-anchored form of interleukin comprises, from the N-terminus to the C-terminus, a signal peptide, interleukin-7, a CD8 hinge, and a CD8 transmembrane domain connected in sequence.

[0078] In some embodiments, the amino acid sequence of the signal peptide comprises the sequence shown in SEQ ID NO:10.

[0079] In some embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID NO:10.

[0080] In some embodiments, the amino acid sequence of the membrane-anchored form of interleukin is as shown in SEQ ID NO:11 or SEQ ID NO:12.

[0081] In some embodiments, the interleukin or its variant in the protein assembly exists separately from the PGC1α or its variant, i.e., the protein assembly contains the interleukin molecule or its variant molecule, and the PGC1α molecule or its variant molecule.

[0082] In some embodiments, the interleukin or a variant thereof in the protein combination is operatively linked to the PGC1α or a variant thereof, and exists as a recombinant protein.

[0083] In some embodiments, the recombinant protein comprises, from N-terminus to C-terminus, a membrane-anchored form of interleukin and PGC1α or a variant thereof.

[0084] In some embodiments, the membrane-anchored form of interleukin and the PGC1α or a variant thereof are operatively linked.

[0085] Unless otherwise specified, the term "operably linked" in this application refers to the functional relationship between two regions of a recombinant protein, namely, interleukin in membrane-anchored form and PGC1α or a variant thereof; wherein the two regions are linked to produce a recombinant protein or a fusion protein.

[0086] In some embodiments, the membrane-anchored form of interleukin and the PGC1α or a variant thereof are linked by a cleavable peptide.

[0087] In some embodiments, the recombinant protein comprises, from the N-terminus to the C-terminus, a membrane-anchored form of interleukin, a cleavable peptide, and PGC1α or a variant thereof.

[0088] In some embodiments, the recombinant protein comprises, from the N-terminus to the C-terminus, PGC1α or a variant thereof, a cleavable peptide, and a membrane-anchored form of interleukin.

[0089] Unless otherwise specified, the term "cleavable peptide" in this application refers to a peptide capable of producing multiple proteins from a single transcript. Such a cleavable peptide may be a self-cleaving peptide or derived from a self-cleaving peptide.

[0090] Unless otherwise specified, the term "autosplicing peptide" in this application refers to a short peptide, typically containing 18–25 amino acids, first discovered in foot-and-mouth disease virus, which can generate multiple proteins from a single transcript. Autosplicing peptides do not actually "self-splice," but rather function by causing the ribosome to skip the synthesis of the glycine and proline peptide bonds at the C-terminus of the 2A element. This process leads to the separation of the 2A sequence terminus from the downstream product, with additional 2A residues added to the C-terminus of the upstream protein and an additional proline residue added to the N-terminus of the downstream protein. Common autosplicing peptides include, but are not limited to, P2A, T2A, E2A, and F2A, and these different types of autosplicing peptides originate from different viruses.

[0091] The cleavable peptide described in this application may include a self-cleaving peptide, such as P2A, T2A, E2A, or F2A. The number of self-cleaving peptides in the cleavable peptide may be one or more. For example, in some embodiments, the cleavable peptide comprises two or more P2As linked sequentially.

[0092] The cleavable peptide of this application may comprise at least two self-cleaving peptides, for example, at least two self-cleaving peptides selected from the group consisting of P2A, T2A, E2A, and F2A, linked sequentially. In some embodiments, the cleavable peptide comprises P2A and T2A linked sequentially. In some embodiments, the amino acid sequence of the cleavable peptide comprises the sequence shown in SEQ ID NO:1. In some embodiments, the amino acid sequence of the cleavable peptide is shown in SEQ ID NO:1.

[0093] In some embodiments, the recombinant protein comprises, from the N-terminus to the C-terminus, a signal peptide, interleukin-7, an IgG hinge, a CD8 transmembrane domain, a cleavable peptide (e.g., P2A-T2A), and PGC1α, connected in sequence.

[0094] In some embodiments, the recombinant protein comprises, from the N-terminus to the C-terminus, a signal peptide, interleukin-7, a CD8 hinge, a CD8 transmembrane domain, a cleavable peptide (e.g., P2A-T2A), and PGC1α, connected sequentially.

[0095] In some embodiments, the recombinant protein comprises, from the N-terminus to the C-terminus, sequentially connected a PGC1α, a cleavable peptide (e.g., P2A-T2A), a signal peptide, interleukin-7, an IgG hinge, and a CD8 transmembrane domain.

[0096] In some embodiments, the recombinant protein comprises, from the N-terminus to the C-terminus, sequentially connected PGC1α, a cleavable peptide (e.g., P2A-T2A), a signal peptide, interleukin-7, a CD8 hinge, and a CD8 transmembrane domain.

[0097] In some embodiments, the recombinant protein comprises, from the N-terminus to the C-terminus, a signal peptide, interleukin-7, an IgG hinge, a CD8 transmembrane domain, a cleavable peptide (e.g., P2A-T2A), and a PGC1α variant, connected in sequence.

[0098] In some embodiments, the recombinant protein comprises, from the N-terminus to the C-terminus, a signal peptide, interleukin-7, a CD8 hinge, a CD8 transmembrane domain, a cleavable peptide (e.g., P2A-T2A), and a PGC1α variant, connected in sequence.

[0099] In some embodiments, the recombinant protein comprises, from the N-terminus to the C-terminus, a PGC1α variant, a cleavable peptide (e.g., P2A-T2A), a signal peptide, interleukin-7, an IgG hinge, and a CD8 transmembrane domain, connected in sequence.

[0100] In some embodiments, the recombinant protein comprises, from the N-terminus to the C-terminus, a PGC1α variant, a cleavable peptide (e.g., P2A-T2A), a signal peptide, interleukin-7, a CD8 hinge, and a CD8 transmembrane domain, connected in sequence.

[0101] In some embodiments, the amino acid sequence of the recombinant protein comprises the sequence shown in SEQ ID NO:13 or SEQ ID NO:14.

[0102] In some embodiments, the amino acid sequence of the recombinant protein is shown in SEQ ID NO:13 or SEQ ID NO:14.

[0103] Furthermore, this application also includes, within the scope of this application, functional variants of the recombinant protein described herein. As used herein, the term "functional variant" refers to a recombinant protein, polypeptide, or protein having a substantial or significant sequence identity or similarity to the parental recombinant protein, wherein the functional variant retains the biological activity of the recombinant protein variant. Functional variants encompass, for example, those variants of the recombinant protein (parental recombinant protein) described herein, which retain the ability to recognize target cells to a degree similar to, the same as, or greater than that of the parental recombinant protein. Regarding the parental recombinant protein, the amino acid sequence of the functional variant may, for example, have at least about 30%, about 50%, about 75%, about 80%, about 90%, about 98%, about 99%, or higher identity with the amino acid sequence of the parental recombinant protein.

[0104] The functional variant may, for example, comprise the amino acid sequence of the parental recombinant protein having at least one conserved amino acid substitution. Alternatively or additionally, the functional variant may comprise the amino acid sequence of the parental recombinant protein having at least one non-conserved amino acid substitution. In this case, non-conserved amino acid substitutions that do not interfere with or inhibit the biological activity of the functional variant are preferred. Non-conserved amino acid substitutions can enhance the biological activity of the functional variant, resulting in increased biological activity of the functional variant compared to the parental recombinant protein.

[0105] The amino acid substitutions in the recombinant protein of this application are preferably conservative amino acid substitutions. Conservative amino acid substitutions are those known in the art, and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is exchanged for another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions can include replacing an acidic / negatively charged polar amino acid with another acidic / negatively charged polar amino acid (e.g., Asp or Glu), replacing an amino acid with a nonpolar side chain with another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Tip, Cys, Val, etc.), replacing a basic / positively charged polar amino acid with another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), replacing an uncharged amino acid with a polar side chain with another uncharged amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), replacing an amino acid with a β-branched side chain with another amino acid with a β-branched side chain (e.g., Ile, Thr, and Val), and replacing an amino acid with an aromatic side chain with another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr, etc.).

[0106] The recombinant protein of embodiments of this application (including the functional portion and functional variants of this application) may comprise synthetic amino acids replacing one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, leucine, α-aminodecanoic acid, homoserine, S-acetaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2- Carboxylic acids, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norborneane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0107] Mutants that have a certain degree of amino acid homology with the recombinant protein as described above, for example, homology between 70% and 99%, further homology between 80% and 99%, further homology between 90% and 99%, and homology of 99%, should also fall within the scope of protection of this application.

[0108] The "homology" (sequence identity percentage) of an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence after sequence alignment, and, where necessary, the introduction of vacancies to achieve the maximum number of identical amino acids (or nucleic acids). In other words, the sequence identity percentage (%) of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) relative to the reference sequence by the total number of amino acid residues (or bases) in the candidate or reference sequence (whichever is shorter). Conservative substitutions of amino acid residues may or may not be considered identical residues. For example, publicly available tools can be used, such as BLASTN, BLASTp (available on the website of the US National Center for Biotechnology Information (NCBI), see also Altschul SF et al., Journal of Molecular Biology 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the website of the European Bioinformatics Institute, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., *Bioinformatics* (Cambridge, UK), 23(21):2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software can be used to perform alignments to determine the percentage of identity between amino acid (or nucleic acid) sequences. Those skilled in the art can use the default parameters provided by the tools or can appropriately customize the parameters as needed for the alignment, for example by selecting a suitable algorithm.

[0109] As used in this application, the term "amino acid" refers to an organic compound that includes amino (-NH2) and carboxyl (-COOH) functional groups, as well as the side chain characteristic of each amino acid. Amino acid names are also represented in this disclosure as standard single-letter or three-letter codes, summarized below.

[0110] Amino acid name Three-letter code Single-letter code alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C glutamic acid Glu E glutamine Gln Q glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F proline Pro P Serine Ser S threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

[0111] In a second aspect of this application, a nucleic acid is provided that encodes a protein combination as described in the first aspect.

[0112] In this application, unless otherwise stated, "nucleic acid" has the well-known meaning in the art and is also called "polynucleotide," which is a molecule formed by multiple nucleotide monomers.

[0113] In some embodiments, the nucleic acid comprises a nucleotide sequence encoding the recombinant protein as described above. In some embodiments, the nucleic acid expresses the recombinant protein as described above. In some embodiments, the sequence of the nucleic acid comprises the sequence shown in SEQ ID NO:15 or SEQ ID NO:16. In some embodiments, the sequence of the nucleic acid is shown in SEQ ID NO:15 or SEQ ID NO:16.

[0114] In some embodiments, the nucleic acid, as a single molecule, simultaneously comprises nucleic acid 1 encoding the interleukin or a variant thereof, and nucleic acid 2 encoding the PGC1α or a variant thereof. Although nucleic acid 1 and nucleic acid 2 are contained within the same nucleic acid molecule, they can be expressed separately.

[0115] In this application, the expression of nucleic acid 1 and nucleic acid 2 can be carried out in a manner that is conventional in the art. For example, nucleic acid 1 and nucleic acid 2 can be connected by an IRES element, or they can be expressed by a dual-initiator subsystem.

[0116] The terms “IRES” and “IRES element” used in this application are used interchangeably. IRES elements, found in eukaryotes and viruses that can infect eukaryotes, are nucleic acid sequences that enable protein translation initiation to be independent of the 5' cap structure, thus making it possible to initiate translation directly from the middle of messenger RNA (mRNA).

[0117] In some embodiments, nucleic acid 1 and nucleic acid 2 are connected via an IRES element.

[0118] In some embodiments, the nucleic acid comprises nucleic acid 1, an IRES element, and nucleic acid 2 sequentially from the 5' end to the 3' end.

[0119] In some implementations, nucleic acid 1 and nucleic acid 2 are expressed via a dual-initiator subsystem.

[0120] In some embodiments, the nucleic acid comprises, from the 5' end to the 3' end, a promoter, nucleic acid 1, a promoter, and nucleic acid 2 in sequence.

[0121] Unless otherwise specified, the term "promoter" in this application refers to a group of transcriptional control modules that cluster around the start site of RNA polymerase II. Many ideas about how promoters are organized have been derived from analyses of several viral promoters, including those concerning the HSV thymidine kinase (tk) and the SV40 early transcription unit. These studies, reinforced by more recent work, have shown that promoters consist of discontinuous functional modules, each approximately 7–20 bp of DNA, and contain one or more recognition sites for transcriptionally activating proteins. At least one module in each promoter acts to localize the start site of RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoters of mammalian terminal deoxynucleotidyltransferase genes and SV40 late genes, discontinuous elements overlapping their own start sites help fix the start site.

[0122] Additional promoter elements regulate transcription initiation frequency. These are typically located in regions 30-110 bp upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between elements is flexible, allowing promoter function to be preserved when elements are inverted or moved relative to each other. In the tk promoter, the spacing between elements can increase to 50 bp before activity begins to decline. Depending on the promoter, it appears that individual elements can act collectively or independently to activate transcription. Any promoter that can guide the initiation of transcription from a sequence encoded by a nucleic acid construct can be used in this invention.

[0123] Examples of promoters include, but are not limited to: constitutive promoters such as: simian virus 40 (SV40) early promoter, mouse mammary tumor virus promoter, human immunodeficiency virus long terminal repeat promoter, Moloney virus promoter, avian leukosis virus promoter, Epstein-Barr virus (EBV) early promoter, Rous sarcoma virus (RSV) promoter, human actin promoter, human myosin promoter, human hemoglobin promoter, cytomegalovirus (CMV) promoter, and human muscle creatine promoter; inducible promoters such as: metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter (tet on or tet off); tissue-specific promoters such as: HER-2 promoter, PSA-associated promoter, and bidirectional promoters, which can initiate transcription from either direction of the promoter.

[0124] In some implementations, the promoter is selected from the group consisting of EF1α, CMV, MSCV, and PGK.

[0125] In a third aspect of this application, a recombinant vector is provided, comprising the nucleic acid as described in the second aspect.

[0126] Unless otherwise specified, the term "vector" in this application refers to a delivery vehicle that can operatively insert a genetic element (such as the aforementioned nucleic acid molecule) therein and enable the expression of that genetic element. Vectors can be, for example, plasmids, granules, viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses), RNA vectors, or linear or circular DNA or RNA molecules, which may include chromosomal, non-chromosomal, semi-synthetic, or synthetic nucleic acid molecules. This term includes vectors that serve as self-replicating nucleic acid structures as well as vectors incorporated into the host cell genome. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked.

[0127] In some embodiments, the backbone of the recombinant vector is a viral vector.

[0128] Unless otherwise specified, "viral vector" in this application refers to a vector obtained through viral modification that carries foreign genes and related gene elements, can be packaged into viral particles, and can mediate the transfer and expression of foreign genes. The viral vector can be a recombinant viral vector or a viral vector without viral genes. Non-limiting examples of viral vectors include adenovirus vectors, adeno-associated virus vectors, lentiviral vectors, etc.

[0129] In some embodiments, the viral vector is selected from the group consisting of lentiviral vectors, retroviral vectors, adenoviral vectors, and adeno-associated virus vectors. As used herein, the term "lentivirus" refers to a genus within the family Retroviridae. Lentivirals are unique among retroviruses in their ability to infect non-dividing cells; they can deliver significant amounts of genetic information into the DNA of host cells, making them one of the most efficient methods of gene delivery vectors. Human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV) are examples of lentiviruses.

[0130] In some embodiments, the backbone of the recombinant vector is an HIV recombinant lentiviral vector. In some embodiments, the backbone of the recombinant vector is an HIV-1 recombinant lentiviral vector.

[0131] In some embodiments, the recombinant vector includes a recombinant lentivirus.

[0132] In some embodiments, in the recombinant vector, nucleic acid 1 and nucleic acid 2 are linked by an IRES element.

[0133] In some embodiments, the recombinant vector comprises nucleic acid 1, an IRES element, and nucleic acid 2 sequentially from the 5' end to the 3' end.

[0134] In some embodiments, nucleic acid 1 and nucleic acid 2 are expressed in the recombinant vector via a dual-promoter subsystem.

[0135] In some embodiments, the recombinant vector contains, from the 5' end to the 3' end, a promoter, nucleic acid 1, a promoter, and nucleic acid 2 in sequence.

[0136] In a fourth aspect of this application, a nucleic acid combination is provided, comprising nucleic acid 1 and nucleic acid 2 as defined above.

[0137] While not wishing to be limited by any theory, the nucleic acid combination here can be considered as a combination of two nucleic acid molecules, each containing nucleic acid 1 and nucleic acid 2. Nucleic acid 1 and nucleic acid 2 are expressed separately in different nucleic acid molecules, using promoters as defined above.

[0138] In a fifth aspect of this application, a recombinant vector is provided, comprising the nucleic acid combination as described in the fourth aspect. The backbone and other components of the recombinant vector described herein are as defined in the fourth aspect.

[0139] In some embodiments, the recombinant vector further includes an IRES element for linking nucleic acid 1 and nucleic acid 2.

[0140] In some embodiments, nucleic acid 1 and nucleic acid 2 are linked via an IRES element in the recombinant vector.

[0141] In some embodiments, the recombinant vector contains nucleic acid 1, an IRES element, and nucleic acid 2 sequentially from the 5' end to the 3' end.

[0142] In some embodiments, nucleic acid 1 and nucleic acid 2 are expressed in the recombinant vector via a dual-promoter subsystem.

[0143] In some embodiments, the recombinant vector contains, from the 5' end to the 3' end, a promoter, nucleic acid 1, a promoter, and nucleic acid 2 in sequence.

[0144] In a sixth aspect of this application, a recombinant vector combination is provided, comprising a recombinant vector 1 containing nucleic acid 1 as defined in the fourth aspect, and a recombinant vector 2 containing nucleic acid 2 as defined in the fourth aspect.

[0145] In some embodiments, the recombinant vector 1 and the recombinant vector 2 use the same backbone or expression vector.

[0146] In some embodiments, the recombinant vector 1 and the recombinant vector 2 use different backbones or expression vectors.

[0147] The types of skeletons or expression carriers involved are as defined above.

[0148] In a seventh aspect of this application, an engineered immune cell is provided that expresses the protein combination described in the first aspect.

[0149] In some embodiments, the engineered immune cells comprise one or more of the following: nucleic acids as described in the second aspect, recombinant vectors as described in the third aspect, combinations of nucleic acids as described in the fourth aspect, recombinant vectors as described in the fifth aspect, and combinations of recombinant vectors as described in the sixth aspect.

[0150] In some embodiments, the engineered immune cells are obtained by transducing one or more of the recombinant vectors described in the third aspect, the fifth aspect, and the sixth aspect, after processing the immune cells to be engineered.

[0151] In some embodiments, the engineered immune cells are tumor-infiltrating lymphocytes (TILs).

[0152] The inventors have creatively designed engineered immune cells, such as engineered TILs, to simultaneously express mIL-7 and PGC1α or its variants, thus addressing the issue of significant upregulation of reactive oxygen species in mitochondria during mIL-7 expression in lymphocytes. Specifically, the engineered immune cells overexpressing membrane-bound IL-7 and PGC1α not only enhance the proliferation capacity of TILs in vivo and in vitro but also increase mitochondrial activity of T cells, thereby enhancing energy metabolism.

[0153] In an eighth aspect of this application, a cell-based pharmaceutical composition is provided, comprising engineered lymphocytes as described in the seventh aspect, and a pharmaceutically acceptable carrier.

[0154] Unless otherwise specified, the term "pharmaceutical acceptable" in this application means those ligands, materials, compositions, and / or dosage forms that are suitable for administration to patients within the bounds of reasonable medical judgment and that are commensurate with a reasonable benefit / risk ratio.

[0155] Unless otherwise specified, the term "pharmaceuticalally acceptable carrier" in this application refers to a pharmaceutically acceptable material, composition, or medium, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials. As used herein, the language "pharmaceuticalally acceptable carrier" includes buffers compatible with drug administration, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, and the like. Each carrier is "pharmaceutically acceptable" in the sense of compatibility with other components in the formulation and harmlessness to the patient.

[0156] In a ninth aspect of this application, the use of recombinant vectors as described in the third or fifth aspect, combinations of recombinant vectors as described in the sixth aspect, or engineered immune cells as described in the seventh aspect is provided in the preparation of a medicament for treating cancer.

[0157] The cancers mentioned include, but are not limited to, liver cancer, lung cancer, ovarian cancer, cervical cancer, melanoma, breast cancer, colorectal cancer, and pancreatic cancer.

[0158] In a tenth aspect of this application, a method for treating cancer is provided, the method comprising administering to a subject engineered immune cells as described in the seventh aspect or a cell-pharmaceutical composition as described in the eighth aspect.

[0159] As used in this application, the term "treatment" refers to a therapeutic intervention aimed at reversing, alleviating, improving, suppressing, slowing, or stopping the progression or severity of a condition associated with a disease or symptom. The term "treatment" includes reducing or alleviating at least one side effect or symptom of a disease or symptom. Treatment is generally "effective" if it reduces one or more symptoms or clinical markers. Alternatively, treatment is "effective" if the progression of the disease is reduced or stopped; that is, "treatment" includes not only improvement of symptoms but also the cessation, or at least slowing, of the expected progression or worsening of symptoms in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, reduction of one or more symptoms, reduction of disease severity, stabilization (i.e., non-worsening) of the disease state, delay or slowing of disease progression, improvement or relief of the disease state, and relief (whether partial or complete), whether detectable or undetectable.

[0160] The following are some examples.

[0161] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0162] Experimental Methods 1. Preparation and titer determination of lentiviruses

[0163] 1) Lentiviral vector packaging

[0164] Each lentiviral expression vector was constructed separately. The plasmids of each vector were mixed with the lentiviral packaging plasmids pMDLg / pRRE, pRSV-Rev, and pMD2.G respectively using polyethyleneimine transfection reagent and co-transfected into 293T cells (purchased from ATCC, CRL-3216). Six hours after transfection, the medium was replaced with complete medium (DMEM medium + 10% fetal bovine serum + 100 U / ml penicillin + 100 μg / ml streptomycin). After 72 hours of culture, the viral supernatant was collected, centrifuged at 3000 rpm for 10–15 min at 4°C, filtered through a 0.45 μm pore size filter, and concentrated. The collected viral concentrate was stored at -80°C.

[0165] 2) Lentiviral titer determination

[0166] Add 300 μl of complete culture medium (DMEM medium + 10% fetal bovine serum + 100 U / ml penicillin + 100 μg / ml streptomycin) and Jurkat cells to each well of a 24-well plate to achieve a cell density of 1 × 10⁻⁶ cells / well. 5 Cells / well. Lentiviral concentrate was serially diluted 5-fold with Opti-MEM medium. Each dilution of lentivirus was added at a rate of 200 μl / well to each well of the 24-well plate to infect Jurkat cells (Jurkat cells in the negative control group were only infused with Opti-MEM medium), and then cultured in a cell culture incubator (37°C, 5% CO2). After 3 days of culture, cellular DNA was extracted from each well, and viral titer was determined using qPCR.

[0167] Experimental Method 2. Lentiviral transduction and detection of TIL

[0168] 1) Preparation of TIL cells

[0169] CD3 is isolated / amplified and cultured from liver cancer / colorectal cancer tumor tissue samples obtained by surgical resection / puncture biopsy. + TIL, or TIL recovered from frozen samples, adjusted to 2 × 10⁻⁶. 6 Cells were cultured at a density of 1 / mL in TIL amplification medium (X-vivo 15 + 5% SR + 600 IU / mL IL-2 + 5 ng / mL IL-7 + 5 ng / mL IL-15; all TIL amplification medium mentioned below refers to this formulation) in a cell culture incubator for 24 h (culture temperature: 37℃, carbon dioxide concentration: 5%).

[0170] 2) Lentiviral transduction of TIL

[0171] The obtained TILs were washed with culture medium, and the cell density was adjusted to 3 × 10⁶ cells / year. 6Cells / mL ~ 4 × 10 6 Lentiviral cells / mL were added at an MOI of 1 IU / mL to 10 IU / mL for transduction, and cultured in TIL amplification medium in a cell culture incubator (culture temperature 37℃, carbon dioxide concentration 5%).

[0172] Four to eight hours later, the transduced TILs were co-cultured with gamma-ray irradiated feeder cells (PBMCs) at a ratio of 1:100, with a TIL cell density of 1×10⁻⁶ cells / year during co-culture. 4 The sample was incubated in X-vivo 15 + 5% SR + 600 IU / mL IL-2 + 5 ng / mL IL-7 + 5 ng / mL IL-15 with the addition of 30 ng / mL OKT3 and 3 ng / mL TGFβ1.

[0173] Seven days later, the cells were rehydrated with an equal volume of fluid, and OKT3 was not added again.

[0174] Ten days later, the cells were centrifuged and the medium was changed; TGFβ1 was no longer added to the culture system. Cells were counted, and based on the cell count, a suitable container was selected for cell expansion. Subsequent cell replenishment or expansion was performed every 3 days. During rapid expansion (days 21-24), cells were harvested and cryopreserved in liquid nitrogen using cryopreservation medium (containing 5% human serum albumin: physiological saline = 1:1) for later use. The obtained TILs were named according to their corresponding molecular structures; TIL cells not transduced with lentivirus were named Mock TILs. After thawing, the cryopreserved cells were used for subsequent in vitro and in vivo experiments.

[0175] 3) Detection of membrane-bound IL-7 expression

[0176] The mock TILs to be tested and the experimental group (such as BWm7P4 cells in the following examples) were washed twice with PBS and resuspended in FACS buffer. Anti-Human IL-7 (mouse) and BV421-labeled anti-human CD3 antibodies were added to the cell suspension according to the antibody manufacturer's instructions and incubated at 4°C for 30 min. Mock TILs not transduced with lentivirus were used as a negative control. The expression rate of IL-7 on the cell membrane surface was detected by flow cytometry. Analysis was performed using FlowJo software.

[0177] The mock TILs to be tested and the experimental groups (such as BWm7-GP4, BNC006P4, or BNC007P4 cells in the following examples) were washed twice with PBS and resuspended in FACS buffer. Anti-Human IgG AF647 (mouse) and BV421-labeled anti-human CD3 antibodies were added to the cell suspension according to the antibody manufacturer's instructions and incubated at 4°C for 30 min. Mock TILs not transduced with lentivirus were used as a negative control. The expression rate of IgG on the cell membrane surface was detected by flow cytometry to reflect the expression rate of membrane-anchored IL-7. Analysis was performed using FlowJo software.

[0178] 4) Detection of membrane-bound PGC1α expression

[0179] The mock TILs to be tested and the experimental groups (such as BN118P4, BN118-NTP4, BN118-MutP4, BNC006P4, or BNC007P4 cells in the following examples) were washed twice with PBS, and then resuspended in BD Cytofix / Cytoperm cell fixation / permeabilization buffer for 30 min. After washing and resuspending the cells with BD Perm / Wash buffer, APC-labeled anti-PGC1α antibody and BV421-labeled anti-human CD3 antibody were added to the cell suspension to be tested according to the antibody instructions, and incubated at 4°C for 30 min. Figure 2B For the detection of PGC1α in the data, anti-His tag-PE antibody was used, and flow cytometry staining was performed in the same manner as described above. Mock TIL cells not transduced with lentivirus were used as a negative control. Intracellular PGC1α expression rates in BN118P4, BN118-NTP4, BN118-MutP4, BNC006P4, or BNC007P4 cells were detected by flow cytometry. Analysis was performed using FlowJo software.

[0180] Experimental Method 3. In vitro functional experiment of TIL

[0181] 1) Killing effect detection (IncuCyte live cell real-time dynamic imaging analysis method)

[0182] The killing effect of TILs was detected using the hepatocellular carcinoma cell lines SK-Hep-1 (internal code SK-BN105) and Hep3B. Cells prepared in part 2) of experimental method 2 above were digested and counted at a ratio of 1×10⁻⁶. 4 The target cells were seeded into 96-well plates at a density of / well, and 1×10⁶ cells were added. 4 T cells were co-cultured in / wells. The long-term killing effect was then assessed using the IncuCyte live-cell real-time dynamic imaging system.

[0183] 2) Flow cytometry detection of intracellular IFNγ expression levels

[0184] Surface labeling detection: Wash each group of cells twice with PBS and resuspend in FACS buffer. Prepare the AF700 (Alexa) antibody according to the antibody manufacturer's instructions. Anti-human CD8 antibody labeled with 700, BV650 (Brilliant Violet) TM Anti-human CD4 antibody labeled with 650 Dye, BV421 (Brilliant Violet) TM 421-Dye-labeled anti-human CD3 antibody and other surface marker-related antibodies were added to the cell suspension to be tested and incubated at 4°C for 30 min. The cells were washed twice with FACS buffer and resuspended in PBS. CD8+ levels in TILs of each group were detected by flow cytometry. + T cells, CD4 + The expression of various surface markers in T cells was analyzed using FlowJo software.

[0185] Intracellular cytokine detection: Mock TILs and TILs from each group were co-cultured with their respective target cells in IL-2-free X-vivo medium (effect-to-target ratio 4:1). After 12 h, cytokine secretion blocking agents were added at a ratio of 1:1000, and the cells were cultured at 37°C with 5% carbon dioxide for 4 h. After culture, the cells in each group were washed twice with PBS and resuspended in FACS buffer. Following the antibody instructions, AF700-labeled anti-human CD8 antibody, BV650-labeled anti-human CD4 antibody, BV421-labeled anti-human CD3 antibody, and PE-Cy7-labeled anti-human PD-1 antibody were added to the cell suspension and incubated at 4°C for 30 min. Cells were washed twice with FACS buffer, fixed and permeabilized using the BD Cytofix / Cytoperm kit, and then incubated at 4°C for 30 min with FITC-labeled anti-human IFN antibody, PE-labeled anti-human IL-2 antibody, FITC-labeled Granzyme B antibody, or APC-labeled perforin antibody. IFNγ levels in TILs of each group were detected by flow cytometry. + CD8 + T cells, IL-2 + CD8 + Cells, Granzyme B + CD8 + T cells or perforin + CD8 + The proportion of T cells was analyzed using FlowJo software.

[0186] Mitochondrial functional flow cytometry assay: Mock TILs and TILs from each group were cultured in TIL-specific amplification medium. MitoTracker Green mitochondrial dye was added at a molar ratio of 1:10000, or MitoSOX mitochondrial reactive oxygen species dye was added at a molar ratio of 1:5000. BV421-labeled CD3 antibody was also added. The cells were incubated at 37°C for 30 min. The cells were washed twice with pre-warmed complete medium, resuspended in PBS, and immediately subjected to flow cytometry to detect CD3. + The staining intensity of mitochondrial dyes in T cells was analyzed using FlowJo software.

[0187] 3) Cell proliferation detection

[0188] Each group was inoculated with the same amount of TILs and transduced with the corresponding lentivirus. Cell counts were performed on days 7, 11, and 14 after transduction. Cell proliferation in each group was statistically analyzed.

[0189] Experimental Method 4. In vivo tumor suppression experiment of TIL

[0190] Target cells in the logarithmic growth phase and in good growth condition were collected using trypsin digestion. After washing once with physiological saline, the cell density was adjusted to 2 × 10⁻⁶ cells / year. 7 / ml. 100 μl of cell suspension was subcutaneously injected into the right axillary region of NOG mice, i.e., 2 × 10⁹ cells per mouse. 6 SK-HEP-1 target cells.

[0191] Day 7 after inoculation with SK-Hep-1 target cells (or when the average tumor volume is approximately 50 mm) 3 At that time, TILs overexpressing mIL-7 and / or PGC1α-related structures (1×10⁻⁶) were injected via the tail vein. 7 / each), Mock TIL (1×10) 7 The test substance was administered to each mouse (400 μl / mouse) and the solvent (400 μl / mouse). The day of injection was recorded as day 0 of treatment. Tumor size and mouse weight were measured 2–3 times per week.

[0192] Example 1: Optimization of overexpression of PGC1α-related structures

[0193] First, this study optimized the structure of the overexpressed PGC1α molecule. Since the DNA sequence corresponding to the complete expression of the PGC1α molecule is 2400 bp, considering the effectiveness and efficiency of expression during TIL modification, point mutations or truncation of PGC1α were performed to optimize the structure.

[0194] The amino acid sequence of wild-type PGC1α (PGC1α-WT, corresponding to lentivirus and cell code BN118P4) is shown in SEQ ID NO:6, and the sequence encoding its nucleic acid is shown in SEQ ID NO:22.

[0195] PGC1α-WT was point-mutated to introduce S571A, resulting in PGC1α-Mut (corresponding to lentivirus and cell code BN118-MutP4), whose amino acid sequence is shown in SEQ ID NO:7 and whose nucleic acid sequence is shown in SEQ ID NO:23.

[0196] PGC1α-WT was truncated, retaining the first 270 amino acids from its N-terminus to obtain the truncated mutant PGC1α-NT (corresponding to the lentivirus and cell code BN118-NTP4), whose amino acid sequence is shown in SEQ ID NO:8 and whose nucleic acid coding sequence is shown in SEQ ID NO:24. Figures 2A to 2G This application provides an embodiment for optimizing the overexpression structure of PGC1α. A schematic diagram of PGC1α and its structure is shown below. Figure 2A .

[0197] Lentiviral vector plasmids containing PGC1α-WT, PGC1α-Mut, and PGC1α-NT were constructed, and a 6×his tag (SEQ ID NO:9) was added to the C-terminus of the target protein.

[0198] Referring to Parts 1) and 2) of Experimental Method 2, the three constructed lentiviral vectors were transduced into TILs to overexpress different PGC1α, resulting in gene-edited TILs, which were named BN118P4 TIL, BN118-NTP4 TIL, and BN118-MutP4 TIL, respectively; the expression of different PGC1α in TILs was detected according to Part 4).

[0199] The expression rates of PGC1α overexpression showed that the expression rates of all three structures were around 50%, with the following rates: BN118P4 (48.5%), BN118-NTP4 (53.0%), and BN118-MutP4 (51.6%). Figure 2B Overexpression of PGC1α reduced mitochondrial activity in TILs. Figure 2C The expression of PGC1α significantly enhanced the scavenging capacity of mitochondrial reactive oxygen species (ROS), as evidenced by a significant upregulation in the proportion of highly positive mitochondrial activity staining (high mitochondrial activity) and a significant downregulation in the proportion of low positive mitochondrial activity staining (low mitochondrial activity). Furthermore, MitoSOX staining results indicated that overexpression of PGC1α significantly enhanced the scavenging capacity of mitochondrial ROS. Figure 2D All of these were significantly enhanced, reflected in a significant decrease in the proportion of MitoSOX-positive individuals.

[0200] Simultaneously, in vitro functional experiments were conducted according to section 1) of Experimental Method 3 to detect the cytotoxic effects of the three TIL structures on liver cancer target cells. Gene-edited TILs were co-cultured with SK-HEP-1 tumor cells at an effector-to-target ratio of 1:3, and the cells were placed in an IncuCyte live-cell imaging analysis system to record the cytotoxic effect of TILs on tumor cells in real time. Since the tumor cells all overexpressed mCherry fluorescent protein, the fluorescence changes in tumor cells could be analyzed using IncuCyte software to characterize the degree of cytotoxicity caused by overexpressed TILs. Lower fluorescence signal values ​​indicated lower tumor cell activity and quantity in that group, indicating a more significant TIL cytotoxic effect. Figure 2E The results showed that BN118-MutP4 TIL and BN118-NTP4 TIL had significantly stronger SK-HEP-1 killing effects than BN118P4 TIL. Furthermore, flow cytometry was used to detect CD8+ cytotoxicity after co-culture. + T cell cytokine secretion; TIL overexpression of BN118P4 can upregulate CD8. + Expression of IFNγ and IL-2 in T cells ( Figure 2F ), while also improving GZMB + Perforin + CTLs proportion ( Figure 2G ).

[0201] Based on the above results, BN118-MutP4 showed significantly stronger cytotoxic effects and cytokine secretion than BN118-NTP4. However, due to structural differences, the truncated mutant PGC1α-NT in BN118-NTP4 expresses only 270 amino acids, significantly reducing the sequence length of PGC1α overexpression. Therefore, BN118-NTP4 was ultimately selected for further research.

[0202] Example 2: Optimization of membrane-bound IL-7 related structures

[0203] Because differences in sequence length used to anchor IL-7 can affect the effect of IL-7 on TILs, this study designed different molecular structures to anchor IL-7 to the cell membrane surface. Figure 3ATwo membrane-anchored forms of IL-7 were constructed: BWm7P4 (amino acid sequence as shown in SEQ ID NO:3, nucleic acid sequence as shown in SEQ ID NO:19) and BWm7-GP4 (amino acid sequence as shown in SEQ ID NO:12, nucleic acid sequence as shown in SEQ ID NO:28) using a shorter hinge region CD8hinge (amino acid sequence as shown in SEQ ID NO:3, nucleic acid sequence as shown in SEQ ID NO:19) and a longer hinge region IgG 4 (Hinge+CH3 domain) (amino acid sequence as shown in SEQ ID NO:2, nucleic acid sequence as shown in SEQ ID NO:18).

[0204] Lentiviral vector plasmids containing BWm7P4 and BWm7-GP4 were constructed.

[0205] Referring to Parts 1) and 2) of Experimental Method 2, the two constructed lentiviral vectors were transduced into TILs to overexpress different mIL-7s, resulting in gene-edited TILs, which were named BWm7P4 TIL and BWm7-GP4 TIL, respectively.

[0206] Referring to section 3) of Experimental Method 2, the expression rates of different forms of IL-7 were detected. The results showed that the positive rate of overexpression of BWm7P4 was significantly higher than that of BWm7-GP4 in the two different batches of TIL. Figure 3B Surprisingly, the killing data showed that, under different target cell types (including hepatocellular carcinoma lines SK-HEP-1 and Hep3B) and with different effector-to-target ratios, BWm7-GP4 TIL exhibited stronger killing activity than BWm7P4 TIL. Figure 3C This indicates that the molecular structure of BWm7-GP4 is more conducive to activating TILs, and it can exert a stronger activating effect at lower expression levels. Based on the above results, BWm7-GP4 and BN118-NTP4 will be co-expressed in TILs for subsequent studies.

[0207] Example 3: Optimization of the combined structure of membrane-bound IL-7 and PGC1α

[0208] Based on the above results, PGC1α-NT (amino acid sequence as shown in SEQ ID NO:1, nucleic acid sequence as shown in SEQ ID NO:17) and membrane-bound IL-7 were tandemly expressed in TILs using the P2A-T2A structure (amino acid sequence as shown in SEQ ID NO:8, nucleic acid sequence as shown in SEQ ID NO:24). Lentiviral transduction was performed according to parts 1) and 2) of Experimental Method 2, using an HIV recombinant lentiviral vector.

[0209] Because the P2A-T2A peptide may affect the expression intensity of the pre- and post-tandem molecules, two molecular structures were designed: BNC006P4 (or simply BNC006; amino acid sequence as shown in SEQ ID NO:13, and nucleic acid sequence encoding it as shown in SEQ ID NO:15) and BNC007P4 (or simply BNC007; amino acid sequence as shown in SEQ ID NO:14, and nucleic acid sequence encoding it as shown in SEQ ID NO:16). In BNC006P4, the BWm7-GP4 sequence is located at the 5' end, and the BN118-NTP4 sequence is located at the 3' end; in BNC007P4, the BN118-NTP4 sequence is located at the 5' end, and the BWm7-GP4 sequence is located at the 3' end. Figure 1 , Figure 4A ).

[0210] The expression rates of BNC006P4 and BNC007P4 were detected according to section 3) or 4) of Experimental Method 2. The expression rate results showed that the mIL-7 expression positivity rate of BNC006P4 was closer to that of BWm7-GP4 alone, while the mIL-7 expression rate of BNC007P4 was slightly lower (53%). Figure 4BBased on the results of killing target cells (SK-HEP-1) (taking an effector-to-target ratio of 1:3 as an example), in the batch experiments, the fluorescence intensity of target cells in the group with only target cells (Med group) was 7.72±0.23, while the fluorescence intensity of target cell growth in the Mock group (5.76±0.44) was downregulated by 1.34-fold (P<0.05); the fluorescence intensity of target cell growth in the group that overexpressed BN118-NTP4 alone (4.95±0.11) was downregulated by 1.56-fold (P<0.05); at the same time, the fluorescence intensity of target cells overexpressed BWm7-GP was also downregulated. The fluorescence intensity of target cells in all four groups was downregulated by 1.71-fold (P<0.05). The killing effect of TILs overexpressing BNC006P4 and BNC007P4 on target cells was significantly enhanced, as evidenced by a further downregulation of the fluorescence intensity of target cells. The fluorescence intensity of the BNC006P4 group was 4.08±0.34, downregulated by 1.89-fold (P<0.05), and the fluorescence intensity of the BNC007P4 group was 2.73±0.12, downregulated by 2.82-fold (P<0.001). Therefore, BNC007P4 TILs have a stronger killing effect on target cells. It is noteworthy that the additive fold reduction in target cell growth inhibition between the BN118-NTP4 and BWm7-GP4 groups was 2.66-fold (the product of the downregulation folds of the two groups), and the BNC007P4 group reached 2.82-fold, which was greater than the additive data of the BN118-NTP4 and BWm7-GP4 groups. This indicates that the simultaneous expression of BN118-NTP4 and BWm7-GP4 (i.e., BNC007P4) has a synergistic enhancing effect (1+1>2), and the results from multiple batches are stable and reproducible. Figure 4C Therefore, BNC007P4 was selected for subsequent research. The results of batch 2 (effect-to-target ratio 1:3) also showed the same trend of synergistic effect, that is, the fluorescence intensity of the Med group was 1.84±0.015, the downregulation factor of the Mock group was 1.08-fold (P<0.05, fluorescence intensity: 1.70±0.010), the downregulation factor of the BN118-NTP4 group was 1.53-fold (P<0.05, fluorescence intensity: 1.20±0.075), the downregulation factor of the BWm7-GP4 group was 1.31-fold (P<0.05, fluorescence intensity: 1.40±0.030), and the downregulation factor of the BNC007P4 group was 4.37-fold (P<0.001, fluorescence intensity: 0.421±0.033), which was greater than the superposition factor of 2.00-fold of the BN118-NTP4 group and the BWm7-GP4 group. On the other hand, co-expression of PGC1α and mIL-7 can compensate for the decline in mitochondrial function caused by mIL-7 expression. Figure 4D ) and the ability to scavenge reactive oxygen species (ROS) Figure 4E ).

[0211] Based on the above data, the molecular structure of BNC007P4 was selected for gene modification of TIL, and the following efficacy and safety evaluation experiments were carried out.

[0212] Example 4: In vitro functional experimental data on co-expression of PGC1α and mIL-7

[0213] Cell proliferation was assessed according to section 3) of Experimental Method 3. The seeding density of TIL cells in each group was 3 × 10⁻⁶. 6 Lentiviral transduction was performed at a concentration of / mL and an MOI of 10. Cell counts were performed on days 7, 11, and 14 post-transduction. Cell proliferation was statistically analyzed in each group.

[0214] First, regarding the in vitro proliferation of TILs from Mock, transduced BN118-NTP4, BWm7-GP4, or BNC007P4 groups, it was observed that after 14 days of culture, the cell count in the Mock group was (3.28±1.40)×10⁻⁶. 8 The cell count in the BN118-NTP4 group was (1.05±0.91)×10⁻⁶. 9 The cell count in the BWm7-GP4 group was upregulated by 3.20-fold compared to the Mock group (P<0.01); the cell count in the BWm7-GP4 group was (1.33±0.75)×10⁻⁶. 9 The cell count was upregulated by 4.05-fold relative to the Mock group (P<0.01); the cell count in the BNC007P4 group was (3.35±2.21)×10⁻⁶. 9 The proliferation rate was upregulated by 10.2-fold compared to the Mock group (P<0.01); in summary, the BNC007P4 group had the highest cell proliferation rate. Figure 5A Furthermore, BNC007P4 can significantly upregulate CD8. + CCR7 in T cells + CD45RA - The proportion of TCM ( Figure 5B ) and CD39 - CD69 - The proportion of stem cell-like T cells ( Figure 5C ).

[0215] Furthermore, co-culturing BNC007P4 TIL with liver cancer cell lines showed that BNC007P4 could enhance the killing effect of TIL on tumor cells. Figure 5DIt is worth noting that a synergistic enhancement effect on tumor cell killing was also observed in the BNC007P4 group. Specifically, the fluorescence intensity of target cells in the Med group was 3.72±0.08; the fluorescence intensity of target cells in the Mock group was 1.72±0.17 (downregulated by 2.16-fold relative to the Med group, P<0.05); and the fluorescence intensity of target cells in the BN118-NTP4 group was 0.98±0.09 (downregulated by 3-fold relative to the Med group). The fluorescence intensity of target cells in the BWm7-GP4 group was 0.96±0.15 (downregulated by 3.87-fold relative to the Med group, P<0.001); the fluorescence intensity of target cells in the BNC007P4 group was 0.23±0.15 (downregulated by 14.88-fold relative to the Med group, P<0.001), which was greater than the combined downregulation value of 14.67-fold of the BN118-NTP4 group and the BWm7-GP4 group. Furthermore, flow cytometry was used to detect CD8+ after co-culture. + T cell cytokine secretion. Compared to other groups, overexpression of BNC007P4 upregulated CD8. + Expression of IFNγ and IL-2 in T cells ( Figure 5E ), while also improving GZMB + Perforin + CTLs proportion ( Figure 5F In summary, co-expression of PGC1α and mIL-7 can enhance the specific killing ability of TILs against autologous tumor cells or tumor cell lines in vitro through synergistic effects.

[0216] Example 5: In vivo functional experimental data of co-expression of PGC1α and mIL-7

[0217] Furthermore, the in vivo antitumor efficacy of PGC1α and mIL-7 expression was examined. Specific experimental procedures are detailed in Experimental Method 4. An NCG mouse model with subcutaneous xenografts of the SK-Hep-1 liver cancer cell line was used. Tumors were approximately 200 mm in diameter. 3 Time-return TIL, 1×10 per group 7 The number of cells, and without concomitant IL-2 injection ( Figure 6 (A). Changes in mouse body weight showed that, in the Veh group without TIL treatment, the tumor-bearing mice experienced a gradual decrease in body weight 4 weeks after tumor formation due to the increasing tumor burden. Figure 6 (Middle B). Correspondingly, mice in groups that received transduction of BWm7-GP4, BN118-NTP4, or BNC007P4 showed a tendency to increase body weight; this result indicates that TIL re-infusion may have an anti-tumor effect, and also that no toxic side effects were observed in this mouse model of TIL treatment. Figure 6 (B). Furthermore, by statistically analyzing the tumor growth in mice, it can be known that ( Figure 6 In the middle C group, compared with the Veh group (Day 38 tumor volume: 1023.7±188.8mm), the tumor volume was 1023.7±188.8mm. 3 The TILs in the Mock group showed a certain anti-tumor effect (Day 46 tumor volume: 848.3±35.8mm). 3 Compared to the veh group, the tumor volume was reduced by 1.21 times (P<0.05); the anti-tumor effect of mIL-7 and PGC-1αTIL expression alone was enhanced (Day 46 tumor volume: mIL-7 group, 568.3±177.8mm). 3 PGC-1α group, 560.7±168.0mm 3 Compared to the Veh group, the tumor volume decreased by 1.80-fold (mIL-7 group, P<0.05) and 1.83-fold (PGC-1α group, P<0.05), respectively; the combined transduction of mIL-7 and PGC-1α TILs significantly improved the anti-tumor efficacy of TILs (tumor volume: 158.7±250.1 mm). 3 The level was 6.45 times lower than that of the Veh group (P<0.001), which was 3.29 times higher than the combined level of the mIL-7 group and the PGC-1α group.

[0218] The above results indicate that, compared with the single transfection group, the combined expression of mIL-7 and PGC-1α has a synergistic effect on the anti-tumor efficacy of TIL.

[0219] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments 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.

[0220] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A protein composition, characterized in that, It contains interleukins or variants thereof, and PGC1α or variants thereof.

2. The protein combination as described in claim 1, characterized in that, The interleukin is a membrane-anchored form of interleukin; Optionally, the membrane-anchored form of interleukin includes an operatively linked interleukin and a transmembrane domain; Further optionally, the membrane-anchored form of interleukin satisfies one or more of the conditions shown in items 1) to 4) below: 1) The interleukin and the transmembrane domain are operatively connected by a hinge; optionally, the hinge is an IgG hinge or a CD8 hinge; 2) The transmembrane domain is a CD8 transmembrane domain; 3) The interleukin is interleukin-7; and, 4) The membrane-anchored form of interleukin also contains a signal peptide; Optionally, the membrane-anchored form of interleukin may include, from the N-terminus to the C-terminus, a signal peptide, interleukin-7, an IgG hinge, and a CD8 transmembrane domain.

3. The protein combination as described in claim 1 or 2, characterized in that, In the PGC1α or its variants, the variants are obtained by mutating or truncating the PGC1α; Optionally, the amino acid sequence of the PGC1α is shown in SEQ ID NO:6; Further, alternatively, the amino acid sequence of the variant is as shown in SEQ ID NO:7 or SEQ ID NO:

8.

4. The protein combination according to any one of claims 1 to 3, characterized in that, The interleukin or its variants and the PGC1α or its variants exist separately in their own forms, are co-expressed via DNA or RNA vectors, or are operatively linked as recombinant proteins. Optionally, in the recombinant protein, the interleukin or a variant thereof and the PGC1α or a variant thereof are linked by a linker peptide; the linker peptide is optionally selected from the group consisting of cleavable peptides P2A, T2A, and F2A, for example, the linker peptide is P2A+T2A; wherein: Further optionally, the recombinant protein comprises, from the N-terminus to the C-terminus, the following: Interleukin or its variants, linker peptides, and PGC1α or its variants; or PGC1α or its variants, linker peptides, and interleukins or their variants; Alternatively, the amino acid sequence of the recombinant protein is shown in SEQ ID NO:13 or SEQ ID NO:

14.

5. A nucleic acid encoding a combination of proteins as described in any one of claims 1 to 4; Optionally, the nucleic acid encodes a recombinant protein as defined in claim 4; Further optionally, the nucleic acid sequence comprises the sequence shown in SEQ ID NO:15 or SEQ ID NO:

16.

6. A recombinant vector comprising the nucleic acid as described in claim 5; Optionally, the recombinant vector satisfies one or more of the conditions shown in 1) to 4) below: 1) The recombinant vector used is selected from the group consisting of lentiviral vectors, γ-retroviral vectors, and lipid nanoparticles; 2) Nucleic acid 1 and nucleic acid 2 are expressed separately using a dual-initiator subsystem; 3) Includes promoters selected from the group consisting of EF1α, CMV, MSCV, and PGK; 4) Nucleic acid 1 and nucleic acid 2 are expressed by being linked through an IRES element.

7. A combination of nucleic acids comprising nucleic acid 1 encoding the interleukin or a variant thereof, and nucleic acid 2 encoding the PGC1α or a variant thereof; in, The interleukin or a variant thereof is defined as in claim 2; the PGC1α or a variant thereof is defined as in claim 3; The sequence of nucleic acid 1 may optionally include the sequence shown in SEQ ID NO:27 or SEQ ID NO:28; The sequence of nucleic acid 2 may optionally include sequences as shown in SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:

24.

8. A recombinant vector comprising the nucleic acid combination as described in claim 7; Optionally, the recombinant vector satisfies one or more of the conditions shown in 1) to 4) below: 1) The recombinant vector used is selected from the group consisting of lentiviral vectors, γ-retroviral vectors, and lipid nanoparticles; 2) Nucleic acid 1 and nucleic acid 2 are expressed separately using a dual-initiator subsystem; 3) Includes promoters selected from the group consisting of EF1α, CMV, MSCV, and PGK; 4) Nucleic acid 1 and nucleic acid 2 are expressed by being linked through an IRES element.

9. A recombinant vector combination comprising a recombinant vector 1 comprising a nucleic acid 1 as defined in claim 7, and a recombinant vector 2 comprising a nucleic acid 2 as defined in claim 7.

10. The recombinant vector combination as described in claim 9, characterized in that, The recombinant vectors 1 and 2 are each independently selected from the group consisting of lentiviral vectors, γ-retroviral vectors, and lipid nanoparticles.

11. An engineered immune cell that expresses a combination of proteins as described in any one of claims 1 to 4; Optionally, the engineered immune cells satisfy one or more of the conditions shown in i) to iii) below: i) It comprises one or more of the nucleic acids as described in claim 5 and the combinations of nucleic acids as described in claim 7; ii) It is obtained by transducing immune cells to be engineered from one or more of the recombinant vectors of claim 6, the recombinant vector of claim 8, and the combination of recombinant vectors of claim 9 or 10; and, iii) It is selected from a group consisting of T cells, NK cells and tumor-infiltrating lymphocytes.

12. A cell-based pharmaceutical composition comprising engineered immune cells as described in claim 11, and a pharmaceutically acceptable carrier.

13. Use of the recombinant vector as claimed in claim 6 or 8, the combination of recombinant vectors as claimed in claim 9 or 10, or the engineered immune cells as claimed in claim 11 in the preparation of a medicament for treating cancer.

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