Gene modification method of fusion cytokine expression element and immunotherapy application of fusion cytokine expression element

Abstract

The invention discloses a gene modification method of a fusion cytokine expression element and an immunotherapy application of the fusion cytokine expression element, and relates to the technical field of gene engineering and tumor immunotherapy. Comprising the following steps that A, a preset site is selected in a genome of a host cell, a first construct AL-B-C-B-AR is provided, AL is a first homologous arm located on the upstream of the preset site, AR is a second homologous arm located on the downstream of the preset site, B is an I-SceI incision enzyme recognition site, and C is a selection marker capable of being screened in the forward direction; inserting the B-C-B sequence into the predetermined loci by homologous directional integration to form a replaceable landing frame; wherein the preset site is preferably a safe port site and a non-essential intron segment of a target functional gene, the length of the homologous arm is preferably one hundred to one thousand bases, and after integration, the correctness of the 5'end and the 3 'end is confirmed through PCR breakpoint amplification and sequencing at the same time. The whole body exposure and toxicity are reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering and tumor immunotherapy, in particular to a gene modification method of fusion cytokine expression element and its immunotherapy use. BACKGROUND

[0002] In the existing immunotherapy strategy, enhancing antigen presentation and T cell efficacy are two key directions. Existing solutions co-express immune adjuvants (such as GM-CSF, B7.1) on the expression vector and introduce signal peptides and GPI membrane anchor regions, aiming to improve antigen presentation and local immune recognition strength. This idea can be achieved at the level of eukaryotic vectors and at the level of antigen construction. On the other hand, the scarless replacement strategy of genome site-specific modification can introduce new functional elements into the target site without leaving foreign operation sites. Among them, the two-step homologous recombination process (first insert I-SceI-selection marker-I-SceI, then replace the module with the desired fragment) has been used to improve the accuracy of modification and the feasibility of multi-site modification.

[0003] In the aspect of cell therapy, gene editing of T cell immune suppression axis (such as PD-1) has shown the potential to enhance anti-tumor activity and prolong survival, and is combined with peripheral antigen stimulation system for function expansion and verification. However, if only the receptors and inhibitory receptors of effector cells are enhanced, without limiting and shaping the space and time of cytokine release, systemic toxicity and fatigue phenotype may still occur.

[0004] The existing technology faces the following risks: cytokine system exposure risk: non-directional and continuous secretion easily leads to systemic toxicity, uncontrollable side effects, and mismatch with tumor microenvironment requirements. Uncontrollable expression module: conventional expression cassette is difficult to achieve self-limited release activated immediately and closed when resting, leading to continuous secretion of immune cells in the tumor environment. Insertion position and repeatable modification: random integration and residual operation sites are not conducive to quality consistency and multi-round editing, affecting process amplification and regulatory compliance. Although the two-step scarless strategy has been proposed, there is a lack of specialized integrated process and safety structure library for cytokine expression elements, and synergy with effector modules: such as only implementing checkpoint editing of PD-1, without superimposing local directional cytokine enhancement and membrane anchoring strategy, which is still insufficient for microenvironment remodeling of stubborn solid tumors. Therefore, it is necessary to design a gene modification method of fusion cytokine expression element and its immunotherapy use. SUMMARY

[0005] The purpose of the present application is to provide a gene modification method of fusion cytokine expression element and its immunotherapy use to solve the problems raised in the background art.

[0006] To solve the above technical problems, the present application provides the following technical solutions: a gene modification method of fusion cytokine expression element and its immunotherapy use, comprising the following steps: A) Select a predetermined site in the genome of a host cell and provide a first construct AL-B-C-B-AR, wherein AL is a first homologous arm upstream of the predetermined site, AR is a second homologous arm downstream of the predetermined site, B is an I-SceI endonuclease recognition site, and C is a positively selectable selection marker; the B-C-B sequence is inserted into the predetermined site by homologous directional integration to form a replaceable landing frame; wherein the predetermined site is preferably a safe harbor site and a non-essential intron segment of a target functional gene, the length of the homologous arm is preferably one hundred to one thousand bases, and the correctness of the 5' end and the 3' end after integration is confirmed by PCR breakpoint amplification and sequencing at the same time; B) Provide a second construct B-TL-E-TR-B, wherein TL and TR are homologous to the left and right side sequences after the landing frame in step A, and E is a cytokine expression element; I-SceI is transiently expressed and transcribed in the host cell to allow the B-C-B module in step A to be excised and replaced by the E element under the mediation of homologous recombination, thereby achieving site-specific integration without residual operation sites at the predetermined site; wherein residual-free means that the target allele after integration does not contain the I-SceI site and the selection marker sequence and the copy number is not more than two copies; C) The obtained cells are subjected to at least copy number detection, double-end sequencing of insertion site, transcriptional check of adjacent gene expression without abnormality, and expression leakage evaluation under induced and non-induced conditions; Wherein, the E element comprises the following three parts: a) a directional domain for realizing secretion and membrane anchoring, selected from at least one of a human-derived signal peptide, a glycosyl phosphatidylinositol anchoring region, and an immunoglobulin Fc fragment, for defining the enrichment of cytokines in the cell surface and the adjacent microenvironment; b) at least one cytokine coding sequence; c) an expression control domain for driving expression only when the cell is in an activated state and specific microenvironment signals exist, and combined with an insulator and a terminator to reduce background expression.

[0007] According to the above technical solutions, the directional domain is specifically defined as: a) The signal peptide is preferably a human-derived leading peptide with verified secretion efficiency and contains a high-efficiency hydrophobic region and a signal peptidase cleavage site, which is connected to the cytokine coding sequence by a flexible polypeptide linker to avoid structural interference; b) The membrane anchoring structure is preferably a transmembrane proximal sequence and a tail signal of a glycosyl phosphatidylinositol anchoring region for positioning the protein outside the plasma membrane, and / or an engineered immunoglobulin Fc fragment with a site substitution to reduce the binding with Fc receptors to reduce non-specific effects; c) optionally set switchable endoprotease sites and self-cleaving peptide segments for releasing soluble cytokines in the presence of specific proteases and changes in acid-base conditions to achieve dual mode of membrane anchoring and controlled release in the same configuration; And by flow cytometry for membrane surface anchoring protein quantitative identification, by extracellular and intracellular cytokine contrast test confirmed membrane anchoring ratio is not less than fifty percent.

[0008] According to the above technical scheme, the cytokine coding sequence is selected from any one and a combination of multiple of the following: interleukin twelve, interleukin fifteen and its receptor alpha chain complex, granulocyte macrophage colony-stimulating factor, interleukin seven; when multiple cytokines are co-expressed, the polypeptide self-cleavage sequence is preferably used to connect the coding sequences in cis and maintain the translation ratio of each protein substantially equivalent; for multi-subunit proteins, the subunits are preferably conjugated by short linker peptides in a single chain to improve secretion efficiency and stability; the coding sequence is optimized by host preferred codons, avoiding the generation of new splice bodies and open reading frames upstream of strong start codons, and verified polyadenylation signals are set at the three ends.

[0009] According to the above technical scheme, the expression control domain is a regulatory module based on activation dependence and microenvironment response, containing any one and multiple of the following: a) a composite promoter composed of T cell activation transcription factor response elements for enhancing transcription in the presence of antigen receptor signals; b) tumor microenvironment hypoxia response elements and other metabolic stress response elements for up-regulating expression in hypoxic and acidic microenvironments to make expression stronger in tumor local; c) insulator elements and transcription termination sequences are set upstream and downstream of the transcription unit respectively to reduce collateral activation and read-through, and microRNA target sequences for reducing non-target tissue background expression are incorporated in the microRNA recognition sites of non-target tissue-specific microRNAs; d) the lengths of the homologous arms TL and TR are limited to one hundred to one thousand bases, and the overlapping coverage range relative to the B site is at least eighty bases, to improve replacement efficiency and reduce inversion recombination; The combination of the expression control domain and the targeting domain makes the leakage expression under non-inducing conditions not higher than ten percent of the induced level.

[0010] According to the above technical scheme, it further includes the steps of synergistically editing and amplifying the function of immune suppression pathway genes in vitro, specifically: a) loss-of-function editing and expression down-regulation of programmed cell death protein one gene, and optionally site-specific modification of endogenous receptor constant region to reduce autoreactivity and improve receptor uniformity; b) short-term expansion in vitro with antigen receptor associated stimulation in combination with co-stimulation to activate the cells under controlled conditions to evaluate the expression response curve of the E-element and the cell killing efficiency; c) quality release testing at the end of the expansion period, including at least specific lysis assay, cytokine background release evaluation, genetic stability assessment and residual foreign nucleic acid detection; wherein the collaborative editing and the site-directed integration of the E-element are performed at the same predetermined site and different sites, preferably the landing frame is established and replaced first, and then the pathway editing is performed, so as to facilitate process tracing and batch consistency control.

[0011] According to the above technical scheme, further comprising an immune effector cell, the target allelic site of the immune effector cell is integrated with a cytokine expression element E without trace, and meets the following limitations: a) the cytokine baseline expression under non-induction conditions is lower than 10% of the induction peak, and can be significantly up-regulated within four to twenty-four hours when the receptor is activated and the microenvironment signal exists; b) the cytokine encoded by the E-element forms an enrichment on the cell surface and the adjacent microenvironment, and the membrane anchoring proportion is confirmed by protein quantification and flow cytometry, and the cytotoxicity and apoptosis rate are not significantly higher than that of the unmodified control; c) there is no integration event at non-target sites at the genomic level, and the frequency is lower than the preset threshold, the copy number is one to two copies, and it is confirmed by digital PCR and high-throughput sequencing; d) in the in vitro standardized functional assay, the target lysis rate, proliferation ability and persistence indicators all meet the qualified range set by the process specification.

[0012] According to the above technical scheme, further comprising a kit: i) a first construct containing AL-B-C-B-AR and a matched screening reagent, used to establish a landing frame at a predetermined site; ii) a second construct containing B-TL-E-TR-B and a vector for transiently providing I-SceI activity and a transcription product; iii) auxiliary factors and culture additives for promoting homologous recombination efficiency, and primer pairs and standard positive control templates for double-end verification of insertion sites; The kit can further include digital PCR reagents for copy number determination, fluorescently labeled antibodies for membrane anchoring detection, and induction and non-induction medium components for leaky expression evaluation, all components are packaged in sterile lyophilized and low temperature storage form respectively, and are accompanied by step-by-step operation instructions and quality control acceptance standards.

[0013] According to the above technical solution, it is used to treat patients with solid tumors and hematologic malignancies to enhance local immune effects in the tumor microenvironment and reduce the risk of systemic cytokine exposure; the uses include infusion and injection of a pharmaceutical composition containing the immune effector cells into patients, and adjuvant therapy in vivo that can induce and enhance the expression of the E element; wherein the treatment effect is comprehensively evaluated by objective response rate, disease control rate and safety indicators, and the systemic cytokine-related adverse reactions are lower than those of the control regimen without the directional domain and without the expression control domain.

[0014] Compared with existing technologies, the beneficial effects achieved by this invention are as follows: This invention utilizes membrane anchoring and targeted secretion of signal peptides, allowing cytokines to be released locally upon activation of effector cells, reducing systemic exposure and toxicity. It also leverages the concept of signal peptides and GPI anchoring to enhance immune recognition, enabling precise and controllable local immune amplification. Furthermore, it achieves seamless integration and reproducible processing: a two-step I-SceI homologous recombination replacement leaves no exogenous manipulation sites, facilitating multiple rounds of modification and process scale-up, improving consistency and compliance. It also synergizes with checkpoint editing: while targeting cytokines, it combines PD-1 and other inhibitory axis gene editing with peripheral antigen stimulation amplification, enhancing the reliability of antitumor activity and in vivo efficacy verification. Finally, it features a modular component library: expression control domains, secretion / anchoring domains, and cytokine coding domains can be combined according to indications to form a reusable engineered component library, reducing project customization cycles and costs. Attached Figure Description

[0015] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 This is a schematic diagram of the overall process of the present invention. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Please see Figure 1 The present invention provides a technical solution: a gene modification method for fusing cytokine expression elements and its use in immunotherapy, comprising the following steps: A) Select a predetermined site in the host cell genome and provide a first construct AL-BCB-AR, where AL is the first homologous arm upstream of the predetermined site, AR is the second homologous arm downstream of the predetermined site, B is the I-SceI endonuclease recognition site, and C is a selection marker for positive selection; insert the BCB sequence into the predetermined site through homologous directional integration to form a replaceable landing framework; wherein the predetermined site is preferably a safe harbor site and a non-essential intron segment of the target functional gene, the homologous arm length is preferably one hundred to one thousand bases, and after integration, the correctness of the 5′ end and 3′ end is confirmed simultaneously by PCR breakpoint amplification and sequencing; B) Provide a second construct B-TL-E-TR-B, wherein TL and TR are homologous to the left and right side sequences after the landing framework is formed in step A, respectively, and E is a cytokine expression element; transient expression and transcription of I-SceI in the host cell cause the BCB module in step A to be excised and replaced by the E element under homologous recombination, thereby achieving site-specific integration at a predetermined site without residue; wherein "without residue" means that the target allele after integration does not contain the I-SceI site and the selection marker sequence and the copy number does not exceed two copies. C) Perform at least the following on the obtained cells: copy number detection, insertion site paired-end sequencing, transcriptomic examination to ensure no abnormality in the expression of neighboring genes, and assessment of expression leakage under induced and non-induced conditions; The E element comprises the following three parts: a) a directional domain for secretion and membrane anchoring, selected from at least one of human signal peptide, glycosylphosphatidylinositol anchoring region and immunoglobulin Fc fragment, used to define the enrichment of cytokines on the cell surface and in the neighborhood microenvironment; b) at least one cytokine coding sequence; c) an expression control domain for driving expression only when the cell is activated and in the presence of specific microenvironment signals, and combined with insulators and terminators to reduce background expression; The specific targeting domain is defined as follows: a) The signal peptide is preferably a human leading peptide with verified secretion efficiency and contains a highly efficient hydrophobic region and a signal peptide cleavage site. It is linked to the cytokine coding sequence by a flexible polypeptide linker to avoid structural interference. b) The preferred membrane anchoring structure is the transmembrane proximal sequence and tail signal of the glycosylphosphatidylinositol anchoring region, which are used to locate the protein on the outer side of the plasma membrane. The other is an engineered immunoglobulin Fc fragment, which has site substitutions that reduce binding to the Fc receptor to reduce non-specific effects. c) Optional switchable protein cleavage sites and self-cleaving peptides can be set to release soluble cytokines in the presence of specific proteases and under varying acid-base conditions, so as to achieve a dual mode of membrane anchoring and controlled release under the same conformation. Furthermore, the membrane surface anchoring proteins were quantitatively identified by flow cytometry, and the membrane anchoring ratio was confirmed to be no less than 50% by a comparison experiment of extracellular and intracellular cytokines. The cytokine coding sequences are selected from any one or a combination of the following: interleukin-12, interleukin-15 and its receptor alpha chain complex, granulocyte-macrophage colony-stimulating factor, and interleukin-7. When multiple cytokines are co-expressed, it is preferred to use a peptide self-cleavage sequence to cis-link each coding sequence and maintain the translation ratio of each protein to be basically equal. For multi-subunit proteins, it is preferred to use a single-chain approach to connect the subunits with short linker peptides to improve secretion efficiency and stability. The coding sequences are optimized with host-preferred codons to avoid generating new splice variants and open reading frames upstream of strong start codons, and verified polyadenylation signals are set at the three ends. The expression control domain is a regulatory module based on activation dependence and microenvironment response, and includes any one or more of the following: a) A complex promoter composed of T cell-activated transcription factor response elements, used to enhance transcription in the presence of antigen receptor signals; b) Tumor microenvironment hypoxia-responsive elements and other metabolic stress-responsive elements, used to upregulate expression in hypoxic and acidic microenvironments, thereby making expression stronger in the tumor local area; c) Insulator elements and transcription termination sequences are set upstream and downstream of the transcription unit to reduce paraphyletic activation and readthrough, and microRNA target sequences for reducing background expression in non-target tissues are incorporated into microRNA recognition sites that are not specifically expressed in the target tissue. d) The lengths of both homologous arms TL and TR are limited to between one hundred and one thousand bases, and the overlap coverage of the relative B site is at least eighty bases, in order to improve substitution efficiency and reduce inversion recombination. The combination of the expression control domain and the orientation domain ensures that the leakage expression under non-induced conditions is no more than 10 percent of the induced level; It also includes steps for the co-editing and in vitro functional amplification of genes involved in immunosuppressive pathways, specifically: a) The programmed cell death protein gene is disfunctionalized and its expression is downregulated, and the endogenous receptor constant region can be selectively modified to reduce autoreaction and improve receptor homogeneity. b) In vitro, a short-term amplification system combining antigen receptor-related stimulation and co-stimulation was used to activate cells under controlled conditions to evaluate the expression response curve of E element and cell killing efficacy. c) Perform quality release tests at the end of the amplification cycle, including at least specific lysis assays, assessment of background cytokine release, evaluation of genetic stability, and detection of residual exogenous nucleic acids; Among them, collaborative editing and fixed-point integration of E-components are implemented at the same predetermined site and different sites. It is preferable to complete the establishment and replacement of the landing frame first, and then implement the path editing to facilitate process traceability and batch consistency control. It also includes immune effector cells that have cytokine expression element E seamlessly integrated at the target allele site and meet the following criteria: a) Under non-induction conditions, baseline cytokine expression is less than 10 percent of the induced peak, and can be significantly upregulated within four to twenty-four hours in the presence of receptor activation and microenvironment signals; b) Cytokines encoded by E-elements accumulate on the cell surface and in the neighborhood microenvironment. The membrane anchoring ratio was confirmed by both protein quantification and flow cytometry. Furthermore, the cytotoxicity and apoptosis rate were not significantly higher than those of the unmodified control. c) There are no integration events at non-target sites at the genome level and their frequency is below the preset threshold, the copy number is one to two copies and confirmed by digital PCR and high-throughput sequencing; d) In the in vitro standardized functional assay, the target lysis rate, proliferation capacity and persistence index all reached the qualified range set by the process specification; Also includes reagent kits: i) A first construct containing AL-BCB-AR and a matching screening reagent, used to establish a landing framework at a predetermined site; ii) A second construct containing B-TL-E-TR-B, and a vector and transcript for transiently providing I-SceI activity; iii) Co-factors and culture additives that promote homologous recombination efficiency, as well as primer pairs and standard positive control templates for verification of the two ends of the insertion site; The kit may further include digital PCR reagents for copy number determination, fluorescently labeled antibodies for membrane anchoring detection, and induction and non-induction culture medium components for leakage expression assessment. All components are packaged separately in sterile lyophilized and cryopreserved form and come with step-by-step instructions and quality control acceptance criteria. This treatment is intended to enhance local immune effects in the tumor microenvironment and reduce the risk of systemic cytokine exposure in patients with solid tumors and hematologic malignancies. Uses include infusion and injection of drug compositions containing immune effector cells, and adjuvant therapy in vivo that can induce and enhance E-element expression. Treatment efficacy is comprehensively assessed through objective response rate, disease control rate, and safety indicators, with systemic cytokine-related adverse events being lower than in control regimens without a targeting domain or expression control domain.

[0018] Example 1: A landing framework was established at the TRAC site of human peripheral blood-derived T cells, and the E element of "signal peptide-GM-CSF-flexible linker peptide-GPI anchoring region" was integrated in a seamless replacement manner. Combined with an activation-dependent complex promoter and insulator, local GM-CSF-releasing phenotype cells with membrane anchoring and extremely low leakage were obtained when T cells were activated. This was used to enhance local tumor immune amplification and reduce the risk of systemic exposure.

[0019] Donor cells: Peripheral blood mononuclear cells from healthy donors, sorted by magnetic beads to obtain CD3-positive T cells with initial viability ≥95%. First construct AL-BCB-AR: Predetermined site: TRAC intron (human genome coordinates selected according to hg38 reference, excluding key coding splicing sites). AL and AR are each 600–900 bp in length, covering both sides of the conserved region of the TRAC intron. B is the I-SceI site. C is a positively selectable puromycin resistance marker with a minimized promoter to reduce background. Second construct B-TL-E-TR-B: TL and TR are homologous to the sequences flanking the insertion in step A, each 400–800 bp. E-element: a) Direction domain: Human IL-2 signal peptide (N-terminal secretion-directed) + human DAF-derived GPI anchoring region (C-terminal membrane anchoring); b) Cytokine sequence: Human GM-CSF coding sequence (codon optimized, no new splicing sites introduced); c) Expression control domain: 6×NFAT response element + minimal IL-2 promoter; cHS4 type insulators are placed upstream and downstream; BGH polyadenylation signal is used at the 3′ end; the terminator is placed on the readthrough risk side. I-SceI expression vector or mRNA (one-time transient expression). Transfection system: Electroporation system (pulse program pre-optimized for human T cells). Reagents: CD3 / CD28 activation beads, IL-2 cytokine, puromycin, PI-PLC (phosphatidylinositol-specific phospholipase C, used to verify GPI anchoring). Detection methods include: PCR primer pairs (5′ / 3′ breakpoints), Sanger sequencing, digital PCR copy number, flow cytometry antibodies (anti-GM-CSF surface and intracellular markers, T cell markers), ELISA kits, RNA-seq or transcriptome panels.

[0020] S1 Pre-determined landing site establishment S1-1 Pre-activation: T cells were seeded at 1×10^6–2×10^6 cells / mL, CD3 / CD28 microbeads were added, and electroporation was performed 24 hours later.

[0021] S1-2 was electroporated into AL-BCB-AR and positively screened with puromycin for 3–5 days after 48 hours (0.5–1.0 μg / mL).

[0022] S1-3 Identification: 5′ breakpoint PCR was performed using the upstream outer primer of TRAC × the inner primer of C, and 3′ breakpoint PCR was performed using the inner primer of C × the downstream outer primer of TRAC; Sanger sequencing of the paired-end products confirmed correct insertion; digital PCR confirmed that the target site copy number was 1–2 copies.

[0023] S2E Component Design and Fabrication S2-1 Constructing the expression cassette: The sequence is signal peptide-GM-CSF-GPI anchoring region, with G4S×2 flexible linker peptides placed between GM-CSF and GPI, and BGH polyA attached to the 3′ end; insulators are placed upstream and downstream respectively.

[0024] S2-2 Promoter Function: The composite promoter used has a strong response after T cell activation and extremely low background in the resting state; a weak minimum promoter is set at the vector level to reduce leakage.

[0025] S2-3 Loading into the second construct: Loading E into the E site of B-TL-E-TR-B.

[0026] S3 Seamless Replacement S3-1 transiently expresses I-SceI in landing cells to cleave the BCB module, while simultaneously electroporating the B-TL-E-TR-B donor.

[0027] S3-2 After 48–72 hours, perform clonal or population amplification; perform breakpoint PCR using 5′ outer primer × E inner primer and E inner primer × 3′ outer primer; confirm that C and B are completely deleted by cross-region PCR covering the B site.

[0028] S3-3 Digital PCR copy number quantification (acceptance standard: 1–2 copies); whole-genome shallow sequencing sampling to check for integration at non-specific sites (threshold <0.5%). S4 Expression and Functional Verification S4-1 activation induction: Cells were divided into an unactivated group and an activated group (CD3 / CD28 microbeads, 24 hours).

[0029] S4-2 Flow cytometry: Surface anti-GM-CSF staining showed that the proportion of positive cells was ≥60%. After PI-PLC treatment, the surface signal decreased significantly and the supernatant GM-CSF increased instantaneously, proving GPI anchoring.

[0030] S4-3 Leakage assessment: The GM-CSF concentration measured by ELISA in the unactivated group was ≤10% of the peak value in the activated group; the peak value was reached 4–24 hours after activation.

[0031] S4-4 Supporting evidence: When engineered T cells were co-cultured with autologous dendritic cells for 24 hours, the upregulation of CD80, CD86, and HLA-DR in dendritic cells was significantly higher than that in the unengineered control.

[0032] S5 Quality and Safety Control S5-1 cell viability ≥85%; K562-transposon capture assay ruled out random high-copy insertion and was negative; S5-2 Transcriptional analysis: No abnormal upregulation of expression in genes adjacent to TRAC; S5-3 Residual Nucleic Acid: I-SceI mRNA residue negative; S5-4 Cryopreservation and thawing: After one freeze-thaw cycle, the functional and surface GM-CSF positivity rates remained at ≥80%.

[0033] Control 1: Randomly integrated secretory GM-CSF-expressing cells (without GPI anchoring and activation control); Control 2: Cells in which only the landing framework was established without replacing the E element; Evaluation indicators: surface anchoring ratio, leakage expression level, co-culture immune activation markers; In this embodiment, the leakage of GM-CSF in the unactivated state of the cells was significantly lower than that in control 1, and the local enrichment after activation was significantly higher than that in control 1, thus supporting the beneficial effects of "precise and controllable local immune amplification" and "reduced systemic exposure".

[0034] This invention utilizes membrane anchoring and targeted secretion of signal peptides, allowing cytokines to be released locally upon activation of effector cells, reducing systemic exposure and toxicity. Drawing on the approach of signal peptides and GPI anchoring to enhance immune recognition, it achieves precise and controllable local immune amplification. It features seamless integration and reproducible processing: a two-step I-SceI homologous recombination replacement leaves no exogenous manipulation sites, facilitating multiple rounds of modification and process scale-up, improving consistency and compliance. It also synergizes with checkpoint editing: while targeting cytokines, it combines PD-1 and other inhibitory axis gene editing with peripheral antigen stimulation amplification, enhancing the reliability of antitumor activity and in vivo efficacy verification. Furthermore, it includes a modular component library: expression control domains, secretion / anchoring domains, and cytokine coding domains can be combined according to indications to form a reusable engineered component library, reducing project customization cycles and costs.

[0035] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another entity or operation, and do not necessarily imply any such actual relationship or order between these entities and operations. Furthermore, the terms "comprising," "including," and any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, and includes elements inherent to such a process, method, article, or apparatus.

[0036] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments and make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method of genetically modifying a fusion cytokine expression element, characterized by: The method comprises the following steps: A) selecting a predetermined site in the genome of a host cell and providing a first construct AL-B-C-B-AR, wherein AL is a first homology arm upstream of the predetermined site, AR is a second homology arm downstream of the predetermined site, B is an I-SceI endonuclease recognition site, and C is a forwardly selectable selection marker; inserting the B-C-B sequence into the predetermined site by homology-directed integration to form a replaceable landing frame; wherein the predetermined site is preferably a safe harbor site and a non-essential intron segment of a target functional gene, the homology arms preferably have a length of one hundred to one thousand bases, and the correctness of the 5' end and the 3' end after integration is confirmed simultaneously by PCR breakpoint amplification and sequencing; B) providing a second construct B-TL-E-TR-B, wherein TL and TR are homologous to the left and right side sequences after the landing frame in step A, and E is a cytokine expression element; transiently expressing and transcribing I-SceI in the host cell to enable the B-C-B module in step A to be excised and replaced by the E element under the mediation of homologous recombination, thereby achieving site-specific integration without a residual operation site at the predetermined site; wherein residual-free refers to that after integration, the target allele does not contain the I-SceI site and the selection marker sequence and has a copy number of no more than two copies; C) performing at least copy number detection, double-end sequencing of the insertion site, transcriptional examination of no abnormal expression of adjacent genes, and expression leakage evaluation under induced and non-induced conditions on the obtained cells; wherein the E element comprises the following three parts: a) a directional domain for realizing secretion and membrane anchoring, selected from at least one of a human-derived signal peptide, a glycosyl phosphatidylinositol anchoring region, and an immunoglobulin Fc fragment, for defining the enrichment of the cytokine in the cell surface and the adjacent microenvironment; b) at least one cytokine coding sequence; and c) an expression control domain for driving expression only when the cell is in an activated state and specific microenvironment signals exist, and combined with an insulator and a terminator to reduce background expression.

2. The method of claim 1, wherein the method is a method of genetically modifying a fusion cytokine expression element. The directional domain is specifically defined as: a) the signal peptide is preferably a human-derived leading peptide with verified secretion efficiency and contains a highly efficient hydrophobic region and a signal peptidase cleavage site, which is connected to the cytokine coding sequence by a flexible polypeptide linker to avoid structural interference; b) the membrane anchoring structure is preferably a transmembrane proximal sequence and a tail signal of a glycosyl phosphatidylinositol anchoring region for positioning the protein outside the plasma membrane, and / or an engineered immunoglobulin Fc fragment with a site substitution to reduce the binding with Fc receptors to reduce non-specific effects; c) an optional switchable endoprotease site and a self-cleavage peptide segment are provided for releasing a soluble cytokine when a specific protease exists and the acid-base condition changes, to realize a dual mode of membrane anchoring and controlled release under the same configuration; and the membrane surface-anchored protein is quantitatively identified by flow cytometry, and the membrane anchoring proportion is confirmed to be no less than fifty percent by extracellular and intracellular cytokine comparison tests.

3. The gene modification method for fusing cytokine expression elements according to claim 2, characterized in that: The cytokine coding sequence is selected from any one and a combination of multiple of the following: interleukin twelve, interleukin fifteen and its receptor alpha chain complex, granulocyte macrophage colony stimulating factor, interleukin seven; when multiple cytokines are co-expressed, preferably polypeptide self-cleavage sequences are used to link the coding sequences in cis and maintain the translation ratio of each protein substantially equivalent; for multi-subunit proteins, the subunits are preferably conjugated by short linker peptides in a single chain manner to improve secretion efficiency and stability; The coding sequence is optimized by host-preferred codons, avoids the generation of new splice bodies and open reading frames upstream of strong start codons, and sets verified polyadenylation signals at the three ends.

4. The gene modification method for fusing cytokine expression elements according to claim 3, characterized in that: The expression control domain is an activation-dependent and microenvironment-responsive regulatory module, which includes any one and multiple of the following: a) a composite promoter composed of T cell activation transcription factor response elements, for enhancing transcription in the presence of antigen receptor signals; b) tumor microenvironment hypoxia response elements and other metabolic stress response elements, for up-regulating expression in hypoxic and acidic microenvironments to make expression stronger in the tumor local; c) insulator elements and transcription termination sequences are respectively set upstream and downstream of the transcription unit to reduce collateral activation and read-through, and microRNA target sequences for reducing non-target tissue background expression are incorporated in the microRNA recognition sites of non-target tissue-specific microRNAs; d) the lengths of the homologous arms TL and TR are both limited to one hundred to one thousand bases, and the overlapping coverage relative to the B site is at least eighty bases, to improve replacement efficiency and reduce inversion recombination; The combination of the expression control domain and the targeting domain makes the leakage expression under non-inducing conditions not higher than ten percent of the inducing level.

5. The method for genetic modification of a fusion cytokine expression element according to claim 4 and its immunotherapeutic use, characterized by the fact that: It also includes a synergistic editing and in vitro functional amplification step for immune suppression pathway genes, specifically: a) loss-of-function editing and expression down-regulation of programmed cell death protein one gene, and optionally site-specific modification of endogenous receptor constant regions to reduce autoimmunity and improve receptor uniformity; b) short-term expansion in an in vitro expansion system with antigen receptor-related stimulation and costimulatory compatibility, so that the cells are in an activated state under controlled conditions to evaluate the expression response curve of the E element and the cell killing efficiency; c) quality release detection at the end of the expansion period, including at least specific lysis test, cytokine background release evaluation, genetic stability evaluation and residual foreign nucleic acid detection; Wherein, the synergistic editing and the site-specific integration of the E element are implemented at the same predetermined site and different sites, preferably the landing frame is established and replaced first, and then the pathway editing is implemented, so as to facilitate process tracing and batch consistency control.

6. The gene modification method for fusing cytokine expression elements according to claim 5, characterized in that: It also includes immune effector cells, which have no trace integration of cytokine expression element E at the target allelic site, and meet the following limitations: a) the cytokine baseline expression under non-inducing conditions is lower than ten percent of the inducing peak, and can be significantly up-regulated within four to twenty-four hours in the presence of receptor activation and microenvironment signals; b) the E-element encoded cytokine forms enrichment at the cell surface and in the immediate microenvironment, the membrane-anchored proportion is confirmed by protein quantification and flow cytometry, and cytotoxicity and apoptosis rate are not significantly higher than the unmodified control; c) there is no integration event at non-target sites at the genomic level and its frequency is lower than the preset threshold, the copy number is one to two copies and is confirmed by digital PCR and high-throughput sequencing; d) in in vitro standardized functional assays, the target cleavage rate, proliferation capacity and persistence indicators all meet the qualified range set by the process specification.

7. The gene modification method for fusing cytokine expression elements according to claim 6, characterized in that: Also included are kits: i) a first construct containing AL-B-C-B-AR and a set of screening reagents matched therewith, for establishing a landing framework at the predetermined site; ii) a second construct containing B-TL-E-TR-B and a vector and transcription product for transiently providing I-SceI activity; iii) auxiliary factors and culture additives to promote the efficiency of homologous recombination, and primer pairs and standard positive control templates for double-end verification of the insertion site; The kit can further include digital PCR reagents for copy number determination, fluorescently labeled antibodies for membrane anchoring detection, and induced and non-induced medium components for leaky expression evaluation, all components are packaged separately in sterile lyophilized and low-temperature storage form, and are accompanied by step-by-step operation instructions and quality control acceptance standards.

8. An immunotherapeutic use of a genetic modification method of fusion cytokine expression elements, characterized by: For treating patients derived from solid tumors and hematological tumors to enhance local immune effect in the tumor microenvironment and reduce the risk of systemic cytokine exposure; the use includes making the patient receive back infusion and injection of a pharmaceutical composition containing the immune effector cells, and in vivo administration of an adjuvant therapy that can induce and enhance the expression of the E-element; wherein the therapeutic effect is comprehensively evaluated by objective response rate, disease control rate and safety indicators, and the systemic cytokine-related adverse reactions are lower than the control scheme without directional domain and without expression control domain.

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