Application of galectin-1 as immune checkpoint in preparation of tumor immunotherapy medicine and medicine

Abstract

The invention relates to application of galectin-1 (Gal-1) as an immune checkpoint in preparation of a tumor immunotherapy drug and the drug, and provides a new strategy for tumor immunotherapy targeting the new immune checkpoint. The lactose and the derivative thereof can inhibit the immune checkpoint by competing Gal-1 on the surfaces of cancer cells and immune cells, and have no obvious toxic or side effect; by knocking out B4GATL1 to reduce protein galactosylation, the Gal-1 level of tumor metastasis to T cells can be remarkably reduced, so that the activation and function of the T cells are enhanced; besides, the lactose synthetase component LALBA is overexpressed in the mouse tumor, so that de novo synthesis of lactose in the tumor microenvironment can be realized, and further the CD8 + T cell mediated immune response is enhanced. In combination with the anti-tumor effect and economic cost of lactose, a wide and feasible thought is provided for cancer treatment.

Description

Application of galectin-1 as an immune checkpoint in the preparation of tumor immunotherapy drugs and the drug

[0001] This application claims priority to Chinese invention patent application No. 2022114354538 filed on November 16, 2022 and Chinese invention patent application No. 2022114593007 filed on November 16, 2022, each of which is hereby incorporated by reference in its entirety for all purposes. Technical Field

[0002] The present invention belongs to the fields of medicine and tumor treatment, and in particular provides the use of galectin-1 as an immune checkpoint in the preparation of a tumor immunotherapy drug and the drug. Background Art

[0003] Tumor immunotherapy is the most effective cancer treatment method to date; in particular, the targeted inhibition of tumor immune checkpoints (such as PD1, PDL1, CTLA-4, etc.) using biological macromolecules or small molecules has shown good results in the treatment of various tumors; the application of tumor immune checkpoint inhibitor therapy has also exposed some key issues that need to be addressed urgently; for example, it is only effective for some tumor types, but ineffective for others; even the efficacy of different patients with the same tumor type varies significantly; in-depth research on the mechanism of tumor immune action will provide new ideas for solving these problems; on the other hand, the high cost of tumor immune checkpoint inhibitor therapy is the main obstacle to its practical application; for example, under the combined effect of the Chinese government's medical procurement policy and fierce competition among manufacturers, the annual cost of anti-PD1 antibody treatment has dropped from hundreds of thousands of US dollars to tens of thousands of RMB, but it is still a heavy burden on the families of most cancer patients; looking at the world, how to provide affordable tumor immunotherapy for cancer patients is an urgent task.

[0004] Antibodies are the main biological macromolecules of tumor immune checkpoint inhibitors, which have very high requirements for production and preservation, resulting in high prices. There are some small molecule inhibitors of tumor immune checkpoints under development, but they are either complex to produce or have many side effects due to poor targeting.

[0005] Galectins, a family of galactoside-binding proteins, possess potent immunomodulatory activity (reviewed by Rabinovich et al., J Leuk Biol., 2002, 71:741-752). In particular, galactin-1 is a negative regulator of T cell responses and can induce T cell apoptosis (Perillo, NL et al., Nature, 1995, 378(6558):736-739). Galectin-1 is also secreted by activated T cells, acting as an autoregulator of T cell activation and inhibiting antigen-induced T cell proliferation (Blaser C. et al., Euro. J Immunal, 1998, 28:2311-2319).

[0006] However, the existing technology has a very broad understanding of galectin inhibitors, believing that they can be used for pathogenic infections, autoimmune diseases, transplant rejection, graft-versus-host disease, allergies, inflammatory diseases, cancer and tumors, and has not found their special significance in tumor immunotherapy.

[0007] In view of this, this invention is proposed.

[0008] Summary of the Invention

[0009] The present invention surprisingly discovered that cancer cells in tumor tissue can transfer abnormally high levels of galectin-1 to the surfaces of neighboring immune cells, particularly tumor-targeting cytotoxic T cells. Tumor-derived galectin-1 binds to galactosylated proteins on the surface of cytotoxic T cells, regulating T cell activation and function. Lactose and N-acetyllactosamine can competitively bind to tumor-derived galectins on these T cells, restoring their cytotoxic capacity. This discovery makes galectins and their inhibitors particularly significant in the treatment of tumors, compared to conventional inflammatory treatments. Lactose, in particular, is not degraded outside the gastrointestinal tract and can competitively bind to Gal-1 binding sites on the surface of T lymphocytes, blocking the binding of abnormally high levels of Gal-1 secreted by tumor cells to neighboring T lymphocytes and restoring T lymphocyte function. This discovery allows galectins, particularly Gal-1, to be used as a new immune checkpoint in tumor treatment.

[0010] This invention starts from the molecular mechanism, innovatively discovers new mechanisms and targets for targeting tumor immune checkpoints, and screens natural products for regulating tumor immunity, ultimately providing a new method for tumor immunotherapy.

[0011] The present invention surprisingly found that CD8 +The activation level of T cells and the target cell killing activity were significantly enhanced; in in vitro and in vivo tumor infiltration experiments, OT-1 T cells with B4GALT1 gene knockout had a faster proliferation rate, which led to significant tumor growth inhibition. β-1,4-galactosyltransferase 1 (β-1,4-galatosyltransferase 1, B4GALT1) is a key enzyme in the N-glycan biosynthesis pathway. Its inactivation can lead to CD8 + It is worth mentioning that the present invention found that in tumor cells, the inactivation of B4GALT1 can promote CD8 + T cell-mediated immune killing effect.

[0012] The present invention further discovered that the activity of B4GALT1 was inhibited in the mouse colon cancer cell line MC38 tumor, and its immune response capacity was investigated in vitro and in vivo (data not shown in this article). The results showed that:

[0013] (1) In MC38 cells with B4GALT1 knockout, IFNγ, TNFα, and IFNα signaling pathways were significantly upregulated. CRISPR / Cas9 screening studies in tumor cells in recent years have demonstrated that these signaling pathways play an important role in tumor immunotherapy. Furthermore, after IFNγ treatment, the expression level of B2m and the presentation of OVA antigen in MC38 cells with B4GALT1 knockout were significantly enhanced.

[0014] (2) In vitro killing experiments showed that knocking out the B4GALT1 gene in MC38 cells can enhance the specific in vitro killing function of OT-1 T cells. During this process, the present inventors noted that the expression level of IFNγ in OT-1 T cells was significantly increased when co-cultured with MC38 cells with B4GALT1 knockout compared to co-cultured with control MC38 cells.

[0015] (3) In the in vivo tumor growth experiment, the present invention found that the growth rate of MC38 tumors in wild-type mice with B4GALT1 knockout was significantly slower than that in the control group, but this phenomenon was not observed in immunodeficient NPG (NOD-PrkdcscidIL2rgnull) mice. In order to further understand which type of immune cells mediates this tumor immune response, the present invention used specific antibodies to eliminate different immune cells in wild-type mice and then implanted them subcutaneously. Finally, it was found that the elimination of CD8 + and CD4 + T cells can significantly restore the growth rate of MC38 tumors with B4GALT1 knockout, among which CD8 +This was particularly evident after T cell depletion; however, depletion of natural killer cells did not restore their growth rate.

[0016] This indicates that B4GALT1 knockout MC38 tumor cells can enhance immune response activity both in vitro and in vivo, and CD8 + T cells play a major role in this process.

[0017] The present invention found that exogenous expression of LALBA can secrete lactose in tumor cells, and this process depends on the activity of B4GALT1. α-lactalbumin (Lactalbumin Alpha, LALBA) can form heterodimers with B4GALT to participate in the function of lactose synthesis. Overexpression of LALBA in tumor cells can promote the response of tumor cells to immune cells, thereby inhibiting tumor growth. Even if only a part of the tumor cells overexpress LALBA, it can also exert a tumor growth inhibitory effect. In addition, in situ injection of tumors with lentiviruses or adeno-associated viruses encoding LALBA can also achieve the effect of inhibiting tumors, and mice with completely regressed tumors can establish immune memory against the tumor, and no visible tumors will be formed when the tumor is implanted again. However, tumor cells that overexpress mutant LALBA do not produce lactose and have no obvious inhibitory effect on tumor growth.

[0018] The present invention creatively discovered that there is a phenomenon in tumor tissues where cancer cells can transfer abnormally high levels of galectin-1 to the surface of adjacent immune cells, especially tumor-targeted cytotoxic T cells. Subsequently, tumor-derived galectin-1 interacts with galactosylated proteins on the surface of cytotoxic T cells to regulate the activation and function of T cells. The present invention confirmed through anti-Gal-1 antibody staining that lactose treatment can remove Gal-1 from the surface of OT-Ⅰ T cells and, at the same time, reduce the level of Gal-1 on the surface of MC38 cells. It was also found that lactose can significantly increase the exhausted CD8 isolated from tumors. + Expression of Ifnγ and Tnfα in PD-1+ T cells.

[0019] The present invention further discovered that by competitively binding to galectins from tumor cells on these T cells, the killing ability of T cells can be restored.

[0020] The present invention also found that lactose, as a β-galactoside, + The function of N-glycosyl groups can be interfered with in both T cells and tumor cells. Lactose was injected into the tail vein and it was found that lactose solution could significantly inhibit the growth of "hot" tumors, and CD8 + T cells play an important role in this suppression process. Transcriptome sequencing analysis showed that lactose treatment can promote tumor-specific CD8+ By constructing humanized mice with human immune systems, the activation effect of lactose on human tumor immunity was confirmed under three-dimensional culture conditions of human tumors.

[0021] The present invention also confirmed the role of lactose in human tumor immunity in humanized mice with reconstituted human immune systems and in vitro culture of human tumors, suggesting the potential for its clinical application. More importantly, because lactose is inexpensive and readily available, with extremely high global production, its use in the clinical treatment of tumors, either directly or in combination with other therapeutic approaches, would provide a new avenue for cancer patients and significantly reduce the cost of tumor treatment.

[0022] However, the present invention found that only intravenously injected lactose could inhibit tumor growth, while no significant effect was observed with oral or intraperitoneal injection. This may be because oral or intraperitoneal injections have difficulty achieving sufficiently high effective concentrations in the tumor. On the other hand, a stable inhibitory effect was only observed when injections were initiated early after tumor implantation. In the late stages of tumor implantation, lactose injections had no significant therapeutic effect, suggesting that the use of lactose alone in clinical applications may not be able to inhibit mid- and late-stage tumors. In addition, intravenously injected lactose is rapidly consumed in the blood through the renal system and excreted through urine. Therefore, the present invention requires lactose injection every two days. This shortcoming may be compensated by coupling lactose to a protein carrier before injection. The present invention is currently testing this approach, hoping to use carrier proteins to maintain lactose concentrations in the blood for a relatively long time, thereby reducing the frequency of injections.

[0023] Galectins are a family of conserved galactosylation-binding proteins that regulate immune cell function in normal physiological processes and various pathological processes, including tumors. The results of the present invention show that cancer cells can transfer abnormally high levels of galectin-1 to the surface of neighboring immune cells, especially tumor-targeting cytotoxic T cells. Subsequently, tumor-derived galectin-1 interacts with galactosylated proteins on the surface of cytotoxic T cells to regulate T cell activation and function. The present invention conducted a series of Gal-1 transfer experiments, and the results showed that intercellular proximity, cell surface galactosylation levels, and cell surface Gal-1 levels are the main driving forces of intercellular Gal-1 transfer. This suggests a previously unappreciated and undefined mode of intercellular communication, namely proximity-dependent intercellular protein spreading (PDICPS). The low-affinity binding of Gal-1 to its galactosylated targets is an intrinsic feature of this proximity-dependent intercellular protein spreading.

[0024] Targeting this novel immune checkpoint will be a new strategy for tumor immunotherapy. On the one hand, knocking out B4GATL1 to reduce protein galactosylation can significantly reduce Gal-1 levels on tumor-metastatic T cells, thereby enhancing T cell activation and function. On the other hand, lactose and its derivatives can inhibit this immune checkpoint by competing for Gal-1 on the surface of cancer cells and immune cells.

[0025] Targeting this novel immune checkpoint with small molecules would be a viable strategy for tumor immunotherapy. Lactose and related derivatives containing galactosides can inhibit this immune checkpoint by competing for Gal-1 on the surfaces of cancer cells and immune cells. In particular, lactose, a structurally mimicking competitive galectin inhibitor, effectively inhibited tumor growth when injected systemically into the circulatory system of mice with varying tumor burdens. However, the present inventors surprisingly found that lactose activated anti-tumor immunity only in highly immunogenic "hot" tumors, but had no significant effect in less immunogenic "cold" tumors. This indirectly confirms that the primary mechanism by which lactose inhibits tumor growth relies on the immune system and suggests that lactose therapy for "cold" tumors may require combined measures to enhance tumor immunogenicity. To further explore the potential of lactose as an immune checkpoint inhibitor, the present inventors implanted human gastric cancer SGC7901 tumors in humanized mice and observed that systemic intravenous lactose significantly inhibited SGC7901 tumor growth. Subsequently, in an in vitro culture model of surgically removed patient tumors, lactose treatment showed an immune response similar to that of anti-PD-1 pembrolizumab treatment. In addition, in the artificial membrane permeability experiment, the low permeability of lactose also showed that it was lactose itself rather than its degradation products that played a tumor suppressive role in vivo. Comprehensive analysis of various experiments found that lactose inhibited the tumor by competing with CD8 + Gal-1 on T cells activates tumor-infiltrating CD8 + T cells, relieving the inhibition of Gal-1 on T cell activation.

[0026] It is known to those skilled in the art that Galon and Bruni expanded the classification of tumors into four categories (see Galon, J. and Bruni, D., Approaches to treat immune hot, altered and cold tumors with combination immunotherapies", Nature Reviews Drug Discovery (18), March 2019, 197-218): hot, altered-rejecting, altered-immunosuppressive, and cold to facilitate research and communication. Specifically, the category stratification is based on the type, density, and location of immune cells within the tumor site. The authors classified tumors according to immune infiltration rather than cancer type, and the scoring system ("Immunoscore") is based on the quantification of two lymphocyte populations (CD3 and CD8) at the center and invasive edge of the tumor. The score ranges from I0 (low density, such as neither cell type is present in either area) to I4 (high density of immune cell types in both locations). I4 tumors are considered "hot" and I0 tumors are considered "cold". It has been reported that tumor progression (T stage) and invasion (N stage) rely on this pre-existing adaptive intratumoral immunity. More often, researchers are now studying the nature, density, immune function direction, and distribution of immune cells in tumors.

[0027] As reported by Galon et al., the essential characteristics of hot immune tumors are (i) high T cell and cytotoxic T cell infiltration and (ii) checkpoint activation or impaired T-cell function.

[0028] Furthermore, to confirm that the galactosyl group in lactose is the primary functional group for binding to lectins and galectins, the present invention tested lactose, sucrose, N-acetyllactosamine (LacNAc), and lactose-BSA (bovine serum albumin) through lectin competition staining experiments. Because it lacks a galactosyl moiety, sucrose exhibits no competitive binding properties for ECL, sWGA, or Gal-1. Compared to lactose, LacNAc more effectively competes with β-galactosides in the cell surface glycome for binding to ECL and Gal-1, but it also binds to sWGA at higher concentrations. Intravenous injection of LacNAc inhibited MC38 tumor growth in mice. Since lactose in the blood is rapidly excreted through the kidneys, to stabilize its blood concentration, the present invention conjugated lactose to BSA at high temperatures, potentially improving its pharmacokinetics and delaying its metabolism and excretion in the body.

[0029] Based on the above findings, this application provides the following technical solutions.

[0030] In one aspect, the present invention provides the use of galectin-1 (Gal-1, galectin-1) as a T cell immune checkpoint in the preparation of a pharmaceutical composition for tumor immunotherapy.

[0031] The tumor is an immunoinflammatory tumor.

[0032] The application includes: selecting at least one galectin-1 targeted binder or a derivative thereof as the active ingredient of the drug; and / or, selecting a natural galectin-1 targeted binder and allowing the tumor cells to overexpress a precursor of the targeted binder; and / or, determining the galectin-1 binding site on the surface of immune cells in the tumor microenvironment, and reducing, deleting or inactivating the binding site.

[0033] The pharmaceutical composition is selected from the following pharmaceutical compositions:

[0034] (1) The active ingredient of the pharmaceutical composition includes at least one galectin-1 targeted binder or a derivative thereof;

[0035] (2) The pharmaceutical composition comprises an immunopotentiator, which comprises a vector for overexpressing a galectin-1 targeted binder, or a promoter for overexpressing a galectin-1 targeted binder precursor;

[0036] (3) The pharmaceutical composition comprises a modified immune cell, wherein the galectin-1 binding site on the surface of the immune cell is reduced, deleted or inactivated.

[0037] The Galectin-1 targeted binder is generally selected from polynucleotides, polypeptides, antibodies, lipids or carbohydrates. In certain advantageous embodiments, the Galectin-1 targeted binder is in a soluble form.

[0038] Preferably, the tumor is a solid tumor. Preferably, the solid tumor is immunogenic. In some embodiments, the interior and / or margin of the solid tumor is highly infiltrated with T lymphocytes. In some embodiments, the tumor is further characterized by impaired T lymphocyte function. Preferably, the T lymphocytes are CD3+ T lymphocytes and / or CD8 + T lymphocytes.

[0039] In some embodiments, the tumor is breast cancer, pancreatic cancer, colon cancer, melanoma, etc.

[0040] In some embodiments, the tumor is a tumor that has become resistant to PD-1 and / or PD-L1 therapy.

[0041] Preferably, the dosage form of the pharmaceutical composition is an injection preparation, such as an intravenous injection preparation, a subcutaneous injection preparation, an intradermal injection preparation, an intramuscular injection preparation, or an intratumoral injection preparation, etc. In a specific embodiment, the dosage form is an intravenous injection preparation.

[0042] In some embodiments, the galectin-1 targeting binding agent is a carbohydrate or carbohydrate-containing molecule, including but not limited to: disaccharides, non-limiting examples of which include lactose, lactulose, lactose sucrose, methyl β-lactoside, D-galactose, 4-O-β-D-galactopyranosyl-D-mannopyranoside, 3-O-β-D-galactopyranosyl-D-arabinose, 2'-O-methyllactose, lacto-N-biose, N-acetyllactosamine, and thiodigalactopyranoside; larger sugars, such as molecules containing polylactosamine; and synthesis inhibitors, such as thiodigalactosides and sugar polymers. Advantageously, the carbohydrate or carbohydrate-containing molecule is not metabolizable in the host to which the composition is administered. In certain embodiments, the galectin-1 targeted binder is selected from N-acetyl-lactosamine, β-lactosyl-sulfoalbumin, citrus pectin, D-lactitol monohydrate, lactobionic acid, benzyl 4-O-β-D-galactopyranosyl-β-D-glucopyranoside, methyl 4-O-β-D-galactopyranosyl-β-D-glucopyranoside, 2-methyl-β-D-galactose (1→4) D-glucose, methoxyethyl Thioethyl 2acetamido-2-deoxy-4-O-β-D-galactopyranoside-β-D-glucopyranoside, carboxyethylthioethyl 2acetamido-2-deoxy-4-O-β-D-galactopyranoside-β-D-glucopyranoside-BSA conjugate, 4-nitrophenyl 2-acetamido-2-deoxy-3-O-β-D-galactopyranoside-β-D-glucopyranoside and N-propyl-β-lactoside.

[0043] Preferably, the carbohydrate or carbohydrate-containing molecule is not metabolizable in the host to which the composition is administered.

[0044] Preferably, the Galectin-1 targeting binding compound is β-galactoside.

[0045] In some embodiments, the β-galactoside is selected from lactose, lactulose, lactose sucrose, methyl β-lactoside, methyl β-lactoside, 4-O-β-D-galactopyranosyl-D-mannopyranoside, 3-O-β-D-galactopyranosyl-D-arabinose, 2'-O-methyllactose, lacto-N-biose, N-acetyllactosamine, β-D-thiogalactopyranoside.

[0046] In some embodiments, the β-galactoside is lactose; in some embodiments, the β-galactoside is lactose-peptide, such as lactose-BSA; in some embodiments, the β-galactoside is LacNAc.

[0047] In some embodiments, the β-galactoside is lactose. The concentration of lactose in the pharmaceutical composition is 5 mM-400 mM.

[0048] In some embodiments, the medicament comprises a mixture of lactose and its derivatives.

[0049] In some embodiments, the lactose is lactose and / or its hydrates. In some embodiments, the lactose is α-lactose and / or β-lactose; in other embodiments, the lactose is a hydrate of α-lactose; in some embodiments, the lactose is a mixture of α-lactose and α-lactose hydrate; in some embodiments, the lactose is β-lactose; in some embodiments, the lactose is a mixture of one or more of α-lactose, β-lactose, and α-lactose hydrate.

[0050] The lactose derivative is a pharmaceutically acceptable derivative that may have the same parent core structure as the compound itself and, during drug administration, may become lactose or a molecule with similar functions to lactose. For example, the drug, through reactions such as hydrolysis, produces a molecule with the same or similar activity as the original compound. The derivative may be a specific covalent modification of lactose, or other disaccharides, polysaccharides, or small molecules with similar galactose functional groups.

[0051] Preferably, the pharmaceutically acceptable derivatives of lactose may refer in particular to simple derivatives thereof, and in particular to one of its lower esters, lower ethers, lower alkyl substituents, pharmaceutically acceptable salts and lower amides, that is, derivatives obtained by condensation of carboxylic acids, alcohols and amines having 1 to 6, preferably 2 to 6 or 2 to 4 carbon atoms with the parent compound.

[0052] In some embodiments, the pharmaceutically acceptable derivative of lactose is a salt thereof, and the pharmaceutically acceptable salt of lactose can be synthesized from the parent compound by conventional chemical methods, such as the salt synthesized by the method described in Pharmaceutical Salts: Properties, Selection and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, the pharmaceutically acceptable salt can be prepared by reacting the free base of the compound with an acid in water, an organic solvent, or a mixed solution of the two; generally, a non-aqueous medium such as diethyl ether, ethyl acetate, ethanol, isopropanol, or acetonitrile can be used.

[0053] Acid addition salts can be prepared with various acids (inorganic and organic). Examples of acid addition salts can include salts prepared from acids selected from acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, butyric acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, hexanoic acid, caprylic acid, cinnamic acid, citric acid, cycloamic acid, dodecylsulfonic acid, ethane 1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, glucoheptanoic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), α-Ketoglutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, isethionic acid, (+)-L-lactic acid, (+)-DL-lactic acid, lactobionic acid, maleic acid, malic acid, (-)-L-marinic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, palmitic acid, phosphoric acid, propionic acid, L-pyroglutamic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid (+)-L-tartaric acid), thiocyanic acid, p-toluenesulfonic acid, undecylenic acid and valeric acid, and acylamino acids.

[0054] The lactose or pharmaceutically acceptable derivatives thereof may contain one or more asymmetric centers and may therefore exist in various stereoisomeric forms, for example, as enantiomers and / or diastereomers. Therefore, the lactose or pharmaceutically acceptable derivatives and combinations thereof of the present invention may be in the form of individual enantiomers, diastereomers or geometric isomers, or may be in the form of a mixture of stereoisomers.

[0055] The drug comprises lactose and / or a pharmaceutically acceptable derivative thereof. In some embodiments, lactose or a pharmaceutically acceptable derivative thereof is the only active ingredient in the drug other than water. In some embodiments, the drug further comprises a pharmaceutically acceptable excipient. In some embodiments, the drug is a pharmaceutical composition.

[0056] In some embodiments, the β-galactoside derivative is a conjugate of lactose and a polypeptide, the polypeptide is an antibody or antibody fragment capable of recognizing tumor surface-specific antigens, and / or the conjugate of the polypeptide and lactose slows down the metabolic rate of lactose and prolongs the maintenance time of the effective lactose concentration in the body. Furthermore, some polypeptides can further enhance the binding ability of lactose to Gal-1 after being coupled to lactose. An exemplary polypeptide is BSA.

[0057] Preferably, the pharmaceutical composition is an injection preparation, and the pharmaceutical composition is injected via at least one route selected from intravenous, subcutaneous, intradermal, parenteral and intramuscular. In a specific embodiment, the pharmaceutical composition is an intravenous injection preparation.

[0058] In some embodiments, the immune enhancer comprises a vector overexpressing a Galectin-1 targeted binder, and the Galectin-1 targeted binder is a Galectin-1 antibody.

[0059] In some embodiments, the Galectin-1 targeted binder precursor is α-lactalbumin (LALBA).

[0060] In some embodiments, the immunopotentiator comprises an α-lactalbumin (LALBA) promoter. The promoter is selected from: substances that increase LALBA levels, substances that enhance LALBA activity, and / or substances that slow LALBA metabolism. For example, the promoter is selected from: natural purified substances, modified natural purified substances, semi-synthetic substances, and chemically synthesized substances; for example, the promoter is derived from mammals; for example, the promoter is derived from: humans, non-human primates (e.g., gorillas, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), and livestock (e.g., horses, cattle, sheep, pigs, rabbits).

[0061] The promoter is selected from: LALBA overexpression vector, nanoparticles carrying LALBA gene, viral vectors carrying LALBA or its overexpression vector, PEG-modified proteins, protein microspheres, liposomes encapsulating LALBA, and extracellular vesicles carrying LALBA.

[0062] LALBA is a substance selected from the group consisting of LALBA gene, LALBA mRNA, cDNA, LALBA protein, or an active fragment of any of the foregoing;

[0063] The LALBA is derived from mammals, such as humans.

[0064] The enhancer is contained in a pharmaceutical composition or a kit, for example, a pharmaceutical composition or a kit in a form suitable for administration by a method selected from the group consisting of oral administration, injection (e.g., direct naked DNA or protein injection, liposome-encapsulated DNA, RNA or protein injection), gold-coated gene gun bombardment, replication-defective bacteria carrying plasmid DNA, replication-defective adenovirus carrying target DNA or target gene-encoded protein, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, and transdermal administration; and / or the pharmaceutical composition or kit further comprises other anti-tumor active ingredients or is used in combination with other anti-tumor active ingredients, for example, the other anti-tumor active ingredients are selected from: tumor immunotherapy agents, chemotherapy drugs; for example, the other anti-tumor active ingredients are selected from: ipilimumab and nivolumab. The chemotherapy drug is selected from the following drugs: cyclophosphamide, trabectedin, temozolomide, melphalan, dacarbazine, oxaliplatin, methotrexate, mitoxantrone, gemcitabine, 5-fluorouracil (5-FU), bleomycin, doxorubicin, daunorubicin, epirubicin, idarubicin, valrubicin, paclitaxel, cabazitaxel, docetaxel, topotecan, irinotecan, etoposide, carboplatin, cisplatin, bortezomib, vinblastine, vincristine, vindesine, vinorelbine, isothiocyanate, nitrogen mustard, mitomycin C, fludarabine, cytosine arabinoside; and combinations thereof.

[0065] The immune enhancer is a tumor immunotherapy enhancer.

[0066] The galactosylation level on the surface of the modified immune cells is reduced, deleted or inactivated.

[0067] Preferably, the modified immune cells do not express active B4GALT1 protein.

[0068] More preferably, the B4GALT1 gene of the modified immune cells is knocked out or inhibited.

[0069] Furthermore, the inhibition of the B4GALT1 gene refers to the silencing, inactivation or inhibition of the expression of the B4GALT1 gene. Specifically, it may mean that the B4GALT1 gene does not initiate transcription, the transcription process is inhibited, the B4GALT1 mRNA is degraded, the B4GALT1 protein is degraded, or the B4GALT1 inhibitory gene is activated, the protein that inhibits the expression of B4GALT1 is activated, or the expression of factors or proteins that promote the expression of the B4GALT1 gene is inhibited.

[0070] In some specific embodiments, the immune cells contain a reagent that inhibits the transcription or expression of B4GALT1, and the B4GALT1 gene inhibition agent contains an siRNA or shRNA that inhibits the transcription or expression of B4GALT1; in some specific embodiments, the immune cells contain a base sequence encoding an shRNA, preferably, the shRNA can be processed by cells into an siRNA that inhibits the transcription or expression of B4GALT1; preferably, the base sequence is an introduced exogenous sequence, preferably, the base sequence is integrated into the genome of the immune cells.

[0071] In one embodiment, the immune cells contain siRNA or shRNA that inhibits the transcription or expression of B4GALT1.

[0072] In some specific embodiments, the genome of the immune cell contains a base sequence encoding the shRNA.

[0073] In some embodiments, the immune cells are from humans or non-human mammals.

[0074] In some embodiments, the cell is a T cell, a B cell, or a NK cell.

[0075] In some embodiments, the immune cells are CD8 + T cells.

[0076] In some embodiments, the cell is a TCR-T cell or a TCR-NK cell, preferably a TCR-T cell.

[0077] In one aspect, the present invention provides a tumor immunotherapy drug, the active ingredient of which comprises at least one galectin-1 targeted conjugate as described above.

[0078] In one aspect, the present invention provides an immunopotentiator, which is the immunopotentiator according to the first aspect of the present invention. In another aspect, the present invention provides a tumor immunotherapy drug comprising the immunopotentiator.

[0079] In one aspect, the present invention provides a modified immune cell, which is the modified immune cell according to the first aspect of the present invention. In another aspect, the present invention provides a tumor immunotherapy drug comprising the modified immune cell. [0079.1] [Incorporated by reference (Rule 20.6) 27.12.2023] In one aspect, the present invention provides the use of lactose or a derivative thereof in the preparation of a medicament, wherein the lactose or a derivative thereof is an active ingredient of the medicament, for example, one of the active ingredients, the main active ingredient, or the sole active ingredient of the medicament. The lactose is as described above. In some embodiments, the active ingredient refers to lactose or a derivative thereof being the final active ingredient or one of the final active ingredients of the medicament. For example, in some embodiments, the medicament comprises an adduct of lactose or a derivative thereof, and during administration of the medicament, the lactose or a derivative thereof is a degradation product of the adduct. In some embodiments, the medicament comprises a mixture of lactose and a derivative thereof. Preferably, the medicament is an anti-tumor drug. In some embodiments, the lactose is lactose and / or a hydrate thereof. In some embodiments, the lactose is α-lactose and / or β-lactose; in other embodiments, the lactose is a hydrate of α-lactose; in some embodiments, the lactose is a mixture of α-lactose and α-lactose hydrate; in some embodiments, the lactose is β-lactose; and in some embodiments, the lactose is a mixture of one or more of α-lactose, β-lactose, and α-lactose hydrate. The lactose derivative is a pharmaceutically acceptable derivative thereof. This derivative may have the same parent core structure as the compound itself and may, during administration of the drug, transform into lactose or a molecule with similar functions to lactose. For example, the drug may produce a molecule with the same or similar activity as the original compound through reactions such as hydrolysis. This derivative may be a specific covalent modification of lactose or another disaccharide, polysaccharide, or small molecule with similar galactose functional groups. In some embodiments, the drug further comprises a pharmaceutically acceptable excipient. In some embodiments, the drug is a pharmaceutical composition. Preferably, the drug is an injectable formulation. Preferably, the drug is administered by subcutaneous injection, intravenous injection, arterial injection, or intratumoral injection. In one embodiment, the drug is administered by intravenous injection. Preferably, the pharmaceutically acceptable derivative of lactose refers specifically to a simple derivative thereof, and in particular to one of its lower esters, lower ethers, lower alkyl substituents, pharmaceutically acceptable salts, and lower amides, i.e., derivatives obtained by condensation of carboxylic acids and alcohols. Amines having 1 to 6, preferably 2 to 6 or 2 to 4, carbon atoms with the parent compound.In some embodiments, the pharmaceutically acceptable derivative of lactose is a salt thereof, and the pharmaceutically acceptable salt of lactose can be synthesized from the parent compound by conventional chemical methods, such as the salt synthesized by the method described in Pharmaceutical Salts: Properties, Selection and Use, P Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, the pharmaceutically acceptable salt can be prepared by reacting the free base of the compound with an acid in water, an organic solvent, or a mixed solution of the two; generally, a non-aqueous medium such as diethyl ether, ethyl acetate, ethanol, isopropanol, or acetonitrile can be used. [0079.2] [Incorporated by reference (Rules 20.6) 27.12.2023] In one aspect, the present invention provides the use of a lactose prodrug in the preparation of a drug, wherein the lactose prodrug is LALBA protein (lactalbumin alpha, α-lactalbumin) or an agent that causes target cells to overexpress LALBA protein. Target cells overexpressing LALBA protein force B4GALT1 protein (beta-1,4-galactosyltransferase 1, β-1,4-galactosyltransferase) to shift its function from participating in glycosylation modification to interacting with LALBA protein to participate in lactose synthesis. Furthermore, the drug is an anti-tumor drug. Furthermore, the target cells are tumor cells. Preferably, the drug is a liquid preparation, and more preferably, the drug is an injectable. Preferably, the drug is administered by in vivo injection. In some embodiments, the drug is administered by intratumoral injection. Preferably, the drug comprises a delivery vector for delivering the lactose prodrug to target cells, wherein the delivery vector is a cell delivery vector, a viral delivery vector, a liposome delivery vector, a nanoparticle delivery vector, or a ferritin delivery vector. Preferably, the cells are erythrocytes or mesenchymal stem cells; preferably, the virus is an adenovirus or a lentivirus. Furthermore, the drug is an anti-tumor drug. In some embodiments, the agent for overexpressing the LALBA protein comprises a nucleic acid molecule, and in some embodiments, the nucleic acid molecule comprises a base sequence encoding the LALBA protein. In some embodiments, the isolated nucleic acid molecule further comprises a cis-acting element. Furthermore, the isolated nucleic acid molecule may also comprise one or more of the same or homologous base sequences of the flanking sequences of the human LALBA gene, a termination signal sequence, an auxiliary sequence, or a resistance screening gene. In some embodiments, the isolated nucleic acid molecule is DNA or RNA. [0079.3] [Added by reference (Rules 20.6) 27.12.2023] In one aspect, the present invention provides the use of a lactose prodrug in the preparation of a medicament, wherein the lactose prodrug is a LALBA protein or an agent that causes target cells to overexpress the LALBA protein. The agent that overexpresses the LALBA protein comprises a base sequence encoding the LALBA protein. The nucleic acid molecule is DNA or RNA. Preferably, the medicament comprises a delivery vector for delivering the lactose prodrug to the target cell, wherein the delivery vector is a cell delivery vector, a viral delivery vector, a liposome delivery vector, a nanoparticle delivery vector, or a ferritin delivery vector. Preferably, the virus is an adenovirus or a lentivirus. In one embodiment, the medicament is an injectable formulation, and in some embodiments, it is an intratumoral injection. In some embodiments, the medicament further comprises a pharmaceutically acceptable excipient. The medicament is preferably an anti-tumor drug. The tumor is preferably an immunoinflammatory tumor. [0079.4] [Incorporated by reference (Rules 20.6) 27.12.2023] In one aspect, the present invention provides a novel use of the β-1,4-galactosyltransferase (B4GALT1) gene / protein, wherein the novel use is for use in the preparation of a medicament. Preferably, the medicament is an anti-tumor drug. Further, the medicament is selected from the following agents: a) an agent that knocks out the B4GALT1 gene or inhibits the B4GALT1 gene / protein; and / or b) an immune cell in which the B4GALT1 gene is knocked out or inhibited. The immune cell is a T cell, a B cell, or a NK cell. Further, the knockout is complete knockout or knockdown of the B4GALT1 gene. Preferably, the knockout is complete knockout.

[0080] On the one hand, the present invention provides a new use of beta-1,4-galactosyltransferase (B4GALT1) gene / protein, which is an application in the preparation of tumor immunotherapy drugs.

[0081] Furthermore, the drug is selected from the following reagents:

[0082] a) an agent for knocking out / inhibiting the B4GALT1 gene or inhibiting the B4GALT1 protein; and / or,

[0083] b) Immune cells in which the B4GALT1 gene is knocked out or suppressed.

[0084] The immune cell is a T cell, a B cell or a NK cell. In some embodiments, the immune cell is a TCR-T cell or a TCR-NK cell. Preferably, the immune cell is a TCR-T cell.

[0085] Furthermore, the knockout is complete knockout or knockdown of the B4GALT1 gene. Preferably, the knockout is complete knockout.

[0086] Furthermore, knocking out the B4GALT1 gene refers to completely or partially knocking out the B4GALT1 gene sequence in the target cell genome, rendering it unable to encode the B4GALT1 protein or unable to correctly encode a functional B4GALT1 protein. For example, in some embodiments, the entire length of the B4GALT1 gene is knocked out; in some embodiments, the sequence from the start codon to the stop codon of the B4GALT1 gene is knocked out or mutated; in some embodiments, all or part of one or more exons of the B4GALT1 gene are knocked out; and in some embodiments, transcriptional regulatory elements of the B4GALT1 gene, such as the promoter sequence, are knocked out.

[0087] In some embodiments, the reagent for knocking out the B4GALT1 gene comprises a nucleic acid molecule targeting the B4GALT1 gene and a nuclease guided by the nucleic acid molecule. Preferably, the nucleic acid molecule is a guide RNA (gRNA). In some embodiments, the nuclease is a CRISPR nuclease. Preferably, the nuclease is Cas9. Optionally, the reagent further comprises a selection marker gene.

[0088] In some embodiments, the agent for knocking out the B4GALT1 gene comprises a nucleic acid molecule targeting the B4GALT1 gene and an mRNA encoding an endonuclease. Preferably, the nucleic acid molecule is an sgRNA. In some embodiments, the endonuclease is a CRISPR nuclease. Preferably, the endonuclease is Cas9.

[0089] In some embodiments, the reagent for knocking out the B4GALT1 gene comprises a recombinant nucleic acid molecule encoding a protein targeting the B4GALT1 gene and an endonuclease. In some specific embodiments, the protein encoding the protein targeting the B4GALT1 gene is a TAL protein, and in other specific embodiments, the protein encoding the protein targeting the B4GALT1 gene is a zinc finger protein. The endonuclease is preferably FokI.

[0090] Preferably, the reagent for knocking out the B4GALT1 gene further comprises a delivery vector, wherein the delivery vector comprises the gRNA and endonuclease targeting the B4GALT1 gene as described above, or the mRNA encoding the same, or delivers the recombinant nucleic acid molecule targeting the B4GALT1 gene as described above into the target cell. The delivery vector is selected from, but not limited to, cells, viruses, liposomes, nanoparticles, vesicles, ferritin, and other vectors. The cells are preferably red blood cells or mesenchymal stem cells. The viral delivery vector is preferably an adenovirus or lentivirus delivery vector.

[0091] Furthermore, the inhibition of the B4GALT1 gene refers to silencing or inhibiting the B4GALT1 gene, and the methods include activating the B4GALT1 inhibitory gene, activating the protein that inhibits B4GALT1 expression, introducing siRNA that inhibits B4GALT1 transcription or expression, activating microRNA that promotes B4GALT1 mRNA degradation, introducing molecules that promote B4GALT1 protein degradation, and inhibiting the expression of factors and proteins that promote B4GALT1 expression.

[0092] The agent for inhibiting the B4GALT1 gene is selected from one or more of an agent that activates an inhibitory gene of B4GALT1, an agent that activates a protein that inhibits the expression of B4GALT1, siRNA and shRNA that inhibit B4GALT1 transcription or expression, microRNA that promotes the degradation of B4GALT1 mRNA, molecules that promote the degradation of B4GALT1 protein, and factors and proteins that inhibit and promote the expression of B4GALT1.

[0093] In some embodiments, the agent for inhibiting the B4GALT1 gene comprises an siRNA or shRNA that inhibits B4GALT1 transcription or expression, or comprises an siRNA expression vector. In some embodiments, the expression vector comprises a base sequence encoding an shRNA. Preferably, the shRNA can be processed by cells into an siRNA that inhibits B4GALT1 transcription or expression. Preferably, the base sequence is capable of integrating into the host cell genome.

[0094] In some embodiments, the agent for inhibiting the B4GALT1 gene further comprises a delivery vector. Preferably, the delivery vector is a plasmid or a viral vector.

[0095] In one embodiment, the immune cells contain siRNA or shRNA that inhibits the transcription or expression of B4GALT1.

[0096] In some embodiments, the genome of the immune cell contains a base sequence encoding the shRNA, and the shRNA can be processed by the cell into an siRNA that inhibits the transcription or expression of B4GALT1.

[0097] In some embodiments, the agent that inhibits the activity of the B4GALT1 protein comprises a small molecule compound that can inactivate the B4GALT1 protein.

[0098] Preferably, the B4GALT1 gene is a human or non-human mammal B4GALT1 gene.

[0099] Preferably, the drug is a liquid preparation. Further preferably, the drug is in the form of an injection.

[0100] Preferably, the pharmaceutical composition is a liquid preparation. Further preferably, the pharmaceutical composition is in the form of an injection.

[0101] The "tumor" of the present invention includes benign tumors or malignant tumors, but preferably refers to malignant tumors. Optionally, the tumor can be a cold tumor or a hot tumor, preferably, the tumor is a hot tumor.

[0102] Preferably, the tumor of the present invention is selected from the group consisting of a hematological tumor, a solid tumor, or a combination thereof. In another preferred embodiment, the hematological tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0103] In some specific embodiments, the solid tumor is selected from the following group: tumor, at least one of lung cancer, esophageal cancer, osteosarcoma, breast cancer, endometrial cancer, gastric cancer, colon cancer, rectal cancer, melanoma, prostate cancer, liver cancer, nasopharyngeal cancer, brain glioma, leukemia, lymphoma, oral cancer, laryngeal cancer, tongue cancer, bladder cancer, kidney cancer, penile cancer, pancreatic cancer, cervical cancer, and ovarian cancer.

[0104] In one embodiment, the solid tumor is colon cancer. In another embodiment, the solid tumor is melanoma.

[0105] In one aspect, the present invention provides a combined pharmaceutical composition for tumor immunotherapy, the active ingredients of which include at least:

[0106] (1) one or a combination of two or more of the aforementioned galectin-1 targeted conjugate or derivative thereof, the aforementioned immunopotentiator, and the aforementioned modified immune cells;

[0107] (2) At least one component that induces the transformation of a cold tumor into a hot tumor.

[0108] Preferably, the galectin-1 targeting binder is lactose. In some embodiments, the derivative is a glycopeptide. In some embodiments, the derivative is a pharmaceutically acceptable salt of lactose.

[0109] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0110] The combination pharmaceutical composition further includes other anti-tumor active ingredients or is used in combination with other anti-tumor active ingredients, for example, the other anti-tumor active ingredients are selected from: tumor immunotherapy agents, chemotherapy drugs; for example, the other anti-tumor active ingredients are selected from: ipilimumab, nivolumab. Wherein the chemotherapy drug is a drug selected from the following: cyclophosphamide, trabectedin, temozolomide, melphalan, dacarbazine, oxaliplatin, methotrexate, mitoxantrone, gemcitabine, 5-fluorouracil (5-FU), bleomycin, doxorubicin, daunorubicin, epirubicin, idarubicin, valrubicin, paclitaxel, cabazitaxel, docetaxel, topotecan, irinotecan, etoposide, carboplatin, cisplatin, bortezomib, vinblastine, vincristine, vindesine, vinorelbine, serquinone, nitrogen mustard, mitomycin C, fludarabine, cytosine arabinoside; and combinations thereof.

[0111] The present invention provides a method for treating tumors, comprising administering a drug to a subject to inhibit tumors or eliminate tumor cells, wherein the active ingredient of the drug comprises a galectin inhibitor. Preferably, the galectin inhibitor is a galectin-1 inhibitor. Further preferably, the galectin-1 inhibitor is a β-D-galactoside. In some embodiments, the β-galactoside has the structural formula shown in Formula I.

[0112] In one aspect, the present invention provides a method for tumor immunotherapy, comprising the steps of delivering the aforementioned Galectin-1 targeted conjugate into the tumor immune microenvironment of a subject.

[0113] In some embodiments, the drug is administered by injection. The therapeutically effective amount of the compound comprises a pharmaceutical composition.

[0114] Preferably, the pharmaceutical composition is activated by at least one route selected from intravenous, subcutaneous, intradermal, parenteral, intramuscular and intratumoral administration. More preferably, the drug is administered by intravenous injection.

[0115] In some embodiments, the pharmaceutical composition is administered in combination with at least one other therapeutic modality.

[0116] In some embodiments, the compound of the other treatment modality is selected from radiation therapy, chemotherapy, and surgery, and combinations thereof.

[0117] In one aspect, the present invention provides a method for tumor immunotherapy, comprising the following steps: contacting the aforementioned immunopotentiator and / or the aforementioned modified immune cells with tumor cells of a subject.

[0118] In some embodiments, the drug is administered by injection.

[0119] Preferably, the pharmaceutical composition is activated by at least one route selected from intravenous, subcutaneous, intradermal, parenteral, intramuscular and intratumoral. More preferably, the injection is intratumoral injection.

[0120] In some embodiments, the pharmaceutical composition is administered in combination with at least one other therapeutic modality.

[0121] In some embodiments, the compound of the other treatment modality is selected from radiation therapy, chemotherapy, and surgery, and combinations thereof.

[0122] The "tumor" of the present invention includes benign tumors or malignant tumors, but preferably refers to malignant tumors. Optionally, the tumor can be a cold tumor or a hot tumor, preferably, the tumor is a hot tumor.

[0123] Preferably, the tumor of the present invention is selected from the group consisting of a hematological tumor, a solid tumor, or a combination thereof. In another preferred embodiment, the hematological tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0124] In some specific embodiments, the solid tumor is selected from the following group: tumor, at least one of lung cancer, esophageal cancer, osteosarcoma, breast cancer, endometrial cancer, gastric cancer, colon cancer, rectal cancer, melanoma, prostate cancer, liver cancer, nasopharyngeal cancer, brain glioma, leukemia, lymphoma, oral cancer, laryngeal cancer, tongue cancer, bladder cancer, kidney cancer, penile cancer, pancreatic cancer, cervical cancer, and ovarian cancer.

[0125] In some embodiments, the tumor is a tumor that has developed resistance to PD-1 / PD-L1 antibodies.

[0126] In one embodiment, the tumor is colon cancer; in one embodiment, the tumor is melanoma; in one embodiment, the tumor is gastric cancer.

[0127] Preferably, the tumor is derived from a subject that responds poorly to immunotherapy or requires improved immunotherapy response.

[0128] More preferably, the object or the subject is selected from the following groups: a tumor patient with acquired immunodeficiency or damage; a tumor patient for whom tumor immunotherapy is ineffective or ineffective or expected to be ineffective; a patient who is receiving, will receive or has received tumor immunotherapy; or a tumor patient with two or more of the foregoing conditions; and / or the object is a mammal, such as a human, a non-human primate (such as an orangutan, ape), a rodent (such as a rat, a mouse, a guinea pig), a pet (such as a cat, a dog), or a livestock (such as a horse, a cow, a sheep, a pig, or a rabbit).

[0129] The invention has the following beneficial effects:

[0130] The present study demonstrates that cancer cells can transfer abnormally high levels of galectin-1 to the surfaces of neighboring immune cells, particularly tumor-targeting cytotoxic T cells. Tumor-derived galectin-1 then interacts with galactosylated proteins on the surface of cytotoxic T cells to regulate T cell activation and function.

[0131] Targeting this novel immune checkpoint will be a new strategy for tumor immunotherapy. On the one hand, knocking out B4GATL1 to reduce protein galactosylation can significantly reduce Gal-1 levels on tumor-metastatic T cells, thereby enhancing T cell activation and function. On the other hand, lactose and its derivatives can inhibit this immune checkpoint by competing for Gal-1 on the surface of cancer cells and immune cells.

[0132] Targeting this novel immune checkpoint with small molecules would be a viable strategy for tumor immunotherapy. Lactose and related derivatives containing galactosides can inhibit this immune checkpoint by competing for Gal-1 on the surfaces of cancer cells and immune cells. Lactose, in particular, as a structurally mimicking competitive galectin inhibitor, effectively inhibited tumor growth when injected systemically into the circulatory system of mice with varying tumor burdens. Conjugating macromolecules (such as BSA) to lactose enhances its binding to Gal-1 and may help prolong the maintenance of effective lactose concentrations in the body. Lactose is a natural product found in dairy products, comprising approximately 4.8% of cow's milk. Lactose can be prepared in large quantities from cow's milk using mature, simple, and low-cost processes. Currently, China produces tens of thousands of tons of high-purity lactose (above 99%) annually, primarily as an additive and preservative in pharmaceuticals. Compared to anti-PD-1 antibodies, lactose is much simpler to produce and its production cost is several orders of magnitude lower, making it a promising candidate for tumor immunotherapy.

[0133] In addition, overexpression of the lactose synthase component LALBA in mouse tumors can achieve de novo lactose synthesis in the tumor microenvironment, thereby enhancing CD8 +T cell-mediated immune response. Combined with the anti-tumor efficacy and economic cost of lactose, it will provide a widely feasible approach for cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0134] Figure 1 shows in vitro and in vivo CRISPR / Cas9 screening and identification of regulatory PD1 expression and CD8 + T cell function genes result in:

[0135] A. Using primary mouse CD8 + Schematic diagram of in vivo and in vitro CRISPR / Cas9 screening;

[0136] B. Volcano plot showing the results of CRISPR / Cas9 whole-genome in vitro screening;

[0137] C. Validation results of candidate genes in the screening results; flow cytometry was used to detect the mean fluorescence intensity (MFI) of PD1 on the cell surface, and RT-qPCR was used to detect the expression of PD1 mRNA;

[0138] D. KEGG pathways enriched in the whole-genome screening by gene enrichment analysis (GSEA);

[0139] E. Volcano plot showing the results of in vitro CRISPR / Cas9 screening using a custom small library;

[0140] F. Volcano plot showing the results of in vivo CRISPR / Cas9 screening using a custom small library;

[0141] Figure 2 shows that knocking out B4GALT1 in T cells can enhance TCR signaling and T cell anti-tumor function

[0142] A.CD8 + Knockout of the B4GALT1 gene in T cells increased PD1 expression. Flow cytometry was used to measure the mean fluorescence intensity (MFI) of cell surface PD1, and RT-qPCR was used to detect PD1 mRNA expression.

[0143] B. Overexpression of the short or long isoform cDNA of mouse B4GALT1 can complement the PD1 overexpression phenotype induced by B4GALT1 knockout;

[0144] C. After co-culture with B16F10-OVA cells, CD8 + B4GALT1 in T cells increases the expression of IL2, TNFα, and IFNγ. RT-qPCR was used to detect the expression of IL2 and IFNγ mRNA. Enzyme-linked immunosorbent assay (ELISA) was used to detect the secreted TNFα and IFNγ levels in the culture medium.

[0145] D. Knockout of B4GALT1 in T cells enhances the ability to kill B16F10-OVA cells in vitro;

[0146] E. Heatmap showing differentially expressed genes in B4GALT1 knockout and control OT-1 cells after co-culture with tumor cells; genes involved in the TCR signaling pathway are annotated on the left side of the heatmap;

[0147] F. Volcano plot showing significantly upregulated and downregulated genes (pvalue < 0.01) after co-culture of B4GALT1-knockout OT-1 cells with tumor cells; genes involved in the TCR signaling pathway are marked in dark gray; the names of genes with the most significant expression differences and some genes involved in the TCR pathway are marked in the volcano plot;

[0148] G. Volcano plot showing KEGG pathways significantly enriched after co-culture of B4GALT1 knockout OT-1 cells with tumor cells; the names of some of the significantly enriched pathways are annotated in the volcano plot;

[0149] H.B4GALT1 knockout OT-1 cells have a stronger ability to inhibit B16F10-OVA tumor growth in vivo;

[0150] I. B16F10-OVA tumors are smaller in OT-1 mice injected with B4GALT1 knockout mice;

[0151] J. Knockout of B4GALT1 in OT-1 cells increases their number in B16F10-OVA tumors;

[0152] The data in the figures are presented as mean ± standard error (SEM);

[0153] Figure 3 shows the expression of B4GALT1 in the surface tumor microenvironment and tumor-infiltrating CD8 + T cells are associated with the prognosis of tumor patients, including:

[0154] A. Kaplan-Meier survival curves of B4GALT1 expression levels of all primary tumor samples in the TCGA cohort (left) and B4GALT1 expression levels normalized by CD8a (right);

[0155] B. Association between CD8a expression levels and overall survival in patients with different B4GALT1 expression levels in all primary tumor samples in the TCGA cohort;

[0156] C. Association between B4GALT1 expression levels and overall survival in all primary adrenocortical carcinoma (ACC), acute myeloid leukemia (LAML), lung adenocarcinoma (LUAD), and rectal adenocarcinoma (READ) tumor samples from the TCGA cohort with different CD8a expression levels;

[0157] D. Association between CD8a expression levels and overall survival in all primary adrenocortical carcinoma (ACC), acute myeloid leukemia (LAML), lung adenocarcinoma (LUAD), and rectal adenocarcinoma (READ) tumor samples from the TCGA cohort with different B4GALT1 expression levels;

[0158] The p-value of all survival curves was calculated by two-sided log-rank test (two-sidedLog-ranktest);

[0159] Figure 4 shows that knocking out B4GALT1 in tumor cells can enhance the body's immune surveillance response to tumor cells, including:

[0160] A. Knockout of the B4GALT1 gene in MC38 cells inhibits their growth in wild-type mice;

[0161] B. Knockout of the B4GALT1 gene in MC38 cells did not affect their growth in immunodeficient NPG mice;

[0162] C. Growth curves of B4GALT1-knockout MC38 cells in wild-type mice depleted of CD8+T, CD4+T, or NK cells;

[0163] D. Heat map showing differentially expressed genes between control MC38 cells and B4GALT1 knockout MC38 cells;

[0164] E. Volcano plot showing significantly upregulated and downregulated genes in MC38 cells after B4GALT1 knockout (pvalue < 0.01); genes involved in the interferon-γ signaling pathway are marked in dark gray; the names of genes with the most significant expression differences and some genes involved in the interferon-γ pathway are marked in the volcano plot;

[0165] F. Volcano plot showing the significantly enriched HALLMARKER gene set in MC38 cells after B4GALT1 knockout; the names of the most significantly differentially expressed pathways are annotated in the volcano plot;

[0166] G. Compared with control MC38 cells, B2m expression was increased in B4GALT1-knockout MC38 cells after IFNγ treatment;

[0167] H. Compared with control MC38 cells, B4GALT1-knockout MC38 cells have enhanced ability to present Ova peptide after IFNγ treatment;

[0168] I. Knockout of the B4GALT1 gene in MC38 cells makes them more susceptible to cytotoxicity by OT-1 T cells;

[0169] J. After co-culture with B4GALT1-knockout MC38 cells, the expression level of IFNγ in OT-1 T cells was significantly increased;

[0170] The data in the figures are presented as mean ± standard error (SEM);

[0171] Figure 5 shows that intratumoral expression of α-lactalbumin (LALBA) can produce lactose in tumor cells and activate tumor immune responses, where:

[0172] A. Overexpression of LALBA in MC38 cells promoted lactose synthesis; LC-MS was used to detect the lactose content in cells;

[0173] B. LC-MS detection of lactose content in the culture medium;

[0174] C. Overexpression of LALBA in MC38 cells inhibits their growth in wild-type mice;

[0175] D. Overexpression of the LALBA gene in MC38 cells did not affect their growth in immunodeficient NPG mice;

[0176] E. Growth curves of MC38-GFP cells in mice with completely resolved MC38-LALBA tumors and in wild-type mice of the same age who had not been injected;

[0177] F. Orthotopic injection of LALBA-encoding lentivirus into MC38 tumors inhibited tumor growth; arrows indicate the time of virus injection;

[0178] G. After orthotopic injection of LALBA-encoding lentivirus into MC38 tumors, the proportion of IFNγ-positive tumor-infiltrating CD8+ T cells increased;

[0179] H. Re-inoculated MC38 cells were unable to grow in mice that had complete tumor regression after injection of lentivirus encoding LALBA;

[0180] I. Orthotopic injection of AAV encoding LALBA into MC38 tumors inhibits tumor growth; arrows indicate the time of virus injection.

[0181] J. After orthotopic injection of LALBA-encoding AAV into MC38 tumors, the proportion of PD1-positive tumor-infiltrating CD8+ T cells increased;

[0182] K. After orthotopic injection of LALBA-encoding AAV virus into MC38 tumors, the proportion of IFNγ-positive tumor-infiltrating CD8+ T cells increased;

[0183] The data in the figures are presented as mean ± standard error (SEM);

[0184] Figure 6 shows that tail vein injection of lactose solution can enhance the anti-tumor function of mice, where:

[0185] A. Effect of tail vein injection of lactose solution on the growth of MC38 tumors in wild-type mice;

[0186] B. After lactose solution was injected into the tail vein of MC38 tumor-bearing mice, the proportion of PD1-positive tumor-infiltrating CD8+ T cells increased;

[0187] C. After lactose solution was injected into the tail vein of MC38 tumor-bearing mice, the proportion of IFNγ-positive tumor-infiltrating CD8+ T cells increased;

[0188] D. Effect of tail vein injection of lactose solution on the growth of MC38 tumor in immunodeficient NPG mice;

[0189] EG. Effects of tail vein injection of lactose solution on the growth of MC38 cells in wild-type mice depleted of CD8+T (E), CD4+T (F), or NK (G) cells;

[0190] H. Effect of tail vein injection of lactose solution on the growth of 4T1 tumors in wild-type BALB / c mice;

[0191] I. Effects of tail vein injection of lactose solution on the growth of CT26 tumors in wild-type BALB / c mice;

[0192] J. Effect of tail vein injection of lactose solution on the growth of B16F10 tumors in wild-type C57BL / 6J mice;

[0193] K. Effect of tail vein injection of lactose solution on the growth of B16F10-OVA tumors in wild-type C57BL / 6J mice;

[0194] L. Effect of tail vein injection of lactose solution on the growth of SGC7901 tumors in NPG mice with humanized immune system;

[0195] After lactose solution was injected into the tail vein of M.SGC7901 tumor-bearing mice with humanized immune systems, the proportion of IFNγ-positive human CD8+ T cells in the tumor increased;

[0196] N. Effects of tail vein injection of lactose solution on the growth of SGC7901 tumors in immune-deficient NPG mice;

[0197] The data in the figures are presented as mean ± standard error (SEM);

[0198] Figure 7 shows that genome-wide screening identified the N-glycan synthesis pathway regulating PD1 expression in CD8+ T cells, where:

[0199] Schematic diagram of the AN-glycan biosynthesis pathway; genes identified in the genome-wide screen are indicated;

[0200] Distribution of genes related to the BN-glycan synthesis pathway in the whole-genome screening results; the solid line represents all genes; the long dashed line represents genes in the N-glycan synthesis pathway; the short dashed line represents the negative control gRNA;

[0201] C. Flow cytometry was used to detect the expression of PD1 in OT-1 cells after knockout of B4GALT1, Mgat2, and Dpm3 genes.

[0202] Figure 8 shows the effect of B4GALT1 knockout on the function of OT-1 cells in tumors, where:

[0203] A. 24 hours after transplantation, the effect of B4GALT1 knockout on the tumor-infiltrating ability of OT-1 cells was detected;

[0204] B. CFSE signaling indicates that B4GALT1 knockout can enhance the proliferation ability of OT-1 cells in tumors;

[0205] The data in the figures are presented as mean ± standard error (SEM); NS indicates no significant difference;

[0206] FIG9 shows that the expression of B4GALT1 in the tumor microenvironment and tumor-infiltrating CD8+ T cells are associated with the prognosis of tumor patients, among which:

[0207] A. Heatmap showing the association between B4GALT1 expression levels in different primary tumor samples in the TCGA cohort (left) and B4GALT1 expression levels normalized by CD8a (right) and overall survival; ALL-TCGA: represents all primary tumor samples in the TCGA cohort;

[0208] B. Heat map showing the association between B4GALT1 expression levels and overall survival in patients with different CD8a expression levels in primary tumor samples from the TCGA cohort (left: high CD8a expression; right: low CD8a expression); ALL-TCGA: represents all primary tumor samples in the TCGA cohort;

[0209] C. Heat map showing the association between CD8a expression levels and overall survival in patients with different B4GALT1 expression levels in primary tumor samples in the TCGA cohort (left: B4GALT1 high expression; right: B4GALT1 low expression); ALL-TCGA: represents all primary tumor samples in the TCGA cohort;

[0210] Figure 10 shows flow cytometry analysis demonstrating successful elimination of CD8+T (A), CD4+T (B) and NK (C) cells in wild-type mice;

[0211] Figure 11 shows that overexpression of LALBA in a subset of MC38 cells can inhibit the growth of MC38 tumors in wild-type mice;

[0212] FIG12 shows the effects of intratumoral injection of lentivirus encoding two LALBA mutants, LALBA-D107A (A) and LALBA-A126K (B), on the growth of MC38 tumors in wild-type mice;

[0213] Figure 13 shows the effects of B4GALT1 knockout and LALBA overexpression on the overall N-glycome of cells as detected by lectin staining, where:

[0214] A. Demonstrates that increasing lactose concentration in the solution competitively blocks ECL staining of MC38 cells (left), but has no effect on sWGA staining (right);

[0215] B. Shows the effects of B4GALT1 knockout and LALBA overexpression on the mean fluorescence intensity (MFI) of ECL and sWGA in MC38 cells;

[0216] C. Shows the effects of B4GALT1 knockout and LALBA overexpression on the mean fluorescence intensity (MFI) of ECL and sWGA on CD8+ T cells;

[0217] Note: MC38 cells were directly scraped from the plate without trypsinization for ECL and sWGA staining experiments;

[0218] FIG14 shows the effects of tail vein injection of different doses of lactose solution on the growth of MC38 tumors in wild-type mice.

[0219] Effects of two-day tail vein injection of 50 mM, 250 mM, and 400 mM lactose solutions on the growth of MC38 tumors in wild-type mice;

[0220] FIG15 shows the effect of tail vein injection of lactose solution and anti-PD1 (PDCD1) combined administration on the growth of MC38 tumors in wild-type mice;

[0221] FIG16 shows the effect of combined tail vein injection of lactose solution and anti-PDL1 (CD274) on the growth of MC38 tumors in wild-type mice;

[0222] Figure 17 shows RNA-seq analysis of the effect of lactose treatment on the MC38 tumor transcriptome in wild-type mice, where:

[0223] A. Heat map showing differentially expressed genes in lactose-treated tumors; genes involved in the interferon-γ signaling pathway are labeled on the left.

[0224] B. Volcano plot showing significantly upregulated and downregulated genes in MC38 tumors after lactose treatment (pvalue < 0.05); genes involved in the interferon-γ signaling pathway are marked in dark gray; the names of genes with the most significant differential expression and some genes involved in the interferon-γ pathway are marked in the volcano plot;

[0225] C. Volcano plot showing the significantly enriched HALLMARKER gene set in MC38 tumors after lactose treatment; the names of the most significantly differentially expressed pathways are annotated in the volcano plot;

[0226] DF: Transcriptional signature analysis of control and lactose-treated tumor samples;

[0227] The data in the figures are presented as mean ± standard error (SEM);

[0228] Figure 18 shows single-cell transcriptome analysis of tumor-infiltrating T cells after control and lactose treatment, where:

[0229] A. Flowchart of single-cell transcriptome analysis and TCR analysis;

[0230] B. 13,839 CD3-positive T cells isolated from control and lactose-treated tumor samples can be divided into 8 clusters (C0-C7) in the t-SNE plot;

[0231] The Ct-SNE plots show the distribution of CD3-positive T cells in eight populations isolated from control (left) and lactose-treated (right) tumor samples.

[0232] Dt-SNE plots show the expression of Cd8a (left) and Cd4 (right) in all cells;

[0233] E. Marker genes that define each T cell population;

[0234] F. Distribution of T cells in different populations isolated from control and lactose-treated tumor samples;

[0235] Figure 19 shows single-cell TCR sequencing analysis of tumor-infiltrating T cells after control and lactose treatment, wherein:

[0236] A. Clonal steady-state space clonotype identification using CDR3 amino acid sequences in control and lactose-treated samples;

[0237] B. Clonotype identification using CDR3 amino acid sequences, relative proportions of specific clonotypes in control and lactose-treated samples;

[0238] C. TCR diversity analysis based on Shannon, InvSimpson, Chao, and ACE (abundance-based coverage estimator) indices in control and lactose-treated samples;

[0239] D. Distribution of the largest T cell clone, CN1, in control and lactose-treated samples;

[0240] E. Distribution of the largest T cell clone, CN1, in different populations in control and lactose samples;

[0241] FIG20 shows that in NPG mice with humanized immune systems, tail vein injection of lactose showed an inhibitory effect on SGC7901 growth similar to that of pembrolizumab (anti-human PD1 antibody);

[0242] Figure 21 Safety and toxicity evaluation of short-term and long-term intravenous lactose administration in mice,

[0243] A. Short-term (24 h post-injection) effects of intravenous lactose on blood / serum biochemical and hematological parameters in wild-type mice;

[0244] B. Long-term effects of intravenous lactose on blood / serum biochemical and hematological parameters in wild-type mice (12 tail vein injections, tested 24 days later);

[0245] C. The effects of intravenous lactose or PBS injection on mouse body weight were monitored over 23 days.

[0246] FIG22 is a structural diagram of a lentiviral vector overexpressing LALBA;

[0247] FIG23 is a structural diagram of an adenoviral vector overexpressing LALBA;

[0248] FIG24 shows the structure of the pMSCV-CD19 scFv-IRES-RFP plasmid;

[0249] Figure 25 CRISPR / Cas9 knockout of B4galt1 in OT-Ⅰ T cells alters lectin binding;

[0250] Figure 26 Expression of Galectins in OT-Ⅰ T cells and MC38 tumor cells;

[0251] Figure 27. Production of Gal-1 on the surface of OT-Ⅰ T cells is independent of TCR activation;

[0252] Figure 28 Gal-1 on the surface of OT-Ⅰ T cells is derived from MC38 cells;

[0253] Figure 29: Transfer of Gal-1 from MC38 cells to OT-Ⅰ T cells requires close contact;

[0254] Figure 29 Antibody activation of B4galt1 knockout T cells is not affected by Gal-1;

[0255] Figure 31 Killing experiment of wild-type and B4galt1 knockout OT-Ⅰ T cells and wild-type and Gal-1 knockout MC38

[0256] Figure 32: Gal-1 is transferred from tumor cells to infiltrating CD8+ T cells in the tumor microenvironment in vivo;

[0257] Figure 33: Growth of Gal-1 knockout MC38 tumors is inhibited by CD8+ and CD4+ T cells of the host immune system;

[0258] FIG34 Flow cytometry analysis of Gal-1 expression on the surface of CD8+ T cells in mouse spleen, peripheral blood and MC38 tumors;

[0259] Figure 35 Gal-1 knockout reduced the level of Gal-1 on the surface of MC38 tumor-infiltrating CD8+ T cells;

[0260] Figure 36: Purification and activity testing of recombinant Gal-1 protein;

[0261] FIG37 Identification and analysis of Gal-1 binding proteins in wild-type and B4galt1 knockout OT-Ⅰ T cells;

[0262] FIG38 Western immunoblotting verifies CD8β and other B4galt substrates in T cell membrane proteins;

[0263] Figure 39 Gal-1 binds to and regulates TCR-CD8 colocalization;

[0264] Figure 40 Effects of B4galt1 and CD8 in OT-Ⅰ T cells and hCD19-CAR T cells, respectively;

[0265] Figure 41 Lactose can significantly enhance the specific killing of tumor cells by OT-Ⅰ T cells;

[0266] Figure 42 Lactose treatment can remove Gal-1 on the surface of OT-Ⅰ T cells in an in vitro killing system;

[0267] Figure 43 Lactose reverses the inhibitory effect of Gal-1 on OT-Ⅰ T cell activity;

[0268] Figure 44 Lactose can remove Gal-1 from the surface of tumor-infiltrating T cells in vivo and enhance T cell cytotoxicity;

[0269] Figure 45 Intravenous lactose injection inhibits the growth of MC38 tumors in wild-type mice;

[0270] Figure 46 Effects of intravenous lactose injection on different types of tumors;

[0271] Figure 47 Parallel artificial membrane permeability experiments of lactose;

[0272] Figure 48 Properties of lactose and its derivatives. DETAILED DESCRIPTION

[0273] The present invention will be further described below with reference to specific examples, but the present invention is not limited to the scope of the examples.

[0274] 1. Experimental materials, reagents, and instruments

[0275] In order to more clearly illustrate the present invention, the following experimental materials, reagents, and instruments used in the present invention are listed hereby. Materials, reagents, or instruments not listed are conventional materials, reagents, or instruments in the art and can be purchased through normal commercial means.

[0276] 1.1 Experimental Materials

[0277] (1) pKLV-U6-sgRNA-PGK-puro2ABFP (Addgene #50946) was purchased from Addgene.

[0278] (2) pMSCV-IRES-GFP (Addgene #20672) was purchased from Addgene.

[0279] (3) pMSCV-CD19 scFv-IRES-RFP plasmid was obtained from Shao Feng's laboratory at the Beijing Institute of Life Sciences.

[0280] (4) Lenti-EF1α related plasmids were constructed using lenti-U6 sgRNA-EF1α-IRES-GFP or lenti-EF1α-puro empty.

[0281] (5) Mouse Gal-1 gene cDNA (mm39ENSMUST00000089377.6) was amplified from a mouse T cell cDNA library.

[0282] (6) Mouse B4galt1 gene cDNA was amplified from mouse T cell cDNA library.

[0283] (7) Trans1-T1 competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd., catalog number CD501-02.

[0284] (8) DH10B-Plus electroporation competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd., catalog number DE1072M.

[0285] (9) Human embryonic kidney HEK293T cell line: purchased from Life Technologies.

[0286] (10) Mouse hybridoma PK136 cell line: purchased from the American Type Culture Collection (ATCC).

[0287] (11) C57BL / 6J mice: purchased from Beijing Weitonglihua Company.

[0288] (12) BALB / c mice: purchased from Beijing Weitonglihua Company.

[0289] (13)B-NDG(NOD.CB17-Prkdc scid Il2rg tm1 Bcgen) mice were purchased from Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd.

[0290] (14) Peptide-N-glycosidase F (PNGase F): purchased from New England Biolabs, catalog number P0704S.

[0291] (15) Recombinant mouse interleukin-2 (rIL-2): purchased from BioLegend, catalog number 575404.

[0292] (16) Recombinant mouse interleukin-7 (rIL-7): purchased from BioLegend, catalog number 577804.

[0293] (17) Recombinant mouse interleukin-15 (rIL-15): purchased from BioLegend, catalog number 566304.

[0294] (18) OVA oligopeptide (SIINFEKL): purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number T510212.

[0295] (19) Polybrene: purchased from Sigma, product number S2667.

[0296] (20) α-Lactose hydrate: purchased from Sigma, product number L3625 (L3625-1KG, Lot#SLCD8654).

[0297] (21) Collagenase IV: purchased from Sigma, catalog number V900893.

[0298] (22) Anti-mouse CD3e antibody (clone number: 145-2C11), purchased from eBioscience, catalog number 14-0031-82.

[0299] (23) Anti-mouse CD28 antibody Ultra-LEAF TM Purified anti-mouse CD28 (clone number: 37.51): purchased from BioLegend, catalog number 102116.

[0300] (24) Anti-mouse PD-1 antibody PE anti-mouse CD279 (clone number: RMP1-30): purchased from BioLegend, product number 109104.

[0301] (25) Anti-mouse PD-1 antibody APC anti-mouse CD279 (clone number: RMP1-30): purchased from BioLegend, product number 109112.

[0302] (26) Anti-mouse Gal-1 antibody PE conjugated: purchased from bio-techne R&D, catalog number IC1245P.

[0303] (27) Anti-mouse interferon γ antibody FITC anti-mouse IFNγ (clone number: XMG1.2): purchased from BioLegend, product number 505806.

[0304] (28) Anti-mouse CD8a APC anti-mouse CD8a (clone number: 53-6.7): purchased from eBioscience, product number 17-0081-83.

[0305] (29) Anti-mouse TCR Vα2 monoclonal antibody PE conjugated (clone number: B20.1): purchased from BD Biosciences, catalog number 553289.

[0306] (30) Biotin anti-mouse CD8a monoclonal antibody (clone number: 53-6.7): purchased from BioLegend, catalog number 100704.

[0307] (31) Anti-mouse CD8β antibody (clone number: EPR22331-54): purchased from abcam, catalog number ab228965.

[0308] (32) Biotinylated Erythrina chinensis agglutinin (Biotin ECL): purchased from Vector Laboratories, catalog number B-1145-5.

[0309] (33) Biotinylated succinylated wheat germ agglutinin (Biotin sWGA): purchased from Vector Laboratories, product number B-1025S-5.

[0310] (34) Biotinylated mouse galectin (Biotin-rGal-1): prepared in the laboratory (Gal-1 is the sequence shown in mm39 ENSMUST00000089377.6).

[0311] (35) PE streptavidin: purchased from BioLegend, catalog number 405204.

[0312] (36) Anti-mouse NK1.1 antibody: purified from mouse hybridoma cell line PK136.

[0313] (37) Anti-mouse CD8α antibody (clone number: 2.43): purchased from BioXCell, product number BE0061.

[0314] (38) Anti-mouse CD4 antibody (clone number: GK1.5): purchased from BioXCell, catalog number BE0003-1.

[0315] (39) Anti-mouse Ly9 antibody: purchased from abcam, catalog number ab252931.

[0316] (40) Anti-mouse Igf2γ antibody: purchased from Sino Biological, catalog number 107533-T40.

[0317] (41) Anti-mouse Itga1 antibody: purchased from Solarbio, catalog number K002895P.

[0318] (42) Anti-mouse Ighg1 antibody: purchased from Solarbio, product number K111394P.

[0319] (43) Anti-mouse Lnpep antibody: purchased from Santa Cruz, catalog number sc-365300.

[0320] (44) Anti-mouse Sell antibody: purchased from Santa Cruz, product number sc-390756.

[0321] (45) Anti-mouse IgG 680RD fluorescent secondary antibody: purchased from LI-COR, catalog number P / N 926-68072

[0322] (46) Anti-mouse IgG 800RD fluorescent secondary antibody: purchased from LI-COR, catalog number P / N 926-32210

[0323] (47) Anti-rabbit IgG 680RD fluorescent secondary antibody: purchased from LI-COR, catalog number P / N 926-68071

[0324] (48) Anti-rabbit IgG 800RD fluorescent secondary antibody: purchased from LI-COR, catalog number P / N 926-32211

[0325] (49) Anti-human CD8α APC anti-human CD8α: purchased from BioLegend, catalog number 300912.

[0326] (50)ImProm-Ⅱ TM Reverse Transcriptase: Promega, catalog number A3803.

[0327] (51) Random Hexamer Primer: purchased from Thermo Scientific, catalog number 51709.

[0328] 1.2 Enzyme-linked immunosorbent assay (ELISA) reagents

[0329] (1) Mouse TNFα Elisa kit: ABclonal, catalog number RK00027.

[0330] (2) Mouse IFNγ Elisa kit: ABclonal, catalog number RK00019.

[0331] (3) Human TNFα Elisa kit: ABclonal, catalog number RK00030.

[0332] (4) Human IFNγ Elisa kit: ABclonal, catalog number RK00015.

[0333] 1.3 Experimental instruments

[0334] (1) Flow cytometer: BD Accuri™ C6, BD FACSAria II, BD FACSAria III, BD FACSAria Fusion.

[0335] (2) Microplate reader: PerkinElmer EnSpire Multimode Plate Reader.

[0336] 2. Experimental methods

[0337] In order to more clearly illustrate the present invention, the following experimental methods used in the present invention are listed hereby. Those not listed are conventional experimental methods known to those skilled in the art.

[0338] (1) Construct the vector MSCV-U6 sgRNA-PGK-PURO-2A-BFP.

[0339] Backbone vector: MSCV-U6gRNA-PGK-Puro-2a-BFP, digested with BbsⅠ.

[0340] Forward primer: 5'-CACCG+20nt sgRNA sequence-3'

[0341] Reverse primer: 5'-AAAC+20nt sgRNA sequence+C-3'

[0342] Cloning method: T4 ligation.

[0343] (2) Construct the vector PKLV-U6 sgRNA-PGK-PURO-2A-BFP.

[0344] Backbone vector: PKLV-U6gRNA-PGK-Puro-2a-BFP, digested with BbsⅠ.

[0345] Forward primer: 5'-CACCG+20nt sgRNA sequence-3'

[0346] Reverse primer: 5'-AAAC+20nt sgRNA sequence+C-3'

[0347] Cloning method: T4 ligation.

[0348] (3) Construct the vector lenti-U6 sgRNA-EF1α-Gal-1-EGFP fusion.

[0349] Step 1: Construct MSCV-EF1α-Gal-1-IRES-GFP.

[0350] Backbone vector: MSCV-EF1α-Lalba-IRES-GFP, digested with BsiWⅠ and EcoRI.

[0351] PCR template: mouse T cell cDNA library.

[0352] Forward primer: 5'-CCATTTCAGGTGTCGTGACGTACGTGCCACCATGGCCTGTGGTCTGGTCG-3' (SEQ ID NO: 6)

[0353] Reverse primer: 5'-ACGTTAGGGGGGGGGGGCGGAATTCTCACTCAAAGGCCACGCAC-3' (SEQ ID NO: 7)

[0354] Cloning method: Gibson assembly.

[0355] Gibson assembly system: 30 ng of enzyme-digested vector, 10 ng of PCR product, 2.5 μL 2× Gibson, add double-distilled water to 5 μL, and ligate at 50°C for 15 minutes.

[0356] Step 2: Construct lenti-U6 sgRNA-EF1α-Gal-1-IRES-GFP.

[0357] Backbone vector: lenti-U6 sgRNA-EF1α-IRES-GFP, digested with BamHI and MluⅠ.

[0358] Fragment template: MSCV-EF1α-Galectin1-IRES-GFP, digested with BamHI and MluⅠ.

[0359] Cloning method: T4 ligation.

[0360] Step 3: Construct the target vector.

[0361] Backbone vector: lenti-U6 sgRNA-EF1α-Gal-1-IRES-GFP, digested with XcmⅠ and MluⅠ.

[0362] First-round PCR template: lenti-U6 sgRNA-EF1α-Gal-1-IRES-GFP

[0363] Forward primer: 5'-AGACTTCAAGATTAAGTGCGTGGCCTTTGAGATGGTGAGCAAGGGCGAGG-3' (SEQ ID NO: 8)

[0364] Reverse primer: 5'-AATCCAGAGGTTGATTGTCG-3' (SEQ ID NO: 9)

[0365] Second round PCR template: previous round PCR product.

[0366] Forward primer: 5'-CCTCAACATGGAGGCCATCAACTACATGGCGGCGGATGGAGACTTCAAGATTAAGTGC-3' (SEQ ID NO: 10)

[0367] Reverse primer: 5'-AATCCAGAGGTTGATTGTCG-3' (SEQ ID NO: 11)

[0368] Cloning method: Gibson assembly.

[0369] (4) Construction of vector: lenti-EF1α-puro-2A-OVA

[0370] Backbone vector: lenti-EF1α-puro empty, digested with BamHI and MluⅠ.

[0371] PCR template 1: MSCV-U6 sgRNA-PGK-PURO-2A-BFP

[0372] Forward primer: 5'-TGGTGCATGACCCGCAAGCC-3' (SEQ ID NO: 12)

[0373] Reverse primer: 5'-TGGGCCAGGATTCTCCTCCA-3' (SEQ ID NO: 13)

[0374] PCR template 2: fully synthetic OVA oligopeptide sequence vector.

[0375] Forward primer: 5'-ACGTGGAGGAGAATCCTGGCCCAAGCACCAGGACACAAATAAATAAGG-3' (SEQ ID NO: 14)

[0376] Reverse primer: 5'-CCAGAGGTTGATTGTCGACTTAACGCGTTTAAGGGGAAACACATCTGCCAAAG-3'(SEQ ID NO:15)

[0377] Cloning method: Gibson assembly.

[0378] (5) Construction of vector: MSCV-U6 sgB4galt1-PGK-PURO-2A-B4GALT1CDS (two transcripts cloned separately, PCR template 2 with different forward primers)

[0379] Backbone vector: MSCV-U6 sgB4GALT1-PGK-Puro-2a-BFP, digested with BamHI and SalⅠ.

[0380] PCR template 1: MSCV-U6 sgB4GALT1-PGK-Puro-2a-BFP

[0381] Forward primer: 5'-GTCCTAGCAATTTTTTTGGATCCAATTCT-3' (SEQ ID NO: 16)

[0382] Reverse primer: 5'-TGGGCCAGGATTCTCCTCC-3' (SEQ ID NO: 17)

[0383] PCR template 2: mouse T cell cDNA library.

[0384] Forward primer: 5'-TGGAGGAGAATCCTGGCCCAATGAGGTTTCGTGAGCAGTTCC-3' (long transcript) (SEQ ID NO: 18)

[0385] 5'-TGGAGGAGAATCCTGGCCCAATGCCGGGCGCGACCCTG-3' (short transcript) (SEQ ID NO: 19)

[0386] Reverse primer: 5'-CCTTTGGAGTATTGGATGCAAATAATACAGCCAGT-3' (SEQ ID NO: 20)

[0387] PCR template 3: mouse T cell cDNA library.

[0388] Forward primer: 5'-CTGGCTGTATTATTTGCATCCAATACTCCAAAGG-3' (SEQ ID NO: 21)

[0389] Reverse primer: 5'-TCGATAAGCTTGGCTGCAGGTCGACCTATCTCGGTGTCCCGATGTCCACTGTG-3' (SEQ ID NO: 22)

[0390] Cloning method: Gibson assembly.

[0391] (6) Construction of vector: pQE-80-Gal-1opt

[0392] Backbone vector: pQE-80-Tn5, digested with BamHI and HindⅡ.

[0393] PCR template: fully synthetic codon-optimized mouse Gelctin-1 sequence vector.

[0394] Forward primer: 5'-TCGCATCACCATCACCATCACGGATCCATGGCCTGCGGCCTGGTGG-3' (SEQ ID NO: 23)

[0395] Reverse primer: 5'-GGAGTCCAAGCTCAGCTAATTAAGCTTTTATTCAAAGGCCACGCATT-3' (SEQ ID NO: 24)

[0396] Cloning method: Gibson assembly.

[0397] (7) Construction of vector: pMSCV-CD19 scFv(FMC63)-IRES-RFP-U6 sgRNA

[0398] Backbone vector: pMSCV-CD19 scFv-IRES-RFP, digested with ClaⅠ.

[0399] PCR template: MSCV-U6 sgB4galt1-PGK-PURO-2A-B4GALT1 CDS.

[0400] Forward primer: 5'-CGTCGACCTGCAGCCAAGCTTATCGATGAGGGCCTATTTCCCATGAT-3' (SEQ ID NO: 25)

[0401] Reverse primer: 5'-CTAAATAAAATCTTTTATTTTATCGATcAAAAAAATTGCTAGGACCGGC-3' (SEQ ID NO: 26)

[0402] Cloning method: Gibson assembly.

[0403] (8) Construction of vector: lenti-SFFV-NY ESO-1-T2A-BFP-U6 shRNA

[0404] Backbone vector: lenti-SFFV-NY-ESO-1-T2A-BFP-U6 sgRNA, digested with BamBI and XhoI.

[0405] PCR Template 1: MSCV-CD19 scFv-IRES-RFP-U6 shRNA.

[0406] Forward primer: 5'-GTCGACCTGCAGCCAAGCTT-3' (SEQ ID NO: 27)

[0407] Reverse primer: 5'-GATTGTCGACGGATCCTCTAGACTCGAGATCGCCATTTGTCTCGAGGT-3' (SEQ ID NO: 28)

[0408] PCR template 2: lenti-SFFV-NY-ESO-1-T2A-BFP-U6 sgRNA.

[0409] Forward primer: 5'-CCAACGGCCCTGTGATGCA-3' (SEQ ID NO: 29)

[0410] Reverse primer: 5'-AAGCTTGGCTGCAGGTCGACTCAATTAAGCTTGTGCCCCAG-3' (SEQ ID NO: 30)

[0411] Cloning method: Gibson assembly.

[0412] (9) Construction of vector: pMSCV-Biggest clone TCRA-2A-TCRN-IRES-GFP

[0413] Backbone vector: pMSCV-IRES-RFP, digested with EcoRI and XhoI.

[0414] Fragment template: fully synthetic Biggest clone vector, digested with EcoRI and XhoⅠ.

[0415] Cloning method: T4 ligation.

[0416] (10) Construction of B16F10-OVA cell line

[0417] Lentivirus is packaged with the lenti-EF1α-puro-2A-OVA vector. The viral fluid is directly infected into the B16F10 cell line without concentration. 48 hours after infection, 2 μg / mL of puromycin is added for positive infection screening. Because dead cells and debris are generated during screening, timely medium replacement or subculture is necessary. After 4-5 days of screening, flow cytometry can be used to detect the presentation of the OVA oligopeptide on the B16F10 cell surface. After approximately 7 days, the B16F10-OVA cell line is sufficiently expanded and can be aliquoted and frozen for future use.

[0418] (11) Construction of MC38-Cas9 cell line

[0419] The lentivirus was packaged using the lenti-EF1α-Cas9-2A-blast vector. Due to the large size of the vector and the low viral titer, the virus needed to be concentrated by centrifugation before infecting the MC38 cell line. 48 hours after infection, 10 μg / mL of blasticidin was added for positive infection screening. During this screening process, dead cells and debris generated required prompt medium replacement or subculture. After approximately 10 days of screening, the MC38-Cas9 cell line was sufficiently expanded and aliquoted and frozen for future use.

[0420] (12) Construction of MC38-B2m KO, Gal-1KO, and B2m / Gal-1DKO cell lines

[0421] Lentiviral vectors (pKLV-U6 sgB2m-PGK-PURO-2A-BFP, pKLV-U6 sgGal-1-PGK-PURO-2A-BFP, pKLV-U6sgB2m / sgGal-1-PGK-PURO-2A-BFP, and pKLV-U6 sgEmpty-PGK-PURO-2A-BFP (empty vector without targeting sgRNA as a control)) were used to package the MC38-Cas9 cell line. The viral fluids were directly infected without concentration. 48 hours after infection, 5 μg / mL puromycin and 10 μg / mL blasticidin were added for positive selection. Dead cells and debris generated during selection required prompt medium replacement or subculture. Approximately 7 days after selection, the knockout and control cell lines were sufficiently expanded and aliquoted and frozen for future use.

[0422] (13) Construction of MC38-Gal-1-EGFP and MC38-IRES-EGFP cell lines

[0423] Lentiviral vectors, lenti-U6 sgRNA-EF1α-Gal-1-EGFP fusion vector and lenti-U6 sgRNA-EF1α-IRES-GFP (control vector), were used to directly infect MC38-Cas9 cells without concentration. GFP-positive cells were sorted by flow cytometry 48 hours after infection. Approximately 7 days after sorting, the Gal-1-expressing and control cell lines were sufficiently expanded and aliquoted and frozen for future use.

[0424] (14) Activation and adoptive transfer of memory T cells

[0425] Days 2 to 7 are the differentiation and culture stage of memory T cells. If the infected virus contains puromycin resistance, puromycin is added for selection starting 48 hours after infection (day 3) at a concentration of 3 μg / mL. If the infected virus does not contain puromycin resistance but contains fluorescent protein expression, fluorescent labeling sorting can be performed by flow cytometry 48 hours after infection (day 3) or later. After 4 days of screening for infected positive cells (day 7), T cells can be co-cultured with tumor cells presenting OVA oligopeptides (without adding puromycin), i.e., activation of memory T cells, in which the ratio of T cells to tumor cells is 3:1 or other specified ratios. Before adoptive transfer of T cells, T cells are first labeled with CFSE to distinguish them from T cells in the mouse body. The labeled cells are injected into B16F10-OVA tumor-bearing mice via the tail vein (day 14 of tumor bearing), with 2×10 per mouse implanted. 6 cell.

[0426] (15) In vitro killing experiment: OT-Ⅰ T cells targeting tumor cells

[0427] B16F10-OVA cells and B16F10 cells were labeled with CFSE, hi (2.5 mM) and CFSE lo (50nM), and then co-cultured with T cells in 96-well plates at the specified ratio. Samples containing only tumor cells without T cells served as controls. After 24 hours of culture, CFSE was detected by flow cytometry. hi and CFSE lo The ratio of the two groups. The specific killing rate is calculated by the formula: [1-(CFSE of the sample containing T cells hi / CFSE lo ) / (CFSE in samples containing only tumor cells hi / CFSE lo )] × 100%. For the MC38 cell killing experiment, OVA oligopeptide was added to a portion of the MC38 cells to a final concentration of 100 ng / mL before cell collection and incubated at 37°C for 3 hours. The subsequent steps were basically the same as the B16F10 cell killing experiment. MC38 cells incubated with OVA oligopeptide, i.e., OVA pulsed MC38, and MC38 cells not incubated with OVA oligopeptide were labeled with CFSE, respectively. hi With CFSE lo , and then mixed with T cells in proportion for co-culture.

[0428] (16) Killing experiment of OT-Ⅰ T cells treated with lactose

[0429] Prepare conditioned medium: 50% fresh T cell culture medium without cytokines and 50% MC38 conditioned medium. Use this conditioned medium to prepare a medium containing 0 mM and 20 mM lactose. This medium is then used for OT-I T cell-targeted tumor cell killing experiments.

[0430] (17) Construction of antigen-specific T cells: Construction of Anti-CD19-CAR T cells

[0431] Mouse CD8 + T cell isolation kit to purify CD8 from the spleen of Cas9-EGFP knock-in mice + T cells.

[0432] On day 0, CD8 T cells were stimulated with anti-CD3 (1 μg / mL) and anti-CD28 (0.5 μg / mL) in RPMI 1640 complete medium containing 20 ng / mL IL-2. + T cells were enriched for 24 hours. On the first day, activated CD8 + T cells were infected by centrifugation in 24-well plates with retrovirus carrying anti-CD19-CAR and sgRNA expression units, and 8 μg / mL Polybrene was added and centrifuged at 2000g at 30℃ for 1 hour, with an acceleration of 1 and a deceleration of 0. On the third day, the cells positive for infection (RFP + ) cells were flow cytometry sorted and further cultured under the same culture conditions as the memory T cells described above. On day 7, T cells expressing anti-CD19-CAR were mixed with Nalm6 cells for in vitro killing experiments.

[0433] (18) Construction of antigen-specific T cells: Construction of NY-ESO-1-specific TCR T cells

[0434] The construction process of NY-ESO-1 specific TCR T cells refers to the isolation, culture and infection methods of human primary T cells. + Seven days after transduction, T cells expressing NY-ESO-1-specific TCR were mixed with A375 cells for in vitro killing assay.

[0435] (19) Construction of antigen-specific T cells: Construction of Biggest clone TCR T cells

[0436] The construction process of Biggest clone TCR T cells is the same as that of anti-CD19-CAR T cells. During infection, the enriched T cells were infected by centrifugation with retrovirus carrying the Biggest clone TCR and sgRNA expression unit. On the 3rd day, the infection-positive (GFP + ) cells were flow cytometry sorted and further cultured under the same culture conditions as the memory T cells described above. On day 7, T cells expressing the biggest clone TCR were mixed with MC38 cells for in vitro killing experiments.

[0437] (20) Establishment of a subcutaneous tumor model in mice

[0438] The cells were collected, washed twice with DPBS to remove the residual culture medium, filtered with a 40 μm filter membrane, and aliquoted according to the required cell number.

[0439] Table 1 Number of cells implanted into subcutaneous tumors in mice

[0440] Shave the hair on one side of the mouse's back the day before tumor implantation. During implantation, mix the cell suspension thoroughly before aspirating it. Use a 1mL syringe to draw 100μL of the cell suspension. Lift the skin off the hairless area of ​​the back and pierce the skin 0.5cm deep for injection. A small bulge will be visible under the skin. Before removing the needle, rotate the pillow half a turn to prevent spillage of the cell fluid. Approximately 7 days after implantation, measure the length and width of the tumor using an electronic vernier caliper, measuring every 2-3 days. Tumor volume is calculated using the formula: 1 / 2 × length × width × width.

[0441] One day after tumor implantation, a 250 mM lactose solution was prepared in DPBS and filtered through a 0.22 μm filter. Each tumor-bearing mouse was injected with 200 μL of lactose solution via the tail vein using a 1 mL syringe. The control group was injected with 200 μL of DPBS every two days until day 21. For LacNAc treatment, LacNAc (20 mM, 200 μL) in DPBS was injected intravenously every other day. The control group was injected with 200 μL of DPBS until day 21.

[0442] (21) Clearance of NK cells, CD4+T cells, and CD8+T cells in mice

[0443] NK cell clearance: Anti-NK1.1 antibody was diluted with DPBS to a final concentration of 100 μg / 200 μL. The day of tumor implantation was designated as day 0. On days -3, -1, 6, and 8, mice were intraperitoneally injected with 100 μg of the diluted antibody. +T cell elimination: Anti-CD4 antibody was diluted with DPBS to a final concentration of 400 μg / 200 μL. The day of tumor implantation was designated as day 0. On days -2, -1, 6, and 7, mice were intraperitoneally injected with 400 μg of the diluted antibody. + T cell depletion: Anti-CD8a antibody was diluted in DPBS to a final concentration of 250 μg / 200 μL. The day of tumor implantation was designated as day 0. On days -2, -1, 6, and 7, mice were intraperitoneally injected with 250 μg of the diluted antibody.

[0444] (22) Recombinant Gal-1 protein purification:

[0445] Clonal expression test: Transform the prokaryotic expression plasmid pQE-80-Gal-1 opti into BL21 competent cells and evenly plate onto agarose plates containing ampicillin. Pick five individual clones and place them in LB medium containing ampicillin and culture overnight at 37°C on a shaker. Inoculate the bacterial suspension at a ratio of 1:100 into LB medium containing ampicillin and culture at 37°C on a shaker. After approximately 2 hours, check the OD value at 600 nm. If it is approximately 0.7-0.8, stop the culture. Cool the suspension on ice for 1-2 minutes, then add IPTG to a final concentration of 0.25 mM and culture at 18°C ​​on a shaker for 17 hours. After induction of expression, harvest the cells, wash them once with DPBS, resuspend them in E. coli lysis buffer, and disrupt them by sonication. A small amount of the lysate is added to 5× SDS loading buffer and boiled at 100°C for 10 minutes. Protein electrophoresis is then performed.

[0446] Purification of recombinant Gal-1 protein: The selected monoclonal strain is cultured and expanded to 500 mL. The activation and induction expression steps are the same as those for the clone expression test. After 17 hours of induction expression, centrifuge at 9600g at 4℃ for 15 minutes to collect the bacteria. From this step on, operate on ice. (1) Wash once with pre-cooled DPBS, add 80mL of E. coli lysis buffer to resuspend, and perform ultrasonic disruption, 15 seconds on, 15 seconds off, and ultrasonicate for half an hour to an hour until the bacteria become clear. During this period, it must be cooled in an ice-water mixture. Transfer the lysed bacteria to an ultracentrifuge tube and centrifuge at 21600g at 4℃ for 20 minutes. During the centrifugation, wash 2mL of Ni-NTA twice with lysis buffer. Low-speed centrifugation is required to prevent damage to the nickel-labeled agarose beads. Mix the supernatant of the lysed bacteria with Ni-NTA and incubate at 4℃ for 2 hours by slow inversion. Transfer the incubated Ni-NTA to the chromatography column. When the liquid is almost drained, wash it once with ten times the column volume of nickel column cleaning buffer. After the liquid has drained away, add 10 mL of nickel column eluent. During this time, use 1.5 mL centrifuge tubes to collect the eluent, with 400-500 μL per tube. Use Nanodrop to test the protein concentration of each tube in sequence. Collect the liquid in the centrifuge tubes containing higher protein concentrations and place them in a dialysis bag. Dialyze three times, the first two times for two hours each, and the third time overnight. Divide the dialyzed protein solution into 1.5 mL centrifuge tubes, determine the concentration using the Bradford protein assay, and store at 4°C for later use or frozen at -80°C.

[0447] Recombinant Gal-1 protein activity test: collect primary mouse T cells cultured in vitro and dispense into 1.5 mL centrifuge tubes, 5×10 5 Cells. Add the recombinant Gal-1 protein to be tested and mix thoroughly. Incubate at room temperature for 10 minutes. Wash twice with DPBS. Stain with anti-Gal-1 using standard cell surface antigen and antibody staining methods. Flow cytometry analysis: Indicator 1 is the intensity of anti-Gal-1 staining, and Indicator 2 is the degree of T cell clumping as indicated by FSC or SSC.

[0448] (23) Mass spectrometry and analysis

[0449] The mass spectrometer was an LTQ ORBITRAP Velos mass spectrometer equipped with a nano-ESI ion source (ThermoFisher Scientific, San Jose, CA, USA). The mass spectrometer was operated in data-dependent mode, performing one MS scan followed by ten HCD (high-energy collision dissociation) MS / MS scans per cycle. Raw data were processed using Proteome Discoverer (version 1.4, available at https: / / www.thermofisher.com / hk / en / home / industrial / mass-spectrometry / liquid-chromatography-mass-spectrometry-lc-ms / lc-ms-software / multi-omics-data-analysis / proteome-discoverer-software.html) to generate a raw intensity table of peptide spectrum matches (PSMs) for all identified proteins. This was further analyzed using the R package DEqMS. The raw intensity values ​​were log2 transformed, and the median log2 intensity was subtracted from each PSM to obtain a relative log2 ratio. The differentially expressed proteins were then calculated using a Bayesian algorithm. Differentially expressed proteins were screened based on a P < 0.05 standard. These proteins were then subjected to functional enrichment analysis using the R package ClusterProfiler (version 3.12.0).

[0450] (24) Gal-1 transfer experiment

[0451] Gal-1 transfer assays were performed using coculture of T cells with MC38 cells. OT-I T cells were cocultured with wild-type or Gal-1-deficient MC38 cells in conditioned medium (50% fresh cytokine-free T cell medium and 50% conditioned medium from wild-type or Gal-1-deficient MC38 cells) at a 2:1 ratio or the indicated ratio for 8 hours. Cell surface Gal-1 levels were then assessed by flow cytometry. Gal-1 transfer assays were performed in a Boyden chamber. OT-I T cells were seeded in the upper compartment of a Boyden chamber and MC38 cells were seeded in the lower compartment, or both OT-I T cells and MC38 cells were seeded in the lower compartment. Cell surface Gal-1 levels were assessed by flow cytometry after 8 hours of culture. Gal-1 transfer assays were performed on low-melting-point agarose gels. MC38 cells were seeded in a well plate in advance. After they attached, low-melting-point agarose dissolved in culture medium was added. After solidification, OT-Ⅰ T cells were added to the agarose layer, or directly added to MC38 cells without agarose layer. After culturing for 8 hours, the level of cell surface Gal-1 was detected by flow cytometry.

[0452] (25) Fluorescence resonance energy transfer (FRET)

[0453] (1) Recombinant Gal-1 treatment: 1.2×10 6 Wild-type or B4galt1 knockout memory T cells were incubated at room temperature for 10 minutes and then washed twice with DPBS.

[0454] (2) Lactose treatment: 20 mM lactose was added during the recombinant Gal-1 treatment and incubation.

[0455] (3) Antibody incubation: After treatment with recombinant Gal-1 and lactose, T cells were incubated with PE-anti-Vα2 and APC-anti-CD8α antibodies at 4°C for 30 minutes. Four staining groups were set up for each sample, including one group with each of the two antibodies, a group with both antibodies, and a group without antibodies.

[0456] (4) After staining, fix the cells with cell fixative at 4°C for 10 minutes, wash once and then use flow cytometry for detection.

[0457] (5) Detect the mean fluorescence intensity values ​​of PE, APC and FRET channels, and calculate the TCR-CD8 FRET unit using the formula.

[0458] (26) Mouse tumor model, drug treatment and adoptive transfer experiments

[0459] 4×10 5B16F10-OVA cells were subcutaneously injected into female C57BL / 6J mice. Seven days after injection, the mice with tumors of similar size were randomly divided into two groups and then injected with 2×10 6 OT-1 T cells were transfected with B4GALT1 sgRNA or control RNA. Tumor size was then measured every two days using a vernier caliper. Tumor volume was calculated as width × width × length × 1 / 2.

[0460] Lactose treatment: MC38 (1×10 6 cells), B16F1O(4×10 5 cells), B16F10-OVA (4×10 5 cells), B16F10-Control (4×10 5 cells), 4T1 (4×10 5 cells), CT26 (4×10 5 cells) and SGC7901 (5×10 6 Cells were implanted subcutaneously into female C57BL / 6J or NPG mice. Unless otherwise specified, each tumor-bearing mouse was injected with 200 μl of 250 mM lactose solution in PBS via the tail vein every other day starting from the second day after tumor implantation (until the 21st day).

[0461] Combination therapy: On days 6, 9, and 12 after tumor implantation, 100 μg of anti-PD1 (CloneRMP1-14, BioXCell, BE0146) or PDL1 (Clone10F.9G2, BioXCell, BE0101) antibodies were injected intraperitoneally with 200 μl diluted in PBS.

[0462] CD8 + Depletion of T cells, CD4+ T cells, and NK cells: 3 days and 1 day before tumor implantation, intraperitoneal injection of 200 μl of 100 μg of NK antibody diluted with PBS; 2 days and 1 day before tumor implantation, intraperitoneal injection of 250 μg of CD8 antibody (Clone2.43, BioXCell, BE0061) diluted with PBS; 2 days and 1 day before tumor implantation, intraperitoneal injection of 400 μg of CD4 antibody (CloneGK1.5, BioXCell, BE0003-1) diluted with PBS;

[0463] MC38 secondary inoculation tumor model: 1×10 6MC38-GFP cells or MC38-LALBA overexpressing cells were subcutaneously injected into female C57BL / 6J mice. 21 days after tumor implantation, mice whose MC38-LALBA tumors had completely disappeared were inoculated with 1×10 6 The control group consisted of female C57BL / 6J mice of the same age that had not been inoculated with MC38-GFP cells.

[0464] MC38-GFP / MC38-LALBA mixed tumor model: MC38-GFP and MC38-LALBA cells were mixed at a ratio of 9:1 or 1:1 and implanted subcutaneously in female C57BL / 6J mice at a total volume of 1×10 6 The control group received subcutaneous injection of 1×10 6 mice of the same age as those for MC38-GFP cells;

[0465] (27) Humanized mice

[0466] 3-4 week old NPG mice were irradiated with a half-lethal dose of 120 cGy and then injected with human CD34+ umbilical cord blood cells via the tail vein. 12 weeks after transplantation, human CD45+ cells in the peripheral blood were detected to confirm the reconstruction of the human hematopoietic system. 13 weeks after hematopoietic stem cell implantation, 5×10 6 SGC7901 cells were subcutaneously implanted into mice. Starting from the second day after tumor implantation, each tumor-bearing mouse was injected with a 200 μl dose of 250 mM lactose solution in PBS via the tail vein every other day. The control group was injected with an equal volume of PBS buffer. Ten days after tumor implantation, two doses of anti-human PD1 antibody (Pembrolizumab, Selleck, Cat#A2005) (10 mg / kg) were injected weekly. Tumor size was measured every 2-3 days.

[0467] (28) In vitro culture, infection, and adoptive transfer of memory T cells

[0468] On day 0, spleen cells were isolated from 6-8 week old female Cas9-EGFP / OT-1 mice and cultured for 24 hours in RPMI complete medium (RPMI1640, 10% FBS, 20 mM HEPES, 1 mM sodium pyruvate, 100 μg / ml penicillin) containing IL-2 (10 ng / ml) and SIINFEKL peptide (10 ng / ml). On day 1, activated Cas9-EGFP / OT-1 was enriched by Percoll density gradient centrifugation. T cells were then added to a 24-well plate with cell suspension, retrovirus, and 8 μg / ml polybrene and centrifuged (2,000 g, 30°C, 1 h, with minimum acceleration). After centrifugation, the culture plate was placed in a 37°C constant temperature CO2 incubator for 5 h, and then the culture medium was replaced with RPMI complete medium containing IL2 (2 ng / ml), IL7 (2.5 ng / ml), and IL15 (10 ng / ml). The cell density was 3 × 10 5 / ml; two days after infection (day 3), selection was performed with 3 μg / ml puromycin for 4 days; on day 7, cells were available for co-culture or adoptive transfer experiments.

[0469] (29) Isolation of tumor-infiltrating lymphocytes (TIL)

[0470] Add 5 ml of RPMI medium containing 2% FBS and 50 U / ml type IV collagenase (Invitrogen, V900893) to a six-well plate, then cut B16F10-OVA, MC38, and SGC7901 tumors into small pieces in the above solution and incubate at 37°C for 1 hour. The digested suspension is filtered through a 70 μm filter membrane and washed three times with PBS before use for antibody staining and flow cytometry analysis.

[0471] (30) Infiltration and proliferation of T cells in tumors

[0472] After 4 days of Puromycin selection, infected OT-1 T cells were labeled with CFSE (carboxyfluoresceindiacetatesuccinimidylester, Invitrogen), and then 2×10 6 The labeled cells were injected into the tail vein of mice bearing tumor (B16F10-OVA) for 14 days; the intensity of CFSE was detected by flow cytometry 24 hours and 6 days after implantation.

[0473] (31) Construction of retroviral whole-genome CRISPR / Cas9 library

[0474] The whole-genome CRISPR knockout library (1000000096) was purchased from Addgene. The gRNA region was amplified by PCR using primer pair F: 5'-GGCTTTATATATCTTGTGGAAAGGACGAAACACCG-3' (SEQ ID NO: 3) and R: 5'-CTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3' (SEQ ID NO: 4) and constructed into the MSCV-gRNA-PGK-PURO-2A-BFP vector via Gibson reaction.

[0475] (32) Construction of custom retroviral small libraries

[0476] Based on the results of the whole-genome knockout screening, a total of 1,398 genes were screened; based on the performance of the gRNA, an average of three gRNAs were selected from the initial library for each gene, and a total of 105 intergenic control gRNAs were added; oligonucleotides containing the gRNA sequence were synthesized (custom sequence chip), amplified by PCR, and cloned into the MSCV-gRNA-PGK-PURO-2A-BFP vector via the Gibson reaction.

[0477] (33) CRISPR / Cas9 screening

[0478] For in vitro PD1 expression screening, 3x10 6 B16F10-OVA cells were plated in 15 cm culture dishes containing DMEM (DMEM + 10% FBS + Pen / Strep); on day 7, OT-1 T cells selected by puromycin were resuspended in complete RPMI1640 medium containing IL2 (2 ng / ml), IL7 (2.5 ng / ml) and IL15 (10 ng / ml) to a final concentration of 6 × 10 cells / ml. 5 cells and added them to B16F10-OVA cells at a ratio of 4:1. The cells were co-cultured at 37°C overnight. CD8 + T cells were stained with PE anti-PD1 in 2% FBS / PBS on ice for 30 minutes; the top and bottom 5% of cells expressing PD1+ were sorted by BD FACSAria.

[0479] For in vivo screening of customized secondary small libraries, 2x10 6 OT-1 T cells infected with the library (2x10 per recipient mouse) were used as the starting input sample (approximately 269X cell coverage for each sgRNA); OT-1 T cells infected with the library (2x10 per recipient mouse) were used as the starting input sample (approximately 269X cell coverage for each sgRNA); 6Cells were transferred intravenously into mice bearing B16F10-OVA tumors; a total of 24 mice were used as recipients; 7 days after transplantation, B16F10-OVA tumors were digested into single-cell suspensions, and tumor-infiltrating CD8 cells were isolated using biotin anti-mouse CD8a (Biolegend, 100704) and streptavidin magnetic beads. + T cells; at the same time, 1 / 20 volume of each tumor single cell suspension was taken for OT-1 staining to estimate the number of OT-1 T cells infiltrating each tumor; a total of 1×104 to 1×10 5 OT-1 T cells.

[0480] (34) Sequencing library preparation

[0481] Genomic DNA was extracted using the phenol / chloroform method. Titanium Taq DNA polymerase (Clontech, 639242) was used for the first round of PCR amplification of the sgRNA region. The second round of PCR added adapter sequences and sequencing tags to each sample. Finally, Nova-seq 150-bp paired-end sequencing (Illumina) was performed.

[0482] (35) Screening result analysis

[0483] The raw data were preprocessed using sequence sorting tools, and the tool Cutadapt (version 3.4) was used to remove the adapter sequences and the tool FLASH (version 1.2.11) was used to merge the double-end sequencing sequences; the CRISPR / Cas9 screening data were first analyzed using MAGeCK (version 0.5.9.5), and the MAGeCK "count" command generated the gRNA counts of the samples and merged their raw reads into the count matrix; next, the default settings of the MAGeCK "test" command (available from https: / / sourceforge.net / projects / mageck / ) were used to identify the top-ranked forward or reverse gRNAs or genes; gene set enrichment analysis was performed using the default parameters of the GSEA() function of the R language clusterProfiler package (version 3.12.0); the KEGG database can be found in the "C2" category of the R language package msigdbr package (version 7.5.1) (https: / / igordot.github.io / msigdbr / ).

[0484] (36) Flow cytometry

[0485] For cell surface antigen staining, cells were stained in 2% FBS / PBS on ice for 30 minutes. Intracellular staining was performed using a fixation / permeabilization kit (BD Biosciences) according to the manufacturer's instructions. The following antibodies were used: APC anti-mouse CD8a (Invitrogen, 17-0081-83), PE anti-mouse CD279 (PD-1) (BioLegend, 109104), APC anti-mouse CD279 (PD-1) (BioLegend, 109112), FITC anti-mouse IFNγ (BioLegend, 505806), APC anti-mouse β2-microglobulin (BioLegend, 154506), APC anti-mouse H2Db (BioLegend, 111513 ), APC anti-mouse OVA257-264 (SIINFEKL) (Invitrogen, 17-5743-82), FITC anti-mouse CD3 (BioLegend, 100204), PE-streptavidin (peolegend, 405203), biotinylated Erythrina chinensis lectin (B-1145-5), biotinylated succinylated wheat germ agglutinin (B-1025S-5), APC anti-human CD8a (BioLegend, 300912), PE anti-human IFNγ (BioLegend, 506507), FITC anti-mouse TCRVα2 (BioLegend, 127806); flow cytometry experiments were performed using a BD FACSAria instrument, and data were analyzed by FlowJo.

[0486] (37) Enzyme-linked immunosorbent assay

[0487] OT-1 T cells (3x10 5 / ml) and B16F10-OVA cells were co-cultured at 37°C for 8 hours in the presence of culture medium containing IL2 (2 ng / ml), IL7 (2.5 ng / ml), and IL15 (10 ng / ml). The supernatant was collected after co-culture. TNFα and IFNγ in the co-culture supernatant were detected using ELISA kits (ABclona, ​​Cat#RK00027, Cat#RK00019). Each sample had two replicates.

[0488] (38) In vitro killing experiment

[0489] For the in vitro OT-1 T cell killing assay, B16F10-OVA cells and B16F10 cells were labeled with CFSEhi (2.5 mM) and CFSElo (50 nM), respectively, and then co-cultured with OT-1 T cells in 96-well plates at the specified ratios. The control group consisted of tumor cells without OT-1 T cells. After 24 hours of incubation, the ratio of CFSEhi and CFSElo cells in the two populations was determined by flow cytometry.

[0490] For the MC38 in vitro killing assay, MC38 cells were treated with 100 ng / ml of Ovapeptide (SIINFEKL) at 37°C for 3 h and then cocultured with OT-1 T cells;

[0491] The specific killing rate was calculated as [1-(CFSEhi / CFSElo containing T cells) / (CFSEhi / CFSElo containing tumor cells only)]×100%.

[0492] (39) Lactose quantification using liquid chromatography-mass spectrometry (LC-MS)

[0493] Cell sample preparation: After collecting cells, wash them twice with PBS; each 2×10 6 Add 400 μl of methanol / acetonitrile mixture (methanol:acetonitrile:water = 4:4:2) to the cells, mix thoroughly, place at -80°C for 1 hour, and then centrifuge at 14,000g at 4°C for 15 minutes. Transfer the supernatant after centrifugation to a new tube and repeat the above steps. The final supernatant can be used for mass spectrometry quantitative analysis.

[0494] Cell culture medium sample preparation: Collect the cell supernatant and filter it through a 0.45 μm filter to remove cell debris. Then, add 2.5 volumes of methanol, mix thoroughly for 2 minutes, and centrifuge at 13,000 rpm for 15 minutes. The resulting supernatant can be used for quantitative mass spectrometry analysis.

[0495] A 100 mM lactose solution was diluted with 50% aqueous methanol to 100, 200, 500, 1000, 2000, 5000, and 10000 nM and used as a standard; the standard solution and the prepared sample were transferred to an LC sample vial for analysis; LC-MS analysis was performed in negative ESI mode on a Thermo Vanquish UHPLC equipped with a Thermo Q Exactive HF-X hybrid quadrupole-Orbitrap mass spectrometer; a Waters Acquity UPLC B E H Amide column (1.7 μm, 2.1×100 mm) was used to achieve separation at a column temperature of 35°C under isocratic elution with 30% mobile phase A (5 mM ammonium formate aqueous solution) and 70% mobile phase B (acetonitrile); the flow rate was 0.3 ml / min, the injection volume was 10 μl, and the run time for each injection was 5 min.

[0496] Full-scan mass spectra were acquired in the range of m / z 66.7 to 1000 using the following ESI source settings: spray voltage: 2.5 kV; auxiliary gas heater temperature: 380°C; capillary temperature: 320°C; sheath gas flow rate: 10 units; MS1 scan parameters included resolution 60000, AGC target 3e6, and maximum injection time 200 ms; data were processed using ThermoXcalibur software (version 4.2), and quantification was performed using the [M+FA]-adduct of lactose (m / z 387.1138).

[0497] (40)RT-qPCR

[0498] use RNA was extracted using ImProm-II TM cDNA was synthesized using a reverse transcriptase system (Promega, Cat# A3801), using 100 ng RNA per reaction; qPCR reactions were performed using Takara (Takara, Cat#RR420A) with 1 μl cDNA per 20 μl reaction volume; relative gene expression levels were normalized using GAPDH.

[0499] (41) TCGA (Cancer Genome Atlas) data analysis

[0500] Transcriptome expression profiles and clinical data for all tumor types were downloaded from the TCGA official website (https: / / gdcportal.nci.nih.gov / ). To normalize B4GALT1 gene expression using CD8a, the B4GALT1 expression value was divided by the CD8a expression value in the corresponding sample. Subsequent survival analysis and HR calculation were performed using the R language survival package (version 3.3.1) and survminer package (version 0.4.9). Patients were grouped using the median gene expression level as the grouping criterion. Survival curves were compared using a two-sided log-rank test and Kaplan-Meier curves were plotted.

[0501] (42) RNA-seq data analysis

[0502] Paired-end 150bp sequencing of the transcriptome was performed on the NovaSeq6000S4 platform; raw data were mapped to the mouse genome (mm10) using TopHat (version 2.1.1)5; gene read counts were calculated using HTSeq (version 1.99.2), and normalized using cufflinks (version 2.2.1); differentially expressed genes were calculated using the DESeq2 package (version 1.22.2) in R, with a P-value of 0.05 or 0.01 as the screening criteria; functional enrichment analysis of differentially expressed genes was performed using the enricher() function in the ClusterProfiler package (version 3.12.0) in R; characteristic gene sets were estimated using the average gene expression levels of genes in the corresponding samples; Supplementary Table 7 lists the complete list of genes involved in each characteristic gene set.

[0503] (43) Single-cell RNA-seq sample preparation

[0504] Three pairs of MC38 tumor cells treated with PBS or lactose were screened using CD3-positive genes. The same number of CD3-positive cells from each sample were mixed and screened by flow cytometry. 5 × 10 5 -1×10 6 CD3-positive cells were resuspended at a density of 100 cells / ml, and the cell viability was 85%. Single-cell libraries were prepared using the 5'V(D)J and gene expression platforms according to the methods provided on the 10×Genomics official website and then sequenced on the NovaSeq6000S4 platform.

[0505] (44) TCR data quality control and analysis

[0506] The raw data were analyzed and processed using the latest mouse reference genome (refdata-gex-mm10-2020-A, 10×Genomics) and the CellRangervdj command (version 7.0.0) with default parameters; the intermediate results were then processed using the R language scRepertoire package (version 1.2.0) to generate an output file (filtered_contig_annotations.csv). Finally, a total of 14,851 positive cells were defined in the present invention, of which 6,914 were from PBS samples and 7,937 were from LAC samples. Further analysis results are shown in Figure S13.

[0507] (45) Quality control, filtering and analysis of single-cell RNA-seq data

[0508] The raw data were assembled and analyzed using the latest mouse reference genome (refdata-gex-mm10-2020-A, 10×Genomics) and the default parameters of the CellRanger command (version 7.0.0); the sequencing saturation was approximately 60%, and the sequencing depth of each cell was approximately 25,000 sequences; as described in previously published articles, the present invention eliminated cells with abnormal gene numbers and abnormal mitochondrial gene counts; ultimately, 19,334 cells were retained from the two libraries, of which 13,839 cells had both T cell receptor sequencing data and single-cell transcriptome sequencing data; the R language Seurat package (version 2.3.4) was used for further analysis; for the cell clustering results, the FindClusters() function was used to divide the cells into 10 groups at a resolution of 0.5.

[0509] (46) Blood sample biochemical analysis

[0510] Blood samples (500 μl) were collected in EDTA-coated tubes; a portion of the blood was taken for the evaluation of hematological parameters: white blood cells (WBC), lymphocytes (LYM), intermediate cells (MID), granulocytes (GRA), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width-standard deviation (RDW-SD), and red blood cell distribution-coefficient of variation ( The remaining samples were centrifuged (1500 g, room temperature for 10 min), and the supernatant was used for biochemical parameter analysis: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatinine (CR), blood urea nitrogen (BUN), lactate dehydrogenase (LDH), creatine kinase (CK), glucose (GLU), and inorganic phosphorus (Pi) were measured using a fully automatic clinical biochemical analyzer (Beckman Coulter AU5800).

[0511] (47) Data Analysis

[0512] All statistical analyses were performed using R language version 4.1.0. Two-tailed paired T-tests were used to determine statistical significance (*P < 0.05, **P < 0.01; ***P < 0.001; NS not significant) using the t.test() function. Two-way ANOVA was performed using the aov() function. The figures show the mean and standard error (SEM), where the standard error was calculated using the sem() function.

[0513] (48) Data Acquisition

[0514] The raw sequencing data used in this article have been deposited in the National Genome Science Data Center with the project number PRJCA010494. The access website is: https: / / ngdc.cncb.ac.cn / search / ?dbId=&q=PRJCA010494; transcriptome data and clinical data of all tumor types were downloaded from the National Cancer Institute and National Human Genome Research Institute Data Center (https: / / gdc-portal.nci.nih.gov / ).

[0515] Lactose treatment of tumor-bearing mice: α-lactose was purchased from Sigma-Aldrich (L3625-1KG, Lot#SLCD8654); 250 mM α-lactose was prepared by dissolving 0.09 g of lactose powder in 1 mL of PBS (C14190500BT, Lot#8121493) and filtering through a 0.22 μm filter membrane until completely dissolved; MC38 (1×10 6 cells), B16F1O(4×10 5 cells), B16F10-OVA (4×10 5 cells), B16F10-Control (4×10 5 cells), 4T1 (4×10 5 cells), CT26 (4×10 5 cells) and SGC7901 (5×10 6 Cells were implanted subcutaneously into female C57BL / 6J or NPG mice; starting from the second day after tumor implantation, each tumor-bearing mouse was injected with 200 μl of 250 mM lactose solution dissolved in PBS via the tail vein every other day (injection until the 21st day).

[0516] Other experimental methods not listed are routine.

[0517] Example 1: Identification of CD8 by in vitro and in vivo CRISPR / Cas9 screening + Genes and pathways regulating PD1 expression and TCR activation in T cells

[0518] The inventors established an in vitro genome-wide CRISPR / Cas9 screening system to identify + Genes and pathways regulating PD1 expression in T cells (Figure 1A); Briefly, splenic CD8 T cells from Cas9-EGFP / OT-1 mice were pre-stimulated with ovalbumin (Ova) peptide. + T cells, and then use retrovirus to infect the cells with a guide RNA (gRNA) library targeting the whole genome; after puromycin selection, memory CD8 +T cells; PD1-overexpressing and PD1-underexpressing cells were enriched by flow cytometry; next-generation sequencing revealed the distribution of these cell subsets and individual gRNAs in the starting cells in the whole-genome library; as shown in Figure 1B, PDCD1 and established PD1 regulatory factors, such as SATB1 and FUT8, were successfully screened as positive controls; most of the top candidate genes were independently verified by FACS and RT-qPCR (Figure 1C); gene set enrichment analysis (GSEA) identified several KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways that were significantly involved in regulating CD8 + PD1 expression in T cells (Figure 1D); known genes in the TCR activation pathway (such as CD3d, Zap70 and Lat) ranked at the top of the list of candidate genes regulated by PD1; unexpectedly, the results showed that genes involved in protein trafficking pathways (such as Srp14, Srp68, Sec16A) and genes for aminoacyl-tRNA biosynthesis (such as Mars, Hars2, Eprs) were also significantly enriched in the PD1 low expression group; finally, the inventors screened and verified various components in N-glycan biosynthesis, including B4GALT1, Mgat2 and Dpm3, which can negatively regulate PD1 expression (Figures 1C-D and Figure 7).

[0519] To validate candidate genes in high-throughput manner and test their potential functions in the tumor microenvironment (Figure 1A), the inventors synthesized a custom gRNA library containing 4617 gRNAs targeting preferred candidate genes obtained from whole-genome screening and 105 intergenic control gRNAs; the gRNA library was infected with Cas9-EGFP / OT-1 memory CD8 + T cells were reactivated in vitro or transplanted into wild-type C57BL / 6J mice subcutaneously inoculated with B16F10-OVA tumors to screen for in vivo regulatory CD8 + Genes encoding T cell function; T cells were collected from tumors 7 days later for gRNA sequencing; gRNAs that inactivated Socs1, Regnase-1 (Zc3h12a), Rc3h1, and CD36 led to an increase in the number of OT-1 T cells infiltrating B16F10-OVA tumors in syngeneic mice (Figure 1F); Figures 1E-F show that inactivation of B4GALT1, encoding β-1,4-galactosyltransferase 1, exhibited significant phenotypes in both in vitro and in vivo screening.

[0520] Example 2: Knockout of CD8 + B4GALT1 in T cells can activate TCR signaling and enhance T cell-mediated tumor immunotherapy

[0521] Infection of Cas9-EGFP / OT-1 CD8 cells with different gRNAs targeting B4GALT1 + 2A and 7C). These phenotypes could be complemented by overexpression of the short or long isoform cDNA of mouse B4GALT1 (Figure 2B), indicating that the response to inhibition of TCR activation was not due to B4GALT1 ligand-induced signal transduction but to its biosynthetic function. In addition, after co-culture with B16F10-OVA, B4GALT1-knockout OT-1 T cells showed enhanced expression of T cell activation and cytotoxicity markers IFN, IL-2, and TNF, as well as enhanced in vitro target cell killing activity (Figures 2C-D). Whole-genome RNA sequencing analysis confirmed that after co-culture with B16F10-OVA cells, B4GALT1-knockout CD8 + T cells enhanced TCR activation (Figure 2E-G); control gRNA and B4GALT1-targeted gRNA infected CD8 + Gene set enrichment analysis (GSEA) of differentially expressed genes (DEGs) between T cells showed that the TCR signaling pathway was at the top of the significantly altered pathways ( Figure 2G ).

[0522] When B16F10-OVA cells were inoculated subcutaneously into wild-type mice, OT-1 T cells infected with B4GALT1gRNA had significantly higher tumor-killing activity than cells infected with control gRNA (Figure 2H-I). Analysis of tumor-infiltrating lymphocytes (TILs) showed that there were more infiltrating OT-1 T cells in the tumor when infected with B4GALT1gRNA (Figure 2J). These results indicate that the inhibitory protein N-glycome can enhance the CD8 + T cell function and B4GALT1 may be potential targets for enhancing CAR-T cell activity.

[0523] In B16F10-OVA tumors, the number of infiltrating OT-1 T cells infected with B4GALT1gRNA 24 hours after intravenous injection was similar to that of cells infected with control gRNA (Figure 8A), indicating that B4GALT1 had no significant effect on the infiltration of OT-1 T cells; on the other hand, CFSE (carboxyfluorescein succinimidyl ester) analysis at a later time point (6 days after injection) showed that B4GALT1 inactivation enhanced the proliferation of OT-1 T cells in the tumor (Figure 8B).

[0524] Example 3: Expression of B4GALT1 in the tumor microenvironment and tumor-infiltrating CD8 + Experiment on the correlation between T cells and prognosis of human patients

[0525] To investigate the potential clinical relevance of B4GALT1 in human cancer patients, the inventors analyzed cancer samples in The Cancer Genome Atlas. Although the expression level of B4GALT1 was not associated with the overall survival benefit of all primary tumor samples collected by TCGA, after standardizing the expression level of CD8a, the Kaplan-Meier curve showed that patients with low B4GALT1 expression had significantly longer survival (Figure 3A and Figure 9A); on the other hand, the inventors first divided all tumor samples into B4GALT1 low expression and B4GALT1 high expression groups; high expression of CD8a in the B4GALT1 low expression group was positively correlated with longer overall survival, while there was no such correlation in the B4GALT1 high expression group (Figure 3B and Figure 9C); in adrenocortical carcinoma (A In the datasets of 365 cancer cells (CC), acute myeloid leukemia (LAML), lung adenocarcinoma (LUAD), and rectal adenocarcinoma (READ), it was found that low B4GALT1 expression levels were positively correlated with longer overall survival in patients in the CD8a high expression group compared with those in the CD8a low expression group (Figures 3C and 9B). Conversely, higher CD8a expression levels were positively correlated with better overall survival in patients in the B4GALT1 low expression group compared with those in the B4GALT1 high expression group (Figures 3D and 9C). The results showed that the expression level of B4GALT1 in the tumor microenvironment and the infiltrating CD8 + T cells are associated with the prognosis of cancer patients.

[0526] Example 4: Inhibition of B4GALT1 activity in tumor cells enhances IFNγ signaling pathway and immune surveillance response

[0527] The inventors believe that targeting the same gene or pathway in immune cells and tumor cells can work synergistically to enhance the efficacy of tumor immunotherapy; in addition, the expression of B4GALT1 in tumors depends mainly on the malignant tumor cells rather than CD8 + T cells; unexpectedly, when the inventors used the CRISPR / Cas9 system to conduct in vivo screening experiments on genes and pathways related to regulating the resistance and sensitivity of tumor cells to immune surveillance in MC38 tumors, the inventors found that B4GALT1 could be screened out; in wild-type C57BL / 6J mice, the growth rate of subcutaneously implanted MC38 tumors with B4GALT1 knockout was significantly reduced compared with the control group cells, while there was no obvious difference in the immunodeficient NPG (NOD-PrkdcscidIL2rgnull) mice (Figure 4A-B); if specific antibodies were used to eliminate different immune cells in wild-type mice and then subcutaneously implanted, the inventors found that elimination of CD8 +The removal of CD4+ T cells significantly restored the growth rate of MC38 tumors with B4GALT1 deletion, but the removal of natural killer (NK) cells could not restore the growth rate (Figure 4C, Figure 10). The above results indicate that in wild-type mice, CD8 + T cells play a major role in controlling the growth of B4GALT1-knockout MC38 tumors.

[0528] The inventors performed RNA sequencing analysis on B4GALT1-knockout MC38 cells and control cells. The results showed that the IFNα, IFNγ, and TNFα signaling pathways were significantly upregulated (Figures 4D-F). After IFNγ treatment, B2m expression levels and Ova antigen presentation were significantly enhanced in B4GALT1-knockout MC38 cells compared with control cells (Figures 4G-H). At the same time, knocking out the B4GALT1 gene in MC38 cells can enhance the specific in vitro killing function of OT-1 T cells (Figure 4I). Notably, compared with co-culture with control MC38 cells, the IFNγ expression level in OT-1 T cells was significantly increased when co-cultured with B4GALT1-knockout MC38 cells (Figure 4J).

[0529] Example 5: Exogenously expressed α-lactalbumin (LALBA) can produce lactose in tumor cells and activate tumor immune responses

[0530] In addition to its binding function to the N-glycosylation group, B4GALT1 can also form heterodimers with LALBA in breast tissue to synthesize lactose. The inventors hypothesized that exogenous overexpression of LALBA in tumor cells can alter the catalytic activity of endogenous B4GALT1 in the lactose biosynthesis process; therefore, LALBA may competitively reduce the normal function of B4GALT1 in tumor cells; in addition, de novo lactose synthesis can also inhibit the activity of B4GALT1 and interfere with tumor cells and intratumoral CD8 + N-glycosylation function of T cells; lactose is a disaccharide composed of galactose and glucose, which can simulate galactosylated oligosaccharides or proteins; in vitro experiments have shown that in glycoproteins and glycolipids, lactose can compete with and interfere with the function of β-galactosidase, such as the interaction with lectins.

[0531] The inventors constructed two LALBA overexpression vectors (LALBAOE), in which the LALBA gene was selected from the cDNA region of the sequence with ID No. 16770 in Genebank: 1918-2349. The structure of the constructed lentiviral vector is shown in Figure 22, and its base sequence is shown in SEQ ID NO: 1. The structure of the constructed adenoviral vector overexpressing LALBA is shown in Figure 23, and its base sequence is shown in SEQ ID NO: 2.

[0532] Lentivirus was packaged by co-transfecting HEK-293T cells with a lentiviral expression vector, psPAX2, and pMD2.G. 48 hours after transfection, cell supernatants were collected and filtered through a 0.45 μm filter to remove cell debris. The virus was either used directly for infection or concentrated by high-speed centrifugation (107,000 g at 4°C for 2.5 hours) and resuspended in PBS for in vivo experiments. The following controls were also included: lentivirus carrying a site-directed mutation in the LALBA gene with either D107A or A126K, with the site-directed mutation of A126K being A126K:GCC→AAG; the site-directed mutation of D107A being D107A:GAT→GCC.

[0533] Adeno-associated virus (AAV) type 8 was prepared using an AAV viral vector expressing a lentiviral expression vector (LALBA-IRES-GFP) or GFP. HEK-293T cells were first co-transfected with the AAV vector, packaging plasmid, and AAV helper plasmid. After 72 h, the culture medium and cells were collected for precipitation and lysis, respectively, followed by density gradient centrifugation with iodixanol. The purified virus was finally ultracentrifuged and diluted with PBS for subsequent experiments.

[0534] 1×10 6 MC38 cells were implanted subcutaneously into female C57BL / 6J mice. Nine days after implantation, mice with tumors of similar size were injected intratumorally with the virus on days 9, 12, and 15 after implantation. The titers of lentivirus and adeno-associated virus were determined by serial dilution in MC38 cells. In each injection, the same unit of lentivirus or adeno-associated virus (the amount of virus that can infect 8×104 MC38 cells in vitro) was injected into the tumor. Tumor size was measured every 3 days until day 24.

[0535] As shown in Figures 5A-B, overexpression of LALBA in MC38 cells using lentivirus can promote de novo synthesis and secretion of lactose, and this process is dependent on the expression of B4GALT1. If D107A or A126K site-directed mutations are performed on the LALBA gene, lactose synthase is inactivated. When MC38 cells overexpressing LALBA are implanted subcutaneously in wild-type mice, the tumor growth rate is significantly lower than that of control cells (expressing only GFP), similar to MC38 tumors with B4GALT1 knockout (Figure 5C). Importantly, when MC38 tumors overexpress LALBA, the inventors observed complete tumor regression in more than 50% of the mice (CR stands for complete response). In contrast, in immunodeficient NPG mice, MC38 tumors overexpressing LALBA showed no significant difference in tumor size compared to control cells expressing only GFP. There was no obvious difference in growth rate (Figure 5D); subsequently, the inventors re-implanted MC38 control cells into the mice with completely regressed tumors. Compared with wild-type mice of the same age who had not been injected, mice with completely regressed MC38-LALBA tumors were completely resistant to MC38 control cells, indicating that tumor-specific immune memory had been established (Figure 5E); at the same time, the inventors also subcutaneously implanted a mixture of overexpressed LALBA and control MC38 cells into wild-type mice, with a mixing ratio of 1:1 or 1:9; as shown in Figure 11, the addition of LALBA-overexpressing MC38 to control cells can significantly inhibit tumor growth (6 / 22 tumors in the 1:1 mixed tumors completely regressed; 3 / 22 tumors in the 1:9 mixed tumors completely regressed); the above results indicate that partial or complete overexpression of LALBA in tumor cells can enhance immune suppression against tumors and form immune memory.

[0536] To achieve overexpression of LALBA in tumors, the inventors injected lentivirus encoding GFP control or LALBA into the tumors; as shown in Figure 5F, after three intratumoral injections of LALBA lentivirus, the growth rate of MC38 tumors in wild-type mice was significantly inhibited (4 / 10 tumors completely regressed), while mutant LALBA (D107A and A126K) had no obvious inhibitory effect (Figure 12, arrows indicate the time of virus injection); flow cytometry analysis showed that the number of IFNγ+CD8+T cells in tumors injected with LALBA lentivirus was significantly increased (Figure 5G); in addition, mice with complete tumor remission after LALBA lentivirus injection were completely resistant to control MC38 cells (Figure 5H); finally, when LALBA was overexpressed in MC38 tumors using adeno-associated virus (AAV), the inventors could observe similar tumor inhibitory effects (Figures 5I-K); flow cytometry analysis showed that tumor-infiltrating CD8 + T cells can express higher levels of IFNγ and PD1.

[0537] The inventors used biotin-labeled lectins for flow cytometry staining, including Erythrinacristagalli lectin (ECL) and succinyl-wheatgerm lectin (sWGA), which can measure the expression levels of terminal β-galactosidase and β-N-acetylglucosamine on the cell surface, respectively. For MC38 cells, lactose blocked ECL staining but had no effect on sWGA staining (Figure 13A); B4GALT1 knockout MC38 cells expressed slightly higher levels of ECL on the surface, while sWGA expression increased significantly; the results showed that LALBA overexpression on the surface of MC38 cells significantly increased both ECL and sWGA (Figure 13B); On the other hand, in CD8 + Knockout of B4GALT1 in T cells significantly reduced the expression of ECL and enhanced the expression of sWGA (Figure 13C).

[0538] Example 6: Lactose, a competitive inhibitor that structurally mimics the N-glycosyl group, can enhance tumor immune surveillance in vivo

[0539] The above results show that in CD8 + Interfering with the function of the N-glycosyl group in T cells and tumor cells with lactose can activate tumor immune responses in a synergistic manner. After subcutaneous implantation of MC38 tumors in wild-type mice, the inventors injected lactose through the tail vein every two days. Compared with the PBS control, lactose significantly reduced the growth rate of MC38 tumors and enhanced the expression of CD8 + The expression of PD1 and IFNγ on the surface of T cells (Figure 6A-C, Figure 14); when the inventors used immunodeficient NPG mice for the same treatment, the growth rate of MC38 tumors injected with lactose was not significantly different from that of PBS controls (Figure 6D); CD8 + After the elimination of CD4+ T cells, lactose lost its inhibitory effect on MC38 tumors, while the elimination of CD4+ T cells or NK cells did not affect the inhibitory effect of lactose on tumor growth (Figure 6E-G); in addition, in MC38 tumors treated with anti-PD1 or anti-PDL1 antibodies, tail vein injection of lactose did not further enhance the inhibitory effect on tumors (Figure 15, Figure 16).

[0540] The inventors treated MC38 subcutaneous tumors in wild-type mice with lactose or control (PBS) and then performed transcriptome sequencing analysis of the cell population. They ultimately found 585 significantly upregulated genes and 528 significantly downregulated genes (Figures 17A-B). Surprisingly, the most significantly enriched HALLMARK pathways (Figure 17C), such as IFNγ, hypoxia pathway, TNFα signaling pathway via NFκB, and Myc targets (V1 and V2), were consistent with the pathways significantly enriched in B4GALT1-knockout MC38 cells (Figure 4F). Gene expression signature analysis (Figures 17E-F) showed that lactose-treated tumors had significantly high expression of Tfh cell characteristics, T cell positive enrichment characteristics, and cytotoxic T cell characteristics.

[0541] To further analyze the potential effects of lactose on the tumor microenvironment, especially tumor-infiltrating T cells, the inventors sorted CD3-positive T cells from MC38 tumor samples treated with lactose and PBS, and then performed single-cell transcriptome sequencing and TCR sequencing (Figures 18 and 19). The results showed that after lactose treatment, effector / exhausted memory CD8 + T cells (C0:CD8 + The proportion of PD1+PRF1+) increased from 26.0% to 40.1%, while the proportion of progenitor T cells / undifferentiated CD8 + T cells (C2:CD8 + TCF7+SELL+) and regulatory CD4+ T cells (C1:CD4+AREG+FOXP3+) decreased from 19.7% to 11.8% and from 23.5% to 14.2%, respectively (Figure 18F); TCR clone type analysis results showed that lactose-treated tumors had significantly increased TCR clones compared with the control group (Figures 19A-C); the largest TCR clone was CN1 (CDR3:TRA; TRB=CAIDPYNQGKLIF; CASSPGQGYAEQFF), which can target the specific neoantigen (ASMTNMELMintheAdpgkgene) of MC38 cells. After lactose treatment, its proportion increased from 2.3% to 35.1% (Figures 19D-E); In summary, the results of cell population and single-cell transcriptome sequencing analysis showed that lactose treatment can promote tumor-specific CD8 + Proliferation of T cells and enhancement of their function.

[0542] In wild-type mice, tail vein injection of lactose had no significant effect on the growth of "cold tumors" such as 4T1 and CT26 (Figure 6H-I); compared with the control group, exogenous overexpression of Ova in B16F10 cells significantly enhanced the sensitivity of the tumor to lactose treatment (Figure 6J-K), because the expression of Ova converted the original B16F10 "cold tumor" into a "hot tumor."

[0543] At the same time, the inventors subcutaneously implanted SGC7901 human gastric cancer cells into NPG mice transplanted with human immune cells, and then injected lactose or PBS into the tail vein. Similar to mice treated with anti-PD1 antibodies (Figure 20), lactose significantly reduced the growth rate of SGC7901 tumors in humanized mice (Figure 6L). After lactose treatment, SGC7901 tumors contained a higher proportion of IFNγ+ tumor-infiltrating human CD8 + In contrast, lactose did not exert a tumor suppressive effect in NPG mice that were not transplanted with human immune cells (Figure 6N).

[0544] Finally, the inventors evaluated the safety of lactose after tail vein injection in wild-type C57BL / 6J mice; as shown in Figure 21, short-term (24 hours) or long-term (24 days) lactose injection every two days did not produce obvious toxic side effects.

[0545] Example 7: B4galt1 regulates CD8 through tumor-derived Gal-1 + T cell killing ability

[0546] 1. B4galt1 catalyzes CD8 galactosylation and regulates TCR-CD8 colocalization via Gal-1

[0547] To further dissect the molecular mechanism by which B4galt1 regulates T cell activation, the present inventors first used biotinylated erythrosine agglutinin (ECL) and succinylated wheat germ agglutinin (sWGA) for flow cytometric analysis to analyze cell surface expression of βGal and βGlcNAc, respectively. B4galt1-deficient OT-I T cells showed a slight decrease in ECL staining intensity, while sWGA staining intensity was significantly increased (Figures 25A-B). Surprisingly, staining with biotinylated recombinant galectin-1 (rGal-1) revealed highly significant differences between control and B4galt1-deficient OT-I T cells (Figure 25C).

[0548] Figure 25: A. B4galt1 knockout cells and control OT-I T cells were incubated with biotinylated ECL and then stained with streptavidin-PE. The dashed line indicates cells not incubated with lectin but stained with streptavidin-PE alone. The mean fluorescence intensity (MFI) of each sample was determined by flow cytometry. B. B4galt1 knockout cells and control OT-I T cells were incubated with biotinylated sWGA and then stained with streptavidin-PE. C. B4galt1 knockout cells and control OT-I T cells were incubated with biotinylated recombinant Gal-1 and then stained with streptavidin-PE. The dashed line indicates cells not incubated with lectin but stained with streptavidin-PE alone.

[0549] RNA sequencing analysis revealed that galectins-1, -3, and -9 (Gal-1, Gal-3, and Gal-9) were the most highly expressed galectins in OT-I T cells and MC38 cells (Figure 26A). Intracellular flow cytometry confirmed that all of these lectins were highly expressed in both OT-I T cells and MC38 cells. However, cell surface staining revealed that Gal-1, -3, and -9 were expressed only on the surface of MC38 cells and not on the surface of OT-I T cells (Figure 26B). Comparison of surface and intracellular staining intensities on MC38 cells revealed that Gal-1 was more prominent on the surface of MC38 cells than Gal-3 and Gal-9 (Figure 26C).

[0550] Figure 26. A. FPKM represents the expression levels of various galectin mRNAs in OT-I T cells and MC38 cells, derived from RNA sequencing analysis. B. Flow cytometric staining and analysis of surface and intracellular galectins-1, -3, and -9 in OT-I T cells and MC38 cells. C. Mean fluorescence intensity of surface and intracellular galectin-1, -3, and -9 staining in MC38 cells, with the ratio of surface to intracellular intensity calculated.

[0551] 2.CD8 + Gal-1 on the surface of T cells comes from adjacent tumor cells

[0552] To explore CD8 +Regarding the source of Gal-1 on the T cell surface, coculture of OT-I T cells with either OVA oligopeptide-treated or untreated MC38 cells significantly increased Gal-1 staining on the OT-I T cell surface, with no significant difference. This result suggests that interaction between the TCR receptor and the MHC-I / OVA complex, or that TCR activation, is not necessary for the appearance of Gal-1 on the T cell surface (Figure 27A). On the other hand, activation of OT-I T cells with anti-CD3 / anti-CD28 antibodies alone in the absence of MC38 cells also failed to increase surface Gal-1 staining (Figure 27A). Consistently, in the absence of MC38 cell coculture, B4galt1-knockout OT-I T cells did not exhibit significantly enhanced T cell activation markers upon stimulation with anti-CD3 / anti-CD28 antibodies compared to wild-type controls (Figure 27B). In summary, the increased Gal-1 staining on the OT-I T cell surface likely originates from the cocultured tumor cells.

[0553] Figure 27, A. OT-I T cells activated or untreated with anti-CD3 / anti-CD28 antibodies, and MC38 cells co-cultured with OVA oligopeptide-pretreated or untreated MC38 cells were stained with anti-Gal-1 antibody. The dashed line indicates staining with an isotype control antibody. B. B4galt1 knockout cells and control OT-I T cells were activated with anti-CD3 / anti-CD28 antibodies. Ifnγ and Tnfα mRNA expression levels were assessed by quantitative RT-qPCR.

[0554] To confirm that the Gal-1 on the surface of OT-I T cells came from co-cultured tumor cells, the present invention co-cultured OT-I T cells with wild-type and Gal-1 knockout MC38 cells, respectively. It was found that Gal-1 staining on the surface of OT-I T cells was significantly increased only when co-cultured with wild-type MC38 cells (Figure 28A). Then, the present invention used conditioned medium cultured with wild-type or Gal-1 knockout MC38 cells as the medium for co-culture of OT-I T cells and MC38 cells. It was found that Gal-1 staining on the surface of OT-I T cells could only be increased in the presence of wild-type MC38 cells, not in the presence of wild-type conditioned medium alone (Figure 28B). By setting co-culture conditions with different ratios of MC38 cells and OT-I T cells, it was found that the intensity of Gal-1 staining on the surface of OT-I T cells increased with increasing MC38 cell ratios (Figures 28C-D).

[0555] Figure 28: A. OT-I T cells co-cultured with Gal-1 knockout MC38 cells, wild-type MC38 cells, or not co-cultured with MC38 cells were stained with anti-Gal-1 antibodies. B. OT-I T cells were co-cultured with wild-type or Gal-1 knockout MC38 cells at a 2:1 cell ratio for 8 hours in conditioned medium from different MC38 cells. Cells were stained with anti-Gal-1 antibodies, and the mean fluorescence intensity of cell surface Gal-1 was measured by flow cytometry. C. OT-I T cells were co-cultured with MC38 cells at ratios of 0:10, 1:10, 1:2, 2:1, 10:1, and 10:0, and then stained with anti-Gal-1 antibodies. D. The mean fluorescence intensity of cell surface Gal-1 on OT-I T cells and MC38 cells after co-culture at different ratios was measured by flow cytometry.

[0556] Further investigation revealed that when OT-I T cells and MC38 cells were physically separated by a membrane in a Boyden chamber (Figure 29A) or a thin low-melting-point agarose membrane (Figure 29B), Gal-1 protein could no longer be effectively transferred from MC38 cells to OT-I T cells. This experimental evidence indicates that close cell-cell contact is necessary for Gal-1 transfer. Unlike previous mechanisms of intercellular transfer of surface proteins, such as endocytosis, exosome transport, and nanochannel shuttling, this process is named proximity-dependent intercellular protein spreading (PDICPS) in this study.

[0557] Figure 29, A. OT-I T cells were seeded in the upper compartment of a Boyden Chamber (i), or simultaneously with MC38 cells in the lower compartment (ii), or together with MC38 cells in the lower compartment (iii). Eight hours later, cells were stained with anti-Gal-1 antibodies, and the mean fluorescence intensity of cell surface Gal-1 was measured by flow cytometry. B. After MC38 cells adhered to the bottom of the culture dish, medium containing low-melting-point agarose was added. OT-I T cells were then added to the bottom of the culture dish (i), above the agarose layer (ii), or directly onto the MC38 cells (iii). Cells were stained with anti-Gal-1 antibodies, and the mean fluorescence intensity of cell surface Gal-1 was measured by flow cytometry.

[0558] 3. B4galt1 regulates CD8 through Gal-1 + T cell killing ability

[0559] By detecting the RNA levels of the cytotoxic factors TNFα and IFNγ, the present invention found that the addition of exogenous recombinant Gal-1 protein to wild-type OT-I T cells can significantly inhibit the activation of T cells by anti-CD3 / anti-CD28 antibodies, while there is no inhibitory effect in B4galt1 knockout OT-I T cells (Figure 29A). In the absence of OVA oligopeptide pretreatment, OT-I T cells co-cultured with Gal-1 knockout MC38 cells showed enhanced activation by anti-CD3 / anti-CD28 antibodies compared to OT-I T cells co-cultured with wild-type MC38 cells (Figure 29B). Similarly, B4galt1 knockout OT-I T cells did not show enhanced T cell activation under these conditions.

[0560] In Figure 29, A. Recombinant Gal-1 treatment (2.5 μg / mL) significantly reduced the number of wild-type OT-Ⅰ T cells (1×10 5 A. Coculture with Gal-1-deficient MC38 cells increased Tnfα and Ifnγ mRNA expression in wild-type OT-Ⅰ T cells activated with anti-CD3 / anti-CD28 antibodies compared with wild-type MC38 cells, whereas coculture with Gal-1-deficient MC38 cells had no significant effect on B4galt1-deficient OT-Ⅰ T cells. (p-values ​​are from two-tailed t-tests.) B. Coculture with Gal-1-deficient MC38 cells increased Tnfα and Ifnγ mRNA expression in wild-type OT-Ⅰ T cells activated with anti-CD3 / anti-CD28 antibodies compared with wild-type MC38 cells, whereas coculture with Gal-1-deficient MC38 cells had no significant effect on B4galt1-deficient OT-Ⅰ T cells.

[0561] In terms of tumor cell cytotoxicity, the present invention compared the specific cytotoxicity of wild-type and B4galt1 knockout OT-I T cells against wild-type MC38 and Gal-1 knockout MC38, respectively (Figure 31A). It was found that wild-type MC38 cells were more sensitive to specific cytotoxicity mediated by B4galt1 knockout OT-I T cells than Gal-1 knockout MC38 cells (Figure 31B). These results demonstrate that Gal-1 transferred from tumor cells to the surface of T cells plays an important role in regulating their cell activation and cytotoxicity, and that B4galt1 knockout can only exert its effect in the presence of Gal-1.

[0562] Figure 31: A. Cytotoxicity of wild-type (left panel) and Gal-1-deficient (right panel) MC38 cells pretreated with OVA oligopeptide by B4galt1-deficient and wild-type OT-I T cells, respectively. p-values ​​are derived from two-tailed T-tests. B. Cytotoxicity of B4galt1-deficient OT-I T cells compared to wild-type OT-I T cells was calculated when targeting wild-type and Gal-1-deficient MC38 cells, respectively. Data were calculated using a 2:1 (T cell:MC38 cell) ratio.

[0563] Subsequently, the present inventors wanted to know whether the same event of tumor cell Gal-1 transferring to the surface of infiltrating T cells and suppressing their immune response could also occur in the tumor microenvironment in vivo. Therefore, the present inventors overexpressed the Gal-1-EGFP fusion protein in MC38 cells to track the transfer of Gal-1 in tumors in vivo. By detecting fluorescence by flow cytometry, the present inventors found that CD8 + T cells had significantly enhanced total GFP signal (Figure 32A) and surface GFP signal (Figure 32B).

[0564] In Figure 32, A. MC38 cells expressing exogenous Gal-1-EGFP fusion protein or control EGFP protein were subcutaneously inoculated into wild-type C57BL / 6J mice. Three weeks later, the tumor-infiltrating CD8 + T cells showed a clear GFP signal only in Gal-1-EGFP tumors. B. Tumor-infiltrating CD8 + Anti-GFP antibody staining on the T cell surface showed obvious GFP signals.

[0565] Furthermore, subcutaneous tumor growth of Gal-1 knockout MC38 cells progressed extremely slowly in wild-type C57BL / 6J mice compared with wild-type MC38 cells ( FIG. 33A ), but was significantly slower in immunodeficient NPG (NOD-Prkdc scid Il2rg null ) mice grew normally (Figure 33B). Different types of immune cells in mice were eliminated by specific neutralizing antibodies (Figure 33C), and then Gal-1 knockout MC38 cells were subcutaneously inoculated into immune-deficient mice. The present invention found that CD8 + and CD4 +Depletion of T cells significantly restored the growth of Gal-1 knockout MC38 tumors, but depletion of NK cells had no effect (Figure 33D). Knockout of MHC-I complex component B2m also restored the growth of Gal-1 knockout MC38 tumors in wild-type mice (Figure 33E). These results indicate that the growth of Gal-1 knockout MC38 tumors is affected by the host immune system CD8 + and CD4 + T cell inhibition, that is, Gal-1 may mediate the inhibition of T cell activity in tumors in vivo.

[0566] In Figure 33, A. Gal-1 knockout MC38 cells and wild-type MC38 cells were subcutaneously inoculated into wild-type C57BL / 6J mice and tumor growth was monitored. p-value is from two-way ANOVA; B. Gal-1 knockout MC38 cells and wild-type MC38 cells were subcutaneously inoculated into immunodeficient NPG (NOD-Prkdc scid Il2rg null ) mice and monitored tumor growth. p-values ​​are from two-way ANOVA; C. Flow cytometry analysis showed that different neutralizing antibodies successfully depleted CD8 in peripheral blood samples of wild-type mice. + T cells (left), CD4 + T cells (middle) and NK cells (right); D. Gal-1 knockout MC38 cells were subcutaneously inoculated into CD8 + T cells, CD4 + Tumor growth was monitored in wild-type mice deficient in T cells and NK cells. p-values ​​are derived from two-way ANOVA. E. Gal-1 / B2m double-knockout and Gal-1 single-knockout MC38 cells were subcutaneously inoculated into wild-type C57BL / 6J mice, and tumor growth was monitored. p-values ​​are derived from two-way ANOVA.

[0567] Based on the results of previous in vitro experiments, we speculated that the distribution of endogenous Gal-1 on immune cells in different tissues in the body may be different. Therefore, the present invention detected the CD8 + Endogenous Gal-1 staining on the surface of T cells. Spleen, peripheral blood, and subcutaneous tumors were collected from wild-type C57BL / 6J mice and stained with anti-CD8 and anti-Gal-1 or isotype control antibodies. CD8 expression in different tissues was detected by flow cytometry. + The expression of Gal-1 on the surface of T cells is shown in Figure 34. The CD8 + Gal-1 is barely detectable on T cells, whereas tumor-infiltrating CD8 + T cells are mainly PD-1 positive, i.e. exhausted CD8+ T cells, whose surface staining is positive for Gal-1.

[0568] Furthermore, wild-type C57BL / 6J mice were subcutaneously inoculated with B2m knockout and Gal-1 / B2m double knockout MC38 cells. Three weeks later, tumors were harvested and stained with anti-CD8a and anti-Gal-1 antibodies. The results showed that compared with B2m knockout MC38 tumors, CD8 + The Gal-1 signal on the surface of T cells was significantly reduced, suggesting that MC38 tumors are CD8 + The source of Gal-1 on the surface of T cells ( FIG. 35 ) yielded similar conclusions to those obtained in vitro.

[0569] Example 7 In vitro and in vivo experiments confirmed that galectin-1 (Gal-1) can be transferred from tumor cells to adjacent CD8 + However, when the B4GALT1 gene inactivated in T cells, Gal-1 transfer was significantly reduced and the effect on CD8 + The inhibitory ability of T cells is reduced. The above results demonstrate a new immune checkpoint mechanism. The present invention may use small molecules to inhibit the binding of tumor-derived Gal-1 to adjacent CD8 + T cell surface, thereby enhancing tumor immunity.

[0570] Example 8: B4galt1 regulates TCR-CD8 colocalization via cell surface Gal-1

[0571] In order to identify the role of B4galt1 in CD8 + The present invention intends to utilize the affinity of β-galactoside and Gal-1 to collect galactosylated proteins on the cell membrane by pulldown, and to identify and compare wild-type and B4galt1 knockout CD8 + Protein abundance in T cells was used to identify the substrate of B4galt1.

[0572] First, a large amount of recombinant Gal-1 protein needs to be purified. Using the E. coli expression system, the His-tagged overexpression vector is transformed into the expression strain BL21. After small-scale test expression, single clones with considerable expression levels are selected, and then large-scale purification is performed. As shown in Figure 36A, the lanes from left to right are the expression strain lysate supernatant, Ni-NTA affinity purification column effluent, Ni-NTA affinity purification column wash solution, protein eluate, protein marker, and quantitative control protein BSA. The target protein size is consistent with the estimate and the bands are clear. A large amount of recombinant Gal-1 (~16kD) is eluted and dialyzed into storage buffer or coupling buffer for later use.

[0573] Figure 36B. OT-I T cells were incubated with 0.5 μg of commercially available recombinant Gal-1 protein or laboratory-purified recombinant Gal-1 protein, followed by staining with anti-Gal-1 antibody and flow cytometry analysis. Figure 36C. OT-I T cells were incubated with recombinant Gal-1 protein from different sources and at different amounts, then stained with anti-Gal-1 antibody, and mean fluorescence intensity was measured.

[0574] Before the formal experiment, the present invention tested the activity of the purified recombinant Gal-1 protein, taking advantage of its ability to bind to T cell surface proteins and be detected by anti-Gal-1 antibodies. 0.5 μg of commercial recombinant Gal-1 protein or laboratory-purified recombinant Gal-1 protein was incubated with OT-I T cells, then stained with anti-Gal-1 antibodies and detected by flow cytometry. Based on the flow cytometric staining results (Figure 36B), OT-I T cells were incubated with recombinant Gal-1 proteins from different sources and in different amounts and then stained with anti-Gal-1 antibodies. The results showed that the binding activity of laboratory-purified recombinant Gal-1 was similar to that of commercial recombinant Gal-1 (Figures 36B-C). Subsequently, NHS-activated Sepahrose was used to incubate and couple with the purified recombinant Gal-1 protein to obtain recombinant Gal-1-Sepharose.

[0575] 1. B4galt1 catalyzes CD8 galactosylation and regulates TCR-CD8 colocalization via Gal-1

[0576] Gal-1-binding proteins were enriched from membrane protein extracts of OT-I T cells using recombinant Gal-1-Sepharose, eluted with lactose solution, and the resulting proteins were analyzed by mass spectrometry to identify their species and abundance. Analysis of proteins with significant differences between wild-type and B4galt1-deficient OT-I T cells revealed that TCRα / β (OT-I) and CD8α / β were both at the top of the list (Figure 37A). KEGG analysis revealed significant enrichment of the TCR signaling pathway (Figure 37B). This suggests that galactosylation of the TCR and CD8 proteins is significantly reduced in B4galt1-deficient OT-I T cells.

[0577] Figure 37A. Volcano plot showing Gal-1-binding proteins identified in wild-type and B4galt1-knockout OT-I T cell membrane proteins. Proteins involved in the TCR signaling pathway are underlined. p-values ​​are from Limma in DEqMS (version 1.8.0). Figure 37B. Bar chart showing KEGG pathways significantly altered in B4galt1-knockout OT-I T cells.

[0578] Western blotting confirmed the reduced binding of Gal-1 to CD8 in B4Galt1-knockout T cells (Figure 38A). Interestingly, the migration rate of CD8β protein in SDS-PAGE differed between membrane proteins of wild-type and B4galt1-knockout T cells, indicating that CD8β is a direct substrate of B4galt1 (Figure 38A). Indeed, treatment with peptide-N-glycosidase F (PNGase F), which cleaves all glycosylation on the protein, abolished the difference in CD8β protein migration (Figure 38B).

[0579] Figure 38A. Western blotting validates the top ten genes from pulldown-MS data analysis. The migration rates of CD8b, Itgal, Ly9, and Lnpep proteins were significantly increased in B4galt1-deficient OT-I T cells compared to wild-type OT-I T cells. Figure 38B. After PNGase F digestion of protein glycans, the migration rates of CD8b, Itgal, Ly9, and Lnpep proteins were consistent in wild-type and B4galt1-deficient OT-I T cells.

[0580] The interaction between TCR and CD8 was measured using fluorescence resonance energy transfer (FRET) assays (Figure 39A). As shown in Figure 39B, addition of recombinant Gal-1 to wild-type OT-I T cells significantly reduced the TCR-CD8 FRET signal, a phenotype that could be reversed by lactose treatment. In contrast, in OT-I T cells deficient in B4galt1, recombinant Gal-1 had no significant effect on TCR-CD8 FRET.

[0581] Figure 39A. Schematic diagram of detecting fluorescence resonance energy transfer between TCR and CD8; Figure 39B. Relative fold change of FRET unit after wild-type and B4galt1 knockout OT-Ⅰ T cells were treated with recombinant Gal-1 or recombinant Gal-1+lactose.

[0582] 2. B4galt1 regulates T cell killing and is dependent on CD8

[0583] Through cell killing experiments, the present invention found that inactivation of B4galt1 affects TCR-T cell-mediated target cell killing, but does not affect CAR-T-mediated target cell killing. Similarly, CD8 knockout does not affect CAR-T killing, which suggests that the phenotype of B4galt1 regulating T cell tumor killing function through Gal-1 is dependent on CD8 (Figure 40A-D).

[0584] Figure 40A. CRISPR / Cas9 knockout of B4galt1 in OT-I T cells significantly enhances in vitro specific cytotoxicity against B16F10-OVA cells. Figure 40B. CRISPR / Cas9 knockout of CD8a in OT-I T cells significantly reduces in vitro specific cytotoxicity against B16F10-OVA cells. Figure 40C. CRISPR / Cas9 knockout of B4galt1 in hCD19-CAR T cells does not affect in vitro cytotoxicity against Nalm6 cells. Figure 40D. CRISPR / Cas9 knockout of CD8a in hCD19-CAR T cells does not affect in vitro cytotoxicity against Nalm6 cells.

[0585] Example 8 Comparison of wild type and B4GALT1 knockout CD8 + The phenotype of T cells in recombinant Gal-1 pulldown-MS and TCR-CD8 FRET. The present invention confirms that CD8, as a catalytic substrate of B4GALT1, and its co-localization with TCR are affected by Gal-1 on the surface of T cells, that is, B4GALT1 inhibits TCR activation mediated by TCR-CD8 interaction through Gal-1.

[0586] Example 9: Lactose enhances CD8 by competing for cell surface Gal-1 + T cell anti-tumor ability

[0587] 1. Lactose can enhance CD8 as a competitive inhibitor of Gal-1 + T cell activity

[0588] In the mammary gland, B4GALT1 interacts with LALBA to form lactose synthase, which transfers galactose to glucose to generate lactose. In vitro experiments have shown that the galactose portion of lactose can compete with and interfere with the function of β-galactosidase in glycoproteins and glycolipids. Therefore, the present invention proposes the hypothesis that lactose and its derivatives may be a new class of immune checkpoint inhibitors, which can compete with CD8 + Gal-1 on the surface of T cells enhances the anti-tumor immune function of T cells.

[0589] Under in vitro culture conditions, lactose significantly enhanced the specific cytotoxicity of OT-I T cells against MC38 cells pretreated with OVA oligopeptide (Figure 41A). Furthermore, in this in vitro cytotoxicity system, lactose significantly increased the expression of cytotoxic factors such as Ifnγ and Tnfα in OT-I T cells, indicating enhanced T cell activation (Figure 41B). On the other hand, lactose treatment did not significantly affect the expression of Ifnγ and Tnfα in OT-I T cells when stimulated with anti-CD3 / anti-CD28 antibodies in the absence of exogenous Gal-1 (Figure 41C).

[0590] Figure 41A. Lactose treatment increased the in vitro specific cytotoxicity of OT-I T cells against MC38 cells pretreated with OVA oligopeptide. Figure 41B. Lactose treatment increased the expression of Tnfα and Ifnγ in OT-I T cells cocultured with MC38 cells pretreated with OVA oligopeptide. The relative expression levels of Tnfα and Ifnγ mRNA were determined by quantitative RT-qPCR. Figure 41C. Lactose treatment had no significant effect on the expression of Tnfα and Ifnγ mRNA in OT-I T cells stimulated with anti-CD3 / anti-CD28 antibodies. The relative expression levels of Tnfα and Ifnγ mRNA were determined by quantitative RT-qPCR.

[0591] In the above-mentioned in vitro killing system, the present invention confirmed through anti-Gal-1 antibody staining that lactose treatment can remove Gal-1 from the surface of OT-IT cells and also reduce the level of Gal-1 on the surface of MC38 cells (Figure 42A-C).

[0592] Figure 42A. Representative flow cytometry analysis of OT-I T cells co-cultured with MC38 cells at a ratio of 2:1 for 8 hours. Figure 42B. OT-I T cells were co-cultured with MC38 cells and then incubated with lactose, followed by staining with anti-Gal-1 antibody, and the mean fluorescence intensity of Gal-1 on the surface of MC38 cells was measured by flow cytometry. Figure 42C. OT-I T cells were co-cultured with MC38 cells and then incubated with lactose, followed by staining with anti-Gal-1 antibody, and the mean fluorescence intensity of Gal-1 on the surface of OT-I T cells was measured by flow cytometry.

[0593] To further verify the mechanism of action of lactose, the present invention allowed OT-I T cells to obtain Gal-1 by adding exogenous recombinant Gal-1 or co-culturing with MC38 cells. It was found that the activity of OT-I T cells was significantly inhibited. At this time, treatment with lactose could reverse the inhibitory effect of Gal-1 on OT-I T cell activity (Figures 43A-B).

[0594] Figure 43A. Recombinant Gal-1 treatment reduces TNFα expression in OT-I T cells stimulated with anti-CD3 / anti-CD28 antibodies, and this inhibitory effect is reversed by lactose treatment. Relative expression of TNFα mRNA was determined by quantitative RT-qPCR. Figure 43B. OT-I T cells were cocultured with wild-type or Gal-1-knockout MC38 cells and then stimulated with anti-CD3 / anti-CD28 antibodies in the presence or absence of lactose. Relative expression of TNFα and Ifnγ mRNA was determined by quantitative RT-qPCR.

[0595] To explore the role of lactose in vivo, the present invention treated cells digested from MC38 tumors in vivo with lactose solutions of different concentrations, and then through anti-Gal-1 staining and flow cytometry, the present invention found that lactose can also significantly reduce the level of Gal-1 on the surface of infiltrating T cells (Figure 44A). In addition, lactose can significantly increase the expression of exhausted CD8 T cells isolated from tumors. + PD-1 + Expression of Ifnγ and Tnfα by T cells ( FIG. 44B ).

[0596] Figure 44A. Cells isolated from MC38 tumors were treated with different concentrations of lactose to compete for the removal of MC38 tumor-infiltrating CD8 + Gal-1 on the surface of T cells. The mean fluorescence intensity of Gal-1 was measured by flow cytometry. Figure 44B. Cells isolated from MC38 tumors were treated with different concentrations of lactose and anti-CD3 / anti-CD28 antibodies were added to stimulate MC38 tumor-infiltrating CD8 + Quantitative RT-qPCR was used to detect the expression of Tnfα and Ifnγ mRNA in T cells.

[0597] 2. Lactose can enhance the activity of CD8+ T cells in the body and inhibit tumor growth

[0598] To explore the effect of lactose on tumors in vivo, we injected lactose solution into the tail vein of wild-type mice subcutaneously inoculated with MC38 tumors every two days and monitored tumor growth. The results showed that lactose injection significantly inhibited the growth of MC38 tumors compared with the PBS control group (Figure 45A) and increased the number of IFNγ-positive CD8 + On the other hand, when the experimental mice were immunodeficient NPG mice, the tumor-suppressing effect of lactose was absent (Figure 45C). Neutralizing antibodies were used to eliminate CD8 T cells in wild-type mice. + T cells can significantly reduce the effect of lactose injection on MC38 tumors, and when CD4 + When T cells and NK cells were treated, the degree of reduction in the tumor suppressive effect of lactose was smaller (Figure 45D). For MC38 tumors with B2m knockout, lactose treatment also had no significant effect (Figure 45E). This suggests that the effect of lactose on tumors in vivo is largely dependent on CD8 + T cells.

[0599] Figure 45: A. Effect of intravenous lactose injection on the growth of subcutaneous MC38 tumors in C57BL / 6J wild-type mice. B. Intravenous lactose injection increases IFNγ infiltration in MC38 tumors. + CD8 + C. Effect of intravenous lactose on the immune-deficient NPG (NOD-Prkdc scid Il2rg null ) Effect of intravenous lactose on the growth of subcutaneous MC38 tumor in mice. + T cells, CD4 + Effects of intravenous lactose on the growth of MC38 tumors in C57BL / 6J wild-type mice with T cell or NK cell deficiency. E. Effects of intravenous lactose on the growth of MC38 tumors in C57BL / 6J wild-type mice with B2m knockout.

[0600] To test whether lactose has an effect on other different types of tumors, the present invention subcutaneously inoculated wild-type syngeneic mice with the breast cancer cell line 4T1 and the mouse colon cancer cell line CT26, and then injected lactose intravenously. The results showed that lactose treatment did not significantly affect the growth of 4T1 and CT26 tumors (Figures 46A-B). These two tumors are considered immune "cold" tumors and have a weak response to immunotherapy. Subsequently, the present invention subcutaneously inoculated two melanoma cell lines, B16F10-Puro and B16F10-OVA, into wild-type syngeneic mice, using B16F10-Puro as a control cell line. Exogenous overexpression of OVA protein caused B16F10 cells to transform from "cold" tumors to "hot" tumors (the latter is considered to have a stronger response to immunotherapy), thereby significantly enhancing sensitivity to lactose treatment (Figures 46C-D).

[0601] Figure 46 shows: A. Effect of lactose on subcutaneous 4T1 tumor growth in BALB / c wild-type mice. B. Effect of lactose on subcutaneous CT26 tumor growth in BALB / c wild-type mice. C. Effect of lactose on subcutaneous B16F10-Puro tumor growth in C57BL / 6J wild-type mice. D. Effect of lactose on subcutaneous B16F10-OVA tumor growth in C57BL / 6J wild-type mice.

[0602] 3. Biological properties of lactose and its derivatives

[0603] To further explore the possibility of lactose as a new immunotherapy drug, the present invention conducted preliminary experiments on the functionality of lactose and its galactosyl groups. The present invention found that in the absence of functional mammalian transporters, lactose could not effectively enter the cell (Figure 47), while the chemical properties of lactose in the circulatory system were stable. Therefore, the present invention infers that the inhibition of tumor immune checkpoints by intravenous injection of lactose is not due to its degraded monosaccharide products (galactose and glucose). Figure 47 uses a parallel artificial membrane permeability assay (PAMPA) to detect the permeability of lactose. Testosterone and methotrexate were used as positive and negative controls, respectively.

[0604] To confirm that the galactosyl group in lactose is the primary functional group for binding to lectins and galectins, the present invention tested lactose, sucrose, N-acetyllactosamine (LacNAc), and lactose-BSA (bovine serum albumin) in a lectin competition staining assay (Figure 48A). Because it lacks a galactosyl moiety, sucrose exhibited no competitive binding properties for ECL, sWGA, or Gal-1 (Figure 48B). Compared to lactose, LacNAc more effectively competed with β-galactosides in the cell surface glycome for binding to ECL and Gal-1, but also bound to sWGA at higher concentrations (Figure 48B). Intravenous injection of LacNAc inhibited MC38 tumor growth in mice (Figure 48C). Because lactose in the blood is rapidly excreted through the kidneys, to stabilize its blood concentration, the present invention conjugated lactose to BSA at elevated temperatures (Figure 48A), potentially improving its pharmacokinetics and slowing its metabolism and excretion in the body.

[0605] Figure 48 shows: A. Structures of lactose, sucrose, N-acetyllactosamine (LacNAc), and lactose-bovine serum albumin (BSA). Coomassie blue staining of an SDS-PAGE gel demonstrates efficient conjugation of BSA to lactose. B. Competition of lactose, sucrose, N-acetyllactosamine, and lactose-BSA for binding to ECL, sWGA, and Gal-1 on the surface of MC38 cells. C. Effect of intravenous injection of LacNAc (20 mM / 250 μL DPBS) on MC38 tumor growth in C57BL / 6J wild-type mice.

[0606] Through in vitro T cell tumor killing experiments, cytotoxic factor detection, and in vivo tumor models, the present invention demonstrates that lactose can enhance TCR-CD8 colocalization and TCR activation by competing for cell surface Gal-1, thereby improving T cell activity and anti-tumor ability. Competitive binding experiments between lactose and its derivatives and lectins indicate that the galactosyl group in these molecules is the primary functional group that binds to Gal-1, providing a molecular basis for further exploring the potential of lactose and its derivatives as anti-tumor drugs.

[0607] The above description is merely a preferred embodiment of the present invention; however, the scope of protection of the present invention is not limited thereto. Any person skilled in the art who, within the technical scope disclosed by the present invention, makes equivalent substitutions or modifications based on the technical solutions and improved concepts of the present invention shall be covered by the scope of protection of the present invention.

Claims

1. Application of galectin-1 as an immune checkpoint in the preparation of tumor immunotherapy drugs.

2. The use according to claim 1, wherein the tumor is an immunoinflammatory tumor.

3. The use according to claim 2, comprising: selecting at least one galectin-1 targeted binder or a derivative thereof as the active ingredient of the drug; And / or, a natural galectin-1 targeting binder is selected, and the tumor cells are allowed to overexpress the precursor of the targeting binder; and / or, the galectin-1 binding site on the surface of immune cells in the tumor microenvironment is determined, and the binding site is reduced, deleted or inactivated.

4. The use according to claim 3, wherein the galectin-1 targeting conjugate is lactose, and the immune cell is CD8 + T lymphocytes.

5. A tumor immunotherapy drug, the active ingredient of which comprises at least one galectin-1 targeted conjugate, wherein the galectin-1 targeted conjugate is a β-galactoside that is soluble and cannot be metabolized outside the host's gastrointestinal tract. The tumor immunotherapy drug according to claim 5 , wherein the tumor is an immunoinflammatory tumor.

7. The tumor immunotherapy drug according to claim 5, wherein the β-galactoside is selected from lactose, lactulose, lactose sucrose, methyl β-lactoside, methyl β-lactoside, 4-O-β-D-galactopyranosyl-D-mannopyranoside, 3-O-β-D-galactopyranosyl-D-arabinose, 2'-O-methyllactose, lacto-N-biose, N-acetyllactosamine or β-D-thiogalactopyranoside, and the derivative of the galectin-1 targeted binder is a conjugate of the β-galactoside and a polypeptide or a pharmaceutically acceptable salt of the β-galactoside.

8. The tumor immunotherapy drug according to claim 5, wherein the galectin-1 targeting binder is lactose or N-acetyllactosamine.

9. The tumor immunotherapy drug according to claim 5, wherein the dosage form of the pharmaceutical composition is an injection preparation. 10 . The tumor immunotherapy drug according to claim 5 , wherein the galectin-1 targeting conjugate is lactose, and the concentration of the lactose in the drug is 50 mM-400 mM.

11. A tumor microenvironment immunoenhancer, comprising at least one expression vector of a galectin-1 antibody or a LALBA promoter. The tumor microenvironment immunity enhancer according to claim 10 , wherein the tumor is an immunoinflammatory tumor.

13. The tumor microenvironment immunity enhancer according to claim 10, wherein the LALBA promoter is selected from the group consisting of substances that increase LALBA levels, substances that enhance LALBA activity, and / or substances that delay LALBA metabolism.

14. The tumor microenvironment immunoenhancer according to claim 10, wherein the LALBA promoter is selected from the group consisting of: a LALBA overexpression vector, nanoparticles carrying the LALBA gene, a viral vector carrying LALBA or its overexpression vector, a PEG-modified protein, protein microspheres, liposomes encapsulating LALBA, and extracellular vesicles carrying LALBA; and LALBA is a substance selected from the group consisting of the LALBA gene, LALBA mRNA, cDNA, LALBA protein, or an active fragment of any of the foregoing. The tumor microenvironment immunity enhancer according to claim 14 , wherein the viral vector carrying LALBA or its overexpression vector is an adenoviral vector or a lentiviral vector.

16. A modified immune cell, wherein the modified immune cell is a T lymphocyte or a NK cell, and the galactosylation level on the surface of the modified immune cell is reduced, deleted or inactivated.

17. The modified immune cell according to claim 16, wherein the modified immune cell is a CD8 + T lymphocytes. The modified immune cell according to claim 16 , wherein the modified immune cell is a TCR-T cell or a TCR-NK cell.

19. The modified immune cell according to claim 16, wherein The galactosylation of the immune cells is significantly reduced, absent or inactivated.

20. The modified immune cell according to claim 16, wherein The immune cells are CD8 + T lymphocytes.

21. The modified immune cell according to claim 16, wherein The immune cells do not express active B4GALT1 protein.

22. The modified immune cell according to claim 16, wherein The B4GALT1 gene of the immune cells is knocked out or inhibited.

23. A pharmaceutical composition for tumor immunotherapy, the active ingredients of which include at least: The galectin-1 targeted conjugate according to any one of claims 5 to 10, and / or the tumor microenvironment immunoenhancer according to any one of claims 11 to 15, and / or the modified immune cell according to any one of claims 16 to 22.

24. The tumor immunotherapy pharmaceutical composition according to claim 23, wherein the active ingredient further comprises at least one substance that induces the transformation of cold tumors into hot tumors.

25. The tumor immunotherapy pharmaceutical composition according to claim 23, further comprising a pharmaceutically acceptable excipient.

26. A combination drug composition for tumor immunotherapy, comprising the tumor immunotherapy drug composition according to any one of claims 23 to 25 and at least one other anti-tumor active ingredient, wherein the other anti-tumor active ingredient is selected from a tumor immunotherapy agent and a chemotherapeutic agent.

27. The combination drug composition according to claim 26, wherein the other anti-tumor active ingredients are selected from 28. The combination composition of claim 26, wherein the chemotherapeutic drug is selected from the group consisting of cyclophosphamide, trabectedin, temozolomide, melphalan, dacarbazine, oxaliplatin, methotrexate, mitoxantrone, gemcitabine, 5-fluorouracil (5-FU), bleomycin, doxorubicin, daunorubicin, epirubicin, idarubicin, valrubicin, paclitaxel, cabazitaxel, docetaxel, topotecan, irinotecan, etoposide, carboplatin, cisplatin, bortezomib, vinblastine, vincristine, vindesine, vinorelbine, isothiocyanate, nitrogen mustard, mitomycin C, fludarabine, cytosine arabinoside; and combinations thereof.

29. A method for tumor immunotherapy, said method being selected from one or both of the following: (a) administering the tumor immunotherapy drug according to any one of claims 5 to 10 to a subject, allowing the drug to enter the subject's tumor immune microenvironment; (b) contacting the tumor microenvironment immunity enhancer according to any one of claims 11 to 15 or the modified immune cells according to any one of claims 16 to 22 with tumor cells of the subject.

30. The tumor immunotherapy method according to claim 29, wherein The tumor is an immunoinflammatory tumor.

31. The tumor immunotherapy method according to claim 29, wherein The tumor is a cold tumor. Before the step, the step further includes converting the tumor into a hot tumor, that is, a tumor with immune inflammation.

32. The tumor immunotherapy method according to claim 29, wherein The administration method described in (a) is intravenous injection.

33. The tumor immunotherapy method according to claim 29, wherein (b) The administration method is intratumoral injection.

34. [Incorporated by reference (Rule 20.6) 27.12.2023] New use of lactose or its derivatives, characterized in that The new use is the use of lactose or its derivatives as an effective ingredient in the preparation of medicines.

35. [Incorporated by reference (Rule 20.6) 27.12.2023] The novel use of lactose or its derivatives according to claim 34, characterized in that: The drug is an anti-tumor drug.

36. [Incorporated by reference (Rule 20.6) 27.12.2023] The novel use of lactose or its derivatives as claimed in claim 34, characterized in that: The medicine is an injection.

37. [Incorporated by reference (Rule 20.6) 27.12.2023] The novel use of lactose or its derivatives as claimed in claim 34, characterized in that: The medicine is for intravenous injection.

38. [Incorporated by reference (Rule 20.6) 27.12.2023] New use of LALBA gene / protein, characterized in that The new use is the application of the LALBA gene / protein in the preparation of a drug, wherein the drug comprises a reagent for overexpressing the LALBA gene / protein.

39. [Incorporated by reference (Rule 20.6) 27.12.2023] The novel use of the LALBA gene / protein according to claim 38, characterized in that: The drug is an anti-tumor drug.

40. [Incorporated by reference (Rule 20.6) 27.12.2023] The novel use of the LALBA gene / protein as claimed in claim 38, characterized in that: The reagent for overexpressing the LALBA gene / protein comprises an isolated nucleic acid molecule comprising a base sequence encoding the LALBA protein.

41. [Incorporated by reference (Rule 20.6) 27.12.2023] The novel use of the LALBA gene / protein according to claim 38, characterized in that: The drug comprises a delivery vector, which is an adenovirus or lentivirus vector.

42. [Incorporated by reference (Rule 20.6) 27.12.2023] The novel use of the LALBA gene / protein according to any one of claims 38 to 41, characterized in that: The medicine is an injection.

43. [Incorporated by reference (Rule 20.6) 27.12.2023] The novel use of the LALBA gene / protein according to claim 42, characterized in that: The injection is an intratumoral injection.

44. [Incorporated by reference (Rule 20.6) 27.12.2023] A novel use of the B4GALT1 gene / protein, characterized in that: The novel use is the use of the B4GALT1 gene / protein in the preparation of a drug, wherein the drug comprises the following reagents: a) an agent for knocking out the B4GALT1 gene or inhibiting the B4GALT1 gene / protein; and / or, b) Immune cells in which the B4GALT1 gene is knocked out or suppressed.

45. [Incorporated by reference (Rule 20.6) 27.12.2023] The novel use of the B4GALT1 gene / protein according to claim 44, characterized in that: The drug is an anti-tumor drug.

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